Preparation and analysis of hydrolysates from whey protein concentrate using the proteases from Bacillus licheniformis and Aspergillus oryzae

The action of proteases from Bacillus licheniformis and Aspergillus oryzae was studied with the aim of preparing hydrolysates from whey protein concentrate with nutritionally appropriate peptide profile. Various enzyme/substrate ratios were used, and the peptides were fractionated by size‐exclusion HPLC followed by their quantification using the rapid method of correct fraction area (CFA). The protease from B. licheniformis (E:S of 8:100) produced the best peptide with a much lower amount of large peptides (44.61%), greater of di‐ and tripeptides (8.79%) and higher sum of the di‐ and tripeptides with free amino acids (9.99%) than the other hydrolysates. The advantage of using a lower E:S ratio to obtain a nutritionally adequate peptide profile was observed for the protease of A. oryzae when it passed from 3:100 to 2:100.     

The effect of bleaching agent on the flavor of liquid whey and whey protein concentrate

The increasing use and demand for whey protein as an ingredient requires a bland-tasting, neutral-colored final product. The bleaching of colored Cheddar whey is necessary to achieve this goal. Currently, hydrogen peroxide (HP) and benzoyl peroxide (BPO) are utilized for bleaching liquid whey before spray drying. There is no current information on the effect of the bleaching process on the flavor of spray-dried whey protein concentrate (WPC). The objective of this study was to characterize the effect of bleaching on the flavor of liquid and spray-dried Cheddar whey. Cheddar cheeses colored with water-soluble annatto were manufactured in duplicate. Four bleaching treatments (HP, 250 and 500 mg/kg and BPO, 10 and 20 mg/kg) were applied to liquid whey for 1.5 h at 60°C followed by cooling to 5°C. A control whey with no bleach was also evaluated. Flavor of the liquid wheys was evaluated by sensory and instrumental volatile analysis. One HP treatment and one BPO treatment were subsequently selected and incorporated into liquid whey along with an unbleached control that was processed into spray-dried WPC. These trials were conducted in triplicate. The WPC were evaluated by sensory and instrumental analyses as well as color and proximate analyses. The HP-bleached liquid whey and WPC contained higher concentrations of oxidation reaction products, including the compounds heptanal, hexanal, octanal, and nonanal, compared with unbleached or BPO-bleached liquid whey or WPC. The HP products were higher in overall oxidation products compared with BPO samples. The HP liquid whey and WPC were higher in fatty and cardboard flavors compared with the control or BPO samples. Hunter CIE Lab color values (L*, a*, b*) of WPC powders were distinct on all 3 color scale parameters, with HP-bleached WPC having the highest L* values. Hydrogen peroxide resulted in a whiter WPC and higher off-flavor intensities; however, there was no difference in norbixin recovery between HP and BPO. These results indicate that the bleaching of liquid whey may affect the flavor of WPC and that the type of bleaching agent used may affect WPC flavor.     

乳清蛋白水解物水解度3种测定方法的比较

选用胰蛋白酶水解乳清浓缩蛋白，对不同时问乳清蛋白水解液的水解度测定表明，pH—stat法与茚三酮比色法获得的乳清蛋白水解液的水解度基本一致．而甲醛滴定法测定的结果偏低。按本文的实验条件，pH—stat法与茚三酮比色法获得的结果最接近实际值。鉴于pH—scat法对酶的类型和水解条件有一定的要求，茚三酮比色法则无此方面限制，因此认为茚三酮比色法是目前三种方法中最适合测定乳清蛋白水解液水解度的方法。     

Impact of whey protein isolates and concentrates on the formation of protein nanoparticles‐stabilised Pickering emulsions

