Identification of Lactic Acid Bacteria Isolated from Tulum Cheese During Ripening Period

Abstract

The fresh Tulum cheese was manufactured and then ripened at 10 ± 2°C for 3 months. Isolation and identification of lactic acid bacteria (LAB) was carried out during the ripening period of 90 days. Lactic acid bacteria were isolated from PCA, M17, and MRS agar. The strains isolated were identified using morphology, colony pigmentation, production of carbon dioxide from glucose, growth at 4 and 40°C, salt tolerance, starch hydrolysis, and sugar fermentation with the API system methods. A total of 253 strains isolated during the storage. Identifying strains belonged to genera of the lactobacilli (133), pediococci (44), enterococci (29), leuconostocs (27), and lactococci (8). 12 of 253 strains were not also identified. Lactobacilli (75.2%) were frequently determined on MRS medium. In the results of this article, lactobacilli were found at high frequencies, while enterococci, lactococci, leuconostocs, and pediococci were found at low frequencies in Tulum cheese. Lactobacilli increased during the ripening period, but the others did not change a significant amount.

Keywords:

• Tulum cheese
• Lactic acid bacteria
• Microflora

INTRODUCTION

Tulum cheese, special a local cheese produced in Turkey, is not well known elsewhere. It is preferred for its characteristic taste and flavor.[1,2] Tulum cheese is produced from Civil cheese and whey curd in small scale dairy plants and is also home made. Civil cheese is manufactured from sour milk with a small amount of rennet and a heating process. The Civil and whey curd mixture is pressed into goatskins or plastic bags and then ripened. During the ripening period, natural contaminating microorganisms, especially moulds, grow and contribute to the ripening process. Tulum is a semi-hard cheese that can be made from whole, semi skimmed or skimmed sheep's, goat's, cow's, and buffalo's milk or their mixture. It has a crumbly texture and a strong flavor. Tulum means the skin of goat or sheep. Traditionally, Tulum cheese is manufactured from skimmed milk (after butter production) for family use, and yogurt is sometimes added for taste.[3,4]

Lactic acid bacteria have been used for the production of fermented dairy products and in meats and vegetable silage fermentations from ancient times. Thousands of strains have been isolated from different food fermentations and hundreds of their selected strains also used as starter cultures in industrial food fermentations.[5] Many species of LAB bacteria play an important role on the ripening cheeses.[6] Their ability to produce lactic acid from lactose is probably the most important property of dairy lactic acid bacteria. It helps to reduce pH, which leads to the development of desirable organoleptic characteristics and prevents the growth of pathogen microorganisms.[7,8,9,10] Determination of the lactic microflora of some traditional cheese varieties, such as Tulum cheese, will contribute to the definition of a starter that allows standardization of product quality and safety without changing the fundamental properties of the product.

The purpose of this article was to isolate and identify lactic microflora of Tulum cheese using the different media. In addition, the selectivity of the media used, as well as the significance and evaluation of species found fresh Tulum Cheese and during the ripening periods, are discussed.

MATERIAL AND METHODS

Sampling

Tulum cheese was manufactured from whole cows milk. Milk was provided from Atatürk University, Faculty of Agriculture, and Department of Food Engineering Pilot Dairy Factory, Erzurum, Turkey. Tulum cheese was produced as described by Akyüz,[11] Kurt and Öztek,[12] Çakmakçi,[13] and Gürses.[14] For this purpose, whole cow milk was firstly separated and pasteurized at 65°C for 30 minutes and then cooled to 30°C. At this temperature, commercial rennet (strength, 1:15,000) was added to coagulate the milk within 90 minutes. The coagulum was cut into 2 cm × 2 cm × 2 cm, and the curd pieces were transferred into vats lined with cheesecloth and pressed using 60 kg weight for 12 hours. The strained coagulum (Teleme) broke up by hand and salted with coarse salt (3.5%). The salted teleme was pressed to remove whey using 30 kg weight for 3–4 hours. After second press operation, it was transferred in glass jars without air holes and then ripened at 10 ± 2°C for 3 months.

