Introductıon

Yogurt is a fermented sour milk product that has been made by humans for centuries and is famous for its unique taste and health benefits ^2,6,7,23. The origin of yogurt can be traced back to ancient civilizations such as the Sumerians and Babylonians, who discovered the transformative effects of natural fermentation on milk. Today, yogurt is produced traditionally and used by the entire population of the world spontaneously at home and for commercial purposes on the basis of  lactic starter culture. The main role in yeast basedyogurt fermentation is played by lactic acid bacteria and yeast fungi. At this time, lactic acid bacteria absorb lactose sugar and produce lactic acid, and yeast fungi produce alcoholic fermentation. Lactic acid bacteria strains are known to produce metabolites with high antimicrobial properties and in our research work we have worked with the spontaneously prepared yogurts of Uchoglan, Tazakend, Guzanli and Imamgulubayli villages of Agdam region ^{1,8,10,11-13,15-18}.

Currently, yeast-based yogurt is being prepared and used in different areas of the Azerbaijan Republic. It is one of the important issues to study which types of microbial cultures are participate in this process ^5,21,22.  This point – the main purpose of presented work in the review was to bring out the microbial strains of spontaneous yeast base yogurt into pure cultures which prepared by the population in some regions of Aghdam district and to study their morpho – cultural and quantitative characteristics.

Materıals and Methods

Spontaneous yogurt samples taken from different areas of Aghdam region: Uchoglan, Tazakend, Guzanli, Imamgulubayli villages were used as the research objects. For the cultivation of yeast fungi, nutrient medium with malt agar was used. Meat – peptone agar medium was used for the cultivation of lactic acid bacteria ¹⁴.

The collected samples were delivered to the microbiology laboratory through special cooling chambers within 6 hours. From the delivered yogurt samples to the laboratory, 1 ml of spontaneous yogurt was taken with a sterile pipette and added into the test bottles containing 9 ml of sterile water, and it diluted up to 5 times. The suspension obtained from the fourth and fifth dilution was taken with a sterile pipette, 0.1 ml from the end suspensiom was added to the meat-peptone agar and malt agar nutrient media in the petri dishes, and spread with a sterile spatula. Cultivation was carried out in four to five replicates. The cultured petri dishes were kept for 3 days in a thermostat at a temperature of 30°C for incubation. Colonies were observed in Petri dishes after the incubation period. Colonies observed in Petri dishes differed in number and morpho-cultural characteristics. The number of microorganisms was determined according to the number of colonies observed in Petri dishes. Then, the color of the colonies, the shape of their edges and surface were determined. In order to determine the shape of the cells, preparations were prepared and looked at under a light microscope, and the strains were removed into pure culture.

It was investigated by Gram staining method whether the isolated bacterial strains were gram positive or gram negative or whether they produce or do not produce spores and pigments. We also tested lactic acid bacteria by using 3% hydrogen peroxide solution. For yeast strains, the forms of asci which formed from vegetative cells, the number and structure of ascospores in asci were determined.

By determining these characteristic features, bacterial strains were assigned  according to Bergey’s designation (Bergeys Mannal, 1997), and yeast strains were assigned according to Lodder (1870), Babyeva and Golubeva (1979) designation for identify the genus of isolated species ^{4, 19}.

Results and Dıscussıons

Morpho-cultural and quantitative characteristics of lactic acid bacteria and yeast fungi isolated from each sample were determined during the research work.

The color of lactic acid bacteria strain BDU-UN1 isolated from Ucoglan village yogurt was in white color, in small size, the edges of them were round and their shape was in coke-shaped  (Tab.1, Fig. 1).

Figure 1: BSU – UN1 lactic acid bacteria strain: a) growing colonies on solid nutrient agar medium, b) cells appearance under a microscope  Click here to view Figure

The colony size of BDU – UN2 lactic acid bacteria was large, its  color was whitish, the surface of it was wrinkled, the cell shape of it was rod – shaped (Tab.1, Fig. 2).

