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. 2022 Jun 27;59(8):3031–3042. doi: 10.1007/s13197-022-05512-2

Impact of a novel probiotic Lactobacillus strain isolated from the bee gut on GABA content, antioxidant activity, and potential cytotoxic activity against HT-29 cell line of rice bran

Mohamed Ghamry 1,2, Ahmed Fathy Ghazal 1, Qais Ali Al-Maqtqri 1, Li Li 1,✉, Wei Zhao 1,✉
PMCID: PMC9304478  PMID: 35872742

Abstract

Rice bran was fermented with Lactobacillus apis, isolated from the bee gut as a novel probiotic strain, and Saccharomyces cerevisiae to investigate the relationship between its metabolites and antioxidant activity, nutraceutical value, and cytotoxic activity against the HT-29 cell line. The findings showed that L. apis improved the antioxidant activity (DPPH of 37.73%) and antioxidant capacity (ABTS of 37.62 mg Trolox/g,), as well as, hydroxyl radical-scavenging activity (91.55%) of rice bran compared to S. cerevisiae. The metabolic analysis of volatile compounds revealed an increase of alcohols and lactones in the samples fermented with S. cerevisiae. While the samples fermented with L. apis displayed an increase of ketones, esters, and thiazoles. On the other hand, L. apis and S. cerevisiae exhibited a significant ability to increase γ-aminobutyric acid during different fermentation times. Compared with non-fermented samples (18.54%), L. apis increased the cytotoxic activity of rice bran against the HT-29 cell line to 34.17%, and S. cerevisiae to 31.34%. These results suggest that the fermentation of rice bran with S. cerevisiae and L. apis provides a promising strategy to improve the antioxidant activity and nutraceuticals of rice bran, and a potential source for plant-based pharmaceutical products.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-022-05512-2.

Keywords: Lactobacillus apis, Saccharomyces cerevisiae, Rice bran, Antioxidants, Cytotoxic activity

Introduction

Nutraceuticals and functional food have received significant interest in developing bioactive components that can play a promising role as therapeutic agents against many diseases (Ashraf et al. 2022). Cereal processing by-products, a low-cost source of nutritionally valuable components, such as dietary fibers, proteins, minerals and antioxidants, are promising materials to be valorised to dietary supplements for the food, nutraceutical, and pharmaceutical industries (Vaitkeviciene et al. 2021). Rice bran and its active compounds, such as γ-oryzanol, tocopherol, adenosine, quercetin, ferulic and coumaric acids, play a role as a functional food having the wild interest in its functional aspects and health qualities. Rice bran contains approximately 10–15% of protein, and the amino acid composition of rice bran is closer to the recommended model of the Food and Agriculture Organization (FAO)/World Health Organization (WHO). Its nutritional value is comparable to soybeans and egg proteins (Yu et al. 2019). In addition, rice bran is considered a good source of healthy oil, containing approximately 12–18% of oil with 47% monounsaturated, 33% polyunsaturated, and 20% saturated fats (Bhosale and Vijayalakshmi, 2015). Rice bran polyphenols, being potential antioxidants, prevent various pathological conditions related to oxidative stress and inflammation, highlighting them as nutraceutical agents (Costa et al. 2016). However, despite the high nutrition value and varied promising components in the rice bran, deep processing technologies are still required to improve their nutraceutical value and bioactivity (Wang et al. 2020).

Fermentation is used as a biotechnological method to enhance the availability of new bioactive nutrients and metabolic components with the most promising health benefits in fermented foods (Ashraf et al. 2022). Lactobacillus is a safe and effective probiotic widely used in fermented foods, contributing to several flavor components, such as organic acids, vitamins, and amino acids, and improving flavor, taste, and other fermentation properties. In addition, many bioactive components are generated during fermentation, such as exopolysaccharides and polyphenols, depending on fermented raw material and the Lactobacillus species (Yang et al. 2020). Since ancient times, Saccharomyces cerevisiae (S. cerevisiae) has been widely used in baking, alcoholic beverages, and waffles. S. cerevisiae and Lactobacillus have also been studied to investigate their positive anticancer effects (Christ-Ribeiro et al. 2021).

Recently, the research of novel probiotic strains from untraditional sources may provide a favorable way to get promising probiotic strains, which may achieve unique health and therapeutic benefits compared to traditional strains and enhance technological properties (Adnan et al. 2021; Plessas et al. 2017). One of the most important potential sources of probiotics is bee gut, such as Lactobacillus strains that are not sufficiently studied as probiotics or applied in fermented food.

