Food for improving muscle quality and function as well as preparation method and application thereof

Abstract

The invention provides a food capable of improving muscle quality and functions as well as a preparation method and application of the food. The food contains lactoferrin and creatine, and the mass ratio of the lactoferrin to the creatine is (3-9): 1. In terms of the total dry matter content of the food, the content of the lactoferrin in the food is 0.1-90 g / 100 g, and the content of the creatine in the food is 0.01-30 g / 100 g. The food disclosed by the invention can play an important role in preventing, treating and improving the sarcopenia syndrome by improving muscle quality, muscle strength and functions, promoting myogenic differentiation and the like in multiple aspects and multiple dimensions.

Description

Technical Field

[0001] The present invention belongs to the field of food technology, and specifically relates to a food containing lactoferrin and creatine, in particular a food for middle-aged and elderly people, a health food, a dietary nutritional supplement or a special medical purpose formula food that can improve muscle attenuation and promote myogenic differentiation and is beneficial to muscle health. Background Art

[0002] Sarcopenia is a syndrome characterized by a progressive age-related decrease in skeletal muscle mass, accompanied by a decrease in muscle strength and / or muscle function. It often results in reduced mobility in middle-aged and elderly people, affecting daily activities such as walking, sitting, climbing, and lifting heavy objects, and may even lead to balance disorders, difficulty standing, and a high risk of falling.

[0003] Epidemiological surveys show that skeletal muscle mass decreases with age. Reports indicate that starting at age 40, the body loses 0.5% to 1% of its muscle mass annually. After age 50, the average annual decrease in skeletal muscle mass is 1% to 2%. Chronic muscle loss reaches over 30% in those over 60, and approximately 50% in those over 80. A 30% decrease in muscle mass can affect normal muscle function, resulting in symptoms such as muscle flab, increased skin wrinkling, weight loss, frailty, and decreased immunity. International epidemiological surveys show that sarcopenia affects 5% to 13% of those aged 60 to 70, increasing to 11% to 52% in those aged 80 and above. Domestic studies have shown that the prevalence of sarcopenia among urban elderly people in Taiwan has reached 14.4%. Data from a Shanghai study indicate that the prevalence of sarcopenia in men over 60 is 12.3% and in women 4.8%.

[0004] With the aging population, the prevention and treatment of sarcopenia has become a major public health issue. Currently, the main treatments for sarcopenia include medication, muscle exercise training, and nutritional support. Medication primarily involves the use of synthetic hormones or other chemicals to stimulate muscle growth, but these drugs have significant side effects and their long-term safety is questionable. Exercise therapy primarily aims to improve muscle mass and function by increasing exercise volume, but its adherence and feasibility are low, making it particularly difficult to implement for elderly individuals with poor physical conditions. Nutritional therapy is one of the main approaches currently advocated for the prevention and treatment of sarcopenia, both domestically and internationally. Nutritional interventions that have been extensively studied and have reached consensus include adequate high-quality protein and essential amino acids (leucine), increased intake of n-3 polyunsaturated fatty acids, adequate vitamin D, increased micro-antioxidant nutrients (vitamin C, vitamin E, selenium, etc.), and HMB supplementation.

[0005] Currently, the nutritional functional foods for sarcopenia on the market mainly improve muscle mass and function by supplementing protein and essential amino acids (especially leucine), supplemented with HMB, antioxidants or medicinal and edible ingredients. Specific complete nutritional foods for sarcopenia are special medical foods specifically for this group of people. They can be used as a single source of nutrition to meet the nutritional needs of the target group, but there are currently no such products approved for marketing in my country. Therefore, there is still a shortage of nutritional functional foods specifically for people with sarcopenia on the market, and there are problems such as many ingredients, high cost, poor taste, high effective dose, and unsatisfactory results. There is an urgent need for nutritional functional foods with simple formula composition, low cost, high safety and significant efficacy to improve sarcopenia. Summary of the Invention

[0006] An object of the present invention is to provide a food that can benefit muscle health.

[0007] Another object of the present invention is to provide a method for preparing the food.

[0008] Another object of the present invention is to provide related applications of the food in improving muscle attenuation and promoting myogenic differentiation.

[0009] In one aspect, the present invention provides a food, comprising lactoferrin and creatine, wherein the mass ratio of lactoferrin to creatine is (3-9):1; based on the total dry matter content of the food, the content of lactoferrin in the food is 0.1-90 g / 100 g, and the content of creatine in the food is 0.01-30 g / 100 g.

