Method for preparing fructo-oligosaccharide through enzymolysis and application of fructo-oligosaccharide in special medical food

Abstract

The invention relates to a method for preparing fructo-oligosaccharide through enzymolysis and application of the fructo-oligosaccharide in special medical food, and belongs to the field of enzyme engineering. The preparation method comprises the steps of raw material pretreatment, step-by-step enzymolysis, enzyme deactivation, separation and purification and the like. The step-by-step enzymolysis is divided into three stages, specific enzymes are respectively added in each stage, and the temperature, the pH value, the stirring speed and the reaction time are accurately controlled. Compared with the traditional method, the method has the advantages of high conversion rate, low by-product, high reaction efficiency and the like. Besides, the experiment of applying the fructo-oligosaccharide prepared by the method to the tumor total nutrient formula food shows that the fructo-oligosaccharide can effectively improve the nutritional status of experimental animals, has an obvious synergistic effect when being used together with related products, and has a good application value in the field of special medical foods.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering, and particularly relates to a method for preparing fructooligosaccharide by enzymolysis and application thereof in special medical food. BACKGROUND

[0002] Fructooligosaccharide is a functional oligosaccharide generated by enzymatic catalysis of sucrose or inulin, mainly composed of sucrose, sucrose and sucrose, and has many physiological functions such as promoting the proliferation of intestinal beneficial flora, inhibiting the growth of harmful bacteria, regulating the balance of intestinal microecology, reducing blood lipids, and promoting mineral absorption, and is widely used in food, health products, medicine and other fields. According to the difference of raw materials and production methods, fructooligosaccharide can be divided into two types of inulin and sucrose. Inulin fructooligosaccharide is prepared by hydrolysis of natural inulin or chicory, and has more impurities; sucrose fructooligosaccharide is prepared by transglycosylation of fructose transferase, and has high production efficiency and high product quality, and becomes the main production method in industry.

[0003] Chinese patent with publication number CN00130200.0A discloses a method for preparing fructooligosaccharide, using aspergillus niger strain, obtaining fructose transferase by secondary culture, adding sucrose solution with a concentration of 30%-50% into the enzyme tank, adjusting the pH value to 5-8, and performing primary enzymatic contact reaction at 62-70℃ for 8-10 hours. The product of primary enzymatic reaction is subjected to primary nanofiltration treatment, and the filtrate syrup after nanofiltration is subjected to secondary enzymatic reaction, and then the enzymatic syrup is subjected to secondary nanofiltration treatment. However, the culture of the strain has certain randomness, and it is difficult to be industrialized.

[0004] Chinese patent with publication number CN113621664A discloses a method for preparing high-purity fructooligosaccharide using sucrose as substrate, adding fructose transferase preparation first to perform partial transglycosylation, and then adding microbial cells capable of consuming glucose to perform transglycosylation of fructose transferase preparation and sugar consumption of microbial cells simultaneously, thereby obtaining fructooligosaccharide.

[0005] Chinese patent with publication number CN102127574A discloses a method for producing fructooligosaccharide by fructose transferase, 30-60% sucrose solution, enzyme amount of fructose transferase 2000-5000u / kg sucrose, pH control at 4.9-7.0, temperature at 27-31℃, stirring condition of 30-49r / min, reaction for 13-40 hours, separation of mycelium by microfiltration membrane to obtain light yellow to yellow liquid, decolorization by macroporous resin, and finally concentrated into fructooligosaccharide.

[0006] However, in the traditional enzymatic process with sucrose as the substrate, the product can only obtain a purity of 55%-60% of fructo-oligosaccharide product, wherein the residual glucose, fructose and unreacted sucrose not only affect the physiological efficacy of the product, but also limit its application in special groups of people such as diabetic patients, and there are problems such as unsatisfactory type of fructo-oligosaccharide product, low conversion rate, and not widely used fructo-oligosaccharide. SUMMARY

[0007] In order to overcome the shortcomings of the prior art, the present application provides a method for preparing fructo-oligosaccharide by enzymatic hydrolysis, which has significant advantages in production efficiency and product quality, and the prepared fructo-oligosaccharide can effectively improve the nutritional status of the body in special medical food, and has important application value.

