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Article

Exploration and Improvement of Acid Hydrolysis Conditions for Inulin-Type Fructans Monosaccharide Composition Analysis: Monosaccharide Recovery and By-Product Identification

by
Xinyan Zong
,
Ningyu Lei
,
Junyi Yin
,
Weiwei He
*,
Shaoping Nie
and
Mingyong Xie
State Key Laboratory of Food Science and Resources, China-Canada Joint Laboratory of Food Science and Technology (Nanchang), Key Laboratory of Bioactive Polysaccharides of Jiangxi Province, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China
*
Author to whom correspondence should be addressed.
Foods 2024, 13(8), 1241; https://doi.org/10.3390/foods13081241
Submission received: 9 March 2024 / Revised: 11 April 2024 / Accepted: 13 April 2024 / Published: 18 April 2024
(This article belongs to the Special Issue Identification, Characterization and Quantification of Food Compounds Using Chromatography and Mass Spectrometry)
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Abstract

:
Acid hydrolysis serves as the primary method for determining the monosaccharide composition of polysaccharides. However, inappropriate acid hydrolysis conditions may catalyze the breakdown of monosaccharides such as fructans (Fru), generating non-sugar by-products that affect the accuracy of monosaccharide composition analysis. In this study, we determined the monosaccharide recovery rate and non-sugar by-product formation of inulin-type fructan (ITF) and Fru under varied acid hydrolysis conditions using HPAEC-PAD and UPLC-Triple-TOF/MS, respectively. The results revealed significant variations in the recovery rate of Fru within ITF under different hydrolysis conditions, while glucose remained relatively stable. Optimal hydrolysis conditions for achieving a relatively high monosaccharide recovery rate for ITF entailed 80 °C, 2 h, and 1 M sulfuric acid. Furthermore, we validated the stability of Fru during acid hydrolysis. The results indicated that Fru experienced significant degradation with an increasing temperature and acid concentration, with a pronounced decrease observed when the temperature exceeds 100 °C or the H2SO4 concentration surpasses 2 M. Finally, three common by-products associated with Fru degradation, namely 5-hydroxymethyl-2-furaldehyde, 5-methyl-2-furaldehyde, and furfural, were identified in both Fru and ITF hydrolysis processes. These findings revealed that the degradation of Fru under acidic conditions was a vital factor leading to inaccuracies in determining the Fru content during ITF monosaccharide analysis.

Graphical Abstract

1. Introduction

Fructans, initially discovered by the German scientist Rose in 1804, are fructofuranose-based compounds primarily composed of fructose linkages. They were first isolated from Inula helenium L., a plant belonging to the Asteraceae family [1]. Plant-derived fructans, due to their isolation from Inula helenium and naming as inulin, are also termed as inulin-type fructans (ITF). IFT predominantly consists of β-1,2-linked fructofuranose units. In recent years, numerous investigations delve into a multitude of aspects concerning of ITF, including encompassing extraction, purification, physicochemical properties, structural characteristics, and physiological activities [2,3]. ITF derived from natural plants exhibits a wide range of bioactive functions and demonstrates considerable edible and medicinal value [4]. Furthermore, research on ITF has leveraged the inherent properties of fructans to drive advancements in food technology, health products, and industrial production [5,6].
The determination of monosaccharide composition plays a pivotal role in elucidating the primary structure of polysaccharides [7]. Furthermore, exploring acid hydrolysis conditions for monosaccharide composition analysis is not only necessary for the structural elucidation of ITF but also holds substantial importance for the identification of polysaccharides sourced from diverse origins, types, or compositions. Acid hydrolysis stands as the cornerstone pretreatment method for polysaccharide samples, facilitating the liberation of constituent monosaccharides for subsequent analysis, often employing the high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method. Key variables such as acid hydrolysis duration, temperature, and acid concentration are meticulously adjusted to ensure the optimal recovery, precision, and reproducibility of monosaccharide data [8]. Tailoring hydrolysis conditions to suit the dissociation of polysaccharides with diverse structural characteristics is crucial to achieving accurate results [9,10]. For instance, distinct ketoses (e.g., D-fructose) and aldoses (e.g., D-glucose) may yield identical alditol products upon reduction, owing to the enolization and dehydration processes converting D-fructose to alditol under harsh hydrolytic conditions. Consequently, milder hydrolysis conditions are preferred for samples rich in fructose [11,12,13].
ITF is a furanose fructan characterized but exhibits lower stability in acid hydrolysis compared to pyranose fructans, with a recovery rate of only 80~90% [14,15]. Therefore, when delving into the analysis of ITF’s monosaccharide composition, researchers are inevitably confronted with the challenge posed by its diminished stability during acid hydrolysis. The accuracy of determining its monosaccharide composition is intricately tied to various factors, including monosaccharide variability, their linkages, and configurations. For instance, in the investigation of ITF extracted from Polygonatum sibiricum, different hydrolysis temperatures exerted notable effects on the structural characterization efforts. Moreover, under strong acid or high-temperature conditions, carbohydrates can undergo enolization and dehydration reactions, leading to the generation of various non-sugar by-products [16]. For instance, in the acid-catalyzed hydrolysis of ITF, the depolymerization of polysaccharides into free Fru may lead to additional hydrolysis, resulting in the formation of certain furfural by-products [17,18]. This process compromises the accuracy and reliability of experimental data, posing challenges to the structural characterization of ITF [19,20].
Additionally, the process yields isomaltol, hydroxyacetylfuran carboxylic acid, formic acid, acetic acid, and propionic acid [21,22]. Inappropriate hydrolysis methodologies result in the degradation of polysaccharides into non-sugar compounds, leading to incorrect estimations of monosaccharide content in ITF. Consequently, proper modification of experimental hydrolysis conditions, considering the unique properties inherent to diverse polysaccharides, constitutes an imperative consideration [23]. The customization and optimization of acid hydrolysis conditions tailored to the specific attributes of a given polysaccharide are pivotal for ensuring accuracy in the analysis of its monosaccharide composition and facilitating structural characterization [24,25].
In this study, we explored optimal acid hydrolysis conditions and analyzed hydrolysis by-products of ITF in varying acid concentrations, hydrolysis time, and temperatures. Using the HPAEC-PAD method to determine the monosaccharide recovery rates of ITF under various acid hydrolysis conditions, we optimized the acid hydrolysis conditions for IFT monosaccharide composition analysis. Furthermore, the impact of acid hydrolysis conditions on Fru recovery rates was validated through Fru standard samples. Subsequently, in order to explore the main reasons for the inaccurate determination of fructose content in the analysis of ITF monosaccharide composition, we identified the hydrolysis by-products in IFT and fructose standard samples using UPLC-Triple-TOF/MS. Based on this study, we aim to provide theoretical basis for the hydrolysis and structural analysis of ITF.

