Application of inonotus obliquus in preparation of feed from lavender residues

Abstract

The invention discloses an application of inonotus obliquus in preparation of a feed from lavender residues. The preparation method comprises the following steps: inoculating a crushed lavender residue with an Inonotus obliquus XJ-1 bacterial agent, and carrying out fermentation so as to obtain a lavender residue fermentation product. According to the fermentation product, fiber components which are not easy to digest by animals are fully decomposed, and meanwhile, a part of flavonoid substances which are embedded in residue cells and are not easy to utilize are released, so that the biological activity effect of the flavonoid substances is better exerted; the content of tannin is obviously reduced after 10 days of fermentation, the palatability of animals is improved to a certain extent, compared with the content of tannin reduced after 2-3 months of silage treatment, the fermented product has the advantage of lower time cost, and by adding the fermented product into daily ration, sufficient digestion of the daily ration, metabolism of nutrient substances and activity of rumen microorganisms can be promoted; when the fermented product is applied to feed, a theoretical basis can be provided for developing and utilizing rare fungi and ground-source resources in Xinjiang to assist green and sustainable development of animal husbandry.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to the technical field of application of Inonotus obliquus in preparing feed using lavender residues. Background Art

[0002] Inonotus obliquus, also known as Chaga, is a rare medicinal fungus that grows on birch trees. It contains a variety of bioactive ingredients, such as polysaccharides, triterpenoids, polyphenols, etc., which have the effects of lowering blood sugar, anti-tumor, anti-oxidation and improving immunity. Inonotus obliquus is scarce and has high medicinal value. It is mainly distributed in cold areas between 45 and 50 degrees north latitude, including North America, Finland, Poland, Western Siberia, Eastern Russia, Kamchatka Peninsula, and Lesser Khingan Mountains in Heilongjiang Province and Changbai Mountain in Jilin Province in northeastern China and Hokkaido, Japan. Wild Inonotus obliquus grows very slowly and is expensive. The sclerotium needs to grow in the wild environment for 10 to 15 years before it has good medicinal value. Liquid fermentation has the advantages of short culture cycle, high yield, and no seasonal and environmental restrictions. The polysaccharides, polyphenols, triterpenoids and other bioactive substances obtained from the fermentation products also have antioxidant, anti-tumor, and blood sugar-lowering functions, which greatly improves the practical value of large-scale production of Inonotus obliquus.

[0003] Lavender (Lavandula angustifolia Mill) belongs to the genus Lavandula of the Lamiaceae family. It is a precious spice plant native to the Mediterranean region. It was introduced to my country in 1952 and is now planted on a large scale in the Yili region of Xinjiang, accounting for more than 90% of the country's total planting area. Lavender is rich in essential oils, tannins, coumarins, flavonoids, polyphenols and other chemical components. The substances extracted from it as raw materials are widely used in medicine, food, cosmetics and other fields. The non-volatile components of lavender contain a large number of active substances, including flavonoids, phenolic acids, cinnamic acids, coumarins and their derivatives. Most of these active ingredients have significant antioxidant and anti-tumor activities and have potential application value. However, these components are ultimately retained in the discarded residues after the lavender essential oil is extracted and are not reasonably utilized, resulting in a great waste of resources. When used as feed, the lavender residues not only contain fiber components that are difficult for animals to digest, but also contain tannins that affect animal eating. Therefore, the feed prepared with lavender residues has serious problems such as low animal digestion and absorption rate and poor palatability. At present, there are no reports on the application of lavender residues in feed additives. Summary of the invention

[0004] In view of the problems that the feed prepared from lavender residues currently has low animal digestion and absorption rate and poor palatability, and the technical problems that the research on the application of lavender residues to feeds has not been reported, the present invention aims to provide an application of Inonotus obliquus in the preparation of feeds from lavender residues. The present invention obtains a strain Inonotus obliquus XJ-1 from the sclerotium tissue of Inonotus obliquus in Altay, Xinjiang, and finds that the strain has the ability to produce cellulase, which can fully decompose the fiber components that are difficult for animals to digest in lavender residues, and release some flavonoids that are not easy to use and are partially embedded in the residue cells, so as to better play its biological activity. Adding lavender residue solid fermentation products to the diet can promote the full digestion of the diet, the metabolism of nutrients and the activity of rumen microorganisms. Using the bacterial agent as a feed additive can provide a theoretical basis for the development and utilization of Xinjiang rare fungi and terrestrial resources to help Xinjiang animal husbandry green and sustainable development.

