Kinetic properties analysis of beta-mannanase from Klebsiella oxytoca KUB-CW2-3 expressed in Escherichia coli.

Endo-1,4-ß-mannanase is an enzyme that can catalyze the random hydrolysis of ß-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and offers many applications in different biotechnology industries. Purification and kinetic properties of the endo-1,4-ß-mannanase from recombinant Escherichia coli strain KMAN-3 were examined. Recombinant ß-mannanase (KMAN-3) was purified 50.5 fold using Ni-NTA Agarose resin and specific activity of 11900 U/mg protein was obtained. Purified KMAN-3 showed a single band on SDS-PAGE with a molecular weight of 43 kDa. Km and Vmax values of KMAN-3 on ivory nut mannan, locust bean gum, defatted copra meal and konjac glucomannan were 243, 3.83 × 105 37 and 2.13 × 106 mg ml-1 and 2940, 61,100, 3930 and 1.56 × 1010 mg-1, respectively. Carboxymethyl cellulose was not digested by KMAN-3.

Secretory expression of ß-mannanase from Bacillus circulans NT 6.7 in Lactobacillus plantarum.

The ß-mannanase gene of Bacillus circulans NT 6.7 was successfully cloned in Lactobacillus plantarum WCFS1 using the pSIP403 expression vector and secreted to the supernatant rather than accumulated in the cells. The highest activity was achieved by controlling the pH at 6 during cultivation. Maximum mannanase activities detected in the supernatant and cell-free extract of 200 ml MRS broth were 8.2 and 0.86 U/ml, respectively. Enzyme activity in the supernatant increased to 27 U/ml by fermentation in a 5-L bioreactor with automatic pH control. The optimum temperature of recombinant ß-mannanase was 50 °C and stable between 30 and 50 °C. The optimum pH was 6 with stability in the range 5-7. Enzyme activity slightly increased with Co2+ but was strongly inhibited by EDTA. The enzyme exhibited high specificity to galactomannan substrates. The main products of copra meal and locust bean gum hydrolysis were manno-oligosaccharides. Therefore, recombinant ß-mannanase produced from a food grade host, L. plantarum WCFS1, showed potential for use in manno-oligosaccharides production and other food-related applications.

Distinct gut microbiota of healthy children from two different geographic regions of Thailand.

In Thailand, food consumption by people from each region is different. This can be an important environmental factor which shapes the gut microbiota further affecting their health. This study aimed to use quantitative PCR (qPCR) to investigate the intestinal microbial community in 60 healthy children (aged 8-11 years) living in specific areas, namely central (CT) and northeastern (NE) Thailand where each region has its own typical food consumption. The children from NE had significantly higher consumption frequency of meat (chicken and beef), a wide variety of carbohydrate sources (noodle, fermented rice and sweet potato) including vegetables and fruit, while in CT, there was a significant preference for rice, breakfast cereal and cow milk. The qPCR analysis resulted in significantly higher abundance of lactobacilli, Clostridium coccoides-Eubacterium rectale, Clostridium leptum, Prevotella and Bacteroides fragilis in children from the NE region. However, no significant difference in the count of Bifidobacterium spp., Enterobacteriaceae and methanogens was observed. Considering the correlation of food sources and microbial groups, the consumption frequency of vegetables showed a moderately positive correlation coefficient of 0.42 and 0.34 to the Lactobacillus group (P = 0.001) and the Prevotella group (P = 0.008), respectively, while a diet of fish and beef showed a moderately negative correlation coefficient of -0.41 (P = 0.001) and -0.33 (P = 0.09) to Bifidobacterium spp., respectively. Our results suggested that high frequency consumption of varieties of carbohydrates, protein sources, fruits and vegetables by the NE children promoted a high abundance of bacterial species in the phyla Firmicutes and Bacteroidetes.

Purification and characterization of a novel plantaricin, KL-1Y, from Lactobacillus plantarum KL-1.

