Method for improving yield of oligosaccharide prepared by liquid fermentation method

Abstract

According to the method, filamentous fungi are used as fermentation strains, high-viscosity polysaccharide is used as an induction carbon source, and the oligosaccharide is prepared in a two-stage fermentation mode. Wherein the fermentation temperature of the first stage is 28-30 DEG C, and the fermentation temperature of the second stage is 30-70 DEG C. According to the method, through a two-stage fermentation mode, the problems that filamentous fungi are highly active in the liquid fermentation process, oligosaccharide can be continuously hydrolyzed into monosaccharide capable of being directly metabolized, and accumulation of oligosaccharide is not facilitated are solved. The yield of the functional oligosaccharide prepared by the method can be obviously improved to about 78%.

Description

Technical Field

[0001] The invention relates to a method for improving the yield of oligosaccharides prepared by liquid fermentation, and belongs to the technical field of microbial culture and bioenzyme hydrolysis. Background Art

[0002] In recent years, with the rise of sugar bioengineering, functional oligosaccharides have attracted much attention as functional additives in food, feed, and cosmetics due to their special biological functions. Functional oligosaccharides refer to incomplete degradation products of high-molecular-weight polysaccharides with a degree of polymerization of 2 to 20. The biological functions of functional oligosaccharides are mainly manifested in the selective proliferation of beneficial bacteria such as bifidobacteria and Lactobacillus acidophilus in the intestines of humans or animals, limiting the colonization of pathogens or conditional pathogens in the digestive tract, stimulating the body to produce immune factors, and regulating the acid-base microenvironment of the intestine. Functional oligosaccharides are often prepared by dilute acid hydrolysis, steam explosion or enzymatic hydrolysis. Previous research results have found that when high-viscosity polysaccharides are used as enzyme inducers, filamentous fungi can secrete related substrate hydrolases to hydrolyze high-viscosity polysaccharides into incomplete degradation products, oligosaccharides and monosaccharides step by step. Our research group has successfully achieved the simultaneous preparation of polysaccharide hydrolases and functional oligosaccharides by regulating liquid fermentation parameters (reference patent: ZL202210741463.8), but the yield of functional oligosaccharides is not ideal. The reason is that under normal fermentation conditions, filamentous fungi will further degrade incomplete degradation products and oligosaccharides into directly usable monosaccharides in order to maintain their normal growth and metabolism, which is not conducive to the accumulation of oligosaccharides. Therefore, if certain means can be used to limit the normal metabolism of filamentous fungi and retain the activity of hydrolases in the fermentation broth, it will help the accumulation of functional oligosaccharides and achieve efficient preparation of oligosaccharides. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a method for improving the yield of oligosaccharides prepared by liquid fermentation.

[0004] Technical solution: The method provided by the present invention for improving the yield of oligosaccharides prepared by liquid fermentation method uses filamentous fungi as fermentation strains, utilizes high-viscosity polysaccharides as induced carbon sources, and prepares oligosaccharides in a two-stage fermentation mode; wherein the fermentation temperature of the first stage is 28-30°C, and the fermentation temperature of the second stage is 30-70°C.

[0005] The first fermentation period is 0 to 48 hours, and the second fermentation period is 48 to 168 hours.

[0006] The filamentous fungus is one or more of Trichoderma reesei (T. reesei), Aspergillus niger (A. niger), and Trichoderma viride (T. viride).

[0007] The high-viscosity polysaccharide includes plant mucopolysaccharide.

[0008] The plant mucopolysaccharide includes galactomannan, glucomannan, arabinoxylan, fucose or carrageenan.

[0009] The inoculation amount of the filamentous fungus is 5%, and the spore content of the spore suspension used for inoculation is 1.0×10 7～8 cfu / mL.

[0010] The fermentation conditions also include setting the shaking speed to 170 rpm.

[0011] The fermentation medium also includes glucose, ammonium sulfate, urea, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, cobalt chloride and citric acid.