2. Materials and Methods

2.1. Materials and Reagents

Inulin-type fructan was obtained from BENEO-Orafti (Oreye, Belgium). Monosaccharide standards, D-glucose (Glc) and D-fructose (Fru), were purchased from Sigma-Aldrich (Bratislava, Slovakia). Oasis HLB DISKS were acquired from Waters Corporation (Milford, MA, USA). Sulfuric acid (H2SO4, GR) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals used in this study were of analytical grade and obtained commercially.

2.2. Hydrolysis and Monosaccharide Composition Analysis

To explore the stability of monosaccharides under different hydrolysis conditions [26], the Fru and ITF were hydrolyzed under the same conditions. The samples (5 mg each) were individually dissolved in 0.5 mL of H2SO4 and stirred for 30 min in an ice bath. Hydrolysis reactions were subsequently conducted under varied conditions, including different temperatures (40, 60, 80, 100, and 120 °C), acid concentrations (0.5, 1 and 2 M), and heating durations (0.25, 0.5, 1, and 2 h). When the reaction was completed, the sample was diluted to a volume of 20 μg/mL before chromatography analysis.
The monosaccharide composition was analyzed by HPAEC (Dionex ICS-6000 system, Thermo Fisher Scientific, Waltham, MA, USA), and pulsed amperometric detector (PAD) equipped with working electrode (gold electrode) and reference electrode (Ag/AgCl) was used for detection. Dionex Carbo PacTM PA-20 (3 mm × 30 mm) guard column and PA-20 (3 mm × 150 mm) analytical column was adopted for separation [13]. Gradient elution was performed using 250 mmol/L NaOH solution, deionized water, and 1 mol/L NaOAc solution as mobile phases at a flow rate of 0.5 mL/min. The column and detector temperatures were 30 °C and 25 °C, respectively, while the injection volume was 10 μL. The detection was carried out using a sugar standard four potential method. The monosaccharide standards and these samples were processed and analyzed in parallel.

2.3. Desalination and Hydrolysis By-Products Identification

To identify the hydrolysis by-products under different hydrolysis conditions, the experiment was divided into three groups with significant differences based on the hydrolysis conditions set in 0: incomplete acid hydrolysis conditions (IN: hydrolyze with 0.5 M H2SO4 at 40 °C for 0.5 h), better acid hydrolysis conditions (BE: hydrolyze with 1 M H2SO4 at 80 °C for 2 h), and excessive acid hydrolysis conditions (EX: hydrolyze with 1 M H2SO4 at 120 °C for 1 h).
After hydrolysis, the sodium hydroxide solution was used to neutralize the residual H2SO4 in the hydrolyzed solution, maintaining the pH of the reaction system at 7.0. Solid phase extraction was carried out using a vacuum solid phase extraction device and an activated Waters Oasis HLB cartridge (200 mg, 6 cc) solid phase extraction column to extract and desalt the neutralized sample. The sample was washed through the extraction column with 2.5 mL of deionized water, followed by washing and collecting the eluent with 1.5 mL of methanol solution. Finally, the eluent was collected and passed through a 0.22 μm organic filter and transferred to a sample vial for subsequent UPLC-Triple-TOF/MS determination and analysis [27,28]. Three parallel eluents were prepared for each sample.
UPLC-Triple-TOF/MS was employed to identify compounds generated in the reaction systems under different hydrolysis conditions and analyze the association between hydrolysis conditions and side reactions based on changes in characteristic differential compounds. The chromatographic column used was an ACQUITY UPLC HSS T3 (100 Å, 50 mm × 2.1 mm, 1.8 μm). The mobile phases were 0.1% formic acid aqueous solution and methanol at a flow rate of 0.25 mL/min. The injection volume was 5 μL. Mass spectrometric detection was performed in positive/negative ionization modes.

2.4. Statistical Analysis

The monosaccharide standards at different concentrations were analyzed by HPAEC-PAD to establish standard curves with peak area integration as the variable. This was used to calculate the monosaccharide recovery rates of the aforementioned samples. Data plotting was performed using Origin Pro 2017C (version B9.4.2.380, Stat-Ease Inc., Minneapolis, MN, USA). The results are expressed as mean ± standard deviation (SD).
The hydrolysis products of Fru and ITF under three different acidic hydrolysis conditions were determined by UPLC-Triple-TOF/MS. Feature compounds were preprocessed using Progenesis QI software (V 2.1 ASMS 2015). After preprocessing, the data were uploaded to MetaboAnalyst 5.0 (www.metaboanalyst.ca, accessed on 8 September 2023) [28,29] for chemometric analysis. The chemical categories of compounds were queried on the PubChem website (https://pubchem.ncbi.nlm.nih.gov/, accessed on 18 September 2023).

