Use of collagen hydrolysate to improve endurance exercise performance and to stimulate lipid metabolism

Abstract

The present invention relates to the use of collagen hydrolysate for improving endurance exercise capacity by increasing mitochondrial activity.Furthermore, the present invention relates to the use of collagen hydrolysate for stimulating lipid metabolism, particularly for weight loss, by increasing mitochondrial activity. [Selected Figure] Figure 1C

Description

[Technical Field]

[0001] The present invention relates to the use of collagen hydrolysate to improve endurance athletic performance.

[0002] Furthermore, the present invention relates to the use of collagen hydrolysate to stimulate lipid metabolism, in particular for weight loss. [Background technology]

[0003] Collagen hydrolysates, produced by enzymatic hydrolysis of collagen-containing starting materials, especially of animal origin, consist of a mixture of peptides with a molecular weight distribution over a specific size range, depending on the starting material and production conditions. The use of collagen hydrolysates as nutritional supplements, especially for preventing and / or treating diseases of bone, joint or connective tissue, has been known for quite some time. This is because it has been shown that collagen peptides have a stimulating effect on the synthesis of the extracellular matrix in these tissue types (see, for example, Bello et al., Curr. Med. Res. Opin. 2006 (22) 2221-2232).

[0004] Surprisingly, collagen hydrolysate was shown to cause an increase in mitochondrial activity in human and animal cells, i.e., an increase in the number of mitochondria per cell and / or an increase in the size of individual mitochondria.

[0005] This finding indicates that the collagen hydrolysate of the present invention can be used to improve endurance exercise capacity and stimulate lipid metabolism in humans and animals in a targeted manner by increasing mitochondrial activity in muscle cells.

[0006] The endurance performance of a human or animal body correlates with the aerobic metabolic capacity of muscle tissue, which provides the necessary energy over time in the form of adenosine triphosphate (ATP). A key factor in aerobic capacity is oxygen uptake, which is determined by three factors: oxygen delivery from the lungs, oxygen transport via the cardiovascular system, and oxygen utilization in muscle cells. While the maximum oxygen supply is essentially predetermined by the individual's anatomical environment (total alveolar surface area), oxygen transport and oxygen consumption can be enhanced by training and other means, with the latter typically representing the decisive limiting factor. For this reason, endurance performance essentially depends on the number of mitochondria (per muscle cell or within the entire muscle tissue) where the respiratory chain reaction, which consumes oxygen and produces ATP, occurs.

[0007] Generally speaking, increasing mitochondrial activity means that the metabolic rate of the body increases, and the amount of nutrients metabolized per unit time for energy recovery increases.However, higher metabolic activity inevitably leads to a greater breakdown of body reserves (if nutrient supply is kept constant), that is, it leads to the stimulation of lipid metabolism.Therefore, since the metabolism of long-chain carboxylic acids from adipose tissue occurs substantially throughout the body, especially in the liver, in this aspect of the present invention - in contrast to improving endurance exercise capacity - it is not only the mitochondria of muscle cells that are relevant.

[0008] The use of collagen hydrolysate according to the present invention particularly includes non-therapeutic use, i.e., administration of collagen hydrolysate to humans or animals that do not require medical treatment for endurance athletic performance or body weight.Rather, its use is carried out for the purpose of generally desirable enhancement of endurance athletic performance on the one hand.This contributes to improving quality of life, and is particularly relevant for athletes.On the other hand, weight loss as a result of stimulating lipid metabolism may be desired mainly for cosmetic reasons, i.e., to improve body proportions.

[0009] However, the present invention also encompasses the therapeutic use of collagen hydrolysates for the prevention and / or treatment of pathological conditions characterized by decreased mitochondrial activity, in particular pathological conditions that may be characterized by decreased endurance exercise capacity and / or weight gain.

[0010] In this therapeutic use context, the pathological condition is preferably selected from obesity, cardiovascular disease, cardiac arrhythmias, heart failure, hypotension, hypertension, metabolic disorders, diabetes, metabolic syndrome, sideroblastic anemia, renal and hepatic dysfunction, neuropathy, ataxia, seizures, dementia, Alzheimer's disease, autism, depression, chronic fatigue syndrome, Parkinson's disease, motor neuron disease, multiple sclerosis, stroke-like symptoms, headache, myoclonus, paralysis, neuralgia, hyperalgesia, hyperesthesia, dysphagia, vomiting, constipation, diarrhea, optic nerve fiber and retinal degeneration, nystagmus, ptosis, night blindness, hearing loss, deafness, and inner ear disorders. In these indications, the therapeutic effect can be achieved by increasing mitochondrial number or activity.

