Modification of wheat bran insoluble and soluble dietary fibers with snail enzyme

Xin Liu, Keke Suo, Pei Wng, Xue Li, Limin Ho*, Jiqing Zhu, Junjun Yi,Qiozhen Kng, Jinyong Hung, Jike Lu,*

a School of Life Sciences, Zhengzhou University, Henan 450001, China

b The Quartermaster Research Institute of Engineering and Technology, Academy of Military Sciences PLA China, Beijing 100010, China

c School of Agricultural Science, Zhengzhou University, Henan 450001, China

Keywords:

Snail enzyme

Insoluble dietary fiber

Soluble dietary fiber

Physicochemical properties

Functional properties

ABSTRACT

Insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) extracted from wheat bran were modified by snail enzyme and their physicochemical properties (water retention capacity and oil retention capacity),functional properties (cholesterol adsorption capacity, glucose adsorption capacity and antioxidant activity)and structural characterizations were evaluated. The results showed that snail enzyme modification led to the significant increase in oil retention capacity of IDF, glucose adsorption capacity and cholesterol adsorption capacity of IDF and SDF. Enzymatic modification also markedly improved the DPPH radical scavenging capacity and reducing power of IDF and SDF. Meanwhile, scanning electron microscopy(SEM) analysis indicated the microstructures of IDF and SDF powders were significantly changed. Fourier transfer-infrared spectrometry (FT-IR) showed that snail enzyme modification could degrade the part of cellulose and hemicellulose of IDF and SDF. All these improved physicochemical and functional properties of IDF and SDF might depend on their structural changes. It suggested that snail enzyme modification could effectively improve physicochemical and functional properties of IDF and SDF from wheat bran.

1. Introduction

Dietary fiber (DF) is involved in health promotive activities and disease preventive, including reducing the levels of glucose and cholesterol in blood, controlling body weight, preventing colon cancer and so on [1,2]. Most of DF is insoluble (insoluble dietary fiber, IDF), and only a small part is soluble (soluble dietary fiber,SDF) [3]. IDF can regulate the growth of gut microbes, reduce the intestinal transport and increase the fecal volume [1]. Compared to IDF, SDF plays an important role in immunomodulatory activity and blood sugar regulation [4,5]. Meanwhile, SDF can more easily form gels and emulsifiers to make it incorporate into foods [6].Therefore, DF gains more attention of the consumers.

In China, wheat is one of the main food crops and the annual output of wheat accounts for about 20% of the world’s total output [7]. A huge amount of wheat bran is produced in flour processing [8]. Wheat bran, which takes up about 25% of the grain weight of wheat, is usually rendered as feed, wine making, vinegars and not further industrialized[9]. Although wheat bran has been reported to be abundant in DF, it has less comprehensive utilization in food industry [10].

Several ways to modify DF from different food sources have been reported, such as physical, chemical and enzymatic methods [11-13].Enzymatic hydrolysis can make the particle of DF smaller by breaking chemical bonds [14,15]. The water retention capacity (WRC)and oil retention capacity (ORC) of carrot pomace IDF were 1.28-fold and 1.09-fold as that before modification with enzymatic hydrolysis,respectively [13]. Modification of oat bran with xylanase could increase the content of SDF and decrease the water binding capacity[14]. Snail enzyme from snail sac and digestive tract is a complex enzyme containing more than 20 kinds of enzymes, such as cellulase,pectinase, amylase, protease, β-glucosidase, etc. [16]. Snail enzyme as a natural enzyme has advantages such as low cost and easy access.Meanwhile, snail enzyme has strong digestive capacity and is widely used in the breaking of yeast cells [17]. However, DF modified by snail enzyme has never been reported to our knowledge.

Whether snail enzyme can modify wheat bran DF to improve the application of wheat bran needs further research. In this study, snail enzyme was applied to modify the wheat bran IDF and SDF, and the physicochemical properties (WRC and ORC), functional properties(cholesterol adsorption capacity (CAC), glucose adsorption capacity(GAC) and antioxidant activity) and characterizations (scanning electron microscopy (SEM) and Fourier transfer-infrared spectrometry(FT-IR)) of unmodified and modified SDF and IDF were investigated.This work will contribute to developing an innovative and effective method to modify the wheat bran DF and providing a theoretical basis for the comprehensive utilization of wheat bran in food industry.