[0010] The food of the present invention can improve muscle quality by regulating the structure of the gastrocnemius muscle, increasing the gastrocnemius muscle mass and the gastrocnemius muscle index, and can improve muscle strength and function by increasing the grid hanging time and grip strength, prolonging the exhaustion time, etc.; it can also increase the expression levels of myogenic differentiation factor 1 (MyoD1), myogenin (MyoG) and myogenic factor 5 (Myf5), and promote myogenic differentiation.

[0011] According to specific embodiments of the present invention, the food of the present invention, including but not limited to elderly food, health food, dietary nutritional supplement food, or special medical purpose formula food, is beneficial to muscle health and can alleviate physical fatigue caused by sarcopenia. It can play an important role in the prevention and treatment of sarcopenia by improving muscle mass, muscle strength and function, and promoting myogenic differentiation. In addition, the food of the present invention can also be a sports nutrition food, more specifically, a sports nutrition food suitable for people who need to repair body tissues and grow muscle.

[0012] According to a specific embodiment of the present invention, the dosage form of the food of the present invention may include powder, tablet, hard capsule, soft capsule, granule, gel candy or oral liquid, etc.

[0013] According to a specific embodiment of the present invention, in the food of the present invention, the specific content of the lactoferrin and creatine can be appropriately adjusted within the range according to the specific type of food. Specifically, based on the total dry matter content of the food, the content of the lactoferrin in the food can be 0.1-0.5g / 100g, 0.5-1g / 100g, 1-5g / 100g, 5-10g / 100g, 10-15g / 100g, 15-20g / 100g, 20-25g / 100g, 25-30g / 100g, 30-35g / 100g. 100g, 35-40g / 100g, 40-45g / 100g, 45-50g / 100g, 50-55g / 100g, 55-60g / 100g, 60-65g / 100g, 65-70g / 100g, 70-75g / 100g, 75-80g / 100g, 80-85g / 100g or 85-90g / 100g. Specifically, based on the total dry matter content of the food, the content of creatine in the food can be 0.01-0.05g / 100g, 0.05-0.1g / 100g, 0.1-0.5g / 100g, 0.5-1g / 100g, 1-5g / 100g, 5-10g / 100g, 10-15g / 100g, 15-20g / 100g, 20-25g / 100g or 25-30g / 100g.

[0014] According to a specific embodiment of the present invention, in the food of the present invention, the source of the lactoferrin includes cow's milk, goat's milk and / or their products, including but not limited to one or more of lactoferrin powder, whole milk powder, skim milk powder, and whey protein powder.

[0015] According to a specific embodiment of the present invention, in the food of the present invention, the creatine is derived from a raw material containing creatine monohydrate.

[0016] According to a specific embodiment of the present invention, in addition to the lactoferrin and creatine, the food of the present invention may further include other components, such as one or more of proteins (preferably proteins, polypeptides, and / or amino acids derived from mammalian milk), fats, vitamins, and minerals. Alternatively, the food may optionally contain auxiliary materials and / or additives used in the food and / or health food fields. The specific types and amounts of these substances added can be determined by reference to the existing technologies in the relevant fields and should comply with the requirements of relevant national or industry standards.

[0017] In this article, unless otherwise specified, when "food" and "health food" appear as parallel terms, "food" generally refers to ordinary food.

[0018] According to a specific embodiment of the present invention, the raw material composition of the food of the present invention includes, by weight: 0-80 parts of protein (other protein raw materials except lactoferrin), 0-20 parts of fat, 0.1-90 parts of lactoferrin, 0.01-30 parts of creatine, 0-4 parts of vitamin mixture, 0-15 parts of mineral mixture, and 0-30 parts of β-hydroxy-β-methylbutyrate (HMB).

[0019] According to a specific embodiment of the present invention, in the food of the present invention, the vitamin mixture contains vitamin A, vitamin D, vitamin E, vitamin C and vitamin B1, vitamin B2, pantothenic acid, niacin, vitamin B6, vitamin B 12 One or more of; the mineral mixture contains one or more of calcium, iron, magnesium, potassium, zinc, and selenium.

[0020] According to a specific embodiment of the present invention, in the food of the present invention, the mass ratio of lactoferrin to creatine is (4-8):1, more preferably (4-6):1.

