Study of the acid production and proteolytic activity of the strains isolated from tulum cheese made by artisanal technology

Scientia Agriculturae www.pscipub.com/sa E-ISSN: 2310-953X / P-ISSN: 2311-0228 DOI: 10.15192/PSCP.SA.2018.21.3.9397 Sci. Agri. 21 (3), 2018: 93-97 PSCI Publications Study of the acid production and proteolytic activity of the strains isolated from tulum cheese made by artisanal technology Katya P. Dimitrova 1*, Atanas S. Vuchkov 2, Yordanka M. Kartalska 1 1. Agricultural University Plovdiv, Faculty of plant protection, Department of microbiology and ecological biotechnologies, 12 Mendeleev blv, Plovdiv, Bulgaria. 2. Agricultural University Plovdiv, Faculty of agronomy, Department of animal science, 12 Mendeleev blv, Plovdiv, Bulgaria. Corresponding author email: katia_dimitrova@au-plovdiv.bg Abstract. Tulum cheese is one of the specific products for the countries of the Balkan Peninsula. Its traditional artisanal production involves the use of raw milk and ripening in an animal skin bag for several months until the cheese obtains its distinctive taste and odor. There are several technologies of production of tulum cheese and just as there are differences among the countries, there are also differences among the regions of each country. The predominant microflora of tulum cheese made by artisanal technology is different strains of lactobacilli and enterococcii. The aim of this work is to study acid production and proteolytic activity of 20 strains lactic acid bacteria with ovoid shape which are isolated from fresh cheese crumbs and ripened tulum cheese. The obtained results show that isolated strains can lower the ph to 4.45 and elevate the titratable acidity up to 110 Тh of reconstituted skim milk. The strains showed similar proteolytic activity and the most active strain has a clear zone of 22.89 mm on 24 h and 32.92 on 48 h on MRS-agar supplemented with 5% milk. Key words: active acidity; enterococci; farmhouse skin bag cheese; proteolysis; titratable acidity Introduction Tulum cheese is among those cheese varieties which are known only in a restricted geographic area in the world and are produced and consumed locally. It has a white or cream color, high fat content and a crumbly and semi-hard texture; it is dispersible in the mouth and has a distinctive buttery and pungent flavour. The word tulum means goat s or sheep s skin bag, which is used for packaging and ripening (Cakmakci et al., 2011). There are several extensive and detailed review articles which deal with the geographical area of tulum cheese production, the different methods of production, the microflora of the cheese and other characteristics (Hayaloglu et al., 2007; Kamber et al., 2008; Kalit 2010). Tulum cheese is generally produced from unpasteurized milk in rural areas. Villagers produce tulum cheese using traditional methods and market it after 15 to 90 days of ripening. Traditionally, the packing material is a container made of goat`s or sheep`s skin. The microbiological, chemical, and physical aspects of the cheese may vary depending on the plant, the experience of the personnel, and quality of the raw milk. Since tulum cheese is produced using traditional methods in small-scale establishments, starter cultures are not usually used in production and the fermentation process depends on the indigenous bacterial flora (Öksüztepe et al., 2005).The main microflora of the cheese during the manufacture and ripening of tulum cheese are lactic acid bacteria (Hayaloglu et al. 2007; Litopoulou-Tzanetaki and Tzanetakis, 2011). Lactic acid bacteria (LAB) comprise a wide range