Functional composition for improving appetite of plateau population and its application

Abstract

The present invention relates to a functional composition for improving the appetite of people in plateaus, the functional composition comprising inulin, codonopsis polysaccharide and bifidobacterium. Experiments show that under the conditions of rapid plateau environment, the combination of these three functional factors can increase the relative abundance of beneficial bacteria such as bifidobacteria in the intestines of mice, and the relative abundance is significantly higher than that of the original control group (P < 0.05), fully exerting the role of regulating intestinal flora; and the synergistic effect of the three functional factors can affect the secretion of appetite hormones and food intake of mice, and has the effect of improving the appetite of people in rapid plateaus. Among them, the appetite-improving effect of the medium- and high-dose inulin group, the low-dose codonopsis polysaccharide group, and the low-dose bifidobacterium group is the most significant. The PYY concentration in the serum of the test group of the composition decreased, and the NPY concentration increased, which alleviated the reduced energy intake and weight loss caused by loss of appetite, and had the functional effect of regulating intestinal flora and improving the appetite of people in rapid plateaus.

Description

Technical Field

[0001] The invention belongs to the technical field of plateau food or medicine, and particularly relates to a functional composition for improving the appetite of plateau people, and also relates to the application of the composition. Background Art

[0002] my country's plateaus are vast and climatically complex, primarily encompassing the Qinghai-Tibet Plateau, the Yunnan-Guizhou Plateau, and the Loess Plateau in central and western China. The Qinghai-Tibet Plateau, known as the "Roof of the World" and the "Third Pole," has an average elevation of over 4,000 meters. In recent years, with the development of tourism, an increasing number of people from the plains have traveled to the plateau for tourism and living. Countless soldiers stationed in the plains have also traveled to the plateau to fulfill their sacred mission of defending the country and guarding the borders. However, plateaus possess unique environmental characteristics, including low pressure, low oxygen levels, low temperatures, dryness, high winds, and intense radiation. The widespread, nonspecific effects of hypoxia on human function and metabolism are the most significant factor affecting the plateau environment. Currently, the mainstream medical view is that plateaus are defined as areas above 2,500 meters above sea level. This is primarily due to the fact that physiological reactions, clinical symptoms, and biochemical changes become more pronounced when a person reaches altitudes above 2,500 meters. When personnel rapidly enter high altitude areas from plains within a short period of time, or when personnel at high altitude enter even higher altitudes, they often experience a series of non-specific clinical symptoms of acute mountain sickness, such as headaches, dizziness, palpitations, shortness of breath, loss of appetite, fatigue, insomnia, memory loss, and blood pressure changes. These symptoms primarily affect the respiratory, digestive, nervous, and circulatory systems. Among these, dietary disturbances caused by loss of appetite are particularly common among those who rapidly enter the plateau. Studies have found that gastrointestinal symptoms are the most common early symptoms among those who rapidly enter the plateau, with nausea, vomiting, and loss of appetite occurring in up to 60% of cases. These symptoms severely impact and restrict the combat effectiveness of troops rapidly entering the plateau. Overcoming the adverse effects of the extreme plateau environment on the health of soldiers and personnel has become an urgent issue. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a functional composition for improving the appetite of people in plateaus, and also to provide the application of the composition in improving the appetite of people who are rapidly entering plateaus.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] 1. A functional composition for improving the appetite of plateau population, comprising inulin, codonopsis polysaccharide and bifidobacteria.

[0006] In the functional composition for improving the appetite of plateau people, the mass ratio of inulin to codonopsis polysaccharide is 15-25:3-7, and the number of viable bifidobacteria is 1.14×10 per gram of codonopsis polysaccharide. 8 CFU~6.67×10 9 CFU.

[0007] In the functional composition for improving the appetite of plateau people, the mass ratio of inulin to codonopsis polysaccharide is 4.31:1, and the number of viable bifidobacteria is 2.47×10 per gram of codonopsis polysaccharide. 9 CFU.

[0008] Furthermore, the functional composition for improving the appetite of plateau population further comprises food-grade excipients or pharmaceutical excipients.

[0009] Furthermore, the bifidobacterium is Bifidobacterium lactis subspecies BLa36.

[0010] Furthermore, the food-grade auxiliary material is selected from a nutrient enhancer, an antioxidant, a colorant, a flavor enhancer, a flavoring agent, a thickener, a preservative, a sweetener, an anticaking agent or an acidity regulator.

[0011] Preservatives include sodium benzoate, potassium sorbate, sodium isoVC, calcium propionate, etc., which are mainly used to extend the shelf life of food.

[0012] Sweeteners include maltitol, acesulfame potassium, cyclamate, saccharin sodium, aspartame, stevia, sucrose or white sugar, etc.

[0013] Colorants include grape purple, sunset yellow, lemon yellow, carmine, tea green, etc., which can change the color of the appearance of food and stimulate appetite.

[0014] Thickeners include gelatin, carrageenan, pectin, gum arabic, xanthan gum, etc., which can be added to food to maintain a good viscosity.

[0015] Acidity regulators: including citric acid, malic acid, tartaric acid, etc., which will give food a refreshing sour taste.

[0016] Anti-caking agents: including talc, silicon dioxide, potassium ferrocyanide, etc., used to prevent granular or powdered food from clumping into lumps.

[0017] Flavor enhancers: including disodium succinate, alanine, aminoacetic acid (glycine), etc., which can supplement or enhance the original flavor of food.

[0018] Edible flavors: natural or artificially synthesized flavors extracted from a variety of animals and plants. They can improve food quality, make up for flavor defects in food, and increase the color, aroma and taste of food.

[0019] Furthermore, the thickener is maltodextrin.

[0020] Furthermore, the nutritional enhancers are vitamins, minerals, dietary fiber, taurine and choline.

[0021] Nutritional enhancers are selected from vitamins (such as vitamins A, B1, B6, B 12 , C, D, E, etc.), minerals (such as magnesium, phosphorus, calcium, iron, zinc, selenium or their derivatives, etc.), dietary fiber, taurine or choline.

[0022] 2. Use of any of the above compositions in the preparation of functional foods or medicines for improving appetite in people experiencing rapid plateauing.

