Hydrolysis of whey protein isolate with Bacillus licheniformis protease: aggregating capacities of peptide fractions

In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating peptides, as a function of concentration, and their aggregating capacities toward added intact proteins. The amount of aggregated material and the composition of the aggregates obtained were measured by nitrogen concentration and size exclusion chromatography, respectively. The results showed that of the four fractions obtained from the aggregating peptides, two were insoluble, while the other two consisted of 1:1 mixture of low and high solubility peptides. Therefore, insoluble peptides coaggregated, assumedly via hydrophobic interactions, other relatively more soluble peptides. It was also shown that aggregating peptides could aggregate intact protein nonspecifically since the same peptides were involved in the aggregation of whey proteins, beta-casein, and bovine serum albumin. Both insoluble and partly insoluble peptides were required for the aggregation of intact protein. These results are of interest for the applications of protein hydrolysates, as mixtures of intact protein and peptides are often present in these applications.     

Hydrolysis of whey protein isolate with Bacillus licheniformis protease: fractionation and identification of aggregating peptides

The objective of this work was to identify the dominant aggregating peptides from a whey protein hydrolysate (degree of hydrolysis of 6.8%) obtained with Bacillus licheniformis protease. The aggregating peptides were fractionated with preparative reversed-phase chromatography and identified with liquid chromatography-mass spectrometry. The results showed that the dominant aggregating peptide, at pH 7.0, was beta-lg AB [f1-45]. In addition, the peptides beta-lg AB [f90-108]-S-S-alpha-la [f50-113], alpha-la [f12-49]-S-S-alpha-la [f50-113], beta-lg AB [f90-108]-S-S-beta-lg AB [f90-108], beta-lg A [f90-157], and beta-lg AB [f135-157/158] were also identified as main aggregating peptides. The results further showed that aggregation, via hydrophobic interactions, prevented further digestion (at pH 8.0), thereby explaining the large size of the aggregating peptides. It is hypothesized that B. licheniformis protease breaks down hydrophilic segments in the substrate and, therefore, preserves hydrophobic segments that aggregate once exposed to the solvent.     

Emulsion properties of casein and whey protein hydrolysates and the relation with other hydrolysate characteristics

Casein and whey protein were hydrolyzed using 11 different commercially available enzyme preparations. Emulsion-forming ability and emulsion stability of the digests were measured as well as biochemical properties with the objective to study the relations between hydrolysate characteristics and emulsion properties. All whey protein hydrolysates formed emulsions with bimodal droplet size distributions, signifying poor emulsion-forming ability. Emulsion-forming ability of some casein hydrolysates was comparable to that of intact casein. Emulsion instability was caused by creaming and coalescence. Creaming occurred mainly in whey hydrolysate emulsions and in casein hydrolysate emulsions containing large emulsion droplets. Coalescence was dominant in casein emulsions with a broad particle size distribution. Emulsion instability due to coalescence was related to apparent molecular weight distribution of hydrolysates; a relative high amount of peptides larger than 2 kDa positively influences emulsion stability.     

Physicochemical and nitrogen solubility properties of Bacillus proteinase hydrolysates of sodium caseinate incubated with transglutaminase pre- and post-hydrolysis

Sodium caseinate (NaCN), hydrolyzed with Protamex, a Bacillus proteinase preparation, to 0.5, 1.3, and 17.5% degrees of hydrolysis, was incubated with transglutaminase (TGase) for 3, 42, and 290 min at enzyme/substrate ratios of 1, 1, and 10% (w/w), respectively, pre- and post-hydrolysis. The electrophoretic, reversed-phase high-performance liquid chromatography (RP-HPLC) and nitrogen solubility profiles of the modified products were investigated. Combinations of hydrolysis and incubation with TGase generated products displaying novel physicochemical and nitrogen solubility properties. Significant changes in sodium dodecyl sulfate (SDS) and urea polyacrylamide gel electrophoresis profiles were apparent in the modified caseinate samples. Extensive TGase cross-linking resulted in polymers that were unable to enter the resolving gel during SDS polyacrylamide gradient gel electrophoresis. Extensive combined enzymatic modification resulted in peptides eluting earlier on RP-HPLC than limited combined enzymatic modification or limited hydrolysis. Combination of enzymatic treatments resulted in significantly (P < 0.005) improved solubility around pH 4.6, compared to incubation with TGase or hydrolysis of NaCN alone.     

