Innovative approaches for amino acid production via consolidated bioprocessing of agricultural biomass.

Agricultural plantations in Indonesia and Malaysia yield substantial waste, necessitating proper disposal to address environmental concerns. Yet, these wastes, rich in starch and lignocellulosic content, offer an opportunity for value-added product development, particularly amino acid production. Traditional methods often rely on costly commercial enzymes to convert biomass into fermentable sugars for amino acid production. An alternative, consolidated bioprocessing, enables the direct conversion of agricultural biomass into amino acids using selected microorganisms. This review provides a comprehensive assessment of the potential of agricultural biomass in Indonesia and Malaysia for amino acid production through consolidated bioprocessing. It explores suitable microorganisms and presents a case study on using Bacillus subtilis ATCC 6051 to produce 9.56 mg/mL of amino acids directly from pineapple plant stems. These findings contribute to the advancement of sustainable amino acid production methods using agricultural biomass especially in Indonesia and Malaysia through consolidated bioprocessing, reducing waste and enhancing environmental sustainability.

Anti-Diabetic Effect of Lactobacillus Paracasei Isolated from Malaysian Water Kefir Grains.

The prevalence of type 2 diabetes mellitus (T2DM) is alarming because it is always linked to the increase in chronic diseases, mortality, and socioeconomic burden. Water kefir has a wide range of functional and probiotic characteristics attributed to the microorganisms present in the kefir grains. The present study aims to evaluate the in vivo anti-diabetic potential of the isolated Lactobacillus paracasei from Malaysian water kefir grains (MWKG) which was reported to have excellent probiotic properties and high antioxidant activities as reported previously. High-fat diet/streptozotocin (HFD/STZ) induction was used to obtain a T2DM model followed by treatment with the isolated L. paracasei from MWKG. The levels of glucose, insulin, and in vivo liver antioxidants were quantified after 14 weeks. Gene expression analysis of the liver was also carried out using microarray analysis, and several genes were selected for validation using quantitative real-time PCR. Insulin tolerance test demonstrated that the L. paracasei isolated from the MWKG alleviated T2DM by improving the area under the curve of the insulin tolerance test whereby low-dose and high-dose concentrations treated groups showed 2424.50 ± 437.02 mmol/L·min and 2017.50 ± 347.09 mmol/L·min, respectively, compared to untreated diabetic mice which was 3884.50 ± 39.36 mmol/L·min. Additionally, treatment with the isolated L. paracasei from MWKG regulated the expression of several genes related to glucose homeostasis and lipid metabolism in diabetic mice. These results suggested that the isolated L. paracasei from MWKG could be a potential dietary supplement for T2DM.

Shelf life of indigenous tengkawang butter: Storage kinetic and effect of antioxidant to oxidation stability index.

Tengkawang butter is an indigenous and traditional butter from Borneo that can be a lipid source for pharmaceutical and food applications. Studies found that Tengkawang butter is a cheaper cocoa butter substitution without compromising its quality. However, the current storage method is still very traditional, resulting in faster deterioration of Tengkawang butter. This study aims to calculate and evaluate the storage kinetics model with the Arrhenius model and the tengkawang butter oxidation stability index analysis. Storage conditions were carried out at temperatures of -5, 5, 24, and 60 °C to predict the tengkawang butter storage kinetics model. The addition of antioxidants such as ascorbic acid, tocopherol, and lignin to tengkawang butter help increase the oxidation stability index. The kinetics of the tengkawang butter acidity and peroxide models followed a zero-order reaction with activation energy values of 11.139 kJ mol-1 and 12.320 kJ mol-1, respectively. The prediction model for acidity is Acidity = 4.417-7.903t exp (-11,139/RT), and the model for peroxide is peroxide = 2.155-10.998t exp (-12,320/RT). The oxidation stability index values at 22 °C and the rate of oxidation when the temperature rises by ten degrees (Q10) of tengkawang butter, tengkawang butter with ascorbic acid, tengkawang butter with tocopherol, and tengkawang butter with lignin were 66,896 and 2.815; 224,680 and 1.993; 106,120 and 2.725; 81,658 and 2.961, respectively. The kinetic and oxidation stability index model data can be used as a reference for storage and preserving products made from tengkawang butter.

Reduction of the acidity and peroxide numbers of tengkawang butter (Shorea stenoptera) using thermal and acid activated bentonites.

