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Table of Content

      
    16 July 2008
    Volume 43 Issue 7
    Articles

    Physiological characteristics of Lactobacillus delbrueckii subsp. lactis
    mutant strains with reduced H+-ATPase activity

    GAO Li-li,MA Xia,ZHAO Hong-fei,PEI Jia-wei,L〖AKU¨〗 Zhao-lin,ZHANG Bo-lin *

    J4. 2008, 43(7):  1-09 .  doi:
    Abstract ( 875 )   PDF (1476KB) ( 773 )   Save
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    Three mutant strains (5M6, 5M1 and 5B1) obtained from Lactobacillus delbrueckii subsp. lactis L5 and grown in MRS medium, which contained a lethal concentration of neomycin sulfate, showed reduced H+-ATPase activity. The membrane-bound H+-ATPase activities of 3 mutant strains were reduced by 51.3% for 5M6, 27.7% for 5M1 and 34.3% for 5B1 respectively. Cell shapes of the mutant strains 5M6, 5M1and 5B1 were slenderer than that of the parental strain L5. Physiological performance indicated that the ATP level of the parental strain (0.63×10-18mol/cell) was lower than that of the mutant strains. The conversion rate of glutamic acid (GA) to γ-aminobutyric acid (GABA) by parental strain L5 cultured in MRS at 37℃ for 12h was 13.91%. The conversion rates of GA to GABA were 16.73% in mutant strain 5M6, 15.6% in mutant strain 5M1 and 15.4% in mutant strain 5B1. The parental strain L5 transformed 13.91% GA to GABA. It was proved that the parental strain showed more acid and salt resistance than its mutant strains in an extended growth period.

    Construction of a controlled autolysis Lactococcus strain
    WANG Shao-hua,KONG Jian*,CHEN Fu-kun
    J4. 2008, 43(7):  10-13 .  doi:
    Abstract ( 776 )   PDF (389KB) ( 900 )   Save
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    To elucidate the characterization of the lysis cassette (Hyb5-Lyb5) encoded by temperate bacteriophage φPYB5 of Lactobacillus fermentum, the hyb5 and lyb5 genes were cloned into the plasmid pSEC under the control of the nisin-inducible promoter, which can generate the recombinant plasmid pSEC-hyb5-lyb5. Then, the pSEC-hyb5-lyb5 was tromsformed into Lactococcus lactis NZ9000 by electroporation, yielding NZphl. Hyb5 and Lyb5 were successfully expressed under the induction of nisin and effectively induced the lysis of NZphl, resulting in rapid reduction of OD600 and leakage of abundant intracellular proteins. These results will facilitate the application of autolysis lactic acid bacteria in fermentation to improve the quality of products.

    Adhesion capacity of the single domain of Lp-1643 protein
    LIU Fang,DU Li-hui,YANG Li-jie,DU Peng,HUO Gui-cheng*
    J4. 2008, 43(7):  14-17 .  doi:
    Abstract ( 770 )   PDF (707KB) ( 704 )   Save
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    The gene coding for the first domain of the N terminus of Lp-1643 protein was amplified from KLDS 1.0320 genome. An expression vector designated pET30a/N1 was constructed. The recombinant protein was highly expressed in the strain E. coli BL21 induced by IPTG. Affinity chromatography with HisTrap FF column was used to separate and purify the recombinant protein of His-N1. Influences of the recombinant protein His-N1 on adhesion capacity of strain KLDS 1.0320 toCaco-2 cells were studied with BSA as a negative control. It was found that the numbers of KLDS 1.0320 adhering to Caco-2 cells pretreated by His-N1 dramatically decreased. This result indicates the single domain of Lp-1643 protein can adhere to Caco-2 cells.

    Diversity of lactic acid bacteria isolated in sour milk from Xinjiang

    ZHAO Rui,HUO Gui-cheng*
    J4. 2008, 43(7):  18-22 .  doi:
    Abstract ( 838 )   PDF (354KB) ( 570 )   Save
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    Seventy one lactic acid bacterial strains were isolated from 15 samples of naturally fermented sour milk collected from the farmyards in Xinjiang. They were identified by physiological and biochemical tests, 28 of them were further analyzed by sequencing their 16S rRNA genes. Based on constructing phylogenetic neighbor-joining trees, the results in combination with traditional taxonomic methods based on phenotypic characteristics showed that the lactic acid bacteria in traditional dairy products covered 5 genera and 11 species or subspecies. Lactobacillus was the predominant genus, of which Lb. helveticus was the most predominant strain. Some strains could adapt to a wide temperature range.

