Improvement of ɣ-Aminobutyric Acid Production and Cell Viability of Lactiplantibacillus plantarum B7 via Whole-Cell Immobilisation in Repeated Batch Fermentation System.

Whole-cell immobilisation technology involving ℽ-aminobutyric acid GABA biosynthesis using lactic acid bacteria (LAB) has been extensively studied owing to its numerous benefits over free-living bacteria, including enhanced productivity, improved cell viability, ability to prevent cell lysis and protect cells against bacteriophages and other stressful conditions. Therefore, a novel LAB biocatalyst was developed using various fruit and fruit waste, immobilising a potential probiotic strain, Lactiplantibacillus plantarum B7, via an adsorption method to improve GABA and cell viability. Apple and watermelon rind have been known to be the ideal natural supports for L. plantarum B7 owing to higher GABA and lactic acid production and improved cell viability among the other natural supports tested and selected to be used in repeated batch fermentation (RBF) to improve GABA production and cell viability. In general, immobilisation of L. plantarum B7 on natural support has better GABA and lactic acid production with improved cell viability via RBF compared to free cells. Watermelon rind-supported cells and apple-supported cells could produce nine and eight successful GABA cycles, respectively, within RBF, whereas free cells could only produce up to four cycles. When using watermelon rind-supported cells and apple-supported cells in RBF, the GABA titer may be raised by up to 6.7 (218.480 ± 0.280 g/L) and 6 (195.439 ± 0.042 g/L) times, respectively, in comparison to GABA synthesis by free cells in single batch fermentation (32.65 ± 0.029 g/L). Additionally, natural support immobilised L. plantarum B7 could retain half of its cell viability even after the 12th cycle of RBF, while no cell was observed in control.

Strategies for improvement of gamma-aminobutyric acid (GABA) biosynthesis via lactic acid bacteria (LAB) fermentation.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid widely distributed in nature and extensively explored for its numerous physiological functions and effects on metabolic disorders. Lactic acid bacteria (LAB) are one of the most important GABA producers, vigorously pursued due to their high GABA content and generally regarded as safe (GRAS) status that allows for direct formulation in various GABA-enriched food products. To meet the strict requirements of the food and nutraceutical industries, the biosynthesis of GABA is typically preferred over the chemical synthesis route. The production of GABA varies among various strains of LAB and is affected by different fermentation conditions. Hence, optimizing the fermentation conditions to enhance the activity of the key enzyme glutamic acid decarboxylase is essential to maximize GABA production. This paper reviews the beneficial effects of GABA on human health and its applications in fermented food products. A particular emphasis is given to the biosynthetic approach for producing GABA by various LAB species via the microbial fermentation route. Efficient strategies for enhancing GABA production through optimization of the fermentation conditions, mode of fermentation, two-step fermentation, co-culturing approach, immobilization technique and genetic engineering are discussed in detail.

Development of In Situ Product Recovery (ISPR) System Using Amberlite IRA67 for Enhanced Biosynthesis of Hyaluronic Acid by Streptococcus zooepidemicus.

High broth viscosity due to the accumulation of hyaluronic acid (HA) causes a limited yield of HA. It is a major problem of HA production using Streptococcus zooepidemicus. Extractive fermentation via in situ product recovery (ISPR) was utilized to enhance the HA production. Resins from Amberlite: IRA400 Cl; IRA900 Cl; IRA410 Cl; IRA402 Cl; and IRA67 were tested for the HA adsorption. IRA67 showed high adsorption capacity on HA. The study of the adsorption via a 2 L stirred tank bioreactor of S. zooepidemicus fermentation was investigated to elucidate the adsorption of HA onto IRA67 in dispersed and integrated internal column systems. The application of a dispersed IRA67 improved the HA production compared to the fermentation without resin addition by 1.37-fold. The HA production was further improved by 1.36-fold with an internal column (3.928 g/L) over that obtained with dispersed IRA67. The cultivation with an internal column shows the highest reduction of viscosity value after the addition of IRA67 resin: from 58.8 to 23.7 (mPa·s), suggesting the most effective ISPR of HA. The improved biosynthesis of HA indicated that an extractive fermentation by ISPR adsorption is effective and may streamline the HA purification.

