Abstract
Chitooligosaccharides (COS) are derived from chitosan, which can be used as nutraceuticals and functional foods. Because of their various biological activities, COS are widely used in the food, medicine, agriculture, and other fields. COS were prepared by chitosanase from Pseudoalteromonas sp. SY39 and their anti-obesity activity was researched in mice in this study. The effects of hydrolysis time, temperature, the ratio of enzyme to chitosan, and pH on the productivity of COS were discussed. Preparation process of COS was established in a 5-L fermenter. COS were characterized and their anti-obesity activity was studied in animal experiments. The results showed that COS could effectively reduce serum lipid levels and obesity in mice, and have a good anti-obesity activity. The preparation technology and remarkable anti-obesity activity of COS further expand their applications in the food and pharmaceutical industries.
Chitooligosaccharides (COS) were effectively prepared by using chitosanase under the optimum conditions, which had excellent anti-obesity activity in mice.
Chitooligosaccharides (COS), also called chitosan oligomers or chitooligomers, are composed of oligomers of β-1,4-linked 2-amino-2-deoxy-d-glucopyranose (GlcN) and 2-acetamido-2-deoxy-d-glucopyranose (GlcNAc) [1–3]. COS are typically produced by partial hydrolysis from chitosan and have attracted a wide attention in food and medicinal applications because of their nontoxic property and various biological activities. They are water-soluble with anti-coagulation, anti-oxidation, inflammatory resistance, immune stimulation, anti-bacterial, anti-tumor, and other functions [4–6]. Additionally, a variety of studies have indicated that COS exert effective anti-obesity effects in obese animals. As an effective lipid-lowering dietary supplement and functional food, COS have attracted a great interest of many researchers. Research suggested that COS significantly reduced the weight gain of rats and effectively inhibited adipose tissue hypertrophy and hyperplasia [7]. Administration with COS significantly lowered serum lipid levels and hepatic lipid accumulation in the high-fat diet-fed C57BL/6 mice [8]. Jiang et al. [9] revealed chitooligosaccharide capsules (COSTC) could reduce cholesterol and ameliorate serum lipid levels. Yang et al. [10] indicated that COS are not only beneficial for inhibiting dyslipidemia but also improve the pathological changes of liver tissue in rats, alleviate liver damage, maintain normal lipid metabolism in the liver.
COS can be prepared by enzymatic hydrolysis methods (specific enzymes and nonspecific enzymes), physical methods (ultrasonic treatment, ultraviolet radiation, and microwave treatment) and chemical methods (acid hydrolysis and hydrogen peroxide oxidation). Specific enzymes mainly refer to chitosanases and nonspecific enzymes include cellulose, lysozyme, lipase, protease, amylase, etc. The chemical method is easy to operate, whereas the difficulty of separating products as well as the environmental pollution limits its application. The yield of physical method is low although the product is easy to purify, by contrast, the enzymatic hydrolysis method is more admirable as its environmental friendliness, higher efficacy, as well as operation and monitor controllable [11,12]. At present, COS are mainly obtained from nonspecific enzymatic method, but its application is greatly limited by its low degradability and productivity. However, the chitosanase hydrolysis method overcomes these disadvantages and is the most ideal and efficient method for preparing COS. Although there are many reports about the anti-obesity of COS, these COS are mostly prepared by non-chitosanase method, which greatly restricts their range of applications [13]. Thus, acquiring an efficient protocol for product bioactive COS would be vastly desirable.
In our previous work, a new chitosanase, CsnM, was purified from the marine bacterium Pseudoalteromonas sp. SY39. Its excellent properties such as cold-adaptation and thermo-tolerance make it suitable for the preparation of COS in large quantities and efficiency [14]. Here, the effects of hydrolysis time, temperature, the ratio of enzyme to chitosan and pH on the productivity of COS were discussed. COS were prepared by using CsnM under the optimal conditions and consisted of the oligosaccharides with degree of polymerization (DP) of 2–5. The efficient protocol for producing COS with anti-obesity activity lays a foundation for future applications.
