Isolation and biochemical characterization of δ-aminolevulinic acid dehydratase from Streptomyces yokosukanensis ATCC 25520

Isolation and biochemical characterization of δ-aminolevulinic acid dehydratase from Streptomyces yokosukanensis ATCC 25520

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   Research & Reviews: A Journal of Dairy Science and Technology  Volume 1, Issue 2, August 2012, Pages  __________________________________________________________________________________________  ISSN: 2319-3409© STM Journals 2012. All Rights Reserved Page 1 Isolation and Biochemical Characterization of  Lactobacillus  species Isolated from Dahi  Aarti Bhardwaj 1  , Monica Puniya  2  , K. P. S. Sangu 1  , Sanjay Kumar  3  , Tejpal Dhewa  4 *   1 Depatment of Dairy Science and Technology, Janta Vedic College, Baraut-250611, Uttar Pradesh, India 2 Dairy Cattle Nutrition Division, 3 Dairy Microbiology Division, National Dairy Research Institute, Karnal-132001, Haryana, India 4 Bhaskaracharya College of Applied Sciences (University of Delhi), New Delhi-110075, India *  Author for Correspondence  Email:, Tel: + 91-8836325454 1.   INTRODUCTION Generally, the healthiness of food has been linked to a nutritionally rich diet recommended by specialists and the role of it in totality has been emphasized instead of emphasizing on individual components. During the past few decades, the lifestyle in the developed and developing countries has been changing fast with regard to living standards, diet, hygiene and usage of antibiotics. The prevalence of chronic diseases like allergies and gut-associated disorders (e.g., Crohn’s disease, ulcerative colitis, and inflammatory bowel disease) are of rising importance in the world nowadays. Hence, the balance in microbiota of gut is considered to provide the colonization resistance against infectious agents and promote anti-allergic processes, stimulate immune system and reduce hypersensitivity   [1  –  4].   Milk, very first food of mammals including humans, is surrounded with emotional and cultural importance in the society. Men have be en habituated to think of milk as nature’s most perfect food for them. Therefore, milk and dairy products have long been recognized as an important constituent of balanced diet for human beings as these products provide a wide range of essential nutrients. ABSTRACT  Dahi (curd) is a fermented milk product, most commonly used by Indian population. Trials are in process to establish dahi as a source of health beneficial organisms (probiotics). Hence, the present study is directed towards the study of prevalence of lactobacillus species in Dahi. A total of 40 samples of dahi were collected for the isolation of Lactobacilli using Lactobacillus selection MRS agar. Thirty-eight colonies were randomly picked based on colonial morphology. All the isolates were subjected to cell morphology, physiology and an array of biochemical characterization. The isolates showed different growth patterns at different temperatures (15 ° C and 45 ° C), oxygen and at different concentrations of  NaCl (2.0, 4.0 and 6.5%). On the basis of physiological tests and sugar utilization pattern, all the seventy-eight isolates were confirmed to the different species of Lactobacillus: Lactobacillus casei (24.35%), Lactobacillus brevis (3.84%), Lactobacillus fermentum (6.41%), Lactobacillus plantarum (7.69%), Lactobacillus helveticus (5.12%), Lactobacillus rhamnosus (6.41%), Lactobacillus viridiscence (5.12%), Lactobacillus lactis (3.84%), Lactobacillus acidophilus (37.17%). Among isolates, L. acidophilus was found to be prevalent in dahi.  Kewords:   Dahi, robiotics, biochemical characteriation, MRS aar      Research & Reviews: A Journal of Dairy Science and Technology  Volume 2, Issue 2, August 2012, Pages  __________________________________________________________________________________________  ISSN: 2319-3409© STM Journals 2012. All Rights Reserved Page 2 In addition, the evidence of health benefits of milk associated with the presence of specific components or beneficial bacteria is gaining scientific credibility at a rapid pace. Therefore, the best known examples of functional foods (i.e., that benefit the consumers beyond nutrition) are fermented milks containing friendly bacteria and are called probiotics   [5, 6].   Milk itself is much more than the total sum of its nutrients as it is also a natural source of biologically active compounds that exert potential impact on human health. Probiotic group of microorganisms that are present in fermented milk products (i.e., dahi, yoghurt, cheese, etc.) beneficially affect the host by improving the intestinal microbial balance. The effect of probiotics includes alleviation of intestinal disorders (i.e., lactose intolerance, acute gastroenteritis due to enteric pathogens, constipation, and inflammatory bowel disease) and a number of food allergies. The therapeutic value of fermented dairy products has been utilized, dating back to Biblical days. These foods confer the added health benefits or disease-prevention characteristics beyond the basic nutrition of the food [7].   