Enzymatic Modification of Milk Flavor - ACS Symposium Series (ACS

4 Enzymatic Modification of Milk Flavor

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 12, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch004

W. F. SHIPE Department of Food Science, Cornell University, Ithaca, N.Y. 14853

Until r e c e n t l y enzymatic a c t i o n i n m i l k has been regarded as undesirable. So most of the a t t e n t i o n has been given to inactivating the enzymes, r a t h e r than u s i n g them. But research in the l a s t few years i n d i c a t e that enzymatic m o d i f i c a t i o n of m i l k may be f e a s i b l e i n some cases. Enzymes reported to a f f e c t m i l k f l a v o r are e i t h e r o x i d a t i v e or h y d r o l y t i c . Table I shows the o x i d a t i v e enzymes and t h e i r r e a l or p o t e n t i a l e f f e c t . Table I.

O x i d a t i v e Enzymes Reported

1. 2. 3.

"Oleinase" Xanthine oxidase Lactoperoxidase

4. 5. 6.

S u l f h y d r y l oxidase Hexose oxidases Lactose dehydrogenase

to Have F l a v o r Impact

Enhances oxidation? Enhances oxidation? Enhances o x i d a t i o n (nonenzymatically). Reduces cooked f l a v o r . Increases a c i d i t y . Increases a c i d i t y .

In 1931 Kende (1) reported that an enzyme which he c a l l e d o l e i n a s e was i n v o l v e d in o x i d i z e d f l a v o r development. T h i s conc l u s i o n was based p r i m a r i l y on the f a c t that o x i d i z e d f l a v o r development was i n h i b i t e d by heat treatment. I t was assumed that the i n h i b i t i o n was a r e s u l t of the i n a c t i v a t i o n of " o l e i n a s e " . I t was not recognized at that time that heat treatment tends to inhibit o x i d a t i o n , presumably by producing a c t i v e s u l f h y d r y l groups. The existence of an o l e i n a s e was never confirmed but people are still searching f o r it. I t has been reported that xanthine oxidase c o n t r i b u t e s to spontaneous o x i d i z e d f l a v o r development (2). Evidence has been presented which shows a good c o r r e l a t i o n between xanthine oxidase activity and production of TBA r e a c t i v e components. Furthermore, thermal and chemical treatments known to i n a c t i v a t e xanthine oxidase a l s o i n h i b i t e d the development of o x i d i z e d f l a v o r . However, there is no d i r e c t evidence to show that m i l k contains a

57 Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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s u b s t r a t e that i s acted on by xanthine oxidase. Recently, i t has been p o s t u l a t e d (3) that xanthine oxidase produces superoxide anion i n milk which may undergo non-enzymatic dismutation t o form s i n g l e t oxygen which could c a t a l y z e l i p i d o x i d a t i o n . Furthermore, i t was p o s t u l a t e d that the superoxide dismutase could i n h i b i t t h i s o x i d a t i o n by c a t a l y z i n g the conversion of superoxide anion to t r i p l e t oxygen and hydrogen peroxide. Superoxide anion has a l s o been detected (4) i n m i l k serum that had been exposed to f l u orescent l i g h t . Regardless of the source of the superoxide anion, superoxide dismutase could p o s s i b l y c o n t r i b u t e to the m i l k a n t i oxidant system. However, more research i s needed to determine i f e i t h e r xanthine oxidase or superoxide dismutase have any impact on m i l k f l a v o r . I n c i d e n t a l l y , i f xanthine oxidase does c a t a l y z e o x i d a t i o n the enzyme can be i n a c t i v a t e d by heating to 94°C f o l l o w i n g normal p a s t e u r i z a t i o n . M i l k p l a n t s with steam i n j e c t i o n - v a c uum p r o c e s s i n g equipment can e a s i l y produce xanthine oxidase f r e e m i l k with only a s l i g h t cooked f l a v o r . E r i k s s o n (5) reported that l a c t o p e r o x i d a s e can c a t a l y z e o x i d a t i o n by v i r t u a l of the heme i r o n that i t c o n t a i n s . In other words, i t i s a non-enzymatic c a t a l y s t . I mention t h i s because there may be other enzymes that have s i g n i f i c a n t non-enzymatic effects. Consequently, when we use enzymes we must not ignore p o s s i b l e non-enzymatic e f f e c t s of enzymes. In 1967 Kiermeier and Petz (6) i n Germany i s o l a t e d a s u l f h y d r y l oxidase from milk. T h i s enzyme o x i d i z e s s u l f h y d r y l groups to d i s u l p h i d e s . S u l f h y d r y l oxidase s t u d i e s have a l s o been conducted at North C a r o l i n a State and C o r n e l l . Table I I shows the e f f e c t of t h i s enzyme on s u l f h y d r y l groups and cooked f l a v o r as reported by Siewright (7). As w i l l be noted t h i s enzyme can s i g n i f i c a n t l y lower cooked f l a v o r . I t has been suggested that s u l f h y d r y l oxidase might be used to e l i m i n a t e the strong cooked f l a vor i n m i l k that i s produced by u l t r a h i g h temperature (UHT) Table I I . E f f e c t of S u l f h y d r y l Oxidase on S u l f h y d r y l Concentrat i o n and Cooked F l a v o r . Sulfhydryl Flavor Sample Level Intensity Control 0.18 3.4 1% enzyme s o l u t i o n 0.09 1.6 2% enzyme s o l u t i o n 0.05 1.1 a

