HOP-MANGO WINE AND PREPARATION METHOD THEREOF

Abstract

Disclosed is a method for preparing hop-mango wine, belonging to the technical field of food processing. Based on the fruit wine brewing process, the invention incorporates different varieties of hops (Fuggle hops, Saaz hops, Cascade hops, Spalt hops) into mango wine to brew a hopmango wine rich in substances such as phenolics. Using mango wine from the same batch of mangoes produced under the same conditions without added hops as a blank control, the effects of different hop varieties on substances in mango wine, including total phenols, flavonoids, hydroxyl radical scavenging rate, and DPPH radical scavenging capacity, are investigated, and the influence of different hops on the antioxidant activity of mango wine is explored. The results indicate that under the experimental conditions, hops can enhance the antioxidant activity of mango wine to varying degrees. Among these, it is found comparatively that under the experimental conditions, a 1:3 addition amount of Fuggle hops resulted in stronger total phenol content and antioxidant activity than the other groups.

Description

TECHNICAL FIELD The present invention belongs to the technical field of food processing, and particularly relates to a hop-mango wine and a preparation method thereof. BACKGROUND Hops, scientifically known as Humu / us lupulus, are a plant with natural antioxidant effects. Among the chemical components of hops, hop essential oil, bitter substances, and polyphenols are the three major components that play important roles in beer brewing, significantly contributing to preventing beer oxidation, extending shelf life, and maintaining beer flavour stability. Polyphenolic substances in hops account for about 20% of the phenolic substance content in beer, serving as natural preservatives; the effective content of flavonoid substances in hop crude extract products ranges between 5% and 20%, holding significant value in food, pharmaceuticals, and health products. In brewing, whole hops are less frequently used due to a utilization rate of less than 30% and inconvenience in transportation, leading to more applied research selecting hop products. The Panzhihua region in China is one of the country's five superior mango development areas, where mango sales are primarily as fresh fruit, accounting for over 95%. However, mangoes are tropical fruits with vigorous physiological metabolism post-harvest, sensitivity to low temperatures, susceptibility to chilling injury, and poor storability. Furthermore, mango sales in China are mainly concentrated in mid- to low-end markets with low prices and concentrated harvest times, leading to seasonal surplus. To prevent significant waste caused by rot, mangoes are often processed into products such as canned mango, mango slices, mango pulp, and fruit wine to extend their shelf life. As a fermented fruit wine, mango wine retains the nutritional value of mango itself, and beneficial microorganisms produced through fermentation can also promote digestion and absorption. Additionally, consuming mango wine in daily life can protect blood vessels and prevent arteriosclerosis. During the mango wine brewing process, an appropriate amount of potassium metabisulfite needs to be added to adjust 802 concentration to protect juice stability, prevent oxidation, and effectively inhibit the growth of miscellaneous bacteria. The WHO also recommends reducing 802 concentration in food to prevent food poisoning caused by illegal addition. This invention selects Kiett mangoes from the Panzhihua region in China and four types of hops as experimental materials, measuring and comparing the total phenol content, flavonoid content, DPPH and hydroxyl radical scavenging capacities in mango wine with different hop addition amounts, providing a theoretical reference for application in mango wine quality improvement, industrial production, and the development and utilization of hops. SUMMARY To address the aforementioned technical problems, the present invention proposes a hop- mango wine and its preparation method. To achieve the above objective, the present invention provides the following technical solutions: The first technical solution of the present invention: A method for preparing hop-mango wine, comprising the following steps: (1) Mango treatment: selecting fresh, ripe Kiett mangoes with yellow skin, free from diseases, pests, and rot, having intact fruit and a sweet aroma, washing them thoroughly under running water, manually peeling and removing pits, then placing the peeled and pitted pulp into a blender to be pulped into mango juice; (2) Pectinase treatment: adding pectinase to the mango juice to increase mango juice yield, and then separating with a centrifuge to obtain clarified juice; (3) Composition adjustment: since fresh mango fruit contains 9.5% 20.9% sugar and using only fresh fruit for fermentation results in low alcohol content, appropriately adding white sugar to increase the fermentation alcohol level; (4) Preparation of extracts from different hop varieties: weighing Spalt hops and preparing hop solutions of the same concentration; (5) Dry yeast activation: taking an appropriate amount of active dry yeast based on the mass of the mango juice and adding the active dry yeast to distilled water for activation, then setting the active dry yeast aside for use; (6) Fermentation: placing a