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. 2020 Nov 9;10(12):515. doi: 10.1007/s13205-020-02488-8

Production and development of vinegar fermentation from broken Riceberry rice using raw starch-degrading enzyme hydrolysis

Nujarin Sangngern 1, Thidarat Puangnark 1, Watsachon Nguansangiam 1, Pramuan Saithong 2, Vichien Kitpreechavanich 3, Thanasak Lomthong 1,✉
PMCID: PMC7652972  PMID: 33194519

Abstract

Broken Riceberry rice was used as a substrate for sugar syrup production by the hydrolysis of raw starch-degrading enzyme as a low-temperature amylase (iKnowZyme® LTAA, Thailand). Response surface methodology (RSM) with a central composite design (CCD) showed that an optimized substrate concentration of 250 g/L yielded 13°Brix of total soluble solid (TSS) content when incubated at 50 °C for 12 h. The major product from the broken Riceberry rice hydrolysis was glucose with lesser amounts of maltose and maltotriose. Maximum alcohol content (16% w/v) for broken Riceberry rice wine was obtained after fermentation with two mixed strains of Saccharomyces cerevisiae for 10 days. Scanning electron micrographs showed that yeast strains could grow on the solid residue of broken Riceberry rice that supported yeast cell survival under stress conditions. Broken Riceberry rice wine was used as the substrate for vinegar fermentation by Acetobacter aceti TISTR 354. Maximum acetic acid concentration was achieved at 5.4% when incubated at room temperature for 6 days, containing 10.92 mg/L and 965.53 ± 7.74 mL sample/g DPPH of anthocyanin content and antioxidant assay, respectively. Our finding revealed the feasibility of broken Riceberry rice substrate for sugar syrup, wine and vinegar production by raw starch-degrading enzyme hydrolysis which increased the value of low-cost agricultural crops through biotechnological processes.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02488-8) contains supplementary material, which is available to authorized users.

Keywords: Broken Riceberry rice, Raw starch-degrading enzyme, Vinegar fermentation, Enzyme hydrolysis

Introduction

Broken Riceberry rice, a low-cost agricultural crop in Thailand, is obtained during the process of polished rice production and classified to a lower grade because of the irregular size and shape of the grains compared to the normal product (Luang-In et al. 2018). Broken Riceberry rice comprises about 20–30% of the total Riceberry rice product yield at approximately 1200–1800 tons per harvest season (Luang-In et al. 2018). The price of broken Riceberry rice in the market is about three times cheaper than commercial Riceberry rice due to customers preferred to consume the rice grains that have a regular shape; however, nutrients and chemical compositions of both rice types are similar (Ngamdee et al. 2019).

Riceberry rice offers increased health benefits over other carbohydrate-based foods, since it contains several nutrients and antioxidative compounds (Leardkamolkarn et al. 2011), and contains high contents of vitamins, antioxidants and mineral salts compared to other crops (Pitija et al. 2013; Liang et al. 2014). Riceberry rice has been used to produce various healthy products including noodles (Sirichokworrakit et al. 2015), rice-based milk (Wattananapakasem et al. 2018), Kao-Taen (Payim 2016) and lip gloss (Vutdhipapornkul et al. 2014); however, utilization as value added to low-cost broken Riceberry rice through biotechnological processes using cold fermentation technology has not yet been reported.

Wine is an alcoholic beverage that is formed by fermentation of various kinds of fruit by yeast strains (Musyimi et al. 2014). Rice-based fermented beverages are called rice wine in Thailand as the generic name given to alcoholic beverages made using rice as the substrate (Chuenchomrat et al. 2008). Vinegar is the second step in the fermentation process after alcoholic fermentation to obtain acetic acid as the final product (Saithong et al. 2017). Various types of vinegars are obtained from different substrates such as pineapple, rice, mango, mushroom and apple (Li et al. 2014; Kulkarni 2015; Saithong et al. 2017; Qi et al. 2017). This process can be applied to increase the value of broken Riceberry rice.

The cold hydrolysis process is used to hydrolyze starch-based substrates at low temperature (40–50 °C) compared to conventional methods at high temperature (90–105 °C) (Cinelli et al. 2015). Complete hydrolysis of starch-based materials at low temperature reduces energy consumption and the cost of the operation (Lomthong et al. 2016). Therefore, this research aimed to produce and develop vinegar fermentation using broken Riceberry rice as substrate, via low-temperature saccharification of raw starch-degrading enzyme, to increase the value of this low-cost agricultural crop and provide an alternative healthy product through a biotechnological process.