Êxpressed as o p t i c a l d e n s i t y . Ellman - DTNB Method Flavor intensity: 1 = s l i g h t -> 4 = very strong cooked treatment. However, one needs to determine whether the s u l f h y d r y l oxidase treatment w i l l make the m i l k more s u s c e p t i b l e to o x i d a t i o n by e l i m i n a t i n g s u l f h y d r y l groups which have anti-oxygenic properties. Since UHT treatment i s u s u a l l y given to prolong storage l i f e and the r i s k of o x i d i z e d f l a v o r reaching the d e t e c t a b l e l e v e l i n c r e a s e s with age, one should avoid lowering the o x i d a t i v e s t a b i l i t y of the UHT milk. To preserve the anti-oxygenic b e n e f i t

Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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and s t i l l e l i m i n a t e cooked f l a v o r , one might wait to add the s u l f h y d r y l oxidase u n t i l a f t e r storage, i . e . j u s t before consumption. Rand (8) has s t u d i e d two methods f o r conversion of l a c t o s e i n m i l k to a c i d . In one method he f i r s t hydrolyzed the l a c t o s e to glucose and galactose w i t h l a c t a s e and then converted the hexoses to the corresponding a l d o b i o n i c a c i d s with hexose oxidases. In the second method he used l a c t o s e dehydrogenase to produce l a c t o b i o n i c d e l t a l a c t o n e which subsequently hydrolyzed to l a c t o b i o n i c a c i d . He reported that t h i s l a t t e r method was l e s s e f f i c i e n t than the f i r s t method. The r a t e of a c i d production with hexose o x i dases was increased by the a d d i t i o n of c a t a l a s e and IÔ^ which s u p p l i e d oxygen f o r regenerating the reduced enzymes. The enzymatic process changes the f l a v o r and a l s o gets r i d of the l a c t o s e which i s an a d d i t i o n a l advantage f o r those with l a c t o s e i n t o l e r ance. Glucose o x i d a s e - c a t a l a s e systems a l s o have p o t e n t i a l value as oxygen scavengers i n products such as m i l k powders, dry i c e cream mixes, c o f f e e and a c t i v e dry yeast (9). Since these products do not c o n t a i n enough moisture f o r the enzymatic r e a c t i o n i t i s necessary to provide water. T h i s problem has been solved by p u t t i n g glucose, glucose o x i d a s e - c a t a l a s e and s u f f i c i e n t moisture i n a p l a s t i c envelope. T h i s envelope should r e a d i l y transmit gaseous oxygen but should r e s t r i c t moisture passage. I t has been reported that these enzymatic oxygen scavenger packets produced lower l e v e l s of r e s i d u a l oxygen than vacuum treatment followed by f l u s h i n g with n i t r o g e n . Although these packages have had only l i m i t e d comm e r c i a l use they do i l l u s t r a t e an ingenious approach to f l a v o r control. Table I I I l i s t s the h y d r o l y t i c enzymes that have a f l a v o r Table I I I . 1. 2. 3. 4. 5. 6.

