Composite antibiotic-replacing microcapsule preparation as well as preparation method and application thereof

Abstract

The invention provides a composite antibiotic-replacing microcapsule preparation as well as a preparation method and application thereof. The composite antibiotic-replacing microcapsule preparation comprises a core material as well as an inner-layer wall material and an outer-layer wall material which are sequentially coated outside the core material, wherein the core material comprises a Chinese herbal medicine extract, plant essential oil and probiotics, the Chinese herbal medicine extract comprises forsythin, astragalus polysaccharide and atractylenolide III, the plant essential oil comprises origanum oil and cinnamyl aldehyde, and the probiotics comprise bacillus subtilis; the inner-layer wall material is prepared by crosslinking sodium alginate and calcium chloride, and the outer-layer wall material comprises chitosan. The traditional Chinese medicine composition has the characteristics of synergistic interaction, high stability and good intestinal tract targeting property, and can improve the resistance replacement effect.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic alternative technology in animal husbandry, specifically relating to a compound antibiotic alternative microcapsule formulation, its preparation method, and its application. Background Technology

[0002] With the large-scale development of livestock farming, antibiotics are widely used in preventing livestock diseases and promoting growth. However, long-term excessive use has led to increasingly prominent problems such as bacterial resistance, drug residues in animal products, and intestinal flora imbalance, seriously threatening food safety and public health. Therefore, developing safe, efficient, and residue-free antibiotic alternatives has become a research hotspot in the livestock industry.

[0003] Currently, antibiotic alternatives on the market have the following problems: First, single-component antibiotic alternatives have limited efficacy. For example, herbal extracts have low bioavailability, plant essential oils are volatile and have poor stability, and probiotics are easily inactivated in feed processing and the gastrointestinal environment. Second, compound preparations are mostly simple mixtures that do not fully utilize the synergistic effect of each component. Third, there is a lack of targeted delivery technology. Functional components are easily destroyed by gastric acid in the stomach and are difficult to effectively colonize the target sites in the intestine, resulting in a significant reduction in the efficacy of antibiotic alternatives. Summary of the Invention

[0004] In view of this, the present invention provides a compound antibiotic alternative microcapsule formulation, its preparation method and application, which has the characteristics of synergistic effect, strong stability and good intestinal targeting, and can improve the antibiotic alternative effect.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a composite anti-antibiotic microcapsule formulation, the composite anti-antibiotic microcapsule formulation comprising a core material, and an inner wall material and an outer wall material sequentially coating the core material; wherein, the core material comprises traditional Chinese medicine extracts, plant essential oils, and probiotics, the traditional Chinese medicine extracts comprising forsythoside, astragalus polysaccharide, and atractylodes lactone III, the plant essential oils comprising oregano oil and cinnamaldehyde, and the probiotics comprising Bacillus subtilis; the inner wall material is obtained by cross-linking sodium alginate and calcium chloride, and the outer wall material comprises chitosan.

[0006] Preferably, the compound antibiotic alternative microcapsule formulation further includes glycerin.

[0007] Preferably, the mass ratio of forsythoside, astragalus polysaccharide, and atractylodes lactone III is (5-8):(3-6):(2-4); and / or, The mass ratio of oregano oil to cinnamaldehyde is 2:1; and / or, The viable count of the Bacillus subtilis was 1 × 10⁻⁶. 10 CFU / g; and / or, The mass ratio of the herbal extract, the plant essential oil and the Bacillus subtilis is (10~15):(2~4):(4~6).

[0008] Preferably, the particle size of the compound antibiotic alternative microcapsule formulation is 100 μm to 200 μm.

[0009] Secondly, the present invention also provides a method for preparing the aforementioned compound antibiotic alternative microcapsule formulation, comprising the following steps: S1. Forsythoside, astragalus polysaccharide and atractylodes lactone III are mixed to obtain a traditional Chinese medicine extract; S2. Mix oregano oil, cinnamaldehyde and emulsifier aqueous solution, emulsify to obtain plant essential oil emulsion; S3. Mix Bacillus subtilis and physiological saline to obtain a probiotic suspension; S4. Mix the herbal extract, the plant essential oil emulsion and the probiotic suspension, and disperse them by ultrasonication to obtain the core material mixture. S5. Mix the sodium alginate aqueous solution and the core material mixture, add it dropwise into the calcium chloride solution, and solidify to obtain primary microcapsules; S6. The primary microcapsules are soaked in a chitosan solution, cross-linked, washed, and vacuum dried to obtain a composite antibiotic alternative microcapsule formulation.

