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.

Dietary fatty acid metabolism: New insights into the similarities of lipid metabolism in humans and hamsters.

Hamsters have been long accepted as animal models to study the lipid metabolism in humans. However, very few scientific works described in detail the fatty acid (FA) composition of plasma and erythrocytes in hamsters in relation to their dietary intake, and none work was found comparing them with that described in humans. Therefore, a study was carried out to compare the effect of ingesting olive oil or dairy fat, as part of an equilibrated diet in healthy subjects, on plasma and erythrocytes FA composition. More than 40 FA were detected in samples of both species. It was demonstrated that plasma total FA (TFA) concentration and FA profiles are similar in humans and hamsters. In both species linoleic, oleic and palmitic acids are the main FA and accounted for the 70% of TFA. Differences found between species can be explained by differences in the dietary intake and differences in the proportion of triglycerides, cholesteryl esters and phospholipid fractions in plasma of both species. Changes in dietary FA intake causes similar changes in FA concentration in the plasma of both species and can be explained by the same metabolic processes. The erythrocyte FA profile differs more between the two species. Moreover, unlike humans, the FA profile of hamster erythrocytes is more sensitive to changes in dietary FA than that of plasma.

The Sum of Plasma Fatty Acids iso16:0, iso17:0, trans11-18:1, cis9, trans11-CLA, and cis6-18:1 as Biomarker of Dairy Intake Established in an Intervention Study and Validated in the EPIC Cohort of Gipuzkoa.

The questioned reliability of 15:0, 17:0, and trans9-16:1 acids as biomarkers of dairy fat intake also questions the relationship between the intake of these products and their health effects. Two studies were conducted in the same geographical region. In an intervention study, volunteers followed a diet rich in dairy products followed by a diet without dairy products. Plasma and erythrocyte fatty acids (FA) were analyzed, and their correlations with dairy product intakes were tested. The FA biomarkers selected were validated in the Gipuzkoa cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) observational study. The correlation coefficients between plasma concentrations of iso16:0, iso17:0, trans11-18:1, cis9, trans11-18:2, and cis6-18:1 and the dairy fat ingested are similar in both studies, indicating that their concentration increases by 0.8 µmol/L per gram of dairy fat ingested. The biomarkers are positively related to plasma triglycerides (r = 0.324 and 0.204 in the intervention and observational studies, respectively) and total cholesterol (r = 0.459 and 0.382), but no correlation was found between the biomarkers and atherogenicity indexes. In conclusion, the sum of the plasma concentration of the selected FAs can be used as biomarkers of dairy product consumption. A linear relationship exists between their plasma concentrations and ruminant product intake. These biomarkers allow for obtaining consistent relationships between dairy intake and plasma biochemical parameters.

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

Rapeseed and sunflower oilcake as supplements for dairy sheep: animal performance and milk fatty acid concentrations.

The influence of different amounts of oilseed cake (rapeseed and sunflower) on animal production parameters and fatty acid (FA) concentrations of the milk was studied in a Latxa dairy sheep experimental flock, both in winter (50% oilcakes; indoor feeding) and in spring (30% oilcakes; part-time grazing). The two different levels of the oilcakes tested did not affect animal production parameters or milk yield. Milk fat content and the fat/protein ratio decreased significantly with 30 and 50% sunflower cake. Yet, fat/protein ratio values were within the range for cheesemaking. Both levels of either type of oilcake tested significantly increased the concentrations of nutritionally interesting FA (CLA isomer C18:2cis-9, trans-11, vaccenic, oleic, and total unsaturated FA), while simultaneously decreasing the concentration of atherogenic FA. The atherogenicity indexes of milks from ewes fed 50 or 30% of either oilcake were significantly lower than those of their corresponding control. The use of cakes in winter increased the concentration of nutritionally interesting FA to the values obtained with part-time grazing.

Interannual and geographical reproducibility of the nutritional quality of milk fat from commercial grazing flocks.

