Genomic and phenotypic imprints of microbial domestication on cheese starter cultures.

Domestication - the artificial selection of wild species to obtain variants with traits of human interest - was integral to the rise of complex societies. The oversupply of food was probably associated with the formalization of food preservation strategies through microbial fermentation. While considerable literature exists on the antiquity of fermented food, only few eukaryotic microbes have been studied so far for signs of domestication, less is known for bacteria. Here, we tested if cheese starter cultures harbour typical hallmarks of domestication by characterising over 100 community samples and over 100 individual strains isolated from historical and modern traditional Swiss cheese starter cultures. We find that cheese starter cultures have low genetic diversity both at the species and strain-level and maintained stable phenotypic traits. Molecular clock dating further suggests that the evolutionary origin of the bacteria approximately coincided with the first archaeological records of cheese making. Finally, we find evidence for ongoing genome decay and pseudogenization via transposon insertion related to a reduction of their niche breadth. Future work documenting the prevalence of these hallmarks across diverse fermented food systems and geographic regions will be key to unveiling the joint history of humanity with fermented food microbes.

Tuning and modeling cheese flavor.

Flavor is a major sensory attribute affecting consumers' preference for cheese products. Differences in cheesemaking change the cheese microenvironment, thereby affecting cheese flavor profiles. A framework for tuning cheese flavor is proposed in this study, which depicts the full picture of flavor development and modulation, from manufacturing and ripening factors through the main biochemical pathways to flavor compounds and flavor notes. Taking semi-hard and hard cheeses as examples, this review describes how cheese flavor profiles are affected by milk type and applied treatment, fat and salt content, microbiota composition and microbial interactions, ripening time, temperature, and environmental humidity, together with packaging method and material. Moreover, these factors are linked to flavor profiles through their effects on proteolysis, the further catabolism of amino acids, and lipolysis. Acids, alcohols, ketones, esters, aldehydes, lactones, and sulfur compounds are key volatiles, which elicit fruity, sweet, rancid, green, creamy, pungent, alcoholic, nutty, fatty, and sweaty flavor notes, contributing to the overall flavor profiles. Additionally, this review demonstrates how data-driven modeling techniques can link these influencing factors to resulting flavor profiles. This is done by providing a comprehensive review on the (i) identification of key factors and flavor compounds, (ii) discrimination of cheeses, and (iii) prediction of flavor notes. Overall, this review provides knowledge tools for cheese flavor modulation and sheds light on using data-driven modeling techniques to aid cheese flavor analysis and flavor prediction.

Molecular identification of Staphylococcus aureus-related enterotoxin genes in cheese samples.

Background: Dairy products are considered some important sources of various nutritional compounds; however, pathogenic bacterial growth is a critical destructive factor to these products leading to consumer health and system financial crises. Aim: The current study was carried out to identify if there is any presence of Staphylococcus aureus-related enterotoxin genes in cheese samples. Methods: The research included the collection of 35 samples. The samples passed through conventional cultivation processes and a PCR method to detect the presence of icaA, sea, hla, and fnbA enterotoxin genes in these samples. Results: The conventional identification revealed the growth of S. aureus from the cheese samples. The PCR findings recorded the presence of the icaA, sea, hla, and fnbA in 31 (88.5%), 27 (77%), 19 (54%), and 12 (34%), respectively, of cheese samples. The sequencing revealed close similarities with global isolates, which reached up to 98.5% of identity. Conclusion: The current results indicate the presence of enterotoxin genes of S. aureus in high rates in the dairy products examined, which reveals critical problems of food safety due to the possible presence of enterotoxins in consumer dairy products.

Microbiological quality and native lactic acid bacteria diversity of artisanal Mexican cheeses: A review.