3. Results and Discussion

3.1. Optimization of Acid Hydrolysis Conditions of ITF

Previous studies have shown that ITF undergoes hydrolysis into Glc and Fru in H2SO4 solution. However, Fru may exhibit poor stability under acidic hydrolysis conditions, potentially resulting in inaccuracies in the determination of monosaccharide composition. To investigate the changes in monosaccharide recovery rate of ITF under varying hydrolysis conditions and determine the optimal hydrolysis conditions, ITF was hydrolyzed at different conditions, including H2SO4 concentrations of 0.5 M, 1 M, and 2 M, temperatures ranging from 40 °C to 120 °C, and reaction times of 0.25 h, 0.5 h, 1 h, and 2 h. The relative percentage of the recovery rate from each experiment is shown in Figure 1.
In general, the recovery rates of these two monosaccharides varied under different hydrolysis conditions. However, compared to the recovery rate of Fru, the recovery rate of glucose from ITF remained relatively stable under different conditions. Specifically, under relatively mild acid condition (0.5 M H2SO4) and temperature rang (from 40 °C to 60 °C), the recovery rate of Fru increased with reaction time. However, when the hydrolysis temperature exceeded 100 °C and the concentration of H2SO4 exceeded 1 M, the Fru recovery rate experienced a dramatic decrease. Therefore, this hydrolysis condition was optimal for the determination of monosaccharides in ITF. In addition, these results indicated that Fru can achieve a relatively ideal range of the hydrolysis recovery rates under mild hydrolysis conditions [30]. Once the hydrolysis conditions meet the requirements for monosaccharide release, the variation in the recovery rate is no longer dependent on whether the hydrolysis intensity is sufficient to support monosaccharide release but rather on the stability of the monosaccharide in the hydrolytic environment [27,31]. However, when hydrolysis conditions (e.g., acid strength or temperature) exceed the optimal parameters, with increasing hydrolysis intensity, Fru may gradually degrade and convert into non-sugar substances, resulting in a significant decrease in the recovery rate, approaching zero [27].
In contrast to Fru, the Glc released from ITF exhibited stable hydrolysis recovery rates under the different hydrolysis conditions set in the experiment. As shown in Figure 1, during the initial hydrolysis process at 40 °C using 0.5 M sulfuric acid hydrolysis for 0.25~1 h and 1 M sulfuric acid hydrolysis for 0.25~0.5 h, the recovery rate of Glc gradually increased, indicating the gradual release of free Glc monosaccharides from ITF, reaching a recovery rate of approximately 15%. Subsequently, even with the increased hydrolysis intensity up to 120 °C with 2 M sulfuric acid for 2 h, no significant trend of Glc loss or degradation was observed, and the recovery rate remained relatively stable within the range of 10% to 16%. These results indicated that Glc may not achieve satisfactory recovery rates under low hydrolysis intensities but exhibits good stability under subsequent severe hydrolysis conditions, with relatively minor variations influenced by the different hydrolysis conditions within the specified range [32,33].
Therefore, under the conditions of 1 M H2SO4 at 80 °C for 2 h, both fructose and glucose exhibit better recovery rates, making it the optimal hydrolysis condition for determining the composition of ITF monosaccharides (Figure 1). However, taking into account practical considerations and efficiency, it can be inferred that a broader range of acid hydrolysis conditions, spanning temperatures from 60 to 100 °C, sulfuric acid concentrations ranging from 0.5 to 1 M, and hydrolysis duration of 0.5 to 2 h, could be deemed as suitable for the hydrolysis of ITF. Subsequently, we employed the optimized acid hydrolysis conditions (1 M sulfuric acid hydrolysis for 2 h at 100 °C) to elucidate the structural characteristics of ITF from Polygonatum sibiricum [34,35].

3.2. Stability of Monosaccharide Standards under Different Hydrolysis Conditions

Previous studies suggest that Fru exhibits poorer stability compared to other monosaccharide such as glucose or galactose [22]. During acid hydrolysis, monosaccharide (e.g., Fru) released from the polysaccharide chain may undergo further degradation to produce by-products such as 5-hydroxymethyl-2-furaldehyde, hydroxyacetylfuran, 5-methyl-2-furaldehyde, hydroxyacetylfuran formic acid, formic acid, acetic acid, and levulinic acid [21,36]. The degradation rate and degree of fructose increase with higher levels of acid concentration, longer reaction time, and elevated temperature [27,31]. To elucidate how these hydrolysis conditions affect the content and stability of free fructose released from ITF, we examined the acid stability of fructose. Figure 2 shows the fluctuation in Fru standard monosaccharide concentration over hydrolysis time under different temperature and H2SO4 concentrations. The recovery rate obtained from each experiment is expressed in relative percentage and listed in Supplementary Data Table S1.
The results showed that Fru underwent significant degradation with the progression of hydrolysis under various conditions, resulting in a decrease in concentration, and generally stabilizes after 1 h (Figure 2). Furthermore, with increasing hydrolysis temperature and H2SO4 concentrations, the Fru concentration exhibited a decrease trends. Specifically, when the concentration of H2SO4 reached 2 M or the temperature exceeded 100 °C, the Fru recovery rate significantly decreased or even approaching 0. This is in accordance with several previous studies [31,37], supporting the hypothesis that excessively strong hydrolysis conditions during the hydrolysis of ITF can lead to fructose degradation, thereby reducing the accuracy of Fru composition results.

