Primary Study On The Microbial Flora Of Zhejiang Traditional Rosy Vinegar Fermentation

Zhejiang rosy vinegar was a traditional condiment with rose-color, sort sweet, soft and sterling aroma, and particular flavor. There was a large of demands in the market for its nutrition and good taste. The most particular characteristic was as a production by surface fermentation with natural microbes in the environment along the season change, but without any artificial microbes inoculation. Zhejiang rosy vinegar was produced only once a year since the climate from May to December was the only fitted to its production process. For a long time, there was just some common sense or experience to the microbe origin, distribution, and action in Zhejiang rosy vinegar fermentation; basal microbe flora is not investigated.Pertinent microbes of Zhejiang rosy vinegar were mainly originated from straw cover, with gram-positive bacillus as main bacteria, Aspergillus and Rhizopus as main molds, and ethanol making yeasts as main yeasts. The microbe floras and counts in the straw cover were great different before and after its solarization treatment. Before the solarization, bacteria and yeast counts were more than molds counts, with respectively 2.7×10~4, 1.3×10~3 and 567 cfu/g straw. After the solarization, however, molds counts were more than bacteria, with respectively 2.5×10~3 and 1.7×10~3 cfu/g straw, and the yeasts were the least whose counts were less than 10~2 cfu/g straw. And the microbe distribution after solarization was more balanced too. The changes of microbe flora and counts before and after solarization were favorable for going on wheels of "Fahua" and steadying brewing mass quality during "Fahua" phase.During "Fahua" phase, alcohol content, total acid and amino nitrogen were raised and pH value was dropped until "Fahua" finished. The temperature, total sugars and reducing sugars rose first and then dropped. During fermentation, alcohol content rose and achieved the peak value about 5.01% at the 5th day, then dropped to about 2.85% at the 18th day. pH value dropped first and then rose softly, after 57 days maintained between 3.15 to 3.20. The acetic acid content rose and achieved the peak value about 5.48g/100ml at the 57th day, then dropped softly, at the 95th day the acetic acid was 4.89g/100ml. But maturation of the vinegar should go down to December in the same year.Corresponding to the change course of chemical and physical indexes above, molds were the most microbes in the prophase (before 9 days) of "Fahua", and yeasts and bacteria were main microbe in the metaphase and anaphase (9 to 15 days) of "Fahua". And the microbial communities of outside and inside the mash were different completely. The process of fermentation was separated into two stages called as alcoholic fermentation and acetic acid fermentation. The results showed that the sedimentation appearance of brewing rice mass could be used as the time point to separate two stages presumably. In the stage of alcoholic fermentation, yeasts and bacteria co-existed on the surface of mash whose counts were 4.8×10~7 and 1.2×10~7 cfu/ml at the second day respectively. Then counts dropped to about 10 and 2.6×10~6 cfu/ml at the 13th day. In the prophase of acetic acid fermentation, bacteria counts keep on drop until acetic acid bacteria become the main bacteria in the mash. Then the counts rose and got the peak value about 5.5×10~5 cfu/ml mash at the 57th day. In the stage of vinegar maturation, acetic acid bacteria counts dropped down, but some acetic acid bacteria were isolated from mature mash on December in 2005. There were acetic acid bacteria in maturation stage the acetic acid content didn't increase; the main effect of maturation was to form special flavor and color of Zhejiang rosy vinegar.43 strains of molds, 9 strains of yeasts and 16 strains of common bacteria were isolated. 32 strains of molds were included in Aspergillus spp., Monascus spp., Rhizopu spp., Penicillium spp., Mucor spp. and Fusarium. spp., and the taxology of the rest 11 molds were not decided. The activities of glucoamylase activity of strain M1 (Rhizopu) and the proteinase activity of strain M38 (Aspergillus flavus) were high; M1 could also produce alcohol and acid. Only 3 strains (Y2, Y3 and Y20) of yeasts had the activity to produce alcohol, while whole yeasts had glucoamylase and proteinase activities. In order to identify 4 strains of molds and 4 strains of yeasts, the Biolog Systems were used. These molds were identified to be Penicillium islandicum Sopp, Penicillium islandicum Sopp, Aspergillus flavus and Penicillium raistrickii G.Sm. and these yeasts were identified to be Saccharomyces boulardii, Rhodosporidium diobovatum, Rhodosporidium diobovatum and Kluyveromyces marxianus。11 strains of acetic acid bacteria were isolated and identified by physiological and biochemical characteristics or 16S rDNA fragment analyse, and they all belonged to Acetobacter pasteurianus. 11 strains of acetic acid bacteria all had the ability to produce acetic acid, and 4 strains (AB3, AB8, AB9 and AB26) better with acid at 4.14, 4.43, 4.28 and 4.38g/100ml respectively. Besides, their fermenation rates were speedier than industrialized strain named "HuNiang 1.01" in the same condition called surface fermentation.