Effects of pH, temperature, supplementation with whey protein concentrate, and adjunct cultures on the production of exopolysaccharides by Streptococcus thermophilus 1275

Effects of pH, temperature, supplementation with whey protein concentrate (WPC), and non-EPS culture on the exopolysaccharide (EPS) production by Streptococcus thermophilus 1275 were studied. The organism was grown in 10% reconstituted skim milk (RSM) in a Biostat B fermenter for 24 h at various pH (4.5, 5.5 and 6.5) and temperatures (30, 37, 40, and 42 degrees C), and supplementation with WPC 392, and non-EPS producing S. thermophilus 1303 and the amount of EPS produced were determined. Bacterial counts were enumerated and the concentrations of lactic acid, lactose, glucose, and galactose were also determined. A maximum of 406 mg/L of EPS was produced in RSM at 37 degrees C after 24 h of fermentation at pH 4.08 when the pH was not controlled. A pH of 5.5 and temperature of 40 degrees C were found to be optimal for EPS production by S. thermophilus 1275, yielding 458 mg/L. The EPS production increased when RSM was supplemented with WPC 392. At optimum pH and at 37 degrees C with WPC supplementation, the level of EPS increased to 1029 mg/L. Co-culturing S. thermophilus 1275 with non-EPS S. thermophilus 1303 increased EPS production at 37 degrees C and pH 5.5 to 832 mg/L. High temperature (42 degrees C) reduced the amount of EPS production, and EPS production ceased at pH 4.5 when maintained constantly at this pH. The level of lactose utilization and lactic acid production depended on growth conditions of the organism. No glucose was detected, while galactose was found to accumulate in the medium.     

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。     

利用冷榨花生饼制备花生多肽饮料

以冷榨花生饼为原料,采用碱法和酶水解法制备花生蛋白,以蛋白质提取率为指标,确定蛋白提取条件,并利用所提取蛋白或蛋白水解物经乳酸菌发酵制备花生多肽饮料。结果表明NaOH溶液提取花生蛋白的最佳条件为：pH9.0、温度55℃、料液比1:8(g/mL)、浸提2h,蛋白提取率80.68%;胰蛋白酶水解蛋白的最佳条件为：酶与底物比1:50(m/m)、底物质量浓度5g/100mL、pH9.0、水解温度50℃,蛋白提取率96.26%。以花生水解蛋白和脱盐乳清粉为原料,采用直投式乳酸菌为发酵剂,发酵条件为：花生水解蛋白质量浓度2g/100mL、乳清粉加入量1g/100mL、发酵剂与发酵液比1:25(g/kg)、42℃发酵5h、4℃后熟15h、蔗糖质量分数9%时的口感最佳。     

水牛奶乳清蛋白制备抗氧化活性肽工艺的研究

实验是以水牛奶为原料,分离纯化后得到乳清蛋白。利用碱性蛋白酶、中性蛋白酶、胰蛋白酶、木瓜蛋白酶和胃蛋白酶5种不同的蛋白酶对水牛奶乳清蛋白酶解以制备抗氧化活性多肽。酶筛选结果显示,中性蛋白酶是最适宜酶解水牛奶乳清蛋白制备抗氧化活性肽,其酶解液的还原能力和DPPH自由基清除率较其他4种酶高。探讨酶解反应时pH、温度、时间、酶浓度对酶解反应的水解度、酶解液的还原能力和DPPH自由基的清除率的影响,在单因素试验基础上,采用响应面法对酶解工艺进行优化。结果表明,中性蛋白酶酶解乳清蛋白的最佳工艺参数为:pH为7.4,温度为50.5℃,酶与底物浓度比为2.1%,酶解时间5.0h,此时2mg/mL酶解物的DPPH自由基清除率为32.58%。实测结果与预测值吻合效果良好。     

文蛤肉复合酶分步酶解工艺的研究

本实验探讨了中性蛋白酶和胰蛋白酶的复合酶与风味蛋白酶分步酶解文蛤肉的技术。 通过q检验法确定中性蛋白酶和胰蛋白酶最佳的复合比例,再通过正交试验探讨复合酶与风味蛋白酶二段酶解的最佳工艺参数,并以水解度、水解得率及风味评分值为指标对分步酶解工艺的最佳条件进行比较验证。结果表明,胰蛋白酶与中性蛋白酶的最佳复合比例为3:1,风味蛋白酶二段酶解的最优工艺参数为水解温度55℃、水解时间5.0h、加酶量1000 U/g(原料)、pH值(5.00±0.05),所得文蛤肉水解液中水解度、水解得率及风味评分值分别为55.97%、87.14%及230.98。     

