Characterization of a lytic Lactobacillus plantarum bacteriophage and molecular cloning of a lysin gene in Escherichia coli

Bacteriophage SC921, which can infect Lactobacillus plantarum specifically, was isolated from a fermented vegetable source, Kimchi. This phage is active against six of 11 strains of L. plantarum tested as hosts. Morphologically, it has an isometric head (60 nm in diameter) and a non-contractile tail (260 nm long and 9-11 nm wide), indicating that it belongs to Bradley's group B or the Siphoviridae family according to the International Committee on Taxonomy of Viruses (ICTV). The bouyant density was 1.58 g/cm3. SDS-PAGE experimentation indicated that the phage particle contains two major structural proteins and several minor proteins. The genome was a double stranded linear DNA molecule with cohesive ends and 66.5 kb long by mapping genomic DNA digested with the restriction endonucleases: KpnI, SmaI, and XbaI. The [G + C] content of the phage DNA is 39.4%. For this lysin gene study, 9.4 kb of KpnI-digested DNA fragment was cloned into pUC19 and expressed in Escherichia coli. The KpnI fragment was considered as the genetic element responsible for the lysis gene of L. plantarum bacteriophage. The cloned fragment in pUC19 was hybridized to a 9.4-kb fragment generated by KpnI digestion of SC 921 as a probe. This confirmed that the fragment in pUC19 originated from phage DNA. The lysin gene was near the middle of the phage genome.     

Characterization and adsorption of a Lactobacillus plantarum virulent phage

Bacteriophage infection of lactic acid bacteria is considered one of the biggest worldwide problems in the food industry. Bacteriophages may cause negative effects on the fermentation of various dairy-based products. A virulent bacteriophage was isolated from an abnormal fermentation liquid of Lactobacillus plantarum IMAU10120. The characterization and influence of temperature, pH, divalent cations, and chloramphenicol on the adsorption ability of this phage were evaluated. The results showed that this phage belonged to the Siphoviridae family. It exhibited a burst time of 135 min and a burst size of approximately 215 counts expressed per milliliter per infective center. No significant effect was shown to influence its viability and adsorption at 10 to 37°C. More than 90% of phages exhibited infectivity from pH 5 to 9. Divalent ions and chloramphenicol did not have a significant influence on the adsorption of this phage. The information obtained in this study will enrich the database of lactobacilli virulent phages and provide a basis of information for the control of phages in the food fermentation industry.     

Preparation of a Lactobacillus plantarum starter culture for cucumber fermentations that can meet kosher guidelines

A method is described for growth of a Lactobacillus plantarum starter culture in jars of commercially available pasteurized fresh-pack kosher dill cucumbers so that jars can be used to inoculate commercial scale cucumber fermentation tanks. A procedure is also described to transfer lactic acid bacteria from frozen storage in MRS broth into cucumber juice and commercial jars of kosher dill cucumbers so that a selected strain of lactic acid bacteria can be kosher certified for commercial fermentations in processing plants that operate under kosher certification. The strain of L. plantarum used in these experiments grew to maximum cell numbers in 4 d at 20 to 25 °C and then maintained viable cell numbers for 2 wk at >10(8) CFU/mL so the culture was suitable for inoculation of fermentation tanks. Refrigeration of jars of culture after they grow to maximum numbers minimizes die-off of cells sufficiently so that a pure culture can be maintained by aseptically transferring brine containing viable bacteria to a new pH-adjusted jar only once every 4 mo. PRACTICAL APPLICATION: This report describes a method to prepare a lactic acid bacteria starter culture suitable for kosher vegetable fermentations.     

