Nova Biotechnol Chim (2022) 21(2): e1269 DOI: 10.36547/nbc.1269 1 Nova Biotechnologica et Chimica Identification, safety, and technological characteristics of Weissella strains from traditional Southwestern Algerian kaddid Kamel Boubakri1,2,, Tayeb Idoui1, Chiara Montanari3, Federica Barbieri3, Fausto Gardini3,4, Graciela Vignolo5 1Laboratory of Biotechnology, Environment and Health (LBEH), University Mohamed Seddik Ben Yahia of Jijel, Jijel 18000, Algeria 2Department of Biology, Faculty of Sciences, University Yahia Farès of Médéa, Médéa 26000, Algeria 3Interdepartmental Center for Industrial Agri-Food Research, University of Bologna, Cesena, Italy 4Department of Agricultural and Food Sciences, University of Bologna, Bologna 33-40126, Italy 5Centro de Referencia para Lactobacilos (CERELA), CONICET, San Miguel de Tucumán, Tucumán 4000, Argentina  Corresponding author: boubakri.kamel@univ-medea.dz Article info Article history: Received: 20th November 2021 Accepted: 7th January 2022 Keywords: Kaddid Starter culture Traditional fermentation Weissella Abstract Kaddid is a dry-fermented meat product traditionally produced in North Africa by spontaneous fermentation. As a reservoir of natural biodiversity, the identification and relevant traits of lactic acid bacteria (LAB) were carried out from South-western Algeria homemade samples. After a preliminary physiological and biochemical screening, 19 presumptive LAB isolates were selected on the basis of antimicrobial compounds production. The isolates were identified by 16S rRNA gene sequencing as Weissella cibaria, W. confusa, W. paramesenteroides, Pediococcusacidilactici, and Enterococcus hirae. As predominant, the safety and technological characterization of Weissella strains were performed. The production of antimicrobial and antifungal compounds was observed, while neither H2S, biogenic amines nor hemolytic activity were detected; antibiotic resistance was exhibited, however several strains were more susceptible to assayed antibiotics. Technological characterization of Weissella strains showed high acidification rates even in the presence of up to 10 % NaCl, autolytic and proteolytic capacity however, no EPS production and lipolytic activity were observed. Strains characterization led to the selection of W. cibaria BK2, W. confusa BK6 and BK11 as well as W. paramesenteroides BK8 to be considered as possible candidates for use as starter culture for kaddid fermentation to improve and standardize this traditional meat product. Introduction Even today, traditional meat products are generally homemade as a mean to preserve meat to be consumed in times of scarcity. Among traditional products, the appreciation of fermented meats is probably related to their unique and specific sensory properties and alleged rootedness in a socio-cultural context. Meat products prepared in North African countries are usually dried or cooked because of the weather and are rarely smoked; they are produced from different meat (beef, lamb, goat and camel) depending on the geographic area. Dried kaddid has been traditionally used as mailto:boubakri.kamel@univ-medea.dz Nova Biotechnol Chim (2022) 21(2): e1269 2 ingredient to prepare different winter dishes as stews and soups (Gagaoua and Boudechicha 2018). In Algeria, it is prepared by adding salt and spices to lamb meat cutting into thin strips, which are then hang on a string and exposed to the air until thoroughly dried and stored at room temperature; salt content in the final product ranges from 7 % to 12 % (Bennani et al. 1995; Benlacheheb et al. 2018). Microbiological survey of dry-salted kaddid showed the presence of lactic acid bacteria (LAB) in addition to microorganisms related to the product hygiene (Bennani et al. 2000; Ben Belgacem et al. 2008, 2010; Essid et al. 2009; Benlacheheb et al. 2018). Physicochemical features of kaddid during ripening such as pH, moisture, salt content and water activity are main modulators of microbiological evolution. It is known that the presence of LAB induces desirable attributes to traditional fermented food products, enhancing their typical characteristics while conferring additional safety and health benefits. Distinctive features derived from LAB metabolic activities in salted meat will determine final quality of fermented products. In view to design novel functional starter cultures, it would be necessary to exploit the autochthonous LAB with technological, functional and safety characteristics. A quick growth and high acidification rate in the presence of high salt content are the main criteria for strains selection by which a safe initial environment can be created and food pathogen and spoilage reduced (Fusco 2015; Fessard and Remize 2017). From a safety point of view, the production of antibacterial and antifungal compounds during fermentation are desired features as these metabolites inhibit pathogen and spoilage proliferation. The lack of antibiotic resistance, virulence factors and aminogenesis among other traits, are also required (Castellano et al. 2017). In addition, the United Nations Food and Agriculture Organization (FAO)/World Health Organization (WHO) stated