AU2020404339A1 - Aeromicrobium reducing zearalenone and use therefor - Google Patents

Abstract

iJ -tP* @h (- 9J)W fI IN -Ef EP il (19) P d PIT, R. ~(10) M KTW/A V4:Y (43) d VTWO 2021/121428 A1 2021 4 6 ) 24 (24.06.2021) WIPO I PCT (51) M pj }3 T A : (72)&fplR:4 MJ1]9(XU,Jianhong); +[1T NM3V fl C12N 1/20 (2006.01) C12R 101 (2006.01) r Ex "kW50 , Jiangsu 210014 (CN) ' A23L 5/20 (2016.01) A 7 (SHI,Jianrong); + l 3P&IT NMAT1TtE (21) ) PCT/CN2020/138613 "tkW150t, Jiangsu 210014 (CN)o rtMi(HOU, Mingxuan); + @SlhENM I ZiAX " 50t, (22) M pj H: 2020 412 ] 23 H (23.12.2020) Jiangsu 210014 (CN)o IENIJ(WANG, Gang); +[ (25) $ i4ig# : IA EU,450 , Jiangsu 210014 (2) _,_j 4 (CN). o t H(HU, Junqiang); +M ilT% MA (26) /Exi 4:50t, Jiangsu 210014 (CN)o (30) fi : (74) R T A: I-'j A 4 !P l -X i (JIANGSU SUNDY 202010664401.2 2020*7A 10 H (10.07.2020) CN LAWFIRM); + l 1T$$ INGSUSUND (71) $Mi.: 5I-U t'AiIl44 pA(JIANGSU ACADEMY 684At6W, WCtr, Jiangsu 210019 (CN)o OF AGRICULTURAL SCIENCES) [CN/CN]; +l fl IT @ M V -f A A X " Q fi 50 P T Jiangsu 210014 (CN)5 gP): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, (54) Title: AEROMICROBIUM REDUCING ZEARALENONE AND USE THEREFOR =~(5 4) &fi - 02 The stainHA1I Awmhicr 5nerras im5SW-57(NRM391) Aorwomnier bia ameslshR18(A00M216)} Afrtntnwn~fm stra17YN1(It4400) Aw..lcrb-a d -ns a*i i4(LT790M68 AemlerngsenIsaind Pge196(MH2127) Nmmr n itrl ranu 161(R0 4132) emmticr~nigefaiA-72(M6415) ( ) b r tAre c g aAermbrnan aulhereftr, KSld-7(R 2t) Aesnmaen o.ngydgsnsequen iCW35EF6n374} - n livstck Ii AeWame WanIbWl WWjairemesimt[f1~ 193ilJ Aemnitoirde sirAR1 CP11fi2) 2 (57) Abstract: AnAeroicrobiumreducingzearalenoneandausetherefor,saidHA- (Aeromibrobium tamlense)strainhavingan accession number of: CGMCC No. 19892, and a16S rDNA nucleotide sequence thereof being as shown in SEQ ID NO.1. The HA Sstrain may be used in reducing zearalenone. In aprocess of resolving toxin contamination, reduction is total, with no secondary conta 1mination, thereby improving the quality level and safety level of agricultural products and feedstuff, ensuring food safety for humans Sand livestock. S(57)IjK: -$d it ¶MI § #4tflM J94Q 2 % &L & , it HA-1(Aeromicrobium tamense) 2$t (g Cl § NJ CGMCC No.19892, A 16S rDNAQ B & r JH SEQ ID NO. fI ; HA-1 I M - PT T C £( t4~ Wfl +, A P A ntUTV+ 4A j rr, T 4- A iX$J %k 1' f ) tFZ¼V r11 W O 2021/2 12 1 428 A |l1||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, Fl, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, IT, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZWc (84)4~~~>~ p AT-~ M': ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), kil (AM, AZ, BY, KG, KZ, RU, TJ, TM), [III (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). 4RAis2I]J4. 17(FR: - t1ts$iEA t91t, itL#21 (2) (a) fTft 1 l'Vf)$$4fR 99 1)111113,1 -. 4(d) (i) fH 48.2 (a) (viii)). - §tMIk ])l 23 (1)]5. 2 (a))

Description

Aeromicrobium Reducing Zearalenone and Use therefor

Technical Field

[0001] The disclosure herein relates to the field of Aeromicrobium, in particular to

an Aeromicrobium for degrading zearalenone and an application thereof.

Background

[0002] Zearalenone (ZEN) was separated from corn polluted by Fusarium

graminearum in 1962 by Stob, et al., also known as F-2 toxin. Its chemical structure

belongs to a dihydroxybenzoic acid lactone compound, and its chemical name is

6-(10-hydroxy-6-oxyhydrogen-undecyl)-3-resorcylic acid lactolide. The pure product

of ZEN is a crystalline substance with a molecular formula of C 18 H 22 05 , a molecular

weight of 318, a melting point of 165°C, and good thermal stability. ZEN is insoluble

in water, but is soluble in organic solvents such as alkaline aqueous solution, ethyl

acetate, acetonitrile, methanol, ether, benzene and chloroform. Its ester bond may

be opened in an alkaline environment and recovered when the concentration of

alkali decreases. The maximum ultraviolet spectral absorption of ZEN is 236nm and

the maximum infrared spectral absorption is 970nm.

