Development of a menaquinone-7 enriched functional food

Food and Bioproducts Processing 1 1 7 ( 2 0 1 9 ) 258–265

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Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp

Development of a menaquinone-7 enriched functional food Yanwei Ma a , Pui Ting Prudence Tang a , Dale D. McClure a , Peter Valtchev a , John F. Ashton b , Fariba Dehghani a , John M. Kavanagh a,∗ a b

The University of Sydney, School of Chemical and Biomolecular Engineering, NSW 2006, Australia Sanitarium Development and Innovation, Cooranbong, Australia

a r t i c l e

i n f o

a b s t r a c t

Article history:

There is increasing interest in the development of fortified foods enriched in menaquinone-7

Received 27 March 2019

as high dietary intakes may reduce the incidence of osteoporosis and cardiovascular calci-

Received in revised form 25 June

fication. In this work we explore the potential of using food ingredients as fermentation

2019

substrates for the development of such products. It was found that a combination of soy

Accepted 26 June 2019

protein and glycerol was the most suitable for MK-7 production. The process was scaled-up;

Available online 1 August 2019

use of a dual feeding strategy was found to avoid foaming and improve MK-7 production.

Keywords:

highest values reported in the open literature. Finally, the resulting product was formu-

The MK-7 titre and productivity were 99 mg L−1 and 2.1 mg L−1 h−1 respectively, amongst the Menaquinone-7

lated into a food (soymilk); it was found that 75% of the MK-7 remained after 24 weeks of

Vitamin K

room temperature storage. These results clearly demonstrate an approach to utilise food

Bacillus subtilis

ingredients for the production of MK-7 enriched functional foods.

Fed-batch culture

© 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Functional foods Natto

1.

Introduction

Vitamin K refers to a group of fat soluble vitamers that share the same 2-methyl-1,4-napthoquinone head structure but differ in the lengths and structures of their isoprenoid tails (Bentley and Meganathan, 1982). Two major vitamers exist; K1 (phylloquinone) which is found in leafy green vegetables and K2 (the menaquinones) which are found in fermented foods and some animal products (Kamao et al., 2007). The chain length of the menaquinone isoprenoid tail varies depending on the bacterial species (Collins and Jones, 1981) and is denoted as MK-n, where n is the number of isoprenoid units. Both phylloquinone (Vitamin K1 ) and the menaquinones (Vitamin

cardiovascular calcification (Knapen et al., 2015, 2013; Shearer et al., 2012). Adequate intakes for Vitamin K are of the order 60–70 ␮g per day (Australian Government National Health and Medical Research Council, 2014; EFSA Panel on Dietetic Products et al., 2017). The most well-known food rich in menaquinones, is natto, a Japanese fermented soy product. Its high content of MK-7 (8–9 ␮g g−1 ) (Kamao et al., 2007) is due to fermentation by Bacillus subtilis natto. Regular dietary intake of natto is thought to be associated with reduced bone loss in postmenopausal women in some parts of Japan (Kaneki et al., 2001; Ikeda et al., 2006). However, due to its unique smell and stringy texture, natto has not been as widely accepted in other parts of the world as a dietary

K2 ) are capable of acting as co-factors for the enzyme ␥-glutamate carboxylase. This enzyme carboxylates Vitamin K dependent proteins which are responsible for a range of functions. The most widely known

source of Vitamin K. Hence, it is desirable to develop a palatable func-

of these is blood coagulation (Dam, 1935); recent studies have found that it plays an important role in the prevention of osteoporosis and

other food matrices. B. subtilis is a Generally Recognised as Safe (GRAS)

tional food enriched in MK-7 from fermentation of food substrates by B. subtilis natto, which can either be consumed directly or fortified into microorganism, which makes it particularly attractive from a food pro-

∗ Corresponding author at: School of Chemical and Biomolecular Engineering, The University of Sydney, Building J01, New South Wales, 2006, Australia. E-mail address: [email protected] (J.M. Kavanagh). https://doi.org/10.1016/j.fbp.2019.06.017 0960-3085/© 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Food and Bioproducts Processing 1 1 7 ( 2 0 1 9 ) 258–265

duction perspective. Such a product may be beneficial in reducing the incidence of cardiovascular disease and osteoporosis, which are diseases of global significance particularly given the aging population. Various efforts have been made to produce MK-7 from a range of substrates, including soy beans and soy bean extract (Sato et al., 2001a, 2001b; Wu and Ahn, 2011a, 2011b), soy peptone and yeast extract (Berenjian et al., 2011; Benedetti, 2007; Song et al., 2014), soy and corn mixtures (Mahanama et al., 2011), soy pulp (Survase et al., 2006) and wheat (Sumi et al., 2005; Sumi, 2001). It has been shown that it is possible to produce high concentrations (up to 226 mg L−1 (Berenjian et al., 2014)) of MK-7 using submerged fermentation techniques and biotechnological media containing soy peptone and yeast extract. From the perspective of producing a functional food it may be desirable to replace such ingredients with food grade substrates which have a lower cost and can be readily formulated into existing products. Additionally, such an approach has the advantage of simplifying any downstream processing as rather than producing pure MK-7 the fermentation medium can be used directly as a food ingredient after appropriate pre-treatment. Hence the aims of this study are threefold; firstly, to screen a range of different food products to identify those most suitable for the production of MK-7; secondly to scale the process up using a bench-top scale bioreactor and thirdly to measure the shelf life of the resulting product to assess its suitability as a functional food ingredient.

