Indigenous Lactobacillus Plantarum Research Articles

Biosurfactant production by Lactobacillus plantarum MGL-8 from mango waste.

Enhancing biosurfactant production from indigenous Lactobacillus plantarum MGL-8 using mango waste substrate, and evaluating its characteristics as food sanitizer. Mango juice (a mixture of mango paste, sucrose, glycerol and deionized water) was used for batch fermentation with L. plantarum MGL-8 (L-MJ) and uninoculated (MC-MJ). Agitation, aeration and temperature were controlled. Maximum lactic acid bacteria (LAB) growth was observed in MC-MJ and L-MJ at 48h, and the L-MJ fermentation provided the highest biosurfactant yield of 4.22gL-1 at 120h. The dried crude biosurfactant (BSF) provided surface tension 36.6mNm-1 , a maximum emulsification index (E24%) of 41% and zone of inhibition of 15.53mm. Preliminary characterization by Gas chromatography-Mass spectrometry (GC-MS) and Fourier transform infrared (FTIR) indicated a multi-component glycolipoprotein BSF associated with fatty dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, proteins and polysaccharides. The BSF also displayed bactericidal activity against Listeria monocytogenes at 400μgml-1 . Mango waste substrate enhanced biosurfactant production by indigenous L. plantarum MGL-8. The study identifies a production process and characteristics of the biosurfactant, which can be employed as a food sanitizer.

Development of probiotic gummy candy using the indigenous Lactobacillus plantarum Dad-13 strain; evaluation of its gastrointestinal resistance and shelflife prediction

Nowadays, functional food especially probiotic products have gained more interest, and gummy candy L. plantarum Dad-13 is an innovation. However, the efficacy of probiotics relies on their viable cells and resistance in the gastrointestinal tract. Therefore, this research aimed to evaluate the gastrointestinal resistance and probiotic cells' shelf life and investigate gummy probiotics' characteristics. A market survey was carried to understand the consumer's knowledge on the health benefit of probiotics and their product, also intend to buy the product. Gastrointestinal simulation with pepsin and the pancreatic enzyme was conducted to evaluate the gastrointestinal tract's probiotic resistance, probiotic shelf life was evaluated with the Accelerated Shelf-life Test (ASLT). Proximate analysis was carried out according to the Association of Official Analytical Chemists (AOAC), and physical characteristics were analysed before and after 90 days of storage at 4oC. The result showed that the supplementation of L. plantarum Dad-13 in the gummy probiotic had an excellent survival during gastrointestinal system simulation with the predicted shelf life was 75.17 days or 2.5 months at 4oC of storage. However, the moisture content of gummy probiotics was more than 20%. Considering the survival of L. plantarum Dad13 during gastrointestinal simulation, it can be concluded that gummy probiotics can be used as a carrier for L. plantarum Dad-13.

Indigenous Yeast, Lactic Acid Bacteria, and Acetic Acid Bacteria from Cocoa Bean Fermentation in Indonesia Can Inhibit Fungal-Growth-Producing Mycotoxins

Cocoa bean fermentation is an important process in the manufacturing of cocoa products. It involves microbes, such as lactic acid bacteria, yeast, and acetic acid bacteria. The presence of mold in cocoa bean fermentation is undesired, as it reduces the quality and may produce mycotoxins, which can cause poisoning and death. Aspergillus niger is a fungus that produces ochratoxin A, which is often found in dried agricultural products such as seeds and cereals. In this study, we applied indigenous Candida famata HY-37, Lactobacillus plantarum HL-15, and Acetobacter spp. HA-37 as starter cultures for cocoa bean fermentation. We found that the use of L. plantarum HL-15 individually or in combination Candida famata HY-37, Lactobacillus plantarum HL-15, and Acetobacter spp. HA-37 as a starter for cocoa bean fermentation can inhibit the growth of A. niger YAC-9 and the synthesis of ochratoxin A during fermentation and drying. With biological methods that use indigenous Lactobacillus plantarum HL-15 individually or in combination with Candida famata HY-37 and Acetobacter spp. HA-37, we successfully inhibited contamination by ochratoxin-A-producing fungi. Thus, the three indigenous microbes should be used in cocoa bean fermentation to inhibit the growth of fungi that produce mycotoxins and thus improve the quality.

Zearalenone Adsorbent Based on a Lyophilized Indigenous Bacterial Lactobacillus plantarum Strain as Feed Additive for Pigs: A Preliminary Study In Vivo.

