Selection and Application of Protease- and Lipase-Producing Bacteria for Fermented Shrimp Paste (Kapi) Production

Abstract

Kapi is a Thai fermented shrimp paste which is widely consumed as a food condiment or flavoring in various Thai dishes. Generally, production of Kapi is mainly by a natural fermentation process which causes variations in the quality of the products. One effort that can be applied for improving the quality of Kapi is applying starter culture. This present study deals with the isolation, screening, and identification of protease- and lipase-producing bacteria from Kapi in conjunction with their technological properties and safety assessment to be applied as an autochthonous starter culture for Kapi production. A total of 195 bacterial isolates exhibiting proteolytic activity on skim milk agar plate were obtained. Five bacterial strains (MSK-3P, MSK-4P, MSK-5P, MSK-7P, and MSK-10P) exhibited the high activity of i protease (>5 U/mL) using Anson method were selected to be investigated for their potential application as starter culture. All the 5 strains were identified as Virgibacillus halodenitrificans by using 16S IRNA sequence analysis. All selected strains exhibited growth in the presence of NaCl up to 25%. Neither strains showed hemolytic activity nor biogenic amines (BAs) formation. Among all selected strains, only strain MSK-10P had no ability to form a biofilm, while other four strains showed a weak ability. Three strains, MSK-3P, MSK-4P, and MSK-10P showed susceptibility to all antibiotics tested however, strain MSK-7P exhibited multiple resistance to tetracyclin and vancomycin. According to the obtained results, V. halodenitrificans MSK-10P was selected to be applied as the starter culture for the production of Kapi. A total of 46 bacterial isolates showing lipolytic activity on agar plate containing olive oil were selected. Among them, 4 selected bacterial strains, namely LSM3, LSM4, LSM15, and LSM16 exhibited the highest lipase activity using copper soap method were selected. Molecular identification by using 16S RNA sequencing and was also confirmed by specific primer for staphylococci revealed that all of them were closely related to Staphylococcus sp. All of the selected isolates exhibited growth in the presence of NaCl up to 20%. In addition, strains LSM3, LSM15, and LSM16 showed non-hemolytic activity (y-hemolysis), while all strains exhibited weak ability in biofilm formation. Strains LSM4, LSM15, and LSM16 displayed antagonistic activity toward only Gram-positive strains, Staphylococcus aureus DMST8840, Bacillus cereus DMST5540, and Listeria monocytogenes ' DMSTI7303. The antibacterial activity against tested Gram-positive bacteria came from bacteriocin-like inhibitory substance (BLIS). Moreover, isolates LSMI5 and LSM16 showed susceptible to all tested antibiotics. Eurthermore, classical staphylococcal enterotoxin (SE) genes were not found in all strains. Therefore, isolate LSM16 which exhibited the highest lipase activity was selected to be applied as the promising starter culture in the production of Kapi. The optimization study for protease production by V. halodenitrificans MSK-10P was conducted by using -one-factor-at a-time- (OFAT) method. The optimum protease production was obtained at temperature 37°C, pH 8.0, 1% (w/v) cascin as substrate, 10% (w/v) NaCI, 2.5% (v/v) of inoculum, and under agitation at 150 rpm. In addition, time course production showed that the highest protease production was attained at 36 h of incubation which gave 3.83-fold increasing production, from 9.47 U/mL to 36.32 U/mL. Moreover, crude protease from V. halodenitrificans MSK-IOP also showed halotolerance, since it can still active in 0- 25% (w/v) NaCI. The lipase production by Staph, xylosus LSM16 was also optimized by using OFAT method. The result showed that lipase production was opfimum at temperature 37°C, pH 7.0, 2% (w/v) olive oil, 10% (w/v) NaCl, 1.5% (v/v) of inoculum, under agitation at 150 rpm. Time course study for lipase production showed that the highest lipase production was achieved at 48 h, which gave 3.24-fold increasing of the lipase production from 0.67 U/mL to 2.27 U/mL. As obtained in stability study towards salt condition, the crude lipase of Staph. xylosus LSM16 was found stable under 0-20% (w/v) NaCl. The application of protease-producing bacteria, v. halodenirificans MSK-10P and lipase-producing bacteria, Staph. xylosus LSMI6, was studied in the Kapi production. Four treatments of Kapi production were performed: i) natural fermentation (as control, KC); (ii) inoculated with V. halodenitrificans MSK-10P (KP); (iii) inoculated with S. xylosus LSM16 (KL); and (iv) inoculated with both selected protease- and lipase-producing strain (KM). Physicochemical charicteristic and microbiological safety of the Kapi were investigated. It was found that all the Kapi products inoculated with starter culture were meet the physicochemical characteristics and microbiological safety required by Thai Industrial Standard (TIS) 1205/2006. Moisture, salt, and total nitrogen content of Kapi products were in the range of 40.02-42.73%, 40.01-42.79%, and 6.33-7.82%, respectively. Microbiological quality of all finished Kapi product was also under limitations of criteria as regulated in TIS 1205/2006. In addition, none of the Kapi products contained the four important BAs (histamine, tyramine, putrescine, and cadaverine) mainly obtained in seafood samples. Moreover, sensory analysis also showed that all Kapi inoculated with starter culture were well-accepted by panelists. These results provided that V. halodenitrificans MSK-10P and Staph. xylosus LSM16 could be applied as promising strater cultures in the production of Kapi.