Nutrient Bioavailability - American Chemical Society

12 Nutrient Bioavailability D. B. THOMPSON and J. W. ERDMAN, JR.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 24, 2016 | http://pubs.acs.org Publication Date: March 6, 1981 | doi: 10.1021/bk-1981-0147.ch012

Department of Food Science, University of Illinois, Urbana, IL 61801

Functional properties, as considered from the point of view of the food processor, are those properties which impart desired physical characteristics to the products. For example, foam stability would be an important functional property to a producer of whipped toppings. From the perspective of the consumer, this functional property may contribute to satisfaction and lead to repeat purchases. Increasingly, however, the consumer is concerned with the nutritional impact of his purchases. Thus, the food industry will be pressured to expand its concept of functional properties to include nutritional considerations. Martinez (1) has recently suggested that functionality be defined as "the set of properties that contributes to the desired color, flavor, texture, and nutritive value of a product". In order to assess the nutritive value of a product, one must evaluate more than the presence of the nutrients; one must evaluate nutrient bioavailability. Bioavailability can be defined as the extent to which a chemically present nutrient can be utilized by animals (humans) (2). B i o a v a i l a b i l i t y can be influenced d i r e c t l y or i n d i r e c t l y by many p h y s i o l o g i c a l , p a t h o l o g i c a l , chemical, n u t r i t i o n a l , and processing c o n d i t i o n s . Discussion in t h i s chapter w i l l be l i m i t e d to unit food processing e f f e c t s upon the b i o a v a i l a b i l i t y of n u t r i e n t s from plant protein foods. The b i o a v a i l a b i l i t y of amino a c i d s , carbohydrates, l i p i d s , vitamins and minerals from processed foods w i l l be s e l e c t i v e l y reviewed. Amino Acids Experimental Procedures. Nutrient b i o a v a i l a b i l i t y is a complex subject when applied to protein components. In f a c t , i t i s an error to speak of the b i o a v a i l a b i l i t y of the p r o t e i n ; rather one must consider the a v a i l a b i l i t y of the i n d i v i d u a l amino acids which make up the p r o t e i n . Amino acid a v a i l a b i l i t y implies s u f f i c i e n t d i g e s t i o n of the p r o t e i n in the i n t e s t i n e s to allow absorption into the t i s s u e . Then, in the case where 0097-6156/81/0147-0243$08.00/0 © 1981 American Chemical Society

Cherry; Protein Functionality in Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


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an amino acid i s l i m i t i n g , there should be no r e s t r i c t i o n to u t i l i z a t i o n of that amino a c i d . When these c r i t e r i a are s a t i s f i e d , the amino acid may be termed a v a i l a b l e . One may chemically analyze the constituent amino acids in a p r o t e i n , but t h i s analysis i s best viewed as an estimate of the p o t e n t i a l n u t r i t i o n a l value of the p r o t e i n . If d i g e s t i o n , a b s o r p t i o n , and u t i l i z a t i o n are unhindered, then the p o t e n t i a l may be r e a l i z e d . However, these processes are u s u a l l y i n e f f i c i e n t to some degree, leading to the important c o n s i d e r a t i o n of amino acid a v a i l a b i l i t y . The subject i s complicated in that the extent of the i n e f f i c i e n c y varies with the animal, other d i e t a r y components, and the treatment to which the protein may have been subjected. The i n f l u e n c e of processing on amino acid a v a i l a b i l i t y may be profound, e s p e c i a l l y under extreme c o n d i t i o n s . Processing can increase amino acid a v a i l a b i l i t y , often by increasing d i g e s t i b i l i t y or i n a c t i v a t i n g a n t i n u t r i t i o n a l f a c t o r s . Processing may also serve to decrease amino acid a v a i l a b i l i t y . Protein h y d r o l y s i s in 6N HC1 and subsequent analysis to determine amino acids (except tryptophan, which is acid l a b i l e ) c h e m i c a l l y present i s a f i r s t step in protein q u a l i t y evalu ation. The chemical score and the EAA index represent attempts to use t h i s information to chemically estimate n u t r i t i o n a l q u a l i t y of p r o t e i n ; t h e i r obvious l i m i t a t i o n is t h e i r disregard for amino acid a v a i l a b i l i t y . The chemical score i s obtained by evaluating the percent of the l i m i t i n g amino acid in comparison to that amino acid in whole egg protein {3). The EAA index i s the geometric mean of the r a t i o s of each of the e s s e n t i a l amino acids to those amino acids occurring in whole egg (4). Since l y s i n e is often the l i m i t i n g amino acid Tn plant p r o t e i n and i s e s p e c i a l l y s e n s i t i v e to processing damage, t h i s amino acid has received much a t t e n t i o n . Carpenter {$) has devised a method of determining "available lysine" using dinitrofluorobenzene (DNFB). On the assumption that a f r e e ε - a m i no group w i l l represent an undamaged, unmodified l y s i n e r e s i d u e , Carpenter measured a v a i l a b l e l y s i n e by the extent to which DNFB r e a c t s with f r e e ε - a m i n o groups. While t h i s approach has considerable merit in assessing damage to l y s i n e , the word a v a i l a b l e i s open to question. Some of Carpenter's " a v a i l a b l e lysine" may not be absorbed ( 6 ) ; thus the residue would not be t r u l y a v a i l a b l e to the organism in v i v o . Further attempts to evaluate n u t r i t i o n a l q u a l i t y of p r o t e i n have been made both in vivo and in v i t r o . In vivo measurements have been performed with humans, experimental animals (often r a t s ) , and with lower organisms such as Tetrahymena p y r i f o r m i s . Experimental design is simpler using the lower organisms; at the same time, the strength of the conclusions that may be extended to humans is lessened. There has been much d i s c u s s i o n regarding the type of u t i l i z a t i o n model to be determined. In the U . S . , the l e g a l l y acceptable


