Recombinant human lactoferrin milk tablet with functions of losing weight and reducing blood sugar and preparation method of recombinant human lactoferrin milk tablet

Abstract

The invention provides a recombinant human lactoferrin milk tablet with weight losing and blood sugar reducing functions and a preparation method thereof, and belongs to the technical field of dairy product processing. The recombinant human lactoferrin milk tablet disclosed by the invention is prepared from the following components in parts by mass: 95 to 105 parts of recombinant human lactoferrin, 3 to 4 parts of zinc gluconate, 1 to 1.5 parts of vitamin E, 0.03 to 0.04 part of folic acid and 1300 to 1400 parts of milk powder. Experiments prove that the recombinant human lactoferrin milk tablet prepared by the invention can slow down the weight gain of mice and relieve the impaired glucose metabolism capability caused by high fat diet, and meanwhile, the recombinant human lactoferrin can regulate the lipid degeneration of the liver to a certain extent, relieve the lipid deposition of the liver and inhibit fat cell hypertrophy. The recombinant human lactoferrin milk tablet has the weight-losing and blood-glucose-reducing effects for the first time, and has a wide application prospect in the field of weight losing and blood glucose reducing in the future.

Description

Technical Field

[0001] This invention relates to the field of dairy processing technology, and in particular to a recombinant human lactoferrin milk tablet with weight loss and blood sugar reduction functions and its preparation method. Background Technology

[0002] Obesity and type 2 diabetes have become major global public health problems, both closely related to insulin resistance, chronic inflammation, and gut microbiota dysbiosis. Current mainstream interventions include chemotherapy (such as metformin and orlistat), strict dietary control, and increased exercise. However, long-term use of chemotherapy drugs often brings side effects such as gastrointestinal discomfort and increased burden on liver and kidney function, leading to poor patient adherence. While dietary and exercise interventions have no side effects, they are difficult to maintain long-term, and their effectiveness varies significantly from person to person. Therefore, developing a safe, effective, easy-to-adhere-to, and nutritionally balanced natural health product has significant market value and clinical significance. Summary of the Invention

[0003] In view of this, the present invention provides a recombinant human lactoferrin milk tablet with weight loss and blood sugar reduction functions and its preparation method, so as to solve the above problems.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of recombinant human lactoferrin in the preparation of hypoglycemic drugs or functional foods with auxiliary weight loss and hypoglycemic effects.

[0005] This invention provides a recombinant human lactoferrin milk tablet with weight loss and blood sugar reduction functions, comprising the following components in parts by weight: 95-105 parts of recombinant human lactoferrin, 3-4 parts of zinc gluconate, 1-1.5 parts of vitamin E, 0.03-0.04 parts of folic acid, and 1300-1400 parts of milk powder; This invention also provides a method for preparing the recombinant human lactoferrin milk tablets, comprising the following steps: S1. Mix lactoferrin, zinc gluconate, vitamin E, folic acid and milk powder, then add 10-15% water by weight of the total materials, and then homogenize to obtain a slurry; S2. After drying the homogenate, crush and sieve it, add lubricant and mix, and then press it into milk tablets using a tablet press.

[0006] Preferably, the homogenization temperature is 70~75℃, the homogenization pressure is 20~30MP, and the homogenization time is 10~20min.

[0007] Preferably, the drying temperature is 50~60℃, and the drying is carried out until the moisture content is 2~4%.

[0008] Preferably, the pressure of the tablet is 0.4~0.6MPa.

[0009] By adopting the above technical solution, the present invention has the following beneficial effects: The recombinant human lactoferrin milk tablets of the present invention comprise the following components in parts by weight: 95-105 parts lactoferrin, 3-4 parts zinc gluconate, 1-1.5 parts vitamin E, 0.03-0.04 parts folic acid, and 1300-1400 parts milk powder. Experiments have demonstrated that the recombinant human lactoferrin milk tablets prepared by the present invention can slow down weight gain in mice and alleviate impaired glucose metabolism caused by a high-fat diet, while bovine lactoferrin milk tablets prepared using the same method do not have this effect. Simultaneously, lactoferrin can regulate hepatic lipid degeneration to a certain extent, reduce hepatic lipid deposition, and inhibit adipocyte hypertrophy. This invention is the first to propose that recombinant human lactoferrin milk tablets have a hypoglycemic effect, and has broad application prospects in the field of hypoglycemia. Attached Figure Description

[0010] Figure 1 The effect of lactoferrin on oral glucose tolerance in mice under HFD stimulation is shown in Figure 1. (A) is the curve of glucose content changing over time, and (B) is the area under the curve.

