Extraction, Physicochemical and Film Properties of Polysaccharides From Highland Barley Bran Fermented by Aureobasidium Pullulans

Qin Liu¹, Pei Liu¹, You Zhou¹, Jinyu Chang¹, Xuan Chen¹, Qingyun Lyu^1,2*, Gang Liu^1,2*

¹School of Food Science and Engineering, Wuhan Polytechnic University, Wuhan 430023, China

²Key Laboratory for Deep Processing of Major Grain and Oil, Wuhan Polytechnic University, Ministry of Education, Wuhan 430023, China

^*Corresponding authors:

Qingyun Lyu. E-mail: lqingy2018@whpu.edu.cn; 

Gang Liu. E-mail: lg820823@163.com; 

Table of Content

It was feasible to make an edible fermented polysaccharide film with improved water solubility.

 

 

Abstract：Plastic packaging films are not recyclable, difficult to be degraded and thus threaten the environment and the health of humans and other animals. Therefore, the purpose of the research is to derive edible polysaccharide films from highland barley bran fermented by Aureobasidium pullulans. Fermentation conditions using UV-mutated A. pullulans were optimized and the rheological, mechanical and barrier properties of edible polysaccharide films were analyzed. The polysaccharides produced by mutagenesis were increased by 8.6% after fermentation. The products before and after fermentation contained α- and β-glycosidic bonds, C-O-C, C=O, O-H, and other characteristic peaks of polysaccharides. Post-fermented polysaccharide films (post-FPF) were more moisture resistant than pre-fermented polysaccharide (pre-FPF) and control group (CGPF). Mechanical properties were better for CGPF than post-FPF, but post-FPF had better water solubility. It was feasible to make an edible polysaccharide film with improved water solubility. Post-fermentation polysaccharide films could help to reduce the cost of edible film production and improve the comprehensive utilization of bran.

 

Keywords: edible film, highland barley bran, pullulan, rheological property, UV-mutation

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1. Introduction

Highland (naked) barley is a major cereal crop in Tibet, Gansu, Yunnan, and other regions in China due to its rich nutritional value conferred by cellulose, protein, vitamins, fat, β-glucan, inorganic salts, and other ingredients.^[1] Highland barley is highly resistant to cold, and has a short growth period, high yield, and strong adaptability.^[2,3] It is also a main source of food, fuel, and a raw material used to produce beer, medicine, and health products.^[4-6] Highland barley bran (HBB), a by-product of barley processing, is mainly used as feed or directly discarded in industrial production. To render HBB into food ingredients would be significantly beneficial to the environment by reducing non-recyclable waste.

Pullulan is a water-soluble, extracellular, amorphous, viscous, and linear polysaccharide secreted by Aureobasidium pullulans that comprises basic maltotriose units connected by α-1, 6 glycosidic bonds. This unique linkage gives pullulan good film-forming properties.^[7] Pullulan is often used as a packaging material as it has good color retention, preservation, and oxidation resistance and has been applied in the food, preservation, pharmaceutical packaging, cosmetics, and petroleum industries because it has good plasticity, as well as film-forming and gas barrier properties.^[8,9] It also has other promising applications, but it has not been produced on a large scale because melanin generated during A. pullulans fermentation firmly adheres to pullulan and is difficult to be removed. In polysaccharide decolorization, the product recovery rate is greatly reduced, and the cost is high. The retained melanin affects the color and yield of products; therefore, it must be removed.

Bacterial strains can be manipulated by physical and chemical means, complex mutagenesis, gene shuffling, or genetic engineering to obtain high-yield or low-pigment strains. For example, Imshenetskiĭ and Kondrat'eva treated wild Pullularia pullulans strains with 0.3% colchicine, and the amount of pullulan produced by a mutant strain was increased by 7–8 mg/l.^[10] Szymańska and Galas irradiated wild P. pullulans strains with ultraviolet to obtain two mutant strains, namely, M-u1 and M-u2, which reduced pigment and increased polysaccharide yields to 10 g/L.^[11] Kang et al. generated the F3-2 strain through genome shuffling the A. pullulans N3.387 strain, and its pullulan yield reached 20.7 g/L.^[12] Ma et al. knocked out the pullulan synthase gene of the original HN6.2 strain to increase polysaccharide yield.^[13] However, the development of the pullulan industry is hindered by many technical problems, such as melanin secretion and low pullulan yield. These technical issues can be solved in several ways, such as medium optimization, fermentation condition control, and mutation breeding. Among these, mutation breeding is the most economical selection.^[14] Mutagenic strains with a high polysaccharide yield and low pigment production have rarely been reported, and polysaccharide extraction and applications have not been studied in details.