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model i s the protein e f f i c i e n c y r a t i o n PER

=


=

(PER):

weight gain protein consumed

which measures u t i l i z a t i o n BV

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for

growth.

B i o l o g i c a l value

(BV):

nitrogen retained nitrogen absorbed

is often determined, accounting for nitrogen u t i l i z a t i o n for both maintenance and growth, and allowing use of either human or animal s u b j e c t s . As with the chemical score approach, a serious l i m i t a t i o n of these feeding studies is that the only amino acid for which a v a i l a b i l i t y can be determined i s the l i m i t i n g amino acid in the t e s t d i e t . This l i m i t i n g amino acid may or may not be of p r a c t i c a l importance in a normal mixed diet. In v i t r o attempts have been made to simulate d i g e s t i o n in order to measure the d i g e s t i b i l i t y of a protein by d i g e s t i v e enzymes. The pepsin-pancreatin digest index i s j u s t one example of an enzyme system which may be applied to a t e s t p r o t e i n p r i o r to analysis of released amino a c i d s . These d i g e s t i b i l i t y values are appropriate to the extent that they simulate the d i g e s t i v e t r a c t , the subtle workings of which are not e a s i l y i m i t a t e d . The greatest value of t h i s approach is not for accuracy but for a s e n s i t i v e method of monitoring changes in the protein due to treatment ( 7 ) . In a d d i t i o n , in vitro nay often provide clues regarding the reason protein q u a l i t y is improved or reduced. A hybrid approach combining in v i t r o and in vivo methods is the everted sac technique of WTTson and Wiseman {S) which has been used to estimate both d i g e s t i b i l i t y and absorption. This method uses the rat small i n t e s t i n e outside of the animal.

w o r k

3

Processes A f f e c t i n g N u t r i t i o n a l Value of Amino A c i d s . Many unit processing operations" have been shown to i n f l u e n c e protein quality. Heat, commonly applied to protein to increase d i g e s t i b i l i t y , can not only make the protein i n t r i n s i c a l l y more d i g e s t i b l e but can i n a c t i v a t e i n h i b i t o r s to protein d i g e s t i o n . Denaturation of protein is thought to be the mechanism for the increased d i g e s t i b i l i t y and the i n a c t i v a t i o n of i n h i b i t o r y substances. Excessive heat can lead to decreased d i g e s t i b i l i t y and absorption, r e s u l t i n g in reduced amino acid a v a i l a b i l i t y . A number of mechanisms i n v o l v i n g c r o s s - l i n k formation have been suggested to account for these changes. The M a i l l a r d r e a c t i o n involves the complexation of a reducing sugar and a free amino group of the p r o t e i n , g e n e r a l l y the ε - a m i n o group of l y s i n e . The A madori compound which forms in the preliminary stages of



