Special microbial compound fertilizer for improving quality and enhancing aroma of pandan leaves and preparation method of special microbial compound fertilizer

Abstract

The invention discloses a special microbial compound fertilizer for improving quality and enhancing aroma of pandan leaves and a preparation method of the special microbial compound fertilizer, and belongs to the technical field of bacterial fertilizers. The microbial compound fertilizer is obtained by utilizing compound bacteria to ferment and treat waste, the compound bacteria are formed by compounding yarrowia lipolytica, trichoderma and bacillus subtilis, and the waste is formed by compounding pineapple leaves and banana stems. In the compound bacteria, yarrowia lipolytica, trichoderma and bacillus subtilis are compounded in the form of bacterial powder, and the mass ratio is (1-5): (1-2): (1-4). The preservation number of the yarrowia lipolytica is ACCC (China General Microbiological Culture Collection Center) 21176, and the preservation number of the trichoderma sp. Is ACCC 31526; the mass ratio of pineapple leaves to banana stems in the waste is 3: 4. When the microbial fertilizer is applied to pandan leaf planting soil, the aroma components of the pandan leaves can be remarkably improved, and a novel bacterial fertilizer is provided for improving the quality and aroma of the pandan leaves.

Description

Technical Field

[0001] This invention relates to the field of microbial fertilizer technology, and in particular to a microbial compound fertilizer specifically for improving the quality and aroma of pandanus leaves and its preparation method. Background Technology

[0002] Pandanus leaves, also known as fragrant pandanus or variegated pandanus, are perennial herbaceous tropical aromatic plants, famous for their unique fragrance. The distinctive aroma of pandanus leaves primarily comes from 2-acetyl-1-pyrrolline and squalene, which are widely used in the food, beverage, and fragrance industries.

[0003] The cultivation method affects the production of aroma components in pandan leaves, and the current traditional methods of cultivating pandan leaves have the following problems:

[0004] Aroma quality is unstable: Traditional cultivation depends on varieties and climate, and the content of aroma components fluctuates greatly, making it difficult to standardize.

[0005] Dependence on chemical fertilizers: Overuse of chemical fertilizers leads to soil compaction and imbalance of microbial communities, which is detrimental to the synthesis of aroma substances in the long run and may also cause residues.

[0006] Existing microbial fertilizers are generally versatile but lack specificity: most microbial fertilizers on the market focus on promoting growth and resisting diseases, but lack functional strains and formulas that specifically regulate the aroma metabolism pathway of pandanus leaves, resulting in insignificant effects on improving quality and enhancing aroma.

[0007] Therefore, there is an urgent need to develop a specialized microbial compound fertilizer that can specifically promote the synthesis of aroma in pandanus leaves, improve plant health, and is environmentally friendly. Summary of the Invention

[0008] The purpose of this invention is to provide a microbial compound fertilizer specifically for improving the quality and aroma of pandan leaves and its preparation method, in order to solve the problems existing in the prior art. The microbial fertilizer containing three specific strains of yeast, Trichoderma and Bacillus subtilis obtained by optimizing the strains and preparation process can significantly improve the aroma components of pandan leaves, providing a new type of microbial compound fertilizer for improving the quality and aroma of pandan leaves.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] This invention provides a microbial compound fertilizer specifically for improving the quality and aroma of pandan leaves. It is obtained by fermenting waste using compound bacteria, wherein the compound bacteria are composed of Yersinia lipolytica, Trichoderma and Bacillus subtilis, and the waste is composed of pineapple leaves and banana stems.

[0011] Preferably, the compound bacteria are composed of Yersinia lipophila, Trichoderma and Bacillus subtilis in the form of bacterial powder, with a mass ratio of (1-5):(1-2):(1-4).

[0012] Preferably, the preservation number of the *Yarrowia lipolytica* is ACCC 21176, and the preservation number of the *Trichoderma* is ACCC 31526.

