IMPROVEMENT OF FOOD QUALITY BY IRRADIATION PROCEEDINGS OF A PANEL, VIENNA, 18-22 JUNE 1973 ORGANIZED BY THE JOINT FAO/IAEA DIVISION M Ш a OF ATOMIC ENERGY IN FOOD ¿ p P * m % AND AGRICULTURE

I w J INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1974

IMPROVEMENT OF FOOD QUALITY BY IRRADIATION

PANEL PROCEEDINGS SERIES

IMPROVEMENT OF FOOD QUALITY BY IRRADIATION i

PROCEEDINGS OF A(PANEL ON IMPROVEMENT OF FOOD QUALITY BY IRRADIATION ORGANIZED BY THE JOINT FAO/IAEA DIVISION OF ATOMIC ENERGY IN FOOD AND AGRICULTURE AND HELD IN VIENNA, 18-22 JUNE 1973

INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 1974 IMPROVEMENT OF FOOD QUALITY BY IRRADIATION IAEA, VIENNA, 1974 STI/PUB/370

Printed by the IAEA in Austria August 1974 FOREWORD

Early work on the treatment of foods with ionizing radiation concen­ trated mainly on food preservation. In the course of these studies it became apparent that irradiation may also enhance the technological and/or hygienic quality of foods and .agricultural commodities. On 17-24 June 1973 a Panel was convened in Vienna on this subject by the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture. From the material presented and discussed at the meeting it is clear that significant improvements in technological and hygienic quality may accrue from the application of radiation to foods. It is found, for example, that starch preparations with desirable new technological properties, flours of better baking qualities, food enzymes possessing unusual quality charac­ teristics, and amino-carbonyl reaction products of enhanced anti-oxidative activity can be obtained by radiation treatment of food or of some of its components. The juice yield of some fruits can be increased; the cooking time required for dried vegetables can be reduced. Microbial contamina­ tion of some non-perishable (dry) food ingredients, e.g. spices and gelatin, can be decreased considerably, thus increasing their hygienic quality and avoiding contamination, introduced by these ingredients, in the perishable (high-moisture, low-acid) foods produced with their aid. The Panel felt that, in view of the promising practical potentialities, there is now a need for a more intensive study of the technological aspects, as well .as the underlying chemical, physical and radiation biological phenom ena. The present book contains the papers presented by various experts at the Panel, together with a Summary and Recommendations.

CONTENTS

Irradiation of food and food constituents: chemical and hygienic co n s e q u e n ce s (I A E A -P L -5 6 1 / 1 ) ...... ; ...... 1 J . Schubert Some theoretical considerations on the chemical mechanism of the radiation-induced depolymerization of high molecular c a rb o h y d ra te s (I A E A -P L -5 6 1 /2 ) ...... 39 H. S c h e r z Influence d'une irradiation gamma sur la salubrité et les propriétés te ch n o lo g iq u e s de l'a m id o n de m a'is (I A E A -P L -5 6 1 /3 ) ...... 51 L . Saint-Lèbe, G. Berger, A. Mucchielli Physico-chem ical changes in irradiated (gamma 60Co) inulin (I A E A -P L -5 6 1 / 4 ) ...... 61 Stefania Bachman, H. Zegota Effects of ionizing radiation on gelatin in the solid state (I A E A -P L -5 6 1 / 5) ...... 77 Stefania Bachman, S. Galant, Z. Gasyna, S. Witkowski, H. Zegota The antioxidative effects of gamma-irradiated amino-sugar r e a c tio n p ro d u cts (I A E A -P L -5 6 1 /6 ) ...... 95 M . Fujimaki, M. Morita, H. Kato Aspects of the effect of ionizing radiation on enzymes (IA E A -P L -5 6 1 / 7) ...... 101 T . Sanner, G iz e lla Kovács-Proszt, S. Witkowski Changes in cell permeability due to irradiation: effect of 60Co gamma rays on the phospholipase D enzyme (IAEA-PL-561/8) A b s tra ct on ly ...... 117 W .S . Sherif Immobilization of enzymes by the radiopolymerization of acryl am ide (I A E A -P L -5 6 1 /9 ) ...... 119 K. Kawashima, K. Umeda Compositional and quality changes in some irradiated foods (IAEA-PL-561/10)...... 129 A . Sreenivasan Effects of irradiation on the technological and hygienic qualities of several food products (IAEA-PL-561/11)...... 157 I. Kiss, J. Farkas, S. Ferenczi, B. Kalman, J. B e c z n e r S u m m a ry and R e co m m e n d a tio n s ...... 179

L is t o f P a n el M e m b e r s ...... 187

IAEA-PL-561/1

IRRADIATION OF FOOD AND FOOD CONSTITUENTS Chemical and hygienic consequences

J . SCHUBERT University of Pittsburgh, Graduate School of Public Health, Pittsburgh, Pa. , United States of America

Abstract

IRRADIATION OF FOOD AND FOOD CONSTITUENTS: CHEMICAL AND HYGIENIC CONSEQUENCES. A major objective of food irradiation is to optimize the hygienic and technological qualities while

m inim izing the radiation doses employed. In order to attain or approach these aims it is necessary to

consider how the radiation chemical changes produced in food ingredients can be m odified, m inim ized, or

m axim ized. Compounds formed upon irradiation which are most likely to prove cytotoxic in vitro but

rarely in vivo are those containing a conjugated double bond, especially a , 6-unsaturated carbonyl compounds. These are formed principally upon irradiation of aqueous solutions of sugars and unsaturated fatty acids. Another group of cytotoxic agents arise from irradiation of certain amino acids and nucleic-acid bases when

oxygen is present, resulting in the formation of adducts with radiolytic peroxides, e .g . histidine, which inhibit

bacterial growth. In some cases, the effects of irradiation on food ingredients parallel those produced by non-irradiation

processes. For example, a , 8- unsaturated carbonyl compounds are produced by autoxidation of lipids independently of irradiation, or by simply heating 2-deoxysugars in aqueous solution. Methods for enhancing

the hygienic effects of certain foods while m inim izing the irradiation doses employed are discussed. These are based on the use of selected, non-toxic scavenging agents which can be chosen so as to increase or decrease

the levels of a given free radical such as OH or е "^ . depending on the objective sought.

I. INTRODUCTION

A major objective of food irradiation is to optimize the desirable hygienic and technological qualities while minimizing the radiation doses employed. The consequences of food irradiation depend on several, often inter-related, factors including: (1) water content; (2) pH; (3) chemical and physical nature of the food; (4) conditions of irradiation, e. g. , presence or absence of oxygen, dose-rate and total dose, hardness of radiation, temperature; (5) types of microbial, fungal and other reproducing organisms present; and (6) the effects of pre- or post-irradiation treatment of the fo o d , e.g., heat, cooking, storage conditions. This paper describes experiments, principally on the indirect effects of gamma irradiation in the low to moderate dose range (0-0.5 M ra d), on three important food constituents — carbohydrates, amino acids and lipids. From the hygienic standpoint, the results of these experiments show that two principal classes of compounds, which are cytotoxic in vitro but practically non-cytotoxic in vivo, are formed. One class, arising from

1 2 SCHUBERT the irradiation of aqueous solutions of sugars and unsaturated fatty acid emulsions are a, (3-un saturated carbonyl compounds:

R 2

a, 13- Unsaturated carbonyl compound <

The discussion is devoted mainly to compounds remaining in the medium or food after irradiation. That is, not the volatile fractions, since these obviously no longer influence the chemical or hygienic properties. The other class of cytotoxic compounds arise from the reaction of hydrogen peroxide — whether formed from radiolysis or added after irradiation — with certain amino acids, especially histidine [1], as well as nucleic acid bases and other common food constituents capable of forming adducts, actually chelates, with hydrogen peroxide:

,R H О - O''

H

Hydrogen peroxide adduct

In some cases we find that heating or autoclaving of food constituents, or combined heating and irradiation, causes antibacterial substances to be formed, either de novo or else in concentrations above that produced by irradiation alone. The formation of a, /3-unsaturated carbonyl compounds arises readily from certain unirradiated compounds as, for example, by the auto-oxidation of lipids or simply by thermalization of 2-deoxysugars or glyceraldehyde [2] . The formation of carbonyl compounds takes place in irradiated foods. In particular, their yields in irradiated strawberries and meat are described. In these cases, the radiolytic carbonyl compounds are not of the a, |3-unsaturated carbonyl class nor are they as cytotoxic. Finally, radiation chemical approaches to attain desired chemical, physical and hygienic changes in foods at minimal radiation doses are discussed. These approaches are based on the use of selected, non-toxic radical scavenging agents which can be chosen so as to increase or decrease the levels of those radicals which are largely responsible for the indirect action of radiation, namely the solvated electron, e¡q, and, in particular, the hydroxyl radical, OH. It is hoped that this paper will stimulate thought and discussion on new or modified procedures for improving the technology of food irradiation and, in line with the objectives of the Panel, will give rise to improved practical possibilities for preserving and improving food quality by ionizing radiations. IAEA-PL-561/1 3

II. MEDIUM EFFECTS . '

Organisms and chemicals in general may be damaged or altered by contact with solutions of substances previously exposed to ultra-violet or ionizing radiations. This ability of radiation to produce profound alterations in organisms and molecules indirectly by prior irradiation of their environ­ ment is called a medium effect, or, alternatively, an indirect or after-effect. Since water is a major component of cells, the chemical transformation induced by the passage of ionizing radiations through animal and vegetable tissues and cell media are due to the formation of free radical and molecular products from water. These in turn react with each other and with other constituents. Ever since medium effects were recognized, it has been repeatedly observed,beginning in 1889 by E. Roux, that the cytotoxic and clastogenic (chromosome-breaking) properties of irradiated media can be traced to the sugar components. The unique involvement of sugar and polysaccharides was demonstrated by numerous irradiation experiments in which investigators systematically irradiated individual components of a growth medium for yeast, bacteria and other organisms of plant or animal origin. The investi­ gations of Stone and his collaborators at the University of Texas beginning in the 1940's also showed that the simple addition of hydrogen peroxide with various amino acids and nucleic acid bases added to non-irradiated cultures of Staphylococcus aureus enhanced the mutation rates. Cytotoxic effects have also been observed in extracts, homogenates, and other modes of preparation from irradiated natural products and foods [3] . The author has previously summarized many of the findings on medium effects, particularly on the mutagenicity and cytotoxicity of irradiated foods and food components [3] . Another extensive review appeared not long thereafter by Kesavan and Swaminathan [4] and by Becking [5] . The latter described the radiosterilization of bacteria in nutrient media including foodstuffs and soils. A tabulation of some of the media, including foodstuffs, which have demonstrated medium-effects following irradiation, is given in Table I.

III. RADIATION CHEMICAL CONSIDERATIONS

The chemical reactions taking place in irradiated media or food are initiated by (1) indirect action of the radiolytic products of water decomposi­ tion, namely three primary free radicals — the hydroxyl radical, OH, the hydrated electron, e^q, and the hydrogen atom, H; and molecular products — hydrogen peroxide, hydrogen gas and the hydrated hydrogen ion; and (2) direct action in which chemical bonds are ruptured by the dissipation or transfer of excess energy within the molecules or atoms traversed by ionization tracks. The final products resulting from indirect or direct action are not necessarily the same. The ensuing chemical transformations depend on the rates at which the radical and molecular products react with various molecules in the media, as well as with each other, to yield stable end-products. . Scavenger molecules are often used to remove or generate a radical preferentially by virtue of an 4 SCHUBERT

TABLE I. FOODS AND FOOD CONSTITUENTS WHICH HAVE PRODUCED MEDIUM EFFECTS (CYTOTOXICITY, MUTAGENICITY, CLASTOGENICITY) FOLLOWING IRRADIATION AND THE TEST ORGANISMS AFFECTED3

I. Food Constituents

(1) Carbohydrate solutions (including mono-, di-, and polysaccharides, e.g., agar,histidine solutions in presence of O2 ,pyrimidine bases.

(2) Nutrient culture media

(3) Blood plasma

II. Foods and Natural Products

(1) Fruit juices (pineapple, apple, orange)

(2) Leaves of Vicia faba plants

(3) Potatoes

(4) Ovarian tissue

Organisms Affected

(1 ) Protozoa (9) Barley and onion root tips

(2 ) Spermatozoa (10) Yeast

(3) Chick embryo cells (11) Mammalian cells (e.g., HeLa

(4) Microorgani sms (12) Human fibroblasts

(5) Carrot cells (13) Neurospora

(6 ) Paramecia (14) Drosophila

(7) Water parsnips (15) Seeds of pea and pumpkin

(8 ) Lymphocytes

a See references [3] and[4]for literature citations relative to the items tabulated here. IAEA-PL-561/1 5 especially high and preferential reaction rate with a given radical concomi­ tantly with a low reaction rate for other radicals. Therefore, it is possible to modify deliberately the course of a radiation-induced reaction by addition of one or more selective scavenging agents.

1. W a ter r a d io ly s is

The transfer of energy to water upon irradiation takes place in about 10“16 s and, as described by Draganic and Draganic [6] and others, produces ionization:

Н20 -л/'л» e + H2O and excitation

H ^ o - ^ v v H2 O*

The electrons become thermalized and hydrated in ~10'12 s:

e '------> e¡------> e ¡ q

The H20 + ions react with water by a proton transfer reaction to produce the hydrogen ion and the OH radical:

H 20 ++H 20 ------>H30 + ~+0H

Finally, the excited water molecules yield mainly the hydrogen atom and hydroxyl radical:

H20 * ------>H + OH

The molecular product, hydrogen gas, arises from several sources in clu d in g:

2H20 e âq + eaq - * H2 + 20H ‘

H + H ------H 2

e aq + H ------* H2

Hydrogen peroxide is produced principally by the recombination of the OH radicals:

OH + O H ------» ttjOr, TAB LE I I . RADIOLYSIS' OF WATER Principal reactions leading to the formation of the primary (earliest detected) free radicals and molecular products

H \ + /о н

w

H2O 2 I A E A -P L - 5 6 1 / 1 7

The radiolytic reactions leading to the primary products in water are summarized in Table II. Within ~10'9s, following the passage of high energy radiation, the state of irradiated water is then represented by the expression:

H20-^vV'-> H30 +, OH, e;q, H, H20, H2

The OH and H20 2 are the oxidizing components and ejq, H and H2 are the reducing components. The radiation chemical yield, G, is defined as:

_ Number of species produced or disappearing 100 eV absorbed

For practical purposes it is convenient to express the chemical yield of a substance X in terms of micromoles per litre per kilorad:

1. 04 ^moles/litre produced or disappearing G(X) ~ krad o r

= 1. 04 D (k ra d )X G (X)

The best known yields of the primary products of water radiolysis at neutral pH are given in Table III. Actually, above pH 3, the yields remain relatively constant. It should be noted [7] that the radical yields given in Table III apply only to an infinitely dilute solution and increase with increasing concentration of a reactive solute. At sufficiently high concentrations the effects may, as Hart [7] points out, merge into direct effect and chain reactions.

TABLE III. YIELDS OF THE PRIMARY RADICAL AND MOLECULAR PRODUCTS OF GAMMA-IRRADIATED 0 2 -FREE WATER AT NEUTRAL pHa

Gred=(GeSq+GH) Gegq GH G0H GH2 GH202

3.2 2.65 0.6 2.7 0.45 0.67

a Adapted from data, p. 140 in R ef. [6 ]. SCHUBERT

TABLE IV. REACTIONS OF PRIMARY PRODUCTS FROM WATER RADIOLYSIS WITH DISSOLVED ORGANIC MOLECULES (R = organic group)

HYDROGEN ABSTRACTION RH + "H ---- ^ R* + H2 -

RH + - O H ---- R- + H20

DIMERIZATION

r . + r ------s, R-R

DISPROPORTIONATION (Original compound reformed plus new product)

ch 3ch2 + ch3ch2 ---- > C H 3-CH3 + CH2=CH2

HYDROGEN PEROXIDE FORMATION

R-OH + 02 -----> R = 0 + H02

2H02 -----H202 + 02

HYDROPEROXIDE FORMATION

R- + 02 -----> R 0 0 - + R H -----> R 0 0 H + R-

HYDROGEN PEROXIDE ADDUCT

HC = C — CH2 — CH — COO' HC = C----CH2 --- CH — COO- I 4 51 |+ + HoOp -----^ ¡ i I, ¡3 J NH3 ' ' h ^ 2 42/N\ H N* С /N\ H--0-0--\ >HH \H

HISTIDINE HISTIDINE-PEROXIDE ADDUCT IAEA-PL-561/1 9

For application of radiation chemistry to practical problems it is necessary to use the concept of material balance. From the equations for water radiolysis it is seen, for example, that to form the oxidizing products two water molecules must be decomposed to give one H20 2 molecule while each OH radical requires only one H^O molecule. Hence:

G - h 2 o ~ G o h + 2 G h 2 o 2

Similarly, for the reducing products:

G -H20 = GH+G e¡q + 2 GH 2

Since eaq and H are interconvertible, depending on pH:

% + Î I f — H the reducing radicals are often treated as a unit, i. e. , Gred - (Ge;q + GH). The chemical yield of a substance, in contrast to that of the primary yield, is usually expressed as G(± substance), as in the given example.

2. Hydrogen peroxide formation

Hydrogen peroxide plays an important chemical and biological role in the irradiation of foods and food constituents. When air or oxygen is present along with organic compounds including sugars, amino acids, etc. , the amount of H20 2 produced increases several-fold, and even more so in acidic media. The cytotoxic and mutagenic actions of H20 2 and organic peroxides are well known and will be discussed later in relation to hygienic effects of irradiation on foods. The calculated yields of G(H20 2) in the presence of organic molecules are usually in fair to good agreement with those expected for the major path­ ways for the reactions. The examples which follow illustrate in general the kinds of radiolytic reactions which take place including hydrogen abstraction, and dimerization (see Table IV). Consider, for example, the reaction of ethanol in water solution at pH 7 in the presence of oxygen [8 ]:

- a q

H + o 2 ------> h o 2

C H 3CH 2OH + O H ------► CHgÔHOH + h 2o

о II CH 3CHOH + 0 2 ----- > CH3CH + H 02

2H 02------0 2 10 SCHUBERT

The calculated yield of hydrogen peroxide as deduced from the reactions is:

G(H 20 2) = G H2q 2 + I (G red + G 0H)

From Table III we calculate that G(H20 2) = 3. 6 while the observed yield V was 3. 2. The yield of acetaldehyde, CH3 CH, should be equal to G0h- = 2. 7 from the reactions given and experimentally was found to be 2. 2, indicating that some other reactions are involved. In the absence of oxygen, the alcohol radicals can react with each other rather than with the more rapid reaction with oxygen. This leads to the formation of a glycol:

2CH3CH O H ------>CH3CHOH I CH3CHOH

3. Scavengers

Scavenger concentrations should be at least 10"4 molar to react with all the diffusing species produced in water radiolysis. When possible, concentrations above 0. 1M are used to obtain optimum effects. Some commonly used OH scavengers are formate, HCOO", the simple alcohols such as methanol, ethanol and 2-propanol, and iodide. The scavenging reactions with formate proceed as follows:

H C O O ' + O H ------* • COO’ + H20

H C O O '+ H ------>-C O O '+H 2

The rate constant of formate with e~a q is far less. When metal ions are involved, it must be kept in mind that the • COO" radical is a strong reducing agent, actually comparable to e^ [9] . The alcohols scavenge OH by the reaction:

ROH + O H ------>ROH + H20

ROH + H ------>ROH + H 2

The ROH radical does not react readily with e^ . For scavenging eaq, the gas, nitrous oxide, is commonly used:

n 2° + e¡ q ------* n 2 + O H ' + OH

Nitrous oxide has the advantage of producing only gaseous products and appears to be independent of dose-rate. Note that it converts the e ¡q to

i IAEA-PL-561/1 11

OH, which means that in radiolytic reactions involving the OH radical, one can approximately double the yield of OH radicals for a given radiation dose in the presence of nitrous oxide. Other scavengers for e^q include chloracetic acid, acetone, chloroform and oxygen.

4. Polymers

It is of interest to point out that polymers undergo relatively large physical changes with relatively low doses of radiation [.10] . This is an important consideration for assessing the possible physical changes produced in food subjected to ionizing radiation since foods contain numerous polymeric constituents such as polysaccharides, proteins, DNA, and structures in which polymers are incorporated, such as cell membranes and chromosomes. Consider a polymer given a dose of 1 Mrad and a G-value for the radiation-induced reaction of 5. Then 3. 2 X 1018 chemical changes are produced per gram of material of unit density. If the substance irradiated were a small molecule with a molecular weight of a 100, then a gram contains 6 X 1021 m olecules and the fraction of molecules affected = 3. 2 X 1018/ 6 X 1021 = 0. 5 X 10 3 = 0. 05%. However, if the molecular weight were 106, then one gram contains 6 X 1017 molecules and hence 3. 2 X 1018/ 6 X 1017 = 5. Thus, each molecule would undergo five chemical re a c tio n s . If irradiation ruptures bonds, i. e. cross-links, then the polymer would undergo partial degradation, "softening", with the molecular weight decreasing six-fold. If irradiation induces coupling, that is, cross-linking, the polymer will become "harder", its molecular weight increasing six-fold.

IV. IRRADIATED FOOD COMPONENTS: CHEMICAL AND HYGIENIC ASPECTS

This section summarizes the results of some experiments derived largely from the investigations of our group at the University of Pittsburgh. Initially, we began our work by checking some of the experimental findings reported in 1965 by Steward and his associates [11] who found, in common with earlier investigators, that mutagenic and cytotoxic effects were observed in plant cells grown in irradiated sucrose solutions. Shortly thereafter, Shaw [12] demonstrated that Steward's irradiated sucrose solutions inhibited the growth of cultured human lymphocytes and were clastogenic. Our primary objectives were to characterize and identify the unknown cytotoxic compounds produced in irradiated food components and to correlate the radiolytically induced chemical changes with their concomitant biological manifestations, mainly their antibacterial activity and ability to produce chromosomal aberrations. A summary of our earlier findings on irradiated aqueous solutions of carbohydrates appeared in 1971 [2] while our investiga­ tion on the chemical, cytogenetic and antibacterial properties of irradiated strawberries were reported more recently [13] . In this section I will introduce some of our more recent observations and the results of some of our previously unreported investigations. 12 SCHUBERT

1. Experimental considerations and techniques

Irradiations were carried out under controlled conditions of temperature (25°C ± 1°C) using a cobalt-60 Gammacell-220 delivering 6 krad/min. Solu­ tions were freed of oxygen by flushing with helium containing less than 1 ppm of oxygen. When solutions were irradiated in the oxygenated form, oxygen gas was passed through the solution continuously at a rate high enough so that the production of H20 2 was independent of flow rate of the helium (> 20 cm 3/min). Dose-rates were measured with the FeS04 dosimeter using the values G(Fe3+) = 15. 6 and eFe3+ = 22. 01 at 304 nm and 25°C. Detection and identification of the actual classes of compounds responsible for observed cytotoxic activity were facilitated by the use of a new equation [14] for cell growth by which a linear relationship is obtained between cell growth and inhibitor concentration, C, expressed in units of (jm ole/cellX 10"8 . Growth is described by TT, the initial doubling time, which is the net time required for cells to double after inoculation of saline-washed log-phase cells into the basal medium containing a given concentration or volume of irradiated solution. A single index of relative inhibition is expressed by the concentration of inhibitor at which Tj = 10 hours or approximately ten times the control value, i. e. when no inhibitor is present. The m icrobial test system consisted of Salmonella typhimurium LT2 grown in phosphate-buffered synthetic glucose medium as the carbon source. r Measurement of growth is made turbidimetrically. Sucrose does not contri­ bute to the growth and neither does it inhibit growth in the concentrations em p lo y e d . More recently, in collaboration with Prof. R. J. Duffin, the equation for cell growth I previously described 1.14] was derived by us in a more general and improved manner, yielding even better agreement with experi­ mental data. The new equation is given by the expression:

lo g (Q + b )= X C + lo g b

w here X is the inhibition constant, b the intercept constant, and С is the concentration of inhibitor expressed as pm oles/cell. The term Q = T - T0/T 0 = Tj/Tq where T is the time needed for log-phase cells to double in number following inoculation into the growth medium con­ taining the added inhibitor, and T0 is the time needed for the cells to double in the absence of added inhibitor. The product, ЛЬ, which is characteristic of the compound, is used for structure-activity correlations. For the present, however, I will employ the earlier formulation because it yields similar results and because most of our data so far have been correlated by it. Cytogenetic effects in vitro in mice and rats were carried out using a modified short-term human lymphocyte culture. The method employs three or four drops of blood from a finger prick incubated in 5 ml of Eagle's minimum essential spinner medium with phytohaemagglutinin at 3 7°C for 2 days. At this time an irradiated preparation is incorporated in the culture medium and the culture incubated for an additional 24 hours, at which time the cells are treated with colcemid and harvested. In the in-vivo experi­ ments cytogenetic measurements are made on cells obtained by serial aspirations of bone marrow from the femur. Complete details on the cytogenetic and scoring techniques employed are given in Ref. [3]. IAEA-PL-561/1 13

Chemical analyses and evaluation included standard gas chromatography- using the trimethylsilyl (TMS) derivatizing agent (Tri-Sil, Pierce Chemical Co. ) and mass spectrometry. Coupled gas chromatograph-mass spectro­ metry analyses were carried out using an LKB 9000 apparatus. Total carbonyls were determined by our modified 2, 4-dinitrophenyl- hydrazone (2,4-DNP) method which could be carried out rapidly in the presence of carbohydrates without prior separation 115]. The method also enables us to analyse for small amounts of dicarbonyls such as glyoxal and methylglyoxal. Other techniques and experimental methods are mentioned where in d ica ted .

2. Irradiated carbohydrates

(a) Aqueous solutions

In Steward's investigations [11] it was found from a systematic study of the individual components of the basal medium that the inhibitory effects on cell growth were due to the sucrose moiety of the cell medium. They also demonstrated that the pH of irradiated (nearly O2 -free) sucrose solu­ tions dropped considerably — from pH 7. 0 to pH 3. 3 after irradiation with 2 Mrad and that autoclaving of the irradiated (note acid) solution enhanced the cytotoxic properties. In order to identify the types of compounds responsible for the cytotoxic effects we tested solutions of irradiated sucrose and a variety of mono­ saccharides for antibacterial activity under different conditions (e. g. , radiation dose, with and without autoclaving, effect of added compounds such as amino acids on the cytotoxic properties of irradiated sugars). The chemical studies included examination of the pH-dependent u. v. spectra, determination of carbonyl yields, gas chromatography, etc. Before proceeding to the presentation of some of the details of our findings it is well to summarize the highlights of our findings to date since we can now explain a good deal of the radiation chemistry of normal aldoses, e. g. , glucose, mannose, ribose, and identify the class of products causing most of the biological effects. Contrary to most reports [16] in the literature, irradiation of sugars in vacuo does not yield significant amounts of small molecules such as formaldehyde, glyoxal, glyceraldehyde, i. e. chain scission scarcely occurs, at least in radiation doses up to 5 Mrad. Rather, the radiolytic products are mainly derived by reaction of OH radicals with the sugar, leading to the oxidation of hydroxyl groups, generally with concomitant loss of a neighbouring hydroxyl group. The predominantly biologically active and ultra-violet absorbing products are dicarbonyl sugars which convert to a, (3-un saturated carbonyl sugars by enolization or dehydration upon heating. Carbonyls are produced in irradiated glucose (5. 8 X 10"2 M) for example, with G(carbonyls) = 1. 0. The yield of biologically active carbonyl sugars is increased or decreased upon irradiation in the presence of appropriate scavengers. The a, ¡3-unsaturated carbonyl sugars are also formed without irradiation when aqueous solutions of 2-deoxy sugars are heated or autoclaved. Two other products formed in relatively good yield in irradiated glucose are 2-deoxygluconolactone (G s 0. 6) and gluconic acid (G a= 0. 2). At pH 7 14 SCHUBERT

the 2-deoxygluconolactone becomes 2-deoxygluconic acid. The splitting of irradiated disaccharides such as sugar is also an OH mediated reaction. Upon irradiation in vacuo the D-sucrose molecule first splits into D-glucose and D-fructose. Immediately following irradiation, more glucose than fructose is present though their concentrations become about equal within a few days. This indicates that glucose reacts more readily for a short period with the secondary products of irradiation — a fact supported by our studies showing that the u. v. absorption spectra of glucose changes more rapidly with time than that of fructose. The slow increase in the concentration of fructose and glucose is due to acid hydrolysis since the solutions had a pH of 3.5. The concentrations of fructose and glucose do not change when the irradiated solution is vacuum evaporated to one-tenth its original volume. Autoclaving doubles the fraction of irradiated sucrose hydrolysed. The amount of glucose-fructose formed immediately after irradiation is less when the sucrose solutions are irradiated oxygenated. In the 02-free irradiated (2 Mrad) sucrose solution containing 20 m g/m l the total amount of fructose plus glucose formed is 0. 87 m g/m l or 4. 3% of the original sucrose present while only 0. 25 m g/m l or 1. 25% of the sucrose was converted to fructose plus glucose in the oxygenated system. Howéver, from direct analyses of sucrose, the total fraction of sucrose decomposed in the oxygenated sucrose system was the same as in the 02-free system, namely 5%. The G(glucose) and G(fructose) yields were determined for irradiated, 02-free sucrose solution in the presence and absence of scavengers for 0H (N20) and eaq 1.17] at doses beginning as low as 100 krad. The results are given in Table V. The radiolytic decomposition was linear up to about 300 krad and then the yield curves of glucose and fructose showed an increased slope, presumably because of the increased acidity and the concomitant

TABLE V a. CALCULATED G-VALUES FOR D-GLUCOSE AND D-FRUCTOSE PRODUCED FROM GAMMA-IRRADIATED D-SUCROSE (0. 028M) Dose-rate = 5.46 krad/min

G(glucose) G(fructose)

Og-free 2.1 1.4

N20 (saturated) 3.6 2.3

KI (0.028 M) 0.4 0.4

a Data from Master's thesis of M. A. Nardi, University of Pittsburgh, Graduate School of Public Health, 1972. IAEA-PL- 561/1 15

TABLE VI. INITIAL G (CARBONYL) VALUES FOR 0.058M SOLUTIONS OF UNBUFFERED SUCROSE, GLUCOSE AND FRUCTOSE IRRADIATED EITHER IN THE ABSENCE OF OXYGEN OR IN THE PRESENCE OF NITROUS OXIDE

Sugar G(carbonyl)_q ^ G(carbonyl)N 0 g n2o/g-o2

Sucrose 1.35 2.13 1 .58

Glucose 1.00 1.70 1 .70

Fructose 1 .35 2.08 1 .54

Time (Hours)

F IG .l. Inhibition of S. typhimurium LT 2 by irradiated (2 Mrad), oxygen-free solutions of sucrose

(2% w t/v o l., 0.058M ) which were autoclaved after irradiation. The numbers in parentheses represent the m illilitres of irradiated, autoclaved sucrose incorporated in the growth medium whose total volume

was 5.0 m l. 16 SCHUBERT conversion of the relatively inactive eàq to the H radical which also is involved in carbonyl formation from sugars [2] . The yields of glucose and fructose depend on the sucrose concentration, increasing until about 1% sucrose. Beyond this value the yields become independent of sucrose concentration, at least to 5% (0. 14M). Since the u. v. spectra of irradiated sugar solutions are characteristic of carbonyls [2] , we ran analyses for carbonyls in irradiated, 0 2-free sucrose, glucose and fructose in the presence and absence of N20. The results illustrate the role of the OH radical since the carbonyl yield increased considerably in its presence. In the presence of OH scavengers such as formate, however, the carbonyl yields drop several-fold (> 5 times). The initial G(carbonyl) values are summarized in Table VI. In the presence of oxygen, the carbonyl yield for irradiated sucrose is much larger than observed for irradiated, 0 2-free sucrose, namely G(carbonyl)o2 =2.1 c o m ­ pared with 1.35. There is little doubt that in oxygenated sugars an appreci­ able fraction of carbonyls arises from hydrolysis of organic peroxides. Our m icrobiological test system provided a good deal of information and contributed to the elucidation of the compounds responsible for the cytotoxic properties of irradiated sugars. As shown in Fig. 1 the inhibition of S. typhimurium growth by irradiated, autoclaved sucrose solution was concentration dependent.

CARBONYL CONCENTRATION x 1 0 8 (/¿mol/cell)

FIG. 2. Inhibition of the growth of S. typhimurium LT, b y 60Co-gam ma-irradiated sucrose solutions

(0 .058M) as a function of the radiolytic carbonyl content. During irradiation at 25°C + 1°C and 6. 7 krads/min the sucrose solutions were kept in nearly full glass-stoppered bottles. All samples, except that given 521 krads

which was N 2 О saturated, were irradiated 0 2 -free and were unbuffered. All solid points represent solutions autoclaved after irradiation:

(♦ - 104 krads); ( ■ ,□ - 313 krads); (А, Д - 417 krads); (© , O - 521 krads); (▼ , V - 2000 krads). IAEA-PL-561/1 17

The growth inhibiting properties of irradiated sugar solutions increase upon heating, especially with time of heating at a given temperature. Note that the irradiated oxygenated sucrose solutions are much more inhibitory than the corresponding 0 2-free solutions, reflecting, most probably, the fact that the carbonyl yields are nearly double, as mentioned previously (Go2 = 2. 1 and G-o2 = 1. 35 for sucrose). The dependence of the growth inhibitory action of sugar on total carbonyl content is strikingly shown in the plot of log T¡ versus carbonyl concentra­ tions in Fig. 2 12]. The data also show the enhanced effect of autoclaving. Note the linear relationship between Tj and carbonyl levels over the range of 104 krad to 2000 krad. Actually the relationship holds for radiation doses at least as low as 50 krad to at least as high as 5000 krad. All other irradiated monosaccharides tested gave results similar to that shown for s u c r o s e . A large number of known compounds were tested for antibacterial activity. A structure-activity relationship using compounds of known structure was obtained by utilizing the intercept at Tj = 10 hours. The resulting data enabled us to identify the class of compounds producing the observed antibacterial activity. From Fig. 3 and from corresponding data on irradiated sugars we were able to identify without ambiguity the fact that the cytotoxic compounds produced in irradiated sugars were a, (3-un saturated carbonyl compounds. This conclusion was verified by u. v. spectrophotometry [2] . The fact that the carbonyl compounds found in irradiated sugars are formed from the original sugar without a change in chain length was demonstrated by the absence of significant amounts of compounds having less than the number of carbon atoms present in the original sugar. Our carbonyl assay is capable of detecting < 2 pg/m l of glyoxal, for example, but none was found. Gas chromatographic analyses also showed the

k e t o n e s } SATURATED 0R NON-CONJUGATED AICOHOLS >1 GLYCOLS______

ALDEHYDES (SATURATED OR NON-CONJUGATED

а . Э -UNSATURATED ACIDS

GLYOXAL DIMETHYLGLYOXAL

a , /3 -UNSATURATED ALDEHYDES I

a,/9-UNSATURATED KETONES I 1 I I 1 I I I I______I I 1 I I I i 11______I I I I II 1 I 0.1 0.2 0.4 0.6 1.0 2 4 6 8 10 20 40 100 Cioh

FIG .3, Relative inhibition of_S. typhimurium LTg growth by different classes of compounds expressed in terms of C -^ , i.e . the concentration of inhibitor at which the initial doubling tim e, Tj, equals 10 hours. 18 SCHUBERT

TABLE VII. FORMATION OF a, /3-UN.SATURATED CARBONYL COMPOUNDS BY THERMAL DEHYDRATION AND/OR ENOLIZATION

0 0 O H 0 Il II enolization i II - .... ->■ u _ r H3C - С - С - H = С - С - H а

METHYL GLYOXAL a, 3-UNSATURATED CARBONYL

OH OH 0 0 1 1 II H20 9 II H2C - с - с - Н ... ~ нзс - C - С - H

DL-GLYCERALDEHYDE METHYL GLYOXAL

H H 0 H 0 i i и H20 У i и H 3 c - с - с - с - н — --- > H3C - С = С - С - H

ALDOL CROTONALDEHYDE

H - С = 0 H - С = 0

н2с H - С i II н - С - ОН ■HpO H - с ^ 1 н - с - он H - с - O H

н2с - он H2C - O H

2-DEOXY-D-RIBOSE a,g-UNSATURATED ALDEHYDE

absence of small compounds. No carbonyls were found upon distillation, thus ruling out the non-hydroxylated monocarbonyls such as propionaldehyde, butylaldehyde, etc. The yield of dimers in irradiated 02-free sugars was surprisingly low. Using gas chromatography, we assumed that any substance which eluted at temperatures above 210°C was dimeric. In irradiated, 02-free D-glucose we found G(dimer) = 0. 05 - 0.1, which accounted for less than 5% of the radiolytic products of D-glucose. We found that many known compounds became antibacterial upon heating. For example, glyceraldehyde, whose antibacterial activity is nearly entirely due to its a, (3-unsaturated carbonyl content (3% at room temperature and 26% after autoclaving). Other compounds which became highly inhibitory with Cioh corresponding to a, /3-un saturated carbonyl compounds included 2-deoxy-D-ribose and aldol as shown in Table VII along with the reactions IAEA-PL-561/1 19 showing that heating causes them to lose water as the first step to the formation of a, |3-un saturated carbonyl compounds. We have identified all the 5-carbon a, ¡3-unsaturated carbonyl sugars formed by the thermaliza- tion of 2-deoxy-D-ribose, 2-deoxy-D-glucose and 2-deoxy-D-galactose [18]. In view of the apparent enolizable nature of the cytotoxic carbonyls from irradiated sugars, we were able to devise a procedure to separate them from glucose and other neutral and non-enolizable products. The procedure for isolation used an anion-exchange column since, at alkaline pH, enolate formation takes place. We were thus able to obtain the biologically active carbonyls free of glucose. The carbonyl fraction gave the u. v. spec­ trum typical of irradiated glucose with the same pH dependency. The m icro­ bial test gave а С щ identical to that of irradiated glucose. When the solution containing the biologically active fraction was frëeze- dried, trimethylsilylated and subjected to gas chromatography a chromato­ gram was obtained showing nine distinct peaks. From gas chromatography, mass spectrometry and chemical synthesis we were able to identify D-gluconic acid and 2-deoxy-D-gluconic acid lactone as products with relatively high yield as well. Since irradiated D-mannose gives 2-deoxy-D-gluconic acid lactone as does glucose but not galactose it was clear that stereochemistry at C-4 had been maintained, while stereochemistry at C-2 had been lost. We were able to obtain clear separations of the gluconic and 2-deoxygluconic acid lactone peaks by adjusting the effluents from anion exchange to pH 7.5 wish sodium carbonate. This converted 2-deoxy-D-gluconic acid lactone to the free acid. Since gluconic acid at low pH exists as a mixture of two lactones and the free acid, it is difficult to resolve the single 2-deoxy­ gluconic acid lactone peak from that of the gluconic acid peaks when derivatization is carried out at low pH.

TABLE VIII. pH OF AQUEOUS SOLUTIONS PREPARED FROM IRRADIATED SOLID SUCROSE3 The sucrose was dissolved in distilled water to give a 2% solution

Irradiation Atmosphere Radiation Dose 0 2 Mrad 5 Mradb

Helium 6.99 5.07 4.44

Air 6.65 5.11 4.48

Oxygen 6.85 4.84 4.54

a The 2 Mrad samples were dissolved in water eleven days post­ irradiation while solutions of the 5 Mrad samples were pre­ pared thirteen days post-irradiation. The pH's were read while helium flowed through the solutions.

b Analyses for H2O2 were made in the 5 Mrad samples by the titanium sulfate method. All of the solutions had about 1 yg/ml of H2O2 . 20 SCHUBERT

After.freeze-drying and trimethylsilylation, only 2 peaks were observed upon gas chromatography, the major peak being 2-deoxygluconic acid and the minor peak D-gluconic acid. These conclusions were confirmed by mass spectrometry. The presence of 2-deoxy-D-gluconic acid lactone in irradiated D-glucose has been independently demonstrated recently by Kawakishi and Namiki [17] though no G-values were determined.

(b) Reaction mechanism

At this point it is indicated that the radiation chemistry of irradiated sugars by which the acids are formed is similar to that proposed by Hartmann et al. [19] for the radiolysis of 2-deoxy-D-ribose. The reactions leading to gluconic acid and 2-deoxy-D-gluconic acid from irradiated glucose are as follows:

CH9OH CH2OH Disproportionation

OH o r H2Q 2______^ (OH >== О

HO I OH OH

D-gluconic -H2o acid lactone v HOH2 С (3) CHoOH ) —О G lu co s e О -> f\p*y— О + [ Glucose]

HO HO

2 - deoxy- D- gluconic acid lactone

We thought that 2-deoxy-D-gluconic acid lactone might undergo thermal dehydration in aqueous solution as do 2-deoxysugars. The compound formed might then be expected to contribute to the observed ultra-violet spectrum of irradiated D-glucose. However, no ultra-violet spectrum was observed under any conditions (autoclaved or non-autoclaved in neutral or acidic solution). The mechanism of the formation of the enolizable, a, ¡3- un saturated carbonyl sugars have been described previously [2] .

(c) Solid irradiated sucrose

Solid sucrose crystals were irradiated to a maximum dose of 5 Mrad in the presence of air, helium and oxygen. TMS derivatives were made from each sample and total carbohydrates were determined by gas chromato­ graphy. Within the limits of experimental error no decomposition could IAEA-PL-561/1 21 be detected from the measurement of the sucrose peaks. However, in all three cases small quantities of fructose and glucose were detected. The actual yields, which were approximately equal in each sample are: (1) fructose — 0. 14 wt%; (2) glucose — 0.17 wt%. If it is assumed that fructose and glucose represent the majority of radiolytic products, then only 0.3% deomposition occurred as a result of a 5-Mrad dose of radiation. The only visual evidence of any radiolytic decomposition was the light amber colour of the irradiated sucrose crystals, which is presumably due to radiation-induced colour centres in the crystal structure. When solid irradiated sucrose is dissolved in water to give a 2% solution, the resulting pH shows that some acid formation took place. Further, the higher the radiation dose the lower the pH (Table VIII). Acid formation in solid, irradiated carbohydrates has been reported by Lofroth [22] . When the 2- and 5-Mrad solid irradiated sucrose samples were dissolved in water to give a 2% solution, 2. 5-m l samples were added to 2. 5-m l volumes of the growth media for testing of the antibacterial action. Only a slight inhibition was observed, though the inhibitory action was greatest in the samples irradiated in oxygen and least in helium. It is likely, of course, that a more sensitive test system, e. g. , reduction in bacterial number, would have produced a more marked inhibition. Irradiation of solid polysaccharides leads to bond rupture with values of G(bond rupture) ~ 5 - 10 but without cross-linking. While degradation appears enhanced in the absence of oxygen, post-irradiation effects can make big modifications. For example, if irradiation is carried out in the absence of oxygen and stored oxygen-free, then no post-irradiation changes occur. However, if stored in dry oxygen, the viscosity falls continuously for about ten days. In the presence of oxygen and water vapour, the after-effect is said to be suppressed L20] . In fact, very complicated changes occur in the irradiation of natural biopolymers. Peroxy derivatives and H20 2 formation can lead to degradation depending on whether irradiation has been carried out in the presence of water vapour, e.g. dextran[2l]. Irradiated simple polycrystalline carbohydrates consume oxygen when dissolved in water. It appears that aqueous solutions formed from the irradiated solid carbohydrates contain organic peroxides and a large variety of neutral and acidic compounds [22]. Phillips [16] has recently reviewed the radiolysis of crystalline carbohydrates.

3. .Irradiated lipids

The oxidation of unsaturated fatty acids produces a, /3-un saturated carbonyl compounds [23] as does the irradiation of compounds such as methyloleate 124]. Irradiation of lipid emulsions in the presence of oxygen proceeds by way of a straightforward mechanism [24, 25] . The majority of the reaction is assumed to take place on the unsaturated portion of the lipids in that abstraction of an allylic hydrogen is greatly favoured over abstrac­ tion of a saturated hydrogen. Reaction is generally assumed to take place via a chain mechanism where the initiating step involves abstraction of an allylic hydrogen by a hydrogen atom or hydroxyl radical followed by addition of oxygen to give a hydroperoxyl radical (Eq. 1). The chain reaction is 22 SCHUBERT propagated by abstraction of an allylic hydrogen by the hydroperoxyl radical (Eq. 2), while termination takes place by destruction of the radicals via dimerization or disproportionation.

H2O ^^H ,O H ,e¡q, H20 2

H R - CH= CH- CH2-R ' ------► R - CH = CH - CH - R 1 (1) OH

R - CH = CH - CH - R ' + 0 2 ------R - CH = CH - CH - R '

O-O’

0 - 0 О-OH I I R - CH = CH - CH -R ' + R - CH= CH- CH 2 -R' R - CH = CH - CH - R ' + R - CH = CH - CH - R '

(2 )

The initially formed peroxides are cytotoxic, and production of peroxides by radiation has been proposed as one of the causes of radiation damage to the living organism. However, peroxides are, in general, unstable so that formation of peroxides, per se, are not likely to be implicated as cytotoxic compounds in irradiated foods. Breakdown products of the hydro­ peroxides, however, are frequently carbonyl compounds and, particularly in the case of doubly unsaturated acids such as linoleic acid (I), a, ¡3-unsaturated

(I) ketones have been observed [25] . Autoxidation of emulsions of methyl linoleate gives three primary hydroperoxides [24] (Eq. 3).