[0011] Figure 2 The effect of lactoferrin on fasting blood glucose in mice under HFD stimulation.

[0012] Figure 3 The effects of lactoferrin on serum cholesterol in mice under HFD stimulation: (A) total serum cholesterol; (B) serum triglycerides; (C) high-density lipoprotein cholesterol; (D) low-density lipoprotein cholesterol; (E) free fatty acids.

[0013] Figure 4 The images show the morphology of the livers of mice in different treatment groups.

[0014] Figure 5 Image of mouse liver stained with hematoxylin and eosin (HE).

[0015] Figure 6 For quantitative analysis of liver fat area.

[0016] Figure 7 Visual representation of the morphology of white adipose tissue in the epididymis of mice in different treatment groups.

[0017] Figure 8 HE-stained white adipose tissue cells of mouse epididymis in different treatment groups.

[0018] Figure 9 HE staining area of ​​fat in different treatment groups. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] The recombinant human lactoferrin in this embodiment of the invention is expressed in a bovine mammary gland bioreactor and purified using liquid chromatography column purification product (prepared in our laboratory). The bovine lactoferrin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0021] Example 1 Recombinant human lactoferrin milk tablets with weight loss and blood sugar lowering functions are composed of the following components by weight: recombinant human lactoferrin 95 mg, zinc gluconate 3 mg, vitamin E 1 mg, folic acid 0.034 mg, and milk powder 1300 mg.

[0022] Preparation method: Lactoferrin, zinc gluconate, vitamin E, folic acid and milk powder are mixed, then water of 10% of the total mass of the materials is added, and then homogenized to obtain a slurry; the homogenization temperature is 70℃, the homogenization pressure is 20MPa, and the homogenization time is 10min.

[0023] The homogenate is then dried at 50°C until the moisture content is 2%, and then compressed into tablets at a pressure of 0.4 MPa.

[0024] Example 2 Recombinant human lactoferrin milk tablets with weight loss and blood sugar lowering functions are composed of the following components by weight: recombinant human lactoferrin 100 mg, zinc gluconate 3.5 mg, vitamin E 1.2 mg, folic acid 0.035 mg, and milk powder 1350 mg. Preparation method: Lactoferrin, zinc gluconate, vitamin E, folic acid and milk powder are mixed, then water of 12% of the total mass of the materials is added, and then homogenized to obtain a slurry; the homogenization temperature is 72℃, the homogenization pressure is 25MPa, and the homogenization time is 15min.

[0025] The homogenate is then dried at 55°C until the moisture content is 3% before being compressed into tablets at a pressure of 0.5 MPa.

[0026] Example 3 Recombinant human lactoferrin milk tablets with weight loss and blood sugar lowering functions are composed of the following components by weight: recombinant human lactoferrin 105 mg, zinc gluconate 4 mg, vitamin E 1.5 mg, folic acid 0.04 mg, and milk powder 1400 mg. Preparation method: Lactoferrin, zinc gluconate, vitamin E, folic acid and milk powder are mixed, then water of 15% of the total mass of the materials is added, and then homogenized to obtain a slurry; the homogenization temperature is 75℃, the homogenization pressure is 30MPa, and the homogenization time is 20min.

[0027] The homogenate is then dried at 60°C until the moisture content is 4%, and then compressed into tablets at a pressure of 0.6 MPa.

[0028] Experiment Example 1. Blood Sugar Lowering and Fat Reduction Experiment The preparation method of the recombinant human lactoferrin (rhLF) milk tablets in this experiment is the same as in Example 2.

[0029] Bovine lactoferrin (bLF) milk tablets are made by replacing recombinant human lactoferrin (rhLF) in Example 2 with bovine lactoferrin, while the other components, their amounts, and preparation methods are the same as in Example 2.

[0030] Experimental materials: 6-week-old male mice (strain C57BL6N) were purchased from Beijing Vital River.

[0031] The control diet (Con, 10% of calories from fat, 20% from protein, 70% from carbohydrates, 3.42 kcal / g) and the high-fat diet (HFD, 60% of calories from fat, 20% from protein, 20% from carbohydrates, 5.24 kcal / g) for mice were purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.

