瑞士乳杆菌LH-G51冻干菌粉生产工艺研究

目的：对瑞士乳杆菌LH-G51菌粉生产工艺进行优化。方法：通过单因素及正交实验设计,确定适合LH-G51生长的发酵培养基、冻干保护剂配方及生产工艺。结果：发酵时间、碳氮源及微量元素等添加量均对LH-G51生长有明显影响。最终确定LH-G51培养基碳氮源及微量元素为：葡萄糖20 g/L,大豆蛋白胨10 g/L,酵母膏15 g/L,MgSO4·7H2O为250 mg/L,MnSO4·H2O为50 mg/L,发酵时间10 h,保护剂为B。结论：根据优化工艺,LH-G51冻干菌粉活菌数可达到4.21×1011 CFU/g。     

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。     

L-色氨酸基因工程菌发酵培养基配方优化研究

[目的]对L-色氨酸基因工程菌液态发酵的培养基进行优化。[方法]通过P-B试验,筛选出对基础发酵培养基的发酵液中L-色氨酸浓度影响显著的因素,进一步通过最陡爬坡试验、B-B试验对影响显著的因素进行优化。在此基础上,确定最佳的发酵培养基配方。[结果]酵母粉、Fe SO4·7H2O、KH2PO4对发酵液中L-色氨酸浓度的影响显著;最佳培养基配方为:Glucose 25.0 g/L,酵母粉4.5 g/L,(NH4)2SO49.0 g/L,Mg SO44.5 g/L,柠檬酸钠2.0 g/L,Fe SO4·7H2O 96.1 m g/L,KH2PO41.2 g/L。[结论]根据此配方进行验证实验,发酵液中L-色氨酸浓度可达2.25 g/L。     

特基拉芽胞杆菌B05高密度发酵培养基与发酵条件优化

特基拉芽胞杆菌(Bacillus tequilensis)B05是1株能显著提高小麦、玉米耐盐、耐旱能力的根际优良菌株,为提高其扩繁效率及使用效果,降低使用成本,以发酵液活菌数为指标,采用单因子预筛选和正交试验方法对菌株发酵培养基及发酵条件进行优化,在10 L发酵罐水平上进行扩大培养。结果表明,菌株B05最适发酵培养基(质量与体积分数)为麦芽浸粉1%、酵母浸粉2%、胰蛋白胨2%、复合无机盐0.3%(质量比为KH_2PO_4:K_2HPO_4:MnSO_4·H_2O=1:1:1)。最佳发酵培养条件为培养温度30℃、初始pH 6.5、摇床转速200 r/min、接种量7%、装液量40%,培养18 h菌液活菌数可达9.3×10~(10) cfu/mL,较优化前活菌数量提高了两个数量级。在含盐0.4%的盐碱土壤上进行了小麦盆栽试验,较空白对照植株生物量提高45.8%。     

猪源屎肠球菌WEI-9的高密度培养

目的对分离自健康仔猪肠道的屎肠球菌(Enterococcus faecium)WEI-9的高密度发酵培养基进行响应面优化,为菌株WEI-9的工业化生产奠定基础。方法首先采用单因素试验确定最适高密度发酵培养基的碳源和氮源,随后采用Plackett-Burman设计筛选出影响菌株WEI-9发酵活菌数的显著因素,利用最陡爬坡试验得出显著因素逼近最大活菌数产量的响应区域,最后应用Box-Behnken设计和响应面分析法确定显著影响因子的最佳浓度。结果优化后的最适高密度发酵培养基成分和配比为:乳清粉21.34 g/L,蛋白胨21.94 g/L,Na AC·3H2O 5 g/L,柠檬酸铵2 g/L,K2HPO4·3H2O 2 g/L,Mg SO4·7H2O 0.2 g/L,Mn SO4·H2O 0.05 g/L,吐温-80 1 g/L,发酵液最高活菌数达到1.6×109CFU/m L,是相同条件下MRS培养基中活菌数的1.98倍。结论本研究实现了猪源屎肠球菌(Enterococcus faecium)WEI-9的高密度培养。     

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。     

发酵中含氮类原料对保加利亚乳杆菌活菌数的影响

目的研究生产发酵过程中培养基含氮原料与发酵的相关性。方法实验通过对不同生产厂家三种原料进行理化指标检测,并通过正交试验,比较不同原料发酵后保加利亚乳杆菌发酵液活菌数。结果最佳条件为酪蛋白胨氨基氮含量高,酵母浸粉的炽灼残渣低,牛肉粉总氮含量高。结论经过相同原料不同生产厂家的配方发酵后的发酵液活菌数存在差异,其理化指标对发酵有一定影响。     

