Improved pullulan production by a mutant of Aureobasidium melanogenum TN3-1 from a natural honey and capsule shell preparation

International Journal of Biological Macromolecules 141 (2019) 268–277

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Improved pullulan production by a mutant of Aureobasidium melanogenum TN3-1 from a natural honey and capsule shell preparation Lu Chen a,1, Zhe Chi a,b,⁎, Guang-Lei Liu a,b,1, Si-Jia Xue a, Zhi-Peng Wang a,1, Zhong Hu c, Zhen-Ming Chi a,b a b c

College of Marine Life Sciences, Ocean university of China, Yushan Road, No. 5, Qingdao, China Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, 266003 Qingdao, China Department of Biology, Shantou University, Shantou 515063, China

a r t i c l e

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Article history: Received 21 July 2019 Received in revised form 25 August 2019 Accepted 31 August 2019 Available online 02 September 2019 Keywords: A. melanogenum TN3-1 High pullulan producer Genome duplication Pullulan Capsule shells

a b s t r a c t Aureobasidium melanogenum TN3-1 isolated from a natural honey was a highly genome-duplicated yeast-like fungal strain and a very high pullulan producer. In this study, simultaneous removal of both duplicated AMY1 genes encoding α-amylase and duplicated PKS1 genes responsible for melanin biosynthesis in A. melanogenum TN3-1 rendered a mutant AMY-PKS-11 to transform 140.0 g/L of glucose to produce 103.50 g/L of pigmentfree pullulan with molecular weight (Mw) of 3.2 × 105 g/mol. α-Amylase activity produced by the mutant AMY-PKS-11 and expression of the AMY1 genes and PKS genes in it was reduced, but expression of various genes responsible for pullulan biosynthesis in the mutant AMY-PKS-11 was up-regulated. The produced pullulan was used to make the capsule shells successfully and the prepared pullulan capsule shells had various advantages such as high strength, good oxygen barrier properties, raw materials availability, tightness, lightness and high water resistance and may be suitable for all the consumers. Therefore, the prepared capsule shells had highly potential applications in food and pharmaceutical industries. © 2019 Elsevier B.V. All rights reserved.

1. Introduction So far, gelatin has been the most commonly used biomaterial for making hard capsule shells due to the facts that the hard capsule shells made of gelatin is the reversible gel formation, the gel is dissolved readily in biological fluids at body temperature, gelatin has good filmforming properties and surface active properties. However, the gelatin capsule shells also have many disadvantages. For example, the gelatin capsule shells may become brittle after exposure to low humidity, undergo cross-linking reactions which reduce water solubility and retard disintegration of the shell and thus slow down the drug release at high temperatures, the amine groups of gelatin can react with aldehyde groups in the drugs, reducing sugars, metal ions, plasticizers, preservative anionic and cationic polymeric materials, are incompatibility with hygroscopic substances and gelatin is an animal-derived ingredient with the mad cow disease, foot-and-mouth disease and transmissible spongiform encephalopathy scare and refused by some religious believers, vegetarians, diabetic persons and patients with restricted diet [1]. Therefore, it is of significance to replace the gelatin capsule shells with the non-animal capsule shells and to make them that are suitable ⁎ Corresponding author at: College of Marine Life Sciences, Ocean university of China, Yushan Road, No. 5, Qingdao, China. E-mail address: [email protected] (Z. Chi). 1 Lu Chen, Guang-Lei Liu and Zhi-Peng Wang made equal contribution to this work.

https://doi.org/10.1016/j.ijbiomac.2019.08.264 0141-8130/© 2019 Elsevier B.V. All rights reserved.

for cultural and dietary requirements of religious believers, vegetarians, diabetic persons and patients with restricted diet. Although hydroxypropyl methyl cellulose (HPMC), starch and modified starch have been successfully used to make capsule shells, they have some limitations due to the leakage of encapsulated drug, poor mechanical properties and poor in vivo disintegration performance [2]. It has been well recognized that the capsule shells made of pullulan and other microbial exopolysaccharides can meet such demands because very high level of pullulan with high Mw can be produced by Aureobasidium melanogenum TN3-1 isolated from the natural honey [3], Mws of the produced pullulan can be genetically edited based on synthetic biology [4] and the microbially produced polysaccharides are green and safe. It is true that the pullulan capsule shells have several distinct advantages over the gelatin capsule shells. Besides no mad cow disease, foot-andmouth disease and transmissible spongiform encephalopathy risk, pullulan is an excellent film-forming, non-toxic, tasteless, nonimmunogenic, non-carcinogenic, non-mutagenic, biodegradable, soluble, oil resistant, transparent and oxygen impermeable polysaccharide and can be widely applied in food and pharmaceutical industries [1]. Pullulan produced by different strains of A. melanogenum is a linear exopolysaccharide with maltotriose repeating units connected by α-(1 → 6) glycosidic bonds and has been widely used to prepare capsule shells suitable for cultural and dietary requirements of vegetarians, diabetic persons and patients with restricted diet [5,6]. However, so far, it has been found that most of strains of Aureobasidium spp. only can

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transform sucrose into high level of pullulan due to their sensitivity to high concentration glucose in the medium, producing b70 g/L pullulan [7]. Furthermore, when glucose is used as the substrate for pullulan production, only b52.5 g/L pullulan is achieved [8,9]. Recently, a novel yeastlike fungal strain A. melanogenum TN3-1 isolated from the natural honey was found to be able to produce over 110 g/L pullulan from 140 g/L glucose because of its bigger cells, thicker cell wall, more small vacuoles, higher intracellular glycerol, trehalose and glycogen than A. melanogenum P16 isolated from a mangrove ecosystem [3,7]. However, this novel yeast-like fungal strain can produce yellow-brown and black pigment and the Mw of the produced pullulan is only 1.6 × 105 g/mol [3]. It has been well documented that A. melanogenum can synthesize dihydroxynaphthalene (DHN)-melanin and a PKS gene is involved the DHN-melanin biosynthesis [10]. It also has been demonstrated that αamylase, glucoamylase and isopullulanase can determine Mw of pullulan produced by A. melanogenum P16 [4]. Therefore, in this study, in order to produce colorless pullulan, increase Mw of the produced pullulan and make it possible for the produced pullulan to be used in food and pharmaceutical industries, the PKS gene and α-amylase gene in the novel yeast-like fungal strain A. melanogenum TN3-1 were abolished and the obtained mutants were used to produce colorless and high Mw pullulan. Then, the produced pullulan was used to develop pullulan based biopolymer hard capsule shells with better physical and mechanical properties than those of the gelatin capsule shells, to develop hard capsule shells as a substitute for gelatin or animal protein based products for pharmaceutical industry and to evaluate the physical and mechanical properties of the pullulan based hard capsule shells (Fig. 1). 2. Materials and methods 2.1. The pullulan producers, media and plasmids A. melanogenum TN3-1 isolated from the natural honey was the very high pullulan (over 110 g/L pullulan) producing yeast-like fungal strain

