Rice (Oryza sativa L) is a staple food for over half
the world's population and supplies as much as 70% of
the dietary energy and protein in certain regions of the
world . During rice milling, the outer brown layer is
separated from the inner rice kernel, yielding white rice
and rice bran. Over 63 million tons of rice bran is produced
world-wide each year, and more than 90% is sold
cheaply as animal feed . Rice bran is often defatted
and heat treated to prevent lipid oxidation. This milling
coproduct is termed heat-stabilized defatted rice bran
(HDRB). HDRB is widely available and inexpensive,
costing about $125 to $195 per metric ton . Although
HDRB is widely used as an animal feed, it has been increasingly
utilized by the food industry in recent decades
. The micronutrient and macronutrient profile of
HDRB makes it well-suited for health foods, breads, cereals,
crackers, and pasta. Its nutrient profile also makes
HDRB a potential media for yeast propagation.
Yeast is vital to leavening of bread and the fermentation
of alcoholic beverages. Saccharomyces cerevisiae (S.
cerevisiae) is the yeast most commonly used in production
of bread, beer, and wine . In brewing, S. cerevisiae
is propagated in wort, composed of malt grain and water.
Wort consists of proteins, free amino acids, fermentable
sugars (fructose, sucrose, glucose, maltose), dextrins, vitamins,
and minerals . This complex media supplies
yeast with the nutrients necessary for aerobic growth. In
the production of baker's yeast, S. cerevisiae is most often
cultivated in a high sugar medium composed of beet
or cane molasses supplemented with inorganic ammonia,
vitamins, and trace minerals .
S. cerevisiae is also used as an alternative source
of proteins, enzymes, vitamins, and yeast extracts for the
food and pharmaceutical industries. Molasses is the most
common substrate for growth of food grade yeast; however,
approximately 40% of molasses is non-fermentable.
This unused fraction increases the cost of yeast production and leads to industrial waste. Therefore, recent efforts
have focused on using food processing coproducts
as substrates for food grade yeast production . To date,
S. cerevisiae has been grown on processing coproducts,
including fruit and vegetable extract, date byproducts,
hydrolyzed cassava waste, shrimp shell waste [8-11].
HDRB has been shown to support the growth of
Lactobacillus acidophilus as well as several Bacillus species
. However, previous research has not fully evaluated
HDRB as a growth media for food grade yeasts,
such as S. cerevisiae.
Therefore, the objectives of this study were to determine
the composition of heat-stabilized defatted rice
bran on the basis of percent moisture, crude protein,
starch, lipid, total dietary fiber, ash, soluble sugars, and
total phenolics (mg/g) and evaluate the growth of S. cerevisiae
ATCC 26603 in heat-stabilized defatted rice bran
at high and low initial inoculum levels.
Materials and Methods
HDRB from Riceland Foods, Inc (Stuttgart, AR,
USA) was ground with IKA M20 (IKA Works Inc.
Wilmington, NC, USA) and passed through a 60 mesh
sieve to obtain a fine homogenous powder. A food-grade
yeast strain, S. cerevisiae ATCC 26603, was purchased
from American Type Culture Collection (ATCC; Manassas,
VA, USA). The substances for preparing yeast
media (YM) agar and phosphate buffer solution (PBS)
were purchased from Difco Laboratories (Detroit, MI,
USA). The reagents and enzymes used for starch determination
were purchased from Megazyme International
Ireland, Ltd. (Bray Business Park, Bray, Co. Wicklow, Ireland).
Other analytical-grade chemicals were purchased
from Fisher Scientific (Pittsburgh, PA, USA) and Sigma
Chemical Co. (St. Louis, MO, USA).
Determination of moisture content
The moisture content of HDRB was determined
by drying 2 g of each sample at 104oC for 3 hr . After
drying, samples were covered, transferred to desiccator,
cooled for 2.5 min, and weighed. Values are expressed as
a mean of three determinations on dry weight basis.
