Submerged Fermentation Process Research Articles

Integration of electronic nose, electronic tongue, and colorimeter in combination with chemometrics for monitoring the fermentation process of Tremella fuciformis

This study proposes an efficient method for monitoring the submerged fermentation process of Tremella fuciformis (T. fuciformis) by integrating electronic nose (e-nose), electronic tongue (e-tongue), and colorimeter sensors using a data fusion strategy. Chemometrics was employed to establish qualitative identification and quantitative prediction models. The Pearson correlation analysis was applied to extract features from the e-nose and tongue sensor arrays. The optimal sensor arrays for monitoring the submerged fermentation process of T. fuciformis were obtained, and four different data fusion methods were developed by incorporating the colorimeter data features. To achieve qualitative identification, the physicochemical data and principal component analysis (PCA) results were utilized to determine three stages of the fermentation process. The fusion signal based on full features proved to be the optimal data fusion method, exhibiting the highest accuracy across different models. Notably, random forest (RF) was shown to be the most accurate pattern recognition method in this paper. For quantitative prediction, partial least squares regression (PLSR) and support vector regression (SVR) were employed to predict the sugar content and dry cell weight during fermentation. The best respective predictive R2 values for reducing sugar, tremella polysaccharide and dry cell weight were found to be 0.965, 0.988, and 0.970. Furthermore, due to its ability to capture nonlinear data relationships, SVR had superior performance in prediction modeling than PLSR. The results demonstrated that the combination of electronic sensor fusion signals and chemometrics provided a promising method for effectively monitoring T. fuciformis fermentation.

Role of Nitrogen Source for L-Glutaminase Production from Fungal Strain using through Submerged Fermentation

L-glutaminase has attracted much attention due its wide range of applications in several fields. The L-glutaminase widely used in pharmaceutical and food industries. L-glutaminase is generally regarded as a key enzyme that controls the delicious taste of fermented foods such as soy sauce. L-glutaminase production was carried out by using supplementation of organic and inorganic nitrogen sources such as yeast extract, malt extract, peptone and urea at concentration ranging from 0.25% to 1.25% with increments of 0.25% and also different inorganic nitrogen sources like ammonium sulphate and ammonium chloride at concentration ranging from 0.025% to 0.125% with increments of 0.025%. The malt extract (1%) produced 399.9 IU, were best organic nitrogen source and ammonium sulphate (0.1%) appear to be good inorganic nitrogen source under submerged fermentation process and showed 546 IU. Current study is an exploring step to industrial sector to upscale their L-glutaminase production and it will useful strategy to commercial sector and alternative to old methods

Production of extracellular Pullulanase by NP 7 bacteria using OFAT methodology

Pullulanase is a debranching enzyme that spilts explicitly α-1,6 glycosidic bonds. It is widely used in starch saccharification, in production of glucose, fructose as well as maltotriose syrup and bioethanol production. Pullulanase is produced by submerged fermentation process that requires optimization of physical and chemical parameters to achieve higher activity and make this process cost effective. The present study aimed the optimization of physical as well as chemical parameters of production medium. The optimization includes physical parameters such as inoculum size, incubation time, temperature, pH and agitation rate while chemical parameters include carbon source, nitrogen source and different metal ions. From the optimization study, it was found that highest pullulanase production was achieved after 48 hours of incubation at 45ºC temperature, pH 7 and 120 rpm. Optimization of chemical parameters reveals that incorporation of starch (1%) and 0.2mMole/L of calcium chloride in the media gave significant increase in enzyme activity. The maximum productivity achieved after optimization was 139.33 U/ml using OFAT method which was four times higher.

