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Probiotic Strain Limosilactobacillus reuteri 29B is Proven Safe and Exhibits Potential Probiotic Traits in a Murine Vaginal Model

Rangasamy P, Foo HL, Yusof BNM, Chew SY, Jamil AAM, Than LTL

Probiotics Antimicrob Proteins, 2023 Jun 14.
PMID: 37314695 DOI: 10.1007/s12602-023-10094-2

Lactobacilli, the most common group of bacteria found in a healthy vaginal microbiota, have been demonstrated to act as a defence against colonisation and overgrowth of vaginal pathogens. These groups of bacteria have sparked interests in incorporating them as probiotics aimed at re-establishing balance within the urogenital ecosystem. In this study, the safety characteristics of Limosilactobacillus reuteri 29B (L29B) strain were evaluated through whole genome sequencing (WGS) and animal study. Cell culture assay and 16S rDNA analysis were done to evaluate the ability of the strain to colonise and adhere to the mouse vaginal tract, and RAST analysis was performed to screen for potential genes associated with probiotic trait. The histological study on the mice organs and blood analysis of the mice showed there was no incidence of inflammation. We also found no evidence of bacterial translocation. The cell culture assay on HeLa cells showed 85% of adhesion, and there was a significant reduction of Candida strain viability in displacement assay. As for the 16S rDNA analysis, there was a significant amount of L29B colonisation of the vaginal microflora. Taken together, the intravaginal administration of L29B significantly reduced the number Enterobacteriaceae and Staphylococcaceae that were present in mouse vaginal tract. It also improved and promoted a balanced vaginal microflora environment without causing any harm or irritation to mice. Limosilactobacillus 29B (L29B) is safe to be administered intravaginally.
Fulltext Brown and Green Seaweed Antioxidant Properties and Effects on Blood Plasma Antioxidant Enzyme Activities, Hepatic Antioxidant Genes Expression, Blood Plasma Lipid Profile, and Meat Quality in Broiler Chickens

Azizi MN, Loh TC, Foo HL, Akit H, Izuddin WI, Yohanna D

Animals (Basel), 2023 May 09;13(10).
PMID: 37238013 DOI: 10.3390/ani13101582

The study was designed to analyze the effects of brown seaweed (BS) and green seaweed (GS) on blood plasma antioxidant enzyme activities, hepatic antioxidant genes expression, blood plasma lipid profile, breast meat quality, and chemical composition in broiler chickens. The dietary treatment groups contained basal diet [negative control (NC)], basal diet + vitamin E (100 mg/kg feed) [positive control (PC)], basal diet + 0.25, 0.50, 0.75, 1, and 1.25% BS and GS supplements separately. The findings showed that both BS and GS exhibited remarkable antioxidant activity. In contrast, the maximum antioxidant activity was recorded by BS (55.19%), which was significantly higher than the GS (25.74%). Results showed that various levels of BS and GS had no significant effects on broiler blood plasma catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) enzyme activities. The hepatic superoxide dismutase 1 (SOD1) gene mRNA expression was significantly higher for birds fed 0.50% and 0.75% BS. Regarding the plasma lipid profile, the total cholesterol (TC) and high-density lipoprotein (HDL) levels were higher (p < 0.05) for birds fed 0.75 and 1% BS compared to the negative and positive control groups. The findings showed that different levels of BS and GS had significantly higher breast meat crude protein (CP) content.
Fulltext Biotransformation of gluten-free composite flour mediated by probiotics via solid-state fermentation process conducted under different moisture contents

