Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.

The Influence of Intermolecular Interactions between Maleic Anhydride, Cellulose Nanocrystal, and Nisin-Z on the Structural, Thermal, and Antimicrobial Properties of Starch-PVA Plasticized Matrix

On behalf of a circular economy, regular plastics have been replaced by biodegradable packagings. Besides, active films have been applied to improve the shelf-life and quality of foods. In this work, blends were developed using starch as a low-cost natural polymer, mixed with poly(vinyl alcohol) due to its physical-chemical and biodegradable properties. Moreover, maleic anhydride (MaAh), cellulose-nanocrystal (CN), and nisin-z (N-Z) were added, respectively, as a compatibilizer, a mechanical-reinforce, and antimicrobial agents. The thermal stability of the films was analyzed, which blends’ melting temperature occurred around 200–207 °C, and it was influenced by CN, N-Z, and MaAh amounts. N-Z and MaAh acted against S. aureus and P. aeruginosa by compound diffusion (inhibition-halo around 1.85 and 2.18 cm); while S. Choleraesuis and E. coli were inhibited by contact. Therefore, these blends presented the potential to be used as active biodegradable packaging in the food industry.

Functionalization of Crosslinked Sodium Alginate/Gelatin Wet-Spun Porous Fibers with Nisin Z for the Inhibition of Staphylococcus aureus-Induced Infections

Nisin Z, an amphipathic peptide, with a significant antibacterial activity against Gram-positive bacteria and low toxicity in humans, has been studied for food preservation applications. Thus far, very little research has been done to explore its potential in biomedicine. Here, we report the modification of sodium alginate (SA) and gelatin (GN) blended microfibers, produced via the wet-spinning technique, with Nisin Z, with the purpose of eradicating Staphylococcus aureus-induced infections. Wet-spun SAGN microfibers were successfully produced at a 70/30% v/v of SA (2 wt%)/GN (1 wt%) polymer ratio by extrusion within a calcium chloride (CaCl2) coagulation bath. Modifications to the biodegradable fibers’ chemical stability and structure were then introduced via crosslinking with CaCl2 and glutaraldehyde (SAGNCL). Regardless of the chemical modification employed, all microfibers were labelled as homogeneous both in size (≈246.79 µm) and shape (cylindrical and defect-free). SA-free microfibers, with an increased surface area for peptide immobilization, originated from the action of phosphate buffer saline solution on SAGN fibers, were also produced (GNCL). Their durability in physiological conditions (simulated body fluid) was, however, compromised very early in the experiment (day 1 and 3, with and without Nisin Z, respectively). Only the crosslinked SAGNCL fibers remained intact for the 28 day-testing period. Their thermal resilience in comparison with the unmodified and SA-free fibers was also demonstrated. Nisin Z was functionalized onto the unmodified and chemically altered fibers at an average concentration of 178 µg/mL. Nisin Z did not impact on the fiber’s morphology nor on their chemical/thermal stability. However, the peptide improved the SA fibers (control) structural integrity, guaranteeing its stability for longer, in physiological conditions. Its main effect was detected on the time-kill kinetics of the bacteria S. aureus. SAGNCL and GNCL loaded with Nisin Z were capable of progressively eliminating the bacteria, reaching an inhibition superior to 99% after 24 h of culture. The peptide-modified SA and SAGN were not as effective, losing their antimicrobial action after 6 h of incubation. Bacteria elimination was consistent with the release kinetics of Nisin Z from the fibers. In general, data revealed the increased potential and durable effect of Nisin Z (significantly superior to its free, unloaded form) against S. aureus-induced infections, while loaded onto prospective biomedical wet-spun scaffolds.

Combinational Antibacterial Activity of Nisin and 3-Phenyllactic Acid and Their Co-production by Engineered Lactococcus lactis

Nisin produced by certain Lactococcus lactis strains is commercially used in meat and dairy industries because of its effective antibacterial activity and food safety characteristics. It has been proved that the antibacterial activity could be enhanced when combined with other antimicrobial agents. In this study, we demonstrated that nisin and 3-phenyllactic acid (PLA) in combination displayed excellent combinational antibacterial activity against foodborne pathogens including S. xylosus and M. luteus. The potential application in food preservation was further verified via microbial analysis during the storage of meat and milk, and determination of strawberry rot rate. Scanning electron microscopy observation indicated a distinct mode of PLA with nisin, which may target at the dividing cell, contributing to their combinational antibacterial effect of nisin and PLA. Considering the positive results, a nisin-PLA co-producing strain was constructed based on the food-grade strain L. lactis F44, a nisin Z producer. By the knockout of two L-lactate dehydrogenase (LDH) and overexpression of D-LDHY25A, the yield of PLA was significantly increased 1.77-fold in comparison with the wild type. Anti-bacterial assays demonstrated that the fermentation product of the recombinant strain performed highly effective antibacterial activity. These results provided a promising prospect for the nisin-PLA co-expressing L. lactis in food preservation on account of its considerable antibacterial activity and cost-effective performance.

