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Cis and trans isomers of the vitamin menaquinone-7: which one is biologically significant?

DOI:10.1007/s00253-020-10409-1
Authors:
Neha Lal at Malaviya National Institute of Technology Jaipur
Neha Lal
Aydin Berenjian at The University of Waikato
Aydin Berenjian
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Recently, several studies have indicated that an adequate intake of menaquinone-7 (MK-7) offers numerous health benefits. However, the low availability of MK-7 in the diet necessitates the development of dietary supplements or functional food products to complement natural food sources and meet the daily intake requirements. Like most biological molecules, MK-7 can exist as geometric isomers that can occur in the cis, trans, and cis/trans forms; however, only the all-trans form is biologically significant. MK-7 is traditionally produced through bacterial fermentation, but various synthetic preparations have lately become available. The isomer composition in the final product is influenced by numerous factors, including the methods of production and purification, as well as particular environmental and storage conditions. The MK-7 profile obtained from the various production methods has not yet been elucidated, and the ideal method for the synthesis of the all-trans form of the vitamin is also debatable. Consequently, the quantification of the MK-7 profile of various products is necessary to develop an understanding of the factors that influence the proportion of isomers that are obtained in different preparations. Several possible methods exist for the quantification of MK-7 isomers, and of these, liquid chromatography in conjunction with mass spectrometry techniques appears to be the most promising. Evaluation of the isomer composition is an important consideration, as only the all-trans form sustains biological activity. Furthermore, knowledge of the prominent factors that influence the MK-7 composition may also enable their manipulation to obtain a more favorable MK-7 profile in the final product.
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MINI-REVIEW
Cis and trans isomers of the vitamin menaquinone-7: which one is
biologically significant?
Neha Lal
1
&Aydin Berenjian
1
Received: 14 November 2019 /Revised: 15 January 2020 /Accepted: 23 January 2020 /Published online: 3 February 2020
#Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Recently, several studies have indicated that an adequate intake of menaquinone-7 (MK-7) offers numerous health benefits.
However, the low availability of MK-7 in the diet necessitates the development of dietary supplements or functional food
products to complement natural food sources and meet the daily intake requirements. Like most biological molecules, MK-7
can exist as geometric isomers that can occur in the cis,trans,andcis/trans forms; however, only the all-trans form is biologically
significant. MK-7 is traditionally produced through bacterial fermentation, but various synthetic preparations have lately become
available. The isomer composition in the final product is influenced by numerous factors, including the methods of production
and purification, as well as particular environmental and storage conditions. The MK-7 profile obtained from the various
production methods has not yet been elucidated, and the ideal method for the synthesis of the all-trans form of the vitamin is
also debatable. Consequently, the quantification of the MK-7 profile of various products is necessary to develop an understanding
of the factors that influence the proportion of isomers that are obtained in different preparations. Several possible methods exist
for the quantification of MK-7 isomers, and of these, liquid chromatography in conjunction with mass spectrometry techniques
appears to be the most promising. Evaluation of the isomer composition is an important consideration, as only the all-trans form
sustains biological activity. Furthermore, knowledge of the prominent factors that influence the MK-7 composition may also
enable their manipulation to obtain a more favorable MK-7 profile in the final product.
Keywords Cis and trans isomers .MK-7 .Biological activity .Synthesis .Quantification
Introduction
Vitamin K collectively refers to a group of fat-soluble com-
pounds that contain a 2-methyl-1, 4-naphthoquinone moiety,
but differ in the structure of a lateral isoprenoid chain at the 3-
position (Sitkowski et al. 2018;Szterketal.2018b). Vitamin
K naturally exists in two forms, phylloquinone and a series of
menaquinones, both of which play an important role in hemo-
stasis (Berenjian et al. 2015; Beulens et al. 2013; Nannapaneni
et al. 2017; Szterk et al. 2018b). The major dietary source of
phylloquinone are green vegetables, whereas menaquinones
are primarily of microbial origin and are present in certain
animal products and fermented foods such as meat, beef liver,
eggs, natto, hard cheeses, curd, and sauerkraut (Berenjian
et al. 2013; Beulens et al. 2013; Szterk et al. 2018b; Vermeer
et al. 2018).
