Freeze-thaw properties of β-glucan gels. [303080]

Freeze-thaw Properties of β-glucan Gels

Running title: Freeze-thaw properties of β-glucan gels.

Abstract

The network structure of β-glucan polymers and the presence of water have significant effects on the properties of β-[anonimizat]. [anonimizat], [anonimizat]-[anonimizat] β-glucan. To characterize β-[anonimizat], [anonimizat] (LF-NMR) to measure water proton transverse relaxation in aqueous β-glucan solutions during storage. [anonimizat]. [anonimizat]. The location of each water component was identified in the porous microstructure of the cryogel. The pore size measured from scanning electron microscopy (SEM) images agreed with the pore size estimated from relaxation time. The formation of cryogel was confirmed by rheological. [anonimizat]-spin relaxation characteristics.

Key words: β-glucan; cryogelation; LF-NMR; water distribution; SEM; rheology.

1. [anonimizat]1, reduce the risk of type 2 diabetes2-3, enhance gut health4, and improve immunity5, β-[anonimizat], barley, rye, and wheat. β-glucan is a linear polysaccharide of d-glucopyranosyl residues linked by β-(1→3) and β-(1→4) glycosidic bonds6, whose molecular weight is reported to be between 60 and 3000 kDa7. Together with lichenase (EC 3.2.1.73), the oat β-[anonimizat] β-glucan chain. [anonimizat], 3-O-β-cellobiosyl-d-glucose (DP3) and 3-O-β-cellotriosyl-d-glucose (DP4)8-10 . [anonimizat], and gelation behavior mainly contribute to the healthcare effects of oat β-glucan. Therefore, it’s quite imperative to thoroughly understand how the β-glucan polysaccharide works in different aqueous environments. With a sufficiently high concentration, β-glucan may change from a solution form to a gel form. There are many studies indicating how aggregate structures of oat β-glucan can be in the solution and in the form of a gel, with static and dynamic light scattering15-16, rheometry17-18, atomic force microscopy19, and confocal microscopy20. [anonimizat], structure and concentration of oat β-glucans on its aggregation state are different. [anonimizat] β-glucans with low molecular weight is more likely to form aggregates and gels21-24 than the one with high molecular weight. Moreover, the gelation rate of β-glucan increases with the increase of the polysaccharide concentration in the solution. Time-domain 1H NMR has been widely applied to the study of polysaccharide solution and gel25-27. Meanwhile, more useful information on polymer dynamics and aggregation structure can be obtained through the transverse relaxation time, T2 28. Adding polysaccharide to water will usually aggrandize aqueous proton transverse relaxation rate, 1/T2. The proton frequently exchange between water and polysaccharide hydroxyl groups, which has a significant effect on the transverse relaxation mechanism of polysaccharide aqueous solution and gel. It is possible to calculate29-31 the water proton transverse relaxation time with the help of a two-site exchange model. Investigations were made to study the hydration of chitosan and the water tightly coordinated with the polysaccharide in chitosan hydrogel. Therefore, it is possible to elucidate the microstructure of native starch granule and the gelatinization of starch through the NMR relaxation and diffusion methods. In the work described in this article, different water components was identified by low-field nuclear magnetic resonance (LF-NMR) and the description of its residue’s microscopic structure was made. In addition, the formation process of oat β-glucan gel was verified by rheological method, providing a theoretical basis for the practical application of β-glucan in food industry and the fine processing of oat and Highland barley, eventually making a contribution to the development of grain science. Moreover, the oat β-glucan gel can also be used as a sustained-release drug carrier. In contrast to the conventional pharmaceutical preparations, these gel carriers have many advantages, such as long treatment period, relatively low drug delivery frequency, little gastrointestinal stimulation, non-toxic side effects, few fluctuations in drugs peak period, no oral administration no drug release in the front of the stomach and small intestine, repeated drug use on gastrointestinal mucosa stimulation and reduced systemic side effects32.

2. Materials and methods

2.1. Isolation and purification of β-glucan

To extract Oat β-glucan respectively from the Weiduyou 1 oat cultivar and barley β-glucan from the Highland barley cultivar, the method described by Lazaridou et al33 was used here with minor modification. Briefly, the bran was treated with hot 80% ethanol in water, then washed with absolute ethanol. After being air-dried, the bran was used for β-glucan extraction with 52 ℃ water as well as a thermostable amylase and a pan creatin digestion. Having be precipitated with ethanol, the β-glucan precipitate was solubilized with water and lyophilized.

