**2. Fermented soybean food products**

prothrombin and partial prothrombin times. Additionally, inadequacy of vitamin K also affects the bone mineral density (BMD) that leads toward higher bone fracture rate among population especially postmenopausal women and increased the risk of coronary artery diseases [1, 2].

Vitamin K deficiency is one of the alarming dilemmas among newborns, teenagers, and postmenopausal women. Neonates and their nursing mothers are 33.3% and 65% deficit in vitamin K, respectively [3]. Moreover, gastrointestinal disorders, unnecessary use of antibiot‐ ics, and alcoholics are some potential causes of vitamin K deficiency that hinder the fat incorporation in the body, and thus absorption of this vitamin is reduced [4]. Similarly, continuous utilization of broad‐spectrum antibiotics suppresses the synthesis of vitamin K in the intestinal gut, whereas antagonist drugs badly effect to vitamin K functioning that delays the blood coagulation and necessary modifies certain protein which are indispensable for bone health [5]. Likewise, inflammatory bowel disease individual also showed vitamin K deficiency prevalence that declined the bone mineral density possibly due to its malabsorption [6].

The ingestion of vitamin rich dietary sources is rational approach to manage the vitamin K deficiency population [7]. Additionally, supplementation of vitamin K in the diet is also improved the serum vitamin K concentration [8, 9]. In this context, pharmacological dose of vitamin K (45 mg/day) is effective to ameliorate problem of bone fractures [10]. Therefore, more osteocalcin carboxylation is attained via consumption of well above recommended dietary

Vitamin K predominantly, naturally, presents in the form of phylloquinone (vitamin K1) and menaquinone (vitamin K2). Primarily, phylloquinone is present in green leafy vegetables such as spinach, kale, broccoli, and certain vegetable oils [12]. However, menaquinones are present

Fermented soybean (natto) is one of the richest sources of menaquinones principally mena‐ quinone‐7 (MK‐7) (882–1034 μg/100 g) among other fermented plant and animal‐based food products [8, 13]. Menaquinone‐7 containing natto is prepared by the fermentation process with *Bacillus subtilis*. Raw soybean is converted into slimy and sticky food predominantly owing to glutamic residues [14]. This menaquinone is also available in other food products such as cheese, meat and meat‐based products, and fermented milk. Menaquinones are also synthe‐ sized by intestinal microflora such as *Escherichia coli*, *Bacteroides vulgatus,* and *Bacteroides fragilis*

From total vitamin K dietary intake, menaquinone‐4 (MK‐4), menaquinone‐7 (MK‐7), and phylloquinone contribute around 7, 24, and 60%, respectively [16]. Humans and laboratory animals have ability to transform phylloquinone into MK‐4 and MK‐7 by rearrangement of integral side chain that has the ability to strengthen the bone [17]. In both phylloquinone and menaquinones, total vitamin K is about 1.16 μg/kg body weight is required for normal functioning. Menaquinone‐7 normally stored around 10% in the liver. However, only 5–25% of ingested vitamin K is catabolized to MK‐4 followed by the conversion of menaquinones in

requirement amount of vitamin K [11].

36 Vitamin K2 - Vital for Health and Wellbeing

[15].

the liver via prenylation [10].

in the fermented soybean and animal products [8].

Traditionally, *Bacillus subtilis* fermented soybean food products are consumed in different parts of world. Among the fermented soybean products, natto is attained special attention which is consumed in Japan, whereas similar products such as kinema is produced in India, Satlyangser and Bari in Bhutan, Thua‐Nao in Thailand, Tao‐si in Philippines, Douchi in China and Dawadawa in Nigeria, Pepok in Myanmar. Kinema is similar natto fermented soybean which is originated from south China and expanded in Nepal, India, Burma, Thailand, and Japan [18– 20].