3.3. Analysis of Hydrolysis By-Products

3.3.1. Trend of Hydrolysis By-Products of ITF under Different Hydrolysis Conditions

The hydrolysis reaction of polysaccharides is a complex process that involves not only the breakdown of polysaccharides into oligosaccharides and monosaccharides but also various side reactions leading to the formation of non-sugar compounds such as furfural [22,28]. To investigate the types and variations in the content of non-sugar compounds generated during the hydrolysis process of ITF, UPLC-Triple-TOF/MS analysis was employed to determine the relevant compound profiles under different acid hydrolysis conditions, including incomplete hydrolysis conditions (IN) with 0.5 M H2SO4 at 40 °C for 0.5 h, better hydrolysis conditions (BE) with 1 M H2SO4 at 80 °C for 2 h, and excessive hydrolysis conditions (EX) with 1 M H2SO4 at 120 °C for 1 h. Furthermore, employing Progenesis QI software for data processing and statistical analysis, a total of 585 characterized compounds were selected from 7456 and 5179 original data in positive and negative ion modes from Fru and ITF hydrolysis solution.
Based on the identification of compounds obtained in three different hydrolysis conditions (the three hydrolysis conditions group IN, BE, and EX are introduced in 0), chemometric analysis methods were performed to investigate the differential features of Fru and ITF. Further clarification of the differences in compound generation under different hydrolysis conditions was carried out by using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) score plots, as shown in Figure 3. The results indicated a certain degree of dispersion among the different groups, suggesting the presence of differences in the compounds generated under different hydrolysis conditions.
Figure 4A and Figure 4B, respectively, display comparative analyses of the hydrolysis by-products of Fru and ITF under varying acid intensities. In general, numerous by-products resulting from the hydrolysis of Fru and ITF underwent significant alterations as the hydrolysis conditions intensified, which is consistent with previous reports [38]. Furthermore, previous studies have documented that the generation of by-products resulting from ITF hydrolysis primarily comprising furfural compounds [36,39,40].
During Fru hydrolysis and ITF hydrolysis, the levels of furfural compounds, such as 5-hydroxymethyl-2-furaldehyde (5-HMF) and 5-methyl-2-furaldehyde (5-MF), varied among different hydrolysis conditions. During Fru hydrolysis, the content of 5-HMF accumulated gradually with increased hydrolysis degree, while 5-MF only exhibited a slight increase in the EX and BE groups compared to the IN group. Interestingly, furfural (FF) exhibited a decrease in the EX group compared to the BE group during Fru hydrolysis. During ITF hydrolysis, both 5-HMF and 5-MF underwent a trend of initially rising and then declining as the acid hydrolysis conditions intensified (IN → BE → EX), while furfural (FF) shows a lower concentration in the EX group compared to the BE group. Due to the intensification of hydrolysis conditions, both in the hydrolysis of Fru and ITF, there was a significant reduction in Fru in the products. Therefore, it can be inferred that intensified hydrolysis conditions may lead to the instability of the furfural compound generated from Fru conversion, resulting in its further decomposition or conversion into various by-products, including 2-hydroxyacetofuran [41], 5-chloromethylfurfural [42], furan formaldehyde, acetylpropionic acid, formic acid, etc. For instance, according to a report by Asghari FS and Yoshida H (2006), it has been observed that at lower pH levels, 5-HMF may convert into levulinic and formic acids [43].

3.3.2. Identification of Characteristic Hydrolysis By-Product

To investigate the similarity between the main by-products generated during the hydrolysis of fructose and ITF, we compiled a list of common by-products potentially presented in the hydrolysis products of both Fru and ITF, totaling 57 types, as shown in Table S2. Among these, the compounds potentially linked to sugar degradation comprise the 13 substances listed in Table 1. The presence of furfural, acids, alcohols, and aldehydes in the sample corroborates with the reported literature on sugar degradation products [44,45,46].
The three common degradation products of Fru, 5-hydroxymethyl-2-furaldehyde, 5-methyl-2-furaldehyde, and furfural, were detected in the products of both Fru and ITF under different hydrolysis conditions, indicating that Fru generated by ITF hydrolysis also underwent degradation through pathways associated with 5-HMF [36]. As illustrated in Figure 5, previous studies have shown that under acidic conditions, Fru can undergo degradation through two pathways, leading to the formation of furfural compounds. This study unveiled notable fluctuations in 5-HMF and 5-MF levels with alterations in hydrolysis conditions (Figure 4), suggesting that, under the current hydrolysis parameters, Fru predominantly degrades through pathways associated with 5-HMF [47,48].
Hence, there is a critical need to concentrate more on methods to limit the conversion of Fru to 5-HMF during the hydrolysis of ITF to enhance the accuracy of fructose content determination in ITF.
Table 1. The common differentiated sugar degradation-associated compounds of Fru and ITF under three acid hydrolysis conditions.
Table 1. The common differentiated sugar degradation-associated compounds of Fru and ITF under three acid hydrolysis conditions.
NoIdentificationm/z aAdductsError (ppm)RT b (min)ScoreFormulaClassification cReferences
12,5-Furandicarboxaldehyde125.02 [M+H]+−3.4933.806 46.1C6H4O3Aldehyde[16,44,46]
25-Hydroxymethyl-2-furaldehyde109.0285[M+H−H2O]+0.4193.660 43C6H6O3
3Furfural97.03 [M+H]+−2.1103.101 40.4C5H4O2
45-Methyl-2-furaldehyde111.0438[M+H]+−4.4084.007 38.7C6H6O2
52-Phenylethanol123.0799 [M+H]+−2.3505.077 37.3C8H10O
62-Hydroxycinnamic acid165.05 [M+H]+−0.8383.540 41.6C9H8O3Acid[44,49,50]
71,4-Dihydroxy-2-naphthoic acid205.05 [M+H]+−2.3774.034 38.3C11H8O4
82-Hydroxy-8-methylchromene-2-carboxylate207.06 [M+H]+−2.3505.077 38.9C11H10O4Ester[51,52]
92-Hydroxy-3-methylbenzalpyruvate207.06 [M+H]+−1.9875.699 41.1C11H10O4Ketone[45,50]
102-Hydroxy-5-methylquinone139.04 [M+H]+−1.9213.819 39.7C7H6O3Others[38,51,53]
112-Hydroxy-6-oxo-6-(2-hydroxyphenoxy)-hexa-2,4-dienoate273.04 [M+H]+, [M+Na]+, [M+H−H2O]+−0.3874.209 39.7C12H10O6
12(R)-(−)-Mellein179.07 [M+H]+−1.9854.847 38.6C10H10O3
132-Hydroxyxanthone213.0541 [M+H]+−2.3676.092 38.5C13H8O3
Note: a, experimental data; b. Retention time; c. Classification, based on Pubmed website query results.

