Characterization of Vitamin B₁₂ Compounds in Fermented Poultry Manure Fertilizers

Abstract

(1) Background: Currently, no data are available on the vitamin B₁₂ content of an organic fertilizer product, viz. fermented poultry manure, or whether the organic fertilizer product contains vitamin B₁₂ or inactive corrinoids (or both). (2) Methods: This study conducted a microbiological assay to determine the vitamin B₁₂ content of various commercially available fermented poultry manure fertilizer products. (3) Results: The results varied from 1.4 μg to approximately 20 μg per 100 g of dry weight. In the bioautography analysis, selected products had two positive spots with identical R_f values of vitamin B₁₂ and pseudovitamin B_12. High-performance liquid chromatography and liquid chromatography/electrospray ionization−mass spectrometry analyses of the selected products indicated that these fertilizers primarily contained vitamin B₁₂. They also contained minor inactive cobamides such as pseudovitamin B₁₂, 2-methyladenyl cobamide, and 2-methylmercaptoadenyl cobamide. (4) Conclusions: These results suggested that edible plants would enrich vitamin B₁₂ using fermented poultry manure organic fertilizer products.

1. Introduction

Vitamin B₁₂ (B₁₂) or cobalamin is synthesized only by certain bacteria and archaea [1]. Animal-derived foods such as fish, shellfish, milk, and meat, are the major dietary B₁₂ sources, whereas plant-derived foods, such as fruits, vegetables, and grains, appear to contain none or trace levels of B₁₂ [2]. The recommended dietary allowance (RDA) for adults is 2.4 μg of B₁₂ per day [3,4], and this value is the lowest among the RDA of all vitamins. Thus, vegetarians and particularly vegans have a low intake of B₁₂. Strict vegetarians are at high risk of developing B₁₂ deficiency [5,6]. B₁₂ deficiency causes hematological, psychiatric, and neurological disorders [7]. Megaloblastic anemia is an early hematological sign. Neuropsychiatric disorders, subacute combined degeneration, and peripheral neuropathy are the most common neurologic manifestations [8]. Hence, production of vegetables containing sufficient quantities of B₁₂ is required to prevent vegetarians from developing B₁₂ deficiency.

Mozafar [9] indicated that the B₁₂ content in vegetables was significantly increased by the addition of cow manure fertilizer. A preliminary study showed that the addition of fermented poultry manure products as organic fertilizers increased the amount of B₁₂ (0.43–0.91 µg/100 g wet weight) in rice bran compared with that in the control (0.03–0.23 µg/100 g wet weight) without the fertilizer treatment. Furthermore, the B₁₂ contents in rice bran increased with increasing B₁₂ content in soils, suggesting that B₁₂ would be enriched in rice bran by the addition of fermented poultry manure products. However, some intestinal bacteria reportedly can synthesize various cobamides with various base moieties in the α-axial ligand of the molecule [10]. Various cabamides that were inactive in humans exist in animal feces [11] and intestinal digesta [12]. Although there are various types of commercially available fermented poultry manure products, there are few reports on the B₁₂ level of these products and on whether these products contain inactive cobamides or B₁₂.

Therefore, we conducted this study to determine the B₁₂ content of commercially available poultry manure organic fertilizer products and characterize the B₁₂ compounds found in selected fertilizers.

2. Materials and Methods

2.1. Materials

B₁₂ was obtained from Sigma (St. Louis, MO, USA). Pseudovitamin B₁₂ (PseudoB₁₂), benzimidazolyl cyanocobamide (BIA), and 5-hydroxybenzimidazolyl cyanocobamide (Factor III) were obtained as described previously [13]. The B₁₂ microbial assay medium for Lactobacillus delbrueckii ATCC7830 was purchased from Nissui (Tokyo, Japan). We used an ultraviolet (UV)-visible spectrophotometer (Shimadzu, Kyoto, Japan) to determine the turbidity of microbial test cultures. Various types of commercially available fermented poultry manure organic fertilizers and rice brans were purchased from local markets in Japan. Samples A, B, C, D, G, H, and I were obtained from fermented poultry manure fertilizer makers located in Okayama, Fukuoka, Saitama, Hokkaido, Hyogo, Hiroshima, and Kagawa prefectures, and samples E and F were from different makers in Aichi prefecture, Japan.

