用生料制备功能性双歧醋生产工艺的实验室研究

目的将双歧杆菌、醋酸菌、酵母菌和粉碎的制醋原料及麸曲共同发酵,通过生料制醋的方法来制备功能性双歧醋。方法将粉碎的玉米与麸曲、酵母液、麸皮和水搅拌均匀,使其经过液态糖化和酒精发酵后,接入醋酸菌和双歧杆菌(二者比例为1∶1),同时加入辅料,进行醋酸发酵,当检测到醋酸酸度为5.0%~7.5%时,加入食盐终止发酵,经过过滤,除菌澄清得到功能性双歧醋。结果双歧醋的最终醋酸度为3.2%,外观红棕色,光泽度好,清澈透明,无沉淀和悬浮物。总菌数:醋酸菌为3.3×1011/m l,双歧杆菌为1.9×107/m l;活菌数:醋酸菌为1.7×1011/m l,双歧杆菌为6.8×106/m l;大肠菌群数3个/100 m l;致病菌:不得检出。结论双歧杆菌及其代谢物可以在双歧醋中存活,生料固态发酵制备双歧醋的方法可行。     

分子信标-实时 PCR法快速检测双歧杆菌的研究

为建立双歧制品中双歧杆菌快速、敏感、特异的检测方法,根据双歧杆菌16SrRNA/16SrDNA基因设计合成了双歧杆菌属特异性引物和分子信标探针,建立了快速检测双歧杆菌的分子信标-实时PCR检测方法,并对反应条件进行优化。检测方法重复性好,批内和批间变异系数均小于5%;特异性强,扩增曲线呈现明显的S型,无非特异性扩增;灵敏度高,是普通PCR的100倍,对纯双歧杆菌DNA的检出限为5.7fg/PCR反应体系,纯双歧杆菌菌液的检出限为2×103CFU/mL;线形范围宽,起始模板数在2×108CFU/mL～2×104CFU/mL之间具有良好的线性关系,相关系数大于97%。该方法具有灵敏、特异、简便和快速的特点,可用于对双歧杆菌原位菌数的定量检测。     

鸡源双歧杆菌定向选育和发酵参数研究

目的从肉仔鸡肠道中筛选出耐酸、耐胆盐和耐消化酶的优良双歧杆菌,研究其生长特性,并优化其发酵参数,为转化生产力提供理论依据。方法通过无菌采样并分离得到多株双歧杆菌,对分离获得的双歧杆菌进行形态学、生化特性研究,然后采用牛津杯法,测定90株双歧杆菌对大肠埃希菌和沙门菌的抑制作用,采用改良MRS培养基,模拟鸡胃肠道逆环境,对其耐消化道特性进行研究,筛选出优良双歧杆菌,再进行生长特性研究及发酵参数优化。结果从肉仔鸡肠道分离出90株双歧杆菌,初步挑选出23株作为候选菌株,抑菌试验测得双歧杆菌B1、B2和B3具有良好的抑菌效果,然后经过耐受消化道逆环境试验,发现B2菌株的耐受能力最好,初步鉴定双歧杆菌B2为小鸡双歧杆菌,并将其定名为Bifidobacterium pullorum B2,对其生长特性的研究发现经18 h发酵细菌总数可以从8.3×105CFU/mL升高到1.3×109CFU/mL,运用优化的发酵培养基进行中试试验,发酵后的活菌数可达1.41×1010CFU/mL。结论本实验从肉仔鸡肠道中分离筛选并初步鉴定了Bifidobacterium pullorum B2,优化了制备Bifidobacterium pullorum B2发酵液的发酵条件,降低了生产成本。     

长双歧杆菌DD98冻干保护剂优化及菌粉保存稳定性研究

以长双歧杆菌DD98为研究对象,通过对冻干保护剂配方的优化,冻干菌粉的存活率提高到90%以上。通过进一步稳定性研究,采用保护剂优化配方制备的冻干菌粉在4℃保存24个月后,活菌数仍在1.0×10^10 CFU/g以上,在25℃条件下可以保存12个月,双歧杆菌的存活率在1.0×10^6CFU/g以上,符合FAO/WHO建议食品益生菌活菌数应在1.0×10^6 CFU/g^1.0×10^7CFU/g的标准。     

