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Solid-state Fermentation of Moringa oleifera Leaf Meal using Bacillus pumilus

CICC 10440

Article  in  Journal of Chemical Technology & Biotechnology · March 2017

DOI: 10.1002/jctb.5203

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Research Article
Received: 25 July 2016 Revised: 10 January 2017 Accepted article published: 19 January 2017 Published online in Wiley Online Library: 1 March 2017

(wileyonlinelibrary.com) DOI 10.1002/jctb.5203

Solid-state fermentation of Moringa oleifera

leaf meal using Bacillus pumilus CICC 10440
Mengmeng Zhang,a,b,c† Yewei Huang,a,b,c† Haichu Zhao,a,b,c Tengfei
Wang,a,b Chuanqi Xie,a,b,c Dengyang Zhang,a,b,c Xuanjun Wanga,b,d*
and Jun Shenga,b*

Abstract
BACKGROUND: Moringa leaf is licensed as a new food ingredient and is rich in nutrients. However, owing to its high cellulose
content, the nutrients are difficult to digest and absorb, which limits the product development and application of Moringa leaf.
RESULTS: Four bacterial strains – Trichoderma reesei CICC 2626, Bacillus pumilus CICC 10440, Bacillus subtilis subsp. CICC 21934,
and Bacillus sp. CICC 21942 – were chosen to ferment with Moringa leaf powder. The results of solid fermentation and liquid
fermentation show that solid-state fermentation using Bacillus pumilus CICC 10440 in a 1:60 ratio with Moringa leaf powder
produced the greatest amount of soluble protein (285 mg g−1 , 2.42 times higher than the control) in 24 h. Using a cellulase
activity assay and western blotting, it was found that Bacillus pumilus CICC 10440 uses endoglucanase to dissolve the cellulose
to release nutrients from the Moringa oleifera leaf. We expanded the fermentation scale of the Moringa oleifera leaf in a 200 L
fermenter and produced a 50 kg batch of Moringa oleifera product.
CONCLUSION: The results showing improvement in the nutrient release of Moringa leaf via microbial fermentation are a
promising start and provide a basis for further product development of Moringa leaf.
© 2017 Society of Chemical Industry

Keywords: Moringa oleifera leaf meal; solid-state fermentation; cellulose; Bacillus pumilus CICC 10440; endoglucanase

INTRODUCTION the improvement of flavor, nutrient absorption and nutrient uti-

Moringa oleifera Lam. (Moringaceae) is a perennial angiosperm
whose genus includes several other species.1 Moringa oleifera is
native to the Himalayan region and is widely cultivated throughout
the tropical and sub-tropical regions of Asia, Africa, North Amer-
ica, and South America.2,3 With a shrub growth form, Moringa
oleifera Lam. ranges from 5–15 m in height, is approximately 30 cm
in diameter, and has thin and brittle stems. The tree is known
by several regional names, including benzolive, drumstick tree,
horseradish tree, kelor, marango, mlonge, mulangay, saijihan, and
sajna.4
Recently, Moringa oleifera has received increased agricultural
and industrial attention for several reasons: it is a perennial that
requires very little water, it is nutrient-cycling efficient, and it is
adaptable to a wide array of environments.5,6 Moringa also has a
high nutritional value and thus may serve as a viable alternative
to traditional food crops that are not perennial and/or must be
planted more frequently.7 Moringa is considered to be the most
nutrient-rich plant on earth, as almost every part of the plant can
be used for food, medication, or industrial purposes.8 Accordingly,
its leaves have been consumed by people in Asia for millennia as
a health food. The leaves have immense nutritional value, as they
have been found to contain phytochemicals, vitamins, minerals,
and amino acids. As such, Moringa leaf products, especially leaf
meal, are becoming increasingly popular.5
While Moringa leaves are known for their health benefits, they
are infrequently consumed, probably owing to their high cellulose
lization is severely lacking for Moringa leaves. Moringa leaves con-
tain a considerable amount of cellulose and pectin, which can be
broken down by cellulase and pectinase, respectively, promoting
nutrient release. The bio-degradation of cellulose has been stud-
ied extensively, and a number of cellulolytic enzymes have been
characterized.10,11 Commercial cellulases are produced mainly by
fungi;12 – 14 however, bacteria have also been considered as robust
and versatile enzyme producers due to their high growth rate, sta-
bility under extreme conditions, and multienzyme complexes.15,16
Bacillus strains are often used in modern biological fermentation
technology, as they are non-toxic and biologically safe.17,18 From a
∗ Correspondence to: J Sheng or X Wang, Key Laboratory of Pu-er Tea Science,
Ministry of Education, Yunnan Agricultural University, Kunming 650201,
China. E-mail: shengj@ynau.edu.cn (Sheng); wangxuanjun@gmail.com
(Wang)
† These authors contributed equally to this work
a Key Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural
University, Kunming, China
b Tea Research Center of Yunnan, Kunming, China
c College of Food Science and Technology, Yunnan Agricultural University, Kun-
ming, China
d State Key Laboratory for Conservation and Utilization of Bio-Resources in
2083

