Casein phosphopeptide and preparation method thereof
The supernatant obtained by fermentation of Aspergillus oryzae RIB40 was treated with enzymatic yak casein, combined with the Ca-ethanol precipitation method, the bitter taste problem during casein hydrolysis was solved, and the extraction rate and taste of casein phosphopeptide were improved.
Patent Information
- Application Number
- CN202411676828.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-02-14
AI Technical Summary
While increasing the content of casein phosphopeptide, how to avoid the bitter taste generated during casein hydrolysis, affecting the tolerance and taste of the product.
The supernatant obtained by fermenting Aspergillus oryzae RIB40 was used to treat the enzymatically dissolved yak casein to improve the extraction rate of casein phosphopeptide and purify it by Ca-ethanol precipitation method to solve the bitter taste problem.
It achieves a high extraction rate of casein phosphopeptide (more than 98%), has no bitter taste, good taste, and is suitable for industrial production.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of dairy product preparation, and in particular relates to a casein phosphopeptide and a preparation method thereof. Background Art
[0002] Yak milk is a rare dairy product with extremely rich nutritional value. It is natural, pure and green and is regarded as "natural concentrated milk". Compared with ordinary cow's milk, yak milk has higher protein content and total amino acid content and is more comprehensive. Casein (CN) is the main protein in cow's milk and contains 8 essential amino acids for the human body, including α s1 -、α s2 Yak milk and its casein have huge economic value. In the future, milk and dairy products will increase significantly. The production and development of yak dairy products has broad market prospects.
[0003] Casein phosphopeptide (CPP) is a structural fragment obtained by enzymatic hydrolysis of bovine casein. Its basic structure is -SerPSerP-SerP-Glu-Glu-, and its molecular weight is 1000-5000Da. This structure plays an important role in exerting the functionality of CPP. CPP can be used as a carrier of calcium, magnesium, zinc, iron and other ions. It can chelate calcium ions in the neutral and alkaline lower intestine of animals, effectively preventing the formation of calcium phosphate precipitation, resulting in an increase in the concentration of soluble calcium in the small intestine, and increasing calcium absorption and utilization. With the promotion and application of calcium fortifiers, CPP has been widely used in calcium-fortified dairy products, nutritional supplements and anti-caries chewing gum.
[0004] To prevent the ingestion of bitter substances, most animals instinctively dislike bitter substances. However, bitterness is produced during the hydrolysis of casein into peptides, which in turn affects the tolerance of the product. Therefore, the presence of bitterness limits the application of casein hydrolysates in food. The bitterness of protein hydrolysates mainly comes from the hydrophobic peptides in the hydrolysate. Peptides containing more hydrophobic groups produce bitterness when they come into contact with taste buds. The degree of hydrolysis of casein is directly related to its bitterness, but in actual production, in order to increase the yield and functional properties of casein peptides, the degree of casein hydrolysis is increased as much as possible, so the generation of bitterness will be inevitable. Therefore, how to increase the content of casein phosphopeptides while avoiding the generation of bitterness is an urgent problem to be solved. Summary of the invention
[0005] In view of this, the object of the present invention is to provide a casein phosphopeptide and a preparation method thereof. The casein phosphopeptide prepared by the method has an extraction rate of more than 98%, and the casein phosphopeptide has no bitterness and good taste.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] The present invention provides a method for preparing casein phosphopeptide, comprising the following steps:
[0008] The spore suspension of Aspergillus oryzae RIB40 was fermented and cultured, the supernatant was collected by centrifugation, and then the enzymatically hydrolyzed yak milk casein was added to the supernatant to obtain crude casein phosphopeptide after reaction treatment.
[0009] Preferably, the method for preparing the enzymatically hydrolyzed yak casein comprises mixing a yak casein solution with trypsin for enzymatic hydrolysis to obtain the enzymatically hydrolyzed yak casein.
[0010] Preferably, the pH value of the yak milk casein solution is 7.5-8.5, the temperature of the enzymatic hydrolysis is 45-55° C., and the time of the enzymatic hydrolysis is 1-3 hours.