4. Conclusions

In this study, the optimal hydrolysis conditions for monosaccharide compositional of ITF were explored, and the results revealed that Fru was more susceptible to degradation compared to Glc, and milder hydrolysis conditions were more suitable for preserving the integrity of ITF. The optimal hydrolysis conditions for achieving relatively high yields and satisfactory stability of ITF in optimized hydrolysis conditions (80 °C, 2 h, and 1 M sulfuric acid). Furthermore, this study investigated the by-products generated during the acid hydrolysis of Fru and ITF, and the results indicated that, with the intensification of hydrolysis conditions, 5-hydroxymethyl-2-furaldehyde, 5-methyl-2-furaldehyde, and furfural were closely associated with fructose degradation. This indicates that the liberated free fructose during ITF hydrolysis undergoes further degradation through pathways associated with 5-HMF. Based on the above conclusions, this study provides some theoretical foundations for the establishment of appropriate hydrolysis conditions and the reduction in the side reactions in the in-depth physicochemical analysis of polysaccharides. In addition, to enhance our comprehension of Fru acid degradation and its influencing factors, it will be crucial to conduct a quantitative analysis of Fru by-product content in future studies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods13081241/s1, Table S1: Recovery rate (%) of ITF monosaccharides composition under different H2SO4 concentration, hydrolysis time and temperature (n = 3); Table S2. Identified compounds potentially presented in the hydrolysis products of both fructose (Fru) and isomaltulose (ITF).

Author Contributions

Writing and original draft preparation, X.Z.; preparation and data collection, N.L.; preparation and investigation, review and editing, W.H.; writing, review and editing, J.Y.; supervision commentary, revision and funding acquisition, S.N.; supervision, M.X. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Key Laboratory of Bioactive Polysaccharides of Jiangxi Province (20212BCD42016).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

