马铃薯淀粉同步糖化发酵制备L-乳酸条件的统计学优化

以马铃薯淀粉为原料,采用同步糖化发酵方法制备乳酸。通过Plackett-Burman实验设计对影响乳酸产量的7个因子进行筛选,结果表明淀粉质量浓度、糖化酶用量和发酵温度3个因素对乳酸产量影响显著。利用最陡爬坡试验逼近最大响应区,采用中心复合实验设计及响应面分析法进行回归分析,建立影响乳酸产量的二次模型。模型求解得出最优淀粉质量浓度为271.89g/L,糖化酶用量为265.09U/g,发酵温度为39.05℃,最大理论乳酸产量为196.99g/L。3批验证实验结果平均值与预测值接近,表明该模型与实际情况拟合良好,实际最大乳酸产量为193.6g/L,较优化前提高了13.9%,L-乳酸的平均纯度达到95.2%。     

基因工程菌发酵生产L-乳酸研究进展

乳酸是重要的工业平台化学品。随着聚乳酸产业的兴起,对高质量L-乳酸的需求量也不断增加。为了进一步降低L-乳酸发酵成本,提高菌株的工业适应性,各种现代生物技术已经应用到L-乳酸发酵菌种的改造上来。文中简要综述了近年来使用乳酸菌、酵母、大肠杆菌及米根霉等基因工程菌株发酵生产L-乳酸的技术进展。     

L-乳酸的发酵生产和聚L-乳酸的化学加工

L-乳酸广泛应用于食品、医药、日化和工业等各个领域。近年来随着石化资源的不断紧缺,众多化学合成的高分子材料的生产受到了限制。以生物质资源为基础的L-乳酸因此被大量用于加工生产成聚L-乳酸等环境友好型生物可降解材料。正是由于L-乳酸需求量的增大,如何高效低成本地生产L-乳酸显得尤为重要。系统综述了L-乳酸生产菌株的选育,用于L-乳酸发酵生产的廉价资源的开发利用,L-乳酸的发酵生产和L-乳酸的分离纯化等方面的研究进展。目前研究的热点和难点正是基于上述四个部分:菌种方面,以可以高效代谢利用廉价底物,且营养需求低的选育目标获得了多个优良的生产菌种,然而具备综合代谢优势的菌种还有待进一步选育;发酵底物方面,已开发利用多种廉价,来源丰富且易于菌种代谢并高效转化成乳酸的底物,但是对这些底物工业规模应用还有待进一步研究;发酵工艺方面,建立了环境友好型,劳动强度低的发酵工艺,然而实际应用中仍然存在成本高的问题;后提取方面,通过选育低营养需求的生产菌种和采用新型发酵工艺有效地简化了后提取过程,但是实际应用方面仍受发酵工艺成本高的制约。最后对聚L-乳酸的化学加工以及聚L-乳酸的生物降解进行了探讨并提出了一些建议。     

L-乳酸发酵的研究

本文报导了L-乳酸产生菌筛选、发酵条件以及发酵产物鉴定的结果。从56株根霉中筛选出10株产L-乳酸较高的菌株,其中根霉R47产L-乳酸最高,产酸稳定。发酵条件试验结果表明,该菌最适发酵培养基组成(％)：葡萄糖15,尿素0．2,KH 2PO40．02,MgSO4·7H2O0．025,ZnSO4·7H2 0 0．0044,CaCO3,6,7;pH6．7。在摇瓶培养条件下,35℃48小时,产L-乳酸达11．84 g／100 ml,对糖的重量转化率达78,9％。发酵液经离子交换等方法纯化,得到无色或微黄色透明糖浆状液体。经纸层析、比旋光度测定、紫外光谱和红外光谱分析证明确系L-乳酸。     

L-乳酸发酵菌株的选育

以初筛得到的 1株干酪乳杆菌鼠李糖亚种突变株R2 (Lactobcilluscaseisubsp .rhamnosusR2 )为出发菌株 ,经紫外线、硫酸二乙酯、复合诱变处理 ,筛选出 1株产乳酸较高的突变株ZY ,L 乳酸含量达 93.9%。以正交试验为基础对发酵培养基进行优化 ,采用优化后的培养基发酵 4d ,残糖降至 0 .1 %以下 ,L 乳酸产量达9.5 7g/ 2 0 0mL ,对糖的转化率达 96 .3%。     