保加利亚乳杆菌发酵乳清的抗氧化性研究

通过控制初始发酵条件:pH6.0、发酵温度39℃,可以提高发酵乳清清除羟基自由基和DPPH.的能力。在发酵20h内,随着乳清蛋白水解度的增加,发酵乳清清除羟自由基和DPPH.的能力也随之增大,在发酵20h时达到最大,分别为48.06%和73.52%,发酵乳清清除羟自由基和DPPH.的能力与未发酵乳清相比分别提高了22.73%和46.09%,与保加利亚乳杆菌菌体相比分别提高了23.75%和40.63%。探讨了蛋白水解度和保加利亚乳杆菌活菌数和抗氧化活性之间的关系。20h后,发酵液自由基清除能力与水解度之间没有正相关性,因此不能采用蛋白水解度作为评价发酵乳清抗氧化性的指标。本研究对可能影响保加利亚乳杆菌发酵乳清抗氧化性的因素进行探讨,为开发功能性乳清产品奠定了理论和实践基础。     

Production and partial purification of proteases from Aspergillus oryzae grown in a medium based on whey protein as an exclusive nitrogen source

Several attempts have been made to incorporate whey proteins into curd to increase cheese yield. For some types of cheese, degradation of whey proteins that have been incorporated into the curd would be required to obtain acceptable flavor and texture. On the basis of the high potential for protease synthesis in Aspergillus oryzae, sodium nitrate as a nitrogen source in a minimal medium for fungi, known as Czapek-Dox medium, was replaced with whey protein isolate to induce the protease to hydrolyze whey protein using A. oryzae AHU7146. A solid-phase medium adjusted to pH 6 was suitable for this purpose when incubation was carried out at 25°C for 2 wk. The application of column chromatography enabled the resolution of 3 proteolytic components (1, 2, and 3). With respect to optimal temperature and zymographic analysis, component 1 was similar to component 3. In contrast, component 2 was less abundant than the other components and exhibited activity in the alkaline pH region. The degradation of β-lactoglobulin and α-lactalbumin in whey protein isolate solution by the crude enzyme was primarily attributed to the action of components 1 and 3, based on HPLC analysis and the N-terminal amino acid sequences; however, zymography demonstrated evident proteolysis due to component 2. Because heat-denatured whey protein aggregates were digestible by the crude enzyme, the proteolytic system from A. oryzae has the potential as an additive to stimulate the ripening of cheese enriched with whey protein.     

Hydrolysis of lactose in whey permeate for subsequent fermentation to ethanol

Fermentation of lactose in whey permeate directly into ethanol has had only limited commercial success, as the yields and alcohol tolerances of the organisms capable of directly fermenting lactose are low. This study proposes an alternative strategy: treat the permeate with acid to liberate monomeric sugars that are readily fermented into ethanol. We identified optimum hydrolysis conditions that yield mostly monomeric sugars and limit formation of fermentation inhibitors such as hydroxymethyl furfural by caramelization reactions. Both lactose solutions and commercial whey permeates were hydrolyzed using inorganic acids and carbonic acid. In all cases, more glucose was consumed by secondary reactions than galactose. Galactose was recovered in approximately stoichiometric proportions. Whey permeate has substantial buffering capacity-even at high partial pressures (>5500 kPa[g]), carbon dioxide had little effect on the pH in whey permeate solutions. The elevated temperatures required for hydrolysis with CO2-generated inhibitory compounds through caramelization reactions. For these reasons, carbon dioxide was not a feasible acidulant. With mineral acids reversion reactions dominated, resulting in a stable amount of glucose released. However, the Maillard browning reactions also appeared to be involved. By applying Hammet's acidity function, kinetic data from all experiments were described by a single line. With concentrated inorganic acids, low reaction temperatures allowed lactose hydrolysis with minimal by-product formation and generated a hexose-rich solution amenable to fermentation.     