Determination of protein content
The protein content of HDRB before and after
heat sterilization (121oC, 30 min) was determined by the
automated Kjel-Foss method 46-08 . Samples were
digested with Kjeldahl Digestion System 6 for 1 hr at 420
oC, and nitrogen content of the samples was determined
with a KjelTech Analyzer 2000 (Tecator Co., Hoganas,
Sweden). Percentage total protein content in HDRB was
calculated with a conversion factor of 5.95 . Values
are expressed as a mean of three determinations on dry
Determination of starch content
The starch content of HDRB before and after heat
sterilization (121oC, 30 min) was determined by Megazyme
total starch assay procedure AACC 76.13 .
For this procedure, 5 mL of aqueous ethanol (80% v/v)
was added to 100 mg HDRB. The mixture was stirred by
vortexing then was incubated at 85oC for 5 min before
mixing in an additional 5 mL of aqueous ethanol. The
mixture was centrifuged for 10 min at 1000 x g. The supernatant
was discarded, and the pellet was resuspended
in 10 mL of aqueous ethanol before centrifuging a second
time at 1000 x g. The supernatant was discarded, and the
precipitate was immediately mixed with 3 mL of thermostable
α-amylase in MOPS buffer (50 mM, pH 7.0).
Samples were incubated in a boiling water bath for 6 min
with vigorous stirring after 2 min and 4 min. Samples
were transferred to a 50oC water bath. Sodium acetate
buffer (4.0 mL, 200 mM, pH 4.5) was added, followed by
amyloglucosidase (0.1 mL, 20 U). Samples were vortexed
and incubated at 50oC for 30 min. An aliquot (1.0 mL)
of each sample was diluted to 10 mL with distilled water
and was centrifuged at 3,000 RCF for 10 min. Three
milliliters of glucose determination reagent (GOPOD)
was added to each sample, including glucose controls.
Glucose controls consisted of 0.1 mL of glucose standard
solution (1 mg/mL) and 3 mL of GOPOD Reagent.
A reagent blank was used, consisting of 0.1 mL of water
and 3 mL of GOPOD Reagent. Absorbance was determined
at 510 nm. Values are expressed as a mean of three
Determination of soluble sugars
Soluble sugars were extracted in triplicate at 50oC
for 15 min using a 10:1 ratio of ethanol-to-sample .
Extracted sugars were quantified by the phenol-sulfuric
acid colorimetric method of Dubois et al.  using a
Shimadzu Model UV-1601 spectrophotometer (Kyoto,
Japan). A standard curve was prepared using sucrose at
concentrations from 3 to 50 μg/mL. Soluble sugars from
triplicate determinations are expressed on a dry weight
Determination of crude lipid content
The crude lipid content of HDRB before and after
heat sterilization (121oC, 30 min) was determined by the
crude fat in soy flours method 30-26 . In brief, 5 g
of HDRB was weighed onto filter paper and enclosed in
second filter. Samples were placed in butt-type extraction
apparatus as described in AACC method 30-26 .
Approximately 250 mL of petroleum ether was added to
each flask. Ether was evaporated from the samples for
3 hr using a water bath. Samples were then dried overnight.
Values are expressed as a mean of three determinations
on a dry weight basis.
Determination of total dietary fiber content
The total dietary fiber content was determined
by enzymatic-gravimetric analysis following AACC
method 32-07 . For the analysis, one gram of HDRB
sample from before and after heat sterilization (121o C,
30 min) was individually subjected to sequential enzymatic digestion by heat stable α-amylase, protease, and
amyloglucosidase. The mixture was precipitated with 4
volumes 95% ethanol. Precipitate was filtered and dried.
The residues were corrected for protein and ash content.
Values are expressed as a mean of three determinations
on a dry weight basis.
Determination of ash content
The ash content was determined by the AACC
method 08-03 for feedstuffs . In brief, triplicate 2 g
samples of HDRB were weighed into crucible and were
heated in an electric furnace at 600oC for 2 hr. Crucibles
were then transferred to desiccator and cooled before
weighing. Values are expressed as a mean of three determinations
on a dry weight basis.
