Journal

J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

Moriinga oleifera S
Seeds Extract
E Activiity on
Ennteropaathogen
nic Esccherichiia coli and Aeeromonas
hyddrophylla Cellss in Aq
quatic Microco
M osm

Claire Sttephane Meetsopkeng

Laaboratory off Hydrobiology and Ennvironment, University of Yaoundee 1, Camero
oon
E-mail: mclaairestephanee@yahoo.co
om

Chretiien Lontsi Djimeli

D
Laaboratory off Hydrobiology and Ennvironment, University of Yaoundee 1, Camero
oon
E-mail:
E lonttsichretien@
@yahoo.com
m

Olive V
Vivien Noah
h Ewoti
Laaboratory off Hydrobiology and Ennvironment, University of Yaoundee 1, Camero
oon
E-mail: nnoahewoti@
@yahoo.fr

Luciennee Marlyse Moungang

M
Laaboratory off Hydrobiology and Ennvironment, University of Yaoundee 1, Camero
oon
E-mail: luumou2000@
@yahoo.fr

Paaul Alain Naana

Laaboratory off Hydrobiology and Ennvironment, University of Yaoundee 1, Camero
oon
E-mail: naanpaul4life@
@yahoo.fr

Antooine Tamsa Arfao

A
Microobiology andd Biotechno
ology Laborratory, Sain
nt Jerome Caatholic Univversity of Douala,
D
Caameroon. E--mail: tamarrfao@yahooo.fr

13 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

Pierreette Ngo Bah

hebeck
Instituut de Recherches Agron
nomique poour le Devélloppement (IRAD),
( Yao
aounde, Cam
meroon
E-mail: nggobahebeck
k@yahoo.fr

Télesphhore Sime-N
Ngando
Laborratoire ‘Microorganism
mes: Génomee et Environ
nnement’, UMR
U CNRSS 6023, Uniiversité
Clermont
C Au
Auvergne, Ceedex, Francce
E-mail: Teelesphore.siime-ngando
o@univ-bpcclermont.fr

Moïse
M Nola (Correspon
nding authorr)
Laaboratory off Hydrobiology and Ennvironment, University of Yaoundee 1, Camero
oon
E-mail: mooise.nola@yahoo.com

Receiveed: June 13, 2019 Acccepted: Auggust 4, 2019 Publish

hed: August 7, 2019
Doi: 100.5296/jab.vv7i2.14917 URL: httpps://doi.org//10.5296/jab
b.v7i2.149117

Abstract
This stuudy aimed tot evaluate in microco sm conditio on, the survvival of Aero
romonas hyd drophila
and Entteropathogeenic Escheriichia coli (E EPEC), in the
t presencee of M. oleififera aqueou us seeds
extract at concentrrations varyying from 1 to 40 g/L L, and undeer 4 °C andd 23 °C inccubation
temperaature.It has been noted d that cell abbundances decrease grradually witth the increeasing in
the seedds extract concentratio
c on. Howeveer, a marked d cells regro
owth was soometimes noted.
n In
monosppecies cell inncubation condition, unnder 4 °C, th he EPEC ceells inhibitioon percentag
ges (CIP)
values vvaried from
m 52.12 to 99.84%.
9 Thhose of A. hydrophila
h varied
v from
m 13.2 to 96 6%. The
lowest CIPs were noted at th he extract cooncentration n 1g/L for EPEC
E and A A. hydroph
hila. The
highest CIP value was registeered at 10 annd 40 g/L for f EPEC an nd at 15 g/LL for A. hyd
drophila.
Under 223 °C incubbation, the EPEC CIPss values varried from 74.04 to 99.99% and tho ose of A.
hydrophhila varied from 21.2 to 97.8%. For E. coli, the loweest and thee highest CIP C were
recordeed at the extract
e conccentration 1g/L and 30 3 g/L, respectively. In bispeciies cells
incubation condition, the CIP Ps were rellatively diffferent. Thesse results sshow the potential
p
exploitaation of M. oleifera exttracts in thee microbiolo
ogical treatm
ment of potaable water.
Keywords: Aerom monas hydrrophila, Aqquatic microcosm, Mo
oringa oleiffera seeds extract,
Escheriichia coli