of genera and include a considerable number of species. Their common traits are: Gram-positive, usually catalase negative, growth under microaerophilic to strictly anaerobic conditions and lactic acid production. These bacteria are the major component of the starters used in fermentation, especially for dairy products, and some of them are also natural components of the gastrointestinal microflora (Coeuret et al., 2003). The study of tulum cheese microflora of Gurses and Erdogan (2003) has revealed the presence of lactic acid bacteria belonging to the genera Lactococcus, Lactobacillus, Pediococcus, Enterococcus and Leuconostoc. Enteroccoci, in particular, were found at high levels, as estimated by the percentage (ca. 19% to 34% of all isolates) during the entire ripening period of the cheese (Öksüztepe, 2005). Therefore, the enterococci are an important part of the fermented food microbiota, especially in cheese (Giraffa, 2003; Gomes et al., 2010) and may play a role in the development of the organoleptic characteristics during the ripening of cheese (Litopoulou-Tzanetaki and Tzanetakis, 2014). Enterococcus faecalis, Enterococcus faecium and Enterococcus durans are the species most frequently found in dairy products (Cogan et al., 1997; Franz et al., 1999). The isolation and screening of microorganisms from naturally occurring processes have always been the most powerful means for obtaining useful cultures for scientific and commercial purposes. The wild lactic acid bacterial flora represents a natural reservoir for cultures that were not exposed to any industrial selection (El Soda et al., 2003). Because of the fact that there is very little information about tulum cheese made in Bulgaria, the aim of the study was to isolate and study ovoid strains of lactic acid bacteria from tulum cheese made from raw sheep milk from

Karakachan`s breed using artisanal technology and examine the acid production and proteolytic activity of the isolated bacteria. Materials and methods Cheese sample Samples of fresh cheese crumbs were taken before filling the skin bag and after ripening period of 4 months. The cheese was made from unpasteurized milk from Karakachan`s sheep which were bred in the area of the southwest mountain region of Bulgaria. The cheese samples (20g) were blended with 180 ml of sterile 2% sodium citrate (40 C) mixed well for 5 min by Star Blender LB400 (VWR, France). This homogenate was decimally diluted using Ringer solution. Isolation of the strains One ml from dilutions 10-6 -10-7 was pour plated in duplicate onto the M17-agar (HiMedia, India). The plates were incubated at 30 C for 48 h. After the incubation, typical colonies were randomly picked from the plates and were subjected to further analysis. The designations of strains used in the current study are listed in table 1 - strains from MS1 to MS14 are isolated from ripened tulum cheese, strains from MS15 to MS20 are isolated from fresh cheese crumbs. The morphological and physiological characteristics of the isolated bacteria were tested according to Cogan et al., (1997) and the following criteria cell morphology, Gram staining, catalase test, development in litmus milk (HiMedia, India). The assessment of physiological characteristics was on M17-broth (HiMedia, India) at 10 C, 30 C and 45 C and with presence of 6.5% NaCl and ph 9.6. Acid production The acid production was determined in heat treated (7 min at 121 C) 10% reconstituted skim milk (producer Volstin, code PL3029160IWE, Poland). The sterilized reconstituted skim milk (RSM) in this condition has mean ph value of 6.36 and titratable acidity of 