foods
Article
Prebiotic, Antipathogenic Bacteria and Hypocholesterolemia
Properties of Fermented Rice Bran Extracts Derived from Black
Rice and Germinated Brown Rice
Khaetthareeya Sutthanut 1,2, *,† , Patcharaporn Tippayawat 3,† , Sukanya Srijampa 3 , Wisitsak Phoksawat 4 ,
Pornchanan Vachirodom 1 and Roongrawee Wandee 1
Citation: Sutthanut, K.; Tippayawat,
P.; Srijampa, S.; Phoksawat, W.;
Vachirodom, P.; Wandee, R. Prebiotic,
Antipathogenic Bacteria and
Hypocholesterolemia Properties of
Fermented Rice Bran Extracts
Derived from Black Rice and
Germinated Brown Rice. Foods 2022,
11, 3704. https://doi.org/10.3390/
foods11223704
Academic Editors: Evandro Leite De
Souza and Xichun Peng
Received: 9 September 2022
Accepted: 12 November 2022
Published: 18 November 2022
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Attribution (CC BY) license (https://
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4.0/).
Foods 2022, 11, 3704. https://doi.org/10.3390/foods11223704
1 Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University,
Khon Kaen 40002, Thailand
2 Human High Performance & Health Promotion Research Institute: HHP&HP Research Institute,
Khon Kaen University, Khon Kaen 40002, Thailand
3 Department of Medical Technology, Faculty of Associated Medical Sciences, Khon Kaen University,
Khon Kaen 40002, Thailand
4 Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
* Correspondence: khaesu@kku.ac.th
† These authors contributed equally to this work.
Abstract: Rice bran is a rich source of health-promoting nutrition and bioactive compounds; never-
theless, the properties of rice brans depend on cultivars, ages, and preparation methods, drawing
the potential of raw materials for health benefits. Therefore, this research aimed to investigate the
health-promoting properties of fermented rice bran extracts from cultivar black rice (H7F) and ger-
minated brown rice (G13F), focusing on their prebiotic, antipathogenic bacteria activity and safety
demonstrated in vitro and in vivo study models, respectively. Here, the screening of metabolites’
change after rice bran fermentation by ATR-FTIR spectra revealed specific peaks corresponding to the
composited components of protein, carbohydrate, and lipid. Then, in the in vitro study, the prebiotic
capability of H7F and G13F extracts was demonstrated by a growth-promoting effect on Lactobacillus
delbrueckii subsp. lactis under specific acidic conditions. Furthermore, antipathogenic bacterial activity
against Escherichia coli and Staphylococcus aureus was presented at 25 mg/mL of MIC values and
50 mg/mL of MBC of both fermented rice bran extracts, eliminating the bacteria by interfering with
the biofilm formation. For safety, an acute and chronic toxicity study using Wistar rats was conducted,
in which changes in the body and organ weights, histopathology of organs, blood chemistry, and
hematological parameters were observed after H7F and G13F treatment. Desirably, they showed no
toxicity, with a significant reduction in blood cholesterol levels in the chronic treatment of H7F and
G13F. Conclusively, the overall results evidenced the health benefits of H7F and G13F related to their
prebiotic and antipathogenic bacteria properties and hypocholesterolemia potential with a high level
of safety. Therefore, the fermented rice bran extracts were demonstrated as potential materials for the
further development of functional ingredients and health products.
Keywords: rice bran; black rice; germinated brown rice; ferment; prebiotic; antibacterial; toxicity
1. Introduction
Rice is one of the most important crops cultivated worldwide, especially in the Asian
region [1,2]. Thailand is one of the top 10 rice-producing countries and a banner exporter of
processed rice products [3]. Rice bran, a rice milling by-product, is a nourishing source of
phytochemical compounds, including oryzanol, γ-amino butyric acid (GABA), phenolics,
flavonoids, arabinoxylan, phytosterols, phytic acid, and vitamins, etc. [4–7]. According to
several previous studies, rice bran extracts from various kinds of colored rice (pink, red,
purple, brown, and black) have been proven to contain health-promoting benefits, e.g.,
https://www.mdpi.com/journal/foods
Foods 2022, 11, 3704 2 of 15

antioxidation, anti-inflammation, and anticancer effects, as well as the improvement of

lipid profiles [6,8,9].
Prebiotics and probiotics are functional health-promoting food ingredients on demand
in the market with highly safe and naturally health-balancing features. Prebiotics are
nondigestible foods that can enhance the propagation and functions of health-beneficial
gut microbiomes—probiotics such as Bifidobacterium and Lactobacillus spp. [10], the two
common genera of probiotic bacteria, with some isolated species: L. plantarum, L. casei, L.
acidophilus, B. animalis, and B. bifidum. Probiotics have antipathogenic activity associated
with producing bioactive compounds, short-chain fatty acids (SCFA), peptides, and bac-
teriocins. Prebiotics possess health-promoting effects by modulating the immune system,
lowering allergy risk, reducing the production of harmful protein metabolites, and sup-
pressing pathogenic bacterial growth in the gastrointestinal tract [11,12]. Prebiotics such
as dietary fiber are food components that enhance the growth of probiotic bacteria; for
instance, the fructooligosaccharides found in onion, asparagus, and wheat are the source
of Bifidobacterium probiotics [12]. In addition, combining prebiotics and probiotics, known
as synbiotics, offers intensive synergistic benefits on multiple health issues: nutrition,
preventing infant diseases, reducing irritable bowel syndrome-relating symptoms and
compromising bacterial vaginosis and urinary tract infection, and lowering cholesterol
blood level [12–15].
Interestingly, the prebiotic and synbiotic properties of rice bran extracts have been evi-
denced. Dietary rice bran enhanced the growth and colonization of Lactobacillus rhamnosus
GG and Escherichia coli Nissle 1917 [16], which was effective against human rotavirus (HRV)
diarrhea, promoted maintenance of gut epithelial health during infection, and improved
innate immune response by increasing intestinal IFN-γ and total IgA level in gnotobiotic
pigs [17]. Moreover, fermented rice bran by Lactobacillus plantarum B2 featured a synbiotic
product in promoting fecal lactic acid bacteria growth and inhibiting pathogens (Escherichia
coli and Salmonella spp.) in Wistar rat intestine [18]. Interestingly, synergistic effects from
combinational treatment of Thai black rice bran extract and yeast beta-glucan showed an
increase in antioxidant capacity and reduced inflammatory cytokines in colitis-induced
rats [6,19]. Moreover, fermentation is a biochemical process to improve rice and rice bran’s
nutritional quality and biological activities [20,21]. Fermentation increased crude protein
contents, phytochemicals, and gamma-butyric acid in fermented rice bran [21,22].
Although many prior studies have reported various health benefits of rice bran extracts,
there is very little information regarding fermented rice bran from KKU ULR0381 black rice
cultivar (H7F) and germinated brown rice (G13F). Thus, this study aimed to investigate
the biochemical components of fermented rice bran extracts by attenuated total reflection–
Fourier transform infrared spectroscopy (ATR-FTIR), the prebiotic property promoting
Lactobacillus dellbrueckii (L. dellbrueckii) subsp. lactis growth, antipathogenic bacteria activity,
and a safety and toxicity test in both in vitro and in vivo models. The obtained information
will deliver a guideline to support health product development from H7F and G13F.

