凝乳酶添加量对牦牛乳硬质干酪蛋白质降解的影响

通过测定不同可溶性氮含量以及采用尿素凝胶电泳法,研究了不同小牛皱胃酶添加量对牦牛乳硬质干酪3个月成熟过程中蛋白质降解的影响,并对干酪苦味进行了感官评价。研究表明:凝乳酶添加量对干酪p H 4.6SN和12%TCASN影响显著(P0.05),在成熟1个月时,不同凝乳酶添加量干酪p H 4.6SN之间差距较大,且凝乳酶添加量与干酪p H 4.6SN间存在较强线性关系。凝乳酶添加量对5%PTASN和游离氨基酸影响不显著(P0.05)。尿素凝胶电泳显示:干酪中αs-酪蛋白降解依赖于凝乳酶添加量,且降解程度大于β-酪蛋白。凝乳酶添加量对干酪苦味影响显著(P0.05),且随着凝乳酶添加量的增多,其苦味程度逐渐加重,但是大部分干酪苦味属于轻微苦味和中等程度苦味之间。干酪苦味与12%TCASN、αs-酪蛋白和β-酪蛋白降解率之间具有较强相关性(Spearman相关系数0.7,P0.01)。     

成熟温度和时间对牦牛乳硬质干酪中生物活性肽的影响

以不同条件成熟的牦牛乳硬质干酪为研究对象,探究成熟温度和时间对牦牛乳硬质干酪中水溶性多肽的DPPH自由基清除能力、抑菌性以及抑制血管紧张素转换酶（ACE）活性的影响。结果表明,5 ℃、10 ℃、15 ℃成熟4～6个月牦牛乳硬质干酪的水溶性多肽对DPPH自由基均有清除能力。5 ℃、10 ℃成熟5个月、15 ℃成熟4个月牦牛乳硬质干酪的水溶性多肽对DPPH自由基清除率最高,分别为（20.38±1.20）%、（28.70±0.67）%和（30.19±0.32）%。5 ℃、10 ℃、15 ℃成熟的牦牛乳硬质干酪在4～6个月成熟过程中,其水溶性多肽对大肠杆菌、金黄色葡萄球菌的抑制程度分别呈增强趋势。成熟温度从5 ℃提高到15 ℃时,牦牛乳硬质干酪的水溶性多肽对大肠杆菌、金黄色葡萄球菌和血管紧张素转换酶的抑制作用呈加强趋势。     

发酵剂添加量和成熟时间对牦牛乳硬质干酪脂肪氧化的影响

为研究发酵剂添加量和成熟时间对牦牛乳硬质干酪中脂肪氧化的影响,试验以添加不同发酵剂质量分数(1%、2%、3%)制得的牦牛乳硬质干酪为材料,对其90 d成熟期内的氧化指标和理化指标进行测定。结果表明:发酵剂添加量和成熟时间对牦牛乳硬质干酪的理化指标和脂肪氧化程度均有显著性影响(P <0. 05),且随发酵剂添加量的增加,干酪中酸度值(acidity value,ADV)、过氧化值(peroxide value,POV)、羰基价(carbonyl value,CV)、硫代巴比妥酸值(thiobarbituric acid value,TBA)增加;随成熟时间的延长,ADV、CV、TBA值增加,POV值先增加后降低。发酵剂添加量为3%时,能够显著增大干酪中脂肪的氧化程度(P <0. 05),同时随成熟时间的延长,脂肪氧化持续进行并且氧化程度也在不断加深。该研究将发酵剂添加量和成熟时间相结合,探讨牦牛乳硬质干酪中脂肪氧化的变化规律,以期从脂肪的氧化机制调控干酪的品质,为实现工业化生产提供理论依据。     