Isolation

From the media used to study the lactic acid bacteria (MRS, M17, PCA) in Tulum cheese, a number of colonies were randomly selected from counting plates (25–30 from PCA plates; 5–15 from the other media).[7]

Identification

A total of 253 strains were submitted to Gram staining and the catalase test.[15] Gram-positive and catalase-negative LAB strains were identified by using the following tests: morphology, colony pigmentation, production of carbon dioxide from glucose (in MRS broth covered with an agar seal trap), growth at 4 and 40°C in BHI broth, salt tolerance (2 and 6.5% NaCl in BHI), starch hydrolysis, and sugar fermentation[16] with the identification method of API system using the API 50CHL (Bio Merieux, Marcy I'Etoile France) for lactobacilli and leuconostocs and the API 20STREP (Bio Merieux) for lactococci and enterococci.[7] Identification to species level was determined according to Schleifer et al.[17] and Facklam and Collins[18] as described by Lopez-Diaz et al.[7]

RESULTS

Isolation and identification of LAB of Tulum cheese was carried out during the ripening period of 90 days. Lactic acid bacteria were isolated from PCA, M17, and MRS agar media. The strains isolated were identified using morphology, colony pigmentation, production of carbon dioxide from glucose, growth at 4 and 40°C, salt tolerance, starch hydrolysis, and sugar fermentation with the API system methods.

The distribution of LAB isolated from different media for Tulum cheese samples is presented in Table 1. In isolates from MRS agar, lactobacilli were significantly dominated (85/113). The majority of enterococci were isolated from PCA agar (19/76). Pediococci (17/64), leuconostocs (15/64) and lactococci (5/64) were found in higher proportions in M17 agar than those the other media. 12 out of 253 isolates obtained all the media was not also identified. All of the LAB strains grown at 4°C and 40°C on 2% NaCl but did not hydrolyzed to starch. Lactobacillus brevis, Lb. parabuhneri strains produced CO₂ from glucose. While the Enterococcus faecalis, En. faecium and En. durans strains grown in pH 9.2 Broth, the other strains did not. Besides, all of the identifying strains were gram-positive and catalase-negative (Table 2). Genus Lactobacillus frequently dominated in Tulum cheese, but enterococci, lactococci, leuconostocs and pediococci were found in low proportions during the ripening period (fig. 1).

Figure 1 The percentages of genus LAB isolated from Tulum cheese during the ripening period.

Table 1 Distribution of LAB isolated from different media during the ripening of Tulum cheese.

	Media
Genus	PCA	M17	MRS	Total
Lactobacillus	29	19	85	133
Pediococcus	14	17	13	44
Enterococcus	19	6	4	29
Leuconostocs	5	15	7	27
Lactococcus	2	5	1	8
ND	7	2	3	12
Total	76	64	113	253

Table 2 Biochemical characteristics of the LAB isolated from Tulum cheese.

Strains	Growth at 4°C	Growth at 40°C	Growth on 2% NaCl	Growth on 6,5% NaCl	CO2 from glucose	Starchhydrolysis	pH 9,2
Lactobacillus casei	+	+	+	+	−	−	−
Lb. curuvatus	+	+	+	+	−	−	−
Lb. brevis	+	+	+	+	+	−	−
Lb. parabuchneri	+	+	+	+	+	−	−
Lb. bifermentas	+	+	+	+	−	−	−
Lactococcus lactis	+	+	+	−	−	−	−
P. acetilactici	+	+	+	+	−	−	−
En. faecalis	+	+	+	+	−	−	+
En. faecium	+	+	+	+	−	−	+
En. durans	+	+	+	+	−	−	+
Ln. mesenteroides	+	+	+	+	−	−	−
+ = Positive reaction; − = negative reaction.

Distribution of LAB isolated from Tulum cheese is presented in Table 3. Lactobacillus parabuchneri, Lb. bifermentas, and Lb. paracasei strains were 13.6%, 13.6% and 9.1% of isolates in fresh Tulum cheese, and 17.6%, 14.7%, and 14.7% of isolates at the end of 90 days, respectively. These strains generally tended to increase during the storage. Lactobacillus. brevis and Lb. curuvatus strains were not change also in significant amounts. Lactococcus lactis strain was 4.5% in fresh cheese and decreased by 3.0% after 90 days. In addition, it was observed that this strain did not excessively change during the storage periods. Pediococcus acetilactici was 18.2% in fresh cheese and decreased by 10.3% at 15 day and increased by 17.6% again after 90 days. Enterococcus feacelis was also 13.6% in fresh cheese and decreased by 3.0% at end of the storage period. Leuconostoc mesenteroides spp strains were 9.1% in fresh cheese and increased by 14.7% after 90 days. It was observed that Ln. mesenteroides spp strain tended to increase during storage (Table 3).

Table 3 The proportions of LAB strains isolated from Tulum cheese during ripening (%).