Xác định đồng thời Nisin A và Nisin Z trong sản phẩm dinh dưỡng bằng sắc ký lỏng khối phổ hai lần (LC-MS/MS)

Nghiên cứu này phát triển phương pháp xác định đồng thời Nisin A và Nisin Z trong các sản phẩm dinh dưỡng bằng LC-MS/MS. Các chất phân tích được chiết siêu âm ra khỏi nền mẫu bằng hỗn hợp dung dịch đệm CH3COONH4 0,1M - NaCl 1M (pH = 2)/MeOH (1/1, v/v) ở nhiệt độ phòng trong 10  phút, được làm sạch qua cột chiết pha rắn SPE C18 (500 mg, 3 mL), dịch chiết sau khi làm sạch được tách bằng sắc ký lỏng sử dụng cột C18 (100 mm × 2 mm × 1,7 µm), định lượng bằng detector khối phổ với chế độ ESI (+). Thẩm định phương pháp cho kết quả đường chuẩn tuyến tính trong khoảng 5,00 - 1.000 µg/L; RSD 2,12 - 5,77%; độ thu hồi 80,1 - 105% đạt yêu cầu AOAC. Ứng dụng phương pháp phân tích 25 mẫu thực phẩm dinh dưỡng thu thập trên thị trường (bao gồm sữa lỏng, sữa bột, bơ, phomat, bột dinh dưỡng) cho thấy 02 mẫu phát hiện đồng thời Nisin A và Nisin Z.

Simultaneous determination of Nisin A and Nisin Z in nutritional food by liquid chromatography tandem mass spectrometry (LC-MS/MS)

A method for the simultaneous determination of Nisin A and Nisin Z in nutritional products by LC-MS/MS has been developed. Sample treatment was performed by ultrasonic extraction using a mixture of 0.1 M CH3COONH4 buffer solution - 1.0 M NaCl (pH 2.0): MeOH (1:1, v/v) at room temperature for 10 minutes. Extracts were then cleaned through C18 solid phase extraction column (500 mg, 3 mL) and analyzed on LC-MS/MS instrument using a C18 column (100 mm × 2 mm × 1.7 µm) with ESI (+) mode. The method was validated for linear calibration curve in the range of 10 - 1,000 µg/L; relative standard deviation (RSD) 2.12 - 5.77%; recovery 80.1 - 105%, meeting AOAC requirements. The method has been applied to analyze 25 samples of nutritious food collected from the local markets (including: liquid milk, powdered milk, butter, cheese, nutritious powder), showing that two samples were detected both Nisin A and Nisin Z.

Antimicrobial Activity of Lactococcus lactis subsp. lactis Isolated from a Stranded Cuvier’s Beaked Whale (Ziphius cavirostris) against Gram-Positive and -Negative Bacteria

In the present study, we isolated and characterized Lactococcus lactis (L. lactis) subsp. lactis from a female Cuvier’s beaked whale (Ziphius cavirostris) stranded in Shizuoka, Japan. Only five isolates (CBW1-5), grown on Lactobacilli de Man Rogosa Sharpe (MRS) agar plates prepared using 50% artificial seawater, were positive in L. lactis species-specific primer PCR. Their 16S rRNA sequences were highly similar to those of L. lactis subsp. lactis JCM 5805T. The Gram reaction, motility, gas production from glucose, catalase production, and growth conditions were consistent with those of the type strain. Additionally, carbohydrate utilization of the strains was consistent with previously reported marine organism-derived strains. The pH-neutralized cell-free culture supernatant of strain CBW2 inhibited the growth of Bacillus subtilis subsp. subtilis ATCC 6051 and Vibrio alginolyticus ATCC 17749, whereas protease treatment eliminated or diminished its inhibitory activity. The strain possesses a precursor of the nisin structural gene (nisA), which showed 100% homology with nisin Z, and nisin biosynthesis-related genes (nisB, nisC, nisT, nisP, nisF, nisI, and nisRK), suggesting that the strain produces a nisin-like substance. This study provides fundamental information on whale-derived L. lactis subsp. lactis which may be useful for reducing the carriage of B. subtilis subsp. subtilis and V. alginolyticus.

Incorporation and antimicrobial activity of nisin Z within carrageenan/chitosan multilayers

An antimicrobial peptide, nisin Z, was embedded within polyelectrolyte multilayers (PEMs) composed of natural polysaccharides in order to explore the potential of forming a multilayer with antimicrobial properties. Using attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR), the formation of carrageenan/chitosan multilayers and the inclusion of nisin Z in two different configurations was investigated. Approximately 0.89 µg cm−2 nisin Z was contained within a 4.5 bilayer film. The antimicrobial properties of these films were also investigated. The peptide containing films were able to kill over 90% and 99% of planktonic and biofilm cells, respectively, against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) strains compared to control films. Additionally, surface topography and wettability studies using atomic force microscopy (AFM) and the captive bubble technique revealed that surface roughness and hydrophobicity was similar for both nisin containing multilayers. This suggests that the antimicrobial efficacy of the peptide is unaffected by its location within the multilayer. Overall, these results demonstrate the potential to embed and protect natural antimicrobials within a multilayer to create functionalised coatings that may be desired by industry, such as in the food, biomaterials, and pharmaceutical industry sectors.