豆酱种曲培养过程中米曲霉Y29与3.042产酶特性比较

将米曲霉Y29菌株和沪酿3.042菌株在豆酱制种曲过程中产生的蛋白酶和淀粉酶的活性进行了比较,结果表明:米曲霉Y29菌株的酸性蛋白酶、中性蛋白酶和碱性蛋白酶活力的峰值分别比米曲霉沪酿3.042菌株高了45.55%,15.13%,9.86%,而α-淀粉酶和糖化酶活力二者无显著差异;并且两菌株蛋白酶和淀粉酶酶活高峰出现的时间也一致。     

Actinomucor elegans、Aspegillus oryzae和Rhizopus oligosporus产蛋白酶条件及蛋白酶性质的比较

比较了腐乳生产菌株Actinomucor elegans、豆酱和酱油生产菌株Aspegillus oryzae以及天培生产菌株Rhizopus oligosporus产生蛋白酶的条件和所产蛋白酶的性质。结果表明,不同的菌株产酶条件及蛋白酶的性质有较大的差异:少孢根霉主要产生酸性蛋白酶,在pH2.5-4.0的酸性介质中、32℃条件下培养时产酶能力较强,所分泌的蛋白酶系在pH5.0时酶活力最高,在pH5.0附近最稳定;米曲霉可以产生酸性、中性及碱性蛋白酶,所产生的蛋白酶活力显著高于少孢根霉和毛霉,米曲霉在酸性条件下产酸性蛋白酶能力强,在中性条件下产中性蛋白酶能力强,在碱性条件下产碱性蛋白酶能力强,在28-32℃时产酶能力强,所分泌的蛋白酶系在pH5.0-9.0的广泛范围内有很强的活力,在pH6.0-8.0的范围内稳定性强;毛霉可以产生酸性、中性及碱性蛋白酶,但酶活力明显低于米曲霉,毛霉在中性偏酸性(pH5.5)的介质中产酸性蛋白酶的能力较强,但介质的酸碱度对毛霉产中性及碱性蛋白酶没有影响,在28℃时产酸性、中性和碱性蛋白酶的能力都比较强,毛霉所分泌的蛋白酶系在pH5.0-9.0的广泛pH范围内有活力,在pH5.0-6.0时酶活力最高,在pH5.0-7.0时稳定强。     

盐浓度对不同米曲霉所产中性蛋白酶活力的影响

试验对酱油酿造过程中，盐浓度对不同米曲霉所产中性蛋白酶活力的影响进行了研究。结果表明盐浓度对不同米曲霉所产中性蛋白酶活力的影响是不同的。所以在酱油酿造过程中，在一定的盐浓度下测定中性蛋白酶活力才是更客观更有效的。     

三种酱油发酵曲霉酶活力的比较

制曲对酱油酿造至关重要,影响着酱油的品质。因此,本研究对酱油制曲过程中的米曲霉A、米曲霉沪酿3.042、米曲霉A100-8 3种曲霉在不同时间的酶活力进行了研究,包括酸性蛋白酶、淀粉酶、糖化酶、纤维素酶、果胶酶、亮氨酸氨肽酶、谷氨酰胺酶、植酸酶。结合酶活力的变化趋势、原料水解度以及酿造后期淋油的综合情况,最终确定在制曲42h对3株曲霉所制备的大曲进行收曲,比较发现米曲霉A100-8在收曲时,各种酶活表现最佳。     

多菌种制曲在原池浇淋酱油制曲工艺中的应用研究

采用原池浇淋工艺,添加糖化增香曲(HQ)、曲精(QJ)与米曲霉沪酿3.042进行混合制曲,以沪酿3.042米曲霉、曲精分别单独制曲进行对照实验,对制曲过程中中性蛋白酶、酸性蛋白酶、淀粉酶、纤维素酶及理化指标的变化进行了研究。结果表明,曲精和糖化增香曲混合制曲,成曲的酸性蛋白酶活力比曲精单独制曲提高42.6%,但是多菌株组合混合制曲较单菌株制曲,成曲的中性蛋白酶酶活力没有增加,淀粉酶酶活力均不高,未检测到纤维素酶活。     

