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Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1954 Degradation of amino acids by Lactobacillus casei in relation to flavor development in cheddar cheese Thorvald Kristoffersen Iowa State College Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, and the Food Science Commons Recommended Citation Kristoffersen, Thorvald, "Degradation of amino acids by Lactobacillus casei in relation to flavor development in cheddar cheese " (1954). Retrospective Theses and Dissertations. 12406. https://lib.dr.iastate.edu/rtd/12406 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overiaps. ProQuest information and Learning 300 North Zeeb Road. Ann Arbor, Ml 48106-1346 USA 800-521-0600 NOTE TO USERS This reproduction is the best copy available. UMI* DSaRADA.TION OF A13N0 ACIDS BY LACTOBACILLUS CASEI IK RELATION TO FLAVOR DEVSLOPliSHT IN CHEDDAR CHSSSE Thorvald Kristoffersen A Dissertation Subciitted to the Graduate Faculty in Partial Fulfillment of the Hequirements for- the Degree of DOCTOR OF PHILOSOPHY l-Iajor Sul3ject: Dairy Bacteriology Approved: Signature was redacted for privacy. in Charge of Major v/orfe Signature was redacted for privacy. Signature was redacted for privacy. lov^a State College l95iv UMI Number: DP11805 UMI UMI Microform DP11805 Copyrigiit 2005 by ProQuest Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Infomnation and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor. Ml 48106-1346 Q, V "-• C ^. A b ii K TABLE OP CONTSNTS , Page INTRODUCTION 1 1 ) REVIEW OF LITERATURE k- Bacterial Deamination ^ Isolation of Phosphopeptones and Phosphoserine from Casein and the Effect of the Phosphoric Acid (rroup on Proteolytic Activity 10 Monoester and Diester Linkages in Casein 13 Hilk Phosphatases 1^ Action of Kilk Phosphatases on Casein IS The Role of Lactobacilli in Cheddar Cheese 19 Amino Acids and Their Degradation Products as Indexes of Development of Characteristic Cheddar Cheese Flavor 22 EXPSRIliSNTAL 26 Methods of Procedure 26 Description of sources and identification of lactobacilli 26 Cell production 27 Determination of deaminase activity 2S Determination of ammonia 2g Determination of pyruvic acid 29 Determination of lactic acid 3^ Determination of pH 32 Determination of nitrogen 3^ Preparation of phospho serine 3|^- Determination of barium 37 Determination of phosphorus 3^ Preparation of idiey globulin 35 Preparation of potato phosphatase 35 Cheese making procedure 37 Scoring of the cheese 37 Bacterial examination of cheese 3^ Hitroprusside test for sulphhydryl groups in cheese 3^ Lead acetate paper test for hydrogen sulfide JLn" cheese 39 Determination of phosphatase activity at pH 5*^ ill milk, vdiey, whey globulins and potato phosphatase solution 39 T11416 Hi TABLE OP COKTEIITS (Continued) Page Detenaination of phosphatase activity of cheese at pH* 5.V 4-1 Preparation of cheese for determination of nitrogen fractions Ul PrQ)aration of cheese extract for paper chromatography ' Paper chromatography procedure ^ Determination of pH on cheese 4*7 RESULTS A, Deamination of Amino Acids by L. casei Preliminary studies and observations ^ Production of cells Selection of a suitable strain of L, easel 4^ DeamTnation temperature 50 Effect of time 50 Formation of pyruvic and lactic acids 53 The choice of phosphate buffer 5^ Phosphoserine cocroosition 55 Deamination of individual amino acids and some related compounds 5^ Factors influencing production of the serine deaminase of L. casei strain 7 59 Effect of glucose in the growth medium on deamination of PL-serine and formation of H2S 59 Deaminase activity of cells harvested at different physiological states as expressed by pH of the medium at harvest time 63 Factors influencing deamination of serine by L. easel strain 7 grown under standard conditions 66 Effect of temperature 66 Effect of pH 67 Effect of gassing -with nitrogen and addition of cysteine, glutathione and adenosine-5-phosphate JO Effect of pasteurization 7 Stability li Occurrence in Cheddar cheese of lactobacllli capable of producing hydrogen sulfide and deaminating serine 35 iv TABLE OP CONTENTS (Continued) Page B, CheesemaJcing Experiments 9® Arrangement SO Phosphatase activity at pH 5.