Application of raw starch degrading enzyme from Laceyella sacchari LP175 for development of bacterial cellulose fermentation using colored rice as substrate

Abstract

Brown and black rice substrates were applied for sugar syrup production by the hydrolysis of raw starch degrading enzyme (RSDE) from Laceyella sacchari LP175 (300 U/mL) and commercial glucoamylase (GA, 2.0 U/mL) at 50 °C for 12 h using a simplex centroid mixture design. Results indicated that 300 g/L of substrates, consisting of 255 g/L Leum Pua glutinous rice and 45 g/L Black Jasmine rice, gave the highest sugar syrup production at 124.6 ± 2.52 g/L with 2.00 ± 0.05 mg GAE/mL of total phenolic content (TPC), equivalent to 0.42 ± 0.01 g/g rice sample and 6.67 ± 0.15 mg GAE/g rice sample, respectively. The obtained sugar syrup was used as the substrate for production of bacterial cellulose (Nata) by Komagataeibacter xylinus AGR 60 in a plastic tray at room temperature for 9 days. The fermentation medium containing 200 mL of rice syrup (25 g/L), 2.0 g of ammonium sulfate [(NH₄)₂SO₄] and 0.4 mL glacial acetic acid yielded 1.1 ± 0.08 cm thickness with 8.15 ± 0.12 g of dry weight. The obtained bacterial cellulose from colored rice was characterized compared with bacterial cellulose from the conventional coconut juice by scanning electron microscope (SEM) and Fourier-transform infrared spectroscopy (FTIR) which demonstrated that the sugar syrup from colored rice could use as substrate for a novel bacterial cellulose as a healthy product in the future through microbial enzyme technological process.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-02673-3.

Introduction

Brown and black rice have attracted significant attention as healthy raw materials for foods, cosmetic ingredients and various biotechnological products in Thailand. Brown and black rice have high nutrition value from contained antioxidants and bioactive compounds compared to traditional white rice (Leardkamolkarn et al. 2011; Pornputtapitak et al. 2018). When compared with white rice, brown and black rice showed higher contents of fiber, nutrients and starch for production of various healthy products (Leardkamolkarn et al. 2011; Phuapaiboon 2017). Utilization of brown and black rice for production of healthy food products has been reported in various publications for noodles (Sirichokworrakit et al. 2015), Kao-Taen (Payim 2016), lip gloss (Vutdhipapornkul et al. 2014) and vinegar (Phuapaiboon 2017; Sangngern et al. 2020).

Nata or bacterial cellulose (BC) (C₆H₁₀O₅)_n is a water-insoluble polysaccharide, derived from the fermentation by Komagataeibacter xylinus (Acetobacter xylinum) (Jagannath et al. 2010; Campano et al. 2016). Nata contains β-1,4 linkages between two glucose molecules, similar to cellulose in plants but with different physical and chemical features (Shoda and Sugano 2005). Applications or potential applications of these different characteristics and properties of Nata were reported in various fields including foods, medicines and cosmetics (Fu et al. 2013; Shi et al. 2014; Esa et al. 2014; Pourali and Yahyaei 2019). Production of Nata or bacterial cellulose was developed with various substrates to obtain products with new characteristics and functions such orange, pineapple, apple, Japanese pear, grape, pomegranate and watermelon (Kurosumi et al. 2009; Hungund et al. 2013) but has not yet been reported from Thai rice.

The cold hydrolysis process hydrolyzes starch-based substrates at low temperature (40–50 °C) compared to conventional methods at high temperature (90–105 °C) (Cinelli et al. 2015) which reduces starch industry energy consumption and operational costs (Cinelli et al. 2015; Lomthong et al. 2016). Raw starch degrading enzyme (RSDE) contains raw starch binding sites (SBDs) in the structure, which adsorbs to the surface of granules under gelatinization temperature. Lomthong et al. (2018) that RSDE showed higher efficiency for hydrolysis of raw cassava starch than commercial α-amylase (Termamyl^®) at 50 °C. Recently, cold hydrolysis by RSDE has been reported for various kinds of fermentation products including bioethanol (Lomthong et al. 2016), lactic acid (Okano et al. 2018) and vinegar (Lomthong and Saithong 2019) but scant information exists for bio-cellulose fermentation. The thermophilic filamentous bacterium Laceyella sacchari LP175 has proved a potent strain for production of RSDE. The RSDE produced from L. sacchari LP175 has been applied for sugar syrup production of cassava chips at 50 °C for 12 h and used as a substrate for bioethanol fermentation (Lomthong et al. 2016). In this study, the RSDE produced from L. sacchari LP175 was utilized to produce sugar syrup from brown and black rice.