[0008] (1) Raw material pretreatment: select edible grade sucrose with a purity of not less than 99%, dissolve it in deionized water to prepare a sucrose solution with a mass concentration of 45%-55%, and reserve it.

[0009] (2) Enzymatic hydrolysis: A stage: add 20-50 U / g of glucose oxidase of Penicillium and 0.01%-0.1% of lecithin to the sucrose solution, and react at 30-40℃, pH=5.0-6.0, 100-150 r / min for 1.5-3h. B stage: add 0.1-0.5 U / g of β-fructosyltransferase of Aspergillus niger, 0.1-0.5 U / g of β-fructosyltransferase of Aspergillus oryzae, and 0.1-0.5 U / g of invertase, and react at 40-50℃, pH=4.5-5.5, 100-150 r / min for 1.5-3h, and then react at 50-60℃, pH=4.5-5.5, 100-150 r / min for 1.5-3h. C stage: add 20-50 U / g of glucose oxidase of Bacillus subtilis, and react at 40-50℃, pH=6.5-7.5, 100-150 r / min for 1.5-3h.

[0010] (3) Enzyme inactivation treatment: after the reaction is completed, the reaction solution is quickly heated to 85-95℃ and kept for 5-10 minutes to inactivate the enzyme.

[0011] (4) Separation and purification: A. Filtration: the reaction solution after enzyme inactivation is filtered through a 0.45 μm microporous filter to remove insoluble impurities that may be produced during the reaction. B. Desalination by ion exchange resin: the filtered solution is desalted by strong acid cation exchange resin and strong base anion exchange resin to remove inorganic ions produced during the reaction. C. Activated carbon decolorization: 0.5%-1.5% activated carbon is added to the desalted solution, stirred at 50-60℃ for 30-60 minutes for decolorization treatment to remove pigments and odor substances in the solution, and the activated carbon is removed by filtration. D. Concentration and crystallization: the decolorized solution is concentrated under reduced pressure at 40-50℃ and a vacuum degree of-0.08 to-0.09 MPa to make the solid content of the solution reach 60%-70%. The concentrated solution is cooled to 20-25℃ and crystallized for 12-24 hours, and the oligofructose crystals are obtained by centrifugal separation. E. Drying: the oligofructose crystals are vacuum dried at 40-50℃ until the moisture content is less than 5% to obtain finished oligofructose.

[0012] The 20-50 U / g in the present application refers to 20-50 U of enzyme added per gram of sucrose. The 0.01%-0.1% lecithin refers to mass / volume ratio, relative to the reaction solution. The strong acid cation exchange resin used in the experiment of the present application is a styrene-based strong acid cation exchange resin (001x7 type), and the strong base anion exchange resin is a styrene-based strong base anion exchange resin (201x7 type). The glucose oxidase of Penicillium refers to glucose oxidase derived from Penicillium, the glucose oxidase of Bacillus subtilis refers to glucose oxidase derived from Bacillus subtilis, the β-fructosyltransferase of Aspergillus oryzae refers to β-fructosyltransferase derived from Aspergillus oryzae, and the β-fructosyltransferase of Aspergillus niger refers to β-fructosyltransferase derived from Aspergillus niger, which are purchased from Shanghai Huashangxiangyang Biotechnology.

[0013] Compared with the prior art, the technical effect of the present application is that: 1. The method for preparing oligofructose by enzymatic hydrolysis has high conversion rate and high product purity The present application adopts a step-by-step enzymatic hydrolysis process, and by optimizing the addition order, types and reaction conditions of enzymes, the conversion efficiency of sucrose to oligofructose can be greatly improved. Comparative experiments show that when the enzymes are not added in stages (Comparative Example 1), two enzymes are added less in stage B (Comparative Example 2), etc., the conversion rate of oligofructose is significantly reduced, fully reflecting the advantages of the method in improving the conversion rate.

