Optimisation of biodegradation conditions for waste canola oil by cold-adapted Rhodococcus sp. AQ5-07 from Antarctica

BackgroundThe potential waste canola oil-degrading ability of the cold-adapted Antarctic bacterial strain Rhodococcus sp. AQ5-07 was evaluated. Globally, increasing waste from food industries generates serious anthropogenic environmental risks that can threaten terrestrial and aquatic organisms and communities. The removal of oils such as canola oil from the environment and wastewater using biological approaches is desirable as the thermal process of oil degradation is expensive and ineffective.ResultsRhodococcus sp. AQ5-07 was found to have high canola oil-degrading ability. Physico-cultural conditions influencing its activity were studied using one-factor-at-a-time (OFAT) and statistical optimisation approaches. Considerable degradation (78.60%) of 3% oil was achieved by this bacterium when incubated with 1.0 g/L ammonium sulphate, 0.3 g/L yeast extract, pH 7.5 and 10% inoculum at 10°C over a 72-h incubation period. Optimisation of the medium conditions using response surface methodology (RSM) resulted in a 9.01% increase in oil degradation (87.61%) when supplemented with 3.5% canola oil, 1.05 g/L ammonium sulphate, 0.28g/L yeast extract, pH 7.5 and 10% inoculum at 12.5°C over the same incubation period. The bacterium was able to tolerate an oil concentration of up to 4.0%, after which decreased bacterial growth and oil degradation were observed.ConclusionsThese features make this strain worthy of examination for practical bioremediation of lipid-rich contaminated sites. This is the first report of any waste catering oil degradation by bacteria originating from Antarctica.How to cite: Ibrahim S, Zahri KNM, Convey P, et al. Optimisation of biodegradation conditions for waste canola oil by cold-adapted Rhodococcus sp. AQ5-07 from Antarctica. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.07.005     

Scaling-up batch conditions for efficient sucrose hydrolysis catalyzed by an immobilized recombinant Pichia pastoris cells in a stirrer tank reactor

BackgroundInvert sugar is used greatly in food and pharmaceutical industries. This paper describes scaling-up batch conditions for sucrose inversion catalyzed by the recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase entrapped in calcium alginate beads. For the first time, we describe the application of a kinetic model to predict the fractional conversion expected during sucrose hydrolysis reaction in both, a model and a prototype bioreactor with 0.5- and 5-L working volume, respectively.ResultsDifferent scaled-up criteria used to operate the 0.5-L bioreactor were analyzed to explore the invert sugar large scale production. After model inversion studies, a 5-L scaled-up reaction system was performed in a 7-L stirred reactor. Both scaled-up criteria, immobilized biocatalyst dosage and stirring speed, were analyzed in each type of bioreactors and the collected data were used to ensure an efficient scale-up of this biocatalyst.ConclusionsTo date, there is not enough information to describe the large-scale production of invert sugar using different scaled-up criteria such as dose of immobilized biocatalyst and stirring speed effect on mass transfer. The present study results constitute a valuable tool to successfully carry out this type of high-scale operation for industrial purposes.     

Bioconversion of mixed free fatty acids to poly-3-hydroxyalkanoates by Pseudomonas putida BET001 and modeling of its fermentation in shake flasks

BackgroundThe paper reports on the utilization of palm kernel oil (PKO) as a low cost renewable substrate for medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) production by Pseudomonas putida BET001. Investigation on the effects of selected key variables on growth, mixed free fatty acids consumption and mcl-PHA production by the bacterial culture in the shaken flask system were carried out along with its kinetic modeling.ResultsThe biomass production, fatty acids consumption and mcl-PHA production were found favorable when the strain was cultured in mineral medium at pH 6–7, 28°C, aeration surface-to-volume ratio of 0.4 × 10⁶ m^- 1, 250 rpm agitation rate for 48 h. Mcl-PHA production by this strain showed mixed growth and non-growth associated components as described by Luedeking–Piret kinetic model.ConclusionThe findings of this study provided add to the literature on key variables in for achieving good microbial growth and mcl-PHA production in shake flasks culture. In addition, suitable kinetic model to describe cultivation in this system was also presented.     

