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Precipitation of inulins and oligoglucoses by ethanol and other solvents
Food Chemistry 81 (2003) 125–132 www.elsevier.com/locate/foodchem
Analytical, Nutritional and Clinical Methods
Precipitation of inulins and oligoglucoses by ethanol and other solvents Yuoh Ku, Olaf Jansen1, Carolyn J. Oles, Esther Z. Lazar, Jeanne I. Rader* Division of Research & Applied Technology, HFS-840, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD 20740-3853, USA Received 4 March 2002; received in revised form 10 August 2002; accepted 10 August 2002
Abstract We investigated the ethanol precipitation step of the gravimetric method of dietary fiber analysis (AOAC Official Method 985.29). Four different solvents: ethanol, propanol, acetone and acetonitrile at four ratios: 1:1, 2:1, 3:1 and 4:1 (solvent : supernatant, v/v) were studied. Using inulins that contain components with a full range of degree of polymerization values (DP), we found that the percents of precipitation by these solvents were proportional to the average DP of the products. Use of ethanol and propanol produced similar results. In general, acetonitrile and acetone precipitated more of the inulins than did ethanol. Supernatant solutions were analyzed by high performance anion exchange chromatography in order to determine the size of the components that were precipitated. Our data showed that components of inulin with DP 1–10 remain in solution after the addition of ethanol at a ratio of 4:1. However, significant amounts of molecules of DP 11 and 12 and smaller amounts of molecules of DP 14– 18 also remain in solution. The precipitation patterns of oligoglucoses with DP 1–DP 7 were also investigated. Our data suggest that the precipitation behavior of oligoglucoses follows a pattern similar to that of inulins. Published by Elsevier Science Ltd.
1. Introduction Association of Official Analytical Chemists (AOAC) Official Method 985.29 (AOAC, 2000b) for the analysis of dietary fiber has been widely used in the USA and abroad. This method does not accurately or reliably quantify certain non-starch oligosaccharides such as inulin. A critical step in AOAC Official Method 985.29 is the precipitation of fiber by addition of 4 volumes of 95% ethanol per volume of sample. Certain non-starch oligosaccharides, fructooligosaccharides (FOS), and indigestible dextrin (e.g., Fibersol-2) are not precipitated by 95% ethanol (Prosky, 2000), and thus are not included among components analyzed as dietary fiber. Specific methods for the analyses of several of these compounds have been developed, e.g. inulin (AOAC, 2000a). This crucial precipitation step in AOAC Official Method 985.29 is neither well documented nor
* Corresponding author. Tel.: +1-301-436-1786; fax: +1-301-4362636. E-mail address: [email protected] (J.I. Rader). 1 Visiting Scientist, University of Hamburg, Hamburg, Germany. 0308-8146/03/$ - see front matter Published by Elsevier Science Ltd. PII: S0308-8146(02)00393-X
clearly understood. It is generally assumed that oligosaccharides above a certain degree of polymerization (DP) will be precipitated by the addition of 95% ethanol and those below that DP will remain in solution. The absence of research in this area is primarily due to the lack of oligosaccharide standards of various DPs. Inulins, which consist of molecules with a wide range of DP values, are appropriate candidates for systematic investigations on the relationship between chain length and precipitation by specific solvents. Inulins and FOS are used as food ingredients and have been reported to show significant ‘‘dietary fiber’’ effects (Hoebrigs, 1997). The question arises as to whether such compounds should be excluded from consideration as dietary fiber simply because they do not precipitate on addition of 4 volumes of 95% ethanol. In this study, we investigated the precipitation of several inulins and oligoglucoses by various concentrations of ethanol, acetonitrile, acetone and propanol. In our earlier work on the determination of sugars and starches in food, we reported that when analyzing sugars by HPLC, the use of acetonitrile at a ratio of 1:1 has some advantages over the use of ethanol at a ratio of 4:1 (Casterline, Ik, & Ku, 2000).
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2. Materials and methods 2.1. Standards and reagents Raftiline ST-Gel and Rafiline were obtained from Tiense Suikerraffinaderij (Belgium). Raftiline GR, Raftiline HP-Gel and Raftiline HP were obtained from Orafti (Malvern, PA, USA). Inulin HD was obtained from Imperial Suiker Unie (Texas, USA) and ‘‘Inulin from Chicorie’’ from Fluka Chemie AG (Switerland). 1-Kestose (GF2), Nystose (GF3) and GF4 were gifts from Dr. Yasuhito Tashiro, Meiji Seika Kaisha Ltd. (Japan). Oligoglucoses with DP2 were purchased from Supelco (USA). UV grade acetonitrile was purchased from Burdick & Jackson (USA). USP 200 proof ethyl alcohol was obtained from Warner-Graham Company (USA). 1-Propanol and acetone were purchased from J. T. Baker (USA). 2-(N-Morpholino)ethanesulfonic acid (MES) and Tris(hydroxymethyl) aminomethane (TRIS) were purchased from Sigma Chemical Co. (USA). 2.2. Analysis of inulin High performance anion exchange (HPAE) chromatography was performed with a DX 500 Dionex System (Sunnyvale, CA, USA) consisting of a GP40 gradient pump, a Dionex eluent organizer and an ED 40 electrochemical detector working in pulsed amperometric mode (PAD). The detector was equipped with a gold electrode and Ag/AgCl reference electrode. The chromatographic conditions and the gradient for the separation of molecules of DP 4–7 (42 min) were set up as described by Durgnat and Martinez (1997). Subsequently, the gradient program was extended by increasing the percent of sodium acetate to cover molecules with a DP range up to 32. Data acquisition was stopped at 103 min. Table 1 lists the HPAE gradient analysis conditions.