酪蛋白水解物替代乳清蛋白的加工性能评价研究

利用复合蛋白酶水解酪蛋白制备适度及深度水解酪蛋白产品,测定酪蛋白水解物的加工性能。结果表明,经过酶解后,适度水解酪蛋白溶解度接近90%,深度水解酪蛋白溶解度接近100%,显著高于酪蛋白和乳清蛋白。此外,适度水解酪蛋白吸油性、起泡性分别约为乳清蛋白的3倍和1.5倍。深度水解酪蛋白在起泡性和乳化性上也显著高于乳清蛋白。可见,两款酪蛋白水解物在起泡性、乳化性、吸油性、溶解性等方面均在一定程度上优于乳清蛋白,可广泛替代乳清蛋白在食品工业中大规模应用。      

Enzymatic proteolysis,under high pressure of soybean whey: Analysis of peptides and the allergen Gly m 1 in the hydrolysates

Soybean (Glycine max) whey was hydrolyzed with Alcalase, Neutrase, Corolase 7089 and Corolase PNL during high pressure (HP) treatment at 100, 200 and 300 MPa and at atmospheric pressure for 15 min. The protein content and the degree of hydrolysis were determined. Furthermore, the allergen Gly m 1 in the treated soybean whey and the hydrolysates was detected. The results showed that HP treatments increased the hydrolysis by the four proteases used. Pressure at 200 and 300 MPa proved to be better pressures to enhance the proteolysis. The immunochemical response of soybean whey to anti-Gly m 1 monoclonal antibodies decreased after the HP treatments and this decrease was greater after the combined treatment of high pressure and enzymatic hydrolysis. Soybean whey proteins hydrolysed at high pressure could be used as sources of peptides with low antigenicity when incorporated as food ingredients.     

Peptides released from acid goat whey by a yeast-lactobacillus association isolated from cheese microflora

Seven lactobacilli and a variety of microflora extracted from twenty five commercial cheeses were grown on unsupplemented acid goat whey and screened for their capacity to hydrolyse whey proteins [alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg)] and to generate peptides. Fermentations were performed aerobically or anaerobically at 37 degrees C using crude or pre-heated whey (10 min at 65, 75 or 85 degrees C). Under aerobic conditions, growth of lactobacilli was poor and protein hydrolysis did not occur. Anaerobic conditions slightly increased lactobacilli growth but neither beta-lg hydrolysis nor peptide generation were observed. More than 50% of alpha-la was digested into a truncated form of alpha-la (+/- 12 kDa) in crude whey and whey pre-heated at 65 degrees C. Twenty-five microflora extracted from raw milk cheeses were screened for their proteolytic activities on acid goat whey under the conditions previously described. Eight of them were able to hydrolyse up to 50% of alpha-la mainly during aerobic growth on crude or pre-heated whey. The corresponding hydrolysates were enriched in peptides. The hydrolysate involving microflora extracted from Comté cheese after or at 18 months ripening was the only one to exhibit hydrolysis of both alpha-la and beta-lg. Microbiological analysis showed that microorganisms originating from Comté cheese and capable of growth on unsupplemented whey consisted of Candida parapsilosis and Lactobacillus paracasei. Fermentation kinetic profiles suggested that peptides were released from alpha-la hydrolysis. The co-culture of both microorganisms was required for alpha-la hydrolysis that occurred concomitantly with the pH decrease. During whey fermentation, Cand. parapsilosis excrete at least one protease responsible for alpha-la hydrolysis, and Lb. paracasei is responsible for medium acidification that is required for protease activation.     

Effect of protein supplementation on the rheological characteristics of milk permeates fermented with exopolysaccharide-producing Lactococcus lactis subsp. cremoris

This research studied the effect of addition of whey proteins on the rheological properties of ultrafiltration permeate fermented with the exopolysaccharide (EPS)-producing strain Lactococcus lactis subsp. cremoris JFR1. Milk permeates containing 8% solids and various levels of added whey proteins (0, 2, 4, 6 and 8%) were fermented for 12 h at 30 °C. The rheological properties of the fermented samples were then evaluated and compared to controls fermented with a non-EPS producing strain. Scanning electron microscopy was also employed to confirm the existence of interactions between whey protein aggregates and EPS. The presence of EPS considerably increased the viscosity and viscoelastic properties of the media, especially in samples containing >2% whey protein added. The results obtained demonstrate the importance of EPS–protein interactions in structure formation and may help explain the viscosifying mechanism of EPS in fermented dairy products. Production of highly viscous material could potentially be employed in the future as a novel fiber-rich functional ingredient in dairy products.     