[0023] The food or medicine is in the form of powder, tablet or solution.

[0024] The beneficial effects of the present invention are as follows: The main innovations of the present invention are: 1. Exploring the mechanism of action of intestinal flora in regulating appetite during rapid altitude exposure; 2. Screening for functional factors and formulas that regulate intestinal flora and improve appetite during rapid altitude exposure; 3. Developing a functional food that regulates intestinal flora and improves appetite during rapid altitude exposure.

[0025] Based on the construction of an animal model of rapid plateau exposure, the present invention studied the effects of different functional factors including inulin, codonopsis polysaccharide and bifidobacterium on the food intake, body weight, intestinal flora and appetite hormones of mice under rapid plateau exposure, and further compared and analyzed the effects of functional factors at different doses. According to the results, it was confirmed that under the conditions of rapid plateau exposure, the combination of these three functional factors can increase the relative abundance of beneficial bacteria such as bifidobacteria in the intestines of mice, and the relative abundance is significantly higher than that of the original control group (P < 0.05), fully exerting the role of regulating intestinal flora; and the synergistic effect of the three functional factors can affect the secretion of appetite hormones and food intake of mice, and has the effect of improving appetite after rapid plateau exposure. Among them, the appetite improvement effect of the high-dose inulin group, the low-dose codonopsis polysaccharide group, and the low-dose bifidobacterium group was the most significant.

[0026] The present invention also developed a ready-to-eat synbiotic functional food with the ability to regulate intestinal flora and improve appetite during acute plateau exposure. Experiments were conducted to verify the effectiveness of this functional food in regulating intestinal flora and improving appetite during acute plateau exposure. Based on human equivalent dose calculations, combined with response surface data and official recommended intakes, the ratio of inulin and codonopsis polysaccharide, as well as the number of viable bifidobacteria, was determined for this ready-to-eat synbiotic military functional food. This functional food is compact and lightweight, allowing for efficient and convenient modular integration with a variety of military foods and fast food options, demonstrating its potential for practical application. Tasting tests demonstrated that this functional food had a generally acceptable taste, a user-friendly packaging design, and demonstrated food safety. Following acute plateau exposure, the experimental group experienced an increase in the relative abundance of probiotic bacteria in their intestinal flora. This enhanced the expression of several intestinal microbial functions, including energy production and conversion, coenzyme transport, and metabolism. Serum PYY levels decreased, while NPY levels increased. This alleviated the reduced energy intake and weight loss associated with decreased appetite. The results showed that this type of functional food has the functional effect of regulating intestinal flora and improving appetite when rapidly entering the plateau. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0028] Figure 1 The comparison of the average body weight differences of mice in each group on days 0, 3, 7, 10, 14, and 15 in Example 4 is shown.

[0029] Figure 2 The figure shows the comparison of the average body weight changes of mice in each group in Example 4.

[0030] Figure 3 The results of agarose gel electrophoresis of the PCR amplification products from mouse feces in Example 4 are shown.

[0031] Figure 4 This is a sample picture of functional food products and packaging. DETAILED DESCRIPTION

[0032] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the preferred embodiment technical solutions of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Experimental methods without specific conditions in the embodiments are generally based on conventional conditions or the conditions recommended by the manufacturer.

[0033] Main reagents:

[0034] Mouse NPY ELISA kit, mouse PYY ELISA kit, mouse Ghrelin ELISA kit: Wuhan Yilai Ruite Biotechnology Co., Ltd.;

[0035] DNA extraction kit (EZNASoil DNA Kit): Omega, USA;

[0036] DNA polymerase (FastPfu Polymerase) Beijing Quanshijin Biotechnology Co., Ltd.;

[0037] DNA gel recovery kit (Axygen Biosciences): Axygen, USA;

[0038] Agarose (biowest Agarose): biowest company, Spain;

[0039] Library construction kit (NEXTFLEX): BIOO Scientific, USA.

[0040] Inulin (90%) Gansu Likang Nutrition Food Co., Ltd.;

[0041] Codonopsis pilosula polysaccharide (50%) Lanzhou Waterless Biotechnology Co., Ltd.

[0042] Bifidobacterium (Bifidobacterium animalis subsp. lactis BLa36) Jiangsu Weikang Biotechnology Co., Ltd.

[0043] Main instruments:

[0044] Plateau environment simulation cabin (5000m): Guizhou Fenglei Aviation Ordnance Co., Ltd.

[0045] Example 1

[0046] Experimental animals: 7- to 8-week-old SPF-grade C57BL / 6J male mice were provided by Hunan Slake Jingda Experimental Animal Co., Ltd., animal license number: SCXK (Xiang) 2019-0004.

[0047] Adaptive feeding: The candidate experimental mice were fed in an animal room (310 m above sea level) with room temperature controlled at 20-25°C and humidity at 50%-70% under simulated natural light (12 h of light and 12 h of dark each day). Distilled water and regular feed were supplied without restriction for a total of 7 days.

[0048] Animal Grouping: Thirty healthy mice weighing 24 ± 2 g were weighed and randomly divided into three groups of 10 mice each, stratified by 1 g weight increments. The three groups were randomly assigned as a control group (P), a group with a 1-day plateau exposure (G1), and a group with a 7-day plateau exposure (G7). Each group of mice was individually numbered on their tails with a marker.

[0049] The experiment lasted 7 days. Group G7 was immediately transferred to a high-altitude environmental chamber (simulating an altitude of 5000 m, the same applies hereafter) at the start of the experiment and continued until the end of the experiment. Group P remained in the animal room for feeding throughout the experiment. Group G1 was fed in the animal room for 3 days and then transferred to the high-altitude environmental chamber for 1 day before sampling. The high-altitude environmental chamber maintained the same temperature, humidity, and lighting conditions as the animal room. All groups were provided with ample water and regular feed throughout the experiment.

[0050] 1. Feces collection: Immediately after the experiment, collect mouse feces. Use a cotton swab moistened with distilled water to wipe the mouse anus. Collect 2 to 3 pieces of fresh feces and place them in corresponding numbered cryovials. Immediately place the cryovials in a liquid nitrogen tank.

