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Novel functional fermented dairy product rich in menaquinone-7
Rachel Southee a
, Saima Haroon a
, Alireza Ebrahiminezad a,b,c
, Younes Ghasemi b
,
Aydin Berenjian a,n
a
School of Engineering, Faculty of Science & Engineering, The University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand
b
Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences,
Shiraz, Iran
c
Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, Fasa, Iran
a r t i c l e i n f o
Article history:
Received 25 January 2016
Received in revised form
26 April 2016
Accepted 30 April 2016
Available online 6 May 2016
Keywords:
Fermented milk
Menaquinone
Functional food
Bacillus subtilis natto
a b s t r a c t
Population based studies have shown a direct correlation between calcium and menaquinone-7 (MK-7)
consumption and improved bone and cardiovascular health. MK-7 could only be produced by fermen-
tation mainly using Bacillus subtilis species. However, this product is not readily available at an affordable
price due to the high recovery and purification costs. Functional foods, therefore, due to the simpler
production and less downstream processing steps can be seen as a low-cost and flexible alternative
approach to address the current challenges in MK-7 production. In this study, we successfully developed
a functional milk-product rich in MK-7 and calcium by using Bacillus subtilis natto. The maximum MK-7
concentration of 11.22 mg/L was achieved for the media consisted of standard milk and 1% (w/v) glycerol
at 200 rpm, 1 vvm, 40 °C after 48 h of fermentation. Based on the results, MK-7 concentration increased
between cell's mid-exponential growth and the stationary phase. The 80% of total MK-7 concentration
was produced after 30 h of fermentation where the concentration of glycerol as a carbon source became
exhausted. This research is the first report on its own which significantly contributes to the initial de-
velopment of a novel functional dairy product high in MK-7. These results provide a basis of knowledge
and new discoveries that can be carried out to develop a product that can act as a preventative in health
issues such as osteoporosis and cardiovascular diseases.
& 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Recent studies have shown that consumption of menaquinone-
7 (MK-7) substantially reduces the risk of bone fractures and car-
diovascular disorders (Hamidi et al., 2013; Brandenburg et al.,
2015). This vitamin can only be produced by bacterial fermenta-
tion and has a key function in the synthesis of two proteins in-
volved in bone metabolism, namely osteocalcin and matrix Gla-
protein (MGP). Osteocalcin is essential for keeping calcium in bone
and MGP is a strong inhibitor of vascular calcification (Schurgers
et al., 2001). Cardiovascular disease and osteoporosis are crucial
health issues worldwide and the health care cost for the man-
agement of these diseases is high. Due to the increasing number of
patients with osteoporosis and cardiovascular diseases, re-
searchers are looking for new approaches to prevent these health
problems (Berenjian et al., 2012; Brandenburg et al., 2015).
Over recent years, intensive studies have been conducted into
the fermentation, extraction and purification techniques to
develop a purified MK-7 for inclusion in human health supple-
ments (Kaneki et al., 2001; Sato et al., 2001; Berenjian et al., 2011;
Berenjian et al., 2012; Santos et al., 2013). However, this product is
not readily available at an affordable price due to the significant
barriers in the production, extraction and purification steps; par-
ticularly substantial high downstream processing costs and low
purity fractions (Berenjian et al., 2015; Ebrahiminezhad et al.,
2016). These issues have resulted in extensive high product cost
(e.g. MK-7 has a market price of US$5 million/kg).
Functional foods due to the simpler production methods and
less downstream processing steps can be seen as a low-cost,
flexible alternative approach to prevent major health complica-
tions (Marina et al., 2014). Functional foods provide significant
benefits beyond basic nutrition and play a key role in reducing or
minimizing the risk of certain diseases and other health condi-
tions. To address the current challenges in MK-7 fermentation
process, therefore, it is desirable to produce a nutritional food
supplement rich in MK-7. This product could be available to a wide
range of consumers at a relatively cheap price.
Milk due to its rich nutrient profile and high calcium level has
the potential to be a potent media for developing a new generation
of functional foods to address cardiovascular disease and
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/bab
Biocatalysis and Agricultural Biotechnology
http://dx.doi.org/10.1016/j.bcab.2016.04.011
1878-8181/& 2016 Elsevier Ltd. All rights reserved.
n
Corresponding author.
E-mail address: aydin.berenjian@waikato.ac.nz (A. Berenjian).
Biocatalysis and Agricultural Biotechnology 7 (2016) 31–35
osteoporosis as well as other health complications. It has been
reported that the synergistic effect between calcium and MK-7,
significantly improves the bone and cardiovascular health (Zitter-
mann, 2001; Yamaguchi and Weitzmann, 2011). The aim of the
present study, therefore, is to investigate and develop a novel
functional dairy product rich in MK-7 by using probiotic bacteria
(e.g. Bacillus subtilis natto). The objective of this investigation is to
optimize a media to improve and provide the greatest yield of MK-
7. Therefore, the aims of the present study are: (i) the potential of
milk as a medium to be utilized by Bacillus subtilis natto and MK-7
production, and (ii) approaches to provide the greatest yield for
MK-7 biosynthesis.