[0010] Preferably, in step S6, the primary microcapsules are soaked in a chitosan solution, crosslinked, washed, glycerol is added, and then vacuum dried to obtain a composite antibiotic alternative microcapsule formulation.

[0011] Preferably, in step S1, the preparation method of forsythoside includes the following steps: mixing forsythia, water and cellulase, ultrasonically extracting, then filtering, concentrating, purifying, and spray drying to obtain forsythoside.

[0012] Preferably, in step S1, the preparation method of the astragalus polysaccharide includes the following steps: mixing astragalus, water and cellulase, ultrasonically extracting, then filtering, concentrating, purifying, and spray drying to obtain astragalus polysaccharide.

[0013] Preferably, in step S1, the preparation method of atractylodes lactone III includes the following steps: mixing Atractylodes macrocephala, water and cellulase, ultrasonically extracting, then filtering, concentrating, purifying, and spray drying to obtain atractylodes lactone III.

[0014] Thirdly, the present invention also provides a feed additive, including the compound antibiotic alternative microcapsule formulation described above or the compound antibiotic alternative microcapsule formulation prepared by the preparation method described above.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the compound anti-antibiotic microcapsule preparation provided by the present invention, the extracts of traditional Chinese medicine, plant essential oils and probiotics complement each other and enhance the effect. Forsythoside, oregano oil and cinnamaldehyde work together to inhibit bacteria, astragalus polysaccharide enhances immunity, and atractylodes lactone III and Bacillus subtilis work together to regulate the intestinal flora, which can greatly improve the anti-antibiotic effect.

[0016] (2) The compound anti-antibiotic microcapsule formulation provided by the present invention has a double-layer microcapsule structure. The extracts of traditional Chinese medicine, plant essential oils and probiotics are embedded in the sodium alginate-chitosan double-layer wall material, so that the compound anti-antibiotic microcapsule formulation does not degrade in the stomach and is precisely released in the intestine, which can achieve targeted intestinal release and improve the bioavailability of functional components.

[0017] (3) The compound antibiotic alternative microcapsule formulation provided by the present invention has strong stability. The volatilization rate of plant essential oil can be reduced after encapsulation, and the survival rate of encapsulated probiotics in feed processing (high temperature, extrusion) can be improved.

[0018] (4) The compound antibiotic alternative microcapsule preparation provided by the present invention is safe and residue-free. All components are natural products or probiotics, which meet the safety standards for feed additives, have no risk of antibiotic residues, and can avoid the development of bacterial resistance.

[0019] (5) The compound antibiotic alternative microcapsule preparation provided by the present invention can be added to feed to increase the daily weight gain of piglets, reduce the feed conversion ratio, and improve production performance. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0021] In a first aspect, the present invention provides a composite anti-antibiotic microcapsule formulation, the composite anti-antibiotic microcapsule formulation comprising a core material, and an inner wall material and an outer wall material sequentially coating the core material; wherein, the core material comprises traditional Chinese medicine extracts, plant essential oils, and probiotics, the traditional Chinese medicine extracts comprising forsythoside, astragalus polysaccharide, and atractylodes lactone III, the plant essential oils comprising oregano oil and cinnamaldehyde, and the probiotics comprising Bacillus subtilis; the inner wall material is obtained by cross-linking sodium alginate and calcium chloride, and the outer wall material comprises chitosan.