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M a i l l a r d browning w i l l y i e l d the constituent amino acid upon 6N HC1 h y d r o l y s i s and a n a l y s i s , but the Amadori amino acid moiety may be unavailable to the rat (9) since i n t e s t i n a l h y d r o l y s i s is less harsh than 6N HC1 h y d r o l y s i s . Thus, we have a c l e a r d i s t i n c t i o n between p o t e n t i a l n u t r i t i o n a l value and actual availability. Another type of c r o s s - l i n k formation r e s u l t i n g from excessive heat is the formation of i s o p e p t i d e s , in which the ε - a m i n o group of l y s i n e i s involved in a new peptide bond, probably by d i s p l a c i n g the amide group of asparagine or glutamine. The l y s i n e moiety of free ε - N - ( β - a s p a r t y l ) - l y s i n e was found to be only very s l i g h t l y a v a i l a b l e to the rat (10), whereas the l y s i n e moiety of free e ( Y - L - g l u t a m y l ) - L - l y s i n e was determined to be almost completely a v a i l a b l e (10,11). It has been hypothesized that the isopeptide i t s e l f may be absorbed and subsequently hydrolyzed by a kidney ε - l y s i n e deacylase, and that only after h y d r o l y s i s could the l y s i n e be u t i l i z e d . It now appears more l i k e l y that gut enzymes may be r e s p o n s i b l e for p a r t i a l isopeptide h y d r o l y s i s (12). Waibel and Carpenter (JJ_) suggest that h y d r o l y s i s may occur within the i n t e s t i n a l wall after absorption of the i s o p e p t i d e . However, these studies on absorption of the free e ( Y - L - g l u t a m y l ) - L l y s i n e may not be relevant to c r o s s - l i n k s in an overheated protein. It may be concluded that c r o s s - l i n k i n g due to isopeptide formation i s probably responsible for decreased o v e r a l l d i g e s t i b i l i t y of the protein (6,12); thus, i f the d i g e s t i v e enzymes are not able to release the smaller peptides, then the question of a v a i l a b i l i t y of the isopeptides per se i s beside the p o i n t . Ford (13) points out that the rate of d i g e s t i b i l i t y of isopeptide l i n k s may preclude the maximum a v a i l a b i l i t y of the l y s i n e as a r e s u l t of the l y s i n e entering the system too l a t e to be e f f e c t i v e l y u t i l i z e d by the t i s s u e . Thus, the l y s i n e in these linkages would be at least p a r t l y u n a v a i l a b l e . Other workers have shown that small q u a n t i t i e s of isopeptides are found in the urine (14). Excessive heat can cause destruction of amino acid residues. The amino acid most s u s c e p t i b l e to d i r e c t heat destruction is cystine. Although not an e s s e n t i a l amino a c i d , c y s t i n e does have a sparing e f f e c t on the d i e t a r y requirement f o r methionine. As a r e s u l t , cystine d e s t r u c t i o n can be n u t r i t i o n a l l y important. In a d d i t i o n , many vegetable proteins are l i m i t i n g in the s u l f u r amino a c i d s . Cystine destruction would be p a r t i c u l a r l y harmful for these p r o t e i n s . Excessive heat due to r o a s t i n g can cause racemization of amino acid residues (15). Most amino acids are only a v a i l a b l e in the L form. Consequently, complete racemization could be equivalent to a 50% decrease in a v a i l a b i l i t y for the residues affected. A l k a l i treatment of protein can be used to increase