Table 1 HPAE gradient analysis conditions Run time (min)
Flow rate (ml/min)
Eluent A (%) water
Eluent B (%) 300 mM NaOH
Eluent C (%) 150 mM NaOH 500 mM NaOAC
1.01 2.50 2.51 1.07 7.10 9.00 13.00 14.00 18.00 18.10 26.00 26.10 30.00 30.10 35.00 35.10 38.00 38.10 42.00 70.00 90.00 100.00 101.10 125.00
1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
56.0 54.5 51.5 51.0 81.0 85.5 60.0 38.0 39.5 73.0 44.0 73.0 64.0 42.0 22.0 42.0 42.0 35.0 32.0 15.0 10.0 9.0 0.0 0.0
44.0 42.5 41.5 40.0 14.0 9.5 30.0 50.0 46.0 19.0 32.0 19.0 22.0 32.0 40.0 32.0 32.0 32.0 32.0 30.0 15.0 10.0 0.0 0.0
0.0 3.0 7.0 9.0 5.0 5.0 10.0 12.0 14.5 8.0 24.0 8.0 14.0 26.0 38.0 26.0 26.0 33.0 36.0 55.0 75.0 81.0 100.0 100.0
2.3. Analysis of oligoglucoses HPLC analysis was performed with a Shimadzu System consisting of LC-600 pumps, CTP-6A column oven, RID-6A RI detector and AST-LC computer using Shimadzu EZChrom chromatography data system. The column used was a Benson Carbohydrate (Ag+) BC200 column (Alltech, Deerfield, IL), 300 mm 7.8 mm, with a Ag+ BC-200 guard column. The mobile phase was isocratic with degassed HPLC grade water. The flow rate was 0.3 ml/min. 2.4. Gravimetric method for determination of precipitation of inulins The analytical scheme is shown in Fig. 1. Approximately 0.5 g of triplicate test portions of inulin products were weighed and the weights were recorded. The samples
Fig. 1. Analytical scheme for the determination of inulins and oligoglucoses.
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were dissolved in 25 ml of MES/Tris buffer (2%). The samples were stirred with a magnetic stirrer until dissolution. Some inulin samples required the application of heat for complete dissolution. One milliliter of the solution was pipetted into pre-weighed test tubes. Solvents (i.e. ethanol, propanol, acetone or acetonitrile) were then added separately. Four ratios of solvent to test sample (i.e. 1:1; 2:1; 3:1 and 4:1, v/v) were investigated. The tubes were vortexed and stored at 4 C overnight. The following morning, the samples were warmed to room temperature and centrifuged for 10 min at 2500 rpm. The samples were then washed once with 4 ml of pure test solvent and centrifuged again. The solvent was discarded and the samples were placed in a drying oven at 110 10 C. for 75 min to remove the excess solvent. The samples were reweighed. Calculation of the difference between two weights gave the amount precipitated by the solvents. 2.5. HPLC analysis of oligosaccharides One percent solutions of inulin or oligoglucose were used for HPLC analysis. The three types of inulin, i.e. Inulin HD, Raftiline GR and Raftiline ST, with a full DP range from simple sugars to an estimated DP of 60, were investigated. The general procedures shown in Fig. 1 were followed. The supernatant solutions were diluted and analyzed by the HPLC procedure.
