Tailoring Enzyme Systems for Food Processing - ACS Symposium

Chapter 3

Tailoring Enzyme Systems for Food Processing Downloaded by KTH ROYAL INST OF TECHNOLOGY on January 24, 2016 | http://pubs.acs.org Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

Joseph E. Spradlin General Foods USA, White Plains,NY10625 Three illustrations are used to review the various approaches taken by the enzyme industry in tailoring enzyme preparations to meet the production and product quality needs of the food industry. Tailored enzyme preparations have been able to convert the corn syrup industry from an acid-based industry to an enzymebased industry, to overcome the problems created in the baking industry as grain technology improved and automation was introduced, and to rescue the cheese industry as the supply of bovine rennet decreased and the demand for cheese and cheese flavor increased. The use o f enzymes by t h e food i n d u s t r y as a p r o c e s s i n g t o o l i s i n c r e a s i n g r a p i d l y due t o t h e t a i l o r i n g o f enzyme p r e p a r a t i o n s t o f i t t h e p r o c e s s i n g needs o f the i n d u s t r y . The p o t e n t i a l o f u s i n g enzymes f o r c o n v e r t i n g a g r i c u l t u r a l p r o d u c t s i n t o food p r o d u c t s i s enormous, and has m i n i m a l l y been tapped as a method o f p r o d u c t i o n . To d a t e , the major modern a p p l i c a t i o n s coming a f t e r t h e a g e - o l d a p p l i c a t i o n s i n b a k i n g , brewing, and cheese have c e n t e r e d on the p r o d u c t i o n o f commodity i n g r e d i e n t s such as c o r n s y r u p s , whey u p g r a d i n g and h y d r o l y z e d p r o t e i n s . Today food p r o c e s s o r s a r e s t a r t i n g t o use enzymes as p r o c e s s i n g t o o l s t o improve the a t t r i b u t e s of t h e i r products. Appearance, t e x t u r e , f l a v o r , c o l o r , s t a b i l i t y , and n u t r i t i o n are a t t r i b u t e s b e i n g i n f l u e n c e d by a p p l y i n g t a i l o r e d enzyme p r e p a r a t i o n s d u r i n g t h e p r o c e s s o f c o n v e r t i n g a g r i c u l t u r a l products i n t o processed foods. Food p r o c e s s o r s a r e a l s o f i n d i n g s e v e r a l p r o c e s s i n g advantages i n u s i n g enzymes, such as i n c r e a s e d y i e l d s and improved p r o c e s s i n g parameters l i k e v i s c o s i t y , f i l t r a t i o n r a t e s , and ease o f h a n d l i n g ( 1 ) . Enzymes o f f e r s e v e r a l advantages t o t h e food p r o c e s s o r o v e r p h y s i c a l and c h e m i c a l methods o f p r o c e s s i n g . Enzymes work under v e r y m i l d c o n d i t i o n s o f pH and temperature w h i l e a c c o m p l i s h i n g the same r e a c t i o n t h a t o t h e r w i s e would r e q u i r e extreme pH and temperature c o n d i t i o n s . Consumers a r e becoming i n c r e a s i n g l y aware o f a r t i f i c i a l and p o t e n t i a l l y t o x i c c h e m i c a l s p r e s e n t i n f o o d s . The consumer, as w e l l as t h e government, i s a s k i n g f o r l e s s use o f chemical a d d i t i v e s t o c o n t r o l product a t t r i b u t e s . Enzymes a r e a l s o 0097-6156/89/0389-0024$06.00/0 © 1989 American Chemical Society

In Biocatalysis in Agricultural Biotechnology; Whitaker, John R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Downloaded by KTH ROYAL INST OF TECHNOLOGY on January 24, 2016 | http://pubs.acs.org Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

3. SPRADLIN

Tailoring Enzyme Systems for Food Processing

25

v e r y s p e c i f i c and c a n s e l e c t o u t one s p e c i f i c r e a c t i o n , such as s t a r c h h y d r o l y s i s , i n a m i x t u r e o f s t a r c h , p r o t e i n , and f a t . Chemical and p h y s i c a l p r o c e s s i n g methods a r e not s e l e c t i v e and i n the s t a r c h , p r o t e i n , f a t m i x t u r e would h y d r o l y z e a l l t h r e e o f the polymers. Thus, by p e r f o r m i n g s p e c i f i c p r o c e s s i n g t a s k s under r e l a t i v e l y m i l d p r o c e s s i n g c o n d i t i o n s , enzymes c a n o f f e r s e l e c t e d chemical r e a c t i o n s i n a heterogeneous m i x t u r e , f r e e o f u n d e s i r a b l e side reactions. The f a c t o r s t o be c o n s i d e r e d i n t a i l o r i n g enzymes f o r the food i n d u s t r y can be d i v i d e d i n t o five areas: a) s e n s i t i v i t y t o p r o c e s s i n g c o n d i t i o n s ; b) c a t a l y t i c s p e c i f i c i t y o r a c t i o n p r o f i l e ; c) p u r i t y ; d) s o u r c e ; and e) a p p l i c a t i o n economics. S e n s i t i v i t y to Processing Conditions. I n most food a p p l i c a t i o n s , the environment i n which an enzyme must work has v e r y narrow limits. The c o m p o s i t i o n o f the raw m a t e r i a l s and the f i n a l p r o d u c t o f t e n d i c t a t e the pH, s a l t c o m p o s i t i o n , and i n h e r e n t enzyme inhibitor level. The temperature ranges a l l o w a b l e a r e o f t e n c o n t r o l l e d by the temperatures r e q u i r e d t o o b t a i n the d e s i r e d p r o d u c t a t t r i b u t e s from temperature-dependent p h y s i c a l and c h e m i c a l changes i n the raw m a t e r i a l s such as s t a r c h g e l a t i n i z a t i o n and heat induced p r o t e i n changes. T h i s temperature range r e s t r i c t i o n i s a l s o c o n t r o l l e d by temperature above o r below which u n d e s i r a b l e p r o d u c t a t t r i b u t e s develop. I t i s o f t e n d e s i r a b l e t o be a b l e t o c o n t r o l the a c t i v i t y o f an enzyme d u r i n g p r o c e s s i n g by a l t e r i n g t h e c o n d i t i o n s o f i t s environment such as pH o r temperature. Catalytic Specificity. The c a t a l y t i c s p e c i f i c i t y o r a c t i o n p r o f i l e o f an enzyme i s the t a i l o r i n g f a c t o r t h a t o f f e r s u n l i m i t e d potential. Being a b l e t o s e l e c t j u s t the e x a c t bond t o c l e a v e i n a polymer, o r t o c o n t r o l w i t h i n a v e r y narrow range the s i z e d i s t r i b u t i o n i n a polymer d i g e s t , o f f e r s enormous p r o d u c t q u a l i t y potential. Purity. The i s s u e o f o b t a i n i n g p u r i t y e c o n o m i c a l l y i s a challenging t a i l o r i n g factor. P u r i t y , i n an i n d u s t r i a l a p p l i c a t i o n sense, i s d e f i n e d by the absence o f s i d e r e a c t i o n s t h a t would cause n e g a t i v e impacts on one o r more p r o d u c t a t t r i b u t e s . U n l i k e most i n g r e d i e n t s used by the food p r o c e s s o r , enzymes a r e s o l d by t h e i r a c t i v i t y r a t h e r than t h e i r w e i g h t . T h e r e f o r e , the a c t i v i t y p e r u n i t weight o f the enzyme p r o d u c t i s o f prime importance. The enzymes used i n i n d u s t r y a r e r a r e l y c r y s t a l l i n e , c h e m i c a l l y pure, o r even single protein preparations. These i m p u r i t i e s may not i n t e r f e r e w i t h enzyme a c t i v i t y but enzyme i n p u r i t i e s c a n c a t a l y z e the formation of side products, present a t o x i c o l o g i c a l hazard, o r d e c r e a s e a d e s i r a b l e a t t r i b u t e o f t h e f i n a l food p r o d u c t such as c o l o r , t a s t e , o r t e x t u r e . What i s b e i n g added t o t h e food as p a r t of the enzyme p r e p a r a t i o n has t o be e v a l u a t e d as t o i t s impact on f i n a l product a t t r i b u t e s . In most a p p l i c a t i o n s the l e v e l o f enzyme used i s v e r y low and i s i n a c t i v a t e d d u r i n g the p r o c e s s i n g , a l l o w i n g the enzyme t o be regarded as a p r o c e s s i n g a i d . There a r e many p o t e n t i a l food a p p l i c a t i o n s f o r enzymes which r e q u i r e a c t i v i t y l e v e l s o r p r o c e s s i n g c o n d i t i o n s which would e i t h e r make the economics u n f a v o r a b l e o r would l e a v e u n d e s i r a b l e l e v e l s o f a c t i v e enzyme i n t h e f i n a l



26

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

product. Immobilized enzymes are a way of overcoming these types of problems. Immobilized enzymes are usually completely recoverable from the reaction mixtures allowing them to be reused repeatedly without any contamination of the f i n a l product. Immobilized enzymes o f f e r the added advantage of eliminating the need to heat the product to inactivate the enzyme. In many applications heating cannot be done due to the temperature s e n s i t i v i t y of the product. Immobilization also allows the use of higher a c t i v i t y levels compared to using free enzyme, resulting i n a shortening of reaction times or reducing the size of the vessel needed to carry out the reaction. The absence of enzyme i n the f i n a l product i s often a major driving force i n using immobilized over soluble enzyme. Source. Enzymes for food applications come from a l l three kingdoms; plant, animal, and microbial. T r a d i t i o n a l l y used plant and animal enzymes are the plant proteases such as papain, f i c i n and bromelain, plant amylases from malt, and animal rennin which i s used i n cheese manufacture. Microbial c e l l s are the usual and most promising future source of i n d u s t r i a l enzymes. Estimates of the number of microorganisms i n the world tested as potential sources of enzymes f a l l around 2% with only about 25 organisms, including a dozen or so fungi, currently used for i n d u s t r i a l enzymes. Application Economics. The bottom line i s always the cost vs. benefit aspect of the process. The goal i s always to be the least cost producer of the highest quality product. Cost i s the most important decision-making aspect i n whether to put an enzyme-based process into production once the technical f e a s i b i l i t y has been demonstrated. The t a i l o r i n g of cost e f f e c t i v e enzyme preparations often becomes the challenge more than the t a i l o r i n g of c a t a l y t i c properties. Besides the direct cost of the enzyme product, enzymes need to be tailored to allow the processing of raw materials without adding long reaction times or costly unit operations such as material separation, cooling, heating, evaporation, or ion exchange, to the normal process flow. T a i l o r i n g Opportunities. There are many methods or approaches available to t a i l o r enzyme products. Early i n the history of enzyme companies, methods such as source selection, microbial s t r a i n selection, growth conditions, media, p u r i f i c a t i o n , and recovery systems, were primarily used to make each enzyme preparation unique. Later, immobilization, encapsulation, and chemical modification of the enzyme molecule i t s e l f were added as methods of t a i l o r i n g enzymes to better f i t i n d u s t r i a l applications. Today, a l l of these methods are s t i l l being used, and now we have added genetic engineering to our t a i l o r i n g expertises. Microbial enzymes o f f e r a much higher potential for being t a i l o r e d into cost e f f e c t i v e highly s p e c i f i c enzyme preparations for use i n food processing than do plant and animal enzymes. They o f f e r a great variety of c a t a l y t i c a c t i v i t i e s and ranges of environmental adaptability. They have the greatest potential for being produced in an inexpensive, regular and abundant supply. Microbial enzymes i n general are more stable than their corresponding plant and animal counterparts having similar a c t i v i t y . Microbial c e l l s also o f f e r an unlimited potential for genetic and environmental manipulation to



