Dietary factor composition for reducing content of propionic acid in intestinal tract of methylmalonic acidmia patient in targeted manner and application of dietary factor composition

Abstract

The invention discloses a dietary factor composition for reducing the content of propionic acid in intestinal tracts of methylmalonic acidmia patients in a targeted manner and application of the dietary factor composition, and belongs to the technical field of microorganisms and the technical field of medicines. In-vitro coprophilous fungus fermentation experiments prove that the combination of two dietary factors, namely arabinogalactan and tremella polysaccharide, can relieve methylmalonic acidmia, which is specifically embodied in that (a) intestinal flora metabolites (propionic acid) are reduced; (b) diet management of methylmalonic acidmia; (c) treating and / or ameliorating methylmalonatemia, and / or treating, ameliorating and / or preventing symptoms associated therewith.

Description

Technical Field

[0001] The present invention relates to the fields of dietary management, microbial technology or medical technology, and in particular to a dietary factor combination and application thereof for targetedly reducing intestinal propionic acid content in patients with methylmalonic acidemia. Background Art

[0002] Methylmalonic aciduria (MMA), also known as methylmalonic acidemia, is a rare and treatable genetic metabolic disease. It is also the most common organic acid metabolic disease in my country, and it often develops in the neonatal period or infant period. MMA is ranked No. 71 in the first batch of rare diseases in my country. The results of the national screening study of 7.82 million newborns showed that the detection rate of MMA was 1 / 15213. MMA was first reported in my country in 2000, and diagnosis, treatment and prevention technologies were gradually established. It was clear that simple MMA caused by methylmalonyl-CoA mutase deficiency accounted for 1 / 3 of MMA patients in my country, and required special diet management, cobalamin and L-carnitine treatment. MMA is caused by the accumulation of harmful substances such as methylmalonic acid in the body due to the deficiency of methylmalonyl-CoA mutase or its coenzyme cobalamin, causing damage to multiple organs such as the brain, blood, and kidneys. Due to the lack of specificity in the clinical manifestations of MMA, diagnosis and treatment are often delayed, and it cannot be cured, with extremely high mortality and disability rates.

[0003] Propionate is a precursor of propionyl-CoA, and the production of propionate in the human body originates from intestinal bacterial fermentation. In addition to acetate and butyrate, propionate is one of the main short-chain fatty acids (SCFA) produced by the microbiota in the intestine. Therefore, any changes in the production of any major SCFA (such as propionate) may have an impact on the substrates used by the microbiota and the metabolites produced by the microbiota. About 50% of propionate production in the body comes from the metabolic breakdown of amino acids, 25% comes from the catabolism of odd-chain fatty acids, and the rest is contributed by the intestinal flora.

[0004] In long-term treatment management, metronidazole can effectively inhibit the production of propionyl-CoA by anaerobic bacteria in the intestines fermenting carbohydrates, or be used alternately with other antibiotics (such as amoxicillin or sulfamethoxazole). However, long-term use of antibiotics can easily lead to the production of drug-resistant flora and cause intestinal flora disorders. Therefore, there is an urgent need to find alternative therapies that can reduce the production of propionic acid by intestinal flora without side effects.

[0005] Common dietary factors include food-derived dietary polysaccharides, dietary polyphenols, flavonoids, triterpenoids and dietary fiber. Food-derived dietary factors have attracted the attention of researchers at home and abroad because of their ability to improve metabolic-related indicators and regulate host health. In addition, dietary factors have unique advantages such as high safety, easy access to food, and reduced side effects of antibiotics. They are potential effective therapies for reducing intestinal propionic acid production and relieving methylmalonic acidemia. However, no dietary factors have been found that can effectively reduce intestinal propionic acid production. Summary of the invention

[0006] In view of the above problems existing in the prior art, the present invention provides a dietary factor combination and its application for reducing the intestinal propionic acid content of patients with methylmalonic acidemia. The present invention can reduce the production of propionic acid in the intestine, and thus can be used for dietary management of patients with methylmalonic acidemia. In this way, the defects of traditional treatment methods can be overcome. The present invention provides an innovative and feasible treatment approach, providing a new solution for the dietary management of methylmalonic acidemia.

