2021 Volume 27 Issue 2 Pages 249-257
β-(1→4)-Mannobiose (Man2) is an attractive oligosaccharide for food and feed additives. Quantifying Man2 is useful for the industrial production of Man2. In this study, we evaluated colorimetric and HPLC-based quantification methods for Man2. For the colorimetric methods, quantification of d-glucose and α-d-mannose 1-phosphate (Man1P) was combined with the epimerization of Man2 to Manβ-4Glc and phosphorolysis of Manβ-4Glc. For the HPLC-based methods, high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and ultra-performance liquid chromatography with P-aminobenzoic ethyl ester labeling (ABEE-UPLC) were applied. The Man1P-based colorimetric method and HPAEC-PAD accurately determined Man2 even in the presence of other sugars tested. The d-glucose-based colorimetric method was unsuitable for samples containing excess concentrations of d-glucose and d-mannose, and the ABEE-UPLC method was unsuitable for samples containing cellobiose and maltose. For quantifying Man2 in mannanase-treated copra meal extract, the analytical values from the four methods corresponded well with each other.
β-(1→4)-Mannobiose (Man2), an oligosaccharide derived from β-mannan, is highly resistant to digestive enzymes (Asano et al., 2003), and exhibits various beneficial physiological functions as an additive to animal feed and human foods.
In chicken feed, Man2 has been reported to be effective at preventing Salmonella enterica infection by improving its clearance through the intestines, which reduces intestinal pathology in the cecal tonsils and increases IgA production (Agunos et al., 2007). Man2 acts as an immune modulator by exerting several effects on the intestinal immune system, and up-regulates antibacterial defenses in macrophages (Ibuki et al., 2010; Ibuki et al., 2011; Kovacs-Nolan et al., 2013). Man2 has also been reported to improve intestinal morphology (Duarte et al., 2014; Ibuki et al., 2014a), to exhibit a therapeutic effect on intestinal inflammation in a porcine model of colitis (Ibuki et al., 2014b), and to suppress muscle proteolysis in growing chickens (Ibuki et al., 2013; Ibuki et al., 2014c). Because of these physiological functions, feeding Man2 is effective for improving growth performance and productivity in the chicken industry (Duarte et al., 2014; Rikimaru et al., 2017). It has also been shown to be effective for enhancing the immune response and resistance against disease-causing bacteria in shrimp production (Elshopakey et al., 2018).
An experiment using a mouse model of intranasally-induced pollen allergy has also illustrated the prophylactic and therapeutic effects of Man2 against pollen allergy. Thus, Man2 can be considered a potential candidate for attenuating the allergic response (Yang et al., 2013a; Yang et al., 2013b). Overall, these studies have demonstrated that Man2 can provide several beneficial physiological functions.
Copra meal, a by-product of the coconut oil production process, is a good source of β-mannan for the production of Man2. Mannanase-hydrolyzed copra meal (MCM™) has a high content of Man2 and is widely used in animal feed, mainly in Asia and Europe, for preventing pathogenic infections and improving the growth performance of livestock (Ibuki et al., 2015; Fukui et al., 2019). The production of Man2 from coconuts by enzymatic hydrolysis has been actively developed to provide healthy food materials.
High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is an official method for analyzing carbohydrates found in foods (AOAC International, 2003). The advantages of this method are the high resolution for various compounds, high sensitivity, and no requirement for derivatization. An alternative method for quantifying carbohydrates is labeling them with P-aminobenzoic ethyl ester (ABEE), which can then be detected sensitively using a fluorescence detector (Yasuno et al., 1997; Yasuno et al., 1999). Combining the use of ABEE-labeling with ultra-performance liquid chromatography (UPLC) provides a rapid and sensitive analytical system for carbohydrates (Sakamoto et al., 2015).
