**3. Functions and biosynthesis of menaquinones in bacteria**

The distribution of isoprenoid quinones has been studied in 900 microbial strains, 56 mold strains, and 88 yeast strains. About half of the studied bacteria contain menaquinone, but no menaquinones have been found in molds and yeast [10]. Menaquinone and demethylmena‐ quinones (DMKs) are found in the cytoplasmic membrane of bacteria. MKs and DMKs function as a reversible redox component of the electron transfer chain [11]. Additionally, reduced MKs exhibit antioxidant properties and can play a role in protecting cellular membranes from lipid oxidation [12]. Menaquinones are also necessary for sporulation and proper regulation of cytochrome formation in some Gram‐positive bacteria such as *Lactobacillus subtilis* [13]. In particular, the food industry uses various lactic acid bacteria (LAB) as starter cultures to produce fermented milk products, meat products, and vegetables. As many LAB lack a heme biosynthesis pathway, which results in an incomplete electron transport chain, the addition of menaquinone to the media facilitates aerobic growth, improves yield, and reduces production costs [14].

Menaquinone synthesis has mostly been described in *Escherichia coli*, *Mycobacterium phlei*, and *Bacillus subtilis*. In *E. coli*, chorismate from the shikimate pathway is converted into the naphthoquinone ring by six enzymes (MenFDHCEB) [11, 15]. The isoprenoid side chain is synthesized separately and is joined to the naphthoquinone ring to form demethylmenaqui‐ none. Prenylation and methylation catalyzed by polyprenyltransferase (MenA) and methyl‐ transferase (MenG) are the last steps of the synthesis of menaquinone [16, 17]. An alternative pathway, called the futalosine pathway, was described in microorganisms that lack *men* genes. In this pathway, chorismate is converted to menaquinone with four enzymes encoded by *mqnABCD* genes and unknown enzymes [17–19]. The majority of the bacteria containing the classical menaquinone pathway are obligately or facultatively aerobic, and the majority of menaquinone in anaerobic bacteria is synthesized via the futalosine pathway [17]. For example, the metabolic pathway of *Lactoccocus lactis*, which is used as a cheese starter, can function through aerobic and anaerobic reactions, and the *men* genes for the synthesis of menaquinone were detected in its genome.

Menaquinones have side chains of different sizes in different organisms and sometimes even within the same organism. Depending on the growing conditions, the basic structure can be modified by demethylation of the naphthoquinone ring to reform DMK or by saturation of the isoprenoid side chain [2, 19].

#### **4. Non-dietary sources of menaquinones**

conversion of PK and/or menadione [5]. However, in the literature, all MKs are mostly grouped under the term vitamin K2 resulting in the assumption that all MKs are similar in origin and function. Moreover, despite the knowledge that MKs are present in the food supply, little is known about their individual synthesis, growth conditions, and interactions of the producing bacteria and the total amounts of the different MKs in fermented foods. Regarding the findings that MKs play an important role in health aspects beyond coagulation, study of the interaction of MKs with other nutrients may lead to a better understanding of the effect of different food

Such a global view could be essential for guiding the development of dietary intake recom‐

Both vitamin K forms have 2‐methyl‐1,4‐naphthoquinone, also called menadione or vitamin K3, as a common ring structure. However, they differ from each other in the length and degree

Phylloquinone (vitamin K1) possesses a phytyl side chain, which consists of four isoprene units, and one of them is unsaturated. Phylloquinone is found primarily in plants in association with chlorophyll, whereas menaquinone (vitamin K2) is principally synthesized by bacteria. Menaquinone contains side chains of varying length, for most the part of a polymer of repeating unsaturated 5‐carbon prenyl units. Depending on the microorganism by which the chain is synthesized, the chain length generally ranges from 4 to 13 prenyl units. Menaquinones are classified according to the number of prenyl units. The number of units is given in a suffix (‐*n*), that is, menaquinone‐*n* and often abbreviated as MK‐*n* [2, 6, 7]. Some bacteria produce isoprenologues in which one or more of the prenyl units are saturated. The additional hydrogen atoms are indicated with the prefix dihydro‐, tetrahydro‐, and so on and are

Vitamin K is fat soluble. The melting points of menaquinones vary from 35°C to 62°C depend‐ ing on the length of the multiprenyl side chain. Menaquinones are stable to heat and air but

The distribution of isoprenoid quinones has been studied in 900 microbial strains, 56 mold strains, and 88 yeast strains. About half of the studied bacteria contain menaquinone, but no menaquinones have been found in molds and yeast [10]. Menaquinone and demethylmena‐ quinones (DMKs) are found in the cytoplasmic membrane of bacteria. MKs and DMKs function as a reversible redox component of the electron transfer chain [11]. Additionally, reduced MKs exhibit antioxidant properties and can play a role in protecting cellular membranes from lipid

items on health aspects, such as bone health or cardiovascular health.

of saturation of the polyisoprenoid side chain attached to the 3‐position.

mendations for vitamin K.

