Osmanthus fragrans and its application in the preparation of biosurfactants by degrading feather powder

Abstract

The invention discloses Luteimonas Wenzhouensis and its application in degrading feather powder to prepare biosurfactants, belonging to the field of microbial technology. The Luteimonas Wenzhouensis DF114 of the invention has a deposit number of CCTCC M 20221181. The Luteimonas Wenzhouensis DF114 strain of the invention can not only degrade feather powder, but also directly convert feather powder into a substance with reduced surface tension, and has application potential. The Luteimonas Wenzhouensis DF114 obtained by screening is applied to the treatment of waste feathers to prepare biosurfactants, which not only alleviates the pressure of environmental pollution, but also reduces the production cost of biosurfactants, making it possible to use it in the detergent industry.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to Osmanthus fragrans and application of Osmanthus fragrans in preparing biosurfactants by degrading feather powder. Background Art

[0002] With the rapid development of the poultry industry, over 10 million tons of feathers are produced worldwide annually. A small portion is used as stuffing, ornaments, and feed, while the vast majority is directly incinerated or discarded, causing not only environmental pollution and disease transmission but also a significant waste of resources. Keratin, the primary component of poultry feathers, possesses a highly stable structure due to disulfide bonds, hydrogen bonds, and interlamellar van der Waals forces. It is insoluble in water, dilute acids, and alkalis, and resists hydrolysis by most proteases. Therefore, finding a suitable method to convert feather waste into usable components is not only economically beneficial but also offers significant social and environmental benefits. Microbial degradation of feathers offers the advantages of mild conditions, low energy consumption, minimal pollution, and high efficiency. Furthermore, it can convert the keratin in feathers into biousable, environmentally friendly products, truly achieving the goal of value-added waste utilization. Microbial treatment primarily utilizes microorganisms such as bacteria, fungi, and actinomycetes to degrade feathers. Bacillus licheniformis, Bacillus subtilis, Bacillus brevis, Bacillus cereus, and Bacillus halodurans are the most commonly reported feather-degrading bacteria. The main feather-degrading fungi are Candida, while research on actinomycetes mainly focuses on the genus Streptomyces. Summary of the Invention

[0003] To this end, the technical problem to be solved by the present invention is to provide a feather-degrading bacterium and its application. The present invention isolates a bacterial strain that directly degrades feathers, and uses the strain to ferment and treat feather waste to obtain surfactants. The reaction conditions are mild, the treatment cost is low, and it is environmentally friendly.

[0004] Based on this, the first object of the present invention is to provide a strain of Luteimonas wenzhouensis DF114, which was deposited with the China Center for Type Culture Collection (CCTCC) on July 26, 2022, at Wuhan University, Wuhan, China, with a postal code of 430072, and a deposit number of CCTCC M 20221181. The strain has brown colonies, a moist surface, and is Gram-negative. It is aerobic, immotile, rod-shaped bacterium with a biosafety level of 1.

[0005] The second object of the present invention is to provide a preparation comprising the above-mentioned Osmanthus fragrans DF114 or its fermentation liquid.

[0006] The third object of the present invention is to provide the use of Osmanthus fragrans DF114 in one or more of the following:

[0007] (1) Degrade feathers;

[0008] (2) Preparation of biosurfactants;

[0009] (3) Preparation of keratinase.

[0010] Preferably, the keratinase is feather keratinase.

[0011] Preferably, the feathers can be duck feathers, chicken feathers or goose feathers, and more preferably, they can be discarded duck feathers, chicken feathers or goose feathers.

[0012] Preferably, the biosurfactant is obtained by fermenting and culturing Osmanthus fragrans DF114 using feathers as substrate.

[0013] The fourth object of the present invention is to provide a method for degrading feathers, comprising inoculating Osmanthus fragrans DF114 into a culture medium containing feathers or feather powder and culturing the culture medium.

[0014] Preferably, the culture medium components include: feathers or feather powder, K2HPO4, KH2PO4, NaCl, MgSO4.

[0015] Preferably, the culture medium components and their contents are as follows: Feather meal 10-160g·L -1 , K2HPO4 1.4g·L -1 , KH2PO4 0.7g·L -1 , NaCl 0.5 g·L -1 , MgSO4 0.1g·L -1 , pH 7.0.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) Osmanthus fragrans DF114 of the present invention can degrade feather powder. -1 Under the conditions of , the degradation rate of puffed feather powder can reach more than 78.34%;

[0018] (2) At the same time, when the Wenzhou osmanthus DF114 of the present invention degraded feather powder at an initial pH of 7.46 and pH 7.97 for 26 days, the surface tension of the degradation liquid decreased from the initial 43.95±0.25 mN / m and 43.71±0.78 mN / m to 31.95±1.58 mN / m and 25.34±1.87 mN / m, respectively; when the sterile blank degraded feather powder at an initial pH of 7.46 and pH 7.97 for 26 days, the surface tension of the degradation liquid decreased from the initial 43.97±0.15 mN / m and 43.81±0.34 mN / m to 42.97±0.58 mN / m and 42.74±0.58 mN / m, respectively.

