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. Author manuscript; available in PMC: 2023 Feb 22.
Published in final edited form as: Food Chem. 2022 Sep 2;401:134071. doi: 10.1016/j.foodchem.2022.134071

Chromatographic fractionation of food-grade oligosaccharides: Recognizing and avoiding sensory-relevant impurities

Toren S Andrewson 1, Laura E Martin 1, Juyun Lim 1,*, Michael H Penner 1,*
PMCID: PMC9945451  NIHMSID: NIHMS1872270  PMID: 36115234

Abstract

Flash chromatography utilizing microcrystalline cellulose (MCC) stationary phases and aqueous ethanol mobile phases have shown promise for the production of food-grade oligosaccharides. The current work extends the scope of these systems by demonstrating their use for the production of food-grade maltooligosaccharide preparations enriched in high degree of polymerization (DP) components. Furthermore, it is shown herein that caution must be exercised when using these MCC-based chromatographic systems in order to avoid sensory-relevant contamination of the final oligosaccharide preparations. Such contamination, most notably off-taste, is shown to arise from impurities common to commercially available MCC that manifest under certain chromatographic scenarios. A mitigation strategy based on washing the stationary phase with appropriate aqueous-ethanol solutions (i.e., accounting for the entire mobile phase concentration range) prior to oligosaccharide fractionation is presented as a means by which to avoid contamination.

Keywords: Separation, Chromatography, Cellulose, Impurities, Oligosaccharides, Off-taste

1. Introduction

Carbohydrates are of great interest to the food and nutritional sciences due to their obvious importance in human diet and health. Of these, starch is typically the most abundant dietary carbohydrate and the greatest source of energy in human diets (Hardy, Brand-Miller et al., 2015). Commercially produced starch hydrolysis products (i.e., maltodextrins) (Shah et al., 2010, Braquehais and Cava 2011, Leemhuis et al., 2014, Kong et al., 2020) as well as prebiotic oligosaccharides (Wang 2009, Gibson et al., 2017, Guimarães et al., 2020) are also widely used as food ingredients due to their functional properties and health benefits. Each class of carbohydrate has unique structures that impact their functional, nutritional, and sensorial properties. One structural property thought to have a significant impact on the functional and sensorial properties of oligosaccharides is their degree of polymerization (DP) (White et al., 2003, Belorkar and Gupta 2016). For example, an increasing body of work has demonstrated that maltooligosaccharides (MOS) with DP greater than 3 elicit a unique, non-sweet taste quality (i. e., “starchy”), while maltose (DP 2) and maltotriose (DP 3) elicit a sweet taste (Lapis et al., 2016, Pullicin et al., 2018).

Investigating the sensorial properties of specific oligosaccharides can be challenging because commercially available food-grade preparations are typically heterogeneous with respect to DP (i.e., polydisperse) and any attempts to prepare DP-defined preparations are limited to the use of verifiable food-grade processes. Recognizing this challenge, our research group has developed reliable, food-grade methods to produce DP-defined oligosaccharides suitable for use in studies that include human testing (Pullicin et al., 2017, Pullicin et al., 2018, Ooi et al., 2022). These methods utilize adsorption chromatography to separate oligosaccharide mixtures based on their relative partitioning between microcrystalline cellulose (MCC) stationary phases and aqueous-ethanol mobile phases.

While we were developing a procedure to expand the applicability of this chromatographic system, we observed conditions that, if not accounted for, can lead to final oligosaccharide preparations containing sensory-relevant contamination. The presence of such contamination can, in turn, result in considerable losses in time and resources. This communication begins by outlining a novel application of the MCC-based chromatographic system during which sensory-relevant contamination was encountered. The primary aim of the current study was to determine the source of the sensory-relevant contamination and to establish a protocol by which such contamination can be avoided.

2. Materials and methods

2.1. Materials

STAR-DRI 10 Maltodextrin (MD; Tate & Lyle Ingredients Americas, Decatur, IL) and a DP 9 + MOS preparation (Balto et al., 2016) were used as MOS starting materials for subsequent DP-based chromatographic fractionation. Pharmaceutical-grade microcrystalline cellulose (MCC; Avicel PH-101, UPI Chemicals, Somerset, NJ) was used as the chromatographic stationary phase. Fractionation solvents were composed of 100 % ACS/USP-grade ethanol (Pharmco Aaper, Shelbyville, KT) and deionized 18.2 Ω water purified with a Millipore Direct-Q 5 UV-R system (Burlington, MA).