发酵剂对牦牛乳硬质干酪成熟过程中生物胺的影响

乳酸菌产生物胺的能力具有菌株特异性,因此,为了探究不同种类发酵剂对牦牛乳硬质干酪中生物胺形成的影响,该试验利用高效液相色谱对3种不同发酵剂制作的硬质干酪成熟过程中生物胺进行了测定和分析。结果表明,嗜热和嗜温发酵剂牦牛乳硬质干酪中检测出2-苯乙胺、腐胺、尸胺、组胺和酪胺,混合发酵剂干酪中检测出腐胺、2-苯乙胺、尸胺和酪胺。各生物胺之间呈现正相关性。3种不同发酵剂干酪在1～6个月成熟过程中,其各生物胺整体呈现增加趋势,嗜热、嗜温和混合发酵剂干酪中总生物胺最高含量分别为(448.3±9.6)、(456.8±58.4)、(293±24.5)mg/kg。组胺和酪胺是2种毒性相对高的生物胺,嗜热发酵剂干酪中组胺和嗜温发酵剂干酪中酪胺最高,其最高含量分别为(20.8±7.9)、(92.9±6.7)mg/kg,混合发酵干酪中未检测出组胺,酪胺含量次之,3种不同发酵剂干酪中组胺、酪胺含量均低于推荐安全剂量50 mg/kg和100 mg/kg。这为合理选择发酵剂和控制干酪中生物胺形成提供了依据。     

发酵剂对牦牛乳硬质干酪蛋白降解和苦味关系的影响

用甘肃天祝新鲜牦牛乳为原料分别添加嗜温、嗜热和混合发酵剂制作硬质干酪,以pH 4.6-可溶性氮（soluble nitrogen,SN）、12%三氯乙酸氮（trichloroacrtic acid-N,TCA-N）、游离氨基酸（free amino acid,FAA）含量和疏水性肽/亲水性肽（S/Q）为蛋白水解度指标,研究3 种牦牛乳硬质干酪在6 个月内成熟过程中苦味和蛋白质降解之间的关系。结果表明：3 种干酪在成熟过程中pH 4.6-SN、12% TCA-N和FAA含量均呈上升趋势,苦味值与pH 4.6-SN、12% TCA-N和FAA含量成正相关,相关系数分别为0.400、0.412和0.458。3 种干酪成熟过程中S/Q的变化趋势和程度不同,嗜温发酵剂干酪中S/Q呈现降低趋势;嗜热和混合发酵剂干酪中S/Q均呈现先降低后增大的趋势,但在这两种干酪中S/Q的变化程度不同,嗜热发酵剂干酪在1～3 个月S/Q略有降低,在3～6 个月S/Q快速增大,而混合发酵剂干酪正好相反。S/Q与苦味值成极显著正相关（r=0.895）,S/Q可很好地反映干酪中苦味的强弱。而干酪中苦味强弱与蛋白质降解强弱密切相关,对蛋白降解程度越大的发酵剂制作的干酪越容易产生苦味,其中,嗜热发酵剂对干酪蛋白降解程度最大,混合发酵剂次之,嗜温发酵剂最小。     

酪蛋白降解对牦牛乳硬质干酪苦味的影响

针对牦牛乳硬质干酪的苦味缺陷,分别以小牛皱胃酶、微生物凝乳酶和木瓜蛋白酶制作的牦牛乳硬质干酪为研究对象,利用尿素聚丙烯酰胺凝胶电泳,研究牦牛乳硬质干酪pH 4.6水不溶性酪蛋白的降解程度,且对成熟过程中的牦牛乳硬质干酪苦味进行感官评价,探究牦牛乳硬质干酪pH 4.6水不溶性酪蛋白降解对其苦味的影响。结果表明：牦牛乳硬质干酪在成熟期间酪蛋白发生了明显的降解,且αs-酪蛋白均比β-酪蛋白降解速率快。经尿素聚丙烯酰胺凝胶电泳分离后,发现木瓜蛋白酶制作的牦牛乳硬质干酪pH 4.6水不溶性酪蛋白在Pre-αs-酪蛋白区域有较强的蛋白带。木瓜蛋白酶制作的牦牛乳硬质干酪pH 4.6水不溶性酪蛋白中αs-酪蛋白和β-酪蛋白降解程度均显著或极显著高于微生物凝乳酶和小牛皱胃酶制作的牦牛乳硬质干酪（P＜0.05或P＜0.01）,木瓜蛋白酶制作的牦牛乳硬质干酪的苦味值极显著高于微生物凝乳酶和小牛皱胃酶制作的牦牛乳硬质干酪的苦味值（P＜0.01）,通过主成分分析得出3 种凝乳酶制作牦牛乳硬质干酪的苦味值和未降解β-酪蛋白和αs-酪蛋白含量成极显著负相关。这为控制牦牛乳硬质干酪品质提供了理论参考。     

