2022 Volume 28 Issue 1 Pages 95-103
Khanomjeen, a Thai fermented rice noodle, is made from fermented rice flour prepared from Indica rice that has traditionally undergone solid- and submerged-state fermentations. In response to changing consumer preferences, some khanomjeen producers have replaced the traditional solid-state fermentation with submerged fermentation, expecting a similar softness and whiteness to that of khanomjeen-style unfermented rice noodles. In the present study, the degree of protein removal in the modified fermentation method was lower than that of the traditional method. The hardness of khanomjeen made by the modified method was 60% of that made by the traditional method but its whiteness was identical to that of the unfermented rice noodles. The metagenomic profiles revealed that the modified method allowed the growth of possibly proteolytic bacteria, but suppressed possibly pigment-producing bacteria. The modified fermentation method could thus help to control the microbiota to improve the texture and color of khanomjeen and meet current consumer demands.
Khanomjeen, a traditional Thai fermented rice noodle widely consumed in the country, is part of a food culture in constant demand nationwide. The elastic texture, pale-yellow appearance, characteristic flavor, and preservative properties of khanomjeen are recognized as unique characteristics. Similar fermented rice noodles are also commonly produced in other Southeast Asian countries and China. The fermented rice flour used for making khanomjeen is prepared from Indica rice. Broken rice, a byproduct of rice milling production is also used as the raw material. Therefore, khanomjeen production is important for the agricultural and food industries of Thailand. In the traditional method for producing fermented rice flour, rice grains soaked in water undergo solid-state fermentation for a few days. After wet-milling, the fermented rice slurry is then precipitated in saline water where the secondary fermentation takes place for a few days. After squeezing out the excess moisture, the fermented rice flour is then collected and used for producing khanomjeen (Fig. 1).
Production and sampling schemes for the traditional and modified rice fermentation methods. Fermented rice flour for preparing khanomjeen was produced by the traditional and modified methods as described in the Materials and Methods section. For the traditional method, the rice grains were moisturized by soaking for 1 h (TR0), drained then subjected to solid-state fermentation for 3 d (TR1 to 3). After wet-milling in saline water (2% w/v), the rice slurry was subjected to submerged fermentation for 2 d (TF1 and 2) with samples collected daily for further analyses. The finished fermented flour (TF2) was immediately used for preparing khanomjeen. For the modified method, the rice grains were directly subjected to the first submerged fermentation for 3 d (MR1 to 3). The moisturized rice grains (MR0) were collected after being submerged in water for 1 h. After wet-milling in saline water (2% w/v), the rice slurry was subjected to the second submerged fermentation for 2 d (MF1 and 2) with samples collected daily for further analyses. The finished fermented flour (MF2) was immediately used for preparing khanomjeen.
The unique elastic texture of khanomjeen is achieved by removing soluble rice protein during the fermentation (Satmalee et al., 2017; Shompoosang et al., 2019). Khanomjeen can also retain its quality without deterioration for a few days at ambient temperature by maintaining the acidic conditions of the noodles derived from the fermented rice flour (Marui et al. 2020). The microorganisms which influence the characteristics of fermented rice noodles originate from the raw materials and production environment. The functions of these microorganisms, their diversity and their succession during the production of fermented rice flour are of scientific interest when modifying processes to improve the quality of khanomjeen. Lactic acid bacteria (LAB) are important for producing the lactic acid and volatile compounds related to the unique flavor and preservative properties of khanomjeen products (Keatkrai and Jirapakkul, 2010). Streptococcus, Lactobacillus species and Pediococcus acidilactici have been isolated and reported as the dominant LAB species in fermented rice flour for khanomjeen (Boonmee, 1989; Sirirote et al., 1991; Uchimura et al., 1991). Some LAB species are also known to produce hydrolytic enzymes such as protease, amylase and lipase to digest rice constituents related to the quality of fermented rice noodles (Sirirote et al., 1991; Yi et al., 2017). As well as lactic acid bacteria, Bacillus and Enterobacteriaceae have been found during the early stages of rice fermentation for khanomjeen, as well as in a similar type of fermented rice noodle produced in Myanmar (Boonmee, 1989; Ikeda et al., 2003). We have previously demonstrated the effectiveness of a proteolytic bacterium, Enterobacter ludwigii strain SK01, as a starter culture for fermented rice flour production in improving the texture and color qualities of khanomjeen (Shompoosang et al., 2019). Bacillus subtilis isolated from the soaked rice and fermented rice flour used for khanomjeen has been determined as a proteolytic bacterium (Boonmee, 1989; Phromraksa et al., 2008), but has also been reported as a cause of brownish fermented rice grains in solid-state fermentation (Boonmee, 1989).