Enhanced alkaline catalase production by Serratia marcescens FZSF01: Enzyme purification,characterization, and recombinant expression

BackgroundCatalase (CAT) is an important enzyme that degrades H₂O₂ into H₂O and O₂. To obtain an efficient catalase, in this study, a new strain of high catalase-producing Serratia marcescens, named FZSF01, was screened and its catalase was purified and characterized.ResultsAfter optimization of fermentation conditions, the yield of catalase produced by this strain was as high as 51,468 U/ml. This catalase was further purified using two steps: DEAE-fast flow and Sephedex-G150. The purified catalase showed a specific activity of 197,575 U/mg with a molecular mass of 58 kDa. This catalase exhibited high activity at 20–70°C and pH 5.0–11.0. Km of the catalase was approximately 68 mM, and Vmax was 1886.8 mol/min mg. This catalase was further identified by LC–MS/MS, and the encoding gene was cloned and expressed in Escherichia coli BL21 (DE3) with a production of 17,267 ± 2037 U/ml.ConclusionsTo our knowledge, these results represent one of the highest fermentation levels reported among current catalase-producing strains. This FZSF01 catalase may be suitable for several industrial applications that comprise exposure to alkaline conditions and under a wide range of temperatures.     

Characterization of Lactobacillus fermentum UCO-979C,a probiotic strain with a potent anti-Helicobacter pylori activity

BackgroundHelicobacter pylori is considered as the main risk factor in the development of gastric cancer. In the present study, we performed a detailed characterization of the probiotic properties and the anti-H. pylori activity of a previously isolated lactobacillus strain — Lactobacillus fermentum UCO-979C — obtained from human gut.ResultsThe strain tolerated pH 3.0; grew in the presence of 2% bile salts; produced lactic acid and hydrogen peroxide; aggregated in saline solution; showed high hydrophobicity; showed high adherence to glass; Caco-2 and gastric adenocarcinoma human cells (AGS) cells; showed an efficient colonization in Mongolian Gerbils; and potently inhibited the growth and urease activity of H. pylori strains. L. fermentum UCO-979C significantly inhibited H. pylori-induced IL-8 production in AGS cells and reduced the viability of H. pylori. With regard to innocuousness, the strain UCO-979C was susceptible to several antibiotics and did not produce histamine or beta-haemolysis in blood agar containing red blood cells from various origins.ConclusionThe results demonstrated that L. fermentum UCO-979C is a very good candidate as a probiotic for the protection of humans against H. pylori infections.     

Respiration in Azotobacter vinelandii and its relationship with the synthesis of biopolymers

Azotobacter vinelandii is a gram-negative soil bacterium that produces two biopolymers of biotechnological interest, alginate and poly(3-hydroxybutyrate), and it has been widely studied because of its capability to fix nitrogen even in the presence of oxygen. This bacterium is characterized by its high respiration rates, which are almost 10-fold higher than those of Escherichia coli and are a disadvantage for fermentation processes. On the other hand, several works have demonstrated that adequate control of the oxygen supply in A. vinelandii cultivations determines the yields and physicochemical characteristics of alginate and poly(3-hydroxybutyrate).Here, we summarize a review of the characteristics of A. vinelandii related to its respiration systems, as well as some of the most important findings on the oxygen consumption rates as a function of the cultivation parameters and biopolymer production.How to cite: Castillo T, García A, Padilla-Córdova C, et al. Respiration in Azotobacter vinelandii and its relationship with the synthesis of biopolymers. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.08.001     

Yield and rheological properties of exopolysaccharide from a local isolate: Xanthomonas axonopodis pv. vesicatoria

BackgroundThe aim of the present study was to evaluate gum productivity of a local strain, Xanthomonas axonopodis pv. vesicatoria, isolated from pepper plant, and its rheological behavior for the first time compared to the standard strain, Xanthomonas campestris DSM 19000 (NRRL B-1459). The influence of operational conditions (agitation rate and inoculum volume) on gum production and rheological properties of gums from the Xanthomonas strains were investigated.ResultsThe isolated strain of Xanthomonas showed similar xanthan yield compared to the standard strain. Furthermore, this study clearly confirmed that gum yield depended on bacterial strain, agitation rate, and inoculum size. The most suitable conditions for the gum production in an orbital shaker in terms of agitation rate and inoculum size were 180 rpm and 5%, respectively, resulting in an average production of 10.96 and 11.19 g/L for X. axonopodis pv.vesicatoria and X. campestris DSM 19000, respectively. Regarding the rheological properties, Ostwald-de-Waele and power law models were used to describe flow and oscillatory behavior of the gum solutions, respectively. Consistency of the novel gum solution remarkably was much higher than the commercial xanthan gum solution. Flow and oscillatory behavior and their temperature ramps showed that weak gel-like structure could be obtained with less gum concentrations when the novel gum was used.ConclusionTherefore, yield and technological properties of the aqueous solutions of the exopolysaccharide synthesized by X. axonopodis pv. vesicatoria were observed to be more suitable for industrial production.     

