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5-1959

The Use of Protease Enzymes in the Manufacture of Cheddar Cheese

Osamu Fujiwara Utah State University

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Recommended Citation Fujiwara, Osamu, "The Use of Protease Enzymes in the Manufacture of Cheddar Cheese" (1959). All Graduate Theses and Dissertations. 4768. https://digitalcommons.usu.edu/etd/4768

This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. THE IBE OF PROTEASE ENZYMES IN THE

MANUFACTURE OF CHEDDAR CHEESE

by

Osamu Fujiwara

A thesis sul:mitted in partial fulfillment of the requirements for the degree

of

MAS"J'ER OF SCIENCE

in

Dairy Manufacturing

lJfAH STATE UNIVERSITY• Logan, Utah 1959 ACKNOWLEOOMENr

I viah to express appreciation to Professor A. J. Morris for his able assistance in directing this research problem and for his help in the periodic scoring of the cheese. I am also grateful to

Professor Paul B. Larsen for his assistance in scoring the cheese . I vish to thank Dr. Harris 0, Van Orden and the Chemistry Department end Dr. Gene W, Miller and the Department of Botany and Plant Pathology at Utah State University for the use of their equipment and laboratory vhere much of the analytical work of this project vas dona.

I vish to thank Mr. R, Z, Roundy of the Central Research

Laboratories, Chicago, Illinois, and Paul-Lewio, Inc., Milwaukee,

Wieconsin for their help in obtaining the commercial enzyme and information.

Osemu Fujiwara TABLE OF CONTENTS

Page

Introduction

Importance of proj ect 1 Purpose of project 1

Review of literature J Importance of enzymes found in milk J Effect of proteolytic enzymes obtained from animsl sources on cheese making J Significance of hydrolysis of pr oteins uring the ripening period 4 Soluble nitrogen products r esulting f rom action of pepsin 5 Effpct of various concentrations of rennet on Cheddar cheese making 6 Activity of rennet and its disadvantage 7

Procedure 8

Measureme nt of enzyme activity 8 Manufacturing of the cheese 9 Results 15 Activity of individual enzyme 15 Results from interpretation of Tables 7 to 10 22 Soluble nitrogen det ermination 28 Chromatograph for amino acids in cheese JJ Summary J8

Conclusions J9 Literature cited 41 LIST OF TABLES

Table Page

1. Activity of rennet by clotting-milk method • 17

2 . Activities of P- enzyme and F-enzyme by clotting-milk method 17

J . Record of pH and acidity developmen t in the manufacture of Cheddar cheese 18

~ . Record of pH and acidity development in the manufacture of Cheddar cheese 19 5 . Record of pH and acidity development in the manufacture of Cheddar cheese 20

6. Record of pH and acidity development in the manufacture of Cheddar cheese 21

7. Scores of Cheddar cheese samples 25

8 . Scores of Cheddar cheese samples 26

9 . Scores of Cheddar cheese samples 27 10 . Soluble nitrogen determination in total solids non fat in Cheddar cheese 30

11. Soluble nitrogen det ermination in total solids non fat 1n Cheddar cheese 31

12. Soluble nitrogen determination in total solids non fat in Cheddar cheese 32

lJ. Paper chromatography 35

1~. Qualitative analysis of amino acids in cheese by paper chromatography 36

15. Qualitative analysis of amino acids in cheese by paper chromatography 37 LIST OF FIGURES

Figure Page 1. Manufacturing procedure for Cheddar cheese 10 2. Enzyme activity of clotting milk 16

), Soluble nitrogen in cheese 29 INI'RODUCTION

Importance ~ ~

The factors governing flavor and body and texture in cheese

making include the quality of the milk used, the heat treatment of

the milk, the quality and quantity of the starter, the treatment

during the ripening period, other biological conditions and manu­ facturing technics.

The ripening period, the period of storage of the cheese after

the manufacturing pr ocess , is one of t he mo re important stops .

Fresh cheese he s little flavor; it has only a very mild taste and

aroma, The body of the cheeae is relativel y firm, tough and curdy, and somewhat mesly. However, during the r i pening process, the body

and t exture of t he cheese loses some of i ts elasticity end f irmness, and curdy nature and becomes mellow and di ssolves easily i n the mouth. It also develops the characteristic flavors end aroma of Cheddar cheeee.

The physical changes occurring in ripen ing are accompanied by the decomposition of proteins and the hydrolysis of fats, with the fermentation of lactose end the formation of volatile fatty acids .

Changes occurring during the ripening period con be controlled by altering the conditions of ripening . Among these conditions is the type snd degree of enzyme activity on the proteins . The enzymes are some of the ripening agents which influence the rste of ripeni ng.

Purpose ~ mJlli

The purpose of this experiment is to attempt to improve t he body and texture end flavor of Chedder cheese end to shorten the 2 ripening period~ the addition of oommerciallr-prepared enzrmes.

The ripening period for average Cheddar cheese is at least three months, and the "aged" Cheddar cheese is ripened for a much longer period. 3

REVIEW OF LITERATURE

Importlnct 2I. ~ ~ 1n J!Jill Although the enzymes protease, lipase, ond phosphatase ort

found in very small quontities in milk, their activities should

not be ignored under fovoroble conditions. According to Ling (6) their specific activities ere&

1. Protease, the proteolytic enzyme stimuloting protein hydrolysis and capable of breoking down the proteins into polypeptides and amino acids. Its activity is often used to meesure the ripe age of the cheaae. 2. Lipase, the enzymt hydrolyzing ~ilk fat under fovorable conditions and responsible for the formation of volatile fatty acids including butyric acid, which gives cheese a rancid flavor. Lipases are used in some of the foreign processes of cheese manufacture but lipase activity does not play an important role in Cheddar cheese making. It is insctivoted by the pasteurization of milk. 3. Phosphotase serves no useful purpose in cheese making, but is of interest because it can serve as a means of checking the proper pasteurizotion of milk. This is accomplished by testing for its presence in the milk, since it is inectiveted by proper pesteurizetion,

Effect g! proteolytic enzY!!!eS obttined from enimel sources 5m

Information concerning the use of proteolytic enzymes in cheese meking was received from Roundy (12), Centrel Research Laboratory,

Chicago, Illinois, where an inactive enzyme (pro-enzyme or zymogen) was prepared, which was activoted at the time of use with a small amount (about one per cent) of an active trypsin or enterokinase.

