Iv o-vvv--v—vvv——vvvwvm-v - . I . _ ‘7" . ’ 2.2. i"- . ~ .K ‘<’ . h >- ., 9.. ' ' - o _ "..- ...O . ~ . ‘ ‘ ~ ‘1--.0 0 . ‘ ' - 3 .: '7.:‘.: . M ‘ .1: .:;:;j - .I: - ' ' ‘ _' ‘ ‘ ' . C . '- . . . - . -‘ -‘ . ' I “.- v — ".-, . ' :- _ : o v‘. n ‘ . - , , . . . . - L . ' » ~~. _' :-‘-:-;-. . a-- ‘. .. . ~ ‘ ' ‘ ' I 0-“ — . v ' . . : ~ ‘ . ‘ " :- ’ ‘ - 9_ ‘ '.' j _. — - - ~ ‘ _ j. . ‘;- . -.. :-: o : .-' ' ‘ ' - ‘. '1 I: I: ‘ . ‘ . - . .-_r .’ 2?- 3' "x n. c n - ‘.._. _ . . ..:_.': ‘-:.-: . ‘ ' ' ' I _- - ‘- I - - - . - . . ~ ‘ - -. - ' - ‘ ;- -l ‘ . ‘ . O . ‘ .- . ' . o ._ - __ . - _ _ . ~ . .‘h‘ ' ’ . . a . ‘ ' ‘0 . . O ' - I .' - I . . H a . ‘ . .- I . . ' ‘O ' - ' - - 0 - - - -' v - - II I . ‘ . ‘ ‘ : .QV . . . ' .c',l 2. ‘ ; . ' -. I v .- -’ . .-.-. -‘ ¢ ' . J C - V ‘ - > ‘ . . . . . - I, - ' o. — . ‘ _ h t ‘ ‘. . . . ' - . . . ‘.'-‘ -': ; I - . I c . ‘ ‘. : . ‘ _‘A"_ - I - t O . l - ~ -. .- ‘, I ’ . I . ._ ‘ _~ 1. I . ‘ ' . -' ‘- _. - . - - ° ' ‘ . i. > ' ' ’. : . . V 19 I5 I . - .— ~ - ; - - _ . l : A: - .. .- .. I ‘ . - - . . _ - . . o ‘ . ' o. ' ~ I . . I - I r ' . . I .'- . . I | . _ . . I _ . I- . 'a -. . I ' . . I a I ' - - ‘: ’ ‘ t..- f - ‘_‘ , . ‘ ’ . 1 . I . r: . 1 ' ‘ £7 . O V '- . A.<' 0' n ‘ ‘. - #3:: .- \ L' i. 0 1...- a. '0 N o V, acumen. .‘ 2. . ' L‘ .T'rfiESIS an 5.; B“ .‘u‘f‘ ".7. L ”r- 3 ‘ I’m, _ l \ ABSTRACT lg VITRO ENZYMATIC DIGESTION OF MILK PROTEINS By Steven R. Dimler The groundwork was laid for an in depth study into an in vigrg enzymatic approach to digesting proteins for the purpose of evaluating their nutritive value. Three types of milk proteins served as model systems: whole casein, aS-casein, and B-lactoglobulin. A static (in flask) digest was used. End product inhibition studies indicated that amino acids do competitively inhibit the action of pancreatin on native proteins. Using relatively high concentrations of pepsin and pancreatin helped to alleviate the adverse effects of product inhibition. Expressed as mg free amino acids per 100 mg of protein digest: whole casein released 22 mg, aS-casein released 30 mg, and B-lactoglobulin released 35 mg. Pepsin Pancreatin Digest index values for whole casein, aS-casein and B-lactoglobulin were 78, 75, and 95 respectively. The above proteins were subjected to prior treat- ment before being enzymatically digested. These consisted of autoclaving, autoclaving with equal amounts of glucose, and 8L protei casein leased and 5- reduct both i Steven R. Dimler and autoclaving with 5X the amount of glucose relative to protein. Only specific essential amino acid residues in casein were reduced and the total free amino acids re- leased remained unchanged in the digests. Both aS-casein and B-lactoglobulin digest fractions showed progressive reductions in all of the amino acids released, implying V both individual and structural damage. in pl IE VITRO ENZYMATIC DIGESTION OF MILK PROTEINS By Steven R. Dimler A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1975 TE appreciati guidance 3 study. A? Dr. R.F. t-I in Prepara: The amino acid mlmeroUS te The xita. for h out the cou ACKNOWLEDGMENTS The author wishes to express his sincerest appreciation to Dr. J.R. Brunner for his counsel, guidance and inspiration during the course of this study. Appreciation and thanks are also extended to Dr. R.F. McFeeters and Dr. W.G. Bergen for their advice in preparation of the manuscript. The technical assistance of Ms. Ursula Koch for amino acid analysis and for her aid and counsel on numerous technical matters is much appreciated. The author is especially grateful to his wife, Nita, for her encouragement and wise suggestions through- out the course of this study. ii ACKN LIST LIST IXTRC TABLE OF CONTENTS ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION . REVIEW OF THE LITERATURE Aspects of In Vivo and In Vitro Proteolysis Relationships of—Various Proteins to Degree of Digestion Methods of Assaying Nutritive Value of Proteins EXPERIMENTAL . Chemicals and Materials Chemical Methods Preparation of Digestion Samples for Analysis . . . . Nitrogen . . Alpha Amino Nitrogen--Ninhydrin Test Free Amino Acids . . . . . . Physical Methods . Preparation of Whole Casein Preparation of aS-Casein . Preparation of B-Lactoglobulin Acrylamide Gel Electrophoresis Treatment of Milk Proteins Enzymatic Digestion pH—Stat Evaluations RESULTS Product Inhibition Trypsin Pancreatin . . . Digests with Varying Amounts of Enzyme Milk Protein Fractions . . . . . . Amino Acid Profile Color Changes During Treatments . Effects of Treatments on the Rate of Digestion iii Page iii vi 004-‘NNH Cl) Enzymatic Digestion of Protein Samples Amino Acid Profile Whole Casein aS-Casein . 3- Lactoglobulin . Pepsin Pancreatin Digest Index DISCUSSION Static vs Dynamic System . . Proteolytic Digestion of Milk Proteins Structural Differences Effects of Treatments Pepsin Pancreatin Digest Index CONCLUSION BIBLIOGRAPHY APPENDIX Reagents for Ninhydrin Test Amino Acids Used for Pancreatin.Inhibitor. Study. Dynamic Digest System . . Enzymatic Digestion of the Dried, Whole E0 {g ' Solids Sample . . . . . . . . . . iv 28 28 28 30 31 32 50 50 53 53 54 56 61 62 7O 7O 71 73 Table LIST OF TABLES Effects of end products on the proteolytic action of trypsin and pancreatin on whole casein . . . The effects of varying amounts of pepsin and pancreatin upon the rate of digestion of whole casein Amino acid composition of proteins studied compared to published values Rate of pepsin-pancreatin digest of milk proteins with and without treatments Free amino acids in pepsin-pancreatin digest of whole casein samples Free amino acids in pepsin-pancreatin digest of aS-casin samples Free amino acids in pepsin-pancreatin digest of Riactoglobulin samples Decreases in enzymatically released free amino acids . . Comparison of the digest and residue fractions in a pepsin-pancreatin digest of untreated milk proteins Page 33 35 37 38 42 44 46 48 49 Figure LIST OF FIGURES Lineweaver-Burke plot illustrating effects of end product inhibition on tryptic (left) and pancreatic (right) digest of whole casein . . . Rates of digestion of casein with varying amounts of pepsin and pancreatin . Rate of digestion of whole casein by pepsin- pancreatin-pancreatin . . . . . . . . Rate of digestion of a -casein by pepsin- pancreatin-pancreati . . . . . Rate of digestion of B-lactoglobu1in by pepsin-pancreatin-pancreatin . Effects of various treatments of whole casein on the enzymatic release of amino acids . Effects of various treatments of aS-casein on the enzymatic release of amino acids Effects of various treatments of B-lactoglobulin on the enzymatic release of amino acids vi Page 34 36 39 40 41 58 59 6O INTRODUCTION The value of new sources of proteins for human food, the effects of various processes and storage condi- tions, and the combination of several protein foods into the human diet to optimize their nutritive value is an area of contemporary interest. The means for analyzing the nutritive value has rested traditionally with indices such as protein efficiency ratio (PER), net protein utilization (NPU), biological value (BV), and the slope assay which are obtained from lengthy, expensive animal assays. Shorter chemical methods such as chemical score and the modified essential amino acid index (MEAAPI) often show poor correlation to animal assays. l2 Vitro enzymatic digests offer a viable alternative to animal assays as an option for monitoring due to their low cost, quickness, and high correlation to animal assays. The objective of this study was to further improve upon the ig.vigrg system, identifying its strengths and weaknesses and to use the method to analyze different native and treated proteins. REVIEW OF THE LITERATURE Apsects of £3 Vivo and £3 Vitro Proteolysis Peters (1970) suggested that the small peptides, three to six amino acid residues in length, released by peptic and pancreatic digestion undergo final hydrolysis to free amino acids and dipeptides by amino peptidases which, according to Matthews (1971), are secreted by the brush border or interior of mucosal cells. However, part of the digestion of small peptides occurs by the pancreatic enzymes which are bound to the brush borders in the rat (Woodley, 1969) and probably also in humans (Goldberg 35 al., 1971). Amino acids and dipeptides are transported into the enterocytes where dipeptide hydrolysis occurs. Holdsworth (1972) reviewed the work concerned with the absorption of dipeptides into the blood and concluded that the only known tripeptide to cross the enterocyte intact ‘was composed of histidine, proline, and glutamic acid, a thyrotrophin releasing factor which is resistant to proteolytic enzymes. It was not clear though what pro- portion of an ingested protein is completely digested to amino acids before absorption. By placing a mixture of eighteen amino acids in the jejenum of a man, Adibi et al. (1967) found that the amino acids were absorbed at varying rates. Delhumeau 35 al. (1962) "fed” different molar ratios of amino acids to the loop of a rat intestine and concluded that there was a higher absorption rate of a mixture simulating whole egg hydrolyzate than one simulating a casein hydrolyzate. Although the literature reports conflicting results, Itoh 3; al. (1973) supported