The potential of in-house pectinolytic enzymes for industrial application

Pectinases are one of the essential groups of enzymes in the field of biotechnology and have applications in various industries including food, textile, paper industries, and waste management. Pectinases, like other industrial enzymes, are usually expensive. This research aimed at producing in-house pectinase from waste fruits, by an inductive mechanism. The study specifically optimized the production of in-house pectinases from mango and pawpaw peels used as substrates and also demonstrated the hydrolytic potential of the substrate-based in-house pectinase enzymes through viscosity analysis. The mango and pawpaw peels obtained from Blue Skies Co. Ltd. were oven-dried and milled to sieve particle sizes depending on the mode of the fermentation process. Measured amounts of the substrates were put into 50 ml Falcon tubes and subjected to both solid-state and submerged fermentation processes. The substrates were inoculated with one milliliter of Aspergillus niger isolate suspension and incubated at 30 °C for 2, 4, 6, 8, and 10 days under both fermentation processes. The crude enzyme extracts (in-house pectinolytic enzymes) obtained from the mango and pawpaw peels of both fermentation processes were used as enzyme sources for the hydrolysis of 1% w/v pectin solution prepared in phosphate buffer, pH 6.8 at 40 °C in viscosity analysis. The viscosities of the various treatments were recorded at different time intervals of 10, 20, and 30 min. Based on the viscosity analysis, it was observed that pectinolytic enzymes from pawpaw peels showed higher pectinolytic potential relative to the mango counterpart for both fermentation processes. However, there were consistent optima at day 4 for the mango peels and day 6 for the pawpaw peels using the submerged fermentation process. Also, there was a linear correlation between the pectinolytic activity and incubation period. The results obtained from this study show a higher potential to produce pectinases from agro-wastes such as mango and pawpaw peels for industrial purposes using an appropriate fermentation process.

Screening and Optimization of Fermentation Medium for Bacillus velezensis BP-1 and Its Biocontrol Effects against Peyronellaea arachidicola

In the submerged fermentation process, the introduction of novel nutrient media as sources of carbon and nitrogen can enhance antifungal activity. In this study, we used a submerged fermentation process to find the optimal media for the Bacillus velezensis BP-1 strain to grow in, and that would boost its antifungal activity against Peyronellaea arachidicola. By using a single-factor test and central composite design (CCD) in the response surface methodology, the optimal fermentation medium for the B. velezensis BP-1 strain was identified. The antagonistic potential of B. velezensis BP-1 was assayed against the P. arachidicola fungus to manage web blotch disease in peanut plant leaves. The LB medium was screened as the best medium, with a maximum antifungal activity of 90% in comparison to the other mediums. Semolina flour as the carbon source, peanut root extract as the nitrogen source, and magnesium sulfate as the inorganic salt were selected as the best nutrient components in comparison to the others. The response surface methodology was optimized by using 15 g/L of semolina flour as the carbon source, 13.68 g/L of peanut root extract as the nitrogen source, and 0.50 g/L of magnesium sulfate as the inorganic salt, achieving 90% inhibition of P. arachidicola. The in vitro bioassays showed that the optimized fermentation broth of B. velezensis BP-1 had significant antifungal activity, with an inhibition rate of 88.34% against P. arachidicola. In the pot experiments on disease control, the management effects revealed that the pre-inoculation spray of the B. velezensis BP-1 broth had significant efficiency (96%) when compared to the post-inoculation spray of the B. velezensis BP-1 broth. These findings suggest that the optimized fermentation broth of the B. velezensis BP-1 strain had strong antifungal activity. This could be a potent biocontrol tool for aiding in the early disease management of web blotch in peanut plants.

Optimization and characterization of alkaliphilic lipase from a novel Bacillus cereus NC7401 strain isolated from diesel fuel polluted soil.

Five Bacillus cereus strains including B. cereus AVP12, B. cereus NC7401, B. cereus BDBCO1, B. cereus JF70 and B. specie JL47 isolated from the diesel fuel polluted soil adhered to the roots of Tagetes minuta were screened for lipase production with phenol red agar method. B. cereus NC7401 strain successfully expressing and secreting lipase with maximal lipolytic activity was subjected to a submerged fermentation process with five different carbon (starch, glucose, maltose, fructose, and lactose) and five different nitrogen (tryptone, ammonium nitrate, peptone, urea, yeast extract) sources to produce lipase enzyme. Maximum enzyme activity was found with starch (30.6 UmL-1), maltose (40 UmL-1), and tryptone (38.6 UmL-1), and the lipases produced using these sources were named lipase A, B, and C respectively. The total protein content of 8.56, 8.86, and 2.75 μg mL-1 were obtained from B. cereus NC7401 cultured using starch, maltose, and tryptone respectively. Lipase was stable between temperature range 30-80°C and pH 5-10 whereas optimally active at 55°C and pH 8.0. The enzyme was relatively stable for 10 days at 4°C and its optimum reaction time with the substrate was 30 minutes. It was tolerant to 1.5% (v/v) methanol as an organic solvent, 1.5% (v/v) Triton X-100 as a media additive and 1.5% (w/v) Ni2+ as a metal ion. SDS, n-hexane, and Ag+ inhibited lipolytic activity. Oil stains were removed from cotton fabric which showed oil removal efficiency enhancement in the presence of a lipase. Fat hydrolysis of 20, 24, and 30% was achieved following 6 hours of incubation of the fat particles with lipase A, B, and C respectively at a concentration of 20 mg mL-1. To as best of our knowledge, this study on lipases extracted from bacteria of Azad Kashmir, Pakistan origin has never been reported before.

