Cloning of Tap Ⅱ Chalcone Isomerases(CHI1 A) Gene and Construction of Lactococcus Lactis Expression Vector

[Objective]The aim was to clone TapⅡchalcone isomerases（CHI1 A） from soybean specially and construct expression vector of PNZ8149-CHI1 A,and then transform it into Lactococcus Lactis NICE systers.[Method]Chalcone isomerases（CHI1 A） was cloned by RTPCR method,and it was sequenced after cloning into pMD18-T vectors,and recombined to expression vector PNZ8149-CHI1 A,then it was transformed into Lactococcus Lactis NZ3900[Result]The sequencing results indicated that the cloned fragment of CHI1 A contained 670 nucleotides,and shared a sequence homology of 92% with that from Genbank accession number AF595413（CHI1 A）.CHI1 A was transformed into NICE expression system successfully by identification of PCR and digestion.[Conclusion]The foundation of using the microorganism fermentation method to produce flavonoids was laid by construction of efficient induction expression vector with chalcone isomerases CHI1 A.

仿刺参(Apostichopus japonicus)肠道源乳酸乳球菌(Lactococcus lactis)的分离鉴定及其益生特性分析

利用MRS固体培养基从南移仿刺参肠道中分离得到1株优势乳酸菌,命名为AJC-XP-15,其最适生长温度为30℃,最适生长pH为6.5;其表面疏水性和自聚率分别为34.56%和35.61%,经16S RNA基因序列分析,并结合形态学和生理生化特性将其鉴定为乳酸乳球菌(Lactococcus lactis)。拮抗实验结果显示,菌株AJC-XP-15及其发酵上清液对塔氏弧菌(Vibriotubiashii)、哈维氏弧菌(V.harveyi)、副溶血弧菌(V.parahaemolyticus,VP)和嗜水气单胞菌(Aeromonas hydrophila)等病原菌具有很好的抑制作用;体外益生实验表明,菌株AJC-XP-15对人工胃肠液的耐受性很好,在pH3.0的模拟人工胃液处理3 h后的存活率为71.43%,在pH 6.8的模拟人工肠液处理3 h后的存活率为92.1%;抗氧化能力结果显示,菌株AJC-XP-15的无细胞提取物对DPPH自由基和羟自由基的清除能力最强,分别为(82.67±6.92)%和(15.36±2.95)%,其发酵上清液对超氧阴离子自由基的清除能力最强,为(26.36±2.58)%;药物敏感性分析结果显示,菌株AJC-XP-15对四环素、恩诺沙星、盐酸多西环素等抗生素敏感,对氟苯尼考、复方新诺明、庆大霉素等抗生素不敏感。研究结果可为仿刺参肠道源乳酸菌的分离鉴定和候选益生菌种筛选提供参考依据。

Non-Fusion and Fusion Expression of β-Galactosidase from Lactobacillus bulgaricus in Lactococcus lactis

Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.

Construction and Secretory Expression of β-Galactosidase Gene from Lactobacillus Bulgaricus in Lactococcus Lactis

Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.

Oral vaccination of mice against rodent malaria with recombinant Lactococcus lactis expressing MSP-1_(19)

AIM： To construct the recombinant Lactococcus/actis as oral delivery vaccination against malaria. METHODS： The C-terminal 19-ku fragments of MSP1 （MSP-119） of Plasmodium yoelii265-BY was expressed in L. lactis and the recombinant L. lact/s was administered orally to BALB/c and C57BL/6 mice. After seven interval vaccinations within 4 wk, the mice were challenged with P. yoelii 265-BY parasites of erythroo/tic stage. The protective efficacy of recombinant L. lactiswas evaluated. RESULTS： The peak parasitemias in average for the experiment groups of BALB/c and C57BL/6 mice were 0.8± 0.4% and 20.8±26.5%, respectively, and those of their control groups were 12.0±0.8% and 60.8±9.6%, respectively. None of the BALB/c mice in both experimental group and control group died during the experiment. However, all the C57BL/6 mice in the control group died within 23 d and all the vaccinated mice survived well. CONCLUSION： The results imply the potential of recombinant L. lactis as oral delivery vaccination against malaria.