3. Results and discussion The weight percents of various inulins precipitated by four solvents at ratios of 4:1 are listed in Table 2. The weight percent of precipitation by ethanol is very similar to that of propanol. In general, acetonitrile and acetone precipitated more of the inulins than did ethanol. Differences in the weights precipitated are dependent on the molecular weight distributions of the various inulins. According to the manufacturer, Raftiline St-Gel and Raftiline GR have an average DP of 10, and Raftiline HP and Raftiline HP-gel have an average DP of > 23. Inulin HD and Inulin IQ have an average DP of 9–12. We performed HPAE chromatography on the supernatants in order to determine the size of the components that were precipitated. Three types of inulin (i.e. Inulin HD, Raftiline GR and Raftiline ST), with a full DP range from simple sugars to an estimated DP of 60, were analyzed separately. The results of analyses of supernatant fractions of these three inulins were similar, therefore the data were averaged and standard deviations were calculated. Fig. 2 shows the ratio of peak areas before and after ethanol precipitation. A ratio of 1 indicates that there is no precipitation. There is no precipitation when 1 part of ethanol was added to 1 part of
Table 2 Weight percent of inulins precipitated by four solvents at ratios of 4:1 (gravimetric method)
Raftiline St-Gel Inulin HD InulinIQ Raftiline GR Chicorie Raftiline HP-Gel Raftiline HP
Ethanol
Propanol
Acetone
Acetonitrile
46.42.2 55.82.5 52.32.7 65.63.9 83.95.8 97.81.9 99.50.9
47.8 1.9 52.4 5.5 52.1 6.1 59.5 1.6 79.1 10.2 95.9 2.1 97.8 1.9
56.82.9 57.52.0 63.03.5 73.95.8 99.11.5 96.22.5 96.92.7
68.7 8.6 71.0 1.7 72.3 1.4 74.7 5.3 95.7 4.3 93.2 2.6 95.7 3.7
Values are means S.D. of three independent measurements. Average degrees of polymerization: Inulin HD), Inulin IQ, 9–12; Raftiline St-Gel, Raftiline GR, 10; Raftiline HP-Gel, Raftiline HP, >23.
supernatant (data not shown). The first panel shows the results with 2 parts of ethanol and 1 part of supernatant (2:1 v/v). At a 2:1 ratio, only molecules with high DP begin to precipitate. For example, for molecules with DP 26, only 60% remained in solution. For molecules with DP 31, only 30% remained in solution. At a 3:1 ratio, most of the molecules with high DP precipitated and the molecules of lower DP began to precipitate. Finally, at a 4:1 ratio, the ratio required by AOAC method 985.29, only molecules with DP 1–10 seem to remain in solution. However, there are still significant amounts of molecules of DP 11 and 12 and small amounts of molecules of DP 14–18 remaining in solution. The recent data of Ohkuma, Matsuda, Katta, and Tsuji (2000) using corn syrup solids as starting materials, suggest that the AOAC method for dietary fiber can only determine water-soluble dietary fiber components with an average DP of 12 or higher. We also investigated the precipitation pattern with solvents other than ethanol. Fig. 3 shows the ratio of peak areas before and after acetone precipitation. Acetone precipitates more inulin than ethanol. The results of acetonitrile precipitation are shown in Fig. 4. Acetonitrile at a 1:1 ratio caused little or no precipitation at least up to DP 20. Significant increases in precipitation occur at a ratio of 2:1. Most of the molecules with high and medium DP were completely precipitated. About 25–30% of simple sugars remained in solution. Further increases in the acetonitrile: supernatant ratio seemed to have no further effect. At a ratio of 4:1, the percent of simple sugars remaining in solution is higher than that observed at ratios of 2:1 or 3:1 acetonitrile:supernatant. Inulin is an unique carbohydrate in that chemically it consists of repeated units of fructose. In this regard, it differs from most other fibers. Biologically, it also appears to behave differently than other fibers. Therefore, our data on the precipitation behavior of inulin may not be representative of that for other fibers. The precipitation patterns of oligoglucoses from DP 1 to 7 were also investigated and the results are shown in Table 3. Oligoglucoses of DP 8 and above are not
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Fig. 2. Analysis of supernatants by HPAE chromatography to determine the size of the components precipitated by ethanol: Inulin HD, Raftiline GR and Raftiline ST with a full DP range from simple sugars to an estimated DP of 60 were analyzed before and after ethanol precipitation. The panels in the figure show the ratios of peak areas before and after precipitation. A ratio of 1 indicates that there was no precipitation.
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Fig. 3. Analysis of supernatants by HPAE chromatography to determine the size of the components precipitated by acetone: Inulin HD, Raftiline GR and Raftiline ST with a full DP range from simple sugars to an estimated DP of 60 were analyzed before and after acetone precipitation. The panels in the figure show the ratios of peak areas before and after precipitation. A ratio of 1 indicates that there was no precipitation.
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Fig. 4. Analysis of supernatants by HPAE chromatography to determine the size of the components precipitated by acetonitrile: Inulin HD, Raftiline GR and Raftiline ST with a full DP range from simple sugars to an estimated DP of 60 were analyzed before and after acetonitrile precipitation. The panels in the figure show the ratios of peak areas before and after precipitation. A ratio of 1 indicates that there was no precipitation.
