Preparation and preservation of lactic acid starter culture concentrates Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/fb494b80g

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  • Streptococcus lactis C2F and Streptococcus cremoris 459 grew to a maximum population of 1 to 2 x 10⁹ colony forming units (cfu)/ml with a generation time of 96 min in 11% nonfat dry milk (NFDM) under static conditions at 22° C. Streptococcus diacetilactis 18-16 grew to a maximum population of 7 x 10⁸ cfu/ml with a generation time of 95 min under the same conditions. Incubation of S. lactis C2F at 28° to 30° C resulted in a reduction of the generation time to 60 min but no significant increase in the maximum population. Maintaining the pH of S. lactis C2F cultures at 6.3 with sodium hydroxide, potassium hydroxide or calcium hydroxide increased the maximum population to 4.5 to 5.5 x 10⁹ cfu/ml. When ammonium hydroxide, sodium carbonate or ammonium carbonate were used as neutralizing agents a maximum population of 8.4 to 8.9 x 10⁹ cfu/ml resulted. No significant change in generation time was observed except when ammonium hydroxide was used as the neutralizer when the generation time was reduced to 44 min. Sodium lactate formed during growth of S. lactis C2F cultures in which the pH was maintained with sodium carbonate was established as a contributing factor in limiting the maximum population. Increasing the percentage of NFDM above 11% (s/v) did not increase the maximum population level. However, there was an increase in the maximum population to 1.0 x 10¹⁰ cfu/ml when pH-controlled S. lactis C2F cultures were sparged with nitrogen gas at a low flow rate (controlled growth). S. cremoris 459 grown under these conditions (pH 6.3, 30° C) in NFDM had a generation time of 66 min and reached a maximum population of 8.7 x 10⁹ cfu/ml. Under the same conditions, S. diacetilactis 18-16 grew to a maximum population of 9.4 x 10⁹ cfu/ml in NFDM but only when the milk was supplemented with 1.5%, yeast extract. Concentrates of S. lactis C2F, S. cremoris 459 and S. diacetilactis 18-16 were prepared by adjusting NFDM cultures in the late logarithmic growth phase under controlled growth conditions, to pH 6.9 and adding sodium citrate to a final concentration of 4.5 %. Cells were harvested from the partially cleared medium by centrifugation. The sedimented cells were reconstituted to one-tenth the original culture volume, lyophilized and stored at -196, -22, +7 and +22° C. Lyophilization of S. lactis C2F cell concentrates (5.5 x 10¹⁰ cfu /ml) in 10% nonfat milk caused a 31% reduction in acid-producing activity of the cells measured immediately after freeze drying. When the cells were suspended in 5% monosodium glutamate, this effect was reduced to a 14% loss and use of 5% trehalose resulted in only a 6% loss in activity. Lyophilization of S. cremoris 459 and S. diacetilactis 18-16 cell concentrates in 5% monosodium glutamate, containing 4.2 x 10¹⁰ and 4.6 x 10¹⁰ cfu/ml respectively, caused a loss in activity of 31% for S. cremoris and 7% for S. diacetilactis 18-16. Using activity of the lyophilized concentrate as 100%, the concentrates of S. lactis C2F cells lyophilized in 10% NFDM and stored at +22 and +7° C showed greater than 80% decrease in acid-producing activity in seven days, 64% at -22° in 21 days and no reduction in 21 days at -196° C. Lyophilization of S. lactis C2F cell concentrate in 5% monosodium glutamate showed a reduction in activity of 70% in 21 days when stored at +22 and +7° C. Lyophilization of S. lactis C2F cell concentrates in 5% monosodium glutamate supplemented with 0.16 M potassium iodide resulted in a 32% and 20% reduction in activity when stored at +22 and +7° C respectively for 21 days; there was no loss when stored at -22 and -196° C. The addition of 0.05% ascorbic acid or 0.05% butylated hydroxyanisole did not appreciably alter the results obtained with the use of glutamate alone. There was no reduction in activity after 28 days of storage of lyophilized cell concentrates of S. lactis C2F, S. cremoris 459 or S. diacetilactis 18-16 when stored under vacuum in 5% monosodium glutamate at +22°, +7°, -22°, or -196° C. Lyophilized concentrates of S. lactis C2F stored in 10% NFDM under vacuum also showed no reduction in activity at +22°, +7°, -22° and -196° C.
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