Limosilactobacillus reuteri Fermented Brown Rice

Limosilactobacillus reuteri Fermented Brown Rice: Comparison

Please note this is a comparison between Version 1 by Deog-Hwan Oh and Version 2 by Vivi Li.

Oxidative stress has been postulated to play a role in several diseases, including cardiovascular diseases, diabetes, and stress-related disorders (anxiety/depression). Presently, natural plant-derived phytochemicals are an important tool in reducing metabolomic disorders or for avoiding the side effects of current medicinal therapies. Brown Rice (Oryza sativa L.) is an important part of Asian diets reported as a rich source of bioactive phytonutrients.

brown rice
fermentation
antioxidants
oxidative stress
untargeted metabolomics
UHPLC-QTOF/MS
health benefits

1. Introduction

Oxidative stress is a condition that is caused by an imbalance between antioxidants and free radicals of living organisms. This imbalance occurs due to the excessive production of reactive oxygen species (ROS) or antioxidant deficiency that leads to the damage of aerobic organisms as well as chronic inflammation; referred to as oxidative stress [1]. Lower ROS concentration is important for normal cellular signalling, while excess ROS can cause oxidative damage to DNA, lipids, proteins, and is associated with several chronic diseases ^[2][3][2,3]. The current definition of oxidative stress includes metabolic stress-related pathways that participate in both cellular and extracellular metabolic events. The biology of oxidative stress is extremely complex, with multiple mechanisms at work [4]. Regardless of the mechanism, oxidative stress causes the onset of many diseases including cardiovascular diseases, diabetes, and anxiety or depression which are considered a major public health issue worldwide. As a result, consuming antioxidants to prevent oxidative stress is becoming important for health. Moreover, an increase in the health-consciousness of consumers has increased the demand for nutritional and disease-preventing functional foods, probiotics, prebiotics, and postbiotics. Numerous studies have focused on probiotics, specifically lactobacilli strains, that have the potential to act as antioxidants to protect the host from oxidative stress [5]. Some lactobacilli strains have been found to quench oxygen free radicals using a chemical antioxidant method.

Many studies have been reported that phytochemicals (e.g., polyphenols and phenolic acids) derived from natural plants have the potential to target oxidative stress and inflammatory pathways ^[6][7][6,7]. Rice is a staple food (in many countries) that belongs to the grass family (Oryza sativa). The total worldwide production of rice was about 769,657,791 tonnes in an area of 167,249,103 ha. Epidemiological studies have shown that the low incidence of chronic diseases in rice-consuming regions can be correlated with rice antioxidants ^[8][9][8,9]. The antioxidant activity and phytochemical content of brown rice (BR) have been recorded in several studies. Components such as γ-oryzanol, phenolic acids, gamma-aminobutyric acid (GABA), flavonoids, and γ-tocotrienol contribute to the health-promoting properties of brown rice [10].

Evidence supports the effect of solid-state fermentation (SSF) techniques using lactic acid bacteria (LABs) and fungal strains on antioxidant levels and bioactive properties in a variety of substrates, including barley [11], pearl millet [12], and rice [2]. Many researchers, food scientists, and industrialists use the SSF process to enhance the nutritional quality of food and food products. Biological methods are environmentally friendly, relatively safe, and rely on the use of appropriate and specific microorganisms [13]. Our study aimed to provide knowledge to quantify the quality of these phytochemical antioxidants in whole brown rice to meet the needs of food producers and consumers of rice: (1) to analyze the antioxidant properties of differently fermented brown (FBR) rice over raw brown rice (BR); (2) detection of bioactive compounds in raw BR and different LABs fermented brown rice using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS), and (3) detection of cellular antioxidant activity of the best LAB fermenting bacterial strain (L. reuteri FBR).

2. Untargeted Metabolomics Using UHPLC Q-TOF-MS/MS in Brown Rice Samples

UHPLC Q-TOF-MS/MS detection is considered a gold standard technique for the precise detection and quantification of a wide variety of components. Therefore, in this study, we have used this detection technique for the identification of phenolic compounds in brown rice.