Construction and Immunogenicity of Recombinant Lactococcus lactis Expressing S1 Protein of Porcine Epidemic Diarrhea Virus(PEDV)

To evaluate the specific immune responses induced by recombinant Lactococcus lactis（L.lactis） which expresses porcine epidemic diarrhea virus（PEDV） S1 protein through oral administration,the spike gene fragment of PEDV was amplified from PEDV SDLY strain to construct p MG36 e-S1 recombinant plasmid.The recombinant plasmid was then electro-transferred into competent cells of L.lactis MG1363,to prepare the recombinant L.lactis expressing S1 protein of PEDV.The expression of target protein was identified by SDS-PAGE and Western-blot.New Zealand white rabbits were orally administered with the recombinant strain;the antibody titer in intestinal mucosa and serum was detected by neutralizing test;and the specific Ig G in serum was evaluated by indirect ELISA.The results showed that the recombinant L.lactis could effectively induce high level of Ig G in serum and high level of mucosal immune antibody.The recombinant L.lactis is qualified to be a potential oral vaccine because it could successfully stimulate both humoral and mucosal immune responses against PEDV.

Inhibition mechanisms of secretome proteins from Paenibacillus polymyxa Kp10 and Lactococcus lactis Gh1 against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus

Objective:To determine the inhibition mechanisms of secretome protein extracted from Paenibacillus polymyxa Kp10(Kp10)and Lactococcus lactis Gh1(Gh1)against methicillin-resistant Staphylococcus aureus(MRSA)and vancomycin-resistant Enterococcus(VRE).Methods:The sensitivity and viability of MRSA and VRE treated with secretome proteins of Kp10 and Gh1 were determined using minimal inhibitory concentration,minimum bactericidal concentration,and time-to-kill assays.The morphological changes were observed using scanning electron microscopy and transmission electron microscopy.To elucidate the antimicrobial mechanism of secretome protein of Kp10 and Gh1 against MRSA and VRE,2D gel proteomic analysis using liquid chromatography-mass spectrometry was run by comparing upregulated and downregulated proteins,and the proton motive force study including the efflux of ATP,pH gradient,and the membrane potential study were conducted.Results:MRSA and VRE were sensitive to Kp10 and Gh1 secretome protein extracts and displayed apparent morphological and internal composition changes.Several proteins associated with cellular component functions were either downregulated or upregulated in treated MRSA and VRE by changing the membrane potential gradient.Conclusions:Kp10 and Gh1 secretome proteins reduce the growth of VRE and MRSA by damaging the cell membrane.Cell division,cell wall biosynthesis,and protein synthesis are involved in the inhibition mechanism.

Improvement of Poplar Bark Extract on Physiological Characters of Lactococcus lactis in Intestine of White Feather Broilers

[Objective]The paper was to study the improvement of poplar bark extract on intestinal Lactococcus lactis of white feather broilers.[Method]Totally 450 Ross 308 white-feather broilers were randomly divided into five groups:control group,low dose group,medium dose group,high dose group,and antibiotic group(oxytetracycline hydrochloride).The feeding duration was 45 d.The probiotics were screened and isolated through homology,and the physiological and biochemical characteristics of chicken intestinal bacteria in different groups were compared to determine the properties of bacterial strain.The drug resistance,antibacterial ability,proliferation ability,acid resistance and bile salt resistance of isolated strain were tested,and a strain of L.lactis was obtained.[Result]The isolated L.lactis was sensitive to other drugs except natural tetracyclines,and there was no significant difference among the four groups except oxytetracycline group;as the concentration of extract increased,the inhibition of L.lactis against Salmonella sp.increased;the medium dose extract had the largest increase in the ability to tolerate the proliferation of L.lactis.[Conclusion]Feeding poplar bark extract will produce positive effects on the physiological characters of intestinal L.lactis in broiler chicken,which will provide potential probiotic strain for chicken production.

Synergistic Effect of Watermelon Powder and Lactococcus lactis subsp lactis Supplemented Diet Partially Ameliorates Aβ42-Dependent Lifespan Shorten and Flight Impairment in Transgenic Drosophila

Oxidative stress has been strongly related with Alzheimer disease pathogenesis. We determined the effects of watermelon powder (WMP) and Lactococcus lactis subsp lactis (LAL) supplementation on the generated Aβ42-induced phenotypes in a Drosophila melanogaster model of AD. Enhanced Aβ42 expression in D. melanogaster neurons can diminish lifespan and flight ability. We have observed longevity methods to assay the effects of WMP and LAL on D. melanogaster survival. Furthermore, flies expressing Aβ42 in their body fed WMP and LAL had up to 90 days, or 35% longer median lifespan than those fed a control diet. In addition, synergistic effect of WMP and LAL improved Aβ42-induced flight impairments in the Drosophila wing tissues. Our microscope experiments revealed that individuals fed synergistic effect of WMP and LAL had ameliorated Aβ42 expression as well as increment of flight ability than Aβ42-induced flies. We propose that WMP is typically rich in L-citrulline and LAL, rich in naturally occurring probiotics and antioxidants, and that it promotes the survival of neurons in brain and wing muscle tissues with increased levels of Aβ42 via a protective cell survival mechanism.

