Submerged Fermentation Conditions Research Articles

New food ingredient via acid-tolerant Rhizopus oligorporus growth

As the global population grows, the consumption of unsustainably produced proteins increases. Researching sustainable food sources with a positive environmental impact is essential. This research provides valuable insights into the production and application of nutrient rich food sources. The potential of the biomass generated through fermentation with R. oligosporus was evaluated as a new protein source with low environmental impact. A fermentation method has been optimized to maximize the biomass production of R. oligosporus mycelium in solid-state and submerged-state fermentation, yielding 5.9 g dry weight/L and 5 g dry weight/L, respectively. The growth medium for these fungi was adapted to acid-tolerant (pH 4-3) conditions to create a food development environment that is more difficult for pathogens to thrive in, both in solid state and submerged state fermentation. The highest protein content obtained was 38.4 % wet weight by solid state fermentation. Fungal biomass exhibited techno-functional properties relevant to food product development tested both on pH 4 and pH 3, showing high water absorption and emulsifying activity, particularly in submerged fermentation at pH 3. In contrast, solid-state fermentation at pH 4 resulted in higher emulsion stability. This research demonstrates the potential of Rhizopus oligosporus as an acid-tolerant, high-quality protein ingredient with valuable functional properties. Its efficient production using minimal resources makes it a valuable asset for the food industry.

Microbial degradation of paddy (Oryza sativa) straw by submerged state fermentation

This study elucidates the effective lignocellulose degrading potential of microbial consortium by using paddy straw as the sole source under submerged fermentation conditions. Straw degradation was enhanced by bioaugmentation procedures where consortia of isolated and standard cultures resulted in maximum enzyme activities of endoglucanase (1 U/ml), exoglucanase (1.31 U/ml), β-glucosidase (2.42 U/ml), xylanase (12.63 U/ml) and manganese peroxidase (5.43 U/ml) after 28 days of incubation along with laccase (4.36 U/ml) and lignin peroxidase (6.71 U/ml) after 21 days of incubation. By the action of consortium, maximum reduction of cellulose, hemicellulose and lignin was found to be 35.31, 22.50 and 44.72%, respectively after 28 days of incubation. A significant increase in percentage loss in dry matter of paddy straw was observed till 4 weeks of fermentation. Field emission scanning electron microscope (FE-SEM) micrographs revealed sufficient loosening of cellulose and hemicellulose showing successful degradation of straw tissue. Overall, the above stated findings emphasize on potential of lignocellulosic microorganisms to be used as an alternative to traditional stubble burning practice. Although, these extracellular enzymes are well equipped in breaking down lignocelluloses to their monomeric or oligomeric subunits, but they may not be exploited at commercial level due to very slow rate of reaction. The efficacy of these cultures for straw degradation in field condition further needs elaborative experimental trials as environmental parameters are quite variable in the field.

Biodegradation of Keratin Waste by Bacillus velezensis HFS_F2 through Optimized Keratinase Production Medium.

Enormous aggregates of keratinous wastes are produced annually by the poultry and leather industries which cause environmental degradation globally. To combat this issue, microbially synthesized extracellular proteases known as keratinase are used widely which is effective in degrading keratin found in hair and feathers. In the present work, keratinolytic bacteria were isolated from poultry farm soil and feather waste, and various cultural conditions were optimized to provide the highest enzyme production for efficient keratin waste degradation. Based on the primary and secondary screening methods, the potent keratinolytic strain (HFS_F2T) with the highest enzyme activity 32.65 ± 0.16 U/mL was genotypically characterized by 16S rRNA sequencing and was confirmed as Bacillus velezensis HFS_F2T ON556508. Through one-variable-at-a-time approach (OVAT), the keratinase production medium was optimized with sucrose (carbon source), beef extract (nitrogen source) pH-7, inoculum size (5%), and incubation at 37 °C). The degree of degradation (%DD) of keratin wastes was evaluated after 35days of degradation in the optimized keratinase production medium devoid of feather meal under submerged fermentation conditions. Further, the deteriorated keratin wastes were visually examined and the hydrolysed bovine hair with 77.32 ± 0.32% degradation was morphologically analysed through Field Emission Scanning Electron Microscopy (FESEM) to confirm the structural disintegration of the cuticle. Therefore, the current study would be a convincing strategy for reducing the detrimental impact of pollutants from the poultry and leather industries by efficient keratin waste degradation through the production of microbial keratinase.

