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Nutritional Enhancement of Barley in Solid State Fermentation by Rhizopus oligosporus ML-10
Rubina Nelofer1*, Muhammad Nadeem1, Muhammad Irfan2 and Quratulain Syed1
1Department of Biotechnology and Food Research Center, Pakistan Council of Scientific and Industrial Research (PCSIR), Pakistan
2Department of Biotechnology, University of Sargodha, Pakistan
Submission: April 09, 2018;Published: June 18, 2018
*Corresponding author: Dr. Rubina Nelofer, Department of Biotechnology and Food Research Center, Pakistan Council of Scientific and Industrial Research (PCSIR), Laboratories Complex, Pakistan, Email: email@example.com
How to cite this article: Rubina N, Muhammad N, Muhammad I Quratulain S. Nutritional Enhancement of Barley in Solid State Fermentation by Rhizopus
oligosporus ML-10. Nutri Food Sci Int J. 2018; 6(5): 555700. DOI:10.19080/NFSIJ.2018.06.555700.
The present study was undertaken for nutritional enhancement of whole barley grains by optimizing solid state fermentation conditions with Rhizopus oligosporus ML-10 in polythene bags. The influence of various soaking time, boiling time, inoculum size, incubation time and temperature on the levels of proteins, fats and carbohydrates were evaluated during fermentation of barley. 500grams whole barley grains after soaking for 12h in 1L distilled water (pH, 4.5) were allowed to boil for 20min followed by drying at 80 °C for 15min. After this pretreatment, dehulled barley grains were inoculated with 1.5 % (v/w) spore suspension containing 1.0 x106 spores per mL of 96h aged R. oligosporus ML-10 in 25 x 25 cm2 polythene bags and incubated at 30 °C for 36h. The nutritional value of barley in terms of protein contents increased significantly from 10.25% to 16.85% after solid state fermentation. However, the level of total carbohydrate and fat after fermentation process was found to decrease from 68.6% to 63.55 and 2.13 % to 1.62 % respectively. From the results of present study it was concluded that the solid state fermentation of whole grain barley resulted in increasing the protein contents up to 64.3 % under optimized conditions by Rhizopus oligosporus ML-10 in polythene bags.
Keywords: Barley; Solid state fermentation; Protein; Fat; Carbohydrates; R. oligosprous; Nutritional value; Protein; Calorie; Hordeum vulgare
Fermented foods either from plant or animal origin are commonly used in all parts of the world as a low cost protein source which fulfill the protein/calorie deficiency problems especially in developing countries where meat products are in poor supply [1- 4]. Barley is one of the most popular staple foods in Pakistan and many other countries. The barley (Hordeum vulgare) is reported to contain (w/w) 6.75 % crude fiber, 11.67% crude protein, 2.31 % crude fat and 2.22 % ash  .The consumption of barley and its products is found to assist in decreasing the risk of type 2 diabetes [6-7]. Moreover, it also helps in reducing the total serum lipids and LDL-cholesterol which in turn reduces the risk of cardiovascular diseases [8-9]. Whole grain barley has also been reported as a rich source of phytochemicals . Therefore, increasing consumption of fermented cereals, would lower food cost and promote better health .
Microbial fermentation is also considered as one of the oldest and most economical methods for value additions and preservation of food. The fermentation process helps in increasing the digestibility and bioavailability of proteins, carbohydrates, lipids, minerals and vitamin contents in cereals. Moreover, it shortens the cooking time and increases the microbial safety .
The present study was undertaken to enhance the nutritional value of barley by optimizing the process parameters such as
soaking and boiling time, inoculum size, incubation time and temperature, in solid state fermentation to study their effect on protein, fat and carbohydrates levels.
The fungal strain of Rhizopus oligosporus ML-10 used in the present study was obtained from Microbiology Lab, Biotechnology and Food Research Centre, PCSIR Labs Complex, Lahore. The culture was grown on potato dextrose agar (Oxoid) slant at 30 °C for 96h.The culture was then preserved at 4 °C in refrigerator and sub-culturing was done after every 4-6 week for further study.
Ten ml of sterilized distilled water was transferred to 96h aged potato dextrose agar slant of Rhizopus oligosporus ML-10. The spores were dislodged by using a sterile inoculation needle under aseptic conditions. The spore suspension containing approximately 1.0 x106 spores per mL was used as inoculum for the fermentation of pretreated barley grains with Rhizopus oligosporus ML-10.
Substrate: The whole barley grains used in the present study was purchased from local
The solid state fermentation of barley was carried out in
accordance with the modified method of Berg et al. . 500g of
whole barley grains were soaked for 12h in 1L distilled water in
plastic beaker at room temperature. After manual dehulling, the
soaked barley grains were allowed to boil in tap water for 20 min.