2. Results
2.1. Comparison of Biochemical Components between Nonfermented and Fermented Rice Bran by
Derivative FT-IR Spectra
In this study, ATR-FTIR was used to monitor two different fermentative rice brans
compared with nonfermentative forms of those rice brans. The second derivative ATR-FTIR
spectra of H7F and G13F showed different compositions of biochemical ingredients from
nonfermented rice bran (H7NF and G13NF) (Figure 1). Both fermented rice bran extracts
exhibited feature peaks corresponding to protein, carbohydrate, and lipid composition. For
the H7F sample, the specific peaks included phosphate and collagen at wavelength number
position 1089 cm−1 , serine, threonine, and tyrosine of proteins at 1167 cm−1 , phosphate I
and amide III at 1234 cm−1 , and fatty acid at 1740 cm−1 of the H7F spectrum (Figure 1A).
On the other hand, at the G13F spectra, the major peaks were found at 1164 cm−1 (ser-
ine, threonine, and tyrosine of proteins), 1268 cm−1 (phosphate I), 1409 cm−1 (fatty acid
Foods 2022, 11, x FOR PEER REVIEW  3  of  17 
 

composition. For the H7F sample, the specific peaks included phosphate and collagen at 
wavelength number position 1089 cm−1, serine, threonine, and tyrosine of proteins at 1167 
Foods 2022, 11, 3704 3 of 15
cm−1, phosphate I and amide III at 1234 cm−1, and fatty acid at 1740 cm−1 of the H7F spec‐
trum (Figure1A). On the other hand, at the G13F spectra, the major peaks were found at 
1164 cm−1 (serine, threonine, and tyrosine of proteins), 1268 cm−1 (phosphate I), 1409 cm−1 
and acid 
(fatty  amino acid),
and  and
amino  1707
acid),  and  −1 (guanine).
cm1707  The The 
cm−1  (guanine).  detailed information
detailed  onon 
information  biochemical
bio‐
chemical compositions’ change after fermentation is shown in Figure 1C. 
compositions’ change after fermentation is shown in Figure 1C.

 
Figure 1. Comparison of the second derivative spectra between H7NF vs. and G13NF vs. 
Figure 1. Comparison of the second derivative spectra between H7NF vs. H7F (A) H7F (A) and G13NF
G13F (B) by ATRFTIR. The prediction of biochemical compositions’ change by using the band posi‐
vs. G13F (B) by ATRFTIR. The prediction of biochemical compositions’ change by using the band
tions at specific wavelength number (cm−1) of IR spectra of H7F and G13F compared to H7NF and 
positions at specific wavelength number (cm−1 ) of IR spectra of H7F and G13F compared to H7NF
G13NF, respectively (C). 
and G13NF, respectively (C).
2.2. Lactic Acid Bacteria Growth Promoting of Fermented Rice Bran Extracts 
2.2. Lactic Acid Bacteria Growth Promoting of Fermented Rice Bran Extracts 
According 
Accordingto to
the the
metabolites’ 
metabolites’change  of  rice 
change of bran  under 
rice bran the  fermentation, 
under the  fer‐the fer-
the fermentation,
mented rice bran extracts (H7F and G13F) were used to prove the prebiotic property for a 
mented rice bran extracts (H7F and G13F) were used to prove the prebiotic property for
probiotic bacterium—L
a probiotic bacterium—L.. delbrueckii subsp
delbrueckii .subsp.
lactis. To determine whether H7F and G13F ex‐
lactis. To determine whether H7F and G13F
tracts  can  promote  the  growth  probiotic  bacteria 
extracts can promote the growth probiotic bacteria in  in  vitro 
in in study,  the  numbers 
vitro study, of  L. of L.
the numbers
delbrueckii subsp. lactis were determined after culture with the extracts. The probiotic was
significantly increased in coculture condition of all concentrations (3.125, 6.25, 12.5, 25, and
  50 mg/mL) of both H7F and G13F extracts (Figure 2A), and it can observe the plateau in
the numbers of lactic acid bacteria at concentrations of 12.5 mg/mL of the G13F extract.
Moreover, the pH of nonfermented rice bran extracts (H7NF and G13NF) and fermented
rice bran extracts (H7F and G13F) were evaluated to indicate optimal acid production to
promote the lactic acid bacteria growth. The reduction in pH values, the higher level of
lactic acid production (Figure 2B) with a dose-dependent trend (decreasing pH) depending
 significantly  increased in coculture  condition  of all  concentrations  (3.125,  6.25, 12.5, 25, 
and 50 mg/mL) of both H7F and G13F extracts (Figure 2A), and it can observe the plateau 
in the numbers of lactic acid bacteria at concentrations of 12.5 mg/mL of the G13F extract. 
Moreover, the pH of nonfermented rice bran extracts (H7NF and G13NF) and fermented 
rice bran extracts (H7F and G13F) were evaluated to indicate optimal acid production to 
Foods 2022, 11, 3704 4 of 15
promote the lactic acid bacteria growth. The reduction in pH values, the higher level of 
lactic acid production (Figure 2B) with a dose‐dependent trend (decreasing pH) depend‐
ing on the extract concentrations. The results supported that the fermented rice bran ex‐
ontracts (H7F and G13F) could promote L
the extract concentrations. The results . delbrueckii subsp
supported that.the fermented rice bran extracts
lactis in the apparent optimal 
(H7F and G13F) could promote L. delbrueckii subsp. lactis in the apparent optimal growth at
growth at acidic pH; optimal pH range between 4.5–5.2. 
acidic pH; optimal pH range between 4.5–5.2.

 
Figure 2. The fermented rice bran extracts (H7F and G13F) promoting L. delbrueckii subsp. lactis
Figure  2.  The  fermented  rice  bran  extracts  (H7F  and  G13F)  promoting L.  delbrueckii  subsp. lactis 
growth determined by flow cytometry compared to the control (A) and a pH gradient during various
growth determined by flow cytometry compared to the control (A) and a pH gradient during vari‐
concentrations of nonfermented rice bran extracts (H7NF and G13NF) and fermented rice bran
ous concentrations of nonfermented rice bran extracts (H7NF and G13NF) and fermented rice bran 
extracts (H7F and G13F) (B); * significant difference when compared to the control (p-value < 0.05).
extracts (H7F and G13F) (B); * significant difference when compared to the control (p‐value < 0.05).  

2.3. Pathogenic Bacteria Growth Inhibition of Nonfermented and Fermented Rice Bran Extracts
2.3. Pathogenic Bacteria Growth Inhibition of Nonfermented and Fermented Rice Bran Extracts   
The antibacterial potential of nonfermented and fermented rice bran extract against
The antibacterial potential of nonfermented and fermented rice bran extract against 
pathogenic bacteria was evaluated by agar well diffusion assay. The 400 mg/mL concentra-
pathogenic bacteria was evaluated by agar well diffusion assay. The 400 mg/mL concen‐
tion of either
tration H7F or G13F extracts inhibited the growth of both S. .aureus
of either H7F or G13F extracts inhibited the growth of both S aureusandandE. E.coli;
coli;the
averages of the inhibition zone of H7F were 12.15 and 15.96 mm, and of G13F were 12.15
the averages of the inhibition zone of H7F were 12.15 and 15.96 mm, and of G13F were 
and 15.50
12.15  mm,
and  respectively.
15.50  Unlike
mm,  respectively . Unlike 
the fermented rice bran
the  fermented  extracts,
rice  bran  the nonfermented
extracts,  the  nonfer‐
rice bran samples (H7NF and G13NF) could not inhibit these two pathogenic bacteria
mented rice bran samples (H7NF and G13NF) could not inhibit these two pathogenic bac‐
teria  (3A,B).
(Figure Figure In3A,B ). In  addition, 
addition, the results from the
the  results  broth
from the microdilution technique
broth  microdilution  showed
technique 
showed  equivalent 
equivalent degrees ofdegrees  of  antimicrobial 
antimicrobial activitiesactivities 
betweenbetween 
H7F andH7F and  G13F  samples. 
G13F samples. The mini-
mum inhibitory concentration (MIC) values of H7F and G13F on E. coli and S.. aureus
The minimum inhibitory concentration (MIC) values of H7F and G13F on E coli and S .
growth
aureus growth were identically detected at 25 and 50 mg/mL concentrations, respectively
were identically detected at 25 and 50 mg/mL concentrations, respectively. For minimum . 
For minimum bactericidal concentration (MBC), 50 mg / mL of both fermented rice bran 
bactericidal concentration (MBC), 50 mg/mL of both fermented rice bran extracts could
inhibit the growth of S. aureus and E. .coli
extracts could inhibit the growth of S aureus and E
(Figure . coli (Figure 3C,D).   
3C,D).