Soybean conquers special attention in the public owing to its protein and fat contents. Numerous cultivars are currently available for household consumption. Among, yellow, green, and black soybeans are commonly used after cooking or fermentation. Bacillus fermented products such as natto and cheonggukjang are also consumed [21, 22]. *B. subtilis* fermented soybean has slimy appearance, softer texture, and white goosy substance with distinctive rotten flavor. Moreover, fermentation also removes the beany odor of raw soybean completely and improves its hedonic response [19]. However, large number of population does not like its slimy appearance and powerful rotten odor of fermented soybean such as kinema, while other has enjoyable experience. Freshly prepared fermented soybean is consumed as a fried curry with boiled rice or mixed with vegetables or consumed with boiled rice, soup, and pickle [20].

Researchers are trying to identify the new strains that may have potential similar *B. subtilis* and have ability to produce ample amount of MK‐7 in the soybean. In this context, *B. amyloliquefaciens KCTC11712BP* under its optimum conditions remarkably enhanced MK‐7 concentration in cheonggukjang (fermented soybean) and has shorter production time than that of natto [23]. Menaquinones are produced by microorganisms during electron transport chain respiration process [24].

Moreover, natto is rich in menaquinones especially MK‐7 followed by other menaquinones such as MK‐5, MK‐6, MK‐8 which are ranged from 882–1034 μg/100 g, 7.1–7.8 μg/100 g, 12.7– 14.8 μg/100 g, 78.3–89.8 μg/100 g, respectively [13]. Similarly, Berenjian et al. [25] also reported that MK‐7 content in fermented soybean is varied from 800–900 μg/100 g. Moreover, Booth [26] also reported that natto contains 998 μg/100 g MK‐7. Previously, Dajanta et al. explicated that low fat (15.6%), high protein (45.8%), and small seed size of soybean are ideal characteristics of soybean for nato preparation [22]. Moreover, fermentation with *B. subtilis* exerts better nutritional attributes and sensory acceptability of the developed natto. Earlier, *B. subtilis* fermented Pakistani soybean also improved the concentration of MK‐7 ranged from 681.35 to 803.82 μg/100 g. Contrary, MK‐7 was not present in freshly cooked or reconstituted spinach [2]. The details of menaquinones concentration in various fermented soybean products are summarized in **Table 1**.


**2.3. Kinema**

**2.4. Hawaijar**

**2.5. Cheonggukjang**

the lack of pathogenicity [20].

it can be stored for several months [18].

Cleaned and pre‐soaked soybeans are autoclaved at 121°C for 35 min and cooled to 30–35°C. The soybean is inoculated with active culture of *B. subtilis*. The inoculated soybeans are placed for fermentation at 37°C for 48 h at 85% RH. The activity of bacteria is reduced by using the refrigeration temperature for 12 h. Fresh kinema is immediately consumed because its shelf life is about 2–3 days in summer and 5–7 days in winter without refrigeration. After sun drying,

Vitamin K2 Rich Food Products http://dx.doi.org/10.5772/63902 39

Generally, small‐sized soybean seeds are used for the production of hawaijar. Soybean seeds are boiled without soaking and packed loosely in bamboo basket lined with fig or banana leaves. Natural fermentation process is completed within 3–5 days. Mixed microflora includ‐ ing *B. subtilis*, *B. cereus*, *B. licheniformis*, *Staphylococcus sciuri*, *Staphylococcus aureus*, *Providencia rettgeri*, *Alcaligenes* sp. are fermented organism present in the final product. Production of

Washed soybean seeds are soaked in tap water for 12 h. They are autoclaved at 121°C for 30  min and cooled up to 40°C prior to inoculation with pre‐culture cell suspension of *Bacillus amyloliquefaciens strain KCTC 11712BP*. General for the preparation of cheonggukjang inocu‐

Menaquinones are commonly synthesized by the action of intestinal microflora *B. vulgatus*, *B. fragilis,* and *E. coli* in human large intestine during electron transport chain reaction of respiration. Commercially, MK‐7 was produced by action of bacteria such as *E. coli*, *Flavobac‐ terium,* and *B. subtilis natto*. Moreover, among the fermented microflora, *B. subtilis* is consider one of the best options for the production of MK‐7 due to its safety aspects as well as potential to synthesis the range of menaquinones such as MK‐4 to MK‐8 [15, 24, 29]. These hydrolytic bacteria are significantly consumed and reduced the indigestible oligo‐ and polysaccharides. The *Bacillus* species also lowered the activity of anti‐nutrients of the soybean that hamper the

