紫外诱变选育米曲霉高产蛋白酶菌株

以从自然发酵黄豆酱中筛选的5株野生米曲霉为供试菌株,以这些菌株产蛋白酶(酸、中、碱)、淀粉酶(α-淀粉酶、糖化酶)活力大小为评价标准,筛选出米曲霉K61作为诱变出发菌株。采用紫外诱变对米曲霉K61菌株进行改造,最终筛选出一株蛋白酶活力高且遗传性能稳定的突变株Y29。将米曲霉Y29菌株应用于黄豆酱的生产中,并与目前工业生产中广泛应用的米曲霉沪酿3.042菌株进行比较。性能实验结果表明米曲霉Y29菌株的蛋白酶(酸、中、碱)活力明显高于米曲霉沪酿3.042菌株,但α-淀粉酶、糖化酶、生长速度和孢子数这4个指标两者的差异并不显著;制酱品质试验结果表明,米曲霉Y29菌株的酱香更浓郁一些,氨基酸态氮含量达到0.77g/100mL,高于米曲霉沪酿3.042菌株,其它指标均符合国家标准GB2718-1996。     

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%。     

中性蛋白酶高产菌株的筛选及产酶酶系分析

目的:满足水产中对中性蛋白酶的需求。方法:以实验室保藏的米曲霉ZW为出发菌株,经Co60定向诱变,通过透明圈法初筛、摇瓶发酵复筛,筛选中性蛋白酶活力高的菌株,并对其进行产酶酶系分析。结果:筛选到的米曲霉ZW-06产中性蛋白酶酶活可达15000U/g干曲,比诱变前酶活提高了74%,是目前国内报道的固体发酵产中性蛋白酶活力最高的菌株;经过10代传代之后,酶活力仍保持稳定。通过对米曲霉ZW-06进行产酶酶系分析,发现发酵产物中除了有较高的中性蛋白酶酶活,还有较高的木聚糖酶和酸性纤维素酶酶活,酶活分别达到49879U/g干曲和21099U/g干曲。结论:米曲霉ZW-06在饲料工业中有很大     

我国部分地区自然发酵豆酱中的曲霉菌及其有性型

从我国42个地区的自然发酵酱醅和酱醪样品中分离得到曲霉Aspergillus及其有性型真菌20种,其中优势曲霉菌种类为A.tubingensis、A.flavus、A.niger、A.ochraceus、A.candidus和A.fumigatus.调查研究发现,从酱醅和酱醪样品中分离出黄曲霉的频率较高,表明自然发酵豆酱存在一定的安全隐患.研究中分离获得的曲霉菌株有望用于纯种制曲或特殊风味豆酱的工业化生产.     

东北传统豆酱发酵过程中微生物的多样性

以东北传统发酵豆酱为研究对象,分析豆酱发酵过程中微生物群落的动态变化。分别选择发酵豆酱0、35、65、75和105 d(成品豆酱)作为研究材料,通过PCR-DGGE分析微生物多样性,检测了豆酱发酵过程中蛋白质和氨基酸态氮的变化。结果表明,豆酱发酵过程中主要优势细菌为芽孢杆菌和乳酸菌,芽孢杆菌包括枯草芽孢杆菌、短小芽孢杆菌、淀粉液化芽孢杆菌、地衣芽孢杆菌的近缘种等;主要乳酸菌为乳球菌、明串珠菌、魏斯氏菌等种属细菌的近缘种;东北豆酱发酵过程中优势真菌为米曲霉、散囊菌和谢瓦氏曲霉的近缘种,随发酵时间延长数量逐渐减少;粗蛋白相对含量先略有平稳上升后下降,最后成品酱粗蛋白含量下降为24.76%;氨基态氮含量一直在增加,成品酱中为101.2 g/kg。     

固态发酵苦荞制备多肽菌种的筛选

【目的】筛选固态发酵苦荞高产多肽及发酵产物液具有抗菌、抗氧化活性的菌株。【方法】采用米曲霉、酱油曲霉、雅致放射毛霉和少孢根霉分别对苦荞进行固态发酵,以蛋白酶活力、水解度、可溶性肽得率、抑菌率和体外自由基清除率作为筛菌指标。【结果】米曲霉固态发酵苦荞的可溶性肽得率最高达38.83%±1.18%,发酵产物液对大肠杆菌和金黄色葡萄球菌的抑菌率分别为96.62%±1.66%和97.54%±0.54%,同时羟自由基(·OH)清除率和二苯基苦味酰基苯肼自由基(DPPH·)清除率分别为55.65%±1.25%和10.84%±1.03%。对米曲霉发酵2 d发酵产物液的不同分子量分布及活性分析表明,分子量大小对抗菌及抗氧化活性有一定的影响。【结论】米曲霉可作为固态发酵苦荞制备多肽且发酵产物液具有抗菌及抗氧化活性的最佳菌株,并在多肽产量提升及抗菌、抗氧化活性的研究上具有巨大空间。     

传统发酵豆酱发酵过程中养分动态及细菌多样性

以山东传统发酵豆酱作为研究对象,测定了发酵不同阶段的总酸、可溶性糖、有机碳、粗蛋白、氨基酸态氮、主要挥发性产物等指标,对样品中细菌进行PCR-DGGE分析,探讨了传统发酵豆酱中养分动态及细菌多样性.结果表明发酵过程中总酸含量先上升后下降而后又上升,最后成品酱中为6.26%;有机碳和可溶性糖含量都逐渐减少;粗蛋白相对含量先略有平稳上升后下降;氨基酸态氮含量一直在增加,成品酱中为101.2g/kg;乳酸和甘油含量随发酵进程而增加,成品酱中分别为5.65g/kg和14.72g/kg.DGGE分析表明发酵15d时细菌种类最多,随后一部分逐渐消失,种类趋于稳定,最后成品酱中出现有几种明显的优势种,主要包括:未培养细菌(Uncultured bacterium)、乳酸乳球菌(Lactococcus lactis)、地衣芽孢杆菌(Bacillus licheniformis)的近缘种.     