2.1. Phenolic Compounds

In the present research, the phenolic compositions of BR treated with different fermentation bacteria were selected and positively or tentatively identified by UHPLCQ-TOF-MS/MS. Phenolic identification and characterization were achieved by comparing our results with mass spectral literature evidence and cross-referencing it with other available spectral databases, such as Metlin and Metabolomics Workbench. A total of 15 phenolic compounds were tentatively found from our soluble extracts of raw BR, L. reuteri FBR, L. fermentum FBR, and L. plantarum FBR respectively, as shown in Table 12. In the ethanol extract, we identified compounds 1 to 14 at different adduct charges [M − H]⁻ and [M − H]⁺ which are identified by comparing with mass spectral libraries, XCMS online (Metlin), and Metabolomics Workbench. Heat map analysis was used for clustering phenolic compounds based on their concentrations (Figure 1) where the colour scheme from blue to red shows concentration in decreasing order.

Figure 1. Heat map showing levels of phenolic compounds in raw and LABs fermented BR samples.

Table 12. Phenolic compounds detected in raw and LABs fermented BR.

S. No	Sample Name	Retention Time (min)	Peak Area	Adduct/Charge	Precursor Mass	Found at Mass	Molecular Formula	Tentative Phenolic Compound
1	Raw BR	Nd	Nd

^[18][32], and vanillic acid ^[19][33] are already reported in the literature for their strong antioxidant capacities.

2.2. Levels of Amino Acid in Brown Rice

In the growth and development of organisms, amino acids play an important role and can also improve the taste of food. In our present study, a total of 18 amino acids were detected in raw and differently fermented BR samples (Figure 2 and Table 23) which shows statistically significant differences from each other after comparing their levels. Raw BR contained the least number of amino acids, which may be due to more bound molecules with the parent, whereas fermentation leads to an increase in amino acid content. The levels of amino acids were detected highest in the L. reuteri FBR sample which might be strain-specific as fermentation microorganisms produce enzymes that lead to the formation of several metabolites and bioactive compounds from the food matrix ^[20][34]. In the ethanol extract, we found levels of some essential amino acids (tryptophan, lysine, methionine, and histidine), as well as certain conditionally essential amino acids (arginine, ornithine, serine, and glutamine), increased drastically after fermentation (Figure 2 and Table 23). The identification was done by comparing with mass spectral libraries, XCMS online (Metlin) and Metabolomics Workbench. In amino acids, L. reuteri FBR also shows the highest number of amino acids content as observed in phenolic compounds.

Figure 2. Heat map showing levels of amino acids in raw and different fermented BR samples.

Table 23. Amino acids detected in raw and LABs fermented brown rice.

S. No	Sample Name	Retention Time (Min)		Peak Area	Adduct/ Charge	Precursor Mass	Found at Mass	Formula Finder Result	Amino Acid

Figure 3. Heat map showing levels of fatty acids in raw and different LABs fermented BR samples.

Table 34. Fatty acids detected in raw and LABs fermented brown rice.

S. No	Sample Name	Retention Time	Peak Area	Adduct/Charge	Precursor Mass	Found at Mass	Formula Finder Result	Fatty Acid
[M − H]−	Nd	Nd
1	Raw BR	C	1.00		₂₅	H	₃₆	O	Beta-carotenol
	1.52 × 10		³	[M + H]+						156.077	156.077	C	₆	H
	1		Raw BR	₉						N	₃				O	₂	Histidine	39.97	1.91 × 10²	[M − H]−
			L. plantarum FBR															45.56	4.53 × 10⁵	[M − H]−	353.268
L. plantarum FBR		1.00			7.31 × 10	353.187
255.234		255.2331	C		⁵	[M + H]+	156.077	156.077	L. fermentum FBR			Nd	Nd	[M − H]−				Nd	Nd
₁₆	H	₃₂		O	₂	Palmitic Acid	L. fermentum FBR	1.02		6.33 × 10⁵	[M + H]+	156.077	156.0771	L. reuteri FBR	45.45	5.19 × 10
							L. plantarum FBR	1.94	5.15 × 10³	[M − H]−	255.234	255.2331	⁵	[M − H]−	353.268	353.284
							2	Raw BR	Nd	Nd	[M + H]+	Nd	Nd	C₁₀H₁₂O₂	Eugenol
			L. plantarum FBR					Nd	Nd
L. reuteri
L. fermentum FBR	28.03	4.28 × 10³	[M − H]−	255.234	255.2332	FBR		1.02		7.45 × 10⁵	[M + H]+	156.077	156.0771
L. reuteri FBR	23.73	1.53 × 10⁴	[M − H]−	255.234	255.2332	2
2	Raw BR	Raw BR	ND		NDND		[M − H]−	ND	ND	C₆H₁₄N₂O₂	ND	[M + H]+	ND	ND	C₅H₁₀O₂Lysine	Valeric acid	[M + H]+	Nd	Nd