References

  1. Marie-Luise, P.; de Vos, W.M. Back to the Roots: Revisiting the Use of the Fiber-Rich Cichorium intybus L. Taproots. Adv. Nutr. 2021, 12, 1598. [Google Scholar]
  2. Mensink, M.A.; Frijlink, H.W.; Maarschalk, K.v.d.V.; Hinrichs, W.L. Inulin, a flexible oligosaccharide. II: Review of its pharmaceutical applications. Carbohydr. Polym. 2015, 134, 418–428. [Google Scholar] [CrossRef]
  3. Apolinário, A.C.; de Lima Damasceno, B.P.G.; de Macêdo Beltrão, N.E.; Pessoa, A.; Converti, A.; da Silva, J.A. Inulin-type fructans: A review on different aspects of biochemical and pharmaceutical technology. Carbohydr. Polym. 2014, 101, 368–378. [Google Scholar] [CrossRef]
  4. Gupta, N.; Jangid, A.K.; Pooja, D.; Kulhari, H. Inulin: A novel and stretchy polysaccharide tool for biomedical and nutritional applications. Int. J. Biol. Macromol. 2019, 132, 852–863. [Google Scholar] [CrossRef]
  5. Shoaib, M.; Shehzad, A.; Omar, M.; Rakha, A.; Raza, H.; Sharif, H.R.; Shakeel, A.; Ansari, A.; Niazi, S. Inulin: Properties, health benefits and food applications. Carbohydr. Polym. 2016, 147, 444–454. [Google Scholar] [CrossRef]
  6. Meyer, D.; Bayarri, S.; Tárrega, A.; Costell, E. Inulin as texture modifier in dairy products. Food Hydrocoll. 2011, 25, 1881–1890. [Google Scholar] [CrossRef]
  7. Cataldi, T.R.I. Recent advances in carbohydrate analysis by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Ann. Chim. 1994, 84, 385–396. [Google Scholar]
  8. Wang, Q.-C.; Zhao, X.; Pu, J.-H.; Luan, X.-H. Influences of acidic reaction and hydrolytic conditions on monosaccharide composition analysis of acidic, neutral and basic polysaccharides. Carbohydr. Polym. 2016, 143, 296–300. [Google Scholar] [CrossRef]
  9. Pico, J.; Vidal, N.P.; Widjaja, L.; Falardeau, L.; Albino, L.; Martinez, M.M. Development and assessment of GC/MS and HPAEC/PAD methodologies for the quantification of α-galacto-oligosaccharides (GOS) in dry beans (Phaseolus vulgaris). Food Chem. 2021, 349, 129151. [Google Scholar] [CrossRef]
  10. Joyce, G.E.; Kagan, I.A.; Flythe, M.D.; Davis, B.E.; Schendel, R.R. Profiling of cool-season forage arabinoxylans via a validated HPAEC-PAD method. Front. Plant Sci. 2023, 14, 1116995. [Google Scholar] [CrossRef]
  11. Li, K.; Liu, S.; Tan, Y.; Chao, N.; Tian, X.; Qi, L.; Powell, W.A.; Jiang, X.; Gai, Y. Optimized GC-MS Method To Simultaneously Quantify Acetylated Aldose, Ketose, and Alditol for Plant Tissues Based on Derivatization in a Methyl Sulfoxide/1-Methylimidazole System. J. Agric. Food Chem. 2013, 61, 4011–4018. [Google Scholar] [CrossRef]
  12. Shi, H.; Wan, Y.; Li, O.; Zhang, X.; Xie, M.; Nie, S.; Yin, J. Two-step hydrolysis method for monosaccharide composition analysis of natural polysaccharides rich in uronic acids. Food Hydrocoll. 2020, 101, 105524. [Google Scholar] [CrossRef]
  13. Zhang, Z.; Khan, N.M.; Nunez, K.M.; Chess, E.K.; Szabo, C.M. Complete monosaccharide analysis by High-performance anion-exchange chromatography with pulsed amperometric detection. Anal. Chem. 2012, 84, 4104–4110. [Google Scholar] [CrossRef]
  14. Yang, Z.; Hu, J.; Zhao, M. Isolation and quantitative determination of inulin-type oligosaccharides in roots of Morinda officinalis. Carbohydr. Polym. 2011, 83, 1997–2004. [Google Scholar] [CrossRef]
  15. Rubel, I.A.; Iraporda, C.; Novosad, R.; Cabrera, F.A.; Genovese, D.B.; Manrique, G.D. Inulin rich carbohydrates extraction from Jerusalem artichoke (Helianthus tuberosus L.) tubers and application of different drying methods. Food Res. Int. 2018, 103, 226–233. [Google Scholar] [CrossRef]
  16. Antal, M.J., Jr.; Mok, W.S.L.; Richards, G.N. Mechanism of formation of 5-(hydroxymethyl)-2-furaldehyde from D-fructose and sucrose. Carbohydr. Res. 1990, 199, 91–109. [Google Scholar] [CrossRef]
  17. Madani-Tonekaboni, M.; Kamankesh, M.; Mohammadi, A. Determination of furfural and hydroxymethyl furfural from baby formula using dispersive liquid–liquid microextraction coupled with high performance liquid chromatography and method optimization by response surface methodology. J. Food Compos. Anal. 2015, 40, 1–7. [Google Scholar] [CrossRef]
  18. Kamalabadi, M.; Ghaemi, E.; Mohammadi, A.; Alizadeh, N. Determination of furfural and hydroxymethylfurfural from baby formula using headspace solid phase microextraction based on nanostructured polypyrrole fiber coupled with ion mobility spectrometry. Food Chem. 2015, 181, 72–77. [Google Scholar] [CrossRef]
  19. Rocklin, R.D.; Christopher, A.P.E. Determination of carbohydrates by anion exchange chromatography with pulsed amperometric detection. J. Liq. Chromatogr. 1983, 9, 1577–1590. [Google Scholar] [CrossRef]
  20. Amelung, W.; Cheshire, M.V.; Guggenberger, G. Determination of neutral and acidic sugars in soil by capillary gas-liquid chromatography after trifluoroacetic acid hydrolysis. Soil Biol. Biochem. 1996, 28, 1631–1639. [Google Scholar] [CrossRef]
  21. Amin, E.S. Action of dilute sulfuric acid on D-fructose. Carbohydr. Res. 1967, 4, 96–98. [Google Scholar] [CrossRef]
  22. Shaw, P.E.; Tatum, J.H.; Berry, R.E. Acid-catalyzed degradation of D-fructose. Carbohydr. Res. 1967, 5, 266–273. [Google Scholar] [CrossRef]
  23. Cataldi, T.R.I.; Campa, C.; De Benedetto, G.E. Carbohydrate analysis by high-performance anion-exchange chromatography with pulsed amperometric detection: The potential is still growing. Anal. Bioanal. Chem. 2000, 368, 739–758. [Google Scholar] [CrossRef]