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[3] and the mutant AMY-PKS-11 in which all the AMY genes encoding α-amylase and PKS1 genes encoding Pks1 were deleted was used to produce colorless and high Mw pullulan. A. melanogenum P16, also a high pullulan producer, was used as a control strain [7]. The wild type strain TN3-1, P16 strain and the mutant AMY-PKS-11 were grown in a YPD medium [7]. The wild type strain TN3-1 and the mutant AMYPKS-11 were also grown in the pullulan production medium containing 140.0 g/L glucose [3]. The plasmid FL4a-nat-loxp carrying the nourseothricin resistance gene (NAT gene) constructed in this laboratory was used to construct the knock-out vectors used in this study. A plasmid pAMCRE1 carrying autonomously replicating DNA sequence (ARS), Cre recombinase gene and hygromycin B resistance gene (HPT gene) constructed in this laboratory was used to remove the NAT gene in the disruptants and was automatically lost during cell cultivation of the disruptants. The disruptants obtained in this study were grown in the YPD medium containing 50.0 μg/mL nourseothricin and both hygromycin B (50.0 μg/mL) and nourseothricin (50.0 μg/mL) [9]. 2.2. Genome sequencing and estimation of the copy numbers of the duplicated genes The purified genomes of A. melanogenum TN3-1 and A. melanogenum P16 were sequenced using a HiSeq™ 2500 sequence platform provided by Illumina Company. The sequenced genomes were annotated using KEGG (Kyoto Encyclopedia of Genes and Genomes) (http://www. kegg.jp/kegg/tool/annotate_sequence.html) for the metabolic pathways [11] and GO (Gene Ontology, http://www.geneontology.org/) for annotation of the homologous genes and their function, location of cellular components and biological processes. Proteins with highly similar sequences and similar functions in the same pathway are grouped together and labeled with the same KO (KEGG Orthology) number (https://www.kegg.jp/kegg/ko.html). For this, the statistics of the duplicated genes were made based on their different KO and KEGG numbers of the predicted proteins through linux codes.

Fig. 1. Genetic modifcation of the TN3-1 strain and hard capsule making using the produced pullulan.

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Additionally, an all-against-all predicted protein sequence similarity search of A. melanogenum TN3-1 and A. melanogenum P16 proteins to the A. melanogenum TN3-1 and A. melanogenum P16 protein databases was performed by using blastp included in the BLAST 2.2.25+ [12]. The number of hits was counted for each query.

centrifuged at 14000 ×g and 4 °C to discard the cells and denatured proteins. The pullulan in the supernatants were isolated using cold 100% ethanol and quantitatively determined according to the methods described by Ma et al. [7]. 2.5. Assay of α-maylase, glucoamylase and isopullulanase activities

2.3. Construction of the knock-out vectors for disruption of the target genes in A. melanogenum TN3-1 A. melanogenum TN3-1 was the highly genome-duplicated yeast-like fungal strain and most of its genes were duplicated. In order to smultaneously abolish both the AMY genes encoding α-amylase and PKS1 genes encoding Pks1 in it, the 5′-arms and 3′-arms of the AMY1 gene, AMY2 gene, PKS1 gene and PKS2 gene were PCR amplified using the primers (AMY1-5-F, AMY1-5-R, AMY1-3-F, AMY1-3-R, AMY2-5-F, AMY2-5-R, AMY2-3-F, AMY2-3-R, PKS1-5-F, PKS1-5-R, PKS1-3-F, PKS1-3-R, PKS2-5-F, PKS2-5-R, PKS2-3-F, PKS2-3-R) (Supplementary file 1) and the genomic DNA of A. melanogenum TN3-1 as the template. The PCR products obtained were treated with the enzymes shown in Supplementary file 1 and the treated PCR products were ligated into the plasmid FL4a-nat-loxp carrying the NAT gene with T4 DNA ligase, respectively, yielding the recombinant plasmids FL4a-nat-loxp-ΔAMY1, FL4a-nat-loxp-ΔAMY2, FL4a-nat-loxp-ΔPKS1, FL4a-nat-loxp-ΔPKS2 (Supplementary file 2). In order to obtain the linear DNAs for disruption of the AMY1, AMY2, PKS1 and PKS2 genes, the PCR products were amplified using the primers (AMY1-5-F/AMY1-3-R, AMY2-5-F/AMY2-3-R, PKS1-5-F/PKS13-R, PKS2-5-F/PKS2-3-R) (Supplementary file 1) and the plasmid FL4a-nat-loxp-ΔAMY1, FL4a-nat-loxp-ΔAMY2, FL4a-nat-loxp-ΔPKS1, FL4a-nat-loxp-ΔPKS2 obtained above as the templates. The linear DNA fragments 5′-arm of the AMY1 gene-PolyA-NAT-Pgk-3′-arm of the AMY1 gene, 5′-arm of the AMY2 gene-PolyA-NAT-Pgk-3′-arm of the AMY2 gene, 5′-arm of the PKS1 gene-PolyA-NAT-Pgk-3′-arm of the PKS1 gene and 5′-arm of the PKS2 gene-PolyA-NAT-Pgk-3′-arm of the PKS2 gene were obtained. The linear DNA fragment 5′-arm of the AMY1 gene-PolyA-NAT-Pgk-3′-arm of the AMY1 gene was transformed into the competent cells of the TN3–1 strain to acquire the single amy1 mutants in which the AMY1 gene was removed based on the methods described by Chi et al. [13]. Then, the plasmid pAMCRE1 was transformed into the competent cells of the mutants to delete the NAT gene and was automatically lost after cultivation of the transformant cells. Furthermore, the linear DNA fragment 5′-arm of the AMY2 genePolyA-NAT-Pgk-3′-arm of the AMY2 gene was introduced into the transformant cells which were sensitive to both hygromycin B and nourseothricin to disable the AMY2 gene, the NAT gene in the double amy1 amy2 mutants was again removed using the same methods as described above. The linear DNA fragment 5′-arm of the PKS1 gene-PolyANAT-Pgk-3′-arm of the PKS1 gene was transformed into the double amy1 amy2 mutants to get the triple pks1, amy1 and amy2 mutants and the NAT gene in the triple pks1 amy1 and amy2 mutants was again removed using the same methods as described above. Finally, the linear DNA fragment 5′-arm of the PKS2 gene-PolyA-NAT-Pgk-3′arm of the PKS2 gene was introduced into the triple pks1, amy1 and amy2 mutants to yield the quadruple pks1, pks2, amy1 and amy2 mutants. 2.4. Pullulan production by the wild type strain TN3-1 and the mutants obtained above The wild type strain TN3-1 and the mutants obtained above were aerobically cultivated in the YPD medium at 28 °C for 24 h. The seed cultures (5.0 mL) were transferred into the pullulan production medium (30.0 mL) with 140.0 g/L glucose in 250-mL flasks and the cultures in the flasks were grown by shaking at 180 rpm and 28 °C for 96 h. The cultures obtained were heated at 100 °C for 15 min to kill all the cells and denature the proteins. After cooling, the treated cultures were

