Screening and application of lactic acid bacteria and yeasts with l‐lactic acid‐producing and antioxidant capacity in traditional fermented rice acid

Abstract In the study, Lactobacillus paracasei H4‐11, Lactobacillus fermentum D1‐1, Lactobacillus casei H1‐8, Lactobacillus reuteri H2‐12, and Kluyveromyces marxianus L1‐1 were screened from traditional fermented rice acid based on several indicators: L‐lactic acid production capacity (13.46 ~ 19.69 g/kg), antioxidant capacity (DPPH clearance ability of 35.36 ~ 56.89%), and savory flavor indicators. Glutinous rice, quinoa, barley rice, and brown rice were selected to carry out rice acid fermentation. Different viable lactic acid bacteria and yeasts were screened, respectively, in the saccharification, acidification, alcoholization, and late acidification stages. Rice acid fermented with L. paracasei H4‐11 and K. marxianus L1‐1 in glutinous rice showed the high L‐lactic acid content (23.09 g/kg). The DPPH free radical scavenging ability in rice acid fermented with L. fermentum D1‐1 and L. paracasei H4‐11, respectively, reached 34.27% and 33.05% in 96 hr. Although quinoa rice acid had the highest L‐lactic acid content (33.74 g/kg) and the DPPH free radical scavenging ability (60.10%), it had the poor taste due to the high astringency intensity and bitter intensity. Rice acid fermented with both L. paracasei H4‐11 and K. marxianus L1‐1 in glutinous rice showed the highest savory flavor and had the lowest astringency and bitter. L. paracasei H4‐11 and K. marxianus L1‐1 were the potential strains for the fermentation of rice acid. These results promote the industrial development of Chinese rice acid. Practical applications Rice acid as a functional nondairy fermented product is widely accepted by Chinese consumers. However, rice acid fermentation is still a traditional spontaneous process, which causes instability in its flavor and quality. Lactobacillus paracasei H4‐11 and Kluyveromyces marxianus L1‐1 screened in this study contributed to the improvements in the flavor and antioxidant capacity of rice acid. In addition, glutinous rice was confirmed as the suitable fermentation material of rice acid in the study.


| INTRODUC TI ON
Sour soup, as one of the traditional foods of Miao and Dong Nationalities, is popular in Guizhou, China. Sour soup mainly includes "white-acid" (rice acid) prepared with glutinous rice or nonglutinous rice and "red-acid" prepared with tomato or red pepper as raw materials. Especially, a composite base prepared with two sour soups is used in cooking a large number of foods with different flavors. Rice acid plays an important role in Chinese acidic grain-fermented soup since it has the antifatigue, anti-aging, and immunoregulatory functions and can lower cardiovascular disease risks (Yuan, 2010).
In general, rice acid was prepared through the fermentation process involving lactic acid bacteria (LAB) and yeasts. Fermentation is a common food-processing method that preserves foods and endows them with special flavors (Shen et al., 2018). Previous studies demonstrated that LAB and yeasts had good probiotic properties and produced a variety of compounds such as organic acids, acetaldehyde, methyl alcohols, and esters (Rai et al., 2019;Yadav et al., 2016). A recent study demonstrated that the combination of LAB and Saccharomyces cerevisiae produced a flavor profile close to that of the product fermented with Obushera (Mukisa et al., 2017). In particular, L-lactic acid was the most important acid since it could be produced with immobilized Lactobacillus casei and agro-industrial wastes as carbon and nitrogen sources (Thakur et al., 2019). The main nutritional component in foods fermented with LAB is lactic acid. Notably, the human body has only L-lactic acid dehydrogenase, which can only catalyze L-lactic acid. The excessive intake of D-lactic acid or DL-lactic acid can cause metabolic disorders in the human body and adverse reactions such as acidosis (Narayanan et al., 2004). Therefore, more fermented foods containing more L-lactic acid should be developed. LAB and yeasts assist in the release and biotransformation of those health-beneficial compounds via fermentation, increase their bioavailability, and reduce sugar contents in fermented foods (Shen et al., 2018). The fermentation process of rice acid was optimized (Yuan, 2010). However, screening strains of LAB and yeasts from in rice acid has not been reported. The fermentation process with LAB and yeasts is a potential method of improving the quality indicators of rice acid, including acid-producing capacity and flavor characteristics.
Fermentation with LAB and yeasts is a common process of fermented foods and can improve the potential quality such as antioxidant capacity (Shen et al., 2018;Wang et al., 2014). It is necessary to screen the strains with high antioxidant ability. Menezes et al. (2020) evaluated the phytate hydrolysis and antioxidant activities of some yeast strains and found that 13 strains showed positive results and might improve the nutrient availability of plant-based foods. Hamidi et al. (2020) indicated that Rhodotorula mucilaginosa sp. GUMS16 had the antioxidant and antiproliferative activities. Shen et al. (2018) demonstrated the increases in phenolic, fatty acid, and phytosterol contents and a special antioxidant activity in sweet potato fermented with Lactobacillus acidophilus. In the study, common assays and methods were used to screen LAB and yeasts with high antioxidant capacity from fermented rice acid.
However, rice acid fermentation is still a traditional process with a long fermentation period and susceptible to the raw material quality, bacterial contamination, and environmental factors. Therefore, the quality or flavor of rice acid is not stable. Our preliminary investigation showed that rice acid fermentation microorganisms mainly included Lactobacillus, yeasts, acetic acid bacteria, and Leuconostoc (Jiang et al., 1997).
Therefore, in order to shorten the fermentation period and improve the quality of rice acid, LAB and yeast strains with L-lactic acid-producing and antioxidant capacity were screened from traditional fermented rice acid and identified in the study. In addition, this study confirmed that glutinous rice was a suitable raw material for rice acid fermentation.