content, low satiating power and strong odor.4,9 Information on Yunnan, Yunnan Agricultural University, Kunming, China

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 www.soci.org
commercial standpoint, Bacillus pumilus, Bacillus sp., Bacillus sub-
tilis subsp. and Trichoderma reesei have been found to be potent
extracellular enzyme producers; as a result, they were chosen for
the fermentation of Moringa oleifera leaf meal in the present study.
The aim of the present study was to improve the nutritional value
of Moringa oleifera leaf meal. In our approach, optimal fermenta-
tion conditions and growth conditions were established to maxi-
mize the release of nutrients from Moringa leaf meal. The comple-
tion of this work has helped overcome the nutritional deficiencies
associated with the consumption of Moringa leaves and drastically
improved their nutritional value. The findings are relevant to the
development of Moringa products and have valuable implications
for the Moringa industry.
MATERIALS AND METHODS
Raw material and microorganisms
Moringa oleifera leaf was obtained from the Dehong Autonomous
Prefecture, Yunnan Province, China. The dried Moringa leaves
were shredded using an ultrafine mill to obtain Moringa leaf
powder to be subjected to fermentation. The bacteria used for
the Moringa oleifera leaf meal fermentation were required to cause
no effects on the human body and had to be widely available in
the surrounding area. Thus, we chose the four strains used in this
experiment from the China Center of Industrial Culture Collection
(Table 1).
Culture of microbial strains
Freeze-dried bacterial cultures from sealed vials were activated
by transferring samples of each culture to nutrient agar plates
and incubating them at 30 ∘ C for 24 h. The four bacterial strains
were initially screened for cellulase activity in cellulose culture
medium containing 0.2% (w/w) carboxymethyl cellulose (CMC,
Nuclear, Diadema, Brazil) on agar medium (sodium chloride, 10 g;
agar, 15 g L−1 ; pH 7.0). The eugonic colonies were then selected,
amplified and cultured in a tube containing 5 mL of nutrient
broth at 30 ∘ C for 24 h. The colonies formed in the nutrient broth
were then transferred to Moringa oleifera medium containing 15%
(w/w) Moringa leaf meal in agar medium (sodium chloride, 10 g;
agar, 15 g L−1 ; pH 7.0) and incubated at 30 ∘ C for 24 h19 before
being inoculated in Moringa oleifera medium.18 The domesticated
mature strains were cryopreserved in 60% glycerol and stored at
−80 ∘ C.
Preparation of seed culture and crude enzyme
The colonies were inoculated in 5 mL of nutrient broth. Aliquots
of this culture (≈0.01 mL, HiMedia SS-4) were then transferred to
500 mL nutrient broth (autoclaved at 1.05 kg cm−2 for 20 min) and
incubated at 30 ∘ C for 18 h while shaking at 200 rpm.20 This cul-
ture was used to prepare subcultures with 2% (v/v) inoculum for
each. The contents were centrifuged at 4000 rpm for 10 min at
4 ∘ C, and the pellets were used for Moringa leaf powder fermen-
tation. The remaining clear and cell-free supernatant was used in
carboxymethyl cellulose assays.21
Fermentation
The strains stored in glycerol at −80 ∘ C were revived by streaking
samples on LB plates and incubating them overnight at 30 ∘ C.22
To determine the optimum medium composition, Bacillus Pumilus
CICC 10440 strains were inoculated in 500 mL shaker flasks, each
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containing 100 mL of a different culture medium. The cultures
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M Zhang et al.
were incubated at 30 ∘ C with shaking at 200 rpm. The bottles
were sealed with either a rubber septum or an aluminum cap.
The inoculum size was 5% at an initial absorbance (at 600 nm) of
approximately 0.12.23 Samples were taken every hour to detect the
absorbance at 600 nm until growth reached the stationary phase.
