Pullulan Production Research Articles

Biofabrication of Functional Pullulan by Aureobasidium pullulans under the Effect of Varying Mineral Salts and Sugar Stress Conditions.

Pullulan is a linear exopolysaccharide, produced in the fermentation media of Aureobasidium pullulans, with a variety of applications in the food and pharmaceutical industries. Pullulan derivatives have growing potential for biomedical applications, but the high cost of pullulan biofabrication currently restricts its commercial use. Better control over pullulan yield, molecular weight and melanin production by altering fermentation conditions could improve the economics. In this study, the effects of sugar and mineral salt stresses on the pullulan production of A. pullulans ATCC 42023 were examined in batch processes. The chemical structure of the recovered pullulan was characterized by FTIR and NMR spectroscopy, and the molecular weight distribution was obtained via SEC. Pullulan yield and melanin production varied when the conditions were adjusted, and pullulans with different molar masses were obtained. Higher-yield pullulan production and a lower polydispersity index were observed when CuSO4 was added to the fermentation in comparison with the control and with the addition of sugars and other salts. Biofabrication of pullulan under stress conditions is a promising strategy to enhance biopolymer yield and to obtain pullulan with a targeted molecular weight.

Effect of add different carbon and nitrogen source on the pullulan polysaccharide production from fungus Aureobasidium pullulans

Ten local isolates of Aureobasidium pullulans were isolated from different plant leaves. The ability of these isolates for pullulans production using production medium was investigated. The highest production of biomass and pullulans were achieved from isolates (A5, A2)with (11.93, 23.5)g/l respactivily.
 The effect of eight carbon and eight nitrogen sources at final concentration (5%) on biomass and pullulan production from local isolates were tested. The results showed that glucose gave maximum production of polysaccharide from isolate (A7) with (15.5g/l).While the highest production of biomass were obtained when sucrose were used as a carbon source with (14.53)g/l from isolate (A5). Lactose and xylose failed to support polysaccharide and biomass production. Also Ammonium nitrate gave highest production of polysaccharide for all isolates tested were (22.9 g/l) from (A9). While the lowest production of polysaccharide and biomass were obtained when urea was used as a nitrogen source(1.15 ,0.28 g/l)from isolates (A6, A9) respectively .The highest production of biomass was obtained in case of using ammonium sulphat as a nitrogen source with (18.97 g/l) from isolate (A9).

High-level production of pullulan and its biosynthesis regulation in Aureobasidium pullulans BL06.

Pullulan has many potential applications in the food, pharmaceutical, cosmetic and environmental industries. However, the yield and molecular properties of pullulan produced by various strains still need to be promoted to fit the application needs. A novel yeast-like strain Aureobasidium pullulans BL06 producing high molecular weight (Mw) pullulan (3.3 × 106Da) was isolated and identified in this study. The remarkable Mw of pullulan produced by A. pullulans BL06 was the highest level ever reported thus far. To further regulate the biosynthesis of pullulan in A. pullulans BL06, three gene knockout strains A. pullulans BL06 ΔPMAs, A. pullulans BL06 Δmel, and A. pullulans BL06 ΔPMAsΔmel, were constructed. The results showed that A. pullulans BL06 ΔPMAs could produce 140.2g/L of moderate Mw (1.3 × 105Da) pullulan after 120h of fermentation. The highest yield level of pullulan to date could vastly reduce its production cost and expand its application scope and potential. The application experiments in food preservation showed that the moderate-Mw pullulan obtained in this work could reduce the weight loss of celery cabbages and mangos by 12.5% and 22%, respectively. Thus, the novel strains A. pullulans BL06 and A. pullulans BL06 ΔPMAs possessed unlimited development prospects in pullulan production at various Mw ranges and pullulan applications in multiple fields.

Whole-crop biorefinery of corn biomass for pullulan production by Aureobasidium pullulans

In the present study, corn starch, cob, and straw were biorefined and used as feedstocks for the production of pullulan. The titer and molecular weight (Mw) of pullulan significantly decreased when corn cob and straw hydrolysates were utilized by the parental strain Aureobasidium pullulans CCTCC M 2012259 (PS). Based on adaptive laboratory evolution of PS, an evolved strain A. pullulans EV6 with strong adaptability to the whole corn biomass hydrolysate and high capability of pullulan biosynthesis was screened. Batch pullulan fermentation results indicated that EV6 produced an increased titer of pullulan with a higher Mw than PS. The underlying reasons for these increases were revealed by assaying key enzymes activities and measuring intracellular uridine diphosphate glucose levels. Subsequently, whole-crop biorefinery of corn biomass was conducted, and the results confirmed that whole corn crop has immense potential for efficient pullulan production.