| Materials
Two types of fermentation samples were selected in the study, including high-temperature fermentation samples (D1, D1, and D3 from Congjiang, Guizhou, China) and low-temperature fermentation sample (H1, H2, H3, H4, L1, M1, and M2 from Huangping and Kaili, Guizhou, China). Figure 1 and Table 1 show the process and sample characteristics of different rice acid. The rice acid fermentation process involves two steps. First, a mixture of rice and water is fermented in a fermentation jar at room temperature for 30 days to obtain the "primary rice acid soup." Second, the primary rice acid soup was subjected to additional fermentation at 28 ~ 35°C for 4 ~ 7 days, thus obtaining in a final rice acid soup product. Samples L1, M1, and M2 came from the enterprise, and the remaining 7 samples were collected from farmers. The samples were produced locally by individuals (farmers) or generated via a standardized process (enterprises) and had different flavors due to different fermentation process. MRS agar medium, potato dextrose agar (PDA) media, MRS liquid medium, and PDB liquid medium were obtained from Bio-way Biotechnology Co., Ltd. (Shanghai, China).
Other chemicals were purchased from Sigma-Aldrich Co., Ltd., USA.

| Screening of LAB strains and yeast strains
Isolation and purification of LAB strains and yeast strains LAB strains were isolated with MRS medium. Modified MRS liquid culture medium was prepared with beef extract (10 g), peptone (10 g), yeast extract (5 g), anhydrous glucose (20 g), Tween-80 (1 ml), dipotassium phosphate (2 g), sodium acetate (5 g), diammonium hydrogen citrate (2 g), magnesium sulfate (0.58 g), and manganese sulfate (0.25 g) and 1 L of distilled water. CaCO 3 (10 g) was added to check whether there was a calcium-soluble circle. After the pH was adjusted to 6.4 ± 0.2, the prepared medium was sterilized at 121°C for 15 min. The modified MRS medium contained 2% agar.
LAB strains were isolated according to the following procedure.
The rice acid samples were obtained on the site, sealed in sterile sampling bottles at 4°C, and immediately carried to the laboratory. The samples were immediately diluted by 10 3 , 10 4 , and 10 5 times with sterile water and spread on modified MRS solid medium, which was incubated at 37°C for 48 hr. The suspected colonies were picked and further purified with streak plate method. The above steps were repeated for 4 to 5 times until a single colony was obtained. The purified single colonies were stored in MRS slant culture medium at 4°C until use. Subcultures were performed every 4 weeks for further analysis.
Yeast strains were isolated according to the following procedure.
A sample of rice acid was diluted and spread on PDA medium. The medium was cultivated at 30°C for 3-5 days. Suspected yeast colonies with different colony characteristics were picked and purified with streak plate method.