    Effect of yeast extract concentration on the batch fermentation kinetics of
    Lactobacillus delbrueckii subsp.bulgaricus KLDS 1.9201

    LI Ai-li,HUO Gui-cheng*,DENG Kai-bo
    J4. 2008, 43(7):  23-27 .  doi:
    Abstract ( 699 )   PDF (623KB) ( 650 )   Save
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    The influence of yeast extract concentrations ranging from 0 to 20g/L on the kinetic parameters of Lactobacillus delbrueckii subsp.bulgaricus KLDS 1.9201 by batch fermentation in whey medium was analyzed by comparing the growth and production rates during various growth phases. The results indicated that lactic acid production rate varied in proportion to that of growth during the whole phase under yeast extract supplemented and non-supplemented conditions. When the yeast extract supplementations increased to 10g/L,the duration of the growth-associated phases decreased from 6h to 4h, the highest growth rates of KLDS 1.9201 attained to 1.28g/L·h after 4h growth, and 39% acid production rate occurred during the slow down phase(3h). Growth inhibition was observed when the concentration of yeast extract was more than 20g/L.

    Identification of α-galactosidase-producing lactic acid bacteria and their fermentation performance

    CHEN Jun-liang,YANG Li-jie,HUO Gui-cheng*
    J4. 2008, 43(7):  28-32 .  doi:
    Abstract ( 822 )   PDF (682KB) ( 788 )   Save
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    Two α-galactosidase-producing strains were obtained from traditional indigenous dairy products. Based on morphological identification, physiological and biochemical characteristics and 16S rRNA gene sequence analysis, these two α-galactosidase-producing strains were identified as Lactobacillus fermentum and Bifidobacterium longum, and coded as LB21 and KLDS2.0509, respectively. Soymilk was fermented with each strain and α-galactosidase activities, production of organic acid, metabolism of oligosaccharides and proteolytic enzymes were assessed during 48h incubation at 37℃. LB21 and KLDS2.0509 exhibited variable α-galactosidase activities, of which the highest activities were 26.8U/mL and 31.5U/mL, and pH values were 5.1 and 5.0 at the end of fermentation respectively. Both LB21 and KLDS2.0509 could effectively degrade soymilk raffinose. The hydrolysis of protein increased with an extension of fermentation time.

    Identification of a bacteriocin-producing Lactobacillus and the characteristics of its antimicrobial substance

    GONG Han-sheng,MENG Xiang-chen*
    J4. 2008, 43(7):  33-39 .  doi:
    Abstract ( 728 )   PDF (738KB) ( 877 )   Save
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    Sixty seven strains of Lactobacillus, isolated from traditional dairy products of Inner Mongolia, were screened for antagonistic activities. One of them, named KLDS 1.0355 showed high inhibitory activity. This strain was identified as Lactobacillus brevis by its carbohydrate fermentation pattern using the API 50 CHL test kit and 16S rDNA gene sequencing. The cell-free supernatant (CFS) of KLDS 1.0355 culture was heat-stable (30min at 121℃) and remained active after incubation at pH 2.0 to pH 10.0. The CFS of KLDS 1.0355 was found to be sensitive to proteolytic enzymes such as pepsin, trypsin, papain, α-chymotrypsin and proteinase K. The results indicated that this antagonistic agent was a kind of protein. The action mode of the CFS was confirmed as bactericidal. CFS of KLDS 1.0355 exhibited a broad spectrum of antagonistic activity against various species of gram-positive and gram-negative bacteria.

    The effects of centrifugal conditions on the viability of Bifidobacterium sp.

    DU Peng,LIU Fang,HUO Gui-cheng*
    J4. 2008, 43(7):  40-44 .  doi:
    Abstract ( 694 )   PDF (746KB) ( 944 )   Save
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    The centrifugal conditions such as centrifugal force, time and temperature were optimized. The results showed that the highest harvesting rates of viable cells of B. longum KLDS 2.0001 and B. infantis KLDS 2.0002 were 94.49% and 91.19% respectively under 6000g at 4℃ for 30min.