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

Xylanases are key biocatalysts in the degradation of the β‐1,4‐glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-β-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications.Key points• A GH10 endo-1,4-β-xylanase (Xyl4) was isolated from a compost metagenomic library• MATLAB’s in-house functions were developed to identify the xylanase-producing clones• Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysisGraphical abstract

Statistical versus neural network-embedded swarm intelligence optimization of a metallo-neutral-protease production: activity kinetics and food industry applications

An integrated approach involving response surface methodology (RSM) and artificial neural network-ant-colony hybrid optimization (ANN-ACO) was adopted to develop a bioprocess medium to increase the yield of Bacillus cereus neutral protease under submerged fermentation conditions. The ANN-ACO model was comparatively superior (predicted r 2 = 98.5%, mean squared error [MSE] = 0.0353) to RSM model (predicted r 2 = 86.4%, MSE = 23.85) in predictive capability arising from its low performance error. The hybrid model recommended a medium containing (gL−1) molasses 45.00, urea 9.81, casein 25.45, Ca2+ 1.23, Zn2+ 0.021, Mn2+ 0.020, and 4.45% (vv−1) inoculum, for a 6.75-fold increase in protease activity from a baseline of 76.63 UmL−1. Yield was further increased in a 5-L bioreactor to a final volumetric productivity of 3.472 mg(Lh)−1. The 10.0-fold purified 46.6-kDa-enzyme had maximum activity at pH 6.5, 45–55 °C, with Km of 6.92 mM, Vmax of 769.23 µmolmL−1 min−1, kcat of 28.49 s−1, and kcat/Km of 4.117 × 103 M−1 s−1, at 45 °C, pH 6.5. The enzyme was stabilized by Ca2+, activated by Zn2+ but inhibited by EDTA suggesting that it was a metallo-protease. The biomolecule significantly clarified orange and pineapple juices indicating its food industry application.

Microbial production of dextran using pineapple waste extract: a two-step statistical optimization of submerged fermentation conditions and structural characterization

Microbial production of dextran using pineapple waste extract: a two-step statistical optimization of submerged fermentation conditions and structural characterization

Production and Optimization of L-glutaminase from Halophilic Fusarium solani-melongenae Strain CRI 24 under Submerged and Solid State Fermentation

L-glutaminase is a unique enzyme with catalytic activity and the ability to modulate glutamine levels, making it a valuable enzyme with numerous potential applications. L-glutaminase triggers a distinctive reaction by converting L-glutamine into glutamic acid while releasing ammonia concurrently. This enzymatic process holds potential applications across diverse industries, notably in food and pharmaceuticals. The primary objective of the present research was to identify and isolate a fungal strain proficient in L-glutaminase production from soil found in maritime environments. The physical and nutritional conditions were optimized for maximum synthesis of L-glutaminase under solid state fermentation (SSF) and submerged fermentation conditions (SmF). The isolated organism was identified as Fusarium solani-melongenae strain CRI 24 by morphological and 18S rRNA analysis. The optimum carbon source under SmF and SSF was found to be starch (0.2% w/v). Wheat bran as solid substrate among others showed optimum enzyme activity. On the seventh day of incubation, at pH 8.0 and 0.7% L-glutamine concentration under SSF and SmF, the highest enzyme activity was detected. The greatest enzyme activity in SSF was seen at a moisture content of 10%. Fusarium solani-melongenae species produced the enzyme under optimal conditions and 4.20 and 4.73-fold increase (from 0.8 U/mL to 3.61 U/mL and from 0.781 U/mL to 3.69 U/mL) was achieved after optimization in submerged and in solid state fermentation, respectively. The selective isolation and optimization processes described in this work are a promising technique for the industrial production of L-glutaminase and can be applied in the pharmaceutical and food industries.

Application of sequential design for enhanced L-asparaginase synthesis from Ganoderma australe GPC191.

With an increasing demand for L-asparaginase in pharmaceutical and food sectors for its cytostatic and acrylamide-reducing qualities, there's a need to discover novel, highly productive enzyme sources with improved pharmacokinetic profiles. Keeping this in mind, the present study aimed at maximizing the potential of Ganoderma australe GPC191 to produce L-asparaginase by fermentation medium optimization using statistical validation. Of the 11 physicochemical parameters evaluated under submerged fermentation conditions through one-factor-at-a-time approach and Plackett-Burman design, only four parameters (inoculum load, L-asparagine, soybean meal, and initial pH) influenced L-asparaginase production, significantly (p < 0.001). The optimal levels and interaction effects of these on the overall production were further evaluated by the central composite rotatable design of response surface methodology. Post-optimization, 27.34 U/mL was predicted as the maximum activity at pH 7 with 5n inoculum load and 15g/L each of L-asparagine and soybean meal. Experimental validation yielded an activity of 28.52 U/mL, indicating an overall 18.17-fold increase from the unoptimized stage. To our knowledge, this is the first report signifying the L-asparaginase production aptitude of G. australe with sequential statistical validation using agricultural waste, which can serve as a model to enhance its yields, offering a sustainable and cost-effective solution for industrial application.