27 Th. The RSM was inoculated with 2% of the activated culture and the incubation was performed at 30 C for 24 h, 48 h and 72 h. The active acidity of the samples was measured at 20 C by the laboratory phmeter inolab ph 730 (WTW GmbH, Germany). The titratable acidity is expressed as Thorner degrees ( Th) and was determined by blending a 10 ml milk sample with 20 ml of distilled water and by titration with 0.1N NaOH with 1% phenolphthalein as an indicator. The quantity (ml) of NaOH used for titration is multiplied by 10 in order to obtain the value of titratable acidity in Th. In the text the titratable acidity is also converted to % of lactic acid on the assumption that 1 Th corresponds to 0.009 % of lactic acid. Proteolytic activity The proteolytic activity of the isolated bacteria was done by agar well diffusion assay according to Phyu et al. (2015) on MRS-agar supplemented with 5% skim milk. The strains were activated in M17- broth for 24 h at 30 C. Wells of 10 mm in diameter were prepared on agar medium and 50 μl of broth for each well was used. After the incubation lasting 24 h and 48 h, the diameter of each clear zone was measured by electronic caliper and recorded in mm. Table 1. The morphological and physiological characteristics of the strains Tests in Strain 10 C 30 C 45 C order Gr Cat LM M17 ph = 9.6 6.5% NaCl 1 MS1 (+) (-) (+) (+) (+) (+) 2 MS2 (+) (-) (+) (+) (+) (+) 3 MS3 (+) (-) (+) (+) (+) (+) 4 MS4 (+) (-) (+) (+) (+) (+) 5 MS5 (+) (-) (+) (+) (+) (+) 6 MS6 (+) (-) (+) (+) (+) (+) 7 MS7 (+) (-) (+) (+) (+) (+) 8 MS8 (+) (-) (+) (+) (+) (+) 9 MS9 (+) (-) (+) (+) (+) (+) 10 MS10 (+) (-) (+) (+) (+) (+) 11 MS11 (+) (-) (+) (+) (+) (+) 12 MS12 (+) (-) (+) (+) (+) (+) 13 MS13 (+) (-) (+) (+) (+) (+) 14 MS14 (+) (-) (+) (+) (+) (+) 15 MS15 (+) (-) (+) (+) (+) (+) 16 MS16 (+) (-) (+) (+) (+) (+) 17 MS17 (+) (-) (+) (+) (+) (+) 18 MS18 (+) (-) (+) (+) (+) (+) 19 MS19 (+) (-) (+) (+) (+) (+) 20 MS20 (+) (-) (+) (+) (+) (+) 94

Statistical analysis Experimental results were obtained from two independent trials for acid production and three trials for proteolytic activity with duplicate measurements. Statistical analyses of the data were carried out using SPSS 20.0.0 (IBM 2011), and the values are given as mean, SD - standard deviation and SE standard error, a variance analysis was performed for determining statistical differences at p = 0.05. Results and discussion The morphological and physiological characteristics of the isolated bacteria are presented in table 1. All strains are ovoid in shape, gram positive and catalase negative. They can develop in the litmus milk at 30 C for 48 h the visible features are the change of the milk color and the formation of a solid coagulum through the glass tube. All stains grow at 10 C and 45 C, with the presence of 6.5% NaCl and ph=9.6. Based on these characteristics, it is assumed that the isolated bacteria belong to the genus Enterococcus. The changes in the active acidity of milk during the incubation of the strains at 30 C can be seen in table 2. The average mean of ph for 24 h is 5.36 and the lowest value is measured at MS16 and MS15-5.20 and 5.21 respectively, and the highest at MS4 and MS5 5.46. The change of active acidity for the first 24 h of incubation is in the narrow range of 1.17 and 1.15 units. On 48 h, the lowest value is again measured at MS16 4.85 and also at MS14 4.95 along with MS15 4.97. The highest value of рн is at MS1 5.08. On 72 h of the strains cultivation, the lowest acidity is measured at MS16 4.45 and also at MS17 4.63 and MS12 4.64, with insignificant difference between them. Table 