Colorimetric analysis can process several samples more easily than HPLC-based methods. Since it does not require any expensive instruments, it can be used for high-throughput analysis. Colorimetric quantification methods for carbohydrates such as monosaccharides and sugar phosphates have been established (Bergmeyer and Michal, 1974; Miwa et al., 1972; Nihira et al., 2007; Nihira et al., 2013; Peterson and Young 1968; Reissig et al., 1955). A combination of monosaccharide or sugar phosphate quantification reactions with the phosphorolysis of oligosaccharides can be used to quantify specific oligosaccharides, because glycoside phosphorylases have very high substrate specificity (Kitaoka et al., 2001; Shirokane et al., 2000). A colorimetric quantification method for Man2, where glucose oxidase-peroxidase colorimetric quantification of d-glucose was combined with the epimerization of Man2 by cellobiose 2-epimerase (EC 5.1.3.11, CE) and the phosphorolysis of 4-O-β-d-mannosyl-d-glucose (Manβ-4Glc) by 4-O-β-d-mannosyl-d-glucose phosphorylase (EC 2.4.1.281, MGP), has been previously reported (Jaito et al., 2014).
During industrial Man2 production, any Man2 included in a raw material has to be quantified. This means that Man2 must be specifically quantified in the presence of other sugars. Therefore, the present study aims to evaluate the specificity of Man2 quantification using colorimetric and HPLC-based methods by measuring a constant concentration of Man2 in the presence of various sugars, and to quantify Man2 in a crude solution, MCM™, so that the methods can be compared. Two colorimetric methods and two HPLC-based methods for Man2 quantification are investigated: the colorimetric quantification of d-glucose and Man1P coupled to CE and MGP reactions (methods 1 and 2, respectively) and HPLC-based methods for quantifying Man2 using HPAEC-PAD (method 3) and ABEE-UPLC (method 4).
Preparation of enzymes for Man2 quantification Recombinant CE (Genbank accession number, ADU20582.1) from Ruminococcus albus (RaCE) was produced in Escherichia coli. The expression plasmid for RaCE with C-terminal six His residues (His-tag) was constructed. To introduce the His-tag, the stop codon before the coding region of the tag in the expression plasmid of RaCE, derived from pET-23a (Novagen, Darmstadt, Germany) (Fujiwara et al., 2013), was eliminated by PCR. The PCR was performed using Primestar Max DNA polymerase (Takara Bio, Kusatsu, Japan), the expression plasmid as the template, and a set of primers: 5′-GATATCCACCACCACCACCACCACTG-3′ (sense orientation) and 5′-GTGGTGGATATCTACTCCGCGTGT-3′ (antisense orientation). The PCR product was introduced into E. coli DH5α, then the plasmid DNA was obtained from the resulting transformant. Recombinant RaCE was produced in the transformant of E. coli BL21 (DE3), which harbored the expression plasmid. The transformant was cultured in 4 L of Luria-Bertani (LB) broth containing 100 µg/mL ampicillin at 37 °C until the A600 value reached 0.5. Production of the recombinant protein was induced by adding 0.1 M isopropyl β-d-thiogalactoside at a final concentration of 0.1 mM, then the culture broth was incubated with vigorous shaking at 18 °C for 24 h. The bacterial cells, harvested by centrifugation at 12 000 × g at 4 °C for 5 min, were suspended in 80 mL of 20 mM imidazole-HCl buffer containing 0.5 M NaCl (pH 7.0), and disrupted by sonication. A cell-free extract was obtained by centrifugation at 5 700 × g at 4 °C for 10 min and applied onto Ni2+ immobilized Chelating Sepharose Fast Flow (GE Healthcare, Uppsala, Sweden), which was equilibrated with the same buffer. After eluting the non-adsorbed protein with the equilibration buffer, the adsorbed protein was eluted using a linear gradient of 20–500 mM imidazole (200 mL in total). The fractions containing highly purified RaCE were collected, then dialyzed against 10 mM Tris-HCl buffer containing 50% (w/v) glycerol. The purity of the enzyme was determined using SDS-PAGE.
