Functional oligosaccharide syrup prepared using crystalline glucose mother liquor

Abstract

This invention discloses a functional oligosaccharide syrup prepared using crystalline glucose mother liquor, belonging to the field of comprehensive utilization of crystalline glucose mother liquor. Using crystalline glucose mother liquor as a substrate, this invention prepares an oligosaccharide syrup mainly composed of gentiosaccharides and maltodextrins through a combination of glucosidase catalysis and yeast fermentation. In vitro fermentation experiments have demonstrated that it can significantly increase the content of short-chain fatty acids in the product and promote the proliferation of beneficial intestinal bacteria such as Bifidobacteria and Lactobacillus, showing potential as a novel prebiotic. The product of this invention has high added value, and the preparation process is simple, facilitating industrial application. It provides a green, low-carbon, and economically beneficial new method for the comprehensive utilization of crystalline glucose mother liquor.

Description

Technical Field

[0001] This invention relates to a functional oligosaccharide syrup prepared using crystalline glucose mother liquor, belonging to the field of comprehensive utilization of crystalline glucose mother liquor. Background Technology

[0002] Crystallized glucose mother liquor is a byproduct of the crystallization process, mainly the residue remaining after centrifugation to separate the crystals from the slurry. It can be categorized as primary, secondary, and tertiary mother liquor based on the number of separation stages. Globally, over 20 million tons of crystalline glucose are produced annually, along with at least 4 million tons of mother liquor, representing a massive output. However, the mother liquor has a high water content and short shelf life; if not utilized promptly, it is prone to spoilage and new contamination, increasing production costs. How to rationally utilize this mother liquor resource has become a major challenge for sugar companies. Existing utilization methods result in low added value, such as direct sale, synthetic pigments, hydrogenation to produce sugar alcohols, and fermentation to produce organic acids, failing to fully explore the components of the glucose mother liquor for high-value utilization. In fact, besides high-concentration glucose, the mother liquor also contains a large amount of oligosaccharides, possessing significant development potential. Therefore, this patent proposes a new approach to utilizing mother liquor resources by preparing an oligosaccharide syrup with intestinal probiotic effects based on crystalline glucose mother liquor.

[0003] Functional oligosaccharides are a class of low-degree polymeric sugars formed by 2 to 7 monosaccharide molecules linked by α, β, or other glycosidic bonds. They are typically not digested and absorbed by the human body, but can directly enter the large intestine, thus playing a certain role in intestinal health. Oligosaccharides linked by α-1,4 and α-1,6 glycosidic bonds are called maltodextrose, including linear and branched structures, such as isomaltose, maltotriose, isomalttriose, panose, and maltotetraose. Oligosaccharides linked by β-1,6 glycosidic bonds are called gentiosaccharides, including gentiobiose, gentiotriose, and gentiotetraose. They exhibit a characteristic mild bitterness, which can have a refreshing and invigorating effect, and possess unique physiological benefits, helping to balance the intestinal environment and promote the growth of beneficial intestinal bacteria. They are high-value-added sugar products with great application potential. Oligosaccharides are usually prepared by enzymatic methods, which utilize β-glucosidase to catalyze a transglycosylation reaction on a high concentration of glucose substrate. However, due to the low conversion rate, the cost is high, and 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 crystalline glucose mother liquor and efficiently produce functional syrups based on gentianose. Summary of the Invention

[0005] To achieve high-value utilization of crystalline glucose mother liquor resources, this invention develops a functional oligosaccharide syrup prepared from crystalline glucose mother liquor. By using a method combining glucosidase catalysis and yeast fermentation, the application value of crystalline glucose mother liquor is significantly improved.

[0006] This invention provides a functional oligosaccharide syrup prepared using crystalline glucose mother liquor, wherein the functional oligosaccharide syrup contains, by mass fraction, 50%–70% gentiobiose, 15%–30% panose, 5%–15% isomaltose, 1%–5% maltotetraose, 1%–5% glucose, 0.5%–5% isomalttriose, 0.2%–5% gentiotriose, 0.1%–5% maltose, and 0.1%–5% maltotriose.

[0007] The present invention also provides products containing the aforementioned functional oligosaccharide syrup, the products including food, pharmaceuticals or health products.

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

[0009] (1) Dilute the crystalline glucose mother liquor to a suitable concentration, then adjust the pH and preheat to the reaction temperature;

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

[0011] (3) Dilute product 1 to a suitable concentration, add yeast for fermentation, then centrifuge to obtain the supernatant, and then pass it through a membrane and concentrate to obtain an oligosaccharide syrup mainly composed of gentiosaccharide and maltodextrin.