64 Vitamin K2 - Vital for Health and Wellbeing

**2. Structure of vitamin K**

abbreviated MK‐*n*(H2), MK‐*n*(H4), etc. [8].

are very sensitive to alkali and ultraviolet (UV) irradiation [9].

**3. Functions and biosynthesis of menaquinones in bacteria**

Bacterially synthesized menaquinones that contribute to human vitamin K2 requirements may be produced by the gut microbiota or by bacteria present in food. In humans, the most important genera of intestinal flora are *Bacteroides* and *Bifidobacteria*. However, only *Bacteroides* can synthesize menaquinone. The major forms produced by *Bacteroides* are MK‐10 and MK‐11. MK‐6 produced by *Eubacterium lentum*, MK‐7 produced by *Veillonella*, and MK‐8 produced by *Enterobacter* were also found in isolates from intestinal flora [2, 7, 8, 20]. Most menaquinones are present in the distal colon, but the most promising site of absorption is the terminal ileum, where there are menaquinone‐producing bacteria and bile salts that are needed for solubili‐ zation of menaquinones [7, 21]. Therefore, although intestinal microflora synthesize large amounts of menaquinones, the bioavailability of bacterial menaquinone is poor, and diet is the major source of functionally available vitamin K2 [3, 7, 8]. Recent studies also showed that a short‐term decrease in dietary vitamin K intake is not compensated by intestinal menaqui‐ nones [22–24].

#### **5. Dietary sources of menaquinones**

Vitamin K2 is mostly synthesized by bacteria; therefore, the highest number of long‐chain menaquinones is found in fermented dairy products, such as cheese and fermented vegetables, such as natto and sauerkraut [16]. One exception is MK‐4, which is formed by a realkylation step from menadione present in animal feed or as a product of tissue‐specific conversion directly from dietary phylloquinone [5]. The extent of the conversion to MK‐4 is estimated to range from 5% to 25% of the ingested phylloquinone [25].

**Menaquinone content (μg/100 g; mean ± SD or range)**

*Fermented milk*

*Yogurt*

*Cheese*

**Food MK-4 MK-5 MK-6 MK-7 MK-8 MK-9 MK-10 Source**  Cream 8 ± 3 nr nr nd nr nr nr [32] Butter 13.5–15.9 nd nd nd nd nd nr [6]

Whole milk, sour 0.6 ± 0.02 0.3 ± 0.002 0.2 ± 0.03 0.4 ± 0.04 2.0 ± 0.1 4.7 ± 0.2 nd [31] Buttermilk 0.2–0.3 0.1–0.2 0–0.2 0.1–0.3 0.5–0.6 1.2–1.6 nr [6] Mesophilic nr nr 4.2 5 25.9 100.8 8.5 [34] Thermophilic nd nd nd nd nd nd nd [34]

Whole 0.4–1.0 nr nr nr nr 0–2.0 nr [29] Whole 0.5–0.7 0–0.2 nd nd nd nd nr [6] Whole 1 ± 0.1 nr nr 0.1 ± 0.2 nr nr nr [32] Plain 0.4 ± 0.03 0.1 ± 0.006 nd nd nd nd nd [31] Skimmed nd nd nd nd 0–0.2 nd nr [6]

