Raw Sheep Milk Research Articles

Influence of the Blends of Sheep's and Goat's Milk on the Functional Properties and Volatile Compounds of Lighvan Cheese During Ripening: A Comparative Study

ABSTRACTIn this study, the influence of different ratios of sheep's to goat's milk (100:0, 75:25, 50:50, 25:75, and 0:100) on the content of functional metabolites and volatiles in Lighvan cheese throughout ripening was investigated. The samples were ripened under brine for 90 days and assessed in terms of variations in chemical composition, proteolysis, angiotensin‐converting enzyme, α‐amylase, and α‐glucosidase inhibitory activities, antioxidant power, total free amino acids, gamma amino butyric acid, free fatty acids, and volatile compounds. Results showed that by increasing the proportion of goat's milk, the samples exhibited higher antioxidant effect and inhibition of the angiotensin‐converting enzyme during ripening. After 90 days, an increase in α‐glucosidase and a slight decrease in α‐amylase inhibitory activities were observed in Lighvan cheese samples that were produced with 50%, 75% and 100% of goat's milk. In contrast, higher contents of sheep's milk led to a significant increase in the concentration of esters, terpenes and alcohols and a slight increase in gamma‐aminobutyric acid. The highest concentration of saturated and unsaturated fatty acids was observed in the cheese prepared with 100% raw sheep's milk after 90 days of ripening. The results revealed the possibility of making a functional white‐brined cheese by monitoring the type and ratio of the used milk and ripening time.

Determination of mycotoxin content, microbiological and physicochemical properties and sensory quality during ripening of Erzincan Tulum cheese produced with strain Penicillium roqueforti 41

Erzincan Tulum cheeses were produced from raw sheep milk with traditional homemade rennet as the uninoculated (control) and inoculated with Penicillium roqueforti 41. The cheese was ripened at 4 ± 1 °C for 120 days. All the cheese samples were drilled to achieve oxygenation during the last two stages of the ripening period. The microbiological and physicochemical properties, presence of mycotoxins and sensory characteristics of the Tulum cheeses, which were filled by pressing into the goat skin bags (called tulums) and plastic barrels, were investigated during the ripening period. The coliform bacteria count of the cheeses decreased, and Staphylococcus aureus was not detected (< 2 log CFU/g) in the cheeses. The lowest and highest levels of penicillic acid (PA), roquefortine C and mycophenolic acid (MPA) in the cheeses during ripening ranged from 0.01–0.08, 0.01–0.06 and ND–0.06 mg/kg, respectively. Patulin was not detected. It was concluded that the Tulum cheeses were safer in terms of the amount of patulin, PA, roquefortine C, and MPA than those previously reported in the literature. P. roqueforti counts considerably increased during the last two stages of ripening. A new variety of credible mouldy cheese can be manufactured using the P. roqueforti 41 strain. However, a drilling process is needed to provide oxygen for P. roqueforti 41 growth during ripening.

Particles in Raw Sheep Milk Can Modulate the Inflammatory Response in THP-1, a Human Monocyte Cell Line, In Vitro

Background: The UK dairy sheep industry is relatively small but growing, particularly for cheese and yogurt products. Anecdotally, sheep milk (SM) may be better tolerated by humans than cows’ milk and could have environmental as well as health benefits. All milk contains sub-micron particles called extracellular vesicles (EVs) which are mainly derived from the mammary epithelium. Physiologically, milk-derived EVs are thought to aid in the development of infant immunity and the microbiome, but may also have health benefits to adult humans. The purpose of this study was to determine whether EVs could be isolated from raw sheep milk and whether they have any effect on inflammatory responses in THP-1, a human monocyte cell line, in vitro. Methods: Using sequential ultracentrifugation, vesicles of &lt;1 µm (LEV) followed by &lt;200 nm (sEVs) were isolated from six individual sheep during mid-lactation. RNA was extracted and microRNA analyzed by RTqPCR for sequences previously identified in cows’ milk. Human THP-1 monocytes were differentiated into macrophages and incubated with SM-derived LEVs and sEVs in the presence of pro-inflammatory LPS to measure the effects on the secretion of the chemokine CCL-2 or in the presence of DMNQ and fluorescent dihydrorhodamine-1,2,3 to measure reactive oxygen species. Results: LEVs induced an increase in ROS in both monocytes and macrophages, whilst sEVs decreased DMNQ-mediated ROS in macrophages but not monocytes. Interestingly, the LEVs did not induce CCL2 release; however, they increased LPS-induced CCL2 secretion in monocytes but not macrophages. miR26a, miR92a, miR125b, miR155 and miR223 were identified in both sEVs and LEVs by RT-qPCR and could be responsible for the modulation of ROS and CCL2 expression. Conclusions: These findings suggest that like cows’ milk, sheep milk contains EVs, and they can influence human monocyte/macrophage responses, and so is worthy of further investigation for its potential human- and non-human-animal health benefits.

