浓香型皇台大曲可培养细菌群落结构及其产淀粉酶的研究

采用稀释平板法,从浓香型皇台大曲中分离纯化出73株细菌菌株,经PCR扩增其16S rDNA并测序,比对分析发现这些纯化的细菌菌株中有23株16S rDNA序列各异的可培养细菌,这些菌株分别为Bacillus licheniformis16株、Bacillus subtilis2株、Bacillus amyloliquefaciens1株、Bacillus cereus1株、Bacillus atrophaeus1株、Bacillus sonorensis1株和Brevibacillus sp.1株;采用透明圈法和液态发酵法对大曲中可培养细菌产淀粉酶性能进行检测,结果表明Bacilluslicheniformis、Bacillus subtilis和Bacillus cereus的一些菌株产淀粉酶的活性较高,其中,Bacillus licheniformis的数量最多,是重要的白酒酿造功能菌。     

Enhanced production of α‐amylase from Bacillus subtilis subsp. spizizenii in solid state fermentation by response surface methodology and its evaluation in the hydrolysis of raw potato starch

This is an attempt to lower the cost of starch hydrolysis by the discovery of new generation α‐amylase. A natural isolate of Bacillus subtilis subsp. spizizenii was capable of producing appreciable amounts of raw potato starch digesting α‐amylase in solid state fermentation of wheat bran. The enzyme productivity has been substantially enhanced by supplementing various nutrients and statistically studying their interactions by response surface methodology. A central composite design for amylase production system elucidated a wheat bran‐based medium supplemented with soybean meal, threonine, and B‐complex vitamins predicting a yield of 521 391 U/g dry solids. The enzyme preparation could effectively digest 5–15% suspension of insoluble potato starch in 6 h revealing the dextrose equivalent of 32–44. The supplementation of a glucoamylase preparation, thereafter, brought about complete saccharification. The yield achieved in the statistically optimized amylase system may be one of the best to date and its capability in directly liquefying raw potato starch granules makes this study novel.     

Bacterial and fungal diversity in the traditional Chinese strong flavour liquor Daqu

In this study, the diversity of bacterial and fungal communities in the strong flavour Daqu production process was investigated using polymerase chain reaction denatured gradient gel electrophoresis (PCR‐DGGE). The band number, dominance, diversity and similarity of the 16S rDNA differed in the DGGE patterns, reflecting the wide diversity of the microbial communities present in the different phases. The band abundance and dominance of the eukaryotic microbial community varied in the different phases. To provide insight into factors contributing to this diversity, the physicochemical properties of Daqu were explored. The core temperature of Daqu gradually increased to a maximum during the Dinghuo phase followed by a decline. The moisture and starch content decreased throughout fermentation. The total acid, reducing sugar, amino nitrogen, α‐amylase activity, glucoamylase activity, protease activity, bacterial and mould number increased gradually to a maximum value at the end of the Peijun phase before decreasing in the Dinghuo phase increasing slightly in the Huanluo phase and then remaining almost unchanged. The esterase activity exhibited a similar change to that of other enzymes and then slightly increased after the Peijun stage. For yeasts, the highest number was obtained at the Peijun stage and then decreased. Correlation analysis revealed that the diversity of both bacteria and fungi was negatively correlated with moisture and starch and positively correlated with other indices. The reducing sugar, amino nitrogen, α‐amylase and protease activities showed significant positive correlation with bacteria, and glucoamylase and esterase showed significant correlation with fungi. © 2019 The Institute of Brewing & Distilling     

晒黄烟调制期叶面可培养细菌的多样性研究

以云南特色晒黄烟烟叶为供试样品, 对调制期三个部位烟叶定期取样, 研究叶面可培养细菌的动态变化和多样性。结果表明, 晒黄烟调制期叶面可培养细菌总量呈先升高后降低再略升高的趋势。细菌种群较为丰富, 主要分布在Bacillus 属、Pseudomonas 属、Arthrobacter 属和Exiguobacterium 属等10 个属。Bacillus 属菌株是整个调制期的优势细菌类群, 与烤烟烟叶的优势种群基本一致。      

Amylolytic Hydrolysis of Native Starch Granules Affected by Granule Surface Area

ABSTRACT: Initial stage of hydrolysis of native starch granules with various amylolytic enzymes, α‐amylase from Bacillus subtilis, glucoamylase I (GA‐I) and II (GA‐II) from Aspergillus niger, and β‐amylase from sweet potato showed that the reaction was apparently affected by a specific surface area of the starch granules. The ratios of the reciprocal of initial velocity of each amylolytic hydrolysis for native potato and maize starch to that for rice with the amylolytic enzymes were nearly equivalent to the ratio of surface area per mass of the 2 starch granules to that of rice, that is, 6.94 and 2.25, respectively. Thus, the reciprocal of initial velocity of each enzymatic hydrolysis as expressed in a Lineweaver–Burk plot was a linear function of the reciprocal of surface area for each starch granule. As a result, it is concluded that amylolytic hydrolysis of native starch granules is governed by the specific surface area, not by the mass concentration, of each granule.     

