Proteins at Interfaces - ACS Publications - American Chemical Society

Chapter 40

Interfacial Behavior of Food Proteins Studied by the Drop Volume Method E. Tornberg

Downloaded by CORNELL UNIV on August 23, 2016 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch040

Swedish Meat Research Institute, P.O. Box 504, S-244 00 Kävlinge, Sweden

Many food items contain emulsions and foams, which are often stabilised by proteins forming a protective membrane at the interface. By preparing the food, adsorption of the available proteins, - by virtue of their surface activity -, is performed at the liquid/air (foams) and/or at the liquid/liquid (emulsions) interface. One way to study the interfacial behaviour of food proteins at those interfaces is to follow the interfacial tension decay accomplished by the adsorption of the proteins. So far a considerable amount of work has been devoted to the study of spread protein films at these types of interface. However, the study of the adsorption of proteins at the interface from a subphase of known concentration is more similar to the conditions prevailing during formation of emulsions and foams.

Methods The interfacial tension decay of food proteins adsorbing from a subphase has, in this study, been monitored with an apparatus based on the drop volume technique ( 1 . 2 . 3 ) . The following procedure was used (for details cf. ref. 2). A drop of a certain volume, corresponding to a certain interfacial tension (γ) value, is expelled rapidly, and the time necessary for the interfacial tension to fall to such a value that the drop becomes detached is measured. This procedure is repeated for differing drop sizes, i.e. for different values of the interfacial tension. A plot of the interfacial tension as a function of time (t) can then be made, as seen in Figure 1. This 0097-6156/87/0343-0647$06.00/0 © 1987 American Chemical Society

American Chemical Society Library 1155 16that St N.W. Brash and Horbett; Proteins Interfaces f

ACS Symposium Series; American Chemical Society: Washington D.C Washington, 20036 DC, 1987.


648

PROTEINS AT INTERFACES

has been done for the adsorption at the air/water interface of an u l t r a f n i t r a t e d and spray-dried whey protein concentrate (WPC) dispersed in 0.2 M NaCl solution at pH 7 for d i f f e r e n t i n i t i a l subphase concentrations. By use of this procedure the advantages of the drop volume method, as opposed to the Wilhelmy plate method can, be exploited; i . e . no problems with the contact angle (1), which makes i t especially suited for studies at the l i q u i d / l i q u i d i n t e r f a c e s , the p o s s i b i l i t y of measurement at elevated temperatures (t > 25°C) (1), and the formation of a clean interface in short time-periods with an i n i t i a l l y uniform concentration of proteins (3). These types of measurement have been used to follow the i n t e r f a c i a l tension decay or the r i s e in surface pressure, π ·£ = γ - γ t ( Ύ = i n i t i a l i n t e r f a c i a l tension of the clean interface) with time for a variety of food protein preparations. They have also been studied at d i f f e r e n t interfaces [air/water (A/W) and soybean oil/water (0/W) interfaces] and when the charge density of the proteins varies (pH, ionic strength). A l l the measurements have been carried out at a temperature of 25°C. 0

0

The kinetics of the i n t e r f a c i a l tension decay In Figure 2 three representative Y-t-curves are demonstrated. The slowest decay is exerted at the A/W-interface by a WPC dispersed in 0.2 M NaCl at pH 7, denoted (0.2-7), at a protein concentration of 10~ wt%. As can be seen from this curve there is an induction period before the i n t e r f a c i a l tension starts to f a l l , which can be even more pronounced at lower concentrations and for other proteins ( 4 ) . J . A . de F e i j t e r (5) has recently suggested a mechanism for this behaviour. He found, when measuring simultaneously the surface pressure and the surface concentration (Γ by ellipsometry), that the Γ-t-measurements did not give r i s e to an induction period, whereas the π - t - c u r v e could. Moreover, the surface concentration at the end of the induction period was about 1-1.5 mg/m for the proteins studied (Lysozyme, BSA and Ovalbumin), i . e . about monolayer coverage. This means there w i l l be no appreciable increase in surface pressure u n t i l almost monolayer coverage, or expressed d i f f e r e n t l y at the beginning of the condensed phase. Rearrangements of the proteins within the adsorbed f i l m w i l l then increase the surface coverage and thereby the i n t e r f a c i a l pressure (6). However, these rearrangements w i l l with time be r e s t r i c t e d by the increased incompressibility of the f i l m . Therefore, the r i s e in surface pressure w i l l f a l l off, as seen from the curves in Figure 2. 3

2

By p l o t t i n g the rate of the surface pressure increase, π

l o g | | , as a function of the change in compressibility of the protein f i l m can more e a s i l y be followed. This is i l l u s t r a t e d in Figure 3 for the three curves in Figure 2. Graham & P h i l l i p s (7) have shown that slow conformational changes occur in a f i l m , when i t is highly incompressible. Therefore, the 'kinks' observed in the log^-^-curves are suggested to arise from a r e l a t i v e l y abrubt increase in the incompressibility of the f i l m .

