Induction of heat tolerance in autochthonous and allochthonous thermotolerant probiotics for application to white shrimp feed

The main aim of this work was to produce autochthonous (host origin) thermotolerant probiotic feeds to have sufficient numbers of viable cells on extended storage and application into commercial shrimp ponds. Thermotolerant lactic acid bacteria and Bacillus sp. preliminarily identified as Lactobacillus plantarum ssp1 (ID1L), Leuconostoc mesenteroides ssp mesenteroides/dextranicum2 (ID2L), Bacillus licheniformis (ID3B) and Bacillus subtilis (ID4B) were isolated under high temperature from healthy white shrimp (Litopenaeus vannamei) grown in natural farming and research ponds, Thailand. These bacteria and introduced allochthonous (external source) L. brevis 860 showed antagonistic effects on Vibrio sp. and some food pathogens tested. Autochthonous and allochthonous lactic acid bacteria (ID1L and L. brevis 860) were further selected to study heat adaptation characteristics under different condition of pH, physiological growth state, temperature and time of heat pretreatment and growth media‏. Cultures at pH 7 and in stationary growth phase when exposed to heat adaptation temperature profile (preheated at 45 °C, 20 min) were found to improve decimal reduction time (D70 value) and survived better than those suspended at pH 3 and in log growth phase. In order to retain 50% cell survival, ID1L could be preheated to 50 °C for a maximum of 15 min and L. brevis 860 to 45 °C for a maximum of 15 to 20 min. Cross protection (treating with 0.3 M NaCl for 30 min) also led to good cell recovery.Preheated and cross protected ID1L and L. brevis 860 were then manually sprayed (top-dressed) on hot white shrimp pellets received from the commercial pellet mill after it had cooled to 60 and 50 °C, respectively from an outlet temperature of 95 °C and then stored in refrigeration for 4 weeks. Lactobacillus plantarum ssp1 (ID1L) grown in De Man, Rogosa, Sharp media with cross protection showed the highest survival percentage (47.8%) while L. brevis 860 had the highest survival of 41.7% for those grown in skim milk. Similar tests were also conducted with heat tolerant mesophilic Bacillus sp., B. licheniformis (ID3B) and B. subtilis (ID4B) without heat adaptation by spraying (top-dressed) at 80 and 70 °C, respectively. Fresh pellets sprayed with organisms at ambient temperature were used as control. While the initial cell count was lower for those sprayed at high temperatures, the cell count was found to decrease rapidly on 4-week refrigerated storage in the case of those sprayed at ambient temperature. Overall spraying probiotics at high temperature is recommended as it also prevents the growth of fungi on the pellets which is a problem for feed storage based on all other characteristics studied.