H y d r o l y t i c Enzymes with F l a v o r Impact

Lipase Lactase M i l k proteases Phospholipase C Phospholipase D Trypsin

Causes h y d r o l y t i c r a n c i d i t y . Increases sweetness. Increases b i t t e r n e s s Inhibits oxidation. Inhibits oxidation. Inhibits oxidation.

impact. Of course, l i p a s e heads the l i s t because i t i s the natur a l l y o c c u r r i n g enzymes that appears to have the g r e a t e s t f l a v o r impact. In f a c t two survey taken i n the past year i n New York C i t y i n d i c a t e s that h y d r o l y t i c r a n c i d i t y was the p r i n c i p a l o f f f l a v o r i n commercial m i l k samples. Therefore, the dairymen needs to know how to c o n t r o l l i p o l y s i s . C o n t r o l i n t h i s case p r i m a r i l y i n v o l v e s keeping the enzyme away from the s u b s t r a t e . At the time of m i l k s e c r e t i o n the f a t globule i s f a i r l y w e l l p r o t e c t e d from the l i p a s e by the f a t g l o b u l e membrane. T h i s membrane can be a l t e r e d by thermal or mechanical abuse. Obviously the a l t e r a t i o n i s increased by i n c r e a s i n g the abuse. The mere c o o l i n g of some


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milk from 37 to 5°C i s enough to cause l i p o l y s i s presumably by l i p a s e l o c a t e d i n the membrane. Heating cooled m i l k up to 30°C and r e c o o l i n g to 5°C causes even greater i n c r e a s e s i n l i p o l y s i s . The e f f e c t of t h i s s o - c a l l e d temperature a c t i v a t i o n i s shown i n Table IV. Apparently, these temperature changes causes d i s r u p t i o n Table IV.

E f f e c t of " A c t i v a t i o n

Temperature °C A c i d degree v a l u e s Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 12, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch004

As determined

3

15 1.0

20 1.3

11

Temperature on L i p a s e A c t i o n 25 2.8

30 3.9

35 2.2

40 0.9

by method of Thomas, N i e l s e n and Olson (10).

of the f a t globule membrane so as to i n c r e a s e the contact between f a t and l i p a s e . Mechanical a g i t a t i o n such as simple s t i r r i n g or pumping can a l s o expose the f a t to the enzyme. The magnitude of t h i s e f f e c t i s dependent on the temperature as i s shown i n Table V. Obviously, the e f f e c t of mechanical abuse i s l e s s i f the m i l k i s kept c o o l . Vigorous a g i t a t i o n i n a Waring Blendor or homogen i z e r produces even more d r a s t i c i n c r e a s e s . The increase produced Table V.

E f f e c t of Temperature and Shaking on L i p o l y s i s Temperature 5°C 26°C % Increase i n l i p o l y s i s on shaking 17 370 As determined

by Kurkovsky and Sharp (11).

