Abstract
Optimization of Carnobacterium divergens V41 growth and bacteriocin activity in a culture medium deprived of animal protein, needs for food bioprotection, was performed by using a statistical approach. In a screening experiment, twelve factors (pH, temperature, carbohydrates, NaCl, yeast extract, soy peptone, sodium acetate, ammonium citrate, magnesium sulphate, manganese sulphate, ascorbic acid and thiamine) were tested for their influence on the maximal growth and bacteriocin activity using a two-level incomplete factorial design with 192 experiments performed in microtiter plate wells. Based on results, a basic medium was developed and three variables (pH, temperature and carbohydrates concentration) were selected for a scale-up study in bioreactor. A 23 complete factorial design was performed, allowing the estimation of linear effects of factors and all the first order interactions. The best conditions for the cell production were obtained with a temperature of 15°C and a carbohydrates concentration of 20 g/l whatever the pH (in the range 6.5-8), and the best conditions for bacteriocin activity were obtained at 15°C and pH 6.5 whatever the carbohydrates concentration (in the range 2-20 g/l). The predicted final count of C. divergens V41 and the bacteriocin activity under the optimized conditions (15°C, pH 6.5, 20 g/l carbohydrates) were 2.4 x 1010 CFU/ml and 819200 AU/ml respectively. C. divergens V41 cells cultivated in the optimized conditions were able to grow in cold-smoked salmon and totally inhibited the growth of Listeria monocytogenes (< 50 CFU g-1) during five weeks of vacuum storage at 4° and 8°C.
Highlights
Microbiological quality of food, especially ready-to-eat food, is a constant concern in the food industry
The interactions of NaCl with carbohydrates (−0.19), pH (−0.16), or ascorbic acid (−0.08) or the interaction of temperature with ascorbic acid (−0.10) had a negative estimated effect, whereas the interaction of sodium acetate with NaCl (+0.13) had a positive effect on the response. Those results indicate that the inhibiting effect of NaCl on growth is reinforced at high value of carbohydrate, pH and ascorbic acid, but lowered in presence of sodium acetate
A different pH optimum for growth and for bacteriocin activity has already been observed for a bacteriocin-producing strain of Lactobacillus curvatus (Messens et al, 2003)
Summary
Microbiological quality of food, especially ready-to-eat food, is a constant concern in the food industry. The preservative hurdles used in such products are salting, drying, smoking, vacuum-packaging, and chilledstorage, in association with good hygienic and manufacturing practices in industry These hurdle could be combined with lactic acid bacteria (LAB) used as an extra hurdle to prevent the growth of spoiling and pathogenic bacteria. Optimization is performed in laboratory culture media such as MRS (De Man et al, 1960) but some studies report cultivation tests on several raw materials, residues or by-products of low- (or zero-) cost for the production of bacteriocins (Todorov and Dicks, 2005, 2007a; Metsoviti et al, 2011; Garsa et al, 2014; Bali et al, 2016). In France, the use of cattle materials (like animals’ flours) is totally prohibited in the feed for animals intended for human consumption (Règlement (CE) n◦999/2001) as well as
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