The activities of α-amylase and glucoamyalse in the cultures of the TN3-1 strain and mutants in which the AMY1 and AMY2 genes had been removed were determined according to the methods described by Liu et al. [14]. An isopullulanase activity in the cultures of the TN31 strain and mutants in which the AMY1 and AMY2 genes had been disabled was examined based on the methods described by Ma et al. [7]. The protein concentrations in the cultures were quantitatively determined using the methods described by Bradford [15], and the bovine serum albumin served as the standard. One α-amylase activity unit, one glucoamylase activity unit and one isopullulanase activity unit were defined as the amount of enzyme that hydrolyzes 1.0 mg of soluble starch per min; the amount of enzyme that produces 1.0 μM of reducing sugar from soluble starch per min and the amount of enzyme that produces 1.0 μM of reducing sugar from pullulan per min under the assay conditions used in this study, respectively. At the same time, the TN3-1 strain and its disruptants were grown on the YPD plates with 20.0 g/L soluble starch at 28 °C for 96 h. Then, an iodine solution was added to the plates and the clear zones formed around the colonies were observed and photographed. 2.6. Melanin extraction and determination and observation of the colonies Melanin in the cell cultures (3 × 107 cells/mL) of the wild type strain TN3-1 and the disruptants were extracted and purified as described by Jiang et al. [10]. Finally, purified melanin was lyophilized, the amount of the dried melanin was determined and calculated according to the methods described by Zou et al. [16]. The wild type strain TN3-1 and the disruptants were grown on a PDA plate which contained 200 g/L potato extract and 20.0 g/L dextrose at 28 °C for 4 days. The colonies formed on the plates were observed and photographed. 2.7. Determination of viscosity of the supernatants The viscosity of the diluted culture supernatants of the wild type strain TN3-1 and the disruptants was determined using an Ubbelohde capillary viscometer at 25 °C. The intrinsic viscosity [η] of the culture supernatants was calculated from the Huggins equation. The relative viscosity was calculated based on the equation [η] = (0.000258) × Mw0.646 [17] and the relative viscosity of the culture supernatants of the wild type strain TN3-1 was regarded as 100%. 2.8. Determination of transcriptional levels of various genes in the wild type strain TN3-1 and its mutants To conduct real-time quantitative RT-PCR, the cells of the wild type strain TN3-1 and its mutants were collected and washed with sterile distilled water by centrifugation. A total RNA in the washed cells was isolated and purified using an E.Z.N.A.TM Fungal RNA Kit (OMEGA 197 Biotech, Shanghai). A reverse transcription was conducted using a Prime Script RT reagent Kit (TaKaRa, Japan) based on the manufacturer's protocol. The fluorescent real-time RT-PCR assay was carried out according to the methods described by Liu et al. [18]. All the primers for the fluorescent real-time PCR shown in Supplementary file 1 were designed and synthesized based on the corresponding genes in the wild type strain TN3-1. The relative expression levels of the different genes were calculated using the formula RATE = 2−ΔΔCt and the relative transcriptional levels of the various genes in the wild type strain TN3-1 were considered as 100%.