The inactiveted enzyme and active enzyme were used in this research under the label of "proteue from animal origin." The enzyme is added immediately before the addition of rennet, and the coagulation of milk by rennet occurs in the normal manner as on page 9 in the 4 section of the preparation of Cheddar cheese. The enzyme is apparently

activated during the curd-making process and during the ripening period. The resulting product is usually relatively soft and elastic, whereas

the product from the use of the fully-activated enzyme showed many undesirable characteristics. The phenomenon were attributed to the

rapid rata of digestion of break-down of the curd aad are1 1. Less in whey of much of the casein. 2. A soft and flocculent finished product. J. Excessive moisture centent in the finished product. 4. Development of bitter and scid flavors.

One desirable characteristic found was that the body of the

finished cheese has a superior quality to that produced using the

inactive enzyme. Roundy (12) suggests that the enzymes be added st

the rate of one to two grams per thousand pounds of ailk and that the

enzymes should be added in the form of a freshly prepared solution. The enzymes should be used at a ratio of five parts of the active

enzyme to ninety-five parts of the inactive enzyme.

Significance 2f hYdrolysis ~ proteins ~ ~ ripening ~ During the ripening of the cheese, the complex protein molecules

are broken down into compounds which become progressively simpler in cheaioal composition and physical structure. The changes may be

represented in the order of insoluble proteins, protease&, peptones, polypeptides and amino acids. All these stages are found in the

ripened cheese and are brought about by the proteolytic enzymes of

rennet and enzymes secreted by the lactic acid-producing bacteria. The specificity of rennin as described by Neiland (10 ) is to cleave

phospho-emides.

The rate of ripening of cheese csn be determined by the amount 5 of water-soluble nitrogen compounds . The investigations of the various ripening phenomenon have placed emphasis on the water-

soluble nitrogen compounds and have not corr~lated the effect of the water-soluble nitrogen compounds with the flavor development

of the cheese. Storgards and Lindquist (14 ) inve~tigated the flavor of each amino acid and its relative concentration in cheese, They also pointed out that peptides may also contribute to the flavor of cheese. Harper and Swanson (4) examined the ripening index (soluble nitrogen divided by total ni trogen in cheese on

per cent basis) and th~ concentrations of various free amino acids

of Cheddar cheese pos~essin g various degrees of Cheddar flavor.

They found that the free amino acid concentration was more closely

related to th~ flavor intensity than was the ripening index. The following chart is the work Storgards and Lindquist (14) conducted:

Fhvor of Group A B c D E Amino acid

FlaYor intensity 0 1 2 3 4 Age in month 0.5 8 8 8 48 Ripening index 9.1 18.7 35.5 32.9 46.9 mg. free amino acid from the water extract of lOg cheese solid

Valine 1 3 14 34 55 Sl. sweet Isol~ucine 1 5 8 13 47 Bitter Glycine 2 4 9 13 52 Sweet Lysine 4 30 41 46 102 Bitter Leucine 13 37 53 56 145 Sl. bitter Glutamic acid 15 37 98 61 269 Broth Proline 4 15 15 16 34 Sweet Phenyl alenine 6 19 19 37 61 Sl. bitter Aspartic eoid 26 75 48 56 102 Bitter broth

Total found 72 225 305 332 867

~ nitrogen products reaultinc !r2m J£11Qn ~ ~ Zittle and Cerbulia (20) worked on the amount of soluble products 6 resulting from the action of pepain on whole casein at pH 6, 5, This was determined by precipitating the solution with oalcium chloride and twelve per cent trichloro-acetic acid and adjusting the pH to 4.7 with hydrochloric acid. The study of the soluble fraction of

the result by chromatography ahowed that it consisted of more than

ten components of which none could be identified as free amino. A

high molecular weight for some of the component was indicated by

retention of the material within a cell ophane dialysis bag and also

by the presence of many amino acids in these components. These

evidences suggest a fairly large polypeptide. The results also

indicate that the soluble fraction arises f rom the rapid hydrolysis

of papain or rennin and from a number of susceptible bonds that give

rise to a number of relatively large peptides. The specificity of

pepsin suggests that the split would occur adjacent to phenylalanine or tyrosine, which corresponds to the table of enzyme specificity given by Neiland (1 0).

Effect 91 ~ concentrations 91 rennet 2n ~ £hi.!.ll! l!IA.Uni Reaearch work done on the effects of various concentrations of rennet on cheese making by MoCulloch (9) showed the following:

1. The chief flavor defecta were bitterness and fruitiness.

Six of the ten high rennet cheeses were de termined to be decidedly bitter.

2. The body and texture defecta were pastiness, mealiness, opennesa of texture, and atickinesa.

J. The addition of large amounta of rennet in the manufacture of Cheddar cheese tends to produce a greater quantity of water­ soluble nitrogen.

4. The firmer curd was obtained during the cooking process of 7 high rennet cheese, owing possibly to the greater coagulating pro­ perty of excess rennet .

5. There was no notice1ble effect of loss or gain of fat and moisture content,

McCulloch (9) also suggested that bitter flavor was produced Qy certain types of organisms and by the rapid hydrolysis of the casein and that they were more pronounced in conditione of high moisture and warm temperatura during the curing process, Sinoe the cheese including the lower rennet cheese also had a body defect, McCulloch concluded that the defects were due to processing, not to a body defect from an additional enzyme.

Actiyity gt ~ ~ i1A disadyantaga Ling (6) stated that the presence of pepsin in rennet has been considered to be a disadvantage in cheese making. Rennin is able to coagulate milk of very low hydrogen in concentration; and its optimum pH is in the region of 5.4, whereas papain is able to sot at much higher acidities which are unfavorable to rennin. Also it is known that rennin is able to break down the protein molecules to much greeter extant than pepsin; one disadvantage of the presence of pepsin in rennet extracts is the tendency to produce peptonea in cheese, These are believed to give a bitter flavor to the ripened product (7), Some work done at the Geneva Station u,y Van Slyke and Price (15, 16) showed that cheese made with larger amounts of rennet furnished greater quantities of soluble nitrogen compounds then did cheese made with smeller amounts of rennet. This conclusion corresponds to work done by Babcock, Russell, and Vivien (1) and Freeman and Dahle (J), 8

PROCEDURE

Measurement of ~ activitY

Enzymes are macromolecular catalysts of biological origin or

can be defined as catalytically active proteins. Like other cata­

lysts, enzymes also accelerate chemical reactions. Thera ere quite

a number of methods of measuring the activity of milk curdling

enzymes such as the curd-tension method, end the chemical analysis

for amino ecids. The proteolytic enzyme has an ability to coagulate

milk; therefore, clotting of milk can be measured against time, and its activity can be interpreted by means of a graph.