ООН А Л Л 0? / W V R' АЛЛА R R 1 ----- 2-* R I + R I R'+R --- V V

ОО Н ОО Н (II) (III) (IV)

R = CH3(CH2)3, R ’ = (CH^gCOOCHg

In polar media, i. e ., water, all of these hydroperoxides break down to give significant amounts of a, /З-unsaturated carbonyl compounds. Of particular interest is hydroperoxide(III) which can break down not only to trans-2- octenal(V) (Eq. 4), but also is degraded to trans-4-hydroxy-2-octenal (VI), probably by way of a second oxidation (Eq. 5). IAEA-PL-561/1 23

н AMA ДА/ АЛАЛ •R i R 1 R R I R' (V) О О ООН н 2о онI л л л л ДА/ (4) R , R 1

О О R' + 'О Н

0-0

Ог ' '— ' '—‘ ' О9 R R 1 — R — — R 1 — 2-» R R 1

RH НО-О ООН /\J\J\ R R R' R'

о 9 (5)

ООН

R, О о Н2Р R 1 R R II Н \ л л Н о - о ‘ I Ь- о .'с = с А е ОН

(VI) Compounds V and VI are both a, (3-unsaturated aldehydes. Moreover, VI has been found to be a potent cytotoxic agent. The same hydroperoxides, II-IV, are formed by gamma irradiation of emulsions and once formed decompose in the same manner to give a, 0-unsaturated carbonyls 124] . Consequently, biologically active radiation products can be assumed to be similar if not identical to the already characterized products from lipid autoxidation. As Schauenstein [23] pointed out, when the lipids are present in a water suspension, the carbonyls accumulate in the water phase. In the absence of water, the carbonyl compounds are formed only in minute yields. When water is present the water-soluble compounds, which include a, ¡3-un saturated carbonyl compounds, constantly pass into the water phase and are not available for further reaction with the unsaturated fatty acid phase, thus enabling them to accumulate to appreciable levels. These 24 SCHUBERT observations are of importance in considerations involving the formation of non-volatile cytotoxic carbonyls in foods containing fats since the hygienic properties of the food after irradiation can be.expected to be influenced. This aspect is discussed in a subsequent section dealing with carbonyls in irradiated foods.

TABLE IX. ANTIBACTERIAL ACTION OF "STAGNANT" IRRADIATED (2 M ra d s) AM INO ACIDS

Amino Acid Room Temp. Autoclaveda (0.02M in H 0) 2 pH 3 pH 7 pH 3 pH 7

Alanine3 0 0 0 0

e-alanine + 0 0 0

Arginine3 0 0 0 0

Aspartic ++ 0 ++ 0

Cysteine n.t.d — n.t. —

Glutamic ++ 0 ++ 0

G1 ycineb 0 0 0 0

Hi stidi ne +++ 0 0a 0a

Lysine + 0 0 0

Phenylalanine n.t. 0 n.t. 0

Proline 0 0 0 0

Serine + 0 0 0

Threonine0 + 0 0 0

Valinec + 0 + 0

a The non-irradiated pH 3, with and without autoclaving, solution delayed growth for an hour.

b When non-irradiated glycine was autoclaved at pH 3, a delay in growth for about one hour was observed.

c Addition of catalase (10 ug/ml ) eliminated observed inhibition.

d n.t. = not tested. IAEA-PL-561/1 25

TABLE X. CARBONYL LEVEL OF IRRADIATED AMINO ACIDS

Total Carbonyls Amino Acid Initial Cone. (M) I Breakdown Produced (yg/ml)

Aspartic Acid 0.0505 30.9 15

Glutamic Acid 0.0507 19.3 24

e-Al a ni ne 0.104 — 25

Threonine 0.104 18 129

4. Irradiated amino acids

Fourteen different amino acids (0. 02M) were irradiated with 2 Mrad at pH 3 and pH 7 when in contact with air. The effect of autoclaving after irradiation was also tested and the results are summarized in Table IX. None of them proved inhibitory to bacterial growth (S. typhimurium) when irradiated at pH 7, whether or not they were subsequently autoclaved. When irradiated at pH 3, however, eight of the amino acids proved inhibitory to some extent. Of these, histidine proved to be outstandingly inhibitory. When the pH 3 irradiated solutions were autoclaved, antibacterial activity of histidine, lysine, j3-alanine, serine and threonine largely dis­ appeared (Table IX). However, autoclaving did not affect the inhibitory action of the two dicarboxylic amino acids, aspartic and glutamic. The degree of decomposition and carbonyl formation were determined on four of the amino acids which proved antibacterial on irradiation. The amino acid assays were carried out by gas chromatography. The results are summarized in Table X. Despite the large concentration of carbonyls in threonine, only a small amount of inhibition was obtained. Upon auto­ claving, the inhibition was lost. It appears, therefore, that the kind of carbonyls produced in threonine are not hydroxylated a, j3-unsaturated carbonyls but volatiles, e. g. , propionaldehyde and non-volatile keto acids. A similar decrease in inhibition is observed when solutions of propion­ aldehyde are autoclaved before testing for antibacterial activity. The produc­ tion of volatile carbonyls from irradiated protein-containing foods is well known and discussed later. It is also clear that the carbonyls produced in irradiated sugars and lipids are largely non-volatile because they are usually hydroxylated. Since irradiated histidine proved highly inhibitory when irradiated in the presence of air or oxygen, we investigated it in some detail. When solutions of histidine are irradiated Ог-free, no antibacterial action is observed. It is known, however, that histidine is particularly susceptible to deamination after irradiation. Several derivatives of imidizole are formed along with amino acids upon irradiation [26]. From the nature of these products formed and their concentration, little or no inhibition would be expected in our test systems. 26 SCHUBERT

FIG, 4. Growth-rates of S. typhimurium LT 2 as affected by the incorporation in the growth medium of gamma-irradiated solutions of oxygenated and oxygen-free solutions of histidine. Aqueous solution of

DL-histidine (free base), 0.058M and pH 7.5, were irradiated with the controls - histidine-free solutions

at the same pH. After irradiation the irradiated solutions were diluted with equal volumes of the growth

m edium , hence the concentration of histidine in the growth medium = 0.029M .

Explanation of symbols:

X 25W O and X 100 WO = oxygenated water irradiated with 25 krads and 100 krads, respectively.

X 2sHisWHe = oxygen-free histidine receiving 25 and 100 krads radiation, respectively.

X 25HÍSWO and X1CoHisWO = oxygenated histidine solutions irradiated with 25 krads and 100 krads, respectively.

5. Peroxide adduct formation

When histidine solutions are irradiated in the presence of oxygen at relatively low doses (~ 20 krad), the solutions become highly inhibitory. The amount of H202 formed is too low to account for the observed inhibition (Fig. 4). We carried out numerous investigations in which the concentrations of H20 2 and of histidine were varied (e. g. Fig. 5). It turned out 113] that the inhibitory action of histidine irradiated in the presence of oxygen is due to the formation of a stable adduct with H20 2 via chelation, i. e. , ring form a­ tion involving hydrogen bonding of the peroxide oxygens. The structure is shown in Table IV. We have verified this structure by synthesis of the adduct and crystallographic analysis. IAEA-PL-561/1 27

FIG. 5. Inhibition of growth of typhimurium LT 2 by different concentrations of DL-histidine in the presence of a fixed concentration of hydrogen peroxide. Initial concentrations: H 2 0 2 = 1.5X10"4M

(5.0 jig/m l), His49 = 1.16 X 10 ”3 M; Hisis = 2.9 X 10~ 3M , HÍS4 = 1.16 x 10' 2 M; HiSi = 2.9 X lO '^M .

Time ( Days)

F I G . 6. Effect of different compounds on the rate of disappearance at 37°C of H 2 0 2 from a phosphate- buffered, pH 7.0, glucose solution. Initial concentrations of components are: glucose = 2. 8 X 1 0 "2 M ; H 20 2 = 5.9 xl0“4 M ; sodium monohydrogen-dihydrogen phosphate = 0.05M ; test compounds = 0.02M , 28 SCHUBERT

TABLE XI. INHIBITION OF GROWTH OF Salmonella typhimurium LTZ BY AMINO ACIDS AND RELATED COMPOUNDS IN THE PRESENCE OF ADDED H20 2 Amino acid = 2. 9 X 10"2; H20 2 = 2.9 X 10"4 (10 j^g/ml); pH = 7

Compound3 + H202 Inhibitory Action

a-alanine + 3-alanine 0. Arginine <0b Asparagi ne <0b Aspartic

a None of the compounds tested were inhibitory in the absence of H2O2 . The H20 2 alone was slightly inhibitory.

b Presence of compound actually reduced inhibition caused by H2O 2 control.

The electrical charge on the adduct is important since this determines the ability to transport peroxide into a cell. The histidine-peroxide adduct is neutral, while that of malonic acid has two negative charges, hence the latter actually acts to eliminate or reduce the inhibitory action of hydrogen peroxide. The most stable adducts are those compounds which can form a ring or chelate structure, while monofunctional compounds such as alanine, imidazole, propionic acid, etc. are much less inhibitory. If the hydrogen bonding ability of histidine is blocked the antibacterial action largely disappears. A summary of the effect of added H202 on the antibacterial action of non-irradiated amino and carboxylic acids and some derivatives is given in Table XI. ^ IAEA-PL'561/1 29

TABLE XII. ACUTE TOXICITY OF CARBONYL COMPOUNDS (I. P. ) (Male Swiss Webster mice)

Minimum Non -Lethal Dose Compound Structure mg/kg mmol/kg

0 0 и 11 Methyl glyoxal CH3-C-C-H 1 ,3000 18

0 II Glycol aldehyde ch2oh-c-h 45 0.75

0 II Ethylvinyl Ketone ch3-ch2 -c-ch=ch2 33 0.39

0

Crotonaldehyde ch3-ch=ch-c-h 45 0.64

0 II Acrolein CH2=CH-C-H 27 0.48

We have developed a simple procedure to detect peroxide adduct forma­ tion and to evaluate the relative stability. The procedure involves the use of buffered glucose at pH 7 in which a competition between an added compound and glucose for H202 develops. The extent of adduct formation is reflected in the rate of disappearance of H20 2 from the medium since glucose destroys the H2C^ by reacting with it to form gluconic acid. Data are given for several compounds, including serum albumin and nucleic acid bases, in Fig. 6.

6. Carbonyl cytogenetics and toxicity

The acute toxicity in mammals of a, /3-un saturated carbonyl compounds is fairly low (Table XII) as might be expected from their enormously high rates of reaction with -SH groups, i. e. , they are largely destroyed before substantial amounts reach the target organs. Schauenstein and Esterbauer (unpublished work) have détermined the LD50 dose for a large number of hydroxylated a, jS-unsaturated carbonyl compounds. The LD50's fell generally in the range of 80 - 250 mg/kg. The oral LD50 values are far higher as, for example, with trans-2-hexenol they are 780-1130 mg/kg for rats and 1550-1950 mg/kg for mice 127]. We have tested, in vitro, the cytogenetic effects of several a, /3-un saturated carbonyls and saturated aldehydes 128]. Varying con­ centrations of a given carbonyl compound were incorporated in the lympho­ cyte culture after 48 hours of incubation, and 24 hours later the cells were harvested. The a, /3-un saturated carbonyls (crotonaldehyde and ethylvinyl ketone) were far more cytotoxic than the saturated aldehydes (glycoaldehyde,

/ CO TABLE XIII. CYTOGENETIC EFFECTS OF CARBONYL COMPOUNDS о

Concentrations (pmol/ml) in Culture Medium Mitotic Inhibition Chromosomal Breaks 1 Threshold Threshold Mi nimum Saturated Carbonyls Structure No Growth (No Breaks) Clastogenic

0 Glycolaldehyde CH20H-C-H 0.42-0.83 0.20 0.20-0.42

0 Glyceraldehyde CH20H-CH0H-C-H 2-3 0.5 1-2

0

Propionaldehyde ch3-ch2-c-h 3 1 1 .7 T R E B U H SC

a,g-Unsaturated Carbonyls

0 Acrolein ch2=ch-c-h 0.1 — 0.05

0 Ethyl vinyl Ketone CH3-CH2-C-CH=CH2 0.12 0.03 0.06-0.1

Carbonyl from Autoclaved 0 2-Deoxyribose CH2OH-CHOH-CH=CH-C-H 0.2 — 0.11

0 Crotonal dehyde CH3-CH=CH-C-H 0.14-0.28 0.036 0.07-0.18 IAEA-PL-561/1 31

glyceraldehyde and propionaldehyde). For example, the minimum concen­ trations of the saturated aldehydes which inhibited mitosis ranged from about 0. 8 to 3 pm ole/m l compared with 0. 1 to 0. 3 ^m ole/m l for the a, j3-unsaturated carbonyls, whereas the minimum concentration producing chromosomal breakage was about 0. 3 ^m ole/m l for saturated aldehydes and about 0. 08 /Limole/ml for the a, /3-un saturated carbonyls (Table XIII). Depending on concentration, the aldehydes completely pulverized chromosomes or produced less severe chromosome breakage. No structural re-arrangements were noted which suggested that interferences with the rejoining mechanisms occurred. The concentration range over which chromosomal damage was observed was very narrow, e. g. , no damage took place with crotonaldehyde or ethylvinyl ketone at concentrations one- half that producing pulverization and breakage. The cytotoxic effects of 6 -c a r b o n a, |3-unsaturated carbonyl sugars produced by radiolysis of hexoses, or autoclaving of 2-deoxyhexoses, as well as the 5-carbon a, 0-unsaturated carbonyl sugar produced by autoclaving solutions of 2-deoxyribose, are equivalent to the model a, ft-un saturated carbonyls. We have also identified 5-hydroxymethylfurfural (HMF) as one of the clastogenic carbonyl compounds formed upon autoclaving of acidified sucrose. This is an important point since irradiated (2 Mrad), unbuffered sucrose solutions (0. 058M) become acid (pH 3. 3 - 3. 5 after irradiation). Therefore, at least part of the clastogenic effect of irradiated sugar is contributed by HMF. In fact, > 75 /^g/ml of HMF is formed and, in the presence of H20 2, even more. We found that a final concentration in the cell culture of about 100 pg/m l (0. 8 ^m ole/m l) of HMF produces above control-level chromosome breaks and exchange aberrations. When taken together with the ~ 200 fjg/m l of radiolytic carbonyls, it becomes apparent that autoclaving of irradiated sugar solutions enhances the clastogenic effects in at least two ways: (1) production of HMF, and (2) increase in the proportion of a, /3-u n satu rated carbonyl sugar fraction of the radiolytic carbonyls. It is of historical interest to note that the control sucrose solutions used in the investigations of Shaw 112] were apparently not treated under conditions at which 5-HMF is formed. If a proper sucrose control were used (pH 3.3) corresponding to the pH of the irradiated sucrose solutions, and treated further in the manner of the irradiated, chromosomal aberra­ tions might have been observed in the controls to the same degree as in the irradiated sample. This is especially probable when it is considered that the 2% irradiated solutions were autoclaved, reduced in volume by evapora­ tion to a 20% concentration and stored at acid pH for several months at room temperature or at 5°C. The unirradiated sucrose solutions appear to have been used without any treatment but m erely added to the culture medium to give the same sucrose concentration as the treated irradiated samples.

V. IRRADIATED FOODS

Numerous monographs and review articles are available on the chemical changes produced in irradiated foods. Most of the detailed assays have been made on the volatile compounds produced. From the hygienic standpoint, particularly as regards human consumption, we are, of course, concerned with the non-volatile compounds remaining in the food after irradiation. 32 SCHUBERT

The formation of potentially harmful substances or unwanted changes such as off-tastes and odours generally increase with increasing radiation dose. However, by combining irradiation with partially or fully cooked food, or by carrying out irradiation at very low temperatures, many of the undesired reactions can be minimized. It must be recognized that while irradiation may cause changes in food, they are no greater, and, often, less than when food is treated by other widely used methods of preservation such as canning. However, what is called low-dose or pasteurization dose treatment of foods, roughly in the 5000 to 200 000 rad range, often produces no noticeable taste or textural changes in food, especially in foods with little moisture content such as wheat, and when "soft" irradiation is used to treat only the surface or skin of a food, such as oranges, there is also no notice­ able taste or textural change. When food is irradiated with high doses (4-6 Mrad) the volatile compounds have been recovered and analysed, usually by gas chromatography and mass spectrometry. A high proportion of the volatile compounds are hydro­ carbons, carbonyls and sulphur-containing compounds, as shown by many investigations [29] . These products arise primarily from the protein and fat. Thus, the aromatic hydrocarbons, benzene and toluene, arise from amino acids in the meat protein having aromatic groupings, namely phenyla­ lanine and tyrosine, while sulphur compounds arise directly from the sulphur- containing amino acids, cysteine and methionine. The aliphatic hydrocarbons and carbonyl compounds are derived from the direct breakage of bonds in the lipids. This section summarizes our findings on the non-volatile carbonyls produced in irradiated strawberries, beef and potatoes. It also summarizes our findings on our in-vitro and in-vivo cytogenetic investigations of irradiated strawberries.

1. Irradiated beef

Irradiation of meat in the 0. 5-1 Mrad range has been shown to signi­ ficantly retard spoilage due both to bacteria [30] and lipid autoxidation [31]. It is interesting to note that despite the known formation of hydroperoxides from irradiated lipids, irradiated bacon showed much lower peroxide values than did unirradiated meat even with no storage [32] indicating that peroxide formation is not a problem for irradiated meats. Since it is known, however, that the initially formed hydroperoxides can break down to give o^jS-unsaturated carbonyls [24] it is particularly important to compare at least the total carbonyl level of irradiated meats with that of unirradiated meats. Because of the presence of enzymes and the possibility of reactions between carbonyls and meat protein, we tested several procedures for the assay of carbonyls present in the meat post-irradiation. Our general approach was to homogenize the meat in a liquid and subject the homogenate to ultra­ filtration or dialysis and then to analyse the protein-free liquids by our modified 2,4-DNP method [15]. However, the carbonyl levels found were not reproducible and often increased in concentration upon storage of the homogenates, possibly due to enzymatic action. The use of an enzyme inhibitor such as sodium fluoride improved the assays. Ultimately, the method involving trichloroacetic acid precipitation proved satisfactory as described: IAEA-PL-561/1 33

To 20 g of cold lean beef brisket was added 80 ml of cold distilled water. The mixture was homogenized in a Waring blender for 10 min in the cold, transferred to a Potter Mill and homogenized for another 10 min. To 30 ml of the homogenate was added 3.3 ml of a 40% trichloro­ acetic acid (TCA) solution to give a final concentration of 10% TCA. The TCA treatment inactivates enzymes and removes proteins and interference from coloured products, e. g. , myoglobin [29]. The acidified homogenate was allowed to stand in the cold for 15 min and was then centrifuged in a refrigerated ultracentrifuge at 10 000 rev/m in for 10 min. A 5-m l aliquot of the supernatant was removed and the pH was adjusted to about neutrality by adding about 1 ml of a 10% sodium hydroxide solution. Total carbonyl assays were carried out on 1 ml of the neutral solution. This method gives at least 80% recovery of added glyceraldehyde and ff-ketoglutaric acid. When the method was applied to irradiated meat a significant increase in total carbonyls could be observed. From carbonyl yields at lower doses (0.3-1 Mrad) a rough value of G(carbonyl) = 0. 8 was obtained. Preliminary evidence indicates that the carbonyls formed, or at least present after homogenization, are not unsaturated in that no differences in ultra-violet spectra could be observed between irradiated and unirradiated beef. The reduction of Salmonella in meat by irradiation is well known [30, 3 3]. Here, the irradiation acts directly on the bacteria. However, since carbonyl compounds are known to inhibit bacterial growth it would be expected that the radiolytic carbonyls would reinforce the antibacterial action of irradiation and continue to act after the cessation of irradiation. At the 5.5-Mrad dose level at least 200 yg of carbonyls per gram of beef were present. Autoclaving of the filtrates did not produce any change in the observed carbonyl contents. At lower doses, the G(carbonyl) = 0.8 would correspond to about 80 ppm, assuming an average molecular weight of 100. In any event, these concentrations of carbonyls would be expected to inhibit the growth of Salmonella if a more sensitive test system had been employed, e. g. a smaller number of cells per millilitre. Batzer et al. [34] measured the production of carbonyl compounds in irradiated meat and meat fats. In this early (1957) investigation, the carbonyls in the food were extracted either by benzene or acid-salt extrac­ tion which were subsequently analysed for carbonyls by 2,4-DNP. Radiation doses were very high, 2 to 10 Mrad. As would be expected, different carbonyl compounds were obtained depending on the method of extraction used. For example, in irradiated ground beef the acid-salt extraction gave 27 to 58 times more carbonyls on assay than the benzene extraction. In fats, the two extraction methods gave nearly equivalent values. Their findings are of interest in that apparently large amounts of carbonyls were produced in irradiated beef with a much smaller contribution from the fat m oiety . More recently, Tajima et al. [35] applied the techniques of Batzer et al. [34] to the determination of carbonyl compounds in wiener sausage. They reported that 262 ц g/ g fresh weight of apparent carbonyls were produced at 0.25 Mrad in unsmoked sausage and 324 pg/g of smoked sausage. At 0.5 Mrad they reported finding 299 and 420 jug/g respectively. On storage for 18 days, the apparent carbonyl contents increased from 10 to 25% while the amount of volatile compounds decreased. No significant changes in free amino acid contents were observed in the dose ranges studied (0-0.5 Mrad). 34 SCHUBERT

2. Irradiated strawberries

Strawberries have been irradiated at about 200 krad (radurization) to inhibit mould formation. Experiments on the cytological and mutagenic effects of the juice or puree from irradiated strawberries have proved negative. Some slight chemical changes, e. g. in niacin and thiamine, in irradiated strawberries have been reported. Above 200 krad some loss in colour occurs as a result of the destruction of the anthocyanin pigments (see Ref. [13] for a review of previous published work on chemical and biological changes). Our investigations included homogenates, centrifugante, distillates and powders prepared from untreated and irradiated whole fresh straw­ berries and freeze-dried strawberries using various chemical and biological procedures. In particular, we made chemical assays for the first time of the total carbonyl compounds remaining in irradiated strawberries after irradiation. We performed in-vitro and in-vivo measurements in rats and mice for chromosomal aberrations. In addition, we tested irradiated straw­ berry homogenates and extracts for antibacterial activity.

Radiation Dose (Krads)

FIG. 7. Carbonyls produced upon irradiation of fresh whole strawberries as a function of radiation dose.

The carbonyl assays were made on the dialysates prepared from an homogenized preparation of fresh whole strawberries. The calculation of carbonyl concentrations was based on a molar absorptivity, e = 18 x 103 , corresponding to that of glyceraldehyde. From Ref. [13]. IAEA'PL-561/1 35

TABLE XIV. CYTOGENETIC a EFFECT OF IRRADIATED AND NON-IRRADIATED STRAWBERRY PUREE ON MICE IN VIVOb-c

No. of aberretions No. of damaged cells No. of Total cells with aberrations Post- cells Aberration- intu­ Chromatid Chromosome scored/ Chromatid Chromosome free cells1 bation, no. of Strawberry puree days Bi B, Bj Cu C( animals No % No. % No. %

( i Q 0 0 0 0 14/3 0 0 0 0 14 • 100 Saline control I 4 1Д 0 0 0 0 28/4 1 4 0 0 27 96 i? 1/1 0 0 0 0 130/7 1 1 0 0 129 99

(1 0 Q 1/1 0 0 54/3 1 2 0 0 53 99 Nonirradiated 0 0 0 0 0 98/5 0 0 0 0 93 100 I 4 ь 0 0 0 0 1/1 99/5 0 0 1 1 93 99

(1 1/1 0 0 0 0 17/3 1 6 0 0 16 94 Irradiated (1.5 Mrad) I4 0 0 0 0 1/1 43/4 0 0 1 2 42 98 I? 3/3 0 0 0 2/2 171/7 3 2 2 1 166 97 “ The cytogenetic analyses were carried out on bone marrow cells obtained by aspiration from the femurs as discussed in the text.b The ll-week*o!d Swiss-Webster mice were given 0.5 cma of strawberry puree three times a day by intubation for 5 days. Each group consisted of seven animals, at least three of which were used to supply bone marrow cells for a given day postintubation. c Table from Ref. [13].

Strawberries are of particular interest because the concentration of mono- and polysaccharides is so high. For example, we found that near half the dry weight and 5% of the fresh weight consists of D-fructose (2.3%), D-glucose (2.6%) and D-sucrose (0.14%). However, the carbohydrates are present in essentially a comparatively rigid structure, unlike that of solu­ tions, which would reduce the extent of indirect action on the sugars since diffusion of OH radicals is required. Doses of 0.15 and 0.3 Mrad produced no detectable change in sugar content. The volatile fractions obtained by distillation from fresh, homo­ genized strawberries under 1-mm pressure and at temperatures up to 50°C were analysed by coupled gas chromatography-mass spectrometry. Irradiation at 0.2 Mrad produced minimal changes in the volatiles compared with the non-irradiated samples, though a small additional amount of isobutyric acid was formed. At 1.5 Mrad we found eight compounds not detected in the non-irradiated samples including a new compound tentatively identified as methoxyacetaldehyde. Assay for carbonyls in the strawberries was carried out at doses beginning at 0.25 Mrad and in incremental doses up to 3 Mrad. The carbonyl yield was linear to 1 Mrad and then levelled off with a G(carbonyl) = 2.2 (Fig. 7). When the corresponding dialysates were autoclaved, the carbonyl levels dropped about 50% at doses > 0.5 Mrad. The carbonyls present in the strawberries were quite stable since the overall carbonyl content de­ creased only 16% after storage for 20 days in a refrigerator. From the spectra, volatility and slight antibacterial activity, little or no enolizable or hydroxylated a, j3-unsaturated carbonyl compounds were produced upon irradiation of fresh, whole strawberries, though small amounts of trans-2- hexenal are known to be naturally present in strawberries [27]. No in-vivo clastogenic properties of irradiated whole strawberries were found in mice or rats fed strawberry puree three times daily for 5 days as shown for mice (Table XIV) and for rats. 36 SCHUBERT

3. Irradiated potatoes

We carried out preliminary experiments on the carbonyls in raw potatoes that had received radiation doses up to 50 krad which is well above that used to inhibit sprouting (~ 10 krad). Each potato was cut into quarters before irradiation so that two quarters would serve as non­ irradiated controls. After irradiation, the samples were peeled, diced and homogenized with half their weight in added water. Subsequently, they were centrifuged and the supernatants analysed. No detectable differences in carbonyl levels between the irradiated and non-irradiated potatoes were found. However, these findings are provisional because of the high background present. Tajima et al. [36] analysed the carbonyls in the volatile fractions from potatoes that had been irradiated with 11 and 89 krad and then cooked. They found no significant differences in the volatile carbonyls from non-irradiated potatoes as compared with those given 11 krad. However, significant increases (~ 2-fold) of volatile carbonyls were found in the potatoes irradiated with 89 krad. No significant effect on volatile compounds after storage for 50 days was found. I have reviewed previously (Ref. [3] pp..880, 884-885) the various cytological effects of preparations from irradiated potatoes. Growth inhibition and sometimes stimulating effects on various m icro-organism s as well as cytological abnormalities in seedlings have been reported at levels of 20 krad and higher, at least for brief periods (24 hours) of post­ irradiation. However, more systematic investigations in depth under a variety of conditions are needed to clarify the somewhat confusing situation.

VI. CONCLUDING COMMENTS

Our investigations indicate that the indirect hygienic effects of food irradiation are mediated by carbonyl compounds, especially the a, /3-un saturated carbonyls, and of hydrogen peroxide which can act directly or, more likely, via the formation of chelated, hydrogen-bonded adducts with bifunctional food constituents [3] . Related to the formation of carbonyls are the organic peroxides [3] which are formed in the presence of oxygen but which hydrolyse to carbonyl compounds when acted upon by w a ter. As far as can be ascertained, the carbonyls and peroxides and peroxide adducts are relatively harmless when ingested since they are so reactive. In fact, the reactive groupings in food and in tissue cells, especially the sulfhydryl groups, react readily and extremely rapidly with both peroxides and a, (3-un saturated carbonyl compounds. Hence, it appears that it would take extremely large, chronic doses to produce harmful effects in mammals. For example, when heated fats and fat fractions were fed daily to rodents for 17 months they were found to lack carcinogenicity [37]. In any event, ordinary cooking and autoxidation of food constituents produces compounds similar to those produced by irradiation. We have also found that several amino acids, including histidine, glutamic acid and cysteine eliminate or reduce the cytotoxic effects of irradiated sugar solutions. One reason appears to be the instability IAEA-PL-561/1 37

(transient life time) of organic peroxide adducts as compared to hydrogen peroxide adducts because of the fact that the energy of the 0 -0 bond in the organic peroxides is much less than that in hydrogen peroxide. Our future investigations include the isolation and identification of the a, /З-unsaturated carbonyl sugars produced upon irradiation of sugars. This will enable us to proceed with a well-defined battery of CC'MT tests (C = carcinogenic, С1 = clastogenic, M = mutagenicity, T = teratogenic) with which to evaluate irradiated foods, at least those in the liquid state. It appears worthwhile to consider the use of selected scavengers for reducing the radiation dosage required to produce a desired physical, chemical or hygienic change in irradiated foods. Thus, in some cases, irradiation of foods sealed in an atmosphere of N20 gas could reduce the radiation doses to half when the OH radical, as is often the case, is the active agent. Nitrous oxide is relatively unreactive and non-toxic being used as a propellant for foods in aerosol cans and as a well-known anesthetic.

ACKNOWLEDGEMENTS

The investigations cited in this paper have been supported for the most part by the United States Atomic Energy Commission, Division of Biology and Medicine, under contract AT(30- 1)-3641 and form erly, in part, by the Division of Food Chemistry, Bureau of Food Science, United States Food and Drug Administration, Department of Health, Education, and Welfare, under contract FDA 68-39. I am indebted to Mrs. D. Stock for technical assistance and my former colleague, Dr. Edward B. Sanders, for numerous helpful discussions on the chemistry of the radiation-induced reactions in food constituents, on the organic chemistry of carbohydrates, and for his collaboration in many of the investigative programmes reported here. I also wish to acknowledge the advice of Dr. E. J. Hart in the preparation of the radiolysis scheme of water shown in Table II and to Drs. N. Wald and S. Pan for the chromo­ somal analyses cited in Table XIII.

REFEREN CES

[1] SCHUBERT, J. , WATSON, J.A ., BAECKER, J.M. , Int. J. Radiat. Biol. 14(1969) 577. [2] SCHUBERT, J., SANDERS, E. B ., Nature New Biology 233 (1971) 199.

[3] SCHUBERT, J. , Bull. World Health Organ. 41 (1969) 873.

[4] KESAVAN, P.C. , SWAMINATHAN, M . S ., Radiat. Botany И (1971) 253. [5] BECKING, J.H., Misc. Papers 9, Landbouwhogeschool Wageningen, The Netherlands (1971) 55-87.

[ 6] DRAGANIC, I.G ., DRAGANIC, Z.D . , The Radiation Chemistry of Water, Academic Press, New York (1971).

[7] HART, E.J. , Radiat. Res. Rev. 3 (1972) 285.

[ 8] O'DONNELL, J.H. , SANGSTER, D. F ., Principles of Radiation Chemistry, American Elsevier Publishing C o ., New York (1970).

[9] KE, C .H ., SCHUBERT, J., Radiat. Res. 49 (1972) 507.

[10] HENLEY, E .J ., JOHNSON, E .R ., The Chemistry and Physics of High Energy Reactions, University Press, Washington, D. C. (1969). [1 1 ] HOLSTEN, R.D. , SUGD , M ., STEWARD, F .C ., Nature (London) 208 (1965) 850. [12] SHAW, М ., HAYES, E. , Nature (London) 211 (1966) 1254. 38 SCHUBERT

[13] SCHUBERT, J. , SANDERS, E. В ., PAN, S.F., WALD, N ., J. Agrie. Food Chem. 21 (1973) 684. [ 1 4 ] S C H U B E R T , J, , J. Gen. Microbiol. 64(1970) 37. [15] SANDERS, E. В. , SCHUBERT, J. , Anal. Chem. 43 (1971) 59.

[16] PHILLIPS, G .O . , Radiat. Res. Rev. 3 (1972) 335.

[17] KAWAKISHI, S ., NAMIKI, M ., Carbohydrate Res. 26 (1973) 252.

[18] SANDERS, E .В ., SCHUBERT, J. , Tetrahedron Letters (submitted for publication).

[19] HARTMANN, V ., SONNTAG, С. V. , SCHULTE-FROHLINDE, D ., Z. Naturforsch. 256 (1970) 1394.

[20] ALEXANDER, P ., LETT, J. T ., in Comprehensive Biochemistry, Ch. VIII, V ol.27 (FLORKIN, М .,

STOTZ, E. H ., Eds), Elsevier Publishing C orp., New York (1967).

[21] FLYNN, J.H ., WALL, L. A ., MORROW, W .L ., J. Res. Natl. Bur. Stand. 71A (1967) 35. [22] LOFROTH, G ., Acta Chem. Scand. 21 (1967) 1997.

[23] SCHAUENSTEIN, E ., J. Lipid Res. 8(1967)417.

[24] HYDE, S.М. , VERDIN, D. , Trans. Faraday Soc. 64 (1968) 144.

[25] WILLS, E.D. , ROTBLAT, J. , Int. J. Radiat. Biol. 8 (1964) 551. [26] MOSEBACK, K.O. , Klin. Wochenschr. 39 (1961) 80. [27] GAUNT, I.F.,et al., Food Cosmet. Toxicol. 9 (1971) 775.

[28] SCHUBERT, J., PAN, S.F. , WALD, N ., 2nd Annual Meeting, Environmental Mutagen Soc.,

March 21-24, 1971.

[29] ROODYN, D. В. , MANDEL, G .H ., Biochim. Biophys. Acta 41 (1960) 80.

[30] LEY, F.J., et a l., J. Hyg. 68 (1970) 293. [31] GREENE, B.E., WATTS, В .М ., Food Technol. 20 (1966) 111.

[32] RHODES, D. N. , SHEPHERD, H.J. , J. Sci. Food Agrie. 18 (1967) 456.

[33] LEY, F. J., et al.. Lab. Anim. 3 ( 1 9 6 9 ) 221. [34] BATZER, O.F. , SKIBNEY, M ., DOTY, D .M ., SCHWE1GERT, B .S., Agrie. Food Chem. 5

(1957) 700.

[35] TAJIMA, M ., MORITA, M ., FUJIMAKI, M. , Agrie. Biol. Chem. (Tokyo) 34 (1970) 1859.

[36] TAJIMA, M ., KIDA, K. , FUJIMAKI, M. , Agrie. Biol. Chem. (Tokyo) 31 (1967) 935. [37] O' GARA, R. W, , STEWART, L ., BROWN, J ., HUEPER, W .C ., J. Natl. Cancer Inst. 42 (1969) 275.

I IA E A -P L - 5 6 1 / 2

SOME THEORETICAL CONSIDERATIONS ON THE CHEMICAL MECHANISM OF THE RADIATION-INDU CED DE PO LYME RIZ AT ION OF HIGH MOLECULAR CARBOHYDRATES

H. SCHERZ Institute of Radiation Technology, Federal Research Centre for Food Preservation, Karlsruhe, Federal Republic of Germany

Abstract

SOME THEORETICAL CONSIDERATIONS ON THE CHEMICAL MECHANISM OF THE RADIATION-INDUCED

DEPOLYMERIZATION OF HIGH MOLECULAR CARBOHYDRATES. Irradiation of high molecular carbohydrates in the solid state, as well as of theiraqueous solutions, causes the breaking of the external ether bridges. Two mechanisms can be assumed which take place simultaneously: (a) Direct action of radiation on the oxygen bridges in the solid state leads to the formation of an -O ' - radical; then the -О С - linkage to the next hexose unit is split off giving a positive ion at Q

and a glycosyl radical. In aqueous solutions H 30 + are formed as primary species with G-values of ~ 2 . 8 which would hydrolyse the glycosidic bond according to the mechanism of the normal acid hydrolysis.

(b) Irradiation causes “ directly or indirectly — alterations of some of the monosaccharide units in the chain m olecule by radiation-induced dehydration and 6-splitting, forming deoxycarbonyl or deoxyacid groups in the chain. It is not difficult to imagine that the ether bridges of these altered groups show an essentially higher

sensitivity towards hydrolysis than the other linkages. In general, it could be stated that irradiation forms

"weak points” in the chain m olecule. External influences (heating, acid treatment etc.) or further energy transfer w ill act preferentially at these points and cause the breaking of the carbohydrate chain.

Irradiation of high molecular carbohydrates like starch, cellulose and pectines causes alterations of some of their physical properties like viscosity, mechanical strength, swelling and solubility which can perhaps enhance the qualities of carbohydrate-containing foodstuffs or can help to improve their technological processing. In all cases these alterations are connected with the radiation-induced depolymerization of the chain m ole­ cules which consist of hexose units linked together by ether bridges. In this paper I want to present some theoretical aspects of the depolymerization reaction. I feel that such considerations may be necessary also for the discussion of the application of irradiation in food technology, because a good theory can often be a great help in practical conclusions. Radiation transforms its energy to matter by three mechanisms:

(a) Photo-effect (b) Compton effect (c) Formation of an electron-positron pair.

39 40 SCHERZ

During this action the molecules undergo a lot of different energetic states; ionization takes place as well as splitting of chemical bonds and formation of free radicals. The reaction can be expressed in the following sch e m e :

n E 1 M -----^ М пх, M *, M + ( + e") where nx are the different energetic states. The life-tim e of such primary species is dependent on the aggregate state, temperature, atmosphere etc. In the solid state, in which the mobility of the molecules is strongly reduced, the primary species are stable over a long time. Free radicals are detectable by EPR spectroscopy. They disappear as follows:

(a) By recombination

M - + M ’ ----- »-(M - M)* — o-M - M

During this reaction excited states are assumed which can lead to structural alterations.

(b) By disproportionation

M ' + M ------► (M . . M)°------

(c) By reaction with accompanying substances under formation of secondary r a d ic a ls

M ‘ + N -----► M + N ‘

Further primary species of the radiation effect on matter are ions which are formed when the energy levels of excitation are exceeded. The electrons are bound to return when their energy is too low to get out of the coulomb field of positive ions. By recombination super-excited states are assumed which can lead to the formation of radicals or undergo cascade degradation over different stages of excitation to the normal state.

M+ + e"— -M xx------M’

M

When the kinetic energy of the electrons is high enough to escape from the coulomb field, the electrons can be stabilized. In liquid medium like water they will be surrounded by solvent molecules. In this state, they are called "solvated electrons" which undergo specific chemical reactions. IAEA-PL-561/2 41

Although the transport of masses is negligible in solid states, charge and energy-transfer reactions take place to a remarkable degree from the primarily attacked molecules or molecule groups to the surrounding parts. Here a distinction must be made between intra- and intermolecular energy transfer. In the case of intramolecular energy transfer, some specific groups of the molecule accept the energy and conduct it over to the whole molecule; it may also occur, however, that unspecific sites of the molecule accept the energy to transfer it to a specific group which solely undergoes the chemical alterations. The intramolecular energy transfer occurs especially with high molecular substances.

FIG. 1. EPR signal of irradiated Schardinger dextrin.

20 gauss

FIG. 2. EPR signal of the irradiated com plex of Schardinger dextrin with benzene. 42 SCHERZ

Intermolecular energy transfer reactions take place with substance mixtures. In this case one of the components in the mixture accepts the energy without altering itself and transfers it to the compound with the greatest lability. For example, when a solution of dextran and penicillamine (substituted cysteine) is freeze-dried and irradiated [ 1 ] in the solid state, the EPR signal shows only the sulphur radical whereas the dextran radical does not appear. Such processes are important for the understanding of some physical and chemical phenomenoma in irradiated food. Energy transfer is dependent on the physical state like crystallinity, water content etc. A single substance in different crystal structures often shows great variation in the decomposition rate. As an example the energy transfer in the carbohydrate systems should be discussed. The main studies on this subject have been done by Phillips and co-workers [ 2]. Irradiation of polycrystalline anhydrous a- or /3-glucose shows an initial G-value of 20, whereas a freeze-dried glucose solution in which the greatest part of the molecules are present in an amorphous state has a G-value of 7; for a-D-glucose monohydrate a value of 11 has been determined. The high degree of radiolytic decomposition of anhydrous glucose can be explained by its high crystallinity which favours the energy transfer effect. It can be assumed that the transfer reaction occurs over hydrogen bridges. In a-D-glucose monohydrate crystals the oxygen atoms at C-3, C-2 and C-6 are connected with water molecules. The distances to the neighbouring molecules are greater than those found in the anhydrous glucose in which four oxygen atoms, namely those at C-6, C-4, C-3 and C-2, are connected by hydrogen bridges. Another example of intermolecular energy transfer is found in the complexes of the Schardinger dextrins with aromatic compounds. Dextrin alone has a total G-value of 15. When a complex of dextrin with benzene or toluene is irradiated only the EPR signal of the benzene is obtained, and the G-value falls to 2. 6 in the case of toluene (Figs 1 and 2).

FIG. 3. Dependence of the breaking strength of cotton cellulose on the degree of substitution of C O -Q H 5 groups (A: 1.2 X 1020 e V / g , B: 1 . 3 X Ю 21 e V / g ) . IAEA-PL-561/2 43

TABLE I. DEPENDENCE OF THE SWELLING TEMPERATURE AND IODINE AFFINITY OF GRANULAR POTATO STARCH ON IRRADIATION DOSE

Irradiation dose Swelling temperature Iodine affinity (M r a d ) C°C)

0 68 - 6 9 - 4 . 5

0.1 6 7 - 6 9 4 . 3

0 . 5 6 7 - 6 9 3 . 6

2 .0 6 4 - 6 5 3 . 1

10.0 6 0 - 6 1 1.1

TABLE II. DEPENDENCE OF THE VjISCOSITY OF A 2% SOLUTION OF CORN STARCH ON IRRADIATION DOSE

Irradiation dose Viscosity, 77 (M ra d ) (centipoise)

0 6 . 3

10 2 . 5

20 2.0

30 1. 0 '

100 0 . 9

w a ter 0 . 4

An example of intramolecular energy transfer is provided by substituted cellulose [3] . When the hydrogens of the OH-groups of cotton cellulose are substituted by benzoyl groups, its mechanical stability against irradiation is much greater than that of the unsubstituted one (Fig. 3). At a dose of 1.2 X 1020 eV/g unsubstituted cellulose has 80% of the breaking strength of the non-irradiated material and at a dose of 1.3 X 1021 eV/g it only has 20%. The benzoylation of OH-groups to a substitution degree of 0.4 enhances, at the first dose, the value to 100% and the substitution degree of 1.2 at the second dose enhances it to 80% of the initial breaking strength1. It is further necessary to distinguish between direct and indirect effects of irradiation. The latter occur in diluted solution; radiation reacts with the solvent forming primary species which attack the dissolved substances in a second step.

1 The aromatic groups accept all the radiation energy and prevent destruction of the carbohydrate chain. 44 SCHERZ

TABLE III. DEPENDENCE OF THE AVERAGE DEGREE OF POLYMERIZATION (DP) OF POTATO-STARCH AMYLOSE ON IRRADIATION DOSE BY MEASURING THE LIMIT VISCOSITY

Irradiation dose Limit viscosity DP

(M ra d ) ( t?)

0 2 3 0 1 7 0 0

0 , 0 5 220 1 6 5 0

0.1 1 50 1100

0.2 110 8 0 0

0 . 5 9 5 7 0 0

1.0 80 6 0 0

2 .0 50 3 5 0

5 . 0 4 0 3 0 0

10. 0 35 2 5 0

The most important action of irradiation on high polymer carbohydrates is the splitting of the external ether bridges which connect the single monosaccharides to chain molecules. Most of the studies in this field have been done with starch, and some interesting results are presented here. Tables I and II show the influence of irradiation on swelling, iodine affinity and viscosity [4] . All these effects lead to the conclusion that the degree of polymerization (DP) is strongly reduced by irradiation. Although for linear chain molecules an exact mathematical relation exists between the limit viscosity (r?) and DP (Staudinger equation), this relation cannot be applied since starch consists of two different species, namely the linear chain molecule amylose and the strongly branched amylopectine. When the two species are separated, the degree of polymerization for amylose can be calculated from viscosity measurements as a function of the irradiation dose (Table III) [5] . Irradiation also causes an enhanced sensitivity towards enzymatic degradation, and Fig. 4 presents a study by Guilbot and Tollier [6] showing the hydrolysis of corn starch by a-amylase to be a function of the irradiation dose. The irradiation was carried out in the native state; the results show that especially the initial velocity of the enzymatic degradation increases strongly with the irradiation dose. The exact chemical mechanism of the depolymerization reaction can be investigated in two ways. First, by evaluating the structure of the radiation-induced radicals by interpretation of the EPR spectra and, secondly, by an exact analysis of the whole radiolysis products. The EPR spectra which had been obtained from irradiated starch were not sufficiently fine-structured to allow an exact analysis of the radicals (Fig. 5). As the main component a doublet was obtained, and the calculated g-factors lead to the conclusion that the free electron is located mainly at the C1-position of the hexose unit [7]. An analysis of the degradation products could be more promising for this purpose. IAEA-PL-561/2 45

FIG. 4. a-Am ylolysis of corn starch irradiated in the solid state with different doses (I: unirradiated,

2: 12.5 Mrad, 3: 19 Mrad, 4: 25 Mrad, 5: 35 Mrad, 6 : 70 Mrad). Ordinate: Ratio of (alcohol-soluble carbohydrate/total carbohydrate) X 100.