Morphological, physiological, and biochemical characteristics
LAB strains were checked through microscopic observations according to the following procedure. Colonies were picked with needles onto clean slides. After the cells were fixed, gram staining was performed according to the following procedure. After staining with crystal violet and iodine, 95% alcohol and saffron solution was, respectively, added on the slides for decoloring and counterstaining. Then, the slides were placed under the microscope for bacterial observation. The observation results were interpreted based on their staining results. Purple and red, respectively, indicated Gram-positive and Gram-negative bacteria.
The photograph was captured to record the bacterial morphology. Catalase-negative and Gram-positive results indicated LAB strains. Catalase test was performed according to the following procedure. Colonies were picked with an inoculation needle and placed on a clean glass slide. Hydrogen peroxide solution (3%) was added. Catalase-positive and Catalase-negative results were determined according to the appearance of air bubbles within 30 s.
Yeast strains were checked through microscopic observations according to the following procedure. Suspected yeasts were transferred into a water-immersed slide for microscopic observations. The strains with typical yeast characteristics and good gas production performance were cultivated in slant tubes, and then, 30% glycerol was added according to ratio of 1:1 in the tubes, which were preserved in a laboratory refrigerator at −80°C.

Acid production assay
The activated strain was inoculated in MRS liquid medium according to 3% inoculation volume and cultured at 37°C for 16 hr, and then, the pH was measured. The total acids were measured according F I G U R E 1 Rice acid processing process to the method by Cao et al. (2017). The purity of L-lactic acid was Survival at pH 2.0 and pH 3.0 According to the method described by Ramos et al. (2013), activated strains were, respectively, inoculated in MRS liquid medium with different pH values (pH 2.0 and pH 3.0) according to the inoculation TA B L E 1 Samples of rice acid obtained with different fermentation methods

Sample Location
High temperature/Low temperature Farmer/ Enterprise Processing process

H1
Huangping Low temperature (8°C-12°C) Farmer Compared to Figure 1, the raw material for the first fermentation is rice and flour.

H2
Huangping Low temperature (6°C-10°C) Farmer Compared to Figure 1, the raw material for the first fermentation is rice and the second fermentation takes 6 d.

H4
Huangping Low temperature (8°C-12°C) Farmer Compared to Figure 1, the raw material for the first fermentation is rice and the second fermentation takes 6 d.

L1 Kaili
Low temperature (10°C-14°C) Enterprise Compared to Figure 1, the raw material for the first fermentation is rice and the second fermentation takes 5 d.

M1 Kaili
Low temperature (13°C-17°C) Enterprise Compared to Figure 1, the raw material for the first fermentation is rice and the second fermentation takes 7 d.

M2 Kaili
Low temperature (13°C-17°C) Enterprise Compared to the processing of M1, the second fermentation 6 d.

D1
Congjiang High temperature (60°C-75°C) Farmer Compared to Figure 1, the second fermentation takes 7 d and its fermentation near the cooktop indoor.

Congjiang
High temperature (50°C-70°C) Farmer Compared to Figure 1, the second fermentation takes 3 d and its fermentation near the cooktop outside.

D3 Congjiang
High temperature (60°C-80°C) Farmer Compared to Figure 1, the second fermentation takes 5 d and its fermentation near the stove indoor.
F I G U R E 2 (a) The morphology of lactic acid bacteria colonies. (b) The microstructures of 15 different lactic acid bacteria under the microscope volume of 3% and cultivated at 37°C for 16 hr, and then, OD 600 nm was measured.