2. Materials and methods
2.1. Preparation of microorganism and inoculum
Bacillus strain was isolated from fermented natto product (Ya-
mada Foods Co. LTD) as described by Joshi (Joshi et al., 2008).
Briefly, soybean samples were placed in a screw-cap bottle con-
taining 5 g sterile diluent (1 g peptone/L) and were shaken for
20 min Serial dilutions were performed in the same diluent and
0.1 mL of each dilution was spread directly onto nutrient agar.
After incubation at 30 °C for 48 h, colonies were identified as Ba-
cillus subtilis natto by morphological and biochemical tests and 16S
rRNA gene sequencing. Luria–Bertani broth was used for pre-
paration of the inoculum. The culture was grown for 10 h at 30 °C
having the final concentration of 6 Â 106
cells/mL.
2.2. Chemicals
Peptone and Luria–Bertani broth were from Becton Dickinson
(Franklin Lakes, New Jersey, USA); iso-propyl and lactose were
supplied by Asia Pacific Speciality Chem Ltd, Wellington, New
Zealand. n-Hexane, methanol, D (-) fructose, D(þ) glucose, D(-)
mannitol were purchased from Merck Pty Ltd., Darmstadt, Ger-
many. Sucrose, maltose, glycerol, yeast extract, potassium dihy-
drogen phosphate (KH2PO4) and agar plates were purchased from
BD, Fort Richard Laboratories, Auckland, New Zealand. Pure MK-7
was supplied from ChromaDex (Irvine, California, USA). Different
types of milk including fat, standard, lite, trim, protein, and cho-
colate milk were purchased from Goodman Fielder, Auckland, New
Zealand.
2.3. Fermentation process
Each media was inoculated with 2% (v/v) Bacillus subtilis natto
and incubated at 40 °C in aerobic conditions for 48 h (at 120 rpm).
A 3-L fermentor (BioFlo/CelliGen 115, New Brunswick Scientific
Co., USA) was equipped with a top-driven stirrer with two six
blade Rushton impellers (i. e. diameter 45 mm, width 14 mm,
length 14 mm) and four baffles (i.e. width 14 mm). Filtered air was
injected through a perforated pipe sparger located under the
bottom impeller. The levels for aeration and stirrer controllers
were set at 1 vvm and 200 rpm, respectively. The DO probe was
calibrated using a standard two-point calibration method. The 0%
calibration was achieved by sparging nitrogen into the medium at
approximately 1 vvm until the value stabilized near zero; the 100%
calibration was achieved by increasing agitation to approximately
50 rpm and increasing airflow to 5 vvm. After calibration, dis-
solved oxygen values were reported based on these calibration set
points.
2.4. Analysis of MK-7
MK-7 was extracted from the fermentation media with the
addition of n-hexane:2-propanol in the following ratios (2:1, v/v)
and 1:4 (milk: organic, v/v) (Berenjian et al., 2011). Briefly, fer-
mented milk, n-hexane and 2-propanol were mixed at 2500 rpm
for one minute, and centrifuged for 5 min at 3000 rpm to separate
the organic and inorganic layers. Consequently, the organic phase
was evaporated to recover the MK-7. High-performance liquid
chromatography (HPLC) with auto-sampler (Waters 717 plus),
Waters 515 pump, Photodiode Array detector (Waters 996) and a
C18 Gemini column (5 mm, 250 Â 4.6 mm, 40 °C) was used to
measure the MK-7 concentration. Methanol (HPLC grade) was
used as a mobile phase at a flow rate of 1 mL/min, and a wave-
length of 248 nm was chosen for the analytical analysis. HP
ChemStation LC software was used for data acquisition. A cali-
bration curve was prepared using pure MK-7 standards.
2.5. Experimental plan and statistical analysis
The suitability of milk as a fermentation media for MK-7 pro-
duction was investigated by using different types of milk including
fat, standard, lite, trim, protein, and chocolate. Each media was
inoculated with 2% (v/v) Bacillus subtilis natto and incubated at
40 °C for 96 h at 120 rpm. A sample from each fermented media
was extracted using organic solvents for HPLC analysis as describe
in “Analysis of MK-7″ section. The Plackett–Burman design was
used for screening the effect of extra nutrients on MK-7 bio-
synthesis at two levels (þ1 and À1). MODDE software version 11
(Umetrics, Sweden) was used to create experimental design ma-
trices. Among the nutrients glycerol, mannitol, glucose, maltose,
lactose, fructose, yeast extract, soy peptone, NaCl, sucrose, KH2SO4
were tested at high (þ) and a low (À) concentration for their
significance in MK-7 production (Table 1). The main effect of each
variable upon MK-7 production was estimated as the difference
between both averages of measurements made of the high and
low levels. The quality of fit for the regression model equation was
expressed as R2
and the statistical significant was determined by
analysis of variance (ANOVA) tests. Statistical significance was
accepted at po0.10 and data was reported as a mean7standard
deviation.
