Preparation method and application of aroma-enhancing enzyme preparation

Abstract

The preparation method comprises the following steps: S1, culturing eurotium amstelodami, collecting spores generated by culture, diluting the spores, inoculating the spores on a sterilized substrate for solid-state fermentation culture, and after spores are produced, diluting a culture medium rich in spores into eurotium amstelodami inoculation liquid; s2, culturing bacillus amyloliquefaciens, collecting thalli, and diluting the thalli into bacillus amyloliquefaciens inoculation liquid; s3, mixing the eurotium amstelodami inoculation liquid and the bacillus amyloliquefaciens inoculation liquid, and inoculating a sterilized substrate with the mixture; s4, fermenting according to preset temperature and time; and S5, collecting the fermentation substrate, and drying to obtain the solid-state fermentation product.

Description

Technical Field

[0001] This application relates to the fields of food and feed, and in particular to a method for preparing and applying a flavoring enzyme preparation. Background Technology

[0002] Food enzymes are playing an increasingly important role in improving the textural properties of food, enhancing its flavor and nutritional value, stabilizing active ingredients and food systems, reducing food safety risks, and simplifying processing techniques. Summary of the Invention

[0003] This application provides a method for preparing a flavor-enhancing enzyme preparation and its application, for enhancing the aroma of food or feed.

[0004] In a first aspect, this application provides a method for preparing a flavoring enzyme preparation, comprising the steps of: S1, culture Amsterdam polycystic spores, collect the spores produced by the culture, dilute them and inoculate them on a sterile substrate for solid-state fermentation culture. After spore production, dilute the spore-rich culture medium to prepare Amsterdam polycystic spore inoculum. S2, culture Bacillus amyloliquefaciens, collect the bacterial cells, and dilute them to prepare Bacillus amyloliquefaciens inoculation solution; S3, mix the Amsterdam cystis inoculation solution and Bacillus amyloliquefaciens inoculation solution and inoculate them onto the sterilized substrate; S4, fermentation at the preset temperature and time; S5. Collect the fermentation substrate and dry it to obtain solid fermentation product.

[0005] Preferably, in step S1, *Isodon spp. Amsterdam* is cultured on a PDA plate at room temperature for 8-15 days.

[0006] Preferably, in step S1, the spore-rich culture medium is diluted to 1×10⁻⁶. 7 Amsterdam spirulina inoculation solution.

[0007] Preferably, in step S2, the culture of Bacillus amyloliquefaciens is carried out in the deep liquid layer inside the fermenter.

[0008] Preferably, in step S2, the collected bacterial cells are diluted to 1×10⁻⁶. 7 Bacillus amyloliquefaciens inoculation solution.

[0009] Preferably, in step S3, the inoculum of *Synthia stenoptera* and the inoculum of *Bacillus amyloliquefaciens* are mixed in a 1:1 ratio.

[0010] Preferably, in step S4, the fermentation start temperature is controlled at 18-22℃, the maximum temperature is controlled below 50℃, and the fermentation time is more than 36 hours.

[0011] Preferably, in steps S1-S4, the sterilization substrate is a bran substrate.

[0012] Secondly, this application provides an application of the preparation method of the flavoring enzyme preparation as described above, for the preparation of feed enzyme preparations.

[0013] Thirdly, this application provides an application of the preparation method of the flavoring enzyme preparation as described above, for the preparation of brewing enzyme preparations.

[0014] The technical solutions provided in this application have the following advantages compared with the prior art: The method provided in this application embodiment exhibits antagonistic competition between the Amsterdam strain of *Synthia spp.* LB102 and the strain of *Bacillus amyloliquefaciens* TM21. The aroma of the liquid co-culture fermented product and the solid co-culture fermented product are significantly increased, and the palatability is good. This solid fermented product and fermentation method are of great significance in the production of feed enzyme preparations and brewing enzyme preparations. Attached Figure Description

[0015] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] One or more embodiments are illustrated by way of example with reference numerals in the accompanying drawings. These illustrations do not constitute a limitation on the embodiments. Elements with the same reference numerals in the drawings are denoted as similar elements. Unless otherwise stated, the figures in the drawings are not to be limited by scale.

