Effect of Preheat Temperature on the Hydrophobic Properties of Milk

Feb 19, 1991 - The emulsifying activity index (EAI) was greatest for II and no significant differences were observed for foam and emulsion stability b...
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Chapter 3

Effect of Preheat Temperature on the Hydrophobic Properties of Milk Proteins N. Parris, J. H. Woychik, and P. Cooke

Downloaded by YORK UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: February 19, 1991 | doi: 10.1021/bk-1991-0454.ch003

Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, PA 19118

Pre-heat treatment of skim milk for the preparation of nonfat dry milk (NDM) powders results in physicochemical changes that effect the functional behavior of the powder. The functional properties of NDM powders heated to 63°C (I), 74°C (II), and 85°C (III) for 30 min before spray drying were different. Comparison of reversed phase chromatographic profiles indicated that milk proteins were altered and a whey­ -casein complex was formed in II and III. Hydrophobic interaction chromatography (HIC) indicated that the complex formed eluted before β - c a s e i n and hence was more hydrop h i l i c than was indicated under reversed phase chromatography conditions. Alkane binding to the milk proteins in the rehydrated powders also indicated that II and III were more hydrophilic than I. Electron micrographs of immuno-gold labeled whey proteins in the rehydrated powders indicated that less than ten percent of the gold particles were associated with the casein micelles for I but greater than f i f t y percent were associated with the micelles for II and III. The appearance of the com­ plex formed in II and III on the surface of casein micelles suggests it may contribute to the increased hydrophilic character of these powders. Examination of their func­ tional properties indicated that III had the lowest s o l u b i l i t y and the greatest percent overrun in a foaming test. The emulsifying a c t i v i t y index (EAI) was greatest for II and no significant differences were observed for foam and emulsion s t a b i l i t y between the three powders. This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Nonfat dry m i l k (NDM) i s f r e q u e n t l y used as a f u n c t i o n a l i n g r e d i e n t f o r d a i r y , bakery, c o n f e c t i o n e r y , and other food a p p l i c a t i o n s . Heat treatment o f skim m i l k before spray d r y i n g i s used widely as a means o f manipulating the f u n c t i o n a l p r o p e r t i e s o f NDM powders i n m i l k produ c t s . As a r e s u l t , the end-use o f such products i s very dependent on the heat treatment the powders r e c e i v e ( 1 ) . Heat treatments can denature whey p r o t e i n s r e s u l t i n g i n aggregation which a l t e r the a b i l i t y of the powders t o rehydrate. Although d i f f i c u l t t o d i s t i n g u i s h , aggregat i o n i s a separate and u s u a l l y i r r e v e r s i b l e process which f o l l o w s denaturation o f whey p r o t e i n . Denaturation i s normally r e v e r s i b l e and can be stopped before aggregation begins (2). L i m i t e d whey p r o t e i n d e n a t u r a t i o n can improve the e m u l s i f y i n g p r o p e r t i e s of whey p r o t e i n i n food s y s tems (3.). Caseins, which account f o r about 80% of t h e t o t a l milk p r o t e i n s , e x i s t i n milk as s o l u b l e complexes or m i c e l l e s . Caseins a r e a m p h i p h i l i c i n nature and a r e p r i m a r i l y r e s p o n s i b l e f o r the e x c e l l e n t s u r f a c t a n t prope r t i e s o r f u n c t i o n a l i t y o f milk i n g r e d i e n t s . U n l i k e whey p r o t e i n s the c a s e i n a t e system i n milk i s very s t a b l e t o heat and tends t o r e s i s t c o a g u l a t i o n . Heat treatment, however, i s known t o i n c r e a s e the a c i d i t y o f m i l k s , p r i m a r i l y through the decomposition of l a c t o s e , and a l s o t o reduce both t o t a l s o l u b l e and i o n i c calcium r e s u l t i n g i n f l o c c u l a t i o n o f c a s e i n m i c e l l e s and a corresponding loss of functional properties. Preheat treatment o f skim milk between 85°C and 100°C f o r 30 min i n the p r e p a r a t i o n o f NDM powders i s commonly used i n the baking i n d u s t r y t o improve the e x t e n s i b i l i t y and water absorption of dough (4). Highheat NDM powders (85°C) absorb more water than lower heated powders and t h i s can improve i t s emulsion s t a b i l i t y and g e l a t i o n p r o p e r t i e s (5). Such f u n c t i o n a l prope r t i e s are important i n comminuted meat, c o n f e c t i o n e r y , as w e l l as i n r e c o n s t i t u t e d concentrated s t e r i l e and baked products. G e n e r a l l y extensive denaturation i s used t o prevent undesired s i d e r e a c t i o n s such as l o a f depress i o n o r c o a g u l a t i o n . Because some water a b s o r p t i o n i s necessary, i c e cream and d a i r y beverages a r e f r e q u e n t l y made with low- and medium-heat NDM (63°C, 74°C). Yogurt t e x t u r e i s g r e a t l y a f f e c t e d by the degree o f whey p r o t e i n denaturation (6). Batch-type h e a t i n g of skim milk between 85-95°C f o r 5 t o 10 min before i n o c u l a t i o n i s an important p r o c e s s i n g v a r i a b l e i n the manufacture o f yogurt. The purpose o f t h i s study was t o assess the heat induced physicochemical changes t h a t occur d u r i n g manuf a c t u r i n g o f NDM powders. Changes i n milk p r o t e i n p r o f i l e s were i d e n t i f i e d using chromatographic methods

