A harmless recycling treatment method for pig manure containing heavy metals and antibiotics

Abstract

The present application relates to biological fermentation and new material preparation technology. The present application provides a harmless recycling treatment method of pig manure containing heavy metals and antibiotics, which comprises the following steps: adding malusrobusta pomace to pig manure, and carrying out composting fermentation; adding water to the composting fermentation product, and carrying out liquid fermentation; carrying out solid-liquid separation on the fermentation product of liquid fermentation, and respectively taking the residue phase product and the liquid phase product; drying the residue phase product, and then reacting in a cellulase aqueous solution, then configuring the reaction product into an aqueous solution, and then adding hyaluronic acid, polyvinyl alcohol, glycerol and glutaraldehyde in sequence, and then carrying out heating reaction after stirring uniformly to obtain a membrane liquid, and then preparing a breathable membrane; adding the liquid phase product to boiled and rotten potatoes, and then carrying out reaction, and then evaporating water in the reaction product to form a mass, and then sterilizing and freeze-drying to obtain a feed additive. The present application can significantly reduce the content of effective state heavy metals and antibiotics, the obtained feed can promote the growth of pigs, and the obtained breathable membrane has excellent performance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of agricultural waste treatment, and relates to a harmless recycling treatment method for pig manure containing heavy metals and antibiotics. BACKGROUND

[0002] The harmless treatment of animal manure has been a focus of research, which helps to alleviate the environmental pollution and epidemic transmission risk caused by animal manure. However, the general harmless treatment technology for animal manure lacks the key treatment of heavy metals and antibiotics in the manure, so that the content of heavy metals and antibiotics in the treated product still exceeds the standard, resulting in that the treated product cannot be recycled and has the risk of environmental pollution.

[0003] In recent years, there have been many research results on the treatment of heavy metals and antibiotics in manure. For example, Wang Qirong [1] achieved effective treatment of heavy metals by using microbial leaching technology, and Zhong Yujian [2] also achieved effective treatment of heavy metals and antibiotics by using microbial leaching technology. However, the main problem of microbial leaching technology is the lack of effective treatment or recycling method for leaching liquor, which leads to poor economic efficiency of industrial application of this method. Fermentation technology as a green technology is increasingly attracting people's attention, and there have been many reports on the treatment of heavy metals and antibiotics in manure, for example, Zhao Chuyue [3] and others used high-temperature aerobic fermentation to treat chicken manure, achieving effective passivation of heavy metals.

[0004] It can be seen that many harmless treatment technologies for heavy metals and antibiotics in manure have been found in the art, and this aspect of harmless technology has been relatively mature if the input-output problem is not considered. However, the animal manure harmless treatment technology is facing the problem of industrialization and popularization, and the reason is that the treated product is difficult to be productized. Generally, animal manure itself or the fermentation product of animal manure can be used as plant fertilizer, but the value of plant fertilizer is usually low, which makes it difficult to realize high value of manure fermentation product. At the same time, affected by the palatability problem, animal manure fermentation product is generally difficult to be used to prepare animal feed with high value.

[0005] One direction to improve the value of animal manure is to use it to prepare functional materials, such as adsorbents based on the porosity of dried manure, or membrane materials based on the rich organic matter in manure. However, when using manure to prepare these materials, it is necessary to reduce the content of heavy metals and antibiotics in the manure. According to the inventors' knowledge, how to solve the above problems while ensuring that functional materials with excellent performance are obtained has not been reported in depth.

[0006] In addition, the current treatment of feces containing heavy metals and antibiotics is generally difficult to achieve complete high-value utilization of solid and liquid materials throughout the treatment process, resulting in solid or liquid waste, making harmless treatment and high-value not comprehensive.

[0007] In summary, the technical field in the treatment of pig manure containing heavy metals and antibiotics faces the shortcomings of low harmless treatment degree, incomplete harmless treatment and difficulty in achieving high-value treatment.