About 65% of the total functional foods market is covered with dairy probiotic products [8]. Although the primary aim of food is to provide enough nutrients to fulfil the body requirements, yet various functions of the host body are modulated by the diet consumed. Hence, in order to compensate for deficiency of certain nutrients in the diet due to changes in nutritional habits of industrialized nations, the concept of functional foods has been developed for the consumers. The microbial ecology in the gastrointestinal tract influences many functions in our body (i.e., digestion, absorption of nutrients, detoxification and ultimately the functioning of immune system). All these aspects make the gut a target organ for development of functional foods that can help in maintaining the relative balance of microorganisms in the gastrointestinal tract. The establishment of microbial balance by shifting it towards a beneficial one with the help of specific dietary components (i.e., probiotics and prebiotics) has opened the gateway for the development of functional foods ensuring more benefits to the host ’s  health   [9]. Probiotics are defined as live microbes which transit the gastro-intestinal tract and in doing so, benefit the health of the consumer [10, 11].   Probiotic microorganisms are found in many food products, especially in the fermented foods. Therefore, the probiotic lactic acid bacteria can be isolated from the fermented milk products like acidophilus milk, yoghurt and dahi. Dahi, naturally fermented milk by lactic cultures, is widely consumed throughout South Asia, especially in the Indian subcontinent. The micro-flora of dahi varies from one geographical region to the other and also with seasonal variations of the year. Apart from the starter lactic cultures, dahi can also have some probiotics like  Lactobacillus acidophilus,  Lactobacillus casei,  Lactobacillus    Research & Reviews: A Journal of Dairy Science and Technology  Volume 2, Issue 2, August 2012, Pages  __________________________________________________________________________________________  ISSN: 2319-3409© STM Journals 2012. All Rights Reserved Page 3 bulgaricus, Streptococcus thermophilus,  etc. Therefore, dahi can be used as a source for isolation of probiotic bacteria [5, 6] and also a product of immense importance for human consumption. In view of the above facts, the present study   has been designed to isolate and identify a  Lactobacillus  species from dahi samples collected from the local market. 2.   MATERIALS AND METHODS 2.1. Isolation of Lactic Acid Bacteria Dahi samples (40) for the isolation of lactic acid bacteria were collected from different rural and urban locations in order to get a wider diversity of lactic acid bacterial strains. Each sample was taken in a sterile container separately and placed in a polyethylene bag during transportation to the laboratory employing standard conditions for sample collection. One gram of curd sample was immediately processed under aseptic conditions by suspending in 9 mL of normal saline (0.85%) and was vortexed for proper mixing. One milliliter of each well-mixed dahi sample was enriched in 9 mL of sterile  Lactobacillus  selection MRS broth for 24 h at 37 °C. Before inoculation of sample, the pH of MRS broth was adjusted to 6.5 ± 0.2. The enriched samples were streaked on the Petri plates containing  Lactobacillus  selection MRS agar with the help of calibrated inoculating loop and incubated aerobically at 37 °C for 48 h and observed for the growth of colonies. 2.2. Identification of Selected  Lactobacilli   The identification and further characterization of  Lactobacilli  isolates grown on MRS agar was done mainly with the help of the following tests: microscopic examination (Gram staining), catalase test, growth at different temperatures 10 + 1 ºC and 42 + 1 ºC), growth under aerobic and anaerobic conditions, growth at different NaCl concentration, fermentation of different carbohydrates, etc. a)   Microscopic Examination: The purity morphological identification of the isolates as  Lactobacilli  was confirmed microscopically by performing Gram staining, for which single colony of each isolate was picked up and stained as per the standard protocol and viewed under oil immersion for similar type of cells. b)   Micrometry: Each isolate after Gram staining was subjected to microscopic measurements employing ocular and stage micrometer. To determine the size of  Lactobacilli  isolates, prepared slides were observed under oil-immersion objective and number of ocular divisions occupied by each bacillus was recorded and interpreted as per the above formula.  2.2.1. Physiological Characterization of  Isolates After confirming the purity of culture, each isolate was further assessed for growth at two different temperatures. a)   Growth of isolates at (10 °C and 42 °C): The isolates were tested for their ability to   Research & Reviews: A Journal of Dairy Science and Technology  Volume 2, Issue 2, August 2012, Pages  __________________________________________________________________________________________  ISSN: 2319-3409© STM Journals 2012. All Rights Reserved Page 4 grow in MRS broth at 10 + 1 °C for 7 days and 42 °C by incubating for 24  –  48 h. For this, 10 mL of MRS broth tubes were inoculated @ 1% of  Lactobacilli  cultures. The development of turbidity in culture tubes was recorded as the ability of isolates to grow at 10 °C and 42 °C and results were noted as positive or negative. b)   Oxygen requirement of the isolates: All the isolates were inoculated in MRS broth and were kept differently under oxygenated condition; in dessicator with burned candle (for micro-aerophilic condition) and in anaerobic jar with gas pack at 37 °C for 24  –  48 h to determine the impact of oxygen on the growth of the  Lactobacilli  isolates and results were noted as positive or negative.  