Anti-bacterial Protein Extracts From Seeds of Marigold and Paprika

8/10/2019 Anti-bacterial Protein Extracts From Seeds of Marigold and Paprika

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Anti-bacterial protein extracts from seeds of marigold and paprika

United States Patent 6086885

The present invention describes the method of extraction and the effect of a

crude protein extract from the seed tissue of two botanical species, Tagetes and

apsicum, on the survival of two economicall! important gram"negative

bacteria Salmonella t!phimurium and #scherichia coli$

US Patent References:

%ethod for preparing a cell free homogenate of hr!santhemum

cinerariaefolium &Trev$' (occ$ containing en)!mes and methods of use

*ito et al$ " +une, -85 " .5/5.55

emicellulase supplement to improve the energ! efficienc! of hemicellulose"

containing animal feed

1odge et al$ " +ul!, --5 " 5./-8/8

(iocidal proteins from plants

(roe2aert et al$ " %a!, --6 " 55.33-

(iocidal proteins

(roe2ert et al$ " +ul!, --6 " 55485/5

Pesticide product derived from the plant Tagetes minuta

2ioga et al$ " September, --3 " 566/-5

nventors7

*iegenfuss, Steve &es %oines, 9'

(rin2haus, 1riedhelm &Urbandale, 9'

:reaves, +ohn &9n2en!, 9'

9pplication ;umber7

0-


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Primar! lass7

./.ent 9$

Parent ase ata7

This application claims the benefit of Provisional 9ppl$ Ser$ ;o$ 60


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collecting the second supernate?

adding amonium sulfate to 30D relative saturation as a precipitating agent

thereb! precipitating a second fraction and forming a third supernate?

dissolving the second precipitate fraction in an aCueous medium?

heating the aCueous medium, which contains the second precipitate fraction, to

80E $ thus forming a third precipitate and a third supernate?

separating the third supernate?

dial!)ing the third supernate using a 000 alton molecular weight cut off

membrane? and

collecting the dial!)ed supernate which does not pass through the membrane$

/$ The proteinaceous extract of claim wherein the gram"negative bacteria are

selected from the group consisting of #scherichia coli, Fisteria and

Salmonella$

4$ The proteinaceous extract of claim , wherein the gram"negative bacteria is

strain 05373 of #scherichia coli$

.$ The proteinaceous extract of claim , wherein the seeds are from Tagetes sp$

and the gram"negative bacteria is Salmonella t!phimurium$

5$ The proteinaccous extract of claim , wherein the gram"negative bacteria is

Fisteria monoc!togenes$

6$ The proteinaccous extract of claim , wherein the Tagetes sp$ is Tagetes

erecta$

3$ The proteinaceous extract of claim , wherein the apsicum sp is apsicumannum$

8$ 9n animal feed composition having anti"bacterial activit! comprising a feed

to which the proteinaccous extract of claim is added in an amount effective to

reduce populations of gram"negative bacteria in an animal$


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-$ 9 method of reducing a population of gram negative bacteria in an animal

comprising adding the proteinaceous extract of claim to a feed and orall!

administering the feed to the animal in an amount effective to reduce the

population of gram"negative bacteria in the animal$

0$ The method of claim -, wherein the proteinaceous extract, after free)edr!ing, is added in an amount of between 0$0 ppm and 000 ppm b! weight of

the untreated feed$

$ 9 method of reducing the bacterial load of meat comprising adding the

proteinaceous extract of claim to a feed and orall! administering the feed to

an animal to be used for meat in an amount effective to reduce the population

of gram"negative bacteria in the animal$

escription7

1#F 1 T# ;=#;T;