2.4. Effect of the Fermented Rice Bran on Biofilm Formation

Following the demonstration of antipathogenic bacterial effects after treatment with the
fermented rice bran extracts (H7F and G13F), the inhibitory mechanism against pathogenic
bacteria of H7F or G13F was further investigated. Biofilm formation is an important viru-
lence factor produced by both S. aureus and E. coli; this mechanism can prevent pathogenic
bacteria destruction by toxic agents [23,24]. In our study, the H7F and G13F at half of
MBC concentration (25 mg/mL) were cultured with S. aureus and E. coli; consequently, the
biofilm formation and bacterial destruction were delineated by imaging the morphology of
S. aureus and E. coli after the sample treatment under a scanning electron microscope (SEM).
Biofilm formation reduction and bacterial cell breakage were detected in the treated groups,
which differed from the control (untreated) group of each bacteria type. The control group
showed colonization and biofilm formation of S. aureus; similarly, the control for E. coli
  presented biofilm formation and colonization with a flagella structure. Based on the results,
the induction of bacterial cell membrane breakage and colony shrinkage was suggested as
a mechanism of antimicrobial activity detected in H7F and G13F (Figure 4).

2.5. Toxicity of the Fermented Rice Bran Extracts In Vitro Study

After fermentation of the rice brans, we demonstrated the metabolites’ change and
acidic increase; thus, before using them as a supplement or functional food/beverage,
toxicity to human and animal models should be investigated. The toxicity to human
peripheral mononuclear cells (PBMCs) was demonstrated in in vitro study. For the result,
Foods 2022, 11, 3704 5 of 15

all the concentrations of either H7F or G13F extract showed no toxicity to human PBMCs
with more than 80% cell viability compared to the control (untreated group) (Figure 5A,B).
The %survival of the cells of both fermented rice bran extracts’ treatment might indicate
Foods 2022, 11, x FOR PEER REVIEW  5  of  17 
  that these metabolic compositions and acidic conditions after the rice bran fermentative
process were nontoxic to human PBMCs [25].

 
Figure 3. Screening of antimicrobial effects of fermented rice bran H7 (H7F) andextract (G13F) 
Figure 3. Screening of antimicrobial effects of fermented rice bran H7 (H7F) and G13 G13 extract (G13F)
compared to nonfermented rice (H7NF and G13NF). According to the well diffusion assay, only 
compared to nonfermented rice (H7NF and G13NF). According to the well diffusion assay, only
H7F and G13F significantly exhibited inhibition zone
H7F and G13F significantly exhibited inhibition zone against Escherichia coli and Staphylococcus au‐
against Escherichia coli and Staphylococcus
reus  (A,B).  A  minimum  inhibition  concentration  (MIC)  and  minimum  bactericidal  concentration 
aureus (A,B). A minimum inhibition concentration (MIC) and minimum bactericidal concentration
(MBC) values were also evaluated by broth microdilution technique (C,D). Various concentrations 
(MBC) values were also evaluated by broth microdilution
of H7F for MIC and MBC were investigated, as follows : lane 1 technique (C,D).
= 200 mg/mL; 2  = Various concentrations
100 mg/mL; 3  = 50
mg/mL; 4  = 25 mg/mL; 5  = 12.5 mg/mL; 6  = 6.25 mg/mL; 7  = 3.12 mg/mL; 8  = 1.56 mg/mL; 9 
of H7F for MIC and MBC were investigated, as follows: lane 1 = 200 mg/mL; 2 = 100= mg/mL;
0.8 
3mg= /50
mL,  respectively
mg/mL; . Distilled 
4 = 25 mg/mL;water  (DW) 
5 = 12.5 was  used 
mg/mL; 6 = for 
6.25noninhibition 
mg/mL; 7 = control  (or  lane +),
3.12 mg/mL; and 
8 = 1.56 5 
mg/mL;
9μg=/mL of Ampicillin drug (Amp) was used for inhibition control
0.8 mg/mL, respectively. Distilled water (DW) was used(or lane −). **** indicates statistical 
for noninhibition control (or lane +),
significance at p‐value < 0.0001. 
and 5 µg/mL of Ampicillin drug (Amp) was used for inhibition control (or lane −). **** indicates
statistical significance at p-value < 0.0001.
2.4. Effect of the Fermented Rice Bran on Biofilm Formation 
2.6. Toxicity in Acute or Chronic Manner of the Fermented Rice Bran Extracts In Vivo Study
Following the demonstration of antipathogenic bacterial effects after treatment with 
the fermented rice bran extracts (H7F and G13F), the inhibitory mechanism against path‐
To confirm the safety and bioavailability of product use, the study in the animal models
ogenic  bacteria 
should of  H7F  or In
be demonstrated. G13F  was  further 
our study, Wistarinvestigated.  Biofilm 
rats were used formation 
for toxicity is  an 
study im‐acute
in the
portant virulence factor produced by both S. aureus and E. coli; this mechanism can pre‐
or chronic manner. The growth and behavior of all the animals after receiving a single dose
vent pathogenic bacteria destruction by toxic agents [23,24]. In our study, the H7F and 
of G13F and H7F extracts at each concentration of 1000, 2500, and 5000 mg/kg/day were
G13F at half of MBC concentration (25 mg/mL) were cultured with S. aureus and E. coli; 
normal over a 14-day period without detection of mortality, change of hematological and
consequently, the biofilm formation and bacterial destruction were delineated by imaging 
biochemical values, or any pathological lesion of organs.
the morphology of S. aureus and E. coli after the sample treatment under a scanning elec‐
For chronic toxicity, both the male and female animals that received fermented rice
tron microscope (SEM). Biofilm formation reduction and bacterial cell breakage were de‐
bran H7F or G13F showed healthiness and no signs of toxicity when compared to the
tected in the treated groups, which differed from the control (untreated) group of each 
control group. In addition, there were no significant differences in body and organ weights
bacteria type. The control group showed colonization and biofilm formation of S. aureus; 
among the treated groups
similarly, the control that received different doses of the extracts. However, there
for E. coli presented biofilm formation and colonization with a fla‐
were changes in
gella structure WBC, platelet, and cholesterol levels after treatment with specific doses of
. Based on the results, the induction of bacterial cell membrane breakage 
H7F or G13F. For hematological values, the increase in WBC levels in all treated groups of
and colony shrinkage was suggested as a mechanism of antimicrobial activity detected in 
H7F and G13F of
H7F and G13F (Figure both4).
genders
   was demonstrated; however, significant differences were
detected at high doses; 2000 mg/kg of H7F and 500 mg/kg of G13F in female rats as well
as of 1000 and 2000 mg/kg of G13F in male rats, when compared to the vehicle group
(Table 1). Furthermore, compared to the untreated group, higher platelet count levels of
 Foods 2022, 11, x FOR PEER REVIEW  6  of  17 
 

Foods 2022, 11, 3704 6 of 15

male rats receiving 500, 1000, and 2000 mg/kg of G13F extract were significantly revealed
(Table 1). Additionally, the reduction in cholesterol levels in 500 and 2000 mg/kg of H7F
Foods 2022, 11, x FOR PEER REVIEW and 1000 mg/kg of G13F-treated groups of male rats was significant compared 6  of  to
17  both
 
the vehicle and untreated groups. In contrast, others presented no statistical significance
(Table 2).

 
Figure 4. Fermented rice bran H7 (H7F) and G13 extract (G13F) inhibited biofilm formation. How‐
ever, biofilm and colonization of Staphylococcus aureus were still observed in the untreated condition 
(A), while they were decreased in the treated groups with either H7F (B) or G13F extracts (C). In 
addition,  while  the  control  (untreated)  group  of  Escherichia  coli still  presented  biofilm  formation, 
flagella structure, and colonization (D), they were decreased in the treated groups with either H7F 
(E) and G13F extracts (F). Interestingly, cell membrane breakage of E. coli (a Gram‐negative bacillus) 
was displayed in the treatment of either H7F or G13F (E,F). 

