Received Date: 01/03/2016; Accepted Date: 20/06/2016; Published Date: 01/07/2016
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Lactic Acid Bacteria, Yoghurt, Total Viable count, Lactobacillus, Streptococcus.
Milk contains high nutritive food value for the new borne mammal and human beings. It is also an ideal growth medium for the microbial proliferation. Fermented milk and milk based products at different formulation in different name are popular throughout the world for their taste as well as health benefits. Popular fermented milk products named, Tarag - China and Mongolia , Masai - East African Rift Valley between southern Kenya and northern Tanzania , Gariss – Sudan , White cheese and Rob – Sudan , Chal – Iran , Katyk – Bulgaria , Laban - Lebanon and some Arab countries , Kefir – Caucasian countries, Koumiss – Russia and Siberia, Mazun – Armenia, Leben – Egypt, Gioddu – Italy, Buttermilk – Western Europe, Villi and langfil – Scandinavian countries , cheese and yoghurts in many countries are produced by the activity of LAB. The nature of the fermented milk products depends on the type and temperature of the milk, pre-treatment steps, reaction conditions, technological approaches, microbial compositions or the added starter culture .
LAB are Gram positive, non-spore former, usually nonmotile, nonacid fast, nonrespiring rods or coccobacilli with frequently in chain, devoid of cytochrome, catalase negative. They grow well under anaerobic conditions but may grow in microaerophilic as well as aerobic conditions. They exhibit optimum growth at slightly lower acidic condition (pH 5.5 – 6.0) while growth is often restricted at neutral or somewhat alkaline condition (pH above 7.0 to 7.5). They are strictly fermentative, with lactic acid as the major end product during sugar fermentation [9,10]. LAB can be classified on the basis of their morphology (cocci or rods, tetrad formation), mode of glucose fermentation, growth at different temperatures and salt concentrations, and configuration of the lactic acid production (D, L or both) . Besides, fatty acid composition and motility are also considered as the identifying tools. They have two different metabolic pathways for hexose fermentation. In homofermentative pathway, lactic acid (more than 85%) is major end product whereas in heterofermentative pathway lactic acid, ethanol/acetone and CO2 are the terminal products [9,10].
Lactic acid bacteria (LAB) are widely distributed in nature, occur as natural adventitious contaminants in raw milk, yoghurt, etc. and sometimes with the coexistence of yeasts, moulds and some other pathogenic microorganisms. LAB as indigenous flora in raw milk acidify the milk very slowly due to their low numbers. Hence most of the dairy industries now a days use LAB as starter culture for the manufacture of fermented products including milk products such as yoghurt, cheese; meat products, bakery products, wine, and vegetables [8,10]. Besides, alive quantified LAB starter culture in some instances resolves the danger of being lost off the raw milk flora in the newer fermentation technology as well as giving conformance of uniformity , contribute to taste, flavor, aroma, viscosity and texture development of the product [11,12], alleviation of lactose intolerance [13,14] as well as preservative activity . At present, LAB are in the focus of intensive research for their ability to promoting probiotic properties and exerting antagonistic effect on the gastro enteric pathogens - Clostridium difficle, Campylobacter jejuni, Helicobacter pylori and rotavirus , antitumoral activity , reduction of serum cholesterol, stimulation of the immune system  and stabilization of the gut microflora .
It is of immense importance to know the total numbers and composition of LAB in raw milk and fermented milk products, to explore the promising and beneficial strains and their usage as starter culture in fermentation technology for the newer and quality product innovation and novel applications as well. Conventional plate count methods using selective media are the gold standard for the determination of total LAB count as well as sophisticated techniques such as real-time quantitative PCR  is also employing now a day. Till date a lot of experimental procedures have been developed for the identification of LAB. Rapid identification through biochemical tests miniature - API 38 CHL strips, API 20 STREP and API 50 CHS [3,21-23]; Random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR)  and RAPD-PCR followed by 16S rDNA gene sequencing , 16S rRNA gene sequences analysis and PCR-DGGE [1,25] are the updated biochemical and molecular techniques now a days employed for the rapid as well as huge number of LAB identification. On the contrary, in spite of having few limitations, the conventional phenotypic expression (cultural and microscopy), biochemical and physiological methods are still being considered the principal methods of LAB identification [2,21,23,26] with limited resources and simple instrumentations. This conventional cultural technique mainly employs selective media for the genus specific LAB, in some cases in combination with specific molecular techniques. This study has been conducted to determine the total load of LAB, isolation and taxonomic determination of the LAB from variety of samples including the fresh raw cow milk, pasteurized milk as well as local and commercially produced yoghurt samples applying the widely accepted cultural techniques using selective agar media, biochemical and physiological tests. This study was further extended to select the best organoleptic characteristic yoghurt producer in terms of color, consistency, quantity of whey, and presence or absence or gas in the yoghurt; to screen the effective lactate producer.
