Probiotic properties and proteomics analysis of ethanol-induced Lactococcus lactis subsp. lactis IL1403.

Our previous study indicated that ethanol-induced intracellular extracts (E-IEs) of Lactococcus lactis subsp. Lactis IL1403 (L. lactis IL1403) alleviated hangovers more effectively in mice than untreated intracellular extracts (U-IEs), but the material basis was unclear. Considering that stress-related proteins might play a significant role, the effects of ethanol induction on probiotic properties of L. lactis IL1403 and the associated stress response mechanism were initially explored in this study. E-IEs of L. lactis IL1403 showed better biological activities, significantly increased bacteria survival rates in oxidative stress environments, increased ADH activity, and enhanced proliferation in RAW264.7 and AML-12 cells. Proteomic analyses revealed that 414 proteins were significantly changed in response to ethanol induction. The expression of proteins involved in the universal stress response, DNA repair, oxidative stress response, and ethanol metabolism was rapidly upregulated under ethanol stress, and quantitative real-time PCR (qRT-PCR) results were consistent with proteomic data. KEGG pathway analysis indicated that citrate metabolism, starch and sucrose metabolism, and pyruvate metabolism were significantly enriched during ethanol stress to increase energy requirements and survival rates of stressed cells. Based on this observation, the active induction is an effective strategy for increasing the biological activity of L. lactis IL1403. Exploring the molecular mechanism and material basis of their functions in vivo can help us understand the adaptive regulatory mechanism of microorganisms.

Protective effect of the stressed supernatant from Lactococcus lactis subsp. lactis and its metabolic analysis.

There are numerous factors restricting wide application of lactic acid bacteria (LAB) in dairy industry, causing urgent demands for novel bioprotectants. Protective effects and metabolites of Lactococcus lactis subsp. lactis (L. lactis) from ultraviolet (UV)-induced supernatant were investigated and the protective mechanism was explored. The strain viability of the group treated with the supernatant of continuous UV irradiation (V1) and the group with intermittent UV irradiation (V2) was 8.45 and 14.13 times of the control group, respectively. Further exploration on the protective of L. lactis supernatant, under different dose of UV treatment, showed it was dose-dependent. The condition for the supernatant with best protective effect was vertical distance 50.00 cm, horizontal distance 25.00 cm, intermittent UV irradiation (30 s interval 30 s) for 4.5 min (V2), which was chose for untargeted metabolite analysis. And that in V1 was for comparative study. There were 181 up-regulated metabolites in V1 and 161 up-regulated metabolites in V2, respectively. Most of the up-regulated metabolites were related to secondary metabolite synthesis, environmental microbial metabolism, antibiotic synthesis and amino acid biosynthesis. Notably, production of dithiothreitol (DTT) in V2 was 65.2-fold higher than that in the control group. Trehalose in ABC transporter pathway was also up-regulated in the metabolites induced by UV. Results indicated that L. lactis could adapt to the UV stress by adjusting metabolic pathways and producing special metabolites to protect itself. This research offers the basis for robust strain development and contributes to initial study on potential bioprotectant.

60Coγ induction improves the protective effect of Acetobacter pasteurianus against ionizing radiation in mice.

Exposure to ionizing radiation (IR) tends to cause serious health concerns. Thus, radioprotective agents are vital for the population exposed to radiation. As microorganisms have the advantages of fast reproduction and no geographical restrictions, direct microbe-based and environmental induction compounds are thriving radioprotectants resources. Oxidative system and oxidase in Acetobacter pasteurianus are unique and intriguing, the radioprotective effect of the cell-free extract from A. pasteurianus (APE) and 60Coγ-treated extract (IRE) were comparatively investigated in the present study. The survival rate of A. pasteurianus with IRE addition was 149.1% in H2O2 damage test, while that with APE was only 10.4%. The viability of 60Coγ-treated AML-12 cells was increased by 18.8% with IRE addition, yet APE showed no significant radioprotective effect. Moreover, in 60Coγ-treated mice, IRE could significantly protect the white blood cell, improve the liver index, and attenuate the injuries of immune organs in mice. Administration of IRE significantly raised the activities of superoxide dismutase (SOD) and reduced the products of lipid peroxidation. These results clarified that gavage with APE and IRE presented notable antioxidant and radioprotective efficacy. A. pasteurianus showed appealing potential to be novel radioprotective bioagents and 60Coγ treatment on microbe could be a new method for the development of better radioprotectant. KEY POINTS: • 60Coγ induction could improve the radioprotective effect of APE. • IRE protected white blood cell in mice under IR. • IRE products have broad application prospects in radioprotection based on microbes.