Glucose and yeast powder were also added to determine the opti-
mum culture medium composition (Table 2). Then, the strains were
collected and centrifuged at 4000 rpm for 10 min. The resulting
precipitant was washed twice with distilled water and then mixed
with Moringa leaf powder at a ratio of 1:20 (w/w). Distilled water
was added to the Moringa leaf powder to generate moisture ratios
of 1:0.1 and 1:0.15 (w/w) during liquid–state fermentation and
1:1 (w/w) during solid–state fermentation. We designed control
experiments using solid-state fermentation and liquid fermenta-
tion to show the changes in the protein content without the inocu-
lation of microorganisms, with the other experimental conditions
remaining unchanged. The soluble protein was detected by uni-
form sampling every 24 h. The optimal strain as well as the optimal
method of fermentation was identified according to the experi-
mental conditions that produced the greatest amount of soluble
protein in the least amount of time.
To study the kinetics of solid-state fermentation, batch fermen-
tation was performed in a 200 L fermentation tank using 100 L of
culture medium, 4% (w/w) carbon source supplement, 2% bacte-
rial inoculation and 1:1.2 ventilation.23,24 Fermentation occurred at
a constant temperature of 30 ∘ C while stirring (200 rpm). After 40 h,
aliquots were collected and centrifuged at 4000 rpm for 10 min.
The resulting precipitant was washed twice with distilled water
and then mixed with Moringa leaf powder at the ratio of 1:60. Dis-
tilled water was then added to obtain a 1:1 leaf powder moisture
ratio (w/v). Sealed fermentation was conducted at 30 ∘ C for 4 days,
and the Moringa proteins were then extracted.25,26
Analytical methods
2 g of fermentation matter was used to determine the protein con-
centration of the samples at 24 h time points. With this approach,
the proteins were extracted from the fermentation matter by
adding approximately 10 volumes of distilled water (w/v, 20 mL) to
2 g of fermentation matter and then extracting the samples for 1 h
at 60 rpm in the shaker incubator. The supernatant remaining after
centrifugation was used to analyze the fermentation parameters.27
The protein levels were quantified using the Bicinchoninic Acid
Kit (Sigma), and the biomass was measured at an optical density
of 600 nm (UNICO UV-2000, USA). The total phenolic content was
determined with the Folin-Ciocalteu method.4 The total polysac-
charide content was determined with the anthrone sulfuric acid
colorimetric method.
The plate assay for lytic activity was performed by culturing
bacteria for 14 h on a 0.1% carboxymethyl cellulose medium
with 2% agar that was flooded with an aqueous solution of
0.1% Congo red followed by the addition of 1 mol L−1 NaCl with
intermittent shaking for 15 min at 25 rpm. The bacterial growth
was stopped by the addition of 1 mol L−1 HCl, and the enzymatic
activity was inhibited by the addition of 1 mol L−1 NaOH28 . The lytic
activity was indicated by the size of the clear zone surrounding
the bacteria located on carboxymethyl cellulose-containing agar
plates.
The carboxymethyl cellulase activity was measured by the
reduction of 3,5-dinitrosalicylic acid in the presence of the
glucose released by the enzymatic hydrolysis of cellulose, as
previously described. Briefly, 25 μL of enzyme solution (super-
natant) was added to 25 μL of CMC (1%) and incubated at
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Table 1. Seed culture media recipe for the four strains of choice

Strain Temperature (∘ C) Product Medium L−1 Source

Trichoderma reesei CICC 2626 24 Cellulase 200 g potato powder + 20 g China Center of Industrial
glucose Culture Collection
Bacillus pumilus CICC 10440 25 Cellulase 5.0 g peptone + 3.0 g beef
extract + 5.0 g NaCl PH 7.0
Bacillus subtilis subsp. CICC 21934 37 Alkaline pectinase
Bacillus sp. CICC 21942 37 Pectinase

