Vitamin B₁₂ sources and microbial interaction

Short abstract

Vitamin B₁₂ is synthesized only by certain bacteria and archaeon, but not by plants. The synthesized vitamin B₁₂ is transferred and accumulates in animal tissues, which can occur in certain plant and mushroom species through microbial interaction. In particular, the meat and milk of herbivorous ruminant animals (e.g. cattle and sheep) are good sources of vitamin B₁₂ for humans. Ruminants acquire vitamin B₁₂, which is considered an essential nutrient, through a symbiotic relationship with the bacteria present in their stomachs. In aquatic environments, most phytoplankton acquire vitamin B₁₂ through a symbiotic relationship with bacteria, and they become food for larval fish and bivalves. Edible plants and mushrooms rarely contain a considerable amount of vitamin B₁₂, mainly due to concomitant bacteria in soil and/or their aerial surfaces. Thus, humans acquire vitamin B₁₂ formed by microbial interaction via mainly ruminants and fish (or shellfish) as food sources. In this review, up-to-date information on vitamin B₁₂ sources and bioavailability are also discussed.

Impact statement

To prevent vitamin B₁₂ (B₁₂) deficiency in high-risk populations such as vegetarians and elderly subjects, it is necessary to identify foods that contain high levels of B₁₂. B₁₂ is synthesized by only certain bacteria and archaeon, but not by plants or animals. The synthesized B₁₂ is transferred and accumulated in animal tissues, even in certain plant tissues via microbial interaction. Meats and milks of herbivorous ruminant animals are good sources of B₁₂ for humans. Ruminants acquire the essential B₁₂ through a symbiotic relationship with bacteria inside the body. Thus, we also depend on B₁₂-producing bacteria located in ruminant stomachs. While edible plants and mushrooms rarely contain a considerable amount of B₁₂, mainly due to concomitant bacteria in soil and/or their aerial surfaces. In this mini-review, we described up-to-date information on B₁₂ sources and bioavailability with reference to the interaction of microbes as B₁₂-producers.

Introduction

Vitamin B₁₂ (B₁₂) or cyanocobalamin is a member of the corrinoids that contain a corrin ring (Figure 1). Hydroxocobalamin, methylcobalamin, and 5′-deoxyadenosylcobalamin are chemically more labile than cyanocobalamin.¹ In particular, methylcobalamin is the cofactor of methionine synthase (EC 2.1.1.13), and 5′-deoxyadenosylcobalamin functions as the coenzyme of methylmalonyl-CoA mutase (EC 5.4.99.2), which catalyzes the conversion of (R)-methylmalonyl-CoA to succinyl-CoA in the catabolic pathway of amino acids and odd-chain fatty acids in mammals.^2,3

Figure 1.

Structural formula of vitamin B₁₂ and partial structures of vitamin B₁₂-related compounds. (1) 5′-Deoxyadenosylcobalamin, (2) methylcobalamin (3) hydroxocobalamin, (4) cyanocobalamin (vitamin B₁₂), (5) pseudovitamin B₁₂, (6) 5-methoxybenzimidazolyl cobamide, and (7) 2-methylmercaptoadenyl cobamide.

B₁₂ is synthesized by certain bacteria and archaeon, but not by plants or animals.⁴ Thus, B₁₂-synthesing bacteria (including archaeon) are sources of B₁₂ compounds found in foods. Both aerobic⁵ and anaerobic⁶ biosynthetic pathways of B₁₂ compounds exist. The lower ligand is attached to the cobalt-coordinated corrin ring via the nucleotide loop, and 5,6-dimethylbenzimidazole is usually found as a base. Anaerobic microorganisms can synthesize corrinoids carrying bases other than 5,6-dimethylbenzimidazole.⁷ Other than B₁₂, pseudovitamin B₁₂ (pseudoB₁₂), which contains adenine as a base, is the only cobamide found commonly in food.⁸ 5-Methoxybenzimidazolyl and 2-methylmercaptoadenyl cobamides are found in escargots.⁹

Ten years has passed since publication of my initial review concerning B₁₂ sources and bioavailability¹⁰ in this journal. For the last 10 years, liquid chromatography/electrospray ionization–tandem mass spectrometry has been widely used to analyze B₁₂ compounds, and various corrinoid compounds have been newly identified from food.⁸ In this mini-review, we describe up-to-date information on B₁₂ sources and bioavailability with reference to the interaction of microbes as B₁₂ producers.

