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Item Factors affecting acid protease production by Mucor circinelloides MG603064.1 through SmF process: characterization and fromage frais making(BioTechnologia, 2023) Bensmail, Souhila; Boudjema, Khaled; Naimi-Fazouane, Fethia; Bensmail, Samira; Djouahra-Fahem, Djamila; Ferhoum, Fatiha; Bourfis, NassimaThe exploitation of food industry wastes and their conversion into value-added products present a promising and continuously growing field, given the diversity of elaborated wastes. The current work aimed to utilize sweet cheese whey as a growth medium for acid protease production by a local fungus strain. The biochemical and physicochemical properties of the cheese whey, such as pH, conductivity, chemical oxygen demand, biological oxygen demand (BOD5), total nitrogen and protein contents, and mineral salts, were assessed using various analytical methods. The effect of certain parameters on acid protease production by Mucor circinelloides MG603064.1 through the SmF process was investigated using the conventional design method "One factor at a time". Subsequent to characterization, the crude extract was used in a trial to create fromage frais, compared to the commercial rennin CHY-MAX® Powder Extra. Cheese whey characterization revealed its richness in total nitrogen (1.044 ± 0.044 g/l), protein content (6.52 ± 0.04 g/l), and principal mineral salts: calcium (1.637 ± 0.037 g/l), phosphorus (1.173 ± 0.023 g/l), and chloride (1.66 ± 0.09 g/l). The optimal values of the SmF process for acid protease production, such as the inoculum size, beef extract, and KH2PO4 supplements, the initial pH of cheese whey, and incubation temperature were, respectively, 11% (v/v), 0.4% (w/v), 0.5% (w/v), 5.5, and 30°C. Under these conditions, the lowest milk-clotting time of 290 s was achieved, representing an 18.41-fold increase compared to the initial step using the unoptimized medium. The enzyme showed maximum milk-clotting activity at pH 5, a temperature of 60°C, and in the presence of 0.025 M of CaCl2. The enzyme activity also significantly improved with sonication (35 kHz) for 10 min. The crude extract of M. circinelloides ensured the production of fresh cheese samples with characteristics roughly similar to those obtained by the control (CHY-MAX® rennin). The acid protease of M. circinelloides could successfully substitute the conventional rennin in the manufacture of fresh cheese.Item Characterization and antibacterial activity of alkaloids and polyphenols extracts from Haplophyllum tuberculatum (Forssk.) A. Juss.(Pamukkale University, 2023) Djouahra-Fahem, Djamila; Bensmail, Souhila; Bouteldja, Razika; Messad, Sara; Ferhoum, Fatiha; Bourfis, Nassima; Acheuk, Fatma; Fazouane, FethiaHaplophyllum tuberculatum is a medicinal plant belonging to the Rutaceae family, is renowned for its various therapeutic properties. This study aims to characterize the alkaloids and polyphenol extracts from this plant and assess their antibacterial activity. Herein, the extraction of polyphenols and alkaloids from this plant was performed by the maceration method. Folin Ciacalteu's method was used to estimate the total phenolic content, and the qualitative characterization of the two extracts was performed by thin-layer chromatography. Whilst, the antibacterial activity of the two extracts was tested with the disk diffusion method on a solid medium and the minimal inhibitory concentration (MIC) of susceptible bacteria was determined using the agar dilution method. Our results indicate respective yields of 8.39% in polyphenols and 0.37% in alkaloid extracts, while the total phenolic content was estimated to be 74.45 mg GAE/g of dry matter in polyphenolic extract. Thin-layer chromatography analysis allowed choosing the system toluene-acetate-ethanol-concentrated ammonia (40:4:8:3, v/v) to separate H. tuberculatum alkaloids, and ethyl acetate-methanol-water (100:13.5:10, v/v/) for the separation of its polyphenols. The same analysis detected traces of quercitin, catechin, and rutin in the polyphenolic extract. Our findings demonstrated good antibacterial activity on Gram-positive strains such as Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633, and moderate activity on Pseudomonas aeruginosa ATCC 27953, with MICs ranging from 0.625 to 10 mg/mL for alkaloids and from 5 to 20 mg/mL for polyphenols.Item Trichoderma spp. isolates from the rhizosphere of healthy olive trees in northern Algeria and their biocontrol potentials against the olive