[0014] 2. The method for preparing oligofructose by enzymatic hydrolysis can effectively control the generation of by-products In the enzymatic hydrolysis reaction process of the present application, the content of by-products such as glucose and fructose is low. It shows that this method can effectively reduce by-products and improve product quality.

[0015] 3. The enzymatic preparation of fructooligosaccharides has high reaction efficiency and short time consumption The oligofructose content of Examples 1-3 has exceeded 89% at 6h, and only a small increase (<0.5%) at 12h, indicating that 6h is close to the reaction endpoint, while the traditional process and some comparative examples (such as Comparative Examples 1-3) have a slow reaction, with oligofructose content less than 65% at 6h, and close to 80% only after 12h. The method can significantly save time and improve production efficiency.

[0016] 4. The enzymatic preparation of fructooligosaccharides has a scientific and reasonable process design The present application uses a process of stepwise enzymatic hydrolysis and condition regulation, specifically three stages of A, B and C for enzymatic hydrolysis, with precise control of temperature, pH, stirring rate and other parameters in each stage, and different enzymes are used in different stages to avoid mutual interference between enzymes and fully exert the catalytic effect of each enzyme.

[0017] 5. The technical advantages of the prepared oligofructose in the application of special medical food In the application experiment of tumor whole nutrition formula food, the body weight maintenance rate of the combined low dose group (adding 3% oligofructose) and the combined high dose group (adding 5% oligofructose) was significantly higher than that of the model control group and the Zuxin group. Among them, the body weight maintenance rate of the combined high dose group reached 108.2%, which was 33.1% higher than that of the model control group. The albumin level of mice is an important indicator of nutritional status. The albumin levels of the combined low dose group and the combined high dose group were 3.35g / dL and 3.68g / dL, respectively, which were significantly improved compared with the model control group (1.85g / dL) (P<0.01), and the combined high dose group was close to the level of the blank group (3.76g / dL), indicating that the oligofructose can effectively improve the nutritional status of tumor mice. Compared with the use of Zuxin alone, the effect of the combination of oligofructose and Zuxin is better. The combined low dose group and the combined high dose group are better than the Zuxin group in terms of body weight maintenance and albumin level improvement, which reflects good synergistic effect and can better meet the nutritional support needs of special populations in special medical food. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 : Rat albumin level. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme of the present application more clear, the following examples are used to further illustrate the present application, but the protection scope of the present application is not limited to these examples, and the examples are only used to explain the present application. Those skilled in the art should understand that any changes or equivalent replacements without departing from the concept of the present application are included in the protection scope of the present application.

[0020] Example 1 Method for preparing fructooligosaccharides by enzymatic hydrolysis 1. Raw material pretreatment (1) Raw material selection: Edible grade sucrose with purity ≥ 99% was used.

[0021] (2) Solution preparation: Sucrose was dissolved in deionized water to prepare a sucrose solution with a mass concentration of 50%, which was used as needed.

[0022] 2. Stepwise enzymatic hydrolysis reaction Table 1. Reaction conditions for three-stage enzymatic hydrolysis in Example 1 3. Enzyme inactivation treatment: After the reaction was completed, the reaction solution was quickly heated to 90°C and maintained for 8 minutes to completely inactivate all enzymes and terminate the reaction.

[0023] 4. Separation, purification and product preparation: (1) Filtration: The enzyme-inactivated reaction solution was filtered through a 0.45 μm microporous filter membrane to remove insoluble impurities (such as bacterial cells and particles).

[0024] (2) Desalination: The filtered solution was sequentially passed through strong acid cation exchange resin and strong base anion exchange resin to remove inorganic ions.

[0025] (3) Decolorization: 1.0% active carbon by mass was added to the desalted solution, which was stirred at 55°C for 45 minutes, and then filtered to remove the active carbon.

[0026] (4) Concentration and crystallization: The decolorized solution was concentrated under reduced pressure at 45°C and a vacuum degree of -0.08 to -0.09 MPa until the solid content reached 65%. The solution was cooled to 20-25°C and allowed to crystallize for 20 hours, after which the fructooligosaccharide crystals were separated by centrifugation.