[0013] Preferably, the mass ratio of pineapple leaves to banana stems in the waste is 3:4.

[0014] This invention also provides a method for preparing the aforementioned microbial compound fertilizer, comprising the following steps:

[0015] (1) After sun-drying pineapple leaves and banana stems, crush them and inoculate half of the crushed material with a mixture of Yeast lipolytica and Trichoderma for aerobic fermentation to obtain the first fermentation product;

[0016] (2) Inoculate the first fermentation product with Bacillus subtilis. After fermentation for 12 hours, add the remaining 1 / 2 of the crushed material into the fermentation system, stir evenly and continue fermentation to obtain the second fermentation product, which is the microbial compound fertilizer.

[0017] Preferably, in step (1), the conditions for aerobic fermentation are: 28-30℃, 100-150rpm fermentation culture for 30-36h.

[0018] Preferably, in step (2), the fermentation conditions are 30-32℃, 60-80rpm for 60-72h.

[0019] The present invention also provides the application of the aforementioned microbial compound fertilizer in the cultivation of pandanus leaves.

[0020] The present invention also provides the application of the aforementioned microbial compound fertilizer in improving the quality and aroma of pandanus leaves.

[0021] The present invention also provides a method for improving the quality and aroma of pandanus leaves, including the step of applying the microbial compound fertilizer to the roots, wherein the root application is carried out by applying the fertilizer once before planting and once during planting.

[0022] The present invention discloses the following technical effects:

[0023] (1) Outstanding effect in targeted enhancement of aroma quality: The microbial compound fertilizer prepared by this invention through the synergistic compounding and step-by-step fermentation process of specific functional strains (Yersinia lipolyticis, Trichoderma and Bacillus subtilis) can target the synthesis of 2-acetyl-1-pyrrolline, a key aroma substance in pandanus leaves. Experimental data show that the optimal embodiment (experimental group 9) can make the content of 2-acetyl-1-pyrrolline in pandanus leaves reach 19.27±0.15μg / g, which is significantly higher than that of conventional fertilizer group (8.14±0.13μg / g) and commercially available ordinary microbial fertilizer group (11.07±0.20μg / g); at the same time, the squalene content is also synergistically increased to 312.15±12.71μg / g, realizing a stable and efficient enhancement of the aroma quality of pandanus leaves.

[0024] (2) Strong synergy between strains and innovative process, resulting in high viable cell count: A step-by-step inoculation and sequential substrate addition process is employed. First, *Yersinia lipolyticis* and *Trichoderma* are used to rapidly form a dominant microbial community and create a favorable microenvironment. Subsequently, *Bacillus subtilis* is introduced and substrate is added, effectively avoiding nutrient competition among the strains and achieving a high-density, stable coexistence of the three types of microorganisms. The viable cell count of the compound fertilizer prepared using the process of this invention can reach 2.41 × 10⁻⁶. 10 CFU / g provides a sufficient basis for active microorganisms to ensure fertilizer effectiveness.

[0025] (3) Resource utilization and environmental friendliness: Using agricultural waste such as pineapple leaves and banana stems as the main fermentation substrates, the resource utilization and high-value utilization of waste are realized, and the production cost is reduced. The product is based entirely on microbial fermentation and does not contain any chemically synthesized substances. After application, it can improve the soil micro-ecology and thus affect the synthesis of pandan leaf aroma components, which is in line with the development direction of green and sustainable agriculture.

[0026] (4) High specificity and easy application: This invention is designed for the aroma synthesis and metabolic pathway of Panax notoginseng leaves. The combination of strains and processes are specifically optimized for this crop, which solves the problem of insufficient specificity of general microbial fertilizers. The prepared microbial compound fertilizer can be directly used as base fertilizer or top dressing for root application. It has good compatibility with existing agronomic practices, is easy to operate, and is easy to promote among growers.