Materials and Methods
Materials
The chitosan (viscosity: 20 mPa·s; degree of deacetylation: 93%) was obtained from Anhui yuanzheng Bioengineering Co., Ltd. (Anhui, China). CsnM (180 U/mL) was characterized in early research and preserved in our laboratory. All other chemicals used in this study were of reagent grade. High-fat diet (HFD) feed and normal fat diet feed were purchased from Darenfucheng Animal Husbandry Company (Qingdao, China). The compositions of the experimental diets are shown in Table 1.
Compositions of experimental diet (g/100 g).
| Components | Normal fat diet | High-fat diet |
| Casein | 20 | 20 |
| Corn starch | 39.7 | 25 |
| Sucrose | 10 | 20 |
| Corn oil | - | 5 |
| Lard | - | 20 |
| Mineral | 3.5 | 3.5 |
| Vitamin | 1 | 1 |
| Cellulose | 5 | 5 |
| Choline bistartrate | 0.3 | 0.3 |
| DL-Methionine | - | 0.2 |
| L-Cystine | 0.3 | - |
| Dextrose | 13.2 | - |
| Soybean oil | 7 | - |
| Components | Normal fat diet | High-fat diet |
| Casein | 20 | 20 |
| Corn starch | 39.7 | 25 |
| Sucrose | 10 | 20 |
| Corn oil | - | 5 |
| Lard | - | 20 |
| Mineral | 3.5 | 3.5 |
| Vitamin | 1 | 1 |
| Cellulose | 5 | 5 |
| Choline bistartrate | 0.3 | 0.3 |
| DL-Methionine | - | 0.2 |
| L-Cystine | 0.3 | - |
| Dextrose | 13.2 | - |
| Soybean oil | 7 | - |
Compositions of experimental diet (g/100 g).
| Components | Normal fat diet | High-fat diet |
| Casein | 20 | 20 |
| Corn starch | 39.7 | 25 |
| Sucrose | 10 | 20 |
| Corn oil | - | 5 |
| Lard | - | 20 |
| Mineral | 3.5 | 3.5 |
| Vitamin | 1 | 1 |
| Cellulose | 5 | 5 |
| Choline bistartrate | 0.3 | 0.3 |
| DL-Methionine | - | 0.2 |
| L-Cystine | 0.3 | - |
| Dextrose | 13.2 | - |
| Soybean oil | 7 | - |
| Components | Normal fat diet | High-fat diet |
| Casein | 20 | 20 |
| Corn starch | 39.7 | 25 |
| Sucrose | 10 | 20 |
| Corn oil | - | 5 |
| Lard | - | 20 |
| Mineral | 3.5 | 3.5 |
| Vitamin | 1 | 1 |
| Cellulose | 5 | 5 |
| Choline bistartrate | 0.3 | 0.3 |
| DL-Methionine | - | 0.2 |
| L-Cystine | 0.3 | - |
| Dextrose | 13.2 | - |
| Soybean oil | 7 | - |
Optimization conditions of preparation of COS
A typical enzymatic reaction for the production of COS was carried out by incubating the chitosan substrate and CsnM. Chitosan substrate was prepared with 1 M acetic acid and 1 M NaOH. Chitosan was dissolved in 1 M acetic acid aqueous solution to a concentration of 10 mg/mL, and the pH was adjusted to 6.0 with 1 M NaOH. The effects of temperature, pH, the optimum ratio of enzyme to chitosan and hydrolysis time were investigated. The specific conditions of each experiment were specified in the footnotes of the figure. The reducing sugars were determined under the specified time with 3,5-dinitrosalicylic acid (DNS) method and expressed as a dextrose equivalent (DE) value [15]. Chitosan is hydrolyzed completely when the reducing sugar content is constant [16].