[0003] ZEN is a secondary metabolite of Fusarium graminearum, which widely

exists in grains and feeds. It does great harm to health after being ingested by

humans and animals. The structure of ZEN and its derivatives is similar to that of

estrogen, which may bind to estrogen receptor (ER), cause reproductive system disorder and have reproductive toxicity. In addition, it also has cytotoxicity, immunotoxicity, genotoxicity, organ toxicity and carcinogenicity. Many studies have proved that ZEN has reproductive toxicity to mice, pigs and other livestock, mainly manifested in estrogen effect, including decreased fertility, increased embryo mortality, reduced litter size, and changes in content of hormone in adrenal gland, thyroid gland, pituitary gland and blood. ZEN has immunotoxicity. High-dose ZEN may cause changes in a series of immune parameters, such as inhibiting the proliferation of lymphocytes and increasing the expression of interleukin-2 (IL-2) and interleukin-5 (IL-5). ZEN may cause cell damage and has cytotoxicity. Ma Yongjiang et al. have found that ZEN may significantly inhibit the activation of mouse spleen lymphocytes cultured in vitro and cause the apoptosis of the cells. ZEN has genotoxicity, mainly manifested in causing chromosome variation, damaging DNA and RNA templates, and thus causing gene mutation. ZEN organotoxicity is mainly manifested in action of ZEN mainly on the liver in the metabolic process, which has a strong toxic effect on hepatocytes and liver. ZEN also has carcinogenicity. Data show that ZEN may increase the incidence rate of brain cancer, liver cancer, breast cancer, esophageal cancer and other diseases. The toxicity mechanism of ZEN mainly includes the following four aspects: 1) many studies have shown that ZEN has estrogen like activity; 2) ZEN may act on the regulatory mechanism of cell membrane, leading to cell apoptosis and tissue necrosis; 3) the metabolites of ZEN may increase the production of MDA, the end product of lipid peroxidation; 4) ZEN may cause sister chromatid exchange and chromosome aberration, and induce the production of polyploidy.

[0004] According to the statistics of the Food and Agriculture Organization (FAO) of

the United Nations, about 25% of the world's agricultural products have been

polluted by mycotoxin, which has caused great safety hazards to livestock and

human health. Cheng Chuanmin et al. performed a ZEN test on 1655 feed samples

from 19 provinces in China and found that the pollution situations were different for

different types of feeds. Among them, the concentrate supplement of dairy cows in

lactation period had the highest detection rate of ZEN, as high as 92.9%. Rong

Xiaoping et al. collected 179 samples from Beijing, Shandong, Henan, Sichuan and

other regions for analysis. The results showed that the detection rate of ZEN was

more than 90%, and the over-standard rate was 23.21%, of which the highest

content was 3387 P g/kg. A series of researches and reports on ZEN pollution have

also been carried out abroad. Rodrigues et al. reported that the pollution rates of

ZEN in corn and wheat samples in the Middle East and Africa were 16% and 25%

respectively, of which the highest content of ZEN reached 310 p g/kg and 392 p g/kg

respectively. In Europe, many grains such as wheat, corn, soybean and rice were also

reported to be polluted by ZEN, and ZEN pollution existed more or less in different

countries. There were also different degrees of ZEN pollution in the Americas, in

which the detection rates of ZEN in corn and DDGS of the United States were 42%

and 71% respectively.

[0005] In order to reduce the harm of ZEN, in addition to strengthening the

prevention and supervision measures of grain mildew, the research on ZEN

detoxification methods is also essential. Any detoxification measure for polluted

feeds should follow the following principles: 1) it may effectively remove and destroy

mycotoxin; 2) it does not produce toxic residues or carcinogenic and mutagenic

residues in the treated products or feeds after feeding treatment; 3) it does not

change the nutritional characteristics and palatability of feed; 4) it is economically

and technically feasible and does not significantly affect the cost of the end product.

At present, common ZEN detoxification methods include physical detoxification

methods, chemical detoxification methods and biological detoxification methods.