Tengkawang fat (Shorea stenoptera), from an indigenous plant of the Kalimantan forest, has excellent potential as an alternative source of vegetable fat because it has a high level of fatty acids composition. Activated natural bentonite can be used as a bleaching agent to improve the quality of tengkawang fat. This research aims to reduce the acidity, peroxide number values and identify the physicochemical properties (fatty acid composition, nutrients, and thermal) of tengkawang butter. Initially, tengkawang samples from Nanga Yen and Sintang were pre-treated using the degumming process with 1% phosphoric acid and the neutralization process with a 1 M NaOH 10% w/w solution. The results show that the acidity (mg NaOH/g) of the tengkawang fat samples was reduced from 11.00 to 3.36 when using bentonite activated at 200 °C. The bentonite activated with 0.5 M HCl reduced the acidity to 3.61. The peroxide number (meq O2/kg) of the tengkawang fat samples was reduced from 9.45 to 4.84 and 3.47 by bleaching with thermal-activated and acid-activated bentonites, respectively. Peroxide value correlates with ß-carotene content. The smaller of the ß-carotene content, the smaller the peroxide value. The acidity, peroxide number, and iodine number values from tengkawang fat after treatment adhere to the SNI 2903: 2016 standard. The main content of fatty acids in tengkawang fat is palmitic acid, stearic acid, and oleic acid. These results show that both products are suitable for the food industry in terms of the acid and peroxide numbers. The application of this research results will assist local people in increasing the economic value of the product from tengkawang plant, which is an indigenous plant from Kalimantan.

Enhanced volatile fatty acid production from sago hampas by Clostridium beijerinckii SR1 for bioelectricity generation using microbial fuel cells.

Sago hampas is a starch-based biomass from sago processing industries consisted of 58% remaining starch. This study has demonstrated the bioconversion of sago hampas to volatile fatty acids (VFAs) by Clostridium beijerinckii SR1 via anaerobic digestion. Higher total VFAs were obtained from sago hampas (5.04 g/L and 0.287 g/g) as compared to commercial starch (5.94 g/L and 0.318 g/g). The physical factors have been investigated for the enhancement of VFAs production using one-factor-at-a-time (OFAT). The optimum condition; 3% substrate concentration, 3 g/L of yeast extract concentration and 2 g/L of ammonium nitrate enhanced the production of VFAs by 52.6%, resulted the total VFAs produced is 7.69 g/L with the VFAs yield of 0.451 g/g. VFAs hydrolysate produced successfully generated 273.4 mV of open voltage circuit and 61.5 mW/m2 of power density in microbial fuel cells. It was suggested that sago hampas provide as an alternative carbon feedstock for bioelectricity generation.

Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a ß-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli.

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM ß-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of ß-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. ß-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant ß-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular ß-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly ß-CGTase by the recombinant E. coli.

In-Silico Characterization of Glycosyl Hydrolase Family 1 ß-Glucosidase from Trichoderma asperellum UPM1.

ß-glucosidases (Bgl) are widely utilized for releasing non-reducing terminal glucosyl residues. Nevertheless, feedback inhibition by glucose end product has limited its application. A noticeable exception has been found for ß-glucosidases of the glycoside hydrolase (GH) family 1, which exhibit tolerance and even stimulation by glucose. In this study, using local isolate Trichoderma asperellum UPM1, the gene encoding ß-glucosidase from GH family 1, hereafter designated as TaBgl2, was isolated and characterized via in-silico analyses. A comparison of enzyme activity was subsequently made by heterologous expression in Escherichia coli BL21(DE3). The presence of N-terminal signature, cis-peptide bonds, conserved active site motifs, non-proline cis peptide bonds, substrate binding, and a lone conserved stabilizing tryptophan (W) residue confirms the identity of Trichoderma sp. GH family 1 ß-glucosidase isolated. Glucose tolerance was suggested by the presence of 14 of 22 known consensus residues, along with corresponding residues L167 and P172, crucial in the retention of the active site's narrow cavity. Retention of 40% of relative hydrolytic activity on ρ-nitrophenyl-ß-D-glucopyranoside (ρNPG) in a concentration of 0.2 M glucose was comparable to that of GH family 1 ß-glucosidase (Cel1A) from Trichoderma reesei. This research thus underlines the potential in the prediction of enzymatic function, and of industrial importance, glucose tolerance of family 1 ß-glucosidases following relevant in-silico analyses.

Production of Biosurfactant Produced from Used Cooking Oil by Bacillus sp. HIP3 for Heavy Metals Removal.