Koyum KA, Foo HL, Ramli N, Loh TC

Front Nutr, 2023;10:910537.
PMID: 36875851 DOI: 10.3389/fnut.2023.910537

Staple foods produced from composite flour are considered feasible to alleviate protein-energy malnutrition (PEM). However, one of the major limitations of composite flour is poor protein digestibility. The biotransformation process mediated by probiotics via solid-state fermentation (SSF) holds a promising potential to address the poor protein digestibility in composite flour. Yet, there is no report established in this regard to the best of our knowledge. Therefore, 4 strains of Lactiplantibacillus plantarum and Pediococcus pentosaceus UP2 isolated from Malaysian foods that were previously reported to produce versatile extracellular hydrolytic enzymes were employed to biotransform gluten-free composite flour derived from rice, sorghum, and soybean. The SSF process was performed under 30-60% (v/w) moisture content for 7 days, where samples were withdrawn at 24 h intervals for various analyses such as pH, total titratable acidity (TTA), extracellular protease activity, soluble protein concentration, crude protein content, and in vitro protein digestibility. The pH of the biotransformed composite flour showed a significant reduction from the initial range of pH 5.98-6.67 to the final pH of 4.36-3.65, corresponding to the increase in the percentage of TTA in the range of 0.28-0.47% to 1.07-1.65% from days 0 to 4 and remained stable till day 7 of the SSF process. The probiotics strains exhibited high extracellular proteolytic activity (0.63-1.35 U/mg to 4.21-5.13 U/mg) from days 0 to 7. In addition, the treated composite flour soluble protein increased significantly (p ≤ 0.05) (0.58-0.60 mg/mL to 0.72-0.79 mg/mL) from days 0 to 7, crude protein content (12.00-12.18% to 13.04-14.39%) and protein digestibility (70.05-70.72% to 78.46-79.95%) from days 0 to 4 of SSF. The results of biotransformation of 50% (v/w) moisture content were mostly comparable to 60% (v/w) moisture content, implying 50% (v/w) moisture content was the most suitable moisture content for the effective biotransformation of gluten-free composite flour mediated by probiotics via SSF since flour quality is better at lower moisture content. As for the overall performance, L. plantarum RS5 was ranked the best strain, attributed to the general improvement in the physicochemical properties of composite flour.
Fulltext Blood lipid profiles, fatty acid deposition and expression of hepatic lipid and lipoprotein metabolism genes in laying hens fed palm oils, palm kernel oil, and soybean oil

Izuddin WI, Loh TC, Nayan N, Akit H, Noor AM, Foo HL

Front Vet Sci, 2023;10:1192841.
PMID: 37519991 DOI: 10.3389/fvets.2023.1192841

The palm oil, palm kernel oil and soybean oil have unique and distinctive fatty acid chain length and saturation profiles, and how they affect lipid peroxidation, fatty acid intake and metabolism is worth exploring in poultry. This study elucidated the influence the dietary oils on lipid peroxidation, blood lipid profiles, fatty acid deposition of liver, serum and yolk and the expression of liver genes related to lipid and lipoprotein metabolism in laying hens. About 150 Hisex brown laying hens were fed diets containing crude palm oil (CPO), red palm oil (RPO), refined palm oil (RBD), palm kernel oil (PKO) or soybean oil (SBO) for 16 weeks. Serum, liver and yolk lipid peroxidation were not different between dietary oils. The PKO increased liver, serum and yolk medium-chain fatty acids (MCFA). There was no difference in liver saturated fatty acids (SFA). The CPO and RPO reduced serum SFA, but the PKO increased yolk SFA. The SBO increased polyunsaturated fatty acids (PUFA) in liver serum and yolk. No difference in liver elaidic acid (C18:1-trans), but SBO lowered elaidic acid (C18:1-trans) in serum. Higher very-low density lipoprotein (VLDL) in CPO than RPO and SBO and greater serum lipase in CPO, RBD and PKO than SBO. There was no difference in sterol regulatory element-binding protein 2 (SREBP-II) between oils. Apolipoprotein VLDL-II (APOVLDL2) was upregulated in palm oils and apolipoprotein B-100 (APOB) in RBD. Downregulation in peroxisome proliferator-activated receptor-alpha (PPAR-α), peroxisome proliferator-activated receptor gamma (PPAR-γ) and low-density lipoprotein receptor (LDLR) was observed in palm oils and PKO. In conclusion, different dietary oils greatly influence several aspects of fatty acid metabolism, deposition and lipoprotein profiles but have no influence on reducing lipid peroxidation.
Effects of Postbiotics and Paraprobiotics as Replacements for Antibiotics on Growth Performance, Carcass Characteristics, Small Intestine Histomorphology, Immune Status and Hepatic Growth Gene Expression in Broiler Chickens

