Method for producing high-digestibility feed based on Penicillium sclerotiorum fermented Zingiberaceae plants and application of high-digestibility feed

Abstract

The invention discloses a method for producing a high-digestibility feed based on Penicillium sclerotiorum fermented Zingiberaceae plants and application of the high-digestibility feed. The preparation method comprises the following steps: inoculating a Penicillium sclerotiorum spore suspension into a fermentation substrate prepared by taking zingiberaceae plants as raw materials, and carrying out solid-state fermentation; the content of bad flavor components (such as aldehyde ketone compounds and fatty acid) in zingiberaceae plants can be remarkably reduced, the content of beneficial components (such as ester derivatives, terpenoid derivatives, long-chain fatty acid, free amino acid and soluble protein) can be increased, and main precursors causing bitter and pungent smells of the raw materials can be removed from the source; the palatability of the feed is fundamentally improved, and the fundamental improvement on the flavor of stems and leaves of zingiberaceae plants is realized.

Description

Technical Field

[0001] This invention relates to the field of high-value utilization of agricultural by-products and microbial fermentation technology, and in particular to a method and application for producing highly digestible feed based on Penicillium sclerotiorum fermentation of ginger plants. Background Technology

[0002] The fruits of Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko, all belonging to the ginger family, are important medicinal and edible raw materials; however, their stem and leaf byproducts have not been effectively utilized. Although these stems and leaves contain potential active ingredients, their high-value application faces two long-standing and unresolved technical bottlenecks: First, the unpleasant flavors are persistent and complex. These stems and leaves contain a variety of inherent small-molecule unpleasant flavor precursors, which are the root cause of their poor palatability. These mainly include: (1) Phenolic compounds: such as 4-hydroxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, etc., which have a strong bitter taste and irritation. (2) Fatty acids with unpleasant odors: such as valeric acid (with a sweaty smell), hexanoic acid (with a sweaty smell), etc. (3) Certain irritating terpenes: such as (-)-myrtenol, etc. These substances are chemically stable and are difficult to remove effectively by conventional physical crushing, drying or soaking in warm water, etc., and cannot fundamentally improve palatability. Furthermore, chemical methods may introduce residues, bringing safety and environmental risks.

[0003] Second, conventional microbial fermentation techniques have limited effectiveness. Existing research on plant by-product fermentation mainly focuses on using white-rot fungi to degrade lignocellulose or using lactic acid bacteria for silage preservation. The former is time-consuming and primarily alters the macroscopic structure, while the latter mainly functions as an acidifier and preservative. These conventional microorganisms lack the specific degradation ability for the aforementioned small-molecule unpleasant flavor compounds, and their own metabolism may even produce new unpleasant odors. Therefore, existing fermentation techniques cannot fundamentally improve the flavor of ginger family stems and leaves.

[0004] Therefore, there is an urgent need in this field to find a green biotechnology that can specifically target and degrade these key undesirable flavor precursors in order to break through the core barrier of high-value utilization of ginger family stems and leaves. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of Penicillium sclerotium and / or Penicillium sclerotium spores in fermented ginger plants.

[0006] Another objective of this invention is to provide a method for fermenting ginger plants based on Penicillium sclerotiorum.

[0007] Another object of the present invention is to provide the application of the method for fermenting ginger plants based on Penicillium sclerotiorum in the preparation of highly digestible feed.

[0008] Another object of the present invention is to provide a method for producing highly digestible feed based on the fermentation of ginger plants by Penicillium sclerotiorum.

[0009] The objective of this invention is achieved through the following technical solution: The application of *Penicillium sclerotium* and / or *Penicillium sclerotium* spores in the fermentation of ginger plants; wherein the application includes reducing the content of undesirable flavor components and increasing the content of beneficial components in ginger plants; specifically including at least one of the following aspects: (1) Degradation of the content of aldehydes and ketones (undesirable flavor precursors) in ginger plants; (2) Reduce the fatty acid content in ginger family plants; (3) Increase the content of esters and terpenoid derivatives in ginger family plants; (4) Increase the content of long-chain fatty acids in ginger family plants; (5) Increase the content of free amino acids in ginger family plants; (6) Increase the content of soluble protein in ginger plants.