[0032] Grouping and treatment of experimental animals: Six-week-old mice were acclimatized for one week and then divided into four groups according to their body weight: (1) CON group (n=7): fed with the control diet and administered physiological saline (100 mg / kg body weight) by gavage. (2) HFD group (n=7): fed a high-fat diet and administered physiological saline (100 mg / kg body weight) by gavage. (3) HFD-rhLF milk tablet group (n=7): fed with a high-fat diet plus rhLF milk tablets, and gavaged with lactoferrin milk tablets rhLF (100 mg protein / Kg body weight) prepared with physiological saline. (4) HFD-bLF milk tablet group (n=7): fed with a high-fat diet plus bLF milk tablets, and gavaged with lactoferrin milk tablets bLF (100 mg protein / Kg body weight) prepared with physiological saline. Mouse body weight and food intake were recorded weekly. Successful modeling was considered achieved when the HFD group's body weight exceeded that of the CON group by approximately 20%. After successful modeling, mice underwent an oral glucose tolerance test. Following a 16-hour fast, mice were euthanized, and blood, liver, epididymal fat, inguinal subcutaneous fat, and palm fat were collected and weighed. Organ coefficient analysis was performed based on the mice's post-fasting body weight. Collected organs were frozen at -80°C for subsequent analysis.

[0033] 1. Oral glucose tolerance test (1) Fast the mice for 16 hours and keep them in normal drinking water. Prepare a 0.15 g / mL glucose solution with physiological saline and let it stand overnight.

[0034] (2) Weigh the mouse and mark it on its tail with a marker.

[0035] (3) Take out the mouse and place it gently on the iron grid of the cage. Insert the blood collection needle into the end of the mouse's tail about 1-2 mm and gently squeeze the tail vein to make the blood accumulate into a drop. Discard the first drop and use the second drop to test the blood glucose. Use a blood glucose meter to measure the fasting blood glucose. The measured value is the blood glucose value at 0 min.

[0036] (4) Mice were administered 10 μL / g body weight (BW) glucose solution via gavage, and the time was recorded. The time after gavage was recorded as 0 s. At 15 min, 30 min, 60 min, 90 min, and 120 min after glucose gavage, a drop of blood was collected from the tail vein of each mouse, and the blood glucose level was measured and recorded using a glucometer. After the experiment, the mice were allowed free access to food. The effect of LF on oral glucose tolerance under HFD stimulation in mice is as follows: Figure 1 As shown, the effect of LF on fasting blood glucose in mice under HFD stimulation is as follows: Figure 2 As shown.

[0037] Figure 1 The results showed that after oral glucose administration, the blood glucose concentration in mice rapidly increased and then gradually decreased to normal levels. Figure 1 (A). After statistically analyzing the area under the curve, the results are as follows: Figure 1 As shown in Figure B, there was a significant difference between the high-fat diet group and the CON group, indicating that a high-fat diet can lead to a decrease in the glucose clearance capacity of mice. However, compared with the HFD group and the HFD-bLF milk tablet group, the area under the curve of the HFD-rhLF milk tablet group was less different from that of the CON group, indicating that the rhLF milk tablets did indeed regulate glucose metabolism and reduce the negative impact of a high-fat diet on glucose metabolism.

[0038] Figure 2 The results showed that rhLF could improve hyperglycemia induced by a high-fat diet in mice. However, bLF showed no significant difference from the HFD group and did not improve hyperglycemia in mice.

[0039] 2. Serum biochemical analysis Blood was collected from the orbital cavity of mice. After standing for 4 hours, the collected blood was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant was collected as mouse serum.

[0040] Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), and non-esterified fatty acids (NEFA) were measured. Results are as follows: Figure 3 As shown.

[0041] like Figure 3 As shown in Figure A, the high-fat diet group had a significantly higher total cholesterol level than the control diet group, while the HFD-rhLF milk tablet group reversed this result, significantly improving the increase in serum TC caused by HFD stimulation, while the HFD-bLF group did not show any improvement. Figure 3 As shown in Figure B, there were no significant differences in triglyceride levels among the groups. Figure 3 As shown in C and D, high-fat feeding led to a significant increase in both high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in mice, but neither of the two LF treatments could reverse these results. Figure 3 As can be seen from E, there was no significant difference in free fatty acids among the groups.

[0042] 3. Organ pathological examination 1. Preparation of paraffin sections The collected mouse liver and adipose tissue were divided into two parts. One part was flash-frozen in liquid nitrogen and stored at -80°C, while the other part was fixed in 4% neutral paraformaldehyde solution. Mouse organs and tissues fixed for more than one week were dehydrated and embedded in paraffin. Sections were developed in a 45°C water bath, dried at 37°C, and baked at 60°C for 2 hours for staining.