Physical detoxification methods mainly include heat treatment, grinding, irradiation

and physical adsorption. At present, adding adsorbents to feeds is the most

commonly used method to solve mycotoxin pollution. Mycotoxin adsorbents may

combine toxins in vivo or in vitro to form complexes, so that the toxins will not be

absorbed when passing through the digestive tract and will be discharged directly

with the adsorbents. However, because the toxins are only adsorbed on the surfaces

of the adsorbents and do not degrade, it may cause secondary pollution to the

environment. Chemical detoxification mainly uses chemical reagents such as alkali,

oxidant and organic solvent to react with ZEN, so as to change its chemical structure

and achieve the purpose ofdetoxification. Chemical detoxification method can

produce significant detoxification effect on ZEN polluted food and feeds, and has the

characteristics of rapid effect. However, due to the certain toxicity of chemical reagents themselves, it is possible to change the properties of food and feeds, the cost is relatively high, and it is easy to cause secondary pollution to the environment, so it is not suitable for practical production. Thedetoxification of mycotoxin by adopting biological methods is a research hotspot at present, mainly including microbial adsorption, microbial degradation and biological enzymes having degradation effect for mycotoxin by cloning and expression. Biological adsorption method uses the special structure of cell walls to adsorb toxins to achieve the purpose of mycotoxin detoxification, but this method is reversible and the toxins have not been transformed or disappeared. Biological degradation refers to the use of extracellular and intracellular enzymes and some secondary metabolites secreted by microorganisms to decompose mycotoxin into non-toxic and harmless substances.

Biodegradable method has the advantages of low cost, high efficiency, strong

specificity, not destroying other components in food and feeds, and not producing

toxic and harmful degradation products, which is currently the most effective and

feasible method for ZEN detoxification.

[0006] The existence of ZEN will cause serious damage to the economy, and has

many toxic effects such as reproductive toxicity, cytotoxicity and immunotoxicity,

which seriously affect the health of animals and humans. Therefore, it is very

necessary to find a safe and efficient way to remove ZEN.

Summary

[0007] The purpose of the disclosure is to develop an Aeromicrobium for degrading

zearalenone and an application thereof, aiming at solving the problem of wide

pollution caused by zearalenone toxin in the process of grain production, feed

processing and preservation.

[0008] In order to realize the purpose, the disclosure provides the following

technical solution:

[0009] The disclosure provides a strain HA-1, with a Latin name of Aeromicrobium

tamlense, preserved in China General Microbiological Culture Collection Center,

address: Institute of Microbiology, Chinese Academy of Sciences, 3#, No.1 Courtyard,

West Beichen Road, Chaoyang District, Beijing 100101, from May 29, 2020, with

preservation number CGMCC No. 19892.

[0010] Preferably, a 16S rDNA nucleotide sequence of the strain HA-1 is as shown

in SEQ ID NO.1.

[0011] An example of the disclosure further provides an application of the strain

HA-1 to degradation of zearalenone.

[0012] Preferably, an example of the disclosure provides a method for applying the

strain HA-1 to degradation of zearalenone in grains or feeds, and the grains include

at least one of corn, wheat, barley, rice, sorghum and millet.

[0013] In the present application, the term "grains" refers to gramineous gain crops

and their seeds, including corn, wheat, barley, rice, sorghum, millet and other

miscellaneous grains, such as sorghum, wild rice, oat and myotonin, the nutrients contained in which mainly include sugars, mainly starch, followed by protein. The term "feeds" refers to substances that can provide the nutritional needs of livestock without harmful phenomena under reasonable feeding.

[0014] An example of the disclosure provides a bacterial agent for degrading

zearalenone, wherein the bacterial agent includes the strain HA-1 with preservation

number CGMCC No. 19892; the bacterial agent is a liquid bacterial agent.

[0015] Preferably, in an example of the disclosure, the concentration of the strain

HA-1 in the liquid bacterial agent is 109-10 bacteria/ml.

[0016] Preferably, in an example of the disclosure, the inoculation amount of the

liquid bacterial agent is 107-8 bacteria/g.

[0017] An example of the disclosure further provides another bacterial agent for

degrading zearalenone, wherein the bacterial agent is a solid bacterial agent

prepared by mixing the liquid bacterial agent and an adsorbent according to a

volume-mass ratio of 1 mL:(0.5-2)g.

[0018] Preferably, in an example of the disclosure, the adsorbent is a mixture

consisting of montmorillonite and yeast cell wall, and in the mixture, the mass ratio

of montmorillonite to yeast cell wall is 4:(0.5-1.5).

[0019] Preferably, in an example of the disclosure, the inoculation amount of the

solid bacterial agent is 3-10 wt%.

[0020] The example of the disclosure has the following advantages: zearalenone is

degraded by Aeromicrobium tamlense HA-1 and is completely degraded in the

process of solving toxin pollution without causing secondary pollution, the quality and safety level of agricultural products and feeds are improved, and the edible safety of human and livestock is ensured; the use of Aeromicrobium tamlense HA-1 strain to produce various preparations for degrading zearalenone has the characteristics of low production cost and safe use.

Brief Description of Figures

[0021] FIG. 1 illustrates a liquid chromatogram of ZEN degradation by

Aeromicrobium tamlense HA-1.