The objective of the present work was to study the differences in the fatty acid (FA) composition of raw sheep milk fat under commercial milk production conditions throughout lactation, in two consecutive years. Particular attention was placed on the C18:2cis-9,trans-11 isomer, C18:1trans-11 acid, and unsaturated FA as the feeding regimen of 10 commercial flocks of latxa dairy sheep changed from indoor feeding to part-time grazing conditions (from early spring) as traditionally practiced in the Basque Country (Northern Spain). Farms located at an altitude of between 600 and 700 m, in two different geographical areas with different rainfall were selected. Milk samples were collected monthly from late January (indoor feeding) until mid-, or end of, June (outdoor feeding), during two consecutive years. In spite of some interannual variability (most likely due to large differences in rainfall), the evolution of individual FA throughout lactation was comparable between years, indicating that it was reproducible under commercial milk production conditions. The average concentrations of C18:2cis-9,trans-11 isomer and C18:1trans-11 acid in milk from the commercial flocks increased about 200% during the transition period (end of March or early April until May), from indoor feeding (late January or early February until the end of March) to the outdoor period (early May to mid-June), remaining constant during the outdoor period (27·53 ± 9·32 µmol/g fat and 71·58 ± 20·53 µmol/g fat, respectively). Non-atherogenic FA comprised approximately 50% of all saturated FA at any time during lactation, whereas the milk atherogenicity index decreased significantly during the outdoor period. The Trolox-equivalent antioxidant capacity of the water-soluble milk fraction did not appear to be influenced by feeding management. The FA composition of cheeses made during the second year with milk from the indoor or outdoor periods reflected those of the corresponding milks. A principal components analysis clearly showed that differences in the milk FA composition were primarily due to outdoor grazing, with very little contribution from the geographical zone or the year.

Effects of seasonal changes in feeding management under part-time grazing on terpene concentrations of ewes' milk.

Terpene composition of ewes' raw milk from nine commercial flocks was analysed from February to July. Ewes' diet consisted of concentrate and conserved forage in winter (indoor feeding) and part-time grazing from spring (transition and outdoor feeding). Regardless of the feeding, limonene and ß-phellandrene were the most abundant monoterpenes and ß-caryophyllene showed the highest concentrations among sesquiterpenes. Terpene content increased in the milks of commercial flocks when animals were reared under grazing management. Monoterpenes were detected in the milks of all the commercial flocks throughout the season, whereas sesquiterpenes were only detected in the milks from flocks grazing on non-cultivated community-owned grasslands in which a higher biodiversity of plant species grew. These preliminary results indicated that ß-caryophyllene could be a potential pasture-diet marker in the case of milks from animals grazing a higher biodiversity of plant species but in-depth studies including information on terpene composition of plants ingested by the animals are necessary to evaluate the suitability of ß-caryophyllene or another terpenoid compound as pasture biomarker.

Seasonal changes in the technological and compositional quality of ewe's raw milks from commercial flocks under part-time grazing.

Rennet coagulation parameters, curd texture and gross compositional variables were studied in ewes' raw milk samples from nine commercial flocks using different concentrate:forage ratios and grazing times. From early lactation to March flocks were fed concentrate pellets and hay whereas from April to the end of lactation flocks were allowed to graze from 6 to 19 h/day receiving concentrate supplementation in the morning and evening. Milk from late-lactation flocks, when allowed to graze, showed higher content of fat, dry matter, protein, casein, soluble protein, total calcium, curd firmness and curd resistance to compression than the milk from early lactation flocks. Higher total calcium content and lower fat content were found when the early lactation flocks were fed high concentrate:forage ratio than when the flocks were fed low ratio. Curd firmness were lower for milk from flocks fed high concentrate:forage ratio, and the curd resistance to compression was greater, than for milk from flocks fed low ratio. At late lactation, when flocks grazed for a long time per day the total calcium content was higher than when the flocks grazed for a short time per day. Principal component analysis showed that protein and fat content were highly correlated with coagulum and curd firmness, whereas total calcium content was highly correlated with curd resistance to compression, and milk pH with rennet coagulation time.

