CN114517169B

CN114517169B - Streptomyces strain SEM-14 and derivative product and application thereof

Info

Publication number: CN114517169B
Application number: CN202210236014.8A
Authority: CN
Inventors: 杨琼; 李庆荣; 邹宇晓; 黎尔纳; 廖森泰; 邢东旭; 肖阳
Original assignee: Sericulture and Agri Food Research Institute GAAS
Current assignee: Sericulture and Agri Food Research Institute GAAS
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2024-03-22
Anticipated expiration: 2042-03-11
Also published as: CN114517169A

Abstract

The invention provides a streptomycete strain SEM-14, a derivative product and application thereof, and belongs to the technical field of functional microorganisms. Streptomyces strain SEM-14 has the characteristic of secreting cellulase with high enzyme activity and has the characteristic of high temperature resistance. Thus, the method is applicable to a variety of applications. The streptomycete strain SEM-14 is used as a fiber biodegradation bacterium, can be used for decomposing cellulose by using straw decomposition agents, improves the feed conversion rate, and simultaneously is used as probiotics to be added into the feed to help animals improve the decomposing capacity of the feed and improve the utilization rate of the feed.

Description

Streptomyces strain SEM-14 and derivative product and application thereof

Technical Field

The invention belongs to the technical field of functional microorganisms, and particularly relates to a streptomycete strain SEM-14, a derivative product and application thereof.

Background

Resource and environmental problems are the most major challenges facing humans in the 21 st century. Biological resources are renewable resources and are the basic material sources for human society to survive. More than 90% of the total polysaccharide is lignocellulose substances, and cellulose is the most abundant polysaccharide substance on the earth. Such substances are the main components of plant cell walls and are the most abundant and cheapest renewable resources on earth. Cellulose resources in China are very rich, but the resources are not fully developed and utilized, and certain pollution is caused to the environment. Along with rapid world population growth and gradual exhaustion of grain and mineral resources, a microbial technology for efficiently converting lignocellulose renewable resources is developed, and fuels, feeds and chemical products which are urgently needed by human beings are produced through fermentation, so that the method has great practical significance and development prospect.

With the rapid advance of the breeding industry to the feed industry, the resource shortage situation is more and more prominent, so that the utilization of plant feeds, especially fibrous feeds, is enhanced, the decomposition and conversion of cellulose by using cellulase is a big subject for developing feed resources and developing animal husbandry, and the development of cellulase additives is an effective way for solving the problem. The cellulose degrading bacteria can supplement endogenous enzymes of animals, promote digestion and absorption of nutrients, eliminate anti-nutritional factors and improve the utilization rate of the animals to fiber feeds.

At present, cellulase-producing strains which are reported at home and abroad and are screened from the nature are mainly fungi, such as trichoderma, aspergillus, penicillium and acremonium strains, and cellulase-producing bacteria are mainly bacillus-producing clostridium, streptomyces flavus-like bacteria, acidophilic cellulose decomposing bacteria and the like, and the research on the cellulase-producing streptomyces is little reported.

Disclosure of Invention

In view of the above, the invention aims to provide a streptomycete strain SEM-14 which has the capability of secreting cellulase with high activity and can efficiently degrade cellulose.

The invention also aims to provide a derivative product prepared based on the streptomycete strain SEM-14 and application thereof.

The invention provides a Streptomyces sp strain SEM-14, which is used for secreting cellulase.

Preferably, the filter paper enzyme activity of the cellulase is 100-124.40 mug/h/mL.

Preferably, the strain SEM-14 grows normally under high temperature conditions;

the high temperature is 35-65 ℃.

Preferably, the strain SEM-14 has a deposit number of GDMCC No:61765.

the invention provides a bacterial agent for degrading cellulose, and an active ingredient comprises the bacterial strain SEM-14.

The invention provides application of the strain SEM-14 or the microbial inoculum in degrading cellulose.

Preferably, the cellulose comprises one or more of the following: straw, filter paper and microcrystalline cellulose.

Preferably, the temperature for degrading the cellulose is 35-65 ℃.

The invention provides an animal feed, which comprises a basic feed and any one of the following components: the strain SEM-14 and the microbial inoculum;

the concentration of viable bacteria of the strain SEM-14 in the feed is 10 ⁸ ～10 ⁹ CFU/g。

The invention provides an application of the feed in animal cultivation.