[0051] 2. Serum collection: After completing the collection of mouse feces, blood collection will begin immediately. The eyeball removal blood collection method is used to collect mouse blood and separate the serum. The separated serum is labeled according to the experimental mouse group and stored in a refrigerator at -20°C for testing.

[0052] 3. Body Weight and Food Intake: Before the experiment began, the body weight of each group of mice and the amount of maintenance feed provided were weighed and recorded. According to the experimental plan, the body weight of each group of mice and the amount of remaining maintenance feed were weighed and recorded during the experiment and before sampling at the end of the experiment. Mouse body weight and food intake data were analyzed using IBM SPSS Statistics 24 software. The data are shown in Table 1.

[0053] Table 1 Statistics of food intake and body weight of mice ( n=10)

[0054]

[0055] During the experiment, the average weight of mice in Group P gradually increased over time, rising from 25.22g at the start to 26.04g at the end. The average weight of mice in Group G7 dropped sharply after entering the simulated high altitude environment of 5,000m, reaching a low of 20.70g on the third day of the plateau ascent. It then gradually increased to 22.40g at the end of the experiment, but was still lower than the 25.01g at the start. This suggests that the hypobaric, hypoxic environment after the plateau ascent significantly suppresses the mice's appetite and causes weight loss, with statistically significant differences between the groups.

[0056] 4. Serum levels of appetite hormones neuropeptide Y (NPY), peptide tyrosine (PYY), and ghrelin were determined according to the ELISA kit instructions. Table 2 shows the changes in NPY, PYY, and ghrelin levels in mouse serum.

[0057] Table 2 Changes of NPY, PYY and Ghrelin levels in mouse serum ( n=10)

[0058]

[0059] Note: * indicates P < 0.05, which is significantly different from the plain control group; ** indicates P < 0.01, which is extremely significant compared with the plain control group.

[0060] Compared with the P group, serum NPY levels in mice in groups G7 and G1 were lower, with no significant difference between the two groups (P < 0.05). Serum PYY levels in mice in groups G7 and G1 were higher, with no significant difference between the two groups (P < 0.05). Serum ghrelin levels in mice in groups G7 and G1 were higher, with no significant difference between the two groups (P < 0.05). Serum ghrelin levels in mice in groups G7 and G1 were higher, reaching 1.49 ± 0.44 ng / mL. Serum PYY levels increased, while NPY levels decreased, with a significant difference between the one-day plateau exposure group and the control group (P < 0.05). Serum ghrelin levels increased in all groups after the acute plateau exposure, indicating that the decrease in hypothalamic NPY secretion during the acute plateau exposure is not primarily due to decreased ghrelin secretion.

[0061] 5. MiSeq library construction: Follow the instructions of the NEXTFLEX Rapid DNA-Seq Kit to complete the MiSeq library construction. The specific steps are as follows: (1) Add adapter links to both ends of the DNA fragments and use magnetic beads to remove the adapter self-linked fragments; (2) Enrich the library template by PCR; (3) Use magnetic beads to recover the PCR products of mouse fecal samples to complete the library construction.

[0062] 6. Illumina sequencing: DNA sequencing was performed using the Miseq PE250 high-throughput sequencing platform.

[0063] Example 2

[0064] In the early stages of this study, we investigated the effects of functional factors, including inulin, Codonopsis polysaccharides, and Bifidobacterium, on a simulated plateau altitude excursion at 5000 m. We established a control group, a plateau control group, a high-dose group, a medium-dose group, and a low-dose group. Ultimately, we screened out three functional factors: inulin, Codonopsis polysaccharides, and Bifidobacterium, which affected the secretion of appetite hormones and food intake in mice, and all showed improved appetite after acute plateau exposure. The appetite-improving effects were most significant in the medium- and high-dose inulin, low-dose Codonopsis polysaccharides, and low-dose Bifidobacterium groups. These three functional factors, inulin, Codonopsis polysaccharides, and Bifidobacterium, modulate the intestinal microbiota by increasing the relative abundance of beneficial bacteria, such as Bifidobacterium, in the mice's intestines. However, using Bifidobacterium alone as a functional factor was not ideal for modulating the intestinal microbiota. The abundance of functional traits related to carbohydrate, amino acid, fat, nucleotide, and energy metabolism in the intestinal microbiota of the medium- and high-dose inulin, low-dose Codonopsis polysaccharides, and low-dose Bifidobacterium groups increased to varying degrees after acute plateau exposure. It shows that under the conditions of rapid entry into high altitude, the above functional factors have a positive promoting effect on the expression of genes related to energy metabolism in the intestinal flora.

[0065] Food intake remained stable across all groups of mice in the plain environment, with no trend over time. However, medium and high doses of inulin resulted in a statistically significant decrease in average food intake. This mechanism may be due to inulin's inability to be metabolized and subsequently binding to fat, protein, and other substances in the intestine, reducing energy metabolism. Furthermore, inulin intake increases gastrointestinal emptying time, thereby suppressing food intake. These two factors combined resulted in a decrease in food intake in the medium and high inulin dose groups compared to the plain control group. After acute exposure to the plateau, average food intake in the medium and high inulin dose groups increased significantly compared to the plateau control group, achieving statistical significance. The low-dose group also experienced a statistically significant increase in food intake compared to the plateau control group, but the difference was not statistically significant. This suggests that medium and high doses of inulin are effective in regulating the appetite of mice exposed to the plateau environment. In the plains, the average weight of mice in the high-dose group initially decreased and then increased, reaching a minimum of 21.83g on the fourth day. It then slowly increased until it exceeded the average weight at the start of the experiment on the ninth day. The difference from the control group was statistically significant from the third day onward. The average weight of mice in the remaining groups gradually increased over time. After the rapid transition to the plateau, the weight of mice in all experimental groups decreased significantly, with significant differences from the PC group (P < 0.001). The magnitude of the weight loss in each dose group was less than that in the plateau control group.

[0066] Example 3

[0067] Further, a single-factor experiment on the appetite-promoting effect of three functional factors, inulin, codonopsis polysaccharide, and bifidobacterium, on mice exposed to rapid altitude exposure was conducted, as well as a single-factor experiment on the gavage cycle of the functional factors. The concentrations of each functional factor and the gavage time in the three-factor three-level response surface experiment were determined.