Figure 2: BSU – UN2 lactic acid bacteria strain:  a)growing colonies on solid nutrient agar medium, b) cells appearance under a microscope Click here to view Figure

The colony surface of  BSU –  QI4 strain isolated from Quzanli village yogurt was wrinkled, it was in whitish colour and the shape of their cells was in coke – shaped (Tab.1, Fig. 3).

Figure 3: BSU – QI4 lactic acid bacteria strain: a) growing colonies on solid nutrient agar medium, b) cells appearance under a microscope. Click here to view Figure

The colony size of  BDU – TD6 lactic acid bacteria strain isolated from Tazakend yogurt was medium, its color was white with slightly smooth and round surface, and the cell shape was  rod – shaped (Tab.1, Fig. 4).

Figure 4: BSU – TD6 lactic acid strain: a) growing colonies on solid nutrient agar medium, b) cells appearance under a microscope Click here to view Figure

The colony size of BDU – II8 lactic acid bacteria strain isolated from Imamgulubayli village yogurt was small with round shape and its surface was slightly convex in white colour, it had a coke – shaped cellular form (Tab.1, Fig. 5).

Figure 5: BSU – İİ8 lactic acid strain: a) growing colonies on solid nutrient agar medium, b) cells appearance under a microscope. Click here to view Figure

Summarizing the above, it can be concluded that the color of the colony of the lactic acid bacteria strains we studied was white, whitish, size – small, medium, large, surface – smooth, slightly convex, cellular shape was straw and coke – shaped.

Table 1: Morpho-cultural features of lactic acid bacteria strains.

 Morpho-cultural features of isolated yeast strains

Morpho-cultural and quantitative characteristics of yeast strains isolated from yogurt samples taken from different settlements of Agdam region were studied.

It was determined that, the colony surface of BSU – UN3 yeast strain isolated from Uchoglan village yogurt had a slightly convex surface, paste-like consistency, light yellow color, and their shapes were mostly in round cell shapes (Tab. 2, Fig. 6).

Figure 6: BSU – UN3 yeast strain: a) growing colonies on solid nutrient agar medium, b) cells appearance under a microscope Click here to view Figure

The colony color of BSU – QI5 yeast strain isolated from Guzanli village yogurt was light yellow, their edges were smooth with paste – like consistency and they had a slightly raised  surface. Their cell shape was elongated lemon – shaped (Tab.2, Fig.7).

Figure 7: BSU – QI5 yeast strain: a) growing colonies on solid nutrient agar medium, b) cells appearance under a microscope. Click here to view Figure

The consistency of  BDU – TD7 yeast strain colony isolated from Tazakend village yogurt was paste – like, colony colour was white with smooth surface and edges and their cells were like as lemon – shaped elongated cells (Tab. 2, Fig. 8).

Figure 8: BSU – TD7 yeast strain: a) growing colonies on solid nutrient agar medium, b) cells appearance under a microscope Click here to view Figure

Based on Figure  9, we can say that the colony shape of BSU – II9 yeast strains isolated from Imamamgulubeyli village yogurt was round, their colonies were in yellowish – white colour with paste – like consistency, colonies were slightly convex on the surface, and slightly larger in size. Cells were elongated and balloon – shaped (Tab. 2, Fig. 9).

Figure 9: BSU – II9 yeast strain: a) growing colonies on solid nutrient agar medium, b) cells appearance under a microscope. Click here to view Figure

The morpho – cultural colony signs of the yeast strains isolated from spontaneous yogurt samples that we used as our study object were tabulated and their cellular structure is also reflected in the following table  (Tab 2). 

Table 2: Morpho – cultural features of yeast strains

As a result of the research, the amount of lactic acid bacteria and yeast fungi obtained from spontaneous yogurt samples taken from the villages of Uchoglan, Imamgulubayli, Tazakend, Guzanli of Agdam region was determined (Tab 3).

The amount of yeast cells in 1 ml of spontaneous yogurt samples taken from Uchoglan village was 15×104, 48×104 in Guzanli village yogurt, 268×104 in Tazakend yogurt, and 95×104 in Imamgulubeyli village yogurt.