Influence of Dietary Biosynthesized Zinc Oxide Nanoparticles on Broiler Zinc Uptake, Bone Quality, and Antioxidative Status.

A total of 180 broiler chickens (Cobb500) were randomly allotted to five experimental groups consisting of six replicates and six birds in each pen. Each group was fed a basal diet supplemented with 100 mg/kg ZnO (control) and 10, 40, 70, and 100 mg/kg ZnO NPs for 35 days. Resultantly, Zn uptake and accumulation in serum, breast muscle, tibia bone, and liver were linearly and significantly (p < 0.05) increased with increasing dietary ZnO NPs supplementation at 100 mg/kg compared to the control group (dietary 100 mg/kg ZnO), implying effective absorption capacity of ZnO NPs. This was followed by lower Zn excretion in feces in broilers fed ZnO NPs compared to controls (p < 0.05). Furthermore, dietary ZnO NPs at 40, 70, and 100 mg/kg levels improved broiler tibia bone morphological traits, such as weight, length, and thickness. Similarly, tibia bone mineralization increased in broilers fed ZnO NPs at 100 mg/kg compared to the control (p < 0.05), as demonstrated by tibia ash, Zn, Ca, and P retention. Antioxidative status in serum and liver tissue was also increased in broilers fed dietary ZnO NPs at 70 and 100 mg/kg compared to the control (p < 0.05). In conclusion, dietary ZnO NPs increased Zn absorption in broiler chickens and had a positive influence on tibia bone development and antioxidative status in serum and liver tissue, with dietary ZnO NPs supplementation at 70 and 100 mg/kg showing the optimum effects.

Antibacterial Potential of Biosynthesized Zinc Oxide Nanoparticles against Poultry-Associated Foodborne Pathogens: An In Vitro Study.

Since the emergence of multidrug-resistant bacteria in the poultry industry is currently a serious threat, there is an urgent need to develop a more efficient and alternative antibacterial substance. Zinc oxide nanoparticles (ZnO NPs) have exhibited antibacterial efficacy against a wide range of microorganisms. Although the in vitro antibacterial activity of ZnO NPs has been studied, little is known about the antibacterial mechanisms of ZnO NPs against poultry-associated foodborne pathogens. In the present study, ZnO NPs were successfully synthesized using Lactobacillus plantarum TA4, characterized, and their antibacterial potential against common avian pathogens (Salmonella spp., Escherichia coli, and Staphylococcus aureus) was investigated. Confirmation of ZnO NPs by UV-Visual spectroscopy showed an absorption band center at 360 nm. Morphologically, the synthesized ZnO NPs were oval with an average particle size of 29.7 nm. Based on the dissolution study of Zn2+, ZnO NPs released more ions than their bulk counterparts. Results from the agar well diffusion assay indicated that ZnO NPs effectively inhibited the growth of the three poultry-associated foodborne pathogens. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were assessed using various concentrations of ZnO NPs, which resulted in excellent antibacterial activity as compared to their bulkier counterparts. S. aureus was more susceptible to ZnO NPs compared to the other tested bacteria. Furthermore, the ZnO NPs demonstrated substantial biofilm inhibition and eradication. The formation of reactive oxygen species (ROS) and cellular material leakage was quantified to determine the underlying antibacterial mechanisms, whereas a scanning electron microscope (SEM) was used to examine the morphological changes of tested bacteria treated with ZnO NPs. The findings suggested that ROS-induced oxidative stress caused membrane damage and bacterial cell death. Overall, the results demonstrated that ZnO NPs could be developed as an alternative antibiotic in poultry production and revealed new possibilities in combating pathogenic microorganisms.

Pulp Enhancement of Oil Palm Empty Fruit Bunches (OPEFBs) via Biobleaching by Using Xylano-Pectinolytic Enzymes of Bacillus amyloliquefaciens ADI2.

