University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange
Masters Theses Graduate School
3-1972
Stability and Acceptability of Intermediate Moisture Textured Vegetable Protein
Helen Sy Yu University of Tennessee - Knoxville
Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes
Part of the Food Science Commons
Recommended Citation Yu, Helen Sy, "Stability and Acceptability of Intermediate Moisture Textured Vegetable Protein. " Master's Thesis, University of Tennessee, 1972. https://trace.tennessee.edu/utk_gradthes/3218
This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council:
I am submitting herewith a thesis written by Helen Sy Yu entitled "Stability and Acceptability of Intermediate Moisture Textured Vegetable Protein." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Food Science and Technology.
J.L. Collins, Major Professor
We have read this thesis and recommend its acceptance:
J. Owen Mundt, Ivan E. McCarty
Accepted for the Council: Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official studentecor r ds.) February 21 , 1972
To the Graduate Council: I am submitting herewith a thesis written by Helen Sy Yu entitled "Stability and Acceptability of Intermediate Mo isture Textured Ve getable Protein ." I recommend that it be accepted for nine quarter hours of credit in partial ful fillment of the requirements for the degree of Master of Science , with a major in Food Technology .
We have read this thesis and recommend its acceptance:
Accepted fo r the Council:
viCe��� ancercJ� Graduate Studies and Research STABILITY AND ACCEPTAB ILITY OF INTERMEDIATE MO ISTURE TEXTURED VE GETABLE PROTEIN
A Thesis Presented to the Graduate Council of The University of Tennessee
In Partial Fulfillment of the Requirements for the Degree Mas ter of Sc ience
by Helen Sy Yu March 1972 ACKNOWLEDGMENTS
The author wishes to express her deepest gratitude to Dr. J. L. Collins for his valuable and patient· guidance and encouragement throughout the planning, conducting and reporting of the pres ent study. Sincere appreciation is extended to Dr. J. 0. Mundt for his valuable advice and assis tance during the course of the research work . Special thanks are due to Dr . M. R. Johnston and Professor I. E. McCarty for their constructive advice , to Dr. W. D. Barber and Dr. V. H. Reitch for their assist ance with the statis tical analysis , to Miss Ruth Hill for her assistance with the laboratory work and to members of the sensory panel for their participation . Special acknowledgment is made to Archer Daniels Midland Company and to Pfizer and Company , Incorporated for their donation of the materials used in this experiment . Deep appreciation is expressed to her parents , Mr. and Mrs . Delfin Yu , and her family for their encouragement and support during the course of her study in the United States.
ii
101554-4 ABSTRACT
This study was des igned to develop a shelf stab le and acceptable intermediate moisture product us ing dry extruded textured vegetab le protein chunks as the food base. The water activity of the protein chunks was adjusted to 0.85 and 0.80 by cooking in solut ions of sorbitol , sodium chloride , propylene glycol , sucrose and potassium sorb ate . The effe cts of calcium lactate and a lower pH on the proper ties of the protein product were determined. Products pre pared at 0.85 Aw cons isted of four treatments : 1) without calcium lactate , pH 7.0; 2) without calcium lactate , pH
5.5; 3) with calcium lactate , pH 7.0, and 4) with calcium lactate , pH 5.5. On the products at 0.80 Aw , only two tre atments were tested: 1) without calcium lactate , pH
7.0 and 2) without calcium lactate , pH 5.5. The samp les were packed in sterile air- tight jars and held at 27.6° C for sixty days . Storage stab ility was studied by determining the microbial , phys ical , and chemical characteristics of the product at ten - day intervals . Overall acceptability was evaluated on the freshly prepared product by the hedonic-preference test. Samples were prepared at water activities of 0.80 and 0.70 and deep- fried before sensory evaluat ion. A difference- preference test was also
iii iv con ducted to evaluate sensory attributes on texture , mo ist ness , and flavor. Products at 0.85 Aw were susceptible to mold growth . The bacterial growth cu rve was fairly rapid during the early storage period and the microb ial flora was predominantly Pseudomonas spp . When water ac tivity was lowered to 0.80, mold growth was completely inhib ited and bacterial grow th was gre atly hindered. Pseudomonas spp . was in the majority at the initial stage; howeve r, members of the Family Ach romob actereceae soon out grew the Pseudomonas spp . and dominated throughout the remaining storage period . Calcium lactate lowered signi ficantly the water activity , mo isture content and pH of the intermediate moisture product . No significant ch ange in color was ob served; however , there was an increase in firmness . The lowering of pH to 5.5 caused a decrease in water act ivity and moisture content of the product with target Aw of 0.85. Shear resistance was significantly increased. Color of the protein chunks was lighter at pH 5.5 than at pH 7.0. Similar trends on water activity, moisture content , firmness and color were observed in products lowered to 0.8 0 Aw; howeve r, no significant difference was established be tween the two pH levels . There was an ove rall increase in water act ivity of the intermediate moisture product at both levels of water v activity as storage time increased. However, moisture content de creased in products at 0.85 Aw while it increas ed at 0.80 Aw . In samp les at 0.85 Aw , a gradual decrease in pH was observed during storage . The stored product had a slightly higher shear value than the fresh product while its color was not changed. Results of the preference test indicated that deep fried texture d vegetable protein chunks were an acceptab le product . It had a predominant sweet taste and a slightly detectable bitter and burning aftertas te ; however, these effects were more pronounced in samples at Aw of 0.70 than at 0.80. Protein chunks at 0.80 Aw were rated slightly toughe r and drier than those at 0.70 Aw . TABLE OF CONTENTS CHAPTER PAGE
I. INTRODUCTION. . 1
II. REVIEW OF LITERATURE . 3
The Fundamentals of Intermediate Moisture Foods . 3 Definition and principle of intermediate
moisture foods ...... 3
The concept of water activity . . 4
Sorption phenomena in foods . • 5
Water activity and microbial growth . • 6
Water activity and chemical reactions . ..8
The Technology of Intermediate Moisture Foods ..11
Development of intermediate moisture foods . . . 11 Requirements and limitations of intermediate
moisture foods ...... 13 Formul ation and preparat ion of intermediate
moisture foods ...... 15 Storage stability of intermediate moisture
foods ...... 17
The Properties of Textured Vegetable Proteins . . 19 Definition and composition of textured
vegetable proteins ...... 19 Phys ical and functional properties of textured
vegetable proteins .. • • • • • 2 0 vi vi i
CHAPTER PAGE
Stability and vers atility of textured
vegetable proteins ... • • 2 2
III. MATERIALS AND METHODS ... . • • • 2 3
Product Specification and Formulation of
Infus ion Solution ...... 23
Experimental Des ign ...... e ••24
Experiment one: storage stab ility at 0.85
water activity ...... 24
Experiment two: storage stability at 0.80
water activi ty . .26
Experiment three: preparat ion of samp les for
sens ory evaluation ...... •...... 30
Methodology and Instrumentation . . . o .33
Water activity determination . o o .35
Moisture determination. . . 35
pH measurement .. .. G •••••• ..36
Protein de terminat ion • . . . o • • • . .36
Sodium chloride determination . o o •• o o • o36
Shear press measurement o o • • • • • • 37
Color me as urement . . o 0 0 • 3 7
Microbiological examination . o ••• o ••••37
Plating pro cedures ... . 0 • 3 7
Study of cultures . .. o o •••••o •••38
Sensory evaluation . o39 viii CHAPTER PAGE
Test methods ...... 39
Sensory panel . . • 39
Test procedures . . . . .40 Statistical analysis. . .40
IV. EXPERIMENTAL RESULTS ••. • • • • 4 2 Properties and Storage Stability of Intermediate Moisture Textured Vegetable Protein Chunks
With Target Water Activity of 0.8 5 ....•..42
Water activity ...... • . . • . . ...42 Moisture content .. .45
pH value . . • • • • 4 5 Firmness. .49
Co lor . • . • . . 54 Correlation between variables .62
Proximate analysis •• . • 66
Microbiology ...•. • • 66 Properties and Storage Stability of Intermediate Moisture Textured Vegetable Protein Chunks
With Target Water Activity of 0.80 ...•...71
Water activity . e • .71
Mo isture content .. . . 71
pH value . • • 7 4
Firmness . • 7 4 .
Color ...... 80 ix
CHAPTER PAGE
Correlation between variables . • • • 8 5
Proximate analys is .. .85
Microbiology .... • • • 8 5
Sens ory Evaluat ion of Deep-Fried Interme diate
Moisture Textured Vegetable Protein Chunks ...89
V. DISCUSS ION...... 9 3
VI . SUMMARY .. . • 10 5
LITERAT URE CITED. 10 8
APPENDIXES. . • • . • . • 115
Appendix A...... 116
Appendix B. 140
• VITA...... 14 5 LIST OF TABLES TABLE PAGE I. Composition (Per Cent) of the Infusion Solutions Used in the Preparation of Intermediate Moisture Textured Vegetable Protein Chunks . .27 II. Compos ition (Per Cent) of the Infusion Solutions Used in the Preparation of Intermediate Moisture Textured Vegetable Protein Chunks for Sensory Evaluation ...... 32 III. F-Table Showing the Effect of Treatments on Water Activity of Intermediate Moisture Textured Vegetabl e Protein Chunks with Target Water Activity of 0.85...... 43 IV. Effect of Treatments on Water Activity of Inter mediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 .... 44 V. F-Table Showing the Effect of Treatments on Mo isture Content of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85...... 46 VI . Effect of Treatments on Moisture Content of Intermediate Moisture Textured Vegetable Pro- tein Chunks with Target Water Activity of 0.85 ..47 VI I. F-Table Showing the Effe ct of Treatments on pH of
X xi TABLE PAGE Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.8So . . .48 VI II. Effect of Treatments on pH of Intermediate Moisture Textured Vegetable Protein Chunks
with Target Water Activity of 0.85 . 0 . . .so IX. Effect of the Interaction of Calcium Lactate and Acidity on pH of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 .. o • . . . 51 X. F-Table Showing the Effect of Treatments on Firmness of Intermediate Mo isture Textured Vegetable Protein Chunks with Target Water
Activity of 0.85 . .5 2 XI . Effect of Tre atments on Firmness of Intermediate Mo isture Textured Vegetable Protein Chunks with Target Water Activi ty of 0.85 .•. .53 XII. Effect of the Interaction of Calcium Lactate and Acidity on Firmness of Interme diate Moisture Textured Vegetable Protein Chunks with Target
Water Activity of 0.85 ...... 55 XIII. F-Table Showing the Effect of Treatments on C.I.E.
Coordinates " X " and "Y" of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 .•...... 56 xii
TABLE PAGE XIV. F-Table Showing the Effect of Tre atments on Ligh tness Index of Intermediate Mo isture Textured Ve getable Protein Chunks with Target Water Activity of 0.8 5 ...... 5 7 XV . Effect of Tre atments on Color Indices of Inter mediate Moisture Textured Ve getable Protein Chunks with Target Water Activity of 0.85 ....58 XVI . Effect of the Interaction of Calcium Lact ate and Acidity on Color Indices of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Act ivity of 0.85 .61 XVI I. Effect of the Interaction of Acidity and Storage Time on Color Indices of Intermedi ate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 ...... 63 XVIII. Correlation Coefficients between Water Activity , Moisture Content , pH , Firmness and C.I.E.
Coordinates "X" and "Y" of Interme diate Moisture Textured Ve getable Protein Chunks with Target Water Activity of 0.85 . . . . .65 XIX. Composit ion (Per Cent) of Intermediate Moisture Textured Vegetable Protein Chunks with Target
Water Activity of 0.85 ...... 6 7 xiii TABLE PAGE XX. F-Tab le Showing the Effect of Treatments on Water Activity of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80...... 72 XXI . Effect of Treatments on Water Activity of Inter mediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80 ...73 XXII. F-Tab le Showing the Effect of Treatments on Mo isture Content of Intermediate Moisture Textured Vegetable Protein Chunks with Target
Water Activity of 0.80...... 75 XXIII . Effect of Treatments on Mo isture Content of Intermediate Moisture Textured Vegetable Pro tein Chunks with Target Water Activity of 0.80 ...... 86 XXIV. F-Table Showing the Effect of Treatments on pH of Intermediate Mo isture Textured Ve getable Protein Chunks with Target Water Activity of 0.80 ...... 77 XXV. Effect of Treatments on pH of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80 ...... 78 XXVI . F-Table Showing the Effe ct of Tre atments on Firmness of Intermediate Moisture Textured xiv TABLE PAGE Vegetable Protein Chunks with Target Water Activity of 0.80...... 79 XXVI I. Effect of Treatments on Firmness of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80 ...... 81 XXVIII. F-Table Showing the Effect of Treatments on
C.I.E. Coordinates "X" and "Y" of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80 ...... 82 XXI X. F-Table Showing the Effect of Tre atments on Lightness Index of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80 ...... 83 XXX. Effect of Tre atments on Color Indices of Inter mediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80 ...84 XXXI . Correlation Coefficients between Water Activity , Moisture Content and pH of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80 ...... 86 XXXI I. Composit ion (Per Cent) of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80 ...... 87 XV
TABLE PAGE XXXIII. F-Table Showing the Effect of Water Activity on Sensory Panel Scores of Deep-Fried Inter mediate Mo isture Textured Vegetable Protein Chunks ...... 9 0 XXXIV. Effect of Water Activity on Sensory Panel Scores of Deep-Fried Intermediate Moisture Textured
Vegetable Protein Chunks ...... 92 XXXV. Formulas for the Calculation of C.I.E. Values from Color-Eye Colorimeter Tri-St imulus Data
(X, Y, Z, X) ••• ••••••••••••••11 6 XXXVI . Effect of the Interaction of Calcium Lactate and Acidity on Water Activity of Intermediate Moisture Textured Vegetable Protein Chunks
with Target Water Activity of 0.85 ...... 117 XXXVII. Effect of the Interaction of Calcium Lactate and Storage Time on Water Activity of Inter mediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 ..118 XXXVI II. Effect of the Interaction of Acidity and Storage Time on Water Activity of Inter- mediate Moisture Textured Vegetable Protein Chunks with Target Water Act ivity of 0.85 ..119 XXXI X. Effect of the Interaction of Calcium Lactate, Acidity and Storage Time on Water Activity xvi TABLE PAGE of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 ...... 120 XL . Effect of the Interaction of Calcium Lactate and Acidity on Moisture Content of Inter- mediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 ..121 XLI. Effect of the Interaction of Calcium Lactate and Storage Time on Moisture Content of Inter mediate Moisture Textured Vegetable Protein
Chunks with Target Water Activity of 0.85 ..122 XLI I. Effect of the Interaction of Acidity and Storage Time on Moisture Content of Inter mediate Moisture Textured Vegetable Protein
Chunks with Target Water Activity of 0�85 I • 123 XLI II. Effect of the Interaction of Calcium Lactate, Acidity and Storage Time on Moisture Content of Intermediate Moisture Textured Vegetab le Protein Chunks with Target Water Activity
of 0.85 I I I I I I I I I I I I I I I I 0 I I 124 XLIV. Effect of the Interaction of Calcium Lactate and Storage Time on pH of Intermediate Moisture Textured Vegetable Protein Chunks
with Target Water Activity of 0�85� 1 1 •• I 125 xvii
TABLE PAGE XLV . Effect of the Interaction of Acidity and Storage Time on pH of Interme diate Mo isture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 ...... 126 XLVI . Effe ct of the Interaction of Calcium Lactate , Acidity and Storage Time on pH of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.8 5 ...... 127 XLVI I. Effect of the Interaction of Calcium Lactate and Storage Time on Firmness of Intermediate Moisture Textured Ve getable Protein Chunks with Target Water Activity of 0.85. . 128 XLVI II. Effect of the Interaction of Acidity and Storage Time on Firmness of Intermediate Moisture Textured Ve getable Protein Chunks with Target Water Activity of 0.85 ...... 12 9 XL IX. Effect of the Interaction of Calcium Lactate , Acidity and Storage Time on Firmness of Intermediate Mo isture Textured Vegetable Protein Chunks with Target Water Activity
of 0.85 . . . • ...... 13 0 L. Effect of the Interaction of Calcium Lactate . and Storage Time on Color Indices of Inter mediate Moisture Textured Vegetable Protein xviii TABLE PAGE Chunks with Target Water Activity of 0.85 ..131 LI . Effect of the Interaction of Calcium Lactate , Acidity and Storage Time on Color Indices of Intermediate Moisture Textured Ve getable Protein Chunks with Target Water Ac tivity of 0.85 ...... 133 LII. Effect of the Interaction of Acidity and Storage Time on Water Activity of Inter- mediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80 ..13 5 LII I. Effect of the Interaction of Acidity and Storage Time on Moisture Content of Inter mediate Mo isture Textured Ve getable Protein Chunks with Target Water Activity of 0.80 ..136 LIV. Effect of the Interaction of Acidity and Storage Time on pH of Intermedi ate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.80...... 137 LV. Effe ct of the Interaction of Acidity and Storage Time on Firmness of Intermediate Mo isture Textured Vegetable Protein Chunks with Target Water Activity of 0.80. . 138 LVI . Effe ct of the Interaction of Acidity and Storage Time on Color Indices of xix TABLE PAGE Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity
of 0.80 ...... 13 9 LIST OF FIGURES FIGURE PAGE 1. Diagram for the Preparation of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 ...... 25 2. Diagram for the Preparation of Intermediate Moisture Textured Ve getable Protein Chunks with
Target Water Activity of 0. 80 ...... 29 3. Diagram for the Preparation of Deep-Fried Inter mediate Moisture Textured Vegetable Protein Chunks for Sens ory Evaluation ...... 31 4. Tot al Plate Count of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 and at pH of 7.0 ...... 68 5. Total Plate Count of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water Activity of 0.85 and at pH of 5.5 ...... 69 6. Total Plate Count of Intermediate Moisture Textured Vegetable Protein Chunks with Target Water
Activity of 0.80...... • • 8 8 7. Effect of Calcium Lactate and pH on the Color of Intermediate Moisture Textured Vegetable Protein Chunks ...... 140
XX CHAPTER I
INTRODUCTION
Intermediate moisture technology has gained consider able recognition recently as a method of food preservation . This is due primarily to the successful commercial develop ment of soft, moist pet foods and to the availab ility of acceptable antimycotics . Essentially , intermediate moisture foods have the moist texture and structural character of natural food materials.and the shelf stability of dehydrated �oods . The properties that permit stability during storage are due to the addition of water soluble solids which impart bacterio static protection to the food. With these attributes , · the development of intermediate moisture human foods was initi ated. However, production of acceptable foods was more difficult than anticipated. Prototype products developed so far have met the storage stability requirement , but their flavor ratings are far from satisfactory . Undesirable flavor characteristics are imparted by the additives used to bring the water activity of the food into the interme diate moisture range. An effort is being made to find acceptable additives which have negligible flavor effects . Another problem encountered in the development of 1 2 intermedi ate moisture human foods is the establishment of a true moisture equilibrium throughout the product. This re quires that the food material be porous and ab sorptive so that the solution containing the additives can penetrate uniformly into the system . Some conventional foods have complex structures which interfere with solute diffusion. However, · textured vegetable protein , an extruded soybean flour product , lends its elf readily to the formation of a stable equilibrium because it is porous and ab sorptive . Furthermore , it has a chewy texture which is desi rable in many food products . This study was conducted to develop an intermediate moisture product using dry extruded textured vegetable pro tein chunks as the food base; to determine the microbial, chemical and phys ical characteristics during storage and to evaluate the overal l acceptab ility of the product . CHAPTER II
RE VIEW OF LITERATURE
I. THE FUNDAMENTALS OF INTERMEDIATE MOISTURE FOODS
Definition and Principle of Intermediate Mo isture Foods
An intermediate mo isture (IM) food is one that is suf- ficiently moist to be eaten without rehydration, ·and yet is shelf stable without refrigeration or heat processing (33)*. Its moisture level, generally in the range of 20 to SO per cent, makes intermediate moisture food acceptable for direct consumption. The lowering of water activity , due to the presence of concentrations of solutes , makes it res istant to microb ial deterioration (13, 53) . Microorganisms require moisture for growth . However, it is the amount of available moisture rather than total moisture which determines their growth limits (24 , 41) . Thus , total moisture content is not an accurate indication of food stability . A factor more clos ely related to this is the water activity of the system , which measures the availability of water for biological and chemical reactions (41, SO, 54) . The control of water activity through the concentrations of solutes is the basic principle of inter- mediate moisture foods .
