Whole-Genome Sequence of Lactococcus lactis Subsp. lactis LL16 Confirms Safety, Probiotic Potential, and Reveals Functional Traits.

Safety is the most important criteria of any substance or microorganism applied in the food industry. The whole-genome sequencing (WGS) of an indigenous dairy isolate LL16 confirmed it to be Lactococcus lactis subsp. lactis with genome size 2,589,406 bp, 35.4% GC content, 246 subsystems, and 1 plasmid (repUS4). The Nextera XT library preparation kit was used to generate the DNA libraries, and the sequencing was carried out on an Illumina MiSeq platform. In silico analysis of L. lactis LL16 strain revealed non-pathogenicity and the absence of genes involved in transferable antimicrobial resistances, virulence, and formation of biogenic amines. One region in the L. lactis LL16 genome was identified as type III polyketide synthases (T3PKS) to produce putative bacteriocins lactococcin B, and enterolysin A. The probiotic and functional potential of L. lactis LL16 was investigated by the presence of genes involved in adhesion and colonization of the host's intestines and tolerance to acid and bile, production of enzymes, amino acids, and B-group vitamins. Genes encoding the production of neurotransmitters serotonin and gamma-aminobutyric acid (GABA) were detected; however, L. lactis LL16 was able to produce only GABA during milk fermentation. These findings demonstrate a variety of positive features that support the use of L. lactis LL16 in the dairy sector as a functional strain with probiotic and GABA-producing properties.

Screening for Antifungal Indigenous Lactobacilli Strains Isolated from Local Fermented Milk for Developing Bioprotective Fermentates and Coatings Based on Acid Whey Protein Concentrate for Fresh Cheese Quality Maintenance.

The demand for healthy foods without artificial food additives is constantly increasing. Hence, natural food preservation methods using bioprotective cultures could be an alternative to chemical preservatives. Thus, the main purpose of this work was to screen the indigenous lactobacilli isolated from fermented cow milk for their safety and antifungal activity to select the safe strain with the strongest fungicidal properties for the development of bioprotective acid whey protein concentrate (AWPC) based fermentates and their coatings intended for fresh cheese quality maintenance. Therefore, 12 lactobacilli strains were isolated and identified from raw fermented cow milk as protective cultures. The safety of the stains was determined by applying antibiotic susceptibility, haemolytic and enzymatic evaluation. Only one strain, Lacticaseibacillus paracasei A11, met all safety requirements and demonstrated a broad spectrum of antifungal activity in vitro. The strain was cultivated in AWPC for 48 h and grew well (biomass yield 8 log10 cfu mL-1). L. paracasei A11 AWPC fermentate was used as a vehicle for protective culture in the development of pectin-AWPC-based edible coating. Both the fermentate and coating were tested for their antimicrobial properties on fresh acid-curd cheese. Coating with L. paracasei A11 strain reduced yeast and mould counts by 1.0-1.5 log10 cfu mL-1 (p ≤ 0.001) during cheese storage (14 days), simultaneously preserving its flavour and prolonging the shelf life for six days.

Exploring the Potential of Sustainable Acid Whey Cheese Supplemented with Apple Pomace and GABA-Producing Indigenous Lactococcus lactis Strain.

This study aimed to utilize two by-products, acid whey and apple pomace, as well as an indigenous Lactococcus lactis LL16 strain with the probiotic potential to produce a sustainable cheese with functional properties. Acid whey protein cheese was made by thermocoagulation of fresh acid whey and enhancing the final product by adding apple pomace, L. lactis LL16 strain, or a mixture of both. The sensory, the physicochemical, the proteolytic, and the microbiological parameters were evaluated during 14 days of refrigerated storage. The supplementation of the cheese with apple pomace affected (p ≤ 0.05) the cheese composition (moisture, protein, fat, carbohydrate, and fiber), the texture, the color (lightness, redness, and yellowness), and the overall sensory acceptability. The addition of the presumptive probiotic L. lactis LL16 strain decreased (p ≤ 0.05) the concentration of glutamic acid, thus increasing γ-aminobutyric acid (GABA) significantly in the acid whey cheese. The supplementation with apple pomace resulted in slightly (p < 0.05) higher counts of L. lactis LL16 on day seven, suggesting a positive effect of apple pomace components on strain survival. The symbiotic effect of apple pomace and LL16 was noted on proteolysis (pH 4.6-soluble nitrogen and free amino acids) in the cheese on day one, which may have positively influenced the overall sensory acceptance.

Application of Edible Coating Based on Liquid Acid Whey Protein Concentrate with Indigenous Lactobacillus helveticus for Acid-Curd Cheese Quality Improvement.

