Method for preparing iron complex of yeast polysaccharide

Abstract

The application discloses a preparation method of a yeast polysaccharide iron complex and belongs to the technical field of deep processing of yeast. The steps are as follows: after a yeast polysaccharide solution is oxidized by hydrogen peroxide, sodium citrate and an iron salt are added in sequence, and the yeast polysaccharide iron is obtained through a complexation reaction. The yeast polysaccharide iron prepared by the method has an iron content of greater than or equal to 10%, a water solubility of greater than or equal to 90%, good stability and no iron rust smell. The slow-release characteristics of the yeast polysaccharide iron can significantly reduce gastrointestinal irritation, realize sustained release in the stomach, and effectively improve liver iron accumulation and promote the organization utilization and storage of iron.

Description

Technical Field

[0001] This invention relates to the field of yeast deep processing technology, and in particular to a method for preparing yeast polysaccharide iron complex. Background Technology

[0002] Iron, an essential trace element for the human body, plays a vital role in maintaining the normal functioning of many life processes. Its core function lies in participating in the synthesis of hemoglobin, a key component of red blood cells responsible for transporting oxygen from the lungs to all tissues and organs. Iron deficiency hinders oxygen transport, leading to symptoms of iron-deficiency anemia such as fatigue and dizziness. In severe cases, it can impair brain oxygen supply, reducing memory and attention. Iron is also an important component of many enzymes, participating in physiological processes such as energy metabolism and immune cell activation. It is crucial for the growth and development of children and adolescents; deficiency can lead to growth retardation. For pregnant women, sufficient iron can prevent fetal hypoxia and ensure the health of both mother and child.

[0003] In our daily diet, we need to supplement our iron intake by consuming iron-rich foods such as red meat, animal liver, and spinach to meet the body's normal physiological needs. However, relying solely on food to supplement iron is sometimes insufficient, perhaps due to low absorption rates of iron from food or other factors, resulting in inadequate iron intake to meet the body's actual requirements. In such cases, additional iron supplements, such as oral or injectable iron supplements, are usually necessary to ensure adequate iron intake and effectively meet the body's iron needs to maintain health. However, common iron supplements, such as ferrous sulfate and ferrous gluconate, have some drawbacks. For example, they may irritate the gastrointestinal tract, causing discomfort such as nausea, vomiting, and diarrhea. In addition, some iron supplements have an unpleasant taste and are difficult for patients to accept.

[0004] In comparison, yeast polysaccharide iron has significant advantages. Yeast polysaccharide iron is an organic iron supplement using yeast as a carrier. As a high-quality iron supplement, it exhibits significant advantages in many aspects. Yeast polysaccharide iron uses yeast as a carrier, and yeast polysaccharides themselves are natural bioactive substances that can activate immune cells such as macrophages and T cells, enhancing immune activity. It is also gentler than chemically synthesized polysaccharides, reducing the risk of adverse reactions. Its excellent antioxidant properties also prevent iron oxidation, ensuring stable product quality. After entering the human body, the yeast polysaccharide iron complex continuously and stably releases iron ions, maintaining a stable blood concentration and ensuring reliable efficacy. In terms of bioavailability, after entering the digestive system, it releases iron ions through the action of gastric acid, and the yeast polysaccharide ligand also assists in iron absorption. Its bioavailability is far higher than that of traditional inorganic iron supplements such as ferrous sulfate, helping to improve iron deficiency anemia. More importantly, yeast polysaccharide iron has minimal gastrointestinal irritation, avoiding the nausea and vomiting caused by traditional iron supplements, thus improving patient adherence. At the same time, its immunomodulatory activity can enhance the body's immunity when supplementing iron, achieving synergistic effects, improving overall health, and broadening its application value.

[0005] However, existing technologies for preparing iron from yeast polysaccharides have significant shortcomings in terms of process, efficiency, cost, and raw material utilization. For example, some methods, such as purifying polysaccharide iron with ethanol, are cumbersome and time-consuming. The ethanol recovery process consumes a large amount of steam and condensate, increasing production costs and posing safety hazards due to the flammability of ethanol. Some methods employ ultrafiltration membrane technology, but due to the small pore size of the membrane, filtration efficiency is low in large-scale production when dealing with polysaccharides with a wide molecular weight distribution, and membrane pore blockage is prone to occur, affecting production continuity. Although the process using starch hydrolysate as raw material has a high iron loading rate, the bioavailability of the raw material itself is low, requiring absorption through the glycolysis pathway in the digestive tract. The absorption rate and efficiency are easily affected by various factors, impacting the iron supplementation effect. Other technologies require a continuous nitrogen supply during production, which significantly limits large-scale production, and the continuous supply of nitrogen further increases production costs, hindering industrial-scale promotion.

[0006] Therefore, it is still very necessary to further optimize the preparation process of yeast polysaccharide iron, improve the iron content and stability of yeast polysaccharide iron, and improve its bioavailability. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing yeast polysaccharide iron complex, in order to improve the iron content, stability and / or bioavailability of yeast polysaccharide iron.