mango wine fermentation tank into a drying oven for primary and secondary fermentation; (7) Aging: aging the mature wine at temperatures below 20°C for 2 3 months to improve its flavour and taste, clarifying the aged wine using a gelatine-tannin method, followed by filtration with gauze, and finally bottling the mango wine and sterilizing it in a 90°C water bath for 20 minutes, thereby obtaining the hop-mango wine. Furthermore, in step (1) of the hop-mango wine preparation method, the pulping into mango juice is performed under a pulp-to-water ratio of 1:3. Furthermore, in step (2) of the hop-mango wine preparation method, pectinase is added to the mango juice at a mass fraction of 0.02%, and the treatment is conducted under a 45 50°C water bath condition for 1.5 hours. Furthermore, in step (3) of the hop-mango wine preparation method, white sugar is appropriately added to increase the fermentation alcohol content, and the sugar addition amount is 19%. Furthermore, in step (4) of the hop-mango wine preparation method, the preparation of extracts of different hop varieties requires the preparation of Fuggle hops, Saaz hops, Cascade hops, and Spalt hops; the preparation method is as follows: 200 mL of distilled water is taken and heated to 60°C, then 0.20 g of hops is added and boiled for 2 minutes, after which the mixture is allowed to cool to 25°C and is filtered. Furthermore, in step (5) of the hop-mango wine preparation method, the yeast inoculation amount is 0.2%, and the activation conditions comprise activation in 1% sucrose water at 35°C for 30 minutes. the active yeast is preferably Angel active dry yeast. Furthermore, in step (6) of the hop-mango wine preparation method, the temperature during primary fermentation is maintained at 25 30°C for 5 7 days, and the temperature during secondary fermentation is maintained at 20 25°C until fermentation concludes after approximately 10 days. The second technical solution of the present invention: A hop-mango wine prepared by the preparation method described above. Compared with the prior art, the present invention has the following advantages and technical effects: (1) The combination of mango's sweet aroma with the bitterness, citrus notes, and herbal aroma of hops may create a unique flavour profile, filling a gap in the market. (2) Mangoes are perishable and difficult to store; deep processing into wine can extend the industrial chain and reduce post-harvest losses, which is particularly suitable for mango producing areas. (3) The development of hop-mango wine in the present invention can enrich the mango processing product line, promote the transition of agriculture from primary production to high value-added processing, extend the storage time of mangoes, reduce losses, and improve economic benefits. BRIEF DESCRIPTION OF THE FIGURES The drawings forming a part of the present invention are used to provide further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an undue limitation of the present invention. In the drawings: Figure 1 is a flow chart of the hop-mango wine preparation method of the present invention. DESCRIPTION OF THE INVENTION Various exemplary embodiments of the present invention are described in detail below. This detailed description should not be construed as limiting the invention, but should be understood as a more detailed description of certain aspects, characteristics, and embodiments of the invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the invention. Furthermore, for numerical ranges in the present invention, each intermediate value between the upper and lower limits of the range is specifically disclosed. Every smaller range between any stated value or intermediate value in a stated range and any other stated value or intermediate value within the said range is also included in the invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described in the present invention, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and / or materials related to the respective documents. In the event of conflict with any incorporated document, the content of this specification shall prevail. Various modifications and changes to the specific embodiments described in the specification of the present invention can be made without departing from the scope or spirit of the invention, which will be obvious to those skilled in the art. Other embodiments derived from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the invention are exemplary only. Regarding the terms used herein, such as "comprising", "including", "having", "containing", etc., they are all open-ended terms, meaning including but not limited to. An embodiment of the present invention proposes a method for preparing hop-mango wine, a flow chart of which is shown in Figure 1, and the method specifically comprises the following steps: (1) Mango treatment is performed, where fresh, ripe Kiett mangoes with yellow skin, free from diseases, pests, and rot, having intact fruit and a sweet aroma are selected; the mangoes are washed thoroughly under running water, and manual peeling and pit removal are adopted; the peeled and pitted pulp is placed into a blender and is pulped into mango juice. (2) Pectinase treatment is carried out, where pectinase is added to the mango juice to increase the juice yield, and then a centrifuge is used for separation to obtain clarified juice. (3) Composition adjustment