Decant off water and kept the grains in oven at 80 °C for 15 min
to remove excess of water. Then the spore suspension of Rhizopus
oligosporus ML-10 at the rate of 1.5 % (V/W) was mixed well
with pretreated barley grains at room temperature. Packed the
inoculated barley grains in pre- holed 25 x 25cm2 polythene bags
and kept at 30 °C for 36h. All the experiments were conducted in
triplicate. The flow sheet of the process is given in Figure 1.
Proximate composition: Moisture, Fat, total carbohydrates
and total nitrogen (microkjeldahl) were determined according to
The soaking time on the fermentation of barley is important
in solid state fermentation due to its effect on the softening of
kernels for smooth mycelial growth of the Rhizopus oligosporus
ML-10. Therefore, soaking time was varied from 6, 12, 18 and
24h in distilled water at room temperature. The data in Figure 2
revealed that the soaking time of 12h was found to be optimum
for the production of nutritionally rich fermented barley with the
increase in protein contents from 10.25 % to 16.55 %. Further
increase in soaking time resulted in decreasing the protein
contents as evident from Figure 2. However, the percentage
of carbohydrates and fat in fermented barley was found to
decrease slightly from 65 to 63.5 % and from 1.72 % to 1.52
%, respectively, with the increase of soaking time. It has been
reported earlier that during soaking of soybean, organic acids are
formed which resulted in lowering the pH from 6.0 to 3.9 .
Moreover, during soaking, the bacterial fermentation occurred
resulting in acidification. Many workers, described the addition
of < 0.5% lactic acid or < 0.25 % acetic acid during soaking stage
which lowered the initial pH, allowing the mould to grow and
suppress the bacterial growth . In the present study, the pH
was also noted to decrease from 6.5 to 4.3. Our results are in good
agreement with earlier investigations that a natural microbial
acidification occurred during the soaking process in Indonesia
soybeans fermentation . According to other investigators, the
changes observed during soaking were due to leaching of soluble
components and also due to enzyme activities during sprouting
and fermentation [17,18].
Partial cooking or boiling of the cereals was also known to
play a vital role in the production of fermented food. Therefore,
boiling time ranging from 15 to 30mints was evaluated for the
production of nutritionally rich barley in solid state fermentation
as given in Figure 3. Boiling of cereals in water has been reported
to destroy contamination bacteria and anti nutritional factors
and also release of some nutrients which are essential for mould
growth . According to Steinkraus et al.  a heat stable and
water soluble mold inhibitor is leached out during boiling process,
which is discarded later. The results in Figure 3 revealed that
combination of soaking and boiling was most favorable for the
growth of Rhizopus oligosporus ML-10. Longer boiling time alone
did not give the same elasticity of the seed as did the combination
of soaking and boiling. For longer boiling the gelatinization of
starch content occurred and the cake formation deteriorated.
Therefore, the boiling time of 15min was found to be suitable
for the production of fermented whole grain cereal by Rhizopus
oligosporus ML-10 in solid state fermentation. Mulyowidaro et al.
 reported that mild boiling (15 min at 95 °C) of soaked and
biologically acidified beans results in sufficient survival of a mixed
flora of lactic acid bacteria and acillus spp. A strong proteolytic
Bacillus spoilage was also observed in beans which had been
heated for 60 min at 95 oC or 15 min at 121 °C . Moreover,
these workers also hypothesized that the lack of beany flavor in
tempeh was due to the result of inactivation of the lipooxygenase
associated with the formation of such flavors during the boiling
stage. According to earlier investigations, the combination of
cooking and fermentation improved the nutrient quality of all
tested sorghum seeds and reduced the content of anti nutritional
factors to a safe level in comparison with other methods of
Homogeneous seedling of substrate with adequate spore
inoculation along with rapid starting of growth is important
to achieve successful fermentation of the cereals for improved
nutritional quality. Rhizopus oligosporus is the most preferred
fungus in tempeh fermentation due to rapid growth rate at high
temperature, high proteolytic and lypolytic activities and strong
antioxidant properties . Therefore, various inoculum sizes of
96hrs aged Rhizopus oligosporus ML-10 containing 106 spores/ml
was used for nutritional enhancement of barley by fermentation.
In Figure 4, the data shows that with low inoculum size 0.5 %
(v/w) the fungus grew slowly and barley dense cake formation
did not form as also indicated from low protein contents 9.01
%. This slow growth may increase the risk of contamination in
barley cereals with microbes. The optimum inoculum size of 1.5
% (v/w) containing 1.0 x106 spores of Rhizopus oligosporus ML-10
per ml was found to enhance protein contents up to 10.55 % in
fermented barley. At high inoculum size (2 %), the fungus grew
rapidly and the dense cake formation was uneven resulting in
slight decrease in protein level, whereas the carbohydrates level
significantly decreased down to 62.5 %.