[0068] 1. Determine the intermediate dose of inulin administered orally to animals in single-factor experiments to be 2,000 mg / kg -1 ·d -1 The dosage range is 1000 to 3000 mg kg -1 ·d -1 The intermediate dose of Codonopsis pilosula polysaccharide in animal experiments was 500 mg·kg -1 ·d -1 The dosage range is 100-900 mg·kg -1 ·d -1 The median oral dose of Bifidobacterium in animal experiments was 4.0×10 7 CFU·d -1 , the dose range was 1.6×10 6 ~10 9 CFU·d -1The variable factor was set to a gradient gavage dose of 5 levels, and the dose of the nonvariable functional factor was uniformly set to the intermediate dose. The gavage volume of the experimental mice was set to 0.1 mL / 10 g (based on a mouse weight of 20 g, the gavage volume was 0.2 mL). The gavage protocols for the functional factor single-factor experiment and the gavage cycle experiment are shown in Table 3.

[0069] Mice were grouped: 150 mice were weighed and randomly divided into 15 groups, each containing 10 mice. These groups included a plain control group (PC group), a plateau control group (HC group), five gradient-dose inulin groups (corresponding to InA, InB, InC, InD, and InE groups), five gradient-dose Codonopsis polysaccharide groups (corresponding to CPPA, CPPB, CPPC, CPPD, and CPPE groups), and five gradient-dose Bifidobacterium groups (corresponding to BA, BB, BC, BD, and BE groups). Because the intermediate-dose groups for each functional factor had consistent gavage doses and test parameters, and to minimize unnecessary animal mortality and adhere to the principles of "reduction, substitution, and optimization" during experimental animal use, the intermediate-dose groups for each functional factor were shared into one group (i.e., InC, CPPC, and BC groups were combined into one group). Cages were numbered according to group, and mice in each group were placed in two cages, with 5 mice in each.

[0070] 2. Grouping of experimental animals during functional factor gavage cycles

[0071] Adaptive feeding was the same as before, and mice were grouped. Mice were stratified by 1 g weight, and 40 mice were weighed and selected. They were randomly divided into five groups of 10 mice each, namely the 3-day group, the 7-day group, the 14-day group (i.e., the InC group), the 21-day group, and the 28-day group. Cages were numbered according to group, and mice in each group were placed in two cages, with 5 mice in each cage.

[0072] For gavage and plateau exposure, according to the experimental animal grouping plan, remove the prepared functional factor solution from the refrigerator daily and thaw in a water bath at 38°C for 15 minutes. Weigh the mice every three days and calibrate the gavage volume. Gavage should be performed between 9:00 and 11:00 AM daily.

[0073] Mice in the PC group were housed in the animal room throughout the experiment, maintained under adaptive feeding conditions, and fasted for 12 hours before sampling, with adequate water. Mice in the HC group were housed in the animal room for 14 days before the experiment, maintained under adaptive feeding conditions, and transferred to a high-altitude environment simulation chamber on the morning of the 14th day. Samples were collected one day later. They fasted for 12 hours before sampling and maintained adequate water. Mice in the other experimental groups were housed in the animal room during gavage administration, maintained under adaptive feeding conditions, and transferred to a high-altitude environment simulation chamber after gavage administration. Samples were collected one day later. They fasted for 12 hours before sampling and maintained adequate water.

[0074] Table 3 Gavage schemes for single-factor experiments of functional factors and gavage cycle experiments

[0075]

[0076] The results of the single-factor inulin dose experiment showed that the serum NPY concentration of mice in the InC group was the highest in all experiments; the serum NPY concentration of mice in the InB, InC, and InD groups was higher than that in the HC group, and the results were statistically significant. The corresponding doses of the InB, InC, and InD groups were determined to be 1500-2500 mg·kg -1 ·d -1 The dose range of inulin in the response surface experiment was 2 000 mg·kg -1 ·d -1 An intermediate dose.

[0077] The results of the single-factor experiment on the dosage of Codonopsis pilosula polysaccharide showed that the serum NPY concentration of mice in the CPPC group was the highest in all experiments; the NPY concentrations in the serum of mice in the CPPC and CPPD groups were higher than those in the HC group, and the results were statistically significant. The corresponding dosages for the CPPB, CPPC, and CPPD groups were determined to be 300-700 mg·kg -1 ·d -1 The dosage range of Codonopsis pilosula polysaccharide in the response surface experiment was 500 mg·kg -1 ·d -1 An intermediate dose.

[0078] The results of the single-factor experiment on Bifidobacterium dosage showed that the serum NPY concentration of mice in the BB group was the highest in all experiments; the serum NPY concentrations of mice in the BA, BB, and BC groups were all higher than those in the HC group, and the results were statistically significant. The corresponding doses for the BA, BB, and BC groups were determined to be 1.6×10 6 ~4.0×10 7 CFU·d -1 The dosage range of Bifidobacterium in the response surface experiment was selected as 2.08×10 7 CFU·d -1 An intermediate dose.

[0079] Results from a single-factor experiment using gavage duration showed that serum NPY concentrations in mice gradually increased with prolonged gavage duration from 3 to 14 days, reaching a significant difference from the HC group by day 14 (P < 0.05). After day 14, serum NPY levels fluctuated with prolonged gavage duration, indicating that the increase in serum NPY reached a plateau and that the functional benefit from prolonged gavage duration was not significant. Therefore, a 14-day gavage duration was maintained for the response surface experiment.

[0080] Based on the results of the single-factor experiments, a response surface experiment was designed to optimize the concentration of the appetite factor NPY. Inulin dose, Codonopsis polysaccharide dose, and Bifidobacterium dose were used as experimental factors, and NPY concentration in mouse serum was used as the response. Using DesignExpert 13 software, a three-factor, three-level response surface design was conducted using the Box-Behnken central composite design principle. The design table is shown in Table 4, and the response surface results are shown in Table 5.