	L. plantarum FBR	1.02		9.16 × 10⁵	[M − H]−
	L. plantarum	145.099	FBR	ND	ND	[M + H]+	ND	L. fermentum FBR	Nd		Nd	[M + H]+	Nd	Nd
145.0982
L. fermentum FBR	1.02		2.17 × 10⁵	[M − H]−	145.099	145.0984	ND	ND	L. reuteri FBR	20.81	2.24 × 10⁵
L. reuteri	[M + H]+	FBR	179.107	179.1067
1.02		1.40 × 10	⁶	[M − H]−	145.099	145.0983
L. reuteri FBR	22.88	1.02 × 10³
ND
C
	₁₁
		H
			₁₂
N
	₂
		O
			₂

Tryptophan

	ND
	L. fermentum FBR	ND	ND	[M + H]+	[M + H]+	185.066	185.0663	3	Raw BR	33.80	2.20 × 10⁵	[M − H]−	293.177	293.1761	C
3	Raw BR	ND		ND	[M + H]+	ND	₁₇		H₂₆O₄	6-Gingerol
	ND	C	₅	H	₁₁	NO					₃	S	Methionine
	3													Raw BR		46.24	5.29 × 10³	[M − H]−	279.234	279.2332	L. plantarum FBR	33.80	2.08 × 10⁶	[M − H]−	293.177	293.176
								C						₁₈	H	₃₂	O	₂	Linoleic Acid	L. plantarum FBR	1.17
L. plantarum FBR							46.26		5.66 × 10⁵	[M + H]+										166.053	6.56 × 10166.0536	⁵	[M − H]−	279.234	279.2332	L. fermentum FBR	33.81	2.03 × 10⁶	[M − H]−	293.177	293.1761
L. fermentum FBR		1.17		5.33 × 10⁵	[M + H]+	166.053	166.0537		L. reuteri FBR	33.80	2.13 × 10⁶	[M − H]−	293.177							293.1762
L. reuteri FBR	1.18		8.41 × 10⁵	[M + H]+	166.053	166.0536	4		Raw BR	Nd
4	Raw BR	Nd	[M + H]+	Nd	1.17			2.56 × 10Nd	²	C₁₅H₁₀O₄	Chrysin
	[M − H]−	146.047	146.0457	C	₅	H		₉	NO
	L. fermentum FBR	46.24	6.16 × 10⁵									[M − H]−	279.234	279.2334
	L. reuteri FBR	46.25	6.64 × 10⁵				[M − H]−					279.234	279.2334	₄
4	Glutamic acid	Raw BR	47.28				1.45 × 10⁵			[M + H]+	271.264	271.2637	C₁₇H₃₄O₂		L. plantarum FBR	Nd	Nd	[M + H]+	Nd	Nd
		Heptadecanoic acid	L. plantarum FBR	1.47		3.18 × 10⁵	[M − H]−	146.047	146.046
			L. plantarum FBR	47.28	1.44 × 10⁶	L. fermentum FBR	14.78	4.09 × 10⁵	[M + H]+	253.052	253.0524
			[M + H]+	271.264	L. fermentum FBR	1.47		4.58 × 10⁴	[M − H]−	146.047	146.046	L. reuteri FBR		14.81	1.01 × 10⁵	[M + H]+	253.052	253.0527
L. reuteri FBR	1.47		2.65 × 10⁶	[M − H]−	146.047	146.0458	5	Raw BR	Nd	Nd
5	Raw BR		ND		[M + H]+	Nd		Nd	C	₁₆H₈N₂O₅	Apigenin
	ND	[M + H]−	ND	ND	C	₄		H				₉NO₂	Gamma-aminobutyric acid	L. plantarum FBR	14.79	4.68 × 10⁵	[M + H]+	269.047	269.0457
	L. plantarum FBR	1.16		1.38 × 10⁵										[M − H]−	102.057	102.056	L. fermentum FBR	14.78	3.38 × 10⁵	[M + H]+	269.047	269.0457
	L. fermentum FBR	1.16		2.76 × 10⁴			[M − H]−							102.057	102.0563	L. reuteri FBR	14.78	5.15 × 10⁵	[M + H]+	269.047	269.0458
