Research Of Crux Technology On Preparing High-activity Concentrated Yoghourt Freeze-dry Starter Culture

With the rapidly development of fermented dairy product industry, It's very important to research and develop high activity concentrated yoghourt starter culture for promoting the industrial process of lactic acid bacteria starter and development of fermented dairy industry. In this paper, the crux technology of high-activity concentrated yoghourt starter was studied including isolating fine lactobacillus which was of fine fermentive character, high in the resistance of freeze-dry and low in the character of post acidification, filtration of the low cost culture medium and fine freeze-dry protestant, optimizing the condition of cell cultivation, concentration and separation, the technology of lyophilization and the storage of starter culture. In addition, a experiment at the level of 5L fermentive vessel and the sample of starter culture was prepared. A yoghourt fermentation experiment was conducted in dairy company. The study provided a scientific foundation for application of high-activity concentrated yoghourt starter in our country.Seven strains of Lactobacillus bulgaricus was isolated from the yoghourt produced by the famous dairy company or the commercial starter culture abroad. By studying on the character of fermentation, resistance of freeze-drying and post acidification character of different strains in milk and mixed proportion to Streptococcus thermophilus in milk, L.bulgaricus L.b-DR,L.b-Sl was screened and the optimum proportion to S.thermophilus S.t-SY was determined at 1:1.The growth experiment of lactobacillus in carrot juice, tomato juice, wort which are apt to gain and separate, was conducted. The result showed that wort was the best base medium for lactobacillus, which promoted not only S.thermophilus but also L.bulgaricus. The effect of adding 9 kinds of different nutrient factors in wort on the growth of S.t-SY was studied. The results showed that Soya peptone, Beef Extract, Yeast Extract could promote the growth of S.t-SY significantly in wort and K2HPO4 could not only promote cell growth, but also protect lactobacillus from being damaged by low pH of medium. The optimum enrichment medium obtained by orthogonality test L₉(3³) consist of adding 0.5% Soya peptone,0.5% Yeast Extract,l% Beef Extract,0.2% K₂HPO₄ and 0.1%Tween80 in the 10° wort medium.In the optimum enrichment medium, the living-cell number of S.t-SY , L.b-DR, L.b-S1 were 2.42×10⁹, 2.85×10⁹ ,4.81×10⁹cfu/mL cultivated for 16h at 37℃, which were increased by 16.13, 9.89, 22.58 times compared with wort base medium and the same with the living number in the MRS used in lab, but the cost was decreased 1000yuan/t compared with MRS medium.The effect of cultivation temperature, mode, initial pH of culture medium, inoculation amount, feed culture on the cell growth of lactobacillus was studied at the level of 300mL flask. The optimum condition to cultivate L.b-Sl and S.t-SY was that the initial pH of medium was 6.5, inoculation 2%, cultivated 37℃ for 16h. In the condition, the living-cell number of L.b-Sl,L.b-DR, S.t-SY was 4.31×10⁹,2.93×l0⁹,2.74×10⁹cfu/mL.On the base of 300mL flask experiment, the experiment of 5L fermentive vessel was conducted. The results showed that the living-cell number of L.b-Sl, L.b-DR, S.t-SY could reach 4.21 *10^ 2.76xlO9> 2.65x 109cfu/mL in the condition that the initial pH of medium was 6.5, inoculation 2%, velocity of stir was lOOrpm and cultivated at 37°C for 16h.The effects of centrifuging force, time on the loss rate, survival rate and harvest rate of lactobacillus was studied. The results showed that the optimum conditions for centrifugation of L.b-Sl was 1632g (4000r) for lOmin or 573 8g (7500r) for 5min and the highest harvesting rate of living cells was 98.12% or 97.81%, 98.35%sï¿¡ 97.89%, which of S.t-SY was 3672g(6000r) lOmin or 5738g(7500r)5min, the harvesting rate of living cells was 95.19% or 97.29%.The effect of freeze temperature, time and starter's moisture content on the livability of L.b-Sl was studied. The results showed that -20—-18°C and -40^-35°C did not effect the livability of L.b-Sl markedly(P>0.05), there was a significant difference in the freeze livability of L.b-Sl between the start-point and the end-point of freezing (P< 0.01) .However, after the culture of strain L.b-Sl was fully frozen, no significant loss in viability was found due to the extension of frozen time (P>0.05) .During freeze-drying, the livability was effected markedly by the moisture content of starter but there was a difference in the livability of cell because of the level of descending of starter's moisture content. It was the most significantly difference (i><0.01) during the course of descending of starter's moisture content from 40% to 5% and the livability of cell descended from 88.68% to 40.11%, about 50% (Z><0.01) .A complex protectant was screened, which could protect cell from damaging during freeze-dry and storage. After freeze-drying, the living-cell number of starter, whether single strain or mixed strains were over 10 cfu/g. The fermentive character of starter was fine and could make milk curdle when it was inoculated at 0.01%,cultivated at 42°C for 4⁵h.The yoghourt was delicious, the titration acidity could reach 80⁹0 °T and pH was about 4.5 4.6. Compared with traditional yoghourt manufacture, inoculation were decreased by 100 1000 times which could reach the international advanced level. Packaged with vacuumize and air, starter culture was storaged at -18°C > 4°C for 1 year, the reduced number didn't amount to one log rank and the living cell number was still over 1010cfu/g. At the same time, the starter's character was stable during storage, which was the same with directed Vat Set(DVS).The freeze-dry starter at 5L was prepared and a yoghourt fermentation experiment was done in some fermented dairy company. The results showed that the freeze-dried starter was of fine fermentive character, which was the same with the starter at the level of cuvette and the commercial starter from Rhodia or Chr.Hansen company which was famous for yoghourt starter culture, but the fermentive cost was decreased by 40yuan/t.The study provided a scientific foundation for industrialization application of high-activity concentrated yoghourt starter culture in our country.