Radioprotective effect of radiation-induced Lactococcus lactis cell-free extract against 60Coγ injury in mice.

Ionizing radiation (IR) is widely used in the diagnosis and treatment of various cancers. However, IR can cause damage to human health by producing reactive oxygen species. Lactococcus lactis is a type of microorganism that is beneficial to human health and has a strong antioxidant capacity. In this study, the protective effect of normal and IR-induced L. lactis IL1403 cell-free extracts (CFE and IR-CFE, respectively) against oxidative damage in vitro and the radioprotective effect of IR-CFE in vivo was evaluated using 60Coγ-induced oxidative damage model in mice. Results showed that IR-CFE exhibited a stronger oxidative damage-protective effect than CFE for L. lactis IL1403 under H2O2 in vitro. Moreover, IR-CFE also showed strong radioprotective effect on hepatocyte cells (AML-12) under radiation condition, and the effect was better than that of CFE. Animal experiment indicated that IR-CFE could reduce the IR-induced damage to the hematopoietic system by increasing the number of white blood cells and red blood cells in peripheral blood of irradiated mice. It was also observed that IR-CFE could markedly alleviate the 60Coγ-induced oxidative stress via increasing the activities of superoxide dismutase and glutathione peroxidase, enhancing the levels of glutathione, and decreasing the contents of malondialdehyde in serum, liver, and spleen. In addition, IR-CFE also could reduce the activities of alanine transaminase and aspartate aminotransferase in serum, thereby reducing radiation damage to the liver. These results suggested that IR-CFE could be considered as potential candidates for natural radioprotective agents. This study provides a theoretical basis for improving the application of lactic acid bacteria.

Hydroxypropyl ß-cyclodextrin improving multiple stresses tolerance of Lactococcus lactis subsp. lactis.

L. lactis is known as industrial starter in the fermentation of dairy and meat products, and it plays an important role in human health as an edible probiotic. During industrial production, L. lactis often experiences different stresses that delay the growth and decrease the survival in some serious conditions. In this study, the protective effects of hydroxypropyl ß-cyclodextrin (HP ß-CD) on L. lactis under multiple stresses were investigated. The microbial cells were treated with different stresses including heat, NaCl, cold, and H2 O2 stresses, and the results were showed by measuring the OD600 or spot plating method. The growth and tolerance were improved when HP ß-CD was added during different stress conditions, better than that of trehalose. Besides, the scanning electron microscopic and fluorescence spectrum studies showed that HP ß-CD could combine with L. lactis to protect the cell structure, suggesting that HP ß-CD may act as a protective agent of L. lactis. Therefore, HP ß-CD could be considered as a potential protective agent to be applied in food industry, and its protective mechanism on L. lactis still needs further investigation.

An innovative method to enhance protease tolerance of nisin in endogenous proteases.