3. SPRADLIN


27

increase y i e l d s , to produce altered enzymes, or to introduce new genes to produce t o t a l l y new enzymes. Several papers i n this symposium use site-directed mutagenesis as a means of t a i l o r i n g the properties of an enzyme. Today, recombinant DNA techniques are being used i n many laboratories to t a i l o r cost-effective enzyme products directed to the food industry. The technologies needed to synthesize a new gene for a new enzyme i n the laboratory, attach i t to an expression vector, and insert i t into a microorganism are available today. It i s only a matter of time before i t ' s done. Three food industries, corn syrup, baking, and dairy, w i l l be used to i l l u s t r a t e how enzyme preparations have been and are being tailored for use i n the food industry. Corn Syrup The corn syrup industry i s a good example of how enzymes took over and improved an industry after i t was established as a profitable and important industry. In the beginning, acid was used as the catalyst to hydrolyze starch into syrups i n order to impart new and desirable attributes t o food products. The process involved treating a 40%-45% solution of refined starch with hydrochloric acid at pH 1.5 and a temperature of 150°C for a few minutes. The acid was not selective and hydrolyzed the protein and fat present i n the starch preparation as well as the starch. This led to extensive undesired browning due to Maillard reactions. The syrup was neutralized, f i l t e r e d , carbon-treated, and ion-exchange treated to become a food product or an ingredient to be used by other food processors i n preparing products. The properties and subsequently the applications of the resulting corn syrup products, were dependent upon the extent of hydrolysis. The extent of hydrolysis i n corn syrup i s expressed i n dextrose equivalents (DE) which i s the percentage of glucosidic bonds hydrolyzed with native starch being 0 and pure glucose being 100. The DE of acid-catalyzed corn syrups depend only upon the time of reaction. A l l acid-catalyzed syrup at any single DE level w i l l always have the same dextrin p r o f i l e . Amylases. Acid hydrolyzed corn syrups were a great innovation f o r the food industry, but both the process and the product had their shortcomings. During the late 1950's, low DE corn syrups were gaining wide acceptance but had two major drawbacks. One was the development of a haze after concentration which limited i t s usefulness. The other was the high v i s c o s i t y of the hydrolyzates which made them d i f f i c u l t to process. The use of enzymes as a processing tool to improve the attributes of an existing food product was introduced to the industry. Up to that time enzymes were used only to enhance products that by their complex nature required enzyme reactions such as brewing and cheese production. In 1960, Corn Products Co. obtained a patent for using an alpha amylase to p a r t i a l l y hydrolyze the high molecular weight dextrins i n low DE acid hydrolyzed syrups without substantially increasing the DE (2). After concentration, the amylase-treated acid hydrolyzed syrups had a l l of the desirable c h a r a c t e r i s t i c s of low DE acid syrup but none of the undesirable features. During this same period of



28


time, h i g h DE a c i d h y d r o l y z e d syrups were b e i n g used as a d j u n c t s i n f e r m e n t a t i o n s but had a major problem of c r y s t a l l i z a t i o n due to the h i g h l e v e l of g l u c o s e . In 1962, Anheuser-Busch o b t a i n e d a p a t e n t d e s c r i b i n g the p r o d u c t i o n o f a h i g h l y f e r m e n t a b l e c o r n syrup i n which 80% to 90% o f the t o t a l c a r b o h y d r a t e i s f e r m e n t a b l e and t h e r e i s no u n d e s i r a b l e tendency toward c r y s t a l l i z a t i o n . T h e i r process i n v o l v e d a c i d c o n v e r s i o n o f s t a r c h to about DE 20 and then adding malt to i n c r e a s e the m a l t o s e l e v e l as h i g h as p o s s i b l e which r e s u l t e d i n a syrup o f about 55 DE. Next, a m i c r o b i a l amylase was added to f u r t h e r reduce the l o n g e r c h a i n d e x t r i n s to g l u c o s e t o g i v e a f i n a l DE i n the low 70's. T h i s was the b e g i n n i n g o f an i n d u s t r y t h a t now o f f e r s p r o d u c t s p r e p a r e d by c o n v e r t i n g s t a r c h i n a v a r i e t y o f ways: a c i d , acid-enzyme, enzyme, o r d u a l enzyme. Today t h e r e a r e two primary types o f enzyme produced h i g h m a l t o s e syrups a v a i l a b l e c o m m e r c i a l l y . One has a DE o f about 42 and c o n t a i n s about 40% m a l t o s e and o n l y about 8% g l u c o s e . This product i s w i d e l y used i n jams and c o n f e c t i o n e r i e s because i t i s r e s i s t a n t to c o l o r f o r m a t i o n , i s n o n - h y g r o s c o p i c and does not c r y s t a l l i z e as r e a d i l y as g l u c o s e s y r u p s . The o t h e r commercial h i g h m a l t o s e syrup has a DE o f about 62 and c o n t a i n s about 35% m a l t o s e and about 40% glucose. T h i s syrup i s h i g h l y f e r m e n t a b l e and s t a b l e to s t o r a g e ; t h e r e f o r e , i t i s used w i d e l y i n brewing and bread making. The p r o p e r t i e s o f low DE c o r n syrup and i t s e f f e c t when used i n a food system i s v e r y dependent upon i t s d e x t r i n p r o f i l e . By t a i l o r i n g the a c t i o n p a t t e r n o f the amylase, c o r n syrups o f unique and h i g h l y v a r y i n g d e x t r i n p r o f i l e s can be p r o d u c e d . Today t h i s i s an undeveloped a r e a . Knowledge i s c u r r e n t l y b e i n g g e n e r a t e d to understand how enzyme d i g e s t e d s t a r c h of c o n t r o l l e d d e x t r i n p r o f i l e can s e r v e as f a t m i m e t i c s , foam and g e l s t a b i l i z e r s and many o t h e r food a p p l i c a t i o n s p r e v i o u s l y f u l f i l l e d by c h e m i c a l a d d i t i v e s o r h i g h l e v e l s o f f a t . The t a i l o r i n g o f the a c t i o n p a t t e r n s of amylases w i l l be a major f i e l d i n the f u t u r e . The a c t i o n p a t t e r n of an amylase and the r e s u l t i n g c o m p o s i t i o n o f i t s d i g e s t are c o n t r o l l e d by the b i n d i n g e n e r g i e s o f the a c t i v e s i t e to the g l u c o s e r e s i d u e s i n the s t a r c h c h a i n . Each amylase has i t s own unique b i n d i n g energy p r o f i l e a c r o s s i t s a c t i v e s i t e . In a d d i t i o n , amylases d i f f e r i n the number o f g l u c o s e u n i t s t h a t w i l l f i t i n t o the a c t i v e s i t e . These two f a c t o r s w i l l c o n t r o l the d e x t r i n d i s t r i b u t i o n p r o f i l e of the r e s u l t i n g syrups. F o r example, the amylase from B a c i l l u s l i c h e n i f o r m i s produces m a l t o s e , m a l t o t r i o s e and m a l t o p e n t o s e as i t s main end p r o d u c t s w h i l e the amylase from B a c i l l u s a m y l o l i q u i f a c i e n s produces maltohexose as i t s major end p r o d u c t . The a c t i v e s i t e of the amylase from B a c i l l u s a m y l o l i q u i f a c i e n s has been e x t e n s i v e l y s t u d i e d t o the p o i n t t h a t i t i s known t h a t the a c t i v e s i t e i n t e r a c t s w i t h t e n g l u c o s e u n i t s a l o n g the s t a r c h c h a i n w i t h b i n d i n g o f the t e n t h g l u c o s e h a v i n g a l a r g e p o s i t i v e v a l u e t h a t h i n d e r s b i n d i n g o f s u b s t r a t e (_3). The c a t a l y t i c s i t e of the enzyme i s i n p r o x i m i t y o f the g l u c o s i d i c bond between g l u c o s e u n i t s s i x and seven, r e s u l t i n g i n the major p r o d u c t b e i n g maltohexose.Many amylases have the a b i l i t y to c l e a v e o f f m u l t i p l e p r o d u c t s from each s u c c e s s f u l enzyme/substrate e n c o u n t e r . This property allows f o r a c c u m u l a t i o n o f s m a l l o l i g o s a c c h a r i d e s i n the e a r l y phases o f amylase h y d r o l y s i s . The amylase has the a b i l i t y to c l e a v e a t the end o f a l a r g e s u b s t r a t e p r o d u c i n g s m a l l d e x t r i n s a f t e r the f i r s t random a t t a c k . A l t h o u g h the number o f r u p t u r e s per e f f e c t i v e