Table 2. Culture media composition

Culture medium per litre

Component A/pH 7.0 B/pH 7.0 C/pH 7.0

MOPS 8.0 g 8.0 g -

Na2 HPO4 · 2H2 O 8.6 g 1.8 g -
KH2 PO4 3.0 g 1.5 g -
NH4 Cl 1.0 g 7.0 g -
K2 SO4 - 0.35 g -
Yeast - 5.0 g -
FeCl3 60 mg 60 mg -
H3 BO3 3.0 mg 3.0 mg -
MnCl3 · 4H2 O 15 mg 15 mg -
CuCl2 · 2H2 O 1.5 mg 1.5 mg -
Na2 MoO4 · 2H2 O 2.5 mg 2.5 mg -
CoCl2 · 6H2 O 2.5 mg 2.5 mg -
ZnAc · 2H2 O 8.0 mg 8.0 mg -
Glucose 10 g 2.0 g -
MgSO4 · 7H2 O 0.6 g 0.17 g -
NaCl 0.5 g - 5.0 g
Tryptone - - 5.0 g
Beef extract - - 3.0 g

50 ∘ C for 2 h. After incubation, 75 μL of 3,5-dinitrosalicylic acid

reagent was added.29,30 The reaction mixture was boiled for
10 min in a boiling-water bath, and 25 μL of 40% sodium potas-
sium tartrate was added. After cooling at room temperature,
the absorbance at 550 nm was measured. The carboxymethyl
cellulase activity was quantified using a calibration curve for
glucose, where one unit of carboxymethyl cellulase activity was
defined as the amount of enzyme that released 1 μmol of glucose
per minute.31
The western blotting method was used to ascertain the cel-
lulases. The domesticated Bacillus pumilus CICC 10440 was cul-
tivated in LB medium at 30 ∘ C for 24 h. The contents were cen-
trifuged at 4000 rpm for 10 min at 4 ∘ C, and the extracellular pro-
teins in the supernatant were precipitated using the DOC-TCA
method.32,33 90 μg of protein per sample was analyzed using 12%
(w/v) SDS-PAGE, and the protein was then transferred to a PVDF
membrane (Whatman) at 200 mA for 90 min. The membrane was
sealed in PBST-prepared blocking buffer containing 0.5% BSA. It
was then incubated at 4 ∘ C overnight in blocking buffer containing
0.01–0.05 μg mL−1 primary antibody (endoglucanase and pecti-
nase, PL Laboratories, Inc.), washed with PBST, and incubated in
blocking buffer containing 0.01–0.05 μg mL−1 of secondary anti-
body (alkaline phosphatase-rabbit anti-mouse IgG, Sigma). Finally,
the membrane was subjected to an exposure treatment under
Figure 1. Growth of Trichoderma reesei CICC 2626, Bacillus pumilus CICC
10440, Bacillus subtilis subsp. CICC 21934 and Bacillus sp. CICC 21942 at
30 ∘ C on medium containing 15% (w/v) Moringa oleifera as the sole carbon
source.
RESULTS AND DISCUSSION
Domestication of cellulolytic strains
To dissolve the cellulose and promote the release of nutrients
from the Moringa leaves, the bacteria that produced cellulase were
selected using the streak plate method.26,34 After 10 generations
of subculture, the bacterial colonies in Moringa oleifera medium
displayed stable growth and acclimation to the domestication
process (Fig. 1). These results suggest that Trichoderma reesei CICC
2626, Bacillus pumilus CICC 10440, Bacillus subtilis subsp. CICC
21934 and Bacillus sp. CICC 21942 secreted the cellulase and
exhibited the cellulose hydrolytic activity needed to support their
growth during the process of domestication.
Liquid-state fermentation
To evaluate the effect of fermentation on nutrient release,
liquid-state fermentation involving various domesticated strains
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dark conditions. of bacteria was used to ferment Moringa leaf meal.35 The protein

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 www.soci.org M Zhang et al.