[0008] [1] Wang Qirong. Microbial leaching of heavy metal pollution in livestock and poultry manure[D]. Changchun University of Science and Technology, 2022. DOI: 10.26977 / d.cnki.gccgc.2022.000318

[0009] [2] Zhong Yujian. Effects of indirect biological leaching of Aspergillus niger on heavy metals, antibiotics and antibiotic resistance genes in chicken manure[D]. Guangdong University of Technology, 2019. DOI: 10.27029 / d.cnki.ggdgu.2019.000769

[0010] [3] Zhao Chao-yue, Wang Zhipeng, Ma Feng, et al. Study on heavy metal passivation in chicken manure during super-high temperature aerobic fermentation[C] / / Chinese Society for Environmental Sciences. Proceedings of the 2022 Annual Scientific and Technological Conference of the Chinese Society for Environmental Sciences (II). Northeastern University, School of Mechanical Engineering and Automation, 2022:8. DOI: 10.26914 / c.cnkihy.2022.016793 SUMMARY

[0011] In view of the shortcomings of the prior art, the purpose of the present application is to realize a full-phase harmless reprocessing method for pig manure containing heavy metals and antibiotics, without solid and liquid waste, and to realize high-value of the treatment products.

[0012] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0013] A harmless recycling treatment method for pig manure containing heavy metals and antibiotics, the treatment method comprising the following steps:

[0014] (1) adding rose fruit residue to pig manure, inoculating compound bacteria 1 for composting fermentation; adding water to the composting fermentation product, inoculating compound bacteria 2 for liquid fermentation, and performing solid-liquid separation on the fermentation product of liquid fermentation, and taking the residue phase product and the liquid phase product for standby respectively;

[0015] (2) drying the residue phase product, reacting the residue phase product in a cellulase aqueous solution, washing the reaction product with water and drying the reaction product, then preparing an aqueous solution of the reaction product, adding hyaluronic acid, polyvinyl alcohol, glycerol and glutaraldehyde in sequence, stirring uniformly and then performing a heating reaction to obtain a coating liquid, and preparing a breathable film through coating and drying;

[0016] (3) adding the liquid phase product to boiled potatoes to perform a reaction, evaporating water from the reaction product until the reaction product can be kneaded into a ball after the reaction is completed, sterilizing the reaction product and then performing freeze-drying to obtain a feed additive;

[0017] The complex bacteria 1 is composed of culture solutions of Aspergillus niger YM33182 and Bacillus subtilis ZK1-18.

[0018] The complex bacteria 2 is composed of culture solutions of Bacillus licheniformis ZJU12 and Bacillus subtilis YDC-001.

[0019] Further, in step (1), the weight ratio of pig manure to roxburgh rose pomace is 13-16:1, and the weight ratio of the composting fermentation product to water is 1:4-5.

[0020] Further, in step (1), the weight ratio of pig manure to roxburgh rose pomace is 13-16:1, and the weight ratio of the composting fermentation product to water is 1:4-5.

[0021] Further, when liquid fermentation is performed, the inoculation amount of the complex bacteria 2 is 2% v / v, the fermentation temperature is 35°C, and the fermentation time is 36 hours.

[0022] Further, in step (2), when the residue phase product is dried, the temperature is 180°C, and the residue phase product is ground through a 50-mesh sieve after drying; in the cellulase aqueous solution, the weight content of cellulase is 5%; the final concentrations of hyaluronic acid, polyvinyl alcohol, glycerol and glutaraldehyde are 1% w / w, 2% w / w, 1% w / w and 0.5% w / w, respectively.

[0023] Further, when the residue phase product is dried and reacted in a cellulase aqueous solution, the reaction temperature is 45°C, and the reaction time is 2 hours; the reaction product is ground through a 50-mesh sieve after drying; when the reaction product is prepared into an aqueous solution, the weight content of solute in the aqueous solution is 5%, and the aqueous solution is kept at 80°C for 30 minutes after preparation; when the heating reaction is performed, the temperature is 90°C, and the reaction time is 3 hours.

[0024] Further, in step (3), the liquid-solid weight ratio of the liquid product and the boiled and rotten potatoes is 1:8, and the reaction temperature of the reaction is 37℃, and the reaction time is 5 hours.

[0025] Further, the ratio of the number of bacterial bodies of Aspergillus niger YM33182 and Bacillus subtilis ZK1-18 in the complex bacteria 1 is 1:1, and the ratio of the number of bacterial bodies of Bacillus licheniformis ZJU12 and Bacillus subtilis YDC-001 in the complex bacteria 2 is 1:1.

[0026] The application also provides a breathable film and a feed additive obtained by the above-mentioned processing method, and a corresponding feed.