Curd 0.3–0.6 0–0.2 0.1–0.3 0.2–0.5 4.8–5.4 18.1–19.2 nr [6] Curd 2–10 nr nr nr nr 40–70 nr [29] Hard 4.2–6.6 1.3–1.7 0.6–1.0 1.1–1.5 14.9–18.2 45.3–54.9 nr [6] Semi‐hard nr nr 1.9 1.1 3.9 17.5 4.7 [34] Soft 3.3–3.9 0.2 –0.4 0.5–0.7 0.9 – 1.1 10.7–12.2 35.1–42.7 nr [6] Soft nr nr 1.7 1.2 7.0 27.3 2.9 [34] Processed 5 ± 2 nr nr 0.3 ± 0.1 nr nr nr [32] Blue cheese nr nr 4.9 12.4 7.7 19.3 2.9 [34] Appenzeller 4.3–5.2 nr nr nr nr nr nr [33] Caerphilly nr nr 1.6 ± 0.1 nd 1.6 ± 0.1 32.4 ± 0.8 nd [34] Cheddar 10.2 nr nr nr nr nr nr [27] Cheddar nr nr 2.2 2.1 3.2 12.9 5.2 [34] Cheshire nr nr 1.6± 0.2 nd 5.8 ± 0.2 24.2 ± 0.4 nd [34] Comté 5.5–8.4 nr nr nr nr nr nr [33] Comté nd nd nd nd nd nd nd [34] Edam 3.3 ± 0.2 1.0 ± 0.1 0.6 ± 0.1 1.3 ± 0.1 10.5 ± 0.8 30.0 ± 2.6 0.9 ± 0.1 [31] Emmental 8.1–8.6 nr nr nr nr nr nr [33] Emmental nr nr nd nd nd nd 4.0 [34]

21 ± 7 nr nr nd nr nr nr [32]

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Searching for information about the concentration of vitamin K2 in food is not very fruitful. Out of more than 70 national food databases, only 12 provide the vitamin K content of food items. Only three of these food databases (the United States, the Netherlands, Turkey) specifically report the vitamin K2 concentration; all others publish only phylloquinone (PK) or total vitamin K or give no further information about the vitamin forms included in the given values. The comparison of the provided concentration of MK in these three databases is not possible because the values are based on different specifications and different processes. The data given in the US database are for MK‐4. However, the Dutch database includes several types of menaquinones, ranging from MK‐4 to MK‐10. For the data from the Turkish database, there is no information concerning the definition of vitamin K2 [16, 26]. In countries where animals are supplemented with menadione as practiced in the United States [27] and the Netherlands [28], the MK‐4 concentration is normally higher in food of animal origin. The supplementation practice used in Turkey is unknown. Last, the process and the bacterial strains used in the production of fermented food determine the concentration and forms of MK in products [16].

Scanning the literature for publications that report the results of vitamin K measurements in food provides additional separate values for different menaquinones. However, information about longer‐chain menaquinones (MK‐5 to MK‐10) is very limited. **Table 1** summarizes the values of vitamin K2 for animal products such as dairy, meat, fish and eggs, and fermented vegetable products such as bread, sauerkraut, and legumes (natto).



**5. Dietary sources of menaquinones**

66 Vitamin K2 - Vital for Health and Wellbeing

products [16].

*Dairy*

range from 5% to 25% of the ingested phylloquinone [25].

Vitamin K2 is mostly synthesized by bacteria; therefore, the highest number of long‐chain menaquinones is found in fermented dairy products, such as cheese and fermented vegetables, such as natto and sauerkraut [16]. One exception is MK‐4, which is formed by a realkylation step from menadione present in animal feed or as a product of tissue‐specific conversion directly from dietary phylloquinone [5]. The extent of the conversion to MK‐4 is estimated to

Searching for information about the concentration of vitamin K2 in food is not very fruitful. Out of more than 70 national food databases, only 12 provide the vitamin K content of food items. Only three of these food databases (the United States, the Netherlands, Turkey) specifically report the vitamin K2 concentration; all others publish only phylloquinone (PK) or total vitamin K or give no further information about the vitamin forms included in the given values. The comparison of the provided concentration of MK in these three databases is not possible because the values are based on different specifications and different processes. The data given in the US database are for MK‐4. However, the Dutch database includes several types of menaquinones, ranging from MK‐4 to MK‐10. For the data from the Turkish database, there is no information concerning the definition of vitamin K2 [16, 26]. In countries where animals are supplemented with menadione as practiced in the United States [27] and the Netherlands [28], the MK‐4 concentration is normally higher in food of animal origin. The supplementation practice used in Turkey is unknown. Last, the process and the bacterial strains used in the production of fermented food determine the concentration and forms of MK in

Scanning the literature for publications that report the results of vitamin K measurements in food provides additional separate values for different menaquinones. However, information about longer‐chain menaquinones (MK‐5 to MK‐10) is very limited. **Table 1** summarizes the values of vitamin K2 for animal products such as dairy, meat, fish and eggs, and fermented