Bile salt tolerance assay
Activated strains were inoculated according to the inoculation volume of 3% in MRS liquid media containing different concentrations of bile salts (0.3 g/L and 0.6 g/L) and cultivated at 37°C for 16 hr, and the OD 600 nm was measured.

Antioxidant property assay
Preparation of the sterile saline suspension of LAB and yeasts. Single colonies of LAB and yeasts with good growth conditions obtained through the above separation steps were respectively inoculated into 3 ml of MRS and PDB liquid medium and respectively cultivated at 37°C for 18-20 hr and 30°C for 48-72 hr. The culture medium was inoculated in 50 ml of MRS and PDB liquid medium according to the inoculation volume of 2% and allowed to stand for 18 hr to obtain the culture medium of the strains. The cells were collected by centrifugation at 1,000 rpm for 6 min at 4°C. After washing twice with sterile physiological saline, the cells were resuspended and the cell density was adjusted to 1.01 ± 0.05 × 10 8 CFU/ml.
Determination of the ability to scavenge 1,1-diphenyl-2trinitrophenylhydrazine (DPPH). LAB strains and yeast strains were tested to explore their ability to scavenge free radicals (DPPH) according to the following steps. First, 2 ml of saline suspension of lactic acid bacteria to be tested was added to 2 ml of 0.4 mmol/L DPPH solution, mixed well, placed in the light-shielding environment at room temperature for 30-min reaction, and then centrifuged at 8,000g for 10 min. Then, the OD 517 nm of the supernatant was measured. In the blank group, DPPH was replaced by an equal volume of absolute ethanol. The control group samples were replaced by an equal volume of distilled water. A mixture of the equal volume of distilled water and absolute ethanol was used as the calibration sample to adjust the OD 517 nm to zero. The experiments were performed in triplicate.
The scavenging rate is calculated according as: where A control is the absorbance of the control group, and A sample is the absorbance of the sample group. Determination of hydroxyl radical scavenging ability (OH -). According to the reported method with minor modifications (Mu et al., 2018), hydroxyl radical scavenging ability was determined. The reaction mixture contained 1.0 ml of PBS (0.02 mol/L, pH = 7.4), 0.5 ml of o-phenanthroline (2.5 mmol/L), 0.5 ml of FeSO 4 (2.5 mmol/L), 0.5 ml of H 2 O 2 (2.0 mmol/L), and 0.5 ml of bacterial suspension. After reaction for 1 hr in water bath at 37°C, the reaction mixture was centrifuged at 4,000 g for 10 min and OD 536 nm of the supernatant was measured as A sample . OD 536 nm of the blank in which the sample was replaced with distilled water was measured as A blank . Distilled water was used to replace H 2 O 2 as the control group to measure OD 536 nm , which was recorded as A control .

Screening of aroma-producing yeasts
After the isolation and purification of the yeast strains, they were respectively cultured in PDB liquid medium in a shaker (30°C, 120 r/min) for 1 d. The strains capable of producing mellow aroma and ester aroma were screened. The aroma profiles were investigated by sniffing and sensory evaluation, and the keyway was to smell the aroma according to the previous method (Wang, 2016).

Bacterial and fungal DNA extraction
Bacterial and fungal DNA extraction kits were, respectively, used for DNA extraction according to the kit instructions of BayGene/ Sigma-Aldrich.
After extracting the fungal DNA, PCR was performed with the primers of NL1 (5ʹ-GCA TAT CAATAAGCG GAG GAA AAG-3ʹ) and NL4 (5ʹ-GGTCCG TGTTTC AAG ACG G-3ʹ). PCR reaction system (25 μl) was composed of 2 μl of DNA template, 1 μl of NL1, 1 μl of NL4 (10 μmol/L) each, 12.5 μl of 2 × Taq PCR MasterMix, and 8.5 μl of ddH 2 O. PCR amplification program was set as follows: predenaturation at 95°C for 5 min, 40 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 40 s, and extension at 72°C for 30 s, followed by the extension at 72°C for 10 min. PCR products were detected by 1% agarose gel electrophoresis and sequenced with the primers (NL1 and NL4). Fungal ITS sequence was completed by Thermo Fisher Scientific Inc.