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2.9. Pullulan production using a 10-liter fermenter The mutant AMY-PKS-11, one of the disruptants in which all the AMY1, AMY2, PKS1 and PKS2 genes had been disabled was aerobically grown in the YPD medium at 28 °C for 36 h. Eight hundred milliliters of the seed culture were transferred to 6.2 L of the pullulan production medium with 140.0 g/L glucose in a 10-liter fermenter [BIOQ-60056010B, Huihetang Bio-Engineering Equipment (Shanghai) CO-LTD, China] equipped with a 10-liter vessel (7.0 L working volume). The fermentation was carried out at 28 °C for 120 h as described by Ma et al. [7]. During the fermentation, only 50.0 mL of the culture was harvested in the interval of 12 h and was centrifuged at 13680 ×g and 4 °C for 10 min, and the pullulan titer, Mw of the pullulan, reducing sugar in the supernatant obtained were measured as described above. The amount of reducing sugar (glucose) in the culture was assayed based on the methods as described by Spiro [19]. The cell dry weight in 10.0 mL of the culture during the 10-liter fermentation was also measured as described by Chi et al. [20]. 2.10. Purification of the pullulan and determination of Mw of the purified pullulan The obtained pullulan from the culture supernatants of the 10-liter fermenter was dissolved in the pure water and protein in the solution was removed by using calcium hydroxide (pH 11) and phosphoric acid (pH 7.5). The treated pullulan was applied to the neutral macroporous adsorption resin D101 column and the pullulan was eluted using pure water at a flow rate of 0.5 mL/min. The pullulan in the eluate was precipitated by adding the cold 100% ethanol and the collected pullulan was dried until its weight was constant. The Mw of the purified pullulan was determined using a Gel Permeation Chromatography (GPC) as described by Liu et al. [4]. 2.11. Preparation of pullulan-based capsule shells The purifed pullulan (18.0 g), gellan (a gelling agent) (0.08 g), glycerol (a plasticizer) (10 mL) and potassium citrate (a gelling promoter) (0.1 g) were dissolved in 100 mL of the pure water, respectively. The solutions were mixed well and the mixture was degassed by ultrasonication during the incubation at 45 °C for 30 min. The conventional dip-coating method was used for hard capsule shell development. The preheated stainless steel mold pins were dipped into the mixture, the pullulan solution thermally gelled on the surface of the pins at 15 °C. When the pins were withdrawn, a film of the gelled pullulan solution remained on the pins. The coated pins were dried in a controlled temperature at 50 °C for 30 min. The dried capsule pieces were then stripped, cut into sizes, and fitted together. 2.12. Characterization of the capsule shells 2.12.1. Appearance of the prepared capsule shells Average shell thickness (μm), max gap between the body and cap (μm), shell surface rough edge, smooth surface, length of the body and cap and fragility were checked according to Chinese Pharmacopeia (CP, 2010) and the mean values were calculated. 2.12.2. Loss on drying and water solubility The films of the prepared capsule shells were weighed and then they were preserved in a desiccator (0% relative humidity) for 10 days until their weights were constant. The weight of the dried film (W0) was measured again and their weight loss was calculated. The dried films were soaked in 5.0 mL of deionized water in a 25 ± 1 °C shaking water bath for 5 min at 50 rpm, followed by a careful separation of each insoluble film. Each film was dried in a vacuum drying oven (100 °C) until constant weights (W1) were obtained. The water solubility

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(WS) was calculated with the equation provided by Wu et al. [21]. All the tests were conducted in triplicate. 2.12.3. Measurement of residue on ignition The dried films obtained above were completely burnt at 700–800 °C and the weights of the residue from each dried film were determined according to Chinese Pharmacopeia (CP, 2010). 2.12.4. Oxygen barrier properties A 60 mL-bottle was filled with 8.0 g of fresh soybean oil and covered tightly with the films of the prepared pullulan capsule shells and stored at a controlled temperature (60 °C) for 15 days. The peroxide value of the soybean oil samples in the bottles was measured by using sodium thiosulfate titration [22] and the untreated fresh soybean oil was used as a control. The peroxide value was calculated based on the Eq. (1): PV ¼ ðV1 −V0 Þ  C  0:1269=M  100  78:8

ð1Þ

PV (meq/kg): the peroxide value of the soybean oil samples; V1: the volume (mL) of the consumed sodium thiosulfate solution during titration for the samples; V0: the volume (mL) of the consumed sodium thiosulfate solution during titration for the control samples; C: the concentration (0.01 M) of sodium thiosulfate solution; M: the weight (g) of the films of the prepared capsules. 2.12.5. Water vapor permeability (WVP) determination After the films of the prepared pullulan capsule shells were equilibrated at 53% relative humidity for 72 h, the treated films were sealed in a small bottle (10-mL) filled with anhydrous CaCl2 (the distance between the anhydrous CaCl2 and the film was 5 mm). Then, the small bottles were placed in a cabinet with a controlled relative humidity of 100% (BaCl2 saturated solution) at 25 °C. Periodical weightings of the small bottles monitored their weight changes for every 24 h. The WVP values were calculated according to the Eq. (2): WVP ¼ Δm  d=A  Δt

ð2Þ

WVP: the water vapor permeability, ng m/m2 s Pa Δm: the net weight increase of the small bottle, g A: the area of the films of the prepared pullulan capsule shells, m2 Δt: the time interval, d d: thickness of the films of the prepared pullulan capsule shells, m 2.12.6. Dissolution of amoxicillin from the capsule shells in vitro Each capsule shell (the total numbers of the capsule shells were 10) was filled with 0.25 g amoxicillin powder. The capsule shells were immersed in dissolution media (degassed by ultrasonication for 30 min) mimicking the stomach condition that was prepared by 0.1 M HCl solution (pH = 2) at 37 ± 0.5 °C, and the solution was stirred at 100 ± 1 rpm paddle speed using dissolution apparatus (RCZ-6C2, HUANGHAI, China). The samples with released amoxicillin were collected after 2, 5, 8, 10, 15, 20, 30, 45 and 60 min of the incubation, and filtered through a 0.22 μm polyethersulfone membrane to remove undissolved amoxicillin before quantification [23]. The OD 205nm value of the filtrated samples was determined using a UV spectrophotometer (TU1810, PERSEE, China) and the amoxicillin concentrations in the filtrated samples were calculated according to the standard curve. 2.12.7. Measurements of the maximum gap between the body and cap by scanning electron microscopy (SEM) Closed empty capsule shells were cut at the closure to expose the cross-section between the body and cap. The cut capsule shells were sputter-coated with platinum vapor. The coated samples were observed using a scanning electron microscope (TESCAN VEGA3,

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Czekh) under high vacuum at a high voltage of 20.0 kV. The shell thickness was measured at 30 different points and the maximum gap between the body and cap was located and measured from the cross-sectioned samples. 2.12.8. Powder leakage test To test for fracture resistance, each empty capsule shell which was tied with a 100 g weight was dropped from a height of 8 cm. A total number of 50 capsule shells was used per test [24]. After that, integrity and tightness of all the capsule shells were examined. 3. Results and discussion 3.1. Genome sequencing of A. melanogenum TN3-1 and P16 The total assembly size of the genome of the TN3-1 strain was 51.6 Mb, much larger than that (26.1 Mb) of the genome of the P16 strain (data not shown). Analysis of their predicted proteins with allagainst-all blastp and aligning the predicted proteins back to the genome found that the genome of the TN3-1 strain appeared to have a whole genome duplication (WGD) and contained two highly identical gene copies of almost half of the proteins while the genome of the P16 strain only contained a small fraction of the duplicated genes (Fig. 2). For example, two copies of the AMY1 and AMY2 genes encoding αamylase and two copies of the PKS1 and PKS2 genes encoding polyketide synthase catalyzing the first step reaction of melanin biosynthesis appeared in the genome of the TN3-1 strain (Fig. 1). Furthermore, the cell sizes, cell walls and the number of small vacuoles of A. melanogenum TN3-1 were also much larger, thicker and higher, respectively, than those of A. melanogenum P16 [3]. So the TN3-1 strain could be a good chassis for biosynthetic biology and metabolic engineering. However, it is challenging how to genetically edit such genomes in the novel eukaryotic cells. It has been reported that the genome of Hortaea werneckii, an extremely halotolerant black yeast also has higher numbers of the duplicated genes (nearly 90% of the genes exist in at least two copies) than those of Saccharomyces cerevisiae and Mycosphaerella graminocpla [25]. This WGD A. melanogenum TN3-1 isolated from natural honey, a very high osmotic environment may benefit from the potential advantages of a large genetic redundancy. For example, it could easily adapt to the harsh fructose and glucose-rich environments in the natural honey and produce very high concentration of pullulan from 140.0 g/L glucose [3]. Therefore, A. melanogenum