2.6. Glycerol addition and measurement
Glycerol concentrations ranging from 1% to 8% (w/v) were used
to study the most effective concentration on improving MK-7
production. The concentration of glycerol was determined en-
zymatically by using free glycerol determination kit (Sigma-Al-
drich Co., St Louis, MO, USA) following its own procedure. Briefly,
800 mL of the glycerol free reagent was inoculated with 10 mL of
fermented media and then incubated for 5 min at 37 °C. The UV-
absorbance of resulting solution was measured at 540 nm using
the absorbance of water as the reference.
Table 1
Milk type composition.
Type Fat (%) Protein (%) Carbohydrate (%)
Standard 3.6 3.1 4.6
Lite 1.5 3.4 4.7
Trim 0.3 3.6 4.8
Full fat 4 3.1 4.6
Protein 0.5 5.8 5.2
Chocolate 1.1 4.3 6.6
R. Southee et al. / Biocatalysis and Agricultural Biotechnology 7 (2016) 31–3532
3. Result and discussion
3.1. Feasibility of milk media
Different types of milk were investigated to determine their
suitability as a fermentation media for Bacillus subtilis natto
growth and MK-7 production. Fat, standards, lite, trim, protein,
and chocolate milk due to their different fat, protein, and carbo-
hydrate concentrations (Table 1) were used for Bacillus subtilis
natto fermentation. The results imply the possible use of milk as
the suitable substrate for developing a functional food rich in MK-
7. As shown in Fig. 1, the standard milk resulted in the highest MK-
7 concentration of 9.78 mg/L as compared to the other used milk
media (po0.1). Based on the results, MK-7 concentration was
higher in the media with higher fat contents. This behaviour could
be due to the fact that Bacillus subtilis natto utilizes fat through a
shikimate pathway for menaquinone biosynthesis (Sonenshein
et al., 2002). However, the possible reason for lower MK-7 pro-
duction in full cream milk as compared to the standard milk could
be due to unhomogenized fat content which in turn reduces the
microorganisms access to the fat. The standard milk was chosen
for further optimization studies to enhance the MK-7 production.
3.2. Effect of nutrients
The effects of the addition of different nutrients namely glycerol,
mannitol, glucose, maltose, lactose, fructose, yeast extract, soy pep-
tone, NaCl, sucrose, and KH2SO4 on enhancing the MK-7 production
by using the standard milk was further investigated. The availability
of various carbon, nitrogen and salt sources could improve the
bacterial metabolism and MK-7 production (Berenjian et al., 2015).
Therefore, it is critical to investigate the effect of additional nutrients
on MK-7 production and establish an optimum fermentation med-
ium which can significantly increase the MK-7 production.
The Plackett-Burman design was used to screen and determine
the key nutrients that can have a significant effect on MK-7 pro-
duction. The levels of investigated variables are listed in Table 2.
While the Placket–Burman design does not evaluate the interaction
terms, it considerably reduces the number of experiments that are
required to evaluate the main effects (Giordano et al., 2011). The
analysis of variance of the model parameters (ANOVA) on MK-7
production are presented in Table 3. The regression coefficient was
0.957, which indicates that the model as fitted explains almost 96%
of the variability around the mean and confirms the good model fit.
Presence of NaCl and KH2SO4 as well as protein sources had a
negligible impact on MK-7 production (p40.1). However, maltose,
fructose, and lactose showed a significantly negative effect on the
MK-7 production. Based on the results, glycerol provided the
greatest yield of MK-7 production. The low P-value of glycerol
(po0.1) reveals that it is the most significant factor on MK-7 bio-
synthesis among all the studied factors. For Bacillus subtilis natto,
glycerol clearly alleviates the pathway for the production of shi-
kimate which could be based on conversion of glycerol into me-
naquinone by condensation of phosphoenolpyruvate and ery-
throse-4-phosphate (Sonenshein et al., 2002). In addition, it has
been reported that glycerol decreases the molecular weight of
biopolymers, such as ε-poly-Llysine and poly (hydroxyalkanoate).
This reduction may result in decreasing the viscosity of the media,
which can have a positive impact on stimulating the uptake of
extracellular substrates and enhancing the mass transfer. The
viscosity affects the diffusion of oxygen, consequently the growth
of Bacillus subtilis natto and MK-7 production. Glycerol is a col-
ourless and odourless liquid which is widely used in the food in-
dustry and food formulations (Santa Cruz 2010). Glycerol is safe for
Table 2
Plackett-Burman design with MK-7 yield.