Pullulan production by Aureobasidium pullulans MTCC 1991 from apple pomace and its characterization

Apple pomace, a by-product of the apple juice processing industry, is significant agro-based waste in the Union Territory of Jammu & Kashmir, India. A considerable amount of apple is processed for juice production, resulting in a surplus of apple pomace, which is generally underutilized. This study utilized apple pomace as a substrate matrix for producing pullulan by applying Aureobasidium pullulans MTCC 1991. Solid-state fermentation was done at a lab scale in Erlenmeyer flasks to produce this exopolysaccharide. The suitable conditions for solid-state fermentation of apple pomace were: a solid-liquid ratio of 1:4, inoculum volume of 3 mL, pH 6 and incubation time of 14 days. A yield of 42 mg/g dw was obtained in the control sample without adding any other nutrients or chemicals. The addition of yeast extract at a concentration of 1% (w/w) and sucrose (5% w/w) significantly (p < 0.05) increased the yield to 62 mg/g dw and 75 mg/g dw, respectively. The purified pullulan had similar characteristics to that of standard pullulan, as confirmed by ATR-FTIR, H NMR and TLC techniques. Pullulan production from apple pomace can be a better way to utilize this by-product from the apple juice industry. It will be a cost-effective technique for pullulan production as an inexpensive substrate and an environmentally friendly approach are also used.

High-level production of pullulan from high concentration of glucose by mutagenesis and adaptive laboratory evolution of Aureobasidium pullulans

The cost of carbon sources and the low efficiency of the fermentation titer limit the industrial application of pullulan. In this study, a hypertonic-tolerant strain with efficient utilization of glucose was obtained using a double strategy. Initially, a strain for efficient synthesis of pullulan from glucose was generated by mutagenesis. Subsequently, the mutant was directionally evolved on the plate containing a high glucose concentration to enhance high osmotic resistance. The enzyme activities and the transcriptional levels involved in pullulan biosynthesis and high osmotic tolerance in mutants were increased. Nitrogen source and inorganic salts also significantly affected the production of pullulan by M233-20 from high concentration of glucose. The pullulan titer of 162.1 g/L was obtained using the response surface methodology in the flask. The strain M233-20 produced 162.3 g/L pullulan in a 30-L bioreactor with a yield of 0.82 g/g glucose. Hence, this work provides a potential industrial pullulan producer M233-20 and a strategy to develop excellent strain.

MAL31, a sugar transporter involved in pullulan biosynthesis in Aureobasidium pullulans

To investigate the role of the sugar transporter MAL31 on pullulan biosynthesis, the coding gene mal31 was respectively disrupted and overexpressed in the parental strain A. pullulans CCTCC M 2012259 to construct mutants of A. pullulans Δmal31 and A. pullulans Mal31. Batch pullulan production significantly decreased by 69.1 % in A. pullulans Δmal31 but increased by 15.9 % in A. pullulans Mal31, as compared to the parental strain. We performed kinetics analysis, assays of key enzymes, determination of intracellular UDPG, NADH, and ATP contents, and measurement of transcriptional levels of genes associated with pullulan biosynthesis and excretion. The results confirmed that the mal31 disruption decreased the glucose consumption rate, decreased the formation rate and titer of pullulan, but increased the intracellular UDPG supply for β-glucan accumulation. In contrast, the mal31 overexpression increased the transcriptional levels of genes associated with pullulan biosynthesis, and accelerated the rates of glucose consumption and pullulan formation, thereby increased pullulan production. Our findings revealed that MAL31 is involved in the transport of precursors for pullulan biosynthesis. This study provides an accurate operating site for genetic modification of A. pullulans for improving pullulan production and also presents a feasible technique route for the overproduction of other polysaccharides.

