Liquid-state Fermentation Research Articles

Combining Transcriptomics and Polyphenol Profiling to Provide Insights into Phenolics Transformation of the Fermented Chinese Jujube.

As an important medicine homologous food, Chinese jujube is rich in nutrition and medicinal value. To enhance the bioactive compounds level of Chinese jujube products, three kinds of fungi strains (Rhizopus oryzae, Aspergillus niger and Monascus purpureus) were firstly selected to evaluate their effects on total soluble phenolic compounds (TSPC) and total soluble flavonoids compounds (TSFC) contents during liquid state fermentation of Chinese jujube. As the best strain, the highest contents of TSPC and TSFC could increase by 102.1% (26.02 mg GAE/g DW) and 722.8% (18.76 mg RE/g DW) under M. purpureus fermentation when compared to the unfermented sample, respectively. Qualitative and quantitative analysis of individual polyphenol compounds indicated that proto-catechuic acid, p-hydroxybenzoic acid and chlorogenic acid showed the highest level in the fer-mented Chinese jujube at the 7th day, which was enhanced by 16.72-, 14.05- and 6.03-fold when compared to the control, respectively. Combining with RNA sequencing, function annotation of CAZymes database and polyphenol profiling, three potential transformation pathways of poly-phenol compounds were proposed in the fermented Chinese jujube by M. purpureus, such as the conversion of insoluble bound phenolic acids, rutin and anthocyanin degradation. These findings would be beneficial for better understanding of the biotransformation mechanism of polyphenol compounds in fungi fermentation.

Production of Single-Cell Protein from Fruit Peel Wastes Using Palmyrah Toddy Yeast

Single-cell protein (SCP) from agro-waste material has gained increased attention in the recent past as a relatively cheap and alternative protein source to meet the nutritional demand generated by the fast-growing population. Furthermore, bioconversion of these wastes into SCP such as value-added products reduce the environmental-related issues. In this study, locally available pineapple (Ananas comosus), watermelon (Citrullus lanatus), papaya (Carica papaya), sour orange (Citrus medica), banana (Musa acuminata) and mango (Mangifera indica) peel wastes were investigated for their suitability to produce SCP using palmyrah (Borassus flabellifer) toddy carrying natural mixed yeast and bacteria culture under liquid state fermentation system. Moreover, this study attempted to select the best substrate and the optimized process condition for SCP production to increase the protein yield. The physicochemical properties of selected fruit peels were analyzed. The sterilized peel extracts (10%, v/v) were inoculated with 5 mL of palmyrah toddy and allowed to ferment in a shaking incubator at 100 rpm for 48 h in triplicate At the end of fermentation, the sediments were collected by centrifugation at 1252× g, oven-dried, and the dry weight was taken to determine the protein content. The biomass yield ranged from 5.3 ± 0.6 to 11.7 ± 0.8 g/L, with the least biomass yield being observed with watermelon peels while the maximum yield was observed with papaya peels. Papaya peel generated a significantly higher (p < 0.05) amount of protein (52.4 ± 0.4%) followed by pineapple (49.7 ± 1.3%), watermelon (45.2 ± 0.7%), banana (30.4 ± 0.6%), sour orange (29.5 ± 1.2%) and mango (24.6 ± 0.2%) peels. The optimum condition for the fermentation of papaya waste was pH 5.0, 25 °C, and 24 h. Nucleic acid reduction treatment significantly reduces dry weight and protein content of biomass. It can be concluded that papaya peel waste is a suitable substrate for protein-rich cell biomass production using the natural toddy mixed culture of palmyrah.