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s o l u b i l i z a t i o n , for the production of i s o l a t e s , and in the spinning process for t e x t u r i z a t i o n . This treatment has important b e n e f i t s in terms of some f u n c t i o n a l p r o p e r t i e s , but i t must be applied with c o n s i d e r a t i o n for amino acid availability. A l k a l i treatment, i f e x c e s s i v e , can r e s u l t in amino acid d e s t r u c t i o n , e s p e c i a l l y to c y s t i n e . It can a l s o cause racemization. C r o s s - l i n k s may form by r e a c t i o n with dehydroalanine, a degradation product of c y s t i n e or s e r i n e . Lysinoalanine (LAL) and lanthionine are two of the p o s s i b l e r e a c t i o n products. Considerable attention has been given to LAL, which has been shown to be nephrotoxic to the r a t (16), the only species so far shown to e x h i b i t s e n s i t i v i t y to LAL. De Groot et a l . (17) found that f r e e LAL is more nephrotoxic than protein-bound LAL. The d i f f e r e n c e may be a b e n e f i c i a l consequence of reduced d i g e s t i b i l i t y of protein c o n t a i n i n g LAL. Woodard and Short (16) found both protein-bound LAL and free LAL to be nephrotoxic. The disagreeing r e s u l t s from these two l a b o r a t o r i e s have not been s a t i s f a c t o r i l y explained, but Struthers et al_. (18) described data which suggests that d i f f e r e n c e s in the s t r a i n s of rats may be r e s p o n s i b l e . Karayiannis et aj_. (19) claimed that d i f f e r e n c e s in d i e t s , not the s t r a i n oT~rats, were responsible for v a r i a b l e n e p h r o t o x i c i t y r e s u l t i n g from feeding protein-bound LAL. They suggested that other n u t r i t i o n a l f a c t o r s modulated development of kidney l e s i o n s , and that the balance of e s s e n t i a l amino acids in the d i e t may have been an important f a c t o r . Gould and MacGregor (20) and Struthers et aj_. (18) have also reviewed the controversy regarding b i o l o g i c a l e f f e c t s of LAL. Not only do species respond d i f f e r e n t l y to LAL, but the extent of LAL formation v a r i e s with the type of protein treated (21). Damage due to a l k a l i treatment is induced and/or exacerbated by heat. The a v a i l a b i l i t y of amino acids from protein may be affected by the refinement of the types of protein molecules. In some protein sources storage protein may be i s o l a t e d from non-storage p r o t e i n , or a c i d - p r e c i p i t a t e d proteins i s o l a t e d from whey p r o t e i n s . In the preparation of c l a s s i c a l protein i s o l a t e s by s o l u b i l i z a t i o n and acid p r e c i p i t a t i o n , the amino a c i d make-up of the iso-late d i f f e r s from that of the e x t r a c t . Consequently, c o n s i d e r a t i o n must be given to the amino acids chemically present in a r e f i n e d p r o t e i n . Another method of protein treatment involves enzymatic modification. Enzymes per se may be added or the protein may be subjected to the m i c r o b i a l enzymes produced from fermentation. There is l i t t l e evidence to i n d i c a t e that the amino acid a v a i l a b i l i t y is influenced by these treatments, although one might expect b e n e f i t s from increased digestibility. The p l a s t e i n r e a c t i o n is a type of enzymatic m o d i f i c a t i o n , in which protein h y d r o l y s i s is followed by resynthesis of peptide bonds. Evidence i n d i c a t e s that amino

American Chemfeaf Society Library 1155 18* t t . N. w.

Cherry; Protein Functionality in Foods ACS Symposium Series; American Chemical Washington, D. G. Society: 20Û36Washington, DC, 1981.