[0018] Figure 1 This is a chromatogram of a single culture fermentation product of *Synthia serrata* in an embodiment of this application; Figure 2 This is a chromatogram of the co-culture fermentation product of *Synthia serrata* and *Bacillus amyloliquefaciens* in the embodiments of this application; Figure 3 This is a chromatogram of a single-culture fermentation product of Bacillus amyloliquefaciens in the embodiments of this application; Figure 4The Venn analysis diagrams (positive ion mode) of each sample in the embodiments of this application are shown. Figure 5 This is a Venn analysis diagram (negative ion mode) of each sample in the embodiments of this application. Figure 6 This is a KEGG enrichment map of differentially metabolites in the embodiments of this application; Figure 7 This is a flowchart illustrating a method according to an embodiment of this application. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] The following disclosure provides numerous different embodiments or examples for implementing various structures of the invention. To simplify the disclosure, specific examples of components and arrangements are described below. These are merely examples and are not intended to limit the scope of the invention. Furthermore, reference numerals and / or letters may be repeated in different examples. Such repetition is for simplification and clarity and does not in itself indicate a relationship between the various embodiments and / or arrangements discussed.

[0021] like Figure 7 As shown in the embodiments of this application, a method for preparing a flavoring enzyme preparation is provided, the preparation method comprising the following steps: S1. Cultivate *Arisaema Amsterdame*, collect the spores produced by the culture, dilute them, and inoculate them onto a sterile substrate for solid-state fermentation. After spore production, dilute the spore-rich culture medium to prepare *Arisaema Amsterdame* inoculum.

[0022] *Eurotium amstelodami* is a beneficial symbiotic in traditional fermentation systems. In recent years, it has been frequently found in various habitats, including high-temperature fermentation starter cultures for soy sauce aroma, wheat koji, Liubao tea, Pu'er tea, Fuzhuan tea, ham, Korean meju (soybean paste), sedimentary environments in the South China Sea, and soils of the Qinghai-Tibet Plateau, highlighting its enormous potential for development and utilization. Under suitable conditions, Pu'er tea and Liubao tea develop small golden spots, similar to the "golden flowers" of Fuzhuan tea. Research has confirmed that the fungi producing the "golden flowers" in Liubao tea are *Eurotium amstelodami*, *Eurotium niveoglaucum*, *Eurotium repens*, *Eurotium rubrum*, and *Eurotium cristatum*, among others. Fungi of the genus *Cynodon* decompose the inherent biological components of tea leaves by secreting a complex and diverse range of extracellular enzymes, such as cellulase, hemicellulase, amylase, polyphenol oxidase, mannanase, and pectinase, transforming them into more potent active ingredients. During this process, they produce abundant metabolites, contributing to the unique characteristics, aroma, flavor, and health benefits of fermented tea. To date, more than a dozen species of *Cynodon* fungi have been isolated from various types of fermented tea. Furthermore, the fermentation mycelium of *Cynodon* fungi contains abundant polysaccharides, superoxide dismutase, nucleosides, ergosterol, vitamins, trace elements, and other active ingredients. In particular, fungal polysaccharides can significantly improve the growth performance of livestock and poultry, enhance meat quality, boost immunity, maintain intestinal microbiota balance, and adsorb aflatoxin. Therefore, *Cynodon d'Amsterdam* is a newly discovered strain with enormous application potential in the feed and food industries.

[0023] In this embodiment, the *Isodon spp.* strain LB102 was selected, which was isolated from Liubao tea in Wuzhou, Guangxi Zhuang Autonomous Region. During cultivation, room temperature culture can be performed on PDA plates for 8-15 days, preferably 10 days. After 10 days, the spore-producing medium is collected, diluted, and inoculated onto a sterilized substrate for solid-state fermentation. The sterilized substrate can be wheat bran. After spore production during solid-state fermentation, the spore-rich medium is diluted to form an inoculum solution (suspension), with a dilution factor of 1×10⁻⁶. 7 .

[0024] S2, culture Bacillus amyloliquefaciens, collect the bacterial cells, and dilute them to prepare Bacillus amyloliquefaciens inoculation solution.