Systematic characterisation, and effect of nutritional and physical parameters on culturability, laccase production and dye decolorisation potential by P. pistillaris from Pakistan

Podaxis pistillaris is neutraceutically, cosmoceutically and medicinally recognised macrofungus. During this research work, this edible mushroom was systematically characterised. Its culturability, laccase production, and dye decolorisation potential were evaluated and optimised. Among the different media tested, PDA proved as most efficient medium for culturability of P. pistillaris. Conditions for laccase production were optimised in submerged state fermentation. Maximum laccase secretion was noted after 14th day of Incubation at 35 °C with 130 rpm and 5 pH of medium. Fructose and ammonium-phosphate was found as best carbon and nitrogen source, while wheat straw revealed as good ligno-cellulosic source for strengthening laccase production. Congo-red dye decolorisation capability by crude laccase enzyme was evaluated and found maximum decolorisation potential (92.2%) after 288h of incubation. From this pilot study, it was confirmed that this edible macrofungus has culturability, laccase production and dye decolorisation attributes that will further contribute in delignification, biosorption and bioremediation.

Actividad antioxidante del extracto acuoso de granos de café usados y fermentados con Pleurotus ostreatus

Background: Coffee residues, including spent coffee grounds (SCG), are an important source of bioactive components like phenolic compounds which are characterized to exert bioactivity. Objective: Evaluate the antioxidant effect of the aqueous extract obtained from SCG, under submerged-state fermentation using Pleurotus ostreatus. Methods: Submerged-state fermentation using Pleurotus ostreatus was used to release phenolic compounds from SCG (0, 5 and 10 %). After fermentation, the qualitative and quantitative phytochemical content was monitored (carbohydrates, polyphenols, flavonoids, and cafeoylquinic acid), as well as the antioxidant properties (antiradical activity and reducing power). In addition, lipid oxidation levels of pork meat homogenates incorporated with SCG extract during storage were evaluated. Results and conclusions: The results showed an increase in phytochemical content and antioxidant properties in the aqueous extract from SCG fermented with P. ostreatus, in dependence of the used substrate concentration. Also, a reduction in lipid oxidation values was showed in pork meat homogenates treated with the fermented SCG extract. Fungal fermentation process of coffee residues using Pleurotus spp., could be a promissory strategy to obtain natural additives.

Integrative addition of sucrose esters and immobilisation technology for enhancing yellow pigment yield of Monascus purpureus HBSD08 under submerged fermentation conditions and its molecular mechanism

In this study, the production of yellow pigment by Monascus purpureus upon the addition of sucrose esters under submerged fermentation was significantly promoted. When the addition of sucrose esters was integrated with immobilised fermentation using 0.75 g of corn cob as the carrier for 5 days, the yellow pigment yield reached 39.24 AU/50 mL and was 4.12 times that of the control group. Monascus of integrative fermentation under five-batch fermentation gave a higher yellow pigment yield than Monascus under free fermentation. The extracellular production of yellow pigment in the fourth batch reached a maximum value of 56.71 AU/50 mL. Transcriptome analysis revealed that the integrative fermentation effectively regulated the pyruvate metabolism process, changed the intrinsic components of the cell membrane and the membrane permeability, and improved the transport capacity of pigment across the membrane. The significantly enriched KEGG metabolic pathways revealed that immobilised fermentation enhanced the glycolysis/gluconeogenesis pathway involved in pigment synthesis and altered the metabolic flow. There was an increase in the precursors of yellow pigment synthesis—such as acetyl-CoA, which is produced during tyrosine metabolism—thereby increasing yellow pigment production. This study provides some potential approaches for improving yellow pigment yield whilst also deepening a theoretical understanding of the underpinning mechanisms.