6	Raw BR
	L. reuteri FBR	1.16		1.28 × 10⁶	[M − H]−	102.057	102.0561	Nd	Nd	[M + H]+	Nd	Nd	C₉H₆O₂	Coumarin
	6	Raw BR	ND		ND	[M + H]+	ND	ND	C₆H₁₄N₄O₂	Arginine	L. plantarum FBR	1.92			1.24 × 10⁵	[M + H]+	147.044
		L. plantarum	147.0444
FBR		1.11		2.81 × 10⁶	[M + H]+	175.118	175.1183	L. fermentum FBR			1.87	2.94 × 10⁵			[M + H]+	147.044	147.0447
L. reuteri FBR		1.90	1.55 × 10⁵
L. fermentum FBR	1.14		9.09 × 10⁵	[M + H]+	175.118	175.1194	[M + H]+	147.044	147.0445
L. reuteri FBR	1.11		4.99 × 10⁶	[M + H]+	175.118	175.1184	7	Raw BR	Nd	Nd	[M + H]+	Nd
271.2636
L. fermentum FBR	47.26	1.44 × 10⁶	[M + H]+	271.264	271.2637
L. reuteri FBR	47.27	1.45 × 10⁶	[M + H]+	271.264	271.2638	7		Raw BR	ND		Nd	ND	C₁₅H	[M − H]−	₁₄	ND	O	ND	C₅H₁₁₇	Epigallocatechin
NO	₂	Valine	L. plantarum FBR	12.26	7.44 × 10⁵		[M + H]+	305.071	305.067
			L. plantarum FBR	1.49			6.15 × 10⁵	[M − H]−	116.073	116.0717	L. fermentum FBR	12.28		1.20 × 10⁶		[M + H]+		305.071			305.067
			L. fermentum FBR	1.50			1.37 × 10⁵	[M − H]−	116.073	116.0718	L. reuteri FBR	12.28		1.38 × 10⁶		[M + H]+		305.071			305.067
			L. reuteri FBR	1.45		7.61 × 10⁵	[M − H]−	116.073	116.0719	8	Raw BR	Nd	Nd	[M + H]+	Nd	Nd	C
8	Raw BR	₇	1.14		H	₁₉	N	₃	Spermidine
	1.19 × 10		³	[M − H]−							132.031	132.0307	C	₄	H₇NO₄	Aspartic acid		L. plantarum FBR	Nd	Nd	[M + H]+	Nd	Nd
	L. plantarum FBR		1.15
	5		Raw BR	27.69						5.28 × 10³	[M − H]−	283.265					283.2644	C	7.26 × 10⁴	[M − H]−	132.031	L. fermentum FBR	Nd	Nd	[M + H]+	Nd	Nd
132.0303		L. reuteri FBR	0.96	8.75 × 10⁵	[M + H]+	188.176	188.1761
9		Raw BR	38.06	3.21 × 10⁴	[M − H]−	277.182	277.1812	C₁₇H₂₆O₃	6-Paradol
		L. plantarum FBR	38.06	4.38 × 10⁵	[M − H]−	277.182	277.1812
	L. fermentum FBR	38.08	4.33 × 10⁵	[M − H]−	277.182	277.1813
	L. reuteri FBR
₁₈	H	₃₆	O	₂	Stearic acid
						L. plantarum FBR	49.10	L. fermentum FBR	1.15		8.76 × 10³	[M − H]−	132.031	132.0305
						1.23 × 10	⁶	[M − H]−	L. reuteri FBR	1.12		4.89 × 10⁵	[M − H]−	132.031	132.0302
						9	Raw BR	ND		ND	[M − H]−	ND	ND	C₉H₁₁NO₂	Phenylalanine
L. plantarum FBR	4.01		2.26 × 10⁶	[M − H]−	164.072		164.0718
L. fermentum FBR	4.03		3.50 × 10⁵	[M − H]−	164.072		164.072	38.06	4.42 × 10⁵	[M − H]−	277.182	277.1812
283.265	283.2645
L. fermentum FBR	49.09	1.20 × 10⁶	[M − H]−	283.265	283.2644
L. reuteri FBR	49.10	1.24 × 10⁶	[M − H]−	283.265	283.2645