Fourteen fresh cow milk samples were collected into the sterilized screw cap conical flask from the nearby village of the University of Chittagong, Bangladesh and transferred to the Microbiology Laboratory within 30 mins of sampling. On the other hand, one commercially available pasteurized liquid milk and four yoghurt samples (local and commercially manufactured) were collected from different shops of the Chittagong city. All the sample were analyzed on the same day of sampling and kept at refrigerated (2-8°C) condition until microbiological analysis.
Enumeration, isolation, storage and maintenance of LAB: 10 mL of sample was homogenized with 90 mL of 0.85% (w/v) sterile sodium chloride solution to make an initial dilution (10−1). Serial dilutions upto 10−6 were made for each sample. 1 mL sample from each of the corresponding dilutions (10−5 and 10−6) were inoculated into various selective media such as MRS agar (Oxoid, UK), Rogosa agar (Oxoid, UK), and Yeast Glucose Lemco Agar (YGLA). MRS agar (pH 6.2-6.6) and Rogosa agar (pH 5.2- 5.6) for total LAB enumeration as well as lactobacilli isolation  while YGLA (pH 7.0) for the isolation of streptococci  of LAB origin. Inoculated plates were then incubated at 37°C for 48-72 hours both at aerobic and anaerobic conditions. After incubation, plates with 30–300 colonies were enumerated and the calculated results were expressed as colony forming unit (cfu) per gm or mL by multiplying the average number of colonies with the reciprocal of dilution factor . Colonies with distinct morphologies such as form, elevation, margin, surface, color and consistency were selected randomly and isolated . Presumptive lactobacilli colonies were purified by inoculating into MRS and Rogosa broth followed by incubation at 37°C for 24 hours as well as streaking on to Rogosa agar and MRS agar. Similar steps and conditions were followed for the purification of the streptococci using Yeast Glucose Lemco Broth (YGLB) and YGLA. Lactobacilli were streaked on MRS agar slant and streptococci on YGLA slant and kept at 4°C. Cultures were maintained by streaking fortnightly on the same media. Prior use, lactobacilli were activated in MRS broth and streptococci in YGLB at 37°C for 24 hours.
Selection of the isolates: Isolated 191 strains were evaluated and categorized into 13 groups on the basis of their colony morphology in media plates and slants, Gram staining, cell morphology, catalase activity, and motility test (summarized in Table 1). Among them, seven were selected and isolated from MRS media for Lactobacillus spp. and hence marked as M18A, M18B, M18C, M16C, M12A, M17B, and M14B. The remaining six colonies were isolated from YGLA for Streptococcus spp. and marked Y1A, Y11A, Y8, Y19B, Y-YL and Y-GW. These 13 representative isolates were identified upto species level by the traditional phenotypic methods.