by homogenization exceeds the amount of i n c r e a s e of f a t g l o b u l e surface area. Apparently, p a r t of the i n c r e a s e i s due to changes i n the nature of the f a t g l o b u l e membrane. F o r t u n a t e l y , most of the l i p a s e i n m i l k i s i n a c t i v a t e d by p a s t e u r i z a t i o n which i s done e i t h e r j u s t before or a f t e r homogenization. Most l i p o l y s i s can be avoided i f m i l k i s not subjected to thermal or mechanical abuse and i s p a s t e u r i z e d soon a f t e r production. However, bulk handling of m i l k and t r a n s p o r t i n g i t over as much as 300 or 400 m i l e s makes i t d i f f i c u l t to f o l l o w simple c o n t r o l measures. Furthermore, p a s t e u r i z a t i o n does not completely i n a c t i v a t e a l l of the l i p a s e (12) and i n the case of homogenized milk, t h i s can cause f l a v o r problems. Consequently, other methods of c o n t r o l l i n g l i p o l y s i s are being sought. Lipase can be i n a c t i v a t e d by adding HÔ^, but such a d d i t i o n s are not l e g a l . Exposure to f l u o r e s c e n t l i g h t can i n h i b i t l i p o l y s i s but t h i s would be a questionable procedure because of the r i s k of producing l i g h t induced o f f - f l a v o r s . Shahani and Chandan (13) observed that non-fat m i l k s o l i d s i n h i b i t e d p u r i f i e d m i l k l i p a s e , presumably by absorbing on the surface of the substrate. The a d d i t i o n of s o l i d s would a l s o i n c r e a s e the n u t r i t i v e value of the product and thus would have a double b e n e f i t . A d d i t i o n a l evidence on the e f f e c t of a d d i t i v e s was revealed i n a recent study (14) on the e f f e c t of l e c i t h i n , c a s e i n and t r y p s i n on l i p o l y s i s (Table V I ) . In t h i s study, emulsions of 2% m i l k f a t were s t a b i l i z e d with 0.5% c a s e i n + 0 . 5 % bovine l e c i t h i n ,


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Table VI.

I n t e r a c t i o n of L i p a s e and T r y p s i n i n D i f f e r e n t Emulsions of M i l k Fat Free F a t t y Acids (peg/ml) Additives Without T r y p s i n With T r y p s i n 1

1

,

1

8

Lecithin h h L e c i t h i n + Casein 3.40 2.50^ Casein 7.44° 15.51 Casein + L e c i t h i n 0.65* 0.87 Numbers with same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) a


1

or 0.5% of each. The emulsions were then incubated a t 21°C f o r 60 min. with l i p a s e or l i p a s e and t r y p s i n . Casein enhanced l i p o l y s i s when added alone or a f t e r l e c i t h i n , but i t appeared to i n h i b i t when added p r i o r t o l e c i t h i n . Since m i l k l i p a s e i s known to a s s o c i a t e with c a s e i n , the c a s e i n absorbed on the f a t may " a t t r a c t " l i p a s e to the f a t surface. The a d d i t i o n of l e c i t h i n a f t e r the c a s e i n may block the absorption of the l i p a s e . The f a c t that t r y p s i n enhances l i p o l y s i s when c a s e i n alone was used, suggest that the unhydrolyzed c a s e i n does p r o t e c t the f a t to some extent. Regardless of the explanation of these r e s u l t s , they c l e a r l y i l l u s t r a t e that l i p o l y s i s i s a f f e c t e d by the m a t e r i a l that i s absorbed on the f a t globule. So f a r I have been t a l k i n g about i n a c t i v a t i o n or i n h i b i t i n g l i p o l y s i s but i n many products f r e e f a t t y a c i d s are d e s i r a b l e . Even good f r e s h m i l k contains some f r e e f a t t y a c i d s . But by comp a r i s o n good b u t t e r contains 6 to 7 times more f r e e f a t t y a c i d s , cheddar cheese 4 t o 5 times more and blue cheese 50 t o 100 times more. Lipases play a dual r o l e i n blue cheese f l a v o r production by r e l e a s i n g v o l a t i l e f a t t y a c i d s , some o f which c o n t r i b u t e d i r e c t l y t o f l a v o r and some serve as precursors of methyl ketones. The methyl ketones are major c o n t r i b u t o r s to blue cheese f l a v o r . Richardson and Nelson (15) have i n d i c a t e d that cheddar cheeses were o r g a n o l e p t i c a l l y p r e f e r r e d when g a s t r i c l i p a s e preparations were included i n t h e i r manufacture. So l i p o l y s i s i s o f t e n d e s i r able i f we can c o n t r o l the amount and kinds of f a t t y a c i d s r e leased. M i l k and p a n c r e a t i c l i p a s e s are f a i r l y n o n - s p e c i f i c i . e . the r a t i o of f a t t y a c i d s i n the hydrolysate and the unhydrolyzed f a t a r e e s s e n t i a l l y the same. By c o n t r a s t p r e - g a s t r i c ( i . e . o r a l ) l i p a s e s from calves and k i d s and fungal sources p r e f e r e n t i a l l y r e l e a s e high proportions of short chain f a t t y a c i d s . But even the fungal enzymes e x h i b i t considerable d i f f e r e n c e s as i s shown i n Table V I I . Arnold et a l . (16) have reviewed the d i f f e r e n c e s i n s p e c i f i c i t y of v a r i o u s l i p a s e s and how d i f f e r e n t l i p a s e s can be used t o produce l i p o l y z e d milk f a t with s p e c i f i c f l a v o r characteristics. They pointed out that s e l e c t i v e l y hydrolyzed f a t s are added to a v a r i e t y of foods i n c l u d i n g c e r e a l products, margarines, s a l a d d r e s s i n g s , c o f f e e whiteners, soups, snack foods and c o n f e c t i o n a r y products such as m i l k chocolates, creams and


