基于CRISPR/Cas9系统对乳酸乳球菌进行绿色荧光蛋白标记

乳酸乳球菌是治疗剂在体内运送的良好载体,研究其在体内的真实运送情况需对其进行标记。该实验利用CRISPR/Cas9系统对乳酸乳球菌(Lactococcus lactis)NZ9000进行增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)标记,用于研究乳酸乳球菌在体内的运送,评价其作为益生菌的功能。基于该实验室已构建的乳酸乳球菌CRISPR/Cas9编辑质粒pLL25构建重组质粒pYSH,其上携带eGFP及同源臂,电转入乳酸乳球菌NZ9000感受态细胞中,将基因组中的乳酸脱氢酶基因(ldh)替换为绿色荧光蛋白基因,从而使Lactococcus lactis NZ9000获得标记,表达绿色荧光蛋白。对绿色荧光标记的Lactococcus lactis NZ9000突变株,酶标仪定量分析eGFP的表达强度。荧光强度定量分析结果表明,在乳酸乳球菌不同生长阶段,eGFP基因均能稳定表达。     

基于CRISPR/Cas9系统对乳酸乳球菌进行绿色荧光蛋白标记

乳酸乳球菌是治疗剂在体内运送的良好载体,研究其在体内的真实运送情况需对其进行标记。该实验利用CRISPR/Cas9系统对乳酸乳球菌(Lactococcus lactis)NZ9000进行增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)标记,用于研究乳酸乳球菌在体内的运送,评价其作为益生菌的功能。基于该实验室已构建的乳酸乳球菌CRISPR/Cas9编辑质粒pLL25构建重组质粒pYSH,其上携带eGFP及同源臂,电转入乳酸乳球菌NZ9000感受态细胞中,将基因组中的乳酸脱氢酶基因(ldh)替换为绿色荧光蛋白基因,从而使Lactococcus lactis NZ9000获得标记,表达绿色荧光蛋白。对绿色荧光标记的Lactococcus lactis NZ9000突变株,酶标仪定量分析eGFP的表达强度。荧光强度定量分析结果表明,在乳酸乳球菌不同生长阶段,eGFP基因均能稳定表达。     

乳酸链球菌素抑菌机理及在食品保鲜中的研究进展

乳酸链球菌素（Nisin）是由乳酸乳球菌乳酸亚种的某些菌株代谢过程中合成的一种天然生物防腐剂,具有良好的抑菌性,可以有效地抑制革兰氏阳性菌（金黄色葡萄球菌、枯草芽孢杆菌、单增李斯特菌、小球菌等）,尤其是细菌芽孢的生长。且因为摄入后可快速被人体中的蛋白酶消化分解为各种氨基酸不会对人体产生毒害作用,因此被广泛的用于食品保鲜领域。本文主要综述了乳酸链球菌素的抑菌机理及其近10年来在食品保鲜领域中的应用,其中着重讨论了在果蔬保鲜中的应用,最后分析了乳酸链球菌素未来的发展方向,以期为乳酸链球菌素在食品保鲜中的进一步研究和应用提供理论参考。     

Screening of dairy yeast strains for probiotic applications

To evaluate the potential of yeasts of dairy origin as probiotics, we tested 8 species including Candida humilis, Debaryomyces hansenii, Debaryomyces occidentalis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Yarrowia lipolytica, isolated from commercial blue cheese and kefir. Strains were randomly selected from each species and tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Among the 8 species, K. lactis showed higher adhesive ability than K. marxianus, K. lodderae, and D. hansenii. The other 4 species were poorly adhesive. All species other than K. marxianus and C. humilis were resistant to acidic conditions. In the presence of bile acid, growth inhibition was undetectable when incubation was carried out at 27 degrees C; however, it was evident for C. humilis and a strain of D. occidentalis when incubated at 37 degrees C. Moreover, the influence of proteinase treatment of living cells of K. lactis and K. lodderae on their adhesion to Caco-2 cells was evaluated. Although a slight reduction was recognized when K. lactis was treated with proteinase K, the influence of intestinal protease treatments of pepsin followed by trypsin was negligible. These results indicated that a proteinaceous factor was unlikely to be involved in adhesion of K. lactis and K. lodderae to Caco-2 cells. No stimulation of IL-8 synthesis by Caco-2 cells was recognized in the presence of K. lactis. In conclusion, K. lactis was the most attractive to continue study for use as probiotic microorganisms.     

Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells: adhesion properties, competition against enteropathogens and modulation of IL-8 production

The human intestinal microbiota plays a pivotal role in human nutrition and health by promoting the supply of nutrients, preventing pathogen colonization and shaping and maintaining normal mucosal immunity. The depletion of the individual microbiota can result in a higher susceptibility to enteropathogenic bacteria infection. In order to reduce this risk, the use of food supplements containing probiotic bacteria has been recently addressed. In this paper, we investigate the protective role toward enteropathogen infection of probiotic strains belonging to Lactobacillus and Bifidobacterium. According to our experimental data, Lactobacillus acidophilus Bar13, L. plantarum Bar10, Bifidobacterium longum Bar33 and B. lactis Bar30 were effective in displacing the enteropathogens Salmonella typhimurium and Escherichia coli H10407 from a Caco-2 cell layer. Moreover, L. acidophilus Bar13 and B. longum Bar33 have been assessed for their immunomodulatory activity on IL-8 production by HT29 cells. Both strains showed the potential to protect enterocytes from an acute inflammatory response. These probiotic strains are potential candidates for the development of new functional foods helpful in counteracting enteropathogen infections.     

重组乳酸乳球菌滴鼻免疫小鼠后粘膜免疫特性及耐

乳酸菌NICE(乳链菌肽控制表达nisin controlled expression,NICE)系统可将治疗性蛋白或保护性抗原蛋白表达于细胞外或锚定于菌体的肽聚糖上,递呈抗原蛋白并激活粘膜免疫系统,刺激特异性s-IgA的产生使其作为粘膜免疫原递呈的活载体成为可能。本试验将构建的重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS鼻腔免疫BALB/c小鼠,检测呼吸道粘膜中抗体s-IgA和血清中特异性抗体IgG含量,评价其动态变化情况,同时检测脾脏细胞因子IL-4和IL-10的活性。结果重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS免疫小鼠后,在测定时间内重组菌试验组小鼠呼吸道粘膜中特异性s-IgA水平高于对照组,差异极显著((P<0.01);血清中特异性抗体IgG水显著高于对照组((P<0.01);脾脏细胞悬液中的IL-10和IL-4含量与对照组无差异性(p>0.05),结果表明重组菌PNZ8149/NZ3900-M/PRRS经粘膜途径免疫小鼠后能能刺激小鼠粘膜特异性抗体s-IgA和血清中特异性抗体IgG的分泌,且能避免粘膜免疫耐受的产生,为该重组疫苗的进一步应用奠定了理论基础。     

Safety assessment of dairy microorganisms: the Lactococcus genus

The Lactococcus genus includes 5 species. Lactococcus lactis subsp. lactis is the most common in dairy product but L. garviae has been also isolated. Their biotope is animal skin and plants. Owing to its biochemical characteristics, strains of L. lactis are widely used in dairy fermented products processing. Cases of human infections due to lactococci are very seldom reported even if Lactococcus garviae can be involved in fish diseases. Then L. lactis can be considered as safe and it is most commonly considered as Generally Recognized as Safe.     

