Immobilization of peroxidase on textile carrier materials and their application in the bleaching of colored whey

Textiles represent promising support materials for enzymes. The goal of the present work was to investigate the immobilization of commercial peroxidase on a polyester needle felt and the repeated use in the gentle degradation of norbixin in whey from dairy cheese as a practical application. High enzyme loads were obtained by a 2-step immobilization procedure. First, the number of functional groups on the textile surface was increased by a modification with amino-functional polyvinylamine. Second, the enzyme was immobilized by using 2 types of crosslinking agents. Due to the iron content of peroxidase, inductively coupled plasma–optical emission spectrometry was used for the quantitative determination of the enzyme load on the textile. The enzyme activity was evaluated using common 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) assay for peroxidases. By the variation of enzyme input and crosslinker concentration, a maximal enzyme load of 80 mg/g of textile was achieved, and a maximum specific activity of 57 U/g of textile. For the visualization of the enzyme on the fiber surface, fluorescence microscopy as well as scanning probe microscopy were used. The immobilized peroxidase showed significant activity, even after 50 reuse cycles. In addition, the potential of the new support and enzyme combination in commercial whey bleaching was demonstrated successfully on a 10-L scale.     

干酪乳清水解产物的抗氧化活性研究

选用新鲜干酪乳清为原料,研究碱性蛋白酶对乳清水解产物的抗氧化活性。以水解产物的亚铁还原能力、对卵磷脂脂质氧化体系的过氧化抑制作用、羟自由基清除能力和超氧阴离子自由基的清除能力为指标评价乳清水解产物的抗氧化能力。结果表明,乳清在水解前经过预热处理并不能增加其水解产物的抗氧化活性,1 h水解物的亚铁还原能力最高,2 h水解产物对卵磷脂脂质氧化体系的过氧化抑制作用最高,抑制率达到24.82%;2 h水解产物羟自由基清除率最高,达到70.28%;2 h水解产物超氧阴离子自由基清除率最高,达到21.4%。但是乳清水解产物的抗氧化能力与水解度没有线性关系。     

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.     

Expanded functionality of modified whey protein dispersions after transglutaminase catalysis

The functionality of whey dispersions, prepared with a modified whey protein concentrate (mWPC) ingredient, was significantly altered after cross-linking with microbial transglutaminase (TGase) upon pH adjustment to 8. Test TGase-mWPC solutions, pH 8, gelled faster than control mWPC dispersions, as measured in real time; whereas, the gelling temperature of pretreated TGase-mWPC samples (37 °C, 2.5 h) increased from 67.8 to 74.8 °C with a minimal change in gel strength. Prolonged prior incubation with the enzyme (37 °C, 20 h) raised the gel strength in both control mWPC and TGase-mWPC dispersions, though these values were approximately 2.7 times lower in TGase-mWPC samples. Furthermore, the gelling temperature was raised by 9 °C after extensive polymerization. The water holding capacity was not impacted by enzymatic processing while emulsions prepared with TGase-mWPC dispersions proved very stable with no evidence of phase separation during storage at room temperature for 1 mo. Moreover, the apparent viscosity of TGase-mWPC emulsions exhibited a 10-fold increase compared to nonenzyme-treated mWPC samples. The particle size was nearly 11 μm in covalently linked TGase-mWPC test fractions compared with 8 μm in nonpolymerized mWPC dispersions. Ultimately, the functional characteristics of TGase-mWPC ingredients may be designed to deliver superior performance, especially with regard to improving heat and emulsion stability.     