observations showing higher growth rates for rats fed whole casein protein than those fed a compositionally identical amino acid counterpart. One explanation contributing to the dif- ference in growth rates is derived from the work of Matthews gt al. (1968) who noted that the transport of methionyl-methionine, methionyl-glycine, and glycyl- methionine was more rapid than the identical free amino acids. lg 3139 conditions of the digestive tract include a pH range of 2 to 6 in the stomach and 4.5 to 6.5 in the small intestine (Nasset, 1957). Using meals containing up to 30 g of protein, Nixon and Mawer (1970a) estimated the secretion of endogenous protein to range from 2 to 8 g. Digestive enzymes, following isolation and purification, exhibit definite pH values for optimum activity. For pepsin (3.4.4.1), the proteinase in the gastic juice, Bovey and Yanari (1960) identified the optimum pH to be 1.8. The pancreatic enzymes released by the pancreas to the small intestine consist primarily of trypsin (3.4.4.4) and chymotrypsin (3.4.4.5), with optimum activities at pH 7 to 8. Smith and Hill (1960) suggested that leucine aminopeptidase (3.4.1.1), identified by its trivial name of erepsin, be given more active consideration as an important enzyme in physiologic proteolysis. Each of the digestive enzymes mentioned above cleave the proteins differently (Hill, 1965). Pepsin, the least specific, cleaves peptide bonds in which phenylalanine, tryosine, glutamic acid, cystine and cysteine contribute either the amino or carboxyl group of the bond. Trypsin has a specificity limited to cleaving peptide bonds with lysine or arginine in the carboxyl position. Chymotrypsin cleaves the peptide bonds with tyrosine, phenylalanine and tryptophan in the carboxyl position. Leucine aminopeptidase, although showing highest hydrolysis rate with leucine residues, will cleave peptide bonds adjacent to any a-amino group. The L- isomeric form of the amino acid residue is preferred for each of the enzymes (Rupp gt gt., 1966). Relationshipsgt Various Proteins tg Degree gt Digestion Proteins differing in structure and composition undergo different digestion and absorption patterns. m... 235.1%? ’l-Ja' 3"" ’1‘! d.- ‘1' "4 ('1) Epstein and Possick (1961) attributed these differences in the strengths of the stresses involved in the tertiary linkages between polypeptide chains. Allison (1964) drew an analogy stating that the degree to which a protein's structure contributes to its proteolysis is as essential and unique as behavior contributes to behavioral reactions. Zebrowski (1968) observed from studies with white rats that casein emptied from the stomach faster than did heated soya protein. The intestine also contained more soluble nitrogen from digested casein than from the heated soya diet. The indigenous proteins of milk form a stable, colloidal suspension. Casein, comprizing 80% of the total proteinaceous material, consists of loosely packed calcium caseinate complex units joined by calcium and calcium phosphate-citrate linkages (Moor, 1967a). Thompson gt gt. (1965) defined casein as a heterogeneous group of phospho- proteins precipitated from skim milk at pH 4.6 and 20 C. Waugh (1961) suggested one form in which the interaction of the casein fractions stablized the micelles in casein. aS-Casein, the major component of the casein fraction, as . described by McKenzie (1967), is defined as a calcium sensitive, disordered protein without intermolecular disulfide bonds. aS-Casein's disordered configuration allows it to undergo rapid digestion particularly by the newborn. tein c0 Moor (1 protein groups 1 Tanford is compa a large that 5-1. structure A little nu products, cereal prc new proces Of Continu Th B-Lactoglobulin represents 10% of the total pro- tein content and 50% of the total whey proteins in milk. Moor (1967b) described B-lactoglobulin as a globular protein containing disulfide linkages and sulfhydryl groups susceptible to reversible disulfide interchange. Tanford gt gt. (1962) indicated that its native structure is compact. Refolding, following denaturation, produces a large number of a-helixes. McKenzie (1967) concluded that B-lactoglobulin exhibits changes in its secondary structure influenced by the environmental conditions. Although normal heat processing procedures induce little nutritional damage to the protein in milk and meat products, it enhances the quality of most legume and some cereal proteins (Bender, 1972). As "new" proteins and new processing techniques gain recognition, assays capable of continuously monitoring the nutritional changes become more significant. The structure and composition of protein species determines the effects of various potentially damaging processes. Menden and Cremer (1966) compared the effects of heat with and without added glucose on casein and on meat protein. The overall analysis indicated that in the casein fraction lysine and arginine showed the largest decline. There was some reduction in methionine and tyrosine. In meat, threonine was the only residue adversely affected. intera absenc 1974a- genera sugars trypto; e a. Bender, degrada tion ree Carpente )buron ( mild app block a lYSine), infrared masked by groups un Eamon (1! Pancreatic to blOcked fI‘EES lysli physiologic Hurreu and the enzyme“: by aCid hYClI Extensive reviews treat the subject of protein interactions as induced by heat both in the presence and absence of a reducing sugar (Janick, 1973; Lien and Nawar, l974a-c; and Bender, 1972). The amino acids which are generally labile to heat in the presence of reducing sugars include lysine, arginine, histidine, methionine, tryptophan, and threonine (Baldwin gtth., 1951; Cook gt gt., 1951; Ford and Salter, 1966; Mauron, 1970; and Bender, 1972). Most studies have focused upon lysine degradation because of the prevalent Maillard condensaf tion reaction (Henry, 1957; Holsinger gt gt., 1972; Carpenter, 1973; and Hurrell and Carpenter, 1974). Mauron (1970) and Carpenter (1973) observed that under mild application of heat the sugar moieties mask or block a free amino group (e.g., the e-amino group of lysine). Baldwin gt gt. (1951) showed with the aid of infrared spectrophotoscropy that the amino groups were masked by masses of hydroxyl groups with the carboxyl groups unchanged in a casein-dextrose heat-treated mixture. Mauron (1970) pointed out that neither pepsin nor the pancreatic enzymes (trypsin can cleave the bond adjacent to blocked lysine amine groups. However, acid hydrolysis frees lysine of the sugar moiety, thus implying physiological availability when analyzed quantitatively. Hurrell and Carpenter (1974) stated that around 50% of the enzymatically unavailable lysine groups are recovered by acid hydrolysis. Increased levels of heating degrade the b6 underg resist amino e.g., hibito: (1973). casein damage, conditi sugars I and gluc total co: acids are Since the Inomehtari a protein '3’ _ a...an aCl( Severa1 va or enzyme (< combination USEd a pept mlcrObiologj the basic amino acids that were blocked. Proteins also undergo structural damage with the development of enzyme- resistant, intramolecular linkages between hydroxyl and amino groups. The byproducts of heat-sugar treatments, e.g., premelanoidins, may in themselves be enzyme in- hibitors in the digestion processes, Adrian and Frangne (1973). Rao and Rao (1972) reported that autoclaving casein in the presence of arabinose caused the most damage, followed by glucose, then lactose. Under similar conditions, Tu and Eskin (1973) found that the following sugars caused damage in decreasing order: xylose, fructose, and glucose. Methods gt Assaying Nutrttive Value gt Proteins Block and Mitchell (1946) noted that not only the total content but also the relative proportion of amino acids are essential considerations for proper growth. Since the pooling of ingested amino acids occurs only momentarily, if at all, the key to the nutritive value of a protein is the presence of an appropriate assay of amino acids present at the site of protein synthesis. Within the framework of an tg ttttg digest, several variables exist such as the type and concentration of enzyme(s) employed, and the method of analysis. Many combinations have been employed. Sheffner gt gt (1956) used a peptic (100 2.5, S:E) digest which was analyzed microbiologically for available amino acids to formulate his Peps of essen pepsin, between Microorg. using mi< various 1 acids, nc sune solt known. B Utilized hiStidine Utilized Closely t. (1964) ob- rat feedil fOIIOwed l which was }k Yamashi ta digestion overall nu performed his Pepsin Digest Ratio. After tabulating the profile of essential amino acids after consecutive digests with pepsin, trypsin, and erepsin, the greatest difference between the digests appeared following the peptic digest. Microorganisms were used to the data. However, when using microorganisms, neither a) the gxtent to which the various peptides contribute to the total "free" amino acids, nor b) the microorganism's specificities to con- sume soluble peptides rather than free amino acids are known. Baldwin (1951) indicated that microorganisms utilized a large proportion of lysine, methionine, histidine and threonine whereas none of these were utilized by the