Materials and methods

Substrates and enzyme

Broken Riceberry rice was obtained from the Nong Luang Enterprise Group, Khampangphet Province, Thailand, reduced to powder using an electric grinder and stored under dry condition until required for use. The chemical compositions of broken Riceberry rice were analyzed. Total starch was determined by a total starch assay kit (Megazyme International Ireland, Wicklow, Ireland) and amylose content was determined by amperometric titration with potassium iodate solution (Takeda et al. 1987; Gibson et al. 1997), performed by the Cassava and Starch Technology Research Unit (CSTRU) at Kasetsart University as described by Lomthong et al. (2016). Protein, fat, and fiber contents were analyzed using AOAC methods (Helrich 1990).

Raw starch-degrading enzyme as a low-temperature amylase (iKnowZyme® LTAA) was obtained from the Reach Biotechnology Co., Ltd., Thailand and stored at -20 °C until required for use.

Microorganisms and inoculum preparation

Two strains of yeast (Saccharomyces cerevisiae var. montache and Saccharomyces cerevisiae var. kyokai) and vinegar-producing bacteria (Acetobacter aceti TISTR 354) were obtained from the Department of Applied Microbiology, Institute of Food Research and Product Development (IFRPD), Kasetsart University, Thailand. The yeast strains were grown separately in YM medium (3 g/L yeast extract, 3 g/L malt extract and 5 g/L peptone, supplemented with 10 g/L glucose) and incubated at 30 °C for 16 h; 10% (v/v) was used as inoculum for broken Riceberry rice wine fermentation. For vinegar fermentation, A. aceti TISTR 354 was grown on broken Riceberry rice inoculum liquid medium containing 7.0 g broken Riceberry rice powder, 7.0 mL of 95% ethanol and 76.0 mL of distilled water, and incubated at 30 °C without shaking for 4 days; 10% (v/v, approximate 108 CFU/mL on the De Man, Rogosa and Sharpe (MRS) agar plate) was used as the vinegar starter (Saithong et al. 2017).

Production of sugar syrup by central composite design (CCD)

Factors affecting sugar syrup production including substrate concentration (X1) and incubation time (X2) were investigated via low-temperature saccharification of a raw starch-degrading enzyme. The experiments were performed in 250-mL Erlenmeyer flasks which contained 49 mL of distilled water and 1.0 mL of low-temperature amylase (iKnowZyme® LTAA). Hydrolysis was performed at 50 °C using different substrate concentrations (108.6–391.4 g/L) and incubation times (7.758–16.242 h) designed by response surface methodology (RSM) with five levels of central composite design (CCD) including 0, + 1, − 1, -α and + α as shown in supplementary Table 1. Experimental design and statistical analysis of the data were performed using STATISTICA 10 for Windows™. Data from the experimental design were subjected to a second-order multiple regression analysis using least-squares regression methodology to obtain the parameter estimators of the mathematical model (Lomthong et al. 2015).

To validate the model obtained from experimental design, the predicted value was calculated from a second-order polynomial equation for maximum sugar syrup production and validated using the optimum parameters in the 5.0-L glass jar chamber (18 × 18 × 28 cm) with a 3.0-L working volume of substrate suspension.

Scanning electron microscopy (SEM) was used to study the morphological changes in native and digested broken Riceberry rice powder after hydrolysis by iKnowZyme® LTAA in a 5.0-L glass jar. All samples were cleaned with distilled water, dried at 50 °C for 24 h, and then examined under a scanning electron microscope (Model SU8020; Hitachi, Tokyo, Japan) at 10.0 kV as reported by Lomthong et al. (2015). The hydrolysis products of broken Riceberry rice powder were qualitatively determined by thin-layer chromatography (TLC) as reported by Sassaki et al. (2008) using glucose, maltose and maltotriose as standards (Lomthong et al. 2016).