Cloning,purification,and characterization of branched-chain α-keto acid decarboxylases from Lactococcus lactis strains with different 3-methylbutanal production abilities

Lactococcus lactis is an important food-grade microorganism that has been successfully applied as a starter to increase the level of 3-methylbutanal produced during the ripening of cheese.Three variants of branched-chain α-keto acid decarboxylase (KADC) were discovered in L.lactis strains with different 3-methylbutanal production abilities.Three genes encoding KADCs of varying lengths (KADC-long,KADC-middle,and KADC-short) were cloned and heterologously expressed into Escherichia coli.KADC activity was only detected in the E.coli cloned with the KADC-long-encoding gene.Homology modeling of the three KADC recombination proteins showed that an active-site residue (Glu462) and an S-pocket structure were necessary for the ability to catalyze substrates.KADC-long showed maximum activity at pH 7.0 and 30 ℃.The substrate hydrolysis and kinetic parameters demonstrated that KADC-long efficiently produces 2-methylbutanal and 3-methylbutanal.The heterologous expression of the full-length kdcA in low-3-methylbutanal-yield L.lactis strains increased their production yields.The results of this study demonstrate the function of the complete KADC in 3-methylbutanal production.

Fermentation of groundnut brittle by Lactococcus lactis producesγ-amino butyric acid and enhances nutritional quality and safety

Objectives:The study aimed to evaluate the anti-nutrient reductions andγ-amino butyric acid(GABA)enrichments of chikki(peanut brittle),a popular of traditional snack food,using Lactococcus lactis subsp.lactis.The nutritional,storage,and sensorial analysis in order to understand the safety and functionality of chikki following fermentation were attempted.Materials and Methods:Partial fermentations of chikki were carried out using overnight grown culture of L.lactis samples.The fermented chikki were further analysed for GABA and anti-nutrients.The antioxidant profile,protein,and sugar were also analysed.The storage studies were carried on up to 2 months for functional property evaluations.Results:Fermentations at 37°C,pH of 5,with 1%inoculum and incubation for 24 h were optimal conditions,and resulted in the GABA concentration of 816 mg/g,respectively,and the GABA concentration did not change significantly(P>0.05)upon storage for upto a period of 2 months.Analysis of the fermented chikki revealed a slightly higher level of phenolic,flavonoid,protein,and sugar contents as compared to those which were not subjected to fermentation.The results of sensorial analysis showed an overall general acceptability on a 5-point hedonic scale to be 8.5±0.01(before storage)and 8.03±0.01(after storage).The fermented chikki also possessed antioxidant properties and significantly(P<0.05)low levels of phytates with complete reductions of other anti-nutrients.Conclusions:Traditionally prepared/manufactured chikki lacks GABA,possesses notable levels of anti-nutrients with lower phenolics,flavonoids,as well as antioxidants.Our study suggested a simple preparation of GABA could enrich the popular ethnic snack through fermentation by L.lactis subsp.lactis.The developed snack is acceptable,economical with good shelf life,and has substantially reduced levels of anti-nutrients originating from groundnuts affording consumer safety.

Prevalence of Clostridium spp.,in Kashar cheese and efficiency of Lactiplantibacillus plantarum and Lactococcus lactis subsp.lactis mix as a biocontrol agents for Clostridium spp.

Clostridium sporogenes and Clostridium tepidium strains were detected in traditional Kashar cheese samples with late blowing characteristics.To control Clostridium spp.,in Kashar cheese,dairy originated Lactic Acid Bacteria(LAB)strains were tested under in vitro conditions and during Kashar production.Two strains,Lactiplantibacillus plantarum and Lactococcus lactis subsp.lactis demonstrated anticlostridial activity in vitro and the co-inoculum of these two strains(107 cfu g^(-1))were tested during the challenge test on Kashar cheese production in which a contamination ratio of 10^(4) cfu g^(-1) with spores of Cl.sporogenes were applied.Kashar samples were stored at 4℃ and 25℃ during 40 days of storage period and microbiological and physicochemical properties of Kashar samples were determined during this period.A decrement of nearly 1 log cfu g^(-1) in Cl.sporogenes numbers was observed in Kashar samples produced with co-inoculum of Lb.plantarum and L.lactis subsp.lactis stored at 4℃ but this was not the case for the Kashar samples stored at 25℃.This study revealed the potential of distinct LAB strains to control Cl.sporogenes spores in semi-hard cheese samples as biocontrol agents at 4℃ storage.

