Lactobacillus Plantarum IIA-1A5 Research Articles

Fatty acid profiles of Lactobacillus plantarum IIA-1A5 and Lactobacillus acidophilus IIA-2B4

Probiotic lactic acid bacteria have emerged as a natural alternative treatment for cancer in recent years. Indigenous probiotic lactic acid bacteria isolated from beef, Lactobacillus plantarum IIA-1A5 and Lactobacillus acidophilus IIA-2B4, have displayed in vitro anticancer activities against cervical and colorectal cancer cells. Recent studies have suggested that fatty acids such as capric, caprylic, and linoleic acids may play a role in inhibiting tumor development, although the precise mechanisms remain unclear. The aim of this study is to analyze the fatty acid profiles of L. plantarum IIA-1A5 and L. acidophilus IIA-2B4. Intracellular and extracellular fractions of bacterial cultures were obtained using centrifugation. The fatty acid composition was identified using GC-FID. Identified fatty acids were subjected to ANOVA for statistical analysis. Notably, the results show that saturated fatty acid contents exhibited significant differences between all samples, whereas unsaturated fatty acid components varied only between extracellular and intracellular fractions, contributing to the observed diverse anticancer activity. Furthermore, specific saturated fatty acids (C8:0 and C10:0) were found exclusively in L. plantarum IIA-1A5 extracellular or L. acidophilus IIA-2B4 intracellular fraction. These findings might provide valuable insights into the potential therapeutic applications in cancer treatment, warranting further investigation into their underlying mechanisms and clinical potential.

Peranan Probiotik Lactobacillus plantarum IIA-1A5 dalam Meningkatkan Ketahanan Tubuh, Performa, dan Kualitas Telur Ayam Komersial

Probiotics are microorganisms that are non-pathogenic and beneficial for health when used in certain doses through the mechanism of increasing the function of the small intestine in chickens. This study aims to analyze the effect of adding the probiotic Lactobacillus plantarum IIA-1A5 to feed to improve the body's immune system, production performance, and egg quality in laying hens aged 49‒52 weeks. The experimental design used was a Completely Randomized Design (CRD). The treatment for adding the probiotic Lactobacillus plantarum IIA-1A5 to feed consists of four treatment levels, namely P0 (without probiotics), P1 (feed + 8.1 x 106 cfu/g or 1 g/kg feed), P2 (feed + 2.43 x 107 cfu/g or 3 g/kg feed), and P3 (feed + 4.86 x 107 cfu/g or 6 g/kg feed). The results of the study showed that administering the probiotic Lactobacillus plantarum IIA-1A5 in feed could reduce Escherichia coli bacteria in the small intestine of laying hens. Giving the probiotic Lactobacillus plantarum also had a significant effect (P<0.05) on feed consumption and egg yolk color. The addition of the probiotic Lactobacillus plantarum IIA-1A5 in feed can increase animal resistance, performance, and quality of chicken eggs. The use of probiotics with a treatment level of 1 g kg-1 feed (8.1 x 106 cfu g-1) showed the best results.
 
 Keywords: heat stress, laying hens, probiotic

In vitro anticancer potentials of Lactobacillus plantarum IIA-1A5 and Lactobacillus acidophilus IIA-2B4 extracts against WiDr human colon cancer cell line

Colorectal cancer is an emerging public health problem in Indonesia that urges more serious attempts to combat the disease. Previous in vitro studies indicated some probiotics offer promising avenues for the prevention and treatment of colorectal cancer. Earlier, two Indonesian meat-based probiotics of Lactobacillus plantarum IIA-1A5 and L. acidophilus IIA-2B4, were isolated and exhibited promising functional properties in food fermentation. Nevertheless, their potential anticancer activity has never been examined. This study aims to determine the inhibitory properties of L. plantarum IIA-1A5 and L. acidophilus IIA-2B4 against the WiDr human colon carcinoma cell line. To address, extracellular and intracellular extracts were firstly harvested from both probiotics at the population of 8.22 and 9.13 log (cfu/mL) for L. plantarum IIA-1A5 and L. acidophilus IIA-2B4, respectively. The extracts from both bacteria were found to have similar pH and titratable acidity values, but different in their protein contents. Further, extra- and intracellular extracts of both strains displayed inhibitory activity towards WiDr cells in a dose-dependent response. The calculated IC50 values of extracellular extracts of both probiotics were significantly lower than that of the intracellular extracts. This activity is predicted to be partly contributed by some protein substances in the extract.