2.

Materials and methods

2.1.

Materials

Food ingredients including soy protein isolate, milk protein isolate, whole almond paste, whole peanut butter, wholegrain wheat, wholegrain rice, wholegrain sorghum, corn syrup solids, sucrose, inulin, glycerol, fructose, and glucose were kindly supplied by Sanitarium Health and Wellbeing Company. Ingredients were chosen on the basis that they are widely available and currently found in foods. Here we have chosen to use a range of sources including dairy, wholegrains, legumes and nuts in order to find the most suitable in terms of both producing a high MK-7 concentration as well as for formulation into foods. Food grade antifoam A (A6582) and yeast extract were purchased from Sigma Aldrich (Castle Hill, NSW, Australia). HPLC standards menaquinone-7 (sc-218691) and menaquinone-9 (sc-211788) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HPLC grade methanol, dichloromethane, isopropanol and hexane were purchased from Merck (Bayswater, VIC, Australia).

2.2.

Isolation of microorganisms

The MK-7 producing strains were isolated from commercially available natto. Samples (typically one fermented soybean) were taken and streaked out on tryptic soy agar plates (tryptone 17 g L−1 , soytone 3 g L−1 , NaCl 5 g L−1 , K2 HPO4 2.5 g L−1 , agar 15 g L−1 ) and grown at 30 ◦ C for 24 h. Single colonies from these plates were isolated and grown in 5 mL tryptic soy broth at 37 ◦ C overnight. Glycerol stocks were prepared by adding sterile glycerol to overnight cultures to make up a final concentration of 10% (v/v) glycerol and stored at −80 ◦ C. The strain types were confirmed by performing 16 s rRNA sequence analysis. Genomic DNA was extracted following a FastPrep purification protocol (Yeates and Gillings, 1998) and was used as the template for the PCR amplification of corresponding 16S rRNA fragments. The 16S rRNA fragments were amplified using universal primer pairs 27 F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 519R (5 -

259

GWATTACCGCGGCKGCTG-3 ). Here, W can be A or T; K is G or T; and M is A or C. The amplified fragments were sequenced using Sanger sequencing service provided by Australian Genome Research Facility NSW Node, and analysed using Basic Local Alignment Search Tool (BLAST) at the NCBI public database.

2.3.

Culture conditions

The culture media for screening high MK-7 producing strains contained 18.9% (w/v) soy peptone, 5% (w/v) yeast extract, 5% (w/v) glycerol and 0.6% (w/v) K2 HPO4 (Berenjian et al., 2011). Screening of B. subtilis natto strains was carried out in 5 mL culture in 25 mL amber vials at 40 ◦ C for 144 h without shaking. Spore solutions were prepared by scraping bacterial growth off a streak plate, resuspending in sterile phosphate buffered saline (0.01 M phosphate buffer, 2.7 mM potassium chloride and 0.137 M sodium chloride, pH 7.4) and incubating at 80 ◦ C for 30 min. These solutions were stored at 4 ◦ C before being used. The culture media for screening nitrogen and carbon source contained 10% (w/v) nitrogen sources and 5% (w/v) carbon sources with supplementation of 0.6% K2 HPO4 . Selection of nitrogen and carbon source was carried out in 10 mL culture in 25 mL amber vials at 40 ◦ C for 144 h without shaking using a previously developed method (Berenjian et al., 2011). These vials were inoculated with 2% (v/v) of the spore solution (4 × 108 CFU mL−1 ). Optimisation experiments were performed using glycerol concentrations between 2–15 % (w/v) and soy protein concentrations between 3.7–13.7 % (w/v) while the concentrations of K2 HPO4 and yeast extract were fixed at 0.6% (w/v) and 0.5% (w/v) respectively. These experiments were performed using 20 mL of medium in 100 mL Erlenmeyer flasks. These were incubated at 40 ◦ C for 144 h with shaking (120 rpm). B. subtilis natto spore solution (4 × 108 CFU mL−1 ) was used to inoculate the flask media at 2% (v/v). All screening and optimisation experiments were performed in triplicate; reported results are the mean ± one standard deviation. bench scale bioreactor (New BrunswickTM A BioFlo® /CelliGen® 115) with a working volume of 2 L was used to carry out the fed-batch experiments with an initial medium volume of 800 mL. The fermenter was equipped with two, six bladed Rushton impellers (55 mm in diameter) and four baffles. The inoculation size was 16 mL (2% (v/v)) of an overnight culture of seed media of the same composition as the initial culture. The incubation temperature was controlled at 40 ◦ C and the dissolved oxygen (DO) level was maintained at 20–50% (of saturation) by using a pre-optimised aeration rate of 5 L min−1 and a stirrer speed of 1000 rpm, corresponding to an impeller tip speed of 2.9 m s−1 . Dissolved oxygen and pH values were measured using an InPro 6830 dissolved oxygen sensor (Mettler Toledo) and 405-DPAS-SC-K8S/325 pH probe (Mettler Toledo); the data was logged using the in-built data logging system at a frequency of once per minute. The feeding speeds of glycerol and soy protein solution for the fed-batch cultures were controlled via speed-adjustable pumps installed in the bioreactor. The feed solutions consisted of 50% or 100% (w/w) glycerol and 13.7% (w/v) soy protein. Additions of soy protein were made in pulses; the pump was turned on for 10 min, then off for 50, with these being repeated over the course of the batch. A similar strategy was used for glycerol addition; 4.4 g of glycerol solution (50% (w/w)) was added in