Recombinant mannose 6-phosphate isomerase (EC 5.3.1.8, M6PI) from E. coli was produced in E. coli. The manA gene (GenBank accession number, CP011343.2) was amplified by PCR from the genomic DNA of E. coli DH5α using KOD Plus DNA polymerase (Toyobo, Osaka, Japan) and primers: 5′-AAAAACATATGCAAAAACTCATTAAC-3′ (sense orientation) and 5 ′-AAAAACTCGAGCAG CTTGTTGTAAACACG-3′ (antisense orientation). The PCR product and pET-23a were digested with NdeI and XhoI (both from Takara Bio), and then these fragments were connected using DNA Ligation Kit Mighty Mix (Takara Bio). The resulting plasmid DNA was introduced into E. coli BL21 (DE3). The recombinant M6PI was produced in a 1-L batch under the same conditions as described above, and then purified in the same manner as that for RaCE.
Recombinant phosphomannomutase (EC 5.4.2.8, PMM) from E. coli was produced in E. coli. The cpsG gene (GenBank accession number, STEB01000002.1) was amplified by PCR from the genomic DNA of E. coli DH5α, and then cloned into pET-23a in the same manner as the manA gene. The PCR used the primers 5′-AAAAACATATGAAAAAATTAACCTGC-3′ (sense orientation) and 5 ′-AAAAACTCGAGCAG CAACGTCAGCAGAGT-3′ (antisense orientation). The recombinant PMM was produced in a 2-L batch and purified by the same method as that for RaCE.
Glucose 6-phosphate dehydrogenase (EC 1.1.1.49, G6PD) and glucose 6-phosphate isomerase (EC 5.3.1.9, G6PI) were purchased from Nacalai Tesque (Kyoto, Japan). The recombinant MGP from R. albus (RaMGP) was prepared as described by Kawahara et al. (2012).
Enzyme assay For the assay of CE, 100 µL of a reaction mixture, containing the enzyme, 10 mM cellobiose (Sigma, St. Louis, USA) and 11 mM sodium phosphate buffer (pH 7.5), was incubated at 30 °C for 20 min. To stop the reaction, 50 µL of 0.1 M HCl was added to the reaction mixture, and then the mixture was boiled for 3 min. The sample was desalted with Amberlite MB-4 (Organo, Tokyo, Japan), and the Glcβ-4Man produced was measured by HPLC under the following conditions: injection volume, 10 µL; column, two Sugar SP0810 in tandem (8.0 mm i.d. × 300 mm; Shodex, Tokyo, Japan); column temperature, 70 °C; eluent, water; flow rate, 0.5 mL/min; and pulsed amperometric detection. Glcβ-4Man (0–2 mM), prepared as described by Hamura et al. (2013), was used as a standard. One U of CE activity was defined as the amount of enzyme required to produce 1 µmol of Glcβ-4Man in 1 min under these conditions.
The M6PI activity was determined from the amount of d-fructose 6-phoshate (Fru6P) produced from d-mannose 6-phosphate (Man6P). A reaction mixture (50 µL), containing enzyme, 10 mM Man6P (sodium salt; Sigma), 10 mM MgCl2, and 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer (pH 7.0), was incubated at 37 °C for 10 min. The reaction mixture was heated at 100 °C for 3 min to stop the reaction, and then the Fru6P concentration was measured. The reaction mixture was mixed with an equal volume of a mixture containing 2 U/mL G6PD, 2 U/mL G6PI, 0.2 µM d-glucose 1,6-bisphosphate (cyclohexylammonium salt; Sigma), 1 mM thio-NAD+ (Oriental Yeast Co., Ltd., Tokyo, Japan), 15 mM MgCl2, and 150 mM HEPES-NaOH buffer (pH 7.0), and then incubated at 37 °C for 20 min. The absorbance was measured at 400 nm and the Fru6P concentration was determined based on a standard curve of 0–0.2 mM Fru6P (sodium salt; Nacalai Tesque). One U of M6PI activity was defined as the amount of enzyme required to produce 1 µmol of Fru6P in 1 min under these conditions.