[0022] In this invention, the extracts of traditional Chinese medicine, plant essential oils, and probiotics complement each other and synergistically enhance each other's effects. Forsythoside, oregano oil, and cinnamaldehyde work together to inhibit bacteria, astragalus polysaccharides enhance immunity, and atractylodes lactone III and Bacillus subtilis work together to regulate intestinal flora, which can significantly improve the antibiotic alternative effect. Moreover, the extracts of traditional Chinese medicine, plant essential oils, and probiotics are encapsulated in a sodium alginate-chitosan double-layer wall material, which prevents the compound antibiotic alternative microcapsule formulation from degrading in the stomach and allows for precise release in the intestine, achieving targeted intestinal release and improving the bioavailability of functional components. At the same time, the plant essential oils encapsulated in the double-layer wall material can reduce the volatility of the plant essential oils, and the probiotics encapsulated in the double-layer wall material can improve the survival rate of probiotics during feed processing (high temperature, extrusion) when the compound antibiotic alternative microcapsule formulation is added to feed. In addition, the components of the compound antibiotic alternative microcapsule formulation are natural products or probiotics, which meet the safety standards of feed additives, have no risk of antibiotic residues, and can avoid the development of bacterial resistance.

[0023] It should be noted that forsythoside has broad-spectrum antibacterial activity, inhibiting Escherichia coli and Salmonella; astragalus polysaccharide can enhance the body's immunity and promote the proliferation of immune cells; atractylodes lactone III can regulate the intestinal mucosal barrier function and improve digestion and absorption; and traditional Chinese medicine extracts can simultaneously inhibit bacteria and regulate immunity. Oregano oil contains carvacrol and thymol, which have strong antibacterial activity; cinnamaldehyde can destroy bacterial cell membranes and synergistically inhibit bacteria; and plant essential oils can broaden the antibacterial spectrum and enhance the antibacterial effect. Bacillus subtilis is acid and bile salt resistant, can colonize the intestine and secrete antimicrobial peptides; probiotics can synergistically regulate the intestinal flora with traditional Chinese medicine extracts and plant essential oils; sodium alginate is stable in an acidic environment (stomach), and chitosan is degraded in the alkaline environment of the intestine, achieving targeted release in the intestine.

[0024] In some embodiments, the compound anti-antibiotic microcapsule formulation further includes glycerol. Glycerol, as a plasticizer, can improve the stability of the compound anti-antibiotic microcapsule formulation.

[0025] In some embodiments, the mass ratio of forsythoside, astragalus polysaccharide, and atractylodes lactone III is (5~8):(3~6):(2~4). The mass ratio of forsythoside, astragalus polysaccharide, and atractylodes lactone III falling within the above range is beneficial for the herbal extract to simultaneously achieve antibacterial and immunomodulatory effects.

[0026] In some embodiments, the mass ratio of oregano oil to cinnamaldehyde is 2:1. A mass ratio of oregano oil to cinnamaldehyde within the aforementioned range is beneficial for broadening the antibacterial spectrum of the plant essential oil and enhancing its antibacterial effect.

[0027] In some embodiments, the viable count of the Bacillus subtilis is 1 × 10⁻⁶. 10 CFU / g. The viable count of the Bacillus subtilis is within the above range, which is beneficial for regulating the intestinal flora.

[0028] In some embodiments, the mass ratio of the herbal extract, the plant essential oil, and the Bacillus subtilis is (10~15):(2~4):(4~6). A mass ratio of the herbal extract, the plant essential oil, and the Bacillus subtilis within the above range is beneficial for improving the anti-antibiotic effect of the compound anti-antibiotic microcapsule formulation.

[0029] In some embodiments, the particle size of the compound anti-alternative microcapsule formulation is 100 μm to 200 μm. A particle size within this range is beneficial for ensuring the targeted release of the compound anti-alternative microcapsule formulation in the gastrointestinal tract.

[0030] Secondly, the present invention also provides a method for preparing the aforementioned compound antibiotic alternative microcapsule formulation, comprising the following steps: S1. Forsythoside, astragalus polysaccharide and atractylodes lactone III are mixed to obtain a traditional Chinese medicine extract; S2. Mix oregano oil, cinnamaldehyde and emulsifier aqueous solution, emulsify to obtain plant essential oil emulsion; S3. Mix Bacillus subtilis and physiological saline to obtain a probiotic suspension; S4. Mix the herbal extract, the plant essential oil emulsion and the probiotic suspension, and disperse them by ultrasonication to obtain the core material mixture. S5. Mix the sodium alginate aqueous solution and the core material mixture, add it dropwise into the calcium chloride solution, and solidify to obtain primary microcapsules; S6. The primary microcapsules are soaked in a chitosan solution, cross-linked, washed, and vacuum dried to obtain a composite antibiotic alternative microcapsule formulation.

