阅读说明：本技术 一种黑曲霉菌株及其在赭曲霉毒素a降解中的应用 (Aspergillus niger strain and application thereof in ochratoxin A degradation ) 是由 孙秀兰 邹东 纪剑 孙嘉笛 叶永丽 张银志 于 2021-09-15 设计创作，主要内容包括：本发明公开了一种黑曲霉菌株及其在赭曲霉毒素A降解中的应用,属于基因工程领域。本发明提供了一株黑曲霉(Aspergillus niger)FS-UV-21,已于2020年11月9日保存于中国微生物菌种保藏中心,保藏编号为CGMCC No.20751。黑曲霉(Aspergillus niger)FS-UV-21对赭曲霉毒素A具有良好的降解效果,降解率达到74.49％,较原菌株提高了18.23％；达到最佳降解效果的时间提前,缩短了18h；黑曲霉属于食品级微生物,具有较高的食品安全性,可以实现真菌毒素的生物高效降解。(The invention discloses an aspergillus niger strain and application thereof in ochratoxin A degradation, belonging to the field of genetic engineering. The invention provides an Aspergillus niger FS-UV-21 strain which is preserved in China center for culture collection of microorganisms in 11 months and 9 days in 2020, and the preservation number is CGMCC No. 20751. Aspergillus niger FS-UV-21 has good degradation effect on ochratoxin A, the degradation rate reaches 74.49%, and is improved by 18.23% compared with the original strain; the time for achieving the optimal degradation effect is advanced, and 18h is shortened; aspergillus niger belongs to food-grade microorganisms, has high food safety, and can realize the high-efficiency biological degradation of mycotoxin.)

1. A strain of Aspergillus niger (Aspergillus niger) is preserved in China general microbiological culture collection center at 11 months and 09 days in 2020 with the preservation number of CGMCC No. 20751.

2. A microbial preparation comprising the aspergillus niger according to claim 1.

3. The microbial preparation of claim 2, wherein the microbial preparation is a microorganismCharacterized in that the content of Aspergillus niger in the microbial preparation is at least 1 x 10⁶CFU/mL。

4. A product comprising the aspergillus niger according to claim 1 or the microbial preparation according to claim 2 or 3.

5. The product of claim 4, wherein the Aspergillus niger content of the product is at least 1 x 10⁶CFU/mL。

6. The product of claim 4 or 5, wherein the product is a feed additive or a chemical.

7. Use of the aspergillus niger according to claim 1, or the microbial preparation according to claim 2 or 3, or the product according to any one of claims 4 to 6 for the degradation of mycotoxins.

8. The use as claimed in claim 7 wherein the mycotoxins include one or more of aflatoxin A, aflatoxin B1, zearalenone, ochratoxin A vomitoxin.

9. A method for degrading ochratoxin A, which is characterized in that Aspergillus niger as defined in claim 1, or a microbial preparation as defined in claim 2 or 3, or a product as defined in any one of claims 4 to 6 is added to an environment containing ochratoxin A for degradation.

10. Use of the aspergillus niger according to claim 1, or the microbial preparation according to claim 2 or 3, for the preparation of a degradable mycotoxin product.

Technical Field

The invention relates to an aspergillus niger strain and application thereof in ochratoxin A degradation, belonging to the field of genetic engineering.

Background

Mycotoxins are secondary metabolites produced by fungi, widely exist in agricultural products and products thereof, and have become one of the major global food-borne risks facing human health due to serious harm to humans and animals. More than 400 fungal toxins are discovered by scientists, and although they all show different physiological and biochemical properties, the fungal toxins have the properties of toxicity, carcinogenesis, mutagenesis and the like, and the relationship between the disturbance of the immune system of human and animals and the fungal toxins is known. Currently, the most common mycotoxins comprise ochratoxin, aflatoxin, zearalenol, vomitoxin and the like, and the mycotoxins are the mycotoxins which are found to be the most harmful to grains, feed, animals and human beings. Ochratoxins, and in particular ochratoxin a (ota), are considered to be one of the five most important mycotoxins that contaminate food and feed. OTA has nephrotoxicity, hepatotoxicity, teratogenicity, immunotoxicity, and genotoxicity, etc., and has been classified as a class 2B carcinogen by the international agency for research on cancer. OTA is a great hazard and there is therefore a need to find an effective and safe technique to reduce ochratoxin levels to a threshold.