Synthetic Gene-Based Heterologous Expression, Proteolytic, and Structural Characterization of Caseinolytic Protease of Lactobacillus plantarum IIA-1A5

Genome sequence of Indonesian probiotic of Lactobacillus plantarum II1A5 contains a gene encoding a proteolytic subunit of caseinolytic protease, designated as ClpP_LP. This study aims to express the Clp gene heterological and apply its proteolytic activity to some livestock products. To address this, the gene encoding ClpP_LP was optimized in silico by improving its Codon Adaptation Index and GC content to 0.94 and 53.62%, respectively. The optimized gene was then inserted into pET28a, transformed into Escherichia coli BL21(DE3), and over-expressed by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside at 37°C. The result showed that ClpP_LP was successfully over-expressed in a fully soluble form with the specific activity towards milk casein was 7739.89 AU mg-1. This activity was significantly greater than that of chymotrypsin. Further, the three-dimensional model of ClpP_LP was built using SWISS MODEL, which showed that this protein formed a homo-tetradecameric (14-mer) structure with each monomer consisting of 7 α-helix and 10 β-sheets. The identification of the active side showed that the active side of ClpP_LP is Ser-97, His-122, Asp-171, and forms a substrate-binding cavity with a size of about 29.5 Ǻ. Overall, our approach can serve as an appropriate platform for the production of ClpP_LP in a large-scale production for various applications in dairy products and derivatives.

Kinetic and Thermodynamic Study of Plantaricin IIA-1A5, a Bacteriocin Produced by Indonesian Probiotic Lactobacillus plantarum IIA-1A5.

Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5, a locally isolated probiotic from Indonesia. Plantaricin IIA-1A5 exhibits antibacterial activity against wide spectrum of pathogenic bacteria, thus promising to be applied in various food products. Nevertheless, thermal stability of this bacteriocin remains to be fully investigated. This study aims to determine thermal stability of plantaricin IIA-1A5 through kinetic and thermodynamic parameters. To address, plantaricin IIA-1A5 was purified from Lactobacillus plantarum IIA-1A5, which was growth under whey media, using ammonium sulfate precipitation followed by ionexchange chromatography. Purified plantaricin IIA-IA5 was then subjected to analysis of its bacteriocin activity. The thermal inactivation of bacteriocin from L. plantarum IIA-1A5 was calculated by incubating the bacteriocin at different temperatures ranging from 60-80 °C for 30 to 90 min, which was then used to calculate its kinetic and thermodynamic parameters. The result showed the inactivation rates (k-value) were ranging from 0.008 to 0.013 min-1. Heat resistance of plantaricin IIA-1A5 (D-value) at constant heating temperature of 60, 65, 70, 75, and 80 °C were 311.6, 305.9, 294.5, 198.9, and 180.2 min, which indicated a faster inactivation at higher temperatures. D-value sensitivity for temperature changes (z-value) was calculated to be 75.76 °C. Further, thermodynamic analysis suggested that plantaricin IIA-1A5 is thermostable, with activation energy (Ea) of 29.02 kJ mol-1. This result showed that plantaricin IIA-1A5 is considerably more heat-stable than plantaricin members and promises to be applied in food industries where heat treatments are applied. Furthermore, a possible mechanism by which plantaricin IIA-1A5 maintains its stability was also discussed by referring to its thermodynamic parameters.