[0081] Table 4. Response surface experiment factor level table

[0082]

[0083] Table 5 Response surface experiment results

[0084]

[0085] The results of variance analysis of the response surface regression equation show that the model's P value is less than 0.000 1, indicating that the established model is extremely significant; the lack-of-fit term P value is 0.416 0, and the lack-of-fit term is not significant, indicating that the degree of fit of the selected quadratic polynomial model is good. 2 =0.984 7, indicating that the correlation of the regression equation is good, R adj 2 =0.9649, indicating that 96.49% of the variability in the experimental data can be explained by this regression equation. The corresponding P values ​​indicate that inulin A and all quadratic terms have extremely significant effects on serum NPY levels in mice exposed to rapid altitude exposure (P < 0.001). Bifidobacterium C and the BC interaction term have extremely significant effects (P < 0.01), and the AB interaction term has a significant effect (P < 0.05). The remaining factors are non-significant. The F values ​​indicate that the order of influence of the three factors on serum NPY levels in mice exposed to rapid altitude exposure is: inulin (A) > Bifidobacterium (C) > Codonopsis (B).

[0086] 1. The predicted value of the optimal functional factor combination formula for improving the appetite of mice rapidly entering high altitude is 2134.13 mg·kg of inulin -1 ·d -1 , Codonopsis pilosula polysaccharide 495.04 mg·kg -1 ·d -1 , Bifidobacterium 2.45×10 7 CFU·d -1 After mice were gavaged with this combination formula for 14 days, the predicted value of serum NPY content in mice under acute plateau conditions was 259.88 pg·mL -1 .

[0087] 2. The effects of various functional factors on serum NPY levels in mice exposed to high altitude conditions were ranked from greatest to least: inulin, Bifidobacterium, and Codonopsis. Significant interactions were observed between inulin and Codonopsis polysaccharides, and between Codonopsis polysaccharides and Bifidobacterium, while the interaction between inulin and Bifidobacterium was not significant.

[0088] Example 4

[0089] According to the results of response surface experiment, the oral doses of functional factor combination formula were determined to be inulin 2134.13 mg·kg -1 ·d -1 , Codonopsis pilosula polysaccharide 495.04 mg·kg -1 ·d -1 , Bifidobacterium 2.45×10 7 CFU·d -1 The oral gavage volume of the experimental mice was set at 0.1 mL / 10 g (calculated based on a mouse weight of 20 g, the oral gavage volume was 0.2 mL), and the functional factor solution of the corresponding concentration was calculated and prepared.

[0090] 1. Adaptive feeding, same as Example 1

[0091] 2. Animal grouping

[0092] Thirty mice were weighed and randomly divided into three groups of 10 mice each: a plain control group, a plateau control group, and a functional factor intervention group, each designated as the PC group, the HC group, and the T group. Cages were numbered according to group, and mice in each group were placed in two cages, with five mice in each.

[0093] 3. Oral administration and rapid plateau admission plan

[0094] Mice in the PC group were housed in the animal room throughout the experiment, maintaining adaptive feeding conditions. Mice in the HC and T groups were housed in the animal room for 14 days before the experiment, maintaining adaptive feeding conditions. On the morning of the 14th day, mice were transferred to a high-altitude environment simulation chamber (simulating an altitude of 5000 m) and samples were collected one day later.

[0095] 4. Obtain materials

[0096] At the end of the experiment, feces were collected from the PC and HC groups. Fecal samples were collected from the T group before and after the simulated plateau environment. These samples were labeled as the T0 group (fecal sample collected before the plateau) and the T group (fecal sample collected after the plateau environment) for intestinal flora diversity testing. Blood collection began immediately after fecal collection. The specific procedures were the same as in Example 1.

[0097] 5. Body weight and food intake measurement

[0098] Same as Example 1. The food intake and body weight of each group of mice were recorded and sorted during the experiment. The average body weight of mice in the Codonopsis polysaccharide functional factor experiment is shown in Table 6.

[0099] Table 6 Statistics of daily food intake and body weight of mice ( n=10)

[0100]

[0101] Note: 1. *** indicates P < 0.001, which is extremely significant compared with the PC group. 2. ### indicates P < 0.01, which is extremely significant compared with the HC group.

[0102] After 14 days of the plain environment experiment, the average daily food intake of mice in the PC group was 3.12±0.32g; the average daily food intake of mice in the HC group was 3.29±0.33g; and the average daily food intake of mice in the T group was 3.00±0.27g. There was no significant difference among the three groups.

[0103] During a 1-day simulated plateau environment experiment at an altitude of 5,000 m, the average daily food intake of mice in the HC group was 0.13±0.04 g; the average daily food intake of mice in the T group was 0.51±0.03 g, both significantly different from the HC group (P<0.001). This indicates that in terms of food intake, the average food intake of mice in the plain environment was relatively stable, with no trend change over time. The average food intake of the functional factor intervention group was lower than that of the two control groups, but the difference was not statistically significant. Under the rapid plateau environment, the average food intake of both the plateau control group and the functional factor intervention group decreased compared with the plain control group, but the average food intake of the functional factor intervention group was higher than that of the plateau control group, with a significant difference between the groups (P<0.001). This indicates that the functional factor combination formula can significantly improve the appetite of mice under rapid plateau conditions and alleviate the adverse effects of rapid plateau exposure on appetite.

[0104] After 14 days of the plains experiment, the average weight of mice in the PC group increased by 2.63g; the average weight of mice in the HC group increased by 2.33g; and the average weight of mice in the T group increased by 1.70g. In terms of weight, the average weight of mice in each group showed a gradual increase over time under plains conditions. Among them, the average weight of mice in the functional factor intervention group was slightly lower than that of the two control groups, and there was no significant difference in the average weight of mice in each group.

[0105] After a one-day simulated rapid ascent to a 5,000-meter altitude, mice in the T group lost an average of 1.99 g in weight, while those in the HC group lost an average of 3.18 g. While all groups experienced significant weight loss after the rapid ascent, the T group experienced a significantly smaller decrease than the HC group (P < 0.001). Figure 1This is a comparison of the average body weight differences of mice in each group on days 0, 3, 7, 10, 14, and 15. Figure 2 Comparison of mean body weight changes in each group of mice on day 15. 1. *** indicates a highly significant difference compared to the PC group (P < 0.001). 2. ### indicates a highly significant difference compared to the HC group (P < 0.001). Following acute plateau exposure, mean body weight decreased in both the plateau control group and the functional factor intervention group. The decrease in the functional factor intervention group was significantly less than in the plateau control group (P < 0.001). Following acute plateau exposure, changes in body weight correlated with changes in average food intake.

