Development Of Culture Starter For Industrial Production Of Sayram Yoghurt

The Sayram yoghurt culture starter (Lactobacillus helveticus MB2-1) from laboratory is extracted from Xinjiang traditional yogurt Ketec. This yogurt is thick brushed, has a unique flavor. As a traditional product, it herited a excellent quality and doesn’t contain any additives. Currently, dairy products have lots of quality concern, at the meantime, the yogurt fermentation agent market is dominated by foreign companies. It is more urgent than ever to carry forward excellent traditional products, as to develope the the yogurt fermentation agent technology in order to lanch its chinese indepent production line. The purpose of this experiment is to develop the Sayram yoghurt culture starter using industrial tank, ceramic membrane and spray drying techniques. In order to count viable cells during the process, the MTT colorimetric methode application is studied as well. In this study, the main findings are as follows:1-MTT colorimetric methode application on Lactobacillus helveticus MB2-1viable cells countingMTT colorimetric methods broth has gone through different operating wavelength scan in order to select the optimum operating wavelength510nm. And further more, influence of different reagents on this optimal working wavelength have been observed. The tendance linair curve established between MTT viable count method reslut, the OD values, and the result obtained with the plate dilution counting(cfu/mL) proves the faisibility of MTT colorimetric method application on Lactobacillus helveticus MB2-1celle counting; relative coefficients of this linear relationship determinated the optimum working conditions of the MTT method as follows:broth1mL, MTT0.2mL, inoculation temperature40℃, the reaction time1H, DMSOlmL; significance analysis of data on different concentrations of broth using MTT colorimetric counting method has showed that this methode is applicable for cell concentration107-109cfu/mL; this study has also confirmed the necessity of the pre-treatment of the MTT assay:saline diluted broth was centrifugated at3500t/min of10min, the washed cells were collected and rewashed for2times; as well as the necessity of a high-speed centrifugal separation of formazan:remove the supernatant after a12000t/min centrifugation for10min in order to isolate formazan; Finally, the MTT colorimetric methode application standard linear for Lactobacillus helveticus MB2-1is determined (the number of viable cells C (cfu/mL) in terms of OD510value):if OD510is less than0.48, the plate dilution counting method result C×109=1.267×OD510+0.044, and when the measured OD value is above0.48, the plate dilution counting method reslut C×109=0.3359×OD510+0.4936. Both of correlation coefficients is above0.99.2. Lactobacillus helveticus MB2-1cultivationBy means of the MTT colometric method, single factor experiments was used to discover best culture medium ingredients and fermentation conditions for Lactobacillus helveticus MB2-1.(ex:the ratio of carbon and nitrogen, growth factor, pH buffer, fermentation temperature, initial pH value, inoculum volume). Then the Plackette Burman experiment was employed to determine ingredients who have a significant role in promoting cell growth. Afterwards, factors orthogonal experiment was used to identify Lactobacillus helveticus MB2-1’s best enrichment medium formulation (L-1):80g whey powder, yeast extract10.7g, wort leaching powder1.8g, distilled water1L; as to determine its optimal culture conditions:inoculation amount of4%, the initial pH value of6.4, incubation temperature of40℃. With this optimal cultivation methode obtained above, a fermentation of Lactobacillus helveticus mb2-1was monitored. The growing curve has showed that the optimal incubation time is10hours. At that time, Lactobacillus helveticus MB2-1concentration reached2.84×109cfu/mL, compared to its intial cultivation result(0.5×109cfu/mL). The cell concentration has raised5.68times. Bacterium cultivated by this optimal methode takes4.5hours to make milk into curd, and the yoghurt obtained in the end has a viscosity of2685.2Mpa.3. First process of industrial culture starter of Lactobacillus helveticus MB2-1production:cultivation in cuve of50LApply the optimal cultivation methode obtained above to cultivate Lactobacillus helveticus MB2-1in cuve of50L:a miniature of industrial equipment, which allows explore the possibility of industrial production.The cultivation program is described as follows:Set the material temperature at60℃, maintained it at this temperature for medium pasteurization for30min. After having cooled materials, set the heating temperature at38℃, the material temperature at40℃. Then inoculte4%(v/v) activated Lactobacillus helveticus MB2-1to ferment during10hours. Besides froms the beginning of fermentation, incubation stirring function should be closed. At the beginning of fermentaion, this funtion is used to mix well the medium and inoculum. The cultivation obtained a cell concentration of0.95×109cfu/mL,4. Second process of industrial culture starter of Lactobacillus helveticus MB2-1production:ceramic membrane ultrafiltrationThe broth obtained from50L cuve is concentrated through the ceramic membrane equipment. By following the amount of cycle flux and bacterial mortality in this ultrafiltration process, the ceramic membrane device parameters were decided as follows: set the working pressure as0.15Mpa, open the valve of ultrafiltration, close the exsit of concentrated solution, lanch the cycle of bactera concentration for about3-4hours in order to get the50L brothe concentrated. The cell survival rate was82.49%, the celle has been concentrated for7.4times. The cell concentration obtained was7.03×109cfu/mL.5.Third process of industrial culture starter of Lactobacillus helveticus MB2-1production:spray dryingAccording to the survival of bacterial cells on the spray-drying step, the moisture content of the dry culture starter obtained and the speed of the spray drying, one protective agent was chosen and so was its ration:a portion proective agent made by10%skim milk,5%sucrose and5%trehalose should be well mixed with the same portion broth. The operating parameters of spray drying have also been determined:Spray pressure was set at0.25Mpa, thimble pressure was set at0.3Mpa, inlet temperature was set at160℃, the outlet temperature of70℃. Pre-cleaning used distilled water, when the setting temperature is reached, hold it for10-15minutes. Then inlet the concentrated broth at the speed of5t/min. The final culture starter dry powder has the bacterial survival rate of84.32%, its viable cells reached1.06×1010cfu/g.6.Application of spray dried Lactobacillus helveticus MB2-lculture starterAccording to curd time in yogurt preparation, the curd pH, acidity as well as the yoghurt sensory quality, the application methode of Lactobacillus helveticus MB2-1culture started was determined:0.8%o(g/g) inoculum was added to pre-heated and then cooled to room temperature milk. Milk was then put under40℃for5.4hours, after confirming the milk pH has dropped to4.9, the fermentation should will be stoped, and curd milk was placed under4℃C for24hours in order to get a smooth texture, thick brushed Ketec yogurt.