Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-1959 The Use of Protease Enzymes in the Manufacture of Cheddar Cheese Osamu Fujiwara Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Food Science Commons 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.