Run Glycerol Mannitol Glucose Maltose Lactose Fructose Yeast extract Soy peptone NaCl Sucrose KH2SO4 MK-7
1 8 2 8 2 2 2 5 8 0.05 2 0.07 10.1370.83
2 8 8 8 8 2 2 1 8 0.05 8 0.05 10.5975.15
3 2 8 8 2 8 2 1 2 0.05 8 0.07 10.0571.03
4 8 2 8 8 2 8 1 2 0.01 8 0.07 7.3575.01
5 8 8 2 8 8 2 5 2 0.01 2 0.07 8.1672.79
6 8 8 8 2 8 8 1 8 0.01 2 0.05 7.8171.35
7 2 8 8 8 2 8 5 2 0.05 2 0.05 3.2770.01
8 2 2 8 8 8 2 5 8 0.01 8 0.05 3.8474.11
9 2 2 2 8 8 8 1 8 0.05 2 0.07 1.4771.38
10 8 2 2 2 8 8 5 2 0.05 8 0.05 10.177.38
11 2 8 2 2 2 8 5 8 0.01 8 0.07 10.3870.84
12 2 2 2 2 2 2 1 2 0.01 2 0.05 10.2370.32
Fig. 1. Effect of different types of milk on MK-7 production.
Table 3
Statistical analysis of variables for screening the nutrients.
MK7 Coefficient Std. Error P-value Conf. int (7)
Constant 8.30766 0.364589 0.000185115 1.16029
Glycerol 1.38042 0.407623 0.0428852 1.29725
Mannitol 0.281251 0.407623 0.539793 1.29725
Glucose À0.734583 0.407623 0.169321 1.29725
Maltose À2.31542 0.407623 0.010812 1.29725
Lactose À1.02375 0.407623 0.086822 1.29725
Fructose À1.19792 0.407623 0.0605696 1.29725
Yeast extract 0.00458299 0.407623 0.991735 1.29725
Soy peptone À0.440417 0.407623 0.359055 1.29725
NaCl À0.0404173 0.407623 0.92727 1.29725
Sucrose 0.789584 0.407623 0.14814 1.29725
KH2SO4 0.112917 0.407623 0.799761 1.29725
R2
¼0.957, R2
(Adj)¼0.801; Significance code: Po0.1.
R. Southee et al. / Biocatalysis and Agricultural Biotechnology 7 (2016) 31–35 33
human consumption, and has a low cost as compared to the other
nutrient sources, thus enabling the possible development of a low
cost functional food. To further improve yield of MK-7, glycerol
concentration was subjected to further concentration optimization.
3.3. Glycerol concentration
The effect of adding various concentrations of glycerol (1–8%
(v/v)) on MK-7 production was investigated. As depicted in Fig. 2,
as compared with the control media (in absence of glycerol) the
MK-7 production was enhanced in the presence of different gly-
cerol concentrations. These data reveals the key role of glycerol in
MK-7 biosynthesis via Bacillus subtilis natto metabolic pathway.
Based on the results, the presence of 1% (w/v) glycerol significantly
improved the MK-7 production by 18% as compared to the control
media (po0.1). However, higher glycerol concentrations showed
no improvement on enhancing the MK-7 biosynthesis.
The concentration of glycerol was measured during the fer-
mentation process. Glycerol concentration in the media was de-
creased, regardless of the initial concentrations throughout the
fermentation period. A further increase in the glycerol con-
centration from 4% to 8% (w/v) resulted in higher glycerol con-
sumption. However, MK-7 biosynthesis showed no significant
change when using higher concentration of glycerol. This clearly
shows that higher concentrations of glycerol was not utilized
through a shikimate pathway (Bentley et al., 1990) to further en-
hance the menaquinone biosynthesis.
3.4. Monitoring process parameters at the optimized condition
Fermentation operational conditions have a key role in both
growth and production of any wished metabolite. These factors are
of critical importance for the design and scale-up of bioreactors. In
order to gain a better understanding on how these factors affect
MK-7 production, bacterial growth, glycerol concentration, dis-
solved oxygen profile and pH at various stages of the fermentation
were investigated for the optimized medium.
MK-7 production was closely coupled with cell growth. As can
be concluded from Fig. 3, MK-7 concentration increased approxi-
mately 3-fold between cell's mid-exponential growth and the
stationary phase, reaching its optimal concentration after 48 h
(11.2270.6 mg/L). According to the results glycerol concentration
in the medium stridently decreased to approximately 0.2% at the
end of the fermentation; coinciding with an increase in biomass
and MK-7 production. Our results show that the great bulk of MK-
7 was mainly produced at the stationary phase where the con-
centration of glycerol as a carbon source was limited. pH of the
media decreased from 6.65 to 5.32 after the cell growth rate
reached its maximum level after 30 h.
To date, number of Bacillus subtilis natto based products are
used as food or nutritional supplements with various positive
impacts on human health which shows this microorganism is not
only non-pathogenic but also has probiotic effects (Barbosa et al.,
2005; Tsukamoto et al., 2001). The newly developed fermented
milk rich in MK-7 can significantly provoke immune system, bone
and cardiovascular health. The changing lifestyles and growing
health awareness are the key factors that affect the market for
functional food ingredients. Given the position of heart and bone
health as a leading health issues worldwide (Brandenburg et al.,
2015; Berenjian et al., 2015), product developers have the oppor-
tunity to use the outcomes of the present study to further develop
a product to prevent bone and cardiovascular diseases.