  24. Bai, W.; Fang, X.; Zhao, W.; Huang, S.; Zhang, H.; Qian, M. Determination of oligosaccharides and monosaccharides in Hakka rice wine by precolumn derivation high-performance liquid chromatography. J. Food Drug Anal. 2015, 23, 645–651. [Google Scholar] [CrossRef]
  25. Liu, Y.; Lei, F.; He, L.; Xu, W.; Jiang, J. Comparative study on the monosaccharides of three typical galactomannans hydrolyzed by different methods. Ind. Crops Prod. 2020, 157, 112895. [Google Scholar] [CrossRef]
  26. Adeogun, A.I.; Idowu, M.A.; Oladeji, O.B.; Ofudje, E.A.; Akinloye, A.O. Kinetic, thermodynamic and optimization studies of dilute acid hydrolysis of Manihot esculenta peels for glucose production. Waste Biomass Valorization 2020, 11, 817–826. [Google Scholar] [CrossRef]
  27. Silva, V.G.; Aguilar, M.S.C.; Ascanio, G.; Aguayo, J.P.; Pérez-Salas, K.Y.; Notario, A.d.C.S. Acid Hydrolysis of Pectin and Mucilage from Cactus (Opuntia ficus) for Identification and Quantification of Monosaccharides. Molecules 2022, 27, 5830. [Google Scholar] [CrossRef]
  28. Świątek, K.; Gaag, S.; Klier, A.; Kruse, A.; Sauer, J.; Steinbach, D. Acid hydrolysis of Lignocellulosic biomass: Sugars and furfurals formation. Catalysts 2020, 10, 437. [Google Scholar] [CrossRef]
  29. Chong, J.; Soufan, O.; Li, C.; Caraus, I.; Li, S.; Bourque, G.; Wishart, D.S.; Xia, J. MetaboAnalyst 4.0: Towards more transparent and integrative metabolomics analysis. Nucleic Acids Res. 2018, 46, W486–W494. [Google Scholar] [CrossRef]
  30. Panahabadi, R.; Ahmadikhah, A.; Farrokhi, N. Genetic dissection of monosaccharides contents in rice whole grain using genome-wide association study. Plant Genome 2023, 16, e20292. [Google Scholar] [CrossRef]
  31. Thani, N.M.; Kamal, S.M.M.; Taip, F.S.; Sulaiman, A.; Omar, R.; Siajam, S.I. Hydrolysis and characterization of sugar recovery from bakery waste under optimized subcritical water conditions. J. Food Sci. Technol. 2020, 57, 3108–3118. [Google Scholar] [CrossRef]
  32. Liu, D.; Tang, W.; Yin, J.-Y.; Nie, S.-P.; Xie, M.-Y. Monosaccharide composition analysis of polysaccharides from natural sources: Hydrolysis condition and detection method development. Food Hydrocoll. 2021, 116, 106641. [Google Scholar] [CrossRef]
  33. Xie, B.; Yi, L.; Zhu, Y.; Chang, X.; Hao, J.; Pang, L.; Ouyang, Y.; Yuan, S.; Zhang, Z. Structural elucidation of a branch-on-branch β-glucan from Hericium erinaceus with A HPAEC-PAD-MS system. Carbohydr. Polym. 2021, 251, 117080. [Google Scholar] [CrossRef]
  34. Wang, S.; Li, G.; Zhang, X.; Wang, Y.; Qiang, Y.; Wang, B.; Zou, J.; Niu, J.; Wang, Z. Structural characterization and antioxidant activity of Polygonatum sibiricum polysaccharides. Carbohydr. Polym. 2022, 291, 119524. [Google Scholar] [CrossRef]
  35. Zong, X.; Wang, Z.; Chen, S.; Li, S.; Xie, M.; Nie, S.; Yin, J. Optimized acid hydrolysis conditions for better characterization the structure of inulin-type fructan from Polygonatum sibiricum. Int. J. Biol. Macromol. 2024, 256, 128030. [Google Scholar] [CrossRef]
  36. Moreau, C.; Durand, R.; Razigade, S.; Duhamet, J.; Faugeras, P.; Rivalier, P.; Ros, P.; Avignon, G. Dehydration of fructose to 5-hydroxymethylfurfural over H-mordenites. Appl. Catal. A Gen. 1996, 145, 211–224. [Google Scholar] [CrossRef]
  37. Uhliariková, I.; Matulová, M.; Capek, P. Optimizing acid hydrolysis for monosaccharide compositional analysis of Nostoc cf. linckia acidic exopolysaccharide. Carbohydr. Res. 2021, 508, 108400. [Google Scholar] [CrossRef]
  38. Philip, E.S.; James, H.T.; Robert, E.B. 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, a degradation product of a hexose. Carbohydr. Res. 1971, 16, 207–211. [Google Scholar]
  39. Cordella, C.; Militão, J.S.; Clément, M.-C.; Drajnudel, P.; Cabrol-Bass, D. Detection and quantification of honey adulteration via direct incorporation of sugar syrups or bee-feeding: Preliminary study using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and chemometrics. Anal. Chim. Acta 2005, 531, 239–248. [Google Scholar] [CrossRef]
  40. Kim, Y.-J.; Choi, J.; Lee, G.; Lee, K.-G. Analysis of furan and monosaccharides in various coffee beans. J. Food Sci. Technol. 2021, 58, 862–869. [Google Scholar] [CrossRef]
  41. Miller, R.E.; Cantor, S.M. 2-Hydroxyacetylfuran from sugars. J. Am. Chem. Soc. 1952, 20, 5236–5237. [Google Scholar] [CrossRef]
  42. Brown, D.W.; Floyd, A.J.; Kinsman, R.G.; Roshan-Ali, Y. Dehydration reactions of fructose in non-aqueous media. J. Chem. Technol. Biotechnol. 1982, 32, 920–924. [Google Scholar] [CrossRef]
  43. Asghari, F.S.; Yoshida, H. Acid-catalyzed production of 5-hydroxymethyl furfural from D-fructose in subcritical water. Ind. Eng. Chem. Res. 2013, 45, 2163–2173. [Google Scholar] [CrossRef]
  44. Fachri, B.A.; Abdilla, R.M.; van de Bovenkamp, H.H.; Rasrendra, C.B.; Heeres, H.J. Experimental and kinetic modeling studies on the sulfuric acid catalyzed conversion of d-fructose to 5-Hydroxymethylfurfural and levulinic acid in water. ACS Sustain. Chem. Eng. 2015, 3, 3024–3034. [Google Scholar] [CrossRef]
  45. Perez, G.P.; Mukherjee, A.; Dumont, M.J. Insights into HMF catalysis. J. Ind. Eng. Chem. 2019, 70, 1–34. [Google Scholar] [CrossRef]
  46. Swift, T.D.; Bagia, C.; Choudhary, V.; Peklaris, G.; Nikolakis, V.; Vlachos, D.G. Kinetics of homogeneous bronsted acid catalyzed fructose dehydration and 5-Hydroxymethyl furfural rehydration: A combined experimental and computational study. ACS Catal. 2014, 4, 259–267. [Google Scholar] [CrossRef]