By the milk-clotting method suggested by Colowick und Kaplan (2)

20 grams of vhole powdered milk was ground to a smooth paste with a

small amount of pH 4.6 acetate buffer (prepared by two volumes of one molar ecatic acid with one volume of one molar sodium hydroxide ). Ten ml. of this buffer was diluted to 85 ml . and added to the powdered milk to give a total volume of 100 ml. The reconstituted milk was filtered through a cheese cloth. One ml. of a series dilution of the enzyme being tested vas incubated in a test tube at 40°C vith

10 ml. of the prepared milk. ~hen the tubes showed a thickening on tilting, the reaction vee considered compl ete. In general, the relation between clotting time and enzyme concentration is a straight line that can be expressed by the equation:

E : k/t E •••• veight of enzyme t •••• time in seconds 9

Hanufocturipg 2! ~ ~

Preparation ~ ~· Frozen starter vas made from Hansen

cultures consisting of a mixture of numbers 23, 24, 25, 32 and 33.

Bulk starter vas prepared ~ edding three per cent of the frozen

starter to the eterilized skim milk and ~ incubating at 70Pf for

15 to 17 hours.

Preparation ~ commercial~ solution 12t ~making. Three kinde of commercial enzymes vere aveileble for use; one from

enimal source, and tvo, F- and P-enzymee, vere obtained from plant

source . The commercial enzymes vere veighed end kept under refriger­

ation in e ?ial until its use. When they vere added individually to

milk, they vere diluted vith 300 ml. of cold voter. (Since animal enzyme ie unactive, five parts of mixase (activator) vas added to

95 parte of the unective enzyme in ordor to make it active.)

!.b! ~method 1:21: ~ ~ ~. Three vats of cheese vare prepared from the semo milk using the Wilson's (18 ) clock method, which is outlined in Figure 1. The control vat did

not contain any commercial enzymes, end the other tvo vere treeted vitb different concentrations of the commercial enzymes. Tho same manufacturing proceduro ves used in each vat.

~ gf ~ anelvsio. Analysis vere mado by the methods

ee recommended ~ Wilster (19) end Van Slyke end Price (17).

Sampling ~. When a sample representative of the entire cheese vas desired, the semple hed to consist of et least three plugs; one taken from the center, another taken from a pcint one inch beneath the outer edge, end the third from a point midway between the firet tvo plugs. All plugs were taken perpendicular to the face of the cheese end extended neerly through the cheese. Aciditittl! !Tocedure at~ Time

0.16 - 0.17 6.5 Adding starter 86- 88~ - hour _ 0.165 - 0.175 6.5 - 6.54 Adding the previously prepared commercial enzyme and rennet 86 - 88~ (90cc per 1000 lb milk)

Setting 25 - .30 ] minutes 0.10 - 0.12 6.4 - 6.52 Cutting 86 - 880f ] 15 llinute hours Steam on 7 houra ] 30 minute Steam off 104~ 0.12 - 0.14 6.1 - 6.15 Dipping 1040f - 0.15 - 0.20 6.0 Packing ] 15 minute] 2f hours 0.45 5.35 "Milling 95 - 100~ ] 15 minute Salting 90- 920f ] 30 minute Hooping 85 - 9o0f Pressing - Onr night Dressirur • Killing vas done b,y titrable acidity, as soon as acidity goes up to 0.45 it is done regardless of time which is suggested about 2t hours. .... Figure 1. Manufacturing procedure for Cheddar cheese (clock method) 0 11 The half-inch portion of the plug adjacent to the rind was not included in the sample. Since an experimental sample repreeentatiYe

of the interior of the cheese was desired, the samples vera taken

from the middle portion of the three plugs. All samples were wrapped

in the non-absorbent material and kept in the small air-tight, stop­

pered containers which were kept in the refrigerator until the analysis were made.

~ M .!.W. in~. 1. The plugs were cut uniformly into strips, and an equal number

of strips from each plug vera ground rapidly in a mortar. Tbe round

cheese was flattened in the mortar with the pestle, removed to a piece

of foil and cut into stripe of suitable size for insertion into test bottles.

2. Exactly nine grsma of the prepared samples were weighed into

a tared, nine gram, 50 per cent-large-bodied-cream-test bottle. J. About 15 ml. of cold water were added, and the bottle was allowed to stand in a water bath at a temperature of about 1400f with frequent shaking until the pieces were well disintegrated.

4. About 15 to 18 ml. of sulphuric acid were added. The acid

was added slowly at first, with intermittent rotating to prevent

boiling over. The proper amount of acid was judged by the rate and the color of the mixture.

5. The bottles were centrifuged for five minutes, and then

water at 18cPF was added to the base of the bottle neok.

6, The bottles were centrifuged for two minutes, and water at

lSODr was added to bring the fat column between the graduation.

7. The bottles were centrifuged for one minute snd placed in the water bath at 1J50f for five minutes end the fat content was read. 12

Mohtur• in ~. 1. The moisture dishes were dried for one hour at 100°C and

allowed to cool for one-half hour in a dessicator.

2. A cover glass was placed on the dish end both were weighed. J, The cheese sample was plsctd in a tumbler end rapidly cut

into Smill pieces by means of a spatula. The pieces were thoroughly mixed while being cut.

4. Approximately two to three grams of the sample were placed in each of two dishes, the cover glass immediately replaced, and a

second weighing made.

5. The samples were dried slowly without covers in an oven at 100°C until the weight became constant,

6. After drying, the samples were pl aced in s dessicator for about one hour, or until they reached room temperature; each was weighed without further delay, with the original cover glass in place.

7. Loss in weight, divided by the weight of the sample, multi­ plied by 100 was equal to the percentage of moisture in cheese.

~ nitrogen determination. The soluble nitrogen deter­ mination was made by taking approximately five grams of cheese samples which were exactly weighed, By the Modified Kjeldahl method as outlined by Hawk, Oser •nd Summerson (5) they were ground with 50 ml. of distilled water by the high speed grinder, and then the liquids were obtained by mesne of high speed centrifuge (13,000 rpm) , The supernatant solution was cooled so the butterfat was solidified, This solution WiS filtered through glass wool.

One ml. of the clear solution, free of any particles wee digested with two ml. of 50 per cent sulphuric acid containing one per cent of 13 selenium dioxide in the sand bath at 100°C for four to five hours until all the soluble proteins vere digested . After digestion, sulfur trioxide fumes came off; the solution vas allowed to cool and vas transferred with washing to a 50 ml.-volumetric flask and vee brought up to the mark vith distilled water. The diluted diges- tion mixture vas shaken then; tvo ml. of this solution vas transferred into a 10 ml.-volumetric flask, and five ml. of distilled water and three ml. of Nesslers' reagent vere added . The mixture vas well mixed, and a part vas transferred to the spectrometer cell after an elapse of 10 minutes. The color vas read at 430 millimicron against a blank consisting of distilled water which vas carried through digestion procedure.