[0022] FIG. 2 illustrates a 16S rDNA phylogenetic tree of Aeromicrobium tamlense

HA-1.

[0023] FIG. 3 illustrates morphology of Aeromicrobium tamlense HA-1 under a

microscope.

Description of Preservation

10024] An Aeromicrobium tamlense HA-1 is preserved in China General

Microbiological Culture Collection Center at Institute of Microbiology, Chinese

Academy of Sciences, 3#, No.1 Courtyard, West Beichen Road, Chaoyang District,

Beijing, from May 29, 2020, with preservation number CGMCC No. 19892.

Detailed Description

[0025] An example of the disclosure provides an Aeromicrobium tamlense HA-1,

which has an effect of degrading zearalenone.

[0026] An example of the disclosure provides an Aeromicrobium tamlense HA-1,

which is preserved in China General Microbiological Culture Collection Center at

Institute of Microbiology, Chinese Academy of Sciences, 3#, No.1 Courtyard, West

Beichen Road, Chaoyang District, Beijing, from May 29, 2020, with preservation

number CGMCC No. 19892.

[0027] In an example of the disclosure, a nucleotide sequence of the

Aeromicrobium tamlense HA-1 is as shown in SEQ ID NO.1.

[0028] An example of the disclosure further provides an application of the

Aeromicrobium tamlense HA-1 to degradation of zearalenone.

[0029] In an example of the disclosure, a method for applying the Aeromicrobium

tamlense HA-1 to degradation of zearalenone in grains or feeds is provided.

[0030] In the disclosure, the grains preferably include at least one of corn, wheat,

barley, rice, sorghum and millet.

[0031] An example of the disclosure provides a bacterial agent for degrading

zearalenone, the bacterial agent includes the Aeromicrobium tamlense HA-1, and the

bacterial agent is a liquid bacterial agent.

[0032] In an example of the disclosure, a method for preparing the liquid bacterial

agent preferably includes the following steps:

[0033] a) activation: activating a preserved Aeromicrobium tamlense HA-1 original

seed on an NB culture medium (its formula is the same as that of seed culture

medium), and determining its degradation performance of zearalenone;

[0034] b) primary extended culture: inoculating the activated Aeromicrobium

tamlense HA-1 into a triangular conical flask containing a seed culture medium, and

performing shaking culture till a logarithmic period to obtain the strain;

[0035] c) secondary extended culture: inoculating the strain into a seed tank

containing a seed culture medium, performing culture till a logarithmic growth

period to obtain Aeromicrobium tamlense HA-1 seed solution;

[0036] d) Tertiary extended culture: inoculating the obtained Aeromicrobium

tamlense HA-1 seed solution into a production tank containing a fermentation

culture medium for fermentation culture, and an HA-1liquid bacterial agent capable

of degrading zearalenone is formed when the solution is removd out of the tank.

[0037] In the disclosure, the seed culture medium preferably consists of the

following components by mass-volume ratio (g/L): 1.0% of peptone, 0.3% of beef

extract, 0.5% of NaCl, and balance of water; the pH is preferably 7.0-7.2.

[0038] In the disclosure, the seed culture medium is preferably cooled to 25-35°C

after high-pressure moist heat sterilization at 1210 C, and is further preferably cooled

to 30° C for standby.

[0039] In the disclosure, preferably the strain after primary extended culture is

inoculated into the seed tank for secondary extended culture, and the inoculation

amount is 3-10% (v/v), preferably 8% (v/v).

[0040] In the disclosure, the conditions for the secondary extended culture are

preferably as follows: the ventilation amount of sterile air is preferably 1:0.4-0.8 (v/v

iM), further preferably 1:0.6 (v/v m); the stirring speed is preferably 100-160 rpm,

further preferably 120-140 rpm, and further preferably 130 rpm; the culture

temperature is preferably 30-32°C, further preferably 31°C; the culture time is

preferably 60-72 hours, further preferably 63-68 hours, and further preferably 66

hours.

[0041] In the disclosure, the fermentation culture medium preferably consists of

the following components by mass-volume ratio (g/L): 1.0% of glucose, 0.5% of

soybean cake powder, 0.2% of K2 HPO 4, 0.02% of MgSO 4 , 0.01% of NaCl, 0.5% of

CaCO 3, and balance of water; the pH of the fermentation culture medium is

preferably 7.0-7.2.

[0042] In the disclosure, preferably the Aeromicrobium tamlense HA-1 seed

solution after secondary extended culture is inoculated into the production tank for

tertiary extended culture, and the inoculation amount is 3-10%, preferably 8% (v/v).

[0043] In the disclosure, the conditions of the tertiary extended culture are

preferably as follows: the ventilation amount of sterile air is preferably 1:0.4-0.8 (v/v

•M), further preferably 1:0.6 (v/v m); the stirring speed is preferably 100-160 rpm, further preferably 120-140 rpm, and further preferably 130 rpm; the culture temperature is preferably 30-32°C, further preferably 31°C; the culture time is preferably 60-72 hours, further preferably 63-68 hours, and further preferably 66 hours.

