Isolation and Identification of Lactic Acid Bacteria from Cow Milk and Milk Products.

Lactic acid bacteria (LAB) have long been consumed by people in several fermented foods such as dairy products. A study was conducted on lactating dairy cows to isolate and characterize LAB from dairy products found in and around Bahir-Dar city, North Western Ethiopia. Milk and milk products were randomly collected from dairy farms, milk vending shops, individual households, and supermarkets for bacteriological investigations. A total of sixteen samples were taken from different sources and cultured on different selective media: de Man, Rogosa, and Sharpe (MRS) agar for Lactobacillus spp.; M17 agar for Lactococcus spp.; Rogasa SL agar for Streptococci spp.; and MRS supplemented with cysteine (0.5%) for Bifidobacteria spp. Different laboratory techniques were implemented for LAB isolation and identification. A total of 41 bacterial isolates were grouped under five different genera of LAB and Bifidobacteria spp. were identified based on the growth morphology on the selective media, growth at a different temperature, gas production from glucose, carbohydrate fermentation, and other biochemical tests. LAB genera such as Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and Bifidobacterium spp. were isolated and identified from raw milk, cheese, and yogurt. Based on the current study, the majority of the LAB (24.38%) was isolated from cheese and yogurt. Among these, Lactobacillus, Lactococcus (21.94%), Streptococcus (19.51%), Leuconostoc (14.64%), Bifidobacteria (12.19%), and Pediococcus (7.31%) spp. were also identified from these products. Furthermore, based on the bacterial load count and different identification methodologies, our study revealed that Lactobacillus spp. were the dominant LAB isolated from milk and milk products. As a result, since there are few studies on the isolation and identification of lactic acid bacteria from dairy products in Ethiopia, more research studies are needed to complete the identification and characterization to species level and their possible role as probiotics.

1. Introduction

People have historically consumed lactic acid bacteria (LAB) in fermented foods such as dairy products. LAB are currently the subject of extensive international research due to their critical involvement in most fermented foods, as well as their potential to create antimicrobial chemicals that promote probiotic characteristics [1] including antitumor activity [2], alleviation of lactose intolerance [3], a decrease of serum cholesterol [4], gut microflora stabilization [5], and stimulation of the immune system [6]. In the production of fermented milk, the LAB strain produced for exopolysaccharides is used to improve their viscosity and smoothness [7]. Some LAB strains are known to produce the presumed effects of mannitol which promote health [8].

Probiotics are live bacteria or yeasts that, when given in sufficient proportions, provide a health benefit to the host [9, 10]. To date, with the growing interest in health consciousness, the concept of probiotic foods has gotten a lot of attention. A large number of probiotic species and strains belong to the genera Lactobacillus and Bifidobacterium. Other groups of the LAB (Streptococcus and Enterococcus), Bacillus, Propionibacterium, and the yeast Saccharomyces are being used as probiotic microorganisms [11, 12].

The gastrointestinal tract (GIT) consists of diverse microorganisms mainly dominated by bacteria. Other archaeal domains, yeasts, and molds are also found in the GIT [13]. Probiotic microorganisms are thought to have a wide range of health benefits. They exert antimicrobial activity against enteric pathogenic bacteria; stimulate mucosal and systemic immune responses of the host; provide anticolon cancer, anticholesterol, and anti-inflammatory effects; and improve nutritional status. They also reduce symptoms of diarrhea, lactose intolerance, and allergic reactions [13]. So, multistrain or multispecies probiotic mixes are becoming more popular because they can give additive or even synergistic effects, as well as a broader spectrum of health advantages [14, 15].

The benefits of probiotic intervention in the gut microbiota in maintaining and restoring health are becoming more well recognized, drawing increased scientific interest [16-18]. The expected probiotic strains should be assessed for possession of physiological and genetic traits and technological properties beneficial to the host. Probiotic organisms must be able to live in the presence of gastric acid and bile, as well as adhere to and colonize the gastrointestinal epithelial layer. They also should be safe and nonpathogenic to the host [19].

Probiotics are the key components of functional meals that account for over 65 percent of the global functional food market. LAB, such as Lactobacillus, Bifidobacterium, and Enterococcus spp., are among the live bacteria included in probiotic supplements. Within the phylum Firmicutes, LAB are made up of various genera. LAB includes the genera of Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Lactosphaera, Melissococcus, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella [20-24].