[0010] The ginger family plant mentioned is at least one of Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko; preferably, it is a by-product of ginger family plants, such as at least one of the stems and leaves of Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko.

[0011] The preferred form of Penicillium sclerotium is Penicillium sclerotium (… Penicillium sclerotiorum )jyscaumcx01.

[0012] (1) The aldehyde and ketone compounds mentioned include at least one of 4-hydroxybenzaldehyde and 2-hydroxy-4-methoxybenzaldehyde; preferably 4-hydroxybenzaldehyde and 2-hydroxy-4-methoxybenzaldehyde from Amomum villosum and Amomum tsao-ko; after fermentation by Penicillium sclerotium, the content of aldehyde and ketone compounds in ginger plants is significantly reduced.

[0013] (2) The fatty acids mentioned include at least one of valeric acid, hexanoic acid and oleic acid; preferably valeric acid and hexanoic acid from cardamom leaves, and oleic acid from amomum stems and leaves.

[0014] The ester derivatives mentioned in (3) include isophytol acetate.

[0015] (3) The terpene derivatives mentioned include at least one of neophytadiene and γ-sitosterol.

[0016] The long-chain fatty acid mentioned in (4) is a beneficial long-chain fatty acid, including unsaturated fatty acids and saturated fatty acids; more preferably, at least one of cis-13-octadecenoic acid (an unsaturated fatty acid) and n-hexadecanoic acid (palmitic acid, a saturated fatty acid).

[0017] A method for fermenting ginger plants based on Penicillium sclerotiorum includes the following steps: S1. Raw material pretreatment: Dry and crush ginger plants, adjust the moisture content to 55% to 65%, sterilize, and obtain the fermentation substrate; S2. Inoculation and fermentation: The suspension of Penicillium sclerotiorum spores is inoculated into the fermentation substrate obtained in step S1, and static solid-state fermentation is carried out at 25-30°C to reduce the content of undesirable flavor components and increase the content of beneficial components in ginger plants; wherein, undesirable flavor components include at least one of aldehydes, ketones and fatty acids; and beneficial components include at least one of ester derivatives, terpenoid derivatives, long-chain fatty acids, free amino acids and soluble proteins.

[0018] The ginger family plant mentioned in step S1 is at least one of Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko; preferably it is a by-product of ginger family plants, such as at least one of the stems and leaves of Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko.

[0019] The pulverization mentioned in step S1 is performed using a plant pulverizer.

[0020] The pulverization mentioned in step S1 is pulverization to 40-60 mesh; preferably pulverization to 60 mesh.

[0021] In step S1, the moisture content can be adjusted by adding an appropriate amount of water or at least one of PDB liquid culture medium; preferably, it is adjusted by using a solution obtained by mixing sterile water and PDB liquid culture medium at a volume ratio of 9:1; a moisture range of 55% to 65% (preferably 60%) is one of the key factors to ensure good growth and enzyme production of Penicillium sclerotiorum.

[0022] The sterilization conditions described in step S1 are: sterilization at 121°C for 25-35 minutes; preferably: sterilization at 121°C for 30 minutes.

[0023] The *Penicillium sclerotium* mentioned in step S2 is preferably *Penicillium sclerotium* (… Penicillium sclerotiorum )jyscaumcx01.

[0024] The Penicillium sclerotiorum spore suspension mentioned in step S2 is preferably prepared by the following method: Penicillium sclerotiorum (… Penicillium sclerotiorum The strain is activated and cultured until a large number of spores are produced. Then, the spores are washed with sterile water containing 0.05% (v / v) Tween-80, and mycelial debris is removed by filtration to obtain a suspension of Penicillium sclerotiorum spores. Before use, the spores can be counted with a hemocytometer and adjusted to the required concentration.

[0025] The cultivation conditions are: constant temperature cultivation at 20-30℃ (preferably 28℃) for 3-5 days.