Comparative Study of Aflatoxin M1 Carry-Over from Feed to Raw ‎Milk in Cow, Buffalo, Sheep, and Goats in Different Areas of ‎Baghdad Province

Mycotoxin aflatoxin B1 (AFB1) is a threat to food safety and human health because it is present in animal feed and is metabolized into aflatoxin M1 (AFM1), a more toxic form, during lactation. The aim of this study was to quantify AFM1 concentrations in raw milk of buffalos, cows, sheep, and goats sampled randomly from four regions within Baghdad ‎Province, Iraq, and to compare these levels with the maximum allowable levels set forth by the ‎European Commission (EC), the Iraqi Standard Specification (IQS), and the food and Drug Administration (FDA). The carry-over of AFB1 from feed to AFM1 in milk were also calculated for each of the ‎studied species. A total of 200 random samples, including 50 each from cows, buffaloes, ‎sheep, and goats, were collected from farms located in Zu'afraniya, Nahrawan, Abu Ghraib, and ‎Fedhalia regions. AFM1 and AFB1 concentrations were determined using the ‎enzyme-linked immunosorbent assay (ELISA). Raw milk samples from cows, sheep, buffaloes, and goats were all found to have AFM1 concentrations that were below the limits set by the EC, IQS, and FDA. Animal feed samples, on the other hand, had AFB1 concentrations of 10.08, 5.95, 4.27, and 7.10 ppb for ‎buffaloes, cows, sheep, and goats, respectively. The observed carry-over rates ranged from 0.36% ‎in goats to 0.78% in buffaloes to 0.66% in cows. Multiple factors, including animal species, ‎are considered, and it is determined that a universal carry-over equation cannot be applied to all cases. ‎Therefore, it is essential to regularly monitor AFM1 levels in milk from various animal species in order to ‎lessen potential health risks. Furthermore, the study suggests enhancing agricultural and veterinary ‎practices to better regulate feed quality for dairy animals‎‎‎‎.

Antilisterial activity of raw sheep milk from two native Epirus breeds: Culture-dependent identification, bacteriocin gene detection and primary safety evaluation of the antagonistic LAB biota