Screening of xylanase producing Bacillus species and optimization of xylanase process parameters in submerged fermentation

One of the main process inputs in the microbial enzyme, an environment friendly and an extraordinary catalyst, production is characteristics of the microorganism. This study aimed to screen newly isolated Bacillus species for the xylanase production potentiality and investigate crucial parameters that affect the xylanase producing process. Bacteria samples were isolated from potential agricultural and food sources such as soil, wheat bran, and sourdough. Eight xylanase producing isolates showed 99% homology to Bacillus species according to 16S rRNA sequence analysis. Isolate A67 has been found the best xylanase producer isolate and identified as Bacillus halotolerans DSM 8802. Incubation temperature, initial medium pH, and agitation speed parameters were optimized by RSM in the shake flask scale. The optimum xylanase production points were found as 30 °C, pH 8, and 127.27 rpm under submerged fermentation conditions. Xylanase activity was obtained experimentally as 23.47 U/mL resulting 4.21-fold increase with the statistical optimization. R2 and predicted R2 values that reflects the significance of the designed model were calculated as 0.9964 and 0.9674, respectively. Incubation temperature has been concluded as the most important parameter that affects the xylanase activity for isolate A67. As it has low incubation temperature demand, metabolizes xylan polymer as a carbon source and most of the Bacillus species do not produce any toxin, isolate A67 could have potential for the usage of the lignocellulosic biomass added media to make fermentation cost effective in food-grade xylanase production process.

Optimization of in situ cellulase production from Penicillium oxalicum P-07 under submerged fermentation conditions with different cellulose types

Biological enzymes have attracted much attention due to their good performance and environmentally friendly characteristics. This case focused on promoting the production of cellulase from a fungus strain (identified as Penicillium oxalicum) was isolated from food waste (FW). To better apply Penicillium oxalicum P-07 to cellulose degradation, the optimization of process parameters is particularly important. The combination of medium substrates sodium carboxymethyl cellulose (CMC-Na) and wheat bran (WB) presented higher filter paper activity (FPase) than the sum of the sole ones. Further optimizing the degradation conditions to pH 5.0 and 37 ℃, the FPase increased by 1.5 times, from 0.30 U/mL to 0.45 U/mL. Meanwhile, endoglucanase (CMCase), cellobiohydrolases (CBHase) and β-glucosidase reached 5.66, 0.25 and 5.59 U/mL respectively. The outcomes demonstrated that Penicillium oxalicum P-07 cellulase production was greatly aided by the optimization of operation parameters.

Optimization of submerged fermentation conditions for glucanase production by Burkholderia pyrrocinia B1213 using Jiuzao

This study optimized the fermentation conditions for producing glucanase from Burkholderia pyrrocinia B1213 using Jiuzao, a residue from Baijiu distillation, as the carbon source. The effects of Jiuzao particle size and concentration, type of nitrogen source, urea concentration, initial pH, inoculum size, loading volume, shaking speed, temperature, surfactant type and incubation time on glucanase production by B. pyrrocinia B1213 were investigated separately through single factor design. Then, five variables, Jiuzao concentration, inoculum size, initial pH, temperature and incubation time, were found to significantly affect glucanase production using the Plackett-Burman design. Following, the optimal conditions for glucanase production by B. pyrrocinia B1213 were found using the steepest ascent path and response surface methodology designs as: particle size, 40-60 mesh; Jiuzao concentration, 58.4 g/L; urea concentration, 8 g/L; initial pH, 6; loading volume, 15 mL/250 mL; inoculum size, 0.63% (v/v); temperature, 26 °C; shaking speed, 160 rpm; and incubation time, 120 h. Under these conditions, the glucanase activity of the B. pyrrcinia B1213 strain was 1336 U/mL, producing the biological enzymes needed in Baijiu making from a by-product. This study has provided experimental data and theoretical information for using B. pyrrcinia B1213 in Baijiu production. Keywords: Burkholderia pyrrocinia; Glucanase; Response surface methodology; Fermentation conditions; Jiuzao

Statistical optimization for the production of bioactive exopolysaccharide by Ganoderma gibbosum under submerged fermentation conditions and in-vitro evaluation of its antioxidant activity

Statistical optimization for the production of bioactive exopolysaccharide by Ganoderma gibbosum under submerged fermentation conditions and in-vitro evaluation of its antioxidant activity

Efficacy of the Fruit and Vegetable Peels as Substrates for the Growth and Production of α-Amylases in Marine Actinobacteria.