[0031] It should be noted that emulsifying the plant essential oil before encapsulation can reduce its volatility and make it easier to disperse. In step S2, the emulsification can be high-speed shear emulsification, and the emulsifier aqueous solution can be a Tween-80 aqueous solution. In step S3, the physiological saline is preferably sterile physiological saline. The order of steps S1 to S3 is not limited. Steps S5 and S6 are a double-layer microcapsule encapsulation process, in which an inner wall material and an outer wall material are sequentially coated on the core material to form a sodium alginate-chitosan double-layer wall material. In step S5, the liquid after mixing the sodium alginate aqueous solution and the core material mixture can be dripped into the calcium chloride solution using a peristaltic pump.

[0032] In some embodiments, in step S6, the primary microcapsules are immersed in a chitosan solution for cross-linking, washed, and then glycerol is added, followed by vacuum drying to obtain a composite antibiotic alternative microcapsule formulation. Adding glycerol after washing, as a plasticizer, can improve the stability of the prepared composite antibiotic alternative microcapsule formulation.

[0033] In some embodiments, the preparation method of forsythoside in step S1 includes the following steps: mixing forsythia, water, and cellulase, ultrasonically extracting, then filtering, concentrating, purifying, and spray drying to obtain forsythoside. Using ultrasound-assisted enzymatic hydrolysis to extract forsythoside can improve the dissolution rate of active ingredients, shorten the extraction time, and retain the activity of the ingredients.

[0034] In some embodiments, the preparation method of Astragalus polysaccharide in step S1 includes the following steps: mixing Astragalus, water, and cellulase, extracting by ultrasound, then filtering, concentrating, purifying, and spray drying to obtain Astragalus polysaccharide. Using ultrasound-assisted enzymatic hydrolysis to extract Astragalus polysaccharide can improve the dissolution rate of active ingredients, shorten the extraction time, and retain the activity of the ingredients.

[0035] In some embodiments, the preparation method of atractylodes lactone III in step S1 includes the following steps: mixing Atractylodes macrocephala, water, and cellulase, extracting by ultrasound, then filtering, concentrating, purifying, and spray drying to obtain atractylodes lactone III. Using ultrasound-assisted enzymatic hydrolysis to extract atractylodes lactone III can improve the dissolution rate of the active ingredient, shorten the extraction time, and retain the activity of the ingredient.

[0036] Thirdly, the present invention also provides a feed additive, including the aforementioned compound antibiotic alternative microcapsule formulation or the compound antibiotic alternative microcapsule formulation prepared by the aforementioned method. Adding the compound antibiotic alternative microcapsule formulation to feed can promote intestinal health in piglets, enhance immunity, increase daily weight gain in piglets, reduce feed conversion ratio, and thereby improve production performance.

[0037] Example 1 A method for preparing a compound antibiotic alternative microcapsule formulation includes the following steps: S1. Take 6 g of forsythoside, 4 g of astragalus polysaccharide and 3 g of atractylodes lactone III, mix them to obtain 13 g of herbal extract; wherein: The preparation method of forsythoside is as follows: take 50 g of forsythia, add 500 mL of water, add 0.5 g of cellulase, extract by ultrasonication (power 200 W, temperature 50 ℃, time 60 min), filter, concentrate, purify and spray dry to obtain forsythoside; The preparation method of Astragalus polysaccharide is as follows: Take 50 g of Astragalus, add 500 mL of water, add 0.5 g of cellulase, and extract by ultrasonication (power 200 W, temperature 50 ℃, time 60 min). After filtration, concentration, purification and spray drying, Astragalus polysaccharide is obtained. The preparation method of atractylodes lactone III is as follows: Take 50 g of Atractylodes macrocephala, add 500 mL of water, add 0.5 g of cellulase, extract by ultrasonication (power 200 W, temperature 50 ℃, time 60 min), filter, concentrate, purify and spray dry to obtain atractylodes lactone III; S2. Take 2 g of oregano oil and 1 g of cinnamaldehyde, add 100 mL of an aqueous solution containing 1% Tween-80, and emulsify by high-speed shearing (10000 r / min, 15 min) to obtain a plant essential oil emulsion. S3. Take 5 g of Bacillus subtilis powder (live count 1×10⁻⁶). 10 Add CFU / g to 50 mL of sterile saline, stir well to obtain a probiotic suspension; S4. Mix the herbal extract obtained in step S1, the plant essential oil emulsion obtained in step S2, and the probiotic suspension obtained in step S3, and disperse them by ultrasonication (power 300 W, time 10 min) to obtain the core material mixture. S5. Take 4 g of sodium alginate, add 90 mL of deionized water, stir and dissolve at 50 °C, add the core material mixture obtained in step S4, stir evenly, and then drip it into 2% calcium chloride solution through a peristaltic pump. Solidify for 30 min, filter, and obtain primary microcapsules. S6. The primary microcapsules obtained in step S5 are soaked in a 1% chitosan solution and crosslinked for 20 min. After washing, 0.8 g of glycerol is added and the mixture is vacuum dried at 40 °C to obtain the composite antibiotic alternative microcapsule formulation.