[0044] In the disclosure, after the finish of tertiary extended culture, the liquid

bacterial agent is obtained.

[0045] In the disclosure, the concentration of Aeromicrobium tamlense HA-1 in the

liquid bacterial agent is preferably 109-10 bacteria/ml, further preferably 5 X 109

bacteria/ml.

[0046] In the disclosure, when using the liquid bacterial agent, the liquid bacterial

agent is uniformly mixed into grains or feeds to degrade zearalenone.

[0047] In the disclosure, the inoculation amount of the liquid bacterial agent is

preferably 107-" bacteria/g, further preferably 5 X 107 bacteria/g.

[0048] An example of the disclosure further provides another bacterial agent for

degrading zearalenone. The bacterial agent is preferably a solid bacterial agent

prepared by mixing the liquid bacterial agent and an adsorbent.

[0049] In an example of the disclosure, the volume-mass ratio of the liquid

bacterial agent to the adsorbent is preferably 1 mL:(0.5-2) g, further preferably 1

mL:(0.8-1.5) g, and further preferably 1 mL:1 g.

[0050] In an example of the disclosure, the adsorbent is preferably a mixture of

montmorillonite and yeast cell wall.

[0051] In an example of the disclosure, the mass ratio of montmorillonite to yeast

cell wall is preferably 4:(0.5-1.5), further preferably 4:1.

[0052] In the disclosure, when the solid bacterial agent is used, the solid bacterial

agent is mixed with grains or feeds to be treated, and then distilled water is added

according to the solid-liquid ratio of 1:1 (mass ratio) to degrade zearalenone.

[0053] In the disclosure, the inoculation amount of the solid bacterial agent is

preferably 3-10 wt%, further preferably 5 wt%.

[0054] The technical solutions provided by the disclosure will be described in detail

below in combination with the examples, which should not be understood as limiting

the scope of protection of the disclosure.

[0055] Example 1: Obtaining ofAeromicrobium tamlense HA-1

[0056] Soil samples were collected from wheat fields infected with wheat scab for a

long time (the collection place is Liuhe District, Nanjing, Jiangsu). 5 g of the collected

soil samples were weighed and placed in a 250 mL triangular conical flask. 95 mL of

PBS buffer were added and shaken in a shaking table at 3 0 ° C and 180 rpm for 30 min.

After 30 min, the triangular conical flask was taken out and stood until the solid and

liquid were layered, 1 mL of supernatant was sucked with a pipette gun and

transferred to a basic culture medium containing 10mg/L of ZEN, the basic culture

medium was placed in a shaking table at 300 C and 180 r/min for shaking culture for

d, the content of ZEN in the culture medium was detected by HPLC, the culture

medium with reduced ZEN was transferred to the basic culture medium containing

mg/L of ZEN again, culture was continuously performed for 5 d, and then the degradation effect of ZEN was detected. The enrichment solution with stable ZEN degradation effect was diluted and coated on LB solid plates according to the concentration gradient of 10-4-10-9, and constant-temperature culture was performed at 30°C for 3 d. Single colonies on the plates were picked to measure the degradation ability of ZEN one by one, and finally the HA-1 strain capable of degrading ZEN was obtained through screening. See FIG. 1 for the degradation chromatogram of ZEN by HA-1.

[0057] Example 2: Strain identification of Aeromicrobium tamlense HA-1

[0058] The colony and cell morphology observation and physiological and

biochemical identification results of the strain HA-1 obtained in example 1 were as

follows: the colony was light yellow and round, had regular edges and smooth and

wet surfaces, was sticky, easy to pick up, was Gram-positive, had an irregular rod

shape under a microscope, had no spore and motility, was positive under contact

enzyme test, glucose oxidation and fermentation test, catalase test, gelatin

hydrolysis test and lipase test were positive, was negative under oxidase test, nitrate

reduction test and methyl red reaction test, and were capable of using citrate,

propionic acid, mannose and succinate.

[0059] The 16S rDNA sequence of HA-1 was cloned and sequenced. BLAST

comparison was performed to the sequencing results in GenBank to determine the

phylogenetic and evolutionary status of HA-1. The phylogenetic tree was shown in

FIG. 2. HA-1 was finally identified as Aeromicrobium tamlense. The morphology of

Aeromicrobium tamlense HA-1 strain was shown in FIG. 3. The 16S rDNA sequence of

Aeromicrobium tamlense HA-1 was shown in SEQ ID No.1.