This review aims to provide an overview of artisanal Mexican cheeses microbiota focused on microbiological quality and safety, as well as native Lactic acid Bacteria (LAB) diversity. For the search, key words of artisanal Mexican cheeses varieties was carried out through several online databases and original articles were screened and data about populations of indicator microorganisms, presence of pathogens, and native LAB identified were extracted. Several artisanal Mexican cheeses exceeded the permissible limit established in Mexican regulation (NOM-243-SSA1-2010) for indicator microorganisms, as well as in some types of cheese, the presence of pathogens was confirmed. However, other varieties of artisanal Mexican cheeses possess unique physicochemical characteristics, and during their manufacturing particular steps are used that contribute to ensuring their quality and safety. Additionally, strains able to control the growth of pathogenic and spoilage bacteria are part of the microbiota of some artisanal Mexican cheeses. About native LAB diversity, it is composed by species of Lactobacillus, Enterococcus, Streptococcus, Leuconostoc, Weisella, Lactococcus, Pediococus, Aerococus, Carnobacterium, Tetragenococus, among others genera. Otherwise, artisanal Mexican cheeses represent an important source of specific LAB with several approaches within human health because they showed potential for the development of functional foods, nutraceutical, and bioprotective cultures.

Antioxidant Peptides Derived from Cheese Products via Single and Mixed Lactobacillus Strain Fermentation.

Probiotics are used in cheese fermentation to endow the product with unique functional properties, such as enhanced flavor and aroma development through proteolysis and lipolysis. In this study, two probiotic Lactobacillus strains, Lactobacillus plantarum A3 and Lactobacillus reuteri WQY-1, were selected to develop new probiotic cheeses in the form of single- and mixed-strain starters. The results demonstrated that the L. plantarum A3 single-strain group and the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group exhibited superior product performance, particularly the release of functional hydrolysates during cheese ripening. Furthermore, Label-free quantitative proteomic analysis revealed 26 unique antioxidant peptides in the L. plantarum A3 single-strain group and 53 in the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group. Among these, CMENSAEPEQSLACQCL (ß-lactoglobulin), CMENSAEPEQSLVCQCL (ß-lactoglobulin), and IQYVLSR (κ-casein) have been found to possess potential antioxidant properties both in vitro and in vivo. This confirmed that milk-derived protein peptides in cheese products exhibit potential antioxidant functions through the hydrolysis of probiotic strains.

A hurdle strategy based on the combination of non-thermal treatments to control diarrheagenic E. coli in cheese.

This study aimed to assess the efficacy of a multi-hurdle process combining mild High Hydrostatic Pressure (HHP) treatments and Thyme Oil (TO) edible films as a non-thermal method to combat pathogenic E. coli (aEPEC and STEC) in raw cow's-milk cheese stored at 7 °C and packaged under modified atmosphere. Changes in headspace atmosphere of cheese packs and treatment effects on Lactic Acid Bacteria (LAB) counts and diarrheagenic E. coli strains (aEPEC and STEC) were evaluated over a 28 d storage period. The results demonstrated that the combined treatment exhibited the most significant antimicrobial effect against both strains compared to individual treatments, achieving reductions of 4.30 and 4.80 log cfu/g after 28 d of storage for aEPEC and STEC, respectively. Notably, the synergistic effect of the combination treatment resulted in the complete inactivation of intact cells for STEC and nearly completed inactivation for aEPEC by the end of the storage period. These findings suggest that the combination of HHP with selected hurdles could effectively enhance microbial inactivation capacity, offering promising alternatives for improving cheese safety without affecting the starter microbiota.

Antibacterial and antibiofilm effect of essential oils on staphylococci isolated from cheese - application of the oil mixture in a cheese model.

The aim of the research was to examine the antimicrobial and antibiofilm effects of angelica, immortelle, laurel, hyssop, and sage plant dust essential oils (EOs) against isolated strains of Staphylococcus spp. from cheeses, in vitro and in the model of white cheese. MALDI-TOF MS analysis confirmed two Staphylococcus aureus strains and two coagulase-negative, identified as S. saprophyticus and S. warneri. All isolates produce biofilm, where the strains of S. aureus showed slightly better adherence. The main component of angelica EO was ß-phellandrene (48.19 %), while α-pinene (20.33 %) were dominant in immortelle EO, in hyssop EO cis-pinocamphone (37.25 %), in laurel EO 1,8-cineole (43.15 %) and in sage EO epirosmanol (26.25 %). The sage EO exhibited the strongest antistaphylococcal activity against all isolates. Synergism was also detected in combination of sage with hyssop or laurel EO. Better antibiofilm activity was confirmed for sage EO compared to hyssop EO. The mixture of sage/laurel EOs reduced the total number of staphylococci in the cheese after 4 days. Results indicate that in vitro applied EOs showed significant antistaphylococcal and antibiofilm activity, while the oil mixture reduced the initial total number of staphylococci.