This study aims to study the impact of Lactobacillus apis isolated from the bee gut on the nutraceuticals of rice bran, compared to Saccharomyces cerevisiae as the most common strain used in cereal fermentation. Further, we also aim to investigate the improvement of pharmaceuticals, antioxidants, and cytotoxicity against colon cancer cell line HT29 of rice bran fermented by S. cerevisiae and L. apis.

Materials and methods

Materials

Rice bran was purchased from Xinji Fuzhiyuan Flour industry Co., Ltd (Hebei province, China). Saccharomyces cerevisiae was purchased from the commerce. The bumblebees (Bombus terrestris) were obtained from Koppert BV (Beijing, China). Chemicals used in this study, including 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Other reagents were analytical grade and purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). As mentioned in our previous study, Lactobacillus apis was isolated from the bee gut and identified by the 16S rRNA sequencing method (Ghamry et al. 2021).

Preparation of fermentation medium

The fermentation procedure was conducted as reported by Wang et al. (2020). The fermentation medium was composed of bran (5 g), CaCl2 (0.1 g), Na2HPO4 (0.1 g), Na2CO3 (0.1 g), KH2PO4 (0.03 g), and distilled water (50 mL). The fermentation medium was sterilized at 121 °C for 30 min. The sterilized rice bran was inoculated with 1% (v/v) of L. apis in MRS medium incubated in MRS medium overnight (OD = 0.8), or with 0.5% of instant baking yeast. Fermentation mediums were incubated at 37 °C with shaking at 200 rpm for different fermentation times (12, 24, 48, and 72 h) (Shaker MQD-S3R, Shanghai Minguan Instrument Co., Ltd, Shanghai, China). The fermentation broths were collected at different fermentation times (0, 12, 24, 48, and 72 h) by centrifugation (4000×g, 30 min, 4 °C) (Centrifuge GL-21MS, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd, Shanghai, China). The collected broths were stored at  − 80 °C for further analysis.

The broths from the samples fermented for 72 h were prepared by ultrafiltration, and then the filtered broths were used for LC-MS analysis and cytotoxic activity test. Fermentation supernatant was added to the upper casing of the ultrafiltration tube (3000 Da MWCO, Amicon Ultra 14 ml 3K, Millipore, USA) and subsequently was centrifuged at 4000×g and 4 °C for 30 min. After ultrafiltration, fractions in the lower casing were collected and stored at -80 °C for further analysis.

Total phenolics and antioxidant activity assays

Total phenolics were determined by the Folin–Ciocalteu colorimetric method, with a calibration curve of gallic acid (Al-Maqtari et al. 2021). The results presented as mg of gallic acid equivalents per mL of fermented broth (mg GAE/mL). DPPH and ABTS scavenging activity, and scavenging activity for metal chelating were measured according to the method described by Wang et al. (2020).

Volatile compounds analysis by SPME/GC–MS

According to Rui-Peng et al. (2020), the volatile compounds were analyzed and identified. The volatile components profile was measured by solid-phase microextraction (SPME) and analyzed by GC–MS (Scion SQ 456-GC, Bruker, USA) with a DB-Wax column 30 m × 0.25 mm, 0.25 μm (J&W Scientific, Folsom, CA, USA). A 0.80 mL/min flow rate of helium was used as a mobile phase with an initial temperature of 40 °C and held for 2 min. Then, the temperature rose to 250 °C at 10 °C/min and held for 6 min. The mass scanning range was 33–400 amu. The identification of volatile compounds was verified by comparing the mass spectral data with those in the NIST 2017 and Wiley 9 standard library.

Analysis of free amino acids and GABA concentration

The free amino acids in fermented and non-fermented samples were determined according to the methods used by Xu et al. (2021). Fermented samples were homogenized and centrifuged at 4000 × g for 10 min. In a 25 mL volumetric flask, 5 mL of the supernatant was transferred with 5% Trichloroacetic Acid (TCA) solution (w/v) used for constant volume. Samples were put in an ultrasonic water bath for one hour, and passed through a 0.22 μm microporous membrane. Subsequently, the amino acids solution was derivatized with O- phthaldialdehyde. An aliquot (1 μL) was injected injected into a high-performance liquid chromatography (HPLC) system (Agilent 1100) coupled with a UV detector (Diode array detector, Agilent Corp., Karlsruhe, Germany) and a reversed-phase column of ODS Hypersil (125 mm × 4.6 mm × 5 μm particle size; Agilent, USA).