Gram-positive lactic acid-producing bacteria that belong to the phylum Actinobacteria are Aerococcus, Microbacterium, and Propionibacterium [25] as well as Bifidobacterium [21, 26]. Gram-positive bacteria that ferment carbohydrates into energy and lactic acid are members of the LAB family [22, 27]. Depending on the organism, the metabolic processes are different if glucose is the major source of carbon: homofermentative bacteria, including Lactococcus and Streptococcus spp., give two lactates from a single glucose molecule, whereas glucose molecules are transformed into lactates with ethanol and carbon dioxide (i.e., Leuconostoc and Weissella spp.) [22, 28, 29].

LAB also produces tiny organic molecules that give the fermented product its aroma and flavor [28]. The study on isolation and characterization of LAB or probiotics was so far not well studied in milk and milk products in Ethiopia. Considering the importance of probiotic bacteria in milk and milk products, only a few works have been done on probiotics in the study area. Therefore, LAB from cow milk and milk products were isolated and identified in this research.

2. Materials and Methods

2.1. Study Animals and Design

Lactating dairy cows were the study animals that were managed in a semi-intensive way. A cross-sectional study was conducted from November 2015 to March 2016 to isolate and characterize LAB from milk and milk products gathered from lactating dairy cows located at different farms of Bahir Dar district.

2.2. Study Methodology

2.2.1. Procedures for Collecting and Handling Samples

For the bacteriological analysis, a total of 16 milk and milk products were collected (7 raw milk samples from dairy farms (3), households (2), and milk samples from vending shops (2); 4 cheese samples from households and supermarkets; and 5 yogurts from households, milk vending shops, and cafeterias). Early in the morning, a total of 300–500 ml of milk and milk products were collected from dairy farms, individual households, supermarkets, milk vending shops, and cafeterias (farmers) by using a sterile glass bottle. Then, the samples were labeled correctly, stored at +4oC, and the samples were cultivated bacteriologically.

2.2.2. Isolation and Identification of LAB

Bacterial isolation was performed by preparing serial dilutions of the samples with sterilized maximum recovery diluents (pH = 7), 0.1 ml of the dilution was spread on de Man, Rogosa, and Sharpe (MRS) agar for LAB isolation, M17 broth + agar powder for Lactococcus isolation (HiMedia, Mumbai, India), Rogasa SL agar for Streptococcus thermophilus, and MRS agar + cysteine (0.5%) for Bifidobacterium. The agar plates were incubated at 30°C and 37°C under aerobic and anaerobic conditions for 48–72 h [30].

Anaerobic jars were used to achieve the anaerobic condition. Viable aerobic and anaerobic counts of the dairy products were determined. The isolation was obtained by morphological characteristics (colony and cell morphology), on the selective media, and biochemical tests used were Gram reaction, catalase test, production of acid from glucose, and growth at different temperatures. Only Gram-positive bacteria with catalase-negative responses were found, and representative isolates were purified by streaking them over the same agar substrate several times. Growth at various temperatures, hetero-, and homofermentative activity (using MRS broth) with inverted Durham tubes in MRS broth was determined for the Gram-positive, catalase-negative rods. Following that, isolates from dairy product samples were selected for further identification based on the previous assays by Harrigan and MacCance [31]. Moreover, all isolates were preserved in brain heart infusion (BHI) with 15% glycerol to detect the carbohydrate fermentation profile of the isolates (Supplementary Materials).

At the end of incubation (37°C for 48 h), the LAB were selected based on their morphology in their selective media such as colony character and biochemical profiles. Moreover, the carbohydrate fermentation profiles of isolates were determined with the micromethod according to Roy and Ward [32]. The details for isolation and identification of the LAB are included in a separate supplementary file.

2.2.3. Enumeration of LAB

Different selective mediums were used to culture the LAB such as MRS, M17, and MRS supplemented with cysteine for Lactobacillus, Lactococcus, and Bifidobacteria, respectively, described by Oxoid Manual [33]. For Bifidobacteria spp., the colony count methodology was used to enumerate presumptive LAB at 30°C and 37°C under both aerobic and anaerobic conditions. The technique of counting was as follows. First, tenfold serial dilution of 9 ml maximum recovery diluents to 1 ml of the sample was done up to 10−8, and accordingly, all of the serially diluted samples were cultured on the MRS agar, M17 agar, Rogasa SL agar, and MRS + 0.5% cysteine for the cultivation of all Lactobacillus, Lactococcus, Lactobacillus, Streptococcus thermophilus, and Bifidobacteria spp., respectively. Then, incubate at 37°C for 48 h, and 10−6 dilutions were selected for colony characterization and counted accordingly. During the test, representative colonies were stored in glycerol culture at 20oC and kept for further investigation.