*The numbers in parentheses repres ent similarly numbered reference in the Literature Cited. 3 4 The Concept of Water Activity Water activity (Aw) is defined as the ratio of the vapor pressure of water (P) in the food to the vapor pres sure of pure water (Po) under identical conditions , Aw = P/Po (13, 36) . The presence of dissolved solids in the food causes a difference between P and Po (33) . Thus , pure water has a Aw of 1.00 whi le water in a food solution has a value less than one (12, 24) . By regulating the composi tion of the moisture phase, the water activity of a sub stance can be controlled (12) . In an ideal solution , the extent to which the vapor pressure of the solvent is decreased is given by Raoult's Law:
where n1 and n2 refer to the number of mo les of solute and solvent in the solut ion , respectively (12, 17) . In practice , however, solutions of electrolytes and non-electrolytes deviate greatly from ideal behavior (12, 17). For example , to obtain a Aw of 0.90 at 25° C, a total solute concentration of 6.17 moles is required for an ideal solution ; for sucrose solution , 4.11 moles ; and for sodium chloride solution , only 2.83 moles . Generally , the ef fective concentration is much greater than its actual con centration (12, 53) . 5 Sorption Phenomena in Foods The water activity of a sys tem is related to the sorption properties of foods (36) . This relat ionship is represented graphically by the water sorption isotherm, which is a plot of the moisture content versus its equilib rium relat ive humidity at a constant temperature (12, 17) . Since the activity of a vapor equals the activity of the corresponding liquid phase at equilibrium, the water ac tivity value is numerically equal to the decimal fraction corresponding to per cent relative humidity (12, 36) . Generally , the isotherm for food is a sigmoid curve . Water content increas es from 0 at 0.00 Aw to infinity at 1.00 Aw ( 17) . The water sorption isotherm can be divided into three sections depending on the state of water present. The first section corresponds to the portion below its first inflec tion point , known as the adsorbed monomolecular layer of water. Here, water is tightly bound and the food is micro biologically stable . Ab ove this point and up to the second inflection point corresponds to the second section. Water exists largely in mul timolecular layers , less tightly held to food constituents. The area toward the higher end of this section is representative of intermediate moisture foods (33, 36, 53) . The food is bacteriologically stab le but may support mold and yeas t growth . Beyond this second 6 section moisture generally is cons idered to be largely free water and food is subject to microbial spoilage (53) . It is apparent that water activity has signifi cant effects on the extent of microbial activity in a food system.
Water Activity and Microbial Growth Each microorganism has its own characteristic opt imal water activity and its own range of water activity for growth . As the water activity is reduced from its opt imum , there is a lengthening of the lag phase of growth , a de crease in growth rate and the amount of cell substance syn thesized (12, 24) . There are six factors that affect the water activity requirements of microorganisms (24 , ·57) : 1) The kind of solute us ed to reduce water activity . For many organisms , the limiting water activi ty for growth is independent of the solute used. However, a few organisms have lower limiting water activity values with some solutes than with others . For example , potassium chloride is less inhibitory than sodium ch loride , and it in turn is less toxic than sodium sulfate . 2) The nut ritive value of the culture medium . The presence of nutrients increas es th e range of water activity over which the organisms can sur vive . 3) Temperature . The greatest tolerance to low water activity occurs at the optimum temperature fo r growth . As temperature is decreased or increased, the range of water 7 activity permitting growth is reduced. 4) Oxygen supply . The presence of oxygen increases the range of water activity over wh ich aerobic organisms can grow . For anaerob ic or ganisms , tolerance to low water activity is greatest in the ab sence of oxygen. 5) pH . Most organisms are more toler ant of low water activity at pH values near neutrality than in acid or alkaline media. 6) Inhibitor. The presence of inh ibitors narrows the range of water activity for microb ial growth . In general, bacteria require more available moisture than do yeasts or molds . A Aw of 0.91 is normally suffi cient to suppress bacterial growth ; howeve r, halophiles may survive even down at Aw of 0.75. Most species of yeasts and molds are inhibited at Aw of 0.88 and 0.80, respectively , although xerophilic molds and osmophilic yeas ts can still grow at Aw between 0.65 and 0.60 (52) . Clos tridium botulinum has been reported to grow only at Aw above 0.94. Growth of Staphylococcus aureus , Escherichia coli, Clos tridium� frigens , and Salmonella can be prevented at Aw below 0.86
( 33) • Bas ed on the ab ove minimal limits , Aw values below 0.65 are required to completely inhibit all microbial growth. However, such a low water activity generally is not applicable in the fabrication of intermediate moisture foods . At a Aw of 0.65, most food would resemble 8 commercially dehydrated products . The total moisture content would be well below 20 per .cent , thus foods lose their nat ural texture and give a sensation of harsh dryness (13, 53) . To retain the semi-moist texture , Aw values between 0.70 and 0.85 are recommended. Although these levels are not suffi ciently low for long-term inhibition of mold growth , addition of antimycotics would contribute some preservative effect and prolong storage stab ility (33, 53) .
Water Activity and Chemical Reactions In addition to controlling microbial growth , water activity also has significant effects on the enzy matic and chemical processes in a food system . Studies have shown that the rates of various deteriorative reactions such as lipid oxidation , non- enzymatic browning and enzymatic browning depend largely on water activity . The dominating effect of water is due to its two important properties: 1) It can act as a solvent for the reactants and 2) it can be a reactant itself and it participates in speci fic reac tions . This is demonstrated in a model system studied by Schobell (56) . A saturated sucrose solution containing citric acid was free ze-dried to a Aw of 0.60 to 0.70. It was found that even at th is low moisture level, hydrolysis of sucrose occurred in the presence of citric acid. Water acts as a solvent of the reaction , dissolves further reactants and transports them to the reaction sites and also 9 participates in the reaction, as shown by the following equation: H+ sucres� + H2o ------� glucose + fructose
The reducing sugars formed in the ab ove model sys tem also indicated the potential sus ceptability of an initially stable system to non-enzymatic browning during storage . This was verified in a study conducted by Karel and Labuza (35) . It was found that browning in all carb onyl- containing system occurs whether they are present initially or formed during storage. The relationship between rate of non en�ymatic browning and water activity was studied by Labuza, et al ., (38) . Results showed that as humidity increas ed, browning rate increas ed up to a maximum point in the range of intermediate moisture foods ; then it decreas ed as humidity was further increas ed. Therefore , to reduce non- enzymatic browning during storage , water activity of intermediate moisture food mus t be held above the point of maximum brown- ing (38). Many studies on freeze-dried and dehydrated foods showed that maximum resistance to lipid oxidation during storage occurs above the monolayer region in the sorption isotherm (48, 55) . Martinez and Labuza (48) found that freeze-dried salmon has best overall stability at 32 per cent relative humidity . This is far in excess of the 10 recommended 2 per cent maximum for most dehydrated foods . The oxidation of methyl linoleate in a freeze-dried model sys tem was investigated by Maloney, et al ., (43) at Aw 's ranging from 0.0 to 0.6. Water was found to have a pro tective effect on oxidation . As its concentrat ion was increased, reaction rate decreas ed up to about 0.5 Aw where a level ing off occurred. Further study on this area was done by Labuza, et al., (38) on the oxidation of linoleic acid. It was observed that as moisture level was increased beyond 0.5 Aw , the protective effect of water was reduced and oxidation increased. This means that within the intermediate moisture range , control of lipid oxidation is more effective at the lower water activity limit. In any food system, chemical reactions do not take place independently; complicating factors always occur. LeRoux and Tan (40) studied the interaction between lipid oxidation and non-enzymatic browning in a system of casein methyl linoleate at several humidities . Results indicated that at high humidities browning occurred at a rapid rate due to lipid oxidation products and at low humidites little browning took place. The interaction between protein and lipid oxidation was investigated by Labuza, et !k, (37) . Lipid oxidation resulted in the production of free radicals wh ich reacted with protein even at low water activity caus ing its degradation. All these interactions have effects 11 on the stability of intermediate moisture foods . Extensive studies have been conducted by Acker (1, 2) on the relation between water activity and enzyme activity . In a model system of barley malt and lecithin (1) , enzymatic reaction was found to proceed rapidly at high humidities . No change was observed at low humidities where water content was below the monomolecular-region . In another study with low moisture foods , Acker (2) ob served that diffus ion and mobility of the substrate determines enzymatic reaction rate . At low water activity , free water is limited; therefore , diffusion of the substrate is hindered and enzymatic activity occurs at a very low rate. That some enzymatic reactions do occur even at very low water activity is verified by the observations of Duckworth, � al ., (21) . In their study on the diffusion of solutes at low moisture level , it was found that substantial diffusion and mobility of substrate occurs even at water activity clos e to the monolayer region . At intermediate moisture range , water serves both as a solvent for the substrate and a medium for enzyme reaction. It was concluded that to increase storage life of intermedi ate moisture foods , enzymes must be inactivated (38).
II. THE TECHNOLOGY OF INTERMEDIATE MOISTURE FOODS
Development of Intermediate Moisture Foods Historical ly , the fundamental principles of intermediate 12 moisture· foods have been applied to the preservation of foods for centuries. Such common items as jams and jellies, dried fruits , dry sausage, jerky and country ham are essentially intermediate moisture foods (13 , 33). Yet , a new series of food products is currently evo lving which represents a maj or breakthrough in intermediate moisture technology. The availability of acceptable ant imycotics which are effective for suppressing the growth of yeas t and mold facilitated this development (20). Initial development of intermedi ate moisture foods was mainly confined to pet foods (33 , 53). Prior to this, pet foods were processed either dry or canned. Dry pet foods have excellent storage characteristics , high nutri tional value , relative low cost but poor pal atability. Canned pet foods , on the other hand , have meat-like texture and appeal but are expensive and deteriorate rapidly once opened. Th e need for a convenient , shelf stable, ready-to eat pet food brought ab out a new look into the basics of water activity and intermediate moisture phenomena (20 , 33). Intermediate mo isture pet food was found to meet all these requirements and commercial manufacture was started. Th e success and excellent storage stability of inter mediate moisture pet foods motivated the extens ion of this technology to human foods . Firs t attempts made on meat spread products were shelf stable; however, the texture did 13 not. resemble that of real meat and flavor was strongly spiced to make it acceptable (33) . Extens ive work conducted by the General Foods Technical Center on possible infusion methods led to increased retention of natural appearance and texture of foods (33) . Such items as diced chicken, coars e ground beef, diced carrots , beef stew, barb ecued pork and apple pie filling had been prepared with Aw ranging from 0.71 to 0.81 (33, 53). ' . Recently , the feas ibility of preparing complex mix tures intermediate moisture items such as chicken a la king , ham in cream sauce , Hungarian goulash and Irish stew was investigated. This involved a number of components in the same system . A trans fer of moisture from the higher water activity to the lower water activity will occur until a single equilibrium water activity is reached. At this point , the components will retain different total moisture con tents , according to their individual water sorption iso- therm (53) . In spite of the considerable progress made , intermediate moisture human foods are still in the proto- type stage (33) . Much research has to be done to meet the requirements and overcome the limitations before commercial development of intermediate moisture human foods can get underway .
Requirements and Limitations of Intermediate Mo isture Foods In pioneering the work on intermediate moisture human 14 foods , General Foods set up three specificat ions: 1) the products should be acceptable after four months ' storage at 38° C, 2) the additives and antimycotics used should meet the Food and Drug Administration (FDA) requirements , and 3) the structure and texture of the natural food material must be retained (33) . As mentioned earlier, indefinite shelf life can be attained by lowering the Aw to below 0.65; how ever, this is impractical in relation to the sens ory at tributes of such products . The moisture content would generally be too low to impart the semi-moist texture and the high concent ration of solutes required would reduce their palatability and acceptability. Th erefore , the Aw need to be raised to between 0.70 and 0.85 (53) . To meet the shelf stability requirement at this water activity range , some antimycotics have to be added to prevent mold and yeast growth . Potass ium sorb ate , sorb ic acid, and propylene glyco l have been approved by the FDA for such use . The principal additive used in controlling water activity in pet food is sugar . Ab out 22 per cent sugar is required to produce a stab le intermediate moisture food with a Aw of 0.78 (33 , 53). While pets can tolerate this amount of sugar in their meat , such a high level would be undesirable to humans . Furthermore , the sweet taste imparted would be abnormal to many meat and vegetable products (13 , 33) . Sodium ch loride (NaCl) , being an 15 electrolyte, is a good agent for depressing water activity . However, it is again limited in use and only a normal leve l of seas oning can be added (13) . The combination of NaCl and glycerol is pres ently the most promising. Glycerol is substituted for sugar because it has less flavor impact and is better tolerated physiological ly ; however, it imparts some bitter aftertaste. Th is limitation of sweetness and bitterness contributed by the additives necessitates further effort to find other sources of water soluble solids (13 , 33) . Th e soft , moist technology employed in the manufacture of intermediate moisture pet foods is not applicable to intermediate moisture human foods . Essentially , it involves the mixing and grinding together of wet and dry materials to produce equilibrium throughout the aqueous phase (20 , 33) . This then limits its application to comminuted products which do not have the texture of real meat (33) . In order to meet the specification on texture , each dis tinct food piece must be infused with additives until the desired water activity is reached. Two infusion methods have been devised to overcome this problem and in addition,· help to retain the appearance and flavor of the natural food ( 33) .
Formulation and Preparation of Intermediate Moisture Foods In fabricating intermediate moisture food, the first 16 step is the selection of an appropriate target water activity
(53) . This differs among different foods , depending on their nature and history. The de gree of processing, type of pack aging and condition of storing must be taken into cons idera tion. Generally , heat processed foods sealed in moisture proof packages and stored under optimum conditions offer longer shelf life even at minimum water activity level (13) . The next step is to consider the formula ingredients neces sary to provide the desired water activity (53) . Since the additives will diffuse into the food until the final concen tration in the food is the same as th at in the solution , the composition of the solution must be based on the amount of water in the food to be treated and the des ired composition of the finished product (33) . A typical formulation for intermediate moisture carrots developed by General Foods is 51.1 per cent glyce rol , 39 .3 per cent water, 2.1 per cent NaCl , 0.9 per cent propylene gly col and 0.3 per cent potas sium sorbate . The final Aw of the product is 0.77 (33) . The principal agents for depressing water activity are glycerol and NaCl. Propylene glycol contributes slightly to lowering water activity . In addition , it provides some antimycotic effect and-increas es the plasti city of the food. Although Raoult's Law applies only to ideal solu tions , it can still be used to approximate the water activity level of the infusion solut ion. Among the three principal 17 additives mentioned, NaCl is most effective in controlling water activity per mole of solute , then glycerol and the least is sucrose (53) . One important consideration in es tablishing water activity is the "salting-in" and "salting out" effects of solutes in foods (12) . Salting-in causes the effective concentration of solutes to be smaller than the actual concentration. For example , the effective con centration of NaCl is reduced when urea is present . Salting out effect gives an opposite reaction. Sucrose and mannitol were found to cause mutual increases in effective concentra tions . The food is then immersed in the infusion solution until equilibrium is reached. It is essential that the aqueous phase present in the food be brought uniformly to the desired level of water activity so that each piece has the proper water activity and moisture content (13 , 33) . The starting food material can either be in the natural form or freeze-dried form. Both have sponge -like struc tures and ab sorb the infusion solution eas ily without caus ing shrinkage . The amount of solution ab sorbed is approximately equal to the water holding capacity of the food (33, 53) .