The PMM activity was determined from the amount of Man6P produced from Man1P. A reaction mixture (50 µL) containing enzyme, 10 mM Man1P (cyclohexylammonium salt), 10 mM MgCl2, and 0.1 M HEPES-NaOH buffer (pH 7.0) was incubated at 37 °C for 10 min. Man1P, prepared as described by Liu et al. (2015), was kindly provided by Dr. Motomitsu Kitaoka (Niigata University, Niigata, Japan). The reaction mixture was heated at 100 °C for 3 min to stop the reaction. To quantify the Man6P concentration, 50 µL of the reaction mixture was mixed with 60 µL of a mixture containing 1.7 U/mL G6PD, 1.7 U/mL G6PI, 2.8 U/mL M6PI, 0.17 µM d-glucose 1,6-bisphosphate, 0.83 mM thio-NAD+, 12.5 mM MgCl2, and 125 mM HEPES-NaOH buffer (pH 7.0), and then incubated at 37 °C for 20 min. The Man6P concentration was determined from the A400 value based on a standard curve obtained using 0–0.2 mM Man6P. One U of PMM activity was defined as the amount of enzyme required to produce 1 µmol of Man6P in 1 min under these conditions.
The MGP activity was determined based on its phosphorolytic velocity to 2 mM Manβ-4Glc in 100 mM sodium phosphate buffer (pH 6.5). The enzyme assay was performed as described by Kawahara et al. (2012). One U of MGP activity was defined as the amount of enzyme required to produce 1 µmol of d-glucose in 1 min under these conditions.
Colorimetric quantification of Man2 based on d-glucose generated by the reactions of CE and MGP (method 1) d-Glucose, produced by the sequential reactions of CE and MGP with Man2, was quantified as described by Jaito et al. (2014) (Fig. 1A). Fifty microliters of the sample were mixed with 100 µL of the enzyme mixture, which contained 0.4 U/mL RaMGP, 2 U/mL RaCE, and 70 mM sodium phosphate buffer (pH 7.0), and 20 µL of d-glucose quantification reagent (Glucose C-II Test Wako; Fujifilm Wako Pure Chemical, Osaka, Japan), and then incubated at 37 °C for 1 h. The absorbance of the resulting solution was measured at 505 nm, and the Man2 concentration was determined based on a standard curve using 0–0.5 mM Man2 (Megazyme, Bray, Ireland). The colorimetric analysis without CE and MGP was performed as a control.
Scheme of colorimetric quantification of Man2.
A, Scheme of colorimetric quantification of Man2 based on d-glucose and Man1P derived from Man2 by the concerted reactions of CE and MGP. B, Scheme of quantification of Man1P.
Colorimetric quantification of Man2 based on Man1P generated by the reactions of CE and MGP (method 2) Man1P, produced by the sequential reactions of CE and MGP with Man2, was quantified as described by Nihira et al. (2013) (Fig. 1B). The concentration of Man1P was determined from the concentration of thio-NADH produced by the sequential reactions of PMM, M6PI, G6PI, and G6PDH on Man1P. Twenty microliters of the sample were mixed with 80 µL of the enzyme mixture, which contained 1.25 U/mL G6PD, 1.25 U/mL G6PI, 1.25 U/mL M6PI, 1.25 U/mL PMM, 0.4 U/mL RaMGP, 2 U/mL RaCE, 0.125 µM d-glucose 1,6-bisphosphate, 0.625 mM thio-NAD+, 12.5 mM MgCl2, and 31 mM sodium phosphate buffer (pH 7.0), then incubated at 37 °C for 30 min. The absorbance of the resulting solution was measured at 400 nm, and then the Man2 concentration was determined based on a standard curve over the range of 0–0.5 mM Man2.