[0008] The method for preparing yeast polysaccharide iron provided by the present invention includes: treating a yeast polysaccharide solution with hydrogen peroxide, adding sodium citrate and iron salt, and reacting to obtain a product containing yeast polysaccharide iron.

[0009] The iron salt is ferric citrate, ferric pyrophosphate, ferric ammonium citrate, ferrous gluconate, and / or ferrous succinate.

[0010] In the preparation method described in this invention, the iron salt can be either ferrous (Fe2+) or ferric (Fe3+), and can be one or more of ferric pyrophosphate, ferric citrate, ferric ammonium citrate, ferric chloride, ferrous gluconate, ferrous succinate, ferrous citrate, or ferrous fumarate. Under suitable conditions, ferric citrate, ferrous gluconate, and / or ferrous succinate exhibit better chelation effects with yeast polysaccharides compared to other iron salts, resulting in yeast polysaccharide iron containing more iron. Ferric citrate shows the best effect among these.

[0011] In the preparation method of the present invention, the solvent of the yeast polysaccharide solution is water, wherein the mass fraction of yeast polysaccharide is 5%~15% and the pH value is 6.5~7.5.

[0012] In this embodiment of the invention, the mass fraction of yeast polysaccharide is 6%~10%, or 8%~12%, or 8%~10%, or 9%~11%. In a specific embodiment, the mass fraction of yeast polysaccharide in the yeast polysaccharide solution is 10%.

[0013] In this embodiment of the invention, the pH value is 6.8 to 7.2, for example, 6.8, 6.9, 7.0, 7.1 or 7.2. Preferably, it is 7.0.

[0014] In the preparation method described in this invention, the yeast polysaccharide was pretreated before chelation in order to oxidize some of the aldehyde groups in the polysaccharide chain to carboxyl groups, thus providing more binding sites for iron ions. After optimization of the conditions, treating the yeast polysaccharide with hydrogen peroxide at a suitable concentration yielded better results. The hydrogen peroxide was a 30% (v / v) aqueous solution, and the mass of the aqueous solution was 3%–6% of the dry matter of the yeast polysaccharide solution.

[0015] In this embodiment of the invention, the mass of the hydrogen peroxide aqueous solution is 4% to 6% or 3% to 5% of the dry matter of the yeast polysaccharide solution. Preferably, the mass of hydrogen peroxide is 5% of the yeast polysaccharide solution.

[0016] In the preparation method described in this invention, the hydrogen peroxide treatment conditions were optimized. Compared with other conditions, the following conditions are more favorable for improving the chelation effect of iron and polysaccharides in the complex. The hydrogen peroxide treatment conditions include reacting at 45~55℃ for 0.5~3 h, followed by reacting at 85~95℃ for 25~35 min.

[0017] In this embodiment of the invention, the temperature is first raised to 45~55℃ before hydrogen peroxide solution is added.

[0018] In this embodiment of the invention, the conditions for hydrogen peroxide treatment include reacting at 50°C for 1.5 to 2.5 h, and then raising the temperature to 90°C and reacting for 30 min.

[0019] The preparation method of this invention optimizes the chelation conditions between iron and yeast polysaccharides. Compared with other conditions, the conditions described below are more conducive to improving the chelation effect between iron and polysaccharides. In a specific embodiment, the mass ratio of sodium citrate to yeast polysaccharides is (0.2~0.8):1. In another specific embodiment, the mass ratio of sodium citrate to yeast polysaccharides is 0.5:1.

[0020] In the preparation method of this invention, the mass ratio of the iron salt to the yeast polysaccharide is (1.5~2):1. In a specific embodiment, the mass ratio of the iron salt to the yeast polysaccharide is 1.5:1 or 2:1.

[0021] In the preparation method described in this invention, sodium citrate and iron salt are added after the temperature is lowered to 50-60°C. In a specific embodiment, the temperature is lowered to 50°C or 60°C.

[0022] The iron salt is a ferric salt, and sodium citrate and iron salt are added. If the iron salt is a ferrous salt, ascorbic acid is also added, and the mass ratio of ascorbic acid to yeast polysaccharide is (0.008~0.012):1. In a specific embodiment, the mass ratio of ascorbic acid to yeast polysaccharide is 0.01:1.

[0023] In the preparation method described in this invention, after adding sodium citrate and iron salt, the pH value is adjusted to 7.5-9.0, and the reaction is carried out for 2-8 hours. In the embodiments of this invention, the pH value is 7.5-8.5, and the reaction is carried out for 4-6 hours. In a specific embodiment, the pH value is adjusted to 8.0, and the reaction is carried out for 5 hours.

[0024] In the preparation method of this invention, the product containing yeast polysaccharide iron is separated by a 3-10 kDa microfiltration membrane. The solution obtained from membrane separation is water or an aqueous solution of ascorbic acid. In a specific embodiment, the microfiltration membrane is a 3 kDa microfiltration membrane or a 10 kDa microfiltration membrane. The ascorbic acid aqueous solution contains 0.1% ascorbic acid by mass.

[0025] In the preparation method described in this invention, the membrane separates Na into the permeate. + The mass fraction is not higher than 0.35%; the mass fraction of ascorbic acid in the aqueous solution of the ascorbic acid is 0.1%.