3. SPRADLIN


29

e n c o u n t e r i s b a s i c a l l y a k i n e t i c phenomenon, the s i z e o f the p r o d u c t s r e l e a s e d w i l l p r o b a b l y be i n f l u e n c e d by monomer-subsite interactions. A f t e r the f i r s t c l e a v a g e , rearrangement o f the enzyme-substrate complex w i l l y i e l d a s e r i e s o f complexes whose r e l a t i v e c o n c e n t r a t i o n p a r t i a l l y depend upon the subsite-monomer i n t e r a c t i o n energies. The degree o f m u l t i p l e a t t a c k has been q u a n t i t a t e d f o r s e v e r a l alpha-amylases ( 4 ) . The h i g h e s t v a l u e i s f o r p o r c i n e p a n c r e a t i c a l p h a amylase w i t h s i x subsequent a t t a c k s p e r e f f e c t i v e e n c o u n t e r a t i t s o p t i m a l pH w h i l e A s p e r g i l l u s oryzae amylase undergoes two subsequent a t t a c k s p e r e f f e c t i v e e n c o u n t e r . I f p o r c i n e p a n c r e a t i c a l p h a amylase i s s t u d i e d a t pH 10, i t s subsequent number o f a t t a c k s drops t o 0.7. T h i s r e p e t i t i v e a t t a c k f e a t u r e i s one t h a t c o u l d be t a i l o r e d i n t o the a c t i o n p r o p e r t i e s o f l a b o r a t o r y d e s i g n e d amylases. In the f u t u r e , t a i l o r e d amylases w i l l be a b l e t o produce a range of p r o d u c t s o f f e r i n g a wide v a r i e t y o f u n i q u e p r o p e r t i e s t h a t c a n be used t o c o n t r o l the a t t r i b u t e s o f food p r o d u c t s . Heat S t a b l e A l p h a Amylase. One o f the key enzymes i n today's c o r n syrup i n d u s t r y i s a heat s t a b l e a l p h a amylase. I t i s now used i n the f i r s t s t e p i n p r o d u c i n g a l l v a r i e t i e s o f c o r n syrups such as low DE, h i g h m a l t o s e , h i g h g l u c o s e , and h i g h f r u c t o s e c o r n s y r u p s . In 1973, enzyme t a i l o r i n g i n t r o d u c e d the c o r n syrup b u s i n e s s t o an enzyme p r e p a r a t i o n t h a t was d e s i g n e d t o r e p l a c e the a c i d s t a r c h converters. The a l p h a amylase from B a c i l l u s l i c h e n i f o r m i s became a v a i l a b l e a t a c o s t e f f e c t i v e p r i c e . The enzyme had t o f u n c t i o n a t 105°C w i t h a h a l f - l i f e l o n g enough t o reduce the c h a i n l e n g t h o f the s t a r c h m o l e c u l e s t o a p o i n t t h a t would p r e v e n t p r e c i p i t a t i o n when the temperature dropped t o 95°C. One r e a s o n the a c i d c a t a l y z e d r e a c t i o n s were c a r r i e d out a t 150°C was due t o the need to open up the i n n e r s t r u c t u r e o f the s t a r c h g r a n u l e to make the bonds available for hydrolysis. A t lower t e m p e r a t u r e s , y i e l d was l o s t due to u n d i g e s t e d s t a r c h In the enzyme p r o c e s s u s i n g heat s t a b l e a l p h a amylase, the r e a c t i o n i s c a r r i e d out i n a j e t c o o k e r where m e c h a n i c a l s h e a r i s a p p l i e d as the temperature i s r a i s e d t o 105°C by i n j e c t i n g p r e s s u r i z e d steam. Under t h e s e c o n d i t i o n s , the enzyme c a t a l y z e s enough h y d r o l y s i s o f the i n n e r c o r e o f the s t a r c h g r a n u l e to p r e v e n t significant yield losses. I f the j e t c o o k i n g p r o c e s s i s r u n a t 95°C, where the enzyme i s much more s t a b l e , a s i g n i f i c a n t y i e l d l o s s i s obtained. A f t e r about f i v e minutes a t 105°C the s t a r c h s l u r r y i s f l a s h c o o l e d to 95°C and h e l d a t that temperature w h i l e the enzyme i n c r e a s e s the DE t o about t w e l v e . The temperature o f t h i s syrup c a n now be lowered t o 60°C o r l e s s and enzyme t r e a t e d i n a v a r i e t y o f ways t o produce a wide range o f c o r n syrup p r o d u c t s o f v a r y i n g p r o p e r t i e s and d e x t r i n d i s t r i b u t i o n s . Today, t h e r e a r e s e v e r a l heat s t a b l e a l p h a amylase p r o d u c t s on the market, many o f which were t a i l o r e d u s i n g m u t a t i o n and recombinant DNA t e c h n i q u e s . A major t a i l o r i n g t a r g e t has been improvements o f y i e l d d u r i n g enzyme p r o d u c t i o n i n o r d e r t o be more c o s t e f f e c t i v e i n the m a r k e t p l a c e . A l l o f the heat s t a b l e a l p h a amylases r e q u i r e c a l c i u m f o r s t a b i l i z a t i o n which must be added to the s t a r c h s l u r r y and then removed by i o n exchange l a t e r i n the process. The c a l c i u m r e q u i r e m e n t s v a r y w i d e l y . F o r example, the amylase from B. l i c h e n i f o r m i s r e q u i r e s o n l y 5 ppm c a l c i u m w h i l e the


30



amylase from B. amyloliquifaciens requires 150 ppm calcium. A t a i l o r i n g need for the future would be a very stable non-calcium requiring amylase obtained i n high y i e l d during i t s production. Amyloglucosidases. Amyloglucosidases or glucoamylases as they are often c a l l e d , catalyze the stepwise hydrolysis of the alpha 1-4 links i n starch releasing beta glucose from the non-reducing end of the chain. Since glucoamylases can only bind at the end of each polymer chain i t s a c t i v i t y i s somewhat controlled by the size of the dextrin or starch molecules i n solution. Most glucoamylases w i l l also hydrolyze alpha 1-6 and alpha 1-3 bonds but do so at very slow rates. The major use of glucoamylases i n the corn starch industry today i s to digest to as near completion as possible the product resulting from the hydrolysis of starch with heat stable alpha amylase. The starting syrup i s usually at about 30% dry solids with a DE of about 12 to 15. Two major things must be done to the syrup as i t exits the heat stable amylase step. F i r s t , i t has to be cooled to 60°C because a heat stable glucoamylase has not yet been t a i l o r e d . Second, the pH must be adjusted to about pH 4.5. A major need of the industry i s a cost e f f e c t i v e heat stable glucoamylase that performs at about pH 6.5. This would eliminate the pH adjustment step as well as to allow both the alpha amylase and the glucoamylase reactions to proceed at the same time. Glucoamylases also require calcium ions for s t a b i l i t y but i n practice the calcium level i s adjusted before the alpha amylase step to s t a b i l i z e i t . The resulting product from glucoamylase treatment, a glucose syrup, usually has a DE of about 97 and a glucose content of 95% to 97% with 3-5% higher saccharides present. The glucose syrup i s then used i n several ways. I t can be used as a syrup or c r y s t a l l i z e d to give pure s o l i d glucose. Today a very large percentage of i t i s used as feed stock to produce high fructose corn syrup. In a l l cases, the higher the glucose level the better and each percent of carbohydrate not converted to glucose equates to m i l l i o n s of pounds of product lost annually for the industry. To push the reaction to as close to completion as possible, glucoamylase i s used batchwise for reaction periods of 48 to 92 hours. At pH 4.5 and 60°C the 30% syrups used for glucoamylase feed stocks are stable microbiologically since microorganisms w i l l not grow under these conditions. In practice, the maximum glucose level can be missed due to the thermodynamics of the system. In a 30% solution of glucose, there i s a small level of alpha 1-4, alpha 1-6, and alpha 1-3 disaccharides produced due to the systems thermodynamics. Since glucoamylase can catalyze a l l three of these reactions, we now have conditions that allow for the formation of alpha 1-6 and alpha 1-3 disaccharides. I f the reaction i s allowed to continue a glucose syrup reaching a level of 97% glucose would slowly decrease with time. Operating the glucoamylase reaction at a lower dry solids level w i l l greatly reduce the reversion reactions but unacceptable evaporation costs r e s u l t . Immobilization i s often suggested as a method of t a i l o r i n g enzymes for more e f f i c i e n t i n d u s t r i a l use. In the case of glucoamylase very superior properties would have to be obtained to warrant immobilizing the enzyme due to i t s r e l a t i v e l y low cost i n the soluble form. Some of the potential benefits would be l o g i s t i c


3. SPRADLIN


31

convenience, lower c a p i t a l and energy cost, reduction i n reaction time from 48 hours to less than one hour ,less color development during processing and lower in-process volume inventories. Glucoamylase has been successfully immobilized with h a l f - l i v e s of a month while operating under conditions that prevent microbial growth. Immobilized glucoamylase systems have a very high enzyme to substrate r a t i o . This causes a higher rate of formation of alpha 1,6 and 1,3 disaccharides leading to lower f i n a l glucose levels than in batch systems. A major problem during the 1970 s with commercial glucoamylase products was the presence of a contaminating enzyme which formed d i and trisaccharides with alpha 1-6 linkages. Thus, r e l a t i v e l y high levels of isomaltose and panose were observed which were much higher than predicted by recombination v i a glucoamylase reversion. T a i l o r i n g of preparations f i r s t by p u r i f i c a t i o n procedures and l a t e r by mutation and media control removed the contaminating transglucosidase from commercial products.


1

Debranching Enzymes. One major source of y i e l d loss i n the production of high fructose corn syrup i s the loss due to incomplete conversion of starch to glucose. When a need exists with a s i g n i f i c a n t d o l l a r value associated with i t , someone w i l l develop a cost effective solution i f given enough time. There are enzymes whose primary reaction i s to catalyze the cleavage of the alpha 1-6 bonds of the amylopectin i n liquefied starch. These enzymes are referred to as debranching enzymes and include pullulanase and isoamylase. Enzyme companies are now t a i l o r i n g products for the primary purpose of obtaining another percent y i e l d of glucose during glucoamylase conversion of starch. One such product currently available commercially i s derived from Bacillus acidopullulyticus (5-6). The application conditions of this product are such that i t can be used at the same time as glucoamylase and results i n higher yields i n shorter time. Another company has taken a different approach to solving the glucose y i e l d problem (7-8). Its s c i e n t i s t s i d e n t i f i e d a new amylolytic enzyme from B a c i l l u s megaterium. The enzyme hydrolyzes alpha 1-4 bonds and does not d i r e c t l y hydrolyze alpha 1-6 bonds. The unique property of this enzyme i s i t s a b i l i t y , v i a a transfer reaction, to convert the glucoamylase resistant saccharides present in a syrup into forms easily hydrolyzed to glucose by the glucoamylase. This new amylolytic enzyme i s produced by B a c i l l u s megaterium as a trace contaminant i n a complex mixture of proteins which also include a large amount of beta amylase. By genetic engineering the B. megaterium gene for this enzyme was transferred into B. s u b t i l i s (9) resulting i n a 10,000-fold increase i n y i e l d and a product free of other amylases. Glucose Isomerase. The biggest success story i n the enzyme industry has to be glucose isomerase. The very f i r s t report of an enzyme that converts glucose to fructose was i n 1957 Q 0 ) . In less than 20 years the enzyme was studied, tailored into a cost e f f e c t i v e immobilized form and put into production to make a commodity corn syrup product. In less than 30 years from i t s f i r s t description, i t became the largest commercially used immobilized enzyme and responsible for making one of the world's major sweeteners. Since