Nisin, a natural peptide produced by Lactococcus lactis cultivation in milk whey, is widely used as a preservative in industrial production. However, nisin can be degraded by endogenous enzymes in foods. In this study, we investigated the antibacterial activity of nisin-soybean protein and nisin-egg white protein and compared them with that of free nisin in cantaloupe juice, which was used as a model of endogenous protease environment. Results showed that endogenous proteases in the model resulted in a loss of nisin activity, but combining nisin with protein (soybean or egg white) resulted in greater protection of its antimicrobial activity by inhibiting endogenous proteases. The microbial addition experiment (Staphylococcus aureus and Micrococcus luteus) and preservation experiment in the food model showed that the antibacterial activity of nisin combined with either of the 2 proteins was higher than that of nisin alone in an endogenous protease environment. In summary, soybean protein and egg white protein improved the protease tolerance of nisin, expanding the application scope of nisin in food.

Protective effect of ß-cyclodextrin on stability of nisin and corresponding interactions involved.

Nisin has been extensively employed in food industry as an antimicrobial peptide, but the proteolytic degradation and interference by food components resulted in the decrease of its antimicrobial activity. This study intends to explore the protective effect of ß-cyclodextrin (ß-CD) on nisin and corresponding interactions involved. The antibacterial experiments indicated that the combination of nisin and ß-CD could provide a better antibacterial effect on cooked pork meat. Spectral analysis with UV-vis and fluorescence spectroscopy showed that the micro-environment of nisin could be influenced by ß-CD. Tricine-SDS-PAGE results exhibited that the combination of nisin and ß-CD could delay the degradation of nisin and improve its stability in the presence of trypsin. In sum, the study provided a potential approach to improve the stability of nisin, especially in the presence of trypsin.

Short communication: Global transcriptome analysis of Lactococcus lactis ssp. lactis in response to gradient freezing.

Lactic acid bacteria are often preserved as starter cultures by freezing to extend shelf stability as well as maintain cell viability and acidification activity. Previous studies showed that the endocyte extracted from gradient-freezing pretreated cells could act as lyoprotectant in the lyophilization process of Lactococcus lactis ssp. lactis. In this study, the molecular mechanisms of L. lactis in response to gradient freezing exposure are described using high-throughput sequencing. Nineteen of 56 genes were upregulated after gradient freezing, whereas 37 genes were downregulated. Further validation results of quantitative real-time PCR experiments were consistent with the RNA sequencing. Gene Ontology (http://www.geneontology.org/) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) pathway were used to analyze the differentially expressed genes. Several pathways, such as glutathione metabolism, ATP-binding cassette transport, metabolism of cell wall and cell membrane components, and stress response-related pathways, were affected by gradient freezing. Six genes relevant to freezing stress response were selected for quantitative real-time PCR, including 3 upregulated genes (hisK, eutD, dukA) and 3 downregulated genes (als, yedF, pepN). The Gene Ontology enrichment and KEGG pathway analyses showed these genes may influence stress response-related pathways, improving the survival of the L. lactis under freezing stress. The identification of these genes deepened an understanding about their response under freezing stress, helping us find potential genes or pathways related to gradient freezing for further research on lyoprotectants.

Protective effects of soybean protein and egg white protein on the antibacterial activity of nisin in the presence of trypsin.

The using of nisin to prevent foodborne pathogens (Staphylococcus aureus and Listeria monocytogenes) from contamination has received broad attentions during meat processing. However, the application of nisin has been limited because its antibacterial activity may be inhibited by trypsin. In this study, the protective effects of soybean protein and egg white protein on antibacterial activity of nisin were evaluated. It could be concluded that exogenous trypsin decreased the antibacterial activity of nisin, soybean protein and egg white protein could keep the nisin activity from enzymolysis of trypsin. Trypsin inhibitors in soybean protein and egg white protein could protect the antibacterial activity of nisin. Nisin with soybean protein or egg white protein in cooked meat product presented better quality preservation effects than nisin alone in the presence of trypsin. The total viable counts (TVC) and total volatile basic nitrogen (TVB-N) of nisin-treated group were significantly higher than these in nisin-soybean protein-treated and nisin-egg white protein-treated groups with trypsin. This study showed the potential of using soybean protein and egg white protein to stabilize the antibacterial activity of nisin under high trypsin conditions.