All differences in the properties of the various forms of
vitamin K are attributable to the length and degree of satura-
tion of the isoprenoid side chain (Beulens et al. 2013; Pucaj
et al. 2011; Sitkowski et al. 2018). Phylloquinone is a single
compound with a side chain comprising four isoprenoid resi-
dues, three of which are unsaturated (Beulens et al. 2013).
Whereas, menaquinones contain side chains of varying length
and degree of saturation, and can generally be represented in
the form MK-n, where n denotes the number of unsaturated
isoprenoid units in a chain, which is usually between four and
thirteen (Beulens et al. 2013;Dainesetal.2003;Szterketal.
2018b).
MK-7 is a class of menaquinones that can be synthesized
through both natural (bacterial fermentation) and chemical
means for a variety of applications, including functional foods
and dietary supplements (Berenjian et al. 2015; Southee et al.
2016; Szterk et al. 2018a; Szterk et al. 2018b; Walther et al.
2013). Lately, MK-7 has gained widespread interest in both
*Aydin Berenjian
aydin.berenjian@waikato.ac.nz
1
School of Engineering, Faculty of Science and Engineering, The
University of Waikato, Hamilton, New Zealand
Applied Microbiology and Biotechnology (2020) 104:2765–2776
https://doi.org/10.1007/s00253-020-10409-1
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... However, the administration of MK-4 is not reflected in an increased serum concentration [6]. By contrast, MK-7 is absorbed efficiently, which is reflected in increased serum MK-7 levels up to several days, thereby contributing to vitamin K status [6,21]. US Pharmacopeia monographs have been developed to establish quality standards for menaquinone-7 as a dietary ingredient at typically recommended levels [22]. ...
... US Pharmacopeia monographs have been developed to establish quality standards for menaquinone-7 as a dietary ingredient at typically recommended levels [22]. Although, there are cis, trans, and cis/trans isomers of MK-7, only the all-trans form is biologically active [21]. It can be produced naturally through fermentation, as in the case of natto, or by organic synthesis. ...
... In the process of organic synthesis of MK-7, cis/trans isomers can be produced at the same time. Thus, the potential for the different isomeric forms of MK-7 to have different cytotoxic or toxic properties is an important consideration [21,23]. Naturally derived MK-7 is Fig. 1 The biosynthesis pathways of menaquinone in isoforms from Bacillus subtilis, Saccharomyces cerevisiae and S. coelicolor [26,37,41]. ...
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Vitamin K2 (menaquinone, MK) is an essential lipid-soluble vitamin with critical roles in blood coagulation and bone metabolism. Chemically, the term vitamin K2 encompasses a group of small molecules that contain a common naphthoquinone head group and a polyisoprenyl side chain of variable length. Among them, menaquinone-7 (MK-7) is the most potent form. Here, the biosynthetic pathways of vitamin K2 and different types of MK produced by microorganisms are briefly introduced. Further, we provide a new aspect of MK-7 production, which shares a common naphthoquinone ring and polyisoprene biosynthesis pathway, by analyzing strategies for expanding the product spectrum. We review the findings of metabolic engineering strategies targeting the shikimate pathway, polyisoprene pathway, and menaquinone pathway, as well as membrane engineering, which provide comprehensive insights for enhancing the yield of MK-7. Finally, the current limitations and perspectives of microbial menaquinone production are also discussed. This article provides in-depth information on metabolic engineering strategies for vitamin K2 production by expanding the product spectrum.
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... 4 Second, the form of MK-7 used was missing. 1 The highest quality MK-7 supplements contain ≤2% cis form 5 ; therefore, products with >98% of the trans form should be used and may be labeled "all trans. " 5 Third, in the early stages of aortic stenosis, endothelial damage and lipid deposition triggered an inflammatory response within the valve. 2 4 An inverse correlation was shown between several subtypes of vitamin K2 intake and risk of coronary heart disease. 4 A combination of vitamins K1 and K2 should be tested in a larger trial. ...
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... However, many secondary metabolites with special structural segments tend to isomerise spontaneously until an equilibrium is formed. For examples, the isomerisation of a form of vitamin K, MK-7, which possesses cis and trans isomers, tend to occurs due to light exposure, purification techniques and storage conditions (Lal and Berenjian 2020). Amphiol, isolated from the culture broth of the fungus Pseudogymnoascus sp. ...