构巢曲霉冻干菌粉的制备及优化

【背景】目前,用以降解园林绿化废弃物中木质素的菌剂多为液体菌剂或固体菌剂,鲜有对粉状菌剂的研究。【目的】研制高活性冻干菌粉,提高其冻干存活率并优化其工艺,以解决液体菌剂或固体菌剂在运输、储藏及使用上存在的问题。【方法】以一株木质素降解菌构巢曲霉(Aspergillus nidulans)为研究对象,利用真空冷冻干燥法制备冻干菌粉。以菌株的冻干存活率为评价指标,通过单因素试验筛选适于菌株冻干过程的保护剂种类及浓度梯度,再通过正交试验优化冻干菌粉复合保护剂配方。获得配方后,进一步探究冻干菌粉的复水条件和储藏条件。【结果】保护效果较优的4种保护剂成分经复配后对冻干存活率的影响顺序为蔗糖>葡萄糖>脱脂乳粉> α-乳糖。经优化后的保护剂配方以蔗糖15%、葡萄糖1%、α-乳糖10%、脱脂乳粉1%为最佳;复水条件以生理盐水为溶剂,复水30 min为最优。在此条件下制备和使用冻干菌粉,菌株的冻干存活率可达83.33%,有效活菌数可达1.2×10¹⁰ CFU/g。最佳储藏温度为-20 ℃,在此温度下保存28 d后,菌粉活性无明显下降。【结论】该研究获得的制备和储藏构巢曲霉冻干菌粉条件,具有菌株损失率低、可长时间保存的特点,对推进木质素降解菌在实际生产中应用具有积极作用。     

极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶发酵条件的优化

对极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶的发酵条件进行优化。结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶。正交试验结果表明:菌株Pseudoalteromonassp. QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2.6H2O 3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%。     

饲用凝结芽孢杆菌高密度发酵及高纯度菌粉的制备

对凝结芽孢杆菌的发酵工艺进行了研究,150 L发酵罐发酵44 h,最终发酵液活菌数达到了2.01×1010cfu/m L,芽孢形成率达到96.82%。3 m3罐中试,发酵时间为44 h,发酵液中的活菌数达到2.10×1010cfu/m L,芽孢形成率达到91.70%。发酵液离心后,收集菌液进行喷雾干燥以收获菌粉(进风温度160℃,出风温度80℃),菌粉收率为2.50%,芽孢收率86.07%。     

Production of probiotic cabbage juice by lactic acid bacteria

Research was undertaken to determine the suitability of cabbage as a raw material for production of probiotic cabbage juice by lactic acid bacteria (Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7). Cabbage juice was inoculated with a 24-h-old lactic culture and incubated at 30 degrees C. Changes in pH, acidity, sugar content, and viable cell counts during fermentation under controlled conditions were monitored. L. casei, L. delbrueckii, and L. plantarum grew well on cabbage juice and reached nearly 10x10(8) CFU/mL after 48 h of fermentation at 30 degrees C. L. casei, however, produced a smaller amount of titratable acidity expressed as lactic acid than L. delbrueckii or L. plantarum. After 4 weeks of cold storage at 4 degrees C, the viable cell counts of L. plantarum and L. delbrueckii were still 4.1x10(7) and 4.5x10(5) mL(-1), respectively. L. casei did not survive the low pH and high acidity conditions in fermented cabbage juice and lost cell viability completely after 2 weeks of cold storage at 4 degrees C. Fermented cabbage juice could serve as a healthy beverage for vegetarians and lactose-allergic consumers.     

植物乳杆菌Lp-2的高密度发酵

高密度培养植物乳杆菌是制作其发酵剂的重要环节。首先,研究了不同的溶氧和pH对植物乳杆菌的分批发酵的影响。在分批发酵的基础上,为进一步提高发酵液中的菌体浓度,进行了补料分批发酵实验。最终通过对蔗糖反馈补料发酵试验对比改造获得了pH反馈补料发酵工艺。此发酵补料工艺可以控制蔗糖残糖量始终处于较低的水平,因此获得了最高的菌体产量。菌体干重达到13.56g/L,较分批培养提高90.05%。     

Development of a quantitative PCR for detection of Lactobacillus plantarum starters during wine malolactic fermentation

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at 3.6 × 10(6) CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above 10(5)/ml for approx. 10 days, after which cell numbers decreased to levels of 10(3) CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. 1 × 10(2) CFU/ml was detected. The minimum detection level for quantitative PCR in this study was 1 × 10(2) to 1 × 10(3) CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.     

