Asian Journal of Biotechnology and Bioresource Technology

Amylases are important industrial enzymes involved in the hydrolysis of starch and have wide applications in food and other biotechnological industries. Amylolytic Lactobacillus species are of particular interest because of their ability to produce extracellular enzymes and their established relevance in food fermentation. This study evaluated selected characterised Lactobacillus strains for extracellular amylase production, optimised culture conditions for enhanced enzyme yield, and characterised the amylases produced by the most active isolates. Three previously characterised isolates were screened for amylase production using the starch agar plate method. The selected isolates were cultivated in de Man, Rogosa and Sharpe broth under submerged shake flask fermentation at 37°C and 150 rpm for 48 h. Crude enzymes were partially purified by 90% ammonium sulphate precipitation and dialysis, while amylase activity was determined using the 3,5-dinitrosalicylic acid method with soluble starch as substrate. The effects of pH, temperature, and fermentation time on enzyme production were investigated using a one-factor-at-a-time approach. All screened isolates demonstrated amylase activity, with Lactobacillus fermentum ON406509 producing the largest starch hydrolysis zone (22 mm) and the highest initial amylase yield (32.31 U/mL), followed by Lactobacillus plantarum KCB4 (18 mm; 23.72 U/mL) and Lactobacillus plantarum ON406571 (14 mm; 22.68 U/mL). Optimisation increased amylase production, with maximum yields of 35.7 U/mL for L. fermentum ON406509 and 35.4 U/mL for L. plantarum KCB4 at pH 6.0. The optimum temperature for production was 40°C, producing 34.83 U/mL and 34.44 U/mL, respectively, while maximum production was obtained after 48 h of fermentation, with yields of 34.11 U/mL and 33.37 U/mL, respectively. Characterisation of the partially purified enzymes showed that amylase activity increased with temperature from 25°C to 40°C, reaching maximum activities of 24.648 U/mL for L. fermentum ON406509 and 27.407 U/mL for L. plantarum KCB4 at 40°C. For pH activity, L. fermentum ON406509 exhibited maximum activity at pH 7.0 (29.074 U/mL), whereas L. plantarum KCB4 showed maximum activity at pH 8.0 (28.888 U/mL). Both enzymes showed their lowest activities at pH 3.0. Temperature stability studies showed that both enzymes retained maximum activity at 40°C, followed by a gradual decline at higher temperatures. Both enzymes exhibited maximum pH stability at pH 4.0. L. plantarum KCB4 retained approximately 96–100% residual activity between pH 3.0 and 5.0, whereas L. fermentum ON406509 demonstrated broader stability, retaining approximately 89–100% residual activity from pH 3.0 to 8.0. These findings demonstrate that the selected Lactobacillus strains are efficient extracellular amylase producers and that optimisation substantially improves enzyme yield. The broad pH stability of L. fermentum ON406509 and the favourable activity profile of both enzymes at 40°C further indicate their potential for applications in food processing and other biotechnological processes.