Feed contamination with naturally occurring mycotoxins is an unavoidable condition of significant concern in intensive productions. The presence of high concentrations of zearalenone >1ppm in the diet can cause major reproductive disorders, particularly in swine. In order to reduce the consequences of intoxication, mycotoxin adsorbents are incorporated into feed. In the present study, zearalenone adsorption capacity of a lyophilized indigenous strain of Lactobacillus plantarum (L4; previously isolated from pig's rectal swabs) was first evaluated in vitro. A preliminary study in vivo was then performed in which the indigenous Lactobacillus plantarum strain was lyophilized and the powder obtained (L-L4) was incorporated into the diet of gilts two gilts received basal diet (control) and two received basal diet containing 2g/kg L-L4 (treated). After an adaptation period, all the feed was contaminated with zearalenone at a dose of 0.93mg ZEA/kg. Results from in vitro assay showed that L-L4 adsorbed 87.9% (SD 3.97) of zearalenone in 0.9% NaCl solution. In the in vivo exploratory study, higher daily weight gain and lower vulva area were observed in gilts that incorporated L-L4 to the diet. Additionally, higher zearalenone concentrations were eliminated in faeces from treated animals. The use of a product based on a lyophilized indigenous Lactobacillus strain to protect gilts from detrimental effects of zearalenone consumption has shown promising results so far. However, further studies are required in order to accurately assess its impact and evaluate doses according to different degrees of mycotoxins contamination.

Optimization of flaxseed milk fermentation for the production of functional peptides and estimation of their bioactivities.

Bioactive peptides are protein fragments which have a positive impact on the functions and conditions of living organisms. Apart from other animal and plant sources flaxseed is an excellent source of bioactive peptides. In recent years, fermentation has been explored as effective way for bioactive peptides generation. Hence, the present study has been carried out to evaluate an indigenous Lactobacillus plantarum strain NCDC 374 for fermentation and peptides generation in flaxseed milk. Optimization of fermentation condition to obtain maximum functional properties (Proteolytic activity, Antioxidant activity and ACE inhibition %) was investigated using response surface methodology. Optimal condition to produce the functional peptides were found to be 4.20% inoculum size with 126 hours of fermentation time. The fermented milk resulted in 67.38% inhibition in DPPH, 41.35% inhibition in ACE and 30.38 micro gram leucine/ml proteolytic activity. Molecular weight cut off membrane (Viva spin) were used to fractionate the peptides. 10 kDa peptides showed optimal results for % DPPH inhibition, ACE inhibition, Antimicrobial activity and DPP-IV inhibition as compared to 5 kDa.

Growth and bioactive peptides production potential of Lactobacillus plantarum strain C2 in soy milk: A LC-MS/MS based revelation for peptides biofunctionality

Bioactive peptides are protein fragments which have a positive impact on the functions and conditions of living organisms. Apart from other animal and plant sources soybean is an excellent source of bioactive peptides. In recent years, fermentation has been explored as effective way for bioactive peptides generation. Hence, the present study has been carried out to evaluate an indigenous Lactobacillus plantarum strain C2 (LP C2) for fermentation and peptides generation in soy milk. Peptides were further tested for important biofunctionalities (antioxidative and antihypertensive) and characterized by LC-MS/MS. Besides its excellent log count increases, protein hydrolysis and α–galactosidase activities, LP C2 reduced soy oligosaccharides (sucrose, raffinose and stachyose) in a strain specific manner. Following fractionation, the 10 kDa fraction not only showed the highest peptide content in comparison to 3 kDa and 5 kDa, but also exhibited the highest antioxidant activity. Conversely, no significant differences were observed in ACE-inhibitory activities of all three fractions of peptides. Moreover, we are the first to report L. plantarum mediated generation, and LC-MS/MS based characterization of 17 biofunctional soy peptides showing both antioxidant and ACE-inhibitory activities. Thus, this strain can be further used for the preparation of soy based functional fermented foods and bioactive food supplements.