Production of the Polysaccharide Pullulan by Aureobasidium pullulans Cell Immobilization

This review examines the immobilization of A. pullulans cells for production of the fungal polysaccharide pullulan. Pullulan is a water-soluble gum that exists structurally as a glucan consisting primarily of maltotriose units, which has a variety of food, non-food and biomedical applications. Cells can be immobilized by carrier-binding or entrapment techniques. The number of studies utilizing carrier-binding as a method to immobilize A. pullulans cells appears to outnumber the investigations using cell entrapment. A variety of solid supports, including polyurethane foam, sponge, diatomaceous earth, ion-exchanger, zeolite and plastic composite, have been employed to immobilize pullulan-producing A. pullulans cells. The most effective solid support that was used to adsorb the fungal cells was polyurethane foam which produced polysaccharide after 18 cycles of use. To entrap pullulan-producing fungal cells, agents such as polyurethane foam, polyvinyl alcohol, calcium alginate, agar, agarose, carrageenan and chitosan were investigated. Polysaccharide production by cells entrapped in polyurethane foam, polyvinyl alcohol or calcium alginate was highest and the immobilized cells could be reutilized for several cycles. It was shown that the pullulan content of the polysaccharide synthesized by cells entrapped in calcium alginate beads was low, which limits the method’s usefulness for pullulan production. Further, many of the entrapped fungal cells synthesized polysaccharide with a low pullulan content. It was concluded that carrier-binding techniques may be more effective than entrapment techniques for A. pullulans cell immobilization, since carrier-binding is less likely to affect the pullulan content of the polysaccharide being synthesized.

A critical review on valorization of food processing wastes and by-products for pullulan production.

Pullulan is a commercially available exopolymer biosynthesized by Aureobasidium pullulans supplemented with nitrogen, carbon and other vital components through submerged and solid-state fermentation. These nutrients are very expensive and it raises the cost for the production of pullulan. Hence, the need of alternative cost-effective raw materials for its production is a prerequisite. Owing to its unique physicochemical features, pullulan has various applications in the food, pharmacological, and biomedical domains. Food industrial wastes generate a considerable number of by-products which accumulates and has a negative influence on the environment. These by-products are made up of proteins, carbohydrates, and other components, can be employed as substrates for the production of pullulan. The present review briefs on the pullulan production using food processing waste and by-products and the elements that impact it. It provides an insight into versatile applications of pullulan in food industries. Various challenges and future prospects in the field of research on pullulan production have been uncovered.

Optimization of hazelnut husk medium for pullulan production by a domestic A. pullulans strain

Hazelnut husk is one of the most abundant agricultural residue in Turkey. Valorization of this lignocellulosic biomass would provide a promoting alternative for economical production of pullulan. In this study, dried hazelnut husk hydrolysate was used directly as fermentation medium for pullulan production by a domestic strain of Aureobasidium pullulans. The aim of this work was the optimization of some fermentation medium parameters by central composite design using response surface methodology (RSM). The effects of (NH4)2SO4 concentration, the volume of concentrated H2SO4 and the amount of ground hazelnut husk on pullulan production were optimized by RSM. The optimum levels of the fermentation parameters defined as 7.2 gL−1, 2.5 mL and 20 g, respectively. The maximum pullulan and exopolysaccharide concentrations were determined as 74.39 and 75.95 gL−1 , respectively in the optimum conditions. Specific growth rate of the strain was found as 0.097 h−1. FTIR spectral attributes confirmed the structure of pullulan. Thermal decomposition temperature of synthesized pullulan was found as 247.15 °C. This study showed that hazelnut husk was one of the novel substrate for production of the pullulan by A. pullulans AZ-6. No previous work was found to utilize dried hazelnut husk as fermentation medium for pullulan production by A. pullulans.

Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins

Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, “yeast-like” ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5′-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.

Physicochemical properties of pullulan/chitosan/graphene oxide composite films

AbstractWe report the production of pullulan (Pul)/chitosan (Chi)/graphene oxide (GO) composite films (CF‐Pul/Chi + GO). We observed that the addition of GO increases the film's roughness while keeping good GO dispersion. Both the strong interactions between Pul and Chi and the formation of hydrogen bonds between Chi and GO seem to be responsible for the formation of the composite film. The dielectric constant, Young's modulus and the tensile strength of the CF‐Pul/Chi + GO films exhibit maximum values at 1 μg mL−1 GO concentration. At GO concentrations higher than 1 μg mL−1, the interfacial layers between the polymer matrix and GO overlap, affecting the value of the dielectric constant and the mechanical properties of the films. Moreover, the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay carried out on LL47 fibroblast cells reveals that the CF‐Pul/Chi + 1 μg mL−1 GO films are not cytotoxic. In this regard, CF‐Pul/Chi 50/50 + 1 μg mL−1 GO has remarkable properties to be considered for a safe film with potential uses in the food, cosmetic and pharmaceutical industries. © 2022 Society of Industrial Chemistry.

Evaluation of Some Agro-Industrial Wastes as Fermentation Medium for Pullulan Production by Aureobasidium pullulans AZ-6.