2.2. Partial hydrolysis with acid

In the Wood et al12, low molecular weight polysaccharide12 was obtained by mild acid hydrolysis. Firstly, two grams of β-glucan was dissolved in 200 mL deionized water. Secondly, the solution was stirred and heated to 70℃. Finally, with the concentrated HCl added to 0.1 M, the samples were hydrolyzed at 85 ℃ for 30, 60 and 90 mins, quickly cooled dwon to the room temperature, and then neutralized with NaOH solution. With two volumes of 100% ethanol added to the partially hydrolyzed oat β-glucan, the precipitates were formed and solubilized in water, then desalted by dialysis in tubing with a molecular weight cut off 10 kDa. The dialyzed solutions were lyophilized.

Samples of β-glucan with different molecular weights (Table 1) were used to prepare 4% (w/w) aqueous solution.. Except that the solvent is deionized water, the preparation method of the solution was the same as that of HPSEC-MALLS analysis. Two mL of each β-glucan solution was transferred into a NMR sample tube at an internal diameter of 15 mm. Sealed with PTFE plugs and stored in a refrigerator at -18 ℃ for 21 hrs, the tubes with β-glucan samples inside were moved into an incubator and thawed at 25 ℃ for 3 hrs. Having been tested after each of five freeze-thaw cycles, these test tubes were also tested after every three additional cycles for 11 total freeze-thaw cycles. For the sake of rheology analysis, 5 mL of β-glucan solution was transferred into a cylindrical mold at an internal diameter of 36 mm. Afterward, these solutions were treated with repeated freeze-thaw cycles and tested after 0, 1, 2, 3, 4, 5, 8 and 11 freeze-thaw cycles.

2.3.Preparation of β-glucan frozen gel

Having been stirred and prepared in an 85 ℃ water bath for 3hrs, the solution of various molecular weight (mass fraction is 4%) oat and barley β-glucan solutions had been cooled down to room temperature, placed in a refrigerator at 18 ℃, then frozen for 21 hrs, and thawed at the room temperature for 3 hrs. The processing time for a freezing and thawing cycle was in accordance with the requirements of test sample solution for the corresponding number of freezing and thawing with the prepared samples labeled as OG0 and BG0.

2.4. Methylation analysis

The test samples were processed in the following steps, (1) β-glucan methylation with CH3I; (2) hydrolysis with 2 mol/L trifluoroacetic acid at 121 °C for 1.5 hrs; (3) post-hydrolysis with sodium borohydride β -glucan reduction; (4) acetylation at 100 °C for 2.5 hrs; (5) quantitative determination of partially methylated sugar alcohol acetate with a 7890B/5977A GC-MS equipped with HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm).

With the initial temperature of the column staying at 140 °C for 2 mins, and then raised to 320 ℃ at 6 ℃/min for another 3 mins, the partially methylated sugar alcohol acetate was characterized by GC-MS and quantified by a gas chromatograph that was equipped with a hydrogen flame ionization detector34.

2.5. LF-NMR measurements

In order to perform the LF-NMR experiments, the MiniMR NMR spectrometer (Niumag, China) was employed for the operation at 23 MHz for 1H resonance. Having been placed in a water bath at 32 ℃ for at least 15 mins, all the NMR tubes with samples were transferred into the NMR probe at a constant temperature of 32 ℃. The spin-spin relaxation time, T2, was obtained using a Carr-Purcell-Meiboom-Gill (CPMG) sequence with a 90°-180° pulse and spacing of 0.1 ms. The 90° pulse was 18 μs, and the number of echoes was 18000, which produced a sampling space of about 4.23 s. With the samples prepared under different conditions and assayed with at least three replicates, each sample was repeatedly scanned eight times with a delay of 10s. Signal collection and analysis were performed with Niumag NMR analysis software (Niumag, China). The relaxation signal decay curves were fitted with an exponential equation.

Among them, Ai is the echo amplitude of the first component at time t and T2i is the corresponding spin-spin relaxation time. A0 is the noise of the curve.

2.6. principal component analysis of LF-NMR

The principal component analysis module in SPSS18.0 was utilized in the test, to make an analysis of the LF-NMR relaxation time distribution data of 4% oat and highland barley β-glucan gel systemins with various mass fractions and different molecular weights. With the score data of principal components obtained, the principal components with eigenvalues greater than 1 were retained, and the score graph was drawn by Origin9.0 and analyzed accordingly.

