Phenotypic and genotypic characterization of antimicrobial resistances reveals the effect of the production chain in reducing resistant lactic acid bacteria in an artisanal raw ewe milk PDO cheese.

Antimicrobial resistance (AMR) is a significant public health threat, with the food production chain, and, specifically, fermented products, as a potential vehicle for dissemination. However, information about dairy products, especially raw ewe milk cheeses, is limited. The present study analysed, for the first time, the occurrence of AMRs related to lactic acid bacteria (LAB) along a raw ewe milk cheese production chain for the most common antimicrobial agents used on farms (dihydrostreptomycin, benzylpenicillin, amoxicillin and polymyxin B). More than 200 LAB isolates were obtained and identified by Sanger sequencing (V1-V3 16S rRNA regions); these isolates included 8 LAB genera and 21 species. Significant differences in LAB composition were observed throughout the production chain (P ≤ 0.001), with Enterococcus (e.g., E. hirae and E. faecalis) and Bacillus (e.g., B. thuringiensis and B. cereus) predominating in ovine faeces and raw ewe milk, respectively, along with Lactococcus (L. lactis) in whey and fresh cheeses, while Lactobacillus and Lacticaseibacillus species (e.g., Lactobacillus sp. and L. paracasei) prevailed in ripened cheeses. Phenotypically, by broth microdilution, Lactococcus, Enterococcus and Bacillus species presented the greatest resistance rates (on average, 78.2 %, 56.8 % and 53.4 %, respectively), specifically against polymyxin B, and were more susceptible to dihydrostreptomycin. Conversely, Lacticaseibacillus and Lactobacillus were more susceptible to all antimicrobials tested (31.4 % and 39.1 %, respectively). Thus, resistance patterns and multidrug resistance were reduced along the production chain (P ≤ 0.05). Genotypically, through HT-qPCR, 31 antimicrobial resistance genes (ARGs) and 6 mobile genetic elements (MGEs) were detected, predominating Str, StrB and aadA-01, related to aminoglycoside resistance, and the transposons tnpA-02 and tnpA-01. In general, a significant reduction in ARGs and MGEs abundances was also observed throughout the production chain (P ≤ 0.001). The current findings indicate that LAB dynamics throughout the raw ewe milk cheese production chain facilitated a reduction in AMRs, which has not been reported to date.

Cross-sectional, commercial testing, and chromatographic study of the occurrence of antibiotic residues throughout an artisanal raw milk cheese production chain.

This study investigated antibiotic utilization in artisanal dairies and residue occurrence throughout the raw milk cheese production chain using commercial testing (Charm KIS and Eclipse Farm3G) and UHPLC-QqQ-MS/MS and LC-QqQ-MS/MS. The cross-sectional survey results revealed gaps in the producers' knowledge of antibiotic use. Commercial testing detected antibiotic levels close to the LOD in 12.5 % of the samples, mainly in raw milk and whey, with 10.0 % testing positive, specifically in fresh and ripened cheeses, indicating that antibiotics are concentrated during cheese-making. Chromatographically, several antibiotics were identified in the faeces of healthy animals, with chlortetracycline (15.7 ± 34.5 µg/kg) and sulfamethazine (7.69 ± 16.5 µg/kg) predominating. However, only tylosin was identified in raw milk (3.28 ± 7.44 µg/kg) and whey (2.91 ± 6.55 µg/kg), and none were found in fresh or ripened cheeses. The discrepancy between commercial and analytical approaches is attributed to compounds or metabolites not covered chromatographically.

Normal-Fat vs. High-Fat Diets and Olive Oil vs. CLA-Rich Dairy Fat: A Comparative Study of Their Effects on Atherosclerosis in Male Golden Syrian Hamsters.