[0012] In one embodiment of the present invention, the crystalline glucose mother liquor mentioned 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 to 6.0, and the reaction temperature range is 40 to 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 self-made enzyme or a commercially available enzyme, the amount added is 10-100 U / g dry base mother liquor, and the reaction time is 24-72 h.

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

[0017] In one embodiment of the present invention, the yeast in step (3) includes, but is not limited to, baker's yeast, high-sugar tolerant yeast, Ayers 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 present invention also provides the application of the method in the treatment of crystallized glucose mother liquor.

[0020] The present invention also provides the application of the functional oligosaccharide syrup or the method described herein in the preparation of food, pharmaceuticals or health products.

[0021] Beneficial effects

[0022] This invention utilizes crystalline glucose mother liquor as a substrate to prepare functional oligosaccharide syrup. The resulting functional oligosaccharide syrup contains 50%–70% gentiobiose, 15%–30% panose, and other oligosaccharides, with glucose content almost depleted. The functional oligosaccharides can significantly promote the production of short-chain fatty acids and increase the relative abundance of probiotics such as Bifidobacteria and Lactobacillus in the intestinal flora, demonstrating their potential as prebiotics and playing a role in alleviating intestinal inflammation and maintaining intestinal homeostasis.

[0023] This invention provides a simple, efficient, and safe method for preparing functional oligosaccharide syrup. The product can be obtained through enzymatic reaction, yeast fermentation, separation, and concentration. Compared with other methods of utilizing crystalline glucose mother liquor, this method requires less production equipment and produces a high-value-added product. It achieves full utilization of the mother liquor components and provides a green, low-carbon, and environmentally friendly new means for the comprehensive utilization of crystalline glucose mother liquor, with the potential to achieve industrial production. Attached Figure Description

[0024] Figure 1 High-performance anion exchange chromatogram of the secondary mother liquor and the functional oligosaccharide syrup prepared using it as a substrate.

[0025] Figure 2 The 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 in vitro fermentation for 48 h.

[0026] Figure 3 The relative abundance of Bifidobacteria and Lactobacillus in the products of different samples (control, secondary mother liquor, product 1, functional oligosaccharide syrup, isomaltooligosaccharide, gentiobiose) after 48 h of in vitro fermentation. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, 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] The secondary mother liquor of crystalline glucose was obtained from Heilongjiang Jinxiang Biochemical Co., Ltd.; glucosidase (product number: Aromase H2, 1000U / g) was purchased from Amano Enzyme Preparations (Jiangsu) Co., Ltd.; and highly active dry yeast was purchased from Angel Yeast Co., Ltd.

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

[0031] (1) Analytical methods for glucose mother liquor and reaction products

[0032] Samples diluted to an appropriate ratio were centrifuged, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. The carbohydrate components of the samples were analyzed using a high-performance anion exchange chromatography system equipped with a pulsed amperometric detector. The analytical conditions were: Thermo Fisher Scientific ICC-5000, CarboPac PA 200 column, gradient elution with 100 mM NaOH and 500 mM NaAc solutions, flow rate 0.5 mL / min, column temperature 35 °C, injection volume 10 μL, and external standard method for quantification.

[0033] (2) Calculation method of product proportion

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

[0035] The relative content of a certain sugar is equal to the content of that sugar divided by the sum of the contents of all sugars in the sample.

[0036] (3) In vitro fermentation method

[0037] First, a carbonate-phosphate fermentation broth was prepared as the basal culture medium for intestinal microorganisms. Then, different sugar samples were dissolved in the basal medium at a ratio of 1:100 (w / v). After sterilization, the solution was placed in an anaerobic workstation (AW400SG, Electrotek, UK) overnight for deoxygenation. Fresh fecal samples were collected from four healthy volunteers (aged 24–28 years, two males and two females), homogenized, and immediately inoculated into the culture medium at a ratio of 1:9 (v / v). The sample bottles were sealed and placed in a shaker at 37°C for anaerobic fermentation. After 24 hours, the samples were removed and centrifuged at 4°C and 10,000g for 15 minutes. The supernatant was used to analyze the short-chain fatty acid content, and the bacterial precipitate was used to analyze the diversity of intestinal microorganisms.

[0038] (4) Methods for determining short-chain fatty acids

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

[0040] Example 1

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

[0042] (2) Add glucosidase at an enzyme dosage of 10 U / g dry base mother liquor, react at 50℃ for 24 h, and then inactivate 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. Fermentation was carried out at 25°C for 24 hours, and then the yeast cells were removed by centrifugation. The supernatant was passed through a 0.45 μm aqueous membrane and concentrated to a solid content of 30% (w / w) to obtain an oligosaccharide syrup. Ion chromatography analysis showed that the content of functional oligosaccharides in the oligosaccharide syrup could reach more than 90%, including isomaltose, gentiobiose, maltotriose, isomalttriose, gentiotriose, panose, and maltotetraose, as shown in Table 1.