Three bacteriocins from Lactobacillus plantarum KL-1 were successfully purified using ammonium sulfate precipitation, cation-exchange chromatography and reverse-phase HPLC. The bacteriocin peptides KL-1X, -1Y and -1Z had molecular masses of 3053.82, 3498.16 and 3533.16 Da, respectively. All three peptides were stable at pH 2-12 and 25 °C and at high temperatures of 80 and 100 °C for 30 min and 121 °C for 15 min. However, they differed in their susceptibility to proteolytic enzymes and their inhibition spectra. KL-1Y showed broad inhibitory activities against Gram-positive and Gram-negative bacteria, including Salmonella enterica serovar Enteritidis DMST 17368, Pseudomonas aeruginosa ATCC 15442, P. aeruginosa ATCC 9027, Escherichia coli O157:H7 and E. coli ATCC 8739. KL-1X and -1Z inhibited only Gram-positive bacteria. KL-1X, KL-1Y and KL-1Z exhibited synergistic activity. The successful amino acid sequencing of KL-1Y had a hydrophobicity of approximately 30 % and no cysteine residues suggested its novelty, and it was designated "plantaricin KL-1Y". Plantaricin KL-1Y exhibited bactericidal activity against Bacillus cereus JCM 2152(T). Compared to nisin, KL-1Y displayed broad inhibitory activities of 200, 800, 1600, 800, 400 and 400 AU/mL against the growth of Bacillus coagulans JCM 2257(T), B. cereus JCM 2152(T), Listeria innocua ATCC 33090(T), Staphylococcus aureus TISTR 118, E. coli O157:H7 and E. coli ATCC 8739, respectively, whereas nisin had similar activities against only B. coagulans JCM 2257(T) and B. cereus JCM 2152(T). Therefore, the novel plantaricin KL-1Y is a promising antimicrobial substance for food safety uses in the future.

Simulated Swine Digestion and Gut Microbiota Fermentation of Hydrolyzed Copra Meal.

This study aimed to compare the effects of hydrolyzed copra meal (HCM) inclusion at 1% on its in vitro digestibility and the microbiota and cecum fermentation using the gut microbiota of weaned swine, targeting microbial community and short-chain fatty acids (SCF). For this reason, three treatments were considered: control (no copra meal), 1% non-hydrolyzed copra meal (CM), and 1% HCM. Non-defatted copra meal was hydrolyzed and analyzed (reducing sugars and total carbohydrates) in our laboratory. For digestion, microbiota identification, and fermentation assays, fresh fecal samples from two weaned pigs (1 month old) were used. Three replicates of each treatment were employed. HCM was more digestible, with approximately 0.68 g of hydrolysate recovered after simulated digestion compared to 0.82 g of hydrolysate recovered from CM. This was shown by Scanning Electron Microscope (SEM) images. Also, the three swine shared the majority of microbial species identified at the phylum and family levels. There were no differences (p > 0.05) between treatments in the microbial community and SCFA during fermentation. However, higher Chao-1 and Shannon indexes were observed in CM and HCM treatments. HCM was also found to be capable of preserving Actinobacterota and Proteobacteria at the phylum level, while at the family level, both treatments may help Lactobacillaceae, Peptostreptococcaceae, Lachnospiraceae, and Ruminococcaceae survive in the long term. Also, there was a potential trend of increasing acetic acid and butyric acid in the CM and HCM treatments. While HCM shows promise in potentially modulating the gut microbiota of weaned swine, additional research is required to investigate the effects of higher doses of HCM on swine performance parameters.

In Silico Analysis and Development of the Secretory Expression of D-Psicose-3-Epimerase in Escherichia coli.

D-psicose-3-epimerase (DPEase), a key enzyme for D-psicose production, has been successfully expressed in Escherichia coli with high yield. However, intracellular expression results in high downstream processing costs and greater risk of lipopolysaccharide (LPS) contamination during cell disruption. The secretory expression of DPEase could minimize the number of purification steps and prevent LPS contamination, but achieving the secretion expression of DPEase in E. coli is challenging and has not been reported due to certain limitations. This study addresses these challenges by enhancing the secretion of DPEase in E. coli through computational predictions and structural analyses. Signal peptide prediction identified PelB as the most effective signal peptide for DPEase localization and enhanced solubility. Supplementary strategies included the addition of 0.1% (v/v) Triton X-100 to promote protein secretion, resulting in higher extracellular DPEase (0.5 unit/mL). Low-temperature expression (20 °C) mitigated the formation of inclusion bodies, thus enhancing DPEase solubility. Our findings highlight the pivotal role of signal peptide selection in modulating DPEase solubility and activity, offering valuable insights for protein expression and secretion studies, especially for rare sugar production. Ongoing exploration of alternative signal peptides and refinement of secretion strategies promise further enhancement in enzyme secretion efficiency and process safety, paving the way for broader applications in biotechnology.

