Development of Freeze-Thaw Tolerant Lactobacillus rhamnosus GG by Adaptive Laboratory Evolution

The industrial application of microorganisms as starters or probiotics requires their preservation to assure viability and metabolic activity. Freezing is routinely used for this purpose, but the cold damage caused by ice crystal formation may result in severe decrease in microbial activity. In this study, adaptive laboratory evolution (ALE) technique was applied to a lactic acid bacterium to select tolerant strains against freezing and thawing stresses. Lactobacillus rhamnosus GG was subjected to freeze-thaw-growth (FTG) for 150 cycles with four replicates. After 150 cycles, FTG-evolved mutants showed improved fitness (survival rates), faster growth rate, and shortened lag phase than those of the ancestor. Genome sequencing analysis of two evolved mutants showed genetic variants at distant loci in six genes and one intergenic space. Loss-of-function mutations were thought to alter the structure of the microbial cell membrane (one insertion in cls), peptidoglycan (two missense mutations in dacA and murQ), and capsular polysaccharides (one missense mutation in wze), resulting in an increase in cellular fluidity. Consequently, L. rhamnosus GG was successfully evolved into stress-tolerant mutants using FTG-ALE in a concerted mode at distal loci of DNA. This study reports for the first time the functioning of dacA and murQ in freeze-thaw sensitivity of cells and demonstrates that simple treatment of ALE designed appropriately can lead to an intelligent genetic changes at multiple target genes in the host microbial cell

Phenotypic and genomic analyses of bacteriocin-producing probiotic Enterococcus faecium EFEL8600 isolated from Korean soy-meju

Enterococcus faecium is a prevalent species found in fermented soybean products, known for its contributions to flavor development and inhibition of pathogenic microorganisms during fermentation. This study aims to provide comprehensive phenotypic and genomic evidence supporting the probiotic characteristics of E. faecium EFEL8600, a bacteriocin-producing strain isolated from Korean soy-meju. Phenotypic analysis revealed that EFEL8600 produced a peptide with inhibitory activity against Listeria monocytogenes, estimated to be 4.6 kDa, corresponding to the size of enterocins P or Q. Furthermore, EFEL8600 exhibited probiotic traits, such as resilience in gastrointestinal conditions, antioxidant and anti-inflammatory activities, and protection of the intestinal barrier. Safety assessments demonstrated no hemolytic and bile salt deconjugation activities. Genomic analysis revealed the presence of several genes associated with probiotic characteristics and bacteriocin production, while few deleterious genes with a low likelihood of expression or transferring were detected. Overall, this study highlights E. faecium EFEL8600 as a potent anti-listeria probiotic strain suitable for use as a starter culture in soymilk fermentation, providing potential health benefits to consumers

Suitability Analysis of 17 Probiotic Type Strains of Lactic Acid Bacteria as Starter for Kimchi Fermentation

The use of probiotic starters can improve the sensory and health-promoting properties of fermented foods. This study aimed to evaluate the suitability of probiotic lactic acid bacteria (LAB) as a starter for kimchi fermentation. Seventeen probiotic type strains were tested for their growth rates, volatile aroma compounds, metabolites, and sensory characteristics of kimchi, and their characteristics were compared to those of Leuconostoc (Le.) mesenteroides DRC 1506, a commercial kimchi starter. Among the tested strains, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Lacticaseibacillus paracasei, and Ligilactobacillus salivarius exhibited high or moderate growth rates in simulated kimchi juice (SKJ) at 37 °C and 15 °C. When these five strains were inoculated in kimchi and metabolite profiles were analyzed during fermentation using GC/MS and 1H-NMR, data from the principal component analysis (PCA) showed that L. fermentum and L. reuteri were highly correlated with Le. mesenteroides in concentrations of sugar, mannitol, lactate, acetate, and total volatile compounds. Sensory test results also indicated that these three strains showed similar sensory preferences. In conclusion, L. fermentum and L. reuteri can be considered potential candidates as probiotic starters or cocultures to develop health-promoting kimchi products

COVID-19 Vaccination, Adverse Effect, and SARS-CoV-2 Infection among Couples in Infertility Clinic: A Prospective Study

Background: Although periconception vaccination is important for maternal and neonatal health, vaccine hesitancy could pose a challenge to achieving a healthy pregnancy in infertile women. We assessed the prevalence of coronavirus disease 2019 (COVID-19) booster vaccination, associated factors, adverse event after vaccination, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among women seeking fertility treatment. Methods: We used the data from participants of the Pregnancy and Urban Environment study (PRUNE study), which is a prospective observational study designed to explore the association between pregnancy and the urban environment in infertile couples. From October 2019 to September 2021, a total of 534 patients provided informed consent and participated in the first and second rounds of a mobile survey conducted at six-month intervals. In the second and third rounds of the survey, we included questions about COVID-19 vaccination and SARS-CoV-2 infection. Adjusted risk ratios (aRR) were calculated for COVID-19 booster vaccination and SARS-CoV-2 infection. Results: All the women participating in the survey had completed the primary series of COVID-19 vaccination. The booster vaccination rate was 38.5%, and the prevalence of SARS-CoV-2 infection was 34.4%. The likelihood of receiving a booster vaccination was higher when women reported alcohol consumption (aRR = 1.90, 95% confidence interval (CI): 1.03–3.51, p = 0.041). SARS-CoV-2 infection was inversely associated with nulliparity (aRR = 0.44, 95% CI: 0.17–1.09, p = 0.077). Among the women vaccinated against COVID-19, 44.3% of respondents reported at least one adverse reaction after receiving the vaccine. Conclusions: All the women participating in the survey had completed primary series of COVID-19 vaccination. The booster vaccination rate was 38.5%, and the prevalence of SARS-CoV-2 infection was 34.4%. Among the vaccinated women, 44.3% reported experiencing at least one adverse reaction after receiving the COVID-19 vaccine. Fever/chilling was the most common (61.8%), followed by abnormal vaginal bleeding/menstrual irregularity (41.8%). A targeted education program by health professionals is needed to promote the benefits of periconception vaccination and reduce the resistance to the COVID-19 vaccine among infertile couples. Clinical Trial Registration: The study was registered at Clinical Research Information Service (https://cris.nih.go.kr/cris/info/dataset.do), registration number: KCT0003560