Danladi Y, Loh TC, Foo HL, Akit H, Md Tamrin NA, Naeem Azizi M

Animals (Basel), 2022 Apr 03;12(7).
PMID: 35405905 DOI: 10.3390/ani12070917

Background: This experiment was designed to investigate how replacing antibiotics with postbiotics and paraprobiotics could affect growth performance, small intestine morphology, immune status, and hepatic growth gene expression in broiler chickens. Methods: The experiment followed a completely randomized design (CRD) in which eight treatments were replicated six times with seven birds per replicate. A total of 336, one-day-old (COBB 500) chicks were fed with the eight treatment diets, which include T1 = negative control (Basal diet), T2 = positive control (Basal diet + 0.01% (w/w) Oxytetracycline), T3 = Basal diet + 0.2% (v/w) postbiotic TL1, T4 = Basal diet + 0.2% (v/w) postbiotic RS5, T5 = Basal diet + 0.2% (v/w) paraprobiotic RG11, T6 = Basal diet + 0.2% (v/w) postbiotic RI11, T7 = Basal diet + 0.2% (v/w) paraprobiotic RG14, T8 = Basal diet + 0.2% (v/w) paraprobiotic RI11, for 35 days in a closed house system. Results: The growth performance indicators (final body weight, cumulative weight gain, and feed conversion ratio) were not significantly (p > 0.05) affected by the dietary treatments. However, feed intake recorded a significant (p < 0.05) change in the starter and finisher phases across the dietary treatments. Paraprobiotic RG14 had significantly (p < 0.05) lower abdominal fat and intestines. Villi heights were significantly (p < 0.05) increased, while the crypt depth decreased significantly due to dietary treatments. The dietary treatments significantly influenced colon mucosa sIgA (p < 0.05). Similarly, plasma immunoglobulin IgM level recorded significant (p < 0.05) changes at the finisher phase. In this current study, the hepatic GHR and IGF-1 expressions were significantly (p < 0.05) increased by postbiotics and paraprobiotics supplementation. Conclusions: Therefore, it was concluded that postbiotics and paraprobiotics differ in their effect on broiler chickens. However, they can replace antibiotics without compromising the growth performance, carcass yield, and immune status of broiler chickens.
Fulltext Chemical Compositions of Brown and Green Seaweed, and Effects on Nutrient Digestibility in Broiler Chickens

Azizi MN, Loh TC, Foo HL, Akit H, Izuddin WI, Shazali N, et al.

Animals (Basel), 2021 Jul 20;11(7).
PMID: 34359273 DOI: 10.3390/ani11072147

This study aimed to analyse the nutritional properties, apparent ileal digestibility (AID) and apparent metabolisable energy (AME) of broiler chickens fed with brown seaweed (BS) and green seaweed (GS). Proximate analysis was performed to determine the nutrient composition of seaweed. The amino acids were determined using high-performance liquid chromatography (HPLC), and atomic absorption spectroscopy was used to determine the minerals content. The gross energy (GE) was determined using a fully automatic bomb calorimeter, and the AME value was calculated. Titanium dioxide (TiO2) was used as an indigestible marker to calculate the AID. A digestibility trial was conducted to investigate the effects of seaweeds on crude protein (CP), crude fibre (CF), ether extract (EE), dry matter (DM), organic matter (OM), amino acids (AA) and minerals digestibility, and AME on broiler chickens. Thirty-six broiler chickens were randomly distributed into two dietary treatment groups with six replicates and three birds per replicate. Results showed that brown and green seaweed was a source of macro and micronutrients. For the AME and AID of seaweed-based diets, the results showed that the AME value for BS and GS was 2894.13 and 2780.70 kcal/kg, respectively. The AID of BS and GS was 88.82% and 86.8% for EE, 82.03% and 80.6% for OM, 60.69% and 57.80% for CP, 48.56 and 44.02% for CF, and 17.97 and 19.40% for ash contents, respectively. Meanwhile, the AID of CP and CF was significantly higher for BS compared to the GS. Findings showed that the AID of various AA was 40.96 to 77.54%, and the AID of selected minerals (Ca, Na, K, Mg, Zn, Cu, Fe) for both BS and GS groups were above 90%.
Fulltext A refined medium to enhance the antimicrobial activity of postbiotic produced by Lactiplantibacillus plantarum RS5