[0027] In summary, this invention not only provides a highly efficient fertilizer for improving the quality and aroma of pandanus leaves, but also establishes specific process methods in terms of strain synergy mechanism, fermentation process innovation and agricultural waste utilization, which have important application value and market prospects. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] 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. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Existing technologies relate to improving the aroma composition of pandanus leaves through fertilization, such as the invention patent "CN118160479A A Method for Fertilizing Pandanus Leaves," which discloses a method for fertilizing pandanus leaves by applying a mixed fertilizer once within one year of planting, followed by applications of the same mixed fertilizer every six months thereafter, and spraying zinc fertilizer during the pandanus leaf harvesting period; the mixed fertilizer includes inorganic and organic fertilizers, with the inorganic fertilizers including nitrogen, phosphorus, and potassium fertilizers. Through years of research, the applicant has discovered that a balanced application of nitrogen, phosphorus, and potassium fertilizers before harvest, and the application of zinc fertilizer during harvest, can significantly promote the growth of pandanus leaves and the accumulation of aroma components, ultimately significantly improving the quality of the pandanus leaves. Although this solution involves a technical approach that combines organic and inorganic fertilizers to enhance the aroma components of pandanus leaves, the reliance on chemical fertilizers leads to the following problems: excessive use of chemical fertilizers causes soil compaction and microbial community imbalance, which is detrimental to the synthesis of aroma substances in the long run and may result in residues; and organic fertilizers such as sheep manure have problems such as slow nutrient release, potential heavy metal content leading to excessive heavy metals in the soil, odor pollution, and difficulty in controlling fertilizer efficiency. In contrast, microbial agents have strong functionality. Therefore, this invention relates to a microbial compound fertilizer with a specific bacterial composition and prepared under a specific process.

[0034] The strains used in the following examples were sourced from the China Agricultural Microbiological Culture Collection Center: Yarrowia lipolytica ACCC21176 and Trichoderma sp. ACCC 31526. Bacillus subtilis was purchased from Jinan Xinchenxu Chemical Co., Ltd., with an active ingredient content of 99% (powder form). This strain can also be obtained through other commercial channels.

[0035] Preparation of Yersinia lipolytica powder: Yersinia lipolytica was activated with PDA medium and then inoculated with malt extract medium (20.0 g malt extract, 20.0 g glucose, 1.0 g peptone and 1 L distilled water) at an inoculation rate of 5% w / v. The mixture was placed in a shaker and fermented at 28℃ and 180 rpm for 120 h. The fermentation broth was collected and freeze-dried to obtain Yersinia lipolytica powder.

[0036] Preparation of Trichoderma powder: Trichoderma was activated with PDA medium and then inoculated with liquid fermentation medium (40g wheat bran, 50g potato, 20g sucrose, 0.5g KH2PO4, 0.5g MgSO4·7H2O, 0.25g CaCl2 and 1L distilled water) at an inoculation rate of 5% w / v. The mixture was placed on a shaker and fermented at 28℃ and 100rpm for 96h. The fermentation broth was collected and freeze-dried to obtain Trichoderma powder.

[0037] Preparation of Bacillus subtilis powder: Bacillus subtilis was activated with LB solid plates and then inoculated into LB liquid medium at an inoculation rate of 5% w / v. The medium was then placed in a shaker and fermented at 30℃ and 120 rpm for 72 h. The fermentation broth was collected and freeze-dried to obtain Bacillus subtilis powder.

[0038] Example 1: A method for preparing a microbial compound fertilizer specifically for improving the quality and aroma of pandanus leaves, comprising the following steps:

[0039] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, half of the powder was inoculated with a mixture of Yersinia lipolytica powder and Trichoderma powder at a 5% w / w inoculation rate (the mass ratio of Yersinia lipolytica powder and Trichoderma powder was 1:1). The moisture content was adjusted to 50% with sterile water and aerobic fermentation was carried out at 28℃ and 100rpm for 30h to obtain the first fermentation product.