Preparation of COS
COS were prepared in a 5-L fermenter (BLBIO, Shanghai, China). Chitosan substrate was firstly prepared by being dissolved in 1 M aqueous acetic acid to a concentration of 1% (w/v), and the pH was adjusted to 6.0 with 1 M NaOH. Two liters of 1% chitosan substrates and 360 U CsnM were then put into the fermenter. The temperature and the stirring speed of the fermenter were set at 40°C and 200 r/min, respectively. The procedure mentioned above provided a better condition for dissolving higher concentration of Chitosan. Afterward, a higher concentration of chitosan substrate (5%, w/v) with higher viscosity was utilized to obtain a higher concentration of COS. Two liters of 5% chitosan substrate (w/v) and CsnM (1,800 U) were added to the fermenter and the pH of the fermenter was adjusted to 6.0 with NaOH. As the time of the enzymatic reaction prolonged, chitosan was gradually degraded to COS with lower viscosity. Part of reaction solution was taken out from the fermenter as the content of reducing sugar was not increased anymore. The reaction solution was terminated by heating at 100°C for 30 min. The pH of the reaction solution was adjusted to 7.0 with 1 M NaOH solution followed by being centrifuged for 20 min at 6,000 rpm. Finally, COS was obtained by collecting the supernatant. Additionally, 2 L of 5% chitosan substrate and CsnM (1,800 U) were added into the remained reaction system. When the content of reducing sugar was no longer increased, all the reaction solution was taken out from the fermenter and processed as before to obtain COS.
Characterization of COS
The average DP and average molecular weight (MW) of COS were conducted using a modified version of the method described by Wu [17]. The hydrolysis products were analyzed by thin-layer chromatography (TLC) and electrospray ionization-mass spectrometry (ESI-MS). TLC analysis using silica gel 60 F254 (Merck KGaA, 64,271 Darmstadt, Germany) and color-developed with 0.5% ninhydrin dissolved in ethanol, while the extending solvent is isopropanol/ammonium water/water (70:3:27, v/v/v) [18]. ESI-MS was performed in positive-ion mode using Thermo Fisher ScientificTM Q ExactiveTM Hybrid Quadrupole-OrbitrapTM (Waltham, MA, USA).
Animal experiments
Twenty-four healthy male KM mice (16–18 g; 4 weeks) were purchased from Darenfucheng Animal Husbandry Company (Qingdao, China). The mice were housed under standard conditions: 21 ± 2°C, a day-night rhythm, and a relative humidity of 45–65%. After a week of acclimation period, the mice were randomly divided into three groups (eight mice per group). (1) normal fat (NF) group, fed with a normal fat diet for 8 weeks; (2) high-fat diet (HFD) group, fed with HFD for 8 weeks; (3) COS group, fed an HFD for 4 weeks and then fed with HFD and COS (350 mg/kg of body weight per day) for another 4 weeks. During the experiment, mice had free access to food and water. The mice were weighed weekly and the energy intake levels for each group were recorded. At the end of the four-week treatment period, 12-h fasted animals were sacrificed. Blood was obtained from the retro-orbital plexus. Serum was obtained from blood by centrifugation (4°C, 12,000 r/min, 20 min), and stored at -80°C. Livers and adipose tissues were quickly stripped and weighed. After pictures were taken, the tissues were stored at -80°C for further analysis. The animals used in these experiments received humane care according to the guidelines of the Qingdao University Animal Care and Use Committee.
Biochemical parameter analysis
Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), free fatty acid (FFA), and high-density lipoprotein cholesterol (HDL-C) were determined by commercial detection kits (Nanjing Jiancheng of Bioengineering Institute, Nanjing, China) according to the instructions of kits.
Histological analysis
For histological study, liver and epididymal adipose tissue were collected and weighed individually and then were rinsed with normal saline at the end of the experimental period. The tissues were immediately fixed in 10% buffered formalin for 24 h and embedded in paraffin, sectioned as a thickness of 5 μm. After stained with hematoxylin and eosin (H&E), the sections were observed under a microscope at 200× magnification.
Data analysis
All data were presented as the mean ± SD. One-way analysis of variance (ANOVA) followed by Dunnett’s test was used to determine whether differences existed between multiple groups and the HFD group using SPSS software (SPSS Inc., Chicago, IL, USA). A p-value <0.05 was considered statistically significant.