[0012] The method also includes a fermentation liquid treatment method: the fermentation liquid is centrifuged at 10,000 rpm for 5 minutes to obtain a supernatant, the supernatant is ultrafiltered, and the permeate is an arabino-oligoxylose solution.

[0013] The ultrafiltration membrane used in ultrafiltration treatment is 3000-5000Da.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The method of the present invention solves the problem that the filamentous fungi are highly active in the liquid fermentation process and will continuously hydrolyze oligosaccharides into monosaccharides that can be directly metabolized, which is not conducive to the accumulation of oligosaccharides through a two-stage fermentation mode. The yield of functional oligosaccharides prepared by this method can be significantly increased to about 78%. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Attached Figure 1 The effect of different fermentation temperatures on the yield of oligosaccharides after 72 hours of fermentation;

[0016] Attached Figure 2 The effect of different temperature change time on the yield of oligosaccharides;

[0017] Attached Figure 3 This study is about the effect of mucopolysaccharides from different sources as inducing substrates on the yield of oligosaccharides in variable temperature fermentation. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0019] Embodiment 1:

[0020] The effect of different temperature changes on the yield of arabinoxalo-oligosaccharides was investigated using plantago polysaccharide as an induction substrate, including the following steps:

[0021] (1) Plantago polysaccharide was used as the induced carbon source. Plantago polysaccharide 20.00 g, glucose 1.00 g, potassium dihydrogen phosphate 20.00 g, urea 3.00 g, magnesium sulfate 1.50 g, ammonium sulfate 14.00 g, calcium chloride 4.00 g, FeSO4·7H2O 5.00 mg, MnSO4·7H2O 1.60 mg, ZnSO4·7H2O 1.40 mg, CoCl2 2.00 mg were fully dissolved in distilled water, and 1 mol / L citric acid buffer 50 mL was added. The volume was fixed to 1000 mL with distilled water. The enzyme production medium was divided and sterilized at 121°C for 15 min. The initial viscosity of the fermentation broth was characterized by a multifunctional rheometer.

[0022] (2) The spore suspension of Trichoderma reesei (RUT-C30) (the spore number was controlled to be 1.0×10 8 cfu / mL) was mixed evenly with the enzyme production medium at an inoculation rate of 5%, and the enzyme production temperature was 28°C within 72 hours. After 72 hours, the enzyme production temperature was adjusted to 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100°C. The culture was placed in a shaker at 170 rpm for 168 hours, and the fermentation liquid was centrifuged at 10,000 rpm for 5 minutes to collect the supernatant.

[0023] (3) Transfer the supernatant to an ultrafiltration centrifuge tube with a molecular weight of 3000Da or 5000Da. Centrifuge at 5000 rpm for 60 min, measure the volume of the permeate, replenish the retentate volume to the initial volume with deionized water, centrifuge again at 5000 rpm for 60 min, and repeat the above steps three times.

[0024] (4) After the three permeates are mixed evenly, the total volume of the permeate is measured. The method for measuring the content of arabinoxooligosaccharides in the permeate is the method for measuring the content of arabinoxooligosaccharides in the permeate after the fermentation supernatant is treated with ultrafiltration. The specific operation is as follows:

[0025] The permeate was mixed with 8% dilute sulfuric acid at a volume ratio (v:v) of 1:1, and hydrolyzed at 121°C for 60 minutes to completely hydrolyze the functional oligosaccharides into monosaccharides. The concentration of monosaccharides in the dilute sulfuric acid hydrolyzate was determined by high performance liquid chromatography. The specific test conditions are as follows: the chromatograph is Agilent high performance liquid chromatograph 1260; the chromatographic column is Bio-Rad Aminex HPX-87H; the detector is a differential detector; the mobile phase is 5mmol / L sulfuric acid solution; the flow rate is 0.6mL / min; the column temperature is 55°C; the injection volume is 20.0μL. The calculation formula for the determination of oligosaccharide content is as follows:

[0026]