From raw milk cheese to the gut: investigating the colonization strategies of Bifidobacterium mongoliense.

The microbial ecology of raw milk cheeses is determined by bacteria originating from milk and milk-producing animals. Recently, it has been shown that members of the Bifidobacterium mongoliense species may become transmitted along the Parmigiano Reggiano cheese production chain and ultimately may colonize the consumer intestine. However, there is a lack of knowledge regarding the molecular mechanisms that mediate the interaction between B. mongoliense and the human gut. Based on 128 raw milk cheeses collected from different Italian regions, we isolated and characterized 10 B. mongoliense strains. Comparative genomics allowed us to unveil the presence of enzymes required for the degradation of sialylated host-glycans in B. mongoliense, corroborating the appreciable growth on de Man-Rogosa-Sharpe (MRS) medium supplemented with 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL). The B. mongoliense BMONG18 was chosen, due to its superior ability to utilize 3'-SL and mucin as representative strain, to investigate its behavior when co-inoculated with other bifidobacterial species. Conversely, members of other bifidobacterial species did not appear to benefit from the presence of BMONG18, highlighting a competitive scenario for nutrient acquisition. Transcriptomic data of BMONG18 reveal no significant differences in gene expression when cultivated in a gut simulating medium (GSM), regardless of whether cheese was included or not. Furthermore, BMONG18 was shown to exhibit high adhesion capabilities to HT29-MTX human cells, in line with its colonization ability of a human host.IMPORTANCEFermented foods are nourishments produced through controlled microbial growth that play an essential role in worldwide human nutrition. Research interest in fermented foods has increased since the 80s, driven by growing awareness of their potential health benefits beyond mere nutritional content. Bifidobacterium mongoliense, previously identified throughout the production process of Parmigiano Reggiano cheese, was found to be capable of establishing itself in the intestines of its consumers. Our study underscores molecular mechanisms through which this bifidobacterial species, derived from food, interacts with the host and other gut microbiota members.

Assessing refrigerated preservation performance using Listeria predictive microbiology models and temperature data: Refrigerator performance indicator and time-temperature equivalent.

Time-temperature data for queso fresco (QF) cheese varieties stored in a residential refrigerator operating at 5°C and a predictive microbiology secondary model for Listeria monocytogenes in QF were used to estimate a refrigerator performance indicator (RPI) of microbial preservation. RPI values were used to assess how compressor technology (single [SS] and variable speed [VS]), ambient temperature (21.1°C [LT] and 32.2°C [HT]), and refrigerator load (22.5 kg regular load and 39 kg higher load) affected preservation performance. All deterministic and probabilistic RPI estimations slightly exceeded the desirable 1.0 value, i.e., the variable temperatures for the QF kept in the refrigerator were worse than keeping it constantly at the temperature recommended by food safety agencies for QF. Furthermore, the mean comparison of estimates of the time-temperature equivalent indicator previously developed by French researchers showed similar behavior to those observed for RPI. Finally, statistical analysis showed that Tambient was the factor with the highest impact on refrigerator performance because of its impact on the sample temperature increase during door openings and when exposed to ambient temperature during product use. This highlights the need to reduce the time for product temperature recovery by improving the compressor operation logic. Also important are consumer behavior changes such as a reduction in product exposure to ambient temperature and in the door opening duration and frequency. PRACTICAL APPLICATION: This study demonstrated how a quantitative tool (RPI) can assess refrigerator preservation performance. Although the findings presented can be applied to any cold chain segment, the data used was collected for its weakest link, the domestic refrigerator. Surveys show that 77% of them operate above the recommended 4°C. The RPI methodology is ready for use by refrigerator designers to assess performance improvements possible by modifications of the compressor operation logic. Moreover, it can be integrated into smart-hubs monitoring the frequency and duration of refrigerator door openings to inform consumers when their habits are compromising the preservation performance of the refrigerator.

