The efficiency of Lactiplantibacillus plantarum S61 strain as protective cultures in ground beef against foodborne pathogen Escherichia coli.

The aim of the study was the bio-control effectiveness of the Lactiplantibacillus plantarum S61 strain, isolated from traditional fermenting green olives, against Escherichia coli B805 in ground beef. The bio-control effect of L. plantarum S61 against E. coli B805 was evaluated in ground meat during storage under refrigeration at 4 °C. The results showed that L. plantarum S61 reduced the biomass of pathogenic bacteria (E. coli) in ground meat during 10 days of storage at 4 °C. Moreover, the treatment with L. plantarum S61 has no adverse effect on the sensory properties of ground meat after 10 days of storage at 4 °C. The treatment with L. plantarum S61 and storage at 4 °C effectively decreases the growth and risk of pathogenic bacteria in ground meat and, consequently, increases the product's shelf life. Therefore, the application of L. plantarum S61 during the storage of ground meat beef may help reduce the use of chemical preservatives in meat products. Consequently, L. plantarum S61 can be applied as a bio-control agent against spoilage and pathogenic bacteria in meat and meat products.

Biochemical characterization of an alkaline and detergent-stable Lipase from Fusarium annulatum Bugnicourt strain CBS associated with olive tree dieback.

This work describes a novel extracellular lipolytic carboxylester hydrolase named FAL, with lipase and phospholipase A1 (PLA1) activity, from a newly isolated filamentous fungus Ascomycota CBS strain, identified as Fusarium annulatum Bunigcourt. FAL was purified to about 62-fold using ammonium sulphate precipitation, Superdex® 200 Increase gel filtration and Q-Sepharose Fast Flow columns, with a total yield of 21%. The specific activity of FAL was found to be 3500 U/mg at pH 9 and 40°C and 5000 U/mg at pH 11 and 45°C, on emulsions of triocanoin and egg yolk phosphatidylcholine, respectively. SDS-PAGE and zymography analysis estimated the molecular weight of FAL to be 33 kDa. FAL was shown to be a PLA1 with a regioselectivity to the sn-1 position of surface-coated phospholipids esterified with α-eleostearic acid. FAL is a serine enzyme since its activity on triglycerides and phospholipids was completely inhibited by the lipase inhibitor Orlistat (40 µM). Interestingly, compared to Fusarium graminearum lipase (GZEL) and the Thermomyces lanuginosus lipase (Lipolase®), this novel fungal (phospho)lipase showed extreme tolerance to the presence of non-polar organic solvents, non-ionic and anionic surfactants, and oxidants, in addition to significant compatibility and stability with some available laundry detergents. The analysis of washing performance showed that it has the capability to efficiently eliminate oil-stains. Overall, FAL could be an ideal choice for application in detergents.

Purification, Biochemical and Kinetic Characterization of a Novel Alkaline sn-1,3-Regioselective Triacylglycerol Lipase from Penicilliumcrustosum Thom Strain P22 Isolated from Moroccan Olive Mill Wastewater.

A novel extracellular lipase from a filamentous fungus Ascomycota strain, P22, was isolated from olive mill wastewater, then purified and characterized. This strain was identified as Penicillium crustosum Thom based on sequencing analyses. Penicilliumcrustosum Thom strain P22 lipase (PCrL) was purified 63-fold to homogeneity using ammonium sulfate precipitation and chromatography on a Q-Sepharose Fast Flow column, with a total yield of 34%. The purified PCrL had a molecular mass of 28 kDa, estimated by SDS-PAGE. The 20 NH2-terminal amino-acid residues showed a high degree of homology with those of other Penicillium lipases. The specific activity of PCrL at pH 9 and 37 °C were found to be 5000 and 10,000 U/mg on olive oil and trioctanoin emulsions, respectively. PCrL exhibited clear regioselectivity toward the sn-1 position of the surface-coated triglycerides which were esterified with α-eleostearic acid at the sn-1/3 position. PCrL was completely inhibited by 53 µM of Orlistat, 5 mM of phenylmethylsulfonylfluoride, and 2 mM of diiodopropyl fluorophosphate, suggesting that it belonged to the serine lipase family. PCrL showed high activity and stability in the presence of water-immiscible organic solvents, surfactant, and oxidizing agents, and showed considerable compatibility with commercial laundry detergents. Washing performance analysis revealed that it could effectively remove oil stains. Hence, PCrL has several attractive properties that make it a promising potential candidate for detergent formulations.