[0027] Where: Yield 低聚糖 is the concentration of arabinoxol in the permeate, g / L; C is the mass concentration of high-viscosity polysaccharide added to the fermentation broth, g / L; 0.05 is the total volume of the fermentation broth, L; C 单糖 is the mass concentration of monosaccharides in the retentate after acid hydrolysis, g / L; C 单糖 is the mass concentration of monosaccharides in the retentate before acid hydrolysis, g / L; V is the total volume of the retentate used for acid hydrolysis, L; 2V is the total volume of the acid hydrolyzate, L; i is the conversion coefficient between monosaccharides and oligosaccharides, 0.88 is the conversion coefficient between oligopentoses and monosaccharides, and 0.9 is the conversion coefficient between oligohexoses and monosaccharides.

[0028] The results are as follows Figure 1 As shown in the figure, after 72h of liquid fermentation with plantago polysaccharide as the induction substrate, the fermentation temperature was adjusted. The adjustment of the fermentation temperature had a significant effect on the yield of oligosaccharides. It can be seen from the results that when the fermentation temperature was adjusted to 50°C, the yield of oligosaccharides (below 3000Da and 5000Da) in the fermentation broth reached the maximum value, which was 46.02% and 78.38% respectively. If the temperature is adjusted too low or too high, although the metabolic activity of Trichoderma reesei is inhibited, the hydrolase efficiency will also be significantly reduced or the enzyme protein will be denatured and inactivated, which is not conducive to the accumulation of oligosaccharides.

[0029] Embodiment 2:

[0030] Using plantago polysaccharide as an induced carbon source, the liquid fermentation temperature was adjusted to 50°C after 24, 48, 72, 96, 120, and 144 hours, respectively, to study the effect of different temperature change times on the yield of arabinooligosaccharides, including the following steps:

[0031] (1) Weigh 20.00 g of psyllium polysaccharide, 1.00 g of glucose, 20.00 g of potassium dihydrogen phosphate, 3.00 g of urea, 1.50 g of magnesium sulfate, 14.00 g of ammonium sulfate, 4.00 g of calcium chloride, 5.00 mg of FeSO4·7H2O, 1.60 mg of MnSO4·7H2O, 1.40 mg of ZnSO4·7H2O, and 2.00 mg of CoCl2, respectively, fully dissolve them in distilled water, add 50 mL of 1 mol / L citric acid buffer, and dilute to 1000 mL with distilled water. After the enzyme production culture medium is divided into portions, sterilize it at 121°C for 15 min.

[0032] (2) A spore suspension of Aspergillus niger (CGMCC 3.3928) (the spore count was controlled to be 1.0×10 8cfu / mL) was mixed evenly with the enzyme production medium at an inoculation rate of 5%, and the initial fermentation temperature was 28°C. The enzyme production temperature was adjusted to 50°C after 24, 48, 72, 96, 120, and 144 hours, respectively. The culture was placed in a shaker at 170 rpm for 168 hours, and the fermentation liquid was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected.

[0033] (3) Other operation steps and result determination were carried out in accordance with the operation steps in Example 1.

[0034] The results are as follows Figure 2 As shown in the figure, the temperature change time has a significant effect on the yield of arabinoxylan. When the temperature is changed within 24-48 hours, the amount of arabinoxylan hydrolase secreted by Aspergillus niger at the beginning of fermentation is small, which cannot meet the needs of efficient hydrolysis of arabinoxylan. However, if the temperature change time is after 120 hours, the yield of arabinoxylan in the fermentation broth decreases due to the longer normal metabolism time of Aspergillus niger.