[0027] (5) Drying: The crystals were dried under vacuum at 45°C until the moisture content was ≤ 5%, resulting in the finished product of fructooligosaccharides.

[0028] Example 2 Method for preparing fructooligosaccharides by enzymatic hydrolysis 1. Raw material pretreatment (1) Raw material selection: Edible grade sucrose with purity ≥ 99% was used.

[0029] (2) Solution preparation: Sucrose was dissolved in deionized water to prepare a sucrose solution with a mass concentration of 45%, which was used as needed.

[0030] 2. Stepwise enzymatic hydrolysis reaction Table 2. Reaction conditions for three-stage enzymatic hydrolysis in Example 2 3. Inactivation treatment: after the reaction is completed, the reaction solution is rapidly heated to 85°C and maintained for 5 minutes to inactivate all enzymes and terminate the reaction.

[0031] 4. Separation, purification and product preparation: (1) Filtration: the inactivated reaction solution is filtered through a 0.45 μm microporous filter membrane to remove insoluble impurities (such as bacterial cells and particles).

[0032] (2) Desalination: the filtered solution is sequentially passed through a strong acid cation exchange resin and a strong base anion exchange resin to remove inorganic ions.

[0033] (3) Decolorization: 0.5% activated carbon is added to the desalted solution, which is stirred at 50°C for 30 minutes, and then filtered to remove the activated carbon.

[0034] (4) Concentration and crystallization: the decolorized solution is concentrated under reduced pressure at 40°C and a vacuum degree of -0.08 to -0.09 MPa until the solid content is 60%. The solution is cooled to 20-25°C and allowed to crystallize for 12 hours. The oligofructose crystals are separated by centrifugation.

[0035] (5) Drying: the crystals are dried under vacuum at 40°C until the moisture content is ≤5%, and the finished oligofructose is obtained.

[0036] Example 3 Method for preparing oligofructose by enzymatic hydrolysis 1. Raw material pretreatment (1) Raw material selection: food-grade sucrose with a purity of ≥99% is used.

[0037] (2) Solution preparation: sucrose is dissolved in deionized water to prepare a sucrose solution with a mass concentration of 55%, which is ready for use.

[0038] 2. Stepwise enzymatic hydrolysis reaction Table 3 Stepwise regulation of reaction conditions in three-stage enzymatic hydrolysis of Example 3 3. Inactivation treatment: after the reaction is completed, the reaction solution is rapidly heated to 95°C and maintained for 10 minutes to inactivate all enzymes and terminate the reaction.

[0039] 4. Separation, purification and product preparation: (1) Filtration: the inactivated reaction solution is filtered through a 0.45 μm microporous filter membrane to remove insoluble impurities (such as bacterial cells and particles).

[0040] (2) Desalination: the filtered solution is sequentially passed through a strong acid cation exchange resin and a strong base anion exchange resin to remove inorganic ions.

[0041] (3) decolorization: 1.5% active carbon was added into the desalination solution, and stirred at 60°C for 60 minutes, and then filtered to remove the active carbon.

[0042] (4) concentration and crystallization: the solution after decolorization was concentrated under reduced pressure at 50°C and vacuum degree of -0.08 to -0.09 MPa until the solid content was 70%; and then cooled to 20-25°C to stand for crystallization for 24 hours, and centrifuged to separate the oligofructose crystals.

[0043] (5) drying: the crystals were dried at 50°C under vacuum until the moisture content was ≤5%, and then the finished oligofructose was obtained.

[0044] Example 1 Method for preparing oligofructose by enzymatic hydrolysis Meanwhile, 35 U / g of Penicillium glucose oxidase, 0.05% of lecithin, 0.3 U / g of Aspergillus niger β-fructosyltransferase, 0.3 U / g of Aspergillus oryzae β-fructosyltransferase, 0.3 U / g of invertase, and 35 U / g of Bacillus subtilis glucose oxidase were added into the sucrose solution.