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acid a v a i l a b i l i t y is not influenced by t h i s treatment ( 2 2 ) . A newer method of protein treatment is chemical m o d i f i c a t i o n of the amino acid r e s i d u e s , e s p e c i a l l y l y s i n e . A c y l a t i o n of the ε-amino group has profound e f f e c t s upon the p r o t e i n structure due to e l i m i n a t i o n of the c a t i o n i c amino group. S u c c i n y l a t i o n has even more profound e f f e c t s since the amino group is e s s e n t i a l l y replaced by an anionic carboxyl group. Functional properties have been shown to be g r e a t l y improved by a c y l a t i o n ( 2 3 ) , but l y s i n e a v a i l a b i l i t y after m o d i f i c a t i o n is a matter of concern. Bjarnason and Carpenter ( 1 2 ) have shown free ε - Ν - a c e t y l l y s i n e to have only 5 0 % o f the a v a i l a b i l i t y of l y s i n e and f r e e ε - Ν - p r o p i o n y l l y s i n e to have no lysine a v a i l a b i l i t y . Since L e c l e r c and Benoiton ( 2 4 ) have shown N-acetyl l y s i n e but not N-propionyl l y s i n e tô~~be a substrate for rat kidney ε - l y s i n e deacylase, these a v a i l a b i l i t y studies suggest that the a c t i v i t y of ε - l y s i n e deacylase in the kidney may determine the a v a i l a b i l i t y of l y s i n e in acylated protein. ε - Ν - s u c c i n y l l y s i n e has not been evaluated as a substrate for ε - l y s i n e deacylase. Finot et ( 1 0 ) found no h y d r o l y s i s of ε - Ν - l e u c y l - l y s i n e nor - N - p â T m i t y l - l y s i n e in homogenates of rat i n t e s t i n a l mucosa, l i v e r , or kidney. Thus, simple ε - Ν - a c y l - l y s i n e with acyl groups greater than two carbons would not appear to be a v a i l a b l e . However, Padayatti and Van Kley ( 2 5 ) have shown that an ε - p e p t i d a s e is capable of hydrolyzing ε - Ν - ( « - 3 $ ρ 3 ^ 1 ) - l y s i n e and ε - N - l e u c y l - l y s i n e ; thus these d e r i v a t i v e s are probably a v a i l a b l e . Since ε - Ν - s u c c i n y l l y s i n e is s t r u c t u r a l l y s i m i l a r to both ε - N - b u t y r y l - l y s i n e and ε - Ν - ^ - a s p a r t y l ) - l y s i n e , i t is d i f f i c u l t to p r e d i c t the a v a i l a b i l i t y of the l y s i n e in ε - Ν - s u c c i n y l l y s i n e . ε-Ν-propionyl l y s i n e from lactalbumin is somewhat a v a i l a b l e (Table I ) , suggesting that the longer action of gut enzymes may r e s u l t in some s i g n i f i c a n t h y d r o l y s i s ( 1 2 ) . ε

TABLE I EFFECT OF ACYLATION ON LYSINE

AVAILABILITY

1

Estimated % A c t i v i t y ^ ε - a c e t y l - L - 1 y s i ne ε-propionyl-L-lysine Bovine plasma albumin Acetyl BPA Lactalbumin Formyl lactalbumin Propionyl ( 4 0 % ) lactalbumin Propionyl ( 9 5 % ) lactalbumin

5ϋ* 0 85 67 122 77 107 43^

From ( J 2 J . Compared to L - l y s i n e hydrochloride = 1 0 0 .



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Conclusions and a p p l i c a t i o n of conclusions regarding amino acid a v a i l a b i l i t y must be kept in p e r s p e c t i v e . A change in a v a i l a b i l i t y may have l i t t l e p r a c t i c a l e f f e c t i f the protein in question is not a s i g n i f i c a n t d i e t a r y source of p r o t e i n . If the l i m i t i n g amino acid in the protein is not the l i m i t i n g amino acid in the d i e t , a change in a v a i l a b i l i t y could be e q u a l l y unimportant. In f a c t , i f proteins are fed in complementary f a s h i o n , the l i m i t i n g amino acid in the mixed d i e t may be d i f f e r e n t than the l i m i t i n g amino acid in e i t h e r protein source fed as the sole source. In a d d i t i o n , the l i m i t i n g amino acid depends upon the animal used in the t e s t . Thus, one must c a r e f u l l y consider the p r a c t i c a l relevance of amino acid a v a i l a b i l i t y determinations.