Enzymes from haloalkaliphilic microorganisms have recently focused attention on their potential and suitability in various applications. In this study, the growth and production of extracellular amylases in the marine actinomycetes, using kitchen waste as the raw starch source, have been investigated. Actinobacteria were isolated from the seawater of the Kachhighadi Coast near Dwarika, Gujarat. Seven Actinobacterial isolates of pre-monsoon, monsoon, and post-monsoon seasons belonging to different strains of Nocardiopsis genera were screened and selected for amylase production. The amylase production was initially assessed on the solid media supplemented with the extracts of different fruits and vegetable peels as a substrate by agar plate assay. The strains Kh-2(13), Kh-2(1), and Kh-3(12) produced maximum amylase with potato peel as a substrate, while no significant differences were found with the media containing other peels. Nevertheless, all strains produced amylases at a significant level with other raw substrates as well. For the optimization of the growth and enzyme production, the selected two isolates Kh-2(13) and Kh-3(12) of the monsoon and winter seasons were cultivated in a liquid medium under the submerged fermentation conditions, with potato peel as a substrate. In both organisms, the optimum amylase production was observed in the stationary phase of growth. For amylase production, the effect of different physical and chemical parameters was evaluated. The optimum growth and amylase production was achieved in 2% inoculum size, at pH 8.0, 28℃, and 5% salt concentration. On the basis of the amylase production index (API) (a ratio of the amylase units and cell growth), both isolates produced significant amylase with the only extract of potato peels, without any other supplements. The trends further indicated that while additional complex sources, such as yeast extract and peptone can enhance the cell growth of the actinobacteria, the amylase production remained unaltered. The study projects the significance of waste raw materials for the production of enzymes in extremophilic microorganisms.

Optimization of Orange Pigment Yield Produced by Monascus ruber under Submerged Fermentation Conditions

Optimization of Orange Pigment Yield Produced by Monascus ruber under Submerged Fermentation Conditions

Production of mycelial biomass, proteases and protease inhibitors by Ganoderma lucidum under different submerged fermentation conditions.

Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.

Solid-state fermentation produces greater stearidonic acid levels in genetically engineered Mucor circinelloides.

Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are important dietary components due to their health benefits and preventative role in cardiovascular disease. Fish-based and plant seed oils are rich in stearidonic acid (SDA; 18:4, n-3), which are readily metabolized into ω-3 PUFAs such as eicosapentaenoic acid. However, these natural sources of SDA are generally low yielding and are unlikely to meet global demands, so new sustainable microbial fermentative sources of SDA need to be identified. Expression of delta15-desaturase in the oleaginous filamentous fungus Mucor circinelloides (McD15D) has been used to construct a recombinant SDA-producing McD15D strain that produces 5·0% SDA levels using submerged fermentation conditions. Switching to solid-state fermentation conditions in the same medium with submerged fermentation resulted in this engineered strain producing significantly higher amounts of SDA. A Box-Behnken design of response surface methodology approach has been used to identify optimal glucose and ammonium tartrate concentrations and temperature levels to maximize SDA production. The use of these optimal solid-state fermentation conditions resulted in the spores and mycelium of the recombinant McD15D producing 19·5% (0·64 mg g-1 ) and 12·2% (1·52 mg g-1 ) SDA content, respectively, which represents an overall increase in SDA yield of 188·0% compared with SDA yields produced using submerged fermentation conditions.

Optimization of Independent Variables for the Production of Extracellular Alpha Amylase by Bacillus subtilis IMD34 Using Plackett-Burman Design

Bacillus species are widely used for thermostable α-amylase production for various applications such as in the detergent and food industries. This research aimed to study the production of α-amylase by B. subtilis IMD34 in submerged fermentation conditions using Plackett-Burman (PB) design, along with the optimization of certain environmental factors for optimal growth of amylolytic bacteria. Plackett-Burman design was used to evaluate the effect of ten independent variables on α-amylase production. Maximum enzyme activity was recorded in a medium containing (g/l); starch (6.0), peptone (12.0), K2HPO4 (1.0), Na2HPO4 (2.0), NaCl (0.6), Na2SO4 (0.1), MgSO4.7H20 (1.0), FeCl3.6H2O (0.02), CaCl2.2H2O (0.02) and with an agitation speed (200 rpm). From the pareto chart it was observed that the most important independent variables for amylase production were starch, peptone, NaCl, Mg2SO4, CaCl2.2H2O and agitation. Optimization of environmental parameters to assess their effect on the growth of bacteria showed that optimal pH, incubation time and temperature were recorded at 6, 48h and 37˚C. Evaluating independent variables statistically using PB design revealed that some of the medium’s components had a positive impact on α-amylase production by B. subtilis IMD34.