[0026] The inoculum size of the *Penicillium sclerotium* spore suspension in step S2 is 1 × 10⁻⁶. 4CFU / g ~ 1×10 8 CFU / g.

[0027] The preferred temperature for static solid-state fermentation in step S2 is 28°C.

[0028] The static solid-state fermentation time described in step S2 is 0 to 28 days (excluding 0); preferably 14 to 28 days. Forced ventilation is not required throughout the fermentation process, but ambient air circulation is maintained.

[0029] The aldehydes and ketones mentioned in step S2 include at least one of 4-hydroxybenzaldehyde and 2-hydroxy-4-methoxybenzaldehyde; preferably 4-hydroxybenzaldehyde and 2-hydroxy-4-methoxybenzaldehyde from Amomum villosum and Amomum tsao-ko; after fermentation by Penicillium sclerotium, the content of aldehydes and ketones in ginger plants is significantly reduced.

[0030] The fatty acids mentioned in step S2 include at least one of valeric acid, hexanoic acid, and oleic acid; preferably valeric acid and hexanoic acid from cardamom leaves, and oleic acid from amomum stems and leaves.

[0031] The ester derivatives mentioned in step S2 include isophytol acetate.

[0032] The terpene derivatives mentioned in step S2 include at least one of neophytadiene and γ-sitosterol.

[0033] The long-chain fatty acid mentioned in step S2 is a beneficial long-chain fatty acid, including unsaturated fatty acids and saturated fatty acids; it is further preferably at least one of cis-13-octadecenoic acid (an unsaturated fatty acid) and n-hexadecanoic acid (palmitic acid, a saturated fatty acid).

[0034] The application of the method for fermenting ginger plants based on Penicillium sclerotiorum in the preparation of highly digestible feed.

[0035] The feed mentioned is feed for livestock, such as herbivorous livestock like cattle, sheep, and horses.

[0036] A method for producing highly digestible feed from ginger plants based on Penicillium sclerotiorum fermentation includes steps S1 and S2 of the above-mentioned method based on Penicillium sclerotiorum fermentation of ginger plants, and the following steps: S3. Post-processing: After fermentation in step S2 is completed, freeze-dry the fermentation product to constant weight to obtain highly digestible feed.

[0037] In step S3, freeze-drying to constant weight means freeze-drying until the moisture content is less than 12%.

[0038] The present invention has the following advantages and effects compared with the prior art: 1. This invention provides a method for solid-state fermentation of ginger family stems and leaves using Penicillium sclerotiorum to improve their flavor, nutritional value, and digestibility. This method can simultaneously overcome the three major bottlenecks in palatability, nutrition, and usability of ginger family stems and leaves, achieving a fundamental improvement in the flavor of ginger family stems and leaves. The resulting fermented products can be used in feed and other fields.

[0039] 2. The method of the present invention can directly remove the main precursors that cause the bitterness and irritating odor of the raw materials from the root, fundamentally improving palatability. For example, the content of 4-hydroxybenzaldehyde in the stems and leaves of Amomum villosum and Amomum tsao-ko is significantly reduced (degradation rate >99%), and the content of 2-hydroxy-4-methoxybenzaldehyde in the leaves and leaves of Amomum villosum and Amomum tsao-ko is greatly reduced (degradation rate up to 94%).

[0040] 3. The method of the present invention can effectively eliminate unpleasant odors such as "sweaty smell" in raw materials. For example, the content of valeric acid and hexanoic acid in cardamom leaves is reduced by more than 90%; the content of oleic acid and other substances in cardamom stems and leaves is also significantly reduced.

[0041] 4. The method of this invention actively constructs a pleasant flavor profile dominated by fruity, sweet, and floral aromas, achieving positive flavor reconstruction. Attached Figure Description

[0042] Figure 1 This is a chromatogram of the volatile substances from the stems and leaves of three ginger family plants (Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko) after fermentation by Penicillium sclerotiorum.