[0038] Comparative Example 1 A method for preparing a compound antibiotic alternative microcapsule formulation differs from Example 1 in that: in step S1, atractylodes lactone III is replaced with licorice extract; the rest is the same as in Example 1.

[0039] Comparative Example 2 A method for preparing a compound antibiotic alternative microcapsule formulation differs from Example 1 in that: in step S2, cinnamaldehyde is replaced with thymol; the rest is the same as in Example 1.

[0040] Comparative Example 3 A method for preparing a compound antibiotic alternative microcapsule formulation differs from Example 1 in that: in step S3, Bacillus subtilis is replaced with lactic acid bacteria; the rest is the same as in Example 1.

[0041] Comparative Example 4 A method for preparing a compound antibiotic alternative differs from Example 1 in that steps S5 and S6 are omitted, and the core material mixture obtained in step S4 is directly spray-dried; the rest is the same as in Example 1.

[0042] Application examples Animal experiments verified 1. Experimental Grouping: 400 weaned piglets aged 21 days were randomly divided into 8 groups of 50 piglets each. There were no significant differences in initial weight and health status among the groups. The experimental period was 42 days. All groups were fed a basal diet (corn-soybean meal type, crude protein 18%, digestible energy 3.2 MJ / kg), with only the additives differing. The specific grouping is shown in Table 1. The formulations prepared in Example 1 and Comparative Examples 1-4 are respectively designated as Formulation A, Formulation B, Formulation C, Formulation D, and Formulation E.

[0043] Table 1 Animal Experiment Grouping

[0044] 2. Detection indicators: average daily weight gain, feed conversion ratio, diarrhea rate, intestinal flora (number of Escherichia coli and lactic acid bacteria), and serum immunoglobulin (IgG and IgA) content. The results are shown in Table 2.

[0045] Table 2 Results of detection indicators for each animal experimental group

[0046] As shown in Table 2, for growth performance, the average daily weight gain of group 4 (474.4 g / d) was higher than that of other groups (P<0.05), with an increase of 18.6% compared to group 1 (blank control group), an increase of 5.6% compared to group 2 (antibiotic control group), and an increase of 4.8% to 13.4% compared to groups 5-8 (comparative groups). The feed conversion ratio of group 4 (2.00) was significantly lower than that of other groups, with a decrease of 13.0% compared to group 1 and a decrease of 5.7% to 12.3% compared to groups 5-8. This indicates that the compound antibiotic alternative microcapsule formulation provided by this invention has the best growth-promoting effect. Regarding gut health, the diarrhea rate in group 4 (3.2%) was only 20.5% of that in group 1, a decrease of 59.0% compared to group 5 (herbal medicine replacement group), and a decrease of 57.3% compared to group 8 (unencapsulated group). The number of intestinal lactic acid bacteria in group 4 increased by 83.8% compared to group 1, and the number of Escherichia coli decreased by 71.2% compared to group 1, both significantly better than the control group (P<0.05). This indicates that the compound antibiotic alternative microcapsule formulation provided by this invention has outstanding effects on gut microbiota balance and disease prevention and control. Regarding immunity, the serum IgG and IgA levels in group 4 increased by 25.3% and 31.7% respectively compared to group 1, indicating that the compound anti-antibody microcapsule formulation provided by this invention has a significant immune-enhancing effect.