FIG. 5. EPR signal of irradiated potato starch.

Analysis of the low molecular sugars in irradiated wheat starch gave the distribution of oligosaccharides shown in Table IV [8]. From the table it can be seen that almost equal concentrations of maltose, maltotriose and maltotetrose are formed, whereas the concen­ tration of glucose is much lower. It seems that the di- tri- and tetra- saccharides are split off from the starch chain as especially stable units. If the depolymerization would occur according to purely statistical laws, a steeper increase in the oligosaccharides could be expected. 46 SCHERZ

TABLE IV. DISTRIBUTION OF THE OLIGOSACCHARIDES AS A FUNCTION OF IRRADIATION DOSE FOR IRRADIATED WHEAT STARCH

D ose Total amount of sugar I II III IV V

(k rad ) ( m g /100 g ) ( m g /100 g )

0 14 - 4 . 6 5 . 4 3 . 4 6

20 19 - 5 . 4 6.0 6.8 6 CO CO 4 0 21 - 6 . 4 6 . 4 2

6 0 4 6 - 1 3 . 8 12.8 10.8 19

200 7 0 3 . 6 1 3 . 2 1 7 . 4 12. 0 3 5

4 0 0 110 4 . 4 1 8 . 4 1 1 .7 2 6 . 4 4 5

6 0 0 1 2 4 5 . 2 22 . 8 3 1 . 8 2 1 . 0 35

8 0 0 1 4 0 6.0 3 1 . 8 3 4 . 8 2 3 . 4 33

1000 1 7 2 7 . 2 5 7 . 0 5 7 . 0 4 0 . 8 12

I: glucose IV: maltotetrose

II: maltose V: maltopentose

III: maltotriose

F IG . 6 . Mechanism of the radiation-induced chain splitting of Schardinger dextrin.

As an explanation of the depolymerization of such high molecular carbohydrates, Phillips et al. [9] postulate that in the solid state the radiation reacts with the oxygen of the ether bridges forming a - Ó - radical. In the next step the O-С linkage to the next hexose unit is split off forming a positive charge at the C-4 atom. This positive ion reacts rapidly with the OH- ions of water which is always present either as natural moisture or when the substance is dissolved in water (Fig. 6). IAEA-PL-561/2 47

In aqueous solutions the OH' radicals react with the dissolved carbo­ hydrates when hydrogen is removed from the C-H bonds. Solvated electrons and H' atoms remain almost unreactive. Protons are also primary species of the water radiolysis which are formed according to the following reaction: ,

h 2° ■H2° + e

H 2o + + H2o -н 3о + + OH’

The protons which are formed at high concentrations in so-called "clusters" may perhaps hydrolyse the dissolved carbohydrate chain according to the normal mechanism of acid hydrolysis:

HgO + ..-R - O - R . . -R - O - R - + н2о I H

,-R - O - R ------R+ + ROH I H

R+ + 2 H20 ■ ROH + H p

H30 + OH 2ЩО where R- are the hexose units. Recently, some new types of chemical reactions caused by radiation have been detected with vicinal polyhydroxy compounds. The first one was the radiation-induced dehydration leading to deoxycarbonyl compounds according to the following scheme [ 10] :

- CH - CH - •-C - CH - С - СН- I I L___I—, II • OH OH 0(H o h ] о We found such deoxycarbonyl compounds among the low molecular weight ' radiolysis products of starch [ 11], and we noticed further that a small amount of such deoxycarbonyl groups remains also in the high molecular carbohydrate chain [ 12] . It had been found that the glycosidic bonds of such altered hexose units are sensitive and hydrolyse more readily than the other o n e s [ 13]. A second type of such reactions was the radiation-induced 0-splitting that occurs with -С -O- linkages according to the following schemes [ 14] : I . I I I I С - О - - С - о - с - ■ c = о 'C - I I I I - О - H ■ -- с O' С' + с = о I I For С - С - linkages a similar mechanism can be assumed, e.g. in the formation of malondialdehyde, which is also a radiolytic degradation product of carbohydrates [ 10]. Н H Н I О = С - С C f ç • o = С С = С - ОН + ' с I I I I I I I н н о н OH Н H Н OH 50 SCHERZ

Radiolysis of solid lactose leads to the formation of 5-deoxylactobionic a cid [ 15], and among the radiolysis products of starch, 5-deoxygluconic acid was detected. For both compounds, Scheme 1 is proposed. The acid groups between the hexose units would also be labile points of the chain where, a break is very likely to occur. Another mechanism is supposed to lead directly to a splitting of the chain. It was found that irradiation of starch causes the formation of 1,4-pyrones [16], and the main component was identified as hydroxymaltol. The formation can be explained by assuming that a radical is formed at C-4 of a glucose molecule which undergoes (3-splitting and dehydration (Scheme 2). According to that scheme the end group of one part of the chain residue is supposed to be a 2-deoxy-3, 4-diketohexaldose. The glycosidic bonds to such compounds are easily hydrolysed and during this reaction the re-arrangement to hydroxymaltol can be assumed. i

REFERENCES

[ 1] HENDRIKS EN, T., SANNER, T., PIHL, A., Radiation Res. 18 (1963) 147.

[2] PHILLIPS, G. O. ( Energy Transfer in Radiation Processes, Elsevier publishing C o ., Amsterdam, London,

New York (1966).

[3] ARTHUR, J .C ., MARES, T ., I. Appl. Polymer. Sci. 9 (1965) 2581. [4] RADLEY, J. A, , Starke 12 (1960) 201.

[5] GREENWOOD, C .T. , MacKENZIE, S ., Starke 15 (1963) 444.

[6] TOLLIER, M. T h ., GUILBOT, A ., Stârke 18 (1966) 305. [7] SAMEC, М ., BLINC, R., HERAK, K ., ADAMIC, I., Starke 16 (1964) 181.

[8] ANATHASWAMY, H .N ., VAKIL, U .K ., SREENIVASAN, A ., J. Food Sci. 35 (1970) 765. [9] PHILLIPS, G .O ., BAUGH, P.J., J. Chem. Soc. (London), (1966) 387.

[10] SCHERZ, H ., Radiation Res. 43 (1970) 12.

[11] SCHERZ, H ., Starke 23 (1971) 259. [12] SCHERZ, H ., (unpublished).

[13] SZEITLI, H ., Starke _19 (1967) 145. [14] HARTMANN, V ., SONNTAG, С', v. , S CHU LT E- FROH LIND E, D. , Z . Naturforsch. 25b (1970) 1384. [15] DIZDAROGLU, М., SONNTAG, C. v. , Z. Naturforsch. (in press). [16] SCHERZ, H ., Z. Naturforsch. (in press). IAEA-PL-561/3

INFLUENCE D'UNE IRRADIATION GAMMA SUR LA SALUBRITE ET LES PROPRIETES TECHNOLOGIQUES DE L'AMIDON DE MAIS

L. SAINT-LEBE, G. BERGER, A. MUCCHIELLI Service de radioagronomie, Département de biologie, CEA, Centre d’ études nucléaires de Cadarache, Saint-Paul-lez-Durance, France P

Abstract-Résumé

INFLUENCE OF GAM MA RADIATION ON THE WHOLESOMENESS AND TECHNOLOGICAL PROPERTIES OF

MAIZE STARCH. In most cases irradiation of starch at a dose of 300 krad enables the desired hygienic qualities to be achieved without changing the other technological properties of the product. Among the induced modifications, the adjustable formation of hydrogen peroxide can be of immediate interest to technologists.

INFLUENCE D'UNE IRRADIATION GAM MA SUR LA SALUBRITE ET LES PROPRIETES TECHNOLOGIQUES DE

L'AMIDON DE MAIS. L’ irradiation de l’ amidon à une dose de 300 krad permet dans la plupart des cas d'obtenir l ’ assainissement escompté sans pour cela perturber la technologie ultérieure du produit. Parmi les modifications induites la formation modulable de peroxyde d'hydrogène peut présenter a court terme un intérêt pour le technologue.

INTRODUCTION

Le programme du Service de radioagronomie dans le domaine de l'irradiation des aliments vise un double objectif: - faire admettre â moyen terme l'irradiation comme un procédé qui garantit dans les meilleures conditions l'assainissement des produits alimentaires en poudre; les études portent actuellement sur un premier modèle, l'amidon de mais [1]; - à court terme imposer l'irradiation au stade industriel en essayant de promouvoir en France la radappertisation des provendes pour animaux de laboratoire; c'est une préoccupation récente, et le programme est en cours de mise en place; l'impact psychologique d'une telle réalisation serait tout aussi important que la somme d'informations scientifiques que l'on pourrait recueillir au niveau des élevages. Dans les deux cas l'amélioration escomptée est essentiellement de nature microbiologique. Toutefois, les recherches toxicologiques axées pour une grande part sur l'identification et le dosage des produits radio- formés et surtout l'étude technologique, qui a pour objet de vérifier que les fabrications ultérieures ne seront pas perturbées, peuvent faire apparaître des modifications de la molécule intéressantes pour l'industrie. Nous ne traiterons dans ce mémoire que des travaux sur l'assainisse­ ment de l'amidon.

51 52 SAINT-LEBE et al.

1. L'ASSAINISSEMENT DES PRODUITS ALIMENTAIRES EN POUDRE

A l'heure actuelle on peut estimer que près des deux tiers des produits agricoles français subissent un traitement industriel avant d'être consommés: de 1959 â 1966 la valeur des denrées com m ercialisées â l'état brut est passée de 26 â 29 milliards de francs, celle des aliments élaborés de 46 â 77 milliards [2]. Cette transformation revêt des formes différentes: pour les pays industrialisés il s'agit de produits caractérisés par la facilité d'emploi et surtout la rapidité du dernier stade de préparation; pour les pays en voie de développement, de denrées nouvelles visant â pallier la malnutrition. Dans les deux cas les aliments se présenteront sous forme liquide ou pâteuse qu'il suffira de réchauffer et exceptionnellement de cuire, ou sous la forme de poudres, plus faciles â stocker et distribuer, qu'il s'agira de réhydrater et de porter á la température de consommation. Les produits alimentaires en poudre peuvent être classés en deux catégories selon qu'ils servent d'aliments directement consommés (potages en sachet, entremets instantanés, etc.) ou de matière première (amidons, fécules, farines, etc. ) utilisée par l'industrie alimentaire. Dans le premier cas l'assainissement, défini en termes d'élimination des germes pathogènes, se posera avec d'autant plus d'acuité que le stade de la cuisson sera supprimé. Dans le deuxième cas il suffira de détruire un maximum de m icro-organism es en vue d'abaisser le plus possible les barèmes de stérilisation du produit fini, d'où résultent un moindre coût et une amélioration de la valeur nutritive et gustative. Dans le cas des poudres la mise en oeuvre des procédés classiques de pasteurisation ou de stérilisation par la chaleur a des conséquences néfastes sur le goût, la couleur et surtout la structure; de plus elle nécessite l'em ploi d'emballages rigides et résistants. Le technologue a donc tendance à utiliser des produits chimiques dont la gamme est très étendue: anhydride sulfureux, chlore, oxyde de propylène, oxyde d'éthylène, etc., mais qui à forte dose risquent d'être toxiques. L'irradiation pourrait ici se substituer â l'utilisation de plus en plus controversée d'ingrédients chimiques et assurer en permanence l'assainissement escompté dans des emballages légers et souples. Le coût du traitement de ces produits de deuxième transformation sera faible en raison du facteur de charge élevé de l'irradia­ teur, ce qui n'est pas le cas pour des produits agricoles frais dont la production est forcément discontinue.

2. ASSAINISSEMENT DE L'AMIDON DE MAIS PAR IRRADIATION GAMMA (COBALT-60)

2 .1 .’ Utilisation de l'amidon

Qu'il soit natif, prégélatinisé ou partiellement modifié l'amidon est utilisé selon le cas directement, en mélange avec d'autres poudres, enfin comme constituant de base d'un grand nombre de préparations alimentaires industrielles. Il entre dans la composition des farines instantanées pour enfants, des farines diététiques, des aliments pour bétail, notamment des aliments pour veaux (20 000 tonnes), des pâtes à tarte surgelées, des potages en sachet [3], etc. Les industries françaises les plus diverses font appel â lui [4]: il constitue pour la brasserie (10 000 tonnes) un grain cru de haute IAEA-PL-561/3 53 qualité permettant d'équilibrer la teneur en protéines du moût; la biscuiterie s'en sert pour «couper» les farines trop riches en gluten; il est en confiserie le constituant de base de certains bonbons tendres et en pharmacie un exci­ pient ou un agent d'agglomération; la charcuterie l'em ploie comme liant ou épaississant, la margarinerie comme traceur ... Il est aussi utilisé en grande quantité par l'industrie de la conserve dans la préparation des potages liquides ou concentrés, des sauces et des plats cuisinés en boîte ainsi que des babyfoods.

2. 2. Assainissement de l'amidon

Il n'existe nulle part de réglementation officielle en matière de salubrité bactériologique des amidons. Aux Etats-Unis les normes pour les farines et les amidons destinés â la conserverie sont parfois celles définies par la National Canners Association, c'est-â-dire moins de 150 spores par gramme [ 5]. En France rien n'est prévu par la législation mais les amidonniers ont un cahier des charges imposé par les utilisateurs qui répercutent au niveau de la matière première les règles de plus en plus sévères qu'ils doivent respecter pour leurs produits finis. L'effort des amidonniers a donc porté sur l'asepsie de la fabrication; en dépit de progrès certains l'am élioration de qualité ne peut en aucun cas être garantie. Il n'est pas concevable que les 500 tonnes d'amidon sec produites tous les jours en France à des fins alimentaires le soient dans des conditions interdisant toute pollution microbienne: une tonne d'amidon requiert 8000 m3 d'air et 3 â 6 m3 d'eau. Ce n'est pas un problème unique­ ment français; il se pose aux Etats-Unis [ 5] où il est dans certains cas résolu par l'utilisation d'oxyde de propylène, 300 ppm autorisées par la Food and Drug Administration, et en URSS où l'on envisage d'employer l'anhydride sulfureux â raison de 1 kg par tonne d'amidon [ 6]. Dans la plupart des cas la contamination sera faible et interviendra pour l'essentiel après le séchage, sans oublier la pollution non négligeable apportée par l'emballage. Toutefois des accidents au cours des différentes étapes de la fabrication peuvent créer des conditions favorables á la survie ou au développement d'espèces présentes sur le grain de ma’is ou dans les ateliers. Très schématiquement l'assainissement de l'amidon se posera en termes d'élimination de spores.

2.3. Irradiation de l'amidon [ 7]

Dans un premier temps, un souci tout particulier a été apporté â l'échantillonnage: les différents prélèvements, â partir de stocks très pollués, sont homogénéisés avant d'être répartis en fractions plus petites, ce processus étant répété jusqu'à l'obtention d'échantillons de 100 g. La population se répartit comme indiqué dans le tableau I. L'irradiation de ce lot d'amidon â la dose de 150 krad (12 000 Ci, cobalt-60, 100 krad/h) détruit approximativement 95% des bactéries aérobies (fig. 1) et des m oisissures (fig. 2). Les spores de Clostridium sulfito-réduc- teurs disparaissent complètement á 300 krad, il en restait environ 8% à 200 krad. Les spores de bactéries aérobies mésophiles prétraitées 10 minutes â 80°C ou 3 minutes â 100°C diminuent de 95% â 200 krad, par contre il reste â cette dose 10% de spores de bactéries aérobies thermophiles, ce qui constitue quand même un résultat très intéressant pour les industriels de la conserve. 54 SAINT-LEBE et al.

TABLEAU I. HOMOGENEITE DE LA CONTAMINATION DE L'AMIDON

Nombre de micro­ N o m b r e de organismes par g E c a r t- ty p e déterminations M o y e n n e

Spores de moisissures 100 15 8

Spores de Clostridium 7 1 23 8 sulfito-réducteurs

Spores de Bacillus 1 0 5 5 2 3 5 8 thermophiles

Bactéries aérobies 6 1 0 4 8 8 m é s o p h ile s

Spores de Bacillus

mésophiles ( 1 0 m in à 5 5 9 2 1 7 8

8 0 °C )

Spores de Bacillus

mésophiles (3 min à 4 4 2 1 26 8

1 0 0 ° Q

L'étude des moisissures, entreprise récemment sur un amidon de mais frais et très pollué puisque prélevé dans un circuit de dépoussiérage, con­ firme le résultat précédent: une irradiation á la dose de 150 krad permet d'élim iner 95% environ de la population fongique (fig. 3) (population initiale moyenne: 8600 germ es/g, écart-type: 4312 pour 14 déterminations). Par contre, il est nécessaire d'appliquer une dose de 250 krad â de la fécule de pomme de terre pour obtenir le même effet qu'une irradiation â la dose de 150 krad sur amidon de mais, aussi bien dans le cas des moisissures (fig. 4) (population initiale moyenne: 1511 germ es/g, écart-type: 418 pour 16 déterminations), que dans le cas des bactéries aérobies mésophiles (fig. 5) (population initiale moyenne: 1412 germ es/g, écart-type: 335 pour 16 déterminations). Cette radiorésistance peut être imputée â une sélection liée au vieillissement du produit: la fécule de pomme de terre est fabriquée pendant 2 â 4 mois par an alors que la production de l'amidon de mais est étalée sur toute l'année. Les résultats spectaculaires obtenus notamment au niveau des spores de Clostridium sulfito-réducteurs et de m oisissures peuvent être imputés d'une part â l'affaiblissement des germes au cours du processus de fabrica­ tion, d'autre part et surtout â l'action des substances formées au cours de l'irradiation de l'amidon. Les expériences en cours ont pour but essentiel de faire la part de l'effet direct du rayonnement gamma du cobalt-60 sur la m icroflore et de l'action des produits de radiolyse, parmi lesquels le peroxyde d'hydrogène [ 8] parait occuper une place de choix. Dans un premier temps les essais portent sur les spores de Clostridium perfringens réensemencées massivement dans de l'amidon à différentes teneurs en anhydride sulfureux afin que les quantités de peroxyde d'hydrogène dosées immédiatement après irradiation â la dose de 200 krad soient comprises en tre 0 et 200 ppm . IAEA-PL-561/3 55

Dose (Krad ) 100 200

FIG. 1. Effets de différentes doses d'irradiation gamma sur les bactéries aérobies de 1* amidon de mais.

Dose (K rad)

100 200

FIG .2. Effets de différentes doses d’ irradiation gamma sur les spores de moisissures de l ’ amidon de mais. 56 SAINT-LEBE et al.

Dose (Krad) 100 200 300

FIG ,3. Effets de différentes doses d'irradiation gamma sur les moisissures d’ un amidon de mais très pollué, prélevé dans un circuit de dépoussiérage (les points manquants à 275 et 300 krad correspondent à une population résiduelle nulle).

Dose (Krad) 100 200 300 400

FIG .4. Effets de différentes doses d'irradiation gamma sur les spores de moisissures d'une fécule de pomme de te rre . IAEA-PL-561/3 57

Dose (Krad) 100 200 300 400

FIG.5. Effets de différentes doses d’ irradiation gamma sur les bactéries aérobies mésophiles d'une fécule de pomme de terre (le point manquant à 300 krad correspond à une population résiduelle nulle).

En conclusion, ces résultats préliminaires permettent d'affirmer que la «stérilité com m erciale» sera obtenue dans un grand nombre de cas avec une dose inférieure â 300 krad. L'effet constaté est beaucoup plus spectacu­ laire que prévu; les essais en cours visent à définir les causes de cette radiosensibilité que l'on peut d'ores et déjà imputer en partie â la présence de produits de radiolyse, notamment au peroxyde d'hydrogène. L'ensemble des travaux reste néanmoins axé sur la détermination spécifique de la m icro­ flore de l'amidon en sac avant irradiation, après irradiation et au cours du stockage, le but final étant de définir sur quelle fraction de la production l'irradiation est techniquement applicable et de moduler la dose en fonction des utilisations ultérieures du produit.

3. MODIFICATIONS DE L'AMIDON IRRADIE

L'objectif des travaux est d'évaluer les conséquences d'une irradiation de l'amidon - appliquée uniquement dans un but d'assainissement microbien - sur sa structure moléculaire et d'en préciser l'impact au niveau technologique mais surtout toxicologique. Toutefois le domaine de dose exploré, 0 à 2 Mrad, est suffisamment large pour qu'il soit possible de déceler le cas échéant des modifications dont l'intérêt pour l'industrie alimentaire justifierait à lui seul l'application de ce traitement, même â des doses plus élevées. Cette deuxième voie de recherche n'est pas pour nous prioritaire, du moins à l'heure actuelle. Il faut signaler â ce sujet les travaux de Korotchenko et al. [ 9], qui ont mis en évidence l ’effet protecteur d'une suspension d'amidon irradiée â 50 Mrad sur la corrosion de l'acier dans une solution acide; la dihydroxyacétone et l'hydroxyméthylfurfural radioformés seraient respon­ sables de cette action anticorrosive. 58 SAINT-LEBE et al.

Nos recherches portent â la fois sur l'identification et le dosage des produits de radiolyse dans le but d'évaluer leur toxicité et de définir un schéma de dégradation de la molécule d'amidon, et sur l'étude de l'évolution des principales propriétés technologiques de l'amidon.

3.1. Produits de radiolyse

Les travaux sont réalisés â l’aide de différentes méthodes allant de la simple colorim étrie â la chromatographie en phase gazeuse couplée â un spectrographe de masse, et â l'utilisation d'amidon uniformément marqué au carbone-14. La concentration des divers produits est étudiée en fonction des paramètres d'irradiation (dose, débit de dose, composition de l'atm os­ phère, température), des caractéristiques de l'amidon (hydratation, impuretés) et des conditions du stockage (température, durée); de plus on compare chaque fois les effets d'un traitement thermique â ceux d'une irradiation. Pour l'instant on a pu identifier et doser avec certitude, c'est-â-dire avec au moins deux méthodes, les produits suivants: - aldéhyde malonique [10], 2 à 3 (/ug/g)/Mrad - aldéhyde formique [11], 20 (¿ig/g)/Mrad

- peroxyde d'hydrogène [ 12], 6, 6 (pg/g)/100 krad à partir de 100 krad et jusqu'à 400 krad - acide formique, 100 (¡jg/ g)/M rad - maltose, glucose, galactose, fructose, ribose, arabinose, xylose.

Dose en Krad

FIG.6 . Formation du peroxyde d’ hydrogène et disparition de l ’ anhydride sulfureux en fonction de la dose d’irradiation: — /jg de peroxyde.d'hydrogène total par gramme d’am idon ; ------Mg d ’ a n h y d rid e su lfu reu x libre total par gramme d ’ amidon. IAEA-PL-561/3 59

De plus la présence de mannose, d'érythrose, d'acétaldéhyde, d'acétone, d'alcools méthylique et éthylique est très probable: les prochains travaux visent à la confirmer. Par ailleurs on a montré l'absence d'un certain nombre de molécules susceptibles de se former par radiolyse: méthylglyoxal, < 0, 2 (p.g/ g)/Mrad; diacétyle, < 0, 1 (pg/ g)/Mrad; aceto'ine, < 0, 1 (jug/g)/Mrad, et furfural, < 0,4 Oug/g)/Mrad. Les recherches sur l'aldéhyde malonique ont abouti à la m ise au point d'un test d'irradiation de l'amidon [ 13, 14J qui permet de déceler une dose de 2 5 krad même après trois mois de stockage. De même, les premiers résultats de l'étude des sucres permettent d'esquisser un schéma de dégradation de la molécule d'amidon: l'irradiation provoque des coupures, de la liaison a 1-4 en donnant du glucose et du maltose, ainsi qu'à l'intérieur de l'unité glucosidique libérant des pentoses et probablement un tétrose. En ce qui concerne les sucres, l'oxygène ne joue aucun rôle et il y a vraisemblablement, bien que le milieu soit acide, des épimérisations, dont celle du glucose en mannose, le galactose restant stable. Le peroxyde d'hydrogène [12] n'apparaît que lorsque tout l'anhydride sulfureux a été oxydé (fig. 6) - et il n'y en a plus après deux jours de stockage â la température de 2 5°C. Cette disparition est certainement due â une interaction du peroxyde avec les sucres, les aldéhydes et les cétones radioformés, ces derniers étant transformés en hydroxyalcoyl-peroxydes, puis en acides. Le technologue se trouve donc en présence de deux possibilités intéressantes: - favoriser la formation du peroxyde, qui peut être un radiosensibilisateur des spores, en diminuant la quantité d'anhydride sulfureux avant irradia­ tion et en augmentant la teneur en eau et la température jusqu'à respective­ ment 20% et 25°C; ceci ne présente aucun danger puisque le peroxyde organique est transformé au bout de 48 heures en acide non toxique; - augmenter la quantité d'anhydride sulfureux au cours de la fabrication de l'amidon pour obtenir un produit le moins pollué possible avant irradiation, ce qui est possible puisque cet anhydride est oxydé au cours du traitement.

3.2. Propriétés technologiques

A 300 krad la viscosité maximale des empois d'amidon diminue de 20% environ. Cette dépolymérisation attendue n'est pas gênante, du moins â cette dose, pour la préparation d'un grand nombre de produits à base d'amidon, comme l'a montré un test de fabrication de crèmes pâtissières effectué récemment. A des doses de 3 â 5 Mrad et dans des conditions qui restent à préciser l'irradiation pourrait permettre la fabrication de dextrines, l'intérêt résidant à la fois dans le coût de l'opération et dans la possibilité de la mécaniser. L'allure des a et |3-amylolyses d'amidon irradié dépend des conditions d'irradiation. Le processus est accéléré car les modifications de structure radioinduites facilitent le contact enzyme-substrat; il peut aussi être ralenti par la présence d'unités anhydroglucose radioformées qui constituent des points de blocage vis-â-vis de l'action enzymatique [15]. La dégradation enzymatique de l'amidon est de plus en plus employée par les industriels, ce qui donne tout son intérêt â une telle étude. 60 SAINT-LEBE et al.

CONCLUSION

L'irradiation apparaît comme une technique d'avenir pour l'assainisse­ ment des poudres alimentaires. Dans le cas de l'amidon, l'objectif escompté sera atteint le plus souvent avec une dose inférieure â 300 krad. Les modifi­ cations radioinduites sont essentiellement abordées ici sous l'angle toxi­ cologique. Toutefois, une étude des modifications sous irradiation de la structure de l'amidon dans le but d'évaluer l'intérêt de produire des m olé­ cules nouvelles ou de favoriser l'action des enzymes au cours des divers processus de fabrication peut constituer une voie féconde.

REFERENCES

[1] SAINT-LEBE, L ., BERGER, G ., in 4th Conf. Int. Util. Energie Atom. Fins Pacif. (Actes Conf. Genève,

1971) 12, AIE A, Vienne, ONU, New York (1972) 347.

[2] DUBOURGNOUS, J ., in Cahiers du CENECA (Centre parisien des Congrès internationaux, Paris, 26-28 février 1969) 2, n” spécial (1969) 60.

[3] VOGEL, W .F., Conserv. 1 (1969) 1.

[4] MARTIN, J.L., Aliment. Vie 54 4-5-6 (1966) 99.

[5] VOJNOVICH, C ., PFEIFFER, V .F ., GRIFFIN, E .L ., Cereal Sci. Today 15 12 (1970) 401.

[ 6 ] DESINOVA, G .I., CHERNIKOVA, G .R., NEKRICH, N .A ., Sakh. Prom. 43 8 (1969) 63.

[71 SAINT-LEBE, L ., MUCCH1ELLI, A ., LEROY, P ., BEERENS, H ., in Radiation Preservation of Food (C.R. Coll. Bombay, 1972), AIEA, Vienne (1973) 155.

[ 8 ] BERGER, G ., SAINT-LEBE, L ., C.R. Acad. Sci. Paris 272 (1971) 455. [9] KOROTCHENKO, K .A ., PUTILOVA, I.N ., CHERNOBAEVA, N. M ., Izv. Vyssh. Uchebn. Zaved. 3 ( 1 9 7 1 ) 5 3 .

[10] BERGER, G ., SAINT-LEBE, L ., C.R. Acad. Sci. Paris 268 (1969) 1620.

[11] BERGER, G ., SAINT-LEBE, L ., Ibid. 271 (1970) 552.

[12] BERGER, G ., SAINT-LEBE, L ., Ibid. 272 (1971) 1455.

[13] BERGER, G ., SAINT-LEBE, L ., Stârke 21 (1969) 205. [1 4 ] BERGER, G ., ROSTAN-WOODHOUSE, D .. SAINT-LEBE, L. , Ibid. 24(1971) 15. [15] TOLUER, T ., GUILBOT, A ., Ibid. 18 (1966) 305. IAEA-PL-561/4

PHYSICO-CHEMICAL CHANGES IN IRRADIATED (GAMMA 60Co) INULIN

Stefania BACHMAN, H. ZEGOTA Institute of Applied Radiation Chemistry, Technical University of-fcodz, ■fcodz, Poland

Abstract

PHYSICOCHEMICAL CHANGES IN IRRADIATED (GAM MA MCo) INUUN.

The effect of gamma 60Co' rays on the formation (radiolysis) of major products of inulin when irradiated in aqueous solution and in the solid state has been investigated. In both cases processes have been found to occur which lead to the degradation of inulin molecules to smaller fragments. Among the products of inulin radiolysis the authors have been able to detect: deoxysugars, formaldehyde, oxalic acid and substances

absorbing between 220 and 320 nm. The use of thin-layer chromatography has established the presence of

fructose, glucose and sucrose. The intermediate products of inulin radiolysis are free radicals which form with the yield Grad¿cai = 5.5 in de-aerated inulin and Gradical = 0.45 in the presence of air. The ESR spectra obtained for inulin resemble those obtained for fructose which points to the similar character of the radicals forming in both cases. The presence of oxygen during inulin irradiation énhances the contribution

of oxidative processes leading to the formation of organic acids.

INTRODUCTION

Plants belonging to the family Compositae that contain inulin and other fructosanes as reserve substances are an important group of plant raw material for the food industry. Among the more important ones are the tubers of dahlias, the tubers of the Jerusalem artichoke (J.a. ) and chicory roots. Utilisation of fructosane-containing plants, such as the J.a., is a recurrent problem that was taken up in the years of poor potato or sugar- beet crops caused by pest dissemination, unfavourable weather conditions etc. The J.a. tuber, whose environmental demands are low, is the cheapest raw material for the food industry, especially for the fermentation industry. Apart from the highly valuable tubers the above-ground portions of the plant are also valuable and could solve the feed problems in agriculture [2, 3, 13]. Apart from a wide range of applications in the food industries (fermentation, confectionary), fructosanes and particularly inulin may be used in the pharmaco-chem ical industry in the production of fructose which is a valuable substitute for sucrose in diabetic conditions. Despite numerous studies, the structure and composition of plant fructosanes, including inulin, are still far from being definitely established [6]. According to Jefford and Edelman [ 14], reserve carbohydrates in the Compositae consist of a series of /З-D-fructofuranosides each of which is described by the general formula G ~ F- (F)n w h ere " G ~ F " represents glucose and fructose respectively and "n" may be any number ranging from 0 for sucrose to about 40 for inalin. All these homologues are normal

61 62 BACHMAN and ZEGOTA chains where monosaccharide residues are linked by 1, 2 bonds. Instability of inulin in natural plant systems is due mainly to inulinase which hydrolyses this oligosaccharide to fructosanes of different chain lengths of different molecular weight [28] . Thanks to improvements in breeding and agriculture, as well as in analytical and technological methods permitting adequate evaluation of the raw material, the problem of inulin-containing plants is of considerable economic significance. Radiation techniques may prove to be very useful both in the tuber storage process (by inhibiting sprouting) and in technological processing, acceleration of hydrolysis or replacement of the acidic method in the course of fructosane hydrolysis, wort sterilization etc. The utilitarian considerations as well as the virtual absence of reported data on inulin radiolysis made us investigate the effect of gamma 60Co rays on this oligosaccharide, irradiated in model systems in aqueous solutions and in the solid state. Studies on radiolysis products of aqueous inulin solutions are connected with the behaviour of this sugar in natural biological systems subjected to irradiation. The aim of investigations on inulin irradi­ ated in the solid state is to gain a deeper insight into the effect of ionizing radiation on carbohydrates.

Characteristics of inulin

Inulin, whose em pirical formula is given as (C6H10O)n or (C6 H10O)n ■ H20, is a white crystalline powder made up of minute birefringent crystals. Its solubility in cold water is low, giving opalescent solutions, whereas its solubility in hot water is good. The plane of polarized light in an inulin solution undergoes rotation to the left, its specific rotation being equal to [a ]D = -40°. Over the years there have been numerous unsuccessful attempts at establishing the existence of reducing properties of inulin. Methyl derivates of the following sugars have been found to occur among the products of exhaustive méthylation of inulin [11]: 3, 4, 6-trimethylfuranose (90.9%), 1, 3, 4, 6-tetramethylfuranose (3.2%), and a mixture of tetramethyl glucopyranose (2.2%) with trimethylglucose (3.2%). The absence of dimethyl- hexoses is evidence of a non-branched structure. The high content of tetra- and trimethylglucose suggests that glucose is linked to the polyfructosane chain. The inulin chain contains about 3 5 fructofuranosane residues and a fructose bonded to the glucopyranose unit by way of a sucrose-type linkage. The glucose to fructose ratio in inulin is assumed to be 1 : 40 [ 12]. Since inulin does not show any evidence of end-groups a ring structure is assumed. Inulin hydrolysis may take place due to heating in dilute acid medium or due to the effect of the enzyme inulinase. During acid hydrolysis about 90% of the cleavage products is fructose, the remaining 10% being difructose anhydrides [30]. Such cleavage is known as reversion in contrast to inversion for sucrose. Starch hydrolysis takes 618 times longer than inulin hydrolysis [31]. Enzymatic degradation of inulin to D-fructose takes place at a tempera­ ture of 50- 60°C, because of the action of the inulinase enzyme which is present in the raw material, and occurs during storage. Susumi Murakami [28] states that inulinase contained in J.a. tubers is inactive and becomes activated in the process of sprouting. It can also be activated by the addition of proteolytic enzymes, such as trypsin, pepsin and papain, to the juice squeezed out of the raw material. IAEA-PL-561/4 63

Effect of radiation on inulin

Inulin was not the subject of detailed radiation investigations and only some estimations of the physico-chemical changes in irradiated solutions of this sugar were made. Khenokh [ 15] observed that exposure of aqueous inulin solutions to radium rays gives rise to reducing power and pH decrease. He also found that the content of organic acids increases with an increase in irradiation time. Khenokh supposed that radium radiation brings about a cleavage of the 1, 2 glycosodic linkage with the formation of a ketone group which causes an increase of reducing power. Further studies [ 16] revealed that compounds absorbing in the range of 220 to 300 nm are formed in inulin solutions. Optical density of the solution increased with an increase in dose. Another observation he made was that the amount of formaldehyde formed in the process is higher than that in irradiated starch. Salunkhe and Moser [25] reported an increase in the yield of acid hydrolysis of irradiated inulin. Kuri et al. [ 18] reported the formation of free radicals in irradiated inulin whose nature, however, has not yet been established. It also seems perti­ nent to mention here the effect of ionizing radiation on simple carbohydrates, and on polysaccharides irradiated in solutions and in the solid state.

Radiolysis of fructose, a monomer of inulin

The effect of ionizing radiation on monosaccharides has been presented using fructose as an example. On irradiation of fructose crystals with doses of several dozen megarads the colour changes and off-flavour develops. Chemical changes of fructose in the solid state induced by ionizing radiation amount to a decrease of reducing power and the formation of formaldehyde and compounds absorbing in ultra-violet [ 17] . The yield of organic acid is dependent on the degree of crystallinity and on the presence of oxygen during irradiation. At least three simultaneous processes leading to molecular degradation take place in irradiated fructose solutions [23]:

1. Oxidation of the first-order alcohol group at carbon C -l to glucosone which then undergoes further transformation to 2-oxogluconic acid; 2. Fission of the linkage C-2 and C-3 leading to two- and four-carbon aldehyde fragments which may still undergo oxidation to acids; 3. Symmetric cleavage of the molecule leading to glyceric, aldehyde and glycerosone.

Other primary processes may comprise oxidation of a first-order alcohol group at carbon C-6 with the formation of 5-oxogluconic acid. The process of radiation degradation of fructose may be schematically represented as shown in Fig. 1. The use of isotope dilution analysis made it possible to discover the presence of other radiolysis products, such as oxalic acid, dihydroxy acetone and formaldehyde, in the irradiated fructose solutions [23]. Radiation yield of acids depends on irradiation conditions and is Gaci(js = 0.4 for de-aerated solutions and Gacids =1.1 for solutions irradiated under oxygen. In com ­ parison to other monosaccharides (e.g. glucose), fructose undergoes more extensive damage when exposed to analogous radiation doses. 64 BACHMAN and ZEGOTA

Fructose

Glycollaldehijde Glucosone Glijcerosone

A- Carbon fragment Glyceraldehyde

FIG. 1. Schematic representation of radiation degradation of fructose [ 23].

-O -О I I СНг CH2

-o I СНг

FIG.2. Schematic representation of radiation degradation of polysaccharide [2 2 ].

Radiolysis of polysaccharides

As no reported data on the radiolysis of oligofructosanes and poly- fructosanes are available, the effect of radiation on polysaccharides is presented using starch as an example. Numerous studies have been devoted to account for the mechanism of the effect of ionizing radiation on polymers of glucose, and especially of starch. Khenokh [ 15] and Bourne et al. [7] investigated the effect of radiation on dilute solutions of starch (0. 25%) and observed that an increase in radiation dose is accompanied by a decrease in solution viscosity, which is related to the decrease in the average mole­ cular weight of starch. The acidity in irradiated starch solutions increases IAEA-PL-561/4 65

the radiation yield of carboxyl groups under oxygen and nitrogen, the G-values being 1.5 and 1.4 respectively [7] . A polysaccharide degradation diagram after Phillips [22] is presented in Fig. 2. The presence of formaldehyde and substances absorbing in u.v. have also been detected among the products of starch degradation [ 15, 26] . Irradiation of solid starch also leads to physico-chemical changes of both the macromolecules as well as the starch granules. The dominating process, however, is the degradation of the molecule. For instance, the molecular weight determined by end-group analysis in starch changes from 462 000 for the control sample to 16 500 for a sample irradiated with a dose of 18 Mrad [ 20] . The occurrence of degradation processes is also indicated by the increased concentration of reducing compounds. Studies on the effect of starch irradiation revealed the existence of a protective effect of the water contained in it [ 10, 21, 24] . Scherz [27] suggests two possible processes to account for the mechanism by which various starch radiolysis products are formed: (a) direct action of gamma radiation on starch molecules, which results in ionization, excitation and formation of radicals; and (b) action of the radiolysis products of the water contained in the starch. The contribution of both these processes to the final effect of radiation on solid-state carbo­ hydrates is hard to determine.

MATERIALS AND METHODS

Investigations were carried out on inulin containing up to 2. 5% water, manufactured by British Drug Houses Ltd. (BDH).

Preparation of solutions for irradiation

Triply distilled water was used in the preparation of the 0.1% inulin solution. Degassing of both the solid-state inulin sample as well as the solutions was carried out by employing the pump-freeze-thaw technique. Samples of 1 g of solid inulin or 25 ml of a 0. 1% solution were subjected to radiation which was done simultaneously for degassed and non-degassed s a m p le s .

Technique of irradiation

The samples in cylindrical glass vessels were irradiated at room temperature in a radiation chamber with gamma 60Co sources of 20 kCi total activity at a dose-rate of 1 M rad/h. Irradiation with doses below 0. 5 Mrad was carried out using a cobalt source Gammacell 200, where the dose-rate was 100 krad/h. Dosimetric measurements were performed using a Fricke dosimeter and a maltose dosimeter of our own design [ 5]. Radiolysis products were analysed immediately after the exposure.

Analytical methods

Electron spin resonance (ESR) investigations were performed on a microwave Russian-made spectrometer, type RE 1301, operating in the 66 BACHMAN and ZEGOTA frequency band X. Quantitative measurements were carried out on fixed polymer standards calibrated according to DPPH (1, l-diphenyl-2- picrylhydrazyl).

Infra-red spectroscopy

Infra-red absorption studies of irradiated inulin samples were performed on a Unicam SP-200 spectrophotometer. Tablets in KBr were prepared for the measurements.

Ultra-violet absorption

Ultra-violet absorption of inulin solutions was studied on a Beckman DK-2 spectrophotometer. Inulin irradiated in the solid state was dissolved in 25% ethanol. This solvent was chosen because of the low solubility of inulin in water. 1. 5% solutions of inulin irradiated in the solid state were prepared for u.v. absorption measurements. Absorption of irradiated 0. 1% aqueous inulin solutions was examined following their four-fold dilution with distilled water.

Thin-layer chromatography (TLC)

TLC studies of sugars were carried out on plates, 200 mm X 200 mm and 200 mm X 100 mm, covered with silica gel G, manufactured by Merck, impregnated with IN boric acid. The following solvents were used: (a) isopropanol:ethyl acetate:water (7:1:2) and (b) n-butanol:ethyl acetate: isopropanol:acetic acid:water (35:100:60:35:30). Colour developing on plates was made using a diphenylamine reagent prepared according to McNally and Overend [ 19]. Deoxysugars were detected according to the method described by Anderson [ 1] . Quantitative determinations of sugars were performed using the anthrone method [4] . Formaldehyde was determined according to the procedure of Tanenbaum and B riefer [29] . Quantitative determinations of deoxysugars were carried out using the colorim etric method described by Waravdekar and Saslaw [32] . Oxalic acid was determined according to the method suggested by Draganic [ 9]. Because of the low solubility of inulin in water all quantitative studies on radiolysis products in samples irradiated in the solid state were perform ed in 2. 5% solutions in 25% ethanol.

RESULTS AND DISCUSSION

S u gars

The anthrone method was used to determine total sugars. Under the influence of the anthrone reagent an acidic hydrolysis of inulin to simple sugars first takes place followed by their condensation with anthrone and the formation of a coloured compound— 1, 4-dianthronylydenopentene. The anthrone method permitted the determination of the changes in the concentration of total sugars that is dependent on the radiation dose. It has been found that in irradiated aqueous solutions the sugar concentration IAEA-PL-561/4 67

Dost

F1G.3. Relative changes in total sugar concentration plotted against radiation dose: о — о irradiated in the absence of air, • — • irradiated in air.

decreases with the increase in dose; in the presence of oxygen this decrease is more pronounced. In the semilogarithmic system the changes in the concentration of total sugars as a function of dose can be presented approxi­ mately in the form of a straight line (Fig. 3). In the case of de-aerated solutions a straight line is obtained which can be described by the equation:

log = -o-D (1) where C0 - initial concentration of inulin, С - inulin concentration after irradiation with dose D, a - coefficient. Since the dose D is the product of dose-rate and time, Eq. (1) may be written as:

С = C0 e'kt (2) where t - period of irradiation, к - constant coefficient.

The above equation is characteristic of first-order chemical reactions. In the case of solutions irradiated in the presence of air, a curve composed of two straight-line segments is obtained (Fig.3). For doses up to 1 Mrad the degradation of inulin is faster than in the de-aerated samples. Possibly, the contribution of oxidative processes leading to the formation of organic 68 BACHMAN and ZEGOTA

TABLE I. THIN-LAYER CHROMATOGRAPHIC DATA FOR 0. 1% INULIN SOLUTIONS IRRADIATED WITH A DOSE OF 1 Mrad

Colour of spots C o m p o u n d r f

0 . 3 0 Brownish-green S u crose

0 . 4 0 Brown F ructose

0 . 4 7 Y e llo w ?

0 . 62 Brownish-blue G lu c o s e

0 . 6 6 O ra n g e ?

0 . 7 0 Brownish-yellow D e o x y su g a r

0 . 7 7 Pink D e o x y su g a r

acids increases. The irradiation of solid-state inulin does not bring about any distinct changes in the concentration of sugars over the range of doses up to 5 M ra d .