4. Conclusions
From the results it is evident that milk is a promising
fermentation media for MK-7 production by using Bacillus subtilis
natto. This research successfully met its objectives of investigating
the effect of fermentation conditions on MK-7 production. Stan-
dard milk proved to be the most effective milk media with a MK-7
concentration of 11.22 mg/L. Among the tested nutrients, only
glycerol showed a significant positive effect on MK-7 yield. Gly-
cerol concentrations were screened and the optimal concentration
of glycerol was found to be 1% (v/v). This study can significantly
contribute to the initial development of a novel functional dairy
product high in MK-7 by providing a basis of knowledge and new
discoveries that can be carried out to develop a functional food
that can act as a preventative in health issues, osteoporosis and
cardiovascular diseases.
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Novel functional fermented dairy product rich in menaquinone-7

  • 1. Novel functional fermented dairy product rich in menaquinone-7 Rachel Southee a , Saima Haroon a , Alireza Ebrahiminezad a,b,c , Younes Ghasemi b , Aydin Berenjian a,n a School of Engineering, Faculty of Science & Engineering, The University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand b Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran c Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, Fasa, Iran a r t i c l e i n f o Article history: Received 25 January 2016 Received in revised form 26 April 2016 Accepted 30 April 2016 Available online 6 May 2016 Keywords: Fermented milk Menaquinone Functional food Bacillus subtilis natto a b s t r a c t Population based studies have shown a direct correlation between calcium and menaquinone-7 (MK-7) consumption and improved bone and cardiovascular health. MK-7 could only be produced by fermen- tation mainly using Bacillus subtilis species. However, this product is not readily available at an affordable price due to the high recovery and purification costs. Functional foods, therefore, due to the simpler production and less downstream processing steps can be seen as a low-cost and flexible alternative approach to address the current challenges in MK-7 production. In this study, we successfully developed a functional milk-product rich in MK-7 and calcium by using Bacillus subtilis natto. The maximum MK-7 concentration of 11.22 mg/L was achieved for the media consisted of standard milk and 1% (w/v) glycerol at 200 rpm, 1 vvm, 40 °C after 48 h of fermentation. Based on the results, MK-7 concentration increased between cell's mid-exponential growth and the stationary phase. The 80% of total MK-7 concentration was produced after 30 h of fermentation where the concentration of glycerol as a carbon source became exhausted. This research is the first report on its own which significantly contributes to the initial de- velopment of a novel functional dairy product high in MK-7. These results provide a basis of knowledge and new discoveries that can be carried out to develop a product that can act as a preventative in health issues such as osteoporosis and cardiovascular diseases. & 2016 Elsevier Ltd. All rights reserved. 1. Introduction Recent studies have shown that consumption of menaquinone- 7 (MK-7) substantially reduces the risk of bone fractures and car- diovascular disorders (Hamidi et al., 2013; Brandenburg et al., 2015). This vitamin can only be produced by bacterial fermenta- tion and has a key function in the synthesis of two proteins in- volved in bone metabolism, namely osteocalcin and matrix Gla- protein (MGP). Osteocalcin is essential for keeping calcium in bone and MGP is a strong inhibitor of vascular calcification (Schurgers et al., 2001). Cardiovascular disease and osteoporosis are crucial health issues worldwide and the health care cost for the man- agement of these diseases is high. Due to the increasing number of patients with osteoporosis and cardiovascular diseases, re- searchers are looking for new approaches to prevent these health problems (Berenjian et al., 2012; Brandenburg et al., 2015). Over recent years, intensive studies have been conducted into the fermentation, extraction and purification techniques to develop a purified MK-7 for inclusion in human health supple- ments (Kaneki et al., 2001; Sato et al., 2001; Berenjian et al., 2011; Berenjian et al., 2012; Santos et al., 2013). However, this product is not readily available at an affordable price due to the significant barriers in the production, extraction and purification steps; par- ticularly substantial high downstream processing costs and low purity fractions (Berenjian et al., 2015; Ebrahiminezhad et al., 2016). These issues have resulted in extensive high product cost (e.g. MK-7 has a market price of US$5 million/kg). Functional foods due to the simpler production methods and less downstream processing steps can be seen as a low-cost, flexible alternative approach to prevent major health complica- tions (Marina et al., 2014). Functional foods provide significant benefits beyond basic nutrition and play a key role in reducing or minimizing the risk of certain diseases and other health condi- tions. To address the current challenges in MK-7 fermentation process, therefore, it is desirable to produce a nutritional food supplement rich in MK-7. This product could be available to a wide range of consumers at a relatively cheap price. Milk due to its rich nutrient profile and high calcium level has the potential to be a potent media for developing a new generation of functional foods to address cardiovascular disease and Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bab Biocatalysis and Agricultural Biotechnology http://dx.doi.org/10.1016/j.bcab.2016.04.011 1878-8181/& 2016 Elsevier Ltd. All rights reserved. n Corresponding author. E-mail address: aydin.berenjian@waikato.ac.nz (A. Berenjian). Biocatalysis and Agricultural Biotechnology 7 (2016) 31–35