[0035] Embodiment 3:

[0036] Galactomannan, glucomannan, arabinoxylan, fucose and carrageenan were used as induced carbon sources, and the addition amount of induced carbon source was 20 g / L. The effects of different induced carbon sources on the yield of oligosaccharides during liquid temperature-variable fermentation were studied, including the following steps:

[0037] (1) Using Trichoderma viride CGMCC3.2942 as the enzyme-producing strain, weigh 20.00 g of inducing carbon source, 1.00 g of glucose, 20.00 g of potassium dihydrogen phosphate, 3.00 g of urea, 1.50 g of magnesium sulfate, 14.00 g of ammonium sulfate, 4.00 g of calcium chloride, 5.00 mg of FeSO4·7H2O, 1.60 mg of MnSO4·7H2O, 1.40 mg of ZnSO4·7H2O, and 2.00 mg of CoCl2, fully dissolve them in distilled water, add 50 mL of 1 mol / L citric acid buffer, and distilled water to make up to 1000 mL. After the enzyme-producing culture medium is divided into portions, sterilize it at 121°C for 15 min.

[0038] (2) The Trichoderma viride spore suspension (the spore count was controlled to be 5.0×10 7 cfu / mL) was mixed evenly with the enzyme production medium at an inoculation rate of 5%, and the initial fermentation temperature was 28°C. After 96 hours, the enzyme production temperature was adjusted to 50°C. The culture was placed in a shaker at 170 rpm for 144 hours, and the fermentation liquid was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected;

[0039] (3) Other operation steps and result determination were carried out in accordance with Example 1, wherein the molecular weight of the oligosaccharide was only 3000 Da.

[0040] The results are as follows Figure 3 As shown, this method is suitable for preparing oligosaccharides from different inducing substrates, but the accumulation of oligosaccharides is greatly affected by the chemical composition and glycosidic bonds of the inducers, among which galactomannan and arabinoxylan have better effects.

Claims

1. A method for improving the yield of oligosaccharides prepared by liquid fermentation, characterized in that: Oligosaccharides were prepared in a two-stage fermentation mode using filamentous fungi as fermentation strains and high-viscosity polysaccharides as induced carbon sources. The fermentation temperature in the first stage is 28-30°C, and that in the second stage is 30-70°C.

2. The method for improving the yield of oligosaccharides prepared by liquid fermentation according to claim 1, characterized in that: The first fermentation period is 0 to 48 hours, and the second fermentation period is 48 to 168 hours.

3. The method for improving the yield of oligosaccharides prepared by liquid fermentation according to claim 1, characterized in that: The filamentous fungus is one or more of Trichoderma reesei (T. reesei), Aspergillus niger (A. niger), and Trichoderma viride (T. viride).

4. The method for improving the yield of oligosaccharides prepared by liquid fermentation according to claim 1, characterized in that: The high-viscosity polysaccharide includes plant mucopolysaccharide.

5. The method for improving the yield of oligosaccharides prepared by liquid fermentation according to claim 4, characterized in that: The plant mucopolysaccharide includes galactomannan, glucomannan, arabinoxylan, fucose or carrageenan.

6. The method for improving the yield of oligosaccharides prepared by liquid fermentation according to claim 1, characterized in that: The inoculation amount of the filamentous fungus is 5%, and the spore content of the spore suspension used for inoculation is 1.0×10 7～8 cfu / mL.

7. The method for improving the yield of oligosaccharides prepared by liquid fermentation according to claim 1, characterized in that: The fermentation conditions also include setting the shaking speed to 170 rpm.

8. The method for improving the yield of oligosaccharides prepared by liquid fermentation according to claim 1, characterized in that: The fermentation medium also includes glucose, ammonium sulfate, urea, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, cobalt chloride and citric acid.

9. The method for improving the yield of oligosaccharides prepared by liquid fermentation according to claim 1, characterized in that: The method also includes a fermentation liquid treatment method: the fermentation liquid is centrifuged at 10,000 rpm for 5 minutes to obtain a supernatant, the supernatant is ultrafiltered, and the permeate is an arabino-oligoxylose solution.

10. The method for improving the yield of oligosaccharides prepared by liquid fermentation according to claim 9, characterized in that: The ultrafiltration membrane used in ultrafiltration treatment is 3000-5000Da.

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