原料乳冷藏时间对牦牛乳硬质干酪理化指标的影响

冷链贮存运输已广泛运用于各大乳制品生产,冷藏时间的长短对原料乳及其制作的干酪理化品质具有重要影响。该研究以冷藏24、48、72 h牦牛乳为原料制作硬质干酪,比较了硬质干酪成熟过程中的理化指标变化规律。结果表明,在0～6个月成熟期中,冷藏24、48、72 h牦牛乳制成的硬质干酪中水分含量、脂肪含量呈现降低趋势,NaCl含量呈现增加趋势。pH值在成熟初期下降,到成熟后期又逐渐升高;pH 4.6可溶性氮、12%三氯乙酸可溶性氮和游离氨基酸总量呈现增加趋势。整个成熟过程中,3组牦牛乳硬质干酪中pH 4.6可溶性氮、12%三氯乙酸可溶性氮以及游离氨基酸总量始终为冷藏72 h组>冷藏48 h组>冷藏24 h组。该研究可为牦牛乳的冷藏及其在干酪加工中的应用提供理论参考。     

冷藏乳制作的牦牛乳硬质干酪成熟过程中生物胺含量的变化

目的:揭示冷藏原料乳制作的干酪成熟过程中生物胺含量变化规律,评价其质量安全性。方法:以冷藏24,48,72 h牦牛乳制作的硬质干酪为研究对象,利用高效液相色谱法对干酪成熟过程中生物胺含量进行测定。结果:在0～6个月成熟过程中,不同冷藏牦牛乳制作的硬质干酪中生物胺含量呈升高趋势。牦牛乳冷藏时间从24 h延长到72 h时,其干酪中总生物胺、2-苯乙胺、尸胺、酪胺和腐胺含量也依次增大。成熟4个月后,冷藏72 h牦牛乳制作的硬质干酪中各生物胺含量明显高于其余两组干酪。成熟6个月时,冷藏72 h原料乳制作的牦牛乳硬质干酪中生物胺总量和酪胺含量分别为(212.94±8.03),(81.04±3.92) mg/kg。结论:随着原料乳冷藏时间和干酪成熟时间的延长,干酪中生物胺含量增多,但是原料乳冷藏时间低于72 h时,其干酪中生物胺含量低于学者建议含量。     

不同发酵剂对牦牛乳硬质干酪品质的影响

以新鲜牦牛乳为原料,分别添加嗜温、嗜热和混合(嗜温∶嗜热=1∶1)三种发酵剂制作硬质干酪,研究在1~180 d成熟过程中,不同类型发酵剂制作的干酪中蛋白降解和ACP(酸性磷酸酶)对其品质的影响。结果表明:牦牛乳硬质干酪成熟过程中发挥作用的ACP主要来自发酵剂,且干酪中蛋白降解受ACP影响显著,ACP与PPN(多肽氮)呈强正相关性(r=0.720),与CN(酪蛋白氮)和PN(蛋白氮)呈强负相关性。三种干酪PPN在60~120 d均保持稳定状态。不同类型的发酵剂对干酪蛋白降解强弱不同,过强或过弱均会影响到干酪的品质,嗜温发酵剂对干酪蛋白降解最弱,该干酪风味比较清淡;嗜热发酵剂对干酪蛋白降解能力最强,该发酵剂制作的干酪苦味较重,但组织状态较好;混合发酵剂对蛋白降解适中,该干酪发酵风味浓郁,组织状态较佳。      