The growth and production of antimicrobial compounds from Lactobacillus plantarum IIA-1A5 on cheese whey medium

Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5 which is isolated from Indonesian beef, and it inhibits activity of Gram negative and positive pathogenic bacteria. However, preparation of the antibacterial agent for further applications or studies is costly due to the usage of a bacterial medium. Therefore, this study was aimed to investigate the feasibility of cheese whey as a growth medium for production of the bacteriocin. The growth curve of Lactobacillus plantarum IIA-1A5 in producing antimicrobial compounds was found to occur in the logarithmic phase with an incubation time of 28 and 32 hours. Purification of plantaricin IIA-1A5 produced peptides with a molecular weight of 9.59 kDa consisting of whey and whey+ (20g/L sucrose, 12.5 g/L tryptone and 7.5 g/L yeast extract); thus, the peptide was grouped as class IIa (<10 kDa) bacteriocin. The protein concentration of plantaricin IIA-1A5 with whey+ (sucrose, tryptone, and yeast extract) treatment and whey treatment was 1883.17 mg/ml and lower than 325.58 mg/ml, respectively. Based on the antimicrobial activity test using a paper disc method, plantaricin IIA-1A5 demonstrated antimicrobial activity against Staphylococcus aureus ATCC 25923; the whey and whey+ treatment yielded 38.02 IU/dL and 321 IU/dL, respectively, while antimicrobial activity against Escherichia coli ATCC 25922 using whey and whey+ treatment yielded 44.85 IU/dL and 172.08 IU/dL, respectively. The effectiveness of the antimicrobial properties of plantaricin IIA-1A5 in the whey medium is proven through the results of this study. In short, the whey is appropriate growth medium for bacteriocin production.

Characterization of plantaricin IIA-1A5 biosynthesized by Lactobacillus plantarum IIA-1A5 in corn steep liquor based medium

Characterization of plantaricin IIA-1A5 biosynthesized by Lactobacillus plantarum IIA-1A5 in corn steep liquor based medium

Expression of Lon-like Protease Gene from Lactobacillus plantarum IIA-1A5 in Escherichia coli BL21(DE3)

Proteases are one of most important and abundant enzymes produced by the biotechnology industry, for scientific, physiological and industrial application and dominates of the whole enzyme market. Lactobacillus plantarum IIA-1A5 is an Indonesian lactic acid bacteria (LAB) isolated from beef Peranakan Ongole cattle. Preliminary analysis on its whole genome sequence indicated that this strain harbours some genes involved in protein degradation and might be promising to be further applied. This study aims to optimize the gene sequence of a lon-like protease of L. plantarum IIA-1A5 for heterologous expression system. The Lon-like gene expression system is made using genes that have been optimized first in silico. pET-28a(+), E. coli BL21(DE3), Nde1 and BamH1 were used in this study as a expression vector, a host and retriction enzyme, respectively. Molecular weight was validated using SDS-PAGE and expasy.org software. The results showed that optimization increased codon adaptation index value (CAI) and GC content to 0.97 and 56.57%, respectively, which were suitable for the E. coli expression system. The Lon-like IIA gene was successfully expressed in the cell cytoplasm by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C. As many as 88% of Lon-like IIA codons were distributed in the 91-100 quality group. Lon-like IIA was successfully expressed in a host cell induced with 1 mM IPTG at 37oC . IPTG induction was performed at the 3rd hour of incubation with OD600 0.59. In addition, Lon-like IIA molecular weight was detected approximately 43 kDa.

Lactobacillus plantarum IIA-1A5 Fermentation Patterns by Using whey, buttermilk and Whey Enriched by Skimmed Milk as Growth Media

Lactobacillus plantarum IIA-1A5 Fermentation Patterns by Using whey, buttermilk and Whey Enriched by Skimmed Milk as Growth Media

Purification of Bacteriocin from Lactobacillus plantarum IIA-1A5 Grown in Various Whey Cheese Media Under Freeze Dried Condition.

Lactobacillus plantarum IIA-1A5 is a lactic acid bacteria (LAB) that has been reported to have capacity to produce bacteriocin, usually called plantaricin. This bacteriocin is usually produced in commercial synthetic media. However, the media is expensive, thus finding a novel source which is less expensive and abundant is necessary. The present work was aimed to compare the use of gouda and mozarella cheese whey as growth media of L. plantarum IIA-1A5 producing plantaricin, as well as to evaluate the effectiveness of freeze dried plantaricin as an antimicrobial agent. The results showed that gouda and mozarella cheese whey were applicable for growth media of L. plantarum IIA-1A5 and production of plantaricin. The plantaricin produced from the whey showed a size of about 9.6 kDa. Freeze dried plantaricin was shown to be relatively stable in the second week of storage, but there was a slight decrease in protein concentration during storage, indicating that the protein was partially denatured and precipitated. However, the freeze dried plantaricin showed inhibitory activities against S. aureus ATCC 25923, B. cereus ATCC 21332, S. thpymurium ATCC 14028, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 during storage at room temperature.