The relationship between milk fat intake (because of its high saturated fatty acid content) and the risk of suffering from cardiovascular diseases remains controversial. Thus, Golden Syrian hamsters were fed two types of fat-sheep milk fat that was rich in rumenic (cis9,trans11-18:2) and vaccenic (trans11-18:1) acids and olive oil-and two doses (a high- or normal-fat diet) for 14 weeks, and markers of lipid metabolism and atherosclerosis evolution were analyzed. The results revealed that the type and percentage of fat affected most plasma biochemical parameters related to lipid metabolism, while only the expression of five (CD36, SR-B1, ACAT, LDLR, and HMG-CoAR) of the studied lipid-metabolism-related genes was affected by these factors. According to aortic histology, when ingested in excess, both fats caused a similar increase in the thickness of fatty streaks, but the high-milk-fat-based diet caused a more atherogenic plasma profile. The compositions of the fats that were used, the results that were obtained, and the scientific literature indicated that the rumenic acid present in milk fat would regulate the expression of genes involved in ROS generation and, thus, protect against LDL oxidation, causing an effect similar to that of olive oil.

Relationship between the dynamics of volatile aroma compounds and microbial succession during the ripening of raw ewe milk-derived Idiazabal cheese.

Cheese microbiota contributes to various biochemical processes that lead to the formation of volatile compounds and the development of flavour during ripening. Nonetheless, the role of these microorganisms in volatile aroma compounds production is little understood. This work reports for the first time the dynamics and odour impact of volatile compounds, and their relationship to microbial shifts during the ripening of a raw ewe milk-derived cheese (Idiazabal). By means of SPME-GC-MS, 81 volatile compounds were identified, among which acids predominated, followed by esters, ketones and alcohols. The ripening time influenced the abundance of most volatile compounds, thus the moments of greatest abundance were determined (such as 30-60 days for acids). Through Odour Impact Ratio (OIR) values, esters and acids were reported as the predominant odour-active chemical families, while individually, ethyl hexanoate, ethyl 3-methyl butanoate, ethyl butanoate, butanoic acid or 3-methyl butanal were notable odorants, which would provide fruity, rancid, cheesy or malt odour notes. Using a bidirectional orthogonal partial least squares (O2PLS) approach with Spearman's correlations, 12 bacterial genera were reported as key bacteria for the volatile and aromatic composition of Idiazabal cheese, namely Psychrobacter, Enterococcus, Brevibacterium, Streptococcus, Leuconostoc, Chromohalobacter, Chryseobacterium, Carnobacterium, Lactococcus, Obesumbacterium, Stenotrophomonas and Flavobacterium. Non-starter lactic acid bacteria (NSLAB) were highly related to the formation of certain acids, esters and alcohols, such as 3-hexenoic acid, ethyl butanoate or 1-butanol. On the other hand, the starter LAB (SLAB) was related to particular ketones production, specifically 3-hydroxy-2-butanone; and environmental and/or non-desirable bacteria to certain ketones, hydrocarbons and sulphur compounds formation, such as 2-propanone, t-3-octene and dimethyl sulphone. Additionally, the SLAB Lactococcus and Psychrobacter, Brevibacterium and Chromohalobacter were described as having a negative effect on aroma development caused by NSLAB and vice versa. These results provide novel knowledge to help understand the aroma formation in a raw ewe milk-derived cheese.

Relationship between the Dynamics of Gross Composition, Free Fatty Acids and Biogenic Amines, and Microbial Shifts during the Ripening of Raw Ewe Milk-Derived Idiazabal Cheese.

This study reports for the first time the relationship between bacterial succession, characterized by high-throughput sequencing (sequencing of V3-V4 16S rRNA regions), and the evolution of gross composition, free fatty acids (FFAs) and biogenic amines (BAs) during cheese ripening. Specifically, Idiazabal PDO cheese, a raw ewe milk-derived semi-hard o hard cheese, was analysed. Altogether, 8 gross parameters were monitored (pH, dry matter, protein, fat, Ca, Mg, P and NaCl) and 21 FFAs and 8 BAs were detected. The ripening time influenced the concentration of most physico-chemical parameters, whereas the producer mainly affected the gross composition and FFAs. Through an O2PLS approach, the non-starter lactic acid bacteria Lactobacillus, Enterococcus and Streptococcus were reported as positively related to the evolution of gross composition and FFAs release, while only Lactobacillus was positively related to BAs production. Several environmental or non-desirable bacteria showed negative correlations, which could indicate the negative impact of gross composition on their growth, the antimicrobial effect of FFAs and/or the metabolic use of FFAs by these genera, and their ability to degrade BAs. Nonetheless, Obesumbacterium and Chromohalobacter were positively associated with the synthesis of FFAs and BAs, respectively. This research work provides novel information that may contribute to the understanding of possible functional relationships between bacterial communities and the evolution of several cheese quality and safety parameters.