[0005] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions.

[0006] In the first aspect, the present invention provides an Inonotus obliquus XJ-1, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC), with a deposit number of CGMCC No.41419, a deposit date of August 5, 2024, and an ITSDNA sequence as shown in SEQ ID NO: 1.

[0007] In a second aspect, the present invention provides a method for preparing an Inonotus obliquus XJ-1 bacterial agent, wherein the method comprises culturing the Inonotus obliquus XJ-1 strain at 30°C and 150 r / min for 3.5 days, inoculating its seed liquid into a fermentation medium at a 10% inoculation rate, and further culturing for 10 days.

[0008] The fermentation medium of the present invention adopts 20.0 g / L glucose, 4.0 g / L peptone, MgSO 4 1.0g / L, KH 2 PO 4 1.0g / L, prepared.

[0009] In a third aspect, the present invention provides a use of Inonotus obliquus XJ-1 in preparing feed using lavender residues.

[0010] In a fourth aspect, the present invention provides a use of an Inonotus obliquus XJ-1 inoculant in preparing feed using lavender residues.

[0011] In the present invention, Inonotus obliquus XJ-1 bacterial agent is inoculated into crushed lavender residue, fermented for 10 days at a water content of 60% and a temperature of 28°C, dried at 50°C, and the obtained lavender residue solid fermentation product is mixed with a diet.

[0012] Through the above technical scheme, the present invention obtains the following technical effects:

[0013] (1) The pure culture strain Inonotus obliquus XJ-1 provided by the present invention is isolated from the sclerotium of Inonotus obliquus in Altay, Xinjiang. The strain has the ability to produce cellulase, which can fully decompose the fiber components in lavender residues that are difficult for animals to digest, and at the same time release some flavonoids that are embedded in the residue cells and are difficult to utilize, so as to better exert their biological activity; on the other hand, the tannin contained in the lavender residues will affect the feeding of animals. After fermentation for 10 days, the tannin content is significantly reduced, which improves the palatability of animals to a certain extent. Compared with the silage treatment for 2-3 months to reduce tannin, it has a lower time cost advantage.

[0014] (2) The present invention provides an application of Inonotus obliquus XJ-1 inoculant in the preparation of feed with lavender residues. Adding lavender residue solid fermentation products to the diet will not affect the normal growth and metabolism of rumen microorganisms, but can also promote the full digestion of the diet and the metabolism of nutrients and the activity of rumen microorganisms. Significant effects can be achieved by using a low dose of 1.0% fermentation product. The application of the fermentation product in feed can provide a theoretical basis for the development and utilization of Xinjiang's rare fungi and terrestrial resources to help Xinjiang's animal husbandry to develop green and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the colony morphology of Inonotus obliquus XJ-1.

[0016] Figure 2 Phylogenetic tree constructed for the ITS rDNA sequence of the XJ-1 gene of Inonotus obliquus. DETAILED DESCRIPTION

[0017] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0018] In this example, a plant flavonoid content detection kit produced by Solebow Biochemical Kit Co., Ltd. was used to determine the flavonoid mass content according to the instructions. A tannin content test kit produced by Suzhou Geruisi Biotechnology Co., Ltd. was used to determine the tannin mass content of the lavender residue sample according to the instructions.

[0019] The complete mixed ration used in this example was purchased from Xinjiang Tiankang Feed Co., Ltd.