Wine fermentation

Sugar syrup, obtained from hydrolysis by raw starch-degrading enzyme (iKnowZyme® LTAA), was varied at four concentrations. These included undiluted and diluted with distilled water in the ratio of 1:1, 1:2 and 1:3. The effect of bioactive compound concentration in rice syrup was investigated on the quality of alcoholic fermentation and the feasibility of using the diluted rice syrup to reduce the cost and operational time for industrial application. The fermentation was operated in a 5.0-L glass jar chamber (18 × 18 × 28 cm) with a 3.0-L working volume in each experiment. Sucrose powder was added to the reactions to ensure that all initial sugar concentrations were 22°Brix. Diammonium phosphate (DAP) at 1.2 g/L and potassium metabisulphite (KMS) at 350 ppm were added to the reaction and the pH was adjusted to 4.0 by citric acid. The reaction tanks were maintained at room temperature for 24 h before adding yeast inoculum. For alcoholic fermentation, 10% (v/v) of both strains were inoculated into the fermentation tank at equal ratios of S. cerevisiae var. kyokai and S. cerevisiae var. montache. The fermentation was incubated at 30 °C for 10 days under static fermentation (without shaking process).

Growth of Saccharomyces strains on the broken Riceberry rice powder after cultivation on the medium for 10 days was studied by scanning electron microscopy (SEM). The solid fermented residue was separated from the bioethanol fermentation by centrifugation at 4032×g for 15 min at 4 °C, packed into Whatman filter paper No. 1 and soaked through a series of 15–100% ethanol for 15 min at each stage (Thirunavukarasu et al. 2016). The substrate was finally placed in a critical point drying (CPD) machine (Polaron Range CPD 7501, Quorum Technologies Ltd., England) with liquid CO2, before examination by a scanning electron microscope (Model SU8020; Hitachi, Tokyo, Japan) at 10.0 kV.

Vinegar fermentation

Vinegar production was performed by surface culture fermentation (SCF) as reported by Saithong et al. (2017) using the Riceberry rice wine obtained from the alcoholic fermentation as describes above as the substrate. The SCF is a static fermentation process that usually requires two steps as vinegar starter culture preparation (within 2 days) and vinegar production (3–4 days) at room temperature. Suitable mixture ratios (100:300:600 mL) followed Saithong et al. (2017) of vinegar starter (A. aceti TISTR 354), Riceberry rice wine and rice residue suspension (4°Brix), respectively, were poured into stainless steel trays and covered with plastic sheets. A hole was punched in the plastic sheet and the presence of water vapor on the sheet was observed for 2 days (Starter culture preparation). Vinegar production was then performed, and 1 L of the clear Riceberry rice wine (10.0% w/v) was added into the same tank (Starter culture) as described above which was then covered with a plastic sheet. A hole was punched in the plastic sheet and the culture was left to stand for 3–4 days.

Analysis

Alcohol and total soluble solid (TSS) content

An ebulliometer (Dujardin-Salleron, Paris, France) was used to assess the total alcohol content (Kocabey et al. 2016). Total soluble solid (TSS) content was investigated using a refractometer (RA-250WE, Kyoto Electronics, Kyoto, Japan).

pH and titratable acidity

Samples were centrifuged (5.0 min at 3500×g), with pH measured at 30 °C using a pH meter (Model 430; Corning, NY, USA). Titratable acidity is a measure of acid content in wine and vinegar. This was determined as acetic acid for vinegar by titration with 1 N NaOH using phenolphthalein as an indicator (Helrich 1990). All measurements were conducted in triplicate.

DPPH radical-scavenging assay

Antioxidant assay was reported as half-maximum effective concentration (EC50) which is defined as unit mL sample/g DPPH, following the modified procedure of Brand-Williams et al. (1995) using 2,2-diphenyl-2-picrylhydrazyl (DPPH) assay. The 50 µL appropriated diluted samples of the broken Riceberry berry rice vinegar were mixed with 950 µL of 0.0394 g/L DPPH solution, and reactions were incubated at room temperature for 30 min in darkness. Absorbance was measured with a UV–Vis spectrophotometer at 515 nm.

Anthocyanin content

Total anthocyanin content was determined by the pH-differential method, following Wrolstad et al. (2005) and Lomthong et al. (2019) with slight modifications. Anthocyanin pigment concentration was calculated and expressed as cyanidin-3-glucoside equivalents per 100 g. All determinations were performed in triplicate.