Storage Stability of Intermediate Mo isture Foods Results of the studies conducted so far seem to indicate that intermediate moisture foods are stab le during 18 storage , . both microbiologically and chemically . All experi mental products developed by the General Foods Technical Center were stored for four months at 32° F and 100.4° F. No significant microbial growth was observed. Cultures of S. aureus were inoculated into two intermediate moisture casserole items and tests showed considerable reduction in viable bacteria during storage . There was no significant change in sensory properties either. Flavor and texture of the stored products were rated comparab le to the freshly prepared full moisture products. The color and flavor of intermediate moisture carrots were retained and celery was found to have a marked crispness (13) . Chen (15) studied the physical and chemical changes of intermediate moisture breaded deep-fried catfish during a five -week storage period at 100° F. Mo isture level and pH were found to decrease at the initial stage . Firmness of the fish flesh increased during the firs t three weeks . No rancidity nor color change was observed in the stored products. Collins , et al ., (1 8) made a similar study; how ever, the storage temperature was lowered to 80° F. Mold and standard plate count revealed no microb ial growth after five weeks of storage. A decrease in moisture content was observed, but there was no overall change in water activity . No rancidity was detected on samples stored for nine months . Since food is a complex system , it is difficult to 19 predict the extent of deterioration as a function of water activity and water content , especially at the intermediate moisture level (38) . Additional studies are needed in the area of enzymatic reactions , chemical interactions and their effects on the sensory attributes of intermediate moisture foods . Although much knowledge has been gained on the rela tion between water activity and microbial growth , stab ility of intermediate moisture foods on an extended basis still needs to be investigated.
III. THE PROPERTIES OF TEXTURED VEGETABLE PROTE INS
Definition and Composition of Textured Vegetable Proteins Textured vegetable protein is a food product derived from edible protein sources and is characterized by an identifiable texture and structure which can withstand cook ing and retorting without physical breakdown (23) . Basic ally, it cons ists of three components ; namely , the protein material , fat , and a binding sys tem. In addition, such ingredients as carb ohydrates , stab ilizers , flavors , colors and nutrients may be added (62) . Its composition and formul ation are varied according to the ultimate utiliza tion. Generally , dried textured protein contains 8 per cent moisture , SO per cent protein, 32 per cent carbohydrate, 1 per cent fat , 3 per cent fiber, and 6 per cent ash . 20 When rehydrat ed, it holds three times its weight in water with a solid content of ab out 30 per cent (7, 66) . When simulated into meat analogs, its fat content is increased to ab out 21 per cent while carbohydrate is reduced to 24 per cent (7). Each component of the textured protein can be precisely contro lled to provide the proper phys ical and functional properties desired.
Phys ical and Functional Prope rties of Textured Vegetable Proteins Textured vegetable protein is available in two forms, depending on the texturizing process employed. The spun protein is made up of protein fibers bound together into meat-like structure while textured protein is extruded protein shaped into fib rous textured form (29, 65, 66) . Since textured ve getable protein is a fabricated product, texture, size, and shape can be bui lt into the protein to meet specific requirements. Its texture may be tende r or tough, crunchy or chewy . It may come as chips, di ces, slices or fl akes with sizes ranging from fines to granules to one- inch chunks (47, 65, 66) . Structurally, textured vegetable protein resembles freeze-dried meat with densities ranging from 9 to 25 lbs ./ ft3 . (61) . It has excellent water and fat ab sorbing prop erties which make it capable of carrying and retaining any flavor, color, or additive . Because of its porous structure, 21 it absorb s liquid easily without causing shrinkage . The rate and amount of ab sorption is determined by its basic structure and particle size (46 , 61) . Since textured vegetable protein is used primarily in the pr�paration of meat analogs , caramel coloring and meat flavorings are incorporated into the product during proces�ing. Mos t of the flavorings used are derived from meat sources since synthetic flavors have not been fully developed to retain the true essence of meat fl avors (29) . In its natural state , textured vegetable protein has a light tan color and bland flavor (61) . However, characteristic beany and bitter flavors are imparted when soy protein is used as the basic material . These flavors are derived from enzymatic and non-enzymatic reactions during macera tion of the soybean (49 , 64). Many methods of eliminating these objectionab le flavors have been investigated. Re cently, Fuj imaki, et al ., (25) applied proteolytic enzymes to soy proteins and reported their effectiveness as de odorizing agents . Arai , et al ., (6) carried this experi ment further and found a combination of two enzyme prep arations capable of deodorizing and debittering soy protein . However, more knowledge and understanding of the flavor components of the soybean is necess ary before this problem can be completely overcome . 22 Stability and Versatility of Textured Vegetable Proteins Freshly prepared textured vegetable protein is a perishable food and must be properly handled and processed for extended storage. Commercially , it is prepared in canned, frozen or dehydrated forms (62, 65) . Dried tex tured vegetab le protein was found to have low bacterial counts and no off-flavor was detected in samples stored for one year (22) . For canned and fro zen samples , shape and texture of the protein products are maintained during storage. Because of its physical properties , textured vege table prqtein is adaptab le to any method of food preserva tion. Its structural integrity makes it capab le of with standing heat during canning and drying (23) . Th e ab sence of cel lular structure makes it suitable for freezing without textural damage due to ice crystal formation (51) . Its porosity and ab sorbing properties make it possible to retain additives and chemical preservatives without causing shrink age or shrivelling (46) . With all these process ing poten tials and the versatility in texture , size , shape , color , and flavor, there is no limit to the kinds of foods that can be fabricated from textured vegetable proteins (45) . CHAPTER III
MATERIALS AND METHODS
I. PRODUCT SPECIFICATION AND .FORMULAT ION OF INFUS ION SOLUTION
The textured vegetable protein chunks were ob tained from Archer Daniels Midland Comp any, Decatur, Illinois . The dry protein chunks were the "fas t hydrating" type, uncolored and unflavored. The size specification was : chunks No . 10 , approximate dimens ion of 3/8" x 3/8" x 1/2" and bulk dens ity
of 27 � 3 lbs ./ft . 3 (7) . A target water activity of 0.85 was selected for the preparat ion of the intermediate moisture textured vegetable protein item. The protein chunks , when rehydrated, had a moisture content of 70.7 per cent . It was as sumed that the same amount of an infusion solution would be absorbed during equilibration and the infus ion solution formulation was based on this moisture level. The two principal water activity depress ing agents utilized were sorbitol and NaCl . Sorbitol was substituted for the formerly studied glyce rol because of the lesser advers e flavor effects from sorbitol. The amount of NaCl used was limited to its highest threshold level. Potas s ium sorb ate, an antimycotic agent, was added up to the maximum 23 24 level permitted by FDA. Because of its sweetness, only a small amount of sucrose was added to provide a familiar mouthfeel and mask some of the flavor effects of sorb itol. Propylene glycol, which imparts an adverse taste , was used for its combined water activity depressing, plasticizing , and antimycotic effects , but in a small amount . With the levels of NaCl, propylene glycol, sucrose and potas s ium sorb ate held constant; the desired 0.85 Aw in the finished product was obtained by regulating the proportional amounts of sorbitol and water in the infus ion solutions .
II. EXPERIMENTAL DES IGN
Experiment One : Storage Stability at 0.85 Water Activity This experiment was conducted to determine storage stability of an intermediate moisture textured vegetable protein product at a Aw of 0.85. The product was prepared according to the procedure outlined in Figure 1. Dry protein chunks were held in boiling water for five minutes , drained and centrifuged at 95 x G for 1-1/2 minutes . During rehydration, 0.25 per cent calcium lactate (CaL) (g/100 ml water) was added as a firming agent to one half of the protein chunks . Each lot of the rehydrated products (without CaL and with CaL) were divided into two sublots . To one sublot from each lot, 0.45 per cent malic acid (g/100 ml solution) was added to lower the pH from 7.0 to 5.5. 25 Dry Protein Chunks
Rehydrated ..(- -boiled in water for 5 min. --),... I Rehydrated I (without CaL) (with 0.25% CaL) I I
I Centrifuged I" f-- at 95 X G for 1-1/2 min .
Centrifuged �at 95 X G for 1-1/2 min .
Finished Product (without CaL , (without CaL , lif 7 . 0) pH 5 .5)
Figure 1. Diagram for the preparation of intennediate moisture textured vegetable protein clnmks with target water activity of 0. 85. 26 The objective was to determine the effect of a lower pH on the chemical and physical properties and the microbial flora of the product. T�e rehydrated protein chunks were held in boiling infusion solution for ten minutes , drained and cen trifuged at 95 x G for 1-1/ 2 minutes to remove excess solu tion. The composition of the infusion solution us ed to lower the Aw to 0.85 is shown in Table I. Weight ratio of the rehydrated protein chunks to the infus ion solution was 3:5. There were · four treatments in this experiment : 1) without CaL, pH 7.0; 2) without CaL, pH 5.5; 3) with CaL , pH 7.0; and 4) with CaL, pH 5.5. Two hundred grams of the cooled, finished product from each treatment were placed in sterile pint size jars , in duplicate , and 70 grams were placed in sterile 2-1/ 2 ounce size jars . The former product was used for phys ical and chemical ·analyses wh ile the latter was used for micro biological examination. In both cases , the jars were filled to capacity . A total of 56 pint size jars and 28, 2-1/ 2 ounce size jars per replication were stored in an incubator at 26.70 C. Three jars from each tre atment were removed from storage for analysis of the product every ten days for a period of sixty days . The experiment was repli cated three times .
Experiment Two : Storage Stability at 0.80 Water Activity A second experiment was conducted to study the phys ical , 27
TABLE I
COMPOS ITION (PER CENT) OF THE INFUS ION SOLUTIONS USED IN THE PREPARATION OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTE IN CHUNKS
� tJ ComInfusponentsion Solu ofti theon Tar et Water A:c tivi t 0. s o.s Water 51.9 40 .6 Sorbitol 35 .4 46.7 Sodium chloride 6.4 6.4 Propylene glycol 2.8 2.8 Sucrose 2.8 2.8 Potas s ium s orb ate 0.7 0.7 Total 100.0 100 .0 28 chemical , and microb ial ch anges of the intermediate mo isture product during sto rage when Aw was lowered to 0.80. The preparatory steps were simil ar to those of Experiment One , except for two modifications (Figure 2) . Calcium lactate was not added during rehydration of the protein chunks . Ins tead of us ing centrifugat ion , the excess solution in the cooked samp les was pressed out with a Carver Press . Samples of 500 g. were filled to the top of the cy lindrical ce ll with alternating layers of cheese cloth and held at 100 lb s . pres sure for one minute . To lower the Aw of the product to 0.80, . the concentration of sorbitol was increased by 11 per cent ove r that amount used to lower Aw to 0.85 (Table I, page 27) . There was a corresponding decrease in the percentage of water wh ile the leve l of the other ingre dients was not ch anged. The pH of one half of the rehydrated product was adj us ted to 5.5 by the addition of 0.45 per cent malic acid (g/ 100 ml solution) to the infusion solution . In this ex periment , there were two tre atments : 1) without CaL , pH 7.0 and 2) without CaL , pH 5.5. Seventy grams of the cooled, finished product from each tre atment were placed in sterile 2-1/2 ounce size jars , in duplicate , and 200 g. were placed in sterile pint size jars . A total of 28, 2-1/ 2 ounce size jars and 16 pint size jars per replicate were stored in an incubator at 26 .7° C . for up to sixty days . Analysis were made on the fresh and 29
Centri fu ed �at 95 X G for 1-1/2 min .
---- �-- boiled in solution for 10 min . -�) I Cooked I dhunks :solution = 3:5 (with 0.45% acid)
Finished Product (pH 5. 5) I
I Stored I .-(--at 26 . 7° C for up to 60 days
Figure 2. Diagram for the preparation of intermediate moisture textured vegetable protein chunks with target water activity of 0.80 . 30 stored samples at ten day interval s. The experiment was rep licated two times .
Experiment Three: Preparation of Samples for Sensory Evalua tion This experiment was conducted to determine the overall acceptability of the intermediate moisture textured vegetable protein product at two levels of water activity , 0.70 and 0.80. The preparatory steps (Figure 3) were essentially similar to those of Experiment Two . The dry protein chunks were dehydrated in boiling water for five minutes , drained and centrifuged at 95 X G for 1-1/ 2 minutes . The rehydrated chunks were divided into two lots and cooked in two differ ent infusion solutions (Table II) to yield intermediate moisture products with initial Aw of 0.80 and 0.90.. In order to permit a more acceptable flavor, artificial flavor ings were added to the infusion solutions . The flavorings were 5 per cent Corral beef pas te (Pfizer, Incorporated, New York) , 1.5 per cent liquid hickory smoke (Hickory Specialties , Incorporated, Ocala, Florida) , and 0.2 per cent Eechee Ban seasoning (Takeda, Osaka , Japan) . All percentages were based on g/100 g. of solution . The excess solution in the cooked products was pressed out by a Carver Press at 100 lbs . pressure for one minute. The products were stored temporarily at 40° F. The intermediate moisture products were deep-fried 31
Dry Protein Chunks
�<---- with the addition of: ----�) 5% beef flavor 1.5% smoke flavor 0. 2% seasoning
Pressed �at 100 lbs . pressure I j for 1 min. l IM Product I lIM ProductI (Aw = 0. 90) (Aw = 0 .80)
Deep-fried at 350° F � for 1 min.
Finished Finished Product Product (Aw = 0. 80) (Aw = 0.70
Figure 3. Diagram for the preparation of deep-fried intermediate moisture textured vegetable protein Chunks for sensory evaluation. 32
TABLE I I
COMPOSITION (PER CENT) OF THE INFUSION SOLUTIONS USED IN THE PREPARAT ION OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS FOR SENSORY EVALUAT ION
Components of the Target Water Ac tivitr oer'ore DeeE-Fried Infus ion Solution o.go O.RO Water 60.4 40 .6 Sorbitol 26.9 46.7 Sodium chloride 6.4 6.4 Propylene glycol 2.8 2. 8 Sucrose 2.8 2.8 Potassium sorb ate 0.7 0.7 Total 100 .0 100.0 33 before sensory evaluat ion. Deep- frying imparted a ch aracter istic flavor to the products and lowered the water activi ty further by approximately 0.10 unit . Before frying , the samples were removed from the cooler and left to stand at room temperature for two hours . They were dipped in a di luted Seal- n - Save stabilizer solution , prepared with one part of stabilizer solution to two parts of water . Seal- n Save (Germantown Manufacturing Company , Broomall, Pennsyl vania) is a concentrated liquid stabilizer wh ich was used to form a moisture and oil barrier on the deep- fried items (27) . Moisture in food is sealed in and there is less oil penetration during deep- frying . The products were deep fried at 350 ° F for one minute , and excess oil was ab sorb ed on paper towels . The two samples , with initial Aw of 0.80 and 0.90 , had a final Aw of approximately 0.70 and 0.80, respect ive ly , after deep- frying . These samp les were used for sensory tests . The experiment was repli cated three times .
III . METHODOLOGY AND INSTRUMENTATION
In Experiment One , determinations of water activity , mo isture content , pH , texture , color, total plate count for bacteria and the count for yeas ts and mo lds were made on the fresh and stored samples at ten- day intervals for up to sixty days . There were four treatments with three jars per treatment per storage period . The contents of two jars 34 (pint siz e) were used for phys ical and chemical analyses and the contents of one jar (2-1/ 2 ounce size) was used for microbiological examinations . The experiment was replicated three times . Within each replicate, the following tests were conducted. For water activity , one determinat ion was conducted from each of the two jars . For moisture content and pH measurement , two samples were taken from each of the two jars . Texture and color measurements were obtained from three observations from each of the two jars . For the microbiological examinat ions , one sample was taken from the one jar and the slurry was used to determine total plate count and yeast and mold counts . Protein and sodium chloride contents were analyzed on the fresh samples and three ob servati ons were obtained for each treatment. In Experiment Two , similar analysis was made on the fresh and stored samples except for those of texture and color wh ich were made on the fresh samples and the samples held for sixty days . There were two treatments with two jars per tre atment per storage period. The contents of the two jars (2-1/2 ounce size) were used for the determinat ion of water activity , moisture content, pH and microbiological tests . For texture and color measurement , each tre atment had four jars (pint size) at zero storage and four jars held for sixty days . The experiment was replicated two times and the following tests were conducted for each replication. 35 For water activity , two determinations were conducted from each of the two jars . For moisture content and· pH measure ment , sampling was carried out in triplicate from each of the two jars . For texture and color, three measurements were conducted from each of the four jars . For micro biological tests , one sampling was made from each of the two jars . Protein and sodium ch loride contents were determined on the fresh samp les , and six observations were obtained from each treatment.
Water Activity Determination The water activity was de termined by an electric hygrometer indicator, Mode l 15-3001 (Hydrodynamics , Incor porated, Silver Spring, Maryland) . Twenty -five grams of samp le were sealed tightly in a half-pint jar. with a hydrosensor inserted through the lid. The hydrosensor was connected to the hygrometer indicator and when equilibrium was reached, the temperature and dial readings were re corded. The readings were converted to per cent relative humidity by the use of calib rated curves provided by the manufacturer. Water activity was calculated by dividing percentage of relative humidity by 100 . Readings were taken at 26.7° C � 0.5o C.
Moisture Determination Moisture content was determined by the vacuum oven 36 method (5) . Five grams of samples were dried at 70° C for sixteen hours at a vacuum of 508 torr. Results were ex pressed as grams of water per 5.00 grams of sample.