Optimasi Ekstraksi DNA Genomik Probiotik Lactobacillus plantarum IIA-1A5 dari Daging Sapi Peranakan Ongole untuk Sekuensing Genom Utuh

Lactobacillus plantarum IIA-1A5 is an Indonesian probiotic bacterium isolated from Ongole grade cattle. This strain has demonstrated series of benefical properties and promising to be further applied in development of functional foods. Attempts to further exploit this strain require comprehensive understanding on its molecular properties, in particular is the genes responsible for its probiotic properties.Whole genome sequencing (WGS) is currently an affordable tool to fully decipher genetic organization of the bacteria, yet often limited by the requirement of high amount of high quality of genomic DNA ofsample. This study aims to develop an optimum method for obtaining high quality genomic DNA of L. plantarum IIA-1A5 in sufficient amount of WGS study. To address, genomic DNA of L. plantarum IIA-1A5 was extracted from growth phase of adaptation, exponential and stationary. The result showed that exponential and stationary phases were able to yield genomic DNA higher than minimum requirement for WGS. Nevertheless, the quality of genomic DNA from both phases were considerably low, according to their 260/280 and 260/230 ratio. The worst quality was found on 260/280 ratio referring to thepresence of high amount of proteinaceous compounds in the sample. Further optimization was done on genomic DNA from stationary phase by adding proteinase K at various incubation time. It showed that the incubation for 7.5 to 10 hours yielded acceptable purity and amount of genomic DNA for WGS. In addition, we also observed a trade-off phenomenon of yield-purity genomic DNA of L. plantarum IIA-1A5. Altogether, the optimized method here should contribute to further molecular studies of this probiotic strain.

Application of Purified Bacteriocin from Lactobacillus plantarum IIA-1A5 as a Bio-preservative of Beef Sausage

Application of Purified Bacteriocin from Lactobacillus plantarum IIA-1A5 as a Bio-preservative of Beef Sausage

Plantaricin IIA-1A5 from Lactobacillus plantarum IIA-1A5 Retards Pathogenic Bacteria in Beef Meatball Stored at Room Temperature

This study aims to demonstrate the efficiency of antibacterial activity of plantaricin IIA-1A5 in meatball when stored at room temperature. The results revealed addition of plantaricin IIA-1A5 to meatball reduced population of contaminant pathogenic bacteria of Staphylococcus aureus and Escherichia coli. Furthermore, the application of 0.3% plantaricin IIA-1A5 extends the shelf life of the meatball to up to 20 h at room temperature based on its microbiology quality, as stated by the Indonesian National Standard for meatball products. The ability of plantaricin IIA-1A5 in retarding the growth of these pathogenic bacteria is comparable to that of 0.3% nitrite, which has been widely used. In addition, Aw and pH values of meatball in the absence (or in the presence) of nitrite or plantaricin did not significantly vary. This implies that reducing the population of pathogenic bacteria was indeed affected by nitrite or plantaricin IIA-1A5. Overall, this result demonstrated that the use of plantaricin IIA-1A5 as a nitrite replacer is promising.