is conducted; since fresh mango fruit contains 9.5% to 20.9% sugar and using only fresh fruit for fermentation results in low alcohol content, white sugar is appropriately added to increase the fermentation alcohol level. (4) Extracts of different hop varieties are prepared, where Spalt hops are weighed, and hop solutions of the same concentration are prepared. (5) Dry yeast activation is performed, where an appropriate amount of active dry yeast is taken according to the mass of the mango juice, the yeast is added to distilled water for activation, and then it is set aside for use. (6) Fermentation is carried out, where a mango wine fermentation tank is placed into a drying oven for primary fermentation and secondary fermentation. (7) Aging is conducted, where the mature wine is aged at temperatures below 20°C for 2 to 3 months to improve its flavour and taste; the aged wine is clarified using a gelatine-tannin method and is then filtered with gauze; finally, the mango wine is bottled and is sterilized by maintaining it in a 90°C water bath for 20 minutes, whereby the hop-mango wine is obtained. In a preferred embodiment of the present invention, step (1) is performed, where the pulping into mango juice is carried out under a pulp-to-water ratio of 1 : 3. In a preferred embodiment of the present invention, step (2) is carried out, where pectinase is added to the mango juice at a mass fraction of 0.02%, and treatment is conducted under a 45 50°C water bath condition for 1.5 hours. In a preferred embodiment of the present invention, step (3) is conducted, where white sugar is appropriately added to increase the fermentation alcohol content, and the sugar addition amount is 19%. In a preferred embodiment of the present invention, step (4) is performed, where the preparation of extracts of different hop varieties requires the preparation of Fuggle hops, Saaz hops, Cascade hops, and Spalt hops; the preparation method is as follows: 200 mL ofdistilled water is taken and heated to 60°C, then 0.20 g of hops is added and boiled for 2 minutes, after which the mixture is allowed to cool to 25°C and is filtered. In a preferred embodiment of the present invention, step (5) is carried out, where the yeast inoculation amount is 0.2%, and the activation conditions are activation in 1% sucrose water at 35°C for 30 minutes. In a preferred embodiment of the present invention, step (6) is performed, where the temperature during primary fermentation is maintained at 2530°C for 57 days; the temperature during secondary fermentation is maintained at 2025°C, and fermentation is concluded after approximately 10 days. The technical solution of the present invention is further explained below through embodiments. Embodiment A method for preparing hop-mango wine is provided, which specifically comprises the following steps: (1) Mango treatment is performed, where fresh, ripe Kiett mangoes with yellow skin, free from diseases, pests, and rot, having intact fruit and a sweet aroma are selected; the mangoes are washed thoroughly under running water, and manual peeling and pit removal are adopted; the peeled and pitted pulp is placed into a blender, and pulping into mango juice is carried out under a pulp-to-water ratio of 1:3. (2) Pectinase treatment is carried out, where pectinase is added to the mango juice at a mass fraction of 0.02%, and treatment is conducted under a 4550°C water bath condition for 1.5 hours to increase the mango juice yield; then a centrifuge is used for separation to obtain clarified juice. (3) Composition adjustment is conducted; since fresh mango fruit contains 9.5% to 20.9% sugar and using only fresh fruit for fermentation results in low alcohol content, white sugar is appropriately added to increase the fermentation alcohol level; mango wine brewed with a 19% sugar content has the most mellow wine aroma; the white sugar is first dissolved in a small amount of mango juice and then added to the fermentation broth, and stirring is performed to mix the fermentation juice uniformly. (4) Spalt hop extract preparation is performed, where Spalt hops are weighed, and hop solutions of the same concentration are prepared; 200 mL of distilled water is taken and heated to 60°C, then 0.20 9 of hops is added and boiled for 2 minutes; after the mixture is allowed to cool to 25°C, filtration is performed, and the filtrate is set aside for use. (5) Dry yeast activation is performed, where an appropriate amount of active dry yeast is taken according to the mass ofthe mango juice, and the yeast is added to distilled water; constant- temperature water bath treatment at 3843°C is conducted for 30 minutes, then cooling is performed to below room temperature, and the activated yeast is set aside for use. (6) Fermentation is carried out, which comprises: (i) primary fermentation, where a mango wine fermentation tank is placed into a drying oven, the temperature is maintained at 2530°C, and fermentation is conducted for 57 days; (ii) secondary fermentation, where the temperature is maintained at 2025°C, and fermentation is concluded after approximately 10 days. (7) Aging is conducted, where the mature wine is aged at temperatures below 20°C for 2 to 3 months to improve its flavour and taste; the aged wine is clarified using a gelatine-tannin method and is then filtered with gauze; finally, the mango wine is bottled and is sterilized by maintaining it in a 90°C