[0027] At the beginning of the research, the inventors referred to the method of fermenting hawthorn powder in the patent CN112715763B applied by the inventors' team before, and used Aspergillus niger YM33182 to ferment the fermentation substrate composed of pig manure and roxburgh rose pomace, but found that the fermentation process could not be completely started, and the fermentation process was passively terminated after 4 days of fermentation. The reason may be that the fermentation substrate is not suitable for the growth of Aspergillus niger YM33182, or the metabolic products in the early stage of fermentation seriously inhibit the growth of the bacterial body. Based on this situation, the inventors considered using complex bacteria for fermentation, and after continuous exploration, the inventors found that the combination of Aspergillus niger YM33182 and Bacillus subtilis ZK1-18 could successfully complete the composting fermentation process. It is worth mentioning that when Bacillus subtilis ZK1-18 is replaced by Bacillus subtilis YDC-001 used by the applicant's other research team in other patents (CN115736103A), the fermentation cannot be successfully completed. In addition, Bacillus subtilis ZK1-18 was first reported in CN104195078A, and it was originally used to decompose phosphorus. Therefore, after mixing it with Aspergillus niger YM33182, it produces the effect of reducing the content of heavy metals and antibiotics, and discovers its new use.

[0028] After the composting fermentation is completed, the inventor finds that the composting fermentation treatment has limited effect on the reduction of heavy metal and antibiotic content. After groping, the inventor attempts to continue liquid fermentation treatment on the composting fermentation product. Similarly, after continuous attempts, it is finally found that the effect of selecting the compound bacteria 2 is the best. Bacillus licheniformis ZJU12 is first reported in CN100352916C, and its original role is antibacterial. It can be known that after mixing Bacillus licheniformis ZJU12 and Bacillus subtilis YDC-001, the effect of reducing heavy metal and antibiotic content is also produced, and it can also be known that it has no obvious antibacterial effect on Bacillus subtilis YDC-001.

[0029] Through further research, it is found that the addition of rose pomace is necessary for the feeding effect of the obtained feed and the performance of the obtained breathable film, which can be known from one comparative example of the present application. In addition, the selection of hyaluronic acid is also important when preparing the breathable film, which can be known from another comparative example of the present application.

[0030] The beneficial effects of the present application are:

[0031] The present application realizes the full-phase, harmless and high-value recycling treatment of pig manure containing heavy metals and antibiotics, can realize more than 99.7% reduction of effective state content of various heavy metals, and more than 96% degradation of various antibiotic contents. The obtained feed can significantly promote the growth of pigs, and the obtained breathable film has excellent mechanical properties and breathability. DETAILED DESCRIPTION

[0032] The present application will be specifically described through the following examples, and it is necessary to point out here that the following examples are only used to further illustrate the present application and cannot be understood as limiting the protection scope of the present application. Some non-essential improvements and adjustments made by skilled persons in the art according to the above application content still belong to the protection scope of the present application.

[0033] Example 1

[0034] 1.1 Raw materials and reagents

[0035] Strains: Aspergillus niger YM33182, the preservation number is CCTCC No: M206H3; Bacillus subtilis ZK1-18, the preservation number is CGMCC No. 9402; Bacillus subtilis YDC-001, the preservation number is CCTCC NO: M201028; Bacillus licheniformis ZJU12, the preservation number is CCTCC M205072; all strains are commercially available, and the present application does not involve the screening and preservation of the foregoing strains.

[0036] Feces: pig feces collected from a pig farm, with a water content of 44%, a C / N of 12.1, an organic matter of 445 g / kg, a total nitrogen of 38.3 g / kg, an available Cu content of 84.92 mg / kg, an available Zn content of 452.35 mg / kg, an available Cd content of 37.15 mg / kg, an enrofloxacin content of 31.4 μg / kg, and a norfloxacin content of 21.7 μg / kg.

[0037] Compound bacteria 1: Aspergillus niger YM33182 and Bacillus subtilis ZK1-18 culture solutions each cultured to the plateau phase were mixed in a ratio of 1:1 according to the number of bacterial bodies to obtain the compound bacteria 1;

[0038] Compound bacteria 2: Bacillus licheniformis ZJU12 and Bacillus subtilis YDC-001 culture solutions were mixed in a ratio of 1:1 according to the number of bacterial bodies to obtain the compound bacteria 2.