双酶法制备混合蛋白肽的研究

利用双酶法同步水解动植物混合蛋白(大豆蛋白和虾粉)制备蛋白肽,最佳酶解条件为:大豆蛋白粉和虾粉混合蛋白(按照3∶2的比例混合)作为底物,底物浓度为12.0%、Flavourzyme风味蛋白酶的添加量为1.27%、Protamex复合蛋白酶的添加量为0.64%、水解温度55℃,起始pH值7.0,酶水解时间4h,此条件下水解度为29.0%。     

Use of chitosan for selective removal of beta-lactoglobulin from whey

A method is described for selective removal of undenatured β-lactoglobulin from cheese whey based on interactions between whey proteins and chitosan. Whey was previously clarified at pH 4.5 with addition of chitosan (25 mg/100 mL), and selective removal of β-lactoglobulin was studied in the pH interval 4.6 to 6.5. Addition of chitosan caused selective precipitation of β-lactoglobulin that increased with pH. The content of β-lactoglobulin in whey decreased as the amount of chitosan added was increased. At pH 6.2, addition of 1.9 to 3.0 mg/mL of chitosan led to complete removal of β-lactoglobulin, whereas at least 80% of the rest of whey proteins remained in solution. The production of cheese whey without β-lactoglobulin could help to expand the applications of dairy by-products in food processing, and to isolate hypoallergenic whey protein concentrates.     

Survival and growth characteristics of Escherichia coli O157:H7 in pasteurized and unpasteurized Cheddar cheese whey

The objective of this study was to determine the survival and growth characteristics of Escherichia coli O157:H7 in whey. A five-strain mixture of E. coli O157:H7 was inoculated into 100 ml of fresh, pasteurized or unpasteurized Cheddar cheese whey (pH 5.5) at 10(5) or 10(2) CFU/ml, and stored at 4, 10 or 15 degrees C. The population of E. coli O157:H7 (on Sorbitol MacConkey agar supplemented with 0.1% 4-methylumbelliferyl-beta-D-glucuronide) and lactic acid bacteria (on All Purpose Tween agar) were determined on days 0, 1, 4, 7, 14, 21 and 28. At all storage temperatures, survival of E. coli O157:H7 was significantly higher (P<0.01) in the pasteurized whey compared to that in the unpasteurized samples. At 10 and 15 degrees C, E. coli O157:H7 in pasteurized whey significantly (P<0.05) increased during the first week of storage, followed by a decrease thereafter. However at the same temperatures, E. coli O157:H7 exhibited a steady decline in the unpasteurized samples from day 0. At 4 degrees C, E. coli O157:H7 did not grow in pasteurized and unpasteurized whey; however, the pathogen persisted longer in pasteurized samples. At all the three storage temperatures, E. coli O157:H7 survived up to day 21 in the pasteurized and unpasteurized whey. The initial load of lactic acid bacteria in the unpasteurized whey samples was approximately 7.0 log10 CFU/ml and, by day 28, greater than 3.0 log10 CFU/ml of lactic acid bacteria survived in unpasteurized whey at all temperatures, with the highest counts recovered at 4 degrees C. Results indicate the potential risk of persistence of E. coli O157:H7 in whey in the event of contamination with this pathogen.     

东海海参胶原蛋白多肽的制备及清除自由基功能研究

以东海海参为原料,采用酶法提取胶原蛋白。选取木瓜蛋白酶、风味蛋白酶以及复合酶(木瓜-风味),在其最佳水解条件下水解,以自由基清除率为指标,确定最佳水解时间及酶种类。研究结果表明,用木瓜蛋白酶水解4 h得到的多肽清除自由基的能力最强,清除率在70%以上;多肽清除羟自由基和超氧自由基的IC50分别为27.8mg/mL和49.3mg/mL。通过超滤处理及DEAE琼脂糖离子交换柱(DEAE Sepharose FF)分离纯化抗氧化肽,得到分子质量小于5kDa的肽,其功能性最强。利用HPLC测定该肽的氨基酸含量,结果显示该肽段以甘氨酸(Gly)、丙氨酸(Ala)、谷氨酸(Glu)、脯氨酸(Pro)、天冬氨酸(Asp)、精氨酸(Arg)、丝氨酸(Ser)和苏氨酸(Thr)为主。     