[0026] The yeast polysaccharide iron prepared by the method provided by this invention has good water solubility and strong stability. The iron content is greater than 10%, the water solubility is greater than 90%, and it has good stability. It has a delicate taste and light aroma, with no obvious metallic taste.

[0027] In some embodiments, the preparation method of the present invention includes: a yeast polysaccharide solution with a mass fraction of 6%~10% and a pH value of 6.8~7.2, mixed with 3%~6% of a 30% hydrogen peroxide aqueous solution at 50°C and reacted for 0.5~3 h, then heated to 90°C and reacted for 30 min, cooled to 50°C and sodium citrate and ferric salt were added to the reaction system so that the mass fraction of sodium citrate was 16.7% and the mass fraction of ferric salt was 50%, the pH value was adjusted to 7.5~9.0 and reacted for 2~8 h, and then separated by a 3~10 kDa micromembrane, the retentate being a product containing yeast polysaccharide iron.

[0028] In other embodiments, the preparation method of the present invention includes: a yeast polysaccharide solution with a mass fraction of 6%~10% and a pH value of 6.8~7.2, mixed with 3%~6% of a 30% hydrogen peroxide aqueous solution at 50°C for 0.5~3 h, then heated to 90°C and reacted for 30 min, cooled to 50°C and then sodium citrate, ascorbic acid and ferrous salt were added to the reaction system so that the mass fraction of sodium citrate was 16.7%, the mass fraction of ferrous salt was 50%, and the mass fraction of ascorbic acid was 0.1%, the pH value was adjusted to 7.5~9.0 and reacted for 2~8 h, and then separated by a 3~10 kDa micromembrane, the retentate being a product containing yeast polysaccharide iron.

[0029] In some specific embodiments, the preparation method of the present invention includes: a yeast polysaccharide solution with a mass fraction of 10% and a pH value of 7.0, mixed with 4%~5% of a 30% hydrogen peroxide aqueous solution at 50°C and reacted for 2 h, then heated to 90°C and reacted for 30 min, then cooled to 50°C~60°C and sodium citrate and ferric salt were added to the reaction system until the mass fraction of sodium citrate was 14.3% and the mass fraction of ferric salt was 57.2%, the pH value was adjusted to 8.0 and reacted for 5 h, and then separated by a 3 kDa or 10 kDa micromembrane. The retentate is a product containing yeast polysaccharide iron.

[0030] In some specific embodiments, the preparation method of the present invention includes: a yeast polysaccharide solution with a mass fraction of 10% and a pH value of 7.0, mixed with 4%~5% of a 30% hydrogen peroxide aqueous solution at 50°C and reacted for 2 h, then heated to 90°C and reacted for 30 min, cooled to 50°C~60°C and then sodium citrate, ascorbic acid and ferrous salt were added to the reaction system so that the mass fraction of sodium citrate was 14.3%, the mass fraction of ferrous salt was 57.2%, and the mass fraction of ascorbic acid was 0.1%, the pH value was adjusted to 8.0 and reacted for 5 h, and then separated by a 3 kDa or 10 kDa micromembrane, the retentate being a product containing yeast polysaccharide iron.

[0031] In the preparation method described in this invention, the retentate after membrane separation is spray-dried to obtain the yeast polysaccharide iron. The spray-drying conditions include an inlet air temperature of not less than 170°C and an outlet air temperature of not less than 90°C.

[0032] Furthermore, the present invention provides yeast polysaccharide iron prepared by the preparation method described above.

[0033] The iron in the yeast polysaccharide provided by this invention has an iron mass fraction of not less than 10%.

[0034] Furthermore, the present invention also provides the application of the aforementioned yeast polysaccharide iron in the preparation of iron supplements.

[0035] Furthermore, the present invention also provides an iron supplement containing the yeast polysaccharide iron and excipients as described above.

[0036] The dosage forms of the iron supplements described in this invention include, but are not limited to, oral solid dosage forms, oral liquid dosage forms, semi-solid dosage forms, and special drug delivery formulations. Oral solid dosage forms include tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, and controlled-release capsules; oral liquid dosage forms include oral solutions, suspensions, drops, and syrups; semi-solid dosage forms include gels, ointments, and pastes; and special drug delivery formulations include at least one of lyophilized powder for injection, sterile powder for injection, transdermal patches, sublingual tablets, and / or orally disintegrating tablets.

[0037] The excipients in the iron supplement formulations described in this invention include, but are not limited to, at least one of the following: fillers, binders, disintegrants, lubricants, flow aids, flavoring agents, sweeteners, preservatives, stabilizers, solvents, suspending agents, plasticizers, matrices, coating materials, pH adjusters, chelating agents, antioxidants, osmotic pressure regulators, transdermal penetration enhancers, and / or colorants.

[0038] Furthermore, the present invention also provides a method for iron supplementation, which includes administering an iron supplement as described above.

[0039] As a feasible example, the methods of administration include, but are not limited to, oral administration, injection (intramuscular injection, intravenous injection, intravenous infusion), transdermal administration, sublingual administration, and mucosal administration (oral mucosal administration, nasal mucosal administration).