Results and discussion
Factors affecting the hydrolysis of chitosan
In order to study the effects of reaction time on the hydrolysis of chitosan, the content of reducing sugars in the reaction solution was determined every 2 h in 12 h (Figure 1(a)). The optimal reaction time was 10 h as the maximum DE value was observed at 10 h (Figure 1(a)). The effects of reaction temperatures on the hydrolysis of chitosan were investigated over the range of 30–50°C as shown in Figure 1(b). When the temperature was 40°C, the DE value reached the maximum value. However, as the temperature raised more than 40°C, the yield of COS decreased with it. The yield decrease at high temperatures was probably due to reversible or irreversible changes in the molecular structure of enzyme. Therefore, the optimum hydrolysis temperature is 40°C, which is the same as the optimal temperature of CsnM (Figure 1(b)). As shown in Figure 1(c), the optimum pH of enzymatic hydrolysis is 6.0 as the yield of COS was found to be maximum when the pH was set at 6.0. The previously reported temperature of 40°C [19], 45°C [20], 50°C [16] and 55°C [13] as well as pH levels of 5.3 [21], 5.5 [15], 5.8 [16] and 7.0 [22] as optimal conditions for the enzymatic hydrolysis of chitosan. The differences may be attributed to the discrepancies in properties of enzymes, sources of chitosan and reaction time. Figure 1(d) shows the effects of the enzyme concentration on the hydrolysis of chitosan. The yield of reducing sugars increased with the increase of enzyme concentration, and reached the maximum yield of 47.8% with no more increase when the enzyme amount was 18 U/g chitosan substrate. There was no obvious difference in reducing sugar production between 18 U/g and 36 U/g chitosan. Therefore, the optimum enzyme concentration was 18 U/g chitosan. The activity of the enzyme was expressed in international units (U).
The effects of hydrolysis time, temperature, the ratio of enzyme to chitosan, and pH on the productivity of COS. (a) Effect of time on hydrolysis of chitosan. Chitosan: 10 mg/mL, 200 mL; crude enzyme: 36 U; pH 6.0; 40°C. (b) Effect of temperature on hydrolysis of chitosan. Chitosan: 10 mg/mL, 200 mL; crude enzyme: 36 U; pH 6.0; at different temperatures for 6 h. (c) Effect of pH on hydrolysis of chitosan. Chitosan: 10 mg/mL, 200 mL; crude enzyme: 36 U; pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0; 40°C; 10 h. (d) Effect of chitosanase concentration on hydrolysis of chitosan. Chitosan: 10 mg/mL, 200 mL; pH 6.0; 40°C for 10 h. Data are shown as mean ± SD (n = 3).
Characterization of COS
According to the acid hydrolysis method, the average DP and average MW of COS were 2.93 and 489.73, respectively. The COS were analyzed by silica gel TLC (Figure 2(a)). The spots on TLC plate were in good agreement with the chitodisaccharide (DP2), chitotrisaccharide (DP3), chitotetrasaccharide (DP4), and Chitopentasaccharides (DP5) markers. Figure 2(b) shows the ESI-MS spectrum of COS. The m/z values of 341.16, 251.62 (z = 2), 502.23, 332.15 (z = 2) and 412.69 (z = 2) correspond to the glucosamine dimers, trimers, tetramers, and pentamers, respectively. Monomers were not detected in COS. Accordingly, the COS were composed of DP2, DP3, DP4, and DP5.
Characterization of the COS. (a) TLC analysis of COS. Lane M: standard chitosan oligomers (GlN, DP1–5); Lane 1: COS. (b) ESI-MS analysis of COS.
Energy intake, body weight, weight gain, and liver index
To understand the possible effects of lipid modification, we determined energy intake, body weight, and the body weight gain of the mice. Energy intake in the HFD group (24.76 ± 0.90 kcal/day) was slightly higher than that in the COS group (23.20 ± 0.24 kcal/day) during the experimental period, but the difference was not significant (Figure 3(a)). However, the energy intake of the HFD group was higher than that of the NF group (17.02 ± 1.56 kcal/day; p < 0.001). The body weight of the mice was measured over an 8-week period, and the results are shown in Figure 3(b). For the body weight gain, the average body weight of the HFD group and NF group increased by 24.86% and 15.51% within 5–8 weeks, respectively. Meanwhile, the four-week administration of COS significantly decreased body weight gain by 4.86% (Figure 3(c)). In addition, the effect of COS on reducing weight gain was greater than that of the HFD group, suggesting that COS has an effective anti-obesity effect in the HFD-induced obese mice. The effect of COS on liver fat deposition was reflected by the liver index (liver mass/body mass × 100%) (Figure 3(d)). The liver index of the NF group was significantly lower than that of the HFD group (p < 0.01), indicating that the liver of the HFD group had high fat content. COS supplementation led to an obvious reduction in the liver index. Similar to the previous study [8], COS showed obvious inhibition of increases in mice body weight and liver index without influence on the appetite, suggesting effective anti-obesity effects in HFD-induced obese mice.