6	Raw BR	34.57	3.18 × 10⁴	[M − H]−	243.161	243.1605	C₁₃H₂₄O₄	Tridecanedioic acid
	L. plantarum FBR	34.58	2.98 × 10⁵	[M − H]−	243.161	243.1606
	L. fermentum FBR	34.58	2.90 × 10⁵	[M − H]−	243.161	243.1604
	L. reuteri FBR	34.57	3.01 × 10⁵	[M − H]−	243.161	Raw BR			ND	ND	[M + H]+	ND	ND	C₁₂H₂₀O₃	Traumatin
L. plantarum FBR	32.80	2.64 × 10⁵	[M + H]+	213.149	213.1491
L. fermentum FBR	32.82	4.26 × 10⁵	[M + H]+	213.149	213.1492
L. reuteri FBR	32.82	5.20 × 10⁵	[M + H]+	213.149	213.1492	L. reuteri
8	Raw BR	ND	ND	[M − H]−	ND	ND	C₁₈H₃₂O₅	Octadecadienoic acid/Corchorifatty acid F
	L. plantarum FBR	ND	ND	[M − H]−	ND	ND			FBR	3.98		7.21 × 10⁶	[M − H]−	164.072	164.0718	10	Raw BR	Nd	Nd
	L. plantarum FBR	7.64	6.19 × 10⁵		[M − H]−	203.084			203.0829
	L. fermentum FBR	7.66	7.77 × 10⁴		[M − H]−	203.084			203.0832
243.1605	L. reuteri FBR	7.61	2.78 × 10⁶
L. fermentum FBR	ND	ND	[M − H]−	ND	ND
L. reuteri FBR	30.87	4.08 × 10⁵	[M − H]−	327.219	327.2181
9	Raw BR	ND	ND	[M − H]−	ND	ND	C₆H₁₂O₄	Mevalonic Acid
	L. plantarum FBR	3.49	7.39 × 10⁴	[M − H]−	147.067	147.0667			10
	L. fermentum FBR	ND	ND	[M − H]−	ND	ND
	L. reuteri FBR	3.47	3.13 × 10⁵	[M − H]−	147.067	147.0667				[M − H]−	Raw BR	ND		ND	[M − H]−Nd	Nd	C₉
10	Raw BR	22.59	3.99 × 10⁴	H	[M − H]−	₈	O₂	Cinnamic acid
	ND	ND	C		₅				H	187.099	₁₂	N₂	187.0979	O	C₉H₁₆O₄₂	Ornithine		Azelaic Acid	L. plantarum FBR	4.01	3.26 × 10⁵	[M − H]−	147.046	147.0455
	L. plantarum FBR	1.01
	L. plantarum FBR	22.50								1.53 × 10⁵			[M − H]−				131.084		2.77 × 10131.0827	⁵	[M − H]−	187.099	187.0979	L. fermentum FBR	4.03	4.46 × 10⁴	[M − H]−	147.046	147.0456
L. fermentum FBR	1.01			6.67 × 10⁴		[M − H]−	131.084	131.0828
L. fermentum FBR	22.61	6.12 × 10⁴		[M − H]−	187.099	187.0978	L. reuteri FBR	3.98	1.09 × 10⁶	[M − H]−	147.046	147.0454
L. reuteri FBR
L. reuteri	1.01		FBR5.35 × 10⁵	22.50[M − H]−	131.084	131.0827	6.25 × 10⁵	[M − H]−	187.099	187.0979	11	Raw BR	Nd
11	Raw BR	1.12		Nd	[M + NH4]+	Nd	Nd	3.31 × 10²	C	[M − H]−		₉H₈	104.036	O₃	p-Coumaric acid
	104.0353	C	₃
	11			Raw BR	ND	ND	[M − H]−	ND		ND			H₇NO₃			CSerine	₉H₁₈O₃	9-Hydroxynonanoic acid	L. plantarum FBR	1.86	1.06 × 10
				L. plantarum FBR	1.12		⁶	[M + NH4]+		182.081	182.0813
3.06 × 10				⁵	[M − H]−	104.036	104.0353
L. plantarum FBR		23.49	3.10 × 10⁴	[M − H]−	173.119	173.1187	L. fermentum FBR	1.92
L. fermentum FBR
L. fermentum FBR	23.50	6.90 × 10⁵	[M + NH4]+	182.081	3.49 × 10182.0813