Nutritional Quality and Biological Application of Mushroom Protein as a Novel Protein Alternative.

PURPOSE OF REVIEW: Global concerns about population growth, economic, and nutritional transitions and health have led to the search for a low-cost protein alternative to animal origins. This review provides an overview of the viability of exploring mushroom protein as a future protein alternative considering the nutritional value, quality, digestibility, and biological benefits. RECENT FINDINGS: Plant proteins are commonly used as alternatives to animal proteins, but the majority of them are low in quality due to a lack of one or more essential amino acids. Edible mushroom proteins usually have a complete essential amino acid profile, meet dietary requirements, and provide economic advantages over animal and plant sources. Mushroom proteins may provide health advantages by eliciting antioxidant, antitumor, angiotensin-converting enzyme (ACE), inhibitory and antimicrobial properties over animal proteins. Protein concentrates, hydrolysates, and peptides from mushrooms are being used to improve human health. Also, edible mushrooms can be used to fortify traditional food to increase protein value and functional qualities. These characteristics highlight mushroom proteins as inexpensive, high-quality proteins that can be used as a meat alternative, as pharmaceuticals, and as treatments to alleviate malnutrition. Edible mushroom proteins are high in quality, low in cost, widely available, and meet environmental and social requirements, making them suitable as sustainable alternative proteins.

Selection of pretreatment method and mannanase enzyme to improve the functionality of palm kernel cake.

Palm kernel cake (PKC) is a by-product of palm kernel oil extraction with moderate nutritional value, containing 30-35% ß-mannan, which is indigestible, slows growth, and reduces feed efficiency. PKC can be improved by mannanase hydrolysis, but the effectiveness of mannanase is dependent on the microbial source. Thus, the effect of steam pretreatment and bacterial mannanases on PKC quality was investigated. PKC was pretreated by steaming and hydrolyzed in the small intestine by various mannanases. The contents of reducing sugar, total sugar, and protein release were measured. Steamed PKC had a significant increase in protein (16.95 ± 0.14 to 20.98 ± 0.13%) and a substantial decrease in hemicellulose (29.52 ± 0.44 to 3.46 ± 0.88%) and lignin (8.94 ± 0.28 to 1.40 ± 0.22%). Mannanases from Escherichia coli-KMAN-3 and E. coli-Man6.7 recorded the highest activities, followed by commercial mannanase, Bacillus circulans NT6.7 and B. amyloliquefaciens NT6.3 mannanases, orderly. B. circulans NT6.7 and B. amyloliquefaciens NT6.3 had multi-activities that include glucanase (3.10 ± 0.04% and 2.47 ± 0.02%) and amylase (1.74 ± 0.03% and 1.38 ± 0.04%), respectively. B. amyloliquefaciens NT6.3 mannanase hydrolyzed steamed PKC to release more reducing sugar, total sugar, and protein than hydrolyzed raw PKC. In raw and steamed PKC, B. amyloliquefaciens NT6.3 mannanase produced the highest reducing sugar release. As a result, steam pretreatment and mannanase hydrolysis, particularly from B. amyloliquefaciens, can be used to increase the functioning of PKC and develop new feed ingredients for monogastric animals at a reasonable cost.

In vitro gastrointestinal digestion of Lentinus squarrosulus powder and impact on human fecal microbiota.