The Streptomyces sp strain SEM-14 provided by the invention has the characteristic of secreting cellulase. The strain SEM-14 can normally grow in a culture medium containing microcrystalline cellulose, and meanwhile, the Streptomyces strain SEM-14 has higher filter paper enzyme activity at 24 hours, 48 hours, 72 hours and 96 hours through detection by a filter paper strip culture method. As can be seen, the Streptomyces strain SEM-14 has the property of secreting highly active cellulases. And from 48 hours on, the enzyme activity of SEM-14 was significantly increased compared to the two Streptomyces strains (X5, X6) and the Bacillus strains (IP 3 and IP 4) identified by applicant's isolation. This shows that strain SEM-14 has higher application value in degrading cellulose.

Meanwhile, the strain SEM-14 provided by the invention has higher temperature tolerance. Experiments prove that the strain SEM-14 grows normally in the range of 35-65 ℃ and hypha or thalli, the highest tolerance temperature of the separated and identified streptomyces strain (X5) of the applicant is 60 ℃, and the highest tolerance temperature of the other streptomyces strain (X6) and bacillus strain (IP 3 and IP 4) is only 55 ℃. Based on the high temperature resistance of the strain SEM-14, the strain can be applied to compost fermentation.

Drawings

FIG. 1 shows the colony morphology of Streptomyces strain SEM-14 provided by the invention;

FIG. 2 is a mycelium characterization of Streptomyces strain SEM-14 provided by the invention;

FIG. 3 shows the spore morphology of Streptomyces strain SEM-14 provided by the invention.

Biological material preservation information

Streptomyces sp SEM-14, deposited in the China center for type culture Collection, GDMCC, with addresses of building 5, no. 59, national institute for microbiology, guangdong province, first middle road 100, and accession number GDMCC No. 7, with a deposition time of 2021, month 1, and a deposition number of GDMCC No:61765.

Detailed Description

In the invention, the strain SEM-14 is derived from silkworm excrement and is obtained by separating a high-temperature phase in the silkworm excrement composting process. Morphological features of the strain SEM-14 were as follows: observing that the bacterial colony is grey white, pink, circular and has radial mycelium surface wrinkles around the bacterial colony, and the bacterial colony forms a concentric circle structure and is opaque; the mycelia are in filament shape and spores are in short column shape when observed by an electron microscope. The sequence of the strain SEM-14 has the similarity of more than 99% with streptomycete through the amplification sequencing of the 16SrRNA gene fragment. Morphological characteristics and molecular identification result show that the strain SEM-14 belongs to one of streptomycete.

In the present invention, the strain SEM-14 was grown normally on cellulose screening medium. The formulation of the cellulose screening medium is preferably as follows: 5g of sodium carboxymethyl cellulose (microcrystalline cellulose), 2g of yeast powder, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 20g of agar, 1000ml of distilled water and natural pH. The strain SEM-14 was able to grow on microcrystalline cellulose as a carbon source. Meanwhile, the strain SEM-14 can also grow in enrichment in a culture medium taking straw and filter paper as carbon sources, which shows that the strain SEM-14 has the characteristic of secreting and degrading cellulase. In addition, the strain SEM-14 has the effect of degrading filter paper for a long time as detected by a filter paper culture method. Thus, the strain SEM-14 has cellulase secretion performance. The filter paper enzyme activity of the cellulase is preferably 100-124.40 mug/h/mL.

In the present invention, the strain SEM-14 preferably grows normally under high temperature conditions. The elevated temperature is preferably 35 to 65 ℃. The experimental result shows that the mycelium of the strain SEM-14 grows normally and a large number of thalli survive after culturing for 72 hours at 65 ℃. As can be seen, the strain SEM-14 has high temperature resistance. In view of the cellulase and high temperature resistant properties of the strain SEM-14, which have high enzyme activity secretion compared to the prior art, the strain SEM-14 is deposited to the Guangdong province microorganism strain collection, the deposit number is preferably GDMCC No:61765.

in the present invention, the method for the expanded culture of the strain SEM-14 preferably comprises the steps of:

the strain SEM-14 is inoculated into enrichment medium and cultured at 28-32 ℃.

In the present invention, the enrichment medium preferably comprises the following components: 5g of cellulose (straw powder and filtered pulp), 5g of peptone, 2g of calcium carbonate, 5g of sodium chloride, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate and 1000ml of distilled water, and the pH is natural. The temperature of the culture is preferably 30 ℃. The time of the culture is preferably 3 to 7 days, more preferably 5 days.

