| Kefir is a kind of compound fermented dairy product,which is fermented by dozens of microorganisms mainly including lactic acid bacteria and yeast.It has unique taste and high nutrition.It has the functions of disease resistant bacteria,regulating intestinal flora,lowering cholesterol,blood pressure,antitoxin,anti-inflammatory,immune regulation,etc.In order to increase and endow kefir with stronger prebiotic characteristics,a probiotic Lactobacillus rhamnosus hsryfm 1301 with multiple functional characteristics was added to the traditional kefir fermentation agent,which was fermented by 1:1(v/v),5:1(v/v)and 25:1(v/v),the effects of adding this exogenous probiotic on the fermentation characteristics,enzyme activity characteristics,antibacterial ability,metabolites and other related indicators of traditional kefir fermentation milk were studied.The results are as follows:1.By measuring kefir fermented milk,L rhamnosus hsryfm 1301 and mixed fermented milk(KL 1:1,KL 5:1,KL 25:1)the indexes of hardness,adhesiveness,cohesiveness,elasticity,stickiness,mastication,viscosity and water holding capacity of kefir fermented milk were found to be significantly improved after adding different proportion of exogenous probiotics hsryfm 1301,which was positively correlated with the addition amount of hsryfm 1301;pH value was negatively correlated with the addition amount of hsryfm 1301;coagulation speed was positively correlated with the addition proportion of hsryfm 1301;mixed fermented milk was more positively correlated with the addition amount of hsryfm 1301 than kefir fermented milk;the hardness,adhesiveness,elasticity,adhesiveness and chewability of kefir fermented milk did not change significantly(p>0.05),the viscosity increased significantly(p<0.05),the water holding capacity increased by 0.54%?5.24%,and the water holding capacity basically reached the level of hsryfm 1301 fermentation.Kefir and hsryfm 1301 mixed fermented milk(KL 1:1)had the best quality,the curdling time was 16 h,the acidity was 89.7°T,viscosity and water holding capacity were respectively 1527.67 cp and 74.06%.2.The activity of LDH,?-gal and ?-gal in kefir fermented milk three key enzymes of lactose fermentation were determined.It was found that the mixed fermentation with exogenous probiotics hsryfm 1301 inhibited the activity of LDH,?-gal and ?-gal in kefir fermented milk.When mixed with 1:1 ratio,the activity of KL 1:1 was the lowest,LDH was only 46%of kefir fermented milk,?-gal was only 0.098 U/mg,?-gal was 0.122 U/mg lower than kefir fermented milk.In addition,through the correlation analysis of the quantity of hsryfm 1301,fermentation index and enzyme activity index,it was found that the longer the curd time of mixed fermented milk was,the stronger the activity of LDH and ?-gal was;the higher the curd acidity was,the stronger the activity of LDH and ?-gal was;the higher the viscosity was,the lower the activity of ?-gal and LDH was;the activity of LDH and ?-gal was negatively correlated with the water holding capacity;the more hsryfm 1301 was added,the weaker the activity of LDH and ?-gal was.3.In order to study the bacteriostatic effect of single bacteria fermented milk and mixed fermented milk,the inhibition of Staphylococcus aureus,Bacillus subtilis,Bacillus cereus,E.coli,Salmonella enterotyphi and Pseudomonas brenneri was determined by co culture and single-layer agar plate diffusion method,it was found that adding hsryfm 1301 enhanced the growth inhibition ability of kefir fermented milk.The results of co culture experiment showed that:when the metabolites of fermented milk were co cultured with pathogenic bacteria 100:1,the inhibition effect of hsryfm 1301 fermented milk was the best,the pathogenic bacteria almost did not grow,but kefir fermented milk was the weakest.The inhibition ability of mixed fermented milk increased with the increase of the proportion of hsryfm 1301.When the proportion of mixed fermented milk was 1:1,the growth rate of six pathogens is the lowest.The maximum growth rate of Staphylococcus aureus,E.coli,S.enterotyphi in 24 hours was only 22.22%,28.40%and 13.55%of the control group in 4 hours.The results of single-layer agar plate diffusion experiment showed that the inhibition ability of kefir fermentation supernatant to six pathogens was lower than that of hsryfm 1301,and the inhibition effect of mixed fermentation milk fermentation supernatant to six pathogens was broad-spectrum;after adding hsryfm 1301,the inhibition effect of kefir fermentation milk to six pathogens was improved in varying degrees,and the inhibition effect intensity was enhanced by the proportion of hsryfm 1301 KL 1:1 is the most prominent,and its bacteriostatic ability is not significantly different from that of hsryfm 1301 fermented milk.It is better than Gram-positive in improving the bacteriostatic ability of Gram-negative bacteria,E.coli,S.enterotyphi and P.brenneri increased by 25.52%,29.10%and 38.98%,respectively.The bacteriostatic ability of KL 1:1 against S.enterotyphi,P.brenneri and Bacillus subtilis was the strongest,and the diameter of inhibition circle was 27.33 mm,26.17 mm and 21.17 mm respectively.In addition,in the neutral condition,the fermentation liquid has no bacteriostatic ability;after heat treatment,the diameter of bacteriostatic circle reduces.Therefore,it is speculated that the existing bacteriostatic substances are acid substances,some substances which cooperate with acid to complete the bacteriostatic effect,and some thermosensitive substances4.The content of acetaldehyde,diacetyl,organic acid and volatile matter in single fermentation milk and mixed fermentation milk were determined.The results showed that the addition of hsryfm 1301 had no significant effect on the diacetyl production capacity of kefir fermentation milk(p>0.05),but increased the acetaldehyde production capacity of kefir fermentation milk(p<0.05).The acetaldehyde production of 1:1 mixed fermentation milk was the highest,27.06%higher than that of kefir fermentation milk,11.88%higher than that of hsryfm 1301.The content of five organic acids in mixed fermented milk also changed in different degrees.KL 1:1 of mixed fermented milk was the most significant(p<0.05).The content of lactic acid,citric acid,fumaric acid and malic acid increased by 27.99%,0.63%,14.55%and 19.95%respectively compared with kefir fermented milk,and the content of acetic acid decreased by 31.15%,among which the content of lactic acid increased by 4.55%compared with hsryfm 1301.The main components of the volatile metabolites of mixed fermented milk and single fermented milk were not significantly different.There were more acids,esters and alkenes in the volatile metabolites of KL 1:1 fermented milk,among which the relative contents of furfuryl alcohol,benzaldehyde and 2,4-di-tert-butylphenol,styrene,4-phenylacetic acid tridecyl ester and acetic acid were higher,and the acetic acid content was 4.36%higher than that of hsryfm 1301.Ethanol,a characteristic product of kefir,was not found,and 0.11%DMSO was detected in KL 1:1 mixed fermented milk.In addition,it was found that there were three potential antibacterial components in KL 1:1 fermented milk besides alcohols and acids:m-xylene,2,4-di-tert-butylphenol and dimethyl sulfone. |
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