2.2. Extraction and Assay of Vitamin B₁₂

We added rice brans or fermented poultry manure fertilizer products (10 g each) were added to 100 mL of 5 mmol/L acetate buffer (pH 4.5) containing 0.01% (w/v) potassium cyanide (KCN). Total B₁₂ was extracted by boiling this mixture for 30 min in a fume hood (Dalton Co., Tokyo, Japan). The B₁₂ content was evaluated by the microbiological assay using L. leichmannii ATCC 7830. The obtained B₁₂ extracts were diluted with distilled water and used as samples. The turbidity (% T) of Lactobacillus test culture was determined at 600 nm using a Shimadzu spectrophotometer. This bacterium can use an alkali-resistant factor (deoxyribosides or deoxyribonucleotides) and B₁₂ [14]. We calculated the amount of B₁₂ by subtracting the values of the alkali-resistant factor from the total B₁₂ values.

2.3. Thin-Layer Chromatography (TLC) Bioautography Assay Using B₁₂-Dependent Escherichia coli 215

The extracts of selected fertilizer A, B, and C products listed in

Table 1. Vitamin B₁₂ contents in nine types of commercially available fermented poultry manure organic fertilizer products. B₁₂ was extracted from various fermented poultry manure products by the KCN-boiling method and determined by the microbiological method as described in the text.

Fertilizer Products	Manufacture’s Location in Japan	Vitamin B12 Contents (µg/100 g Dry Weight)
A	Okayama prefecture	20.6 ± 0.8
B	Fukuoka prefecture	12.6 ± 0.5
C	Saitama prefecture	8.5 ± 0.2
D	Hokkaido prefecture	7.4 ± 0.2
E	Aichi prefecture	4.8 ± 0.2
F	Aichi prefecture	12.5 ± 0.5
G	Hyogo prefecture	10.1 ± 0.3
H	Hiroshima prefecture	6.8 ± 0.1
I	Kagawa prefecture	1.4 ± 0.0
Mean ± SD		9.4 ± 5.5

were placed on C18 Cartridges (Sep-Pak ^®Vac 20 cc (5 g), Waters Corp., Milford, MA, USA), which was washed with 30 mL of distilled water. The B₁₂ compounds were eluted with 30 mL of 75% (v/v) ethanol. We evaporated the eluate to dryness under reduced pressure and dissolved the residual fraction in a small amount of distilled water. These extracts (1 μL) were placed onto silica gel 60 TLC plates and treated with 2-propanol/NH₄OH (28%)/water (7:1:2 v/v/v) as a solvent in the dark at room temperature (25 °C). B₁₂ compounds on the TLC plate was visualized with 2,3,5-triphenyltetrazolium salt using the method of Tanioka et al. [15].

2.4. High-Performance Liquid Chromatography (HPLC)

The selected fertilizer extracts were treated with Sep-Pak ^®Vac 20 cc (5 g) C18 Cartridges (Waters Corp.) under the same above-described conditions. The eluate (approximately 30 mL) was evaporated to dryness under reduced pressure, dissolved in 5 mL of distilled water, and centrifuged for 10 min at 10,000 g. We loaded the supernatant onto an EASI-EXTRACT ^® B₁₂ Immunoaffinity Column (P80) (R-Biopharm AG, Darmstadt, Germany) and then purified the B₁₂ compounds according to the manufacturer’s recommended protocol. The purified samples were dissolved in distilled water and filtered using a Nanosep MF centrifuge device (0.4 µm, Pall Corp., Tokyo, Japan) to remove small particles. The purified compounds were dissolved in 80 µL of Milli-Q water, filtered using a membrane filter, and then analyzed using an HPLC apparatus (SPD-10AV UV-Vis detector, SCL-10A VP system controller, DGU-20A₃ degasser, LC-10Ai liquid chromatography, and CTO-20A column oven). An aliquot (35 µL) of the samples was placed on a reversed-phase HPLC column (Wakosil-II 5C18RS, φ 4.6 × 150 mm; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) equilibrated with 20% (v/v) methanol containing 1% (v/v) acetic acid. The corrinoids were isocratically eluted with the same solution at a flow rate of 1.0 mL/min at 40 °C and monitored by determining the absorbance at 361 nm. We analyzed authentic B₁₂, BIA, Factor III, and PseudoB₁₂ were analyzed under the same conditions. The retention times of the authentic Factor III, BIA, PseudoB₁₂, and B₁₂ were 7.7, 8.0, 8.5, and 10.3 min, respectively.