Techno-economic analysis of Cellulase Production by Trichoderma reesei in Submerged Fermentation Processes using a Process Simulator

Enzymes are valuable in many biofuels production processes, and their wide application has generated significant market demand in recent years. To develop lignocellulosic fuels in a biorefinery, high cost and low catalytic efficiency of cellulase are a major obstacle in the industrial use of this enzyme. A techno-economic assessment of cellulase production by Trichoderma reesei in submerged fermentation (SmF) processes was carried out in this study. SuperPro Designer Software was used to develop a process model that estimated the economic analysis of the fermentation processes by creating alternative schemes (batch and fed-batch) to improve the process. Sensitivity analysis study was carried out using Monte Carlo Software. Economic profitability indicators were used to assess cellulase yield, productivity, aeration rate, and specific power input on the unit production cost (UPC) of cellulase enzyme. Results show that the batch process had a higher aeration requirement when compared to the fed-batch process. Fed-batch process route had a shorter payback time (1.88 years), higher internal rate of return IRR (36.95%), and a more positive Net present value ($140,328,000) than the batch process. In addition, the Fed-batch process route is more economically viable than the batch process route, since the certainty of achieving its base case value of UPC is at the optimum. This study has been able to design a process route that reduces the production cost of cellulase, while increasing its availability in the market for sustainable fuel production in a biorefinery.

Parametric optimisation for detoxification of non-oil Jatropha residual material using Taguchi method

ABSTRACT Phorbol esters are the major toxic compounds found in Jatropha seed cake and oil. Removal of phorbol esters from Jatropha seed cake would allow the utilisation of detoxified seed cake as a protein-rich supplement for animal feed. Research was performed to determine the effects of submerged fermentation (SMF) on the phorbol ester (PE) degradation rate. In Taguchi design of experiments, the L18 orthogonal array was used for optimising the SMF process for detoxification of Jatropha seed cake. Process parameters selected for the study were pH, percentage of seed cake, time, temperature and rpm. The response parameter for detoxification was measured in terms of phorbol ester degradation. Analysis of variance (ANOVA) was performed for predicting the optimum process parameters. The percentage contribution of the process parameters with reference to phorbol ester degradation has been predicted. The hierarchy of significant process parameters is in the order of seed cake (1%), temperature (35 °C), pH (7.5) and time (12 h).

Production and Partial Purification of Xylanase with Special Application in Pharmaceutical and Food Industries

Enzymes are used in a variety of ways in the biopharmaceutical industry. Enzymes are the focal point of metabolic and biochemical processes. Xylanase is one of them which are widely used in various purpose. Xylanase is a naturally occurring enzyme present in bacteria and fungi. Xylanases are members of the CAZymes (carbohydrate-active macromolecular biocatalyst) community and are classified as glycoside hydrolases, further divided into clans and families. Xylanases are produced by solid-state or submerged fermentation (SMF) techniques. SSF processes have many advantages over SMF processes, including lower fermentation cultivation costs, lower risk of contamination, improved enzyme stability. The microorganism Aspergillus spp. was used to produce xylanase, and this organism was isolated from soil. Solid-state fermentation is done with 80% moisture content for the production of this extracellular enzyme. After fermentation, purification is done in 70% saturated ammonium sulfate salt fractionation because the best result is found in this condition. After production and purification of xylanase, which is stored in desiccators cause, the enzyme is hygroscopic.