Four pairs of tautomers from rhizomes of Acorus calamus and fruits of Cornus officinalis
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A pair of new guaiane-type sesquiterpene tautomers (1) was isolated from rhizomes of Acorus calamus. Meanwhile, three pairs of known compounds, including a pair of dihydroflavone glycoside tautomers (2), a pair of 4-hydroxybenzoic acid ester glycoside tautomers (3), as well as a pair of secoiridoid glycoside tautomers (4) were isolated from fruits of Cornus officinalis. Their structures were elucidated by extensive spectroscopic and computational methods. Furthermore, the tautomeric mechanisms were discussed.
View
... The chemical structure of cis and trans MK-7 isomers (adapted from Lal and Berenjian[40]) ...
Optimisation of the fermentation media to enhance the production of the bioactive isomer of vitamin menaquinone-7
Article
Full-text available
Menaquinone-7 (MK-7) offers significant health benefits; however, only the all-trans form is biologically active. MK-7 produced through fermentation can occur as all-trans and cis isomers, and the therapeutic value of the resulting MK-7 is exclusively determined by the quantity of the all-trans isomer. Therefore, this study aimed to investigate the effect of the media composition on the isomer profile obtained from fermentation and determine the optimum media combination to increase the concentration of the all-trans isomer and diminish the production of cis MK-7. For this purpose, design of experiments (DOE) was used to screen the most effective nutrients, and a central composite face-centred design (CCF) was employed to optimise the media components. The optimum media consisted of 1% (w/v) glucose, 2% (w/v) yeast extract, 2% (w/v) soy peptone, 2% (w/v) tryptone, and 0.1% (w/v) CaCl2. This composition resulted in an average all-trans and cis isomer concentration of 36.366 mg/L and 1.225 mg/L, respectively. In addition, the optimised media enabled an all-trans isomer concentration 12.2-fold greater and a cis isomer concentration 2.9-fold less than the unoptimised media. This study was the first to consider the development of an optimised fermentation media to enhance the production of the bioactive isomer of MK-7 and minimise the concentration of the inactive isomer. Furthermore, this media is commercially promising, as it will improve the process productivity and reduce the costs associated with the industrial fermentation of the vitamin.
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... Due to the identification of a high content of the cis/trans MK7 isomers in reference to all trans MK7 in some dietary supplements, 18 the determination of the biological activity as well as the toxicity of the cis/trans forms of MK7 is needed. 32 Since the vitamin K supplementation is essential to reach the recommended daily intake in certain conditions such as during developmental stages, CVD, and osteopenia, and, considering that the cis isomers have lower biological effects, the isomeric composition must be taken into consideration for the supplement formulation and its efficacy. ...
Carboxylative efficacy of trans and cis MK7 and comparison with other vitamin K isomers
Article
Full-text available
  • May 2022
Carboxylative enzymes are involved in many pathways and their regulation plays a crucial role in many of these pathways. In particular, γ‐glutamylcarboxylase (GGCX) converts glutamate residues (Glu) into γ‐carboxyglutamate (Gla) of the vitamin K‐dependent proteins (VKDPs) activating them. VKDPs include at least 17 proteins involved in processes such as blood coagulation, blood vessels calcification, and bone mineralization. VKDPs are activated by the reduced form of vitamin K, naturally occurring as vitamin K1 (phylloquinone) and K2 (menaquinones, MKs). Among these, MK7 is the most efficient in terms of bioavailability and biological effect. Similarly to other trans isomers, it is produced by natural fermentation or chemically in both trans and cis. However, the efficacy of the biological effect of the different isomers and the impact on humans are unknown. Our study assessed carboxylative efficacy of trans and cis MK7 and compared it with other vitamin K isomers, evaluating both the expression of residues of carboxylated Gla‐protein by western blot analysis and using a cell‐free system to determine the GGCX activity by HPLC. Trans MK7H2 showed a higher ability to carboxylate the 70 KDa GLA‐protein, previously inhibited in vitro by warfarin treatment. However, cis MK7 also induced a carboxylation activity albeit of a small extent. The data were confirmed chromatographically, in which a slight carboxylative activity of cis MK7H2 was demonstrated, comparable with both K1H2 and oxidized trans MK7 but less than trans MK7H2. For the first time, a difference of biological activity between cis and trans configuration of menaquinone‐7 has been reported.