[0011] Because increased mitochondrial activity reduces glucose levels, the administration of collagen hydrolysate can also have a positive effect on the prevention and / or treatment of cancer, or malignant tumors. This is based on the knowledge that the energy required by tumor cells is mainly provided by anaerobic metabolism (lactic acid fermentation), and that metabolism necessarily depends on glucose. The higher the glucose consumption within the body's cells, the more tumor cells will decline.

[0012] In the present invention, it has been discovered that collagen hydrolysate induces an increase in the expression of AMP-activated protein kinase (AMPK) enzyme. This regulatory enzyme also has an effect on cellular energy metabolism, so increasing the amount of AMPK has a positive effect on endurance exercise capacity and lipid metabolism. There is likely a positive correlation between the increase in mitochondrial activity caused by collagen hydrolysate and the increase in AMPK expression.

[0013] In all uses according to the invention, the collagen hydrolysate is preferably administered enterally, in particular orally.

[0014] In a preferred embodiment of the present invention, collagen hydrolysate is administered in the form of a nutritional supplement.Particularly advantageously, it is administered in the form of a solution, for example, in the form of an ampoule, or in the form of a powder.Due to its easy solubility, collagen hydrolysate can also be added to various beverages without causing turbidity.The use of tasteless and odorless collagen hydrolysate can increase user acceptance.

[0015] According to a preferred embodiment of the present invention, the nutritional supplement does not contain any other protein or protein hydrolysate other than collagen hydrolysate. In known nutritional supplements for muscle building and muscle maintenance, various proteins are used, especially by athletes, to replace a large portion of carbohydrates and fats with proteins as an energy source. However, the use according to the present invention is not based on the function of collagen hydrolysate as an energy source, but on its specific effect on mitochondrial activity as described above.

[0016] Thus, in a further aspect of the invention, the nutritional supplement does not contain any further physiologically active ingredients other than collagen hydrolysate.

[0017] However, in another embodiment, the present invention also encompasses the administration of collagen hydrolysate as a component of a (nutritional) supplement containing various additional ingredients, in particular collagen hydrolysate can be added to or processed into foods and beverages such as chocolate bars, protein bars or cereal bars (so-called functional foods) or milk, dairy products (e.g. yogurt) and milk substitutes (e.g. soy milk, almond milk and coconut milk).

[0018] Regardless of the dosage form, collagen hydrolysate is typically administered in an amount of 1 to 40 g / day, preferably 2.5 to 30 g / day, more preferably 10 to 25 g / day, especially 12.5 to 20 g / day.

[0019] In the use of the present invention, collagen hydrolysate is not used as the only physiologically active ingredient of nutritional supplements, but can be combined with one or more additional ingredients that have a positive effect on general health, especially on endurance athletic performance.This type of ingredient is preferably selected from vitamins C, B, D, E and K, linoleic acid, caffeine and its derivatives, guarana extract, green tea extract, epigallocatechin gallate, creatine, L-carnitine, L-citrulline, L-arginine, α-lipoic acid, N-acetylcysteine, NADH, D-ribose, magnesium aspartate, antioxidants such as anthocyanins, carotenoids, flavonoids, resveratrol, glutathione, superoxide dismutase and xanthans such as mangiferin, minerals such as iron, magnesium, calcium, zinc, selenium and phosphorus, and additional proteins, hydrolysates or peptides such as soybean, wheat or whey protein.

[0020] A further advantageous embodiment of the present invention is to prepare collagen hydrolysates by converting them into ubiquinone-10 and / or ubiquinol, i.e., coenzyme Q. 10 Regarding the combination of collagen hydrolysate with the oxidized or reduced forms of ubiquinol, ubiquinol is preferred here due to its good bioavailability. Since a daily intake of 50-100 mg of ubiquinol has been observed to have a positive effect on physical performance, it is assumed that the antioxidant properties of ubiquinol promote mitochondrial activity. This supports the efficacy of collagen hydrolysate in the above-mentioned indications associated with mitochondrial dysfunction. Alternatively or additionally, collagen hydrolysate can be combined with pyroquinoline quinone (PQQ), which has recently been discovered as an important redox cofactor.