Although these flavor, texture, color and preservative qualities of khanomjeen produced by traditional rice fermentation have long been favored by consumers, producers are now also trying to modify the product quality to meet the demands of current consumers. For example, some consumers now expect khanomjeen noodle products to be of a similar softness and whiteness as the khanomjeen-style unfermented rice noodles, now becoming common in Thailand. Unlike the traditional khanomjeen production process, for the production of unfermented rice noodles the rice grains are wet-milled immediately after soaking in water overnight to prepare the rice flour. The resulting noodle product generally exhibits a low elasticity, soft texture and white appearance. Therefore, to incorporate such product features, some producers have modified the traditional method of producing fermented rice flour by replacing solid-state rice fermentation with the submerged method of fermentation (Fig. 1) to produce khanomjeen with a soft texture and whiteness similar to that of unfermented rice noodles, although the advantages of this modification have not yet been scientifically proven.
The present study will therefore use chemical, texture and color analyses to investigate the advantages of the modified rice fermentation method for improving the quality of khanomjeen products to meet the current consumer demand. The bacterial metagenomic profiles arising during the traditional and modified fermentation processes for producing fermented rice flour will be compared to better understand the possible mechanisms for improving the quality of khanomjeen products at the microbial level.
Preparation of fermented rice flour and khanomjeen Indica rice (variety Leuang Pratew) was used to produce the fermented rice flour as shown in Fig. 1. For the traditional method, rice grains (10 kg) were soaked in sterilized tap water for 1 h then transferred to a plastic sieve, drained then covered with a plastic sheet for the solid-state fermentation at ambient temperature (30–35 °C). The fermented rice grains were washed with sterilized tap water daily for 3 d during this solid-state fermentation. The fermented rice grains were then wet-milled in sterilized saline water (2% NaCl w/v), followed by filtration through cheese cloth to remove the grain residue. The fermented rice flour slurry was then precipitated in sterilized saline water for the submerged fermentation stage at ambient temperature. The clear upper phase of the saline water was changed daily. After 3 d of first submerged fermentation, the clear upper phase of water was removed, followed by wet milling with saline water (2% NaCl w/v). The wet milled slurry was then subjected to the second submerged fermentation. Fermented rice flour was collected in the same manner as the traditional method. Samples (100 g) collected from each fermentation process were stored at −80°C until analysis. Approximately 8.5 kg of fermented rice flour was obtained from each fermentation method.
The khanomjeen was produced as described by Shompoosang et al. (2019) using fermented rice flour made by the traditional or modified fermentation methods. The fermented rice flour (approx. 200 g) was heated in boiling water for 4 min, kneaded into a dough, kneaded again with a small amount of boiling water until the dough became creamy, then the flour slurry was extruded into noodles in boiling water for 1 min. Pore size of the extruding device was approximately 2.0 mm. The noodles were immediately cooled down using water then drained in a plastic sieve container. In reference to the practical commercial practice of khanomjeen before selling, the noodle products were covered with a plastic film then stored at ambient temperature for 18 h, followed by analyzing their texture and color.
As a reference sample for the texture and color analyses in the present study, khanomjeen-style unfermented rice noodles were prepared as follows: Indica rice grains (variety Leuang Pratew) were soaked in sterilized tap water for 12 h. The moisturized rice grains were then wet-milled in sterilized saline water (2% NaCl w/v). The rice flour was collected then immediately used for preparing noodles in the same manner as the fermented rice flour products.