成熟温度对牦牛乳硬质干酪中乳酸菌自溶及氨肽酶活性的影响

通过动态监测5、10、15℃成熟牦牛乳硬质干酪在1~6个月成熟过程中乳酸菌总数、乳酸脱氢酶和氨肽酶活性等变化,探究提高成熟温度对其乳酸菌自溶和氨肽酶活性的影响。研究结果表明:成熟温度对p H值影响显著(P0.05)。干酪中乳酸菌在1~6个月成熟过程中其数量呈现降低趋势。p H值与乳酸菌总数之间存在负相关性。5、10、15℃成熟干酪中乳酸脱氢酶活力依次呈现增加趋势,15、10℃成熟干酪中氨肽酶活性高于5℃成熟干酪中氨肽酶活性。因此,15℃成熟干酪中乳酸菌自溶程度最强,其次为10℃成熟干酪,5℃成熟干酪中乳酸菌自溶程度最弱。15℃成熟干酪中广谱性氨肽酶(broad-specificity aminopeptidase,Pep N)和脯氨酸氨肽酶(X-prolyl-dipeptidlylaminopeptidase,Pep X)活性分别是10℃成熟干酪中Pep N和Pep X活性的2.19和2.64倍。5℃成熟干酪中Pep N、Pep X活性较低,分别没有超过0.29 U/g和0.4 U/g。     

Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese

We applied capillary electrophoresis, liquid chromatography coupled with tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to determine the composition of water-insoluble and water-soluble proteinaceous fractions of the cheese and to study in detail the degradation of caseins during 8 mo of ripening of Estonian high-temperature cooked hard cheese Old Saare. The application of high-resolution and high-accuracy MS/MS enabled identification of more than 3,000 small peptides, representing a fairly full casein peptidome containing peptides of 4 to 25 AA in length: 1,049 from β-casein (CN), 944 from α_S1-CN, 813 from α_S2-CN, and 234 from κ-CN. The majority of β-CN- and α_S1-CN-derived peptides originated from the N-terminal parts of the molecule, f6-93 and f1-124, respectively; peptides from α_S2-CN arose predominantly from the C-terminal end f100-162. At the beginning of ripening, we found a relatively high amount of peptides originating from the glycomacropeptide part of κ-CN, whereas peptides from para-κ-CN prevailed during the later stages of ripening of the cheese. The cleavage patterns of β-CN, α_S2-CN, as well as α_S1-CN, showed that primary proteolysis was started mainly by plasmin, although a low proteolytic activity of chymosin was also evident. Based on the analysis of cleavage sites, we observed a significant participation of proteolytic enzymes, including amino- and carboxypeptidases, of both mesophilic and thermophilic starter bacteria in further hydrolysis of oligopeptides during the ripening. Several new phosphopeptides were detected in the result of MS/MS data analysis. The profiles of the estimated concentrations of phosphopeptides revealed that those originating from β-CN and α_S1-CN accumulated during cheese maturation. In contrast, we did not notice any generation of phosphopeptides from the highly phosphorylated part of α_S2-CN, f25-80, presumably due to the inaccessibility of this region to the action of plasmin and chymosin. The analysis of cleavage sites and the combination of principal component and clustering analyses provided a characterization of the complex dynamics of formation and degradation of peptides during cheese maturation. We made an attempt to obtain a comprehensive picture of proteolysis during Old Saare cheese ripening on the basis of the detailed peptidomic data, including also the less abundant peptides determined by MS/MS, and complemented by the data on intact caseins and free AA and reported the results in the paper.     

Effects of high pressure on proteolytic enzymes in cheese: relationship with the proteolysis of ewe milk cheese

Ewe milk cheeses were submitted to 200, 300, 400, and 500 MPa (2P to 5P) at 2 stages of ripening (after 1 and 15 d of manufacturing; P1 and P15). The high-pressure-treated cheeses showed a more important hydrolysis of β-casein than control and 2P1 cheeses. Degradation of α_s1-casein was more important in 3P1, 4P1, and P15 cheeses than control and 2P1 cheeses. The 5P1 cheeses exhibited the lowest degradation of α_s-caseins, probably as a consequence of the inactivation of residual chymosin. Treatment at 300 MPa applied on the first day of ripening increased the peptidolytic activity, accelerating the secondary proteolysis of cheeses. The 3P1 cheeses had extensive peptide degradation and the highest content of free amino acids. Treatments at 500 MPa, however, decelerated the proteolysis of cheeses due to a reduction of microbial population and inactivation of enzymes.     