Microbiome signatures associated with flavor development differentiate Protected Designation of origin water Buffalo Mozzarella cheese from different production areas.

Water Buffalo Mozzarella (BM) is a typical cheese from Southern Italy with unique flavor profile and texture. It is produced following a traditional back-slopping procedure and received the Protected Designation of Origin (PDO) label. To better understand the link between the production area, the microbiome composition and the flavor profile of the products, we performed a multiomic characterization of PDO BM collected from 57 different dairies located in the two main PDO production area, i.e. Caserta (n = 35) and Salerno (n = 22). Thus, we assessed the microbiome by high-throughput shotgun metagenomic sequencing and the Volatile Organic Compounds (VOCs) by gas chromatography/mass spectrometry (GC/MS). Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. delbrueckii were identified as the core microbiome present in all samples. However, the microbiome taxonomic profiles resulted in a clustering of the samples based on their geographical origin, also showing that BM from Caserta had a greater microbial diversity. Consistently, Caserta and Salerno samples also showed different VOC profiles. These results suggest that the microbiome and its specific metabolic activity are part of the terroir that shape BM specific features, linking this traditional product with the area of production, thus opening new clues for improving traceability and fraud protection of traditional products.

Onopordum platylepis (Murb.) Murb. as a novel source of thistle rennet: First application to the manufacture of traditional Italian raw ewe's milk cheese.

In this study, for the very first time, aqueous extracts obtained from flowers of spontaneously grown or cultivated Onopordum platylepis (Murb.) Murb. thistles were used as coagulating agents for the pilot-scale manufacture of Caciofiore, a traditional Italian raw ewe's milk cheese. Cheese prototypes were compared to control cheeses curdled with a commercial thistle rennet obtained from flowers of Cynara cardunculus L. After 45 days of ripening under controlled conditions, both the experimental and control cheese prototypes were analyzed for: cheese yield, physico-chemical (pH, titratable acidity, aw, proximate composition), morpho-textural (color and texture), and microbiological parameters (viable cell counts and species composition assessed by Illumina sequencing), as well as volatile profile by SPME-GC-MS. Slight variations in titratable acidity, color, and texture were observed among samples. Based on the results overall collected, neither the yield nor the proximate composition was apparently affected by the type of thistle coagulant. However, the experimental cheese prototypes curdled with extracts from flowers of both spontaneous or cultivated thistles showed 10 % higher values of water-soluble nitrogen compared to the control prototypes. On the other hand, these latter showed slightly higher loads of presumptive lactococci, thermophilic cocci, coliforms, and eumycetes, but lower counts of Escherichia coli. No statistically significant differences were revealed by the metataxonomic analysis of the bacterial and fungal biota. Though most volatile organic compounds (VOCs) were consistent among the prototypes, significant variability was observed in the abundance of some key aroma compounds, such as butanoic, hexanoic, and octanoic acids, ethanol, propan-2-ol, isobutyl acetate, 2-methyl butanoic acid, and 3-methyl butanal. However, further investigations are required to attribute these differences to either the type of coagulant or the metabolic activity of the microorganisms occurring in the analyzed cheese samples. The results overall collected support the potential exploitation of O. platylepis as a novel source of thistle coagulant to produce ewe's milk cheeses.

Microbiome mapping in dairy industry reveals new species and genes for probiotic and bioprotective activities.

