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ABSTRACT: It is desirable to obtain high levels of viable Lacticaseibacillus paracasei, a widely used food probiotic whose antibacterial activity and potential application in milk remain largely uninvestigated. Here, we isolated and purified the L. paracasei strain XLK 401 from food-grade blueberry ferments and found that it exhibited strong antibacterial activity against both gram-positive and gram-negative foodborne pathogens, including Staphylococcus aureus, Salmonella paratyphi B, Escherichia coli O157, and Shigella flexneri. Then, we applied alternating tangential flow (ATF) technology to produce viable L. paracasei XLK 401 cells and its cell-free supernatant (CFS). Compared with the conventional fed-batch method, 22 h of ATF-based processing markedly increased the number of viable cells of L. paracasei XLK 401 to 12.14 ± 0.13 log cfu/mL. Additionally, the CFS exhibited good thermal stability and pH tolerance, inhibiting biofilm formation in the abovementioned foodborne pathogens. According to liquid chromatography-mass spectrometry analysis, organic acids were the main antibacterial components of XLK 401 CFS, accounting for its inhibition activity. Moreover, the CFS of L. paracasei XLK 401 effectively inhibited the growth of multidrug-resistant gram-positive Staph. aureus and gram-negative E. coli O157 pathogens in milk, and caused a reduction in the pathogenic cell counts by 6 to 7 log cfu/mL compared with untreated control, thus considerably maintaining the safety of milk samples. For the first time to our knowledge, ATF-based technology was employed to obtain viable L. paracasei on a large scale, and its CFS could serve as a broad-spectrum biopreservative for potential application against foodborne pathogens in milk products.

ABSTRACT: Phenyllactic acid (PLA) has been demonstrated to possess antibacterial activity and capacity to prolong food shelf life. However, studies on the performance of PLA in inhibiting Staphylococcus aureus and its effectiveness when applied to dairy products are largely lacking. Here, antibacterial activity (planktonic and biofilm states) of PLA against S. aureus CICC10145 (S. aureus_45) were investigated. The results showed that PLA inhibited growth of S. aureus_45 and formation of S. aureus_45 biofilm. Next, the antibacterial action target of PLA was uncovered from both physiological and phenotypic perspectives. The results showed that PLA decreased cell metabolic activity and cell viability, damaged cell membrane integrity, triggered leakage of intracellular contents (DNA, proteins, and ATP), and caused oxidative stress damage and morphological deformation of S. aureus_45. In practical application, the antibacterial activity of PLA against S. aureus_45 cells was further confirmed in skim milk and cheese as dairy food models, and the antibacterial effects can be adequately maintained during storage for 21 d, at least at 4°C. These findings suggested that PLA could be a potential candidate for controlling S. aureus outgrowth in dairy foods.

ABSTRACT: Staphylococcus aureus and its biofilm have emerged as a significant threat to the safety of dairy products. In recent years, lactic acid bacteria (LAB) bacteriocins have been widely acknowledged as the potential natural antibacterial substance in food biopreservation due to their excellent antibacterial effects. However, few LAB bacteriocins with antibacterial and antibiofilm activity against S. aureus have been reported in dairy products. In the present study, a novel bacteriocin LSX01 of Lactobacillus paracasei LS-6 isolated from a traditional fermented yogurt produced in Yunnan, China, was purified and characterized extensively. The LSX01 possessed a molecular weight of 967.49 Da and an AA sequence of LDQAGISYT. The minimum inhibitory concentration of LSX01 against S. aureus_45 was 16.90 μg/mL, which was close to or lower than the previously reported bacteriocins. The LSX01 exhibited an extensive antimicrobial spectrum against both gram-positive and gram-negative bacteria. Moreover, LSX01 exhibited excellent tolerance to heat and acid-base treatments, and sensitivity to the proteolytic enzymes, such as pepsin and proteinase K. Furthermore, the treatment of S. aureus_45 planktonic cells with LSX01 significantly reduced their metabolic activity and disrupted the cell membrane integrity. Scan electron microscopy results demonstrated that LSX01 induced cytoplasmic content leakage and cell deformation. Additionally, biofilm formation of S. aureus_45 was also significantly inhibited by LSX01. Overall, the results suggested that the novel LAB bacteriocin LSX01 possessed antibacterial activity and antibiofilm activity against S. aureus and, hence, could have potential for improving safety of dairy products.

Foodborne pathogens and their biofilms pose a risk to human health through food chain. However, the bacteriocin resources combating this threat are still limited. Here, Lacticaseibacillus rhamnosus, one of the most used probiotics in food industry, was prepared on a large scale using alternating tangential flow (ATF) perfusion-based technology. Compared to the conventional fed-batch approach, ATF perfusion remarkably increased the viable cells of L. rhamnosus CLK 101 to 11.93 ± 0.14 log CFU/mL. Based on obtained viable cells, we purified and characterized a novel bacteriocin CLK_01 with a broad spectrum of activity against both Gram-positive and Gram-negative foodborne pathogens. LC-MS/MS analysis revealed that CLK_01 has a molecular mass of 701.49 Da and a hydrophobic amino acid composition of I–K–K–V-T-I. As a novel bacteriocin, CLK_01 showed high thermal stability and acid-base tolerance over 25–121 °C and pH 2–10. It significantly reduced cell viability of bacterial pathogens (p