**Food MK-4 MK-5 MK-6 MK-7 MK-8 MK-9 MK-10 Source** 

Whole milk 0.7–0.9 0.0–0.1 nd nd nd nd nr [6] Whole milk 0.8–1.0 nr nr nr nr nr nr [27] Whole milk 2 ± 0.3 nr nr nd nr nr nr [32] Whole milk 0.4–1.0 nr nr nr nr 0–2 nr [29] Milk 1% fat 0.3–0.4 nr nr nr nr nr nr [27] Milk 2% fat 0.4–0.5 nr nr nr nr nr nr [27] Whipped cream 5.2–5.6 nd nd nd nd nd nr [6]

vegetable products such as bread, sauerkraut, and legumes (natto).

**Menaquinone content (μg/100 g; mean ± SD or range)**


**Menaquinone content (μg/100 g; mean ± SD or range)**

Chicken meat, leg and thigh

Rainbow trout, cultivated

*Fish*

*Eggs*

*Bread*

*Plant products*

Hikiwari natto (chopped natto)

**Food MK-4 MK-5 MK-6 MK-7 MK-8 MK-9 MK-10 Source** 

Chicken muscle 8.9 nr nd nd nr nr nr [30]

Pike perch 0.2 ± 0.025 0.05 ± 0.0044 0.05 ± 0.0008 0.5 ± 0.13 nd nd nd [31] Baltic herring 0.21 ± 0.002 nr nd nd nd nd nd [31] Horse mackerel 0.6 ± 0.1 nr nr nd nr nr nr [32] Mackerel 1 ± 0.2 nr nr nd nr nr nr [32] Mackerel 0.3–0.5 nd nd nd nd nd nr [6] Salmon 0.2–0.3 nr nr nr nr nr nr [27] Plaice 0.1–0.3 nd 0.2–0.3 0.0–0.1 1.3–1.8 nr nr [6] Eel 1.4–2.1 nd 0.0–0.2 0.2–0.6 nd nd nr [6] Salmon 0.4–0.6 nd nd nd nd nd nr [6]

Egg yolk 29.1–33.5 nd 0.6–0.8 nd nd nd nr [6] Egg albumen 0.8–1.0 nd nd nd nd nd nr [6] Whole egg 7 ± 3 nr nr nd nr nr nr [32] Egg white 1 ± 1 nr nr nd nr nr nr [32] Egg yolk 64 ± 31 nr nr nd nr nr nr [32] Whole egg 5.6 nr nr nr nr nr nr [27] Egg white 0.4 nr nr nr nr nr nr [27] Egg yolk 15.5 nr nr nr nr nr nr [27]

Bread 0 nr nr nr nr 0.9–2 nr [29] Buckwheat nd nd nd 1.0–1.2 nd nd nr [6]

Sauerkraut 0.3–0.5 0.6–1.0 1.4–1.6 0.1–0.3 0.6–0.9 0.9–1.3 nr [6] Natto nd 7.1–7.8 12.7–14.8 882–1034 78.3–89.8 nd nr [6]

2 ± 3 nr nr 939 ± 753 nr nr nr [32]

nd nr nr 827 ± 194 nr nr nr [32]

60 ± 8.2 nd nd nd nd nd nd [31]

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3.1 ± 0.2 0.09 ± 0.019 nd 0.2 ± 0.058 nd nd nd [31]


**Menaquinone content (μg/100 g; mean ± SD or range)**

68 Vitamin K2 - Vital for Health and Wellbeing

*Meat*

**Food MK-4 MK-5 MK-6 MK-7 MK-8 MK-9 MK-10 Source**  Aged 90 d 5.2 ± 0.1 nd trace trace nd nd nd [31] Aged 180 d 6.1 ± 0.5 nd trace nd nd nd nd [31] Gamalost 1.0 ± 0.0 0.6 ± 0.0 0.3 ± 0.0 0.9 ± 0.1 4.8 ± 0.7 42.3 ± 7.0 2.1 ±0.4 [35] Jarlsberg 8.4 nr nr nr nr nr nr [33] Gruyère 8.1–9.6 nr nr nr nr nr nr [33] Leicester nr nr 2.0 ± 0.1 2.1 ± 0.1 4.8 ± 0.2 16.2 ± 0.3 4.4 ± 0.2 [34] Mozzarella 3.1–4.0 nr nr nr nr nr nr [27] Mozzarella nd nd nd nd nd nd nd [34] Norvegia 5.1 ± 0.9 nd 0.3 ± 0.1 1.3 ± 0.2 5.3 ± 0.5 29.6 ± 3.6 nd [35] Raclette 5 nr nr nr nr nr nr [33] Swiss cheese 6.2–8.8 nr nr nr nr nr nr [27]