	Ripeninng periods (days)
Strains	Fresh cheese	15	30	45	60	90
Lactobacillus	10 (45.5)	13 (56.5)	16 (55.2)	19 (57.6)	18 (62.1)	21 (61.7)
Lactobacillus paracasei	2 (9.1)	3 (13.0)	4 (13.8)	4 (12.1)	5 (17.2)	5 (14.7)
Lactobacillus casei	1 (4.5)	1 (4.3)	1 (3.4)	2 (6.1)	2 (6.9)	3 (8.8)
Lactobacillus curuvatus	1 (4.5)	1 (4.3)	2 (6.9)	2 (6.1)	1 (3.4)	2 (5.9)
Lactobacillus brevis	–	–	–	1 (3.0)	1 (3.4)	–
Lactobacillus parabuchneri	3 (13.6)	5 (21,7)	5 (17.2)	6 (18.2)	4 (13.8)	6 (17.6)
Lactobacillus bifermentas	3 (13.6)	3 (13.0)	4 (13.8)	4 (12.1)	5 (17.2)	5 (14.7)
Lactococcus	1 (4.5)	1 (4.3)	–	1 (3.0)	1 (5.0)	1 (3.0)
Lactococcus. lactis	1 (4.5)	1 (4.3)	–	1 (3.0)	1 (5.0)	1 (3.0)
Pediococcus	4 (18.2)	3 (13.0)	6 (20.7)	7 (21.2)	6 (20.7)	6 (17.6)
Pediococcus acetilactici	4 (18.2)	3 (13.0)	6 (20.7)	7 (21.2)	6 (20.7)	6 (17.6)
Enterococcus	5 (22.7)	6 (26.1)	3 (10.3)	2 (6.1)	1 (3.4)	1 (3.0)
Enterococcus faecalis	3 (13.6)	4 (17.4)	3 (10.3)	2 (6.1)	1 (3,4)	1 (3.0)
Enterococus faecium	1 (4.5)	–	–	–	–	–
Enterococcus durans	1 (4.5)	2 (8.7)	–	–	–	–
Leuconostocs	2 (9.1)	2 (8.7)	4 (13.8)	4 (12.1)	3 (15.0)	5 (14.7)
Leuconostoc mesenteroides spp	2 (9.1)	2 (8.7)	4 (13.8)	4 (12.1)	3 (15.0)	5 (14.7)
Total	22 (100)	23 (100)	29 (100)	33 (100)	29 (100)	34 (100)

DISCUSSION

As can be seen from Table 1, many of LAB isolated cheese samples are belong to genus lactobacillus. Lactobacilli were found in higher proportion (25.0%) in MRS than those in the other media. This situation has confirmed the useful of this medium for the isolation of these genera.[8,10] In addition, Garcia-Fontan et al.[19] stated that the M17 agar is a selective medium for lactococci. Biochemical characteristics of identifying LAB in our study were also harmonious with results from Toma piemontese cheese by Fortina et al.[20]

While the lactobacilli increased during the ripening period, the other genera did not change in a significant amount (Fig. 1). This pattern seems to be common in most cheese varieties. It was also reported that lactobacilli was commonly found in cheeses with long-term ripening.[5,20] As a matter of fact, lactococci develop at the beginning of the process and carry out the acidification of the milk, which would lead to the formation of the curd; subsequently, the lactic acid flora changes and lactobacilli become dominant and participate in the ripening.[7] In addition, it was stated that lactobacilli grown at low pH more than the other,[10] and leuconostoc and lactococci that are aciduric microorganisms, has a slower metabolism in low pH.[19] Leuconostoc may also contribute to the development of texture inside the cheese due to their heterofermentative character. Besides, it was explained that Pediococci might have contributed to the flavor and acceleration of cheese ripening.[21]

While the lactobacilli isolated from Tulum cheese frequently increased, enterococci decreased during the storage period. Lactobacilli dominated during the storage period (Table 3). Durlu-Ozkaya[8] stated that Lb. paracasei was found in significant amount in Beyaz cheeses. Gerasi et al.[21] also reported that Leu. mesenteroides was 41.6% on surface parts of Manura cheese and, Lb. paracasei (21.8%) in inner parts at the end of ripening period. L. Lactis was not found in significant amounts during the storage of Tulum cheese in our study.

CONCLUSION

In the results of this article, it was determined that genus lactobacillus dominated, and the others were also found in low proportions in Tulum cheese. Lactobacillus paracasei strain was also found in high levels in fresh Tulum cheese and during ripening period, more so than other strains. This strain, therefore may be suggested as starter culture for industrial production of this cheese. This study was an examination of the possibility of using certain LAB species as starters, as suggested by their quantitative importance for this cheese variety. However, further investigations should be carried out for the improvement and standardization of Tulum cheese.