[0020] Example 1: Taxonomic identification of Inonotus obliquus XJ-1 based on ITS sequence analysis

[0021] The present invention separates and purifies the sclerotium tissue of Inonotus obliquus in Altay, Xinjiang, and obtains a strain of Inonotus obliquus numbered XJ-1. The colony morphology of the strain is shown in the attached Figure 1 As shown in the figure, the color of the colony gradually changed from light yellow to dark brown at the beginning. The mycelium grew slowly on the PDA plate. The optimal temperature was 28℃ and the mycelium grew to a diameter of 8 cm after 15 days of cultivation. The Inonotus obliquus strain numbered XJ-1 was identified by ITS. The fungal ITS universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS

[0022] 4(5'-TCCTCCGCTTATTGATATGC-3'). The ITS rDNA sequence analysis results were submitted to the GenBank database, and the BLAST online comparison analysis was performed. The phylogenetic tree was constructed using MEGA 11.0 software and the Neighbor-joining method (1000bootstrap) for homology analysis.

[0023] The results of ITSDNA sequence phylogenetic analysis and microbiological characteristics analysis showed that the strain numbered XJ-1 belongs to the genus Inonotus obliquus. The gene of the strain was sequenced and the obtained sequence was compared and analyzed on the NCBI website. The sequence was submitted to the GenBank database and obtained the accession number PQ285365. The phylogenetic tree is shown in the attached Figure 2 As shown, after comparison and analysis, it was found that the ITSDNA gene sequence of the Inonotus obliquus strain numbered XJ-1 had the highest homology with that of Inonotus obliquus HS819, which was 99.86%. It was named Inonotus obliquus XJ-1, and the fungus has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the strain deposit number CGMCC No.41419 and the deposit date: August 5, 2024.

[0024] Example 2: Preparation of Inonotus obliquus XJ-1 bacterial agent

[0025] The Inonotus obliquus XJ-1 strain stored on a 4°C PDA slant was inoculated onto a PDA plate and cultured at 30°C for 7-14 days. After activation twice, four bacterial cakes with a diameter of 1 cm were punched out on the PDA plate using a hole puncher and inoculated into a seed culture medium containing 30.0 g / L glucose, 5.0 g / L peptone, and MgSO 4 0.5g / L, KH 2 PO 4 2.0g / L, pH natural. After culturing at 30℃, 150r / min for 3.5 days, the seed liquid was inoculated into the fermentation medium at a 10% inoculation rate and cultured at 150r / min, 30℃ for 10 days. The fermentation medium used: glucose 20.0g / L, peptone 4.0g / L, MgSO 4 1.0g / L, KH 2 PO 4 1.0g / L, pH natural, prepared.

[0026] Example 3: Lavender residue solid fermentation product

[0027] Inonotus obliquus XJ-1 inoculum was inoculated into crushed lavender residue at a rate of 10%, and fermented for 10 days at a water content of 60% and a temperature of 28°C. The fermented lavender residue was dried at 50°C to obtain a lavender residue solid fermentation product.

[0028] Example 4: Determination and analysis of the contents of acidic detergent fiber, neutral detergent fiber, total flavonoids and tannins in lavender residue before and after fermentation

[0029] This embodiment refers to the national standard GB / T 20806-2022, and adopts the washing method to determine the neutral detergent fiber content, and refers to the agricultural industry standard NY / T 1459-2022, and adopts the washing method to determine the acid detergent fiber content.

[0030] As can be seen from the data in Table 1, the acidic detergent fiber and neutral detergent fiber content of lavender residues are significantly reduced, the total flavonoid content is significantly increased, and the tannin content is significantly reduced after fermentation of lavender residues by Inonotus obliquus XJ-1. It shows that the pure culture strain of Inonotus obliquus XJ-1 has the ability to produce cellulase, which can fully decompose the fiber components that are difficult for animals to digest in lavender residues, and release some flavonoids that are not easy to use in cells, so as to better play its biological activity. In existing actual production, lavender residues contain a large amount of tannin components, which greatly reduces the palatability of animals, and the tannin content can be effectively reduced by silage treatment. While silage production usually requires fermentation for several months, the fermentation time of the present invention is only 10d after optimization of the experiment, which greatly reduces the time cost.

[0031] Table 1: Comparison of the contents of four components in lavender residue before and after fermentation

[0032]

[0033] Note: t-test, different capital letters on shoulders indicate extremely significant differences at p<0.01, and different lowercase letters indicate significant differences at p<0.05.