2. The changes of the active acidity (ph) during the incubation of the strains in RSM at 30 C Active acidity, рн Strain 24 h 48 h 72 h Mean SD SE Mean SD SE Mean SD SE MS1 5.44 0.21 0.15 5.08 0.18 0.13 4.73 0.06 0.04 MS2 5.42 0.16 0.12 5.06 0.16 0.12 4.73 0.04 0.03 MS3 5.43 0.22 0.16 5.00 0.21 0.15 4.74 0.05 0.03 MS4 5.46 0.23 0.17 5.05 0.22 0.16 4.75 0.09 0.06 MS5 5.46 0.18 0.13 5.00 0.17 0.12 4.75 0.20 0.14 MS6 5.35 0.27 0.19 5.04 0.23 0.16 4.74 0.18 0.13 MS7 5.38 0.25 0.18 5.03 0.25 0.18 4.80 0.11 0.08 MS8 5.36 0.30 0.21 5.03 0.21 0.15 4.78 0.14 0.10 MS9 5.39 0.27 0.19 5.02 0.17 0.12 4.80 0.18 0.13 MS10 5.36 0.23 0.17 5.02 0.22 0.16 4.78 0.14 0.10 MS11 5.39 0.16 0.12 5.05 0.21 0.15 4.67 0.18 0.13 MS12 5.31 0.28 0.20 4.98 0.31 0.22 4.64 0.24 0.17 MS13 5.38 0.31 0.22 5.08 0.29 0.21 4.69 0.29 0.21 MS14 5.34 0.19 0.14 4.95 0.23 0.17 4.67 0.21 0.15 MS15 5.21 0.08 0.06 4.97 0.23 0.16 4.67 0.24 0.17 MS16 5.20 0.30 0.22 4.85 0.37 0.27 4.45 0.27 0.19 MS17 5.36 0.18 0.13 5.02 0.27 0.19 4.63 0.16 0.12 MS18 5.32 0.23 0.17 5.00 0.29 0.21 4.75 0.27 0.19 MS19 5.29 0.22 0.16 4.99 0.20 0.14 4.74 0.22 0.16 MS20 5.37 0.25 0.18 4.99 0.26 0.19 4.67 0.20 0.14 Table 3. The changes of the titratable acidity ( Th) during the incubation of the stains in RSM at 30 C Titratable acidity, Th Strain 24 h 48 h 72 h Mean SD SE Mean SD SE Mean SD SE MS1 57 9.4 4.7 73 9.6 4.8 92 6.4 3.2 MS2 56 4.4 2.2 73 8.1 4.1 94 2.6 1.3 MS3 55 6.2 3.1 74 6.6 3.3 94 2.2 1.1 MS4 54 7.3 3.6 72 7.5 3.8 95 4.1 2.1 MS5 54 6.5 3.2 74 12.1 6.0 94 13.0 6.5 MS6 56 7.2 3.6 73 9.9 4.9 92 8.7 4.3 MS7 55 6.1 3.1 73 9.0 4.5 92 6.7 3.4 MS8 55 7.6 3.8 72 9.3 4.6 92 8.5 4.3 MS9 55 7.4 3.7 71 8.4 4.2 90 6.6 3.3 MS10 57 7.0 3.5 74 9.2 4.6 93 7.0 3.5 MS11 57 6.1 3.5 71 9.3 4.7 96 9.5 4.8 MS12 58 9.0 5.2 76 10.1 5.8 95 9.6 4.8 MS13 55 6.7 3.8 69 9.3 4.6 92 10.5 5.2 MS14 58 6.9 4.0 74 7.8 3.9 94 9.3 4.7 MS15 59 6.4 3.7 75 9.0 4.5 94 6.2 3.1 MS16 63 11.0 6.4 85 15.7 7.8 110 10.1 5.1 MS17 59 7.6 4.4 73 10.1 5.1 100 5.5 2.8 MS18 61 6.7 3.8 72 9.6 4.8 95 6.4 3.2 MS19 60 8.5 4.9 74 8.7 4.3 93 9.0 4.5 MS20 60 8.7 5.0 73 9.6 4.8 96 5.8 2.9 95

At the end of the cultivation period, strains MS9 and MS7 - рн 4.80 have the highest value of the active acidity. The statistical analysis revealed that there is no significant difference between the values of ph of the different strains during the same period. The results conform to the data of other researchers (Olbrich dos Santos et al., 2015) who have studied acidifying capacity of the strain Enterococcus faecium, which is able to decrease ph of milk to 5.2-4.8 for 24 h and further have lowered ph to 4.8 during the next 48 h. In general, the enterococci exhibit a low acidifying activity in milk (Jamaly et al., 2010). The results about the titratable acidity are presented in table 3. The data show that at 24 h, the highest values are at strains MS16 with 63 Th (0.56% lactic acid) and MS18-61 Th (0.55% lactic acid), the least active strains are MS4 and MS5 with 54 Th (0.48% lactic acid). On 48 h, the highest value of the titratable acidity was measured again at MS16-85 Th, the same strain has the highest increase of the parameter comparing the mean value of 24 to 48 h - 22 Th. On 48 h, the lowest titratable acidity was measured at strains MS13, and also MS11 and MS9 respectively with 69 and 71 Th. At the end of the studied period, strain MS16 again has the highest value of the parameter - 110 Th (0.99% lactic acid), and the lowest value is at MS9-90 Th. Despite the mentioned mean value of the measured acidity, the statistical analysis does not indicate any significant difference between the tested