Vitamin B₁₂ in animal-derived foods

Many studies concerning the association between dietary B₁₂ sources and serum (or plasma) B₁₂ levels (as a marker of B₁₂ status) indicate that meat, milk, and fish are associated with higher serum (or plasma) B₁₂, particularly in western countries.¹¹ Indeed, milk has been reported as the most important source of B₁₂ for increasing serum B₁₂ levels.^11–13 Various types of animal meats (e.g. beef, veal, mutton, and lamb) are derived from the muscles of ruminant animals (e.g. cattle and sheep). The remaining major meats (pork and poultry) are derived from omnivorous animals (pig and chicken) (Figure 2). Bovine milk and fermented milk (e.g. yogurt and cheese) are widely available dairy products and good B₁₂ sources.¹¹

Figure 2.

Vitamin B₁₂ sources and microbial interactions in meat, eggs, milk, and milk products. Cattle are herbivorous ruminant animals and their stomachs contain various microorganisms including B₁₂-synthesizing bacteria. The synthesized B₁₂ is absorbed in the intestine and stored in the liver and muscles of cattle or secreted into milk. Bovine milk and fermented milk (yogurt and cheese) are major dairy products for humans. Chickens are omnivores and eat both plants and animals that contain considerable amounts of B₁₂. The B₁₂ contents of raw meats are generally higher in cattle than in chicken.

Cattle and sheep are herbivores and eat plants like grass, which is free of B₁₂. These ruminants have stomachs consisting of four chambers that contain various microorganisms, including B₁₂-synthesizing bacteria.^14,15 The B₁₂ synthesized in the stomach is absorbed in the intestine, transferred into the blood and stored in the liver and muscles of the animal or secreted into the milk. The cobalt content of the diet is the most important factor affecting the synthesis of B₁₂ in ruminant microorganisms.¹⁶ Thus, cobalt-deficiency readily induces B₁₂-deficiency in ruminants.¹⁷ To enrich the B₁₂ content of meat and milk, various methods for increasing ruminant B₁₂ synthesis have been investigated.^18–20 Pigs and chickens are omnivores and eat both plants and animals, which are B₁₂ sources. The B₁₂ content of raw meat is generally higher in these ruminants than in pig or chicken,²¹ although the B₁₂ content of poultry meat may be increased by the administration of lactic bacteria.²² Chicken egg consumption does not appear to significantly contribute to higher serum B₁₂ in humans.¹¹

Meat

Raw livers of beef, pork, and chicken contain high B₁₂ (52.8, 25.2, and 44.4 μg/100 g wet weight, respectively)²¹ and are excellent sources of B₁₂. The B₁₂ content of raw meats (approximately 1.0–2.0 μg/100 g wet weight) is higher in beef than in pork (approximately 0.5 μg/100 g wet weight) or chicken (<0.5 μg/100 g wet weight),^21,23 suggesting that the meats and livers of ruminant animals contain higher amounts of B₁₂ relative to those of omnivorous animals. A considerable loss of B₁₂ has been reported after cooking beef, pork, and chicken meats.^23–25 The retention of B₁₂ in vacuum-cooked meats has been reported to be 100% for veal, lamb, and pork, and 87% for beef.²⁶ For more detailed information on animal sources of B₁₂, such as meat and dairy products, please refer to an excellent review cited in Gille and Schmid.²³