[0007] The technical solution of the present invention is as follows:

[0008] The first object of the present invention is to provide a dietary factor composition, comprising arabinogalactan and tremella polysaccharide, wherein the mass ratio of arabinogalactan to tremella polysaccharide is 1:(0.6-1).

[0009] In one embodiment of the present invention, the mass ratio of arabinogalactan to tremella polysaccharide is 1:0.6.

[0010] The second object of the present invention is to provide an application of the above-mentioned dietary factor composition for preparing foods, special medical foods, health products or medicines for reducing the content of propionic acid, a metabolite of intestinal flora in methylmalonic acidemia.

[0011] In one embodiment of the present invention, the content of the dietary factor composition is not less than 0.06 g / g.

[0012] In one embodiment of the present invention, the drug is administered to a human subject at least 3 years old for the following conditions or at least one aspect:

[0013] (a) Reduce propionic acid, a metabolite of intestinal flora;

[0014] (b) Dietary management of methylmalonic acidemia;

[0015] (c) treating and / or alleviating methylmalonic acidemia, and / or treating, alleviating and / or preventing symptoms associated therewith.

[0016] In one embodiment of the present invention, the human subject is 3-10 years old.

[0017] The third object of the present invention is to provide a food containing the dietary factor composition.

[0018] The fourth object of the present invention is to provide a special medical food containing the above-mentioned dietary factor composition.

[0019] The fifth object of the present invention is to provide a health product containing the above dietary factor composition.

[0020] The sixth object of the present invention is to provide a medicine containing the above dietary factor composition.

[0021] The beneficial technical effects of the present invention are:

[0022] The present invention discovered two dietary factors that can significantly reduce the intestinal flora metabolite propionic acid of patients with methylmalonic acidemia through in vitro fecal flora fermentation experiments: arabinogalactan and tremella polysaccharide.

[0023] The present invention conducted an in vitro fecal flora fermentation experiment by compounding dietary factors and found that compared with a single dietary factor, the combination of arabinogalactan + tremella polysaccharide dietary factors has a synergistic promoting effect, and the content of the metabolite propionic acid in the fecal flora fermentation supernatant is significantly lower than that of a single dietary factor.

[0024] The present invention verifies through animal experiments that the combination of arabinogalactan + Tremella polysaccharide dietary factors has the effect of significantly reducing the intestinal flora metabolite propionic acid. Therefore, the combination of arabinogalactan and Tremella polysaccharide dietary factors has a good effect in reducing the intestinal flora metabolite propionic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The propionic acid content in feces of healthy people and people with methylmalonic acidemia;

[0026] Figure 2 The effect of dietary factors on short-chain fatty acids in the supernatant of in vitro fermentation of fecal bacteria in people with methylmalonic acidemia (fermentation for 6 hours);

[0027] Figure 3 The effect of dietary factors on short-chain fatty acids in the supernatant of in vitro fermentation of fecal bacteria in people with methylmalonic acidemia (fermentation for 12 hours);

[0028] Figure 4 The effect of dietary factors on short-chain fatty acids in the supernatant of in vitro fermentation of fecal bacteria in people with methylmalonic acidemia (fermentation for 24 hours);

[0029] Figure 5 The effect of dietary factors on the content of propionic acid in the supernatant of fecal bacteria in vitro fermentation at different time points of 6h, 12h, and 24h;

[0030] Figure 6The effect of dietary factors on the total short-chain fatty acids in the supernatant of 24h in vitro fermentation of fecal bacteria in people with methylmalonic acidemia;

[0031] Figure 7 To investigate the effects of dietary factors on the propionic acid content in the supernatant of in vitro fermentation of fecal bacteria in people with methylmalonic acidemia.

[0032] Figure 8 Flow chart for modeling animal experiments;

[0033] Fig. 9 The changes of propionic acid content in rat feces metabolites by different dietary factor combinations at 14, 21 and 28 days;

[0034] Fig.10 The changes of propionic acid content in rat fecal metabolites after different dietary factor combinations on the 28th day. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0036] The methylmalonic acid involved in the following examples was purchased from Sigma.

[0037] The arabinogalactan (article number: PN01485) involved in the following examples was purchased from Perfemiker, and the Tremella polysaccharide was purchased from Xi'an Shengqing Biotechnology Co., Ltd.