Y. Ku et al. / Food Chemistry 81 (2003) 125–132 Table 3 Percent of oligoglucoses (DP 1–7) precipitated by various solvents (HPLC method) Degree of polymerization
DP1 DP2 DP3 DP4 DP5 DP6 DP7
Solvents and ratios Ethanol
Acetone
Acetonitrile
(4:1)
(4:1)
(4:1)
(1:1)
0 0 0 0 0 0 0
0 0 0 0 14.30.6 25.70.6 46.71.1
57.02.0 87.01.0 70.01.7 67.71.5 68.31.5 100 100
0 0 0 0 0 0 0
DP, degree of polymerization. Values are means S.D. of three independent measurements.
commercially available and thus, could not be studied. Ethanol at a 4:1 ratio and acetonitrile at a 1:1 ratio did not precipitate any of the low DP oligoglucoses. However, we found that acetone at a 4:1 ratio begins to precipitate oligoglucose with DP 5. The percent of precipitation increases with increasing DP. Acetonitrile at a ratio of 4:1 does precipitate glucose and shows complete precipitation of oligoglucoses with DP 6 and 7. Our data suggest that the precipitation behavior of inulins and oligoglucoses follow a similar pattern. It should be emphasized that our precipitation data are based on inulin and oligoglucose, and that their chemical structures differ from those of other fibers, which may respond differently. These studies were undertaken in order to better understand the precipitation step in AOAC official method 985.29 for the analysis of dietary fiber. Our findings of marked differences between solvents in precipitating specific carbohydrate components are significant because precipitability by 95% alcohol ‘‘defines’’ dietary fiber by this method. At the present time, there are a wide variety of definitions for dietary fiber. Some are based on specific analytical methods for isolating and quantifying dietary fiber while others emphasize physiologic attributes of dietary fiber or its components. In the United States, dietary fiber is defined by a number of official methods of the Association of Official Analytical Chemists. The current situation with respect to isolating and hence defining dietary fiber on the basis of specific methods has been criticized because it does not consider the possible role(s) of the isolated compounds in health. Similarly, new fiber or fiber-like components that may be developed in the future and which may be found to provide beneficial effects may be excluded as ‘‘dietary fiber’’ by the current methods-based definition. As efforts to make food labeling more uniform throughout the world continue, the existence of a single definition of dietary fiber becomes more important.
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For these reasons, there have been a number of recommendations in the United States to modify the definition of dietary fiber (Gordon, 1999). Recently, both the American Association of Cereal Chemists (AACC, 2001) and a Panel of the Food and Nutrition Board of the Institute of Medicine (FNB/IOM, 2001) reviewed current definitions and proposed new definitions of dietary fiber. Both the AACC and IOM proposed definitions include criteria regarding digestibility and beneficial physiologic effects. Discussions about what components might be included or excluded by the new proposed definitions and about the availaility of current validated methods to address such components are continuing. Areas of particular interest include questions of which physiologic functions and which DP should be used to distinguish oligosaccharides from dietary fiber. With respect to determining DP, chromatographic analysis such as that described here provides an effective means of separating mixtures of molecules of differing DP. One of the most important physiologic characteristics of components defined as dietary fiber is that such components are not digested in the upper GI track. Inulins with DP 3 and above are not digested in the upper GI track. Therefore, using only a digestibility criterion, such components might be considered as dietary fiber. However, with our current knowledge, we do not know whether these small DP (perhaps 3 and above) molecules have any other fundamental physiological characteristics of dietary fiber (e.g. causing laxation). Furthermore, most of the inulins possess a full range of molecules of various DP. In foods such as cereals, we have little or no information regarding the distribution and relative quantities of the various DP molecules. Therefore, the net functionality of these molecules is still incompletely understood. In conclusion, more research is needed, particularly on the relationship between the specific DP of fiber components and their physiological functions. Such knowledge will assist in developing methods that are applicable to the identification and quantitation of these components.
Acknowledgements We thank Dr. Michael McLaughlin of the Center for Food Safety and Applied Nutrition, Food and Drug Administration, for his technical assistance in performing the HPAEC analysis.
References American Association of Cereal Chemists (AACC). (2001). The definition of dietary fiber. Cereal Foods World, 46, 112–116.
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Association of Official Analytical Chemists (AOAC). (2000a). Fructans in food products, ion exchange chromatographic method. In Official methods of analysis (17th ed. Official Method 997.08, Chapter 45.4.06A). Gaithersburg, MD: Association of Official Analytical Chemists. Association of Official Analytical Chemists (AOAC). (2000b). Total dietary fiber in foods—enzymatic gravimetric method. In Official methods of analysis (17th ed. Official Method 985.29, Chapter 45.4.07). Gaithersburg, MD: Association of Official Analytical Chemists. Casterline, J. L., Oles, C. J., & Ku, Y. (2000). Measurement of sugars and starches in foods by a modification of the AOAC total dietary fiber method. JAOAC International, 82, 759–765. Durgnat, J. M., & Martinez, C. (1997). Determination of fructooligosaccharides in raw material and finished products by HPAE-PAD. Seminars in Food Analysis, 2, 85–97.