Glucose monohydrate and maltose monohydrate were purchased from Spectrum Chemicals (Gardena, CA) as analytical standards; maltopentaose, maltonanaose, maltododecaose, maltotetradecaose, maltoheptadecaose and maltoicosaose standards were from Carboexpert (Daejeon, South Korea). Silica gel 60 thin layer chromatography (TLC) plates, HPLC-grade isopropanol, and sulfuric acid were purchased from EMD Millipore (Billerica, MA). Anthrone was from Alfa Aesar (Ward Hill, MA) and ReagentPlus 1-napthol from Sigma Aldrich (St. Louis, MO). HPLC/ACS-grade acetonitrile was from Fischer-Scientific (Fair-lawn, NJ). pH strips were purchased from VWR Scientific (Radnor, PA).

2.2. Methods

2.2.1. Chromatographic fractionation

2.2.1.1. Column-ready preparations.

Prior to each chromatographic fractionation, starting materials were processed to make MOS preparations enriched in specific DP components (Balto et al., 2016). The process makes use of the DP-dependence of MOS solubility in aqueous ethanol solutions; short-chain MOS have greater solubility in ethanol than long-chain MOS (Defloor et al., 1998, Hu and Goff 2018). Two column-ready preparations were developed; preparation I was enriched in DP 5 – 12 and preparation II in DP 12 – 25. For preparation I, 5 g MD were washed twice with 80 % aqueous ethanol (ethanol volume/total volume) based on the procedure in Balto et al. (2016). The washed precipitate was then re-suspended in a 70 % ethanol solution and allowed to settle overnight at 5 °C. The resulting supernatant constituted column-ready preparation I. For preparation II, 8 g DP 9 + MOS were suspended in a 50 % ethanol solution and allowed to settle overnight at 5 °C. The resulting supernatant constituted column-ready preparation II.

2.2.1.2. Column chromatography.

Fractionation of column-ready preparations occurred by adapting the method described by Pullicin et al. (2018). The 73 mm (ID) × 305 mm glass column (Synthware; Pleasant Prairie, WI) was packed with 200 g MCC suspended in 700 mL 70 % ethanol and then rinsed with an additional 500 mL 70 % ethanol. After packing to a final height of ~15 cm, 8–12 mL of column-ready sample containing ~1 g MOS was loaded and allowed to percolate into the column prior to starting the elution gradient. Mobile phase flow rates of ~15 mL/min were maintained by keeping the headspace of the column at 2–5 psi using compressed air.

Chromatography employed stepwise descending aqueous ethanol gradients. Distinct methods were developed to fractionate column-ready preparations I and II, targeting medium- and high-DP MOS, respectively. Preparation I was fractioned using a stepwise gradient consisting of 1.5 L 75 % ethanol, 1.5 L 65 % ethanol, and 1.5 L 55 % ethanol. Preparation II was separated in an analogous manner. The stepwise gradient series used 500 mL 65 % ethanol, 500 mL 60 % ethanol, 1 L 55 % ethanol, 1 L 50 % ethanol, and 500 mL 45 % ethanol. Fractions were collected in 100–120 mL volumes. Knowledge of what fractions to pool for optimal recovery of the target MOS was based on TLC results relative to analytical standards. TLC was done using silica gel 60 plates in an enclosed chamber saturated with an 18 1-propanol: 57 ethanol: 25 water solvent system (Huber et al., 1968). Developed plates were stained using the method previously described in our work (Ooi et al., 2022). In general, fractions containing a total of 360–400 mL of eluent were pooled. For example, for preparation I, the following were pooled: 360 mL after 500 mL of 65 % ethanol had eluted (fraction A) and 360 mL after 500 mL of 55 % ethanol had eluted (fraction B). For preparation II: 400 mL pooled after 700 mL 55 % ethanol had eluted (fraction C) and 400 mL after 800 mL 50 % ethanol had eluted (fraction D) was typical.

Rotary evaporation (Buchi Rotavapor R-300 with 150 torr Welch DryFast Collegiate Dry Vacuum Pump) was used to concentrate samples and eliminate residual ethanol. Each sample was reduced to a viscous liquid in a 60 °C water bath, washed with DI water, and re-concentrated. The resulting samples were frozen and lyophilized with a Labconco Freezone Freeze Dryer (Hampton, NH). Each chromatographic run resulted in 20–50 mg of the desired MOS fractions.