In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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and m i c e l l a r a s s o c i a t i o n s o f whey p r o t e i n s o r complexes were examined by immuno-labeled e l e c t r o n microscopic techniques. M a t e r i a l and Methods

Downloaded by YORK UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: February 19, 1991 | doi: 10.1021/bk-1991-0454.ch003

Nonfat Dry M i l k . Raw pooled herd milk from H o l s t e i n , A y r s h i r e , and Brown Swiss cows was skimmed a t 39°C and preheated a t 63°C, 74 °C, and 85°C f o r 30 min; concen­ t r a t e d ; then spray d r i e d t o y i e l d NDM powders I, I I , and I I I according t o a p r e v i o u s l y published procedure (7). Hydrophobic I n t e r a c t i o n Chromatography (HIC). Chromatographic separations o f milk p r o t e i n s were c a r r i e d out on a Spectra Physics (San Jose, CA) SP-8800 HPLC system, equipped with a LC-HINT column, 0.46 χ 10 cm, (Supelco, Inc., B e l l e f o n t e , PA); mobile phase, s o l v e n t B: 0.05M sodium phosphate, pH 6.0, i n 3.75M urea, s o l v e n t A: 2.0M ammonium s u l f a t e i n s o l v e n t B; g r a d i e n t , 0%-100%B, 30 min; flow r a t e , 0.8 mL/min; d e t e c t o r g a i n 0.1 aufs. Sample, 1.0 g NDM was d i s s o l v e d i n 10 mL o f water, and c e n t r i f u g e d a t 100,000 χ g, 4°C, 40 min. The supernatant was f i l t e r e d (0.45 μΜ pore s i z e ) and 200 μΙ> i n j e c t e d onto the column. The p e l l e t , 3 mg, was d i s s o l v e d i n 1 ml o f s o l v e n t B, H 0, s o l v e n t A, s e p a r a t e l y , and 100 i n ­ j e c t e d onto the column. ?

Gel E l e c t r o p h o r e s i s . SDS- and urea-PAGE o f skim milk p r o t e i n s was c a r r i e d out on a PhastSystem (Pharmacia, Piscataway, NJ) as p r e v i o u s l y d e s c r i b e d (8 and Van Hekken, D. e t a l . J . Dairy S c i . i n p r e s s ) . The molecular weight standards from Bio-Rad, (Richmond, CA) and t h e i r corresponding molecular weights f o r SDS-PAGE were: phosphorylase b, 97,400; bovine serum albumin (BSA), 66,200; ovalbumin, 42,699; carbonic anhydrase, 31,000; soybean t r y p s i n i n h i b i t o r , 21,500; lysozyme, 14,000. E l e c t r o p h o r e t i c B l o t t i n g . A m o d i f i c a t i o n o f the method of Towbin e t a l . (9) was used f o r the e l e c t r o p h o r e t i c t r a n s f e r o f milk p r o t e i n s from the polyacrylamide g e l t o n i t r o c e l l u l o s e (NC) membrane. H a l f o f the g e l c o n t a i n i n g the skim milk p r o t e i n s (39°C) was s t a i n e d with Coomassie R350 dye and the other h a l f was removed from the c e l l o ­ phane backing f o r e l e c t r o b l o t t i n g . The g e l i s o l a t e d f o r immunoblotting was allowed t o e q u i l i b r a t e i n t r a n s f e r b u f f e r ; 25mM T r i s , 192mM g l y c i n e , and 20% methanol, pH 8.3, f o r 5 min. The p r o t e i n s were t r a n s f e r r e d t o a NC membrane using a M i n i Trans-Blot c e l l (Bio-Rad, Richmond, CA) a t 60 V, 0.15 A, f o r 3 h a t room temperature with e l e c t r o d e s 8 cm apart. Immunological Detection. To avoid n o n s p e c i f i c b i n d i n g , the NC membrane was incubated i n b l o c k i n g b u f f e r ; 50mM T r i s , 150mM NaCl, pH 7.4, c o n t a i n i n g 2.5 g g e l a t i n and