The main index of mice. (a) energy intake. (b) body weight. $ p < 0.05: NF group vs. HFD group; # p < 0.05, ## p < 0.01: COS group vs. HFD group. (c) weight gain. (d) liver index. The data are presented as the means ± SD (n = 8). Compared to HFD group, ** p < 0.01; *** p < 0.001.
Serum lipid concentrations of mice
Research has shown that the high-fat diets lead to increases in TG, TC, LDL levels and a reduction in HDL levels [10]. Serum lipid concentrations of mice are shown in Table 2. Serum TC, TG, FFA levels of the HFD group were significantly higher than those of the NF group (serum TC: p < 0.05, serum TG and FFA: p < 0.01), showing that hyperlipidemia mice models were established successfully. Compared to the HFD group, there was a decrease in TG and TC (p < 0.05), but no significant difference in HDL of the COS group. Research showed that COSTC improved lipid metabolism via upregulating the gene expression and activity of cholesterol 7α-hydroxylase (CYP7A1), liver X receptor alpha (LXRA) and peroxisome proliferation activated receptor-α (PPARα) [9].
Serum lipid concentrations of mice in different experimental groups.
| Groups | NF | HFD | COS |
| TG (mM) | 1.13 ± 0.09** | 1.66 ± 0.22 | 1.08 ± 0.15* |
| TC (mM) | 2.94 ± 0.17* | 3.54 ± 0.36 | 2.94 ± 0.14* |
| HDL (mM) | 6.67 ± 0.28** | 8.53 ± 0.40 | 8.05 ± 0.56 |
| LDL (mM) | 0.58 ± 0.15 | 0.59 ± 0.12 | 0.61 ± 0.11 |
| FFA (mM) | 0.57 ± 0.09** | 0.84 ± 0.05 | 0.70 ± 0.15 |
| Groups | NF | HFD | COS |
| TG (mM) | 1.13 ± 0.09** | 1.66 ± 0.22 | 1.08 ± 0.15* |
| TC (mM) | 2.94 ± 0.17* | 3.54 ± 0.36 | 2.94 ± 0.14* |
| HDL (mM) | 6.67 ± 0.28** | 8.53 ± 0.40 | 8.05 ± 0.56 |
| LDL (mM) | 0.58 ± 0.15 | 0.59 ± 0.12 | 0.61 ± 0.11 |
| FFA (mM) | 0.57 ± 0.09** | 0.84 ± 0.05 | 0.70 ± 0.15 |
Results are expressed as mean ± SD (n = 8), * p < 0.05 versus HFD group; ** p < 0.01 versus HFD group.
Serum lipid concentrations of mice in different experimental groups.
| Groups | NF | HFD | COS |
| TG (mM) | 1.13 ± 0.09** | 1.66 ± 0.22 | 1.08 ± 0.15* |
| TC (mM) | 2.94 ± 0.17* | 3.54 ± 0.36 | 2.94 ± 0.14* |
| HDL (mM) | 6.67 ± 0.28** | 8.53 ± 0.40 | 8.05 ± 0.56 |
| LDL (mM) | 0.58 ± 0.15 | 0.59 ± 0.12 | 0.61 ± 0.11 |
| FFA (mM) | 0.57 ± 0.09** | 0.84 ± 0.05 | 0.70 ± 0.15 |
| Groups | NF | HFD | COS |
| TG (mM) | 1.13 ± 0.09** | 1.66 ± 0.22 | 1.08 ± 0.15* |
| TC (mM) | 2.94 ± 0.17* | 3.54 ± 0.36 | 2.94 ± 0.14* |
| HDL (mM) | 6.67 ± 0.28** | 8.53 ± 0.40 | 8.05 ± 0.56 |
| LDL (mM) | 0.58 ± 0.15 | 0.59 ± 0.12 | 0.61 ± 0.11 |
| FFA (mM) | 0.57 ± 0.09** | 0.84 ± 0.05 | 0.70 ± 0.15 |
Results are expressed as mean ± SD (n = 8), * p < 0.05 versus HFD group; ** p < 0.01 versus HFD group.