Analysis of organic acids and water-soluble vitamins

The organic acid content was measured by HPLC (Agilent 1100, USA), using C18 column (150 mm × 4.6 mm, Agilent, USA), under the following conditions: column temperature, 25 °C; detector wavelength, 215 nm; mobile phase: 0.01 mol /mL potassium dihydrogen phosphate solution at a flow rate of 1.0 mL/min. Approximately 20 mL of fermented samples were centrifuged at 4000 × g for 10 min, and then the supernatant was removed and filtered through a 0.22 µm microporous membrane for organic acid determination. Standard solutions of malic acid, lactic acid, acetic acid, citric acid, fumaric acid, propionic acid, and butyric acid were prepared and determined under similar conditions for quantitative analysis.

Water-soluble vitamins in fermented and non-fermented samples were determined according to the method described by Zafra-Gómez et al. (2006). Five mL of the fermented supernatant was mixed with 1 mL of the precipitation solution, vortexed for 1 min, centrifuged after 15 min in the dark at 4000 × g for 10 min. The supernatant was filtered through 0.22 µm nylon millipore filters before injection into the HPLC system (Agilent 1100 Technologies, USA). The precipitation solution consisted of 5.46 g of phosphotungstic polyhydrated, 9.10 g of zinc acetate, and 5.8 mL of glacial acetic acid in 100 mL Milli-Q water.

Liquid chromatography-mass spectrometry (LC–MS) analysis

The LC–MS system was used to analyze the differences of metabolic components in purified broth fermented for 72 h compared to non-fermented broths (0 h). UPLC system (Waters ACQUITY) with BEH C18 column (2.1 × 150 mm 1.7 um, Agilent, USA) was used for separation. Under two ion modes (positive-ion and negative-ion mode), the eluent was analyzed by a mass spectrometer (Thermo Fisher Scientific). The scan range covered 50–1000 m/z in negative-ion mode and 50–2000 m/z in positive-ion mode. The gradient elution procedure was conducted with 0.1% formic acid (mobile phase A) and ACN (mobile phase B). The program was performed as follows: 0–40 min, 100 A; 40–45 min, 70% A; 45–50 min, 20%A; 50 − 55 min, 100% B. The flow rate was maintained at 0.3 mL/min. The column temperature was kept at 45 °C, and the sample injection volume was set at 5 μL. The mzCloud and ChemSpider databases were used to obtain accurate qualitative and relative quantitative results.

Cell lines and culture conditions

The cytotoxicity of the purified broths from fermented and non-fermented samples was evaluated on human colon cancer cells (HT29 cell line), HT29 cells were purchased from Shanghai Cybertron Biotech (Shanghai, China). HT29 cells were seeded in 96-well plates during the logarithmic growth phase at a concentration of 1 × 104 in McCoy's 5A medium mixed with fetal bovine serum at a ratio of 9:1. The cell line was cultured in an atmosphere of 5% CO2 at 37 °C until the cells adhered to the wall. Then, the cells were treated with the purified samples at 50, 100, 500, and 1000 µg/mL, and incubated for 72 h. Cells grown in normal conditions without any addition were used as a control. The medium containing the sample was removed, washed with PBS three times, then 100 μL of medium containing 0.5 mg/mL MTT was added to each well and incubated for 4 h. The supernatant was discarded, and 100 μL of DMSO was added to each well. After gently shaking for 10 min, the absorbance at 570 nm was measured (Ju et al. 2004).

Statistical analysis

Every experiment was performed in triplicate, and the average values were expressed as mean ± standard deviation (SD). A significant difference among values was calculated based on Tukey's procedure at p < 0.05 using the SPSS statistics 22 (SPSS, USA).

Results and discussion

Total phenolics and antioxidant activity

The impact of S. cerevisiae and L. apis on total phenolics was measured at different fermentation times (0, 12, 24, 48, and 72 h) to determine the release of phenolics in fermented broths during fermentation. As shown in Table 1, the impact of L. apis on total phenolics and antioxidant activity of rice bran showed a significant enhancement compared to S. cerevisiae at different fermentation times. Rice bran samples fermented with L. apis strain showed a progressive increase of TPC during fermentation till 48 h. On the other hand, the S. cerevisiae showed a significant decrease of TPC during the first two days of fermentation, then showed a significant increase in the samples fermented for 48 and 72 h. L. apis increased TPC in fermented broths from 72.94 GAE/g in non-fermented samples (0 h) to 109.43 GAE/g in those fermented for 48 h. Otherwise, the samples fermented with S. cerevisiae for 72 h showed the highest impact on increased TPC compared to other fermentation times and non-fermented samples (85.99 GAE/g). Our results showed that L. apis was an effective strain to enhance the rice bran properties compared with S. cerevisiae. Additionally, L. apis strain indicates a promising effect compared with the results reported for other common strains used in fermentation, such as L. plantarum and L. lactic (Nisa et al. 2019).