2.5. Toxicity of the Fermented Rice Bran Extracts In Vitro Study   
After fermentation of the rice brans, we demonstrated the metabolites’ change and 
Figure 4. Fermented rice bran H7 (H7F) and G13 extract (G13F) inhibited biofilm formation.  However,
acidic increase; thus, before using them as a supplement or functional food/beverage, tox‐
Figure 4. Fermented rice bran H7 (H7F) and G13
biofilm and colonization of Staphylococcus aureus extract (G13F) inhibited biofilm formation
were still observed in the untreated condition
icity to human and animal models should be investigated. The toxicity to human periph‐ . How‐ (A),
ever, biofilm and colonization of Staphylococcus aureus were still observed in the untreated condition 
eral mononuclear cells (PBMCs) was demonstrated in in vitro study. For the result, all the 
while they were decreased in the treated groups with either H7F (B) or G13F extracts (C). In addition,
(A), while they were decreased in the treated groups with either H7F (B) or G13F extracts (C). In 
concentrations of either H7F or G13F extract showed no toxicity to human PBMCs
while thewhile 
addition,  control
the (untreated)
control  (untreated)  of Escherichia
group group  coli still
of  Escherichia  coli presented biofilm
still  presented 
with 
formation,
biofilm  flagella
formation, 
more than 80% cell viability compared to the control (untreated group) 
structure, and colonization (D), they were decreased in the treated groups(Figure
with 5A,B
either
flagella structure, and colonization (D), they were decreased in the treated groups with either H7F  ). The 
H7F (E) and
%survival of the cells of both fermented rice bran extracts’ treatment might indicate that 
G13F
(E) . Interestingly, cell membrane breakage of E. coli
extracts (F). Interestingly,
and G13F extracts (F) cell membrane breakage of E. coli (a(a Gram‐negative bacillus) 
Gram-negative bacillus) was
these metabolic compositions and acidic conditions after the rice bran fermentative pro‐
was displayed in the treatment of either H7F or G13F (E,F). 
displayed in the treatment of either H7F or G13F (E,F).
cess were nontoxic to human PBMCs [25]. 
2.5. Toxicity of the Fermented Rice Bran Extracts In Vitro Study   
After fermentation of the rice brans, we demonstrated the metabolites’ change and 
acidic increase; thus, before using them as a supplement or functional food/beverage, tox‐
icity to human and animal models should be investigated. The toxicity to human periph‐
eral mononuclear cells (PBMCs) was demonstrated in in vitro study. For the result, all the 
concentrations of either H7F or G13F extract showed no toxicity to human PBMCs with 
more than 80% cell viability compared to the control (untreated group) (Figure 5A,B). The 
%survival of the cells of both fermented rice bran extracts’ treatment might indicate that 
these metabolic compositions and acidic conditions after the rice bran fermentative pro‐
cess were nontoxic to human PBMCs [25]. 
 
Figure 5. Toxicity test of fermented rice bran H7 (H7F) and extract (G13F) to human PBMCs by 
Figure 5. Toxicity test of fermented rice bran H7 (H7F) and G13 G13 extract (G13F) to human PBMCs by
MTT assay.Both H7F (A)
MTT assay. Both H7F (A) and G13F (B)
and G13F (B) extracts showed no toxicity to human PBMCs, expressing 
extracts showed no toxicity to human PBMCs, expressing
more than 80%of cell viability at all tested concentrations. 
more than 80% of cell viability at all tested concentrations.

   
Table 1. Effects on hematological values in male (A) and female Wistar rats (B) of fermented rice bran
H7 (H7F) and G13 extracts (G13F) in chronic toxicity study.

  Male (n = 5 in each group)

(A) Doses of H7F (mg/kg) Doses of G13F (mg/kg)
Parameters Vehicle Untreated
500 1000 2000 500 1000
  2000
6
RBC (10 /µL) 9.04 ± 0.49 Figure 5. Toxicity test of fermented rice bran H7 (H7F) and G13
8.75 ± 0.84 8.05 ± 2.04 8.80 ± 0.06 8.16 ± 1.42 9.16extract (G13F) to human PBMCs by 
± 1.29 9.07 ± 0.61 9.04 ± 0.60
HGB (g/dL) 15.90 ± 0.32 MTT assay.
14.96 ± 1.22 Both H7F (A)
14.16 ± 3.57 and G13F (B)
15.55 ± 0.66 extracts showed no toxicity to human PBMCs, expressing 
14.73 ± 2.55 15.88 ± 1.71 15.60 ± 0.58 15.60 ± 0.42
HCT (%) 48.26 ± 1.62 44.76 ± 3.78
more than 80% 41.04 ± 10.63 45.10 ± 2.16 42.18 ± 7.61
of cell viability at all tested concentrations.  44.48 ± 4.38 43.96 ± 1.59 44.38 ± 1.62

   
Foods 2022, 11, 3704 7 of 15

Table 1. Cont.