羊奶乳清蛋白水解物的酶解制备

采用Alcalase与Flavourzyme两种酶对羊奶乳清蛋白进行水解,以水解度为指标,对两种酶单独使用及复合使用水解羊奶乳清蛋白的工艺条件进行了研究。试验结果显示:采用Alcalase与Flavourzyme复合水解羊奶乳清蛋白的效果较好,特别是采用先添加Flavourzyme后加入Alcalase进行水解,不仅能提高羊奶乳清蛋白的水解度,使其达到32.81%,而且对改善水解液的口感有较大的作用。     

FTIR spectra of whey and casein hydrolysates in relation to their functional properties

Mid-infrared spectra of whey and casein hydrolysates were recorded using Fourier transform infrared (FTIR) spectroscopy. Multivariate data analysis techniques were used to investigate the capacity of FTIR spectra to classify hydrolysates and to study the ability of the spectra to predict bitterness, solubility, emulsifying, and foaming properties of hydrolysates. Principal component analysis revealed that hydrolysates prepared from different protein sources or with different classes of proteolytic enzymes are distinguished effectively on basis of their FTIR spectra. Moreover, multivariate regression analysis showed satisfactory to good prediction of functional parameters; the coefficient of determination (R(2)) varied from 0.60 to 0.92. The accurate prediction of bitterness and emulsion forming ability of hydrolysates by using only one uncomplicated and rapid analytical method has not been reported before. FTIR spectra in combination with multivariate data analysis proved to be valuable in protein hydrolysate fingerprinting and can be used as an alternative for laborious functionality measurements.     

Rheological and physical properties of derivitized whey protein isolate powders

Pregelatinized starch is employed in many food applications due to the instantaneous nature of thickening and stability imparted by modification. Proteins, however, have been excluded as a viscosifying agent due to requisite thermal treatments required to create structure. Whey protein isolate gels were produced while manipulating heating time, pH, and mineral type/content, producing a variety of gel types/networks. Gels were frozen, freeze-dried, and ground into a powder. Once reconstituted in deionized water, gel powders were evaluated based on solubility studies, rotational viscometry, and electrophoresis. The protein powder exhibiting the largest apparent viscosity, highest degree of hydrolysis, and greatest solubility was selected for pH and temperature stability analyses and small amplitude oscillatory rheology. This processing technique manipulates WPI into a product capable of forming cold-set weak gel structures suitable for thickening over a wide range of temperature and pH food systems.     

Rheological properties and characterization of polymerized whey protein isolates.

Whey protein polymers were formed by heating whey protein isolate solutions at 80 degrees C. Flow behaviors of whey protein polymers produced from different protein concentrations and heating times were comparable to various flow behaviors of hydrocolloids. Polymer formation was found to be a two-phase process. The initial protein concentration was a significant factor that determines the size and/or shape of the primary polymer in the first phase as shown by intrinsic viscosity. Heating time was a factor in determining the aggregation in the second phase as shown by apparent viscosity. Intrinsic viscosity of whey protein polymers was as high as 141.7 +/- 7.30 mL/g, compared to 5.04 +/- 0.20 mL/g for native whey proteins. The intrinsic viscosity and gel electrophoresis data suggested that disulfide bonds played an important role in whey polymer formation.     