2.5. Liquid Chromatography–Mass Spectrometry (LC–MS)

We analyzed an aliquot (2 μL) of the compounds purified using the above-described immunoaffinity column by a LCMS-IT-TOF system (Shimadzu) using the method of Okamoto et al. [14]. The identification of B₁₂ or cyanocobalamin (C₆₃H₈₈CoN₁₄O₁₄P) (m/z 678.2914), BIA (C₆₁H₈₄CoN₁₄O₁₄P) (m/z 664.2748), Factor III (C₆₁H₈₄CoN₁₄O₁₅P) (m/z 672.2715), adenyl cyanocobamide or PseudoB₁₂ (C₅₉H₈₃CoN₁₇O₁₄P) (m/z 672.7749), 2-methyladenyl cyanocobamide or Factor A (C₆₀H₈₅CoN₁₇O₁₄P) (m/z 679.7834), 2-methylmercaptoadenyl cyanocobamide or Factor S (C₆₀H₈₅CoN₁₇O₁₄PS) (m/z 695.7657), and p-cresolyl dicyanocobamide (C₆₂H₈₆CoN₁₃O₁₅P) (m/z 659.2783) and representing [M + 2H]²⁺ was confirmed by comparison of the observed molecular ions.

3. Results and Discussion

The amount of B₁₂ in nine types of commercially available fermented poultry manure fertilizer products was determined using a microbiological method (Table 1). The B₁₂ content of the fermented poultry manure fertilizer products varied from 1.4 μg to approximately 20 μg per 100 g of dry weight. The B₁₂ contents of organic wastes (cow dung and sheep feces) used as organic fertilizers were 14–100 and 186 μg/100 g, respectively [9]. The The B₁₂ contents of the fermented poultry manure fertilizers tested in the present study were lower than those of these organic wastes. During the fermentation process of the poultry manure, uric acid present in the manure is degraded into urea and ammonia, and heat is generated at approximately 60 °C. B₁₂ is unstable under alkaline and heat conditions [16]. Low B₁₂ contents of the poultry manure fertilizers might be due to the loss of B₁₂ during the fermentation process.

Human feces reportedly contain various inactive cobamides such as Factor A (60.6%), p-cresol cobamide (16.3%), PseudoB₁₂ (12.5%), Factor S (15.5%), Factor III (1.8%), and phenol cobamide (0.1%) [11]. To clarify whether the fermented poultry manure fertilizer products contained B₁₂ or inactive cobamindes, the corrinoids observed in the selected fertilizer products (A, B, and C) listed in Table 1 were analyzed using an E. coli 215 bioautogram after separation by silica gel 60 TLC. The corrinoids observed in fertilizer A and B products produced obvious spots with R_f values identical to those of B₁₂ and PseudoB₁₂. However, the fertilizer C product showed only the spot with an R_f value identical to that of B₁₂ (Figure 1).

Corrinoids were purified from the extracts of the selected fertilizer A, B, and C products using a B₁₂ immunoaffinity column and then identified by C18 reversed-phase HPLC (Figure 2). The retention times of Factor III, BIA, PseudoB₁₂, and B₁₂ were 7.7, 8.0, 8.5, and 10.3 min, respectively. The retention times of the three main peaks found in the fertilizer A product were similar to those of Factor III (peak d), PseudoB₁₂ (peak b), and B₁₂ (peak a). Two peaks with retention times of 8.9 (peak e) and 11.9 (peak f) min were detected as unidentified compounds. We obtained similar results in the fertilizer B product. However, the fertilizer C product showed a single major peak with a retention time of 10.2 min, which was the identical retention time of B₁₂.