Ooi MF, Foo HL, Loh TC, Mohamad R, Rahim RA, Ariff A

Sci Rep, 2021 Apr 07;11(1):7617.
PMID: 33828119 DOI: 10.1038/s41598-021-87081-6

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.
Fulltext Supplementation of postbiotic RI11 improves antioxidant enzyme activity, upregulated gut barrier genes, and reduced cytokine, acute phase protein, and heat shock protein 70 gene expression levels in heat-stressed broilers

Humam AM, Loh TC, Foo HL, Izuddin WI, Zulkifli I, Samsudin AA, et al.

Poult Sci, 2021 Mar;100(3):100908.
PMID: 33518339 DOI: 10.1016/j.psj.2020.12.011

The aim of this work was to evaluate the impacts of feeding different levels of postbiotic RI11 on antioxidant enzyme activity, physiological stress indicators, and cytokine and gut barrier gene expression in broilers under heat stress. A total of 252 male broilers Cobb 500 were allocated in cages in environmentally controlled chambers. All the broilers received the same basal diet from 1 to 21 d. On day 22, the broilers were weighed and grouped into 7 treatment groups and exhibited to cyclic high temperature at 36 ± 1°C for 3 h per day until the end of the experiment. From day 22 to 42, broilers were fed with one of the 7 following diets: negative control, basal diet (0.0% RI11) (NC group); positive control, NC diet + 0.02% (w/w) oxytetracycline (OTC group); antioxidant control, NC diet + 0.02% (w/w) ascorbic acid. The other 4 other groups were as follows: NC diet + 0.2% cell-free supernatant (postbiotic RI11) (v/w), NC diet + 0.4% cell-free supernatant (postbiotic RI11) (v/w), NC diet + 0.6% cell-free supernatant (postbiotic RI11) (v/w), and NC diet + 0.8% cell-free supernatant (postbiotic RI11) (v/w). Supplementation of different levels (0.4, 0.6, and 0.8%) of postbiotic RI11 increased plasma glutathione peroxidase, catalase, and glutathione enzyme activity. Postbiotic RI11 groups particularly at levels of 0.4 and 0.6% upregulated the mRNA expression of IL-10 and downregulated the IL-8, tumor necrosis factor alpha, heat shock protein 70, and alpha-1-acid glycoprotein levels compared with the NC and OTC groups. Feeding postbiotic RI11, particularly at the level of 0.6%, upregulated ileum zonula occludens-1 and mucin 2 mRNA expressions. However, no difference was observed in ileum claudin 1, ceruloplasmin, IL-6, IL-2, and interferon expression, but downregulation of occludin expression was observed as compared with the NC group. Supplementation of postbiotic RI11 at different levels quadratically increased plasma glutathione peroxidase, catalase and glutathione, IL-10, mucin 2, and zonula occludens-1 mRNA expression and reduced plasma IL-8, tumor necrosis factor alpha, alpha-1-acid glycoprotein, and heat shock protein 70 mRNA expression. The results suggested that postbiotics produced from Lactiplantibacillus plantarum RI11 especially at the level of 0.6% (v/w) could be used as an alternative to antibiotics and natural sources of antioxidants in poultry feeding.
Fulltext Anti-cancer and anti-inflammatory effects elicited by short chain fatty acids produced by Escherichia coli isolated from healthy human gut microbiota

Nakkarach A, Foo HL, Song AA, Mutalib NEA, Nitisinprasert S, Withayagiat U

Microb Cell Fact, 2021 Feb 05;20(1):36.
PMID: 33546705 DOI: 10.1186/s12934-020-01477-z