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Food and Bioproducts Processing 1 1 7 ( 2 0 1 9 ) 258–265

10 min pulses with 50 and 20 min gaps between additions for average feed rates of 1.3 g L−1 h−1 and 2.4 g L−1 h−1 respectively. To achieve an average feeding rate of 3.0 g L−1 h−1 pure glycerol was used as the feed; 2 g was added over a period of 5 min; with 25 min gaps between pulses. Average feed rates were calculated on the basis of the final culture volume. A built-in foam sensor was used to prevent foam formation by adding food grade antifoam A to the culture as demanded.

with a 35 ◦ C cell temperature, an excitation wavelength of 243 nm and an emission wavelength of 430 nm. The mobile phase contained methanol:dichloromethane (9:1, v/v), ZnCl2 1.37 g L−1 , sodium acetate 0.41 g L−1 and glacial acetic acid 0.30 g L−1 and was run at a flowrate of 0.3 mL min−1 and an oven temperature of 40 ◦ C. The calibration curve was determined between five standard concentrations between 156 ␮g L−1 and 2500 ␮g L−1 (linear regression, R2 = 0.9999).

2.4.

2.6.

Determination of viable cell counts

Viable cell counts were determined by performing standard serial dilutions of the homogenised fermented samples with sterile phosphate buffered solution. Serial dilutions were performed to a final count of 30–300 colonies per plate (typical samples were diluted by 4–8 orders of magnitude). The number of colonies were counted after 24 h incubation at 30 ◦ C and expressed as colony forming units (CFU) per mL sample.

2.5. Measurement of glycerol and menaquinone-7 concentrations The concentration of glycerol was assayed with a free glycerol determination kit from Sigma-Aldrich (Castle Hill, NSW, Australia). To determine the amount of glycerol in the culture media, 10 ␮L of water, glycerol standard (0.26 g L−1 , G7793, Sigma Aldrich) and sample were added to 800 ␮L free glycerol reagent (F6428, Sigma Aldrich) and incubated at 37 ◦ C for 5 min. The UV absorbance of water, standard and sample at 540 nm were recorded as Ablank , Astandard and Asample . The glycerol concentration was calculated as: glycerol concentration =

Asample − Ablank Astandard − Ablank

× standard concentration (1)

MK-7 was extracted from the fermentation medium using 2-propanol and n-hexane which were added in a 1:2:5 volume ratio (culture medium: 2-propanol: n-hexane). Menaquinone9 (in isopropanol) was added as an internal standard to a final concentration of 50 mg L−1 before extraction. The mixture was vigorously vortexed after addition of each solvent and then centrifuged to facilitate phase separation. The organic phase was then separated and evaporated under vacuum to recover the extracted MK-7. This was then redissolved in the mobile phase before being syringe filtered (0.22 ␮m) into amber HPLC vials for analysis. An Agilent HPLC HP1050 (Hewlett-Packard, USA) equipped with a single channel UV detector and a Gemini C18 110A column (150 mm × 4.60 mm × 5 ␮m, Phenomenex, USA) was used for the analysis of MK-7 concentration. Methanol:dichloromethane (9:1, v/v) was used as the mobile phase with a flow rate of 1 mL min−1 ; the column temperature was 40 ◦ C. A single channel UV wavelength of 248 nm was used for quantitation. The calibration curve was determined by performing linear regression to 5 standard points between 5 mg L−1 to 100 mg L−1 (linear regression, R2 = 0.9999). The MK-7 titres were also confirmed by analysis using a separate HPLC system (Shimadzu Prominence-i LC-2030) using a C18 column (150 mm × 2.0 mm × 3 ␮m particle size, 12 nm pore size, YMC-Pack ODS-AM), a reducing column (4.6 × 30 mm) which was packed with zinc dust (<63 ␮m, EMPLURA, Merck). A fluorescence detector (Shimadzu RF-20Axs) was used

Shelf life study

To assess the shelf life of the MK-7 rich product it was formulated into two food matrices (regular soymilk containing sunflower and canola oil as well as fat free soymilk) at a concentration of 177 ␮g L−1 . Samples were UHT treated and aliquots were stored under sterile conditions in the dark at room temperature (25 ◦ C) or in a 35 ◦ C incubator. The samples were stored in 15 mL Falcon tubes with screw tops, the headspace contained air. Samples were analysed at the initial time point as well as after 6, 12 and 24 weeks of storage. MK-7 concentrations were measured using the method previously described; the MK-7 concentration at day 0 was expressed as 100% and the MK-7 concentration after storage was expressed as a percentage of the original concentration at day 0. Experiments were performed in triplicate; reported results are the average concentration measurement of three aliquots while error bars denote one standard deviation about the mean.

3.