Application of Antimicrobial Agents Produced by <em>Lactobacillus plantarum</em> IIA-1A5 as Natural Preservative on Beef during Room Temperature Storage

Lactobacillus plantarum IIA-1A5 is indigenous lactic acid bacteria isolated from Indonesian beef. Lactobacillus plantarum IIA-1A5 was reported could produce bacteriocin, called plantaricin IIA-1A5. The aims of this research was to analyze application of plantaricin IIA-1A5 as a natural preservative on beef. Based on antagonistic test, plantaricin IIA-1A5 had good moderate antimicrobial activity against pathogenic bacteria isolated from human's feces that cause diarrhea such as Salmonella 38, Enteropathogenic Escherichia coli K11 and Shigella A33. Application of plantaricin IIA-1A5 was effective as a natural preservative on beef stored at room temperature by inhibiting the growth of Escherichia coli and Staphylococcus aureus. Plantaricin IIA-1A5 could kill all of the Escherichia coli after 5 h storage. Plantaricin IIA-1A5 could reduce the population of Staphylococcus aureus in beef during room temperature storage. Interestingly, plantaricin produced by Lactobacillus plantarum IIA-1A5 was effective against gram negative and positive bacteria. According to physichochemichal and microbiology quality, plantaricin IIA-1A5 was recommended as biopreservative agents for beef.

Plantaricin IIA-1A5 from Lactobacillus plantarum IIA-1A5 displays bactericidal activity against Staphylococcus aureus.

Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5 isolated from Indonesian beef. This research aimed to identify the genes involved in plantaricin IIA-1A5 production and examine its mode of action against Staphylococcus aureus. It has been reported that a bacteriocin structural gene, plnW, is present in genome of L. plantarum IIA-1A5. Here, we reported the presence of additional genes responsible for plantaricin precursor (plnA and plnEF) and a gene encoding the quorum sensor of histidine kinase (plnB). It indicates that genes involved in production of plantaricin IIA-1A5 are organized in at least two bacteriocin operons (plnABCD, plnEFI) and a structural plnW gene. Purified plantaricin IIA-1A5 yielded a single band in SDS-PAGE with apparent size of 6.4 kDa. Amino acid composition of purified plantaricin IIA-1A5 was mainly composed of cationic glutamic acid and cysteine that allowed the formation of disulphide bonds, suggesting plantaricin IIA-1A5 belongs to the pediocin-subclass of class II bacteriocins. Plantaricin IIA-1A5 displayed remarkable antibacterial activity against S. aureus, which was initiated by the adsorption of plantaricin IIA-1A5 onto the cell membrane of S. aureus. The adsorption is hypothesised to be facilitated by non-ionic interactions as it is reduced by the presence of organic solvents or detergents. This adsorption promoted leakage of cellular metabolites through the cell membrane of S. aureus, as indicated by the release of genetic and proteinaceous material of S. aureus observed at 260 and 280 nm, respectively. The leakage also promoted the release of divalent (Ca(2+), Mg(2+)) and monovalent (K(+)) cations. The release of these intracellular components might be due to pores formed in the cell membrane of S. aureus by plantaricin IIA-1A5 as shown by scanning electron microscopy. Altogether, the mode of action of plantaricin IIA-1A5 against S. aureus seems to be bactericidal as indicated by lysis of the cell membrane.

English

Bacteriocins produced by Indonesian lactic acid bacteria Lactobacillus plantarum IIA-1A5, IIA-1B1, IIA-2B2 were purified and characterized. Plantaricin W gene had been successfully amplified from all strains. This amplicon showed the expected 200 bp size of plantaricin W gene. This bacteriocins purified from L. plantarum IIA-1A5, IIA-1B1, and IIA-2B2 were named plantaricin IIA-1A5, IIA-1B1, and IIA-2B2. Purification by cation exchange chromatography increased the purity (fold) and activity of plantaricins. Purity of plantaricin IIA-1A5 was increased by 3.13 fold with specific activity 13.40 AU/mg. Plantaricin IIA-1B1 had 2.98 fold purity with specific activity 5.12 AU/mg, while purity of plantaricin IIA-2B2 was 1.37 fold with specific activity 7.70 AU/mg. All plantaricins could inhibit the growth of pathogenic bacteria, such as Escherichia coli, Salmonella typhimurium, Bacillus cereus, and Staphylococcus aureus. Plantaricins could be digested by trypsin. Stability of plantaricins at 80 oC for 30 min and at 121 oC for 15 min were affected by type of plantaricin and species of pathogenic bacteria. Generally, plantaricin IIA-1A5 was better as antimicrobial agent than plantaricin IIA-1B1 and plantaricin IIA-2B2.