[0019] (3) Osmanthus wenzhouensis DF114 is a harmless bacterium (level 4) with a biosafety level of (BSL-1) that exists naturally in nature. The relevant enzymes produced during its growth process effectively degrade feathers. At the same time, using the feather degradation products as substrates, it not only completely converts feathers into usable products, but also produces environmentally friendly proteins and surfactants;

[0020] (4) The present invention proposes for the first time that the fungus species Wenzhou Osmanthus fragrans DF114 has the ability to degrade feathers and simultaneously produce surfactants;

[0021] Luteimonas wenzhouensis DF114 was deposited in the China Center for Type Culture Collection (CCTCC) on July 26, 2022, at Wuhan University, Wuhan, China, Postal Code: 430072, with the deposit number: CCTCC M20221181. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The phenomenon before and after the degradation of feather powder by Luteimonas wenzhouensis DF114 of the present invention;

[0023] Figure 2 This is the growth curve of Luteimonas wenzhouensis DF114 of the present invention under different temperature conditions;

[0024] Figure 3 is the degradation rate of feather powder by Luteimonas wenzhouensis DF114 of the present invention;

[0025] Figure 4 Surface tension of Luteimonas wenzhouensis DF114 after feather meal degradation at pH 7.46 and pH 7.97 for 26 days. DETAILED DESCRIPTION

[0026] The following is a further explanation of the contents of the present invention in combination with implementation cases, but is not intended to limit the present invention.

[0027] Example 1 Isolation, identification and cultivation of bacterial strains

[0028] 1. Separation

[0029] Source of strain: This strain was derived from compost and was purified to obtain strain DF114.

[0030] 2. Identification

[0031] (1) Colony morphology, physiological and biochemical identification: Observe the strain DF114 by streaking on LB plates and incubate at 37°C for 48 h. Observe and record the colony shape, color, surface texture, and colony edge. The colony is brown, moist, and Gram-negative.

[0032] (2) Identification by molecular biology methods

[0033] The strain was pure cultured, total DNA of the strain was extracted, 16S rDNA was PCR amplified (primers 27F: AGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT), and the amplified product was sent to the Guangzhou branch of Beijing Qingke Biotechnology Co., Ltd. for sequence determination. The forward and reverse sequences were spliced ​​to obtain a 16S rDNA gene sequence with a length of 1472 bp, as shown in SEQ ID NO.1. The determination results were compared by BLAST on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The strain had a homology of 98.52% with the Gram-negative bacteria-Luteimonas wenzhouensis strain YD-1. The screened strain was finally named Luteimonas wenzhouensis DF114 and was deposited in the China Center for Type Culture Collection (CCTCC) on July 26, 2022. The address is Wuhan University, Wuhan, China, Postal Code: 430072, and the deposition number is: CCTCC M20221181.

[0034] (3) Culture: Activate the bacteria by streaking on LB solid medium, then inoculate into LB liquid medium for 20 h. Then, inoculate 100 μL of the bacterial solution into four 50 mL bottles of LB medium, culture at 25°C, 30°C, 35°C, and 40°C, respectively, at a speed of 180 rpm, and measure the OD at different times. 600 .

[0035] The growth curve of Osmanthus fragrans DF114 is as follows Figure 2 .

[0036] SEQ ID NO.1

[0037]

[0038] Example 2 Degradation of Feather Powder by Osmanthus fragrans DF114

[0039] 1. Preparation process of expanded feather powder

[0040] Preheat the extruder for 30 minutes with a 75kW motor and a current of about 150A. Then evenly load the duck feathers, which have been free of impurities and have a moisture content of about 13%, at 400°C. After starting the machine, reduce the temperature to about 350°C. If the discharge is beige, this is normal.