water bath for 20 minutes, whereby the Spalt hop-mango wine is obtained. Performance Testing (1) The basic physicochemical indicator test results for hop-mango wines of different varieties in the embodiment are shown in Table 1. Table 1 Analysis Results of Physicochemical Tests for Hop-Mango Wines of Different Varieties Total Phenols Alcohol Content Flavonoids _ Total Acid Sample Clarity (10'2) (mg / L) (%voL) (mg / L) (g / L) Control 264.9710.11 12.1110.29 256.1110.12 1.0210.41 17.331011 Group Fuggle 299.1210.29 13.1410.13 277.8310.78 1.0110.21 15.011011 Hops Saaz 274.2210.31 12.991033 274331031 11710.47 14.831056 Hops Cascade H 284.071030 12.3410.71 289.5610.47 0.841083 16.8410.32 ops Spalt 291.2110.56 12.921038 286.1810.71 0.951052 15.6110.71 Hops (2) The correlation analysis results between total phenols and antioxidant indicators for hop- mango wines of different varieties in the embodiment are shown in Table 2. Table 2 Correlation Analysis Between Total Phenols and Antioxidant lndicators in Hop Wine Samples Antioxidant Method Wine Sample Hydroxyl Radical Method DPPH Method Control Group 0.829* 0.887" Fuggle Hops 0.915" 0.925" Cascade Hops 0.879* 0.908* Spalt Hops 0854* 0.911" Note: "**" indicates a highly significant difference (P < 0.01 ), and "*" indicates a significant difference (P < 0.05). (3) The sensory evaluation results for hop-mango wines of different varieties in the embodiment are shown in Table 3. According to the results from Tables 1 to 3, it can be observed that the total phenol content in hop-added wine samples shows a significant positive correlation with antioxidant indicators (P < 0.05). The correlation coefficient R between total phenol content and hydroxyl radical scavenging capacity ranges from 0.854 to 0.916, and the correlation coefficient R between total phenol content and DPPH radical scavenging capacity ranges from 0.908 to 0.925. Table 3 Sensory evaluation results for hop-mango wines of different varieties Wine Comprehensive Comprehensive Colour Aroma Taste Style _ Sample Evaluation Score The colour is relatively Control 14 22 31 4 transparent, with fruit 71 Group _ and Wine aromas The colour is relatively Fu le trans arent, with ood gg 15 25 34 p g Hops fruit and wine aromas, and a pure taste The colour is relatively transparent, with Saaz Hops 14 25 34 certain fruit and wine 79 aromas, and a good taste The colour is relatively Cascade transparent, with fruit 14 24 32 5 75 Hops and wine aromas, but the body is thin The colour is relatively transparent, with slight Spalt Hops 15 26 34 7 _ _ 82 frUIt and Wlne aromas, but the style is poor Note: 85-100, Excellent; 80-84, Good; 70-79, Average; 50-70, Poor; <50, Unacceptable. Thus, it can be concluded that the key factor affecting the antioxidant activity of hop-mango wine is the total phenol content. Compared with the control group, the treated wine samples (added with Fuggle hops, Saaz hops, Cascade hops, and Spalt hops) exhibit relatively higher correlation coefficients between total phenol content and antioxidant activity, while the control group shows relatively lower correlation coefficients. Since an increased correlation may enhance the contribution of phenolic compounds to antioxidant capacity, the explanation for the improved antioxidant capacity of wine due to the presence of hops may be that hops, which are rich in phenolic compounds, increase the total phenol content of mango wine during the brewing process. Among these, the wine sample added with Spalt hops demonstrates the best effect. The above describes the preferred specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention shall fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method for preparing a hop-mango wine, which includes the following steps: (1) the processing of mangoes, by selecting fresh, ripe Kiett mangoes immediately yellow peel, free from diseases, pests and rot, with intact fruit and a sweet scent, the Wash the mangoes thoroughly under running water, peel them by hand and removing the pits, and then placing the peeled and pitted pulp in a blender to turn the peeled and pitted pulp into mango juice grind; (2) treating with pectinase, by adding pectinase to the mango juice to the to increase juice yield, followed by separation using a centrifuge to clarified juice available; (3) adjusting the composition, due to the use of only fresh fruit for fermentation, which results in a low alcohol content since fresh mango fruits contain 9.5% 20.9% sugar, by appropriately adding white sugar to add to increase the alcohol content of the fermentation; (4) preparing extracts of different hop varieties by weighing out aroma hops and to prepare hop solutions with the same concentration; (5) activating dry yeast by taking an appropriate amount of active dry yeast based on the mass of the mango juice and the active dry yeast to be added to distilled water for activation, then setting aside the active dry yeast for use; (6) ferment, by placing a mango wine fermentation tank in a drying oven for primary and secondary fermentation; (7) ripening, by ripening for 2 - 3 months at temperatures below 20°C of the wine to improve the taste and aroma, the clarification of the aged wine with using a gelatin-tannin method, followed by filtration with gauze, and finally bottling the mango wine and sterilizing the mango wine for 20 minutes in a 90°C water bath to obtain the hop-mango wine.