Heavy metals from industrial effluents and sewage contribute to serious water pollution in most developing countries. The constant penetration and contamination of heavy metals into natural water sources may substantially raise the chances of human exposure to these metals through ingestion, inhalation, or skin contact, which could lead to liver damage, cancer, and other severe conditions in the long term. Biosurfactant as an efficient biological surface-active agent may provide an alternative solution for the removal of heavy metals from industrial wastes. Biosurfactants exhibit the properties of reducing surface and interfacial tension, stabilizing emulsions, promoting foaming, high selectivity, and specific activity at extreme temperatures, pH, and salinity, and the ability to be synthesized from renewable resources. This study aimed to produce biosurfactant from renewable feedstock, which is used cooking oil (UCO), by a local isolate, namely Bacillus sp. HIP3 for heavy metals removal. Bacillus sp. HIP3 is a Gram-positive isolate that gave the highest oil displacement area with the lowest surface tension, of 38 mN/m, after 7 days of culturing in mineral salt medium and 2% (v/v) UCO at a temperature of 30 °C and under agitation at 200 rpm. An extraction method, using chloroform:methanol (2:1) as the solvents, gave the highest biosurfactant yield, which was 9.5 g/L. High performance liquid chromatography (HPLC) analysis confirmed that the biosurfactant produced by Bacillus sp. HIP3 consists of a lipopeptide similar to standard surfactin. The biosurfactant was capable of removing 13.57%, 12.71%, 2.91%, 1.68%, and 0.7% of copper, lead, zinc, chromium, and cadmium, respectively, from artificially contaminated water, highlighting its potential for bioremediation.

Direct Bioelectricity Generation from Sago Hampas by Clostridium beijerinckii SR1 Using Microbial Fuel Cell.

Microbial fuel cells offer a technology for simultaneous biomass degradation and biological electricity generation. Microbial fuel cells have the ability to utilize a wide range of biomass including carbohydrates, such as starch. Sago hampas is a starchy biomass that has 58% starch content. With this significant amount of starch content in the sago hampas, it has a high potential to be utilized as a carbon source for the bioelectricity generation using microbial fuel cells by Clostridium beijerinckii SR1. The maximum power density obtained from 20 g/L of sago hampas was 73.8 mW/cm2 with stable cell voltage output of 211.7 mV. The total substrate consumed was 95.1% with the respect of 10.7% coulombic efficiency. The results obtained were almost comparable to the sago hampas hydrolysate with the maximum power density 56.5 mW/cm2. These results demonstrate the feasibility of solid biomass to be utilized for the power generation in fuel cells as well as high substrate degradation efficiency. Thus, this approach provides a promising way to exploit sago hampas for bioenergy generation.

Comparison of hydro-distillation, hydro-distillation with enzyme-assisted and supercritical fluid for the extraction of essential oil from pineapple peels.

Pineapple peel is a potential feedstock for the extraction of essential oil due to the presence of aromatic compounds. To extract the essential oil from pineapple peels, three different methods were applied, i.e., (1) hydro-distillation (HD); (2) hydro-distillation with enzyme-assisted (HDEA); and (3) supercritical fluid extraction (SFE). SFE had successfully produced an essential oil with the yield of 0.17% (w/w) together with 0.64% (w/w) of concrete, whereby the HD and HDEA had only produced hydrosols with the yield of 70.65% (w/w) and 80.65% (w/w), respectively. Parameters' optimization for HD (substrate to solvent ratio, temperature, and extraction duration) and HDEA (cellulase loading and incubation duration) significantly affected the hydrosol yield, but did not extract out the essential oil. This is because only SFE had successfully ruptured the oil gland after observed under the scanning electron microscope. The essential oil obtained from SFE composed of mainly propanoic acid ethyl ester (40.25%), lactic acid ethyl ester (19.35%), 2-heptanol (15.02%), propanol (8.18%), 3-hexanone (2.60%), and butanoic acid ethyl ester (1.58%). In overall, it can be concluded that the SFE had successfully extracted the essential oil as compared to the HD and HDEA methods.

Improved Biobutanol Production in 2-L Simultaneous Saccharification and Fermentation with Delayed Yeast Extract Feeding and in-situ Recovery.

Simultaneous saccharification and fermentation (SSF) with delayed yeast extract feeding (DYEF) was conducted in a 2-L bioreactor equipped with in-situ recovery using a gas stripping in order to enhance biobutanol production from lignocellulosic biomass of oil palm empty fruit bunch (OPEFB). This study showed that 2.88 g/L of biobutanol has been produced from SSF with a similar yield of 0.23 g/g as compared to separate hydrolysis and fermentation (SHF). An increase of 42% of biobutanol concentration was observed when DYEF was introduced in the SSF at 39 h of fermentation operation. Biobutanol production was further enhanced up to 11% with a total improvement of 72% when in-situ recovery using a gas stripping was implemented to reduce the solvents inhibition in the bioreactor. In overall, DYEF and in-situ recovery were able to enhance biobutanol production in SSF.

