| Lactic acid bacteria(LAB)is a kind of beneficial bacteria that relate to human closely,and it is used more and more widely.Lactic acid bacteria can synthetize and secrete exopolysaccharide(EPS)that can increase fermented milk viscosity,improve the taste and flavor of dairy products,even these products' nutritional value.LAB EPS has the function of adhesion,this function can improve the structure of the intestinal microbial colony.moreover,EPS also have series of probiotic functions,such as antioxidant,imunoregulation,antitumor,etc.First this study separates 42 strains from the Yunnan endemic fermented foods,such as yogurt,yak milk,sour pickles and soil.Then the morphology of all strains were determined by Gram stain and the phylogenetic trees were constructed after 16s rRNA and dnaK molecular identification.It found that there are 36 Lactobacillus plantarum strains,4 Lactobacillus pentosus strains,1 Pediococcus pentosaceus strain and 1 Lactobacillus casei strain through 16s rRNA molecular identification,and there are 26 Lactobacillus plantarum strains,9 Lactobacillus plantarum subsp.plantarum strains,4 Lactobacillus pentosus strains,1 Lactobacillus paracasei strain,1 Lactobacillus paraplantarum strain through dnaK molecular identification.There are some differences between 16s rRNA and dnaK molecular identificaton.The strains YM-2 and SP8 are selected as the research objects in the study.It comprehensively consider that their differences about EPS yields?relative genes and EPS antioxidant properties.The range of EPS yields produced by 38 LAB strains from 14 mg/L to 135 mg/L.The minimum EPS yield of SP8 strain is 14.779 mg/L,the max EPS yield of JHM6-30 strain is 133.864 mg/L,and the EPS yield of YM-2 strain is 25.45 mg/L.The EPS yields of most strains between 80 mg/L to 130 mg/L,only 4 strains' EPS yields below 70 mg/L,and one strain's EPS yield is higher than 130 mg/L.In subsequent trials,it found that the EPS yields of those strains are not stable,which may relate to their metabolic activity,and all strains'EPS yield are about 200 mg/L.The response surface analysis is used to optimize EPS biosynthetic conditions of YM-2 and SP8 strains.There are 4 culture conditions,carbon source,nitrogen source,cultural time and temperature respectively.It found that the EPS yield of 2 strains increase with the increase of nitrogen source;but there is little effect on EPS yield when glucose concentration change,the EPS increase less when glucose concentration increase,but when the glucose concentration is too large,the EPS yield decline,higher glucose concentration may inhibit EPS biosynthesis;The effects of cultural time and temperature are significant.At initial culture,EPS yield increase with increase of incubation time,when it reaches a certain stage,EPS yield decline with increasing time.This is because that at the culture initiation stage,bacteria mainly synthetize EPS,but when environmental conditions become deteriorate,bacteria mainly decompose EPS to obtain energy.when the initial cultural temperature is low,the EPS yield increases with the increase of temperature,but the temperature is too high,EPS yield can decrease with the increase of temperature.This is because that the temperature is too low or too high will inhibit the enzyme activity that relate to EPS synthesis,which makes the EPS yield decrease.According to response surface optimization method obtains optimal EPS synthesis conditions respectively were:carbon source 30 g/L,nitrogen source 30 g/L,culture time 30.05 h,culture temperature 36.36? of YM-2 strain,the theoretical EPS yield is 255.254 mg/L;carbon source 21.97 g/L,nitrogen source 30 g/L,culture time 22.17 h,culture temperature 35.61 ? of SP8 strain,the theoretical EPS yield is 282.494 mg/L.The results are similar to actual yields,257.362 mg/L and 280.105 mg/L respectively.The response surface experimentations are credible.This study also determines antioxidant activity of EPS produced by 6 LAB strains.Samples and vitamin C gradient concentrations are 50 pg/mL?100 ?g/mL?150 ?g/mL and 200 ?g/mL,vitamin C is used as a positive control.It found that antioxidant activities of EPS are weak compare to Vc,but all EPS have some certain antioxidant activities.Antioxidant activity and EPS concentration are positive correlations.The concentration of EPS is 50 g/mL,there are no significant differences about DPPH free radical scavenging ability among all kinds EPS,but when the concentration of EPS are 100?g/mL?150 pg/mL?200 ?g/mL,there are very significant differences.The antioxidant activity of EPS produced by YM-2 strain is the best,and significantly higher than EPS produced by other strains,following are strains KY1-2L?SP8?QB3-4?HH4-7L and JHM6-30.The DPPH scavenging ability of EPS produced by JHM6-30 enhanced at higher EPS concentration.The thin-layer chromatography and gas chromatography are used to determine monosaccharide components of EPS produced by two LAB strains.In TLC analysis,it found that YM-2 EPS is mainly composed of lactose?sucrose and a little of galactose;and SP8 EPS is mainly composed of lactose and galactose.The analysis shows that the monosaccharide of both EPS are composed of glucose?galactose and fructose,respectively.Gas chromatographic analysis found that the EPS produced by YM-2 and SP8 strains are composed of Rhamnose,Arabinose,N-acetyl glucosamine and Galactose,but no glucose component.This is remarkably different from component of other heteropolysaccharides had reported.In recent years,more and more gene clusters related to EPS synthesis was cloned.The EPS of Lactococcus lactis NIZOB40 produced by 14 genes including eps RXABCDEFGIJKL,each gene has its clear physiological function.Lb.rhamnosus ATCC9595 EPS biosynthesis gene cluster is 18.7Kb in length,containing 18 genes.In those 18 genes,wzd and wze are response for determining polysaccharide chain length,wel F?wel G?wel H?well?wel J?wel E are response for encoding glycosyltransferase,wzm may code protein that transfer sugar,rml A?rml B?rml C?rml D are response for coding rhamnose precursor.As the standard strain,Lb.plantarum WCFS1 has 4 gene clusters related to EPS synthesis in its genome,including cps1(ABCDEFGHI)?cps2(ABCDEFGHIJK)?cps3(ABDEHIJ)? cps4(ABDEHIJ).Lb.plantarum WCFS1 is used as the standard strain in this study,the standard strain has gene clusters and gene related to EPS biosynthesis,such as cps2,cps3,cps4 and glf2.According to PCR reactions and sequence determination,it found that genes' differences and similarities,which related to EPS biosynthesis in the 39 LAB strains.The 28 and 33 strains have same gene components;the gene components of SP8 strain are similar to SP8-7L's genes,only cps2(C)included,other genes are deleted;strains 9?HH4-1L?HH4-2L have same gene components;and strains HH4-7L?7?22?24?27?30 and 31 have same gene components.The most strains delete their cps2 gene cluster,but contain cps2(D),there are a part of cps2 components in strains YM4-3?KY1-2L?28?33?32 and YM4-5,and only cps2(AB)exists in strain S11;cps3 cluster appear different degree deletion in some strains,but strains QB3-3?QBLB3-1?QB3-2 are lack of cps3 cluster;the most strains have a complete cps4 cluster relatively,but there is only cps4 cluster in strains 23 and KY6-2L.Moreover,there is generally contain glf2 in every strain. |
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