Whey protein nanoparticles (NPs) were prepared by heat‐induced method. The influences of whey protein isolates (WPIs) and concentrates (WPCs) on the formation of NPs were first investigated. Then Pickering emulsions were produced by protein NPs and their properties were evaluated. After heat treatment, WPC NPs showed larger particle size, higher stability against NaCl, lower negative charge and contact angle between air and water. Dispersions of WPC NPs appeared as higher turbidity and viscosity than those of WPI NPs. The interfacial tension of WPC NPs (~7.9 mN/m at 3 wt% NPs) was greatly lower than that of WPI NPs (~12.1 mN/m at 3 wt% NPs). WPC NPs‐stabilised emulsions had smaller particle size and were more homogeneous than WPI NPs‐stabilised emulsions. WPC NPs‐stabilised emulsions had higher stability against NaCl, pH and coalescence during storage.     

The effect of acidification of liquid whey protein concentrate on the flavor of spray-dried powder

Off-flavors in whey protein negatively influence consumer acceptance of whey protein ingredient applications. Clear acidic beverages are a common application of whey protein, and recent studies have demonstrated that beverage processing steps, including acidification, enhance off-flavor production from whey protein. The objective of this study was to determine the effect of preacidification of liquid ultrafiltered whey protein concentrate (WPC) before spray drying on flavor of dried WPC. Two experiments were performed to achieve the objective. In both experiments, Cheddar cheese whey was manufactured, fat-separated, pasteurized, bleached (250 mg/kg of hydrogen peroxide), and ultrafiltered (UF) to obtain liquid WPC that was 13% solids (wt/wt) and 80% protein on a solids basis. In experiment 1, the liquid retentate was then acidified using a blend of phosphoric and citric acids to the following pH values: no acidification (control; pH 6.5), pH 5.5, or pH 3.5. The UF permeate was used to normalize the protein concentration of each treatment. The retentates were then spray dried. In experiment 2, 150 μg/kg of deuterated hexanal (D₁₂-hexanal) was added to each treatment, followed by acidification and spray drying. Both experiments were replicated 3 times. Flavor properties of the spray-dried WPC were evaluated by sensory and instrumental analyses in experiment 1 and by instrumental analysis in experiment 2. Preacidification to pH 3.5 resulted in decreased cardboard flavor and aroma intensities and an increase in soapy flavor, with decreased concentrations of hexanal, heptanal, nonanal, decanal, dimethyl disulfide, and dimethyl trisulfide compared with spray drying at pH 6.5 or 5.5. Adjustment to pH 5.5 before spray drying increased cabbage flavor and increased concentrations of nonanal at evaluation pH values of 3.5 and 5.5 and dimethyl trisulfide at all evaluation pH values. In general, the flavor effects of preacidification were consistent regardless of the pH to which the solutions were adjusted after spray drying. Preacidification to pH 3.5 increased recovery of D₁₂-hexanal in liquid WPC and decreased recovery of D₁₂-hexanal in the resulting powder when evaluated at pH 6.5 or 5.5. These results demonstrate that acidification of liquid WPC80 to pH 3.5 before spray drying decreases off-flavors in spray-dried WPC and suggest that the mechanism for off-flavor reduction is the decreased protein interactions with volatile compounds at low pH in liquid WPC or the increased interactions between protein and volatile compounds in the resulting powder.     

Process development for a novel milk protein concentrate with whey proteins as fibrils

Milk protein concentrate (MPC) is a preferred ingredient to provide nutritional and functional benefits in various dairy and food products. Altering the protein configuration and protein-protein interactions in MPC can provide a novel functionality and may open doors for new applications. The fibrilization process converts the globular structure of whey proteins to fibrils and consequently increases viscosity and water holding capacity compared with the native protein structure. The objective of the current work was to selectively convert the whey proteins in MPC as fibrils. For this purpose, simulated control model MPC was prepared by combining solutions of micellar casein concentrate (MCC) and milk whey protein isolate (mWPI) to give casein and whey protein in an 80:20 ratio. The mWPI solution was converted to fibrils by heating at low pH, neutralized, and combined with MCC solution similar to control model MPC and termed “fibrillated model MPC.” Thioflavin T fluorescence value, transmission electron microscopy, and gel electrophoresis confirmed the fibril formation and their survival after neutralization and mixing with MCC. Further, the fibrillated mWPI showed significantly higher viscosity and consistency coefficient than nonfibrillated mWPI. Similarly, fibrillated model MPC showed significantly higher viscosity and consistency coefficient compared with control model MPC. Hence, the fibrillated model MPC can be used as ingredient to increase viscosity. Heat coagulation time was found to be significantly higher for control model MPC compared with fibrillated model MPC.     

