| Kefir originated in the Caucasus,the world’s longest-lived region.It is a fermented dairy product with a small amount of alcohol and a variety of probiotic functions.Due to the complex and unstable conformation of the Kefir granule flora,most of the strains are unknown,and it is not easy to be subcultured for a long time.Therefore,screening the main strains that constitute the Kefir granule to prepare a variety of probiotic mixed fermented milk has become a research hotspot.At the same time,the preparation of starter cultures with clear bacterial phase structure and fixed bacterial composition can promote the industrial production of Kefir milk.In this study,a representative strain(Lactobacillus plantarum)、(Saccharomyces cerevisiae)and(Acetobacter)were selected from the three main microflora in Kefir grains.A new type of Kefir fermented dairy product was prepared by response surface experiment,and its flavor substances were detected and the mechanism of fermentation metabolism was preliminarily explored.The structure of different groups of fermented milk was characterized,and amino acid analysis was used for nutritional evaluation to explore the differences between them.GC-MS non-targeted metabolomics analysis was performed on different groups of mixed fermented milk,and the mechanism leading to the difference in flavor of different groups of fermented milk was preliminarily studied by differential metabolites.Finally,on the basis of high-density fermentation of Lactobacillus plantarum L1 and Acetobacter A3,Saccharomyces cerevisiae TN1 was compounded to prepare Kefir milk direct-vat starter.The experimental results are as follows:(1)Four yeast strains were screened from the traditional starter melon koji,and their acid resistance,high temperature resistance and alcohol resistance were investigated by Duchenne test.Among them,strains TN1 and N2 grew vigorously and had higher biomass.The aroma components of fermented milk of TN1 and N2 were analyzed by solid phase microextraction-gas chromatographymass spectrometry(SPME-GC-MS).It was found that there were many kinds of aroma substances produced by TNi fermented milk,and the content of typical flavor substances was moderate,which was suitable for the subsequent development of Kefir milk.The strain TN1 was preliminarily identified as Saccharomyces cerevisiae by morphological observation,26S rDNA sequencing and phylogenetic tree construction.(2)Lactobacillus plantarum,acetic acid bacteria and self-selected Saccharomyces cerevisiae preserved in the laboratory were used to ferment milk together,and the effects of different strains on product quality and flavor substances were discussed.Based on the single-factor test of single strain and two-two compound strains,the Box-Behnken central composite design and response surface analysis were used to optimize the strain ratio of three strains mixed fermented yoghurt with sensory evaluation as the index.The physical and chemical indexes,texture and gas chromatography-mass spectrometry(SPME-GC-MS)were used to explore the sensory quality and main flavor substances of the products in each experimental group,and the ratio of Kefir starter was determined.In order to facilitate large-scale production,the fermentation process was studied,and the response surface optimization test was carried out with white sugar,fermentation temperature,fermentation time and inoculation amount as factors.The results showed that the optimal ratio of Kefir main starter culture was L1:TN1:A3=2.0:0.3:2.0(the initial number of L1 bacteria was 4.1×108 cfu/mL,the number of TN1 bacteria was 8.1 ×107 cfu/mL,and the number of A3 bacteria was 2.9×108 cfu/mL).The sensory score of the optimized group was the best,the product flavor was better,and the flavor substances such as L(-)-lactic acid ethyl ester,2-methylpropanol and phenylethyl alcohol were unique in traditional Kefir.The suitable fermentation time was 12 h,the fermentation temperature was 37℃,the total inoculation amount was 7%(V/V),and the addition amount of white granulated sugar was 5%(W/W).The test provided a basis for the development of Kefir starter culture and related products.(3)The fermented milk of Kefir group and single Lactobacillus plantarum L1 group were characterized by scanning electron microscopy(SEM),laser confocal microscopy(CLSM)and lowfield nuclear magnetic resonance(LF-NMR)to explore the relationship between texture,rheology,microstructure and water distribution and the suitable tissue state.The results showed that the addition of acetic acid bacteria and yeast increased the water content of kefir,resulting in a decrease in its water holding rate,and with the increase of acidity,protein aggregation,whey precipitation and structural instability;rheological analysis shows that with the increase of shear force,the apparent viscosity decreases faster.CLSM,LF-NMR and SEM showed that the hydration and degree of freedom of Kefir yoghurt protein decreased,resulting in an increase in protein network density and a weak cheese structure.Therefore,Kefir milk is not suitable for the preparation of set yoghurt,and is more suitable for making refreshing yoghurt drinks.In addition,the analysis of amino acid content showed that the change of amino acid content was closely related to the synthesis of flavor substances.(4)The differential metabolites in yogurt of different fermentation groups were identified by gas chromatography-mass spectrometry(GC-MS)non-targeted metabolomics analysis technology,and the results were analyzed by multivariate statistical analysis.The results showed that there were differences in the metabolites of yoghurt with different combinations of strains.Based on the results of principal component analysis(PCA)orthogonal partial least squares-discriminant analysis(OPLS-DA),it was found that the four groups of samples had good separation,suitable parallel sample spacing,unfitted model,and good predictability.The main differential metabolites were 2,3butanediol and acetal.The former was slightly sweet and sticky,mellow and long aftertaste,which gave Kefir fermented milk a unique taste.The latter is an important substance to regulate the flavor and softness of the wine.Through KEGG pathway enrichment,a total of 28 significantly enriched pathway pathways and 10 very significant enrichment pathways were obtained(p<0.01).Based on topological analysis,the key metabolic pathways were screened,and eight important metabolic pathways were screened.These pathways include:galactose metabolism,glyceride metabolism,butyric acid metabolism,glyoxylic acid and dicarboxylic acid metabolism,nicotinic acid and nicotinamide metabolism,tyrosine metabolism,methane metabolism,pentose and glucuronic acid mutual transformation.This study provides some theoretical guidance for the regulation of flavor substances and the formation of flavor substances in Kefir fermented milk.(5)In order to develop a kefir direct-vat starter culture,the high-density culture conditions of Acetobacter A3 were as follows:the ratio of yeast powder to peptone was 1:2,the ratio of carbon to nitrogen was 5:1(that is,the mass fraction of carbon source was 3%,the mass fraction of nitrogen source was 0.6%),and the inoculation amount was 4%(V/V).The growth curves of Lactobacillus plantarum L1 strain at the ratio of yeast powder to peptone of 1:2,1:1 and 2:1 all reached the stable period after 18 h,and the bacterial content is not much different.Therefore,the same ratio of yeast powder to peptone as A3 was selected.At this time,the optimal carbon-nitrogen ratio was 10:1(carbon source mass fraction was 3%,nitrogen source mass fraction was 0.3%),and the inoculation amount was 4%(V/V);YPD medium was used for high-density culture of saccharomyces cerevisiae TN1 to a stable period.Finally,the three strains were compounded to prepare Kefir mixed starter culture.The freeze-dried protective agent formula was skimmed milk powder 12%(W/W),trehalose 14%(W/W),L-cysteine hydrochloride 0.15%(W/W).In conclusion,the Kefir milk produced by the fixed strain and the fermentation process has a unique flavor and sand taste.After GC-MS detection,it is determined that the main flavor substances in the traditional Kefir milk are reproduced,with alcohols that are not available in the market.The structural characterization of the prepared Kefir milk was carried out to explore the reasons for the structural changes and provide ideas for the type of fermented milk produced by subsequent factories.A classical research pathway was found through non-targeted metabolomics;through high-density fermentation experiments and factory design,it has practical guidance for Kefir milk production. |
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