[0106] 6. Determination of appetite hormone content in serum

[0107] The determination method is the same as in Example 1. The levels of appetite hormones such as NPY and PYY in the serum of mice are shown in Table 7.

[0108] Table 7 Changes in the levels of appetite hormones in mouse serum ( n=10)

[0109]

[0110] Note: * indicates P < 0.05, which is significantly different from the HC group.

[0111] After rapid exposure to the plateau, the NPY content in the serum of each group of mice decreased compared with the PC group, while the PYY content increased compared with the PC group. Among them, the serum PYY content in the T group was lower than that in the HC group; the serum NPY content was higher than that in the HC group, and the difference between the groups was significant (P < 0.05). After rapid exposure to the plateau, the NPY content in the serum of each group of mice decreased compared with the PC group, while the PYY content increased compared with the PC group. Under the action of the combined formula, the change in appetite hormones in the T group after rapid exposure to the plateau was smaller than that in the HC group, and the difference in serum NPY content between the two groups was significant (P < 0.05). The serum NPY concentration of mice after rapid exposure to the plateau was 243.17 pg·mL -1 , and the response surface model predicted concentration value of 259.88 pg·mL -1 The results were close, indicating that the response surface model can better predict the efficacy of the functional factor combination formula, and the combination formula can alleviate the impact of the rapid plateau environment on the appetite hormone content in the serum of mice.

[0112] 7. Intestinal flora diversity detection

[0113] 7.1 Sequencing Quality

[0114] PCR amplification was performed on mouse fecal samples, and the product quality inspection was qualified. The PCR product was detected by agarose gel electrophoresis, and the target band size was correct and the concentration was appropriate, which met the requirements of subsequent sequencing analysis. Figure 3 .

[0115] After Illumina sequencing and data optimization, 3,140,705 valid sequences were obtained from mouse fecal samples, with an average sequence length of 418 bp. To ensure the quality of subsequent microbial diversity and composition analysis, the samples were leveled to a minimum number of sequences (19,866 sequences per sample). After excluding chloroplast and mitochondrial sequences, a total of 28,074 ASVs were obtained. The rarefaction curve shows that when the number of sequenced sequences exceeds 12,000, the curve enters a plateau and rises slowly, indicating that the number of newly added ASVs is limited with increasing sequencing load. This indicates that the sequencing was sufficient to cover the majority of ASVs in the samples, and the sequencing results can reflect the majority of microbial species in the samples.

[0116] Pan-Core species analysis was performed on the four sample groups at the genus level. Genus-level Pan-Core species analysis curves of the mouse gut microbiota were generated based on the number of Pan and Core species in the four sample groups. These curves showed that when the sample size exceeded 6, the curves gradually flattened, indicating that the number of Pan and Core species in each group changed more gradually with increasing sample size. The sample size selected for this sequencing was sufficient to cover the vast majority of species in the samples. The results showed that the T0 group had extremely significant differences in Pan species compared to the PC group (P < 0.01). Compared to the HC group, the T group had extremely significant differences in Pan species (P < 0.001) and significant differences in Core species (P < 0.05).

[0117] 7.2 Alpha Diversity Analysis

[0118] Alpha diversity analysis of mouse intestinal flora was performed at the genus level, and the results are shown in Table 8.

[0119] Table 8 Alpha diversity of mouse intestinal flora ( n=10)

[0120]

[0121] Note: 1. ** 2. ### indicates that the difference is extremely significant when compared with the PC group, P < 0.01. 3. Indicates significant difference compared with T0 group, P < 0.05; Compared with the T0 group, P < 0.01, the difference is extremely significant.

[0122] The Ace and Chao indices, which are positively correlated with species richness, were higher in the HC group compared with the PC group, with no significant differences; lower in the T0 group compared with the PC group, with no significant differences; and lower in the T group compared with both the HC and T0 groups, with extremely significant differences (P < 0.001) and extremely significant differences (P < 0.01), respectively. The Shannon index, which is positively correlated with species diversity, was lower in the HC group compared with the PC group, with no significant differences; there was a downward trend in the T0 group compared with the PC group, but the difference was not significant; and lower in the T group compared with both the HC and T0 groups, with an extremely significant difference (P < 0.01). The Simpson index, which is negatively correlated with species diversity, was higher in the HC group compared with the PC group, with an extremely significant difference (P < 0.01); higher in the T0 group compared with the PC group, with no significant differences; lower in the T group compared with the HC group, with no significant differences; and higher in the T group compared with the T0 group, with an extremely significant difference (P < 0.01). The Shannoneven index, which is positively correlated with species evenness, was significantly lower in the HC group compared to the PC group (P < 0.01). The T0 group showed a nonsignificantly lower index compared to the PC group, but the difference was not significant. The T group showed a nonsignificantly higher index compared to the HC group, and a significant lower index compared to the T0 group (P < 0.05). The species coverage index for each group was 1, indicating that the sequencing results are representative of the true microbial population in the samples.

[0123] Alpha diversity analysis showed that the ace index and chao index of the functional factor intervention group decreased significantly after acute plateau exposure compared with the control group, with statistically significant differences. This result may be due to the significant increase in the relative abundance of Bifidobacterium in the gut microbiota of mice exposed to the functional factor combination formula. Beta diversity analysis showed that samples from the HC and PC groups were the farthest apart, clustering at the upper and lower ends of the dendrogram, respectively. Samples from the T and T0 groups clustered in the middle of the dendrogram, with the T group closer to the HC and the T0 group closer to the PC group. This suggests that the changes in the gut microbiota of mice exposed to the functional factor combination formula after acute plateau exposure were minimal and closer to the plain control group than to the plateau control group. Analysis of microbial composition and inter-group differences showed that the relative abundance of probiotics, such as Bifidobacterium and Lactobacillus, in the gut microbiota of mice exposed to the functional factor combination formula increased compared with the control group before and after acute plateau exposure, with statistically significant differences in Bifidobacterium between groups.