2.7. Dynamic rheometry

The study of the rheological properties of the β-glucan solution and cryogelThe was made with usage of parallel plate geometry (PP25/P2, 25 mm diameter, 1 mm gap) and a Physica MCR-301 rheometer (Anton Paar, Austria) at 25 ℃. With the solution or cryogel transferred from the cylindrical mold to the lower plate of the rheometer before the test, G′ (storage modulus), G″ (loss modulus), and tan δ (G″/G′) were obtained from oscillatory measurements with 0.1% strain and frequency from 0.1 to 10 Hz. Repeated analysis was performed at least three times.

2.8. Scanning electron microscopy

With the β-glucan solutions and cryogel lyophilized and a thin layer was cut cautiously out from it with a sharp blade, the microstructure of a cross section of the sample was acquired with a JSM-7500 F scanning electron microscope (JEOL, Japan). A small piece of cross section was fixed onto an aluminum stub with double-sided conductive tape and sputter-coated with gold. Scanning electron microscopy (SEM) was used to observe the effect of acceleration voltage of 3 kv..

Results and discussion

3.1. Analysis of glycosidic linkage

Fig. 1. shows gas chromatography-mass spectrometry measurements of partially methylated glycolic acetate of β-glucan. Figure 1(a): the mole percent of OG0 (Glcp)1→ is 1.60%, the mole percent of OG0 →3(Glcp)1→ is 27.10%, and the mole percent of OG0 →4(Glcp)1→ is 71.30%. Figure 1 (b), the mole percent of →(Glcp)1→ to BG0 is 2.22%, the mole percent of (Glcp)3→ to 1 is 27.68%, and the mole percent of (Glcp)4→ to 1 is 70.10%. From these data, it could be calculated that the ratio of bonds (1→4) to (1→3) in oat β-glucan was 2.63, and that the ratio of bonds (1→4) and (1→3) in highland barley β-glucan was 2.53.

3.2. LF-NMR measurements

It was measured that both spin-lattice relaxation time (longitudinal relaxation time) and spin-spin relaxation time (transverse relaxation time) could reflect the combination of water and matrix. A higher sensitivity of the transverse relaxation time T2 could be noticed in characterizing the fluidity of diverse moisture and determining the type of moisture in regard to the longitudinal relaxation time T1. Generally speaking, the shorter the relaxation time T2 was , the better the combination of water and the matrix could become; the longer the relaxation time T2 was, the weaker the combination of water and matrix could become. Proton density M2 represented the relative moisture content of different transverse relaxation times.

Fig. 2 (a) and (b) shows the lateral relaxation time distributions OG0 and BG0 (see Table 1 in Methods) after 8 freeze-thaw cycles. The gel system of β-glucan shows three sets of proton relaxation peaks T21, T22 and T23. Thereinto, T21 corresponds to the water located in the physical cross-linking zone formed between the β-glucan molecules with a short transverse relaxation time; T22 corresponds to the moisture located in the gel skeleton with a relatively longer transverse relaxation time; T23 corresponds to the water in the macropores formed by the melting of ice crystals with the longest transverse relaxation time. In the gel system, the diffusion of water molecules in various microenvironments is largely influenced by the gel network, and there merges multiple T2 distributions, i.e., multiple peaks (Fig. 2) with the diffusion exchange between the microenvironments blocked. The relaxation time T23 closely depends on the mass fraction corresponding to the peak of the largest proportion in the gel. With the increase of the mass fraction, the corresponding relaxation time decreases, which indicates a change in the moisture state and the environment. This process can be qualitatively regarded as the relationship between the concentration of β-glucan and the transverse relaxation time of water. The higher the β-glucan concentration is, the more active hydrogen can be exchanged in the system, which has an effect on the transverse relaxation time of water. Moreover, because of the short relaxation time of the active hydrogen on the sugar chain, the transverse relaxation time of the water protons undergoing chemical exchange becomes shorter with the increase of the polysaccharide mass fraction. Therefore, with a short relaxation time, the gel network formed by the higher mass fraction of β-glucan in aqueous solution becomes denser, which interferes the water molecules diffusion and reduces the lateral relaxation time35.