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... Microbial fermentation can also produce the final products more economically from inexpensive sources of energy, carbon, electrons, and trace nutrients [36]. Even though the chemical synthesized vitamin K can be made for high purity of all-trans vitamin K2 which has biological activity [37,38], it still has product safety concerns for use as a GRAS food supplement [39]. ...
Production of Vitamin K by Wild-Type and Engineered Microorganisms
Article
Full-text available
  • Mar 2022
Vitamin K is a fat-soluble vitamin that mainly exists as phylloquinone or menaquinone in nature. Vitamin K plays an important role in blood clotting and bone health in humans. For use as a nutraceutical, vitamin K is produced by natural extraction, chemical synthesis, and microbial fermentation. Natural extraction and chemical synthesis methods for vitamin K production have limitations, such as low yield of products and environmental concerns. Microbial fermentation is a more sustainable process for industrial production of natural vitamin K than two other methods. Recent advanced genetic technology facilitates industrial production of vitamin K by increasing the yield and productivity of microbial host strains. This review covers (i) general information about vitamin K and microbial host, (ii) current titers of vitamin K produced by wild-type microorganisms, and (iii) vitamin K production by engineered microorganisms, including the details of strain engineering strategies. Finally, current limitations and future directions for microbial production of vitamin K are also discussed.
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... Long-chain menaquinones can be produced naturally through biosynthesis, or by organic synthesis; the former is more favoured due to its non-toxic nature [12], and several bacteria have been reported to synthesize long-chain menaquinones. They are constituents of the bacterial cytoplasmic membrane, and act as an electron-delivery vector of the respiratory chain due to their ability to mediate electron transport between enzymes [13]. ...
Optimised “green solvent” extraction of long-chain menaquinones (Vitamin K2) from wet Lactococcus lactis biomass
Article
Menaquinone (vitamin K2) is an essential fat-soluble vitamin for bone and cardiovascular health. Long-chain menaquinones are exclusively synthesised by bacteria, and Lactococcus lactis appears promising as a microbial production platform. Menaquinone exists in the bacterial cytoplasmic membrane protected by a thick cell wall, thus requiring efficient cell lysis and extraction for its recovery. In this work, several potential solvents that are ranked as “recommended” for pharmaceutical manufacture were screened for their effectiveness, with the aid of external heating, to simultaneously disrupt cells and extract menaquinone from wet biomass. These results were compared with those obtained by traditional two-step strategies and extraction approaches using dry biomass. Ethanol appeared to be the most effective extractant, and the technique was then optimised using response surface methodology. Under optimal conditions (75 °C, biomass concentration of 0.199 g mL⁻¹ and extraction time of 36.8 min), the extraction yields for menaquinone-7 and menaquinone-8 were 6.73 ± 0.17 and 179.6 ± 3.59 µg g⁻¹ dry cell weight, respectively. In addition, the use of ethanol as the extraction solvent coupled with microwave heating and bead-beating was investigated. Enhanced extraction was achieved using microwave-assisted extraction at an elevated temperature of 125 °C for 5 min, with a 1.15- and 1.2-fold increase in menaquinone-7 and menaquinone-8 yields, respectively. This work proposes the development of an integrated single-step process for the sustainable and efficient recovery of menaquinone from biomass, with the advantage of using ethanol as a green extraction solvent.
View
... Menaquinones (MK-n), the vitamin K 2 class of compounds with a 2-methyl-1,4-naphthoquinone moiety connected with n isoprenyl units, are crucially involved in diverse biological functions and insufficient levels of vitamin K result in diseases (Schurgers and Vermeer 2002;Halder et al. 2019;Sato et al. 2020;Farhadi Moghadam and Fereidoni 2020). Indeed, only the all-trans form of MK-7 is biological active (Lal et al. 2020). Recently, the acid-base and the redox properties of all-trans MK-4, -7, and -9 in 1, 2-dimyristoyl-snglycero-3-phosphocholine (DMPC) monolayers on mercury electrodes have been studied (Dharmaraj et al. 2020). ...
