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European Journal of Applied Sciences – Vol. 9, No. 6

Publication Date: December 25, 2021

DOI:10.14738/aivp.96.11227. Çay, M., Oğuzkan, S. B., & Kılıç, I. H. (2021). Isolation and Molecular Characterization of Legume Microorganisms Used in Yogurt

Fermentation. European Journal of Applied Sciences, 9(6). 342-348.

Services for Science and Education – United Kingdom

Isolation and Molecular Characterization of Legume

Microorganisms Used in Yogurt Fermentation

Mesut Çay

Department of Biology, Faculty of Art and Science

University of Gaziantep, 27060, Gaziantep, Turkey

Sibel Bayıl Oğuzkan

Department of Medical Services and Techniques

Health Services, University of Gaziantep, 27060, Turkey

İbrahim Halil Kılıç

Department of Biology, Faculty of Art and Science

University of Gaziantep, 27060, Gaziantep, Turkey

ABSTRACT

Yogurt is generally defined as a fermented dairy product which is produced by using

the symbiotic cultures of Streptococcus thermophilus and Lactobacillus delbrueckii

subsp. bulgaricus. The positive effects of yogurt and other similar fermented dairy

products on human diet and health continue to be proven and this has led to

increased yogurt consumption in the world which in turn demands more

production. In addition, there are works in progress to improve yogurt quality and

produce yogurt with various specifications using newly developed techniques. In

this study, four distinct types of legume (lentil, bean, cow pea and broad bean) were

used to ferment yogurt from milk of without UHT. Yogurt samples were diluted and

then each sample was transferred to a M17 Agar medium and bacteria were isolated

from these samples. Fresh pure cultures were obtained. The new generation

sequence was subjected to a 16s rRNA array analysis and characterization using

MALDI-TOF methods. It was determined that the bacteria in the yogurt which were

produced were Streptococcus thermophilus and Lactobacillus delbrueckii subsp.

bulgaricus. In accordance with the data obtained, the bacteria which was

reproduced in the MRS and M17 mediums proved to be preliminary examples of

cultivated bacteria which can be utilized in commercial yogurt production.

Key words: Yogurt, 16S rRNA, MALDI-TOF, Isolation, Molecular characterization.

INTRODUCTION

It is not known exactly how far back yogurt can be traced in history, but the general belief is

that it was first produced by nomadic tribes living in the Middle Asian region. In their slow

transition from a life of hunting and gathering to permanent settlement, these nomadic tribes

also started domesticating wild animals. It was reported that animals such as sheep, goats,

cattle and water buffalo were domesticated between 6000-8000 B.C. The domestication of

these animals increased the diversity of animal products. Animal milk became a primary food

source but, for that, it had to be preserved for a longer time. For this purposethe first method

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Çay, M., Oğuzkan, S. B., & Kılıç, I. H. (2021). Isolation and Molecular Characterization of Legume Microorganisms Used in Yogurt Fermentation.

European Journal of Applied Sciences, 9(6). 342-348.

URL: http://dx.doi.org/10.14738/aivp.96.11227

developed was to boil the milk. This led to a reduction in the density of the milk, denaturation

in casein and degradation of the pathogenic bacterium. Therefore, an effective preservation

method was developed at that time. It is believed that yogurt formation is triggered by the

reaction between heat-resistant lactic acid bacteria(LAB) in partly processed milk and

fermentative bacteria nourished as a result of other conditions (Tamime and Robinson 2007;

Özden 2008).It was determined that according to TSE standards, to produce yogurt,

Streptococcusthermophilus and Lactobacillusbulgaricusare added to homogenized milk to start

lactic acid fermentation, which then produces a fermented milk product (TS 1330\ T4). Yogurt

is rich in calcium, phosphor, magnesium, vitamin A and riboflavin (Erkaya and Şengül, 2012).

Beside its importance in nutrition, yogurt preserves its fresh state for a long time when stored

at cold temperatures (3-10°C) and has a low pH value, which creates a hostile environment for

pathogen microorganisms to survive.

This is one of the main reasons why yogurt is one of the most widely known and consumed

dairy products in Turkey (Tekinşen et al., 2008). Contemporary studies in the fields of science,

including health and technology, differing preferences of taste and the extensive variety of

yogurt products indicate a public confidence in and a high demand for yogurt products. This

has also led to a need for domestic starter cultures for yogurt. In our country, yogurt production

by milk fermentation is made possible by using imported starter cultures (Güven, 2008).The

objective was to determine, at molecular level, the species of the microorganisms in yogurt,

which are produced using microbiological methods by making use of some of the legume types

identified for the study.

The objective was to identify the microorganism species by microorganism characteristics, to

enable yogurt production in controlled conditions using selected isolates, as well as to obtain

preliminary data for the industrial utilization of various strains.

MATERIALS AND METHODS

Yogurt Production with using legumes

Commercially obtained legume types were named at the species level in the Arts and Sciences

Faculty, Biology Department at Gaziantep University. Commercially accessible daily

pasteurized milk was used in the yogurt production. After production, the yogurt was stored at

+4°C degrees. On the 2nd day following the storage, yoghurt products were diluted and

transferred to the medium. Milk was heated to 80°C degrees and then cooled down to 45°C.

Then the pre-sorted legumes were added to the milk. After this addition, the product was

subjected to 6 hours of incubation. The end products obtained at the end of the 6-hour

incubation were then stored at +4°C degrees for later stages.

LAB Isolation

Sterile dilutions were used to isolate lactic acid bacteria and yogurt samples were diluted to a

10-6 dilution rate. Tubes at 10-5 and 10-6 dilution rates were used for transfer. Intransfer, MRS

Agar at pH 5.4 (Difco 288210) was used for Lactobacillus delbrueckii subsp. bulgaricus isolation

and M17Agar mediums and the addition of a lactose solution at 50ml 10% (m/v) per medium

liter was used to enable the isolation of the Streptococcus thermophilus(Klein, 2003; Zamfir,

2005; Rosaria, 2006). After the yogurt samples were inoculated into the medium, the bacteria

which were developed in the MRS medium for Lactobacillus isolation were titled as Xbas, Ybas,