[0011] Preferably, the fermentation medium is PDA medium; the inoculation amount of the spore suspension is 5% to 7%; the fermentation temperature is 27 to 31°C, and the fermentation time is 36 to 50 hours; the reaction temperature is 45 to 55°C, and the reaction time is 4 to 6 hours; the supernatant has a pH of 7.0 to 8.0; and the volume ratio of the supernatant to the enzymatically hydrolyzed yak casein is 1:1 to 4.
[0012] Preferably, the preparation method further comprises: after obtaining the crude casein phosphopeptide, sterilizing the crude casein phosphopeptide to obtain the sterilized casein phosphopeptide.
[0013] Preferably, the preparation method further comprises: after obtaining the sterilized casein phosphopeptide, mixing the sterilized casein phosphopeptide with CaCl2, adding ethanol solution, precipitating with alcohol, filtering and collecting the precipitate, to obtain the product.
[0014] Preferably, the sterilization temperature is 80-90° C., and the sterilization time is 25-35 min.
[0015] Preferably, the sterilized casein phosphopeptide is a sterilized casein phosphopeptide with a pH of 5 to 6, and the mass volume ratio of the CaCl2 to the sterilized casein phosphopeptide is 0.1 to 0.15 g:100 mL; the volume ratio of the sterilized casein phosphopeptide to the ethanol solution is 1:0.5 to 1.5, and the volume percentage of the ethanol solution is 85% to 95%; the temperature of the alcohol precipitation is 3 to 6°C, and the time of the alcohol precipitation is 4 to 6 hours.
[0016] Preferably, the preparation of the yak casein solution comprises the step of mixing yak casein with water.
[0017] The present invention also provides a casein phosphopeptide prepared by the above preparation method.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The present invention provides a casein phosphopeptide and a preparation method thereof. The present invention uses Aspergillus oryzae RIB40 fermentation to obtain a supernatant, and treats the yak milk casein after enzymatic hydrolysis, which can further improve the yield of casein phosphopeptide, and the supernatant obtained by using Aspergillus oryzae RIB40 fermentation can also solve the bitterness caused by the enzymatic hydrolysis of yak milk casein, thereby improving the taste of casein phosphopeptide and making it more acceptable to consumers. The preparation method of the present invention has simple process, low cost, and is suitable for industrial production. DETAILED DESCRIPTION
[0020] The present invention provides a method for preparing casein phosphopeptide, comprising the following steps:
[0021] The spore suspension of Aspergillus oryzae RIB40 was fermented and cultured, the supernatant was collected by centrifugation, and then the enzymatically hydrolyzed yak milk casein was added to the supernatant to obtain crude casein phosphopeptide after reaction treatment.
[0022] In the present invention, the Aspergillus oryzae RIB40 is Aspergillus oryzae RIB40. The present invention does not specifically limit the source of Aspergillus oryzae RIB40, and any product known in the art or commercially available can be used. The fermentation medium is a PDA medium, and the raw materials of the PDA medium include potatoes and glucose in a mass ratio of 10:1. For example, based on 1L, the preparation method of the PDA medium is to mix 200g of potatoes, 20g of glucose and 1000mL of distilled water, and sterilize at 121°C for 30min. The inoculation amount of the spore suspension is preferably 5% to 7%, more preferably 5.5 to 6.5%, and the inoculation amount refers to the ratio of the volume of the seed solution transferred to the volume of the culture solution after inoculation; the fermentation temperature is preferably 27 to 31°C, more preferably 28 to 30°C, more preferably 28, 29 or 30°C, and the fermentation time is preferably 36 to 50h, more preferably 40 to 49h, more preferably 48h; the reaction temperature is preferably 45 to 55°C, more preferably 46 to 54°C, more preferably 50°C; the reaction time is preferably 4 to 6h, more preferably 4.5 to 5.5h, more preferably 5h; the pH of the supernatant is preferably 7.0 to 8.0, such as 7.0, 7.5 or 8.0; the volume ratio of the supernatant to the enzymatically hydrolyzed yak casein is preferably 1:1 to 4, more preferably 1:2 to 3, more preferably 1:2. The density of the spore suspension is 1 to 3×10 8 / mL. The preparation method of the spore suspension is to inoculate Aspergillus oryzae RIB40 in a malt juice medium with an inoculation amount of 6-10% to obtain a spore suspension. The culture temperature is preferably 27-31°C, further 28-30°C, more preferably 28, 29 or 30°C, and the culture time is preferably 3-5d, further preferably 3-4d, more preferably 4d. The supernatant obtained by fermentation culture of Aspergillus oryzae RIB40 in the present invention can further improve the yield of casein phosphopeptide, and the obtained casein phosphopeptide has no bitter taste.
