Purification of Aspergillus sp. S1-13 chitinases and their role in saccharification of chitin in mash of solid-state culture with shellfish waste

In a suspension of solid-state culture of Aspergillus sp. S1-13 containing a lactic acid-treated crab shell as the substrate, the saccharification of chitin in the shell proceeded to form N-acetylglucosamine (GlcNAc): the culture was the source of chitin and chitinases. The analysis of chitinases in the water-extract of the solid-state culture indicated occurrence of an exochitinase (Exo, MW 73 kDa) and two endochitinases. The amounts of the endochitinases suggested that one of them (Endo-1, MW 45 kDa) might be the main species in the chitin-saccharification. The amount of GlcNAc released from the LA-treated crab shell by the combined action of isolated Exo and Endo-1 was very small, predicting participation in the saccharification of other enzyme species, which might be hardly extracted with water from the solid-state culture. The re-extraction of the solid-state culture using 2 M KCl, which was extracted with water beforehand, demonstrated another endochitinase (Endo-2, MW 51 kDa). Endo-2 isolated from the salt-extract can adsorb to chitin, and can hydrolyze the chitin in the adsorbed state. The roles of these chitinases in the chitin-saccharification based on their properties and combined action were discussed.     

Utilization of shrimp shellfish waste as a substrate for solid-state cultivation of Aspergillus sp. S1-13: evaluation of a culture based on chitinase formation which is necessary for chitin-assimilation

The utilization of shrimp shellfish waste as a substrate for solid-state cultivation of a filamentous fungus, Aspergillus sp. S1-13, was investigated. The organism was selected from among 220 isolates based on the productivity of its chitinolytic enzyme (chitinase), which might reflect microbial growth. The enzyme was produced only when the organism was grown on medium containing the shellfish waste. The addition of 58-65% water (w/w) to the medium was effective in enhancing production, and a certain amount of enzyme was observed in media of higher water content (up to about 75%). The initial pH and nitrogen source (ammonium sulfate) of the solid-state medium also affected the amount of enzyme. The amount of enzyme increased 2-fold in an optimum solid-state medium: 5 g of shrimp shellfish waste and 3 ml of basal medium (pH 5) containing 0.1% (NH4)2SO4 was inoculated with 4 ml of spore suspension; static cultivation at room temperature. The amount increased further (1.5-fold) when the cultivation was carried out at 37 degrees C, with 1.85 units of the enzyme formed from 1 g of shrimp shellfish waste. An analysis by ion-exchange column chromatography suggested the presence of at least two colloidal chitin-hydrolyzing enzymes and one p-nitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in an extract of the solid-state culture. The elution profile was similar to that obtained with a liquid culture filtrate.     

黄酒发酵醪中特有乳酸杆菌检测和培养基的研究及初步鉴定

通过可溶性淀粉的添加量、培养温度、"X"物质的添加量三因素三水平对黄酒发酵醪中乳酸杆菌培养基配方和培养方法的正交试验研究,以及性能的测定和初步鉴定,表明:黄酒发酵醪中乳酸杆菌培养、分离的培养基配方和培养方法为:加饭酒稀释至酒精度14.5%voL、加"X"物质的量1.3%、加可溶性淀粉0.5%或不加、培养温度18℃或30℃、压盖密封培养;黄酒后发酵醪中的优势菌,在上述培养基中18℃生长良好,25℃及以上生长不良或生长极慢,初步鉴定为乳酸杆菌的新种,命名为黄酒乳酸杆菌Ⅱ-1(Lactobacillus chinese-rice-wineⅡ-1)。黄酒前发酵醪中的优势菌,在上述培养基中30℃生长良好,20℃及以下生长缓慢,初步鉴定为乳酸杆菌的新种,命名为黄酒乳酸杆菌Ⅱ-2(Lactobacillus chinese-rice-wineⅡ-2)。     

Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium

A strictly anaerobic, mesophilic and chitinolytic bacterial strain, M-21, was isolated from a soil sample collected from Mie University campus and identified as Clostridium paraputrificum based on morphological and physiological characteristics, and 16S rRNA sequence analysis. C. paraputrificum M-21 utilized chitin and N-acetyl- -glucosamine (GlcNAc), a constituent monosaccharide of chitin, to produce a large amount of gas along with acetic acid and propionic acid as major fermentation products. Hydrogen and carbon dioxide accounted for 65% and 35% of the gas evolved, respectively. The conditions for 1 l batch culture of C. paraputrificum, including pH of the medium, incubation temperature and agitation speed, were optimized for hydrogen production with GlcNAc as the carbon source. The bacterium grew rapidly on GlcNAc with a doubling time of around 30 min, and produced hydrogen gas with a yield of 1.9 mol H₂/mol GlcNAc under the following cultivation conditions: initial medium pH of 6.5, incubation temperature of 45°C, agitation speed of 250 rpm, and working volume of 50% of the fermentor. The dry cell weight harvested from this culture was 2.0 g/l.     

一株强化发酵甜瓣子的乳酸片球菌特性及应用效果评价

甜瓣子是以蚕豆制曲后加盐水发酵而成的,是郫县豆瓣风味形成的关键阶段。筛选适用于强化甜瓣子发酵的专用乳酸菌,以期应用于强化甜瓣子发酵。以甜瓣子为原料筛选生长速度快、产酸能力强的高效乳酸菌,研究其生长特性。利用筛选得到的高效乳酸菌制备菌剂并用于强化甜瓣子发酵,分析甜瓣子品质。该研究筛选得到1株乳酸片球菌ND1,37℃培养6 h、OD610达1. 375,培养24 h发酵液中乳酸含量达到1. 296 g/100 g。ND1在4～50℃条件生长良好,高温更利于其生长,在37～45℃生长速度最快;能耐受10%的盐,在盐含量(质量分数)为0%～8%的条件生长良好;在pH 3. 5～6生长速度快。利用菌株ND1发酵得到的乳酸菌剂活菌数可达1. 07×10¹²CFU/g,利用该菌剂发酵甜瓣子,可有效提高甜瓣子中有机酸(尤其是乙酸、苹果酸、乳酸)含量,强化甜瓣子风味。该研究筛选的乳酸片球菌ND1具有良好的温度、酸度、盐度适应性,能改善甜瓣子中有机酸含量,有望被用作强化甜瓣子发酵增香的乳酸菌,改善郫县豆瓣品质。     

The simultaneous saccharification and fermentation of malt dust and use in the acidification of mash

In brewing, the mash or wort is frequently acidified by the addition of lactic acid or the bioacidification of the mash. The present study provides an alternative approach for mash or wort acidification by the simultaneous saccharification and fermentation (SSF) of malt dust. In this method, fermentable carbohydrates released by the enzymatic breakdown of the cellulosic portion of the malt dust are converted to lactic acid by lactic acid bacteria. The effect of temperature, ranging between 45 and 51°C, solid loading of malt dust at 2, 5 and 10% (w/v) on a dry basis, and enzyme loading at 0.65, 2.6 and 6.5 filter paper units (FPU) per gram malt dust on SSF and change in pH in mash acidification were examined. The final pH and lactic acid concentration and final glucose concentration of the SSF media were significantly affected by the temperature of the process (p < 0.05). The highest lactic acid titre (9.7 g/L) and the lowest pH (3.12) were obtained by SSF of 10% (w/v) malt dust at 45°C with 6.5 FPU/g. The pH of the mashing solution [containing 20% (w/v) ground malt] decreased to around 5.4 and 5.2 after adding 1.9 and 2.9% of SSF media with pH 3.39. © 2019 The Institute of Brewing & Distilling     

黄酒发酵醪抑制霉菌生长的初步探讨

通过酒精、浆水、乳酸、pH值、发酵醪液对霉菌生长抑制作用的研究发现：低酒精含量50％（v／v）、一定量乳酸、pH值4．0和3．8并不能抑制霉菌生长繁殖。黄酒发酵醪中高密度的乳酸杆菌、发酵产生和积累的乳杆菌素可以抑制霉菌生长，并根据相关研究的报道初步确定了黄酒发酵醪抑制霉菌生长的主要因素。     