[0043] Figure 2 This is a box plot comparing the effects of Penicillium sclerotiorum fermentation on the in vitro dry matter digestibility (IVDMD) of the stems and leaves of three ginger family plants (Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko).

[0044] Figure 3 This is a bar chart showing the total amino acid content of Amomum villosum leaves (SRY) and stems (SRJ) after fermentation by Penicillium sclerotium. In the bar chart, A and B represent the effects of different inoculum amounts of Penicillium sclerotium on the total amino acid content of Amomum villosum leaves (SRY) and stems (SRJ), respectively; C and D represent the effects of different fermentation times of Penicillium sclerotium on the total amino acid content of Amomum villosum leaves (SRY) and stems (SRJ), respectively.

[0045] Figure 4 This is a bar chart showing the soluble protein content of Amomum villosum (SRY) and Amomum villosum stem (SRJ) after fermentation by Penicillium sclerotium. A and B represent the effects of different fermentation times of Penicillium sclerotium on the soluble protein content of Amomum villosum leaves (SRY) and Amomum villosum stem (SRJ), respectively. C and D represent the effects of different inoculum amounts of Penicillium sclerotium on the soluble protein content of Amomum villosum leaves (SRY) and Amomum villosum stem (SRJ), respectively. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0047] The sclerotium involved in this embodiment of the invention is derived from the sclerotium of purslane (Penicillium sclerotium). Penicillium sclerotiorum The strain jyscaumcx01 is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 60249), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 11, 2017. This strain has been disclosed in a Chinese patent (patent number: 201711279355.9, title: *Penicillium sclerotium* var. *purslane* and its application in the preparation of anti-Ralstonia solanacearum drugs).

[0048] The components and formulations of the culture medium involved in the embodiments of the present invention are as follows: The components and proportions of PDA medium are as follows: 200g potato, 15-20g glucose, 15-20g agar, add distilled water to 1000mL, adjust pH to 7.0, and autoclave at 121℃ for 20min.

[0049] The components and proportions of PDB liquid culture medium are as follows: 200g potato, 15-20g glucose, distilled water added to 1000mL, pH adjusted to 7.0, and autoclaved at 121℃ for 20min.

[0050] Example 1: Evaluation of the comprehensive effects of Penicillium sclerotiorum fermentation on stems and leaves of ginger plants. 1. Fermentation Experiment Design 1.1 Materials and Methods 1.1.1 Raw materials and strains: Stems and leaves of three ginger family plants—Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko (waste products after harvesting mature plants), and Penicillium sclerotiorum (… Penicillium sclerotiorum ).

[0051] 1.1.2 Methods (1) Raw material pretreatment (fermentation substrate): Dry, mold-free stems and leaves of ginger family plants were pulverized using a plant pulverizer and passed through a 60-mesh sieve. 15g (dry weight) portions of powder were weighed and dispensed into fermentation tanks. The required water volume was calculated, and a solution of sterile water and PDB liquid culture medium at a volume ratio of 9:1 was sprayed on the mixture, adjusting the moisture content of the stems and leaves to 80%. The mixture was sterilized at 121℃ for 30 minutes, cooled to room temperature, and ready for use.

[0052] (2) Preparation, inoculation and fermentation of microbial strains Penicillium sclerotiorum preserved on PDA medium ( Penicillium sclerotiorum Activate the spores by incubation at 28°C for 3–5 days until a large number of spores are produced. Rinse the spores with sterile water containing 0.05% (v / v) Tween-80, filter to remove mycelial debris, and adjust the spore concentration using a hemocytometer. Finally, dilute the spore solution at 1×10⁻⁶. 6 Inoculate the pretreated fermentation substrate with an inoculum of CFU / g, ensuring thorough mixing to guarantee inoculation consistency. Ferment at 28℃ for 0, 7, 14, 21, and 28 days. Forced ventilation is not required throughout the fermentation process, but ambient air circulation should be maintained. The experiment was conducted in triplicate. The products from the fermentation of Amomum villosum stems (SRJ) for 0 and 28 days were named SRJ0 and SRJ28, respectively. Similarly, the products from the fermentation of Amomum villosum leaves (SRY), Alpinia oxyphylla stems (YZJ), Alpinia oxyphylla leaves (YZY), Amomum tsao-ko stems (CGJ), and Amomum tsao-ko leaves (CGY) for 0 and 28 days were named SRY0, SRY28, YZJ0, YZJ28, YZY0, YZY28, CGJ0, CGJ28, CGY0, and CGY28, respectively.