the importance to conduct a minimum safety assessment including several specific metabolite productions, toxin production, and potential hemolysis, even for microbial populations classified as GRAS (FAO/WHO 2002). Moreover, changes in meat and fat are responsible for the flavour in fermented meat. Proteolysis and lipolysis by meat enzymes and LAB will impact on the development of typical sensory characteristics by peptides and amino acids generation (Fadda et al. 2010; Vignolo et al. 2019). Thus, the aim of this study was the identification of the LAB population from Algerian kaddid samples and traits related to technological and safety features of autochthonous isolated LAB were investigated. Experimental Samples collection and LAB isolation Samples of homemade kaddid were collected from the Southwestern region of Algeria, namely Tindouf (one sample from camel meat) and Béchar (6 samples from lamp meat) provinces. Approximately 400 g of ready to consume kaddid pieces from each producer were placed in sterile plastic bags and transported refrigerated to the laboratory for analyses. Each sample (10 g) was suspended in 90 mL of sterile tryptone-salt diluents (tryptone 1 g.L-1; NaCl 0.85 g.L-1; Tween 80 1 mL.L-1), homogenized for 3 min (Stomacher 400, Seward, Worthing, UK) and serially diluted. Dilutions were then plated in duplicate on MRS and M17 (Merck, Darmstadt-Germany) media, and microaerobically incubated at 30 °C during 72 h. An average of 20 – 25 colonies per kaddid sample were randomly picked from both media plates containing 100 – 300 colonies. Gram-positive and catalase-negative bacteria (presumptive LAB) were sub-cultured on the corresponding medium and stored in 25 % of glycerol at -80 °C for further use. Physiological and biochemical characterization of LAB Sixty-three presumptive LAB isolates were preliminarily identified according to Von Wright and Axelsson (2012) and the scheme described by Carr et al. (2002). Growth at 10 ºC and 45 ºC during 7 d and 24 h, respectively, at pH 4 to 9.6 and in the presence of 4 to 10 % NaCl were evaluated in MRS (bacilli) and M17 (cocci). Production of gas (CO2) from glucose (Gibson and Abdelmalek 1945) and arginine hydrolysis (Møller 1955) were analyzed. For carbohydrate Nova Biotechnol Chim (2022) 21(2): e1269 3 fermentation MEVAG medium (Biokar Diagnostic, Allonne, France) supplemented with different sugars (Table 1) was used, LAB suspensions being inoculated by a central puncture and covered by a vaseline layer to promote anaerobiosis; after incubation (30 °C; 24 h) color changes due to the sugars utilization by bacteria were recorded. Molecular identification Genomic DNA was extracted from pure LAB cultures using InstaGene matrix (Bio-Rad Laboratories Inc., Hercules, USA). Molecular identification was performed as described by Montanari et al. (2015). The partial 16S rRNA gene sequence was amplified using the primers LpigF/LpigR (5’-TACGGGAGGCAGCAGTAG- 3’ and 5’-CATGGTGTGACGGGCGGT-3’; Eurofins Genomics Germany GmbH, Ebersberg, Germany). PCR amplifications were performed using Taq DNA polymerase kit from Thermo Fisher Scientific (Waltham, USA). Reaction mixtures consisted of buffer 5X, 2 mM MgCl2, 50 mM each of the 4 deoxynucleoside triphosphates (dNTP), 1.25 U of Taq-polymerase, 0.5 mM of each primer, and 0.5 μL of appropriately diluted template DNA in a final volume of 50 μL. Amplification was performed on a Biometra T3000 thermal cycler (Analytik Jena GmbH, Jena, Germany) with initial denaturation at 94 °C for 5 min, then 34 cycles at 94 °C for 1 min, 55.5 °C for 2 min, and 72 °C for 2 min, followed by final extension at 72 °C for 10 min. PCR products were separated by electrophoresis on 1.5 % (w/v) agarose gel (Lonza, Italy) stained with ethidium bromide (0.5 μg.mL-1). The 600 bp amplicons were eluted from an agarose gel, purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced at the BMR Genomics Srl. sequencing facility (Padova, Italy) using the same primers as for amplification. Sequence similarity searches were performed using BLAST network service (http://blast.ncbi.nlm.nih.gov/) and Ez-Taxon Server (http://147.47.212.35:8080/). Antimicrobial activity LAB (63 strains) inter-species inhibitory capacity was first investigated by using the disc method described by Tadesse et al. (2004) with modifications. Overnight LAB cultures (0.5 mL) used as indicators were mixed with 12 mL of melted and cooled MRS agar media, poured into plates, let solidify and dried. LAB cultures were centrifuged (7,000 × g; 15 min) and the obtained cell free supernatants were used to impregnate sterile filter paper discs that were deposited on the seeded agar. On the other hand, the antibacterial activity of identified Weissella isolates was evaluated by a semi-quantitative agar-spot-test as described by Fontana et al. (2015) against Gram positive and Gram-negative bacteria (Table 3). Overnight LAB cultures were centrifuged (7,000 × g; 15 min), and supernatants (5 µL) were spotted onto 10 mL of BHI agar (Britania, Argentina) plates (0.7 %) previously inoculated with 50 µL of each indicator strain. Plates were incubated at 30 °C for 48 h and the presence