Abstract: Objective:To optimize a cost-effective medium for high cell density fermentation of Lactobacillus plantarum KLDS 1.0391 and produce probiotic direct-in-vat starter culture. Methods:The number of viable bacteria and antimicrobial activity of fermentation broth were determined,fermentation effects of skim milk powder,whey powder,wheat bran with different hydrolysis degrees and corn steep powder were compared. The direct-feeding starter of Lactobacillus plantarum in freeze-dried form was prepared and added to yogurt to evaluate its effects on fermentation time,pH,titration acidity,viscosity and sensory evaluation,and determine its feasibility in yogurt application. Results:The optimum hydrolysis degrees of skim milk powder,whey powder and wheat bran were determined to be 15%,15% and 20%,respectively. The best fermentation results were obtained when Lactobacillus plantarum KLDS 1.0391 was cultured in corn steep powder medium,with the number of viable bacteria achieved 9.53 lg CFU/mL and the diameter of bacteriostatic circle achieved 12.69 mm. The addition of direct-in-vat starter culture of Lactobacillus plantarum exhibited no significant effect on the fermentation time and sensory evaluation of yogurt(P>0.05),while the viscosity of yogurt decreased and the post-acidification of yogurt could be delayed. Conclusion:The low-cost natural medium for high cell density fermentation of Lactobacillus plantarum KLDS 1.0391 was obtained in this study,its components were 4% corn steep powder,supplemented with 2% glucose and 0.1% Tween-80. Conclusion:The direct-in-vat starter culture of Lactobacillus plantarum produced in this study could be used in yogurt production.