Response surface methodology based optimization for degradation of align in Laminaria japonica feedstuff via fermentation by Bacillus in Apostichopus japonicas farming

BackgroundThe alga Laminaria japonica is the most economically important brown seaweed cultured in China, which is used as food and aquatic animal feedstuff. However, the use of L. japonica as a feedstuff in Apostichopus japonicas farming is not ideal because A. japonicas does not produce enough enzyme activity for degrading the large amount of algin present in L. japonica. In this study, semi solid fermentation of the L. japonica feedstuff employing a Bacillus strain as the microbe was used to as a mean to degrade the algin content in L. japonica feedstuff.ResultsThe Bacillus strain, Bacillus amyloliquefaciens WB1, was isolated by virtue of its ability to utilize sodium alginate as the sole carbon source. Eight factors affecting growth and algin-degrading capacity of WB1 were investigated. The results of Plackett–Burman design indicated that fermentation time, beef extract, and solvent to solid ratio were the significant parameters. Furthermore, the mutual interaction between the solvent to solid ratio and beef extract concentration was more significant than the other pairs of parameters on algin degradation. Optimal values obtained from Central-Composite Design were 113.94 h for fermentation time, 0.3% (w/v) beef extract and 44.87 (v/w) ratio of solvent to feedstuff. Under optimal conditions, 56.88% of the algin was degraded when a 50-fold scale-up fermentation was carried out, using a 5-L fermenter.ConclusionsThis study provides an alternative and economical way to reduce the algin content in L. japonica through degradation by WB1, making it a promising potential source of feed for cultured L. japonica.     

Highly efficient enzymatic preparation of isomalto-oligosaccharides from starch using an enzyme cocktail

BackgroundCurrent commercial production of isomalto-oligosaccharides (IMOs) commonly involves a lengthy multistage process with low yields.ResultsTo improve the process efficiency for production of IMOs, we developed a simple and efficient method by using enzyme cocktails composed of the recombinant Bacillus naganoensis pullulanase produced by Bacillus licheniformis, α-amylase from Bacillus amyloliquefaciens, barley bran β-amylase, and α-transglucosidase from Aspergillus niger to perform simultaneous saccharification and transglycosylation to process the liquefied starch. After 13 h of reacting time, 49.09% IMOs (calculated from the total amount of isomaltose, isomaltotriose, and panose) were produced.ConclusionsOur method of using an enzyme cocktail for the efficient production of IMOs offers an attractive alternative to the process presently in use.     

Xylitol production and furfural consumption by a wild type Geotrichum sp.

BackgroundXylitol is a five carbons polyol with promising medical applications. It can be obtained from chemical d-xylose reduction or by microbial fermentation of Sugarcane Bagasse Hemicellulosic Hydrolysate. For this last process, some microbial inhibitors, as furfural, constitute severe bottleneck. In this case, the use of strains able to produce xylitol simultaneously to furfural neutralization is an interesting alternative. A wild-type strain of Geotrichum sp. was detected with this ability, and its performance in xylitol production and furfural consumption was evaluated. Furthermore, were analyzed its degradation products.ResultsGeotrichum sp. produced xylitol from d-xylose fermentation with a yield of 0.44 g·g^-1. Furfural was fully consumed in fermentation assay and when provided in the medium until concentration of 6 g·L^-1. The furfural degradation product is not an identified molecule, presenting a molecular weight of 161 g·mol^-1, an uncommon feature for the microbial metabolism of this product.ConclusionThis strain presents most remarkable potential in performing furfural consumption simultaneous to xylitol production. Subsequent efforts must be employed to establish bioprocess to simultaneous detoxification and xylitol production by Geotrichum sp.     