� Measurement A Beckman Zeromatic pH Meter was used to measure the pH of the samples . The slurry, prepared by blending 10 g. of samp le with 100 ml. of distilled water for one minute, was stirred continuously with a magnetic stirrer as the pH was being measured.
Protein Determination Total nitrogen content was determined by the modified Kjeldahl method (4) . Concentrated sulfuric acid and Kelpac package containing 10 g. potas s ium sul fate and 0.3 g. cupric sulfate were used for product digestion. Two grams of samp le were used for each test. Percentage of protein was calculated with the 6.25 fac tor and the amount of protein was based on the dry weight.
Sodium Chloride Determination The amount of NaCl was determined by the Volhard thiocyanate method described in Jacobs (32) . Two grams of sample were ashed in a muffle furnace at 500° C for five hours . The ash was dissolved in water and an aliquot was taken for analysis. The percentage of NaCl was calculated on the dry weight basis . 37 Shear Press Me asurement Res istance to shear was measured by the Allo- Krame r Shear Press, Model SP- 12, us ing a 1000-lb. proving ring and a 20- second thrust. Fifty grams of samp le were placed in a standard shear - compression cell , and the maximum force (dial readings) required to shear the samp le was recorded. The dial readings were converted to the equivalent pounds force by the us e of a standard curve .
Color Me asurement The Color Eye Colorimeter, Model D - 1 (Ins trument Development Laboratory , Division of Kollmo rgan Company , Attleboro , Massachusetts) , was us ed to measure the tristim ulus value s of the samples . Thirty grams of chopped sample were packed as uniformly as possible into a 2 - 1/2 inch diameter plexi- glass cylinder. The bottom, which was made of optical glass , was fitted over the samp le port and held tightly with rubb er bands . The colorimeter values were converted to the Commission Internationale de l'Ecl ainage (C. I.E.) X, Y, and Z values , from which were calculated the chromaticity coordinates x and y and lightness index (Table XXXV, App endix) (42) . Purity and dominant wavelength were obtained by reference to the C.I.E. chromaticity dia gram (30) .
Microbiological Examination Plating procedures . Plating was accomplished according 38 to the procedures recommended by the American Pub lic Health Association (58) . Twenty grams of sample were taken from each jar and blended with 180 g. of steri le water for one minute. Seri al dilutions were made and appropriate aliquots were plated. Plate count agar was used to determine total bacterial count with plates incubated at 32° C for 48 hours . Yeast and mold counts were determined by us ing acidified potato dextrose agar and the plates were incubated at 32° C for four days . The colonies were counted with the aid of a Spencer colony counter and results were expressed as the number of microorganisms per gram of sample.
Study of cultures . Test with the oxidas e reagent indicated that the colonies developing during the earlier storage. period were Pseudomonas spp . Representat ive colonies from the so- and 60-day storage periods were isolated onto plate count agar slants and incubated at 32° C for 48 hours . Pre liminary differentiation of the isolates were made according to cultural and morphological char acteristics and gram-stain reactions . Further identificat ion was cond ucted by the oxidase test, aerobic fermentation of glucose broth and production of ketogluconic acid. Pseudo monas spp . were identified by gelatin liquefaction and nitrate reduction tests . The scheme of identification of isol ates is shown in Appendix (8, 26, 31) . Molds were identified with the aid of Gilman's manual (28) . 39 Sensory Evaluation Test methods . Two test methods were us ed to evaluate the sensory attributes and overal l acce�tability of the sampl es. The difference-pre ference test (3, 39) was con ducted to de termine if there was a significant difference between the two samples and to provide an indicat ion of the degree of acceptability . Three factors were evaluated: texture , mo istness , and flavor. A seven -point descriptive graduated scale was designed for each factor, with "neither nor" as the neutral base (Appendix) . For flavor evaluation , in addition to the hedonic scale , five characteristic flavors were described and the panelis ts were asked to indicate their degree of perceptib ility. In conjunction with the scoring test, a hedonic preference test (3, 39) was de signed to determine the de gree of acceptance for each sample . A nine point hedonic scale was used with the description ranging from "like extremely" to "dislike extremely" (Appendix) . For analysis of the data, numerical values were assigned to each descriptive term. Values ranging from one to seven were us ed in the scoring test while one to nine were used in the hedonic scale . The sensory tests were replicated three times .
Sensory panel. A panel of ten judges , composed of graduate students and staff of the Food Technology Depart ment , participated in the sens ory tests . Two training 40 sessions were conducted to acquaint the panel members with the product . Samples cons isting of products of intermediate and the extreme ch aracteristics of texture and mo istness we re evaluated. A general agreement on the me aning of the de scriptive �erms used in the scoring test was established. The panel , consisting of three women and seven men , was us ed throughout the test sessions .
Test procedures. The two deep-fried samples , each cons isting of two chunks selected at random , were placed on paper plates wh ich were divided into two sections and identified by three- digit numbers . Two sets of simi lar samples were presented during each sitting of the panel . The firs t set was used for the diffe rence- pre ference test of. texture , mo istness and flavor while the second set, having different codes , was evaluated for taste preference . 'Al l samples we re tested at amb ient room temperature and at the same time of day . The judges were seated at opposite ends of long tables . The tests were carried out in a room lighted uniformly with cool white fluorescent lights .
Statistical Analys is The Statistical Analysis System (10) computer program at the University Computer Center was used for the calcula tion of analys is of variance . Data on water activity , moisture , pH , texture and color determinat ions in Experiments 41 One and Two were analy zed as factorial arrangements . of a split-plot design. The sens ory data from Experiment Three were analyzed as a one -way classification by the analysis of variance. Correl ation coefficients between the variables were determined . Differences between means we re analyzed by the Duncan 's Multiple Range test (60) . CHAPTER IV
EXPERIMENTAL RE SULTS
I. PROPERTIES AND STORAGE STABILITY OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85
Water Activity The analysis of variance summary for the · effect of calcium lactate , acidity and storage time on water activity of the intermedi ate mo isture product is shown in Tab le III. The factors were significant at the 0.01 level of prob a bility while the interactions were not significant . The effect of tre atments on water activity is pre sented in Table IV. Water activity of the protein chunks treated with 0.25 per cent calcium lactate was 0.008 unit lower than that in samp les without adde d calcium. When pH was adjus ted from 7.0 to 5.5, water activi ty was reduced by 0.006 unit. There was a significant increase in water activity dur ing the firs t ten days of storage . The water activity was maintained at a cons tant level through fifty days . The water activity increased at sixty days ' storage to make the leve l significantly higher than that at ten days ' storage . 42 43
TABLE III
F - TABLE SHOWING THE EFFE CT OF TREATMENTS ON WATER ACTIVI TY OF INTERMEDIATE MOI STURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACT IVITY OF 0.85
Source Degrees of Freedom Me an Squares x lo -S f . Total 167 A. Calcium lactate 1 231 .03** B. Acidity 1 190 .01**
A x B 1 2.21 Error i 6 9.75 c. Time 6 5.54**
A X c 6 0.90
B X C 6 0.77
A X B X C 6 1.24 Error ii 48 1.42 Replication.!/ 2 171. 43**· Re sidual error 84 13.36
**Signifi cant at the 0.01 leve l of prob ability . !/Error i us ed to test significance of replications . 44
TABLE IV
EFFECT OF TREATMENTS ON WATER ACTIVITY OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.8 5
�aicium [act ate Aci !/Means of 84 observations . �/ Me ans of 24 observations . Means within a column followed by the same letter are not significantly different at the 0.05 level of probability. 45 For the interactions that were not significant , tables showing the appropriate means are presented in the Appendix (Tables XXXVI through LI) . Moisture Content The summary of the analysis of variance for the effect of treatments on moisture content is shown in Table V. Cal cium lactate , acidity and storage time caused a significant change in the moisture content at the 0.01 level of proba bility while the interactions had no influence . The change in mo isture content as affected by the treatments is presented in Table VI . The protein chunks with 0.25 per cent calcium lactate had less moisture than those without adde d calcium . The adj us tment of pH to 5.5 caus ed a decre ase of 0.1 8 g. in moisture . Signifi cant moisture losses occurred between the zero and ten days ' storage and the twenty and thi rty days ' interval . No change in moisture was observed beyond thi rty days . � Value The analysis of variance summary for the effect of tre atments on pH value of the intermediate mo isture product is shown in Table VI I. Calcium lactate , acidity and storage time caused significant changes in pH at the 0.01 level of prob ability. The interaction of calcium lactate and acidi ty was significant at the 0.01 level . 46 TABLE V F - TABLE SHOWING THE. EFFECT OF TREATMENTS ON MO ISTURE CONTENT OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVI TY OF 0.85 Source Degrees o f FreedOin Me an Squares x lo-2 Total 335 A. Calcium lactate 1 131. 19** B. Acidity 1 247 .49** A x B 1 0.05 Error i 6 3.01 c. Time 6 0.73** A X c 6 0.01 B X C 6 0.04 A X B X C 6 0.03 Error ii 4 8 0.08 Rep lication.!/ 2 36 . 40** Residual error 252 0.0 4 **Significant at the 0.01 leve l of prob ability. !/Error i used to test significance of replications . 47 TABLE VI EFFECT OF TREATMENTS ON MOISTURE CONTENT OF INTERMEDIATE MOISTURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85!/ �a!cium Lactate Xc iait:t: T1me Per Cent Mean!/ pH Mean�} Days Mean17 o.oo · 2.1s a 7.0 2.18f 0 2.11P 0.25 2.03b 5.5 2.oog 10 2.og rs 20 2.loPr 30 2.Q 8 S 40 2.09s so 2.Q 8 S 60 2.Q 7S 1/ Grams of water per 5.0 0 grams of sampl e. 1/Me ans of 168 observations . 1/Me ans of 48 ob servations . Means within a column fol lowed by the same letter are not signifi cantly di fferent at the 0.05 level of probability. 48 TABLE VII F - TABLE SHOWING THE EFFECT OF TREATMENTS ON pH OF INTERMEDIATE MOISTURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACT IVITY OF 0.85 Source Degrees of Freedom Mean Squares x lo-2 Total 335 A. Calcium lactate 1 35 . 16** B. Acidity 1 16678.90** A x B 1 22.27** Error i 6 0.56 c. Time 6 55. 43** A X c 6 0.3 6 B X C 6 5.73 A X B X C 6 1.54 Error ii 48 3.00 Replicationl/ 2 73. 10** Res idual error 252 0. 0 7 **Significant at the 0.01 leve l of probability. !/Error i used to test 'igniticance of replications . 49 The mean pH values of the protein chunks as influenced by tre atments are presented in Table VIII. There was a re duction of 0.06 pH unit with the addition of 0.25 per cent calcium lactate . When 0.45 per cent malic acid was added, pH shifted from 6.81 to 5.40. The pH of the samp les was significantly de cre ased after twenty days comp ared to zero storage . There was a further decre as e at thi rty days but on subsequent days the pH had inc reas ed to the level as that at twenty days . The interaction means between cal cium lactate and acidity on pH are presented in Table IX. Calc ium lactate decreas ed the pH of the product significantly at normal pH (7.0) ; however, it had no signifi cant effect on the pH value of the samples at the adj us ted pH (5.5). Firmness The summary of the analys is of variance for the effect of cal cium lactate , acidity and storage time on firmness of the product is shown in Table X. The factors and the interaction between cal cium lactate and ac idity were significant at the 0.01 level of probability. The effect of treatments on firmness of the product is presented in Table XI . The me an shear value of the pro tein chunks treated with 0.25 per cent calcium lactate was 33 per cent greater than samp les without added calcium . The lowering of pH to 5.5 caus ed an 80 per cent increase in so TABLE VI II EFFECT OF TREATMENTS ON pH OF INTERME DIATE MO ISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.8S Calcium Lactate Ac idi tr Time Per Cent Meanl7 pH Meani7 Days Mean!7 0.00 6.14a 7.0 6.8l f 0 6.27P 0.2S 6.o8b s.s S.4o& 10 6.21Pr 20 6.11 rs 30 S.94t 40 6.11 rs so 6.0 75 60 6.045 1/Means of 168 ob servations . �/Me ans of 48 observations . Means within a column followed by the same letter are not significantly different at the O.OS leve l of prob ability. 51 TABLE IX EFFECT OF THE INTERACTION OF CALCIUM LACTATE AND AC IDITY ON pH OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACT IVITY OF O.Bsl! Ac 1dity (pH) Calcium Lactate (Per Cent) 7.0 5.5 0.00 5.41c 0.25 5.4o c !/Me ans of 84 ob servations . Me ans fo llowed by the same letter are not signifi cantly diffe rent at the 0.05 level of probability. 52 TABLE X F-TABLE SHOWING THE EFFECT OF TREATMENTS ON FIRMNESS OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85 Source Degrees of Freedom Mean Squares Total 503 A. Calcium lactate 1 3097615.4** B. Acidity 1 12298125.9** Ax B 1 151112.2** Error i 6 4960 .4 c. Time 6 5941.9** A X c 6 410.3 B X C 6 549 .6 A X B X C 6 81.5 Error ii 48 89 8. 3 Replication!/ 2 83159 .4** Residual error 420 310.7 **Significant at the 0.01 level of probability. !/Error i used to test signifi cance of replicat ions . 53 TABLE XI EFFECT OF TREATMENTS ON FIRMNESS OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACTIVITY OF O.Ssl/ �aicium [act ate Aciaitx: Time Pe r Cent Mean27 pH Mean�.? Days Mean�.? 0.00 473b 7.0 395 g 0 535r 0.25 63o a 5.5 7o7 f 10 sssP 20 556P 30 546Pr 40 547Pr so 561P 60 556P !/ Firmness was expressed as the pounds of force re quired to shear the sample . 1/Means of 252 ob servations . �/Means of 72 observations . Me ans within a column followed by the same letter are not significantly different at the 0.05 level of prob ability. 54 shear values . Firmn e ss of the samp les taken after ten days ' storage we re fi rmer than samp les me as ured at zero days ' storage . · After this increase in fi rmn ess , no fu rther ch anges were found . Th e interaction means of calcium lactate and acidity on firmne ss are presented in Table XI I. The addition of calcium lactate incre ased the firmness of the product sig nificantly at both pH levels ; howeve r, the firming effect of cal cium was greater at pH 5.5 than at pH 7.0 . Color Th e analys is of variance summa ries for the effect of treatments on co lor of the product are shown in Table XI II and Table XIV. Calc ium lactate had no significant effect on chromaticity coordinates x and y; howeve r, the effe ct was significant on lightness index at th e 0.0 1 leve l of prob ability. Acidity influenced ch romaticity coordinates x and y, and lightness index of th e protein ch unks . Storage time had no signi ficant effe ct on al l th e th ree color me a surements at the 0.0 1 leve l of prob ability. Th e interac tion between calcium lactate and acidity was signif icant on ch romaticity coordinate x at the 0.05 level of prob ability. Interaction of acidity and time was significant on chr oma ticity x and lightnes s index . The effect of tre atments on various color indices is presented in Table XV . With the addition of 0.25 per cent 55 TABLE XII EFFECT OF THE INTERACTION OF CALCIUM LACTATE AND ACIDITY ON FIRMNESS OF INTERMEDIATE MOISTURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.851,11 · .Xciait l Calcium Lactate �Per Centl 7.0 tERJ s.s 0.00 334d 612b 0.25 456c 803a !/Firmness was expressed as the pounds of force re quired to shear the sample. �/Means of 126 observations . Means followed by the same letter are not signifi cantly different at the 0.05 level of probability. 56 TABLE XIII F-TABLE SHOWING THE EFFECT OF TREATMENTS ON C.I.E. COORDINATES "X" AND "Y" OF INTERMEDIATE MO ISTURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85 Mean Sguares x lo-5 coordinate Coordinate Source Desrees of Freedom X y Total 503 A. Calcium lactate 1 0.02 0.72 B. Acidity 1 1523. 59** 9.37* A x B 1 18. 24** 3.66 Error i 6 2.49 0.70 c. Time 6 4.65** 2.72** A X c 6 1.69 0.19 B X C 6 3.79* 0.27 A X B X C 6 2.23 0.39 Error ii 48 1.40 0.73 Replication!