    Growth performance of Lactobacillus strains isolated from sour milk

    ZHAO Rui,SU An-fen,XU Ting-yu,HUO Gui-cheng*
    J4. 2008, 43(7):  45-50 .  doi:
    Abstract ( 907 )   PDF (319KB) ( 839 )   Save
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    Abilities of growth, acid-production and proteolysis performance of 38 Lactobacillus strains isolated from sour milk in Xinjiang at different temperatures were measured. Some strains could live in a wider temperature range than the control strain of Lb.acidophilus. The abilities of growth and production of acid at different temperatures and proteolysis performance were dependent on the species. Great differences were observed among strains of the same species. Some Lactobacillus strains exhibited excellent growth performance and are potential prospects in the application in dairy products.

    Screening of lactic acid bacteria with high-acid adaptive capacity and characterization of their acid-adaptive conditions
    LIU Huai-long,MENG Xiang-chen*
    J4. 2008, 43(7):  51-55 .  doi:
    Abstract ( 839 )   PDF (898KB) ( 764 )   Save
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    he acid tolerance of 24 lactic acid bacteria including Bifidobacterium, Lactobacillus plantarum, Lactobacillus bulgaricus and Lactococcus lactis were evaluated. Twenty out of 24 lactic acid bacteria were sensitive to acid except 4 L.bulgaricus. When these 20 lactic acid bacteria were exposed to a moderate acid environment for acid adaptation, the acid tolerance of 13 strains could be improved. Among them, L. lactis KLDS4.0312 had the highest acid adaptive capacity and the acid tolerance was increased by 7?542 times. The optimal conditions for acid adaptation in KLDS4.0312 were pH 4.5 and 30min. But the acid adaptation of the stationary phase cells of KLDS4.0312 could only be enhanced up to 27-fold compared with the parental strains, and the acid adaptive capacity in KLDS4.0312 stationary phase cells was lower than that in log phase cells.

    Application of PCR-DGGE technique to monitor the species changes of microecological preparation after successive cultures

    MA Jun-xiao ,KONG Jian * ,JI Ming-jie

    J4. 2008, 43(7):  56-60 .  doi:
    Abstract ( 729 )   PDF (579KB) ( 801 )   Save
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    Denaturing gradient gel electrophoresis (DGGE) technique combined with classical plate culture and sequencing of partial 16S ribosomal RNA (rRNA) genes were applied to examine microbial community changes of microecological preparation, which was coded as WS-401, composed of lactic acid bacteria and Bacillus sp., and subjected to successive sub-cultured processes. The population of bacteria with a diverse morphology in WS-401 preparation was 2×107cfu/mL. Two types of band patterns appeared on DGGE gel for three strains of isolates, implying two different species. These isolates were identified as Bacillus cereus and Streptococcus thermophillus by comparison with identification ladder and sequence analysis. When the microecological preparation WS-401 was continuously sub-culturing for five times in medium LB and MRS medium and at 37℃ or 30℃, respectively, microbial communities largely changed. The results show that Bacillus cereus is dominant in LB medium, while L. plantarum and L. paracasei are dominant in MRS medium. It was concluded that medium components and culture conditions have significant influences on microbial communities and species dynamic changes of microecological preparation.

    Screening of broad-spectrum bacteriocin-producing lactic acid bacteria

    HU Shu-min,KONG Jian,JI Ming-jie*
    J4. 2008, 43(7):  61-64 .  doi:
    Abstract ( 1003 )   PDF (427KB) ( 954 )   Save
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    LAB211 and LAB30 strains, which showed obvious micro-biostatic effects, were selected from 70 Lactic acid bacteria isolated from fresh milk using the agar diffusion method. The supernatants of LAB211 and LAB30 cultures eliminated the effects of organic acid and hydrogen peroxide, and still exhibited inhibitor activity against the gram-positive bacteria. However, the inhibitory activity of LAB30 stain decreased after treatment with protease K and trypsin, while the inhibitory activity of LAB211 strain decreased by treatment with protease K, which showed that this inhibitory material had the feature of protein. Thus, the inhibitory material produced by LAB211 and LAB30 could be classed as bacteriocin. PCR analysis showed that the inhibitory materials were not nisin. The inhibitory spectrum proved that the supernatants of LAB211 and LAB30 could inhibit not only gram-positive bacteria but also partial gram-negative bacteria. Therefore, these two wide inhibitory spectrum bacteriocin-producing Lactic acid bacteria strains were isolated.