[0039] Rosa roxburghii fruit residue: fresh Rosa roxburghii fruit flesh was crushed into residue.

[0040] Apple residue: apple flesh was crushed into residue.

[0041] Potatoes: purchased from a local farmers' market.

[0042] Basic feed: formula (w / w): corn 48%, broken rice 10%, bran 10%, bean cake 10%, fish meal 8%, green material 2.5%, calcium carbonate 0.5%, calcium phosphate 0.5%, salt 0.5%.

[0043] Cellulase solution: cellulase (enzyme activity 50000 U / g, purchased from Chengdu Wanxiang Hongrun Biological Technology Co., Ltd.) was added to distilled water to prepare a cellulase solution with a cellulase weight content of 5%;

[0044] Hyaluronic acid: purchased from Sigma-Aldrich;

[0045] Sodium alginate: purchased from Sigma-Aldrich;

[0046] Polyvinyl alcohol: purchased from Sigma-Aldrich;

[0047] Glycerol: purchased from Sigma-Aldrich;

[0048] Glutaraldehyde: purchased from Sigma-Aldrich;

[0049] Sterile water: self-made.

[0050] 1.2 Preparation method

[0051] 1.2.1 Fermentation

[0052] Primary fermentation:

[0053] According to the proportion of 15:1 by weight, add the Malus halliana residue to the pig manure, then add sterile water, stir evenly to 59-61% w / w, inoculate compound bacteria 1 according to the inoculation amount of 6% v / v, and compost fermentation for 25 days. During the period, turn over and aerate when the compost temperature reaches 50°C.

[0054] Secondary fermentation:

[0055] According to the weight ratio of 1:4.5, mix the primary fermentation product and sterile water, inoculate compound bacteria 2 according to the inoculation amount of 2% v / v, and carry out liquid fermentation at 35°C for 36 hours. After that, the fermentation is subjected to solid-liquid separation, and the residue phase and liquid phase products are taken respectively for standby.

[0056] 1.2.2 Product preparation

[0057] 1.2.2.1 Preparation of breathable film

[0058] (1) The residue phase product separated out after secondary fermentation is subjected to high-temperature (180°C) drying treatment, ground and passed through a 50-mesh sieve, then placed in a cellulase solution, reacted at 45°C for 2 hours, washed with sterile water 3 times after centrifugation, then centrifuged to take the residue phase, dried in the shade, ground and passed through a 50-mesh sieve;

[0059] (2) According to the proportion of 5% by weight content, configure the water solution of the product obtained in step (1), heat to 80°C and keep for 30 minutes, then add hyaluronic acid, polyvinyl alcohol, glycerol and glutaraldehyde in turn, so that the final concentrations of the four substances added are 1% w / w, 2% w / w, 1% w / w and 0.5% w / w respectively, stir evenly, then heat to 90°C, react under stirring condition (350 rpm) for 3 hours to obtain film solution, then evenly pour the film solution on a glass plate, spread with a plastic spatula, then place in an oven (60°C) to dry into a film.

[0060] 1.2.2.2 Preparation of feed

[0061] (1) The liquid phase product separated from the two-stage fermentation was added to the boiled potatoes at a liquid-solid weight ratio of 1:8. After 5 hours of reaction at 37°C, the temperature was raised to 100°C, and the water was evaporated under stirring until the mixture formed a lump. The lump was sterilized by high-temperature sterilization (121°C, 103.4 kPa, 30 minutes), and then freeze-dried to obtain a powder, which was a feed additive.

[0062] (2) The feed additive was added to the basal feed at an amount of 4% w / w to obtain a feed.

[0063] Example 2

[0064] In the first-stage fermentation, the weight ratio of pig manure to rose pomace was 13:1; in the second-stage fermentation, the weight ratio of the first-stage fermentation product to sterile water was 1:4; and the rest of the scheme was consistent with Example 1.

[0065] Example 3

[0066] In the first-stage fermentation, the weight ratio of pig manure to rose pomace was 16:1; in the second-stage fermentation, the weight ratio of the first-stage fermentation product to sterile water was 1:5; and the rest of the scheme was consistent with Example 1.

