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.

This work describes the immobilization and the characterization of purified Penicillium crustosum Thom P22 lipase (PCrL) using adsorption, encapsulation, and adsorption–encapsulation approaches. The maximum activity of the immobilized PCrL on CaCO₃ microspheres and sodium alginate beads was shifted from 37 to 45 °C, compared with that of the free enzyme. When sodium alginate was coupled with zeolite or chitosan, the immobilization yield reached 100% and the immobilized PCrL showed improved stability over a wide temperature range, retaining all of its initial activity after a one-hour incubation at 60 °C. The immobilization of PCrL significantly improves its catalytic performance in organic solvents, its pH tolerance value, and its thermal stability. Interestingly, 95% and almost 50% of PCrL’s initial activity was retained after 6 and 12 cycles, respectively. The characteristics of all PCrL forms were analyzed by X-ray diffraction and scanning electron microscopy combined with energy dispersive spectroscopy. The maximum conversion efficiency of oleic acid and methanol to methyl esters (biodiesel), by PCrL immobilized on CaCO₃, was 65% after a 12 h incubation at 40 °C, while free PCrL generated only 30% conversion, under the same conditions.

The present work reports the controlled fermentation of heat-shocked, unsalted and inoculated green olives. The effects of heat-shock (60, 70 and 80 °C three times for 5 min), inoculation with the oleuropeinolytic strain of L. plantarum FSO175 (L.p-FSO175) and the addition of Cell-Free Supernatant of C. pelliculosa L18 (CFS of C.p-L18) on the fermentation process of unsalted green olives were examined. The results showed a drastic reduction in the initial indigenous Enterobacteria, and an improvement in the acidification of heat-shocked olives at 70 and 80 °C, when compared to 60 °C. The inoculation with L.p-FSO175 and addition of CFS of C.p-L18 enhanced the fermentation and preservation of unsalted green olives, indicated by a significant decrease in pH, increase in free acidity and total disappearance of Enterobacteria. The heat-shock treatment at high temperature (80 °C), inoculation with L.p-FSO175 and addition of CFS of C.p-L18 led to the best reduction in bitterness, and favorable color changes (L, a, and b) in fermented olives. This sequential method led to more appreciated sensory characteristics (mainly bitterness and color) of fermented olives, lower spoilage incidence in olives, and reduced fermentation time to 50 days, and therefore may be suitable to control the fermentation of unsalted green olives of the Moroccan picholine variety.

Biological preservatives are increasingly in demand as an alternative to chemicals, because of consumer’s health and environmental concerns. Lactic acid bacteria isolated from fermented olives, considered as extreme environment, may encompass significant antimicrobial activity against microorganisms involved in foodborne diseases and food spoilage. Three LAB strains (Levilactobacillus brevis S27, Lactiplantibacillus pentosus S42, and Lactiplantibacillus plantarum S62), isolated from fermented olive, were studied for their antimicrobial activity against pathogenic bacteria (Listeria innocua and E. coli) and yeast (Rhodotorula glutinis), involved in foodborne diseases and food spoilage. The microbial culture, cell-free supernatant (CFS) of all LAB strains showed inhibition zone (IZ) values ranges of 26.33-28.83 mm, 14.16-16.33 mm and 10-12.16 mm against R. glutinis, L. innocua and E. coli, respectively. The IZ values ranges obtained with NCFS (neutralized CFS at pH 7) against R. glutinis and L. innocua were respectively of 17.83-20.50 mm and 13.16-14.50 mm, whereas no inhibition was obtained against E. coli. Their bioactive molecules of proteinaceous nature showed that among the four peptide bands obtained, two bands of approximately 42 and 56 kDa are common for the three LAB strains (S27, S42 and S62). Two additional bands of 46 and 89kD are observed in S42 and S27, respectively. The minimum inhibitory concentration of 10-fold concentrated CFS (10xCFS) obtained was 5% against E. coli and 4.5% against L. innocua and R. glutinis. The 10xCFS of Lb. plantarum S62, selected as the most efficient strain against pathogens, demonstrated its effectiveness in decontamination of milk at concentrations of 0.45% (v/v) and 0.5% (v/v) against L. innocua and E. coli. The CFS allowed, at 0.5 mL/piece of 5 cm diameter, a total inhibition of R. glutinis, without affecting the color of Pastilla leaf. The CFS of Lb. plantarum S62 can be used as a bio-preservative against pathogenic bacteria (L. innocua and E. coli) in milk, and against R. glutinis in Pastilla leaf.

In this study, the characterization, production, and application of antifungal metabolites obtained from the Levilactobacillus (L. brevis S27) and two Lactiplantibacillus (L. pentosus S42 and L. plantarum S62) strains were evaluated. The lactic acid bacteria (LAB) cells showed antifungal activity against molds. The cell-free supernatant (CFS) of Levilactobacillus and Lactiplantibacillus presented antimicrobial activity against fungi and foodborne pathogenic bacteria, and the antifungal activity was significantly (p < 0.05) higher than that of the antibacterial activity. Moreover, the antifungal metabolites were characterized as proteinaceous compounds that remained stable under both high and low temperatures and demonstrated activity across a broad pH range. Additionally, metabolite production was significantly higher (p < 0.05) at an initial pH of 5 when incubated at either 25 °C or 37 °C. All strains and their CFSs exhibited strong bio-preservative effects against Penicillium digitatum in yogurt and against Aspergillus niger on orange fruit. Consequently, these Lactobacilli strains and their antifungal metabolites represent a novel approach to biocontrol in the agri-food industry and agricultural products.