Table 1.

Total phenolic and antioxidant activity in the rice bran fermentation broths

TPC (mg Gallic acid equivalent/g) DPPH (%) ABTS (mg Trolox equivalent/g) HRCA (%)
0 h 72.94 ± 0.73 g 20.38 ± 0.31e 27.59 ± 0.38 g 39.69 ± 1.41 h
S. cerevisiae (12 h) 59.35 ± 0.91i 18.89 ± 1.15 g 29.23 ± 0.76f 48.26 ± 0.67e
S. cerevisiae (24 h) 65.58 ± 0.52 h 18.16 ± 0.95 h 29.57 ± 0.74f 40.09 ± 1.02 g
S. cerevisiae (48 h) 79.02 ± 1.96f 19.13 ± 1.9f 32.85 ± 0.69d 46.12 ± 2.15f
S. cerevisiae (72 h) 85.99 ± 0.51e 22.84 ± 1.08d 32.07 ± 0.59e 51.01 ± 1.21d
L. apis (12 h) 88.61 ± 0.89d 29.02 ± 1.16c 32.76 ± 0.78c 60.28 ± 1.14c
L. apis (24 h) 106.85 ± 1.07b 37.73 ± 0.48a 36.17 ± 0.17b 90.76 ± 1.52a
L. apis (48 h) 109.4 ± 1.09a 37.17 ± 1.24a 37.62 ± 0.11a 91.55 ± 2.22a
L. apis (72 h) 102.95 ± 1.11c 33.85 ± 0.73b 36.04 ± 0.12b 89.92 ± 1.95b
P-value  < 0.01  < 0.01  < 0.01  < 0.01
F-value 564.875 109.297 126.69 370.790
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Total phenolic content (TPC) (mg Gallic acid equivalent/g), DPPH radical scavenging activity (%), ABTS scavenging activity (mg Trolox equivalent/g sample), and hydroxyl radical scavenging activity (HRCA) (%). Values are expressed as the mean ± SD (n = 3). Means with different small letter superscripts in the same column are significantly different at p < 0.05

DPPH and ABTS radical-scavenging assays were used to determine the impact of fermentation on antioxidant activity, which depends on different antioxidants' ability to convert free radicals into non-radical components by contributing an electron or hydrogen atom. Such assays are widely used to evaluate the free radical-scavenging potential of antioxidants and are considered standard colorimetric methods (Cho, 2020). Compared with non-fermented rice bran (0 h), S. cerevisiae and L. apis significantly increased DPPH radical-scavenging activity and ABTS radical-scavenging activity at different fermentation times. The samples fermented with S. cerevisiae for 72 h showed a significant enhancement of DPPH radical-scavenging activity (22.84%). However, the samples fermented with L. apis for 48 h exhibited the highest DPPH radical-scavenging activity (37.17%) compared to 0 h (20.38%) (Table 1). ABTS radical-scavenging activity significantly increased in the samples fermented with S. cerevisiae or L. apis for 48 h, 37.62 and 32.85 mg Trolox/g, compared with 27.59 mg Trolox/g in the non-fermented samples (Table 1). The highest hydroxyl radical-scavenging activity (HRCA) was observed in the samples fermented with S. cerevisiae for 72 h (51.01%) and samples fermented with L. apis for 48 h (91.55%) (Table 1).

The increasing DPPH and ABTS activity in fermented rice bran is due to producing certain metabolites by S. cerevisiae and L. apis, such as organic acids, amino acids and peptides, ketones, and saccharides. Moreover, that may be because some phytochemicals release from bound to free forms, such as phenolic acids. The increase of the HRCA ability is due to the release of certain products that can chelate metal ions, including saccharides, organic acids, and phenols. A similar impact by other Lactobacillus strains was reported by Wang et al. (2020), showing that the hydroxyl radical-scavenging activity increased significantly in rice bran during fermentation with L. plantarum due to increased specific antioxidant components in the fermentation broths, including organic acids saccharides, ketones, and phenols. Due to the positive effect of L. apis on antioxidant activity, this strain provides a promising probiotic strain compared to S. cerevisiae, lactic acid bacteria, and some fungi that are commonly used in cereals fermentation (Punia et al. 2021).