[0021] On the other hand, the present invention also provides a method for preparing the food, the method comprising:

[0022] The composition of the food raw material is adjusted so that it contains lactoferrin and creatine, and the content of the lactoferrin in the food is 0.1-90g / 100g, the content of the creatine in the food is 0.01-30g / 100g, and the mass ratio of lactoferrin to creatine is (3-9):1, based on the total dry matter content of the food product, to prepare the food.

[0023] According to some specific embodiments of the present invention, the food of the present invention comprises the following components, by weight: 40-80 parts of concentrated whey protein powder, 10-25 parts of lactoferrin powder, 1.1-8.3 parts of creatine, 2.0-4.5 parts of calcium lactate, 2.5-5.0 parts of soybean lecithin, and 0.03-0.15 parts of sucralose. The preparation method of the food comprises: weighing the raw materials and auxiliary materials according to the proportions, pouring them into a dry mixer, mixing them evenly, and sealing and packaging them for later use.

[0024] According to some specific embodiments of the present invention, the food of the present invention is composed of the following components by weight: 30-65 parts of isolated whey protein powder, 5-15 parts of hydrolyzed whey protein powder, 2-4 parts of fish oil, 30-40 parts of lactoferrin, 2-13 parts of creatine, 2-4 parts of a vitamin mixture, 3-6 parts of a mineral mixture, and 2-4 parts of HMB. The preparation method of the food comprises: weighing the raw materials and auxiliary materials according to the proportions, pouring them into a dry mixer and mixing them evenly, optionally taking the prepared products and sieving and mixing them, adding an appropriate amount of water to prepare a soft material, granulating them using a one-step granulator, and then drying, sorting, and sieving them before packaging them into bags, with each bag containing 5g.

[0025] According to some specific embodiments of the present invention, the food of the present invention comprises the following components, by weight: 15-35 parts of skim milk powder, 30-70 parts of lactoferrin, 3-25 parts of creatine, 1-4 parts of a vitamin mixture, 7-15 parts of a mineral mixture, 0.5-1.5 parts of magnesium stearate, 0.5-1.5 parts of silicon dioxide, and 0.5-1.5 parts of sodium carboxymethylcellulose. The preparation method of the food comprises: weighing the raw materials and auxiliary materials according to the proportions, pouring them into a dry mixer and mixing them uniformly, placing them into a tablet press, punching them into tablets, and sealing and packaging them for later use.

[0026] According to some specific embodiments of the present invention, the food comprises the following components, by weight: 0-25 parts of concentrated whey protein powder, 65-90 parts of lactoferrin, and 10-25 parts of creatine. The food is prepared by weighing the ingredients according to a specific ratio, mixing them in a dry mixer, and then preparing a 0.5g capsule.

[0027] In the food of the present invention, the specific amount of each component can be appropriately adjusted within the above range according to the specific type of food, and should comply with the provisions of relevant national standards or industry standards.

[0028] On the other hand, the present invention also provides the use of the food in preparing a food for improving muscle attenuation and / or promoting myogenic differentiation.

[0029] According to some specific embodiments of the present invention, in the application of the food of the present invention, the improvement of muscle attenuation includes improving muscle strength, muscle quality, muscle function, etc., specifically regulating the structure of the gastrocnemius muscle, increasing the gastrocnemius muscle mass and gastrocnemius muscle index, increasing the body's lean body mass, increasing the grid hanging time and grip strength, and prolonging the exhaustion time; the promotion of myogenic differentiation includes increasing the expression levels of myogenic differentiation factor 1 (MyoD1), myogenin (MyoG) and myogenic factor 5 (Myf5).

[0030] In some specific embodiments of the present invention, the present invention has found through various experiments on model mice treated with lactoferrin and creatine that administering an effective concentration of lactoferrin and creatine combined intervention can significantly improve muscle strength, muscle mass, muscle function, etc. in mice, thereby effectively delaying the aging of skeletal muscle in mice. Through changes in mRNA expression levels, it was found that after the combined intervention of lactoferrin and creatine, the combined intervention group of lactoferrin and creatine (LF+CR group) showed varying degrees of improvement in the levels of some myogenic differentiation-related factors, such as myogenic differentiation factor 1 (MyoD1), myogenin (MyoG), myocyte enhancer factor 2C (MEF2C), and myogenic factor 5 (Myf5), compared with the aging group (MOD group), the lactoferrin group (LF group), and the creatine group (CR group).