WBC (103 /µL) 3.16 ± 1.63 2.43 ± 1.80 3.71 ± 1.78 4.63 ± 1.59 4.15 ± 1.39 5.03 ± 1.69 6.08 ± 1.08 a,b 6.13 ± 1.01 a,b
PLT (103 /µL) 502.8 ± 327.2 482.0 ± 156.1 587.6 ± 193.2 565.0 ± 235.4 558.8 ± 378.4 838.6 ± 120.2 b 719.0 ± 99.4 b 826.8 ± 124.2 b
MPV (fL) 8.0 ± 0.30 7.6 ± 0.10 7.7 ± 0.36 7.7 ± 0.14 7.6 ± 0.31 7.6 ± 0.20 7.4 ± 0.13 7.5 ± 0.11
NE (%) 15.62 ± 3.64 22.82 ± 18.06 17.70 ± 8.87 15.48 ± 4.67 12.23 ± 3.35 15.42 ± 4.41 12.14 ± 3.08 12.18 ± 2.25
LY (%) 76.42 ± 5.46 70.80 ± 15.27 77.52 ± 7.35 77.48 ± 3.49 83.50 ± 2.65 77.72 ± 4.07 83.50 ± 3.36 82.80 ± 4.03
MO (%) 4.88 ± 3.66 4.16 ± 2.42 2.84 ± 1.80 3.63 ± 1.56 3.28 ± 1.67 5.14 ± 0.42 2.90 ± 0.88 3.68 ± 1.99
EO (%) 3.04 ± 3.33 2.12 ± 1.90 1.86 ± 1.23 3.23 ± 1.9 0.93 ± 0.25 1.66 ± 0.50 1.44 ± 0.30 1.28 ± 0.33
BA (%) 0.00 ± 0.00 0.10 ± 0.14 0.08 ± 0.11 0.00 ± 0.00 0.00 ± 0.00 0.06 ± 0.13 0.02 ± 0.05 0.06 ± 0.09
MCV (fL) 53.44 ± 1.17 51.16 ± 0.65 50.92 ± 0.87 51.50 ± 2.23 51.73 ± 2.73 48.76 ± 1.94 48.54 ± 1.12 49.22 ± 2.40
MCH (pg) 17.62 ± 0.63 17.10 ± 0.28 17.58 ± 0.28 17.75 ± 0.65 18.05 ± 0.31 17.40 ± 0.55 17.22 ± 0.55 17.30 ± 0.81
MCHC (g/dL) 32.98 ± 0.46 33.42 ± 0.24 34.54 ± 0.58 34.48 ± 0.49 34.95 ± 1.49 35.68 ± 0.40 35.50 ± 0.37 35.18 ± 0.56
RDW (%) 21.88 ± 0.84 21.76 ± 1.22 20.20 ± 3.43 20.88 ± 0.41 20.70 ± 2.98 22.42 ± 2.24 21.92 ± 1.20 22.32 ± 1.18
Female (n = 5 in each group)
(B) Doses of H7F (mg/kg) Doses of G13F (mg/kg)
Parameters Vehicle Untreated
500 1000 2000 500 1000 2000
RBC (106 /µL) 8.06 ± 1.78 7.57 ± 1.10 8.37 ± 0.34 8.19 ± 0.30 7.94 ± 1.06 8.04 ± 0.33 7.72 ± 0.76 8.33 ± 0.35
HGB (g/dL) 15.30 ± 2.69 13.92 ± 1.96 15.44 ± 0.71 15.28 ± 0.50 14.62 ± 2.02 15.00 ± 0.86 14.10 ± 1.57 15.56 ± 0.72
HCT (%) 43.62 ± 8.36 41.14 ± 5.50 44.06 ± 2.35 43.86 ± 1.59 42.56 ± 5.50 43.26 ± 2.32 41.00 ± 40.00 44.66 ± 1.85
WBC (103 /µL) 1.85 ± 0.67 1.97 ± 1.77 2.83 ± 1.71 2.90 ± 0.89 3.27 ± 0.98 a 3.46 ± 1.02 a 2.26 ± 0.68 2.96 ± 1.17
PLT (103 /µL) 514.6 ± 283.2 484.4 ± 402.4 560.0 ± 49.7 575.4 ± 235.4 568.8 ± 238.2 682.2 ± 133.4 722.0 ± 91.5 703.8 ± 110.8
MPV (fL) 7.8 ± 0.54 7.4 ± 0.28 7.3 ± 0.3 7.4 ± 0.23 7.4 ± 0.48 7.4 ± 0.15 7.5 ± 0.23 7.4 ± 0.13
NE (%) 14.87 ± 7.74 12.62 ± 7.00 8.52 ± 2.25 7.45 ± 2.95 12.42 ± 2.79 14.80 ± 9.93 20.23 ± 5.24 11.78 ± 3.41
LY (%) 76.53 ± 6.74 80.52 ± 11.81 85.20 ± 7.12 82.12 ± 3.74 76.90 ± 8.88 81.48 ± 9.62 75.50 ± 3.47 82.86 ± 5.38
MO (%) 3.73 ± 0.84 2.00 ± 1.59 2.90 ± 1.72 4.68 ± 1.53 5.26 ± 3.79 2.52 ± 1.56 2.23 ± 1.72 4.12 ± 2.20
EO (%) 4.86 ± 4.80 4.76 ± 4.52 3.34 ± 4.02 4.40 ± 6.53 5.30 ± 3.34 1.16 ± 0.49 2.03 ± 0.32 1.30 ± 0.60
BA (%) 0.82 ± 1.65 0.10 ± 0.14 0.04 ± 0.09 0.54 ± 1.00 0.12 ± 0.16 0.04 ± 0.09 0.00 ± 0.00 0.12 ± 0.16
MCV (fL) 54.38 ± 1.61 54.50 ± 1.90 52.64 ± 1.64 53.58 ± 1.60 53.64 ± 0.17 53.82 ± 0.99 53.17 ± 0.96 53.68 ± 2.16
MCH (pg) 19.36 ± 4.04 18.40 ± 0.38 18.46 ± 0.43 18.66 ± 0.35 18.42 ± 0.71 18.64 ± 0.45 18.23 ± 0.35 18.70 ± 0.59
MCHC (g/dL) 35.52 ± 6.82 33.82 ± 0.96 35.08 ± 0.40 34.86 ± 0.54 34.34 ± 0.49 34.70 ± 0.25 34.37 ± 0.55 34.82 ± 0.47
RDW (%) 18.48 ± 2.75 17.38 ± 2.12 18.52 ± 1.10 18.78 ± 0.55 18.28 ± 1.57 18.04 ± 1.27 18.37 ± 0.84 19.00 ± 0.55
Data are represented as mean ± standard deviation (SD). N = number of samples; RBC = red blood cell;
HGB = hemoglobin; HCT = hematocrit; WBC = white blood cell; PLT = platelet; MPV = mean platelet vol-
ume; NE = neutrophils; LY = lymphocyte; MO = monocyte; EO = eosinophils; BA = basophile; MCV = mean
cell volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration;
RDW = red blood cell distribution width; statistical significance at p-value < 0.05 when compared to vehicle group
(A) and untreated group (B).

Table 2. Effects on biochemical parameters in male (A) and female Wistar rats (B) of fermented rice
bran H7 (H7F) and G13 extracts (G13F) in chronic toxicity study.

Male (n = 5 in each group)

(A) Doses of H7F (mg/kg) Doses of G13F (mg/kg)
Parameters Vehicle Untreated
500 1000 2000 500 1000 2000
BUN (mg/dL) 20.34 ± 0.78 26.32 ± 2.84 23.36 ± 1.62 17.50 ± 1.10 20.23 ± 2.63 26.3 ± 0.55 24.58 ± 0.40 25.48 ± 0.95
Cr (mg/dL) 0.32 ± 0.03 0.31 ± 0.06 0.30 ± 0.02 0.29 ± 0.05 0.27 ± 0.04 0.28 ± 0.29 0.31 ± 0.02 0.27 ± 0.03
Na (mmol/L) 145.6 ± 2.97 145.0 ± 1.00 142.0 ± 2.12 144.8 ± 1.71 144.5 ± 1.92 141.2 ± 1.92 142.2 ± 0.84 141.8 ± 2.28
K (mmol/L) 5.89 ± 0.26 5.60 ± 0.12 5.72 ± 0.58 5.69 ± 0.27 5.52 ± 0.70 6.06 ± 0.85 5.58 ± 0.33 6.35 ± 1.03
Cl (mmol/L) 97.3 ± 3.30 97.3 ± 2.32 96.0 ± 1.66 98.8 ± 1.50 99.5 ± 0.93 97.8 ± 1.35 97.7 ± 1.04 97.2 ± 1.88
HCO3 -
24.8 ± 3.35 27.8 ± 1.30 26.4 ± 2.19 27.5 ± 0.58 26.0 ± 0.82 27.8 ± 1.64 28.8 ± 1.30 26.4 ± 0.55
(mmol/L)
Cholesterol a,b a,b a,b
113.8 ± 25.28 90.8 ± 7.79 74.8 ± 8.87 81.5 ± 11.27 76.2 ± 6.34 90.8 ± 9.01 78.6 ± 5.32 81.6 ± 14.77
(mg/dL)
Triglyceride
235.0 ± 82.85 181.8 ± 31.15 150.2 ± 34.27 175.7 ± 40.96 185.8 ± 55.87 182.4 ± 32.55 174.2 ± 42.10 167.2 ± 38.47
(mg/dL)
Total protein
6.66 ± 0.43 6.42 ± 0.18 6.32 ± 0.30 6.25 ± 0.17 6.38 ± 0.34 6.22 ± 0.34 6.16 ± 0.17 6.38 ± 0.32
(g/dL)
Albumin
4.16 ± 0.40 4.32 ± 0.13 4.04 ± 0.25 4.23 ± 0.22 4.23 ± 0.29 4.02 ± 0.26 4.10 ± 0.12 4.22 ± 0.23
(g/dL)
Total bilirubin
0.08 ± 0.02 0.07 ± 0.01 0.08 ± 0.02 0.10 ± 0.03 0.10 ± 0.02 0.09 ± 0.29 0.08 ± 0.02 0.07 ± 0.02
(mg/dL)
ALT (U/L) 76.6 ± 55.32 99.6 ± 21.78 66.4 ± 25.23 44.2 ± 16.64 47.2 ± 5.74 73.8 ± 19.31 86.4 ± 48.52 55.2 ± 12.09
AST (U/L) 121.6 ± 39.87 162.0 ± 31.68 109.6 ± 15.63 147.2 ± 30.87 143.0 ± 23.02 136.2 ± 39.15 135.6 ± 20.31 87.4 ± 26.54
ALP (U/L) 97.8 ± 16.68 79.0 ± 19.33 79.2 ± 16.45 62.0 ± 8.76 94.0 ± 28.04 98.2 ± 26.78 74.4 ± 5.77 69.8 ± 6.50
Female (n = 5 in each group)
(B) Doses of H7F (mg/kg) Doses of G13F (mg/kg)
Parameters Vehicle Untreated
500 1000 2000 500 1000 2000
BUN (mg/dL) 20.48 ± 1.86 19.26 ± 1.86 12.38 ± 1.73 21.12 ± 3.78 15.04 ± 3.25 26.94 ± 1.66 26.77 ± 5.33 24.64 ± 2.29
Cr (mg/dL) 0.32 ± 0.01 0.33 ± 0.07 0.33 ± 0.05 0.35 ± 0.05 0.31 ± 0.05 0.33 ± 0.03 0.25 ± 0.02 0.34 ± 0.05
Na (mmol/L) 141.4 ± 1.14 145.4 ± 1.34 143.2 ± 1.30 141.6 ± 1.50 141.8 ± 2.59 141.2 ± 1.30 141.0 ± 0.70 144.0 ± 1.00
K (mmol/L) 4.93 ± 0.36 5.07 ± 0.33 4.84 ± 0.23 5.22 ± 0.16 5.79 ± 0.94 5.87 ± 0.22 5.66 ± 0.59 5.46 ± 0.17
Cl (mmol/L) 98.3 ± 0.66 100.9 ± 1.22 102.8 ± 0.89 98.9 ± 1.64 101.7 ± 1.90 98.7 ± 1.14 100.0 ± 1.83 100.8 ± 1.51
Foods 2022, 11, 3704 8 of 15