Palmyrah (<em>Borassus flabellifer</em> L.) Toddy as a Source of Vinegar Production and its Chemical Analysis

Purpose: Vinegar is a condiment prepared by alcoholic and consequent acetic acid fermentation using different sugary and starchy materials. This can be utilized as a flavoring agent, preservative, medicine, cleaning agent and herbicide. Palmyrah toddy contains around 4-6% of alcohol which is produced in huge amounts during their season. So, this research was designed to increase the utilization of toddy by means of vinegar. Research Method: Vinegar was prepared with toddy as a substrate (5.0% of alcohol) by using Orleans, generator and optimized submerged fermentation processes. The chemical characteristics of vinegar produced from Palmyrah were determined based on SLS 168:1999. Findings: Orleans process significantly (p<0.05) reduced the time required to produce the maximum acid (4.01%) contained corn cob and activated bacteria covering gel-like structure. During the generator process over oxidation of ethanol was observed due to the high rate of aeration with the pump. Optimized conditions of submerged fermentation was exhibited as 10% inoculum size (5, 10, 20 and 30%), 30o C temperature (28, 30 and 32o C)and with shaking showed 3.96, 4.23 and 4.53% of acid production at 8th, 7th, and 5th day of fermentation respectively. Chemical characteristics of produced palmyrah vinegar complied with SLS 168:1999 as total acidity (4.32%), total solids (4.08 (w/v), alkaline oxidation value (89.6), iodine value (556) and residual ethyl alcohol (0%). The efficiency of acetic acid production by using an optimized submerged fermentation process was 92.4%. Originality/ Value: Palmyrah toddy could be used for the production of natural vinegar under optimum conditions, and further field study has to be done to apply this finding at the industrial level.

Fermentation of the psyllid-pathogenic fungus Pandora sp. nov. inedit. (Entomophthorales: Entomophthoraceae)

ABSTRACT Pandora sp. nov. inedit. (ARSEF13372) is a recently isolated entomophthoralean fungus with high potential for psyllid pest control. The use of entomopathogenic fungi as biocontrol agents in large-scale field application requires biomass production in a sufficient quantity and quality. This work is the first attempt to explore the biomass production of the novel entomophthoralean Pandora species in a liquid medium. This study aims at establishing a submerged fermentation process with potential for mass production of the fungus. Three different complex nutrient sources were evaluated for maximising biomass in a liquid shaking culture. Pandora sp. nov. grew best in media containing skimmed milk, but even faster in a mixture of skimmed milk, yeast extract and a low-cost protein hydrolysate from animal by-products. Subsequently, the produced biomass can be encapsulated in biopolymer beads or granules in order to transfer the fungus into an easily applicable form. Therefore, growth as finely dispersed mycelium was promoted by increased media osmolality through the addition of sodium chloride. After the medium had been transferred to a stirred tank bioreactor with a working volume of 8 L, a maximum biomass dry weight of 21 g·L−1 was reached after 48 h. These promising results can pave the way for large-scale fermentation and formulation processes of this novel Pandora species for biological psyllid pest control.

Citric acid production by Aspergillus spp. through submerged fermentation using different production mediums containing agroindustrial residues

The production of secondary metabolites of high added value by microorganisms has been extensively studied, mainly in the formulation of medium containing agro-industrial residues. Citric acid is a tricarboxylic organic acid obtained through submerged fermentation processes. The Aspergillus genus is considered an excellent producer of bioactive substances of industrial interest. This study aims to determine the rate of citric acid production using four strains of the microorganism (UCP 1099, 1356, 1357, and 1463) in alternative culture mediums containing citrus pomace (pineapple, orange and lemon) from the food industries in the state of Pernambuco, and to compare the results with the production obtained with the standard production medium using computational statistical tools. Assays were carried out with four samples of Aspergillus isolated from Caatinga in three conventional mediums, for 144 h, 37°C, and 180 rpm. The pH variation, sugar consumption, and citric acid production were analyzed. The results showed that the medium-called two e and the sample UCP1357, presented a citric acid production of 7.22g.L-1. After selecting the medium and the best producer microorganisms, production tests were carried out with a medium formulated with citrus fruit residues. The results revealed that all the residues produced citric acid, with the lemon peel being the best of the residues tested (6.91 g.L-1). It appears that there is a possibility of using residues in the formulation of means of production, thus contributing to the reuse of the present nutrients and the reduction of the environmental impact.