of a clear inhibition zone around the spots was considered as a positive antagonistic effect. Inhibitory activity was expressed as + (halos presence) or – (no halos) around the spot. Positive antibacterial activity LAB supernatants were neutralized (4N NaOH) and treated with catalase (1,000 U.mL-1) (Sigma- Aldrich, St Louis, USA) to determine the chemical nature of the inhibitory substance. Antifungal activity against Aspergillus flavus, Penicillium expansum, and Fusarium oxysporum albedinis was investigated by a modified agar diffusion assay (Magnusson et al. 2003). Petri plates containing Potato Dextrose Agar (PDA) were inoculated with the fungus and incubated at 25 °C during 48 h. After visible formation of conidiospores, they were collected and adjusted to 105 spores/ml of sterile saline solution. LAB selected strains were streak- inoculated on MRS agar plates and after incubation (30 °C; 48 h), plates were overlaid with 10 mL of PDA soft agar (0.7 % agar) containing fungal spore suspensions (104.mL-1) and incubated aerobically (30 °C; 48h). Plates were then examined for clear inhibition zones around the LAB streaks and scored as – (no growth inhibition), + (1 – 5 mm growth inhibition), ++ (5 – 10 mm growth inhibition) and +++ (˃ 10mm growth inhibition). 3 http://blast.ncbi.nlm.nih.gov/ http://147.47.212.35:8080/ Nova Biotechnol Chim (2022) 21(2): e1269 2 Biogenic amines and H2S production The ability to decarboxylate amino acids used as precursor was tested according to Bover-Cid and Holzapfel (1999). Briefly, the plates with the agar medium, supplemented with histidine, tyrosine, ornithine, and lysine 1 % (w/v) were spotted with the active Weissella (7 strains) and incubated anaerobically (30 °C; 2 – 5 d). Growth of decarboxylating strains was easily recognizable because of a purple halo in the yellow medium. Production of H2Swas investigated on TSI (Triple sugar iron) agar medium by a central puncture inoculation. After incubation (30 °C; 48 – 72 h), H2S production was confirmed by the blackening of the medium and gas bubbles in the agar (Guiraud 2003). Antibiotic resistance Antibiotics recommended for the European Food Safety Authority (EFSA 2012) to identify bacterial strains with potential acquired resistance were used. Ampicillin (AMP; 0.032 – 16 g.mL-1), vancomycin (VAN; 0.25 – 128 g.mL-1), chloramphenicol (CHL; 0.125 – 64 g.mL-1), gentamycin (GEN; 0.5 – 256 g.mL-1), streptomycin (STR; 0.5 – 256 g.mL-1), kanamycin (KAN; 2 – 1024 g.mL-1), tetracycline (TET; 0.125 – 64 g.mL-1), erythromycin (ERY; 0.016 – 8 g.mL-1) and clindamycin (CLI; 0.032 – 16 g.mL- 1) were tested. The minimum inhibitory concentration (MIC) of antibiotics was determined by the broth micro-dilution method reported by the ISO 10932/IDF 233 standard. The strains were classified as susceptible or resistant according to the cut-off values proposed by EFSA (2012). A bacterial strain was defined as susceptible or resistant when it was inhibited or not, at a specific antimicrobial concentration equal or lower than the established cut-off value. Hemolysin and gelatinase activity Hemolysin activity was determined on Columbia Blood Agar (Oxoid) containing 5 % defibrinized horse blood after 48 h of incubation at 37 °C, both under aerobic and anaerobic conditions. The type of hemolysis (α, β or ɣ) was determined. Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 were used as positive control for β- and α–hemolysis, respectively. Zones of clearing around colonies indicated -hemolysin production. Gelatinase production was detected by inoculating LAB onto freshly prepared peptone yeast extract agar containing gelatin (30 g.L-1; Difco). Plates were incubated overnight at 37 °C and cooled at room temperature for 2 h. The presence of turbid zone around the colonies was considered as positive result. Growth and acidification Selected Weissella strains were inoculated (1 %) into MRS broth, incubated at 10, 30, 37 and 44 °C for 48 h and growth at OD620 was measured. Similarly, the growth of each strain was evaluated in MRS with pH adjusted to pH 4.5, 5, 6, 7, 8 and 9.6 and supplemented with 4, 6.5 and 10 % NaCl. The OD620 was measured at 0, 2, 4, 6, 24 and 48 h. Acidification ability was evaluated as reported by Ammor et al. (2005), using Sausage-Broth (SB) medium. Eighty (80) ml of SB medium was inoculated with an overnight culture of each strain. The pH values and OD620 were recorded after 0, 3, 6, 24, 48, 72 and 96 h of incubation at 30 °C using a pH meter and a spectrophotometer. Autolytic activity and thermoresistance Each strain was suspended in PBS buffer (pH 7) at a DO620 : 0.2 and subjected to a freeze cycle (-20 °C for 24 h) and after thawing, strains were incubated at 30 °C for 24 h. Autolytic activity was determined by the decrease percentage in absorbance at D620 after time interval (Piraino et al. 2008) as %AA: (Ai-At) × 100/Ai, where AA: autolytic activity, Ai: initial Absorbance and At: Absorbance after 24 h of incubation. Autolysis was classified according to lactobacilli genus (Ayad et al. 2004), ranged from 70 – 96 % (good), 40 – 69 % (low) and 0 – 39 % (poor). Thermo-resistance was evaluated as reported by Badis et al. (2004); LAB strains inoculated in MRS broth were heated at 60.5 °C during 30 min, and then incubated at 30 °C for 24 to 48 h and the colonies in MRS agar were enumerated. 