[0040] (2) Inoculate the first fermentation product with Bacillus subtilis powder (the mass ratio of Yersinia lipolyticis powder and Bacillus subtilis powder is 1:1), ferment at 30℃ and 60rpm for 12h, then add the remaining 1 / 2 of the crushed material into the fermentation system, stir evenly at 100rpm, and continue to ferment at 30℃ and 60rpm for 60h to obtain the second fermentation product, which is the microbial compound fertilizer.

[0041] Example 2: A method for preparing a microbial compound fertilizer specifically for improving the quality and aroma of pandanus leaves, comprising the following steps:

[0042] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, half of the powder was inoculated with a mixture of Yersinia lipolytica powder and Trichoderma powder at an inoculation rate of 8% w / w (the mass ratio of Yersinia lipolytica powder and Trichoderma powder was 1:2). The moisture content was adjusted to 55% with sterile water and aerobic fermentation was carried out at 28℃ and 120rpm for 30h to obtain the first fermentation product.

[0043] (2) Inoculate the first fermentation product with Bacillus subtilis powder (the mass ratio of Yersinia lipolyticis powder and Bacillus subtilis powder is 1:1), ferment at 30℃ and 70rpm for 12h, then add the remaining 1 / 2 of the crushed material into the fermentation system, stir evenly at 100rpm, and continue to ferment at 30℃ and 70rpm for 65h to obtain the second fermentation product, which is the microbial compound fertilizer.

[0044] Example 3: A method for preparing a microbial compound fertilizer specifically for improving the quality and aroma of pandanus leaves, comprising the following steps:

[0045] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, half of the powder was inoculated with a mixture of Yersinia lipolytica powder and Trichoderma powder at a mass ratio of 2:1. The moisture content was adjusted to 55% with sterile water and aerobic fermentation was carried out at 30℃ and 120rpm for 30h to obtain the first fermentation product.

[0046] (2) Inoculate the first fermentation product with Bacillus subtilis powder (mass ratio of Yersinia lipolyticis powder to Bacillus subtilis powder 2:1), ferment at 32℃ and 70rpm for 12h, then add the remaining 1 / 2 of the crushed material into the fermentation system, stir evenly at 100rpm, and continue fermenting at 32℃ and 70rpm for 60h to obtain the second fermentation product, which is the microbial compound fertilizer.

[0047] Example 4: A method for preparing a microbial compound fertilizer specifically for improving the quality and aroma of pandanus leaves, comprising the following steps:

[0048] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, half of the powder was inoculated with a mixture of Yersinia lipolytica powder and Trichoderma powder at a 5% w / w inoculation rate (the mass ratio of Yersinia lipolytica powder and Trichoderma powder was 2:1). The moisture content was adjusted to 50% with sterile water and aerobic fermentation was carried out at 30℃ and 120rpm for 30h to obtain the first fermentation product.

[0049] (2) Inoculate the first fermentation product with Bacillus subtilis powder (mass ratio of Yersinia lipolyticis powder and Bacillus subtilis powder 2:2), ferment at 30℃ and 70rpm for 12h, then add the remaining 1 / 2 of the crushed material into the fermentation system, stir evenly at 100rpm, and continue fermenting at 30℃ and 70rpm for 60h to obtain the second fermentation product, which is the microbial compound fertilizer.

[0050] Example 5: A method for preparing a microbial compound fertilizer specifically for improving the quality and aroma of pandanus leaves, comprising the following steps:

[0051] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, half of the powder was inoculated with a mixture of Yersinia lipolytica powder and Trichoderma powder at an inoculation rate of 8% w / w (the mass ratio of Yersinia lipolytica powder and Trichoderma powder was 2:1). The moisture content was adjusted to 55% with sterile water and aerobic fermentation was carried out at 30℃ and 150rpm for 36h to obtain the first fermentation product.