Raw milk from native small ruminant breeds in Epirus, Greece, is a valuable natural source of autochthonous lactic acid bacteria (LAB) strains with superior biotechnological properties. In this study, two bulk milks (RM1, RM2) from two local sheep yards, intended for traditional Kefalotyri cheese production, were preselected for bacteriocin-like antilisterial activity by in vitro tests. Their antagonistic LAB biota was quantified followed by polyphasic (16S rRNA gene sequencing; IGS for Enterococcus; a multiplex-PCR for Leuconostoc) identification of 42 LAB (RM1/18; RM2/24) isolates further evaluated for bacteriocin encoding genes and primary safety traits. Representative isolates of the numerically dominant mesophilic LAB were Leuconostoc mesenteroides (10) in both RMs, Streptococcus parauberis (7) in RM2, and Lactococcus lactis (1) in RM1; the subdominant thermophilic LAB isolates were Enterococcus durans (8), E. faecium (6), E. faecalis (3), E. hirae (1), E. hermanniensis (1), Streptococcus lutetiensis (2), S. equinus (1) and S. gallolyticus (1). Based on their rpoB, araA, dsr and sorA profiles, six Ln. mesenteroides strains (8 isolates) were atypical lying between the subspecies mesenteroides and dextranicum, whereas two strains profiled with Ln. mesenteroides subsp. jonggajibkimchii that is first-time reported in Greek dairy food. Two RM1 E. faecium strain biotypes (3 isolates) showed strong, enterocin-mediated antilisterial activity due to entA/entB/entP possession. One E. durans from RM1 possessed entA and entP, while additional nine RM2 isolates of the E. faecium/durans group processed entA or entP singly. All showed direct (cell-associated) antilisterial activity only, as also both S. lutetiensis strains from RM2 did strongly. Desirably, no LAB isolate was β-hemolyrtic, or cytolysin-positive, or possessed vanA, vanB for vancomycin resistance, or agg, espA, hyl, and IS16 virulence genes. However, all three E. faecalis from RM2 possessed gelE and/or ace virulence genes. In conclusion, all Ln. mesenteroides strains, the two safe, enterocin A-B-P-producing E. faecium strains, and the two antilisterial S. lutetiensis strains should be validated further as potential costarter or adjunct cultures in Kefalotyri cheese. The prevalence of α-hemolytic pyogenic streptococci in raw milk, mainly S. parauberis in RM2, requires consideration in respect to subclinical mastitis in sheep and the farm hygiene overall.

Comparative lipidomics analysis of in vitro lipid digestion of sheep milk: Influence of homogenization and heat treatment

This study investigated the changes in sheep milk lipids during in vitro gastrointestinal digestion in response to heat treatment (75°C/15 s and 95°C/5 min) and homogenization (200/50 bar) using lipidomics. Homogenized and pasteurized sheep milk had higher levels of polar lipids in gastric digesta emptied at 20 min than raw sheep milk. Intense heat treatment of homogenized sheep milk resulted in a reduced level of polar lipids compared with homogenized-pasteurized sheep milk. The release rate of free fatty acids during small intestinal digestion for gastric digesta emptied at 20 min followed the order: raw ≤ pasteurized < homogenized-pasteurized ≤ homogenized-heated sheep milk; the rate for gastric digesta emptied at 180 min showed a reverse order. No differences in the lipolysis degree were observed among differently processed sheep milks. These results indicated that processing treatments affect the lipid composition of digesta and the lipolysis rate but not the lipolysis degree during small intestinal digestion.

Bioactivity and volatile compound evaluation in sheep milk processed by ohmic heating

Ohmic heating may improve bioactive compounds and processing, ensuring food safety of beverages, liquid and pasty food, or liquid with solid pieces. Due to those traits, this study conducted a comparison between ohmic heating technology and conventional heating (CH), with a focus on assessing the impact of both methods on functional compounds (such as angiotensin-converting enzyme inhibition, α-amylase and α-glucosidase inhibition, and antioxidant activity) in both fresh and thawed raw sheep milk, which had been frozen for up to 3 mo. Different ohmic heating conditions were applied and compared to CH (3.33-8.33 V/cm vs. CH [73°C/15 s]). A total of 18 peptides with some functional activities were identified by MALDI-TOF mass spectrometry analysis. Ohmic heating samples presented the highest activities related to health, followed by CH and raw milk samples; antioxidant activity range was from 0.11% to 0.71%, antihypertensive activity ranged from 0.20% to 0.72%, and antidiabetic activity ranged from 0.21% to 0.79%. Of 51 volatile compounds detected, some were degraded by freezing, storing, and heating the sheep milk. This study showed for the first time that ohmic heating processing improved sheep milk bioactive peptides and preserved volatile compounds.

Genomic and Phenotypic Characterization of Mastitis-Causing Staphylococci and Probiotic Lactic Acid Bacteria Isolated from Raw Sheep's Milk.