[0034] Example 5: In vitro fermentation of lavender residue solid fermentation product on sheep rumen liquid

[0035] (1) Preparation of artificial rumen fluid

[0036] Composition of artificial rumen nutrient solution: trace element solution A: MnCl 2 ·4H 2 O 10.0g, CaCl 2 ·2H 2 O13.2g, FeCl 3 6H 2 O 8.0g, CoCl 2 6H 2 O 1.0g, 100mL distilled water; buffer solution B: NH 4 HCO 3 4.0 g, NaHCO 3 35.0g, distilled water 1000mL; constant element solution C: KH 2 PO 4 6.2g, Na 2 HPO 4 5.7 g MgSO 4 7H 2 O0.6g, distilled water 1000mL; Resazurin solution: Resazurin 0.1g, 100mL distilled water; Reducing agent solution: Na 2S·9H 2 O625.0mg, 1mol / LNaOH 4.0mL, distilled water 95mL. The composition ratio of the nutrient solution: trace element solution A 0.1mL, buffer solution B 200mL, major element solution C 200mL, resazurin solution 1mL, reducing agent solution 40mL, distilled water (H 2 O)400mL.

[0037] The prepared solution was added into the bottle in the proportion of nutrient solution and mixed well. CO 2 , preheat in a 39°C water bath until the mixture becomes colorless and transparent.

[0038] (2) Preparation of fermentation broth

[0039] The sheep rumen fluid was collected using a vacuum stomach tube sampler and filtered through 4 layers of gauze to remove impurities. 2 The mixture was placed in a sealed thermos bottle preheated to 39°C and immediately brought back to the laboratory and transferred to a 39°C constant temperature water bath with continuous CO 2 Keep anaerobic.

[0040] Artificial rumen nutrient solution and fresh sheep rumen fluid were mixed in a ratio of 2:1 to prepare in vitro fermentation liquid, which was then placed in a 39°C chamber and aerated with CO. 2 Stir evenly in a volumetric flask and set aside.

[0041] (3) Fermentation outside rumen fluid

[0042] Accurately weigh 0.4g of total mixed diet, control group: no addition of lavender residue solid fermentation product; low-dose treatment group: addition of 1% lavender residue solid fermentation product; high-dose treatment group: addition of 1.5% lavender residue solid fermentation product; put the three treatment groups into a preheated culture bottle, quickly add 60ml of fermentation liquid and seal for fermentation. Cultivate at a constant temperature of 39℃ for 48h, with 3 replicates in each group. Measure gas production at 8, 24 and 48h of fermentation, and detect pH, rumen liquid ammonia nitrogen, microbial protein (MCP), and volatile fatty acid content after fermentation. Use a portable acidity meter to measure the pH of the fermentation liquid; use a colorimetric method to measure the ammonia nitrogen (NH 3 -N) content; Coomassie brilliant blue method was used to determine the MCP content; gas chromatography was used to determine the volatile fatty acids content in rumen fluid.

[0043] Example 6: Effect of solid fermentation product of lavender residue on in vitro fermentation of sheep rumen fluid

[0044] pH is a comprehensive reaction of microorganisms in rumen fluid to the metabolism and utilization of nutrients, and the normal range is 6.0-7.5. As shown in Table 2, the pH of the fermented liquid remained within the suitable pH range of the rumen after 48 hours of fermentation, indicating that adding lavender residue solid fermentation products to the diet will not affect the normal growth and metabolism of rumen microorganisms. The pH of the treatment group in the experiment was significantly lower than that of the control group, indicating that the fermentation products promoted rumen digestion, fully degraded the diet to form more volatile fatty acids, and provided more energy for the animal body.

[0045] Gas production is an important indicator for evaluating gastric microbial fermentation in the rumen fermentation system of ruminants, and to a certain extent indicates the overall trend of rumen microbial fermentation activity. The gas production of the experimental treatment group at three time points was significantly higher than that of the control group, and the gas production was the highest in the first 24 hours, indicating that the solid fermentation of lavender residue promoted the full fermentation of the diet and the metabolic function of its nutrients and the activity of rumen microorganisms.

[0046] However, the solid fermentation product of lavender residue had no significant effect on the contents of ammonia nitrogen, MCP and most volatile fatty acids, and the 1.5% high-dose treatment group significantly reduced the content of isobutyric acid.