[0023] In the present invention, the method for preparing the enzymatically hydrolyzed yak casein comprises mixing a yak casein solution with trypsin for enzymatic hydrolysis to obtain the enzymatically hydrolyzed yak casein. The pH value of the yak casein solution is preferably 7.5-8.5, and further 8.0; the enzymatic hydrolysis temperature is preferably 45-55°C, further preferably 46-54°C, and more preferably 50°C; the enzymatic hydrolysis time is preferably 1-3h, further preferably 1.5-2.5h, and more preferably 2h. The volume mass ratio of the yak casein solution to trypsin is 1000mL:0.1g. The preparation of the yak casein solution comprises the step of mixing yak casein with water, and the mass volume ratio of yak casein to water is preferably 1:8-10, and further preferably 1:9. The yield of casein phosphopeptides obtained by enzymatic hydrolysis of yak casein by the above method of the present invention is high.
[0024] The preparation method of the present invention further comprises: after obtaining the crude casein phosphopeptide, sterilizing the crude casein phosphopeptide to obtain the sterilized casein phosphopeptide. The sterilization temperature is preferably 80-90°C, more preferably 82-88°C, and more preferably 85°C; the sterilization time is preferably 25-35min, more preferably 26-34min, and more preferably 30min. After obtaining the sterilized casein phosphopeptide, the sterilized casein phosphopeptide is mixed with CaCl2, an ethanol solution is added, alcohol precipitation is performed, and the precipitate is collected by filtration. The sterilized casein phosphopeptide is a sterilized casein phosphopeptide with a pH of 5-6, such as a sterilized casein phosphopeptide with a pH of 5.5. The mass volume ratio of the CaCl2 to the sterilized casein phosphopeptide is 0.1-0.15g:100mL, more preferably 0.12-0.14g:100mL; the volume ratio of the sterilized casein phosphopeptide to the ethanol solution is preferably 1:0.5-1.5, and more preferably 1:1. The volume percentage of the ethanol solution is preferably 85% to 95%, such as a 90% ethanol solution; the temperature of the alcohol precipitation is 3 to 6°C, preferably 4 to 5°C, and the time of the alcohol precipitation is preferably 4 to 6 hours, more preferably 4.5 to 5.5 hours, and more preferably 5 hours. The casein phosphopeptide purified by the Ca-ethanol precipitation method has high purity.
[0025] The present invention also provides a casein phosphopeptide prepared by the above preparation method.
[0026] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.
[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0028] In the following examples, the yak milk casein was purchased from Luhuo Kangba Shengde Yak Industry Co., Ltd. The trypsin of the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd.
[0029] Example 1
[0030] A method for preparing casein phosphopeptide, comprising the following steps:
[0031] (1) adding 500 g of yak casein to 4500 mL of water to obtain a yak casein solution;
[0032] (2) adjusting the pH value of the yak milk casein solution to 8.0 with 1 mol / L NaOH solution, then adding 0.5 g of trypsin to 5000 mL of the pH 8.0 yak milk casein solution, and performing enzymatic hydrolysis at 50° C. for 2 h to obtain enzymatically hydrolyzed yak milk casein;
[0033] (3) Aspergillus oryzae RIB40 was inoculated into malt extract medium at an inoculum rate of 8% and cultured at 30°C for 4 days to obtain a spore suspension. 5 mL of 2 × 10 8 The spore suspension of 1000 spores / mL was inoculated into PDA medium at an inoculum amount of 6%, and fermented at 30°C for 48 hours, centrifuged at 4000 rpm for 15 minutes, and the supernatant was collected. The pH value of the supernatant was adjusted to 7.5, and then 100 mL of enzymatically hydrolyzed yak milk casein was added to 50 mL of the supernatant at pH 7.5, and the reaction was treated at 50°C for 5 hours to obtain crude casein phosphopeptide;
[0034] (4) adjusting the crude casein phosphopeptide to 85° C., keeping the temperature for 30 min, and sterilizing the crude casein phosphopeptide to obtain sterilized casein phosphopeptide;
[0035] (5) The pH of the sterilized casein phosphopeptide was adjusted to 5.5, 0.14 g of CaCl2 was added to 100 mL of the sterilized casein phosphopeptide with a pH of 5.5, and the mixture was stirred for 20 min. Then, 100 mL of a 90% ethanol solution was added, and the mixture was precipitated at 5°C for 5 h. The precipitate was collected by filtration and freeze-dried to obtain the dried casein phosphopeptide.
