酱油曲霉蛋白酶活力的提高

蛋白酶在工业上被广泛地应用于皮革、毛皮、丝绸、医药、食品和酿造等行业。霉菌生产的蛋白酶在豆酱和酱油生产上的应用已经具有相当悠悠的历史。曲霉菌,例如米曲霉、黄曲霉、栖土曲霉等,一般能分泌2～3种蛋白酶(酸性、中性及碱性),其中以中性和碱性酶为主。黑曲酶则以产酸性蛋白酶为主。酱油曲霉所产的蛋白酶也以中性     

米曲霉酸性蛋白酶基因在毕赤酵母中的异源表达及酶学性质

酸性蛋白酶作为一类重要的天冬氨酸蛋白酶,被广泛应用于食品、医药和皮革等领域。为推动酸性蛋白酶的研究及应用,通过对发酵豆制品样品进行宏基因组测序,从中获得米曲霉酸性蛋白酶基因pepA,在毕赤酵母GS115中进行异源表达,并对重组酶PepA进行酶学性质分析。结果显示毕赤酵母发酵上清液中酸性蛋白酶的活性为50.62 U/mL。SDS-PAGE验证PepA的分子量约为50 kDa,且发酵上清液几乎无杂蛋白。PepA的最适pH值为4.5,最适温度为50℃,Mn~(2+)和Cu~(2+)对其具有激活作用,而Fe~(3+)、Fe~(2+)与Ca~(2+)则具有抑制作用。上述研究结果可为米曲霉酸性蛋白酶的异源表达及其相关工业应用提供指导。     

毛霉蛋白酶的研究

蛋白酶是食品工业中最重要的加工配料(辅料)之一,蛋白酶在食品加工中的应用主要是利用了其对蛋白质所产生的多样化水解作用[1-2]。许多受人欢迎的食品,如干酪、啤酒和酱油等的生产过程都包含蛋白酶催化蛋白质水解这一关键性的反应。对这些食品加工技术的改进体现在,基于对这些加工过程所涉及的各种     

酱油生产中用固定化乳酸细菌进行快速连续乳酸发酵

在酱油分批生产中,酱油醪的酶水解,乳酸发酵和乙醇发酵时间需要6个月。乳酸发酵在控制酱醪的pH和为酵母菌的乙醇发酵准备适合的条件是很重要的。乳酸发酵过程对酱油醪的质量控制也很重要,它能影响酱油的风味。研究表明,在酱油醪中加乳酸菌,对快速发酵是有效的。但需要在酱油醪发酵之前大量培养乳酸细菌。此外,乳酸细菌和酵     

玉米原料无蒸煮酒精发酵工艺的研究

在玉米原料无蒸煮酒精发酵过程中,添加少量的纤维素酶,酸性蛋白酶可提高糖化酶对生淀粉的糖化作用,减少糖化酶用量。在料水比１：２．５,糖化酶加量２００ｕ／ｇ,纤维素酶加量５ｕ／ｇ,酸性蛋白酶加量０．０１％,３０℃,ｐＨ３．５条件下,Ｎｏ．２１４菌株经９６ｈ发酵,醪液酒精度达１２．８％,淀粉利用率达９２．１％。     

以玉米淀粉糖渣为原料制备米曲发酵酱油

以玉米淀粉糖渣为原料制备米曲,米曲中的中性蛋白酶活力能够达到4000 U/g(干基),糖化酶比活力可达到120 U/g左右,满足酿造酱油所需要求。用糖渣米曲发酵酱油,氨基态氮和总氮质量浓度分别能够达到7.1 g/L和11.9 g/L,还原糖质量浓度为13.2 g/L,各项理化指标均可达到国家二级酱油标准。     

利用黑曲霉固态发酵啤酒糟生产饲料复合酶的研究

以啤酒糟为主要基质,利用黑曲霉固态发酵生产酸性蛋白酶、木聚糖酶和纤维素酶等多种饲料复合酶,研究了黑曲霉固态发酵培养基组成对复合酶酶活的影响,确定最优培养基配方为:啤酒糟75%,麸皮25%,硫酸铵1%,KH_2PO_4 0.2%,MnSO_4 0.1%、ZnSO_4 0.2%,料水比1:2。在适宜的发酵条件下,经30℃发酵5 d,烘干后得到的复合酶制剂中,具有多种酶活性(以干基计)。其中酸性蛋白酶活力3 800 U/g,木聚糖酶活力12 00 U/g和纤维素酶活力18 U/g。     