The present work reports the biobleaching effect on OPEFB pulp upon utilisation of extracellular xylano-pectinolytic enzymes simultaneously yielded from Bacillus amyloliquefaciens ADI2. The impacts of different doses, retention times, pH, and temperatures required for the pulp biobleaching process were delineated accordingly. Here, the OPEFB pulp was subjected to pre-treatment with xylano-pectinolytic enzymes generated from the same alkalo-thermotolerant isolate that yielded those of higher quality. Remarkable enhanced outcomes were observed across varying pulp attributes: for example, enzyme-treated pulp treated to chemical bleaching sequence generated improved brightness of 11.25%. This resulted in 11.25% of less chlorine or chemical consumption required for obtaining pulp with optical attributes identical to those generated via typical chemical bleaching processes. Ultimately, the reduced consumption of chlorine would minimise the organochlorine compounds found in an effluent, resulting in a lowered environmental effect of paper-making processes overall as a consequence. This will undoubtedly facilitate such environmentally-friendly technology incorporation in the paper pulp industry of today.

A refined medium to enhance the antimicrobial activity of postbiotic produced by Lactiplantibacillus plantarum RS5.

Postbiotic RS5, produced by Lactiplantibacillus plantarum RS5, has been identified as a promising alternative feed supplement for various livestock. This study aimed to lower the production cost by enhancing the antimicrobial activity of the postbiotic RS5 by improving the culture density of L. plantarum RS5 and reducing the cost of growth medium. A combination of conventional and statistical-based approaches (Fractional Factorial Design and Central Composite Design of Response Surface Methodology) was employed to develop a refined medium for the enhancement of the antimicrobial activity of postbiotic RS5. A refined medium containing 20 g/L of glucose, 27.84 g/L of yeast extract, 5.75 g/L of sodium acetate, 1.12 g/L of Tween 80 and 0.05 g/L of manganese sulphate enhanced the antimicrobial activity of postbiotic RS5 by 108%. The cost of the production medium was reduced by 85% as compared to the commercially available de Man, Rogosa and Sharpe medium that is typically used for Lactobacillus cultivation. Hence, the refined medium has made the postbiotic RS5 more feasible and cost-effective to be adopted as a feed supplement for various livestock industries.

Effects of degree of substitution and irradiation doses on the properties of hydrogel prepared from carboxymethyl-sago starch and polyethylene glycol.

Carboxymethyl starch (CMS) was produced from sago starch via carboxymethylation. The CMS with different degree of substitution (DS) ranges from 0.4 to 0.8 were mixed with polyethylene glycol (PEG) of different molecular weight and distilled water and the hydrogel was cured by electron beam irradiation with doses ranging from 25 to 35 kGy. The results revealed that CMS-PEG hydrogels with DS 0.4 give the optimum gel content when radiated at 30 kGy and with PEG 600. Thermogravimetric analysis (TGA) revealed that there are two phases exist in CMS with DS 0.4 in contrast to the three steps decomposition occurs in DS 0.6 and 0.8. It shows that the CMS with DS 0.4 is more thermally stable. Surface morphology revealed crosslinking among the blends when subjected into the radiation dose. The study shows both radiation and PEG addition improved most of the properties of CMS irrespective of the DS value.

Interrelations of Synthesis Method, Polyethylene Glycol Coating, Physico-Chemical Characteristics, and Antimicrobial Activity of Silver Nanoparticles.

Silver nanoparticles (AgNPs) have been found to have extensive biomedical and biological applications. They can be synthesised using chemical and biological methods, and coated by polymer to enhance their stability. Hence, the changes in the physico-chemical characteristics of AgNPs must be scrutinised due to their importance for biological activity. The UV-Visible absorption spectra of polyethylene glycol (PEG) -coated AgNPs displayed a distinctive narrow peak compared to uncoated AgNPs. In addition, High-Resolution Transmission Electron Microscopy analysis revealed that the shapes of all AgNPs, were predominantly spherical, triangular, and rod-shaped. Fourier-Transform Infrared Spectroscopy analysis further confirmed the role of PEG molecules in the reduction and stabilisation of the AgNPs. Moreover, dynamic light scattering analysis also revealed that the polydispersity index values of PEG-coated AgNPs were lower than the uncoated AgNPs, implying a more uniform size distribution. Furthermore, the uncoated and PEG-coated biologically synthesised AgNPs demonstrated antagonisms activities towards tested pathogenic bacteria, whereas no antagonism activity was detected for the chemically synthesised AgNPs. Overall, generalisation on the interrelations of synthesis methods, PEG coating, characteristics, and antimicrobial activity of AgNPs were established in this study.