The effects of the chemical environment of menaquinones in lipid monolayers on mercury electrodes on the thermodynamics and kinetics of their electrochemistry
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  • Mar 2021
The effects of the chemical environment of menaquinones (all- trans MK-4 and all -trans MK-7) incorporated in lipid monolayers on mercury electrodes have been studied with respect to the thermodynamics and kinetics of their electrochemistry. The chemical environment relates to the composition of lipid films as well as the adjacent aqueous phase. It could be shown that the addition of all- trans MK-4 to TMCL does not change the phase transition temperatures of TMCL. In case of DMPC monolayers, the presence of cholesterol has no effect on the thermodynamics (formal redox potentials) of all- trans MK-7, but the kinetics are affected. Addition of an inert electrolyte (sodium perchlorate; change of ionic strength) to the aqueous phase shifts the redox potentials of all- trans MK-7 only slightly. The formal redox potentials of all- trans MK-4 were determined in TMCL and nCL monolayers and found to be higher in nCL monolayers than in TMCL monolayers. The apparent electron transfer rate constants, transfer coefficients and activation energies of all- trans MK-4 in cardiolipins have been also determined. Most surprisingly, the apparent electron transfer rate constants of all- trans MK-4 exhibit an opposite pH dependence for TMCL and nCL films: the rate constants increase in TMCL films with increasing pH, but in nCL films they increase with decreasing pH. This study is a contribution to understand environmental effects on the redox properties of membrane bond redox systems. Graphical abstract
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The impact of key fermentation parameters on the production of the all-trans isomer of menaquinone-7
Article
  • Nov 2022
Menaquinone-7 (MK-7) offers many health benefits and is the most superior form of vitamin K2 from a health and nutritional perspective. MK-7 can exist as geometric isomers; however, only the all-trans isomer is bioactive. Consequently, the therapeutic value of MK-7 products is determined by the proportion of the all-trans isomer. The fermentation-based synthesis of MK-7 is preferred, and the resulting product comprises both the all-trans and cis forms of the vitamin. The fermentation conditions influence the isomer profile. Hence, the objective of this investigation was to assess the significant fermentation parameters to enhance the concentration of the all-trans isomer and diminish the production of cis MK-7. Accordingly, a central composite face-centred (CCF) design was used to optimise the inoculum size, fermentation temperature, agitation speed, and fermentation time. The optimum conditions comprised an inoculum size of 2% (v/v), a fermentation temperature of 40 ° C, an agitation speed of 200 rpm, and a fermentation period of 7 days. This resulted in an all-trans MK-7 concentration of 53.292 mg/L and a cis isomer concentration of 1.222 mg/L. This study was the first to evaluate the impact of essential fermentation parameters on the MK-7 isomer profile to boost the production of the bioactive MK-7 isomer and reduce the concentration of the ineffectual isomer. The ideal fermentation conditions determined from this investigation present a valuable opportunity to develop a fermentation process that selectively favours the production of all-trans MK-7, which is commercially attractive, as it will refine the production process and decrease the concomitant expenses.
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The impact of a key nutraceutical complex on chondrocyte cells and matrix Gla protein expression
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Osteoarthritis (OA) is the most common type of arthritis among adults, which is associated with cartilage breakdown. Chondrocytes are the only type of cell in articular cartilage that secrete extracellular matrix (ECM). Therefore, to prevent and manage the occurrence of arthritic cartilage, it is important to consider approaches to increase the survival of chondrocyte cells. The production of reactive oxygen species (ROS), the release of pro-inflammatory cytokines (e.g. interleukin-1β (IL-1β)), and cartilage calcification play an imperative role in OA pathophysiology. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) is an enzyme that regulates the production of ROS in chondrocyte cells. ROS and cytokines lead to ECM degradation and chondrocyte apoptosis. Moreover, the matrix γ-carboxyglutamic acid (Gla) protein (MGP) acts as a decalcification agent in chondrocytes, which can inhibit the pathogenic mineralisation of cartilage. The correct balance of nutrients may potentially have a protective effect against OA. In the present study, the effect of essential nutrients on Nox4 activity in chondrocyte cells, chondrocyte viability, and MGP expression was investigated. Chondrocyte cells were pre-treated with the nutrients, and the cells were subsequently assessed for Nox4 activity using the chemiluminescence technique. It was found that the production of ROS significantly decreased after preincubation in the presence of nutrients in comparison to the control samples. Furthermore, it was found that the presence of nutraceuticals improved the viability of chondrocyte cells and the expression of MGP, which acts as a mineralisation inhibitor in the chondrocyte growth plate. The results of this study suggest that the correct balance of key nutrients at the molecular level plays an essential role in supporting chondrocyte cells and promoting cartilage decalcification.