[0040] The present invention provides a method for preparing yeast polysaccharide iron, comprising: treating a yeast polysaccharide solution with hydrogen peroxide, adding sodium citrate and iron salt, and reacting to obtain a product containing yeast polysaccharide iron. The polysaccharide iron prepared by this method has an iron content of over 10%, water solubility of over 90%, good stability, no obvious rusty taste, and a significant sustained-release effect in the gastrointestinal tract, releasing in the stomach and reducing gastrointestinal irritation during iron supplementation. Attached Figure Description

[0041] Figure 1 Iron content standard curve;

[0042] Figure 2 Changes in soluble iron content after simulated in vitro digestion of yeast polysaccharide iron and ferrous sulfate in the gastrointestinal tract;

[0043] Figure 3 Changes in serum iron levels in different groups under a gavage dose of 20 mg / kg;

[0044] Figure 4 Changes in liver iron content in different groups under gavage administration of 20 mg / kg;

[0045] Figure 5 A schematic diagram of the mouse experiment process. Detailed Implementation

[0046] This invention provides a method for preparing yeast polysaccharide-iron complex. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0047] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0048] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0049] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0050] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0051] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0052] The embodiments and comparative examples of this invention describe some examples. These embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples. In fact, good preparation results can be achieved at any point value between the two endpoint values ​​shown in the embodiments.

[0053] The preparation method described in this invention includes:

[0054] Preparation: Prepare a solution of the above yeast polysaccharide with a mass concentration of 6%-10%, preferably 10%-12%.

[0055] Hydrogen peroxide: Adjust the pH of the mixed solution to 7.0, heat to 50℃, then add 3%-6% of 30% hydrogen peroxide solution (on a dry basis) and react for 0.5-3 h. After the reaction is complete, heat the solution to 90℃ and maintain for 30 min to decompose excess hydrogen peroxide.

[0056] Chelated iron: Add 50%-80% sodium citrate to the solution (if the iron is ferrous, add 0.1%-0.4% ascorbic acid), then adjust the pH of the solution to 7.5-9.0, heat the solution to 50-80℃, add 100%-200% (based on polysaccharide) of iron supplement and react for 2-8 hours.

[0057] Membrane separation: The above mixed solution is separated by a 3-10 kDa membrane separation device to remove unbound free iron, sodium ions and other small molecule impurities, and the retentate is collected for later use.

[0058] Spray drying: The above-mentioned retentate is spray dried with an inlet air temperature of not less than 170°C and an outlet air temperature of not less than 90°C to obtain yeast polysaccharide iron powder.

[0059] All test materials used in this invention are common commercially available products. The yeast polysaccharide used in the examples (obtained from yeast cell walls through thermal extraction and enzymatic hydrolysis) has a polysaccharide content of over 60% and a nucleic acid content of less than 5%. Information on reagents and instruments used in the examples is shown in Table 1.

[0060] Table 1: Experimental Reagents

[0061]

[0062] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0063] Example 1

[0064] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. The pH was adjusted to 7.0 by adding acid and alkali. The temperature was raised to 50°C, and 0.5% (based on the mass fraction of dry matter in the yeast polysaccharide solution) of 30% H₂O₂ was added. The reaction proceeded for 2 h, then the temperature was raised to 90°C and maintained for 30 min. The temperature was then lowered to 50°C, and 0.5 kg of anhydrous sodium citrate and 1.5 kg of ferric pyrophosphate were added. The pH was adjusted to 8.0, and the reaction proceeded for 5 h. The solution was then cooled and filtered through a 10 kDa microfiltration membrane to the permeate (Na₂O₃). + If the content is less than 0.35%, the yeast polysaccharide iron complex is obtained by spray drying the retained retentate.

[0065] Example 2

[0066] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. The pH was adjusted to 7.0 by adding acid and alkali. The temperature was raised to 50°C, and 5% (based on the mass fraction of dry matter in the yeast polysaccharide solution) of 30% hydrogen peroxide was added. The reaction proceeded for 2 h. The temperature was then raised to 90°C and maintained for 30 min. The temperature was then lowered to 60°C, and 0.5 kg of anhydrous sodium citrate and 1.5 kg of ferric ammonium citrate were added. The pH was adjusted to 8.0, and the reaction proceeded for 5 h. The solution was then cooled and filtered through a 10 kDa microfiltration membrane to the permeate (Na₂O₃). + If the content is less than 0.35%, the yeast polysaccharide iron complex is obtained by spray drying the retained retentate.

[0067] Example 3

[0068] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. Sodium hydroxide was added to adjust the pH to 7.0, and the temperature was raised to 50°C. 4% (based on the mass fraction of dry matter in the yeast polysaccharide solution) of 30% hydrogen peroxide was added, and the reaction proceeded for 2 h. The temperature was then raised to 90°C and maintained for 30 min, followed by cooling to 50°C. 0.5 kg of anhydrous sodium citrate and 2 kg of ferric citrate were added, and the pH was adjusted to 8.0. The reaction proceeded for 5 h. The solution was then cooled and filtered through a 10 kDa microfiltration membrane to the permeate (Na₂O₃). + If the content is less than 0.35%, the yeast polysaccharide iron complex is obtained by spray drying the retained retentate.