TN3-1 is an excellent producer of pullulan, one of the most commercially important carbohydrate polymers. 3.2. Enzyme activity and disruption of the α-amylase gene It has been well confirmed that α-amylase, glucoamylase and isopullulanase can determine the Mw of the pullulan produced by A. melanogenum P16 because these enzymes can hydrolyze the produced pullulan [4]. However, the Mw of the produced pullulan is always inversely proportional to the concentration of the produced pullulan [14]. The results in Table 1 indeed showed that activities of α-amylase, glucoamylase and isopullulanase produced by the P16 strain were much lower than those of α-amylase, glucoamylase and isopullulanase produced by the TN3-1 strain. Therefore, it is necessary to increase Mw of the pullulan produced by A. melanogenum TN3-1 by deletion of the α-amylase genes. Simultaneous removal of the two copies of the α-amylase genes in A. melanogenum TN3-1 by using the Cre-Loxp editing system as described in Materials and methods rendered the mutants AMY2-4, AMY2-5 and AMY2-6 to produce less pullulan and lower α-amylase activity than their wild type strain TN3-1, but increased the viscosity of pullulan solution compared to that of pullulan solution produced by their wild type strain TN3-1 (Fig. 3A and Table 2). Meanwhile, their cell growth was not affected (Fig. 3A). For example, the disruptant AMY2-4 only produced 63.58 ± 3.21 g/L of pullulan from glucose while its wild type strain TN-3-1 produced 71.53 g/L of pullulan from glucose under the same conditions. The relative activity of α-amylase produced by the disruptant AMY2-4 were only 32.72% of that of α-amylase produced by its wild type strain TN3-1 and the relative viscosity of the pullulan solution produced by the disruptant AMY2-4 was increased by 68.43% compared to that of the pullulan solution produced by its wild type strain TN3-1 (Table 2 and Fig. 3A). In our previous studies [4], it was found that the disruption of all the α-amylase, glucoamylase and isopullulanase genes in A. melanogenum P16 also caused decrease in pullulan production and increase in Mw of the produced pullulan. Furthermore, when the disruptant AMY2-4 and its wild type strain TN3-1 were grown on the YPD medium containing soluble starch, the clear zone was formed around the wild type strain TN3-1 colony whereas no such clear zone was formed around the mutant AMY2-4 colony (Fig. 4), indicating that amylase activity produced by the disruptant AMY2-4 was indeed greatly reduced compared to that produced by the TN3-1 strain. 3.3. Pigment production and the knock-out of the PKS genes All the strains of Aureobasidium spp. are usually called black yeasts because they can produce DHN-melanin [26] and the DHN-melanin biosynthesis is controlled by the PKS gene encoding a protein consisting of one ketosynthase (KS), one acyl transferase (AT), two acyl carrier proteins (ACP), one thioesterase (TE) and one cyclase (CYC) [10]. The TN3-1 strain used in this study also produced a yellow brown and dark pigment [3]. Therefore, based on the disruptant AMY2-4, the two copies of the PKS1 and PKS2 genes were continuously abolished by using the Cre-Loxp system shown in Materials and methods. It can be clearly observed from the results in Fig. 3 that the simultaneous abolishment of the two copies of the PKS1/2 genes in the disruptant AMY2-4 made all the mutants (AMY-PKS-2, AMY-PKS-11 and AMY-PKS-12)

Table 1 Comparison of the relative enzyme activities produced by the P16 strain and TN3-1 strain.

Fig. 2. The number of the duplicated genes in the genomes of A. melanogenum TN3-1 and A. melanogenum P16. All the predicted proteins were aligned to the genomes by Exonerate and the number of possible alignment locations was counted for each protein. The numbers of single copy genes and the duplicated genes are represented as columns of different heights.

Enzymes

P16 (%)

TN3-1 (%)

α-Amylase Glucoamylase Isopullulanase

100 100 100

510.40 ± 7.59⁎⁎ 304.90 ± 5.27⁎⁎ 244.27 ± 14.12⁎⁎

⁎⁎ (p b 0.01) means the significant difference. Data are given as mean ± SD, n = 3.

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Fig. 4. The clear zone formed by the TN3-1 strain and the disruptant strain. A: Control TN31 strain; B: Disruptant strain AMY2-4. The strains were grown on the YPD plate with 20.0 g/L soluble starch at 28 °C for 96 h.

Fig. 3. Pullulan production and cell growth by different disruptants in which the two copies of the α-amylase genes had been removed and their wild type strain A. melanogenum TN3-1 (A); Pullulan production from glucose and cell growth of different disruptants (the quadruple pks1, pks2, amy1 and amy2 mutants) and the mutant AMY2-4 (the double amy1 and amy2 mutant) (B) and melanin production by the wild type strain TN3-1, the mutant AMY2-4 and the mutant AMY-PKS-11 (C). Data are given as mean ± SD, n = 3.

Table 2 Relative activity of α-amylase and relative viscosity of pullulan solution produced by different disruptants and A. melanogenum TN3-1.