The invention provides a bacterial agent for degrading cellulose, and an active ingredient preferably comprises the strain SEM-14. The microbial inoculum preferably further comprises auxiliary materials. The auxiliary materials of the bacterial agent are not particularly limited, and the auxiliary materials of the bacterial agent well known in the art can be adopted. In the microbial inoculum, the viable count of the strain SEM-14 is preferably 10 ⁸ ～10 ⁹ CFU/g, more preferably5×10 ⁸ CFU/g. The preparation method of the microbial inoculum is not particularly limited, and the microbial inoculum can be prepared by the preparation method of the microbial inoculum well known in the art.

Based on the dual performances of the strain SEM-14, namely the cellulase with high enzyme activity and high temperature resistance, the invention provides the application of the strain SEM-14 or the microbial inoculum in degrading cellulose.

In the present invention, the degrading cellulose preferably includes cellulose compost fermentation or promotion of digestion and utilization of cellulose. The cellulose preferably comprises one or more of the following: agricultural waste, filter paper and microcrystalline cellulose. The agricultural waste preferably includes crop straw and the like. The temperature is preferably 35-65 ℃ during the fermentation of the cellulose compost. When cellulose compost is fermented, the inoculation amount of the strain SEM-14 is 5-15%, more preferably 10%. The method of operation of the cellulose compost fermentation is not particularly limited in the present invention, and a cellulose compost fermentation scheme well known in the art may be employed.

The invention provides an animal feed, which comprises a basic feed and one of the following components: the strain SEM-14 and the microbial inoculum.

In the invention, the strain SEM-14 or the microbial inoculum is used as gastrointestinal probiotics to improve the decomposing capability of animals on cellulose in feed, thereby improving the utilization rate of the feed. The concentration of viable bacteria of the strain SEM-14 in the feed is preferably 10 ⁸ ～10 ⁹ CFU/g, more preferably 5X 10 ⁸ CFU/g. The composition of the basic feed is not particularly limited in the present invention, and the preparation method of the basic feed known in the art may be adopted. The method of preparing the animal feed of the present invention is not particularly limited, and the preparation schemes of animal feeds containing probiotics, which are well known in the art, may be adopted.

The invention provides an application of the feed in animal cultivation.

In the present invention, the animal farming includes livestock farming and/or aquaculture. The livestock breeding comprises breeding of animals such as cattle, sheep, horses, donkeys and the like which mainly eat grass. The aquaculture preferably comprises grass carp. The feeding amount and the feeding method of the feed are in a conventional feeding mode in the field.

The following examples are provided to illustrate a Streptomyces strain SEM-14 and its derivatives and applications, but should not be construed as limiting the scope of the invention.

Example 1

Separation and identification method of Streptomyces sp SEM-14 strain

(1) Sample dilution: sampling fermented silkworm excrement compost, weighing 5g, dissolving in a triangular flask containing 45ml of sterile water, and oscillating at 150rpm to obtain uniform 10 ^-1 Diluting, sucking 1ml of the diluted solution into a test tube containing 9ml of sterile water by using a 1ml pipette, and shaking thoroughly to obtain 10 ^-2 And sequentially diluted to 10 ^-6 。

(2) Preparation of a culture medium:

cellulose screening medium: microcrystalline cellulose sodium 10.0g/L, KH ₂ PO ₄ 2.0 g/L，MgSO ₄ · 7H ₂ O 0.5g/L，(NH ₄ )2SO ₄ 4.0 g/L, naCl 0.5g/L, agar 18.0g/L, pH 7.2.

Screening and identifying the culture medium: 5g of sodium carboxymethyl cellulose (microcrystalline cellulose), 2g of yeast powder, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 20g of agar, 1000ml of distilled water and natural pH.

Enrichment medium: 5g of cellulose (straw powder and filtered pulp), 5g of peptone, 2g of calcium carbonate, 5g of sodium chloride, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 1000ml of distilled water and natural pH.

Filter paper strip disintegration medium: (NH) ₄ ) ₂ SO ₄ 1.0g/l, 7 hydrated magnesium sulfate 0.5g/l, monopotassium phosphate 1.0g/l, yeast extract 0.1g/l, filter paper strips (1 cm multiplied by 6 cm) 3 strips/triangular flask, and the pH value is natural.