strains. The proteolytic activity of the strains is an important indicator for their ability to take part in the ripening process of the cheese. Most of the researchers assumed that the lactobacilli play the major role in this process (Coeuret et al., 2003), but the presence of a large number of enterococii in the cheese may also contribute to the overall proteolysis of the product (Čanžek Majhenič, 2006). Table 4. The proteolytic activity of the isolated bacteria, mm Proteolytic activity, (mm) Strain 24h 48h Mean SD SE Mean SD SE MS1 18.72 1.83 0.91 25.54 1.96 0.98 MS2 19.33 1.93 0.96 24.95 4.43 2.21 MS3 19.18 3.84 1.92 23.56 2.03 1.01 MS4 19.42 3.79 1.90 26.18 5.47 2.74 MS5 19.03 2.19 1.09 26.66 5.43 2.72 MS6 18.60 1.97 0.98 24.99 3.67 1.83 MS7 17.68 1.96 0.98 24.05 3.20 1.60 MS8 17.85 1.87 0.93 23.23 3.14 1.57 MS9 22.89 4.66 1.65 32.92 13.87 6.94 MS10 18.35 1.04 0.52 24.90 2.98 1.49 MS11 19.32 0.73 0.36 27.99 2.24 1.12 MS12 18.31 1.52 0.76 23.83 2.34 1.17 MS13 19.72 2.67 1.34 27.44 1.02 0.59 MS14 18.60 3.09 1.54 24.67 3.13 1.81 MS15 18.28 3.66 1.83 22.53 3.20 1.85 MS16 18.81 2.36 1.18 26.86 0.78 0.55 MS17 18.28 3.22 1.61 22.95 3.28 1.64 MS18 17.57 1.76 0.88 24.16 5.80 2.90 MS19 17.42 1.91 0.96 22.23 2.13 1.07 MS20 17.92 1.98 0.99 24.60 2.31 1.16 The proteolytic activity of the strains examined in the current study is presented as a clear zone (mm) on MRSagar supplemented with 5% milk (the data are in table 4). After 24 h of incubation, the most visible zone is at strain MS9-22.89 mm, and the minimum is at MS19-17.42 mm. The increase of the zone in MS9 happens at the same pace up for the first 24 h and then up to 48 h and for that reason it again has the largest zone in the measurement made at 48 h - 32.92 mm. For the strain MS19 on 48 h proteolytic zone is the smallest - 22.23 mm. The studied strains show a good proteolytic activity according to Abubakr and Al-Adiwish (2017), who defined the proteolytic activity as good when the clear halo is more than 6 mm. The comparison between the increases of the proteolytic zone indicates that in 70% of the studied strains, the proteolytic zone expands at the same rate for the two measurements up to 24 h and up to 48 h. For the rest 30% of the strains, the zone increases more visibly during the first 24 h in comparison to the consecutive incubation up to 48 h. The used in the current study 20 strains of ovoid gram-positive bacteria were isolated from fresh cheese crumbs and also from the cheese ripened in the skin bag made from artisanal technology. On the basis of the morphological and physiological characteristics of the isolated bacteria we assumed that the isolated bacteria belong to the genus Enterococcus. After 72 hour the cultivation in sterile reconstituted skim milk (10%) one of the strains (ML16) is capable of lowering the active acidity to 4.45, and elevates the titratable acidity to 110 Th (0.99% lactic acid). The proteolytic activity of the strains examined in the current study is presented as a clear zone (mm) on MRS-agar supplemented with 5% milk has a maximum value of 22.89 mm at 24 hour of incubation and 32.92 mm at 48 hour. The comparison between the increases of the proteolytic zone indicates that in 70% of the studied strains, the proteolytic zone expands at the same rate for the two measurements up to 24 h and up to 48 h. For the rest 30% of the strains, the zone increases more visibly during the first 24 h in comparison to the consecutive incubation up to 48 h. 96