SPME/GC–MS analysis

Principal components analysis (PCA) and heatmap showed the differences in volatile composition in the samples fermented by S. cerevisiae and L. apis at different fermentation times, as shown in Fig. 1, and GC/MS spectra are shown in Figs. S1 and S2. The samples fermented with L. apis showed a clear and distinct distribution between samples at different fermentation times. Otherwise, the samples fermented with S. cerevisiae showed a discreate distribution between samples fermented for 48 h with the other samples (Fig. 1A). Based on the heatmap (Fig. 1B), the samples fermented with S. cerevisiae for 48 h exhibited a significant increase of acids. In addition, the alcohols, phenols, and lactones increased significantly in the samples fermented for 24 and 72 h. Ketones, furans, and thiazoles were increased significantly in the samples fermented with L. apis for 12 h, and a significant increase of terpenes was observed in the samples fermented for 72 h.

Fig. 1.

Fig. 1

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Principle component analysis (PCA) A, and heatmap B of volatile components in rice bran fermentation broths

The volatile components in the fermented samples with S. cerevisiae or L. apis during different fermentation times are shown in Table S1. The results showed that 92 volatile compounds were detected as the most abundant in the fermented and non-fermented rice bran, including 18 alcohols, 10 acids, 13 aldehydes, 12 ketones, 11 esters, 7 phenols, 6 benzenes, 5 lactones, 3 thiazoles, 3 terpenes, 2 furans, 1 pyridine, and 1 pyrazine. The most abundant alcohols in the samples fermented with S. cerevisiae were 3-methyl-1-butanol, phenyl ethyl alcohol, 1-hexanol, and ethanol compared with 1-hexanol, 1-nonanol, and 1-decanol in samples fermented with L. apis. The pathways of biosynthesis and degradation of amino acids, especially aromatic amino acids and branched-chain amino acids, are the main pathways to produce amino acids and alcohols in the samples fermented by S. cerevisiae. S. cerevisiae produces 3-methyl-1-butanol through the degradation pathway of branched-chain amino acids, while phenyl ethyl alcohol is produced by the phenylalanine degradation pathway (Kim et al. 2014). The main pathways to produce alcohols in Lactobacillus spp. are lipid oxidation pathways such as 1-hexanol and 1-nonanol (Liu et al. 2021). Butanoic acid, hexanoic acid and acetic acid were the most abundant acids in the samples fermented with S. cerevisiae or L. apis. During fermentation, aldehydes are converted into alcohols and acids due to the effects of the microorganisms’ enzymes, showing the decrease of aldehydes and increase of acids and alcohols with S. cerevisiae or L. apis fermentation. In addition, the release of free fatty acids during fermentation leads to the formation of esters and ketones by reacting with alcohols (Folashade et al. 2019). Our findings presented some remarkable changes in the formation of volatile compounds during fermentation of rice bran with L. apis compared with some other Lactobacillus strains.

Effect of fermentation on free amino acid composition and GABA content

The impact of S. cerevisiae and L. apis on free amino acid composition and GABA concentration in rice bran during fermentation are presented in Fig. 2. Principal component analysis (PCA) (Fig. 2A) showed a clear separation in FAAs distribution between samples fermented with S. cerevisiae and L. apis, and between different fermentation times. Principal components 1 and 2 accounted for 75.09% and 12.56% of the total variance, respectively. The PCA analysis displayed an evident separation between the samples fermented with L. apis for different fermentation times compared with S. cerevisiae, which showed a clear separation between samples fermented for 48 h and samples with the other fermentation times and non-fermented samples (0 h).

Fig. 2.