Human scFv SIgA expressed on Lactococcus lactis as a vector for the treatment of mucosal disease

The gastrointestinal tract is a complex niche and the main port of entry of many pathogens that trigger a wide range of diseases like inflammatory bowel disease (IBD) and colon cancer. Antibodies are effective for treating such diseases, but a system capable of local delivery at the site of the pathology, thus avoiding systemic side effects, is not yet available. Here we report a novel recombinant scFvSIgA1 protein produced by Lactococcus lactis, anchored to the bacterial membrane, which retains its full immuno-recognizing potential. This scFv fragment employed was specific for a colon cancer epitope, epithelial glycoprotein protein-2 (EGP-2). Accordingly L. lactis expressing this chimeric protein was capable of binding cells expressing this epitope. Expression of specific antibodies on bacteria may allow local delivery of anticancer agents produced by such bacteria in conjunction with the antibody and provides a new avenue in the quest for targeted drug delivery.     

Lactococcus lactis subsp. lactis HFY14对狼疮性肾炎小鼠肾功能的改善作用

本实验研究分离自四川红原自然发酵牦牛酸乳的Lactococcus lactis subsp. lactis HFY14(LLSLHFY14)对降植烷诱导狼疮性肾炎小鼠肾功能改善的效果。采用不同剂量LLSLHFY14灌胃小鼠，分析小鼠血清白细胞介素(interleukin,IL)-2、IL-12等免疫指标的变化情况，然后通过苏木精-伊红(hematoxylin-eosin,H&E)切片观察肾脏组织的病变情况，通过定量聚合酶链式反应(quantitative polymerase chain reaction,qPCR)和Western blot测定肾脏组织中的IL-4、IL-1β和转化生长因子-β(transforming growth factor beta,TGF-β)mRNA相对表达量和蛋白相对表达量。结果显示，LLSLHFY14能够提升狼疮性肾炎小鼠胸腺指数和脾脏指数。血清实验显示LLSLHFY14能够抑制狼疮性肾炎造成的小鼠IL-2、IL-12、总蛋白(total protein,TP)、白蛋白(albumin,ALB)质量浓度下降和干扰素γ(interferon ga...     

Lactococcus lactis subsp.lactis IL1403谷氨酸脱羧酶的克隆表达、分离纯化及活性研究

谷氨酸脱羧酶(GAD)是功能因子γ-氨基丁酸(GABA)生物合成过程中的重要酶。为了得到一种有高效活性的GAD基因工程菌,克隆到一种GAD基因,来源于微生物Lactococcus lactis subsp.lactis IL1403。以pET-22b(+)为载体质粒,Escherichia coli BL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的重组GAD过量表达;经亲和层析纯化的重组蛋白质样品进行SDS-PAGE分析,在约54 000处出现显著的特征蛋白质条带;活性检测结果表明,该重组GAD的转化活性比野生菌株有明显提高,野生菌株经5h细胞转化,反应底物转化率为55.8%,而工程菌20min后转化率达到82.1%,30min后转化可达到98.3%。     

Emerging risk of infestation and contamination of dried fruits by mites in the Czech Republic

The introduction of live insects into human food is rare in developed countries. However, we report, for the first time, an emerging risk that exists from dried fruit in Central Europe. Recently, massive and frequent infestation of dried fruit imported from the Mediterranean region by the mite, Carpoglpyhus lactis L. (Acarina: Carpoglyphidae), has been found. In 180 samples taken from supermarkets, 13% were contaminated; the contamination levels ranged from 0 to 660 mites per g of dried fruit. The contamination was found in dried apricots, figs, plums and raisins. To estimate the risks and food preferences of C. lactis, its growth rate was examined under laboratory conditions. Starting with a hypothetical population of 10 mites per g of dried fruit, the risk level of 1000 mites per g of dried fruit is reached at 42 days for dried figs, 49 days for dried pineapple and 63 days for dried apricots, dates and plums at 25 °C and 85% relative humidity. We found that mites are able to enter every dried fruit packing material tested, including polypropylene and aluminum foils. This indicates that mites can move from package to package in supermarkets. Mites are known as allergen producers and vectors of mycotoxin-producing fungi. These findings indicate that an increased risk of C. lactis contamination exists in dried fruit.     

