动物微生态制剂猪肠源乳球菌的分离与鉴定

试验的菌种是从宁夏平罗县边远农村基本自然生长的健康肉猪的小肠、大肠和盲肠中分离获得的,共分离出64株菌株。通过对这64株菌株的菌落形态观察和革兰氏染色镜检。筛选出9株进行了乳球菌属的生理生化鉴定,初步确定这9株属于乳球菌属(Lactococcus)。再通过糖醇类发酵产酸鉴定,确定2株为乳酸乳球菌乳酸亚种(L.lactissubsplactis),3株为植物乳球菌(L.plantarum),4株为棉籽糖乳球菌(L.raffi-nolactis)。各项鉴定结果均符合乳球菌属和相应种鉴定标准。     

30周龄笼养海蓝褐壳蛋鸡盲肠中优良乳酸菌的筛选

目的对笼养蛋鸡盲肠中的优良乳酸菌进行筛选与鉴定。方法以30周龄笼养海蓝褐壳蛋鸡盲肠分离纯化得到10株乳酸菌为试验菌,通过发酵产酸力、耐酸性和耐胆盐的筛选试验,获得产酸能力高、对酸和胆盐有高耐受力的乳酸菌,进行生长曲线和产酸曲线及对抗生素的耐受力的测定,并采用API CHL50试剂条对选出的乳酸菌进行鉴定。结果 10株乳酸菌在发酵起始时间点时p H在6.10~6.40,经过48 h的发酵阶段,发酵液的p H明显降低;酸性环境和胆盐对各菌株存活率均有抑制。CL1、CL12和CL54生长较快,最佳生长和产酸发酵时间是24 h,CL26和CL31生长相对较慢,到32 h时生长曲线达到最高点;对抗生素的耐受力检测结果显示,CL1、CL12和CL54对抗生素的耐受力很高,而CL26和CL31对抗生素的耐受力较弱。结论对CL1、CL12和CL54这三株菌进行种属鉴定。根据API CHL50生化鉴定条说明进行接菌发酵试验,经对照鉴定,CL1为Lactococcus Lactis ssp Lactis1(乳酸乳球菌亚种1),CL12为Pediococcus Acidilactici(乳酸片球菌),CL54为Lactobacillus Brevis1(短乳杆菌1)。     

氨水中和Lactobacillus delbrueckii subsp.lactis BME5-18M发酵生成L-乳酸铵的研究

实验确定了Lactobacillus delbrueckiisubsp.lactis BME5-18M接种的最佳种龄为24h.以氨水取代传统的中和剂碳酸钙中和发酵生成的乳酸、调控发酵液的pH,考察了不同pH值对菌体生长和产酸的影响,确定了菌种生长和产酸的较适pH值为6.5.考察了底物流加速度对菌种生长和产酸的影响,对间歇和流加发酵时菌体的生长量和产酸量进行了动力学关联.在较适pH值6.5和较佳流加速度25mL/h条件下,乳酸的产量可达到136.8g/L,产率为1.71 g/(L·h).     

产D-乳酸重组大肠杆菌ptsG基因的敲除及其混合糖同步发酵

为构建能够同时高效利用五碳糖和六碳糖发酵产D-乳酸的重组大肠杆菌工程菌,以能高效利用五碳糖发酵产D-乳酸的大肠杆菌工程菌E.coli JH13为出发菌株,通过Red同源重组技术敲除葡萄糖跨膜转运基因pts G。实验结果表明,pts G缺陷菌株E.coli JH15在10%混合糖(5%葡萄糖和5%木糖)培养基中发酵,可同时利用五碳糖和六碳糖以完成发酵;而对照菌葡萄糖消耗完才利用木糖,发酵结束还有18 g/L木糖残留;JH15乳酸产量为83.04 g/L,相比于对照菌株提高了25.86%;在稻草秸秆水解液中发酵,JH15同时利用葡萄糖、木糖和L-阿拉伯糖,乳酸产量为25.15 g/L,转化率为86.42%。JH15作为能利用混合糖同步发酵产D-乳酸的大肠杆菌工程菌,它的成功构建为利用廉价的木质纤维素水解物为原料发酵生产D-乳酸提供参考依据。     

氨水中和Lactobacillus delbrueckiisubsp. lactis BME5-18M发酵生成L-乳酸铵的研究*

实验确定了Lactobacillusdelbrueckiisubsp lactisBME5-18M接种的最佳种龄为24h。以氨水取代传统的中和剂碳酸钙中和发酵生成的乳酸、调控发酵液的pH ,考察了不同pH值对菌体生长和产酸的影响 ,确定了菌种生长和产酸的较适pH值为 6.5。考察了底物流加速度对菌种生长和产酸的影响 ,对间歇和流加发酵时菌体的生长量和产酸量进行了动力学关联。在较适pH值 6.5和较佳流加速度 25mL/h条件下 ,乳酸的产量可达到 136.8g/L ,产率为1.     