1.13		4.70 × 10	⁴	[M − H]−	104.036	104.0356	³	[M − H]−	173.119	173.1188	L. reuteri
L. reuteri FBR	23.48	1.39 × 10⁵	[M − H]−	173.119	173.1186
12	Raw BR	39.06	2.45 × 10³	[M − H]−	313.24	313.2389	C₁₈H₃₄O₄	Octadecanedioic acid
	L. plantarum FBR	39.07	[M − H]−	203.084	203.0829
	3.52 × 10	⁵	[M − H]−	18	Raw BR	ND			ND		[M + H]+	ND	ND	C₅H₉NO₂	Proline
	313.24	313.2388
L. fermentum FBR	39.06	3.22 × 10⁵	[M − H]−		313.24	313.2389
L. reuteri FBR	39.06	3.83 × 10⁵	[M − H]−		313.24	313.2386
13	Raw BR	ND	ND	[M − H]−	ND	ND	C₁₈H₃₄O₅	Pinellic acid
	L. plantarum FBR	33.48	3.12 × 10⁵	[M − H]−	329.234	329.2337			L. plantarum FBR
	L. fermentum FBR	ND	ND		[M + H]+	ND			ND
	7	FBR	1.87	2.13 × 10⁶	[M + NH4]+	182.081			182.0812
12
		L. reuteri FBR	1.12		5.73 × 10⁵	[M − H]−	104.036	104.0353	Raw BR	Nd	Nd	[M − H]−	Nd	Nd	C
		12	Raw BR	ND		ND	[M − H]−	₉	H	₁₀O₃	Methoxyphenylacetic acid
	ND		ND	C	₆	H	₁₃					NO	₂	Leucine		L. plantarum FBR
L. plantarum	15.28		FBR													2.46		5.20 × 10⁶	1.95 × 10[M − H]−	⁶165.057	165.0558
[M − H]−	130.088		130.0874												L. fermentum FBR	15.29	4.09 × 10⁵	[M − H]−	165.057	165.056
L. fermentum FBR	2.48		3.24 × 10⁵					[M − H]−	130.088	130.0875	L. reuteri FBR				15.27	1.92 × 10⁷	[M − H]−	165.057	165.0557
L. reuteri FBR	2.41		6.63 × 10⁶	[M − H]−	130.088	130.0875	13	Raw BR	Nd	Nd	[M − H]−	Nd	Nd	C₇H₆O₃	Sesamol/2-Hydroxybenzoic acid
13	Raw BR	ND		ND	[M − H]−	ND		ND	C₅H₁₀N₂O₃	Glutamine	L. plantarum FBR	Nd	Nd			[M − H]−	Nd	Nd
	L. plantarum FBR	ND		ND	[M − H]−	ND		ND			L. fermentum FBR	Nd	Nd			[M − H]−	Nd	Nd
	L. fermentum FBR	ND		ND	[M − H]−	ND		ND			33.48	L. reuteri FBR	19.63			3.24 × 10⁵	[M − H]−	137.025	137.0249
	L. reuteri FBR	1.10		1.62 × 10⁴	[M − H]−	145.063	145.0619	14			Raw BR	Nd	Nd	[M − H]−	Nd	Nd	C₈H₈O	Vanillic acid
14	Raw BR	ND		ND	[M − H]−	ND	ND		C₉H₁₁NO₃	Tyrosine	L. plantarum FBR	15.28	3.18 × 10⁵	[M − H]−	119.051	119.0504
	L. plantarum FBR	1.98		3.38 × 10⁵	[M − H]−	180.068	180.0667				L. fermentum FBR	Nd	Nd
	L. fermentum	[M − H]−	FBR	ND		Nd	Nd
	ND	[M − H]−	ND	ND	L. reuteri FBR	15.27	1.27 × 10⁶	[M − H]−			119.051
L. reuteri FBR	1.89		4.08 × 10⁵	[M − H]−	180.068	119.0504
180.0667	15	Raw BR	Nd	Nd	Nd	Nd	Nd	C₆H₈O₆	Ascorbic acid (Vitamin C)
L. plantarum FBR		Nd	Nd	Nd	Nd	Nd
L. fermentum FBR		Nd	Nd	Nd	Nd	Nd
L. reuteri FBR		1.02	3.05 × 10³	[M + H]+	209.009	209.0107