Effects of COS on liver and epididymal adipose tissue
The liver weight of the mice is shown in Figure 4(a). The liver weight of the HFD group had increased compared with the NF group. In contrast to the HFD group, the liver weight of the COS group was dramatically decreased (p < 0.001) (Figure 4(a)). The liver images of the mice are shown in Figure 4(b). The livers of the NF group were small in volume, bright red, supple texture and had sharp edges. The livers of the HFD group were larger, slightly swollen and yellow, and the margins were thick, indicating that severe fatty liver-like disease occurred. However, this fatty liver status of the COS group is alleviated, which means that COS protect the liver by reducing fat formation. Figure 4(c) shows the result of liver sections, the NF group showed no histological abnormalities with fewer fat droplets, whereas the hepatocytes in the HFD group showed severe fat vacuoles, indicating that the mice had developed hepatic steatosis induced by HFD. Treatment in the COS group markedly decreased fat vacuoles and lipid droplets in hepatocytes whose cell morphology and arrangement were similar to that in the NF group. The epididymal fat weight of mice in HFD was increased (Figure 4(d)). In contrast to the HFD group, the epididymal fat weight of COS group was dramatically decreased (p < 0.001). The result was in agreement with the previous study of COS [10], which alleviated the accumulation of epididymal fat and hepatic steatosis. Tao et al. [8] reported that COS ameliorated liver lipid accumulation via downregulating the lipogenic mRNA expression of sterol regulatory element-binding protein-1 c (SREBP-1 c) and fatty acid synthase (FAS) in the liver. Figure 4(e) shows the images of epididymal adipose tissue. The mice fed with a HFD showed hypertrophy of epididymal adipose tissue, while COS administration decreased the size of epididymal adipose tissue. The images of epididymal adipose tissue sections are exhibited in Figure 4(f). The epididymal fat cells of HFD were significantly hypertrophic compared to the NF group. The sizes of adipocytes were prominently diminished after administering COS, suggesting that COS have anti-obesity activity by reducing the growth and accumulation of adipocytes. Pan et al. [23] showed that chitosan oligosaccharide capsules suppress the hyperplasia or hypertrophy of adipocytes via upregulating the gene expressions of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and adipose differentiation-related protein (ADRP) in the epididymal adipose tissues.
Effects of COS on liver and adipose tissue. (a) Liver weight. (b) The whole liver. (c) Liver histopathological. (d) Epididymal adipose weight. (e) The epididymal adipose tissue. (f) The epididymal adipose tissue slices. The data are presented as the means ± SD (n = 8). Compared to HFD group, * p < 0.05; *** p < 0.001.
Conclusions
Chitosanase from marine bacteria Pseudoalteromonas sp. SY39 (CsnM) is very suitable for the preparation of COS. COS were prepared by hydrolyzing chitosan using CsnM under the optimal conditions of pH 6.0, temperature 40°C, time 10 h and 18 U/(g chitosan) chitosanase. COS were prepared in the 5-L fermenter and consisted of the oligosaccharides with DP2-5. For the first time, COS were prepared by CsnM and used in anti-obesity studies. Results indicated that COS were capable of decreasing serum TC and TG levels of the mice fed with high-fat diet, and have an action of inhibiting the increase of body weight and an action of anti-obesity. The efficient protocol for producing COS with anti-obesity activity further expand their application in the food and pharmaceutical industries.
Author Contributions
A. Li and Y. Han designed the experiments. Y. Zhou, S. Wang, W. Zhao, Z. Lv and H. Li performed the experiments. Y. Zhou and D. Li analyzed the data. Y. Zhou and X. Li wrote the main manuscript text. All authors reviewed the manuscript.
Disclosure statement
The authors state that there is no conflict of interest.
References