Dairy products play a crucial role in human nutrition as they provide essential nutrients. However, the presence of diverse microorganisms in these products can pose challenges to food safety and quality. Here, we provide a comprehensive molecular characterization of a diverse collection of lactic acid bacteria (LAB) and staphylococci isolated from raw sheep's milk. Whole-genome sequencing, phenotypic characterization, and bioinformatics were employed to gain insight into the genetic composition and functional attributes of these bacteria. Bioinformatics analysis revealed the presence of various genetic elements. Important toxin-related genes in staphylococci that contribute to their pathogenic potential were identified and confirmed using phenotypic assays, while adherence-related genes, which are essential for attachment to host tissues, surfaces in the dairy environment, and the creation of biofilms, were also present. Interestingly, the Staphylococcus aureus isolates belonged to sequence type 5, which largely consists of methicillin-susceptible isolates that have been involved in severe nosocomial infections. Although genes encoding methicillin resistance were not identified, multiple resistance genes (RGs) conferring resistance to aminoglycosides, macrolides, and fluroquinolones were found. In contrast, LAB had few inherently present RGs and no virulence genes, suggesting their likely safe status as food additives in dairy products. LAB were also richer in bacteriocins and carbohydrate-active enzymes, indicating their potential to suppress pathogens and effectively utilize carbohydrate substrates, respectively. Additionally, mobile genetic elements, present in both LAB and staphylococci, may facilitate the acquisition and dissemination of genetic traits, including RGs, virulence genes, and metabolic factors, with implications for food quality and public health. The molecular and phenotypic characterization presented herein contributes to the effort to mitigate risks and infections (e.g., mastitis) and enhance the safety and quality of milk and products thereof.

Effect of cooking temperature on alkaline phosphatase in the production of raw-milk Pecorino cheese

Alkaline phosphatase (ALP) is a native raw-milk enzyme used in many countries as the standard assay for rapidly validating the milk pasteurization process. Due to the increased restrictions on the production or import of cheeses produced from unpasteurized milk, ALP activity (<10 mU/g) in cheese was measured as a simple and reliable method to check proper milk pasteurization in cheese for both safety inspection and trading controls. In Sicily, the artisanal cheesemaking of the Protected Denomination of Origin (PDO) semi-hard cheeses made with raw sheep's milk, includes the cooking of the curd, after the whey separation, in the wooden vat under hot Scotta-whey (≥80°C), for 3 to 4 h and finally is left to cool at ambient temperature. Thus, the temperatures adopted during cheesemaking may inactivate the ALP enzyme. To this purpose, the aim of this study was to demonstrate how different temperatures of Scotta-whey (T35, T60, T70, T80, T90, and T100) used during the second cooking of Pecorino cheeses after molding for 3 h, influence the ALP activity in fresh and 3-mo aged cheese, both at core and outside. The results highlight that the rate of reduction of ALP was greater with increasing temperature of the second cooking, in particular for T 80°C curd indicating that the use of Scotta-whey >80°C, could be a breakpoint able to reduce the ALP activity to <10 mU/g values. Different effects between the core and the outside portions of the experimental cheeses were found, with a decrease in ALP activity more on the outside than in the core portions, both in fresh and 3-mo aged cheeses, for T80, T90, and T100 treatments. Care must be taken in using the ALP to control the use of pasteurized milk in the production of PDO cheeses without considering the cheesemaking processes, such as the second cooking, which could be equal to pasteurization and an adequate interaction time/temperature can reduce the ALP activity to values comparable with cheeses produced with pasteurized milk.

Molecular surveillance of Toxoplasma gondii in raw milk and Artisan cheese of sheep, goat, cow and water buffalo origin

This study is aimed at investigating the molecular prevalence of Toxoplasma gondii in raw milk and cheese of different animal species (sheep, goat, cow and water buffalo) in Kayseri Province, Türkiye, to provide a preliminary assessment for contamination risk. A total of 200 milk and cheese samples were analysed by real‐time PCR. Toxoplasma gondii DNA was detected in two (8%) ewes and one (4%) goat raw milk sample, while none of the cheese samples were positive. These results indicated that the presence of T. gondii DNA in raw milk samples sold in Kayseri Province might be a risk factor for public health.

Evaluation of Enterotoxins and Antimicrobial Resistance in Microorganisms Isolated from Raw Sheep Milk and Cheese: Ensuring the Microbiological Safety of These Products in Southern Brazil.