Agro-industrial wastes are rich sources of some nutrients. Thus, utilization of wastes seems to be ecologically sound and economically advantageous. The aim of this work was to investigate the potential usage of various agro-industrial wastes as fermentation medium for pullulan production by a domestic strain; Aureobasidium pullulans AZ-6. In this study, different agro-industrial wastes; various citrus peels, grape pomace, the hydrolysates of hazelnut and chestnut shells, sugarcane molasses residue, dried and fresh hazelnut husks and pumpkin peel, were used as fermentation media without adding any extra nutritional component for pullulan production by A. pullulans AZ-6. As a result, among the tested media, the maximum pullulan concentration was obtained as 33.59 gL-1 using the sugarcane molasses residue, and followed by the corresponding value of 30.02 gL-1 obtained in the dried hazelnut husk hydrolysate medium. Therefore, the usage of agro-industrial wastes as fermentation media is considered to make pullulan production cost effective. In addition, waste treatment from this aspect solves a relevant environmental problem. In this study, sugarcane molasses residue and dried hazelnut husk hydrolysate were used directly as fermentation media for pullulan production for the first time. Pullulan production from sugarcane molasses residue and dried hazelnut husk hydrolysate media might be a promising substrate for economical point of view.

Almond hulls waste valorization towards sustainable agricultural development: Production of pectin, phenolics, pullulan, and single cell protein

This research aimed to valorize almond hulls based on a zero-waste strategy towards sustainable agricultural developments for the recovery and production of valuable compounds. For this purpose, the potential to produce four products, including pectin (AHP), phenolic compounds (AHPC), pullulan (PUL), and single-cell protein (SCP), was examined. The acidic extraction factors were optimized using a Box-Behnken design for the simultaneous extraction of AHP and AHPC, and the obtained results showed that the maximum AHP (26.32% w/w) and AHPC (6.97% w/w) yields were achieved at 90 °C, pH of 1.4, 58.65 min, and liquid–solid ratio (LSR) of 20.13 v/w as the optimum point. In the next step, the solid residues that remained from the AHP and AHPC extraction process (PESR) were treated with cellulase enzyme and ultrasound and were used for simultaneous microbial production of PUL (34.29–24.56 g/L) and biomass containing SCP (19.31–13.44% w/w). Furthermore, the obtained results showed that AHP was low methylated (26.40%), rich in galacturonic acid (67.88%), and high in molecular weight (595.299 kDa). Also, the investigations of structural properties of AHP and PUL confirmed the presence of chemical structures of these polysaccharides in the formed supernatants. In addition, the AHPC showed considerable antioxidant activity compared with ascorbic acid (ASC) and BHA.

Microbial Exploitation of Biofertilizer, Pullulan and Biochar from Floral Waste

In diversified India, there are different traditions and cultures and their spirituality to believe in God is unconditional. People used to go temple and offer several things to God, and mainly the item they offer is the flower. Heaps of flowers flooded in temples are served as waste in nearby areas, or they dumped it into water. Flower waste has abundant properties with several nutrients (macro as well as micro) in them. In this article, we have emphasized the production and application of fertilizer, Pullulan and Biochar with the help of microbes present on floral waste enriched samples. Test samples were collected in the nearby areas of the temple, and after collection, serial dilution of the enriched sample was prepared. 1mL of each serial dilution were outspread on sterile nutrient agar plates, and bacterial isolation was done. Properties of degradation of soil isolates were evaluated. Flowers collected were then dried, and 1% was then transferred in minimal medium (without C source). Streaking was carried out, and isolated possessing tremendous growth was choosen for the development of the consortium. Different combinations of soil isolates were used. Five per cent inoculum of various consortia was spread on flower waste chambers. It was incubated aerobically. Degradation was analyzed after a specific time interval. The microbial consortium supports minimizing the time required for degrading an enormous amount of flower waste. With this perspective, we can prepare good quality Biopolymer Pullulan and Biochar without causing any harm to the environment.

Improved production of β-glucan by a T-DNA-based mutant of Aureobasidium pullulans.