^!- of milk, whey and cheese 92 Changes in pH during ripening 95 Bacteriological examination 97 Sulphhydrygroups and "free" hydrogen sulphide 99 Protein breakdoim 102 Free amino acids 102 Flavor score lOS DISCUSSION 111 SUia^AHI AND CONCLUSIONS 125 BIBLIOGHAPKZ 12g ACKNOVfliSDGEtlSNT 139 INTRODUCTION The problems of flavor development and flavor constituents in Cheddar cheese and the reason(s) for the difference in flavor between Cheddar cheese made from raw and pasteurized milk are still largely tinsolved. Lactobacillus casei, -fcrhich increases in numbers during ripening, generally is considered to be responsible for the deeper changes in Cheddar cheese, but additions of L. casei to pasteurized milk for Cheddar cheese have not resulted in reproduction of the typical flavor of raw milk Cheddar cheese. This failure could indicate that lack of development of true flavor in such cheese either is not directly related to the action of L. casei or is not as much due to the reduction in the numbers of L, casei resulting from pasteuri­ zation as to a change in their metabolism. The sources from which L. casei derives energy for multiplication have not been established. The production of NH^ by L. casei in chalk milk on prolonged standing and the increase in nitrogen as Cheddar cheese matures indicate that amino acids could possibly be the source from tdiich energy is derived, « deamination being involved in the degradation. Serine is an amino acid which particularly would lend itself as a source of energy for L, casei. The pathway for 2 deamination by L. easel, vdiich is considered facultatively anaerobic, vrauld be non-oxldative (hydrolytic). The result­ ing keto acid possibly should not be more than a three-carbon keto acid In order to be further metabolized by L. easel. These requirements are met by serine, vdiich can be deamlnated non-oxidatlvely and yields NH^ and pyruvic acid upon deamination. Cysteine, -Bihlch resembles serine in structure and can be deamlnated in a similar manner, also could possibly be a source of energy. Casein, the major protein constituent of cheese, x^oizld be the most in5>ortant source of serine and cysteine. Serine makes up about 6 per cent of the amino acids in casein but a large part is present as phosphoserine. The presence of a phosphoric acid group has been shoim to inhibit deamination of serine by other bacteria and greatly reduce hydrolysis by pancreatic enzymes. Presumably the inhibition by the phosphoric acid group also would apply to L, easel. This raises the question of the role of phosphatases, a group of milk enzymes tdilch so far has not been considered inroortant in the ripening of Cheddar cheese. The possibility ims con­ sidered that the acid milk phosphatase ifith an optimum about pH 5,7 could be of in^jortance. Preliminary studies showed pasteurization did not destroy this enzyme but resulted in a considerable reduction of activity. The purpose of this study v«.s to investigate the ability 3 of L. easel to deamlnate amino acids. A fev: practical cheese making experiments were conducted to Investigate the possible Importfiuice of degradation of amino acids by L. easel and the role of acid phosphatase activity In Cheddar cheese ripening, hoping some of the problems concerning flavor development and flavor constituents of Cheddar cheese could be elucidated. RETIEif OP LITERATURE Because several somei-.iiat diverse subjects are reviewed In this section, the material has been divided into sub­ sections, each of which covers closely related material. Bacterial Deaminatlon The ability of gram positive dairy organisms, such as streptococci and lactobacilli, to deaminate amino acids is very restricted. Orla-Jensen (I919) reported the true lactic acid bacteria were Incapable of splitting amino acids, Ayers ^ (19 21) were the first to point out the value of NH^ production by certain streptococci as an aid in differenti­

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