The CRISPR‒Cas system acts as a bacterial defense mechanism by conferring adaptive immunity and limiting genetic reshuffling. However, under adverse environmental hazards, bacteria can employ their CRISPR‒Cas system to exchange genes that are vital for adaptation and survival. Levilactobacillus brevis is a lactic acid bacterium with great potential for commercial purposes because it can be genetically manipulated to enhance its functionality and nutritional value. Nevertheless, the CRISPR‒Cas system might interfere with the genetic modification process. Additionally, little is known about the CRISPR‒Cas system in this industrially important microorganism. Here, we investigate the prevalence, diversity, and targets of CRISPR‒Cas systems in the genus Levilactobacillus, further focusing on complete genomes of L. brevis. Using the CRISPRCasFinder webserver, we identified 801 putative CRISPR-Cas systems in the genus Levilactobacillus. Further investigation focusing on the complete genomes of L. brevis revealed 54 putative CRISPR-Cas systems. Of these, 46 were orphan CRISPRs, and eight were CRISPR‒Cas systems. The type II-A CRISPR‒Cas system is the most common in Levilactobacillus and L. brevis complete genomes. Analysis of the spacer's target showed that the CRISPR‒Cas systems of L. brevis mainly target the enterococcal plasmids. Comparative analysis of putative CRISPR-Cas loci in Levilactobacillus brevis.
Lactic acid bacteria (LAB) can produce γ-aminobutyric acid (GABA) with antioxidant properties and sedative effects when it binds to the GABA receptor in the human brain. LAB can also produce bacteriocin-like inhibitory substances (BLIS) with antimicrobial capabilities during carbohydrate fermentation. GABA and BLIS are natural compounds with potential health benefits and food preservation properties. Lactobacillus brevis C23 was co-cultured with three different LABs as inducers, which produced the highest GABA content and BLIS activity. They were cultured in various plant-based media to obtain an edible and better-tasting final product over commercially available media like MRS broth. A coconut-based medium with additives was optimized using response surface methodology (RSM) to increase GABA and BLIS production. The optimized medium for maximum GABA production (3.22 ± 0.01 mg/mL) and BLIS activity (84.40 ± 0.44%) was a 5.5% coconut medium containing 0.23% glucose, 1.44% Tween 20, 0.48% L-glutamic acid, and 0.02% pyridoxine. Due to the presence of GABA, the cell-free supernatant (CFS) as a postbiotic showed higher antioxidant activity than other food preservatives like nisin and potassium sorbate. Finally, microbiological tests on food samples showed that the postbiotic was more effective than other preservatives at combating the growth of LAB, molds and coliform bacteria, making it a possible food preservative.
Kefir is a fermented milk obtained by fermenting milk with kefir grains. The chemical composition ofdairy and non-dairy sources may affect the growth and characterisation of lactic acid bacteria (LAB). In this study, different sources of milk (cow milk) and non-dairy milk (soymilk and coconut milk) were used as the fermentation media for kefir products. The objectives of the study were to isolate and characterise LAB from kefir drink produced from dairy and non-dairy milk. LAB was isolated using different cultural methods, such as MRS Agar, MRS with 0.8% CaCO3, and M17 Agar. The characteristics of the LAB isolates were determined using morphological, biochemical tests and the API 50 CHL kit. The physicochemical composition of the samples was determined using titratable acidity and pH level. Sensory evaluation of the kefir drink samples was also carried out. Results confirmed that the isolates were identified as Lactobacillus buchneri, Lactobacillus brevis 1, Leuconostoc mesenteroides, Lactobacillus acidophilus 3and Lactobacillus plantarum 1. The L. buchneri, L. brevis, Leu. mesenteroides and L. acidophilus are heterofermentative bacteria, whereas L. plantarum is a homofermentative bacterium. Four LAB isolates have the potential to be used as probiotic strains due to their high resistant to low pH and bile salt. The sensory scores of these products range between 5.00 and 8.00 in the 9-point hedonic scale. Most of the sensory panelists preferred cow milk kefir (p < 0.05) compared with coconut milk kefir and soy milk kefir during the sensory evaluation of all attributes. Meanwhile, the preference between coconut milk kefir and soy milk kefir was similar (p>0.05) in all attributes. Therefore, this study will be useful for probiotic manufacturers in the production of alternative probiotic drinks using dairy and non-dairy milk.