TN3-1 AMY2-4 AMY2-5 AMY2-6

Relative activity of α-amylase (%)

Relative viscosity of pullulan (%)

100 32.72 ± 0.39⁎⁎ 37.41 ± 0.64⁎⁎ 36.26 ± 0.53⁎⁎

100 168.43 ± 3.42⁎ 154.92 ± 2.56⁎ 156.73 ± 4.21⁎

⁎ (p b 0.05) means the difference. ⁎⁎ (p b 0.01) means the significant difference. Data are given as mean ± SD, n = 3.

increase pullulan concentration. But their cell growth was not influenced (Fig. 3B). For example, pullulan concentration produced by the mutant AMY-PKS-11 (the quadruple pks1, pks2, amy1 and amy2 mutant) reached 73.65 ± 3.12 g/L, higher than that (63.58 ± 3.21 g/L) produced by the mutant AMY2-4 (the double amy1 and amy2 mutant) (Fig. 3A and B). Meanwhile, it can be obviously seen from the results in Fig. 5 that color of the cell culture (A2), the produced pullulan (B2) and colonies (C2) of the mutant AMY-PKS-11 disappeared whereas that of the cell culture (A1), the produced pullulan (B1) and colonies (C1) of the wild type strain TN3-1 was yellow-brown or black, suggesting that pigment biosynthesis of the mutant AMY-PKS-11 was indeed inhibited. Determination of the extracted melanin from the wild type strain TN3-1 and different disruptants found that no melanin production by the mutant AMY-PKS-11 was observed while the amount of the melanin produced by the wild type strain TN3-1 and the mutant AMY2-4 reached 0.57 ± 0.02 g/L and 0.49 ± 0.04 g/L, respectively (Fig. 3C). This indicated again that melanin biosynthesis in the mutant AMYPKS-11 was totally stopped. These results implied that stop of the melanin biosynthesis could promote increase in pullulan biosynthesis by the mutant AMY-PKS-11. The reasons for these results may be due to the facts that the melanin biosynthesis consumed a considerable amount of carbon sources and cellular energy so that pullulan biosynthesis was reduced because the melanin biosynthesis is a ATP-consuming process [27] and no melanin biosynthesis could be helpful to pullulan biosynthesis and secretion because most of melanin is located in cell wall [10]. The results in Table 3 also showed that specific activity of α-amylase produced by the mutant AMY2-4 and the mutant AMY-PKS-11 were much lower than that of α-amylase produced by the wild type strain TN3-1. However, specific activities of glucoamylase and isopullulanase produced by the mutant AMY2-4 and the mutant AMY-PKS-11 were almost the same as those of glucoamylase and isopullulanase produced by the wild type strain TN3-1. This meant that α-amylase activity in both the mutant AMY2-4 and the mutant AMY-PKS-11 was indeed greatly reduced. Liu et al. [4,14] also showed deletion of α-amylase, glucoamylase and isopullulanase genes or changes in compositions of the pullulan production medium greatly increased Mw of pullulan and reduced pullulan titer and expression of the various genes responsible for pullulan biosynthesis. In our previous study [10], it was found that disruption of the same PKS1 gene in A. melanogenum XJ5-1 produced an albino colony of the mutant. All the results shown above demonstrated that the highly genome duplicated A. melanogenum TN3-1 could be easily edited using the efficient Cre/loxp site-specific recombination system used in this study by continuous abolishment of several genes in the genome. This was the first

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Fig. 5. Color of the cultures of the TN3-1 strain (A1) and the mutant AMY-PKS-11 (A2), of the precipitated pullulan from the supernatants of the TN3-1 strain (B1) and the mutant AMYPKS-11 (B2) cultures and of the colonies of the TN3-1 strain (C1) and the mutant AMY-PKS-11 (C2).

report that the highly genome duplicated yeast-like fungus which can produce very high level of pullulan from glucose was genetically edited.

3.4. Expression of various genes in the mutants AMY2-4 and AMY-PKS-11 and their wild type strain TN3-1 Total RNAs were extracted from the cells of the mutants AMY2-4 and AMY-PKS-11 and their wild type strain TN3-1 and transcriptional levels of various genes related to pullulan synthesis and degradation and melanin biosynthesis were examined as described in Materials and methods. Table 4 showed that the relative transcriptional levels of the α-amylase gene in the mutants AMY2-4 and AMY-PKS-11 and of the PKS1 gene in the mutant AMY-PKS-11 were 0, demonstrating that the α-amylase genes in the mutants AMY2-4 and AMY-PKS-11 and the PKS1 genes in the mutant AMY-PKS-11 were totally disabled. Moreover, the relative transcriptional levels of the Gluk, PGM, PUL1 and AGS2 genes which were closely related to pullulan biosynthesis in the mutant AMYPKS-11 were greatly up-regulated (Table 4). In contrast, the relative transcriptional levels of the IPU, Gluk, PGM and UGP genes in the mutant AMY2-4 were down-regulated (Table 4). That was why pullulan biosynthesis in the mutant AMY2-4 was declined (Fig. 3A), pullulan viscosity

Table 3 Comparison of the specific activities of different enzymes in different disruptants and A. melanogenum TN3-1. Specific enzyme activity (U/mg)

α-Amylase Glucoamylase Isopullulanase

TN3-1

AMY2-4

AMY-PKS-11

40.56 ± 2.12 0.35 ± 0.07 0.24 ± 0.02

8.52 ± 3.55⁎⁎ 0.25 ± 0.02 0.21 ± 0.05

11.67 ± 2.54⁎⁎ 0.33 ± 0.02 0.23 ± 0.02

⁎⁎ (p b 0.01) means the significant difference. Data are given as mean ± SD, n = 3.