Determination of Man2 by HPAEC-PAD (method 3) A CarboPac PA1 column (5 mm i.d. × 200 mm, Thermo Fisher Scientific, Waltham, USA) was used. A sample of the solution (10 µL) was injected into the column (column temperature, 30 °C), then eluted using a linear gradient of NaOH (20–200 mM) at a flow rate of 1.0 mL/min. The carbohydrates were detected by a pulsed amperometric detector (ICS-6000, Thermo Fisher Scientific). A standard curve for Man2 (0–0.5 mM) was used. The limit of determination (LOD) and limit of quantification (LOQ) were 0.042 µM and 0.141 µM, respectively, calculated at a signal-to-noise (S/N) ratio of 3 and 10 of the samples nearest to the detection limits.
Determination of Man2 by ABEE-UPLC (method 4) Man2 was quantified as described by Sakamoto et al. (2015) with some modifications. The saccharides were labeled using an ABEE Labeling Kit (J-Chemical, Tokyo, Japan). Forty microliters of ABEE reagent were added to 10 µL of the sample solution, and then the mixture was heated at 80 °C for 1 h. Six hundred microliters of water and 600 µL of chloroform were added to the sample, and then mixed vigorously. The water phase was collected by centrifugation at 800 × g at 4 °C for 1 min. The solution was passed through a syringe filter unit (0.22 µm, Millipore Sigma, Burlington, USA), and then analyzed using an Ultra Performance LC Acquity™ UPLC system with an Acquity UPLC FLR detector (Waters, Milford, USA). The analytical conditions were as follows: column, Acquity UPLC BEH18 column (1.7 µm, 2.1 mm i.d. × 100 mm, Waters); column temperature, 50 °C; elution, 10%–30% (v/v) methanol in water; flow rate, 0.3 mL/min; detection, fluorescence monitoring (emission at 305 nm and excitation at 360 nm). A standard curve of Man2 (0–0.5 mM) was used. The LOD and LOA of method 4 were 0.0109 mM and 0.0362 mM, respectively, calculated in a similar way as described above.
Sample preparation for Man2 quantification in the presence of various sugars To determine the influence of the presence of other saccharides on the analytical results for Man2, 16 types of 0.1 mM Man2 solution were prepared, each containing one of the following saccharides: 1 mM mannooligosaccharides [α-(1→2)-mannobiose (Sigma), β-(1→4)-mannotriose, β-(1→4)-mannotetraose, β-(1→4)-mannopentaose and β-(1→4)-mannohexaose (Megazyme)], 10 mM monosaccharides (d-glucose, d-fructose, d-galactose, d-mannose, d-xylose, and l-arabinose [(Fujifilm Wako Pure Chemical)], and 10 mM disaccharides [(maltose (Kishida Chemical, Osaka, Japan), trehalose, cellobiose (Sigma-Aldrich), sucrose, and lactose (Fujifilm Wako Pure Chemical)]. A control 0.1 mM Man2 solution with no added saccharide was also prepared.
Preparation of extract of mannanase-treated copra meal An extract of MCM™ (Fuji Oil Co., Ltd., Osaka, Japan), which contains Man2, was prepared. One gram of MCM™ was suspended in 100 mL of water, heated at 80 °C for 10 min, filtered through a 0.45-µm cellulose acetate filter (Advantec, Tokyo, Japan), then diluted appropriately to prepare 36 samples. The protein concentration of these extracts was measured by the Bradford method (Bradford, 1976), using bovine serum albumin (Nacalai Tesque) as the standard protein.
Statistical analysis The analytical values obtained by methods 1–4 were analyzed using Spearman's correlation coefficient by ranking available in IBM SPSS Statistics version 22 (IBM, Armonk, USA).
Colorimetric quantification of Man2 based on Man1P generated by the reaction of CE and MGP As an alternative colorimetric quantification method for Man2, the method for quantifying Man1P described by Nihira et al. (2013) was combined with the reactions of CE and MGP with Man2. The absorbance at 400 nm for thio-NADH, generated in the reaction of G6PDH with Glc6P and thio-NAD+, showed a good linear relationship with the concentration of Man2 (Fig. 2). The absorbance values of the standard solutions of Man2 were consistent with those of the equivalent concentrations of Glc6P. This indicated that the d-mannosyl residues at the non-reducing end of Man2 had been completely converted to Glc6P through the sequential reactions for the quantification of Man2.
