Effect of type and quality of milk on heat induced protein–protein interactions in khoa (2024)

Materials

Sodium bicarbonate, Comassie brilliant blue, Bromophenol, Glycerol, 1-anilinonapthalene-8-sulfonate (ANS) and Ethylenediamine tetra acetic acid (EDTA) were procured from Sigma Aldrich, St. Louis, Missouri, USA. Dinitrophenyle hydrazine and Resorcinol were procured from Rankem, RFCL Ltd., New Delhi, India. Acrylamide, N,N′-methylene-bis-acrylamide, N,N,N′,N′-tetramethylenediamine (TEMED) and Ellman’s reagent from Thermo Fisher Scientific India Pvt. Ltd., Delhi, India. Sodium dodecyl sulphate (SDS) from Sisco Research Laboratories (SRL) Pvt. Ltd., Mumbai, India.

Table1

Chemical characteristic of cow and buffalo milks

Data represented as mean ± SEM, n = 3

Table2

Gross composition and physico-chemical characteristics of fresh, acidic and neutralized buffalo milk khoa (a) buffalo milk khoa (b) cow milk khoa (Choudhary et al. 2016)

Surface hydrophobicity

Surface hydrophobicity of milk and khoa samples was determined by the method of Yuksel et al. (2010). Analysis of hydrophobic sites was carried out in milk samples diluted 1:10 (v/v) with 50mM phosphate buffer (pH 7.0). In case of khoa, 2g of the product was made into slurry with 5ml water and volume was made up to 10ml in a volumetric flask (This reconstituted 10ml khoa sample is equivalent to 10ml of milk). One ml of reconstituted khoa sample was diluted 1:10 (v/v) with phosphate buffer as in the case of milk. The fluorescent probe used was 1-anilinonapthalene-8-sulfonate (ANS). Relative fluorescence of the samples was measured by using a fluorescence spectrophotometer at excitation wavelength 390nm, emission wavelength 480nm with slit width 5nm. Ten µM of ANS concentration was prepared in phosphate buffer and used for ANS titration. Final concentration of ANS in the protein solution was between 0 and 140µM during titration. The main aim of this parameter was to measure the maximum fluorescence which corresponded to level of saturation of the fluorescent marker (ANS) binding. Fluorescence of the samples was also measured before ANS titration and designated as blank.

Sulfhydryl content

Sulfhydryl groups in milk and khoa samples were measured using the protocol of Beveridge et al. (1974). Raw and boiled milk samples were acidified at 20°C with 1N HCl to pH 4.6 in order to fractionate casein from whey. In case of khoa, 10ml of reconstituted khoa samples was acidified to pH 4.6 in order to fractionate casein from whey. Whey protein fractions were lyophilized. Lyophilized whey protein was reconstituted in tris–glycine buffer [TGB, 10.4g tris, 6.9g glysine, 1.2g ethylene diaminetetraacetic acid (EDTA)/l, pH 8.0] to obtain 10% whey protein solution. Ellman’s reagent (5–5′-dithiobis-2-nitrobenzoic acid (DTNB) was prepared in tris glycine buffer (TGB) at a concentration of 4mg/ml. 0.5ml of protein solution was added to 2.5ml of 8M urea in TGB and vortexed. Twenty µl of ellman’s reagent was added, vortexed and absorbance read at 412nm. Sulfhydryl content was expressed as µmoles SH/g of protein.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of milk and Khoa proteins

SDS-PAGE was performed according to the method described by Simon (2004) on 12.5% gel concentration. Casein samples were prepared from milk and khoa samples by the method of Gupta and Ganguli (1965). Lyophilized casein samples were directly dissolved in sample buffer containing 20µl mercaptoethanol/ml to obtain a concentration of 1mg/ml. In case of khoa, lyophilized casein samples were dissolved in sample buffer and kept for 24h at room temperature. Proteins were denatured by keeping it in a boiling water bath for 4min before being injected for electrophoresis. Cooled the contents and centrifuged at 600rpm. Twenty µl of this sample was injected in the wells of the gel. The working solutions were filtered and stored at 6°C. Solutions were brought to room temperature before use.