[0044] Table 1. Sugar composition and relative content (%) of the substrates and reaction products used in Example 1.

[0045]

[0046] Example 2

[0047] Adjust the reaction substrate type by replacing the secondary mother liquor of crystalline glucose in step (1) of Example 1 with the primary mother liquor, tertiary mother liquor or chromatographic separation tail liquor, while keeping other steps unchanged.

[0048] Example 3

[0049] Adjust the reaction pH by replacing the pH adjustment to 4.0 in step (1) of Example 1 with 4.5, 5.0, 5.5, or 6.0, while keeping the other steps unchanged.

[0050] Example 4

[0051] Adjust the reaction preheating temperature, replacing the preheating temperature of 50°C in step (1) and the reaction temperature in step (2) of Example 1 with 40°C, 45°C, 55°C, and 60°C, respectively, while keeping other steps unchanged.

[0052] Example 5

[0053] Adjust the amount of enzyme added to the reaction. Replace the amount of glucosidase added in step (2) of Example 1, which was 10 U / g of dry base mother liquor, with 25 U / g, 50 U / g, 75 U / g, and 100 U / g, while keeping the other steps unchanged.

[0054] Example 6

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

[0056] Example 7

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

[0058] Example 8

[0059] Adjust the amount of yeast added, replacing the addition of yeast powder at 5% of solid content in step (3) of Example 1 with 1%, 2%, and 10%, while keeping other steps unchanged.

[0060] Example 9

[0061] Adjust the fermentation temperature of the yeast, replacing the fermentation at 25°C in step (3) of Example 1 with 28°C, 30°C, and 37°C, while keeping the other steps unchanged.

[0062] Example 10

[0063] Adjust the fermentation time of the yeast, replacing the 24h fermentation in step (3) of Example 1 with 8h, 12h, or 16h, while keeping the other steps unchanged.

[0064] Testing revealed that the functional oligosaccharide syrups obtained in Examples 2-10 contained 50%-70% gentiobiose, 15%-30% panose, 5%-15% isomaltose, 1%-5% maltotetraose, 1%-5% glucose, 0.5%-5% isomalttriose, 0.2%-5% gentiotriose, 0.1%-5% maltose, and 0.1%-5% maltotriose, achieving the same probiotic effect as the product in Example 1. If the preparation method deviates from the above range, for example, if the amount of glucosidase added is less than 10 U / g dry base mother liquor, the reaction time is less than 24 h, or the yeast fermentation temperature is less than 25°C and the fermentation time is less than 8 h, resulting in a gentiobiose mass fraction of less than 50% or a glucose and maltose mass fraction exceeding 10%, the resulting oligosaccharide syrup may not 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. The total short-chain fatty acid content in the fermentation product was determined by gas chromatography and compared with the control (basal culture medium without additional sugar samples), secondary mother liquor, product 1, and 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 metabolic products of intestinal microorganisms, mainly including acetic acid, propionic acid, and butyric acid. They have significant physiological regulatory functions, such as providing energy for intestinal epithelial cells, protecting the intestinal mucosa, and inhibiting intestinal inflammation. The total acid concentration shown in this example is the sum of the contents of acetic acid, propionic acid, and butyric acid. It can be seen that the functional oligosaccharide syrup prepared in this invention exhibits significantly higher concentrations of acetic acid, propionic acid, and total acid after 48 hours of in vitro fermentation compared to other groups. Compared with the control, untreated secondary mother liquor, product 1, and commercially available isomaltooligosaccharide, the functional oligosaccharide syrup prepared in this invention shows a significant increase in acid production, indicating that it can promote the fermentation of intestinal flora to produce short-chain fatty acids, thus playing a beneficial role in intestinal health.