一株戊糖片球菌在低盐固态酱油工艺中的应用

酱油发酵酱醅中筛选到一株戊糖片球菌,对该菌的生长温度、生长pH值、食盐耐受度等生长特性进行研究。采用离子交换色谱法对该菌株产有机酸的情况进行测定;将该菌株添加到低盐固态酱油后酵过程中,以不添加乳酸菌的实验组作对照,对酱油样品理化指标、经济指标进行分析,并采用顶空固相微萃取-气相色谱-质谱法(HS-SPME-GC-MS)对酱油挥发性香气成分进行对比分析。结果表明,该乳酸菌的生长条件与酱油酱醅发酵环境相适应,其发酵产酸以乳酸和乙酸为主,与酱油中有机酸主成分相一致,将该菌株用于低盐固态酱油发酵,对酱油香气和其他指标均有不同程度的促进作用。     

淡紫拟青霉固态发酵黄芩药渣工艺优化

为了降低淡紫拟青霉（Paeciloids lilacinus）的固态发酵成本,以黄芩药渣废弃物为基质,对其培养基组成及发酵条件进行优化,并考察不同几丁质含量对菌株产几丁质酶活性的影响。结果表明,淡紫拟青霉固态发酵的最佳培养基组成为黄芩药渣20 g,玉米粉0.8 g,尿素0.02 g,含水量80%,固态发酵最佳的培养条件为培养温度28℃,接种量10%,培养时间7 d,在此优化条件下,分生孢子数可达8.98×109 CFU/g。添加0.9%几丁质时,可显著增加菌株产几丁质酶酶活性（P<0.05）。     

清香型小曲白酒酒醅中乳酸菌的筛选鉴定及其发酵特性研究

该研究采用传统分离方法从不同季节清香型小曲白酒酒醅中分离乳酸菌,通过形态及分子生物学技术对其进行菌种鉴定,并探究其发酵特性。结果表明,共分离纯化出225株乳酸菌,经鉴定,归属于3属15种。通过高粱汁培养基初筛、固态发酵小试实验复筛,得到两株增酯降杂效果较好的乳酸菌,分别为纳格里乳杆菌（Lactobacillus nagelii）X29和布氏乳杆菌（Lactobacillus buchneri）X9。将强化乳酸菌X29和X9麸皮种加入桂花曲,最佳添加量为20%,此添加量下酒样中乙酸乙酯含量分别提升32.84%和16.79%,乳酸乙酯含量分别提升37.38%和36.44%,正丙醇含量分别降低2.61%和5.40%,杂醇油含量分别降低21.33%和26.71%。乳酸菌强化麸皮种糖化前加入能明显提升酒体中乙酸乙酯含量;糖化后加入则对乳酸乙酯含量和出酒率提升效果明显;延长发酵时间也可提升乙酸乙酯和乳酸乙酯含量。     

多菌混合发酵产纤维素酶及生物法预处理秸秆的研究

对实验室分离筛选的3株绿色木霉和6株枯草芽孢杆菌的生长及产酶情况进行了研究,综合确定绿色木霉绿2与芽孢杆菌S3为混合菌中产纤维素酶酶活最高的组合。在此基础上,采用解脂假丝酵母处理高粱秸秆。先将解脂假丝酵母的种子培养液按照3%的接种量接种到发酵产酶培养基中,隔24 h将绿色木霉绿2的孢子悬浮液按照2.67%的接种量接种到发酵产酶培养基中,再隔12 h将芽孢杆菌S3的种子培养液按照5.33%的接种量接种到发酵产酶培养基中,测定出的滤纸酶活（FPA）最高,为389.89 U/mL,比优化前提高了14.30%。     

白曲霉用于食醋糖化过程的研究

通过正交试验确定了白曲霉SICC3.917三角瓶与糖化曲的最佳培养基成分及最佳培养条件、糖化醪液的最佳制备工艺。结果表明白曲霉的三角瓶最佳培养条件为：麸皮添加量8.5g,玉米粉添加量1.5g,水添加量10.5g,培养温度30℃。白曲霉SICC3.917糖化曲的最佳培养条件为种曲接种量0.5%,熟料含水量52%,培养温度32℃,空气湿度90%。糖化醪液的最佳制备工艺为白曲霉糖化曲的添加量1.3%,大米粉浓度20.7%,糖化温度65℃,糖化时间3h。     