Processing of Soy foods and Amino Acid A v a i l a b i l i t y . Soybeans are an e x c e l l e n t vegetable source of l y s i n e . The f i r s t l i m i t i n g amino acid is methionine. Despite i t s g e n e r a l l y f a v o r a b l e amino acid p r o f i l e , unheated soy protein is an undesirable source of amino acids due to poor d i g e s t i b i l i t y . This poor d i g e s t i b i l i t y has often been ascribed to t r y p s i n i n h i b i t o r s (TI), which are r e a d i l y heat-denatured p r o t e i n s . However, Liener (26) found no c o r r e l a t i o n between the level of TI a c t i v i t y and PER. His in v i t r o studies showed that only about 40% of the d i f f e r e n c e in d i g e s t i b i l i t y between unheated and o p t i m a l l y heated soy extract was due to TI a c t i v i t y ; the r e s t was a t t r i b u t a b l e to the p r o t e i n being in the undenatured state. Even f u l l y denatured legumenous protein is incompletely attacked by d i g e s t i v e enzymes, and the a v a i l a b i l i t y of s u l f u r amino acids may be quite low. Heat treatment is the preferred method of reducing t r y p s i n i n h i b i t o r a c t i v i t y and increasing d i g e s t i b i l i t y . Several i n v e s t i g a t o r s have demonstrated a d e l i c a t e balance between the heat treatment necessary to increase d i g e s t i b i l i t y and reduce t r y p s i n i n h i b i t o r a c t i v i t y and the excessive heat which can reduce d i g e s t i b i l i t y and with i t the amino acid a v a i l a b i l i t y . Hackler et al_. (27) i n v e s t i g a t e d the e f f e c t of heating soy milk at 1 2 1 ° C ~ T o r varying p e r i o d s . They showed that the optimum time of heating was 4 to 8 minutes. Longer times decreased the PER. Wing and Alexander (28) found that a maximum PER for soybean meal was achieved with microwave heating for 2.5 to 3.0 minutes; the PER decreased d r a m a t i c a l l y at 5.0 minutes heating. I r i a r t e and Barnes (29) showed that cystine d e s t r u c t i o n by heat made t h i s amino acid~Tirst l i m i t i n g for the r a t . However, c y s t i n e supplementation did not return the n u t r i t i o n a l value to the optimum l e v e l . These workers were unable to determine the second l i m i t i n g amino acid and could not rule out the p o s s i b l e development of a t o x i c i t y f a c t o r . T a i r a (30) determined that only cystine was destroyed under heating conditions commonly



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employed in soybean p r o c e s s i n g . He also found (31) that c y s t i n e d e s t r u c t i o n was independent of moisture content. K e l l o r (32) points out that overheating adversely a f f e c t s palatability. Precise control of heat for d e s o l v e n t i z i n g and in subsequent processing steps is needed. Stott and Smith (33) showed no negative e f f e c t s to l y s i n e or methionine f o r a t y p i c a l commercial d e s o l v e n t i z i n g - t o a s t i n g process. Soy protein is r e f i n e d in production of concentrates and isolates. The process of refinement of these two products is considerably d i f f e r e n t (34). Concentrates are produced by leaching defatted f l a k e s or f l o u r to remove most of the sugars and ash, leaving a protein and polysaccharide mixture of about 70% p r o t e i n . I s o l a t e s are c l a s s i c a l l y prepared by a l k a l i e x t r a c t i o n of defatted meal, followed by p r e c i p i t a t i o n of p r o t e i n curd at the i s o e l e c t r i c p o i n t . Washing of the curd produces an i s o l a t e of at l e a s t 90% p r o t e i n . Although i s o l a t e s are a higher percentage of p r o t e i n , r e l a t i v e l y more of the o r i g i n a l seed protein w i l l be found in concentrates. I s o e l e c t r i c p r e c i p i t a t i o n r e s u l t s in loss of whey protein as well as undesirable non-protein components. G i l l b e r g (35) has shown that the cystine composition of the i s o l a t e is lower than for the meal extract because n o n - p r e c i p i t a t e d whey protein has a r e l a t i v e l y larger proportion of the t o t a l s u l f u r amino a c i d s . This observation is r e i n f o r c e d by M a t t i l (36), who showed that the range for amino acid composition was for several commercial concentrates as to commercial i s o l a t e s . He found methionine to be lower for i s o l a t e s than for concentrates. M a t t i l (36) emphasized the d i f f e r e n c e in amino acid composition within the two c l a s s i f i c a t i o n s for commercial products. He pointed out that some of the d i f f e r e n c e s are d e l i b e r a t e ; a high s o l u b i l i t y i s o l a t e could well have a d i f f e r e n t amino acid composition than an i s o l a t e with low solubility. M a t t i l evaluated i s o l a t e PER's, which ranged from 1.1 to 1.75. These values were in apparent c o r r e l a t i o n with methionine analyses (from 0.9 to 1.2 g/16 g Ν ) . Heat treatment of commercial i s o l a t e s was studied by Cogan et a l . (37) who found that heat could be applied e i t h e r before or l i f t e r i s o l a t e formation without a f f e c t i n g PER. However, Longenecker et al_. (.38) point out apparent marked d i f f e r e n c e s in manufacturing processes regarding heat treatment of commercial concentrates and i s o l a t e s . They found that in most cases heat treatment had apparently not been optimal and that r e s u l t i n g losses in n u t r i t i o n a l value could be quite l a r g e . Longenecker and Lo (39) analyzed the e f f e c t of excessive heat treatment on soy i s o l a t e and found only a 14% decrease in methionine by chemical a n a l y s i s but a PER drop from 2.10 to 1.13. Using a plasma amino acid technique, they concluded that methionine a v a i l a b i l i t y was reduced 46% by the excess heat, a f i n d i n g which more reasonably explains the large PER drop.