微贮用植物乳杆菌固定化菌剂的制备

乳酸菌与纤维素降解菌因其可防止微贮饲料酸败、增加秸秆饲料的营养价值等优点,在秸秆微贮过程中起重要作用。但由于乳酸菌的繁殖会抑制纤维素降解菌的活性,如何实现微贮过程中两种微生物分时发挥功能是解决上述问题的关键。文中利用固定化技术将乳酸菌制备成含有玉米秸秆粉的固定化菌剂以达到缓释的目的。首先制作固定化空白小球得出复合固定化载体成球的最佳浓度,利用玉米芯吸附植物乳杆菌S1得到复合固定化载体,以对S1的包埋率、成球效果等为指标,通过对比两种固定化方法 (包埋法与包埋-交联法),得到固定化植物乳杆菌S1的最佳条件。研究表明,使用6%PVA+0.4%SA+0.3%CMC-Na进行包埋-交联时成球效果最好,使用1.2%SA+0.5%CMC-Na进行直接包埋时成球效果最好。通过对比5种固定化工艺,将1.2%SA+0.5%CMC-Na和吸附玉米粉组成的固定化载体混合物逐滴滴入4%氯化钙中直接包埋24 h得到的固定化小球其机械强度以及包埋率均优于其他工艺。因此,利用玉米芯吸附-海藻酸钠包埋的方法可以有效提高植物乳杆菌包埋效率,为使用固定化技术制备微贮饲料菌剂奠定基础。     

来自詹氏乳杆菌的耐热D-乳酸脱氢酶的酶学性质研究

【目的】D-乳酸脱氢酶是催化丙酮酸合成D-乳酸的关键酶。由于其不耐热,从而限制了D-乳酸高温发酵菌株的构建。本文从詹氏乳杆菌中克隆新型D-乳酸脱氢酶研究其酶学性质,为构建D-乳酸高温发酵菌株,进一步降低D-乳酸生产成本奠定基础。【方法】通过克隆詹氏乳杆菌的D-乳酸脱氢酶,将其进行体外表达,并与来自植物乳杆菌中的D-乳酸脱氢酶的最适温度、最适pH、动力学参数及热稳定性和热失活性相比较,研究詹氏乳杆菌D-乳酸脱氢酶的耐热性。【结果】詹氏乳杆菌的D-乳酸脱氢酶最适温度(45 °C)比植物乳杆菌中的D-乳酸脱氢酶的最适温度(30 °C)高很多,热失活的时间和温度均要比植物乳杆菌中D-乳酸脱氢酶高很多。同时其催化效率(kcat/Km)是植物乳杆菌D-乳酸脱氢酶的3倍左右。【结论】詹氏乳杆菌的D-乳酸脱氢酶具有更好的耐热性和更高的催化活力。     

Response Surface Optimization of Lyoprotectant for Lactobacillus bulgaricus During Vacuum Freeze-Drying

The individual and interactive effects of skimmed milk powder, lactose, and sodium ascorbate on the number of viable cells and freeze-drying survival for vacuum freeze-dried powder formulation of Lactobacillus bulgaricus were studied by response surface methodology, and the optimal compound lyoprotectant formulations were gained. It is shown that skim milk powder, lactose, and sodium ascorbate had a significant impact on variables and survival of cultures after freeze-drying. Also, their protective abilities could be enhanced significantly when using them as a mixture of 28% w/v skim milk, 24% w/v lactose, and 4.8% w/v sodium ascorbate. The optimal freeze-drying survival rate and the number of viable cells of Lactobacillus bulgaricus were observed to be (64.41 ± 0.02)% and (3.22 ± 0.02) × 10¹¹ colony-forming units (CFU)/g using the optimal compound protectants, which were very close to the expected values 64.47% and 3.28 × 10¹¹ CFU/g.     

Combined effect of alkali pretreatment and sodium chloride addition on the olive fermentation process

Green olives of the Tunisian variety "Meski" were treated according to a Spanish-style green olive preservation process by using an alkaline treatment (1.5, 2 and 2.5% (w/v) NaOH) to eliminate bitterness, combined with different brine concentrations (6, 9 and 12% (w/v) NaCl). A spontaneous fermentation by the environmental microflora took place. Results showed that 2% NaOH solution and 9% sodium chloride brine was an optimal combination inducing the best growth of Lactobacillus species (10(8) CFU/ml) and acidity of 0.726 g lactic acid/100 ml brine. In all trials and independently of the treatment, Lb. plantarum was the most dominant strain of Lactobacillus. Moreover, pretreatment with lye and lactic fermentation of olives contributed to coliform elimination.     