[0021] In a particular embodiment of the present invention, collagen hydrolysate is administered in combination with endurance training or high altitude training.Endurance training can increase the aerobic capacity of metabolism.It is known that physical training in a relatively oxygen-deficient environment (hypoxia training) has a significant effect on endurance exercise capacity, so the simultaneous administration of collagen hydrolysate is expected to produce a synergistic effect.This is particularly interesting for athletes.

[0022] On the other hand, within the scope of the present invention, it is equally possible and indeed useful if the administration of collagen hydrolysate is carried out in the absence of endurance, altitude or muscle training, in particular as the effects according to the present invention on mitochondrial activity etc. have already been observed in combination with normal physical activity.

[0023] According to the present invention, the molecular weight of the collagen hydrolysate used can vary over a wide range, but the upper limit is set in that the degree of hydrolysis is sufficiently high so that the collagen hydrolysate does not gel at room temperature and is water-soluble, unlike denatured collagen or gelatin. The water-soluble peptides of the collagen hydrolysate have an average molecular weight of 200 to 25,000 Da, preferably 1,000 to 6,000 Da, more preferably 1,200 to 4,000 Da, even more preferably 1,500 to 3,500 Da, and especially 2,800 to 3,300 Da.

[0024] Preferably, the collagen hydrolysate is produced by enzymatic hydrolysis of a collagen-containing starting material, in particular using endopeptidases and / or exopeptidases of microbial or plant origin.

[0025] The collagen-containing starting material is typically selected from the skin or bone of vertebrates, preferably mammals, in particular bovine or porcine skin (cattle bone or pigskin). Collagen hydrolysates can be produced from these starting materials either in a single step or via an intermediate gelatin process, in which case both type A and type B gelatin can be used.

[0026] Alternatively, the collagen hydrolysate used in accordance with the present invention may be produced by recombinant gene expression. By utilizing natural collagen sequences, particularly of bovine or porcine origin, and their expression in genetically modified cells (e.g., yeast, bacteria, or plant cells, especially tobacco), it is possible to produce products that are substantially identical to the hydrolysis products of collagen-containing raw materials. Thus, it is possible to obtain a relatively narrow or precisely defined molecular weight distribution. Alternatively, the sequences may be modified by mutation to obtain certain effects on the specific properties of the product.

[0027] The present invention further relates to a method for improving endurance exercise capacity and / or stimulating lipid metabolism by increasing mitochondrial activity, in particular a method for reducing body weight.The method preferably involves enteral administration, in particular oral administration, of collagen hydrolysate to humans or animals.The method may be a therapeutic method or may be non-therapeutic in nature. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows fluorescence microscopy images of SH-SY5Y cells incubated in the presence of collagen hydrolysate. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is further described in detail with respect to in vitro and in vivo experimental results, which are described within the context of the following examples. [Example]

[0030] 1. Increasing Mitochondrial Number with Collagen Hydrolysate The effectiveness of collagen hydrolysate for increasing mitochondrial number in vitro on human neuronal cells (neuroblastoma SH-SY5Y) could be demonstrated.

[0031] SH-SY5Y cells were incubated in medium with collagen hydrolysate at different concentrations (0.05%, 0.2%, and 2.5% by weight) from pig skin gelatin produced by enzymatic hydrolysis and having an average molecular weight in the range of 3,000 Da (designated collagen hydrolysate A below). The molecular weight distribution of the peptides was determined by gel permeation chromatography and is shown in Table 1 below. Table 1: Molecular weight distribution of collagen hydrolysate A

[0032] [Table 1]

[0033] To enable direct assessment of mitochondrial number, we fluorescently labeled the mitochondrial protein component TOM20. TOM20 is a subunit of a receptor complex in the outer membrane of mitochondria and functions to translocate cytoplasmic precursor proteins (pre-peptides) into mitochondria. Here, cleavage of the pre-sequence activates enzymes in the respiratory chain or the citric acid cycle.