Table 2. Cont.

HCO3 -
28.2 ± 0.84 26.2 ± 1.30 26.2 ± 1.30 26.6 ± 0.89 24.8 ± 3.42 27.4 ± 1.82 27.0 ± 1.40 27.0 ± 1.23
(mmol/L)
Cholesterol
80.6 ± 13.85 79.8 ± 18.42 80.2 ± 18.31 84.4 ± 15.24 89.2 ± 19.72 82.6 ± 8.79 99.3 ± 33.56 89.4 ± 21.13
(mg/dL)
Triglyceride
187.2 ± 49.78 166.4 ± 26.12 125.8 ± 40.68 152.4 ± 45.10 128.4 ± 49.92 139.2 ± 35.27 124.3 ± 57.29 153.8 ± 50.49
(mg/dL)
Total protein
6.40 ± 0.17 6.62 ± 0.61 6.08 ± 0.15 6.40 ± 0.43 6.24 ± 0.40 6.28 ± 0.32 6.57 ± 0.35 6.36 ± 0.21
(g/dL)
Albumin
4.48 ± 0.11 4.58 ± 0.35 4.38 ± 0.13 4.44 ± 0.33 4.34 ± 0.23 4.58 ± 0.26 4.83 ± 0.15 4.60 ± 0.14
(g/dL)
Total bilirubin
0.07 ± 0.02 0.07 ± 0.02 0.09 ± 0.02 0.08 ± 0.03 0.09 ± 0.02 0.06 ± 0.01 0.07 ± 0.04 0.04 ± 0.01
(mg/dL)
ALT (U/L) 41.0 ± 7.45 42.2 ± 21.98 34.6 ± 18.19 40.4 ± 7.37 30.6 ± 6.19 43.2 ± 0.09 45.0 ± 2.20 44.6 ± 3.58
AST (U/L) 113.0 ± 19.53 126.0 ± 47.21 57.0 ± 21.24 86.2 ± 13.95 71.8 ± 21.23 114.2 ± 28.72 98.0 ± 29.10 115.0 ± 42.86
ALP (U/L) 34.6 ± 7.16 33.4 ± 8.96 22.0 ± 1.52 32.2 ± 4.49 30.0 ± 13.29 58.4 ± 17.14 47.0 ± 10.58 59.8 ± 29.94
Data are represented as mean ± standard deviation (SD). N = number of samples; BUN = blood urea nitrogen;
Cr = creatinine; Na = sodium; K = potassium; Cl = chloride; HCO3 − = bicarbonate; ALT = alanine aminotransferase;
AST = aspartate aminotransferase; ALP = alkaline phosphatase; statistical significance at p-value < 0.05 when
compared to vehicle group (A) and untreated group (B).

3. Discussion
Rice bran—the outer layer fractions of rice grains produced from rice milling—is a
rich source of fiber, vitamins, amino acids, and phenolic compounds. The high bioactivity
and antioxidative properties of rice are potentially fortified through germination and fer-
mentation [20,21,26–29]. Therefore, evidence of the genuine benefits of germinated rice and
fermented rice and their extracts is pivotal in supporting the rationale for health-promoting
applications. The present study has aimed to examine the properties of fermented rice
bran of selected rice cultivars, focusing on the screening of biochemical composition by
ATR-FTIR, prebiotic properties, antipathogenic effects, safety, and toxicity in both acute
and chronic manners. This is to obtain scientific evidence that will serve as guidelines for
further developing new functional food supplementary products from these ingredients.
The technology of ATR-FTIR was selected in this study to enable direct analysis of
the chemical composition of the sample based on a total internal reflection via evanescent
waves [30,31]. The ratios of prominent biochemical compounds, including amino acids
(serine, threonine, and tyrosine) and polysaccharides, of fermented-rice bran derived from
fermentation by L. delbrueckii subsp. lactis of both H7 and G13 were increased when com-
pared to nonfermented rice bran samples. The results agreed with those of Russo et al. [32],
who reported an increasing level of gross energy and crude protein after the fermentation
of rice bran. Macek et al.’s study reported that the fermented rice can produce amino acids
such as serine, threonine, and tyrosine. These amino acids are essential factors of protein
biosynthesis in organism, including boosting the growth of microorganism [33]. In addition,
fatty acid was increased after both rice fermentation. These results are in accordance with
previous research that showed a slight increase in fatty acid after fermenting chia and
sesame seeds [34]. An increase in fatty acids such as short-chain fatty acids in the fermented
product cloud be attributed to microorganisms’ metabolism [35], producing SCFA (acetate,
propionate, and butyrate) from carbohydrates and protein catabolism [36,37]. Through
biosynthetic and metabolic versatility, fermentation by lactic acid bacteria is a value-added
form of food processing to fortify the products with macronutrients, micronutrients (vita-
mins), or non-nutrition such as bacteriocin and phytochemical derivatives [20,32].
To answer whether H7F and G13F extracts could promote the growth and lactic acid
production of L. delbrueckii subsp. lactis, a broth dilution technique was employed. The
results revealed that both extracts of fermented rice bran could virtually enhance the
propagation of probiotic bacteria. In addition, the acidic property of the fermented rice
bran extracts, particularly at pH range 4.5–5.2, could be suitable to promote L. delbrueckii
subsp. lactis, resulting in organic acid production under the growth of lactic acid bacteria.
This result has implied the health impact of H7F and G13F following the principle of
prebiotic-fortified probiotic functions. Probiotic bacteria in the gastrointestinal tract have
various beneficial effects, e.g., maintenance of intestinal microbial balance, production of
Foods 2022, 11, 3704 9 of 15

antimicrobial substances against pathogens, induction of mucin production, stimulation