Production of pectinases towards the extraction of natural pectin from orange peels using fungal sources

Pectin is a polysaccharide, which is present in middle lamella of cell wall of higher plants. Almost all fruits have a part of pectin in their cell walls. Pectinases are used in the degradation of the plant material and promptly extract juices from different fruits. For this purpose, identified fungi were screened qualitatively and quantitatively for the production of pectinase at 30 ºC for 92 hours of incubation time. In liquid state fermentation, identified strains of Trichoderma asperellum, Aspergillus niger and Thermomyces were treated in which Trichoderma asperellum produced maximum pectinase activities (9.2 U/ml) after 24 hours of incubation by using orange peels as a sole carbon source. Furthermore, pectinases produced from fungal strains were used to produce natural pectin from fruit waste like orange peels. During fermentation process, pectinase produced from fungal strains were inoculated to dried orange peels for 24 hours of incubation time at 30 ºC. The filtered extract was treated with 3 volumes of 80% ethanol for washing to extract pectin. Trichoderma asperellum gave maximum pectin yield [(11.7%)], Aspergillus niger and Thermomyces gave minimum pectin yield [(9.7%, 7.5%)] from orange peels respectively and the amount of pectin yield extracted from orange peels control was [(3.5%)]. Pectinases and pectin has found huge industrial applications in soft drinks, dairy products, pharmaceuticals and food industries, therefore, the present work has focused on the cost effective production of pectinase enzyme and extraction of natural pectin by using potential indigenous fungal strains.

Lactobacillus fermentum as a new inhibitor to control advanced glycation end-product formation during vinegar fermentation

The inhibitory activity of lactic acid bacteria (LAB) toward advanced glycation end-products (AGEs) during vinegar fermentation was studied, and its relationships with the substrate consumption, antioxidant capacity, total phenolic content, total flavonoid compounds, α-glucosidase, and α-amylase activity inhibition were evaluated. The vinegar was made from rice powder flour by liquid-state fermentation (LSF). The selected LAB strains were separately co-cultivated with Saccharomyces cerevisiae and Acetobacter pasteurianus 1.41 in alcoholic and acetic acid fermentation, respectively. Among 3 strains, Lactobacillus fermentum showed the strongest inhibitory effect on the formation of total fluorescent AGEs and carboxymethyl lysine (CML)/carboxyethyl lysine (CEL) in the fermentation process. The corresponding mechanisms included the acceleration of substrate consumption, improvement of antioxidant activities, and inhibition of α-glucosidase and α-amylase. In addition, the fluorescent AGEs and the CML/CEL were negatively correlated with the antioxidant activities, while the α-glucosidase and α-amylase activities were positively correlated with the total phenols and total flavonoids. Moreover, the variety of main flavor compounds increased, including esters, alcohols, phenols and acids. The results of the study support the potential use of screened LAB strains to inhibit the formation of fluorescent AGEs, CML and CEL on fermented products and in the food processing industry, without associated risks to consumers.

Preparation of instant dark tea by liquid-state fermentation using sequential inoculation with Eurotium cristatum and Aspergillus niger: Processes optimization, physiochemical characteristics and antioxidant activity

Preparation of instant dark tea (IDT) by liquid-state fermentation (LF) is an effective procedure, of which the process is more easily controlled than solid-state fermentation (SSF). The present study focused on developing a sequential inoculation liquid-state fermentation (SILF) procedure using the Eurotium cristatum and Aspergillus niger to prepare IDT. The key parameters (turbidity, redness, and tea pigments) were optimized by response surface methodology (RSM). Optimum conditions were modeled to be a water-tea ratio of 24.0 mL/g, inoculum size of 8.7% (v/v), and time of 9 d. The predicted and experimental values of redness, turbidity and tea pigments content were within a 5% difference using the optimized conditions. Bench scale was achieved in a 5-L fermenter, which generated IDT with a redness value of 43.50, a turbidity of 5.67 NTU and tea pigments content of 23.53%, w/w. The evaluation by E-nose and E-tongue showed that IDT had a similar aroma and mouthfeel with a commercial instant Fu-brick tea (IFT), and the antioxidant activities in vitro, color and clarity of tea infusion of IDT were higher than those of the IFT, respectively. These results indicate that preparation of IDT by SILF is feasible, this work expect to set up a new aspect for preparation of IDT.

Screening of Bacillus subtilis and Optimization of Liquid Fermentation Conditions for Nattokinase Production

Screening of Bacillus subtilis and Optimization of Liquid Fermentation Conditions for Nattokinase Production

Comparison of Liquid and Solid-State Fermentation Processes for the Production of Enzymes and Beta-Glucan from Hulled Barley.

