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Comparative analysis of Staphylococcus aureus strains by molecular microbiology methods
34-39Views:167Staphylococcus aureus is a very important pathogen for dairy farms and milk processing plants. Subclinical mastitis is often caused by this species, and it can contaminate bulk tank milk when milking cows are suffering from mastitis. Additionally, thermostable enterotoxins (SE) produced by some types of this bacterium can cause food poisoning.
The aim of our research was to examine the number of S. aureus in bulk tank milk in two dairy farms and the enterotoxin-producing ability, genetic relation (pulsotype) and antibiotic resistance of S. aureus strains from different sources (bulk tank milk, udder quarter milk and environment).
The results show that the mean number of S. aureus of bulk tank milk of two farms significantly differed (P<0.05). Fourteen isolates were selected for further molecular genetic studies (five isolates were from bulk tank milk and nine isolates were from udder quarter milk). S. aureus was not recovered from the environmental samples. Three of the fourteen isolates (21.4%) tested by multiplex PCR were positive for SE genes. Two isolates carried one gene (seb) and one isolate carried two genes (seg and sei). The fourteen strains were classified into three pulsotypes and two subtypes at 86% similarity level. Isolates from bulk tank milk (n=5), were divided into 2 pulsotypes (A, C) and one subtype (C1). The isolates from udder quarter milk (n=9) belonged to three different pulsotypes (A, B, C) and two subtypes (A1, C1). The distribution of pulsotypes in the present study revealed genetic relationship between S. aureus isolated from udder quarter milk and bulk tank milk. This could be explained by the fact that in farms with a high number of infected cows, these cows could represent the main source of contamination. The results of the antibiotic resistance investigations show, that all strains were susceptible to methicillin, cefoxitin, lincomycin, tetracycline, erythromycin and sulfamethoxazole/trimethoprim. Thirteen out of fourteen strains were resistant to penicillin (A and C pulsotypes, A1 and C1 subtypes) and just one isolate was susceptible (B pulsotype) to all antibiotics tested. -
Microbiological status of bulk tank milk and different flavored gomolya cheeses produced by a milk producing and processing plant
73-78Views:277The microbiological quality of milk is important not only for food safety, but it can also influence the quality of dairy products. In this study, our aim was to assess the microbiological status of the bulk milk of a milk-producing farm, and some natural and flavored (garlic, dill, onion) gomolya cheeses made from pasteurized milk produced by their own processing plant. We determined the number of coliform bacteria, Escherichia coli, Staphylococcus aureus, and molds of three milk and eight cheese samples. The tests were conducted between July and September, 2017.
In bulk milk, the mean coliform count was 3.83±0.17 log10 CFU/ml; the mean E. coli count was 1.38±0.14 log10 CFU/ml; the mean mold count was 3.74±1.30 log10 CFU/ml; and the S. aureus count was <1.00 log10 CFU/ml, respectively. The mean coliform count in gomolya cheeses was 3.69±1.00 log10 CFU/g; the mean E. coli count was 2.63±0.58 log10 CFU/g; the mean S. aureus count was 3.69±1.35 log10 CFU/g and the mean mold count was 1.74±0.37 log10 CFU/g. The amount of coliforms detected in different flavored gomolya cheeses were significantly different (P<0.05). More than 10 CFU/g of E. coli was found only in the dill flavored cheeses, and S. aureus was found only in dill (3.66±1.86 log10 CFU/g) and onion (3.71±0.52 log10 CFU/g) flavored gomolya cheeses. Based on the obtained results, it was found that the amount of coliform bacteria and E. coli in bulk milk exceeded the limit set in regulation of the Hungarian Ministry of Health (MoH) 4/1998 (XI. 11.) and the amount of S. aureus was below the limit. For gomolya cheeses, the S. aureus count exceeded the limit. The amount of coliform bacteria remained above the limit in cheeses, except for the garlic flavored gomolya cheese. In cheeses, a larger E. coli count was detected than in the bulk milk, but there is no specific limit for cheeses in the regulation. The mold count exceeded the limit specified in the regulation in cheeses, but a lower value was detected relative to milk.
The results show that, in the case of bulk milk and gomolya cheeses, certain detected quantities exceeded the limit values set forth in regulation of MoH 4/1998 (XI. 11.). The results indicate an inadequate microbiological state of the raw material and the finished products. The reasons for these are due to reduced technological hygiene or the inappropriate handling of raw material and finished products. In this study, we have summarized the results of our preliminary studies, which can provide a basis for further hygiene studies.