[0069] Example 4

[0070] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. Sodium hydroxide was added to adjust the pH to 7.0, and the temperature was raised to 50°C. 4% (based on the mass fraction of dry matter in the yeast polysaccharide solution) of 30% hydrogen peroxide was added, and the reaction proceeded for 2 h. The temperature was then raised to 90°C and maintained for 30 min, followed by cooling to 50°C. 0.5 kg of anhydrous sodium citrate and 2 kg of ferric citrate were added, and the pH was adjusted to 8.0. The reaction proceeded for 5 h. The solution was then cooled and filtered through a 3 kDa microfiltration membrane to the permeate (Na₂O₃). + If the content is less than 0.35%, the yeast polysaccharide iron complex is obtained by spray drying the retained retentate.

[0071] Example 5

[0072] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. Sodium hydroxide was added to adjust the pH to 7.0, and the temperature was raised to 50°C. 5% (based on the mass fraction of dry matter in the yeast polysaccharide solution) of 30% hydrogen peroxide was added and reacted for 2 h. Then, the temperature was raised to 90°C and held for 30 min. Then, the temperature was lowered to 60°C, and 0.5 kg of anhydrous sodium citrate, 1.5 kg of ferrous gluconate, and 10 g of ascorbic acid were added. The pH was adjusted to 8.0 and the reaction was allowed to proceed for 5 h. After cooling, the solution was filtered through a 10 kDa microfiltration membrane until the Na content of the permeate was less than 0.35%. 0.1% ascorbic acid was added to the purified water added during the filtration process. The retained retentate was spray-dried to obtain the yeast polysaccharide iron complex.

[0073] Example 6

[0074] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. Sodium hydroxide was added to adjust the pH to 7.0, and the temperature was raised to 50°C. 5% (based on the mass fraction of dry matter in the yeast polysaccharide solution) of 30% hydrogen peroxide was added, and the reaction proceeded for 2 h. The temperature was then raised to 90°C and maintained for 30 min, followed by cooling to 60°C. 0.5 kg of anhydrous sodium citrate, 1.5 kg of ferrous succinate, and 10 g of ascorbic acid were added, and the pH was adjusted to 8.0. The reaction proceeded for 5 h. The solution was then cooled and filtered through a 10 kDa microfiltration membrane to the permeate (Na₂O₃). + If the content is less than 0.35%, 0.1% ascorbic acid should be added to the pure water added during the membrane process, and the retentate should be spray-dried to obtain the yeast polysaccharide iron complex.

[0075] Comparative Example 1: No hydrogen peroxide

[0076] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. Sodium hydroxide was added to adjust the pH to 7.0, and the temperature was raised to 50°C. 0.5 kg of anhydrous sodium citrate and 1 kg of ferric citrate were added, and the pH was adjusted to 8.0. The reaction was allowed to proceed for 4 hours. After cooling, the solution was filtered through a 10 kDa microfiltration membrane to the permeate (Na₂O₃).+ If the content is less than 0.35%, the yeast polysaccharide iron complex is obtained by spray drying the retained retentate.

[0077] Comparative Example 2: Polysaccharide: Sodium citrate = 3:1

[0078] 0.9 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. Sodium hydroxide was added to adjust the pH to 7.0, and the temperature was raised to 50°C. 5% (based on the mass fraction of dry matter in the yeast polysaccharide solution) of 30% hydrogen peroxide was added, and the reaction proceeded for 2 h. The temperature was then raised to 90°C and maintained for 30 min, followed by cooling to 50°C. 0.3 kg of anhydrous sodium citrate and 0.9 kg of ferric citrate were added, and the pH was adjusted to 8.0. The reaction proceeded for 4 h. The solution was then cooled and filtered through a 10 kDa microfiltration membrane to the permeate (Na₂O₃). + If the content is less than 0.35%, the yeast polysaccharide iron complex is obtained by spray drying the retained retentate.

[0079] Comparative Example 3: Sodium Citrate-Free

[0080] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. Sodium hydroxide was added to adjust the pH to 7.0, and the temperature was raised to 50°C. 5% (based on the mass fraction of dry matter in the yeast polysaccharide solution) of 30% hydrogen peroxide was added, and the reaction proceeded for 2 h. The temperature was then raised to 90°C and maintained for 30 min, followed by cooling to 50°C. 1 kg of ferric citrate was added, and the pH was adjusted to 8.0. The reaction proceeded for 4 h. The solution was then cooled and filtered through a 10 kDa microfiltration membrane to the permeate (Na₂O₃). + If the content is less than 0.35%, the yeast polysaccharide iron complex is obtained by spray drying the retained retentate.

[0081] Comparative Example 4: Ferrous iron without ascorbic acid

[0082] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. Sodium hydroxide was added to adjust the pH to 7.0, and the temperature was raised to 50°C. 5% (based on the mass fraction of dry matter in the yeast polysaccharide solution) of 30% hydrogen peroxide was added, and the reaction proceeded for 2 h. The temperature was then raised to 90°C and maintained for 30 min, followed by cooling to 60°C. 0.5 kg of anhydrous sodium citrate and 1.5 kg of ferrous gluconate were added, and the pH was adjusted to 8.0. The reaction proceeded for 5 h. The solution was then cooled and filtered through a 10 kDa microfiltration membrane to the permeate (Na₂O₃). + If the content is less than 0.35%, the yeast polysaccharide iron complex is obtained by spray drying the retained retentate.