produced by the mutant AMY2-4 was rose (Table 2) and pullulan biosynthesis in the mutant AMY-PKS-11 was promoted (Fig. 3B). 3.5. Pullulan production from high concentration of glucose by the mutant AMY-PKS-11 grown in the 10-liter fermenter The results mentioned above revealed that the mutant AMY-PKS-11 could produce pigment-free pullulan (Fig. 5) and pullulan biosynthesis in it was enhanced (Fig. 3B). Therefore, pullulan production by the mutant was conducted in the 10-l fermenter using the pullulan production medium containing 140.0 g/L glucose. The results in Fig. 6A demonstrated that during the fermentation, the mutant AMY-PKS-11 could produce 103.50 ± 1.02 g/L pullulan and its cell mass reached 15.75 ± 0.67 g/L within 120 h, leaving 1.383 g/L glucose in the fermented medium, suggesting that 88.78% of glucose was used for pullulan biosynthesis and cell growth by the mutant AMY-PKS-11. In this case, the yield was 0.75 g/g of glucose and the productivity was 0.86 ± 0.02 g/L/h. It has been reported that A. melanogenum TN3-1, the wild type strain used in this study, could yield 110.3 g/L of pullulan from 140.0 g/L glucose within 132 h during the same 10-l fermentation [3]. During the 10-l fermentation, the disruptant DG41, a glucose derepressed mutant, produced 64.93 ± 1.33 g/L pullulan from 120 g/L of glucose, while its wild-type strain P16 produced only 52.0 ± 1.95 g/L pullulan within 132 h [9]. An osmotolerant and nonpigmented isolate A. pullulans RBF-4A3, isolated from flowers of Caesulia axillaris, produced 34.68–66.79 g/L pullulan from up to 150 g/L glucose [28]. In fact, most strains of Aureobasidium spp. obtained so far, transform glucose only to produce b53 g/L pullulan [8,9]. Therefore, the process for high level pullulan production in a large scale fermentation could increase plant efficiency, reduce energy for pullulan production and recovery, increase pullulan yields per unit of working volume in the fermenter and greatly reduce labor force. This meant that like its wild type strain TN3-1, the mutant AMY-PKS-11 and the process developed in this study had highly potential applications various sections.

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Table 4 The relative transcriptional levels of the genes related to pullulan biosynthesis and degradation and melanin biosynthesis. Gene name

Function

TN3-1 (%)

AMY2-4 (%)

AMY-PKS-11 (%)

AMY GLY IPU GluK PGM UGP UGT1 PUL1 AGS2 PKS

Hydrolysis of α-1,4-glucosidic linkages Hydrolysis of α-1,4- and α-1,6-glucosidic linkages Hydrolysis of α-1,4-glucosidic linkages Phosphorylation of glucose Phosphoglucose mutase UDPG-pyrophosphorylase UDP-glucosyltranferase Pullulan synthetase alpha-glucan synthase polyketide synthase

100 100 100 100 100 100 100 100 100 100

0 97.37 ± 2.84 87.73 ± 13.24 80.06 ± 2.92⁎ 88.88 ± 9.80⁎ 73.77 ± 2.80⁎ 105.32 ± 6.09 102.87 ± 1.24 104.22 ± 1.09 117.40 ± 0.40

0 92.67 ± 3.86 109.29 ± 8.87 129.53 ± 8.47⁎ 126.05 ± 8.40⁎ 100.06 ± 1.21 94.24 ± 3.68 169.32 ± 5.44⁎ 146.50 ± 8.91⁎ 0⁎⁎

Data are given as mean ± SD, n = 3. ⁎ (p b 0.05) means the difference. ⁎⁎ (p b 0.01) means the significant difference.

3.6. Purification of the pullulan and determination of its mw

3.7. The capsule shell preparation and characterization

The pullulan produced by the mutant AMY-PKS-11 during the fermentation was purified as described in Materials and methods. Then, Mw of the purified pullulan was determined using GPC. The results in Supplementary file 3 indicated that Mn = 2.61 × 105 g/mol; Mw = 3.251 × 105 g/mol; Mz = 4.82 × 105 g/mol; Mp = 2.75 × 105 g/mol and Mw/Mn = 1.25 while Mw of the pullulan produced by its wild type strain TN3-1 was 1.6 × 105 g/mol [3]. This demonstrated that deletion of the α-amylase gene and the PKS1 gene greatly improved Mw of pullulan and pullulan production and the produced pullulan stayed in a very narrow range of Mw. However, removal of all the α-amylase gene, glucoamylase gene and isopullulanase gene in A. melanogenum P16 could make the triple mutant DT15 produce 46.2 g/L of pullulan with a Mw of 3.02 × 106 Da from sucrose [4].

Because the gelatin capsule shells that are being widely used in this world have many drawbacks and are refused by vegetarian, those whose religion prohibits the consumption of animal derived products and those who are diabetic persons and patients with restricted diet, it is urgent to develop the green and healthy capsule shells which can be warmly welcome by all the people in the world [29]. The photos of the prepared pullulan capsule shells are shown in Fig. 6B. It can be clearly observed from Fig. 6B that the prepared pullulan capsule shells were appealing in appearance (apparent, tasteless, dull, glossy, even, smooth, tough, tight and no bubbles). These photos also highlighted that the average shell body and cap thicknesses were 0.088 ± 0.005 (mm), and length of the cap and body were 9.82 and 16.63 mm, respectively (data not shown). These meant that all the appearances of the prepared pullulan capsules (shell surface, rough edge, smooth surface, length of the body and cap, tightness degree and fragility) met the requirements of the Chinese Pharmacopeia (CP, 2010). It should be stressed that the capsule shells were made of the blending of the pullulan produced in this study and gellan in order to improve the film properties of pullulan-based films with high-quality mechanical characteristics. In fact, pullulan blend with whey protein, caseinate, rice protein concentrate, starch, alginate, carboxymethylcellulose and chitosan also have been tried [21]. Meanwhile, a division of Warner Lambert (later as Pfizer), developed a different HPMC gelling system and obtained an US patent for a HPMC capsule using gellan gum (hydrocolloids) as the gelling agent and either ethylenediamine tetra acetic acid (EDTA) or sodium citrate as a gelling promoter (sequestering agents). However, the slow dissolution of this Shell 3 in acidic buffer makes it difficult for formulation development for the highly regulated prescription drugs [30]. Powder leakage is another issue of the prepared capsule shells because the presence of powder outside the capsule shells can lead to quality and safety concerns at clinical study site [31,32]. Our powder leakage test did not find any leaking capsule shells used in this study (data not shown). At the same time, the scanning electron photomicrograph by comparison of the joint gap between the body and cap by scanning electron microscopy was presented in Fig. 7. The results showed that the gap between the body and cap of the prepared pullulan capsule was 38.01 ± 1.07 μm. In contrast, Ku et al. [24] reported the gap between the body and cap of gelatin capsule was 66.86 μm. This meant that the gelatin capsule shells had a large gap between the body and cap, twice as much as the gap for the prepared pullulan capsule shells in this study. After determination of loss on drying and residue on ignition, the percentages of residue weights after loss on drying of the pullulan capsule shells and ignition at high temperature were 7.21 ± 0.47% and 1.10 ± 0.01%, respectively (data not shown). All these issues also met the requirements of the Chinese Pharmacopeia (CP, 2010). It has been

Fig. 6. The time course of pullulan production, cell growth and changes in glucose concentration during the 10-liter fermentation. DCW: dried cell weight (A); The photographs of the capsule shells made of the produced pullulan (B); Data are given as mean ± SD, n = 3.