蔬菜发酵菌种的筛选及发酵特性

为了选择适合蔬菜发酵的发酵菌种,本研究从四川泡菜老汤中分离的6 种乳酸菌Lactobacillus plantarum、Lactobacillus fermentum、Lactobacillus brevi、Lactobacillus pentosus、Leuconostoc mesenteroides、Lactobacillus lactics筛选发酵菌种。6 种乳酸菌接入亚硝酸盐的MRS培养液中,6 种乳酸菌还原亚硝酸盐的大小顺序为Lactobacilluspentosus＞Lactobacillus plantarum＞Leuconostoc mesenteroides＞Lactobacillus fermentum＞Lactobacillus lactics＞Lactobacillus brevis。以pH值降低的速率为发酵速率,Lactobacillus plantarum、Lactobacillus pentosus、Leuconostocmesenteroides这3 种发酵剂的发酵速率较其他3 种快,并且通过乳酸菌的全细胞蛋白电泳实验得出此3 种乳酸菌在甘蓝发酵过程中能够成为优势菌。从乳酸菌还原亚硝酸盐的能力、发酵速率、发酵菌种的生存能力（是否能成为优势菌）的实验结果表明Lactobacillus plantarum、Lactobacillus pentosus、Leuconostoc mesenteroides这3 种乳酸菌作为蔬菜发酵的发酵剂。通过对3 种发酵菌种发酵特性的研究可知,Leuconostoc mesenteroides较另两种生长周期短,稳定期维持时间短,很快进入衰退期。Lactobacillus plantarum菌较Lactobacillus pentosus、Leuconostoc mesenteroides耐酸,Leuconostoc mesenteroides对酸敏感。Leuconostoc mesenteroides最适生长温度为30 ℃,Lactobacillus plantarum、Lactobacillus plantarum两种菌的最适生长温度是37 ℃。15 ℃条件下Leuconostoc mesenteroides的光密度（OD600 nm）值很低,说明Leuconostoc mesenteroides较Lactobacillus plantarum、Lactobacillus pentosus对低温敏感。     

Malic Acid as a Source of Carbon Dioxide in Cucumber Juice Fermentations

The degradation of malic acid to lactic acid and CO₂ during fermentation of cucumber juice was investigated. This malolactic reaction was the major source of CO₂ when cucumber juice was fermented by Lactobacillus plantarum. It may also be an important CO₂ source in controlled cucumber fermentations. In addition to CO₂ production, the degradation of malate served to buffer the fermentation and increase sugar utilization. The pH after 7 days’fermentation was 2.8 when 13 mM malic acid was present in the juice and 4.1 with 135 mM malic acid. In the same fermentations, 52% of the sugars were degraded with the low malic acid concentration while complete sugar utilization was observed with the highest malic acid level.     

荧光假单胞菌噬菌体KS461-2的分离及其生物学特性研究

从2-酮基-D-葡萄糖酸产生菌荧光假单胞菌A46异常发酵液中分离得到1种噬菌体,将其命名为KS461-2。电镜观察表明,噬菌体KS461-2呈蝌蚪状。根据ICTV对病毒的分类标准,噬菌体KS461-2属长尾噬菌体科,为烈性噬菌体。噬菌体KS461-2对宿主菌的最佳感染复数为0．01,温度和pH对其稳定性具有显著的影响。一步生长曲线显示,噬菌体KS461-2的潜伏期约30min,裂解期约135min,裂解量为24。SDS-PAGE显示,噬菌体KS461-2有5种主要的结构蛋白,其分子量分别为66．0、61．0、42．5、34．7和14．4kDa。     

蔬菜汁的乳酸菌发酵

为制备不含任何动物性来源成分的纯植物性的乳酸菌发酵产品,采用双歧乳杆菌、植物乳杆菌、保加利亚乳杆菌、嗜酸乳杆菌4种乳酸菌,在豆角、香菇、番茄、青瓜、胡萝卜和包菜6和蔬菜培养液中进行发酵。实验结果证明,4种乳酸菌可以在不添加其他营养物质的纯蔬菜培养液正常生长,但是不同菌种适合的蔬菜培养液也不相同,且混合蔬菜培养液培养效果要好于单一蔬菜培养液。番茄和青瓜混合培养液适合双歧乳杆菌生长,包菜和青瓜混合培养液适合植物乳杆菌生长,包菜和番茄混合培养液适合保加利亚乳杆菌生长,豆角和青瓜混合培养液适合嗜酸乳杆菌生长。蔬菜的乳酸菌发酵产品,在4℃下冷藏2周后,4种菌体的存活率均在106CFU/mL以上。在不添加其他营养物质的条件下,蔬菜汁可以用来生产活性乳酸菌产品,满足素食主义者、乳糖不耐症者的消费需求。     

蔬菜汁乳酸菌发酵过程中产酸量预测的研究

本文以胡萝卜、番茄为试验原料,经过加工处理,将所得的蔬菜汁用嗜酸乳杆菌和植物乳杆菌进行发酵。采用四元二次通用旋转组合试验将发酵温度、La、Lp两菌种接种量、发酵时间四因素对发酵蔬菜汁的产酸量进行回归,得到方程Y=0.4394＋0.102x1＋0.0338x3＋0.1107x4-0.0163x1^2,其中x1为发酵温度、x3为植物乳杆菌接种量、x4为发酵时间、Y为发酵蔬菜汁的产酸量。     