Milk

The B₁₂ concentrations in milk from ruminants such as sheep (0.71 μg/100 g of milk), cow (0.35 μg/100 g of milk), and goat (0.06 μg/100 g of milk) are higher than those found in human milk (0.04 μg/100 g of milk).²⁷ While the B₁₂ content of bovine milk is not high relative to beef meats, bovine milk and fermented milk (e.g. yogurt and cheese) are major B₁₂ sources because the intake of milk or dairy products is high in various populations.²⁸ The B₁₂ concentration of bovine milk varies according to many factors such as the cow type, breeding state, and milking time.^29,30 B₁₂ concentrations in milk from Holstein cows appears to be generally higher than those in milk from Jersey cows.^29,30 Rutten et al.³¹ found that a single nucleotide polymorphism (SNP) along the cow genome affects the B₁₂ concentration in milk (Figure 3). Although a significant association was found between 68 SNP and B₁₂ content in the raw milk of 487 first-lactation cows, this SNP was not found in the genes known to be involved in B₁₂ uptake or transport, implying that there are associations related to genes involved in unknown processes such as the ruminant production of B₁₂ or the secretion of B₁₂ by the mammary gland.³¹

Figure 3.

Single nucleotide polymorphism (SNP) and the vitamin B₁₂ content of bovine milk. A significant association was found between 68 SNPs and the B₁₂ content of raw milk in 487 first-lactation cows, but these SNPs were not in the following genes involved in B₁₂ uptake and transport systems.

GIF: gastric intrinsic factor; CUBN: cubilin; AMN: amnionless; ABCC1: ATP-binding cassette; sub-family C; member 1; CD320: transcobalamin receptor; TCN1: transcobalaminI (haptocorin); TCN2: transcobalaminII; LMBRD1: methylmalonic aciduria cblF type 1; MMACHC: methylmalonic aciduria cblC type; MMADHC: methylmalonic aciduria cblD type; MMAB: methylmalonic aciduria cblB type; MUT: methylmalonyl CoA mutase; MMAA: methylmalonic aciduria cblA type; MTR: 5-methyltetrahydrofolate-homocysteine methyltransferase; MTRR: 5-methyltetrahydrofolate-homocysteine reductase; LRP2: low-density lipoprotein receptor-related protein 2 (megalin).

The B₁₂ found in bovine milk mainly binds to transcobalamin, one of the mammalian B₁₂-binding proteins located in blood,³² whereas haptocorrin is the predominant B₁₂-binding protein in human milk.³³ The bioavailability of B₁₂ in cow’s milk appears to be higher than that of cyanocobalamin.³⁴

When the B₁₂ contents of 26 types of commercially available natural cheeses were determined,³⁵ the B₁₂ content was higher in hard and semi-hard cheeses (approximately 2.8 μg/100 g dry basis) and washed rind cheeses (approximately 4.2 μg/100 g dry basis) than in fresh (approximately 1.2 μg/100 g dry basis) or soft (approximately 1.8 μg/100 g dry basis) cheeses. Liquid chromatography/electrospray ionization—tandem mass spectrometry analysis has indicated that B₁₂ is the predominant corrinoid compound in the tested natural cheeses, but traces of unidentified corrinoid compounds were found in some of the tested cheeses.³⁵

An appreciable loss of B₁₂ occurs during the storage, thermal processing, and fermentation of milk.^23,25 Recently, Johns et al.³⁶ found that the rate of B₁₂ loss was three times greater in chocolate-flavored milk (approximately 33.5%) than in unflavored milk (approximately 15%) during heat treatment (1 h at 100°C). The increased loss of B₁₂ in chocolate-flavored milk was attributable to cocoa polyphenols that readily form peroxides.³⁶

The photodegradation of vitamin B₂ is well known to occur in milk during light exposure.³⁷ On exposure to light, vitamin B₂ forms free radicals, which cause the color change in milk.³⁸ A light exposure experiment of B₁₂ indicated that B₁₂ is decomposed by singlet oxygen formed in an aqueous solution.³⁹ In addition, a B₁₂ loss of 1–27% in commercially available milk products is caused by exposure to fluorescent light for 24 h at 4°C.⁴⁰ These observations suggest that storage in light accelerates the degradation of both vitamin B₂ and B₁₂ in milk.