Solid-state fermentation using hulled barley was carried out to produce enzymes and β-glucan. The one-factor-at-a-time experiments were carried out to determine the optimal composition of the basal medium. The modified synthetic medium composition in liquid-state fermentation was determined to be 70 g/l hulled barley, 0 g/l rice bran, 5 g/l soytone, and 6 g/l ascorbic acid. Optimal pretreatment conditions of hulled barley by solid-state fermentation were evaluated in terms of maximum production of fungal biomass, amylase, protease, and β-glucan, which were 1.26 mg/g, 31310.34 U/g, 2614.95 U/g, and 14.6% (w/w), respectively, at 60 min of pretreatment condition. Thus, the solid-state fermentation process was found to enhance the overall fermentation yields of hulled barley to produce high amounts of enzymes and β-glucan.

Anaerobic digestion of rice straw pretreatment liquor without detoxification for continuous biogas production using a 100 L internal circulation reactor

The production of biogas from lignocellulose by anaerobic digestion (AD) has attracted much attention recently. However, the over-acidification phenomenon and low biogas yield often occur during the AD of lignocellulose. In this work, the liquid-state fermentation mode using pretreatment liquor from dilute acid treated rice straw without detoxification for continuous biogas production in a 100 L internal circulation reactor was studied. The highest biogas yield reached 0.66 m3/kg CODconsumption while the methane yield was 0.38 m3/kg CODconsumption. The pH values of outlet were maintained at 6.9–7.4, and even the inlet pH values were lower than 6.0. Furthermore, from the second day to the end of fermentation, the COD removal rates were more than 90%, even though the inlet COD was close to 20000 mg/L. The organic substances from acid pretreated rice straw were efficiently degraded by AD, which was confirmed by substrate metabolism analysis. These results indicated that the whole fermentation process of AD was very stable and showed great potential for industrial application.

Induced effect of Ca2+ and Al3+ on chaetominine synthesis by Aspergillus fumigatus CY018 under submerged fermentation.

Chaetominine (CHA), an alkaloid with a biological activity obtained from Aspergillus fumigatus CY018, has strong anticancer activity against the human leukemia cells. However, its physiological and biochemical research is limited by CHA yield in the liquid-state fermentation, which is a problem that urgently needs effective biological solution. In this work, Ca2+ and Al3+ were found to have a strong promoting effect on CHA production after multiple metal ions screening. Then, the addition condition of Ca2+ and Al3+ was, respectively, optimized CHA production and dry cell weight. The intermediate metabolites were increased with coaddition of Ca2+ and Al3+ . The activities of key enzymes of DAHPs, AroAs, and TrpCs in the CHA biosynthesis pathway were improved by 3.58-, 3.60-, and 3.34-fold, respectively. Meanwhile, the transcription level of laeA, dahp, cs, and trpC was upregulated by 3.22-, 12.65-, 5.58-, and 6.99-fold, respectively, by coaddition of Ca2+ and Al3+ . Additionally, the fermentation strategy was successfully scaled up to a 5-L bioreactor, in which CHA production could attain 75.6 mg/L at 336 h. This work demonstrated that Ca2+ and Al3+ coaddition was an effective strategy for increasing CHA production, and the information obtained might be useful in the fermentation of filamentous fungi with the addition of metal ions.

Influence of Sugars, Sugar Alcohols and Their Combinations on Environmentally Significant Cellulase Production Under Liquid State Fermentation

To expand the range of soluble carbon sources for enzyme production by the microbial system, we investigated the ability of different sugars in cellulase production. Carbon sources play a vital role in cell metabolism and the synthesis of cellulase. Although the insoluble cellulosic materials are considered the most effective natural inducers for cellulase production by microorganisms in terms of both enzyme yield and productivity, their insolubility causes many problems and presents a major drawback that is partly responsible for the high cost of cellulase production. Mostly, the insolubility of sugar polymer leads to difficult and complex fermentation operations, including sterilization, cell biomass measurement, mixing and aeration of the fermentation broth, continuous feeding/sampling, and subsequent enzyme purification. Second, cellulase gets absorbed into the solid cellulose surface, leading to enzyme loss. The present paper gives a comparative view on the utility of pure sugars (lactose, CMC, sucrose, maltose, cellobiose, xylose, trehalose, arabinose) and sugar alcohols (sorbitol, mannitol) on cellulase production by various fungal strains. It also describes the blending effect of sugars as well as sugar alcohols on cellulase production by fungal strains.