[0034] The amount of fluorescently labeled TOM20 visible under a fluorescent microscope is a measure of the number of mitochondria in the cells. Cells incubated with 0.05 wt%, 0.2 wt%, and 2.5 wt% collagen hydrolysate are shown in Figures 1A, 1B, and 1C. As the collagen concentration increases, a faint increase in fluorescence (originally green) is clearly visible in the area surrounding the cell nucleus. These collagen hydrolysates result in an increase in the number of mitochondria in SH-SY5Y cells, thereby increasing overall mitochondrial activity.

[0035] 2. Activation of the enzyme AMPK by collagen hydrolysates in vitro AMP-activated protein kinase (AMPK) is involved in the energy supply of both adipose and muscle tissues. AMP can be considered an indicator of energy deficiency, as it is produced when ATP is consumed. Therefore, AMPK expression activates energy storage from stored fat and through glycolysis.

[0036] To investigate the effect of collagen hydrolysate on AMPK expression, human muscle cells The cells were incubated in a medium containing 0.5 mg / mL collagen hydrolysate for 24 hours. After removing the medium, RNA was extracted from the cell layer, and the amount of AMPK RNA was determined by PCR using specific primers.

[0037] Compared to the collagen hydrolysate-free control, AMPK-RNA levels were significantly increased (>600-fold increase), again demonstrating the stimulatory effect of collagen hydrolysate on cellular energy metabolism.

[0038] 3. Activation of the enzyme AMPK by collagen hydrolysates in vivo The positive effect of collagen hydrolysate on AMPK expression could be confirmed using in vivo animal studies.

[0039] To this end, mice were fed 10 g of collagen hydrolysate equivalent to a human diet daily for three months. After sacrifice, the quadriceps muscles were completely excised, flash-frozen, and pulverized. Soluble protein was extracted from the muscle tissue, and AMPK levels were measured by immunoassay (ELISA).

[0040] Compared to the control group that did not receive collagen hydrolysate, AMPK levels increased by 1.5 to 2 times.

[0041] 4. Effect of collagen hydrolysate on in vitro NADH production Low-energy NAD +The high-energy form of nicotinamide adenine dinucleotide, NADH+H, from + The formation of ATP is equivalent to energy production in the form of ATP, and is therefore an indirect measure of mitochondrial activity in muscle cells.

[0042] In this study, in a medium containing 0.5 mg / mL collagen hydrolysate human muscle cells The cells were incubated for 6 days. After removing the medium, triglycerides were extracted using glycerol kinase and glycerol-3-phosphate dehydrogenase enzymes, and the energy contained in the triglycerides or glycerol was measured in the form of NADH+H+ released from them.

[0043] The amount of NADH increased by approximately two-fold compared to the control without collagen hydrolysate.

[0044] 5. Increasing Mitochondrial Density in Rats In Vivo In preclinical studies, a significant increase in mitochondrial density (i.e. an increase in the number and / or size of mitochondria) in the skeletal muscle of rats could be demonstrated as a result of the administration of different collagen hydrolysates.

[0045] The study was carried out in male rats of the CD® IGS line (Charles River Laboratories, Sulzfeld), which were 64 days old and weighed 300-400 g at the start of the study period. The test and control groups each contained 6 animals.

[0046] At the start of the study (t=0), a biopsy was collected from the quadriceps muscle of each rat by fine needle aspiration. Over a 4-week period, animals in the test group received a daily dose of 200 mg of each collagen hydrolysate (see below) per kg of current body weight (equivalent to a daily dose of 15 g for a 75 kg human). The collagen hydrolysate was dissolved in an appropriate volume of tap water at a concentration of 20 mg / mL and administered via stomach tube. Animals in the control group received the same volume of tap water without collagen hydrolysate.

[0047] In addition to collagen hydrolysate A, collagen hydrolysate B of bovine split gelatin with an average molecular weight of 2,000 Da and collagen hydrolysate C of bovine split gelatin with an average molecular weight of 3,500 Da were used as additional test groups. They were in each case produced by enzymatic hydrolysis. The molecular weight distributions of all three hydrolysates are shown in Table 2 below. Table 2: Molecular weight distribution of collagen hydrolysates in wt%

[0048] [Table 2]

[0049] At the end of the study period (t = 4 weeks), all rats were sacrificed and quadriceps biopsies were again taken by fine needle aspiration. Over the 4-week period, the rats showed an average weight gain of approximately 30%, which was not significantly different between the test and control groups.