丙酮酸对细菌L-乳酸发酵的促进作用

研究了丙酮酸在不同浓度和添加时间对LactobacilluscaseiL 乳酸发酵过程中葡萄糖转化率和L 乳酸产量的影响。结果表明 ,当丙酮酸的添加量为 30g·L- 1 时 ,L 乳酸的产量达到 74g·L- 1 。在 72h的发酵周期内 ,丙酮酸在 2 4h和 42h添加的效果好于其他时间添加。     

乳杆菌Lactobacillus sp.lxp发酵高产L-乳酸研究

筛选得到一株乳杆菌Laetobaeillus sp．,进行发酵生产高浓度L-乳酸的研究。考察了种龄、接种量、温度和不同pH调节剂对乳酸发酵的影响。结果表明：最佳种子培养时间为15h;最佳接种量为15％;最适培养温度为42℃;与氨水和氢氧化钠相比,碳酸钙更适于作为发酵过程的pH调节刺;以葡萄糖为碳源,添加豆粕水解液和玉米浆作为辅料,2L罐培养120h,L-乳酸质量浓度可达202 g/L,糖转化率91．3％,L-乳酸占发酵液中总酸含量98％以上。     

米根霉发酵生产L-乳酸

报道了Ｌ－乳酸菌株的分离与筛选,探讨了不同碳源、氮源、通气量、温度等发酵条件对产Ｌ－乳酸的影响,从７８株米根霉中筛选出１３株产Ｌ－乳酸较高的菌株,其中米根霉（Ｒｈｉｚｏｐｕｓ ｏｒｙｚａｅ）Ｒｓ９２８产Ｌ－乳酸最高,产酸最稳定。试验结果表明,该菌株最适发酵培养组成（％）：淀粉水解糖１６,ＭｇＳＯ４ ０．０８,ＫＨ２ＰＯ４ ０．０５,ＺｎＳＯ４ ０．０１,ＣａＣＯ３ ７,ｐＨ自然。在６０ｔ发酵罐中,     

耐氨米根霉发酵生产L-乳酸的研究

传统的L-乳酸发酵法生产以CaCO3为酸中和剂,在乳酸后提取中产生的大量石膏废渣不仅在过滤时造成较大的乳酸损失,而且由于废渣不易处理,对L-乳酸万吨级规模的生产将形成巨大的环保压力和废渣处理成本。为此,为了降低L-乳酸生产成本,该文采用氨水为酸中和剂,用筛选得到的一株米根霉RhizopusoryzaeJS-N02-02进行以氨水为中和剂的L-乳酸摇瓶、15L自动发酵罐的发酵试验。以玉米粉双酶水解糖为碳源,接种孢子浓度1×105个ml,以0.01%(NH4)2SO4为氮源,30℃,15L自动发酵罐连续5批发酵,平均总糖浓度为136.8gL,平均产酸达100.6gL,L-乳酸纯度达95.3%,糖酸转化率达71.6%。     

PURIFICATION AND CHARACTERIZATION OF PULLULANASE FROM Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co²⁺ and completely inhibited by Hg²⁺. The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.     

Multiple competitive PCR-DGGE as a tool for quantifying and profiling defined mixed cultures of lactic acid bacteria during production of probiotics from complex polysaccharides

AIMS: To apply a denaturing gradient gel electrophoretic (DGGE) method to quantify and profile individual strains during a mixed culture fermentation. METHODS AND RESULTS: DNA was extracted during the culture of lactic acid bacteria (LAB) and amplified in a multiple competitive PCR (cPCR) using general primers targeting 16S rDNA and DNA from Streptococcus salivarius as competitive DNA. Subsequently the 200-kb amplified fragments were separated by DGGE. The method was validated in pure cultures and used to profile a mixture of three LAB grown on glucose, soluble starch and glycogen from mussel processing waste. The inclusion of a starch- and glycogen-degrading strain (Lactobacillus plantarum) and a weakly amylotic nisin-resistant strain (Lact. paracasei) allowed proliferation of the nisin producing Lactococcus lactis which in itself is unable to grow on complex carbohydrates. cPCR-DGGE permitted the monitoring of a different strain succession on the different carbohydrates, related to amylolytic activity and substrate consumption, acid production and nisin production. CONCLUSIONS: cPCR-DGGE is a useful tool for profiling defined mixed cultures of bacteria and hence allows their interaction to be studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Provided validation of the method for each specific case, it may be appropriate to monitor and control the reproducibility of any defined mixed culture of bacteria, with the advantage that an increase in the strain numbers to be monitored does not yield an increase in the labour of the procedure.     