The Nesalers' reagent vsa prepared by adding 100 grams of mercuric iodide end 70 grams of potassium iodide to 400 ml. of water in e liter volumetric flask. It vas well mixed . One hundred grams of sodium ., hydroxide vas dissolved in about 500 ml . of veter, end the solut i on ., vas cooled thoroughly. This sodium hydroxide solution vas added vi th constant shaking to the mixture in the flask. The mixture was made up with water to a liter mark. When the small amount of brownish-red precipitate which formed settled out, the supernatant fluid vas ready to be poured off and used.

Preparation of nitrogen standard solution wea as follows:

Using Ammonium di-hydrogen phosphate NH4H2~(Molecular weight 115.04g) 14.380g per 250 ml. of water 500 micro mol• per ml. 40 al. or above solution make up to 50 ml . 400 u mole per ml. 30 300 20 200 10 100 5 50 2 20 1 10 14 The reaction of digested soluble nitrogen wi th the Nesslers' reagent is as follows (11)1

2Mgi4=1- 2 NH3 - NH2Hg2I3 1 NH/1 sr- ~ ChromstograpbY f2r ~ ~ determination gualitatiytly, Approximattly 10 grams of each cheese sample are ground with 20 ml.

of water and centrifuged. The supernatant solution was used for the paper chromatography. According to the procedure of Smith (13) a

small amount of this solution vas spotted on the Whstmsn No, 1 paper in one centimeter diameter.

The paper was placed in the vessel with the desired solvent and

set for 20 to 24 hours. It vas taken from the vessel and dried.

After drying was complete, ninhydrin solution vas spread over the paper, The ninhydrin solution was prepared by dissolving 0.2 per cent ninhydrin (triketo-hydrindene) in acetone.

Zittle, C, A, and Cerbulia, J, (20) suggested the few solvents which art most satisfactor,r.

The solvents used on this experiment were as follows:

Houra for Hours for Component. 8-inch riu complete dry BuA n-butanol 120 10 hours half hour Acetic acid .30 Glacial Water 50

BuP n-butanol 60 10 hours half hour Pyridint 60 Water 60

The above solvents were chosen because they have the singular property of compacting the spots so that no diffusion or elongation occurs during the chromatograp~. 15

RESULTS Activity R! individual lDJlal The results regarding enzyme activity are presented in Figure 1. The dotted lines above the solid line shows thet at this greet dilu­ tion the enzymes did not have enough activity to coagulate the milk casein even if the milk and enzymes solutions were incubated in the water bath at 40°C for two or more hours.

The extension below the solid line shows that higher concentra­ tion of the enzyme solution coagulated the milk immediately. It ia almost impossible to measure the time required for coagulation when such a high concentration was used. The line nearest to the left "y" axis indicates the strongest activity end the line furthest to the right indicates the least activity. A concentration of 0,0002425 grams of rennet in 10 ml. of the prepared milk sample took 840 seconds to show the physical appearance of coagulation of the milk; however, other enzymes did not show any sign of coagulation even when they were heated in the beth at 40°C for two hours. The 0.002 gram of P-enzyme solution took 720 seconds, while the rAnnet solution took only 100 seconds. However, the other enzymes, F end animal source proteases, showed a weaker activity than either P-enzyme or rennet.

The concentration of animal source enzyme at 0.04 gram took 525 seconds for the coagulation of milk, while rennet and P-enzyme took 65 end 290 seconds respectively.

At the concentration of 0,02 grams the F-enzyme solution caused coagulation in 240 seconds while rennet showed reaction instantaneously. 16

J No indioation or coagulation

Rennet log t 0

a

2

Q ' ' .... , ..

l immediate coagulation

l 2 Concentration log o Figure 2, Enzyme activit7 of clotting milk 17

Table 1. Activity of rennet by clotting-milk method

Rennet Cosguzatiol Concentration LollC Time :sec" Lo"T 1 ml/10 ml prepared milk xlcf 0.015500 2.190.3 15 1.1761 0.007750 1.889.3 45 1.65.32 0.00.3870 1.5877 95 l.9'ffl 0.001940 1.2878 105 2.0212 0.000970 0.9868 210 2 • .3222 0.000485 0.6857 .380 2.5798 0.0002425 0 • .3856 840 2.924.3

Tabla 2. Activities of P-enzyme and F-enzyme by clotting-milk method8

Animal Source EnsVlltea P-enz•~n• F ...,nzV1o8 Prete "e Concentra- Coag. Coeg. tion Loll C Timel~ec, Loll T imec~(~· sec Loll T Time(sec Lo~r T gram~lO ml prep 1 d mil~ xlo4 0 .10 J.OOOO immediate 50 1.6990 .30 1.4771 0.08 2.9031 20 1..3010 80 1.9031 40 1.6021 0.06 2.7782 30 1.4771 120 2.0792 60 1.7782 0.04 2.6021 40 1.6021 150 2.1761 80 1.90.31 0.02 2.)010 60 1.7782 240 2.3802 150 2.1761 0.01 2.0000 90 1.9542 na 270 2.4314 o.oos 1.9030 120 2.0792 coag , 315 2.4983 0.006 1.7782 180 2.255.3 435 2.6385 0.004 1.6021 290 2.4624 525 2.7202 0.002 1.3010 720 2.8573 no coag. a Enzyme activity on casein is plotted on the graph. Log T (Time in second on log scale) versus Log C (Concentration on log scale). Table J. Record of pH and acidity development in the manufacture of Cheddar cheese

Let 6 Animal Type of enzyme added Control Control Protease Protease

Amount of enz added 1000 lb per cent per cent per Acidity of starter 1.02 1.02 1.02 1.10 1.10 1.10

Acidity vhen starter added 0.20 0.20 0.20 0.18 0.18 0.18

Acidity vhen rennet added 0.205 0.205 0.205 0.19 0.19 0.19 Acidity of whey at cutting 0.115 o.uo o.uo o.u o.u o.u Acidity of vhey at dipping 0.125 0.122 0.120 0.13 0.13 0.13 Acidity of vhST at packing 0.200 0.190 0.190

pH at cutting 6.5 6.45 6.48 6.50 6.50 6.49 pH at dipping 6.1 6.10 6.18 6.25 6.25 6.25

Percentaga yield 11.5 11.5 11.5 11.5 11.5 11.5

..... 00 Tabl e 4. Record of pH and acidity development in the manufacture of Cheddar cheese

Lot c D 8 Animal Protease Type of enzyme added Control Protease F Amount of e per cent

Acidity of starter 1.05 1.05 1.05 0.80 0.80 0.80 Acidity when starter added 0.18 0.18 0.18 0.17 0.17 0.17 Acidity when rennet added 0.19 0.19 0.19 0.175 0.175 0.175 Acidity of whey at cutting 0.115 0.115 0.115 0.12 0.12 0.12