Preparation, characterization, immobilization, and molecular docking analysis of a novel detergent-stable subtilisin-like serine protease from Streptomyces mutabilis strain TN-X30.

We recently described the production of a detergent-biocompatible crude protease from Streptomyces mutabilis strain TN-X30. Here, we describe the purification, characterization, and immobilization of the serine alkaline protease (named SPSM), as well as the cloning, sequencing, and over-expression of its corresponding gene (spSM). Pure enzyme was obtained after ammonium sulphate precipitation followed by heat-treatment and Sephacryl® S-200 column purification. The sequence of the first 26 NH2-terminal residues of SPSM showed a high sequence identity to subtilisin-like serine proteases produced by actinobacteria. The spSM gene was heterologously expressed in Escherichia coli BL21(DE3)pLysS and E. coli BL21-AI™ strains using pTrc99A (rSPSM) and Gateway™ pDEST™ 17 [(His)6-tagged SPSM] vectors, respectively. Results obtained indicated that the (His)6-tagged SPSM showed the highest stability. The SPSM was immobilized using encapsulation and adsorption-encapsulation approaches and three different carriers. Features of SPSM in soluble and immobilized forms were analyzed by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode, X-ray diffraction (XRD), zeta potential measurements, and field emission scanning electron microscopy (FE-SEM). The white clay and kaolin used in this study are eco-friendly binders to alginate-SPSM and show great potential for application of the immobilized SPSM in various industries. Molecular modeling and docking of N-succinyl-l-Phe-l-Ala-l-Ala-l-Phe-p-nitroanilide in the active site of SPSM revealed the involvement of 21 amino acids in substrate binding.

Antifungal activity of lactic acid bacteria and their application in food biopreservation.

Lactic acid bacteria (LAB) are ubiquitous bacteria associated with spontaneous lactic fermentation of vegetables, dairy and meat products. They are generally recognized as safe (GRAS), and they are involved in transformation of probiotic lacto-fermented foods, highly desired for their nutraceutical properties. The antifungal activity is one of the exciting properties of LAB, because of its possible application in food bio-preservation, as alternative to chemical preservatives. Many recent research works have been developed on antifungal activity of LAB, and they demonstrate their capacity to produce various antifungal compounds, (i.e. organic acids, PLA, proteinaceous compounds, peptides, cyclic dipeptides, fatty acids, and other compounds), of different properties (hydrophilic, hydrophobic and amphiphilic). The effectiveness of LAB in controlling spoilage and pathogenic fungi, demonstrated in different agricultural and food products, can be due to the synergistic effect between their antifungal compounds of different properties; where the amphiphilic-compounds allow the contact between the target microbial cell (hydrophilic compartment) and antifungal hydrophobic-compounds. Further studies on the interaction between compounds of these three properties are to de be developed, in order to highlight more their mechanism of action, and make LAB more profitable in improving shelf life and nutraceutical properties of foods.

Characterization of antimicrobial compounds obtained from the potential probiotic Lactiplantibacillus plantarum S61 and their application as a biopreservative agent.

