**7. Dietary recommendations for menaquinone**

Dietary recommendations for vitamin K are still based on knowledge of phylloquinone and its classic role as an enzyme cofactor for coagulation proteins. The recommendations do not consider the differences in bioavailability and bioactivity between the different forms of vitamin K or the possibly higher requirements for health effects apart from coagulation, such as bone or cardiovascular health [16].

Depending on country, sex, and age, the recommendations for vitamin K range from 50 to 120 μg per day for adults 19 years and older. These recommendations are generally presented as adequate intake or estimated values, and no tolerable upper intake level has been established for vitamin K [16, 25, 48]. Research for valuable biomarkers to measure the status of vitamin K in the population is ongoing. A recent study from Maastricht University compared the biomarkers for coagulation with those of bone and vascular health in 896 healthy volunteers. Whereas all coagulation proteins were completely carboxylated by vitamin K, and a high concentration of undercarboxylated Gla proteins (osteocalcin and matrix Gla protein) was found in the majority of the blood samples, indicating that most of the volunteers in this study had an inadequate supply of vitamin K [23]. As long as robust physiological endpoints are missing to differentiate the contribution of MKs to human health from that of PK, it is unlikely that specific dietary recommendations for MKs will be widely adopted in the near future. In the meantime, a preferred recommendation could be to consume a wide variety of foods which are good sources of PKs and MKs, respectively, such as green leafy vegetables and fermented dairy products [16, 49].

#### **8. Dietary intake of menaquinones**

In soft cheese, the average total menaquinones range 40.1 μg/100 g to 61 μg/100 g depending on the source, analytical method, and type of cheese (**Table 1**). Manoury and coauthors reported a soft cheese and a blue cheese with very high concentrations (up to 4.110 μg/100 g and 70 μg/100 g, respectively), but the researchers could not explain why these two cheeses

One cheese with mold was also analyzed for menaquinone content. Gamalost, a Norwegian mold (*Mucor mucedo*) ripened autochthonous cheese, contains more menaquinone than Norvegia, a semihard Norwegian cheese, but the mold did not contribute to the production of vitamin K in Gamalost. The low pH in Gamalost and a higher fermentation rate may explain

Some work has been conducted to improve the content of different menaquinones in dairy products. New research demonstrated that strains of *Lactobacillus fermentum* LC 272 isolated from raw milk could be a starter culture for fermented milk with a high level of vitamin K2 (MK‐4) production [41]. This strain can produce 185 μg/L in Rogosa medium and 64 μg/L in reconstituted skim milk. Morishita and coworkers published a study in 1999 that showed the possibility of producing MK‐8 and MK‐9 with *Lactocouccus lactis* ssp. *cremoris* YIT2011 and MK‐9 and MK‐10 with *Lactococcus lactis* YIT 3001 (29–123 μg of menaquinone/L of the fer‐ mented medium) [38]. Additionally, several patents for *Lactococcus* capable of producing a

In contrast to fermented animal products, fermented vegetable products contain mainly MK‐7 (**Table 1**). Natto, a traditional Japanese food produced with *Bacillus subtili*s natto, contains the highest amount of menaquinone. The highest measured value is almost 1000 μg/100 g. *B. subtilis* natto is the key microorganism for industrial production of MK‐7, and much work has been done to improve the production. Optimization of the fermentation medium, mutations of the strains, and biofilm formation have been described as means for improving the yield of MK‐7 [42–46]. The use of organic solvents to extract vitamins is one of the major issues of the bulk production of MK‐7. Berenjian and coworkers demonstrated that the addition of vegeta‐ ble oil during a dynamic fermentation process could be a good process for producing an oil rich in MK‐7. In that study, the oil contained 724 mg/L of MK‐7, and they suggested using the

Dietary recommendations for vitamin K are still based on knowledge of phylloquinone and its classic role as an enzyme cofactor for coagulation proteins. The recommendations do not consider the differences in bioavailability and bioactivity between the different forms of vitamin K or the possibly higher requirements for health effects apart from coagulation, such

Depending on country, sex, and age, the recommendations for vitamin K range from 50 to 120 μg per day for adults 19 years and older. These recommendations are generally presented

are so rich in menaquinones [34].