Construction of phylogenetic tree
After sequencing PCR products, the obtained gene sequences were checked with Chromas 2.33 software and compared with the known sequences in GenBank by BLAST software for strain identification.
The sequences were aligned with the corresponding model strains and outgroup strains in Dnaman 5.0 software, and the UPGMA method was used for 1000 bootstrap tests to construct a phylogenetic tree (Suzuki et al., 2002).

| Sensory evaluation and electronic tongue measurement
Quantitative descriptive sensory analysis method was used to evaluate the rice acid samples according to a ten-point interval scale (0 = none, 9 = extremely strong). The sensory evaluation panel was composed of 15 females and 5 males (20-30 years old).
Sensory sessions took place in a sensory laboratory, which met international standards for a test room. According to the discussion and pre-experiment results with the panel, the sensory evaluation was performed in coded tasting cups containing 100 ml of water with 1.5 g of samples. Samples were presented in a random sequence (Lin et al., 2018). Meanwhile, the aroma profiles were investigated by sniffing (Wang, 2016).

| Statistical analysis
All experiments were performed in triplicate. The data are expressed as mean ± SD. The mean and standard deviation (SD) were calculated, and other statistical analyses were carried out in Microsoft R Excel 2010 software package and SPSS 20.0.  TA B L E 2 Acid-producing capacity of lactic acid bacteria and yeasts were rod-shaped (long rod, medium rod, and short rod). The minority of them were oval, and only a few bacteria were spherical. Figure 3a shows the diverse colony morphology, and red, yellowish, and white colonies were observed. The colonies of yeast strains were spherical or wrinkled. The microstructures of yeast strains under the microscope showed significant differences. Figure 3b shows 12 yeast strains with the shapes of spheroids or ellipsoids.

| Acid-producing capacity of different LAB and yeasts
In the study, 86 potential LAB strains were selected for the analysis of physiological and biochemical characteristics (the data were not shown). Sixty-seven strains were Gram-positive and catalasenegative strains. Nineteen strains were Gram-negative and catalase-positive strains, indicating that 19 strains were not lactic acid bacteria. Based on the calcium-soluble circle, 67 strains of LAB were screened in terms of pH and acid production ( Table 2).
The acid content in different strains was 5.57-19.69 g/kg. The strain H4-11 had the highest acid content (19.69 g/kg), and the strains of H2-1, H1-8, H1-6, H1-3, H3-9, H3-5, and H3-10 had the relatively high acid content. Consistently, in the previous study (Moon et al., 2012), Lactobacillus paracasei subcasei CHB2121 produced 192 g/L-lactic acid in a medium containing 200 g/L glucose. The trends of pH value of LAB showed the significant differences. The pH range of different strains was 3.85-5.92. The pH and total acid content were significantly different among different strains (p < .05). Thirty-three yeast strains had a weaker acid production capacity than LAB strains. The acid contents of yeast strains were 0.92-11.33 g/kg. The acid contents of the strains h1-1, h1-2, h2-2, h3-2, h3-3, l1-1, l1-3, m1-2, m2-1, and m2-2 were higher than 6.00 g/kg, indicating that the acid production capacity of LAB was significantly different from that of yeast strains (p < .05). Consistently, in the previous study (Domizio et al., 2016), LAB strains showed the higher production of lactic acid than yeast strains in fermented beer.

| Analysis of the aroma-producing capacity of yeast strains
Different yeast strains produced different flavor characteristics (Table 3). The volatile flavors produced by yeast strains screened from rice acid were alcoholic flavor, ester flavor, fruit sweet flavor, and acidity formed in the acid production process. In terms of the aroma-producing performance, 30 yeast strains were selected for the comparative analysis through the sensory evaluation and sniffing method (Wang, 2016). Finally, 6 yeast strains with good aromaproducing capacity (h22, h31, h32, h33, l11, and m21) were selected for antioxidant experiments. In the future study, we will focus on volatile flavor components such as ethyl acetate and acetic acid, which promote the flavor accumulation of rice acid inoculated with yeast strains.