32


glucose isomerase i s an i n t r a c e l l u l a r enzyme, stable above 60°C and a l l of i t s reactants are small molecules, glucose isomerase i s well suited for immobilization. Many approaches have been used to immobilize glucose isomerase using both whole c e l l s and c e l l free preparations. Commercial products of glucose isomerase are constantly being improved and today the enzyme cost per unit of fructose produced i s only a small f r a c t i o n of what i t was during the mid and late 1970's. In spite of the great commercial success of glucose isomerase, there i s s t i l l room for improvement. There are two major environmental problems that have been present since the beginning that s t i l l elude the researcher. A l l glucose isomerases require one or more heavy metal ions for a c t i v i t y and s t a b i l i t y . To make matters worse, calcium ions i n h i b i t the a c t i v i t y . In practice this means that calcium required for s t a b i l i z a t i o n of the alpha amylase and the glucoamylase must be removed by ion exchange before the magnesium ions, which are needed for glucose isomerase a c t i v i t y , can be added to the syrup. Of course, after isomerization the magnesium ions also have to be removed by ion exchange. Thus i n the production of high fructose corn syrup the syrup must be treated with ion exchangers twice. An enzyme not inhibited by calcium or not requiring a metal ion would find a ready market. The other big environmental problem i s pH. A l l currently available glucose isomerase products have to be used at a pH above 7.0 for cost e f f e c t i v e operation. Color formation and other alkaline catalyzed reactions increase refining cost and cause a loss of y i e l d . An enzyme capable of prolonged operation at a pH below 7 i s needed. Baking Enzymes play a very c r i t i c a l role in the baking industry. Traditional methods of baking are based on the presence of enzymes in the flour and i n the microorganisms used to ferment the dough. The endogenous alpha amylase hydrolyzes the low level of starch made available by the rupture of starch granules during the m i l l i n g process to dextrins. These dextrins become the substrate for the flour's endogenous beta amylase which produces maltose. The yeast i n the dough then uses i t s alpha glucosidase to convert the maltose to glucose, which the yeast can now use to produce carbon dioxide via i t s g l y c o l y t i c pathway. But this i s just the gas formation aspect of baking. Wheat flour also contains low levels of proteases, pentosanases, isoamylases, lipoxygenases, lipases, and esterases i n addition to the two types of amylases. A l l of these enzymes play a role i n the quality of the baked goods produced. Therefore, the enzyme content of flour i s of major importance i n the baking industry. Flour Amylases. Beta amylase i s produced i n wheat during the ripening stage and i s stored i n the aleurone layer u n t i l needed during germination and therefore i s present i n ungerminated grain. Plant alpha amylase, i n contrast, i s not present i n ungerminated grain to any s i g n i f i c a n t extent but i s synthesized de novo upon germination (11). Prior to 1920, the growing and harvesting of wheat were poorly controlled. F i e l d sprouting was routine and produced varying levels



3.

SPRADLIN


33

of a l p h a amylase i n the r e s u l t i n g f l o u r . With the i n t r o d u c t i o n o f m e c h a n i c a l h a r v e s t i n g of wheat i n the 1920's, f i e l d s p r o u t i n g became a rare occurrence. The l e v e l of a l p h a amylase i n f l o u r , produced from wheat t h a t has been c a r e f u l l y and e x p e r t l y grown, h a r v e s t e d , and m i l l e d , w i l l be too low t o produce a c c e p t a b l e b r e a d by the t r a d i t i o n a l system of dough p r e p a r a t i o n . The h i s t o r y of the use o f exogenous enzymes i n the food i n d u s t r y has n e a r l y p a r a l l e l e d the h i s t o r y o f the development of c e r e a l t e c h n o l o g y . The b e n e f i t s o f a d d i n g malt d i a s t a s e (amylase) t o bread to i n c r e a s e volume and q u a l i t y were known i n the 1800's. The p r a c t i c e o f adding malt to f l o u r became an e a r l y a r t i n the m i l l i n g and b a k i n g i n d u s t r y . I f the a l p h a amylase l e v e l was low, poor f e r m e n t a t i o n s were o b t a i n e d . I f the a l p h a amylase l e v e l was h i g h , the r e s u l t i n g bread had a s t i c k y , m o i s t , i n e l a s t i c crumb s t r u c t u r e . With p r o p e r a l p h a amylase s u p p l e m e n t a t i o n o f f l o u r , dough v i s c o s i t y i s reduced and the r a t e o f f e r m e n t a t i o n i n c r e a s e d r e s u l t i n g i n a p r o d u c t of improved volume and t e x t u r e . As bread making became more and more automated, r e p l a c i n g the bakers d e c i s i o n s on c r i t i c a l p r o c e s s i n g end p o i n t s w i t h p r e s e t p r o c e s s i n g parameters, the amylase l e v e l i n the f l o u r became a c r i t i c a l f a c t o r i n the i n d u s t r y . R e s p o n s i b i l i t y f o r the amylase l e v e l f e l l t o the m i l l e r t o s u p p l y f l o u r t h a t would perform t o the baker's s t a n d a r d s . The m i l l e r o r i g i n a l l y added the a l p h a amylase i n the form of m a l t . But i n 1884, Takamine d i s c o v e r e d t h a t the fungus A s p e r g i l l u s o r y z a e c o u l d be grown on wheat b r a n by s o l i d s t a t e f e r m e n t a t i o n t o produce a h i g h l e v e l o f a l p h a amylase. T h i s enzyme would prove to be an i d e a l amylase f o r supplementing f l o u r . Today i t i s the major method used by m i l l e r s to s t a n d a r d i z e f l o u r amylase activity. M a l t supplemented f l o u r i s d a r k e r than f u n g a l amylase supplemented f l o u r . T h i s d a r k e r c o l o r a d v e r s e l y a f f e c t s the c o l o r of the b r e a d . M a l t a l s o c o n t a i n s a r e l a t i v e l y h i g h l e v e l of p r o t e o l y t i c a c t i v i t y which can r e s u l t i n a r e d u c t i o n o f g l u t e n q u a l i t y i n low p r o t e i n f l o u r s . A. o r y z a e amylase p r o d u c t s c o n t a i n a n e g l i g i b l e protease content. Another major d i f f e r e n c e between malt a l p h a amylase and f u n g a l a l p h a amylase i s i n t h e i r t h e r m o s t a b i l i t y . M a l t amylase i s more s t a b l e and i f too much i s added can l e a d t o the formation of s t i c k y bread. The f u n g a l amylase i s i n a c t i v a t e d a t about 60°C b e f o r e a p p r e c i a b l e amounts o f s t a r c h a r e g e l a t i n i z e d , thus making the c o n t r o l o f the r e a c t i o n e a s i e r . A. o r y z a e amylase can be added to low a l p h a amylase f l o u r w i t h r e l a t i v e s a f e t y t o o b t a i n an i n c r e a s e d l o a f volume and l e s s f i r m crumb w i t h l i t t l e danger o f o v e r t r e a t m e n t . Flour Proteases. In the b a k i n g p r o c e s s , the q u a l i t y and q u a n t i t y of g l u t e n p r o t e i n determines the r h e o l o g i c a l c h a r a c t e r i s t i c s o f the dough. Wheat f l o u r c o n t a i n s 7% to 15% p r o t e i n w i t h about 80% b e i n g gluten. Ungerminated f l o u r c o n t a i n s low l e v e l s o f p r o t e a s e which p l a y a minor r o l e i n bread making. S i n c e the 1920's, p r o t e o l y t i c enzymes have been used as a d d i t i v e s i n b a k i n g t o improve dough h a n d l i n g p r o p e r t i e s . At t h a t time, the main s o u r c e was m a l t and i t was o f t e n d i f f i c u l t t o c o n t r o l the r a t i o o f a l p h a amylase and protease i n malt products. The a r t of t a i l o r i n g malt p r o d u c t s f o r the b a k i n g i n d u s t r y f o c u s e d p r i m a r i l y on the time and the c o n d i t i o n used f o r m a l t i n g . The use of p r o t e a s e s i n b a k i n g i n c r e a s e d s i g n i f i c a n t l y i n the 1950's a f t e r the i n t r o d u c t i o n o f f u n g a l



34


proteases from A. oryzae. Today the supplementation of flour made from hard, high protein wheat i s routinely practiced i n order to improve dough handling properties, to control mixing times, and to increase loaf volume. Without protease addition, high gluten doughs produce small loaves with b l i s t e r e d or cracked crusts. The level of protease a c t i v i t y i n a flour i s p a r t i c u l a r l y important today due to the use of automated baking operations that require s t r i c t control of mixing time. The controlled use of proteolytic enzymes can reduce the dough mixing time by 30% without adverse effect on dough properties and on bread quality. Excess proteolytic a c t i v i t y w i l l cause slack, sticky doughs and poor quality bread. Today the baking industry uses protease products tailored for their use from fungal proteases. Fungal proteases are less stable than malt proteases and are inactivated at a lower temperature. This feature allows for r e l a t i v e l y good tolerance to overusage. Fungal protease products contain both endopeptidase and exopeptidase a c t i v i t i e s . The endopeptidases affect the v i s c o e l a s t i c properties of gluten. The exopeptidases produce free amino acids which improve both bread flavor and crust color through increased Maillard reactions during baking. Bread Staling. Staling i n the baking industry i s a problem begging to be solved and the enzyme industry i s starting to t a i l o r products directed toward solving the problem. Staling of bakery products i s a general term used to cover a large number of changes that occur during the normal storage of bakery products. For the consumer, the staleness of a bakery product i s the sum of several perceptive judgements about the changes i n several product attributes. The major attributes used by a consumer to judge staleness are loss of perceived moisture and the firming or texture deterioration of the crumb. Up to very recently the dogma regarding the mechanism of s t a l i n g has focused on the rétrogradation of starch as the primary cause. The change i n the c r y s t a l l i n e structure of starch over time does play a role i n the s t a l i n g processes. Current theories on staling are focusing more on the level of free water i n the product with starch rétrogradation being one of the major mechanisms through which water becomes bound during s t a l i n g . It may not be the presence of retrograded starch that causes the firmness but the reduction of free water content below a c r i t i c a l l e v e l . Soon after the practice of supplementing flour with alpha amylase started, observations were made that the higher the amylase l e v e l , the longer the shelf l i f e . But, as discussed e a r l i e r , excess alpha amylase a c t i v i t y can lead to a gummy bread texture, d i f f i c u l t to s l i c e and unacceptable to the consumer. In 1953, a comprehensive study (12^) was reported i n which fungal, cereal and b a c t e r i a l alpha amylases were investigated at equivalent levels for their a b i l i t y to reduce firmness development. A l l three amylase products retarded firmness development and t h e i r effectiveness to do so was p o s i t i v e l y correlated to their heat s t a b i l i t i e s . The b a c t e r i a l amylase, having the highest heat s t a b i l i t y , produced the biggest effect while the fungal amylase showed the least having been inactivated before the starch was gelatinized. Theories were proposed that the amylase produced dextrins that interfered with the c r y s t a l l i z a t i o n of starch. However, i n 1959, based on X-ray studies of bread (13), i t was found that b a c t e r i a l alpha amylase treatment increased rather