Optimisation of Simultaneous Saccharification and Fermentation (SSF) for Biobutanol Production Using Pretreated Oil Palm Empty Fruit Bunch.

This study was conducted in order to optimise simultaneous saccharification and fermentation (SSF) for biobutanol production from a pretreated oil palm empty fruit bunch (OPEFB) by Clostridium acetobutylicum ATCC 824. Temperature, initial pH, cellulase loading and substrate concentration were screened using one factor at a time (OFAT) and further statistically optimised by central composite design (CCD) using the response surface methodology (RSM) approach. Approximately 2.47 g/L of biobutanol concentration and 0.10 g/g of biobutanol yield were obtained after being screened through OFAT with 29.55% increment (1.42 fold). The optimised conditions for SSF after CCD were: temperature of 35 °C, initial pH of 5.5, cellulase loading of 15 FPU/g-substrate and substrate concentration of 5% (w/v). This optimisation study resulted in 55.95% increment (2.14 fold) of biobutanol concentration equivalent to 3.97 g/L and biobutanol yield of 0.16 g/g. The model and optimisation design obtained from this study are important for further improvement of biobutanol production, especially in consolidated bioprocessing technology.

Pre-treatment of Oil Palm Biomass for Fermentable Sugars Production.

Malaysia is the second largest palm oil producer in the world and this industry generates more than 80 million tonnes of biomass every year. When considering the potential of this biomass to be used as a fermentation feedstock, many studies have been conducted to develop a complete process for sugar production. One of the essential processes is the pre-treatment to modify the lignocellulosic components by altering the structural arrangement and/or removing lignin component to expose the internal structure of cellulose and hemicellulose for cellulases to digest it into sugars. Each of the pre-treatment processes that were developed has their own advantages and disadvantages, which are reviewed in this study.

Combination of Superheated Steam with Laccase Pretreatment Together with Size Reduction to Enhance Enzymatic Hydrolysis of Oil Palm Biomass.

The combination of superheated steam (SHS) with ligninolytic enzyme laccase pretreatment together with size reduction was conducted in order to enhance the enzymatic hydrolysis of oil palm biomass into glucose. The oil palm empty fruit bunch (OPEFB) and oil palm mesocarp fiber (OPMF) were pretreated with SHS and ground using a hammer mill to sizes of 2, 1, 0.5 and 0.25 mm before pretreatment using laccase to remove lignin. This study showed that reduction of size from raw to 0.25 mm plays important role in lignin degradation by laccase that removed 38.7% and 39.6% of the lignin from OPEFB and OPMF, respectively. The subsequent saccharification process of these pretreated OPEFB and OPMF generates glucose yields of 71.5% and 63.0%, which represent a 4.6 and 4.8-fold increase, respectively, as compared to untreated samples. This study showed that the combination of SHS with laccase pretreatment together with size reduction could enhance the glucose yield.

Corrigendum to "Safety and Clinical Usage of Newcastle Disease Virus in Cancer Therapy".

[This corrects the article DOI: 10.1155/2011/718710.].

Harnessing the potential of ligninolytic enzymes for lignocellulosic biomass pretreatment.

Abundant lignocellulosic biomass from various industries provides a great potential feedstock for the production of value-added products such as biofuel, animal feed, and paper pulping. However, low yield of sugar obtained from lignocellulosic hydrolysate is usually due to the presence of lignin that acts as a protective barrier for cellulose and thus restricts the accessibility of the enzyme to work on the cellulosic component. This review focuses on the significance of biological pretreatment specifically using ligninolytic enzymes as an alternative method apart from the conventional physical and chemical pretreatment. Different modes of biological pretreatment are discussed in this paper which is based on (i) fungal pretreatment where fungi mycelia colonise and directly attack the substrate by releasing ligninolytic enzymes and (ii) enzymatic pretreatment using ligninolytic enzymes to counter the drawbacks of fungal pretreatment. This review also discusses the important factors of biological pretreatment using ligninolytic enzymes such as nature of the lignocellulosic biomass, pH, temperature, presence of mediator, oxygen, and surfactant during the biodelignification process.