| Survival test at pH 2.0 and 3.0 and bile salt tolerance assay
Based on the acid-producing properties of LAB, 37 LAB strains with large acid production and low pH were selected for acid and bile salt resistance experiments ( Except M1-6, all strains grew well under 0.3 g/L bile salt. The previous study (Kaktcham et al., 2018) demonstrated that the bile salt tolerance was a fundamental property required for probiotic LAB to survive. In our study, we screened the potential functional LAB strains for rice acid fermentation.

| Antioxidant capacity
The 16 LAB strains and 6 yeast strains were selected for antioxidant capacity tests with DPPH, FRAP, and hydroxyl radical (  (Czyżowska et al., 2017), some strains in our study had the higher antioxidant activity including DPPH scavenging ability and FRAP reducing ability and will be applied in rice acid fermentation in the future study.

| Growth curves of different LAB strains and yeast strains
The high-quality LAB strains H4-11, H4-12, M1-8, and D1-1 were selected in terms of acid production performance and antioxidant indicators. The growth curves were plotted with the colony count and pH of the 4 LAB strains (Figure 4a-d). In fact, a minimum level of 10 6 CFU of viable probiotic bacteria per milliliter or gram of foods is accepted. The growth trend of LAB strain H4-11 was consistent with that of Gallo et al. (2018). In a similar report, Lactobacillus helveticus KLDS1.9204 had the highest acid production among 27 LAB strains (Ai et al., 2015). Based on comprehensive acid production, alcoholic and ester-producing properties, and antioxidant capacity, a high-quality yeast L1-1 strain was selected as OD 600 nm in the growth curves ( Figure 4e) indicated that yeast strain L1-1 had the better growth ability than other yeast strains.

| Identification of different LAB strains and yeast strains with molecular biological tools
The high acid production in Chinese traditional fermented rice acid is ascribed to the strains with the high acid-producing capacity. Due to the interactions among different strains, the unique taste and aroma are formed in fermented foods (Qin & Ding, 2007). A phylogenetic tree was constructed based on 16S rRNA and ITS rRNA sequences of strains ( Figure 5). The identified strains included TA B L E 4 Determination of acid at pH 2.0 and 3.0 and bile salt at 0.3 g/L and 0.6 g/L tolerance of lactic acid bacteria  (Kerry et al., 2018). Although some strains were reported, the strains screened by different systems had different physiological and biochemical characteristics, such as L. paracasei H4-11 in this study. When the glucose content in the medium was not high, the acid content was 19.69 g/kg in our study. Consistently, in the previous study (Hassan & Bullerman, 2008), L. paracasei isolated from sourdough bread had the unique acid-producing capacity and antifungal activity.
Based on the consideration of all the indicators in this study, four LAB strains (H4-11, H1-8, D1-1, and H4-12) with the good characteristics were finally selected to establish a phylogenetic tree. Strain H4-11 was L. paracasei whose sequence homology with L. paracasei FX-6 reached 99.79%. Strain H1-8 was L. casei whose sequence homology with L. casei 20130913 reached 99.58%. The sequence homology between L. fermentum D1-1 and L. fermentum lfh25 was 98.50%, and the sequence homology between L. reuteri H4-12 and L. reuteri JCM1112 was 92.18%. H4-12 might be a new strain, but it requires to be verified in subsequent experiments. Based on the results of fungal ITS sequences, the identified yeast strains included K. marxianus, P. fermentans, Rhodotorula spp., Candida, P. pastoris, P. mansurica, and C. albicans. L1-1 selected for the construction of a phylogenetic tree was an ester-producing and alcohol-producing strain. Strain L1-1 was determined as K.