Development of Beauveria bassiana (Ascomycota: Hypocreales) as a mycoinsecticide to control green peach aphid, Myzus persicae (Homoptera: Aphididae) and investigation of its biocontrol potential

• Biological control of Myzus persicae using EPF B. bassiana was studied. • Oil-based formulation was achieved by liquid-state fermentation. • AFIDISIDAL-OD Bbas -TR61 was determined to be most efficacious against Myzus persicae . Long-term and intensive use of synthetic insecticides to control the green peach aphid, Myzus persicae (Homoptera: Aphididae) in agricultural production in the world has resulted in pest resistance and environmental pollution. The aim of the study was to develop an environmentally-friendly and effective mycoinsecticide from a local fungal isolate against M. persicae . According to the results of the screening experiments using 15 isolates (6 × Metarhizium , 5 × Beauveria , 2 × Isaria , 2 × Lecanicillium ) at 1 × 10 7 conidia ml −1 concentrations and the dose–response experiments at 1 × 10 5 –1 × 10 9 conidia ml −1 concentrations against M. persicae nymphs, Beauveria bassiana (KTU-24) was identified as the most promising isolate. The strain KTU-24 is also had tolerance to ecological conditions such as temperature, and UV-B. KTU-24 with advantageous and superior properties was used to develop a test mycoinsecticide. Mass production of spores by KTU-24 was conducted out by liquid-state fermentation using liquid medium. Spores harvested from the sporulated biomass were used to develop an oil-based mycoinsecticide, and the product was designated as AFIDISIDAL-OD Bbas -TR61. The product had lethal effect on M. persicae nymphs at a concentration of 1 × 10 8 conidia ml −1 in leaf-disc (82.5%) and pot (84.33%) experiments in a climate chamber. The oil-based mycoinsecticide developed in this study could be profitable when used in aphid-IPM prgrams by reducing crop loss and synthetic pesticides use.

Enhancement of triterpene production via in situ extractive fermentation of Sanghuangporus vaninii YC-1.

There have been many studies on the activities and polysaccharide production of Sanghuangporus vaninii. However, few studies have looked at triterpene production from S. vaninii using liquid-state fermentation. A method for enhancing the production of triterpenes by in situ extractive fermentation (ISEF) was studied. Eight solvents were investigated as extractants for triterpene production in the ISEF system. The results showed that using vegetable oil as an extractant significantly increased the yield of total triterpenes and biomass of S. vaninii YC-1, reaching 18.98±0.71 and 44.67±2.21g/L, respectively. In 5L fermenter experiments, the added vegetable oil improved the dissolved oxygen condition of the fermentation broth and promoted the growth of S. vaninii YC-1. Furthermore, adding vegetable oil increased the expression of fatty acid synthesis-related genes such as FAD2 and SCD, thereby increasing the synthesis of unsaturated fatty acids in the cell membrane of S. vaninii YC-1. Therefore, the cell membrane permeability of S. vaninii YC-1 increased by 19%. Our results indicated that vegetable oil increased the permeability of S. vaninii YC-1 cell membranes to promote the production of total triterpenes. The use of vegetable oil as an extractant was thus effective in increasing the yield of triterpenes in the ISEF system.