[0053] (3) Post-processing After fermentation, all fermented materials are freeze-dried in a freeze dryer until constant weight (moisture content below 12%). Finally, they are sealed and packaged to obtain the final fermented ginger plant stem and leaf product.

[0054] 1.2 Analysis and Testing: (1) GC-MS: Analysis of volatile flavor compounds. GC-MS pretreatment: Take 100 mg of the final fermented ginger plant stem and leaf products and place them in a vial. Add 1 mL of dichloromethane containing 5 ppm ethyl decanoate and sonicate for 30 min, twice. Add an appropriate amount of anhydrous sodium sulfate and place in a -4℃ refrigerator for 12 h. Centrifuge and collect the supernatant (10000 g, 10 min), and filter it through a Millipore filter with a pore size of 0.22 μm. Each species contains 3 biological replicates. GC-MS detection conditions: GC-MS: SHIMADZU GCMS-TQ8040, instrument specifications: triple quadrupole, column: Agilent DB-5MS gas chromatographic column (30 m × 0.25 mm × 0.25 μm), injection port temperature 250℃, detector 260℃, initial temperature 60℃ held for 2 min, then increased to 260℃ / min at 6℃ / min and held for 15 min. The injection volume was 1 μL, the split ratio was 3:1, and the MS conditions were: EI ion source, ionization energy 70 eV, scan range m / z 50~600 amu, ion source temperature 200℃, and GC-MS interface temperature 250℃.

[0055] (2) In vitro digestibility (IVDMD): determined by the two-step method of pepsin-secretin. Experimental Procedure: Weigh 0.50 g of the final fermented ginger plant stems and leaves (using a 60-well filter bag) and place it in a 250 mL Erlenmeyer flask. Add 30 mL of 0.04 mol / L HCl buffer (prepared as follows: weigh 3.00 g of glycine (MW=75.07) into a beaker, then add approximately 800 mL of ultrapure water and stir to dissolve; monitor with a pH meter and slowly add concentrated hydrochloric acid until the pH stabilizes at 2.0; finally, transfer the solution to a 1 L volumetric flask, bring to the mark, mix well, and store at 4℃), 1 mL of 40 mg / mL pepsin HCl buffer (prepared as follows: weigh 400 mg of pepsin, add approximately 8 mL of 0.04 mol / L HCl buffer, and bring to a final volume of 10 mL with the same HCl buffer. Prepare fresh before use and store on ice), and add 0.5 g of bacterial growth inhibitor. mL (0.5% (w / v) chloramphenicol), sealed in an Erlenmeyer flask, placed at 37°C on a shaker at 80 r / min for 12 h. The Erlenmeyer flask was removed, titrated with 1 mol / L NaOH solution to adjust the pH to 8.0, then 10 mL of 0.05 mol / L PBS buffer and 1 mL of PBS buffer containing 8 mg / mL trypsin were added, and incubation continued for 12 h. After cooling in an ice-water bath, the filter bag was rinsed twice with distilled water and dried at 65°C for 24 h to constant weight. The absolute digestibility (DW) of dry weight was calculated using the following formula. Three parallel studies were conducted. The calculation formula is as follows: ; In the formula, m0: dry weight of the sample before digestion; m1: dry weight of the sample after digestion.

[0056] (3) Free amino acids and soluble proteins: determined by ninhydrin reagent colorimetric method (national standard GB / T 8314) and Coomassie brilliant blue method.

[0057] 1.3 Results and Discussion 1.3.1 Creative transformation of volatile flavor compounds The results are as follows Figure 1 As shown in Tables 1-6.

[0058] Table 1. Changes in the relative content of key compounds before and after fermentation of Amomum villosum stems.