Egg

Raw and boiled whole chicken eggs contain 0.9 µg of B₁₂ per 100 g wet weight of the edible portion,²¹ and most of the B₁₂ is located in the egg yolk.⁴¹ Although hens have been fed B₁₂-supplemented diets to enrich B₁₂ in eggs, egg yolk B₁₂ levels were reportedly not changed.⁴² Thus, the bioavailability of B₁₂ in egg dishes is considered very low (∼10%) due to the poor absorption of B₁₂ of eggs.^43,44 Accordingly, egg intake does not significantly contribute to higher serum B₁₂ in humans.¹¹

An egg product called a century egg (“Pidan” in Chinese) is an alkaline-fermented ethnic food in China. The egg yolks of these eggs contain 1.9 ± 0.6 µg of B₁₂ per 100 g wet weight. The B₁₂ present in the yolk of century eggs was recovered completely in macromolecular fractions.⁴⁵ However, approximately 52% of the free B₁₂ was formed from the century egg yolk during in vitro gastric digestion,⁴⁵ suggesting that century eggs may be a good source of B₁₂.

Fish and shellfish

People from Japan and France obtain most (84% and 64%, respectively) of their daily B₁₂ intake from fish and shellfish.^46,47 Scheers et al.⁴⁸ indicated that serum B₁₂ levels were significantly increased in subjects ingesting fish diets compared to meat diets, suggesting that B₁₂ is suitable as a marker for fish intake. Several studies have also indicated that fish and shellfish are important contributors to human B₁₂ status.^11,13,48

In aquatic environments, B₁₂ produced by certain bacteria and archaea is taken up by B₁₂-requiring bacteria, as well as eukaryotic phytoplankton,⁴⁹ most of which fall as easy prey to zooplankton. Metagenomic analysis has suggested that Thaumarchaeota is the major B₁₂ producer in aquatic environments.⁵⁰ Indeed, relative to unsupplemented phytoplankton, B₁₂-supplemented phytoplankton can significantly stimulate the growth of rotifers as a food of larval fish.⁵¹ Thus, the bacterial B₁₂ is transferred to fish bodies via plankton and concentrated in the bodies of bigger predatory fishes in the ocean food chain (Figure 4). Thus, meat B₁₂ content is generally higher in bigger carnivorous fish than in small body fish²¹: in particular, substantial amounts of B₁₂ have been shown to be accumulated in the liver or kidney of tuna⁵² and salmon.⁵³

Figure 4.

Vitamin B₁₂ sources and microbial interactions in fish and shellfish. In aquatic environments, the B₁₂ produced by certain bacteria and archaea is taken up by B₁₂-requireing bacteria as well as eukaryotic phytoplankton, most of which fall as easy prey to zooplankton. The bacterial B₁₂ is transferred to fish bodies via plankton and concentrated in the bodies of bigger predatory fishes in the ocean food chain. B₁₂ levels were significantly higher in edible bivalves that can siphon phytoplankton than in edible snails, most of which are herbivorous, eating seaweed.

The amounts of B₁₂ are three times greater in the viscera (approximately 37.5 μg/100 g wet weight) than in the meat (approximately 12.2 μg/100 g wet weight) of round herring.⁵⁴ Approximately 73% of total B₁₂ found in the whole fish body (except for head and bones) were recovered in the meats (approximately 5.1 μg of B₁₂ per one body).⁵⁴ Serum B₁₂ levels of subjects consuming herring diets are significantly increased compared to meat (poultry and pork) diets⁴⁸: because poultry and pork meats (less than 1.0 μg/100 g wet weight) are not high in B₁₂.

The B₁₂ contents of round herring and skipjack tuna meats decrease up to approximately 62% and 85% by various conventional cooking.^25,48,55 However, the retention of B₁₂ in vacuum-cooked fish has been reported to be 92% for salmon and 72% for cod.²⁶