米醋生产用米酒发酵工艺优化

以大米为试验原料,葡萄糖值（DE值）和酒精度为考察指标,研究大米酒精发酵工艺对米醋生产过程的影响。通过正交试验确定大米液化的最佳工艺条件为料水比1∶2.5（g∶mL）,液化酶0.3%,氯化钙0.1%,液化温度97 ℃,液化时间90 min;糖化的最佳工艺条件为糖化酶0.2%,糖化温度65 ℃,糖化时间为60 min;酒精发酵的最佳工艺条件为酵母接种量0.25%,发酵温度33 ℃,发酵时间12 d。在此最佳条件下,最终发酵前醪液的还原糖含量和DE值分别达到19.8 g/100 mL和75.8%,发酵后酒精度达到12.0%vol,出酒率为37.67%。     

以酱渣为原料生产蛋白饲料的研究

本文介绍了以酱渣为原料，生产蛋白饲料的研究成果，试验获得一适合于酱渣固体发酵的组合菌种Ａ３＋Ｅ（３１１）＋Ａ（Ｓ７７７）。结果表明，培养基中的初始含水量为６５％，在２８℃固体发酵３天，产品的粗蛋白含量小试可达到３０％。中试和大试分别达到２５％和２４％，发酵结果比较理想。     

β-葡聚糖酶[EC. 3. 2. 1. 73]的研究 Ⅰ.产酶菌种的筛选和培养

β-葡聚糖酶主要用于啤酒工业,它能专一地分解粘度很高的各种大麦β-葡聚糖中β-1.3和β-1.4糖苷键,使麦汁粘度降低,从而缩短麦汁和啤酒的过滤时间,增加产量,并改善啤酒的质量。 试验所用菌种Bacillus Subtilis是从34株细菌中筛选并经诱变获得,它具有较高的产β-葡聚糖酶的能力。 在以淀粉、蔗糖和硫酸铵为主的培养基、pH7.2～7.5、温度为37℃条件下,进行搅拌通风培养两天。酶活力约为30U/ml。 发酵液采用絮凝剂处理,经硅藻土过滤,再将滤液经超滤或真空低温浓缩,即获得了深棕色的液体酶制剂。若进一步用酒精沉淀或进行喷雾干燥,即可制得粉末状酶制剂产品。 β-葡聚糖酶应用于啤酒糖化,对加速麦汁过滤、提高麦汁收得率,均有明显效果。     

米酒酿造的糖化条件优化

采用复合的纯种糖化曲和自然糖化曲酿造米酒,考查了糖化温度、糖化时间、糖化曲用量和两种糖化曲的质量比对米酒糖化液品质的影响,利用正交试验优化了米酒酿造的糖化条件。结果表明,米酒糖化的优化条件为糖化温度36 ℃、糖化时间40 h、糖化曲用量0.35%、两种糖化曲质量比1∶1。优化条件下,糖化液的总酸含量为5.24 g/L,淀粉转化率为63.53%,感官评分为88分。     

Reduction of Campylobacter jejuni on chicken wings by chemical treatments

Eight chemicals, including glycerol monolaurate, hydrogen peroxide, acetic acid, lactic acid, sodium benzoate, sodium chlorate, sodium carbonate, and sodium hydroxide, were tested individually or in combination for their ability to inactivate Campylobacter jejuni at 4 degrees C in suspension. Results showed that treatment for up to 20 min with 0.01% glycerol monolaurate, 0.1% sodium benzoate, 50 or 100 mM sodium chlorate, or 1% lactic acid did not substantially (< or = 0.5 log CFU/ml) reduce C. jejuni populations but that 0.1 and 0.2% hydrogen peroxide for 20 min reduced C. jejuni populations by ca. 2.0 and 4.5 log CFU/ml, respectively. By contrast, treatments with 0.5, 1.0, 1.5, and 2.0% acetic acid, 25, 50, and 100 mM sodium carbonate, and 0.05 and 0.1 N sodium hydroxide reduced C. jejuni populations by >5 log CFU/ml within 2 min. A combination of 0.5% acetic acid plus 0.05% potassium sorbate or 0.5% acetic acid plus 0.05% sodium benzoate reduced C. jejuni populations by >5 log CFU/ml within 1 min; however, substituting 0.5% lactic acid for 0.5% acetic acid was not effective, with a reduction of C. jejuni of <0.5 log CFU/ml. A combination of acidic calcium sulfate, lactic acid, ethanol, sodium dodecyl sulfate, and polypropylene glycol (ACS-LA) also reduced C. jejuni in suspension by >5 log CFU/ml within 1 min. All chemicals or chemical combinations for which there was a >5-log/ml reduction of C. jejuni in suspension were further evaluated for C. jejuni inactivation on chicken wings. Treatments at 4 degrees C of 2% acetic acid, 100 mM sodium carbonate, or 0.1 N sodium hydroxide for up to 45 s reduced C. jejuni populations by ca. 1.4, 1.6, or 3.5 log CFU/g, respectively. Treatment with ACS-LA at 4 degrees C for 15 s reduced C. jejuni by >5 log CFU/g to an undetectable level. The ACS-LA treatment was highly effective in chilled water at killing C. jejuni on chicken and, if recycled, may be a useful treatment in chill water tanks for poultry processors to reduce campylobacters on poultry skin after slaughter.     