4 Nova Biotechnol Chim (2022) 21(2): e1269 3 Proteolytic and lipolytic activities Five microliters (5 μL) of each LAB strain suspended in PBS buffer (pH 7) were spot inoculated onto tryptone soy agar (TSA) supplemented with sterile skim milk (10 %) as described by Guiraud (2003). After incubation at 30 °C for 5 d, the caseinolytic activity (measured in mm) was determined by the presence of a clear area around the spot. For lipolytic activity, the technique reported by Mauriello et al. (2004) with minor modifications was used. One mL of LAB overnight cultures was inoculated into 10 mL of a broth containing tryptone (1 % (w/v)), yeast extract (0.5 % (w/v)), NaCl (3 % (w/v)), pH 7.0, supplemented with 4 % (w/v) lamb fat previously homogenized by vigorous shaking. After incubation at 30 °C for 7 d, free fatty acids were then determined. The lipids were extracted into 10 ml of petroleum ether by shaking for 1 min. The fatty acids of the upper phase were titrated with NaOH (0.1 M) in ethanol using 1 % phenolphthalein-ethanol solution as indicator. Results were expressed as % of oleic acid by a × N × 28.2/g, where a: mL NaOH used for titration, N: NaOH normality, 28.2: % of oleic acid equivalent weight and g: amount of lamb fat used. Exopolysaccharides (EPS) production Active cultures of LAB strains were spotted on MRS agar in which glucose was replaced by sucrose and incubated at 30 °C for 2 – 7 d. Ropiness was examined by the presence of a ropy condition after touching the colony with a loop. Statistical analysis Agar assays were performed by duplicate and growth curves by triplicate. In the case of antibiotic resistance, media values were compared with cut- off points. The media and SD were calculated for growth data, results (means OD ± SD) being evaluated by the application of ANOVA to define differences and statistical significances were determined by the Tukey test. Results and Discussion Physiological and biochemical characterization of LAB isolates Sixty-three presumptive LAB isolates (42 cocci and 21 bacilli) were subjected to a preliminary characterization. Results showed that 10 % of the isolates produced gas from glucose (Table 1). Among homofermentative cocci/coccobacilli isolates, those tetrads-forming cocci (23.8 %) that grew at 10 °C and 40 – 45 °C, up to 10 % NaCl but not at pH > 8.0 were presumed as Pediococcus. However, chains-forming homofermentative cocci (47.7 %) that developed at the same temperatures, in a wider pH range (4.0 to 9.6) and up to 6.5 % NaCl were assigned to Enterococcus genus, differing from lactococci in that the latter are not able to grow at 40 – 45 °C. On the other hand, heterofermentative cocci/coccobacilli isolates (28.6 %) able to grow at 10 °C, up to 6.5 % NaCl and pH between 4.5 and 8.0 (arginine mostly positive and variable growth at pH > 6.0, between 40 – 45 °C and NaCl > 6.5 %) have been characterized as presumptive Leuconostoc or Weissella. Concerning bacilli (21 %) involving homo and heterofermentative isolates would be assigned to Lactobacillus or Weissella genus with variable arginine hydrolysis, ability to grow between 40 – 45 °C in a wide range of pH and resistant to NaCl and sugar fermentation capacity except for xylose. Sugars fermentation for cocci/coccobacilli isolates exhibited variable carbohydrates fermentation; heterofermentative coccobacilli fermented cellobiose, fructose, maltose, mannose, sucrose, and xylose but not melibiose, raffinose, sorbitol and trehalose. Based on phenotypic, morphologic and biochemical characterization, pH, temperature, salt tolerance and sugars fermentation, it can be preliminary suggested that cocci/coccobacilli isolates belong to Pediococcus, Enterococcus, Leuconostoc, Weissella and Lactobacillus genera. These results agree with the LAB from high salt- containing fish and meat products (Najjari et al. 2008; Ben Belgacem et al. 2010; Belfiore et al. 2013). 5 Nova Biotechnol Chim (2022) 21(2): e1269 2 Molecular identification of isolates exhibiting antimicrobial activity After the first inter-LAB species inhibition assay, selected isolates (19) were subjected to molecular identification. Table 1. Physiological and biochemical characterization of LAB isolated from kaddid. Physiological traits Cocci/Coccobacilli (42) Bacilli (21) Homofermentative Heterofermentative Homo/Hetero Microscopy Cocci/ tetrads Cocci/ chains Coccobacilli single/pairs Chains/ pairs CO2 from glucose – – + +/– Arginine hydrolysis +(v) +/– – +/– Growth at 10 °C + + + + 45 °C + + +/– v NaCl 4% + + + + 6.5% + + +/– + (v) 10% + +/– – (v) + (v) pH 4.5 + + + + 6.5 + + + (v) + 8.0 – + +/– v 9.6 – + – – D-Glucose + + + + D-Arabinose +/– +/– +/– +/– D-Cellobiose – +/– + + D-Fructose +/– + + + D-Galactose +/– + +/– + D-Lactose + + + + D-Maltose – – + + D-Mannose – + + + D-Mannitol + – +/– + D-Melibiose – +/– – + D-Ribose + +/– + + D-Raffinose +/– – – +/– D-Sorbitol +/– +/– – +/– D-Sucrose +/– + + +/– D-Trehalose – + – + D-Xylose +/– +/– + – + = Positive; – = negative; +/– = more