An analysis of inter- and intraspecific genetic variabilities in the Kluyveromyces marxianus group of yeast species for the reconsideration of the K. lactis taxon

In the present work, we analyse the sequences of the 5.8S rRNA gene and the two internal transcribed spacers 1 and 2 (5.8S-ITS region), obtained from 39 strains belonging to the species Kluyveromyces aestuarii, K. dobzhanskii, K. lactis and K. marxianus, K. nonfermentans and K. wickerhamii, to solve the phylogenetic relationships among these species and also to determine the possible genetic basis for the delimitation of the two currently accepted K. lactis varieties: lactis, including lactose-positive strains isolated from dairy products, and drosophilarum, comprising lactose-negative strains isolated from insects and plant exudates. The determination of the phylogenetic relationships within the species K. lactis, together with the examination of the electrophoretic karyotypes and phenotypic characterization of strains representatives of K. lactis var. lactis and var. drosophilarum, allowed differentiation of two groups of strains. The first, and ancestral, group comprises lactose-negative strains isolated from natural habitats in North America. The second, and derived, group includes both lactose-negative strains isolated from natural habitats in Europe and wine fermentation in South Africa, and lactose-positive strains associated with dairy products. These results suggest that the present taxon K. lactis is a complex of different species, subspecies or, at least, genetically structured populations.     

Age-related decline in macrophage and lymphocyte functions in mice and its alleviation by treatment with probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum

This study evaluated the effects of probiotic Dahi administration in ageing mice on macrophage and lymphocyte functions. Probiotic Dahi were prepared by co-culturing in buffalo milk (3% fat) Dahi bacteria (Lactococcus lactis ssp. cremoris NCDC-86 and Lc. lactis ssp. lactis biovar diacetylactis NCDC-60) along with Lactobacillus acidophilus LaVK2 (La-Dahi) or combined Lb. acidophilus and Bifidobacterium bifidum BbVK3 (LaBb-Dahi). Four groups of 12 mo old mice were fed for four months, with the supplements (5 g/day) of buffalo milk (3% fat), Dahi, La-Dahi and LaBb-Dahi, respectively, in addition to basal diet, and a fifth group that received no supplements served as control. The immune functions of young mice (4 mo old) were also compared with those of ageing adult mice (16 mo old). The production of nitric oxide and cytokines IL-6 and TNF-α declined and that of immunosuppressive prostaglandin E2 (PGE2) increased by stimulated peritoneal and splenic macrophages in ageing mice, compared with their young counterparts. The proliferation of stimulated splenocytes diminished and the production of IL-2 decreased and that of IL-6 and TNF-α enhanced in ageing compared with young mice. Feeding ageing mice with La-Dahi or LaBb-Dahi improved peritoneal macrophage functions stimulating nitric oxide and IL-6 and diminishing PGE2 production. Feeding La-Dahi or LaBb-Dahi also improved lymphocyte functions stimulating their proliferation and production of IL-2 in ageing mice. To conclude, the probiotic La-Dahi and LaBb-Dahi are effective in reversing age related decline in immune functions in mice.     

Physical separation and functional interaction of Kluyveromyces lactis and Saccharomyces cerevisiae ARS elements derived from killer plasmid DNA

Two DNA fragments which have autonomously replicating sequence (ARS) activity in both Saccharomyces cerevisiae and Kluyveromyces lactis have been isolated from the K. lactis kl killer plasmid. One fragment (Kla1) is 700 base pairs (bp) in length and plasmids carrying it are mitotically unstable in both hosts. In K. lactis, this instability leads to colonies having a 'nibbled' phenotype when grown on selective media and appears to be the result of inefficient plasmid segregation. The other fragment (Kla2) is an artificial junction fragment of 1100 bp which was produced during the cloning procedure. Kla2 has been divided into two sub-fragments Kla2A and Kla2B which have, respectively, ARS activity in K. lactis and S. cerevisiae but not the other species. This indicates that these two closely related yeasts have different sequence requirements for ARS activity. Kla2B contains a perfect match to the S. cerevisiae ARS consensus but Kla2A does not. Both Kla2A and Kla1 share a 10 bp sequence as the sole region of homology between them. This sequence, 5'TCATAATATA3', is tentatively offered as defining the ARS consensus sequence for K. lactis.     