Thin-layer chromatography

TLC was used in the qualitative identification of radiolysis products which is useful as an illustration of the data obtained by analytical methods. Results of chromatographic studies of irradiated aqueous solutions of inulin have shown that a dose as small as 0. 5 Mrad brings about considerable degradation of the molecule. In the first stage, the main process is the radiation-chemical hydrolysis of 1, 2 glycosidic bonds in inulin. An increase in the dose is accompanied by an increase in the degree of degradation (a greater number of low-molecular fragments is formed). Seven distinct spots appeared on the chromatogram (Table I). A comparison with the Rf for standard substances revealed the presence of sucrose (RF = 0.30), fructose (RF = 0.40) and glucose (RF = 0.62). On the basis of reported data it could be expected that deoxysugars should be among the products of inulin radiolysis. It was found by Anderson's method that the spots of Rp = 0.7 0 and 0.77 are deoxycompounds of, as yet, undetermined composition. Chromatographic studies of inulin irradiated in the dry state have shown no degradation of the molecule over the range of doses employed up to 5. 0 M ra d .

Determination of deoxysugars

Results of quantitative analysis of deoxysugars, calculated as 2-deoxyglucose, are presented in Fig. 4. In irradiated solutions of inulin, for doses below 2 Mrad, there is first a rapid increase in the concentration of deoxysugars till the curve reaches its maximum and for higher doses the concentration of deoxysugars diminishes. This dependence of the concen­ tration of deoxysugars on dose points to the existence of two competitive processes. One process is the formation of deoxysugars which are the IAEA-PL-561/4 69

I

к 8. 3

Q

Dose

FIG.4. Deoxysugar formation in irradiated inulin plotted against radiation dose, о— о irradiation of 0.1% aqueous solutions, •— • irradiation of solid inulin which was then diluted to obtain a 3% solution (see materials and methods).

С MO CHO : u > Thiobarbituric С Нг ¡o CH2 a c id H-C-OH CHO + H-C-OM н-оо

Red product

ch2oh H;CH0 И-С-0Н CHO Thiobarbituric CH 2 I0«~ с н г acid H-Ç-0H ¿HO H-C-OH + H-OO

FIG. 5. Schematic representation of the oxidation reaction of deoxysugars to malone dialdehyde, and condensation of dialdehyde with thiobarbituric acid. 70 BACHMAN and ZEGOTA primary products of inulin radiolysis. The other process, which is com­ petitive in relation to the first, leads to the conversion of deoxysugars. The analytical method of deoxysugar determination used in this study depends on their oxidation to malone dialdehyde (Fig. 5) which gives a coloured product with thiobarbituric acid (ТВA). As a result of ionizing radiation the oxidation of the aldehyde groups to carboxyl groups probably takes place. As a result of this the malone dialdehyde is not formed but appropriate acid which does not undergo condensation with TBA. This should be held responsible for the diminished amounts of deoxysugars on inulin irradiation with doses above 2 Mrad. The radiation yield of deoxysugars, calculated for the initial slope of the curve in Fig. 4, is G = 0.50. Inulin irradiated in the solid state also revealed the presence of deoxy­ sugars but, in the range of doses under discussion, the yield of deoxy- compounds is markedly lower and the characteristic maximum of the curve is not observed. The effect of oxygen on the yield of deoxycompounds was found to be negligible both for inulin irradiated in aqueous solutions as well as in the solid state.

Formaldehyde determination

One of the products of carbohydrate radiolysis is formaldehyde. Its presence'in irradiated inulin was reported by Khenokh [ 16]. In the study reported here the radiation yield GHCHOwas determined which, for irradi­ ated inulin solutions (calculated from the initial slope of the concentration versus dose curve), is 0.08. Small quantities of formaldehyde were also detected in the solutions prepared from inulin irradiated in the solid state.

Oxalic acid determination

Formation of organic acids is one of the main directions of carbohydrate radiolysis, both in solution as well as in the solid state. The presence of acids is evidenced by the diminishing pH values of irradiated sugars. It was found in our study that one of the products of inulin radiolysis is oxalic acid whose quantity increases with the increase in the radiation dose absorbed. In the case of inulin irradiated in solution as well as in the solid state the concen­ tration of oxalic acid is higher when samples are irradiated in the presence o f ox yg en .

Electron spin resonance spectra (ESR)

The formation of free radicals in irradiated biological systems, both models and natural, has troubled sanitarians for a long time, the cause of concern being their carcinogenic properties. Pertinent investigations in radiation chemistry are also interesting from the theoretical point of v ie w . In the course of carbohydrate irradiation in the solid state, stable radicals are observed to arise which may be investigated by means of ESR spectroscopy. In contrast to many other carbohydrates producing doublet resonance [ 10], inulin irradiated in the presence of oxygen gives a four- line ESR spectrum (Fig. 6). A more simple spectrum is obtained for inulin irradiated under oxygen-free conditions (Fig. 6). The presence of oxygen and moisture during irradiation of non-degassed samples brings about the IAEA-PL-561/4

Лг

50Cs

F IG . 6 . ESR spectra for inulin irradiated in air (a) and in the absence of air (b), and for polycrystalline fructose irradiated in air (c).

Dose

FIG. 7. Concentration of the most stable free radicals in inulin as a function of radiation dose: о ------о

irradiated in the absence of air, • ------# irradiated in air. 72 BACHMAN and ZEGOTA formation of other radicals. The reaction below is thought to play a significant part:

+ 02 ->ROO'-> radicals + non-radical products

To supplement the above studies, ESR measurements of polycrystalline fructose samples have been performed under the same experimental conditions. The spectrum obtained for fructose is presented in Fig. 6. It corresponds to the one described by Williams [33] . Collins [ 8] observed a similar characteristic of ESR spectra and reported approximate radical yields for sugars of the glucosane series such as glucose, cellobiose and cellulose. On the basis of the results presented here it can be concluded that there is also an analogous similarity in the fructosane series (fructose and inulin). It is well known, that gamma radiation has sufficient energy to bring about a cleavage of all chemical bonds in a carbohydrate molecule, and it seems probable that the energy absorbed in a number of definite places in a furanose or pyranose ring is transferred to the bond which undergoes cleavage. The number of radicals induced by gamma radiation in inulin is dependent on the radiation dose and presence of oxygen (Fig. 7). Since oxygen facilitates the recombination and radical degradation processes, the number of stable radicals formed in its presence is lower. The yields of radiation radicals calculated from the slope of the curves in Fig. 7 are G = 5. 5 for degassed samples and 0.45 for samples irradiated in the air. Studies of the kinetics of the radical degradation in samples both in the presence and in the absence of oxygen have shown that in the form er case the life-tim e of the radicals was several hours, whereas in the latter case radicals weredetected even 100 days after exposure had been stopped. At the moment when carbohydrates irradiated in the solid state are dissolved, the radicals stabilized in the crystalline lattice react immedi­ ately with water, oxygen, another carbohydrate molecule or another radical. These reactions may be schematically represented as:

R^ + Rg - p rod u ct

R' + 0 2 -* ROO ->• product

R- + H 20 - Rj

R' + carbohydrate -*■ Rj:

Radicals R' and Rjj may enter subsequent reactions till a stable product is fo r m e d .

Infra-red spectroscopy

Infra-red absorption studies have not shown any significant differences between inulin irradiated with doses below 5 Mrad and the control sample. It was only when the doses exceeded 10 Mrad that weak absorption at 1740 cm-1 appeared which corresponds to a carbonyl group. Other fragments of the spectrum do not reveal any distinct changes which indicates that irradiation of solid-state inulin with a dose of up to 5 Mrad does not produce any significant chemical or structural changes in most of the furanose rings in the molecule. IAEA-PL-561/4 73

Wavelength [n m ]

F IG . 8 . Ultra-violet spectra for irradiated inulin: (a) irradiation of 0 .1°]o aqueous solutions, (b) for 1. 5°]o solutions of inulin irradiated in the solid state.

Radiation doses: 1, Control; 2, 0.5 Mrad; 3, 1 Mrad; 4, 3.0 Mrad; 5, 5.0 Mrad.

Ultra-violet absorption

Carbohydrates exposed to radiation display characteristic absorption in ultra-violet at wave lengths (X) between 220 and 320 nm. The absorption peak for most sugars occurs at wave lengths between 265 and 275 nm. For inulin, irradiated in solution and in the solid state, the maximum absorption is at A. = 270 nm, and for doses exceeding 1 Mrad it shifts towards shorter wave lengths of Xmax = 265 nm (Fig. 8). The shift of absorption peak in the u.v. spectrum was also observed by Khenokh [ 16] . In the case of irradiation of aqueous solutions of inulin the absorption intensity is considerably higher than for inulin irradiated in the solid state. In order to obtain comparable absorption values the measurments were performed on 1. 5% solutions of inulin irradiated in the dry state, on the one hand, and on 0. 1% aqueous solutions diluted four times after irradiation, on the other. No effect of the presence of oxygen during inulin irradiation on the position of the absorption maximum and on the character of the u.v. absorption spectrum was observed.

CONCLUSIONS

It appears from the studies perform ed so far and from reported data [ 22] that carbohydrates irradiated in the solid state are more resistant to radiation than those irradiated in solution. 74 BACHMAN and ZEGOTA

In aqueous solutions, apart from processes of direct absorption of energy by the molecule, additional processes take place that are brought about by the presence of the products of water radiolysis, such as OH and H radicals as well as e¡olv>. The attack of OH radicals seems to be par­ ticularly effective since their reaction rate with carbohydrates, e.g. glucose, is of the order 1 X 109 litres/m ole • s [ 22]. For doses below 0. 5 Mrad the main process occurring in irradiated aqueous solutions of inulin is the radiation-chemical radiolysis of glycosidic bonds. Thin-layer chromatography studies show that products of different molecular weight are obtained, ranging from monosaccharides (fructose, glucose) through disaccharides (sucrose) to undegraded inulin molecules. The content of fructose among degradation products is small similarl to the share of glucose among the products of starch radiolysis [ 20] . Inulin irradiated in the solid state undergoes less degradation than it does when irradiated with the same dose in solution. In both cases similar radiolysis products are obtained, such as deoxysugars, formaldehyde, organic acids, substances absorbing in ultra-violet etc., though their concentration is significantly lower in the case of inulin irradiated in the solid state. The presence of oxygen at the time of irradiation enhances the contri­ bution of oxidative processes leading to oxidation of the first-order alcohol groups to aldehydes and acids. The intermediate products of inulin radiolysis are free radicals, the number of which is dependent on the radiation dose. The structure of the ESR spectra and the kinetics of radical decay are affected by the presence of oxygen at the time of irradiation and storage of samples. The studies we have perform ed supplement the scarce reported data on the effect of radiation on fructosanes, and inulin in particular. An understanding of the processes that take place in irradiated carbo­ hydrates contributes to a better knowledge of radiolysis of oxygen-containing organic compounds and makes it possible to study their behaviour in the course of irradiation of natural systems, raw materials or food.

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Dahlia tubers, J. Chem. Soc. (1950) 1297.

[13] ISO, I., A czicsoka (die Topinambur), Budapest (1955).

[ 14] JEFFORD, T. G ., EDELM AN, J ., Changes in content and composition of the fructose polymers in

tubers of Helianthus tuberosus during growth of daughter plants, J. Exp. Bot. 12 (1961) 177. [ 15] KHENOKH, M . A ., Cleavage of macromolecules of natural high-molecular substances under the

action of у-rays, Zh. Obshch. Khim. 20_(1950) 1560.

[16] KHENOKH, M .A ., Action of у-radiation of cobalt-60 on carbohydrates, Dokl. Akad. Nauk SSSR

104(1955) 746.

[17] KHENOKH, M .A ., KUZICHEVA, E .A ., EVDOKIMOV, V .F ., Action of Go6" у-radiation on dry carbohydrates, Dokl. Akad. Nauk SSSR 135 (1960) 471.

[18] KURI, Z ., FUIWARA, Y ., UEDA, H ., Behaviour of the free radicals trapped in gamma-irradiated compounds which contain hydrogen bonds, J. Chem. Phys. 33 (1960) 1884.

[19] McNALLY, S ., OVEREND, W .G ., The chromatographic differentation of some deoxy-sugars,

J. Chromatogr. 21 (1966) 160.

[20] ORESHKO, V .F ., KOROTCHENKO, K .A ., Destruction of starch depending on the dose of ionizing

y-radiation, Dokl. Akad. Nauk SSSR 133 (1960) 1219. [21] ORESHKD, V .F ., Protective action of water in the radiolysis of starch, Zh. Fiz. Khim. 34(1960) 2369.

[22] PHILLIPS, G .O ., Radiation chemistry of carbohydrates, Adv. Carbohydr. Chem. 16 (1961) 32.

[23] PHILLIPS, G .O ., MOODY, G .J., Radiation chemistry of carbohydrates. Part III. The effect of

gamma-radiation on aqueous solutions of D-fructose, J. Chem. Soc. (1960) 754.

[24] REUSCHL, H., GUILBOT, A ., Der Einfluss von Gammastrahlen auf Kartoffelst'árke in Abhangigkeit

von ihrem Wassergehalt, Starke 18 (1966) 73. [25] SALUNKHE, D .K ., MOSER, .F. V ., Cheaper fructose on way ?, Food Eng. 30 (1958) 110.

[26] SAMEC, М ., Verânderungen der Kartoffelstarke unter dem Einfluss ionisierender Strahlen, St'árke 11

(1959) 285.

[27] SCHERZ, H ., Bestrahlung von Stârke. Untersuchung der niedermolekularen Spaltprodukte, Stârke 23 (1971) 259.

[28] SUSUMI MURAKAMI, The inulinase of Helianthus tuberosus, Science Reports, Saitama U niv., Ser. В,

1 (1954) 153. [29] TANENBAUM, М ., BRICKER, C .E ., Microdetermination of free formaldehyde, Analyt. Chem. 23

(1951) 354.

[30] THIES, H ., SOUCI, S .W ., KALLINICH, G ., Der Verlauf der Sáurehydrolyse des Inulins. Ein Beitrag

zur quantitativen Bestimmung des Inulins in Kohlenhydratgemischen, Z . Lebensm.-Untersuch. Forsch. 96 (1953) 41. [31] THIES, H ., SOUCI, S .W ., KALLINICH, G ., Über die Abhangigkeit der Sáurehydrolyse des Inulins,

der Saccharose und der Starke vom pH-W ert und von der Temperature, Z . Lebensm. -Untersuch.

Forsch. 96 (1953) 83. [32] WARAVDEKAR, V .S ., SASLAW, L .D ., A sensitive colorimetric method for the estimation of 2-deoxysugars with the use of the malondialdehyde-thiobarbituric acid reaction, J. Biol. Chem.

234(1959) 1945. [33] WILLIAMS, D ., SCHMID, B ., WOLFROM, M .L ., MICHAEUS, A ., McCABE, L.J., Paramagentic

resonance spectra of free radicals trapped on irradiation of crystalline carbohydrates, Proc. Natl.

Acad. Sci. U.S. 45 (1959) 1744.

IAEA-PL-561/5

EFFECTS OF IONIZING RADIATION ON GELATIN IN THE SOLID STATE

Stefania BACHMAN, S. GALANT, Z. GASYNA, S. WITKOWSKI, H. ZEGOTA Institute of Applied Radiation Chemistry, Technical University ofiódz, ■fcódz, Poland

Abstract

EFFECTS OF IONIZING RADIATION ON GELATIN IN THE SOLID STATE.

The effect of gamma s0Co radiation on some technological and physico-chem ical properties of gelatin irradiated in the solid state was investigated. Doses of up to 2 .5 Mrad did not give rise to any negative changes in irradiated gelatin. Doses between 0.5 and 1.0 Mrad were sufficient to sterilize gelatin with insignificant surface contamination. The effect of radiation between 0.5 and 3 .5 Mrad on degradation of gelatin molecules and increase in polydispersity was demonstrated on the basis of studies on viscosity and molecular sieving on Sepharose 4 В gel, as well as by determinations of sedimentation velocity constant and optical rotatory dispersion. Irradiation of gelatin did not produce any distinct changes in amino acid composi­ tion though it did give rise to an increase in the content of carbonyl groups. Ionizing radiation induced the formation of free radicals, producing ESR spectra in the form of a symmetrical doublet. The kinetics of free- radical decay in gelatin while stored at room temperature in the presence of air were also studied. The studies performed may serve as evidence and arguments in favour of legalizing the process of radiation sterilization of gelatin.

INTRODUCTION

Since gelatin is a perfect nutrient substance for m icro-organism s there exists, in the process of its production and storage, a constant hazard of its microbial contamination which, together with gelatin, may be introduced in the final product bringing about spoilage and diminished durability of canning products, non-sterility of pharmaceutical gelatin and technological problems in the photochemical industry. With the increasing demand for gelatin, intensive studies are under way in search of methods for sterilizing gelatin which would not give rise to any undesirable changes in its physico-chemical properties. Apart from considerable effectiveness in microbial inactivation, radappertization of gelatin in plastic packages provides the possibility of protecting the material from incidental contamination [ 2 ].

Physico-chemical properties of gelatin

Gelatin is a product obtained from animal materials containing collagen. It is an industrial product of collagen conversion, is soluble in water and is obtained by degradation of collagen bonds and by hydrolysis of cross- linking bonds. Gelatin has an amorphous structure of variable molecular size. According to several authors, the molecular weight of gelatin from different origins ranges from 20 000 to 110 000. Smaller molecules, and even peptides, may be present as well as very large protein aggregates.

77 7 8 BACHMAN et al.

TABLE I. FUNCTIONAL GROUPS IN COLLAGEN [21]

K ind o f F o rm u la Derived from: group

- С ОО Н C a r b o x y l- Aspartic acid, glutamic acid

- n h 2 e - a m i n o Lysine, hydroxylysine

NH

- N H - С G u a n id in e - A r g in in e \ n h 2

- С - N II It I m i d a z o l e - H istid in e H C C H \ / N

H

- O H H y d r o x y l- Hydroxyproline, serine,

threonine, hydroxylysine

- CO - NH - P e p tid e - Am ino- and imino-acid residues — c o - n C linked by peptide bonds

It is assumed that in solution the gelatin molecule has the form of randomly branched chains which may assume a variety of shapes, making a random coil [4]. In comparison to globular proteins of approximately the same molecular weight, protein molecules present a less compact structure [ 5] and, consequently, a significantly greater volume. The chemical reactivity of gelatin is determined by its molecular structure and its amino acid composition. Weakening of the molecular structure gives rise to a great number of reactive acidic and basic functional groups as shown in Table I [21]. Chemical reactivity is also brought about by components of non-collagenous origin such as sugars, nucleic acids, other tissue proteins and impurities resulting from the technological production of gelatin. The diversity of the functional groups in gelatin makes it highly susceptible to modification and introduction of additional substances like sulphur-containing compounds. The gel strength and intrinsic viscosity of gelatin are important properties determining its technological value as a commercial product.

Effect of radiation on gelatin: some reported data

The effect of radiation on gelatin, and its sols in particular, has been investigated for quite a long time. Fernau and Pauli [ 8] found that irra­ diation with radium rapidly reduced the gelatin sol viscosity which became stable at a definite dose level. Zhukov and Unkowskaja [24] found that the effect of radium rays on gelatin manifests itself in greater internal changes. Khenokh [ 14] irradiated a 0. 5% sol of thermally sterilized gelatin and observed reduced viscosity, protein decomposition and changes in pH which grew in intensity with increasing radiation dose. According .to Putilova [20], IAEA-PL-561/5 79 dénaturation of part of gelatin may be caused by irreversible coagulation of the macromolecular fraction. In contrast to this, the fraction with the lowest molecular, weight, being the most resistant to coagulation during gamma irradiation, is retained in the solution. Khenokh [14] irradiated some amino acids or their derivatives which enter into the composition of gelatin and found that the amino group in glycol and the imino group in proline and acetylglycol, as well as the peptide bond in diketone piperazine, undergo decomposition yielding am m on ia. Prusak and Sciarrone [ 19] subjected two kinds of gelatin in solution (1 and 5%) and as a film to radiation doses of between 0. 41 and 2. 1 Mrad. On the basis of carbonyl-group determinations and the differences in ESR spectra before and after irradiation they drew conclusions about the changes in molecular weight and physico-chemical properties. The concentration of carbonyl groups in irradiated gelatin was found to increase with dose, the effect being stronger for gelatin irradiated in solution than in film s . The effect of radiation on gelatin was also investigated by Máteles and Goldblith [ 16]. Samples were irradiated in a source of gamma 60Co and above 2 Mrep in a linear accelerator, and studied for viscosity and gel strength. For doses up to 2 Mrad they observed a decrease in viscosity and no changes in gel strength. The change in colour of irradiated gelatin was pointed out by Sheppard [23] and Arnev [ 1] and attributed to the presence of an unidentified photo- and radio-sensitive component. Frank and Grünewald [9] irradiated solid gelatin containing 12% water by using, a Van de Graaff (1 MeV) accelerator and a linear one (10 MeV). A dose of 4. 5 Mrad was considered sufficient to sterilize the product. At this dose the gel strength decreased by about 30%. Radicals which formed as a result of irradiation, disappeared when the gelatin was immersed in water or heated [ 9]. The aim of the studies undertaken was to establish the effect of gamma 60Co ionizing radiation in the range of radappertizing doses up to 3. 5 Mrad on major physico-chemical and technological properties of gelatin. The starting point in the choice of dose was to determine their effect on inactivation of contaminating organisms. To make the changes in the physico-chemical properties more prominent, doses above 3. 5 Mrad were a ls o u sed .

MATERIALS AND METHODS

Investigations were carried out on Polish-made commercial product, the samples being taken from different production batches. The composition of the gelatin was: water, 10. 8 to 13. 7%; mineral matter, 0. 5 to 1. 69%; and total nitrogen, 16. 9 to 18. 4%.

Irradiation technique

Samples were irradiated in a MITR radiation facility in Lodz equipped with sources of gamma 60Co of 20 kCi total activity. Gelatin was irradiated in the solid state in the form of powder or granular at room temperature in the presence of air. Most of the samples were irradiated in closed 80 BACHMAN et al. polyethylene bags in amounts ranging from 60 to 250 g with 0. 5 Mrad/h. A number of samples for special determinations were irradiated in glass test-tubes or in ESR tubes. The time of analysis after irradiation was not strictly limited and was from several hours to several dozen hours, but always the same for a series of samples.

M icrobiological studies

M icrobiological studies for control samples and irradiated ones were carried out according to Polish Regulation PN-63/A-82245. Sporotests containing Bacillus pumilus E 601 were employed as biological controls of radiation sterilization. Inactivation of spores was tested on "universal medium" [22]. Control reading of spores was made after cultivation at 32°C.

Technological studies

In order to determine the effect of radiation on the technological value of gelatin, canning trials were performed. To do this, slices of meat 15 cm x 15 cm were powdered with irradiated and control gelatin, rolled up, bound up with yarn, and, after a lapse of 3 h, boiled for 30 min. Irradiated gelatin samples were also used in the production of canned ham "Oblong", adding 30 g to each can. Standardization estimation was made 10 days later. Chunks of sirloin weighing 31-0 g were put into 99 cm x 51 cm cans and covered with 20% gelatin solution which had previously been irradiated with 0. 0, 2. 0 and 5. 0 Mrad. The cans were examined after 5 days, 3 months and 6 months. After the cans had been opened, the colour of the jelly, its consistency and the colour of the meat bulk surface were examined. The organoleptic tests were performed by a group of 7 people by means of the triangular test according to the Polish Regulations.

Physico-chemical methods

Hydroxyproline (Pro-OH) was determined according to Stegemann's method as modified by Hurych and Chvapil [ 13]. The amino acid content was determined by the method of Stein and Moore [ 17]. Column chromato­ graphy of amino acids in gelatin hydrolysates was performed with a Japanese ILC-3BC Jeol automatic analyser. Carbonyl group estimations were made as described by Hamilton [11]. The viscosity of the gelatin solution was estimated in a HSppler visco­ meter in accordance with Polish Regulations PN-63/A-82245 and the gel strength was determined according to Bloom's method. The degree of poly- dispersity was estimated by elution on Sepharose 4 В according to Chevé [ 6]. Determinations of sedimentation velocity constant were made using a Hungarian-made analytical ultracentrifuge, type G-120 (MOM) with a Philpot-Svensson optical system. Measurements of sedimentation coefficients were made at 35°C for 46 000 rev/m in. The optical rotation of gelatin solutions was measured on a VSU-2P Carl Zeiss spectrophotometer equipped with a polarim etric attachment built according to the design of Dirkx and co-workers [7]. Polaroids manufactured by Carl Zeiss, Jena, were used in this equipment. Measurements were taken in the spectral range of 320 - 700 nm at 20°C. IAEA-PL-561/5 81

Electron paramagnetic resonance studies of irradiated gelatin were carried out on a RE 1302 spectrometer operating in the band X. Measure­ ments were taken at the temperature of liquid nitrogen (77 K), 15 min after exposure had been finished. The ESR spectrum was recorded in the form of a first derivative, and the areas below the curve were calculated by the double integration method. Quantitative determinations of free radicals were performed by using polymer standards calibrated with DPPH (1, 1- diphenyl-2-picrylhydrazyl).

RESULTS AND DISCUSSION

M icrobiological studies

The gelatin used in m icrobiological studies was drawn from different production batches certified for trade and production in the canning industry and, as such, it was not markedly contaminated, especially with anaerobic bacteria. On average, the studies revealed the presence of 2. 5 x 103 to 1.8 x 104 total bacterial count (TBC), The basic material used in the m icro­ biological studies on the effectiveness of radiation sterilization of the contaminated product was semitechnical gelatin, or drawn from some gyles, especially selected as problem material. The results obtained are given in Table II. Doses of 0. 75 to 1. 5 Mrad were found to be sufficient to bring about sterilization of the product. A post-effect of irradiation was also observed. Determinations made after 2 and 6 weeks revealed the number of marked m icro-organism s in irradiated gelatin to be smaller than in the determinations performed after 3 days. These results are important from the point of view of production technology. They show that storage for several weeks of irradiated gelatin, which is well protected against secondary infection, .will make it possible to lower the radiation dose [ 3].

Technological studies

Organoleptic estimation (using the triangular test) of a 10% gelatin solution prepared from samples irradiated with doses up to 3 Mrad did not reveal any changes in colour or clarity. With doses higher than 0. 5 Mrad a peptone odour is produced which changes into a broth odour (above 2. 5 Mrad) and then to the odour and taste of bone glue (above 5 Mrad). Tests for colour change did not reveal any negative properties of irradiated gelatin. Tests with canned meat (ham, sirloin, or tongue) showed no sign of discolouration or of protein precipitation. In all samples the jelly was equally compact. In the process of heat treatment of the above products containing gelatin irradiated with doses up to 5 Mrad no changes in viscosity or gel strength were observed. Neither were any perceptible changes in the odour of irradiated gelatin detected. The quality of the taste, though, did improve. The broth taste in gelatin irradiated with doses higher than 2. 5 Mrad may be an undesirable property in jellies and confectionery which confirms the results obtained by Frank and Grünewald [9]. TABLE II. EFFECT OF DIFFERENT DOSES OF GAMMA60 Co RADIATION ON THE CONTAMINATION DEGREE “ OF SEMITECHNICAL GELATIN Determinations made 3, 14 and 42 days after irradiation

Anaerobes A erobic G elatin- Total bacterial count Proteolytic Enter o- Pathogenic B. c o li thermophilic liquefying per gram bacteria c o c c i staphylocci Thermophilic Gas producing bacteria bacteria in 10 ml Days after Dose 3 irradiation (Mrad ) Spore- Heated at Heated at Heated at H eated at bearing Unheated 80*C for Unheated 70*C for Negative Unheated 70’ C for Unheated Heated Unheated 70*C for 80aC for 10 m in 2 b 2 h 2 h 10 min)

0 18 720 12 000 1 : 10 1 :1 0 - 1: 10 1 : 10 1: 10 1 : 10 1: 10 1: 10 16 000 10 000 28 -

0 .5 1 600 1000 1: 10 1: 10 - - 1 : 10 1 : 10 - - 1: 10 1 : 10 1400 1000 18 -

0 .7 5 800 - 1 : 10 - - - - - 1 : 10 1 : 10 1000 - 15 -

3 l, a et BACHMAN 1. 00 - 1: 10 ------1: 10 - - - 6 -

1.2 5 ------1: 10 - - - -

1. 50 ------

0 2 1 0 0 0 14 000 1 : 10 1: 10 - - 1 : 10 1 : 10 1: 10 1 : 10 1 : 10 1: 10 17 000 14 000 30 -

0 .5 1000 700 1 : 10 1: 10 - - 1 : 10 - - - 1: 10 1: 10 800 500 16 -

0 .7 5 480 - 1 : 10 1: 10 ------1 : 10 - 300 - 12 - 14 1. 00 ------1 : 10 - - 2 -

1.25 ------

1,5 0 ------

0 20 000 12 000 1 : 10 1: 10 - - 1: 10 1 : 10 1 : 10 1: 10 1 : 10 1 : 10 16 000 11000 30 -

0 .5 800 300 1: 10 1: 10 - - 1 : 10 - - - 1 : 10 1: 10 700 300 18 -

0.7 5 500 - 1: 10 ------1: 10 - - - 12 - 42 1 .0 * ' “ - " ■ - • ■ * ■ ‘ ■

1.25 ------

1.5 0 ------

a Dose-rate, 1.27 Mrad/h. - Absent IAE А-PL-561/5 83

TABLE III. EFFECT OF GAMMA 60Co RADIATION ON THE CONTENT OF HYDROXYPROLINE IN GELATIN

D o s e 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 (M ra d )

P r o -O H

( g / 1 0 0 g 1 3 . 1 5 1 2 . 9 1 2 . 8 5 1 2 . 9 1 3 . 0 5 . 1 2 . 7 5 dry wt)

TABLE IV. AMINO ACID CONTENT OF GELATIN AFTER IRRADIATION3

Dose (Mrad) A m in o a c id C o n tr o l ------

1.0 2.5 3.5

Hydroxyproline 1 1 . 9 1 0 . 6 0 1 1 .0 0 1 3 .0 0

Aspartic acid 5 . 4 0 6 . 0 0 5 . 7 0 5 . 5 0

T h re o n in e 1 . 6 5 2 . 1 2 1 . 7 0 1 . 7 0

S erin e 3 . 1 3 3 . 1 4 3 . 1 5 3 . 3 0

Glutamic acid 1 0 .1 0 1 0 . 5 0 1 0 .3 0 1 0 . 00

P ro lin e 1 3 . 5 5 1 3 . 3 0 1 3 . 2 0 1 4 .4 0

G ly c in e 2 2 . 3 0 2 1 . 2 0 2 1 . 3 0 2 1 . 5 0

A la n in e 8 . 6 0 9 . 3 5 9 . 2 0 8 . 6 0

V a lin e 2 . 4 1 2 . 5 3 2 . 50 2 . 5 0

A r g in in e 8 . 2 8 7 . 7 5 7 . 0 0 7 . 1 0

M e th io n in e 0 . 5 3 0 . 90 0 . 5 3 0 . 4 0

Is o le u c in e 1 . 1 5 1 .2 3 1 .1 6 1 . 00

L e u c in e 2 . 8 0 2 . 8 0 2 . 7 4 2 . 7 0

T h y ro sin e tr a c e s tra c e s

Phenylalanine 2 . 0 4 1 . 9 0 1 .8 7 1 . 9 0

Hydroxylysine 0 . 9 0 0 . 9 1 0 . 7 7 1 . 1 0

L y sin e 3 . 4 5 3 . 8 7 3 . 4 8 2 . 7 0

H is tid in e 0 . 7 7 0 . 7 3 0 . 6 4 0 . 6 0

n h 4+ 1 . 1 2 1 . 0 1 0 . 7 7 0 . 90

a Expressed as per cent of amino acid (g/g) in the sample. 84 BACHMAN et al.

Physico-chemical studies

Hydroxyproline determinations

Hydroxyproline is a specific amino acid for collagen and gelatin. It was thus considered appropriate to determine its radioresistance. Table III shows the Pro-OH content of gelatin in relation to the radiation dose. On the basis of these data it can be concluded that the Pro-OH content of gelatin irradiated in the solid state does not undergo any change in the range of doses employed. These data may be confirmed by the results of deter­ minations made with an automatic amino acid analyser presented in Table IV.

£

Cr О v ■ о

4 .

Oose

FIG. 1. Carbonyl group content in irradiated gelatin.

Dose

FIG. 2. Effect of radiation on the viscosity of gelatin sol. IAEA-PL-561/5 85

Analysis of amino acid content of gelatin

Studies on amino acid content were made to extend the information on the alteration of irradiated gelatin. The aim was to find out whether other amino acid residues of gelatin show similar radioresistance as hydroxy­ p r o lin e . It appears from Table IV that the amino acid content of gelatin irradiated with doses of 1. 0 - 3. 5 Mrad does not undergo any marked changes in comparison with the control sample.

Determination of carbonyl groups

Figure 1 shows the effect of irradiation on the content of carbonyl groups in gelatin. There is a distinct increase in the concentration of carbonyl groups as a function of the doses used. A similar dose dependence has been observed in the studies on collagen irradiated in de-aerated solutions [25].

Determination of viscosity changes

The dependence of viscosity on radiation dose is presented in Fig. 2. It follows from the plot that the degree of gelatin degradation stays at a constant level between the doses of 1.0 and 2. 5 Mrad and increases markedly when the dose exceeds the latter value.

Gel strength determination

Figure 3 shows the dependence of gel strength measured in Bloom's degrees on radiation dose. The hardness of the gel prepared from gelatin irradiated in the solid state decreases with radiation dose between 0. 5 and 2. 0 M rad.

FIG.3. Gel strength of irradiated gelatin. 8 6 BACHMAN et al. f TABLE V. EFFECT OF GAMMA 60 Со RADIATION ON CHANGES IN GELATIN POLYDISPERSITY

Dose (Mrad) C a lc u la te d C o n tro l v a lu e s 0 . 0 1 . 0 1 . 5 2 . 5 3 . 5

8 7 . 4 9 5 .2 9 6 .6 9 7 . 0 9 8 . 8 V e

o 2 1 1 6 0 0 1 5 2 3 0 2 2 2 5 0 1 6 4 7 0 2 2 170

°¡0 O Z 1 0 0 .0 1 3 1 .3 1 9 1 . 8 1 4 2 . 0 1 9 1 . 2

FIG.4. Elution curves for control (— ) and the sample irradiated with a dose of 3.5 Mrad (— ). IAEA-PL-561/5 87

FIG.5. Dependence of the sedimentation velocity constant on the radiation dose.

Havelengtà jnm]

FIG. 6. Optical rotatory dispersion curves of control and irradiated gelatin. 88 BACHMAN et al.

Changes in polydispersity

By plotting elution curves in the system of co-ordinates, optical density versus column eluate volume (Ve, D/Ve) [6], the following values were calculated:

Ve - average volume of elution which corresponds to the average size of the gelatin molecule, cr2 - variance, i. e. the square of standard deviation which may be the measure of gelatin polydispersity, cr2% - denotes an increase in polydispersity.

The values calculated for particular doses and for control samples are given in Table V. It appears from Table V that ionizing radiation reduces the average molecular size, which is evidenced by the increasing Ve values with radiation dose. The marked rise in a2 values for particular doses is the measure of increase in polydispersity of solutions of gelatin irradiated in the solid state. The results are illustrated by the two elution curves presented in Fig. 4.for the 3. 5-Mrad dose and for the control sample.

Sedimentation velocity constant estimation

The quantity that is strongly affected by ionizing radiation is the sedi­ mentation velocity constant. The dependence of this constant on radiation dose is shown in Fig. 5. It can be concluded from the graph that an increase in radiation dose is accompanied by a distinct, linear decrease in the values of sedimentation velocity constant. Such a dependence points to the degrada­ tion of gelatin molecules irradiated in the solid state.

Optical rotatory dispersion (ORD) of gelatin

The optical rotatory dispersion of gelatin solutions may be described by Drude's simple law: К “ " X2 - X2 w h ere a is the optical rotation, K, Xc are constants, and X is the wavelength of linearly polarized light.

The results of ORD measurements for gelatin are presented in Fig. 6. The value for the specific rotation is formulated in the system of co-ordinates resulting from a re-arrangement of Drude’s equation [a] X2 • 10'6 = К + X2 [о-] -lO"6 from which the parameters К and Xc were calculated (Fig. 7). The results of calculations in Table VI illustrate the values of optical activity of different gelatins and the changes brought about by irradiation. Optical activity of irradiated gelatin diminishes for nearly all gelatins, without any significant change in the Xc value. Different kinds of gelatin show differences in activity. The drop in optical activity on irradiation shows that the content of collagen structures present in gelatin solution diminishes at the temperature at which the measurements were taken. With other methods it was found that such a degradation leads to a rise in polydispersity and reduces the average molecular weight of gelatin. IAEA-PL-561/5 89

FIG.7. Drude's plots of optical rotatory dispersion data.

TABLE VI. CHANGES IN THE PARAMETERS К AND Xc OF GELATIN AS A RESULT OF IRRADIATION

No. of gelatin n i II I b a tc h

Dose (Mrad ) 0 3 . 5 .0 0 . 5 1 . 5 2 . 5 3 . 5 0 to 3 . 5

К - 5 6 . 8 - 5 0 . 0 - 8 7 - 8 7 - 7 7 - 7 7 - 6 8 -61.5 to 0.5 ? с о 2 0 7 2 0 5 1 97 197 1 97 1 97 - 2 0 1 90 BACHMAN et al.

- H

FIG.8 . ESR spectra for irradiated gelatin.

Electron spin resonance spectroscopy

Ionizing radiation induces the formation of stable free radicals in gelatin which can be investigated by using the electron spin resonance (ESR) method. The ESR spectra we obtained for gelatin (Fig. 8) are of the nature of a symmetrical doublet which is characteristic of proteins that do not contain any significant amounts of sulphur-containing amino acids like cysteine and cystine [ 10, 12, 18]. No other signal typical of sulphur radi­ cals has been detected in the gelatin ESR spectrum which means that sulphur-containing amino acids either do not appear there or that they are present in infinitesimal quantities. This is confirmed by studies on amino acid composition. Independently of the radiation dose absorbed and the gelatin batch studied, .we have obtained ESR spectra of sim ilar hyper- ' fine structure. Particular spectra differed only with respect to the magnitude of the signal. The area beneath the ESR signal integral curve is proportional to the number of radicals in the sample [ 15]. Results of quantitative measurements of the concentration of free radicals in irradiated gelatin for different production batches are plotted against the radiation dose in Fig. 9. Particular batches of gelatin differed among themselves with respect to the content of impurities and the degree of granulation. Radiation yields (Gradicals) for particular kinds of gelatin calculated from the initial slopes of the curves in Fig. 9 are given in Table VII. IAEA-PL-561/5 91

FIG. 9. Content of free radicals as a function of the dose used for different gelatin batches.

TABLE VII. RADIATION YIELD OF FREE RADICALS FOR DIFFERENT BATCHES OF GELATIN

Gelatin batch No. I II III IV

1 . 6 2 1 . 3 0 0 . 55 0 . 31 R a d i c a ls 92 BACHMAN et al.

FIG. 10. Kinetics of free radical decay in the irradiated gelatin.

The kinetics of free-radical decay during storage of gelatin have been studied at room temperature in the presence of air. After exposure has been finished there is a gradual decay of the free radicals. Curves representing this are shown in Fig. 10. The presence of oxygen makes the processes of recombination and free-radical decay easier, as an important role is played by the reaction:

R* + O 2 HO2

The peroxide radical RO2 that'is formed may then decay in the reactions leading to the formation of peroxide bonds of the type R1OOR2 or ROOH. The presence of free radicals in irradiated gelatin samples could be detected as many as forty days after the exposure.

CONCLUSIONS

On the basis of investigations on different production batches of gelatin used in the canning and pharmaceutical industries, it can be stated that the overall extent of its contamination is relatively small and that it is possible to sterilize it with quite low doses of ionizing radiation. In the case of insignificant surface contamination the dose needed for its sterilization is IAEA-PL-561/5 93

0. 5 to 1. 0 Mrad. The sterilizing effect of the doses employed increases with the time of sample storage, as a post-effect of irradiation. For a product put to use after a certain period of storage (more than two weeks) the sterilizing radiation doses can be lowered. Organoleptic changes appearing in gelatin at doses above 0. 5 Mrad (e. g. the production of a broth taste) are not detectable in canned meat and probably improve its taste. The technological process of gelatin production does not ensure uniform chemical composition nor identical molecular weight of the protein. This gives rise to differences in the properties of gelatin of different origin (from different raw material and from different technological processes). The chemical composition of irradiated gelatin does not undergo any changes. Hydroxyproline determinations did not show any changes in the range of doses considerably higher than the sterilizing ones. Amino acid determinations have shown that doses of up to 3. 5 Mrad do not give rise to any far-reaching changes in the amino acid composition. For doses exceeding 0. 5 Mrad distinct changes are observed in the content of carbonyl groups. The physico-chem ical changes occurring in the range of sterilizing doses suggested by us do not diminish its technological value. The drop in the viscosity of irradiated gelatin is evidence of protein molecular degradation which is confirmed by the results of elution on agar gel, Sepharose 4 В and by measurements of the sedimentation velocity constant. Preliminary measurements of the optical rotatory dispersion of gelatin have also shown protein degradation which leads to an increase in gelatin polydispersity and a decrease in its molecular weight. The radiation yield of free radicals in gelatin irradiated in the solid state varied from 0. 3 to 2. 6 depending on the origin and granulation degree of gelatin used. Free-radical decay in gelatin while stored at room tempera­ ture and in the presence of air has been observed. However, the radicals were still detectable by the ESR method 30 days after the exposure had been finished. On the basis of our studies it can be said that, in view of the protein character of gelatin, radiation sterilization has an advantage over conventional methods, producing the desired effect and leaving the physico­ chemical properties intact. After the results arrived at in this study have been supplemented with data from feeding tests they will be used in support of legalizing the process of radiation sterilization.

REFERENCES

[1] ARNEV, J., J. Biol. Chera. 110 (1935) 43. [2] BACHMAN, S ., Badania nad zastosowaniem promieni у 60Co do sterylizacji zelatyny konserwowej, Contract ofiZM , 1968/69. [3] BACHMAN, S ., GIESZCZYNSKA, J ., Wtosnesci fiz-chem zelatyny napromienianej dawkami radaperty-

zacyjnymi promieniowania у 60Co, Mat. II Sesji Kom. Techn. i Chem. Zywn. PAN (1971). [4] BOETKER, H ., DOTY, P., A study of gelatin molecules, aggregates and gels, J. Am. Chem. Soc. 58

(1954) 968.

[5] CHEVE, J .-L ., Contribution à l’étude de la structure des gélatines, C.R. Acad. Sci., Paris 267 (1968) 1566.

[ 6 ] CHEVE, J .-L ., Etude de la polydispersité des gélatines au moyen des tamis moléculaires, J. Chim. Phys. 6 8 (1971) 258. [7] DIRKX, J.P ., Van der HAAK, P. J., SIXMA, F .L ., Evaluation of simple method for optical rotatory

dispersion measurements in the visible and ultraviolet regions. Anal. Chem . 36 (1964) 1988. 94 BACHMAN et al.

[ 8 ] FERNAU, A ., PAULI, W ., The effect of penetrating radium rays on organic and inorganic colloids and biocolloids (in Russian) Colloid. Zhurn, 30 (1922) 6 . [9] FRANK, H ., GRÜNEWALD, T h ., Untersuchungen über die Môglichkeiten einer Strahlensterilisierung

(Radappertisation) von Gelatine, Fleischwirtschaft 49 (1969) 74.

[10] GORDY, W ., SHIELDS, H ., Proc. Natl. Acad. Sci. U.S. 46 (1960) 1124. [11] HAMILTON, L .D .G ., The estimation of side chain groups in the protein, cited from P. Alexander's

Laboratory Manual of Analytical Methods of Protein Chemistry, Pergamon Press, N. Y . (1960) 96. [12] HENRIKSEN, T ., Radiat. Res. 18 (1963) 147.

[13] HURYCH, J., CHVAPIL, М ., Cited from Reich, G ., Kolagen (1970) 57. [ 14] KHENOKH, M. A ., The effect of penetrating radium rays on the colloid-chem ical properties of gelatin

sols, Zh. Obshch. Khim. Cl 10 (1941) 776.

[15] KROH, J., Wolne rodniki w chemii radiacyjnej, PWN, W-wa (1967).

[16] MATELES, R .I., GOLDBL1TH, S. A ., Some effects of ionizing radiations on gelatin, Food Technol. 12 (1958) 633.

[17] MOORE, S., STEIN, W .H ., Methods in Enzymology 6^(1963) 819. [18] PATTEN, F., GORDY, W ., Radiat. Res. 14 (1961) 573.

[19] PRUSAK, L. P ., SCIARRONE, B.J., Effect of ionizing radiation on two gelatin fractions. III. Carbonyl

group analysis and electron spin resonance studies, J. Pharm. Sci. 55 (1966) 407.

[20] PUTILOVA, K .M ., Zh. Obshch. Khim. 0 10(1941) 176.