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Biofilm reactors as a promising method for vitamin K (menaquinone-7) production
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Menaquinone-7 (MK-7) is the most potent subtype of vitamin K with extraordinarily high half-life in the circulatory system. Therefore, MK-7 plays a critical role in promoting human wellbeing today. Studies on MK-7 every year show more and more magnificent benefits of it in preventing cardiovascular diseases and osteoporosis to battling cancer cells, Alzheimer’s and Parkinson’s diseases. Thus, it needs to be supplemented to daily diet for accumulative and long-term benefits. Chemical synthesis of MK-7 produces a significant cis-isomer form of it, which has no biological activity. Fortunately, due to its key role in electron transfer in bacteria, trans-MK-7 is biosynthesized by especially Gram-positive strains mainly Bacillus genus. Concordantly, MK-7 could be produced via solid or liquid state fermentation strategies. In either regime, when static fermentation is applied in the absence of agitation and aeration, operational issues arise such as heat and mass transfer inefficiencies. Thus, scaling up the process becomes a challenge. On the other hand, studies have indicated that biofilm and pellicle formation that occur in static fermentations are key characteristics for extracellular MK-7 secretion. Therefore, this review covers the most recent discoveries of the therapeutic properties of MK-7 and optimization attempts at increasing its biosynthesis in different media compositions and effective growth parameters as well as the cutting-edge use of biofilm reactors where B. subtilis cells have the infrastructures to form mature biofilm formations on plastic composite supports. Biofilm reactors therefore can provide robust extracellular MK-7 secretion while simultaneously enduring high agitation and aeration rates, which then address the scale-up and operational issues associated with static fermentation strategies.
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Effects of medium components in a glycerol-based medium on vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors
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Menaquinone-7 (MK-7) as the most important form of Vitamin K has been reported to have miraculous benefits such as preventing cardiovascular diseases and osteoporosis along with antitumor effects. Therefore, there have been numerous studies in the past decades to improve MK-7 production via microbial fermentation. Unfortunately, both solid and liquid state fermentation strategies that are utilized for MK-7 production, face fundamental operational and scale-up issues as well as intense heat and mass transfer problems during fermentation. In this regard, biofilm reactors seem to be a practical solution to overcome these issues and enhance the production in agitated liquid fermentation. Therefore, this study was undertaken to utilize biofilm reactors in investigating and optimizing different media components in a glycerol-based medium. Using response surface methodology, the effects of glycerol, yeast extract, and soytone were studied in the fermentation medium on MK-7 production in biofilm reactor. With a composition of 48.2 g/L of glycerol, 8.1 g/L of yeast extracts, 13.6 g/L of soytone and 0.06 g/L of K2HPO4, MK-7 concentrations could reach 14.7 ± 1.4 mg/L in biofilm reactors, which was 57% higher compared to the MK-7 concentration achieved in suspended-cell reactors under similar conditions, while glycerol was depleted by the end of the fifth day in biofilm reactors, but glycerol was never depleted in suspended-cell reactors. Evidently, biofilm reactors present a reliable strategy to address the operational issues that occur during MK-7 biosynthesis on an industrial scale production.