[0052] (2) Inoculate the first fermentation product with Bacillus subtilis powder (mass ratio of Yersinia lipolyticis powder to Bacillus subtilis powder 2:3), ferment at 32℃ and 80rpm for 12h, then add the remaining 1 / 2 of the crushed material to the fermentation system, stir evenly at 100rpm, and continue fermenting at 32℃ and 80rpm for 72h to obtain the second fermentation product, which is the microbial compound fertilizer.

[0053] Example 6: A method for preparing a microbial compound fertilizer specifically for improving the quality and aroma of pandanus leaves, comprising the following steps:

[0054] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, half of the powder was inoculated with a mixture of Yersinia lipolytica powder and Trichoderma powder at a 5% w / w inoculation rate (the mass ratio of Yersinia lipolytica powder and Trichoderma powder was 2:1). The moisture content was adjusted to 55% with sterile water and aerobic fermentation was carried out at 28℃ and 100rpm for 32h to obtain the first fermentation product.

[0055] (2) Inoculate the first fermentation product with Bacillus subtilis powder (mass ratio of Yersinia lipolyticis powder to Bacillus subtilis powder 2:4), ferment at 30℃ and 80rpm for 12h, then add the remaining 1 / 2 of the crushed material to the fermentation system, stir evenly at 100rpm, and continue fermenting at 30℃ and 80rpm for 60h to obtain the second fermentation product, which is the microbial compound fertilizer.

[0056] Example 7

[0057] The difference from Example 6 is that the mass ratio of *Yersinia lipolyticis* powder to *Trichoderma* powder is 2:1, and the mass ratio of *Yersinia lipolyticis* powder to *Bacillus subtilis* powder is 2:3.5. All other method steps are the same.

[0058] Example 8

[0059] The difference from Example 6 is that the mass ratio of *Yersinia lipolyticis* powder to *Trichoderma* powder is 3:1, and the mass ratio of *Yersinia lipolyticis* powder to *Bacillus subtilis* powder is 3:3.5. All other method steps are the same.

[0060] Example 9

[0061] The difference from Example 6 is that the mass ratio of *Yersinia lipolyticis* powder to *Trichoderma* powder is 4:1, and the mass ratio of *Yersinia lipolyticis* powder to *Bacillus subtilis* powder is 4:3.5. All other method steps are the same.

[0062] Example 10

[0063] The difference from Example 6 is that the mass ratio of *Yersinia lipolyticis* powder to *Trichoderma* powder is 5:1, and the mass ratio of *Yersinia lipolyticis* powder to *Bacillus subtilis* powder is 5:3.5. All other method steps are the same.

[0064] Comparative Example 1

[0065] The difference from Example 9 is that Bacillus subtilis powder is not added. The specific steps are as follows:

[0066] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, half of the powder was inoculated with a mixture of Yersinia lipolytica powder and Trichoderma powder at a 5% w / w inoculation rate (the mass ratio of Yersinia lipolytica powder and Trichoderma powder was 4:1). The moisture content was adjusted to 55% with sterile water and aerobic fermentation was carried out at 28℃ and 100rpm for 32h to obtain the first fermentation product.

[0067] (2) Add the remaining 1 / 2 of the crushed material to the fermentation system, stir evenly at 100 rpm, and continue to ferment at 30℃ and 80 rpm for 72 hours to obtain the second fermentation product, which is microbial compound fertilizer.

[0068] Comparative Example 2

[0069] The difference from Example 9 is that no Yersinia lipolyticis powder is added. The specific steps are as follows:

[0070] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, half of the powder was inoculated with Trichoderma powder at a 5% w / w inoculation rate. The moisture content was adjusted to 55% with sterile water and aerobic fermentation was carried out at 28℃ and 100rpm for 32h to obtain the first fermentation product.