Evaluation of malolactic-deficient strains of Lactobacillus plantarum for use in cucumber fermentations

In the fermentation of cucumbers, naturally occurring strains of Lactobacillus plantarum decarboxylate malic acid (MDC⁺) to form lactic acid and CO₂. Since CO₂ buildup in the brine contributes to bloater damage of the cucumbers, it is desirable to have strains of L. plantarum that do not decarboxylate malic acid (MDC^-1). Two MDC^- mutants, obtained by N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis of different MDC⁺ strains, and their parent strains of L. plantarum wre evaluated in laboratory fermentations of filter-sterilized cucumber juice and of whole cucumbers as to growth rate, end-products, residual malic acid, and competitiveness with the natural flora. Effects of temperature (15 to 40°C) and NaCl concentration (0-6%) on growth in cucumber juice were determined. One MDC^-1 strain, designated MOP3-M6, was selected for further development because of its relative dominance in cucumber ferementations, high residual malic acid concentration after fermentation and greater salt tolerance as compared to its closest rival mutant culture. Growth lag and generation times averaged 1·6 and 1·2 times greater, respectively, for the MOP3-M6 mutant than its parent. However, this mutant may still have application as a starter culture for cucumber fermentation, particularly under relatively aseptic conditions.     

Selection and characterization of mixed starter cultures for lactic acid fermentation of carrot, cabbage, beet and onion vegetable mixtures

An evaluation of various lactic acid bacteria (LAB) for the fermentation of cabbage, carrot and beet-based vegetable products was carried out. As part of a screening process, the growth of 15 cultures in a vegetable juice medium (VJM) was characterized by automated spectrophotometry. Acidification patterns as well as viability during storage of the LAB were also established. There were greater differences between the pure cultures than the mixed ones with respect to growth in VJM and viability during storage. Reductions in viable cell counts during storage of the fermented VJM occurred more rapidly with a Leuconostoc strain than for pediococci or lactobacilli. Inoculation of vegetables was carried out with cultures of Lactobacillus plantarum NK-312, Pediococcus acidilactici AFERM 772 and Leuconostoc mesenteroides BLAC which were rehydrated in a brine. This rehydration procedure was not detrimental to viability. During fermentation of a carrot/cabbage vegetable mix, sugar metabolism was characterized by the assimilation of both glucose and fructose, but sugars remained in the fermented vegetables when acidification stopped. The pH in the LAB-inoculated vegetables after 72 h at 20 degrees C was significantly lower (by 0.2 units) than the uninoculated control. Inoculation with LAB designed for silage fermentation resulted in the inhibition of acetic acid production, and reduced the production of ethanol during fermentation. The selection process on VJM enabled the preparation of a mixed culture that was more rapid than the silage inoculants in acidifying the medium and was more effective in reducing the production of gas during the fermentation and storage of the fermented vegetables.     

一株产细菌素植物乳杆菌高密度发酵培养基的筛选及其在酸奶中的应用

目的:优化用于高密度发酵产细菌素的植物乳杆菌KLDS 1.0391的经济有效培养基,旨在生产具有益生作用的辅助发酵剂。方法:以发酵液中活菌数量和抑菌效果为指标,比较不同水解度的脱脂乳粉、乳清粉、麦麸,以及玉米浆粉的发酵效果,制备冻干形式的植物乳杆菌直投式发酵剂,将其添加至酸奶中,评价其对酸奶发酵时间、pH、滴定酸度、粘度和感官评价的影响,进而确定其在酸奶中应用的可行性。结果:确定脱脂乳粉、乳清粉和麦麸培养基的最佳水解度分别为15%、15%和20%;植物乳杆菌KLDS 1.0391在玉米浆粉培养基中获得的发酵效果最佳,活菌数量达到9.53 lg CFU/mL,抑菌圈直径达到12.69 mm。植物乳杆菌直投式菌种的添加对酸奶发酵时间和感官评价无显著影响(P>0.05),酸奶粘度下降,但可以缓解酸奶的后酸化程度。通过优化得到植物乳杆菌KLDS 1.0391高密度发酵的廉价培养基为:4%玉米浆粉,同时补充2%葡萄糖和0.1% Tween-80。结论:制得的植物乳杆菌直投式发酵剂可应用于酸奶生产中。     