[0060] Example 3: Preparation of Aeromicrobium tamlense HA-1 liquid bacterial

agent

[0061] The preserved Aeromicrobium tamlense HA-1 strain was activated on an NB

culture medium, and its biodegradability to zearalenone was determined; the

activated Aeromicrobium tamlense HA-1 was inoculated into a conical flask

containing a seed culture medium (the seed culture medium consisted of the

following components by mass-volume ratio (g/L): 1.0% of peptone, 0.3% of beef

extract and 0.5% of NaCl, had pH of 7.0-7.2, was subjected to high-pressure moisture

heat sterilization at 1210C and was cooled to 300 C for standby). The controlled

culture conditions were as follows: sterile air was fed according to the ventilation

ratio of 1:0.6 (v/v • m), the stirring speed was 130 rpm, the culture temperature

was 30-32° C, the culture time was 66 hours, and shaking culture was performed till a

logarithmic period to obtain a strain; the strain was inoculated into a seed tank

containing a seed culture medium according to the inoculation amount of 8% (v/v),

and culture was performed till a logarithmic growth period to obtain Aeromicrobium

tamlense HA-1 seed solution; according to the inoculation amount of 8% (v/v), the

obtained Aeromicrobium tamlense HA-1 seed solution was inoculated into a

production tank containing a fermentation culture medium (the fermentation

medium preferably consisted of the following components by mass-volume ratio

(g/L): 1.0% of glucose, 0.5% of soybean cake powder, 0.2% of K 2 HPO 4, 0.02% of

MgSO4 , 0.01% of NaCl and 0.5% of CaCO 3, and the pH of the fermentation culture

medium was preferably 7.0-7.2) for fermentation culture. The culture conditions

were the same as those for the preparation of Aeromicrobium tamlense HA-1 seed

solution. When the concentration of Aeromicrobium tamlense HA-1 in the

fermentation solution was detected as 5 X 10 9/ml, an HA-1 liquid bacterial agent

capable of degrading zearalenone was formed when the solution was removed out

of the tank.

[0062] The above ZEN toxin detection method was a conventional method in the

art. The specific detection steps were also shown in the content disclosed in the

document "Determination of seventeen mycotoxins in barley and malt in the Czech

Republic. Food Control, 2015(47):108-113".

[0063] Example 4: Preparation of Aeromicrobium tamlense HA-1 solid bacterial

agent

[0064] The liquid bacterial agent prepared in example 3 was mixed with an

adsorbent according to a volume-mass ratio of 1 mL:1 g to obtain a solid bacterial

agent. The adsorbent in this example is an adsorbent prepared by mixing

montmorillonite and yeast cell wall according to a mass ratio of 4:1.

[0065] Experimental example 1: Degradation of ZEN in corn powder by

Aeromicrobium tamlense HA-1liquid bacterial agent

[0066] Corn inoculated with Fusarium graminearum and harvested in the

experimental base of Jiangsu Academy of Agricultural Sciences was roasted at 70°C

for 24 hours and then crushed into powder. The powder was divided into two groups

(one group for control and the other group for experiment), each group had three

repetitions. The weight of the powder in each repetition was 50 g. The powder was

respectively placed into 250 mL conical flasks. 2.5 mL of HA-1 liquid bacterial agent

(prepared in example 3) were added to each conical flask for processing samples, 2.5

mL of NB culture medium were respectively to the other three control conical flasks,

mL of deionized water were added to each conical flask, uniform mixing was

performed, and then the mixture was placed in a shaking table at 30°C for culture

for 24 hours, that is completing the degradation of ZEN toxins in the samples.

[0067] The degradation rate of ZEN in each group was detected according to the

following steps: 2.0 g of each treated sample were respectively weighed and placed

into a 50 mL centrifuge tube, 20 mL of extract (80% of acetonitrile-0.1% of formic

acid water) were added, the centrifuge tube was placed in a shaker for shaking for

min, a salt bag (2.0 g of FeSO 4 , 1.0 g of NaCl, and 1.0 g of Na 3 CH 5 O 7 • 2H 2 0) was

added, manual shaking was performed for 2 min, centrifugation was performed for

min at 10000 rpm, 2 mL of supernatant were taken and placed into a test tube,

blow drying was performed by using a nitrogen blower, 1 mL of 50% acetonitrile

water was added for re-dissolution, the solution was enabled to pass through a 0.22

im filter membrane, then the solution was placed into a liquid vial, and the content

of ZEN in the sample was detected by LC-MS. The test results are as shown in Table 1.

From the results, it can be seen that the HA-1liquid bacterial agent can well degrade

ZEN in corn powder, and the degradation rate can reach 98.19%.