长双歧杆菌发酵培养基的优化

目的对长双歧杆菌液态发酵培养基进行优化。方法以长双歧杆菌（Bifidobacteriumlongum）为发酵菌株,以MRS培养基为基础培养基,以发酵液活菌数为指标,通过单因素添加实验考察发酵培养基的碳源和氮源的种类,并验证优化后培养基的效果。结果优化后培养基的最适碳源为葡萄糖,最适氮源为酪蛋白胨、牛肉蛋白胨、水解乳蛋白,发酵液活菌数达到2．09×10^9CFU／mL,比原MRS培养基（1．22×10^9CFU／mL）提高了71．30％。结论优化后培养基优于原MRS基础培养基,可应用于长双歧杆菌的液态发酵。     

思连康对小鼠腹腔巨噬细胞吞噬率和吞噬指数的影响

目的研究双歧杆菌四联活菌片(商品名:思连康)对小鼠腹腔巨噬细胞吞噬鸡红细胞吞噬率及吞噬指数的影响。方法将SPF小鼠30只随机分成三组,每组10只,Ⅰ组灌胃生理盐水,Ⅱ组灌胃婴儿双歧杆菌菌悬液,Ⅲ组灌胃双歧杆菌四联活菌片菌悬液,每天给药0.5 mL,菌液浓度为1.0×10~8 CFU/mL,连续给药10 d后小鼠腹腔注入2%鸡红细胞悬液1 mL(红细胞数量为2×10~8个/mL),30 min后处死,取小鼠腹腔洗液,观察并记录吞噬鸡红细胞的巨噬细胞数及被吞噬的鸡红细胞数,计算吞噬率及吞噬指数。结果与Ⅰ组相比,Ⅱ组和Ⅲ组小鼠腹腔巨噬细胞吞噬鸡红细胞的吞噬率和吞噬指数均显著升高(Ps0.05),其中Ⅲ组高于Ⅱ组(P0.05)。结论双歧杆菌四联活菌片及其婴儿双歧杆菌通过提高小鼠腹腔巨噬细胞的吞噬率和吞噬指数提高机体的免疫力。     

双歧杆菌与辐射损伤的拮抗效应

目的探讨双歧杆菌拮抗辐射损伤的时效、量效和理想的拮抗分子。方法实验动物均选用SPF级C57BL小鼠,用不同药物和不同照射剂量处理后,建立急性放射损伤模型。WBC计数:用不同数量级浓度的双歧杆菌菌悬液对小鼠灌肠28 d后,5.0 Gy照射后3 d、6 d和10 d采取小鼠外周血检测WBC数。30 d小鼠生存率:用1×109CFU/m l双歧杆菌菌悬液灌肠28 d后,用一次性8.0 Gy60Coγ射线全身照射,建立急性放射损伤模型,每日上下午各观察小鼠1次,记录死亡动物数和死亡日期。抗氧化指标检测:用双歧杆菌菌体3种不同成分LTA、WPG和EPS,连续7 d腹腔注射小鼠后6.0 Gy照射,检测肝组织CAT和SOD的活性。结果WBC计数结果显示:双歧杆菌提高了辐射损伤WBC的最低值(P<0.05),随着剂量增加有上升趋势,理想的双歧杆菌菌悬液浓度为1×109CFU/m l。30 d小鼠生存率试验结果显示:双歧杆菌将小鼠生存率提高了43.3%。抗氧化结果显示:双歧杆菌菌体3种成分均具有抗氧化作用,尤其LTA抗氧化作用最为明显(P<0.05)。结论双歧杆菌对辐射损伤具有拮抗作用,其较为理想的双歧杆菌菌悬液浓度数量级为109CFU/m l,双歧杆菌细胞壁的3种成分中抗辐射较为理想的效应分子是LTA。     

利用乳清培养基生产乳品发酵剂的研究

通过测试保加利亚乳杆菌和嗜热链球菌在乳清培养基中的生长能力,以期获得新型乳品发酵剂工业用培养基。试验结果表明:研制的pH值内控型乳清培养基缓冲能力强,增殖效果佳:嗜热链球菌经42℃培养3h后的活菌数达到0.29×109CFU/mL,培养6h后的活菌数达到0.42×109CFU/mL。在此基础上流加20%氨水恒定pH值,保加利亚乳杆菌和嗜热链球菌的活菌数达到了1.85×109CFU/mL和3.65×108CFU/mL,分别是不加缓冲液培养的7倍和3.5倍。     