Food and Nutrition Board, Institute of Medicine, National Academy of Sciences. (2001). Dietary reference intakes: proposed definition of dietary fiber [Unedited proof]. A report of the Panel on the Definition of Dietary Fiber and the Standing Committee on the Scientific Evaluation of Dietary References Intakes, National Academy Press, Washington, DC. Gordon, D. T. (1999). Defining dietary fiber. Cereal Foods World, 44, 74. Hoebrigs, H. (1997). Fructans in foods and food products, ionexchange chromatographic method: collaborative study. JAOAC International, 80, 1029–1036. Ohkuima, K., Matsuda, I., Katta, Y., & Tsuji, K. (2000). New method for determining total dietary fiber by liquid chromatography. JAOAC International, 83, 1013–1019. Prosky, L. (2000). What is dietary fiber? JAOAC International, 83, 985–987.
Analytical, Nutritional and Clinical Methods
Precipitation of inulins and oligoglucoses by ethanol and other solvents Yuoh Ku, Olaf Jansen1, Carolyn J. Oles, Esther Z. Lazar, Jeanne I. Rader* Division of Research & Applied Technology, HFS-840, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD 20740-3853, USA Received 4 March 2002; received in revised form 10 August 2002; accepted 10 August 2002
Abstract We investigated the ethanol precipitation step of the gravimetric method of dietary fiber analysis (AOAC Official Method 985.29). Four different solvents: ethanol, propanol, acetone and acetonitrile at four ratios: 1:1, 2:1, 3:1 and 4:1 (solvent : supernatant, v/v) were studied. Using inulins that contain components with a full range of degree of polymerization values (DP), we found that the percents of precipitation by these solvents were proportional to the average DP of the products. Use of ethanol and propanol produced similar results. In general, acetonitrile and acetone precipitated more of the inulins than did ethanol. Supernatant solutions were analyzed by high performance anion exchange chromatography in order to determine the size of the components that were precipitated. Our data showed that components of inulin with DP 1–10 remain in solution after the addition of ethanol at a ratio of 4:1. However, significant amounts of molecules of DP 11 and 12 and smaller amounts of molecules of DP 14– 18 also remain in solution. The precipitation patterns of oligoglucoses with DP 1–DP 7 were also investigated. Our data suggest that the precipitation behavior of oligoglucoses follows a pattern similar to that of inulins. Published by Elsevier Science Ltd.
1. Introduction Association of Official Analytical Chemists (AOAC) Official Method 985.29 (AOAC, 2000b) for the analysis of dietary fiber has been widely used in the USA and abroad. This method does not accurately or reliably quantify certain non-starch oligosaccharides such as inulin. A critical step in AOAC Official Method 985.29 is the precipitation of fiber by addition of 4 volumes of 95% ethanol per volume of sample. Certain non-starch oligosaccharides, fructooligosaccharides (FOS), and indigestible dextrin (e.g., Fibersol-2) are not precipitated by 95% ethanol (Prosky, 2000), and thus are not included among components analyzed as dietary fiber. Specific methods for the analyses of several of these compounds have been developed, e.g. inulin (AOAC, 2000a). This crucial precipitation step in AOAC Official Method 985.29 is neither well documented nor
* Corresponding author. Tel.: +1-301-436-1786; fax: +1-301-4362636. E-mail address: [email protected] (J.I. Rader). 1 Visiting Scientist, University of Hamburg, Hamburg, Germany. 0308-8146/03/$ - see front matter Published by Elsevier Science Ltd. PII: S0308-8146(02)00393-X
clearly understood. It is generally assumed that oligosaccharides above a certain degree of polymerization (DP) will be precipitated by the addition of 95% ethanol and those below that DP will remain in solution. The absence of research in this area is primarily due to the lack of oligosaccharide standards of various DPs. Inulins, which consist of molecules with a wide range of DP values, are appropriate candidates for systematic investigations on the relationship between chain length and precipitation by specific solvents. Inulins and FOS are used as food ingredients and have been reported to show significant ‘‘dietary fiber’’ effects (Hoebrigs, 1997). The question arises as to whether such compounds should be excluded from consideration as dietary fiber simply because they do not precipitate on addition of 4 volumes of 95% ethanol. In this study, we investigated the precipitation of several inulins and oligoglucoses by various concentrations of ethanol, acetonitrile, acetone and propanol. In our earlier work on the determination of sugars and starches in food, we reported that when analyzing sugars by HPLC, the use of acetonitrile at a ratio of 1:1 has some advantages over the use of ethanol at a ratio of 4:1 (Casterline, Ik, & Ku, 2000).
