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• The mycelium of F. velutipes was sensitive to bacterial contamination in cultivation. • Two natural biopolymers, ε-poly- l- lysine and chitosan, can effectively inhibit bacteria co-cultured with F. velutipes. • The antibacterial activity was enhanced by combining ε-poly- l - lysine with chitosan. • Hurdle technology was first applied to the control of bacteria in a submerged culture of F. velutipes for liquid spawn production. • The models were established to predict the growth of bacteria in the submerged production of F. velutipes liquid spawn. Flammulina velutipes is a commercially important mushroom that widely cultivated in East Asia. Sawdust spawn is extensively adopted in the cultivation of mushrooms including F. velutipes. Recently, the use of liquid spawn has become increasingly popular in mushroom cultivation in China and many other countries. Owing to the high risk of bacterial contamination in a submerged culture of mushroom mycelium, an attempt was made to examine the feasibility of using the hurdle technology to prevent and control bacteria with Escherichia coli and Bacillus subtilis as indicator strains during the production of F. velutipes liquid spawn. Two food-grade natural biopolymers, i.e. ε-poly-lysine (ε-PL) and chitosan, were selected as antimicrobials. Results showed that the mycelial colonization of F. velutipes was poor or inhibited completely by both indicator strains at the density higher than 10 colony-forming units (CFU)/mL present in the liquid spawn. In submerged cultures, F. velutipes was greatly suppressed by ε-PL at 0.25 mg/mL, and this was 0.30 mg/mL for chitosan. When combined, ε-PL and chitosan did not result in a significant enhancement of activity against the fungus. The antibacterial effects were enhanced when ε-PL and chitosan were combined at an ε-PL/chitosan mass ratio of 3:2. For effectively inhibiting the bacteria co-cultured with F. velutipes , ε-PL, chitosan, and temperature were designed as hurdles. Two mathematical models relating inhibition of E. coli and B. subtilis , respectively, were established by response surface methodology, and could be used to predict the behavior of the culture system. This study provides useful guidelines for the production of a reliable and high-quality liquid spawn of F. velutipes or other mushrooms. [ABSTRACT FROM AUTHOR]