In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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2.5 mL 10% NP-40. The membrane was incubated f o r 1 h with primary immune antiserum t o cow whey p r o t e i n Cat. #AXL-306 (Accurate Chemical and S c i e n t i f i c Corp., Westbury, NY) and secondary antibody, goat a n t i r a b b i t IgG conjugated t o horse r a d i s h peroxidase (Behring D i a g n o s t i c , La J o l l a , CA) f o r 1 h. Both a n t i s e r a were d i l u t e d 1:1000 i n b l o c k i n g b u f f e r . The peroxidase sub­ s t r a t e was developed i n 4 - c h l o r o - l - n a p h t h o l , 10 mg i n 3.3 ml methanol 16.7 ml b l o c k i n g b u f f e r and 10 μΙ> of 30% H0.

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E l e c t r o n Microscopy. Samples of heated skim m i l k and rehydrated NDM powders were prepared s e p a r a t e l y f o r immunolabeling as whole mounts by negative s t a i n i n g and as t h i n s e c t i o n s , embedded i n p l a s t i c . For l a b e l i n g with negative s t a i n i n g , a l i q u o t s of m i l k c o n t a i n i n g 0.1% g e l a t i n were incubated f o r 1 h a t room temperature with r a b b i t antiserum t o cow whey p r o t e i n i n phosphateb u f f e r e d s a l i n e (PBS). The mixture was absorbed t o Formvar-carbon coated Ni Specimen g r i d s f o r 10 min, washed with 5-10 drops of 1% g e l a t i n i n PBS, and i n c u ­ bated f o r 1 h on a 100 M L drop of goat a n t i r a b b i t IgG, conjugated t o 10 nm c o l l o i d a l g o l d p a r t i c l e s GAR 10, (Jannsen L i f e Sciences Products, Beerse, Belgium). The samples were washed s e q u e n t i a l l y with: 1% g e l a t i n i n PBS, PBS, water, and f i n a l l y s t a i n e d with 2% u r a n y l a c e t a t e solution. For immunolabeling of t h i n s e c t i o n s , f r e s h samples of m i l k were embedded i n the low temperature embedment, L o w i c r y l K4M, according t o the procedure of Altmann e t a l . (10). Thin s e c t i o n s on Formvar-carbon coated N i specimen g r i d s were incubated s e q u e n t i a l l y with: 1% g e l a t i n i n PBS, r a b b i t antiserum t o cow whey p r o t e i n , gelatin/PBS, and goat a n t i r a b b i t IgG, conjugated t o c o l l o i d a l gold p a r t i c l e s . Sections were then washed with gelatin/PBS, PBS, and water before post s t a i n i n g the s e c t i o n s with 2% u r a n y l a c e t a t e . Hydrophobicity. P r o t e i n h y d r o p h o b i c i t y was determined by measuring the extent of alkane b i n d i n g t o NDM p r o t e i n by a m o d i f i c a t i o n of the procedure of Mangino e t a l . (11). Two mL of aqueous NDM s o l u t i o n (10%) and heptane (1.5 mL) were added t o 5 mL v i a l s , sealed with p a r a f i l m , and mixed sideways a t 3 rev/min a t 25.0 ± 0.2°C f o r 18 h with a model R-7636-00 Roto-torque r o t a t o r , (Cole-Palmer, I n t e r ­ n a t i o n a l Chicago, IL, USA). The alkane l a y e r was d i s ­ carded and the aqueous phase (1.0 ml) was e x t r a c t e d with undecane (0.6 mL) c o n t a i n i n g an i n t e r n a l standard (octane) f o r 2 h a t 3 rev/min. Samples (0.5 ML) of the undecane phase were i n j e c t e d i n t o a model 5710A gas chromatograph (Hewlett-Packard Co., Palo A l t o , CA) equipped with a f l a m e - i o n i z a t i o n d e t e c t o r . Separations were c a r r i e d out on a column of Chromosorb W coated with 10% (w/w) OV-101.