Winter/spring changes in fatty acid composition of farmhouse Idiazabal cheese due to different flock management systems.

Typically, two different flock managements are employed by basque sheepherders in winter and spring. Thus, seasonal changes in the fatty acid (FA) composition of Idiazabal PDO farmhouse cheeses were studied. Ewe's raw milk cheeses elaborated in winter and spring were collected after 120 days of ripening from 10 Idiazabal PDO farmhouses. In winter, concentrate and conserved forages were fed, whereas a part-time grazing system was adopted from spring onward. Spring cheeses had less (P

Hygienic quality of ewes' milk cheeses manufactured with artisan-produced lamb rennet pastes.

The use of artisan-produced lamb rennet pastes, but not any of the other commercial animal rennets, imparts a characteristic flavour to the cheese, so most Mediterranean ewes' milk cheeses are coagulated with this kind of rennet paste. In contrast to the advantages of using lamb or kid rennet pastes from the sensory point of view, questions are still raised as to their hygienic quality. The goal was to examine the microbiological and enzymic quality of lamb rennet pastes prepared by cheese manufacturers for their own use, and evaluate the hygienic quality of raw sheeps' milk cheeses made with them, using Idiazabal cheese as a model. Lamb rennet pastes prepared by artisan cheese makers from the Basque region of Spain (27), and Italy (8) were evaluated. For cheese making experiments 5 different lamb rennet pastes were selected among the 27 samples from the Basque Country region of Spain. Microbiological analyses were carried out on samples from rennet pastes, rennet extracts, milks and cheeses during ripening. Enzymic activities studied in rennet paste were: total coagulating strength and lipase. Analysis of variance and Student's t-tests was performed. The results show that the artisan-produced rennet pastes contain high levels of a variety of microorganisms. After 60 ripening days, which is the minimum ripening period required for Idiazabal cheese prior to its commercialization, no Eschericia coli, Clostridium, Salmonella spp. or Listeria monocytogenes were detected, and levels for the rest of the microorganisms were below the limits of the European legislative standards for cheese manufactured with raw milk. We can conclude that the use of artisan-produced lamb rennet pastes of questionable hygienic quality for the manufacture of raw milk hard cheeses yields products of good hygienic quality.

Changes in lipid fractions and sensory properties of Idiazabal cheese induced by lipase addition.

This work studied the addition of an adequate lipase to enhance lipolysis reactions and the development of piquant flavour and sharp odour in Idiazabal cheese, as an alternative to the use of lamb rennet paste. Cheeses were manufactured from bulk raw ewes' milk in 50 l vats with commercial bovine rennet and 80 lipase units of pregastric or 180 lipase units of fungal lipase and ripened for 180 days. A higher lipolytic activity was induced by lipase addition promoting strong changes in odour and flavour attributes. Both fungal and pregastric lipases increased the content of total free fatty acids (FFA), but the fungal lipase released mainly medium- and long-chain FFA. In contrast, the pregastric lipase preferably released short-chain FFA. Diglyceride (DG) content was considerably higher in cheeses made with added pregastric lipase compared with those made with fungal lipase or with no lipase. Monoglycerides (MG) were detected only in cheeses made with either lipase added, reaching comparable concentrations after ripening for 180 days. The cheeses made with pregastric lipase had the highest scores for odour and flavour intensity, and sharp and rennet odours, desirable attributes for the Idiazabal cheese made with lamb rennet paste. None of the texture attributes were significantly influenced by the concentrations of MG and DG in the cheeses made with either lipase. Thus, the pregastric lipase was more appropriate than the fungal lipase to develop a more traditionally-flavoured Idiazabal cheese.