Nd—not detected, BR—brown rice, and FBR—fermented brown rice.

Results showed that the highest phenolic compounds were detected in the L. reuteri FBR sample. Because phenolic compounds are not readily available, they typically occur in cereals in esterified linkages to the cereal wall matrix ^[14][28]. Fermentation is considered to be a possible strategy to release insoluble or bound phenolic compounds and thus leading to improve the poor bioavailability of grain phenolics. Comparing different fermenting bacteria in the present study we found that L. reuteri fermentation releases most of the phenolic compounds compared with other bacterial strains and thus improves the bioavailability and bioaccessibility of cereal grains such as brown rice phenolics ^[15][29]. Many phenolic compounds detected in the current study such as p-coumaric acid ^[16][30], ascorbic acid ^[17][31], cinnamic acid

15

	Raw BR

ND
ND
[M − H]−
ND
ND
C
	₄
		H
			₉
NO
	₃

		Threonine


L. plantarum
FBR
ND
ND
[M − H]−
ND
ND
L. fermentum
FBR
ND
ND
[M − H]−
ND
ND
L. reuteri
FBR
1.14
1.14 × 10
⁵
[M − H]−	118.052	118.051
16	Raw BR	ND	ND		[M − H]−	ND	ND	C₄H₈N₂O₃	Asparagine
	L. plantarum FBR	1.10	6.8 × 10⁴		[M − H]−	131.047	131.0462
	L. fermentum FBR	1.13	3.41 × 10⁴		[M − H]−	131.047	131.0464
	L. reuteri FBR	1.13	3.01 × 10⁵		[M − H]−	131.047	131.0461
17	Raw BR	ND	ND		[M − H]−	ND

	L. fermentum
	FBR
ND
ND		[M + H]+	ND	ND
L. reuteri FBR	0.86	7.90 × 10⁵		[M +H]+	116.07	116.0704

ND—not detected, BR—brown rice, and FBR—fermented brown rice.

2.3. Level of Fatty Acid in Brown Rice Samples

In particular, fermentation has been proposed as a tool for enhancing foods’ nutritional values, both in terms of enhanced bioavailability of bioactive components as well as the production of health-promoting end-products. Due to their proven benefit, in the last decade, short-chain fatty acids (SCFAs) have emerged as some of the most researched compounds. In the present study, 13 fatty acids were detected in raw and different LABs fermented BR samples (Table 34) and fatty acid levels were found to be significantly different in all samples. The results show that the highest levels of fatty acids were found in L. reuteri FBR. Heat map analysis was used for separating fatty acids based on the different concentrations, represented in different shades of green (dark to light) in decreasing order (Figure 3).

3.54 × 10

⁴

[M − H]−

329.234

329.2339

L. reuteri

FBR

32.82

7.37 × 10

⁶

[M − H]−

329.234

329.2331

ND—not detected, BR—brown rice, and FBR—fermented brown rice.

3. Cell Viability Assay and Cellular Antioxidant Activity (CAA)

3.1. Cell Viability Assay

Cytotoxicity is regarded as an important step in determining the suitability and further applications of any food extract. Using the Ez cytox assay kit, the cytotoxic effect of L. reuteri FBR extracts at 0.3–10 mg/mL concentrations was investigated using Caco-2 cell lines. Figure 4 depicts the cell viability results of the extract after 12 h of incubation. It was observed that cell viability was not much decreased after increasing the concentration up to 10 mg/mL. No significant differences were observed in cytotoxicity assay by using 0.3–10 mg/mL concentrations (Figure 4). The extract was observed to be non-toxic after 12 h assay as the extract still shows about 97 per cent of cell viability. Our results were found similar to the results presented by Yue et al. ^[21][35].