14 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

1. Introoduction
In mostt regions arround the word,
w and AAfrica in paarticular, hee increasingg need for water is
related to populatiion increasee. Unfortunnately, potab ble water is unavailabble in most regions
around the world. These
T situattions obligee the populaation to relieed on water sources of doubtful
d
quality to satisfy their
t daily needs whicch exposes them to microbiologi
m ical contamminations
(UN/W WWD, 2006;; WHO, 201 17a; 2017b)). To remed dy this situattion, severall solutions are
a often
recomm mended. Thhese could be chemicaal treatmen nt of water,, of which the residu ues have
negative effects onn health, or filtration annd boiling. Some of these methodds are laboriious and
are not always withhin the reacch of the poopulations. Alternative
A methods off water disinfection
by plannt extracts are
a also prop posed (Adrriana et al., 2007; Weaathers & R Reed 2014). In fact,
several studies repoorted on thee antimicrobbial propertiies of plantss (Sunda et aal., 2008). Statistics
S
show thhat over 80% % of African n and Asiann household ds use mediccinal plants to treat theemselves
(WHO 2002). Huundreds of plant speccies can bee used for therapeuticc purposes by the
indigennous populaation (Tamsaa Arfao et aal., 2013). For F example, aqueous extracts of LantanaL
camaraa, Cymbogonn citratus an nd Hibiscuss rosa-sinen nsis present a bactericiddal effect inn aquatic
environnment (Adriana et al., 2007).
2 In thee same way, Eucalyptuss microcorys ys extract showed an
inhibitinng effect with
w respect to certain ppathogenic germs g in aq
quatic microocosm (Weaathers &
Reed, 22014).
The bioological treeatment of water usinng seeds of o Moringa oleifera ccould consttitute an
alternattive or integgrated solutiion for the improvemeent of water quality. It is the mostt widely
cultivatted species of the gen nus Moringaa, and its young
y seed pods and leaves are used as
vegetabbles. All parrts of the Moringa
M tree are edible and
a have loong been cononsumed by humans
(Prabhuu et al., 20111; Kuete, 2017).
2 Morringa is useed worldwidde in traditiional mediccine, for
various health coonditions, suchs as skkin infectioons, anemiaa, cholera, fever, resspiratory
disorderrs, tubercullosis, and inntestinal woorms (Sairaam, 1999; Fuglie,
F 20011; Mahmoo od et al.,
2010).
Phytochhemical anaalyses have shown thatt M. oleiferra is a rich source of ppotassium, calcium, c
phosphoorous, iron,, vitamins A and D, esssential amiino acids, as a well as kknown antio oxidants,
such ass β-carotenee, vitamin C,C and flavoonoids (Ben nnett et al., 2003; Mbikkay, 2012). A wide
variety of polyphennols and ph henolic acidds as well as flavonoids, glucosinoolates, and possibly
p
alkaloidds are believved to be reesponsible fo
for the effects of the plaant (Ferreiraa et al., 200
08; Stohs
& Hartm man, 2015)..
Seed poowder has been
b indicatted as very effective inn clarifying polluted annd dirty water from
rivers. T
The floc conntained in th
he seeds or ccakes is a baasic polypep
ptide, more specifically
y a set of
active ccationic pollyelectrolytees with a m
molecular weight betweeen 6 and 117 KDa (Jah hn & Al
Azbaia,, 1988). Theese positiveely charged ppolypeptidees neutralizee colloids inn murky waaters that
are gennerally negaatively charrged. In adddition, the seeds contain 4L-rhaamnosyloxy y benzyl
isothioccyanates, which
w could be an antiimicrobial agent (Caceres, 1991)). The cultivability
allows the growthh and dev velopment of planktonic microo organisms iin the watter.Little
informaations relateed to the imppact of the vvarious conncentrations of aqueouss extract of seeds of
M. oleiffera on the bacterial grrowth are avvailable, wh hether cells are in monoospecies or belongs
to manyy species. Also,
A the im
mpact of envvironmentall temperaturre on this pllant extractt activity