[0045] Table 4. Three-stage enzymatic hydrolysis of Example 1 The other steps were the same as those of Example 1.

[0046] Example 2 Method for preparing oligofructose by enzymatic hydrolysis Table 5. Three-stage enzymatic hydrolysis of Example 2 The other steps were the same as those of Example 1.

[0047] Example 3 Method for preparing oligofructose by enzymatic hydrolysis Table 6. Three-stage enzymatic hydrolysis of Example 3 The other steps were the same as those of Example 1.

[0048] Example 4 Method for preparing oligofructose by enzymatic hydrolysis Table 7. Three-stage enzymatic hydrolysis of Example 4 The other steps were the same as those of Example 1.

[0049] Example 5 Method for preparing oligofructose by enzymatic hydrolysis Table 8. Three-stage enzymatic hydrolysis of Example 5 The other steps were the same as those of Example 1.

[0050] Example 1 Method for enzymatic preparation of fructooligosaccharides Table 9 Three-stage enzymatic hydrolysis of Example 1 with stepwise adjustment of reaction conditions Other steps are the same as Example 1.

[0051] Example 2 Method for enzymatic preparation of fructooligosaccharides Table 10 Three-stage enzymatic hydrolysis of Example 2 with stepwise adjustment of reaction conditions Other steps are the same as Example 1.

[0052] Example 3 Method for enzymatic preparation of fructooligosaccharides Table 11 Three-stage enzymatic hydrolysis of Example 3 with stepwise adjustment of reaction conditions Other steps are the same as Example 1.

[0053] Example 4 Method for enzymatic preparation of fructooligosaccharides 1. Raw material pretreatment (1) Raw material selection: Edible grade sucrose with purity ≥ 99% was used.

[0054] (2) Solution preparation: Sucrose was dissolved in deionized water to prepare a sucrose solution with a mass concentration of 50%, ready for use.

[0055] 2. Stepwise enzymatic hydrolysis reaction Table 12 Three-stage enzymatic hydrolysis of Example 4 with stepwise adjustment of reaction conditions 3. Enzyme inactivation treatment: After the reaction was completed, the reaction solution was quickly heated to 90°C and maintained for 8 minutes to completely inactivate all enzymes and terminate the reaction.

[0056] 4. Separation, purification and product preparation: (1) Filtration: The enzyme-inactivated reaction solution was filtered through a 0.45 μm microporous filter membrane to remove insoluble impurities (such as bacterial cells and particles).

[0057] (2) Desalination: The filtered solution was passed through strong acid cation exchange resin and strong base anion exchange resin in sequence to remove inorganic ions.

[0058] (3) Decolorization: 1.0% activated carbon was added to the desalted solution, stirred at 55°C for 45 minutes, and filtered to remove the activated carbon.

[0059] (4) Concentration and crystallization: the decolorized solution was concentrated under reduced pressure at 45°C and vacuum degree of -0.08 to -0.09 MPa until the solid content reached 65%; the solution was cooled to 20-25°C and crystallized for 20 hours, and then the oligofructose crystals were separated by centrifugation.

[0060] (5) Drying: the crystals were dried under vacuum at 45°C until the moisture content was ≤5%, and the finished oligofructose was obtained.

[0061] Preparation of oligofructose by enzymatic hydrolysis 1. Pretreatment of raw material (1) Selection of raw material: edible-grade sucrose with purity ≥99% was used.

[0062] (2) Preparation of solution: sucrose was dissolved in deionized water to prepare a sucrose solution with a mass concentration of 50%, which was used for later experiments.

[0063] 2. Stepwise enzymatic hydrolysis reaction Table 13 Stepwise enzymatic hydrolysis reaction conditions of Example 10 3. Enzyme inactivation treatment: after the reaction was completed, the reaction solution was quickly heated to 90°C and maintained for 8 minutes to completely inactivate all enzymes and terminate the reaction.