The resident microbiome in food industries may impact on food quality and safety. In particular, microbes residing on surfaces in dairy industries may actively participate in cheese fermentation and ripening and contribute to the typical flavor and texture. In this work, we carried out an extensive microbiome mapping in 73 cheese-making industries producing different types of cheeses (fresh, medium and long ripened) and located in 4 European countries. We sequenced and analyzed metagenomes from cheese samples, raw materials and environmental swabs collected from both food contact and non-food contact surfaces, as well as operators' hands and aprons. Dairy plants were shown to harbor a very complex microbiome, characterized by high prevalence of genes potentially involved in flavor development, probiotic activities, and resistance to gastro-intestinal transit, suggesting that these microbes may potentially be transferred to the human gut microbiome. More than 6100 high-quality Metagenome Assembled Genomes (MAGs) were reconstructed, including MAGs from several Lactic Acid Bacteria species and putative new species. Although microbial pathogens were not prevalent, we found several MAGs harboring genes related to antibiotic resistance, highlighting that dairy industry surfaces represent a potential hotspot for antimicrobial resistance (AR) spreading along the food chain. Finally, we identified facility-specific strains that can represent clear microbial signatures of different cheesemaking facilities, suggesting an interesting potential of microbiome tracking for the traceability of cheese origin.

Beneficial effect of GABA-producing Lactiplantibacillus strain LPB145 isolated from cheese starters evaluated in anxiety- and depression-like behaviours in rats.

In a previous study, we reported the in vitro potential probiotic and gamma-aminobutyric acid (GABA) production, of several strains from a collection of Lactiplantibacillus (Lpb) strains within the community of natural whey starters from the artisanal cheese industry. GABA is a non-protein amino acid widely distributed in nature and produced in animals, plants, and microorganisms. However, the best known role of GABA is its function as the major inhibitory neurotransmitter of the central nervous system. Preclinical and clinical evidence suggests that the GABAergic system has a relevant role in mental health disorders, such as anxiety and major depression. The modulation of the GABAergic system has been suggested as a potential strategy for treatment, one such mechanism of modulation is the influence of the microbiota-gut-brain axis through probiotic treatments. The present study was designed to investigate the in vivo probiotic potential of LPB145, a Lactiplantibacillus strain previously characterised as a GABA-producing potentially probiotic strain. Therefore, we evaluated the behavioural effects of chronic oral administration of LPB145 on rats' anxiety- and depression-like behaviours, using the elevated plus maze, open field, and the forced swimming test. The impact of LPB145 strain treatment on the gut microbiota structure and diversity was assessed to discern a possible mechanism of action of the LPB145 treatment through the microbiota-gut-brain axis. Our results showed that LPB145 administration induced an antidepressive-like behaviour without changes in locomotor activity. In contrast, the treatment did not modify the experimental anxiety. The structure and diversity of the intestinal microbiota remained unaffected by the treatment when compared to the control. However, specific clades that could be implicated in the behavioural changes did show differences in their relative abundance. These findings provide evidence regarding the potential of probiotic strains isolated from alimentary sources, to modulate the microbiota-gut-brain axis and positively impact mental health.

Differential adaptation of the yeast Candida anglica to fermented food.

A single strain of Candida anglica, isolated from cider, is available in international yeast collections. We present here seven new strains isolated from French PDO cheeses. For one of the cheese strains, we achieved a high-quality genome assembly of 13.7 Mb with eight near-complete telomere-to-telomere chromosomes. The genomes of two additional cheese strains and of the cider strain were also assembled and annotated, resulting in a core genome of 5966 coding sequences. Phylogenetic analysis showed that the seven cheese strains clustered together, away from the cider strain. Mating-type locus analysis revealed the presence of a MATa locus in the cider strain but a MATalpha locus in all cheese strains. The presence of LINE retrotransposons at identical genome position in the cheese strains, and two different karyotypic profiles resulting from chromosomal rearrangements were observed. Together, these findings are consistent with clonal propagation of the cheese strains. Phenotypic trait variations were observed within the cheese population under stress conditions whereas the cider strain was found to have a much greater capacity for growth in all conditions tested.

Analysis of composition and molecular characterization of mycobiota occurring on surface of cheese ripened in Dossena's mine.