[0067] Comparative Example 2

[0068] The functional oligosaccharide syrup obtained in Example 1 was used for in vitro fermentation. The microbial diversity of its fermentation products was further analyzed and compared with the control (basal culture medium without additional sugar samples), secondary mother liquor, product 1, and commercially available isomaltooligosaccharide (same as comparative example 1). The results are as follows: Figure 3As shown in the figure, Bifidobacteria and Lactobacillus are important core and beneficial bacteria in the human gut. Bifidobacteria help improve blood sugar control, lower blood lipid levels, and enhance immunity, while Lactobacillus regulates the balance of the gut microbiota, inhibits the production of inflammatory factors, and improves digestive function. Therefore, their abundance is often used as a physiological indicator to evaluate the efficacy of prebiotic products. It can be seen that the relative abundance of Bifidobacteria and Lactobacillus varies significantly in different samples. The content is extremely low in the control group, but increases somewhat in the secondary mother liquor group. In the functional oligosaccharide syrup prepared in this invention, the relative abundance of both probiotics increases significantly, indicating that this product has the ability to promote the proliferation of probiotics such as Bifidobacteria and Lactobacillus. Compared with the effects of commonly available functional oligosaccharides on the market—isomaltooligosaccharides—it only promotes the proliferation of Bifidobacteria and has no effect on Lactobacillus, and its ability to promote the proliferation of Bifidobacteria is weaker than that of the oligosaccharide syrup prepared in this invention. The above results all indicate that the functional oligosaccharide syrup developed in this patent has certain intestinal probiotic effects and has the potential to be used as a novel prebiotic in food, pharmaceuticals, or health products.

[0069] Comparative Example 3

[0070] Oligosaccharides were prepared using a 50% glucose solution (w / w) as the substrate, following the same process as in Example 1. The composition of the obtained products is shown in Table 2. Product 2 was defined as the product obtained after adding glucosidase and reacting for 24 hours followed by enzyme inactivation, and product 3 was defined as the product obtained after further yeast fermentation. It can be seen that using pure glucose solution as the substrate, compared to using the secondary mother liquor of crystalline glucose, reduces the conversion rate of glucosidase treatment. The relative content of gentiobiose decreased by 22.27%, and no gentiotriose was generated. This indicates that other sugar components in the crystalline glucose mother liquor help to weaken the inhibitory effect of glucose on glucosidase, thereby promoting the formation of oligosaccharides. Product 3 was further obtained by yeast fermentation of product 2, in which the relative content of gentiobiose exceeded 99%. In vitro fermentation experiments were also conducted, and the results are as follows: Figure 2 and Figure 3 As shown in the image, "gentiobiose". (The text appears to be incomplete and contains several tyrosine sac Figure 2 It was found that gentiobiose promoted the formation of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid more effectively than the secondary mother liquor, and its effect was comparable to that of isomaltooligosaccharide, but significantly weaker than that of functional oligosaccharide syrup. This indicates that gentiobiose, gentiotriose, and maltodextrin in functional oligosaccharide syrup can exert a certain synergistic effect, helping to enhance the intestinal probiotic efficacy of the product. Figure 3It is known that while gentiobiose can increase the relative abundance of beneficial bacteria such as Bifidobacteria and Lactobacillus in the gut microbiota, its effect is still weaker than that of functional oligosaccharide syrups prepared using the mother liquor as a substrate. Therefore, the functional oligosaccharide syrup prepared in this patent has a better intestinal probiotic effect than isomaltooligosaccharide and gentiobiose, and its production cost is lower, showing great potential for future market applications.

[0071] Table 2 shows the sugar composition and relative content (%) of the substrates and reaction products used in Comparative Example 3.

[0072]

[0073] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing functional oligosaccharide syrup, characterized in that, Includes the following steps: (1) After diluting the mother liquor of crystalline glucose, adjust the pH and preheat to the reaction temperature; (2) Add glucosidase to the crystalline glucose mother liquor after step (1) to inactivate the enzyme and obtain product 1. (3) After diluting product 1, yeast is added for fermentation, then the supernatant is collected by centrifugation, and then the product is obtained by membrane treatment and concentration. In step (1), adjust the pH to 4.0~6.0 and preheat to 40~60℃; In step (2), 10~100 U / g of dry base mother liquor of glucosidase is added and the reaction is carried out for 24~72 h; the glucosidase includes, but is not limited to, α-glucosidase and β-glucosidase. In step (3), product 1 is diluted to a mass fraction of 10%~30%; In step (3), the amount of yeast added is 1% to 10% w / w of the solid content in product 1, and fermentation is carried out at 25 to 37°C for at least 8 hours. The functional oligosaccharide syrup contains 50%~70% gentiobiose, 15%~30% panose, 5%~15% isomaltose, 1%~5% maltotetraose, 1%~5% glucose, 0.5%~5% isomalttriose, 0.2%~5% gentiotriose, 0.1%~5% maltose, and 0.1%~5% maltotriose by mass fraction. The crystalline glucose mother liquor is a secondary mother liquor.

2. The method according to claim 1, characterized in that, The yeasts include, but are not limited to, baker's yeast, high-sugar tolerant yeast, Ayers yeast or Pichia pastoris.

3. The application of the method according to claim 1 or 2 in the treatment of crystalline glucose mother liquor.

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