Biosynthesis of zinc oxide nanoparticles by cell-biomass and supernatant of Lactobacillus plantarum TA4 and its antibacterial and biocompatibility properties.

This study aims to utilize the cell-biomass (CB) and supernatant (CFS) of zinc-tolerant Lactobacillus plantarum TA4 as a prospective nanofactory to synthesize ZnO NPs. The surface plasmon resonance for the biosynthesized ZnO NPs-CFS and ZnO NPs-CB was 349 nm and 351 nm, respectively, thereby confirming the formation of ZnO NPs. The FTIR analysis revealed the presence of proteins, carboxyl, and hydroxyl groups on the surfaces of both the biosynthesized ZnO NPs that act as reducing and stabilizing agents. The DLS analysis revealed that the poly-dispersity indexes was less than 0.4 for both ZnO NPs. In addition, the HR-TEM micrographs of the biosynthesized ZnO NPs revealed a flower-like pattern for ZnO NPs-CFS and an irregular shape for ZnO NPs-CB with particles size of 291.1 and 191.8 nm, respectively. In this study, the biosynthesized ZnO NPs exhibited antibacterial activity against pathogenic bacteria in a concentration-dependent manner and showed biocompatibility with the Vero cell line at specific concentrations. Overall, CFS and CB of L. plantarum TA4 can potentially be used as a nanofactory for the biological synthesis of ZnO NPs.

An Improved Nanoemulsion Formulation Containing Kojic Monooleate: Optimization, Characterization and In Vitro Studies.

Tyrosinase inhibitors have become increasingly important targets for hyperpigmentation disease treatment. Kojic monooleate (KMO), synthesized from the esterification of kojic acid and oleic acid, has shown a better depigmenting effect than kojic acid. In this study, the process parameters include the speed of high shear, the time of high shear and the speed of the stirrer in the production of nanoemulsion containing KMO was optimized using Response Surface Methodology (RSM), as well as evaluated in terms of its physicochemical properties, safety and efficacy. The optimized condition for the formulation of KMO nanoemulsion was 8.04 min (time of high shear), 4905.42 rpm (speed of high shear), and 271.77 rpm (speed of stirrer), which resulted in a droplet size of 103.97 nm. An analysis of variance (ANOVA) showed that the fitness of the quadratic polynomial fit the experimental data with large F-values (148.79) and small p-values (p < 0.0001) and an insignificant lack of fit. The optimized nanoemulsion containing KMO with a pH value of 5.75, showed a high conductivity value (3.98 mS/cm), which indicated that the nanoemulsion containing KMO was identified as an oil-in-water type of nanoemulsion. The nanoemulsion remains stable (no phase separation) under a centrifugation test and displays accelerated stability during storage at 4, 25 and 45 °C over 90 days. The cytotoxicity assay showed that the optimized nanoemulsion was less toxic, with a 50% inhibition of cell viability (IC50) > 500 µg/mL, and that it can inhibit 67.12% of tyrosinase activity. This study reveals that KMO is a promising candidate for the development of a safe cosmetic agent to prevent hyperpigmentation.

Enhancement of Versatile Extracellular Cellulolytic and Hemicellulolytic Enzyme Productions by Lactobacillus plantarum RI 11 Isolated from Malaysian Food Using Renewable Natural Polymers.