[0036] Example 2
[0037] A method for preparing casein phosphopeptide, comprising the following steps:
[0038] (1) adding 500 g of yak casein to 4000 mL of water to obtain a yak casein solution;
[0039] (2) adjusting the pH value of the yak milk casein solution to 7.5 with 1 mol / L NaOH solution, then adding 0.5 g of trypsin to 5000 mL of the pH 7.5 yak milk casein solution, and performing enzymatic hydrolysis at 45° C. for 3 h to obtain enzymatically hydrolyzed yak milk casein;
[0040] (3) Aspergillus oryzae RIB40 was inoculated into malt extract medium at an inoculum rate of 10% and cultured at 30°C for 3 days to obtain a spore suspension. 5 mL of 2 × 10 8 The spore suspension of 1000 ng / mL was inoculated into PDA medium at an inoculum amount of 7%, and fermented at 30°C for 50 h, centrifuged at 4000 rpm for 15 min, and the supernatant was collected. The pH value of the supernatant was adjusted to 7.0, and then 150 mL of enzymatically hydrolyzed yak milk casein was added to 50 mL of the supernatant at pH 7.0, and the reaction was treated at 50°C for 5.5 h to obtain crude casein phosphopeptide;
[0041] (4) adjusting the crude casein phosphopeptide to 80° C., keeping the temperature for 35 min, and sterilizing the crude casein phosphopeptide to obtain sterilized casein phosphopeptide;
[0042] (5) The pH of the sterilized casein phosphopeptide was adjusted to 5, 0.12 g of CaCl2 was added to 100 mL of the sterilized casein phosphopeptide at pH 5, the mixture was stirred for 20 min, and then 100 mL of 90% ethanol solution was added, the mixture was precipitated at 5°C for 4.5 h, the precipitate was collected by filtration, and freeze-dried to obtain the dried casein phosphopeptide.
[0043] Example 3
[0044] A method for preparing casein phosphopeptide, comprising the following steps:
[0045] (1) adding 500 g of yak casein to 5000 mL of water to obtain a yak casein solution;
[0046] (2) adjusting the pH value of the yak milk casein solution to 8.5 with a 1 mol / L NaOH solution, then adding 0.5 g of trypsin to 5000 mL of the pH 8.5 yak milk casein solution, and performing enzymatic hydrolysis at 55° C. for 1 h to obtain enzymatically hydrolyzed yak milk casein;
[0047] (3) Aspergillus oryzae RIB40 was inoculated into malt extract medium at an inoculum rate of 6% and cultured at 30°C for 5 days to obtain a spore suspension. 5 mL of 3×10 8The spore suspension of 1000 ng / mL was inoculated into PDA medium at an inoculum amount of 5%, and fermented at 30°C for 36 h, centrifuged at 4000 rpm for 15 min, and the supernatant was collected. The pH value of the supernatant was adjusted to 8.0, and then 150 mL of enzymatically hydrolyzed yak milk casein was added to 50 mL of the supernatant at pH 8.0, and the reaction was treated at 55°C for 4.5 h to obtain crude casein phosphopeptide;
[0048] (4) adjusting the crude casein phosphopeptide to 90° C., keeping the temperature for 25 min, and sterilizing the crude casein phosphopeptide to obtain sterilized casein phosphopeptide;
[0049] (5) The pH of the sterilized casein phosphopeptide was adjusted to 6, 0.15 g of CaCl2 was added to 100 mL of the sterilized casein phosphopeptide at pH 6, and the mixture was stirred for 20 min. Then, 100 mL of 90% ethanol solution was added, and the mixture was precipitated at 3°C for 6 h. The precipitate was collected by filtration and freeze-dried to obtain the dried casein phosphopeptide.