[0124] 7.3 Prediction of Functional Characteristics of Mouse Gut Microbiota

[0125] Based on the analysis of mouse gut microbiome sequencing results, PICRUSt2 software was used to predict and analyze the functional characteristics of the gut microbiota in each sample group. The annotated functional genes were then classified according to the COG gene function classification. The COG functions of the mouse gut microbiota primarily include translation, ribosome structure and biogenesis, transcription, replication, recombination and repair, energy production and conversion, carbohydrate transport and metabolism, amino acid transport and metabolism, nucleotide transport and metabolism, fat transport and metabolism, and inorganic ion transport and metabolism. The abundance values ​​corresponding to the COG functional characteristics of the gut microbiota are shown in Table 9.

[0126] Table 9 Abundance values ​​of functional characteristics of mouse intestinal flora ( n=10)

[0127]

[0128] Note: 1. * Indicates P < 0.05, compared with the PC group, the difference is significant; ** Indicates that P < 0.01 compared with the PC group, the difference is extremely significant; *** Compared with the PC group, P < 0.001 indicates that the difference is extremely significant. 2. # Compared with the HC group, P < 0.05 indicates a significant difference.

[0129] After the rapid plateau excursion, the abundance of functional traits closely related to energy metabolism, such as energy production and conversion, in the gut microbiota of mice in the control group decreased statistically significantly compared to the plain control group. However, under the influence of the functional factor combination formula, the abundance of functional traits closely related to transcription, energy production and conversion, nucleotide transport and metabolism, and carbohydrate transport and metabolism in the gut microbiota of mice increased statistically significantly. This suggests that the functional factor combination formula has a positive effect on promoting the expression of some genes closely related to energy metabolism in the gut microbiota of mice under the conditions of rapid plateau excursion.

[0130] Example 5

[0131] According to the single factor experiment, the dose range of inulin response surface is 1500~2500mg·kg -1 ·d -1 The dose range of the response surface of Codonopsis pilosula polysaccharide is 300~700mg·kg -1 ·d -1 The dose range of the response surface of Bifidobacterium was 1.6×10 6 ~4.0×10 7 CFU·d -1 .

[0132] According to the response surface experiment, the optimal dosage of functional ingredients was inulin 2134.13 mg·kg -1 ·d-1 , Codonopsis pilosula polysaccharide 495.04 mg·kg -1 ·d -1 Based on the concept of human equivalent dose, the human dose that may produce equivalent efficacy is inferred from animal experiments. The dose calculation of inulin and codonopsis polysaccharide is shown in formula (1):

[0133] D b = D a ×R ab (1)

[0134] Where D b is the equivalent human dose, D a is the mouse dose, R ab The conversion factor between the two is 0.081. Calculation shows that the equivalent dose of inulin for humans is 121.50 mg kg -1 ·d -1 ~202.50 mg·kg -1 ·d -1 , preferably 172.86 mg·kg -1 ·d -1 The human equivalent dose of Codonopsis pilosula polysaccharide is 24.30 mg·kg -1 ·d -1 ~56.70 mg·kg -1 ·d -1 , preferably 40.10 mg·kg -1 ·d -1 Calculated based on a standard adult weight of 60 kg, the daily intake of inulin is 7.29 g to 12.15 g, preferably 10.37 g, and the daily intake of Codonopsis polysaccharide is 1.46 g to 3.4 g, preferably 2.41 g.

[0135] The mass ratio P of inulin and codonopsis polysaccharide in the functional food formula is calculated as shown in formula (2):

[0136]

[0137] Where M In is the mass of inulin raw material, M CPP is the quality of Codonopsis pilosula polysaccharide raw material, D In The human equivalent dose of inulin is 172.86 mg·kg -1 ·d -1 , D CPP The human equivalent dose of Codonopsis pilosula polysaccharide is 40.10 mg·kg -1 ·d -1 , k In is the percentage of inulin in the inulin raw material, the raw material in this embodiment is 90%, k CPPThe percentage of codonopsis polysaccharide in the codonopsis polysaccharide raw material is 50%. Substituting the above values ​​into formula (2), the mass ratio P of inulin and codonopsis polysaccharide in the present embodiment is calculated to be 2.39.

[0138] The dose range of the response surface of Bifidobacterium in mouse animal experiments was 1.6×10 6 ~4.0×10 7 CFU·d -1 The optimal dose is 2.45×10 7 CFU·d -1 Based on the weight of 20g mice and the standard weight of 60kg adults, the human equivalent dose of Bifidobacterium is calculated by combining formula (1) to be 3.89×10 8 CFU·d -1 ~9.72×10 9 CFU·d -1 , best 5.95×10 9 CFU·d -1 .

[0139] After factory production and debugging, the net weight of each package is set at 3.2g, and the proportion of maltodextrin auxiliary material added is 10%. Based on 3 packages per person per day, the number of viable bifidobacteria added to each package is 1.98×10 9 CFU·d -1 Each package of functional food contains 2.03g of inulin, 0.85g of codonopsis polysaccharide, 0.32g of maltodextrin, and about 0.003g of bifidobacterium powder (animal Bifidobacterium lactis subspecies BLa36, Jiangsu Weikang Biotechnology Co., Ltd.). The combined use of prebiotics and probiotics achieves the superposition and synergy of functional factors, further promoting the efficacy of functional factors; no refrigeration is required during storage and use, and there are no special requirements for supply guarantee conditions and usage scenarios, which facilitates storage and use under field conditions; there is no high temperature environment during the processing of ready-to-eat functional foods, which can effectively ensure the activity and efficacy of probiotics in the finished functional food products.

[0140] According to the ratio, the raw materials such as inulin, codonopsis polysaccharide, bifidobacterium powder and maltodextrin are weighed and added into the three-dimensional mixer for thorough mixing for 20 minutes. Then the finished products are packaged. The mixed raw materials are added into the powder packaging machine, and the finished product packaging operation is performed after adjusting the relevant parameters. Among them, the mass of each package of synbiotic powder is set to 3.2g, and the packaging speed is set to 40 packages / min. Tasting food: Instant synbiotic powder functional food commissioned by Sichuan Gaofuji Biotechnology Co., Ltd. The finished food and packaging samples are shown in the figure below. Figure 4 shown.