微生态白酒糟饲料发酵条件的优化

为优化微生态白酒糟饲料发酵条件,以米曲霉、黑曲霉和酵母菌组合发酵白酒糟,共设计16个发酵组合,根据感官特征初筛出3个组合进行后续发酵实验。测定发酵前、后常规营养成分及发酵后的酶活力,确定最优组合并检测氨基酸含量。结果显示,以米曲霉(0.05%)、黑曲霉(0.05%)和酵母菌(0.05%)组合发酵(80%酒糟+10%玉米+10%麦麸)的效果最好:与发酵前相比,粗蛋白含量、真蛋白含量、酸性蛋白酶活力、中性蛋白酶活力和纤维素酶活力分别提高了30.39%、38.06%、41.69%、67.00%和103.84%,总氨基酸含量提升17.74%,酸性洗涤纤维和中性洗涤纤维含量分别降低23.64%、20.40%。     

酱醪细菌菌株的分离及功能分析

【目的】分离获得来源于酱醪的细菌,考察菌株与酱油品质相关的特性,初步评价其应用于酱油发酵的潜力。【方法】从日式酱油发酵的酱醪体系中分离和筛选优势或特征细菌菌株,比较它们的耐盐性及其在高盐条件下产蛋白酶、有机酸、挥发性物质和氨基酸等的能力。【结果】从日式酱油酱醪中共分离得到9株细菌,分别属于魏斯氏菌(Weissella)、乳酸足球菌(Pediococcus)、乳酸杆菌(Lactobacillus)、芽孢杆菌(Bacillus)、四联球菌(Tetragenococcus)和葡萄球菌(Staphylococcus)属。其中耐盐的细菌有类肠膜魏斯氏菌(Weissella paramesenteroides)CQ03、嗜酸乳酸足球菌(Pediococcus acidilactici)JY07、戊糖乳酸足球菌(Pediococcus pentosaceus)JY08、葡萄球菌(Staphylococcus sp.)JY09和嗜盐四联球菌(Tetragenococcus halophilus)MRS1。在高盐条件下,对它们的特性分析表明:解淀粉芽孢杆菌(Bacillus amyloliquefaciens)B2产蛋白酶和糖化酶的能力较强,W.paramesenteroides CQ03可水解原料产生较多鲜味氨基酸,T.halophilus MRS1产有机酸能力较强,它和S.sp.JY09代谢产生的挥发性物质较多。【结论】筛选得到9株在促进原料水解和提高风味物质合成方面有潜力的菌株,如果应用到酱油工业生产中,将有利于缩短发酵周期,提高酱油品质。     

酱香型白酒发酵中地衣芽孢杆菌与酿酒酵母的相互作用

【目的】为解析酱香型白酒酿造群体微生物的发酵过程, 研究了酱香型白酒酿造中重要微生物地衣芽孢杆菌与酿酒酵母之间的相互作用, 并对它们之间的作用机制进行初步探讨。【方法】通过地衣芽孢杆菌与酿酒酵母共培养体系的构建, 认识了两者的相互作用, 初步分析了酿酒酵母产生抑制物的分子量, 耐热性及对蛋白酶敏感性等特性。【结果】研究表明, 酿酒酵母发酵造成的酸性环境以及某些代谢物质能够抑制地衣芽孢杆菌的生长, 这些物质分子量大于10 kD, 对热和蛋白酶敏感。【结论】白酒酿造中酿酒酵母通过产酸以及大分子的蛋白质类物质对地衣芽孢杆菌生长形成抑制, 该研究促进了对白酒酿造群体微生物发酵过程的解析。     

Substrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine

To elucidate the mechanism of hydrolysis of fish muscle proteins by fish proteinases in fish sauce production, each pure preparation of three alkaline proteinases and two acid proteinases from sardine was tested for its ability to hydrolyze various proteins and its stability in the presence of 0 to 25% of NaCl. Each of the alkaline proteinases hydrolyzed casein more rapidly than other proteins. A major alkaline proteinase (III) hydrolyzed sarcoplasmic protein from sardine 5-times faster than other alkaline proteinases. Each of two acid proteinases hydrolyzed hemoglobin and myoglobin more rapidly than the other proteins. After preincubation with 25% NaCl, an alkaline proteinase (III) and an acid proteinase (II) were stable although the other proteinases became unstable. The two proteinases, alkaline proteinase III and acid proteinase II, were also stable for three months after the beginning of fish sauce production. The proteolytic activity of each of alkaline and the acid proteinases was strongly inhibited by more than 15% NaCl; however, minimum inhibition was observed when sardine muscle proteins were used as the substrate.     