c o m p a r e d

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A l k a l i treatment is used to s o l u b i l i z e and i s o l a t e p r o t e i n s , to improve foaming and emulsifying p r o p e r t i e s , and to obtain protein s o l u t i o n s s u i t a b l e for spinning f i b e r s (17). De Groot and Slump (40) studied the influence of a l k a l i on soy p r o t e i n i s o l a t e s , monitoring the production of l y s i n o a l a n i n e and changes in amino acid content. They found that above pH 10, treatment at 4 0 ° C for 4 hours r e s u l t e d in decreased c y s t i n e and increased LAL (Figure l a ) . They also found that at pH 12.2 f o r 4 hours, l y s i n e and c y s t i n e content s t e a d i l y decreased with increasing temperatures from 20° to 8 0 ° C , and LAL content increased d r a m a t i c a l l y . At pH 12.2 and 4 0 ° C they reported that the greatest loss in c y s t i n e and increase in LAL occurred in the f i r s t hour (Figure l b ) . Thus they concluded that exposure of soy protein i s o l a t e at pH 12.2 for only a short time would destroy some c y s t i n e and decrease the n u t r i t i v e value. Decreased threonine a v a i l a b i l i t y (not shown) in the pH 1 2 . 2 , 4 0 ° C , 4 hour samples was apparent from methionine supplementation s t u d i e s . The authors speculate that racemization of threonine could be r e s p o n s i b l e . Pepsin-pancreatin d i g e s t i o n of the pH 1 2 . 2 , 4 0 ° C , 4 hour samples showed decreased d i g e s t i b i l i t y . Absorption was measured with everted i n t e s t i n a l sacs and was seen to vary according to the amino a c i d . Because LAL and nitrogen u t i l i z a t i o n showed a negative c o r r e l a t i o n , these authors suggested measurement of LAL as an estimation of a l k a l i n e processing damage in soy. In l a t e r work, de Groot et jal_. (17) suggested that c y s t i n e i s a s e n s i t i v e i n d i c a t o r of "Tosses in n u t r i t i v e v a l u e , while safety considerations would be r e l a t e d to LAL content. Sternberg et aj_. (41) analyzed numerous foods and food ingredients f o r LAL. They found LAL to be commonly o c c u r r i n g at greater than 100 yg/g protein in cooked (but not raw) f r a n k f u r t e r , chicken and egg white. Of food ingredients t e s t e d , commercial soy protein i s o l a t e samples varied from 0-370 y g/g p r o t e i n . These data i n d i c a t e that while processing can increase LAL l e v e l s in soy, LAL l e v e l s in soy products may be comparable to l e v e l s encountered in everyday cooked foods. Gould and MacGregor (20) concluded that human intake of LAL i s low compared to l e v e l s needed to cause kidney damage in the rat. They point out, however, that c e r t a i n infant milk formulas have been shown to have LAL at 200-600 ppm, and that t h i s food may c o n s t i t u t e 100% of the d i e t . They see cause for concern, considering the unknown r e l a t i v e s e n s i t i v i t y of man to LAL. F i n l e y and Kohler (42) noted that oxygen i s apparently required for the formation of high l e v e l s of LAL in soy i s o l a t e and sodium c a s e i n a t e . They were able to control LAL formation by l i m i t i n g the amount of oxygen e i t h e r by mixing under nitrogen or by the addition of reducing agents. Even with severe a l k a l i treatment ( 6 0 C , 8 hours, 0.1 Ν NaOH) the LAL e