or less positive; v = variable. In parallel with the physiological/biochemical characterization, amplification of partial 16S rRNA gene sequence allowed the identification of 15 isolates as Weissella (W.) cibaria/confusa with a similarity level of 99 to 100 %, one isolate as W. paramesenteroides (similarity 99.87 %), two isolates as P. acidilactici (similarity > 99 %) and one isolate as E. hirae (similarity 99.77 %), (Table 2). This result indicated that most of LABs were heterofermentative, while only three strains were homofermentative. Because Weissella species constituted the major population among identified LAB, they were used for further studies. High relatedness of W. cibaria and W. confusa observed in this study agrees with that described by Lynch et al. (2015). However, based on differential capacity to ferment carbohydrates, W. cibaria was described to ferment arabinose but not galactose and ribose, while these latter were utilized by W. confusa (Björkroth et al. 2002; Quattrini et al. 2019). Results from carbohydrates fermentation were used to complement molecular identification (Table 1). Thus, strains were distinguished as W. cibaria (BK1, BK2, BK3, BK9, BK10, BK13, BK18, BK19), W. confusa (BK4, BK5, BK6, BK7, BK11, BK12, BK16), W. paramesenteroides (BK8), P. acidilactici (BK14, BK17) and E. hirae (BK15) as shown in Table 2. Species of this genus have been isolated from a wide range of ecological niches including soil, plants, breast milk, oral cavity, urogenital and GIT tract of humans and animals as well as a huge variety of fermented foods (Abriouel 6 Nova Biotechnol Chim (2022) 21(2): e1269 3 et al. 2015; Fusco et al. 2015; Maldonado et al. 2018). From a technological point of view, Weissella plays a key role in food fermentation based on vegetables and to a lesser extent in meat. However, the identification of W. cibaria, W. confusa and W. paramesenteroides from dry-salted Algerian kaddid agrees with those reported during dry-cured sausages fermentation (Fusco et al. 2015). Indeed, W. cibaria and W. confusa were previously retrieved from Portuguese fermented sausages, Thai pork sausages (nham), morcilla de Burgos and fermented fish (Santos et al. 2005; Srionnual et al. 2007; Albano et al. 2009; Kopermsub and Yunchalard 2010; Wongsuphachat et al. 2010). Likewise, W. paramesenteroides was identified from Italian fermented sausages (Urso et al. 2006; Papagianni and Papamichael 2011). Although W. cibariahas been first isolated from Thai fermented meat (Björkroth et al. 2002), together with W. confusa have been associated with a wide range of vegetable fermented products and their ability to use plant carbohydrates was reported (Fusco et al. 2015). Besides plant sugars utilization, W. confusa and W. paramesenteroides showed to use ribose, suggesting they also may grow in meat. Table 2. Lactic acid bacteria (LAB) strains identified from dried salted Algerian kaddid. LAB isolates Isolation source Closest relative Identity [%] Accession No.* BK1 SK1 Weissella cibaria 99.41 % MT158598.1 BK2 SK1 Weissella cibaria 99.87 % MT012260.1 BK3 SK1 Weissella cibaria 99.87 % MT012260.1 BK4 SK2 Weissella confusa 100 % MK503640.1 BK5 SK2 Weissella confusa 100 % MK503640.1 BK6 SK3 Weissella confusa 99.76 % MK503640.1 BK7 SK3 Weissella confusa 100 % MK503640.1 BK8 SK4 Weissella paramesenteroides 99.87 % MN994365.1 BK9 SK4 Weissella cibaria 99.75 % MT012260.1 BK10 SK5 Weissella cibaria 99.55 % MT012260.1 BK11 SK5 Weissella confusa 99.50 % MK503640.1 BK12 SK6 Weissella confusa 99.88 % MK503640.1 BK13 SK6 Weissella cibaria 99.65 % MT012260.1 BK14 SK6 Pediococcus acidilactici 99.74 % CP050079.1 BK15 SK7 Enterococcus hirae 99.77 % MT197246.1 BK16 SK7 Weissella confusa 99.05 % MK503640.1 BK17 SK7 Pediococcus acidilactici 99.88 % CP050079.1 BK18 SK7 Weissella cibaria 100 % MT012260.1 BK19 SK7 Weissella cibaria 99.75 % MT012260.1 SK – sample of kaddid. Kaddid Southwestern Algerian samples were from: SK1, SK2, SK3 (Béchar city), SK5 (BeniOunif), SK6 and SK7(Igli) from Béchar province, and SK4 from Tindouf province. *Sequence similarity searches were performed using BLAST networkservice (http://blast.ncbi.nlm.nih.gov/). Antimicrobial activity In a first attempt to distinguish those isolates exhibiting inhibitory activity, from 63 LAB isolates preliminarily assigned to different LAB genera, 19 of them showed inter-species antagonistic activity (Result not shown) suggesting the presence of antibacterial metabolite/s in the supernatant. Indeed, when neutralized supernatants and catalase addition were evaluated, the inhibitory activity was suppressed indicating that organic acids or some compound of protein nature would be responsible for the antibacterial effect. The production of inhibitory metabolites active against food pathogens could be an important improvement for starter cultures and might be of interest in controlling meat fermentation, which naturally contain competing food-borne pathogens. Therefore, as a dominant population, sixteen LABs assigned to Weissella genus were investigated for their antibacterial and antifungal activity against a range of pathogens and contaminants (Table 3). Results showed Weissella isolates BK2, BK3 and BK19 as the highest inhibitory strains against the assayed pathogens. Bacteriocin production by Weissella species was widely reported, especially 7 https://www.ncbi.nlm.nih.gov/nucleotide/MT158598.1?report=genbank&log$=nucltop&blast_rank=2&RID=89BPJF6N01R https://www.ncbi.nlm.nih.gov/nucleotide/MT012260.1?report=genbank&log$=nucltop&blast_rank=1&RID=89DNR0XF016 https://www.ncbi.nlm.nih.gov/nucleotide/MT012260.1?report=genbank&log$=nucltop&blast_rank=1&RID=89EFXJ7Z014 https://www.ncbi.nlm.nih.gov/nucleotide/MK503640.1?report=genbank&log$=nucltop&blast_rank=2&RID=89EPEGHV014 https://www.ncbi.nlm.nih.gov/nucleotide/MK503640.1?report=genbank&log$=nucltop&blast_rank=2&RID=89F7YZYH014 https://www.ncbi.nlm.nih.gov/nucleotide/MK503640.1?report=genbank&log$=nucltop&blast_rank=2&RID=89FP23GY01R https://www.ncbi.nlm.nih.gov/nucleotide/MK503640.1?report=genbank&log$=nucltop&blast_rank=2&RID=89FXM8BY014 https://www.ncbi.nlm.nih.gov/nucleotide/MN994365.1?report=genbank&log$=nucltop&blast_rank=1&RID=89GNJHAW01R https://www.ncbi.nlm.nih.gov/nucleotide/MT012260.1?report=genbank&log$=nucltop&blast_rank=1&RID=89MTEMFX014 https://www.ncbi.nlm.nih.gov/nucleotide/MT012260.1?report=genbank&log$=nucltop&blast_rank=1&RID=89N8WEMV014 https://www.ncbi.nlm.nih.gov/nucleotide/MK503640.1?report=genbank&log$=nucltop&blast_rank=2&RID=89NJNT5G016 https://www.ncbi.nlm.nih.gov/nucleotide/MK503640.1?report=genbank&log$=nucltop&blast_rank=2&RID=89PJR8FZ014 https://www.ncbi.nlm.nih.gov/nucleotide/MT012260.1?report=genbank&log$=nucltop&blast_rank=1&RID=89RV1PYC01R https://www.ncbi.nlm.nih.gov/nucleotide/CP050079.1?report=genbank&log$=nucltop&blast_rank=1&RID=89ST2GRE014 https://www.ncbi.nlm.nih.gov/nucleotide/MT197246.1?report=genbank&log$=nucltop&blast_rank=1&RID=89T640S2016 https://www.ncbi.nlm.nih.gov/nucleotide/MK503640.1?report=genbank&log$=nucltop&blast_rank=2&RID=89TJ232X014 https://www.ncbi.nlm.nih.gov/nucleotide/CP050079.1?report=genbank&log$=nucltop&blast_rank=1&RID=89TSX2W701R https://www.ncbi.nlm.nih.gov/nucleotide/MT012260.1?report=genbank&log$=nucltop&blast_rank=1&RID=89U1554W016 https://www.ncbi.nlm.nih.gov/nucleotide/MT012260.1?report=genbank&log$=nucltop&blast_rank=1&RID=89U7CP5H016 http://blast.ncbi.nlm.nih.gov/ Nova Biotechnol Chim (2022) 21(2): e1269 2 with activity against other LAB species (Fusco et al. 2015). The exceptional inhibitory ability against E. coli, S. typhimurium and Pseudomonas was in coincidence with that reported for Weissella species (Woraprayote et al. 2015; Fessard and Remize 2017). In addition, supernatants of the examined presumptive Weissella exhibited a high inhibitory activity against L. monocytogenes Scott A, GM1 and GM2 and L. innocua ATCC51742 and DSM20649 as well as S. aureus ATCC 29213 indicator strains, with inhibition zones between 8 to 20 mm, whereas Enterococcus and Pediococcus were not inhibited. The ability of Weissella isolates to prevent Listeria and Staphylococcus growth agree with those reported for W. paramesenteroides, W. hellenicaand W. viridescens from pickles, sea foods and meat fermented products (Papagianni and Papamichael 2011; Masuda et al. 2012; Leong et al. 2013; Chen et al. 2014; Castilho et al. 2019). Moreover, the antifungal activity of 10 out of 16 Weissella isolates against fungal indicators (Table 3) coincide with that reported for W. cibaria, W. confusa and W. paramesenteroides against other phytopathogenic or food fungal strains (Trias et al. 2008; Valerio et al. 2009; Ndagano et al. 2011; Bianchini et al. 2015; Quattrini et al. 2019). The production of lactic and acetic acids by heterofermentative Weissella may account for their great inhibitory activity, in agreement to fungal inhibitory compounds production reported by Gerez et al. (2013). Due to their antimicrobial activity, Weissella have been found to act as foodbiopreservatives and probiotics in humans and animals (Abriouel et al. 2015; Fusco et al. 2015). The production of antimicrobial compounds is desired, thus the proliferation of pathogens or spoilage microorganisms can be controlled during fermentation. Based on these antimicrobial features, W. cibaria BK2, BK3 and BK19, W. confusa BK4, BK6 and BK11, and W. paramesenteroides BK8 were selected to investigate their major safety and technological properties. Safety evaluation Although many LAB species have been recognized as GRAS organisms by FDA (1999) or have attained the QPS status by EFSA (2004), no Weissella species were included. Studies on antibiotic resistance profile of this genus are limited, and MIC cut-off has not still defined by EFSA. When Weissella strains resistance/sensitivity to clinical antibiotics was investigated (Table 4), a multiresistance pattern was found, this being in correlation with that described by Abriouel et al. (2015). Similar to other LAB, W. cibaria, W. confusa and W. paramesenteroides exhibited intrinsic resistance to VAN (Ouoba et al. 2008). However, additional high resistance to KAN, GEN and TET was exhibited for all Weissella strains, strains BK3 and BK19 were also resistant to STR and CHL, this being in coincidence with those reported