Salami 8.2–10.1 nd nd nd nd nd nr [6] Calf liver 1.1–8.9 nr nr nr nr nr nr [27] Beef liver 0.4 ± 0.4 nr nr nr nr nr nr [27] Bovine liver 6.8 ± 1.03 nd 9.44 ± 0.118 25.6 ± 0.59 13.8 ± 0.55 9.8 ± 0.7 14±1.7 [31] Beef liver 0.8 nr 2.5 18.2 4.8 1.5 6.6 [30] Pork liver 0.3–0.4 nd nd nd nd nd nd [6] Pork liver 10.8 ± 1.44 nd nd 16 ± 2.7 25 ± 5.2 6 ± 1.8 8±2.9 [31] Pork liver 0.6 nd 0.04 0.6 0.5 0.3 0.5 [30] Chicken liver 14.1 ± 2.0 nr nr nr nr nr nr [27] Chicken liver 4 nr 0.03 nd 0.09 0.04 0.03 [30] Beef kidney 2.1 nr 0.08 0.2 0.01 nd 0.1 [30] Pork kidney 1.3 nr 0.02 0.07 0.05 0.22 0.24 [30] Chicken kidney 5 nr nd nd nd nd nd [30] Beef muscle 3.4 nr 0.03 0.03 nr nr nr [30] Pork thigh 6 ± 2 nr nr nr nr nr nr [32] Pork steak 1.7–2.4 nd nd 0.4–0.7 0.9–1.2 nd nd [6] Pork chop 3.1 ± 0.46 nd nd 0.12 ± 0.035 nd nd nd [31] Pork muscle 0.9 nr 0.03 0.03 nr nr nr [30] Chicken breast 6.4–11.3 nd nd nd nd nd nd [6] Chicken leg 5.8–10.5 nd nd nd nd nd nd [6] Chicken thigh 27 ± 15 nr nr nd nr nr nr [32]


found a correlation between MK‐9 and MK‐8. For most dairy, the MK‐9 level was four times higher than that of MK‐8, and the authors suggested that microorganisms that produce MK‐9 could also produce MK‐8. Astonishingly, the level of MK‐9 was not dependent on the fat level of the dairy products. Moreover, the authors found no link between pH and the MK‐9 content. The highest amounts of MK‐10 are usually found in hard cheese, with the exception of one

In Swiss Emmental cheese, *Propionibacterium* strains are added to the milk to improve the formation of holes inside the cheese body. During propionic acid fermentation, lactic acid is transformed into propionic acid, acetic acid, and carbon dioxide. Various studies showed the ability of *Propionibacterium* to produce menaquinone MK‐9(4H) in anaerobic conditions [39, 40]. The highest amount of MK‐9(4H) has been detected in Swiss Emmental (up to 31.4 μg/ 100 g MK‐9(4H)) and Norwegian Jarlsberg (65.2 μg/100 g MK‐9(4H)); both cheeses have a high propionic acid concentration. Smaller amounts are also found in Appenzeller (up to 2 μg/

In contrast, dairy products fermented with thermophilic lactic acid bacteria, such as Comté cheese, mozzarella, or yogurt products, contain only small amounts of menaquinone or none (**Table 1**). These thermophilic species include *Streptococcus thermophilus*, *Lactobacillus delbrueck‐*

*ii*, and *Bifidobacterium*, and they are known to be non‐vitamin K producers [2, 34].

Cheese, buttermilk, sour cream, cottage cheese, cream cheese, kefir, yogurt

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semihard cheese [34].

*lactis* and *Lactococcus lactis* ssp.

*Lactococcus lactis* ssp.

*cremoris*

**Species/subspecies Food use** 

*Lactococcus raffinolactis* Cheese *Leuconostoc lactis* Cheese

*Brevibacterium linens* Cheese *Brochothrix thermosphacta* Meat *Hafnia alvei* Cheese

*Propionibacterium shermanii* Cheese *Propionibacterium freudenreichii* Cheese

Adapted from Walther et al. [16].