[0068] Degradation rate (%)= 100 (average concentration of control group-average

concentration of experimental group)/average concentration of control group

[0069] Table 1 Degradation effect of HA-1 liquid bacterial agent on ZEN in corn

powder

Control (ZEN Experiment (ZEN Degradation rate Treatment concentration concentration (%) ug/kg) ug/kg)

1 1495.78 9.29

2 1391.12 41.39

3 1487.23 28.32

Average 1458.04 26.33 98.19

[0070] Experimental example 2: Degradation of ZEN in corn powder by

Aeromicrobium tamlense HA-1 solid bacterial agent

[0071] Corn inoculated with Fusarium graminearum (preserved in the laboratory of

Jiangsu Academy of Agricultural Sciences) and harvested in the experimental base of

Jiangsu Academy of Agricultural Sciences was roasted at 70°C for 24 hours and then

crushed into powder. The powder was divided into two groups (one group for

control and the other group for experiment), each group had three repetitions, The

weight of the powder in each repetition was 50 g. The powder was respectively

placed into 250 mL conical flasks. 2.5 g of HA-1 solid bacterial agent (prepared in

example 4) were added to each conical flask for processing samples, 2.5 g of mixture

of montmorillonite and yeast cell wall (the mass ratio of montmorillonite to yeast cell wall was 4:1) were respectively added to the other three control conical flasks, mL of deionized water were added to each conical flask, uniform mixing was performed, then the mixture was placed in a shaking table at 30°C for culture for 24 hours, 2.0 g of each sample were respectively weighed and placed into a 50 mL centrifuge tube, 20 mL of extract (80% of acetonitrile-0.1% of formic acid water) were added, the centrifuge tube was placed in a shaker for shaking for 30 min, a salt bag (2.0 g of FeSO 4 , 1.0 g of NaCl, and 1.0 g of Na 3 C6 H5 O 7 • 2H2 0) was added, manual shaking was performed for 2 min, centrifugation was performed for 5 min at 10000 rpm, 2 mL of supernatant were taken and placed into a test tube, blow drying was performed by using a nitrogen blower, 1 mL of 50% acetonitrile water was added for re-dissolution, the solution was enabled to pass through a 0.22 Im filter membrane, then the solution was placed into a liquid vial, and the content of ZEN in the sample was detected by LC-MS. The test results are as shown in Table 2. From the results, it can be seen that the HA-1 solid bacterial agent can well degrade ZEN in corn powder, and the degradation rate can reach 94.58%.

[0072] Table 2 Degradation effect of HA-1 solid bacterial agent on ZEN in corn

powder

Control (ZEN Experiment (ZEN Degradation rate Treatment concentration concentration ug/kg) (%) ug/kg)

1 1234.12 78.76

2 1013.24 47.31

3 1165.34 58.91

Average 1137.57 61.66 94.58

[0073] Experimental example 3: Degradation of ZEN in feed by Aeromicrobium

tamlense HA-1liquid bacterial agent

[0074] Pig feeds produced by three different companies in the market (pig feeds in

treatments 1-3 were respectively purchased from Zhengda, New Hope Liuhe and

Dabeinong, and their main components were rice bran, wheat bran, rapeseed cake,

peanut cake, distiller's grains, bran dreg, corn, wheat, fish meal, bone meal, etc.)

were purchased, the content of ZEN in the feeds was detected, samples polluted by

ZEN were selected and divided into two groups (one group for control and the other

group for experiment). Each group had three repetitions. The weight in each

repetition was 50 g. The feeds were respectively placed into 250 mL conical flasks.

2.5 mL of HA-1 liquid bacterial agent (prepared in example 3) were added to each

conical flask for processing samples, 2.5 mL of NB culture medium were respectively

to the other three control conical flasks, 50 mL of deionized water were added to

each conical flask, uniform mixing was performed, and then the mixture was placed

in a shaking table at 300 C for culture for 24 hours, 2.0 g of each sample were

respectively weighed and placed into a 50 mL centrifuge tube, 20 mL of extract (80%

of acetonitrile-0.1% of formic acid water) were added, the centrifuge tube was

placed in a shaker for shaking for 30 min, a salt bag (2.0 g of FeSO 4 , 1.0 g of NaCl, and

1.0 g of Na 3 C6 H5 O 7 • 2H 2 0) was added, manual shaking was performed for 2 min, centrifugation was performed for 5 min at 10000 rpm, 2 mL of supernatant were taken and placed into a test tube, blow drying was performed by using a nitrogen blower, 1 mL of 50% acetonitrile water was added for re-dissolution, the solution was enabled to pass through a 0.22pm filter membrane, then the solution was placed into a liquid vial, and the content of ZEN in the sample was detected by LC-MS.

The test results are as shown in Table 3. From the results, it can be seen that the

HA-1liquid bacterial agent can well degrade ZEN in feeds, and the degradation rate

can reach 89.37%.

