Lactic acid bacteria are recognized as important industrial bacteria due to their fermentative capacity and their health and nutritious benefits. Yogurt is popular and familiar dairy product that is consumed in many countries around the world [1]. Mediterranean, Asian countries and in central Europe are regarded as the main areas where yogurt is produced and consumed [2]. Part of yogurt consumed in Gaza Strip is manufactured by conventional methods where 1-day old yogurt is used as starter culture for fermenting the fresh milk. During the traditional homemade process, spontaneous uncontrolled fermentation conditions dominate. Such type of fermentation is considered as the primary method used to preserve milk.

Yogurt production mostly denotes the use of mixed starter (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus) [3] [4]. Starters for yogurt production grow in a symbiotic association [5]. The bacteria play vital roles during fermentation, including lactose conversion to lactic acid, partial proteolysis of casein, development of aromatic compounds, developing characteristic viscosity and binding of water [1]. The high acidity of yogurt (0.85% - 0.95%) contributes to preservation of the product by inhibition growth of pathogenic or saprophytic bacteria [6]. A number of adopted manipulating processes offered to bring yogurt processing more attractive and give affordable product. Those processes include choice of competent starters that act to increase gelling capacity and bring reduction in syneresis. Due to variations in properties of lactic acid bacteria (LAB), it is important to make assessment of the strains from different origins to characterize efficient and effective strains having desirable properties relevant for yoghurt industry. Therefore, it is very important to screen, identify, classify and choose stains for upcoming applications in the industry [1]. For industrial applications, it is vital to identify the bacteria used for fermentation. Use of phenotypic and biochemical characterization is mostly unreliable for providing quality data about the bacteria and in some cases, the data and results are non-reproducible. These obstacles directed efforts to depend molecular approaches for characterization and subtyping (on strain level) of the isolated bacteria. Therefore, it is mandatory to create efficient and time-saving techniques to categorize the isolated bacteria regularly. Molecular methods are applicable as identification tools as they improve the efficiency of detection and enumeration [7] [8].

The present work aimed to isolate thermophilic yogurt bacterial strain’s Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus from homemade traditional fermented milk and identify them. PCR-based techniques were employed together with the phenotypic categorization methods. Some relevant properties of the isolated bacteria were examined for biotechnological applications as candidate starters for production of yogurt.

Traditional homemade fermented milk samples: Six samples of cow’s homemade traditional yoghurt were collected from three geographical areas of Gaza strip. Samples were collected in sterile cups and stored at 4˚C until used. Ten grams of each sample homogenized and diluted by 90 ml sterile saline solution (0.85% NaCl) followed by 7-fold serial dilution for isolation [9].

Isolation of lactic acid bacteria strains

A 0.1ml taken from the dilutions and plated twice in M17 and MRS agar media used to detect cocci and bacilli, respectively. To prevent yeast contaminants, the media was complemented with 100 mgl⁻¹ of cycloheximide antibiotic. The cultured MRS and M17 agar media were incubated an aerobically by using the gas pack system to provide 42˚C optimum temperature for L. bulgaricus and S. thermophilus, respectively. Colonies were randomly selected, then streaked on agar plates (MRS & M17) and incubated at 42˚C to obtain pure cultures. For short-term preservation, isolated bacteria agar plates (MRS & M17) were kept at 4˚C. For longer preservation, the isolates were stored in M17 and MRS broths supplemented by 20% glycerol as cryoprotectant [9].

Identification of the isolated lactic acid bacteria: Genus of the isolated bacteria was determined by employing morphological, phenotypic and biochemical techniques. All isolates were subjected to gram staining, catalase test, gas production from glucose test, grown at 15˚C & 45˚C, grown at 2%, 4% and 6.5% NaCl concentrations [10].

Profile of sugar fermentation of the isolates:

The API 5o CH system (Biomerieux, France) was used to demonstrate the carbohydrate fermentation profiles of the isolated bacteria. The database of ABIS online for bacterial identification was employed for elucidations of the results obtained from the fermentation profile tests [11].

Molecular identification for isolated bacteria:

Identity of the species confirmed by using species-specific primers by PCR using 16S rDNA sequences [12].

DNA isolation:

Genomic DNA of the isolated bacteria was extracted by Genomic DNA Purification Kit Brand WISARD Promega^Ò for isolation of gram-positive bacteria according to instructions of the manufacturer. Until being used as template for PCR, the extracted DNA was stored at -20˚C.

Primers

PCR conditions

BioRad, (USA) thermal cycler used for amplification of PCR. For synthesis of methionine gene, conditions of PCR and reaction of the mixture were as follows:

Volume of PCR reaction was 30 μl while the volume of DNA was 1 µl.