Statistical analysis

Data reported were expressed as mean values with standard errors. Mean and standard error mean (SEM) were calculated using Microsoft excel (2007) (Microsoft Corp., Redmond, WA). Significant difference between values was verified by one way or two way analysis of variance and comparison between means was made by critical difference value (Snedecor and Cochran 1994).

Surface hydrophobicity

Surface hydrophobicity of milk and khoa samples was analyzed in terms of F_max using ANS titration curve. F_max represented the maximum fluorescence that could be attained under the given conditions and also the maximum numbers of surface sites allowable, to which ANS could be bound (Yuksel et al. 2010). One percent milk or hom*ogenized khoa samples in phosphate buffer were used for ANS titration. F_max values of fresh, acidic and neutralized cow and buffalo milk and their respective khoa samples at various stages i.e. raw, boiled and khoa stages are presented in Table3 and Fig.1 Significant difference (p < 0.05) was observed in F_max values of fresh, acidic and neutralized cow and buffalo milk samples at raw as well as boiled milk stage. However, non-significant difference (p < 0.05) was observed in the resulting khoa samples. It was observed from the titration curves (Fig.1) that as the ANS concentration increased, fluorescence intensity increased up to 307.82, 344.12, 317.81 and 332.12, 388.14, and 343.84 in case of fresh, acidic and neutralized raw buffalo and cow milk samples, respectively. Similarly, in case of boiled milk, F_max increased with the addition of ANS up to maximum level. Carbonaro et al. (1996) reported that interactions between κ-casein and β-lg cause changes in structure of dominant casein which further increases surface hydrophobicity of milk protein.

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Fig.1

ANS titration curves of fresh, acidic and neutralized milk, a buffalo milk, b cow milk, at various stages during preparation of khoa. (i) Raw milk (ii) boiled milk (iii) Khoa. Where (i) FBM fresh buffalo milk, ABM acidic buffalo milk, NBM neutralized buffalo milk; BFBM boiled fresh buffalo milk, BABM boiled acidic buffalo milk, BNBM boiled neutralized buffalo milk; FBMK fresh buffalo milk Khoa, ABMK acidic buffalo milk Khoa, NBMK neutralized buffalo milk Khoa. (i) FCM fresh cow milk, ACM acidic cow milk, NCM neutralized cow milk; BFCM boiled fresh cow milk, BACM boiled acidic cow milk, BNCM boiled neutralized cow milk; FCMK fresh cow milk Khoa, ACMK acidic cow milk Khoa, NCMK neutralized cow milk Khoa

However, further addition of ANS did not increase the fluorescence intensity; which indicated that the protein solution had attained the saturation point. In case of different milk and khoa samples higher values of F_max were observed for acidic samples followed by neutralized and fresh samples, these variations could be due to the structural modification of protein during acidification and neutralization of milk. Fluorescence intensity was drastically reduced in khoa samples (Fig.1) in comparison to raw and boiled milk samples; this could be due to protein–protein interactions as influenced by the heating of milk. Lower surface hydrophobicity values were observed in raw milk as hydrophobic groups were buried inside the native structure of protein molecule. Milk, when heated at higher temperature (> 70°C) results in unfolding of protein molecules which causes the exposure of hydrophobic sites and thus an increase in surface hydrophobicity (Parnell-Clunies et al. 1988). However, with the increase in severity of heat treatment, the protein–protein aggregation phenomena and structural collapse occurs resulting in decrease of surface hydrophobicity (Bonomi et al. 1988). Alais (1984) reported that the very first impact of heat treatment on milk protein was denaturation of serum proteins followed by their aggregation with casein micelle. Our results correlated well with Bonomi et al. (1988) who reported an increase in F_max at 85 and 90°C and a decrease at 120°C as compared to raw milk. Similarly, our results were supported by the work of Pagliarini et al. (1990) who reported lower surface hydrophobicity values for in-bottle sterilized milk as compared to UHT milk. Carbonaro et al. (1996) reported a decrease in surface hydrophobicity of whey protein with heat exposure and ascribed this phenomenon to protein–protein interactions. Parnell-Clunies et al. (1988) analyzed surface hydrophobicity in casein and whey fractions of vat (85°C/10–40min), HTST (98°C/0.5–1.87min) and UHT heating (140°C/2–8s) processed milks and observed a negative correlation between surface hydrophobicity and whey protein denaturation.