[0031] In summary, the present invention provides a food containing lactoferrin and creatine, which improves muscle mass, strength and function by controlling the ratio of lactoferrin and creatine in the product, synergistically increasing the gastrocnemius muscle mass and gastrocnemius muscle index, increasing the grid hanging time and grip strength, prolonging the exhaustion time, etc., thereby effectively delaying the aging of skeletal muscle; at the same time, it also produces a synergistic effect on the expression levels of myogenic differentiation factor 1 (MyoD1), myogenin (MyoG) and myogenic factor 5 (Myf5), thereby promoting myogenic differentiation; and the above technical effects are better than those of the lactoferrin and creatine intervention groups alone, and improve the skeletal muscle quality, strength and function. The results show that foods containing a nutritional composition of lactoferrin and creatine have a significant synergistic effect on improving muscle attenuation and promoting muscle differentiation. The intervention dose is lower than the current recommended daily amount of sports nutrition foods (1-3g / day) and the amount used in clinical trials related to improving muscle wasting syndrome (5-20g / day). It can improve muscle wasting syndrome in multiple aspects and dimensions such as muscle mass, muscle strength and function, and promoting myogenic differentiation. It provides a food with a simple formula, good taste, controllable cost, high safety and significant efficacy for the prevention and treatment of muscle wasting disease, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the grouping and drug administration of animal experiments.

[0033] Figure 2 The weight changes of mice in each group during the intervention process.

[0034] Figure 3 This is the mass of gastrocnemius muscle after sampling from mice in each group.

[0035] Figure 4 This is the gastrocnemius muscle index of mice in each group after sampling.

[0036] Figure 5 Lean meat content and fat content of mice in each group.

[0037] Figure 6 is the grid hanging time of mice in each group.

[0038] Figure 7 is the grip strength of mice in each group.

[0039] Figure 8 is the exhaustion time of mice in each group.

[0040] Figure 9 These are the H&E staining results of mice in each group.

[0041] Figure 10 Generate statistical histograms of relevant gene expression for muscles of each group of mice. DETAILED DESCRIPTION

[0042] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0043] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0044] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in this technical field.

[0045] In the examples, all raw materials are commercially available, wherein the percentage of lactoferrin in the total protein mass in the lactoferrin raw material is ≥95%, and the mass fraction of creatine monohydrate in the creatine raw material is ≥99.97%.

[0046] Example 1

[0047] This embodiment provides a food for improving muscle attenuation and promoting muscle differentiation, which is made from the following raw materials in parts by weight: 69 parts of concentrated whey protein powder, 18 parts of lactoferrin powder, 5.5 parts of creatine, 3.4 parts of calcium lactate, 4 parts of soy lecithin, and 0.1 part of sucralose.

[0048] Preparation method: Weigh all raw materials and auxiliary materials according to the ratio, pour them into a dry mixer and mix for 25 minutes. After mixing evenly, seal and package for later use.

[0049] Example 2

[0050] This embodiment provides a food for improving muscle attenuation and promoting muscle differentiation, which is made from the following raw materials in parts by weight: 40 parts of isolated whey protein powder, 10 parts of hydrolyzed whey protein powder, 3 parts of fish oil, 30 parts of lactoferrin, 5 parts of creatine, 4 parts of vitamin mixture, 5 parts of mineral mixture, and 3 parts of HMB.

[0051] The preparation method includes: weighing each raw material and auxiliary material according to the ratio, pouring them into a dry mixer and mixing for 25 minutes, after mixing evenly, taking 1000g of the prepared product, sieving and mixing, adding appropriate amount of water to prepare a soft material, granulating through a one-step granulator, and then drying, sorting, sieving, and canning into bags, 5g per bag.

[0052] Example 3

[0053] This embodiment provides a food for improving muscle attenuation and promoting muscle differentiation, which is made from the following raw materials in parts by weight: 24 parts of skim milk powder, 50 parts of lactoferrin, 10 parts of creatine, 3 parts of vitamin mixture, 10 parts of mineral mixture, 1 part of magnesium stearate, 1 part of silicon dioxide, and 1 part of sodium carboxymethyl cellulose.

[0054] The preparation method comprises: weighing various raw materials and auxiliary materials according to a ratio, pouring them into a dry mixer and mixing them for 25 minutes, mixing them evenly, putting them into a tablet press and punching them into tablets, and sealing and packaging them for later use.

[0055] Example 4

[0056] This embodiment provides a food for improving muscle attenuation and promoting muscle differentiation, comprising the following ingredients, by weight: 3 parts concentrated whey protein powder, 85 parts lactoferrin, and 12 parts creatine. The food preparation method comprises weighing the ingredients according to a specific ratio, mixing them in a dry mixer for 25 minutes, and then preparing a 0.5 g capsule after uniform mixing.