[21] REICH, G ., KOLAGEN, WNT, W-wa (1970) 94 (translated from German).

[22] RYBICKA, J., ALBRYCHT, H ., WYSOKINSKA, T ., Med. Dosw. Mikrobiol. 19 (1967) 93. [23] SHEPPARD, S.E., Soc. Pract. Phot. 12 (1925)332.

[24] ZHUKOV, J.J., UNKOVSKAJA, V .A ., J. Russ. Phys. Chem. Soc. 62 (1930) 581.

[25] JELENSKA, M. M ., DANCEWICZ, A .M ., Thermal aggregation of tropocollagen solutions irradiated with

low doses of ionizing radiation. Int. J. Radiat. Biol. 16 (1969) 193. IAE А-PL -561/6

THE ANTIOXIDATIVE EFFECTS OF GAMMA-IRRADIATED AMINO-SUGAR REACTION PRODUCTS

M. FUJIMAKI, M. MO RITA, H. KATO The University of Tokyo, Tokyo, Japan

Abstract

THE ANTIOXIDATIVE EFFECTS OF GAMMA-IRRADIATED AMINO-SUGAR REACTION PRODUCTS.

Experiments were carried out on the products from the gamma-irradiated amino acid-sugar reaction.

It is shown that the antioxidative activity of melanoidin is not much affected by the irradiation. The develop­ ment of the antioxidative activity of the amino acid-sugar solution on heating was markedly accelerated when the solution was pre-irradiated. Pre-irradiation of just the sugar solution accelerated the development of the antioxidative activity, as was not the case when the amino acid solution alone was pre-irradiated.

With one exception, all combinations of amino acids with sugars gave almost the same antioxidative results.

INTRODUCTION

Antioxidative activity has been observed in the products of amino- carbonyl reaction. We have revealed that there exists a strong anti­ oxidative activity in non-dialysable melanoidin. However, when melanoidin is chemically degraded, the low molecular and less-coloured fraction also shows the activity. On the other hand, it has been known that browning of a solution of amino acid and sugar is strongly accelerated by gamma irradiation. M oreover, we previously found that the one-less carbon aldehyde of the amino acid was much generated by gamma irradiation, without heating, from the amino acid-sugar solution, e.g. 3-methylbutanal from leucine-glucose solution. Experiments were carried out to examine the antioxidative activity of the products frond the gamma irradiated amino acid-sugar reaction.

EXPERIMENTAL

Melanoidin solution

A solution (pH 6.7) containing D-xylose (2M), glycine (2M) and NaHCOs (0.05M) was heated in boiling water for 10 h. The resultant brown solution was dialysed against distilled water for 3 days. The inner solution was concentrated and dried under reduced pressure. This melanoidin sample was dissolved in a buffer solution at pH 3.0, 7.0 or 11.0 and irradiated with gamma rays in the presence or absence of oxygen.,

Browning reaction solution

An aqueous solution of amino acid (0.1M) and sugar (0.3M) at pH 5.5- 6 was irradiated with 60Co-rays. After irradiation, the amino acid-sugar solution was heated at 100°C.

95 96 FUJIM AKI e t al.

Measurement of antioxidative activity

Methyl linoleate (1 ml), an aqueous solution to be tested (1 ml), and N acetate buffer (0.2 ml, pH 5.5) were shaken in an open test-tube at 30°C for 40-44 h. After the aqueous phase was removed, the peroxide value (POV) of the oil phase was measured by the usual iodide-acetic acid method.

RESULTS

First, the effect of gamma irradiation on the melanoidin solution was examined. As shown in Table I, gamma irradiation on the degassed melanoidin solution little influenced the antioxidative activity at the three different pH values, 3.0, 7.0 and 11.0. In the presence of oxygen, gamma irradiation somewhat destroyed the antioxidative activity. Next, the effect of gamma irradiation on the sugar-amino acid solution followed by heating was investigated. As shown in Table II, pre-irradiation with gamma rays markedly promoted both the browning and the development of antioxidative activity of the leucine-glucose mixture during heating at 100°C. However, in comparison with the control mixture, which was not irradiated, the effect of gamma irradiation on the development of anti­ oxidative activity was much larger than that on the browning.

TABLE I. EFFECT OF GAMMA IRRADIATION ON ANTIOXIDATIVE ACTIVITY OF MELANOIDIN (Gly-Glc)

Thê peroxide value, POV

Control (buffer) 335

Not irradiated 74

D ose D e -a e r a te d W i th 0 2 (M r a d )

Irradiated at pH 3 0 . 5 55 1 0 7

2 4 7 1 3 9

5 58 1 8 2

Irradiated at pH 1 0 . 5 7 9 1 5 3

2 56 1 7 2

5 7 4 1 8 4

Irradiated at pH 11 0 . 5 7 1 2 1 3

2 98 120

5 68 1 0 9

Irradiation was carried out on a 1 °Jo melanoidin solution. Antioxidative activity was measured in a 0 . 5 °]o melanoidin solution containing 0 .1M acetate at pH 5. 0. IAEA-PL-561/6 97

TABLE II. ANTIOXIDATIVE ACTIVITY OF LEUCINE-GLUCOSE SOLUTIONS AFTER GAMMA IRRADIATION AND HEATING

Time of heating at 100°C (h) D ose (M r a d ) 0 1 . 5 4 . 5 1 1 . 5 1 9 . 0

0 POV 3 9 6 4 0 3 4 5 8 2 2 8 1 3 9

0 0 . 0 0 1 0 .0 1 2 0 . 120 0 . 3 7 6 O d 4 7 0 n m

0 . 83 POV 4 3 0 4 4 4 3 4 1 9 5 6 0

OD 4 7 0 nm 0 . 0 03 0 . 0 1 6 0 . 0 4 6 0 . 120 0 . 3 2 4

1 . 9 2 POV 4 2 3 2 2 2 82 4 3 3 9

0 . 0 0 5 0 . 0 4 4 0 .0 8 8 0 . 2 0 8 0 . 4 3 6 n m

5 POV 4 6 1 9 5 4 4 2 9 3 1

0 . 0 1 0 0 . 1 1 7 0 .2 0 2 0 . 4 8 4 0 . 8 4 0 O D 4 7 0 n m

Antioxidative activity was measured in solutions diluted four-fold.

TABLE III. EFFECT OF PRE-IRRADIATION ON THE DEVELOPMENT OF ANTIOXIDATIVE ACTIVITY IN A LEUCINE + GLUCOSE SYSTEM

Time of heating at 100°C (h)

0 1 . 5 4 . 5

Sample without irradiation POV 3 9 6 4 0 3 4 5 8

. o d 4 7 0 n m 0 0 .0 0 1 0 012

0 . 0 1 0 0 .0 2 2 0 . 1 3 5 O D 3 4 0 n m

Irradiation of glucose + leucine POV 4 2 3 2 2 2 82

0 . 0 0 5 0 . 0 4 4 0 . 0 8 8 O D 4 7 0 n m

0 .0 8 3 0 . 3 9 9 0 . 6 5 0 O D 3 4 0 n m

Irradiation of glucose with POV 4 4 4 2 3 2 1 0 6

leucine added later 0 . 0 6 4 0 . 1 4 3 OD 4 7 0 n m 0

0 . 0 5 2 0 . 5 1 5 0 . 9 6 5 O D 3 4 0 n m

Irradiation of leucine with POV 3 5 4 3 9 2 2 8 6

glucose added later O D 4 7 0 n m 0 0 . 0 0 7 0 . 0 3 1

. 0 . 0 8 4 0 . 3 2 4 O D 3 4 0 n m 0 0 1 0

Dose: 1.92 Mrad. 98 FUJIMAKI et al.

TABLE IV. ANTI OXIDATIVE ACTIVITY OF VARIOUS COMBINATIONS OF AMINO ACIDS AND SUGARS AFTER GAMMA IRRADIATION AND HEATING

Dose (Mrad)

A m in o a c id

sugar 5 0

contribution POV POV O D 3 4 0 n m o d 3 4 0 n m

G l y - G l c 98 2 . 6 2 6 5 10 0 . 1 4 8

A l a - G l c 1 0 4 / 1 . 7 7 2 4 8 6 0 . 0 6 1

P h e -G lc 70 2 . 1 0 8 4 8 9 0 . 2 6 0

T r p -G lc 4 4 3 . 3 6 0 2 7 4 1 . 2 4 5

S e r -G lc 9 0 2 . 3 0 6 5 1 1 0 . 0 6 8

L y s -G lc 68 3 . 1 7 2 5 1 6 0 . 4 1 7

A r g -G l c 6 4 3 . 7 7 6 4 5 6 0 . 8 8 5

H i s -G lc 7 4 7 . 2 7 2 4 1 3 1 . 8 5 0

G l u -G l c 78 2 . 4 9 0 5 02 0 . 0 8 2

A s p -G l c 70 2 . 3 6 8 4 2 1 0 . 0 4 6

C y S H -G l c 5 7 5 0 . 4 7 7 1 0 8 0 0 . 0 36

M e t - G l c 1 4 8 2 . 2 7 2 4 9 8 0 . 1 3 2

L eu -F ru 6 4 2 . 7 9 8 4 8 5 0 . 3 4 9

L e u -X y l 9 5 2 . 6 9 6 3 9 4 1 . 0 9 6

Heating: 100°C, 4. 5 h.

Antioxidative activity was measured in solutions diluted four-fold.

A relationship was found between the antioxidative activity (which is indicated by a lower peroxide value) and the optical density. Sample solutions were diluted four-fold for the measurement of antioxidative activity (Table II). When the glucose solution was irradiated alone, leucine added later and the mixture then heated, the development of its antioxidative activity was almost the same as that of the sample which contained both glucose and leucine when irradiated, as shown in Table III. Conversely, pre-irradiation of the solution containing leucine alone had a much sm aller effect on the development of antioxidative activity. Table IV lists the pre-irradiation effects of various combinations of amino acids and sugars on the development of antioxidative activity. Except , for the combination of cysteine and glucose, all combinations tested showed strong effects on the development of antioxidative activity sim ilar to that of the glucose-leucine combination. IAEA-PL-561/6 99

CONCLUSIONS

1. The antioxidative activity of melanoidin was not much affected by gamma irradiation. 2. Development of the antioxidative activity of the amino acid-sugar solution on heating was markedly accelerated when the solution was pre­ irradiated with gamma rays, and the development of the antioxidative activity was much more marked than that of the browning of the colour. 3. Previous separate irradiation of the sugar solution accelerated the development of antioxidative activity on later heating with the amino acid, but heating of pre-irradiated amino acid solution with the sugar did not increase the antioxidative activity. 4. Except for the cysteine-sugar combination, all combinations of amino acids with sugars gave almost the same antioxidative results.

IAEA-PL-561/T

ASPECTS OF THE EFFECT OF IONIZING RADIATION ON ENZYMES

T. SANNER, Gizella KOVACS-PROSZT* S. WITKOWSKI** Norsk Hydro’s Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway

Abstract

ASPECTS OF THE EFFECT OF IONIZING RADIATION ON ENZYMES.

The yield of inactivation of enzymes irradiated in the dry state is nearly the same for most enzymes

(G = 1-3), In contrast, the yield of inactivation in dilute aqueous solution varies by a factor of more than 100 and is, in general, considerably lower than the yield in the dry state. The relative role of the direct and indirect action when food or food constituents are irradiated depends on the micro-environment of the enzyme molecules. The extent of the indirect action is greatly reduced when the radiation is carried out in the frozen state, since then only water radicals formed in the bound water of the protein will participate. In many cases considerable reduction in radiation damage can thus be achieved by irradiation at low temperatures.

This may be of some practical importance as the radiosensitivity of bacterial spores is only slightly affected by the irradiation temperature. When enzymes are exposed to ionizing radiation, the activity will continue to decrease after the end of the irradiation. This after-effect can be of considerable magnitude for certain enzymes. Thus, for Mucor pusillus protease it has been found that after doses which reduced the activity to 50 °Jo m o re than half of the remaining activity disappeared in the after-effect. If irradiation is used for sterilization of enzyme preparations, the after-effect will add to the inactivation occurring during irradiation. Also, a reduction in the heat-sensitivity of enzymes is observed after irradiation.

The kinetic properties of an enzyme are in many cases altered after exposure of the enzyme prepara­ tion. Thus, the Km for substrate may be affected, the requirements for co-factors may be increased or reduced, and the specificity of an enzyme may be altered. The possibility should be considered óf using ionizing radiation for specific modification of enzyme properties.

Specific protection of an enzyme can frequently be achieved by irradiation of the enzyme in the presence of ligand molecules. This specific protection is probably caused by sm all conformational changes in the protein. The possibility is considered of using ionizing radiation combined with specific protection to reduce undesired enzymatic activities of enzyme preparations used in the food industry.

INTRODUCTION

During recent years it has been found that ionizing radiation can, in some cases, be used to improve the quality of raw materials and food products [1]. Some of the improvements resulting from radiation treatment, such as increased yield of juice by radiation of fruit prior to pressing [2] and delayed ripening of fruit by small doses of irradia­ tion [3], may in part be caused by an influence on enzymatic processes occurring after the end of the exposure. Moreover, on the basis of radiation studies on pure enzymes it seems likely that ionizing radiation may also be used to improve the quality of enzyme preparations used in the food industry. The present paper deals with some radiation effects on enzymes which may be of interest in food technology.

* Present address: Central Food Research Institute, Budapest, Hungary.

Present address: Institute of Applied Radiation Chemistry, Technical University of Lódé, Poland.

1 0 1 1 0 2 SANNER et al.

The possibility of using combined treatment of radiation and heat in order to increase storage time of different types of food is at present studied in a number of laboratories. Recently, it has been found that radiation decreases the heat stability of the enzymes [4,5]. Moreover, the activity of irradiated enzymes continues to decrease for some time after the end of the exposure [4-8]. These effects are also of interest if radiation is used for sterilization of enzyme preparations as the inactiva­ tion occurring after the end of the exposure will add to that occurring during irradiation. A second type of combined treatment which may be of practical importance is to reduce the temperature to below the freezing point during irradiation. Such treatment decreases significantly the amount of enzyme inactivation caused by the indirect action [9 - 12]. On the other hand, this treatment seems to have little effect on the radiation sensitivity of bacterial spores [ 13]. In studies of pure enzymes it has been found that the catalytic proper­ ties of an enzyme may be altered after irradiation. For example, the requirement of pectin methyl esterase for calcium ions is considerably reduced after irradiation [4], and the substrate inhibition of glutamate' dehydrogenase is abolished by irradiation [14]. In some cases alteration of the kinetic properties of an enzyme will also affect its specificity. The possibility should be considered that ionizing radiation may be used in order to alter the properties of an enzyme to make it more suitable for use in the food industry. ■ In the case of allosteric enzymes, irradiation of the enzymes in the presence of ligand molecules may protect their functions to different extents [15-17]. It seems that this information can be utilized for enzymes used in the food industry. Thus, the crude enzyme preparation may contain undesirable enzymatic activities. By irradiation of the preparation in the presence of certain ligands, it may be possible to protect preferentially the enzyme of interest, while reducing strongly the undesired activities. In order to understand the mechanism of enzyme inactivation and to utilize this, it is necessary to understand the difference between inactiva­ tion by the direct and indirect action.

YIELD OF ENZYME INACTIVATION

Enzymes irradiated in the dry state under anoxic conditions at room temperature are inactivated with G-values of 1 - 3 (number of enzyme molecules inactivated per 100 eV absorbed) [18]. The loss of activity is mainly due to energy absorption in the enzyme molecules proper, although interaction with radicals or radical fragments formed from neigh­ bouring molecules is of importance [19-21]. The presence of protective or sensitizing substances will in most cases affect the radiosensitivity by a factor of less than 2. The fact that the G-values are nearly the same for most enzymes implies that the D31 dose decreases with increasing molecular weight of the enzyme [18]. The G-values of inactivation of enzymes by the indirect action are considerably smaller than by the direct action and, moreover, the yield IAEA-PL-561/7 103 \ TABLE I. RADIATION SENSITIVITY OF ENZYMES IRRADIATED IN DILUTE SOLUTION IN THE ~PRESE1S¡CE OF A IR

E n zy m e s G -v a lu e R ef.

Papain 1 . 2 8

D N a s e I 0 . 7 22

Chymotrypsin 0 . 5 23

RNase 0 . 5 2 4

Heart lactic dehydrogenase 0 . 5 25

Isocitrate dehydrogenase 0 . 5 2 6

D N a se 11 0 . 4 27

Pectyl methyl esterase 0 . 4 4

Mucor pusillus protease 0 . 4 5

Rennin a 0 . 4

E n olase 0 . 3 2 8

Glutamate dehydrogenase 0 . 3 1 4

DNA-dependent RNA polymerase 0 . 2 2 9 ■

T rypsin 0 . 1 4 3 0

A ld o la s e 0 . 1 4 3 1

Phosphorylase b 0 . 1 9 ' 3 2

A m y la s e 0 . 0 8 33

Glyceraldehyde 3-phosphate

dehydrogenase 0 . 07 3 4

Phosphofructokinase 0 . 06 3 5

Alcohol dehydrogenase 0 . 0 6 3 4

Aspartokinase-homoserine

dehydrogenase (kinase act.) 0 . 0 2 17

Fructose-1, 6 -diphosphatase 0 . 02 16

L y s o z y m e 0 . 0 2 3 6

U rea se 0 . 0 1 5 3 7

Aspartate transcarbamylase 0 . 010 3 8

Aspartokinase-homoserine dehydrogenase (dehydrogenase act. ) 0 . 0 0 6 ' 17

a Unpublished results. 104 SANNER et al.

varies by a factor of more than 100 (Table I). The inactivation of enzymes in dilute solution in the presence of air can generally be accounted for by interaction of OH radicals, formed by energy absorption in the water, with the enzyme molecules [4, 5, 39 -42]. If OH radical scavengers are added, considerable protection of the enzyme can be obtained. Thus, there is no difficulty in obtaining dose reduction factors in solution of the order of 100 or more with most substances just by finding the appropriate conditions. Furthermore, small alterations of the conformation of an enzyme which changes the reactivities of the surface groups will generally influence the radiosensitivity of the enzyme [15 - 17, 32]. There is no correlation between the molecular weight or number of subunits of an enzyme and its radiosensitivity. Nor is there any relation­ ship between the type of chemical groups, the destruction of which is responsible for inactivation and the radiosensitivity. In the case of sulfhydryl enzymes the sensitivity depends on the number of sulfhydryl groups which have to be destroyed and their reactivity [43, 44]. This implies that papain, which contains one essential and very reactive sulfhydryl group, is inactivated with a high yield [8] whereas glyceral- dehyde 3-phosphate dehydrogenase, in which 3-4 SH-groups have to be destroyed before the activity is lost and where the sulfhydryl groups possess a low reactivity, is inactivated with a low yield [34]. It is often claimed that although the G-value of inactivation in the dry state is higher than in solution, enzymes are considerably more sensitive to irradiation in solution. In order to clarify this point, it may be appropriate to illustrate the inactivation in the dry state and in solution by an example. Suppose that we have an enzyme with molecular weight of 25 000 and that the enzyme is inactivated with G = 0.2 in solution and G = 2.0 in the dry state. If we irradiate 1 ml of the enzyme in a concentration of 2X 10"5 M, the D37 dose will be 60 krad. This dose will inactivate 7.5 X 1015 molecules. If we expose 1 g of the enzyme to the same dose in the dry state, we will destroy 10 times as many enzyme molecules. However, as 1 g of the dry enzyme contains 2.4 X 1019 molecules, the inactivated enzyme molecules will only represent about 0.3%, which will be difficult to detect. Let us on the other hand suppose another experiment. We will assume that we make a molecular mixture of 1 g of inert protein and the same amount of enzymes which we had in solution (0.5 mg). If this dry mixture is irradiated, we will still lose 0.3% of the activity, but this will represent only 0.04 X 1015 enzyme molecules, which is much less than was found in solution. If we double the amount of active enzymes in the dry mixture the number of enzyme molecules inactivated will also be doubled, i.e. the percentage inactivation is in all cases the same. This, together with the fact that the G-value of inactivation is nearly the same for all enzymes, is the basis of the use of ionizing radiation for determination of molecular weight of enzymes in cells or crude extracts. On the other hand, if we double the enzyme concentration in the solution, we will observe that the same number of enzyme molecules is inactivated after 60 krad, as in the case of the dilute solution. The reason for this is that the water radicals formed are only able to inactivate a certain number of enzyme molecules. IAEA-PL-561/7 105

FIG. 1. Qualitative ESR spectra (A) and yield of ESR centres (B) observed after irradiation of frozen

Sephadex suspensions in vacuum at 77°K with X-rays. The spectra (A) represent the first derivative of the actual absorption curves and were recorded at 77”K immediately after irradiation. The quantitative yield of ESR centres (B) has been determined in Sephadex suspension with different extents of cross-linking.

The dashed line represents the weighted average of the yield for the pure components. Data taken from Ref. [1 2 ].

ROLE OF INDIRECT ACTION IN THE FROZEN STATE

When foods are exposed to ionizing radiation both the direct and the indirect mechanism of action contribute to the radiation effects observed. In certain cases it may be desirable to alter the relative extent of the direct and indirect action. An easy way to reduce the indirect effect which does not require addition of any additives, is to irradiate at temperatures below the freezing point. The effects of ionizing radiation on aqueous systems are considerably less upon irradiation in the frozen state than at temperatures above 0°C. It has consequently been assumed that the contribution of the indirect effect is of little or no importance in the frozen state. However, several lines of evidence are available showing that an indirect effect also operates in the frozen state although it is under most conditions considerably less than at temperatures above the freezing point [9 - 12, 45, 46]. When ice is irradiated at the temperature of liquid nitrogen, water radicals are stabilized with G = 1 [47]. If a solute is added, the ESR spectrum observed after irradiation at low temperature represents a composite spectrum due to water radicals and radicals stabilized in the solute (Fig.lA). The G-value of radicals stabilized in a frozen solution (Fig.IB) is significantly higher than the weighted average of the yield in 106 SANNER et al.

pure water and in the solute, which represents the expected'yield if no interaction has occurred. By decomposition of the spectra, evidence has been obtained that the "excess radicals" are stabilized on the solute m o le c u le s . When the frozen solutions are heated the water radicals disappear at about 150°K in combination reactions. They interact only to a very small extent with the solute [10, 12]. The radicals present above 150°K represent only radicals stabilized on the solute molecules. Results similar to those shown in Fig.l have been obtained with a number of different compounds [10, 12, 47]. The simplest explanation of the results is that only water radicals formed in the bound water of the solute are able to interact with the solute molecules [10].' It can be calculated that in the case of Thiogel, the yield of water radicals is approximately the same as in the liquid solution. However, only 0.3 g of water participate per gram of Thiogel in the indirect effect [10]. The fact that only bound water participates in the indirect action implies that protection by a radical scavenger mechanism is of little importance in the frozen state. Several lines of evidence indicate, however, that protection by hydrogen-transfer mechanisms are of considerable importance in the frozen state [48 - 50]. In experiments where the frozen solutions have been irradiated at higher temperatures, it has been found that the extent of the indirect action increases with increasing irradiation temperature. Thus, the extent of the indirect action appears to be approximately 50% greater at

id cc

>■ H- > *— ¡Л z ÜJ

IRRAD. TEMP. (°K) IRRAD. TEMP. (°K)

FIG. 2. (A) Effect of irradiation temperature on the observed yield of ESR centres in a 10<7o Thiogel solution and in dry Thiogel. The samples were pretreated at 250°K before the measurements were taken. The number of ESR centres present after irradiation at 77°K were set equal to 1. Data taken from Ref. [1 0 ].

(B) Effect of irradiation temperature on the sensitivity of E. coli B. The relative sensitivity is determined

from the slope of the dose-effect curves. The sensitivity at 0°C was set equal to 1. Data taken from Ref. [4 8 ]. IAEA-PL-561/7 107

200°K than at 77°K (Fig.2A). Oksmo and Brustad [11] have obtained results with trypsin which support the above conclusion. They found that the D37 dose in the frozen solution at 200°K was about half of that in the dry state, indicating that an indirect action did occur. When the frozen solution was irradiated at -10°C, the sensitivity was increased and the D37 dose of only 1/3 to 1/4 of that observed in the dry state. The effect of the irradiation temperature on the survival of Escherichia coli is shown in Fig.2B. It is apparent that in the bacteria, as in most cells and tissues, the radiation effects decrease when the temperature is lowered below the freezing point [48, 51-54]. On the other hand, the sensitivity of spores is not affected to any large extent by the irradiation temperature [13]. Since the sensitivity of bacterial cells is greater than the sensitivity of bacterial spores, a reduction of the sensitivity of a bacterial cell by a factor of 2 is of relatively little importance. It is suggested that attempts may be made to irradiate food at low temperature in cases where destruction of bacterial spores are of major importance. From the present discussion it appears that although the indirect action is not abolished when the temperature is lowered below the freezing point, a considerable reduction takes place. The reduction in the indirect action is greater, the more dilute the solution is. If enzyme extracts for use in-the food industry are irradiated to sterilize the preparation, irradiation at low temperature will decrease the radiosensitivity of the enzymes to a much larger extent than the radiosensitivity of the contaminating m icro­ organisms .

ROLE OF AFTER-EFFECT AND INCREASED HEAT SENSITIVITY

For a number of enzymes it has been found that the heat sensitivity increases after irradiation [4, 5] and that the activity continues to decrease after the end of the exposure [4-8]. The latter effect is usually referred to as the after-effect. If ionizing radiation is used for sterilization of enzyme preparations, the loss of activity caused by the after-effect will add to that occurring during irradiation. Furthermore, in combined treatment of radiation and heat, the increased heat-sensitivity of enzymes may be of importance. The after-effect and increased heat sensitivity after irradiation are illustrated for Mucor pusillus protease in Fig.ЗА. This enzyme represents one of the most promising substitutes for rennin from calf stomach in cheese production [55, 56]. When the native enzyme was heated in solu­ tion at 52.5°C a decrease in activity took place. This decrease represents the normal heat dénaturation of the enzyme. When the activity is plotted against time in a semilogarithmic scale, a straight line is obtained. The heating time which reduces the activity to 37% of the initial activity is called the dénaturation time. When the enzyme is irradiated before heat-treatment, biphasic curves are obtained for the activity as a function of heating time. The biphasic curves can be decomposed into two first-order reactions. The initial rapid inactivation is due to the after-effect. The rate of the after-effect as well as the extent of the after-effect can be obtained from the curve. Under the conditions used here the after-effect is finished after 108 SANNER et al.

INACTIVATED ENZYME (%;

FIG. 3. After-effect and reduced heat-stability after irradiation of Mucor pusillus protease, (A) Effect of heating-time at 52. 5°C of the activity. The enzyme was irradiated at 0°C with doses giving 62 °jo and 3 2 % remaining activity before heat-treatment. (B) AH5’iiand A S* for heat dénaturation and after-effect as a function of the extent of radiation inactivation. The results are calculated on the basis of experiments similar to those in (A) carried out at different temperatures. Data taken from Ref. [5 ].

10-20 minutes. The after-effect can also be observed when the enzyme is kept at room temperature. In that case, however, several hours are needed before the after-effect is finished. The final slope of the curve represents the heat-denaturation. The fact that the slope of the final curve is steeper after irradiation shows that the heat-sensitivity increases upon irradiation. In subsequent experiments the rate of the after-effect as well as the dénaturation time was determined at different temperatures and for different extents of inactivation. The rate constants obtained were plotted in a logarithmic scale against the reciprocal of the absolute temperatures. From the Arrhenius plot, the enthalpy of activation as well as entropy of activation for heat dénaturation and after-effect were derived. It is apparent (Fig.3B) that AH* and AS* associated with heat dénaturation decrease with increasing extent of radiation inactivation of the enzyme. The AH* and AS* associated with the after-effect are considerably smaller than those for the heat dénaturation. Interestingly, the changes in the thermodynamic parameters for the after-effect are dependent on the extent of enzyme inactivation. The low value of AH* is expected as the after-effect also occurs at quite low temperatures. It is of interest to compare the extent of the after-effect and decrease in heat-sensitivity for different enzymes. In the comparison, pectin methyl esterase from tomatoes and rennin from calf stomach were chosen as these two enzymes have approximately the same molecular weight as the protease lAEA-PL-561/7 109

INACTIVATED ENZYME (%) INACTIVATED ENZYME (*/•)

FIG. 4. Effect of radiation on the extent of the after-effect and on the heat sensitivity of Mucor pusillus protease [5 ], pectin methyl esterase [4] and rennin (unpublished). The enzymes were irradiated to different extents of inactivation and subsequently heat-treated. The sensitive fraction represents the extent of the after-effect calculated as the percentage of the activity remaining immediately after irradiation which is lost during the after-effect. The dénaturation time was determined as the time needed to reduce the activity to 37 °Jo of the initial activity, calculated from the final linear part of the heat-inactivation curve (see Fig. ЗА). The dénaturation time for the unirradiated enzyme is in all cases set equal to 100.

from Mucor pusillus, and the three enzymes are inactivated at nearly the same G-values, upon irradiation in solution. However, the three enzymes differ greatly in their heat sensitivity. The results (Fig.4A) demonstrate that the extent of the after-effect, the sensitivity fraction (the percentage of the remaining enzyme activity immediately after irradiation which is lost during the after-effect), increases nearly proportionally with the extent of enzyme inactivation. The sensitive fraction is larger for the protease from Mucor pusillus than for the two other enzymes. In the case of Mucor pusillus protease, approximately half of the remaining activity is destroyed during the after-effect when the enzyme is irradiated to 60% remaining activity. The dénaturation time decreased nearly linearly with the extent of enzyme inactivation for all three enzymes studied (Fig.4B). The decrease was significantly greater for pectin methyl esterase than for the two other enzymes. For pectin methyl esterase the dénaturation time was reduced to approximately 50% of that for the unirradiated enzyme after destroying half of the enzyme activity during irradiation. It has been found that cysteamine, when added after irradiation, is able to protect enzymes against both the after-effect and the increased heat sensitivity. However, the concentrations of cysteamine needed to offer protection is rather high (of the order 10 mM) ■ Compounds such 1 1 0 SANNER et al. as ethanol, glycyl-glycine and cystamine as well as catalase have no effect on the after-effect nor on the heat sensitivity [4, 5]. It should be noted, however, that in the case of sulfhydryl enzymes with reactive sulfhydryl groups, hydrogen peroxide may participate in the after-effect ■ as is found for papain [8], and in such instances addition of catalase after irradiation will strongly reduce the after-effect.

MODIFICATION OF ENZYME PROPERTIES

The data in Table I, showing that the kinase activity of aspartokinase- homoserine dehydrogenase is inactivated with a yield of about 3 times that of the dehydrogenase activity, clearly demonstrate that different functions of an enzyme may be destroyed at different rates upon irradia­ tion. For a number of allosteric enzymes we have found, as discussed later, that their catalytic and allosteric functions are destroyed at widely different rates. The possibility should be considered that ionizing radia­ tion may be used to alter the properties of an enzyme in such a way that it becomes more suitable for industrial use. We will here discuss radia­ tion effects on the kinetic properties of enzymes, and on their specificity. The effect of radiation on the kinetic parameters of enzymes has been studied in considerable detail. In the simplest' case an enzyme reaction can be described by the equation

ki k3 E + S ES ------» E + P кг

V = k 3E Km = (k2 +k3)/k1

If irradiation results in partially damaged enzyme molecules the different rate constants involved in Km may be influenced to different extents, resulting in a change in Km. The results in Fig.5 show the effects of radiation on the СаС1г dependence of pectin methyl esterase. The fact that different dose inactivation curves are obtained when the enzyme is assayed with different concentrations of СаС1г (Fig.5A) is due to the fact that Kcaci, decreases upon irradiation (Fig.5B). Thus, the enzyme molecules present after irradiation have properties different from those of the native enzyme. If for some reason we want to utilize the enzyme under conditions with very small concentrations of Ca++ the activity could be increased by irradiating the enzyme. The effects of irradiation on the kinetic properties of different enzymes are summarized in Table II. In the case of papain no effect on Km was found. This is due to the fact that the inactivation of this enzyme can be completely accounted for by destruction of its essential sulfhydryl group and no partially inactivated enzyme molecule was found [8]. In the case of DNase I, phosphorylase b and chymotrypsin the reduction in V is followed by a change in Km. Depending on the effect of irradiation on the individual rate constants, K m may increase or decrease. It is apparent from Table II that in cases where two parameters have been measured one parameter may change, while the other is unaffected. In the case of isocitrate dehydrogenase, Km for the substrate increased while Km for IAEA-PL-561/7 1 1 1

i------1------i------r q # Unirradiated enzyme D О Irradiated 000 kR) h _ _ •

. / * v Q.

I 0A

? 0.2

_1_ 50 100 INACTIVATED ENZYME (Vo) ____ I______.______I 25 50 DOSE (kR) CaCI, CONC. (mM)

FIG. 5. Effect of radiation on the requirement of pectin methyl esterase for CaCl2. (A) Dose response curves obtained with different concentrations of CaCl2 in the assay mixture. (B) Effect of CaCl2 concentration on the activity of native enzyme and enzyme irradiated with 100 kR. The inserted figure shows the reduction in К c a Q 2 as a function of the extent of enzyme inactivation. Data taken from Ref. [4 ].

TABLE II. EFFECT OF IRRADIATION ON THE KINETIC PROPERTIES OF ENZYMES

Change in per cent

E n z y m e Ref. K,m

D N a s e 1 - 2 5 - 13 57

P ap ain - 2 5 0

Phosphorylase b - 3 0 + 2 5 32

Chymotrypsin - 21 + 2 6 5 8

Pectin methyl esterase, pectin 0 - 5 0 C a C l 2 - 47

Isocitrate dehydrogenase, isocitrate + 6 5 - 50 26 NADP - 3

Aspatokinase-homoserinedehydrogenase

(dehydrogenase activity)

C a t a ly t i c - 4 0 - 7 0 17 A llo s te r i c - 6 0 0 1 1 2 SANNER et al. the cofactor NADP is nearly unaffected. This is probably due to the fact that the sulfhydryl groups are necessary for binding of the cofactor and when the sulfhydryl groups are destroyed the enzyme loses its ability to bind the cofactor, and thus its activity. In cases where Km is affected to different extents for different substrates upon irradiation this may result in an apparent change in the specificity. Such changes in specificity may be of considerable importance for proteolytic enzymes, as the ability to split different peptides may be altered and consequently the reaction products. In agreement with this, Lynn [59] compared the proteolytic effect of native and irradiated chymotrypsin on the release of peptides from proteins and found that its specificity changed upon irradiation. We have recently compared the macropeptides released on incubation of protease from Mucor pusillus with acid casein and к-casein. In this case the results indicate that the specificity of the enzyme was unaffected by irradiation [5]. The effect of radiation on the specificity of enzymes is, however, a subject which has only been studied to a limited extent and which needs further exploration.

SPECIFIC PROTECTION OF CERTAIN ENZYMATIC PROPERTIES

Most of the work on specific protection of certain properties of an enzyme has been done with allosteric enzymes. As the catalytic activity for these enzymes is controlled by the level of certain effector molecules, the radiation effects on the loss of the control mechanism and the loss of the catalytic activity can easily be compared. The possibility should be considered of utilizing the results to improve enzymes for the food industry. Thus, the crude extracts which are usually used in the food industry contain a number of different enzymatic activities. In cases where undesired activity is present, this may be removed by irradiating the enzyme in the presence of substances which afford a preferential protection of the enzymes of interest. The procedure used for studies of preferential protection of allosteric enzymes is illustrated for aspartokinase homoserine dehydrogenase in Fig.6. The activity of this enzyme is controlled by threonine which acts as an allosteric inhibitor molecule [60]. If we measure the enzyme activity in the absence of threonine a normal linear dose response curve is obtained when the remaining activity is plotted against the dose in a sem i-logarithmic plot (Fig.6A). On the other hand, if the activity is assayed in the presence of threonine, the activity increases after small doses and then decreases again. This increase in activity is due to the fact that the enzyme loses its ability to respond to the threonine inhibition. On the basis of the reduction in threonine inhibition the loss of the allosteric function of the enzyme has been determined. It is apparent that the allosteric function of this enzyme is 2 times as sensitive as the catalytic function. If the enzyme is irradiated in the presence of the inhibitor molecule, threonine, the catalytic function is only slightly protected (Fig.6B), while the allosteric function is greatly protected. In fact, it turns out that the protection of the allosteric function is 3.5 times as great as the protection of the catalytic function. Thus, we have here obtained a specific protection of a certain property of an e n z y m e . lAEA-PL-561/7 113

20 ДО 60 80 DOSE (kR) DOSE (kR)

F IG , 6 . (A) Effect of radiation on the catalytic and allosteric activity of aspartokinase homoserine dehydrogenase from E. coli. The enzyme was irradiated and the activity measured in the absence and presence of threonine. The inhibition, expressed in per cent of the inhibition of the unirradiated enzyme,

is taken as a measure of the remaining allosteric activity (dashed line). (B) Preferential protection of the

allosteric function of the dehydrogenase activity by threonine. The enzyme was irradiated in the presence

and absence of 2 mM DL-threonine and the catalytic and allosteric activity determined as in part A.

Data taken from Ref. [1 7 ].

TABLE III. SPECIFIC PROTECTION OF THE CATALYTIC AND ALLOSTERIC FUNCTIONS OF DIFFERENT ENZYMES

Protected function

E n z y m e Added Ugand Gall/Gcat AUosterlc Ref.

(rel. DRF) (rel. DRF)

Fructose-1, 6 -diphosphatase 0.6

AMP 0 . 0 9 16

Fructose-1, 6 -diphosphate 1 . 7

Aspartokinase-homoserine

dehydrogenase

(dehydrogenase act.) 2.0 17

DL-Threonine 0.6 3 . 5

Aspartate transcarbamylase 2.0

Carbamyl phosphate 0.6 3 . 3

CTP 1.0 2. 0 15

PP. 4 . 8 2 . 4 114 SANNER et al.

The described procedure has been used for a number of enzymes to study preferential protection and some results are summarized in Table .III. In all cases studied preferential protection of one enzymatic function was obtained when the enzyme was irradiated in the presence of substrate or allosteric effector molecules. Thus, irradiation of fructose-1, 6-diphosphatase in the presence of the allosteric effector molecule AMP resulted in a 7 times as effective protection of the allosteric function as of the catalytic function. On the other hand, irradia­ tion in the presence of substrate resulted in a 3 times as efficient protec­ tion of the catalytic site as of the allosteric site. Similarly, in the case of aspartate transcarbamylase specific protection was obtained [15]. It is quite astonishing that dose reduction factors of one function of the enzyme may be more than 5 times as high as dose reduction factors of the other function. It clearly demonstrates that small alterations in the conformation of an enzyme molecule strongly influence its sensitivity to the water radicals. It should also be pointed out that in the case of some sulfhydryl enzymes where the inactivation is due to destruction of its SH-group, it is possible to protect the enzyme preferentially by adding sulfhydryl blocking agents. In the case of papain a dose reduction factor of nearly 9 has been obtained by blocking its essential sulfhydryl group during irradiation by mercaptide formation or by mixed disulfide [8].

ACKNOWLEDGEMENTS

This work was supported by the Norwegian Cancer Society. The fellowships to G.K.-P. and S.W. from the International Atomic Energy Agency, Vienna, are gratefully acknowledged.

REFERENCES

[1 KOHN, R .M ., Lebensm.-Wiss. u.Technol. 4 (1971) 69. [2 KISS, I., FARKAS, J., FERENCZY, S ., KALMAN, B ., BECZNER, J., in Improving Food Quality by Irradiation, these Panel Proceedings.

[ 3 SREENIVASAN, A ., these Panel Proceedings,

[ 4 SANNER, T ., KO VACS-PROS ZT, G ., VAS, К ., Radiat. Res. 49 (1972) 300. [ 5 KOVACS-PROSZT, G ., SANNER, T ., Radiat, Res. 53 (1973) 444. [6 MCDONALD, M .R ., Br. J. Radiol. 27 (1954) 62. [ 7 EIDUS, L. Kh., GANASSI, E .E ., Biofizika 4 (1959) 215. [8 PIHL, A ., SANNER, T ., Radiat. Res. 19 (1963) 27. [ 9 LATARJET, R,, EPHRUSSI-TAYLOR, H ., REBEYROTTE, W ., Radiat. Res. Suppl. 1 (1959) 417. [10 SANNER, T ., Radiat. Res. 25 (1965) 586. [11 OKSMO, 0 ., BRUSTAD, T ., Z. Naturforsch. 23B (1968) 962. [12 SANNER, T ., Radiat. Res. 44 (1970) 313. [ 1 3 HOUTERMANS, T ., Z. Naturforsch. Ш (1956) 636, [ 1 4 SANNER, T ., PIHL, A ,, Radiat. Res. 51 (1972) 155.

[ 1 5 KLEPPE, K ., SPAEREN, U .. Biochem. 6 (1967) 3497.

[ 1 6 LITTLE, С ., SANNER, T ,, PIHL, A ., Biochim. Biophys, Acta 178 (1969) 83.

[ 1 7 D1MARGO, G ,, SANNER, T ,, PIHL, A ., Biochim. Biophys, Acta 220 (1970) 1, [ 1 8 HUTCHINSON, F ., Cancer Res. 26 (1966) 2045.

[ 1 9 BRAAMS, R ., Nature (London) 200 (1963) 752.

[20 PIHL, A. # SANNER, T ., Prog, Biochem. Pharmacol. 1_ (1965) 85. [21 COPELAND, E .S., SANNER, T ., PIHL. A ., Radiat, Res. 35 (1968) 437, IAEA-PL-561/7 115

[221 OKADA, S ., Archs. Biochem. Biophys. 6]7 (1957) 102.

[23] BUTLER. J.A .V., ROBINS. A.B., ROTBLAT, J., Proc. R. Soc. (London) Set. A 256 (1960) 1. [24] HUTCHINSON, F., ROSS, D .A., Radiat. Res. 10 (1959) 477.

[25] ADELSTEIN, S.J., Biochem. 4 (1965) 891.

[26] HOLLAND, P., LITTLE, C .. Can. J. Biochem. 49 (1971) 510. [27] ARMSTRONG, R. C ., CHARLESBY, A .. Int. J. Radiat. Biol. 12 (1967) 523.

[28] WINSTEAD, J.A ., Radiat. Res. 30 (1967) 832.

[29] SUMEGI. J.. SANNER, T .. PIHL, A ., Biochim. Biophys. Acta 262 (1972) 145.

[30] SANNER. T ., Radiat. Res. 26 (1965) 95.

[31] QUINTILIANI, М ., BOCCACCI, М ., Int. J. Radiat. Biol. 7 (1963) 255. [32] DAMJANOVICH, S., SANNER. T ., PIHL. A ., Eur. J. Biochem. 1 (1967) 347.

[33] GORIN, G ., WANG, S .-F ., SETH, T .D ., Int. J. Radiat. Biol. 16 (1969) 93.

[34] LANGE, R., PIHL, A ., ELDJARN, L ., Int. J. Radiat. Biol. 1 (1959) 73.

[35] CHAPMAN, A ., SANNER, T ., PIHL, A ., Biochim. Biophys. Acta 178 (1969) 74.

[36] GORIN, G ., PAPAPAVLOU, L .. TAI, L .W ., Int. Radiat. Biol. _1§ (1969) 33.

[37] GORIN. G ., SETH, T .D ., TAI, L.W ., KOLENBRANDER, H. M .. Int. J. Radiat. Biol. 15 (1969) 23.

[38] KLEPPE, K ., SANNER, T ., PIHL. A .. Biochim. Biophys. Acta 118 (1966) 210.

[39] SANNER, T ., PIHL, A ., Biochim. Biophys. Acta 146 (1967) 298.

[40] LYNN, K.R., ORPEN, G .. Int. Radiat. Biol. 14 (1968) 363. [41] ADAMS, G.E., WILLSON. R.L., ALDRICH. J.E., CUNDALL, R.B., Int. J. Radiat. Biol. 16 (1969) 333.

[42] ADAMS, G.E., BISBY, R. H ., CUNDALL, R.B., WILLSON, R. L., Int. J. Radiat. Biol. 20 (1971) 405.

[43] SANNER. T ., PIHL, A ., ''Fundamental aspects of enzyme inactivation by ionizing radiation ”,

Enzymological Aspects of Food Irradiation (Proc. Panel Vienna, 1968), IAEA, Vienna (1969) 23.

[44] SANNER, T ., PIHL, A ., Scand. Lab. Invest. Suppl. 106 (1969) 53.

[45] SHALEK, R.J., SMITH, C. E., HUNTER, J., Radiat. Res. 31 (1967) 467.

[46] ALLAN, J.T ., HAYON, E .M ., WEISS, J., J. Chem. Soc. (1959) 3913.

[47] HENRIKSEN, T ., Radiat. Res. 17 (1962) 158.

[48] SANNER. T ., PIHL. A ., Radiat. Res. 37 (1969) 216.

[49] SANNER, T ., HENRIKSEN, T ., PIHL, A ., Radiat. Res. 32 (1967) 463. [50] PIHL, A ., HENRIKSEN, T ., SANNER. T ., Radiat. Res. 35 (1968) 235.