    Protection of salt-induced whey protein cold-set gels on Bifidobacterium sp.

    ZHANG Jiu-long,MENG Xiang-chen*
    J4. 2008, 43(7):  65-68 .  doi:
    Abstract ( 851 )   PDF (770KB) ( 553 )   Save
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    The protection of salt-induced whey protein isolates (WPI) on Bifidobacterium was investigated. The protection on Bifidobacterium was determined by experiments of acid tolerance, and the gel structure was observed by scanning electron microscope and texture analyser. The results show that the network cold-set gels of 8% WPI solution induced by 7.5mmol/L CaCl2 can give the best protection on bifidobactetrial cells with an acid condition of pH 1.5. The number of bifidobacterial cells decreases by three order of magnitude, and the survival rate of bifidobacteria is 0.42% by treatment in the acid condition of pH 1.5 for 120 minutes, which suggests better protection on bifodobacterial cells by cold-set gels of WPI can be achieved, and can provide a strategy in the development and application of probiotics.

    Cloning and expression of the beta-galactosidase gene of Paenibacillus sp. K1 in E.coli

    LU Wen-wei,KONG Wen-tao,SUN Zhi-lan,KONG Jian*,JI Ming-jie

    J4. 2008, 43(7):  69-73 .  doi:
    Abstract ( 863 )   PDF (503KB) ( 783 )   Save
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    A bacteria strain with β-galactosidase activity was isolated from fresh milk on LB plates containing X-gel. It was identified as Paenibacillus sp. K1 by 16S rDNA analysis. The genomic DNA library of Paenibacillus sp. K1 was constructed in Escherichia coli DH5α with the vector pUC18 (lac-). A gene of β-galactosidase was obtained by sequencing a positive clone with potential β-galactosidase activity from the DNA library. The full length of the gene is 2028 bp. A constructive promoter was found upstream of the ORF (open reading frame). The overproduction of the β-galactosidase was carried out in E.coli BL21 (DE3), and the β-galactosidase activity in E.coli was 25.06U/mL, which was more than 4.55U/mL in the wild strain of Paenibacillus sp. K1. This enzyme was purified using affinity chromatography.

    Expression of lactase gene bga from Paenibacillus sp. K1 in Lactococcus lactis

    SUN Zhi-lan,KONG Wen-tao,KONG Jian*
    J4. 2008, 43(7):  74-77 .  doi:
    Abstract ( 913 )   PDF (570KB) ( 827 )   Save
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    To increase the ability of hydrolyzing lactose of lactococci, the recombinants with overproduction of lactase from Paenibacillus sp. K1 in  
    Lactoc
    occus lactis were constructed. The bga gene was amplified by PCR using the plasmid pUC-bga as templates. PCR products were ligated with L.lactis/E.coli shuttle vector pSEC and pMG36e, which resulted in the formation of plasmids pSEC-bga and pMG36e-bga, respectively. The plasmids pSEC-bga and pMG36e-bga were respectively electroporated into Lactococcus lactis NZ9000 and Lactococcus lactis MG1614. The expression of Lactase in L. lactis NZ9000-Bga and L. lactis MG1614-Bga were carried out in the cytoplasm under the nisin inducible promoter or without induction. Analysis of the residual lactose in the medium by high performance liquid chromatography (HPLC) showed that the ability of utilizing the lactose by constructs L. lactis NZ9000-Bga was significantly higher than that of the control strain, which suggests potential application in dairy fermentation with low lactose.

