Method for optimizing extraction conditions of inonotus obliquus polysaccharide through response surface

Abstract

The invention discloses a method for optimizing extraction conditions of inonotus obliquus polysaccharide through a response surface, and belongs to the technical field of polysaccharide extraction and production. The extraction conditions of the inonotus obliquus polysaccharide after response surface optimization are as follows: the extraction time is 3.06 h, the extraction temperature is 80.16 DEG C, and the ratio of water to materials is 30.81: 1. The yield of the inonotus obliquus extracted under the condition is 5.39 + / -0.06%, and the method has a relatively high extraction rate. And the method has certain promotion significance on expanded production of the inonotus obliquus polysaccharide.

Description

Technical Field

[0001] The invention relates to optimizing extraction conditions of inonotus obliquus polysaccharide by using response surface methodology, and belongs to the technical field of polysaccharide extraction and production. Background Art

[0002] Inonotus obliquus, also known as birch mushroom, also known as oblique tube fiber mushroom, belongs to the Basidiomycetes, Agaricomycetes, Rusty Lepidoptera, Rusty Lepidoptera. The extract of birch inonotus has biological activities such as promoting lipid metabolism and regulating cardiac function, as well as antibacterial, anti-inflammatory, antioxidant and anti-tumor. Inonotus obliquus is rich in polysaccharides and oligosaccharides. Polysaccharides are mainly extracted from the fruiting bodies of birch inonotus and are one of the main functional active ingredients of birch inonotus. At present, the extraction methods of birch inonotus polysaccharides mainly include hot water extraction, ultrasound-assisted extraction, microwave-assisted extraction and enzymatic hydrolysis. Chen et al. used ultrasound combined with microwave extraction to react for 19 minutes under the conditions of ultrasonic power of 50W, water-to-material ratio of 20:1 (mL:g), frequency of 40kHz, and microwave power of 90W, and obtained birch inonotus polysaccharide with a purity of 73.16%, with a yield of 3.25%. Although the use of ultrasound combined with microwave extraction saves time, the equipment is expensive, the extraction cost is too high, and ultrasound and microwaves may have a destructive effect on the structure of polysaccharides. In addition, microwaves consume a lot of energy and may cause harm to the human body. Therefore, hot water extraction is a commonly used extraction method for birch polysaccharides. Properly increasing the water temperature helps to promote the dissolution of polysaccharides, which is safe, effective and convenient to operate. Zhang et al. obtained birch polysaccharides with anti-fatigue properties under the conditions of extraction temperature of 100°C, water-to-material ratio of 25:1 (mL:g), and extraction time of 3h. In addition, Yu et al. obtained birch polysaccharides with weight loss effects under the conditions of extraction temperature of 90°C, water-to-material ratio of 30:1 (L:kg), extraction time of 3h, and deproteinization by Sevage method.

[0003] Inonotus birch polysaccharide is a new type of natural lipid-lowering and blood sugar-lowering substance. Ping et al. used high sugar and high insulin to act on HepG2 cells to establish an insulin resistance cell model, and co-cultured Inonotus birch polysaccharide with HepG2 cells, and detected the glucose consumption of HepG2 cells before and after culture, and found that Inonotus birch polysaccharide can significantly increase the glucose consumption of cells. Xu et al.'s study showed that after daily gavage of 30 and 60 mg / kg.bw Inonotus birch polysaccharide in diabetic mice, blood sugar decreased by 22.51% and 24.32% after three weeks. Inonotus birch polysaccharide can not only reduce the blood sugar of diabetic mice, but also reduce the content of low-density lipoprotein cholesterol in serum, increase the content of high-density lipoprotein cholesterol, and regulate the lipid metabolism of diabetic mice induced by streptozotocin. Yu et al. found that Inonotus birch polysaccharide effectively improved obesity in mice, reduced the weight of mice, accelerated liver glucose metabolism and triglyceride metabolism, and significantly increased the relative abundance of lactobacilli in the cecum. It can be seen that Inonotus obliquus polysaccharide has shown obvious blood sugar and lipid lowering effects both in vivo and in vitro.