Screening of Natural Dyes from Selected Fungal Species

Some fungi are observed as effective pigments. Its importance in the production of natural pigments has grown significantly. The pigment-producing fungi were developed and evaluated for application in dyeing cotton fabric. In the research, five fungal strains were identified as Aspergillus terreus S10, Talaromyces atroroseus WW5A3, Penicillium oxalicum WW3A4 (DG), WW5C2 and WW31DG. These strains were incubated for 21 days under static and non-static conditions using MSM and PDB media. Under liquid state fermentation conditions, the production of the pigments by the fungus was improved by altering temperatures (25-35°C) and pH (4.5-6.5). T. atroroseus WW5A3 showed pinkish color, A. terrreus S10 displayed yellow color, P. oxalicum WW3A4 (DG) presented yellowgreenish, WW5C2 exhibited light yellow color and WW31DG demonstrated greenish color. The results showed the maximum percentage absorbance of T. atroroteus WW5A3 showed 90.36 % at 600 nm, A. terrreus S10 showed 88 % at 500 nm, P. oxalicum WW35A4 showed 46.04 % at 550 nm, WW5C2 showed 59.60 % at 550 nm, and WW31DG showed 81.9 % at 550 nm. The natural fungal pigments were tested against bacterial pathogens to check the antibacterial activity. The results indicated that S. aureus and E. coli exhibited antibacterial activity in terms of maximum zone of inhibition. In conclusion, out of five pigments producing fungi, Aspergillus terreus S10 and Talaromyces atroroseus produced maximum pigment and highest percentage absorbance under liquid state fermentation conditions. Potential applications in the textile and leather industries have been discovered as a result of this research.

Transcriptome analysis revealing molecular mechanisms of enhanced pigment yield by succinic acid and fluconazole

This study aimed to elucidate the molecular mechanisms through which succinic acid and fluconazole stimulate Monascus pigment biosynthesis under liquid fermentation conditions. The pigment yield was significantly improved by adding 0.35 g·L−1 succinic acid or 1.5 g·L−1 fluconazole. Transcriptome sequencing and RT-qPCR confirmation were performed to reveal transcriptome changes. The results indicated that the addition of succinic acid significantly decreased mRNA expression of genes involved in fatty acid biosynthesis while increasing expression of genes involved in pyruvate metabolism. Fluconazole significantly down-regulated transcripts involved in branched-chain amino acid metabolism, fatty acid metabolism, glycolysis/gluconeogenesis, and pyruvate metabolism, as well as the generation of acetyl-CoA for pigment biosynthesis. On the other hand, nitrogen metabolism and lysine degradation pathways were significantly enriched, which could stimulate the generation of acetyl-CoA. Therefore, the mechanism for enhancing pigment yield may be attributed to the competitive regulation of metabolic pathways toward acetyl-CoA biosynthesis. Additionally, up-regulation of some different key genes in the presence of fluconazole or succinic acid was involved in improving pigment production. This study deepens the theoretical understanding for enhancing pigment biosynthesis and provides a few potential approaches for improving pigment yield.

Production and Optimization of Xylanase by Penicillium chrysogenum through Liquid State and Solid State Fermentation

Production and Optimization of Xylanase by Penicillium chrysogenum through Liquid State and Solid State Fermentation

Efficacy of Aspergillus sp in the Production of Protease Enzyme with Different Substrates

In the present study, the soil samples were collected from marine environment of Arichalmunai, Dhanushkodi, Ramnad District, Tamilnadu ,India. Fungal species were isolated by plating method, in 50% sea water containing potato dextrose agar medium .Totally 16 fungal species were isolated and identified from the soil sample. The production of protease from Aspergillus niger , A.flavus and A. terreus by liquid state fermentation. The production of protease enzyme was optimized by using fermentation medium containing different substrates. The maximum protease production was observed on wheat bran, containing medium. The protease production was maximum in temperature 35ºC were recorded. Wheat bran produced the maximum level protease. The optimization work also carried out. This study revealed that coastal environment provides impressive density of fungi in the East Coast of India and are unexplored for microbial resources can be useful in industry.

Solid-State Fermentation With Aspergillus cristatus Enhances the Protopanaxadiol- and Protopanaxatriol-Associated Skin Anti-aging Activity of Panax notoginseng.

A metabolomics approach was used to profile metabolites of Panax notoginseng fermented with Aspergillus cristatus in two ways, liquid-state fermentation (LF-P) and solid-state fermentation (SSF-P) and examine metabolite markers representing antioxidant activity and skin anti-aging. Protopanaxadiol (PPD) and protopanaxatriol (PPT) contents were higher in SSF-P than in LF-P and showed a multiplicative increase over the fermentation period of four days. PPD and PPT levels also correlated with antioxidant and anti-aging effects in skin, based on the mRNA expression of dermal extracellular matrix components. In the bioactivity validation assays, PPD and PPT significantly improved the expression of type-I collagen, fibrillin-1, and elastin in human dermal fibroblasts from both young and old subjects; these were comparable with the effects of the SSF-P extracts. Overall, our results suggest that changes in the metabolites of P. notoginseng fermented with A. cristatus enhance the quality and availability of bioactive compounds associated with skin anti-aging.

