Functional oligosaccharide syrup prepared from crystalline glucose mother liquor

Abstract

The invention discloses functional oligosaccharide syrup prepared from crystalline glucose mother liquor, and belongs to the field of comprehensive utilization of crystalline glucose mother liquor. According to the invention, crystalline glucose mother liquor is used as a substrate, and glucosidase catalysis is combined with saccharomycetes fermentation treatment to prepare the oligosaccharide syrup mainly comprising gentiooligosaccharide and maltooligosaccharide. An in-vitro fermentation experiment proves that the content of short-chain fatty acid in a product can be remarkably increased, proliferation of intestinal probiotics such as bifidobacteria and lactic acid bacteria is promoted, and the probiotics have the potential of serving as novel prebiotics. The product added value is high, the preparation technology is simple, industrial application is convenient to achieve, and a novel green, low-carbon and good-economic-benefit means is provided for comprehensive utilization of the crystalline glucose mother liquor.

Description

Technical Field

[0001] The invention relates to functional oligosaccharide syrup prepared by utilizing crystallized glucose mother liquor, and belongs to the field of comprehensive utilization of crystallized glucose mother liquor. Background Art

[0002] Crystallized glucose mother liquor is a by-product in the process of producing crystallized glucose. It is mainly the remaining part after the crystals are centrifuged from the crystal slurry. It can be divided into primary mother liquor, secondary mother liquor, tertiary mother liquor, etc. according to the number of separations. Globally, more than 20 million tons of crystallized glucose are produced each year, accompanied by at least 4 million tons of mother liquor, and the output is very huge. However, the mother liquor has a high moisture content and a short storage period. If it is not used in time, it is easy to corrupt and cause new pollution, resulting in increased production costs. How to achieve the rational utilization of mother liquor resources has become a problem that plagues the development of sugar-making enterprises. The added value of existing utilization methods is low, such as direct external sales, synthetic pigments, hydrogenation to prepare sugar alcohols, fermentation to produce organic acids, etc., and the components of glucose mother liquor have not been fully explored to achieve its high-value utilization. In fact, in addition to high-concentration glucose, the mother liquor also contains a large amount of oligosaccharide components, which has a high development potential. Therefore, this patent prepares an oligosaccharide syrup with intestinal probiotic effects based on crystallized glucose mother liquor, providing new ideas for the utilization of mother liquor resources.

[0003] Functional oligosaccharides are a type of low-polymer sugars formed by 2 to 7 monosaccharide molecules connected by α-, β- and other glycosidic bonds. They are usually not digested and absorbed by the human body, but can directly enter the large intestine and thus play a certain role in intestinal probiotics. Among them, oligosaccharides connected by α-1,4 and α-1,6 glycosidic bonds are maltooligosaccharides, including linear and branched structures, such as isomaltose, maltotriose, isomaltotriose, panose, maltotetraose, etc. Oligosaccharides connected by β-1,6 glycosidic bonds are gentiooligosaccharides, including gentiobiose, gentiotriose, gentiotetraose, etc. They have a characteristic soft bitter taste and can play a refreshing role. At the same time, they have unique physiological effects, which help to balance the intestinal environment and promote the reproduction of intestinal probiotics. They are high-value-added sugar products with great application prospects. Gentiano oligosaccharides are usually prepared by enzymatic method, that is, using β-glucosidase to catalyze the transglycosidation reaction on a high-concentration glucose substrate to obtain it. However, due to the low conversion rate and high cost, industrial production has not yet been achieved in China.

[0004] Therefore, there is an urgent need for a method that can increase the added value of crystallized glucose mother liquor and efficiently produce a functional syrup mainly composed of gentioose. Summary of the invention

[0005] In order to achieve high added value utilization of crystallized glucose mother liquor resources, the present invention develops a functional oligosaccharide syrup prepared from crystallized glucose mother liquor, and greatly improves the application value of the crystallized glucose mother liquor through a method of glucosidase catalysis combined with yeast fermentation.

[0006] The invention provides a functional oligosaccharide syrup prepared by using crystallized glucose mother liquor. The functional oligosaccharide syrup contains 50% to 70% gentiobiose, 15% to 30% panose, 5% to 15% isomaltose, 1% to 5% maltotetraose, 1% to 5% glucose, 0.5% to 5% isomaltotriose, 0.2% to 5% gentiotriose, 0.1% to 5% maltose and 0.1% to 5% maltotriose in mass fraction.

[0007] The present invention also provides products containing the functional oligosaccharide syrup, and the products include food, medicine or health care products.