Preparation of whey protein hydrolysates using a single- and two-stage enzymatic membrane reactor and their immunological and antioxidant properties: characterization by multivariate data analysis

An initial 5% (w/v), followed thereafter with replacement aliquots of 3% (w/v), whey protein isolate (WPI) (ca. 86.98% Kjeldahl N x 6.38), was hydrolyzed using Protease N Amano G (IUB 3.4.24.28, Bacillus subtilis) in an enzymatic membrane reactor (EMR) fitted with either a 10 or 3 kDa nominal molecular weight cutoff (NMWCO) tangential flow filter (TFF) membrane. The hydrolysates were desalted by adsorption onto a styrene-based macroporous adsorption resin (MAR) and washed with deionized water to remove the alkali, and the peptides were desorbed with 25, 50, and 95% (v/v) ethyl alcohol. The desalted hydrolysates were analyzed for antibody binding, free radical scavenging, and molecular mass analysis as well as total and free amino acids (FAA). For the first time a quantity called IC50, the concentration of peptides causing 50% inhibition of the available antibody, is introduced to quantify inhibition enzyme-linked immunosorbent assay (ELISA) properties. Principal component analysis (PCA) was used for data reduction. The hydrolysate molecular mass provided the most prominent influence (PC1 = 57.35%), followed by inhibition ELISA (PC2 = 18.90%) and the antioxidant properties (PC3 = 10.43%). Ash was significantly reduced in the desalted fractions; the protein adsorption recoveries were high, whereas desorption with alcohol was prominently influenced by the hydrophobic/ hydrophilic amino acid balance. After hydrolysis, some hydrolysates showed increased ELISA reactivity compared with the native WPI.     

Oxygen permeability and mechanical properties of films from hydrolyzed whey protein

The effects of whey protein hydrolysis on film oxygen permeability (OP) and mechanical properties at several glycerol-plasticizer levels were studied. Both 5.5% and 10% degree of hydrolysis (DH) whey protein isolate (WPI) had significant effect (p 0.05) occurred for film OP between unhydrolyzed WPI, 5.5% DH WPI, and 10% DH WPI films at the same glycerol content. Hydrolyzed WPI films of mechanical properties similar to those of WPI films had better oxygen barrier. Therefore, use of hydrolyzed WPI allowed achievement of desired film flexibility with less glycerol and with smaller increase in OP.     

Charge modifications to improve the emulsifying properties of whey protein isolate

Whey protein isolate was modified by ethylene diamine in order to shift its isoelectric point to an alkaline pH. The extent of the modification was studied using SDS-PAGE and MALDI-TOF mass spectrometry. The modified whey proteins were used as an emulsifier to stabilize oil-in-water emulsions at acidic and neutral pH ranges, and their emulsifying properties were compared with that of the unmodified whey proteins and with the previously studied ethylene diamine modified sodium caseinate. The emulsifying activity of the modified whey proteins was similar to that of the unmodified ones, but the stability of an emulsion at pH 5 was significantly improved after the modification. Charge and coverage of droplet surface and the displacement of the interfacial proteins by surfactant Tween 20 were further studied as a function of pH. As compared with the unmodified whey proteins, the modified ones were proven to cover the interface more efficiently with extensive surface charge at pH 5, although the interfacial layer was less resistant to the surfactant displacement.     

Mechanical and barrier properties of cross-linked soy and whey protein based films

Sterilized biofilms based on soy protein isolate (SPI, S system) and a 1:1 mixture of SPI and whey protein isolate (WPI, SW system) were achieved through the formation of cross-links by means of gamma-irradiation combined with thermal treatments. The effect of the incorporation of carboxymethylcellulose (CMC) and poly(vinyl alcohol) was also examined. gamma-Irradiation combined with thermal treatment improved significantly the mechanical properties, namely, puncture strength and puncture deformation, for all types of films. Irradiated formulations that contain CMC behave more similarly as elastomers. CMC showed also significant improvements of the barrier properties, namely, water vapor permeability, for irradiated films of the S system and for non-irradiated films of the SW system.     

Hydrophobicity of whey protein concentrates measured by fluorescence quenching and its relation with surface functional properties

Surface hydrophobicity of whey protein concentrate (WPC) under heated (85 degrees C for 5, 10, 20, 30, 40, and 60 min) and unheated conditions was measured using cis-parinaric acid (CPA), 1-anilino-8-naphthalenesulfonate (ANS), and a fluorescence quenching method using acrylamide as a quencher. This last method evaluates the degree of exposure of tryptophanyl residues in proteins to the solvent. The initial slope of Stern-Volmer plots, K(app), was used as an index of protein hydrophobicity. Surface hydrophobicity of WPC exhibited good relation with surface functional properties such as emulsifying and foaming. Analysis of the data obtained in this work showed that the fluorescence quenching method gave results similar to those obtained using CPA and ANS. Therefore, this simple technique is satisfactory in effectively obtaining information about the hydrophobicity of whey proteins.     