15 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

against microorgannisms is lesss documenteed. Higher environmen ntal temperratures can increase
maintennance energgy demand and reducee carbon use efficiency y (Devêvre & Horwáth h, 2000;
Steinweeg et al., 20008; Allison
n et al., 20110). The aimm of this stu
udy was to aassess the im
mpact of
aqueouss extract off seeds of M.
M oleiferaa on the cu ultivability of
o Aeromonnas hydroph hila and
enteroppathogenic Escherichia
E coli cells inn aquatic microcosm,
m under
u 4 °C aand 23 °C.
2. Mateerials and Methods
M
1.1 Sam
mpling and Preparation
P n of Moringga oleifera Seeds
S Extracct
The seeeds of Moriinga oleiferra were colllected in Maroua
M (Cameroun, Ceentral Africca). This
localityy is at latitude 10°35′27
7″ North, loongitude 14
4°18′57″ Eaast and at 4006 m altitude. This
region iis characteriized by the clayed-sanndy and sanndy-loamy soils
s (Martinn & Segalenn 1966).
The clim mate is Suddano-Saheliian, characteerized by a dry season
n of 8 to 9 m
months and d a rainy
season of 3 to 4 months. Prrecipitation is fairly lo ow with ann annual avverage of 8008 mm
(M’bianndoun et al., 2002).
The seeeds of Moriinga oleifera
a were harvvested, dried d at laborato
ory at a tem
mperature (2
23±2 °C)
for 2 m
months and removed fromf their hhull. They were then crushed annd the powder was
weighedd and then mixed with h sterile disttilled water.. The concentrations coonsidered were
w 1, 5,
10, 15, 20, 30, 40 g/L.Homog
g enized extraacts was lefft to settle fo
or 5 minute s. The pH solutions
s
were adjusted at 7 using NaOHN and HCl solutiions and th he supernaatant filtereed using
successsively whattman filter paper firsstly, nitroceellulose meembrane off 0.45μm porosity
secondlly and the Millex
M memb brane of 0.222µm porossity thirdly (Rodier,
( 20009; APHA, 2012).
2.2 Baccteria Strainns and Cell’’S Suspensioons
The baacterial cellls considerred were eenteropathog genic Esch herichia cooli and Aerromonas
hydrophhila strains. They were selected beecause of theeir high imp portance andd strong occcurrence
as indiccator of miccrobiologicaal quality off water used
d for consummption (Laccasse, 2004; WHO,
2011). TThe enteroppathogenic E. E coli straiin was provvided by thee Laboratorry of Micro obiology
and Ennvironment of Centre Pasteur (Caameroon, Central C Africa). A. hyddrophila strrain was
isolatedd from grouundwater in n Yaounde using mem mbrane filtrration methhod and Am mpicillin
Dextrinn agar culturre medium. Both strainns cells weree then identiified using bbiochemical criteria
(Holt ett al., 2000). Cells weree then storedd in glycero
ol at -15 °C for later usse.2.3 Experrimental
Protocool
Experimments were done in 2 series. Thee first was done
d using cells of onne bacterial species.
The seccond was done
d using cells of bboth bacteriial species under conssideration. At each
extract concentratiion for each
h series of experimentt, 2 groups of glass flaasks A and B were
used. W
With each grroup being made
m up of 3 glass flasks.
For thee first seriess, prior to the
t experim ments, a frozzen vial containing eaach cells strrain was
defrosteed at room temperaturre. The cullture (300 μL) μ was th hen transferrred into 100 mL of
nutrientt broth (Oxxford) and incubated
i aat 37 °C forr 24 hours. Cells weree then colleected by
centrifuugation (80000 rpm for 10 min at 110 °C) and washed twiice with steerile NaCl (8.5 ( g/L)
solutionn. The sediment was then t dilutedd in 10 mL L of sterile NaCl soluttion. Homo ogenized
bacteriaal suspensioon was adju usted to a deensity of 0.5
5 Mac Farlaand (BaCl2 and of H2SO4 1%).
Bacteriaa concentrattion of the original
o susppension wass about 108 CFU/ml.
C Affter dilution
n, 1ml of

16 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

the cellls suspenssion was added to the glass flasks con ntaining 1000ml of different
d
concenttrations of seeds extracct solutionss filtered as indicated ab
bove. The rreally conceentration
8
that the control conntain is 2× 10
1 CFU/mll.
The sammples were then
t incubated for 6houurs. The incuubation period of 6h waas considereed based
on the sstudies carriied out by Weathers
W annd Reed (20 014), which h indicated tthat after 3h
h contact
betweenn a bacteriaal cell and th
he plant exttract, a metaabolic interaactions resuult is observ
ved. Two
temperaature incubaation 4 °C and
a 23 °C w were chosen. The tempeerature of 233 °C was ch hosen to
simulate the ambieent temperaature in moost househo olds in the equatorial rregion, and d 4 °C is
usually used to incuubate or store bacterial strains. Thee glass flask
ks of group A were incu ubated at
4 °C, annd those of group B weere incubateed at 23 °C.
Analysees were carrried out usiing Endo annd Ampicillin Dextrin agar
a culture media resp pectively
for E. coli and A. hydrophila.
h Petri dishess were then incubated at
a 44 °C andd 37 °C resp
pectively
for 24 hhours (Marcchal et al., 1991;
1 APHA A, 2012), and
a the colony formingg units (CFU Us) were
then coounted. The bacterial density was eexpressed in Colonies Forming U Units (CFU)//100 mL
of sampple.
For thee second serries of expeeriments, bbacteria conccentration of
o original ssuspension of about
8
10 CFU U/mL for eaach cells speecies. 100 µL
L of E. coli and the sam
me of A. hydr
drophila werre mixed.
After ddilution, 1000 µL of thee cells susppension wass added to the tubes ccontaining different
d
concenttrations (1, 5, 10, 15, 20, 30, 40 g/L) of seeeds extract solutions ffiltered as inndicated
above ffollowing thhe same prottocol. The eexperiment was perform med in triplilicate.
2.4 Datta Analysis
The varriations of cell
c abundances (N ) aas well as cells
c inhibitiion percenta
tages (CIP) after 6h
accordinng to the concentratio
c on of the exxtract of Mo
oringa oleiffera at eachh temperatu
ure were
illustratted by histtograms. Th
he CIP weere calculatted according to the following formula
(Weatheers & Reed,, 2014; Edimma et al., 20010):
𝑁 𝑁
𝐶𝐼𝑃 100
𝑁
N0 = nnumber of CFU/100
C mL
m before aadding the seeds extraact solutionn; Nn = number of
CFU/1000 mL after incubation in a given condition in
n the seeds extract soluution of M. oleifera.
o
The Sppearman coorrelation teest "r" has been usedd to assess the relatioonship betw ween the
consideered parameeters. The comparison between baacteria abunndances werre carried out
o using
the testt H of Krusskal-Wallis and U of M
Mann Withney. This analysis
a wass done usinng SPSS
version 16.0 prograam.
3. Resu
ults and Disscussion
3.1 Tem
mporal Variaation of Celll Abundancces
When eenteropathoggenic E. colli cells weree the only ceells species in
i solutions , their abundance in
differennt extract cooncentration
ns varied frrom 500×10 0 to 0.92×103 CFU/1000 mL. At 4 °C, it
3
3 3
varied ffrom 224.48×10 to 3.58 ×10 C CFU/100 mL L (Figure 1). The low west abundan nce was
registerred at the concentratio
c on 10 g/L, and the hig ghest at 1gg/L. At 23 °C, it varied from
3 3
129.7×110 to 0.92 ×10 CFU/1 100 mL the lowest abun ndance was registered aat the conceentration