[0043] 2. Hematoxylin-eosin staining Paraffin sections (5 μm thick for adipose tissue and 3.5 μm thick for liver) were stained with hematoxylin and eosin.

[0044] The prepared paraffin sections were then processed as follows: (1) Dewaxing procedure: Xylene I 10 min; Xylene II 10 min; 100% ethanol I 5 min; 100% ethanol II 5 min; 95% ethanol 5 min; 80% ethanol 2 min; 70% ethanol 2 min; Distilled water 2 min.

[0045] (2) Staining: stain with hematoxylin for 5 min; rinse with tap water for 10 min; soak in distilled water for 10 s; saturate with hydrochloric acid-ethanol for 10 s; rinse with tap water for 10 min; stain with eosin for 30 s.

[0046] (3) Immediate dehydration: 70% ethanol for 10s; 80% ethanol for 10s; 90% ethanol for 10s; 100% ethanol for 10s.

[0047] (4) Xylene becomes transparent in 5 minutes.

[0048] (5) Mount the slide with neutral resin. After drying, observe the slide under a regular optical microscope.

[0049] ImageJ was used to statistically analyze the diameter and area of ​​adipocytes in the sections, and to analyze the number of adipocytes. The results are as follows: Figures 4-9 As shown.

[0050] like Figure 5 As shown, the CON group did not exhibit vacuolar degeneration of fat around the central vein, while the HFD diet induced significant lipid deposition and vacuolar degeneration in the mouse liver. In the group supplemented with LF, fat vacuolar degeneration was improved, and lipid droplets near the central vein decreased. This indicates that a high-fat diet leads to significant fat accumulation in the liver. However, LF may have mitigated this damage. Figure 6 It appears that bLF has some effect on hepatic steatosis, but it is not significant, while rhLF can significantly improve lipid deposition in the liver.

[0051] Depend on Figure 7 It was observed that, compared to the CON group, the white adipose tissue of the epididymis in HFD-fed mice showed varying degrees of growth. Furthermore, adipocytes were observed through HE staining (…). Figure 8 The study found that the cell area in the HFD diet group was larger than that in the CON group. Figure 9 It can be seen that rhLF and bLF are significantly lower than HFD, indicating that rhLF and bLF inhibit the hypertrophy of adipocytes.

[0052] In summary, this experiment found that milk tablets prepared using recombinant human LF significantly reduced blood glucose, serum total cholesterol, and fat accumulation in obese mice; while bovine LF milk tablets did not have the effects of reducing blood glucose or serum total cholesterol, only reducing lipid accumulation in adipocytes. This indicates that different LFs have significant differences in their effects on lowering blood glucose and blood lipids.

[0053] As can be seen from the above embodiments, the present invention provides a recombinant human lactoferrin milk tablet with weight loss and blood sugar reduction functions and its preparation method. The recombinant human lactoferrin milk tablet of the present invention has a good blood sugar reduction effect.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made 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. Application of recombinant human lactoferrin in the preparation of hypoglycemic drugs or functional foods with auxiliary weight loss and hypoglycemic effects.

2. A recombinant human lactoferrin milk tablet with weight loss and blood sugar lowering functions, characterized in that, It includes the following components in parts by weight: 95-105 parts lactoferrin, 3-4 parts zinc gluconate, 1-1.5 parts vitamin E, 0.03-0.04 parts folic acid, and 1300-1400 parts milk powder.

3. The recombinant human lactoferrin milk tablets according to claim 2, characterized in that, The milk powder is whole milk powder.

4. The recombinant human lactoferrin milk tablets according to claim 2 or 3, characterized in that, The recombinant human lactoferrin milk tablets have a mass of 1.4~1.6g / tablet.

5. The method for preparing recombinant human lactoferrin milk tablets according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Mix lactoferrin, zinc gluconate, vitamin E, folic acid and milk powder, then add 10-15% water by weight of the total materials, and then homogenize to obtain a slurry; S2. After drying the homogenate, crush and sieve it, add lubricant and mix, and then press it into milk tablets using a tablet press.

6. The preparation method according to claim 5, characterized in that, The homogenization temperature is 70~75℃, the homogenization pressure is 20~30MPa, and the homogenization time is 10~20min.

7. The preparation method according to claim 5, characterized in that, The drying temperature is 50~60℃, and the drying is carried out until the moisture content is 2~4%.

8. The preparation method according to claim 5, characterized in that, The pressure of the tablet is 0.4~0.6MPa.

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