The present study aims to promote in-depth research and practical applications of extracellular polysaccharides generated by A. pullulans by optimizing the fermentation conditions of less pigmented polysaccharides and identifying the properties of the purified polysaccharide films.

 

2. Material and methods

2.1 Materials

The following materials were used in this study: HBB (Tibetan Crystal Barley Food Co., Ltd., Tibetan, China); A. pullulans As.3.837 strain (Guangdong Provincial Fungus Preservation Center, Guangdong, China); Congo red (Shanghai Yuming Industry Co., Ltd., Shanghai, China); total starch (AA/AMG) assay kits (Megazyme International lreland Ltd., Bray, Ireland); and other reagents (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China).

2.2 Preparation of seed and fermentation broth

Powdered HBB was mixed with distilled water to prepare a turbid fermentation broth. The seed liquid medium comprised 1000 mL of distilled water containing sucrose 50 g, dipotassium hydrogen phosphate 5 g, sodium chloride 1 g magnesium sulfate heptahydrate, 0.2 g, ammonium sulfate 0.6 g and yeast extract 3 g. Flat medium comprised 1000 mL of H₂O containing potato 200 g, glucose 20 g and agar 20 g at pH 6.5.

These media were sterilized at 121 °C for 30 min using a YXQ-LS-70A vertical pressure steam sterilization pot (Shanghai Boxun Medical and Instrument Corp., Shanghai, China). A single-cell suspension (1 mL) of A. pullulans at the logarithmic growth phase was diluted 1,000-fold and added to sterile culture dishes. Flat medium was inoculated with bacterial solution (0.1 mL) via a coating method, stirred for 1 min under a 15 Watt UV light located 40 cm from the sample on an SW-CJ-1G clean bench (Purification equipment Corporation, Suzhou, China), then incubated at 28 °C for 72 h in a GHP-9050 Water-Jacketed Thermostatic Incubator (Qixin Scientific Instruments Co., Ltd., Shanghai, China). Three rings of A. pullulans at the logarithmic growth stage were inoculated into seed liquid medium and incubated at 28 °C for 36 h to prepare bacterial suspensions.

2.3 Determination of HBB component content

Total fat, total protein, moisture, and β-glucan in HBB were determined using GB/T 5009.6-2015, GB/T 5009.5-2016, GB 5497-1985, and Congo red methods. The total starch content was evaluated using Total Starch HK test kits (Megazyme).

2.4 Optimization of fermentation conditions

2.4.1 Standard curves of β-glucan and pullulan

Phosphate buffer solution (0.1 mol/L, and pH 8.0) was prepared, and then Congo red solution (0.1 mg/mL) was configured with this solution.

Standard curve for β-glucan: Standard β-glucan (10 mg) was dissolved in deionized water (100 mL) in a volumetric flask then 1 mL of standard β-glucan solution was diluted to 2 mL with deionized water and reacted with Congo red (4 mL) at 20 °C for 10 min in darkness (the blank: deionized water (2 mL) and Congo red (4 mL). The maximum absorption at 545 nm in a wavelength range of 200–700 nm was measured using an Evolution 220 scanning ultraviolet spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Solutions with specific graded concentrations were prepared similarly. Absorbance was measured using an Enspire microplate reader (PerkinElmer Life and Analytical Sciences Inc., Waltham, MA, USA) at the maximum absorption wavelength. Concentration was the abscissa (X), and absorbance was the ordinate (Y) to draw the standard curve. The regression equation of the standard curve was Y = 0.0103X + 0.0075 (R² = 0.9972; linear range: 0–31.25 μg/mL).