Fig. 2

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Principle component analysis A and heatmap plot B for free amino acids concentrations in fermented rice bran broths. GABA concentrations in fermented rice bran broths C. Values are expressed as the mean ± SD (n = 3)

The concentration of individual free amino acids is presented in a heatmap, as shown in Fig. 2B. In total, free amino acids decreased significantly in the samples fermented with S. cerevisiae and L. apis compared with non-fermented samples (Fairbairn et al. 2017). Otherwise, some free amino acids increased significantly during fermentation. As shown in Table S2, glycine increased from 15.77 mg/L in non-fermented samples to 36.14 mg/L in the samples fermented with L. apis for 72 h, and 23.76 mg/L in the samples fermented with S. cerevisiae for 48 h. Alanine increased from 32.97 mg/L in non-fermented samples to 48.65 and 63.72 mg/L in the samples fermented with L. apis for 72 h and S. cerevisiae for 48 h, respectively. Valine concentration showed a significant increase from 12.17 mg/L in non-fermented samples to 27.31 mg/L in the samples fermented with L. apis for 24 h, and 23.09 mg/L in the samples fermented with S. cerevisiae for 72 h. Samples fermented with S. cerevisiae showed a significant increase in phenylalanine and isoleucine, which increased from 10.86 and 10.86 mg/L in non-fermented samples to 23.07 and 13.61 mg/L in the samples fermented for 72 h, respectively. While L. apis significantly increased tryptophan in the samples fermented for 12 h (14.05 mg/L), which decreased significantly during the long fermentation periods. The content of different free amino acids in fermented foods is affected by many factors, such as used strain, nutrition content in fermented food, additives, and fermentation conditions (Cui et al. 2020).

As shown in Fig. 2C, the samples fermented with S. cerevisiae showed a significant and gradual increase in GABA concentration with increasing fermentation time, which increased from 21.06 mg/L in non-fermented samples to 38.38 mg/L in the samples fermented for 72 h. S. cerevisiae is reported as a capable yeast strain to produce several bioactive components, and displays a significant ability to produce GABA compared with other common strains used in food fermentation (Li et al. 2021). GABA concentration was significantly improved during fermentation of rice bran by L. apis, which increased to 38.02 mg/L in the samples fermented for 24 h. The impact of L. apis on increased GABA during fermentation was similar to several Lactobacillus strains (Cui et al. 2020).

Organic acids and water-soluble vitamins analysis

The formation of organic acids in the samples fermented with S. cerevisiae or L. apis is shown in Fig. 3. Principal component analysis (PCA) showed a clear separation between samples fermented with S. cerevisiae for 24 h, and L. apis fermented for 24 and 72 h compared with the other samples (Fig. 3A). As shown in Fig. 3B, the most abundant organic acids detected in fermented samples were malic acid and lactic acid. Meanwhile, acetic, fumaric, propionic, and butyric acids were detected at some fermentation times. Acetic acid was detected in the samples fermented with S. cerevisiae for 24 h at 13.75 mg/100 mL, and 12.98 mg/100 mL was detected in the samples fermented with L. apis for 72 h. While propionic acid and butyric acid were not detected in non-fermented rice bran broths, S. cerevisiae significantly increased propionic acid in the samples fermented for 12 h (1.47 mg/100 mL). On the other hand, L. apis significantly increased butyric acid in the samples fermented for 72 h (2.02 mg/100 mL) compared to 0.785 mg/100 mL in the samples fermented with S. cerevisiae for 12 h. As secondary fermentation metabolites, S. cerevisiae releases specific organic acids that form the product aroma and act as a preservation agent. Some previous studies reported the ability of S. cerevisiae and L. apis to produce short-chain fatty acids in their fermented foods, such as acetic, propionic, and butyric acids (Aoki et al. 2021; Kang et al. 2021).

Fig. 3.

Fig. 3

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Principle component analysis (PCA) A and column plot B of organic acids in fermented rice broths. The concentration of water-soluble vitamins in fermented rice broths C. The results are expressed as the mean ± SD (n = 3)

The impact of S. cerevisiae and L. apis on increased vitamins B and C during rice bran fermentation is shown in Fig. 3C. S. cerevisiae and L. apis significantly improved the concentration of some vitamins during rice bran fermentation. The samples fermented with S. cerevisiae exhibited a significant increase of vitamins compared to those fermented with L. apis at different fermentation times. The concentration of vitamin C increased from 5.685 mg/L in non-fermented samples to 23.046 and 50.062 mg/L in the samples fermented with S. cerevisiae for 48 and 72 h, respectively. S. cerevisiae fermentation showed a significant increase in riboflavin, pyridoxine, folate, and nicotinamide compared to non-fermented samples. The riboflavin concentration increased from 26.676 mg/L to 43.789 mg/L in the samples fermented with S. cerevisiae for 24 h. The highest concentration of vitamins detected in the samples fermented with S. cerevisiae was pyridoxine, which increased from 27.130 mg/L in non-fermented samples to 78.383 mg/L in the samples fermented for 72 h. The samples fermented with S. cerevisiae exhibited a significant increase of folate, with four folds increase in the samples fermented for 24 h. In this study, nicotinamide as a form of vitamin B3 was detected only in the samples fermented with S. cerevisiae for 72 h at 8.020 mg/L. Nicotinamide is used for NAD+ biosynthesis across species, spanning from yeast to humans (Garofalo et al. 2021). On the other hand, the most abundant vitamin detected in the samples fermented with L. apis was pyridoxine, which increased from 27.130 mg/L in non-fermented samples to 37.544 mg/L in the samples fermented for 48 h. The impact of Lactobacillus and yeast on the enhancement of vitamins during fermentation has been reported, and several factors affect the production of vitamins in the fermentation medium. Fermentation was reported as the most effective method to improve cereals' nutritional value and bioactivity, cereals by-products, and other foods (Zhu et al. 2020).