/ 2 21. 38* 3.28 Res idual error 420 0.95 0.39 *Significant at the 0.05 level of probability. **Significant at the 0.01 level of prob ability. !/Error i used to test significance of replications . 57 TABLE XIV F-TABLE SHOWING THE EFFECT OF TREATMENTS ON LIGHTNESS INDEX OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85 Source Degrees of Freedom Mean Squares Total 503 A. Calcium lactate 1 1.65** B. Ac idity 1 62. 62** A x B 1 0.1 2 Error i 6 0.05 c. Time 6 0.24** A X c 6 0.02 B X C 6 0.08** A X B X C 6 0.05 Error ii 48 0.02 Replication!/ 2 0.59** Residual error 420 0.01 **Significant at the 0.0 1 level of probability. !/Error i used to test significance of replications . 58 TABLE XV EFFECT OF TREATMENTS ON COLOR INDICES OF INTERME DIATE MOISTURE TEXTURED VE GETAB LE PROTEIN CHUNKS WITH TARGET WATER ACT IVITY OF 0.85 c. I.E. Dom1.nantl/ Coo rdinates w.�vel ength Purity Lightness Treatments X l (nml (Per Centl Index Calcium lacta e (Per Cent).?.j 0.0 0 0.39 6 a 0.377h 582 38.8 4.S6S 0.25 0.39 6 a 0.377h 582 38.8 4.6sr Ac idity ( pH) �/ 7.0 0.40lc 0.377j 583 40 .5 4.28u 5.5 0.390d 0.37 6k 582 37. 9 4.97 t 5 9 TABLE XV (continued) e. 1 . E. Dom1nant!/ Coordinates Wavelength Purity Lightness � � t-reatments X l nml Per Centl Index Time (day s)�/ z 0 0.39 4 g 0.376Q 582 38 .8 4 .s2 f w 10 0 . 3 96 g 0 .377PQ 582 38 .8 4 .s8 z W 20 o.39sfg 0.377PQ 582 38 .8 4 .64V f vw 30 0.3 96 g 0.378P 582 39.4 4 . 62 £ v 4 0 0.39 7 0.37 8P 582 39 . 4 4 .62 w W so o.39sfg 0.377PQ 582 38.8 4 .67V v 6 0 0.396fg 0.377Pq 582 38.8 4 .69 !/ Dominant wavelength was expressed in nanometers . !./Me ans of 2 5 2 observations . 1/Means of 72 ob servat ions . Under each factor , means within a column followed by the same letter are not significantly di fferent at the 0.0 5 level of prob ability. 60 calcium lactate, there was no ch ange in ch romaticity co ordinates x and y, dominant wave length and purity ; howeve r, lightness index was increas ed by 0.08 unit . Th e chromaticity coordinates x and y were significantly diffe rent between samples of pH 7.0 and pH 5.5. Purity and lightness index were higher in samples of pH 5.5 than pH 7.0. Chromaticity x and y values were not di ffe rent after sixty days ' storage when compared to zero days . Howeve r, signifi cant ch anges did take place between some intermediate periods of storage. Lightness index incre as ed with stor�ge so th at samp les taken after sixty days were lighter th an samp les taken at zero and ten days . Th ere was no apparent change in dominant wave length and purity during storage. Th e dominant wave l ength of the product was between 582 and 583 nm and purity ranged from 37 .9 to 40 .5 per cent . The color of the protein chunks was yel low-orange . (Figure 7, Appendix) . Mean values of the interaction between calcium lactate and acidity are given in Table XVI . There was a mean 0.012 decre ase in chromaticity coordinate x when pH was adj us ted from 7.0 to 5.5 at both levels of calcium lactate . The interaction had no significant effect on chromatici ty coordinate y, dominant wave length and lightness index . In samples with or without calcium lactate , purity was 1.6 per cent higher in samp les at pH 5.5 th an at pH 7.0. Means for the color measurements as affected by the 61 TABLE XVI EFFECT' OF THE INTERACTION OF CALCIUM LACTATE AND AC IDITY ON COLOR INDICES OF INTERMEDIATE MOISTURE TEXTURE D VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.851/ Calcium C.I.E. Dominant27 Purity Light- Lact ate Acidity Coordinates Wavelength (Per ness �Per Centl teHl X r �nml Centl Index 0.00 7.0 o.4ooa 0.377f 583 40 .5 4.22P 5.5 0.39lb 0.377f 582 37. 9 4.9 0P 0.25 7.0 o.4 o2a 0.3 78f 583 40 .5 4.31P 5.5 0.38 9b 0.376 f 5 82 37.9 s .o4P l/Means of 126 observations . �/ Dominant wavelength was expressed in nanometers . Means within a column followed by the same letter are not signi ficantly different at the 0.05 level of prob ability. 6 2 acidity and storage time interaction are presented in Table XVI I. No signifi cant changes in chromaticity coordinate x was observed in samples at pH 7.0 until the forty - day storage period wherein an incre as e over the zero - day was evident . The level was held cons tant through sixty days . Protein chunks at pH 5.5 showed no significant change in chroma ticity coordinate x during storage , but the values were lower than those of pH 7.0 samples . The interaction had no significant effect on chromaticity coordinate y and dominant wave length . At pH 7.0, lightness index was constant up to thi rty days at which time it was significantly higher than· at zero -day ; no additional change occurred up to sixty days . No change in lightness index was observed at pH 5.5 during storage ; however, the samples were significantly lighter than samples at pH 7.0. Correlat ion between Variables The corre lation coefficients between the diffe rent variables are shown in Table XVIII. There was a signifi cant correlation between each pair of variables except for chromaticity coordinate y whe rein no significant correlation was found with water activity and moisture content . The correlation coefficient between moisture and firmness and between pH and coordinate x were greater than � 0.90. When water activity was correlated with moisture , and pH correlated with firmness , the coefficients were between 63 TABLE XVI I EFFECT OF THE INTERACTION OF ACIDITY AND STORAGE TIME ON COLOR INDICES OF INTERMEDIATE MOISTURE TEXTURED VEGETABLE PROTEIN CHUNKS WIT� TARGET WATER ACTIVITY OF 0.85-/ C. I.E. DominantV Acidity Time Coordinates Wavelength Purity Ligh�ness teH� �Dars ) X l �nm� �Per Cent� Index 7.0 0 0.398b 0.3 77£ 583 40 .5 4.135 10 o.4olab 0.3 77£ 583 40 .5 4.zz rs 20 0.401 ab 0.377£ 583 40.5 4.23rS 30 0.401 ab 0.378£ 583 40 .5 4.zg r 40 o.4oza 0.378£ 583 40.5 4.zg r so 0.40 1ab 0.3 77£ 583 40 .5 4.36 r £ 60 o . 4 oza 0.3 77 583 40 .5 4.3sr 6 4 TABLE XVI I (continued) C.I.E. Dominantll Acidity Time Coordinates Wavelength Purity Lightness �EHl (Dax:s) X l �nml �Per Centl Index 5.5 0 0.39 Q C 0.376 f 582 37. 9 4.91P 10 0.39lc 0.377f 5 82 37. 9 4.9 4 P 20 0.389 c 0.3 76f 582 37.9 s.o4P 30 0.39lc 0.377£ 582 37 .9 4.94P 4 0 0.39lc 0.3 77f 582 37.9 4.94P so 0.39 Q C 0.3 76f 582 37.9 4.99 P 60 0.390c 0.3 76f 582 37.9 s.o4P �/Me ans of 36 obs ervations . �/Dominant wavelength was expressed in nanometers . Me ans within each column fol lowed by the same letter are not significantly different at the 0.05 level of prob ability. 65 TABLE XVI II CORRELAT ION COEFFICIENTS BETWEEN WATER ACTIVITY , MOISTURE CONTENT , pH , FIRMNESS AND C.I.E. COORDINATES "X" AND "Y" OF INTERME DIATE MOISTURE TEXTURE D VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85 Mo isture Coordinate Coordinate Variable s Content ;eH Fi rmne ss X l Water activity 0.84 ** 0.4 1 ** - 0.66** 0.36 ** 0 ·.10 Moisture content 0.71 ** - 0.90 ** 0.61 ** 0.1 8 pH - 0.87** 0.90** 0.30 ** **Significant at the 0.01 level of probability. 66 ! 0.80 and ± 0.90 . Significant correlation between all other variables were below ± 0.80. Proximate Analys is Composition of the intermediate moisture textured vegetable protein chunks is presented in Table XIX. Crude protein content ranged from approximately 26 .5 to 32 per cent . The product at pH 5.5 had a higher level of protein than that at pH 7.0. The average NaC l content was 8.10 per cent. Total solids ranged from 54.8 to 60.8 per cent . The product tre ated with 0.25 per cent cal cium lactate and having a pH of 5.5 had the highest total solids . Microbiology The bacterial counts of the intermediate moisture product during the 60-day storage period are shown in Figures 4 and 5. Bacterial count at pH 7.0 (Figure 4) in creased a hundredfold during the first thi rty days of storage ; howeve r, between the twenty and thirty days ' interval rate of growth was greater in samples without calcium lactate than in samples with added calcium lactate . A decrease in bacterial count for both tre atments was ob served between the thirty- and forty- day interval , fol lowed by a paral lel increase through sixty days . The bacteri al count at the end of the sixty days ' storage period in samples with or without calcium lactate was 1 x 105 per gram of sample . 6 7 . TABLE XIX COMPOSITION (PER CENT) OF INTERME DIATE MO ISTURE TEXTUREP. VE GETAB LE PROTEIN CHUNKS WITH TARGET WA TER ACTIVITY OF 0.85 Calcium / Lactate Acidity Crude.!., l/ Sodiuml•Y Tot all (Per Cent) (pH) Protein Chloride Solids 0.0 0 7.0 26.46 8.31 54.83 5.5 31.91 8.43 58. 34 0.25 7.0 27.23 8.52 57.37 5.5 31.77 8.10 60 .83 !/Percentage based on dry weight. / � Me ans of 9 observations . · l/Means of 12 observations . 68 7 I I I I I I I I I i/ /y 0 z I 4-1 0 without bO 0 calcium � lactate with ------0.25% calcium lactate 0 10 20 30 40 so 60 Storage Time (Days) Figure 4. Total plate count of intermediate moisture textured vegetable protein chunks with target water activity of 0.85 and at pH of �.0. 69 _ _. ...... - -- ./ / / v / / � I I I I I I I I I I f I . I 0 z I � I 0 without I calcium bO 0 I lactate � I I with I ------0 .25% I calcium I lactate I I 1 1 0 �------�------�------�------L------�------� 0 10 20 30 40 so 60 Storage Time (Days ) Figure 5. Total plate count of intermedi ate mo isture textured ve getable protein chunks with target water activity of 0.85 and at pH of 5.5 70 At pH 5.5 (Figure 5, page 69) , bacteri al growth was rapid during the first twenty days of storage for samples at both cal cium levels . In samples with calcium lactate, growth continued to increase through thi rty days , followed by a decre as e at the forty - day period. Samples without calcium lactate showed a gradual decrease from twenty through forty days . Between forty and sixty days , bacterial count in creas ed gradually and the growth rate was slightly more rapid in samp les without calcium lactate than in samples with adde d calcium lactate. At the end of the storage period , bacterial count was tenfold lower at pH 5.5 (Figure 4, page 68) than at pH 7.0 (Figure 5) . Cultural and morphological examination of the co lonies showed the presenc� of micrococci , lactob aci lli , Pseudomonas spp . and Bacillus subtilis during the early stage of growth . After thirty days of storage , the micro flora was predominantly Pseudomonas . Mo ld growth was observed on samples at pH 7.0 after twenty days of storage . The molds , which usually appeared near the surface of the j ar , were identified as Penicillium commune . Ob jectionab le odors were detected on samp les having mo lds . No mo ld growth or obj ect ionable odor was ob served on samples at pH 5.5. Cal cium lactate had no apparent effect on mold growth . 71 II. PROPERTIES AND STORAGE STABILITY OF INTERME DIATE MO I STURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 . Water Activity The analys is of variance summary for the effect of treatments on water activity of the intermediate moisture product is shown in Table XX. Acidity had no significant effect on water activity while storage time was significant at the 0.01 level of probability. The acidity and time interaction had no influence . The change in water activity of the product as af fected by treatments is presented in Table XXI . The lower ing of pH from 7.0 to 5.5 did not significantly reduce water activity . During storage , water activity remained constant until the forty- day period at which time a signifi cant decrease ove r the ten-day period was observed. The leve l was maintained from forty through fifty days , fol lowed by a significant increas e at sixty days . The mean water activity values for the interaction between acidity and storage time are presented in Tab le LII in the Appendix. Effects of all non-significant inter actions are found in the Appendix (Tables LIII through LVI) . Moisture Content The summary of the analysis of variance for the effect 72 TABLE XX F-TAB LE SHOWING THE EFFECT OF TREATMENTS ON WATER ACTIVITY OF INTERMEDIATE MOI STURE TEXTURE D VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 Source Degrees of Freedom Me an Squares x lo - S Total 111 A. Acidity 1 502.90 Error i 1 154.14 B. Time 6 17.91** A X B 6 4.46 Error ii 12 2.97 Replication.!/ 1 352.6 9 Residual error 84 14.75 **Significant at the 0.01 level of probability. !/Error i used to test significance of repli cations . 73 TABLE XXI EFFECT OF TREATMENTS ON WATER ACTIVITY OF INTERMEDIATE MOISTURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 Ac1a1tl T1me pH Meani7 Days Mean!/ 7.0 0.813a 0 0.806prs 5.5 0.7 99b 10 o.80 8Pr 20 o.8o8Pr 30 o.8 o sPrs 40 0.801s s o 0.8Q 3 rS 60 0.811P !/Means of 56 ob servati ons . I/Means of 16 observations . Means within a column followed by the same letter are not significantly different at the 0.05 level of prob ability. 74 of acidity and storage time on moisture content is shown in Table XXII. Acidity and its interaction with time were not signifi cant while storage time was significant at the 0.01 level of probability. Mo isture change as a result of tre atment effects is presented in Table XXIII . There was no significant reduction in moisture content of the product when pH was lowered to 5.5. Moisture level remained constant through fi fty days of stor age . At the sixty- day period , a significant increas e over the zero - day was observed. � Value The analysis of variance summary for the effe ct of tre atments on pH value is shown in Table XXIV. Acidity was significant at the 0.01 leve l of probability. Storage time and its interaction with acidity were not significant . The effect of treatments on pH of the product is presented in Table XXV. The addition of 0.45 per cent malic acid lowered significantly the · pH of the samples by 1.2 4 units . Although storage time had no significant influence on pH , there was a trend for a decre as e as time increased . Firmness The summ ary of the analysis of variance for the effect of acidity and storage time on firmness of the inter mediate moisture product is shown in Table XXVI . The factors 75 TABLE XXI I F -TAB LE SHOWING THE EFFECT OF TREATMENTS ON MOISTURE CONTENT OF INTE RMEDIATE MO ISTURE TEXTURED VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 Source Degrees of Free dom Me an Squares x lo-2 Total 167 A. Acidity 1 60 .71 Error i 1 9.81 B. Time 6 0.61* A x B 6 0.1 8 Error ii 12 0.16 Rep lication.!/ 1 34 . 39 Residual error 140 0.0 4 **Significant at the 0.05 level of probability. l/Error i used to test significance of replications . 76 TAB LE XXI I I EFFECT OF TREATMENTS ON MOISTURE CONTENT OF INTERME DIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF o.sol/ Ac 1a1tl T1me p� Mean!/ Days Mean�/ 7.0 1.74 a 0 1.66 r 5.5 1.62a 10 1 .67Pr 20 1.69Pr 30 1.66Pr 40 1 .6sPr so 1.67Pr 60 1 . 1 o P !/ Grams of water per 5.0 0 grams of samples . �/Means of 84 observations . �/Means of 24 ob servati ons . Means within a column followed by the same letter are not signifi cantly different at the 0.05 level of probability. 77 TABLE XXIV F-TABLE SHOWING THE EFFECT OF TREATMENTS ON pH OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 Source Degrees of Freedom Mean Squares x lo- 2 Total 167 A. Acidity 1 6423.25** Error i 1 18.14 B. Time 6 9.14 A x B 6 8.46 Error ii 12 6.34 Replication!/ 1 53.49 Residual error 140 0.09 **Significant at the 0.01 level of probability. l/Error i used to test significance of replications . 78 TABLE XXV EFFECT OF TREATMENTS ON pH OF INTERMEDIATE MOISTURE TEXTURE D VE GETAB LE PROTE IN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 Aciaiti Time s pH · Mean!7 Day Mean.�.? 7.0 6.8 4 a 0 6.2oP 5.5 s.6ob 10 6.31P 20 6.29P 30 6.23P 40 6.19P so 6.20P 60 6.13P 1/Means of 84 observations . !/Means of 24 observations . Means within a column followed by the same letter are not significantly diffe rent at the 0.05 level of probability . 79 TAB LE XXVI F-TABLE SHOWING THE EFFECT OF TREATMENTS ON FIRMNESS OF INTERME DIATE MO ISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 Source Deg rees of Freedom Mean Squares Total 95 A. Acidity 1 2053203.75 Error i 1 66913.44 B. Time 1 25594. 34 A x B 1 712 .32 Error ii 2 2711.69 l Replicationl 1 221232 .00 Res idual error 88 497.00 l/Error i used to test significance of replications . 80 and the interaction were not significant . The mean shear value of the product resulting from treatment effects are given in Table XXVII. Color The analysis of variance summaries for the effect of treatments on color of the intermedi ate moisture product are shown in Tables XXVIII and XXI X. Acidity and its interaction with storage time had no significant effect on chromaticity coordinate x while storage time was sig nificant at the 0.05 level of probability . The two factors and their interaction had no significant influence on chromaticity coordinate y. Acidity and storage time caused a significant change in lightness index at the 0.05 level of probability while the interaction had no influence . The means for the effect of acidity and storage time on the color indices are presented in Table XXX . The mean chromaticity x for the products at both pH levels was 0.396; chromaticity y, 0.378. Storage time caused a significant increase for the chromaticity x after sixty days , but no �hange occurred for the _ chromaticity y. Purity was higher at the adjusted pH level than at normal pH ; there was a slight decrease in purity after sixty days of storage. Samples at pH 5.5 were significantly lighter than samples at pH 7.0 (4.31 vs . 