Light-addressable electrodeposition of cell-encapsulated alginate hydrogels for a cellular microarray using a digital micromirror device

This paper describes a light-addressable electrolytic system used to perform an electrodeposition of calcium alginate hydrogels using a digital micromirror device (DMD). In this system, a patterned light illumination is projected onto a photoconductive substrate serving as a photo-anode to electrolytically produce protons, which can lead to a decreased pH gradient. The low pH generated at the anode can locally release calcium ions from insoluble calcium carbonate (CaCO₃) to cause gelation of calcium alginate through sol-gel transition. By controlling the illumination pattern on the DMD, a light-addressable electrodeposition of calcium alginate hydrogels with different shapes and sizes, as well as multiplexed micropatterning was performed. The effects of the concentration of the alginate and CaCO₃ solutions on the dimensional resolution of alginate hydrogel formation were experimentally examined. A 3 × 3 array of cell-encapsulated alginate hydrogels was also successfully demonstrated through light-addressable electrodeposition. Our proposed method provides a programmable method for the spatiotemporally controllable assembly of cell populations into cellular microarrays and could have a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery.     

Screening of cellulose degradation bacteria from Min pigs and optimization of its cellulase production

BackgroundCellulose as a potential feed resource hinders its utilization because of its complex structure, and cellulase is the key to its biological effective utilization. Animal endogenous probiotics are more susceptible to colonization in the intestinal tract, and their digestive enzymes are more conducive to the digestion and absorption of feed in young animals. Min pigs are potential sources of cellulase probiotics because of the high proportion of dietary fiber in their feed. In this study, the cellulolytic bacteria in the feces of Min pigs were isolated and screened. The characteristics of enzymes and cellulase production were studied, which provided a theoretical basis for the rational utilization of cellulase and high-fiber food in animal production.ResultsIn our study, 10 strains of cellulase producing strains were isolated from Min pig manure, among which the M2 strain had the best enzyme producing ability and was identified as Bacillus velezensis. The optimum production conditions of cellulase from strain M2 were: 2% inoculum, the temperature of 35°C, the pH of 5.0, and the liquid loading volume of 50 mL. The optimum temperature, pH and time for the reaction of cellulase produced by strain M2 were 55°C, 4.5 and 5 min, respectively.ConclusionsMin pigs can be used as a source of cellulase producing strains. The M2 strain isolated from feces was identified as Bacillus velezensis. The cellulase from M2 strain had a good activity and the potential to be used as feed additive for piglets.How to cite: Li F, Xie Y, Gao X, et al. Screening of cellulose degradation bacteria from Min Pigs and optimization of its cellulase production. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.001     

Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

BackgroundIn industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze–thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain.ResultsElite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze–thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield.ConclusionsA multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze–thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation.How to cite: Zhang Q, Jin Y, Fang Y, et al. Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high gravity bioethanol fermentation. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.06.003     

Effect of synthetic and natural media on lipid production from Fusarium oxysporum

BackgroundDependence on fossil resources, for the production of fuels and energy, has resulted in environmental and financial problems, which require our immediate action in order to reverse the situation. Use of renewable sources for the production of fuels and energy is an important alternative with biodiesel remains as one of the promising options. Aim of this work is to evaluate the fungus Fusarium oxysporum for its potentials to accumulate microbial lipids when grown on synthetic media and saccharified sweet sorghum stalks.ResultsThe effect of different carbon sources, nitrogen sources and C/N ratio on the lipid production was initially examined, which resulted in a lipid concentration of 4.4 g/L, with lipid content of 42.6% w/w. Sweet sorghum stalks were able to support growth and lipid production of the fungus, both as carbon source and as nitrogen source. It was also shown that saccharification of the dried stalks is an important step to increase lipid production. Removal of the remaining stalk solids enabled the lipid production during cultivation in increased initial solids of up to 16 w/w. This resulted in a lipid production of 3.81 g/L.ConclusionsIt was demonstrated that F. oxysporum can be used as an efficient oleaginous microorganism, with sweet sorghum serving as an excellent raw material for the cultivation of the fungus. The lipids obtained during this work were also found to have a fatty acid profile with good potentials to be used for biodiesel production.     

Effect on growth and productivity of lutein from the chlorophyta microalga,strain MCH of Muriellopsis sp., when grown in sea water and outdoor conditions at the Atacama Desert