Fatty acids of Pleurotus florida mushroom: Potential molecules for blood glucose control

In Vitro-In Vivo antidiabetic effect of Pleurotus florida mycelia was evaluated using enzymes, yeast cell and in streptozotocin induced type 2 diabetic (T2D) animal model. P. florida mycelia was prepared by submerged fermentation process. The fungal biomass was organically extracted using mixture of ethanol and water. Highest extractives yield of 14.3% w/w was obtained in hydroalcoholic solvent with dielectric constant-δ value of 78.2 (EX78). P. florida mycelia extract (EX 78) shows higher antioxidant power with 86.87% free radical scavenging activity and with 75.29 reducing power. In-vitro α-amylase inhibition, α-glucosidase inhibition and glucose uptake activity of EX 78 shows higher effectiveness. The P. florida mycelia extract (EX 78) is also found to be effective in controlling of blood glucose and insulin level in streptozotocin-nicotinamide induced T2D animals. The blood glucose was reduced to 216 ± 6.21 mg/dl from 357 ± 0.34 mg/dl and the insulin level was increased to 0.423 ± 0.006 ng per mL from 0.164 ± 0.03 ng/mL in P. florida treated animals. The higher fatty acids, C20-eicosane; C24-tetracosane and C30-squalene present in P. florida mycelium extract was found to be effective in inhibiting human intestinal α-glucosidase and pancreatic α-amylase. The P. florida mushroom is found to be effective in controlling high blood glucose elevated liver and cardiac enzymes. Consumption of this mushroom is suitable for controlling blood glucose level in person with hyperglycaemia.

Cellulase Enzyme Production from Filamentous Fungi Trichoderma reesei and Aspergillus awamori in Submerged Fermentation with Rice Straw.

Fungi are a diverse group of microorganisms that play many roles in human livelihoods. However, the isolation of potential fungal species is the key factor to their utilization in different sectors, including the enzyme industry. Hence, in this study, we used two different fungal repositories—soil and weed leaves—to isolate filamentous fungi and evaluate their potential to produce the cellulase enzyme. The fungal strains were isolated using dichloran rose bengal agar (DRBA) and potato dextrose agar (PDA). For cellulase enzyme production, a rice straw submerged fermentation process was used. The enzyme production was carried out at the different incubation times of 3, 5, and 7 days of culture in submerged conditions with rice straw. Fungal identification studies by morphological and molecular methods showed that the soil colonies matched with Trichoderma reesei, and the weed leaf colonies matched with Aspergillus awamori. These species were coded as T. reesei UMK04 and A. awamori UMK02, respectively. This is the first report of A. awamori UMK02 isolation in Malaysian agriculture. The results of cellulase production using the two fungi incorporated with rice straw submerged fermentation showed that T. reesei produced a higher amount of cellulase at Day 5 (27.04 U/mg of dry weight) as compared with A. awamori (15.19 U/mg of dry weight), and the concentration was significantly different (p < 0.05). Our results imply that T. reesei can be utilized for cellulase production using rice straw.

An optimization approach to the bioconversion of flour mill waste to α-amylase enzyme by Aspergillus oryzae

Alpha-amylase is one of the most employed enzymes in the food industry because of its capacity to degrade starch, improving the organoleptic and nutritional properties of food products. Thus, it is very important to develop novel industrial production processes for its production. A submerged fermentation process using Aspergillus oryzae was optimised to modulate the most important factors affecting alpha-amylase production. Flour mill waste, an abundant worldwide agro-industrial residue, was used as substrate due to its starch-rich composition. This residue was able to avoid catabolite repression during the fermentation, acting as a slow-release substrate. The process optimization led to a maximum yield of alpha-amylase production of 14076 ± 2346 U/L. The secretome of the fungi in the tested conditions was analysed by LC-MS, showing that two isoforms of alpha-amylase (amy-1 and amy-3) were produced. Finally, a two-step process was developed to purify alpha-amylase, consisting of fractional precipitation using (NH4)2SO4 followed by hydrophobic interaction chromatography. The purification allowed a four-time concentration of alpha-amylase, with an eleven-time purification factor and 65 % recovery and a reduction of the proteolytic activity to 14%.