[0025] Bacillus amyloliquefaciens is a facultative anaerobic bacterium widely found in soil, plants, and various fermentation systems such as soy sauce-flavored liquor, sweet bean sauce, and Pixian chili bean paste. It produces abundant amylase, protease, and cellulase, exhibiting a particularly strong enzyme-producing capacity. It also produces polypeptides, lipopeptides, and antibacterial proteins, enhancing the body's immune function and the antioxidant function of the intestinal mucosa. However, the probiotic characteristics vary greatly among different strains. Bacillus amyloliquefaciens promotes the improvement of the intestinal microenvironment in livestock and poultry by competing for pathogen adhesion sites, lowering the pH of the digestive tract, and secreting various digestive enzymes and certain antibacterial substances, thereby enhancing nutrient absorption and improving the overall health of livestock and poultry. Studies have shown that adding certain strains of Bacillus amyloliquefaciens to feed significantly improves the growth performance of weaned piglets and broilers, benefits the intestinal health of laying hens, improves egg quality, and can partially replace antibiotics. However, spore-based fermented products often have an unpleasant smell and a slight odor under conventional fermentation conditions, resulting in poor palatability. Improving the aroma of spore-based products and enhancing the quality of fermented products in the food and feed industries is a key aspect of microbial product development.

[0026] In this embodiment, the Bacillus amyloliquefaciens strain selected was TM21, which was isolated from the traditional Chinese sweet bean sauce fermentation system. The Bacillus amyloliquefaciens was cultured in a high-density liquid submerged culture tank. The bacterial cells were collected by centrifugation and then diluted to 1×10⁻⁶. 7 Inoculation solution (suspension).

[0027] S3, mix the Amsterdam cystis inoculation solution and Bacillus amyloliquefaciens inoculation solution and inoculate them onto the sterilized substrate.

[0028] Specifically, the inoculum of *Synthia spp. Amsterdam* and the inoculum of *Bacillus amyloliquefaciens* were mixed in a 1:1 ratio and then inoculated onto a sterilized substrate, which was also selected as a bran substrate.

[0029] S4 ferments at a preset temperature and time.

[0030] In this embodiment, the fermentation start temperature is controlled at 18-22℃, the maximum temperature is controlled below 50℃, and the fermentation time is more than 36 hours. In this embodiment, the fermentation start temperature is controlled at 20℃, the maximum temperature is around 45℃, and the fermentation culture lasts for 48 hours.

[0031] S5. Collect the fermentation substrate and dry it to obtain solid fermentation product.

[0032] Under conventional fermentation conditions, most secondary metabolic biosynthetic gene clusters in microbial genomes are silenced, and the synthetic potential of the new compounds encoded by these silent gene clusters far exceeds our expectations. In this embodiment, a silent gene activation strategy is employed to induce the production of active ingredients and aroma substances, which can improve product quality and enhance market competitiveness.

[0033] In this embodiment, the activation of silent biosynthetic gene clusters through microbial co-culture helps to discover new compounds, enhance product aroma, increase the content of antibacterial substances in Bacillus amyloliquefaciens, and also provides new research areas and opportunities for the utilization of Acinetobacter Amsterdam and Bacillus amyloliquefaciens in the fields of food, feed enzyme preparations and fermented feed.

[0034] Specifically, such as Figure 1-3 As shown, based on the above examples, HPLC analysis of the fermentation products from the co-culture of *Armillaria stenoptera* and *Bacillus amyloliquefaciens* revealed five new absorption peaks: peak 1 (10.097 min), peak 2 (21.690 min), peak 3 (22.315 min), peak 4 (25.679 min), and peak 5 (32.472 min). This indicates that the interaction between *Armillaria stenoptera* and *Bacillus amyloliquefaciens* during co-culture resulted in the production of new secondary metabolites, and the activation of silenced gene clusters.

[0035] To further identify the new metabolites, this embodiment uses UPLC / Q-TOF MS metabolomics to further analyze the new metabolites.

[0036] The analysis used four groups of samples: a mixed culture group of *Cyclocarya Amsterdam* and *Bacillus amyloliquefaciens* (Control), a co-culture group of *Cyclocarya Amsterdam* and *Bacillus amyloliquefaciens* in the early stage (CE, fermentation 24 h), a co-culture group of *Cyclocarya Amsterdam* and *Bacillus amyloliquefaciens* in the middle stage (CM, fermentation 36 h), and a co-culture group of *Cyclocarya Amsterdam* and *Bacillus amyloliquefaciens* in the late stage (CL, fermentation 48 h). Each group had six biological replicates, for a total of 24 samples.