BackgroundMicroalgae are microorganisms that produce various products, for example, pigments, mainly carotenoids. This study aimed to used the strain of Muriellopsis sp. and to evaluate their behavior when grown in freshwater and seawater, along with indoor and outdoor conditions for both cultures. Growth of the strain was evaluated by determining its biomass, lutein productivity with high-performance liquid chromatography (HPLC), and antioxidant activity by using the 2,2-diphenyl-1-picrilhydrazil (DPPH method).ResultsMuriellopsis sp. strain in indoor cultures showed an increased antioxidant activity. In outdoor conditions, both cultures showed increased cells number, concentration of biomass, and lutein productivity. The percentage of lutein obtained from the strain MCH in indoor conditions was 25 times higher than that reported for calendula, reaching 0.75% of lutein in Muriellopsis sp. cultured in seawater, followed by 0.6% in Muriellopsis sp., cultures in freshwater at day 12 of both cultures. These values exceed that of microalgae Scenedesmus almeriensis, which reaches 0.53% lutein.ConclusionsThe results show that the native strain of the Atacama Desert is one of the largest producers of lutein as compared to those reported to date. The study demonstrated the feasibility of producing this carotenoid with well-known properties to prevent some diseases due to its high nutritional value. Muriellopsis sp. cultivation in open-air seawater is a good precedent for developing mass production of this species in an area where freshwater is scarce and costly.How to cite: Cruz-Balladares V, Marticorena P, Riquelme C. Effect on growth and productivity of lutein from the chlorophyta microalga, strain MCH of Muriellopsis sp., when grown in sea water and outdoor conditions at the Atacama Desert. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.09.001     

Antiproliferative activity of biomass extract from Pseudomonas cedrina

BackgroundThe study of plant-associated microorganisms is very important in the discovery and development of bioactive compounds. Pseudomonas is a diverse genus of Gammaproteobacteria comprising more than 60 species capable of establishing themselves in many habitats, which include leaves and stems of many plants. There are reports of metabolites with diverse biological activity obtained from bacteria of this genus, and some of the metabolites have shown cytotoxic activity against cancer cell lines.Because of the high incidence of cancer, research in recent years has focused on obtaining new sources of active compounds that exhibit interesting pharmacodynamic and pharmacokinetic properties that lead to the development of new therapeutic agents.ResultsA bacterial strain was isolated from tumors located in the stem of Pinus patula, and it was identified as Pseudomonas cedrina. Extracts from biomass and broth of P. cedrina were obtained with chloroform:methanol (1:1). Only biomass extracts exhibited antiproliferative activity against human tumor cell lines of cervix (HeLa), lung (A-549), and breast (HBL-100). In addition, a biomass extract from P. cedrina was fractioned by silica gel column chromatography and two diketopiperazines were isolated: cyclo-(l-Prolyl-l-Valine) and cyclo-(l-Leucyl-l-Proline).ConclusionsThis is the first report on the association of P. cedrina with the stems of P. patula in Mexico and the antiproliferative activity of extracts from this species of bacteria against human solid tumor cell lines.How to cite: Sánchez-Tafolla L, Padrón JM, Mendoza G, et al. Antiproliferative activity of biomass extract from Pseudomonas cedrina. Electron J Biotechnol 2019;40. https://doi.org/10.1016/j.ejbt.2019.03.010.     

Systematic engineering of the rate-limiting step of β-alanine biosynthesis in Escherichia coli

BackgroundLarge amounts of β-alanine are required in fine chemical and pharmaceutical synthesis and other fields. Profitable and green methods are required for the industrial production of β-alanine.ResultsReplacing endogenous panD of Escherichia coli with heterologous CgpanD from Corynebacterium glutamicum enabled β-alanine synthesis of 0.67 g/L by strain B0016-082BB. Overexpressing CgpanD on both plasmids and chromosomes to enhance the rate-limiting step improved the β-alanine titer to 4.25 g/L in strain B0016-083BB/pPL451-panD with a slighter metabolic burden. Growth factors were introduced by addition of yeast extract, and 6.65 g/L of β-alanine was synthesized by strain B0016-083BB/pPL451-panD in the M9-3Y medium.ConclusionsEnhancement of the rate-limiting steps in the β-alanine biosynthetic pathway, recruitment of the temperature-sensitive inducible p_L promoter, and optimization of the fermentation process could efficiently increase β-alanine production in E. coli.How to cite: Xua J, Zhua Y, Zhou Z. Systematic engineering of the rate-limiting step of β-alanine biosynthesis in Escherichia col. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.03.002.     

Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake,and investigations into its prebiotic activity

BackgroundManno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain.ResultsGalactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose.ConclusionsThe effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum, which would have potential application in the biotechnological applications.How to cite: Zhang R, Li X-Y, Cen X-L, et al. Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity. Electron J Biotechnol 2021;49. https://doi.org/10.1016/j.ejbt.2020.11.001