2.2.1.3. Characterization of oligosaccharide fractions.

High performance liquid chromatography (HPLC) was used to assess the DP profiles of the final MOS preparations. These chromatographic runs made use of a Shodex HILICpak VN-50 4D analytical column coupled with a HILICpak VN-50G 4A guard column (New York, NY). Mobile phases were acetonitrile/water gradients ranging from 70/30 % to 50/50 % and were optimized for each fraction. Chromatography was performed using the Prominence UFLC-HPLC system (Shimadzu, Columbia, MD) with system controller (CMB-20A), autosampler (SIL-10A), degasser (DGU-20A), solvent delivery module (LC-20AD), column oven (CT20-A) kept between 40 and 60 °C, and an evaporative light scattering detector (ELSD-LT II) with a nitrogen gas pressure of 350 kPa. Chromatogram analyses, including peak integration, were done using LC-solution software (Shimadzu, Kyoto, Japan).

2.2.2. Analysis of contamination

2.2.2.1. Assessing Sensory-Relevant character of oligosaccharide fractions.

Color was analyzed both visually and by absorption spectrophotometry at 420 nm. All pHs were estimated using pH strips. As off-taste was of preeminent concern, our in-house trained panel (n = 4) was asked to taste the target samples. These participants have extensive experience with respect to the taste of MOS (e.g., “starchy”) and identifying any off-taste in stimuli. The samples were tasted by rolling a cotton swab saturated with the stimuli across the tip of the tongue while wearing nose clips (Pullicin et al., 2017). Each participant was asked to verbally describe the taste qualities perceived from each stimulus. At times, participants were given triangle tests (a set of 3 stimuli; 1 target and 2 controls) to investigate if they could discriminate the target stimulus from controls. These combined approaches were used to evaluate and compare the taste of all oligosaccharide preparations, starting materials, and column-ready preparations. When possible, stimuli were given as 75 mM aqueous solutions, as MOS have been shown to elicit a recognizable taste sensation in most subjects at this concentration (Lapis et al., 2016).

2.2.2.2. Assessing MCC as source of Sensory-Relevant contamination.

Two approaches were used to assess the possibility of contaminants in MCC. First, 100 g MCC was mixed with 300 mL 40 % aqueous ethanol and stirred for 30 min. The suspension was allowed to settle overnight at 4 °C, after which approximately 200 mL of the supernatant was collected for further analysis. Second, eluent was collected and analyzed from a packed, but unloaded column. Here, 125 g of MCC was mixed with 500 mL 70 % ethanol and packed as described above (see section 2.2.1.2). A gradient of 1.5 L 70 % ethanol followed by 1.5 L 50 % ethanol was used. Four fractions were collected: the first and last 400 mL of both the 70 % eluent and 50 % eluent. Samples were rotary evaporated to remove residual ethanol and concentrate the solutions to a final volume of approximately 10 mL. The resulting concentrates were tested for sensory properties as described in section 2.2.2.1.

3. Results and discussion

3.1. Oligosaccharide fractionation

A novel application of the chromatographic system was tested in order to extend current understanding of the MCC/aqueous ethanol system’s separatory power. Specifically, this preparation has extended previous work by targeting narrow-DP ranges of high-DP oligosaccharides, which were previously difficult to separate. The specific goal of this phase of work was to prepare food-grade MOS samples differing with respect to their contiguous DP ranges. HPLC chromatograms demonstrating the achievement of that goal are shown in Fig. 1. The chromatograms depict the results of fractionating column-ready preparation I (A and B) and column-ready preparation II (C and D); these chromatographic runs differed with respect to MOS starting materials and aqueous ethanol gradients (see section 2). The resulting MOS preparations have clearly different DP profiles (A, DP 5–8; B, DP 7–12; C, DP 10–18; and D, DP 15–23). These data provide new insights into potential applications of MCC-based chromatographic systems for the fractionation of higher DP MOS preparations; previously published applications of this system were limited to MOS having DP ≤ 8.

Fig. 1.

Fig. 1.

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Chromatograms from HPLC-ELSD depicting degree of polymerization (DP) profiles of final oligosaccharide preparations. A and B were fractioned with a microcrystalline cellulose/aqueous ethanol chromatographic system using column-ready preparation I; C and D used column-ready preparation II (see methods). Acetonitrile/water gradients between 70% and 50% were optimized to obtain each chromatogram. DP is represented as integers above the corresponding peaks.