BACKGROUND: Extracellular metabolites of short chain fatty acids (SCFA) excreted by gut microbiota have been reported to play an important role in the regulation of intestinal homeostasis. Apart from supplying energy, SCFA also elicit immune stimulation in animal and human cells. Therefore, an attempt was conducted to isolate SCFA producing bacteria from healthy human microbiota. The anti-cancer and anti-inflammatory effects of extracellular metabolites and individual SFCA were further investigated by using breast, colon cancer and macrophage cells. Toxin, inflammatory and anti-inflammatory cytokine gene expressions were investigated by RT-qPCR analyses in this study.
RESULTS: Escherichia coli KUB-36 was selected in this study since it has the capability to produce seven SCFA extracellularly. It produced acetic acid as the main SCFA. It is a non-exotoxin producer and hence, it is a safe gut microbiota. The IC50 values indicated that the E. coli KUB-36 metabolites treatment elicited more potent cytotoxicity effect on MCF7 breast cancer cell as compared to colon cancer and leukemia cancer cells but exhibited little cytotoxic effects on normal breast cell. Furthermore, E. coli KUB-36 metabolites and individual SCFA could affect inflammatory responses in lipopolysaccharide-induced THP-1 macrophage cells since they suppressed inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α well as compared to the control, whilst inducing anti-inflammatory cytokine IL-10 expression.
CONCLUSION: SCFA producing E. coli KUB-36 possessed vast potential as a beneficial gut microbe since it is a non-exotoxin producer that exhibited beneficial cytotoxic effects on cancer cells and elicited anti-inflammatory activity simultaneously. However, the probiotic characteristic of E. coli KUB-36 should be further elucidated using in vivo animal models.
Fulltext Is Palm Kernel Cake a Suitable Alternative Feed Ingredient for Poultry?

Azizi MN, Loh TC, Foo HL, Teik Chung EL

Animals (Basel), 2021 Jan 29;11(2).
PMID: 33572711 DOI: 10.3390/ani11020338

Palm kernel cake (PKC), a by-product of oil extracted from palm nuts through expeller press or solvent extraction procedures is one of the highest quantities of locally available and potentially inexpensive agricultural product. PKC provides approximately 14-18% of crude protein (CP), 12-20% crude fiber (CF), 3-9% ether extract (EE), and different amounts of various minerals that feasible to be used as a partial substitute of soybean meal (SBM) and corn in poultry nutrition. Poultry's digestibility is reported to be compromised due to the indigestion of the high fiber content, making PKC potentially low for poultry feeding. Nevertheless, solid-state fermentation (SSF) can be applied to improve the nutritional quality of PKC by improving the CP and reducing CF content. PKC also contains β-mannan polysaccharide, which works as a prebiotic. However, there is a wide variation for the inclusion level of PKC in the broiler diet. These variations may be due to the quality of PKC, its sources, processing methods and value-added treatment. It has been documented that 10-15% of treated PKC could be included in the broiler's diets. The inclusion levels will not contribute to a negative impact on the growth performances and carcass yield. Furthermore, it will not compromise intestinal microflora, morphology, nutrient digestibility, and immune system. PKC with a proper SSF process (FPKC) can be offered up to 10-15% in the diets without affecting broilers' production performance.
Fulltext Promising discovery of beneficial Escherichia coli in the human gut

