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Fermentation of vitamin K2 (menaquinone-7): Development of an optimal fermentation process to selectively enhance the production of the biologically significant all-trans isomer

Vitamin K is a lipid-soluble vitamin first discovered in 1929 by the Danish nutritional biochemist Carl Peter Henrick Dam as an antihaemorrhagic factor capable of correcting dietary-induced bleeding disorders in chicks. The vitamin K family encompasses a series of structurally related compounds, namely vitamin K1 (phylloquinone), vitamin K2 (menaquinones), and vitamin K3 (menadione), which share a common 2-methyl-1,4-naphthoquinone moiety but differ in the structure of a lateral isoprenoid chain at the 3-position. Despite their structural similarities, all K vitamers have different pharmacokinetic properties attributable to the length and degree of saturation of their isoprenoid side chain. All vitamin K isoforms are involved in the activation of hepatic and extrahepatic vitamin K-dependent proteins (VKDPs) and play a central role in blood coagulation and haemostasis. Additionally, it has been established that the potential health benefits of vitamin K are far more diverse. In particular, menaquinones (MKs) offer more significant health gains than phylloquinone (PK) and have been associated with the prevention of osteoporosis and cardiovascular diseases (CVDs) and decreasing the risk of and improving the outcomes associated with several illnesses and health conditions, including cancer, neurological diseases, type 2 diabetes mellitus, chronic kidney disease, immune disorders, obesity, and coronavirus disease 2019 (COVID-19). Of the various MKs, menaquinone-7 (MK-7) has superior bioavailability due to its long plasma half-life. Thus, it is considered the most notable form of vitamin K2 and has the greatest efficacy with respect to extrahepatic biological functions. However, MK-7 exists at low concentrations in limited foods, especially those with universal appeal. This has incentivised the development of MK-7 dietary supplements and functional food products to complement natural sources and satisfy the daily intake requirements of this essential vitamin. Recently, it has been recognised that MK-7 exists as geometric isomers, comprising the bioactive all-trans isomer and various cis forms of the compound, which lack or have considerably compromised biological significance. This is a salient aspect worthy of attention, as the effectiveness of MK-7 nutritional supplements is primarily determined by the content of all-trans MK-7, and all other isomers of the vitamin are essentially impurities that lack therapeutic value. MK-7 can be produced synthetically from chemical reaction methods or naturally from bacterial fermentation. The latest market trend promoting natural and organic alternatives over synthetic products has rendered natural fermentation-based synthesis more favourable from a consumer’s perspective. Furthermore, microbial production is a more sustainable approach for the large-scale synthesis of MK-7; hence, natural fermentation techniques can satisfy both the market demand and sustainable development goals. The MK-7 isomer composition of the final product is influenced by many factors, predominantly the methods used for its production and the purification of the post-reaction mixture, as well as exposure to certain environmental factors and storage conditions. It is important to appreciate that while studies analysing the isomer profile of MK-7 dietary supplement preparations of various origins have been carried out, the isomer composition resulting from fermentation processes has not been elucidated. In light of the fact that only all-trans MK-7 sustains biological activity and fermentation-based synthesis is superior from both the viewpoint of consumers and the environment, the MK-7 isomer profile attained from fermentation warrants further investigation. Therefore, the fundamental aim of this research was to evaluate the MK-7 isomer composition resulting from fermentation processes employing different synthesis conditions to explicate the optimal fermentation method for the production of the biologically significant all-trans form of the vitamin. A holistic approach was adopted to systematically follow a typical fermentation process from its foundational stages to the final fermented product while considering the MK-7 isomer profile obtained from fermentation under various conditions and the bioactivity and therapeutic value of fermented MK-7 consumer end products. Accordingly, key aspects of upstream and downstream fermentation were explored, along with nanobiotechnological approaches, to potentially address the major challenges accompanying the fermentation and downstream processing of MK-7. The isomer composition and stability of all-trans MK-7 resulting from exposure to possible conditions and environmental factors encountered during the manufacture, transportation, and storage of fermented bioactive MK-7 goods available on the market were also assessed. The selection of nutrients, particularly carbon, nitrogen, and salt sources, and