植物乳杆菌噬菌体Lpla的分离鉴定及抗噬菌体菌株的筛选

从泡菜中分离得到1 株以植物乳杆菌WCFS1为宿主菌的烈性噬菌体,命名为Lpla。一步生长曲线结果表明Lpla的潜伏期为15 min,裂解期为180 min,裂解量为43 PFU/cell。根据透射电镜观察的形态将其归为长尾病毒科,B1类。噬菌体的理化性质结果表明,Lpla对酸、碱的耐受性较强,对乙醇、温度和紫外射线的耐受性较低。吸附实验结果显示,Lpla在4 ℃时吸附率最大。Ca2＋、Mg2＋均可促进Lpla对宿主菌的吸附,热灭活宿主菌可使Lpla的吸附率下降20%。本研究筛选出6 株对噬菌体Lpla具有稳定抗性的植物乳杆菌,为制定出有效的噬菌体防治策略提供理论依据,并为进一步将抗性菌株应用于生产实践奠定基础。     

一株地衣芽孢杆菌噬菌体的分离及其性质分析

从生产碱性蛋白酶的车间附近的下水道淤泥和后处理车间空气中分离得到了一株噬菌体。电镜观察表明该噬菌体有多面体的头部和细长的尾部组成,有着长尾病毒科噬菌体的形态特征。在供试菌种中,该噬菌体以地衣芽孢杆菌为宿主,对其生理特性研究发现,该噬菌体在pH 5-10之间有着很好的稳定性,在高温下有一定的稳定性,且金属离子可以促进其对宿主菌的侵染。根据实验结果绘制出噬菌体的一步生长曲线,可知感染宿主菌的潜伏期约为40 min,爆发期约为110 min。对噬菌体核酸性质的分析发现,其遗传物质为双链线状DNA,其分子量大约为36 kb。     

Selection and use of autochthonous mixed starter for lactic acid fermentation of carrots, French beans or marrows

Strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Weissella soli/Weissella koreensis, Enterococcus faecalis, Pediococcus pentosaceus and Lactobacillus fermentum were identified from raw carrots, French beans and marrows by partial 16S rRNA gene sequence. L. plantarum M1, Leuc. mesenteroides C1 and P. pentosaceus F4 were selected based on the rates of growth and acidification in vegetable juice media, and used as the autochthonous mixed starter for the fermentation of carrots, French beans or marrows. An allochthonous starter, consisting of the same species, was also used for fermentation. A two-step fermentation process (1 day at 25 degrees C and 7 days at 15 degrees C) in brine (1% w/v) followed by storage at room temperature in olive oil until 40 days was set up. Unstarted vegetables subjected to the same treatments were used as the controls. Cell numbers of lactic acid bacteria in the started vegetables were ca. 10,000 (autochthonous starter) and 1000 (allochthonous starter) times higher than unstarted samples throughout the process. When fermented with the autochthonous starter, carrots, French beans or marrows were characterized by the rapid decrease of pH (<4.5), marked consumption of fermentable carbohydrates, and inhibition of Enterobacteriaceae and yeasts. Fermentation with the allochthonous starter did not acidify and inhibit bacteria and yeasts so rapidly. After 40 days, carrots, French beans and marrows fermented with the autochthonous starter had significantly (P<0.05) higher total concentration of vitamin C (ascorbate+dehydroascorbate) with respect to those fermented with the allochthonous starter and, especially unstarted vegetables. The same was found for the indexes of color. Firmness of both started vegetables was higher than unstarted vegetables. Sensory analysis differentiated started vegetables. Carrots and French beans fermented with the autochthonous starter were, especially, appreciated for fragrance. Appearance was the sensory attribute that mainly distinguished marrows fermented with the autochthonous starter.     

响应面法优化植物乳杆菌发酵混合蔬菜汁

为了研究一种利用植物乳杆菌发酵的由紫甘蓝、胡萝卜和西红柿混合的饮品。文章确定了由紫甘蓝、胡萝卜和西红柿按照6∶3∶1的比例配制的蔬菜汁,以此为培养基,利用植物乳杆菌发酵产酸。并在单因素实验的基础上,以总酸为指标,利用响应面法对接种量、温度和时间进行发酵优化实验。结果表明,植物乳杆菌在接种量为4.2%,温度为36℃,发酵时间为77 h时达到最佳条件。在该条件下进行验证实验,得到产酸总量为11.238 g/L,与模型预测值11.242 g/L接近,可用于预测植物乳杆菌的发酵条件,为进一步制备饮品提供了依据。     