|Group||Colony morphology||Slant characteristics||Microscopic observation||Staining Properties||Catalase test||Motility test|
|Form||Color||Elevation||Margin||Surface||Cell form and arrangement||Gram||Acid fast||Capsule||Spore|
|1||Circular||White||Pulvinate||Entire||Smooth||Echinulate||Rod, single & in chain||+||-||-||-||-||-|
|2||Circular||White||Flat||Undulate||Rough||Spreading||Rods with rounded end, single and in short chain||+||-||-||-||-||-|
|3||Circular||White||Raised||Entire||Smooth||Filiform||Rods with rounded end, single, and short to long chain||+||-||-||-||-||-|
|4||Circular||White||Light Convex||Entire||Smooth||Beaded||Cocco bacilli, sinlge, pair or in short chain||+||-||-||-||-||-|
|5||Circular||Gray||Contoured||Undulate||Wrinkled||Echinulate||Rounded end rod,ÃÂ single, pair & in short-long chain||+||-||-||-||-||-|
|6||Circular||White||Convex||Entire||Rough||Echinulate- spreading||Rounded end rods, single, and in short chain||+||-||-||-||-||-|
|7||Circular||White||Raised||Entire||Smooth||Spreading||Rounded end rods Single, rarely in pair & in short chain||+||-||-||-||-||-|
|8||Circular||White||Convex||Entire||Smooth||Filiform||Cocci, single, mostly in pairs or short chains||+||-||-||-||-||-|
|9||Circular||White||Convex||Entire||Smooth||Filiform||Cocci, single, mostly in pair or short chain||+||-||-||-||-||-|
|10||Circular||White||Light Convex||Entire||Smooth||Filiform||Cocci, single, pair & in long chain||+||-||-||-||-||-|
|11||Circular||White||Flat||Undulate||Smooth||Echinulate||Cocci, single, mostly in pair & occasionally in short chain||+||-||-||-||-||-|
|12||Circular||Gray||Convex||Entire||Smooth||Echinulate||Cocci, single, pair, medium-long chain||+||-||-||-||-||-|
|13||Circular||White||Convex||Entire||Smooth||Echinulate||Cocci, single, pair & in chain||+||-||-||-||-||-|
Table 1: Cultural, morphological and biochemical characteristics of bacterial isolates.
Characterization of the isolates
Gram-positive, catalase negative rods were characterized according to the methods and criteria of Bergey’s Manual of Determinative Bacteriology, 8th Edition , Axelsson , Kandler and Weiss , Schleifer and Kilpper-Bälz , and Bottazzi . Streptococcus were characterized according to the criteria used by Bergey’s Manual of Determinative Bacteriology, 8th Edition .
All strains were subjected to the common biochemical tests such as Voges-ProsKauer (VP), Methyl Red (MR) reaction test, starch hydrolysis, deep glucose agar test, CO2 gas from glucose in Gibson’s semisolid tomato juice medium, gas from citrate in semisolid citrated milk agar, NH3 from arginine , and gelatin liquefaction. Carbohydrates (glucose, maltose, lactose, arabinose, rhamnose, raffinose, dextrose, sucrose, inulin, xylose, galactose, fructose, mannitol, starch) fermentation test was performed in fermentation broth medium containing 1% (w/v) carbohydrate, bromothymol blue 0.1% as pH indicator, and keeping Durhum’s tube at inverted position. Results were recorded after 48 hours of incubation at 37°C.
Presumptive lactobacilli inoculated in MRS broth and streptococci in YGLB media were further characterized by conducting the following physiochemical tests. Growth response at different temperatures 5°C, 10°C, 27°C, 37°C and 45°C for 24-48 hours, pH 4.5, 6.5, and 7.2 at 37°C for 48 hours, tolerance to NaCl by growth in MRS broth containing NaCl at concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 6.5%, 7% at 37°C for 48 hours, heat resistance by keeping the inoculated media in the boiling water bath at 55°C and 60°C for 30 minutes were evaluated for all of the selected 13 LAB isolates [28,36,37].
Screening of the efficient LAB strain
Potential LAB isolate in terms of yoghurt formation and acid production were studied. 10% reconstituted skimmed milk was inoculated with 0.2 mL (2%) starter culture suspension of Lactobacillus spp. and Streptococcus spp. separately and in different combinations. Inoculated tubes were incubated at 45°C. Characteristics of the yoghurts formed by the individual LAB and mixed inoculums were studied by visual observations. To get the potential lactic acid producer, 100 ml of sterile skimmed milk medium was inoculated with 1mL starter culture of the identified individual species of Lactobacillus and Streptococcus and incubated at 30°C in a water bath for 6 hours and 72 hours respectively. Total acidity was determined by titration of 10 mL of incubated sample cultures titrating with 0.1N NaOH using 0.5% phenolphthalein as indicator. Total acidity was measured after 6 hours and 72 hours of incubation period respectively and expressed in percentage as well as milligram equivalent to lactic acid [23,35].