Monitoring the cell number of Lactococcus lactis subsp. cremoris FC in human feces by real-time PCR with strain-specific primers designed using the RAPD technique

Strain-specific PCR primers for Lactococcus lactis subsp. cremoris FC were developed using the randomly amplified polymorphic DNA (RAPD) technique. RAPD was used to generate strain-specific markers. A 1164-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific for L. lactis subsp. cremoris FC was designed. The specificity of this primer pair was tested with 23 L. lactis subsp. cremoris strains and 20 intestinal bacterial species, and was found to be strain-specific. Subsequently, this primer pair was subjected to the quantification of L. lactis subsp. cremoris FC in the feces of subjects fed fermented milk containing this strain. After administration, L. lactis subsp. cremoris FC was detected in the feces of all 7 subjects, with the maximum number being between 10(5) and 10(9) cells g(-1) of feces. Furthermore, this strain was detected in only one feces sample 2 weeks after administration was stopped. These results suggest that L. lactis subsp. cremoris FC can survive passage through the gastrointestinal tract.     

Growth stimulation of a proteinase positive Lactococcus lactis strain by a proteinase negative Lactococcus lactis strain

Lactococcus lactis AMP15/pAMP31(D471R) is a proteinase negative, lactose negative strain with a modified oligopeptide transport system, and potential as a debittering agent due to its efficient utilization of hydrophobic peptides. Five wild L. lactis strains of dairy origin, which produced cheeses of high flavour quality, were cocultured with L. lactis AMP15/pAMP31(D471R) in an attempt to select adequate combinations of strains for use as defined cheese starters with potential debittering ability. Four of these strains, L. lactis B6, K16, M21 and P21, inhibited growth of L. lactis AMP15/pAMP31(D471R) at a level of 10(6) to 10(7) cfu mL(-1) after 24 h of incubation, even though production of bacteriocin-like compounds could only be proven for L. lactis M21. When L. lactis AMP15/pAMP31(D471R) was cocultured with the fifth strain, L. lactis N22, its growth was significantly (P<0.001) inhibited whereas growth of L. lactis N22 was significantly stimulated. The nature of the interaction was studied and it was established that L. lactis N22 is auxotrophic for folate, a compound produced and excreted by L. lactis AMP15/pAMP31(D471R).     

Nisin-producing organisms during traditional 'Fior di latte' cheese-making monitored by multiplex-PCR and PFGE analyses

In this work we studied using different molecular methods the population dynamics of nisin-producing organisms and the persistence of such organisms within a complex ecosystem, 'Fior di latte' cheese, a traditional high-moisture pasta filata cheese. Using the primers targeting the eubacterial 16S-23S rRNA spacer region, together with those amplifying the nisA or nisZ gene, we were able to provide a rapid species identification of the isolates. Inhibitors of Lactococcus lactis subsp. lactis DSM 20481T used as indicator occurred during the whole process of cheese manufacture as a significant part of lactic microflora; however, only 12 among 109 isolates of bacteriocin producers were nisin producers. Amplification of the nisA or nisZ gene, using DNA extracted directly from dairy samples as templates, showed that the nisin structural gene was detected during cheese-making from milk samples up to the end of curd ripening but not in the final cheese. In order to monitor nisin-producing strains during cheese manufacturing, the 12 Lactococcus lactis nis+ strains were analysed by low frequency restriction fragment and PFGE. Nine isolates among the 12 nisin-producers exhibited an unique and distinct DNA banding pattern and are considered to be genetically diverse. The other three isolates from curd after ripening showed the same restriction pattern and could be the same strain. In fact, it was also isolated 2 months after the first analysis of cheese-making of 'Fior di latte'.     