The use of lactoperoxidase for the bleaching of fluid whey

Lactoperoxidase (LP) is the second most abundant enzyme in bovine milk and has been used in conjunction with hydrogen peroxide (H₂O₂) and thiocyanate (SCN^–) to work as an antimicrobial in raw milk where pasteurization is not feasible. Thiocyanate is naturally present and the lactoperoxidase system purportedly can be used to bleach dairy products, such as whey, with the addition of very little H₂O₂ to the system. This study had 3 objectives: 1) to quantify the amount of H₂O₂ necessary for bleaching of fluid whey using the LP system, 2) to monitor LP activity from raw milk through manufacture of liquid whey, and 3) to compare the flavor of whey protein concentrate 80% (WPC80) bleached by the LP system to that bleached by traditional H₂O₂ bleaching. Cheddar cheese whey with annatto (15 mL of annatto/454 kg of milk, annatto with 3% wt/vol norbixin content) was manufactured using a standard Cheddar cheesemaking procedure. Various levels of H₂O₂ (5–100 mg/kg) were added to fluid whey to determine the optimum concentration of H₂O₂ for LP activity, which was measured using an established colorimetric method. In subsequent experiments, fat-separated whey was bleached for 1 h with 250 mg of H₂O₂/kg (traditional) or 20 mg of H₂O₂/kg (LP system). The WPC80 was manufactured from whey bleached with 250 mg of H₂O₂/kg or 20 mg of H₂O₂/kg. All samples were subjected to color analysis (Hunter color values and norbixin extraction) and proximate analysis (fat, protein, and moisture). Sensory and instrumental volatile analyses were conducted on WPC80. Optimal LP bleaching in fluid whey occurred with the addition of 20 mg of H₂O₂/kg. Bleaching of fluid whey at either 35 or 50°C for 1 h with LP resulted in >99% norbixin destruction compared with 32 or 47% destruction from bleaching with 250 mg of H₂O₂/kg, at 35 or 50°C for 1 h, respectively. Higher aroma intensity and increased lipid oxidation compounds were documented in WPC80 from bleached whey compared with WPC80 from unbleached whey. Monitoring of LP activity throughout cheese and whey manufacture showed that LP activity sharply decreased after 30 min of bleaching (17.01 ± 1.4 to <1 U/mL), suggesting that sufficient bleaching takes place in a very short amount of time. Lactoperoxidase averaged 13.01 ± 0.7 U/mL in unpasteurized, fat-separated liquid whey and 138.6 ± 11.9 U/mL in concentrated retentate (11% solids). Lactoperoxidase may be a viable alternative for chemical whey bleaching.     

Efficient encapsulation of fish oil: Capitalizing on the unique inherent characteristics of whey cream and hydrolyzed whey protein

The effects of protein concentration and of blending a phospholipid-rich whey coproduct, Procream (Salibra 700 Procream, Glanbia Nutritionals), with intact or hydrolyzed whey protein concentrate, on fish oil microencapsulation efficiency and oxidative stability were assessed. Trypsin and protease M, from Aspergillus oryzae, were used to produce 2 unique hydrolysates. All microcapsules had excellent encapsulation efficiencies (>92%) and good physical properties, regardless of protein content and Procream inclusion. Intact α-lactalbumin and β-lactoglobulin and their peptides were involved in stabilizing oil droplets. Disulfide interchange resulted in formation of protein aggregates, which were more pronounced in samples containing Procream. Although all microcapsules had relatively good oxidative stability, most had better stability at 2 versus 0.5% protein. Protease M hydrolysate + Procream microcapsules had the highest stability, regardless of protein content. Results demonstrated that Procream, at a reduced protein inclusion level, can partially replace more expensive whey protein ingredients in microencapsulation, when blended with a select hydrolysate.     

乳清多肽的制备及乳清多肽酒的研制

采用蛋白酶水解乳清粉，对乳清多肽进行酵母菌发酵，并对发酵所得乳清酒进行风味调配。乳清粉最佳的水解条件为：酶浓度与底物浓度之比为1％、温度60℃、pH9．0、时间120min，水解度21．22％。乳清多肽酒的最优发酵条件：接种量5％、起始pH7．5、温度22℃、发酵60h，酒精度可达到3．9％。多肽乳清酒的最佳基本调配是总酸（苹果酸：柠檬酸=1：1）为0．1％、蔗糖为7％、环状糊精为0．6％。     

The effect of bleaching agents on the degradation of vitamins and carotenoids in spray-dried whey protein concentrate