微射流均质预处理提高大豆分离蛋白酶解效率及酶解产物乳化性能

为了探讨微射流均质预处理对大豆分离蛋白酶解效率及酶解产物乳化性能的影响,该文研究比较了微射流均质预处理前后大豆分离蛋白酶解产物的理化性质(水解度、亚基组成、蛋白溶解性、表面疏水性和分子量分布)和乳化性能(通过测定分析样品乳状液的平均粒径和微观结构评估样品的乳化性能)的变化。研究表明:大豆分离蛋白经过微射流均质预处理后采用木瓜蛋白酶水解,其酶解产物(水解度为1.7%)与对照大豆分离蛋白和未经预处理的酶解产物相比,在较低浓度下(30 g/L)制备出粒径细小的稳定乳状液(体积平均粒径≈1.6μm)。微射流均质预处理提高了大豆分离蛋白中α-7S和A-11S亚基的酶解敏感性,使酶解产物在水解度1.3%~1.7%范围内蛋白溶解性显著增加(P0.05),同时保持较高的表面疏水性值,与未经预处理的酶解产物相比形成了更多具有界面活性的可溶性多肽(分子量主要分布在11.3 k Da左右),在乳化过程中可有效防止乳液滴间发生桥联絮凝。因此微射流均质预处理是一种辅助提高大豆蛋白酶解效率和酶解产物乳化性能行之有效的方法。研究结果可为大豆蛋白深加工蛋白乳化剂提供理论和方法参考。     

Aging of whey protein films and the effect on mechanical and barrier properties

This work focuses on the aging of whey protein isolate (WPI) films plasticized with glycerol (G) and sorbitol (S). The films were cast from heated aqueous solutions at pH 7 and dried at 23 degrees C and 50% relative humidity (RH) for 16 h. They were stored in a climate room (23 degrees C, 50% RH) for 120 days, and the film properties were measured at regular intervals. The moisture content (MC) of the WPI/G films decreased from 22% (2 days) to 15% (45 days) and was thereafter constant at 15% (up to 120 days). This affected the mechanical properties and caused an increased stress at break (from 2.7 to 8.3 MPa), a decreased strain at break (from 33 to 4%), and an increased glass transition temperature (T(g)) (from -56 to -45 degrees C). The barrier properties were, however, unaffected, with constant water vapor permeability and a uniform film thickness. The MC of the WPI/S films was constant at approximately 9%, which gave no change in film properties.     

Correlations between biochemical characteristics and foam-forming and -stabilizing ability of whey and casein hydrolysates

Whey protein and casein were hydrolyzed with 11 commercially available enzymes. Foam properties of 44 samples were measured and were related to biochemical properties of the hydrolysates using statistical data analysis. All casein hydrolysates formed high initial foam levels, whereas whey hydrolysates differed in their foam-forming abilities. Regression analysis using the molecular weight distribution of whey hydrolysates as predictors showed that the hydrolysate fraction containing peptides of 3-5 kDa was most strongly related to foam formation. Foam stability of whey hydrolysates and of most casein hydrolysates was inferior to that of the intact proteins. The foam stability of casein hydrolysate foams was correlated to the molecular weight distribution of the hydrolysates; a high proportion of peptides >7 kDa, composed of both intact casein and high molecular weight peptides, was positively related to foam stability.     