乳酸乳球菌表达重组牛乳铁蛋白肽的抑菌活性分析

牛乳铁蛋白肽是由牛乳铁蛋白经消化酶水解产生的一类具有广谱抑菌活性的短肽;乳酸乳球菌作为食品级微生物,既有天然的益生作用,又是理想的表达牛乳铁蛋白肽的载体。【目的】探究重组乳酸乳球菌pAMJ399-LFcinBA/MG1363表达牛乳铁蛋白肽的抑菌活性。【方法】利用牛乳铁蛋白肽标准品绘制定量标准曲线来确定重组牛乳铁蛋白肽的含量,利用牛津杯法及微量肉汤稀释法测定重组牛乳铁蛋白肽对大肠杆菌、金黄色葡萄球菌等35株细菌的抑菌活性及最小抑菌浓度,利用扫描电镜、透射电镜、荧光显微镜、凝胶阻滞试验、黏附试验来探究重组牛乳铁蛋白肽对菌体结构、细菌DNA及黏附力的影响,利用CCK-8检测其对RAW 264.7细胞的毒性作用,并对小鼠红细胞溶血率进行测定。【结果】重组乳酸乳球菌上清中牛乳铁蛋白肽的浓度为24.39μg/mL,重组牛乳铁蛋白肽对测试的25株致病菌均有不同程度的抑制作用,抑菌浓度范围在16–128μg/mL,但对9株乳酸菌以及粪肠球菌没有明显的抑制作用,对大肠杆菌、金黄色葡萄球菌、多杀性巴氏杆菌、鸡白痢沙门菌的菌体完整性具有不同程度的破坏作用,其主要作用靶点为细菌的细胞膜,可以与细菌DNA结合...     

氨水中和Lactobacillus delbrueckii subsp．1actis BME5-18M发酵生成L-乳酸铵的研究

实验确定了Lacobacillus delbrueckii subsp．lactis BME5-18M接种的最佳种龄为24h。以氨水取代传统的中和剂碳酸钙中和发酵生成的乳酸、调控发酵液的pH,考察了不同pH值对菌体生长和产酸的影响,确定了菌种生长和产酸的较适pH值为6．5。考察了底物流加速度对菌种生长和产酸的影响,对间歇和流加发酵时菌体的生长量和产酸量进行了动力学关联。在较适pH值6．5和较佳流加速度25mL／h条件下,乳酸的产量可达到136．8g／L,产率为1．71g/(L．h)。     

重组大肠杆菌利用废纸水解液发酵产L-乳酸

前期通过基因工程手段,构建了一株大肠杆菌工程菌E.coli WL204,该菌株可以有效利用木糖为底物发酵产L-乳酸。以废纸为发酵原料,研究该菌株利用木质纤维素发酵产乳酸的特性。原料以稀硫酸预处理后,经纤维素酶酶解,得到的水解液用Ca(OH)2脱毒后,接种E.coli WL204,在7L发酵罐中发酵72h,每100g废纸可以产生31g乳酸,糖酸转化率为79%。结果表明,E.coli WL204可以木质纤维素原料为底物发酵生产L-乳酸,具有一定的工业化开发潜力。     

