With this objective, a new strain of Lactobacillus plantarum DM5 was isolated from an ethnic fermented drink Marcha of Sikkim, India and explored for its future application in the food industry as a bio-preservative probiotic. The present researches are carried out on “Bacteriocin, antioxidant and novel glucan from probiotic Lactobacillus plantarum DM5 isolated from Marcha of Sikkim”.
Introduction
LAB produce various types of exopolysaccharides that have numerous applications in the food and pharmaceutical industries (Purama and Goyal, 2005; Das and Goyal, 2012). The most diverse group of LAB are Lactobacillus species and according to the carbohydrate metabolism (Kandler and Wiess, 1986; Todorov and Franco, 2010), they can be divided into three groups;.
Lactic acid bacteria in food industry
Microbial diversity of India
Assam, Arunachal Pradesh, Sikkim, Manipur, Mizoram, Meghalaya, Tripura and Nagaland are rich in numerous endemic species of microorganisms (Singh et al., 2009). Marcha is used to prepare alcoholic beverages such as Kodo ko jaanr (fermented finger millet liquor), Bhaati jaanr (fermented rice liquor) and Raksi (distilled liquor) in Sikkim and the Darjeeling hills (Tamang et al., 2012).
LAB as probiotic
Streptococci, enterococci and lactococci can be used as probiotics (Schrezenmeir and de Vrese, 2001; Ouwehand et al., 2002). LAB are also potentially used as starter cultures for the production of milk-based probiotic foods (Saarela et al., 2002).
Antioxidants from LAB
Antioxidants are considered important nutraceuticals due to their many health benefits (Droge, 2002; Valko et al., 2007). Naturally occurring antioxidants from LAB have received much attention in the past decade (Terahara et al., 2001).
Some reports showed the presence of GAD activity in lactic acid bacteria (Komatsuzaki et al., 2005; Cho et al., 2007). It was observed that three Lactobacillus strains isolated from Italian cheese could survive and synthesize GABA under simulated gastrointestinal conditions (Siragusa et al., 2007).
Bacteriocin from LAB
To date, only nisin and pediocins have been used as biopreservatives in food systems (Rodríguez et al., 2002; Cotter, 2012). Immobilized bacteriocins can also be applied for the development of bioactive food packaging (Galvez et al., 2007).
Exopolysaccharides from LAB
Dextran from Pediococcus pentosaceus offers potential for use as a gelling agent in food formulations and as carriers for drug delivery (Patel et al., 2010). The prebiotic effect of low molecular weight dextran with branched α-(1 → 2) bonds has also been reported (Sarbini et al., 2013).
Glucansucrase from LAB
Lactobacillus plantarum can improve intestinal integrity, metabolic activity of intestinal cells and stimulate the immune response (Nissen et al., 2009). Antioxidant activity of Lactobacillus plantarum C88 isolated from traditional Chinese fermented food has been reported (Li et al., 2012).
Objectives of the present study
Glucan-producing lactic acid bacteria and exopolysaccharide characterization. 2010) Plantaricin MG activity against gram-negative bacteria produced by Lactobacillus plantarum KLDS1.0391 isolated from "Jiaoke", a traditional fermented cream from China. 2003) Isolation and characterization of two exopolysaccharides produced by Lactobacillus plantarum EP56. 2007) Lactic acid bacteria in Hamei and Marcha of Northeast India. 2005) Identification of predominant lactic acid bacteria isolated from traditional fermented vegetable products of the Eastern Himalayas.
2006) Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation. 1952). 2013) Antioxidant activity of an exopolysaccharide isolated from Lactobacillus plantarum C88. 2011) Antioxidative activity of lactic acid bacteria in yogurt.
Introduction
The bacteriocin from lactic acid bacteria may provide a new approach to food preservation because they inhibit food spoilage and pathogenic microorganisms. Several in vitro and in vivo experiments have revealed the antagonistic effect of bacteriocin from lactic acid bacteria against important foodborne pathogens such as Helicobacter pylori, Camphylobacter jejuni, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus (Spelhaug and Harlander, 1989; Aymerich). et al., 2000; Thokchom and Joshi, 2012). In addition, lactic acid bacteria of the genera Leuconostoc, Pediococcus, Lactobacillus and Weisella also produce exopolysaccharides (EPSs), with inimitable rheological properties capable of improving the texture of fermented products (Cerning, 1990).