The microbial control has the advantages of safety, high efficiency, economy, strong applicability, environmental friendliness and the like, and becomes a control method which is highly advocated in recent years. Research shows that various microorganisms have a removing effect on OTA, including lactobacillus, acinetobacter, yeast, rhizopus, aspergillus and the like. The study of the weekly breeding and the like shows that the acinetobacter phenylatus and the acinetobacter calcoaceticus in the soil have the capacity of degrading OTA (see the weekly breeding, the jeffersus, the Li Wen. 2012, the study progress of the pollution, the harm and the biological detoxification technology of the aspergillus mycotoxin [ J ]. Chinese veterinary science report, 32(11):1741 and 1746); the screened Luogalus Liuzhou strain has the double degradation functions of OTA and AFB1, and the degradation strain can effectively degrade the OTA and the AFB1 under the condition of low-concentration toxin pollution (see the degradation characteristics of Luogalus Liuzhou Xanthomonas CW574 on AFB _1 and OTA and the application thereof in biological detoxification [ D ] Anhui agricultural university, 2019 ]); researches of the family bear and the like find that the Aspergillus niger M00988 strain has the effect of degrading OTA (see the family bear, Acheuma, Wangchenyi, and the like. prokaryotic expression of a carboxypeptidase gene for degrading ochratoxin A from the Aspergillus niger M00988 strain and immobilization thereof [ J ]. Chinese food science, 2019,019(005): 66-75.). The strains capable of degrading OTA are various, but the strains are good in effect and high in safety, and the strains capable of being effectively applied to toxin degradation in actual samples are few. In addition, strains that can degrade multiple mycotoxins are of greater utility than strains that degrade a single toxin. Therefore, the separation of the strain which has good effects of preventing and treating and degrading the mycotoxins such as ochratoxin and the like is of great significance.

Previous researches show that aspergillus niger has a good degradation effect on OTA toxin. However, the aspergillus niger strains currently used for degrading OTA are poor in degradation effect and require a long time. For example, in the Aspergillus niger W35 disclosed in CN 107312719B, the 48-hour degradation rate of the fermentation liquid is 56.0% at pH 6.5, but the preparation time of the fermentation liquid is longer. Moreover, the existing aspergillus niger only has a single effect, namely only can act on the mycotoxin OTA singly, and in the practical application environment, the aspergillus niger can be a mixed environment of a plurality of mycotoxins, such as: ochratoxin a, aflatoxin B1(AFB1), Zearalenone (ZEN) and vomitoxin (DON). In addition, many studies are only stopped by degradation in the medium, and there is a lack of biodegradation effect in the actual sample. Therefore, Aspergillus niger capable of efficiently degrading various mycotoxins has a good application prospect in the industry.

Disclosure of Invention

In order to solve the problem that a strain which can degrade a plurality of mycotoxins simultaneously and can be applied to the degradation of ochratoxin A in grains is lacked in the prior art, the invention provides an Aspergillus niger FS-UV-21 strain which is preserved in China center for culture collection of microorganisms in 11-9 th 2020 and has the preservation number of CGMCC No. 20751.

The Aspergillus niger FS-UV-21 is characterized in that the Aspergillus niger FS-Z1 is mutagenized in an ultraviolet irradiation mode and repeated for 5 times, 40 irradiated strains are selected, OTA degradation rate of each strain is compared, the strain with the highest degradation rate is selected for storage and identification, and the strain is determined to be Aspergillus niger by combining morphological characteristics and molecular biology identification of the strain. A phylogenetic tree based on the complete 18S rDNA sequence (SEQ ID NO. 1) was named Aspergillus niger FS-UV-21 and sent to the depository.

The Aspergillus niger FS-Z1 is described in the Chinese patent application with publication number CN 103937681B.

The preservation conditions of the Aspergillus niger are as follows: inoculating 3-ring mutagenic Aspergillus niger from solid plate culture medium PDA with good growth to fermentation culture medium, culturing at 28 deg.C with shaking table (180rpm) for 36 hr, transferring 0.45mL into glycerol tube containing 0.45mL sterile glycerol, and storing in-80 deg.C ultra-low temperature refrigerator.