their respective concentrations in the fermentation media are a central aspect of any fermentation process, as they influence microbial growth and metabolism and, ultimately, the process productivity and product yield. Thus, the first step was to consider the impact of the media composition on MK-7 isomer production and ascertain the optimum media combination to favour the synthesis of all-trans MK-7. Several nutrient sources were screened, and the concentration of the effective media components was optimised to obtain the ideal fermentation media, which contained 1% (w/v) glucose, 2% (w/v) yeast extract, 2% (w/v) soy peptone, 2% (w/v) tryptone, and 0.1% (w/v) calcium chloride (CaCl2). The optimal fermentation media resulted in an all-trans isomer concentration of 36.37 mg/L and a cis isomer concentration of 1.23 mg/L. It was also established that only a single cis isomer is obtained from fermentation under the investigated conditions. Moreover, different concentrations of the cis isomer were achieved when the selection and concentration of nutrients constituting the fermentation media were varied, implying that the media composition is instrumental in determining the nature of the extracellular environment in the fermentation broth, and this likely influences the extent to which all-trans MK-7 isomerises to the cis form. Various fermentation parameters and operating conditions also play an indispensable part in fermentation processes, as they contribute to the microbial growth environment and, hence, impact product formation and the efficacy of the fermentation system. Consequently, the subsequent focus was to explore the effect of crucial fermentation parameters, specifically the inoculum size, fermentation temperature, agitation speed, and length of fermentation, on the MK-7 isomer profile. These factors were optimised to enhance the synthesis of the biologically important all-trans isomer and minimise the concentration of cis MK-7 while using the previously developed fermentation media. The optimum fermentation conditions consisted of 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 and enabled an all-trans and cis isomer concentration of 53.29 mg/L and 1.22 mg/L, respectively. An approximately 46.5% greater all-trans MK-7 concentration was attained from fermentation utilising the optimal media, inoculum size, temperature, agitation speed, and duration compared to fermentation with only the optimum media composition, which emphasises the pivotal role of the fermentation environment in regulating the production of the desired isomer. Although fermentation is the preferred method for MK-7 synthesis, its low yield and large number of downstream processing steps increase production expenses and the cost of the final product. The high price of fermented MK-7 supplements reduces their widespread accessibility. While optimisation of various aspects of the fermentation process, such as the fermentation format, nutrient selection and media composition, and value of key fermentation parameters and operating conditions, serves to enhance the production and yield of MK-7, it offers little opportunity to refine the fermentation system through process intensification. Therefore, there is a need for innovative approaches to not only boost the process yield but also streamline the overall fermentation procedure by decreasing the complexity and number of unit operations involved in the downstream processing of the vitamin. It is also essential to ensure that all-trans MK-7 is produced almost exclusively or in the most significant proportion during fermentation. In this regard, bacterial cell immobilisation with biocompatible magnetic iron oxide nanoparticles (IONs) was investigated to assess their influence on the MK-7 isomer profile obtained from fermentation. Three types of IONs, including one uncoated (naked) and two coated (amine-functionalised) IONs, were synthesised using the co-precipitation method and characterised using several techniques, and their effect on microbial growth and the production and yield of MK-7 isomers was considered. Naked IONs were initially examined, and the results were compared with the findings for the 3-aminopropyltriethoxysilane (APTES)- and L-lysine (L-Lys)-coated IONs (IONs@APTES and L-Lys@IONs). The optimum concentration of naked IONs was 300 μg/mL, and it improved the process output and resulted in an all-trans MK-7 concentration of 28.78 mg/L and a 1.6-fold greater all-trans isomer yield relative to the control. In comparison to the naked IONs, the amine-functionalised IONs had superior properties and provided more positive outcomes. The optimal IONs@APTES and L-Lys@IONs concentrations were 300 μg/mL and 400 μg/mL, enabling a maximum all-trans MK-7 concentration of 41.93 mg/L and 32.08 mg/L, respectively. In addition, the yield of the biologically effective isomer compared to the control increased by 3.1-fold for the IONs@APTES and 2.1-fold for the L-Lys@IONs. Of all three forms of IONs that were explored, it was determined that 300 μg/mL of IONs@APTES was the most beneficial to enhance the all-trans isomer concentration and minimise the synthesis