2. The method for preparing a hop-mango wine according to conclusion 1, whereby in step (1) the grinding into mango juice is carried out with a pulp-to-water ratio of 1:3 to to form mango juice.

3. The method for preparing a hop-mango wine according to conclusion 1, whereby in step (2) pectinase is added to the mango juice in a mass fraction of 0.02% and the Treatment for 1.5 hours is performed in a water bath of 45 - 50 °C.

4. The method for preparing a hop-mango wine according to conclusion 1, whereby in step (3) a suitable amount of white sugar is added to increase the alcohol content by to increase fermentation, whereby the amount of added sugar is 19%.

5. The method for preparing a hop-mango wine according to conclusion 1, whereby in step (4) preparing extracts from various hop varieties preparing Fuggie hops, comprises Saaz hops, Cascade hops and Spalt hops, and the method involves: taking 200 ml distilled water, heating to 60°C, adding 0.20 g hops, the Boil for 2 minutes, let it cool to 25°C, and then filter.

6. The method for preparing a hop-mango wine according to conclusion 1, whereby in step (5) the amount of added yeast is 0.2%, where the activation conditions include activation for 30 minutes at 35 °C in 1% sucrose water.

7. The method for preparing a hop-mango wine according to conclusion 1, whereby in step (6) during the primary fermentation the temperature is kept at 25 - 30°C for 5 - 7 days kept and during secondary fermentation the temperature is kept at 20 - 25°C until the fermentation is complete after about 10 days.