[0050] Comparative Example 1
[0051] The difference between this comparative example and Example 1 is that this comparative example 1 does not include step (3) of Example 1.
[0052] A method for preparing casein phosphopeptide, comprising the following steps:
[0053] (1) adding 500 g of yak casein to 4500 mL of water to obtain a yak casein solution;
[0054] (2) adjusting the pH value of the yak milk casein solution to 8.0 with 1 mol / L NaOH solution, then adding 0.5 g of trypsin to 5000 mL of the pH 8.0 yak milk casein solution, and performing enzymatic hydrolysis at 50° C. for 2 h to obtain enzymatically hydrolyzed yak milk casein;
[0055] (3) adjusting the temperature of the enzymatically hydrolyzed yak milk casein to 85° C., keeping the temperature for 30 min, and sterilizing the casein to obtain sterilized casein phosphopeptide;
[0056] (4) The pH of the sterilized casein phosphopeptide was adjusted to 5.5, 100 mL of the sterilized casein phosphopeptide with pH 5.5 was added with 0.14 g of CaCl2, stirred for 20 min, and then 100 mL of 90% ethanol solution was added, and the mixture was precipitated at 5°C for 5 h. The precipitate was collected by filtration and freeze-dried to obtain the dried casein phosphopeptide.
[0057] Test Example 1
[0058] (1) Determination of CPP content in dried casein phosphopeptides prepared in Example 1 and Comparative Example 1
[0059] Determination of casein phosphopeptide content: The CPP content was determined by the barium-ethanol precipitation method according to GB 31617-2014 "National Food Safety Standard Food Nutrition Fortifier Casein Phosphopeptide", the method is as follows:
[0060] Accurately weigh 1.5g of dried casein phosphopeptide and place it in a 50mL centrifuge tube, add 15mL of water to completely dissolve it. Adjust the pH value of the sample solution to 4.6 with hydrochloric acid solution, and centrifuge at 6000rpm and 4℃ for 30min. Take the supernatant and place it in a 50mL centrifuge tube that has been dried and weighed constant. Adjust the pH value to 6.8 with sodium hydroxide, add 1.5mL of barium chloride solution, and then add anhydrous ethanol that has been pre-refrigerated to 4℃ to 50mL, shake well, and refrigerate at 4℃ overnight (at least 12h). Centrifuge at 6000rpm and 4℃ for 30min, discard the supernatant to obtain the precipitate, place the precipitate in a 65℃ oven for 1h, and then place it in a 105℃ oven to dry to constant weight.
[0061] The yield of casein phosphopeptide content (%) = (the mass of the precipitate dried to a constant weight / the mass of the casein phosphopeptide after initial drying) × 100%.
[0062] The results of the measurement were based on the average value, which was repeated four times.
[0063] The measurement results of Example 1 and Comparative Example 1 are shown in Table 1.
[0064] Table 1 CPP content determination results of casein phosphopeptides obtained by different preparation methods
[0065]
[0066]
[0067] As shown in Table 1, compared with Comparative Example 1, the casein phosphopeptide content prepared in the present invention is high in yield. Therefore, the present invention increases the casein phosphopeptide content by treating the enzymatically hydrolyzed yak milk casein with the supernatant obtained by fermentation with Aspergillus oryzae RIB40.