Statistical mixture designs were used to investigate appropriate combinations of substrates to give maximum target production yield (de Castro and Sato 2013). Statistical mixture design is attractive because it reduces the research time required and number of experiments (Lomthong et al. 2020). Therefore, this study aimed to maximize sugar syrup production from brown and black rice using RSDE produced by L. sacchari LP175 by a simplex centroid mixture design. The optimum medium for bacterial cellulose fermentation by K. xylinus AGR 60 was also investigated.

Materials and methods

Substrates and chemical composition analyses

Five varieties of brown and black rice (Riceberry rice, Leum Pua glutinous rice, Black Jasmin rice, Sangyod rice and Tubtim Chumphae rice) were purchased from a local fresh market (Ying Charoen Market), Bangkok, Thailand. Each substrate was ground to powder using an electric grinder and stored at dry condition until required for use. Total starch assay (Megazyme International, Wicklow, Ireland) was performed by the Cassava and Starch Technology Research Unit (CSTRU) at Kasetsart University. Protein, fat and fiber contents were determined following the methods of the Association of Official Analytical Chemists (AOAC) (Helrich 1990).

Microorganism and enzyme preparation

Raw starch degrading enzyme (RSDE) was produced by the thermophilic filamentous bacterium L. sacchari LP175 (TISTR 2280) and cultivated in an optimized medium consisting of 4.93 g/L cassava starch, 2.8 g/L yeast extract, 0.5 g/L dipotassium hydrogen phosphate (K₂HPO₄), 0.5 g/L magnesium sulfate heptahydrate (MgSO₄.7H₂O), and 1.0 g/L calcium chloride (CaCl₂) (pH 6.5) (Lomthong et al. 2015). Fermentation was conducted in a 3.0 L airlift fermenter at 45 °C with aeration rate of 0.5 vvm for 36 h (Lomthong et al. 2016). RSDE activity was determined according to Mitsuiki et al. (2005) by analyzing the reducing sugars released during hydrolysis of raw cassava starch, as previously described by Lomthong et al. (2015). One unit of RSDE activity is defined as the amount of enzyme releasing 1 µg of glucose equivalent per min under standard assay conditions.

Commercial glucoamylase (GA) was obtained from the Reach Biotechnology Co., Ltd., Thailand and stored at − 20 °C until required for use. GA activity was determined by analyzing liberated glucose measured by the glucose oxidase method (Kingsley and Getchell 1969). One unit of GA activity is defined as the amount of enzyme releasing 1 µg of glucose per minute under standard assay conditions.

Bacterial cellulose producing strain K. xylinus AGR 60, obtained from the Institute of Food Research and Product Development (IFRPD), Kasetsart University, Thailand was used for bacterial cellulose (Nata) fermentation. For inoculum preparation, The AGR 60 strain was grown in a 750 mL glass bottle containing sterilized coconut juice (500 mL) with 1.0% ammonium sulfate [(NH₄)₂SO₄] supplementation. The inoculum was incubated at room temperature for a week, and 10% of the culture volume was used for experimental medium fermentation.

Hydrolysis of brown and black rice with raw starch degrading enzyme produced from L. sacchari LP175

Each 300 g/L of brown and black rice powder was hydrolyzed by RSDE produced from L. sacchari LP175 in a 250 mL Erlenmeyer flask containing 100 mL of 300 U/mL RSDE activity with 20 U/mL of commercial glucoamylase in 0.1 M phosphate buffer (pH 6.5) (Lomthong et al. 2016). All experiments were incubated at 50 °C for 12 h without the shaking condition. Samples were withdrawn at 6 and 12 h for determination of reducing sugars and total phenolic content (TPC). Three of the five substrates that showed higher reducing sugars and total phenolic compounds were selected for the statistical mixture design experiments. The 3,5-dinitrosalicylic acid (DNS) method (Miller 1959) was used to determine the amount of reducing sugars released from the hydrolysis of broken rice powder at 540 nm. TPC was determined using the Folin-Ciocalteu method as described by Butsat and Siriamornpun (2010). A 200 mL sample was added with 1.0 mL of diluted (1:10 with distilled water) Folin-Ciocalteu reagent, after mixing with 800 µL of sodium carbonate (Na₂CO₃), and final volume was made up to 5 mL with distilled water. After 2 h of reaction, absorbance at 760 nm was determined. Gallic acid was used as a standard, with results calculated as mg gallic acid equivalents per mL (mg GAE/mL) and mg gallic acid equivalents per g rice sample (mg GAE/g rice sample).

