The research team, led by Abdelkader El Amine Dahou of Abdelhamid Ibn Badis University in Mostaganem, conducted twelve independent cheese-making trials between April 2024 and March 2025, following an optimized version of the traditional Sfisifa protocol. Rather than relying on pasteurized milk and commercial starters, the researchers worked with raw milk from a single Hamra ewe herd, deliberately preserving the native microbial ecosystem. They sampled the product at five critical stages: after coagulation and lactic fermentation, at demolding after 24 hours, after drying and salting at 48 hours, and on days 4 and 10 of ripening. This longitudinal design allowed them to track not just which bacteria were present, but how the entire community shifted as the cheese matured.

The enumeration results revealed a striking microbial succession. Coccal lactic flora, counted on M17 medium, dominated early fermentation, starting at 8 million colony-forming units per gram during coagulation and peaking at 12 million after demolding before declining to 800,000 by the end of ripening. The bacillary flora on MRS medium followed the opposite trajectory, rising from a modest 2,000 units per gram during fermentation to 2 million at full maturation. In proportional terms, Lactococcus species held roughly 49 percent of the isolates during fermentation but slipped to 38 percent by the end, while Lactobacillus climbed from 18 to 41 percent and Enterococcus fell from 33 to 21 percent. This choreographed handoff, from fast acidifiers to slower proteolytic organisms, mirrors patterns documented in traditional raw-milk cheeses across the Mediterranean and Balkans.

In total, the team isolated 32 bacterial strains and subjected them to a dual identification strategy combining classical biochemistry with molecular genetics. Phenotypic profiling used API 50 CHL and API 20 STREP galleries, standardized biochemical test panels that fingerprint bacterial metabolism. For molecular confirmation, the researchers extracted genomic DNA and ran multiplex polymerase chain reactions, a technique that amplifies several target genes simultaneously, generating species-specific DNA fragments of predictable lengths: 290 base pairs for Lactobacillus acidophilus, 390 for Lactococcus lactis subsp. lactis biovar diacetylactis, 420 for Lactococcus lactis, and 730 for Enterococcus faecium. Sequencing of the 16S rRNA gene, the workhorse marker of bacterial taxonomy, followed by comparison against the NCBI BLAST database, confirmed 99 to 100 percent homology with reference species, validating every phenotypic assignment.

With the identity of the players established, the team turned to the central question: what can these bacteria actually do? Acidification, the ability to convert lactose into lactic acid, is the single most important technological trait for any starter culture, because dropping the pH both inhibits spoilage organisms and triggers the coagulation that transforms liquid milk into curd. Here the Lactococcus strains proved unequivocally superior. Lactococcus lactis produced 11 grams of lactic acid per liter over 24 hours, and the diacetylactis biovar pushed that figure to 12.8 grams per liter, driving the final pH down to between 4.17 and 4.36. Enterococcus faecium delivered a moderate 9.7 grams per liter at pH 4.42, while Lactobacillus acidophilus lagged at 7.2 grams per liter and pH 4.81. This built-in variability, the authors note, is actually an asset, because it allows cheesemakers to fine-tune the pace of acid development during curd formation and early ripening.

Environmental tolerance testing painted a picture of strains superbly adapted to the harsh conditions of both their ecological niche and the cheesemaking process. Enterococcus faecium displayed the broadest thermotolerance, growing from 6 to 55 degrees Celsius, while Lactobacillus acidophilus, a confirmed thermophile, initiated growth only at 32 degrees but remained viable up to 55 degrees. The Lactococcus strains operated comfortably between 8 and 42 degrees. Salt tolerance varied just as dramatically: Lactococcus grew in sodium chloride concentrations up to 3 percent, E. faecium tolerated the full 1 to 6 percent range tested, and L. acidophilus sustained growth between 3 and 6 percent. The strains also survived remarkably acidic conditions, with L. acidophilus remaining viable down to pH 2.3 and E. faecium tolerating up to pH 7.3. Such robustness, the researchers argue, reflects genuine ecological fitness shaped by the regional milk substrate rather than laboratory convenience.