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R e l a t i v e Release of Free F a t t y Acids by Two Fungal Lipases Source of L i p a s e Short chain Total free f a t t y acids fatty acids (μ Equiv.) (%) Pénicillium r o q u e f o r t i 38 110 2 Achromobacter 96


Table V I I .

fudges. Lactase i s the next h y d r o l y t i c enzyme on the l i s t but s i n c e t h i s t o p i c i s covered i n the next chapter, I w i l l only take time to p o i n t out a unique use of t h i s enzyme. A commercial company i s c u r r e n t l y producing packets of l a c t a s e which bears the f a n c i f u l name " L a c t i - A i d " . The consumer i s i n s t r u c t e d to add the L a c t Aid to a quart of m i l k and to s t o r e i t i n the r e f r i g e r a t o r f o r 24 hours. During t h i s time the l a c t o s e w i l l be hydrolyzed and the sweetness w i l l be i n c r e a s e d . C e r t a i n l y , the concept of having the consumer c a r r y out the enzymatic treatment i n c r e a s e s the pot e n t i a l use of enzymes. Since consumers are a l r e a d y u s i n g enzymatic meat t e n d e r i z e r s , home use of enzymes may become q u i t e commonplace. Perhaps, we should consider preparing enzyme packets for use by the farmers. For example, we might be able to e l i m i nate the l i p a s e problem by having the farmer add the r e q u i r e d amount of ^2®2 l i p a s e and then have him add s u f f i c i e n t c a t a l a s e to get r i d of the excess ^2^2' l a r i l y , app r o p r i a t e amounts of s u l f h y d r y l oxidase might be packaged f o r use i n bulk-dispensed m i l k such as i s used i n c a f e t e r i a s . The s u l f h y d r y l oxidase could be added f a r enough i n advance of s e r v i n g time to allow the enzyme to e l i m i n a t e cooked f l a v o r . t

0

i

n

a

c

t

i

v

a

t

e

t

n

e

s i m i

M i l k has been known to c o n t a i n proteases f o r some time, but there i s no d i r e c t evidence to i n d i c a t e that they c o n t r i b u t e to f l a v o r i n f l u i d milk. Some s c i e n t i s t s b e l i e v e that they c o n t r i b ute to the f l a v o r of raw m i l k cheeses. In f a c t , a recent r e p o r t (17) i n d i c a t e d that m i l k proteases survived p a s t e u r i z a t i o n and t h e r e f o r e , was a l s o important i n p a s t e u r i z e d products. S i m i l a r proteases from b a c t e r i a l o r i g i n are known to produce b i t t e r f l a v o r s , and perhaps i n some cases b i t t e r f l a v o r i n m i l k may be produced by them. T h i s might p a r t i c u l a r l y be true i n m i l k that has been stored f o r s e v e r a l days. The l a s t three h y d r o l y t i c enzymes on the l i s t have been shown to i n h i b i t o x i d a t i o n . The a c t i o n of these enzymes are of e s p e c i a l i n t e r e s t because they i l l u s t r a t e how r a t h e r l i m i t e d enzymatic a c t i o n can have a s i g n i f i c a n t f l a v o r impact. Furthermore, i t i s worth n o t i n g that these three enzymes c a t a l y z e three d i f f e r e n t h y d r o l y t i c r e a c t i o n s y e t a l l i n h i b i t o x i d a t i o n . T h i s immediately r a i s e s two questions: F i r s t , why should h y d r o l y s i s i n h i b i t oxidation? and second, do they have some common i n h i b i t o r y mechanism? Before t r y i n g to answer these two question, l e t s consider the i n d i v i d u a l enzymes, s t a r t i n g w i t h phospholipases C and D.