Results and discussion

3.1. Screening of high MK-7 productivity B. subtilis natto strains In order to screen for a high producer of MK-7 for future optimisation, pure B. subtilis natto colonies were isolated from 18 different commercial natto products. The model lab strain B. subtilis 168 was also included for comparison. It was found that the majority of the isolates produced similar concentrations of MK-7; values ranged between 25–47 mg L−1 . In comparison the lab strain B. subtilis 168 produced 15 mg L−1 of MK-7. Isolate B11 produced the highest amount of MK-7 (47 mg L−1 ) with the least variation within the three replicates, so it was chosen for future media optimisation and scale-up. All of the strains isolated were confirmed to be B. subtilis by 16 s rRNA sequence analysis.

3.2.

Medium development

The effect of food nutrients on MK-7 production was examined in order to determine the most suitable substrates for fermentation. A diverse group of carbon sources including simple sugars, an oligosaccharide (inulin) and corn syrup were screened, as shown in Fig. 1. They were paired to investigate the best combination between the carbon and nitrogen sources. It was found that glycerol was the best carbon source compared to the others examined. This finding was in agreement with the studies conducted by Sato et al. (2001a) and Berenjian et al. (2011). It is interesting that significantly (p = 0.0003 using an unpaired t-test) more MK-7 (23 mg L−1 ) was produced by B. subtilis natto when inulin (a fructose polymer) was used with soy protein, compared to fructose itself (9 mg L−1 ). Qian et al. (2015) had a similar finding when they compared inulin and fructose as a sole carbon sources for bacillomycin D biosynthesis in

Food and Bioproducts Processing 1 1 7 ( 2 0 1 9 ) 258–265

261

Fig. 1 – MK-7 production utilising food nutrients as carbon and nitrogen sources. Reported values are mean values ± standard deviations are shown (n = 3).

Fig. 2 – MK-7 titres obtained using wholegrains as the protein source. Reported values are mean values ± standard deviations are shown (n = 3).

B. subtilis. To utilise inulin as a carbon source the microorganism needs to produce an enzyme (inulinase/levanase) to break down inulin into simple sugars (Vijayaraghavan et al., 2009). A possible explanation is that the production of these enzymes induced the oxidative phosphorylation activities within the microorganism, where MK-7 is essential as the electron carrier in the prokaryotic electron transport chain in the cytoplasmic membrane (Bentley and Meganathan, 1982). Qian et al. (2015) also observed an upregulated expression level of the Spo0A gene, which facilitated the sporulation in B. subtilis, when inulin was used as a carbon source rather than fructose. This could be another possible reason for the high MK-7 titre as production has been linked to Bacillus sporulation (Farrand and Taber, 1974; Wagner et al., 1999; Pelchovich et al., 2013). In contrast, simple sugars such as glucose, sucrose, fructose and corn syrup solids (which largely contain glucose and fructose) did not give high titres of MK-7 across the range of nitrogen sources tested (all below 12 mg L−1 as seen in Fig. 1). In terms of the food proteins studied, soy protein was found to be the nitrogen source that yielded the highest titre when coupled with glycerol. Considering the fact that B. subtilis natto has been used to ferment soybeans industrially for centuries, it is possible that these strains will also produce high MK-7 titres when grown using other legume proteins as the substrate. In light of this hypothesis, yellow pea protein was also tested as a nitrogen source in addition to produce MK7 with glycerol. It was found that yellow pea protein isolate yielded 25 mg L−1 MK-7, a comparable amount to soy protein at 27 mg L−1 . Hence, pea protein can serve as an alternative nitrogen source for MK-7 production in cases where it may not be desirable to use soy (e.g. to avoid soy allergens). Milk protein isolate produced less than 5 mg L−1 MK-7 across the range of carbon sources studied. This could be potentially because the amino acid profile of milk protein powder is rich in aromatic tyrosine (311 mg g−1 total nitrogen) compared to soybean protein and pea protein (196 mg g−1 total nitrogen and 170 mg g−1 total nitrogen respectively) (FAO, 1970). The naphthoquinone ring structure of MK-7 is synthesised via the shikimate pathway, which is also used to synthesise the precursors for aromatic amino acids (Bentley and Meganathan, 1982). The shikimate pathway is regulated

by a feedback inhibition mechanism; the presence of aromatic amino acids (i.e. phenylalanine, tyrosine and tryptophan) represses this pathway (Tsukamoto et al., 2001; Xu and Zhang, 2017). It is likely that the high content of tyrosine in milk protein inhibited the carbon flux through the shikimate pathway. Experiments were also performed using wholegrain wheat, rice and sorghum (both red and white) as the protein sources. These did not favour MK-7 production, with the maximum titre being 8 mg L−1 for wholegrain wheat (Fig. 2). As a result of this nitrogen and carbon source study, soy protein and glycerol were chosen for further media optimisation in shake flasks. Various initial concentrations of glycerol from 20 g L−1 to 150 g L−1 were tested to investigate their effects on cell growth and MK-7 titres. It was found that using 50 g L−1 glycerol resulted in the highest MK-7 concentration (30 mg L−1 ), as well as the highest cell density (1.5 × 109 CFU mL−1 ). At the end of the incubation period, the glycerol concentration in the media was less than 0.5 g L−1 , indicating that most glycerol was consumed. In contrast, it was found that 20 g L−1 glycerol did not give as high MK-7 titre and cell density in the 6-day fermentation period, while 80 g L−1 glycerol produced slightly less MK-7 and had large amount of residual glycerol. As the initial glycerol concentration increased to 100 g L−1 and 150 g L−1 , it further supressed cell growth, giving lower viable cell counts of 2.7 × 108 and 3.3 × 108 CFU mL−1 ; 10 times less than a glycerol concentration of 50 g L−1 . It was thought the high concentrations of glycerol inhibited the growth due to osmotic stress. As shown in Fig. 3 the final pH was related to the residual glycerol concentration; when the glycerol was consumed the soy protein was used as a carbon source which increased the medium pH. Similar experiments were performed with a glycerol concentration and soy protein concentrations of 37, 74, 100 and 137 g L−1 (Fig. 3). It was found that all the glycerol was consumed with the final pH being approximately 7. Comparisons made using an unpaired t-test showed that reducing the soy protein concentration below 100 g L−1 significantly reduced the MK-7 titre to 22 mg L−1 (p = 0.02) and 24 mg L−1 (p = 0.04) at soy protein concentrations of 37 and 74 g L−1 respectively. On this basis it was decided to use a fed-batch strategy for glyc-