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2. Materials and methods 2.1. Standards and reagents Raftiline ST-Gel and Rafiline were obtained from Tiense Suikerraffinaderij (Belgium). Raftiline GR, Raftiline HP-Gel and Raftiline HP were obtained from Orafti (Malvern, PA, USA). Inulin HD was obtained from Imperial Suiker Unie (Texas, USA) and ‘‘Inulin from Chicorie’’ from Fluka Chemie AG (Switerland). 1-Kestose (GF2), Nystose (GF3) and GF4 were gifts from Dr. Yasuhito Tashiro, Meiji Seika Kaisha Ltd. (Japan). Oligoglucoses with DP2 were purchased from Supelco (USA). UV grade acetonitrile was purchased from Burdick & Jackson (USA). USP 200 proof ethyl alcohol was obtained from Warner-Graham Company (USA). 1-Propanol and acetone were purchased from J. T. Baker (USA). 2-(N-Morpholino)ethanesulfonic acid (MES) and Tris(hydroxymethyl) aminomethane (TRIS) were purchased from Sigma Chemical Co. (USA). 2.2. Analysis of inulin High performance anion exchange (HPAE) chromatography was performed with a DX 500 Dionex System (Sunnyvale, CA, USA) consisting of a GP40 gradient pump, a Dionex eluent organizer and an ED 40 electrochemical detector working in pulsed amperometric mode (PAD). The detector was equipped with a gold electrode and Ag/AgCl reference electrode. The chromatographic conditions and the gradient for the separation of molecules of DP 4–7 (42 min) were set up as described by Durgnat and Martinez (1997). Subsequently, the gradient program was extended by increasing the percent of sodium acetate to cover molecules with a DP range up to 32. Data acquisition was stopped at 103 min. Table 1 lists the HPAE gradient analysis conditions.
Table 1 HPAE gradient analysis conditions Run time (min)
Flow rate (ml/min)
Eluent A (%) water
Eluent B (%) 300 mM NaOH
Eluent C (%) 150 mM NaOH 500 mM NaOAC
1.01 2.50 2.51 1.07 7.10 9.00 13.00 14.00 18.00 18.10 26.00 26.10 30.00 30.10 35.00 35.10 38.00 38.10 42.00 70.00 90.00 100.00 101.10 125.00
1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
56.0 54.5 51.5 51.0 81.0 85.5 60.0 38.0 39.5 73.0 44.0 73.0 64.0 42.0 22.0 42.0 42.0 35.0 32.0 15.0 10.0 9.0 0.0 0.0
44.0 42.5 41.5 40.0 14.0 9.5 30.0 50.0 46.0 19.0 32.0 19.0 22.0 32.0 40.0 32.0 32.0 32.0 32.0 30.0 15.0 10.0 0.0 0.0
0.0 3.0 7.0 9.0 5.0 5.0 10.0 12.0 14.5 8.0 24.0 8.0 14.0 26.0 38.0 26.0 26.0 33.0 36.0 55.0 75.0 81.0 100.0 100.0
2.3. Analysis of oligoglucoses HPLC analysis was performed with a Shimadzu System consisting of LC-600 pumps, CTP-6A column oven, RID-6A RI detector and AST-LC computer using Shimadzu EZChrom chromatography data system. The column used was a Benson Carbohydrate (Ag+) BC200 column (Alltech, Deerfield, IL), 300 mm 7.8 mm, with a Ag+ BC-200 guard column. The mobile phase was isocratic with degassed HPLC grade water. The flow rate was 0.3 ml/min. 2.4. Gravimetric method for determination of precipitation of inulins The analytical scheme is shown in Fig. 1. Approximately 0.5 g of triplicate test portions of inulin products were weighed and the weights were recorded. The samples
Fig. 1. Analytical scheme for the determination of inulins and oligoglucoses.
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were dissolved in 25 ml of MES/Tris buffer (2%). The samples were stirred with a magnetic stirrer until dissolution. Some inulin samples required the application of heat for complete dissolution. One milliliter of the solution was pipetted into pre-weighed test tubes. Solvents (i.e. ethanol, propanol, acetone or acetonitrile) were then added separately. Four ratios of solvent to test sample (i.e. 1:1; 2:1; 3:1 and 4:1, v/v) were investigated. The tubes were vortexed and stored at 4 C overnight. The following morning, the samples were warmed to room temperature and centrifuged for 10 min at 2500 rpm. The samples were then washed once with 4 ml of pure test solvent and centrifuged again. The solvent was discarded and the samples were placed in a drying oven at 110 10 C. for 75 min to remove the excess solvent. The samples were reweighed. Calculation of the difference between two weights gave the amount precipitated by the solvents. 2.5. HPLC analysis of oligosaccharides One percent solutions of inulin or oligoglucose were used for HPLC analysis. The three types of inulin, i.e. Inulin HD, Raftiline GR and Raftiline ST, with a full DP range from simple sugars to an estimated DP of 60, were investigated. The general procedures shown in Fig. 1 were followed. The supernatant solutions were diluted and analyzed by the HPLC procedure.