[0038] The present invention relies on the stool samples of 8 volunteers with methylmalonic acidemia recruited by the Children's Medical Center of Peking University First Hospital (age range 3 to 10 years old), and at the same time, the stool samples of 6 healthy volunteers (age range 3 to 10 years old) are collected. The stool samples are pre-treated to make a stool homogenate, inoculated in a culture medium containing dietary factors, and after 24 hours of anaerobic fermentation, the fermentation supernatant is taken to determine the content of intestinal flora metabolite propionic acid. Based on the in vitro fermentation-targeted intestinal flora metabolite propionic acid analysis results, the dietary factors are verified in vivo by animal experiments, and the dietary fiber combination that can effectively reduce the intestinal flora metabolite propionic acid is further screened through animal experiments.

[0039] Diagnostic criteria for methylmalonic acidemia: patients with clinically confirmed methylmalonic acidemia who meet the measurement methods specified in the "Guidelines for the Diagnosis and Treatment of Rare Diseases (2019 Edition)".

[0040] Propionic acid was extracted from fecal samples with ether, and the content of metabolite propionic acid in feces was detected by gas chromatography-mass spectrometry.

[0041] The results of targeted metabolite propionic acid analysis in fecal samples showed that the content of propionic acid in the supernatant of in vitro fermentation of feces in people with methylmalonic acidemia was significantly higher than that in normal people.

[0042] Fecal samples were pretreated by mixing feces and sterile PBS buffer at a ratio of 1:10 w / v (g / mL), and gauze was used to remove large particles in the fecal mixture.

[0043] The fecal homogenate was inoculated into the culture medium at a 20% (v / v) inoculum.

[0044] The culture medium formula is: GAM culture medium (1L): Peptone 10.0g, soy peptone 3.0g, yeast extract powder 5.0g, beef powder 2.2g, digested serum powder 13.5g, beef liver extract powder 1.2g, potassium dihydrogen phosphate 2.5g, sodium chloride 3.0g, L-cysteine ​​0.3g, sodium thioglycolate 0.3g, vitamin K1 10mg, hemin chloride 5mg; add distilled water 1L, pH7.3±0.1, sterilize at 115℃ for 20min, and add dietary polysaccharides to a final concentration of 1% (w / v). The culture conditions are 37℃ and anaerobic (80% N2, 10% CO2, 10% H2).

[0045] Detection method of short-chain fatty acid propionic acid:

[0046] (1) The content of propionic acid metabolite in fecal supernatant was determined by gas chromatography-mass spectrometry (GC-MS).

[0047] (2) Chromatographic detection conditions: Rtx-Wax column (30m*0.25μm*0.25μm) and mass spectrometer (GC-MS-QP2010Vitta, system) were used to separate different short-chain fatty acids; the carrier gas was N2 with a flow rate of 1mL / min; the injection volume was 1μL and the split ratio was 10:1; the initial column temperature was 100℃, which was increased to 140℃ at a heating rate of 7.5℃ / min, and then increased to 200℃ at a heating rate of 60℃ / min. The temperature was maintained at 200℃ for 3min, the injection temperature was 240℃, and the ionization temperature was 220℃.

[0048] (3) Draw a standard curve: Take 10 μL of acetic acid, propionic acid, and butyric acid in a 2 mL centrifuge tube, dilute to 1 mL with ether, mix thoroughly, take 100 μL, dilute 10 times with ether, mix well, then take 200 μL, 100 μL, 50 μL, 25 μL, 15 μL, and 10 μL, respectively, dilute to 1 mL with ether to obtain mixed standards of different concentrations, pass through the membrane and wait for detection on the machine. Draw a standard curve with the peak area at the corresponding concentration as the horizontal axis and the corresponding concentration as the vertical axis.

[0049] The propionic acid-lowering effect of the dietary factor composition was verified based on the content of propionic acid, a metabolite of intestinal flora in 70 experimental mice (blank group, model group, galacto-oligosaccharide + arabinogalactan group, galacto-oligosaccharide + tremella polysaccharide group, arabinogalactan + tremella polysaccharide group, 7 mice in each group).

