Study On Preparation And Application Of Direct Investment Type Bacillus Natto Starter Culture

Bacillus natto starter culture is an indispensable strain for industrial production of natto.This paper aimed to employ a simple and efficient method to prepare a highly active direct investment type Bacillus natto starter culture which could ferment nutrient-rich"colorful"natto.Pilot scale experiments were carried out and the main researches were as follows:?1?Through the screening of Bacillus natto,the Bacillus natto Y3 with the capacity of high-yield amylase and protease,heat resistant,hereditary stability and good fermenting property was obtained.The multiplication culture conditions of the Bacillus natto were optimized by single factor and orthogonal test and the optimal culture medium formula was obtained as:soluble starch 2%,yeast extract 1.5%,MnSO4 0.08%,MgSO4 0.08%,NaCl 0.8%and CaCl₂ 0.3%.The optimum culture condition was obtained as:initial pH 7.0,temperature 40°C,inoculation amount 5.0%,and shaker speed 170 r/min.After culturing24 h,the viable count and spore rate increased significantly which were up to 5.68×10¹⁰cfu/mL and 88.65%respectively.They were 96.3%and 30.7%higher than they were unoptimized..?2?The direct investment type Bacillus natto starter culture was determined by optimizing the low temperature drum wind drying method,and the optimum technology was:Bacillus natto collection condition was that centrifuged 5000 r/min for 20 min,the compound dry protective agents were that dried skim milk 11.07%,sodium glutamate6.41%and sodium citrate 14.76%,the mixing ration of bacterial sludge and protective agent?w/v?was 1:7 at pH 6.0 for 35 min,added rice flour as the carrier substrate of Bacillus natto,and the optimum mixing ration?v/w?of bacterial suspension and rice flour was 1:2,the drying condition of starter culture was at 50°C for 9 h.Under these conditions the survival rate of Bacillus natto reached 82.66%,and the bacterial content was up to7.93×10¹⁰ cfu/g.The viable bacterial number and fermentation activity had no obvious change after storing at 4°C or-20°C for 180 d.?3?The direct investment type Bacillus natto required rehydration activation before using.The best activation condition was:8%skim milk as the rehydration activation medium of Bacillus natto agent,the starter culture and the rehydration activation medium were mixed in the proportion of 1:100?W/V?at 36°C for 60 min,and viable count of Bacillus natto and nattokinase activity reached 1.01×10⁹ cfu/mL and 415.87 U/g,respectively after solid state fermentation which were significantly higher than the control group without activation.The optimum technology of"colorful"natto was:the proportion of soy bean,mung bean,black bean,red bean and bridled bean was 3:1:1:1:1,high pressure steamed for 40 min,bean amount with 70 g/250 g?the thickness of the beans was about 3 cm?,inoculation amount 4.0%,fermentation temperature 32°C,fermentation time38 h,and after-ripening time 24h.The"colorful"natto had good flavor,high sensory quality score of 97.2 point,the nattokinase activity was up to 611.53 U/g by this technology.The"colorful"natto had red bean fragrance which was more in line with the tastes of Chinese consumers.More importantly,the nutritional value of natto was more abundant.?4?According to the best"colorful"natto preparation technology of laboratory experiment,6 batches of pilot-scale production test were carried out.The results showed that this preparation technology was stable and feasible,and fit for pilot-scale production with great prospects for development.