The EPS are used as viscosifying, stabilizing, emulsifying, sweetening, gelling and water-binding agents in both the food and non-food industries (Wellman and Maddox, 2003; Purama et al., 2009). The classification of lactic acid bacteria into different genera is largely based on phenotypic methods such as morphology, mode of glucose fermentation, growth at different temperatures, ability to grow at high salt concentrations, acid or alkaline tolerance and configuration of the lactic acid produced (Corsetti et al) ., 2001).
Material and Methods .1 Chemicals and reagents .1 Chemicals and reagents
- Preparation of MRS medium
- Preparation of enzyme production medium
- Sources and origins of the fermented food and beverage
- Screening of new Lactic acid bacterium
- Glucansucrase assay
- Morphological, biochemical and physiological characterization of isolate DM5 DM5
- Antibiotic susceptibility profile of isolate DM5
- Carbohydrate fermentation profile of isolate DM5
- Molecular characterization of the isolate DM5 .1 Plasmid DNA profile of isolate DM5 .1 Plasmid DNA profile of isolate DM5
- Antimicrobial spectrum of isolate DM5
A loopful culture of the isolate DM5 was spiked into the semi-solid agar medium and incubated at 37°C for 24-48 hours. The isolate DM5 was subjected to nitrate reduction test using a nitrate agar slant from Hi-Media Pvt. Temperature tolerance of isolate DM5 was determined by growing 1.0% of the isolate DM5 in MRS medium as described in Section 2.2.2 and incubated at different temperatures ranging from 10°C-50°C for 24-48 hours.
An overnight culture of 100 µl of isolate DM5 grown in MRS medium was mixed in MRS soft agar (0.8%, w/v agar) and plated on MRS. For plasmid DNA extraction, the isolate DM5 was grown in 5 ml liquid MRS medium at 37ºC for 20-24 hours.
Results and Discussion
- Selection of the isolate DM5
- Morphological, biochemical and physiological characterization of the isolate DM5 DM5
- Antibiotic susceptibility profile of isolate DM5
- Carbohydrate fermentation profile of isolate DM5
- Molecular characterization of the isolate DM5 .1 Plasmid DNA Profile of isolate DM5 .1 Plasmid DNA Profile of isolate DM5
- Antimicrobial spectrum of isolate DM5
Isolate DM5 was sensitive to almost all β-lactam antibiotics (ampicillin, amoxicillin, carbenicillin, cephalothin, penicillin-G, pipericillin, ticarcillin) like other Lactobacillus spp. Isolate DM5 could not use glycerol and the naturally occurring deoxy sugar rhamnose like Lb. The genomic DNA profile of isolate DM5 was consistent with previous reports where Lactobacillus spp.
The BLAST analysis of the isolate DM5 using the 16S rRNA consensus sequence data showed 100% similarity to several Lactobacillus plantarum strains. The partial sequence of the rpoA gene from the isolate DM5 also showed the highest homology to Lb.
Conclusions
This is the first report of Lactobacillus plantarum showing antimicrobial activity as well as glucansucrase activity. 2008) Phenotypic and genotypic identification of lactic acid bacteria isolated from ethnically fermented bamboo tender shoots of Northeast India. 1993) Plantaricin S and T, two new bacteriocins produced by Lactobacillus plantarum LPC010 isolated from green olive fermentation.
2001) Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain. 2007) Identification of lactobacilli by pheS and rpoA gene sequence analyses. 1991) Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations.
Introduction
Various synthetic and natural antioxidants have been reported such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ); however, there are doubts about the safety and long-term effects of synthetic antioxidants on health (Kumar et al., 2008). It has been shown that some lactobacilli have antioxidative activity and are able to reduce the risk of accumulation of ROS (Kaizu et al., 1993) and the breakdown of the. Lactobacillus plantarum 7FM10 isolated from the traditional Japanese food narezushi (Kanno et al., 2012) and Lactobacillus plantarum isolated from Chinese fermented food (Li et al., 2012) exhibited DPPH, hydroxyl and superoxide radical scavenging ability.
Several Lactobacillus spp have been reported to increase the GABA content of fermented food products (Li and Cao, 2010; Lee et al., 2010). The traditional fermented food sources enriched in glutamate are important sources for isolation of GABA-producing lactic acid bacteria (Di Cagno et al., 2010; . Lee et al., 2010).