Humans have long-used mushrooms as food and medicine, but digestion and colonic fermentation of most mushrooms, including Lentinus squarrosulus is markedly unknown. Here, nutritional profile, digestion and colonic fermentation of L. squarrosulus powder (LP) were determined. The powder contained mainly carbohydrate and protein. SEM and F-TIR analysis of the resistant hydrolysate (RH) revealed that the structure and ratio of carbohydrate and protein components were altered, and released known immunomodulation agents; beta-glucans and mannose. Both LP and RH promoted selected probiotic bacteria, especially Bifidobacterium strains. Using fecal microbiota of five volunteers (V1, V2, V3, V4 and V5), RH stimulated the microbiota of all used volunteers, via decreasing the ratio of Firmicutes/Bacteroidetes ranging from 1.3 to 8.2 times. Also, RH increased the relative abundance of vital immunomodulators; Bacteroides, Bifidobacterium, Clostridium cluster XIVa and IV, and Sutterella. Additionally, RH fermentation enriched the content of branch-chain fatty acids (BCFA) and short-chain fatty acids (SCFA), indicating protein and carbohydrate usage. Notably, propionic and butyric acids were abundant in V1, V2 and V3, while in V4 and V5, acetic and butyric acids were most enriched. Suggesting L. squarrosulus as functional mushroom to improve health and prevent diseases by enhancing gut health.

Bioactive composition and modulatory effects of Hed-Tean-Rad Mushroom, Macrocybe crassa on gut microbiota.

Macrocybe crassa (or Tricholoma crassum) is a nutrient-dense wild edible mushroom native to Thailand. The mushroom extract and its constituents have remarkable biological characteristics, but the influence of the powder on the human gut microbiota is unknown. This study investigated the bioactive composition and modulatory properties of M. crassa powder on gut microbial composition and short-chain fatty acids (SCFA) production. The fermentation of M. crassa powder by human intestinal microbiota released SCFA, mainly acetic acid, propionic acid and butyric acid. M. crassa powder significantly modulated the microbiota by increasing the relative abundances of Bifidobacterium, Lactobacillus/Enterococcus group, Atopobium, Bacteroidaceae/Prevotellaceae, and C. coccoides. F. prausnitzii, Roseburia genus, C. histolyticum and C. cluster IX, similar to that of Fructooligosaccharides (FOS). With M. crassa powder, high content of propionic acid was observed, as well as a number of Bacteroidaceae/Prevotellaceae and C. cluster IX. On the other hand, FOS caused a high acetic acid concentration and a population of Bifidobacterium spp., Atopobium cluster, Bacteroidaceae/Prevotellaceae, and C. coccoides. Therefore, this work will significantly contribute to filling the knowledge gap and revealing the significance of M. crassa in the pharmaceutical industry.

Analyzing Predominant Bacterial Species and Potential Short-Chain Fatty Acid-Associated Metabolic Routes in Human Gut Microbiome Using Integrative Metagenomics.

Gut microbiome plays an essential role in host health, and there is interest in utilizing diet to modulate the composition and function of microbial communities. Copra meal hydrolysate (CMH) is commonly used as a natural additive to enhance health. However, the gut microbiome is largely unknown at species level and is associated with metabolic routes involving short-chain fatty acids (SCFAs). In this study, we aimed to analyze, using integrative metagenomics, the predominant species and metabolic routes involved in SCFAs production in the human gut microbiome after treatment with CMH. The effect of CMH treatment on the Thai gut microbiome was demonstrated using 16S rRNA genes with whole-metagenome shotgun (WMGS) sequencing technology. Accordingly, these results revealed that CMH has potentially beneficial effects on the gut microbiome. Twelve predominant bacterial species, as well as their potential metabolic routes, were involved in cooperative microbiome networks under sugar utilization (e.g., glucose, mannose, or xylose) and energy supply (e.g., NADH and ATP) in relation to SCFAs biosynthesis. These findings suggest that CMH may be used as a potential prebiotic diet for modulating and maintaining the gut microbiome. To our knowledge, this is the first study to reveal the predominant bacterial species and metabolic routes in the Thai gut microbiome after treatment with potential prebiotics.

Isolation and Characterization of Mannanase-Producing Bacteria for Potential Synbiotic Application in Shrimp Farming.