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I f phosphotidylcholine i s the s u b s t r a t e , phospholipase C y i e l d s a d i g l y c e r i d e plus phosphorycholine whereas D y i e l d s phosphatidic a c i d and c h o l i n e . Skukla and Tobias (18) claimed that the phosp h a t i d i c a c i d produced by phospholipase D was r e s p o n s i b l e f o r i t s anti-oxygenic e f f e c t . On the b a s i s of our r e s u l t s we have assumed that the anti-oxygenic e f f e c t of phospholipase C was due to a r e arrangement of one or more of the f a t globule membrane components. Presumably t h i s rearrangement decreases the s u s c e p t i b i l i t y of the l i p i d s to o x i d a t i o n by reducing the exposure of the substrate to pro-oxidants such as copper or i r o n . The i n h i b i t i o n of copper induced o x i d a t i o n by phospholipase C, as reported by Young (19), i s shown i n Table V I I I . Whereas, phospholipase C i n h i b i t s oxidat i o n , Chrisope and M a r s h a l l (20) reported that i t enhances l i p o l y sis. Therefore, t h i s enzyme should not be used i n raw m i l k unless Table V I I I .

3

E f f e c t of Phospholipase C A c t i o n on Development of TBA Reacting Components^ Cow Number

Sample I d e n t i f i c a t i o n Control Milk M i l k +0.05 ppm Copper M i l k + Copper + Enzyme

326 .02 .07 .03

362 .03 .09 .04

377 .04 .09 .04

Incubated f o r 1 hr at 37°C, followed by thermal i n a c t i v a t i o n and ^storage f o r 3 days at 5°C. TBA values expressed as absorbance at 532 nm. Samples w i t h TBA values greater than 0.04 u s u a l l y have detectable o x i d i z e d f l a v o r s . one

i s i n t e r e s t e d i n i n c r e a s i n g the f r e e f a t t y a c i d content. The use of t r y p s i n to i n h i b i t o x i d a t i o n i s perhaps the most i n d i r e c t method f o r c o n t r o l l i n g milk f l a v o r . Ever s i n c e the use of t r y p s i n f o r t h i s purpose was f i r s t reported (21) i n 1939, s c i e n t i s t s have been t r y i n g to e l u c i d a t e the mechanism of i t s a c t i o n . Since p r e l i m i n a r y s t u d i e s had i n d i c a t e d that t r y p t i c a c t i o n a f f e c t e d the f a t globule membrane we i s o l a t e d the membrane using the method of B a i l i e and Morton (22). Our r e s u l t s i n d i c a t e d that approximately 25% of the membrane m a t e r i a l was released i n t o the serum phase by the enzymatic a c t i o n . Furthermore, SDS-polyacrylamide e l e c t r o p h o r e t i c separations revealed (23) that the membrane m a t e r i a l had been p a r t i a l l y hydrolyzed by the t r y p s i n . This suggests that the t r y p s i n e f f e c t may be due to both a r e l e a s e of membrane m a t e r i a l and p r o t e i n h y d r o l y s i s . I t has been suggested that p r o t e i n h y d r o l y s i s i n h i b i t s o x i d a t i o n by i n c r e a s i n g the copper binding c a p a c i t y of the milk. Results of our s t u d i e s (24) i n d i c a t e d that t r y p t i c a c t i o n does increase the copper b i n d i n g c a p a c i t y of the milk. Although there was some v a r i a b i l i t y i n the amount bound, a l l samples showed s i g n i f i c a n t increases. Heat i n a c t i v a t e d t r y p s i n d i d not cause any increase i n copper b i n d i n g thus i n d i c a t i n g that the e f f e c t was due to h y d r o l y t i c a c t i o n r a t h e r than d i r e c t binding to the enzyme.