Sulfhydryl content

Total Sulfhydryl content (–SH) was estimated in both cow and buffalo milks during preparation of khoa at various stages i.e. raw and boiled milks, and khoa stage and the results are presented in Fig.2. It was observed that total –SH content of fresh, acidic and neutralized samples were significantly different (p < 0.05) from each other and the –SH content decreased significantly (p < 0.05) with the intensity of heating, which might be due to the conversion of –SH group into –SS bonding between k-casein and β-lg resulting in protein–protein interactions. Among the three types of samples i.e. fresh, acidic and neutralized milk and khoa, total –SH was highest in fresh followed acidic and neutralized samples. Lower values in acidic milk might be due to the fact that low pH causes modification in whey protein structure resulting in lower availability of –SH groups while in case of neutralized samples the conversion of –SH bond into –SS bond increased with increase in pH up to 6.9 (Wanatabe and Klostemeyer 1976). Presence of –SH groups in isolated casein sample from boiled milk and khoa is due to heat induced entrapped whey proteins indicating heat induced casein–whey interactions. Vakaleris and Pofahl (1968) reported 3 times higher measurable –SH groups in high heat skim milk cheese as compared to pasteurized milk cheese. Since pasteurized milk curd is entirely casein, which has theoretically almost zero or very low measurable –SH groups therefore any significant amount of –SH in pasteurized cheese curd was due to the presence of whey proteins trapped in the curd. Monahan et al. (1995) reported that even in the absence of heating, alkaline pH values affect the denaturation and sulphydryl-mediated polymerization of whey proteins. Guyomarc’H (2006) reported that the heat-treatment of skim milk at alkaline pH generated aggregates of denatured whey proteins and κ-casein in the serum phase of milk, rather than on the surface of the casein micelles which might be the reason for lower sulfhydryl content in neutralized samples. Otte et al. (1999) also reported that the level of whey protein denaturation was largely affected by pH of milk and at lower pH values, the denaturation rate increased. However, Onwulata et al. (2006) reported that alkaline conditions increased denaturation in the extruded whey protein isolate.Cao et al. (2015) reported up to 8.48 µmoles of –SH/g of protein in evaporated milk with protein content of 17.99% which was in range of protein content in khoa samples i.e. 17–19%. Our results were supported by Parnell-Clunies et al. (1988) who reported that –SH content was negatively correlated with whey protein denaturation.

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Fig.2

–SH content of fresh, acidic and neutralized milk during preparation of khoa. a Buffalo milk, b cow milk. Data are presented as mean ± SEM (n = 3). ^a–cDifferent lowercase letters denote significant difference (p < 0.05) between groups (raw milk, boiled milk and Khoa). Error bars show the variations of three determinations in terms of standard error of mean. ^A–CDifferent uppercase letters denote significant difference (p < 0.05) across subgroups [(FBM, ABM and NBM) and (FCM, ACM and NCM)]. FBM fresh buffalo milk, ABM acidic buffalo milk, NBM neutralized buffalo milk, FCM fresh cow milk, ACM acidic cow milk, NCM neutralized cow milk

SDS PAGE

Figure3 depicts the electrophoretic patterns of casein proteins of buffalo and cow milk and khoa samples.