Prebiotics such as mannan-oligosaccharides (MOS) are a promising approach to improve performance and disease resistance in shrimp. To improve prebiotic utilization, we investigated the potential probiotics and their feasibility of synbiotic use in vitro. Two bacterial isolates, Man26 and Man122, were isolated from shrimp intestines and screened for mannanase, the enzyme for mannan digestion. The crude mannanase from both isolates showed optimal activities at pH 8 with optimum temperatures at 60 °C and 50 °C, respectively. The enzymes remained stable at pH 8−10 for 3 h (>70% relative activity). The thermostability range of Man26 was 20−40 °C for 20 min (>50%), while that of Man122 was 20−60 °C for 30 min (>50%). The Vmax of Man122 against locust bean gum substrate was 41.15 ± 12.33 U·mg−1, six times higher than that of Man26. The Km of Man26 and Man122 were 18.92 ± 4.36 mg·mL−1 and 34.53 ± 14.46 mg·mL−1, respectively. With the addition of crude enzymes, reducing sugars of copra meal, palm kernel cake, and soybean meal were significantly increased (p < 0.05), as well as protein release. The results suggest that Man26 and Man122 could potentially be used in animal feeds and synbiotically with copra meal to improve absorption and utilization of feedstuffs.

Functional composition and antioxidant property of crude polysaccharides from the fruiting bodies of Lentinus squarrosulus.

Lentinus squarrosulus (Hed Khon Khao) is a source of bioactive polysaccharides. Three L. squarrosulus crude polysaccharides (LSPs) were subjected to cold water (LSP-CP), hot water (LSP-HP), and aqueous alkaline (LSP-AP) extractions, and their functional compositions and radical scavenging activities were compared. Synchrotron radiation FTIR (SR-FTIR) spectra and PCA plot analysis in the bio-regions (4000-400 cm-1) revealed that functional composition LSPs differ significantly (P < 0.05). All LSPs had lipids, protein, and polysaccharides such as ß-glucan. The major monosaccharides in LSP-CP and LSP-HP are d-galactose, d-glucose, and d-mannose at different proportions, while LSP-AP contained mainly d-glucose. Also, fucose and xylose were present in all the LSPs. LSP-CP, LSP-HP and LSP-AP induced maximum 1,1-diphenyl-2- picrylhydrazyl (DPPH) radical scavenging activity of 78.93 ± 0.42% at 3 mg/mL, 79.16 ± 1.43% at 3 mg/mL and 65.26 ± 1.74% at 5 mg/mL, whiles on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), their maximum activities were 98.94 ± 0.16% at 3 mg/mL, 97.42 ± 0.76% at 3 mg/mL and 47.24 ± 0.045% at 5 mg/mL, respectively. The results showed that LSP-CP and LSP-HP are good ABTS scavengers, whiles LSP-AP is poor ABTS scavenger. In overall, LSPs consist of essential functional compositions and could be used as natural antioxidants. This exploitation of fungal fruiting body extracts increased the potential use of L. squarrosulus in food and medicinal industries.

Pyrodextrins from waxy and normal tapioca starches: Molecular structure and in vitro digestibility.

Pyrodextrins were prepared from acidified waxy and normal tapioca starches (pH∼3) at 3 temperatures (130, 150 and 170 °C) and 3 times (1, 2 and 4 h) to determine their in vitro digestibility and molecular structure. Pyrodextrin from waxy tapioca starch produced at 170 °C/4 h had 5% higher total indigestible carbohydrate than pyrodextrin from normal tapioca starch (45.2 % and 40.4 %, respectively) as determined by a modified AOAC Method 2011.25. The low-molecular weight indigestible carbohydrate content at this condition was also higher for waxy tapioca starch than normal tapioca starch (40.6 % and 34.9 %, respectively). Gel permeation chromatography and nuclear magnetic resonance spectroscopy were used to study changes in molecular structure and correlate with digestibility of the pyrodextrins. Molecular size distribution indicated that waxy tapioca starch underwent thermal modification more readily than normal tapioca starch. Non α-1,4/α-1,6 glucosidic linkages were increased in the pyrodextrins with increasing in indigestible carbohydrate content.

A randomized trial to evaluate the impact of copra meal hydrolysate on gastrointestinal symptoms and gut microbiome.