64

E N Z Y M E S IN FOOD A N D

B E V E R A G E PROCESSING

Results by Babish (25) showed that t r y p t i c a c t i o n reduced oxygen uptake i n samples c o n t a i n i n g added copper (Table IX). The l i n e a r i t y of the data r e v e a l s the l a c k of an i n d u c t i o n p e r i o d which suggests a heme c a t a l y z e d o x i d a t i o n . T h i s theory i s Table IX.

E f f e c t of T r y p s i n Treatment on Oxygen Intake


Time (hours)

Control Milk M i l k + Copper M i l k + T r y p s i n + Copper

0 6.4 6.2 5.7

12

24

36

5.5 5.4

4.0 4.9

3.4 4.7

48 5.2 2.7 4.4

supported by the observation that both h i s t i d i n e and antimycin A i n h i b i t e d o x i d a t i o n i n milk. These two substances are capable of complexing w i t h heme p r o t e i n . T r y p t i c a c t i o n may a l s o a f f e c t the c a t a l y t i c a c t i o n of heme i r o n . Regardless of the mechanism of a c t i o n t r y p s i n does i n h i b i t o x i d i z e d f l a v o r development. The r e s u l t s of a t y p i c a l experiment i s shown i n Table X. Our r e s u l t s (23) a l s o i n d i c a t e that t r y p s i n treatment increased Vitamin A stability. T h i s i n d i c a t e s a f r i n g e b e n e f i t of the enzymatic treatment. Table X. Sample

E f f e c t of T r y p s i n Treatment on the O x i d a t i v e of m i l k TBA V a l u e s Trial 1 Trial 2 3

Control Milk M i l k + Copper M i l k + T r y p s i n + Copper a

Stability

0.03 1.00 0.03

V a l u e s represent absorbance at 532 nm,

0.03 0.70 0.02 3 days a f t e r treatment

The foregoing r e s u l t s were obtained with s o l u b l e t r y p s i n but we have a l s o used immobilized t r y p s i n (26,27). Inasmuch as we only want to hydrolyze a small f r a c t i o n of the p r o t e i n i n t h i s treatment, the immobilized enzyme has the advantage of enabling us to c a r e f u l l y c o n t r o l the extent of h y d r o l y s i s . We have bound t r y p s i n to both porous g l a s s and tygon tubing. Although porous g l a s s has much greater s u r f a c e area f o r b i n d i n g enzymes, the tygon tubing i s e a s i e r to s a n i t i z e and does not plug up. Incid e n t a l l y , we have used 10% ethanol to s a n i t i z e the r e a c t o r or tubing a f t e r each use. By bubbling a i r or n i t r o g e n i n t o the flow stream through the tygon tubing, turbulence i s increased and b e t t e r substrate-enzyme contact i s obtained (26). The tygon tubing s t i l l does not provide as much contact but i t i s l e s s l i k e l y to be plugged by the f a t i n whole milk. Skim milk i s l e s s apt to plug a column r e a c t o r and whey passes through q u i t e readily. So i t i s more f e a s i b l e to t r e a t these two products with immobilized enzymes than whole milk. Swaisgood and a s s o c i a t e s (28,29) a t North C a r o l i n a State have used immobilized s u l f h y d r y l



4.