[0064] 4. Separation, purification and preparation of finished product: (1) Filtration: the enzyme-inactivated reaction solution was filtered through a 0.45 μm microporous filter membrane to remove insoluble impurities (such as bacteria and particles).

[0065] (2) Desalination: the filtered solution was sequentially passed through a strong acid cation exchange resin and a strong base anion exchange resin to remove inorganic ions.

[0066] (3) Decolorization: 1.0% (mass fraction) activated carbon was added to the desalted solution, which was stirred at 55°C for 45 minutes, and then the activated carbon was removed by filtration.

[0067] (4) Concentration and crystallization: the decolorized solution was concentrated under reduced pressure at 45°C and vacuum degree of -0.08 to -0.09 MPa until the solid content reached 65%; the solution was cooled to 20-25°C and crystallized for 20 hours, and then the oligofructose crystals were separated by centrifugation.

[0068] (5) Drying: the crystals were dried under vacuum at 45°C until the moisture content was ≤5%, and the finished oligofructose was obtained.

[0069] Detection of enzymatic hydrolysis reaction progress During the enzymatic hydrolysis reaction, the reaction progress was detected by analyzing the content changes of sucrose, oligofructose, glucose and fructose in the reaction solution by high performance liquid chromatography (HPLC) (based on the total sugar mass, unit: %). Table 14 Changes in the contents of sucrose, fructo-oligosaccharide, glucose, and fructose in the reaction solution From the reaction liquid component data of examples 1-3 and comparative examples 1-10 in table 14, the following conclusions can be drawn: (1) Stepwise enzymatic hydrolysis significantly improves the conversion rate of fructooligosaccharides. The content of fructooligosaccharides in examples 1-3 reaches 89.60%-90.45% at 6h, and stabilizes at 90.16%-90.80% at 12h, with only 2.01%-2.68% sucrose remaining, indicating that the conversion rate of sucrose to fructooligosaccharides is extremely high. Comparative example 1 (enzymes added simultaneously) has only 62.34% fructooligosaccharides at 6h, and 76.78% at 12h, which is much lower than the examples; comparative example 2 (two enzymes added in stage B) has 72.95% fructooligosaccharides and 11.76% sucrose remaining at 12h, indicating that the incomplete enzyme system leads to insufficient conversion; comparative example 3 (constant temperature and pH) has 67.68% fructooligosaccharides at 12h, proving that stepwise temperature and pH control is the key to high conversion rate. (2) The present preparation method can effectively control the accumulation of by-products glucose and fructose. In examples 1-3, the content of glucose is always less than 3%, and the content of fructose is less than 5%, with 2.01%-2.48% glucose and 4.71%-5.00% fructose at 12h, and the total amount of by-products is less than 8%. However, the total amount of glucose and fructose in comparative examples 1-3 is generally 15%-20%, such as 9.87% glucose and 11.56% fructose at 6h in comparative example 3, indicating that the improper enzymatic hydrolysis conditions lead to by-product accumulation; the total amount of by-products (about 8%-10%) in comparative example 9 (without lecithin) and comparative example 10 (with Tween-80) is higher than that in the examples, indicating that lecithin can reduce the generation of by-products. (3) In terms of reaction time, examples 1-3 have higher reaction efficiency, reaching high conversion rate at 6h, which can save time and improve efficiency. The content of fructooligosaccharides in examples 1-3 exceeds 89% at 6h, and only increases slightly (<0.5%) at 12h, indicating that 6h is close to the reaction endpoint, and there is no need for excessive time. However, the reaction of comparative examples is slow, with less than 65% fructooligosaccharides at 6h in comparative examples 1-3, and close to 80% only after 12h, indicating low reaction efficiency; comparative examples 4-8 (temperature and pH conditions deviate from the optimized range) have 65%-78% fructooligosaccharides at 6h, and 85%-87% at 12h, which is still slower than the examples, proving that stepwise temperature and pH control can shorten the reaction time. In addition, the conversion rate of comparative example 1 (enzymes added simultaneously) is much lower than that of the examples, indicating that different enzymes play a role in different stages, avoiding mutual interference, proving the necessity of stepwise addition of enzymes. The low conversion rate of comparative example 2 (two β-fructofuranosidases added) proves that the synergistic effect of β-fructofuranosidases from Aspergillus niger and Aspergillus oryzae and invertase can improve the efficiency, proving the synergistic effect of the complex enzyme system. The low conversion rate of comparative example 3 (constant temperature and pH), comparative example 5 (deviation of temperature), and comparative example 6 (deviation of pH) verifies the key influence of the optimization of temperature and pH parameters in each stage on enzyme activity, proving the importance of stepwise temperature and pH control.The stepwise enzymatic hydrolysis process of this invention achieves high conversion rate (>90%), low by-products (<8%) and short reaction time (6h) of fructooligosaccharides by optimizing the order of enzyme addition, temperature and pH conditions and auxiliary agent (lecithin), which is significantly better than the traditional process.