[0008] The present invention also provides a method for preparing the functional oligosaccharide syrup, comprising the following steps:

[0009] (1) diluting the crystallized glucose mother solution to an appropriate concentration, then adjusting the pH and preheating to the reaction temperature;

[0010] (2) adding glucosidase to the pretreated crystallized glucose mother liquor to react and inactivate the enzyme to obtain product 1;

[0011] (3) Product 1 is diluted to an appropriate concentration, yeast is added for fermentation, and then the supernatant is obtained by centrifugation. The supernatant is then passed through a membrane and concentrated to obtain an oligosaccharide syrup mainly composed of gentiooligosaccharides and maltose oligosaccharides.

[0012] In one embodiment of the present invention, the crystallized glucose mother liquor described in step (1) includes but is not limited to primary mother liquor, secondary mother liquor, tertiary mother liquor and chromatographic separation tail liquor.

[0013] In one embodiment of the present invention, in step (1), the pH is adjusted to 4.0-6.0, and the reaction temperature ranges from 40-60°C.

[0014] In one embodiment of the present invention, the glucosidase in step (2) includes but is not limited to α-glucosidase and β-glucosidase.

[0015] In one embodiment of the present invention, the glucosidase in step (2) is a homemade enzyme or a commercially available enzyme, the addition amount is 10 to 100 U / g dry mother liquid, and the reaction time is 24 to 72 hours.

[0016] In one embodiment of the present invention, in step (3), the product 1 is diluted to a mass fraction of 10% to 30%.

[0017] In one embodiment of the present invention, the yeast described in step (3) includes but is not limited to baker's yeast, high-sugar-tolerant yeast, Ayr yeast or Pichia pastoris.

[0018] In one embodiment of the present invention, the amount of yeast added in step (3) is 1% to 10% (w / w) of the solid content in product 1, the fermentation temperature is 25 to 37° C., and the fermentation time is at least 8 hours, preferably 8 to 24 hours.

[0019] The invention also provides application of the method in treating crystallized glucose mother liquor.

[0020] The present invention also provides the application of the functional oligosaccharide syrup or the method in the preparation of food, medicine or health care product.

[0021] Beneficial Effects

[0022] The present invention uses crystallized glucose mother liquor as a substrate to prepare functional oligosaccharide syrup, and the obtained functional oligosaccharide syrup contains 50% to 70% gentiobiose, 15% to 30% panose and other oligosaccharides, and the glucose content is almost exhausted; the functional oligosaccharides can significantly promote the production of short-chain fatty acids, and increase the relative abundance of probiotics such as bifidobacteria and lactic acid bacteria in intestinal flora, have the potential to be used as prebiotics, and play a role in alleviating intestinal inflammation and maintaining intestinal homeostasis.

[0023] The present invention provides a simple, efficient and safe method for preparing functional oligosaccharide syrup. The product can be obtained through enzymatic reaction, yeast fermentation, separation, concentration and other operations. Compared with other utilization methods of crystalline glucose mother liquor, the present method requires less production equipment and has high product added value, realizes full utilization of mother liquor components, provides a new green, low-carbon and environmentally friendly means for the comprehensive utilization of crystalline glucose mother liquor, and has the potential to realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : High performance anion exchange chromatograms of secondary mother liquor and functional oligosaccharide syrup prepared with it as substrate.

[0025] Figure 2 : Content of short-chain fatty acids (a. acetic acid; b. propionic acid; c. butyric acid; d. total acid) in the products of different samples (control, secondary mother liquor, product 1, functional oligosaccharide syrup, isomaltooligosaccharide, gentiobiose) after 48 h of in vitro fermentation.

[0026] Figure 3 : Relative abundance of bifidobacteria and lactic acid bacteria in the products of different samples (control, secondary mother liquor, product 1, functional oligosaccharide syrup, isomaltooligosaccharide, gentiobiose) after 48h of in vitro fermentation. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0028] The materials involved in the following embodiments are as follows:

[0029] Crystallized glucose secondary mother liquor was obtained from Heilongjiang Jinxiang Biochemical Co., Ltd.; glucosidase (catalog number: Aromase H2, 1000U / g) was purchased from Amano Enzyme Preparation (Jiangsu) Co., Ltd.; high-activity dry yeast was purchased from Angel Yeast Co., Ltd.