Enumeration of LAB
LAB were enumerated from the fresh raw milk, commercial liquid milk and yoghurt samples. Table 2 shows the total viable aerobic and anaerobic count of milk and yoghurt samples. Yoghurt samples showed higher LAB count than the freshly drawn raw milks both at aerobic and anaerobic conditions. Aerobic bacterial count in milk sample ranges from 1.1 × 105 to 7.2 × 106 while anaerobic bacterial count ranges from 6.1 × 105 to 5.3 × 107. In yoghurt samples, total anaerobic bacterial count (2.2 × 107 - 5.4 × 108) is higher than that of total aerobic bacterial count (1.0 × 106 - 5.6 × 107). It has been found that both in milk and yoghurt samples, anaerobic bacterial count (6.1 × 105 to 5.4 × 108 cfu/mL) are greater than that of aerobic count (1.1 × 105 to 5.6 × 107 cfu/mL). These findings correlate with previously examined few studies. For example, Kacem et al. provided that total LAB count in milk and yoghurt shows higher than the cow’s, goat’s and sheep’s milk samples in Western Algeria . Similar LAB count were also reported for the traditional koopeh cheese in Iran , camel’s milk , yoghurt , and goat’s yoghurt .
|No. of Sample||Name and source of Sample||Total LAB count|
|1||Cow milk, CU campus||2.5 x 106||1.2 x 107|
|2||Cow milk, CU campus||1.8 x 106||5.8 x 106|
|3||Cow milk, CU campus||3.0 x 106||7.0 x 106|
|4||Cow milk, CU campus||2.8 x 106||6.8 x 106|
|5||Cow milk, CU campus||2.1 x 106||5.1 x 106|
|6||Pasteurized liquid milk, commercial||1.1 x 106||1.1 x 107|
|7||Cow milk, CU campus||2.1 x 105||1.2 x 106|
|8||Cow milk, CU campus||2.4 x 105||6.2 x 106|
|9||Cow milk, CU campus||1.7 x 105||8.7 x 105|
|10||Cow milk, CU campus||1.1 x 105||6.1 x 105|
|11||Cow milk, Jobra Village||3.5 x 106||1.9 x 107|
|12||Cow milk, Jobra Village||5.3 x 105||5.3 x 107|
|13||Cow milk, Jobra Village||7.2 x 106||5.0 x 107|
|14||Cow milk, CU campus||2.4 x 106||6.9 x 106|
|15||Cow milk, CU campus||8.0 x 105||7.7 x 106|
|16||Yoghurt, commercial||7.2 x 106||3.8 x 107|
|17||Yoghurt, CU campus||1.0 x 106||2.2 x 107|
|18||Yoghurt, Halisahar, Chittagong||5.6 x 107||2.5 x 108|
|19||Yoghurt, CU campus||1.5 x 107||5.4 x 108|
Table 2: Total LAB count at aerobic and anaerobic condition.
Identification of the isolates
The 13 selected representative isolates were chosen among 191 isolates on the basis of their colony characteristics in selective media plates, various staining properties, catalase reactions, and motility (Table 1). These 13 isolates were categorized into two main genus Lactobacillus and Streptococcus using the selective media. Isolates were then studied extensively for various biochemical and physiochemical studies which are shown in Table 3. All of the Lactobacillus species shared the common staining - Gram negative, noncapsulated, nonsporing, nonacid fast and microscopic features - short rod to coccobacilli with single, pair and in chain arrangement with different colony morphology in MRS or Rogosa agar and in MRS slant. Besides, they also showed non-motility, negative result in catalase reaction, MR and VP tests, unable to hydrolyze citrate, starch, and liquefy gelatin. Isolated and preliminary identified Lactobacillus strains were primarily differentiated in some of the biochemical tests, their physiological properties and patterns of carbohydrate fermentation. However, when the fermentation patterns of these isolates were compared with the identification key in Bergey’s Manual of Determinative Bacteriology, 8th edition few deviations were noted.
|VP test||CO2 in Gibson medium||Hydrolysis of||Gelatin Lique-faction||Growth at different pH||Growth at different temperature (°C)||Growth at different NaCl
|Glucose Phosphate Broth||Skim milk media||Cit-rate||Argi-nine||Starch||4.5||6.5||7.2||4||10||27||37||45||1||2||3||4||5||6||6.5||7.0||55||60|
Table 3: Biochemical and physiological characteristics of the LAB isolates.