Determination of total phenolic contents
The total phenolic content of HDRB before and
after heat sterilization (121 oC, 30 min) was determined
using Folin-Ciocalteu's reagent according to the method
of Swain and Hillis  and Joslyn . Fifty milligrams
of HDRB samples from before and after heat sterilization
were weighed into screw-cap test tubes and were
vortexed with 10 mL of methanol. The mixtures were
heated in a water bath at 65oCfor 2 hr, then removed
and allowed to cool to room temperature. An aliquot (0.5
mL) of the methanol extract was diluted to 7 mL with
deionized water and was vortexed for 5 s. A volume of
0.5 mL of Folin-Ciocalteu's reagent was added. Samples
were then vortexed for 5 s and were allowed to stand for
3 min. One milliliter of saturated sodium carbonate was
added to the solution, and the total volume was brought
to 10 mL with water before vortexing for 5 s. After 2 hr,
absorbance was read at 725 nm using a spectrophotometer
(Shimadzu Model UV-1601, Kyoto, Japan). Protocatechuic
acid was serially diluted and used as a standard
curve. Values are expressed as a mean of three determinations
on dry weight basis. Results are expressed as
protocatechuic acid equivalents (PAE) in milligram per
gram dry material.
Growth of Saccharomyces cerevisiae with high initial
In order to optimize growth of S. cerevisiae in
HDRB, the proportion of HDRB-to-water was evaluated
in duplicate at five levels: 2.5, 5, 7.5, 10 and 15% (w/v).
Rice bran slurries were prepared by mixing 1.25, 2.5,
3.75, 5, and 7.5 g HDRB with 50 mL of DI water in a 250
mL flask. A control sample was also included; it consisted
of 50 mL of DI water in a 250 mL flask. HDRB slurries
were not completely sterilized by standard autoclaving
at 121oC, 20 min; however, 121oC for 30 min was sufficient
to kill all organisms. Therefore, the water control
and HDRB slurries were autoclaved for 30 min at 121oC.
Yeast media (50 mL) was prepared in a 250 mL flask and
was autoclaved for 20 min at 121oC.
After cooling to room temperature, HDRB slurries,
YM media, and water control were inoculated with
5 mL of activated (12 hr, 30oC, YM) S. cerevisiae ATCC
26603 at a high initial inoculum level (6.5 Log CFU/mL).
Samples were incubated with orbital shaking (100 rpm)
at 30oC for a total of 2 days. Yeast growth was measured
at 4, 8, 12 24, 36, 48 hr by serial dilution in PBS buffer
and plating on YM agar. Plates were incubated at 30 oC
for 48 hr to enumerate colonies (CFU/mL).
Growth of Saccharomyces cerevisiae ATCC 26603
in HDRB at low initial inoculum level
S. cerevisiae growth was not significantly affected
by the proportion of bran-to-water used in high inoculum
experiments. Because industrial production costs
increase with increasing substrate utilization, a 2.5%
HDRB-water slurry was selected for subsequent study.
Next, growth of S. cerevisiae was measured after a lowlevel
inoculation (1.5 Log CFU/mL) into HDRB slurry
and YM media. Duplicate rice bran samples were prepared
for inoculation by mixing 1.25 g HDRB with 50
mL DI water then sterilizing at 121oC for 30 min. A volume
of 50 mL of YM was prepared as described earlier.
To prepare the inoculum, ten milliliters of activated (12
hr, 30oC) S. cerevisiae ATCC 26603 in YM broth was
centrifuged (5,000 x g, 10 min), and the cell pellet was
washed twice with PBS buffer solution. The pellet was
transferred into the HDRB slurry (2.5% HDRB), YM,
and water control (0 g HDRB) at the low initial inoculation
level of 1.5 Log CFU/mL. Each of these samples was
incubated with orbital shaking (100 rpm) at 30oC for 3
days. Samples were taken at regular intervals, serially diluted
(PBS, pH 7.0), and plated on YM agar. Plates were
incubated (48 hr, 30oC) and counted (Log CFU/mL).
These procedures were repeated and results are reported
as an average of duplicate procedures.
Student's t test was used to determine significant
difference among the before and after autoclaved HDRB
for each component tested using the fit y by x platform
in JMP version 5.0.1 (SAS Institute Inc., Cary, NC).