[0070] Application of fructooligosaccharides in special medical foods and tumor-specific nutritional formula foods Experimental materials: Experimental animals: Several SPF-grade BALB / c mice aged 6-8 weeks and weighing 18-22g were selected, with half males and half females.

[0071] Tumor cell line: Mouse hepatocellular carcinoma cell line H22 was selected.

[0072] Intervention substances: fructooligosaccharides and succinic acid (commercially available products, according to the standard ingredients in the instruction manual) prepared in Example 1 of this invention.

[0073] Main reagents: Mouse albumin ELISA kit, physiological saline, sodium pentobarbital (for anesthesia), etc.

[0074] Main instruments: electronic balance (accuracy 0.01g), low-temperature centrifuge, microplate reader, clean bench, CO2 incubator, etc.

[0075] Experimental methods Control group: No tumor inoculation was performed; instead, 10 animals were given a basic complete nutritional formula.

[0076] Model control group: Inoculated with H22 tumor cells and given a basic complete nutritional formula food, 10 animals per group.

[0077] The Su Yi Su group: Inoculated with H22 tumor cells, and given a complete nutritional formula food with added Su Yi Su (added according to the product's recommended dosage), 10 animals per group.

[0078] Combined low-dose group: Inoculated with H22 tumor cells, and given a complete nutritional formula food supplemented with fructooligosaccharides (3% of the weight of the complete nutritional formula food) and Su Yi Su (50% of the recommended dose of the product), 10 animals per group.

[0079] Combined high-dose group: H22 tumor cells were inoculated and fed a complete nutritional formula food supplemented with fructooligosaccharides (5% of the weight of the complete nutritional formula food) and Su Yi Su (recommended dosage of the product), with 10 animals in each group.

[0080] Except for the control group, mice in all other groups were subcutaneously inoculated with H22 tumor cell suspension (concentration of 1×10⁻⁶) in the right axilla. 7 (0.2 mL per mouse, with a tumor volume of 1 / mL). After inoculation, the mice were observed. Experimental intervention was initiated when the tumor volume reached 50-100 mm³.

[0081] Feeding and intervention All mice were bred in SPF level animal room, temperature 22±2℃, humidity 50±10%, 12h light and dark alternation, free diet and water. The experimental intervention period was 4 weeks, during which the food intake and fecal condition of mice were recorded every day, and the body weight of mice was weighed by electronic balance at fixed time every week and recorded.

[0082] Sample collection and detection After 4 weeks of intervention, all mice were taken blood from the eye socket, and the blood was placed in a centrifuge tube. After standing at room temperature for 30 min, centrifugation was performed at 3000 rpm for 15 min, and the serum was separated. The content of albumin in the serum was detected by ELISA method, and the operation was strictly in accordance with the instructions of the kit.

[0083] Data processing and analysis Body weight maintenance rate calculation: body weight maintenance rate = (body weight at the end of the experiment / body weight at the beginning of the experiment) x 100%.

[0084] Data statistics: SPSS software was used for data analysis, and the measurement data were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparison between groups. If the result of ANOVA was statistically significant (P<0.05), Tukey's multiple comparison test was further used for pairwise comparison between groups. P<0.05 was considered to have statistically significant difference.