Acidity of whey at dipping O.lJ O.lJO 0.140 O.lJ 0.13 O.lJ Acidity of whey at packing 0.34 0.32 0.40 pH at cutting 6.60 6.60 6.60 6.48 6.50 6.50 pH at dipping 6.20 6.20 6.10 6.25 6.21 6.21

Percentage yield 11.5 11.5 11.5 a There vas not enough whey to test...... 0 Table 5. Record of pH and acidity development in the msnufaeture of Cheddsr cheese

Lot E J' 1

Type of enzyme added Contrel Amoun per cent per cent per cent per cent per cent per cent Acidity of starter 0.70 0.70 0.70 0.87 O.?rl 0.87 Acidity when starter added 0.175 0.175 0.175 0.18 0.18 0.18 Acidity when rennet added 0.185 0.185 0.185 0.195 0.195 0.195 Acidity of whey at cutting 0.115 0.115 0.115 0.120 0.120 0.125 Acidity of whey et dipping 0.145 0.140 0.140 0.130 0.130 0.130 Acidity of whey at peeking 0.47 0.470 0.470 0.140 0.160 0.155 0 pH st cutting 6.45 6.45 6.45 6.46 6.45 6.50 pH st dipping 6.05 6.15 6.15 6.40 6.40 6.40 0 Percentage yield 11.5 11.5 11.5 12.0 12.3 12. 0

"'0 Table 6. Record of pH and acidity development in the lll8Dufacture of Cheddar cheese

~t G 20 2 Animal Source Animal Source Type of enzyme added Control Enzyme Enzyme Amount of added per cent per cant per cent Acidity of starter 0.69 0.69 0.69 Acidity when starter added .165 .165 .165 Acidity when rennet added .175 .175 .175 Acidity of whey at cutting .uo .uo .uo Acidity of whey at dipping .1)5 .134 .135 Acidity of whey at packing .210 .209 .211 0 pH at cutting 6.49 6.50 6.50 pH at dipping 6.24 6.24 6.25

Percentage yield 10.5 10.5 10.5 "'.... 22 The P-enzyme solution indicated a reaction at 50 seconds, and animal source protease at 150 seconda.

From this graph it is eaay to see the order of the activities of clotting the milk•

Rennet P-enzyme Animal source en~e F-enzyme

The following results were obtained from the date of cheese manufacturing (see Tables 3 to 6):

1. During the manufacturing of the cheese, there were no notice­ able physical changes observed as expected from the interpretation of

the enzyme activity graph. Such a small dilution showed neither any

particular visible result in clotting time nor tYPe of curd during cheese making. Flavors of the whey expelled by the formation of the curd and the curd itself did not show any changes. The flavor was normal during manufacturing.

2. Whey at cutting, dipping, packing, and milling was titrated with sodium hydroxide, using the indicator phenolphthalein. All the acidities of twenty-one samples came out very close to each other with exception of the whey sample at milling. Thera was an error

introduced at this point due to the following factor. The whey was

obtained by pressing the curd. This allowed s alight amount of curd to enter the test which increased the acidity reeding.

~ f[Qm interpretation ~ ~ 1 iQ lQ:

121 l ~ ~ ~ ~ ~ Jl. These three cheese samples tasted for flavor, body, and texture were approximately the same.

However, a high acidity teste developed during 24-dsy end 107-day

ripening periods, This cause might not be due to enzyme activity,

but to the high acidity at the milling, snd to the inability to maintain identical conditions of manufacturing for all three cheeses. 23

Between 24 end 107 days there vere no significant changes in flavor, body, and texture scores, No noticeable changes in moisture content in the cheese and production yield were observed, which proved that there vas no appreciable protein loss during the curd formation when such s smell amount of additional enzyme vas added to the milk. At

the age of 138 days there vas no sign of special characteristics developed by the addition of the enzyme,

~ .!! ~ l!E.... ~ 2... m 22.. These three samples were evaluated in the same way as the above mentioned samples, However, flavor scores of samples No, 5 end No, 6 were improved by one to two points after the 103-dey ripening period. At the age of 134 days the flavor, body, end texture seemed almost the same as scores taken at the 10)-dsy period,

1:21 £ ~ .!!2,. L.a._ Aru1 _u, The results on the flavor, body, end texture scores above were the same at the 19-dsy and 102-dsy ripening periods, The same comments as Lots A and B were given, At 130 days these cheeses gained an aged flavor, and it seemed that sample No, 8 end 9 improved their flavor, body, and texture. There was no sign of any of the defects or superior qualities expected.

12112 ~ l!E.... .J.Q.. J.L. ,mg ill· The moisture content of sample No, 11 was a little higher then the rest of the samples, but higher moisture was not due to the enzyme activity in this case. If this were true, sample No, 12 would also be higher.

There were no protein losses since the production yield percentages were shown to be identical,

All three samples at 16-day ripening showed there were some defects of body end texture, The characteristics of these samples were pastiness, weak body, end stickiness. At 99 days, Nos, 10 and 11

remained almost the semeJ however, No. 12 showed a characteristic of bitter taste, and a crumbly body was observed.

~ ~ ~ ~ lli l!.... .a,ng J.il, There were some differences in moisture content; but the results were other than expected as the

enzyme aida the coagulation of milk and forms a thick film around the

curd. It is expected to have a higher moisture content if more enzyme were used, Therefore, this was due to the technique of cheese making and some evaporation which took place during the preparation of the sample for the moisture teat.

Flavor, body, and texture scores at 15-dsy ripening period were fair grade cheese, but short and mealy body was observed. However, at

98 days two to four points were dropped on flavor end body score. Definite bitterness was observed at this time. Even when such e small amount of this type of commercial enzyme was used.

~ l ~ ~ ~ ~ ~ 1§1. Within 25 days there were not any significant characteristics of flavor, body, and texture in samples

No. 1 to 15. Therefore, these samples Nos. 17 and 18, with such a high concentration of enzymes, were used for detecting changes taking place in a short time.