2. Materials and methods

2.1 Materials

Wheat bran was bought from local market in Zhengzhou (Henan,China). Wheat bran powder was obtained by drying, crushing and sieving (40 mesh sieve). Snail was obtained from Lver Agricultural Technology Co., Ltd. (Henan, China) and fed in the lab.

2.2 Extraction of snail enzyme

Snail enzyme was extracted in the lab. Firstly, the snails were starved for 3 days, then they were washed and dissected. The brown liquid located in snail sac was suctioned and removed in the centrifugal tube with the syringe. The sac and digestive tract were stripped, sheared and mixed with the brown liquid. Then the mixture was added with citric acid-phosphate buffer (pH 5.0)and placed on the ice. After 2 h, the mixture was centrifuged at 10 000 r/min for 20 min at 4 °C. Finally, the supernatant was filtered with 0.45 μm filter and freeze-dried to obtain the powder. The powder was snail enzyme and stored at -20 °C for further use. The activity of snail enzyme was measured by the cellulase activity, and the enzyme activity was 267.6 U/g with colorimetric method.

2.3 Preparation of total dietary fiber (TDF)

TDF was extracted from wheat bran powder according to the method reported previously [11]. The wheat bran powder was defatted with n-hexane extraction. The defatted wheat bran powder was gelatinized with distilled water (1:10, m/V) in water bath at 95-100 °C for 15 min. The pH was adjusted to 5.5 with HCl. To remove starch, α-amylase was added to the system and oscillated at 95 °C for 1 h. After reaction, the pH of the system was adjusted to 7.0 with NaOH. The mixture was catalyzed by neutral protease at 55 °C for 2 h to remove the protein. Finally, the mixture was put into a boiling water bath for 15 min to denature the enzyme. When cooled to room temperature, the system was added with 4-fold volumes of 95% ethanol. After the alcohol precipitation was carried out overnight at 4 °C, the residue obtained by suction and filtration was dried to obtain TDF.

2.4 Enzymatic modification

Enzymatic modification was conducted according the method of Luo et al. [11] with slight modifications. TDF solution (10%, m/V)was prepared by citric acid-phosphate buffer (0.05 mol/L, pH 4.5),and was treated with snail enzyme (1.5% on the weight of TDF) in oscillator at 45 °C for 4 h. Then the system was reacted at 100 °C for 10 min to inactivate the enzymes. The mixture was separated by centrifugation of 4 000 r/min for 25 min, and the residue was dried in an oven at 70 °C to obtain the IDF after enzymatic modification (E-IDF).The supernatant was added with 4-fold volumes of 95% ethanol and reacted overnight. Then the solution was pumped, filtered, and dried in an oven to obtain the SDF after enzymatic modification (E-SDF). The control group (not treated with snail enzyme) was named SDF and IDF.They were stored at -20 °C for subsequent experiments.

2.5 WRC

WRC was measured following the former method with some modifications [12]. The dry sample was hydrated with deionized water at room temperature for 1 h. The deionized water was filtered with filter paper, and the residue and centrifuge tube were immediately weighted. WRC was measured by Equ (1):

Where m1is the weight of the dry fiber (g), m2is the weight of the centrifuge tube (g) and m3is the weight of the residue and centrifuge tube (g).

2.6 ORC

ORC was assayed by adopting the method reported previously with minor modifications [13]. The dry sample was mixed with hemp seed oil for 1 h, and then the residue was obtained by centrifugation at 3 000 r/min for 15 min. The residue and centrifuge tube were immediately weighted. ORC was evaluated on the basis of Equ (2):

Where w1is the weight of the dry sample (g), w2is the weight of the centrifuge tube (g) and w3is the weight of the residue and centrifuge tube (g).