[0037] like Figure 4-5As shown, Venn analysis was performed on four groups of samples. The mixed culture group of *Armillaria Amsterdam* and *Bacillus amyloliquefaciens* (Control), the early co-culture group (CE), the mid-co-culture group (CM), and the late co-culture group (CL) detected 861, 864, 867, and 868 metabolites, respectively. Among these, each group had 2, 1, 1, and 1 metabolites unique to it, respectively. The metabolites unique to the mixed culture group of *Armillaria Amsterdam* and *Bacillus amyloliquefaciens* (Control) were rutin and delphinidin-3-[galactosyl-(1->4)-glucosinolate]; the metabolite unique to the early co-culture group (CE) was ganoderic acid F; the metabolite unique to the mid-co-culture group (CM) was stearaldehyde; and the metabolite unique to the late co-culture group (CL) was epigallocatechin 3-(3-methyl-gallic acid). Compared to the Control, CE, CM, and CL had 9, 10, and 10 metabolites unique to them, respectively, with a total of 6 metabolites across the three groups. Furthermore, in terms of co-culture time, the CM in the middle stage of co-culture and the CL in the later stage have more shared metabolites. Co-culture between microorganisms increases the variety and number of metabolites, accompanied by the production of "new" metabolites and the disappearance of "old" metabolites.

[0038] Comparative analyses were conducted across different groups: CE vs Control, CM vs Control, and CL vs Control. Additionally, CM vs CE, CL vs CE, and CL vs CM comparison groups were established to further clarify the changes in metabolites during co-culture fermentation. Compared to the single-culture mixed group of *Cytosporum canis* and *Bacillus amyloliquefaciens* (Control), 263, 238, and 203 differentially expressed metabolites were identified in the early co-culture group (CE), mid-co-culture group (CM), and late co-culture group (CL), respectively. As shown in Table 1 below, CE showed 185 upregulated and 78 downregulated metabolites; CM showed 39 upregulated and 199 downregulated metabolites; and CL showed 170 upregulated and 33 downregulated metabolites (Table 1). Furthermore, compared to the early co-culture group, the mid-co-culture group showed 152 differentially expressed metabolites, of which 91 were upregulated and 61 were downregulated. Compared to the early stage of co-culture, 190 differentially expressed metabolites were observed in the later stage of co-culture, of which 103 were upregulated and 87 were downregulated. Compared to the middle stage of co-culture, 44 differentially expressed metabolites were observed in the later stage of co-culture, of which 12 were upregulated and 7 were downregulated.

[0039] Table 1 - Expression of differentially expressed metabolites In this embodiment, compared with the control group, the mid-term group (CM) of co-culture of Acinetobacter Amsterdam and Bacillus amyloliquefaciens showed that 132 metabolites were annotated out of 219 differential metabolites, which were mainly distributed in 59 KEGG metabolic pathways.

[0040] like Figure 6 As shown, using a P-value ≤ 0.05 as the threshold, there are 20 KEGG pathways that are significantly enriched in the metabolic set, such as... Figure 6As shown. Among them, the 18 metabolic pathways with the most significant enrichment of differentially expressed metabolites mainly include: (1) ABC transporter pathway, with metabolites mainly including 14 metabolites: maltose, L-glutamate, lysine, guanosine, L-proline, L-phenylalanine, isoleucine, L-histidine, pramine, uridine, taurine, sucrose, glutathione, and L-aspartic acid; (2) Aminoacyl-tRNA biosynthesis, with metabolites mainly including 9 metabolites: L-glutamate, L-lysine, L-proline, L-phenylalanine, isoleucine, histidine, L-tryptophan, L-tyrosine, and L-aspartic acid; (3) Purine metabolism, with metabolites mainly including 5 metabolites: guanosine, guanine, hypoxanthine, adenine, and xanthine; (4) Cyanoamino acid metabolism. The metabolites mainly include five types: L-glutamic acid, L-phenylalanine, isoleucine, L-tyrosine, and L-aspartic acid; (5) Arginine and proline metabolism, the metabolites mainly include four types: L-glutamic acid, L-glutamine-semialdehyde, L-proline, and 4-guanidinobutyric acid.(6) Phenylalanine metabolism, with metabolites mainly including 2-hydroxycinnamic acid, phenylacetaldehyde, L-phenylalanine, and L-tyrosine; (7) Galactose metabolism, with metabolites mainly including stachyose, raffinose, sucrose, and uridine diphosphate glucose; (8) Lysine biosynthesis, with metabolites mainly including L-lysine, (Z)-But-1-ene-1,2,4-tricarboxylate, diaminopimelic acid, and L-aspartic acid; (9) β-Alanine metabolism, with metabolites mainly including β-alanyl-L-arginine, uracil, L-histidine, and L-aspartic acid; (11) Tyrosine metabolism, with metabolites mainly including carbolic acid, maltine, and tyrosine; (12) Biosynthesis of various secondary metabolites (part 2), with metabolites mainly including L-phenylalanine, L-tryptophan, and tyrosine; ⒀ Pyrimidine metabolism, with metabolites mainly including uracil, uridine, and uridine diphosphate glucose; ⒁ Histidine metabolism, with metabolites mainly including L-glutamate, L-histidine, and L-aspartic acid; ⒂ Staurosporine biosynthesis, with metabolites mainly including L-proline, L-histidine, and L-tryptophan. ⒃ Biosynthesis of phenylalanine, tyrosine and tryptophan, with metabolites mainly including L-phenylalanine, L-tryptophan and tyrosine; ⒄ Biosynthesis of carbapenem, with metabolites mainly including L-glutamate, L-glutamine-semialdehyde and L-proline; ⒅ Biosynthesis of pantothenate and CoA, with metabolites mainly including uracil, panthenol and L-aspartic acid.