[0057] Example 5 Experimental study on improving muscle attenuation and promoting muscle differentiation

[0058] 1 Materials and Methods

[0059] 1.1 Experimental animals

[0060] Six-week-old, SPF-grade male C57BL / 6 mice weighing 20 ± 1 g were purchased from Speffler. They were housed at 22 ± 2°C with a 12-h light-dark cycle and allowed to acclimate for one week.

[0061] 1.2 Preparation of modeling drugs and interventions

[0062] aD-Galactose: Precision weigh D-galactose powder (Sigma, A3134) at a dose of 500 mg / kg / day based on mouse body weight for modeling. Dissolve in distilled water and inject 100 μL intraperitoneally into each mouse.

[0063] b. Lactoferrin: Accurately weigh lactoferrin powder and administer 500 mg / kg / day to mice based on their body weight. Slowly dissolve the powder in distilled water and magnetically stir at 55°C for 5 minutes to fully dissolve. The powder is then divided into portions for use. Each mouse is gavaged with a volume of 100 μL.

[0064] c. Creatine: Accurately weigh creatine powder (MCE, HY-12270) and administer 83 mg / kg / day to mice based on their body weight. Dissolve the powder in distilled water and magnetically stir at 85°C for 5 minutes to fully dissolve. The powder was then dispensed for use. Each mouse received a 100 μL oral dose.

[0065] d. Lactoferrin and Creatine Mixed Solution: Accurately weigh appropriate amounts of lactoferrin and creatine powder (MCE, HY-12270) respectively, with a lactoferrin to creatine weight ratio of 4-6:1. Heat distilled water to approximately 85°C. Slowly add creatine until the solution becomes clear. Adjust the temperature to 55°C. Once the temperature drops, slowly add the lactoferrin powder and stir magnetically until completely dissolved. Aliquot for use. Each mouse should receive 100 μL of the solution by gavage.

[0066] 1.3 Animal grouping

[0067] Six-week-old male SPF C57BL / 6 mice were randomly divided into five groups, with seven mice in each group, namely normal control group (CON group), aging model group (MOD group), lactoferrin group (LF group), creatine group (CR group), and lactoferrin and creatine combined intervention group (LF+CR group). The CON group was intraperitoneally injected and gavaged with normal saline daily, while the other four groups were intraperitoneally injected with D-galactose daily, the MOD group was gavaged with normal saline daily, the LF group was gavaged with lactoferrin solution daily, the CR group was gavaged with creatine solution daily, and the LF+CR group was gavaged with a mixed solution of lactoferrin and creatine daily for 8 weeks ( Figure 1 Each group of mice received intraperitoneal and oral administration of the drug once daily for 56 days. During the experiment, mice in each group had free access to food and water, and their body weight was measured regularly every week.

[0068] 2 Experimental methods

[0069] 2.1 Experimental materials

[0070] After the last oral administration of the drug to the mice, behavioral experiments were performed immediately the next day. After the test, the skeletal muscles of the hind limbs, including the extensor tibialis anterior (TA), soleus (SOL), extensor digitorum longus (EDL), and gastrocnemius (GAS), were obtained and weighed. The skeletal muscles of the left leg of the mice were preserved in tissue fixative, and the skeletal muscles of the right leg were quickly frozen in liquid nitrogen and placed in a -80°C refrigerator for use.

[0071] 2.2 Body composition analysis

[0072] After the intervention, the mice's body composition was measured using the Awake Small Animal Body Composition Analysis and Imaging System (Animal Fat Meter). Before testing, the mice were weighed and placed in the instrument after a self-check. After the measurement, physiological parameters such as lean mass and fat content were obtained while the mice were awake. Each mouse was measured two to three times.

[0073] 2.3 Behavioral experiments

[0074] a. Suspension experiment

[0075] The mice are suspended on an inverted grid 60 cm high, with a thick mat placed underneath. Each mouse is placed in the center of the grid, which is then gently turned upside down with the mouse's head facing downward. The mouse is kept hanging for the time it falls. Each mouse is tested twice, with an interval of >30 minutes between each test. The hanging time is recorded and each mouse is scored according to the scoring criteria. If the mouse falls in less than 10 seconds, the test is repeated immediately to ensure the accuracy of the test results. This test is based on the mouse's instinctive fear of falling. The mouse itself is not accustomed to this experiment, so it is best to let it complete this test in an unknown state with limited repetitions.