2.7 GAC

GAC was determined using the previous method with some modifications [3]. Briefly, 0.5 g fiber was mixed with the 20 mL of glucose solution (50 mmol/L) and incubated in a concussion incubator (160 r/min) at 37 °C for 2 h. The supernatant was obtained by centrifugation at 3 000 r/min for 20 min and the glucose content was determined according to the glucose standard curve. GAC was expressed as Equ (3):

Where C1is the glucose concentration before adsorption (mmol/L),C2is the glucose concentration after adsorption (mmol/L), V is the volume of solution (mL) and m is the weight of sample (g).

2.8 CAC

CAC was measured by the method of previous study with minor modifications [18]. The egg yolk was added to deionized water in the mass ratio of 1:9. The sample was mixed with 30 mL egg yolk solution, and the pH was adjusted to 2 and 7, respectively.The mixture was shaken in the incubator at 37 °C for 2 h. Then the mixture was weighted and centrifuged to obtain the supernatant. The content of cholesterol was determined by O-phthalaldehyde method,and CAC was calculated according to Equ (4):

Where g1is the weight of cholesterol before adsorption (mg), g2is the weight of cholesterol after adsorption (mg), and m is the weight of sample (g).

2.9 Antioxidant activity

The DPPH radical scavenging capacity and the reducing power of IDF, SDF, E-IDF and E-SDF were performed by the method previously reported in our lab [19,20].

2.10 SEM analysis

The surface features of IDF, SDF, E-IDF and E-SDF were examined at magnification of 20 and 3 k with SEM (Zeiss/Auriga FIB SEM Germany) as described by Niu et al. [21] with minor modification. The dried fibers were placed on conducting adhesive and coated with gold. Then the sample was examined at an accelerating voltage of 5 kV [12].

2.11 FT-IR spectroscopy

FT-IR spectrometer (Nicolet iS10, Thermo Scientific, USA) was used to estimate the changes in the molecular structure of IDF and SDF according to the procedure given by Liu et al. [22]. The DFs (IDF, E-IDF,SDF and E-SDF) were mixed with KBr powder, ground and pressed into pellets for FT-IR determination in the rang of 400-4 000 cm-1.

2.12 Statistical analysis

All determinations were performed in triplicate. Statistical analysis was carried out by t-test and Tukey’s multiple-range test using GraphPad Prism 5.0. All the results were represented as the means ± SD (n = 3).

3. Results and discussion

3.1 Physicochemical properties

The chemical structure of DF has hydrophilic groups like carboxyl groups and hydroxyl groups, which can adsorb water of several times its own mass. The WRC shows that DF have the ability to hold water under external pressure, such as centrifugal force [23]. Meanwhile,the ORC represents the capacity of DF to adsorb fat, and high ORC values could decrease cholesterol contents in serum by adsorbing fat [24]. It relies on the surface porosity, overall charge density, and hydrophobic nature of DF [25,26]. So WRC and ORC are important indicators to measure the quality of DF.

The WRC and ORC were presented and compared in Fig. 1.The WRC of SDF and E-SDF was 0, which was due to the high solubility in water. Meanwhile, the WRC of IDF had no obvious change between modified and unmodified groups (P ＞ 0.05). In addition, there were no significant differences in ORC between E-SDF(1.16 g/g) and SDF (1.28 g/g) (P ＞ 0.05, Fig. 1B). It suggested that snail enzyme hydrolysis could not change the ORC of SDF.However, the ORC value of E-IDF was 3.95 g/g, which was 1.17-fold as that before modification (3.38 g/g). Snail enzyme has strong digestive capacity and might degrade some IDF, so some functional groups were exposed after enzymatic modification, which remarkably boosted the ORC value of IDF. Another reason was that the surface porosity of IDF was changed by snail enzyme, and it could combine more oil molecules. Similar result was observed in previous study that the ORC of carrot DF was improved significantly after enzymatic modification [13].