Isolation, screening, and identification of potential cellulolytic and xylanolytic producers for biodegradation of untreated oil palm trunk and its application in saccharification of lemongrass leaves.

This study presents the isolation and screening of fungi with excellent ability to degrade untreated oil palm trunk (OPT) in a solid-state fermentation system (SSF). Qualitative assay of cellulases and xylanase indicates notable secretion of both enzymes by 12 fungal strains from a laboratory collection and 5 strains isolated from a contaminated wooden board. High production of these enzymes was subsequently quantified in OPT in SSF. Aspergillus fumigates SK1 isolated from cow dung gives the highest xylanolytic activity (648.448 U g(-1)), generally high cellulolytic activities (CMCase: 48.006, FPase: 6.860, beta-glucosidase: 16.328 U g(-1)) and moderate lignin peroxidase activity (4.820 U/g), and highest xylanolytic activity. The xylanase encoding gene of Aspergillus fumigates SK1 was screened using polymerase chain reaction by a pair of degenerate primers. Through multiple alignment of the SK1 strain's xylanase nucleotide sequences with other published xylanases, it was confirmed that the gene belonged to the xylanase glycoside hydrolase family 11 (GH11) with a protein size of 24.49 kD. Saccharification of lemongrass leaves using crude cellulases and xylanase gives the maximum reducing sugars production of 6.84 g/L with glucose as the major end product and traces of phenylpropanic compounds (vanillic acid, p-coumaric acid, and ferulic acid).

Preparation of bioactive peptides with high angiotensin converting enzyme inhibitory activity from winged bean [Psophocarpus tetragonolobus (L.) DC.] seed.

Winged bean [Psophocarpus tetragonolobus (L.) DC.] seed is a potential underexploited source of vegetable protein due to its high protein content. In the present work, undefatted and defatted winged bean seed hydrolysates, designated as UWBSH and DWBSH, respectively were produced separately by four proteolytic enzymes namely Flavourzyme, Alcalase, Bromelain, and Papain using pH-stat method in a batch reactor. Enzymatic hydrolysis was carried out over a period of 0.5 to 5 h. UWBSH and DWBSH produced were tested for their ACE inhibitory activity in relation to the hydrolysis time and degree of hydrolysis (DH). Maximum ACE inhibitory activity, both for UWBSH and DWBSH, were observed during 3 to 5 h of hydrolysis. Both, UWBSH (DH 91.84 %), and DWSBH (DH 18.72 %), produced by Papain at 5 h hydrolysis, exhibited exceptionally high ACE inhibitory activity with IC50 value 0.064 and 0.249 mg mL(-1), respectively. Besides, papain-produced UWBSH and DWBSH were further fractionated into three fractions based on molecular weight (UWBSH-I, <10 kDa; UWBSH-II, <5 kDa; UWBSH-III, <2 kDa) and (DWBSH-I, <10 kDa; DWBSH-II, <5 kDa; DWBSH-III, <2 kDa). UWBSH-III revealed the highest ACE inhibitory activity (IC50 0.003 mg mL(-1)) compared with DWBSH-III (IC50 0.130 mg mL(-1)). The results of the present investigation revealed that winged bean seed hydrolysates can be explored as a potential source of ACE inhibitory peptides suggesting their uses for physiological benefits as well as for other functional food applications.

Immunomodulatory effects of Newcastle disease virus AF2240 strain on human peripheral blood mononuclear cells.

Immunotherapy has raised the attention of many scientists because it hold promise to be an attractive therapeutic strategy to treat a number of disorders. In this study, the immunomodulatory effects of low titers of Newcastle disease virus (NDV) AF2240 on human peripheral blood mononuclear cells (PBMC) were analyzed. We evaluated cytokine secretion and PBMC activation by cell proliferation assay, immunophenotyping and enzyme linked immunosorbent assay. The proliferation of the human PBMC was measured to be 28.5% and 36.5% upon treatment with 8 hemaglutinin unit (HAU) and 2 HAU of NDV respectively. Interestingly, the percentage of cells with activating markers CD16 and CD56 were increased significantly. Furthermore, the intracellular perforin and granzyme levels were also increased upon virus infection. Human PBMC treated with NDV titer 8 HAU was found to stimulate the highest level of cytokine production including interferon-γ, interleukin-2 and interleukin-12. The release of these proteins contributes to the antitumor effect of PBMC against MCF-7 breast cancer cells. Based on the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, activated human PBMC showed high cytolytic efficiency towards human breast tumor cells. In summary, NDV was able to stimulate PBMC proliferation, cytokine secretion and cytolytic activity.