The present invention relates to a botanical extract of protein from marigold or

papri2a that has anti"bacterial activit!$ %ore specificall!, the present invention

relates to proteins having anti"bacterial activit! against the gram"negative

bacteria Salmonella sp$ and #scherichia coli$ The present invention describes

the method of extraction and the effect of a protein extract from the seed tissue

of Tagetes sp$ or apsicum sp$ on the survival of two economicall! important

bacteria Salmonella t!phimurium and #$ coli strain 0533$ The presentinvention includes a method for reducing bacteria in animals and animal

products, a method of extraction of the genes encoding for the subGect proteins,

and a method of use of such genes transformed and expressed in grain products

such that the resultant grain has anti"bacterial activit!$

SU%%9@H 1 T# ;=#;T;

The present invention describes the method of extraction and the effect of a

crude protein extract from the seed tissue of two botanical species on the

survival of two economicall! important gram"negative bacteria Salmonellat!phimurium and #scherichia coli$ (oth bacterial organisms are extremel!

common in animal feed and production animals and are of grave concern in

animal agriculture$ Transmitted through the food chain, both bacterial

organisms cause food poisoning in humans, and control of these organisms is a

public health issue of high priorit!$ The! also cause economic losses in

production animal agriculture through pathogenic effects on the gastrointestinal


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tracts of monogastric animals, such as poultr! and swine$ The anti"bacterial

proteins are derived from the seeds of marigold &Tagetes sp$' or papri2a

&apsicum sp$'$

There are several s!nthetic chemicals such as organic acids and formaldeh!de

which can be used to control Salmonella and #$ coli in feeds$ owever, thesechemicals are not without their own concerns on the health of animals and

humans$ 9lso, secondar! plant products contained in an essential oil fraction

have been isolated from man! botanical species, among them Tagetes sp$, and

are 2nown to exhibit strong antimicrobial as well as antifungal activities$

(roe2aert et al$, U$S$ Pat$ ;os$ 5,548,5/5 and ,5.,33- &--6' demonstrated

the use of a range of specific peptides derived from botanical species belonging

to the (rassicaceae, ompositae and Feguminosae families including

@aphanus, (rassica, Sinapis, 9rabidopsis, ahlia, nicus, Fath!rus, litoria,

9maranthus, apsicum, (ri)a and related species on several fungal organisms

and on gram"positive bacteria$ owever, the! did not describe an! effect on

gram"negative bacteria$ To our 2nowledge there are no documented examples

of natural, plant"derived proteins or peptides which have a significant biocidal

effect on gram"negative bacteria such that the! could be used to control these

organisms in a range of applications$ n addition, there are no reports on

antimicrobial or antifungal proteins characteri)ed in Tagetes$ The present

invention describes novel proteins from two botanical sources and their effect

on more important gram"negative bacteria$

The proteins extracted from the seeds of such botanical species can be used to

control Salmonella and #$ coli in animal feeds and human foods$ 1urthermore,

the proteins and the genes that code for them could be seCuenced and

manipulated via genetic engineering techniCues for expression in production

microorganisms via fermentation$ n addition, the genes could be expressed in

the seeds of economicall! important crops such as corn, so!bean, wheat and

rice for inclusion of such anti"bacterial proteins in the downstream processing

and use of these grains in animal feed and human food$ Production of proteins

via genetic engineering of microbes and plants followed b! fermentation or

agronomic production is now common place and established practice$

verproduction of secondar! plant products via pathwa! engineering, on theother hand, is still technicall! a challenge$

(@#1 #S@PT; 1 T# @9B;:S

1:$ is a graphical representation of anti"bacterial inhibition data of the

proteins of the present invention$


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#T9F# #S@PT; 1 T# ;=#;T;

The present invention relates to a crude protein extract, the purified protein&s',

and the gene&s' encoding for the protein that are extracted from marigold and

papri2a$ The protein inhibits the activit! of #$ coli &strain 0533' and possibl!

also S$ enteritidis and S$ choleraesuis$ The protein also inhibits Salmonellat!phimurium and Fisteria monoc!togenes$

The present invention is protein&s', and the gene encoding such proteins, which

inhibit the growth of the gram"negative bacteria$ These proteins are naturall!"