Relationship between the microstructure and the mechanical and barrier properties of whey protein films

This work was focused on the relationship between the microstructure and the mechanical and barrier properties of whey protein isolate (WPI) films. Sorbitol (S) and glycerol (G) were used as plasticizers and the pH was varied between 7 and 9. The films were cast from heated aqueous solutions and dried in a climate room at 23 degrees C and 50% relative humidity for 16 h. The microstructure of the films was found to be dependent on the concentration, the plasticizers, and the pH. When the concentration increased, a more aggregated structure was formed, with a denser protein network and larger pores. This resulted in increased water vapor permeability (WVP) and decreased oxygen permeability (OP). When G was used as a plasticizer instead of S, the microstructure was different, and the moisture content and WVP approximately doubled. When the pH increased from 7 to 9, a denser protein structure was formed, the strain at break increased, and the OP decreased.     

Transcriptome profiling of heat-resistant strain Bacillus licheniformis CGMCC3962 producing Maotai flavor

Although Maotai flavor liquor is exclusive due to its soy sauce flavor, knowledge of its key compound and production mechanism is still scarce until now. To gain insight into the production mechanism of soy sauce flavor, a soy sauce flavor producing strain with high efficiency and heat-resistant capability was obtained, and the metabolic mechanism of the strain was investigated with the technique of microarray profiling. Because high temperature was a key factor for soy sauce flavor production, the global gene expression of this heat-resistant strain fermented at 55 °C was analyzed. Except for the responsive increase of heat shock proteins, which maintained cell survival during heat stress, biosynthesis of cysteine was also up-regulated. In addition, some metabolites were significantly increased when cysteine was added to the fermentation medium, such as 2,3-butanediol, 3-hydroxy-2-butanone, and tetramethylpyrazine, which were important flavor compounds in soy sauce flavor liquor and might be related with soy sauce flavor. The results indicated that cysteine might play an important role in the formation of soy sauce flavor compound, and it might act as an indirect precursor or stimulator of soy sauce flavor formation. This was the first use of the microarray profiling tool to investigate the fermentative strains for Chinese traditional liquor, which would allow a deeper insight into the mechanism of the formation of soy sauce flavor compound.     

Interactions of whey proteins during heat treatment of oil-in-water emulsions formed with whey protein isolate and hydroxylated lecithin

The interactions of proteins during the heat treatment of whey-protein-isolate (WPI)-based oil-in-water emulsions with and without added hydroxylated lecithin were studied by examining the changes in droplet size distribution and the quantity and type of adsorbed and unadsorbed proteins. Heat treatment at 90 degrees C of WPI emulsions resulted in an increase in total adsorbed protein; unadsorbed beta-lactoglobulin (beta-lg) was the main protein interacting with the adsorbed proteins during the first 10 min of heating, but after this time, unadsorbed alpha-lactalbumin (alpha-la) also associated with the adsorbed protein. In emulsions containing hydroxylated lecithin, the increase in total adsorbed protein during heat treatment was much lower and the unadsorbed beta-lg did not appear to interact with the adsorbed proteins during heating. However, the behavior of alpha-la during heat treatment of these emulsions was similar to that observed in the emulsions containing no hydroxylated lecithin. In the presence of NaCl, the particle size of the emulsion droplets and the quantities of adsorbed protein increased markedly during heating. Emulsions containing hydroxylated lecithin were less sensitive to the addition of NaCl. These results suggest that the binding of hydroxylated lecithin to unfolded monomers or intermediate products of beta-lg reduces the extent of heat-induced aggregation of beta-lg and consequently decreases the interactions between unadsorbed beta-lg and adsorbed protein. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated whey protein and hydroxylated lecithin solutions.     

Physical properties, molecular structures, and protein quality of texturized whey protein isolate: effect of extrusion temperature

Although extrusion technology has contributed much to increasing the effective utilization of whey, the effect of extrusion conditions on the functional properties of the proteins is not well understood. In this work, the impact of extrusion temperature on the physical and chemical properties, molecular structures, and protein quality of texturized whey protein isolate (WPI) was investigated at a constant moisture content and compared with WPI treated with simple heat only. The Bradford assay methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high-performance liquid chromatography techniques were used to determine protein solubility and to analyze compositional changes in the two major whey proteins, α-lactalbumin and β-lactoglobulin. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic techniques were applied to study the secondary and tertiary structures of the proteins. This study demonstrated that extrusion temperature is a critical but not the sole determining factor in affecting the functional properties of extruded WPI.