In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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F u n c t i o n a l P r o p e r t i e s . S o l u b i l i t y measurements were performed as p r e v i o u s l y described (7). Emulsions were prepared by homogenizing 3 mL o f p r o t e i n s o l u t i o n (10%) and 1 mL corn o i l f o r 30 sec u s i n g a P o l y t r o n homogenizer at s e t t i n g #5 and determining the emulsion a c t i v i t y index (EAI) by t u r b i d i t y measurements a t p r o t e i n concentrations of 0.1, 0.5 and 1.0% and pH values o f 6, 7, 8, and 9 according t o the procedure o f Pearce and K i n s e l l a (12). Foam formation (% overrun) and foam s t a b i l i t y were d e t e r ­ mined according t o the procedure o f P h i l l i p s e t a l . (13) with the m o d i f i c a t i o n t h a t foam s t a b i l i t y was determined by measuring the time r e q u i r e d f o r drainage equal t o 50% of the weight o f foam (approximately 100 mL). R e s u l t s and D i s c u s s i o n HPLC Separations. E a r l i e r r e s u l t s have shown t h a t e l u t i o n p r o f i l e s f o r s o l u b l e d i a l y z e d m a t e r i a l from rehydrated medium- and high-heat NDM powders (II and I I I , r e s p e c t i v e l y ) were s i g n i f i c a n t l y d i f f e r e n t from the low-heat powder (I) p r o f i l e s (8). The p r o f i l e s f o r I and I I I are d e p i c t e d together with the SDS-PAGE p a t t e r n s o f the p r o t e i n s present i n corresponding peaks from t h e a n a l y t i c a l column (inset) i n Figure 1. The whey-casein complex formed i n I I I was comprised o f many p r o t e i n s i n c l u d i n g BSA, κ-, and ct -caseins, β-lactoglobulin (β-LG), and a-lactalbumin (α-LA) (Figure l b , lane 6 ) . Although RP-HPLC p r o f i l e s and SDS-PAGE g e l s o f reduced p r o t e i n s i n d i c a t e t h a t the complex i s s t a b i l i z e d through d i s u l f i d e l i n k a g e , some o f the n a t i v e whey p r o t e i n s reformed during storage suggesting t h a t whey complexes are a l s o present which are s t a b i l i z e d through l e s s s p e c i f i c i n t e r a c t i o n s (hydrophobic o r calcium-dependent linkages) with the c a s e i n m i c e l l e s . Comparison o f r e t e n t i o n times f o r RP-HPLC s e p a r a t i o n o f m i l k p r o t e i n s from I I I i n d i c a t e d t h a t the whey-casein complex i s more hydrophobic under the c o n d i t i o n s o f chromatography than the other milk p r o t e i n s i n c l u d i n g β-casein (Figure l b ) . E l u t i o n o f milk p r o t e i n s from the same rehydrated powders using hydrophobic i n t e r a c t i o n chromatography (HIC) i n d i c a t e d t h a t the whey-casein complex was l e s s hydrophobic than β-casein. The whey-casein complex was not present i n the supernatant from I but was present i n the e l u t i o n p r o f i l e o f the supernatant from I I I and e l u t e d before β-casein (Figure 2). The complex was a l s o found i n the p e l l e t from I I I and e l u t e d before the c a s e i n s (Figure 3b). SDS-PAGE p a t t e r n s o f the wheyc a s e i n complex ( l e f t i n s e t ) Figure 3b, lane 1, i n d i c a t e d t h a t the sample contained aggregates t h a t d i d not enter the running g e l . Casein m i c e l l e s apparently were not completely d i s s o c i a t e d i n the HIC b u f f e r s i n c e β- and α