Coagulation and proteolysis assays revealed a complementary division of labor among the four species. Monitored with a HÖPPLER falling-ball viscometer, the lactic coagulation tests showed that both Lactococcus strains curdled milk within 12 hours, reaching viscosities of 810 to 980 centipoise, squarely within the 850 to 1100 range considered ideal for cutting the curd and retaining its structure. E. faecium coagulated slowly, taking more than 24 hours and reaching only 720 centipoise, while L. acidophilus managed a mere 410 centipoise, essentially failing as a coagulant. But the story flipped when the team measured proteolytic activity, the capacity to break down milk proteins. On agar plates supplemented with skimmed ewe milk, L. acidophilus carved out clear zones of casein hydrolysis measuring 12 to 14 millimeters across, and E. faecium produced zones of 7 to 9 millimeters, both within the 5 to 15 millimeter threshold associated with meaningful proteolysis. These enzymes liberate the peptides and amino acids that feed ripening microbes and generate flavor compounds, making these two species the flavor architects of the cheese rather than its structural engineers.

Perhaps the most visually dramatic finding concerned texture. On hypersaccharose agar, Lactobacillus acidophilus formed large, mucoid colonies, the classic signature of exopolysaccharide production, long-chain sugar molecules that bacteria secrete to thicken and stabilize their surroundings. Quantitative monitoring confirmed the observation: EPS concentration climbed steadily from 0.8 grams per liter on day 1 to 7.2 grams per liter by day 10 of ripening. Exopolysaccharide-producing lactic acid bacteria are prized in dairy technology because they reinforce protein-polysaccharide interactions in the curd, improving firmness, cohesiveness, and water retention without any artificial additives. In a semi-refined cheese like Hamra, where texture is a defining quality, this single metabolic capability could prove decisive for product consistency.

Taken together, the results describe a naturally regulated fermentation ecosystem in which each species plays a defined and sequential role. Lactococcus strains fire the starting gun, rapidly acidifying and coagulating the milk. Enterococcus faecium takes the baton during the transition, contributing moderate acidification and early proteolysis. Lactobacillus acidophilus finishes the race, degrading proteins and weaving exopolysaccharides through the curd as ripening proceeds. Statistical analysis across triplicate experiments confirmed that these functional differences among strains were significant at p below 0.05, ruling out random variation. The authors propose that this orchestrated consortium could be deployed as natural starter or adjunct cultures, offering artisanal producers a way to standardize quality while keeping the regional identity that industrial starter blends tend to erase.

The study also carries broader implications for the preservation of food heritage. As the International Dairy Federation has emphasized, traditional know-how and microbial diversity are key underpinnings of the sensory and nutritional quality of cheese, yet both are threatened by the homogenizing pressure of commercial cultures and pasteurization. By documenting and characterizing the native flora of Hamra cheese with modern molecular tools, the researchers have effectively created a microbial archive of a cheesemaking tradition from the Algerian steppe. The team’s stated next steps, genomic and metabolomic profiling of the isolated strains, could reveal the complete biochemical pathways behind the cheese’s distinctive character and open the door to controlled fermentation systems that reproduce artisanal quality at scale. For now, the microscopic workforce behind one of Algeria’s oldest cheeses has finally been identified, named, and measured, and it turns out to be a team of four specialists working in remarkable harmony.

Subject of Research: Technological and molecular characterization of indigenous lactic acid bacteria in traditional Algerian Hamra ewe's milk cheese

Article Title: Technological and molecular profiling of lactic flora in Hamra ewe’s milk cheese from the Algerian steppe

Article References: Dahou, A. E. A., Bouchibane, M., Meskini, Z., Aguedal, H., Touzout, N., Elfalleh, W., Bendif, H., & Tahraoui, H. (2026). Technological and molecular profiling of lactic flora in Hamra ewe’s milk cheese from the Algerian steppe. Food Science of Animal Resources, 46(1), Article 59. https://doi.org/10.1007/s44463-026-00064-x