Modelling of the Enzymatic Hydrolysis of Potato (Solanum tuberosum) Using Response Surface Methodology

Starch hydrolysates containing glucose can be obtained from potato (Solanum tuberosum). Acid hydrolysis generates a solution that must be neutralized to be used for fermentative purposes while an enzymatic hydrolysis generates a glucose solution which can be used without further treatments. This work deals with the modelling of the enzymatic hydrolysis of dried potatoes with α‐amylase and glucoamylase with temperature, time, activity units/substrate ratio and α‐amylase activity fraction as operational variables and released glucose concentration as dependent variable. The model obtained showed that the optimal conditions were 70ºC, 60 min; 74 activity units/g substrate and 0.13 α‐amylase activity U/total U fraction. Using these conditions, a glucose concentration of 38.9 g/L was predicted which corresponded well with an experimental value of 38.0 ± 0.5 g/L. The model obtained can be used in the development of processes for the hydrolysis‐fermentation of potato for the production of food additives or ethanol.     

Thermal and pressure‐temperature denaturation kinetics of bacillus subtilis α‐amylase: A study based on gel electrophoresis

Abstract

The commercially available enzyme Bacillus subtilis α‐amylase was characterized by a molecular weight of about 55 kDa and contained isozymes with pI values ranging in a narrow zone (4.6–5.3). Furthermore, the sample was confirmed to contain no disulfide bonds. Upon thermal denaturation in the presence of sodium dodecyl sulfate, a band at half molecular weight was noticed, indicating that the native enzyme might be a dimeric form. Thermal as well as pressure‐temperature denaturation kinetics were investigated using gel electrophoresis and both could accurately be described by a first order kinetic model. For thermal denaturation an activation energy of 283 kJ/mole was calculated. As far as pressure‐temperature denaturation is concerned, activation energy and activation volume at a constant pressure of 5500 bar and a constant temperature of 40°C were calculated respectively as 77.6 kJ/mole and ‐23.2 cm³/mole. These values were compared with those for thermal and pressure‐temperature inactivation of Bacillus subtilis α‐amylase, as determined from residual enzyme activity measurements. No significant differences between the activation energy and volume characterizing denaturation and inactivation of Bacillus subtilis ct‐amylase were observed.     

Benefits of Bacillus megaterium Amylase in Dextrose Production

A Bacillus meguterium amylase (BMA) has the ability to convert branched saccharides to a form that is easily hydrolyzed by glucoamylase. Concequently, dextrose production can be improved by using a combination of BMA and glucoamylase in saccharification. Benefits provided by BMA are similar to those achieved with a debranching enzyme. For instance, BMA is effective in increasing dextrose yield, while at the same time, nearly eliminating tetra- and higher saccharides. BMA can also be used to increase saccharification rate, reduce glucoamylase dosage, or maintain normal dextrose level at higher solids. Benefits of BMA have been quantified by evaluating the enzyme in combination with glucoamylase using typical industrial saccharification conditions.     

汾酒大曲酯酶和淀粉酶同工酶的分析

利用同工酶技术进行了清茬、后火和红心3种汾酒大曲酯酶和淀粉酶同工酶的比较研究。大曲电泳参数研究表明:酯酶和淀粉酶同工酶电泳分离胶浓度分别为12%和10%,上样蛋白浓度分别为5.5～11 mg/mL和0.34～0.69 mg/mL,连续抽提10批储存9 d,同工酶电泳图谱一致稳定。3种大曲同工酶酶谱分析表明:每种大曲都含有丰富的酯酶和淀粉酶同工酶酶带。3种大曲酯酶同工酶谱主带的数量和位置基本相同,曲皮、曲心和整曲也基本一致,仅在弱带区有所不同。3种大曲淀粉酶同工酶谱明显不同,后火曲有一条特征主带。曲心明显不同于曲皮和整曲,曲心的淀粉酶同工酶酶谱明显将3种大曲鉴别,其中有一条酶带可作为后火曲的同工酶标记。10批混合生产用曲的同工酶分析,酯酶和淀粉酶同工酶酶谱基本一致,淀粉酶同工酶谱分析混合曲中有后火曲存在。     