Chemical analyses of fermented rice flour The samples were dried at 60°C overnight then crushed into a powder using an Iwatani Foods Millser model IFM-140 (Iwatani Corp., Tokyo, Japan). The protein was then analyzed by the Kjeldahl method (AOAC International, 2012).
The fermented rice and rice flour samples were diluted 1/10 (w/v) with distilled water, then the pH was measured using a pH meter (model CG 842, Schott AG, Mainz, Germany). The acidity was analyzed by titration using 0.1 mol L−1 NaOH as the titrant.
Texture and color analyses of khanomjeen The texture and color of the noodles were analyzed at ambient temperature 18 h after preparation. The texture was analyzed using a TA-XT plus texture analyzer (Stable Micro Systems, Godalming, UK). The instrument was equipped with spaghetti tensile grips (A/SPR) for measuring the tensile strength (maximum force, in g) and breaking length (distance at maximum force, in mm) at a test speed 3.0 mm s-1. A cylindrical probe (P/36R) was used to determine the hardness (g) by compression subjected to 60% deformation. Six replicates per sample were analyzed. Six replicates per sample were analyzed to obtain the mean value.
The color of the noodle samples was measured using a Datacolor spectrophotometer (Datacolor, Lawrenceville, NJ, USA) according to the Commission Internationale de l'Eclairage (CIE) color system. The parameters L* (lightness), a* (redness) and b* (yellowness) were recorded using a D65 illuminant (daylight, 65° light angle). All samples were measured in triplicate to obtain the mean value. The whiteness (W) was calculated according to Eq. 1 as described by Lu et al. (2005):
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Statistical analysis The statistical analyses for testing the significance of differences in the mean values of properties between the fermentation methods were conducted using IBM SPSS Statistical software version 25 (IBM Corp., Armonk, NY, USA). The chemical properties of the fermented rice flour made by the traditional and modified methods were compared using the paired t-test (p < 0.05). Significant differences in the texture and color of the noodles made between each fermentation method and the unfermented khanomjeen were determined using one-way ANOVA (p < 0.05).
Metagenomic analysis Bacterial DNA was extracted from the fermented rice and rice flour samples using the Dneasy PowerSoil Kit (Qiagen Sciences, Germantown, MD, USA) according to the manufacturer's protocol. The 16S rRNA gene library was constructed using a Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2 (Agilent Technologies, Santa Clara, CA, USA). The methodology is described in the instructions provided by Illumina Inc. (2020)i) on amplicon PCR, clean-up PCR and index PCR. The V3 and V4 regions of the 16S rRNA gene were the target regions amplified by a pair of DNA primers as follows: 16S Amplicon PCR Forward Primer (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′) and 16S Amplicon PCR Reverse Primer (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′). The PCR products were sequenced using a MiSeq sequencer (Illumina Inc., San Diego, CA, USA). The clustering of the Operational Taxonomic Units (OTUs) was determined using the CD-HIT-OTU programii) and rDnaToolsiii) with a 97%-similarity cut-off. Otherwise, those bacterial taxonomy were defined as “Other”. The OTUs and taxonomy composition were analyzed by QIIMEiv).
Process-dependent changes in protein content, pH and acidity in the traditional and modified rice fermentation methods The removal of rice protein from fermented rice flour has been considered to be closely related to the textural properties of the resulting noodle products (Lu et al., 2003; 2008; Satmalee et al., 2017; Yi et al., 2017; Shompoosang et al., 2019). The rice protein content decreased during both the traditional and modified rice fermentation processes (Fig. 2A and B). The highest rate of decrease was observed during the first day of solid-state fermentation for the traditional method (Fig. 2A, TR1) but in the modified method the rate of decrease was nearly constant during the fermentation process. For the traditional and modified methods, the protein content decreased over the 5 days of the process to 44% and 59% of the initial content, respectively (Fig. 2A and B). These differences suggested that proteolytic bacteria were active under both conditions, although the variety and strength of their proteolytic activity might be different.