(3) Separating, purifying and screening strains: respectively sucking 0.1ml of diluents with different dilution factors, uniformly coating the diluents on a cooled cellulose screening culture medium, placing the cooled cellulose screening culture medium in a 30 ℃ incubator, inversely preserving moisture and culturing for 5 days, observing and selecting a colony with good growth vigor for culturing in an enrichment culture medium, placing the colony in a filter paper strip disintegration culture medium for culturing, and taking bacteria with good filter paper separation effect as SEM-14 strain.

(4) Identification of strains

A. Morphological identification

The colony of SEM-14 strain is in the form of grey white powder, round, concentric structure, and opaque (FIG. 1).

SEM-14 mycelia were observed to be in the form of filaments (FIG. 2), and SEM-14 spores were observed to be in the form of short columns (FIG. 3).

B. 16SrRNA sequence alignment assay

Inoculating SEM-14 strain into cellulose screening culture medium according to 1% proportion, culturing in shaking table at 37 deg.C for 48 hr, centrifuging, extracting whole genome according to bacterial genome extraction kit method, using this as template, carrying out 16SrRNA gene PCR amplification, and sequencing. Blast analysis was performed on the obtained 16SrRNA gene fragment sequence (SEQ ID NO: 1) at NCBI, and it was found that the sequence was 99% or more similar to a partial fragment sequence of 16SrRNA of Streptomyces.

Conclusion of the authentication

Based on the results of 16SrRNA data alignment analysis and morphological characteristics of strain SEM-14, SEM-14 was identified as Streptomyces sp and deposited at the Guangdong province microbiological strain collection center (GDMCC) at month 7 and 1 of 2021 under accession number GDMCC No:61765. preservation address: building 5 of national institute No. 59, mitsui No. 100, guangzhou City, china, postal code 510075.

Example 2

High temperature tolerance test of Streptomyces strain SEM-14

The Streptomyces SEM-14 strain is inoculated into a cellulose screening culture medium for activation culture, single colonies are picked, inoculated into 100ml of the cellulose screening culture medium liquid culture medium (at 121 ℃ and sterilized for 20 min), and shake cultivation (with the rotation speed of 4000 rpm) is carried out at 35 ℃, 40 ℃, 50 ℃, 55 ℃ and 65 ℃ respectively, and the culture is inspected at 24 hours, 48 hours and 72 hours. If spherical mycelia are present in the medium or a large number of cells are observed under a 200X microscope, it is indicated that the strain can grow under this condition.

As a result, it was found that the strain SEM-14 of the present invention was able to normally grow hyphae under the environmental conditions of 35 ℃, 40 ℃, 50 ℃, 55 ℃ and 65 ℃ while the observation by a microscope revealed that a large amount of cells were present in the culture broth. This shows that strain SEM-14 can still grow normally under the high temperature condition of 65 ℃.

Example 3

Detection of cellulase Activity secreted by Streptomyces sp SEM-14 Strain

And (3) inoculating activated SEM-14 strain bacterial liquid into a filter paper liquid culture medium according to an inoculum size of 10% by adopting a filter paper bar culture method, culturing at 30 ℃, taking supernatant at intervals of 24 hours, and detecting the activity of the filter paper enzyme by adopting a filter paper enzyme kit manufactured by Suzhou Gray biotechnology Co.

The results are shown in Table 1.

TABLE 1 results of cellulase Activity secreted by SEM-14 Strain

The results show that the strain SEM-14 has high activity of filter paper enzyme activity in 24h,48h, 72h and 96h, and the filter paper enzyme activity in the supernatant is increased along with the increase of the co-culture time, and the strongest activity is detected in 72 h.

Comparative example 1

Two strains X5 and X6 isolated from faeces Bombycis were identified as in example 1, and the identification showed that X5 and X6 were Streptomyces sp. Two strains of bacteria IP3 and IP4 separated from silkworm excrement are identified according to the method of example 1, and the identification results show that the strains IP3 and IP4 are Bacillus sp.

Comparative example 2

The strain X5 and the strain X6 isolated in comparative example 1 and the strain IP3 and the strain IP4 were inoculated into 100ml of a cellulose screening medium liquid medium (sterilized at 121 ℃ C. For 20 minutes) by the method of example 2, and shake-cultured at 35 ℃ C., 40 ℃ C., 50 ℃ C., 55 ℃ C., and 65 ℃ C., respectively (rotational speed: 150 rpm), and the growth conditions of the strain were examined at 24 hours, 48 hours, and 72 hours, and if spherical mycelia were present in the medium, or a large amount of mycelia were observed under a 200X microscope, indicating that the strain could grow under the conditions.