[0067] Comparative Example 1

[0068] The complex bacteria 1 was replaced by Aspergillus niger YM33182 single bacterium agent. The first-stage fermentation scheme in Example 1 was carried out, and after 4 days, the fermentation could not continue, the temperature decreased, and the fermentation failed.

[0069] Comparative Example 2

[0070] Bacillus subtilis ZK1-18 in the complex bacteria 1 was replaced by Bacillus subtilis YDC-001. The first-stage fermentation scheme in Example 1 was carried out, and after 7 days, the fermentation could not continue, the temperature decreased, and the fermentation failed.

[0071] Comparative Example 3

[0072] Bacillus subtilis YDC-001 in the complex bacteria 2 was replaced by Bacillus subtilis ZK1-18, and the rest was consistent with Example 1.

[0073] Comparative Example 4

[0074] The rose pomace was replaced by apple pomace, and the rest was consistent with Example 1.

[0075] Comparative Example 5

[0076] The hyaluronic acid is replaced by sodium alginate, and the rest is consistent with Example 1.

[0077] Comparative Example 6

[0078] The rest is consistent with Example 1 except that only one-stage fermentation is carried out.

[0079] Experimental Example 1

[0080] The residue phase and liquid phase in the two-stage fermentation products of Examples 1-3 and Comparative Examples 3-4 are detected for heavy metal and antibiotic content, and the compost fermentation product of Comparative Example 6 is detected for heavy metal and antibiotic content. It is found through detection that the heavy metals and antibiotics in Examples 1-3 are almost not transferred to the fermentation liquid phase, and the content of each heavy metal is not detected (less than 0.005 μg) in the liquid phase, and the content of each antibiotic is not detected (less than 0.0001 μg) in the liquid phase. The heavy metal and antibiotic content in the residue phase is shown in Table 1.

[0081] Table 1

[0082]

[0083] As shown in Table 1, the present application can reduce the effective state Cu content by 99.9%, reduce the effective state Zn content by 99.7%, and reduce the effective state Cd content by 98.9%, having excellent effect of reducing the effective state content of heavy metals. Comparative Examples 3-4, relative to Comparative Examples 1-2, although can also ferment smoothly, also have a certain reducing effect on the effective state content of heavy metals, but the heavy metal content level after treatment is still high, and the obtained product is difficult to use for preparing other products. Similarly, the degradation effect of the present application on enrofloxacin and norfloxacin is also very obvious, and the degradation rates are more than 96% and 98%, respectively. In addition, as shown in Comparative Example 6, without two-stage fermentation, only one-stage fermentation, the reducing effect on the effective state content of heavy metals and antibiotic content is extremely limited.

[0084] Since the effective state of heavy metals and antibiotic content of the fermentation product obtained after two-stage fermentation is very low, the obtained fermentation product can be used to prepare other products to improve the economic value of the fermentation product.

[0085] Experimental Example 2

[0086] The experimental animals were taken from 240 Jiangxiang piglets, which were weaned on the 12th to 14th day after birth and were fed with breast milk. The piglets were divided into 6 groups, 40 piglets in each group. Among them, examples 1-3 and comparative examples 3-4 were each taken as a group, and each group was fed with the obtained feed. The remaining group was fed with the basic feed as a control group. The feeding time was 50 days, twice a day, free water, and the daily weight gain and diarrhea incidence were investigated. The results are shown in Table 2.

[0087] Table 2

[0088]

[0089] Note: The daily weight gain data in the table is the average value, and the last digit is taken.

[0090] As shown in Table 2, the present application can significantly improve the daily weight gain of the test pigs and promote their growth. At the same time, the obtained feed can enhance immunity and significantly reduce immunity. It is worth noting that the feed obtained in comparative example 3 is not as good as the control group in promoting growth. The reason may be that after the change of the complex bacteria, the fermentation metabolites contain substances that inhibit the growth of pigs.

[0091] Experimental example 3

[0092] The mechanical properties and air permeability of the breathable films obtained in examples 1-3 and comparative examples 3-5 were tested. The mechanical properties were tested according to GB / T 1040.3-2006, and the air permeability was tested according to GB / T 1038-2000. The experimental results are shown in Table 3.