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Fig. 3 – Effect of different initial glycerol and soy protein concentrations on MK-7 production and viable cell counts (A) and (C) as well as residual glycerol concentrations and the final pH (B) and (D). Where the glycerol concentration was varied (A) and (B) the soy protein concentration was fixed at 10% (w/v); while the glycerol concentration was fixed at 5% (w/v) for experiments where the soy protein concentration was varied. Mean values ± standard deviations are shown (n = 3). erol addition and to use medium with a high (100 g L−1 ) protein concentration for further scale-up.

3.3.

Scale-up

Based on the medium screening results experiments were performed using a 2 L bench-top bioreactor with the aim of scaling-up the process and further improving the MK-7 titre. In these experiments relatively high levels of aeration (5 L min−1 ) and stirring (1000 rpm, impeller tip speed of 2.9 m s−1 ) were used to maintain a relatively high DO level (50% of saturation). This was done as high dissolved oxygen levels have been shown to lead to high MK-7 titres (Berenjian et al., 2014). It was found in the previous media optimisation experiments that a high soy protein concentration (up to 100 g L−1 ) was required for maximum MK-7 production. However, excessive foaming became a difficult issue when a high initial protein concentration was used in the bioreactor with the high aeration and agitation rates needed to achieve high oxygen transfer rates. To overcome this issue, two options were available: one option was to increase the amount of antifoam added and the second was to reformulate the medium. Excessive addition of silicone based antifoam reduced the MK-7 titre (less than 50 mg L−1 , results not shown) most likely due to a decrease in the oxygen transfer rate. Addition of vegetable oil based antifoam was shown to be useful for providing an extra carbon source for the fermentation (Berenjian et al., 2014). However, as vegetable oil was not an effective antifoaming agent, a large amount of oil addition (at least 15% v/v) was

required to successfully overcome the foaming issue. Addition of oil also resulted in the fermentation broth separating into two phases which is undesirable from a formulation perspective. It was observed that high degree of foaming usually occurred at the beginning of the fermentation cycle when the protein concentration in the broth was high. It was therefore proposed that reducing the initial soy protein concentration would reduce the severity of foaming. A dual feeding strategy was designed to overcome the foaming issue while meeting the substrate demand for achieving high MK-7 titre. In this new strategy, the batch was started with 20 g L−1 of soy protein and 20 g L−1 of glycerol in an initial working volume of 800 mL medium. As the culture entered the exponential growth phase, the dissolved oxygen concentration started to decrease (Fig. 4). As the glycerol was exhausted, the pH of the medium increased when the culture started to metabolise soy protein as carbon source. The feeding of glycerol and soy protein was triggered when the pH reached 7 again which usually occurred around 5–6 h after inoculation. It was found that the dual feeding strategy implemented was sufficient to maintain the pH at a suitable level without the need to control the pH using acid or base solutions. Glycerol was pulse fed into the fermenter at three different average rates, 1.3 g L−1 h−1 , 2.4 g L−1 h−1 and 3.0 g−1 L−1 h−1 , over the entire fermentation period (detailed information about the addition strategy is given in Section 2.3), while the pulse feeding rate of soy protein was maintained at an average rate of 1.5 g L−1 h−1 . It can be seen in Fig. 4 that

Food and Bioproducts Processing 1 1 7 ( 2 0 1 9 ) 258–265

263

Fig. 5 – Results for the shelf life study. Reported values are the average of three measurements while error bars are one standard deviation about the mean.