米曲霉40188产中性蛋白酶、α-淀粉酶特性的研究

通过对米曲霉40188产酶特征及中性蛋白酶及α-淀粉酶酶学特征的研究,得出米曲霉40188制曲最佳时间为50～72 h,中性蛋白酶和α-淀粉酶酶活在40℃稳定,随着温度升高酶活降低,但α-淀粉酶的热稳定性较中性蛋白酶强,中性蛋白酶的最适pH为7～8,α-淀粉酶的最适pH为6～8,α-淀粉酶的耐盐性较中性蛋白酶强,中性蛋白酶酶活随着盐浓度的增加呈下降趋势。     

豆酱发酵用工业米曲霉菌种的复壮

采用马铃薯葡萄糖培养基,从种曲生产车间的地面及墙壁上分离到24株霉菌菌株。依据菌落形态相似性和差异,将其归纳为7组并选择7株作为试验菌株进一步进行分析。经菌落形态特征及小室培养形态学鉴定,结合其ITS序列分子水平分析,此与米曲霉代表株的相似性为99%,且未检出黄曲霉毒素,因此7株霉菌被鉴定为米曲霉(Aspergillus oryzae)。经麸皮培养基制曲后,菌株ZJGS-LZ-12和ZJGS-LZ-21分泌的中、酸性蛋白酶、淀粉酶及糖化酶活力(U/g干基)较高,产酶特性较好,不产生黄曲霉毒素,适用工业化应用。     

清酒酒曲生产用最适霉菌的筛选

对最适宜制作清酒酒曲的霉菌进行了筛选。对米曲霉3.5232、米曲霉3.800、黑曲霉3.4309、黑曲霉3.1858、根霉3.866分别进行耐酒精试验、耐温试验、耐酸试验,初筛得到米曲霉3.5232、黑曲霉3.1858、根霉3.866三株菌株,分别制成酒曲,通过酒曲α-淀粉酶活力、蛋白酶活力和糖化酶活力的测定最终筛选出米曲霉3.5232是最适宜制作酒曲的霉菌菌株。     

7株米曲霉与沪酿3.042发酵制备郫县豆瓣用甜瓣子的对比研究

为了筛选高效的具有潜在应用价值的郫县豆瓣生产专用米曲霉菌株,研究了7株高产蛋白酶、α-淀粉酶的米曲霉试验菌株(24M-1、21M-2、25M-1、19M-2、BM-2、18M-1、DM1)与沪酿3.042米曲霉在制备甜瓣子中的发酵性能差异,考察了不同菌株发酵的种曲中中性蛋白酶、酸性蛋白酶、α-淀粉酶的酶活力差别;进一步评价了由试验菌株制作的种曲发酵的甜瓣子的氨基酸态氮、总酸、挥发性风味物质、感官评价等发酵特性指标。结果表明,菌株24M-1、DM1发酵甜瓣子的各项发酵特性指标表现突出,甜瓣子的综合品质较佳,明显优于沪酿3.042米曲霉发酵的甜瓣子,可进一步优化菌株培养条件及发酵工艺参数,为其在郫县豆瓣加工中的应用奠定了基础。     

几株米曲霉成曲胞外蛋白鉴定及发酵结果分析

采用SDS-PAGE电泳和MALDI-TOF-MS技术,鉴定4株米曲霉成曲胞外蛋白表达量最高的4种蛋白,其中2种确定为淀粉酶和碱性蛋白酶,这2种酶在4株米曲霉成曲胞外蛋白中的表达量和都超过胞外蛋白总量的50%,但不同菌株间淀粉酶与碱性蛋白酶表达量比例不同。同时分析蛋白电泳凝胶酶蛋白表达量、酶活与发酵结果的关系,结果表明酶蛋白表达量与酶活力具有较好的一致性。蛋白酶活力低的菌株发酵氨基酸态氮水平并不低,淀粉酶高的菌株发酵生成还原糖较低。     

ISOLATION AND CHARACTERISATION OF THE AMYLOTIC SYSTEM OF SCHWANNIOMYCES CASTELLII

The amylolytic system of Schwanniomyces castellii has been isolated and purified by means of ultrafiltration followed by polyacrylamide gel electrophoresis. Both α-amylase and glucoamylase were purified. α-Amylase activity was stable from pH 5·5 to 6·5 and glucoamylase activity was stable at a more acidic range of pH 4·2 to 5·5. The optimal temperature of α-amylase activity was between 30 and 40°C with rapid deactivation at 70°C. The optimal temperature of glucoamylase was 40 to 50°C with rapid decline of activity at 60°C. The K_m of α-amylase with soluble starch as the substrate was 1·15 mg/ml and the K_m of glucoamylase with the same substrate was 10·31 mg/ml. Glucoamylase was able to hydrolyze α-1, 4 and α-1,6 glucosidic linkages, as demonstrated by its ability to hydrolyse maltose and isomaltose respectively, whereas α-amylase could hydrolyse α-1,4 glucosidic linkages only. α-Amylase was shown to be a glycoprotein, whereas no carbohydrates were associated with glucoamylase.     