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Coagulase-negative staphylococci in ewe udder surface and raw milk samples
221-225Views:182Coagulase-negative staphylococci (CNS) are among the major causes of subclinical mastitis in dairy ewe flocks. This has a financially significant impact on the ewe dairy sector and consumer health. The determination of the bacterial count, particularly CNS, is critical in terms of the quantity and quality of ovine milk. Thus, the purpose of this study was to quantify the CNS count in the udder surface and raw milk samples of the ewe, in addition to identifying CNS strains (n=8) collected from udder surface and individual raw milk samples by 16S rRNA gene sequencing. A total of 164 samples of udder surface and raw milk originated from four sheep farms were tested. The obtained values were compared between the different farms. Furthermore, values during 2018 and 2019 in the case of Farm I were compared. Significantly higher (p<0.05) average count was observed in udder surface samples taken from Farm I (2.8±1.0 lg CFU/cm2) than that of Farm III samples (2.3±0.6 lg CFU/cm2). In the case of individual raw milk, the higher value was observed in samples derived from Farm III (3.5±0.9 lg CFU/mL), while the obtained value was significantly lower (p<0.05) in samples originated from Farm IV (1.8±0.4 lg CFU/mL). In the bulk tank milk samples, the highest mean value was 5.3±0.4 CFU/mL, and there was no significant (p>0.05) variation between farms. Coagulase-negative staphylococci counts were decreased to a certain extent in both sample types tested during 2019 except for individual raw milk derived from the Tsigai breed. The correlation between the mean CNS counts of udder surface and individual raw milk was very weak (r=0.048). Staphylococcus simulans, Staphylococcus auricularis, and Staphylococcus equorum were identified by molecular sequencing and Staphylococcus simulans were the most frequently identified CNS species. A higher CNS count of bulk tank milk than individual raw milk indicates possible contamination during milking and storage. Therefore, further studies are required to investigate the other sources of bulk tank milk contamination to improve the hygienic quality of milk.
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Microbiological quality of bulk tank raw milk from two dairy farms in Hajdú-Bihar County, Hungary
105-112Views:327Two main channels have been identified to be responsible for microbiological contamination of raw milk and milk products. Firstly, contamination has occurred due to udder infection from the cow or the blood which harbours most bacteria that come in contact with the raw milk. Secondly, via external factors (may include faeces, skin, contaminated water, environment etc.) which are associated with the operation of milking. There is direct contact with the milk and/or surfaces before, during or after the milking, posing public health risk and economic decline. The aim of this study was to examine the bacteriological quality of bulk tank raw milk samples collected from two different size dairy farms (Farm 1 and Farm 2) of different housing forms (cubicle loose and deep litter) in Hajdú-Bihar County, Hungary in July, 2017. Three samples were taken from each farm, and the total plate count, coliform count, Escherichia coli count, Staphylococcus aureus count, and yeast and mould count were determined in them.
The results clearly showed low level of all measured bacteria group load in Farm 1 samples in comparison to Farm 2 with the exception of coagulase-negative Staphylococcus (CNS) which represented high level in general, indicating significant difference (P<0.05). The mean value of total plate count in Farm 2 samples was higher (1.0 × 105 CFU/mL) than Farm 1 samples (2.8 × 104 CFU/mL). There was a significant difference (P<0.05) in mean count of coliforms in raw milk samples between Farm 1 and Farm 2. Similarly, results of E. coli were significantly different (P<0.05) with mean count of 1.44 × 102 CFU/mL and 2.02 × 103 CFU/mL for Farm 1 and Farm 2 respectively.
Results of Staphylococcus aureus also showed significant difference (P<0.05) with mean count of 9.7 × 101 CFU/mL for Farm 1 and 6.28 × 102 CFU/mL for Farm 2. The mean of mould count recorded was 1.07 × 102 CFU/mL and 4.93 × 102 CFU/mL for Farm 1 and Farm 2 respectively. The recorded mean of yeast count was 1.68 × 103 CFU/mL and 3.41 × 103 CFU/mL for Farm 1 and Farm 2 respectively; however, both farms showed no significant difference (P>0.05) in terms of mean of mould and yeast count. Although Farm 2 produced six times lower milk quantity than Farm 1, the measured microbial parameters were high. Both farms’ microbiological numbers were higher above the permitted limit values as stated by Regulation (EC) No 853/2004, Hungarian Ministry of Health (MoH) 4/1998 (XI. 11.).
This could be an indication of non-conformance to effective GMP, ineffective pre–milking disinfection or udder preparation, poor handling and storage practice, time and temperature abuse and inadequate Food Safety Management System Implementation. Therefore, our recommendation is as follows; establish control measures for pre- and postharvest activities involved in the milking process which would be an effective approach to reduce contamination of the raw milk by pathogenic microorganisms from these farms, strict sanitation regime and hygiene protocol be employed and applied to cows, all equipment, contact surfaces and minimize handling of the milk prior, during and after milking. This will also serve as scientific information to the producers for continual improvement in their operations.