Fig. 4 – Fed-batch fermentation in 2 L bioreactor using glycerol feeding rates (A) 1.3 g L−1 h−1 (B) 2.4 g L−1 h−1 (C) : MK-7 titre; : viable cell counts; : 3.0 g L−1 h−1 . residual glycerol; dotted line: pH; solid line: dissolved oxygen.

different feeding rates of glycerol resulted in minimal differences in the final cell densities in three fed-batch runs. In all runs, the viable cell counts reached a magnitude of 1010 CFU mL−1 , which was approximately 10 times greater than the highest cell density achieved in the shake flask scale. During all three runs, the cell densities reached the maximum after around 12 h of fermentation. After 12 h, the cell densities remained almost constant as the culture growth entered the stationary phase. During the first 12 h, only a small amount of MK-7 was produced. More MK-7 was produced after the cells entered the stationary phase. This showed the same trend as other secondary metabolites produced by B. subtilis reported in the literature such as the lipopeptides iturin A and surfactin (Jin et al., 2015; Marahier et al., 1993). The production of secondary metabolites usually accompanies the microorganism’s responses to a nutrient limited environment, including motility, competence for genetic transformation and sporulation (Smith, 1993). It has been reported that MK-7 pro-

duction is associated with sporulation of B. subtilis, therefore an increase in MK-7 has been observed during the sporulation stage (Farrand and Taber, 1974). The same trend was observed in the last two runs (Fig. 4B and C), where MK-7 concentration more than doubled from 24 h to 48 h; it was hypothesised that this is related to sporulation. The MK-7 titre increased from 20 mg L−1 at 24 h to 58 mg L−1 at 48 h when glycerol was fed at 2.4 g L−1 h−1 . Similarly, the MK-7 titre increased from 34 mg L−1 at 24 h to 99 mg L−1 at 48 h when glycerol was fed at a rate of 3.0 g L−1 h−1 . The residual glycerol concentration decreased rapidly in the first 5–6 h of fermentation before more glycerol was fed into the fermenter. While glycerol was fed at different rates in these three runs, the glycerol concentration was maintained at around 2 g L−1 for all runs. Similarly, dissolved oxygen concentrations dropped swiftly as the carbon source was consumed in the first 5–6 h of the fermentation cycle. As the glycerol was fed in by pulses, it can be clearly seen that the dissolved oxygen level served as a good indication of the residual glycerol level in the media. The DO rose as the glycerol was exhausted, and decreased immediately as another pulse of glycerol was fed into the fermenter. By comparing different feeding rates of glycerol in three fed-batches, it was found that feeding glycerol at a rate of 3.0 g L−1 h−1 could give higher MK-7 concentrations (99 mg L−1 ), this was ten times more than feeding glycerol at 1.3 g L−1 h−1 . In comparison to other MK-7 production processes in the literature, this study is the first to examine the potential use of a range of food ingredients as fermentation substrates to produce MK-7. It also has the highest productivity of all reported processes (Table 1) achieving 2.1 mg L−1 h−1 . Therefore, this dual feeding strategy of carbon and nitrogen source is a promising method for the utilisation of food based substrates for industrial production of MK-7 enriched functional foods.

3.4.

Shelf life study

Recent studies have revealed that the instability of MK-7 in storage could lead to large discrepancy between the nominal

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Table 1 – Comparison of MK-7 titre and productivity in published submerged fermentation processes. Microorganism

Major Substrates (>10 g L−1 )

MK-7 Titre (mg L−1 )

MK-7 Productivity (mg L−1 h−1 )

Reference

B. subtilis DSM17766 B. subtilis D200-41

Soy peptone, yeast extract, dextrin Soybean extract, yeast extract, glycerol Peptone, yeast extract, glycerol, sucrose Soy peptone, yeast extract, glycerol Soy peptone, glycerol Soy peptone, glycerol Soymeal extract, maize meal hydrolysate Tryptone, maltose, glycerol Soybean flour, yeast extract, bovine albumin, glucose Soytone, yeast extract, glycerol Soy protein, glycerol

68 60

0.49 0.42

Benedetti (2007) Sato et al. (2001b)

3.6

0.05

Song et al. (2014)

226 36 41 61

1.88 0.60 0.57 0.42

Berenjian et al. (2014) Luo et al. (2016) Hu et al. (2017) Xu and Zhang (2017)

72 29

0.33 0.10

Wu (2018) Mahdinia (2018)

15 99

0.10 2.1

Mahdinia et al. (2019) This study

B. subtilis natto B. subtilis natto B. subtilis natto B. subtilis natto B. amyloliquefaciens H.␤.D.R.-5 B. subtilis KCTC 12392BP B. subtilis natto B. subtilis natto B. subtilis natto

and actual content of MK-7 in formulations (Szterk et al., 2018; Orlando et al., 2019). Therefore, a shelf-life study of MK-7 in fortified soymilk was performed to demonstrate the stability of MK-7 in food formulation. Results from the shelf life study are presented in Fig. 5. It was found that at room temperature the MK-7 concentration was approximately 75% of the original concentration after 24 weeks. Unsurprisingly, higher temperatures led to an increased rate of degradation, after 24 weeks at 35 ◦ C the final concentration was 50–80% of the original. These results demonstrate that the product is relatively stable in a food matrix and hence can be readily applied to fortified foods in order to increase their MK-7 concentration.

4.

Conclusions

This study demonstrated the feasibility of utilising food based nutrients for development of a vitamin K2 enriched food ingredient. Soy protein and glycerol were identified as the optimal carbon and nitrogen source for MK-7 production. It was also shown that pulse feeding the nitrogen and carbon source concurrently was a useful strategy to scale up the process, particularly to avoid any issues with foam formation. MK7 concentrations were improved by 3.3-fold, from 30 mg L−1 in shake flask scale to 99 mg L−1 in a 2 L fed-batch bioreactor. One millilitre of the broth provides approximately 1.4 times the daily recommended intake of Vitamin K. The process developed in this work uses a GRAS microorganism and food grade ingredients, meaning that the resulting MK-7 rich broth can be readily formulated into food products. To demonstrate this, the broth was formulated into soy milk and the effect of storage time and temperature on the MK-7 concentration was quantified. It was found that after 24 weeks at room temperature approximately 75% of the MK-7 was remaining; this decreased to 50–80% at 35 ◦ C. The results from this work demonstrate a novel, high-productivity process for MK-7 fermentation using safe, readily available ingredients which can be used to produce a MK-7 rich ingredient for formulation into functional foods.