[0083] Comparative Example 5: Acidic pH value

[0084] 1 kg of yeast polysaccharide was added to purified water to prepare a 9 kg solution. Citric acid was added to adjust the pH to 5.5, and the temperature was raised to 50°C. 0.5 kg of anhydrous sodium citrate and 1 kg of ferric citrate were added, and the pH was adjusted to 8.0. The reaction was allowed to proceed for 4 hours. After cooling, the solution was filtered through a 10 kDa microfiltration membrane to the permeate (Na₂O₃). + If the content is less than 0.35%, the yeast polysaccharide iron complex is obtained by spray drying the retained retentate.

[0085] Effect verification

[0086] 1. Iron content detection

[0087] Preparation of ferrous ion standard solution (10 μg / mL): Accurately weigh 0.7022 g of ferrous ammonium sulfate hexahydrate and dissolve it in 20 mL of 6 mol / L HCl and a small amount of deionized water, then dilute to 1 L to obtain a Fe standard solution with a concentration of 100 μg / mL. 2+ Solution. Take 10 mL from the above solution, add 2 mL of 6 mol / L HCl solution, and dilute to 100 mL to prepare a 10 μg / mL iron ion standard solution. Store in the dark.

[0088] (1) Preparation of 10% hydroxylamine hydrochloride solution: Add 5 g of hydroxylamine hydrochloride powder to a beaker, dissolve it in deionized water, and make up to 50 mL.

[0089] (2) Preparation of 0.1% o-phenanthroline solution: Accurately weigh 0.1 g o-phenanthroline into a beaker, add a small amount of concentrated hydrochloric acid, stir until o-phenanthroline is completely dissolved, transfer to a 100 mL volumetric flask, and dilute to the mark with deionized water to obtain o-phenanthroline indicator.

[0090] (3) Acetic acid-sodium acetate buffer: accurately weigh 16.4 g of sodium acetate, dissolve it in deionized water, add 12 mL of glacial acetic acid, and make up to 100 mL.

[0091] Preparation of the standard curve: Add 0, 2, 4, 6, 8, and 10 mL of ferrous ion standard solution to 50 mL volumetric flasks, respectively. Then add 2 mL of 10% hydroxylamine hydrochloride solution and 5 mL of acetate-sodium acetate buffer solution, respectively. Finally, add 5 mL of 0.1% o-phenanthroline indicator. Dilute to volume with deionized water, shake well, and let stand for half an hour. Using the blank without ferrous ion solution, measure the absorbance at 510 nm (results are shown below). Figure 1 ).

[0092] Calculation of iron content in the chelate: Accurately pipette 1 mL of sample solution into a 50 mL volumetric flask, add 2 mL of 10% hydroxylamine hydrochloride solution and 5 mL of acetate-sodium acetate buffer solution, shake well and let stand for 30 min. Then add 5.0 mL of 0.1% o-phenanthroline indicator, dilute to volume and shake well. Using deionized water as a reference solution, measure the absorbance at a wavelength of 510 nm, and then calculate the total iron content of the sample.

[0093] Calculation of ferrous content in the chelate: Accurately pipette 1 mL of sample solution into a 50 mL volumetric flask, add 5 mL of acetate-sodium acetate buffer solution, shake well and let stand for 30 min. Then add 5.0 mL of 0.1% o-phenanthroline indicator, dilute to volume and shake well. Using deionized water as a reference solution, measure the absorbance at a wavelength of 510 nm, and then calculate the ferrous content of the sample.

[0094] Fe 3+ Content = Total iron content - Fe 2+ content

[0095] Identification of trivalent and divalent iron content: The o-phenanthroline method was used to quantitatively analyze the iron content in yeast polysaccharide-Fe(III) samples. The principle is that under acidic conditions, the iron content in yeast polysaccharide-Fe(III) is... 3+ Reduced to Fe by ascorbic acid 2+ o-phenanthroline and Fe 2+ A complexation reaction occurs, and the complex turns red. Based on the color of the reaction product, the iron content of the sample can be preliminarily determined. Then, the absorbance of the sample can be measured by an ultraviolet spectrophotometer for more accurate quantitative analysis.

[0096] The results are shown in Table 2:

[0097] Table 2: Iron content of the examples and comparative samples

[0098]

[0099] (p < 0.05, different letters indicate differences between groups)

[0100] As shown in Table 2, the iron content of the polysaccharide iron samples in the examples was all above 10%, which was significantly higher than that of the comparative samples under the single-factor experimental conditions.

[0101] 2. Stability test

[0102] After placing the sample in a stability test chamber at 40℃ and 70% humidity for a period of time, compare the changes in the color and odor of the sample, and use a colorimeter to measure the color change of the sample powder.

[0103] The L value (brightness) indicates the lightness or darkness of a color: the L value ranges from 0 to 100, where 0 represents pure black and 100 represents pure white. The larger the L value, the brighter the color; the smaller the L value, the darker the color.