Higher than the critical overlap concentration, the 4% concentration of β-glucan in all the solutions belongs to the concentrated solution regime36. In concentrated solutions where β-glucan chains overlap and interwine with each other, there are more likely to form aggregates. As shown in Fig. 3., the T21 of different molecular weight OG and BG polymers gradually rose during the freeze-thaw cycles, which indicated the formation of gel structure. The fast relaxation component (Component 1) had a spin-spin relaxation time, T21, of about 5 mins for OG and 1-15 mins for BG. It was not expected that the inert protons on oat β-glucan would contribute to Component 1 because the CPMG pulse sequence could hardly collect the rapid decaying signal. Component 1 could be reasonably regarded as stemming from water molecules trapped within the cross-links which was formed by consecutive DP3 units and stable physical entanglement points of β-glucan chains. Once the freeze-thaw cycles formed cross-links and stable physical entanglements, they became stable structures so further freeze-thaw treatments could not be easily changed. Therefore, T21 remained essentially constant during the cryogelation process, despite some fulctuation of the values. The spin-spin relaxation of this group of water protons is mainly modified by chemical exchange with labile protons of polysaccharide in the cross-links and stable entanglement points. A low mobility of oat β-glucan chains could be reflected from the relatively small value of T21 in the network skeleton. To some extent, these water molecules can be regarded as an integral part of the network skeleton structure.

How Component 2 of different molecular weight β-glucan changed could be indicted as follows, The spin-spin relaxation time, T22, distributed from 35 to 160 ms for OG and 10 to 120ms for BG, which means higher mobility of this group of water protons than that of Component 1. Thus, Component 2 could be considered as water confined in the interstitial space between β-glucan chains. β-glucan aqueous solutions experienced a slow freezing process at -18 °C. During the freezing process, ice crystals gradually grew larger and the concentration of β-glucan increased in the liquid phase, thereby enabling the interaction between β-glucan molecules. During the subsequent thawing process, temperature increase enhanced the dynamics of β-glucan to interact with each other; meanwhile, the temperature was still below the freezing point for a period of time with the solid state of ice crystal and a high concentration of β-glucan in the liquid phase. The freeze-thaw cycles result in the formation of water pools surrounded by thin walls of concentrated cryogel, which was similar to a cell structure. Trapped in the interstitial space between aggregates of oat β-glucan chains, water molecules were aimed to Component 2. The spin-spin relaxation time, T22, first increased and then showd a declining trend with freeze-thaw cycles increased. The increase of T22 might be an indication of more open association of β-glucan aggregates, and the decrease of T22 was probably caused by a denser gel phase compressed by ice crystals. At the least, the change of T22 indicates the porosity and heterogeneity during the cryogelation. Even though no clear relationship was found between the molecular weight of β-glucan and the corresponding T22, β-glucan of lower molecular weight was more likely to form a gel.

Fig. 3(e) shows the variation of spin-spin relaxation time of Component 3, T23, which reflects how the melting of ice crystals form the water in the pores in the gel. The initial values of T23 approached the values in fresh β-glucan solutions, thus,Component 3 was regard as bulk water in the aqueous systemins. How T23 of each molecular weight at different stages changed could be indicated as follows: Freeze-thaw treatment caused a rapid increase of T23 at the first stage, which made fewer labile protons on the polysaccharide available to exchange with bulk water protons. The decrease of labile protons made T23 increase because T23 is mainly determined by chemical exchange. More smaller-sized β-glucan produced by higher mobility entered the gel microphase, causing a further rise in T23. Therefore, T23 began to present an obvious rise after approximate four freeze-thaw cycles, and the rise was more obvious for lower molecular weight samples.

As more freeze-thaw treatments appear, the β-glucan concentration in bulk water gradually increases. Except for OG0, freeze-thaw treatment caused a slight increase in T23 of OG30, OG60, OG90 at the first stage. The T23 of OG0 kept rising when it was frozen and thawed from two to four times. During the process of freezing and thawing from two to four times, T23 basically reached equilibrium; after four freeze-thaw cycles, the T23 of various glucans was in balance. The lighter the molecular weight, the shorter the T23 of OG; and the less the molecular weight, the longer the T23 of BG. This difference was caused mainly by the differences in molecular weight and molecular structures of the two polysaccharides. The initial values of T23 approached the values for fresh oat β-glucan solutions, so Component 3 was considered as bulk water in the aqueous systems. It is surprising that T23 was shorter in the aqueous system consisting of small-sized β-glucan at the beginning of freeze-thaw treatment. Although the lower molecular weight β-glucan aqueous system appeared less viscous, the mobility of water therein seemed to be lower than the water mobility in the more viscous solution with a larger-sized oat β-glucan. The reason for this abnormal result is likely in the self-aggregation of oat β-glucan; β-glucan of smaller size is more prone to aggregate than larger polysaccharide molecules. The aggregation may result in a suspension or a network, which makes the mobility of oat β-glucan molecules lower with the slight lowering of the T23 of water interacting with these polysaccharide aggregates after the first several freeze-thaw cycles were completed. The leveling of T23 (Fig. 3e) and T21 (Fig. 3a) indicates that the cryostructure had changed little after 10-13 freeze-thaw cycles.

