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