酶解对乳清蛋白抗原性影响的研究

研究了酶解对乳清蛋白抗原性的影响。选择了7种常见蛋白酶在同一水解模式下水解乳清蛋白，用竞争ELISA法测定水解物的残留抗原性，从而间接测定其过敏性变化。结果表明，酶解能有效降低乳蛋白抗原性，但水解物仍能与特异抗体反应，保留一部分抗原性。不同酶对乳清蛋白过敏原的影响不同，酶的特异性对乳清蛋白水解物的抗原性有较大的影响，碱性蛋白酶降低乳蛋白抗原性的效果最佳，对抗β-乳球蛋白（β-LG）和抗α-乳白蛋白（α-LA）抗体的抗原性分别降低了50．02％和99．72％。     

Production of a high gel strength whey protein concentrate from cheese whey

In order to develop a process for the production of a whey protein concentrate (WPC) with high gel strength and water-holding capacity from cheese whey, we analyzed 10 commercially available WPC with different functional properties. Protein composition and modification were analyzed using electrophoresis, HPLC, and mass spectrometry. The analyses of the WPC revealed that the factors closely associated with gel strength and water-holding capacity were solubility and composition of the protein and the ionic environment. To maintain whey protein solubility, it is necessary to minimize heat exposure of the whey during pretreatment and processing. The presence of the caseinomacropeptide (CMP) in the WPC was found to be detrimental to gel strength and water-holding capacity. All of the commercial WPC that produced high-strength gels exhibited ionic compositions that were consistent with acidic processing to remove divalent cations with subsequent neutralization with sodium hydroxide. We have shown that ultrafiltration/diafiltration of cheese whey, adjusted to pH 2.5, through a membrane with a nominal molecular weight cut-off of 30,000 at 15 degrees C substantially reduced the level of CMP, lactose, and minerals in the whey with retention of the whey proteins. The resulting WPC formed from this process was suitable for the inclusion of sodium polyphosphate to produce superior functional properties in terms of gelation and water-holding capacity.     

陶瓷膜超滤技术浓缩乳清的工艺参数研究

采用孔径为20nm的无机陶瓷膜超滤干酪副产物乳清,浓缩乳清蛋白。通过对膜过滤压力、温度以及乳清pH三个因素进行单因素分析以及正交实验优化,得到最佳工艺条件:操作压力0.25MPa,温度51℃,pH6.1,此条件下超滤膜渗透通量达到169.37L/m2.h,乳清蛋白可浓缩至5.4%,经喷雾干燥制得WPC蛋白质含量为38.2%。     

酶法生产乳清多肽饮料

采用碱性蛋白酶水解乳清多肽,制备具有较好热稳定性的可溶性多肽混合物,然后以其为母液,生产乳清多肽饮料。获得最优水解工艺的最适pH为9.0、最适温度为35℃,乳清中水解度高达25.4%。所研制的乳清多肽饮料无苦味,且具有良好的稳定性。     

近红外反射光谱法测定浓缩乳清蛋白品质的新方法

比较了用近红外反射光谱（NIRS）直接测定和用传统的化学方法测定乳清蛋白的蛋白质、脂肪、水分的效果。结果发现,用近红外反射光谱法测定3种成分均有很好的效果,完全可以代替传统的化学方法测定。该方法快速、简便、准确。     