As Fig. 4a and 4b shows, the spin density, A21, of Component 1 rose with the increasing number of freeze-thaw cycles, which reflected a growth in cross-links and stable physical entanglements. Forming a network skeleton earlier compared with their larger counterparts, smaller BG seemed to form more network skeleton during cryogelation. After 11 freeze-thaw cycles, it was clear that BG0 generated a much smaller network skeleton than these of the other small-sized samples. It is generally accepted that a small-sized barley β-glucan will obtain higher mobility of chains which is conducive to the formation of cryogel structure. The formation and increase of cryostructure deduced from the presence of Component 1 and increase in A21 are in good agreement with the results obtained through traditional methods.

As shown in Fig. 4c and 4d, spin density, A22, augmented with the number of freeze-thaw cycles, reflecting the increase in the cryogel microphase, which showed an inverse relation with the molecular weight of oat β-glucan at all cycles and demonstrated that oat β-glucan of small size produces more cryogel microphase compared with larger polysaccharides. With this result verified by others, A22 increased sharply and then leveled, which indicated that cryogel microphase quantity approached a maximum and remained constant after eight freeze-thaw cycles. Meanwhile, A21 also showed the same trend, so it is assumed that eight could be a critical freeze-thaw cycles number.

After 11 freeze-thaw cycles, the spin density, A23, of the bulk water declined from nearly 98% to about 80% for OG and from 100% to about 75% for BG in β-glucan samples with decreasing molecular weight. At the same time, the increase of cryogel microphase and increasing amount of water entrapped in the gel microphase could cause the decrease of bulk water ratio . That is to say, the bulk water gradually converted into entrapped water during the process of cryogelation.

3.3.principal component analysis

Fig. 5. shows the scores of OG(a) and BG(b) with different molecular weights after eight freeze-thaw cycles. Each circle stands for the overall properties of a particular molecular weight gel. The differences in gel samples of different molecular weights can be scored by principal component analysis. On the other hand, the spacing distances in Fig. 5. marked the tendency that the greater the difference in molecular weight of β-glucan gel, the greater the difference in gel properties. Gel samples of different molecular weights can better differentiated which principal component analysis. For OG and BG, on the left side of the PC1 axis there was the concentrated β-glucan gel samples hydrolyzed for 0 min and 30 mins ; on the right side of the PC1 axis there was the concentrated β-glucan gel hydrolyzed for 60 mins and 90 mins. The comparison indicated the critical molecular weight difference between the hydrolyzed β-glucan samples for 30 mins and 60 mins. Although there was no significant difference on the PC2 axis, there was a significant one in the PC1 axis, indicating that the formation of β-glucan gel37was significantly affected by the molecular weight.

3.4. Analysis of rheological properties

The effect of an applied stress on a fluid can be expressed in two ways, namely, elasticity and viscosity, which can be respectively denoted as G' and G". G' is the storage modulus or elastic modulus, which illustrates the ability of a polymer to deform with the change in external force. On the other hand, G" is the loss modulus or the viscous modulus (Pa) which illustrates the energy loss caused by either the internal or inter-molecular stretching of a polymer when the external force changes. Tan δ = G"/G' (δ is the loss angle) is an index of the viscoelastic properties of the system. Generally, tan δ = 1 is the limit. The larger the tan δ, the more dominant the viscous component, showing more liquid solid properties38.

As shown in Fig. 6, the viscoelastic properties of gels was formed by various molecular weight OG(a) and BG(b) at 4% concentration and after eight freeze-thaw 8 cycles. The values of G' and G" was in connection with the molecular weights of the β-glucan gels; that is, the smaller the molecular weight, the larger the values of G' and G". Although G' of the BG0 gel increased with the increase in oscillation frequency, the G' and G" of the other molecular weight β-glucan gels did not alter with increased oscillation frequency. This discrepancy may be caused by the larger molecular weight of BG0. The molecular weight of the hydrolyzed BG was small, whose gel form has better gel properties in comparison with unhydrolyzed BG0. Thus, since the viscosity behavior of the fluid decreases, the characteristics of elastic behavior enhances with a decrease in the molecular weight of β-glucan, which further confirms that β-glucan with a smaller molecular weight is more likely to form a gel.