利用五碳糖产高纯度L-乳酸的大肠杆菌基因工程菌的构建

[目的]本研究以已敲除多个产杂酸酶基因的大肠杆菌(Escherichia coli)乙醇工程菌SZ470(△frdBC △ldhA △ackA △focA-pflB △pdhR::pflBp6-pflBrbs-aceEF-lpd)为起始菌株,进一步敲除其乙醇脱氢酶(alcohol dehydrogenase,ADH)基因,同时插入带有自身启动子的乳酸片球菌(Pediococcus acidilactici)的L-乳酸脱氢酶(L-lactate dehydrogenase,LLDH)基因,构建可利用五碳糖同型发酵L-乳酸重组大肠杆菌.[方法]利用λ噬菌体Red重组系统构建乙醇脱氢酶基因(adhE)缺失菌株Escherichia coli JH01,并克隆P.acidilactici的ldhL基因,利用染色体插入技术将其整合到JH01基因组,构建产L-乳酸大肠杆菌基因工程菌Escherichia coli JH12,利用无氧发酵15 L发酵罐测定重组菌株L-乳酸产量.[结果]工程菌JH12在15 L发酵罐中以6％的葡萄糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为1.46 g/(L·h),乳酸生产强度为1.14 g/(L·h),乳酸的产量达到41.13 g/L.发酵产物中未检测到琥珀酸、甲酸的生成,仅有少量乙酸生成,L-乳酸纯度达95.69％(L-乳酸在总发酵产物的比率).工程菌JH12以6％的木糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为0.88 g/(L·h),乳酸生产强度为0.60 g/(L·h),乳酸的产量达到34.73 g/L.发酵产物中杂酸少,乳酸的纯度高达98％.[结论]本研究通过基因敲除、染色体插入及无氧进化筛选获得一株产L-乳酸的大肠杆菌工程菌JH12,该菌株不需利用外源质粒,稳定性好,可利用五碳糖进行发酵,发酵产物中杂酸少,L-乳酸的纯度高.本研究为L-乳酸大肠杆菌工程菌的构建提供一定的技术支持,同时也为大肠杆菌L-乳酸的工业化生产提供了参考依据.     

薏苡仁多菌发酵液的菌种优选及其抑制黑色素生成的作用研究

以薏苡仁作为发酵基质,确定利于提高发酵液体外活性的较优乳酸菌种,并分析优势乳酸菌种薏苡仁发酵液对斑马鱼胚体黑色素生成的抑制作用。通过比较分析乳酸乳球菌（Lactococcus lactis）、嗜热链球菌（Streptococcus thermophilus）和保加利亚乳杆菌（Lactobacillus bulgaricus）3种单一乳酸菌和三者复合乳酸菌的薏苡仁发酵液的还原糖、总酚、游离氨基酸、蛋白、总酸和乳酸含量等理化指标及体外羟自由基清除能力和酪氨酸酶活抑制率确定较优发酵菌种,采用高通量测序测定发酵过程中微生物菌群结构;利用斑马鱼模型研究发酵液对黑色素生成的抑制作用。研究结果表明,采用乳酸乳球菌、嗜热链球菌和保加利亚乳杆菌3种乳酸菌复合发酵比单一乳酸菌发酵更具优势。使用以上菌种复合发酵薏苡仁过程中,乳酸乳球菌和嗜热链球菌为发酵前期优势菌群,发酵中后期则以保加利亚乳杆菌为优势菌群。经复合乳酸菌发酵后,薏苡仁发酵液的羟自由基清除率和酪氨酸酶活抑制率分别提高了20.82%和87.26%;斑马鱼模型实验结果表明,薏苡仁发酵液可以显著减少斑马鱼体表黑色素分布,当使用含量为2.0%时,对黑色素...     

Shear treatment of starter culture medium improves separation behavior of Streptococcus thermophilus cells

A central step in the production of starter cultures is the separation of the cells from the fermentation medium, which is usually achieved by disk centrifuges. In case of microorganisms which produce exopolysaccharides (e.g., various strains of lactic acid bacteria), the properties of the respective exopolysaccharides may interfere with this separation step. By using six strains of Streptococcus thermophilus the hypothesis was tested that a shear treatment of the fermented culture medium improves subsequent cell separation markedly. Depending on the type of exopolysaccharides (freely present in the medium, or as capsules around the cells) an energy input of up to 2.5 kJ/mL generated with an Ultra‐Turrax affected cell chain length of the strains and viscosity of fermentation medium differently. For bacteria producing capsular exopolysaccharides, space‐ and time‐resolved centrifugation experiments revealed an increase of sedimentation velocity after shear treatment. In general, viability of the microorganisms, detected by flow cytometry measurements and fermentation experiments, was not affected by the shearing procedure. The results therefore indicate that strain‐targeted shearing is helpful to improve the separability of cells from the fermented media.     