Standard curve for pullulan: The QBT 4485-013 Pullulan method was the reference. Concentration was the abscissa (X), and absorbance was the ordinate (Y) to draw the standard curve. The regression equation of the standard curve was Y = 3×10⁷X − 2×10⁶ (R² = 0.9931; linear range: 0–1.00 mg/mL).

2.4.2 Single-factor test

Fermentation broth (30, 50, 100, and 150 mL) at ratios of 1:10, 1:15, 1:20, and 1:25 was placed in 250-mL Erlenmeyer flasks and sterilized at 121°C for 15 min. The initial pH of the broth mixed with HBB and distilled water was adjusted to 5.5, 6, 7, 8, and 8.5, and the inoculum volume was 2 mL. The mixture was disrupted at 200 rpm using a DDHZ-300 shaker (Suzhou Peiying Experiment Equipment Ltd., Co., Suzhou, China) at a constant fermentation temperature of 28 °C for 1, 2, 3, 4, and 5 days.

Cells in the fermentation broth were pelleted by centrifugation at 4,000 rpm for 10 min. The supernatant mixed with absolute ethanol (2 volumes) was passed through 0.22-µm filters and incubated at 4 °C overnight to precipitate polysaccharides. Polysaccharide precipitate was obtained, and then residual crude polysaccharides were sedimented by centrifugation at 4000 rpm for 5 min and then the two parts of polysaccharides were dried at 50 °C. The absorbance of β-glucan was measured using a TU-1900 UV-visible spectrophotometer (Beijing Puxi General Instrument Co., Ltd., Beijing, China), and the peak area of pullulan was measured with an UltiMate-3000 HPLC (Thermo Fisher Scientific Inc.) in crude polysaccharide solution diluted with deionized water.

2.5 Physicochemical properties of polysaccharides before and after fermentation

2.5.1 Purification of polysaccharide

Polysaccharides from bran before fermentation and fermented broth were purified, respectively. After removing the starch and protein using the Sevag method, and salting out method, ^[15] the polysaccharides were lyophilized and concentrated in vacuo.

2.5.2 Scanning electron microscopy (SEM)

Pre- and post- fermented bran, pre-fermented (from unfermented bran), and post-fermented (from fermented broth) polysaccharides were dried, and their morphology was assessed using a 160 S-3000N SEM (Hitachi Ltd., Tokyo, Japan). Samples were flattened with double-sided tape, then sputter-coated with gold. Samples were magnified ×1000 at an accelerated voltage of 15 kV.^[16]

2.5.3 Fourier transform infrared spectroscopy

The infrared absorption spectra of samples were determined in the range of 650–4000 cm⁻¹ using a Nexus 670 Thermo Nicolet Fourier Transform Infrared Spectrometer (Thermo Fisher Scientific Inc.) under the following conditions: resolution, 4 cm⁻¹; beam precision, 0.01 cm, number of scans, 16 times; ambient temperature, 25 °C.^[17]

2.5.4 Rheological properties

Polysaccharides (1.0%, 1.5% and 2.0%) were stirred, heated at 60 °C and completely dissolved in distilled water, then viscosity was measured as shear rates using a Kinexus Pro+ rheometer (NETZSCH-Gerätebau GmbH., Selb, Germany). The test conditions were as follows: parallel plate clamp, 40 mm; gap setting, 150 μm; test temperature, 25 °C; and shear rate range, 1–1000 s^-1. Solutions of polysaccharide (1.0%, 2.0%, 2.5%, and 3.0%) were prepared as described above. The dynamic viscoelasticity of each polysaccharide solution was measured as angular frequency using a rheometer under the following test conditions: parallel plate clamp, 40 mm; gap setting, 1 mm; test temperature, 25 °C; angular frequency range, 0.1–100 r/s.^[8,18]