The results are shown in Table 2.

TABLE 2 maximum tolerance temperatures for different strains

Strain numbering	X5	X6	IP3	IP4
					Highest withstand temperature (. Degree. C.)	60	55	55	55

As a result, it was found that the highest tolerance temperature of Streptomyces strains X5 and X6 and Bacillus species IP3 and IP4 was 55 to 60℃lower than that of Streptomyces strain SEM-14.

Comparative example 3

The filter paper enzyme activities of the strains X5, X6, IP3 and IP4 were examined by the filter paper bar culture method as described in example 3.

The results are shown in Table 3.

TABLE 3 cellulase enzymatic hydrolysis of different strains

The results show that: from 48h onwards, the enzyme activities of strains X5, X6, IP3, IP4 were significantly reduced (P < 0.05) compared to Streptomyces SEM-14. This shows that Streptomyces SEM-14 has a significant advantage over the other two strains X5, X6 belonging to Streptomyces in degrading cellulose, and is also superior to other conventional cellulase secreting strains.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> institute for processing silkworm industry and agricultural products at the national academy of agricultural sciences in Guangdong province

<120> Streptomyces strain SEM-14 and its derivative and application

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tccctcccac aaggggttgg gccaccggct tcgggtgtta ccgactttcg tgacgtgacg 60

ggcggtgtgt acaaggcccg ggaacgtatt caccgcagca atgctgatct gcgattacta 120

gcgactccga cttcatgggg tcgagttgca gaccccaatc cgaactgaga ccggcttttt 180

gagattcgct ccacctcgcg gtatcgcagc tcattgtacc ggccattgta gcacgtgtgc 240

agcccaagac ataaggggca tgatgacttg acgtcgtccc caccttcctc cgagttgacc 300

ccggcggtct cccgtgagtc cccagcacca caagggcctg ctggcaacac gggacaaggg 360

ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga cagccatgca 420

ccacctgtac accgaccaca aggggggcac tatctctaat gctttccggt gtatgtcaag 480

ccttggtaag gttcttcgcg ttgcgtcgaa ttaagccaca tgctccgccg cttgtgcggg 540

cccccgtcaa ttcctttgag ttttagcctt gcggccgtac tccccaggcg gggcacttaa 600

tgcgttagct gcggcacgga cgacgtggaa tgtcgcccac acctagtgcc caccgtttac 660

ggcgtggact accagggtat ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc 720

agtatcggcc cagagatccg ccttcgccac cggtgttcct cctgatatct gcgcatttca 780

ccgctacacc aggaattccg atctccccta ccgaactcta gcctgcccgt atcgactgca 840

gacccggggt taagccccgg gctttcacaa ccgacgtgac aagccgccta cgagctcttt 900

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gttagccggc gcttcttctg caggtaccgt cactttcgct tcttccctgc tgaaagaggt 1020

ttacaacccg aaggccgtca tccctcacgc ggcgtcgctg catcaggctt tcgcccattg 1080

tgcaatattc cccactgctg cctcccgtag gagtctgggc cgtgtctcag tcccagtgtg 1140

gccggtcgcc ctctcaggcc ggctacccgt cgtcgccttg gtgagccatt acctcaccaa 1200

caagctgata ggccgcgggc tcatcctgca ccgccggagc tttcgaaccg cttggatgcc 1260

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Claims

1. Streptomyces strainStreptomycessp.) strain SEM-14, which strain SEM-14 secretes cellulase and has deposit No. GDMCC No.: 61765.

2. a bacterial agent for degrading cellulose, characterized in that the active ingredient comprises the streptomyces strain SEM-14 according to claim 1.

3. The use of the streptomycete strain SEM-14 according to claim 1 or the microbial inoculum according to claim 2 for degrading cellulose, wherein the cellulose is filter paper, and the temperature of degrading cellulose is 50-65 ℃.

4. An animal feed comprising a basal feed and any one of the following ingredients: the streptomyces strain SEM-14 of claim 1 and the microbial agent of claim 2;

the active bacteria concentration of the streptomycete strain SEM-14 in the feed is 10 ⁸ ～10 ⁹ CFU/g。

5. Use of the animal feed of claim 4 in animal farming.

6. Use of the streptomyces strain SEM-14 according to claim 1 or the microbial inoculum according to claim 2 in compost fermentation.