occurring in the seeds of marigold$ These proteins can be used in the raw form,

as a crude protein extract, and applied to animal feed or human foodstuff

&particularl! those foodstuff that are high in #$ coli or Salmonella

t!phimurium$'$ The proteins can be purified and applied to feed for animals and

foodstuff for humans$ 9lternativel!, the proteins can be applied directl! to

places in which bacteria multipl!$

The genes encoding for these proteins can be transformed into bacteria, fungi,

!easts, plants, or mammals &pro2ar!otic or eu2ar!otic cells', and the protein

can be harvested and applied directl! to the bacteria$ 9dditionall!, the proteins

can be produced within the cells of the material that carries the bacteria$ 1or

example, the proteins can be produced in the grains of material in animal foods$

n chic2en feed, the proteins can be expressed in the petals or seeds of the

marigold flower and supplied as marigold meal &that is supplied for its lutein'

which can be added as a feed additive$ 9 number of cereal grains could be

transformed so that the protein was expressed in the seed$ The transgenic corn,so!bean, canola, peanut, oat, wheat, barle!, rice, sugarbeet, cotton, or tobacco

seed can have the protein expressed therein$ Thus, when the grain is prepared

for animal feed or for human food products, the protein is released from the

seed and is active in decreasing the bacteria present in the grain or feed

product, in the animal that is fed the feed product, and in the meat of animals

fed the feed product$ The proteins can also be placed in soaps, face, and hand

cleansers, as an antimicrobial, and in surface cleansers to decrease bacterial

contamination$

Transgenic corn, so!bean, canola, peanut, oat, wheat, barle!, rice, and other

transformable fruits, vegetables, and plants can have the protein expressed in

tissue other then the seed$ Thus, when the fruit, vegetable, or other plant tissue

or forage is prepared for feeding, the bacteria are decreased$ This is particularl!

useful for the preparation of silage from corn and sorghum$


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9dditionall!, the present invention has usefulness in the pharmaceutical field

and the fields of veterinar! science$ learl!, if the bacteria are present in the

digestive s!stem, the mammal can ingest the protein to decrease the bacteria

present$ (ecause man! bacteria are in the gut, b! encapsulating the protein

material or forming the protein in material that is not bro2en down until it

reaches the gut, the protein material can be useful as a method of decreasing thebacteria in the human or animal digestive s!stem$ 9lternativel!, the protein

could be inGected directl! into the mammalsI intestines or other digestive

organs$

The crude protein extract of the present invention is run on a protein gel to

separate the proteins in the extract$ The proteins are then purified, and each

protein is tested against the listed gram"negative bacteria for the inhibition

effects$ The protein, and


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The vector for transformation would preferabl! comprise a nucleotide seCuence

that corresponds to the protein amino acid seCuence that ma! be optimi)ed for

the selected host$ The vector would also be designed with regard to codon

selection, the initiation of translation, the promoter, and the targeting

seCuences, if needed, to maximi)e the expression of recoverable amounts of the

protein$ =ectors for hosts such as plants, algae, insects, animals, !easts, fungi,bacteria, and humans are commerciall! available from companies such as

;ovagon, and a number of other sources$ =ectors for different hosts are

described in the in urrent Protocols n %olecular (iolog!$

There are a large number of 2nown methods to transform plants$ owever,

certain t!pes of plants are more amenable to transformation than are others$

Tobacco is a readil! transformable plant, and its transformation is well"

published$ %ost dicots can be transformed b! the methods used to transform

tobacco$ %onocots are transformed b! different methods which are also

widel!"published, and, thus, the basic steps of transforming plants, including

monocots, are 2nown in the art$

These steps are concisel! outlined in U$S$ Pat$ ;o$ 5,.8.,-56 A1ertile

Transgenic *ea ma!s Plants omprising eterologous ;9 #ncoding (acillus

Thuringiensis #ndotoxinA, issued +an$ 6, --6, and in U$S$ Pat$ ;o$ 5,.8-,5/0

AProcess of Producing 1ertile *ea ma!s Plants and Progen! omprising a :ene

#ncoding Phosphinothricin 9cet!l TransferaseA, issued 1eb$ 6, --6$

Plant cells, such as mai)e, can be transformed b! a number of different

techniCues$ Some of these techniCues, which have been reported on and are2nown in the art, include mai)e pollen transformation &see, U$S$ Pat$ ;o$