Nakkarach A, Foo HL, Song AA, Nitisinprasert S, Withayagiat U

3 Biotech, 2020 Jul;10(7):296.
PMID: 32550113 DOI: 10.1007/s13205-020-02289-z

Ingested dietary fibres are hydrolysed by colon microbiota to produce energy-providing short-chain fatty acids (SCFA) that stimulate anti-inflammatory effects. SCFA-producing bacteria were screened from bacteria isolated from human faeces using bromothymol blue as an acid indicator and gas chromatography for SCFA profiling. The beneficial functions (antagonistic activity, haemolytic activities, antibiotic susceptibility, mucus adherent percentage and toxin gene detection) were evaluated for the top five SCFA-producing bacteria isolated from three healthy volunteers that identified as Escherichia coli strains. They produced acetic, propionic, isobutyric, butyric, isovaleric, valeric and caproic acids at average concentrations of 15.9, 1.8, 1.1, 1.9, 1.8, 2.7 and 3.4 mM, respectively. The SCFA production by E. coli strains was rapidly increased during the first 8 h of incubation and gradually decreased after 16 h of incubation. All E. coli strains showed acid and bile tolerance, resulting in a survival rate greater than 70% with no haemolytic activity, mucus adherence greater than 40% and susceptibility to conventional antibiotics. Hence, the selected E. coli strains exhibited promising probiotic properties with neither enterotoxin nor LPS producibility was detected. The present results confirm the existence of friendly and harmless E. coli strains in human microbiota as potential probiotics.
Fulltext Dietary Supplementation of Postbiotics Mitigates Adverse Impacts of Heat Stress on Antioxidant Enzyme Activity, Total Antioxidant, Lipid Peroxidation, Physiological Stress Indicators, Lipid Profile and Meat Quality in Broilers

Humam AM, Loh TC, Foo HL, Izuddin WI, Awad EA, Idrus Z, et al.

Animals (Basel), 2020 Jun 05;10(6).
PMID: 32516896 DOI: 10.3390/ani10060982

The purpose of this work was to evaluate the impacts of feeding different postbiotics on oxidative stress markers, physiological stress indicators, lipid profile and meat quality in heat-stressed broilers. A total of 252 male Cobb 500 (22-day-old) were fed with 1 of 6 diets: A basal diet without any supplementation as negative control (NC); basal diet + 0.02% oxytetracycline served as positive control (PC); basal diet + 0.02% ascorbic acid (AA); or the basal diet diet + 0.3% of RI11, RS5 or UL4 postbiotics. Postbiotics supplementation, especially RI11 increased plasma activity of total-antioxidant capacity (T-AOC), catalase (CAT) and glutathione (GSH), and decreased alpha-1-acid-glycoprotein (α1-AGP) and ceruloplasmin (CPN) compared to NC and PC groups. Meat malondialdehyde (MDA) was lower in the postbiotic groups than the NC, PC and AA groups. Plasma corticosterone, heat shock protein70 (HSP70) and high density lipoprotein (HDL) were not affected by dietary treatments. Postbiotics decreased plasma cholesterol concentration compared to other groups, and plasma triglyceride and very low density lipoprotein (VLDL) compared to the NC group. Postbiotics increased breast meat pH, and decreased shear force and lightness (L*) compared to NC and PC groups. The drip loss, cooking loss and yellowness (b*) were lower in postbiotics groups compared to other groups. In conclusion, postbiotics particularly RI11 could be used as an alternative to antibiotics and natural sources of antioxidants for heat-stressed broilers.
Fulltext Enhancement of Versatile Extracellular Cellulolytic and Hemicellulolytic Enzyme Productions by Lactobacillus plantarum RI 11 Isolated from Malaysian Food Using Renewable Natural Polymers

Mohamad Zabidi NA, Foo HL, Loh TC, Mohamad R, Abdul Rahim R

Molecules, 2020 Jun 03;25(11).
PMID: 32503356 DOI: 10.3390/molecules25112607

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

Lim YH, Foo HL, Loh TC, Mohamad R, Abdul Rahim R

Molecules, 2020 Feb 11;25(4).
PMID: 32054138 DOI: 10.3390/molecules25040779

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.
Fulltext Growth Performance, Cytokine Expression, and Immune Responses of Broiler Chickens Fed a Dietary Palm Oil and Sunflower Oil Blend Supplemented With L-Arginine and Varying Concentrations of Vitamin E