Lactobacillus plantarum RI 11 was reported recently to be a potential lignocellulosic biomass degrader since it has the capability of producing versatile extracellular cellulolytic and hemicellulolytic enzymes. Thus, this study was conducted to evaluate further the effects of various renewable natural polymers on the growth and production of extracellular cellulolytic and hemicellulolytic enzymes by this novel isolate. Basal medium supplemented with molasses and yeast extract produced the highest cell biomass (log 10.51 CFU/mL) and extracellular endoglucanase (11.70 µg/min/mg), exoglucanase (9.99 µg/min/mg), ß-glucosidase (10.43 nmol/min/mg), and mannanase (8.03 µg/min/mg), respectively. Subsequently, a statistical optimization approach was employed for the enhancement of cell biomass, and cellulolytic and hemicellulolytic enzyme productions. Basal medium that supplemented with glucose, molasses and soybean pulp (F5 medium) or with rice straw, yeast extract and soybean pulp (F6 medium) produced the highest cell population of log 11.76 CFU/mL, respectively. However, formulated F12 medium supplemented with glucose, molasses and palm kernel cake enhanced extracellular endoglucanase (4 folds), exoglucanase (2.6 folds) and mannanase (2.6 folds) specific activities significantly, indicating that the F12 medium could induce the highest production of extracellular cellulolytic and hemicellulolytic enzymes concomitantly. In conclusion, L. plantarum RI 11 is a promising and versatile bio-transformation agent for lignocellulolytic biomass.

Rapid Evaluation and Optimization of Medium Components Governing Tryptophan Production by Pediococcus acidilactici TP-6 Isolated from Malaysian Food via Statistical Approaches.

Tryptophan is one of the most extensively used amino acids in livestock industry owing to its effectiveness in enhancing the growth performance of animals. Conventionally, the production of tryptophan relies heavily on genetically modified Escherichia coli but its pathogenicity is a great concern. Our recent study demonstrated that a lactic acid bacterium (LAB), Pediococcus acidilactici TP-6 that isolated from Malaysian food was a promising tryptophan producer. However, the tryptophan production must enhance further for viable industrial application. Hence, the current study evaluated the effects of medium components and optimized the medium composition for tryptophan production by P. acidilactici TP-6 statistically using Plackett-Burman Design, and Central Composite Design. The optimized medium containing molasses (14.06 g/L), meat extract (23.68 g/L), urea (5.56 g/L) and FeSO4 (0.024 g/L) significantly enhanced the tryptophan production by 150% as compared to the control de Man, Rogosa and Sharpe medium. The findings obtained in this study revealed that rapid evaluation and effective optimization of medium composition governing tryptophan production by P. acidilactici TP-6 were feasible via statistical approaches. Additionally, the current findings reveal the potential of utilizing LAB as a safer alternative tryptophan producer and provides insight for future exploitation of various amino acid productions by LAB.

Sustainable microbial cell nanofactory for zinc oxide nanoparticles production by zinc-tolerant probiotic Lactobacillus plantarum strain TA4.

BACKGROUND: The use of microorganisms in the biosynthesis of zinc oxide nanoparticles (ZnO NPs) has recently emerged as an alternative to chemical and physical methods due to its low-cost and eco-friendly method. Several lactic acid bacteria (LAB) have developed mechanisms in tolerating Zn2+ through prevention against their toxicity and the production of ZnO NPs. The LAB's main resistance mechanism to Zn2+ is highly depended on the microorganisms' ability to interact with Zn2+ either through biosorption or bioaccumulation processes. Besides the inadequate studies conducted on biosynthesis with the use of zinc-tolerant probiotics, the understanding regarding the mechanism involved in this process is not clear. Therefore, this study determines the features of probiotic LAB strain TA4 related to its resistance to Zn2+. It also attempts to illustrate its potential in creating a sustainable microbial cell nanofactory of ZnO NPs. RESULTS: A zinc-tolerant probiotic strain TA4, which was isolated from local fermented food, was selected based on the principal component analysis (PCA) with the highest score of probiotic attributes. Based on the 16S rRNA gene analysis, this strain was identified as Lactobacillus plantarum strain TA4, indicating its high resistance to Zn2+ at a maximum tolerable concentration (MTC) value of 500 mM and its capability of producing ZnO NPs. The UV-visible spectroscopy analysis proved the formations of ZnO NPs through the notable absorption peak at 380 nm. It was also found from the dynamic light scattering (DLS) analysis that the Z-average particle size amounted to 124.2 nm with monodisperse ZnO NPs. Studies on scanning electron microscope (SEM), energy-dispersive X-ray (EDX) spectroscopy, and Fourier-transform infrared spectroscopy (FT-IR) revealed that the main mechanisms in ZnO NPs biosynthesis were facilitated by the Zn2+ biosorption ability through the functional groups present on the cell surface of strain TA4. CONCLUSIONS: The strong ability of zinc-tolerant probiotic of L. plantarum strain TA4 to tolerate high Zn2+ concentration and to produce ZnO NPs highlights the unique properties of these bacteria as a natural microbial cell nanofactory for a more sustainable and eco-friendly practice of ZnO NPs biosynthesis.