Edible coatings as carriers for protective lactic acid bacteria (LAB) can enhance hygienic quality to dairy products. Thus, the aim of this study was to improve the quality of artisanal acid-curd cheese by applying liquid acid whey protein concentrate based edible coating with entrapped indigenous antimicrobial Lactobacillus helveticus MI-LH13. The edible fresh acid-curd cheese coating was composed of 100% (w/w) liquid acid whey protein concentrate (LAWPC), apple pectin, sunflower oil, and glycerol containing 6 log10 CFU/mL of strain biomass applied on cheese by dipping. The cheese samples were examined over 21 days of storage for changes of microbiological criteria (LAB, yeast and mould, coliform, enterobacteria, and lipolytic microorganism), physicochemical (pH, lactic acid, protein, fat, moisture content, and colour), rheological, and sensory properties. The coating significantly improved appearance and slowed down discolouration of cheese by preserving moisture during prolonged storage. The immobilisation of L. helveticus cells into the coating had no negative effect on their viability throughout 14 days of storage at 4 °C and 23 °C. The application of coating with immobilised cells on cheeses significantly decreased the counts of yeast up to 1 log10 CFU/g during 14 days (p < 0.05) of storage and suppressed growth of mould for 21 days resulting in improved flavour of curd cheese at the end of storage. These findings indicate that LAWPC-pectin formulation provided an excellent matrix to support L. helveticus cell viability. Acting as protective antimicrobial barrier in fresh cheeses, this bioactive coating can reduce microbial contamination after processing enabling the producers to extend the shelf life of this perishable product.

Indigenous Lactococcus lactis with Probiotic Properties: Evaluation of Wet, Thermally- and Freeze-Dried Raisins as Supports for Cell Immobilization, Viability and Aromatic Profile in Fresh Curd Cheese.

Indigenous Lactococcus lactis enriched raisins were incorporated in fresh curd cheese in wet, thermally dried, and freeze-dried form to produce a novel probiotic dairy product. Symbiotic cheese represents a rising trend in the global market. The viability of L. lactis cells was assessed in the cheeses during storage at 4 °C for 14 days and the effect of the added enriched raisins on physicochemical parameters, microbiological characteristics, and sugar content, aromatic profile, and sensory acceptance of cheeses were evaluated. Immobilized L. lactis cells maintained viability at necessary levels (>6 log cfu/g) during storage and significantly increased the acceptability of cheese. The addition of raisins enhanced the volatile profile of cheeses with 2-furanmethanol, 1-octanol, 3-methylbutanal, 2-methylbutanal, 2-furancarboxaldehyde, 1-(2-furanyl)-ethanone, 5-methyl-2-furancarboxaldehyde. The obtained results are encouraging for the production of novel fresh cheeses with improved sensorial and nutritional characteristics on industrial and/or small industrial scale.

Effect of liquid whey protein concentrate-based edible coating enriched with cinnamon carbon dioxide extract on the quality and shelf life of Eastern European curd cheese.

Fresh unripened curd cheese has long been a well-known Eastern European artisanal dairy product; however, due to possible cross-contamination from manual production steps, high moisture content (50-60%), and metabolic activity of present lactic acid bacteria, the shelf life of curd cheese is short (10-20 d). Therefore, the aim of this study was to improve the shelf life of Eastern European acid-curd cheese by applying an antimicrobial protein-based (5%, wt/wt) edible coating. The bioactive edible coating was produced from liquid whey protein concentrate (a cheese production byproduct) and fortified with 0.3% (wt/wt, solution basis) Chinese cinnamon bark (Cinnamomum cassia) CO2 extract. The effect of coating on the cheese was evaluated within package-free (group 1) and additionally vacuum packaged (group 2) conditions to represent types of cheeses sold by small and big scale manufacturers. The cheese samples were examined over 31 d of storage for changes of microbiological (total bacterial count, lactic acid bacteria, yeasts and molds, coliforms, enterobacteria, Staphylococcus spp.), physicochemical (pH, lactic acid, protein, fat, moisture, color change, rheological, and sensory properties). The controlled experiment revealed that in group 1, applied coating affected appearance and color by preserving moisture and decreasing growth of yeasts and molds during prolonged package-free cheese storage. In group 2, coating did not affect moisture, color, or texture, but had a strong antimicrobial effect, decreasing the counts of yeasts and molds by 0.79 to 1.55 log cfu/g during 31 d of storage. In both groups, coating had no effect on pH, lactic acid, protein, and fat contents. Evaluated sensory properties (appearance, odor, taste, texture, and overall acceptability) of all samples were similar, indicating no effect of the coating on the flavor of curd cheese. The edible coating based on liquid whey protein concentrate with the incorporation of cinnamon extract was demonstrated to efficiently extend the shelf life of perishable fresh curd cheese, enhance its functional value, and contribute to a more sustainable production process.