Figure 4. Effect of L. reuteri FBR extracts on viability of Caco-2 cells analyzed by Ez cytox assay kit. Cells were treated with an increased concentration of L. reuteri FBR extracts for 12 h. Data are represented as means ± standard deviations (n = 3).

3.2. Cellular Antioxidant Activity (CAA)

The effect of pretreatment of Caco-2 cells with L. reuteri fermented extract of brown rice on intracellular reactive oxygen species (ROS) was determined using a cell-based assay. The fluorescent probe DCFH-DA is used as an indicator of ROS and oxidative stress in our study. The nonionic and nonpolar DCFH-DA probe diffuses passively into cells before being hydrolyzed by intracellular esterases to form nonfluorescent 2′,7′-dichlorofluorescein (DCFH). Later in the presence of ROS, DCFH that is trapped inside cells is oxidized into fluorescent 2′,7′-dichlorofluorescein (DCF) ^[22][36]. When the cellular antioxidant defence system fails to compensate for ROS production, oxidative stress occurs. This reaction can be slowed down using bioactive compounds, preventing the generation of DCF. Following the uptake of antioxidant compounds can be accomplished on the cell membrane surface or within the cell ^[23][37]. We evaluated the effect of our L. reuteri FBR extract against oxidative stress in Caco-2 cells. In our study, ABAP was chosen as an intracellular oxidizing agent to simulate oxidative stress in cells. 600 µmol L⁻¹ ABAP was chosen as the optimal concentration to induce oxidation. As represented in Figure 5A, CAA values in L. reuteri FBR extract were observed to be 5.7 times higher than the raw BR sample at a concentration of 1mg/mL. Our results indicate that extracts reduced ROS levels at rest in a dose-dependent manner (Figure 5B); CAA values were increased with concentration (0.5 mg/mL to 5 mg/mL) from 49.50 ± 1.67% to 72.49 ± 1.23%. The strength of inhibition strongly followed a curvilinear pattern as L. reuteri FBR extract concentrations increased. A similar effect was previously observed in the study of Grauzdytė et al., where they observed Phyllanthus phillyreifolius extracts in HEK-293 cells ^[24][38], and the study of Kellett et al. ^[25][39] in Caco-2 cells.

Figure 5. In Caco-2 cells, peroxyl radical-induced oxidation of DCFH to DCF and ROS inhibition by raw BR and L. reuteri FBR extract (A,B) showing the effect of dose-dependent inhibition of L. reuteri FBR extracts (0.5–5 mg/mL). Data were represented as means ± standard deviations (n = 3) with one way ANOVA. The columns with different letters (a–d) show significant differences using Tukey’s test at p < 0.05.

4. Conclusions

In our study, we discovered that L. reuteri FBR had higher antioxidant activity as well as a higher concentration of phenolics and flavonoids among all LABs used for the study. This shows the ability of L. reuteri as a promising fermentation strain to increase the bioavailability of cereals or grains in producing health-promoting functional materials. L. reuteri fermentation improves phenolic constituents and antioxidant activity of BR, improves food quality, and confers organoleptic characteristics. Furthermore, we discovered that L. reuteri FBR enhanced the production of essential amino acids and fatty acids using untargeted metabolomics. The present study has provided information on bioactive compounds and antioxidant activities as well as the cellular antioxidant capacities of L. reuteri FBR. These data are required for the processing of the whole BR and its products for the pharmaceutical and food markets. As a result, new strategies and collaborations among industry, researchers, and relevant agencies are required to publicize whole grain consumption. Additionally, the current research is part of ongoing efforts to increase the added value of brown rice production and use in the prevention of human chronic diseases caused by oxidative stress. Moreover, these findings also make this sample a promising material for the development of health-promoting functional food. Whereas, it is necessary to perform more research into the mechanisms of different types of fermentation (solid and liquid-state) on single/pure phenolic compounds and antioxidant properties. Furthermore, in vivo models should be used to study the bioavailability and absorption of phenolic compounds in the gut.