  • 2. osteoporosis as well as other health complications. It has been reported that the synergistic effect between calcium and MK-7, significantly improves the bone and cardiovascular health (Zitter- mann, 2001; Yamaguchi and Weitzmann, 2011). The aim of the present study, therefore, is to investigate and develop a novel functional dairy product rich in MK-7 by using probiotic bacteria (e.g. Bacillus subtilis natto). The objective of this investigation is to optimize a media to improve and provide the greatest yield of MK- 7. Therefore, the aims of the present study are: (i) the potential of milk as a medium to be utilized by Bacillus subtilis natto and MK-7 production, and (ii) approaches to provide the greatest yield for MK-7 biosynthesis. 2. Materials and methods 2.1. Preparation of microorganism and inoculum Bacillus strain was isolated from fermented natto product (Ya- mada Foods Co. LTD) as described by Joshi (Joshi et al., 2008). Briefly, soybean samples were placed in a screw-cap bottle con- taining 5 g sterile diluent (1 g peptone/L) and were shaken for 20 min Serial dilutions were performed in the same diluent and 0.1 mL of each dilution was spread directly onto nutrient agar. After incubation at 30 °C for 48 h, colonies were identified as Ba- cillus subtilis natto by morphological and biochemical tests and 16S rRNA gene sequencing. Luria–Bertani broth was used for pre- paration of the inoculum. The culture was grown for 10 h at 30 °C having the final concentration of 6 Â 106 cells/mL. 2.2. Chemicals Peptone and Luria–Bertani broth were from Becton Dickinson (Franklin Lakes, New Jersey, USA); iso-propyl and lactose were supplied by Asia Pacific Speciality Chem Ltd, Wellington, New Zealand. n-Hexane, methanol, D (-) fructose, D(þ) glucose, D(-) mannitol were purchased from Merck Pty Ltd., Darmstadt, Ger- many. Sucrose, maltose, glycerol, yeast extract, potassium dihy- drogen phosphate (KH2PO4) and agar plates were purchased from BD, Fort Richard Laboratories, Auckland, New Zealand. Pure MK-7 was supplied from ChromaDex (Irvine, California, USA). Different types of milk including fat, standard, lite, trim, protein, and cho- colate milk were purchased from Goodman Fielder, Auckland, New Zealand. 2.3. Fermentation process Each media was inoculated with 2% (v/v) Bacillus subtilis natto and incubated at 40 °C in aerobic conditions for 48 h (at 120 rpm). A 3-L fermentor (BioFlo/CelliGen 115, New Brunswick Scientific Co., USA) was equipped with a top-driven stirrer with two six blade Rushton impellers (i. e. diameter 45 mm, width 14 mm, length 14 mm) and four baffles (i.e. width 14 mm). Filtered air was injected through a perforated pipe sparger located under the bottom impeller. The levels for aeration and stirrer controllers were set at 1 vvm and 200 rpm, respectively. The DO probe was calibrated using a standard two-point calibration method. The 0% calibration was achieved by sparging nitrogen into the medium at approximately 1 vvm until the value stabilized near zero; the 100% calibration was achieved by increasing agitation to approximately 50 rpm and increasing airflow to 5 vvm. After calibration, dis- solved oxygen values were reported based on these calibration set points. 2.4. Analysis of MK-7 MK-7 was extracted from the fermentation media with the addition of n-hexane:2-propanol in the following ratios (2:1, v/v) and 1:4 (milk: organic, v/v) (Berenjian et al., 2011). Briefly, fer- mented milk, n-hexane and 2-propanol were mixed at 2500 rpm for one minute, and centrifuged for 5 min at 3000 rpm to separate the organic and inorganic layers. Consequently, the organic phase was evaporated to recover the MK-7. High-performance liquid chromatography (HPLC) with auto-sampler (Waters 717 plus), Waters 515 pump, Photodiode Array detector (Waters 996) and a C18 Gemini column (5 mm, 250 Â 4.6 mm, 40 °C) was used to measure the MK-7 concentration. Methanol (HPLC grade) was used as a mobile phase at a flow rate of 1 mL/min, and a wave- length of 248 nm was chosen for the analytical analysis. HP ChemStation LC software was used for data acquisition. A cali- bration curve was prepared using pure MK-7 standards. 