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 log 10 cfu·g -1 , yeasts and molds 3.63 log 10 cfu·g -1 , coliforms 5.16 log 10 cfu g -1 , and microorganisms from the group Staphylococcus spp. 4.49 log 10 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.


Introduction
Traditional Travnik/Vlašić cheese is a very popular cheese in Bosnia and Herzegovina.It belongs to the group of white-brined cheeses.The origin of whitebrined cheeses is from the Mediterranean region and the Middle East where ovine, caprine or cow milk was used traditionally [1,2].Production of traditional Travnik/Vlašić cheese is located on the mountain Vlašić and surrounding mountains (near the town Travnik), situated in the middle of the country.It is produced from raw sheep milk (autochthonous sheep Pramenka) on small farms.Production of traditional Travnik/Vlašić cheese is seasonal, usually starts in mid-May and lasts until early October.During this period, sheep are fed only on the pastures [3].
Cheese is produced in low-grade hygienic conditions where a high quantity of salt is added to prevent the rapid development of cheese microflora.Adding a high amount of salt is aimed to control indigenous microflora, especially the growth of pathogens [4].Autochthonous production continues to this day, and causes non-standardized production and very high variability in cheese quality [5].
Traditional cheeses are part of the cultural and historical heritage.They are usually made from raw milk without the addition of starter cultures.In modern milk processing, cheese produced from raw milk can contain a pool of wild microorganisms.Today, artisanal raw-milk cheese is considered to be an important source of lactic acid bacteria genetic diversity [6].At the same time, the traditional way of cheese making is often associated with poorer hygienic production conditions, and sometimes these cheeses do not comply with the food safety regulations.The safety of food products, including cheese, for the health of consumers, is one of the most important aspects of the production, storage, and preservation of products.The quality of the raw material in production and maintaining standards of hygiene are the main factors on which microbiological quality and safety of traditional cheeses depends [7].Traditional cheeses are commonly produced from raw milk because of its enhanced organoleptic properties, as well as because of tradition.However, pathogenic microorganisms present in raw milk, can remain viable in cheese even when they are ripened for several weeks [8].
Traditional production of this cheese is still not standardized.The safety of the food products, including traditional food products, is extremely important for public health.Very few studies have been carried out on the microbiological aspects of traditional Travnik/Vlašić cheese.
This study aimed to examine the microbiological aspects of traditional Travnik/Vlašić cheese produced from raw sheep milk on Vlašić mountain (Bosnia and Herzegovina).

Samples collection
The traditional Travnik/Vlašić cheese used in this study was made on three small farms (A, B, C) from raw sheep milk of autochthonous race Pramenka, using traditional cheese-making technology on Mountain Vlašić (near the town Travnik in the central part of Bosnia and Herzegovina).After production and placement of the cheeses in wooden wats that are filed with brine, the cheeses were transported in the wooden wats to the maturation room in the premises of the Faculty of Agriculture and Food Sciences University of Sarajevo for a ripening period of up to 60 days under optimal conditions.Cheese samples were taken on the 5th, 30th, and 60th day of ripening from every wooden wat.The same procedure was repeated for every stage over 3 years and a total of 27 cheese samples were taken.Microbiological analyses of these samples were performed immediately after the samples were taken from the wats.

Microbiological analysis
The following groups of microorganisms were examined to analyze the hygienic quality of autochthonous Travnik/Vlašić cheese: (i) the total number of aerobic mesophilic bacteria on Milk Plate Count Agar (Liofilchem, Roseto degli Abruzzi, Italy) at 30 °C for 72 h; (ii) yeasts and molds on Rose Bengal Agar with Chloramphenicol Supplement (Liofilchem, Roseto degli Abruzzi, Italy) at 25 °C for 5 days; (iii) coliforms on Violet Red Bile Agar (Liofilchem, Roseto degli Abruzzi, Italy) at 37 °C for 24 h; (iv) number of Staphylococcus spp. on Baird Parker Agar supplemented with aseptically added 50 ml Egg Yolk Telurit at 37 °C for 48h.