This study emphasizes the importance of monitoring the microbiological quality of animal products, such as raw sheep's milk and cheese, to ensure food safety. In Brazil, there is currently no legislation governing the quality of sheep's milk and its derivatives. Therefore, this study aimed to evaluate: (i) the hygienic-sanitary quality of raw sheep's milk and cheese produced in southern Brazil; (ii) the presence of enterotoxins and Staphylococcus spp. in these products; and (iii) the susceptibility of the isolated Staphylococcus spp. to antimicrobial drugs and the presence of resistance genes. A total of 35 samples of sheep's milk and cheese were examined. The microbiological quality and presence of enterotoxins were accessed using Petrifilm and VIDAS SET2 methods, respectively. Antimicrobial susceptibility tests were conducted using VITEK 2 equipment and the disc diffusion method. The presence of resistance genes tet(L), sul1, sul2, ermB, tetM, AAC(6)', tetW, and strA were evaluated through PCR. In total, 39 Staphylococcus spp. were obtained. The resistance genes tetM, ermB, strA, tetL, sul1, AAC(6)', and sul2 were detected in 82%, 59%, 36%, 28%, 23%, 3%, and 3% of isolates, respectively. The findings revealed that both raw sheep's milk and cheese contained Staphylococcus spp. that exhibited resistance to antimicrobial drugs and harbored resistance genes. These results underscore the immediate need for specific legislation in Brazil to regulate the production and sale of these products.

A comprative study of chemical qualities of raw milk of goat and sheep

A comprative study of chemical qualities of raw milk of goat and sheep was conducted at Livestock production and management (unit), Department of NRM, Faculty of agriculture, MGCGV Chitrakoot – Satna (M.P.) during January to April 2022. The objective was to find out the comparative chemical qualities of raw milk of goat and sheep for three animal each viz., goat and sheep for ten days as replication different parameter were subject to statistical analysis applying the technique of analysis of variance (f-test) the most widely used method for determining protein content by kjeldahi method for nitrogen determination since nitrogen is a characteristic can be finding. In view of the finding and results presented above, it may be concluded that the chemical quality of milk of sheep was superior than goat milk, due to higher protein, specific gravity, fat content, lactose, total solid and solid not fat, and lower ash and water content in sheep milk.

CONCENTRATIONS OF CHEMICAL ELEMENTS IN SHEEP MILK FROM THREE FARMS IN SLOVAKIA FROM AREAS WITH DIFFERENT ENVIRONMENTAL BURDEN

Milk is considered as a basic food, highly nutritious, and recommended for everyday consumption during the whole human life. However, due to environmental pollution, milk is not just a source of essential minerals, but potentially can contain heavy metals and trace elements as well which can threaten human health. The study aimed to determine the occurrence of 22 elements: Ag, Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Sb, Se, Sr, Zn in raw sheep milk collected from areas with different environmental burden in Slovakia. Samples were collected from three farms, one from Orava region (potentially undisturbed area) and two from Spiš region (slightly and heavily disturbed area) during the period of lactation. Analysis of the elements was performed using an inductively coupled plasma-optical emission spectrometry with axial plasma configuration and with auto-sampler SPS-3. Statistical significant differences (P &lt; 0.05) were found in concentrations of following elements: Al, As, Ba, Ca, K, Li, Sr, and Zn. Calcium, potassium, sodium, and magnesium were most represented in samples from all monitored farms. The highest element variation was found in sheep milk samples from the heavily disturbed area. This fact confirmed the quality of monitored areas according to environmental regionalization by The Ministry of the Slovak Republic and the Slovak Environmental Agency. Heavy metals found in the heavily disturbed area do not represent any concerns according to European legislation, except lead, for which the mean concentration represents 0.02 mg/kg. This value was set by EC Regulation no. 1881/2006 and Codex Alimentarius as the level of the permitted limit of concentration of Pb in milk.

Influence of the storage temperature of raw sheep milk on the spoilage potential of Pseudomonas spp.