    Cloning of a nisin resistance gene from Lactococcus lactis and
    its application in food-grade selection marker

    GUO Ting-ting,KONG Wen-tao,KONG Jian*,JI Ming-jie
    J4. 2008, 43(7):  78-82 .  doi:
    Abstract ( 811 )   PDF (485KB) ( 733 )   Save
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    Five nisin-resistant strains were isolated from fresh milk on GM17 plates supplemented with bromocresol purple and nisin at a final concentration of 500U/mL. By morphological, physiological, biochemical and 16S rDNA
    sequence analys
    is, all of the isolates were identified as Lactococcus lactis. A pair of primers was designed on the basis of the DNA sequences of a reported nisin resistance gene. PCR amplification was carried out with chromosome and plasmid DNA as templates from five strains, respectively. An expected PCR product amplified from one of the five strains was obtained. After being sequenced, the amplicon was
    confirmed as nsr by BLAST analysis. The nsr gene was cloned into the
    E.coli-L.lactis shuttle vector pTRKH2, resulting in the plasmid pT-nsr. The construct was obtained when the plasmid pT-nsr was electroporated into L.lactis MG1614 competent cells. When the medium contained a maximum of 500U/mL nisin, the construct carrying pT-nsr showed the same growth curve as L.lactis MG1614, which suggests that the nsr gene could be used as a marker for constructing a food-grade vector.

    Transgalactosylation activity of beta-galactosidase produced by Lactobacillus fermentum K4
    LU Wen-wei,KONG Wen-tao,KONG Jian,JI Ming-jie*
    J4. 2008, 43(7):  83-87 .  doi:
    Abstract ( 878 )   PDF (519KB) ( 647 )   Save
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    lactobacilli strain producing beta-galactosidase with high transgalactosylation activity was isolated from Chinese traditional fermented milk. It was identified as Lactobacillus fermentum K4 (EU621851) by sequence analysis of its 16S rDNA. The conditions for transgalactosylation activity were optimized, and the large yield of galactooligosaccharides with various kind of oligosaccharides was obtained by using 20% lactose as a substrate at pH 5.5, 50℃ for 48h. Moreover, the beta-galactosidase produced by Lb. fermentum K4 exhibited obvious transgalactosylation activity even at conditions of low lactose concentration (5%) and pH in the range of 5 to 7. The fresh milk was mixed with beta-galactosidase extracted from Lb. fermentum K4 at 50℃ for 8h, the content of lactose in the milk was reduced and galactooligosaccharides were produced. Therefore, Lb.fermentum K4 will have potential use in fermented diary products.

    Exopolysaccharide production and lyophilization preparation from Streptococcus thermophilus ST

    WU Rong-rong,WANG Li-ping,GAO Li-li,LIU Bai-qu,ZHANG Bo-lin *

    J4. 2008, 43(7):  88-96 .  doi:
    Abstract ( 815 )   PDF (257KB) ( 600 )   Save
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    ST strain, isolated from yogurt purchased from a local market and identified as Streptococcus thermophilus based on physiological-biochemical characteristics and 16S rDNA sequence, produced 55.62mg·L-1 of exopolysaccharides (EPS). The EPS level produced by strain ST depended upon incubation time (24h), tem(20g·L-1). The major monosaccharide of EPS formed by this strain was confirmed to be glucose. Moreover, data from the lyophilization preparation of strain ST showed that the optimal medium containing NFMS (100.0g·L-1), yeast extract (3.0?g·L-1), CaCO3(9.0g·L-1) and whey powder (20.0g·L-1) promoted its viable cells to a level of 1.05×109CFU·mL-1. The combination of NFMS (160.0g·L-1), glycerol (30.0g·L-1), sodium glutamate (30.0g·L-1) and Tween 80 (5.0g·L-1) was proved to be a good cytoprotectant for the protection of strain ST against the stresses of freeze-drying. Temperature, pH and rotation speed were important factors for gaining high viable counts in pilot-plant production of strain ST lyophilizator. Theoptimal combination including fermentation temperature (37℃), rotation speed (90r·min-1) and pH (5.9), resulted in a direct-vat-inoculation preparation of strain ST that contained viable cells of 1.7×1011CFU·g-1 after lyophilization. Use of lyophilized strain ST preparation to directly ferment 80.0g·L-1 reconstituted skim milk to yogurt produced lower whey separation than that of yogurt made with non-EPS strains used as controls.