Methods for the evaluation of antibiotic resistance in Lactobacillus isolated from fermented sausages

ABSTRACT: The present study aimed to assess the antibiotic resistance in 54 indigenous Lactobacillus plantarum isolated from artisanal fermented sausages. The confirmation of the strain species was performed by multiplex-PCR assay. Antibiotic resistance was assessed by disk diffusion (DD) and Minimum Inhibitory Concentration (MIC) methods. Of 54 L. plantarum, 44 strains were genotypically confirmed as L. plantarum and 3 as Lactobacillus pentosus. The highest resistance rates were to ampicillin and streptomycin. The highest susceptibility rates were shown to tetracycline, chloramphenicol and penicillin G. None of the strains showed multidrug resistance. Resistance rates by DD and MIC were not different (P>0.05) for ampicillin, chloramphenicol, erythromycin and penicillin G. Future research should assess the genetic mechanisms underlying the phenotypic resistance in Lactobacillus strains to screen the potential probiotic strains for the development of functional meat products.

PURIFIKASI PARSIAL DAN KARAKTERISASI ß-GALAKTOSIDASE Lactobacillus plantarum B123 INDIGENOS DAN HIDROLISIS LAKTOSA UNTUK PRODUKSI SUSU ULTRA HIGH TEMPERATURE RENDAH LAKTOSA

β-Galactosidase is enzyme which hidrolyze lactose to glucose and galactose. This enzyme is used in production low lactose milk for consumption human which have lactose intolerance. Partial purification of β-galactosidase is important to be conducted to increase β-galactosidase activity in order to its hydrolysis potency on UHT milk lactose increased.This research was aimed to production by partially purification and characterization indigenous β-galactosidase from Lactobacillus plantarum B123, and lactose hydrolysis for production low lactose UHT milk. Partially purification were precipitation following dialysis. Characterization included optimazion and stabilization of enzyme, while lactose hydrolisis for production low lactose UHT milk was detected by enzymatic GOD-POD kit. The results showed that production of β-galactosidase by using partial purification increased from 21.51 ± 0.23 U/mL (crude) to 106.34 ± 0.56 U/mL (dialysis). The optimum crude β-galactosidase activity was reached in precipitation by using 60 % ammonium sulphate. The purity of crude β-galactosidase increased 3.71 times after precipitation, and 14.28 times after dialysis. Characterization of β-galactosidase showed that optimum activities of crude and dialyzed β-galactosidase were at pH 6.5 and 50 oC, respectively. Stability of crude β-galactosidase incubated for 1 h were at pH: 5.0-8.5 and 25-50 °C. Specific activity of crude β-galactosidase was 15.05 U/mg protein, while that dialyzed β-galactosidase was 109.58 U/mg protein. Lactose hidrolysis to produce low lactose UHT milk showed that glucose concentration increased with the increase of hidrolysis time. Time needed to hidrolyze lactose 50 % with 4.8 U/mL β-galactosidase at 50°C was 6.08 h. In conclusion that indigenous β-galactosidase from Lactobacillus plantarum B123 purified partially can be used as lactose hidrolyzer in production of low lactose UHT milk.Key words : b-galactosidase, indigenous Lactobacillus plantarum B123, purification, lactose hidrolysis, UHT Milk

Adhesion of indigenous Lactobacillus plantarum to gut extracellular matrix and its physicochemical characterization.

Adhesion to the human intestinal epithelial cell is considered as one of the important selection criteria of lactobacilli for probiotic attributes. Sixteen Lactobacillus plantarum strains from human origins were subjected for adhesion to extracellular matrix (ECM) components, and their physiochemical characterization, incubation time course and effect of different pH on bacterial adhesion in vitro were studied. Four strains showed significant binding to both fibronectin and mucin. After pretreatment with pepsin and trypsin, the bacterial adhesion to ECM reduced to the level of 50 % and with lysozyme significantly decreased by 65-70 %. Treatment with LiCl also strongly inhibited (90 %) the bacterial adhesion to ECM. Tested strains showed highest binding efficacy at time course of 120 and 180 min. Additionally, the binding of Lp91 to ECM was highest at pH 6 (155 ± 2.90 CFU/well). This study proved that surface layer components are proteinaceous in nature, which contributed in adhesion of lactobacillus strains. Further, the study can provide a better platform for introduction of new indigenous probiotic strains having strong adhesion potential for future use.