Due to their quality, high yield and nutritional value, sheep dairy products have been gaining market space and attracting new producers. Pseudomonas spp. is the most common psychrotrophic genus present in bovine refrigerated raw milk and is characterized by intense lipoproteolytic activity. Metalloprotease aprX is one of the enzymes produced by Pseudomonas spp. which has great industrial importance, since it has specific activity in the deterioration of casein, resulting in significant changes in physicochemical properties and sensorial characteristics of raw milk and dairy products. The aim of this study was to observe the occurrence of Pseudomonas spp. in raw sheep milk and evaluate the influence of storage temperature on the spoilage potential of the strains. Raw sheep milk samples were stored at 4 °C or 9 °C (72 h) and plated in Pseudomonas Agar Base supplemented with CFC (25 ºC / 48 h). The isolates characteristic of Pseudomonas had their genus, species (P. putida, P. fluorescens and P. aeruginosa), and presence of gene aprX confirmed by Polymerase Chain Reaction (PCR). The lipoproteolytic capacity and the spoilage potential of the isolates were evaluated in Milk Agar (10%) and Tributyrin Agar Base (1%) (21 °C / 72 h). Initial (2 h) populations of Pseudomonas spp. ranged from 1.0 to 4.2 log CFU/mL; after 72 h, the values observed in milk samples stored at 9 °C (3.0–7.0 log CFU/mL) were higher than those obtained in samples at 4 °C (1.6–6.0 log CFU/mL). A total of 255 strains of Pseudomonas spp. were isolated: 30.2% (77) from milk stored at 4 °C and 69.8% (178) from milk at 9 °C. At both temperatures, P. fluorescens was predominant, followed by P. putida. P. aeruginosa strains, isolated only from samples stored at 9 °C, showed high lipoproteolytic capacity and were characterized by higher proteolytic potential than lipolytic. P. fluorescens was the only species to present high proteolytic (halos > 2 cm) potential. Considering a single storage temperature (4 °C and 9 °C), only P. fluorescens showed higher proteolytic potential than lipolytic. The aprX gene was predominant in P. fluorescens and P. putida isolates (4 °C and 9 °C); strains obtained from milk stored at 4 °C showed higher proteolytic potential than those isolated from milk at 9 °C. All strains of P. fluorescens and only 46.42% of P. putida presented proteolytic capacity at 9 °C. The aprX gene was observed in all P. aeruginosa strains, but only 95.65% of them showed proteolytic capacity. The lipolytic potentials of P. fluorescens and P. putida were not influenced by milk storage temperature. P. fluorescens was the only species to present high lipolytic potential. P. putida isolated from milk at 9 °C was the only species to present higher lipolytic potential than proteolytic. According to the microbiota and the storage temperature, there may be a predominance of proteolytic or lipolytic enzymes in milk and consequently, defects in sheep dairy products. It should also be considered that most of the isolates of Pseudomonas spp. presented lipoproteolytic capacity, which emphasizes the importance of low initial populations of this microorganism in raw sheep-chilled milk, mainly when used in the production of dairy products.

Occurrence and molecular surveillance of pathogenesis risk-associated factors in Staphylococcus aureus recovered from raw sheep's milk cheese

The aims of our study were to ascertain the presence of different accessory gene regulator (agr) groups, to monitor the occurrence of 12 biofilm- and 5 enterotoxin- related genes and moreover to explore the biofilm-forming ability and antibiotic susceptibility in Staphylococcus aureus isolated from raw sheep’s cheese.From 92 samples of raw sheep’s cheeses, 32% (29 cheeses) harboured Staphylococcus aureus; among those 89.6% were biofilm producers, 55% had enterotoxigenic potential and 17.2% were phenotypically resistant to oxacillin. Five oxacillin resistant isolates were positive for blaZ and were classified into 5 different spa types (t179, t216, t1773, t12029, t13487). In the present study, 79% of Staphylococcus aureus isolates were agrI or agrII-positive. Four biofilm-associated genes (clfA, cna, bbp and fnbpA) were detected only in agr-positive strains.