  47. Antonetti, C.; Melloni, M.; Licursi, D.; Fulignati, S.; Ribechini, E.; Rivas, S.; Parajó, J.C.; Cavani, F.; Galletti, A.M.R. Microwave-assisted dehydration of fructose and inulin to HMF catalyzed by niobium and zirconium phosphate catalysts. Appl. Catal. B Environ. 2017, 206, 364–377. [Google Scholar] [CrossRef]
  48. Ruan, Y.; Niu, C.; Zhang, P.; Qian, Y.; Li, X.; Wang, L.; Ma, B. Acid-catalyzed water extraction of two polysaccharides from Artemisia argyi and their physicochemical properties and antioxidant activities. Gels 2022, 8, 5. [Google Scholar] [CrossRef]
  49. Rice, F.H.; Fishbein, L. Effect of Aqueous Sulfuric Acid on Sugars. II. Spectrophotometric Studies on the Hexoses; Identification of the Ether-soluble Products Formed. J. Am. Chem. Soc. 1956, 78, 3731–3734. [Google Scholar] [CrossRef]
  50. James, C.G.; John, E.H.; David, W. Preparation and structure of an unusual dimeric furan from the acid decomposition of isomaltol. Carbohydr. Res. 1986, 146, 107–116. [Google Scholar]
  51. Akien, G.R.; Qi, L.; Horváth, I.T. Molecular mapping of the acid catalysed dehydration of fructose. Chem. Commun. 2012, 47, 5850–5852. [Google Scholar] [CrossRef] [PubMed]
  52. Rice, F.A.H.; Fishbein, L. Spectrophotometric Studies on the Action of Sulfuric Acid on Reducing Sugars and the Isolation and Identification of the Ether-soluble Substances Produced from Pentoses under Acid Conditions. J. Am. Chem. Soc. 1956, 78, 1005–1009. [Google Scholar] [CrossRef]
  53. Hoang, T.M.C.; van Eck, E.R.H.; Bula, W.P.; Gardeniers, J.G.E.; Lefferts, L.; Seshan, K. Humin based by-products from biomass processing as a potential carbonaceous source for synthesis gas production. Green Chem. 2015, 17, 959–972. [Google Scholar] [CrossRef]
Foods 13 01241 g001aFoods 13 01241 g001b
Figure 1. The fluctuation of monosaccharide (Fru and Glc) recovery rates (%) and total recovery rates (%) over time during the hydrolysis of inulin-type fructans (ITF) under different temperature conditions (ranging from 40 °C to 120 °C) at (A) 0.5 M, (B) 1.0 M, and (C) 2.0 M H2SO4 concentrations.
Figure 1. The fluctuation of monosaccharide (Fru and Glc) recovery rates (%) and total recovery rates (%) over time during the hydrolysis of inulin-type fructans (ITF) under different temperature conditions (ranging from 40 °C to 120 °C) at (A) 0.5 M, (B) 1.0 M, and (C) 2.0 M H2SO4 concentrations.
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Figure 2. The fluctuation in fructose (Fru) standard monosaccharide concentration over hydrolysis time under different temperature conditions (ranging from 40 °C to 120 °C), while H2SO4 concentrations were (A) 0.5 M, (B) 1.0 M, and (C) 2.0 M.
Figure 2. The fluctuation in fructose (Fru) standard monosaccharide concentration over hydrolysis time under different temperature conditions (ranging from 40 °C to 120 °C), while H2SO4 concentrations were (A) 0.5 M, (B) 1.0 M, and (C) 2.0 M.
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Figure 3. PCA score plots and PLS-DA score plots in three acidic hydrolysis conditions. (A) PCA score plots of Fru; (B) PCA score plots of ITF; (C) PLS-DA score plots of Fru; (D) PLS-DA score plots of ITF.
Figure 3. PCA score plots and PLS-DA score plots in three acidic hydrolysis conditions. (A) PCA score plots of Fru; (B) PCA score plots of ITF; (C) PLS-DA score plots of Fru; (D) PLS-DA score plots of ITF.
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Figure 4. Dot map of differentiated compounds from Fru (A) and ITF (B) under different hydrolysis conditions. The color and size of the bubbles represent the Log2FC and VIP values, respectively. IN: incomplete acid hydrolysis conditions (0.5 M H2SO4, 40 °C, 0.5 h); BE: better acid hydrolysis conditions (1 M H2SO4, 80 °C, 2 h); EX: excessive acid hydrolysis conditions (1 M H2SO4, 120 °C, 1 h).
Figure 4. Dot map of differentiated compounds from Fru (A) and ITF (B) under different hydrolysis conditions. The color and size of the bubbles represent the Log2FC and VIP values, respectively. IN: incomplete acid hydrolysis conditions (0.5 M H2SO4, 40 °C, 0.5 h); BE: better acid hydrolysis conditions (1 M H2SO4, 80 °C, 2 h); EX: excessive acid hydrolysis conditions (1 M H2SO4, 120 °C, 1 h).
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Figure 5. Pathways related to the formation of furfural from fructose.
Figure 5. Pathways related to the formation of furfural from fructose.
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Zong, X.; Lei, N.; Yin, J.; He, W.; Nie, S.; Xie, M. Exploration and Improvement of Acid Hydrolysis Conditions for Inulin-Type Fructans Monosaccharide Composition Analysis: Monosaccharide Recovery and By-Product Identification. Foods 2024, 13, 1241. https://doi.org/10.3390/foods13081241

AMA Style

Zong X, Lei N, Yin J, He W, Nie S, Xie M. Exploration and Improvement of Acid Hydrolysis Conditions for Inulin-Type Fructans Monosaccharide Composition Analysis: Monosaccharide Recovery and By-Product Identification. Foods. 2024; 13(8):1241. https://doi.org/10.3390/foods13081241

Chicago/Turabian Style

Zong, Xinyan, Ningyu Lei, Junyi Yin, Weiwei He, Shaoping Nie, and Mingyong Xie. 2024. "Exploration and Improvement of Acid Hydrolysis Conditions for Inulin-Type Fructans Monosaccharide Composition Analysis: Monosaccharide Recovery and By-Product Identification" Foods 13, no. 8: 1241. https://doi.org/10.3390/foods13081241

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