不同包装方式对半硬质干酪成熟期间蛋白质降解的影响

对真空包装和涂蜡包装的半硬质干酪成熟过程中蛋白降解进行了研究。结果表明:2种包装的干酪在成熟过程中pH 4.6SN含量和12%TCA-SN含量都随着时间的延长逐渐增大,且2组数据之间差异显著(P<0.05);2种包装的干酪中游离氨基酸总量随成熟时间的延长而逐渐增加,各种氨基酸含量变化的显著性不同;SDS-PAGE电泳图谱显示2种干酪在成熟期内蛋白质都发生了明显的降解,且涂蜡包装的干酪蛋白降解程度较真空包装的深,在成熟45 d后较为明显。     

Proteolytic and lipolytic changes during the ripening of León raw cow's milk cheese, a Spanish traditional variety

Proteolytic and lipolytic changes were studied throughout ripening of five batches of León cow's milk cheese, a traditional variety made in the north of Spain. Total soluble nitrogen, non-protein nitrogen, oligopeptides nitrogen, amino nitrogen and ammonia nitrogen fractions increased slightly during the ripening process. The final values of these nitrogen fractions indicate that this cheese undergoes a very slight proteolysis as much in extent as in depth. This weak protein degradation is corroborated when the caseins and their degradation products were quantified by electrophoresis. β-Casein stayed practically intact throughout the ripening process and only 10% of α_s-casein became degraded. The content of total free amino acids increased progressively but in a slightly increased way during ripening, reaching final average values of 592 mg (100 g)⁻¹ of total solids. The most abundant free amino acid at the end of ripening was lysine, followed by leucine, glutamic acid, tryptophan, valine and phenylalanine. The acidity index of the fat values increased during ripening by a factor of 4.39. The final values of this parameter are in the range of those observed in other cow's milk cheeses ripened by bacteria. The content in total free fatty acids underwent an increase throughout ripening reaching final average values of 6669 ppm. The most abundant free fatty acid at the end of ripening was oleic acid followed by butyric and palmitic acids. The high content of short-chain fatty acids is outstanding, specially that of butyric acid.     

Effect of proteolysis during Cheddar cheese aging on the detection of milk protein residues by ELISA

Cow milk is a common allergenic food, and cow milk-derived cheese retains an appreciable level of allergenicity. The specific and sensitive detection of milk protein residues in foods is needed to protect milk-allergic consumers from exposure to undeclared milk protein residues contained in foods made with milk or milk-derived ingredients or made on shared equipment or in shared facilities with milk or milk-derived ingredients. However, during cheese ripening, milk proteins are degraded by chymosin and milk-derived and bacterial proteases. Commercial allergen-detection methods are not validated for the detection of residues in fermented or hydrolyzed products. The objective of this research was to evaluate commercially available milk ELISA kits for their capability to detect milk protein residues in aged Cheddar cheese. Cheddar cheese was manufactured at a local dairy plant and was aged at 5°C for 24 mo, with samples removed at various time points throughout aging. Milk protein residues and protein profiles were measured using 4 commercial milk ELISA kits and sodium dodecyl sulfate-PAGE. The ELISA data revealed a 90% loss of milk protein residue signal between the youngest and oldest Cheddar cheese samples (0.5 and 24 mo, respectively). Sodium dodecyl sulfate-PAGE analysis showed protein degradation throughout aging, with the highest level of proteolysis observed at 24 mo. Results suggest that current commercial milk ELISA methods can detect milk protein residues in young Cheddar cheese, but the detection signal dramatically decreases during aging. The 4 evaluated ELISA kits were not capable of detecting trace levels of milk protein residues in aged cheese. Reliable detection of allergen residues in fermented food products is critical for upholding allergen-control programs, maintaining product safety, and protecting allergic consumers. Furthermore, this research suggests a novel use of ELISA kits to monitor protein degradation as an indication of cheese ripening.