烟草种子附生细菌群落结构与多样性分析

为揭示烟草种子附生细菌的群落结构和多样性,采用分离培养法分析了6个烟草品种（烤烟K326、云烟85、云烟87及红花大金元,雪茄烟301和白肋烟L8）种子可培养附生细菌种类,并采用Illumina高通量测序技术研究了4个品种（K326、云烟85、雪茄烟301和白肋烟L8）种子附生细菌的群落结构与多样性。结果表明:6个品种种子上共分离鉴定出细菌35株,可培养优势附生细菌有假单胞菌（Pseudomonas）、芽胞杆菌（Bacillus）、肠杆菌（Enterobacte）、泛菌（Pantoea）、鞘氨醇单胞菌（Sphingomonas）等菌属。高通量测序结果表明:K326、白肋烟L8和云烟85种子的附生细菌结构上存在共性与差异,共有的优势菌有假单胞菌、泛菌、鞘氨醇杆菌（Sphingobacterium）、肠杆菌、糖芽胞杆菌（Saccharibacillus）、寡养单胞菌（Stenotrophomonas）、根瘤菌（Rhizobium）等菌属,烟草品种附生细菌多样性排序依次为云烟85＞K326＞L8＞301。      

Suppression of Fusarium graminearium growth by differently structured starch types

We investigated the growth behavior and amylolytic enzymes of Fusarium graminearum cultivated on different types of native starch granules including barley (A‐type crystalline polymorph), potato and Curcuma zedoaria (B‐type crystalline polymorph), cassava (C‐type crystalline polymorph), and high AM maize (A + Vh‐type crystalline polymorphs). F. graminearum grew poorly on B‐type starches and the accumulation of biomass was similar to that obtained for fungi cultivated under carbohydrate starvation conditions. In comparison, three‐ to fivefold higher accumulation of fungal biomass was observed for growth on the A‐, C‐ and A + Vh‐type starches. Fungal glucoamylase and α‐amylase activity increased over time in the presence of native starch granules. Interestingly, resistant B‐type starches induced the highest amylolytic activity indicating that F. graminearum interacts with B‐type granules although only limited degradation occur. Starch degradation products maltose and malto‐oligosacharides was found to increase glucoamylase and α‐amylase activity, whereas glucose acted as a catabolite repressor.     

Optimisation of conditions for glucose syrup production from banana (Musa paradisiaca L.) pulp using response surface methodology

A central composite design was applied to assess some basic factors that affect profoundly the production of dextrose equivalent from banana pulp. Optimal conditions for the conversion of starch banana pulp to glucose were found to be a banana pulp concentration of 8.5%, reaction time of 90 min, and α‐amylase concentration of 0.04% for liquefaction and reaction time of 24 h with a glucoamylase concentration of 0.05% for saccharification. Only 16 and 12 experiments, respectively, were needed to assess these conditions. The adequacy of model was highly satisfactory, because the coefficients of determination (R²) were 0.93 and 0.91, respectively. The glucose syrup (GS) obtained was slightly yellowish with a hint of brown and slightly sweet. The syrup had a protein of 0.63% and an ash content of 0.60%, with a pH of 4.8. The characteristics of the GS produced met all the recommended standards, except for that of colour.     

Rapid molecular methods for enumeration and taxonomical identification of acetic acid bacteria responsible for submerged vinegar production