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The examination of presumed Escherichia coli count of raw milk samples on several milk production farms
31-37Views:175For dairy farms, it is of great importance to insure the appropriate hygienic status of milk and to examine it regularly. Escherichia coli, belonging to the coliform bacteria type of, is a good indicator of contamination, and therefore suitable for characterising the hygienic condition of milk production.
The aim of our research was to examine the connection between the Escherichi coli count in bulk tank milk and housing and milking technologies of different-sizes farms. We examined the relation using various statistical methods.
Analysing the connection between the E. coli count and the farm size we found no significant difference between the farms. On the basis of the mean values of the E. coli count, we can say that the hygienic conditions are appropriate for mid-sized farms, and tolerable for large farms. We found differences in the hygienic status among the small farms. Half of the eight small farms, had no adequate hygiene. The results of the analysis of the quality categories show that the probability of inadequate quality milk was the largest on small farms (37.5%).
Comparing the various housing and milking methods with each other, there were numerical differences in the E. coli count, but these differences were not significant. We got higher E. coli count values on those farms using tied stall barn and bucket milking installation. The reason for this could be that in cases of farms using bucket milking installation, it is harder to meet the requirements.
After forming groups by farm size, housing and milking methods, we found that the E. coli counts are adequate on mid-size farms using various housing and milking methods; and tolerable on those large farms using loose housing stable and a milking parlour. At the same time, we found inadequate E. coli counts on the smaller farms using tied stall barns and bucket milking installation.
The results show that if there is suitable attention, independent of farm size, housing and milking procedure, it is possible to produce milk with low E. coli counts, and to insure appropriate hygienic conditions.
Further detailed examinations are needed to decide which factors of housing and milking technologies influence the E. coli count of bulk tank milk. -
The effect of season on the microbiological status of raw milk
95-99Views:182Many factors can influence the microbiological quality of raw cow’s milk. In this study, our aim was to determine whether there was any difference between the microbiological statuses of milk produced in different seasons. Samples were collected and analysed from five dairy farms in Hajdú-Bihar County, from February to November in 2019. During our studies, total plate count (TPC), coliform count and Staphylococcus aureus count of raw cow’s milk samples were determined.
There was no significant difference (P>0.05) between the mean TPC values detected in the milk collected in winter and autumn, but that values were significantly (P<0.05) lower than in the milk samples collected in spring and summer. Similarly to the TPC, in the case of coliform bacteria the lowest mean colony count was detected in the samples collected in winter. The difference was significant (P<0.05), compared to the values observed in the samples collected in summer. S. aureus was detected in bulk milk of only two farms in excess of 1.0 log10 cfu/ml. Also in case of S. aureus, there was a significant difference (P<0.05) between the values observed in the samples collected in winter and in summer. Samples from spring and summer contained the highest amount of S. aureus.
Based on the results of our studies, in the case of almost all farms the mean TPC, coliform and S. aureus counts were lower in the samples collected in winter, than in the samples collected in summer. The fact that the samples collected in winter contained the lowest amount of colonies could be attributed to the inhibition of growth of mesophilic microorganisms below 8 °C. Furthermore, the fact that we observed the highest colony counts in samples collected in summer, can be related to the heat stress of cows during the summer due to unfavorable weather conditions (high temperature and humidity).
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Microbial assessment of potential functional dairy products with added dried herbs
59-63Views:133The market of dairy products is a dynamically developing sector of the food industry. Potential, functional dairy products, made by adding herbs or spices, will have antimicrobial and antioxidant effect due to the active biochemical agents of the plant additives. Furthermore, these active components will widen the storage life of food products and enhance their organoleptic properties too. We worked out a technology for creating fresh cheeses using a gentle pasteurizing method by treating the mixture of raw milk and 1.5% fat contained in commercial milk. As herb additives, we used citronella (Melissa officinalis), and peppermint (Mentha x piperita) harvested by us and dried them via Tyndall-method in convective dryer on 40 °C for 5.5 hours per day. The drying period took three days. We bought dried citronella and mint from the supermarket, which were dried by ionizing radiation, to compare the microbiological pollution with the herbs dried by us.
The main target of this research was to create a microbiologically stable, potential functional dairy product. However, because of the bad quality of the raw milk and the gentle heat treatment we used for sterilizing bulk milk, or else, cheeses were not safe for human consumption. As a consequence, we need further studies to modify our technology and get a microbiologically stable product.