耐受性枯草芽孢杆菌的脉冲强光诱变筛选及产酶活力分析

采用脉冲强光对枯草芽孢杆菌进行诱变处理,通过耐受性测试,在抗性菌株中筛选出优良耐受菌株,并与原始菌株对比分析产酶活力及遗传稳定性,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳比较分析菌体蛋白质表达的差异。结果表明,在脉冲电压2?450?V,照射距离5?cm,脉冲40?次条件下,筛选出8?株抗性菌株;经初筛和复筛得到兼具高温耐受、强酸耐受和高浓度胆盐耐受性变异菌株B3和B7,产α-淀粉酶、蛋白酶、脂肪酶活力分别比原始菌株提高了67%和77%（P＜0.01）、56%和71%（P＜0.01）、34%和42%（P＜0.05）,变异菌株产酶稳定性较高（P＞0.05）,可遗传变异;诱变前后菌体蛋白表达出现差异;结果证明脉冲强光用于枯草芽孢杆菌的诱变具有可行性。     

Intensification of Amylase Synthesis with Aspergillus niger by Way of Multistage Mutagenization

Conidia of amylolytically active Aspergillus niger C strain were subjected to four-stage mutagenization, using different combinations of mutagens in each stage (stage I – UV irradiation + ethyleneimine, stage II – UV irradiation + nitrosomethylurea, stage III – UV irradiation + N-methyl-N′-nitro-N-nitrosoguanidine, stage IV – acryflavine). In all, 378 strains were isolated after mutagenization, which were initially evaluated for the total amylolytic activity by the method of test-tube microculture. Nine most active mutants were then tested in submerged culture determining the activity of glucoamylase and α-amylase in post-culture liquid. The activity of α-amylase increased from over 7 to nearly 31%, and that of glucoamylase from 4 to over 61%.     

双菌种制曲改善黄酒麦曲品质的研究

以实验室分离得到的真菌为出发菌株,经平板初筛和制曲复筛,得到用于强化麦曲的功能微生物GC3、ZW12,经分子鉴定分别为米根霉和米曲霉。以糖化酶,α-淀粉酶,酸性蛋白酶为指标,通过不同比例混合接种试验,确定用于制作熟麦曲的菌种为米曲霉苏-16和米曲霉ZW12,混合比例为1∶2。采用响应面分析法对制曲条件进行优化,结果表明:最佳接种量为2.75 mL/50 g原料,原料含水量为81.01%,培养时间47.21h,较传统熟麦曲,3种酶活分别提高了3.5%,77.3%和59.7%。     

PRODUCTION OF AMYLOLYTIC ENZYMES BY SEVERAL YEAST SPECIES

Several amylolytic yeast species, endomycopsis spp, schwanniomyces spp, pichia spp and saccharomyces spp have been compared for their ability to synthesise α-amylase (E.C. 3.2.1.1), glucoamylase (E.C. 3.2.1.3) and pullulanase (E.C. 3.2.1.9). Endomycopsis fibuligera strain 240 possessed the highest glucoamylase activity (208 nmoles glucose/min) and second highest α-amylase activity (128 nmoles maltose/min) and produced the largest amount of biomass. Schwanniomyces spp were the only yeast species studied which exhibited significant debranching activity. It was found that in endomycopsis spp, schwanniomyces spp and pichia spp, glucose, at a concentration above 3.0 × 10^?3 m , appeared to repress α-amylase activity. However, glucoamylase activity was not repressed at that glucose concentration.     