The solid plate culture medium PDA is (w/v): 300g of potato, 20g of glucose, 20g of agar, 0.1g of chloramphenicol, 1L of distilled water, natural pH, and autoclaving at 121 ℃ for 15 min.

The liquid fermentation medium PDB is (w/v): 300g of potato, 20g of glucose and 1L of distilled water, and sterilizing at 121 ℃ for 15min under high pressure in natural pH. The culture conditions of the mutagenic aspergillus niger are as follows: inoculating the mutagenized Aspergillus niger of the preserved glycerin pipe into a liquid culture medium, culturing at 28 ℃ and 180rpm for 48h by a shaking table.

The invention also provides a microbial preparation containing the Aspergillus niger FS-UV-21.

In the inventionIn one embodiment, the microbial preparation comprises Aspergillus niger FS-UV-21 at a concentration of at least 1X 10⁶CFU/mL。

In one embodiment of the invention, the microbial preparation is a solid, liquid microbial inoculum or a lyophilized powder.

The invention also provides a product which contains the Aspergillus niger FS-UV-21 or contains the microbial preparation.

In one embodiment of the invention, the Aspergillus niger FS-UV-21 is present in the product in a concentration of at least 1X 10⁶CFU/mL。

In one embodiment of the invention, the product is a feed additive or a chemical.

The invention also provides the Aspergillus niger FS-UV-21, or the microbial preparation, or the application of the product in the degradation of the mycomycin.

In one embodiment of the invention, the mycotoxins include one or more of ochratoxin a, aflatoxin B1, zearalenone, vomitoxin.

The invention also provides a method for simultaneously degrading ochratoxin A (OTA), aflatoxin B1(AFB1), Zearalenone (ZEN) and vomitoxin (DON), wherein the method comprises the step of adding the Aspergillus niger FS-UV-21, the microbial preparation or the product into an environment containing aflatoxin B1, zearalenone, ochratoxin A and vomitoxin at the same time for degradation.

In one embodiment of the invention, the Aspergillus niger FS-UV-21 is added in an amount of at least: 1X 10⁶CFU/mL。

In one embodiment of the present invention, the Aspergillus niger FS-UV-21 is added to the reaction system in the form of a bacterial suspension which is inoculated into the reaction system in an amount of 2% (v/v).

In one embodiment of the present invention, the degradation conditions are: aspergillus nigerSub-concentration of 1X 10⁶CFU/mL, the addition amount is 2% (v/v), in PDB liquid medium, the content of OTA, AFB1, ZEN, DON in PDB liquid medium is 1ppm respectively, the reaction condition is: shaking and culturing at 28 deg.C and 180rpm for 48 h.

The invention also provides a method for degrading ochratoxin A, which is to add the Aspergillus niger FS-UV-21, the microbial preparation or the product into an environment containing ochratoxin A for degradation.

In one embodiment of the invention, the Aspergillus niger FS-UV-21 is added in an amount of at least: 2% (v/v).

In one embodiment of the present invention, the degradation conditions are: the concentration of Aspergillus niger spore is 1 × 10⁶CFU/mL, the addition amount is 2% (v/v), in PDB liquid culture medium, OTA content in PDB liquid culture medium is 1ppm, the reaction condition is: shaking and culturing at 28 deg.C and 180rpm for 48 h.

The invention also provides application of the Aspergillus niger FS-UV-21 or the microbial preparation in preparation of degradable mycotoxin products.

In one embodiment of the invention, the product is a chemical or feed additive.

In one embodiment of the invention, the mycotoxins are one or more of ochratoxin a (ota), aflatoxin B1(AFB1), Zearalenone (ZEN), vomitoxin (DON).

In one embodiment of the invention, the Aspergillus niger FS-UV-21 is added in an amount of at least: 1X 10⁶CFU/mL。

Advantageous effects

(1) Aspergillus niger FS-UV-21 has good degradation effect on ochratoxin A, the degradation rate reaches 74.49%, and is improved by 18.23% compared with the original strain; the time for achieving the optimal degradation effect is advanced, and the time is shortened by 18 hours.

(2) Aspergillus niger FS-UV-21 has good passaging stability.

(3) Aspergillus niger FS-UV-21 has good degradation effect in an environment simultaneously containing ochratoxin A, aflatoxin B1, zearalenone and vomitoxin, and the degradation rates are 66.09%, 74.61%, 88.29% and 37.76% respectively.

