Note: Descriptions are shown in the official language in which they were submitted.
CA 02206660 1997-06-02
PROCESS FOR PRODUCING A BIOLO~ICAL RODE~TICID~
~chnical S~ctor
~he pre~en~ invsntion is r~lsted to t~ field of
Bioclde~ and p~r~lcu~arl~ ~ith a proce~ for ~he ob~elnmen~.
on an indu~rial scale, of a roden~i~ld~l subs~anc~ of
microbiological origin. useful for harmful rodent conrrol.
~ell a~ with the subst~nce obtain~d through this method.
Pr~iou~ Techn~que
Ha~mful rodnnts c~u~e slgnificsn~ economic losse~
everywhere in the ~orld They do not only destroy crop~.
food and induficrlal re6erves ~ut are also ~h~ c~rriers of
lnfec~lou~ di~ea~es.
Theref~re, ~he flght a~in~ th~e anlmal~ con~titu~es a
~ignificant econo~ic benefit and also plays ~n lmpor~an~ rol~
in ~he prophylaxi~ of the di~ea~s thot they tr~ns~lt
Thore arY differ~n~ m~thod~ for rodont control, ~ut th~
rno~t ~ommon on~3~ are the mechanlcul. chemlcal elnd blo~ogical
2~ method~ l~he microbiologic~l methods ~r~ included within the
latter~.
Msch~nlc~l metho~ ba~ed ~n trapQ, bai~s e~c. are
gucce~fully u~od ln ~mall area~ however, ~hey are le
e~ctive ln l~rger area~.
Chomical ~0thod~ are b~d on the ~ff of bal~
cont~ining differ~nt typos of poi~on. Som~ of the~o products
hav~ sh~rp actlôn ~For exampl~ zinc phosphide. sodium
monofluoracstate 1080) ~nd cau~e death immed~ntely a~tor
~ng~st~on. ho~ever, they are very toxlc for non-~hite
~o ~pecle~, due ~o the high concentrations of non-se~ectiv~
poison~ th~t mos~ ~e u~ed.
Antlcoagulant ~ubstance are among the co~only used
chemical m~thod~. Th~ first generation o~ tho~e ~t~ncoc
appea~3d in the 50 s ~nd among the nlost uced are th~ 3~
Bonzyl~cetonil-4-hydroxycoumar~n (~rfarin) coumate~ral~l
chlorophac~one and dephacinonone. When ~ing ~hss~
cornpo~lndS~. the death o~ the rodsnts occur~ betw~3en 5 ~nd 7
days aftur lnge~ion E;o thero i~; no rejaction or r~action ~o
the bsll. However, for a ~ore effectlve control lt is
nec~YAry to ke~p large amoun~ of ~it in the ~r~tm~nt
are~. slncs ~hexe ~u~3t~nce~ ~Yert thelr actlon only ~fter
rep~ted do3e~ and ~he rodent must ingest the bait during
~ev~ral d~yx to rsach the leth~l dose~
In ~e mid ~o~ the pro~lem of reslstance ~o tradi~lonal
~5 antlco~gulsn~ began to emergo. Thl~ led to the dsvelopm~nt
~f ~ ~econd gonsralioh ~f ~nticoagulan~g cuch as bro~odiaLone
and brod~iacum (klorat).
In g~nsr~l, the d$~atvantago of the chemical mothod~ is
tha~ af~er their repeatod use rodent6 do not instincti~ely
CA 02206660 1997-06-02
ing~t ~h~ bai~. wAlcn reduce~ the off~c~lvene~s of the
product. On ~he o~h~r hand. the~e produc~ ars h~zardou~ for
~an. doms~tic ani~als ~nd th~ ~s~ful fauna
~lologienl met~odg ~rY bssod on ~h~ uso of th~ natural
en~mies of tho rodont~ for controlli~g thoir population. In
goner~l, t~y are ~a~ed on the ~rtifie1al oont~mination of
rodent~ wlth microb~~ cau~ ng infoctiou~ dl~a~e~ ln them
th~ le8d ~o lethal epizooti~s. In ~he~8 methods a ~pecific
organl~m 1~ u~ed for ~ch anlmal ~poci~
lo ~or the obt~lnment of monopathogenic str~ln~ again~t
murine~ difforont culture-medla h~s boen u~od, among whlch
are m~t pep~one both, bro~d and bre~er~ y~t. ~il, poa,
blood. hydrolytic sera tme~t .flour hytroly~Ato and yeast
broth hydrolysato, whi~h are u~d for gro~lng tho mlcrobi~l
inoculu~ that ~fterwards ~ill be usod in Ihe prod~c~lon o~
the ~ain ~ltur~ to obt~in grnln pr~p~r~ion~.