Student's t test was used to detect significant difference
among the five concentrations of HDRB, water control,
and YM at high initial inoculum level for S. cerevisiae
ATCC 26603 using the fit y by x platform in JMP. The
significant difference among the growth population at
the time of maximum cell population for the low initial
inoculum level in the 2.5% HDRB, 0% HDRB (water
control) and YM were also evaluated and analyzed
in comparison to the high initial inoculum level (for S.
cerevisiae ATCC 26603) at 12 hr (time of maximum cell
population) using the previously mentioned statistical
Results and Discussion
HDRB after heat sterilization was composed of
35.9% total dietary fiber, 19.1% crude protein, 14.9%
starch, 16.4% ash, 9.4% moisture, 1.9% soluble sugars, 1.8% crude lipid, and 8.9 mg/g total phenolics (Table 1).
Autoclaving resulted in statistically significant decrease
in extractable crude lipids from 3.1% before autoclaving
to 1.8% after autoclaving. None of the other measured
components were significantly affected by heat sterilization
The modest reduction in extractable crude lipids that accompanied
autoclaving may have been caused by formation
of amylose-lipid complexes, which are less amenable
to ether extraction. This explanation is consistent with
work by Szezodrak and Pomeranz , which demonstrated
that autoclave heating of starch for 1 hr in the
presence of lipids led to the formation of amylose-lipid
The composition of HDRB in this study was similar
to the composition of defatted rice bran given in literature.
Newman et al.  report a much lower level
of total dietary fiber (24.1%) in defeated rice bran; however,
the level of protein in their samples (16.2%) was
only slightly lower than the protein level (19.1%) in the
present study. Results from Pan and Cathcart  were
similar to those reported here. They found defatted rice bran to contain 16.6% protein, 11.28% ash, 7.0% moisture,
and 3.7% fat. Moreover, data from Claye et al. 
is quite similar to our own work. Their analysis showed
defatted rice bran to contain 19.3% protein, 11.9% ash,
5.1% moisture, and 1.3% crude fat.
Growth of Saccharomyces cerevisiae at high initial
Inoculation with a high level of S. cerevisiae ATCC
26603 produced a very similar growth pattern among the five
concentrations of HDRB and YM (Figure 1). After 12 hr. of
incubation, there was no significant difference (p > .05) in
growth among HDRB conditions or between HDRB media
and YM (Table 2). The data suggests that differences in the
carbon source and available nutrients between YM and HDRB
did not significantly affect the yeast growth. Marginal growth
was also observed in the water control (0.7 Log CFU/mL);
however, this can be attributed to the high inoculum . S.
cerevisiae, ATCC 26603 was selected for study because its ability
to grow on hydrolyzed bagasse pith, which is a byproduct
from sugar cane processing . The strain appears to be well
adapted to growth in complex substrates, and may be a good
candidate for industrial applications.
Growth of Saccharomyces cerevisiae at low initial
Inoculation of YM with a low level of S. cerevisiae produced
a growth curve with a brief lag phase followed by typical
exponential and stationary phases (Figure 2). When grown in
YM, S. cerevisiae reached a stationary phase after 24 hr, and
a maximum cell population of 7.8 CFU was observed at this
time point (Table 3). Inoculation of 2.5% HDRB with a lowlevel
of S. cerevisiae led to population growth that extended for 42 hr before reaching a maximum of 7.2 CFU after 42 hr. Yeast
grew more slowly in HDRB media compared to YM; however,
there was no significant difference in maximum cell population
(Table 3 between YM and 2.5% HDRB (p > 0.05). These
results demonstrate that when a low initial inoculum level (1.5
Log CFU/mL) and minimal amounts of HDRB (2.5%) are used
S. cerevisiae ATCC 26603 can achieve maximum cell populations
comparable to that of standard medium (YM), and thus,
can sufficiently utilize the nutrients available in HDRB for its
Heat-stabilized defatted rice bran was composed
of 9.4 ± 0.2% moisture, 19.1 ± 0.7% crude protein, 1.8 ±
0.4% crude lipid, 14.9 ± 0.8% starch, 1.9 ± 0.2% soluble
sugars, 16.4 ± 0.2% ash, 35.9 ± 0.9% total dietary fiber,
and 8.9 ± 0.1 mg/g total phenolics. The data from this
study showed that when compared to YM, HDRB was
a comparable growth medium for S. cerevisiae ATCC
26603. Therefore, HDRB may serve as a low-cost alternative
for industrial propagation of S. cerevisiae.