Optimization Of High Density Cultivation Process Of Lactobacillus Delbrueckii Subsp.bulgaricus QH38-1

With the continuous expansion of the scale of starter culture industry,improving the density and activity of starter culture has become the key to bring considerable economic benefits in related fields.In this study,a Lactobacillus delbrueckii subsp.bulgaricus QH38-1 with excellent fermentation characteristics was used as the research object.The optimal medium and static culture conditions were optimized by single factor,compound,orthogonal test and response surface design.At the same time,from the perspective of symbiotic relationship between Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus,the metabolites of Streptococcus thermophilus which can promote the growth of Lactobacillus delbrueckii subsp.bulgaricus in co-culture were explored,and the high density fermentation of single bacteria was achieved by exogenous addition.The main conclusions of this study are as follows:（1）Through the screening and optimization of carbon sources,nitrogen sources,total and proportion of carbon and nitrogen,buffer system,trace elements and growth factors and other components and dosage of the medium,we obtained the best medium formula of Lactobacillus delbrueckii subsp.bulgaricus QH38-1:Sucrose 14.81g/L,fish peptone 46.07g/L,triammonium citrate/sodium acetate/K₂HPO₄ buffer system 9.23g/L,Mg SO₄·7H₂O 0.25g/L,Mn SO₄·5H₂O 0.05g/L,Zn SO₄·7H₂O 0.25g/L,Vc 0.5g/L.Using the optimized medium,the number of viable bacteria could reach（1.49±0.30）×10⁹CFU/m L,which was 1.69 times higher than that using MRS Medium（8.80±0.22）×10⁸CFU/m L,and the buffer capacity was 1.82 times higher than that using MRS Medium.In addition,the optimized medium uses sucrose and fish peptone as carbon and nitrogen sources,which effectively reduces the cost of fermentation medium.（2）Based on the optimal culture formula of Lactobacillus delbrueckii subsp.bulgaricus QH38-1,the static culture conditions such as static fermentation temperature,initial p H of fermentation and inoculation amount were optimized,and the optimal static culture conditions were obtained as follows:The culture temperature was 37℃,the initial p H was 6.50,the inoculation amount was 5%,and the number of viable bacteria reached（1.67±0.15）×10⁹ CFU/m L at the end of logarithm,which was 1.70 times higher than that before the optimization（9.82±0.32）×10⁸ CFU/m L.These results indicated that the number of viable bacteria of Lactobacillus delbrueckii subsp.bulgaricus QH38-1 could be significantly increased by optimizing the static culture conditions.（3）Based on the optimal culture formula and static fermentation conditions,pyruvic acid,glutathione and folic acid,which can significantly promote Lactobacillus delbrueckii subsp.bulgaricus QH38-1,were selected as exogenous additives,and the optimal proportion of each component was obtained through the response surface:Pyruvate 656.85 mg/L,glutathione 765.01 mg/L and folic acid 76.62 mg/L were added to the fermentation matrix by one-time feeding method.Fermentation parameters were set as follows:inoculum volume 5%,initial p H 6.50,culture temperature 37℃,rotation speed 200 rpm/min,neutralizer 25%ammonia water,constant control p H 5.90,nitrogen pressure preservation（0.025 MPa）.The high density fermentation was performed on the optimized medium and culture condition,and the fermentation was terminated after 8h.The results showed that the viable bacteria count of the fermentation liquid in the optimized medium supplemented with supplemental feed could reach（8.32±0.17）×10⁹CFU/m L,which was 2.30 times higher than that in the optimized medium supplemented without supplemental feed.Compared with the control group of MRS Medium without supplement,the logarithmic growth period was extended by 1 hour,the number of viable bacteria was increased by 4.28 times,and the activity of phosphofructokinase,a key metabolic enzyme,was increased by 1.89 times.The optimal medium formula and static culture condition of Lactobacillus delbrueckii subsp.bulgaricus QH38-1 have been determined,and the addition of exogenous additives such as pyruvate,glutathione and folic acid can significantly increase the viable bacteria count and key enzyme activities in the fermentation broth.These results provide important guidance for the mass production of this strain,and provide reference for the optimization of culture conditions of similar strains.In the future,the growth,acid-producing ability and metabolic characteristics of the strain under different culture conditions can be further studied and optimized to meet the needs of industrial production and application.