| Strain characteristics in rice acid fermented with different raw materials
The traditional fermentation way of rice acid was simulated. After four days of fermentation, the quantities of LAB and yeast in rice acid fermented with different strains in different raw materials showed significant differences (p < .01, Figure 6). The quantity of L. paracasei H4-11 peaked after 24-hr fermentation and reached 2.37 × 10 9 CFU/ml in barley rice acid. In rice acid fermented with the other three cereals (glutinous rice, quinoa and brown rice), the quantity of viable bacteria peaked in 24 hr when only Lactobacillus was inoculated in the rice acid fermentation. Rice acid fermented with four grains showed the consistent growth curves when L. paracasei H4-11 was inoculated in rice acid. However, the quantities of colonies were different, indicating that L. paracasei H4-11 had the different growth mechanism among different raw materials. Rice acid, respectively, inoculated with L. fermentum D1-1 and L. paracasei TA B L E 5 Antioxidant capacity included scavenging ability of DPPH, FRAP, and OH-of lactic acid bacteria and yeasts H4-11 showed significant differences in the quantities of LAB, indicating that the fermentation mechanisms of the strains were different. Interestingly, L. fermentum D1-1 was more suitable for the fermentation of barley rice and brown rice. The colonies in rice acid fermented with barley rice and brown rice at 24 hr were significantly more than those in rice acid fermented with other grains, but rice acid fermented with four cereals showed the consistent trend of the quantity of colonies after 96-hr fermentation. In rice acid fermented with L. paracasei H4-11 (or L. fermentum D1-1) and K. marxianus L1-1, the quantities of LAB and yeasts showed the similar changing trends.
In the first day, the quantities of LAB and yeasts showed an upward trend since LAB strains and yeast strains could supplied nutrients to each other. In the second day, the quantities of LAB and yeasts declined because the two strains competed substrates. After three days, an upward trend appeared again since the accumulation of various nutrients in the fermentation system provided a suitable growth environment for LAB and yeasts. However, after 4 days, LAB and yeasts declined since the nutrients generated by yeasts were mainly utilized by LAB (Mugula et al., 2003). Interestingly, no growth promotion phenomenon was observed in the second and third days. The symbiosis and competition phenomenon was observed in the early fermentation stage. Ishii et al. (1999) had discovered that L. paracasei subsp. tolerans and K. marxianus promoted the symbiosis with each other during the fermentation of Chigo. Until the fourth day of the late fermentation period, yeasts appeared to promote the growth of LAB. Therefore, the taste and aroma of rice acid showed a more stable flavor after four days, indicating that 4 days could be the proper end of rice acid fermentation.

| L-lactic acid-producing performance characteristics in rice acid fermented with different raw materials
L. paracasei H4-11 and L. fermentum D1-1 showed different L-lactic acid production properties in the fermentation with different raw materials: glutinous rice, quinoa, barley rice, and brown rice ( Figure 7). Lactobacillus had been reported to produce L-lactic acid (Bernardo et al., 2016). The purity of L-lactic acid was 96% in our study. Quinoa had the highest L-lactic acid production performance. could increase the production of L-lactic acid. Consistently, the previous study (Plessas et al., 2008)