[0071] (2) Inoculate the first fermentation product with Bacillus subtilis powder (the mass ratio of Trichoderma powder to Bacillus subtilis powder is 1:3.5). After fermenting at 30℃ and 80rpm for 12h, add the remaining 1 / 2 of the crushed material into the fermentation system, stir evenly at 100rpm, and continue fermenting at 30℃ and 80rpm for 60h to obtain the second fermentation product, which is the microbial compound fertilizer.

[0072] Comparative Example 3

[0073] The difference from Example 9 is that Trichoderma powder is not added; the specific steps are as follows:

[0074] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, half of the powder was inoculated with Yersinia lipolytica powder at a 5% w / w inoculation rate. The moisture content was adjusted to 55% with sterile water and aerobic fermentation was carried out at 28℃ and 100rpm for 32h to obtain the first fermentation product.

[0075] (2) Inoculate the first fermentation product with Bacillus subtilis powder (mass ratio of Yersinia lipolyticis powder and Bacillus subtilis powder 4:3.5), ferment at 30℃ and 80rpm for 12h, then add the remaining 1 / 2 of the crushed material into the fermentation system, stir evenly at 100rpm, and continue fermenting at 30℃ and 80rpm for 60h to obtain the second fermentation product, which is the microbial compound fertilizer.

[0076] Comparative Example 4

[0077] The difference from Example 9 is that no Yersinia lipolyticis powder or Trichoderma powder is added. The specific steps are as follows:

[0078] Pineapple leaves and banana stems were sun-dried and then crushed into granules (2.5mm × 2.5mm). They were mixed in a mass ratio of 3:4 to form a powder. Half of the powder was inoculated with Bacillus subtilis powder at an inoculation rate of 5%. After fermentation at 30℃ and 80rpm for 12 hours, the remaining half of the powder was added to the fermentation system and stirred evenly at 100rpm. Fermentation was then continued at 30℃ and 80rpm for 60 hours to obtain microbial compound fertilizer.

[0079] Comparative Example 5

[0080] The difference from Example 9 is that Trichoderma powder and Bacillus subtilis powder are not added. The specific steps are as follows:

[0081] Pineapple leaves and banana stems were sun-dried and then pulverized into granules (2.5mm × 2.5mm). They were mixed in a mass ratio of 3:4 to form a powder. Half of the powder was inoculated with 5% w / w Yersinia lipolytica powder and the moisture content was adjusted to 55% with sterile water. The mixture was then subjected to aerobic fermentation at 28℃ and 100 rpm for 32 hours to obtain the first fermentation product. The remaining half of the powder was added to the fermentation system and stirred evenly at 100 rpm. Fermentation was then continued at 30℃ and 80 rpm for 60 hours to obtain the second fermentation product, which is the microbial compound fertilizer.

[0082] Comparative Example 6

[0083] The difference from Example 9 is that Yersinia lipolyticis powder and Bacillus subtilis powder are not added. The specific steps are as follows:

[0084] Pineapple leaves and banana stems were sun-dried and then pulverized into granules (2.5mm × 2.5mm). They were mixed in a mass ratio of 3:4 to form a powder. Half of the powder was then inoculated with Trichoderma powder at a 5% w / w inoculation rate. The moisture content was adjusted to 55% with sterile water, and the mixture was aerobic fermented at 28℃ and 100 rpm for 32 hours to obtain the first fermentation product. The remaining half of the powder was added to the fermentation system and stirred evenly at 100 rpm. Fermentation was then continued at 30℃ and 80 rpm for 60 hours to obtain the second fermentation product, which is the microbial compound fertilizer.

[0085] Comparative Example 7

[0086] The difference from Example 9 is that the pineapple leaves and banana stems were sun-dried and then pulverized into granules (2.5mm × 2.5mm) and mixed together in a 1:1 mass ratio to form a powder. All other methods and steps are the same.