Acknowledgement This research was financially supported by the Science Research and Technology Transfer Centre of Agricultural University Plovdiv under project 04-15. References Abubakr MAS, Al-Adiwish WM. 2017. Isolation and identification of lactic acid bacteria from different fruits with proteolytic activity. International Journal of Microbiology and Biotechnology 2 (2), 58-64. Cakmakci S, Gurses M, Gundogdu E. 2011. The effect of different packaging materials on proteolysis, sensory scores and gross composition of tulum cheese. African Journal of Biotechnology 10 (21), 4444-4449. Čanžek Majhenič A.2006. Enterococci: yin - yang microbes. Mljekarstvo 56(1), 5-20. Coeuret V, Dubernet S, Bernadeau M, Gueguen M, Vernoux JP. 2003. Isolation, characterization and identification of lactobacilli focusing mainly on cheeses and other dairy products. Lait 83, 269-306. Cogan TM, Barbosa M, Beuvier E, Bianchi-Salvadori B, Cocconcelli PS, Fernandes I, Gomes J, Gomes R, Kalantzopoulos G, Ledda A, Medina M, Rea MC, Rodrigues E.1997. Characterization of the lactic acid bacteria in artisanal dairy products. Journal of Dairy Research 64, 409-421. El Soda M, Ahmed N, Omran N, Osman G, Morsi A. 2003. Isolation, identification and selection of lactic acid bacteria cultures for cheesemaking. Emir. J. Agric. Sci. 15(2), 51-71. Franz CMAP, Holzapfel WH, Stiles ME. 1999. Enterococci at the crossroads of food safety. International Journal of Food Microbiology 47, 1-24. Giraffa G. 2003. Functionality of enterococci in dairy products. International Journal of Food Microbiology 88, 215-222. Gomes BC, Gombossy de Melo Franco BD, Pereira De Martinis EC. 2010. Dualistic aspects of Enterococcus spp. in foods. In: Mendes-Vilas A, editor. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. 1st ed., Badajos, Spain: Formatex Research Center. 1119-1125. Gurses M, Erdogan A. 2006. Identification of lactic acid bacteria isolated from tulum cheese during ripening period. International Journal of Food Properties 9(3), 551-557. Hayaloglu AA, Cakmakci S, Brechany Y, Deegan KC, McSweeney PLH. 2007. Microbiology, biochemistry and volatile composition of tulum cheese ripened in goat`s skin or plastic bags. Journal of Dairy Science 90, 1102-1121. Hayaloglu AA, Fox PF, Guven M, Cakmakci S. 2007. Cheeses of Turkey: 1. Varieties ripened in goat-skin bags. Lait 87, 79 95. Jamaly N, Benjouad A, Comunian R, Daga E, Bouksaim M. 2010. Characterization of enterococci isolated from Moroccan dairy products. African Journal of Microbiology Research 4 (16), 1768-1774. Kalit MT, Kalit S, Havranek J. 2010. An overview of researches on cheeses ripening in aminal skin. Mljekastvo 60 (3), 149-155. Kamber U, Terzi G. 2008. The traditional cheeses of Turkey: Middle and Eastern Black sea region. Food Reviews International 24, 95-118. Litopoulou-Tzanetaki E, Tzanetakis N. 2011. Microbiological characteristics of Greek traditional cheeses. Small Ruminant Research 101, 17-32. Litopoulou-Tzanetaki E, Tzanetakis N. 2014. The microfloras of traditional Greek cheeses. Microbiology Specrtrum 2(1), 1-34. Öksüztepe G, Patir B, Alicioúlu M. 2005. Identification and distribution of lactic acid bacteria during the ripening of şavak tulum cheese. Turk J Vet Anim Sci 29, 873-879. Olbrich dos Santos KM, Silva Vieira AD, Salles HO, da Silva Oliveira J, Costa Rocha CR, Borges MF, Bruno LM, Gombossy de Melo Franco BD, Todorov S.D. 2015. Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses. Brazilian Journal of Microbiology 46(1), 237-249. Phyu HE, Oo ZK, Aye KN. 2015. Screening of proteolytic activity of lactic acid bacteria from various yogurts and fermented milk, Proceedings of The IRES 11th International Conference, Bangkok, Thailand, ISBN: 978-93-85832-07-9, 28-31. 97