[0076] The scoring standard is: assign points based on the actual hanging time, with a full score of 100 points. For example, 120s is scored as 12 points, and >1000s is scored as a full score of 100 points.

[0077] b. Grip strength test

[0078] The basic process of the grip force measurement experiment can be divided into the following steps:

[0079] (1) Install the elastic metal strip onto the sensor and secure it firmly.

[0080] (2) Turn on the sensor and select peak mode. Do not apply a force greater than the sensor's strength tolerance.

[0081] (3) The display sensor is reset to zero.

[0082] (4) Take the mouse out of the cage and grab the middle of the mouse's tail with your thumb and index finger.

[0083] (5) Limb measurement: Place the mouse on the elastic metal bar and allow the mouse's front and hind paws to touch the elastic metal bar before measurement. Keep the torso and the elastic metal bar horizontal. Gently pull the mouse's tail from the top of the elastic metal bar until the mouse can no longer maintain its grip and the metal bar falls off its paws. During the test, the measuring device will record the maximum grip strength value and display it on the instrument screen. Record the maximum grip strength value displayed. Repeat 3 times.

[0084] c. Treadmill test

[0085] (1) Adaptation stage

[0086] Table 1 Treadmill parameters during the adaptation phase

[0087] Speed (m / min) Acceleration time (s) Speed duration (min) Initial velocity 12 5 4 First speed 16 5 4 Secondary speed 20 5 2

[0088] The treadmill parameters for the adaptation phase are shown in Table 1. The treadmill stimulation current was set to 0.5 mA. This gradual training approach allowed the animals to acclimate to the treadmill. This acclimatization process lasted 2–3 days, with one session per day for approximately 10 minutes.

[0089] (2) Testing phase

[0090] Table 2 Treadmill parameters during the test phase

[0091] Speed (m / min) Acceleration time (s) Speed duration (min) Initial velocity 12 5 4 First speed 20 5 to exhaustion

[0092] Treadmill parameters for the testing phase are shown in Table 2. The treadmill stimulation current was set to 0.5 mA. Exhaustion was indicated by the mouse stopping running for 10 seconds, followed by an electrical or noise stimulus for another 10 seconds, and then failing to run. Time to exhaustion was recorded. This procedure was repeated daily for 2-3 days.

[0093] 2.4 Evaluation of pathological indicators

[0094] Muscle slice preparation:

[0095] Dehydration and wax impregnation: Remove the GAS from the tissue fixative and trim it. Place it in a labeled embedding cassette and rinse it in running water for 2 hours. After rinsing, neatly arrange the cassettes and place them in a fully automatic biological dehydrator. Set the dehydration program and remove them after 12 hours of dehydration.

[0096] Tissue embedding: First, inject melted paraffin into the mold, carefully remove the tissue with heated tweezers, and quickly place it into the mold with the cross section facing upwards. After the paraffin has solidified slightly, cover the base of the embedding box. Continue to add wax until the base of the embedding box is submerged, and let it cool at room temperature to solidify.

[0097] Sectioning and spreading: Remove the embedded wax block from the mold and place it on a biological tissue freezing table. After the wax block cools down, fix it on a paraffin slicer. Adjust the slice thickness and trim the slice first. When complete muscle tissue appears, start continuous sectioning. The thickness of each slice is 5 μm. Remove the slice with tweezers and spread it flat on the water surface of the spreader. Use curved tweezers to unfold the folds. The spreading temperature is 42°C.

[0098] Pick up and bake the slices: After the slices are fully unfolded, quickly pick them up with a glass slide, absorb excess moisture with filter paper, mark them with numbers, and place them on a slide baker. Bake the slices at 42°C for 2 hours. After baking, place the slices in a 60-70°C oven overnight to prevent them from falling off.

[0099] At this point, the sections can be dewaxed and rehydrated.

[0100] Reagents time Xylene Ⅰ 20min Xylene Ⅱ 20min 100% ethanol I 5min 100% ethanol Ⅱ 5min 95% ethanol 5min 85% ethanol 5min 75% ethanol 5min Wash with tap water 3min

[0101] Pathological H&E staining was then performed.