Tejsavbaktérium-izolátumok egyes probiotikus tulajdonságainak in vitro vizsgálata

Bacteria carrying genes responsible for antibiotic resistance cannot be used in food production. For this reason,exploring the antibiotic resistance profile of probiotic candidates and the antimicrobial substances theyproduce are essential for probiotic strain selection. The aim of this study was to develop and evaluate additionalelements of a complex in vitro test system for rapid and efficient selection of a large number of putativeprobiotic isolates. In a previous work, we had tested bacterial strains (n=217) isolated from Transylvanian rawsheep milk, cultured sheep milk, and sheep cheese samples and we reduced the sample number to a totalof six Gram-positive, non-hemolytic, catalase-negative, well-aggregating, good acid and bile acid toleratingstrains. In this research, we investigated the antibiotic resistance and antimicrobial production capacity of thepre-selected strains (n=6). The antimicrobial activity of the isolates was determined by the agar well diffusionassay. Strains E15, E66, E173, E198, and E216 were found to inhibit the growth of both Salmonella EnteridisATCC 13076 and the control strain (i.e., Lactobacillus acidophilus ATCC 4356). Antibiotic resistance testswere performed by the agar disk diffusion method. All six isolates belonging to the species of Levilactobacillusbrevis and Lactiplantibacillus plantarum were found to be resistant to several antibiotics and, therefore,cannot be used for the manufacture of commercial probiotic products. In conclusion, our in vitro test systemproved to be capable of effectively screening out unsafe isolates.

Comparison of Activity of Commercial Protective Cultures and Thermophilic Lactic Acid Bacteria against Listeria monocytogenes: A New Perspective to Improve the Safety of Sardinian PDO Cheeses.

Listeria monocytogenes contamination that occurs during and post-processing of dairy products is a serious concern for consumers, and bioprotective cultures can be applied to control the growth of the pathogen in sheep milk cheeses. However, to respect specifications provided for protected designation of origin (PDO) cheeses, only autochthonous microorganisms can be used as bioprotective cultures in these products. This in vitro study aimed to evaluate thermophilic lactic acid bacteria (LAB) isolated from sheep milk as bio-preservative agents to control L. monocytogenes growth in PDO cheese. Results were compared with those obtained with a commercial protective culture (cPC) composed of a Lactiplantibacillus plantarum bacteriocin producer designed to inhibit L. monocytogenes growth in cheese. The in vitro antilisterial activities of n.74 autochthonous LAB and a cPC were tested against 51 L. monocytogenes strains using an agar well diffusion assay. In addition, 16S rRNA sequencing of LAB isolates with antilisterial activity was conducted and strains of Lactobacillus helveticus, Lactobacillus delbrueckii subsp. indicus, Lactobacillus delbrueckii subsp. sunkii, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecalis were identified. In this study, 33.6% (74/220) bacterial strains isolated from milk had characteristics compatible with thermophilic LAB, of which 17.6% (13/74) had in vitro antilisterial activity. These results demonstrate that raw sheep milk can be considered an important source of autochthonous thermophilic LAB that can be employed as protective cultures during the manufacturing of Sardinian PDO cheeses to improve their food safety. The use of bioprotective cultures should be seen as an additional procedure useful to improve cheese safety along with the correct application of good hygienic practices during manufacturing and the post-processing stages.

The effect of seasonality on the biogenic amines, free amino acids, and physico-chemical composition of raw milk Fiore Sardo cheese produced in Sardinia (Italy)