To improve β-1,3-1,6-D-glucan (β-glucan) production by Aureobasidium pullulans, an Agrobacterium tumefaciens-mediated transformation method was developed to screen a mutant A. pullulans CGMCC 19650. Based on thermal asymmetric-interlaced PCR detection, DNA sequencing, BLAST analysis, and quantitative real-time PCR assay, the T-DNA was identified to be inserted in the coding region of mal31 gene, which encodes a sugar transporter involved in pullulan biosynthesis in the mutant. The maximal biomass and β-glucan production under batch fermentation were significantly increased by 47.6% and 78.6%, respectively, while pullulan production was decreased by 41.7% in the mutant, as compared to the parental strain A. pullulans CCTCC M 2012259. Analysis of the physiological mechanism of these changes revealed that mal31 gene disruption increased the transcriptional levels of pgm2, ugp, fks1, and kre6 genes; increased the amounts of key enzymes associated with UDPG and β-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored β-glucan production. However, the T-DNA insertion decreased the transcriptional levels of ags2 genes, and reduced the biosynthetic capability to form pullulan, resulting in the decrease in pullulan production. This study not only provides an effective approach for improved β-glucan production by A. pullulans, but also presents an accurate and useful gene for metabolic engineering of the producer for efficient polysaccharide production. KEY POINTS: • A mutant A. pullulans CGMCC 19650 was screened by using the ATMT method. • The mal31 gene encoding a sugar transporter was disrupted in the mutant. • β-Glucan produced by the mutant was significantly improved.

Efficient pullulan production by Aureobasidium pullulans using cost-effective substrates

In this study, cost-effective substrates such as cassava starch, corn steep liquor (CSL) and soybean meal hydrolysate (SMH) were used for pullulan production by Aureobasidium pullulans CCTCC M 2012259. The medium was optimized using response surface methodology (RSM) and artificial neural network (ANN) approaches, and analysis of variance indicated that the ANN model achieved higher prediction accuracy. The optimal medium predicted by ANN was used to produce high molecular weight pullulan in high yield. SMH substrates increased both biomass and pullulan titer, while CSL substrates maintained higher pullulan molecular weight. Results of kinetic parameters, key enzyme activities and intracellular uridine diphosphate glucose contents revealed the physiological mechanism of changes in pullulan titer and molecular weight using different substrates. Economic analysis of batch pullulan production using different substrates was performed, and the cost of nutrimental materials for CSL and SMH substrates was decreased by 46.1% and 49.9%, respectively, compared to the control using glucose and yeast extract as substrates, which could improve the competitiveness of pullulan against other polysaccharides in industrial applications.

Pullulan production in stirred tank reactor by a colour-variant strain of Aureobasidium pullulans FB-1

A colour-variant strain of Aureobasidium pullulans FB-1 was used for the production of pullulan in a stirred tank reactor in a medium composed of sucrose, ammonium sulphate, yeast extract, K2HPO4, NaCl and MgSO4. Maximum pullulan production (4.8%, w/v) was obtained at an agitation rate of 300 rpm and aeration of 0.75 vvm in a stirred tank reactor (1 L) with a fermentation time of 6 days. Fourier transformed infrared and NMR spectrum of crude pullulan from A. pullulans FB-1 revealed solely α-(1→6) linked maltosyl units, in accord with the generally accepted structure of pullulan. Hydrolysis of crude pullulan, i.e. digestion with pullulanase also confirmed the identity as well as homogeneity of the produced pullulan. XRD analysis of crude pullulan verified its amorphous nature. Thermal analysis showed melting range of crude pullulan from 245.99–295.71 °C.

Optimization of cultivation conditions for pullulan production from Aureobasidium pullulans MR by response surface methodology

Optimization of cultivation conditions for pullulan production from Aureobasidium pullulans MR by response surface methodology

Pullulan-Apple Fiber Biocomposite Films: Optical, Mechanical, Barrier, Antioxidant and Antibacterial Properties.

More than 150 million tons of synthetic plastics are produced worldwide from petrochemical-based materials, many of these plastics being used to produce single-use consumer products like food packaging. The main goal of this work was to research the production and characterization of pullulan–apple fiber biocomposite films as a new food packaging material. The optical, mechanical, and barrier properties of the developed biocomposite films were evaluated. Furthermore, the antioxidant and antibacterial activities of the biocomposite films were additionally studied. The results show that the Tensile Index and Elastic Modulus of the pullulan–apple fiber films were significantly higher (p-value < 0.05) when compared to the pullulan films. Regarding the water vapor permeability, no significant differences (p-value < 0.05) were observed in water vapor transmission rate (WVTR) when the apple fiber was incorporated into the biocomposite films. A significant increase (p-value < 0.05) of water contact angle in both sides of the films was observed when the apple fiber was incorporated into pullulan, indicating an increase in the hydrophobicity of the developed biocomposite films. It is worth noting the hydrophobicity of the (rough) upper side of the pullulan–apple fiber films, which present a water contact angle of 109.75°. It was possible to verify the microbial growth inhibition around the pullulan–apple fiber films for all the tested bacteria.