[0085] Experimental data Table 15 Body weight record of mice Table 16 Albumin level of mice Compared with the model control group, **P<0.01.

[0086] From the above data, it can be seen that the body weight maintenance rate of the combined high-dose group of mice increased by 33.1% compared with the model control group. At the same time, the albumin level of the combined high-dose group of mice was significantly improved (P<0.01) compared with the model control group.

Claims

1. A method for preparing fructooligosaccharides via enzymatic hydrolysis, characterized in that, The method includes the following steps: (1) Raw material pretreatment: Prepare a 45%-55% sucrose aqueous solution for later use; (2) Stepwise enzymatic hydrolysis: Phase A: Add 20-50 U / g Penicillium glucose oxidase and lecithin, and react for 1.5-3 hours at 30-40℃, pH 5.0-6.0, and 100-150 r / min. Phase B: Add 0.1-0.5 U / g Aspergillus niger β-fructosyltransferase, 0.1-0.5 U / g Aspergillus oryzae β-fructosyltransferase, and 0.1-0.5 U / g invertase. First, react at 40-50℃, pH 4.5-5.5, and 100-150 r / min for 1.5-3 hours, then raise the temperature to 50-60℃ and continue the reaction for another 1.5-3 hours. Phase C: Add 20-50 U / g Bacillus subtilis glucose oxidase and react for 1.5-3 hours at 40-50℃, pH 6.5-7.5, and 100-150 r / min. (3) Inactivate enzymes; (4) Separation and purification: filtration, desalting, decolorization, concentration and crystallization, drying.

2. The method for preparing oligofructose by enzymatic hydrolysis according to claim 1, characterized in that, The amount of lecithin added is 0.01%-0.1%.

3. The method for preparing oligofructose by enzymatic hydrolysis according to claim 1, characterized in that, The stepwise enzymatic hydrolysis is as follows: Phase A: Add 35 U / g Penicillium glucose oxidase and lecithin, and react for 1.5 h at 35℃, pH 5.5, and 120 r / min. Phase B: Add 0.3 U / g Aspergillus niger β-fructosyltransferase, 0.3 U / g Aspergillus oryzae β-fructosyltransferase, and 0.3 U / g invertase. First, react at 45℃, pH 5.0, and 120 r / min for 1.5 h, then raise the temperature to 55℃ and continue the reaction for 1.5 h. Phase C: Add 35 U / g Bacillus subtilis glucose oxidase and react for 1.5 h at 45℃, pH 7.0, and 120 r / min.

4. The method for preparing oligofructose by enzymatic hydrolysis according to claim 1, characterized in that, The pH is adjusted using citrate buffer or phosphate buffer.

5. The method for preparing oligofructose by enzymatic hydrolysis according to claim 1, characterized in that, The filtration process uses a 0.45μm filter membrane to remove insoluble impurities.

6. The method for preparing oligofructose by enzymatic hydrolysis according to claim 1, characterized in that, The desalination process involves removing inorganic ions using a strong acidic cation exchange resin and a strong basic anion exchange resin.

7. The method for preparing oligofructose by enzymatic hydrolysis according to claim 1, characterized in that, The decolorization process involves adding 0.5%-1.5% activated carbon, stirring at 50-60℃ for 30-60 minutes, and then filtering.

8. The method for preparing oligofructose by enzymatic hydrolysis according to claim 1, characterized in that, The concentration and crystallization steps are as follows: concentration to 60%-70% solids at 40-50℃ and vacuum degree of -0.08 to -0.09MPa, cooling to 20-25℃ and standing for crystallization for 12-24 hours, and centrifugation to obtain crystals.

9. The method for preparing oligofructose by enzymatic hydrolysis according to claim 1, characterized in that, The drying process involves vacuum drying at 40-50℃ until the moisture content is ≤5%.

10. The application of the fructooligosaccharide prepared according to claim 1 in special medical foods.

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