Acknowledgements This work was partially funded by Sanitarium Health and Wellbeing as part of the ARC Training Centre for the Australian Food Processing Industry in the 21st Century (IC140100026).

This process has been patented by the authors (provisional patent #2018903171).

References Australian Government National Health and Medical Research Council, Available: https://www.nrv.gov.au/nutrients/vitamin-k. (Accessed 5 June 2018) 2014. Nutrient Reference Values for Australia and New Zealand Including Recommended Dietary Intakes — Vitamin K. Benedetti, A., EP1803820A1 2007. Process for the Preparation of Vitamin K2. Bentley, R., Meganathan, R., 1982. Biosynthesis of vitamin K (menaquinone) in bacteria. Microbiol. Rev. 46, 241–280. Berenjian, A., Mahanama, R., Talbot, A., Biffin, R., Regtop, H., Valtchev, P., Kavanagh, J., Dehghani, F., 2011. Efficient media for high menaquinone-7 production: response surface methodology approach. N. Biotechnol. 28, 665–672. Berenjian, A., Mahanama, R., Talbot, A., Regtop, H., Kavanagh, J.M., Dehghani, F., 2014. Designing of an intensification process for biosynthesis and recovery of Menaquinone-7. Appl. Biochem. Biotechnol. 172, 1347–1357. Collins, M.D., Jones, D., 1981. Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implication. Microbiol. Rev. 45, 316. Dam, H., 1935. Cliv. The antihaemorrhagic vitamin of the chick. Nutr. Rev. 31, 121. EFSA Panel on Dietetic Products, N., Allergies, Turck, D., Bresson, J.-L., Burlingame, B., Dean, T., Fairweather-Tait, S., Heinonen, M., Hirsch-ernst, K.I., Mangelsdorf, I., Mcardle, H.J., Naska, A., Nowicka, G., Pentieva, K., Sanz, Y., Siani, A., Sjödin, A., Stern, M., Tomé, D., Van Loveren, H., Vinceti, M., Willatts, P., Lamberg-Allardt, C., Przyrembel, H., Tetens, I., Dumas, C., Fabiani, L., Ioannidou, S., Neuhäuser-Berthold, M., 2017. Dietary reference values for vitamin K. EFSA J. 15. FAO, 1970. Amino-acid Content of Foods and Biological Data on Proteins. FAO, Rome. Farrand, S.K., Taber, H.W., 1974. Changes in menaquinone concentration during growth and early sporulation in Bacillus subtilis. J. Bacteriol. 117, 324–326. Hu, X.-C., et al., 2017. Enhancing menaquinone-7 production by bacillus natto R127 through the nutritional factors and surfactant. Appl. Biochem. Biotechnol. 182 (4), 1630–1641. Ikeda, Y., Iki, M., Morita, A., Kajita, E., et al., 2006. Intake of fermented soybeans, natto, is associated with reduced bone loss in postmenopausal women: Japanese Population-Based Osteoporosis (JPOS) Study1. J. Nutr. 136, 1323–1328. Jin, H., Li, K., Niu, Y., Guo, M., Hu, C., Chen, S., Huang, F., 2015. Continuous enhancement of iturin A production by Bacillus