益生乳酸菌Lactobacillus casei Zhang和Lactobacillus plantarum P8对全价饲料pH及微生物类群变化的研究

研究了益生乳酸菌干酪乳杆菌Zhang(Lactobacillus casei Zhang)和植物乳杆菌P8(Lactobacillus planta-rum P8)对全价饲料pH及微生物类群变化的影响。分别将L.casei Zhang、L.plantarum P8单一菌种及复合菌种(11)以6.30 lg cfu/g的接种总量发酵全价饲料,测定25℃10 d发酵期间全价饲料pH和微生物类群的变化,应用选择培养基测定发酵饲料中的乳酸菌及杂菌(酵母菌、霉菌、大肠菌群、芽胞杆菌和梭状芽胞杆菌)的动态变化,应用RT-PCR技术测定试验组中的L.casei Zhang和L.plantarum P8的动态变化。结果显示,试验组pH下降显著,发酵10 d时,L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料的pH分别为4.23、4.24和4.22,显著低于对照组(P0.05);L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料中的L.casei Zhang、L.plantarum P8活菌数分别为8.91、8.89、6.58和8.69 lg cfu/g。发酵期间,试验组中酵母菌、霉菌、大肠菌群、芽胞杆菌及梭状芽胞杆菌活菌数显著低于对照组(P0.05),其中L.plantarum P8单一菌种发酵和复合菌种发酵对杂菌抑制效果显著优于L.casei Zhang单一菌种发酵(P0.05)。结果表明,全价饲料经L.casei Zhang、L.plantarum P8发酵可以显著降低其pH,抑制其中杂菌的生长,同时L.casei Zhang、L.plantarum P8在饲料中具有良好的稳定性。     

Modeling of lactic acid fermentation of leguminous plant juices

Lactic acid fermentation of leguminous plant juices was modeled to provide a comparative efficiency assessment of the previously selected strains of lactic acid bacteria as potential components of starter cultures. Juices of the legumes fodder galega, red clover, and alfalfa were subjected to lactic acid fermentation in 27 variants of experiment. Local strains (Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, and Lactobacillus sp. RS 4) and the collection strain Lactobacillus plantarum BS 933 appeared the most efficient (with reference to the rate and degree of acidogenesis, ratio of lactic and acetic acids, and dynamics of microflora) in fermenting fodder galega juice; Lactobacillus sp. RS 1, Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, Lactobacillus sp. RS 4, and L. plantarum BS 933 were the most efficient for red clover juice. Correction of alfalfa juice fermentation using the tested lactic acid bacterial strains appeared inefficient, which is explainable by its increased protein content and a low level of the acids produced during fermentation.     

水稻秸秆低温复合菌系多样性及发酵动态

【目的】为解决中国寒冷地区水稻秸秆大面积废弃问题,加快低温地区水稻秸秆饲料转化,本文筛选了可以低温下加速秸秆发酵过程的微生物复合菌系,研究其微生物组成并跟踪其发酵动态。【方法】通过5℃下连续定向富集筛选,获得低温复合菌系。采用克隆文库方法分析复合菌系的组成。将复合菌系和商业接种剂(由Lactobacillus plantarum,Enterococcus faecium,L.salivarilus,Pediococcus acidilactici组成)分别接入稻秸进行10℃发酵。气质联机(GC-MS)测定发酵产物的同时,通过变性梯度凝胶电泳检测微生物在发酵体系的定殖情况。采用定量PCR方法追踪复合菌系组成菌在发酵过程中的动态。【结果】16S rDNA克隆文库分析结果表明复合系主要由两种微生物组成,一种属乳酸杆菌(Lactobacillus),一种属乳酸球菌(Leuconostoc)。10℃稻秸发酵结果表明,在发酵第6天接种复合菌系处理的pH已经下降到4.3,乳酸菌菌落形成单位为2.9×109CFU/g鲜样,而接种商业接种剂的处理pH为5.3,乳酸菌菌落形成单位为3.6×108 CFU/g鲜样;在发酵30 d时,接种复合菌系处理的乳酸含量为8.1 g/kg鲜样,接种商业接种剂处理的乳酸含量为2.0 g/kg鲜样。变性梯度凝胶电泳结果表明,在接种复合菌系的稻秸中,从发酵的第6天开始,检测到的微生物主要为L.sakei和Leuconostoc inhae,在整个发酵过程中,两菌一直存在;在商业接种剂处理中,发酵第6天检测到的微生物除其四种组成菌外,还包括Uncultured bacterium;而在发酵第16天和第30天,只检测到组成菌中的L.plantarum和E.faecium。定量PCR结果显示,接种复合菌系处理中,L.sakeiDNA在发酵第6天达到41.0%,在发酵第16天已达到65%,Le inhae在发酵的第6天达到整个发酵过程中的最大值(5.5%)。【结论】接种复合菌系,可以有效促进水稻秸秆的低温发酵进程。复合菌系组成菌可以定殖在发酵体系中,并占据优势。复合菌系的关键菌为L.sakei。