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Fig. 7. Scanning electron photomicrographs of the cross-sections shown in red arrow at the closure between the body and cap of the capsule shells.

reported moisture content can be used as a measure of the water absorbing capacity of films and the moisture contents were 12.79% for GK-0, 10.98% for GK-10, 7.74% for GK-15, and 7.15% for GK-20. As reported in a previous study, the moisture content range for the HPMC capsule shells was 2–6%, but for gelatin capsule shells this value ranged from 13 to 15% [33]. The conventional gelatin hard capsule shells are produced with moisture content between 13% and 16% [34]. Usually, the water content of 7.0% is suitable for capsule shell manufacturing. Therefore, the moisture content (7.21 ± 0.47%) (data not shown) of the prepared pullulan capsule shells in this study was acceptable. Water barrier properties and oxygen barrier properties are very important properties of the prepared capsule shells in order to keep stable activity of the drugs in the capsule shells. Therefore, water barrier properties and oxygen barrier properties of the prepared capsule shells were examined as described in Materials and methods. The results in Table 5 witnessed that the oxygen permeability (OP) and water vapor permeability (WVP) of the prepared pullulan films were 41.32 meq/kg and 1.865 ± 0.48 ng m/m2 s Pa, indicating the prepared pullulan films had excellent (high) oxygen barrier properties and water barrier properties and were suitable to keep stable activity of the drugs in the capsule shells. In contrast, the OP value of pullulan-chitosan blend film prepared by Wu et al. [21] was 88.24 meq/Kg and higher than that of the prepared pullulan film in this study. However, the starch films also had excellent (high) oxygen barrier properties (0.048 ± 0.008 to 0.070 ± 0.009 fl m/m2 s Pa) and water barrier properties (1.1 ± 0.5 to 1.8 ± 0.4 ng m/m2s Pa). The oxygen permeability (OP) of the starch films was significantly lower than that of the gelatin and HPMC films [23]. It has been reported that protein-based films had superior mechanical properties compared to polysaccharide-based films and polysaccharide-based films were reported to have better oxygen barrier properties than protein-based films [35]. Park and Chinnan [36] reported that the oxygen permeabilities of methyl cellulose and hydroxypropyl cellulose films were 2.17 and 3.57 fl m/m2 s Pa while the oxygen permeabilities of low density polyethylene, polyethylene, polyvinyl chloride, and polyester were 22.50, 8.30, 0.09–17.99 and 0.13–0.30 fl m/m2 s Pa, respectively. It also has been reported that the water vapor permeabilities of starch samples

were lower than those of gelatin (2.5 ± 0.2 ng m/m2 s Pa) and HPMC (2.4 ± 0.5 ng m/m2 s Pa) films [23]. This meant the WVP and PV values of the pullulan based films prepared in this study were lower than those of the gelatin film and stable activity of the drugs in the capsule shells could be kept. In order to simulate the release of drugs in the prepared capsule shells in the stomach, amoxicillin was used as a model drug, and amoxicillin release kinetics from the pullulan capsule shells stored for different length of time were investigated as described in Materials and methods. The release kinetics of the amoxicillin-containing capsules at 37 ± 0.5 °C and 0.1 M HCl are shown in Table 6. The results in Table 6 indicated that 85.12% of the amoxicillin was released from the commercial gelatin capsule shells within 10 min while 87.9% of the amoxicillin was released from the capsule shells prepared in this study within 30 min. This revealed that the amoxicillin was more quickly released from the commercial gelatin capsule shells than from the prepared capsule shells in this study. According to the Chinese Pharmacopeia (CP, 2010), over 80% of the drugs in the capsule shells must be released within 45 min. This meant the capsule rupture time within 30 min can fulfill the requirements of Chinese Pharmacopeia (Table 6). However, it has been reported that 80% of the amoxicillin was released from the hard capsule shells made of tilapia scale gelatin by 10 min and the curves stabilized after 20 min [2] and the rupture time for mammalian gelatin capsule shells and the capsule shells made of HPMC or TEMPO oxidized Konjac glucomannan is 2–4 min [23,37,38]. In contrast, the release kinetics of erythromycin stearate from DO30% capsule shells under stimulated stomach condition showed that 80% of the drugs were released within 120 min. The rupture time of the capsule shell is about 45–60 min [23]. Thus, the prepared capsule shells in this study are a good candidate to be used as gastric soluble hard capsule shells. 4. Conclusions The mutant AMY-PKS-11 obtained in this study could transform 140.0 g/L of glucose to produce 103.50 g/L of pigment-free pullulan with Mw of 3.2 × 105 g/mol and the obtained pullulan could be used as a good biomaterial. Because the pullulan based capsule shells prepared in this study have various advantages such as high strength, elongation, oxygen barrier properties, good film forming properties, mechanical strength, raw materials availability, biodegradability,

Table 6 Percentage of amoxicillin dissolution in the prepared capsule shells and the commercial gelatin capsule shells. Time (min)

Amoxicillin release (%) Commercial gelatin capsule shells

The prepared pullulan capsule shells

0

0

0

0

19.78 ± 0.72

22.73 ± 0.85

67.15 ± 0.26

42.33 ± 0.20

85.12 ± 0.81

53.68 ± 0.53

87.57 ± 0.56

78.30 ± 0.39

87.39 ± 0.49

78.75 ± 0.21

87.54 ± 0.43

87.95 ± 0.59

87.62 ± 0.47

87.73 ± 0.59

87.80 ± 0.38

87.78 ± 0.65

0 2 5 8 10 15 20

Table 5 Determination of WVP and OP values of the pullulan-based capsule shells. The WVP value (ng m/m2 s Pa)

The OP value (meq/kg)

1.865 ± 0.48

41.32 ± 3.35

Data are given as mean ± SD, n = 3.

30 45 60 Data are given as mean ± SD, n = 3.

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