Tho bacteri~l prep~r~tion obtalnod from cor~al graln~ 1
a ~ae~eri~l culture of strsln~ c~uslng ~yphu~ in rotents,
which ar~ gro~n on di~fe~nt coroal~, among uhlch are ~hs~t
barloy, rice, osts or ry~. Thic formontation on ~ ~olid
modi~m allow~ a largor yiold in th~ gro~th of the
microorg~nism th~n th~t ~hich was ~tt~inod by using llq~id
culture mYdi~.
Dl~clo~uro of the In~ntio~
~atc~ ~he purpo~e of ~ho pr~Qnt inv~ntlon 1~ to pro~ide
a mothod for manufacturing a highly effecti~e mlcro~lologic~l
rodonticide to be u~d in tho control of h~mful rod~nt~.
Thi~ mothad ~llov~ us to obtain ~ high tlter of the dlsea~0
gorm th~t i~ u~od, which assure~ tho hig~ ~ffoctiv~ne~s of
th~ finl~h~3d prodllct. ag w~ ~ 8 reduction of tho ~
noce~sary for its m~nufncturs. AnothYr ob~oc~ of ~his
~ppl~c~tion is tho rodentlcidal compound obt~in~d u~lng thls
m~thod. ~hich has a high p~thogsnicity and virulence ag~in~t
murine~ cau~ing lo~hal ~plzoo~le~.
The pr~nt inven~ion ~on~ists in obtalning ~
propar~tion with biocidal acti~ity, ~ith three maln
component~:
1. Str~in of Salmonella entYritldis.
2. PotYntisting ~olution.
3. Suhst~at~ for ferment~tion co~pos~d of ~ole
rlce.
Th~ three element~ con~titutlng ~h~ pr~paration are
int~grat~d ~y mean~ of though two confluent opora~lon~:
a. Pr~3parlng thl3 :3t~ril8 ric~3 bc~3 ~ub~tr;~t-3
~5 containing the potontlstlng ~bstanc~ ~nd ~d~u~t~d pH.
~. O~t~ining the S~l~onsll~ ~nt~ritldi~ inoculum
and lnoculAting i~ ln a.
In gener~l the proc~ b~gin~ by ~elo~tlng ~nd ~hing
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CA 02206660 1997-06-02
the rice ~hat 1~ going ~o be ~ed as su~stra~e. The firsl
step is ~o ~eparat~ ~he ~mp~ri~ies contslned in ~he s~l~c~ed
rice Rice ~ith a 13~; humidity content ls first wached wi~h
hot water ~t ~o~c during 8 peri~d of nearly 20 minut~3s. and
S tho wat~r uPed fcr the ~ashing i~ drained. La~eer on thi~
substr~e i~ ~u~ject to ~ ~leriliz~ion proc~s~ at 121~C and
a pre~sure o~ 1.2 b~r for ~ period of ~i~e betw~en 35 and ~5
mlnute~. Immedi~ely af~erwards. the ~bs~rate i~ cooled an~
trlod untll it r~ache~ a humidity conren~ be~een 20 ~nd 30%
~nd a tsmperature of 37nC, and ~hen ls b~snded with a mix~d
s~lution which contalns 0.9~ ~odlu~ hydroxide and O.lX
warfarin sodic salt (3-~-Benzyl-acetonil-4-hydroxycoumarin)
which i~ u~sd ~s ~ potentl~tor of the le~hal ~ctlon of ~h~
prepar~ion and added for 1~ minu~e~ ~hile sheking.
At thl~ time ~n sd~ustment ~f ~he pH of the mix~ure i~
t~rrled ou~ hould be bet~een 7 4 and 8.