[0059] Table 2. Changes in the relative content of key compounds before and after fermentation of Amomum villosum leaves.

[0060] Table 3. Changes in the relative content of key compounds before and after fermentation of Alpinia oxyphylla.

[0061] Table 4. Changes in the relative content of key compounds before and after fermentation of Alpinia oxyphylla leaves.

[0062] Table 5. Changes in the relative content of key compounds before and after fermentation of cardamom stems.

[0063] Table 6. Changes in the relative content of key compounds in cardamom leaves before and after fermentation.

[0064] like Figure 1 As shown: GC-MS results indicate that fermentation caused a fundamental change in the volatile composition spectrum of the stems and leaves of ginger family plants.

[0065] As shown in Tables 1-6, after solid-state fermentation by Penicillium sclerotiorum, the key chemical components in the stems and leaves of three ginger family plants (Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko) underwent significant and regular changes.

[0066] Overall, the fermentation process exhibits a clear trend of compound spectrum reconstruction: a large number of undesirable flavor precursors are effectively degraded, such as 4-hydroxybenzaldehyde and 2-hydroxy-4-methoxybenzaldehyde in Amomum villosum and Amomum tsao-ko, as well as a significant decrease in the content of various fatty acids (valeric acid, hexanoic acid) and aldehydes and ketones with grassy and astringent tastes; simultaneously, a series of pleasant flavor compounds are synthesized or enriched, such as a significant increase in the content of cis-13-octadecenoic acid, n-hexadecanoic acid, and some esters and terpenoid derivatives in Alpinia oxyphylla stems and leaves. This "elimination of inferior and enhancement of superior" chemical transformation explains the fundamental reason for the improved flavor of the fermented product at the molecular level. In addition, the changing trends of some compounds (such as neophydediene and certain terpene oxides) differ in different materials. The above data fully demonstrate that the Penicillium sclerotium and its fermentation process used in this invention do not indiscriminately change the composition, but can specifically attack and transform those related molecules that cause palatability problems. This targeted and efficient degradation of phenols, short-chain fatty acids, and specific terpenoids is a function of Penicillium sclerotiorum that has not been disclosed in existing technologies, further demonstrating the effectiveness and application potential of this fermentation method in the targeted improvement of the flavor and quality of ginger family plant by-products.

[0067] 1.3.2 Results of in vitro digestibility The results are as follows Figure 2 As shown in Table 7, the data clearly demonstrate that after 28 days of fermentation using the method of this invention, the IVDMD of the stems and leaves of the three ginger family plants was significantly increased (P<0.01), with the stems of the ginger family plants showing the greatest increase. This fully proves the universality and effectiveness of the process of this invention.

[0068] Table 7. Effects of Penicillium sclerotiorum fermentation on the in vitro dry matter digestibility (IVDMD) of stems and leaves of three ginger family plants.

[0069] 1.3.3 Analysis of changes in free amino acid content (1) Effect of different inoculum amounts on the content of free amino acids: Following the method described in 1.1.2 above, adjust the inoculum size of the spore solution to 1×10⁻⁶. 4 CFU / g, 1×10 6 CFU / g, 1×10 8 CFU / g was used to ferment Amomum villosum leaves (SRY) and Amomum villosum stems (SRJ) for 14 days, with Amomum villosum stems and leaves without added spore liquid serving as the control (CK).

[0070] The results showed that the content of free amino acids in Amomum villosum leaves (SRY) increased significantly with increasing inoculum size, from approximately 3.2% in the control to 10%. 8 4.2% of the inoculated dose ( Figure 3 (A) In Amomum villosum stems (SRJ), the content of free amino acids also showed an increasing trend with increasing inoculum size, increasing from 3.2% in the control to 10%. 8 4.6% of the vaccination volume ( Figure 3 (B in the text). Among them, 10 6 and 10 8 The difference in vaccination volume between the inoculation group and the control group was significant (P<0.05).