rat. Although Sheffner's values agreed closely to the biological values, Akeson and Stahmann (1964) obtained indices showing closer correlations with rat feeding trials. They used pepsin (100:1.5, S:E) followed by pancreatin (25:1, S:E) to produce a digest which was analyzed with an amino acid analyzer. Many researchers, DeBaun and Connors (1954) and Yamashita gt gt. (1970), to cite two, used only a trypsin digestion as a relative gauge of lysine destruction for overall nutritive value. Valaris and Harper (1973a, b) performed inhibitory studies of the effects of carboxy- methyl cellulose on aS-casein and concluded that data obtained from a tryptic hydrolysis can be misleading if the prior peptic treatment was omitted. Menden and Cremer (1966) used relatively high concentrations of pancreatin 10 (2:1, S:E) and avoided using pepsin before hand to limit possible hydrolysis of the pepsin by the pancreatin. The digest ran for 15 h, a time they felt was short enough to avoid autolysis of the pancreatin enzymes. Ford and Salter (1966) reported that prolonged digestion with pepsin (10:1, S:E), pancreatin (10:1, S:E), and erepsin (3:1, S:E) released between 80-85% of the amino nitrogen as free amino acids when assayed microbiologically. The above mentioned tg ytttg digestions were conducted in a flask under static environmental conditions. The results thus obtained are suspect when compared to the process of an tg tttg digestion. Two aspects of the static-digest technique seem worthy of examination. First, the build-up of products of digestion may progressively inhibit further digestion. Second, the build-up of these products may induce trypsin and chymotrypsin to function as trans-peptidases. Ford and Salter (1966) constructed a Sephadex-gel filtration device to simulate a dynamic digest in which the end products were removed into the column during the course of digestion. Mauron gt gt. (1955) used a dialysis-sac digest as another means of implementing a dynamic system. He calculated a Pepsin- Pancreatin Dialysis Digest ratio index, similar to Sheffner's (1956) original index, for several proteins and found quite close agreement to the rats' biological values (Mauron, 1970). A discrepency occurred when a heat-damaged milk protein sample was assayed. 11 In calculating Sheffner's index, both profiles of the free amino acids in the digest and the remaining amino acids not entirely released are compared to a whole egg digest. The index shows much better correlation to rat assays than the modified essential amino acid index (Block and Mitchell, 1946), because it emphasizes the importance of having proper release and balance of essential amino acids throughout the entire digest. Supporting this concept, Melnick gt gt. (1946) noted that in a digest of raw soy protein, the methionine was not released until the end of the digest period. However, in the digest of heated soy, methonine was released through- out the entire course of digestion. They postulated that the disproportionate release may be a factor in lowering the nutritive value. The general conclusion drawn from the major contributors to tg_gtttg digestion techniques supports the use of such a method as a guide toward looking at the value of native proteins as a source of human food. The index may overestimate a proteins' value, especially in treated proteins, because the total digest values have thus far been obtained by acid hydrolysis. As stated previously, acid hydrolysis releases more amino acids than appear to be released under physiological conditions. The final criteria of the outcome of any iE.X£E£9 digestion technique rests upon its correlation with biological assays. The rat has been the test animal used most fr ratio ( of prot benefit. However of all ; require: for an a requirem simply g value (3 Nitrogen digestib: €Stimatic pr0t61n L the Hitrc it is muc the most VaIUe Of L cereal COr balanCeS C AISmeyEr e V~ equatiOns 12 most frequently for biological assays. Protein efficiency ratio (PER) equals the weight gain divided by the amount of protein consumed, both expressed in grams. The benefits of the PER index are its ease and simplicity. However, PER cannot be used to gauge the nutritive value of all proteins for mature humans, because the amino acid requirements for a growing animal are much greater than for an adult (e.g. a growing rat has much higher lysine requirements than an adult human) whose intake of protein simply goes for maintanence and not growth. Biological value (BV) is the retained nitrogen divided by the abosrbed nitrogen. Since the BV index fails to account for digestibility differences between proteins, gross over- estimations of aprotein'snutritive value can occur. Net protein utilization (NPU) is the retained nitrogen over the nitrogen intake or BV times digestibility. Although it is much more difficult to determine, NPU is overall the most accurate index for determining the nutritive value of a wide variety of proteins (Hegarty, 1975). Inglett gt gt. (1969)used a computer to optimize cereal combinations where not only deficiencies but over- balances of essential amino acids were considered. Alsmeyer gt gt. (1974) investigated whether regression equations from a protein's amino acid composition could accurately predict the respective PER value. His equa- tions failed to predict PER values with accuracy. Of the many foods investigated, bean foods especially high in l3 leucine were greatly overestimated and marine foods and noodles also gave poor estimations. Womack gt gt. (1974) used a rat assay to determine the effect of processing on the availability of essential amino acids. They compared an amino acid fortified casein control diet to identical diets containing a 20% reduction in one of the essential amino acids. PER values were used to follow the effects. The critical level was defined as that level of each amino acid which would maintain good PER values but when reduced 20% would yield significant drops in PER. However, their results showed that at the 20% reductions, no significant decreases in PER occurred. Abrahamsson gt gt. (1974) presented the ”ultimate” assay for protein quality studies. They ran PER values on seven individual sources of protein as well as on many various combinations of these seven proteins. Thus, they were able to identify the most nutritive combination. EXPERIMENTAL Chemicals and Materials Pepsin, hog stomach mucosa, was 1-10,000 purified. Pancreatin, hog pancreas, was 5X crystallized. Trypsin was 2X crystallized and salt free. The three enzymes were obtained from Nutritional Biochemical Corporation. All other chemicals used in this study were obtained commercially. Chemical Methods Preparation of Digestion Samglgs for Anatysis The samples removed at various times throughout the digest were first mixed with sufficient trichloroacetic acid (TCA) to obtain a final concentration of 15% TCA. After setting overnight at 4°C, the samples were centrifuged at IOOOX g for 5 min to remove precipitated material. The supernatant was clear and used for further analysis of its nitrogen, alpha amino nitrogen and free amino acids. Nitrogen A micro-Kjeldahl apparatus was used to determine nitrogen in both protein and deproteinated samples (Mangino, 1973). The digestion mixture contained 5.0 g CuSOA-SHZO and 5.0 g SeO2 in 500 ml concentrated H2804. 14 15 Either 15 mg of dried protein or an equivalent concentration of the TCA soluble peptides were digested in duplicate by adding 4 ml of the digestion mixture and boiling for 1 h. After the flasks cooled, 1 m1 of 30% H202 was added and the digestion continued by boiling another hour. After cooling the flasks, the sides were rinsed with 10 m1 of deionized water. Neutralization was accomplished by adding 25 m1 of a 40% NaOH solution. The free ammonia was steam distilled into 15 ml of a 4% boric acid solution containing 5 d of indicator consisting of 400 mg bromcresol green and 40 mg of methyl red dissolved in 100 ml of 95% ethanol. The termination of distillation occurred when the receiving beaker reached a volume of 60 ml. The ammonium borate complex was titrated with 0.0230 N HCl previously standardized by tris-hydroxymethylaminomethane as a primary standard. (ml sample - m1 b1ank)(N)(14.007)(100) mg sample %N = Alpha Amino Nitrogen -- Ninhydrin Test Appropriate aliquot volumes of the deproteinated samples were pipetted into test tubes for further dilution. The dilution factors required ranged from 5, 50, to 100 for the samples from the enzyme blanks, the peptic digest, and the second pancreatic digest respectively. When the proper dilution was obtained the samples were assayed by the ninhydrin test (Clark, 1964). One-half milliliter of the sample was mixed with 1.5 m1 of the ninhydrin reagent 16 solutiona, then set in a boiling water bath for 20 min while topped with glass marbles to prevent evaporation losses. Following the heat treatment, the reaction mixtures were cooled to room temperature followed by the addition of 8.0 ml of 50% n-propanol. Color development stabilized after 10 min. Absorbance readings were re- corded at 570 nm wavelength using a Beckman DK—2, double- beam spectrophotometer. Glycine was used to prepare a standard curve. Free Amino Acids The samples analyzed were deproteinated by TCA and corresponded to a peptic-pancreatic digestion of a total of 26.5 h. The precipitated proteins and large peptides were centrifuged and filtered through a 0.22 um Millipore filter apparatus, yielding a crystal clear, final solution. An aliquot of this solution was mixed with a standard con- centration of nor-leucine which served as an internal standard. The particular nor-leucine solution reacted with the salts in the TCA solution, producing a cloudy appearance. An additional filtering using the Millipore apparatus removed the cloudiness. The samples were then ready for analysis in a Beckman/Spinco Model 121 C Amino Acid Analyzer according to the procedure of Spackman gt gt. (1958), Moore and Stein (1954) and Moore gt gt. (1958) aSee Appendix for the list of ninhydrin reagents. 