Shellfish, such as edible bivalves (e.g. clams, oysters, and mussels) are well known to contain substantial amounts of B₁₂.^56,57 B₁₂ compounds have been purified from these bivalves and identified as B₁₂.^58–61 However, trace pseudoB₁₂ and/or unidentified corrinoid compounds are rarely detected in edible bivalves⁶¹ using liquid chromatography/electrospray ionization–tandem mass spectrometry. Tanioka et al.⁶² have reported that B₁₂ contents are considerably higher in edible bivalves (approximately 60 μg/100 g wet weight) than in edible snails (approximately 20 μg/100 g wet weight). There are three types of snails: sea, freshwater, and land snails.⁶³ Most snails are herbivorous, eating plants and seaweed, while some sea snails are omnivores or carnivores. The differences in the content and B₁₂ compounds between these edible sea snails appear to be attributable to their dietary habitats, because ivory shell (Babylonia japonica; B₁₂ content of meat and viscera, approximately 27.2 and 92.8 μg/100 g wet weight, respectively) and turban shell (Turdo Batillus cornutus; B₁₂ content of meat and viscera, approximately 3.0 and 15.1 μg/100 g wet weight, respectively) are carnivorous and herbivorous sea snails, respectively.⁶⁴

The B₁₂ content (0.2–0.5 μg/100 g dry weight) of seaweeds as foods of herbivorous sea snails (turban shell, T. cornutus) is very low.²¹ Moreover, wakame predominantly contains certain B₁₂ analogues.⁶⁵ Other herbivorous sea snails (such as abalone) mainly contain pseudoB₁₂.⁶⁶ Escargot products contain a small amount (approximately 2.2 µg/100 g wet weight) of B₁₂ and two inactive corrinoids, which have been identified as factor IIIm (methoxymensimidazolyl cobamide) and factor S (2-methylmercaptoadenyl cobamide) using liquid chromatography/electrospray ionization–tandem mass spectrometry.⁶⁷ In particular, 2-methylmercaptoadenyl cyanocobamide is reportedly the predominant corrinoid in human feces.⁶⁸ These results suggest that these edible bivalves and carnivorous sea snails are good sources of B₁₂ for humans.

Vitamin B₁₂ in plant-derived food

Most plants neither produce nor require B₁₂.⁶⁹ Methylotrophys inhabit soil, water, and plants^70,71: in aerial surfaces of plants, Methylobacaterium sp. utilizes methanol emitted by plants; in aquatic environments, methanotrophys colonize macrophytic algae; and in soil, methonotrphs require B₁₂ supplied from rhizobial bacteria. Furthermore, some species of Methylobacterium such as Methylobacterium extroquences NR-1⁷² and the Methylobacterium aquaticum strain 22A⁷³ have B₁₂ biosynthetic pathways. Thus, plant–bacterial interactions play important roles in plant growth because B₁₂ deficiency inhibits plant growth under nitrogen-limited conditions.^70,74

B₁₂ has also been detected in the fruiting bodies of various mushrooms that cannot synthesize B₁₂.⁷⁵ High B₁₂ was detected in mushrooms with enhanced contact with B₁₂-synthesizing bacteria in the soil,⁷⁶ suggesting that B₁₂ found in mushroom fruiting bodies was derived from B₁₂ sources outside the mushrooms, such as concomitant B₁₂-synthesizing bacteria.

As described above, in aquatic environments, phytoplankton–bacterial interactions play important roles in algal growth because half of all algae require B₁₂.⁷⁷ Even in phytoplankton or microalgae without the dependence of B₁₂ for growth, B₁₂ was absorbed, accumulated, and used as a cofactor of B₁₂-dependent methionine synthase (or MetH), which has more efficient catalytic ability than B₁₂-independent methionine synthase (or MetE)^77,78 (Figure 5).

Figure 5.

Vitamin B₁₂ sources and microbial interactions in edible plants, mushrooms, and algae. Most plants neither produce nor require B₁₂. Methylotrophys inhabit soil, water, and plants (aerial surfaces of plants) and some of them have B₁₂ biosynthetic pathways. Plant–bacterial interactions play important roles in plant growth. Mushrooms cannot synthesize B₁₂, but B₁₂ found in mushroom fruiting bodies is derived from B₁₂ sources outside the mushrooms, including concomitant B₁₂-synthesizing bacteria. In aquatic environments, phytoplankton–bacterial interactions play important roles in algal growth because half of all algae require B₁₂. Even in phytoplankton or microalgae without a dependence on B₁₂ for growth, B₁₂ accumulates and is used as a cofactor of B₁₂-dependent methionine synthase (MetH).