Comparison of A400 values of Man2 and Glc6P after the quantification reaction of method 2.
Open and closed symbols refer to Man2 and Glc6P, respectively.
Specificity of colorimetric and HPLC-based quantification of Man2 The effect of the presence of 16 types of saccharides on the determination of Man2 was evaluated. The values of Man2 (0.1 mM) obtained by methods 2 and 3 were 0.0944–0.111 mM and 0.0961–0.105 mM, respectively (Table 1). An excess of mixed sugar had a slight effect on the values depending on the sugar. However, these values were close to 0.1 mM and the variation range was less than one tenth; thus, the sugars would not have substantially affected the quantification of Man2 using these two methods.
| Sugar | Method 2 | Method 3 |
|---|---|---|
| None | 0.107 ± 0.003 | 0.0992 ± 0.0017 |
| 10 mM d-Glucose | 0.102 ± 0.002 | 0.103 ± 0.003 |
| 10 mM d-Fructose | 0.109 ± 0.001 | 0.101 ± 0.001 |
| 10 mM d-Galactose | 0.107 ± 0.002 | 0.102 ± 0.004 |
| 10 mM d-Mannose | 0.106 ± 0.006 | 0.103 ± 0.002 |
| 10 mM d-Xylose | 0.100 ± 0.003 | 0.102 ± 0.001 |
| 10 mM l-Arabinose | 0.0944 ± 0.0049 | 0.103 ± 0.002 |
| 10 mM Maltose | 0.0996 ± 0.0017 | 0.0986 ± 0.0048 |
| 10 mM Trehalose | 0.105 ± 0.005 | 0.101 ± 0.000 |
| 10 mM Sucrose | 0.106 ± 0.002 | 0.105 ± 0.003 |
| 10 mM Cellobiose | 0.0949 ± 0.0033 | 0.0987 ± 0.0038 |
| 10 mM Lactose | 0.106 ± 0.004 | 0.0961 ± 0.0053 |
| 1 mM α-(1→2)-Mannobiose | 0.104 ± 0.001 | 0.102 ± 0.003 |
| 1 mM β-(1→4)-Mannotriose | 0.111 ± 0.002 | 0.101 ± 0.002 |
| 1 mM β-(1→4)-Mannotetraose | 0.109 ± 0.006 | 0.102 ± 0.002 |
| 1 mM β-(1→4)-Mannopentaose | 0.112 ± 0.003 | 0.101 ± 0.002 |
| 1 mM β-(1→4)-Mannohexaose | 0.107 ± 0.001 | 0.102 ± 0.001 |
Values are means ± standard deviation (n = 3).
The Man2 concentrations of the samples mixed with d-glucose and d-mannose were not accurately determined by method 1, although the correct values were obtained for the samples with other saccharides. That is, in method 1, the mean analytical value of Man2 of samples mixed with sugars other than d-glucose and d-mannose was 0.111 mM, while those of samples with d-glucose and d-mannose were 0.137 mM and 0.403 mM, respectively. For quantifying samples that included d-mannose, an accurate value was not obtained because of the epimerization activity of CE to d-mannose to produce d-glucose (Park et al., 2011; Saburi et al., 2015). Method 2 could determine the Man2 concentration accurately and was minimally affected by other saccharides included in the samples; thus, method 2 was more specific for Man2 than method 1. For method 4, the ABEE-labeled oligosaccharides, other than maltose and cellobiose, did not affect the quantification of Man2. The derivatives of maltose and cellobiose could not be separated from the derivative of Man2 under the analytical conditions used in the present study; therefore, the Man2 concentration could not be determined in the presence of these oligosaccharides. Retention times for HPLC analysis of the derivatives of maltose, cellobiose and Man2 were 10.4 min, 10.5 min, and 10.4 min, respectively. Those of the other sugars were as follows, and they were clearly separated from the derivative of Man2 (β-(1→4)-mannohexaose, 8.7 min; β-(1→4)-mannopentaose, 9.0 min; β-(1→4)-mannotetraose, 9.2 min; d-fructose, 9.3 min; α-(1→2)-mannobiose, 9.6 min; β-(1→4)-mannotriose, 9.7 min; lactose, 9.8 min; d-glucose, d-galactose, trehalose, and sucrose, 10.9 min; d-mannose, 11.2 min; d-xylose, and l-arabinose, 11.5 min). Of the two HPLC-based quantification methods for Man2, method 3 was more specific than method 4.