[0030] The detection methods involved in the following embodiments are as follows:

[0031] (1) Glucose mother solution and reaction product analysis method

[0032] Take the sample diluted to an appropriate multiple and centrifuge it, then take the supernatant and filter it through a 0.22um water filter membrane, and use a high-performance anion exchange chromatograph equipped with a pulsed amperometric detector to analyze the sugar components of the sample. The analysis conditions are: Thermo Fisher Scientific ICC-5000, chromatographic column CarboPac PA 200, gradient elution with 100mM NaOH solution and 500mM NaAc solution, flow rate 0.5mL / min, column temperature 35℃, injection volume 10μL, and external standard method quantification.

[0033] (2) Calculation method of product ratio

[0034] The content of a certain sugar = the peak area of ​​the sugar / the peak area of ​​the corresponding standard * the concentration of the standard;

[0035] The relative content of a certain sugar = the content of that sugar / the sum of the contents of all sugars in the sample.

[0036] (3) In vitro fermentation method

[0037] First, carbonate-phosphate fermentation broth was prepared as the basic culture medium for intestinal microorganisms, and then different sugar samples were dissolved in the basic culture medium at a ratio of 1:100 (w / v). After the above solution was sterilized, it was placed in an anaerobic workstation (AW400SG, Electrotek, UK) for deoxygenation overnight. Fresh fecal samples from 4 healthy volunteers (aged 24 to 28 years old, two men and two women) were collected and immediately inoculated into the culture medium at a ratio of 1:9 (v / v) after homogenization. After the sample bottle was sealed, it was placed in a 37°C shaker for anaerobic fermentation. After 24 hours, it was taken out and centrifuged at 4°C and 10,000g for 15 minutes. The supernatant was separated for the analysis of short-chain fatty acid content, and the bacterial precipitate was used to analyze the diversity of intestinal microorganisms.

[0038] (4) Determination of short-chain fatty acids

[0039] Take 0.5mL of fermentation broth and mix it with 20uL of 10% H2SO4 solution, add 1mL of anhydrous ether, shake for 60s and centrifuge, collect the supernatant and add 0.25g of Na2SO4 to remove residual water, collect the supernatant again after centrifugation. The gas chromatograph measurement conditions are: DB-WAX column, hydrogen ion flame detector, injection volume 1uL, carrier gas N2 flow rate 3mL / min; fuel gas H2 flow rate 44mL / min, combustion air flow rate 400mL / min.

[0040] Example 1

[0041] (1) Take 100 g of crystallized glucose secondary mother liquor as the reaction substrate, dilute to a solid content of 50% (w / w), adjust the pH to 4.0, and preheat to 50° C.;

[0042] (2) adding glucosidase at an enzyme dosage of 10 U / g dry mother liquor, reacting at 50° C. for 24 h, and then inactivating the enzyme to obtain product 1;

[0043] (3) Product 1 was diluted to a mass concentration of 20%, and yeast powder was added at 5% of the solid content, and fermented at 25°C for 24 hours, and then the yeast cells were removed by centrifugation. The supernatant was passed through a 0.45um aqueous membrane and concentrated to a solid content of 30% (w / w) to obtain an oligosaccharide syrup. The results of ion chromatography showed that the functional oligosaccharide content in the oligosaccharide syrup could reach more than 90%, including isomaltose, gentiobiose, maltotriose, isomaltotriose, gentiotriose, panose, and maltotetraose, as shown in Table 1.

[0044] Table 1 Sugar composition and relative content of the substrate and reaction product used in Example 1 (unit: %)

[0045]

[0046] Example 2

[0047] The reaction substrate type was adjusted, and the crystallized glucose secondary mother liquor in step (1) of Example 1 was replaced with the primary mother liquor, the tertiary mother liquor or the chromatographic separation tail liquor, while the other steps remained unchanged.

[0048] Example 3

[0049] The reaction pH was adjusted by replacing the pH adjustment to 4.0 in step (1) of Example 1 with 4.5, 5.0, 5.5, and 6.0, while the other steps remained unchanged.

[0050] Example 4

[0051] The reaction preheating temperature was adjusted by replacing the preheating to 50°C in step (1) of Example 1 and the reaction temperature in step (2) with 40°C, 45°C, 55°C, and 60°C, while the other steps remained unchanged.

[0052] Example 5

[0053] The amount of enzyme added in the reaction was adjusted, and the amount of glucosidase added in step (2) of Example 1 at 10 U / g dry mother liquor was replaced with 25 U / g, 50 U / g, 75 U / g, and 100 U / g, while the other steps remained unchanged.

[0054] Example 6

[0055] The reaction time was adjusted by replacing the reaction time of 24 h in step (2) of Example 1 with the reaction time of 36 h, 48 h, 60 h, or 72 h, while the other steps remained unchanged.