[0050] Example 1: Determination of propionic acid content in feces of people with methylmalonic acidemia and healthy people

[0051] Eight volunteers with methylmalonic acidemia were recruited, and they were required to meet the diagnostic criteria specified in the "Guidelines for the Diagnosis and Treatment of Rare Diseases (2019 Edition)". At the same time, six healthy volunteers were recruited, who had not received antibiotic treatment in the past month, had not used probiotic products in the past month, had no gastrointestinal diseases, and were children between the ages of 3 and 10 years old. The stool of 14 volunteers was taken into a 35mL stool collection tube, 50 mg of the stool sample was weighed and placed in a 2mL EP tube, 500μL of saturated sodium chloride solution was added, and it was allowed to stand for 30 minutes. It was crushed with a tissue crusher at 60Hz for 30s. This step was repeated 3 to 5 times until the sample was crushed to no obvious lumps. 20μL of sulfuric acid solution was added for acidification, and the mixture was shaken and mixed. Anhydrous ether was added for extraction in a fume hood, and the supernatant was taken after dehydration with anhydrous sodium sulfate. The supernatant was filtered through an organic phase filter membrane and added to a gas phase vial for analysis on the machine. The results are shown in Figure 1 .

[0052] The results show that: Figure 1 It can be seen that compared with healthy people, the content of propionic acid in the feces of people with methylmalonic acidemia is significantly higher (P<0.001).

[0053] Example 2: Effect of dietary factors on the production of propionic acid metabolites in fecal fermentation supernatant of patients

[0054] Eight volunteers with methylmalonic acidemia were recruited, and they were required to meet the diagnostic criteria specified in the "Guidelines for the Diagnosis and Treatment of Rare Diseases (2019 Edition)". At the same time, six healthy volunteers were recruited, who had not received antibiotic treatment in the past month, had not used probiotic products in the past month, had no gastrointestinal diseases, and were children aged between 3 and 10 years old. The feces of 14 volunteers were collected in 35mL fecal collection tubes, 30% glycerol was added, and stored in a -80℃ refrigerator. The fecal samples were thawed and centrifuged before use, the upper glycerol was discarded, and the fecal samples were mixed evenly in equal amounts under anaerobic conditions. The filtered homogenate was inoculated into GAM medium containing dietary factors (the amount of dietary factors added was as follows: the final concentration of dietary polysaccharides was 1% (w / v), and the culture was carried out at 37℃ anaerobically (80% N2, 10% CO2, 10% H2) for 24 hours to obtain a fermentation broth containing intestinal microorganisms.

[0055] The fecal homogenate preparation method is as follows: equal amounts of fecal samples are weighed and mixed evenly under anaerobic conditions, and then diluted with sterile phosphate buffer saline (PBS) at 1:10 w / v (g / mL), and then filtered with sterile gauze to remove large particles. The filtrate is inoculated into a GAM medium containing dietary factors at a volume ratio of 20%.

[0056] The fermentation liquid after 24 h of anaerobic fermentation was transferred to a 2 ml sterilized centrifuge tube and the supernatant was separated by centrifugation.

[0057] Take 500 μL of fermentation supernatant, then add 20 μL of 10% sulfuric acid to acidify, shake for 30 seconds to mix. Add 1 mL of anhydrous ether to extract SCFAs in a fume hood. Centrifuge after mixing, transfer the supernatant to a centrifuge tube containing anhydrous sodium sulfate to remove water, let it stand for 15 minutes, centrifuge under the same conditions, transfer the supernatant to a gas phase vial, take the supernatant and test the content of propionic acid metabolites on the machine. The results of propionic acid content of fecal fermentation supernatant metabolites of different dietary factors at 6h, 12h, and 24h are shown in Table 1.

[0058] Table 1

[0059]

[0060] Note: * Indicates P < 0.05, *** P<0.001, compared with the Yin ginseng group

[0061] From Table 1 and Figures 2 to 6 Comprehensive analysis showed that after 24 hours of in vitro fermentation, compared with the group without carbon source (Yin ginseng), arabinogalactan and Tremella polysaccharide significantly reduced the content of propionic acid in the fermentation supernatant (P<0.001).