for strains isolated from Chinese dry fermented meat product (Wang et al. 2018) and fermented salted squid (Le and Yang 2018). Similar resistance to GEN and KAN of W. cibaria strain from goat milk was described (Elavarsi et al. 2014), and STR resistance of W. cibaria and W. confusa from fruit/juices was also reported (Xu et al. 2018). However, the investigated strains were sensitive to ERY, which disagree with that found for W. cibariaof vegetable origin (Xu et al. 2018; Dentice Maidana et al. 2019). Antibiotic resistance patterns found here are related to the controversial nature of Weissella genus reported by Abriouel et al. (2015), and the lack of use as commercial starter so far (Fessard and Remize 2017). Despite the resistance to aminoglycosides KAN and GEN, W. cibaria BK2 was the more sensitive among assayed strains. Within the framework of food safety, Weissella strains were also investigated for their hemolytic and gelatinase activity as well as biogenic amines production. Results showed neither gelatinase nor -hemolytic activity was exhibited by Weissella strains, only -hemolysis being observed. Similarly, biogenic amines were not produced by the analyzed strains (data not shown). Due to their controversial status, determination of these safety traits for Weissella strains help in carefully selecting strains lacking pathogenic potential. Technological characterization In view to select Weissella strain/s to be used for kaddid fermetnation, several technological traits 8 Nova Biotechnol Chim (2022) 21(2): e1269 3 were evaluated. The strains tested showed a good adaptation towards cultural stresses, such as temperature, NaCl concentration and pH (Table 4). Acidification showed pH values between 2.10 and 2.37 units after four days, showing a decrease to 4.03 – 4.27 after 6 h of incubation at 30 °C, reaching final values (96 h) in the range of 3.80 – 4.10 (data not shown). The pH reduction is in correlation with the acid production by LAB strains, W. paramesenteroides BK8 being the most acidogenic. The average acidification rate of assayed strains resulted in 0.55 units/day, which was higher than that of traditional kaddid (Benlacheheb et al. 2018). As expected, optimal temperature for Weissella strains was 30 °C reaching OD620 between 1.96 and 2.11 at 48 h with an average growth rate of 0.47/h. When NaCl concentration increased from 4 to 10 % a decrease in Weissella growth from OD620 of 1.68 to 0.06 was produced, W. paramesenteroides BK8 being the most resistant to 10 % of NaCl (Table 4). Osmotic adaptation of strains correlated with their growth under the high salt concentration of kaddid. To prevent spoilage/pathogens proliferation, quick growth/acidification capacity is an important criterion for the selection of LAB starter. In addition, the evaluation of enzymatic activities that could play a role in the flavor development, such as the release of intracellular enzymes by cell lysis (autolysis) showed values in the range of 4.23 – 8.08 %, while proteolytic activity was only exhibited by W. confusa BK11.Autolysis of Weissella strains at 24 h here obtained showed lower values compared to that reported for W. confusa strain isolated from Indian fermented foods (Sharma et al. 2018). All Weissella strains showed to be thermoresistant when heated at 60.5 ºC during 30 min while no lipolytic activity was detected (data not shown). Similarly, a lack of lipolytic activity was also reported for Lactobacillus plantarum isolated from Tunisian kaddid (Essid et al. 2009), but were able to hydrolyze casein, contrarily to the results for W. cibaria strains in this study. W. cibaria has been reported to have an extensive peptidolytic activity (Lynch et al. 2015); perhaps the use of casein as a protein source did not allow evidencing this activity. Furthermore, the lack of H2S and EPS production favor their use as starter culture in meat fermentation; even when the ability to produce EPS is a common trait for W. cibaria and W. confusa (Fusco et al. 2015; Lynch et al. 2015; Quattrini et al. 2019), formation of these compounds in meat products would lead to an indication of sensory spoilage. Therefore, based on antimicrobial activity, antibiotic resistance patterns, growth, and acidification, NaCl tolerance and moderate protein hydrolysis as well as the lack of virulence factors and adverse sensory traits, the strains W. cibaria BK2, W. confusa BK6 and BK11 as well as W. paramesenteroides BK8 may be selected as candidates to be used in the fermentation of Algerian kaddid. Conclusion Microbiological examination of Southwestern Algeria dried and salted kaddid samples, was performed. Antimicrobial activity and other safety traits of LAB isolates were used to select those inhibitory against food pathogens and contaminants, with low antibiotic resistance, unable to produce virulence factors and not aminogenic. Molecular identification showed Weissella species as dominant population and in a lesser extent P. acidilactici and E. hirae. In view to use these strains as autochthonous starter and functional culture, Weissella strains showing technological and safety traits allowed selecting W. cibaria BK2, W. confusa BK6 and BK11 as well as W. paramesenteroides BK8 as valuable candidates which may contribute not only to improve overall quality but also preserve typicality, which benefits for both producers and consumers. Acknowledgements The authors thank the University Yahia-fares in Médéa- Algeria, as well as the fellowship grant in Italy, to perform some trials as molecular identification of strains. Also, a particular thanks to Algerian Ministry of Hight Education and Scientific Researche (MHESR) for the scholarship longstay (PNE 2018/2019, N° 256) at CERELA-CONICET, Tucumán, Argentina. Conflict of Interest The authors declare that they have no conflict of interest. 