3. SPRADLIN


35

than decreased starch c r y s t a l l i z a t i o n . In 1980, another X-ray study (14) looked at the starch c r y s t a l l i z a t i o n pattern i n control bread and breads that had been treated with alpha amylases from malt, fungal and b a c t e r i a l sources. The basic findings agreed with the 1959 study i n that starch c r y s t a l l i n i t y and bread firming were not synonymous and that c r y s t a l l i z a t i o n increased upon amylase treatment. The news i n 1980 was that the type of crystals found i n amylase treated bread were different than the type of crystals found in the control bread. Previous to this 1980 study, i t was known that starches found i n nature are c r y s t a l l i n e and that they exist i n two forms, A and B. The A form contains less than 7% water i n i t s c r y s t a l l i n e structure while the Β form contains over 25% water i n i t s structure (L5). In the study, the control bread formed Β type crystals while the amylase treated bread formed A type c r y s t a l s . Due to the potential of producing gummy bread due to excess alpha amylase a c t i v i t y , the baking industry never adopted for regular use any of the commercially available amylase products for the primary purpose of freshness extension. Recently the enzyme industry set out to t a i l o r products for the baking industry that would decrease the rate of firming i n baked goods without the r i s k of producing a gummy texture. During the last two years, at least four enzyme companies have developed special products and are promoting them for this application. The two major challenges were to develop an enzyme product that would a) allow bread to r e t a i n i t s moisture as free water for as long as possible and b) would have a b u i l t - i n application tolerance l e v e l that would allow wide v a r i a t i o n i n use level without causing production or product problems. The approach one company i s employing i s to use a debranching enzyme along with a low level of b a c t e r i a l alpha amylase (16). They had developed a debranching enzyme from the bacterium Bacillus acidopullulyticus for use i n the corn syrup industry. By blending their debranching enzyme with an alpha amylase, an enzyme preparation was obtained with the a b i l i t y to greatly reduce the formation of Β starch crystals while maintaining a "forgiveness factor" before product or production problems are experienced. A second company also focused i n on changing the structure of the starch. Their approach i s to prevent Β type starch c r y s t a l l i z a t i o n by rearranging the branched portions of the starch chain v i a a transfer reaction. They developed a debranching system for the corn syrup business by transferring a unique amylase gene from Bacillus megaterium into Bacillus s u b t i l i s . This unique amylase has the a b i l i t y to transfer branched dextrin to glucose. In their a n t i - s t a l i n g enzyme preparation they have a blend of the B. megaterium amylase and a glucoamylase. Their l i t e r a t u r e claims that their new product w i l l improve moulding properties, crust color, height, flavor, and rate of s t a l i n g . Another component of flour, the pentosans, have a very high capacity to bind water. Pentosans are present i n wheat to about 4-5% and i n patent wheat flour to about 2.5-3%. Pentosans contribute to about one quarter of the adsorption of dough water which i s disproportionately more than the starch or protein fractions. Pentosans can absorb about 6.5 times their weight of water. A s i g n i f i c a n t a n t i - s t a l i n g effect has been claimed by the addition of pentosanase to bread (17-18).



36


An enzyme company has now i n t r o d u c e d i n t o the market an enzyme p r e p a r a t i o n produced by T r i c h o d e r m a r e e s e i t h a t c o n t a i n s a m i x t u r e of c e l l u l a s e , b e t a - g l u c a n a s e and p e n t o s a n a s e . The c l a i m i s t h a t t h i s new p r o d u c t i s t a i l o r made f o r the b a k i n g i n d u s t r y t o extend the s o f t n e s s l i f e o f b r e a d . A f o u r t h enzyme company has added c e l l u l a s e , h e m i c e l l u l a s e and pentosanase t o f u n g a l a l p h a amylase as t h e i r e n t r y i n t o the a n t i - s t a l i n g market. They c l a i m t h a t the added enzymes work s y n e r g i s t i c a l l y w i t h the amylase t o produce a p r o d u c t w i t h reduced tendency t o become f i r m e r w i t h time. Dairy The d a i r y i n d u s t r y i s v e r y h e a v i l y dependent on the enzyme industry. Without enzymes t h e r e would be no cheese i n d u s t r y . The p r i n c i p a l enzymes used i n d a i r y t e c h n o l o g y a r e p r o t e a s e s , l i p a s e s / e s t e r a s e s , and l a c t a s e s . Cheese p r o d u c t i o n i s the l a r g e s t consumer o f p r o t e a s e s and has been the t a r g e t i n d u s t r y f o r t a i l o r e d enzyme p r e p a r a t i o n s f o r many y e a r s . Besides t a i l o r i n g protease p r e p a r a t i o n s , the enzyme i n d u s t r y has d e v e l o p e d many l i p a s e / e s t e r a s e p r e p a r a t i o n s s p e c i f i c a l l y f o r the r i p e n i n g and f l a v o r development o f cheese p r o d u c t s . The whole c o n c e p t o f r a p i d r i p e n e d cheeses and cheese f l a v o r i n g p r o d u c t s i s based on the use o f t a i l o r e d enzyme preparations. The f l a v o r l e v e l s o f enzyme m o d i f i e d cheese (EMC) p a s t e s and powders a r e 5-100 times g r e a t e r than cheeses aged by conventional procedures. The p o t e n t i a l uses o f l a c t a s e t o overcome n u t r i t i o n a l and p r o d u c t a t t r i b u t e problems have l e d t o the t a i l o r i n g o f many l a c t a s e p r o d u c t s , s e v e r a l i n an i m m o b i l i z e d state. Milk Coagulation. The f i r s t s t e p i n cheese manufacture i s t h e coagulation of milk. T r a d i t i o n a l l y , t h i s c o a g u l a t i o n step i s c a t a l y z e d by the enzyme r e n n e t . Rennet i s a s a l i n e e x t r a c t o f the 4th stomach o f c a l v e s , u s u a l l y s l a u g h t e r e d b e f o r e they a r e 30 days old. The p r i n c i p a l p r o t e a s e i n r e n n e t i s r e n n i n . In an attempt t o a v o i d c o n f u s i o n w i t h the hormone p e p t i d e r e n i n , t h e I n t e r n a t i o n a l Enzyme Nomenclature Committee has a s s i g n e d t h e name chymosin t o t h e protease i n c a l f rennet. D u r i n g the growth o f c a l v e s , chymosin i s r e p l a c e d by p e p s i n , the a c i d p r o t e a s e o f the mature stomach. Chymosin i n i t i a t e s t h e c l o t t i n g o f m i l k by s e l e c t i v e l y c l e a v i n g a s i n g l e p h e n y l a l a n i n e - m e t h i o n i n e bond i n k a p p a - c a s e i n . Cleavage o f t h i s one bond causes the l o s s o f a p e p t i d e from the m i l k ' s micelles. T h i s p e p t i d e i s t h e p a r t o f the n a t i v e m i c e l l e t h a t m a i n t a i n s a n e t n e g a t i v e c h a r g e on the m i c e l l e ' s s u r f a c e c a u s i n g the m i c e l l e s t o r e p e l one a n o t h e r . Once t h i s p e p t i d e i s l o s t the m i c e l l a r s u r f a c e c h a r g e s ( z e t a p o t e n t i a l ) a r e reduced t o about half. The m i c e l l e s c a n now a s s o c i a t e t o form a network s t r u c t u r e o f protein. On e l e c t r o n m i c r o s c o p y e x a m i n a t i o n the i n d i v i d u a l m i c e l l e s can s t i l l be c l e a r l y d i s t i n g u i s h e d i n d i c a t i n g t h a t chymosin does not d i s r u p t the m i c e l l e s t r u c t u r e . Chymosin i s h i g h l y s p e c i f i c i n i t s action. By s p l i t t i n g a s i n g l e p e p t i d e bond i n kappa c a s e i n , which i s o n l y about 10% o f the t o t a l c a s e i n i n m i l k and even l e s s than t h a t o f t h e t o t a l p r o t e i n , chymosin can produce d r a m a t i c changes i n the p h y s i c a l p r o p e r t i e s o f m i l k . Chymosin i s an e x c e l l e n t example of t h e marked e f f e c t enzymes c a n have on t h e f u n c t i o n a l p r o p e r t i e s of f o o d .



3. SPRADLIN


37

U n f o r t u n a t e l y , cheese consumption i s c o n s t a n t l y i n c r e a s i n g and v e a l consumption i s c o n s t a n t l y d e c r e a s i n g . F o r s e v e r a l y e a r s now, t h e r e has been a major s h o r t a g e i n c h y m o s i n . Chymosin i s an a c i d protease. More s p e c i f i c a l l y i t i s an a s p a r t a t e p r o t e a s e . I t i s so named because i t b e l o n g s to a c l a s s of a c i d p r o t e a s e s t h a t have two a s p a r t i c a c i d r e s i d u e s i n v o l v e d i n the a c t i v e s i t e . The t a s k f o r enzyme m a n u f a c t u r e r s i s to d e v e l o p m i c r o b i a l a c i d p r o t e a s e p r e p a r a t i o n s t h a t come as c l o s e as p o s s i b l e to the c a t a l y t i c and s t a b i l i t y p r o p e r t i e s of c h y m o s i n . Nearly a l l a c i d proteases w i l l c a t a l y z e the r a p i d h y d r o l y s i s o f the key p h e n y l a l a n i n e - m e t h i o n i n e bond i n kappa c a s e i n . The problem i s t h a t o t h e r bonds are c l e a v e d and the r e l a t i v e r a t e s o f t h e i r h y d r o l y s i s i s h i g h compared to the r e l a t i v e r a t e f o r chymosin. S u c c e s s f u l m i l k c o a g u l a n t s have been produced from m i c r o b i a l s o u r c e s f o r use i n cheesemaking. The major organisms used to produce m i c r o b i a l rennet p r o d u c t s a r e Mucor p u s i l l u s , Mucor m e i h e i , and E n d o t h i a p a r a s i t i c a w i t h Mucor p u s i l l u s b e i n g the most w i d e l y u s e d . Mucor p u s i l l u s was the f i r s t m i c r o b i a l rennet i d e n t i f i e d t h a t s u c c e s s f u l l y produced a good q u a l i t y c h e e s e . In the 20 y e a r s s i n c e t h e i r f i r s t d i s c o v e r y , m i c r o b i a l r e n n e t s have been t a i l o r e d to improve t h e i r f u n c t i o n a l i t y i n cheese making to more c l o s e l y match the f u n c t i o n a l i t y of c h y m o s i n . M i c r o b i a l rennets i n g e n e r a l have the d i s a d v a n t a g e of b e i n g too s t a b l e . The organisms have been mutated and the enzymes c h e m i c a l l y m o d i f i e d to i n c r e a s e t h e i r s p e c i f i c i t y and to d e c r e a s e t h e i r s t a b i l i t y . In the p r o c e s s o f t a i l o r i n g t h e r m a l l y d e s t a b i l i z e d m i c r o b i a l r e n n e t s , an unexpected b e n e f i t was o b t a i n e d as an i n c r e a s e i n the s p e c i f i c i t y of the m i c r o b i a l enzyme making i t s performance v i r t u a l l y i n d i s t i n g u i s h a b l e from c a l f r e n n e t (20) . Of c o u r s e , the u l t i m a t e t a i l o r i n g t a s k s t i l l needed to be done. U s i n g recombinant DNA t e c h n i q u e s , the b o v i n e chymosin gene needed to be e x p r e s s e d i n a m i c r o o r g a n i s m . Commercial p r o d u c t i o n o f chymosin u s i n g recombinant DNA t e c h n i q u e s became the t a r g e t o f a number of biotechnology firms. At f i r s t the gene was i n s e r t e d i n t o b a c t e r i a and y e a s t c e l l s m a i n l y because the s t a t e of the a r t i n e x p r e s s i o n v e c t o r s was b e s t u n d e r s t o o d i n these o r g a n i s m s . The recombinant DNA p r o c e d u r e s worked g r e a t but the chymosin t h a t was formed was i n an i n a c t i v e form and r e q u i r e d a c o s t l y r e n a t u r a t i o n p r o c e s s to o b t a i n an a c t i v e enzyme. The problem was one t h a t i s common to a number o f complex p r o t e i n s whose c o n f o r m a t i o n i s determined i n p a r t by f o r m a t i o n o f one o r more d i s u l f i d e bonds. When t h e s e p r o t e i n s are produced i n a m i c r o o r g a n i s m , the p r o p e r d i s u l f i d e bonds o f t e n do not form and the p r o t e i n assumes an i n a c t i v e c o n f o r m a t i o n . One way around t h i s problem i s to produce the p r o t e i n i n an o r g a n i s m t h a t w i l l s e c r e t e i t i n i t s a c t i v e form. M i c r o b i a l r e n n e t s , c u r r e n t l y produced and used i n cheese p r o d u c t i o n , are a s p a r t y l p r o t e a s e s l i k e c h y m o s i n . T h e r e f o r e , i f the b o v i n e gene f o r chymosin c o u l d be t r a n s f e r r e d to and e x p r e s s e d i n the same type o f organism t h a t c u r r e n t l y produces an a s p a r t y l p r o t e a s e , then the p r o b a b i l i t y of s u c c e s s i s much higher. S i n c e a l l m i c r o b i a l r e n n e t s a r e produced by f i l a m e n t o u s f u n g i , i n v e s t i g a t o r s t u r n e d to f i l a m e n t o u s f u n g i (19) i n o r d e r to s u c c e s s f u l l y o b t a i n e x p r e s s i o n and s e c r e t i o n of b o v i n e c h y m o s i n . Today, a c t i v e b o v i n e chymosin has been s u c c e s s f u l l y produced from s e v e r a l f i l a m e n t o u s f u n g i and they are i n d i s t i n g u i s h a b l e from the n a t u r a l enzyme o b t a i n e d from c a l v e s ' stomachs.