Image Credits: AI Generated

DOI: 10.1007/s44463-026-00064-x

Keywords: lactic acid bacteria, Hamra cheese, Algeria, ewe's milk, Lactococcus lactis, Enterococcus faecium, Lactobacillus acidophilus, 16S rRNA sequencing, exopolysaccharides, starter cultures, food microbiology, traditional cheesemaking

The research team, led by Fatemeh Shayesteh and colleagues at Shahrekord University of Medical Sciences in Iran, set out to test whether these two probiotic strains could modulate inflammasome signalling, a pathway increasingly implicated in chronic mucosal inflammation across species. Inflammasomes are cytosolic protein complexes that act as intracellular alarm systems, sensing microbial or endogenous danger signals and activating the enzyme caspase-1. Once activated, caspase-1 cleaves and matures the potent inflammatory cytokines interleukin-1β and interleukin-18, which drive neutrophil recruitment, tissue damage, and the self-perpetuating cycle of inflammation characteristic of colitis. When the NLRP3 inflammasome becomes overactive, as has been documented in both human and veterinary cases of inflammatory bowel disease, the result is persistent mucosal injury and poor healing.

To model the disease, the researchers induced ulcerative colitis in 40 male Wistar rats by administering a single intrarectal dose of 4% acetic acid under anaesthesia, a well-established chemical method that reproduces key histopathological features seen in naturally occurring colitis of dogs, cats, and cattle, including mucosal ulceration, epithelial barrier dysfunction, and infiltration of inflammatory cells. The animals were randomly divided into five groups of eight: a healthy control group receiving saline, an untreated colitis group, a group treated with the standard anti-inflammatory drug mesalazine at 300 mg/kg per day, and two probiotic groups receiving daily oral gavage of either B. coagulans or L. acidophilus at a concentration of 10⁹ colony-forming units per millilitre for seven consecutive days. On day ten, the rats were euthanized and colonic tissues were collected for molecular, biochemical, and histological analysis.

The molecular results were striking. Compared with healthy controls, colitis induction caused dramatic upregulation of inflammasome-related genes in colonic tissue: interleukin-18 expression rose roughly 7.6-fold, NLRP3 about 11-fold, caspase-1 more than 12-fold, and tumour necrosis factor-alpha more than 6-fold. Treatment with either probiotic strain significantly reversed these changes. In the L. acidophilus group, expression of all four genes fell substantially relative to untreated colitis animals, and B. coagulans produced reductions of similar magnitude. Notably, the probiotic effects were comparable to those achieved with mesalazine, the benchmark therapy, which itself suppressed the same gene set. The convergence of probiotic and pharmacological outcomes at the transcript level suggests that these bacteria engage the same inflammatory circuitry that conventional drugs target, but through biological mechanisms rather than chemical inhibition.

Protein-level measurements reinforced the gene expression data. Using enzyme-linked immunosorbent assays on colonic tissue homogenates, the team found that colitis induction caused enormous increases in interleukin-1β and interleukin-17 protein concentrations. Both probiotics significantly lowered these cytokines, again paralleling the effect of mesalazine. Interleukin-1β is the direct downstream product of caspase-1 activation within the inflammasome, so its reduction provides functional confirmation that probiotic treatment genuinely dampens inflammasome output rather than merely altering gene transcription. Interleukin-17, meanwhile, is a signature cytokine of the Th17 immune axis that drives neutrophil recruitment and barrier disruption, meaning the probiotics appeared to suppress inflammation at multiple points along the pathological cascade.

The structural evidence was equally persuasive. Macroscopic examination of the colons, scored independently by two blinded observers using the Wallace and Keenan system, revealed severe ulceration, hyperaemia, inflammation, and oedema in untreated colitis animals, with a mean wound score of 3.25. Both probiotic groups showed marked improvement, with L. acidophilus achieving a mean score of 1.50, identical to the mesalazine group, and B. coagulans scoring 1.83. Colon length and weight, which serve as indirect measures of tissue oedema and shortening, were significantly restored in both probiotic groups compared with the untreated colitis group, indicating that the bacteria helped preserve gross tissue morphology.