To confirm RSDE hydrolysis ability at low temperature from L. sacchari LP175, both native and digested brown and black rice powders were examined by scanning electron microscope (SEM). All native rice powders and digested samples were washed with distilled water after hydrolysis at 50 °C for 12 h, dried at 50 °C overnight, and then examined under SEM (model SU8020; Hitachi, Tokyo, Japan) at 10.0 kV.

Hydrolyzed products of brown and black rice were qualitatively detected by thin-layer chromatography (TLC) on silica gel chromatography plates (Merck, 20 × 20 cm) with a solvent mixture of ethyl acetate/2-propanol/glacial acetic acid/water (4:2:2:1, v/v) as described by Sassaki et al. (2008) and Lomthong et al. (2016). TLC plates were run and developed by spraying with a solution of orcinol-sulfuric acid and heating at 100 °C for 10 min. Glucose and maltose were used as standards.

Statistical mixture design for sugar syrup production

A simplex centroid mixture design was used to investigate the optimum combination of each substrate that maximized liberated reducing sugars and total phenolic content, following the method of de Castro and Sato (2013) and Lomthong et al. (2020). In total, seven mixture design experiments using the three selected brown and black rice powders were set at four levels, with different percentages as 0 (0%), 1/3 (33%), 1/2 (50%) and 1 (100%). The experiments were optimized in 250 mL Erlenmeyer flasks containing 100 mL of 300 U/mL RSDE activity with 20 U/mL of commercial GA in 0.1 M phosphate buffer (pH 6.5) as described above and incubated at 50 °C for 12 h. Amounts of liberated reducing sugars and total phenolic compounds were determined for each run. A mixture model was then constructed (de Castro and Sato, 2013), and represented as Eq. (1):

$${\text{Y}}_{\text{i}}={\beta }_{\text{i}}{X}_{\text{i}}+{\beta }_{\text{ij}}{X}_{\text{i}}{X}_{\text{j}}+{\beta }_{\text{ijk}}{X}_{\text{i}}{X}_{\text{j}}{X}_{\text{k}}$$ 1

where Y_i is the predicted response, β_i is the regression coefficient for each linear effect term, β_ij and β_ijk are binary and ternary interaction effect terms, and X_i, X_j and X_k are the coded independent variables. Design-Expert^® Version 12 Software (Stat-Ease, USA) and Statistica^™ 10 for Windows were employed for experimental design, data analysis, and model building.

Production of rice sugar syrup in a 5.0 L glass jar

To upscale sugar syrup production, optimum rice combination was applied in a 5.0 L glass jar chamber (18 × 18 × 28 cm) using a 3.0 L working volume of the optimum rice suspension. The reaction was incubated at 50 °C for 12 h without shaking. Samples were taken at interval times to determine reducing sugars and TPC. The obtained sugar syrup at the end of incubation was used as substrate for bacterial cellulose fermentation.

Bacterial cellulose fermentation

The obtained sugar syrup from hydrolysis by RSDE produced from L. sacchari LP175 and commercial GA was used as substrate for bacterial cellulose fermentation with K. xylinus AGR 60. Fermentation was modified from the method of Jagannath et al. (2008). A total volume of 200 mL of rice syrup at different concentrations (5–50 g/L) was supplemented with 2.0 g (NH₄)₂SO₄ and 0.4 mL glacial acetic acid. Fermentation was carried out in a plastic tray (15 × 25 × 6 cm) with 200 mL of working volume, incubated at room temperature for 9 days. Coconut juice was used as a control experiment by measuring the thickness of each sample. Obtained bacterial cellulose samples were harvested, washed repeatedly with water to remove glacial acetic acid, and determined for thickness and TPC content. Dry weight of bacterial cellulose samples was determined after drying at 50 °C for 24 h. A scanning electron microscope (SEM) was used to determine the structure of freeze-dried cellulose as described above. The crystallinity of dried BC samples was measured by X-ray Diffraction (XRD), collected on a Bruker D8 Advance diffractometer operating in the reflection mode with Cu-Kα radiation at 40 kV and 30 mA. Data were collected in reflection mode in the 5–60° 2θ-range and 0.5 s per step (Moukamnerd et al. 2020; Saleh et al. 2020). The crystallinity index (CI) was calculated using the Segal’s equation (Segal et al. 1959). The Fourier-Transform Infrared Spectroscopy (FTIR) was used to determine the functional groups and chemical bonds of the obtained bacterial cellulose samples. Bacterial cellulose samples were dried at 50 °C for 24 h and cut into 1 × 1 cm pieces, before subjecting to FTIR (Thermo Scientific Nicolet is5, USA). For each sample, the scanning range was 4000 to 400 cm⁻¹ (Saleh et al. 2020).