Cellulase Producing Bacteria Isolation, Screening and Media Optimization from Local Soil Sample

Cellulase enzyme earns consecutively increasing demand in industries of Bangladesh. This experiment has been planned to develop the enzyme industry in our country rather than import enzymes from other countries and conclude with isolating and screening cellulose-degrading bacteria from local land samples and ensuring maximum enzyme production through media optimization. After collecting soil samples from various Tangail areas, a typical dilution of series was performed on a Carboxymethylcellulose plate (CMC) to produce cellulase-producing microorganisms. Based on the hydrolysis zone of the ratio, four isolates were chosen and labeled CBM21, CSG8, CSR35, and CHT8 for further morphological and biochemical analysis. Then subjected to cellulase production in 250 ml of Erlenmeyer flask using CMC broth for 48 hours of fermentation at 37°C at an agitation rate of 140rpm, and cellulase enzyme assay methods evaluated crude enzymes. Eventually, those confirmed isolates were optimized using different parameters by submerged fermentation process for enhanced cellulase production. Among four isolates, CSG8 and CBM21 exhibited the maximum clearance zone with the hydrolytic values 6.96 and 8.46. These isolates were identified as Pseudomonas spp. (CSG8). According to the screening process, CBM21 (0.156U/ml), CSG08 (0.106U/ml), and E. coli (negative control) have been used for optimization with their initial enzyme productivity. After optimization, an amazing enhancement was noticed for CBM21 (1.35 U/ml) and CSG8 (1.23 U/ml) compared to E.coli (0.44 U/ml). The enzyme was characterized in isolates with an effective temperature of 40 and 35°C for CBM21 and CSG8, respectively, and pH 7, glucose 5% with the incubation time 24h was optimal. Aftermath, this type of optimization can be a significant initiative in the enzyme industries of Bangladesh.

Lignin peroxidase: Optimization of media for higher productivity by single factor optimization method

Lignin peroxidase belongs to ligninolytic enzyme group and is one of the industrial important enzymes as it has wide applications in different sectors. Lignin peroxidase is produced by submerged fermentation process which requires optimization of physical and chemical parameters to achieve higher activity and make the process cost effective. The present study aimed at the optimization of physical as well chemical parameters of production medium. The optimization includes physical parameter such as incubation time, inoculum size, temperature, pH, RPM (Rotation per minute) while chemical parameters include carbon source, nitrogen source and different mineral elements. Form the optimization study, it was observed that highest lignin peroxidase production was achieved after 72 hours of incubation at temperature 300C, pH 6 and RPM 120. Optimization of chemical parameters reveals that incorporation of sodium nitrite (9g/L) in the media gave significant increase in enzyme activity. It was found that the maximum productivity achieved after optimization was 2214 U/ml which was four times higher than process without optimized parameters.

Lactic acid production: Utilization of yam peel hydrolysate as a substrate using Rhizopus orysae in kinetic studies

AbstractThe prospect of utilizing yam peel hydrolysate (YPH) as a carbon source for lactic acid (LA) production using Rhizopus orysae was investigated in batch surface and submerged fermentation processes. The kinetics of biomass growth, substrate utilization and LA production were studied using models of modified Luedeking–Piret, logistic and simple Luedeking–Piret equations, respectively, for the surface and submerged processes. The YPH containing 80 g L−1 of reducing sugars (glucose, xylose, ribose and others) was obtained by acid hydrolysis (H2SO4) of 100 g L−1 of yam peel and used for fermentation. The LA yield from the YPH was 80.03% and 75.63% for the surface and submerged fermentation, respectively. These values were higher than those of synthetic glucose syrup (SGS), which were 77.36% and 38.96% for surface and submerged fermentation, respectively. The models were satisfactory for the fermentation processes with high accuracy of R2 ((0.89 to 0.96), (0.95 to 0.99) and (0.86 to 0.99)) for biomass growth, substrate (SGS/YPH) utilization and LA production, respectively. The models also elucidated that the fermentation process was growth associated (α) instead of non‐growth associated (β) because the value of α/β was greater than 1. This justified the other kinetic parameters obtained for the growth phase. Therefore, Rhizopus oryzae effectively converted YPH to LA with a high yield and purity. © 2021 Society of Industrial Chemistry and John Wiley &amp; Sons Ltd