Fig. 1 Effect of snail enzyme modification on the WRC (A) and ORC (B)of SDF and IDF obtained from wheat bran. All values represent the mean ±standard deviations (n = 3). # P ＜ 0.05 compared with IDF. SDF, represents the unmodified SDF; E-SDF, represents the SDF after snail enzyme modification;IDF, represents the unmodified IDF; E-IDF, represents the IDF after snail enzyme modification.

3.2 GAC

DF can bind the glucose in intestinal juice, thereby reducing blood glucose levels [27]. Therefore, DF has special effects in preventing or treating diabetes [28]. As shown in Fig. 2, the GAC values of SDF and IDF were 0.365 and 0.53 μmol/g, respectively. After snail enzyme modification, the GAC values of E-SDF and E-IDF were 2.19-fold and 2.12-fold to SDF and IDF, respectively. The IDF and SDF were found to be effective in adsorbing glucose after snail enzyme modification (P ＜ 0.05). In the previous study, similar results were reported that the GAC of IDF from bamboo shoot shell was increased significantly under the hydrolysis of cellulose and xylanase [11]. In this study, these results could be explained as following aspects. Snail enzyme is a complex enzyme containing more than 20 kinds of enzymes, mainly cellulase and pectinase.Cellulase can partially degrade the cell wall and loosen the fiber structure, which is beneficial to enhance the intramolecular force of DF and glucose, thereby adsorbing more glucose molecules [29].Therefore, snail enzyme modification could increase the GAC of SDF and IDF from wheat bran.

Fig. 2 Effect of snail enzyme modification on the GAC of SDF and IDF obtained from wheat bran. All values represent the mean ± standard deviations(n = 3). * P ＜ 0.05 compared with SDF; # P ＜ 0.05 compared with IDF.

3.3 CAC

DF can reduce cholesterol levels in the serum and risk of cardiovascular disease [3]. In the present study, the small intestine environment (pH 7.0) and stomach environment (pH 2.0) were simulated, to research the CAC of the fibers. As represented in Fig.3, the CAC of SDF was higher than that of IDF at pH 2.0 and the result was reversed at pH 7.0, which suggested that SDF and IDF had different CAC values at different pH. Meanwhile, the snail enzyme hydrolysis significantly increased the CAC value compared to the untreated sample (P ＜ 0.05). The CAC values of SDF were 32.975 and 5.31 mg/g at pH 2.0 and pH 7.0, respectively. Compared with that of SDF, the CAC values of E-SDF were increased markedly by 1.13 and 4.32 times after snail enzyme hydrolysis, respectively.Meanwhile, the CAC of E-IDF (23.27 mg/g) increased 30.44%compared with IDF (17.84 mg/g) at pH 2.0. Due to the surface features and structural properties, DF can bind with cholesterol molecules by physical and chemical chelation. Snail enzyme might change the molecular weight of DF, which made macromolecules into small ones and more binding sides expose. In this research, the higher CAC values of E-IDF and E-SDF were mainly related to the exposure of more binding sites to cholesterol after snail enzyme modification[3]. The combination of cellulose and xylanase was used to improve the CAC of DF in previous study [11]. But in this study, snail enzyme as a compound enzyme can achieve the similar improvement effect and is easy to operate.

Fig. 3 Effect of snail enzyme modification on the CAC of SDF and IDF obtained from wheat bran at pH 2.0 (A) and pH 7.0 (B). All values represent the mean ± standard deviations (n = 3). * P ＜ 0.05 compared with SDF;# P ＜ 0.05 compared with IDF.

3.4 Antioxidant activity

Fig. 4 Effect of snail enzyme modification on the DPPH radical scavenging capacity (A) and assay of reducing power (B) of SDF and IDF obtained from wheat bran.All values represent the mean ± standard deviations (n = 3). * P ＜ 0.05 compared with SDF; ## P ＜ 0.01 compared with SDF.