These findings coincide with the earlier investigations
regarding tempeh production by using different substrates
[14, 21,22]. Some workers reported that 3.35x104cfu/gm is
the optimum level of inoculum for quinoa tempeh . Similar
findings have also been reported by Xin-Mei Feng . According
to them Rhizopus oligosporus was inoculated at approximately
104 spores/g moist substrate. The data in Figure 4 shows that
when Rhizopus oligosporus was inoculated at approximately
1.5x104spores/g moist barley, the time for obtaining dense
mycelial growth was shortened to 36 h. However, the growth was
uneven, probably due to oxygen limitation in the center. Similar
results have also been reported for tempeh fermentation of soy
bean by Nout . Earlier investigation showed the widening
effect of oxygen on hyphae grown in a gradient of limiting oxygen
The incubation temperature was found to be considerable
importance for the production of nutritionally rich fermented
barley. The best mycelia growth was reported at 30 °C as shown
in Figure 5 in which maximum protein contents i.e. 16.75 % were
found. These results are in agreement with the work reported
by Steinkraus et al. . The workers observed good mycelial
growth at 30 °C. Reu et al.  also supported that inoculation
of beans with Rhizopus oligosporus at various temperatures
followed by incubation at 30 °C resulted in both increased and
decreased periods for the lag phase of fungal growth. According
to them maximum difference of 3 h lag phase was found between
initial bean temperatures of 25 and 37 °C. However, in another
investigation, the optimum growth of Rhizopus oligosporus was
reported as 35 °C . The data in Figure 5 also revealed that with
the increase in temperature the fat and carbohydrates contents
did not show significant variation.
The results shown in Figure 6 demonstrate that the 36h of
incubation time is optimum for the fungal mycelia growth because
after this period , the sporulation would be started which had
adverse effect on the production of a fermented cereal product
in respect of protein contents, and in particular fermented whole
grain barley based product of tempe type. It was found that during
this time period the fungus growth was very good, and the fungus
mycelia evenly distributed in the entire grains. As the time of the
incubation was increased the sporulation would be started.
The fermented barley in solid state fermentation with 1.5 %
(v/w) spore suspension of 96h aged R. oligosporus ML-10 in 25 x
25cm2 polythene bags at 30 °C for 36h was analyzed for proximate
composition to evaluate the level of protein, fat and carbohydrates
as compared with unfermented barley.
The results in Table 1 revealed that protein contents increased
significantly whereas the carbohydrates and fats were found to
decrease after fermentation with Rhizopus oligosporus ML-10
in pretreated barley. It was reported by other workers that the
analysis of fermented cereals showed significant decreased in ash
contents after fermentation . In fermented soybean, a slight
increase was found in crude protein and crude fiber content of
Tempe as compared with unfermented soybeans . It was also
observed that the growth of fungus reduced the concentration
of low molecular carbohydrates and increased the dietary fiber
content in fermentation . A decrease in carbohydrate level
after fermentation might be due to the partial removal of non
starch constituents during solids state fermentation process .
It has been reported that during the fermentation process of
cereals, proteases, lipases, phytases and a variety of carbohydrases
are produced resulting in the degradation of macromolecules
into lower molecular weight products thereby improving the
nutritional quality of fermented product .
Nutritionally rich fermented barley has earlier been reported
by Hesseltine et al. . A patented barley tempe product
has been described by fermenting pearled barley kernel with
selected strains of Rhizopus sp . The barley fermentation
process on a new barley genotype has been reported by Eklund-
Jhonson et al. . Variations in total fat contents of fermented
and unfermented samples were observed under the influence of
different parameters as presented in (Figure 2-6). According to
Khaterpaul  that natural fermentation increases whereas the
pure culture fermentation decreased the fat contents in cereals. Fatty acids present in glycerides have been reported to decrease
during fermentation of soy bean from 30% natural lipid by the
action of lipases activity . The protein contents increased
initially as a result of fermentation carried out from 6 to 12h.
This enhancement in protein level can be attributed to microbial
synthesis from metabolic intermediate during the growth .
Similar findings were reported regarding the level of protein in the
pearl millet after fermentation . It has also been reported that
the water soluble protein increased from two to three times after
soy fermentation and four to six times after miso fermentation
[36,37]. However, in another investigation, it was found that the
fermentation process either decreased or did not change the
protein contents of pearl millet flour .
From the results of present study it was concluded that
the solid state fermentation of whole grain barley resulted in
increasing the protein contents up to 64.3 % under optimized
conditions by Rhizopus oligosporus ML-10 in polythene bags.