The Microbial Flora Structure And Degradation Of Biogenic Amines During Rice Wine Brewing

Rice wine is a major alcoholic beverage consumed globally.The unique brewing technology involved in rice wine production elicits a nutrient-rich product with a mellow flavor.A drawback of rice wine is its high content of biogenic amines which poses potential food safety risks.The formation of biogenic amines originates from fermentation microorganisms in rice wine.There is limited understanding of the composition and structure of biogenic amines.In this research,the formation and degradation of biogenic amines during the fermentation,structure of microbial community of the raw materials and fermented liquid,and correlation between formation/degradation of biogenic amines and microorganisms was studied.A Lactobacillus plantarum strain with the capability of biogenic amine degradation was screened from the fermented mash of rice wine and its growth characteristics,amine oxidase production capability,effect on degradation of biogenic amines,and on the quality of rice wine were investigated.Finally,a pilot experiment of rice wine brewing was done in a brewery.Twenty commercial rice wine samples were used to determine the contents of biogenic amines.The contents of biogenic amines were significantly different among all samples.The total biogenic amines content was in the range of 2.8～142.3 mg/L;high content of histamine and tyramine in some samples might cause potential food safety risk.One hundred and seventy-one samples including raw materials,pre-fermentation stage mash,post-fermentation stage mash and maturity stage mash collected from the production lines during winter brewing,spring brewing and autumn brewing in a Shaoxing rice wine factory was utilized to systematically investigate the formation and degradation characteristics of biogenic amine during the fermentation of rice wine.The biogenic amine content of the raw materials was 7.0～35.0 mg/kg.Biogenic amine content in rice wine brewed during winter was very high.This was due to their formation during the pre-fermentation stage and the post-fermentation stage（402.6 mg/kg）,however,they were degraded during the post-fermentation.Biogenic amine content did not show significant change at the mature stage.The total biogenic amine content was 210.2 mg/kg in the end product.The biogenic amine content in rice wine brewed during spring（28.9 mg/kg）was lower than those of the other two types of rice wines.The biogenic amines were accumulated in the pre-fermentation stage（30.3 mg/kg）and degraded in the post-fermentation stage（23.3 mg/kg）,but biogenic amine content slightly increased at the mature stage.Biogenic amine content in rice wine brewed during autumn increased gradually at pre-fermentation stage and reached the maximum value（186.7 mg/kg）at post-fermentation stage.The biogenic amine content was degraded at late fermentation and maturity stages.The biogenic amine content in final autumn-brewed wine was 53.6 mg/kg.Putrescine,tyramine and tryptamine were the main biogenic amines in rice wine,accounted for over 90% of the total biogenic amines.Seventy-two samples were collected from raw materials and fermentation mash brewed in winter,spring and autumn,and their microbial community structures were analyzed using Mi Seq sequencing method.Raw materials,such as yeast,wheat koji and steamed rice,had high biodiversity,which were the main sources of microorganisms in the fermented mash.In fermented mash,the compositions of bacteriophyta and eumycophyta were consistent with those of raw materials.The structure of bacteria and fungi significantly changed during the fermentation.In the pre-fermentation and post-fermentation stages,the bacterial biodiversity gradually increased,and then decreased at the end of fermentation.The dominant bacteria were Lactobacillus,Lactococcus and Leuconostoc at the late stage of fermentation.The diversities of fungi and filamentous fungi（aspergillus,rhizopus,etc.）were gradually decreased,but the yeast kept high abundance during the fermentation.Based on the correlation analysis,there was a significant correlation between the formation/ degradation of biogenic amines and bacteria.Lactic acid bacteria was the major bacteria involved in the formation and degradation of biogenic amines.Yeasts and molds were related to the formation of some biogenic amines,but were not the main microorganisms that produced biogenic amines.Forty-five strains of lactic acid bacteria which could not produce biogenic amines were screened from forty-five mash samples fermented in spring,autumn and winter in a Shaoxing rice wine factory.According to the degradation rate of biogenic amines,nine strains of lactic acid bacteria with degradation rate more than 30% were selected.According to the morphology of lactic acid bacteria,16 S r RNA sequencing,safety of bacteria,acid tolerance,ethanol tolerance and biodegradation rate of biogenic amines,a Lactobacillus plantarum DN2 strain was identified from nine strains.The strain originated from the pre-fermentation stage of autumn rice wine,and could degrade all eight biogenic amines,at a degradation rate of 48.3%.Growth properties and amine oxidase characteristics of Lactobacillus plantarum DN2 in the conditions of rice wine brewing were evaluated.Lactobacillus plantarum DN2 had good growth ability at fermentation temperature（28～33 ℃）,p H range（4.0～6.5）,and the ethanol content（≤14%）.Lactobacillus plantarum DN2 secreted seven kinds of amine oxidase,among which amine oxidase A（containing flavin）and monoamine oxidase had relatively stable protein structure,whereas amine oxidase B（containing flavin）and histamine oxidase were relatively unstable proteins.For the degradation reaction of biogenic amines by amine oxidase,the optimum temperature was 28 ℃,and the optimum p H was from 5.2 to 5.8.Small amount of Cu²⁺,Fe²⁺,Zn²⁺,Ca²⁺,Mg²⁺ had a slight inhibition for the activity of amine oxidase.The effects of Lactobacillus plantarum DN2 added in pre-fermentation,post-fermentation,and both in pre-fermentation and post-fermentation on degradation of biogenic amines and on the quality of rice wine were investigated.When 1.0×10⁶ CFU/g Lactobacillus plantarum DN2 was added in the pre-fermentation mash,the degradation rate of total biogenic amines could reach 49.5%,which did not affect the quality of rice wine.When the same amount of Lactobacillus plantarum DN2 was added in the post fermentation mash,the degradation rate of total biogenic amines（37.0%）and the quality of rice wine all decreased.Adding Lactobacillus plantarum DN2（1.0×10⁷ CFU/g）in pre-fermentation and post-fermentation mash respectively,the degradation rate of total biogenic amine reached 61.4%,but the quality of rice wine was lower than the standard.A pilot test of 360 kg fermenting mash was carried out in a Shaoxing factory.1.0×10⁶ CFU/g Lactobacillus plantarum DN2 added in the pre-fermentation mash leaded to a total biogenic amines degradation rate of 50.3%,and the quality of final product matched with the standard of Shaoxing rice wine.