[51] HOUTERMANS, T ., Z. Naturforsch. 9B (1954) 600.

[52] STAPLETON, G .E ., EDINGTON, C .W ., Radiat. Res. 5 (1956) 39.

[53] WOOD, Т .Н ., TAYLOR. A. L ., Radiat. Res. 7 (1957) 99. [54] TANAKA, Y ., RIXON, R. H ., Int. J. Radiat. Biol. 9 (1965) 503. [55] A RIMA, К ., IWASAKI, S., TAMURA, G .. Agr. Biol. Chem. 31 (1967) 540. [56] MATSUBARA, H ., FEDER, J., in The Enzymes, 3rd Edn, Vol. 3 (BOYER, P .D ., Ed.), Academic

Press, New York (1971) 721. [57] OKADA, S ., FLETCHER, G ., Radiat. Res. 16 (1962) 646.

[58] MEE, L. К ., Radiat. Res. 21 (1964) 501. [59] LYNN. K .R ., Radiat. Res. 45 (1971) 25. [60] TRUFFA-BACHI, P., LE BRAS, G ., COHEN, G .N .. Biochim. Biophys. Acta 128 (1966) 440.

IAEA-PL-561/8

CHANGES IN CELL PERMEABILITY DUE TO IRRADIATION Effect of 60Co gamma rays on the phospholipase D enzyme

W .S . SHERIF Royal Scientific Society, Am m an, Jordan

Abstract

CHANGES IN CELL PERMEABILITY DUE TO IRRADIATION: EFFECT OF 60Co GAMMA RAYS ON THE

PHOSPHOLIPASE D ENZYM E.* Radiation-induced changes in artificial model plant tissues were studied using a two-compartment system separated by various membranes produced by impregnating sheets of filter paper with lipid substances

(including tripalmitin, lecithin and cholesterol) either singly or in various combinations. Changes in permeability of these membranes to alkali ions, glucose, proteins and DNA were studied when one of the above compartments (cells) contained the lecithin-hydrolysing enzyme phospholipase D. The permeability to Na+ , K4- and glucose of membranes containing mixtures of the above lipids was decreased in the presence of phospholipase D. It was found that irradiation decreases the SH content of the enzyme and lowers its lecithin-hydrolysing ability, thereby reducing the decrease in permeability of the artificial membrane.

* This is part of the author's Ph. D. Thesis, University of Bordeaux, France, 1971. Details of the work can be found in the above thesis entitled: "Etude expérimentale de la perméabilité aux ions alcalins, glucose, protéines et ADN, de diverses membranes lipidiques artificielles. Modifications résultant d*une hydrolyse enzymatique avant et après irradiation de l'enzym e par les R du 60C o " .

117 r IAEA-PL-561/9

IMMOBILIZATION OF ENZYMES BY THE RADIOPOLYMERIZATION OF ACRYL AMIDE*

K. KAWASHIMA, K. UMEDA National Food Research Institute, Ministry of Agriculture and Forestry, Tokyo, Japan

Presented by M. Fujimaki

Abstract

IMMOBILIZATION OF ENZYMES BY THE RADIOPOLYMERIZATION OF ACRYL AMIDE.

Several enzymes were imm obilized by radiopolymerization of acryl amide. A spongy membrane

entrapping enzymes was obtained by irradiation treatment in the frozen state. The polymerization rate was increased by the addition of starch and by lyophilization treatment.

Glucose oxidase (with an enzyme activity recovery of 12.3- 33.7%), invertase (69.2%), D-am ino acid

oxidase (25.0, 70.5%), acylase (39.2, 43.7%), mould ct-amylase (18.0%), malt 8- amylase (4.1%), gluco- a m y la s e ( 6 .5%), alkaline protease (5.3%), neutral protease (10.5%) were im m obilized. Invertase entrapped by this method had a wider optimum range of pH but little change was observed in the temperature-activity

p r o file .

Water-insoluble enzyme derivatives are useful not only for medical and pharmaceutical purposes but for various processes in the food industry. The potential advantages of immobilized enzymes are expected to play an important role in the development of automated continuous-flow processes and thereby reduce the cost of enzymes and consequently that of the final p r o d u c ts . Many methods of immobilizing enzymes have been described in the past decade. Among them, gel entrapment is widely applied for various enzymes because it neither alters the enzyme nor requires special radicals in the enzyme. Thus, the method is applicable to crude enzymes as well as to refined ones, to microorganisms and even culture broth which contains enzymes. Enzyme entrapment is usually conducted by the chemical polymerization of acryl amide. The authors have been investigating radiopolymerization of water-soluble monomers and polymers such as acrylic acid, acrylate, acrylic acid derivatives, acrylonitrile, propylene glycol and polyvinyl alcohol. On polymerization of these compounds by irradiation, enzymes can be entrapped in the polymer matrix. Compared with the chemical method, radiopolymerization has the following advantages:

1. The polymerization reaction can be started or stopped freely at a predetermined time. 2. Polymerization is done at the desirable temperature. 3. There is no enzyme inactivation due to chemicals.

* An outline of this work was presented at the annual meeting of the Agricultural Chemical Society

of Japan in Tokyo in April 1973.

119 120 KAWASHMA and UMEDA

4. A highly pure form of polymer is obtainable compared with that obtained by the chemical method where the catalyzer always remains bound to the end of the polymer chains. 5. Several monomers or polymers are co-polymerized and various types of polymer are formed depending on the combination of monomers and p o ly m e r s . 6. A frozen solution can be polymerized and the resulting water-insoluble membrane has a spongy texture with a high surface area.

This paper describes a method for the immobilization of several enzymes by radiopolymerization of acryl amide.

MATERIALS AND METHODS

M a te ria ls

Acryl amide (AA) and N, N1-methylene-bis acryl amide (BA) were purchased from Seikagaku Kogyo Co. Ltd. The former was recrystallized from acetone before use. The following enzymes were immobilized (some of them were crude but they were applied without further refining): Glucose oxidase from Boehringer, Mannheim, invertase and glucoamylase from Seikagaku Kogyo Co. Ltd., acylase and mould a-amylase from Amano Seiyaku Co. Ltd., D-amino acid oxidase from M iles-Serva Ltd., alkaline and neutral protease from Nagase Sangyo Ltd., and malt /З-amylase from a domestic brewery.

Irradiation

Generally a solution of a mixed sample was frozen and irradiated with 60Co (in a Gamma Cell 220, with about 10 000 Ci) at a dose-rate of 800 to 900 krad/h.

Preparation of immobilized enzyme

Two millilitres of standard monomer solution (AA 30 g, BA 1.6 g, distilled water 100 ml), 2 ml of 5% soluble starch (Merck, Zurkovsky) solution and 1 ml of enzyme solution were mixed quickly in a 300-ml flask which was then submerged in a solution of dry ice and acetone at -86°C. Thus, a thin frozen film was formed on the inner wall of the flask. The flask was irradiated with 50 to 70 krad of gamma rays followed by lyophilization or by thawing at room temperature after being kept at -15°C for one night. As the effect of oxygen on the solid-state polymerization of AA is negligible, irradiation was carried out in air without nitrogen gas exchange.

Determination of polymerization rate

The immobilized enzyme membrane was washed repeatedly and kept in a beaker with distilled water over night. The membrane was then washed thoroughly with methyl alcohol, dried with air and further dried at 65 to 70°C for 3 to 4 h before weighing. The rate of polymerization was calculated from the weight. IAEA-PL-561/9 121

Electron microscopy

To observe the structure of the membrane the lyophilized preparation was prepared for electron microscopy. A Nihondenshi JSM-U3 scanning electron microscope was used.

RESULTS AND DISCUSSION

Polymerization rate of acryl amide

1. Effect of soluble starch

From preliminary experiments it was known that radiopolymerization of AA required high radiation doses (over 400 krad for a 12% solution), but in the presence of BA the dose was reduced considerably and the polymer became more rigid. Consequently, the standard monomer solution contained a small amount of BA. The maximum polymerization rate of AA in the membrane which was thawed without lyophilization after irradiation was about 89% at 80 krad when soluble starch was added and 23.5% at 80 krad when it was not added

FIG. 1. Effect of soluble starch on the radiation polymerization rate of acryl amide (not lyophilized).

The AA solution was prepared by dissolving 30 g of acryl amide and 1.6 g of bis acryl amide in 100 m l of distilled water and adding either a 5°¡o solution of soluble starch or distilled water in the ratio of 1 : 1 by volum e. The solution was then frozen, irradiated and thawed at room temperature.

• ------• starch added; * ------* starch not added. 122 KAWASHIMA and UMEDA

(Fig. 1). Thus, starch was effective in increasing the polymerization rate of AA and in decreasing the dose which gave the maximum polymerization ra te. As starch is known to produce radicals on ionizing irradiation, the polymerization of AA was expected to be accelerated by such radicals.

2. Concentration of starch

The effect of the starch concentration on the polymerization of AA is shown in Fig. 2. The polymerization rate reached a maximum at a 5 to 10% concentration of soluble starch. The weight of the polymer increased steadily with the starch concentration, so the apparent decrease of poly­ merization rate above 5 to 10% of starch seemed to occur due to the washing out of soluble starch (Merck, Zurkovsky) which did not participate in the polymerization of AA.

3. Effect of lyophilization

The AA solution was frozen, irradiated and lyophilized as described in Fig. 3. The maximum polymerization rate was about 98% at 60 krad when soluble starch was added and 67% at 60 krad when it was not added. On comparing Fig. 3 with Fig. 1 it can be seen that the polymerization rate was markedly increased by the lyophilization treatment.

CONCENTRATION OF ADDED SOLUBLE STARCH (%)

FIG.2. Effect of soluble starch concentration on the radiation polymerization rate of acryl am ide. The

AA solution (prepared by dissolving 30 g of acryl amide and 1.6 g of bis acryl amide in 100 m l of distilled water and adding the soluble starch solution in a ratio of 1 : 1 by volume) was frozen, irradiated with 60 krad of gamma rays and lyophilized or thawed at room temperature.

• — — ~ • ly o p h iliz e d ; ■ ------■ not lyophilized. IAEA-PL-561/9 123

FIG. 3, Polymerization rate of acryl amide which was irradiated and lyophilized. The AA solution (prepared as described in F ig.l) was frozen, irradiated and lyophilized. • ------• soluble starch was added; » ------* soluble starch was not added.

Radiopolymerization is generally caused by ion polymerization at low temperatures, but AA is known as one of the monomers which polymerizes in the solid state. During lyophilization the moisture was lost and the solid-state polymerization seemed to progress at the same time as the ordinary ion polymerization.

Immobilization of various enzymes

1. Glucose oxidase

Two millilitres of standard monomer solution,in which Tris glycerol buffer was used instead of distilled water, 2 ml of a 10% starch solution and 1 ml of an enzyme solution (0.5 mg/ml) were mixed and tested as show n in T a b le I. In trial 1, the mixture of A and С was directly lyophilized without irradiation and showed only a very little activity. In trial 2, the mixed solution of A, В and С was irradiated at 0°C in an ice-water bath. In trial 3, the same solution mixture was irradiated at -86°C and the activity recovered was about twice that in trial 2. After irradiation at -86°C, lyophilization was carried out in trial 7. The amount of activity was further in c r e a s e d . An immobilized enzyme preparation could be prepared without direct irradiation of the enzyme as shown in trial 4. This would be important when the enzyme is highly radio-sensitive. Pre-irradiation of the solution should 124 KAWASHIMA and UMEDA

TABLE I. VARIOUS METHODS TO IMMOBILIZE GLUCOSE OXIDASE AND THEIR RETAINED ACTIVITY

A c t iv it y T r ia l M ix tu re T r e a t m e n t (%)

1 AC L 0 . 3

2 ABC Irr. with 50.4 krad 1 2 . 3

3 ABC F^Irr. with 50.4 krad 2 5 . 8

4 AB Irr. with 18.6 krad -> С -*■ L 2 4 . 8

5 AB Pre-irr. with 18.6 krad -*■ С -* F

post-irr. with 4 0.5 krad -*• L 3 0 . 5

6 ABC Irr. with 18.2 krad-^L 2 0 . 9

7 ABC F -*■ Irr. with 50.4 krad L 3 3 . 7

A: 2 ml of the AA solution (acryl amide 30 g, bis acryl amide 1.6 g, 100 m l of Tris glycerol buffer a t pH 7 .0 )

B: 2 m l of a 10% solution of soluble starch

C: 1 ml of enzyme solution (0.5 mg/ml)

Irr. : Irra d ia tio n ,

F: Freezing with a mixture of dry ice and acetone

L: Lyophilization

not cause polymerization. To this solution, enzyme was added and the mixture was immediately frozen and lyophilized. The irradiation dose could also be reduced as in trials 5 and 6. The activity of the enzyme was determined colorim etrically according to Bergmeyer. The immobilized enzyme membrane was cut into small pieces (1 mm X 2 mm) and 1/20 to 1 / 50 of the total weight was prepared for the activity determinations. The membrane was washed several times with distilled water. For comparison, the same amount of membrane was inactivated and used for the colouring reaction.

2. Invertase

Two millilitres of standard monomer solution in which acetate buffer at pH 4.6 was used instead of distilled water and 2 ml of a 10% soluble starch solution were mixed with 1 ml of enzyme solution. The mixture was frozen at -86°C, irradiated with 42.8 krad and thawed at room temperature. The resultant enzyme membrane was cut into small pieces and 0.96 g was used to measure its activity. The amount of reducing sugar that was produced was titrated according to the method of Somogyi. For comparison, 0.96 g of membrane was boiled for 3 min and treated in the same way. The recovery of enzyme activity was calculated to be 69.2% of the original activity. Some enzymes are known to shift their apparent optimum pH during immobilization. This can be explained by the changes in hydrogen ion concentration in the micro-environment of the fixed enzymes. Bernferd reported that this phenomenon did not occur in the enzymes which were IAEA-PL-561/9 125

pH

FIG.4. pH-activity profile of invertase. One millilitre of 0.3M sucrose, 4 ml of each buffer, 4 m l of distilled water and 1 m l of free enzyme (3 fig) or 960 mg of immobilized enzyme membrane were shaken at 40°C for 60 min. • ------• free enzyme; о ------о immobilized enzyme.

о <

< _! LU

FIG. 5. Temperature-activity profile of invertase. One millilitre of 0.3M sucrose, 4 m l of distilled water, 5 ml of 0.04M acetate buffer (pH 4.6), 1 m l of free enzyme (3 Mg) or 960 mg of immobilized enzyme were shaken for 60 min at each given temperature.

• free enzyme; ° immobilized enzyme.

i 126 KAWASHIMA and UMEDA

entrapped by chemical polymerization of AA. Invertase that was immobilized by radiopolymerization of AA also did not show any shift in pH, but the range of the optimum pH became wider (Fig. 4). A citrate phosphate buffer was used for pH values from 2.5 to 3.0, an acetate buffer for pH values from 3.5 to 5.5, a phosphate buffer for pH values from 6.0 to 7.0 and boric acid and a borax buffer for pH values from 8.0 to 9.0. The final concentration of buffer in the reaction mixture was 0.02M. There was little change in the reaction temperature-activity profile of the immobilized invertase, as shown in Fig. 5.

3. D-amino acid oxidase

Two millilitres of standard monomer solution, in which 0.05M boric acid and borax buffer at pH 8.3 was used instead of distilled water, containing 5% starch, and 0. 5 ml of enzyme solution (50 mg/ml) was mixed, frozen immediately, gamma irradiated with 49.9 krad and lyophilized. Part of the membrane obtained (120 mg) was washed thoroughly and mixed with 5 ml of 0.05M boric acid and borax buffer (pH 8.3), 1 ml of dl-alanine (17.8 /jg/m l) and 1 ml of crude catalase (120 fj.g/ml). The mixture was allowed to react at 30°C for 60 min and the pyruvic acid formed was titrated iodometrically. The immobilized enzyme membrane retained 70.5% of the original activity. The activity of this membrane was determined four more times in succession and found to be 63.1, 65.6, 70.0 and 66.5% of the original activity. Thus, the immobilized enzyme kept its activity fairly well and the leakage of entrapped enzyme was not considered to be serious. The immobilized enzyme preparation which was not lyophilized after irradiation but kept at -15°C over night and thawed at room temperature retained 25.0% of the original enzyme activity.

4. A c y la s e

One m illilitre of enzyme solution (7 mg/m l), 2 ml of a 10% starch solution and 2 ml of standard monomer solution were mixed, frozen, irradiated with 55.2 krad and lyophilized. Six m illilitres of Veronal buffer (0.1M, pH 8.0), 2 ml of 0.5 X 10"3M CoCl2, 60 mg of immobilized enzyme and 2 ml of 0.1M n-acetyl-dl-methionine were mixed and allowed to react at 37°C for 30 min. The methionine produced was assayed colorim etrically with Ninhydrin and the immobilized acylase was found to have 43.7% of the original enzyme activity. On the other hand, the immobilized acylase preparation which was not lyophilized after radiation but thawed at room temperature exhibited 39.2% of the original enzyme activity.

5. A m y la se

(i) Mould a-amylase. To 4 ml of standard monomer solution was added 1 ml of crude mould a-amylase solution (50 mg/ml) and 2 ml of 0.4M acetate buffer at pH 5.6. The mixture was then frozen, irradiated with 57.2 krad and lyophilized to give a white water-insoluble membrane. The membrane was thoroughly washed with distilled water and 23 mg of it was mixed with 5 ml of citrate buffer at pH 5.6, 4 ml of distilled water IAEA-PL-561/9 127 and 1 ml of 1% starch solution. The final mixture was allowed to react at 30°C for 10 min. The amount of reducing sugar produced was determined by the Somogyi. titration method. The preparation was found to retain 18.0% of the initial enzyme activity. (ii) Malt 0-amylase. Two millilitres of standard monomer solution were mixed with 1 ml1 of a powdered malt solution (50 mg/ml) and treated as described in (i). The substance obtained showed 4.1% of the original enzyme activity. (iii) Gluco-amylase. Two millilitres of standard monomer solution, 1 ml of 10% soluble starch solution and 1 ml of enzyme solution (10 mg/ml) were mixed quickly in an ice-water bath. The mixture was immediately frozen at -86°C, irradiated with 65.5 krad and lyophilized. Part of the white membrane (31.2 mg) was washed with ice water and the activity assayed in 10 ml of 0. 04M acetate buffer at pH 5.0 containing 1 ml of a 1% soluble starch solution as the substrate. The mixture was allowed to react at 45°C for 30 min. The amount of reducing sugar produced was determined by the Somogyi titration method. For comparison, the same amount- of immobilized preparation was boiled for 3 min and prepared for titration. The immobilized gluco-amylase preparation contained 6.5% of the original enzyme activity.

6. Protease

(i) Alkaline protease. Two millilitres of monomer solution containing 40 g of AA, 1.6 g of BA and 100 ml of distilled water were cooled in an ice-water bath and irradiated with 21.5 krad. The solution did not polymerize but remained in the liquid state. To this solution was added 1.5 ml of crude enzyme solution (50 m g/m l), 0.5 ml of 0.4M boric acid and borax buffer (pH 10.0), and 1 ml of a 50% glucose solution. The mixture was frozen immediately at -86°C. After irradiation with 43.0 krad and lyophilization, 24.5 mg of enzyme membrane were weighed, washed thoroughly with water and assayed for activity in 10 ml of 0.04M boric acid and borax buffer (pH 10.0) containing 1 ml of 1.5% casein solution. The mixture was allowed to react at 3 7°C for 60 min. The enzyme activity was determined colorim etrically and the immobilized alkaline protease preparation was found to retain 5.3% of the original enzyme activity. (ii) Neutral protease. To 2 ml of standard monomer solution were added 1.5 ml of crude enzyme solution (50 mg/m l), 1 ml of 50% glucose solution and 0.5 ml of 0.4M phosphate buffer (pH 3.0). The mixture was then frozen at -86°C and irradiated with 52.0 krad. After thawing at room temperature, 0.1 g of the membrane was weighed, washed thoroughly with distilled water and mixed with 10 ml of 0.02M phosphate buffer (pH 8.0) containing 1 ml of a 1.5% casein solution. The activity was determined as described for alkaline protease. The recovery of immobilized enzyme activity was 10. 5%. The results from enzymes 2 to 6 are summarized in Table II. It can be seen that an immobilized enzyme requiring a small molecular substrate has a high recovery and that the one requiring a large molecular substrate has a low recovery. As the enzymes are entrapped in the matrix of the AA polymer, the diffusion of substrate would greatly influence the activity of immobilized e n zy m e s. 128 KAWASHIMA and UMEDA

TABLE II. IMMOBILIZATION OF SEVERAL ENZYMES AND THEIR RETAINED ACTIVITY

Retained activity Enzyme and treatment (%)

1. In vertase ABC -> F Irr. with 42.8 krad 6 9 . 2

2 . d-Am ino acid oxidase ABC -» F Irr. with 49.9 krad L 7 0 . 5 A B C -*■ F -*■ Irr. with 49.9 krad 2 5 . 0

3 . А с у lase ABC F Irr. with 55.2 krad L 4 3 . 7 A B C -*■ F -»• Irr. with 53.5 krad 3 9 . 2

4 . A m y la s e

(i) Mould a-amylase

AC -► F -*■ Irr. with 57.2 krad L 1 8 . 0

(ii) M a lt 6 -a m y la s e A C -> F -* Irr. with 57.2 krad L 4 . 1 (iii) Gluco-amylase

ABC -> F -»■ Irr. with 65.5 krad L 6 . 5

5 . Protease

(i) Alkaline A pre-irr. with 21.5 krad BC F -*• post-irr. with 43.0 krad -* I 5 . 3 (ii) Neutral

ABC -*• F Irr. with 52.0 krad 1 0 . 5

Abbreviations the same as for Table I.

Configuration of immobilized enzyme membrane

An immobilized enzyme was prepared according to the method described in which an enzyme solution was used instead of distilled water. It is a membrane unless it is cut or crushed. When the monomer solution was frozen quickly it can be inferred that the water formed small crystals of ice and that the soluble substances, such as monomer, starch and enzyme, were concentrated around the crystals. Radiopolymerization was brought about under this condition and after thawing or lyophilization numerous small holes appeared within the membrane. This gave the immobilized enzyme preparation a large surface area which would be effective in increasing the activity of the entrapped enzyme prepared by this method. The membrane was spongy and easily swelled up on absorbing water. The membrane which was prepared in the presence of starch was fairly elastic. On the other hand, the membrane prepared without starch was also spongy but rather tuff and rigid. There was a big difference in the appearance of the membrane surface. The membrane containing starch had a smooth and regular surface. This would suggest that AA started polymerization from the various parts of the starch molecule where radicals were produced by irradiation so that a type of "graft" polymerization occurred and a polymer chain was developed. IAEA-PL-561/10

COMPOSITIONAL AND QUALITY CHANGES IN SOME IRRADIATED FOODS

A . SREENIVASAN Bhabha Atom ic Research Centre, Trombay, Bombay, India

Abstract

COMPOSITIONAL AND QUALITY CHANGES IN SOME IRRADIATED FOODS; In gamma-irradiated wheat there is molecular degradation of starch and protein components which influences the Theological properties like gelatinization viscosity of starch, dough development and stability as well as elasticity of gluten. The baking quality of wheat that has been irradiated at disinfestation dose levels is improved, resulting in a product with increased loaf volum e, soft and uniform cell structure and higher acceptability. Enrichment of wheat flour with soya flour beyond 5% adversely affects the Theological properties and breads made with such dough mixes are poor in quality. However, with irradiated wheat, there are no significant changes in bread quality up to 10% replacement with soya' flour; this could be increased to 15% with the use of trace amounts of surfactants like soya lecithin.

A combination of low-dose irradiation and mild heat successfully extends shelf-life of bread chapaties (unleavened, Indian bread) by controlling fungal spoilage. The efficacy of this combination of radiation and heat treatment has been confirmed from studies with fungi isolated from naturally infected, baked products. Irradiated red gram (Cajanus cajan) shows reduction in cooking tim e, improvement in texture and better retention of some of the В vitamins on cooking. Oligosaccharides, reported as flatulence factors in legumes, are decreased in irradiated cooked red gram. There is degradation of proteins due to irradiation and hence increased susceptibility to proteolytic action in vitro.

Low-dose irradiation effectively delays ripening of bananas and mangoes, resulting in better texture of

the fruits over longer periods. In mangoes, there is also improvement in external appearance on ripening, due to formation of anthocyanins. Flavour in mangoes is influenced by the ratio of palmitic to palmitoleic

acid of the lipid fraction which is slightly influenced by irradiation.

Sprout inhibition by irradiation augments flavour in carrots which is reflected in changes in the composition of the volatiles when examined by gas chromatographic-mass spectrometric methods of separation.

In potatoes, syntheses of chlorophyll and solanin are inhibited when irradiated and stored at sub-room temperatures. Post-harvest fungal decay in tropical fruits like figs, grapes and bananas, can be controlled by use of m ild, moist heat and low-dose irradiation. Thermal treatment in the canning of fruits and vegetables can be considerably reduced by resorting to

sterilisation with combined heat and low-dose irradiation. Products so obtained have better texture and

flavour with less loss of nutrients.

I. INTRODUCTION

The importance of radiation as a food preservation process has been successfully demonstrated in various applications including disinfestation of stored grains, elimination or control of spoilage organisms in perishable foods and inhibition of physiological processes such as sprouting in tubers and bulbs and ripening in fruits. Related work carried out at Trombay has been reported in recent publications [1-10]. A vast amount of work has also accumulated on short- and long-term feeding tests with experi­ mental animals which have generally established the wholesomeness of low-dose radiation-processed foods (see Refs [11-16]).

129 130 SREENIVASAN

It is known that, during irradiation of foods, radiolysis of water gives rise to short-lived and transient but chemically reactive free radicals. These in turn bring about various reactions which modify the chemical and physical properties of the major food constituents. There are several investigations to suggest that, in the dose ranges employed, these changes frequently improve the quality of the product (see Refs [17-20]). This paper reviews some of the work carried out at Trombay on compositional and quality aspects of wheat, red gram (Cajanus cajan) and certain fruits and vegetables (mango, banana, carrot and potato) which have been radiation-processed for improved stabilization on storage or to confer other benefits.

II. COMPOSITIONAL CHANGES IN IRRADIATED WHEAT

The use of fast electrons or gamma rays for control of insect infesta­ tion in stored grains has great potentialities and, based on comprehensive long-term feeding studies with experimental animals, some countries have cleared radiation-disinfested wheat for human consumption [21]. Although, at dose levels necessary for insect control, irradiation does not alter the nutritive value of wheat, it is known that subtle changes do occur in the physico-chemical properties of macro-nutrients like starch and proteins either by direct or indirect action [22-24]. These changes may be expected to influence the visco-elastic properties and hence the inherent bread- making quality of wheat, which, in fact, shows improvement in terms of loaf-volume and crumb structure (Rao, Sudha V., Vakil, U.K. and Sreenivasan, A., unpublished). Some relevant results from our studies are summarized in this and the two following sections.

II. 1. Radiation effects on wheat starch

Samples of hard, red winter variety wheat (Triticum aestivum) were exposed at room temperature to 60Co gamma rays at dose levels varying from 20 to 200 krad. Absorption of radiation was checked with ferrous sulphate and eerie sulphate dosimetry [25].

TABLE I. RADIATION EFFECT ON WHEAT STARCH

Maltose value as mg D o se Initial reducing maltose liberated per l e v e l sugars as mg maltose per 10 g wheat Hour at % increase in (kracf) 10 g wheat flour 30°C for lh maltose value

0 9 0 1 5 0 -

20 95 1 7 2 2 8

4 0 1 0 5 1 9 0 4 1 '

6 0 110 1 96 4 3

200 1 2 5 211 4 3

Values are averages of 3 experiments. Aqueous extracts of wheat flour were analysed for reducing-

sugar values. 10 g of wheat flour (60 mesh) was suspended in 46 ml of 0. IN acetate buffer at pH 4 . 8, resulting in a 50- m l total volume and incubated at 30°C for 1 hour to measure diastatic activity. IAEA-PL-561/10 131

Data on initial levels of the water-soluble reducing sugars (Table I) show an increase in irradiated samples. Diastatic activity also increased significantly in irradiated wheat. In other experiments, we also observed that the sensitivity of wheat starch to the action of a- and (3-am ylases is increased due to radiation treatment; this is attributable to their fragmenta­ tion to low molecular weight entities which are more easily attacked by amylolytic enzymes [22].

II.2. Effects of irradiation on wheat proteins

The levels of free tyrosine in irradiated wheat increased by about 13% at 200 krad compared with control unirradiated samples (Table II). Self­ digestion or autolysis of wheat flour showed about 6.2 to 31.4% increase in tyrosine values in irradiated samples over their unirradiated control. The results thus give an indication of molecular fragmentation of proteins to smaller peptides which are more susceptible to proteolytic enzymes. These observations were further substantiated by studying the elution patterns of extracted proteins from unirradiated and irradiated (1 Mrad) wheat, resolved by gel filtration on a Sephadex G-200 column (Fig.l). Though the pattern was the same in both cases, a shift in the molecular weight distribution to lower values was observed with irradiated wheat proteins. About 17% lower protein values were obtained in the glutenin peak with a concomitant increase in the non-protein peak in irradiated wheat. Since glutenin molecules are formed primarily by intermolecular disulphide bonding of gliadin components, thus contributing to the unique visco-elastic properties of wheat proteins [26], the observed changes in their distribu­ tion pattern may influence the rheological properties of irradiated wheat [23]. It was also ascertained that when wheat samples were subjected to prolonged action, sequentially, of pepsin and trypsin, increasingly more «-am ino nitrogen was liberated from irradiated wheat samples (Srinivas, H., Vakil, U.K. and Sreenivasan, A., unpublished). This indicates that in-vitro digestibility of wheat proteins is increased by irradiation.

TABLE II. AUTOLYSIS OF IRRADIATED WHEAT

Initial free Tyrosine liberated D o s e le v e l % in c rea se tyrosin e after 5?-h digestion (kcad ) over control (m g/g wheat) (m g/g wheat flour)

0 1 45 3 0 4 -

20 1 53 3 2 2 6.2

4 0 1 53 3 7 5 2 8 . 3

6 0 1 53 3 8 1 3 0 . 2

200 1 6 4 3 9 6 3 1 . 4

2 .5 g of wheat flour (60 mesh) suspended in 50 m l of 0 .1M citrate buffer at pH 5.5 was

incubated under toluene for h at 40°C with appropriate controls. Reaction was stopped by the addition of 20 °]o trichloroacetic acid. Tyrosine liberated by the action of proteases was estimated in the supernatant by a colorimetric method. Values are averages of 3 experi­

m e n ts . 132 SREENIVASAN

EFFLUENT VOLUME IN m l

F IG .l. Elution pattern of irradiated wheat proteins on Sephadex G -200. 2 m l (25 mg protein) of wheat flour extract [22] was applied on the column (0.5 cm x 50 cm ). 2.5 ml fractions were collected and the OD was measured in a Beckman DB spectrophotometer at 280 nm . ------c o n tr o l; ------irrad iated (1 M r a d ).

Peaks 1, 2, 3 and 4 represent the four main components of wheat proteins, namely, glutenin, gliadin, albumin and non-protein nitrogen, respectively.

TABLE III. EVALUATION OF AMYLOGRAM OF IRRADIATED WHEAT

Dose level (krad)

0 20 200

Gelatinization viscosity 9 8 0 8 3 0 5 5 0 (amylogram units, A .U . )

Gelatinization

temperature (°C) 8 9 8 4 8 4

Gelatinization time 4 2 3 9 3 7 ( m in )

To measure gelatinization viscosity, water-flour slurry was heated in a Brabender amylograph

revolving container with a steady rise in temperature of 1 .5°C/m in until com plete gelatinization o c c u rr e d .

III. RHEOLOGICAL PROPERTIES OF IRRADIATED WHEAT

III. 1. Gelatinization viscosity

The above-mentioned compositional changes are also directly reflected in the physical properties of irradiated wheat measured with different Brabender instruments. When measured at a constantly increasing tempera­ ture it can be seen (Table III) that the gelatinization property of starch in wheat, which is a process similar to the first stage of baking and is governed by modifications brought about by amylases during the heating IAEA-PL-561/10 133

TABLE IV. IMPROVEMENT IN DOUGH DEVELOPMENT PROPERTIES IN IRRADIATED WHEAT

Wheat irradiation (krad)

Experimental dctailo 0 20 200

Water absorption (%) 6 2 6 5 6 9

Farinogram units (F .U .) after

12 min mixing 4 2 0 4 5 0 4 6 0

Dough development time (min) 3 . 5 3 . 5 3 . 0

Dough stability (min) 2 .0 2 . 5 2 . 5

Resistance tim e (min) 5 . 0 5 . 5 6 .0

300 g of wheat flour (60 mesh) was taken in a Farinograph mixing chamber and distilled water was

added till a dough of 500 F. U. consistency was obtained.

period, is markedly affected by radiation treatment. Maximal gelatiniza- tion viscosity in wheat flour, irradiated at 200 krad, was very low (550 A.U.) compared with the control (980 A.U.). The time and temperature to reach the amylogram peak were also less with irradiated samples.

111.2. Dough d ev elop m en t p r o p e rtie s o f irra d ia te d w heat

A dough development curve of wheat flour was obtained in a farino­ graph and individual factors determining the baking strength of flour, such as water absorption (titration curve), dough development capacity (normal curve) and drop of consistency after a one-hour rest period (rest period curve), were evaluated. Results compiled in Table IV show that, to obtain dough consistency of 500 F.U., unirradiated wheat required 62% water, whereas wheat, irradiated at 20 and 200 krad dose levels, absorbed 65 and 69.5%, respectively. The differences in dough consistency at the start of softening (500 F.U.) and 12 min later in control (420 F.U.) and irradiated (450-460 F.U.) wheat samples, also suggest that the mixing tolerance of irradiated wheat was increased, which may result in better dough strength and blending value. A lower mixing requirement of dough from irradiated wheat is highly desirable and economical when mechanical mixing is operated on a commercial basis.

111.3. E valuation o f e x te n so g ra m o f irra d ia te d wheat

Extensometer parameters, evaluated from typical extensograms obtained with irradiated wheat samples, are summarized in Table V. The total energy used in stretching the dough was increased and irradiated samples offered better resistance compared with unirradiated ones. The 'ratio figure' , which indicates the behaviour of the dough, its stability and potential baking volume, was higher in irradiated samples (4.7 and 5.1 for 20- and 200-krad samples, respectively) compared with the control (2.7). These factors show that the elasticity of wheat gluten and potential baking volume could be improved by irradiation. 134 SREENIVASAN

TABLE V. EVALUATION OF EXTENSOGRAM OF IRRADIATED WHEAT

Wheat irradiation (krad)

Experimental factors

0 20 200

A. Energy (cm 2) 4 5 7 8 92

B. Resistance to stretching 3 1 0 5 2 0 6 2 0 (extensogram units)

C. Extensibility (min) 1 1 5 110 120

D . Ratio figure (B/C) 2 , 7 4 . 7 5 . 1

The stretchability or extensibility of the dough, adjusted to 500 F. U. was determined in an

extensograph with fermentation at 30°C for 135 m in, The total force required in stretching the dough to the tearing point was recorded.

FIG.2 . Breads prepared from unirradiated and irradiated wheat. Bread was prepared by straight dough lean formula [5] containing yeast (2.5% ), sugar (1.0% ), shortening (4.0% ), salt (1.5% ), and water as determined by a farinograph. The dough was fermented (2.5 h), proofed (45 min) and moulds baked at 425°F (20 min). IA E A -P L - 5 6 1 / 1 0 135

It was observed (Fig.2) that total loaf volume in yeast-leavened bread, prepared from dough containing appropriate ingredients, increased up to 8% with irradiated wheat samples, compared with the control. This slight but significant increase in loaf volume may be attributed to amylase - susceptible starch degradation in irradiated wheat. This may either result in stimulation of gas production or in increased ability of the dough to retain the gas produced during fermentation. Physically damaged smaller particle-sized starch may help in increasing the available surface area of- hydration as confirmed by increased water absorption capacity of irradiated wheat flour (Table IV). It has been suggested that bread-staling may also be controlled by moderate radiation treatment [27]. The farinograph and extensograph tests also support the view that the overall bread-making characteristics of wheat flour are improved in the dose ranges studied though, at higher doses, these can be adversely affected [28].

IV. IMPROVEMENT IN BAKING QUALITY OF SOYA-ENRICHED BREAD-MIXES

Utilization of defatted flours from oil seeds like soyabean and peanut may help in effectively upgrading protein quality of prepared products and is especially of value to the developing regions of the world with their inadequate intakes of protein [29]. However, optimal use of oil-seed meals in combination with cereal products will have to reckon with factors of acceptability based on sensory and other quality attributes. It is known that enrichment with oil-seed flours changes the rheological properties of doughs. Incorporation cf only small amounts (up to 5% of flour or protein isolate) is generally satisfactory and gives good volume in the finished bread (Rao, Sudha V., Vakil, U.K. and Sreenivasan, A., unpublished). However, up to 10% replacement of wheat flour could be possible in bread mixes containing irradiated wheat. In these studies, the physical properties of soya-enriched bread mixes, using unirradiated and irradiated wheat, were compared and acceptability of bread was evaluated by taste-test panels. When flour from unirradiated wheat was substituted with 5 - 30% of defatted soybean flour, the protein content of the mixes increased from 13.6 to 27.4%. However, the rheological and baking properties of the resulting doughs were adversely affected. Typical results are given in Table VI. Evaluation of amylographs of the different mixes showed that gelatinization viscosity of suspensions at 20 or 30% replacement levels attained much lower values (240 - 300 A.U.) than the desirable range (400 - 600 A.U.) for getting higher loaf volume and took longer time (48 min) to reach maximum viscosity compared with the control (42 min). The highest temperature for peak viscosity for soya flour mixture (20%) was 95°C compared with 89°C for the control. The effects of increasing the level of replacement of wheat flour with 5 to 30% soya flour were also evident on farinogram's. Water absorption capacity increased from 60% in unsupplemented to 62 - 68% in the supplemented mixes. The narrow farinograph band width at the higher level of oil-seed flour replacement suggested poor dough stability. There was a sudden drop in dough consistency and stability after a 60-min fermentation in the mixed dough. Similarly, 136 SREENIVASAN

TABLE VI. RHEOLOGICAL AND BAKING PROPERTIES OF SOYA- ENRICHED BREAD MIXES

Gelatinization W a ter Per cent replacement Extensogiam v is c o s ity a b sorp tion Specific loaf volume with soya flour r a tio (A.U.) (°/o)

Unirradiatéd wheat

N il 4 8 0 6 0 6.2 3 . 3

5 4 4 0 6 0 5 . 8 3 . 3

10 3 8 0 6 2 5 . 8 3 . 2

20 3 0 0 6 4 4 . 9 2 . 9

3 0 2 4 0 68 4 . 4 2 . 3

Irradiated (20 krad)

w h ea t

N il 4 4 0 6 1 7 . 7 3 . 9

5 4 4 0 6 2 6 .1 3 . 5

10 4 2 0 6 2 6 .0 3 . 6

Rheological properties of the dough mixes were determined as described in Section III. Bread was

prepared by the straight dough lean formula. Specific loaf volume or volume/weight (m l/g) of loaf was measured by the seed displacement method.

dough extensibility decreased and its resistance increased after fermenta­ tion for 135 min in oil-seed meal-supplemented doughs. The ratio between these two dropped from 6.2 in the control.to 4.4 in 30% soya-flour supple­ mented dough. The oil-seed flour-containing breads prepared by the straight lean dough formula were low in volume. Sensory scoring of breads with more than 10% soya flour was much lower compared with the ones without it; they were not acceptable to panel judges because of dark crum b-colour, inferior texture and 'nutty' flavour. A comparison of the results for 5% and 10% replacement with soya meal of unirradiated and irradiated (20 krad) wheat flour (Table VI) clearly shows quality improvement in the latter case. Replacement in the bread mixes with up to 10% soybean flour can therefore be achieved without any significant changes in the rheological properties of the resulting dough and in bread volume. The protein content of the bread could be raised from 13 to 17%. Thus, the adverse effects of soya flour addition on the rheological properties of the mix may be compensated by using irradiated wheat flour which modifies mixing tolerance and water absorption capacity of the m ix e s . Recent preliminary experiments have shown that, with the addition of 0.05% soya-lecithin, a known surfactant, the dough could be enriched with up to 15% soyabean flour (Fig.3). Cell structure, crumb texture, flavour and other bread characteristics were improved. Sensory evalua­ tion of these breads revealed that they scored higher compared with those IAEA-PL-561/10 137

FIG .3. Improvement in loaf volume by soya-lecithin. Irradiated wheat flour was replaced by 1-5% soyabean flour. 0 .057» soya-lecithin was added to dough mixes.

without lecithin and were quite acceptable. It would seem, therefore, that bread with 19.3% protein could be had by using irradiated wheat flour with replacement by 15% of soya flour and with addition of soya lecithin in small amounts.

V. RADIATION PRESERVATION OF BREAD

A combination of mild heat and low dose radiation has been success­ fully employed to extend the shelf-life of bread and chapati (Indian un­ leavened bread) which are normally very susceptible to fungal spoilage and do not keep for more than 2-3 days [30]. Chapati and bread slices, packed in polycell pouches and subjected to 50 krad followed by dry heat (65°C, 30 min) remain free of mould growth and are shelf-stable for 2 months at ambient temperature. Inoculated pack studies using spores of Aspergillus sp., isolated from naturally infected bread and chapaties, confirmed the efficacy of the treatment (Fig. 4). It has been reported [31] that bread slices could be kept for a long time free from mould attack by a combination of dry heating and low dose radiation. 138 SREENIVASAN

FIG.4 . Effect of irradiation-heat treatment on inoculated bread slices and chapa ties. Bread slices or chapaties were inoculated at four different spots with 0.2 m l spore suspension (about 106 spores/ml) of a mixture of Aspergillus niger and A . flavous otyzae and packed in poly cell pouches. The sealed packs were incubated at

30°C for 24 h and subsequently subjected to the various treatments. A combination of 50 krad followed by heating at 65°C for 35 min totally inactivated the spores.

FIG .5. Approximate dose response curves for fungi of Aspergillus group isolated from bread and chapaties.

Fungal spores suspended in saline (1.5 x 10 6 spores/ml) were irradiated at a dose-rate of 110 krad per hour. Colony-forming ability was determined by plating and counting after incubation for 24 h and again after 3 and

5 days. Each point is an average of 6 replicates. IA E A -P L - 5 6 1 / 1 0 139

o— o IRR Д------д HEAT* IRR *------к IRR+HEAT

F I G . 6 a. Effect of heat-radiation combination on survival of fungal spores. Experimental details are as given in Fig. 5. Heat treatments were carried out either before or after irradiation by placing the test-tubes containing spores in a water bath (50°C) for 5 min. The test-tubes were kept constantly agitated.

o— o IRR Д------¿ HEAT* IRR *-----* IRR*HEAT

FIG.6b. Effect of heat-radiation combination on the survival of fungal spores. Experimental details are as given in legends for Figs 5 and 6 a . 140 SREENIVASAN

At present, there are no effective methods to preserve chapaties either at ambient or sub-room temperatures. The use of chemical preservatives reduces the organoleptic qualities of chapaties [32]. It has been reported that chapaties could be stored longer if 4% salt or 1. 5% salt and 0. 3% sorbic acid are incorporated into the dough [ 33]; however, acceptability ratings are low in such products. The efficacy of heat-radiation combination treatment was further confirmed in studies with fungi isolated from the cereal products [30]. The radio-sensitivity of spores of the Aspergillus group, isolated from bread and chapaties, are shown in Fig. 5. From these results, it can be seen that A. niger is the most resistant strain, whereas A. flavus oryzae is the most sensitive species. However, noticeable variation in germinating and colony-forming abilities of these fungi was observed when spores were subjected to heat and irradiation (Fig. 6a and b). Maximum sensitization of both the strains of A. flavus (Aflatoxin-producirîg and non-toxic) occurred when heating preceded irradiation, whereas in A. niger, heating after irradiation was more effective. In A. tereus, the sequence of treatments did not show any appreciable differences in their synergistic effect.

VI. IMPROVEMENT IN TEXTURAL QUALITIES AND COMPOSITION OF IRRADIATED RED GRAM (Cajanus cajan)

Next to cereals, dry legumes form the major and widely used food item of average Indian diets. Because of the low or negligible amounts of foods of animal origin in these dietaries, these legumes (pulses) or dahls, as they are called, form the chief source of proteins which, in quality and quantity, supplement the cereal proteins. Most of the legumes take rather a long time to cook and practices such as soaking in water, cooking under pressure and use of chemical additives like cooking soda, are resorted to for hastening the cooking time. Recently, efforts are also being directed to obtain, by mutation breeding, selection and hybridisation techniques, seed strains with better nutritive value and improved cooking qualities. In our work (Nene, S. P ., Vakil, U.K. and Sreenivasan, A ., unpublished), we have examined the potential for use of radiation processing to improve texture, hydration and cooking quality of pulses, particularly red gram or tur dahl, as it is locally known.