3.2. Contamination with Off-Taste in MOS preparations

Off-taste associated with column-fractionated MOS can result in lost preparations, time, and resources. Therefore, the remainder of this communication focuses on the nature of this annoyance and means by which to avoid it. During a pilot study where our trained panel tasted target stimuli, they described MOS preparations A and B as “sweet” or “starchy.” These descriptors were expected for low and medium DP MOS based on previously published work (Lapis et al., 2016). However, preparations C and D were described as “sour” or “bitter” or “off”. Further sensory testing using the same trained panel confirmed that preparations C and D could easily be discriminated from the MOS 9 + starting material, which contained no detectable off-taste. Column-ready preparations (i.e., those resulting from crude fractionation of the starting material based on differential solubility in aqueous-ethanol solutions) also contained no identifiable off-taste and were indiscernible from the MD starting material. The results of these preliminary sensory tests suggested that 1) MOS preparations C and D contained sensory-relevant contaminants with off-taste that made them unsuitable for sensory studies, and 2) the genesis of the contamination was related to chromatographic fractionation.

3.3. Sources of Off-Taste contamination

Our initial inquiry into the source of contamination addressed whether the pharmaceutical-grade MCC products used in the chromatographic fractionations contained impurities. For reference, MCC itself is known to be tasteless (Westermarck et al., 1999, Nsor-Atindana et al., 2017, Santos et al., 2021). However, when a representative MCC sample was extracted with an aqueous-ethanol solvent (40 % ethanol) and that extract was subsequently reduced by rotary evaporation (analogous to that performed for MOS preparations), the resulting concentrate had a mild off-taste. Aqueous 70 % ethanol eluent passed through a freshly packed, but unloaded MCC column also contained distinct contamination, similar to that referred to as “off-taste” in preparations C and D.

A subsequent literature search unearthed reports of impurities in pharmaceutical-grade MCC preparations analogous to those used in this work (Wu et al., 2011); this study was evaluating MCC that was used as an excipient, not as a chromatographic stationary phase as is the case in the current work. The implication from these observations is that the MCC preparations used for MOS fractionation indeed contain sensory-relevant impurities that are amenable to leaching into oligosaccharide fractions during chromatographic runs. The identities of the contaminants are currently unknown. They could be natural compounds present in the starting material prior to the production of MCC or compounds generated during MCC production. Relevant compounds may include those derived from lignin and other plant cell wall constituents (Landin et al., 1993, Kian et al., 2020, Hachaichi et al., 2021, Ventura-Cruz and Tecante, 2021).

3.4. Additional Sensory-Relevant contamination

Off-taste is not the only sign of sensory-relevant contamination seen in MOS preparations fractioned using MCC-based chromatographic systems. Some preparations were found to have off-colors as well. The MD starting material is a white powder and forms clear aqueous solutions or white opaque suspensions, depending on solubility. In contrast, some MCC-fractionated oligosaccharides have possessed a yellow tinge, which is especially noticeable in concentrated solutions (see Fig. 2 for representative examples). This off-color also appears to originate from the MCC stationary phase; the concentrated 70 % ethanol extract of MCC (as discussed in section 3.3) also had a yellow tinge. However, the presence of off-color and off-taste are not always correlated. MOS preparation C (Fig. 1), which presented with off-taste, had the darkest coloring. Yet, preparations B and D possessed similar coloring while only D possessed a notable off-taste. The extent of this yellow coloration can be quantified spectrophotometrically (see data in Table 1).

Fig. 2.

Fig. 2.

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Photograph showing the range of colors in final oligosaccharide preparations (from left to right: A, B, C, and D of Fig. 1). Yellow tinge is considered to be off-color. All samples are pictured at 75 mM concentrations, which is appropriate for sensory testing.

Table 1.

Analysis of contamination markers in final oligosaccharide preparations.

Preparation pHa Colora Absorbanceb (420 nm)
A 6 Light Yellow 0.192
B 7 Flaxen 0.805
C 8 Mustard Yellow 1.026
D 8 Flaxen 0.787
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a

Evaluated at 75 mM concentration.

b

Samples diluted to 5 mM concentration for spectrophotometric analysis.

It was also observed that some MOS preparations resulting from MCC-based fractionation had experienced an alkaline pH shift. Aqueous solutions of 75 mM MOS starting material have pHs of ~5. Equimolar solutions of the fractionated MOS preparations had pH values between 6 and 8 (see Table 1). Equimolar solutions having discernable off-taste tended to have higher pHs than those that did not. In those cases where sensory-relevant contamination was present, the more concentrated the MOS solution the greater the intensity of the off-taste. It was determined that yellow color and higher than expected pH were indicative of potential contamination, but these characteristics could not be used to reliably determine the extent of off-taste contamination.