At 12 days a bitter flavor was detected in sample No. 18, which was treated with P-enzyme. This enzyme activity is shown in Figure 2 and is much stronger than the F-enzyme. At 62 days, both Nos, 17 and

18 showed extremely bad flavor and body texture. These cheeses were not edible after 12 days. Enzymes F and P, even in small amounts, were not suitable for use in cheese production,

h21 ~ ~ ~ ~ ~ ~ lJl. At the ripening age of 8 days, their flavor, body, and texture were fair. There was no sign of bitter Table 7. Scores or Cheddar cheese samples

A.D.S,A, Sllm A.D.S,A, SllQI:• Lot Vat Ripening Flavor Body end Critici sm Ripening Flavor Body and Criti cism No. No. oeriod 1.'5 Texture 10 oeriod 1.'5 Texture 10 1 39.0 28.5 sl. acid 38.0 28.0 sl. acid sl. mealy sl. ferm. sl. ferm. weak A 2 24 )8.0 28.0 high acid 107 37.5 27.5 high scid days sl. mealy days sl. farm . crumbly J )8.0 27.5 high acid 37.5 27.5 high acid mealy crumbly crumblv sl. ferm 4 )8.5 28.5 sl. acid 38.0 27.5 sl. acid sl. gassy sl. farm. weak B 5 20 )7.5 27.5 high scid 103 )9.0 28.5 sl. acid days soft, short days open mealy 6 )8.5 29.0 sl. acid 40.0 28.5 sl. scid sweet holes open

7 39.0 29.0 sl. scid )9 .0 29.0 sl. acid open open

c 8 19 )8.5 28.5 high acid 102 )8.0 27.5 sl. acid days sl. mealy days open, al. crumbly 9 )9.0 29.0 high .acid 39 . 0 29.0 sl. scid open sl. farm. open "' Table 8. Scores of Cheddar cheese samples

A,D,S,A, S~t! t..D,S,A, S~Qt! Lot Vat Ripening Flavor Body and Criticism Ripening Flavor Body and Criticism neriod. No No 1.'5 Texture 10 neriod. 1.'5 Texture 10

10 )8.5 28.0 high acid 39.0 28.5 sl. acid soft crumbly weak D weak 11 16 38.0 26.0 high acid '11 38.0 :n.o high acid days pasty days crumbly sl. pasty 12 38.0 28.5 high acid 36.5 28.0 high acid, sl. mealy bitter, short crumblv 13 )8.0 28.0 high acid )6.0 25.0 high acid abort bitter, abort mealy E crumbly 14 15 38 .0 28.0 high acid 98 35.5 25.0 high acid days short days bitter, short mealy crumbly 15 38.0 28.0 high acid 35.0 25.0 high acid short bitter, short mealY crumblv 16 40.0 30.0 39.5 30.0

F very bitter 17 12 38.0 28.5 high acid 62 Below Below high acid days mealy days 35 25 short, pasty crumbly 18 38.0 26.0 high acid Below Below very bitter bitter, short 35 25 high acid crumbly short, pasty ..,,.umblv Table 9. Scores of Cheddar cheese samples

A.D.S . A. Ss;o[! Lot Vet Ripening Flavor Body and CriticiSII lio. lio oruQ!l 45 Texture 30

19 38 28 high acid slightly mealy color bleach G 20 21 38 29 high acid days slightly mealy 21 39 29.5 high acid 28 flavor detected even when a high concentration was used. Of the three

samples compared, No. 20 seemed to have the better body and texture.

At this time samples No, 20 and 21 showed no different characteristics when compared to No. 19. After a ripening period of 21 days, there

were no defects detected from the additional enzyme. Among the three samples, sample No. 21 which was treated with 30 grams of animal

source protease showed an improvement in the body and texture and slightly better flavor than the others.

~ nitrogen determination

As shown in Figure 3, there are some relationships between addi­ tional enzyme concentration and the amount of soluble nitrogen. Lower concentration of enzyme did not increase the soluble nitrogen appreci­ ably in 80 days; however, when s higher concentration was used, there was a marked increase in nitrogen content. This corresponds with the work done by McCulloch (1), and VanSlyke and Price (15) on various concentrations of rennet for cheese making.

The bitter flavor does not necessarily agree with the amount of soluble nitrogenous compounds present in cheese. Probably the par­ ticular compounds produced in cheese during the ripening period, both soluble and insoluble in form, are responsible for bitterness. Samples No , 17 and 18 gave extreme bit terness, and here is the probable evidence that a great amount of bitterness comes definitely from soluble matter caused by rapid hydrolysis of proteins. Bitter­ ness of soluble matter could be from the particular tYPes of amino acids, ss Harper and Swanson suggested (4), and from peptones.

Soluble nitrogeneous compounds increase as the ripening period increases, but the true meaning of wholesome ripening characteristics cannot be entirely dependent on it. The ripening index can be used .. ~ --- Contrel c --Treated with commercial enzymes c0 1.0 A ••• Aniaal source protease ... F ••• F-enzyme ...... "' 0.9 P ••• P-enzyma 30 ds7s .,0 B • • • Bitter flavor developed in cheese ...... 0.8 ..0 ...c 0.7 ..,~ 0.6 Ripening age ~ 75 days "i:l 0.5 C> :d 0.4 80 da,-s ...... "'0 .. o • 85 da,-s 84 da,.a 81 da711

.... I 0 I '):; o•• ' I' i C> I 0 I' : 'I '"' o. : I .:: ' ' ' 0 ~~~----~~~--~~~--~~~~--~~~--~~~=------­ Sample No. l 2 J 4 5 6 7 8 9 10 11 12 l J 14 l 5 16 17 18 0*1 *2* 0*1*2* O* 4*6* 2* 4* 6* 2* 4* 6* 0*50*50* A A A A A A F F F p p p F p B B B B B B • Concentration of enz,-me (gram per 1000 lb. milk). Figure ). Soluble nitrogen in ebease Table 10. Soluble nitrogen determination in total solids non fat 1n Cheddar cheese

Samole No (vAt no.) _l _2 ~ _1._ _i _Q_

Semple weight 3.0712 g 3.2115 g 3.0183 g 5.0673 g 5-4182 g 4-4230 g Per cent moisture 39.0 40.0 40.0 39.0 39.5 38.5 Per cent fat 30.0 30.0 30.0 30.5 32.0 32.0

Per cent dry matter 31.0 30.0 30.0 30.5 28.5 29.5

Dry metter weight 0.9540 g 0.9635 g 0.9058 g 1.51.5 g 1.543 g 1.304 g Ripening period 75 days 75 days 75 days 85 days 85 days 85 days

A.P. A.P. A.P. A.P. Enzyme treatment Control lg/1000 lb 2g/1000 lb Control lg/1000 lb 2g/1000 lb

Spectrograph reading 22x10-6 22xltr6 30x10-6 33.5x1o-6 33.5x10-6 28.8x10-6 mole/1111

Gram of soluble nitrogen 0.00)6 g 0.0036 g 0.00456 g 0.00418 g 0.00418 g 0.00)60 g Per cent soluble nitrogen in T.S.N.F.a 0.378 0.374 0.503 0.270 0.271 0.276 a T.S.N.F.- Total solids non fat in Cheddar cheese.

w 0 Table 11. Soluble nitrogen determination in total solids non fet in Cbedder cheese

Semole No. (vat no .) 7 8 9 10 11 12 Sample weight 4.8964 g 4.60?5 g 4.8298 g 4.7240 g 5.2740 g 4.8355 g Per cent moisture 38.7 38.3 38.5 41.6 42.6 41.8 Per cent ret 33.0 32.5 33.0 30.0 30.0 30.0 Per cent dry matter 28.3 29.2 28.5 28.4 27.4 28 .2 Dry matter weight 1.384 g 1.343 g 1.375 g 1.341 g 1.443 g 1.361 g Ripening period 84 days 84 days 84 days 81 days 81 days 81 dey 11

A.P. A.P. F F F Enzyme treatment Control 4g/1000 1b 6g/1000 1b 2g/1000 lb 4g/1000 lb 6g/1000 lb

Spectrograph reading 28.8xla-6 27 .5x10-b 33.5xla-b 31.8x10-b 32.7xla-b )2.5xla-6 mole/mJ.