[0050] To measure mitochondrial density, biopsies taken before and after the test period were prepared for scanning electron microscopy analysis as described (see A. Glauert & P. ​​Lewis: Biological Specimen Preparation for Transmission Electron Microscopy, Princeton Legacy Library, 2014). In each case, the mitochondrial density was measured at 15.17 × 15.17 μm (230 μm 2 The surface area of ​​each muscle biopsy was digitized and semiquantitatively evaluated, where the average mitochondrial surface area was calculated as a percentage of the total surface area using 10 samples from each biopsy. Mitochondria could be localized based on their clearly visible characteristic inner membrane structure with cristae.

[0051] Table 3 shows the mitochondrial density (total surface area 230 μm) in the animals in the test group treated with collagen hydrolysate A. 2 Mitochondria per μm2 ) indicates an increase. Table 3: Gain over time in study group A

[0052] [Table 3]

[0053] The results showed that 4 weeks of administration of collagen hydrolysate A resulted in a highly significant increase in mitochondrial density, on average 78.5%, which was statistically significant (p=0.001).

[0054] Comparison of mitochondrial density in each case between the collagen hydrolysate A, B and C test groups and the control group after 4 weeks according to Table 4 below demonstrated similar findings. Table 4: Comparison of test groups A, B and C with the control group

[0055] [Table 4]

[0056] In the test groups, mitochondrial density was on average 56.6% (A), 59.5% (B), and 90.4% (C) higher than in the control group, also statistically significant (p=0.001). Cohen's d value, a measure of effect size, was above 2.5 in all test groups, thus indicating a highly significant effect in each case.

[0057] In summary, this study provides conclusive evidence that administration of collagen hydrolysate leads to a significant increase in mitochondrial density in muscle cells, resulting in a corresponding increase in mitochondrial activity. This effect can be confirmed using collagen hydrolysates of different origins (porcine and bovine) and molecular weight distributions.

Claims

1. A composition for non-therapeutic use comprising a collagen hydrolysate for improving endurance exercise capacity by increasing mitochondrial activity, wherein the collagen hydrolysate is produced by enzymatic hydrolysis of a collagen-containing starting material selected from mammalian skin or bone.

2. The composition of claim 1 , wherein the collagen hydrolysate is administered enterally.

3. 3. The composition of claim 2, wherein the collagen hydrolysate is administered in the form of a nutritional supplement.

4. 4. The composition of claim 3, wherein the nutritional supplement does not contain any additional proteins or protein hydrolysates other than collagen hydrolysate.

5. 5. The composition of claim 3 or 4, wherein the nutritional supplement does not contain any further physiologically active ingredients other than collagen hydrolysate.

6. The composition according to any one of claims 2 to 5, wherein the collagen hydrolysate is administered in an amount of 1 to 40 g / day.

7. 7. The composition of any one of claims 1 to 4 or 6, wherein the collagen hydrolysate is administered in combination with one or more ingredients selected from vitamins of the C, B, D, E and K type, linoleic acid, caffeine and its derivatives, guarana extract, green tea extract, epigallocatechin gallate, creatine, L-carnitine, L-citrulline, L-arginine, alpha-lipoic acid, N-acetylcysteine, NADH, D-ribose, magnesium aspartate, antioxidants, minerals, and additional proteins, hydrolysates or peptides.

8. 8. The composition of any one of claims 1 to 4, 6 or 7, wherein the collagen hydrolysate is administered in combination with ubiquinone-10 and / or ubiquinol administered in an amount of 50 to 100 mg / day, and / or pyrroloquinoline quinone (PQQ).

9. The composition of any one of claims 1 to 8, wherein the administration of the collagen hydrolysate is combined with endurance training or high altitude training.

10. The composition according to any one of claims 1 to 8, wherein the administration of the collagen hydrolysate is carried out in the absence of endurance training, high altitude training or resistance training.

11. The composition according to any one of claims 1 to 10, wherein the collagen hydrolysate has an average molecular weight of 200 to 25,000 Da.

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