Statistical analysis
All statistical analyses, including ANOVA, were performed by using SPSS software Statistics 20 (IBM, 2013).Tukey test was performed in a case when the experimental factor had an impact on the observed feature.A p value of < 0.05 was considered statistically significant.Results of the analysis are given in a form of means and standard deviations (SD) for cheeses from all three small farms.

Results
Microbiological aspects during ripening of traditional Travnik/Vlašić cheese at the three small farms throughout the three stages are shown in Table 1.The results represent means and standard deviations.
The average value determined for the number of certain groups of microorganisms in all cheese samples at the three farms during the three seasons were: aerobic mesophilic bacteria 8.03 log10 cfu•g -1 , yeasts and moulds 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 analysis indicated that the cheese producer (farm) had no significant effect (p > 0.05) on any of examined groups of microorganisms, while the ripening stage (days) had a significant effect (p < 0.05) on all.The count for aerobic mesophilic bacteria, as well as count for yeasts and moulds, was significantly lower on day 60 of ripening compared to all other ripening stages.Counts of coliforms and Staphylococcus spp.were significantly higher on day 5 compared to day 30, and the count on day 30 was significantly higher compared to the count on day 60 of ripening.Based on these results, it is obvious that the count of examined groups of microorganisms changed during the ripening time and was in decline from day 5 till day 60 of ripening.Despite all the investigated groups of microorganisms decreasing from the beginning till the end of ripening, all the groups are detected even after 60 days.
A strong negative correlation (A = -0.98;B = -0.99;C = -0.97), between the count of aerobic mesophilic bacteria and ripening days, was determined while a correlation between the count of yeasts and moulds and ripening days was strong negative in the cheese samples from the farms A (-0.88) and B (-0.86) and medium in cheese samples from farm C (-0.65).Count of coliforms and days of ripening had high negative correlation coefficients in cheeses from farms B and C (-0.99) and A (-0.91).Correlation between the count of Staphylococcus spp.and days of ripening was very strong negative in A (-0.91) and strong negative in B (-0.89) and C (-0.80) farm cheese samples (Figures 1-4).