of a heightened immune response, and modulation of cytokine production by intestinal
epithelial cells (IECs) [38–40]. Therefore, H7F and G13F are likely effective prebiotic
agents containing bioactive compounds and nutrients for probiotic microorganism growth
promotion. Moreover, the plateau of the L. delbrueckii subsp. lactis population at a G13F
concentration greater than 6.25 mg/mL was demonstrated; this reflected the saturation of
the L. delbrueckii subsp. lactis population in the systems affected by high concentrations
of G13F extract (12.5–50 mg/mL) (Figure 2A). This has suggested the high potential and
attribution of the feature biochemical composition in G13F—the combination of fatty acids,
amino acids, and guanine (Figure 1).
Furthermore, the bactericidal property of H7F and G13F against pathogenic bacteria
(S. aureus and E. coli)—usually causative microorganisms of intestinal infectious diseases
such as acute diarrhea and inflammatory bowel disease (IBD)—was investigated [41].
This study showed that H7F and G13F had a gainful property of inhibiting the growth
of these intestinal pathogens, while nonfermented extract (H7NF and G13NF) failed to
demonstrate this capability. The antibacterial activities could be attributed to some non-
nutrition metabolites, such as bacteriocins, coordinating with low pH conditioning from
lactic acid bacteria after fermentation [12]. This presumption is supported by the previous
reports. L. delbrueckii is a nonmotile Gram-positive rod bacterium that biochemically
transforms sugars into lactic acids via anaerobic fermentation; therefore, many subspecies,
including L. delbrueckii subsp. delbruecki, L. delbrueckii subsp. bulgaricus, and L. delbrueckii
subsp. lactis, have been widely employed in the yogurt, cheese, and dairy products industry.
Interestingly, the health benefits of L. delbrueckii subsp. lactis have been potentiated by
several reports on the ability in bacteriocin production—namely lacticin, which has a broad
spectrum of activity against Gram-positive bacteria and other microorganisms with high
tolerance to intestinal antimicrobial molecules [42–45]. Moreover, this was supported by
the report of Li, Lee, and Cho [24]; the effective microorganism-fermented extract (EM)-YU,
which is a new version of the refreshment drink derived from fermented rice bran, seaweed,
and kiwifruit, was found to exhibit antibacterial activity against E. coli O157:H7 and P.
aeruginosa PAO1 under low pH (at 3.5) conditions. However, apart from fermentation
extracts, nonfermented rice bran extracts of Song-Yod and jasmine rice from different
extraction techniques also reported their antimicrobial activity against Salmonella spp.,
Shigella spp., E. coli, V. cholera, V. vulnificus, and S. aureus [41]. Consequently, a further
suggestion from the present study is that the efficiency of the antibacterial activity of rice
bran may depend on other factors such as rice species, the extraction method of rice bran,
and the strain of bacteria.
Biofilm is an extracellular polymeric conglomeration of shading proteins, capsular
polysaccharides, and slime covering the surface of their colonization. It is a naturally pro-
tective armor and contributes to the encouraging environment for bacterial growth [46,47].
In this study, biofilm formation, observed by SEM, in both S. aureus and E. coli reduced
remarkedly after treatment of either H7F or G13F. Notably, both H7F and G13F could
promote breakage of the cell membrane of E. coli. As supporting evidence, a biofilm in-
hibitory effect against E. coli O157:H7 by crystal-violet biofilm assay of diluted EM-YU
was previously reported [24]. Moreover, Bermudez-Brito et al. [48] reported that lactic
acid could destroy Gram-negative bacteria by inducing cell perforation and inhibiting cell
membrane procreation. This was attributed to pH decreasing in bacterial cytoplasm and
promoting bacteriocin production from lactic acid bacteria [48].
Safety and toxicity information on H7F and G13F is necessary to foster the devel-
opment of new supplementary food products. This present research work is conducted
in in vitro and in vivo test models. Human PBMCs were cocultured with fermented rice
bran extracts before the percentages of live and dead cells were investigated by MTT as-
say. It was revealed that all concentrations of H7F and G13F showed more than 80% of
cell viability, indicating no cytotoxicity to human PBMCs [25]. Relatively, their safety for
acute and chronic administration was significantly agreed upon and evidenced. For acute
Foods 2022, 11, 3704 10 of 15

toxicity, the fermented rice bran extracts showed no toxic effects on body weight, organ
weight, blood chemical and hematological values, and histopathological analysis. These
obtained results indicated the safety of these extracts in the acute phase at all dosing levels.
Moreover, the chronic toxicity test showed no sign of weight loss or pathological lesions of
vital organs in either male or female rats. Moreover, PBMC numbers increased significantly
in many concentrations of H7F and G13F consumption. Presumably, the increase in innate
immune cells (macrophages) may result from the presence of peptidoglycan, which is a
part of the lactic acid bacteria cell wall via inducing the secretion of proliferative-associated
cytokines [49]. Furthermore, G13F seemed to stimulate platelet production. However, the
mechanism at this point remains unclear.
Interestingly, a significant reduction in cholesterol levels at 500 and 2000 mg/kg of H7F
and 1000 mg/kg of G13F in male rats was revealed, compared to those in both vehicle and
untreated groups. These results support health homeostasis through hepatic metabolism.
Although the amount of short-chain fatty acid (SCFA) increases in prebiotic fermentation,
the lipid blood level is maintained through hepatic metabolism of the SCFA as propionate
and butyrate from the portal circulation to prevent systemically high SCFA concentra-
tions [50]. Therefore, the contribution of dietary fibers and biochemical constituents from
rice bran and lactic acid bacteria is presumed, which can disturb lipid digestion, absorption,
and metabolism [11,12]. Dietary fibers can bind to some minor molecules, bile salt, or
enzymes, resulting in reduced lipid digestibility and absorption. The binding property
of fiber to bile acids, fatty acids, and cholesterol has been suggested as an underlining
mechanism [51]. Besides the nutrition, phytochemicals such as anthocyanin-pigmented
rice extract (black rice) could partially contribute to decreased plasma triglyceride, total
cholesterol, and low-density lipoprotein cholesterol in rats [52]. Additionally, lactic acid
bacteria probably disturb lipid metabolism via deconjugating bile salt and suppressing
cholesterol absorption, which agrees with the findings of Pereira and Gibson [53]. Germi-
nated brown rice improves lipid parameters and ameliorates cardiovascular disease (CVD)
risk via transcriptional regulation of hepatic metabolic genes [54]. The hypocholestero-
laemia effect of rice bran in high-fat diet-fed mice has been evidenced in association with
the phytochemical composition of oryzanol, anthocyanins, GABA, and vitamin E [55,56].
Interestingly, the nutrition-fortified final products with GABA and phenolics from the
processes of fermentation and germination have widely been evidenced [57–61]. Therefore,
our study revealed the potential properties of both fermented rice bran extracts derived
from black rice and germinated brown rice, including prebiotic properties, antipathogenic
bacteria, and safety with hypocholesterolemia effect demonstrated in in vitro and in vivo
models, respectively. The obtained information could emphasize that fermented black rice
(H7F) and germinated brown rice (G13F) are the potential functional ingredients for the
further development of health products such as probiotics, prebiotics, and synbiotics, as
well as fermented foods.

4. Materials and Methods

4.1. Preparation of the Rice Bran Extracts
Rice bran of Thai cultivar black rice (H7; KKU ULR0381) and germinated brown rice
(G13) were purchased from local rice farmers in Khon Kaen province and Sakon Nakhon
province of Thailand, respectively. The pure cultures of Aspergillus sojae (TISTR3037) and
Lactobacillus delbrueckii (L. delbrueckii) subsp. lactis were purchased from Thailand Institute
of Scientific and Technological Research (TISTR). The extracts of rice bran were separately
prepared. Firstly, rice bran sample was pasteurized in sterile distilled water (DW) at
63 ± 1 ◦ C for 30 min. Then, rice bran was fermented by Aspergillus sojae (TISTR3037), which
was used as starter culture at 30 ◦ C for 2–5 days and then fermented by L. delbrueckii subsp.
lactis for another 2–5 days. Afterward, the fermented sap was collected by centrifugation
and vacuum filtration and dried using lyophilization (Martin Christ, Osterode am Harz,
Germany) under the following conditions: freezing at −80 ◦ C, followed by a primary
freeze-drying at 0.4 mbar to eliminate free water (ending at 3 ◦ C) and by two subsequent
Foods 2022, 11, 3704 11 of 15

cycles of secondary freeze-drying with a pressure variation of 1.6 mbar at 0.001 mbar to
remove the bound water (ending at 10 ◦ C). Finally, the dried powder of nonfermented rice
bran (H7NF and G13NF) and fermented rice bran (H7F and G13F) were kept at −20 ◦ C
until used in experiments. For the preparation of extracts, the powder of each rice bran
(nonfermentation and fermentation) was dissolved in DW for the stock of the extracts
and kept in −20 ◦ C prior to use in further experiments. To investigate probiotic growth,
antibacterial activity and cytotoxicity, the extracts were prepared in the diluents, including
de Man–Rogaosa–Sharpe (MRS) broth, DW, and RPMI 1640 medium, respectively.