2.2.2. Effect of NaCl Concentrations on Growth of Isolates The isolates were inoculated in MRS broth having different NaCl concentration (2.0%, 4.0% and 6.5%) and incubated at 37 °C for 24  –  48 h. The culture tubes were observed for the presence or absence of growth.  2.2.3. Biochemical Characterization of  Isolates a)   Catalase Test: The test was performed in order to determine the ability of the isolated cultures to degrade the hydrogen peroxide by producing the enzyme catalase. The test was carried out as the slide method, using an inoculating needle. For this, culture from a typical colony was placed onto a clean grease-free glass slide and drop of 3% hydrogen peroxide solution was added onto the culture and closely observed for the evolution of bubbles. The production of bubbles indicated positive catalase reaction and was recorded accordingly for the presence or absence of enzyme. b)   Gas from Glucose:   Sterile test tubes of 10 mL glucose broth containing Durham’s tube (inverted and dipped), were inoculated with  Lactobacilli  cultures at the @1% and incubated at 37 °C for 24  –  48 h. Gas production that appeared in the form of a hollo w space in Durham’s tube was recorded as a positive result. c)   Arginine Hydrolysis: Autoclaved arginine hydrolysis broth tubes were inoculated with the isolated cultures (1%) and incubated at 37 °C for 48 h. After incubation, 3  –  5 drops of the Nessler's reagent were added to each test tube and observed for the change in color (yellow to orange color), indicating a positive result for arginine hydrolysis. d)   Aesculin Hydrolysis: The isolates were also assessed for their ability to hydrolyze glycoside aesculin to aesculetin and glucose. For this, bile aesculin agar plates were streaked with the isolated cultures and incubated at 37 °C for 24  –  48 h. After incubation, the plates were examined for the presence of a dark brown to black halo around the bacterial growth, showing a positive result for aesculin hydrolysis.   Research & Reviews: A Journal of Dairy Science and Technology  Volume 2, Issue 2, August 2012, Pages  __________________________________________________________________________________________  ISSN: 2319-3409© STM Journals 2012. All Rights Reserved Page 5 e)   Nitrate Reduction Test: Nitrate reduction is an important criterion for differentiating and characterizing different types of bacteria. Therefore, the isolates were incubated at 37 °C for 24 h in trypticase nitrate broth. After incubation, 0.5 mL each of sulphanilic acid (0.8%, in 5N Acetic acid) and  -naphthylamine (0.5%, in 5N Acetic acid) were added into the tubes. The appearance of red or pink color indicated the positive test for nitrate reduction and was recorded accordingly   for the isolates tested in the present study. f)   Citrate Utilization Test : The isolates were inoculated in Simmons citrate agar incubated at 37 °C for 24 h. After incubation, the appearance of blue coloration indicated the positive test for citrate utilization and was recorded accordingly for the isolates tested.  2.2.4. Carbohydrate Fermentation Pattern by  the Isolates Most microorganisms obtain their energy through a series of orderly and integrated enzymatic reactions leading to the bio-oxidation of a substrate, frequently a carbohydrate. Thus, different sugars were used for determining the fermentation profile and further characterization of  Lactobacilli  isolates. For this,  Lactobacilli  cultures were subjected to sugar fermentation reactions using CHL medium for knowing their fermentation pattern. CHL medium was used as basal medium. Four milliliter of the medium was taken in each tube and sterilized by autoclaving. One sugar disc was aseptically added to each tube. Each tube was inoculated with 0.1 mL of inoculum, incubated at 37 °C for 24  –  48 h and the results of color change were recorded as positive or negative. A control using 0.1 mL sterile water as inoculum was used to compare the color change. Sugars used to determine the fermentation profile of  Lactobacilli  isolates were arabinose, cellobiose, fructose, galactose, lactose, maltose, mannitol, mannose, melibiose, raffinose, rhamnose, trehalose, salicin, sorbitol, sucrose and xylose. The cultures were identified based on the pattern of sugar utilization [12]. 2.3. Maintenance and Propagation of Cultures For their further assessment of probiotic attribute and other analysis all the isolated  Lactobacilli cultures were maintained in chalk litmus milk at refrigeration temperature after their growth at 37 °C overnight. The cultures were sub-cultured at regular intervals in chalk litmus milk and stored under refrigeration conditions. Before use, the cultures were activated in MRS broth. All the isolates were also maintained at − 70 °C in glycerol stock in triplicates for use in experiment at different stages. 2.4. Purity of Cultures The cultures of  Lactobacilli  species and indicator strains were regularly tested for their purity by microscopic examination and/or
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