The most yeast cells were observed in Tazakend yogurt, and a relatively small amount was observed in spontaneous yogurt brought from the village of Imamgulubayli.

The amount of yeast cells in 1 ml of Guzanli village yogurt was 3.2 times more than the yeast cells in Uchoglan village yogurt, 5.5 times less than the yeast cells in Tazakend yogurt, and 2.1 times less than the yeast cells in Imamgulubeyli village yogurt.

The amount of lactic acid bacteria was 71×104 in 1 ml of yogurt sample taken from Uchoglan village, 142×104 in Guzanli village yogurt sample, 181×104 in Tazakend yogurt sample, and 102×104 in Imamgulubeyli village yogurt sample. The most number of lactic acid bacteria cells were observed in Tazakend village yogurt sample, and the least amount of lactic acid bacteria was observed in Uchoglan village yogurt compared to other settlements.

It was determined that, the amount of lactic acid bacteria in 1 ml of Tazakend village yogurt was 2.5 times more than Uchoglan village yogurt, 1.3 times more than Guzanli village yogurt and it was 1.8 times more than Imamgulubayli village yogurt.

The amount of yeast cells contained in Uchoglan, Guzanli and Imamgulubayli village yogurts was less than lactic acid bacteria contained in those yogurts. The amount of lactic acid bacteria was 4,7 times more than yeast cells in Uçoğlan village yogurt, 2,9 times more in Guzanli village yogurt, and it was 1,1 times more in Imamgulubayli village yogurt.  The number of yeast cells was 1.5 times more than the number of bacteria only in Tazakend village yogurt.

Certain tests were carried on for bacterial strains isolated from spontaneously prepared yogurt samples in 4 settlements of Agdam region and it was determined that they do not form spores and pigments, they are gram positive and cannot produce catalase enzyme.

In yeast strains, diploid cells undergo vegetative reproduction. Asci are mainly formed from vegetative diploid cells. In them, the asci are round or oval. Up to 2 – 4 round colorless and smooth ascospores are located in the ascus. The establishment of  these characteristic signs and tests has helped us to determine bacterial and fungal strains in a genus level.

Table 3: The amount of microorganisms (per 1 ml) in spontaneous yogurts used in the territory of Aghdam region

Conclusıon

Thus, it was determined that 5 lactic acid bacteria and 4 yeast strains were isolated from the studied yogurt samples. The highest amount of isolated lactic acid bacteria and yeast fungi was determined in Tazakend village yogurt.

According to the morpho – cultural characteristic features of the investigated lactic acid bacteria strains, their colony size was small, medium, colonies were white, whitish, yellowish – white with round or coke – shaped, rod – shaped, the colony surface was slightly smooth, sometimes slightly convex. Lactic acid bacteria strains were belonged to 2 genera; Streptococcus spp. (BSU – UN1, BSU – QI4) and Lactobacillus spp. (BSU – UN2, BSU – TD6, BSU – II8) genera.

The colony sizes of the isolated yeast strains were medium, relatively large, their shapes were  mostly round, their consistency was paste-like, the edges were wavy, flat, their surface was wrinkled, convex. The shape of the cells was mainly elongated –  lemon – shaped or egg – shaped. In these strains, asci are mainly formed from vegetative diploid cells. Asci are round or oval. There are 1 – 4 round, colorless and smooth ascospores in the asket. By taking account these signs, all 4 yeast strains (BSU – UN3, BSU – Qİ5, BSU – TD7, BSU – II9) were belonged to the genus Saccharomyces.

In future studies, because we have no conditions for genetic analysis (PCR), the isolated strains will be identified at species level by studying their physiological and biochemical characteristics. After studying the antimicrobial activity of strains, their active ones will be applied for certain purposes in industry.

Acknowledgment

Prof. Khudaverdi G. Ganbarov + 3

Conflict of interest

There are no conflict of interest.

Funding sources

There is no funding sources.

Authors’ Contribution

Contribute all author equally

Data Availability Statement

None

Ethics Approval Statement

None

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