Comparison of quantification of Man2 in a crude solution using methods 1–4 We quantified Man2 in 36 samples of extracts from the functional feed material MCM™ using methods 1–4, and performed a correlation analysis by plotting all combinations between each method. A typical HPAEC-PAD chromatogram of an MCM™ extract is shown in Fig. 3. As well as Man2, MCM™ contains d-glucose, d-mannose, d-fructose, sucrose, Man3, and longer oligosaccharides. The contents (% of solid material, w/w) of Man2, d-glucose, d-mannose, d-fructose, and sucrose were determined from the peak areas of the chromatogram as 11.4%, 1.6%, 2.4%, 1.9%, and 4.5% by comparison to calibration curves prepared from each standard, respectively. This extract contained 0.100–0.158 mg/mL of protein. The linear correlation coefficient (R value) between any two of the four methods was at least 0.983 (p < 0.01), with a slope between 0.947 and 0.999, indicating that similar values were obtained for all methods tested (Fig. 4A–F). As the concentrations of d-glucose and d-mannose in MCM™ were sufficiently low, accurate Man2 concentrations could also be determined by method 1. The procedure for method 1 is simpler than that for method 2; thus, method 1 is considered to be practical for analyzing samples without high concentrations of d-glucose and d-mannose. R values between method 2 and the other methods were relatively low (0.983, 0.985 and 0.985, respectively, with methods 1, 3, and 4). The reason is presumed to be the complexity of method 2. In method 2, four more kinds of enzymes have to be added to the analytical samples than in method 1. In the case of analysis of materials containing large amounts of other sugars, such as d-glucose and d-mannose, the appropriate use of method 2 enhances the accuracy of the analysis. The ABEE-labeled sugars derived from sugars other than Man2 in the extract were successfully separated from the ABEE-labeled Man2; thus, the analytical values of Man2 from method 4 agreed well with those of the other methods.
Typical HPAEC-PAD chromatogram of MCM™ extract. Names of compounds are shown at the top of peaks.
Correlation of analytical values of extract of mannanase-treated copra meal by methods 1–4. ** p < 0.01
The general advantage of the colorimetric methods (1 and 2) compared to the HPLC methods (3 and 4) is that it is possible to analyze a large number of samples (30–40 in this case) at the same time, resulting in the capacity to simultaneously process many samples. Further, these colorimetric methods do not require equipment other than a spectrophotometer.
The analytical challenge of processing a large number of samples efficiently has been attracting attention. Colorimetric quantification has an advantage over HPLC methods in that it can process many samples in a short time. In the present study, two colorimetric and two HPLC-based methods for Man2 quantification were evaluated. Methods 2 and 3 were more specific for quantifying Man2 than the other two methods. For quantifying Man2 contained in MCM™, the results from all of the analytical methods tested in this study were highly correlated with each other. Therefore, these methods could be fully applied for analyzing the quality of products containing Man2 derived from copra meal, which is a major source of β-mannan. In particular, these rapid colorimetric methods for quantifying Man2 would be useful for the high-throughput analysis of samples containing Man2.
Acknowledgements The authors would like to thank Dr. Motomitsu Kitaoka from Niigata University for providing us the Man1P.
Conflicts of interest The authors declare that they have no conflicts of interest.