Statistical analysis

All data were analyzed using SPSS for Windows version 20 (SPSS Inc., USA). Differences among the mean values were tested using the least significant difference multiple range test and significance was taken at the p < 0.05 level.

Results and discussion

Hydrolysis of brown and black rice with raw starch degrading enzyme produced from L. sacchari LP175

Five varieties of brown and black rice including Riceberry rice, Leum Pua glutinous rice, Black Jasmin rice, Sangyod rice and Tubtim Chumphae rice were chosen for this study because the brown and black color seeds are commonly found in various markets in Thailand. The chemical compositions of brown and black rice including starch, protein, fat and fiber contents are shown in Table 1. Luem Pua glutinous rice contained the highest starch content at 86.55 ± 0.16%, while Sangyod, Tubtim Chumphae, Black Jasmine and Riceberry recorded 73.60 ± 0.49, 70.49 ± 0.18, 69.38 ± 0.02 and 68.62 ± 0.22%, respectively. Ito and Lacerda (2019) reported that colored rice contained starch content in the range 73.9–87.5% as a good source of carbohydrate for various biotechnological applications. Protein and fat contents of brown and black rice in this study varied in the range of 9.17 ± 0.05 to 11.19 ± 0.01% and 2.69 ± 0.01 to 6.38 ± 0.01%, respectively while fiber contents were found in the range of 1.19 ± 0.00 to 1.83 ± 0.01. Sompong et al. (2011) reported the chemical compositions of colored rice from Thailand, China and Sri Lanka with protein and fat contents in the range of 8.17 ± 0.41 to 10.85 ± 0.09% and 1.15 ± 0.03 to 3.72 ± 0.06%, respectively. RSDE from L. sacchari LP175 showed synergistic hydrolysis with commercial GA at 50 °C, and less energy required than the conventional starch hydrolysis process in the liquefaction step for gelatinization at 80–105 °C (Lomthong et al. 2016). Hydrolysis results of each rice powder are shown in Table 2. Results revealed that Riceberry, Black Jasmine and Luem Pua glutinous rice gave maximum liberated reducing sugars at 110.50 ± 1.05, 124.16 ± 3.90 and 124.39 ± 3.80 g/L, respectively, equivalent to 0.37 ± 0.01, 0.41 ± 0.01 and 0.41 ± 0.01 g/g rice sample, respectively and higher than Sangyod rice at 90.00 ± 3.80 g/L (0.30 ± 0.01 g/g rice sample) and Tubtim Chumphae rice at 98.90 ± 1.00 g/L (0.33 ± 0.01 g/g rice sample) (Table 2). Sangyod rice and Tubtim Chumphae rice contained higher starch content than Riceberry and Black Jasmine rice but lower sugar contents due to different chains forming in the starch structure, such as linear and branched macromolecules that affected the hydrolysis ability of the enzyme (Lomthong et al. 2015; Ito and Lacerda 2019). To compare with other studies, Photphisutthiphong and Vatanyoopaisarn (2020) hydrolyzed cooked colored rice including Homnil rice, Riceberry rice, jasmine rice, brown jasmine rice and red jasmine rice by commercial α-amylase at 90 °C, yielding 91.7 ± 0.670 g/L, 87.34 ± 0.609, 85.83 ± 0.168, 85.61 ± 0.405 and 74.79 ± 0.842 g/L, respectively. RSDE from L. sacchari LP175 functioned as α-amylase which randomly hydrolyzed starch granules to various oligosaccharides, while commercial GA continued to degrade oligosaccharides to glucose (Lomthong et al. 2015). In case of TPC, the Riceberry, Black Jasmine and Luem Pua glutinous rice gave maximum TPC at 1.68 ± 0.10, 1.96 ± 0.10 and 1.90 ± 0.10 mg GAE/mL, respectively as shown in Table 2, equivalent to 5.61 ± 0.25, 6.53 ± 0.35 and 6.33 ± 0.33 mg GAE/g rice sample respectively. From previous studies, various phenolic compounds in rice and cereal grains were reported such as phenolic acid, tannins and flavonoids as anthocyanins, flavanols and flavanones (Dykes and Rooney 2007). The TPC content represented the amount of phenolic contents in the samples which covered the anthocyanin or phenolic acid as described above; therefore, the TPC content was used as one of the responses in the statistical mixture design experiment. Pang et al. (2018) reported TPC from black rice in the range 1.16–3.32 mg GAE/g rice, while Pramai and Jiamyangyuen (2016) reported that colored rice contained TPC content in the range 1.38–6.34 mg GAE/g rice. Hydrolysis at low temperature protects some natural substrates such as vitamins and bioactive compounds compared to the conventional process that requires boiling or steaming the substrate before hydrolysis and destroys almost all the beneficial nutrients in the natural substrates. Therefore, Riceberry, Black Jasmine and Luem Pua glutinous rice were used for the statistical mixture design experiment.