2.3. Data Analysis

Data from the laboratory were stored and transferred into Microsoft Excel 2016 Spreadsheet analyzed using STATA version 13. The outputs (bacterial isolates) were presented using frequency and percentage.

3. Results

3.1. LAB Isolation

According to this study, identification of the LAB was performed through morphological characteristics. Besides, other biochemical tests and all of the LAB were identified to its generic level in that Lactobacillus spp. were found as whitish, small to large size, the circular margin on MRS media (Figure 1(a)), and Lactococcus spp. were found to be creamy white to yellowish color, small to large in size, and circular margin on M17 media (Figure 1(b)).

Figure 1

(a) Typical isolated colonies of Lactobacillus spp. on MRS media. (b) Lactococcus spp. on M17 media.

Also, Gram stain characteristics of the bacterium revealed that all of the bacteria were Gram-positive, cocci shaped, and have different arrangements: Streptococcus spp. were found in chain arrangement; Leuconostoc spp. were found more in paired arrangement; Pediococcus observed were having tetrads and Bifidobacteria spp. were observed as pleomorphic filamentous-like flagella which were observed under oil immersion. Besides, Bifidobacteria spp. were identified based on the growth morphology on the selective media, growth at a different temperature, gas production from glucose, and carbohydrate fermentation. Our result revealed that a total of 41 bacterial genera were isolated and categorized into five different genera of LAB (Table 1).

Table 1

LAB isolated from various milk and milk products.

Genus	Raw milk	Cheese	Yoghurt	Total
Lactobacillus spp.	5	2	3	10
Lactococcus spp.	4	3	2	9
Streptococcus spp.	4	2	2	8
Leuconostoc spp.	2	2	2	6
Pediococcus spp.	3	0	0	3
Bifidobacteria spp.	3	1	1	5

Moreover, 5 (12.19%) of the bacteria isolated from the raw cow milk were Lactobacillus spp., followed by Lactococcus and Streptococcus spp., but Leuconostoc spp. were found in low percentage. However, in cheese, Lactococcus spp. were found in a higher proportion than the other bacteria, and in the case of yogurt, Lactobacillus spp. were found in a higher proportion (Table 2).

Table 2

Proportions of LAB isolated from milk and milk products.

Sample	Lactobacillus spp.	Lactococcus spp.	Streptococcus spp.	Leuconostoc spp.	Pediococcus spp.	Bifidobacteria spp.	Total
Raw milk	5 (12.19)	4 (9.75%)	4 (9.75%)	2 (4.88%)	3 (7.3%)	3 (7.31%)	21 (51%)
Cheese	2 (4.88)	3 (7.3%)	2 (4.88%)	2 (4.88%)	0 (0%)	1 (2.44%)	10 (24.38%)
Yoghurt	3 (7.31%)	2 (4.88%)	2 (4.88%)	2 (4.88%)	0 (0%)	1 (2.44%)	10 (24.38%)
Total	10 (24.38%)	9 (21.94%)	8 (19.51%)	6 (14.64%)	3 (7.31%)	5 (12.19%)	41

3.2. Enumeration of LAB from Milk and Milk Products

According to the enumeration of LAB, bacterial load was counted from different milk and milk products. Thus, Lactobacillus spp. were found to be high (2.3 × 108 CFU/ml) in yogurt and low (4.5 × 107 CFU/ml) in raw milk; similarly, a higher load of Lactococcus and Bifidobacteria spp. was counted from yogurt (Table 3).

Table 3

Bacterial load in different milk and milk products.

Genus of bacteria	Media	Df	Colony-forming unit (CFU/ml)
			Raw milk	Cheese	Yoghurt
Lactobacillus	MRS	106	4.5 × 107	7.7 × 107	2.3 × 108
Lactococci	M17	106	1.12 × 107	8.01 × 107	2.75 × 109
Bifidobacteria	MRS + cys	106	2.34 × 107	2.84 × 107	1.72 × 107

Df = dilution factor.