38


Cheese R i p e n i n g . Rennet p l a y s a major r o l e i n the t e x t u r e and f l a v o r development o f cheese d u r i n g the r i p e n i n g p r o c e s s . Besides the r a p i d c l e a v a g e of the key p h e n y l a l a n i n e - m e t h i o n i n e bond to c o a g u l a t e m i l k , chymosin has been shown t o h y d r o l y z e a t l e a s t 22 o t h e r bonds i n the c a s e i n m o l e c u l e s . The f a v o r e d amino a c i d s a t the p o i n t of c l e a v a g e are l e u c i n e , i s o l e u c i n e and p h e n y l a l a n i n e . Chymosin produces cheese f r e e o f b i t t e r f l a v o r and o f e x c e l l e n t texture. The use o f o t h e r a c i d p r o t e a s e s such as p e p s i n and many o f the m i c r o b i a l r e n n e t s i n the c o a g u l a t i o n s t e p o f t e n l e a d s to b i t t e r f l a v o r and a s o f t t e x t u r e a f t e r r i p e n i n g . In t r a d i t i o n a l cheese making, w h i l e m i l k i s b e i n g c o a g u l a t e d u s i n g a rennet p r e p a r a t i o n , s t a r t e r c u l t u r e s of m i c r o o r g a n i s m s a r e added which a r e c a r e f u l l y s e l e c t e d f o r the type o f cheese b e i n g produced. Most v a r i e t i e s o f cheese undergo some degree of a g i n g which improves f l a v o r and t e x t u r e . C e r t a i n f r e e amino a c i d s appear w i t h i n a few hours a f t e r the c u r d i s h a r v e s t e d i n d i c a t i n g the presence of exopeptidases. In m o z z a r e l l a cheese d e s i r a b l e changes i n f l a v o r and t e x t u r e are e v i d e n t w i t h i n two weeks. Both p r o t e o l y s i s and l i p o l y s i s are i n v o l v e d i n the cheese ripening process. The r a t e and e x t e n t o f t h e i r i n t e r a c t i o n s a r e i n f l u e n c e d by the rennet p r e p a r a t i o n used, c h a r a c t e r i s t i c s of the s t a r t e r c u l t u r e , pH, m o i s t u r e range, s a l t i n g p r a c t i c e s , temperature, and the a c t i v i t y of a d v e n t i t i o u s m i c r o o r g a n i s m s p r e s e n t i n o r on the cheese. Rapid Ripened Cheese. Hard cheeses r e q u i r e p r o l o n g e d r i p e n i n g times thus the cheese m a n u f a c t u r e r must a c c e p t c e r t a i n time l a g s between the i n i t i a l c o n v e r s i o n o f m i l k and the r e a l i z a t i o n o f the v a l u e o f the r e s u l t i n g c h e e s e . The time l a g i s dependent upon the cheese variety. For example, mature cheddar r e q u i r e s up to one y e a r t o achieve f u l l maturity. The m a t u r a t i o n p e r i o d t i e s up l a r g e amounts o f c a p i t a l and i s a c o s t l y p r o c e s s . The c a p a c i t y of a cheese p r o d u c t i o n f a c i l i t y i s o f t e n l i m i t e d by the space a v a i l a b l e f o r m a t u r i n g the c h e e s e . Being a b l e to a c c e l e r a t e the a g i n g p r o c e s s would be a major c o s t e f f i c i e n c y improvement. S i n c e many v a r i e t i e s o f cheese a r e matured by the a c t i o n o f i n d i g e n o u s enzymes r a t h e r than by t h e i r v i a b l e m i c r o f l o r a , a g i n g o f these v a r i e t i e s has the p o t e n t i a l of b e i n g a c c e l e r a t e d by a r t i f i c i a l l y i n c r e a s i n g the c o n c e n t r a t i o n o f those enzymes t h a t have a d e f i n i t e r o l e i n the r i p e n i n g p r o c e s s . The t a i l o r i n g o f enzyme p r e p a r a t i o n s to a c c e l e r a t e the r i p e n i n g p r o c e s s , w h i l e g i v i n g e x c e l l e n t f l a v o r and t e x t u r e p r o p e r t i e s , i s a major c h a l l e n g e b e i n g t a c k l e d by the enzyme i n d u s t r y today. P r o t e a s e s f o r A c c e l e r a t i n g Cheese R i p e n i n g . P r o t e o l y s i s p l a y s such a major r o l e i n d e v e l o p i n g the f l a v o r and t e x t u r e o f hard cheese t h a t the a d d i t i o n o f exogenous m i c r o b i a l p r o t e a s e s has been attempted u s i n g every c o m m e r c i a l l y a v a i l a b l e p r o t e a s e p r e p a r a t i o n . By v e r y c a r e f u l l y c h o o s i n g the p r o t e a s e and i t s l e v e l , v e r y s t r o n g f l a v o r e d cheese can be produced i n a s h o r t time. However, i t i s v e r y d i f f i c u l t to produce the p r o p e r b a l a n c e of f l a v o r and t o a v o i d b i t t e r defects i n real applications. To v a r y i n g d e g r e e s , a l l p r o t e a s e treatments w i l l i n c r e a s e the s o f t n e s s o f the f i n a l p r o d u c t , thus a d d i n g a n o t h e r d e f e c t the cheese m a n u f a c t u r e r has to m i n i m i z e



3. SPRADLIN


39

i n t h e r a p i d r i p e n e d cheese p r o d u c t s . The l i m i t o f the magnitude by which cheese f l a v o r s c a n be i n c r e a s e d w i t h p r o t e a s e treatment i s l i m i t e d by t h e o n s e t o f d e f e c t s . When f a c e d w i t h a problem t h a t c a r r i e s a b i g bottom l i n e p r o f i t p o t e n t i a l , t e c h n o l o g i e s w i l l be developed t o s o l v e t h a t problem. A new approach now b e i n g used t o t a i l o r enzyme p r e p a r a t i o n s f o r the cheese i n d u s t r y i s a s p i n - o f f o f the m e d i c a l / p h a r m a c e u t i c a l i n d u s t r i e s technology. Liposome t e c h n o l o g y i s t h e a r t o f p r o d u c i n g m u l t i l a y e r e d l i p i d p a r t i c l e s r a n g i n g i n s i z e from l e s s than one m i c r o n t o s e v e r a l microns w i t h n o n - l i p i d m a t e r i a l s t r a p p e d between layers. I n one c u r r e n t liposome enzyme p r e p a r a t i o n b e i n g t e s t e d now i n cheese making, p r o t e a s e s a r e e n c a p s u l a t e d i n a system o f p h o s p h o l i p i d b i l a y e r membranes ( 2 1 ) . T h i s liposome enzyme system i s a c o m b i n a t i o n o f an e n d o p e p t i d a s e and an e x o p e p t i d a s e . The enzymes a r e s l o w l y r e l e a s e d a f t e r the cheese i s put i n t o the a g i n g chamber. U s i n g these liposomes f o r cheddar cheese p r o d u c t i o n showed t h a t the liposomes were e v e n l y d i s t r i b u t e d and 90% o f the added enzyme was r e t a i n e d i n the cheese curd. The r e s u l t i n g cheese r i p e n e d i n h a l f the normal time w i t h e x c e l l e n t f l a v o r and t e x t u r a l p r o p e r t i e s . The t a i l o r i n g o f p r o t e a s e p r e p a r a t i o n s t o r a p i d l y produce h a r d cheeses i n a c o s t e f f e c t i v e manner and w i t h good f l a v o r and t e x t u r a l properties, i s progressing rapidly. I n t h e next few y e a r s s e v e r a l new and i n n o v a t i v e l y t a i l o r e d enzyme p r e p a r a t i o n s w i l l be a v a i l a b l e f o r a c c e l e r a t i n g the p r o d u c t i o n of hard cheeses. L i p a s e s f o r A c c e l e r a t i n g Cheese R i p e n i n g . Acceleration of l i p o l y s i s by the a d d i t i o n o f e i t h e r a n i m a l o r m i c r o b i a l l i p a s e s has been s u c c e s s f u l l y a p p l i e d to the r e l a t i v e l y s t r o n g f l a v o r e d cheeses. L i p a s e s d e r i v e d from the p r e g a s t r i c g l a n d o f k i d , c a l f and lamb a r e c u r r e n t l y b e i n g added t o a c c e l e r a t e r i p e n i n g i n I t a l i a n type cheeses where t h e c h a r a c t e r i s t i c r a n c i d f l a v o r o f low m o l e c u l a r weight f r e e f a t t y a c i d s , f o r example b u t y r i c a c i d , i s d e s i r a b l e . However, when these p r e g a s t r i c l i p a s e s a r e used t o a c c e l e r a t e m i l d f l a v o r e d cheese such as cheddar, t o o much o f the low m o l e c u l a r weight f a t t y a c i d s a r e produced and a r a n c i d f l a v o r d e v e l o p s . When the a n i m a l pancreas l i p a s e s a r e used i n a h i g h c o n c e n t r a t i o n , e x c e s s i v e amounts o f l a u r i c a c i d a r e produced g i v i n g the cheese a soapy t a s t e . Also, a major d i s a d v a n t a g e o f u s i n g any o f these l i p a s e p r e p a r a t i o n s t a i l o r e d from a n i m a l g l a n d s i s the p r o t e a s e t h a t i s always p r e s e n t . A l t h o u g h the p r o t e a s e i s p r e s e n t i n s m a l l amounts, cheese s o f t e n i n g o c c u r s and a t times b i t t e r o f f - f l a v o r s a r e produced. To d a t e , t h e use o f m i c r o b i a l l i p a s e has a l s o been p r i m a r i l y i n t h e v e r y s t r o n g f l a v o r e d cheeses, l i k e b l u e cheese where r a n c i d and soapy f l a v o r notes a r e l e s s n o t i c e a b l e . When t h e r e i s a need, someone w i l l s o l v e i t . A p a t e n t was i s s u e d i n 1987 (^2) d e s c r i b i n g a new and n o v e l l i p a s e produced by a mutant s t r a i n o f A s p e r g i l l u s t h a t has an a c c e l e r a t i n g e f f e c t on cheese f l a v o r development w i t h o u t l y p o l y t i c enzyme a s s o c i a t e d rancidity. The p a t e n t c l a i m s t h a t t h i s new l i p a s e w i l l be u s e f u l as a r i p e n i n g a c c e l e r a t o r i n t h e p r o d u c t i o n o f m i l d f l a v o r e d cheeses such as cheddar. Enzyme M o d i f i e d Cheese. I f the t e x t u r e i s s u e i s removed from the d e s i r a b l e a t t r i b u t e s o f cheese p r o d u c t i o n and the o n l y a t t r i b u t e