[0075] Table 3 Degradation effect of HA-1liquid bacterial agent on ZEN in feed

Control (ZEN Experiment (ZEN Degradation rate Treatment concentration concentration (%) ug/kg) ug/kg)

1 367.12 29.98

2 298.32 37.34

3 312.76 36.65

Average 326.07 34.66 89.37

[0076] Experimental example 4: Degradation of ZEN in feed by Aeromicrobium

tamlense HA-1 solid bacterial agent

[0077] Pig feeds produced by three different companies in the market (pig feeds in

treatments 1-3 were respectively purchased from Zhengda, New Hope Liuhe and

Dabeinong, and their main components were rice bran, wheat bran, rapeseed cake, peanut cake, distiller's grains, bran dreg, corn, wheat, fish meal, bone meal, etc.) were purchased, the content of ZEN in the feeds was detected, samples polluted by

ZEN were selected and divided into two groups (one group for control and the other

group for experiment). Each group had three repetitions. The weight in each

repetition was 50 g. The feeds were respectively placed into 250 mL conical flasks.

2.5 g of HA-1 solid bacterial agent (prepared in example 4) were added to each

conical flask for processing samples, 2.5 g of mixture of montmorillonite and yeast

cell wall mixed according to a mass ratio of 4:1 were respectively added to the other

three control conical flasks, 50 mL of deionized water were added to each conical

flask, uniform mixing was performed, then the mixture was placed in a shaking table

at 300 C for culture for 24 hours, 2.0 g of each sample after being crushed were

respectively weighed and placed into a 50 mL centrifuge tube, 20 mL of extract (80%

of acetonitrile-0.1% of formic acid water) were added, the centrifuge tube was

placed in a shaker for shaking for 30 min, a salt bag (2.0 g of FeSO 4 , 1.0 g of NaCl, and

1.0 g of Na 3 CH 5 O 7 • 2H2 0) was added, manual shaking was performed for 2 min,

centrifugation was performed for 5 min at 10000 rpm, 2 mL of supernatant were

taken and placed into a test tube, blow drying was performed by using a nitrogen

blower, 1 mL of 50% acetonitrile water was added for re-dissolution, the solution

was enabled to pass through a 0.22pm filter membrane, then the solution was

placed into a liquid vial, and the content of ZEN in the sample was detected by LC-MS.

The test results are as shown in Table 4. From the results, it can be seen that the

HA-1 solid bacterial agent can well degrade ZEN in feeds, and the degradation rate

can reach 80.58%.

[0078] Table 4 Degradation effect of HA-1 solid bacterial agent on ZEN in feed

Control (ZEN Treatment (ZEN Degradation rate Treatment concentration concentration ug/kg) (%) ug/kg)

1 421.21 78.81

2 389.34 72.23

3 403.91 84.76

Average 404.82 78.60 80.58

[0079] It can be seen from the above examples that Aeromicrobium tamlense HA-1

has a good degradation effect on zearalenone in corn and feeds.

[0080] What are described above are only preferred examples of the disclosure. It

should be noted that those skilled in the art may make several improvements and

modifications without departing from the principle of the disclosure, which, however,

should also be regarded as included in the scope of protection of the disclosure.

Claims (10)

What is claimed is:

1. An Aeromicrobium HA-1, with Latin name Aeromicrobium tamlense,

preserved in China General Microbiological Culture Collection Center at Institute of

Microbiology, Chinese Academy of Sciences, 3#, No.1 Courtyard, West Beichen Road,

Chaoyang District, Beijing, from May 29, 2020, with preservation number CGMCC No.

19892.

2. The Aeromicrobium tamlense HA-1 according to claim 1, wherein a

nucleotide sequence of the Aeromicrobium tamlense HA-1 is as shown in SEQ ID

NO.1.

3. An application of the Aeromicrobium tamlense HA-1 according to claim 1 or 2

to degradation of zearalenone.

4. The application according to claim 3, wherein the application comprises

applying the Aeromicrobium tamlense HA-1 to degradation of zearalenone in grains

or feeds, and the grains comprise at least one of corn, wheat, barley, rice, sorghum

and millet.

5. A bacterial agent for degrading zearalenone, wherein the bacterial agent is a

liquid bacterial agent of the Aeromicrobium tamlense HA-1 according to claim 1 or 2.

6. The bacterial agent for degrading zearalenone according to claim 5, wherein

the concentration of Aeromicrobium tamlense HA-1 in the liquid bacterial agent is

109-10 bacteria/ml.

7. The bacterial agent for degrading zearalenone according to claim 5 or 6,

wherein the inoculation amount of the liquid bacterial agent is 107-8 bacteria/g.

8. A bacterial agent for degrading zearalenone, wherein the bacterial agent is a

solid bacterial agent prepared by mixing the liquid bacterial agent according to claim

or 6 and an adsorbent according to a volume-mass ratio of 1 mL:(0.5-2)g.

9. The bacterial agent for degrading zearalenone according to claim 8, wherein

the adsorbent comprise montmorillonite and yeast cell wall, and the mass ratio of

montmorillonite to yeast cell wall is 4 :(0.5-1.5).

10. The bacterial agent for degrading zearalenone according to claim 8 or 9,

wherein the inoculation amount of the solid bacterial agent is 3-10 wt%.

Fig.1 1/3

Fig.2

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