[0041] It is worth noting that the vast majority of these differential metabolites are aroma and flavor compounds, which endow the co-cultured fermented products with rich aroma and good palatability, making them competitive in the market when applied to food and feed production.

[0042] Based on the above, the method for preparing flavor-enhancing enzyme preparations provided in this application embodiment can be used for the preparation of brewing enzyme preparations in food and feed enzyme preparations in feed.

[0043] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0044] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0045] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0046] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms should not be construed as necessarily referring to the same embodiment or example.

[0047] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a flavor-enhancing enzyme preparation, characterized in that, Including the following steps: S1, culture Amsterdam polycystic spores, collect the spores produced by the culture, dilute them and inoculate them on a sterile substrate for solid-state fermentation culture. After spore production, dilute the spore-rich culture medium to prepare Amsterdam polycystic spore inoculum. S2, culture Bacillus amyloliquefaciens, collect the bacterial cells, and dilute them to prepare Bacillus amyloliquefaciens inoculation solution; S3, mix the Amsterdam cystis inoculation solution and Bacillus amyloliquefaciens inoculation solution and inoculate them onto the sterilized substrate; S4, fermentation at the preset temperature and time; S5. Collect the fermentation substrate and dry it to obtain solid fermentation product.

2. The method for preparing the flavor-enhancing enzyme preparation according to claim 1, characterized in that, In step S1, the Amsterdam strain of Acinetobacter LB102 is selected and cultured on a PDA plate at room temperature for 8-15 days.

3. The method for preparing the flavor-enhancing enzyme preparation according to claim 2, characterized in that, In step S1, the spore-rich culture medium is diluted to 1×10⁻⁶. 7 Amsterdam spirulina inoculation solution.

4. The method for preparing the flavor-enhancing enzyme preparation according to claim 3, characterized in that, In step S2, the Bacillus amyloliquefaciens TM21 strain is selected and cultured in the deep liquid layer of the fermenter.

5. The method for preparing the flavor-enhancing enzyme preparation according to claim 4, characterized in that, In step S2, the collected bacterial cells are diluted to 1×10⁻⁶. 7 Bacillus amyloliquefaciens inoculation solution.

6. The method for preparing the flavor-enhancing enzyme preparation according to claim 5, characterized in that, In step S3, the inoculum of *Synthia stenoptera* and the inoculum of *Bacillus amyloliquefaciens* are mixed in a 1:1 ratio.

7. The method for preparing the flavor-enhancing enzyme preparation according to claim 5, characterized in that, In step S4, the initial fermentation temperature is controlled at 18-22℃, the maximum temperature is controlled below 50℃, and the fermentation time is more than 36 hours.

8. The method for preparing the flavor-enhancing enzyme preparation according to claim 5, characterized in that, In steps S1-S4, the sterilization substrate is a bran substrate.

9. The application of a method for preparing a flavor-enhancing enzyme preparation as described in any one of claims 1-8, characterized in that, Used in the preparation of feed enzymes.

10. The application of a method for preparing a flavor-enhancing enzyme preparation as described in any one of claims 1-8, characterized in that, Used for the preparation of brewing enzyme preparations.

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