Under the microscope, the differences were just as clear. Haematoxylin and eosin stained sections from untreated colitis rats displayed pronounced thickening of the mucosal, submucosal, and muscularis layers, extensive epithelial cell loss, a marked reduction in goblet cells, severe leukocyte infiltration, and lymphoid hyperplasia across all tissue layers. In contrast, sections from probiotic-treated animals showed reduced layer thickness, fewer inflammatory cells, milder crypt damage, and tissue involvement confined to the 1 to 25% range, compared with 51 to 75% in the colitis group. Histopathological scores fell from 4 in the colitis group to 2 in both probiotic groups, matching the mesalazine-treated animals. The restoration of goblet cells, which produce the protective mucus layer lining the gut, points to genuine epithelial repair rather than simple suppression of visible inflammation.

The mechanistic story behind these findings is likely multifaceted. Probiotics are known to reinforce epithelial integrity, compete with pathogenic bacteria for adhesion sites and nutrients, produce antimicrobial compounds, and regulate immune cell activity through Toll-like receptor pathways and the production of short-chain fatty acids. The authors suggest that suppression of inflammasome signalling may occur indirectly through these routes: by modulating Toll-like receptor activation, probiotics can shift the danger-sensing environment of the gut mucosa away from the chronic alarm state that keeps NLRP3 primed. Each strain also brings distinct practical advantages. B. coagulans is a spore-forming lactic acid bacterium whose resilient spores survive harsh storage conditions and the acidic gauntlet of the gastrointestinal tract, a trait particularly valuable in farm settings. L. acidophilus, by contrast, is a strong colonizer of the intestinal tract with well-documented immunomodulatory properties, making it well suited to companion animals with chronic enteropathies.

The veterinary implications extend well beyond the laboratory. Chronic inflammatory gut diseases reduce productivity in livestock, impair growth in young animals, and increase morbidity in pets, while long-term drug therapy raises concerns about cost, side effects, and, in food animals, drug residues in the food supply. Probiotics that measurably reduce inflammasome activation and pro-inflammatory cytokines could serve as adjuncts that allow lower doses of conventional drugs, or as standalone interventions in milder cases. The study also fits into a broader trend in veterinary microbiology toward multi-strain formulations, since combinations of Bacillus and Lactobacillus species are thought to target different aspects of gut ecology and immune regulation simultaneously, potentially offering synergistic benefits over single-strain products.

Important caveats remain. Some inflammation and leukocyte infiltration persisted even in probiotic-treated animals, indicating that these bacteria alleviate disease severity but may not fully replace pharmacological therapy in severe cases. Moreover, the murine model, while translational, differs from dogs, cats, and livestock in microbiota composition and immune function, so clinical trials in target veterinary species are the necessary next step, along with optimization of dosing regimens and formulation of multi-strain products tailored to different animal populations. Still, by connecting a practical, shelf-stable probiotic pair to a specific molecular pathway central to colitis pathology, the study provides a mechanistic bridge between the empirical use of probiotics in animal health and the modern immunology of inflammatory disease, and it does so with the kind of rigorous, multi-level evidence, from gene expression to protein to tissue architecture, that could finally move veterinary probiotics from folklore to pharmacology.

Subject of Research: Probiotic modulation of NLRP3 inflammasome signalling in experimental ulcerative colitis

Article Title: Probiotic Intervention With Bacillus coagulans and Lactobacillus acidophilus in Small Animals Experimental Ulcerative Colitis: Insights From a Small Animal Model

Article References: Shayesteh, F., Shahini Shams Abadi, M., Rouhi, L., Doosti, A., & Bagheri, N. (2026). Probiotic Intervention With Bacillus coagulans and Lactobacillus acidophilus in Small Animals Experimental Ulcerative Colitis: Insights From a Small Animal Model. Veterinary Medicine and Science, 12(6), Article e70883. https://doi.org/10.1002/vms3.70883

Image Credits: AI Generated

DOI: 10.1002/vms3.70883

Keywords: probiotics, Bacillus coagulans, Lactobacillus acidophilus, ulcerative colitis, NLRP3 inflammasome, veterinary medicine, inflammatory bowel disease, interleukin-1beta, mesalazine, rat model, gut microbiota, cytokines