[0068] (2) Determination of bitterness value of dried casein phosphopeptides prepared in Example 1 and Comparative Example 1
[0069] The bitterness value was determined by sensory evaluation, referring to the method of Lv Jindi (Study on the Debittering Process of Casein Phosphopeptide, Lv Jindi et al., Food and Fermentation Technology, 2017), and training sensory analysis evaluators according to GB / T 14195-93. After screening, 15 people (male to female ratio of 7:8, all non-smokers) formed an evaluation team. The evaluators rinsed their mouths with distilled water, took about 2mL of the product solution and placed it in their mouths, spit it out after 5-10s, and tasted the next sample after rinsing their mouths. The tasting interval of each sample was 3-5min. The solutions with quinine sulfate mass concentrations of 0, 0.05%, 0.1%, 0.2%, 0.3%, and 0.4% were selected as reference solutions. The bitterness levels were non-bitter, slightly bitter, weakly bitter, generally bitter, bitter, and extremely bitter, and the corresponding bitterness values were 1, 2, 3, 4, 5, and 6, respectively.
[0070] Table 2 Casein phosphopeptide bitterness score results obtained by different methods
[0071] Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 average value Comparative Example 1 5 6 5 5 5 5 6 6 5 5 4 6 5 5 6 5.27 Example 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 1.13
[0072] It can be seen from the results in Table 2 that, compared with Comparative Example 1, the casein phosphopeptide prepared by the present invention has no bitter taste. The present invention treats the enzymatically hydrolyzed yak milk casein with the supernatant obtained by fermentation with Aspergillus oryzae RIB40, thereby solving the bitterness caused by casein enzymatic hydrolysis, improving the taste of casein phosphopeptide, and making it more acceptable to consumers.
[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing casein phosphopeptide, characterized in that: The following steps are involved: The spore suspension of Aspergillus oryzae RIB40 was fermented and cultured, the supernatant was collected by centrifugation, and then the enzymatically hydrolyzed yak milk casein was added to the supernatant to obtain crude casein phosphopeptide after reaction treatment.
2. The preparation method according to claim 1, characterized in that: The method for preparing the enzymatically hydrolyzed yak casein comprises mixing a yak casein solution with trypsin for enzymatic hydrolysis to obtain the enzymatically hydrolyzed yak casein.
3. The preparation method according to claim 2, characterized in that: The pH value of the yak milk casein solution is 7.5-8.5, the temperature of the enzymatic hydrolysis is 45-55° C., and the time of the enzymatic hydrolysis is 1-3 hours.
4. The preparation method according to claim 1, characterized in that: The fermentation medium is a PDA medium; the inoculation amount of the spore suspension is 5% to 7%; the fermentation temperature is 27 to 31° C., and the fermentation time is 36 to 50 hours; the reaction temperature is 45 to 55° C., and the reaction time is 4 to 6 hours; the pH value of the supernatant is 7.0 to 8.0; and the volume ratio of the supernatant to the enzymatically hydrolyzed yak casein is 1:1 to 4.
5. The preparation method according to claim 1, characterized in that: The preparation method further comprises: after obtaining the crude casein phosphopeptide, sterilizing the crude casein phosphopeptide to obtain the sterilized casein phosphopeptide.
6. The preparation method according to claim 1, characterized in that: The preparation method further comprises: After obtaining the sterilized casein phosphopeptide, the sterilized casein phosphopeptide is mixed with CaCl2, an ethanol solution is added, alcohol precipitation is performed, and the precipitate is collected by filtration to obtain the product.
7. The preparation method according to claim 5, characterized in that: The sterilization temperature is 80-90° C., and the sterilization time is 25-35 minutes.
8. The preparation method according to claim 6, characterized in that: The sterilized casein phosphopeptide is a sterilized casein phosphopeptide with a pH of 5 to 6, the mass volume ratio of the CaCl2 to the sterilized casein phosphopeptide is 0.1 to 0.15 g:100 mL; the volume ratio of the sterilized casein phosphopeptide to the ethanol solution is 1:0.5 to 1.5, and the volume percentage of the ethanol solution is 85% to 95%; the temperature of the alcohol precipitation is 3 to 6° C., and the time of the alcohol precipitation is 4 to 6 hours.
9. The preparation method according to claim 2, characterized in that: The preparation of the yak milk casein solution comprises the step of mixing yak milk casein with water.
10. Casein phosphopeptide prepared according to the preparation method according to any one of claims 1 to 9.