This work aimed to characterize the antimicrobial compounds obtained from the potential probiotic Lactiplantibacillus plantarum S61, isolated from traditional fermented green olive, involved in their activity against fungi and bacteria responsible for food spoilage and poisonings. Their application as a biopreservative agent was also investigated. The culture of L. plantarum S61 showed substantial antifungal and antibacterial activity against yeasts (Rhodotorula glutinis and Candida pelliculosa), molds (Penicillium digitatum, Aspergillus niger, Fusarium oxysporum, and Rhizopus oryzae), and pathogenic bacteria (Listeria monocytogenes ATCC 19,117, Salmonella enterica subsp. enterica ATCC 14,028, Staphylococcus aureus subsp. aureus ATCC 6538, Pseudomonas aeruginosa ATCC 49,189), with inhibition zones > 10 mm. Likewise, the cell-free supernatant (CFS) of L. plantarum S61 showed an essential inhibitory effect against fungi and bacteria, with inhibition diameters of 12.25-22.05 mm and 16.95-17.25 mm, respectively. The CFS inhibited molds' biomass and mycelium growth, with inhibition ranges of 63.18-83.64% and 22.57-38.93%, respectively. The antifungal activity of the CFS was stable during 4 weeks of storage at 25 °C, while it gradually decreased during storage at 4 °C. Several antimicrobial compounds were evidenced in the CFS of L. plantarum S61, including organic acids, ethanol, hydrogen peroxide, diacetyl, proteins, and fatty acids. The protein fraction, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), demonstrated important antifungal activity, in relation to the fraction with molecular weight between 2 and 6 kDa. L. plantarum S61 and its CFS, tested in apple and orange fruit biopreservation, demonstrated their protective effect against P. digitatum spoilage. The CFS exhibited effectiveness in reducing Salmonella enterica subsp. enterica ATCC 14,028 in apple juice. L. plantarum S61 and/or its bioactive compounds CFS represent a promising strategy for biocontrol against pathogens and spoilage microorganisms in the agro-industry.

Bio-preservation Effect of Probiotic Lactiplantibacillus plantarum S61 Against Rhodotorula glutinis and Listeria monocytogenes in Poultry Meat.

The objective of this work is the study of the antifungal and antibacterial activity of Lactiplantibacillus plantarum S61 strains, isolated from traditional fermenting green olives against Rhodotorula glutinis UMP 22 and Listeria monocytogenes ATCC 19117, and its application in meat as bio-preservative agent. The cell-free supernatant (CFS) of Lpb. plantarum S61 shows high inhibition zones, which are 22.45 ± 0.49 and 17.75 ± 0.35 mm, against Rhodotorula glutinis and Listeria monocytogenes. The minimum fungicidal and bactericidal concentrations of the CFS obtained are 8% (v/v) and 10% (v/v), respectively. The competition assay, realized in liquid medium by co-culture of Lpb. plantarum S61 with Rho Rhodotorula glutinis and L. monocytogenes, led to inhibition percentages of 77.72% and 89.52%, respectively. However, the antimicrobial activity of Lpb. plantarum S61 was revealed a proteinaceous nature. Lpb. plantarum S61 strain allowed the reduction of L. monocytogenes in minced poultry meat during 7 days of storage at 4 °C. In addition, Lpb. plantarum S61 improved the physicochemical and color parameters of poultry minced meat. Lpb. plantarum S61 and/or its antimicrobial compounds can be applied as bio-preservative agent in meat product and food industry.

Characterization of ß-glucosidase of Lactobacillus plantarum FSO1 and Candida pelliculosa L18 isolated from traditional fermented green olive.

BACKGROUND: Oleuropein, the main bitter phenolic glucoside responsible for green olive bitterness, may be degraded by the ß-glucosidase enzyme to release glucose and phenolic compounds. RESULTS: Lactobacillus plantarum FSO1 and Candida pelliculosa L18 strains, isolated from natural fermented green olives, were tested for their ß-glucosidase production and activity at different initial pH, NaCl concentrations, and temperature. The results showed that strains produced extracellular and induced ß-glucosidase, with a molecular weight of 60 kD. The strains demonstrated their biodegradation capacity of oleuropein, associated with the accumulation of hydroxytyrosol and other phenolic compounds, resulting in antioxidant activity values significantly higher than that of ascorbic acid. The highest production value of ß-glucosidase was 0.91 U/ml obtained at pH 5 and pH 6, respectively for L. plantarum FSO1 and C. pelliculosa L18. The increase of NaCl concentration, from 0 to 10% (w/v), inhibited the production of ß-glucosidase for both strains. However, the ß-glucosidase was activated with an increase of NaCl concentration, with a maximum activity obtained at 8% NaCl (w/v). The enzyme activity was optimal at pH 5 for both strains, while the optimum temperature was 45 °C for L. plantarum FSO1 and 35 °C for C. pelliculosa L18. CONCLUSIONS: L. plantarum FSO1 and C. pelliculosa L18 strains showed their ability to produce an extracellular and induced ß-glucosidase enzyme with promising traits for application in the biological processing of table olives.