The effect of seasonality on the content of biogenic amines (BA), compounds that could threaten food safety at a high level, were evaluated in Fiore Sardo, a typical artisanal raw sheep's milk cheese produced in Sardinia. The cheese samples collected from six farms, after four months of ripening, were manufactured from January to June. The relationship between the BA content, the free amino acid (FAA) concentration, and the physico-chemical composition (pH, a w , NaCl, humidity, and the fat and protein content) was considered. The FAA and BA contents were determined by HPLC-FL. The BA content and the other parameters analysed were significantly affected (p ≤ 0.000) by seasonality and the farm of origin. Despite the differences attributable to the producers, the seasonal variations of all the parameters considered followed the same trend. As the season changed, the level of total BA grew, reaching a mean content of 1402 ± 207 mg/kg in the samples produced in June, almost five times the mean value of those manufactured in January. The most represented biogenic amine was tyramine, followed by putrescine and cadaverine. Histamine was determined almost exclusively in some samples produced in the warmer months, while β-phenylethylamine and tryptamine were found in a low concentration or not detected. The winter products did not exceed the threshold of 900 mg/kg proposed for the total BA in food, while most of the samples manufactured in the hottest months exceeded this limit, posing a potential risk to consumers' health. The total FAA and BA content were correlated (p = 0.01), indicating that with the change of the season, proteolysis phenomena increased and made the precursor amino acids available for microbial decarboxylase activity. In addition, from January to June, the pH and fat levels grew, while those of a w , humidity and NaCl tended to decline. The study highlighted that as the season changed, the rise in temperature, probably combined with the increment of fat and decrement of NaCl content, could favour proteolysis phenomena and bacterial decarboxylase activity and, as a result, the escalation of BA production. Interventions in the production protocol to reduce the negative effect of seasonality on cheese safety are needed. In addition, in light of these results, it is possible to speculate that the rising temperature and the predicted lengthening of the warm season due to climate change, which also seems to affect the spring periods, could create the ideal conditions for the production of biogenic amines for an increasingly extended phase of the year.

Microbiological aspects of the traditional Travnik/Vlašić cheese manufactured in Bosnia and Herzegovina.

The microbiological aspects of traditional Travnik/Vlašić cheese was investigated. The cheese was made traditionally, from raw sheep milk at three small farms (A, B, C) on Mountain Vlašić. The microbiological quality of the cheese was examined during three stages of ripening (5, 30, 60 days) and followed during three seasons (3 years). Twenty-seven samples of cheese were collected and analyzed for the aerobic mesophilic count, yeasts and molds, coliforms and microorganisms from the group Staphylococcus spp. Average values determined for the number of investigated groups of microorganisms of all cheese samples throughout three different stages, seasons, and small farms were: aerobic mesophilic bacteria 8.03 log10 cfu·g-1, yeasts and molds 3.63 log10 cfu·g-1, coliforms 5.16 log10 cfu g-1, and microorganisms from the group Staphylococcus spp. 4.49 log10 cfu g-1. ANOVA showed that experimental factor ripening stage (days) had a significant effect on all testing parameters. Results obtained with this study indicate that hygiene during the production of traditional products must be increased in order to assure high quality of the final products.

Comparative Genomic Analysis Reveals the Functional Traits and Safety Status of Lactic Acid Bacteria Retrieved from Artisanal Cheeses and Raw Sheep Milk.

Lactic acid bacteria (LAB) are valuable for the production of fermented dairy products. We investigated the functional traits of LAB isolated from artisanal cheeses and raw sheep milk, assessed their safety status, and explored the genetic processes underlying the fermentation of carbohydrates. Lactiplantibacillus plantarum had the largest and more functional genome compared to all other LAB, while most of its protein-encoding genes had unknown functions. A key finding of our analysis was the overall absence of acquired resistance genes (RGs), virulence genes (VGs), and prophages, denoting that all LAB isolates fulfill safety criteria and can be used as starter or adjunct cultures. In this regard, the identified mobile genetic elements found in LAB, rather than enabling the integration of RGs or VGs, they likely facilitate the uptake of genes involved in beneficial functions and in the adaptation of LAB in dairy matrices. Another important finding of our study was that bacteriocins and CAZymes were abundant in LAB though each species was associated with specific genes, which in turn had different activity spectrums and identified applications. Additionally, all isolates were able to metabolize glucose, lactose, maltose, and sucrose, but Lactiplantibacillus plantarum was strongly associated with the fermentation of rhamnose, mannose, cellobiose, and trehalose whereas Levilactobacillus brevis with the utilization of arabinose and xylose. Altogether these results suggest that to fully exploit the beneficial properties of LAB, a combination of strains as food additives may be necessary. Interestingly, biological processes involved in the metabolism of carbohydrates that are not of direct interest for the dairy industry may yield valuable metabolites or activate pathways associated with beneficial health effects. Our results provide useful information for the development of new probiotic artisanal cheeses and probiotic starter cultures.