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Fig.3

SDS-PAGE pattern of fresh, acidic and neutralized buffalo and cow milks and their khoa casein separated on 12.5% gel at various stages, a buffalo milk, b cow milk. a Lane 1 molecular weight markers ranging from 3.5 to 205 kDa; lane 2 fresh raw buffalo milk; lane 3 acidic raw buffalo milk; lane 4 neutralized raw buffalo milk; lane 5 boiled fresh buffalo milk; lane 6 boiled acidic buffalo milk; lane 7 boiled neutralized buffalo milk; lane 8 fresh buffalo milk Khoa; lane 9 acidic buffalo milk Khoa: lane 10 neutralized buffalo milk Khoa. b Lane 1 molecular weight markers ranging from 3.5 to 205 kDa; lane 2 fresh raw cow milk; lane 3 acidic raw cow milk; lane 4 neutralized raw cow milk; lane 5 boiled fresh cow milk; lane 6 boiled acidic cow milk; lane 7 boiled neutralized cow milk; lane 8 fresh cow milk Khoa; lane 9 acidic cow milk Khoa: lane 10 neutralized cow milk Khoa

Casein proteins of raw milk resolved into four major bands which were assigned their respective labels (α-S₁, α-S₂, β-casein, κ-casein) based on comparison of their molecular weight with molecular weight markers (lane 1). However, casein protein samples prepared from boiled milk resolved only into α-S₁, α-S₂, β-casein, κ-casein and β-lg. No visible difference was observed in the SDS pattern of fresh, acidic, neutralized cow and buffalo milk samples. However, in case of their respective khoa samples, particularly for khoa from neutralized cow and buffalo milk, bands of β-lg and α-la did not resolve clearly indicating intense denaturation of whey proteins under alkaline conditions resulting in strong binding between casein and whey proteins since whey protein denaturation increases under alkaline conditions (Onwulata et al. 2006). It was concluded from SDS PAGE that casein samples from raw milk resolved into basic casein fractions while the casein samples from boiled milk also contained β-lg fraction of whey protein, this extra band evolved due to the heat induced protein–protein interaction occurring between β-lg and κ-casein resulting in a disulfide bonded protein complex. Addition of β-mercaptoethanol to the sample buffer in SDS PAGE resulted in breakdown of disulphide bond and thus the appearance of β-lg fraction in casein sample. During khoa preparation, intense heating of milk resulted in aagglomerated protein complex of casein and whey protein. Hence, it was evident that heating of milk during preparation of khoa resulted in protein–protein interactions. Doi et al. (1981) also confirmed the formation of heat induced complex between β-lg and κ-casein after heating at 90°C for 10min. SDS PAGE of casein-whey protein complex (in the presence of 2-mercaptoethenol) resulted in resolution of κ-casein and β-lg.

Parnell-Clunies et al. (1988) conducted SDS PAGE of casein fraction of heated milk and indicated the presence of a high molecular weight component in the gel. Similarly, Prasad and Balachandran (1989) conducted PAGE of protein fractions in freshly prepared sterilized concentrated buffalo milk. They observed changes in number of bands and their mobility in the gel matrix of polyacrylamide gel. They also indicated distinct changes in aggregation and disaggregation of casein micelles through the appearance and disappearance of different peaks in elution profile. Havea (2006) conducted two dimensional PAGE of di-sulphide linked protein aggregates in milk protein concentrate powders and observed that these aggregates consisted of κ-casein, β-lg and some αs1-casein indicating protein interactions. Reports are also available on the effect of addition of whey protein or of purified κ-casein to the milk samples. The addition of whey proteins caused an increase in the amount and the size of the complexes, however, addition of κ-casein to milk had little or no effect on the complex formation nor did the added κ-casein could react with the whey protein in milk (Donato et al. 2007).

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