17 Physical Methods Prepgration of Whole Casein Whole casein was precipitated from fresh skim milk at 38 C by the addition of 4N NCl to pH 4.6. The pre- cipitated protein was collected with double layered cheesecloth and squeezed dry. The casein was redissolved in distilled water using 1 N NaOH to maintain a constant pH of 7.5. The procedure was repeated until a caseinate fraction washed and reprecipitated a total of four times was obtained. After the final dissolution, lyophiliza- tion was begun and the dried protein stored at -20 C. Henceforth, the sodium caseinate fraction will be re— ferred to as whole casein. Preparation of aS-Casein The method used closely followed that of El-Negoumy (1966). Approximately 350 g of frozen whole casein was dissolved in l l of a 6.6 N urea solution. An addition of 200 m1 of 7 N H2804 lowered the pH to 1.4. Following the addition of 2 1 of distilled water, which diluted the urea to 2.2 N, an aS-casein-rich fraction precipitated. The centrifuged precipitate underwent purification steps by redispersion in 1 l of a 6.6 N_urea solution. Diluting ‘with distilled water to attain a 4.8 N urea concentration caused some precipitation of residual aS-casein, however ‘most of the further enriched fraction was precipitated at a 3.3 N_urea concentration. After redissolving in a 18 4.8 N urea plus 21.8 g NaCl per 1, an electrophoretically pure fraction was obtained upon dilution with distilled water to both 3.3 N and 1.7 N urea. The precipitate was washed several times with distilled water, then redis- solved in water maintained at pH 7.5 with NaOH, dialyzed for 36 h against distilled water at 4 C, lyophilized, and stored at -20 C as sodium aS-caseinate. Preparation of g-Lactoglobulig The method used followed that suggested by Fox gt gt. (1967). The whey portion from the first precipita- tion of whole casein was carefully decanted into another beaker. Enough trichloroacetic acid (TCA) was added to the acid whey to give a final concentration of 3% TCA. After standing for 30 min, the precipitate was removed by centrifugation and discarded. The clear supernatant was poured into cellulosic, dialysis sacs and pervaporated to one-tenth its original volume. Ammonium sulfate was added to yield a 0.4 saturated solution. The residual precipitate was discarded. Ammonium sulfate was added to saturation. After standing overnight at 4 C, the pre- cipitated fraction was collected by centrifugation and purified by a second suspension in a saturated ammonium sulfate solution. The precipitate was dissolved in 400 m1 of distilled water with l N NaOH to keep a constant pH of 7.5, placed in a dialysis sac and dialyzed against distilled water at 4 C. The solution was lyophilized and stored at -20 C. 19 Acrylamide Gel Electrophoresis Homogeneity of the milk protein fractions was assessed electrophoretically by a modified version of the method by Melachouris (1969). This procedure called for two gel systems, but only a running gel and spacer buffer were used. The running gel was prepared by dissolving 45 g of Cyanogum-41 in 0.380 N trisJHCl buffer at pH 8.9 then making up to 500 m1. A spacer buffer consisted of a 0.062 N tris-HCI pH 6.7 buffer. These solutions were used for B-lactoglobulin. The same solutions containing 5 N urea were used to assay the casein fractions. The stock, electrode-vessel buffer contained 0.046 N_tris- glycine at pH 8.3. Protein samples were dissolved in the spacer buffer, which served to "stack" the proteins during electro— phoresis. Several sucrose crystals and bromophenol blue provided the necessary density and marker dye, respectively. To 20 m1 of the running gel, 0.02 ml of N,N,N',N'- tetramethylethylenediamine was added followed by 0.07 ml of 5% ammonium persulfate. A small amount of water was layered over the top of the gels to insure a level surface. The gel set in 20 min. During the run, a constant instru- ment voltage of 150v was applied. The gels were removed,. stained with amido black, and destained in a methanol- acetic acid solution. 20 Treatment of Milk Proteins The milk proteins were analyzed under four different treatments: a) native, untreated, b) autoclaved for 30 min at 15 psi, c) autoclaved with equal amounts of glucose, and d) autoclaved with 5X the amount of glucose. The protein samples were prepated in concentrations of 12 mg of protein sample per ml water at pH 7.0. The solutions were poured into 125 m1 flasks and covered with aluminum foil. Heat treatments were conducted in a Masco Cyclomatic Sterilizer. Evaporation losses were corrected after cooling. The samples were stored at 4 C. Enzymatic Digestion The pH of untreated and treated protein samples was lowered to 1.8 with 1 N HCl. The volume was increased from 25 to 30 ml, representing a sample concentration of 1.0%. Twenty-five mg of pepsin was dissolved in the solu- tion. After swirling, the flask was placed in a 37 C oven for 20 h of incubation. Peptic digestion was terminated by the addition of a 0.5 NNaHCO3 buffer (pH8) and 1 N NaOH until a stable pH of 8 was obtained. A final increment addition of distilled water brought the total volume added to 5.0 m1. Thirty mg of pancreatin was dissolved into the reaction mixture. Reincubation at 37 C lasted for 3 h followed by a second addition of 30 mg of pancreatin for an additional 3.5 h incubation. 21 Enzyme blanks were treated identically except for the absence of the specimen protein. pN:Stat Evaluations A sargent pH-Stat was calibrated to maintain each sample at 37 C at pH 8.0. Standard volumes used were 10 m1 of substrate solution and 0.2 m1 of enzyme solution. A magnetic stirring bar maintained continuous mixing. The addition of the proton-neutralizing 0.05 N NaOH solution was monitored on the recorder chart. A slight buffering action helped to insure stable monitoring by the pH-Stat. The buffer selected contained 0.605 g of TRIS and 2.34 g of NaCl/l plus a concentration 0f 0.02% Na azide to inhibit microbial growth. A stock protein solution of 1% whole casein dissolved in the buffer was prepared. The stock protein solution was diluted to vary substrate concentrations. Initially, 0.05 NCaCl2 was used to stabilize the trypsin solution against autolysis. However, the Ca (II) caused the casein to precipitate, thus inducing an extraneous drop in pH. Keeping the enzyme solution in an ice bath re- tained its stability and using 0.20 ml solution per reaction did not affect the stabilized temperature in the reaction vessel. A mixture of nine of the water soluble amino acids: glycine, arginine, serine, alanine, histidine, lysine, valine, phenylalanine and methionine at a total concentration of 48 mg/ml, comprized one inhibitor solu— tion. Other inhibitor solutions included a 1% casein 22 solution digested to completion, as monitored by the pH-Stat, with either trypsin or pancreatin. The substrate was allowed to achieve pH and temper- ature stability before the enzyme was injected into the reaction vessel. The injection caused no change in the two parameters. The pH-Stat measured the extent of enzymatic hydrolysis of peptide bonds by maintaining a constant pH through the continuous addition of base. An attached chart recorded the amount of base released over time. Initial velocity rates were measured from the slope of the reaction curve with the aid of a mirror as described by Bergmeyer (1963). The pH-Stat had a maximum release of base over time of 6 units base/unit time. It was found that the initial rate of digestion of 0.010 g/ml of substrate by 2 mg of trypsin was at the maximum value of 6. Hence, the pH-Stat could not record any higher initial reaction rates with digestions containing higher substrate con- centrations. The method used in this study to analyze the data from the reaction rate study was by plotting reciprocal vElocity 2g reciprocal substrate concentration (Line- weaver-Burke plot). By definition, the y-coordinate would represent the recipr0cal maximum initial velocity of the reaction. Thus during the tryptic digestion in this study, the 1/Vmax value of .17 corresponded to a l/(S) value of 100. For the purpose of clarity, the graph was 23 plotted using a 1/(S) value of 100 at the y-coordinate position. However, in order to calculate a linear regression analysis on the plotted points, the l/(S) values (x-axis) had to be all subtracted by 100. The data from pancreatic digestion study was handled similarly, where 1/(S) assumed the value of 68 at a -f l/Vmax of .17. The data was also plotted on an Augustinsson plot ((S)/v 2g (8)). Again, the actual substrate concentra- tion value used had to be adjusted to reflect the physical limitation of the pH-Stat's monitoring capacity. The method used to adjust the values included 1) sub- tracting the 1/(S) value corresponding to the 1/Vmax value of .17 from all of the l/(S) values in order to obtain "adjusted” l/(S) values, 2) inverting them to obtain "adjusted" (S) values, and then 3) using the "adjusted" (S) values to plot the graph of the (S)/v pg (S). Identical slopes between the control digestion and those with inhibitors would imply identical Vmax values. Although not submitted into this thesis, Augustinsson plots were drawn and agreed with the con- clusions of the Lineweaver-Burke plots. RESULTS Product Inhibition Trypsin The purpose of the experiments with the pH-Stat was to explore any evidence of product inhibition on the activity of trypsin and pancreatin. The inhibitors employed were selected in an attempt to simulate varying degrees of proteolysis, ranging from large peptides to free amino acids. They included 1) a tryptic digest, 2) a peptic-tryptic digest, and 3) a mixture of lysine and arginine. The data obtained are tabulated (Table 1) for application to a Lineweaver-Burke plot interpreta- tion. A 0.15% concentration of the inhibitor yielded interpretable results. Higher concentrations of the pre- digested fraction produced greater inhibition, to the point where virtually no enzymatic hydrolysis was detected in a 0.50% protein plus 0.50% pre-digested fraction. Data in Figure 1 illustrates the effect of the inhibitors. As indicated, the Vm of each "digestion" remained constant, ax thus implying a condition representative of competitive inhibition. 24 25 Pancreatin The data collected from the pancreatin study is listed in Table l. The inhibitors employed consisted of different concentrations of a mixture of nine, water soluble amino acidsa. The ratio of whole, intact protein to free amino acids was 2:1 and 1:1. Figure 1 indicates a uniform Vmax implying the inhibition as a competitive type. The latter reaction ratio produced an initial velocity half that of the control rate when using a 0.50% substrate concentration. Various observations in this study merit attention. After increasing the concentration of pancreatin to 10 mg/0.20 ml, the initial velocity was one-half that of trypsin at 2 mg/0.20 m1. However, the total amount of protons released over a longer time span was more than in the tryptic digest. Pancreatic digest of a pepsin- digested substrate produced lower reaction rates than in an intact substrate sample. The peptic digested sample would contain a smaller number of peptide bonds and the increase in free amino acids contribute to enzyme inhibi- tion. Digests with Varying Amounts of Enzyme The purpose in varying enzyme concentration was to obtain maximum digestion with minimum enzyme contribution. aSee Appendix for the contents of each specific amino acid. 26 Figure 2 illustrates the relative extent of digestion arising from varying enzyme concentrations. A Uniform 1% whole casein solution served as the substrate. The peptic and pancreatic digestions were for 3 h and 6.3 h, respectively. The rate of digestion was monitored by the ninhydrin test on TCA-soluble supernatants. Four different digestions were conducted, each with different enzyme concentrations regarding pepsin and pancreatin. The digests were initiated with 4.5 mg, 10 mg, 10 mg, and 25 mg of pepsin. After 3 h, the amount of pancreatin added to the aforementioned digestions was 12 mg, 12 mg, 30 mg, and 30 mg, respectively. Three hundred mg of whole casein was present. The flask with the lowest enzyme concentration typlified that of the digestion procedure of Akeson and Stahmann (1964). As illustrated in Table 2, the pepsin blanks showed a minimum contribution to the ninhydrin positive reaction of the system. The pancreatin blanks induced a greater response. However, in both cases, the increase in absorbance, or the degree of cleavage, was slight and uniform over the time observed. Figure 2 shows the patterns of the protein digests with the enzyme blank values subtracted. The final points indicate that the degree of digestion achieved with the higher concentration of enzyme was at least twice that of the digestion obtained with the lowest enzyme concentra- tion. 27 Milk Protein Fractions Amino Acid Profile The amino acid composition of each of the three proteins studied was obtained after acid hydrolysis in evacuated, sealed ampules for 22 h at 110 C. Compositions were compared to published values as reported in Table 3. Examination of the results indicates close agreements. Deviations may stem from differences in preparative techniques, purity of specimens, analytical methods and genetic variations. Color Changes During Treatments Prior to the heat treatment, all of the protein samples were white in color. Neither whole casein nor as-casein solutions showed indications of browning after autoclaving. However, autoclaving turned the B-lacto- globulin solution slightly tan in color. Heating the proteins with 1% glucose altered the appearances as follows: whole casein became amber-brown, aS-casein turned tan, and B-lactoglobulin was tannish yellow. By increasing the glucose concentration to 5%, heating produced a reddish-brown whole casein, a brownish—tan aS-casein, and a yellowish-orange B-lactoglobulin solution. Effects of Treatments on the Rate of Digestion Table 4 lists the data for all of the digestion experiments monitored by the ninhydrin test. The 28 preceeding Figures 3, 4 and 5 illustrate the relative degree of digestion with time. Preliminary experiments in this study revealed that the reduced degree of diges- tion in a 3 h compared to 20 h peptic digest was overcome by the pancreatic digest. Kjeldahl analysis indicated that after 20 h pepsin and 6.5 h pancreatin (twice added), the TCA-soluble peptides represented 95% or more of the total amount of protein present. Engymattc_Dtggsttgn of Prgtein Samples ——.~—...-——-— ~——— Amino Acid Profile The amino acids liberated by the pepsin-pancreatin sequential proteolysis of the milk protein samples are listed in Tables 5, 6, and 7. Each table shows a compar- ison of the amino acid profile enzymatically released as amino acids per m1 of digest, corrected for the enzyme blank, with the theoretical concentration based on the amount of protein recovered by acid hydrolysis. A high voltage electrophoretic spot test supported the evidence that few large peptides were present in the final digest. As previously mentioned, Kjeldahl analysis showed that at least 95% of the digest was soluble in TCA. “111.919-95'1126319. Data for the digestions of the whole casein samples are presented in Table 5. Due to destruction by acid hydrolysis of the native protein, values for % cystine 29 and tryptophan represent estimations obtained from Block and Weiss (1956). As expected, according to the specificities of the enzymes used, over 40% of arginine, tyrosine, leucine, phenylalanine, lysine, tryptophan, and methionine were released from the untreated substrate. Of the essential amino acids, isoleucine and valine ranked the lowest in amount, accounting for only 10.0% and 9.1%, respectively, of the theoretical total. Low yields of free aspartic acid, glutamic acid, glycine, and proline were noted. Table 8 lists the essential amino acids showing a reduction in amount released compared to the digestion of untreated protein. Autoclaving caused a reduction of 7% for lysine, 22% for arginine, and 5% for methionine. Including 1% glucose into the heat treatment caused further reductions; 12% for lysine, 22% for histidine, 46% for arginine, 7% for methionine, and 28% for trypto- phan. Increasing the glucose concentration to 5% in- duced decreases of 48% for lysine, 45% for histidine, and 24% for methionine. Arginine and tryptophan showed slight increases in relation to the percent decrease following the heat plus 1% glucose treatment. As the glucose concentration was increased further decreases in the total amount of amino acids enzymatically released were slight. The total free amino acids released was approximately 22 mg/100 mg protein. Since the total 30 amount was practically unchanged, selected amino acids showed increases compared to the digest of treated whole casein. The residues involved represent the non-polar, uncharged amino acids: valine, isoleucine, leucine, and phenylalanine. aS-Casein Table 6 lists the results for the digestions of the aS-casein samples. Due to the destruction of tryptophan by acid hydrolysis of the native protein, its value was estimated from Dayhoff (1972). The release of individual, essential amino acids in the digest of the untreated sample expressed as a percentage of the total amount present in the intact protein ranged from 91% for lysine, 70% to 80% for methionine, arginine and tryptophan, 60% for phenylalanine, 45% for leucine, 33% for histidine, and less than 15% for valine and isoleucine. Overall, the digest of the untreated aS-casein fraction contained 3 % by weight of free amino acids. Table 8 lists the essential amino acids reflecting a reduction in amount released compared to the digestion