*B. subtilis* is rod shaped, aerobic, heat resistant, spore forming Gram‐positive bacteria. It has potential to synthesis number of enzymes such as proteolytic, amylolytic, and lipolytic enzymes during fermentation. The colonies of *B. subtilis* are irregular with hair‐like structure having somewhat sliming appearance. Food Drug Administration granted the status of generally *recognized* as safe (GRAS) to the *Bacillus subtilis* and its close relative strains due to

mucilage and ammonia flavor is the indicator of quality hawaijar [18].

availability of nutrients such as proteins and bioactive molecules [30].

lated soybean seeds were placed in fermented for 48 h [28].

**3. Menaquinones synthesizing organisms**

**Table 1.** Menaquinones content of various fermented soybean (μg/100 g or mL).

However, the level of MK‐7 in natto is still less the recommended daily amount of 180 μg/day that needs the utilization of 20–22 g natto/day. Many consumers find natto unpalatable; therefore, the ingestion of sufficient MK‐7 is impractical. In some countries such as Japan, China, Thailand, and India fermented soybean products are commonly consumed, whereas other consumers do not like the fermented soybean products due to unpalatable, slimy, and sticky in nature, so the ingestion of MK‐7 is insufficient among the population. Furthermore, the digestion and utilization of MK‐7 from fermented soybean products are less efficient in humans with aging. Hence, there is a need for production of concentrated, supplementary MK‐ 7 in the diet [25]. The extracted MK‐7 with oil is consumed in various functional foods to fulfill the recommended requirement of vitamin K.

#### **2.1. Natto**

Natto is a Japanese traditional food prepared by fermentation of soybean with *B. subtilis natto*. The sterilized soybeans are fermented at 40°C for 14–18 h till it was fully covered with a viscous, sticky, and string‐like material on surface. The resultant fermented soybean has characteristic odor and musty flavor [28].

#### **2.2. Thua nao**

For thua nao preparation, washed soybeans are soaked in water at ambient temperature for 16 h. Subsequently, soaked soybeans are autoclaved at 121°C for 40 min and cooled to 55°C after removing water. Powder culture of *B. subtilis* is mixed and incubated at 42 °C about 24 h for fermentation. Afterwards, fermented soybeans are placed at 4°C to hamper the bacterial activity.

#### **2.3. Kinema**

**Food Type Country MK‐4 MK‐5 MK‐6 MK‐7 References** Natto Fermented soybean Netherlands 7.5 13.8 998 84.1 [13] Natto Fermented soybean Japan – – – 939 [12] Hikiwari natto Chopped natto Japan ND – – 827 [12] Natto Fermented black soybean Japan – – – 796 [12] Natto Fermented soybean Japan ND – – 87–102 [23] Natto Fermented soybean Pakistan – – – 668–881 [2] Cheonggukjang Fermented soybean Korea ND – – 112–461 [23] Cheonggukjang Fermented soybean Korea 74–76 – – 271–1171 [23] Cheonggukjang Fermented soybean extract Korea – – – 1674–3438 [23] Sauerkraut Fermented vegetables Netherlands 0.4 0.8 1.5 0.2 [13] Cotton tofu Hard type Japan 0.04 – – – [12]

However, the level of MK‐7 in natto is still less the recommended daily amount of 180 μg/day that needs the utilization of 20–22 g natto/day. Many consumers find natto unpalatable; therefore, the ingestion of sufficient MK‐7 is impractical. In some countries such as Japan, China, Thailand, and India fermented soybean products are commonly consumed, whereas other consumers do not like the fermented soybean products due to unpalatable, slimy, and sticky in nature, so the ingestion of MK‐7 is insufficient among the population. Furthermore, the digestion and utilization of MK‐7 from fermented soybean products are less efficient in humans with aging. Hence, there is a need for production of concentrated, supplementary MK‐ 7 in the diet [25]. The extracted MK‐7 with oil is consumed in various functional foods to fulfill