[0062] The above experimental results show that the propionic acid content in the supernatant of in vitro fermentation of feces of people with methylmalonic acidemia is significantly higher than that of normal people, and arabinogalactan and tremella polysaccharide have a good effect on reducing the content of propionic acid in fecal flora after fermentation.

[0063] Example 3: Effect of dietary factor combination on the production of propionic acid metabolites in fecal fermentation supernatant of patients

[0064] The stool samples were the same as those used in Example 2. The specific implementation method was as shown in Example 2. The combination of dietary factors (arabinogalactan: Tremella polysaccharide mass ratio was 1:1) and the dietary factor grouping information was shown in Table 2:

[0065] Table 2

[0066]

[0067] After 24 h of fermentation, the results of metabolite contents in the fecal fermentation supernatant of different dietary factor combinations are shown in Table 3. Figure 7 .

[0068] Table 3

[0069] Group Propionic acid / (μmol / L) No carbon source (healthy) 4.483±0.156 No carbon source (Yin ginseng) 5.575±0.323 A 2.647±0.048 B 2.154±0.098 C <![CDATA[1.463±0.041 *** ]]> D 3.095±0.047 Yang Shen 3.427±0.133

[0070] Note: *** indicates P < 0.001, compared with the Yin ginseng group

[0071] From Table 3, Figure 7 It can be seen that after 24 hours of in vitro fermentation, compared with the blank control group, the combination of arabinogalactan + tremella polysaccharide dietary factors significantly reduced the content of propionic acid in the fermentation supernatant (P<0.0001), and the effect was significantly better than that of A, B, D and Yangshen groups (Note: a. The formula of mixture B in Table 1 of CN 112805010 A is consistent with that of group D in the experiment; a. The formula of the Yangshen group uses the ratio of mixture F in Table 1 of CN 112805010 A).

[0072] The above experimental results show that the content of propionic acid in the supernatant of in vitro fermentation of feces of people with methylmalonic acidemia is significantly higher than that of normal people, and the combination of arabinogalactan + tremella polysaccharide dietary factors has the best effect on reducing the content of propionic acid after fermentation of fecal flora.

[0073] Example 4: Effects of different dietary factor combinations on the production of fecal metabolites of propionic acid in rats with methylmalonic acidemia

[0074] The experimental animal modeling process is shown in Figure 8 ,The information of animal experiment groups is shown in Table 4.

[0075] Table 4

[0076]

[0077]

[0078] The specific method is as follows:

[0079] Seventy SPF Wistar mice were randomly divided into 10 groups, with 7 mice in each group, namely male blank group, male model group, male galacto-oligogalactan + arabinogalactan group, male galacto-oligogalactan + tremella polysaccharide group, male arabinogalactan + tremella polysaccharide group, female blank group, female model group, female galacto-oligogalactan + arabinogalactan group, female galacto-oligogalactan + tremella polysaccharide group, and female arabinogalactan + tremella polysaccharide group.

[0080] Rats were housed in the Experimental Animal Center of Jiangnan University in an environment with a room temperature of 24-26°C, a humidity of 40-70%, a noise level of 60 dB or less, and an illumination of 15-20 LX. The experimental animals followed a 12-h day and 12-h night schedule, were fed with a standard commercial feed, and had free access to food and water. (All animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University, approval number: JN.No20241015W0121204

[530] ). Preparation of methylmalonic acid solution: Methylmalonic acid was dissolved in saline and the pH was adjusted to 7.4 with 6 mmol / L NaOH.

[0081] The experimental process is shown in Table 4 and Figure 8 As shown, the model was established after the rats were 5 days old;

[0082] Intervention treatment experimental process:

[0083] Male blank group and female blank group: healthy mice without modeling, intragastric administration of normal saline, intervention time (15-28 days);

[0084] Male model group and female model group: methylmalonic acidemia rats, intragastrically administered with normal saline, intervention time (15th-28th day);

[0085] Male galacto-oligosaccharide + arabinogalactan group, female galacto-oligosaccharide + arabinogalactan group: methylmalonic acidemia rats, intragastrically administered 1mL / 100g 800mg / kg galacto-oligosaccharide + 500mg / kg arabinogalactan, intervention time (15th-28th day);

[0086] Male galacto-oligosaccharide + Tremella polysaccharide group, female galacto-oligosaccharide + Tremella polysaccharide group: methylmalonic acidemia rats, intragastrically administered 1mL / 100g 800mg / kg galacto-oligosaccharide + 300mg / kg Tremella polysaccharide, intervention time (15th-28th day);

[0087] Male arabinogalactan + Tremella polysaccharide group and female arabinogalactan + Tremella polysaccharide group: methylmalonic acidemia rats, intragastrically administered 1mL / 100g 500mg / kg arabinogalactan + 300mg / kg Tremella polysaccharide, intervention time (15th-28th day).