VI. 1. Reduction in cooking time of irradiated legumes

The influence of 60Co-gamma radiation (0. 5 - 3. 0 Mrad) on cooking time of legumes as peas (Pisum sativum), field beans (Dolichos labab), bengal gram (Cicer arietinum) and red gram has been investigated. The extent of softening on cooking was measured in a texturometer which was devised in this laboratory (Fig. 7). In this appliance, the weight required for complete extrusion of the cooked material through a perforated disc is monitored by an electro-mechanical device, and recorded; this is then plotted against the different cooking periods of time employed. The legume is taken as cooked when the weight added to the pan is almost constant even after further cooking. A typical curve obtained with red gram, unirradiated and irradiated at 1 Mrad dose and showing exponential relationship of cooking time with the softening of the pulse, is shown in Fig. 8. It was observed that the IAEA-PL-561/10 141

FIG.7. Sketch of a texturometer. This consists of a stainléss-steel cylindrical chamber fitted with a perforated disc at the bottom end and resting on a tripod stand. A movable piston operates from the top. The test material is placed between the disc and the piston and, to extrude it out, pressure is applied on the piston by an arbor press.

texture of the irradiated samples, as judged by their shear press values (kg/ g pulse) at any given time, was softer and that there was a reduction in cooking time of about 40% to achieve the same degree of softness. Stress-strain curves of the cooked samples were also obtained using the Instron Universal Testing Machine (table model). The shape of the curves closely resembled those obtained with the texturometer. An interesting observation was with respect to the standard deviations in the texturometer readings for each interval of time (Table VII) which were much less for the irradiated pulse compared with those for the control. 142 SREENIVASAN

F IG . 8 . Measurement of softening of cooked red gram. 5-g lots of red gram were cooked in 100 m l of water. At different tim e intervals, the cooked pulse was transferred to the container of the texturometer and the drained m aterial extruded through the perforated disc of the instrument (Fig. 7) with addition of weights to the

pan. The mean recorded weights of ten experiments were plotted against cooking tim e.

TABLE VII. TEXTUROMETER MEASUREMENT OF IRRADIATED COOKED RED GRAM

Texturometer readings Standard deviation Cooking time (k g )

C o n tr o l Irra d ia ted C o n tr o l Irra d ia ted

4 1 5 . 7 6 9 . 0 4 1 . 8 3 0 . 1 6

6 1 3 . 6 7 6 . 0 6 2 . 0 5 1 . 0 4

8 1 0 . 2 4 4 . 8 8 2 .2 2 0 . 1 6

10 8.12 3 . 4 0 1 . 2 6 0 . 1 5

12 7 . 7 0 2 . 4 4 1 . 5 1 0.12

1 4 6 . 1 4 1 . 4 4 0.20 0.02

16 4 . 5 8 0 . 9 0 1.10 0.01

5-g lots of red gram were cooked in boiling water for different periods and the extent of softening

was measured in each case using the texturometer. Results are means of 10 readings with standard deviations calculated.

This reflects uniformity in. the texture of the irradiated, cooked pulse samples, a desirable attribute in the development of a good quality product. This was further confirmed in sensory evaluation tests carried out by a panel of judges. IAEA-PL-561/10 ИЗ

TEMP. °C FIG. 9a. Solubility of red gram starch. Isolated red gram starch (5 g) was dispersed in water (100 ml) and heated for 30 min in a water bath maintained at the indicated temperature with gentle stirring. The suspension

was centrifuged and solubility was determined after drying the supernatant and weighing the residual dissolved

sta rch .

TEMP. °C

FIG. 9b. Swelling power of isolated red gram starch. 5 g of starch was cooked as described in Fig. 9a. The sample was then centrifuged. The swelling capacity was measured by determining the water retention

capacity of undissolved starch, after making appropriate corrections. 144 SREENIVASAN

VI. 2. Hydration properties of irradiated red gram

The rate of hydration of unirradiated and irradiated (1 Mrad) samples on soaking (during 3 h) and on cooking (during 30 min) in water were compared. It was observed that hydration rate of the irradiated sample was higher up to 1 hour of soaking after which there were no significant differences. Water absorption capacity of the irradiated legume was also higher during 5 - 10 min of cooking after which this was about 10% less. Prolonged cooking, for attaining desirable softness, resulted in rupture of unirradiated samples while the irradiated samples remained intact. This correlated with the decreased swelling power of the starch isolated from irradiated red gram.

VI. 3. Changes in physical and chemical characteristics of starch from red g ra m

Starch was isolated from control and irradiated red gram and its solubility and swelling power were determined at intervals of 5° over a temperature range from 70 to 95°C. It can be seen (Fig. 9a) that solubility of irradiated starch increased with concomitant decrease in swelling (Fig. 9b). The increase in solubility may be attributed to the observed greater breakdown of irradiated starch to more soluble oligosaccharides due to irradiation. The degree of swelling directly influences the course of gelatinization viscosity which was further ascertained with the Brabander amylogram (Fig. 10), using both the red gram, flour and its isolated starch.

IO n O N 4 , (OCBONVU)(DO

- FIG.10. Amylograms of irradiated red gram. To determine the gelatinization viscosity, water-flour or water-

starch slurry was heated as described for Table III, The peak viscosity temperatures were recorded in each case. IAEA-PL-561/10 145

The maximum amylogram units were much lower in irradiated samples than in the co n tro ls; amylogram peaks were obtained at 95°C and 88°C, respectively. Increased additional maltodextrins may thus decrease the v is c o s it y . Non-reducing sugars were separated by paper chromatography using n-butanol:acetic acid:water (4:1:5) as a developing solvent system. Sugars detected were sucrose, raffinose, stachyose and verbascose. Preliminary experiments showed that their quantities were- considerably less in the cooked, irradiated sample. This observation is of practical interest since beans are known to possess flatulence factors causing intestinal disturbances and gas formation in humans after their ingestion [ 34]. These factors have limited the acceptability and consumption of beans, in spite of their high protein content. It has been shown [ 35] that the oligosaccharides - sucrose, raffinose and stachyose - are associated with the gas-producing factor when

TABLE VIII. EFFECT OF IRRADIATION ON TOTAL AND FREE AMINO ACID COMPOSITION OF RED GRAM

Total amino acids, g/16 g N Free amino acids (m g/g N)

A m in o a c id Irrad iated Irrad iated C o n tr o l C o n tro l (1 M rad) ( 1 M ra d )

Aspartic acid 7 . 7 3 7 . 7 6 2 . 5 2 3 . 1

T h re o n in e . 7 3 . 3 6 3 . 3 4 1 3 . 9 6 1 4 . 7 . У "■ Serin e 4 . 3 6 4 . 3 1 -

Glutamic acid 2 3 . 7 1 2 3 . 5 2 5 3 . 0 3 5 8 . 9 5

P ro lin e 3 . 5 4 3 . 4 1 - -

G ly c in e 3 . 0 2 2 . 9 6 1 0 . 8 9 1 1 .3 2

A la n in e 3 . 8 8 ■ 3 . 3 3 1 0 .2 7 1 0 . 0 4

C y s te in e - - T r a c e s T r a c e s

V a lin e 3 . 5 5 3 . 1 7 2 . 1 6 2 . 5 8

M e th io n in e 0 . 7 9 ■ 0 . 8 5 4 . 8 9 4 . 9 3

Iso-leucine 3 . 1 4 3 . 1 4 1 0 0 . 9 0 112.12

L e u c in e 6 . 0 3 5 . 7 7 11.88 1 3 . 3 6

T y r o s in e 1 . 4 5 • 1.68 T r a c e s 1.02

Phenyl alanine 8 . 7 9 8 . 2 8 T r a c e s 1.11

Lysine 6 . 2 9 6.22 2 . 2 6 3 . 4 9

H istid in e 3 . 2 2 2 . 8 5 4 . 5 1 3 . 7 8

A r g in in e 4 . 8 7 4 . 9 3 2 3 . 3 1 20.20

T ry p to p h a n 1 , 2 5 1.22 - -

100 mg of ted gram flour ( = 20 mg protein) was hydrolysed with 6N HC1 at 110°C for 24 h. Excess HC1 was evaporated under vacuum at 50°C. The residue was taken into 20 m l of 0.2M citrate buffer

(p H 5 . 6) and aliquots analysed for total amino acids using á Beckman automatic amino acid analyser. Corrections were made in the values for serine and threonine to account for losses during acid hydrolysis. For free amino acid estimation, 7 0 % ethanol extract of red gram flour was used. 146 SREENIVASAN incubated in thioglycollate media with anaerobic bacteria of the intestinal tract of dogs; this property has been observed with soyabean, cotton seed and peanuts. Thus, the acceleration in the degradation of these oligosaccha­ rides to monosaccharides such as glucose and fructose, which are easily digested and absorbed in the gastro-intestinal tract, may be beneficial in promoting the use of legumes. Further work is in progress.

VI. 4. Effect of irradiation on red gram proteins

Total proteins (2 2. 97%) in red gram were not changed appreciably by radiation treatment. There were also no significant changes in the total amino acid profiles of unirradiated or irradiated (1 Mrad) red gram (Table VIII). However, free (non-protein) amino acids, estimated in alcohol extracts, increased by about 15% at 1 Mrad. The radiosensitivity of red gram proteins was studied by determining the changes in their distribution pattern according to molecular weights by gel filtration. The proteins were extracted with water and fractionated on a Sephadex G-200 column. The elution curve of the control sample showed two main peaks with 46% and 54% proteins, respectively, representing the profile of protein distribution according to molecular weights. In the irradiated (1 Mrad) sample, about 29% more protein value was obtained in the second peak, pointing to the fragmentation of red gram proteins to lower molecular weight protein entities.

DIGESTION TIME (h)

FIG. 11. In-vitro digestibility of red gram proteins. Red gram flour (5 g proteins) was digested with pepsin

(5 mg Sigma preparation) followed by trypsin (5 mg Sigma product) for 24 h each at 37°C. Alpha-amino nitrogen liberated was measured at indicated time intervals and expressed in terms of leucine equivalent.

The lower set of curves are for peptic digests and the upper for peptic followed by tryptic digests. IAEA-PL-561/10 147

When the in-vitro enzymic digestibility of red gram proteins was studied, it was observed that the susceptibility of red gram proteins to successive 24-hour action of pepsin and trypsin was increased due to irradiation. Comparatively more amino nitrogen was liberated from irradiated samples (Fig. 11). The differences due to irradiation are much more pronounced in the peptic digests than in those where peptic action is followed by trypsin digestion. The enhanced proteolytic digestion may be attributed either to the partial destruction of trypsin inhibitor or to the degradation of proteins present in the legumes, thus making them more susceptible to enzyme action. It was observed, however, that the activity of trypsin inhibitor was comparable in control and irradiated red gram samples. This suggests that the observed degradation of proteins in the irradiated pulse is responsible for the increased - enzymic digestibility, largely by pepsin rather than by trypsin, the presence of the trypsin inhibitor masking the trypsin action.

TABLE IX. RETENTION OF В VITAMINS IN RED GRAM ON COOKING

°jo r e te n tio n

R ib o fla v in T h ia m in e N ia c in

Irradiated (1 Mrad, uncooked) 9 8 . 7 9 2 . 7 9 3 . 3

Control (cooked) 88 7 6 . 1 8 3 . 3

Irradiated (cooked) 9 5 8 2 . 7 8 9 . 3

Retention of vitamins was calculated on 100% basis in control unirradiated sample which analysed

to (Mg/g air-dry basis) 6.2 riboflavin; 4 .6 thiamine; and 46 niacin.

VI. 5. Effect of irradiation in В vitamins in red gram

Results on the effects of gamma irradiation and cooking on the retention of some of the water-soluble vitamins are given in Table IX. In the uncooked, irradiated (1 Mrad) sample, destruction of riboflavin was negligible, whereas thiamine and niacin showed 7% losses. However, the vitamins were retained better in the sample irradiated and then cooked than in the corresponding controls. As prolonged heating is known to destroy В vitamins, the reduction in cooking time presumably accounts for the better retention of the vitamins in the radiation-processed, cooked samples.

VII. QUALITY-IMPROVE MENT OF FRUITS AND VEGETABLES BY GAMMA IRRADIATION

The effects of gamma irradiation on post-harvest physiology of fruits and vegetables have been extensively studied from the points of view of delay in ripening, control of rot, disinfestation to meet quarantine needs in international trade and sprout inhibition during storage (for a recent review, see Ref. [36]; see also Refs [ 3, 7, 13 and 16]). 148 SREENIVASAN

TABLE X. EFFECT OF GAMMA IRRADIATION ON FRUIT TEXTURE

’ Instron' reading Fruit T r e a tm e n t (force in kg)

M a n g o C o n tr o l 4 . 4 ± 0 . 3

(A lp h o n s o ) Irradiated

( 2 5 krad) 6 . 0 ± 0 . 3

Skin-coated ( 6%

myvacet) and 7 . 1 ± 0 . 5 irrad iated

( 2 5 krad)

B anana C o n tr o l 3 . 0 ± 0 . 4 (D w a r f Irrad iated c a v e n d is h ) ( 3 5 krad) 10.0 ¿ 0 .8

An Instron Universal Testing machine (table model) was used with a probe of 4 m m in

diameter for pressure testing. The values are the force required to penetrate fruit with

skin to a depth of 5 mm and are averages of five independent determinations. The pressure test was carried out after 13 and 15 days of storage at ambient temperature

(25 - 30”C) in bananas and mangoes, respectively.

VII. 1. Textural changes in mangoes and bananas

Experiments carried out at Trombay have shown that low-dose irradiation of tropical fruits like bananas and mangoes could extend their shelf-life by- delaying ripening and senescence [37-39]. Though irradiation, at the levels employed for delaying the ripening of bananas (2 5-40 krad, depending on the variety) and mangoes (2 5 krad), results in an initial slight reduction in fruit texture (15 - 20%), the subsequent delayed ripening brings about slower changes in the rate of tissue softening which make them more resistant to normal stresses involved during transportation and handling. The data given in Table X show that irradiated fruits remain hard for a longer time, more force being required to penetrate them. Shipment studies conducted within India by rail and to distant countries by air have shown that mangoes, either irradiated or irradiated and skin-coated, could withstand transporta­ tion better than unirradiated fruits [ 7, 40].

VII. 2. Colour changes in mango

Apart from delaying the ripening process, gamma irradiation at low dose levels has been found to increase the pigment content in the skin tissues of mangoes (Thomas, P ., unpublished). Irradiated Totapuri variety mangoes (15 - 75 krad) develop a deep pink colouration around the shoulder regions due to formation of anthocyanins, the maximum pigmentation being at 50 krad (Table XI). Thus, irradiation can bring about an improvement in the external appearance of these mangoes (Fig. 12). Similar radiation- induced formation of anthocyanin pigments has been reported in peaches [ 4 1 ,4 2 ] . IAEA-PL- 561/10 149

TABLE XI. EFFECT OF IRRADIATION ON ANTHOCYANIN FORMATION ,IN T o ta p u ri M ANGOES

Irradiation dose Optical density of pigment

(k rad ) extract at 520 nm

C o n tro l 0 . 0 4 -

15 0 . 4 6

2 5 0 . 4 3

5 0 0 . 5 0

7 5 0 . 2 6

Anthocyanin pigments were extracted from mangoes exposed to the different

doses of gamma rays and stored for 10 days at 20eC. Five grams of skin

tissue from the pigmented area was blended with 100 m l of acidified methanol (pH 1.5) in an Omnimixer for 5 min and the clear supernatant,

after centrifuging, was taken for optical density determination at 520 nm

in a Beckman DB spectrophotometer.

FIG .12. Effect of gamma irradiation on anthocyanin formation in Totapuri mangoes. The photograph was

taken on the 10th day after irradiation. Maximum pigmentation was noticed at 50 krad. 150 SREENIVASAN J

VII. 3. Lipid composition and flavour changes in irradiated mango

A correlation between the aroma and flavour characteristics of the mango (variety Alphonso) and the fatty acid composition of the pulp lipid has been reported from this laboratory [43, 44]. When the ratio of palmitic acid to palmitoleic acid was greater than one, mangoes had a mild aroma and flavour, the reverse being the case when this index was less than one. A study was made (Bandyopadhyay, C. and Gholap, A. S ., unpublished) to ascertain the effects of irradiation on aroma and flavour characteristics of mango as reflected in the fatty acid composition of the pulp. For this, freshly picked, uniformly mature, unripe mangoes, variety Alphonso, were divided into two lots, one lot being irradiated (2 5 krad) immediately. Both lots were kept at ambient temperature (25 - 30°C) for ripening under normal conditions. Samples were taken at definite intervals, peeled and the lipid extracted repeatedly from the pulp with sufficient volume of peroxide-free diethyl ether in a Waring blender. The lipid, obtained after removal of ether in a flash evaporator, was analysed for its glyceride and phospholipid contents as well as for fatty acid composition by gas liquid chromatography [43] Results showed that ripening of both control and irradiated mangoes was accompanied by changes in glyceride content as well as in fatty acid com posi­ tion of the pulp. However, the rate of change was relatively slower in the irradiated samples than in the unirradiated ones. Thus, the glyceride content (% weight of pulp lipid) of 6- and 12-day stored, unirradiated mangoes were 56 and 71, respectively, compared with the corresponding values of 48 and 64 for the irradiated fruits. This could be attributed to the delay in ripening in the latter case. Also, there was a shift in the ratio of palmitic acid to palmitoleic acid as a result of irradiation. For example, in 12-day stored (table-ripe), unirradiated samples, the index was 0. 89 while, in the irradiated samples, this value was obtained after 15 days of storage. Organoleptic evaluation of irradiated samples after 15 days of storage (table-ripe) revealed a slight reduction in aroma and flavour in comparison with 12-day stored, unirradiated samples.

VII. 4. Effect of irradiation on carrot flavour

Experiments carried out in this laboratory (Roy, A. N. and Bandyopadhyay, C., unpublished) showed that an optimum dose of 250 krad inhibited sprouting in carrots. Repeated organoleptic tests on freshly irradiated carrots showed that there was considerable increase in flavour profile, particularly with respect to odour in the irradiated sample compared with the unirradiated one. In further experiments, the odorous compounds trapped in the head space of a simple glass apparatus [45] containing freshly irradiated carrots (250 krad) were analysed by gas chromatographic-mass spectrometric methods and compared with those of unirradiated samples (Bandyopadhyay, C ., unpublished work carried out at the U. S. Army Natick Laboratories, Natick, M ass., USA). Some of the odorous components identified in the head-space gas were ethyl methyl ketone, secondary butyl alcohol, diethyl ether, benzene, ethanol, acetaldehyde, n-hexane, acetone, methyl alcohol and toluene. There was a quantitative increase of all these components in the irradiated sample compared with the unirradiated one. Of significance also was a considerable increase in CO2 content of irradiated carrots. IAEA-PL-561/10 151

VII. 5. Effect of irradiation on quality improvement in potatoes

Apart from its effect on sprout inhibition in potatoes, gamma irradiation has been found to inhibit the synthesis of chlorophyll and solanin in tubers stored at sub-room temperatures [ 46]. Chlorophyll formation leads to higher peeling losses, while the alkaloid solanin is a potential toxin. It has also been observed that irradiated potatoes, stored at ambient or sub-room temperatures (10 - 15°C), give better quality chips (fried) or fresh fries, as compared to conventionally cold-stored (3 - 4°C) tubers. This is attribu­ table to the decreased levels of reducing sugars formed in irradiated tubers under the respective storage conditions.

VII. 6. Control of post-harvest decay in fruits and vegetables

Recent studies have shown that the radiation dose required for control of fungi causing post-harvest rot in strawberry [47], citrus fruits [ 48], pears [49] and stone fruits [50] could be brought down by prior sensitization to moist heat treatment. While these observations relate to disease control in fruits of temperate zones, relatively little work has been reported in the case of tropical and sub-tropical fruits. Studies at Trombay have shown that fungal pathogens causing spoilage of several tropical and sub-tropical fruits could be controlled to some extent by a combination of mild heat and low-dose irradiation [30]. Hot water dip (50°C for 5 min) followed by 150-krad irradiation extended the shelf-life of fresh figs by 3 - 4 days at ambient temperature (28 - 32°C) and 8-10 days at 15°C by delaying the incidence of. rot caused by Rhizoppus sp. and Aspergillus sp. Regardless of the sequence of treatments, combination of similar heat treatment and 100 krad extended the shelf-life of grapes both at ambient and refrigerated storage temperatures. In mangoes, moist heat (50°C, 5 min) followed by 50 krad was effective in controlling stem-end rot caused by Botryodiplodia theobromae, whereas in bananas irradiated (2 5 - 35 krad) for delayed ripening, similar moist-heat (50°C, 5 min) treatment reduced the incidence of stem-end rot caused by Colletotrichum gloesporioides. Though gamma irradiation at 10 krad completely inhibits sprouting of potatoes [ 3, 46], spoilage due to microbial rot is a serious problem when these tubers are stored under high ambient temperatures (2 5 - 35°C) prevailing during most part of the year in tropical regions like India. Recent preliminary studies in our laboratories (Thomas, P. and Padwal-Desai, S.R'., unpublished) have indicated that hot water treatment (55°C, 10 min) of potatoes could be a possible means to reduce the rottage during prolonged storage under such conditions.

VII. 7. Thermal cum irradiation processing of fruits and vegetables

Thermal sterilization of fruits and vegetables often results in undesirable changes in quality attributes, especially texture and flavour. Combination of heat treatment and irradiation have been successfully employed for sterilization of mangoes, guavas, sapotas and apples where, usually, canned products with better texture, flavour, and retention of nutritive value, could be obtained with treatment at 70°C for 10 min and 400 krad [51]. Likewise, excellent quality canned peas with better retention of chlorophyll (Table XII) and texture (Fig. 13) were obtained with the combined use of irradiation 152

TABLE XII. PERCENTAGE CONVERSION OF. CHLOROPHYLL TO PHEOPHYTIN IN PEAS SUBJECTED TO VARIOUS TREATMENTS ON STORAGE AT ROOM TEMPERATURE

N o . of days of storage T r e a t m e n t B la n ch e d C u t -o u t S te r iliz e d

N o . in m e d i u m by 5 20 4 0 6 0 7 5 9 0 1 0 5 120 1 3 5 1 4 5

1 A l k a l i B u ffer Combination 2 5 . 2 4 5 . 3 5 2 . 2 6 4 . 7 6 9 . 8 7 4 . 0 8 0 . 4 8 5 . 6 9 2 . 4 ' 100

2 A l k a l i B uffer H e a t 4 5 . 3 68.0 8 0 . 9 9 0 , 7 100 - - - - - REENI N A S A IV N E E SR

3 A l k a l i B rin e Combination 2 6 .3 6 4 . 6 7 5 . 5 8 0 . 9 100 - - - - -

4 A l k a l i B rine H e a t 6 7 .3 8 2 . 3 9 0 . 7 100 ------

5 W a ter B uffer Combination 3 7 . 6 5 0 . 3 7 5 . 5 100 - -, - - - -

6 W a te r Buffer H e a t 5 6 .7 - ‘ 8 1 . 6 100 ------

7 W a te r Brine Combination 5 4 . 0 7 1 . 4 8 0 . 2 100 ------

8 W a te r Brine H e a t 7 5 . 5 100 ------

Heat process: 121вС for 35 min. Combination process: 1 Mrad + 5 min moist heat treatment at 100°C. Alkali blanching: Green peas were dipped in 27o sodium carbonate solution for 30 min followed by blanching in 0.0005M Ca(OH )2 at 100°C for 5 min. Water blanching: 100°C for 5 min. Buffet; Q.2M ciuate-phosphate buffet (pH 7.5} when filled. B r in e : 2°¡o N a Cl + 3% sugar. Chlorophyll was estimated by standard analytical methods. IAEA-PL-561/10 153 20

I 6 -

I 2 - o> z H X

12 3 4 5 6 7 8 TREATMENT NO-

FIG. 13. Texture of canned peas subjected to different treatments. Texture was evaluated on the 135th day of storage by determining the force (in kg) required to extrude com pletely 10 g of peas through the specially designed extrusion apparatus (Fig. 7). Details of treatments are given in Table XII.

TABLE XIII. ASCORBIC ACID RETENTION ON STORAGE OF PEAS ( m g /100 g)

No. of days of storage T r e a t­ m e n t 2 4 1 5 5 7 5 9 0

1 11.6 8.0 7 . 0 6 . 5 5 . 9

2 1 3 . 0 8.6 7 . 8 7 . 2 6 .8

3 10.1 5 . 4 5 . 1 4 . 9 4 . 6

4 11.2 6 .8 7 . 0 6 .6 6 .1

5 11.2 8.8 8 . 4 8.2 7 . 8 CO 6 1 1 . 9 10.6 0 0 8 . 4 7 . 9

7 1 1 . 7 ' 6.1 6 .1 6.0 5 . 6

8 1 2 . 3 7 . 2 7 . 0 6 .8 6 .1

Treatments 1-8 as in Table XII. Ascorbic acid was estimated by visual titration against 2 : 6 dichlorophenol indophenol. 154 SREENIVASAN

TABLE XIV. NIACIN AND RIBOFLAVIN RETENTION IN PEAS (M g/100 g)

T r e a tm e n ts

V ita m in 1 2 3 4 5 6 7 8

N ia c in 6 0 2 7 8 7 5 8 1 5 5 6 6 2 5 7 7 5 6 8 1 7 5 0

R ib o fla v in 85 103 73 8 5 100 98 100 103

Niacin content of fresh peas,- 1 875 /ig/100 g. Riboflavin content of fresh peas: 210 /ig/100 g. Treatments 1-8 as in Table XII. Niacin and riboflavin were estimated by standard microbiological

methods. The values are for peas stored for 1 month after subjecting them to the respective treatments.

(1 Mrad) and heat processing (100°C, 5 min) (Shrikhande, A. J. , unpublished). The retention of ascorbic acid (Table XIII) and of niacin and riboflavin (Table XIV) in combination-treated peas was comparable to that of the heat- sterilized product.

ACKNOWLEDGEMENTS

Thanks are due to my several colleagues to whose unpublished observa­ tions I have made references in this review. I am especially grateful to Drs. Urmila K. Vakil and Paul Thomas for much valuable assistance in the preparation of this paper.

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(1966) 870. [39] THOMAS, P.. DHARKAR, S.D ., SREENIVASAN. A ., J. Food Sci. 36 (1971) 243. [40] FARKAS, J.. DHARKAR, S.D .. SREENIVASAN, A ., J. Acta Alimentaria 1 (3-4) (1972) 401.

[41] MAXIE, F .C ., JOHNSON, C .F ., BOYD, C ., RAE, H. L ., SOMMER, N .F., Proc. Am. Soc. Hort. Sci. 89 (1966) 91.

[42] AHMED, E .M ., DENNISSON, R .A ., MERKLEY, M .S ., Annual Report - April 1968 to June 1969, Dept, of Food S ci., Florida University, Gainesville, Florida, USA, (1969).

[43] BANDYOPADHYAY, C ., GHOLAP, A. S ., J. Agrie. Food Chem. (in press). [44] BANDYOPADHYAY, C ., GHOLAP, A. S ., J. Sci. Food Agrie, (communicated).

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[47] SOMMER, N .F ., FORTLAGE, R.J., BUCKLEY, P .M ., MITCHELL, E .G ., Phytopathol. 57 (1967) 832.

[48] BARKAI-GOLAN, R ., KAHAN, R. S ., PADOVA, R., Phytopathol. 59 (1969) 922.

[49] BEN-ARIE, R ., BARKAI-GOLAN, R., Int. J. Appl. Radiat. Isot. 20 (1969) 657.

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IAEA-PL-561/11

EFFECTS OF IRRADIATION ON THE TECHNOLOGICAL AND HYGIENIC QUALITIES OF SEVERAL FOOD PRODUCTS

I. KISS, J. FARKAS, S. FERENC ZI*, B. KALM AN, J. BECZNER Central Food Research Institute, Budapest, Hungary

Abstract

EFFECTS OF IRRADIATION ON THE TECHNOLOGICAL AND HYGIENIC QUALITIES OF SEVERAL FOOD

PRODUCTS.

The juice yield of grapes may be increased by irradiation. This is achieved by changing the technological properties of grapes and increasing the permeability of the cell walls. Thus, the energy requirement of pressing may be reduced or, by using the same energy, more juice may be obtained. The chem ical analysis and organoleptic evaluation of the wine fermented from radiation-treated grapes has shown that the application of doses above 0 .8 Mrad is not desirable. According to results achieved by interpolation it was found that a 10- 12*70 increase in juice yield may be obtained by radiation doses of 0 .4 -0 .5 Mrad, which are considered optimal.

In experiments where dried string beans, carrots, celery and parsley root were irradiated in an electron accelerator or in a 60Co gam ma radiation source it was found that the consistency obtained by cooking untreated samples for 10 min may be achieved by cooking for only 2 min if the samples have been previously irradiated with 1 to 3 Mrad.

On the basis of the results of studies on weight increase during rehydration and on change of consistency it seems possible that the reduced cooking time requirement is due not so much to increased water absorption but rather to the effect of irradiation on structural polysaccharides, thus determining the consistency of vegetables. Radiation doses improving consistency did not cause undesirable changes of taste and colour in dried root vegetables and string beans. When onions were irradiated the colour of the product darkened with increasing radiation dose. In relation to ground paprika the protective effect of irradiation was manifested mostly under unfavourable storage conditions, whereas with adequate packaging material radiation reduces the cell count which remains at a low level. The seasonings used in the preparation of industrial m eat products and certain canned goods are mostly highly contaminated and contain also a high proportion of heat-resistant cells (bacterial spores). The cell count of seasonings and additives can be very efficiently reduced with ionizing radiations. A radiation dose of about 0 .3 Mrad seems sufficient to achieve pasteurization and after-treatmentwith 1 .5 Mrad gives a practically sterile product. The spoilage percentage of pork liver paste prepared with irradiated additive mixture was not quite one-seventh of that prepared with untreated additives even when given a heat treatment of F0 = 1 . 4 7 . sterilization equivalent. From this one can conclude that by using radappertized additive mixture a heat treatment of Fq = 2 .5 equivalent would be sufficient to achieve sterility even with respect to Clostridium botulinum. This heat treatment is just above one-sixth of the treatment applied in industry. Such a substantial reduction in the heat requirement without changing the sanitary safety would result in a substantial improvement in the product, in a saving of energy and in an increase in production capacity. Experiences with combination treatment have shown that not quite one-tenth of the present industrial heat treatment was sufficient to achieve m icrobiological stability in samples irradiated with 500 krad.

Meat products can safely be manufactured with a Fq = 2 .5 heat treatment-which is about one-fourth of the treatment applied at present,when irradiated with 500 krad. The quality characteristics of the products are substantially improved and the energy requirement reduced.

* National Research Institute of Enology and Viticulture, Budapest, Hungary.

157 158 KISS et al.

INTRODUCTION

The quality grading of agricultural produce, foods and industrial food products presents a very intricate task. Attention has to be paid not only to their composition and sensory quality, but to their technological characteristics as well. Thus, a treatment which improves one of the important characteristics of a food item may ensure upgrading of its qu ality. Such an improvement is achieved by increasing the yield of a certain amount of raw material or by the maintenance of the nutritive value, taste or colour, etc. of the food product by the improvement of the processing technology. The same applies to the m icrobiological quality, which is the condition of use in various foods and ingredients. In the course of these experiments the pressing quality of grapes, as affected by radiation treatment, was investigated. The improvement of the pressing quality of grapes and other fruit, or the increase of the permeability of the cell walls may be of importance from the point of view of technology. The quality of dehydrated vegetables as a function of rehydration and radiation dose was also studied. The effect of radiation treatment on the stability of the colour and m icrobiological quality of ground paprika under various storage conditions was investigated. A study was made of the effect of radiation-treated ingredients (seasoning) on the quality of canned products prepared with these ingredients. A further subject of investigation was the quality characteristics of preserved products as affected by various combinations of heat and radiation treatment.

MATERIALS AND METHODS

1. Increase of grape juice yield

The juice yield may be increased by improving the pressing quality of grapes. The pressing quality depends, among others, on the quality and quantity of the interlamellar pectin content which seriously affects the textural consistency. Ionizing radiation facilitates the breaking down of pectin, particularly if it is present in a "pure" state. Radiation sensitivity is however reduced by some materials of protective effect. Apart from breaking down the pectin, ionizing radiation probably furthers the leakage of juice, helps the pressing and increases the juice yield by the destruction of the tissues.

1.1. Grape varieties studied

Place of cultivation V a rie ty

E g e r "Egri csillagok" "Olaszrizling"

Miklostelep "Kocsis Irma" "Kovidinka" "Hárslevelü" I A E A -PL" 5 6 1 / 1 1 159

100 kg of grapes of similar quality were picked from each variety and placed in 10-kg crates. 20 kg of grapes were irradiated at each dose level and from these three 5-kg samples were used for pressing (three replicates).

1.2. Radiation treatment

The freshly picked grapes were transported to the Institute and immediately irradiated so they were irradiated between 3 and 5 hours after picking. A 60Co radiation source was used of 60 kCi nominal activity at 1 Mrad h-1 dose-rate. The doses applied were: 0; 0.05; 0.2; 0.8 and 1.6 M rad.

1.3. Methods of investigating the effect of irradiation

1.3.1. Investigation of the juice yield

The 5-kg samples were separated from the stem and pressed on small uniform presses for the same length of time. In each test the weight of the stems, the grapes and the cake, the volume of the juice and its specific gravity were established. The yield and percentage yield (litres per 100 kg grapes) were calculated and the data obtained for the treated samples compared with that of the control.

1.3.2. Observation of the process of fermentation and chemical and sensory analysis of the wine obtained

The juices obtained from the three replicates were combined and 4 litres of the mixture were fermented. The fermentation process was kept under observation in order to establish the effect of radiation treatment on the commencement and course of fermentation, to see whether it was retarded or inhibited. Chemical analysis involved determining the alcohol content, total extract, invert sugar, sugar-free extract, ash, ash alkalinity, titratable acid content, pH value, free and total SO2, -total colloid content, colour value and polyphenol content. Simultaneously, sensory evaluation was also carried out.

2. Investigation of the cooking time of dried vegetables

Recently, preservation methods involving dehydration underwent important developments and the demand for dehydrated vegetables has grown, too. In addition to the increase in output the quality requirements have also developed. Specifications and quality requirements of the users require the application of modern technologies. Low microbial counts, absence of insects, good rehydration capacity and short cooking time are the basic requirements. Since the available literature on radiation treatment of dehydrated products is only of a qualitative character, it seemed desirable to investigate the possibility of the use of radiation treatment for improving the quality of Hungarian dehydrated foods. 160 KISS et al.

The aims of the study were:

(a) To determine the water absorption capacity of dried vegetables as a function of radiation dose and rehydration time; (b) To make an instrumental investigation of the consistency of irradiated and untreated dried vegetable during cooking or soaking at 30°C as a function of rehydration time and dos e-rate; (c) To make a sensory evaluation of the rehydrated vegetables; (d) To study the m icrobiological effect of irradiation by determining the total viable cells count and the coli count.

2. 1. Dried vegetables used in the experiments

The following industrially produced dried vegetables were used:

Moisture content, %

string beans, halved 8.0 carrot strips 9. 2 cellery flakes 8.0 parsley root 8.0 sliced onions 16. 1

40-g samples of the dried vegetables were closed in cellothene1 pouches (70 Mg thick) and the pouches were irradiated and stored at room temperature.

2. 2. Radiation sources

The radiation sources used were:

(a) The 60Co radiation source of 80 000 Ci nominal activity of the MTA (Hungarian Academy of Sciences) Isotope Institute. The dose-rate in the position of the samples was 0. 5 ± 0. 1 Mrad h-1. (b) The Van de Graaff system electron accelerator of 2-MeV maximum energy of the Research Institute for Plastics, Budapest. During irradiation the voltage was 1. 7 MeV, while the dose-rate in the position of the samples was 10.8 Mrad h-1.

The thickness of the pouches containing dried vegetables irradiated in the electron accelerator did not exceed 0. 5 cm. Irradiation was carried out at room temperature.

2.3. Microbiological studies

The viable cell count of the aerobic mesophiles on the dried products was determined by the plating-dilution assay technique in which the cultures were grown on universal nutrient agar at 30°C.

1 2 0 цт cellophane (on the outside) laminated to low-density polyethylene of 50 (im (on the inside). IAEA-PL-561/11 161

2. 4. Rehydration capacity and cooking time requirement of the dried p rod u cts

Rehydration during cooking. On the basis of several preliminary experiments the rehydration of samples during cooking was determined by ' adding 5 g of dried vegetables to 100 ml of boiling water or a 1. 5% salt solution and boiling the vegetables for a predetermined time during which the evaporated water was replaced. At the end of the boiling period the vegetable and broth were poured over a strainer and the vegetable weighed after a desiccation period of 15 min. Instrumental testing of the consistency of rehydrated samples. The consistency of the cooked samples was measured at room temperature with a "texturometer", manufactured by Zenken Co. Ltd., Japan. 2-g samples of the rehydrated vegetables, prepared for weighing, were placed on the grooved measuring pan of 30 mm diameter, and evenly distributed. The sample was then pressed with the plastic measuring head of 23 mm diameter. The arising forces were registered on the instrument. The firmness of the sample was expressed in the scale divisions of maximum deflection. Each cooking series was carried out in two replicates and the consistency was determined in two parallel measurements. Rehydration at 30°C. In some cases the rehydrating capacity of dried carrots was studied by soaking them in water or 1. 5% salt solution at 30°C. The consistency was measured after 0.5; 1.0 and 2.0 hours, respectively.

2. 5. Sensory evaluation of the cooked samples

For the sensory evaluation the samples were cooked in a 1.5% salt solution. 5-g samples were cooked in 100 ml of the salt solution and evaluated by Kramer's ranking system. The panel consisted of 10 members. The colour, taste and consistency of the coded samples was scored according to a 7-score scale. The scale for taste corresponded to the following terms:

excellent 7 scores very good 6 scores good 5 scores medium (not bad, not good) 4 scores slightly objectionable 3 scores highly objectionable 2 scores unpalatable 1 score

On the scale for consistency, 7 scores corresponded to completely pulpy, 1 score to an inedible stringy consistency and the scores in between stood for the consistency between these two poles. On the scale for colour of string beans, 7 scores stood for the most intense green colour and 1 score for complete discolouration (greyish, brownish). Samples were then ranked and the rank sums calculated according to Kramer (1960).

3. Ground paprika

Seasonings used in the canning and meat industries have a very high viable cell count by which they endanger the safety of canned foods and 162 KISS et al.

various unpreserved meat products. Apart from its microbicidal effect, radiation treatment in the dose range of radurization ensures the stability of colour in ground paprika, even when stored under unfavourable conditions. Thus, the colour and cell count in ground paprika as a function of storage time was studied under various conditions of storage and packaging.

3.1. Irradiation and storage of ground paprika

Noble sweet ground paprika of 9.2% moisture content was used in the experiments. The packaging methods applied were as follows:

(a) A conventional paper bag for 50 g of paprika; (b) Loosely filled in aluminium tubes of 70 g capacity at a concentration of 0.5 g/cm 3; (c) Compactly filled in aluminium tubes of 70 g ca p a city at a concentration of 0.7 g/cm 3; (d) Filled in aluminium tubes under N2 flow; (e) Filled in polyester pouches (40 g) ; (f) Filled in polyethylene pouches (40 g).

Irradiation was carried out in an RH-gamma-3 0 60Co radiation source of 20 000 Ci nomina] activity at a dose-rate of 1 Mrad h”1. The samples were treated with 0.5 Mrad. The storage conditions applied were:

Temperature, °C ER H , %

0 . . . 5 > 95 1 0 - 1 2 80 -9 0 20 - 25 40 - 50 30 40 - 50

The aim of storage in high humidity was to study the stability of the irradiated ground paprika under these conditions.

3.2. Quality tests

The total cell count of viable aerobic mesophiles was established by the plating technique on universal nutrient agar. 1 g of ground paprika was suspended in 9 ml of sterile water containing 0.02% Tween 80 and tenfold dilutions of the suspension were applied. Moulds were counted on malt slant agar by the method of the most probable viable cell count on the basis of Hoskins' table (1934). The moisture content was determined in a drying oven at 95 ± 2°C by drying to constant weight. The total pigment content was determined according to Benedek (19 58). Objective colour testing of the ground paprika was performed with Lovibond Flexible Optic Tintometer, type AF 751 from Tintometer Ltd., Salisbury, UK. Sensory testing of the colour was started on the 120th day of storage. All the samples stored at the same temperature (generally 11 samples) were tested at the same time according to the following scoring.scale: IAEA-PL-561/11 163

5 scores brightest flaming red colour typical of paprika 4 scores beautiful deep red colour typical of paprika 3 scores slight darkening (browning) or fading of tint 2 scores intense browning or fading of tint 1 score completely faded or mouldy paprika.

The panel of judges always consisted of the same 9 members. Scores were evaluated according to the ranking method of Kramer (1960).

4. Reduction of the heat-treatment requirement of canned goods by irradiation of additives or combination of heat and radiation treatment

Preservation of meat products in cans may be achieved only at the expense of great losses in nutritive value, over-cooking or other ways of quality deterioration owing to the low heat transfer capacity of meat or to highly contaminated additives. Similar results were obtained with canned green peas. A combination of heat and radiation treatment appears to be a promising method of reducing heat requirement or of improving the quality of the products and, at the same time, ensures high storage stability and a product unobjectionable from the hygienic aspect.

4. 1. Cell-count reduction in food additives

4.1.1. Irradiation of the samples

Pork liver paste was prepared with an additive consisting of ground rice, salt, French seasoning and powdered onion. Irradiation was performed in the' LMB-gamma-1M self-shielding 137Cs source of 3000 Ci nominal activity at a dose-rate of 280 krad h~l. The additive mixture was packed in cellothene pouches and irradiated with 1.5 Mrad. The pouches were stored at room temperature (20-22°C).

4.1.2. Microbiological tests

Determination of viable mesophile aerobic cell count. In some of the experiments the method of the most probable cell count was applied for the determination of surviving microorganisms, making three parallel inocu­ lations at each dilution level. After an incubation period of 48 h at 30°C the most probable cell counts were determined using Hoskins' tables (Hoskins, 1934). Determination of anaerobic viable cell count. The most probable cell count of anaerobic bacteria and their sulphite reducing capacity was determined. Determination of heat-resistant bacterial spores. The microorganisms surviving a 10-min heat treatment at 80°C were considered heat resistant. Of the microorganisms forming the microbial flora of the seasonings, only the bacterial spores have a high probability to survive this heat treatment.

4. 1. 3. Canning of pork liver paste prepared with untreated and irradiated additive mixture

The pork liver paste was prepared according to the formula used in the Hungarian canning industry. Half of the paste mixture was untreated and 164 KISS et al. the other half had seasoning irradiated with 1.5 Mrad added to it. The paste was then filled into cans of 100 g capacity and sealed. The experimental batches, consisting of 43 to 45 cans each, were placed in an oil batch at 110°C and subjected to heat treatment equivalent to the following sterilization v a lu e s :

(a) Pork liver paste prepared with untreated additive mixture, Fo = 13.2; (b) Pork liver paste prepared with untreated additive mixture, Fo = 1.47; (c) Pork liver paste prepared with an additive mixture treated with 1.5 Mrad, Fo = 1.47; (d) Pork liver paste prepared with untreated additive mixture, Fo =0.14; (e) Pork liver paste prepared with an additive mixture irradiated with 1.5 Mrad, Fo = 0.14.

The sterilization equivalents were calculated on the basis of heat penetration measurements carried out with an Elektrolaboratoriet temperature-measuring instrument to four places of measurement, applying the general method of thermal process calculations (Stumbo, 1965).

,4. 2. Combined heat and radiation treatment of canned meats

Combination treatments were carried out on meat hash and pork liver paste. The material filled in the cans was received directly from the factory. The mixtures had a pH of 5.4 - 6.3 and an equilibrium relative humidity of 9 7%. 100-g portions were weighed into each can. The combination treatment of the canned meat hash consisted of irradiation with 500 krad and heat treatment at 100°C for 120 min. Heat treatment was performed in an oil batch at 100°C and heat penetration was observed. The sterilization equivalent was found to be Fo = 0.65. Canned meat hash prepared from the same raw material at the factory served as control sample. Treatments were as follows:

1. 100°C, 120 min (F0 = 0.65) + 500 krad; 2. 500 krad + 100°C, 1 20 min (F0 - 0.65) ; 3. 500 krad 4. 100°C, 120 min (F0 = 0.65); 5. Industrial sterilization (F0 =10).

Irradiation was carried out in a 60Co gamma source of 60 kCi activity at a dose-rate of 0.3 Mrad h '1.