芽孢杆菌dhs-330-021菌粉的制备及稳定性研究

目的:利用喷雾干燥工艺制备芽孢杆菌dhs-330-021菌粉,并研究菌粉的活性及稳定性。方法:以脱脂乳、海藻糖、β-环糊精和谷氨酸钠为保护剂,采用喷雾干燥(条件为:进口温度100℃,出口温度50～60℃,进样速度2～4mL/min)制备芽孢杆菌菌粉,以喷干存活率和菌粉活菌数为指标,选择最佳制备条件。结果:获得喷干保护剂配方为脱脂乳10.0%、海藻糖6.0%、β-环糊精13.0%、谷氨酸钠15.0%,喷干存活率为65.9%,菌粉活菌数为1.38×109CFU/g,存放180 d后菌粉活菌数为1.03×10~9CFU/g。结论:喷雾干燥工艺可以用于芽孢杆菌dhs-330-021菌粉的制备,获得的菌粉稳定性较好。     

分离自婴儿粪便的嗜酸乳杆菌和长双歧杆菌的常温保存探究

目的:试验不同的保护剂成分配方,探究分离自婴儿粪便的2种益生菌(嗜酸乳杆菌和长双歧杆菌)在常温条件下保持较高活性的可行方法。方法:利用均匀设计实验方法设计保护剂配方,用于保存特定高活性高稳定性的菌种,在不同时间点检测活菌数,分析数据得出最佳保护剂成分比例。结果:嗜酸乳杆菌10A31保护剂最佳配方为:海藻糖∶谷氨酸∶天冬氨酸=5∶24∶24;长双歧杆菌11A11保护剂最佳配方为:海藻糖∶谷氨酸∶麦芽糖=34∶11∶10;用最佳保护剂配方保存后,嗜酸乳杆菌10A31在200 d时的菌落计数结果为3.87×109CFU/g,长双歧杆菌11A11在160 d时为1.53×109CFU/g,均具有较高的活性。结论:最佳保护剂配方使2个菌种常温保存期至160 d。     

Wine vinegar production using a noncommercial 100-litre bubble column reactor equipped with a novel type of dynamic sparger

This paper describes batch and semicontinuous acetic acid fermentations for wine vinegar production carried out with Acetobacter pasteurianus, and an industrial strain using a noncommercial 100-L bubble column reactor equipped with a novel type of gas-liquid dynamic sparger. Results showed acetification rates with this fermentor (i.e., an overall acetic acid productivity of 1. 8 g/L/h and yield of 94%) similar to that of the Frings acetator and higher as compared to others fermentors in current industrial use in Spanish wine vinegar factories, and a linear relationship between overall productivity and kLa with different operating conditions and fermentation scales. Copyright 1999 John Wiley & Sons, Inc.     

The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine

AIMS: The objective of this study was to investigate the effects of free molecular and bound forms of sulphur dioxide and oxygen on the viability and culturability of a selected strain of Acetobacter pasteurianus and a selected strain of Brettanomyces bruxellensis in wine. METHODS AND RESULTS: Acetic acid bacteria and Brettanomyces/Dekkera yeasts associated with wine spoilage were isolated from bottled commercial red wines. One bacterium, A. pasteurianus strain A8, and one yeast, B. bruxellensis strain B3a, were selected for further study. The resistance to sulphur dioxide and the effect of oxygen addition on these two selected strains were determined by using plating and epifluorescence techniques for monitoring cell viability in wine. Acetobacter pasteurianus A8 was more resistant to sulphur dioxide than B. bruxellensis B3a, with the latter being rapidly affected by a short exposure time to free molecular form of sulphur dioxide. As expected, neither of these microbial strains was affected by the bound form of sulphur dioxide. The addition of oxygen negated the difference observed between plate and epifluorescence counts for A. pasteurianus A8 during storage, while it stimulated growth of B. bruxellensis B3a. CONCLUSIONS: Acetobacter pasteurianus A8 can survive under anaerobic conditions in wine in the presence of sulphur dioxide. Brettanomyces bruxellensis B3a is more sensitive to sulphur dioxide than A. pasteurianus A8, but can grow in the presence of oxygen. Care should be taken to exclude oxygen from contact with wine when it is being transferred or moved. SIGNIFICANCE AND IMPACT OF THE STUDY: Wine spoilage can be avoided by preventing growth of undesirable acetic acid bacteria and Brettanomyces/Dekkera yeasts through the effective use of sulphur dioxide and the management of oxygen throughout the winemaking process.     