Comparative Study of Extracellular Proteolytic, Cellulolytic, and Hemicellulolytic Enzyme Activities and Biotransformation of Palm Kernel Cake Biomass by Lactic Acid Bacteria Isolated from Malaysian Foods.

Biotransformation via solid state fermentation (SSF) mediated by microorganisms is a promising approach to produce useful products from agricultural biomass. Lactic acid bacteria (LAB) that are commonly found in fermented foods have been shown to exhibit extracellular proteolytic, ß-glucosidase, ß-mannosidase, and ß-mannanase activities. Therefore, extracellular proteolytic, cellulolytic, and hemicellulolytic enzyme activities of seven Lactobacillus plantarum strains (a prominent species of LAB) isolated from Malaysian foods were compared in this study. The biotransformation of palm kernel cake (PKC) biomass mediated by selected L. plantarum strains was subsequently conducted. The results obtained in this study exhibited the studied L. plantarum strains produced versatile multi extracellular hydrolytic enzyme activities that were active from acidic to alkaline pH conditions. The highest total score of extracellular hydrolytic enzyme activities were recorded by L. plantarum RI11, L. plantarum RG11, and L. plantarum RG14. Therefore, they were selected for the subsequent biotransformation of PKC biomass via SSF. The hydrolytic enzyme activities of treated PKC extract were compared for each sampling interval. The scanning electron microscopy analyses revealed the formation of extracellular matrices around L. plantarum strains attached to the surface of PKC biomass during SSF, inferring that the investigated L. plantarum strains have the capability to grow on PKC biomass and perform synergistic secretions of various extracellular proteolytic, cellulolytic, and hemicellulolytic enzymes that were essential for the effective biodegradation of PKC. The substantial growth of selected L. plamtraum strains on PKC during SSF revealed the promising application of selected L. plantarum strains as a biotransformation agent for cellulosic biomass.

Microbial synthesis of zinc oxide nanoparticles and their potential application as an antimicrobial agent and a feed supplement in animal industry: a review.

In recent years, zinc oxide nanoparticles (ZnO NPs) have gained tremendous attention attributed to their unique properties. Notably, evidence has shown that zinc is an important nutrient in living organisms. As such, both prokaryotes and eukaryotes including bacteria, fungi and yeast are exploited for the synthesis of ZnO NPs by using microbial cells or enzyme, protein and other biomolecules compounds in either an intracellular or extracellular route. ZnO NPs exhibit antimicrobial properties, however, the properties of nanoparticles (NPs) are depended upon on their size and shape, which make them specific for various applications. Nevertheless, the desired size and shape of NPs can be obtained through the optimization process of microbes mediated synthesis by manipulating their reaction conditions. It should be noted that ZnO NPs are synthesized by various chemical and physical methods. Nonetheless, these methods are expensive and not environmentally friendly. On that account, the microbes mediated synthesis of ZnO NPs have rapidly evolved recently where the microbes are cleaner, eco-friendly, non-toxic and biocompatible as the alternatives to chemical and physical practices. Moreover, zinc in the form of NPs is more effective than their bulk counterparts and thus, they have been explored for many potential applications including in animals industry. Notably, with the advent of multi-drug resistant strains, ZnO NPs have emerged as the potential antimicrobial agents. This is mainly due to their superior properties in combating a broad spectrum of pathogens. Moreover, zinc is known as an essential trace element for most of the biological function in the animal's body. As such, the applications of ZnO NPs have been reported to significantly enhance the health and production of the farm animals. Thus, this paper reviews the biological synthesis of ZnO NPs by the microbes, the mechanisms of the biological synthesis, parameters for the optimization process and their potential application as an antimicrobial agent and feed supplement in the animal industry as well as their toxicological hazards on animals.