of the untreated protein. Autoclaving alone resulted in reductions of 29% for valine, 14% to 19% for methionine, isoleucine, and lysine, and 8% to 9% for phenylalanine and arginine. Autoclaving with 1% glucose caused greater reductions of 38% to 45% for lysine, valine, isoleucine, methionine, and arginine, 22% for leucine, 16% for phenyl~ alanine, and 7% for histidine. Increasing the glucose 31 to 5% in the autoclaved sample induced little additional change in the amounts released for valine, methionine, isoleucine, leucine, and phenylalanine. However, lysine, arginine, histidine, and tryptophan dropped to 61%, 55%, 23%, and 19%, respectively. The amount of free amino acids released per 100 mg of protein dropped from 30% for the untreated, 27.5% for the autoclaved, 23% for the auto- claved plus 1% glucose, and 21% for the autoclaved plus 5% glucose. B-Lactoglogplin Table 7 lists the results for the digestions of the B-lactoglobulin samples. Due to the destruction of tryptophan and partial destruction of methionine by acid hydrolysis, their values represent estimations from Dayhoff (1972). The release of individual, essential amino acids in the digest of the untreated sample expressed as a percentage of the total amount present in the protein ranged from 70% for arginine, 55% to 60% for lysine, methionine, leucine and tryptophan, 39% to 43% for isoleucine, valine, and phenylalanine, and 31% for histidine. Overall, the free amino acids in the total digest was 34.6%. Table 8 lists the essential amino acids reflecting a reduction in amount released compared to the digest of the untreated protein. Autoclaving caused reductions from 24% to 29% for lysine, methionine, tryptophan and leucine. The remaining values represent from 10% to 20% reductions. The effect of 1% glucose plus heat caused decreases of 47% for methionine, 44% for arginine, 36% to 39% for lysine, leucine and phenylalanine, 28% to 30% for valine, isoleucine and tryptophan, and 23% for histidine. Only lysine, tryptophan and arginine de- creased further with the treatment of heat plus 5% glucose, they were reduced to 48%, 42%, and 55%, respectively. The amount of free amino acids released per 100 mg of protein dropped from 34.6% for the un- treated, 28% for the autoclaved, 22.5% for the auto- claved plus 1% glucose, and 22% for the autoclaved plus 5% glucose. Pepsin Pancreatin D1899911R§E§ Pepsin Pancreatin Digest (PPD) index were deter- mined for each of the untreated, milk proteins by using the method of Akeson and Stahmann (1964) which was a modification of Sheffner's gt gt. (1955) original method. The values for the whole egg digest were obtained by digesting dried whole egg solids exactly as with the milk proteins. A 22 h acid hydrolysis produced a composition of amino acids considered to represent the total amount. The milk proteins gave PPD index values of 78 for casein, 75 for aS-casein, and 95 for B-lacto- globulin. Whole egga was estimated to be 96.5 (Akeson Stahmann, 1965). 8See Appendix for the amino acid composition of the enzymatic digest and the acid hydrolyzate of whole egg. 33 Table 1. Effects of end products on the proteolytic action of trypsin and pancreatin on whole casein -—————..—.-_.__—_— .. .--- --_ _- .-....» ..—. -—-—. ~. .._.. 41.. .,._,~-‘. ... ..i .--4-. .—- Trypsin:t Pancreatine b 17(3) 1/v° (If: b 1/(S) l/vC (Dd .0100 100 .17 - .0100 100 .25 - .0067 150 .18 - .0070 .143 .37 - .0050 200 .19 - .0050 200 .48 - '0040 250 '21 ' .0085 118 .28 .0024z '0030 330 '24 ‘ .0070 143 .40 .00242 .0085 118 .21 .0015W .0050 200 .50 .0024z .0070 143 .26 .0015W .0085 118 .31 .00482 .0050 200 .35 .0015w .0070 143 .45 .00482 .0085 118 .20 .0015x .0050 200 .73 .00482 .0070 143 .25 .0015X .0050 200 .30 .0015X .0070 143 .26 .0020y .0050 200 .34 .0020y 8Used 2 mg for each digest. bSubstrate concentration expressed as g/ml. 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In . . .tll :Hommo oHOSS mo coaumowflp mo dump msu com: Cwumouocma paw Gammon mo mucnoam wcwhum> mo muoommm 039 .N manme 35 .cfiumouocmm paw awmmom pom moauwu Am mv Huoa paw HHNH on mpcommounoo >H adouom .cflumouocmm paw Gammon How mowumu Am mv HHOH paw Huom ou mpcommouuoo HHH msouwp .cflumouocmm new Camden Mom mowumu Am mv Humm was anom ou mucommmuuoo HH anouoo .cHummuocma mam Gammon Mom moaumy Am mv Humm paw H100 on mpcoamouuoo H anouun .maaamm ummwwp oHASHOm mo muommmm one .N magma 36 cwumouocmm paw Gammon mo monsoEm mafihum> £uw3 aflommu mo cowummwwp mo mound .N gunman AIV mz_» zoupmmwza S. m o _m 5 “1H.“ Hmfim ~ >Hm 0 V 8 S H a mu. 3 Wm . mH “N 11 mm om mu mm 0 >1 om 37 Table 3. Amino acid composition of proteins studied compared to published values (expressed as 8/168 N) Amino Whole Casein a -Casein B-Lactoglobulin acid This Tfiis This residue Study (1) Study (2) Study (2) Lys 7.8 7.1 4.4 8.9 9.4 11.0 His 3.3 2.6 2.6 3.4 . 2.7 1.6 Arg 3.8 3.7 3.8 4.6 2.3 2.7 Asp 6.2 5.6 8.2 4.0 10.3 9.9 Thr 3.8 3.8 2.3 2.5 4.6 4.6 Ser 3.9 5.2 5.3 6.9 3.8 3.5 Glu 21.2 20.2 11.4 15 8 16.0 14.0 Pro 10.0 10.3 8 l 8.2 4.4 4.4 Gly 1.4 1.6 3 l 2." 0.9 1.0 Ala 2.5 2.4 2 4 3 2 5.5 5.7 1/2 Cys - 0.3 - - 1.8 2.9 Val 6.8 6.3 7 4 5.4 5.8 5.7 Met 2.4 2.9 2.1 3.2 3.0* 3.0 Ile 5.0 5.7 6 0 6.2 ".7 6.5 Leu 9.0 8.7 12.3 9.5 13.9 14.3 Tyr 5.4 5.7 10.4 8.1 3.9 3.7 Phe 5.0 5.2 7 3 5.8 3.8 3.4 Try 2.5* 2.5 2.5* 1.8 2.1* 2.1 TOTAL N 15.4 14.1 15.2 (1) Block and Weiss (1956) (2) Dayhoff (1972). * Value was est1mated. .moamemm awououa NH you Aummu :Hu0%scwa oomv mums: oucmnhomnm Hmuou mm commondxm mozam> ummwfip ucmumcumasm <09 mnu soum pmuumuunsm mm3 xcman machmm 38 00.0m 00.00 00.0m 00.00 0N.mm 00.0N 00.0w 00.00 0N.0N 0m.wm 0m.om 0m.0N 0.00 00.0w 0N.0N 0m.wm 0N.qm 00.nm mm.q~ 00.0w 00.0w 00.NN 00.0w 00.0w 00.0w 0.0m 00.5N 00.0w mm.0m 5N.Hm 00.mm NN.0N 00.0N Nq.mN 00.0w 00.HN 05.0w 00.NN H.0N u cwummuocmm 00.0w 00.0N 00.0w 00.0m 00.NN 0m.mm 00.0m mq.qm mm.HN 0m.NN 0m.mm 0m.NN 0.0m 00.0 00.0w 00.H~ 0w.mm 00.NN 00.NN 00.HN 00.N~ 00.0H 00.0H 00.0H 0m.0m n.0N :Hummuocmm N0.NH 0N.mH 00.0H 00.0H 00.0H 00.0H aq.qH 0m.mH No.0 n.0H H0.0H 00.0 0.0N 00.0 00.0 0~.0 00.0 mn.~a 00.HH mm.HH ma.HH 0H.0 00.0 <0.n 00.5 0.0 00.H 0N.H mm.H m0.H 0m.H m0.H 00.H mm.H 0m.H mm.a 00.H 00.H 0.0 cflmaom omooaao mmooaaw umoz Houucou mmOUSHo mmoosHU umo: Houucou omoodao omoosao umm: Houucou sz Nm + NH + Mm + .5 + Nm + .5 + 053 ummm ummm umom awo: ummm ummm umowfia m aHowMUI a cfiHsnoawOuomgl 0 awommo oaonz mmucwEummpu unonuwz 0cm cuHB mCMQuoua waE 00 umowwp cwumouocmalcfimaoa mo mumm .0 maan 39 cflummuocmmucHuwouocmaucwmama mp Gwommo mHOLB mo coaumowwp mo mumm A10 mz_h zozpwmwzn 0: 0m 0m 0H 3838 Nm + :m: a. “5830 5 + Sm: 0 Eu: 4 1 50555 oz 0 \. u .\ .m munwwm 0m 0m 0m 0: SlINfl EDNVSHOSHV 40 cfiummhocmmncflumohocmaucHmama ma Gwommoums mo coaumowflp mo mumm .q muswflm sz mzzh zo_pmmwla 0: 0m 0m 0H 0 o mmooano mm + 44m: AV .8830 NH + in: 0 0H ._. n u u n n n u u n mum 0.0a 00.0 0.0H Nq.0 0.0a Nq.0 H.0H mm.0 wq.m mH< q.m m0.0 0.0 00.0 m.m 00.0 0.H H0.0 qq.H haw . u u u u n u . H0.0H oum 5.0 43.H u.m “0.0 H.m 00.0 3.N om.o NN.HN 5H0 0.0H 0H.H m.NN mm.H m.HN 00.H 0.0m 00.H 0m.m uomupse 0.0 00.0 H.m 00.0 0.0 mm.0 0.0 mm.0 0N.0 am< m.mm mm.H H.0m 0H.H N.Nq 00.H 0.00 m0.N mm.m wu< 0.NH Nq.0 0.0a 00.0 «.mm 00.0 ¢.mm mm.0 0N.m mam «.mm 0m.H 0.mm qm.m 0.00 0H.m N.mq 0m.m mm.n mug . w o w . ummww w umowflm Hmuou mapflmmu oAmv puma H0 ofisv 00mm 00 oAmv n 0 oA\V n mamofi 0Hom omOUSHw wmoonfiw 000mm: Hopucou numuomna ocHE¢ gm + 0660 NH + 0600 .0 maan moHaEmm cfiommo mHOLB mo ummwwp swumouocmancwmaom CH mwflom ocflEm moum (I. 44» I..- I» Hgiiu 43 .ummwflp w 00H\w mm pmmmouaxo modam> .ommwae w oon\w .Hmuou Hmowuouoonu ozu mo udoo umm mm 06mmmuaxo modam>o 3 mm pommmumxo modam> .3 00 How Hum 20 CH Cwouonm m>Humc pmmmaoupzn wwo - - - - - - - - - es 0.0a 00.0 0.HH 00.0 H.0H 00.0 0.0a 00.0 00.0 mH< H.m 0H.o m.m 0H.o 0.0 om.o m.k mm.o mo.m Raw u n u u u u u n 00.0 oum 0.0 00.0 0.0 00.0 0.0 00.0 0.0 00.0 0q.HH 5H0 0.0a 0H.H 0.0a 00.H \.ma d0.H 0.0a 00.0 00.0 000-058 m.m 00.0 8.0 km.o N.a 0~.o N.3 mm.o mm.m am< 3.3m Hm.H w.N3 m0.H H.0k 40.N 0.04 ma.~ Hm.m 00¢ N.0N 00.0 0.00 00.0 0.00 00.0 0.00 00.0 00.0 was 0.00 m0.H 0.00 00.0 0.00 00.0 0.H0 00.0 00.0 m04 A00 hammno A30 ommwfla A00 ommwna A00 ummwaa H6060 6204666 o o o n o n «Hmow 000m omoonaw omOUSHw poummm Houucou uuouooSH OGHE< $0 + 0mmm 0H + 0800 moHaEmm wammon a mo umowwp cflummuocmansflmaom cw mpwom ocHEm noun .0 mHQmH .umowfip w 00H\w mw 00mmouaxm mosfim> .Hmuou Hmofiuouomsu mnu mo ucoo Mom mm 00mmmuaxo moDHm>o n .0mmwnc w oon\w mm wommmuaxo mosam> .5 00 How H0: Z0 CH afiououa m>wuma pouhaouvhs vwo q.mm wq.o ~.oa mm.o m.mq mm.o H.Ho mo.H om.H mmo w N.mH mo.H m.oN HH.H o.mm oq.a o.wm mm.H mq.m mH< 0.x mo.o o.HH OH.o o.HH OH.o m.MH mH.o qm.o mam - u - u u n n . mq.¢ cum m.m mm.o n.m om.o w.m mm.o ¢.o No.H mo.oH :Hu o.oH cm.H q.na mq.H w.qm mo.m N.wm om.N mm.w ummuyna N.oH mo.H o.m mm.o m.o oo.H q.“ 05.0 mm.oa am< ©.Hm m”.o w.mm om.o ~.om Rm.H 0.05 No.H mm.m wu< H.mm mo.o o.qN mo.o o.q~ No.0 m.om ¢w.o NN.N ma: H.mm mm.