[0102] ① First, place the slide in hematoxylin stain for 10 minutes;

[0103] ②Then wash with water for 5 to 10 minutes;

[0104] ③ Then place in 1% eosin solution for staining for 30 seconds to 1 minute;

[0105] ④Ethanol dehydration;

[0106] ⑤Finally, seal the slide with neutral resin

[0107] 2.5 Detection of myogenesis-related gene expression levels

[0108] Take some muscle, add 1ml of Trizol to the grinding tube, and grind it using a tissue grinder at 8000 rpm for 30 seconds, three times. After each grind, let it stand on ice for 1 minute to extract tissue RNA:

[0109] ① Add 200 μl of chloroform to the ground tube and centrifuge at 12,000 rpm for 15 min at 4°C.

[0110] ②Pipette 500 μl of supernatant into an enzyme-free 1.5 ml EP tube to which an equal volume of pre-cooled isopropanol has been added and let it stand for 10 minutes.

[0111] ③ Centrifuge at 12000r / min for 10min at 4℃.

[0112] ④Discard the supernatant, add 75% ethanol prepared with pre-cooled DEPC water, and centrifuge at 12000r / min at 4℃ for 5min.

[0113] ⑤Repeat step ④.

[0114] ⑥Discard the supernatant, dry at room temperature for 30 minutes, add DEPC water at 4°C to dissolve RNA, and measure the concentration.

[0115] The obtained tissue RNA was reverse transcribed and the amplification system was as follows:

[0116]

[0117] β-actin was selected as the internal reference gene, and the levels of myogenic differentiation factor 1 (MyoD1), myogenin (MyoG), myocyte enhancer factor 2C (MEF2C), and myogenic factor 5 (Myf5) were analyzed.

[0118] The real-time PCR system is as follows:

[0119] Element 10μL rxn 2×qPCR Mix 5μL Primer 1 (10mM) 0.5μL Primer 2 (10mM) 0.5μL Template DNA 2μL Enzyme-free water 2μL Total volume 10 μL

[0120] 3 Experimental results

[0121] 3.1 Weight changes during the intervention

[0122] from Figure 2 It can be seen that the weight changes of mice in each group showed a similar upward trend during the intervention, but the weight change curve of mice in the combined group (LF+CR group) was closer to that of the normal group compared with the LF group and the CR group, indicating that the combined intervention had a better effect in delaying skeletal muscle aging.

[0123] 3.2 Effect of nutritional composition on muscle mass

[0124] from Figure 3 and Figure 4 As can be seen, compared with the normal (CON) group, the muscle weight of the hind limb gastrocnemius muscles of the aged (MOD) group of mice was significantly reduced; the muscle index of the hind limb gastrocnemius muscles was also significantly reduced. The gastrocnemius muscle index is a criterion for judging muscle atrophy, indicating that aging-related muscle atrophy occurred in the aged mice and the experimental model was successfully established. Compared with the aged (MOD) group of mice, after the combined intervention of lactoferrin and creatine, the muscle weight of the hind limb gastrocnemius muscles of the lactoferrin and creatine combined group (LF+CR group) was significantly increased, and was significantly higher than that of the LF and CR groups. The muscle index was also significantly improved. This indicates that the combined intervention significantly improved the muscle quality of the mice.

[0125] from Figure 5 It can be seen that compared with the aging group (MOD group) mice, after the combined intervention of lactoferrin and creatine, the lean meat content of the mice in the lactoferrin and creatine combined group (LF+CR group) was significantly increased, and was significantly higher than that of the LF group and CR group; but the fat content did not change significantly, and the increase in the lean meat content of the mice indicated that the proportion of their muscle tissue increased, and the skeletal muscle development and growth conditions were improved.

[0126] 3.3 Effects of nutritional composition on muscle strength and function

[0127] from Figure 6 、 Figure 7 It can be seen that compared with the aging group (MOD group), after the combined intervention of lactoferrin and creatine, the grid hanging time and grip strength of mice in the lactoferrin and creatine combined group (LF+CR group) were significantly improved, and were significantly higher than those in the LF group and CR group, indicating that the combined intervention can better enhance the muscle strength of mice.

[0128] from Figure 8 It can be seen that compared with the MOD group, after the combined intervention of lactoferrin and creatine, the treadmill test exhaustion time of mice in the LF+CR group was prolonged and significantly higher than that of the LF group and the CR group, indicating that the muscle function of the mice was improved.

[0129] 3.4 Effects of nutritional composition on skeletal muscle structure

[0130] from Figure 9 H&E staining results from each group of mice revealed that the normal group (CON) exhibited neatly arranged skeletal muscle fibers with uniform diameters, clear cell membranes, polygonal cross-sections, and nuclei located at the cell edges. In the aging group (MOD), skeletal muscle fibers showed localized looseness and disorganized arrangement, with increased interstitial density. Compared with the aging (MOD) group, the lactoferrin and creatine combined intervention group (LF+CR) exhibited significantly larger muscle cells, more regular cell arrangement, and reduced interstitial components, significantly outperforming both the LF and CR groups.