Accurate identification of the fungal community spontaneously colonizing food products, aged in natural and not controlled environments, provides information about potential mycotoxin risk associated with its consumption. Autochthonous mycobiota colonizing cheese aging in Dossena mines, was investigated and characterized by two approaches: microbial isolations and metabarcoding. Microbial isolations and metabarcoding analysis were conducted on cheese samples, obtained by four batches, produced in four different seasons of the year, aged for 90 and 180 days, by five dairy farms. The two approaches, with different taxonomical resolution power, highlighted Penicillium biforme among filamentous fungi, collected from 58 out of 68 cheeses, and Debaryomyces hansenii among yeasts, as the most abundant species (31 ÷ 65%), none representing a health risk for human cheese consumption. Shannon index showed that the richness of mycobiota increases after 180 days of maturation. Beta diversity analysis highlighted significant differences in composition of mycobiota of cheese produced by different dairy farms and aged for different durations. Weak negative growth interaction between P. biforme and Aspergillus westerdijkiae by in vitro analysis was observed leading to hypothesize that a reciprocal control is possible, also affected by natural environmental conditions, possibly disadvantageous for the last species.

Preparation and characterization of carboxymethylated pullulan/butyric acid-modified chitosan active sustainable bi-layer coatings intended for packaging of cheese slices.

Dependence of the food industry on conventional plastic and the generation of enormous amounts of food waste caused by microbiological spoilage have been imposed as inspiration for this work, to develop active sustainable packaging for sliced cheese using the bi-layer design. Pullulan was modified using a green approach to obtain a polyanionic character in the coating formulation. Chitosan, which has a cationic character in an acidic environment, has been modified using a butyric acid to obtain an amphiphilic character. The formed active bi-layer has demonstrated an improved barrier (decreased permeability for moisture vapor 72.2 and 77.7 times) and mechanical properties (increased tensile strength value up to 3.9 and 9.4 times) compared to the monolayer films. A novel approach to microbiological control of sliced cheese has been established, which implies a synergistic effect of Helichrysum italicum essential oil (EO) and corresponding hydrolate (HY) incorporated in separated layers. This design has ensured avoiding surfactants and preserving cheese's sensory properties, prolonging its shelf-life by 50 % at least. Improvements in cheese storage conditions using this packaging lie in the improved barrier, mechanical and antimicrobial properties, the order of lamination, and a good covering of the cheese surface by spraying.

Influence of Cheese Composition on Aroma Content, Release, and Perception.

The quality of a cheese is determined by the balance of aroma compounds primarily produced by microorganisms during the transformation of milk into ripened cheese. The microorganisms, along with the technological parameters used in cheese production, influence aroma formation. The perception of these compounds is further influenced by the composition and structure of the cheese. This study aimed to characterize how cheese composition affects aroma compound production, release, and perception. Sixteen cheeses were produced under controlled conditions, followed by a quantitative descriptive analysis post ripening. Aroma composition was analyzed using HS-SPME-GC-MS, and a dynamic sensory evaluation (TCATA) was combined with nosespace analysis using PTR-ToF-MS. Image analysis was also conducted to characterize cheese structure. Cheese fat and whey lactose contents were identified as key factors in the variability of sensory attributes. GC-MS analyses identified 27 compounds correlated with sensory attributes. In terms of aroma compound release, 23 ions were monitored, with fat, salt, and lactose levels significantly affecting the release of most compounds. Therefore, cheese fat, salt, and whey lactose levels, as well as the types of microbial strains, play a role in influencing the composition, structure, release of aroma compounds, and sensory perception.

Nutritional and microbial profiles of ripened plant-based cheese analogs collected from the European market.

Plant-based cheese analogs have emerged as a novel global market trend driven by sustainability concerns for our planet. This study examines eleven soft ripened plant-based cheese analogs produced in Europe, primarily with bloomy rinds and cashew nuts as the main ingredient. First, we focused on exploring the macronutrients and salt content stated on the labels, as well a detailed fatty acid analysis of the samples. Compared to dairy cheeses, plant-based cheeses share similarities in lipid content, but their fatty acid profiles diverge significantly, with higher ratio of mono- and polyunsaturated fatty acids such as oleic and linoleic acids. We also investigated the microbiota of these analog products, employing a culture-dependent and -independent approaches. We identified a variety of microorganisms in the plant-based cheeses, with Lactococcus lactis and Leuconostoc mesenteroides being the dominant bacterial species, and Geotrichum candidum and Penicillium camemberti the dominant fungal species. Most of the species characterized are similar to those present in dairy cheeses, suggesting that they have been inoculated as culture starters to contribute to the sensorial acceptance of plant-based cheeses. However, we also identify several species that are possibly intrinsic to plant matrices or originate from the production environment, such as Pediococcus pentosaceus and Enterococcus spp. This coexistence of typical dairy-associated organisms with plant associated species highlights the potential microbial dynamics inherent in the production of plant-based cheese. These findings will contribute to a better understanding of plant-based cheese alternatives, enable the development of sustainable products, and pave the way for future research exploring the use of plant-based substrates in the production of cheese analogues.