Organic acid analysis

Organic acids including acetic acid, lactic acid, citric acid, malic acid and tartaric acid in the broken Riceberry vinegar were determined using high-performance liquid chromatography (HPLC) with a refractive index detector (RI). The conditions were performed following the method of Mullin and Emmons (1997) by the Food Quality Assurance Service Center (FQA), Institute of Food Research and Product Development (IFRPD), Kasetsart University, Thailand.

Statistical analysis

All results were calculated as mean ± SD (standard deviation). Mean values, standard deviation, and analysis of variance (ANOVA) were computed using a commercial statistical package SPSS 21.0 (USA). Differences among mean values were tested using the least significant difference multiple range test. Values were considered significant when p < 0.05.

Results and discussion

Chemical composition of broken Riceberry rice powder

The chemical composition of broken Riceberry rice powder is shown in Table 1. The major component was starch (65.77%) with amylose content 9.75%, while protein and lipid contents were 8.87 and 3.26%, respectively. Various publications have reported applications of broken Riceberry rice to increase value through a biotechnological process. Luang-In et al. (2018) studied the bioactive compounds of broken Riceberry rice and reported the potential for application cosmetic ingredients in hair product development. Rakkhumkaew et al. (2019) reported the application of broken Riceberry flour for production of gluten-free bread. Ngamdee et al. (2019) extracted anthocyanin from broken Riceberry as a supplement for chocolate milk products. They demonstrated the possibility of applying broken Riceberry rice powder as a substrate for vinegar fermentation via raw starch-degrading enzyme hydrolysis.

Table 1.

Chemical composition of broken Riceberry rice powder

Component (%) Analysis (%)
Starch 65.77 ± 0.94
Protein 8.87 ± 0.03
Fat 3.26 ± 0.03
Fiber 1.19 ± 0.01
Ash 1.68 ± 0.03
Moisture 11.32 ± 0.11
Amylose 9.75 ± 0.07
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Values are averages of three determinations

Production of sugar syrup using response surface methodology with central composite design

Factors affecting sugar syrup production from broken Riceberry rice powder including substrate concentration (X1) and incubation time (X2) were investigated using response surface methodology (RSM) with five levels of central composite design (CCD) as described above. An experimental matrix and results for sugar syrup production are shown in Table 2. The second-order polynomial equation was constructed as shown in the following equation to predict the production of sugar syrup from broken Riceberry rice powder by raw starch-degrading enzyme hydrolysis:

Y=-59.4042+0.1813X1+7.6054X2-0.0003X12-0.2967X22-0.0011X1X2. 1

Table 2.

Experimental design used in response surface methodology of two independent variables as substrate concentration (X1) and incubation time (X2) with three center points, and observed and predicted sugar syrup production

Treatment number Level Actual level TSS (°Brix)
X1 X2 X1 X2 Predicted Observed
1 0 − 1.414 250 7.758 6.63 6.5 ± 0.5
2 1 − 1 350 15 9.70 8.5 ± 1.32
3 − 1 − 1 150 9 4.12 4.0 ± 0.5
4 1.414 0 391.4 12 10.08 10.5 ± 1.5
5 − 1.414 0 108.6 12 3.91 4.5 ± 0.5
6 1 1 350 15 9.7 10.0 ± 0.5
7 − 1 1 150 15 6.01 5.0 ± 0.5
8 0 1.414 250 16.242 8.36 9.5 ± 0.5
9 0 0 250 12 12.83 12.8 ± 0.29
10 0 0 250 12 12.83 12.5 ± 0.5
11 0 0 250 12 12.83 12.8 ± 0.29
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Values are averages of three determinations

where Y is the predicted response of sugar syrup production from broken Riceberry rice powder, and X1 and X2 are coded values of substrate concentration and incubation time, respectively.

Statistical significance of the regression model was checked by t test and analysis of variance (ANOVA). Corresponding p values of X1 (substrate concentration) and X2 (incubation time) were 0.002 and 0.001, respectively, suggesting that among the two independent variables broken Riceberry rice powder had a significant effect on sugar syrup production at a 95% significance level (p < 0.05). The coefficient of determination (R2) was 95.93% for sugar syrup production from broken Riceberry rice powder. Contour and three-dimensional plots of the interactions among substrate concentration (X1) and incubation time (X2) are shown in Fig. 1. Maximum sugar syrup production was achieved with substrate concentration and incubation time up to 250 g/L and 12 h, respectively. After this point, sugar syrup production declined to below the optimum level.