Optimized medium via statistical approach enhanced threonine production by Pediococcus pentosaceus TL-3 isolated from Malaysian food.

BACKGROUND: Threonine is an essential amino acid that is extensively used in livestock industry as feed supplement due to its pronounced effect in improving the growth performance of animals. Application of genetically engineered bacteria for amino acid production has its share of controversies after eosinophils myalgia syndrome outbreak in 1980s. This has urged for continuous search for a food grade producer as a safer alternative for industrial amino acid production. Lactic acid bacteria (LAB) appear as an exceptional candidate owing to their non-pathogenic nature and reputation of Generally Recognized as Safe (GRAS) status. Recently, we have identified a LAB, Pediococcus pentosaceus TL-3, isolated from Malaysian food as a potential threonine producer. Thus, the objective of this study was to enhance the threonine production by P. pentosaceus TL-3 via optimized medium developed by using Plackett-Burman design (PBD) and central composite design (CCD). RESULTS: Molasses, meat extract, (NH4)2SO4, and MnSO4 were identified as the main medium components for threonine production by P. pentosaceus TL-3. The optimum concentration of molasses, meat extract, (NH4)2SO4 and MnSO4 were found to be 30.79 g/L, 25.30 g/L, 8.59 g/L, and 0.098 g/L respectively based on model obtained in CCD with a predicted net threonine production of 123.07 mg/L. The net threonine production by P. pentosaceus TL-3 in the optimized medium was enhanced approximately 2 folds compared to the control. CONCLUSIONS: This study has revealed the potential of P. pentosaceus TL-3 as a safer alternative to produce threonine. Additionally, the current study has identified the key medium components affecting the production of threonine by P. pentosaceus TL-3, followed by optimization of their concentrations by means of statistical approach. The findings of this study could act as a guideline for the future exploration of amino acid production by LAB.

Extracellular Proteolytic Activity and Amino Acid Production by Lactic Acid Bacteria Isolated from Malaysian Foods.

Amino acids (AAs) are vital elements for growth, reproduction, and maintenance of organisms. Current technology uses genetically engineered microorganisms for AAs production, which has urged the search for a safer food-grade AA producer strain. The extracellular proteolytic activities of lactic acid bacteria (LAB) can be a vital tool to hydrolyze extracellular protein molecules into free AAs, thereby exhibiting great potential for functional AA production. In this study, eight LAB isolated from Malaysian foods were determined for their extracellular proteolytic activities and their capability of producing AAs. All studied LAB exhibited versatile extracellular proteolytic activities from acidic to alkaline pH conditions. In comparison, Pediococcus pentosaceus UP-2 exhibited the highest ability to produce 15 AAs extracellularly, including aspartate, lysine, methionine, threonine, isoleucine, glutamate, proline, alanine, valine, leucine, tryptophan, tyrosine, serine, glycine, and cystine, followed by Pediococcus pentosaceus UL-2, Pediococcus acidilactici UB-6, and Pediococcus acidilactici UP-1 with 11 to 12 different AAs production detected extracellularly. Pediococcus pentosaceus UL-6 demonstrated the highest increment of proline production at 24 h of incubation. However, Pediococcus acidilactici UL-3 and Lactobacillus plantarum I-UL4 exhibited the greatest requirement for AA. The results of this study showed that different LAB possess different extracellular proteolytic activities and potentials as extracellular AA producers.

Comparative studies of versatile extracellular proteolytic activities of lactic acid bacteria and their potential for extracellular amino acid productions as feed supplements.