3.5. Scanning electron microscopy

In β-glucan aqueous solutions, a great many hydrogen bonds are formed between water and hydroxyl groups of polysaccharides, and the folding and crimping movemet of hydrated β-glucan macromolecule in an aqueous solution forms a circular, helical, or double helix structure. Then, as hydrated β-glucan macromolecule stretches in an aqueous solution, a certain part of the molecules is arranged in a straight line and a sugar chain is formed. At the ssame time, a large number of β-glucan molecules are bonded at different points so that a three-dimensional network is formed and gradually filled with water molecules to shape a gel. Fig. 7. shows SEMS of different molecular weight oat β-glucan and barley β-glucan gels. It is clearly that β-glucan gel is a type of three-dimensional network, and that the formed porous structure has a certain regularity related to molecular weight. It is easier for samples with smaller molecular weights to form a three-dimensional network, since they have higher mobility and diffusivity. In addition, while the small molecule β-glucan chains contain a small proportion of inactive fragments and a lower degree of intramolecular action, the probability of effective collision between chains is enhanced, and hence the chances of collision and entanglement between molecules increase.

4. Conclusions

In this study, the imperative effects of the network structure of β-glucan polymers and the presence of water has been found on the properties of β-glucan gels after specific freeze-thaw cycles. What has also been found is that three groups of relaxation components with transverse relaxation times T21, T22 and T23; the corresponding signal peak proportions were A21, A22 and A23. With the increase of freezing and thawing cycles, T21, T22 and T23 of different β-glucan mass fractions and molecular weights generally increased at first and then did not change noticeably. However, T22 and A22 of Highland barley β-glucan increased after either three or five cycles of freezing and thawing. A21 and A22 of different β-glucan mass fractions first increased and then did not change noticeably, and A23 first decreased and then did not change noticeably. The variation of gels with different molecular weight β-glucan and different numbers of freeze-thaw cycles was distinguished by principal component analysis of the relaxation distribution curve. Rheological studies showed that the larger the mass fraction and the less the molecular weight, and that the larger the elastic modulus were and the more viscosity modulus it shows, indicating that more cross-linked structures were formed in the gel. The result of scanning electron microscopy indicated that the gel structure was being denser with the molecular weight becoming smaller, and the gel network was being looser with the molecular weight becoming larger.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the National Science Foundation of China (31101224). The authors would like to thank AiMi (www.aimieditor.com) for providing linguistic assistance.

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Table captions:

Table 1 Molecular characteristics of oat β-glucan samples.

Figure captions:

Figure 1 GC-MS chromatograms of partially methylated alditol acetates of OG0 (a) and BG0 (b).

Figure 2 T2 distribution of OG0 (a) and BG0 (b) with different concentrations after eight freeze-thaw cycles.

Figure 3 Effects of freeze-thaw cycles on T2 of 4% OG (a, c, e) and BG (b, d, f) with different molecular weights.

Figure 4 Effects of freeze-thaw cycles on peak area proportion of 4% OG (a, c, e) and BG (b, d, f) with different molecular weights.

Figure 5 PCA scores plots (PC1, PC2) for 4% OG (a) and BG (b) with different molecular weight β-glucan after eight freeze-thaw cycles.

Figure 6 PCA scores plots (PC1, PC2) for 4% OG (a) and BG (b) with different molecular weight β-glucan after eight freeze-thaw cycles.

Figure 7 SEM images of 4% OG and BG with different molecular weights after eight freeze-thaw cycles.

Table 1

Chocolate formulations studied in this work

Figure 1 GC-MS chromatograms of partially methylated alditol acetates of OG0 (a) and BG0 (b)

Figure 2 T2 distribution of OG0 (a) and BG0 (b) with different concentrations after eight freeze-thaw cycles.

Figure 3 Effects of freeze-thaw cycles on T2 of 4% OG (a, c, e) and BG (b, d, f) with different molecular weights.

Figure 4 Effects of freeze-thaw cycles on peak area proportion of 4% OG (a, c, e) and BG (b, d, f) with different molecular weights.

Figure 5 PCA scores plots (PC1, PC2) for 4% OG (a) and BG (b) with different molecular weight β-glucan after eight freeze-thaw cycles.

Figure 6 Rheology of 4% OG (a) and BG (b) gels with different molecular weight β-glucans after eight freeze-thaw cycles.

Figure 7 SEM images of 4% OG and BG with different molecular weights after eight freeze-thaw cycles.