Effect of protein concentration, pH, lactose content and pasteurization on thermal gelation of acid caprine whey protein concentrates

The influence of pH (4.5-6.5), sodium chloride content (125-375 mM), calcium chloride content (10-30 mM), protein concentration (70-90 g/l) and lactose content on the gel hardness of goat whey protein concentrate (GWPC) in relation to the origin of the acid whey (raw or pasteurized milk) was studied using a factorial design. Gels were obtained after heat treatment (90 degrees C, 30 min). Gel hardness was measured using texture analyser. Only protein concentration and pH were found to have a statistically significant effect on the gel hardness. An increase in the protein concentration resulted in an increase in the gel hardness. GWPC containing 800g/kg protein formed gels with a hardness maximum at the pHi, whereas GWPC containing 300 g/kg protein did not form true gels. Whey from pasteurized milk formed softer gels than whey from raw milk. A high lactose content (approximately 360 g/kg) also reduced the gelation performance of GWPC.     

Composition and calcium status of acid whey from pasteurized, UHT-treated and in-bottle sterilized milk

Whey extracts were obtained from pasteurized, UHT-treated and in-bottle sterilized milks. After acidic precipitation of casein the concentration of protein, NPN, lactose, lipid, calcium, magnesium and potassium was determined. Among the parameters examined, protein content was significantly reduced in the whey extracts from UHT-treated and in-bottle sterilized milks compared with that from pasteurized milk, while lactose content was increased. Calcium extracted in whey was at least 80% of total calcium of the milk. The total calcium to protein ratio of whey was increased as a function of the thermal treatment of milk, while ionic calcium was about 50% of total calcium in all whey extracts. In vitro protein digestibility was found to be significantly lower in whey from UHT-treated and in-bottle sterilized milks than in that from pasteurized milk. Parallel estimation of the percentage of ionic calcium and of the solubility of proteins in the pH range 2-10 indicated that calcium was not involved in the pH-dependent solubility of proteins extracted in the whey, the extent of solubility being essentially a function of the thermal treatment of milk. The results suggest that calcium was not responsible for the formation of soluble protein macroaggregates with impaired digestibility that are present in whey from milk subjected to heat treatment of increasing intensity.     

分步酶解法制备黄浆水活性肽

黄浆水是传统豆制品点脑成型过程压榨出的废弃物,富含低聚糖、蛋白质等营养成分。该研究通过比较酶种类及用量、酶解温度、酶解时间对黄浆水蛋白质水解度的影响,采用正交试验优化获得黄浆水短肽最佳分步酶解工艺：（1）酸性蛋白酶加酶量2 000 U/g,pH 4.0,温度55 ℃,水解2 h;（2）中性蛋白酶8 000 U/g,pH 6.0,温度50 ℃,水解6 h。在此条件下进行验证,水解度可达25.95%,血管紧张素转化酶体外抑制活性达92.0%。采用酸性蛋白酶和中性蛋白酶分步酶解黄浆水制备短肽,制备条件温和,水解度高,可为豆制品加工废弃物的高值化利用奠定基础。     

Composition and calcium status of acid whey from pasteurized,UHT‐treated and in‐bottle sterilized milks

Whey extracts were obtained from pasteurized, UHT‐treated and in‐bottle sterilized milks. After acidic precipitation of casein the concentration of protein, NPN, lactose, lipid, calcium, magnesium and potassium was determined. Among the parameters examined, protein content was significantly reduced in the whey extracts from UHT‐treated and in‐bottle sterilized milks compared with that from pasteurized milk, while lactose content was increased. Calcium extracted in whey was at least 80% of total calcium of the milk. The total calcium to protein ratio of whey was increased as a function of the thermal treatment of milk, while ionic calcium was about 50% of total calcium in all whey extracts. In vitro protein digestibility was found to be significantly lower in whey from UHT‐treated and in‐bottle sterilized milks than in that from pasteurized milk. Parallel estimation of the percentage of ionic calcium and of the solubility of proteins in the pH range 2–10 indicated that calcium was not involved in the pH‐dependent solubility of proteins extracted in the whey, the extent of solubility being essentially a function of the thermal treatment of milk. The results suggest that calcium was not responsible for the formation of soluble protein macroaggregates with impaired digestibility that are present in whey from milk subjected to heat treatment of increasing intensity.