For 16S rRNA gene, PCR conditions and reaction mixture were as follows:

Restriction analysis of 16S rRNA gene product

The restriction reaction of the 16S rRNA gene product was achieved by endonuclease enzyme Eco RI. Mixture of the reaction composed from 1 X of the restriction enzyme buffer, 10 µl of PCR product and 0.5 µl Eco RI [13].

Separation of Amplification Products

For preparation of separation gel, 1.5% agarose was dissolved in 100 µl 1 X TAE buffer, temperature was lowered to 45˚C and 15 µl of ethidium bromide solution added with stirring. The agarose mixture was poured in casting stand then combs were placed and left to form wells [14]. PCR products (5 µl) were mixed with gel loading dye (2 μl). Starting from the second well, the samples loaded. In the first well, 100 bp DNA ladder was loaded as marker for molecular weight of the separated DNA. Electrophoresis at 80 V for 1 hour applied for the PCR products and the marker. The gel documentation system (CSL-MICRODOC, UK) was used to visualize the migrated amplification products. Successful amplification was confirmed by presence 1500 - 2000 bp DNA fragments [15] [16].

Assessment of fermentation performance of the isolates:

Preparation of fermented milk

To make fermented milk, pasteurized mixtures of 60 g/L of powdered milk were dissolved in 5 liters of cow milk [17]. Pasteurization process was carried out by heating the mixture at 80˚C for 15 min in water bath, homogenized and then rapidly cooled to 45˚C [18]. Each identified isolated types (Lactobacillus bulgaricus &Lactobacillus thermophillus) was examined for fermentation performance separately and in combined manner. For separated performance test, 1% of activated inoculums (10⁶ - 10⁷ cfu/ml) of isolated yogurt starters were added to the mixture. Activated inoculums were prepared by incubation in 10% (w/v) sterilized reconstituted skimmed milk and incubated at 37˚C for 18 - 24 h before using pasteurized milk as the growth medium. For combined fermentation performance assessment, the milk mixture was inoculated with 5% (10⁶ - 10⁷ cfu/ml) of S. thermophilus, L. bulgaricus (1:1) with stirring after inoculation and incubated at 42˚C in a shaker water-bath for 8 h. 50 ml of samples were taken in aseptic sterile cups for microbiological and biochemical tests every 1.5 - 2 hrs. Each experiment repeated three times [19] [20].

Measurement of pH and titration of acidity

The pH meter probes directly into a homogenized sample of the fermented milk to determine the pH. For determination of acidity (lactic acid %), 0.1 N NaOH was used to titrate 1 ml 1 ml of homogenized fermented milk while phenolphthalein used as indicator (0.1%, 3 drops). Titration process was performed with continuous stirring until the color of the solution appeared clearly as pink color lasting for 30 seconds. Volume of NaOH recorded and acid concentration calculated by using the formula:

LA % = 10 ∗ VNaOH ∗ 0.009 ∗ 0.1 W ∗ 100 %

where 10 is the dilution factor; W is weight of titrated sample; V NaOH is the volume of sodium hydroxide consumed for neutralizing the lactic acid and the 0.1 is normality of the sodium hydroxide [21].

The biochemical tests included catalase and gas production from glucose fermentation. Catalase test was negative (no babbles) for both the isolates. Additionally, when the isolates were examined for gas production test from glucose

fermentation, there was no gas accumulation in test tubes. This means that the isolates behave as homofermenters of glucose and this result agreed with those