SHiPE


65

oxidase, rennin and a fungal protease to t r e a t skim milk. A number of workers have prepared immobilized l a c t a s e f o r t r e a t i n g skim m i l k and whey. In c o n c l u s i o n , enzymes can be used to improve m i l k f l a v o r i n three ways. Namely 1. By producing or i n c r e a s i n g d e s i r a b l e f l a v o r s such as sweetness. 2. By removing o f f - f l a v o r s such as strong cooked f l a v o r . 3. By preventing development of o f f - f l a v o r s , such as o x i dized f l a v o r . In most cases o x i d i z e d f l a v o r development i s not a c r i t i c a l problem at the present time. However, i t could become one i f we adopt wholesale f o r t i f i c a t i o n of m i l k with i r o n or i n c r e a s e the polyunsaturated f a t t y a c i d content by feeding encapu l a t e d unsaturated o i l s . Whether any of these treatment are used w i l l depend on the seriousness of the f l a v o r problem and the cost of the treatment. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Kende S. Proc. IX Intern. Dairy Congr. (1931), Subject 3, paper 137. Aurand, L. W. and Woods, A. E. J . Dairy S c i . (1959) 42, 1111. Hicks, C. L., Korycka-Dahl, M. and Richardson, T. J . Dairy S c i . (1975) 58, 796 (Abstr.). Korycka-Dahl, M. and Richardson, T. Abstr. 71st Ann. Mtg. Amer. Dairy S c i . Assn. (1976) p54,D49. E r i k s s o n , C. E. J . D a i r y S c i . (1970) 53, 1649 (Abstr.). Kiermeier, F. and Petz, Ε. Z. Lebensmittelunters u-Forsch. (1967) 132, 342. Sievwright, C. A. M.S. T h e s i s , C o r n e l l Univ., Ithaca, NY (1970). Rand, A. G., J r . and Hourigan, J . A. J . D a i r y S c i . (1975) 58, 1144. Reed, G. "Enzymes i n Food P r o c e s s i n g " pp386-390. Academic Press, New York (1966). Thomas, E. L., N i e l s e n , A. J . and Olson, J . C., J r . Amer. M i l k Rev. (1955) 17, 50. Krukovsky, V. N. and Sharp, P. F. J . D a i r y S c i . (1938) 21, 671. Harper, W. J . and Gould, I. Α. XV I n t e r n . D a i r y Congr. (1959) 1455. Shahani, Κ. M. and Chandan, R. C. J . D a i r y S c i . (1963) 46, 597. M a r s h a l l , R. T. and Charoen, C. Abstr. 71st Ann. Mtg. Amer. D a i r y S c i . Assn. (1976) p51,D40. Richardson, G. H. and Nelson, J . H. J . D a i r y S c i . (1967) 50, 1061. Arnold, R. G., Shahani, Κ. M. and Dwivedi, Β. K. J . Dairy S c i . (1975) 58, 1127.


66 17. 18. 19. 20. 21. 22. 23.


24. 25. 26. 27. 28. 29.

E N Z Y M E S IN FOOD A N D B E V E R A G E

PROCESSING

Noomen, A. Netherlands M i l k Dairy J . (1975) 29, 153. Shukla, T. P. and Tobias, J . J . D a i r y S c i . (1970) 53, 637. Young, M. H. M.S. T h e s i s , C o r n e l l Univ., Ithaca, NY (1969). Chrisope, G. L. and M a r s h a l l , , R. T. J . D a i r y S c i . (1975) 58, 794 (Abstr.). Anderson, E. O. M i l k Dealer (1939) 29(3)32. Bailie, M. J . and Morton, R. K. Biochem. J . (1958) 69,35. Gregory, J . F. and Shipe, W. F. J . D a i r y S c i . (1975) 58, 1263. Lim, Diana and Shipe, W. F. J . Dairy S c i . (1972) 55, 753. Babish, J . E. M.S. T h e s i s , C o r n e l l Univ., Ithaca, NY. (1974). Senyk, G. F., Lee, E. C., Shipe, W. F., Hood, L. F. and Downes, T. W. J . Food S c i . (1975) 40, 288. Lee, E. C., Senyk, G. F. and Shipe, W. F. J . Dairy Sci. (1975) 58, 473. Brown, R. J., Poe, L. B., Kasper, G. A. and Swaisgood, H. E. Abstr. 71st Ann. Mtg., Amer. D a i r y S c i . Assn. (1976) p49,D35. J a n o l i n o , V. G. and Swaisgood, H. E. Abstr. 71st Ann. Mtg. Amer. Dairy S c i . Assn. (1976) p50,D37.


Enzymatic Modification of Milk Flavor - ACS Symposium Series (ACS

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