乳清浓缩蛋白在酸奶生产中的应用

以鲜奶，奶粉，乳清蛋白等乳成分为主要原料，研究了乳清浓缩蛋白在酸奶生产中的制做方法，对乳清浓缩蛋白代替部分高档脱脂奶粉生产酸奶产品的保水率，粘度，口感及组织状态进行了比较分析。结果表明，在酸奶生产中，添加一定的浓缩乳清蛋白代替高档脱脂奶粉是可行的，产品较为理想。     

Hydrolysis degree,peptide profile and phenylalanine removal from whey protein concentrate hydrolysates obtained by various proteases

The action of various proteases was tested for preparing whey protein concentrate (WPC) hydrolysates with high degree of hydrolysis (DH), appropriate peptide profiles and reduced phenylalanine (Phe) content. The peptide profile analysis included the fractionation of hydrolysates by size‐exclusion HPLC. The rapid correct fraction area method was used to quantify the components of the chromatographic fractions. Activated carbon (AC) was used to remove Phe, and its efficiency was evaluated by measuring the amount of Phe by second‐derivative spectrophotometry. The results showed that the DH of WPC hydrolysates increased and that the protease from Aspergillus oryzae yielded the highest DH value. This protease also produced the best peptide profile, that is, the highest di‐ and tripeptide content (16.14%), the highest amounts of free amino acids (18.43%) and the lowest amount of large peptides (18.76%). The proteases from both A. oryzae and Bacillus subtilis produced the highest Phe removals (79.0 and 77.8%, respectively).     

A comparison of drying operations on the rheological properties of whey protein thickening ingredients

An existing procedure for the alteration of whey proteins into a cold‐set thickening agent was modified by developing a spray‐drying operation to replace the prohibitively expensive freeze‐drying step. The original and the modified derivatization procedures were used with a commercial whey protein concentrate (WPC). The freeze‐dried and spray‐dried derivatized WPC powders, along with polysaccharide thickeners, were reconstituted in water and evaluated by using a range of rheological studies. The effects of temperature, concentration, and shear on viscosity as well as the mechanical spectra were assessed to characterize the ability of the powders to function in food systems. Rheological characterization revealed the modified derivatization procedure yielded an ingredient having the same cold‐set thickening and gelling ability as the original derivatized powder. The modified whey proteins were also able to achieve, at higher usage levels, textural properties similar to several polysaccharide thickeners. Use of a spray‐drying technique created a more economical process for the production of a whey protein ingredient that was suitable for contributing viscosity and texture to a wide range of food systems.     

胰蛋白酶限制性修饰乳清浓缩蛋白纤维聚合物表面性质的研究

为了研究胰蛋白酶限制性修饰对乳清浓缩蛋白(WPC)热致聚合物的微观形态及表面性质的影响,本文制备了胰蛋白酶在不同水解度(DH为0.2%、0.6%和1%)限制性修饰后的WPC在p H 2.0、90℃下热致聚合物,利用透射电镜分析了聚合物的微观形态特征,测定了不同聚合物的表面性质。结果表明,纤维聚合物较常规p H条件下形成的无定形聚合物具有较差的乳化活性和乳化稳定性以及较优的起泡和泡沫稳定性。胰蛋白酶修饰促进WPC纤维聚合物的形成,乳化活性较天然WPC形成纤维稍有提高;起泡性和泡沫稳定性显著提高,在DH为0.6%时起泡提高幅度最大,较天然WPC纤维提高了11.76%;在DH为1%时,泡沫稳定性较天然WPC纤维提高了12.59%。WPC经胰蛋白酶修饰后所形成更优的纤维结构以及表面疏水性的提高有利于其界面性质的提高。     

Comparison of functional properties of 34% and 80% whey protein and milk serum protein concentrates