[0004] In addition, birch polysaccharides have a variety of biological activities, such as anti-tumor, antioxidant and immune regulation, and are safe and non-toxic. For example, the water-soluble polysaccharide ISP2a of birch not only shows anti-tumor activity in mice, but also can significantly enhance the immune response of mice. Birch polysaccharides have an inhibitory effect on the proliferation of SMMC7721 liver cancer cells. Gavage of birch water extract to mice can effectively inhibit the content of TNF-α and IL-6, indicating that it has the effect of regulating immunity. Summary of the invention

[0005] The present invention is based on a single factor experiment and optimizes the extraction conditions of Inonotus birch polysaccharide by response surface methodology to improve its yield. The present invention optimizes the extraction conditions of Inonotus birch polysaccharide and provides a method for large-scale extraction and production application of Inonotus birch in the food industry in the future.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A method for optimizing the extraction conditions of Inonotus obliquus polysaccharide using a response surface method comprises the following steps:

[0008] Step 1: Extraction of birch polysaccharide by hot water extraction. Crush birch polysaccharide and pass through an 80-mesh sieve. Use hot water extraction to extract birch polysaccharide. After extraction for a period of time under certain temperature and water-to-material ratio conditions, centrifuge at 6000r / min for 10min, and concentrate the supernatant under reduced pressure to a certain volume. Mix the supernatant with 95% ethanol in a volume ratio of 1:3, fully precipitate for 12h, and centrifuge at 6000r / min for 10min. Dry the precipitate in an oven at 60℃ until constant weight to obtain birch polysaccharide. The yield of crude polysaccharide is the ratio of the mass of crude polysaccharide to the mass of raw material. After the crude polysaccharide is purified by deproteinization, decolorization and other purification treatments, birch polysaccharide is finally obtained.

[0009] Step 2: Single factor test. The yield of birch inonotus polysaccharide is affected by three main factors: extraction time, extraction temperature and water-to-material ratio. Therefore, single factor experiments were carried out for different influencing factors. Different extraction temperatures (50℃, 60℃, 70℃, 80℃ and 90℃), extraction times (1h, 2h, 3h, 4h and 5h) and water-to-material ratios (10:1, 20:1, 30:1, 40:1 and 50:1) were used to evaluate the effects of the three factors on the yield of birch inonotus. Time, temperature, water-to-material ratio. The extraction time was selected as 3h, water-to-material ratio as

[0010] Step 3: Optimize the extraction conditions by response surface. The extraction temperature, extraction time and water-to-material ratio are the three factors affecting the yield of birch polysaccharide, and the yield of birch polysaccharide is the response value. Combined with the results of the single factor experiment, the Box-Behnken model in the response surface method was used to design 17 groups of experiments with three factors and three levels to optimize the extraction rate of birch polysaccharide. The yield of birch polysaccharide is equal to the ratio of crude polysaccharide to raw material mass.

[0011] The invention adopts response surface method to optimize the extraction conditions of Inonotus obliquus polysaccharide, and the yield is 5.39±0.06%.

[0012] The beneficial effects achieved by the present invention are:

[0013] 1. The polysaccharide of Inonotus birchii was extracted by hot water extraction. The single factor experiment determined that the extraction conditions of polysaccharide were extraction temperature 80℃, extraction time 4h, and water-to-solid ratio of 40:1.

[0014] 2. Response surface optimization was carried out on the basis of single factor experiment. The extraction conditions of Inonotus birchii polysaccharide were optimized by response surface. The extraction time was 3.06h, the extraction temperature was adjusted to 80℃, and the water-to-solid ratio was 30.81:1. The IOP yield under this extraction condition was 5.39±0.06%, and the extraction rate of Inonotus birchii polysaccharide was improved to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The process flow chart of extracting polysaccharide from Inonotus obliquus

[0016] Figure 2 The effects of extraction conditions on the yield of polysaccharides from Inonotus obliquus, (a) the effect of extraction temperature on the yield, (b) the effect of extraction time on the yield, (c) the effect of water-to-solid ratio on the yield;

[0017] Figure 3 Response surface analysis of the interaction effect of extraction temperature, time and water-to-solid ratio. (a) Three-dimensional response surface plot of extraction temperature on the yield of polysaccharides from birch fungus, (b) Three-dimensional response surface plot of extraction time on the yield of polysaccharides from birch fungus, (c) Three-dimensional response surface plot of water-to-solid ratio on the yield of polysaccharides from birch fungus, (d) Contour plot of extraction temperature and extraction time on the yield of polysaccharides from birch fungus, (e) Contour plot of extraction temperature and water-to-solid ratio on the yield of polysaccharides from birch fungus, (f) Contour plot of extraction time and water-to-solid ratio on the yield of polysaccharides from birch fungus. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0019] A response surface method for optimizing the extraction conditions of Inonotus obliquus polysaccharides, such as Figure 1 As shown, the following steps are included: Step 1: Extracting birch inonotus polysaccharide by hot water extraction. Crush birch inonotus and pass through an 80-mesh sieve. Birch inonotus polysaccharide is extracted by hot water extraction. After extraction for a period of time under certain temperature and water-to-material ratio conditions, centrifuge at 6000r / min for 10min, and concentrate the supernatant under reduced pressure to a certain volume. The supernatant is mixed with 95% ethanol in a volume ratio of 1:3, and after sufficient precipitation for 12h, centrifuge at 6000r / min for 10min. The precipitate is dried in an oven at 60°C until constant weight is obtained to obtain birch inonotus crude polysaccharide. The yield of crude polysaccharide is the ratio of the mass of crude polysaccharide to the mass of raw material. After the crude polysaccharide is purified by deproteinization, decolorization and other purification treatments, birch inonotus polysaccharide is finally obtained.