The changes in rice protein content (A and B), and pH (grey bars, left hand scale) and acidity (white bars, right hand scale) (C and D) during the different stages of the traditional and modified rice fermentation methods. All experiments were performed in triplicate. Error bars represent standard deviations from the mean. Where error bars are not visible, they are within the symbol. The sampling points are described in Fig. 1.
For the traditional method, the pH increased from 5.69 to 7.24 during the first day of solid-state rice fermentation (Fig. 2C, TR1), then decreased to reach 3.81 at the end of fermentation. This decrease in pH coincided with the increase in acidity during fermentation (Fig. 2C). A similar pH profile was observed for the fermented rice flour production process in our previous study (Shompoosang et al. 2019). The increase in the pH of the fermented rice could have been caused by hydrolysis of the rice protein with the liberation of ammonia by proteolytic bacteria as reported by Boonmee (1989). A similar transient increase in pH was not observed during the modified method (Fig. 2D) where the pH decreased continuously from 5.64 to 3.67 during fermentation (Fig. 2D). The final pH values for the traditional and modified methods were comparable, indicating that the fermented rice flour produced by the modified method could retain the acidic conditions necessary to give the resulting khanomjeen products their characteristic storage stability.
Improvement of texture and color properties of khanomjeen by the modification of rice fermentation method The tensile strength and breaking length, which represent the elastic properties of noodles, were 40% and 30% higher, respectively, in khanomjeen made by the modified rice fermentation method than that of the unfermented rice noodles, but 19% and 26% lower compared with the traditional method, respectively (Table 1). The hardness of khanomjeen made by the modified method was approximately 60% of that made by the traditional method (Table 1), but was still harder than that of the unfermented rice noodles (Table 1). This agreed with studies reporting that the removal of rice protein by fermentation resulted in an increase in the elasticity and hardness of fermented rice noodles compared with the unfermented type. The protein decrease observed in the traditional and modified rice fermentation should result in the selective removal of cluster-like structures of the rice protein body-II in the fermented rice flour products and resultant khanomjeen noodles, that was thought to be important for the creation of characteristic texture of the noodles by forming the uniform gel microstructure consisting of only spherical rice protein body-I (Satmalee et al., 2017). Although no significant change in the starch content was observed in both traditional and modified rice fermentation methods in the present study (data not shown), starch hydrolysis in rice fermentation and/or noodles making processes by amylolytic bacteria could also be indirectly related to the protein degradation and texture formation. Indeed, relationship of rice starch breakage and possible protein degradation to texture features of the noodles was recently reported in Chinese fermented rice noodles (Li et al., 2019). Further investigation is needed to fully elucidate the involvement of rice starch hydrolysis in the texture formation of khanomjeen. Lower elasticity and hardness of khanomjeen made of the modified rice fermentation than that of traditional style in the present study (Table 1), presumably because of the higher protein content of the fermented rice flour (Fig. 2A and B). The negative correlation between protein content in fermented rice flour and the elasticity of khanomjeen products was also suggested in our previous study (Shompoosang et al. 2019).
| Fermentation method | Tensile strength‡ (g) | Breaking length‡ (mm) | Hardness‡ (g) | Whiteness‡ |
|---|---|---|---|---|
| Traditional | 8.51 ± 0.71a | 21.21 ± 1.19a | 831 ± 15a | 73.58 ± 0.13a |
| Modified | 6.92 ± 0.64b | 15.64 ± 1.41b | 476 ± 14b | 81.36 ± 0.06b |
| Unfermented† | 5.10 ± 0.44c | 11.86 ± 0.57c | 364 ± 21c | 81.39 ± 0.12b |
The whiteness of khanomjeen made from fermented rice flour prepared by the modified method was identical to that of the unfermented rice noodles (Table 1). However, the noodles made by the traditional method were pale-yellow (data not shown) and significantly less white than the other two types (Table 1).