40


d e s i r e d i s f l a v o r , then a whole new a r e a becomes a v a i l a b l e f o r t a i l o r e d enzyme p r e p a r a t i o n s . The importance of f r e e f a t t y a c i d s to the c h a r a c t e r i s t i c f l a v o r o f v a r i o u s t y p e s of cheese and to d a i r y p r o d u c t s i n g e n e r a l , becomes v e r y apparent as t e c h n o l o g i s t s e v a l u a t e and study the c h e m i s t r y o f food f l a v o r s . The mechanisms and c h e m i s t r y by which r e l a t i v e l y t a s t e l e s s cheese c u r d s become d i s t i n c t i v e l y f l a v o r e d cheeses have been under r e s e a r c h s c r u t i n y f o r many decades. L i p o l y s i s i s d e f i n e d as the enzyme-catalyzed h y d r o l y t i c c l e a v a g e o f t r i g l y c e r i d e s r e s u l t i n g i n the r e l e a s e o f f r e e f a t t y a c i d s . But l i p a s e s d i f f e r g r e a t l y i n t h e i r c a t a l y t i c s p e c i f i c i t y and a r e r e s p o n s i b l e f o r many o f the d i f f e r e n c e s among cheese f l a v o r s . The m i c r o b i a l l i p a s e s , produced by the s t a r t e r c u l t u r e m i c r o o r g a n i s m s , a r e o f t e n the d e c i d i n g f a c t o r on the type o f cheese b e i n g produced. The f l a v o r o f the I t a l i a n cheeses - romano, parmesan, and p r o v o l o n e - a r e e n t i r e l y dependent on f r e e f a t t y a c i d s f o r t h e i r d i s t i n c t i v e flavor. The c a t a l y t i c 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 d i f f e r i n f o u r major a r e a s : f a t t y a c i d c h a i n l e n g t h ; p o s i t i o n o f the f a t t y a c i d i n the g l y c e r i d e s t r u c t u r e ; degree of e s t e r f i c a t i o n i n the g l y c e r i d e ( t r i - , d i - , o r mono-); and the p h y s i c a l s t a t e of the g l y c e r i d e . L i p o l y t i c enzymes r e s p o n s i b l e f o r v a r i o u s cheese f l a v o r s demonstrate p r e f e r e n t i a l r e l e a s e of s p e c i f i c f a t t y a c i d s from one p o s i t i o n i n the b u t t e r f a t when the b u t t e r f a t i s i n the c o r r e c t p h y s i c a l form. Enzyme p r e p a r a t i o n s a r e now b e i n g t a i l o r e d by p r e c u l t u r i n g the a p p r o p r i a t e organism under c o n d i t i o n s t h a t a l l o w the a c c u m u l a t i o n o f the d e s i r e d enzymes f r e e i n s o l u t i o n . Enzyme p r o d u c t s a r e now a v a i l a b l e t h a t a r e c a p a b l e o f p r o d u c i n g s p e c i f i c cheese f l a v o r s i n s l u r r i e s of b u t t e r f a t or i n s l u r r i e s of cheese c u r d . F o r example, a romano cheese f l a v o r was r e c e n t l y r e p o r t e d (^3) t h a t was produced from a v e r y s i m p l e b u t t e r f a t e m u l s i o n . An e m u l s i o n o f water washed b u t t e r and Tween 80 i n phosphate b u f f e r was t r e a t e d w i t h a c r u d e enzyme m i x t u r e i s o l a t e d from Candida r u g o s a . Today, cheese f l a v o r e d p a s t e and powders, c o n t a i n i n g from 5 to 100 times the f l a v o r l e v e l o f n a t i v e c h e e s e , a r e a v a i l a b l e i n the m a r k e t p l a c e f o r most cheese v a r i e t i e s . N e a r l y a l l o f these p r o d u c t s a r e made by u s i n g added enzymes w i t h most o f them u s i n g a b l e n d o f enzymes and m i c r o o r g a n i s m s . S e l e c t e d cheese f l a v o r s a r e produced i n a l i q u i d o r s l u r r y form i n a r e l a t i v e l y s h o r t time compared to making the t r u e cheese and a t a f l a v o r l e v e l many f o l d above the maximum f l a v o r o b t a i n a b l e i n the t r u e c h e e s e . The s y n e r g i s t i c use of enzymes and m i c r o o r g a n i s m s t o produce n a t u r a l f l a v o r s f o r foods i s an i n d u s t r y o f i t s own j u s t now b e i n g born. The t a i l o r i n g of enzyme p r o d u c t s w i t h and w i t h o u t m i c r o o r g a n i s m s , w i l l be a major market a r e a f o r the enzyme i n d u s t r y i n the f u t u r e . Lactase. L a c t o s e , p r e s e n t i n m i l k , i s a d i s a c c h a r i d e sugar composed o f g l u c o s e and g a l a c t o s e . L a c t o s e i s a major problem t o the d a i r y industry. I t i s non-sweet, v e r y low i n s o l u b i l i t y , cannot be absorbed d i r e c t l y from the i n t e s t i n e o f humans and i s m e t a b o l i z e d v e r y p o o r l y by m i c r o o r g a n i s m s . In c o n t r a s t , i t s components ( g l u c o s e and g a l a c t o s e ) have a sweetening power of about 0.8 r e l a t i v e to s u c r o s e , a r e 3 t o 4 times more s o l u b l e than l a c t o s e , a r e e a s i l y absorbed from the i n t e s t i n e and the g l u c o s e i s h i g h l y f e r m e n t a b l e by microorganisms.



3. SPRADLIN


41

L a c t a s e i s an enzyme t h a t s e l e c t i v e l y h y d r o l y z e s l a c t o s e i n t o g l u c o s e and g a l a c t o s e thus h a v i n g the p o t e n t i a l f o r the t a i l o r i n g of enzyme p r e p a r a t i o n s f o r wide use i n the d a i r y i n d u s t r y . Lactase treatment p r o v i d e s a method o f p r e v e n t i n g l a c t o s e c r y s t a l l i z a t i o n i n f r o z e n m i l k and whey based p r o d u c t s as w e l l as i n l i q u i d concentrated milk products. Humans i n t o l e r a n t of l a c t o s e can p r e v e n t s u f f e r i n g from m i l k i n d u c e d abdominal p a i n and o t h e r g a s t r o i n t e s t i n a l d i s t r e s s e s by u s i n g l a c t a s e - t r e a t e d m i l k . Today the l a r g e s t market f o r l a c t a s e i s by i n d i v i d u a l s s u f f e r i n g from lactose intolerance. S e v e r a l y e a r s ago the enzyme i n d u s t r y looked a t the cheese i n d u s t r y and saw l a c t a s e as a b i g o p p o r t u n i t y . B e s i d e s the l a c t o s e c r y s t a l l i z a t i o n problem i n f r o z e n and c o n c e n t r a t e d m i l k p r o d u c t s , the enzyme i n d u s t r y saw a n o t h e r p o t e n t i a l l y b i g p r o f i t a p p l i c a t i o n . Over h a l f o f the cheese whey produced i n making cheese i s d i v e r t e d to waste treatment o r waste d i s p o s a l systems r a t h e r than b e i n g p r o c e s s e d f o r food and feed u s e . U p g r a d i n g cheese whey became the hot development i t e m f o r the enzyme i n d u s t r y . The f i r s t problem f a c e d by the enzyme i n d u s t r y was to p r e p a r e p r e p a r a t i o n s h a v i n g the same c a t a l y t i c p r o p e r t i e s and y e t be a b l e to work a t e x t r e m e l y d i f f e r e n t pH v a l u e s . The pH o f m i l k i s 6.4 to 6.8 w h i l e the pH o f cheese whey i s c l o s e r to 4. A d j u s t i n g the pH o f e i t h e r feed stream i s not a f e a s i b l e answer to the p r o b l e m . Enzyme p r e p a r a t i o n s were d e v e l o p e d from d a i r y y e a s t t h a t a r e a c t i v e around pH 6.7 f o r use i n m i l k systems and o t h e r enzyme p r e p a r a t i o n s were d e v e l o p e d from f u n g a l organisms a c t i v e around pH 4 . 0 f o r use i n cheese whey. The use of s o l u b l e p r e p a r a t i o n s o f a c i d l a c t a s e was never d e v e l o p e d i n t o a c o s t e f f e c t i v e p r o c e s s f o r u p g r a d i n g whey. The low l e v e l of l a c t o s e i n the whey c r e a t e d a l a r g e expense i n c o n c e n t r a t i n g the p r o d u c t s . T h i s p l u s the h i g h c o s t o f food approved l a c t a s e made the p r o d u c t i o n o f sugar from whey uneconomical. The next t a i l o r i n g s t e p , i n an attempt to upgrade whey, was to i m m o b i l i z e the f u n g a l l a c t a s e and use the h y d r o l y z e d p r o d u c t as f e e d s t o c k to grow b a k e r ' s y e a s t . S e v e r a l companies spent y e a r s and a l o t o f money d e v e l o p i n g i m m o b i l i z e d l a c t a s e p r o d u c t s . The one system t h a t was put i n t o commercial p r o d u c t i o n i n the U n i t e d S t a t e s was t a i l o r e d by i m m o b i l i z i n g f u n g a l l a c t a s e onto m i c r o p o r o u s beads o f g l a s s - l i k e c a r r i e r . A f t e r s e v e r a l attempts to make the o p e r a t i o n a p r o f i t a b l e one, the f a c i l i t y was c l o s e d . Today, the l a c t a s e market i s p r i m a r i l y f o r s o l v i n g the l a c t o s e i n t o l e r a n c e problem i n human d i e t s . The t a i l o r i n g o f enzyme p r e p a r a t i o n s c o n t i n u e s and l a c t a s e may s t i l l be developed i n t o a b i g p r o f i t item. T a i l o r i n g Enzymes f o r F u t u r e Food A p p l i c a t i o n s The b i g t a i l o r i n g p r o j e c t s i n the f u t u r e w i l l be i n d e s i g n i n g enzymes u s i n g gene s y n t h e s i s , s i t e - d i r e c t e d mutagenesis and o t h e r genetic engineering technologies. The enzyme's r e s p o n s e s to i t s environment w i l l be the p r i m a r y t a r g e t s . Responses to t e m p e r a t u r e , pH, i o n s , and c h e m i c a l s w i l l be a d j u s t e d to b e s t t a i l o r an enzyme for i t s intended use. To i l l u s t r a t e the c h e m i c a l b a s i s f o r p o t e n t i a l changes, the t a i l o r i n g o f an enzyme's response to temperature w i l l be u s e d .