Food and Bioproducts Processing 1 1 7 ( 2 0 1 9 ) 258–265

subtilis with a stepwise two-stage glucose feeding strategy. BMC Biotechnol. 15, 53. Kamao, M., Suhara, Y., Tsugawa, N., Uwano, M., Yamaguchi, N., Uenishi, K., Ishida, H., Sasaki, S., Okano, T., 2007. Vitamin K content of foods and dietary vitamin K intake in japanese young women. J. Nutr. Sci. Vitaminol. 53, 464–470. Kaneki, M., Hedges, S.J., Hosoi, T., Fujiwara, S., Lyons, A., Crean, S.J., Ishida, N., Nakagawa, M., Takechi, M., Sano, Y., Mizuno, Y., Hoshino, S., Miyao, M., Inoue, S., Horiki, K., Shiraki, M., Ouchi, Y., Orimo, H., 2001. Japanese fermented soybean food as the major determinant of the large geographic difference in circulating levels of vitamin K2: possible implications for hip-fracture risk. Nutrition 17, 315–321. Knapen, M.H.J., Drummen, N.E., Smit, E., Vermeer, C., Theuwissen, E., 2013. Three-year low-dose menaquinone-7 supplementation helps decrease bone loss in healthy postmenopausal women. Osteoporos. Int. 24, 2499–2507. Knapen, M.H.J., Braam, L.A.J.L.M., Drummen, N.E., Bekers, O., Hoeks, A.P.G., Vermeer, C., 2015. Menaquinone-7 supplementation improves arterial stiffness in healthy postmenopausal women. A double-blind randomised clinical trial. Thromb. Haemost. 113, 1135–1144. Luo, M., Ren, L., Chen, S., et al., 2016. Biotechnol Bioproc E 21, 777 https://doi.org/10.1007/s12257-016-0202-9. Mahanama, R., Berenjian, A., Valtchev, P., Talbot, A., Biffin, R., Regtop, H., Dehghani, F., Kavanagh, J.M., 2011. Enhanced production of menaquinone 7 via solid substrate fermentation from Bacillus subtilis. Int. J. Food Eng. 7. Mahdinia, E., 2018. Implementation of fed-batch strategies for vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors. Appl. Microbiol. Biotechnol. 102, 9147–9157. Mahdinia, E., Demirci, A., Berenjian, A., 2019. Effects of medium components in a glycerol-based medium on vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors. Bioprocess Biosyst. Eng. 42, 223–232. Marahier, M.A., Nakano, M.M., Zuber, P., 1993. Regulation of peptide antibiotic production in Bacillus. Mol. Microbiol. 7, 631–636. Orlando, P., Silvestri, S., Marcheggiani, F., Cirilli, I., Tiano, L., 2019. Menaquinone 7 stability of formulations and its relationship with purity profile. Molecules (Basel, Switzerland) 24, 829. Pelchovich, G., Omer-bendori, S., Gophna, U., 2013. Menaquinone and iron are essential for complex colony development in Bacillus subtilis. PLoS One 8, e79488. Qian, S., Lu, Z., Lu, H., Meng, P., Zhang, C., Lv, F., Bie, X., 2015. Effect of inulin on efficient production and regulatory biosynthesis of bacillomycin D in Bacillus subtilis fmbJ. Bioresour. Technol. 179, 260–267. Sato, T., Yamada, Y., Ohtani, Y., Mitsui, N., Murasawa, H., Araki, S., 2001a. Efficient production of menaquinone (vitamin K2) by a menadione-resistant mutant of Bacillus subtilis. J. Ind. Microbiol. Biotechnol. 26, 115–120.

265

Sato, T., Yamada, Y., Ohtani, Y., Mitsui, N., Murasawa, H., Araki, S., 2001b. Production of menaquinone (vitamin K2)-7 by Bacillus subtilis. J. Biosci. Bioeng. 91, 16–20. Shearer, M.J., Fu, X., Booth, S.L., 2012. Vitamin K nutrition, metabolism, and requirements: current concepts and future research. Adv. Nutr. 3, 182–195. Smith, I., 1993. Regulatory Proteins That Control Late-growth Development. Bacillus subtilis and Other Gram-positive Bacteria. American Society of Microbiology. Song, J., Liu, H., Wang, L., Dai, J., Liu, Y., Liu, H., Zhao, G., Wang, P., Zheng, Z., 2014. Enhanced production of vitamin K2 from Bacillus subtilis (natto) by mutation and optimization of the fermentation medium. Braz. Arch. Biol. Technol. 57, 606–612. Sumi, H., 2001. Method for Producing Menaquinone-7. Sumi, H., Asano, T., Yatagai, C., 2005. Additional effect of some nutrients and biological-active substances by Bacillus natto treatment of raw wheat. J. Brew. Soc. Jpn. 100, 449–453. Survase, S.A., Bajaj, I.B., Singhal, R.S., 2006. Biotechnological production of vitamins. Food Technol. Biotechnol. 44, 381–396. Szterk, A., Zmysłowski, A., Bus, K., 2018. Identification of cis/trans isomers of menaquinone-7 in food as exemplified by dietary supplements. Food Chem. 243, 403–409. Tsukamoto, Y., Kasai, M., Kakuda, H., 2001. Construction of a Bacillus subtilis (natto) with high productivity of vitamin K2 (menaquinone-7) by analog resistance. Biosci. Biotechnol. Biochem. 65, 2007–2015. Vijayaraghavan, K., Yamini, D., Ambika, V., Sravya Sowdamini, N., 2009. Trends in inulinase production — a review. Crit. Rev. Biotechnol. 19, 67–77. Wagner, W.P., Nemecek-Marshall, M., Fall, R., 1999. Three distinct phases of isoprene formation during growth and sporulation of Bacillus subtilis. J. Bacteriol. 181, 4700–4703. Wu, W.J., 2018. Statistical optimization of medium components by response surface methodology to enhance menaquinone-7 (Vitamin K2 ) production by Bacillus subtilis. J. Microbiol. Biotechnol. 28, 902–908. Wu, W.-J., Ahn, B.-Y., 2011a. Improved menaquinone (Vitamin K2) production in cheonggukjang by optimization of the fermentation conditions. Food Sci. Biotechnol. 20, 1585–1591. Wu, W.-J., Ahn, B.-Y., 2011b. Isolation and identification of Bacillus amyloliquefaciens BY01 with high productivity of menaquinone for Cheonggukjang production. J. Korean Soc. Appl. Biol. Chem. 54, 783–789. Xu, J.Z., Zhang, W.G., 2017. Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance. J. Zhejiang Univ. Sci. B 18, 462–473. Yeates, C., Gillings, M.R., 1998. Rapid purification of DNA from soil for molecular biodiversity analysis. Lett. Appl. Microbiol. 27, 49–53.