Overall, the modified rice fermentation method investigated in the present study produced whiter and softer khanomjeen products compared with the traditionally-made products, while the elasticity was higher than that of the unfermented rice noodles. These color and textural properties would be expected to be preferred by the current consumers of khanomjeen. These quality improvements could be attributed to changes in the microflora, especially in the proteolytic and pigment-producing species in the modified rice fermentation process. We therefore conducted a metagenomic analysis to compare the bacterial composition of the traditional and modified rice fermentation methods investigated in the present study.
Comparison of bacterial composition during the traditional and modified rice fermentation methods The metagenomic analyses revealed a clear difference in bacterial composition and the representative species between the traditional and modified rice fermentation processes (Fig. 3). In particular, Bacillus wiedmannii and Weissella confusa were dominant on the first day of solid-state fermentation (Fig. 3A, TR1), although the relative abundances of these two species decreased markedly during the following fermentation processes (Fig. 3A). Simultaneously, there was a significant decrease in protein content (Fig. 2A) and a significant increase in pH. Thus, these species might play important role in the hydrolysis of the rice protein during the initial stage of fermentation. The pH increase could be attributed to ammonia released by the bacterial amino acid metabolism. As a representative example, ammonia-releasing reactions such as glutamate deamination and urea degradation have been revealed at the genetic level in a Bacillus subtilis strain for producing natto, a Japanese traditional fermented soybean (Kada et al. 2008). It would be interesting to investigate the ammonia release of Bacillus and other bacterial species involved in the fermented rice flour production in the present study. Acinetobacter baumannii, Kurthia gibsonii and Enterobacter tabaci became dominant on the second day of solid-state fermentation (TR2), with Chryseobacterium oranimense being detected on the third day of solid-state fermentation (TR3) (Fig. 3A). These bacteria may also have been involved in the hydrolysis of rice protein, because proteolytic strains of these species or genus have been reported previously (Priest, 1977; Bouvet and Grimont, 1986; Hantsis-Zacharov et al., 2008; Duan et al., 2015; Miller et al., 2016; Júnior et al., 2018; Dey et al., 2019). Intensive culture-based approaches are necessary for the foregoing bacteria to provide direct evidence of the proteolytic activity in rice fermentation process examined in the present study.
Succession of bacterial composition during the rice fermentation processes for the traditional (A) and modified (B) methods. The bacterial metagenome was analyzed by NGS. The legend shows the bacterial species detected by the NGS metagenomic analysis with a relative abundance ≥ 1.0%. The sampling points are described in Fig. 1.
It should also be noted that the yellowish-orange color from the pigment-producing strain C. oranimense has been reported previously (Hantsis-Zacharov, 2008). The Gluconacetobacter liquefaciens species detected in the solid-state fermentation process (TR1 to 3, Supplementary Table S1) includes a strain producing reddish-brown pigments (Navarro and Komagata, 1999). As well as these aerobic bacteria, a facultative anaerobic bacterium, Bacillus subtilis, which could be involved with brownish fermented rice grains (Boonmee 1989), was also detected on the first day of the solid-state fermentation process (Fig. 3A, TR1). Therefore, the growth of these possibly pigment-producing bacteria during the solid-state rice fermentation could be the major cause of the formation of the pale-yellow color in the traditional style khanomjeen products.
Lactic acid bacteria (LAB), such as Enterococcus camelliae, Enterococcus faecalis, Lactobacillus fermentum, Lactobacillus ultunensis and Lactococcus lactis, were detected in the traditional method (TR2 to 3) from the second or third days of solid-state fermentation through to the following submerged fermentation (TF1 and 2) (Fig. 3A). The anaerobic conditions during the submerged fermentation might be preferential for these LAB species which are thought to be involved in acidifying the fermentation environment as shown in Fig. 2C. The acidic environment should help to suppress the growth of non-acid-resistant microorganisms, such as Bacillus species, that are undesirable in fermented rice flour. Many Bacillus species are known to be potent producers of not only proteolytic but also of amylolytic enzymes (Priest, 1977) which may liquefy the khanomjeen product. Indeed, our results have shown that the relative abundance of Bacillus species decreased from the second day of solid-state fermentation (TR2) while LAB started to be detected at around the same time.