| Electronic tongue measurement in rice acid fermented with different raw materials
The sweetness intensity showed no significant difference among rice acid fermented with different raw materials (Figure 9), indicating that the rice acid fermentation system was mature so that there was no excess starch to be converted into glucose. The most important flavor was sour, which showed significant differences among various samples. Among rice acid fermented with L. fermentum D1-1, the sourness intensity of barley rice and quinoa was more prominent than that of glutinous rice and brown rice inoculated with L. paracasei H4-11. The sourness intensity in rice acid synergistically fermented with L. paracasei H4-11, and K. marxianus L1-1 was more prominent than that in rice acid inoculated with L. paracasei H4-11, indicating that L. paracasei H4-11 and K. marxianus L1-1 played a crucial role in the sourness formation in the rice acid fermentation. The sourness intensity measured by electronic tongue was consistent with the determined concentration of L-lactic acid in this study. Interestingly, in terms of the savory intensity, the F I G U R E 6 Dynamic changes in the quantity of (a) Lactobacillus and (b) K. marxianus of L. paracasei H4-11 and K. marxianus L1-1 fermented rice acid compared with L. fermentum D1-1 and K. marxianus L1-1 fermented rice acid by different raw materials included glutinous rice, quinoa, barley rice, and brown rice flavor in rice acid synergistically fermented with LAB and yeasts was stronger than that in rice acid fermented with single LAB, indicating that K. marxianus L1-1 could improve the flavor of rice acid. Consistently, a recent study (Martínez et al., 2017)  proved that K. marxianus could produce ethanol during saccharification and fermentation in lignocellulosic materials. The bitterness intensity and astringent taste intensity showed the significant differences between rice acid of quinoa and barley rice mainly due to the taste of the raw materials themselves. Both quinoa and barley F I G U R E 7 (a) The capacity of L-lactic acid production of L. paracasei H4-11 and K. marxianus L1-1 fermented rice acid by different raw materials included glutinous rice, quinoa, barley rice, and brown rice. (b) The capacity of L-lactic acid production of L. fermentum D1-1 and K. marxianus L1-1 fermented rice acid by different raw materials included glutinous rice, quinoa, barley rice, and brown rice F I G U R E 8 The DPPH free radical scavenging ability in different strains fermented rice acid (L. paracasei H4-11, L. fermentum D1-1, L. paracasei H4-11, and K. marxianus L1-1, L. fermentum D1-1, and K. marxianus L1-1) by 4 raw materials included glutinous rice, quinoa, barley rice, and brown rice rice had a high astringent taste intensity. Glutinous rice exhibited a unique flavor characteristic, indicating that glutinous rice was more suitable for the joint fermentation of Lactobacillus and K.
marxianus L1-1. The different intensities of sourness, sweetness, bitterness, astringent taste, and savory indicated that glutinous rice was more suitable for the fermentation system of L. paracase H4-11 and K. marxianus L1-1. The flavors produced by the single inoculation with L. paracasei or K. marxianus had been reported (Crafack et al., 2013). However, the mixed fermentation flavors of L. paracasei and K. marxianus were not reported. Therefore, the collaborative fermentation of L. paracase H4-11 and K. marxianus L1-1 selected in this study provides a theoretical support for the unique flavors of Chinese traditional fermented rice acid.

| CON CLUS ION
In this study, we screened L-lactic acid-producing lactic acid bacteria and aroma-producing yeasts from rice acid and applied them improving the quality of rice acid for the first time. According to conventional morphological, physiological, and biochemical characteristics, L-lactic acid production capacity, antioxidant capacity, and flavor characteristics, 16S rRNA, and fungal ITS sequence analysis was performed to screen the strains with high L-lactic acid production performance. Four LAB strains were, respectively, identified as L. paracasei H4-11, L. fermentum D1-1, L. casei H1-8, and L. reuteri H2-12, and a yeast strain was identified as K. marxianus L1-1. Glutinous rice, quinoa, barley rice, and brown rice were selected to perform fermentation experiments. Rice acid synergistically fermented with L. paracasei H4-11 and K. marxianus L1-1 in glutinous rice showed the highest L-lactic acid concentration (23.09 g/kg). Rice acid fermented with both L. paracasei H4-11 and K. marxianus L1-1 in glutinous rice showed a unique flavor and high antioxidant capacity. Therefore, L.
paracasei H4-11 and K. marxianus L1-1 were potential strains for the fermentation of rice acid. We will further explore the mechanism of synergistic fermentation of L. paracasei H4-11 and K. marxianus L1-1 in the future study.

ACK N OWLED G M ENTS
This work was financially supported by Technology platform and tal-

CO N FLI C T S O F I NTE R E S T
All authors declare that they have no conflicts of interest.

E TH I C A L S TATEM ENTS
This study does not involve any human or animal testing. F I G U R E 9 The analysis of taste substances in different strains fermented rice acid (L. paracasei H4-11, L. fermentum D1-1, L. paracasei H4-11 and K. marxianus L1-1, L. fermentum D1-1, and K. marxianus L1-1) by 4 raw materials included glutinous rice, quinoa, barley rice, and brown rice