Edible plants

Sea buckthorn (Hippophae rhamnoides) berries and granulate products, sidea couch grass (Elymus repens) products (dry extract and grinded), and elecampane (Inula helenium) reportedly contain considerable amounts of B₁₂ (approximately 11–37 μg/100 g of dry weight),⁷⁹ suggesting that B₁₂ found in these plant and plant products is due to a symbiosis with B₁₂-synthesizing bacteria.

B₁₂-enriched vegetables

Organic fertilizers such as cow manure appear to slightly increase the B₁₂ content of spinach leaves (approximately 0.14 μg/100 g fresh weight).⁸⁰ Our published⁸¹ and unpublished data have indicated that organic fertilizers mainly contain inactive corrinoids. B₁₂-enriched vegetables have been prepared by treating them with a B₁₂ solution,^82,83 suggesting that free B₁₂-supplemented vegetables may be beneficial to elderly persons because the malabsorption of protein bound B₁₂ is most commonly seen in the elderly.⁸⁴

Mushroom

Trace levels (<0.1 μg/100 g dry weight) of B₁₂ have been found in the dried fruiting bodies of black morels, oyster mushrooms, parasol mushrooms, and porcini mushrooms.⁷⁵ However, the fruiting bodies of black trumpet (Craterellus cornucopioides) and golden chanterelle (Cantharellus cibarius) contain slightly higher levels of B₁₂ (1.09–2.65 μg/100 g dry weight).⁷⁵ In addition, the B₁₂ contents of commercially available dried shiitake mushroom (Lentinula edodes) fruiting bodies significantly varied, with the average B₁₂ value approximately 5.6 μg/100 g dry weight.⁸⁵ B₁₂ found in shiitake mushroom fruiting bodies has not been attributed to the de novo biosynthesis of B₁₂, but appears to be derived from B₁₂ sources outside the mushrooms, presumably concomitant B₁₂-synthesizing bacteria or those existing in bed logs.⁸⁵ Similarly cultivated white button mushroom (Agaricus bisporus) fruiting bodies contain approximately 0.2 μg of B₁₂ per 100 g dry weight,⁸⁶ with the highest B₁₂ content found in the peel portion. B₁₂ was also detected at similar levels in their composts. These results suggest that white button mushroom can absorb B₁₂ from the compost or B₁₂-synthesizing bacteria on the mushroom surface. Truffles (Tuber sp.) live in a close mycorrhizal association with the roots of specific host trees and their fruiting bodies grow underground. Indeed, B₁₂ contents (approximately 11.5 μg of B₁₂ per 100 g dry weight) of several truffle fruiting bodies are higher than those reported for other edible mushroom fruiting bodies.⁷⁶ There is no information available on the physiological function of B₁₂ in these mushrooms. Dried shiitake mushroom fruiting bodies rarely contain the inactive corrinoid, B₁₂[c-lactone].⁸⁵ Lion’s mane mushroom (Hericium erinaceus) fruiting bodies contain considerable amounts of B₁₂[c-lactone].⁸⁷ B₁₂[c-lactone] binds weakly to the intrinsic factor, which is involved in the gastrointestinal absorption of B₁₂ and inhibits the B₁₂-dependent enzymes.⁸⁸ These results suggest that these mushroom fruiting bodies are not good sources of B₁₂ for vegetarians because of their lower B₁₂ content and the occurrence of harmful B₁₂[c-lactone] even in rare cases.