obtained by authors [22] [23] [24]. The cocci isolate show no growth at 2% NaCl while bacilli isolates grew at that concentration. This showed that bacilli isolates were sensitive to 4% and 6.5% concentration and this is in agreement with results obtained by others [25] [26] [27]. Cocci isolates were unable to grow 10˚C while they grow very well at 45˚C and this result agreed with those in literature [28] [29]. Bacilli isolates did not grow at 15˚C but grew 45˚C. According to the results obtained from online ABIS software, API 50 CH fermentation tests showed that the cocci isolates belong to S. thermophilus and the bacilli isolates belong to L. delkbrueckii ssp. bulgaricus and Streptococcus thermophilus strains with high percentage of reliability. This study applied quick and selective identification method for characterization of both L. bulgaricus and S. thermophilus from mixed cultures using a specific PCR based method of methionine biosynthesis gene and restriction analysis method of 16S rRNA gene using Eco RI as previously reported [30]. Molecular diagnosis of the isolates showed that lactobacilli produced a different profile than that of streptococci when performing restriction analysis (Figure 4(a) & Figure 4(b)) and this result agreed with a previous study [13]. The present work seeks to identify yoghurt starters isolated from traditional homemade yoghurt in Gaza Strip. As stated in previous studies, the amplicons of the methionine biosynthesis genes are present in L. lactis and L. helveticus thus, it is applicable for discrimination of these cells. The discrimination of L. bulgaricus, L. lactis and L. helveticus, attained by ARDRA as devised previously [13]. From our findings L. bulgaricus A2 isolate showed the fastest growth pattern among all single cultures. Abu-Tarboush (1996) contradicted this finding by stating that lactobacilli were always less numerous than the streptococci during fermentation of camel milk for 4 h at 42˚C. However, our results agree with Lore et al. 2005, who found that the total LAB counts in suusac (a Kenyan traditional fermented camel-milk) were 6.8 log₁₀cfu/ml and the prominent genus was Lactobacillus ssp. Likewise, Abdel Moneim et al. 2006, stated the main genus was Lactobacillus in garris product (Sudanese traditional fermented camel milk). Our results showed that the final pH after 24 h fermentation of the mixed cultures were lower than pH of single cultures. This was supported by Carrasco et al. (2005) who stated that the pH of blend cultures were much lower than pH of the pure cultures. Also when Abu-Tarboush (1996) studied the action of various strains of commercial cultures in whole camel milk, he displayed that the last pH of the blends L. bulgaricus with the other isolates of S. thermophilus gave lower values comparing with isolates alone at 42˚C for 4h. The results obtained from this work showed that the pH of the fermented raw milk by the isolated starters ranged from 4.20 to 4.98 which is compatible with (Lore et al., 2005) results that described for suusac with pH around 4.30, but were higher than the results described in garris (pH 3.25 - 3.40) which is Sudanese traditional fermented camel milk.

Two isolates of L. delbrueckii ssp. Bulgaricus and S. thermophilus were selected to produce starters. These starters are low in cost and are easily affordable for production of yoghurt at small and large scale. The whole information obtained from the study proved the need to conjoint phenotypic, biochemical and molecular techniques for identifying screened L. bulgaricus and S. thermophilus from natural sources for approving them as starters for manufacturing of yogurt as they were mostly imprecisely identified by phenotypic and biochemical tests only. PCR-ARDRA was found to be beneficial tool for identification of homofermentative and thermophilic dairy bacterial species or subspecies. Distinguishing between isolated cultures by PCR-ARDRA is recommended for identification of lactic acid bacteria in combination with API 50 CHL. After testing and assessing the isolated strains on yogurt production, the data indicated that certain microbial and biochemical changes happen during fermentation of milk. The results showed that microorganisms differed in growth, acid production and titration of acidity. Generally, mixed yogurt culture manifests superior growth, acid production and titrable acidity than single starter cultures. Finally, identifying the starters of yogurt is the first step for using these starters for production in the dairy industry. It is important also to screen all parameters of the isolated LAB concerning their suitability as starter cultures, including acidifying activity, aroma compounds production, particularly acetaldehyde and diacetyl, exopolysaccharides production and resistance toward bacteriophage invasion. Determination of such characteristics would be helpful for industrial applications in future. Analysis of yoghurt starters is a complicated process and involves various techniques, especially phenotypic, biochemical, molecular-based and even genomic techniques.

The authors would like to give thanks to Department of Biology & Biotechnology, Islamic University-Gaza for continuous support and offering exceptional research accommodations.

All stages of research and writing of the manuscript are partially funded by self-efforts from the researchers and partially by Islamic University, Gaza, Palestine.

Dr. Kamal El Kahlout and Dr. Tarek El Bashiti designed the study, analyzed and interpreted the data, supervised all experiments, identified lactobacilli using 16S-ARDRA and drafted and reviewed the manuscript. Ismail El Quqa was in charge of isolating the lactobacilli and their identification by Biochemical tests, isolated DNA from lactobacilli and was responsible for 16S rDNA. Mahmoud El Hindi revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.

Sampling process was part of the master duties of the student and no legal or ethical permissions found needed from the ethics commission for such type of sampling work.

The authors declare that they have no conflicts of interest regarding the publication of this paper.

El Kahlout, K.E.M., El Quqa, I.M., El Hindi, M.W. and El Bashiti, T.A. (2018) Isolation, Biochemical Characterization and DNA Identification of Yogurt Starters Streptococcus thermophilus & Lactobacillus delbrueckii ssp. bulgaricus in Gaza Strip. Advances in Microbiology, 8, 1005-1020. https://doi.org/10.4236/aim.2018.812068

LAB: Lactic Acid Bacteria, TAE: Tris Acetate EDTA, Cfu: Colony for unit, LA: lactic acid, NaOH: sodium hydroxide, MRS: de-man, Rogosa and sharpe medium, NaCl: sodium chloride, CHL assays: Carbohydrate fermentation kit, PCR: polymerase chain reaction, ARDRA: Amplified Ribosomal DNA Restriction Analysis, Eco RI, rRNA: ribiosomal ribonucleic acid, EPS: Exopolysaccaride.