[0141] Participants: 80 male subjects, primarily from organized units at Army Base XX, aged 18 to 23 (mean age 21.08), were randomly divided into a test group (Group T) and a control group (Group C), 40 subjects in each group. All subjects had no history of colds, fevers, or other illnesses within the first two weeks of participation. Test locations: 1. Camp A, Army Base XX (320m above sea level); 2. Camp B, Army Base XX (3650m above sea level).

[0142] Trial time: Early to mid-June 2023, from June 1st to 7th, nutritional surveys and sample collection work will be mainly carried out in Camp A before the rapid entry into the plateau; from June 5th to 13th, functional food tasting (T group) will be organized, 3 packs per person per day, which can be eaten directly with meals in the morning, noon and evening, or brewed with warm water after meals. The control group C will be guaranteed normal diet throughout the whole process; from June 7th to 9th, go to Camp B; from June 10th to 13th, nutritional surveys and blood and fecal sample collection will be carried out in Camp B after the rapid entry into the plateau. Among them, the T group samples collected in Camp A in the low-altitude area are marked as PT group, and the C group samples are marked as PC group; the T group samples collected in Camp B in the plateau area are marked as HT group, and the C group samples are marked as HC group.

[0143] Test results

[0144] 1. Questionnaire Survey: 40 questionnaires were distributed, 40 of which were returned, with 39 valid responses, for a return rate of 97.5%. The survey results are summarized in Table 10. 100% of respondents approved of the taste and 100% approved of the ease of use. No adverse reactions were reported.

[0145] Table 10 Evaluation results of new military functional food tasting (%, n = 39)

[0146]

[0147] 2. Serum appetite hormone content

[0148] After acute exposure to high altitude, serum PYY levels increased in Group C, while NPY levels decreased. However, after the functional food supplementation, serum PYY levels decreased in Group T, while NPY levels increased. Comparison of the two data groups revealed a negative correlation between serum NPY and PYY levels, consistent with previous animal studies. The results are shown in Table 11.

[0149] Table 11: Serum appetite hormone levels ( n=40)

[0150]

[0151]

[0152] Note: * indicates P < 0.05, which is significantly different from the HC group.

[0153] 3. Gut microbiome sequencing results showed significant differences in the core species between the HT and HC groups (P < 0.05); there were no significant differences in the pan and core species between the other groups. The abundance values ​​corresponding to the main functional characteristics of the gut microbiome are shown in Table 12.

[0154] Table 12 Abundance values ​​of functional characteristics of intestinal flora

[0155]

[0156] Note: 1. * indicates P < 0.05, significant difference compared with the PT group. 2. # indicates P < 0.05, significant difference compared with the HC group.

[0157] There were significant differences in the abundance of coenzyme transport and metabolism functional characteristics between the HT group and the PT group (P < 0.05); there were significant differences in the abundance values ​​of three functional characteristics, including coenzyme transport and metabolism, cell wall / membrane / membrane structure biosynthesis, and secondary metabolite biosynthesis, transport and catabolism, between the HT group and the HC group (P < 0.05).

[0158] 4. Nutrition Survey: Statistical analysis of dietary survey results from Camps A and B revealed energy intake before and after the rapid plateau arrival. Energy intake in the low-altitude camps was 3637.87 kcal / day, while in the plateau, Group C had 3498.35 kcal / day and Group T had 3546.76 kcal / day. After the rapid plateau arrival, energy intake in Groups C and T decreased to varying degrees compared to that in the low-altitude camps, with the decrease in Group T being less pronounced than in Group C.

[0159] Statistical analysis of participants' weight, body mass index, and other physical indicators before and after the rapid plateau excursion is shown in Table 13. The results show that Group C experienced a mean weight loss of 0.98 kg and a mean body mass index decrease of 0.32 after the rapid plateau excursion. Group T experienced a mean weight loss of 0.16 kg, a significant difference from Group C (P < 0.05), and a mean body mass index decrease of 0.05, also a significant difference from Group C (P < 0.05).

[0160] Table 13 Average weight and body mass index of participants (n=40)

[0161]

[0162]

[0163] Note: * indicates P < 0.05, which is significantly different from group C.

[0164] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A functional composition for improving the appetite of people who urgently go to high altitude, characterized in that: The functional composition consists of inulin, codonopsis polysaccharide, bifidobacteria and excipients; the mass ratio of inulin to codonopsis polysaccharide in the functional composition is 15-25:3-7, and the human equivalent dose of codonopsis polysaccharide is 24.30 mg·kg -1 ·d -1 ~56.70 mg·kg -1 ·d -1 The number of viable bifidobacteria per gram of Codonopsis polysaccharide was 1.14×10 8 CFU~6.67×10 9 CFU; the bifidobacterium is Bifidobacterium lactis subspecies BLa36.

2. The functional composition for improving appetite of people who rapidly enter plateau according to claim 1, characterized in that: The mass ratio of inulin to codonopsis polysaccharide in the functional composition is 4.31:1, wherein the number of viable bifidobacteria is 2.47×10 9 CFU.

3. The functional composition for improving the appetite of people who rapidly enter plateau according to any one of claims 1 to 2, characterized in that: The auxiliary materials are food grade auxiliary materials or pharmaceutical auxiliary materials.

4. The functional composition for improving appetite of people who rapidly enter plateau according to claim 3, characterized in that: The food grade auxiliary material is selected from nutritional enhancers, antioxidants, colorants, flavor enhancers, flavoring agents, thickeners, preservatives, sweeteners, anticaking agents or acidity regulators.

5. The functional composition for improving appetite of people who rapidly enter plateau according to claim 4, characterized in that: The sweeteners are maltitol, acesulfame potassium, cyclamate, saccharin sodium, aspartame, stevia, sucrose or white sugar.

6. The functional composition for improving appetite of people who rapidly enter plateau according to claim 4, characterized in that: The thickener is maltodextrin.

7. The functional composition for improving appetite of people who rapidly enter plateau according to claim 4, characterized in that: Nutritional enhancers include vitamins, minerals, dietary fiber, taurine and choline.

8. Use of the functional composition according to any one of claims 1 to 7 in the preparation of functional food or medicine for improving appetite in people who rapidly enter plateau.

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