传统发酵豆酱发酵过程中养分动态及细菌多样性

以山东传统发酵豆酱作为研究对象, 测定了发酵不同阶段的总酸、可溶性糖、有机碳、粗蛋白、氨基酸态氮、主要挥发性产物等指标, 对样品中细菌进行PCR-DGGE分析, 探讨了传统发酵豆酱中养分动态及细菌多样性。结果表明发酵过程中总酸含量先上升后下降而后又上升, 最后成品酱中为6.26%; 有机碳和可溶性糖含量都逐渐减少; 粗蛋白相对含量先略有平稳上升后下降; 氨基酸态氮含量一直在增加, 成品酱中为101.2 g/kg; 乳酸和甘油含量随发酵进程而增加, 成品酱中分别为5.65 g/kg和14.72 g/kg。DGGE分析表明发酵15 d时细菌种类最多, 随后一部分逐渐消失, 种类趋于稳定, 最后成品酱中出现有几种明显的优势种, 主要包括：未培养细菌(Uncultured bacterium)、乳酸乳球菌(Lactococcus lactis)、地衣芽孢杆菌(Bacillus licheniformis)的近缘种。     

传统郫县豆瓣酱中高产蛋白酶曲霉的筛选及应用

【背景】米曲霉通过蛋白酶的作用将大分子蛋白质分解为蛋白胨、多肽及氨基酸等营养物质。豆瓣酱的一个重要指标氨基态氮含量则直接由蛋白酶决定,因此米曲霉在豆瓣酱发酵中有着至关重要的作用。【目的】筛选一种高产蛋白酶豆瓣酱专用菌株,研究其发酵豆瓣酱品质的改良效果。【方法】以自然发酵成曲为原料,筛选高产蛋白酶的霉菌。以筛选菌株为曲种发酵制曲,进一步发酵甜瓣子,测定制曲过程中霉菌增长速度和蛋白酶增长速度,通过发酵甜瓣子氨基态氮、挥发性风味物质等指标的测定分析菌株发酵甜瓣子品质的改进效果。【结果】筛选得到17株高产蛋白酶的霉菌,其中菌株PCSM002制曲生长速度最快,PCSM001和PCSM002号菌株产蛋白酶能力最强,麸皮培养基中培养3 d蛋白酶酶活分别为1 450.25、1 703.25 U/g。PCSM001和PCSM002发酵24 d甜瓣子氨基态氮含量达到0.86μg/kg以上,且发酵的甜瓣子香气浓郁、口感丰富。两株菌均已送至广东省微生物菌株保藏中心保藏,保藏编号分别为GDMCC 60198和GDMCC 60199。【结论】筛选得到的高产蛋白酶霉菌有望用于豆瓣酱的发酵,作为豆瓣酱发酵专用菌株,以提高豆瓣酱品质。     

米曲霉发酵纯化无患子皂苷的工艺研究

采用微生物发酵法对无患子皂苷水提取液进行纯化.比较了采用自然发酵、接种酵母菌发酵和接种米曲霉发酵纯化无患子皂苷的效果.结果表明,提取液不灭菌,接种米曲霉发酵纯化效果较为明显,优化后的发酵条件为:温度30℃、接种龄12 h、接种量为3％、摇床转速150 r/min,发酵7d后,皂苷含量稍有下降,但皂苷纯度可从48.71％提高到82.47％.米曲霉发酵法明显优于水提醇沉法、絮凝法和正丁醇萃取法.     

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.     

γ—射线对酱油中细菌总数及营养成分影响的初步研究

用~(60)Co—γ—射线以不同剂量(30,50,70,90万拉德)照射瓶装酱油,然后对酱油中细菌总数及总酸和氨基酸态氮进行分析。结果证明:γ—射线具有良好的杀菌作用,剂量30万拉德已能使细菌总数降低到标准含量以下。而各个剂量的γ—照射对总酸及氨基酸态氮的数值几乎没有影响。     

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。     

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。     

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

Summary The effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h^–1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)^–1·h^–1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as -amylase, indicating that the protease production was selectively stimulated by the oils. Offprint requests to: Y. Fukushima     

Non-enzymic Browning of Soy Sauce

Ion exchange resins have been used to separate soy sauce into three fractions of distinctly different composition: a cation fraction, a neutral fraction and an anion fraction. Almost all of the constituents responsible for browning were recovered in these three fractions.

Storage experiments show that when the three fractions were stored separately, only the cation fraction darkened considerably. When they were combined and stored, the color of the mixture increased at nearly the same rate as that of the original soy sauce. Neutral sugars are important constituents of the neutral fraction with respect to browning. The browning rate of a sugar-amino acid mixture (simulated soy sauce), was about 10% of soy sauce. The effect of the anion fraction (mainly caused by organic acids) and the ashed cation fraction on the over-all browning of soy sauce is calculated to be 1O~12% and 20%, respectively.

The sum of the contribution rate of the anion fraction, the neutral fraction, the amino acids and the ashed cation fraction in the browning of soy sauce was concluded to be approximately 40%. Compounds responcible for residual part of 60% should be considered to exist in the cation fraction. It was suggested that such compounds have strong reducing power and 0₂-uptaking ability.     

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.     

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.     

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).     

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.