酸奶弱后酸化发酵剂乳酸菌育种技术研究进展

后酸化是影响酸奶贮藏期间感官品质和风味特性的重要因素,是决定产品货架期的重要指标。弱后酸化发酵剂菌种的选育是优良发酵剂开发的重要技术手段。通过诱变育种筛选出具有弱后酸化特性的菌株,是延缓酸奶贮藏期间后酸化的技术途径之一。国内外围绕弱后酸化发酵菌株选育,采用诱变育种方法开展了大量卓有成效的研究。本研究针对诱变育种的方法、原理与研究现状,结合本课题组近几年关于发酵剂菌种弱后酸化的研究工作,对弱后酸化乳酸菌菌种的诱变育种进行了简要阐述与展望。     

Influence of encapsulation on the survival of probiotics in food matrix under simulated stress conditions

The main objective of the present study was to evaluate the influence of encapsulation by extrusion technique using two hydrogels, namely; sodium alginate (Na-ALG) and whey protein isolate (WPI) on Bifidobacterium bifidium viability and stability of yoghurt under simulated gastrointestinal conditions. Probiotic bacteria (free or encapsulated) were added to yogurt for four weeks to test their viability and stability. Physicochemical and sensory analysis of yoghurt were conducted. Viability of B. bifidium in the simulated gastrointestinal conditions pH 2 and pH 7.5 was determined. Also, the efficiency of encapsulated final yield of the microcapsules was determined. With storage time, the pH of yoghurt containing encapsulated bacteria increased more than that of yoghurt containing free probiotic bacteria, resulting in a decrease in acidity. When compared to yoghurt containing encapsulated bacteria, the lactose level of yoghurt containing free probiotic bacteria decreased over time. The viscosity of yoghurt containing encapsulated WPI remained stable over the storage period, with syneresis remaining stable. The sensory properties of yoghurt containing free probiotics deteriorated over time. Cell viability was significantly reduced in yoghurt-containing free probiotics compared to other treated yoghurts. Cell viability in free probiotics yoghurt was lower than in encapsulated ones when exposed to simulated gastric and intestinal juice. In conclusion, WPI- encapsulated probiotics showed better stability over 28 days of storage in both yoghurt and gastrointestinal conditions, followed by sodium alginate.     

甜瓜黑斑病菌的拮抗细菌筛选、鉴定及发酵条件优化

从健康甜瓜植株的根、茎、叶和果实分离内生细菌,明确优良拮抗菌株的分类地位及其最优发酵条件。利用平板稀释法分离内生细菌,平板对峙法筛选黑斑病菌(Alternaria tenuissima)的优良拮抗细菌,通过形态学和分子生物学鉴定,并使用单因素和正交试验方法优化发酵条件。分离共得到50株内生细菌,21株对细极链格孢具有拮抗作用,11株的抑制率达到70%以上,其中菌株G2的抑菌率最高,达76.41%,室内离体防效达44.90%,经形态特征和基因序列分析鉴定为枯草芽孢杆菌(Bacillus subtilis),其最优发酵培养基为葡萄糖20 g、硝酸钠30 g、酵母膏10 g和水1 000 mL,最优发酵条件为摇床转速150 r/min,温度32℃,150 mL摇瓶装液50 mL,最佳接种量为0.2%,培养基初始pH为7.5,其最佳发酵时间为18 h。该结果为甜瓜黑斑病生物防治提供了菌种资源,也为甜瓜内生细菌G2的产业化开发提供了依据。     

Lactic acid production by mixed cultures of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus helveticus

Lactic acid production using Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) individually or as mixed culture on cheese whey in stirred or static fermentation conditions was evaluated. Lactic acid production, residual sugar and cell biomass were the main features examined. Increased lactic acid production was observed, when mixed cultures were used in comparison to individual ones. The highest lactic acid concentrations were achieved when K. marxianus yeast was combined with L. delbrueckii ssp. bulgaricus, and when all the strains were used revealing possible synergistic effects between the yeast and the two lactic acid bacteria. The same synergistic effects were further observed and verified when the mixed cultures were applied in sourdough fermentations, proving that the above microbiological system could be applied in the food fermentations where high lactic acid production is sought.     

Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages

J.M. RODRÍGUEZ, L.M. CINTAS, P. CASAUS, N. HORN, H.M. DODD, P.E. HERNÁNDEZ AND M.J. GASSON. 1995. A total of 4608 lactic acid bacteria (LAB) were isolated from 24 Spanish fermented sausages and screened for bacteriocin production. Two strains, BB24 and G18, produced bacteriocins that inhibited a broad spectrum of Gram-positive bacteria. BB24 and G18 were tentatively identified as Lactococcus lactis by carbohydrate fermentation patterns and other biochemical characteristics. The characterization of their bacteriocins suggested that both could be the well-known lantibiotic nisin. This was confirmed by PCR analysis of their genomic DNA. Nucleotide sequencing revealed that they produced nisin A. The fact that BB24 and G18 were isolated from sausages produced in two different regions of Spain suggests that nisin-producing L. lactis strains may be more widespread in meat products than previously thought. Nisin produced by L. lactis BB24 has been purified to homogeneity by a procedure that included ammonium sulphate precipitation and cation-exchange, hydrophobic-interaction and reverse-phase chromatography. The purification procedure was simple, rapid and reproducible.     