浓香型成品大曲中三类水解酶的分离纯化研究

采用盐析、离子交换、分子筛筛选等方法分离、纯化浓香型大曲中糖化淀粉酶、液化淀粉酶和酸性蛋白酶,研究了3种酶的相关酶学特性。结果表明,成品浓香型大曲中,糖化淀粉酶活力均值为441.0 U/g干曲,液化淀粉酶活力为5.68 U/g干曲,酸性蛋白酶活力均值达51.2 U/g干曲。糖化淀粉酶纯化倍数平均达2.44倍,酶活回收率为2.51%,表观分子量2.5×104,液化淀粉酶的纯化倍数平均达3.24倍,酶活回收率28.28%。酸性蛋白酶纯化倍数平均达4.05倍,酶活回收率可达35.62%。     

Raw Starch Digestion by α-Amylase and Glucoamylase from Chalara paradoxa

Glucoamylase and α-amylase of Chalara paradoxa were separated by hydrophobic column chromatography using butyl-Toyopearl 650M. The α-amylase showed the highest activity at pH 5.5 and 45°C, and was stable in the pH range of 5.5–6.5 and at temperatures lower than 40°C. The glucoamylase showed the highest activity at pH 5.0 and 45°C, and was stable in the pH range of 4.0–7.5 and at temperatures lower than 45°C. The molecular mass of the α-amylase and glucoamylase estimated by SDS polyacrylamide gel electrophoresis was 80,000 and 68,000, respectively. Both glucoamylase and α-amylase could digest more effectively raw rice starch and raw corn starch than raw sago starch and raw potato starch. 2% raw rice starch in 10 ml solution was digested by more than 90% by 100 units of each amylase. When these amylases were used combined, raw corn starch was more effectively digested than they were used singly. This cooperative action in raw corn starch digestion was also observed when. C. paradoxa α-amylase and R. niveus glucoamylase were combined.     

淀粉酶产生菌MSP13筛选及其产酶条件初步优化

从优质白酒窖泥中分离筛选出13 株产淀粉酶的兼性厌氧细菌,通过测定α-淀粉酶、β-淀粉酶、糖化酶和脱支酶酶活力,最终筛选出一株生产4 种淀粉酶活力均相对较高的菌株,并对其进行形态观察,生理生化指标和16S rDNA鉴定,确定该菌株为解淀粉芽孢杆菌,并将其命名为MSP13。对菌株MSP13产酶条件进行初步优化,确定其产酶的较佳条件是：CaCl2质量浓度0.15 g/L、MgSO4·7H2O质量浓度0.3 g/L,pH 5.5和温度37 ℃。优化后的菌株MSP13生产4 种酶的酶活力平均提高了31.645%。     

燕麦发芽过程中淀粉及其相关酶活性的动态变化

研究了裸燕麦发芽过程中淀粉及其相关酶活性的动态变化。结果表明,发芽过程中,燕麦还原糖和可溶性糖含量及α-淀粉酶、β-淀粉酶和总淀粉酶活力明显地先增加后降低;直链淀粉、支链淀粉和总淀粉的含量均随着发芽的进行呈下降趋势,发芽72 h分别降低了25.86%、11.08%和17.31%。相关性分析表明,燕麦发芽期间还原糖、可溶性糖含量分别与α-淀粉酶、β-淀粉酶及总淀粉酶活力呈显著正相关,而直链淀粉、支链淀粉及总淀粉含量均与淀粉酶活力呈显著负相关。     

Genetic Engineering of white Shochu-Koji to achieve Higher Levels of Acid-Stable α-Amylase and Glucoamylase and other Properties when used for Shochu Making on a Laboratory Scale

The genes for acid-stable α-amylase and glucoamylase were cloned from white shochu-koji, Aspergillus kawachii. Both genes were used to transform the parent strain of white shochu-koji which carried a dominant selective marker gene, amdS, that originated from A. oryzae. Three lines of transformants were identified that secreted about 6-fold more acid-stable α-amylase activity and about 7-fold more glucoamylase activity than the parent strain in liquid culture. In solid culture, all three transformants had 2-fold higher acid-stable α-amylase activity and 2.4-fold higher glucoamylase activity than the parent strain. When koji was prepared on a laboratory scale, acid-stable α-amylase activity was 5.7-fold higher and glucoamylase activity was 3.8-fold higher than when the parent strain was used. Shochu was produced with a koji ratio of 33% or 10% using one line of transformants on a laboratory scale. Even with a koji ratio of 10%, the weight of the mash obtained with the transformant decreased to almost the same extent as with a koji ratio of 33% and the parent strain. Levels of flavour compounds in shochu produced with koji of the transformant were higher than in the shochu prepared with koji of the parent strain. In particular, levels of isoamyl acetate and β-phenethyl acetate were as high as 12.9 mg/litre and 3.8 mg/litre, respectively.