Gram of soluble nitrogen 0.00360 g 0.00344 g 0.00418 g 0.00398 g 0.00409 g 0.00406 g Per cent soluble nitrogen in T.S.N.F.8 0.260 0.256 0.304 0.296 0.284 0.299 e T.S.N.F.- Total solids non fet in Cheddar cheese. Table 12. Soluble nitrogen determination in total solids non fat in Cheddar cheese

Samo1e No (vatno,) 13 14 15 16 l'Z_ _18 Sample weight 5.1942 g 5.6920 g 5.1640 g 3.4634 g 3.2000 g 3.5630 g

Per cent moisture 40.6 39-3 38.8 38.6 38.6 39.3

Per cent fat 32.5 32.7 32.5 33-5 33.5 3).5 Per cent dry matter 26.9 28.0 28.7 27.9 27.9 27.2

Dry matter weight 1.395 g 1.592 g 1.480 g 0.9660 g 0.8930 g 0.9700 g

Ripening period 80 days 80 days 80 days 30 days 30 days 30 days p p p F p Enzyme treatment 2g/1000 lb 4g/1000 lb 6g/1000 lb Control 50g/1000 lb 50g/1000 lb

Spectrograph reading )1.6x10-6 47.2x10-6 38.0x10-6 5 X 10-6 34xlo-6 55xlo-6 mole/ml

Gram of soluble nitrogen 0.00395 g 0.00590 g 0.00475 g 0.00076 g 0.00076 g 0.0086) g Per cent soluble nitrogen in T .S . N.F. a 0.283 0.)70 0.321 o.rns7 0.587 0.890 a T.S.N.F.- Total solids non fat in Cheddar cheese. JJ as a measurement of cheese aging; however, it does not correspond to ita flavor and body.

All samples were ground and centrifuged. The vater-soluble matter vas then separated from the insoluble matter and tested for flavors.

Somple No. Water soluble Insoluble

1 2 Goof flavor 1 Cheese flavor J it contained not detected. 4 cheese flaTor 5 6 7 8 9 10 11 Stronger flavor 12 Stronger flavor lJ Slightly bitter Slightly bitter 14 Bitter Bitter 15 Bitter Bitter 16 Good cheese flavor 17 Very bitter Bitter 18 Very bitter Bitter

It can be said that cheese flavors come mostly from vater-aoluble compounds. As Harper and Svenson (4) suggested, the amino acids ere responsible for the cheese flaTors.

Bitterness vas found in both vater soluble and insoluble com- pounds. This indicates that the characteristic of bitter flavor may come from particular amino acids end from peptonea.

Chromatograph f2.t ~ ~ 1n ~ The results of the paper chromatography shoved that the samples from Nos. 1 to 16 to be nearly identical. Samples No. 17 and 18 shoved some differences from the other samples tested, and they con- tained much higher concentrations of amino acids end soluble proteins as indicated by more intense color spots. The intensities of the color spota were predominant on lyain, leucine, glutamic acid, and aspartic acid, which were identified by comparing Rr values of samples with atendard Rr values. It was quite difficult to detect any changes taking place or aqy particular materials

produced ~ just looking at the resulting chromatogram of samples No, 1

to 15 (a ripening period 118 to 126 daya). No. 16, which had a ripen­

ing period of only 82 days, showed a definite degree of hydrolysis of proteins by a lighter color intensity on the paper than the rest of

the samples. On the other hand, Nos. 17 end 18 had the same period of ripening aa No, 16, but they showed that the rapid hydrolysis took place ~ the action of additional enzymea,

There were epots detected from Rr zero value to Rr 3 ~ the uae of the solvent butanol-acetic acid, and zero to 8.5 using solvent butanol-pyridine. At Rf value zero, a very intense color was shown which indicated a fairly large amount of higher molecular-weight­ water-soluble substance present in cheese. The Rr between zero to 3 and zero to 8.5, respectively, were not identified as amino acids because of such low mobility. However, they could be peptonea and polypeptidea. 35

Table 13. Paper chromatography Amino acid - Standard Rr values for two solvents

Solvent Comoound BuA BuP

Aspartic acid 23 20 Glutamic acid 28 20 Glycine 23 29 Alanine 30 37 Valine 51 48 Ieo-leuchine 67 56 Leucine 70 60 Histidine 11 24 Lysine 12 13 Arginine 15 15 Phenylelaine 60 63 Tyrosine 45 60 Tryptophan 50 62 Proline 34 34 Table 14. Qualitative analysis of amino acids in cheese by paper chromatography Rr values (per cent basis) Solvent: BuA

Possible C:oi!!POWldB Sample Hea'Yier Histidine Lysine or Vsline or Phenyl- Number comn'_d or other Lvsine arozinin Asnartic Glutamic T -·~ ... alanine Leucine 1 0 10.6 1J.9 15.6 21.8 26.8 49.5 59-J 66.4 2 .; 9.1 12.1 15.5 20.4 26.7 49.5 59.2 66.4 .....::> J 8.3 10.9 19.6 24.2 I>"' 14-J 49.2 59.2 66.5 7 or 7.9 10.6 13.2 18.9 24.9 50.0 59.2 68.0 8 "'.,0 7.7 9.8 1).4 19.2 25.0 50.5 59.) 68 .0 N 9 7.7 9.9 1).2 18.9 26.7 50.5 .,0 59.) 68.1 10 r:l. ::> 7.9 9.9 1).2 18.1 26.7 50.5 59.2 68.8 c 11 .... 7.2 9.8 12.8 ...... 18.1 2).4 50.5 59.6 68.7 12 0 8.4 11.0 14 . 3 19.6 23.4 50.4 59.6 68.8 a 0 13 t 7.9 14.3 19.5 24.9 50.6 59.5 68.7 14 " ..., 7.7 9.8 13.1 18.0 25.0 48.2 56.6 66.4 ~ 15 "' 9.1 u.o 14.4 19.6 25 .9 49.2 56.5 64.5 16 8.4 11.1 15.6 19.6 24.9 49.4 57.4 65.J 17 Smear from the origin up to Rf value 70 which indicates higher concentration of amino acids and/or similar coa- w 18 ooUnds havinoz R.. value 0 to 70 a- Tabll! 15. QualitatiYe sns1yais of amino acids in cheese by pspl!r chrOI!llltograpb;y Rr Yalul! (per cent basis) So1Yent: BuP