*Leuconostoc mesenteroides* Vegetables, dairy

*Staphylococcus xylosus* Dairy, sausage *Staphylococcus equorum* Dairy, meat *Arthrobacter nicotianae* Cheese

*Bacillus subtilis "natto"* Natto (fermented soybean)

**Table 2.** Menaquinone‐producing bacteria in fermented food.

100 g), Comté (up to 6.0 μg/100 g), and Raclette (4.7 μg/100 g) [33].

**Table 1.** Representative ranges of measured menaquinone concentration in food.

Values for MK‐4 to MK‐10 are available. MK‐4 is found in all reported products except buckwheat, hikiwari natto, and black bean natto [6, 27, 29–35]. In non‐fermented dairy products and in eggs, hardly any longer‐chain menaquinones have been reported [6, 27, 29, 32]. Long‐ chain menaquinones are also rare in the muscle meat of beef, pork, and chicken [6, 30–32]. However, in offal, such as the liver and kidney, small‐to‐moderate concentrations of MK‐6 to MK‐10 have been detected [6, 27, 30, 31]. In fish, vitamin K2 concentrations are in general very low, and menaquinones other than MK‐4 have been found in only a few fish species [6, 27, 31, 32]. These small amounts of longer‐chain menaquinones are said to originate from the bacteria in decomposing organic material that serves as food for fish that live at the bottom of the sea such as eel and plaice [36]. In sour milk and buttermilk and in curd and hard and soft cheese, MK‐8 and MK‐9 mainly account for the total concentration of vitamin K followed by MK‐6 and MK‐7 [6, 27, 29, 31–35]. Fermented plant products are characterized by a high concentra‐ tion of MK‐7 (up to 1000 μg/100 g) [6, 32].

Almost no data are available about the stability and changes in vitamin K concentrations during storage of food in general and during ripening of fermented food in particular.

#### **6. Production of different menaquinones by microorganisms in food**

Fermentation is traditionally used to increase shelf life, to inhibit pathogens, and to improve organoleptic properties [37]. Additionally, the microbial production of vitamins provides a very attractive approach for improving the nutritional composition of fermented foods. A number of MK‐producing species are commonly used in industrial food fermentation applications (**Table 2**). The main microorganisms used in fermented dairy products are lactic acid bacteria, which transform lactose into lactic acid. *Lactococcus lactis* ssp. *cremoris*, *Lactococcus lactis* ssp. *lactis*, and *Leuconostoc lactis* are used as starter cultures in semihard and soft cheeses. It was reported that these species produce menaquinone and MK‐7 to MK‐9 in particular for *Lactococcus* and MK‐7 to MK‐10 for *Leuconostoc* [2, 38]. For example, the starter cultures CHN211 and CHN22 from Hansen, which contain these species, produce MK‐4 to MK‐10; MK‐9 is the main menaquinone with 472.4 ± 22.6 μg/100 g cells and 390.3 ± 10.4 μg/100 g cells, respectively [35]. Accordingly, the highest amounts of MK were detected in semihard and soft cheese and in Caerphilly and Cheshire, a crumbly cheese specialty, known for higher num‐ bers of *Lactococcus* species (**Table 1**). In semihard cheese, menaquinones in amounts up to 29.1 μg/100 g have been detected. The main quantified form of menaquinone in dairy is MK‐9 (usually more than 50%), and the second major form is MK‐8. Manoury and coauthors also found a correlation between MK‐9 and MK‐8. For most dairy, the MK‐9 level was four times higher than that of MK‐8, and the authors suggested that microorganisms that produce MK‐9 could also produce MK‐8. Astonishingly, the level of MK‐9 was not dependent on the fat level of the dairy products. Moreover, the authors found no link between pH and the MK‐9 content. The highest amounts of MK‐10 are usually found in hard cheese, with the exception of one semihard cheese [34].


**Table 2.** Menaquinone‐producing bacteria in fermented food.

**Menaquinone content (μg/100 g; mean ± SD or range)**

tion of MK‐7 (up to 1000 μg/100 g) [6, 32].

**Table 1.** Representative ranges of measured menaquinone concentration in food.

nd, not determined; nr, not reported.