The aim of the present study was to search for a rapid and reliable method to enumerate viable acetic acid bacteria (AAB) and to identify to genera and species level AAB isolates from vinegars in full acetic fermentation elaborated by the submerged method from cider, wine and spirit ethanol in industrial bioreactors. Results showed that the rapid epifluorescence staining method using the LIVE/DEAD BacLight bacterial viability kit and direct counts in Neubauer chamber rendered consistent and reliable data for viable cell counts of bacteria in all the studied vinegars. A linear correlation was shown between viable cell counts and fermentation rates. The highest fermentation rates and viable cell counts were found in cider vinegars, whereas spirit vinegars showed the lowest values for both parameters. Eighty-four AAB pure isolates were recovered from 41 different vinegar samples and were submitted to DNA extraction. PCR amplification of the 16S–23S intergenic spacer region of rDNA and subsequent sequencing were carried out to identify isolates to species level. Results showed that Gluconacetobacter europaeus was the predominant cultivable species, appearing in 79% of the total isolates. This was the unique species found in spirit vinegars, and this is the first time that AAB from spirit vinegars are taxonomically identified. Ga. europaeus was as well the predominant cultivable species in white wine vinegars. Cider vinegars presented the highest variability of species: Ga. europaeus (35.3% appearance among cultivable isolates), Ga. xylinus (35.3%), Acetobacter pasteurianus (17.6%) and Ga. hansenii (11.8%). Red wine vinegars showed cultivable isolates of the species Ga. xylinus (71.4%) and Ga. europaeus (28.6%). Summarising, both described methods for AAB enumeration and taxonomical identification proved to be fast and reliable methods, and results revealed Ga. europaeus as the cultivable major species in vinegars in full fermentation conducted by the submerged method, suggesting that Ga. europaeus strains can constitute excellent starter cultures for the elaboration of vinegars by the submerged method.     

Enzymatic synthesis of glycosylated puerarin using maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis

BACKGROUND: The maltogenic amylase from Bacillus stearothermophilus (BSMA) is a valuable biocatalyst that has been used to transglycosylate natural glycosides to improve solubility. To ensure safety, BSMA was produced in Bacillus subtilis, using new shuttle vector‐based expression vectors. The transglycosylation of puerarin was also conducted with crude BSMA and analyzed. RESULTS: Two expression systems, each containing one of the promoters from the genes encoding Bacillus licheniformis maltogenic amylase (BLMA) and an α‐amylase from B. subtilis NA64 (amyR2), were constructed. The amyR2 promoter system was chosen as the best system; it yielded 107 mg of pure BSMA from a 2 L culture. In the transglycosylation reactions of puerarin using crude BSMA, relative amounts for maltosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 3)‐puerarin, and puerarin were determined as 26:18:7:49. A two‐step purification process, including gel permeation chromatography, yielded 1.7 g of the transfer products from 3 g of puerarin. CONCLUSION: The crude BSMA produced from a host generally recognized as safe (B. subtilis) can be used to transglycosylate various functional compounds. The expression system developed in this study will be helpful for the production of other food‐grade enzymes by B. subtilis. Copyright © 2010 Society of Chemical Industry     

Seed coat tannins and bruchid resistance in stored cowpea seeds

Seeds of wild species and varieties of Vigna were screened for their tannins and α‐amylase inhibitor contents as defensive compounds against cowpea weevil. Seed coats contained condensed tannins that were positively correlated to their colour but not to their resistance against the insect. The α‐amylase inhibitors were present in different amount in cotyledons of all species analysed. Amongst the cultivated lines assayed, Vigna unguiculata TVu 2027, an accession identified as moderately resistant, was found to contain the higher amount of α‐amylase inhibitor. When wild species were considered, V luteola and V vexillata (two resistant species) showed the highest content of α‐amylase inhibitors. In addition, two cultivated accessions (Vita 7 and IT 84E‐1‐108) of cowpea seeds, both classified as susceptible accessions, showing a different degree of bruchid damage in storage, were also analysed. No α‐amylase inhibitory activity was found in cotyledons of undamaged Vita 7 seeds, while the seed coat tannin content was found to be 13 times higher in undamaged Vita 7 seeds than in IT 84E‐1‐108 infested seeds. These latter results support the hypothesis that seed coat tannins must also be considered in biochemical defence mechanisms, which can deter, poison or starve bruchid larvae that feed on cowpea seeds. Copyright © 2004 Society of Chemical Industry     

Inhibitory potential of phenylpropanoid glycosides from Ligustrum purpurascens Kudingcha against α‐glucosidase and α‐amylase in vitro

The leaves of Ligustrum purpurascens are used in a Chinese traditional tea called small‐leaved kudingcha, which is rich in phenylpropanoid glycosides (PPGs) and has many beneficial properties. Two critical exoacting glycoside hydrolase enzymes (glucosidases) involved in carbohydrate digestion are α‐glucosidase and α‐amylase. We investigated the properties of PPGs from L. purpurascens for inhibiting α‐amylase and α‐glucosidase activity in vitro and found IC₅₀ values of 1.02 and 0.73 mg mL^?1, respectively. The patterns of inhibiting both α‐amylase and α‐glucosidase were mixed‐inhibition type. Multispectroscopy and molecular docking studies indicated that the interaction between PPGs and α‐amylase and α‐glucosidase altered the conformation of enzymes, with binding at the site close to the active site of enzymes resulting in changed enzyme activity. Our studies may help in the further health use of small‐leaved kudingcha.     