Metabolomic profiles of the liquid state fermentation in co-culture of A. oryzae and Z. rouxii

Aspergillus oryzae and Zygosaccharomyces rouxii are the perquisites microorganisms in food fermentation due to the broad application prospects of their secondary metabolites. The co-culture strategy simulates the naturally occurring ecology by developing artificial microbial communities. This strategy has also been widely adopted to investigate the novel secondary metabolites. In the present study, the effects of co-culture on extracellular and intracellular secondary metabolites of fungi in liquid culture were investigated through UPLC-QTOF-MS. Notably, A. oryzae could significantly inhibit the growth of Z. rouxii when A. oryzae and Z. rouxii were co-cultured. The results further indicated that the co-culture of fungi could affect the secondary metabolites and produce various metabolic pathways. Overall, this study will provide insights into fungal interactions and cell metabolism mechanisms, contributing to supplementing and enriching the fermentation potential of fungi.

Genetic evidence for the requirements of antroquinonol biosynthesis by Antrodia camphorata during liquid-state fermentation.

The solid-state fermentation of Antrodia camphorata could produce a variety of ubiquinone compounds, such as antroquinonol (AQ). However, AQ is hardly synthesized during liquid-state fermentation (LSF). To investigates the mechanism of AQ synthesis, three precursors (ubiquinone 0 UQ0, farnesol and farnesyl diphosphate FPP) were added in LSF. The results showed that UQ0 successfully induced AQ production; however, farnesol and FPP could not induce AQ synthesis. The precursor that restricts the synthesis of AQ is the quinone ring, not the isoprene side chain. Then, the Agrobacterium-mediated transformation system of A. camphorata was established and the genes for quinone ring modification (coq2-6) and isoprene synthesis (HMGR, fps) were overexpressed. The results showed that overexpression of genes for isoprene side chain synthesis could not increase the yield of AQ, but overexpression of coq2 and coq5 could significantly increase AQ production. This is consistent with the results of the experiment of precursors. It indicated that the A. camphorata lack the ability to modify the quinone ring of AQ during LSF. Of the modification steps, prenylation of UQ0 is the key step of AQ biosynthesis. The result will help us to understand the genetic evidence for the requirements of AQ biosynthesis in A. camphorata.

Antibacterial Activities and Toxicity of Streptosporangium sp. SM1P

This study aims to explore the anti-bacterial and toxicity activities from a rare actinobacterium isolated from mangrove, Mempawah District, West Kalimantan. The mangrove mud sample from Mempawah district was inoculated on ISP4 agar using a pour plate method. After 4 days of incubation, a colony of suspected actinobacterium was appeared, then isolated and coded as SM1P. SM1P was characterized based on morphological and biochemical traits and identified as a genus of Streptroporangium then called Streptroporangium sp. SM1P. Streptroporangium sp. SM1P was carried out anti-bacterial assay on both ISP1 agar and ISP4 agar media using the cross-streak method for the solid-state fermentation. The result showed that Streptroporangium sp. SM1P could inhibit Streptococcus sp. and Salmonella typhi on ISP1 agar and treptococcus sp., Escherichia coli, Vibrio cholerae, Staphylococcus aureus and Salmonella typhi on ISP4 agar. Streptroporangium sp. SM1P was cultivated on ISP1 broth and extracted using ethyl acetate, then evaporated to obtain crude extract. The crude extract was used for anti-bacterial assay (well-diffusion method for liquid-state fermentation) and toxicity assay (brine shrimp lethality test). The crude extract was active against 2 of the test bacteria (Streptococcus sp. and E. coli). The best medium and state fermentation for anti-bacterial assay were ISP4 agar with the condition of solid-state fermentation. The extract SM1P prepared on ISP1 broth showed toxic activity based on LC50 (106.094 µg/mL). Therefore, Streptroporangium sp. SM1P have a potential source to explore secondary metabolites having anti-bacterial and toxicity activities.