[0093] Table 3

[0094]

[0095] As shown in Table 3, the breathable film obtained by the present application not only has high tensile strength, which meets the general conditions of use, but also has good air permeability, which is very suitable for agricultural films. Although the film obtained in comparative example 3 also has certain strength and air permeability, due to the growth inhibition effect of the feed obtained in comparative example 3, the overall treatment method of comparative example 3 cannot achieve full high value utilization, and lacks economic efficiency. Comparative examples 4-5 lack both strength and air permeability, and the obtained film is difficult to use as a breathable film for actual use.

Claims

1. A method for the harmless reuse of pig manure containing heavy metals and antibiotics, characterized in that, The processing method includes the following steps: (1) Add prickly pear fruit residue to pig manure, inoculate compound bacteria 1 for composting fermentation; add water to the composting fermentation product, inoculate compound bacteria 2 for liquid fermentation, and separate the solid and liquid fermentation product from the liquid fermentation product, and take the residue product and liquid product for later use. (2) The residue product is dried, placed in a cellulase aqueous solution for reaction, the reaction product is washed with water and the reaction product is dried in the shade, and then the reaction product is prepared into an aqueous solution. Hyaluronic acid, polyvinyl alcohol, glycerol and glutaraldehyde are added in sequence, stirred evenly and heated to obtain a membrane solution. A breathable membrane is prepared by coating and drying. (3) The liquid product is added to the cooked potatoes to react. After the reaction is completed, the water content of the reaction product is evaporated until it can be kneaded into a ball. After sterilization, it is freeze-dried to obtain the feed additive. The compound bacteria 1 consists of a culture medium of Aspergillus niger YM33182 and Bacillus subtilis ZK1-18; The compound bacteria 2 consists of a culture medium of Bacillus licheniformis ZJU12 and Bacillus subtilis YDC-001; In step (1), the weight ratio of pig manure to prickly pear pomace is 13~16:1; the weight ratio of composting fermentation products to water is 1:4~5.

2. The method for harmless reuse of pig manure containing heavy metals and antibiotics according to claim 1, characterized in that, During composting fermentation, the moisture content of the fermentation substrate is 59-61% w / w, the inoculation amount of compound bacteria 1 is 6% v / v, the fermentation time is 25 days, and the compost is turned when the temperature reaches 50℃.

3. The method for harmless reuse of pig manure containing heavy metals and antibiotics according to claim 2, characterized in that, During liquid fermentation, the inoculum size of compound bacteria 2 was 2% v / v, the fermentation temperature was 35℃, and the fermentation time was 36 hours.

4. The method for harmless reuse of pig manure containing heavy metals and antibiotics according to claim 1, characterized in that, In step (2), the slag phase product is dried at a temperature of 180°C and then ground through a 50-mesh sieve; the cellulase aqueous solution contains 5% cellulase by weight; the final concentrations of hyaluronic acid, polyvinyl alcohol, glycerol and glutaraldehyde are 1%w / w, 2%w / w, 1%w / w and 0.5%w / w, respectively.

5. The method for harmless reuse of pig manure containing heavy metals and antibiotics according to claim 4, characterized in that, The residue product was dried and placed in a cellulase aqueous solution for reaction at a temperature of 45°C for 2 hours. After air-drying, the product was ground through a 50-mesh sieve. When preparing the product into an aqueous solution, the solute content in the aqueous solution was 5% by weight, and the solution was kept at 80°C for 30 minutes. When heating the product, the temperature was 90°C and the reaction time was 3 hours.

6. The method for harmless reuse of pig manure containing heavy metals and antibiotics according to claim 1, characterized in that, In step (3), the liquid-solid weight ratio of the liquid product and the cooked potato is 1:8, the reaction temperature is 37°C, and the reaction time is 5 hours.

7. The method for harmless reuse of pig manure containing heavy metals and antibiotics according to claim 1, characterized in that, The ratio of Aspergillus niger YM33182 to Bacillus subtilis ZK1-18 in compound bacteria 1 is 1:1; the ratio of Bacillus licheniformis ZJU12 to Bacillus subtilis YDC-001 in compound bacteria 2 is 1:

1.

8. A breathable membrane, characterized in that, The breathable membrane is prepared by the harmless reuse treatment method according to any one of claims 1 to 7.

9. A feed, characterized in that, The feed comprises feed additives and basic feed prepared by the harmless reuse treatment method according to any one of claims 1 to 7.

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