[0088] Modeling process: male model group, male galacto-oligosaccharide + arabinogalactan group, male galacto-oligosaccharide + Tremella polysaccharide group, male arabinogalactan + Tremella polysaccharide group, female model group, female galacto-oligosaccharide + arabinogalactan group, female galacto-oligosaccharide + Tremella polysaccharide group, female arabinogalactan + Tremella polysaccharide group were subcutaneously injected with methylmalonic acid on days 5-28. Methylmalonic acid was dissolved in normal saline and the pH was adjusted to 7.4 with 6mmol / L NaOH. The dose of methylmalonic acid was as follows: 0.72μmol / g methylmalonic acid was given by gram body weight on days 5-12 after birth; 0.89μmol / g was given on days 13-19 after birth; and 1.67μmol / g was given on days 20-34 after birth. Rats were injected with 10μl / g of the solution, and the male blank group and the female blank group were injected with the same amount of normal saline.

[0089] Specific implementation method Referring to Example 1, 50 mg of rat feces samples were taken on the 14th, 21st and 28th days, placed in a 2mL EP tube, and 500 μL of saturated sodium chloride solution was added. The sample was allowed to stand for 30 minutes, and the sample was broken with a tissue crusher until there was no obvious block. 20 μL of sulfuric acid solution was added to acidify, shaken and mixed, extracted with anhydrous ether, and after dehydration with anhydrous sodium sulfate, the liquid was added to a gas phase vial and analyzed on the machine. The results of the effects of different dietary factor combinations on the content of propionic acid metabolites in rat feces at 14d, 21d and 28d are shown in Table 5. Fig. 9 .

[0090] Table 5

[0091]

[0092]

[0093] Note: *** P < 0.001

[0094] like Fig. 9 As shown in the results, compared with the model group, the combined intervention of arabinogalactan + Tremella polysaccharide dietary factors can significantly reduce the level of propionic acid production by rat intestinal flora (p<0.001), which is consistent with the results of in vitro fecal fermentation experiments. Fig.10 As shown, we unexpectedly found that when one of the dietary factors in the combination was replaced with galactoligosaccharides, and animal experiments were conducted with galactoligosaccharides + arabinogalactan groups and galactoligogalactan + tremella polysaccharide groups, the effects of the arabinogalactan + tremella polysaccharide group were more significant in both male and female rats.

[0095] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A dietary factor composition, characterized in that The invention comprises arabinogalactan and tremella polysaccharide, and the mass ratio of arabinogalactan to tremella polysaccharide is 1:(0.6-1).

2. The dietary factor composition according to claim 1, characterized in that The mass ratio of arabinogalactan to tremella polysaccharide is 1:0.

6.

3. An application of the dietary factor composition according to claim 1, characterized in that: Used for preparing foods, special medical foods, health products or medicines for reducing the content of propionic acid, a metabolite of intestinal flora in methylmalonic acidemia.

4. The use according to claim 3, characterized in that: The content of the dietary factor composition is not less than 0.06g / g.

5. The use according to claim 3, characterized in that: For use in human subjects aged at least 3 years for at least one of the following: (a) Reduce propionic acid, a metabolite of intestinal flora; (b) Dietary management of methylmalonic acidemia; (c) treating and / or alleviating methylmalonic acidemia, and / or treating, alleviating and / or preventing symptoms associated therewith.

6. The use according to claim 5, characterized in that: The human subjects were aged 3-10 years.

7. A food containing the dietary factor composition according to claim 1.

8. A special medical food containing the dietary factor composition according to claim 1.

9. A health product containing the dietary factor composition according to claim 1.

10. A medicine containing the dietary factor composition according to claim 1.

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