Ss111ple Hesvi"r Aspartic and Trace of pheny1aln1ne Number eomn'd Lisine G1utamie aeid Glvaine V,.line and i.,ucine 1 0 8.8 14.3 20.2 2?.6 41.0 52,6

2 " s.o 13.1 18.0 26.2 39.8 51.8

3 8.0 u.s 17.0 23.6 38.2 51.0

7 7.3 11.8 17o3 24.2 38.4 51.1 8 7.3 ll.S 17.3 22.5 39.2 51.1

9 8.0 11.1 16.7 23.4 J9.2 51.2 10 7.0 11.1 16 .5 22.4 36.9 50.5 ll 7.7 16 .5 21.0 39.5 56.5 12 8 , 8 lL. .o 21.0 2?.8 42.2 55 . 0 13 8.4 13.7 18.6 )8.2 50.6

14 8.1 lL.. 7 19.2 24.8 41.2 52.6

15 8.1 15,6 25.8 41.8 53,0 vt!ry faint 16 trace 10.4 17.7 21.8 54.0 color indi- estes low 17 Sml!sred out from the origin which indiestes it con centra- contains higher concentration. tion 18 w ..;) SUMMARY

SeTen lots (21 vats) of Cheddar cheese were manufactured. They were manufactured using various concentrations of animal and plant proteases individually. Tbe Clock Method for making Cheddar cheese was followed in each of the lots.

The cheeses were scored for flavor, body, and texture after approximately one month and three months ripening period on the average, Soluble nitrogen determinations were made and the amino acids were determined qualitatively by chromatographic technique. 39

CO NCLUS I ONS

From the experiments conducted, the following conclusions were reached :

l . During the rna nufectur~ of the cheese making, there were no

noti ceable physical changes observed by the action of the additional comm ercial enzyme used .

2. Acidity development during the cheese making was not influenced by the acti on of add itional commercial enzyme .

3 . There was no appreciable protein loss during the manufacture

of the cheese and no appreciable change in moisture content of cheese a few weeks ol d as compared with the contrel sample.

4. F- and P-enzymes of plant origin were not suitable for cheese

ma king according t o t he results of tbese trials. Such active enzymes

t end to produce bitter flavor and pasty body by the rapid hydrolysis

of protei n at the c o nc~ntrations used in this project.

5. The additional enzyme f or manufacturing of cheese t ends to cause production of a greater quantity of water-soluble nitrogen compo unds by comparison with the control cheese.

6 . Bitter flavor is probably caused by amino acids and peptones produced by hydrolysis of proteins.

7. The soluble nitrogen content in cheese does not necessarily

agree with the degree of ripening, but may be a means of estimation.

8. An animal-source protease can be used for Cheddar cheese ma king, I n this experiment it seemed t o gi ve a softer body and a little more aged type flavor as compared to the control. However, 40 there vas no sign of defects observed by the action of this enzyme at the concentration of 30 grams per 1000 lb. of milk. (Flavor, body and texture scores vere taken 21 days after the cheese vas produced. 41

LITERATURE CITED

(1) Ba bcock, S.M., H. L. Russell, and A. Vivian. Wisconsin: Agricultural Experiment Station. 17th Annual Report. pp. 102-122. 1900. (2) Colowick, S. P., and N. 0. Kaplan. Clotting Method in Enzymology II, p. 58. New York: Academic Press Inc. 1955.

(J) Freeman, T. R., and C. D. Dahle. Pennsylvania: Agricultural Experiment Station, Bul. )62. 1938 .

(4) Harper, W. J., and A.M. Swanson. Studies of Amino Acids in Cheddar Cheese During Ripening. Journal of Dairy Science Abstract MJJ )1:715. 1948.

(5) Hawk, Oser, and Summerson. Practical Physiological Chemistry. l)th Ed. (Personal correspondence with Dr. L. E. Olsen. ) (6) Ling, E. R. Theoretical Dairy Chemistry l(J): 46. New York: Philosphicel Library. 1957. (7) Theoretical Dairy Chemi s try l(J): 167. New York: Philoaphical Library. 1957. (8) Theoretical Dairy Chemistry l(J): 181. New York: Philosphical Library. 1957. (9) McCulloch, C. G. A Study of Some of the Effects of Different Amounts of Re nnet in Cheddar Cheese Making. Unpublished Thesis, Utah State University Library, Logan, Utah. 19)6.

(10) Neilands, J. B., and P. K. Stumpt. Outlines of Enzyme Chemistry. New York: John Wiley and Sons, Inc. pp. 192. 195 5. (11 ) Pierce, W. C., E. L. Haenisch, end D. T. Sawyer. Quantitative Analysis. 4th Ed. pp. 410. New York: John Wiley end Sons, Inc. 1948.

(12) Roundy, R. Z. Utilization end Application of Commercial Enzymes in Cheese Making. (Personal correspondence) Chicago, Illinois: Central Research Laboratory. (lJ) Smith, I. Chromatographic Techniques . Clinical and Biochemical Application. London: Heinemann. 1958. 42 (14) Storgards, T., and B. Lindquist. Assay of Amino Acid in Different Types of Cheese by Chrolll8tography. Journal of Dairy Science Abstract 543 J61 A85 . 1953. (15) VanSlyke, L. L., and W. V. Price. Rennet Enzyme as a Factor in Cheese Making. New York: Experiment Station, Bul. 233. 1903.

(16) and Cheese. New York: Orange Judd Publishing Co. , Inc. pp. 332. 1952.

(17) and Cheese. New York: Orange Judd Publishing Co . , Inc. pp. 461. 1949. (18) Wilson, H. L. Making American Cheddar Cheese of Uniformly Good Quality. U. S. Dept. Agr. B.D.I.M . 947. Nov. 1942.

(19) Wilster, G. H. Practical Cheese-Making. 8th Ed. Oregon: O.S.C. Cooperative Association . pp. V-58. 1955.

(20) Zittle, C. A. and J. Cerbulis. Clott ing of Casein with Pepsin; Amount and Nature of Soluble Products. Journal of Dairy Science 41: 241. 1958.