[0104] The α value (red-green tint) represents the red-green tint of a color: the α value ranges from -128 to +127. Positive values ​​indicate a reddish tint, while negative values ​​indicate a greenish tint. The larger the absolute value, the stronger the red-green tint of the color.

[0105] The b-value (b-value) indicates the yellow-blue tint of a color: the b-value ranges from -128 to +127. Positive values ​​indicate a yellowish tint, while negative values ​​indicate a bluish tint. The larger the absolute value, the stronger the yellow-blue tint of the color.

[0106] Table 3: Color Comparison of Polysaccharide Iron Samples Before and After Placement in the Stability Test Chamber

[0107]

[0108] As shown in Table 3, after being placed in a stability test chamber at 70% humidity and 40°C for two months, the polysaccharide (ferrous) content of comparative examples 1-5 was low, and the color of the samples was relatively unstable. After being placed for a period of time, their brightness increased, while their red and blue values ​​decreased, showing significant fading. In contrast, the color of the sample from the example was relatively stable, and after being placed in the stability test chamber for two months, the color did not change significantly.

[0109] 3. In vitro simulated digestion experiment

[0110] The primary site of iron absorption in the body is the small intestine. Oral iron supplements are typically hydrolyzed by gastric acid and enzymes in the stomach before entering the small intestine, releasing some iron ions. Once in the small intestine, these ions have reduced solubility and are difficult for the intestinal epithelial cells to absorb, thus lowering bioavailability. Therefore, assessing the soluble iron content of iron supplements in gastric and intestinal digestive fluids is an important method for determining their bioavailability.

[0111] The in vitro digestion protocol was slightly modified from the INFOGEST 2.0 improved by Brodkorb et al., and the sample was the polysaccharide iron prepared in Example 3. The in vitro digestion consisted of two parts: in vitro gastric digestion and in vitro intestinal digestion. The inorganic salts in the simulated gastric juice (SGF) included: 6.9 mM KCl, 0.9 mM KH2PO4, 25 mM NaHCO3, 47.2 mM MgCl2, 0.12 mM (NH4)2CO3, 0.5 mM CaCl2, 0.15 mM NaCl, and 15.6 mM HCl; the inorganic salts in the simulated intestinal juice (SIF) included: 6.8 mM KCl, 0.8 mM KH2PO4, 85 mM NaHCO3, 0.33 mM MgCl2, 0.6 mM CaCl2, 38.4 mM NaCl, and 8.4 mM HCl.

[0112] 3.1 Stomach digestion

[0113] Mix 1.6 mL of SGF with 2 mL of sample, then adjust the pH to 2.5 with 6 M HCl solution, add 1 μL of 0.3 M CaCl2 solution and 0.1 mL of pepsin solution to make the enzyme activity in the mixture 2000 U / mL, and add a certain amount of deionized water to make the final volume of the mixture 4 mL. Then immediately incubate in a constant temperature stirrer at 37 ℃ at 100 r / min for 2 h.

[0114] 3.2 Intestinal Digestion

[0115] After 2 h of in vitro gastric digestion, the gastric phase was mixed with 1.6 mL of SIF, and the pH was adjusted to 7.6 with 6 M NaOH. Then, 76 μL of 0.03 M CaCl2 solution, 1.5 mL of bile salt solution (dissolved in SIF), and 0.1 mL of trypsin solution (dissolved in SIF) were added, and deionized water was added to bring the final volume of the mixture to 8 mL, with a bile salt concentration of 10 mM and a trypsin concentration of 100 U / mL. The intestinal digestion solution was placed in a 6000-8000 Da dialysis bag and immediately incubated at 37 °C with a stirrer at 100 r / min for 2 h.

[0116] The results are as follows Figure 2 After simulating gastrointestinal digestion, the soluble iron content of yeast polysaccharide iron was significantly higher than that of FeSO4. FeSO4 is easily precipitated as ferric hydroxide at physiological pH (4.8~8.0), which is difficult to be digested and absorbed in the intestine; in contrast, yeast polysaccharide iron is stable and highly soluble throughout the process, and may have higher bioavailability.

[0117] 4. Polysaccharide Iron Metabolism Experiment in Mice

[0118] 4.1 Materials and Instruments

[0119] Physiological saline, 75% alcohol, 1.5 ml centrifuge tubes, 2 mL syringes, surgical scissors, surgical forceps, rat gavage needles, serum iron assay kit (Nanjing Jiancheng A039-1-1), and ELISA reader (Shanghai Flash Spectrum).

[0120] 4.2 Experimental Methods

[0121] The experimental procedure is as follows Figure 5 Specifically:

[0122] 4.2.1 Laboratory Animals

[0123] Male SD rats (6-7 weeks old) were acclimatized for one week, and then aged 7-8 weeks for experimental treatment.

[0124] 4.2.2, Concentration and Preparation of Test Substance

[0125] Preparation of gavage solution for polysaccharide iron group A (iron content 15.5%): Weigh 12903 mg of polysaccharide iron prepared in Example 3, dissolve it in physiological saline, and bring the volume to 1 L. In actual experiments, 20 mL is sufficient, i.e., 258.1 mg / 20 mL. Administer 2.0 mL to each mouse by gavage.