Figure 2. Soluble protein concentration in liquid fermentation supernatant. A and B represent the concentrations of protein in samples containing 10%
and 15% (w/v) Moringa oleifera leaf meal, respectively. All assays were performed in triplicate. The standard deviation for the average protein concentration
in each sample is indicated by the error bars.

content of the control group remained almost unchanged. Upon

completion of the fermentation, the concentration of soluble pro-
teins in the samples containing either 10% or 15% (w/v) Moringa
leaf meal was determined to be approximately 18 mg mL−1 .
We did not find evidence for a significant increase in protein
concentration over the 7-day fermentation period (Fig. 2).

Solid-state fermentation of single strains

In addition to liquid fermentation, we examined the effect of
solid-state fermentation on the release of nutrients from Moringa
leaf meal.13,36 The soluble protein content of the control group
did not substantially change and was approximately 124 mg g−1 .
Trichoderma reesei CICC 2626 exhibited a soluble protein concen-
tration of approximately 277 mg g−1 on day 7 of the solid-state
fermentation. This value represents almost 2.35 times more pro-
tein than the samples from the control group. Bacillus pumilus CICC
10440 displayed the highest concentration of soluble protein in
the middle of the fermentation period, approximately 285 mg g−1 ,
which represents 2.42 times more protein than the control group.
The soluble protein contents in Bacillus subtilis subsp. CICC 21934
and Bacillus sp. CICC 21942 were highest towards the end of the
fermentation, approximately 235 mg g−1 on day 11. This value
is approximately twice the amount of protein present in the
control group (Fig. 3). Compared with liquid fermentation, the
solid-state fermentation was simpler and less expensive and pro-
duced greater amounts of protein.
Solid-state fermentation of double strains
The effect of the fermentation differed for each of the bacterial
strains examined, probably because each strain secretes a different
2086
Figure 3. Soluble protein content produced during solid-state fermenta-
tion, measured every 24 h. The time point at which the maximum protein
content was detected differed among Trichoderma reesei CICC 2626, Bacil-
lus pumilus CICC 10440, Bacillus subtilis subsp. CICC 21934 and Bacillus sp.
CICC 21942. All assays were performed in triplicate, and the standard devi-
ation of each group of the biological replicates is represented by error bars.
Bacillus sp. CICC 21942 secrete pectinase, whereas Trichoderma
reesei CICC 2626 and Bacillus pumilus CICC 10440 secrete cellulase.
This difference led us to conduct solid-state fermentation using
two strains of bacteria per assay: one that secreted cellulase and
another that secreted pectinase. The results of this analysis showed
that assays containing Trichoderma reesei CICC 2626 and Bacillus
sp. CICC 21942 displayed the greatest soluble protein content
(242 mg g−1 ). The ratio of the two strains in this assay was 1:1
(w/w) (Fig. 4(B)). There was no obvious peak value displayed by the
double fermentation assays, which may be due to the competition
for growth between the different bacteria (Fig. 4).
On comparing the single and double bacteria fermentations, we
found that the single bacteria fermentation had more advantages

cellulose.36 – 38 Specifically, Bacillus subtilis subsp. CICC 21934 and and that the operation was simpler. We conclude that the use

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Figure 4. Results from fermentation assays containing (A) Bacillus subtilis subsp. CICC 21934 and Trichoderma reesei CICC 2626, (B) Bacillus sp. CICC 21942
and Trichoderma reesei CICC 2626, (C) Bacillus pumilus CICC 10440 and Bacillus subtilis subsp. CICC 21934 and (D) Bacillus pumilus CICC 10440 and Bacillus
sp. CICC 21942. Strains used for the double fermentation assays were added in varying proportions: 1:0.5 (w/w), 1:1 (w/w) and 1:1.5 (w/w). All assays were
performed in triplicate, and the standard deviation for each group of biological replicates is represented by error bars.