Khatun J, Loh TC, Foo HL, Akit H, Khan KI

Front Vet Sci, 2020;7:619.
PMID: 33195499 DOI: 10.3389/fvets.2020.00619

This study set out to examine the combined effects of the supplementation of a dietary palm oil (PO) and sunflower oil (SO) blend, 0. 25% L-Arginine (L-Arg), and different levels of vitamin E (Vit E) on growth performance, fat deposition, cytokine expression, and immune response in broilers. A total of 216 1-day-old male broiler chicks (Cobb500) were randomly distributed into six dietary groups as follows: Diet 1: 6% palm oil (negative control); Diet 2: PO and SO blend (4% palm oil and 2% sunflower oil) + 0.25% L-Arg (positive control); Diet 3: (PO and SO blend + 0.25% L-Arg) + 20 mg/kg Vit E; Diet 4: (PO and SO blend + 0.25% L-Arg) + 50 mg/kg Vit E; Diet 5: (PO and SO blend + 0.25% L-Arg) + 100 mg/kg Vit E; and Diet 6: (PO and SO blend + 0.25% L-Arg) + 150 mg/kg Vit E. Weight gain and serum IgG and IgM increased while feed conversion ratio, fat deposition, and plasma cholesterol decreased in broilers fed Vit E with the oil blend and L-Arg, compared to those fed the negative control (Diet 1). Expression of IFN and TNF-α were reduced, whereas TGF-ß1 was up-regulated as the level of Vit E increased in the broiler diets. In summary, the combination of oil blend, L-Arg, and Vit E at a level of 50 mg/kg increased the performance and altered the expression of cytokines that may positively influence immune function in broiler chickens.
Fulltext Comparative Studies of Inhibitory and Antioxidant Activities, and Organic Acids Compositions of Postbiotics Produced by Probiotic Lactiplantibacillus plantarum Strains Isolated From Malaysian Foods

Chang HM, Foo HL, Loh TC, Lim ETC, Abdul Mutalib NE

Front Vet Sci, 2020;7:602280.
PMID: 33575277 DOI: 10.3389/fvets.2020.602280

Despite inflammation being a protective natural defense against imbalance stressors in the body, chronic inflammation could lead to the deterioration of immune response, low production, and poor performance in livestock as well as severe economic losses to the farmers. Postbiotics produced by Lactiplantibacillus plantarum has been reported recently to be a natural source of antioxidant, promoting growth performance, anti-inflammation, and immune responses. However, the effects of fermentation media on the compositions of L. plantarum postbiotic have not been reported elsewhere. Hence, a comparative study was conducted to compare the volatile compounds, organic acid composition, and antioxidant and antimicrobial activities of postbiotics produced by six strains of L. plantarum cultivated by using formulated media and the commercial de Man, Rogosa, and Sharpe (MRS) medium as a control. Postbiotics RG14, RI11, and UL4 produced by using formulated media exhibited higher inhibitory activity against Pediococcus acidilactici 446, Escherichia coli E-30, Salmonella enterica CS3, and vancomycin-resistant Enterococci except for Listeria monocytogenes LS55. As for the antioxidant activity, hydroxyl radical scavenging activity was enhanced in formulated media, whereas reducing power activity was the highest in postbiotic RI11. Three organic acids, namely, acetic acid, caproic acid, and lactic acid, were detected in the postbiotic produced by various L. plantarum strains. The concentration of acetic acid was influenced by the fermentation media, whereas caproic acid was detected as the highest in postbiotic RG11. Lactic acid was the predominant compound detected in all the postbiotics and had the significantly highest concentration in postbiotic RS5 when produced by using the MRS medium. Intermediary and pyrrole compounds were the other main compounds that were detected by using GC-MS. Positive correlations were found between organic acid production and inhibitory activity, as well as antioxidant activity exhibited by postbiotics. In conclusion, the compositions and functional characteristics of postbiotics produced by the six strains of L. plantarum were strain-dependent and affected greatly by the fermentation medium. The effects of postbiotic composition on the functional characteristics of postbiotics were elucidated in this study to warrant their applications as a promising beneficial natural growth promoter for the livestock industry.
Fulltext 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

Lee FH, Wan SY, Foo HL, Loh TC, Mohamad R, Abdul Rahim R, et al.