[0047] Table 2: Effect of solid fermentation product of lavender residue on in vitro fermentation of sheep rumen liquid

[0048] CK 1% low dose 1.5% High Dose pH <![CDATA[7.00±0.01 a ]]> <![CDATA[6.90±0.01 b ]]> <![CDATA[6.86±0.01 b ]]> Cumulative gas production (ml) 8h <![CDATA[8.00±1.41 b ]]> <![CDATA[13.50±0.71 a ]]> <![CDATA[16.00±1.24 a ]]> 24h <![CDATA[12.50±2.12 b ]]> <![CDATA[22.00±0.00 a ]]> <![CDATA[26.00±2.83 a ]]> 48h <![CDATA[13.50±3.54 b ]]> <![CDATA[24.50±0.71 a ]]> <![CDATA[29.00±2.83 a ]]> Ammonia nitrogen (mg / dL) 48.15±0.57 52.32±5.83 50.07±0.89 MCP (mg / dL) 27.74±2.41 29.43±0.74 40.99±1.65 Volatile fatty acids (mmol / L) Acetic acid 16.39±1.94 17.04±2.74 19.34±0.13 Propionic acid 11.20±1.40 11.76±1.73 13.23±0.10 Butyric acid 5.71±0.45 5.80±0.45 6.26±0.03 Isobutyric acid <![CDATA[1.49±0.01 a ]]> <![CDATA[1.51±0.04 a ]]> <![CDATA[1.39±0.00 b ]]> Valeric acid 2.90±0.11 2.94±0.04 2.86±0.01 Isovalerate <![CDATA[1.73±0.04 ab ]]> <![CDATA[1.76±0.06 a ]]> <![CDATA[1.63±0.00 b ]]> Ethylene-propylene ratio 1.46±0.04 1.45±0.02 1.46±0.00

[0049] In summary, the pure culture strain of Inonotus obliquus XJ-1 has the ability to produce cellulase, which can fully decompose the fiber components in lavender residues that are difficult for animals to digest, and at the same time release some flavonoids embedded in cells that are difficult to utilize, so as to better exert their biological activity; fermentation of lavender residues by Inonotus obliquus XJ-1 can significantly reduce the tannin content and improve the palatability of animals, and has a lower time cost compared with silage treatment.

[0050] The results of the in vitro fermentation experiment of lavender residue solid fermentation products in sheep rumen liquid showed that the fermentation products not only did not affect the normal growth and metabolism of rumen microorganisms, but also promoted digestion and full fermentation of diets and the metabolic function of their nutrients and the activity of rumen microorganisms. The 1.0% low-dose treatment group could achieve significant effects.

[0051] The above-described embodiments are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but rather represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. An Inonotus obliquus XJ-1, characterized in that: The strain has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number CGMCC No.41419 and the deposit date August 5, 2024. Its ITSDNA sequence is shown in SEQ ID NO:

1.

2. A method for preparing an Inonotus obliquus XJ-1 bacterial agent, characterized in that: The method is to culture the Inonotus obliquus XJ-1 strain at 30°C and 150 r / min for 3.5 days, then inoculate its seed liquid into a fermentation medium at a 10% inoculation rate, and then culture for another 10 days.

3. The method for preparing the Inonotus obliquus XJ-1 bacterial agent according to claim 2, characterized in that: The fermentation medium is prepared by using 20.0 g / L glucose, 4.0 g / L peptone, 1.0 g / L MgSO4 and 1.0 g / L KH2PO4.

4. An application of Inonotus obliquus XJ-1 in preparing feed from lavender residue.

5. Application of Inonotus obliquus XJ-1 inoculant in preparing feed from lavender residue.

6. The use of an Inonotus obliquus XJ-1 inoculant in preparing feed from lavender residues as claimed in claim 5, characterized in that: Inonotus obliquus XJ-1 inoculum was inoculated into crushed lavender residues, fermented at a moisture content of 60% and a temperature of 28°C for 10 days, dried at 50°C, and the obtained solid fermentation product of lavender residues was mixed with the diet.

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