5,33,00, Universit! of Toledo &--4''? biolistic gun technolog! &see, U$S$

Pat$ ;o$ 5,.8.,-56'? whis2ers technolog! &see, U$S$ Pat$ ;os$ 5,.6.,365 and

5,40/,5/4'? electroporation &see, P#: on %ai)e'? 9grobacterium &see, --6

article on transformation of mai)e cells in ;ature (iotechnolog!, =olume .,

+une --6'? and numerous other methods which ma! have slightl! lower

efficienc! rates than those listed$

Some of these methods reCuire specific t!pes of cells, and other methods can be

practiced on an! number of cell t!pes$ The use of pollen, cot!ledons,

meristems, and ovum as the target tissue can eliminate the need for extensive

tissue culture wor2$ owever, the present state of the technolog! does not

provide ver! efficient use of some of this material$

:enerall!, cells derived from meristematic tissue are useful for transformation

as the! are ver! regenerable$ *!gotic embr!os can also be used$ The method of


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transformation of meristematic cells of cereal is taught in the PT application

B-6


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gunning ;9 into cells, tissue, callus, embr!os, and the li2e, are well"2nown

in the prior art$

9fter the transformation of the plant material is complete, the next step is

identif!ing the cells or material which has been transformed$ n some cases, a

screenable mar2er is emplo!ed, such as the beta"glucuronidase gene of the uidalocus of #$ coli$ Then, the transformed cells expressing the colored protein are

selected for either regeneration or further use$ n man! cases, the transformed

material is identified b! a selectable mar2er$ The putativel! transformed

material is exposed to a toxic agent at var!ing concentrations$ The cells which

are not transformed with the selectable mar2er that provides resistance to this

toxic agent die$ ells or tissues containing the resistant selectable mar2er

generall! proliferate$ t has been noted that although selectable mar2ers protect

the cells from some of the toxic affects of the herbicide or antibiotic, the cells

ma! still be slightl! affected b! the toxic agent b! having slower growth rates$

f the transformed material was cellular, then these cells are regenerated into

plants$ The plants created from either the transformation process or the

regeneration process, or crossed to either of such plants or a progen! of such

plants, are transgenic plants$

The protein of the present invention was extracted from the plant seeds via the

following process7

#J9%PF#

Preparation of Protein #xtract from %arigold and Papri2a Seeds

9Cueous extraction was followed b! ammonium sulfate precipitation in the

interval of 40D to 30D relative saturation, heat precipitation at 80E $, and

dial!sis for 4 da!s$ The detailed methods are described below$

1ive hundred grams of marigold seeds &Tagetes erecta' or /50 g of papri2a

seeds &apsicum sp$' were ground in a coffee mill and extracted for / hours in

/$0 liters &marigold' or $0 liters &papri2a' of extraction buffer at .E $ The

extraction buffer contained 0$0 m% ;a / P . , 5$0 m% ;a / P0 . , 00$0

m% >l, /$0 m% #T9 &eth!lenediaminetetraacetic acid' disodium salt, /$0m% thiourea and $0 m% P%S1 &phen!lmeth!lsulfon!l fluoride' &dissolved in

%e'$ The homogenate was sCuee)ed through cheesecloth and centrifuged

for 40 min$ at 3000K g$ The pellet was discarded, and the supernatant was

retained$ The supernatant was brought to 40D relative saturation with

ammonium sulfate and precipitated overnight$ The precipitate was removed b!