Characterization of Microbial Shifts during the Production and Ripening of Raw Ewe Milk-Derived Idiazabal Cheese by High-Throughput Sequencing.

In this study, we used high-throughput sequencing technologies (sequencing of V3-V4 hypervariable regions of 16S rRNA gene) to investigate for the first time the microbiota of Latxa ewe raw milk and the bacterial shifts that occur during the production and ripening of Idiazabal cheese. Results revealed several bacterial genera not reported previously in raw ewe milk and cheese, such as Buttiauxella and Obesumbacterium. Both the cheese making and ripening processes had a significant impact on bacterial communities. Overall, the growth of lactic acid bacteria (LAB) (Lactococcus, Lactobacillus, Leuconostoc, Enterococcus, Streptococcus and Carnobacterium) was promoted, whereas that of non-desirable and environmental bacteria was inhibited (such as Pseudomonas and Clostridium). However, considerable differences were observed among producers. It is noteworthy that the starter LAB (Lactococcus) predominated up to 30 or 60 days of ripening and then, the growth of non-starter LAB (Lactobacillus, Leuconostoc, Enterococcus and Streptococcus) was promoted. Moreover, in some cases, bacteria related to the production of volatile compounds (such as Hafnia, Brevibacterium and Psychrobacter) also showed notable abundance during the first few weeks of ripening. Overall, the results of this study enhance our understanding of microbial shifts that occur during the production and ripening of a raw ewe milk-derived cheese (Idiazabal), and could indicate that the practices adopted by producers have a great impact on the microbiota and final quality of this cheese.

La microbiota del queso y su importancia funcional / Cheese microbiota and its functional importance

Actualmente, los alimentos funcionales con probióticos son muy populares y existe cada vez un interés mayor en la sociedad por incluirlos en la dieta. Hasta el momento, la investigación científica se centraba en probar los efectos beneficiosos de determinadas cepas bacterianas, independientemente del origen, en humanos. No obstante, los estudios que describen la importancia funcional de especies presentes naturalmente en alimentos no son tan abundantes. Por ello, este trabajo tiene como objetivo analizar la microbiota de uno de los ecosistemas microbianos alimenticios más destacados, como es el queso; y relacionarla con sus propiedades funcionales. Tras analizar la composición microbiana de diferentes quesos, como Cheddar, Emmental, Gruyere, Camembert etc., se ha observado que muchas especies predominantes (por ejemplo, Lactobacillus acidophilus, Bifidobacterium infantis o Streptococcus thermophilus) están relacionadas con el tratamiento de problemas de salud. Entre otros, son efectivas para aliviar la intolerancia a la lactosa, prevenir de alergias, reducir el colesterol, inhibir patógenos intestinales, tratar afecciones inflamatorias o reducir el riesgo asociado a mutagenicidad y carcinogenicidad. Además, se ha comprobado que la mayoría de las cepas mantienen la viabilidad durante la elaboración del queso y tras el proceso digestivo. En definitiva, estaríamos ante un alimento funcional muy beneficioso para la salud

Currently, functional foods with probiotics are very popular and there is an increasing interest in society to include them in diet. Until now, scientific research was focused on testing the beneficial effects of certain bacterial strains, regardless of origin, in humans. However, studies that describe the functional importance of species that are naturally present in food are not as abundant. Therefore, this work aims to analyse the microbiota of one of the most prominent microbial food ecosystems, that is cheese; and relate it to its functional properties. After analysing the microbial composition of different cheeses, such as Cheddar, Emmental, Gruyere, Camembert etc., it has been observed that many predominant species (for example, Lactobacillus acidophilus, Bifidobacterium infantis or Streptococcus thermophilus) are related to the treatment of health problems. Among others, they are effective in relieving lactose intolerance, preventing allergies, lowering cholesterol, inhibiting intestinal pathogens, treating inflammatory diseases or reducing the risk associated to mutagenicity and carcinogenicity. In addition, it has been proven that most strains maintain viability during cheese making and after the digestive process. In short, we would be faced with a functional food that is very beneficial for health