(4) Aspergillus niger FS-UV-21 is used for removing OTA toxin from wheat bran in a biological fermentation mode, and the removal rate is 59.74% at most.

(5) Aspergillus niger belongs to food-grade microorganisms, has high food safety, and can realize the high-efficiency biological degradation of mycotoxin.

Biological material preservation

An Aspergillus niger FS-UV-21 strain is classified and named as Aspergillus niger, is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 09 days 2020, has the preservation number of CGMCC No.20751, and has the preservation address of No. 3 Hospital No. 1 of Xilu, North Cheng of the sunny region in Beijing, China academy of sciences microbial research institute.

Drawings

FIG. 1: inducing Aspergillus niger to degrade OTA.

FIG. 2: a phylogenetic tree of Aspergillus niger FS-UV-21.

FIG. 3: the degradation effect of the original strains Aspergillus niger FS-Z1 and Aspergillus niger FS-UV-21 on OTA is dynamically changed.

FIG. 4: subculture stability of Aspergillus niger FS-UV 21.

FIG. 5: aspergillus niger FS-UV21 degrades the effects of multiple toxins simultaneously.

FIG. 6: aspergillus niger FS-UV21 was used to remove OTA from wheat bran.

Detailed Description

The starting strain Aspergillus niger FS-Z1 in the following examples is described in the Chinese patent publication No. CN 103937681B.

The media involved in the following examples are as follows:

PDA solid medium: 300g of potato, 20g of glucose, 20g of agar, 0.1g of chloramphenicol, 1L of distilled water, natural pH, and autoclaving at 121 ℃ for 15 min.

PDB liquid fermentation medium: 300g of potato, 20g of glucose and 1L of distilled water, and sterilizing at 121 ℃ for 15min under high pressure in natural pH.

The detection methods referred to in the following examples are as follows:

extraction of ochratoxin a (ota) in liquid medium:

and adding 3mL of trichloromethane into 1mL of liquid to be detected, fully oscillating, centrifuging at 5000rpm for 10min, taking a lower trichloromethane layer, and transferring to a new centrifuge tube. The extraction is repeated twice, the extract is subjected to vacuum centrifugation, freezing and volatilizing, 1mL of acetonitrile-2% acetic acid is added for dissolving in water, and the mixture is stored at 4 ℃ after being filtered and used for high performance liquid chromatography detection.

Extraction of ochratoxin A (OTA) in wheat bran:

adding 10mL of 60% acetonitrile water solution into 1g of wheat bran, performing vortex oscillation for 30s, performing ultrasonic treatment for 30min, centrifuging at 8000rpm for 10min, taking supernatant, performing vacuum centrifugation, freezing and volatilizing, adding 1mL of acetonitrile-2% acetic acid for dissolving, filtering, and storing at 4 ℃ for high performance liquid chromatography detection.

Detecting ochratoxin A (OTA) conditions by high performance liquid chromatography: the types of high performance liquid chromatography: agilent Technologies 1260 limits; a chromatographic column: c18 column, column length: 150mm, inner diameter: 4.6mm, viscosity: 3 μm; mobile phase: acetonitrile: water: acetic acid 96:102: 2; flow rate: 1.0 mL/min; detection wavelength: wavelength of excitation light: 333nm, emission wavelength: 460 nm.

Extraction and detection of aflatoxin B1(AFB1) in liquid media:

sucking 1ml from the solution to be detected, adding 3ml of trichloromethane, extracting twice, centrifuging in vacuum, freezing and volatilizing, redissolving with 20% acetonitrile, adding 100ul of trifluoroacetic acid and 200ul of n-hexane, derivatizing for 30min, and detecting by using a high performance liquid chromatography. The chromatographic condition is C18 column 6mm × 150mm × 5 um; mobile phase acetonitrile: water 20:80 detection temperature: 30 ℃; flow rate: 1 ml/min; detection wavelength: excitation wavelength: 360 nm; emission wavelength: 440 nm.