4. 2. 1. Storage and sensory tests

The canned meat was stored at 30-32°C for 30 days during which time it was kept under observation. The consistency of the samples was measured with the texturometer. The cans of meat hash and pork liver paste were kept for 24 h beside the instrument before being measured so that they would be at the same tempera­ ture, since the consistency of semisolid, pasty substances is highly dependent on temperature. The content of the cans was extruded in one piece and then cut into discs of 12 mm thickness. Each disc was cut into four sectors and these were then IAEA-PL-561/11 165 tested in the texturometer. The consistency of the samples was characterized by the height of the first peak in the record as related to 1 V instrument v o lta g e . The sensory evaluation of the meat hash and pork liver paste samples, preserved by different methods, was carried out according to Kramer's ranking method (Kramer, 1960). The scoring panel consisted of 9 or 10 members. The samples were ranked separately for the following properties: colour, consistency, smell and taste.

RESULTS

1. Increase of the juice yield of grapes

The possibility of increasing the pressing capacity or juice yield of grapes by irradiation was investigated. On the basis of experiments with five grape varieties it was established that the yield increased by 2-28% with increasing doses in the range of 0.05 to 1.6 Mrad (Table I). Taking into consideration all the available data, a direct correlation was found between the increase of juice yield and the square root of the radiation dose (Fig. 1). Based on the average of the results an increase of 2. 9% was achieved by irradiation with 0.05 Mrad, and irradiation with 1.6 Mrad effected an increase of 21.6%. With doses of 0.8 and 1.6 Mrad a significant change was observed in the condition of the grapes; the skin of the grapes and the stem became brown, thin and discoloured and the texture underwent a change. The grape cells were highly damaged by these doses. Fermentation of all the juices, even of those obtained from grapes exposed to higher dose levels, could be considered practically normal. The composition of the wine from grapes treated with 1.6 Mrad differed only from the untreated, wine insomuch that it had a higher sugar-free extract content, ash content and pH. No evaluable differences were found in the total colloid content. However, the polyphenol index and colour intensity increased with increasing doses (Table II). According to the sensory evaluation, wine fermented from grapes irradiated with 0.8 Mrad showed signs of deterioration whereas wines treated with 0.05 or 0.2 were not impaired. Summing up the results it seems that doses below 0.8 Mrad are suitable for increasing juice yield because they do not affect the sensory quality of wine. Thus, by improving the technological characteristics of the grapes with a 0.4 - 0.5 Mrad treatment an increase of about 10 - 12% may be achieved.

2. Influence of irradiation on the rehydrating capacity of dried vegetables

The reduction of cooking time and of the heat energy requirement results in time saving, in reduced labour and energy requirement both in the household as well as in the catering business. A particularly important requirement is that every component of a multicomponent soup mixture should have an equally short cooking time. Literary data (Schroeder, 1962; Staden, 1966; Markakis et al., 1965; Metlitsky et a l., 1967; Umeda and Sugawara, 1969) show that irradiation reduces the cooking time of dried vegetables. Irradiated vegetables become 166

TABLE I. PERCENTAGE INCREASE IN JUICE YIELD OF VARIOUS GRAPE VARIETIES COMPARED WITH THE CONTROL, AS A FUNCTION OF RADIATION DOSE

V a r ie t y

R a d ia tio n Cultivated at Eger Cultivated at Miklóstelep dose A v e r a g e (M ra d ) a a Egri O la s z - K o c sis Hárslevelüa Hárslevelii H á r sle v e lü al. et KISS K ô v id in k a c s illa g o k r iz lin g Irm a 1 2 3

0 . 0 5 7 . 1 2.1 4 . 0 2 . 3 0 2 .6 2 . 5 2 . 9

0 .2 9 . 1 5 . 0 7 . 6 1 1 . 5 1 . 9 10.2 5 . 9 4 . 3

0 .8 1 6 . 5 4 . 0 1 4 . 9 1 8 . 8 1 3 . 3 1 3 . 3 20.8 1 4 . 5

1.6 2 0 . 9 4 . 4 2 5 . 0 2 8 . 6 1 8 . 5 2 5 . 8 2 8 . 1 21.6

a First, second and third harvest of grapes. IAEA-PL-561/11 1 6 7

oEgri es¡IIад о к. У= - 0,в9* 0, SSâx п = 26 A bKariainka ñ = Qt6fâ 9 9Hárslei/elü /. t>tiársíe*elil ¿. Ф

♦ 9 ■O 10 20 30 ЬО ~\jdose (krad)

F IG .l. Percentage juice yield related to the square root of the radiation dose with several grape varieties.

cooked in a shorter time than untreated ones. The cell-count reducing effect and the insecticide effect of irradiation has been shown in earlier experiments in this laboratory. Investigations on the weight increase during cooking or soaking showed no substantial difference between untreated samples and those irradiated with doses below 1.2 Mrad. Weight increase in carrots irradiated with 2 or 4 Mrad was somewhat lower than that of the control when they were rehydrated by soaking. No such difference was observed when the samples were cooked. The addition of 1.5% salt to the water reduced the water absorbing capacity of carrots treated with gamma rays. The water absorbing capacity of onions was not changed by irradiation. The textural changes occurring during rehydration were followed by texturometer tests. The firmness-rehydration time correlation was found to approximately fit a linear regression line (Fig. 2). The two-phase analysis of variance of the results has shown irradiation to have a significant effect in all the samples on the textural changes occurring during cooking and soaking. According to the Duncan test the average firmness values determined in treated and untreated dried vegetables, differed, at least at the 9 5% probability level, significantly from one another when 1 Mrad or higher radiation doses were used: after equal cooking time the irradiated samples were more tender than the control samples and the difference increased with increasing radiation doses. The extrapolation of firmness-cooking time regression curves has given cooking times equivalent to different radiation doses or, in other words, has shown how far the cooking time requirement was reduced. This is illustrated in Fig. 3. 168 TABLE И. CHEMICAL ANALYSIS OF WINES OBTAINED FROM THE GRAPE VARIETIES IRRADIATED AT DIFFERENT DOSE LEVELS

T o t a l In v e r t S u g a r- A lk a lin it y T it r a t a b le D o se A lc o h o l A sh F ree SO e T o t a l SOg P o ly p h e n o l C o lo u r Place and variety e x tr a c t sugar free extract o f ash a c id pH in d e x in te n sity (M ra d ) (VOl.^/o) ( g / 1) (m g / ) ) ( m g / 1) ( m e q / ) ( g / 1) ( g / 1) ( g / 1) 1 ( g / 1)

E g e r

Egri csillagok 0 1 1 . 5 2 2 2 . 1 8 0 2 2 . 1 8 1 . 8 4 2 2 . 7 1 0 . 7 2 . 9 2 5 4 3 7 . 6 0 0 . 1 4 0

0 . 0 5 12.11 2 1 . 3 0 0 2 1 .3 0 1 . 8 0 2 1 . 9 9 . 2 3 . 0 5 4 3 8 8 . 9 0 0 . 1 5 4 0 .2 1 2 . 3 2 2 1 .8 0 ' 0 2 1 .8 0 1 . 8 2 21.2 9 . 0 3 . 0 4 5 56 7 . 5 6 0 . 2 0 3

0 .8 - 1 1 . 7 6 2 2 .1 2 0 22.12 1 . 9 7 22.0 9 . 5 3 . 0 0 5 37 8.20 0 . 1 7 9 1.6 12.22 2 8 . 2 1 0 2 8 .2 1 2.10 2 3 . 6 7 . 8 3 . 1 6 7 57 1 0 . 0 6 0 . 2 8 6

Olaszrizling 0 1 0 . 3 0 2 0 . 1 9 0 2 0 . 1 9 1 . 5 1 2 1 .2 8 , 5 3 . 0 1 2 2 3 8.20 0.122 0 . 0 5 1 0 . 7 2 2 1 . 3 3 0 2 1 . 3 3 1.68 22.8 8 . 5 3 . 0 9 3 1 8 8 . 4 0 0 . 1 6 8

0 .2 1 0 . 2 5 2 1 . 0 7 0 2 1 . 0 7 1 . 6 1 2 1 . 4 8.6 3 . 0 0 3 2 8 9 . 7 2 0 . 1 6 0 IS t . l a et KISS 0 .8 1 0 . 7 1 2 0 . 2 4 0 2 0 . 2 4 1 . 6 3 20.2 7 . 9 3 . 0 3 4 2 9 8 . 4 2 0 . 1 5 3

1.6 9 . 9 9 2 0 . 6 7 0 2 0 .6 7 1 . 7 3 21.0 7 . 4 3 . 0 7 5 21 9 . 4 4 0 . 1 8 2

Miklóstelep 0

Kocsis Irma 0 1 2 . 1 9 1 8 . 8 9 0 1 8 . 8 9 2 . 5 7 3 0 . 4 4 . 6 3 . 6 0 5 11 5 . 7 0 0 . 1 8 5 0 . 0 5 1 1 . 9 3 1 7 .0 6 0 1 7 .0 6 2 . 1 7 2 6 . 8 4 . 1 3 . 6 4 4 8 5 . 5 8 0 . 1 9 4 0.2 1 1 .1 7 1 7 . 9 6 0 1 7 .9 6 2.20 2 6 . 8 4 . 8 3 . 5 6 5 1 3 5 . 5 6 0 . 2 1 9

0 .8 1 0 .6 7 1 7 . 5 0 0 1 7 .5 0 2 . 0 8 2 6 . 0 4 . 5 3 . 5 6 5 1 6 5 . 9 6 0 . 2 2 3

1.6 8 . 8 4 7 7 . 2 1 4 1 . 0 4 3 6 .1 7 3 . 3 2 3 3 . 2 6 . 4 3 . 7 6 8 3 4 8 . 7 2 0 . 3 8 1

K ô v id in k a 0 1 1 . 3 1 1 8 ,7 7 0 1 8 .7 7 1 . 7 2 21.0 7 . 4 3 . 1 6 7 3 8 9 . 0 0 0 . 1 1 5

0 . 0 5 1 1 . 4 4 1 9 . 0 1 0 1 9 .0 1 1 . 7 9 2 3 . 0 7 . 9 3 . 0 5 5 7 8 9 . 2 4 0 . 1 2 5

0.2 1 1 . 7 9 1 7 . 6 8 0 1 7 . 6 8 1.86 22.0 6 .6 3 . 1 9 6 4 4 9 . 0 0 0 . 1 3 6 0 .8 1 0 . 9 8 1 9 .5 0 0 1 9 .5 0 2 2 . 0 ' 2 6 . 4 6 .0 3 . 4 4 5 3 9 1 0 . 4 0 0 . 1 8 5 1.6 1 0 . 7 8 2 1 . 5 5 0 2 1 . 5 5 3 . 0 5 3 4 . 6 5 . 9 3 . 6 3 3 1 9 12.20 0 . 3 9 0

Hárslevelü Ia 0 0 1 9 0 .4 0 1 6 2 .0 0 2 8 . 4 0 3 .Ó 2 3 5 . 6 1 3 . 2 3 . 0 3 7 3 8 6 . 5 8 0 . 0 9 7

0 . 0 5 8 . 3 8 2 2 . 4 2 0 2 2 . 4 2 3 . 5 8 3 2 . 8 1 3 . 2 3 . 0 5 3 3 2 6 . 1 8 0 . 1 0 4

0 .2 8 . 4 6 21.21 0 21.21 2 . 4 0 2 8 . 6 1 2 . 5 3 . 1 0 5 2 8 6 . 1 8 0 . 0 9 2

0 .8 7 . 8 5 2 8 . 8 5 7 . 5 1 2 1 . 3 4 2 . 5 3 2 7 . 8 1 0 . 8 3 . 2 1 7 66 8 . 5 2 0 . 2 0 6 1.6 8.02 2 1 . 2 7 0 j 2 1 . 2 7 j 2 . 5 9 3 5 . 2 1 0 . 3 j 3 . 1 9 4 2 7 9 . 4 0 0 . 4 2 6 TABLE II (cont.)

Hárslevelü 2a 0 10.12 2 0 . 8 9 0 2 0 . 8 9 1 . 8 0 2 3 . 8 10.6 2 . 9 8 6 4 1 7 . 4 0 0 . 0 9 4

0 . 0 5 9 . 7 0 21.10 0 21.10 2 . 0 9 2 8 . 4 11.6 2 . 9 9 8 3 4 6.10 0 . 0 8 7 PL- 11 1 /1 1 6 -5 L -P A E A I 0 .2 9 . 1 5 2 4 . 7 3 0 2 4 . 7 3 1 . 9 3 2 4 . 6 10.8 3 . 0 8 9 2 5 6 . 2 4 0 . 1 1 8 0 .8 1 0 . 0 4 2 0 . 0 9 0 2 0 . 0 9 1 . 8 0 2 5 . 6 8.6 3 . 1 2 7 4 2 8 . 0 4 0 . 1 5 0

1.6 9 . 5 0 2 2 . 4 3 0 2 2 . 4 3 2 . 2 3 2 7 . 6 со со 3 . 2 7 10 39 8 . 8 2 0 . 2 8 0

Hárslevelü 3a 0 1 1 . 3 3 2 0 . 6 3 0 2 0 . 6 3 1.88 2 4 . 6 9 . 7 3 . 0 9 10 4 2 6 . 5 8 0 . 1 3 5 0 . 0 5 1 0 .9 1 2 2 . 7 6 0 2 2 . 7 6 2 . 0 5 2 6 . 4 9 . 6 3 .10 10 5 8 6 . 8 0 0 . 1 0 4

0 .2 1 1 . 0 4 2 1.02 0 2 1.02 1 . 8 9 2 4 . 4 9 . 0 3 . 2 0 1 3 4 8 7 . 3 6 0 . 0 9 8 0 .8 1 0 . 7 8 2 0 . 9 6 0 2 0 . 9 6 2 . 4 5 2 8 . 4 7 . 7 3 . 3 8 11 4 1 9 . 1 0 0 . 1 8 5

1.6 1 0 .6 9 2 2 . 3 2 0 2 2 . 3 2 3 . 1 5 3 5 . 2 7 . 4 3 . 5 6 8 37 10.00 0 . 3 6 4

a Grapes from the first, second or third harvest. 169 170 KISS et al.

cannot pansley root

70- Ю-.. «a y> i № £ 'к . <0

C5 o i г í s è w У French beans 4 IU Чч<

S

'fc «3 3 :

о r г t e в w s 8 w ------— cooking tim e {min) ■ OHrad-*-1,0 Nr ad -*- ko ft rad

FIG .2. Softening of dried vegetable lots treated with electron-accelerator, during cooking in distilled water.

Equations of the regression lines and correlation coefficients:

1 y = 57.24 - 0.467x r = -0.2372 7 y - 51.86 ” 3.365x г = -0.9142 2 y = 35.70 + 0.416x r= 0.2134 8 y = 26.43 - 0.580X г = -0.3699 3 y = 24.30 - 0.394X r = -0 .3 6 0 2 9 y = 13.31 - 0.423X г = - 0 . 4 5 8 6 4 y = 71.82 - 1.846x r = - 0 . 6 5 2 1 10 y = 60.62 - 2 . 054x г = -0.6893 5 y = 65.30 - 2.120X r = - 0 . 6 0 4 5 11 y = 52.32 - 1.793x г = -0.6882 6 y = 37.37 + 0.344x r = - 0 . 1 2 5 6 12 y = 34.29 - 0.777x г = -0.5525

Radiation doses improving the cooking quality did not cause undesirable changes in the taste or colour of dried root vegetables or string beans. Dried onions, however, darken in proportion to increasing^,radiation doses. Browning becomes significant on irradiation with 1 Mrad or above. Radiation doses between 0.5 and 2.5 Mrad reduced the total viable cell count of the dried products by one to three orders of magnitude. IAEA-PL-561/11 171

FIG .3. Measure of the reduction of cooking time as a function of radiation treatment (electron radiation and cooking in distilled water).

3. Influence of irradiation on the colour of ground paprika

One of the oldest methods of reducing the m icrobial contamination of seasonings is heat treatment. However, this causes a substantial loss in the volatile oil and other aromatic substance content of seasonings (Proctor et al., 1950;Gisske, 1954; Lerke and Farber, 1960). Good results were achieved by the use of ethylene oxide (Coretti, 19 57) which is not objectionable from the point of view of food hygiene either (Hill, 19 70). However, this agent is not absolutely reliable, it has a synergetic effect as well and the residues found in the food cause problems (Székely, 1968; Murányi and Halász, 1971), therefore it seemed necessary to investigate the possibility of applying radiation treatment. In accordance with the results of other researchers (Tôrok and Farkas, 1961) we found that ground paprika irradiated with 0.3 - 0.4 Mrad retained its m icrobiological stability even in a space of high humidity. In the present study the effect of various packaging methods and storage temperatures on the viable cell count, pigment content, moisture content and colour changes, as observed by sensory evaluation and objective methods, was investigated in samples irradiated and untreated. The total pigment content expressed in capsantine was determined by the method of Benedek (1958) as a function of packaging, treatment, time and conditions of storage (Fig. 4). It was shown that the pigment content of ground paprika depends on storage temperature and time and is not affected substantially by irradiation. Whatever method of packaging was applied the pigment content was most extensively reduced by a storage 172 KISS et al.

Dose at diff. storage temps Paper bags о 0 krad loi Loosely filled in Al tubes •5 0 0 " Г "Sí <► ■ a 0 krad I oQ- •5 0 0 * I 2SC° I' &0 bo- 3 I 5 A500 I ISC’ «V* \o-*5C‘ 5 a . £ 2 .&>

5 i . 1

60 Ш Ш Г o 65 Ш 1SÔ Compactly filled in A/ tubes Polyethylene bags *■ !I s * £ s. <

60 ISO 180 60 190 ISO

Polyester bags Filled in Al tubes under flow

e

Í 3

IО.

60 ISO 160 0 60 ISO lâO Storage time(days) Storage ttmefdayr)

FIG.4. Benedek number (total pigment content expressed in capsantin, g/kg) as a function of radiation

dose, storage temperature and tim e.

1. The experiment with polyethylene pouches was started only on the 60th day of storage.

2 . In the experiments with polyester pouches a control sample was only available on 0 day of storage. The first test was carried out on the 60th day of storage. IAEA-PL-561/11 173 temperature of 30°C, independent of irradiation. The slightest change in the pigment content was observed upon storage at 5 and 10°C. The colour of the paprika stored in paper bags at 0 . . . 5°C faded completely, while that of the sample treated with 500 krad did not change at all. Fading of the sample in a paper bag stored at 5°C may be traced back to several factors such as extensive moulding as an effect of high humidity and good permeability of the paper bag to air. The colour protective effect of irradiation manifests itself just under such unfavourable storage conditions, whereas in a more adequate packaging material it appears in the reduction of cell count and its maintenance at this low level. The characteristic properties of ground paprika — the pigment content and solids content — are not affected by irradiation. Colour may be maintained by the choice of appropriate packaging material and storage conditions. Metal packaging material seems to be the most suitable from this aspect and a storage temperature of 10 - 12°C.

4. M icrobiological stability of heat-processed liver paste as affected by the radurization of the seasoning used in its manufacture

Because of the high m icrobial contamination of additives and ingredients used in the canning and meat industries a much higher safety factor is applied in their heat treatment than necessary or desirable. Thus, frequently, the quality, nutritive value and hedonic value of sterilized products is low. We found in earlier experiments that beyond the reduction of cell count the heat sensitivity of the microorganisms increases after irradiation and thus a lower heat treatment is sufficient to ensure their stability. The effect of radiation treatment of the additive mixture used in the manufacture of pork liver paste was studied on samples heat treated at the level of one-tenth (F0 =1. 47) and of one-hundredth (F0 = 0.14) of the treatment applied in the industry (Fo = 13 to 15 sterilization equivalent). The composition of the microbial flora of the additive mixture before radiation treatment was as follows:

total aerobic viable cell count 4.4 X i o 5/g count of aerobic spores (surviving 10 min at 80°C) 1.8 X i o 3/g

total anaerobic cell count 1.7 X i o 2/g viable cell count of sulphite-reducing anaerobic cells 1.0 X i o 2/ g anaerobic cell count (surviving treatment for 10 min at 80°C) 1.0 X ioVg

After treatment with 1.5 Mrad the additive m ixtu re: appeared to be practically sterile, the total aerobic viable cell count was lower than estimable by the method applied (1 cell per 3 g). The total viable aerobic cell count of the product before heat treatment was 7.0 X 107/can, of which the total aerobic cell count introduced with the untreated seasoning amounted to 2.9 X 106/can. The additive mixture formed 6.6% of the weight of the product and the total viable cell count introduced with the untreated seasoning formed about 4% of the total viable I

174 ' KISS et al.

TABLE III. DISTRIBUTION OF SPOILAGE SYMPTOMS IN CANS OF PORK LIVER PASTE PREPARED WITH UNTREATED AND RADIATION- TREATED ADDITIVE MIXTURE, ON THE 80th DAY OF STORAGE AT 30°C

Sterilization

e q u iv a le n t Acidification of the heat T o t a l A d d itiv e F la t sour w ith ga s H2S and g as fo rm a tio n tr e a tm e n t s p o ila g e m ix tu r e (°Io) fo r m a tio n (°Jo) g iv e n the CSS») С% p ork liv e r

p a ste a F0

U n trea ted 3 2 3 5 3 3 100 a d d itiv e

0 . 1 4 R a d ia tio n -

s te r iliz e d 22 7 2 6 100

a d d itiv e

U n trea ted 9 53 5 67 a d d itiv e

1 . 4 7 R a d ia tio n -

ste r iliz e d 4 5 0 9 a d d itiv e

a z = io°c.

F0 = heat processing equivalent to a heat treatment of Fo min at 121eC .

cell count of the material before sterilization. In spite of this, the experience gained during incubation at 30°C of the samples exposed to reduced heat treatment, as described above, showed the efficiency of preservation by heat treatment to depend to a large extent on the m icrobial contamination of the additive mixture. On the 80th day of storage all the cans, whether blown or not, were opened and the spoilage ratio was established taking into account also the pH, and the sensory properties (odour, consistency, colour) and the distribution of the individual symptoms of spoilage were also evaluated. These results are shown in Table III. In the batch treated at Fo = 13.2 sterilization equivalent not a single blown can was found. The spoilage percentage of pork liver paste prepared with irradiated additive mixture was not quite one-seventh of that prepared with untreated additives even when given a heat treatment of F0 = 1.47 sterilization equivalent. This permits the conclusion that by using radappertized additive mixture a heat treatment of F0 = 2.5 equivalent would be sufficient to achieve sterility. This heat treatment is just above the one-sixth of the treatment applied in the industry. Such a substantial reduction of the heat requirement at unchanged sanitary safety would result in a substantial improvement of the product, in a saving of energy and in increase of production capacity. IAEA-PL-561/11 175

Odour C o lo u r

Treatments: I . 100°C, 120 + 500krad(fQ^ 0,65) 2.500krad H00°C, /10' (Гд х 0,65) 3. t00° C, 120' (F0- 0,65) ¡¿ Sterilization at plant scale f a ,0)

FIG. 5. Sensory evaluation by ranking of canned m eat hash preserved by combination treatment and individual treatments. Evaluation was carried out one month after treatment by a panel of 10 members.

The zone of rank sums not differing at the a ^ 0 .0 5 o r a ^ 0 .0 1 probability level of error is marked in the figure according to Kramer's (1960) table.

С 90 ■ Ç so- 5 ю íi 60 i А a so íi ^ho ч 30 £ 20 Ю Ca û С /. г. 3. ь. s.

Treatments!. I00°C,I20‘*500krad (Г0 a 0,85) 2. 500 krad* /00% tS0'(Fg ^ 0,65) 3.500 krad 4.100% ISO’ (F0 a 0,65) 5. Sterilization al plant scale (Гв ЛЮ)

FIG.6 . Results of the texturometer tests of canned m eat hash subjected to combination treatment or individual treatments. Measurements were carried out 24 h after treatment. The figure shows the average of

20 measurements per sample and the standard deviations.

5. Combined effect of heat and radiation treatment on the quality of canned meat

In order to reduce the heat requirement the possibility of combining it with irradiation was investigated. The aim of these experiments was also to reduce the changes occurring in products subjected to extensive heat treatment or high radiation doses. 176 KISS et al.

In the experiments with meat hash the cans were given a heat treatment of 120°C for 120 min and a radiation dose of 500 krad. The sterilization equivalent of this treatment (Fo = 0. 65) was many times that of the treatment at 100°C for 60 min, as described above. Thus, it was hoped to achieve adequate cooking of the meat while this treatment was still substantially lower than the one applied in industry (Fo -1 0 ). The odour of the product was evaluated by ranking on the day after treatment. The aim of this investigation was to find out whether any off- odour was formed as a result of radiation treatment. The sensory evaluation was repeated one month after treatment and the ranking according to odour was complemented by ranking according to colour. The results are summarized in Fig. 5. The consistency of canned meat hash given combined treatment was compared with that of meat hash prepared by the traditional industrial sterilization method. Results are shown in Fig. 6. The spoilage percentages of meat hash samples given combined treatment or individual treatments and stored for one month at 30°C, are given in Table IV. Experiments carried out with pork liver paste have proved that by reducing the heat-treatment requirement the danger of caramelization was substantially reduced. As regards the combination treatment of meat hash, the application of a 500-krad radiation dose permitted heat treatment below one-tenth of the industrial treatment to achieve m icrobiological stability. Taking into consideration safety measures in relation to the presence of Clostridium botulinum, the application of a radiation treatment with 500 krad would permit an Fo = 2.5 heat treatment to achieve m icrobiological stability. With meat hash the reduction in heat treatment is limited by the cooking requirement of the components. It is noteworthy that irradiation with 500 krad did not induce the formation of off-odour in canned meat hash.

TABLE IV. ST OR A BILIT Y OF CANNED MEAT HASH GIVEN COMBINED TREATMENT OR INDIVIDUAL TREATMENTS Storage: 1 month at 30°C

Sterilization N u m b e r o f N u m b e r o f S p o ila g e

T r e a t m e n t equivalent, s a m p le s s a m p le s ra tio

Fo stored s p o ile d Cfc)

100°C, 120 min 0 . 6 5 15 0 0 + 5 0 0 krad

5 0 0 krad 0 . 6 5 15 0 0 + 100°C, 120 min

100°C, 120 min 0 . 6 5 5 3 6 0

5 0 0 krad - 5 5 1 00

In d u stria l 10 5 0 0 sterilization IAEA-PL-561/il 177

BIBLIOGRAPHY

BENEDEK, L ., 1958, Untersuchungsverfahren zur Bestimmung des Farbstoffgehaltes in Paprikamahlgut,

Z . Lebensm.-Untersuch. Forsch. 107, 228.

CORETTI, K ., 1957, Kaltentkeimung von Gewiirzen m it Aethylenoxid, Fleischwirtschaft_9, 183.

GISSKE, W ., 1954, Keimfreie Gewürzprâparate für die Herstellung von Fleischgerichten und Wurstwaren,

Fleischwirtschaft_7, 2 8 0 .

HILL, E .G ., 1970, Pesticides for insect control in food premises, Food Manufacture 45, 70.

HOSKINS, J .K ., 1934, Most probable numbers for evaluation of coli-aerogenes tests by fermentation tube method, Public Health Repts 49, 393.

KRAMER, A ., 1960, A rapid method for determining significance of differences from rank sums, Food

Technol. 19, 576.

LERKE, P. A ., FARBER, L ., 1960, Effect of electron beam irradiation on the microbial content of spices and teas, Food Technol., _M, 266.

MARKAKIS, P ., NICHOLAS, R .C ., SCHWEIGERT, B .S ., 1965, Irradiation on inland fish, fruits and vegetables. Radiation Pasteurization of Foods, Summaries of Accomplishments Presented at Fifth Annual

Contractors Meeting, Oct. 20-21, CONF-651 024. USAEC, Washington, D .C ., pp.49-52.

METLITSKY, L .V ., ROGACHEV, V .N ., HRUSHCHEV, V. G. , 1967, Radiatsionnaya obrabotka pishchevykh produktov (Radiation treatments of food products), in Russian, Izdatyelstvo "Ekonom ika", Moscow, 31 p.

MURANYI-NAGY, J., HALÁSZ, K ., 1971, Tartósitott élelmiszerek higiénés kérdései (Sanitary problems related to preserved foods), in Hungarian, Conference of the Microbiological and Canning Sections of METE,

May 3-7, 1971, Nagykor6s. ■

PROCTOR, B .E ., GOLDBLITH, S .A ., FRAM, H ., 1950, Effect of supervoltage cathode rays on bacterial flora of spices and other dry food materials, Food Res. JL5, 490.

SCHROEDER, C .W ., 1962, Dehydrating vegetables, U .S. Patent No. 3025171.

STADEN, O .L ., 1966, "Experiences with the irradiation of vegetables in the Netherlands", Food Irradiation.

IAEA, Vienna, 609.

STUMBO, C .R ., 1965, Thermobacteriology in Food Processing, Academic Press, New York, London, pp.126-129.

SZÉKELY, Á ., 1968, Szinergetikus mérgezések (Synergetic poisonings), in Hungarian, Nôvényvédelem 4, 85.

TÔRÔK, G ., FARKAS, J., 1961, Kisérletek füszerpaprika-ôrlemények ionizáló sugárzásos csiraszám- csôkkentésére (Experiments into the reduction of cell count in ground paprika by ionizing radiations), in Hungarian, Kôzponti Elelmiszeripari Kutató Intézet Kôzlem ényei 3, 1.

UMEDA, К ., SUGAWARA, К ., 1969, Radiation effects on plant tissue. Parti. Effects of cathode ray on the dehydration of raw carrot and rehydration of dried carrot, Nippon Shokuhin Gakkaishi _16, p. 15, Ref.

Nucl. Sci. Abstr. 25 (15), No. 29942, 3061 p.

SUMMARY AND RECOMMENDATIONS

1. SU M M AR Y

The Panel recognized that treatment of foods with ionizing radiation not only serves the purpose of stabilizing them in their original state, but may also enhance the technological and/or hygienic properties of certain foodstuffs. Hitherto, emphasis has mainly been placed on preservation of already existing desirable quality characteristics of wholesome food. In view of the evidence presented, it was thought timely to consider the quality improvement aspects of food irradiation as well. The papers submitted to the Panel presented evidence that ionizing radiation can significantly enhance the technological characteristics and the hygienic quality of many raw materials and food products. The hygienic improvements resulting from radiation treatment already show that it is possible to eliminate or reduce the need for the use of chemical preservatives and pesticides in the storage of some foods and agricultural produce. The technological improvements achieved by irradiation facilitate the preparation of some foods of higher nutritive value. The yields of edible fractions of other food sources may be improved as a result of induced physico-chemical and biochemical changes.

1.1. Applications of practical importance

1.1.1. Reports on work on carbohydrate-rich foods such as wheat, potatoes, artichokes and maize, as well as on isolated starch and inulin have shown that, in the course of irradiation, the polysaccharides degrade in part to lower-molecular-weight entities and oligosaccharides of commercial interest, the extent of this degradation depending on the radiation dose employed. The susceptibility to amylolysis of the irradiated products is also increased. The possibility exists for using irradiation to obtain modified starches which could find application in the food, textile, paper and other industries.

1.1.2. Degradation of proteins and a decrease in their viscosity may result from irradiation. Such protein disaggregation and dénaturation with increased susceptibility to proteolytic action is observable, for example, with irradiated wheat or with the legume red gram ( Cajanus с ajan ), leading to improvements in baking and cooking qualities, respectively,

1.1.3. As a result of radiation-induced changes (at the disinfestation dose level) in the starch and protein components of wheat, rheological properties (gélatinisation viscosity, dough development, elasticity of wheat gluten, loaf volume) of wheat flour improve considerably from the point of view of brea d " m aking.

1.1.4. Protein enrichment with up to 10% of defatted soya flour becomes possible with flour from irradiated wheat, without loss in baking quality. The enrichment can be increased to 15% when trace amounts of soya lecithin are included in the dough mix.

179 180 S U M M A R Y and RECOMM ENDATIONS

1.1.5. In the complex enzyme systems, preferential protection against radiation destruction of specific enzymatic functions can be obtained. It was suggested that, in the crude enzyme preparations used in the food industry, attempts may be made to irradiate under conditions where preferential protection is obtained and, thus, undesirable enzyme activities are eliminated. The properties of an enzyme are in many cases altered by irradiation. Such radiation-induced changes may result in enzymes more suitable for specific purposes.

1.1.6. Enzymes could be immobilized by radiopolymerization with monomers such as acrylamide. Such entrapment is effectively increased by copoly­ merization with soluble starch and lyophilization treatment (or in the frozen s t a t e ).

1.1.7. The anti-oxidant activity of amino acid-sugar solutions on heating is markedly accelerated by pre-irradiation of the mixture or of the sugar solution in the mixture. In addition to anti-oxidative effects, the amino- carbonyl reaction products can affect aroma and microbial growth. Together with the fact that the anti-oxidative activity of the products formed is much stronger than the discolouration by browning, the observations can have significance in food technological practices.

1.1.8. Juice yield in grapes and other juicy fruits was reported to be considerably increased by irradiation of the fruit before pressing. This could be of economic value in the fruit juice, fermentation and distillation industries.

1.1.9. There is considerable reduction in rehydration and cooking time of dehydrated vegetables and legumes after irradiation. Structural changes in carbohydrates and proteins result from irradiation, and contribute to the improved rehydration and/or cooking quality. There are no significant losses in nutritive value.

1.1.10. Ground paprika, appropriately packed and irradiated, stores well because of a reduction in microbial cell count. Similarly, with irradiated seasonings used in processed meat preparations, thermal treatment for sterilizing can be greatly reduced. As a result, there is substantial improvement in product quality.

1.1.11. Gelatin can be radurized for use in confections and in the pharma­ ceutical and photofilm industries.

1.1.12. Combination treatment with heat and irradiation can sterilize a variety of fruits, vegetables and meat products with a resulting reduction in thermal treatment and improvement in quality characteristics of the canned products.

1.1.13. It is recognized and described in the Panel reports that, from both the theoretical and applied aspects, combined treatment of foods, such as temperature plus irradiation, can and has led to marked hygienic and technological improvements. Thus, irradiation permits lower temperature for heat treatment and, likewise, mild heating permits the use of smaller SUMMARY and RECOMMENDATIONS 181 radiation doses to attain desired effects such as a decrease in post-harvest fungal damage to tropical fruits and to marked prolongation of the storage time of both leavened and unleavened bread.

1.1.14. Fruits like mango and banana can be delayed in their ripening by low doses of radiation, thus permitting better texture of the fruits over longer periods. Flavour characteristics and external appearance can also be influenced by irradiation.

1.1.15. There is flavour enhancement in carrots, irradiated for inhibition of root hair growth, which is reflected in the composition of the volatiles as characterized by gas chromatographic and mass spectrometric analyses.

1.2. Fundamentals of quality improvement by irradiation

It was recognized by the Panel that most of the basic mechanisms of favourable changes in food quality are insufficiently known at present, in spite of the fact that important progress has been made in many areas.

1.2.1. Upon irradiation, the primary products formed are free radicals — the "dry" and solvated electron, the hydroxyl radical, OH, and the hydrogen radical, H — as well as molecular products, hydrogen gas, hydrogen peroxide, and the hydrogen ion. These primary products may react with food constituents or with each other. The nature and degree of the inter­ actions depend on many factors including the water content, pH, and chemical composition. The relative degree to which direct and indirect effects of the primary radiation products occur depend on the capacity for diffusion and on the micro-environment. Many methods are available for influencing the relative concentrations of the different primary products and to control the degree to which direct or indirect effects predominate. These are important considerations since, for example, the nature and yield of radio­ lytic products depend on the conditions of irradiation.

1.2.2. It was pointed out that the most important chemical reaction brought about by irradiation of carbohydrates of high molecular weight was depoly­ merization of the chain molecules for which two mechanisms were discussed.

1.2.3. Studies on model systems reconfirmed the importance of changes in membrane permeability as a factor in radiation-modification of the properties of plant or animal tissue portions used as human food.

1.2.4. As to the mechanism of antibacterial actions of radiations, model systems have revealed that four types of substances produced by irradiation are responsible for much of the observed in-vitro biological effects. These are carbonyl compounds, hydrogen peroxide, organic peroxides, and peroxide complexes formed by hydrogen bonding with normal food constituents such as small molecules like histidine and macromolecules like proteins. Of particular importance, because of their growth-inhibiting properties, are the alpha, beta-unsaturated carbonyl compounds. It was recognized, however, that all of the aforementioned compounds are so reactive, that, in the amounts

/ i 182 SUMMARY and RECOMMENDATIONS produced upon irradiation, they do not appear to produce any harmful toxic or genetic effects upon ingestion because they are destroyed before they reach critical cellular sites in sufficiently high concentration.

1.2.-5. Specifically in the case of starch, it was reported that radiation- induced hydrogen peroxide could exert a radiation-sensitizing effect on the bacterial flora!of starch.

1.2.6. It was èmphasized that the chemical substances produced by irradiation are 'the same types already present naturally in foods, or else formed by non-irradiation treatment such as, cooking, canning, or simply upon storage or exposure to light and air. In fact, the increments resulting from radiation doses are normally far less than those already consumed d a ily.

1.2.7. The after-effects of irradiation in foods and food constituents can be modified by various substances or treatments (freezing, heating) depending on the compounds responsible.

1.3. Research areas

In the course of the discussions some broad and a few specific areas emerged to be of interest for a systematic clarification of phenomena under­ lying the improvement of quality of irradiated foods and food constituents. These areas can be listed as follows.

1.3.1. Physico-chemical and technological effects. These include the determination of the nature and yield of the radiolytic products: ESR measurements on irradiated solid materials to determine the concentrations and life-tim es of free radicals, and the modification of the properties of polymeric constituents. The change with time under different conditions of storage (e.g. temperature, gas atmosphere, moisture) should be determined. For food in particular, these include the determination of physical and chemical changes, e.g. rheological, mechanical structure, filtration characteristics, processing modifications, solubility, water-binding capacity and formation of compounds contributing to aroma and flavour. The study of model systems includes the most important food constituents: carbohydrates, mono- and polysaccharides, lipids and nitrogen-containing biological micromolecules (including enzymes). The concentrations to be investigated should range from aqueous solutions to the dry state. In the case of the latter, it is necessary to specify water content, purity, and type of structure, i.e. crystalline, amorphous, glassy, etc.

1.3.2. Biological effects. Whenever possible, certain short-term biological testing should be done in vivo. Further, evaluation of the significance of any effects uncovered should be based on the findings from a battery of such tests. The biological testing should include (1) cytogenetic tests: the chromosomal breaking properties in bone marrow and germ cells-, (2) host-mediated assay to detect point mutations; (3) dominant lethal and (4) bacteriostatic and bactericidal assay. SU M M AR Y and RECOMM ENDATIONS 183

1.3.3. Combination treatments. For fundamental investigations of model systems, these would include the chemical and biological manifestations of combinations of food constituents, e.g. varying molar ratios of a carbo­ hydrate with a lipid, or of an enzyme with added carbohydrates, lipids, or proteins. Foods and food constituents should be investigated systematically using temperature adjustments made before, during, or after irradiation. Systematic studies on low-temperature irradiation are needed.

1.3.4. Scavenging actions. These would include the use of nitrous oxide gas which converts the electrons to hydroxyl radicals. Irradiation can also be done in vacuo or in an atmosphere of an inert gas.

1.3.5. Mechanisms underlying the increased yield of juices and softening in some foods following irradiation. To what extent are these effects due to primary degradation processes and/or to influences on the enzymatic action?

1.3.6. Treatment of certain contaminated food additives (e.g. agar, dyes, sugar, spices) to reduce or eliminate their contamination with the lowest possible dose.

1.3.7. The nature, cause, and modification of radiation after-effects.

1.3.8. The use of radiation for preferential protection or modification of e n z y m e s .

1.3.9. Influence of carbonyl compounds and peroxide complexes on the hygienic and technological properties of irradiated foods and the mechanism of their actions on enzymes and m icro-organism s.

1.3. 10. Determination of the nature and yields of alpha, beta-unsaturated carbonyl compounds produced by irradiation as a function of temperature, and concentration of food constituents under investigation.

1.3.11. Studies on the comparative efficacy of different immobilizing systems, the scope for use of such supported enzymes in industrial practice, and the nature of the binding between the enzyme and the supporting structures.

1.3.12. Investigations of the differences, if any, in the degree of breakdown of the proteins in their natural state and in isolated-form . There also exists a need to use irradiation for the preparation of gelatin suitable for use as a plasma extender.

1.3.13. The properties of natural and artificial membranes as affected by irradiation warrant systematic investigation. For example, mixed membranes consisting of fatty acids and cholesterol or protein can be prepared as monolayers on water or filters and the change in enzymatic susceptibility and permeability could be investigated.

1.3.14. Thermalization and auto-oxidation produce compounds similar to those found upon irradiation. The formation of these compounds as well as the biological effects of different processing conditions, not involving irradiation, on the hygienic and technological properties of foods and food constituents warrant investigation. 184 SU M M A R Y and RECOMM ENDATIONS

2. RECOMMENDATIONS

Based on the papers presented, the discussions held at the meeting, and on the possible research areas enumerated above, the Panel found it necessary to make a number of proposals aiming at their ultimate inclusion into the FAO/IAEA programme on food irradiation.

2.1. Work on those foods for which radiation treatment has already been found to be superior to conventional treatments for the improvement of the hygienic, sensory and technological properties, should continue with a view to their commercialization after completion of the required wholesomeness t e s t s .

2.2. As the possibility exists for using irradiation to produce modified starches which could find application in the food, textile, paper, pharma­ ceutical and other industries, further work is recommended to control and characterize depolymerization of carbohydrates so as to obtain products which may have specific advantages in the above industries.

2.3. There is scope for applying irradiation to obtain gelatin for use as a meat product additive and a plasma extender.

2.4. The possibility of using radiation under conditions where preferential protection is obtained, as a means of eliminating undesirable enzymatic activities in industrial enzyme preparations, merits further study. More­ over, the possibility of using radiation for changing the enzymatic properties to make the enzymes more suitable for industrial use should be investigated. / 2.5. The comparative efficacy of different immobilized enzyme systems, the scope for use of such supported enzymes in industrial practice and the nature of the binding between the enzyme and the supporting structures need to be studied.

2.6. Further technological experiments on the use of radiation to improve the juice yield in pressing of fruits and vegetables are economically promising and highly recommended by the Panel.

2. 7. Improvement in rehydration capacity of dried fruits and vegetables should be studied in more detail, with special reference to its technological advantages and potential beneficial consequences to the national economy of various countries producing larger amounts of the above products.

2.8. Improvement of the hygienic quality of spices and condiments by irradiation appears to be of great importance to spice-producing countries and intensification of work along these lines is to be supported.

2.9. As improvement in the overall microbiological quality of food ingredients by irradiation has been shown to have important consequences in the technology of conventional food processing, it is recommended that studies in this field be promoted.

2.10. Combination treatments involving irradiation and conventional processes, like heat treatment, could, in many cases, reduce the severity of the SU M M AR Y and RECOMM ENDATIONS 185 individual treatment components, and studies on the resulting quality improvement are to be encouraged.

2.11. The use of low temperatures for irradiation and selected scavengers of free radicals like N20 for reducing the radiation dosage required to effect a desired hygienic change in irradiated foods was found to be promising and work along these lines is recommended.

2.12. Products of radiolysis of food components should be carefully analysed, identified and determined quantitatively under a variety of external conditions, like temperature, gas atmosphere, humidity, etc. These studies should first be carried out in model systems, including carbohydrates, lipids, proteins, enzymes and bacteria, in a wide concentration range from dilute solutions to the solid state and in various combinations, e.g. mixtures in various molar ratios of a carbohydrate with a lipid, or an enzyme, etc.

2.13. Radiation effects should be investigated also by measuring the physical properties, e.g. viscosity, texture, rehydratability, membrane permeability, etc. of the product.

2.14. It is recommended that comparative investigations on the effect of other processing methods on food should always be carried out in parallel with food irradiation studies.

2.15. Finally, it was recommended that the economic and/or hygienic consequences of the results which can be obtained by the procedures advocated here be carefully examined in a quantitative manner before actual research programmes are started.

LIST OF PANEL MEMBERS

Stefania BACHMAN Technical University, Institute of Applied Radiation Chemistry, tódá, Poland

G. B E R G E R CEA, Centre d'études nucléaires de Cadarache, 13 Saint-Paul-lez-Durance, France

M. FUJIMAKI The University of Tokyo, Department of Agricultural Chemistry, Bunkyo- ku, Tokyo, Japan

I. KISS Central Food Research Institute, Hermann Ottó-út 15, Budapest II, Hungary

T. SANNER Norsk Hydro's Institute for Cancer R e s e a r ch , The Norwegian Radium Hospital, Montebello, Oslo 3, Norway

H. SC H E R Z Institut für Strahlentechnologie, Bundesfors chungs anstalt für Lebensmittelfrischhaltung, Karlsruhe, Federal Republic of Germany

J. SCHUBERT Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pa. 16261, USA

W. S. SHERIF Royal Scientific Society, Food Technology Department, Amman, Jordan

A. SREENIVASAN Bhabha Atomic Research Centre, (C hairm an) Trombay, Bombay 85, India 188 LIST OF PANEL MEMBERS

REPRESENTATIVE OF AN INTERNATIONAL ORGANIZATION

T. FAWI ABDU Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture, IAEA, Vienna

SCIENTIFIC SECRETARY

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