The metabolite compositions in purified fermented broth

LC–MS was used to detect the metabolic components in rice bran samples fermented with S. cerevisiae or L. apis for 72 h compared to non-fermented samples (0 h). Rice bran broths fermented for 72 h were chosen for the study because of the great nutraceutical value and antioxidant activity, as mentioned previously. As shown in Fig. 4A and Table S3A, the samples fermented with L. apis showed 62 abundant components, including acids (69.35%), saccharides (14.52%), ketones (4.84%), and other types (11.29. Otherwise, %). Otherwise, the results showed 40 abundant components in the samples fermented with S. cerevisiae, including acids (60%), saccharides (15%), ketones (5%), and other types (20%) (Fig. 4B and Table S3B). The samples fermented with L. apis had a significant increase of some acids, such as γ‐aminobutyric acid, hydroferulic acid, dihydrosinapic acid, 2-hydroxy-1-naphthoic, leucic acid, cis-aconitic acid, and pyruvic acid. In addition, the purified broths from rice bran fermented with L. apis showed a significantly higher abundance of some dipeptides compared with non-fermented samples and samples fermented with S. cerevisiae, such as leucyl-alanine, leucyl-glycine, and glycyl-lysine (Fig. 4A). The samples fermented with S. cerevisiae exhibited a significant abundance of cinnamic acid, cis-cinnamic acid, hydroxy propionic acid, isobutyric acid, succinic acids, pyruvic acid, hesperidin, and stachyose compared with non-fermented samples (0 h) (Fig. 4B). Rice bran fermented with S. cerevisiae presents a unique source of some highly active antioxidant components, released during fermentation as metabolites or due to the transformation of rice bran phytochemical components (Chaiyasut et al. 2017). Pyruvic acid, pantothenic acid, stachyose, alanine, vitamin c, and rutinose have an effective antioxidant activity, and most of them are present in products fermented with Lactobacillus strains (Wang et al. 2020). Compared with S. cerevisiae, the metabolic components from L. apis fermentation were more diverse with high antioxidant activity. Our findings indicate that the unique diversity and multiplicity of metabolic compounds in the rice bran broth fermented by S. cerevisiae and L. apis may provide exceptional health and therapeutic resource. The differences in mass spectra of metabolic components in fermented and non-fermented rice bran broths are shown in Figs. S3 and S4.

Fig. 4.

Fig. 4

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Heatmap of metabolic components in fermented rice bran broths, fermented by L. apis A and S. cerevisiae B

Cytotoxic activities of purified fermented broth

The purified broths from fermented and non-fermented rice bran showed significant cytotoxic activity against colon cancer cells (HT-29 cells), as shown in Fig. 5. Otherwise, the fermented samples with L. apis or S. cerevisiae showed significantly higher cytotoxic activity compared with non-fermented samples. The increase in fermented rice bran broth concentration caused a significant increase of cytotoxic activity. On the other hand, the increase in non-fermented broth concentration did not cause a significant increase of cytotoxic activity. The highest cytotoxic activity was observed when cells were treated with 1000 µg/mL of L. apis (34.17%) or S. cerevisiae (31.34%) fermented broth, compared with 18.54% in non-fermented samples (Fig. 5A). As shown in Fig. 5B, the fermented rice bran broths significantly impacted the shape of the HT-29 cells and increased the cell death rate compared to non-fermented rice bran broths. Fermentation of rice bran with S. cerevisiae and L. apis has a favorable impact on increasing the bioactive components in fermented broths, which can affect their cytotoxic activity against HT-29 cells. In this study, the effect of L. apis on increased cytotoxic activity was higher than that of S. cerevisiae, which may be related to the superiority of increased antioxidant activity by L. apis compared with S. cerevisiae. In contrast, a study reported that the fermented black rice showed a significant increase of anticancer activity, and decreased antioxidant activity (Yoon et al. 2015). Lee et al. have investigated and reported the positive effect of S. boulardii on survival and reproduction rates of the HT-29 cells by using MTT (Lee et al. 2005). Moreover, the supernatant from Lactobacillus casei medium prevented growth and initiated apoptosis in the HT-29 cells (Tiptiri-Kourpeti et al. 2016). The increase of active principles in the soymilk after lactic fermentation leads to high anticancer activity (Lai et al. 2013). Our result suggests that other metabolic components might positively impact cytotoxic activity during the fermentation of rice bran.