Natto is a Japanese traditional food prepared by fermentation of soybean with *B. subtilis natto*. The sterilized soybeans are fermented at 40°C for 14–18 h till it was fully covered with a viscous, sticky, and string‐like material on surface. The resultant fermented soybean has

For thua nao preparation, washed soybeans are soaked in water at ambient temperature for 16 h. Subsequently, soaked soybeans are autoclaved at 121°C for 40 min and cooled to 55°C after removing water. Powder culture of *B. subtilis* is mixed and incubated at 42 °C about 24 h for fermentation. Afterwards, fermented soybeans are placed at 4°C to hamper the bacterial

ND = not detected; (–) = unknown or not reported.

38 Vitamin K2 - Vital for Health and Wellbeing

the recommended requirement of vitamin K.

characteristic odor and musty flavor [28].

**2.1. Natto**

**2.2. Thua nao**

activity.

**Table 1.** Menaquinones content of various fermented soybean (μg/100 g or mL).

Cleaned and pre‐soaked soybeans are autoclaved at 121°C for 35 min and cooled to 30–35°C. The soybean is inoculated with active culture of *B. subtilis*. The inoculated soybeans are placed for fermentation at 37°C for 48 h at 85% RH. The activity of bacteria is reduced by using the refrigeration temperature for 12 h. Fresh kinema is immediately consumed because its shelf life is about 2–3 days in summer and 5–7 days in winter without refrigeration. After sun drying, it can be stored for several months [18].

#### **2.4. Hawaijar**

Generally, small‐sized soybean seeds are used for the production of hawaijar. Soybean seeds are boiled without soaking and packed loosely in bamboo basket lined with fig or banana leaves. Natural fermentation process is completed within 3–5 days. Mixed microflora includ‐ ing *B. subtilis*, *B. cereus*, *B. licheniformis*, *Staphylococcus sciuri*, *Staphylococcus aureus*, *Providencia rettgeri*, *Alcaligenes* sp. are fermented organism present in the final product. Production of mucilage and ammonia flavor is the indicator of quality hawaijar [18].

#### **2.5. Cheonggukjang**

Washed soybean seeds are soaked in tap water for 12 h. They are autoclaved at 121°C for 30  min and cooled up to 40°C prior to inoculation with pre‐culture cell suspension of *Bacillus amyloliquefaciens strain KCTC 11712BP*. General for the preparation of cheonggukjang inocu‐ lated soybean seeds were placed in fermented for 48 h [28].

#### **3. Menaquinones synthesizing organisms**

Menaquinones are commonly synthesized by the action of intestinal microflora *B. vulgatus*, *B. fragilis,* and *E. coli* in human large intestine during electron transport chain reaction of respiration. Commercially, MK‐7 was produced by action of bacteria such as *E. coli*, *Flavobac‐ terium,* and *B. subtilis natto*. Moreover, among the fermented microflora, *B. subtilis* is consider one of the best options for the production of MK‐7 due to its safety aspects as well as potential to synthesis the range of menaquinones such as MK‐4 to MK‐8 [15, 24, 29]. These hydrolytic bacteria are significantly consumed and reduced the indigestible oligo‐ and polysaccharides. The *Bacillus* species also lowered the activity of anti‐nutrients of the soybean that hamper the availability of nutrients such as proteins and bioactive molecules [30].

*B. subtilis* is rod shaped, aerobic, heat resistant, spore forming Gram‐positive bacteria. It has potential to synthesis number of enzymes such as proteolytic, amylolytic, and lipolytic enzymes during fermentation. The colonies of *B. subtilis* are irregular with hair‐like structure having somewhat sliming appearance. Food Drug Administration granted the status of generally *recognized* as safe (GRAS) to the *Bacillus subtilis* and its close relative strains due to the lack of pathogenicity [20].