2.5. Experimental plan and statistical analysis The suitability of milk as a fermentation media for MK-7 pro- duction was investigated by using different types of milk including fat, standard, lite, trim, protein, and chocolate. Each media was inoculated with 2% (v/v) Bacillus subtilis natto and incubated at 40 °C for 96 h at 120 rpm. A sample from each fermented media was extracted using organic solvents for HPLC analysis as describe in “Analysis of MK-7″ section. The Plackett–Burman design was used for screening the effect of extra nutrients on MK-7 bio- synthesis at two levels (þ1 and À1). MODDE software version 11 (Umetrics, Sweden) was used to create experimental design ma- trices. Among the nutrients glycerol, mannitol, glucose, maltose, lactose, fructose, yeast extract, soy peptone, NaCl, sucrose, KH2SO4 were tested at high (þ) and a low (À) concentration for their significance in MK-7 production (Table 1). The main effect of each variable upon MK-7 production was estimated as the difference between both averages of measurements made of the high and low levels. The quality of fit for the regression model equation was expressed as R2 and the statistical significant was determined by analysis of variance (ANOVA) tests. Statistical significance was accepted at po0.10 and data was reported as a mean7standard deviation. 2.6. Glycerol addition and measurement Glycerol concentrations ranging from 1% to 8% (w/v) were used to study the most effective concentration on improving MK-7 production. The concentration of glycerol was determined en- zymatically by using free glycerol determination kit (Sigma-Al- drich Co., St Louis, MO, USA) following its own procedure. Briefly, 800 mL of the glycerol free reagent was inoculated with 10 mL of fermented media and then incubated for 5 min at 37 °C. The UV- absorbance of resulting solution was measured at 540 nm using the absorbance of water as the reference. Table 1 Milk type composition. Type Fat (%) Protein (%) Carbohydrate (%) Standard 3.6 3.1 4.6 Lite 1.5 3.4 4.7 Trim 0.3 3.6 4.8 Full fat 4 3.1 4.6 Protein 0.5 5.8 5.2 Chocolate 1.1 4.3 6.6 R. Southee et al. / Biocatalysis and Agricultural Biotechnology 7 (2016) 31–3532
  • 3. 3. Result and discussion 3.1. Feasibility of milk media Different types of milk were investigated to determine their suitability as a fermentation media for Bacillus subtilis natto growth and MK-7 production. Fat, standards, lite, trim, protein, and chocolate milk due to their different fat, protein, and carbo- hydrate concentrations (Table 1) were used for Bacillus subtilis natto fermentation. The results imply the possible use of milk as the suitable substrate for developing a functional food rich in MK- 7. As shown in Fig. 1, the standard milk resulted in the highest MK- 7 concentration of 9.78 mg/L as compared to the other used milk media (po0.1). Based on the results, MK-7 concentration was higher in the media with higher fat contents. This behaviour could be due to the fact that Bacillus subtilis natto utilizes fat through a shikimate pathway for menaquinone biosynthesis (Sonenshein et al., 2002). However, the possible reason for lower MK-7 pro- duction in full cream milk as compared to the standard milk could be due to unhomogenized fat content which in turn reduces the microorganisms access to the fat. The standard milk was chosen for further optimization studies to enhance the MK-7 production. 3.2. Effect of nutrients The effects of the addition of different nutrients namely glycerol, mannitol, glucose, maltose, lactose, fructose, yeast extract, soy pep- tone, NaCl, sucrose, and KH2SO4 on enhancing the MK-7 production by using the standard milk was further investigated. The availability of various carbon, nitrogen and salt sources could improve the bacterial metabolism and MK-7 production (Berenjian et al., 2015). Therefore, it is critical to investigate the effect of additional nutrients on MK-7 production and establish an optimum fermentation med- ium which can significantly increase the MK-7 production. The Plackett-Burman design was used to screen and determine the key nutrients that can have a significant effect on MK-7 pro- duction. The levels of investigated variables are listed in Table 2. While the Placket–Burman design does not evaluate the interaction terms, it considerably reduces the number of experiments that are required to evaluate the main effects (Giordano et al., 2011). The analysis of variance of the model parameters (ANOVA) on MK-7 production are presented in Table 3. The regression coefficient was 0.957, which indicates that the model as fitted explains almost 96% of the variability around the mean and confirms the good model fit. Presence of NaCl and KH2SO4 as well as protein sources had a negligible impact on MK-7 production (p40.1). However, maltose, fructose, and lactose showed a significantly negative effect on the MK-7 production. Based on the results, glycerol provided the greatest yield of MK-7 production. The low P-value of glycerol (po0.1) reveals that it is the most significant factor on MK-7 bio- synthesis among all the studied factors. For Bacillus subtilis natto, glycerol clearly alleviates the pathway for the production of shi- kimate which could be based on conversion of glycerol into me- naquinone by condensation of phosphoenolpyruvate and ery- throse-4-phosphate (Sonenshein et al., 2002). In addition, it has been reported that glycerol decreases the molecular weight of biopolymers, such as ε-poly-Llysine and poly (hydroxyalkanoate). This reduction may result in decreasing the viscosity of the media, which can have a positive impact on stimulating the uptake of extracellular substrates and enhancing the mass transfer. The viscosity affects the diffusion of oxygen, consequently the growth of Bacillus subtilis natto and MK-7 production. Glycerol is a col- ourless and odourless liquid which is widely