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Implementation of fed-batch strategies for vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors
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Recent studies show the essential health benefits associated with vitamin K, especially menaquinone-7 (MK-7). These benefits include reducing risks of cardiovascular diseases, osteoporosis, and even cancer. However, MK-7 production on an industrial level is only possible through bacterial fermentation and also current static fermentation strategies are not potent enough with difficulties to scale up. Biofilm reactors, however, may be a practical alternative. Biofilm reactors provide a controlled environment for the microorganisms to form mature and robust biofilms that enable them to produce value-added products with enhanced efficiencies. In this study, fed-batch addition of glucose and glycerol were investigated to the base media in biofilm reactors, as carbon source addition seemed crucial in batch fermentations. Results indicated that fed-batch strategies can be significantly effective in glucose-based medium, increasing the end-product concentrations to 28.7 ± 0.3 mg/L of MK-7 which was 2.3 fold higher than the level produced in suspended-cell bioreactors and renders the biofilm reactors as a potential replacement for static fermentation strategies. Moreover, morphological changes of B. subtilis were tracked during the 12-day long fermentation runs and finally, SEM investigations confirmed significant biofilm and extracellular matrices formed on the plastic composite support (PCS) in the biofilm reactors. In conclusion, biofilm reactors especially with fed-batch fermentation regimes seem to be an effective tool for MK-7 production at industrial scales.
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Microbial production of vitamin K2: current status and future prospects
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Vitamin K2, also called menaquinone, is an essential lipid-soluble vitamin that plays a critical role in blood clotting and prevention of osteoporosis. It has become a focus of research in recent years and has been widely used in the food and pharmaceutical industries. This review will briefly introduce the functions and applications of vitamin K2 first, after which the biosynthesis pathways and enzymes will be analyzed in-depth to highlight the bottlenecks facing the microbial vitamin K2 production on the industrial scale. Then, various strategies, including strain mutagenesis and genetic modification, different cultivation modes, fermentation and separation processes, will be summarized and discussed. The future prospects and perspectives of microbial menaquinone production will also be discussed finally.
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Development of a Menaquinone-7 enriched product through the solid-state fermentation of Bacillus licheniformis
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  • May 2019
Bone fracture is a significantly prevalent issue in both large and small animals, in particular cats, dogs, and horses. Recent studies have shown that adequate consumption of the vitamin Menaquinone-7 (MK-7) has the ability to reduce the incidence of bone fractures, by increasing the bone mineral density. Therefore, the development of an MK-7 enriched animal feed poses as a promising concept that is likely to significantly benefit the health and wellbeing of animals, and improve their quality of life. The aim of this investigation was to build on the different aspects of previously conducted studies, and apply the relevant findings and experimental procedures to enhance the production of MK-7 through the solid-state fermentation of Bacillus licheniformis, for the unique application of an MK-7 enriched animal feed, in order to limit the incidence of bone fractures in animals. An optimisation study was performed using composite face-centered experimental design. The most ideal combination of the experimental variables for the biosynthesis of MK-7 was determined to be 10% (w/w) tryptone, 20% (w/w) glucose, and 2% (w/w) K2HPO4, when the soybean substrate is inoculated with 5% (v/w) of B. licheniformis and incubated at a temperature of 32 °C, for a fermentation period of 72 hours. The optimisation process was then validated and the highest concentration of MK-7 (12.507 mg/kg) that was obtained was fairly similar to the value predicted by the model. The results and observations from this research are a valuable step towards achieving a better understanding of the ability of feed grade bacterial cultures to enhance the production of MK-7 through solid-state fermentation for the development of functional animal feed in the future.
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Enhanced Vitamin K (Menaquinone-7) Production by Bacillus subtilis natto in Biofilm Reactors by Optimization of Glucose-based Medium
Article
Background: Benefits of vitamin K have been reported by many studies recently, due to its ability to reduce the risk of cardiovascular diseases and its potential benefits against osteoporosis. Specifically, menaquinone-7 (MK-7), being the most potent form of vitamin K, has definitely received most of the attention. Currently, solid or static liquid fermentation strategies are utilized for industrial production of MK-7 by Bacillus strains. However, these strategies face fundamental operational and scale-up issues as well as intense pellicle and biofilm formations which is problematic in static liquid fermentation, due to heat and mass transfer inefficiencies they create. Objective: The purpose of this study was to demonstrate that biofilm reactors will overcome the issues associated with suspended cell reactors when using Bacillus strains to produce MK-7. The expectation is that the use of biofilm reactors will result in a significant increase in the production of MK-7. Method: Vitamin K production by Bacillus subtilis natto when grown in a biofilm reactor was evaluated at various concentrations of the three major nutrients, glucose, yeast extract and casein. The data was analyzed using response surface methodology (RSM). Results: The maximum concentration of MK-7 in the biofilm reactors was 20.5±0.5 mg/L, which was a 344 % increase when compared to the amount produced in suspended cell reactors containing the same optimum media composition. Conclusion: These results demonstrate the potential of utilizing biofilm reactors for MK-7 production on an industrial scale.