Applications of bacteriophage in combination with nisin for controlling multidrug-resistant Bacillus cereus in broth and various food matrices.

This study focused on the isolation and characterization of bacteriophages with specific activity against toxin-producing and multidrug-resistant strains of Bacillus cereus sensu stricto (B. cereus s. s.). Ten different samples yielded six bacteriophages by utilizing the double-layer agar technique. The most promising phage, vB_BceS-M2, was selected based on its broad host range and robust lytic activity against various B. cereus s. s. strains. The phage vB_BceS-M2 had a circular double-stranded DNA genome of 56,482 bp. This phage exhibited stability over a wide range of temperatures and pH values, which is crucial for its potential application in food matrices. The combined effect of phage vB_BceS-M2 and nisin, a widely used antimicrobial peptide, was investigated to enhance antimicrobial efficacy against B. cereus in food. The results suggested that nisin showed synergy and combined effect with the phage, potentially overcoming the growth of phage-resistant bacteria in the broth. Furthermore, practical applications were conducted in various liquid and solid food matrices, including whole and skimmed milk, boiled rice, cheese, and frozen meatballs, both at 4 and 25 °C. Phage vB_BceS-M2, either alone or in combination with nisin, reduced the growth rate of B. cereus in foods other than whole milk. The combination of bacteriophage and nisin showed promise for the development of effective antimicrobial interventions to counteract toxigenic and antibiotic-resistant B. cereus in food.

Application of rosemary oil nano-emulsion as antimicrobial and antioxidant natural alternative in pasteurized cream and Karish cheese.

Essential oils possess significant antimicrobial and antioxidant properties and are increasingly used as natural substitutes for food preservation. Therefore, this study investigated the potential application of rosemary essential oil (REO) and REO nano-emulsion in the dairy plant. The antimicrobial effects of REO and REO nano-emulsion were determined by an agar well diffusion assay after chemical profiling by Gas Chromatography-Mass Spectrometry (GC-MS). The REO nano-emulsion was characterized by a Transmission Electron Microscope (TEM). The REO chemical profile revealed the presence of 42 chemical compounds, including 1, 8-cineole (9.72 %), and α-pinene (5.46 %) as major active components. REO nano-emulsion demonstrated significant antimicrobial activity compared to REO (P < 0.05) with a MIC value of 0.0001 mg/ml against Listeria monocytogenes and Aspergillus flavus and 0.001 mg/ml against Pseudomonas aeruginosa and Bacillus cereus. REO nano-emulsion enhanced the oxidative stability of pasteurized fresh cream, revealing a non-significant difference compared with that inoculated with butylated hydroxy anisol (BHA; synthetic antioxidant) (P˃ 0.05). Fortified cream and Karish cheese with REO nano-emulsion were evaluated organoleptically, and the results showed higher grades of overall acceptability when compared to control samples with a statistically significant difference (P < 0.05). Viability studies were estimated using the previously mentioned microorganisms in fortified fresh cream and Karish cheese with REO nano-emulsion. Results of the fortified cream showed a complete reduction of L. monocytogenes, A. flavus, and B. cereus on days 5, 7, and 10, respectively, and a 96.93 % reduction of P. aeruginosa by the end of the storage period. Regarding Karish cheese viability studies, C. albicans, A. flavus, and P. aeruginosa exhibited complete reduction on days 10, 10, and 15 of storage, respectively. In conclusion, REO nano-emulsion was recommended as a natural, safe, and effective antimicrobial and antioxidant additive in the dairy industry.