Degradability of Different Phosphorylated Starches and Thermoplastic Films Prepared from Corn Starch Phosphomonoesters

Different starch types (corn, rice, potato, corn amylose and corn amylopectin) were phosphorylated to varying degrees of substitution (DS) and tested both for acid hydrolysis during 3 h in a boiling bath and for enzymatic hydrolysis with a thermostable bacterial α‐amylase (Bacillus licheniformis) for 30 min at 95 °C. Generally, phosphorylated starches showed a reduced degree of acid hydrolysis during the entire time of hydrolysis (3 h) as well as reduced susceptibility to α‐amyIase hydrolysis. The enzyme action was inhibited by the presence of phosphate groups in the modified starch molecules and the extent of inhibition increased with increasing degree of phosphate substitution, regardless of the starch type. Thermoplastic films were fabricated by blending modified corn starches of different DS with polyacrylate, urea and water at a ratio of 4:5:1:50, heating for 30 min at 95 °C before casting and allowing to cool, stand and dry at room temperature. The plastic films prepared from phosphorylated corn starch showed both higher disintegration rate and a greater degradability by thermostable bacterial α‐amylase than the ones prepared from non‐phosphorylated starch. These new acquired properties can meet the increasing demand for biodegradable disposable plastic bags.     

Present knowledge of the bacterial microflora in the extreme environment of sugar thick juice

The diversity of the bacterial population in sugar thick juice, an intermediate product in the production of beet sugar, which exhibits an extreme, osmophilic environment with a water activity value (a_w) less than 0.86, was assessed with both culture-dependent and -independent 16S ribosomal RNA (rRNA) gene-based analyses. In comparison with previous studies, the number of different thick juice bacterial species increased from 29 to 72. Remarkably, a limited, gram-positive, culturable flora, encompassing species of Bacillus, Staphylococcus and mainly Tetragenococcus dominated thick juice during storage, while a more heterogeneous and unculturable fraction of Acinetobacter, Sporolactobacillus and Thermus species could be detected in freshly produced thick juice. Notably, almost all bacteria detected in the thick juice were also detected in the air, emphasising the importance of further investigation and assessment of strategies to reduce (air) contamination during processing and storage. The discovery of the contamination source may be used for the development of management strategies for thick juice degradation resulting from microbial activity.     

Characterization of the microbial community in three types of fermentation starters used for Chinese liquor production

This study examined and compared the microbial community in three typical fermentation starters (called as Daqu, Xiaoqu, and Fuqu in China) used for liquor production by analysing the 16S and 18S rRNA gene clone library. The results show that the microbial diversity in the three types of fermentation starters (JiuQu) differs significantly. The bacterial species in Daqu and Fuqu were mainly thermophilic or thermotolerant. In Daqu, the dominant bacterial species were Thermoactinomyces sanguinis (53.85%) and Pantoea agglomerans (19.23%), followed by uncultured bacteria (15.39%). The lactic acid bacterium Weissella cibaria (50%) and a member of Enterobacteriaceae, Enterobacter ludwigii (10%), were the dominant bacterial species in Xiaoqu. Low abundances of other bacteria, including Deinococcus radiodurans, Corynebacterium variabile and Acinetobacter baumannii, were reported for Xiaoqu. Enterococcus faecium, Clostridium beijerinckii and Bacillus cereus were observed in Fuqu and accounted for 46.67, 23.33 and 16.67% of the total bacteria identified, respectively. Fungal diversity was high in Daqu and consisted exclusively of thermophilic moulds, such as Aspergillus glaucus (62.5%), Thermomyces lanuginosus (12.5%) and Thermoascus crustaceus (12.5%). Only two fungal species were reported for Fuqu and Xiaoqu and both contained the mould Rhizopus oryzae. Saccharomyces cerevisiae and the non‐Saccharomyces yeast (Saccharomycopsis fibuligera) were also identified in Fuqu and Xiaoqu, respectively. This finding suggests that microbial community structure in JiuQu starters is the key factor to determine the variety of flavours. Copyright © 2015 The Institute of Brewing & Distilling     

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%。