[0131] 3.5 Effect of nutritional composition on the expression of myogenic differentiation-related factors

[0132] from Figure 10 The statistical bar chart of the expression of myogenic differentiation-related factors in each group of mice showed that compared with the normal control group (CON group), the mRNA expression of myogenic differentiation-related factors MyoD1, MyoG, MEF2C, and Myf5 in the aging group (MOD group) was significantly decreased, indicating that in the aging state, the expression of myogenic differentiation-related factors decreased and the differentiation ability of myoblasts decreased; after 8 weeks of combined intervention experiment of lactoferrin and creatine, the mRNA expression of myogenic differentiation-related factors in the lactoferrin and creatine combined group (LF+CR group) increased compared with the aging group (MOD group), and the differentiation ability was improved, while compared with the LF group and CR group, the mRNA expression of MyoD1 and MyoG in the combined intervention group (LF+CR group) were increased to varying degrees. The above experiments verified that the combination of lactoferrin and creatine can adjust the changes in gene expression caused by aging, thereby delaying aging.

[0133] The above embodiments are intended to illustrate the present invention only and are not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions fall within the scope of protection of the present invention. Any content not described in detail in this specification is considered prior art known to those skilled in the art.

Claims

1. A food, comprising lactoferrin and creatine, wherein the mass ratio of lactoferrin to creatine is (3-9):1; based on the total dry matter content of the food, the content of the lactoferrin in the food is 0.1-90g / 100g, and the content of the creatine in the food is 0.01-30g / 100g.

2. The food according to claim 1, which is a sports nutrition food, a food for the elderly, a health food, a dietary nutrition supplement food or a special medical purpose formula food.

3. The food according to claim 1 or 2, wherein The dosage form of the food includes tablets, hard capsules, soft capsules, granules, gel candies, powders or oral liquids.

4. The food according to claim 1, wherein The sources of the lactoferrin include cow's milk, goat's milk and / or their products, including but not limited to one or more of lactoferrin powder, whole milk powder, skim milk powder and whey protein powder; the creatine comes from a raw material containing creatine monohydrate.

5. The food according to any one of claims 1 to 4, wherein The food may further comprise one or more of protein, fat, vitamins, and minerals, or may selectively contain auxiliary materials and / or additives used in the field of food and / or health food.

6. The food according to any one of claims 1 to 5, wherein the raw material composition comprises, by weight: Protein 0-80 parts, fat 0-20 parts, lactoferrin 0.1-90 parts, creatine 0.01-30 parts, vitamin mixture 0-4 parts, mineral mixture 0-15 parts, β-hydroxy-β-methylbutyrate (HMB) 0-30 parts; Preferably, the vitamin mixture contains vitamin A, vitamin D, vitamin E, vitamin C and vitamin B1, vitamin B2, pantothenic acid, niacin, vitamin B6, vitamin B 12 One or more of; the mineral mixture contains one or more of calcium, iron, magnesium, potassium, zinc, and selenium.

7. The food according to any one of claims 1 to 6, wherein The mass ratio of lactoferrin to creatine is (4-8):1, preferably (4-6):

1.

8. A method for preparing the food according to any one of claims 1 to 7, comprising: The composition of the food raw material is adjusted so that it contains lactoferrin and creatine, and the content of the lactoferrin in the food is 0.1-90g / 100g, the content of the creatine in the food is 0.01-30g / 100g, and the mass ratio of lactoferrin to creatine is (3-9):1, based on the total dry matter content of the food product, to prepare the food.

9. Use of the food according to any one of claims 1 to 7 in the preparation of a food for improving muscle attenuation and / or promoting myogenic differentiation.

10. The use according to claim 9, wherein: The improvement of muscle attenuation includes improving muscle strength, muscle quality, muscle function, etc., specifically regulating the structure of the gastrocnemius muscle, increasing the gastrocnemius muscle mass and gastrocnemius index, increasing the body's lean body mass, increasing the grid hanging time and grip strength, and prolonging the time to exhaustion; The promoting myogenic differentiation includes increasing the expression levels of myogenic differentiation factor 1 (MyoD1), myogenin (MyoG) and myogenic factor 5 (Myf5).

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