Im~ediately afterward6. the ~u~strate ~hus o~tained ls
inocula~Qd ~i~h a ~train of ~lmonella enteritidi~ ~n~eric
sub~pecios ~ubgroup l. group D, ~hich is deposlt~d in
~ilable co~l~ctions of th~se microorganis~s. This ino~ul~m
is c~rried out in such a ~y ~h~t the final concen~ration of
the microorg~nism ~n the ~ixture ~hlch origin~lly l~ 10~
Colony Forming Uni~6 tCFU) per mL. af~or a ~rief 4-hour
fermenra~ion period ln solid ~t~te. ~ecome~ 109 CFU per ~L.
as ~0~ obrsining ~hs inoc~lum o~ th~ pre~iou~ st~p we start
a precul ture a f the microo~gani~m which is transferred
t~ a 100 L ferm~nt~or con~ining nutrient brosh in a r~tio
from 1 to 1.5X of the ~olume to be f~rmont~d ad;hsting ths
f~rmentation cond~tion~ at a tomperatur~ of 37~C a pH
3~ butween 7.4 ~nd 7.6. ~h~l~lng bet~een 400 and 600 r. p m and
aoratlon betveen 0.1 and l.o v.~.m. The ferment~tion tlmo
r~n~es from 3 to 5 hours and an lnoculum with tl~r be~weeh
1 and 2 x lolO colony forming uni~ (CFU3 lc flnally
obtsined.
We finally obtai~ 8 batch of a rodantic~da~ product
roady to be packed ant stored a tQmp8r~ture between 5 and
9~C: in these conditlons ~hs ~tB~ility of th~ product is
preservod ~or ~ g perlod of tlme.
The above-~en~ioned produc~ is ~ rodehticldal compo~nd
con~ining grains of cereal ~hat are u~;ed a~ a bait (such a~
wheat, rico, ryo, o~t~ millet, etc~) lt al~o contalns a
s~bstance u~d a~ po~entia~or compo~ed of a mixed solu~on
o~ sodium hydroxide wlth 3-a-b~nzylac~tonil-s-
hydroxycoumarin ~nd an lnoculu~ of ~ s~rain o~ Salmonslla
snterit~di~ snterlc subspeci~3~ subgroup 1 group D with a
flnal ~l~r~ be~een lo6 and 1~ CFU
~h~ mat~i~l thus obtalned ls late~ packed ~nd 3~0r~d.
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CA 02206660 1997-06-02
~ h~ biolog~c~l test~ of ~he flnl~hed product ~howed lOO~
mortali~y rato. wi~h dea~h taking place from ~ to 12 day~
af~er inge~ion by ~he m~in~ fa~nily, ~hl3r~forl3 ~howing a
cignif~cant incr~se of its biological proper~ie~ ~i~h r~g~rd
S ~o ~ff~cti~en~ss, ~hich is due malnly to the ~irul~nce ~d
p~thogeniclty of th~ rexulting product.
S~ld product ha~, ~s it~ m~in foa~u~ex its high
eff~ctl~enes~ ~8 ~ell ~ i~s s~bi~i~y ~or one y~r ~hen
f~ozun ~ mon~hs if stor~d ~etwsen 5 ~nd 15~C. and 30 days if
~tor~d a~ room tempe~ture.
Slnce th~ invent$on has ~o d~ ~ith a proce~ that can
bo ~c~l~d-up to industri~ v81 for the o~toln~ent of a
rod~nticld~l compo~nd~ ~here ~he ~dequate opYrations and
para~t~rs h~ve been de~ined to obtdin b~tches of th~ product
lS ~ith n proper qu~lity ~nd st~billty it can b~ mechaniz~d
u~in~ ~ll-k~oun facilities wit~ stAte-of-the-ar~ ~echnology
~h1ch allo~s the op~imlza~ion of s~id 6cal~-up ct indust~ial
l~vel.
EXAMPLES:
EXA~~LE 1: OBTEIN~E~r OF THE IN~IJM
A microdrop of a deep-froz~n cult~r~ of a strain of
Ssl~onells ~n~eritidis enteric s~b~pecles subgroup 1 group D
i8 ~dded to an enrich~d liquid ~edium, which is incubated
for 4 nour~ at 37~C in a sifter st 180 r.p.m. ~ro~ thls
2~ incubs~ion a prucul~u~e ~ith a titre of 10~ C~U is ob~ained.