Int J Mol Sci, 2019 Oct 09;20(20).
PMID: 31600952 DOI: 10.3390/ijms20204979

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, β-glucosidase, β-mannosidase, and β-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.
Fulltext Effects of postbiotic supplementation on growth performance, ruminal fermentation and microbial profile, blood metabolite and GHR, IGF-1 and MCT-1 gene expression in post-weaning lambs

Izuddin WI, Loh TC, Samsudin AA, Foo HL, Humam AM, Shazali N

BMC Vet Res, 2019 Sep 02;15(1):315.
PMID: 31477098 DOI: 10.1186/s12917-019-2064-9

BACKGROUND: Postbiotics have been established as potential feed additive to be used in monogastric such as poultry and swine to enhance health and growth performance. However, information on the postbiotics as feed additive in ruminants is very limited. The aim of this study was to elucidate the effects of supplementation of postbiotics in newly-weaned lambs on growth performance, digestibility, rumen fermentation characteristics and microbial population, blood metabolite and expression of genes related to growth and volatile fatty acid transport across the rumen epithelium.
RESULTS: Postbiotic supplementation increased weight gain, feed intake, nutrient intake and nutrient digestibility of the lambs. No effect on ruminal pH and total VFA, whereas butyrate and ruminal ammonia-N concentration were improved. The lambs fed with postbiotics had higher blood total protein, urea nitrogen and glucose. However, no difference was observed in blood triglycerides and cholesterol levels. Postbiotics increased the population of fibre degrading bacteria but decreased total protozoa and methanogens in rumen. Postbiotics increased the mRNA expression of hepatic IGF-1 and ruminal MCT-1.
CONCLUSIONS: The inclusion of postbiotics from L. plantarum RG14 in newly-weaned lambs improved growth performance, nutrient intake and nutrient digestibility reflected from better rumen fermentation and microbial parameters, blood metabolites and upregulation of growth and nutrient intake genes in the post-weaning lambs.
Fulltext Optimized medium via statistical approach enhanced threonine production by Pediococcus pentosaceus TL-3 isolated from Malaysian food

Lim YH, Foo HL, Loh TC, Mohamad R, Abdul Rahim R, Idrus Z

Microb Cell Fact, 2019 Jul 22;18(1):125.
PMID: 31331395 DOI: 10.1186/s12934-019-1173-2

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.
Fulltext Postbiotic L. plantarum RG14 improves ruminal epithelium growth, immune status and upregulates the intestinal barrier function in post-weaning lambs

Izuddin WI, Loh TC, Foo HL, Samsudin AA, Humam AM

Sci Rep, 2019 Jul 09;9(1):9938.
PMID: 31289291 DOI: 10.1038/s41598-019-46076-0

We investigate the effects of postbiotic Lactobacillus plantarum RG14 on gastrointestinal histology, haematology, mucosal IgA concentration, microbial population and mRNA expression related to intestinal mucosal immunity and barrier function. Twelve newly weaned lambs were randomly allocated to two treatment groups; the control group without postbiotic supplementation and postbiotic group with supplementation of 0.9% postbiotic in the diet over a 60-day trial. The improvement of rumen papillae height and width were observed in lambs fed with postbiotics. In contrast, no difference was shown in villi height of duodenum, jejunum and ileum between the two groups. Lambs received postbiotics had a lower concentration of IgA in jejunum but no difference in IgA concentration in serum and mucosal of the rumen, duodenum and ileum. In respect of haematology, postbiotics lowered leukocyte, lymphocyte, basophil, neutrophil and platelets, no significant differences in eosinophil. The increase in of IL-6 mRNA and decrease of IL-1β, IL-10, TNF mRNA were observed in the jejunum of lambs receiving postbiotics. Postbiotics also improved the integrity of the intestinal barrier by the upregulation of TJP-1, CLDN-1 and CLDN-4 mRNA. Postbiotic supplementation derived from L. plantarum RG14 in post-weaning lambs enhance the ruminal papillae growth, immune status and gastrointestinal health.

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