Strains M18A and M18B showed common biochemical reactions such as NH3 from arginine, acid and gas from glucose fermentation, and physiological properties with few exceptions - M18B produced CO2 from Gibson’s semisolid media, growth at 45°C while M18A were unable (Table 3). They reflected identical fermentation pattern with few variations as per Bergey’s Manual of Determinative Bacteriology, 8th edition. On the other hand, M18C and M16C both produced CO2 in Gibson’s semisolid media with no citrate utilization, NH3 from arginine, liquefaction of gelation. Both of the strain could not tolerate high salt concentration (6.0%, 6.5% and 7%), low temperatures (4°C and 10°C). M16C produced scanty growth at 45°C while M18C could not grow at all (Table 3). Both of the strains produced almost typical carbohydrate fermentation results in respect to Bergey’s Manual of Determinative Bacteriology. Isolates M12A, M17B and M14B produced similar biochemical reactions except NH3 from arginine by M17B strain, showed optimum growth at 37°C - 45°C with capability to grow at 6%, 6.5% and 7% of NaCl concentrations, heat resistance at 55°C and 60°C for 30 minutes. These three isolates did not produce gas from glucose fermentation and showed characteristic carbohydrate fermentation pattern with few exceptions. Hence, according to the Bergey’s Manual of Determinative Bacteriology, 8th Edition , Axelsson , Kandler and Weiss , Schleifer and Kilpper-Bälz , and Bottazzi  strain M18A is considered as Lactobacillus cellobiosus, M18B - Lactobacillus delbrueckii, M18C - Lactobacillus hilgardii, M16C - Lactobacillus coryniformis subsp. coryniformis, M12A - Lactobacillus salivarius, M17B - Lactobacillus leichmannii, M14B - Lactobacillus plantarum
Strain Y1A was isolated using YGLA medium. Cells of this strain were G+ve cocci, mostly in pairs or short chains, utilized the citrate and produced NH3 from arginine, showed optimum growth at 37°C and 45°C with little growth at 10°C, heat resistance at 60°C for 30 minutes. They produced both acid and gas from glucose fermentation and showed similar sugar fermentation pattern like Streptococcus faecalis mentioned in Bergey’s manual. Strain Y11A and Y-GW shared the almost common biochemical features including CO2 production from Gibson semisolid medium, citrate utilization and NH3 from arginine, growth at low temp. 10°C with optimum growth at 37°C. Strain Y-GW showed luxuriant growth at 45°C strain while Y11a failed to grow. Both strains produced acid and gas from glucose fermentation, as well as reflected similar carbohydrate fermentation pattern with exception in arabinose and inulin fermentation. According to Bergey’s Manual of Determinative Bacteriology (8th edition)  Y11A is considered as Streptococcus lactis and Y-GW resembled to Streptococcus uberis. Isolates Y8 and Y19B produced luxuriant growth at 27°C - 45°C, pH (4.5 to 7.2) while Y8 showed positive growth at 10°C and Y19B produced no growth. Both of them showed heat resistance at 55°C and 60°C for 30 minutes, gelatin liquefaction, difference in citrate utilization and arginine hydrolysis . Strain Y19B could grow at 6.5% and 7% NaCl but strain Y8 could not. These two strains were different in carbohydrate fermentation (Table 3). According to the Bergey’s Manual , strain Y8 is considered as Streptococcus thermophiles and Y19B as Streptococcus faecium. Cells of the strain, Y-YL were cocci, pair and medium to long chain in arrangement, liquefied the gelatin and utilized arginine with NH3 production. They could not grow at low temperatures (10°C and 27°C), high salt concentrations (6.5% and 7.0%), and no heat resistance at 55°C and 60°C for 30 minutes. Acid but no gas was generated from glucose fermentation with identical carbohydrate fermentation like Streptococcus sanguis according to Bergey’s Manual of Determinative Bacteriology, 8th Edition .