~ o.mm ma.m H.Nq «m.m m.nm om.m om.o qu UANV ummwflo oAxv nummwflm ofixv ummwfla oAxV nummwflo mamuou mnwflmmu HmUH uflom mmousaw mmoosaw‘ Umummm HOMucou aumuomsa OGHE< Nm + uwm: Na + uwmm mmaqawm CHHSQOHwOuumanm mo ummwww Cwummuocmanchamm CH mvwom OGHEm mmum .n magma 47 .Hmuou Hmuwumuomzu msu mo puma uma mm 0mmmmuaxm mmsam>o .ummmwv w ooa\w mm 0mmmmuaxm mmaam>n .ummwflu w coa\w mm 0mmmmuaxm mmdam> .2 mm How a0: 20 Cw cflmuoum m>wumc Ummxaouwxn ww0m Nd om mm ma 0 n ma mN 0 Que 0m 0m ma ma 0H m n n @ mfim m 0m mm mm mm A Q 0 n 304 mm mm OH 00 00 NH 9 a n mHH 00 mq mm N0 «0 «H «N m m um: mm mm NH mm mm am a n n Hm> u n u u s u n u n Hmmause mm qq mH mm nq m Rm oq NN wu< mm mm om mm u n 00 mm a mam 00 mm «m H0 mm ma 00 NH m mmq IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII o\b IIIIIIIIIIIIIIIllllllllllllllllllllll mmoosaw mmoodaw umm: mmousaw mmoosfiw umm: mmoonaw mmoudaw umm: mdvfimmn N0 + Na + N0 + Na + N0 + Na + Uflum .:I;mmmm ummm ;:.ua .Iwmwme-s>:wme,a;arzn. ummmir ummml. - ocfla< GHHDQOHwOUUmAum cwmmmoumc awmmmo mHonz mmvwom ocHEm mmum 0mmmmamu hafimowumEANcm CH mmmmmuomm .0 maan 49 .qummua muGSOEm Hmuou unmmmuamu mmmmxucmuma CH mmsaw> .w>H0mmH cu wmaflmm mxmma mcwpmmnmawcomhzeo .mumwmaouvzn 000m msu mo mmosu Eonm mmsam> ummwwv msu wcwuomuundm >0 wwdwfiumummo .mflmwaouvmn 0000 umumw wmum>oomu wwwom ocHEm Hmuou we ooa\wa Ca 0mmmmumxm muwam .DI 00.00 00.00 00.00 00.00 00.00 04.00 00.00 00.0H 00<000 00.0 00.H 00.0 00.H 00.H 0N.H 00.0 00.H 0H0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0000 +.m;0 “0.0 00.0 00.0 00.0 00.0 00.0 NH.0 00.0 :00 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 mHH 00.0 NH.N 00.0 00.0 00.0 00.0 00.0 N0.Hm%0 +.umz Hm.m 00.0 00.0 00.0 0H.0 00.0 40.0 NR.H 00> A00.00 A0N.Nv A00.00 A00.00 0000 00.H 00.0 0N.H 00.0 00.0 00.0 N0.H 00.0 000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000 nmswwwmm ummwfia nmswflmmm umwwwm mmsuwmmm uwmwwm @mdwflwmm ummwwm omew cflasnonouomq|m Gammmou 0 Cwmmmo maosz wwm maonz . mawmuoum xaflfi vmummHuCD mo ummwwv awuwmuoamm nmwmawm m Ca mcowuumum mdvflmmu 0cm ummwwv mSu mo acmwumgaoo .m manme m DISCUSSION Static vs Qynamic System A static digest is represented‘by a system where the substrate and enzymes are mixed and allowed to react to completion in a flask or beaker. Aliquots from the reaction mixture was extracted for analysis. The advan- tages are l) ease and simplicity, 2) low cost and 3) not limited by equipment resource. The problems as outlined by Mauron (1955) are the possibilities of 1) end product inhibition and 2) the action of chymtrypsin as a trans— peptidase. A dynamic digest is represented by a system simulating the intestinal tract where the end products of enzymatic digestion are continually removed from the site of reaction. Mauron (1955) and Ford and Salter (1966) used different devices to achieve a dynamic digestion. The advantages appear to be that the system 1) more closely resembles the functioning of the digestive tract, it 2) avoids the disadvantages of a static digest, and 3) offers the possibility of having a nearly complete digestion. However, the disadvantages are 1) increased sophistication of instruments and manipulation, 2) higher cost, 3) a probable restriction on the number of samples 50 51 digested at one time, and 4) the current impossibility of removing only small peptides and/or free amino acids. At the beginning of this study, a hollow fiber beaker "Osmolyzer" manufactured by Dow Chemicals was tested in a dynamic mode. Because the results of the study supported discontinuing its use, the findings have been relegated to incorporation into the Appendix. Arai gt El- (1975) claims to have induced a trans- peptidase activity with chymotrypsin by using a 30% solution of oligopeptides. The driving force of the reaction is the extreme scarcity of free water. Diehl (1975) proposed that hydrophobic aggregation of the peptides represents a more realistic mechanism for the apparent increase in peptide weight. Therefore, at the 1% concentration of peptides in this study, there is no indication that chymotrypsin acts as a transpeptidase. This study has shown that free amino acids do inhibit the proteolytic action of pancreatin on whole protein substrate. Because inhibition is competitive in nature, the adverse effects can be partially overcome by lengthening the time of reaction and/or by increasing the enzyme concentration. Since pancreatin loses two-thirds of its activity after 4 h at 37 C, the latter option was selected. Akeson and Stahmann (1965) digested whole egg and casein with 15 mg of pepsin and 40 mg of pancreatin/g 52 protein. The amount of free amino acids recovered for whole egg and casein was 24 mg and 16.5 mg/lOO mg diges- tion mixture, respectively. In his dynamic system, Mauron (1970) digested whole egg with 12.5 mg of pepsin and 25 mg pancreatin/g protein. The amount of free amino acids recovered was 22 mg/lOO mg of digestion mixture. Under the static conditions of this study, whole egg and whole casein were digested by 83 mg of pepsin and two additions of 100 mg portions of pancreatin/ g protein. Twenty-five mg and 22 mg of free amino acids were released/100 mg digestion mixture from the whole egg and whole casein digestion, respectively. Hence, the dynamic system used by Mauron (1970) fails to release any higher percentage of free amino acids than a static system. This study's high levels of enzyme compared to the amounts used by Akeson and Stahmann (1965) also failed to release significantly more amounts of free amino acids. However, the advantage to using the higher levels of enzyme is the subsequent breakdown of large peptides to smaller ones (see Table 2 and Figure 2). This further digestion aids greatly in simplifying the preparative steps necessary for analysis by an amino acid analyzer. Akeson and Stahmann's (1965) procedure required deproteiniza- tion with picric acid (which destroyed tryptophan), further separation of the large peptides by gel filtration, and then evaporation in order to obtain a prOper concentration. 53 Although researchers (e.g. Sheffner et al., 1956; Akeson and Stahmann, 1964; and Mauron, 1970) seemed quite hesitant about using large dosages of enzyme, in 3139 enzyme secretions, according to Nixon and Mawer (1970a), range from 66 to 280 mg/g ingested protein. The argument, Menden and Cremer (1966), favoring low enzyme additions in Vitro was the speculation that large amounts of enzyme without a substrate would undergo autolysis, thus pro- ducing a digestion unrepresentative of the enzyme's be- havior in the presence of a substrate. From the results of this study, increasing the enzyme concentrations does not initiate an increase in autolytic behavior. Rather, the increase in enzyme produces an increase in response (absorptivity or free amino acids) which is proportional to its concentration and the very slight proteolytic autolysis which occurs is uniform irrespective of the amount of enzyme. Proteolytic Digestion of Milk Proteins Strucutral Differences Using the amount of free amino acid released as a gauge of digestibility, B-lactoglobulin, aS-casein, and whole casein ranked from most to least. Probably, their structural difference attribute to the observed differences. Mellander (1955) recognized that an enzymatic digest of casein left a considerable residue of high molecular 54 weight peptides which he attributed to phosphorylated residues. McKenzie (1967) noted that aS-casein's dis- ordered configuration would allow for rapid hydrolysis. Tam and Whitaker (1972) found the initial rates of peptic hydrolysis on aS-casein was twice that of whole casein. Fox and Guiney (1973) observed that aS-casein was quite susceptible to proteolysis, but that its susceptibility decreased in heterogenous aggregated systems. Tanford gt gt. (1962) indicated B-lactoglobulin has a compact native structure but can refold following denaturation and produce a large number of a-helicies. Effect of Treatments Reduction in amounts of amino acids released in the heat treated proteins may result from 1) blocking, 2) destruction, and 3) tertiary rebonding leading to segments resistant to hydrolysis. Many investigators (e.g. Bender, 1972; Hurrell and Carpenter, 1974; and Groux, 1974) cite the amino acids with charged side groups as potential sources of interaction with each other or with compounds such as reducing sugars. Lien and Nawar (1974a, b) identified some thermal decomposition products of alanine, leucine, isoleucine, and valine. The effects of the treatments on the release of amino acids in the casein digest agree with those of Erbersodobler (1969), Menden and Cremer (1966) and Rao and Rao (1972). The principal exception would be 55 increased amounts of isoleucine and leucine released compared to significant decreases observed by others. The difference may be due to differences in type of enzymes used. It would be worthwhile noting from Figures 6, 7, and 8 that the more easily digested proteins of B-lacto- globlin and aS-casein experience degradation at a more extreme degree than does whole casein. Because all of the free amino acids released in the 8-1actoglobulin and aS-casein digests decreased systematically with increases in added glucose, it can be deduced that structural changes are significant and produce a molecular configura- tion which isresistant to hydrolysis. Mills and Creamer (1974) observed that heating of B-lactoglobulin solutions caused marked irreversible changes to both the orientation of the proteins backbone as well as the amino acid side chains. Nakanishi