Figure 5. Carboxymethyl cellulose digestion assay results: (A) control

sample; (B) screening of Bacillus pumilus CICC 10440.
of a single bacterial strain for solid-state fermentation is more
convenient and effective and that fermentation with the Bacillus
pumilus CICC 10440 strain produced the greatest soluble protein
content, approximately 285 mg g−1 (which is 2.42 times higher
than the control group), in the shortest time, specifically on day
1 of the fermentation. As result, Bacillus pumilus CICC 10440 was
selected for the Moringa fermentation.
Plate assay for lytic activity
Bacillus pumilus CICC 10440 was tested for lysis on carboxymethyl
cellulose. Lysis was revealed by Congo red staining and quantified
Figure 6. Detection of cellulases using western blotting. The antibodies
used during western blotting were specific to endoglucanase. (A) Marker.
(B) The fermented sample using Bacillus pumilus CICC 10440. The molecu-
lar weight indicated by the arrow in the B-band is about 50 KDa.
The Bacillus pumilus CICC 10440 crude supernatant was used in
this experiment. A large lysis zone was observed on day 4 of the
incubation, consistent with the enzyme activity in the presence of
carboxymethyl cellulose (Fig. 5(B)).
Characteristics of cellulose
Two different antibodies were used to characterize the type
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by examining the clear lysis zone surrounding the colonies.16,26 of cellulase produced by Bacillus pumilus CICC 10440. The

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 www.soci.org M Zhang et al.

Figure 7. Growth curves for Bacillus pumilus CICC 10440. (A) Growth curve for Bacillus pumilus CICC 10440 grown in the presence of media A, B and C. (B)
Influence of the addition of 2% glucose and yeast extract to media B on the growth of Bacillus pumilus CICC 10440. All assays were performed in triplicate,
and the standard deviation of each group of biological replicates is represented by error bars.

Table 3. Changes in nutritional composition of Moringa before and after fermentation

Soluble protein content Scavenging free radicals Total polyphenol content Total polysaccharide content
Sample mg g−1 μmol L−1 g−1 mg g−1 mg g−1

Moringa leaf powder 124 ± 2.5 52 ± 1.1 32.73 ± 0.6 60.19 ± 1.4
Moringa enzyme 265 ± 5.5 145 ± 2.9 33.94 ± 0.7 128.47 ± 2.6

The soluble nutrient composition was increased in the Moringa enzyme samples. All assays were performed in triplicate, and the standard deviations
of the mean are presented above.

endoglucanase expression was high, but the pectinase expression
was virtually absent (Fig. 6). The molecular weight of the detected
protein was approximately 50 kDa, consistent with its known
molecular weight.23 The endoglucanase secreted by Bacillus
pumilus showed high activity in carboxymethyl cellulose (10.12
units). When Moringa leaf powder was used as a substrate, enzyme
activity was again detected (8.34 units). Collectively, these find-
ings suggest that the endoglucanase secreted by Bacillus pumilus
CICC 10440 degrades cellulose in Moringa oleifera, resulting in the
release of nutrients from the Moringa oleifera leaf meal.
Optimization of culture medium
To characterize the culture medium consistent with the optimal
bacterial growth, three types of media, denoted A, B and C, were
tested (Table 2). For the growth of Bacillus pumilus CICC 10440 in
the presence of medium A, B or C, growth curves were calculated
using aliquots from each culture. Bacillus pumilus CICC10440 grew
the fastest in the presence of medium B (Fig. 7(A)), with the OD600
reaching a maximum of 5.94 in 11 h. 2% glucose and yeast extract
were each added to culture media B, and the growth curve of
Bacillus pumilus CICC 10440 was determined (Fig. 7(B)).
Batch fermentation experiment
To evaluate the economic feasibility of the scale-up of Moringa
oleifera leaf meal production using the fermentation conditions
established in this study, a batch fermentation experiment was
carried out on a scale of 50 kg.36,39 Through a series of fermentation
technologies, the levels of water-soluble substances, such as the
nutrients in the Moringa oleifera leaf meal fermentation that can
be fully absorbed by the human body, were obviously improved.
The soluble protein content was increased by a magnitude of 2.14,
2088
and the abundance of scavenging free radicals was increased by a
magnitude of 2.79 (Table 3).
CONCLUSION
Bacillus pumilus CICC 10440 and solid-state fermentation were suc-
cessfully utilized to increase the nutrient release in Moringa oleifera
leaf meal. Moreover, a new, environmentally friendly, economical
scale-up of Moringa leaf fermentation was introduced. These find-
ings provide insight into the development of Moringa products
and have valuable implications for the Moringa industry.
Conflicts of interest The authors have declared no conflicts of
interest.
Ethical approval The manuscript does not describe experi-
ments using animals. The manuscript does not describe human
studies.
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