[0087] Comparative Example 8

[0088] The difference from Example 9 lies in adjusting the timing of adding the pulverized material and the fermentation conditions. The specific steps are as follows:

[0089] (1) After sun-drying pineapple leaves and banana stems, they were crushed into granules (2.5mm×2.5mm) and mixed into a powder at a mass ratio of 3:4. Then, the powder was inoculated with a mixture of Yersinia lipolytica powder and Trichoderma powder at a 5% w / w inoculation rate (the mass ratio of Yersinia lipolytica powder and Trichoderma powder was 4:1). The moisture content was adjusted to 55% with sterile water and aerobic fermentation was carried out at 28℃ and 100rpm for 32h to obtain the first fermentation product.

[0090] (2) Inoculate the first fermentation product with Bacillus subtilis powder (mass ratio of Yersinia lipolyticis powder to Bacillus subtilis powder 4:3.5) and ferment at 30℃ and 80rpm for 72h to obtain the second fermentation product, which is the microbial compound fertilizer.

[0091] Comparative Example 9

[0092] The difference from Example 9 lies in adjusting the timing of adding the bacterial powder. The specific steps are as follows:

[0093] Pineapple leaves and banana stems were sun-dried and then pulverized into granules (2.5mm × 2.5mm). They were mixed in a mass ratio of 3:4 to form a powder. The powder was then inoculated with a mixture of Yersinia lipolytica powder, Trichoderma powder, and Bacillus subtilis powder at a 5% w / w inoculation rate (mass ratio of Yersinia lipolytica powder, Trichoderma powder, and Bacillus subtilis powder 4:1:3.5). The moisture content was adjusted to 55% with sterile water. The mixture was first fermented at 28℃ and 100rpm for 32 hours, and then at 30℃ and 80rpm for 72 hours to obtain a microbial compound fertilizer.

[0094] The viable count of microbial compound fertilizers prepared in Examples 1-10 and Comparative Examples 1-9 was determined using the plate count method. The results are shown in Table 1.

[0095] Table 1 Results of viable cell count determination

[0096]

[0097] The experimental data above show that the fermentation efficiency of microbial compound fertilizer directly depends on the type of microorganisms selected, the ratio of microorganisms, the timing of addition, and the ratio and timing of substrate addition (pineapple leaves and banana stems). Improper control of the microorganism ratio and timing of addition will disrupt the dynamic balance of the microbial community in the fermentation system, leading to a decrease in the total viable count. The substrate, as the main nutrient source for microbial growth, is rich in protein, lipids, carbohydrates, and various vitamins and minerals. By precisely controlling the combination of microorganisms, inoculation strategies, and timing of addition, yeast and Trichoderma can be guided to quickly form dominant populations. The secondary products produced by their metabolism can create a more suitable growth environment for the subsequently introduced Bacillus subtilis. Furthermore, to avoid excessive competition for nutrient resources between yeast and Trichoderma, adding the substrate in batches can provide sufficient usable substrate for Bacillus subtilis after the two fungi reach stable growth, promoting its rapid proliferation and ultimately achieving stable coexistence of the three types of microorganisms in the same system, resulting in a nutritionally complete microbial compound fertilizer with a high viable count.

[0098] Experimental Example 1

[0099] Twenty-two plots were selected, characterized by flat terrain, uniform fertility, consistent previous crop, and convenient irrigation and drainage. Healthy, disease-free Panax notoginseng leaf tissue culture seedlings or tillers of the same variety and uniform size were used. Planting spacing was set at 40cm × 60cm, with 100 seedlings planted in each plot. The seedlings were randomly divided into 22 groups: a blank control group, a conventional fertilizer group, a common microbial fertilizer group, experimental groups 1-10, and control groups 1-9.

[0100] Blank control group: No fertilizer was applied.

[0101] Conventional fertilizer group: Apply NPK fertilizer at the conventional dosage, once before transplanting and once at the root one month after transplanting.

[0102] Common microbial fertilizer group: Commercially available general-purpose microbial fertilizer (purchased from Shijiazhuang Baijiaxing Fertilizer Co., Ltd. in this experiment) was applied once to the soil before transplanting and once to the roots one month after transplanting. The application rate was 2.5 kg / mu each time.