72 Vitamin K2 - Vital for Health and Wellbeing

the differences in menaquinone content [35].

significantly increased amount of vitamin K2 have been deposed.

oil in supplementary and dietary food products [47].

as bone or cardiovascular health [16].

**7. Dietary recommendations for menaquinone**

As shown in **Table 1**, the most important sources of menaquinones are cheese, curd, offal, and fermented soybeans (natto). Based on regional differences in dietary patterns, the form and amount of specific menaquinones consumed may vary widely between populations. For example, in Japan, as a result of natto consumption, MK‐7 is the most frequently consumed form of menaquinones. The contribution of MK‐7 to total vitamin K intake is 25% among young women living in eastern Japan. Nearly all of the MK‐7 intake originates from pulses, including fermented soybean natto [32]. The mean daily intake of MK‐7 in this study was 57.4 μg with a range from 0 to 340 μg.

In countries with a traditional high intake of dairy products, such as the Netherlands, Ger‐ many, and the United Kingdom (UK), MK‐7 to MK‐10 contribute mostly to the menaqui‐ none supply. Beulens and coauthors compiled the results from several European studies that estimated menaquinone intake using Food Frequency Questionnaires (FFQs). The self‐ reported mean daily intake of menaquinones in adults ranged from 20.7 μg for women in the Rotterdam Study to 43 μg in men in the UK National Dietary and Nutrition Survey. In all of these studies, cheese was the most important food source of menaquinones [49]. How‐ ever, these data should be interpreted carefully because they were collected by FFQs that are designed to estimate the relative dietary intake of large populations but not to estimate absolute dietary intake. A seasonal survey in postmenopausal women in Tehran, Iran, used a monthly food record for 1 year. The researchers found a significantly higher intake of vita‐ min K in the spring, summer, and autumn compared to the winter. Unfortunately, these au‐ thors did not further specify vitamin K and did not provide any information about consumption of different food items containing vitamin K [50]. A study in older individuals to calculate the desired duration of a diet recording to estimate the individual vitamin K intake concluded that 13 24‐hour recalls are ideal to record intraindividual variance. As this would not be realistic in most studies, the authors proposed a minimum of six nonconsecu‐ tive days of diet recording [51]. Another possible approach for estimating nutrient status is to use biomarkers. Biomarkers for menaquinones are undercarboxylated vitamin K‐depend‐ ent proteins in the circulatory system. However, in addition to vitamin K availability, these biomarkers depend on the total amount of protein. To be sure that protein status does not confound vitamin K status, the measurements must be corrected for the total amount of the protein under study [52].

**10. Bone health, menaquinones, and fermented dairy products**

to bone health [56].

production [59].

One of the most important research fields in the past and present is the study of the factors that influence the formation and conservation of strong bones. Osteopenia, including osteo‐ porosis, is one of the most prevalent diseases in elderly individuals and is a large social, medical, and economic burden throughout the world. One out of three women and one out of five men older than 50 years are at risk of experiencing an osteoporotic fracture [54]. Low bone mineral mass is the main factor that causes osteoporotic fractures. Bone mass in later life is the result of the peak bone mass achieved during growth and the rate of age‐related bone loss. Consequently, a high peak bone mass at maturity and a low bone loss during aging are the most promising factors in the prevention of osteoporosis and fractures. In addition to factors that influence bone health such as gender, age, body size, genetics, and ethnicity that are not changeable, other factors, especially lifestyle factors such as physical activity, smoking, alcohol consumption, and dietary patterns, can be modified [55]. Different dietary factors are known to positively influence bone health. They range from minerals (e.g., calcium, magnesium, phosphorus, potassium, and various trace elements) and vitamins (A, D, E, K, C, and certain B vitamins) to macronutrients such as proteins and fatty acids and finally to bioactive food components (e.g., peptides) that in recent years have been proposed to be beneficial for bone health [55]. All these elements are involved in bone metabolism. Currently, researchers are trying to identify and understand the mechanisms and interactions of these factors in relation