This study compared the functional properties of serum protein concentrate (SPC) with whey protein concentrate (WPC) made from the same milk and with commercial WPC. The experimental SPC and WPC were produced at 34% or 80% protein from the same lot of milk. Protein contents of WPC and SPC were comparable; however, fat content was much lower in SPC compared with WPC and commercial WPC. The effect of drying methods (freeze vs. spray drying) was studied for 34% WPC and SPC. Few differences due to drying method were found in turbidity and gelation; however, drying method made a large difference in foam formation for WPC but not SPC. Between pH 3 and 7, SPC was found to have lower turbidity than WPC; however, protein solubility was similar between SPC and WPC. Foaming and gelation properties of SPC were better than those of WPC. Differences in functional properties may be explained by differences in composition and extent of denaturation or aggregation.     

Influence of proteolysis of milk on the whey protein to total protein ratio as determined by capillary electrophoresis

Capillary electrophoresis (CE) was used to determine the whey protein to total protein ratio in raw and UHT milk samples with different degrees of proteolysis caused by storage. In raw milks, the analysis of samples taken at regular times demonstrated the influence of proteolysis in the whey protein to total protein determination, which was overestimated after 4 d of storage. In UHT milks, the overestimation of the whey protein to total protein ratio took place after 30 or 60 d of storage. However, the ratios alphaS1-CN/beta-CN and alphas1-CN/kappa-CN permitted detection of the samples of raw or UHT milk with degraded proteins. The distorted capillary electrophoretic pattern obtained for UHT milks made necessary an integration of the electropherograms in a "valley-to-valley" way. Results for raw milk samples were identical when "valley-to-valley" was compared to standard integration techniques. This CE method could be considered an alternative method to derivative spectroscopy for the determination of the whey protein to total protein of milk and could be used to detect samples with proteolysis.     

风味酶水解乳清蛋白生产蛋白胨的工艺条件

为了提高原料乳清蛋白(Whey Protein Concentrate,WPC)的产品附加值,利用风味蛋白酶对WPC进行水解,生产蛋白胨产品。结果表明:最优工艺条件为E/S(18 000 U/100 mL),温度为55℃,pH值6.0,水解时间为4.0 h,底物质量分数为8%,水解度为46.4%。利用此工艺生产的蛋白胨产品在理化指标、氨基酸组成及质量分数上均要好于市售的同类产品。     

Low-fat mozzarella as influenced by microbial exopolysaccharides, preacidification, and whey protein concentrate

Low-fat Mozzarella cheeses containing 6% fat were made by preacidification of milk, preacidification combined with exopolysaccharide- (EPS-) producing starter, used independently or as a coculture with non-EPS starter, and preacidification combined with whey protein concentrate (WPC) and EPS. The impact of these treatments on moisture retention, changes in texture profile analysis, cheese melt, stretch, and on pizza bake performance were investigated over 45 d of storage at 4°C. Preacidified cheeses without EPS (control) had the lowest moisture content (53.75%). These cheeses were hardest and exhibited greatest springiness and chewiness. The meltability and stretchability of these cheeses increased most during the first 28 d of storage. The moisture content in cheeses increased to 55.08, 54.79, and 55.82% with EPS starter (containing 41.18 mg/g of EPS), coculturing (containing 28.61 mg/g of EPS), and WPC (containing 44.23 mg/g of EPS), respectively. Exopolysaccharide reduced hardness, springiness, and chewiness of low-fat cheeses made with preacidified milk in general and such cheeses exhibited an increase in cohesiveness and meltability. Although stretch distance was similar in all cheeses, those containing EPS were softer than the control. Cocultured cheeses exhibited the greatest meltability. Cheeses containing WPC were softest in general; however, hardness remained unchanged over 45 d. Cheeses made with WPC had the least increase in meltability over time. Incorporation of WPC did not reduce surface scorching or increase shred fusion of cheese shreds during pizza baking; however, there was an improvement in these properties between d 7 and 45. Coating of the cheese shreds with oil was necessary for adequate browning, melt, and flow characteristics in all cheese types.