Table 1.

Chemical compositions of brown and black rice

Rice	Component
	Starch (%)	Protein (%)	Fat (%)	Fiber (%)
Riceberry	68.62 ± 0.22a	9.26 ± 0.02b	6.38 ± 0.01e	1.83 ± 0.01d
Tubtim Chumphae	70.49 ± 0.18c	11.19 ± 0.01e	4.22 ± 0.02c	1.59 ± 0.01c
Sangyod	73.60 ± 0.49d	9.72 ± 0.06c	3.76 ± 0.01b	1.34 ± 0.00b
Black Jasmine	69.38 ± 0.02b	10.18 ± 0.03d	4.55 ± 0.03d	1.59 ± 0.03c
Leum Pua	86.55 ± 0.16e	9.17 ± 0.05 a	2.69 ± 0.01a	1.19 ± 0.00a

Values are averages of three determinations

Table 2.

Reducing sugar and TPC from hydrolysis of brown and black rice by RSDE from L. sacchari LP175 and commercial GA at 50 °C for 6 and 12 h

Rice	Reducing sugars (g/L)		TPC (mg GAE/mL)
	6 h	12 h	6 h	12 h
Riceberry	72.50 ± 0.30b	110.50 ± 1.05c	1.64 ± 0.09c	1.68 ± 0.10c
Tubtim Chumphae	65.50 ± 0.00a	98.90 ± 1.00b	1.21 ± 0.04a	1.53 ± 0.08a
Sangyod	65.62 ± 2.40a	90.00 ± 3.80a	1.31 ± 0.02b	1.64 ± 0.06b
Black Jasmine	81.90 ± 2.10c	124.16 ± 3.90d	1.75 ± 0.11d	1.96 ± 0.10d
Leum Pua	85.35 ± 3.50d	124.39 ± 3.80e	1.70 ± 0.13e	1.90 ± 0.10d

Values are averages of three determinations

Different letters within the same column are statistically different at p < 0.05

Scanning electron micrographs confirmed that RSDE from L. sacchari LP175 and commercial GA could hydrolyze all brown and black rice powders at low temperature (Fig. 1). RSDE from L. sacchari LP175 had the ability to hydrolyze starch granules at low temperature from the surface to the center of the granules by the function of starch binding domain (Lomthong et al. 2018). Degraded starch granules were loosened from the structure, forming pits and deep holes on the surface as shown in Fig. 1. The TLC chromatogram confirmed that the hydrolysis product from the reaction was glucose (Supplementary Figure 1) and appropriate for use as a fermentable sugar to produce various kinds of fermentation products.

Fig. 1.

Scanning electron micrographs of brown and black rice before and after hydrolysis by RSDE from L. sacchari LP175 at 50 °C for 12 h.: a Riceberry, b Tubtim Chumphae, c Sangyod, d Black Jasmine and e Leum Pua

Statistical mixture design for sugar syrup and TPC production

Using the one factor at a time method, the three selected substrates were applied for a statistical mixture design experiment which contained seven runs as described above. Results of mixture design for each substrate and combination of substrates for sugar syrup production and TPC are shown in Table 3. Reduced sugar syrup production at 109.87 ± 1.80 g/L (0.37 ± 0.01 g/g rice sample) was obtained when Riceberry rice powder was used as the individual substrate, while individual Black Jasmine and Luem Pua glutinous rice enabled sugar syrup production at 124.30 ± 2.14 and 124.56 ± 2.95 g/L, equivalent to 0.41 ± 0.01 and 0.42 ± 0.01 g/g rice sample, respectively.