Menaquinones, Bacteria, and Foods: Vitamin K2 in the Diet

http://dx.doi.org/ 10.5772/63712

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Most studies that have investigated the relationship between dairy and bone health have shown a beneficial effect of dairy consumption, even if the reason for this link is still unclear [56, 57]. After many years of focusing on calcium as the beneficial element for bone health in dairy, recent evidence suggests that other macro‐ and micronutrients, as well as food compo‐ nents such as bioactive peptides, milk fat globule membrane, prebiotics, and probiotics present in milk and dairy products, play an important role in this health outcome [56]. Many of these nutrients support the bioavailability (phosphorus, vitamin D, magnesium, zinc, potassium), absorption (casein phosphopeptides, phosphorus, lactose, protein) and homeostasis (magne‐ sium, potassium, vitamin D) of calcium and contribute to bone‐building properties (phospho‐

rus, magnesium, potassium, zinc, vitamin D, vitamin B12, and vitamin K) [56–58].

organisms (i.e., bioactive peptides, vitamin B12, or vitamin K2).

Most of these components are not or are positively affected by fermentation. That means their concentration remains the same in the fermented product compared with milk or even increases either by processing (i.e., fat‐soluble vitamins in cheese) or by the activity of micro‐

The role of vitamin K2 in bone health is strongly bound to osteocalcin (OC), a key regulator of calcium usage. This small Ca2+‐binding protein is involved in the mineralization of bones and teeth, and its potential to bind calcium is dependent on carboxylation with vitamin K2.

Only the fully carboxylated OC is able to strongly bind calcium and to consolidate calcification of the hydroxyapatite crystal lattice that requires a sufficient supply with vitamin K2 and other nutrients, such as retinoic acid and vitamin D, all involved in the regulation of osteocalcin

These limitations, together with the scarce and widely varying data on concentrations of different menaquinones in food items, show how fragmentary our knowledge of the supply of vitamin K2 in the general population remains.

#### **9. Pharmacokinetics of menaquinones**

Although the forms of vitamin K are classified as fat‐soluble nutrients, the lipophilicity of the different forms changes with side‐chain length. Whereas menadione is water soluble, phylloquinone and MK‐4 are mildly lipophilic. Long‐chain menaquinones are strongly lipo‐ philic and soluble only in apolar organic solvents [36]. This lipophilicity also influences the absorption of vitamin K, which varies greatly depending on the food matrix. As long‐chain menaquinones are found mainly in the fat fraction of dairy products, the absorption of these menaquinones is almost 100% in contrast to PK, where the poor uptake of only 5–10% from cooked vegetables can be improved only slightly by concomitant fat intake [6]. As a conse‐ quence, even the dietary intake of phylloquinone is much higher, menaquinones are equally important for vitamin K status, because of their better intestinal absorption. Independently of their form and origin, all K vitamins are transported to the liver, incorporated in triglycer‐ ide‐rich lipoproteins. Unlike phylloquinone, which mostly remains in the liver to be used for clotting factor synthesis, menaquinones are released to the bloodstream incorporated in low‐density lipoproteins and transported to the target tissue such as bone and arteries for Gla‐protein carboxylation. Absorbability is further supported by a longer half‐life, up to several days for long‐chain menaquinones compared to phylloquinone, which normally dis‐ appears from the bloodstream after 8 hours. This longer postprandial presence in the blood‐ stream leads to a more constant circulating level of vitamin K2 and, as a consequence, longer availability of these long‐chain menaquinones for uptake by extrahepatic tissues [36, 53]. Although there is some evidence that menaquinones with medium‐chain length like MK‐7 are better absorbed than short‐ (MK‐4) or long‐chain menaquinones (MK‐8 and MK‐9) [6], human data on the bioavailability, absorption, and kinetics of K2 vitamins from food are limited to MK‐7 and MK‐9 and have not been systematically tested for all menaquinones thus far [36, 49].

As researchers have found that MKs play an important role in health aspects beyond coagu‐ lation, the cooperation with other nutrients in vitamin K‐rich food such as fermented dairy products may lead to a better understanding of the effect of different food items on health aspects, for example, bone health or cardiovascular health.