[0071] (2) Effect of different fermentation times on the content of free amino acids: Following the method described in 1.1.2 above, the inoculum size is 1 × 10⁻⁶. 6 CFU / g, fermenting Amomum villosum leaves (SRY) and Amomum villosum stems (SRJ) at 28℃ for 0, 7, 14, 21, and 28 days.

[0072] The results showed that the free amino acid content in Amomum villosum leaves (SRY) peaked at 14 days of fermentation (approximately 9%), then decreased slightly, but remained significantly higher than the pre-fermentation level. Figure 3 C in the form of Amomum villosum stem (SRJ). In the Amomum villosum stem, the content of free amino acids increased steadily with prolonged fermentation time, from approximately 3.3% at day 0 to 4.6% at day 28, showing a steady upward trend. Figure 3 (D in the middle).

[0073] The increase in free amino acid content from 0 to 14 days is likely due to the hydrolysis of plant proteins into free amino acids by proteases and peptidases produced by *Penicillium sclerotiorum* during fermentation. The slight decrease in free amino acid content in *Amomum villosum* leaves after 14 days may be due to some amino acids being utilized by microorganisms as a nitrogen source for growth and metabolism. Overall, *Penicillium sclerotiorum* fermentation can significantly increase the free amino acid content in the stems and leaves of ginger plants, thereby improving the flavor and nutritional value of the fermentation products.

[0074] 1.3.4 Analysis of changes in soluble protein content (1) Effect of different inoculum amounts on soluble protein content: Following the method described in 1.1.2 above, adjust the inoculum size of the spore solution to 1×10⁻⁶. 4 CFU / g, 1×10 6 CFU / g, 1×10 8 CFU / g was used to ferment Amomum villosum leaves (SRY) and Amomum villosum stems (SRJ) for 14 days, with Amomum villosum stems and leaves without added spore liquid serving as the control (CK).

[0075] The results showed that in cardamom leaves (SRY), as the inoculation amount increased from the control (CK) to 1×10⁻⁶, the incidence of cancer decreased. 8 CFU / g, the soluble protein content increased significantly from approximately 12 mg / g to 33 mg / g, of which 10 6 CFU / g and 10 8 The CFU / g inoculation group was significantly different from the control group (P<0.01). Figure 4 (C) In Amomum villosum stem (SRJ), the soluble protein content increased from approximately 21 mg / g in the control to 10 mg / g. 8 The CFU / g inoculation dose of 34 mg / g also showed a significant increasing trend with increasing inoculation dose. Figure 4 (D in the middle).

[0076] (2) Effect of different fermentation times on soluble protein content: Following the method described in 1.1.2 above, the inoculum size is 1 × 10⁻⁶. 6 CFU / g, fermenting Amomum villosum leaves (SRY) and Amomum villosum stems (SRJ) at 28℃ for 0, 7, 14, 21, and 28 days.

[0077] The results showed that in Amomum villosum leaves (SRY), as the fermentation time increased from 0 days to 28 days, the soluble protein content continuously increased from approximately 6 mg / g to 20 mg / g, with highly significant differences in content at 21 days and 28 days compared to the earlier stages (P<0.01). Figure 4(A) In Amomum villosum stem (SRJ), the soluble protein content increased steadily from approximately 12 mg / g at day 0 to 18 mg / g at day 28, with a significant difference between day 28 and day 21 (P<0.05). Figure 4 (B in the middle).

[0078] These results indicate that Penicillium sclerotiorum fermentation can significantly increase the content of soluble protein in the stems and leaves of ginger family plants, and within a certain range, higher inoculum size and longer fermentation time are conducive to the accumulation of soluble protein. This may be because the proteases secreted by Penicillium sclerotiorum break down large protein molecules in plant stems and leaves into smaller soluble proteins and peptides, thereby improving protein solubility and digestibility.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of *Penicillium sclerotium* and / or *Penicillium sclerotium* spores in the fermentation of ginger family plants, characterized by: The applications include at least one of the following aspects: (1) Degradation of the content of aldehydes and ketones in ginger family plants; (2) Reduce the fatty acid content in ginger family plants; (3) Increase the content of esters and terpenoid derivatives in ginger family plants; (4) Increase the content of long-chain fatty acids in ginger family plants; (5) Increase the content of free amino acids in ginger family plants; (6) Increase the content of soluble protein in ginger plants.

