| Luzhou laojiao(a kind of daqu liquor)with a long-standing reputation.It is famous for "aroma is strong,the ancestors of the wine" and is one of the top ten famous wine in our country.In the process of traditional distilling liquor by solid fermentation.The cellar is a huge treasure-house of breeding wine-making microbes and the basis of brewing liquor flavor.Different type and quantity of microbes in pit mud have different inter-specific interaction and metabolic product,it directly influences the richness of fonning the characteristic flavor of luzhou-flavor liquor.The main acid compositions of luzhou-flavor liquor—acetic acid?propionic acid?butyric acid and caproic acid which is produced by acid forming bacteria in pit mud.The caproic acid produced by caproic acid bacteria and alcohol produced by Daqu react to ethyl caproate which is the main fragrances of Luzhou-flavor liquor.To a certain extent,caproic acid bacteria and pit mud determine the quality and style of luzhou-flavor liquor.So it is of great significance to study the diversity of the mud acid forming bacteria in different pit mud of luzhou laojiao.In this study,eight samples were collected from different pit ages in Luzhou Laojiao.Acid forming bacteria were obtained by separation and purification.Their morphological features and acid production capacity were measured.We used methods of BOXAIR-PCR?16S rDNA PCR-RFLP and 16S rDNA to study tested strains' genetic diversity and phylogeny.And we studied the metabolite component of tow high-producing strains(LZ104 and LZ802)by GC-MS method.The following results were obtained:(1)A total of 43 tested strains were screened from different pit muds.The main colony morphology was:round shape,white or yellow color,opaque,wet,central bulge,late colony fold.The results of microscopic examination was:drumstick,fusiform short rods and elongated bacteria-based,neatly arranged,all most was gram stain positive.(2)The acid-producing ability of the tested strains was detennined by copper sulfate method.The results showed that all strains fennentation liquid after adding copper sulfate solution presented a layered phenomenon and produced a floccus,and the ether layer became blue.Indicating that 43 tested strains all had acid producing capacity,but in different strains their capacity was different.Strain LZ104 had the strongest acid production ability,strain LZ602 had the weakest acid production capacity.(3)BOXAIR-PCR results showed that there was significant genetic diversity among the tested strains.All the strains were clustered at the 640,%similarity level.At 82.20.%similar level,the tested strains were divided into eight BOX genetic groups,and the tested strains constructed into seven genetic groups except strain LZ710.Group l was the largest,composed of 17 strains,followed by the genetic group ? and ?,respectively,by seven and six tested bacteria composing.(4)16S rDNA PCR-RFLP analysis was performed on the tested strains according to the results of BOXAIR-PCR.The results showed that the tested strains showed abundant genetic types,including nine kinds of Hue ?.thirteen kinds of M.spl and eight kinds of HinfI.The strains were divided into eight groups at 82.2%,and LZ710 was clustered separately,and the other strains formed seven genetic groups.The clustering results were in good agreement with the results of BOXAIR-PCR.(5)Based on the acid production in the early screening results,11 strains were screened and their titrable acid production was measured.The results showed that most of the strains reached the highest level on the fifth day,and then decreased gradually.The highest acidity of LZ104 was 2.75 g/mL,followed by LZ802,2.54 g/mL,LZ209 the lowest,0.76 g/mL.According to the results of titrable acid production,two high-yielding strain were selected:the strain LZ104 and the strain LZ802.(6)Based on the above results,11 representative strains were sequenced.The phylogenetic status and taxonomic status of the strains were established.Clostridium had four strains.The similarity of 16S rRNA gene sequence between LZ104 and Clostridium fallax(M59088)was 92%.The similarity of 16S rRNA gene sequence between LZ802?LZ401 and Clostridium tyrobutyricum(M59113)was 99%.The similarity of 16S rRNA gene sequence between LZ209 and Clostridium novyi(AB045606)was 96%.Bacillus sp.had six strains,The similarity of 16S rRNA gene sequence between LZ102 and Bacillus simplex(AJ439078)?LZ312 and Bacillus circulans(AY724690)?LZ508 and Bacillus thuringiensis(D16281)?LZ707 and Bacillus altitudinis(AJ831842)?LZ502 and Bacilus licheniformis(CP000002)?LZ301 and Bacillus subtilis(AJ276351)was all 99%;Pseudomonas had one strain.The similarity between LZ304 and Pseudomonas mendocina(D84016)was 99%.In all,tested strains were mainly distributed in Clostridium?Bacillus and Pseudomonas.among which Bacillus were dominant.(7)High yield strain LZ104:rod shape cells,G.drumstick of spores,had a flagella,size 0.8-1.0?m ×0.25-0.27 ?m,generation time was 4.34 h.In semi-solid medium,the third day began to produce acid,which lasted until the eleventh day.High yield strain LZ802:rod or curve slightly shape cells,G-,drumstick or fusifom of spores,had a flagella,size 2.5-4.5 ?m x 1.0-1.5 ?m,generation time was 3.68 h.In semi-solid medium,the third day began to produce acid,which lasted until the thirteenth day.Most of the physiological and biochemical reaction of two strains was positive reaction.(8)The results of the metabolites for high yield strain LZ104 and strain LZ802(GC-MS)turned out that:the fermented liquid of strain LZ104 mainly had eight metabolites,there were three kinds of acids,two kinds of alcohols,three kinds of ketones and one kind of pyrazine.The fermented liquid of strain LZ802 mainly had eleven metabolites,there were four kinds of acids,two kinds of alcohols,two kinds of ketones,one kind of ester,one kind of pyrazine and one kind of decyl eneacyl.Glycerol content in the product of two strains metabolites component was the highest,and their ability to produce butyric acid and 2,6-dimethyl pyrazine was also strong.For the capacity of producing acid,strain LZ104 was strongger than LZ802.Area normalization method analysis results were as follows:percentage composition of acids?alcohols?ketones in LZ104 's total aroma components were respectively 19.46%,43.10%and 36.60%.For LZ802 were respectively 13.54%?32.42%.13.50%and esters accounted for 0.20%.Showed that the content was:alcohol>acid>ketone>esters. |
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