LC-MS analysis precisely identified corrinoid compounds in the selected fertilizer products. As described previously [17,18], the MS results of authentic B₁₂, Factor III, and PseudoB₁₂ indicated major doubly charged ions of m/z 678.2937, m/z 672.2715, and m/z 672.7749, respectively. We eluted selected ion monitoring chromatograms of various cobamides with different base moieties (Figure 3) revealed that PseudoB₁₂ and B₁₂ at retention times of 7.3 and 7.4 min, respectively, whereas Factor III was undetectable. Furthermore, Factor A (m/z 679.7834) occurred at the identical retention time of PseudoB₁₂. We eluted Factor S (m/z 695.7657) at a retention time of 7.5 min. However, there is no information regarding the retention times of Factors A and S as these authentic compounds were not available. These results indicated that the corrinoid compounds presented in the fertilizer A product were completely identified as B₁₂ and PseudoB₁₂ and tentatively identified as Factor A and S. We obtained similar results in the fertilizer B product. However, the fertilizer C product contained only B₁₂, judging from the selected ion monitoring chromatogram data.

TLC-bioautography, HPLC, and LC-MS analyses indicated that B₁₂ was the predominant corrinoid in all the selected fertilizer products, and minor inactive cobamides such as PseudoB₁₂, Factor A, and Factor S appeared in the fertilizer A and B products.

As depicted in Figure 2, the peak d, showing the identical retention time (7.7 min) of Factor III, was detected during the HPLC of fertilizer A and B products. However, Factor III (m/z 672.2715) was not detectable by LC-MS (Figure 4), suggesting that the peak with a retention time of 7.7 min does not come from corrinoid compounds. However, the unidentified compounds with retention times of 8.9 (peak e) and 11.9 (peak f) min detected by HPLC (Figure 2) appear to be derived from Factors A and S, respectively, judging from the data of selected ion monitoring chromatograms by LC-MS (Figure 4). The relative contents (%) of B₁₂ (56.9% and 61.5%), PseudoB₁₂ (15.4% and 13.2%), Factor A (7.2% and 8.0%), and Factor S (20.5% and 17.3%) were calculated in fertilizer A and B products, respectively, using HPLC data. These inactive cobamides would be synthesized by poultry intestinal bacteria and/or formed during the fermentation process of the manure by concomitant bacteria [19]. Differences in the corrinoid compounds found in fertilizers A, B, and C might be due to the different types of corrinoid-synthesizing bacteria in the poultry intestine and fermentation process.

Biofertilizers containing purple photosynthetic bacteria reportedly contain B₁₂ (main) and Factor III (minor) [18,20]. Studies have also reported that cyanobacteria that are responsible for biological nitrogen fixation in flood rice fields primarily contained PseudoB₁₂ [21,22]. However, no information is available on regarding other organic fertilizers and wastes such as cow dung and sheep feces. Several plants, such as lettuces, can absorb B₁₂ from soils [9] or hydroponic solutions [23].

4. Conclusions

A preliminary study indicated that the addition of fermented poultry manure fertilizers increased the amount of B₁₂ in rice bran. However, the B₁₂ content of the fermented poultry manure fertilizers varied from 1.4 μg to approximately 20 μg per 100 g of dry weight. LC-MS analyses showed that the fermented poultry manure fertilizers A, B, and C primarily contained B₁₂.

Therefore, by utilizing such fermented poultry manure fertilizers, edible plants could be enriched in B₁₂. Even if the fertilizer products contain PseudoB₁₂, Factor A, and Factor S, these inactive corrinoids would not be absorbed into the human intestine because of their low affinity to the human intrinsic factor, a gastric B₁₂ transport protein [24]. Furthermore, there is a fertilizer–soil–microbial ecosystem in nature. Further studies are required to clarify the effects of these fermented fertilizers in edible plants by enriching them with B₁₂.