9 Nova Biotechnol Chim (2022) 21(2): e1269 3 Table 3. Antimicrobial activity of Weissella strains isolated from Algerian kaddid. CFS – cell free supernatant. Kaddid Southwestern Algerian samples SK1, SK2, SK3 (Béchar city), SK5 (BeniOunif), SK6 and SK7 (Igli) from Béchar province, and SK4 from Tindouf province. *Antibacterial activity is expressed by inhibition zone diameter (mm); ** Antifungal activity expressed as: – no growth inhibition), + (1 – 5 mm growth inhibition), ++ (5 – 10 mm growth inhibition) and +++ (˃ 10 mm growth inhibition). Indicator microorganism Isolated bacteria Antibacterial and antifungal activity of bacterial CFS BK1 BK2 BK3 BK4 BK5 BK6 BK7 BK8 BK9 BK10 BK11 BK12 BK13 BK16 BK18 BK19 Samples source SK1 SK1 SK1 SK2 SK2 SK3 SK3 SK4 SK4 SK5 SK5 SK6 SK6 SK7 SK7 SK7 Gram (-)Bacteria* Escherichia (E.) coli Algerian meat - 10.5 2.5 - - - - - - - - - - - - 6 E. coli ATCC25922 - - - - - - - - - - - - - - - - Salmonella (S.) bongori Algerian meat - 2.5 5.5 7.5 - - - 6.5 - - - - - - - 3 S. typhimurium ATCC2572 - 11.5 5.5 7.5 - 7.5 - 6.5 - - 8 - 7 4.5 - 12 Klebsiella pneumoniae Algerian kaddid (SK4) - 10.5 4.5 - - 2.5 - - - - - - - - - 10 Citrobacter farmeri Algerian kaddid (SK1) - 5.5 - - - - - - - - - - - - - 4 Pseudomonas (P). frederiksbergensis Algerian kaddid (SK2) - 11.5 4.5 - - - - - - - 6 - - - 7 14 P. aeruginosa ATCC27853 - 9.5 8.5 - - - - - - - - - - - 4 5 Acenitobacter baumanii ATCC 19606 - 5 - - - - - - - - 4 - - - - 6 Gram (+) Bacteria* Listeria (L.) monocytogenes Scott A 8 9 11 14 8 9 11 15 10 9 12 19 18 15 16 16 L. monocytogenes ATCC13932 - - - - - - - - - - - - - - - - L. monocytogenesGM1 Italian chicken meat 9 15 14 15 15 13 10 12 13 14 9 8 12 15 12 14 L. monocytogenes GM2 Italian chicken meat 6 7 11 14 13 10 13 14 14 14 12 8 10 7 13 15 L. monocytogenes ATCC 15313 - 7.5 - - - - - - - - - - - - - 10 L. innocua ATCC51742 10 16 13 11 12 13 10 13 13 12 13 13 13 10 15 15 L.innocua DSM20649 - 13 8 12 - 16 15 15 9 16 20 13 15 11 14 19 Bacillus cereus ATCC 10876 - - - - - - - - - - - - - - - - Enterococcus (En.) faecalis ATCC 49452 - 9 - - - - - - - - - - - - - 7 En. faecalis ATCC 29212 - - - - - - - - - - - - - - - - En. hirae BK15 Algerian kaddid (SK5) - - - - - - - - - - - - - - - - Staphylococcus (S.) aureus ATCC 25923 - 10 - - - 7 - - - - - - - - - - S. aureus ATCC 29213 9 18 16 16 15 15 13 13 15 14 14 14 14 13 13 14 Staphylococcus sp. Algerian kaddid (SK3) - 5 - - - - - - - - - - - - - - Pediococcus acidilactici BK14 Algerian kaddid (SK6) - - - - - - - - - - - - - - - - Molds** Penicillium expansum Algerian wheat - - - - - - - - - - - - - - - - Aspergillus flavus Algerian wheat + +++ ++ - - - - - - - - - +++ + + ++ Fusarium oxysporum albedinis Algerian dates + +++ - - - +++ - +++ - - +++ - - ++ ++ +++ 10 Nova Biotechnol Chim (2022) 21(2): e1269 3 Table 4. Safety and technological characterization of Weissella strains from Algerian kaddid. *pH was determined in SB as pH (96 h) – pH (0 h). VAN – vancomycin; CHL – chloramphenicol; GEN – gentamycin; STR – streptomycin; KAN – kanamycin; TET – tetracycline. Weissella strains Antibiotic resistance pH* Growth (OD620) at 48 h Autolytic activity [%] Temperature [°C] NaCl [%] 10 30 44 4 6.5 10 W. cibaria BK2 VAN/KAN/GEN 2.10 ± 0.02 0.13 ± 0.01 2.10 ± 0.40 1.10 ± 0.3 1.63 ± 0.12 1.23 ± 0.14 0.04 ± 0.00 6.03 ± 0.05 BK3 VAN/KAN/CHL/STR 2.10 ± 0.01 0.15 ± 0.02 2.10 ± 0.08 1.87 ± 0.33 1.63 ± 0.32 1.23 ± 0.00 0.02 ± 0.01 6.06 ± 0.17 BK19 VAN/KAN/GEN/TET/CHL 2.10 ± 0.00 0.15 ± 0.02 2.08 ± 0.11 1.85 ± 0.26 1.63 ± 0.17 1.23 ± 0.11 0.02 ± 0.0 8.08 ± 0.2 W. confusa BK4 VAN/KAN/GEN/TET 2.10 ± 0.03 0.15 ± 0.00 1.96 ± 0.23 1.87 ± 0.70 1.57 ± 0.09 1.23 ± 0.03 0.06 ± 0.01 4.23 ± 0.37 BK6 VAN/KAN/GEN 2.10 ± 0.11 0.19 ± 0.03 2.11 ± 0.07 1.85 ± 0.29 1.63 ± 0.44 1.23 ± 0.10 0.05 ± 0.00 4.23 ± 0.10 BK11 VAN/KAN/GEN 2.15 ± 0.07 0.13 ± 0.01 2.10 ± 0.17 1.68 ± 0.03 1.70 ± 0.05 1.35 ± 0.12 0.04 ± 0.01 5.75 ± 0.44 W. paramesenteroides BK8 VAN/KAN/GEN 2.37 ± 0.03 0.07 ± 0.00 2.10 ± 0.04 1.68 ± 0.15 1.99 ± 0.12 1.40 ± 0.14 0.18 ± 0.01 5.33 ± 0.21 11 Nova Biotechnol Chim (2022) 21(2): e1269 3 References Abriouel H, Lavilla Lerma L, Casado Muñoz M DelC, Pérez Montoro B, Kabisch J, Pichner R, Cho G-S, Neve H, Fusco V, Franz ChMAP, Gálvez A, Benomar N (2015) The controversial nature of the Weissella genus: technological and functional aspects versus whole genome analysis-based pathogenic potential for their application in food and health. 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