used in the food in- dustry and food formulations (Santa Cruz 2010). Glycerol is safe for Table 2 Plackett-Burman design with MK-7 yield. Run Glycerol Mannitol Glucose Maltose Lactose Fructose Yeast extract Soy peptone NaCl Sucrose KH2SO4 MK-7 1 8 2 8 2 2 2 5 8 0.05 2 0.07 10.1370.83 2 8 8 8 8 2 2 1 8 0.05 8 0.05 10.5975.15 3 2 8 8 2 8 2 1 2 0.05 8 0.07 10.0571.03 4 8 2 8 8 2 8 1 2 0.01 8 0.07 7.3575.01 5 8 8 2 8 8 2 5 2 0.01 2 0.07 8.1672.79 6 8 8 8 2 8 8 1 8 0.01 2 0.05 7.8171.35 7 2 8 8 8 2 8 5 2 0.05 2 0.05 3.2770.01 8 2 2 8 8 8 2 5 8 0.01 8 0.05 3.8474.11 9 2 2 2 8 8 8 1 8 0.05 2 0.07 1.4771.38 10 8 2 2 2 8 8 5 2 0.05 8 0.05 10.177.38 11 2 8 2 2 2 8 5 8 0.01 8 0.07 10.3870.84 12 2 2 2 2 2 2 1 2 0.01 2 0.05 10.2370.32 Fig. 1. Effect of different types of milk on MK-7 production. Table 3 Statistical analysis of variables for screening the nutrients. MK7 Coefficient Std. Error P-value Conf. int (7) Constant 8.30766 0.364589 0.000185115 1.16029 Glycerol 1.38042 0.407623 0.0428852 1.29725 Mannitol 0.281251 0.407623 0.539793 1.29725 Glucose À0.734583 0.407623 0.169321 1.29725 Maltose À2.31542 0.407623 0.010812 1.29725 Lactose À1.02375 0.407623 0.086822 1.29725 Fructose À1.19792 0.407623 0.0605696 1.29725 Yeast extract 0.00458299 0.407623 0.991735 1.29725 Soy peptone À0.440417 0.407623 0.359055 1.29725 NaCl À0.0404173 0.407623 0.92727 1.29725 Sucrose 0.789584 0.407623 0.14814 1.29725 KH2SO4 0.112917 0.407623 0.799761 1.29725 R2 ¼0.957, R2 (Adj)¼0.801; Significance code: Po0.1. R. Southee et al. / Biocatalysis and Agricultural Biotechnology 7 (2016) 31–35 33
  • 4. human consumption, and has a low cost as compared to the other nutrient sources, thus enabling the possible development of a low cost functional food. To further improve yield of MK-7, glycerol concentration was subjected to further concentration optimization. 3.3. Glycerol concentration The effect of adding various concentrations of glycerol (1–8% (v/v)) on MK-7 production was investigated. As depicted in Fig. 2, as compared with the control media (in absence of glycerol) the MK-7 production was enhanced in the presence of different gly- cerol concentrations. These data reveals the key role of glycerol in MK-7 biosynthesis via Bacillus subtilis natto metabolic pathway. Based on the results, the presence of 1% (w/v) glycerol significantly improved the MK-7 production by 18% as compared to the control media (po0.1). However, higher glycerol concentrations showed no improvement on enhancing the MK-7 biosynthesis. The concentration of glycerol was measured during the fer- mentation process. Glycerol concentration in the media was de- creased, regardless of the initial concentrations throughout the fermentation period. A further increase in the glycerol con- centration from 4% to 8% (w/v) resulted in higher glycerol con- sumption. However, MK-7 biosynthesis showed no significant change when using higher concentration of glycerol. This clearly shows that higher concentrations of glycerol was not utilized through a shikimate pathway (Bentley et al., 1990) to further en- hance the menaquinone biosynthesis. 3.4. Monitoring process parameters at the optimized condition Fermentation operational conditions have a key role in both growth and production of any wished metabolite. These factors are of critical importance for the design and scale-up of bioreactors. In order to gain a better understanding on how these factors affect MK-7 production, bacterial growth, glycerol concentration, dis- solved oxygen profile and pH at various stages of the fermentation were investigated for the optimized medium. MK-7 production was closely coupled with cell growth. As can be concluded from Fig. 3, MK-7 concentration increased approxi- mately 3-fold between cell's mid-exponential growth and the stationary phase, reaching its optimal concentration after 48 h (11.2270.6 mg/L). According to the results glycerol concentration in the medium stridently decreased to approximately 0.2% at the end of the fermentation; coinciding with an increase in biomass and MK-7 production. Our results show that the great bulk of MK- 7 was mainly produced at the stationary phase where the con- centration of glycerol as a carbon source was limited. pH of the media decreased from 6.65 to 5.32 after the cell growth rate reached its maximum level after 30 h. To date, number of Bacillus subtilis natto based products are used as food or nutritional supplements with various positive impacts on human health which shows this microorganism is not only non-pathogenic but also has probiotic effects (Barbosa et al., 2005; Tsukamoto et al., 2001). The newly developed fermented milk rich in MK-7 can significantly provoke immune system, bone and cardiovascular health. The changing lifestyles and growing health awareness are the key factors that affect the market for functional food ingredients. Given the position of heart and bone health as a leading health issues worldwide (Brandenburg et al., 2015; Berenjian et al., 2015), product developers have the oppor- tunity to use the outcomes of the present study to further develop a product to prevent bone and cardiovascular diseases. 4. Conclusions From the results it is evident that milk is a promising fermentation media for MK-7 production by using Bacillus subtilis natto. 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