The patterns of scavenging DPPH radical and reducing power could well reflect the antioxidant activities of varied materials [30,31].As shown in Fig. 4A, the DPPH radical scavenging capacity increased after the snail enzyme treatment. The DPPH radical scavenging capacity of E-SDF was 13.15%, which was 2.40-fold to SDF(P ＜ 0.05), and the E-IDF had the similar trend. In the reducing power assay, the antioxidant activity is expressed by measuring the absorbance value of Perl’s Prussian at 700 nm [32]. The assay of reducing power of E-SDF was increased significantly compare with SDF (P ＜ 0.05), but the E-IDF and IDF showed no significant difference (P ＞ 0.05). DF contains polysaccharides and the antioxidant capacity of the polysaccharides is associated with their molecular weight [33]. Snail enzyme might reduce the molecular weight of SDF and IDF, which makes more function groups expose.So the antioxidant activity of E-IDF and E-SDF were significantly increased after the snail enzyme treatment. The result was consistent with the previous report which showed that the antioxidant activity of wheat bran IDF was improved after enzymatic hydrolysis [10].

3.5 SEM

SEM is a common method to investigate the modification of DF[10]. SEM micrographs of SDF and IDF before and after modification were shown in Fig. 5. It can be seen from Fig. 5A and 5C that the structure of SDF was loose, while the IDF was compact. Snail enzyme modification boosted the rupture of E-SDF and E-IDF surface and the inner structure was largely exposed. It was easy to form a network structure with oil molecules and glucose molecules, which could contribute to improvement of the ORC, GAC and CAC of E-IDF and E-SDF. Similar result had been reported in former research,where the morphology of corn bran was changed from ribbon matrix to threadlike structures by cellulase treatment [34]. Snail enzyme has strong digestive capacity and may degrade the wall polysaccharides to make the intact wall damage, which lead to changes of the microstructure of E-SDF and E-IDF.

Fig. 5 The scanning electron micrograph (SEM) of SDF and IDF before and after enzymatic modifications at magnification values of 20 k (A and B) and 3 k (C and D). A, SDF; B, E-SDF; C, IDF; D, E-IDF.

3.6 FT-IR spectroscopy

The FT-IR spectroscopy can detect different chemical bonds and functional groups in substances. The FT-IR spectroscopy from 400 cm-1to 4000 cm-1was shown in Fig. 6, which reflected functional groups of IDF, E-IDF, SDF and E-SDF. It was reported that the absorption band at 3 400 cm-1assigned to the —OH stretching [35].Compared with SDF, E-SDF had distinct blue shift at this peak. The reason might be that the hydrogen bonds in cellulose and hemicellulose were destroyed by snail enzyme. The small peaks at 2 920 cm-1represented the C—H stretching vibration of cellulose and hemicellulose.The deep peaks at 1 633 cm-1were reported to the —OH between cellulose and water molecules [12]. The E-SDF and E-IDF showed weaker peak at this wavelength than the SDF. The enzymatic modification might decrease hydrogen bonding, which resulted in the degradation of cellulose and the reduction of combined water. The peak of E-SDF at 1 040 cm-1assigned to oligosaccharide was weaker than SDF. IDF, E-IDF,SDF and E-SDF exhibited the similar spectral peaks which suggested snail enzyme modification had not destructed the fundamental chemical structure of IDF and SDF. Meanwhile, the differences among the peak intensity suggested that snail enzyme modification could degrade the part of cellulose and hemicellulose of IDF and SDF.

Fig. 6 The FT-IR of SDF and IDF before and after snail enzyme modifications.

4. Conclusions

In the present research, the physiochemical and functional properties of wheat bran IDF and SDF before and after modification were studied. Compared with unmodified fibers, enzymatic modification could not only enhance ORC of IDF, but also increase the functional properties, containing the capacities to bind glucose and cholesterol and antioxidant capacity. After the enzymatic modification, the structure of IDF and SDF became loose and porous.These results demonstrated that snail enzyme as a compound enzyme can effectively modify IDF and SDF from wheat bran. Therefore,snail enzyme modified IDF and SDF from wheat bran have potential applications in food industry as a functional ingredient.

conflict of interest

The authors declare they have no competing interests.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (81870093), the Research Project of People’s Liberation Army (BXP20C006, BX115C007), the Special Subject Funding of Zhengzhou University and the Natural Science Foundation of Henan Province for Outstanding Youth (202300410365).