centrifugation at 3000K g for 40 minutes$ The supernatant was adGusted to 30D


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relative saturation with ammonium sulfate and precipitated overnight$ The

precipitate was collected b! centrifugation at 3000K g for 40 min$ The pellet

was re"suspended in .00$0 ml @$$ &reverse osmosis' water and heat

precipitated in a water bath for 40 minutes at 80E $ The precipitate was

collected b! centrifugation at 3000K g, and the supernatant was dial!)ed

against @$$ water for 4 da!s using cellulose ester dial!sis tubing with amolecular weight cut"off of 000 daltons &SpectralPor, Spectrum, US9'$ The

water was changed 4 times on da! , and once each da! for da!s / and 4$ The

dial!)ed supernatant was then fro)en to "60E $ and free)e"dried$

#J9%PF# /

9ntibacterial 9ctivit! 9ssa!

This method was repeated for the marigold and papri2a extracts$ 9

spectrophotometrical method was used where the optical densit! of bacterialcultures was determined when grown in micro"titer plates$

1or appropriate dilution of the protein extracts from marigold and papri2a, 0$

gram of the protein extract was blended with 0$0 grams dextrose to achieve a

homogenous, free"flowing mixture$ ne gram of the protein extract


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#xample /$ The three bacteria were tested at 4 different treatment levels, 0

ppm, 00 ppm and 000 ppm, and each treatment level was replicated / times$

9fter 8 hours incubation, growth of bacteria was measured as optical densit!

as described above$ The results, in percentage of the optical densit! of the

control as mean values for the / replicates, are presented in Table $ The

studentIs t"test was conducted to test for statistical significance with PM0$0indicating statistical significance$

T9(F#

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

nhibition of S$ t!phimurum, #$ coli and F$ monoc!togenes b! protein extracts from

marigold and papri2a as D of control Papri2a &D (acterium


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54$0 M0$0

00 ppm 48$0 M0$0

.6$0 M0$0

000 ppm 65$0 M0$0

68$0 M0$0

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

The results of the gramnegative bacteria stud! clearl! show that there is over a

50D bacterial inhibition at the 00 ppm level$ The data of T9(F# is

illustrated graphicall! in 1:$ $

#J9%PF# .

solation and Purification of eat Stable Protein 1raction &SP1'

Upon isolation of the heat stable protein fraction &SP1', microbial inhibitor!

assa!s were performed to assa! the growth of gram"negative bacteria$ These

tests verified the inhibiting effects of the SP1$ nce the efficac! was

established, the functional protein, or proteins, of interest were isolated from

the SP1 fraction$ 9bout 00 mg of the heat"stable protein fraction dissolvedin 50 mm %#S &p 6$0' was applied on a To!opearl Sp"550 cation exchange

column &0K$6 cm', previousl! eCuilibrated with %#S buffer$ The column was

eluted at 5$0 ml


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antibacterial activit! were further anal!)ed b! reversed phase chromatograph!$

9bout mg amounts of pea2 material from the Sephacr!l column purification

run were loaded onto a silica 8 column &/5K-4 cm' in eCuilibrium with 0$D

T19$ The column was eluted at 5 ml


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express the protein include mai)e, so!bean, sunflower, wheat, barle!, sorghum,

canola, peanut, oats, sugar beet, rice, and tobacco$

9 similar transformation of fungi or bacteria can be performed$ The starting

plasmid would include a promoter recogni)ed b! the host and the selectable

mar2er is often an antibiotic$ The protein product is l!sed from the cell$ Toproduce large Cuantities a fermentation process for producing the protein can be

used$

9lthough the invention has been described with respect to a preferred

embodiment thereof, it is to be also understood that it is not to be so limited

since changes and modifications can be made therein which are within the full

intended scope of this invention as defined b! the appended claims$

Anti-bacterial Protein Extracts From Seeds of Marigold and Paprika

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