Fig. 1.

Fig. 1

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Response plot and contour plot of the combined effects between substrate concentration (g/L) and incubation time (h) for sugar syrup production using broken Riceberry rice as substrate by raw starch-degrading enzyme hydrolysis at 50 °C for 12 h

To validate the model, the hydrolysis was performed in a 5.0-L glass jar chamber (18 × 18 × 28 cm) with a 3.0-L working volume of substrate suspension using the optimum conditions from the predicted model as described above. Results indicated maximum sugar syrup production at 13°Brix while the predicted value was 12.8°Brix, suggesting that the model obtained from response surface methodology with central composite design fitted and was suitable to apply for sugar syrup production from broken Riceberry rice powder by raw starch-degrading enzyme hydrolysis.

Scanning electron micrographs showed that broken Riceberry rice powder was hydrolyzed by raw starch-degrading enzyme at 50 °C. After digestion with raw starch-degrading enzyme, the rice powder surface developed pits with deep holes and loss of structure as shown in Fig. 2b. Lomthong et al. (2015) reported that raw starch-degrading enzyme has a raw starch-binding domain which could adsorb on the surface of starch granules and digest from the outside to the inside of the granules. This result confirmed that raw starch-degrading enzyme as a low-temperature amylase (iKnowZyme® LTAA) could be applied for production of sugar syrup at a lower temperature compared to the conventional enzyme which acted at high temperature (Cinelli et al. 2015). The major product of broken Riceberry rice powder hydrolysis was glucose with lesser amounts of maltose and maltotriose as determined by thin-layer chromatography (TLC) (Fig. 2c).

Fig. 2.

Fig. 2

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Scanning electron micrographs of native broken Riceberry rice (a) and digested by raw starch-degrading enzyme at 50 °C for 12 h (b). TLC chromatogram of broken Riceberry rice syrup after hydrolysis by raw starch-degrading enzyme at 50 °C for 24 h. G1: glucose, G2: maltose and G3: maltotriose (c)

Wine fermentation

Broken Riceberry rice wine fermentation results are shown in Table 3. Yeast strains utilized the liberated sugar from the hydrolysis of broken Riceberry rice and supplemented sucrose for growth and fermentation to alcohol. Maximum alcohol content was achieved using undiluted Riceberry rice syrup residue as substrate (16.2%, w/v), while dilution with distilled water at 1:1, 1:2 and 1:3 gave alcohol contents at 14.8, 14.7 and 13.7%, respectively. High alcohol fermentation was obtained using undiluted broken Riceberry rice sugar syrup because large amounts of hydrolysis residue of broken Riceberry rice in the reaction liberated fermentable sugar by the synergistic effect of the remaining raw starch-degrading enzyme. This was confirmed by the total soluble solids (TSS) content at the end of fermentation at 16°Brix. For the other dilutions, total soluble solids content was 11°Brix. Moreover, glucose content from enzymatic hydrolysis in undiluted rice syrup was higher than in the other treatments, and easier for consumption and conversion to alcohol as compared to sucrose. Koguchi et al. (2010) reported that two strains of Saccharomyces species (S. cerevisiae var. montache and S. cerevisiae var. kyokai) synergistically fermented sugar to alcohol and were suitable for rice wine fermentation. The pH value of the reactions was similar and decreased from 4.0 to 3.6–3.7 due to metabolite products of the yeast strain produced during growth and fermentation. Figure 3a, b reveals that yeast cells could grow on the solid fiber residue of broken Riceberry rice wine to protect against stress conditions at high osmotic pressure, leading to an increase in ethanol production. Trakarnpaiboon et al. (2017) and Wattanagonniyom et al. (2017) reported that the hydrolysis residue of cassava chips increased alcohol fermentation compared to fermentation without solid residue.

Table 3.

Chemical composition of broken Riceberry rice wine after fermentation with yeast strains at room temperature for 10 days

Experiment Chemical analysis
Alcohol content (%) TSS (°Brix) pH DPPH (mL sample/g DPPH)
1 (Undiluted) 16.2 ± 0.16c 16.0 ± 0.5b 3.6 ± 0.06a 1,702.53 ± 7.76d
2 (Diluted 1:1) 14.8 ± 0.15b 11.0 ± 0.5a 3.6 ± 0.06a 858.94 ± 5.28c
3 (Diluted 1:2) 14.7 ± 0.17b 11.0 ± 0.5a 3.7 ± 0.03a 497.66 ± 5.04b
4 (Diluted 1:3) 13.7 ± 0.15a 11.0 ± 0.3a 3.7 ± 0.05a 222.74 ± 0.72a
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Values are averages of three determinations

Different letters within the same column indicate significant difference at p < 0.05

Fig. 3.