3. Results and discussion The weight percents of various inulins precipitated by four solvents at ratios of 4:1 are listed in Table 2. The weight percent of precipitation by ethanol is very similar to that of propanol. In general, acetonitrile and acetone precipitated more of the inulins than did ethanol. Differences in the weights precipitated are dependent on the molecular weight distributions of the various inulins. According to the manufacturer, Raftiline St-Gel and Raftiline GR have an average DP of 10, and Raftiline HP and Raftiline HP-gel have an average DP of > 23. Inulin HD and Inulin IQ have an average DP of 9–12. We performed HPAE chromatography on the supernatants in order to determine the size of the components that were precipitated. Three types of inulin (i.e. Inulin HD, Raftiline GR and Raftiline ST), with a full DP range from simple sugars to an estimated DP of 60, were analyzed separately. The results of analyses of supernatant fractions of these three inulins were similar, therefore the data were averaged and standard deviations were calculated. Fig. 2 shows the ratio of peak areas before and after ethanol precipitation. A ratio of 1 indicates that there is no precipitation. There is no precipitation when 1 part of ethanol was added to 1 part of
Table 2 Weight percent of inulins precipitated by four solvents at ratios of 4:1 (gravimetric method)
Raftiline St-Gel Inulin HD InulinIQ Raftiline GR Chicorie Raftiline HP-Gel Raftiline HP
Ethanol
Propanol
Acetone
Acetonitrile
46.42.2 55.82.5 52.32.7 65.63.9 83.95.8 97.81.9 99.50.9
47.8 1.9 52.4 5.5 52.1 6.1 59.5 1.6 79.1 10.2 95.9 2.1 97.8 1.9
56.82.9 57.52.0 63.03.5 73.95.8 99.11.5 96.22.5 96.92.7
68.7 8.6 71.0 1.7 72.3 1.4 74.7 5.3 95.7 4.3 93.2 2.6 95.7 3.7
Values are means S.D. of three independent measurements. Average degrees of polymerization: Inulin HD), Inulin IQ, 9–12; Raftiline St-Gel, Raftiline GR, 10; Raftiline HP-Gel, Raftiline HP, >23.
supernatant (data not shown). The first panel shows the results with 2 parts of ethanol and 1 part of supernatant (2:1 v/v). At a 2:1 ratio, only molecules with high DP begin to precipitate. For example, for molecules with DP 26, only 60% remained in solution. For molecules with DP 31, only 30% remained in solution. At a 3:1 ratio, most of the molecules with high DP precipitated and the molecules of lower DP began to precipitate. Finally, at a 4:1 ratio, the ratio required by AOAC method 985.29, only molecules with DP 1–10 seem to remain in solution. However, there are still significant amounts of molecules of DP 11 and 12 and small amounts of molecules of DP 14–18 remaining in solution. The recent data of Ohkuma, Matsuda, Katta, and Tsuji (2000) using corn syrup solids as starting materials, suggest that the AOAC method for dietary fiber can only determine water-soluble dietary fiber components with an average DP of 12 or higher. We also investigated the precipitation pattern with solvents other than ethanol. Fig. 3 shows the ratio of peak areas before and after acetone precipitation. Acetone precipitates more inulin than ethanol. The results of acetonitrile precipitation are shown in Fig. 4. Acetonitrile at a 1:1 ratio caused little or no precipitation at least up to DP 20. Significant increases in precipitation occur at a ratio of 2:1. Most of the molecules with high and medium DP were completely precipitated. About 25–30% of simple sugars remained in solution. Further increases in the acetonitrile: supernatant ratio seemed to have no further effect. At a ratio of 4:1, the percent of simple sugars remaining in solution is higher than that observed at ratios of 2:1 or 3:1 acetonitrile:supernatant. Inulin is an unique carbohydrate in that chemically it consists of repeated units of fructose. In this regard, it differs from most other fibers. Biologically, it also appears to behave differently than other fibers. Therefore, our data on the precipitation behavior of inulin may not be representative of that for other fibers. The precipitation patterns of oligoglucoses from DP 1 to 7 were also investigated and the results are shown in Table 3. Oligoglucoses of DP 8 and above are not
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Fig. 2. Analysis of supernatants by HPAE chromatography to determine the size of the components precipitated by ethanol: Inulin HD, Raftiline GR and Raftiline ST with a full DP range from simple sugars to an estimated DP of 60 were analyzed before and after ethanol precipitation. The panels in the figure show the ratios of peak areas before and after precipitation. A ratio of 1 indicates that there was no precipitation.