Indigenous Lactobacillus plantarum as probiotic for larviculture of blue swimming crab, Portunus pelagicus (Linnaeus, 1758): Effects on survival, digestive enzyme activities and water quality

Portunus pelagicus larvae in hatcheries experience high mortality during the early stages of development, primarily due to bacterial infections. A study was performed to determine the effectiveness of the indigenous probiotic Lactobacillus plantarum as a water additive in P. pelagicus larviculture. Three treatments and the control were tested in triplicate. The treatments consisted of three levels of L. plantarum were added daily at designated concentrations 1.0×106 (T-1), 5.0×106 (T-2) and 1.0×107cfuml−1 (T-3) and control without any probiotic. The addition of the probiotic significantly enhanced (p<0.05) survival rate of crab larvae for all treatments over the control. L. plantarum population (cfu ml−1) progressively increased (p<0.05) in treated tanks over the time. Total bacteria and Vibrio counts found significantly lower (p<0.05) in treated tanks over the control. The addition of L. plantarum influenced the nitrogen content and pH of culture water. Supplementation of L. plantarum significantly increased (p<0.05) the protease and amylase activities in the treated groups over the control. The significantly highest protease activity was observed in T-2; while the amylase activity was higher in T-2 and T-3. The increased concentration of probiotic level (1×107) did not increase the enzyme activities and survival of larvae. These results signify that L. plantarum addition at 5.0×106cfuml−1 can effectively enhance the larval survival rate, enzyme activity and improve the water quality of P. pelagicus larviculture.

Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen

Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored.

Bile Salt Hydrolase (Bsh) Activity Screening of Lactobacilli: In Vitro Selection of Indigenous Lactobacillus Strains with Potential Bile Salt Hydrolysing and Cholesterol-Lowering Ability

The bile salt hydrolase (Bsh) activity of probiotic bacterium residing in gastrointestinal tract has often being associated with its cholesterol-lowering effects. Hence, Bsh activity was explored in this study as the criterion for the selection of most potential Bsh-active and cholesterol-lowering indigenous Lactobacillus strains. Forty lactobacilli were adjudged Bsh active after a preliminary screening of 102 lactobacilli and occurrence of Bsh activity correlated well with their natural habitats. Of the 40 shortlisted lactobacilli, fifteen putative Lactobacillus strains were selected and further tested for their comparative Bsh activity. In the end, indigenous Lactobacillus plantarum strains Lp91 and Lp21 were emerged as the promising Bsh-active lactobacilli with their substrate preference inclined more towards glycocholate than other bile acid amino conjugates. In addition, strains Lp91 and Lp21 also exhibited significantly high bile salt deconjugation, cholesterol assimilation and cholesterol co-precipitation ability in vitro. In conclusion, indigenous L. plantarum strains Lp91 and Lp21 may be the promising candidate probiotics to elucidate the ecological significance of probiotic Bsh activity in vivo.

Molecular Identification and Typing of Putative Probiotic Indigenous Lactobacillus plantarum Strain Lp91 of Human Origin by Specific Primed-PCR Assays.

In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase (bsh) and collagen-binding protein (cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles.

Relative gene expression of bile salt hydrolase and surface proteins in two putative indigenous Lactobacillus plantarum strains under in vitro gut conditions

Probiotic bacteria must overcome the toxicity of bile salts secreted in the gut and adhere to the epithelial cells to enable their better colonization with extended transit time. Expression of bile salt hydrolase and other proteins on the surface of probiotic bacteria can help in better survivability and optimal functionality in the gut. Two putative Lactobacillus plantarum isolates i.e., Lp9 and Lp91 along with standard strain CSCC5276 were used. A battery of six housekeeping genes viz. gapB, dnaG, gyrA, ldhD, rpoD and 16S rRNA were evaluated by using geNorm 3.4 excel based application for normalizing the expression of bile salt hydrolase (bsh), mucus-binding protein (mub), mucus adhesion promoting protein (mapA), and elongation factor thermo unstable (EF-Tu) in Lp9 and Lp91. The maximal level of relative bsh gene expression was recorded in Lp91 with 2.89 ± 0.14, 4.57 ± 0.37 and 6.38 ± 0.19 fold increase at 2% bile salt concentration after 1, 2 and 3 h, respectively. Similarly, mub and mapA genes were maximally expressed in Lp9 at the level of 20.07 ± 1.28 and 30.92 ± 1.51 fold, when MRS was supplemented with 0.05% mucin and 1% each of bile and pancreatin (pH 6.5). However, in case of EF-Tu, the maximal expression of 42.84 ± 5.64 fold was recorded in Lp91 in the presence of mucin alone (0.05%). Hence, the expression of bsh, mub, mapA and EF-Tu could be considered as prospective biomarkers for screening of novel probiotic lactobacillus strains for optimal functionality in the gut.

Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum.

BackgroundProbiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt.Methods and FindingsAn indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus.ConclusionThe indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.