Extraction and detection of Zearalenone (ZEN) in liquid culture medium:

sucking 1ml from the solution to be detected, adding 3ml of trichloromethane, extracting twice, centrifuging in vacuum, freezing and volatilizing, redissolving by 50% acetonitrile, and carrying out content detection by high performance liquid chromatography. The chromatographic conditions are chromatographic column: c18 column 6mm × 150mm × 5 um; mobile phase: acetonitrile: 50:50 of water; detecting the temperature: 25 ℃; flow rate: 1 ml/min; detection wavelength: excitation wavelength: 240 nm; emission wavelength: 440 nm.

Extraction and detection of vomitoxin (DON) in liquid medium:

then sucking 1ml of the extract, adding 3ml of trichloromethane for extraction twice, carrying out vacuum centrifugation, freezing and volatilizing, re-dissolving with 20% methanol, and carrying out content detection by high performance liquid chromatography. Chromatographic conditions are as follows: the chromatographic conditions are chromatographic column: c18 column 6mm × 150mm × 5 um; mobile phase: methanol: water 20: 80; detecting the temperature: 30 ℃; flow rate: 0.8 ml/min; detection wavelength: 218 nm.

The method for calculating the degradation rate of ochratoxin A (OTA), aflatoxin B1(AFB1), Zearalenone (ZEN) and vomitoxin (DON) comprises the following steps: (1000-residual concentration)/1000X 100%.

Example 1: preparation of Aspergillus niger FS-UV-21

The method comprises the following specific steps:

carrying out mutagenesis on Aspergillus niger FS-Z1 by adopting an ultraviolet irradiation mode, repeating for 5 times, selecting 40 irradiated strains, comparing the degradation rate of each strain on OTA, and selecting the strain with the highest degradation rate for storage and identification.

The specific mutagenesis steps are as follows:

1. inoculating Aspergillus niger parent strain selected from soy sauce mash into PDA solid culture medium, culturing at 28 deg.C for 120 hr, washing spores with 50mL physiological saline, shaking to obtain monospore suspension, and making into 1 × 10⁶CFU/mL of bacterial suspension.

2. 5mL of the spore suspension is taken, irradiated by 200W ultraviolet for 30min and repeated for 5 times.

3. Selecting 40 strains subjected to mutagenesis, respectively numbering 1-40, inoculating the 40 strains into PDB solid culture medium, selecting appropriate spores from bacterial colonies, and making into 10⁶CFU/mL bacterial suspension, and mixing the bacteriaThe suspension was inoculated in 50mL of a medium containing 1ppm OTA at an inoculum size of 2% (v/v), and shake-cultured at 28 ℃ and 180rpm for 48 hours.

4. Comparing the OTA degradation effect of the original strain and the novel mutant strain (the result is shown in figure 1), selecting the strain FS-UV-21 with the highest degradation efficiency and storing.

Through colony characteristic observation and individual morphology observation, 18S rDNA (the sequence is shown as SEQ NO. ID 1) of the strain FS-UV-21 is compared with a known sequence in a GenBank database, and the homology with Aspergillus niger (Aspergillus niger) is up to 99 percent.

The strain Aspergillus niger can be determined by combining morphological characteristics and molecular biological identification of the strain. A phylogenetic tree based on its 18S rDNA full sequence is shown in FIG. 2 of the specification. As can be seen from FIG. 1, the degradation rate of the strain FS-UV-21 to OTA is 74.49% at most, so the strain is named as Aspergillus niger FS-UV-21 and sent to a preservation center for preservation.

Example 2: dynamic changes in Aspergillus niger FS-UV-21 degradation of OTA

The method comprises the following specific steps:

1. respectively inoculating Aspergillus niger FS-UV-21 and Aspergillus niger FS-Z1 into PDA solid culture medium, culturing at 28 deg.C for 120h, washing spores with 50mL of physiological saline containing 0.02% (v/v) Tween 80, and shaking uniformly to form monospore suspension, wherein the concentration of the spore suspension is as follows: 1X 10⁶CFU/mL。

2. The spore suspensions were inoculated into 50mL PDB medium containing 1ppm OTA at an inoculum size of 2% (v/v), and shake-cultured at 28 ℃ for 72 hours, and a corresponding sample was taken every 6 hours to detect the change in the concentration of OTA, as shown in Table 1 and FIG. 3.