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content was 0.003 g LAL/16 g Ν or below when mixed under nitrogen after addition of b i s u l f i t e , b i s u l f i d e , or c y s t e i n e . Soy protein may be t e x t u r i z e d in two ways: through f i b e r spinning or thermoplastic e x t r u s i o n . Fiber spinning involves treatment of soy i s o l a t e with a l k a l i , s p i n n i n g , and coagulation in a c i d , whereas extrusion subjects soy f l o u r to high temperature and pressure, r e l e a s i n g the thermoplastic material through a die (34). Van Beek et a^. (43) examined spun soy i s o l a t e and found that as the sole" p r o t e i n source to rats i t is f u l l y capable of supporting normal growth when methionine i s added. Earlier, Bressani et _al_. (44) tested spun soy products + egg albumin + wheat gluten and T ô u n d the protein s l i g h t l y less adequate than milk at lower nitrogen l e v e l s , but equivalent to milk at higher levels. These experiments suggest that spun soy i s o l a t e may be an e x c e l l e n t p r o t e i n source. Kies and Fox (45) examined e x t r u s i o n - t e x t u r i z e d soy p r o t e i n in comparison to beef p r o t e i n . At 8.0 g nitrogen per day they found that both products met the human adult male requirements ( i . e . , they gave a p o s i t i v e nitrogen b a l a n c e ) , whereas at 4.0 g nitrogen per day beef was superior to the extended soy ( i . e . , i t had a less negative nitrogen b a l a n c e ) . Methionine supplementation p a r t i a l l y overcame the d i f f e r e n c e at the lower l e v e l . This study shows the adequacy of p r o t e i n q u a l i t y of e x t r u s i o n - t e x t u r e d soy protein for humans fed adequate q u a n t i t i e s of p r o t e i n . The authors point out that applying the 6.25 conversion f a c t o r to both proteins might well put the soy p r o t e i n at a disadvantage i f the true conversion f a c t o r for soy i s lower, as has been suggested. Mustakas et ^1_. ( 46) evaluated the e f f e c t s of e x t r u d e r - p r o c e s s i n g on n u t r i t i o n a l q u a l i t y , f l a v o r , and s t a b i l i t y of the product in an attempt to describe extruder c o n d i t i o n s which would be acceptable in a l l three r e s p e c t s . Urease a c t i v i t y was used as an estimation of t r y p s i n i n h i b i t o r a c t i v i t y ; thus the area between the two urease curves in Figure 2 i n d i c a t e s processing c o n d i t i o n s which s t r i k e a balance between too much and too l i t t l e heat treatment, showing optimal nutritional quality. Using the f l a v o r and peroxide value isograms, processing c o n d i t i o n s may be chosen such that acceptable f l a v o r and s t a b i l i t y may also be achieved. Recently, Jeunink and Cheftel (47) have attempted to i l l u m i n a t e the mechanism of extrusion t e x t u r i z a t i o n . They a t t r i b u t e d the low product s o l u b i l i t y to new d i s u l f i d e bonds and non-covalent i n t e r a c t i o n s , but they could not r u l e out a c o n t r i b u t i o n due to formation of isopeptide l i n k s . The small observed increase in unavailable l y s i n e would be consistent with isopeptide formation. A c y l a t i o n of soy protein has been suggested as a way of improving i t s f u n c t i o n a l p r o p e r t i e s . Franzen and K i n s e l l a (18 ) studied both s u c c i n y l a t i o n and a c e t y l a t i o n of soy p r o t e i n .


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lysine

50|

lysine Ζ Ο SÉ>40j-


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401 20Ί 1 pH 12.2 , 40°c]

4hrs,40°C

Nutrient Bioavailability - American Chemical Society

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