and Wada (1974) identified some de- composition products of heated B-lactoglobulin. Evidence of increasing amounts of furfurals, Samuelsson and Nielson (1970), and protein bound pigments, Groux (1974), would support the concept of the formation of large peptide segments more resistant to further enzymatic hydrolysis. Reducing sugar can condense with the functional side groups of lysine and arginine preventing trypsin from hydrolyzing the adjacent bonds. Pepsin Pancreatin Digest Index Sheffner gt gt. (1956) originally formulated the mathematical concept for analyzing an tg ytttg digest while he termed the Pepsin Digest Ratio (PDR). This index compared favorably to the protein's biological value. Akeson and Stahmann (1964) used both pepsin and pancreatin in.theirdigestion mixture and termed their index Pepsin Pancreatin Digest (PPD) which followed the basic formula of Sheffner gt gt. (1956). Mauron's (1970) only altera- tion to the PPD was the incorporation of a dialysis apparatus, hence the index became Pepsin Pancreatin Dialysis Digest (PPDD). Both the PPD and PPDD show excellent correlation to the biological value of proteins studied. The formula for the basic index considers separately the free amino acids and the rest of the digest, i.e., the residue. Both are compared to a similar digest of whole egg. Sheffner gt gt. (1956) gives a full descrip- tion of the procedure. The data required to calculate PPD indicies are given in Table 9. Values calculated for casein, aS-casein, and B-lactoglobulin were 78, 75 and 95, respectively, where egg protein was assumed to be 96.5. Published biological values for casein range from 69 (Block and Mitchell, 1946), 73 (Mitchell and Block, 1946), to 78 (Rippon, 1959). The PPD indicies for the treated protein digests were not calculated due to Mauron's (1970) observation 57 that such a determination does not reveal a close approxima- tion to animal assays. One explanation suggests that acid used to hydrolyze the digest in order to obtain the composition of the residue fraction releases amino acids that are not released tg ttyg. Simply using the free amino acids present in the digestion mixture to determine an index does not appear to yield a representative index. After incubating intestinal contents of a milk meal, Nixon and Mawer (1970b) suggested that the rate of libera- tion of glycine, proline, and the dicarboxylic acids was so slow that it was necessary to postulate absorption as peptides or hydrolysis at the mucosal surface. 100 % AMINO ACIDS RELEASED U1 25 Figure 6. 58 MET\ PHE .1 LEU .4I""‘ MET PHE LYS ‘\ TRY ‘ HISo—— LYS HIS VAL ILE ILE VAL CONTROL HEAT HEAT + 1% HEAT + 5% GLUCOSE GLUCOSE Effects of various treatments of whole casein on the enzymatic release of amino acids 100 75 5: AMINO ACIDS RELEASED % 25 Figure 7. 59 LYS MET - ARG ‘ TRY \“..Il~ PHE TRY *-0PHE LEU MET I..~ LYS ARG HISce—r LEU HIS VAL ILE 0* + aVAL 41LE CONTROL HEAT HEAT + 1% HEAT + 5% GLUCOSE GLUCOSE Effects of various treatments of aS-casein on the enzymatic release of amino acids 60 100 75 ARG LEU MET TRY LYS VAL \ PHE ‘ ILE ‘~\\ LEU TRY HIS _. VAL,ARG,MET LYS 0—4IPHE 25 . HIS,ILE z AMINO ACIDS RELEASED U1 0 CONTROL HEAT HEAT + 1% HEAT + 5% GLUCOSE GLUCOSE Figure 8. Effects of various treatments of B-lactoglobulin on the enzymatic release of amino acids CONCLUSION Differences can be noted between the three selected milk proteins and the effects of treatments on those pro- {#3 teins by using this particular method of tg Vitro enzymatic I digestion. The authenticity of the data obtained from such a digest must be verified by animal studies. Once accomplished, this sensitive technique could offer in- j valuable application in the area of the development of protein foods. Investigators interested in l) the effects of various processes on the protein's nutritive value, 2) the quality of new sources of protein for human food, and 3) the optimization of the contribution of several protein foods to the total diet would find the thVitro enzymatic digest an economical, rapid method for monitoring the protein's nutritive value. 61 BIBLIOGRAPHY Abrahamsson, L., Forsum, E., Hambraeus, L. 1974. Nutritional evaluation of emergency food mixtures based on wheat supplemented by dif- ferent protein concentrates. Nutrition Reports International, 9(2):l69. Adibi, S.A. and Gray, S.J. 1967. Intestinal absorption of essential amino acids in man. Gastroenterology, 5tz837. Adrian, J. and Frangne, R. 1973. Maillard reaction VIII. Role of premelanoidins in nitrogen digestibility in vivo and proteolysis in vitro. Annales de la Nitrition et de ITAlimentation, 21:111. Akeson, W.A. and Stahmann, M.A. 1964. A pepsin pancreatin digest index of protein quality evaluation. J. 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The course of digestion of different food proteins in the rat. Fractionation of the nitrogen in intestinal contenst. Brit. J. Nutrition, 22:483. APPENDIX Reagents for ninhydrin test 1. Citrate buffer contains 4.3 g citrLc acid and 8.7 g Na-citrate 2H20 in 250 ml, adjust to pH 5. 2. Add 400 mg SnC12-2H20 in 250 ml citrate buffer (0.2 M) pH 5. 3. Add the above solution to 250 m1 methyl Cellosolve containing 10 g dissolved ninhydrin. Amino Acids Used for Pancreatin Inhibitor Study Amino acid mg1mt glycine 5.28 L-arginine 5.27 DL—serine 5.22 L-alanine 6.26 L-histidine 5.68 L-lysine 5.40 DL-valine 4.61 DL-phenylalanine 4.84 DL—methionine 5.49 TOTAL 48.04 70 71 Dynamic Digest System A.Wkaker Osmolyzer” with cellulose acetate, hOUOWIibmm, produced by Dow Chemicals, with a molecular Imiym mmOHEOf 200 was tested for use in an tg vitro enzymatic digestion. The potential advantage of this apparatus was its capacity to remove free amino acids «I continuously from the site of digestion. Acxmcentration solution of CaCl2 was passed dupugh Hmatubing to remove the water and free amino acids:fixm1the beaker by reverse osmosis. To conduct iiuther analysis of gross-amino acids by the ninhydrin test, it was necessary to remove the Ca (11) from the This was best accomplished by precipitating The super- diffusate. the Ca (11) as Ca(0H)2 with NaOH at pH 12-14. natant was readjusted to pH 5 by the addition of formic acid and pH 5 phosphate buffer. Table A lists the other reagents tested: Applicability of various reagents Table A. for the removal of Ca (II) Reagent Observations NHa-oxylate Good precipitory agent HNaZPO4 Poor precipitory agent NaBO3 Poor precipitory agent Na-oxylate Highly insoluble NaZSOQ Highly insoluble 72 The quickest method to detect the presence of ixndividual amino acids proved to be by dansylation (Hartley, 1970). It was not essential to remove the Ca (11) in order to visualize the spots. The one re- quirenmnt was to adjust the reaction mixture to pH 10-11. The NaOH treatment, previously described, caused an excessive DNS-OH reaction. Table B lists other pro- cedures employed to yield clear, dansylated amino acid spots on polyamide sheets: Table B. Effectiveness of various chemicals for the qualitation of amino acids in a Ca (11) solution by dansylation Reagent Observations Triethylamine Produced too large DNS—OH spot NaHCO3 Precipitated out with Ca (11) NHa-oxylate Produced too large DNS-NH2 spot Acetone + Produced a drop in pH NaHCO 3 Pryidine Raised pH to only 8.4 Acetone + Produced the best plate, triethylamine reaction conditions were at pH 10 2 h at 40 C The amino acids lysine, glycine, histidine, phenylalanine, alanine, valine, and tyrosine were observed to pass through the tubing. Not all 18 amino acids were tested, e.g. the carboxylic acid group. The major draw- back to the hollow fiber device was the poor rate of transfer of the amino acids and preferential transfer 73 rates. With a mixture of the water soluble amino acids in the beaker, two concentrations of CaCl2 were passed through the tubing. The percentage of total amino acids and water recovered was determined by the ninhydrin test after 50 min. Using a 7% CaCl2 solution, 2.4% amino acids in 7% of the water was recovered. With a 15% CaCl2 solution, 17.5% amino acids in 25% of the water was recovered. Qualitiative determinations by dansylation showed the recovery was proportional to the amino acids remaining. The poor rate of recovery of the amino acids merited its discontinuation as a viable mode for a dynamic digestion. Chloride ions passed into the beaker from the tubing network, which, potentially, could inhibit the activity of trypsin, a component of pancreatin. Enzymatic Dtgestion of the Dried, Whole Egngolids Sample Amino aCid b d residue Literaturea Theoretical DigestC % LyS 6.6 7.1 2.7 37.8 His 2.3 2.8 0.8 28.6 Arg 6.4 6.6 3.1 47.0 Asp 9.7 9.8 0.2 2.0 Th Ser 7.1 7.4 Glu 12.6 12,6 0.5 3 9 Pro 3.7 4.7 - - Met Ile Leu Tyr Phe Try TOTAL 74 3.1 2.8 — - 5.5 5.0 0.6 12.0 2.3 1.9 0.3 15.8 6.1 6.8 1.8 26.5 3.3 2.4 1.6 66 6 5.0 5.3 0.9 17.0 8.2 9.0 4.8 53.4 4.0 4.1 2.7 65 8 4.8 5.3 3.1 58.5 1.9 1.9 1.1 58.0 97.4 100.0 25 2 aValues stated as g/l6 g N after acid hydrolysis, Mauron (1970). bValues used in this study, stated as g/100 g total amino acids recovered by acid hydrolysis. CDigested with pepsin-(2X)pancreatin used in this study, values expressed as g/100 g digest. dValues expressed as percent of the theoretical total. Il' l '5 I'll Ill |||| Ill