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Modeling of Vitamin K (Menaquinoe-7) Fermentation by Bacillus subtilis natto in Biofilm Reactors
Article
  • Nov 2018
Menaquinoe-7 (MK-7) is the most potent form of vitamin K with numerous benefits for human health such as reducing the risks of cardiovascular diseases and osteoporosis in addition to having antitumor characteristics. Therefore, MK-7 production via bacterial fermentation has been studied intensely in the last few decades. Recently, biofilm reactors were implemented to enhance production levels in our lab. The specific objective of this study was to mathematically model the findings in batch biofilm reactors and thus further deepen our understanding of the conditions governing fermentation to pave the path to scaling up the production to pilot scale fermenters. The modified logistic equation was used to correlate substrate consumption with fermentation time and to model the substrate consumption in the four batch fermentations. Results indicated that this modified logistic model fits well with the experimental data for substrate consumption. Therefore there was no need for the other more complex models. Furthermore, this successfully modified-logistic equation was inserted into the Luedeking-Piret model to estimate MK-7 production based on substrate consumption, because of the typical MK-7 profiles in batch fermentations. There is sufficient evidence in the literature that MK-7 biosynthesis follows a mixed-metabolite pattern in Bacillus subtilis natto. Similarly, the modified-Gompertz model was used also for the same purpose. Results indicated that the modified Luedeking-Piret model fits more accurately for MK-7 production (R² = 0.971, 0.943, 0.970, and 0.959) compared to the modified-Gompertz (R² = 0.914, 0.943, 0.949 and 0.860).
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Determination of vitamin K composition of fermented food
Article
A rapid ultra-high performance liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometric (UHPLC-APCI-MS/MS) method was developed for the analysis of vitamin K compounds: phylloquinone (PK) and menaquinones (MK-n). Non-chlorinated mobile phase composition was optimized for separation of eight vitamin K compounds on a reversed phase column in 10 min. Sample treatment with liquid and solid phase extractions and by the use of MK-4 as an internal standard enabled the quantitation of microgram level of vitamin K compounds in food. The method was used to screen and quantitate vitamin K from 17 fermented food products. The highest amount of PK was detected in kimchi (42 µg/100 g), whereas the highest MK-7 content was detected in natto (902 µg/100 g). Some MK-9 was present in kefir (5 µg/100 g). Two Chinese fermented soybean pastes contained significant amount of MK-6 (5–36 µg/100 g), MK-7 (12–86 µg/100 g), and MK-8 (22–44 µg/100 g).
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The application of multidimensional NMR analysis to cis/trans isomers study of menaquinone-7 (vitamine K2MK-7), identification of the (E,Z3,E2,ω)-menaquinone-7 isomer in dietary supplements
Article
The aim of this paper was to determine the chemical structure of cis/trans isomers of vitamin K2MK-7 found in dietary supplements. Vitamin K2MK-7 was extracted, along with contamination in the form of cis/trans isomers of menaquinone-7, from dietary supplements through tetrahydrofuran and then subjected to fractionation using the semi-preparative chromatography column with cholesteryl group phase (strong stereoselectivity for hydrophobic compounds). The obtained fractions of cis/trans isomers of menaquinone-7 underwent analysis with the use of multidimensional NMR methods optimized for maximum signal separation. NMR analysis made it possible to identify the first geometric isomer of (E,Z3,E2,ω)-menaquinone-7, which underwent NMR comparative analysis with a known “full trans” isomer. It was also possible to confirm the presence of other cis/trans isomers in menaquinone-7 in the collected fractions. Due to the fact that the other fractions indicate a mixture of at least two geometric isomers of menaquinone-7, it was not possible to conduct full structural identification.
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