wilt pathogen, Verticillium dahliae(Springer, 2021) Reghmit, Abdenaceur; Benzina‑tihar, Farida; López Escudero, Francisco Javier; Halouane‑Sahir, Fatma; Oukali, Zahia; Bensmail, Souhila; Ghozali, NourelhoudaTrichoderma species are known for their ability to control many plant diseases. In this study, fifteen (T1–T15) indigenous strains of Trichoderma spp. isolated from rhizosphere soils of olive in northern Algeria were investigated for their biocontrol potential against eleven strains of Verticillium dahliae, the causal agent of wilting on olive (Olea europea cv. europea L.). Direct and indirect confrontation assays were performed to investigate if isolates of Trichoderma affect mycelium growth. In addition, gas chromatography–mass spectroscopy (GC–MS) was applied to identify the volatile organic compounds (VOCS) produced by the T2 isolate. Besides, Trichoderma isolates were screened for the production of extracellular enzymes on solid medium including chitinases, proteasesand cellulases. Results showed that Trichoderma isolates demonstrated an effective potential in reducing mycelium growth against V. dahliae in vitro. T12 was the highest antagonistic of the Trichoderma isolates by direct confrontation assay method. This isolate exhibited an average inhibition rate of 68.86% against V. dahliae isolates, whereas the evaluation of volatile metabolites effect revealed that inhibition percentage of radial growth varies between 55.26 and 65.93%. Importantly, results showed the presence of 33 compounds detected in the methanolic and hexanic fraction of T2 isolate, including important volatile compounds with antifungal activities such as Undecane, Octadecane, Eicosane, 13-Docosenamide, (Z), Hexadecanamide, 9-Octadecenamide, (Z), Cyclopentanone, 2-methyl, Tetradecanoic acid, propyl ester, Tetradecanoic acid, oleic acid, and n-Hexadecanoic acid. Interestingly, all isolates tested showed chitinolytic, proteolytic and cellulolytic activity. T2 and T12 were the effective Trichoderma isolates showing high chitinase production on solid medium. Besides that, isolates T2 and T6 demonstrated the highest chitinolytic and proteolytic activity with values 0.043 µmol/ml/min and 0.019 µmol/ml/min, respectively. Overall, the isolates, T2, T6, and T12 are the most effective agents against olive wilt pathogen V. dahliae in vitro. Hence, they could be further investigated for biocontrol of olive wilt in vivoItem Production of Extracellular Rennin-Like Enzyme by a Newly Isolate Mucor circinelloides (von Tieghem) and its Application in Camembert Cheese Making(Baqiyatallah University of Medical Sciences, 2020) Bensmail, Souhila; Boudjema, Khaled; Naimi-Fazouane, FethiaIntroduction: Aspartic proteases produced by different non-pathogenic fungi belonging to mucorales, are commonly used as milk-coagulants. The present study aims to optimize conditions for milk-clotting protease production by a new strain of Mucor genus and to assess its ability in soft cheese making. Materials and Methods: About 20 fungal strains isolated from soil were investigated for their potential to produce milk-clotting proteases for further applications in cheese making. The hyper producer strain Mucor circinelloides 2095-2047 was selected for optimization of rennin-like enzyme production under solid-state fermentation (SSF) using the stepwise modifications of the selected parameters. The enzyme produced under the optimal conditions was partially purified and then applied in Camembert cheese making trials compared to the crude extract and commercial rennet. Results: The maximum milk-curdling activity achieved after optimization (571.43 SU/mL) was obtained using wheat bran (10 g) as the mainly source of carbon containing 1% galactose; moistened with the M-9 solution (pH 6.0) and incubated at 30°C for 96 hours. The enzyme of M. circinelloides was partially purified with a high recovery of 105% and 6.23-fold purity after (NH4)2SO4 fractionation and dialysis. The physicochemical properties of the three produced cheeses were very close. A low sensory quality of cheese was obtained with the crude extract which was getting better using the pre-purified enzyme. This extract was able to develop a very close or even a similar sensory quality to that obtained by the commercial rennet. Conclusions: According to findings, it is possible to propose the purified enzyme of M. circinelloides as a new alternative for rennet, but further optimization, purification and cheese production tests are required. © 2020 The Author(s)