8. A hop-mango wine prepared according to the preparation method according to any of the conclusions 1 through 7. Figure 1 PATENT APPLICATION NO.: NO 200311 RESEARCH REPORT CONCERNING THE RESULT OF THE STATE OF THE ART RESEARCH RELEVANT LITERATURE 1 Literature with, where necessary, indication of of particular importance for Classification (IPC) Category text passages or figures. conclusion(s) no: X CN 107 858 243 A (CHANGSHA XINYU XINGYUAN 1-8 INV. BIOLOGICAL TECH CO LTD) C12G3 / 024 30 March 2018 (2018-03-30) C12G3 / 026 * conclusions 1-9 * C12G3 / 05 ----- C12G3 / 06 X CN 110 157 571 A (WU WEIJUN) 8 C12G3 / 08 23 August 2019 (2019-08-23) * example 8 * ----- A CN 103 497 867 A (ZHANG YUERONG) 1-8 8 January 2014 (2014-01-08) * paragraph [0002]; conclusions 1-2 * ----- A Anonymous: "Fining agents", 1-8 , 29 July 2025 (2025-07-29), XP093379753, Found on the Internet: URL:https: / / web.archive.org / web / 2025072922 5328 / https: / / www.awri.com.au / industry_supp ort / winemaking_resources / frequently_asked_ Surveyed areas questions / finding_agents / of technology [found on 2026-03-24] * page 5, paragraph 1 - paragraph 2 * C12G ----- A WO 2011 / 079455 A1 (DING ZHENGRAN [CN]; QIN 1-8 JIE [CN]) July 7, 2011 (2011-07-07) * page 3 - page 4; 1-3 * ----- - / -- If amended conclusions have been submitted, this report relates to the conclusions submitted on: Place of investigation: Date on which the investigation was conducted Competent official: completed: Munich March 24, 2026 Shadid, Rania 1 CATEGORY OF THE SIGNED LITERATURE X: the conclusion is deemed not new or not inventive T: after the filing date or the priority date considered in relation to this literature published literature that is not detrimental to the patent application, but is mentioned for clarification of Y: the conclusion is considered non-inventive at 2 the theory or principle underlying the in relation to the combination of this literature with other cited literature of the same category, invention where the combination is obvious to the skilled person E: earlier patent (application), published on or after the is deemed to be the filing date on which the same invention is described A: literature not belonging to category X or Y that the D: stated in the patent application describes the state of the art O: non-written state of the art L: literature mentioned for other reasons P: between the priority date and the filing date &: member of the same patent family or corresponding published literature patent publication page 1 of 2 EOB FORM 02.83 (P0414B) NO 200311 RELEVANT LITERATURE 1 Literature with, where necessary, an indication of of particular interest. Of importance to Category text sections or figures. conclusion(s) no: A ROMAN-BENN ANALESE ET AL: “Pectin: An 1-8 overview of sources, extraction and applications in food products, biomedical, pharmaceutical and environmental issues", FOOD CHEMISTRY ADVANCES, part 2, October 1, 2023 (2023-10-01), page 100192, XP093379317, ISSN: 2772-753X, DOI: 10.1016 / j.focha.2023.100192 * the entire document * ----- 1 CATEGORY OF THE REFERENCED LITERATURE 2 X: the conclusion is deemed not new or not inventive T: after the filing date or the priority date published literature that is not objectionable to the considered in relation to this literature Y: the claim is considered non-inventive for the patent application, but is included for the clarification of in relation to the combination of this literature with the theory or principle underlying the other cited literature of the same category, invention where the combination is obvious to the skilled person E: earlier patent (application), published on or after the filing date, on which the same invention is is considered to be lying down A: literature not belonging to category X or Y that the described state of the art describes D: stated in the patent application O: non-written state of the art L: literature mentioned for other reasons P: between the priority date and the filing date &: member of the same patent family or corresponding patent publication published literature page 2 of 2 EOB FORM 02.83 (P0414C) APPENDIX TO THE REPORT CONCERNING THE RESEARCH INTO THE STATE OF THE ART, NO 200311 CARRIED OUT IN PATENT APPLICATION NO. The appendix contains a list of patent applications or patents published elsewhere (so-called members of the same patent family), that correspond to patent specifications mentioned in the report. The statement has been compiled based on data from the European Patent Office's computer file as of The accuracy and completeness of this statement is guaranteed neither by the European Patent Office nor by the Industrial Office. Property guaranteed; the data is provided for informational purposes. 24-03-2026 In the report Date of Corresponding Date of mentioned patent document publication document(s) publication CN 107858243 A 30-03-2018 NONE ----------------------------------------------------------------------- CN 110157571 A 23-08-2019 NONE ----------------------------------------------------------------------- CN 103497867 A 08-01-2014 NONE ----------------------------------------------------------------------- WO 2011079455 A1 07-07-2011 NONE ----------------------------------------------------------------------- General information regarding this appendix has been published in the 'Official Journal' of the European Patent Office No. 12 / 82, pp. 448 et seq. WRITTEN OPINION FILE NUMBER SUBMISSION DATE PRIORITY DATE APPLICATION NUMBER NO200311 20.10.2025 CLASSIFICATION INV. C12G3 / 024 C12G3 / 026 C12G3 / 05 C12G3 / 06 C12G3 / 08 APPLICANT Sichuan Tourism University This written opinion contains an explanation of the following sections: Part I Basis of the written opinion Part II Priority Part III Determination of novelty, inventiveness and industrial applicability not possible Part IV The application relates to more than one invention Part V Reasoned statement regarding novelty, inventiveness and industrial applicability Part VI Other cited documents Part VII Other defects Part VIII Other remarks THE COMPETENT OFFICIAL Shadid, Rania

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