尾菜液体青贮菌剂制备及应用

对植物乳杆菌(Lactobacillus plantarum)SD2、发酵乳杆菌(Lactobacillus fermentum)SD4在5 L立瓶中分别进行培养,制备尾菜液体青贮菌剂。结果表明,在MRS液体培养基中37 ℃条件下,调控pH值,菌株SD2和SD4最适收获期分别为24 h、20 h,菌株SD2与SD4配比为2∶1时青贮效果最佳,尾菜液体青贮菌剂总活菌数＞1×109 CFU/mL。按0.5%接种量,进行废弃白菜叶、青笋叶青贮发酵应用试验,生产的尾菜青贮饲料品质良好,颜色淡黄色或黄绿色,酸香味浓郁,质地较松散,pH值＜4.0。研制的尾菜青贮菌剂适用于尾菜青贮。     

Effects of indigenous starter cultures on the microbial and physicochemical characteristics of Urutan, a Balinese fermented sausage

Urutan is a Balinese traditional dry fermented sausage prepared from lean pork and various kinds of spice. Urutan is different from the European sausages, because it is fermented under warm condition with fluctuating temperatures of approximately 25 degrees C at night to 50 degrees C during sun drying. In this study, two of the 71 strains of lactic acid bacteria (LAB) isolated from natural urutan fermentation were used as starter cultures: Lactobacillus plantarum U201, the dominant LAB, and Pediococcus acidilactici U318, a bacteriocin producer. A soft urutan with yellowish brown color was produced using these strains as multiple starters. The starter cultures grew in characteristic succession which reconstructed the natural fermentation process. Lactobacilli were dominant until 48 h fermentation and pediococci dominated at the later stage of fermentation. Proliferation of starter cultures produced lactic acid which resulted in the decrease in pH and coagulation of soluble protein in urutan. Both strains could eliminate the Enterobacteriaceae in urutan after 24 h fermentation, and could suppress and eliminate the occurrence of micrococci at 120 h fermentation. By using a single starter culture, no succession was observed to occur in urutan and the time of elimination of Enterobacteriaceae was delayed. Thus, the strains of L. plantarum U201 and P. acidilactici U318 have great potential for use as multiple starter cultures in urutan fermentation.     

胡萝卜、番茄、黄瓜和西芹制汁过程中酶解工艺的优化

目的:优化胡萝卜、番茄、黄瓜和西芹这四种蔬菜制汁的酶解工艺。方法:榨汁过程中分别添加果胶酶或纤维素酶对蔬菜汁进行酶解处理,以出汁率和浊度为指标对酶解条件(酶解时间、酶添加量、酶解温度)进行单因素分析和正交实验优化。结果:四种蔬菜汁的最佳酶解工艺条件为:胡萝卜汁酶解时间60 min,果胶酶添加量0.4%,酶解温度40 ℃,在此条件下出汁率达到84.7%,浊度为54.3 NTU;番茄汁酶解时间40 min,果胶酶添加量0.2%,酶解温度40 ℃,在此条件下出汁率达到95.1%,浊度为36.3 NTU;黄瓜汁酶解时间60 min,果胶酶添加量0.5%,酶解温度40 ℃,在此条件下出汁率达到93.2%,浊度为60.7 NTU;西芹汁酶解时间60 min,纤维素酶添加量0.4%,酶解温度40 ℃,在此条件下出汁率达到92.1%,浊度为33.3 NTU。结论:在最佳酶解工艺条件下制得的蔬菜汁色泽清亮、甘甜爽口,具有一定的开发价值,可用于制备复合果蔬或蔬菜饮料的原料。     

响应面法优化鸡肝发酵工艺

采用益生菌对鸡肝进行发酵。从3种菌株(植物乳杆菌LP1、发酵乳杆菌LF1、枯草芽孢杆菌BS1)中选择生长能力和产酸能力最强的植物乳杆菌LP1作为发酵剂,以发酵初始pH值、葡萄糖添加量、接种量、发酵时间和料液比5个因素进行单因素试验。在此基础上,以发酵鸡肝A_(280 nm)为指标,采用响应面分析法进行3因素3水平响应面优化试验,结果表明:各因素对发酵鸡肝A_(280 nm)的影响大小为发酵初始pH值>料液比>接种量,经过优化得到的最佳发酵工艺为:发酵初始pH 5.57、料液比1∶3.55(m/V)、接种量1.5%,最终发酵鸡肝A_(280 nm)为1.537;发酵鸡肝游离氨基酸和必需氨基酸总含量均显著增加(P<0.05),说明发酵能促使蛋白质分解,进一步提升鸡肝的营养价值。