Previous research has shown that bleaching affects flavor and functionality of whey proteins. The role of different bleaching agents on vitamin and carotenoid degradation is unknown. The objective of this study was to determine the effects of bleaching whey with traditional annatto (norbixin) by hydrogen peroxide (HP), benzoyl peroxide (BP), or native lactoperoxidase (LP) on vitamin and carotenoid degradation in spray-dried whey protein concentrate 80% protein (WPC80). An alternative colorant was also evaluated. Cheddar whey colored with annatto (15 mL/454 L of milk) was manufactured, pasteurized, and fat separated and then assigned to bleaching treatments of 250 mg/kg HP, 50 mg/kg BP, or 20 mg/kg HP (LP system) at 50°C for 1 h. In addition to a control (whey with norbixin, whey from cheese milk with an alternative colorant (AltC) was evaluated. The control and AltC wheys were also heated to 50°C for 1 h. Wheys were concentrated to 80% protein by ultrafiltration and spray dried. The experiment was replicated in triplicate. Samples were taken after initial milk pasteurization, initial whey formation, after fat separation, after whey pasteurization, after bleaching, and after spray drying for vitamin and carotenoid analyses. Concentrations of retinol, a-tocopherol, water-soluble vitamins, norbixin, and other carotenoids were determined by HPLC, and volatile compounds were measured by gas chromatography-mass spectrometry. Sensory attributes of the rehydrated WPC80 were documented by a trained panel. After chemical or enzymatic bleaching, WPC80 displayed 7.0 to 33.3% reductions in retinol, β-carotene, ascorbic acid, thiamin, α-carotene, and α-tocopherol. The WPC80 bleached with BP contained significantly less of these compounds than the HP- or LP-bleached WPC80. Riboflavin, pantothenic acid, pyridoxine, nicotinic acid, and cobalamin concentrations in fluid whey were not affected by bleaching. Fat-soluble vitamins were reduced in all wheys by more than 90% following curd formation and fat separation. With the exception of cobalamin and ascorbic acid, water-soluble vitamins were reduced by less than 20% throughout processing. Norbixin destruction, volatile compound, and sensory results were consistent with previous studies on bleached WPC80. The WPC80 colored with AltC had a similar sensory profile, volatile compound profile, and vitamin concentration as the control WPC80.     

Influence of storage, heat treatment, and solids composition on the bleaching of whey with hydrogen peroxide

The residual annatto colorant in liquid whey is bleached to provide a desired neutral color in dried whey ingredients. This study evaluated the influence of starter culture, whey solids and composition, and spray drying on bleaching efficacy. Cheddar cheese whey with annatto was manufactured with starter culture or by addition of lactic acid and rennet. Pasteurized fat-separated whey was ultrafiltered (retentate) and spray dried to 34% whey protein concentrate (WPC34). Aliquots were bleached at 60 °C for 1 h (hydrogen peroxide, 250 ppm), before pasteurization, after pasteurization, after storage at 3 °C and after freezing at -20 °C. Aliquots of retentate were bleached analogously immediately and after storage at 3 or -20 °C. Freshly spray dried WPC34 was rehydrated to 9% (w/w) solids and bleached. In a final experiment, pasteurized fat-separated whey was ultrafiltered and spray dried to WPC34 and WPC80. The WPC34 and WPC80 retentates were diluted to 7 or 9% solids (w/w) and bleached at 50 °C for 1 h. Freshly spray-dried WPC34 and WPC80 were rehydrated to 9 or 12% solids and bleached. Bleaching efficacy was measured by extraction and quantification of norbixin. Each experiment was replicated 3 times. Starter culture, fat separation, or pasteurization did not impact bleaching efficacy (P > 0.05) while cold or frozen storage decreased bleaching efficacy (P < 0.05). Bleaching efficacy of 80% (w/w) protein liquid retentate was higher than liquid whey or 34% (w/w) protein liquid retentate (P < 0.05). Processing steps, particularly holding times and solids composition, influence bleaching efficacy of whey. PRACTICAL APPLICATION: Optimization of whey bleaching conditions is important to reduce the negative effects of bleaching on the flavor of dried whey ingredients. This study established that liquid storage and whey composition are critical processing points that influence bleaching efficacy.     

乳清多肽的制备及其发酵饮料的开发

研究了乳清多肽的制备、性质及其发酵饮料的开发,结果表明,碱性蛋白酶比中性蛋白酶水解乳清蛋白的能力强,且更经济,水解最佳条件为加酶量为7000(U/g蛋白)、底物添加量为5%、水解温度为60℃、水解初始pH值为8.5,最大乳清蛋白水解度可达到22.45%。最优酒精发酵条件为接种量5%、初始pH7.5、温度22℃、时间45h。乳清多肽发酵饮料的配方为酸量0.1%,蔗糖量为8%,-β环状糊精量为0.5%。     

Fractionation and characterisation for antioxidant activity of hydrolysed whey protein