Table 1: degradation Rate (%) of OTA at different reaction times

As can be seen from FIG. 3, the degradation efficiency is significantly improved compared to the original strain; the time for achieving the optimal degradation effect is advanced from 60 hours to 42 hours, the time is shortened by 18 hours, and the mycotoxin can be degraded more quickly and efficiently.

Example 3: passage stability of Aspergillus niger FS-UV-21

The method comprises the following specific steps:

1. inoculating Aspergillus niger FS-UV-21 into a PDA solid culture medium, and carrying out passage 5, wherein the method comprises the following steps: culturing at 28 deg.C for 7 days to obtain the first generation, transferring the first generation strain to new PDA solid culture medium, culturing at 28 deg.C for 7 days to obtain the second generation, repeating the operation to the fifth generation, and respectively detecting degradation effect.

2. The procedure is as in example 2 (concentrate 10)⁶CFU/mL, with OTA concentration of 1ppm), treating the OTA-containing solution with Aspergillus niger FS-UV-21 for 48h, and respectively detecting the degradation rate of the Aspergillus niger FS-UV-21 after each passage after the determination reaction of the OTA.

As can be seen from FIG. 4, the mutagenic Aspergillus niger has a degradation rate stabilized at about 75% after passage 5, and has good genetic stability.

Example 4: aspergillus niger FS-UV-21 can degrade ochratoxin A, aflatoxin B1, zearalenone and vomitoxin simultaneously

The method comprises the following specific steps:

1. inoculating Aspergillus niger FS-UV-21 into a PDA (potato dextrose agar) solid culture medium, culturing at 28 ℃ for 120h, taking 50mL of physiological saline containing 0.02% (v/v) Tween 80 to wash down spores, and uniformly oscillating to form a monospore suspension, wherein the concentration of the spore suspension is as follows: 1X 10⁶CFU/mL。

2. The spore suspensions are respectively taken and inoculated into 50mL PDB culture medium containing 1ppm OTA, 1ppm AFB1, 1ppm ZEN and 1ppm DON according to the inoculation amount of 2% (v/v), the culture is carried out for 42h at 28 ℃ and 180rpm by shaking table, and the degradation rates of different toxins after the reaction is finished are measured.

As can be seen from FIG. 5, the degradation rates of ochratoxin A, aflatoxin B1, zearalenone and vomitoxin are 66.09%, 74.61%, 88.29% and 37.76%, and Aspergillus niger FS-UV-21 can degrade various mycotoxins simultaneously and has a good degradation effect.

Example 5: degradation of aspergillus niger FS-UV-21 on wheat bran

The method comprises the following specific steps:

1. will be provided withInoculating Aspergillus niger FS-UV-21 into a PDA (potato dextrose agar) solid culture medium, culturing at 28 ℃ for 120h, taking 50mL of physiological saline containing 0.02% (v/v) Tween 80 to wash down spores, and uniformly oscillating to form a monospore suspension, wherein the concentration of the spore suspension is as follows: 1X 10⁶CFU/mL。

2. Treating wheat bran: pulverizing testa Tritici, sieving with 40 mesh sieve, mixing, and adding OTA standard substance to make the content of OTA in testa Tritici be 1 μ g/g.

3. Weighing 10g of the wheat bran treated in the step 2, subpackaging in 50mL triangular conical bottles, sealing with four layers of gauze newspaper, and sterilizing at 121 ℃ for 20 min.

4. Transferring the sterilized wheat bran obtained in the step 3 into a fermentation container, adding sterile water until the material-water ratio is 1:3 (the ratio of the mass of the wheat bran to the volume of the sterile water, g/mL), adding the FS-UV-21 spore suspension prepared in the step 1 according to the inoculum size of 15% of the percentage of the volume of the bacteria liquid to the mass of the wheat bran, uniformly stirring, fermenting at constant temperature of 28 ℃, sampling after fermenting for 1d, 2d, 3d, 4d and 5d respectively, detecting the residual amount of OTA and calculating the degradation rate, wherein the results are shown in Table 2 and figure 6.

Table 2: degradation rate of Aspergillus niger on OTA in wheat bran in different fermentation time

As can be seen from FIG. 6, different fermentation times have an important effect on the removal of OTA from wheat bran by Aspergillus niger, and the degradation rate of OTA increases with the increase of the fermentation time. At the time of fermentation 5d, the degradation rate was up to 59.74%.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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