4. Discussion

Based on the current study, the majority (24.38%) of the LAB isolated from the different milk and milk products was containing Lactobacillus, Lactococcus (21.94%), Streptococcus (19.51%), Leuconostoc (14.64%), Bifidobacteria (12.19%), and Pediococcus (7.31%) spp. Savadogo et al. [34] and Harun-ur-Rashid et al. [35] also found six genera of LAB from traditional fermented milk, including Leuconostoc, Lactococcus, Lactobacillus, Enterococcus, Streptococcus, and Pediococcus spp. Similarly, Abd El Gawad et al. [36] have also identified five different genera of LAB : Lactobacillus (30%), Leuconostoc (26%), Enterococci (20%), Streptococcus (18%), and Aerococcus (18%) spp. Also, a comparable figure was reported by Abdullah and Osman, [37] where the genus Lactobacillus was dominant in all samples tested (69.23%) followed by the genus Lactococcus (19.23%) and the genus Pediococcus (11.53%).

Moreover, this study has similar findings with El-Shafei et al. [38] and Abd El Gawad et al. [36] who identified a higher proportion of Lactobacillus spp. from milk products. Lactobacillus spp. can survive in a highly acidic environment with a pH of 4 to 5 or even lower, and it is Lactobacillus that is responsible for the final stages of fermentation in the goods as a result of these traits. This also demonstrated that Lactobacillus spp. survive in low pH environments [39].

Based on the current study of LAB enumeration from different milk and milk products, it revealed that Lactobacillus spp. were presumptively counted as 4.5 × 107, 7.7 × 107, and 2.3 × 108 CFU/ml from raw cow's milk, cheese, and yogurt, respectively. However, a load of Lactococcus spp. was counted as 1.12 × 107, 8.01 × 107, and 2.75 × 109 CFU/ml from raw cow's milk, cheese, and yogurt, respectively. In the same way, Bifidobacteria spp. were counted as 2.34 × 107, 2.84 × 107, and 1.72 × 108 CFU/ml from the mentioned milk and milk products. These findings were in accordance with the earlier report of Khedid et al. [40] from Morocco, who reported that Lactobacillus spp. counting load were 7.5 × 106 CFU/ml, from camel milk. However, a Lactococcus spp. counting load in the present study was higher than the report of Khedid et al. [40], which is 7.6 × 106 CFU/ml.

Moreover, the finding of Savadogo et al. [34] revealed that LAB were found in large numbers in all samples from various governorates, ranging from 1.02 × 10−2 to 9.89 × 10−2 CFUg−1. All these populations rose from around 102 CFUg−1 to 104 CFUg−1. These findings might be due to the employment of primitive manufacturing procedures in the production of many traditional foods, with the primary risk factors being the use of contaminated raw materials that have not been pasteurized, as well as insufficient fermentation and storage conditions. Despite the fact that the LAB group dominated the microbiota, a final population of roughly 106 to 107 CFUg−1 was achieved in all samples.

The most frequent microbial group detected in milk was LAB, which is relevant given its role in fermentation and the generation of lactic acid and antibacterial compounds, as well as its potential use as a milk starter in standardized production. Gram-stain reaction, cell shape, cellular organization, generation of acid from glucose and lactose, production of gas from glucose, and catalase activity were used to isolate, identify, and classify LAB strains [36, 41].

El-Shafei et al. [38] also reported the preponderance of Lactobacillus spp. was higher among isolated LAB because milk and milk products constitute a varied mixture of diverse microorganisms. Furthermore, cow milk, which is widely used to make fermented milk, may promote the growth of these species. Lactobacillus spp. can survive in a highly acidic environment with a pH of 4 to 5 or even lower, and it is Lactobacillus spp. that is responsible for the final stages of fermentation in products because of these qualities. This also demonstrated that Lactobacillus spp. survive in low pH environments [39].

5. Conclusion

The current study revealed that most of the isolated LAB species were grouped into five genera including Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus, and Bifidobacteria spp. and Lactobacillus spp. were the most common LAB isolated from various milk and milk products among the identified genera. Moreover, Lactobacillus spp. were the commonly isolated bacterial species from raw cow milk and yogurt; however, Lactococcus and Bifidobacteria spp. were found in a higher proportion in cheese. Based on the enumeration of LAB, Lactobacillus, Lactococcus, and Bifidobacteria spp. were counted in higher proportion from yogurt and raw milk. Thus, further study on the identification and characterization of LAB to strain level should be done. The health and economic benefits of the identified LAB should be given more attention in order to improve community health. The possible application of these LAB as probiotics should be further tested on different species of experimental animals.