Characterization of acetic acid bacteria in traditional acetic acid fermentation of rice vinegar (komesu) and unpolished rice vinegar (kurosu) produced in Japan

Bacterial strains were isolated from samples of Japanese rice vinegar (komesu) and unpolished rice vinegar (kurosu) fermented by the traditional static method. Fermentations have never been inoculated with a pure culture since they were started in 1907. A total of 178 isolates were divided into groups A and B on the basis of enterobacterial repetitive intergenic consensus-PCR and random amplified polymorphic DNA fingerprinting analyses. The 16S ribosomal DNA sequences of strains belonging to each group showed similarities of more than 99% with Acetobacter pasteurianus. Group A strains overwhelmingly dominated all stages of fermentation of both types of vinegar. Our results indicate that appropriate strains of acetic acid bacteria have spontaneously established almost pure cultures during nearly a century of komesu and kurosu fermentation.     

Acetobacter intermedius,sp. nov

Strains of a new species in the genus Acetobacter, for which we propose the name A. intermedius sp. nov., were isolated and characterized in pure culture from different sources (Kombucha beverage, cider vinegar, spirit vinegar) and different countries (Switzerland, Slovenia). The isolated strains grow in media with 3% acetic acid and 3% ethanol as does A. europaeus, do, however, not require acetic acid for growth. These characteristics phenotypically position A. intermedius between A. europaeus and A. xylinus, DNA-DNA hybridizations of A. intermedius-DNA with DNA of the type strains of Acetobacter europaeus, A. xylinus, A. aceti, A. hansenii, A. liquefaciens, A. methanolicus, A. pasteurianus, A. diazotrophicus, Gluconobacter oxydans and Escherichia coli HB 101 indicated less than 60% DNA similarity. The important features of the new species are described. Acetobacter intermedius strain TF2 (DSM11804) isolated from the liquid phase of a tea fungus beverage (Kombucha) is the type strain.     

Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis," was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).     

乙醇脱氢酶（ADH）产酶菌株的选育

从食醋生产企业的醋醅中采集样品,以乙醇为唯一碳源,用碳酸钙透明圈平板法分离出185株菌株,然后以产酸量和耐乙醇能力为标准,瓶发酵选育出20株ADH产酶菌株;A5-2产酸量为49.85 g/L,耐乙醇能力强,A5-2的菌种形态学和16S rDNA序列分析初步鉴定为巴斯德醋酸杆菌（ Acetobacter pasteurianus）;A5-2乙醇脱氢酶酶学性质研究表明：最适作用温度和pH分别为45℃和pH 4.0,具有一定的耐热性和良好的耐酸碱性;A5-2乙醇脱氢酶粗酶制备条件为硫酸铵饱和度70%~80%,回收率84%。     

Acetobacter senegalensis isolated from mango fruits: Its polyphasic characterization and adaptation to protect against stressors in the industrial production of vinegar: A review

It has been more than a decade since Acetobacter senegalensis was isolated, identified and described as a thermotolerant strain of acetic acid bacteria. It was isolated from mango fruits in Senegal and used for industrial vinegar production in developing countries, mainly in sub-Saharan Africa. The strain was tested during several spirit vinegar fermentation processes at relatively high temperatures in accordance with African acclimation. The upstream fermentation process had significant stress factors, which are highlighted in this review so that the fermentation process can be better controlled. Due to its high industrial potential, this strain was extensively investigated by diverse industrial microbiologists worldwide; they concentrated on its microbiological, physiological and genomic features. A research group based in Belgium proposed an important project for the investigation of the whole-genome sequence of A. senegalensis. It would use a 454-pyrosequencing technique to determine and corroborate features that could give this strain significant diverse bio-industrial applications. For instance, its application in cocoa bean fermentation has made it a more suitable acetic acid bacterium for the making of chocolate than Acetobacter pasteurianus. Therefore, in this paper, we present a review that summarizes the current research on A. senegalensis at its microbial and genomic levels and also its specific bio-industrial applications, which can provide economic opportunities for African agribusiness. This review summarizes the physiological and genomic characteristics of Acetobacter senegalensis, a thermotolerant strain isolated from mango fruits and intended to be used in industrial vinegar fermentation processes. It also explores other bio-industrial applications such as cocoa fermentation. Vinegar fermentation is usually performed with mesophilic strains in temperate regions of the world. Developing countries, such as Senegal, import vinegar or make ‘fake’ vinegar by diluting acetic acid obtained from petrochemicals. The use of a thermotolerant Acetobacter senegalensis strain as a solid functional starter culture, as well as the design of a new adapted bioreactor, has significantly contributed to food security and the creation of small- to medium-sized enterprises that produce mango vinegar in West Africa.