The impact of copra meal hydrolysate (CMH) on gut health was assessed by conducting a double-blinded, placebo-controlled study. Sixty healthy adult participants, aged 18-40 years were assigned to daily consume 3 g of CMH, 5 g of CMH or placebo in the form of drink powder for 21 days. Consumption of CMH at 3 g/d improved defecating conditions by reducing stool size and also relieved flatulence and bloating symptoms. Fecal samples were collected serially at the baseline before treatment, after the treatment and after a 2-week washout period. The gut microbiomes were similar among the treatment groups, with microbial community changes observed within the groups. Intake of CMH at 3 g/d led to increase microbial diversity and richness. Reduction of the ratio between Firmicutes to Bacteroidetes was observed, although it was not significantly different between the groups. The 3 g/d CMH treatment increased beneficial microbes in the group of fiber-degrading bacteria, especially human colonic Bacteroidetes, while induction of Bifidobacteriaceae was observed after the washout period. Intake of CMH led to increase lactic acid production, while 3 g/d supplement promoted the present of immunoglobulin A (IgA) in stool samples. The 3 g daily dose of CMH led to the potentially beneficial effects on gut health for healthy individuals.

Crystallization, structural characterization and kinetic analysis of a GH26 ß-mannanase from Klebsiella oxytoca KUB-CW2-3.

ß-Mannanase (EC 3.2.1.78) is an enzyme that cleaves within the backbone of mannan-based polysaccharides at ß-1,4-linked D-mannose residues, resulting in the formation of mannooligosaccharides (MOS), which are potential prebiotics. The GH26 ß-mannanase KMAN from Klebsiella oxytoca KUB-CW2-3 shares 49-72% amino-acid sequence similarity with ß-mannanases from other sources. The crystal structure of KMAN at a resolution of 2.57 Šrevealed an open cleft-shaped active site. The enzyme structure is based on a (ß/α)8-barrel architecture, which is a typical characteristic of clan A glycoside hydrolase enzymes. The putative catalytic residues Glu183 and Glu282 are located on the loop connected to ß-strand 4 and at the end of ß-strand 7, respectively. KMAN digests linear MOS with a degree of polymerization (DP) of between 4 and 6, with high catalytic efficiency (kcat/Km) towards DP6 (2571.26 min-1 mM-1). The predominant end products from the hydrolysis of locust bean gum, konjac glucomannan and linear MOS are mannobiose and mannotriose. It was observed that KMAN requires at least four binding sites for the binding of substrate molecules and hydrolysis. Molecular docking of mannotriose and galactosyl-mannotetraose to KMAN confirmed its mode of action, which prefers linear substrates to branched substrates.

Analysis of Human Gut Microbiome: Taxonomy and Metabolic Functions in Thai Adults.

The gut microbiome plays a major role in the maintenance of human health. Characterizing the taxonomy and metabolic functions of the human gut microbiome is necessary for enhancing health. Here, we analyzed the metagenomic sequencing, assembly and construction of a meta-gene catalogue of the human gut microbiome with the overall aim of investigating the taxonomy and metabolic functions of the gut microbiome in Thai adults. As a result, the integrative analysis of 16S rRNA gene and whole metagenome shotgun (WMGS) sequencing data revealed that the dominant gut bacterial families were Lachnospiraceae and Ruminococcaceae of the Firmicutes phylum. Consistently, across 3.8 million (M) genes annotated from 163.5 gigabases (Gb) of WMGS sequencing data, a significant number of genes associated with carbohydrate metabolism of the dominant bacterial families were identified. Further identification of bacterial community-wide metabolic functions promisingly highlighted the importance of Roseburia and Faecalibacterium involvement in central carbon metabolism, sugar utilization and metabolism towards butyrate biosynthesis. This work presents an initial study of shotgun metagenomics in a Thai population-based cohort in a developing Southeast Asian country.

The role of salicylic acid and benzothiadiazole in decreasing phytoplasma titer of sugarcane white leaf disease.