Microbial dynamics-linked properties and functional metabolites during Miang fermentation using the filamentous fungi growth-based process

The microbial community in ethnic fermented tea, “Miang” prepared from Camellia sinensis var. assamica in northern Thailand was investigated during the 10 days of a filamentous fungi growth-based process (FFP) using both culture-dependent and culture-independent techniques. During the aerobic phase (days 1–3), filamentous fungi were visually observed growing on steamed tea leaves during which yeast and lactic acid bacteria (LAB) viable cell numbers substantially increased up to 108 and 109 CFU/g Miang, respectively, and became the main microbial population both in semi-submerged fermentation (SSF) and submerged fermentation (SMF) processes. Next generation sequencing indicated the high diversity of the filamentous fungal population in the initial stage of aerobic fermentation. Aspergillus niger was the predominant fungus on day 3 and was the most important microbe in association with yeast, LAB and Bacillus sp. in both the SSF and SMF processes. Pectinases, β-mannanase and cellulases were mainly found during the aerobic fermentation phase, suggesting they were important during fermentation. The organic acids found in Miang were acetic, gallic, glucuronic, lactic, oxalic and succinic acids, while FFP Miang showed similar bioactive compounds including total tannin, total flavonoids and total polyphenols with both SSF and SMF processes. However, SMF resulted in higher antioxidant activity than SSF, while the hardness of both was similar. This study showed an important product processing platform for assessing the relationship between microbes, biotransformation and also functional metabolites accumulated in Miang fermented using the FFP.

Investigation on spectral and biomedical characterization of rhamnolipid from a marine associated bacterium Pseudomonas aeruginosa (DKB1).

Bio-surfactants are a principal group of significant molecules obtained from the microbial sources expressed with distinctive characteristics like biodegradation of hydrocarbons and also have different biomedical properties. The present investigation aims to assess the biomedical properties of synthesized bio-surfactant, rhamnolipid from Pseudomonas aeruginosa (DKB1) under in vitro conditions. The candidate bacterium P. aeruginosa (DKB1) was isolated from oil-polluted fishing harbors of Kanyakumari coast. Initially, the bio-surfactant production by this candidate strain was confirmed by oil displacement assay, hemolytic assay, drop collapse assay and emulsification index. Further, the production of bio-surfactant was achieved through submerged fermentation process using Bushnell-Haas mineral salts medium supplemented with 2% olive oil. The yield of the bio-surfactant was attained as 2.4g/l and confirmed as rhamnolipid through blue agar plate assay; further, the extracted rhamnolipid was purified and characterized through standard procedures. In stability studies, the rhamnolipid could withstand up to pH 12, temperature 100°C and 15% of NaCl concentration. The biomedical application of rhamnolipid (30μgml-1) was determined by antibacterial, antioxidant and cytotoxic studies. It exhibited a maximum growth inhibition against Bacillus subtilis (26mm) with the MIC value of 8μgml-1. In antioxidant test, rhamnolipid expressed significant (P < 0.0001) inhibition of total reducing power (44.11%), DPPH activity (61.60%), hydroxyl radical (83.30%) and nitric oxide (51.86%) scavenging ability at 100μgml-1with the respective IC50 values of 130.50, 77.18, 52.08 and 95.43μgml-1. The anticancer activity of the rhamnolipid was assessed with the help of MTT test against MCF-7, HT-29 and E-143 cancer cell lines individually, and the viability of the cells was observed, respectively, as 10.24, 17.66 and 13.50% at 250μgml-1concentration with the respective IC50 values of 140.2, 81.02 and 138.9μgml-1. From the results, it could be concluded that the rhamnolipid produced by P. aeruginosa (DKB1) isolated from oil-polluted area has effective biomedical properties.