[0020] Step 2: Single factor test. The yield of birch polysaccharide is affected by three main factors: extraction time, extraction temperature and extraction water-to-solid ratio. Through single factor test, the extraction conditions of birch polysaccharide were determined to be extraction temperature 80℃, extraction time 4h, and water-to-solid ratio of 40:1.

[0021] Step 3: Optimize extraction conditions using response surface analysis. Figure 2As shown in the figure, the extraction temperature, extraction time and water-to-solid ratio are the three factors affecting the yield of birch polysaccharide, and the yield of birch polysaccharide is the response value. Combined with the results of single factor experiments, the Box-Behnken model in the response surface methodology was used to design 17 groups of experiments with three factors and three levels to optimize the extraction rate of birch polysaccharide.

[0022] Table 1 Box-Behnken experimental design

[0023]

[0024]

[0025] Based on the experimental data obtained by Box-Behnken design, the IOP yield (between 2.55% and 5.36%) and the predicted response value Y can be fitted into the following second-order polynomial equation as follows: (In the equation, A represents the extraction temperature; B represents the extraction time; and C represents the water-to-material ratio.)

[0026] Y=5.37+0.034A+0.11B+0.12C+0.028AB+0.18AC+0.17BC-1.49A2-1.1B2-0.82C2.

[0027] The variance analysis of the Box-Behnken design experimental results is shown in Table 1. The P value (P<0.0001) and the F value of 60.96 indicate that the regression model is significant. The multiple correlation coefficient R-Squared = 0.9874 indicates that the accuracy of the response surface model (98.74%) is sufficiently accurate. The modified multiple correlation coefficient Adj R-Squared = 0.9712 means that 97.12% of the experimental results are consistent with the model, indicating that the fit between the measured value and the predicted value of IOP yield is good, and can be used for theoretical prediction of IOP yield. The P values ​​of A2, B2 and C2 are all less than 0.0001, indicating that the three factors of extraction temperature, extraction time and extraction water-to-material ratio have extremely significant effects on polysaccharide yield. According to the F value A2>B2>C2, the influence of the three factors on IOP yield is extraction temperature>extraction time>water-to-material ratio.

[0028] Response surface interaction analysis: In order to clarify the interactive relationship between extraction temperature, extraction time and water-to-solid ratio on IOP yield, multiple regression analysis was performed on the data in Table 1. The three-dimensional response surface plot and the corresponding contour plot show the interaction between variables and their effects on IOP yield. The degree of influence of each factor on IOP yield is shown in Figure 2. Figure 3 As shown. By observing Figure 3 The slope of the surface in the three-dimensional response surface plots in (a), 3(b) and 3(c) determines the degree of influence of the influencing factors on the IOP yield. Figure 3The surface in (b) has the highest inclination and the steepest slope, indicating that the interaction between extraction temperature and water-to-solid ratio is the most significant. Figure 3 The shape of the contour plots in (d), 3(e), and 3(f) can determine the strength of the interaction effect. Figure 3 The contour lines in (f) are close to ellipses, which produces a strong interaction effect, indicating that the interaction between extraction time and water-to-material ratio is significant. The IOP extraction conditions were optimized by response surface analysis. The model predicted the optimal extraction conditions to be 3.06h extraction time, 80.16℃ extraction temperature, and 30.81:1 water-to-material ratio. The IOP yield was 5.73%. In the actual experiment, the extraction time was 3.06h, the extraction temperature was adjusted to 80℃, and the water-to-material ratio was 30.81:1. The yield of birch polysaccharide under this extraction condition was 5.39±0.06%. This parameter is close to the simulation prediction parameter, and the error is within a reasonable range, which proves that this parameter is suitable for the extraction of birch polysaccharide.

[0029] In summary, in the study on response surface optimization of Inonotus obliquus polysaccharide, the extraction conditions optimized by response surface in the present invention have a higher yield for Inonotus obliquus polysaccharide.

Claims

1. A method for optimizing the extraction conditions of Inonotus obliquus polysaccharide using a response surface method, characterized in that: The extraction time was 3.06 h, the extraction temperature was adjusted to 80 °C, and the water-to-material ratio was 30.81:

1.

2. The method for optimizing the extraction conditions of Inonotus obliquus polysaccharides using a response surface method according to claim 1, characterized in that: The yield of Inonotus obliquus polysaccharide was 5.39±0.06%.

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