4.2. Chemical Composition Profile by Attenuated Total Reflection–Fourier Transform Infrared

Spectroscopy (ATR-FTIR)
The chemical compositions of both nonfermented and fermented rice bran extracts of
H7 and G13 were investigated using ATR-FTIR (4500 Series, Agilent Technologies, Santa
Clara, CA, USA). The spectra were recorded in the wave number range of 500–4000 cm−1
to manipulate the operating software of the ATR-FTIR instrument to transform the data
into derivative spectra.

4.3. Prebiotic Property on L. delbrueckii Growth Using Broth Dilution Technique and the Potential
of Hydrogen Ion (pH) Measurement
Various concentrations (3.125, 6.25, 12.5, 25 and 50 mg/mL) of fermented rice bran
H7 (H7F) and G13 (G13F) extracts were cocultured with L. delbrueckii (2 × 104 cfu/mL) in
27.6 g/L de Man–Rogaosa–Sharpe (MRS) broth under anaerobic conditions at 37 ◦ C for 24 h.
Bacterial numbers were analyzed by flow cytometric analysis (BD FACSCantoTM II flow
cytometer, BD Biosciences, San Jose, CA, USA) employing counting beads calculation (BD
Biosciences, CA, USA) followed with pH value measurement, compared to nonfermented
rice bran (H7NF and G13NF) extracts, by using pH meter (Starter 3100, Ohaus, Parsippany,
NJ, USA).

4.4. Antimicrobial Activity Assays

4.4.1. Agar Well Diffusion and Broth Microdilution Technique
Agar well diffusion test was employed to test the inhibitory effect of fermented and
nonfermented rice bran extracts against pathogenic bacteria. Mueller–Hinton (MH) agars
were drilled with 6 mm diameter cork-borer and then streaked with Escherichia coli (E. coli)
ATCC25922 or Staphylococcus aureus (S. aureus) ATCC29213. A total of 100 µL of 400 mg/mL
(excessive concentration dose) fermented (H7F and G13F) and nonfermented rice bran
(H7NF and G13NF) extracts dissolved in DW were added in holes and then incubated at
37 ◦ C for 24 h before determining the diameter of inhibition zone. Distilled water (DW) was
used for noninhibition control, whereas bacteria cocultured with 5 µg/mL of Ampicillin
(Amp) were used for inhibition control.
Moreover, the solution of fermented rice bran extracts was diluted into various concen-
trations, ranging between 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.8 mg/mL, and used in
the experiment to define a minimum inhibition concentration (MIC) as well as a minimum
bactericidal concentration (MBC) by broth microdilution technique. The fermented rice
bran extract solutions were cocultured with pathogenic bacteria in microtiter plate at 37 ◦ C
for 24 h, and subsequently, 10 µL of each was dropped onto MH agar and followed with a
24 h incubation. Finally, the colonies in each unit were observed. The colony numbers of the
treated group were compared to that of noninhibition control (untreated group; distilled
water (DW) + microbial + medium). The MIC was indicated at the lowest concentration, re-
sulting in colony density and size reduction as evidenced by the bacterial growth inhibitory
effect. In contrast, the MBC was accounted for at the lowest concentration, resulting in no
bacterial colony formation [62].

4.4.2. Biofilm Formation by Scanning Electron Microscopy (SEM)

E. coli and S. aureus were cultured in trypticase soy broth (TSB) in 6-well plates. The
concentrations of fermented rice bran extract solution dissolved in TBS were adjusted
Foods 2022, 11, 3704 12 of 15

according to MIC. Bacteria were incubated at 37 ◦ C for 18 h before washing and fixation
with 4% formaldehyde. Finally, samples were dehydrated with absolute ethanol and
observed under a scanning electron microscope (SEM) (S-3000N Hitachi, Tokyo, Japan).

4.5. Safety and Toxicity Assays

4.5.1. Toxicity Test of Human Peripheral Blood Mononuclear Cells (PBMCs) in In
Vitro Model
Human peripheral blood mononuclear cells (PBMCs) from the leftover buffy coat
permitted by the Khon Kaen University Ethical Committee Review Board (HE651334) were
cultured in RPMI 1640 media supplemented with 10% fetal bovine serum at 37 ◦ C for 24 h
with presenting of fermented rice bran extracts at various concentrations of 5, 10, 20, 25, and
50 mg/mL r DW for the control. Compared to the control, %viability of cell was analyzed
by MTT assay.

4.5.2. Study Population in In Vivo Model

A total of 160 Wistar rats, including 80 males and 80 females, aged between 6–8 weeks,
were employed in this experiment. The animals were provided by the National Laboratory
Animal Center, Mahidol University, and they were later treated at the Northeast Laboratory
Animal Center, Khon Kaen University, Thailand. This study was approved by the Ethical
Committee of Khon Kaen University (AEKKU-NELAC 7/2557), whose acute and chronic
toxicity study was performed in respect to OECD guideline 420 [63] and OECD guideline
452 [64], respectively.

4.5.3. Acute Toxicity Test

A total of 80 rats were used and divided into 8 groups with 10 rats, including 5 females
and 5 males in each. Those in groups I, II, and III received an orally fed single dose of G13F
for 14 days at 1000, 2500, and 5000 mg/kg/day; those in group IV, V, and VI received oral
feeding of H7F in the same dose; while those in group VII and VIII were vehicle (DW) and
untreated group, respectively. Each rat’s body weight was recorded daily, while the weight
of organs, blood chemical values, hematological values, and histopathology of organs were
examined after completion of a 14-day term of study.

4.5.4. Chronic Toxicity Test

Similar to the acute toxicity test, 80 rats were used and divided into 8 groups with
10 rats, including 5 females and 5 males, in each. For 6 months, the assigned groups I, II,
and III daily received an orally fed dose of G13F at 500, 1000, and 2000 mg/kg/day; group
IV, V, and VI received H7F; and VII and VIII groups were vehicle and untreated group,
respectively. Each rat’s body weight was recorded weekly, while the weight of organs,
blood chemical values, hematological values, and histopathology of organs were examined
after 6-month term of treatment.

4.6. Statistical Analysis

The data are presented in mean ± standard deviation (SD) from at least triplicates of
independent studied unit. The flow cytometry data were analyzed by the BD FACSDivasTM
software (BD Biosciences, Franklin, CA, USA). The statistical analysis was performed by
SPSS (version 17, SPSS Inc., San Diego, IL, USA) and GraphPad Prism software (GraphPad
Software Inc., Chicago, CA, USA). The nonparametric data were analyzed by Mann–
Whitney U test. Statistical significance was considered at p-value less than 0.05.

5. Conclusions
The health benefits of fermented rice bran extracts from black rice cultivar (H7F) and
germinated brown rice cultivar (G13F) have been evidenced. They exhibited capacity in
controlling bacterial growth with different potencies: growth-promoting for beneficial bac-
teria via the prebiotic property, while inhibiting pathogenic bacterial growth by diminishing
Foods 2022, 11, 3704 13 of 15

biofilm formation and promoting cell membrane breakage. In addition, their high degree
of safety was demonstrated. Coincidently, the reduced cholesterol levels and modulating
immune cell response were manifested. Conclusively, H7F and G13F are suggested as
promising functional ingredients for health-promoting products.

Author Contributions: Conceptualization, K.S.; methodology, P.T. and P.V.; software, S.S. and W.P.;
formal analysis, K.S. and P.T.; investigation, K.S., P.T., S.S., W.P. and P.V.; writing—original draft
preparation, K.S., P.T. and R.W.; writing—review and editing, K.S.; visualization, P.V. and P.T.;
supervision, K.S.; project administration, K.S.; funding acquisition, K.S. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded and supported by Research and Graduate Studies, Khon Kaen
University: 2563-2565; the Fundamental Fund of Khon Kaen University: 2565, and the National
Science, Research and Innovation Fund (NSRF): 2565.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board (or Ethics Committee) of Khon Kaen University (protocol code AEKKU-NELAC 7/2557,
approval in July 2014).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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