70 Vitamin K2 - Vital for Health and Wellbeing

**Food MK-4 MK-5 MK-6 MK-7 MK-8 MK-9 MK-10 Source**  Black bean natto nd nr nr 796 ± 93 nr nr nr [32]

Values for MK‐4 to MK‐10 are available. MK‐4 is found in all reported products except buckwheat, hikiwari natto, and black bean natto [6, 27, 29–35]. In non‐fermented dairy products and in eggs, hardly any longer‐chain menaquinones have been reported [6, 27, 29, 32]. Long‐ chain menaquinones are also rare in the muscle meat of beef, pork, and chicken [6, 30–32]. However, in offal, such as the liver and kidney, small‐to‐moderate concentrations of MK‐6 to MK‐10 have been detected [6, 27, 30, 31]. In fish, vitamin K2 concentrations are in general very low, and menaquinones other than MK‐4 have been found in only a few fish species [6, 27, 31, 32]. These small amounts of longer‐chain menaquinones are said to originate from the bacteria in decomposing organic material that serves as food for fish that live at the bottom of the sea such as eel and plaice [36]. In sour milk and buttermilk and in curd and hard and soft cheese, MK‐8 and MK‐9 mainly account for the total concentration of vitamin K followed by MK‐6 and MK‐7 [6, 27, 29, 31–35]. Fermented plant products are characterized by a high concentra‐

Almost no data are available about the stability and changes in vitamin K concentrations during storage of food in general and during ripening of fermented food in particular.

Fermentation is traditionally used to increase shelf life, to inhibit pathogens, and to improve organoleptic properties [37]. Additionally, the microbial production of vitamins provides a very attractive approach for improving the nutritional composition of fermented foods. A number of MK‐producing species are commonly used in industrial food fermentation applications (**Table 2**). The main microorganisms used in fermented dairy products are lactic acid bacteria, which transform lactose into lactic acid. *Lactococcus lactis* ssp. *cremoris*, *Lactococcus lactis* ssp. *lactis*, and *Leuconostoc lactis* are used as starter cultures in semihard and soft cheeses. It was reported that these species produce menaquinone and MK‐7 to MK‐9 in particular for *Lactococcus* and MK‐7 to MK‐10 for *Leuconostoc* [2, 38]. For example, the starter cultures CHN211 and CHN22 from Hansen, which contain these species, produce MK‐4 to MK‐10; MK‐9 is the main menaquinone with 472.4 ± 22.6 μg/100 g cells and 390.3 ± 10.4 μg/100 g cells, respectively [35]. Accordingly, the highest amounts of MK were detected in semihard and soft cheese and in Caerphilly and Cheshire, a crumbly cheese specialty, known for higher num‐ bers of *Lactococcus* species (**Table 1**). In semihard cheese, menaquinones in amounts up to 29.1 μg/100 g have been detected. The main quantified form of menaquinone in dairy is MK‐9 (usually more than 50%), and the second major form is MK‐8. Manoury and coauthors also

**6. Production of different menaquinones by microorganisms in food**

In Swiss Emmental cheese, *Propionibacterium* strains are added to the milk to improve the formation of holes inside the cheese body. During propionic acid fermentation, lactic acid is transformed into propionic acid, acetic acid, and carbon dioxide. Various studies showed the ability of *Propionibacterium* to produce menaquinone MK‐9(4H) in anaerobic conditions [39, 40]. The highest amount of MK‐9(4H) has been detected in Swiss Emmental (up to 31.4 μg/ 100 g MK‐9(4H)) and Norwegian Jarlsberg (65.2 μg/100 g MK‐9(4H)); both cheeses have a high propionic acid concentration. Smaller amounts are also found in Appenzeller (up to 2 μg/ 100 g), Comté (up to 6.0 μg/100 g), and Raclette (4.7 μg/100 g) [33].

In contrast, dairy products fermented with thermophilic lactic acid bacteria, such as Comté cheese, mozzarella, or yogurt products, contain only small amounts of menaquinone or none (**Table 1**). These thermophilic species include *Streptococcus thermophilus*, *Lactobacillus delbrueck‐ ii*, and *Bifidobacterium*, and they are known to be non‐vitamin K producers [2, 34].

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 are so rich in menaquinones [34].

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

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

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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

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

dairy products [16, 49].

a range from 0 to 340 μg.

**8. Dietary intake of menaquinones**

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 the differences in menaquinone content [35].

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 significantly increased amount of vitamin K2 have been deposed.

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 oil in supplementary and dietary food products [47].