Table 3.

Matrix mixture design results for sugars syrup and TPC at 12 h of incubation

Run	Coded level			Uncoded level (g/L)
	Riceberry (X1)	Black Jasmine (X2)	Luem Pua (X3)	Riceberry (X1)	Black Jasmine (X2)	Luem Pua (X3)	12 h
							Reducing sugars (g/L)	TPC (mg GAE/mL)
1	1	0	0	300	0	0	109.87 ± 1.80	1.62 ± 0.05
2	0	1	0	0	300	0	124.30 ± 2.14	1.96 ± 0.10
3	0	0	1	0	0	300	124.56 ± 2.95	1.92 ± 0.13
4	1/2	1/2	0	150	150	0	117.43 ± 0.80	1.69 ± 0.07
5	1/2	0	½	150	0	150	117.85 ± 3.96	1.79 ± 0.15
6	0	1/2	½	0	150	150	124.50 ± 3.42	1.99 ± 0.20
7	1/3	1/3	1/3	100	100	100	119.76 ± 5.60	1.83 ± 0.06

Values are averages of three determinations

Contour plots of sugar syrup production and TPC for the three substrates after hydrolysis with RSDE and commercial GA at 50 °C for 12 h are shown in Fig. 2. Maximum sugar syrup production was found at the corner of one side of the triangle, between Luem Pua glutinous rice and Black Jasmine rice (Fig. 2a). Similar to the contour plot of TPC (Fig. 2b), maximum TPC was found at one side of the triangle between Luem Pua glutinous rice and Black Jasmine rice. Our results indicated that Luem Pua glutinous rice had a major effect on sugar syrup production with a small amount of Black Jasmine rice and without Riceberry rice powder.

Fig. 2.

Contour plots of reducing sugar and total phenolic content (TPC) of brown and black rice combinations after hydrolysis by RSDE from L. sacchari LP175 at 50 °C for 12 h. a reducing sugar; b TPC

The response variables of sugar syrup and TPC production were analyzed using multiple regression analysis to determine the fitted model. The quadratic model was the best-fitted model for sugar syrup and TPC production. The models had coefficients of determination (R²) of 0.9997 and 0.9995 for sugar syrup and TPC production, respectively while adjusted R² values were 0.9983 and 0.9968, respectively. de Castro and Sato (2013) proposed that coefficients of determination (R²) values higher than 0.90 indicated that all response functions adequately fitted the experimental data. The ANOVA results of models for sugar syrup and TPC production are shown in Supplementary Tables 1 and 2. Results indicated that these quadratic models were significant. The predicted equations of sugars syrup and TPC after 12 h of hydrolysis with RSDE and commercial GA at 50 °C are represented as Eqs. (2), (3):

$$Y_1=109.89X_1+124.32X_2+124.58X_3+0.9936X_1{X}_2+2.15X_1{X}_3-0.1064X_2{X}_3$$ 2

$$Y_2=1.62X_1+1.96X_2+1.92X_3-0.3864X_1{X}_2+0.0936X_1{X}_3+0.2136X_2{X}_3$$ 3

where Y₁ and Y₂ are the predicted responses of sugars syrup production and TPC content, and X₁, X₂ and X₃ represent Riceberry rice, Black Jasmine rice and Luem Pua glutinous rice, respectively.