Administration of high single dose of MK‐7 (2000 mg/kg) did not impart any toxic effect in animal modeling study in both genders. Moreover, prolonged treatment of MK‐7 is considered also safe for human consumption due to its non‐toxic effect on biochemical, hematological, urinary, and histopathological parameters [31].

Likewise, natto has 40% protein and 24.68% fat contents. Controlled fermentation process improves the nutritional composition of natto than that of natural fermentation process [36]. Earlier, Wei and Chang delineated that the natto was prepared from four different soybean cultivars using *B. natto "Itobiki"* for moisture, protein, lipid, ash and carbohydrates as 59–61, 37–43, 20–23, 4–5, and 31–34%, respectively [37]. However, the natto synthesis through *B. natto NRRL B‐3393* exhibited the values ranged from 61–62, 39–44, 25–27, 4.72–4.86, and 25–29%, for

Vitamin K2 Rich Food Products http://dx.doi.org/10.5772/63902 41

Similarly, free amino acid contents were enhanced 60‐fold in fermented products [38]. Free fatty acids impart significant role in the development of unique flavor in fermented soybean. Earlier, Kiuchi et al. reported that carbon length of fatty acids was momentously increased during fermentation [39]. Moreover, the amount of vitamin B complex such thiamine, riboflavin, and niacin is momentously improved during *B. subtilis* fermentation process of cooked soybeans. Additionally, total folate concentration was enhanced during fermentation process of Tempeh (fermented soybean). Fermented soybeans such as kinema and natto have appreciable amount of mineral contents such as iron, magnesium, copper, zinc, sodium, and

Fermented soybean had higher amount of total phenolic, antioxidant capacities, and flavonoid contents in thua nao produce by black soybeans by pure culture of *B. subtilis TN51* compared with yellow soybeans and cooked non‐fermented soybeans. Fermented black soybean and yellow soybean with *B. subtilis* TN51 exhibited higher amount of total phenolics 27.05 and 35.88 mgGAE/g extract than that of non‐fermented products. Whereas total flavonoids contents of fermented black and yellow soybean were 3.50 and 1.38 mg catechin eq/g metha‐ nolic extract. However, contents of total phenolics and flavonoids were significant less in non‐ fermented black and yellow cooked soybeans. Likewise, 2,2‐diphenylpicrylhydrazyl (DPPH)‐ antiradical activity (IC50, mg/mL), and lipid peroxidation inhibition potential were higher in fermented black and yellow soybean compared with non‐fermented cooked soybean [22].

Total phenol content of kinema was 144% higher than the raw soybean. Moreover, kinema has better free radical scavenger ability and metal chelating power and improved reducing power than that of non‐fermented soybean. Therefore, it is suggested that kinema may has potential used as designer foods to alleviate oxidative stress [43]. Likewise, *B. subtilis* fermentation inclines to improve total polyphenolic and anthocyanins content about 10 and 250% during natto preparation from black soybeans, respectively. Moreover, DPPH radical scavenging activity has positive association with fermentation time and concentration of black soybean

Fermentation increases about 58% content of phytosterol in kinema [43]. Later, Moktan et al. [44] reported that the kinema had 144%, 44%, 147%, and 92% higher total phenolics content, antioxidant activity, DPPH scavenging activity, and Fe2+‐chelating activity, respectively, compared to non‐fermented cooked soybean. Similarly, better total phenolic contents and

these attributes.

calcium [40–42].

[19].

**6. Antioxidant potential of fermented soybean**

Recently, Indian researcher Puri et al. identified a *B. subtilis* strain (*Bacillus subtilis MTCC 2756*) for the biosynthesis of menaquinone‐7 for commercial purposes [32]. They found that addition of Tween 80 in fermentation medium significantly boost the MK‐7 yield. Likewise, for the production of MK‐7, groundnut meal is also used as a substrate owing to its protein and oil quality with strong minerals and vitamins profile using *Flavobacterium* sp. *SP‐L‐01* [33].