Harun-ur-Rashid et al.  conducted a study in Bangladesh on the identification of the dominant LAB species from the traditional fermented milk dahi. Their findings are very relevant with our current study. Besides, identified same LAB species have been reported in other fermented products such bushera – a Ugandan traditional fermented beverages . L. plantarum identified from sorghum powder, its corresponding fermented and cooked fermented samples , cereal fermented products – such as maize-derived products Nigerian ogi  and vegetable fermentations . The prevalence of S. lactis is greater in the raw milk due to the free amino acids and the peptides initially present in the milk  but they can also be isolated from other sources . S. lactis is able to produce acid from casein, hence it can be used as the starter culture for making different products including cheese . L. delbrueckii is one best known starter for fermented dairy products because of the capability of producing large amounts of acid in milk, flavor compounds, folic acid and EPS which has effect on the rheological properties of yoghurt .
Determination of the efficient strain
Efficient LAB species was screened considering the parameters: formation and color of yoghurt, amount of whey and presence of gas. L. cellobiosus was the highest amount of whey producer (Table 4). Yoghurt production in different combinations were also tried and summarized in Table 5.
Table 4: Carbohydrates fermentation of LAB.
|Isolate No||Color of Yoghurt||Yoghurt Formation||Amount
|L. coryniformissubsp. Coryniformis||White||+||++||-|
|S. faecium,||Light yellowish||+||+++||-|
|S. sanguis||Cream-light yellowish||+||++||-|
|L. cellobiosus + L. delbrueckii||Cream||+||++||-|
|L. delbrueckii + L. salivarius||Cream||+||++||-|
|L. delbrueckii + L. leichmannii||Cream||+||+++||-|
|L. coryniformissubsp. coryniformis + L. leichmannii||White||+||+||-|
|S. lactis + S. uberis||Cream||+||+++||++|
|All bacteria + S. lactis||White||+||+||-|
|All bacteria + S. uberis||Cream||+||+++++||+|
|+ S. lactis + S. uberis||Cream||+||++++||++|
Table 5: Screening of the efficient LAB isolate.
Among the identified Lactobacillus species, none of the potential species was identified in terms of acid production both in 6 hours and 72 hours of incubation period. L. cellobiosus was found to be a little bit higher acid producer than the others. S. lactis and S. uberis were found as the highest acid producer both after 6 hours (0.14 and 0.18) and 72 hours (0.46 and 0.47) of incubation period (Figure 1). From the following data, it can be summarized that after 6 hours of fermentation period almost all the identified species of the two genus Lactobacillus and Streptococcus produced near about the equal quantity of acid except S. uberis (0.18). Interestingly, S. lactis and S. uberis species kept continue in acid production at longer incubation period. The total acidity equivalent to lactate (mg) for S. uberis was the highest 370 mg while S. lactis was the immediate highest lactic acid producer (360 mg) after 72 hours of incubation period at 35°C (Figure 2). Considering the total acidity, conversion of total acidity in equivalent to lactic acid, it is proved S. lactis and S. uberis are the homofermentative acid producer. Result from the combined activity of the LAB isolates in acid producing capability, again reflected that S. lactis and S. uberis in combination and mixed with all other lactobacilli and the remaining streptococci produced a little higher lactate than the individual species (Figures 1 and 3).
The results obtained in this study revealed the presence of a wide variety of LAB from milk and yoghurt samples. Some of the isolated and identified LAB species show outstanding performances in lactic acid production. The results of this study have indicated that several different species of LAB can be used as the starter culture for the production of yoghurt. Hence, the selection and potential use of appropriate beneficial strain or mixture of strains from the identified isolates as the starter organism for yoghurt and lactate production need more studies about the optimization process among the temperature, time, ingredient compositions as well as inoculum concentrations. Extensive study must be done to improve the technological properties of yoghurt as well as for the development of small-scale dairy industries in Bangladesh.