W. confusa and Lc. lactis were predominant in the early stage of the first submerged fermentation of the modified method (MR1) but their relative abundance decreased as the fermentation process progressed (Fig. 3B). Instead, Lactobacillus plantarum was detected from the second day of the first submerged fermentation (MR2), but its relative abundance was lower than that of the other LAB species. More importantly, the relative abundance of L. fermentum increased significantly at the same time (MR2) then continued to be dominant until the end of the fermentation process (MR3, MF1 and 2). A. baumannii was the second most dominant species from the late stages of the first submerged fermentation (MR3) and the second submerged fermentation (MF1 and 2) (Fig. 3B).
Overall, replacing the solid-state fermentation used in the traditional method with submerged fermentation was effective not only for enhancing the growth of LAB but also for suppressing the growth of possibly pigment-forming aerobic bacteria and Bacillus species that can damage the perceived quality of khanomjeen products. Indeed, the relative abundances of possibly pigment-producing bacteria, such as C. oranimense and G. liquefaciens, and Bacillus species in the modified method, were considerably lower than of those in the traditional method (Supplementary Table S1). Enhancing the growth of LAB from the initial stage of the first submerged fermentation enabled the pH to decrease continuously as shown in Fig. 2D. The change in the dominant bacteria from W. confusa and Lc. lactis (MR1) to L. fermentum and A. baumannii (MR2 to MF2) could be attributed to the difference in acid tolerance of each species. The growth of B. wiedmannii, which might be involved in the hydrolysis of rice protein on the first day of solid-state fermentation in the traditional style (Fig. 3A, TR1), was significantly suppressed in the modified method (Fig. 3B). However, LAB such as W. confusa, Lc. lactis, L. plantarum, and L fermentum, as well as A. baumannii detected in the modified method could be involved in the hydrolysis of rice protein that is necessary to provide the khanomjeen product with its elastic texture as shown in our analysis (Table 1), because proteolytic strains of these species have been reported previously (Bouvet and Grimont, 1986; Khalid and Marth 1990; Sasaki et al., 1995; Flambard et al., 1998; Miller et al. 2016).
It can be concluded that the submerged fermentation processes used in the modified method were advantageous for providing the khanomjeen product with a white appearance and soft texture. Bacillus species and possibly pigment-producing bacteria that may undermine the perceived quality of the khanomjeen product were effectively suppressed in the modified rice fermentation process, although it still allowed the growth of bacteria that may be responsible for the hydrolysis of rice protein that is necessary for maintaining the elastic texture of the khanomjeen noodles. Thus, the modified rice fermentation method should be recommended to khanomjeen producers for improving their product quality to meet current consumer demands. The metagenomic analysis was also useful in obtaining a better understanding of the difference between the traditional and modified rice fermentation regarding bacterial composition and the representative species possibly involved in the special characteristics of khanomjeen, its texture and color. However, further comprehensive study is necessary to clarify the succession of bacterial diversities during the traditional and modified rice fermentation processes and examine their correlations with the fermentation condition such as pH, as well as with the fluctuation of fermentation-related components such as organic acids and rice protein hydrolyzing activities. It will lead to the identification of key factors controlling the bacterial activities in the fermentation processes. Also, isolation and characterization of the bacterial species from the rice fermentation processes as well as their growth and enzymatic property evaluations are needed to fully elucidate their influence on the texture, color and other sensory qualities of fermented rice flour and the khanomjeen products. Such knowledge will help improve the control of the fermentation processes required for producing khanomjeen that can meet changing consumer preferences.
Acknowledgements This work was financially supported by Pichai F and B Partnership, Limited, Nakhon Pathom, Thailand. We thank Philip Creed, PhD, from Edanz Group (https://en-author-services.edanz.com/) for editing a draft of this manuscript.
Conflict of interest There are no conflicts of interest to declare.