5.03 lightness index) . An increase in lightness was observed in samples stored for sixty days . 81 TABLE XXVI I EFFECT OF TREATMENTS ON FIRMNESS OF INTERME DIATE MO I STURE TEXTURE D VE GETABLE PROTE IN CHUN!S ITH TARGET WATER ACTIVITY OF 0.80_,_2, Acidity Time pH . Me an Days Me an 7.0 0 588P 5.5 60 621P !/ Firmne ss was expressed as the pounds of force re quired to she ar the sample. !/Me ans of 48 ob servati ons . Means within a column followed by the same letter are not significantly different at the 0.05 level of probability. 82 TABLE XXVIII F-TABLE SHOWING THE EFFECT OF TREATMENTS ON C.I.E. COORDINATES "X" AND "Y" OF INTERMEDIATE MOI STURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 Me an Squares x lo- S Coordinate Coordinate X Source Desrees o f · Freedom l Total 95 � . A. Acidity 1 225.98 3.56 Error i 1 5.01 0.23 B. Time 1 20 .43* 1.62 A x B 1 0.43 0.25 Error ii 2 0.34 0.23 Replicationl/ 1 0.31 0.56 Res idual error 88 1.54 0.46 *Significant at the 0.05 level of prob ability. !/Error i used to test significance of replications . 83 TABLE XXIX F-TABLE SHOWING THE EFFECT OF TREATMENTS ON LIGHTNESS INDEX OF INTERME DIATE MOISTURE TEXTURED VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 Source Degrees .. o:(·F.reedom Mean Squares Total 9 5 A. Acidity 1 12. 25* Error i 1 0.0 6 B. Time 1 0.99* A x B 1 0.08 Error ii 2 0.03 Rep lication.!/ 1 0.05 Res idual error 88 0.02 *Significant at the 0.05 level of prob ability. !/Error i used to test significance of replications . 84 TABLE XXX EFFECT OF TREATMENTS ON COLOR INDICES OF INTERMEDIATE MOI STURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80!1 c. I.E. DominantZ/ Coordinate Wavelength Purity Lightness Tre atments X l 'nm� �Per Cent� Index Acidity · (pH) 7.0 0.4 0la 0.3 79k 583 40 .5 4.31S 5.5 0.392a 0.3 78k 582 39 .4 5.o3r Time (days) 0 0.395f 0.37 8P 582 39 .4 4.57V 60 0.398& 0.379 P 582 . 40. 5 4.77U !/Me ans of 48 observations . �/ Dominant wavelength was expressed in nanometers . Under each factor, means within a co lumn followed by the same letter are not signifi cantly different at the 0.05 level of probability. 85 The dominant wavelength of the product was between 582 and 583 nm and the color was yellow-orange . Correlation between Variables The correlation coe fficients between water activity , moisture content , and pH are shown in Table XXXI . All cor relations were signifi cant at the 0.0 1 level of prob ability. Mo isture content was highly corre lated to water activity , with a coefficient of 0.94. Correlation coefficients be tween pH and water activity and mo isture con tent were wi thin 0.70 to 0.80. Proximate Analys is Composition of the intermediate mo isture textured vegetable protein chunks is presented in Table XXXI I. The average crude protein content was 25.3 per cent . Products at pH 5. 5 had a slightly higher protein level than those at pH 7.0. On the average , the intermediate moisture product con tained 7.18 per cent NaCl. Total sol ids was cons ide rab ly high, ranging from approximately 65 to 68 per cent . Microb iology Results of the total plate count of the product during storage are shown in Figure 6. Bacterial growth was hindered during the first ten days of storage at both pH levels . On samp les at pH 7.0, the lag phase was followed by a hundred fold incre ase at twenty -day period and a sharp de crease at 86 TABLE XXXI CORRE LAT ION COEFFICIENTS BETWEEN WATER ACTIVITY , MOI STURE CONTENT AND pH OF INTERME DIATE MO ISTURE TEXTURED VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACTIVITY OF 0.80 Variables Mo isture Content pH Water activity 0.9 4 ** 0.71** Mo isture content 0.79 ** **Signifi cant at the 0.01 level of prob ability. 87 TABLE XXXI I COMPOSITION (PER CENT) OF INTERME DIATE MOISTURE TEXTURED VE GETAB LE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF o.so!/ . Acidity (pH) Crude: Protein£1 Sodium. Ch loride£1 Total Solids 7.0 24 .27 7.22 65.50 5.5 26.32 7.13 68 .05 !/Me ans of 12 observations . £/Percentage based on dry weights . 88 ---- pH 7.0 pH 5. 5 0 0 so 60 0 10 2 3 0 4 Storage Time (Days) Figure 6. Total plate count of intermediate moisture textured vegetable protein chunks with target water activity of 0.80. 89 thirty days , after which the bacterial count increas ed steadily to 2 x 104 per gram of sample by the end of the storage period. Samples at pH 5.5 showed a gradual increas e from ten through fifty days , followed by a tenfold decrease during the final ten- day storage interval . Bacteri al count was a hundredfold lower at pH 5.5 than at pH 7.0 at the end of the sixty- day storage period. The dominant flora observed from zero to thi rty days of storage were Pseudomonas spp . On samples held for forty days , . Flavobacterium , Achromob acter, Alcaligenes, and Erwinia were observed. Soon Flavobacterium spp . began to outgrow all other organisms and dominated during the remaining storage time . No visible mold growth was observed and mold count remained zero throughout the storage period . III. SENSORY EVALUATION OF DEEP-FRIED INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS The analysis of variance summaries for the effect of water activity on the sensory panel scores of deep-fried intermedi ate moisture protein chunks are given in Table XXXIII . Water activity had a significant influence on tex ture at the 0.01 level of prob abil ity and on moistness at the 0.05 level. Flavor and overall acceptabil ity were not significantly affected by water activity . The mean sensory panel scores of the protein chunks 90 TABLE XXXIII F-TABLE SHOWING THE EFFECT OF WATER ACTIVITY ON SENSORY PANEL SCORES OF DEEP-FRIED INTERMEDIATE MOISTURE TEXTURED VEGETABLE PROTEIN CHUNKS Re an Squares Degrees of Accept'a- Source Freedom Tex ture Moistness Flavor bilitr Total 59 Water activity 1 10 . 42** 4.27* 0.15 1.07 Replication 2 1.2 7 1.72 0.7 2 0.15 Residual error 56 1.43 0.81 0.94 1.41 *Significant at the 0.05 leve l of probability. **Significant at the 0.01 level of probability. 91 as influenced by water activity are presented in Table XXXIV. The texture and moistness scores were significantly higher at water activity of 0.80 than at 0.70. This indi cated that products at 0.80 Aw were slightly tougher and drier than at 0.70 Aw . No signifi cant diffe rence was found between the flavor and overall acceptability of the two samples ; howeve r, me an panel scores indicated that samp le at 0.80 Aw was slightly preferred to that at 0.70 Aw . 92 TABLE XXXIV EFFECT OF WATER ACTIVITY ON SENSORY PANEL SCORES OF DEEP- FRI ED INTERMEDIATE MOI STURE TEXTURED VE GETABLE PROTEIN CHUNKSl/ Water overall�/ Activity Texture!/ Mo istness�/ Flavor�/ Acceptability f 0.80 4.73 a 4.80 4.8 3P 5.53 s 0.70 3.9ob 4.27 g 4.73P 5.27 S !/Me ans of 30 observations . 3_/scores based on seven-point hedonic scale. Scores Texture Moistness Flavor 7 Ve ry tough Ve ry dry Like very much 4 Neither tough Neither dry nor Neither like nor soft excess moisture nor dislike 1 Very soft Extreme excess Dislike ve ry much mo isture �/Scores based on nine -point hedonic scale . Scores Overall Acce tability 9 L1ke extremel y 5 Neither like nor dislike 1 Dislike extremely Means within a column followed by the same letter are not significantly diffe rent at the 0.05 level of prob ability. CHAPTER V DISCUSSION This experiment was des igned to develop an intermedi ate mo isture product that would be microbio logically stab le during sixty days of storage at 27.60 C and organoleptically acceptable for human consumption . Storage stability of intermediate mo isture foods depends primarily on their water activity ; the lower the water activity , the more re sis tant is the food to microbial deterioration . However , as the water activity is decreased, the amount of solutes required to control water activity mus t be increased and at high concentration of solutes , undesi rab le flavor effects are imparted by the additives wh ich lower the palatability and acceptability of the product substantially . In an effort to maintain a balance betwe en these two factors , selection of an appropriate water activity in the preparat ion of the intermediate moisture textured vegetab le protein chunks was based primarily on the nature of the food, the process ing tre atment and the packaging condition. Textured vegetab le protein is a dry , extruded protein which has been sub j ected to high temperature (300 - 350° F) and pressure during the extrusion process (19) . Micro biological tests conducted on the dry protein chunks by 93 94 one manufacturer showed total plate count of not more than 5000/ g. , flat sour spore count of 1 / g. and yeas t and mo ld count of 50/ g. No coliform bacteria or enteric pathogens were detected (22) . During the preparation of the inter me di ate moisture product , the protein chunks were first rehydrated in boiling water for five minutes , then held in boiling infus ion solution fo r ten minutes . The heat pro cessing was sufficient to des troy ve getative microorganisms . The finished product was packed and sealed in ster�le jars to prevent subsequent contamination. With a low initial bacterial count in the raw material , sufficient heat pro cess ing to des troy vegetative cells , packaging in sterile and air-tight containers and the incorporation of an anti mycotic agent , a Aw of 0.85 was selected as the level for product deve lopment ·and for storage study . The appearance of molds , identified as Penicillium commune , on samples of pH 7.0 after a period of storage indicated that a Aw of 0.85 was not low enough to inhibit the ir growth . Contamination of mold spores might have oc curred during centrifugat ion of the cooked product and during packing . Studies conducted by Snow (59) on the germination of mold spores at controlled humidities showed that spores of Penicillium sp� germinated at Aw range of 1.0 to 0.8. As the water activity was decreased, the latent period of spore germination increas ed and the rate of growth decreas ed. 95 Visible mold growth firs t appeared on samples he ld for twenty days . This indicated th at the lowe ring of Aw to 0.85 was effective in lengthening the latent period of spore germination. In a study on the rate of germination of Aspergi llus spores at 20° C as influenced by humidity , Snow (59 ) found that at humidities over 88 per cent , spore germina tion was rapid, between two to three days . At humidities below 88 per cent , the latent period was much longer, between ten to twenty days and at 76 per cent humidity , germinat ion did not occur unt il after thi rty days . Mold growth was ob served only on samples at pH 7.0 and not on those at pH 5.5. This indicated that the effective ness of potassium sorb ate as an antimycotic was nil at pH near opt imum . Beneke and Fab ian (11) studied the effe ct of sorb ic acid on mold growth at diffe rent pH leve ls . Results showed that no fungi growth occurred at pH 3.0 when 0.01 to 0.1 0 per cent of sorb ic acid was added. Howeve r, as pH was raised to 7.0, no inh ib ition of fungus growth occurred at any of the concentrations of sorb ic acid employed. Since products prepared at 0.85 Aw were sus ceptib le to molds , a second experiment was conducted wherein the Aw was lowered to 0.80. The ab sence of any vis ib le mold growth and the zero mo ld count obtained during the sixty - day storage period indicated th at a Aw of 0.80, with the addi tion of 0.7 per cent potassium sorb ate , was effective in suppressing mold growth . 96 The lowering of Aw to 0.80 also had an effect on bacterial growth . After sixty days ' storage at 27.6° C, the bacterial count was tenfold lower at Aw of 0.80 than at 0.85 in the products with pH of 7.0. At pH 5.5, the lowering of water activity caused a comparab le hundredfold reduction in bacterial count . As water activity was lowered, th e ap parent lag phase was lengthened and growth rate was de creased. The lowering of pH from 7.0 to 5.5 had some in hibitory effe ct on bacterial growth . This indicated that the tolerance of bacteria at low water activity was less at advers e pH values than within the optimal pH range . Differences between the micro flora of the intermediate mo isture protein chunks at 0.85 Aw and at 0.80 Aw were ob served during the storage period. At 0.85 Aw , the initial micro flora were predominantly Micrococci and Lactob acilli. Soon after, the Pseudomonas dominated and pers isted through out the remaining storage period. At 0.80 Aw , the maj ority of the microorganisms during the early stage were the Pseudomonas . After some time , members of the family Achromobacterece ae : Flavob acterium, Achromobacter and Al caligenes began to dominate and survived until the end of the sixty- day storage period . The predominance of Pseudomonas at 0.85 Aw and of Flavob acterium, Achromobacter and Alcaligenes at 0.80 Aw indicated that members of the Achromob actereceae were ab le to grow at a lower water 97 activity range than Pseudomonas . This is supported by the findings of Scott (57) which showed that the lower water activity limit was higher for Ps eudomonas than for Achromob acter. Results of total plate count at 0.80 Aw showed that between the fifty and sixty days ' interval , there was a tenfold increase in bacterial count at pH 7.0 while at pH 5.5, there was a tenfo ld decrease. Microbiological tests on a more extended storage period need to be conducted be fore any conclus ion can be made on the final trend of bacterial growth . However, the bacterial curve up to the fifty days ' storage time indicated that under such adverse environmental conditions , growth at 0.80 Aw was greatly hindered, as compared to that at 0.85 Aw . Studi es on water activity requirements of micro organisms have shown that the minimum Aw for most bacteria is 0.9 0 (52, 57) . However, the gradual increase in bac terial count observed in this experiment indicated that growth was possible at Aw as low as 0.80. One explanation could be the kind of solute employed to reduce water ac tivity . Recently Calh oun and Frazier (14) compared the effect of glucose and NaCl, at concentrations giving the same water activity values , on growth of £. coli and P. flourescens . Sodium chloride was found to inhib it their growth more than glucose. Baird- Parker and Freame (9) 98 reported that for Cl. pe rfringens , the lowest Aw supporting growth was 0.95 to 0.97 when sucrose or NaCl was us ed, but 0.93 to 0.95 when glycerol was used. They also stated that the minimal Aw permitting germination of Cl ..bo tulinum spores was 0.9 3 in NaCl and 0.89 in glycerol . Marquis (44) studied the phys iological action of ionic and non- ionic solutes on bacterial cells and found that NaCl had a spe cific effect on cell permeability and that bacterial re sponse to salts was different from their response to sugars . It is apparent that bacterial inh ib ition cannot be entirely as cribed to lowered water activity ; di fferent solutes exert specific effects . Another explanation for the growth of bacteria at Aw 's of 0.85 and 0.80 could be the nutrit ive value of the food material . In his work with Salmonella oranienburg , Christian (16) observe d that the water activity range for growth was much wider in a minimal medium than in rich sub strate . The high nutritive value of the textured vegetable protein chunks , which contain twe lve amino acids , might have lowered the limit ing water activity for these organisms . Results of the phys ical and chemical analyses of the intermediate mois ture textured vegetable protein chunks indicated that calcium lactate , acidity and storage time have significant effects on the properties of the product . The addition of 0.25 per cent calcium lactate and the 99 lowering of pH to 5.5 caused a decreas e in water activity · and moisture content of the protein chunks with target Aw of 0.85. This ch ange is due to the appreciab le electro static force created between the ions and the water molecules which resul ted in . the hydration of the ionized groups (63) . Since water activity is a me asure of available water, the bonding of water molecules to the ions caus ed a lowering of water activity and moisture content . An increas e in firmness of the protein product was observed when calcium lactate was added. Calcium ions reacted with the polysaccharides to form a gel which sup ported and maintained the structure of the tissues (20) . The lowering of pH also had some firming effect. Results indicated th at samples tre ated with calcium lactate at a pH of 