Immunomodulatory and cytotoxic effects of various Lactococcus strains on the murine macrophage cell line J774.1

In a series of in vitro culture experiments using the murine macrophage-like cell line, J774.1, we investigated the ability of 46 different Lactococcus lactis strains to induce production of the cytokines interleukin (IL)-6, IL-12 and tumor necrosis factor (TNF)-alpha. The extent of induction of IL-6, IL-12 and TNF-alpha was strain-specific and was not related to subspecies, biovariety, or the source of the isolate. When incubated with a high concentration of viable cells of some lactococcal strains, J774.1 cells hardly produced cytokines in which case the percentage of J774.1 cells that were double-stained with the apoptosis probe FITC-labeled annexin V and propidium iodide was significantly increased. This finding suggests that perturbation of cytokine induction is due to the cytotoxic effects of these strains. On the other hand, when incubated with living cells of other strains, even at a high concentration, J774.1 cells produced IL-6, IL-12 and TNF-alpha. In these cases, FITC-labeled annexin V interacted with these cells, suggesting that incubation with these strains causes phosphatidylserine to be exposed at the cell surface. The ability of these strains to induce TNF-alpha, but not IL-6 and IL-12, was lost after heat treatment, suggesting that the stimulus required for TNF-alpha induction is heat sensitive and is different from those required for IL-6 and IL-12 induction. The specificity of cytokine induction by different lactococci is discussed in terms of interaction of non-pathogenic bacteria with macrophages, as well as the implications for the use of lactococci as probiotics.     

Extracellular Expression of a Recombinant Ganoderma lucidium Immunomodulatory Protein by Food-Grade Lactococcus lactis System

Lactococcus lactis is a food-grade microorganism of major commercial importance in food industry. For commercial application of genetically modified L. lactis, a food-grade expression system is strongly recommended. In this study, two food-grade selection markers, nisin immunity gene nisI and nisin resistance gene nsr, were evaluated as dominant markers for the L. lactis food-grade expression system. By using an efficient P_SlpAl promoter fused to a signal peptide from subtilisin YaB (SP_YAB), a functional recombinant Ganoderma lucidium immunomodulatory protein rLZ8 was expressed extracellularly in L. lactis. Replacing the antibiotic marker gene into the proper food-grade selection marker nsr gene, the rLZ8 was expressed extracellularly in the food-grade L. lactis system. This study provides a rationale basis for a food-grade system to express functional peptides extracellularly as an important tool for oral administration of genetically modified L. lactis.     

Antimicrobial potential of immobilized Lactococcus lactis subsp. lactis ATCC 11454 against selected bacteria

Immobilization of living cells of lactic acid bacteria could be an alternative or complementary method of immobilizing organic acids and bacteriocins and inhibit undesirable bacteria in foods. This study evaluated the inhibition potential of immobilized Lactococcus lactis subsp. lactis ATCC 11454 on selected bacteria by a modified method of the agar spot test. L. lactis was immobilized in calcium alginate (1 to 2%)-whey protein concentrate (0 and 1%) beads. The antimicrobial potential of immobilized L. lactis was evaluated in microbiological media against pathogenic bacteria (Escherichia coli, Salmonella, and Staphylococcus aureus) or Pseudomonas putida, a natural meat contaminant, and against seven gram-positive bacteria used as indicator strains. Results obtained in this study indicated that immobilized L. lactis inhibited the growth of S. aureus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus curvatus, Lactobacillus sakei, Kocuria varians, and Pediococcus acidilactici. Only 4 h of incubation at 35 degrees C resulted in a clear inhibition zone around the beads that increased with time. With the addition of 10 mM of a chelating agent (EDTA) to the media, results showed growth inhibition of E. coli; however, P. putida and Salmonella Typhi were unaffected by this treatment. These results indicate that immobilized lactic acid bacteria strains can be successfully used to produce nisin and inhibit bacterial growth in semisolid synthetic media.