2.6 Properties of polysaccharide films

2.6.1 Preparation of polysaccharide film

Experimental (pre- or post- fermented polysaccharide (0.3 g) with glycerol (0.1 g)) and control (β-glucan (0.08 g), pullulan (0.22 g), and glycerol (0.1 g)) groups were dissolved in 20 mL of distilled water, then polysaccharide films were prepared as described by Chang et al.^[19]

2.6.2 Oxygen barrier properties (OBP)

The conical flasks containing 3 g of vegetable oil were sealed with a film and set in a thermostat at 50 °C for 5 days. The OBP (g/100g) was expressed by the peroxide value of the oil and was calculated according to the method of Chang.^[19] The calculated formula was as follows:

where 0.1269 is mass of iodine equivalent to 1mL sodium thiosulfate standard titration solution; V represents the volume of sodium thiosulfate consumed by the sample (mL); V₀ represents the volume of sodium thiosulfate consumed by the blank sample (mL); C means concentration of sodium thiosulfate standard solution (mol/L) and M is the mass of the sample (g).

2.6.3 Water vapor transmission (WVT)

The WVT (g/m²·d) of the films was measured in accordance with method of Chang.^[19] The calculated formula was as follows:

where ΔM (g) represents the increased mass of the desiccant after 24 hours; A (m²) means the area of water vapor permeation; T (d) means the interval time.

2.6.4 Tensile strength (TS)

Parameter was measured using a TA.XT2i instrument (Testometric, S.M.S. Corporation, USA). The TS (MPa) of films was measured in accordance with the method of Chang.^[19] Three samples (width of 10 mm and length of 20 mm) cut from each film were evaluated. Test conditions were set as follows: the measurement speed, 10 mm/min; the measurement speed, 1 mm/min; the initial grip interval, 26 mm; the force, 5 g. The calculated formula was as follows:

in which, F means the maximum force (N); A represents the cross-sectional area of the sample film (mm²).

2.6.5 Water dissolution time (WDT)

Film samples (15 mm wide and 15 mm long) were set in boiling water and the time for the films to completely dissolve was recorded.^[19]

 

Fig. 2b shows that the amount of polysaccharide initially increased then decreased at pH 5.5–8.5. More β-glucans were produced when the initial pH of the fermentation broth was < 6.0, but the pullulan yield was very low. Pullulan production was maximal when the initial pH of the fermentation broth was 7.0. Thus, the initial pH should be 7.0 to ensure the total yield of polysaccharides.

3.3.3 Effects of liquid volume on polysaccharide extraction

Aureobasidium pullulans is an aerobic bacterium, and the volume of liquid affects the contents of dissolved oxygen and substrate. Pullulan production was initially increased, then decreased when liquid volumes ranged from 30 to 150 mL (Fig. 2c). The amount of β-glucan was increased initially, then decreased, and increased again. The value was maximal when the volume was 50 mL. The production of β-glucan decreased substantially at 100 mL because it was utilized by A. pullulans in the presence of oxygen under this condition. Very little polysaccharide was produced, because the amount of liquid and substrates that could be utilized by A. pullulans was insufficient. As the liquid volume was increased, the amount of substrate and dissolved oxygen became sufficient to promote pullulan production. When the volume of liquid was increased again, the decreased amount of dissolved oxygen per unit volume resulted in the ability of A. pullulans to be inhibited.

3.3.4 Effects of duration of fermentation on amount of extracted polysaccharide

The strain started to grow and proliferate at the beginning of fermentation when the amount of substrate was sufficient, and total polysaccharides were increased, reaching the maximum on day 3 (Fig. 2d). The amount of produced polysaccharide was gradually decreased over time, perhaps due to a nutrient deficiency and the breakdown of polysaccharides by bacterial enzymes.^[24]

3.4 Physicochemical properties of polysaccharides before and after fermentation

3.4.1 Scanning electron microscopy

Bran particles became finer with more diamond-shaped fragments after fermentation (Fig. 3a and b). These findings (bran particles with more diamond-shaped fragments) indicated that A. pullulans secreted a large amount of enzymes to degrade and utilize the bran during fermentation. Nordlund found using microscopy that the structures of stained cell walls in the aleurone layer of fermented rye bran are almost completely degraded and the cell contents are largely released.^[25]