泡菜中优良乳酸菌的分离、鉴定及发酵特性

从几种泡菜中分离出86株菌,对其在适温和低温下产酸速率及硝酸盐降解能力进行测定,筛选出5株产酸速率快、硝酸盐降解能力强的菌株。经形态学鉴定及生理生化反应试验,初步鉴定为：植物乳杆菌2株,短乳杆菌1株,戊糖乳杆菌1株,肠膜明串珠菌葡聚糖亚种1株,并对5株菌的发酵性能进行了测定。     

Genomics of lactic acid bacteria

Lactic acid bacteria (LAB) are found to occupy a variety of ecological niches including fermented foods as well as mucosal surfaces of humans and other vertebrates. This review is based on the genomic content of LAB that is responsible for the functional and ecological diversity of these bacteria. These genomes reveal an ongoing process of reductive evolution as the LAB have specialized to different nutritionally rich environments. Species-to-species variation in the number of pseudogenes as well as genes directing nutrient uptake and metabolism reflects the adaptation of LAB to food matrices and the gastrointestinal tract. Although a general trend of genome reduction was observed, certain niche-specific genes appear to be recently acquired and appear on plasmids or adjacent to prophages. Recent work has improved our understanding of the genomic content responsible for various phenotypes that continue to be discovered, as well as those that have been exploited by man for thousands of years.     

产黏乳酸菌的发酵特性研究

通过对2株产黏乳酸球菌的发酵特性比较研究,以感观、酸度、活菌数对数值、培养时间、粘度及乳清析出率为考察指标,结果表明:乳酸球菌Q26具备较好的产黏、产香特性,遗传性质较稳定.通过与乳酸杆菌G18进行发酵应用试验,确定G18和Q26存在共生关系,混合发酵的酸牛乳乳清析出率为0.1％,粘度为4 528mPa·s.通过保质期试验,产品在28、20、4℃下保质期分别为3、6、15d.确定Q26和G18为最佳酸牛乳生产用菌种.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

白菜乳酸菌混菌发酵的研究

研究了大白菜乳酸菌乳链球菌DM 2 2和植物乳杆菌UM 2 2混合发酵中的生长和产酸以及环境因子的影响 ,并对混菌发酵的风味物质作了分析。结果表明 :混菌发酵中 ,DM2 2在发酵前期生长迅速 ,是优势菌群 ,而UM2 2在发酵后期占主导 ;发酵温度、发酵剂组成以及发酵液盐浓度都会显著影响混合发酵中菌的产酸代谢 ;发酵风味物质与单菌发酵区别明显。     

Monitoring the bacterial community of maize silage stored in a bunker silo inoculated with Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri

Aims: To monitor variations in the bacterial community and fermentation products of maize silage within and between bunker silos. Methods and Results: Silage samples were collected in 2008 and 2009 from three dairy farms, wherein the farmers arranged for a contractor to produce maize silage using bunker silos. Silage was prepared using a lactic acid bacteria (LAB) inoculant consisting of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri. Eight samples were collected from each bunker silo; 4 ‘outer’ and 4 ‘inner’ samples were collected from near the top and the bottom of the silo. The dry matter, lactic acid, acetic acid, ethanol, 1‐propanol and 1,2‐propanediol contents differed between bunker silos in both sampling years. Higher acetic acid, 1‐propanol and 1,2‐propanediol contents were found in the bottom than the top layers in the 2008 samples, and higher lactic acid content was found in the top than the bottom layers in the 2009 samples. The bacterial community varied more between bunker silos than within a bunker silo in the 2008 samples, whereas differences between the top and the bottom layers were seen across bunker silos in the 2009 samples. The inoculated LAB were uniformly distributed, while several nonconventional silage bacteria were also detected. Lactobacillus acetotolerans, Lactobacillus panis and Acetobacter pasteurianus were detected in both years. Stenotrophomonas maltophilia was detected in the 2008 samples, and Lactobacillus reuteri, Acinetobacter sp. and Rahnella sp. were detected in the 2009 samples. Conclusions: Although differences were seen within and between bunker silos, the bacterial community may indicate a different relationship between bunker silos and sampling locations within a bunker silo from that indicated by the fermentation products. Significance and Impact of the Study: Analysis of bacterial community can help understand how diverse non‐LAB and LAB species are involved in the ensiling process of bunker‐made maize silage.