Fig. 3

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Scanning electron micrographs of yeast cells grown on solid fiber residue of broken Riceberry rice powder after alcoholic fermentation at 30 °C for 10 days under static fermentation a 1500x, b 3000x

Vinegar fermentation

The Riceberry rice wine obtained from the previous step was used as the substrate for vinegar fermentation, following the method of Saithong et al. (2017) by surface culture fermentation (SCF) with A. aceti TISTR 354. Fermentation results of broken Riceberry rice vinegar are shown in Fig. 4. Alcohol content decreased to 0.5%, while the acidity as acetic acid increased up to 5.4% after 6 days of incubation at room temperature (30 °C). Vinegar obtained from the broken Riceberry rice corresponded to the standard set by the Thai Ministry of Public Health Notification, (No. 204) B.E. 2543 (2000) Re: Vinegar (Thai Ministry of Public Health 2018), with over 4 g of acetic acid per 100 mL. Maximum residue of alcohol did not exceed 0.5%. As compared to the high-temperature heating process, Phuapaiboon (2017) applied alpha-amylase for hydrolysis of Riceberry rice with heating at 121 °C for 30 min. Subsequent fermentation to vinegar by S. cerevisiae E1118 and A. pasteurianum TISTR 102, respectively, yielded 3.18% of titratable acidity. The anthocyanin content of broken Riceberry rice vinegar was 10.92 mg/L at the end of fermentation, while the initial anthocyanin content of broken Riceberry rice wine was 18.73 mg/L. Lomthong and Saithong (2019) reported that anthocyanin content in Leum Pua glutinous rice vinegar was 25.4 mg/L, while Phuapaiboon (2017) reported that anthocyanin content in brewed Riceberry rice vinegar was 1.62 mg/L. The antioxidant assay was also investigated in this study as half-maximum effective concentration (EC50) as described above. The half-maximum effective concentration (EC50) of broken Riceberry vinegar was 965.53 ± 7.74 mL sample/g DPPH. To confirm the organic acid composition in broken Riceberry vinegar, organic acid analysis was conducted as described above. The results showed that the major organic acid in broken Riceberry vinegar was found only acetic acid while other organics acids (lactic acid, citric acid, malic acid and tartaric acid) were not detected in the sample (Supplementary Table 2). This result showed the possibility of producing healthy vinegar using broken Riceberry rice as the substrate without high-temperature heating, as an alternative healthy product from a low-cost agricultural crop in Thailand through a biotechnological process.

Fig. 4.

Fig. 4

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Time course of broken Riceberry rice vinegar fermentation using surface culture fermentation (SCF) by A. aceti TISTR 354 after 6 days of incubation at room temperature (30 °C) (filled square: alcohol content, filled triangle: %acidity as acetic acid)

Conclusions

Broken Riceberry rice is a low-cost agricultural crop in Thailand that is suitable for production of alternative healthy products such as Riceberry rice vinegar. Broken Riceberry rice was hydrolyzed via low-temperature saccharification of raw starch-degrading enzyme at 50 °C. The optimum conditions for sugar syrup production were obtained by response surface methodology (RSM) with a central composite design (CCD), yielding 13°Brix total soluble solid (TSS) content using substrate concentration at 250 g/L and incubation at 50 °C for 12 h. The obtained sugar syrup was used as fermentable sugar with a two-step fermentation process to produce vinegar with 5.4% acetic acid. The results in this research indicate the possibility that broken Riceberry rice could be used as substrate for alternative product production via raw starch-degrading enzyme hydrolysis which low-cost and high nutritional bioactive compounds.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 18 kb) (17.3KB, docx)

Acknowledgements

This research was supported by Institute of Research and Development Rajamangala University of Technology, Thanyaburi (DRF63D0606) and the Department of Applied Microbiology, Institute of Food Research and Product Development, Kasetsart University, Thailand.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

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

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