2. The application according to claim 1, characterized in that: The ginger family plant mentioned is at least one of Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko; The aforementioned Penicillium sclerotium is Penicillium sclerotium ( Penicillium sclerotiorum )jyscaumcx01.

3. The application according to claim 1, characterized in that: (1) The aldehydes and ketones mentioned are at least one of 4-hydroxybenzaldehyde and 2-hydroxy-4-methoxybenzaldehyde; The fatty acid mentioned in (2) is at least one of valeric acid, hexanoic acid and oleic acid; The ester derivative mentioned in (3) is isophytol acetate; (3) The terpene derivatives mentioned are at least one of neophytadiene and γ-sitosterol; The long-chain fatty acid mentioned in (4) is at least one of cis-13-octadecenoic acid and n-hexadecanoic acid.

4. A method for fermenting ginger plants based on Penicillium sclerotiorum, characterized in that, Includes the following steps: S1. Raw material pretreatment: Dry and crush ginger plants, adjust the moisture content to 55% to 65%, sterilize, and obtain the fermentation substrate; S2. Inoculation and fermentation: The suspension of Penicillium sclerotiorum spores is inoculated into the fermentation substrate obtained in step S1, and static solid-state fermentation is carried out at 25-30°C to reduce the content of undesirable flavor components and increase the content of beneficial components in ginger plants; wherein, undesirable flavor components include at least one of aldehydes, ketones and fatty acids; and beneficial components include at least one of ester derivatives, terpenoid derivatives, long-chain fatty acids, free amino acids and soluble proteins.

5. The method according to claim 4, characterized in that: The ginger family plant mentioned in step S1 is at least one of Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko; The *Penicillium sclerotium* mentioned in step S2 is *Penicillium sclerotium* (… Penicillium sclerotiorum jyscaumcx01; The aldehyde or ketone compound mentioned in step S2 is at least one of 4-hydroxybenzaldehyde and 2-hydroxy-4-methoxybenzaldehyde; The fatty acid mentioned in step S2 is at least one of valeric acid, hexanoic acid, and oleic acid; The ester derivative mentioned in step S2 is isophytol acetate; The terpene derivative mentioned in step S2 is at least one of neophytadiene and γ-sitosterol; The long-chain fatty acid mentioned in step S2 is at least one of cis-13-octadecenoic acid and n-hexadecanoic acid.

6. The method according to claim 5, characterized in that: The ginger family plant mentioned in step S1 is at least one of the stems and leaves of Amomum villosum, Alpinia oxyphylla, and Amomum tsao-ko.

7. The method according to claim 4, characterized in that: The inoculum size of the *Penicillium sclerotium* spore suspension in step S2 is 1 × 10⁻⁶. 4 CFU / g ~ 1×10 8 CFU / g; The temperature for static solid-state fermentation in step S2 is 28°C; The static solid-state fermentation time mentioned in step S2 is 0 to 28 days, excluding 0 days.

8. The method according to claim 4, characterized in that: The pulverization mentioned in step S1 refers to pulverizing to 40-60 mesh; In step S1, the moisture content is adjusted by adding at least one of water and PDB liquid culture medium; The sterilization conditions described in step S1 are: sterilization at 121°C for 25–35 minutes.

9. The application of the method for fermenting ginger plants based on Penicillium sclerotiorum according to any one of claims 4 to 8 in the preparation of highly digestible feed.

10. A method for producing highly digestible feed from ginger plants based on Penicillium sclerotiorum fermentation, characterized in that, Includes steps S1 and S2 in the method for fermenting ginger plants based on Penicillium sclerotiorum according to any one of claims 4 to 8, and the following steps: S3. Post-processing: After fermentation in step S2 is completed, freeze-dry the fermentation product to constant weight to obtain highly digestible feed.

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