PROCEDE D'OBTENTION D'UN RODENTICIDE BIOLOGIQUE

PROCESS FOR PRODUCING A BIOLOGICAL RODENTICIDE

Abrégé français

L'invention concerne le domaine des biocides et en particulier elle concerne un procédé d'obtention à échelle industrielle d'une substance rodenticide d'origine microbiologique. Le procédé décrit dans la présente invention se base sur la standardisation de l'obtention du substrat que l'on utilise, ainsi que celle du processus de fermentation qui emploie une souche de Salmonella enteritidis, cette souche s'utilisant comme substance biocide dans la lutte contre les rongeurs. De plus, on utilise un composé anticoagulant que renforce l'effet léthal dudit micro-organisme.

Abrégé anglais

The present invention relates to the field of biocides and particularly to a
process for producing in large quantities a rodenticide substance of
microbiological origin. The disclosed process of the invention is based on the
standardization of the production of the substrate which is used, as well of
the fermentation process using a strain of Salmonella enteritidis, which
strain is used as a biocide substance for regulating the population of
rodents. Additionally, an anticoagulant compound is used to potentiate the
lethal effect of said microorganism.

Revendications

CLAIMS
1. Method for the obteinment of a microbiological
rodenticidal compound which uses grains of cereals as bait
and, as active principles, a monopathogenic microorganism and
an anticoagulant characterized by a highly concentrated
inoculum obtained through fermentation in a liquid nutrient
medium of the Salmonella enteritidis enteric subspecies
subgroup 1 group D, which is subsequently used to inoculate
the bait that had been previously mixed with a mixed solution
composed of sodium hydroxide and warfarin sodic salt.
(3-.alpha.-benzylacetonil-4-hydroxycoumarin), which acts as a
potentiating substance in said compound.
2. Method according to claim 1 wherein the highly
concentrated inoculum is obtained through fermontation at
37°C, at a pH between 7.4 and 7.6, shaking between 400 and
600 r.p.m. and aeration between 0.1 and 1.0 v.v.m. of the
strain of Salmonella enteritidis enteric subspecies subgroup
1 group D, during 3 and 5 hours using liquid nutrient medium
for this purpose.
3. Method according to claim 1, wherein the rice
previously selected and washed with hot water, and with a 13%
humidity content, is subjected to a sterilization process at
121°C and a pressure of 1.2 bar from 35 to 45 minutes, and
after it has been cooled and dried until a humidity content
between 20 and 30% and a temperature of 37°C are reached, it
is then blended with a mixed solution containing 0.9% sodium
hydroxide and 0.1% warfarin sodic salt
(3-.alpha.-benzylacetonil-4-hydroxycoumarin) which is added for 15
minutes while shaking.
4. Method according to claim 3, wherein the mixture is
later on cooled and dried again until a humidity between 20
and 25% and a temperature close to 37°C are reached: its pH
is adjusted between 7 and 8.
5. Method according to claim 4, wherein the inoculation
of the substrate is carried out in such a way that the final
concentration of the microorganism in the mixture, which
originally is 108 CFU/mL, after a brief 4-hour
fermentation period in solid state, becomes 109 CFU per mL.
6. - Rodenticidal compound obtained using the method
described in the above claim, wherein it includes about 62.5%
of a cereal with a humidity content between 50 and 56% which
is used as a bait, about 12.5% of an inoculum of Salmonella
enteritidis enteric subspecies subgroup 1 group D diluted
until about 109 CFU are obtained in the final compound, and
about 25% of the mixed solution containing a warfarin sodic
salt as potentiating agent.
-6-

Description

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
--2--

-
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.
--3--

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~
--4--

CA 02206660 1997-06-02
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.
.