Fig. 5.

Fig. 5

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A Cell viability of HT-29 cell line; B Fluorescence microscopy of live/dead HT-29 cells treated with fermented and non-fermented rice bran broths. Different capital letters A–C and small letters a–d in the same columns represent significant differences (p < 0.05) between control and other doses in the same treatment and between the various treatments at various doses, respectively. * Arrows in the fluorescence microscope picture indicate dead cells B

Conclusion

This study investigated the use of uncommon Lactobacillus strain (L. apis) isolated from the bee gut compared with common yeast used in fermentation (S. cerevisiae) to improve antioxidant activity, nutritional value, and anticancer activity of rice bran. S. cerevisiae and L. apis significantly improved fermented rice bran broth, increased total phenolic content (TPC), and antioxidant activity compared with non-fermented samples. L. apis showed a significant increase of TPC, DPPH, and ABTS radical-scavenging activity and ions chelating ability (HRCA) compared with S. cerevisiae. SPME/GC–MS analysis showed differences in volatile profiles between rice broths fermented with S. cerevisiae and L. apis. S. cerevisiae showed a significant ability to increase GABA in the broths compared to L. apis. Along the same lines, S. cerevisiae exhibited a significant enhancement of the water-soluble vitamins. Metabolic analysis of purified broths showed a high diversity of metabolites with L. apis compared to S. cerevisiae. The cytotoxic activity against colon cancer cells (HT-29 cells) significantly increased with fermentation broths compared to the non-fermented broths. Otherwise, L. apis brought significantly higher cytotoxic activity than S. cerevisiae. Compared with S. cerevisiae, L. apis presents a promising strain that may amplify rice bran's active compounds and produce functional ingredients for human health promotion.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1365 kb) (1.3MB, docx)

Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2017YFC1601704), the National Natural Science Foundation of China (No. 1522044, 31671909 and 31772034), the Program of the Jiangsu Key Laboratory of Advanced Food Manufacturing Equipment & Technology (No. FMZ201904), the National First-Class Discipline Program of Food Science and Technology (No. JUFSTR20180205) to W.Z.

Abbreviations

ABTS

2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonate)

DPPH

2,2-Diphenyl-1-picrylhydrazyl; di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium)

Trolox

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

TPC

Total phenolic content

HPLC

High performance liquid chromatography

LC–MS

Liquid chromatography–mass spectrometry

SPME/GC–MS

Solid-phase microextraction/ gas chromatography-mass spectrometry

HT-29

Human colorectal adenocarcinoma cell line with epithelial morphology

PCA

Principal component analysis

VC

L-ascorbic acid

VB2

Vitamin B2 (Riboflavin)

VB6

Vitamin B6 (Pyridoxine)

Authors' contributions

Conception and design of study: MG, LL, WZ. Acquisition of data: MG, LL, AFG, QAA-M. Analysis and/or interpretation of data: MG, LL. Drafting the manuscript: MG, LL. Revising the manuscript critically for important intellectual content: LL, WZ. Approval of the version of the manuscript to be published: MG, LL, WZ, AFG, QAA-M. The supervision: WZ.

Funding

This work was supported by the National Key Research and Development Program of China (No. 2017YFC1601704), the National Natural Science Foundation of China (No. 1522044, 31671909 and 31772034), the Program of the Jiangsu Key Laboratory of Advanced Food Manufacturing Equipment and Technology (No. FMZ201904), the National First-Class Discipline Program of Food Science and Technology (No. JUFSTR20180205) to W.Z.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Li Li, Email: lili.zz@jiangnan.edu.cn.

Wei Zhao, Email: zhaow@jiangnan.edu.cn.

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