Identification and Quantitation of New Glutamic Acid Derivatives in Soy Sauce by UPLC/MS/MS

Glutamic acid is an abundant amino acid that lends a characteristic umami taste to foods. In fermented foods, glutamic acid can be found as a free amino acid formed by proteolysis or as a non‐proteolytic derivative formed by microorganisms. The aim of the present study was to identify different structures of glutamic acid derivatives in a typical fermented protein‐based food product, soy sauce. An acidic fraction was prepared with anion‐exchange solid‐phase extraction (SPE) and analyzed by UPLC/MS/MS and UPLC/TOF‐MS. α‐Glutamyl, γ‐glutamyl, and pyroglutamyl dipeptides, as well as lactoyl amino acids, were identified in the acidic fraction of soy sauce. They were chemically synthesized for confirmation of their occurrence and quantified in the selected reaction monitoring (SRM) mode. Pyroglutamyl dipeptides accounted for 770 mg/kg of soy sauce, followed by lactoyl amino acids (135 mg/kg) and γ‐glutamyl dipeptides (70 mg/kg). In addition, N‐succinoylglutamic acid was identified for the first time in food as a minor compound in soy sauce (5 mg/kg).     

Characterization of Aspergillus oryzae aspartyl aminopeptidase expressed in Escherichia coli

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.     

Isolation and Characterization of Acidic Peptides in Soy Sauce

A soy sauce sample was fractionated by gel filtration on a Sephadex G–15 column, then the fractions were subfractionated on the basis of acidity by ion exchange chromatography on a QAE-Sephadex A–25 column. The acidic subfractions with various acidities were further fractionated, using a preparative amino acid analyzer and by paper chromatography to separate the acidic peptide components.

Four dipeptides and sugar derivatives of ten dipeptides and two tripeptides were isolated and characterized as the major acidic peptides in soy sauce. However, it was difficult to anticipate any direct contribution of these peptides to the flavor construction in soy sauce on the basis of their contents and taste intensities.     

Hydrolysis of acid-treated protein by a preparation of proteases

Because of less glutaminase activity, soy sauce made with a preparation of proteases from yellow-green Aspergilli contains less glutamic acid than soy sauce made by the traditional shoyu koji method. Thus, an acid treatment was developed to increase this amino acid in enzyme-made shoyu. Amide bonds of glutamine and asparagine in protein molecules were hydrolyzed at 100°C for 30 min with 1.3 N HCl (acid treatment). Using this method, glutamic acid per total nitrogen freed from various proteins by the concerted action of proteinases and peptidases of yellow-green Aspergillus increased to 1.0 to 3.8 times that of control (no acid treatment). An increase of about 31% of glutamic acid per total nitrogen resulted from the acid treatment method in soy sauce made with an enzyme preparation of proteases.     

Involvement of meso-α,∊-Diamino-pimelate D-Dehydrogenase in Lysine Biosynthesis in Corynebacterium glutamicum

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.     

Cloning and functional expression of a Boophilus microplus cathepsin L-like enzyme

A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.     

Isolation and characterization of a hemorrhagic proteinase from timber rattlesnake venom

A protein isolated from timber rattlesnake (Crotalus horridus horridus) venom by ion-exchange and high-pressure liquid chromatography is hemorrhage inducing and lethal to mice (LD50 of 10 micrograms/g of body weight). It is a Ca2+- and Zn2+-containing proteinase and has the ability to hydrolyze hide powder azure. Atomic absorption spectroscopy shows 2.5 Ca2+ and 1 Zn2+ per protein monomer. The proteinase activity is destroyed by incubation with disulfide-reducing agents and by dialysis against ethylenediaminetetraacetate. Coincident with the loss of proteinase activity is a corresponding loss of lethal and hemorrhagic activities, suggesting that all three are related. Attempts to replace the metals and restore activity have been unsuccessful. Amino acid analysis and isoelectric focusing reveal that this component is an acidic protein (pI = 5.1) containing about 20 disulfide bonds and 507 residues. Reduction of one disulfide bond per molecule decreases proteinase activity by 50% while reduction of eight disulfide bonds decreases activity by 80%. Loss of hemorrhagic activity parallels the decrease in proteinase activity.     

Use of glutaminase for soy sauce made by Koji or a preparation of proteases from Aspergillus oryzae

Although a small amount of glutamic acid was released in the hydrolysis of protein or soy sauce made by a preparation of proteases containing little glutaminase, a large amount of glutamic acid was formed in such hydrolyzate or soy sauce made by the addition of mycelia of black Aspergilli or glutaminase from Cryptococcus albidus. The former effect was caused mainly by glutaminase produced by black Aspergilli. The former crude enzyme showed an optimum pH of 5.0, broad pH stability and salt tolerance. The addition of glutaminase from C. albidus ATCC 20293 in soy sauce manufacture using a preparation of proteases resulted in a 42% increase in glutamic acid per total nitrogen content.