Identification, Pilotscale DVS Starter Manufacture And Fermentation Technology Of Lactobacillus Casei Gbhm-21

Lactobacillus casei is a lactic acid bacteria and probiotic ordinarily used in foods. Due to its outstanding fermentation characteristics of generating acid and fragrant, Lactobacillus casei has been widely used in fermented foods, and the studies on its application focused on metabolites and flavour compounds analysis during fermentation. This study systematically investigated Lactobacillus casei GBHM-21(L.casei GBHM-21), a strain isolated from Japanese tratidtional fermented beverages. The strain were identified to subspecies, and were produced to DVS(Direct Vat Set) in a 200 L pilotscale experiment. Furthermore, the fermentation characteristics and the application in fermented milk flavour concentration(FMFCs) of L. casei GBHM-21 were investigated, which will provide the theory basis for the development of L. casei GBHM-21. The main conclusions are as follows:(1) Strain GBHM-21 were accurately identified to subspecies as Lactobacillus casei by its cell morphological, physiological and biochemical characteristics, its 16 S r RNA gene sequence and housekeeping gene phe S gene sequence. This strain is allowed to be appllied in ordinary foods as lactic acid bacteria and health-care products as probiotic bacteria.(2) DVS with high viable cell number and stability is necessary and important for the application of L.casei GBHM-21. The high density cell cultures(HDCCs) of L.casei GBHM-21 were optimized and adjusted in a 200 L pilotscale experiment: separating the glucose from other materials of the medium to avoid nutrient loss resulting from Maillard reaction during cooling hysteresis; optimizing the HDCCs process timing parameter by index p H changing rate. The final viable cell count was up to(1.44-6.42) ×1010 cfu/m L. The industrial continuous-feeding high-speed centrifugation conditions were optimized as: 20000 rpm, feed rate 2 L/min, by exploring the relationship between the centrifugal speed and feed rate.(3) The refrigerated concentrated Direct-Vat-Set(DV of L.casei GBHM-21 were prepared by mixing bacterial centrifugation and protective agent(trehalose saturated solution in glycerol) at the rate of 1:10. The viable cell count of refrigerated concentrated DVS kept under 4℃ within two months could stabilized at 1010 cfu/g. The viable cell count of frozen-dried DVS of L.casei GBHM-21 kept under-20℃ for 24 months could stabilized at 1011 cfu/g. The stability of viable cell count in frozen-dried DVS had a linear, inverse relationship with water activity(Best lower than 0.300).(4) Milk fat level is an important factor of flavour of fermented dairy products. The effects of milk fat level(0–90 g/L) on the changes in flavour-related compounds in milk bases fermented by L. casei GBHM-21 for 72 h were investigated. The free fatty acids(FFAs) and free amino acids(FFAs) selectively, and quality and intensity of volatile compounds(VCs) significantly increased with fat concentration increase. No significant effect on SCFAs was observed, while substantial increase in long-chain fatty acids was obseved. The positive relationship between total FFAs and FAAs was found to be a linear one. The most abundant group of VCs was ketones for low-fat samples and carbonyl acids for high-fat samples. The increase in FFA-derived ketones was first dramatic and then tapered, while increase in FFA-derived carbonyl acids was linear. These results would be useful to better expand fat application in fermented industry.(5) We evaluated the enzyme activity and the relationship between β- galactosidase and H+-ATPase of L.casei GBHM-21 during fermentation, discovering that L.casei GBHM-21 having an outstanding fermentation characteristics on acidity producing ability and high resistance of acid. Furthermore, L.casei GBHM-21 was applied in FMFCs fermentation with high acidity, mor metabolites and rich flavour compounds.(6) The synergistic effects of different levels of lipase(0.01–0.05%) and L. casei GBHM-21 were investigated on the dynamics of strain, FFAs, FAAs, volatile compounds and microstructure during ripening of fermented milk flavour concentrates(FMFCs). The addition of lipase significantly affected acidity and viable cell count, and increased the FFA, volatile compounds, and FAA content considerably, in a concentration-dependent manner. Addition of lipase increased the ester content, while addition of L. casei GBHM-21 brought ketone and others content. 2-Tridecanone and 2-butanone are two important parameters that distinguish the differences in volatile compounds of FCMCs.(7) Lipase addition concentration significantly affected L.casei GBHM-21 fermentation metabolism: suitable lipase concentration(≤0.03%) promoted L.casei GBHM-21 fermentation in FMFCs; and when lipase added amount was over 0.03% to 0.05%, the high acid inhibited L.casei GBHM-21 proliferation and intracellular and extracellular enzymes activity. Therefore, FFAs accumulation rate decreases, the FAAs concentration reduced, microstructure became uneven for over hydrolysis. The principal components of VCs differed significantly with lipase concentration and prolonged ripening.