The predicted Eq. (2) indicated that a total of 300 g/L Leum Pua glutinous rice without Riceberry rice and Black Jasmine rice gave the highest predicted value of sugar syrup production at 124.6 g/L, while the polynomial Eq. (3) indicated that 255 g Leum Pua glutinous rice and 45 g Black Jasmine rice gave the highest predicted value of TPC content, yielding 1.95 mg GAE/mL. Validation of the models in predicted Eqs. (2), (3) was performed under the same conditions in 250 mL Erlenmeyer flasks. Results found that 300 g/L Leum Pua glutinous rice gave yield of sugar syrup at 124.5 ± 3.28 g/L with 1.90 ± 0.1 mg GAE/mL of TPC content, equivalent to 0.42 ± 0.01 g/g rice sample and 6.33 ± 0.33 mg GAE/g rice sample, respectively. The ratio of 255 g Leum Pua glutinous rice and 45 g Black Jasmine rice gave reducing sugar at 124.6 ± 2.52 g/L and TPC content of 2.00 ± 0.05 mg GAE/mL, equivalent to 0.42 ± 0.01 g/g rice sample and 6.67 ± 0.15 mg GAE/g rice sample of sugar syrup and TPC, respectively. Validation results corresponded to the predicted value of mixture design. This demonstrated that the application of statistical mixture designs using different brown and black rice accurately fitted the production of sugar syrup and TPC by hydrolysis of RSDE from L. sacchari LP175 and commercial GA at low temperature. However, the experiment was repeated as this model varied depending on conditions of substrates, such as starch composition.

Results revealed that both conditions gave similar predicted values of sugar syrup production and TPC content. However, the combination of 255 g Leum Pua glutinous rice and 45 g Black Jasmine rice was chosen for production in a 5.0 L glass jar reactor due to the result of sensory evaluation on color of syrup products (data not shown), which gave attractive and innovative products from the combination of two rice substrates.

Production of rice sugar syrup in a 5.0 L glass jar

To upscale for production of sugar syrup and TPC, the optimum combination of Leum Pua glutinous rice and Black Jasmine rice was performed in a 5.0 L glass jar reactor as described above. Time courses of sugar syrup and TPC are shown in Supplementary Figure 2. Maximum sugar syrup liberation (125 ± 1.0 g/L with 0.42 ± 0.01 g/g rice sample) was found at 12 h of incubation at 50 °C. Maximum TPC was recorded at 2.01 ± 0.05 mg GAE/mL equivalent to 6.71 ± 0.16 mg GAE/g rice sample at 12 h of incubation. These results showed the possibility to apply the model obtained in this study at an industrial scale.

Bacterial cellulose (Nata) fermentation

Bacterial cellulose from the fermentation of microorganisms can replace plants as an alternative source of cellulose through oxidative fermentation (Esa et al. 2014). In the production medium, different concentrations of sugar syrup (5–50 g/L) were provided as the carbon source for bacterial cellulose formation. Results showed that K. xylinus AGR 60 utilized rice sugar syrup as a substrate for bacterial cellulose production. Thickness of bacterial cellulose at each syrup concentration is shown in Table 4. Highest thickness of bacterial cellulose was 1.1 ± 0.08 cm with 8.15 ± 0.12 g of dry weight for rice sugar syrup at 25 g/L, after incubation at room temperature for 9 days. High concentrations of rice syrup inhibited growth of K. xylinus (A. xylinum) due to substrate inhibition of glucose and bioactive compounds in the obtained rice syrup or acetic acid which is the main organic acid produced by K. xylinus (Lestari et al. 2014). However, during the fermentation of bacterial cellulose, gluconic acid occurred as a by-product in the culture medium, and this inhibited formation of bacterial cellulose as described by Esa et al. (2014). For the positive control, which used coconut juice as the substrate, thickness of bacterial cellulose was 1.3 ± 0.05 cm with 8.30 ± 0.12 g of dry weight and higher than using rice syrup as substrate. This may affect some nutrients in coconut juice which promote fermentation of bacterial cellulose by K. xylinus (Prades et al. 2012). This finding concurred with Kongruang (2007) who found that coconut juice was better for bacterial cellulose production than pineapple juice since it was rich in carbohydrates, proteins, and trace elements. Prades et al. (2012) reported that coconut juice was rich in amino acids (alanine, arginine, cysteine and serine) and minerals [potassium (K), sulfur (S), calcium (Ca), sodium (Na), magnesium (Mg), iron (Fe) and copper (Cu)], which may be important for growth and bacterial cellulose production by K. xylinus. TPC values of BC obtained from rice syrup and coconut juice were 0.13 ± 0.002 and 0.09 ± 0.002 mg GAE/g fresh weight, respectively. This revealed that bacterial cellulose obtained from rice syrup was a good alternative healthy product than conventional coconut bacterial cellulose in terms of nutrition as an attractive novel product. Photographs and scanning electron micrographs of bacterial cellulose from K. xylinus AGR 60 using coconut juice and rice syrup are shown in Fig. 3. The obtained bacterial cellulose from rice syrup had a pink color after washing, however the color faded after boiling at 100 °C. The XRD pattern analyses of BC samples are shown in Fig. 4, with the range of main peaks at 14.2–14.4°, 16.6–16.7° and 22.5–22.6°. Results concurred with Moukamnerd et al. (2020) who found main peaks of BC produced from fruit processing waste in the range 14.5–15.3°, 16.0–17.0° and 22.0–23.0°, while Saleh et al. (2020) reported main peaks of BC produced from glucose medium at 14.76°, 17.81° and 23.54°, similar to patterns in this study. The crystallinity index (CI) values of BC produced from coconut juice and rice syrup were between 44.01% and 40.60%, respectively which corresponding to the result of Naloka et al. (2020) reported that BC produced from K. xylinus MSKU 12 in a low-cost coconut water containing acetic acid and ethanol has crystallinity index at 44.7%. The FTIR spectra of bacterial cellulose produced from coconut juice and rice syrup by K. xylinus AGR 60 are shown in Fig. 5. Both samples showed similar patterns, indicating that bacterial cellulose produced from coconut juice and rice syrup in this study had a comparable chemical structure. The peak of stretching vibration at wavenumber 3300–3350 cm⁻¹ was attributed to the presence of hydroxyl group (OH), as reported by Moukamnerd et al. (2020) and Saleh et al. (2020). A strong absorption band at 2900 cm⁻¹ was attributed to the presence of CH₂ stretching vibrations (Saleh et al. 2020), while absorption at 1600 cm⁻¹ was assigned to the carboxyl (C = O) functional group (Saleh et al. 2020), and peaks observed at 1425–1420 cm⁻¹ to C–H bonds (Moukamnerd et al. 2020; Saleh et al. 2020). The peaks of both samples at 1300–1500 cm⁻¹ were different. This range of wavenumber was recorded to C–H bonds, as reported by Saleh et al. (2020). An absorption peak around 1000–1100 cm⁻¹ corresponds to C–O–C functionality (Kamarudin et al. 2018; Moukamnerd et al. 2020). Our results showed that production of alternative bacterial cellulose was feasible using Thai rice as the substrate by hydrolysis of RSDE to provide economically healthy products from natural resources.