5.5 had the greatest resistance to shear. Significant reduction in the chromaticity coordinates x and y was found when pH was adjusted to 5.5. The color of the protein chunks was yel low - orange . Samples at pH 5.5 were lighter in color than those at pH 7.0 . Calcium lactate had no signifi cant effect on the color indices of the intermediate moisture product . There was a significant but slight overall increase in water activity during storage. This indicated that equilib ration was taking place and the water activity of the product was modified. Since the product was sealed in 100 airtight j ars , there could be no exchange of mo isture with the environment . Twenty-five grams of samp le were trans ferred from the storage jar to a half-pint jar with the hydrosensor inserted through the lid. Since no attempt was made to control the ambient relative humidity of the air during sample transfer and since the j ar was only filled to one -third of its capacity , the headspace in the jar was filled with air wh ich might have possessed a relat ive humidity different from that surrounding the product in the storage jar. The increase in water activity as me asured for the product after it was trans ferred to the half-pint jar suggested that the re lative humidity of the air might have been higher than that of the original jar containing the product . By placing the product in an atmosphere of an incre ased relat ive humidity , the product might have sorb ed a small amount of moisture thereby causing the moisture con tent of the product to be raised. This relationship was obs erved in the product with Aw of 0.8 0 but not at 0.8 5 . . The gradual decreas e in pH during storage at 0.85 Aw indicated that microb ial and chemical activit ies were taking place . Recently , it was recognized that optimum stability of low moisture foods occurs when the mo isture is least sensit ive to a change in equilib rium relative humidity (ERH) (48, 54) . In a water sorption isotherm wherein the graph is a smooth sigmo id curve , the optimum mo isture leve l 101 corresponds to the inflection point (54) . The increase in firmness of the intermediate moisture product at 0.85 Aw during storage could be due to many factors . Kapsalis (34) stated that relative humidity , · residual moisture , temperature and chemical composi tion af fect textural stability during storage . Although there were some significant differences in the chromaticity coordinates x and y values of the samples with 0.85 Aw between certain storage intervals , there was no significant overall change in the color of the stored product. Having established the minimal water activity require ment for storage stability of the intermediate moisture pro tein product, a third experiment was conducted to determine its overall acceptability to a sensory panel. In an effort to mask some of the flavor effects of the solute additives , the products with a Aw of 0.80 were prepared by the addi tional incorporation of flavoring substances and deep-fried. During frying, the water activity was found to decrease further by approximately 0.10 unit. This led to the possi bility of cooking the protein chunks in an infusion solution to provide an initial Aw of 0.90 and the final required Aw of 0.80 could be attained by subsequent deep- frying . To obtain a Aw of 0.90 before deep- frying , the amount of sorbitol in the infus ion solution was reduced to 26.9 per cent . 102 This was ab out 20 per cent less than th at required to bring Aw down to 0.80. To determine the effect of water activity on sensory attributes, two samples were evaluated. In one , the product was infused in a solution of solutes to yield a Aw of 0.90 , followed by deep- frying to provide a final Aw of 0.80. In the other sample, Aw was brought down di rectly to 0.80 by infusion and with subsequent deep- frying, its value was reduced to 0.70. Results of the panel evaluation indicated that the deep-fried intermediate moisture product was acceptab le at both levels of water activity . Texture of the product at 0.70 Aw was rated "neither soft nor tough" while th at at 0.80 was rated just below the "slightly tough" category . Both samples were scored between "slightly dry" and "nei ther dry nor excess ively moist;" however, the protein chunks at 0.80 Aw were slightly drier than those at Aw of 0.70. One explanation for these effects on texture and moistness could be the relative amount of sorbitol present in the product. Samples at 0.70 Aw contained a greater proportion of sorb itol than those at 0.80. Since sorbitol is highly hygroscopic and its humectancy and softening ef fect increas ed with an increase in concentration, then samples at 0.70 Aw could be softer and more moist than samp les at 0.80 Aw . The flavor and overall acceptability of the two 103 samples were scored between "like slightly" and "neither like nor dislike." Although no significant difference was es tablished, mean panel scores indicated a slight preference for samples at the final 0.80 Aw . A slight beany flavor due to the soy protein was detected by some of the panel members . Both samples imparted a slightly bitter and burning af�er tas te ; however, this was more pronounced in samples at 0.70 Aw than at 0.80. Sweetness was the predominant flavor per ceived in the products . With an incre ase in the amount of sorbitol in the infusion solution , there was a corresponding increase in intensity of sweetness in the intermediate moisture product . Results of this study indicated that a minimum Aw of 0.80 is required for microbial stability. At the sixty day storage period, microbial count of the product was ten fold lower at pH 5.5 than at pH 7.0. In the product with target Aw of 0.85, slight changes in water activity , mois ture content and pH occurred during storage . Color of the protein chunks was maintained throughout the storage period while firmness was increased. In the products with target water activity of 0.80, there was a slight increas e in water activity and moisture content during storage . No change in pH , texture or color was observed in the stored product. Deep-fried intermediate mo isture protein chunks were rated as an acceptable product . It imparted a sweet 104 and slight bitter aftert as te wh ich was more pronounced in samp les at 0.7 0 Aw than at Aw of 0.80. Th ese results cle arly j us tify continued research on a more extende d stor age period not only on the microbial as pect but also the chemical and sens ory stability of the product . Inves tiga tion on the storage ch aracteristics of deep-fried inter mediate moisture protein chunks is a promising area of study . CHAPTER VI SUMMARY Dry extruded textured vegetable protein chunks were infused with solutions containing sorbitol , sodium chloride , propylene glycol , sucrose and potas s ium sorb ate to yield an intermediate moisture product with water activities of 0.85 and 0.80. Calcium lactate was added and pH was lowered to determine their effects on the properties of the protein chunks . The product was kept at 27.6° C fo r sixty days . Microbiological , phys ical and chemical analyses were con ducted on the stored product at ten- day intervals . Sensory evaluation was performed on the freshly prepared samp les with water activities of 0.80 and 0.70. Based on the results of this study , th e following conclus ions were made : 1. A water activity of 0.8 5 was inadequate to in hibit mold growth . Bacterial growth was fai rly rapid initially ; however, its rate decre as ed as storage time increas ed. 2. Th e product with a water activi ty of 0.80 was microbiologically stable. The apparent lag phas e was con siderab ly lengthened and growth rate was decreas ed. Mold growth· was completely suppressed. 105 106 3. Calcium lactate caused a decreas e in water activity and moisture content of the intermediate moisture product at Aw of 0.85. It had a firming effect on the pro tein chunks but no influence on the color. 4. The lowering of pH resulted in a reduction in water activity and moisture content and an increas e in shear value of the product at 0.85 Aw . The color of the protein chunks was yellow-orange. Samples at pH 5.5 were lighter than samples at pH 7.0. 5. Although no significant difference between pH 7.0 and pH 5.5 was established in the product with 0.80 Aw , mean values indicated that acidity had some effect on water activity , moisture content, firmness and color of the prod uct . The direction of change was similar to that observed in products lowered to 0.8 5 Aw . 6. In the product with 0.85 Aw , water activity slightly increased while moisture content decreas ed during storage . At Aw of 0.80, there was an overall increas e in water activity and moisture content. 7. pH of the product at 0.85 Aw decreas ed while firmness increased during storage . No overal l change in color was observed in the stored product. · 8. Deep -fried intermediate moisture protein chunks were rated between "like slightly" and "neither like nor dislike ." The product had a predominant sweet taste and a 107 slight bitter and burning aftertaste . 9. The sensory panel indicated a slight preference for samples at 0.80 Aw than those at Aw of 0.70; however, the difference was not significant . 10. Samples at 0.80 Aw were slightly tougher and drier than those at Aw of 0.70. LITERATURE CITED LITERATURE CITED 1. Acker, L. W. 1963. Enzyme activity at low water cont ents . In "Recent Advances in Food Science ," eds . Ha�thorn , J. , and J. M. Le itch, val . 3, . pp . 239-247� Butter worths Book Comp any , London . 2. Acker, L. W. 1969. Water activity and enzyme activity . Food Techno! . �� 1257-1270 . 3. Amerine , M. A.·, R. M. Pangborn, and E. B. Roessler. 1965 e "Principles of Sensory Evaluation of Foods ," pp e 275- 281, 354-374, 451-472. Academic Press , New York . 4. Anderson, A. K. 1946 . "Laboratory Experiments in Phys io logical Chemistry ," pp . 142-148. John Wiley and Sons , Incorporated, New York . 5. A.O.A. C. 1965. "Official Methods of Analysis ," lOth ed. , p. 486 . Association of Official Chemists , Washing- _ · ton , · D. · C. 6. Arai , S. , M. Noguchi, S. Kuros awa, H. Kato , and M. Fuj i maki . 1970 . Applying proteolytic enzymes on soy bean. VI . Deodorization effe ct of as pergillopep tidas e A and debittering effe ct of aspergil lus acid carboxypeptidase. �· Food Sci . �� 392-395. 7. Archer Daniels Midland Company . Undated. TVP , product types and general information. Decatur , Illinois . 8. Ay res , J. C. 1960. The relat ionship of organisms of the genus Pseudomonas to the spoilage of meat , poultry and eggs . J. Appl. Bacterial. �� 471-486 . 9. Baird-Parker, A. C. , and B. Freame . 1967. Comb ined ef fect of water activity , pH and temperature on the growth of Clostridium botulintim from spore and ve ge tat ive cell 1no cu!a . I· Appl. Bacterial. �� 420-429. 10 .· Barr, A. J. , and J. H. Goodnight . 19 71 . Statistical Analysis Sys tem. North Carol ina State Un iversity. Raleigh , North Carolina. 11. Beneke , E. S., and F. W. Fabian. 1955. Sorbic acid as a fung istatic agent at different pH leve ls for mo lds isolated from strawberries an d tomatoes . Food Techno!. �� 486-488. 109 110 12. Bone , D. P. 1969. Water activity--its chemistry and applications . Food Pr·oduct DeVel'o·pment lC 5) , 81-9 5. 13. Brockmann , M. C. 1970. Deve lopment of intermediate moisture foods for military use. Food Technol . �' 896-900. 14. Calhoun, C. L. , and W. C. Frazier. 1966. Effect of available water on thermal resistance of three non sporef�rming species of bacteria. � Microbial. 14, 416-420. · 15. Chen, C. C. 1970 . Intermediate moisture breeded, deep fried catfish. Master's thesis , The Unive rsity of Tennessee , Knoxville , Tennessee. 16. Christian, J. H. B. 1955. The influence of nutrition on the water relations of Salmonella oranienburg. Aus t. �· Biol. Sci. �' 75-82. 17. Christian, J. H. B. 1963. Water activity and the growth of microorganisms . In "Recent Advances in Food Science," eds . Hawthorn , J., and J. N. Leitch , vol . 3, pp. 248-255. Butterworths Book Company , London . 18. Collins , J. L. , C. C. Chen , J. R. Park , J. 0. Mundt , I. E. McCarty , and M. R. Johnston . 1972. Pre liminary studies on some properties of intermediate moisture , deep-fried fish flesh. J. Food Sci. In press. 19. Conway , H. F. 1971. Extrus ion cooking of cereals and soybeans - part II. Food Product Development �(3) , 14- 22. 20. Desrosier, N. W. 1970 . "The Technology of Food Pre servation ," pp. 365-383. The AVI Publishing Company , Incorporated, Wes tport, Connecticut . 21. Duckworth , R. B., and G. M. Smith. 1963. Diffus ion of solutes at low moisture levels . In "Recent Ad vances in Food Science ," eds . Hawthorn , J., and J. N. Leitch, vol. 3, pp. 230-238. Butterworths Book Company , London . 22. Far-Mar-Co , Incorporated. Undated. Ultra- soy, product information sheet. Technical Bulletin Foods Division� Hutchinson , Kansas . 111 23. Federal Register. 1970. Textured protein products , proposed standard of identity. ·Federal Register 35 (236) , 18530 . 24. Frazier, W. C. 1967. "Food Microbiology ," 2nd ed. , pp . 2-8, 39-41, 164-166. McGraw-Hill Book Company , New York . 25. Fuj imaki, M. , H. Kate, S. Arai , and E. Tamaki. 1968. Applying proteolytic enzymes on soybean o I. Proteo lytic enzyme treatment of soybean protein and its effect on the flavor.· Food Techno!. �' 889-893. 26. Gaby, W. 1.·, and E. Free. 1958. Differential diagnosis of Pseudomonas-like microorganisms · in the clinical laboratory. �· Bacteriol. �' 442-444. 27. Germantown Ma�ufacturing Company . Undated. Seal-n-Save in deep fat-fried foods . Product Information G-470 . Broomall, Penn�ylvania. 28. Gilman , J. C. 19 57. "A Manual of Soil Fungi," 2nd ed. , . pp . 2 3 5 - 2 8 6 • The Iowa State College Press , Ames , Iowa. 29. Glicksman , M. 1971. Fabricated foods . CRC .Critical Reviews in ·Food Techno!. �(1) , 21-39. 30 . Hardy , A. C. 1936. "Handb ook of Colorimetry ," pp . 61, 80 . The Technology �ress, Cambridge, Massachusetts. 31. Haynes , W. C. 1951. Pseudomonas aeruginosa-its . char acterization and ident1ficat 1on. !!_•. Gen. Microbiol . �' 939-950 . 32. Jacobs , M. B. 1958. "The Chemical Analysis of Foods and Food Products ," 2nd ed. , pp . 757-758. D. Van Nostrand Company , · Incorporated, Princeton , New Jersey . 33. Kaplow , M. 1970 . Commercial development of interme diate moisture foods . -Food Techno!. -24, 889-893. 34. Kapsalis, J. G. 1969. Textural qualities of freeze dried and intermediate moisture foods . Abstract No. 55. Presented at the 29th annual meeting of the Institute of Food Technology , Chicago. 35. Karel, M� , and T. P. Labuz a. 1968. Non-enzymatic browning in model systems containing sucrose. J. Agr. Food Chern . 16, 717-719 . 112 36. Labuza, T. P. 1968. Sorption phenomena in ' foods . Food - Technol. _£, 263-272 e 37. Labuza, T. P. , H. Tsuyuki, and M. Karel . 1969. Kinetics of linoleate oxidation in model systems . J. Amer. Oil Chern. Soc . �' 409- 416. 38. Labuza,. T . P., S. R. Tannenbaum , and M. Karel. 1970. Water content and stability of low moisture and intermediate-moisture foods . -Food Technol . -24, 543-549 . 39 . Larmond, E. � 19 70 . Methods for sensory evaluation of food. Pub lication 1284 , Revised ed. , Canada Depart ment of Agriculture , Ottawa, Canada. 40. LeRoux , J. P. , and S. R. Tannenb aum . 1969. Determina tion of the extent of browning in model systems simulating fatty foods . Ab stract No . 9. Presented at the 29th annual meeting of the Ins titute of Food Technology, Chicago. 41. Loncin, M. , J. J. Bimbenet, and J. Lenges . ' 1968. Influ ence of the activity of water on the spoilage of food stuffs . �· Food Technol. �' 131-142. 42. MacKinney , G. , and A. C. Little. 1962. "Color of Foods ," pp . 104-114 . T�e AVI · Publishing Company , Incorporated, Westport, Connecticut . 43. Maloney , T. F. , T. P. Labuza, D.· H. Wallace , . and M. Karel. 1966. Autoxidation of methyl linoleate in freeze-dried model sys tem. I. Effect of water on autocatalyzed oxidat ion. �· Food Sci . �' 878-884 . 44. Marquis , R •.Eo 1968. Salt-induced contraction of bacterial cell walls . �· Bacteriol. �' 775-781 . 45. Martin, R. E. 19 11. Edi&le soy pToteins . Soybean Digest Blue Book Issue �(6) , 44- 51. 46. Martin, R. E., and D. V. LeClair. 1967. Texturized proteins for creating products . Food Eng. l2_(4) , 66-69 . 47. Martin , R. E., R. Wigg ins , and J. V. Ziemba. 1971 . Textured vegetable proteins come of age. � Eng. 43(5) ' 80-82 . 113 48. Martinez, F. , and T . P. Labuz a. 1968. Rate of deteriorat ion of freeze-dried salmon as a function of rel ative humidity . · !!_.. Fo od Sci . �, \2 41-24 7. 49 . Mattick , L. R. , and D. BG Hand . 1969 . Identification of a volatile component in soyb e ans that contributes to the raw bean flavor. �· Agr . Food Chern. !l, 15-17. SO . Matz, .S. A. 1965. "Water in Foods ," pp . 249-261. The AVI Publishing Comp any , We stport , Connecticut . 