~hich i~ after~ards tran~erred to a fer~entator co~aining
thu llqui~ msdlum of nu~rient broth, ~hose c~ ~ition i5-
Bacterlol~gical pep~one - 5.O g~L
3~ Nu~rient extr~ct - 1.0 g~L
Ye~st extract - 2.0 g~L
Elna - 5 0 g~L
EX~MPLF 2 ~ ZATION 0~ ~E PR~PARA'rIO~ OF T~l~
3 5 ~OD~NTI CIP~ POU~
Th~ rlce us~d a~ ba~t and su~strat~ for the grow~h of
the pathog~nlc microorg~ni~m c~n be ~uppll~t in bulk or clean
in b~gs.
After Ihe impuritie~ ~re sep~rsted from ~h~ rlce. it is
~0 carried to the ~nk~ for storing clean rice. cach wlt~ a
capacity of 17 M.T.
If rice i~ ~upplled clean in baga. these are carried on
whaelbarrows to the freight ~eleva~or. which ~a~es them
~trsight ~o the r~ceiYing mlll-hoppers ~here the rice ~n ~lk
~5 fro~ the 51108 ~ill also get there, u~ing elovator~ and
COnVQyer belts.
The do3ifying mlll-hoppern ~ith ~ cnpacity of 650 kg of
rice control and gu~r~ntee the weight of the rice mas~
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n~cessary ~or the proco~, e~ery 1 and 5 hour~ alternatlvely
p~r.
Rlce with a l~X humldl~y content 1~ unload~d from t~e
dosifying mll~-hoppsrs i~to a contalner ~srY th~ whole
pr~ces~ing o~ tho dlfferent co~ponents th~t con~titute the
rodsnt~cidal compound will t~e plac~.
Immodi~tely ~ft~3r~rdx d~3mlner~l11zed ~ter ~8 injectod
and ~-eat~d at ~~C u~til co~ring the entlre ~olume o~ the
~Ice for ~5 mlnut~s. Slmultaneo~ly with ln~e~llon ~f hot
1~ water ~ mlxing mechani.~m 1~ aetlvsted
Th~3 action of thls msch~ni~m n-ake~ of po~:61bll3 tb ~ash
tho rlc~ for 20 minut~. Later on the wat~r th~t ~88 used
for t~ w~hlng i~ dr~inod.
A vacuum is applied ag~ln ~or 5 minu~es ~nd 225 L of hot
~t~r a~ 80~ ~re inject~d. Durlng thls tlm~ the mech~nism
of t~e mix~r i~ kept w~rklng At the ~me ~lme v~por
in~ec~ed ~ ~h pre~ur~ regulated to 1.~ b~r ~nd tompera~ure
of 121~C. dir0c~1y to ~he cham~er of g~id contsin~. ~hen
~he~e par~metor~ are reach~ ln~ide the con~alner,
storlliza~ion procexx take~ pl~co for ~out ~5 mlnuto~.
~ ater on tho ric~ i~ ~oolYd and dried untll rea~hing
humidlty he~een 27 and 28X ~nd ~ tempe~ture of 37DC.
Imm~diately aft~rw~rd~. a ~ixed golution con~ining 0 9%
~odium hydroxide and O.lX Warf~rin Sodle Salt 1~ lniected
25 ln~ ~he container. Once the 282 L of ~aid mlxed ~olut10n
ha~ beon ~n~ected~ this opsration eon~lnu~ for 60 minuto~.
The cooling proc0s~ of the ~ico mass ~s~ng mlx~d solu~lon
continu0~3 un~ t~3mpor~lurc of 37~c i8 re~ch~3d. I:atsr on,
an adju~tmont of the p~I of th~3 mixturo i8 c~rrl~3d out until
3Q v~lue betwoen 7.4 and 8 1~ re~ched.
I~media~ely aftorwards, l~l L of tho dilulod inoculu~
~re in~ect~ ~n~ mixed ~ith th~ ri~e m~s pre~iou~ly treat~d
~lth th~ mix~d ~olu~ion, ~n~il the homogenlza~lon of the
fin~l mixt~r~, At tho end of thl~ ~tep, ~amplss of th~
3~ product are taken for qu~lity analysic.
La'c~r on. the product cont~lin6~d ln th~ cont~inor i8
c~rri~d to th~ mill-hopper of the ~illing ~ch~n~. Th~
on~ire ~ove-mentioned cycle must be fulf~ll~d ln a 6-hour
per1od making lt po~ le to ob~sin 1125 kg of ~h~
~0 rod~3nticidQl compo~nd.
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