[0047] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0048] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound antibiotic alternative microcapsule formulation, characterized in that, The composite antibiotic alternative microcapsule formulation includes a core material, and an inner wall material and an outer wall material sequentially coating the core material; wherein, the core material includes traditional Chinese medicine extracts, plant essential oils, and probiotics, the traditional Chinese medicine extracts include forsythoside, astragalus polysaccharide, and atractylodes lactone III, the plant essential oils include oregano oil and cinnamaldehyde, and the probiotics include Bacillus subtilis; the inner wall material is obtained by cross-linking sodium alginate and calcium chloride, and the outer wall material includes chitosan.

2. The compound antibiotic alternative microcapsule formulation according to claim 1, characterized in that, The compound antibiotic alternative microcapsule formulation also includes glycerin.

3. The compound antibiotic alternative microcapsule formulation according to claim 1, characterized in that, The mass ratio of the forsythoside, the astragalus polysaccharide, and the atractylodes lactone III is (5~8):(3~6):(2~4); and / or, The mass ratio of oregano oil to cinnamaldehyde is 2:1; and / or, The viable count of the Bacillus subtilis was 1 × 10⁻⁶. 10 CFU / g; and / or, The mass ratio of the herbal extract, the plant essential oil and the Bacillus subtilis is (10~15):(2~4):(4~6).

4. The compound antibiotic alternative microcapsule formulation according to claim 1, characterized in that, The particle size of the compound antibiotic alternative microcapsule formulation is 100 μm to 200 μm.

5. The method for preparing the compound antibiotic alternative microcapsule formulation according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Forsythoside, astragalus polysaccharide and atractylodes lactone III are mixed to obtain a traditional Chinese medicine extract; S2. Mix oregano oil, cinnamaldehyde and emulsifier aqueous solution, emulsify to obtain plant essential oil emulsion; S3. Mix Bacillus subtilis and physiological saline to obtain a probiotic suspension; S4. Mix the herbal extract, the plant essential oil emulsion and the probiotic suspension, and disperse them by ultrasonication to obtain the core material mixture. S5. Mix the sodium alginate aqueous solution and the core material mixture, add it dropwise into the calcium chloride solution, and solidify to obtain primary microcapsules; S6. The primary microcapsules are soaked in a chitosan solution, cross-linked, washed, and vacuum dried to obtain a composite antibiotic alternative microcapsule formulation.

6. The method for preparing the compound antibiotic alternative microcapsule formulation according to claim 5, characterized in that, In step S6, the primary microcapsules are soaked in a chitosan solution, crosslinked, washed, glycerol is added, and then vacuum dried to obtain the composite antibiotic alternative microcapsule formulation.

7. The method for preparing the compound antibiotic alternative microcapsule formulation according to claim 5, characterized in that, In step S1, the preparation method of forsythoside includes the following steps: mixing forsythia, water and cellulase, ultrasonically extracting, then filtering, concentrating, purifying, and spray drying to obtain forsythoside.

8. The method for preparing the compound antibiotic alternative microcapsule formulation according to claim 5, characterized in that, In step S1, the preparation method of Astragalus polysaccharide includes the following steps: mixing Astragalus, water and cellulase, ultrasonically extracting, then filtering, concentrating, purifying, and spray drying to obtain Astragalus polysaccharide.

9. The method for preparing the compound antibiotic alternative microcapsule formulation according to claim 5, characterized in that, In step S1, the preparation method of atractylodes lactone III includes the following steps: mixing atractylodes macrocephala, water and cellulase, extracting by ultrasonication, then filtering, concentrating, purifying, and spray drying to obtain atractylodes lactone III.

10. A feed additive, characterized in that, This includes the compound anti-antibiotic microcapsule formulation according to any one of claims 1 to 4, or the compound anti-antibiotic microcapsule formulation prepared by the preparation method according to any one of claims 5 to 9.

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