[0056] Example 7

[0057] The dilution concentration of the reaction product was adjusted by replacing the diluted product 1 in step (3) of Example 1 to a mass concentration of 20% with 10%, 15%, 25%, and 30%, while the other steps remained unchanged.

[0058] Example 8

[0059] The amount of yeast added was adjusted by replacing the 5% of yeast powder added at solid content in step (3) of Example 1 with 1%, 2%, or 10%, while the other steps remained unchanged.

[0060] Example 9

[0061] The fermentation temperature of the yeast was adjusted, and the fermentation at 25°C in step (3) of Example 1 was replaced with 28°C, 30°C, or 37°C, while the other steps remained unchanged.

[0062] Example 10

[0063] The fermentation time of yeast was adjusted, and the fermentation time of 24 h in step (3) of Example 1 was replaced by 8 h, 12 h, and 16 h, while the other steps remained unchanged.

[0064] After testing, the components and mass fractions contained in the functional oligosaccharide syrups obtained in Examples 2 to 10 are 50% to 70% gentiobiose, 15% to 30% panose, 5% to 15% isomaltose, 1% to 5% maltotetraose, 1% to 5% glucose, 0.5% to 5% isomaltotriose, 0.2% to 5% gentiotriose, 0.1% to 5% maltose and 0.1% to 5% maltotriose, which can achieve the same probiotic effect as the product in Example 1. If the preparation method exceeds the above range, for example, the amount of glucosidase added is less than 10U / g dry mother liquor, the reaction time is less than 24h, or the yeast fermentation temperature is less than 25°C, and the fermentation time is less than 8h, so that the mass fraction of gentiobiose in the obtained product is less than 50%, or the mass fractions of glucose and maltose exceed 10%, it may result in the obtained oligosaccharide syrup failing to achieve the same probiotic effect as the product in Example 1.

[0065] Comparative Example 1

[0066] The functional oligosaccharide syrup obtained in Example 1 was used for in vitro fermentation, and the content of total short-chain fatty acids in the fermentation product was determined by gas chromatography, and compared with the control (basal culture medium without additional sugar sample), the secondary mother liquor, the product 1, and the commercially available isomaltooligosaccharide (IMO-90 type, purchased from Baolingbao Biotechnology Co., Ltd.). The results are as follows: Figure 2 As shown. Short-chain fatty acids are important products of intestinal microbial metabolism in vivo, mainly including acetic acid, propionic acid and butyric acid, and have significant physiological regulation functions, such as providing energy for intestinal epithelial cells, protecting intestinal mucosa, inhibiting intestinal inflammation, etc. The total acid concentration shown in this embodiment is the sum of acetic acid, propionic acid and butyric acid contents. It can be seen that the functional oligosaccharide syrup prepared by the present invention has significantly better acetic acid, propionic acid and total acid concentrations than other groups after 48h of in vitro fermentation. Compared with the control, untreated secondary mother liquor, product 1 and commercially available oligomaltose, the acid production of the functional oligosaccharide syrup prepared by the present invention is significantly increased, indicating that it can promote the fermentation of intestinal flora to produce short-chain fatty acids, and play an intestinal prebiotic role.

[0067] Comparative Example 2

[0068] The functional oligosaccharide syrup obtained in Example 1 was used for in vitro fermentation, and the microbial diversity in the fermentation product was further analyzed and compared with the control (basal medium without additional sugar sample), the secondary mother liquor, the product 1, and the commercially available isomaltooligosaccharide (same as comparative example 1). The results are as follows: Figure 3As shown. Bifidobacterium and lactobacillus are important core flora and beneficial flora in the human intestine. The former helps to improve blood sugar control, reduce blood lipid levels, and improve immunity. The latter has the effects of regulating the balance of intestinal microbial communities, inhibiting the production of inflammatory factors, and improving digestive function. Therefore, its abundance is often used to evaluate the physiological indicators of the efficacy of prebiotic products. It can be seen that the relative abundance of bifidobacteria and lactobacillus has significant changes in different samples, wherein the content in the control group is very small, and the content in the secondary mother liquor group has a certain increase. And in the functional oligosaccharide syrup prepared by the present invention, the relative abundance of two probiotics is significantly increased, indicating that this product has the ability to promote the proliferation of probiotics such as bifidobacteria and lactobacillus. Compared with the effect of functional oligosaccharide-isomaltooligosaccharide, which is common in the current market, it can only promote the proliferation of bifidobacteria and has no effect on lactic acid bacteria, and the proliferation ability of bifidobacteria is weaker than the oligosaccharide syrup prepared by the present invention. The above results all indicate that the functional oligosaccharide syrup developed by this patent has certain intestinal prebiotic effects and has the potential to be used as a new type of prebiotic in food, medicine or health care products.