BACKGROUND: Increasing understanding on the functions of amino acids (AA) has led to new commercial applications and expansion of the worldwide markets. However, the current technologies rely heavily on non-food grade microorganism and chemical synthesis for the production of AA. Several studies reported that lactic acid bacteria (LAB) have the capability of producing AA owing to their well-established proteolytic system and amino acid biosynthesis genes. Hence, the objectives of this study were to explore the extracellular proteolytic activity of LAB isolated from various Malaysian fermented foods and their potential to produce AA extracellularly as feed supplements. RESULTS: All the studied LAB isolates were versatile extracellular protease producers, whereby extracellular protease activities were detected from acidic to alkaline pH (pH 5, pH 6.5, pH 8) using qualitative and quantitative proteolytic assays. The highest proteolytic activity at pH 5 (15.76 U/mg) and pH 8 (19.42 U/mg) was achieved by Lactobacillus plantarum RG14, while Lactobacillus plantarum RS5 exhibited the highest proteolytic activity of 17.22 U/mg at pH 6.5. As for the results of AA production conducted in de Man, Rogosa and Sharpe medium and analysed by high pressure liquid chromatography system, all LAB isolates were capable of producing an array of AA. Generally, Pediococcus sp. showed greater ability for AA production as compared to Lactobacillus sp. Moreover, the studied LAB were able to produce a few major feed supplement AA such as methionine, lysine, threonine and tryptophan. P. pentosaceus TL-3 recorded the highest methionine and threonine productivity of 3.72 mg/L/h and 5.58 mg/L/h respectively. However, L. plantarum I-UL4 demonstrated a lysine productivity of 1.24 mg/L/h, while P. acidilactici TP-6 achieved up to 1.73 mg/L/h of tryptophan productivity. CONCLUSION: All the 17 studied LAB isolates possessed versatile extracellular proteolytic system and have vast capability of producing various amino acids including a few major feed supplement AA such as methionine, lysine, threonine and tryptophan. Despite AA production was strain dependent, the studied LAB isolates possessed vast potential and can be exploited further as a bio-agent or an alternative amino acids and bioactive peptide producers.

In vitro molecular study of wound healing using biosynthesized bacteria nanocellulose/silver nanocomposite assisted by bioinformatics databases.

BACKGROUND: In recent years, bacterial nanocellulose (BNC) based nanocomposites have been developed to promote healing property and antibacterial activity of BNC wound dressing. Molecular study can help to better understanding about interaction of genes and pathways involved in healing progression. OBJECTIVES: The aim of this study was to prepare bacterial nanocellulose/silver (BNC/Ag) nanocomposite films as ecofriendly wound dressing in order to assess their physical, cytotoxicity and antimicrobial properties. The in vitro molecular study was performed to evaluate expression of genes involved in healing of wounds after treatment with BNC/Ag biofilms. STUDY DESIGN MATERIALS AND METHODS: Silver nanoparticles were formed by using Citrullus colocynthis extract within new isolated bacterial nanocellulose (BNC) RM1. The nanocomposites were characterized using X-ray diffraction, Fourier transform infrared, and field emission scanning electron microscopy. Besides, swelling property and Ag release profile of the nanocomposites were studied. The ability of nanocomposites to promote wound healing of human dermal fibroblast cells in vitro was studied. Bioinformatics databases were used to identify genes with important healing effect. Key genes which interfered with healing were studied by quantitative real time PCR. RESULTS: Spherical silver nanoparticles with particle size ranging from 20 to 50 nm were synthesized and impregnated within the structure of BNC. The resulting nanocomposites showed significant antibacterial activities with inhibition zones ranging from 7±0.25 to 16.24±0.09 mm against skin pathogenic bacteria. Moreover, it was compatible with human fibroblast cells (HDF) and could promote in vitro wound healing after 48h. Based on bioinformatics databases, the genes of TGF-ß1, MMP2, MMP9, CTNNB1, Wnt4, hsa-miR-29b-3p and hsa-miR-29c-3p played important role in wound healing. The nanocomposites had an effect in expression of the genes in healing. Thus, the BNC/Ag nanocomposite can be used to heal wound in a short period and simple manner. CONCLUSION: This eco-friendly nanocomposite with excellent antibacterial activities and healing property confirming its utility as potential wound dressings.