[0103] Experimental groups 1-10 were each treated with the microbial compound fertilizer prepared in Examples 1-10. The fertilizer was applied to the soil once before transplanting and to the roots once one month after transplanting. The application rate was 2.5 kg / mu each time.

[0104] Control groups 1-9 were each applied the microbial compound fertilizer prepared in comparative examples 1-9. The fertilizer was applied to the soil once before transplanting and to the roots once one month after transplanting. The application rate was 2.5 kg / mu each time.

[0105] After transplanting, each group underwent routine water and fertilizer management. Two months after the second fertilization, the main aroma components (2-acetyl-1-pyrrolidone and squalene) in the leaves of Panax notoginseng were measured, and the results are shown in Table 2.

[0106] Table 2 Results of Aroma Component Determination

[0107]

[0108] The data above shows that after application, the microbial compound fertilizers prepared in experimental groups 1-10 of this invention had higher contents of 2-acetyl-1-pyrroline and squalene than the blank control group, the conventional fertilizer group, and the ordinary microbial fertilizer group, with experimental group 9 showing the best results. In contrast, control groups 1-9 generally showed that a combination of three or two types of bacteria was superior to a single bacteria. However, due to the influence of process conditions and strain selection, the proportion of various bacteria in coexistence could be affected. Therefore, even with comparable viable cell counts, differences in aroma components might occur, or even with a high viable cell count, the aroma component content might be reduced. In summary, this invention achieves optimal results only under specific process conditions using a combination of *Yersinia lipolytica*, *Trichoderma*, and *Bacillus subtilis*.

[0109] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial compound fertilizer specialized for improving the quality and flavor of Gynura japonica, characterized in that, It is obtained by using complex bacteria to ferment waste, wherein the complex bacteria are composed of Yarrowia lipolytica, Trichoderma and Bacillus subtilis, and the waste is composed of pineapple leaves and banana stems.

2. The compound microbial fertilizer according to claim 1, characterized in that, The Yarrowia lipolytica, Trichoderma and Bacillus subtilis in the complex bacteria are compounded in the form of bacterial powder, and the mass ratio is (1-5):(1-2):(1-4).

3. The compound microbial fertilizer according to claim 1, characterized in that, The preservation number of the Yarrowia lipolytica is ACCC 21176, and the preservation number of the Trichoderma is ACCC 31526.

4. The compound microbial fertilizer according to claim 1, characterized in that, The mass ratio of the pineapple leaves to the banana stems in the waste is 3:

4.

5. A method for preparing the microbial compound fertilizer according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: (1) After the pineapple leaves and banana stems are dried by exposure to sunlight and crushed, 1 / 2 of the crushed material is inoculated with a mixed bacteria of Yarrowia lipolytica and Trichoderma for aerobic fermentation to obtain a first fermentation product; (2) Bacillus subtilis is inoculated into the first fermentation product, and after 12 h of fermentation, the remaining 1 / 2 of the crushed material is added to the fermentation system, stirred uniformly and continuously fermented to obtain a second fermentation product, i.e., the microbial compound fertilizer.

6. The production method according to claim 5, wherein In step (1), the aerobic fermentation conditions are 28-30℃, 100-150 rpm, and 30-36 h of fermentation culture.

7. The production method according to claim 5, wherein In step (2), the fermentation conditions are 30-32℃, 60-80 rpm, and 60-72 h of fermentation culture.

8. The microbial compound fertilizer according to any one of claims 1-4 is applied to planting Calathea.

9. The microbial compound fertilizer according to any one of claims 1-4 is applied to improving the quality and fragrance of Calathea.

10. A method of enhancing the flavor of piper betle leaves, the method comprising, The method comprises the following steps of root application of the microbial compound fertilizer according to any one of claims 1-4, wherein the root application is performed once before planting and once during planting.

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