Table 4.

Thickness and dry weight of bacterial cellulose at different concentrations of rice syrup

Rice syrup concentration (g/L)	Bacterial cellulose
	Thickness (cm)	Dry weight (g)
5	0.75 ± 0.05a,b	6.25 ± 0.11a
10	0.8 ± 0.1b,c	7.35 ± 0.11b
20	1.0 ± 0.1c,d	8.04 ± 0.13c
25	1.1 ± 0.08d	8.15 ± 0.12c
40	0.9 ± 0.1b,c	7.78 ± 0.09b
50	0.7 ± 0.1a	6.14 ± 0.16a

Values are averages of three determinations

Different letters within the same column are statistically different at p < 0.05

Fig. 3.

Photographs and scanning electron micrographs of bacterial cellulose of coconut juice and rice sugar syrup by fermentation of K. xylinus AGR 60 at room temperature for 9 days. a, b photographs of bacterial cellulose from coconut juice and rice syrup, respectively. c, d scanning electron micrographs of bacterial cellulose from coconut juice and rice syrup, respectively

Fig. 4.

The XRD spectrum of BC samples obtained from coconut juice and rice syrup by K. xylinus AGR 60

Fig. 5.

FTIR spectra of bacterial cellulose produced from coconut juice and rice syrup by K. xylinus AGR 60 at wavenumber 4000–400 cm⁻¹

Conclusions

Brown and black rice varieties have attracted significant attention as healthy raw materials for production of various foodstuffs. RSDE from the thermophilic filamentous bacterium, L. sacchari LP175 showed synergistic hydrolysis with commercial GA at 50 °C and achieved high concentration of sugar syrup (125 g/L) using statistical mixture design experiments. The obtained sugar syrup could be used as a substrate to produce bacterial cellulose (Nata) as a new innovative product in the food and cosmetic industries. The contained bioactive substances and attractive color compared with coconut bacterial cellulose. Our results indicated the feasibility of increasing the value of local rice through biotechnological processes as a healthy product for future applications in the food and cosmetic industries.

Supplementary Information

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Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

This article does not contain any studies with human or animal subjects.