玉米粉液化及糖化工艺条件优化

以玉米粉为原料,葡萄糖当量（DE）值作为评价指标,研究料液比、时间、酶添加量、温度、pH值对玉米粉液化及糖化效果的影响,采用单因素及正交试验对液化、糖化工艺参数进行优化。结果表明,将玉米粉加水配制成料液比1∶4（g∶mL）的浆料,调pH 6.2,最佳液化工艺条件为α-淀粉酶添加量8 U/g、液化温度80 ℃、液化时间60 min、液化液调pH 4.3;最佳糖化条件为糖化酶添加量250 U/g、糖化温度60 ℃、糖化时间12 h。在此最佳条件下,葡萄糖当量值达到93.1%。     

pH值及乳酸菌对米曲霉固态制曲过程的影响

考察了在固态培养条件下,不同的初始pH值及乳酸菌对米曲霉固态制曲过程的影响。以豆粕和麸皮(质量比为55∶45)为原料,1∶0.6的料水比,0.3%～0.5%的接种量接入沪酿3.042米曲霉种曲,33℃下制曲,定时取样测定固体曲的蛋白酶活力及孢子数。结果表明:适当偏酸性的环境有利于成曲蛋白酶的提高,培养基初始pH调节为6.5时米曲霉中性蛋白酶活力获得最大值(4 080±61)U/g干基,较对照组提高33%～37%;添加1.0%～2.0%乳酸菌,也同样起到改善米曲霉制曲过程的效果。     

Solid substrate production of Epicoccum nigrum conidia for biological control of brown rot on stone fruits

Production of conidia of Epicoccum nigrum, a biocontrol agent of the fungal pathogen Monilinia laxa, was tested in liquid- and solid-state fermentation. Liquid fermentation was conducted in 250 ml Erlenmeyer flasks containing 50 ml of a mineral medium (containing per litre: 20 g lactose, 10 g NO3K, 1 g K2HPO4, 0.5 g MgSO4.7H2O, and 1 ml of a minor-element solution), inoculated with 2 x 10(5) E. nigrum conidia ml(-1), and incubated at 20-25 degrees C and 150 rpm for 7 days. Solid-state fermentation was carried out in specially designed plastic bags (600 cm3) (VALMIC) containing either 50 g of peat/vermiculite (1:1, w/w), or 50 g of peat/vermiculite/lentil meal (1:1:1, w/w/w) with 40% (v/w) initial moisture content. Substrate was inoculated with a conidial suspension of E. nigrum to give 10(5) conidia g(-1) substrate, and bags were incubated at 20-25 degrees C for 7 days in darkness. The amount of conidia of E. nigrum obtained in solid-state fermentation with substrate based on peat/vermiculite/lentil meal was 10-fold higher than with substrate based on peat/vermiculite or in liquid fermentation. Conidial production under these conditions was maintained in the range of 10(8) conidia g(-1) substrate from 10 to 150 days after inoculation. Germinability of these conidia was >90%. Addition of other nutrients than lentil meal to peat/vermiculite did not enhance production of conidia. Presence of peat in the substrate was necessary for good conidia production, but change in the kind of peat or vermiculite did not improve conidial production. Conidial production was similar when the substrate was inoculated with 10(5), 10(6) or 10(7) conidia g(-1) dry substrate. Incubation of bags in light conditions did not enhance conidial production. Fresh conidia produced in this solid-state fermentation system reduced the incidence and lesion diameter induced by M. laxa on peaches.