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Fig. 3. Analysis of supernatants by HPAE chromatography to determine the size of the components precipitated by acetone: Inulin HD, Raftiline GR and Raftiline ST with a full DP range from simple sugars to an estimated DP of 60 were analyzed before and after acetone precipitation. The panels in the figure show the ratios of peak areas before and after precipitation. A ratio of 1 indicates that there was no precipitation.
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Fig. 4. Analysis of supernatants by HPAE chromatography to determine the size of the components precipitated by acetonitrile: Inulin HD, Raftiline GR and Raftiline ST with a full DP range from simple sugars to an estimated DP of 60 were analyzed before and after acetonitrile precipitation. The panels in the figure show the ratios of peak areas before and after precipitation. A ratio of 1 indicates that there was no precipitation.
Y. Ku et al. / Food Chemistry 81 (2003) 125–132 Table 3 Percent of oligoglucoses (DP 1–7) precipitated by various solvents (HPLC method) Degree of polymerization
DP1 DP2 DP3 DP4 DP5 DP6 DP7
Solvents and ratios Ethanol
Acetone
Acetonitrile
(4:1)
(4:1)
(4:1)
(1:1)
0 0 0 0 0 0 0
0 0 0 0 14.30.6 25.70.6 46.71.1
57.02.0 87.01.0 70.01.7 67.71.5 68.31.5 100 100
0 0 0 0 0 0 0
DP, degree of polymerization. Values are means S.D. of three independent measurements.
commercially available and thus, could not be studied. Ethanol at a 4:1 ratio and acetonitrile at a 1:1 ratio did not precipitate any of the low DP oligoglucoses. However, we found that acetone at a 4:1 ratio begins to precipitate oligoglucose with DP 5. The percent of precipitation increases with increasing DP. Acetonitrile at a ratio of 4:1 does precipitate glucose and shows complete precipitation of oligoglucoses with DP 6 and 7. Our data suggest that the precipitation behavior of inulins and oligoglucoses follow a similar pattern. It should be emphasized that our precipitation data are based on inulin and oligoglucose, and that their chemical structures differ from those of other fibers, which may respond differently. These studies were undertaken in order to better understand the precipitation step in AOAC official method 985.29 for the analysis of dietary fiber. Our findings of marked differences between solvents in precipitating specific carbohydrate components are significant because precipitability by 95% alcohol ‘‘defines’’ dietary fiber by this method. At the present time, there are a wide variety of definitions for dietary fiber. Some are based on specific analytical methods for isolating and quantifying dietary fiber while others emphasize physiologic attributes of dietary fiber or its components. In the United States, dietary fiber is defined by a number of official methods of the Association of Official Analytical Chemists. The current situation with respect to isolating and hence defining dietary fiber on the basis of specific methods has been criticized because it does not consider the possible role(s) of the isolated compounds in health. Similarly, new fiber or fiber-like components that may be developed in the future and which may be found to provide beneficial effects may be excluded as ‘‘dietary fiber’’ by the current methods-based definition. As efforts to make food labeling more uniform throughout the world continue, the existence of a single definition of dietary fiber becomes more important.
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For these reasons, there have been a number of recommendations in the United States to modify the definition of dietary fiber (Gordon, 1999). Recently, both the American Association of Cereal Chemists (AACC, 2001) and a Panel of the Food and Nutrition Board of the Institute of Medicine (FNB/IOM, 2001) reviewed current definitions and proposed new definitions of dietary fiber. Both the AACC and IOM proposed definitions include criteria regarding digestibility and beneficial physiologic effects. Discussions about what components might be included or excluded by the new proposed definitions and about the availaility of current validated methods to address such components are continuing. Areas of particular interest include questions of which physiologic functions and which DP should be used to distinguish oligosaccharides from dietary fiber. With respect to determining DP, chromatographic analysis such as that described here provides an effective means of separating mixtures of molecules of differing DP. One of the most important physiologic characteristics of components defined as dietary fiber is that such components are not digested in the upper GI track. Inulins with DP 3 and above are not digested in the upper GI track. Therefore, using only a digestibility criterion, such components might be considered as dietary fiber. However, with our current knowledge, we do not know whether these small DP (perhaps 3 and above) molecules have any other fundamental physiological characteristics of dietary fiber (e.g. causing laxation). Furthermore, most of the inulins possess a full range of molecules of various DP. In foods such as cereals, we have little or no information regarding the distribution and relative quantities of the various DP molecules. Therefore, the net functionality of these molecules is still incompletely understood. In conclusion, more research is needed, particularly on the relationship between the specific DP of fiber components and their physiological functions. Such knowledge will assist in developing methods that are applicable to the identification and quantitation of these components.
Acknowledgements We thank Dr. Michael McLaughlin of the Center for Food Safety and Applied Nutrition, Food and Drug Administration, for his technical assistance in performing the HPAEC analysis.
References American Association of Cereal Chemists (AACC). (2001). The definition of dietary fiber. Cereal Foods World, 46, 112–116.
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