The objective of this research was to study the effect of Benzothiadiazole (BTH) and Salicylic acid (SA) on the systemic acquired resistance (SAR) of sugarcane the phytoplasma associated with the sugarcane white leaf (SCWL) disease. The experiment was conducted on plants of the sugarcane variety Khon Kaen 3 (KK3) infected with SCWL phytoplasma using insect vectors. Biochemical changes related to the SAR such as SA and total phenolic compounds were followed according to 4 different timepoints: 7, 14, 21 and 28 days after inoculation. Together, phytoplasma were quantified by RT-qPCR using the secA gene of phytoplasma. According to our results, the spraying of BTH and SA tended to increase the amounts of SA, total phenolic compounds and a lower presence of phytoplasma in the plants in comparison with the inoculated control. Spraying BTH at a concentration of 2.4 mM and SA at a concentration of 2.4 mM exhibited the best efficiency to reduce the concentration of phytoplasma. According to RT-qPCR results, the inoculated plants sprayed with BTH displayed a significantly lower concentration of phytoplasma compared to the inoculated controls. Overall, our results indicated that the spray of BTH and SA could induce an efficient SAR response to the phytoplasma associated with the SCWL disease. We expect these results will give support to the development of new products for controlling white leaf disease in sugarcane.

Copra meal hydrolysis by the recombinant ß-mannanase KMAN-3 and MAN 6.7 expressed in Escherichia coli.

Hydrolysis products of defatted copra meal (DCM) hydrolysis were investigated with either recombinant ß-mannanases from Klebsiella oxytoca KUB-CW2-3 (KMAN-3) or Bacillus circulans NT 6.7 (MAN 6.7). Morphological changes and functional groups of solid residues were also determined by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. Results revealed that the Michaelis-Menten constant (K m) and maximum velocity (V max) values of KMAN-3 on DCM were 2.4 mg/ml and 5.4 U/mg, respectively, while MAN 6.7 recorded K m and V max at 2.0 mg/ml and 4.3 U/mg, respectively. Both enzymes efficiently randomly hydrolysed DCM and produced a range of different manno-oligosaccharides (MOS). The profile of hydrolysis products was different for each enzyme used. Main products from hydrolysis of DCM by KMAN-3 and MAN 6.7 were various MOS including mannobiose (M2), mannotriose (M3), mannotetraose (M4), and mannose, whereas mannopentaose (M5) was only found from KMAN-3. Amount of M3 produced by KMAN-3 was about three times higher than from MAN 6.7. Total MOS yield for KMAN-3 was 1.5-folds higher than for MAN 6.7. SEM analysis showed that enzymatic hydrolysis with KMAN-3 and MAN 6.7 resulted in deconstruction of the DCM structure which generated a variety of MOS products. FTIR spectra revealed that the properties of both hydrolysed solids were not significantly different compared to the original DCM. Results suggested that KMAN-3 was a promising candidate for production of high MOS content from copra meal.

Nutritional improvement of copra meal using mannanase and Saccharomyces cerevisiae.

Copra meal is a by-product of coconut milk extraction, contained 4.60 ± 0.01 g/100 g DM and 62.19 ± 0.53% of protein and fiber, respectively. The optimal condition for quality improvement of copra meal was investigated using Box-Behnken design combined with response surface methodology (RSM). The simultaneous saccharification and fermentation (SSF) of Copra meal was performed by mannanase enzyme and yeast, Saccharomyces cerevisiae. The concentration of mannanase was determined as the most important factor to increase protein content in copra meal. The protein content was increased by 64% when 0.7% of enzyme per copra meal dry weight, the ratio of copra meal to water at 1:4.56 and fermentation time of 90.25 h at 30 °C were used. The program predicted an increase of 3.06 g of protein/100 g dry matter; however, the experimental result showed an increase of 3.35 g/100 g DM of protein in copra meal. The 10 kg of copra meal SSF in Koji reactor, the protein content increased to 4.18 g/100 g DM, while fiber content decreased 49%. Moreover, amino acids were increased by 64.05% and oligosaccharides, especially mannohexaose, were increased to 0.708 g/g DM. Results showed that fermentation of copra meal with mannanase and yeast offers a potential method to improve the nutrition of copra meal as animal feed.