[0041] 2. Wenzhou Osmanthus DF114 degraded feather powder

[0042] Wenzhou osmanthus DF114 was selected from LB solid medium and added to LB liquid medium. After culturing at 180 rpm and 37 °C for 20 h, 1 mL of bacterial culture solution was added to 50 mL of feather powder containing different culture media (80.0 g·L -1 , K2HPO41.4g·L -1 , KH2PO4 0.7g·L -1 , NaCl 0.5 g·L -1 , MgSO4 0.1g·L -1 , pH 7.0, water as solvent). Under the same conditions, three replicates were set up, with no bacteria added as a control, and cultured at 180 rpm and 37°C. After degradation, the solids in the culture medium were collected, cleaned, dried at 80°C, and weighed. The degradation rate of feather meal was calculated using the following formula:

[0043] Feather powder degradation rate (%) = (M0-M t ) / M0×100% formula (1)

[0044] Where, M0: initial mass of feather meal (g); M t : The mass of remaining feather meal at reaction time t (g).

[0045] The results are as follows Figure 3 As shown, the degradation rate of Osmanthus fragrans DF114 of the present invention on puffed feathers can reach over 78.34%.

[0046] Example 3 Determination of surface tension of fermentation liquid of feather powder degraded by Osmanthus fragrans DF114

[0047] In the presence of 160g L -1 Culture medium of puffed feather powder (prepared as in Example 2) (puffed feather powder 160.0 g L-1 , K2HPO4 1.4g L -1 , KH2PO4 0.7g L -1 , NaCl 0.5 g L -1 , MgSO4 0.1g L -1 , pH 7.46, solvent is water), (expanded feather powder 160.0 g L -1 , K2HPO4 1.4g L -1 , KH2PO4 0.7g L -1 , NaCl 0.5 g L -1 , MgSO4 0.1g L -1 The surface tension of feather meal degradation by Osmanthus fragrans DF114 was measured at the initial stage (0d) and the 26th day (26d) under the conditions of 4% inoculum (pH 7.97, water as solvent), an initial inoculum size of 20% and a temperature of 30℃. The same expanded feather meal culture medium without bacterial solution was used as a control, and three replicates were set up.

[0048] The results are as follows Figure 4 As shown, after 26 days of degradation of feather meal by Osmanthus fragrans DF114 at initial pH 7.46 and pH 7.97, the surface tension of the degradation solution decreased from 43.95±0.25mN / m and 43.71±0.78mN / m to 31.95±1.58mN / m and 25.34±1.87mN / m, respectively. The surface tension of the sterile blank group with initial pH 7.46 and pH 7.97 feather meal solutions was 43.97±0.15mN / m and 43.81±0.34mN / m, respectively. After 26 days of shaking, the surface tension remained essentially unchanged at 42.97±0.58mN / m and 42.74±0.58mN / m, respectively.

[0049] The present invention utilizes Luteimonas wenzhouensis DF114 to degrade feather powder to prepare biosurfactants, which can not only economically solve the environmental problems caused by discarded feathers, but also meet the industrial demand for biosurfactants, and has important research significance and application value.

[0050] The above embodiments describe the present invention in detail in different implementation processes, but the embodiments of the present invention are not limited thereto. A person skilled in the art can achieve the purpose of the present invention based on the contents disclosed in the present invention.

[0051] It is apparent to those skilled in the art that several improvements and modifications may be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Osmanthus fragrans Luteimonas wenzhouensis )DF114, the accession number is: CCTCC M 20221181.

2. A preparation, characterized in that The invention comprises the Osmanthus fragrans DF114 or its fermentation liquid as claimed in claim 1.

3. Use of the Osmanthus fragrans DF114 according to claim 1 in one or more of the following: (1) Degradation of feathers; (2) Preparation of biosurfactants.

4. The use according to claim 3, characterized in that: The biosurfactant is prepared by fermenting and culturing Osmanthus fragrans DF114 with feathers as substrate.

5. The use according to claim 4, characterized in that: The feathers are duck feathers, chicken feathers or goose feathers.

6. The use according to claim 5, characterized in that: The feathers are discarded duck feathers, chicken feathers or goose feathers.

7. A method for degrading feathers, characterized in that: The Osmanthus fragrans DF114 described in claim 1 is inoculated into a culture medium containing feathers or feather powder for cultivation.

8. The method according to claim 7, characterized in that The culture medium components include: feathers or feather powder, K2HPO4, KH2PO4, NaCl, and MgSO4.

9. The method according to claim 8, characterized in that The culture medium components and their contents are as follows: Feather meal 10-160 g·L -1 , K2HPO4 1.4 g·L -1 , KH2PO4 0.7 g·L -1 , NaCl 0.5 g·L -1 , MgSO40.1g·L -1 .

10. The method according to claim 7, characterized in that The feathers are chicken feathers, duck feathers or goose feathers.

11. The method according to claim 7, characterized in that The feathers are discarded duck feathers, chicken feathers or goose feathers.

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