Fermentation conditions affecting the bacterial growth and exopolysaccharide production by Streptococcus thermophilus ST 111 in milk-based medium

AIMS: To study the effect of different fermentation conditions and to model the effect of temperature and pH on different biokinetic parameters of bacterial growth and exopolysaccharides (EPS) production of Streptococcus thermophilus ST 111 in milk-based medium. METHODS AND RESULTS: The influence of temperature and pH was studied through fermentation and modelling. Fermentations under non-pH controlled conditions with S. thermophilus ST 111 indicated that the EPS production was low in milk medium, even if additional nitrogen sources were supplemented. Under pH-controlled conditions, addition of whey protein hydrolysate to the milk medium resulted in a fivefold increase of the EPS production. This medium did not contain polysaccharides interfering with EPS isolation. Primary and secondary modelling of different fermentations revealed an optimum temperature and pH of 40 degrees C and constant pH 6.2, respectively, for growth in milk medium supplemented with whey protein hydrolysate. Maximum EPS production was observed in the range of 32-42 degrees C and constant pH 5.5-6.6. Whereas growth and maximum EPS production were clearly influenced by temperature and pH, the specific EPS production was only affected by stress conditions (T = 49 degrees C). CONCLUSIONS: Addition of whey protein hydrolysate to milk medium resulted in an increased growth and EPS production of S. thermophilus ST 111 under pH-controlled conditions. A modelling approach allowed studying the influence of temperature and pH on the kinetics of both growth and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an appropriate milk-based medium and a combined model of temperature and pH can be of practical importance for the production of yoghurt or other fermented milks as well as for process optimization of the large-scale production of starter strains to be used for their EPS production.     

Safety evaluation of sous vide-processed ready meals

AIMS: To assess survival, growth and toxin production of spore-forming bacteria in sous vide products exposed to a relatively high heat treatment. METHODS AND RESULTS: During a three-year period, 2,168 sous vide-processed, commercially available ready-made meals with a shelf life of 3-5 weeks were examined. The products were stored at 4 degrees C for the first 1/3 and at 7 degrees C for the remaining 2/3 of their shelf life period. Three-fourths of the samples had less than 10 bacteria per gram the day after production, and none had more than 1,000. Similar numbers were found at the end of the shelf life when stored as described above. At abuse temperature (20 degrees C), the number of bacteria increased to 10(6)-10(7) cfu g(-1) 7 d after production. A total of 350 isolates of Bacillus spp. were collected, but no Clostridium strains were detected. Only 11 of the 113 tested strains were able to grow at 7 degrees C in broth, and none of the psychrotrophic strains were able to produce substantial amounts of toxins causing food poisoning. CONCLUSION: The health risk of these products is small as long as the temperature during storage is low. For microbial testing of the end products, traditional plating will suffice.     

Use of RAPD and 16S rDNA sequencing for the study of Lactobacillus population dynamics in natural whey culture

The development of communities of the thermophilic microflora of natural whey culture for Parmigiano Reggiano cheese production was studied by means of molecular techniques. RAPD analysis facilitates the identification of the Lactobacillus strains involved in this microbial association and permitted the study of population dynamics during two cycles of whey fermentation. Analysis of RAPD fingerprints revealed the presence of four biotypes that dominate the whey fermentation process. Sequence analysis of 16S rDNA demonstrated that the strains isolated from whey belong to Lact. helveticus and Lact. delbrueckii ssp. lactis species.     

Detection of interactions between yeasts and lactic acid bacteria isolated from sugary kefir grains

Different techniques were tested for studying the synergism between the micro-organisms of sugary kefir grains. Agar cultures in Petri dishes did not give reproducible results. In sequential cultures, i.e. growing one organism, sterile filtering and then inoculating the other, 10 of 18 selected lactic acid bacteria/yeast pairs revealed stimulation of bacterial growth in a poor glucose medium. In mixed culture, Saccharomyces florentinus supported better survival of Lactobacillus hilgardii and a significant increase in lactic acid production; at the same time, the growth and alcoholic fermentation of S. florentinus were drastically reduced. The inter-relationships between these two strains were the same when immobilized in calcium alginate beads, even though total metabolite production was always lower than with free cells. The stimulation of Lact. hilgardii by Candida lambica in sequential culture was not confirmed in mixed culture, where the two organisms grew as in pure culture, and bacterial growth and lactic acid production were inhibited in the immobilized system.