3.5. Method development to avoid sensory-relevant contamination

Commercially-available MCC preparations are known to exhibit batch-to-batch differences in purity (Wu et al., 2011). Hence, the incidence of off-taste contamination is difficult to predict. Therefore, it is prudent to make allowances for the presence of such impurities; this includes employing both mitigation and tracking tactics. Mitigation is best achieved by appropriately washing the MCC prior to oligosaccharide fractionation. This can be done prior to packing the column or by flushing the packed column. The key point is that the washing should be done using ample solvent that spans the entire range of the mobile phase gradient composition to be used in the chromatographic run. The optimum approach when using stepwise gradients is to wash the freshly packed column with an excess (~1 L) of the final mobile phase to be used in the stepwise gradient until the eluent runs clear. Then, a second wash of the column is advisable using the initial mobile phase that is to be used in the chromatographic run. Upon completion of the second wash, the column is ready for oligosaccharide fractionation. These mitigation recommendations are based on findings showing that a stepwise gradient resulted in pulses of impurities eluting at distinct steps, as in the cases of preparations C and D.

Alternatively, one can carefully avoid collecting eluent immediately after making a step change in the mobile phase gradient, since this is when impurities are most likely to elute. However, we do not endorse this approach because determining the amount of eluent to void following changes in gradient composition is difficult to determine and there is always the potential of losing significant amounts of oligosaccharides with the voided eluent.

The mitigation approach advocated herein (i.e., appropriately washing the packed column prior to beginning fractionation), is designed to account for the possibility of multiple sensory-relevant impurities being present in the chosen MCC preparation and that those impurities likely differ with respect to their relative partition co-efficients within this chromatographic system. It is prudent to combine a monitoring scheme with the mitigation strategy; this can be done by assessing the pH, color, and taste of eluents during fractionation. This will provide an initial assessment of the likelihood of sensory-relevant contamination; but as explained previously, these parameters are not fool-proof predictors of off-flavor contamination in the final MOS preparations. The latter is best assessed using sensory tests following concentration and drying of collected column eluent. If sensory-relevant contamination is further identified, the contaminated fractions can be “washed” in aqueous ethanol until markers of contamination are no longer present. This is achieved using the principles of ethanol fractionation described above (see section 2.2.1.1) to precipitate oligosaccharides while leaving contaminants in the supernatant to be discarded.

To demonstrate the effectiveness of this mitigation approach, we have included a clean sample, analogous to fraction C, which was the sample with the most severe contamination. This sample was prepared with the approach described herein, both with alterations to chromatographic preparation and washing in aqueous ethanol. The color of the sample is white or off-white, as shown in Fig. 3. Additionally, the pH of the sample is 6, and spectrophotometric analysis (at 420 nm) revealed an absorbance of 0.096. Critically, sensory analysis by the trained sensory panel revealed that the cleaned sample no longer elicited “sour” or “bitter” perceptions, and was instead described as “mild” and “starchy”.

Fig. 3.

Fig. 3.

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Photograph of final oligosaccharide preparation C, produced using the “clean” methodology described in this manuscript. Sample is pictured at 75 mM concentrations, which is appropriate for sensory testing.

4. Conclusion

Chromatographic systems employing MCC stationary phases and aqueous ethanol mobile phases are highly effective for fractionating oligosaccharides in a food-grade, DP-dependent manner. This is demonstrated by the current study, which obtained MOS fractions with average DP ranging from ~6 to ~20. The resulting oligosaccharides may be used for human psychophysical testing or in targeted commercial applications. However, herein we show that commercially available MCC preparations used as the stationary phase may contain impurities that can leach into the oligosaccharide preparations during fractionation. If this happens, the resulting fractionated preparations may elicit “off-taste,” which is described as “sour” or “bitter.” To avoid such contamination, MCC stationary phases should be appropriately washed with aqueous ethanol solutions that cover the entire concentration range to be used in the subsequent chromatographic run; all washing is to be done prior to oligosaccharide fractionation. It is also prudent to monitor for potential sensory-relevant contamination based on the pH and color of eluted fractions. The recommended procedures should allow consistent fractionation of food-grade oligosaccharides even when the purity of the MCC cannot be guaranteed.

Acknowledgements

This research was supported by grant R01DC017555 from the NIH/NIDCD. The authors would like to thank Tate & Lyle Ingredients Americas for providing starting materials.

Footnotes

CRediT authorship contribution statement

Toren S. Andrewson: Investigation, Formal analysis, Visualization, Writing – original draft. Laura E. Martin: Formal analysis, Visualization. Juyun Lim: Conceptualization, Methodology, Resources, Supervision, Writing – review & editing. Michael H. Penner: Conceptualization, Methodology, Resources, Supervision, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

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Data Availability Statement

Data will be made available on request.

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