51. Miles Lab or�tories , Incorporated. Undated. Temptein spun vegetable protein meat analogs � Elkhart , India��· 52 . Mossel, D. A. A. , an d H . . J. L. Van Kuijk. 1955. A new and simp le: technique for the direct determination of the equilih rium rel ative humidity of foods . Food · Research '*' 415-:423� 53. Potter, N� N� 1970. Intermediate mois ture foods : principles and technolo�y . Food Product Development 1_(7) , 38-48. 54 . Rockland , L. B. 1969 . Water activity and storage stabil�ty. Food ) Technol . 23, 1241-1249 . ; -- - 55. Salwin , H. 1959 . Defining minimum mo isture require ments for qehydrated foods . Food Techno!. 13, 594- 595. 56. Schoebel , T. , s. R. Tannenb aum , and T. P. Labuza. 1969 . Re action at limited water concentrat ion . I. Sucrose hydro 1 y s i s . !!,•; Food S c i . 3 4 , 3 2 4 - 3 2 9 . 57. Sco!t, W. J. : 1957. Water relations of food spoilage m1croorgan1sms . In "Advances in Food Research ," eds . Chichester, C. 0. , E. M. Mrak , and G. F. Stewart, val . 7, pp . 83-127. Academic Press, New York . 58. Sharf , J. M. , ed. 1966 .. "Recommended Me thods for the Microbiologi cal Examination of Foo TAB LE XXXV FORMULAS FOR THE CAL CULAT ION OF C. I.E. VA LUES FROM COLOR-EYE COLORIMETER TRI -STIMULUS DATA X, Y, Z, ( r) Fo rmulas 1. C. I.E. X = (0 . 783) X + (0 . 197) X 2. C. I.E. y · = y 3 . C. I.E. z = (1. 18) z 4. . Chromaticity coordinates : X = C.I.E. X + v + z t.I.E. c.I.E. y = C.I .E. Y c.!�E. · + . + .x c. 1 E. Y c.I.E. Z 5. Lightness index = � 116 117 TABLE XXXVI EFFE CT OF THE INTERACTION OF CALCIUM LACTATE AN D ACIDITY ON WATER ACTIVITY OF INTERMEDIATE MO ISTURE TEXTURE D VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85!/ Acidity (pH) Calcium Lactate (Per Cent) 7.0 5.5 0.00 0.858 0.852 0.25 0.851 0.844 !/Me ans of 42 observati ons . No significant differences among the means at the 0.05 level of probability. 118 TABLE XXXVI I EFFECT OF THE INTERACTION OF CALCIUM LACTATE AND STORAGE TIME ON WATER ACTIVITY OF INTERMEDIATE MOISTURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85_1 / Ca�c�um I:actate tl'er �entl Time �Dals ) O.OD 0.�� 0 0.852 0.844 10 0.854 0.847 20 0.854 0.849 30 0.8 56 0.847 40 0.855 0.848 so 0.856 0.848 60 0.857 0.849 !/Me ans of 12 ob servations . No significant differences among the means at the 0.05 level of probability. 119 TABLE XXXVIII EFFECT OF THE INTERACTION OF ACIDITY AND STORAGE TIME ON WATER ACTIVITY OF INTERMEDIATE MOI STURE TEXTURED VEGETABLE PROTEIN CHUNKS WIT TARGET WATER ACTIVITY OF 0.85�-/ Xc 1a1ix: tERJ Time �Dals) z .� �.� 0 0.851 0.845 10 0.854 0.846 20 0.854 0.848 30 0.8 55 0.848 40 0.855 0.848 so 0.854 0.850 60 0.856 0.850 !/Means of 12 observations . No significant differences among the means at the 0.05 level of probability. 120 TABLE XXXIX EFFE CT OF THE INTERACT ION OF CALCIUM LACTATE , ACI DITY AND STORAGE TIME ON WATER ACT IVITY OF INTERMEDIATE MOI STURE . TEXTURED VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACTIVITY OF 0.8s l/1 � r T i e ija�c1um tactate l'e 5entJ m �DalsJ lJ.OO �.2� pH 7. 0 0 0.8SS 0.847 10 0.8S7 0.8S2 20 0.8S7 0.8S2 30 0.8S8 0.8S2 40 0.8S8 0.8S3 so 0.8S9 0.8SO 60 0. 860 0.8 S2 pH S.S 0 0.849 0.8 41 10 0.8SO 0.842 20 0.8Sl 0.84S 30 0.8S3 0.842 40 0.8S2 0.843 so 0.8S2 0.847 60 0.8S4 0.84S l/Means of 6 observations . No significant diffe rences among the means at the o.os level of prob ability. 121 l TABLE XL EFFECT OF THE INTERACTION OF CALCIUM LACTATE AND ACIDITY ON MOISTURE CONTENT OF INTERMEDIATE MOI STURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85!,�/ Calcium Lactate (Per Cent) 7�0 5.5 0.00 2.24 2.0 6 0.25 2.11 1.94 li Grarns of water per 5.00 grams of sample. !/Means o·f 84 observations . No significant differences among the me ans at the 0.05 level of �robabi1ity. 122 TABLE XLI EFFECT OF THE INTERACTION OF CALCIUM LACTATE AND STORAGE TIME ON MO ISTURE CONTENT OF INTERMEDIATE MOI STURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85!,_2/ �a�c1um [actate tPer �entJ Time (Dax:s) • 0 .2� 0 2 .17' 2.0 4 10 2 . 15. 2 . 02 20 2.16 2.04 30 2.14 2.01 40 2.15 2.02 50 2.14 2. o 2 · 60 2.13 2.01 !/Grams of water per 5.00 grams of sample. !/Means of 24 observations . No significant differences among the means at the ·o.os level of probability. 123 TABLE XLI I EFFECT OF THE INTERACTION OF ACIDITY AND STORAGE TIME ON MOISTURE CONTENT OF INTERMEDIATE MOI STURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85!,1/ Xcia:iti Time �Dars ) 7.0 �ERJ�.� 0 2.19 2. 0 2 . 10 2.17 2.00 20 2.19 2.01 30 2.16 2.00 40 2.16 2.00 so 2.17 1.99 . 60 2.15 1.98 !/Grams of water per 5.00 grams of sample. �/Means of 24 observations . No significant differences among the means at the 0.05 level of probability. 124 TABLE XLI II EFFECT OF THE INTERACTION OF CALCIUM LACTATE , ACIDITY AND STORAGE TIME ON MOI STURE CONTENT OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF o.ssl,�/ [act ate rPe r entJ Time (Darsl �glum �-� 2� pH 7.0 0 2.25 2 .13' 10 2.23 2.11 20 2.25 . 2.12 30 2.23 2.09 . 40 2.23 2 .10 . so 2.23 2 .10 '. 60 2.2 2 2.09 pH 5.5 0 2.03 1.95 10 2.06 1.93 20 2.0 7 ' 1.9 5 . 30 2.06 1.94 40 2.07 1.94 so 2.05 1.93 60 2.04 1.92 .!/Grams of water per 5.00 grams of sample . '!_/Me ans of 12 observations . No significant diffe rences among the means at the 0.05 level of probability. 125 TABLE XLIV EFFECT OF THE INTERACTION OF CALCIUM LACTATE AND STORAGE TIME ON pH OF INTERMEDIATE MOI STURE TEXTURED VEGETAB LE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.8sl/ a c1um I:actate �Pe r �ent Time (Dars) ij . �0 0.2� 0 6.31 6.23 10 6.25 6.16 20 6.15 6.08 30 5.98 5.91 40 6.13 6.08 so 6.09 6.05 60 6.06 6.0 1 !/Means of 24 observations . No significant differences among the means at' the 0.05 level of probability. 126 TABLE XLV EFFECT OF THE INTERACTION OF ACIDITY AND STORAGE TIME ON pH OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.85!/ Xc1C11tl Time (Dalsl 7.D [ERJ�.� 0 7.01 5.53 10 6.97 5.45 20 6.82 5.4 1 30 6.66 5.23 40 6.78 5.44 so 6.75 5.38 60 6.70 5.37 !/Means of 24 observations . No significant diffe rences among the me ans at the 0.05 level of probability. 127 TABLE XLVI EFFECT OF THE INTERACTION OF CALCIUM LACTATE , ACIDITY AND STORAGE TIME ON pH OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVI TY OF 0.8sl/ Time (Days ) pH 7. 0 0 7.09 6.92 10 7.0S 6.88 20 6.88 6.7S 30 6.72 6.S9 40 6.83 6.73 so 6.77 6.74 60 6.7S 6.66 pH S.S 0 S.S2 S.S4 10 S.46 S.44 20 S.42 S.40 30 S.23 S.23 40 S.44 S.44 so S.42 S.3S 60 S.38 S.3 7 !/Means of 12 ob servations . No significant differences among the means at the O.OS level of prob ability. 128 TABLE XLVII EFFECT OF THE INTERACTION OF CALCIUM LACTATE AND STORAGE TIME ON FIRMNESS OF INTERMEDIATE MOISTURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.851,�/ �a&c1um J:actate �Per �entJ Time (Dars) • D .2� 0 460 610 10 4 77 638 20 4 79 633 30 469 623 40 466 628 so 480 641 60 4 78 634 !/Firmness was express ed as the pounds of force required to shear the sample. �/Means of 36 obs ervations . No significant differences among the means at the 0.05 level of probability. 129 TABLE XLVIII EFFECT OF THE INTERACT ION OF ACIDITY AND STORAGE TIME ON FIRMNE SS OF INTERME DIATE MOISTURE TEXTURED VE GETAB LE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF o.ss !.�/ �c 1a1tl �EHJ : Time (Dals) 7.0 �.� 0 379 691 10 400 716 . 20 398 713 30 393 699 40 394 . 700 so 400 . 722 60 400 712 !/ Firmness was expressed as the pounds of force re quired to shear the sample . �/Me ans of 36 ob servations . No significant differences among the means at the 0.05 level of prob abil ity . 130 TAB LE XLIX EFFECT OF THE INTERACT ION OF CALCIUM LACTATE , AC IDITY AN D STORAGE TIME ON FIRMNESS OF INTERMEDIATE MO ISTURE TEXTURE D VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACT IVITY OF 0.8S! ,�/ � n tJ T i m §a c1um I:actate rPe r ffe e (Dax:sl o.Dn �. 2� pH 7.0 0 321 437 10 338 462 20 338 4S9 30 334 4S3 40 331 4S6 so 33S 46S 60 340 460 pH S.S 0 S99 783 10 616 81S 20 620 80 7 30 60S 792 40 601 800 so 62S 818 60 616 808 !/ Firmness was expressed as the pounds of force re quired to shear the sample. �/Means of 18 observations . No significant differences among the means at the O.OS level of prob ability. 131 TAB LE L EFFECT OF THE INTERACT ION OF CALCIUM LACTATE AND STORAGE TIME ON COLOR INDICES OF INTERME DIATE MO ISTURE TEXTURED VE GETABLE PROTE IN CHUNKS WITH TARGET WATER ACT IVITY OF 0.85-1 / Calc 1um C.I.E. �/ Lactate Time Coordinates WaDominave lentngth Purity L ightness r n t X (Pe Ce ) (Days) y �nm) �Per Cent) Index 0.00 0 0.394 0.376 582 38.8 4.45 1 0 0.396 0.377 582 38.8 4.52 20 0.395 0.377 582 38.8 4. 59 30 0.39 7 0.3 78 582 39 .4 4.54 40 0.397 0.378 582 39 .4 4.54 so 0.396 0.377 582 38.8 4.65 60 0.396 0.377 582 38 .8 4.64 0.25 0 0.394 0.376 582 38 .8 4.59 1 0 0.397 0.377 582 38.8 4.64 20 0.396 0.3 77 582 38.8 4.68 30 0.395 0.377 582 38 .8 4.69 40 0.396 0.378 582 39 .4 4.70 so 0.395 0.376 582 38 .8 4. 70 60 0.396 0.376 582 38.8 4.74 132 TABLE L (continued) !/Me ans of 36 observati ons . �/Dominant wavelength was expressed in nanometers . No significant differences among the means within each variable at the 0.05 level of probability. 133 TABLE LI EFFECT . OF THE INTERACTION OF CALCIUM LACTATE , ACIDITY AND STORAGE TIME ON COLOR INDICES OF INTERMEDIATE MOI STURE TEXTURED VEGETABLE PROTEIN CHUNKS WITH TARGET WATER ACTIVITY OF 0.8sl/ Calcium Lactate Acid- C. I.E. Domin an t3./ Purity (Per ity Time Coordinates Wavelength (Per Ligh tne s s Cent) (;eH) (DarsJ X l 'run) Cent� Inde x 0.0 0 7.0 0 0.398 0. 3 76 S83 40.S 4.09 10 0.400 0.377 S83 40.S 4.19 20 0.400 0.377 S83 40.S 4.2 1 30 0.402 0.378 S83 40.S 4.22 40 0.401 0.378 S83 40.S 4.24 so 0.402 0.377 S83 40.S 4.29 60 0.401 0.377 S83 40.S 4.34 0.00 s.s 0 0.392 0.376 S82 37.9 4.81 10 0.391 0.377 S82 37.9 4.86 20 0.389 0.376 S82 37.9 4.97 30 0.392 0.378 S82 39 .4 4.87 40 0.392 0.378 S82 39.4 4.83 so 0.389 0.376 S82 37.9 S.Ol 60 0.391 0.377 S82 37.9 4.94 0.2S 7.0 0 0.399 0.3 77 S83 40 .S 4.17 10 0.402 0.377 S83 40.S 4.2S 134 TABLE LI (cont inued) Calcium Lactate Acid- C. I.E. Domin an t'l:./ Purity (Per ity T ime Coordinates Wavelength (Per Lightness Cent� �;eH) �DaiS� X I �nm) Cent� Index 20 0.402 0.377 S83 40.S 4.2S 30 0.400 0.378 S83 40 .S 4.36 40 0.403 0.378 S83 43.0 4.34 so 0.401 0.377 S83 40.S 4.42 60 0.404 0.378 S83 43.0 4.36 0.2S s.s 0 0.388 0.37S S82 37.9 s.01 10 0.392 0.377 S82 37.9 s.0 2 20 0.389 0.3 76 S82 37.9 S.ll 30 0.390 0.377 S82 37.9 S.02 40 0.390 0.377 S82 37.9 s.os so 0.390 0.376 S82 37.9 4.98 60 0.388 0.37S S82 37.9 S.l3 !/Means of 18 observations . �/Dominant wavelength was expressed in nanometers . No significant differences among the means within each variable at the O.OS level of probability. 135 TABLE LII EFFECT OF THE INTERACTION OF ACIDITY AND STORAGE TIME ON WATER ACTIVITY OF INTERMEDIATE MOI STURE TEXTURED VEGETABLE PROTEIN CHUNKS WIT TARGET WATER ACTIVITY OF 0.80-�/ Aci so 0.808 0.799 60 0.816 0.805 !/Means of 8 observations . No significant differences among the means at the 0.05 level of probability. 136 TABLE LI II EFFECT OF THE INTERACTION OF AC IDITY AND STORAGE TIME ON MO ISTURE CONTENT OF INTERMEDIATE MO ISTURE TEXTURED VE GETABLE PROTE IN CHUNKS WIIH rARGET WATER ACTIVITY OF 0.80-'-'2 R� Xciaitr [E Time �Dalsl 7.0 �.s 0 1.7 2 1.60 10 1.73 1.60 . 20 1.75 1.62 30 1.7 2 1.59 40 1.73 1.6 2 . so 1.71 1.63 60 1.76 1. 64· !/ Grams of water per 5.00 grams of sample. �/Me ans of 12 ob servations . No significant diffe rences among the means at the 0.05 level of probability. 137 TABLE LIV EFFECT OF THE INTERACTION OF ACIDITY AND STORAGE TIME ON pH OF INTERMEDIATE MO ISTURE TEXTURED VEGETABLE PROTE IN CHUNKS WITH TAR ET WATER ACTIVITY OF 0.80-�/ Xcia1tl Time {Dars ) 7.tJ tERl�.� 0 6.94 5.47 10 6.96 5.67 20 6.92 5.66 30 6.83 5.63 40 6.78 5.60 so 6.75 5.65 60 6.71 5.5 4 !/Me ans of 12 observations . No significant differences among the means at the 0.05 level of probability. 138 TAB LE LV EFFECT OF THE INTERACT ION OF AC IDITY AND STORAGE TIME ON FIRMNESS OF INTERMEDIATE MOISTURE TEXT URE D VE GETABLE PROTE IN CHUNKS WITH TA�G� T WATER ACT IVITY OF 0.80-'-/ (Days) 7.0. Time 5.5 0 444 731 60 471 769 !./ Firmness was expressed as the pounds of force required to shear the sample. �/Me ans of 24 observations . No significant diffe rences among the means at th e 0.05 leve l of probability . 139 TABLE LVI EFFECT OF THE INTERACTION OF ACIDITY AND STORAGE TIME ON COLOR INDICES OF INTERMEDIATE MOISTURE TEXTURED VE GETABLE PROTEIN CHUNKS WIT TARGET WATER ACTIVITY OF 0.80¥-/ C. I.E. Dominant!/ Acidity Time Coordinates Wavelength Puri:ty Lightness (pH) (Days) X y �nm) �Per Cent) Index 7.0 0 0.400 0.379 583 40.5 4.18 60 0.403 0.379 583 43.0 4.44 5.5 0 0.390 0.3 77 582 37.9 4.96 60 0.393 0.378 582 39 .4 5.10 !/Means of 24 observations . �/Dominant wavelength was expressed in nanometers . No significant differences among the means within each variable at the 0.05 level of probability. APPENDI X B x - without calcium lactate and w:j.th 0.25% calcium lactate ·- �I \'��'· •- pH 7.0 I \ --� A- pH 5. 5 - ����-+--�--*-�--�=-+-�� - �-+--�--+-��-+--��� ·�·�--�--+-��-+--�--T-�r--+- -1 \ "" ------� -- .�� �-4 � 4--4,�-4--��� ..�--� --�� �--�--�� . eRIE� YEL OWISH � Km ''"'" N a... -�·,..,.. r \ MEE I I itt' / Ml ;· ---r---.�--"-· , The (x, y ) - Ch romat icity Diag ram of the C.I .E. Sys tem Fi gure 7. Effe ct of calcium lactate and pH on the color of intermedi at e moisture textured vegetable protein chunks . 140 141 Scheme for the Identification of Gram Negative Rods and Pseudomonads I. Identification of Gram Negative Rods A. Produce acid or acid and gas strongly at 37° C Oxidase positive : Aeromonas Oxidase negative : Enterobacteriaceae B. Do not produce strong acid, if any , at 37° C Yellow pigmentation Produce ketogluconic acid : Xanthomonas Does not produce ketogluconic ac1d : Flavobacterium No yellow p1gment Oxidase pos itive Pseudomonas Oxidas e negative Not fermentative : Alcaligenes Fermentative Weakly acid : Achromob acter Curved rods : Vibrio spp . Produce HAc on EtoH-Ca carbonate agar: Acetobacter II. Identi fication of Pseudomonas A. Grow at 4Z° C : �· Aeruginosa B. Gelatin liquefied Grow better at 37° C than at Z0° C Nitrate reduced : P. Caviae Nitrate not reducea : �· re tivorum Grow better at zoo than at �7o C Nitrate reduced : c·P. fluorescens , P. myxogenes Nitrate not reducea : �· fragi1 C. Gel atin not liquefied . Grow better at than at ° Nitrate reduced370 : c P. put ida Z0 c Nitrate not reducea : P. amb i8ua Grow better at zoo C than at 37 C Nitrate reduced : P. convexa Nitrate not reducea : P. taetrolens Reaction not known : P7 incogn1to 142 SCORING SHEET PRODUCT DATE NAME · ------A. Evaluate each samp le for the quality factor lis ted below. Use the appropriate scale to show your evaluation and check the point that best describes your feeling ab out the sample. Texture Code Code Ve ry tough Moderately tough Slightly tough Neither tough nor soft Slightly soft Moderately soft Ve ry soft Mo istness Very dry Moderately dry Slightly dry Neither dry nor excess mo isture Slightly excess mo isture Moderately excess mois ture Extreme excess moisture Overall Flavor Like very much Like moderately 143 (Overall Flavor) Code Code Like slightly Neither like nor dislike Dislike slightly Dislike moderately Dislike very much B. Please indicate the perceptibility of the following . flavors in each of the samples . Mark a double check (��) if the flavor is intens ely perceptib le and a single check (V) if it is moderately or slightly perceptible . Check as many flavors as applicable . Flavor Code Code Beany flavor Salty Sweet Bitter Burning or biting aftertaste 144 SCORING SHEET PRODUCT DATE NAME · ------Please evaluate the se samples for overall accepta bility. Show how much you like or dislike each samp le by checking at the point that best describes your feeling . CODE CODE Like extremely Like extremely Like very much Like very much Like mode rately Like moderately Like slightly Like slightly Neither like nor dislike Neither like nor dislike Dislike slightly Dislike slightly Dislike moderately Dislike moderately Dislike very much Dislike very much Dislike extreme ly Dislike extreme ly COMMENTS COMMENTS VITA The author was born on December 5, 1945, in Mani la, Philippines . From 1951 to 1959 she attended the Hope Christian High School. In 1959 she entered the University of the Philippines High School and was graduated in 1963. In June , . 1963, she attended The Univers ity of the Philip pines and was graduated with a Bachelor of Science de gree in Food Technology in 1968. She was a recipient of the National Science Development Board Scholarship during the course of her undergraduate study. From May , 1968 to July, 1969 she worked with the Reliance Commercial Enter prise, Incorporated, in the Food Product Deyelopment Division. Since Fall of 1969 she has been studying at The University of Tennessee , Knoxville , in the Department of Food Technology and working to complete the requirements for the degree of Master of Science. 145