[0126] Preparation of gavage solution for ferrous sulfate group B (ferrous sulfate heptahydrate with an iron content of 20%): Weigh 10000 mg of ferrous sulfate heptahydrate, dissolve it in physiological saline, and bring the volume to 1 L. In actual experiments, 20 mL is sufficient, i.e., 200 mg / 20 mL. Administer 2.0 mL to each rat by gavage. This is equivalent to 20 mg Fe / kg WB in rats.

[0127] 4.2.3 Experimental Environment

[0128] Temperature: Approximately 26℃;

[0129] Lighting: 12 hours of light / 12 hours of darkness;

[0130] Diet: Animals are fed feed in the animal house and have free access to food and water.

[0131] 4.2.4 Experimental Procedure

[0132] ① After one week of acclimatization, the SD rats were divided into two groups of 7 rats each according to their body weight: polysaccharide iron group A and ferrous sulfate group B.

[0133] ②The experiment was conducted with fasting at 17:00 the day before the experiment, but water was allowed. At 9:00 the morning of the experiment, different iron solutions were administered by gavage.

[0134] ③ Rats were anesthetized with isoflurane inhalation at 0, 1, 2, 4, and 6 hours after gavage. Blood was collected through the ophthalmic venous plexus, with 600-800 μL collected at each time point. After blood collection, the rats were returned to their cages. The rats were sacrificed 6 hours after blood collection, and liver tissue was dissected and collected.

[0135] 4.3 Experimental Results

[0136] 4.3.1 Changes in serum iron levels in different groups as follows: Figure 3 In the two iron supplement groups, the initial serum iron levels in rats were high, increased 1 hour after gavage, and then gradually decreased, all falling below the initial levels. Ferrous sulfate showed a faster rate of iron increase, but the final serum concentration showed no significant difference. This indicates that both iron supplements are rapidly absorbed into the bloodstream in a short period, with similar absorption rates and peak values.

[0137] 4.3.2 Changes in liver iron content in different groups as follows Figure 4 In the study of liver iron content changes among different groups administered 20 mg / kg via gavage, the liver iron content in the polysaccharide iron group was significantly higher than that in the ferrous sulfate group. Although serum iron levels were similar in the short term, the increase in liver iron content may indicate that an iron supplement can better maintain iron balance with long-term use. The liver is an important organ for iron storage, and the increase in liver iron content suggests that polysaccharide iron may have better iron storage capacity and higher bioavailability, meaning that iron is not only absorbed into the bloodstream but also better utilized and stored by tissues.

[0138] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing yeast polysaccharide iron, including: After treating the yeast polysaccharide solution with hydrogen peroxide, sodium citrate and iron salt were added, and the reaction was carried out to obtain a product containing yeast polysaccharide iron. The iron salt is ferric citrate, ferric pyrophosphate, ferric ammonium citrate, ferrous gluconate and / or ferrous succinate; The yeast polysaccharide solution is in water as the solvent, wherein the mass fraction of yeast polysaccharide is 5%~15%, and the pH value is 6.5~7.5; The hydrogen peroxide is a 30% (v / v) aqueous solution of hydrogen peroxide, and the mass of the aqueous solution of hydrogen peroxide is 3% to 6% of the dry matter of the yeast polysaccharide solution; the hydrogen peroxide treatment conditions include reacting at 45 to 55°C for 0.5 to 3 h, and then reacting at 85 to 95°C for 25 to 35 min. The mass ratio of sodium citrate to yeast polysaccharide is 0.5:1; the mass ratio of iron salt to yeast polysaccharide is (1.5~2):1; If the iron salt is a ferrous salt, ascorbic acid is also added, and the mass ratio of ascorbic acid to yeast polysaccharide is (0.008~0.012):

1.

2. The preparation method according to claim 1, characterized in that, After cooling to 50-60℃, add sodium citrate and iron salt.

3. The preparation method according to claim 1, characterized in that, After adding sodium citrate and iron salt, adjust the pH to 7.5-8.5 and react for 4-6 hours.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The product containing yeast polysaccharide iron is separated by a 3-10 kDa microfiltration membrane, and the solution obtained by membrane separation is water or an aqueous solution of ascorbic acid.

5. The preparation method according to claim 4, characterized in that, The membrane separates Na into the permeate. + The mass fraction is not higher than 0.35%; the mass fraction of ascorbic acid in the aqueous solution of the ascorbic acid is 0.1%.

6. The preparation method according to claim 5, characterized in that, The retentate after membrane separation is spray-dried to obtain the yeast polysaccharide iron.

7. The yeast polysaccharide iron prepared by the preparation method according to any one of claims 1 to 6.

8. The yeast polysaccharide iron according to claim 7, characterized in that, The iron content shall not be less than 10% by mass.

9. The use of the yeast polysaccharide iron according to claim 7 or 8 in the preparation of iron supplements.

10. An iron supplement, characterized in that, It contains the yeast polysaccharide iron and excipients as described in claim 7 or 8.

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