NisP Is Related to Nisin Precursor Processing and Possibly to Immunity in Lactococcus Lactis

In this study, a plasmid was integrated into nisP, creating the first defined mutation in a nisin biosynthetic gene. The mutant strain secreted fully modified nisin with the N-terminal leader still attached.The presence of the leader was confirmed by N-terminal sequencing of the purified precursor. The dehydration and lanthionine formation of the precursor were already completed as active nisin could be formed by cleaving the leader from the inactive precursor by a trypsin treatment or by incubation with wild type cells. Nisin immunity of the NisP mutant strain was lowered to about 10% of the wild type immunity. The results show that NisP is needed for precursor processing and for development of high immunity of nisin.

Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.

Cloning and Expression of Bile Salt Hydrolase Gene from Lactobacillus plantarum M1-UVS29

We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH, pNZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol· min^-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.

The Citrate Metabolism in Homo-and Heterofermentative LAB:A Selective Means of Becoming Dominant over Other Microorganisms in Complex Ecosystems

The citrate metabolism has been extensively studied in lactic acid bacteria (LAB) for its aroma compound production. Among the 4-carbon (C4) by-products obtained from citrate fermentation, diacetyl is one of the better known products for its contribution to the buttery aroma of dairy products. A lot of documents deal with ways to improve diacetyl concentration in food matrices. Apart from these organoleptic advantages, in a microbial ecosystem, the citrate metabolism gives selective advantages to citrate positive microorganisms. Citrate metabolism allows the LAB to use another carbon source for their growth, withstand acidic conditions and generate a “proton motive force” (PMF). Moreover, the citrate/glucid co-metabolism leads to the fast release of organic compounds known for having bacteriostatic effects. Under specific conditions, the C4?pathway liberates diacetyl which is bacteriostatic. In this review we first describe the citrate metabolism and the enzymes involved in the two homo- and heterofermentative LABLc diacetylactisandLeuconostocspp. Moreover, the way to shift the metabolic pathway toward the production of aromatic compounds is discussed for both of these fermentative types of bacteria. Finally, the selective advantages of citrate metabolism for LAB in complex microbial ecosystems are delineated.

Plant-based,adjuvant-free,potent multivalent vaccines for avian influenza virus via Lactococcus surface display

Influenza epidemics frequently and unpredictably break out all over the world,and seriously affect the breeding industry and human activity.Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety.Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines,especially in low immunogenicity.In this study,we developed a new subunit vaccine platform for a potent,adjuvant-free,and multivalent vaccination.The ectodomains of hemagglutinins(HAs)of influenza viruses were expressed in plants as trimers(tHAs)to mimic their native forms.tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus(iLact)to produce iLact-tHAs,an antigen-carrying bacteria-like particle(BLP).tHAs BLP showed strong immune responses in mice and chickens without adjuvants.Moreover,simultaneous injection of two different antigens by two different formulas,t^(HAH5N6+H9N2) BLP or a combination of t^(HAH5N6) BLP and t^(HAH9N2) BLP,led to strong immune responses to both antigens.Based on these results,we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

Development of Zn^(2+)-controlled expression system for lactic acid bacteria and its application in engineered probiotics

Lactococcus lactis and Streptococcus thermophilus are considered as ideal chassis of engineered probiotics,while food-grade genetic tools are limited in those strains.Here,a Zn^(2+)-controlled gene expression(ZICE)system was identified in the genome of S.thermophilus CGMCC7.179,including a transcriptional regulator sczAst and a promoter region of cation transporter czcD(PczcDst).Specific binding of the SczAst to the palindromic sequences in PczcDst was demonstrated by EMSA analysis,suggesting the regulation role of SczAst on PczcDst.To evaluate their possibility to control gene expression in vivo,the sczAst-PczcDst was employed to drive the expression of green fluorescence protein(GFP)gene in L.lactis NZ9000 and S.thermophilus CGMCC7.179,respectively.Both of the transformants could express GFP under Zn^(2+)induction,while no fluorescence without Zn^(2+)addition.For optimal conditions,Zn^(2+)was used at a final concentration of 0.8 mM in L.lactis and 0.16 mM in S.thermophilus at OD600 close to 0.4,and omitting yeast extract powder in the medium unexpectedly improved GFP expression level by 2.2-fold.With the help of the ZICE system,engineered L.lactis and S.thermophilus strains were constructed to secret cytokine interleukin-10(IL-10)with immunogenicity,and the IL-10 content in the supernatant of the engineered L.lactis was 59.37%of that under the nisin controlled expression system.This study provided a tightly controlled expression system by the food-grade inducer Zn^(2+),having potential in development of engineered probiotics.