[0069] Comparative Example 3

[0070] Using 50% glucose solution (w / w) as the reaction substrate, oligogentiosaccharides were prepared using the same process as in Example 1, and the composition of the obtained products is shown in Table 2. The product obtained after adding glucosidase for 24 hours and inactivating the enzyme is defined as product 2, and the product obtained after further yeast fermentation is defined as product 3. It can be seen that compared with the use of crystallized glucose secondary mother liquor as a substrate, the use of pure glucose solution as a substrate will reduce the conversion rate of glucosidase treatment, wherein the relative content of gentiobiose decreased by 22.27%, and no gentiotriose was produced. This shows that other sugar components in the crystallized glucose mother liquor help to weaken the inhibitory effect of glucose on glucosidase, thereby promoting the formation of oligogentiosaccharides. Product 2 was further subjected to yeast fermentation to obtain product 3, in which the relative content of gentiobiose exceeded 99%. The same in vitro fermentation experiment was carried out, and the results are as follows Figure 2 and Figure 3 As shown in the "gentiobiose" in Figure 2 It can be seen that gentiobiose has a better effect on promoting the production of short-chain fatty acids such as acetate, propionate and butyrate than the secondary mother liquor, and is equal to the effect of oligosaccharide isomaltooligosaccharide, but significantly weaker than the functional oligosaccharide syrup. This shows that gentiobiose, gentiotriose and oligosaccharide in the functional oligosaccharide syrup can play a certain synergistic role and help improve the intestinal probiotic effect of the product. Figure 3It can be seen that although gentiobiose can increase the relative abundance of probiotics such as bifidobacteria and lactic acid bacteria in the intestinal flora, the effect is also weaker than that of the functional oligosaccharide syrup prepared using the mother liquor as the substrate. Therefore, the functional oligosaccharide syrup prepared by this patent has better intestinal probiotic effects than oligosaccharides and gentiobiose, and has a lower production cost, and has great potential for market application in the future.

[0071] Table 2 Sugar composition and relative content of the substrate and reaction product used in Comparative Example 3 (unit: %)

[0072]

[0073] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A functional oligosaccharide syrup, characterized in that: The functional oligosaccharide syrup contains 50% to 70% gentiobiose, 15% to 30% panose, 5% to 15% isomaltose, 1% to 5% maltotetraose, 1% to 5% glucose, 0.5% to 5% isomaltotriose, 0.2% to 5% gentiotriose, 0.1% to 5% maltose and 0.1% to 5% maltotriose in mass fractions.

2. A product containing the functional oligosaccharide syrup according to claim 1, characterized in that: The products include food, medicine or health products.

3. The method for preparing the functional oligosaccharide syrup according to claim 1, characterized in that: The following steps are involved: (1) diluting the crystallized glucose mother solution, adjusting the pH, and preheating to the reaction temperature; (2) adding glucosidase to the crystallized glucose mother liquor treated in step (1) to react and inactivate the enzyme to obtain product 1; (3) After diluting the product 1, yeast is added for fermentation, and then the supernatant is obtained by centrifugation, and then the supernatant is passed through a membrane and concentrated to obtain the functional oligosaccharide syrup.

4. The method according to claim 3, characterized in that: In step (1), the pH is adjusted to 4.0-6.0 and preheated to 40-60°C.

5. The method according to claim 4, characterized in that In step (2), 10 to 100 U / g of dry mother solution of glucosidase is added and the reaction is carried out for 24 to 72 hours; the glucosidase includes but is not limited to α-glucosidase and β-glucosidase.

6. The method according to claim 5, characterized in that In step (3), the product 1 is diluted to a mass fraction of 10% to 30%.

7. The method according to claim 6, characterized in that In step (3), the amount of yeast added is 1% to 10% w / w of the solid content in product 1, and the fermentation is carried out at 25 to 37° C. for at least 8 hours.

8. The method according to claim 7, characterized in that The yeast includes but is not limited to baker's yeast, high sugar tolerant yeast, ale yeast or Pichia pastoris; the crystallized glucose mother liquor includes but is not limited to primary mother liquor, secondary mother liquor, tertiary mother liquor or chromatographic separation tail liquor.

9. Use of the method according to any one of claims 3 to 8 in treating crystallized glucose mother liquor.

10. Use of the functional oligosaccharide syrup according to claim 1 or the method according to any one of claims 3 to 8 in the preparation of food, medicine or health care products.

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