Material and Methods .1 Chemicals and reagents .1 Chemicals and reagents
- Microorganism and culture medium
- Mammalian cell line and maintenance
- Determination of protein concentration
- In vitro assessment of antioxidant activity of Lactobacillus strains
- Identification of GABA producing ability of Lb. plantarum DM5 by thin layer chromatography layer chromatography
The absorbance of the bacterial suspension was measured at A600 after mixing and after 6 hours of incubation at 37ºC. The protein concentration of the cell-free extract was estimated according to the method of Lowry et al., (1951). The specific activity (U/mg) of the cell-free extract containing bile salt hydrolase activity was calculated using the following equation;
The absorbance of the mixture was then determined at 517 nm using a microplate reader (Tecan, Infinite 200 Pro). The superoxide anion radical scavenging activity of the sample was calculated using the following equation:
Results and Discussion
GABA producing ability of isolate Lb. plantarum DM5
MSG and GABA production in the cell-free supernatant was detected by TLC (Fig. 3.3.13). The GABA standard (lane 1) and the intense spot obtained in lane 3 show the same retention factor Rf=4.4 mm. The chromatogram showed a clear and strong spot for GABA from the culture supernatant of Lb.
Previous studies showed that the doses of GABA (0.5 mg/kg) significantly decreased the systolic blood pressure of hypertensive rats after administration (Hayakaw et al., 2004).
Conclusions
In vitro screening of Lactobacillus plantarum as probiotic bacteria and their fermented characteristics in soy milk. 2009) Treatment of irritable bowel syndrome with probiotics: finally an etiopathogenic approach. Radical scavenging capacities of saba-narezushi, fermented Japanese mackerel and its lactic acid bacteria. 2009) Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum. 2000). 1999) Antioxidant capacity of lactic acid bacteria. 2005) Acid and bile tolerance and cholesterol scavenging ability of lactobacilli strains. 2010) Lactobacillus plantarum intake reduces some gastrointestinal symptoms during antibiotic treatment. 1951).
Probiotic strain Lactobacillus plantarum MBIMCC 2415 with antioxidant activity as starter cultures in the production of dried fermented meat products. 2011) Assessment of probiotic properties of Lactobacillus plantarum ZLP001 isolated from the gastrointestinal tract of weaned pigs. 2006) Antioxidative activities of soy milk fermented with lactic acid bacteria and bifidobacteria.
Introduction
There is a renewed interest in antimicrobial peptides (bacteriocins and bacteriocin-like compounds) produced by lactic acid bacteria due to their potential use as antimicrobial agents for improving the quality and safety of the food product (Galvez et al., 2007). These antimicrobial proteins are generally purified by ammonium sulfate precipitation followed by gel filtration, ion exchange chromatography, and reversed-phase high-performance liquid chromatography (Carolissen-Mackay et al., 1997; Vera Pingitore et al., 2007). Bacteriocins from Lactobacillus plantarum are generally classified as a small, heat-stable, listeria-active peptides (Messi et al., 2001; Todorov and Dicks, 2005; De Vuyst and Leroy, 2007) belonging to class IIa bacteriocin (Klaenhammer , 1993).
Bacteriocin can be added to foods as food preservatives, additives and as shelf life extenders (Ross et al., 2002). In addition, the bacteriocin was purified by ammonium sulfate precipitation, cation exchange chromatography, and gel filtration.
Material and Methods .1 Chemicals and reagents .1 Chemicals and reagents
- Microorganism and culture medium
- Production of bacteriocin from Lb. plantarum DM5
- Antimicrobial activity assay of Lb. plantarum DM5
- Estimation of protein concentration
- Fermentation profile of Lb. plantarum DM5 in MRS medium
- Effect of temperature and pH on bacteriocin activity
- Effect of enzymes on bacteriocin activity
- Effect of organic solvents, surfactants, salts and detergents on bacteriocin activity activity
- Mode of action of bacteriocin from Lb. plantarum DM5
- Purification of bacteriocin
- In situ assay of bacteriocin activity by agar gel overlay method
The cell-free supernatant was then used for bacteriocin purification as described in section 4.2.10.1. The specific activity (AU/mg) of the cell-free supernatant was calculated using the following equation; The thermal stability of the antimicrobial compound was determined by incubating 5 ml of cell-free supernatant (pH 6.0) of Lb.
The residual antimicrobial activities of the cell-free supernatant were then tested using the agar well diffusion method as previously described in section 4.2.4. The above-mentioned reagents were added to 5 ml of cell-free supernatant (pH 6.0) of Lb.