42


Enzymes respond to temperature i n two ways. F i r s t the enzyme's s t a b i l i t y i s a f f e c t e d by temperature. As the temperature i s i n c r e a s e d , i n a c t i v a t i o n o f the enzyme due t o thermal d e n a t u r a t i o n o f the p r o t e i n i n c r e a s e s r e s u l t i n g i n a d e c r e a s e i n the c o n c e n t r a t i o n of a c t i v e enzyme. The second e f f e c t of temperature i s on the reaction rate d i r e c t l y . F o r c h e m i c a l r e a c t i o n s , the A r r h e n i u s e q u a t i o n d e s c r i b e s the r e l a t i o n s h i p between the r a t e c o n s t a n t o f the r e a c t i o n and the a b s o l u t e temperature. T h i s e q u a t i o n , which i s e m p i r i c a l and has no t h e o r e t i c a l b a s i s , was developed i n 1889 by A r r h e n i u s to d e f i n e the a c t i v a t i o n energy o f a r e a c t i o n . The r o l e o f enzymes i n c h e m i c a l r e a c t i o n s i s to lower the a c t i v a t i o n energy. In f a c t , the a c t i v a t i o n energy o f e n z y m e - c a t a l y z e d r e a c t i o n s appear to be more c h a r a c t e r i s t i c o f the enzyme than o f the s u b s t r a t e s i n v o l v e d i n the reaction. For most e n z y m e - c a t a l y z e d r e a c t i o n s , the a c t i v a t i o n e n e r g i e s a r e between 6,000 and 15,000 c a l / m o l ( 2 4 ) . S i n c e the r a t e of r e a c t i o n i s dependent upon the a c t i v a t i o n energy, the g e n e t i c e n g i n e e r i n g t a s k i s to d e s i g n i n t o the enzyme a temperature s e n s i t i v e c o n f o r m a t i o n change. T h i s c o n f o r m a t i o n change needs to cause a major change i n the a c t i v a t i o n energy a t a d e s i r e d temperature. T h i s c o u l d be t o e i t h e r t u r n on o r t u r n o f f the a c t i v i t y depending upon the a p p l i c a t i o n . F o r example, i n cheese making i t may be d e s i r a b l e to t u r n o f f the a c t i v i t y as the temperature r i s e s , w h i l e i n bread b a k i n g i t may be d e s i r a b l e t o have enzyme a c t i v i t y o n l y a t h i g h temperatures. The s w i t c h can work i n either direction. The o t h e r t e m p e r a t u r e - r e l a t e d p r o p e r t y o f enzymes needing a t t e n t i o n i s thermal i n a c t i v a t i o n . T a i l o r i n g enzymes f o r the cheese i n d u s t r y has a l r e a d y produced l e s s s t a b l e p r o t e a s e s . T a i l o r i n g a l p h a amylase p r e p a r a t i o n s f o r the c o r n syrup i n d u s t r y has produced amylases w i t h long enough h a l f - l i v e s a t 105°C to a l l o w t h e i r use i n p r o d u c t i o n at t h a t temperature. The c u r r e n t p r o c e s s i n g t r e n d i n the food i n d u s t r y i s toward s h o r t e r p r o c e s s i n g t i m e s . One o f the major p r o c e s s i n g methods i n the food i n d u s t r y today i s r e f e r r e d t o as h i g h temperature and s h o r t time (HTST) p r o c e s s i n g . P r o d u c t s made under these c o n d i t i o n s o f t e n have p h y s i c a l p r o p e r t i e s v e r y s i m i l a r to p r o d u c t s made by much l o n g e r p r o c e s s i n g methods. However, most of the time the f l a v o r development i s not a c c e p t a b l e . Enzymes c a p a b l e o f working a t h i g h temperatures, even f o r s h o r t time p e r i o d s , are needed by the food i n d u s t r y . Microwave c o o k i n g i s a b l e to cook food r a p i d l y p r o d u c i n g the p h y s i c a l changes d e s i r e d but o f t e n f a l l s s h o r t i n f l a v o r development. Enzyme p r e p a r a t i o n s a r e needed to s o l v e the f l a v o r g e n e r a t i o n and t e x t u r a l problems a s s o c i a t e d w i t h microwave c o o k i n g and r e h e a t i n g . Conclusion The p a s t s u c c e s s o f t a i l o r i n g enzyme p r e p a r a t i o n s f o r a p p l i c a t i o n i n the food i n d u s t r y can be e x p r e s s e d by summarizing a few major a p p l i c a t i o n s o f enzymes i n the i n d u s t r y today. Almost a l l c l e a r f r u i t j u i c e s a r e enzyme t r e a t e d . A h i g h p r o p o r t i o n o f grape j u i c e , grape must and grape wine i s enzyme t r e a t e d . A l l p r o d u c t i o n o f g l u c o s e from s t a r c h i s done u s i n g the d u a l enzyme p r o c e s s . Corn syrups a r e produced by e i t h e r an acid-enzyme p r o c e s s o r by a d u a l enzyme p r o c e s s . A l l i s o m e r i z a t i o n s f o r the p r o d u c t i o n o f h i g h f r u c t o s e syrups a r e done e n z y m a t i c a l l y . Most bread i s p r o c e s s e d


3. SPRADLIN


w i t h enzyme a d d i t i v e s . Egg w h i t e s a r e g e n e r a l l y desugared enzymatically p r i o r to drying. Uses o f commercial enzymes i n the d a i r y and brewing i n d u s t r i e s a r e growing r a p i d l y . The a b i l i t y o f enzyme m a n u f a c t u r e r s t o d e s i g n enzyme p r e p a r a t i o n s and p r o c e s s e s f o r s p e c i f i c food a p p l i c a t i o n s i s l i m i t e d o n l y by t h e a b i l i t y o f t h e food p r o c e s s o r t o i d e n t i f y and communicate s p e c i f i c a p p l i c a t i o n needs t o the enzyme m a n u f a c t u r e r s .


Literature Cited

1. Cheetham, P. S. J. In Handbook of Enzyme Biotechnology; Wiseman, Α., Ed.; Ellis Horwood Limited: 1985, pp 323-361. 2. Snyder, E. C.; Kovi, E. R. U. S. Patent 2 965 520, 1960. 3. Thoma, J. Α.; Brothers, C.; Spradlin, J. Biochemistry 1970, 1768. 4. Robyt, J. F.; French, D. Arch. Biochem. Biophys. 1967, 122, 8. 5. Nielsen, G. C.; Dier, I. V.; Outtrup, N.; Norman, Β. E. U. K. Patent Appl. GB 2 097 405 A, 1982. 6. Jensen, B. F.; Norman, Β. E. Process Biochem. 1984, 19, 129. 7. David, M. H.; Gunther, H.; Roper, H. Starch/Starke 1970, 39, 436. 8. Hebeda, R. E.; Styslund, C. R.; Teague, M. Starch/Starke 1988, 40, 33. 9. Colson, C. Α.; Walon, C.; Lejeume, P.; Willinot, K. Eur. Patent Appl. 0 165 002, 1985. 10. Marshall, R. O.; Kooi, Ε. T. Science 1957, 125, 648. 11. Thoma, J. Α.; Spradlin, J. E.; Dygert, S. In The Enzymes; Boyer, P., Ed.; Academic Press: New York, 1971; Vol. 5, pp 115-189. 12. Miller, B. S.; Johnson, J. Α.; Palmer, D. L. Food Techno1 1953, 7, 38. 13. Zobel, H. F.; Senti, F. R. Cereal Chem. 1959, 36, 441. 14. Dragsdorf, R. D.; Varriano-Marston, E. Cereal Chem. 1980, 57, 310. 15. Sarko, Α.; Wu, H. C. H. Starch/Starke 1978, 30, 73. 16. Carroll, J. O.; Boyce, C. O. L.; Wong, T. M.; Starace, C. A. U. S. Patent 4 654 216, 1987. 17. Cook, Α.; Johannson, H. U. S. Patent 3 512 992, 1970. 18. Kulp, K.; Cereal Chem. 1968, 45, 339. 19. Cullen, D.; Gray, G. L.; Wilson, L. J.; Hayenga, K. J.; Lamso, M. H.; Rey, M. W.; Norton, S.; Berko, R. M. Biotech. 1987, 5, 369. 20. Branner-Jorgensen, S. U. S. Patent 4 386 160, 1983. 21. Kirby, C. J.; Brooker, Β. E.; Law, B. A. Int. J. Food Sci. Tech. 1987, 22, 355. 22. Arbige, M. V.; Newbeck, C. E. U. S. Patent 4 636 468, 1987. 23. Lee, D. -C. M.; Shi, H.; Huang, A. -S.; Carlin, J. T.; Ho, C. -T.; Chang, S. S. In Biogeneration of Aromas; Parliment, T. H.; Croteau, R., Eds.; ACS Symposium Series No. 317; American Chemical Society: Washington, DC, 1986; pp 370-378. 24. Sizer, I. W. Adv. Enzymol. 1943, 3, 35. RECEIVED

November 2, 1988


Tailoring Enzyme Systems for Food Processing - ACS Symposium

Recommend Documents