Whey protein isolate (WPI) was hydrolysed for 1 h using Alcalase, Protamex and Flavourzyme. Native WPI, hydrolysed WPI and two commercial WPI hydrolysates were subjected to fractionation by size exclusion chromatography. Antioxidant activity of WPI fractions was measured with a liposome‐oxidising system (50 µM FeCl₃/0.1 µM ascorbate, pH 7.0). Lipid oxidation was measured as thiobarbituric acid‐reactive substances (TBARS). Gel electrophoresis and amino acid analysis were run to identify the peptide composition. The influence of amino acid composition on antioxidant activity was evaluated using multivariate analysis methods (correlation analysis, principal component analysis, multiple linear regression and discriminant analysis). TBARS assays indicated the presence of antioxidant activity in all protein fractions, including non‐hydrolysed WPI. For native and hydrolysed WPI samples the first fraction (> 45 kDa) showed a higher TBARS inhibition effect (24–27%) when compared with lower‐molecular‐weight fractions and hydrolysate mixtures. In contrast, for commercial WPI hydrolysates a higher inhibitory effect was found in most of the lower‐molecular‐weight fractions (30–55%). The ability of WPI fractions to delay lipid oxidation was found to be related to the prevalence of histidine and hydrophobic amino acids. Copyright © 2004 Society of Chemical Industry     

乳清蛋白-甘油二酯纳米乳液制备及其质量评价的研究

采用高压均质法制备乳清蛋白-甘油二酯纳米乳液,以粒径和包埋率为综合指标,在单因素实验的基础上,采用响应面分析法优化纳米乳液的制备条件,并对纳米乳液的表面性质、表征、温度、氧化及贮藏稳定性进行研究。结果表明,乳清蛋白-甘油二酯纳米乳液的最佳工艺条件为:壁材浓度15.83%,壁芯比3.35∶1,乳化剂添加量4.02%,此时,纳米乳液的包埋率最高,为75.5%。纳米乳液带负电,分布均匀,平均粒径在142 nm左右,有明显的壳核结构,包被效果较好。纳米乳液在80℃以下具有较好的稳定性,且能有效延缓甘油二酯的氧化,最佳贮藏温度为4℃。     

乳清蛋白功能活性的研究

乳清蛋白是牛乳乳清中存在的一类蛋白质,其必需氨基酸种类齐全、数量充足且比例适当,是一种营养价值较高的优质蛋白,在维持机体肠道、肌肉组织的健康和补充体内的谷胱甘肽数量、抗氧化等方面有重要作用。乳清蛋白中还含有β-乳球蛋白、α-乳白蛋白、血清白蛋白、免疫球蛋白、乳铁传递蛋白以及乳过氧物酶等多种生物活性蛋白。这些物质参与构成机体非特异性防御屏障,在抗菌、抗病毒、免疫调节等方面也发挥积极作用。     

Effects of native and denatured whey proteins on plasminogen activator activity

The plasmin system native to bovine milk consists of the caseinolytic serine proteinase plasmin; its inactive zymogen, plasminogen; plasminogen activators; and inhibitors. Evidence in the literature indicates that whey proteins may inhibit plasmin activity, but there is very little mention of their effect on plasminogen activators. The objective of this research was to determine the effect of both unheated and heat-denatured beta-lactoglobulin (beta-LG), alpha-lactalbumin (alpha-LA), and BSA on plasminogen activators. Plasminogen activator activity was significantly stimulated by non-heat treated and denatured alpha-LA as well as by denatured beta-LG. The stimulation effect by these whey proteins was kinetically characterized, which showed that all 3 significantly increased the rate of plasminogen activation. The stimulation effect was shown to be independent of any effect of the whey proteins on plasmin activity by testing 2 different substrates, d-Val-Leu-Lys p-nitroanilide (S-2251) and Spectrozyme PL (Spec PL), in a plasmin assay. Results using S-2251 confirmed the inhibitory effect of whey proteins on plasmin observed by several researchers. However, use of SpecPL did not suggest inhibition. Ligand binding studies showed this discrepancy to be due to significant interaction between S-2251 and the whey proteins. Overall, this study indicates that whey protein incorporation into cheese may not hinder plasmin activity and may stimulate plasminogen activation. Furthermore, the results indicate the need for careful consideration of the type of synthetic substrate chosen for model work involving whey proteins and the plasmin system.     

乳清蛋白可食用膜抑菌性、溶解性的研究

主要研究了Nisin、纳塔霉素和溶菌酶不同组合添加下乳清蛋白可食用膜的抑菌性和溶解性,结果表明:随着抑菌剂浓度的增加,膜的抑菌效果增强,不同抑菌剂组合下膜的抑菌效果不同,0.35%Nisin和0.08%溶菌酶组合下抑菌效果更好。在酸性环境中,当添加0.03%溶菌酶和0.25%纳塔霉素时,可有效降低膜的溶解性;在中性环境中,各组膜的溶解性几乎相当,在碱性环境中,大部分膜的溶解性要低于其在酸性环境中。