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


40. TORNBERG

Interfacial Behavior of Food Proteins

Time

649

(minutes)

Figure 1. Time-dependence of i n t e r f a c i a l tensions at the air-water (A/W) interface for WPC at different subphase concentrations. The WPC is dispersed in 0.2 M NaCl solution at pH 7 (0.2-7).(Reproduced with permission from Ref. 4. Copyright 1978 Blackwell S c i e n t i f i c Publications.) WPC (0.2-7)

A/W 0.001 %

WPC (0.2-7)

Oft* 1 % '

SOYA ( 0 - 7 )

A/W 0.1 %

7T(mN/m)

20 -

TIME (MINUTES)

Figure 2. Time-dependence of the i n t e r f a c i a l p r e s s u r e , 4 0 , for three curves. They represent WPC dispersed in (0.2-7) adsorbing at the A/W and the soya bean oil/water (0/W) interfaces at a subphase concentration of 10~ and 10" wt%, respectively, and a soya protein i s o l a t e adsorbing at the A/Winterface at a subphase concentration of 10" wtX. π

3

1


650


WPC (0.2-7) A/W Ô.001 %

WPC (0.2-7) O/W 1 %

SOYA (0-7) 0.1 % PROT. A/W


LOG(cffr/dt)

10

15

20

ΠΤ

(mN/m)

Figure 3. Log°j^ as a function of in Figure 2.

π

25

30

35

for those curves given


40


40. TORNBERG


Consequently, t h i s w i l l be followed by a decrease in the rate of the surface pressure build-up. Another interesting Y - t - c u r v e , observed for extracted meat proteins at certain concentrations is a p e c u l i a r , stepwise behaviour not e a r l i e r shown for proteins (8). This can be seen in the middle diagram in Figure 4. To be able to elucidate this behaviour we have to find out what the extracted j u i c e of meat proteins consists of. The beef muscle is heavily comminuted (Moulinex), centrifuged (25,000 χ g) and the supernatant is collected as a meat j u i c e of about 10% of the o r i g i n a l weight and with a protein content ranging from 11 to 15%. Through TCA-precipitation i t was found that the non-protein nitrogen content of this meat j u i c e was about 25%. A beef muscle consists on average of 75% water, 18% protein, 3% fat and 4% other substances, which to 45% of t h e i r content are composed of non-protein nitrogen ( c r e a t i n , amino acids and dipeptides). The proteins in the meat j u i c e were i d e n t i f i e d by electrophoretic separation as consisting of sarcoplasmic proteins to 96% and high molecular weight proteins to 4%, mainly t i t i n (MWa* 1000 kdalton). The sarcoplasmic proteins, which are the soluble proteins of the sarcoplasm (mostly the enzymes of the g l y c o l y t i c pathway), constitute about 30 to 35% of the t o t a l muscle protein. Evidently, the molecular weight d i s t r i b u t i o n of the components within the meat j u i c e covers such a wide range as 1000 kdalton down to 100-200 da1ton. The upper diagram in Figure 4 gives the lowering of the i n t e r f a c i a l tension at the A/W-interface of the meat j u i c e in (0.2-7) at a concentration of 10~ wt%. The i n t e r f a c i a l tension decay is r e l a t i v e l y rapid and high. By lowering the protein concentration of the meat j u i c e by one decade to 10"·* wt% the stepwise character of the Ύ - t - c u r v e emerges. A very quick lowering of the i n t e r f a c i a l tension is followed by an induction period and l a t e r on another, slower decrease in surface tension is obtained, more l i k e the usual behaviour of proteins at these low concentrations. Therefore, the f i r s t quick decay in surface tension was suspected to originate from the non-protein nitrogen f r a c t i o n . This was confirmed by TCA-precipitation of the meat j u i c e and thereafter r e g i s t r a t i o n of the Y - t - c u r v e of the supernatant at the concentration of 10~ wt%. The result can be seen in the lower diagram in Figure 4. Furthermore, measurements of the i n t e r f a c i a l tension decay of pure amino acids (for example ot-alanine) at the same concentration give s i m i l a r results (8). Therefore, i t is suggested that the stepwise character of the Y - t - c u r v e originates from competitive adsorption between the amino acids and dipeptides in the non-protein nitrogen fraction and the high molecular weight proteins in the protein f r a c t i o n . It i s not so much that the p r o b a b i l i t y of adsorption is much higher for the proteins than for the smaller peptides, but rather that the rate of desorption decreases markedly with increasing molecular weight (9). The proteins w i l l then remain for longer time-periods at the interface compared to the smaller peptides, resulting in exclusion of the l a t t e r and a larger i n t e r f a c i a l tension decay. 2

3


651



652

Figure 4. The lowering of the interfacial tension at the air-water interface of meat juice in (0.2-7) at a concentration of 10" wt% (upper) and 10" (lower). Continued on next page. 2

3


40.

TORNBERG

653



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Proteins at Interfaces - ACS Publications - American Chemical Society

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