Red algae

The red algae Porphyra sp. is one of the most commercially available marine crops and well known as a sea vegetable.⁸⁹ Various species of Porphyra are widely consumed as dried nori sheet products, which contain substantial amounts of B₁₂ (approximately ∼77.6 μg/100 g dry weight).⁹⁰ Our results⁹¹ and unpublished data have indicated that dried Chinese nori (zicai), dried New Zealand nori (karengo), dried Korean nori (kim), and canned Welsh nori (laverbread) contain approximately 60.2, 28.5, 66.8, and 2.8 μg of B₁₂ per 100 g weight, respectively. The characterization of B₁₂ compounds found in edible algae including Porphyra sp. have been described in the literature.^10,90 Genomic analyses of Porphyra umbilicalis have suggested the physiological function of B₁₂ as well as evolutionary insights in red algae.⁹² Our studies of naturally occurring plant-based foods with high B₁₂ contents suggests that nori is the most suitable B₁₂ source presently available for vegans.⁹³ B₁₂ from dried nori is significantly absorbed and functional in B₁₂-depleted rats.^94,95

Green algae

The green alga Chlorella sp. is used in human food supplements and contains biologically active B₁₂.^96–99 Recently, we analyzed B₁₂ compounds in 19 dried Chlorella health supplements. Chlorella B₁₂ contents varied from <0.1 μg to approximately 415 μg per 100 g of dry weight.¹⁰⁰ Chlorella cell types of the low B₁₂ group were aseptically grown in large culture vessels (closed culture conditions), and the other Chlorella cell types were openly grown in large culture tanks (open culture conditions). Among the Chlorella species, B₁₂ contents were much higher in Chlorella pyrenoidosa than in Chlorella vulgaris under open culture conditions.¹⁰⁰ Chlorella cells reportedly have an uptake system of exogenous B₁₂.¹⁰¹ Thus, B₁₂ compounds in Chlorella cells are likely derived from B₁₂-synthesizing bacteria that are present under open culture conditions or from the addition of crystalline B₁₂ or from B₁₂-containing organic ingredients in the culture medium.

The coenzyme forms of B₁₂, 5′-deoxyadenosylcobalamin (approximately 32%) and methylcobalamin (approximately 8%), were considerably present in Chlorella tablets,¹⁰⁰ whereas cyanocobalamin was present at the lowest concentrations. Chlorella NC64A reportedly expresses homologous genes that encode B₁₂-dependent and -independent methionine synthases and methylmalonyl-CoA mutase.¹⁰² Indeed, B₁₂-dependent methionine synthase and methylmalonyl-CoA mutase activities were detected in cell homogenates of C. pyrenoidosa.¹⁰⁰

We stress that if Chlorella tablets are to be consumed as a sole B₁₂ source, Chlorella tablets with moderate or high levels of B₁₂ must be identified using liquid chromatography/electrospray ionization–tandem mass spectrometry, because inactive corrinoid compounds (a cobalt-free corrinoid and 5-methoxybenzimidazolyl cyanocobamide) were rarely detected in some high B₁₂-containing Chlorella tablets.¹⁰⁰

Conclusion

B₁₂ is synthesized by certain bacteria and archaeon, but not by plants or animals. The synthesized B₁₂ is transferred and accumulates in animal tissues, and even in certain plant tissues via microbial interaction. Meats and milks of herbivorous ruminant animals are good sources of B₁₂ for humans. Ruminants acquire the essential nutrient B₁₂ through a symbiotic relationship with bacteria inside the body. In a broad sense, we (except vegetarians) also depend on B₁₂-producing bacteria located in ruminant stomachs. In aquatic environments, most phytoplankton acquire B₁₂ through a symbiotic relationship with bacteria. Even algae that have no requirement of B₁₂ for growth can accumulate substantial amounts of B₁₂ and have the ability to use B₁₂ as a cofactor in B₁₂-dependent methionine synthase. Then, phytoplankton becomes food for fish and bivalves in the natural food chain. Thus, humans acquire B₁₂ formed by a microbial interaction via mainly ruminants and fish (or shellfish) as foods. Recently, it was reported that B₁₂ is a modulator of gut microbial ecology.¹⁰³ The bioavailability of food B₁₂ is approximately 50% in healthy humans¹⁰⁴ and unabsorbed B₁₂ would affect intestinal microbial ecology, which is expected to have a substantial impact on human health.

Author contributions

All authors contributed equally to the preparation of this manuscript and have approved the final version.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article

Funding

This work was supported by JSPS KAKENHI Grant number 25450168 (FW) and 16K07736 (FW).