of cis MK-7. The magnetic nature of IONs also increases the scope for process intensification through magnetic separation technology, which has the potential to improve the productivity of MK-7 fermentation. It is of interest to note that the greatest all-trans MK-7 concentration obtained in the presence of IONs (41.93 mg/L) was less than that achieved in their absence when applying the same fermentation media and operating conditions as previous experiments (53.29 mg/L). This disparity can be attributed to the different fermentation volumes used in the two studies, as even the untreated (control) samples in the experiments involving IONs resulted in considerably lower concentrations of the all-trans isomer (less than 15 mg/L). All prior investigations were carried out on a small scale (6 mL), whereas the studies employing IONs required a slightly greater volume (approximately 20 mL) to accommodate the inclusion of IONs in the fermentation medium and the related measurements. It is apparent that even a small increase in the volume and, consequently, the scale of the fermentation process has a noticeable impact on the concentration of the target product. These observations highlight the central role of the fermentation volume in determining the concentration of all-trans MK-7 attained on a larger scale and present basic knowledge of likely challenges associated with the scale-up of fermentation processes targeting the production of the bioactive isomer. It has been proposed that exposure to various environmental conditions influences the isomer composition of MK-7 products. Awareness of these factors and their impact on the proportion of MK-7 isomers is vital to preserve the content of the biologically significant all-trans isomer and prevent its transformation to cis MK-7 during the storage of fermented MK-7 supplements and fortified or functional foods. In this respect, the effect of short-term exposure to common environmental factors and storage conditions, such as light, atmospheric oxygen, and different temperatures, on the MK-7 isomer profile was considered to establish the optimum conditions to conserve the quantity of the all-trans isomer during the storage of fermented MK-7 preparations. It was ascertained that the vitamin is reasonably heat-stable but extremely light-sensitive. Long-term storage at a low temperature with minimal oxygen exposure in the absence of light resulted in a negligible change in the concentration of the all-trans isomer and was, thus, the optimal environment for the prolonged storage of fermented all-trans MK-7. These findings provide a deeper understanding of the impact of different environments on the stability of the biologically efficacious isomer and are a valuable step forward in establishing ideal storage conditions to maintain the concentration of all-trans MK-7 in fermented nutritional supplements. Overall, this research presents novel insights into the MK-7 isomer profile achieved from fermentation in various contexts, a previously unexplored field, and has laid the foundation for future studies. Although it is desirable to exclusively produce the bioactive isomer from fermentation, the collective experimental findings have revealed that obtaining small quantities of the cis isomer alongside all-trans MK-7 is largely unavoidable under the circumstances considered. However, it is possible to maximise the concentration of the all-trans isomer and minimise the amount of the biologically insignificant isomer, which is a reasonable compromise. Appreciation of the key factors that influence the MK-7 isomer composition resulting from fermentation will allow their manipulation to attain a more favourable MK-7 profile in the final preparation, which can be included in nutraceuticals, dietary supplements, and functional food products or used in purified form. Furthermore, the outcomes of this study have the potential to aid the development of an industrial fermentation process that specifically targets the production of the biologically active and therapeutically valuable all-trans MK-7 isomer. The prospect for process intensification through the innovative use of magnetic IONs will likely streamline the production system and decrease the related expenses. This will help reduce the price and increase the accessibility of fermented bioactive MK-7 supplements and fortified or functional foods. Moreover, a greater understanding of the environmental factors and storage conditions that affect the isomer composition of fermented MK-7 preparations is likely to improve the standard of MK-7 nutritional products by preserving the quantity of the all-trans isomer. The widespread availability and consumption of high-quality bioactive MK-7 products by a range of consumers are expected to raise the vitamin K status of individuals and decrease the risk and progression of several age-related disorders and diseases of global significance. This holds great promise for reducing the disease burden and alleviating the socioeconomic consequences of an ageing population.
Type of thesis
The University of Waikato
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