Discussion
Traditional cheeses represent a special group of food products and unique microbial ecosystems.Since indigenous microflora from raw milk is included in the process of production of traditional cheeses, safety of the product is important to safeguard and protect health of the consumers.Safe food improves population health and saves lives, while strengthening economic growth    in the region [9].Poor hygiene is the main problem in the production of such products, because natural microbial flora is composed of not only lactic acid bacteria, but also pathogens.Due to this, study on the microbiological quality of traditional Travnik/Vlašić cheese from Bosnia and Herzegovina is important not only to show us diversity of pathogens in raw milk cheese, but also to give us a new knowledge on the quality of the cheese.
Based on the results obtained with this study, counts of aerobic mesophilic bacteria were higher in the beginning of ripening period, and similar at the end of ripening when compared to counts in traditional white brined Greek cheese Batzos made from raw goat milk [10], where the count of aerobic mesophilic bacteria was 7.69 log 10 cfu.g -1 on day 1 of ripening, 7.56 log10 cfu.g -1 on day 30 and 6.95 log10 cfu.g -1 on day 60.The counts in this research were higher than that in Urfa cheese made in Turkey.In addition, aerobic mesophilic bacteria were also present during all ripening period up to day 60 in Urfa cheese [11].The results obtained with this study show that aerobic mesophilic bacteria were higher in traditional Travnik/Vlašić cheese in comparation with other white brined cheeses, which can be due to low sanitary conditions during milking and milk handling.
Since yeasts and moulds can cause alterations in sensory parameters of dairy products their presence is undesirable [12].The appearance of yeasts in some acidified dairy products in high numbers may be caused by their ability to grow in low water activities (aw) and low temperatures, and ability to tolerate low pH and high salt concentrations [13].The presence of yeasts can lead to various quality defects, especially when grown in high numbers 5-6 log CFU/g, and this can result in softening of the cheese texture, discoloration, off-flavours, and swelling of the cans [14].The mean and minimum counts for yeasts and moulds, obtained in our study, were slightly higher than those for artisanal Turkish White Pickled cheese [15], while the maximum count was similar.The reason for this may be poor hygienic conditions and non-standardized production, and the ability of yeasts to resist high salt concentration as well as low pH.
As coliforms are highly sensitive to chemical sanitisers, they are useful to indicate inadequate sanitation of equipment and if they are present in high numbers that is a sign for inappropriate cleaning and sanitation [16].The coliform counts in this study were higher than that presented for Greek Feta cheese made of sheep pasteurized milk [17], but the decline in counts were similar.Lactic acid bacteria consume sugars for their growth resulting in low pH and lack of oxygen where the population of coliforms is decreasing, so coliforms can grow only in the beginning stages of cheese making when conditions (e.g.pH and temperature) are favourable [18,19].It seems that the maximum levels of yeasts and coliforms for most cheeses are at day 4 [20], and this study showed that the highest levels of these microorganisms were in the beginning of the ripening period.
Counts of Staphylococcus spp.during the ripening of Feta cheese from raw ewe's milk were 5.91 ± 1.0 log 10 cfu.g -1 on day 4, 3.54 ± 3.1 log10 cfu.g -1 on day 30, and 4.88 ± 0.2 log10 cfu.g -1 on day 60 [20].Traditional Travnik/Vlašić cheese had an initial higher count when compared to results of Feta cheese, and at the end of ripening count of Staphylococcus spp. was notably lower in Travnik/Vlašić cheese.The count of Staphylococcus spp. was in accordance with the results in Turkish feta cheese [21] where the count of Staphylococcus aureus increased on the first day of production, but salting of the cheese, as well as lower pH, resulted in inhibition of this pathogen.

Conclusions
This study on the microbiological aspects of traditional Travnik/Vlašić cheese is important because this cheese is one of the most important traditional cheeses in Bosnia and Herzegovina and the study provided useful knowledge on the diversity of pathogens present in the ecosystem of this cheese.The results obtained from this research indicates that cheese is produced in poor hygienic and sanitary conditions, with low level of cleaning of materials used in production.Although the total count of each of the examined groups of microorganisms was decreasing during ripening, these microorganisms were detected up to the end of the ripening period, which shows that microbiological quality of the cheese must be improved.This study highlights that it is vital to introduce and sustain good hygiene practice in the production of this traditional cheese in order to have product of high quality which is safe for consumers.One way to create safe final products is to organize trainings on good milking practice, cheese production and ripening, and storage practices for small producers.Research on microbiological quality of traditional Travnik/Vlašić cheese will lead to improvement in product safety.

Figure 3 .
Figure 3.Total coliforms count during the ripening period of traditional Travnik/Vlašić cheese throughout three different stages, seasons and farms.

Figure 4 .
Figure 4. Total Staphylococcus count during the ripening period of traditional Travnik/Vlašić cheese throughout three different stages, seasons and farms.

Figure 1 .
Figure 1.Total count of aerobic mesophilic bacteria during the ripening period of traditional Travnik/Vlašić cheese throughout three different stages, seasons, and farms.

Figure 2 .
Figure 2. Total yeast and molds during the ripening period of traditional Travnik/Vlašić cheese throughout three different stages, seasons and farms.
indigenous bacterial cultures and technological parameters as the basis for industrial production", financed by the Ministry of Foreign Affairs of the Kingdom of Norway through the HERD program (ref.nr.332160UC).The authors of this paper appreciate all the help from colleagues at Norwegian University of Life Sciences NMBU (Ass, Norway) and Faculty of Agriculture and Food Sciences University of Sarajevo (Bosnia and Herzegovina).

Table 1 .
Average value of microbiological parameters of traditional Travnik/Vlašić cheese from three small farms throughout three stages of ripening and seasons.