17 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

30 g/L, and the hiighest at 1g

g/L. Cells cconcentratio
ons in the control (soluution without seeds
3
extract)) was 500×110 CFU/100 mL at 23 °C and 4 °C C, respectiv
vely.
When A A. hydrophilla cells were the only ccells speciess in solution
ns, their abun
undance variied from
3 3
500×100 to 11×100 CFU/100 mL. At 4°°C, it varied d from 434×10 to 200×103 CFU//100 mL
3

(Figure 1). The lowwest abundaance was reggistered at thhe concentration 15 g/L L, and the highest at
3 3
1g/L. AAt 23 °C, itt varied from
m 394×10 to 11×10 CFU/100 mL m the low west abundaance was
registerred at the concentratio
c on 15 g/L, aand the hig ghest at 5g/L. Cells cooncentration ns in the
3
control (solution without
w seed
ds extract) w
were 500×10 0 CFU/100 0 mL.
hila and entteropathogeenic E. coli cells, it waas observed in most
In the ppresence off A. hydroph
cases thhat cell abuundances decrease graadually with h increasingg seeds exttract concen ntration.
Howeveer, a slight regrowth of o E. coli w was perceptible at thee extract cooncentrationn 40 g/L
(Figure 1). It was also
a noted att each extracct concentraation in each
h of the incuubation tem
mperature,
that E. coli cells abbundances as
a well as th
those of A. hydrophila
h were relativvely higherr at 4 °C
comparred to those recorded att 23 °C (Figgure 1).
When ccells of the 2 bacterial species weere present simultaneo ously, the abbundance of E. coli
3
under 4 °C varied from
f 179.4 ×10 to 0 CF FU/100 mL L. That of A. hydrophilaa under 23 °C C varied
3
from 1331.6×10 too 0 CFU/10 00 mL (Figuure 1). Thee lowest abu undance waas registereed at the
concenttration 40 g//L for both E.
E coli and AA. hydrophiila. The high hest abundaances were recorded
r
at 5g/L and 1g/L for
f E. coli and
a A. hydroophila, resp pectively (Figure 1). At 23 °C, thee lowest
abundannce was reggistered at th
he concentraation 40 g/L L for E. coli and A. hydrrophila. Thee highest
abundannces were recorded at 1g/L
1 for botth E. coli an
nd A. hydrop phila (Figurre 1). In most cases,
cell abuundances deecreased graadually withh increasing g seeds extrract concentntration. Howwever, a
markedd regrowth of
o E. coli waas noted at thhe extract co
oncentration n of 20 g/L. In contrast with the
case whhen cells bellonging to one
o species w were used, E.E coli cells abundancees as well as those of
A. hydrrophila weree sometimes relativelyy lower at 4 °C comparred to thosee recorded at a 23 °C
(Figure 1).

18 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

Figuree 1. Variation in E. coli abundance with respecct to the con

ncentration of seeds ex xtract in
monosppecies cellss incubationn condition ((A) and bisp
pecies cells incubation condition (B),
( and
thatt of A. hydroophila in moonospecies cells incubaation condittion (C) andd bispecies cells
incubaation condition (D)

19 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

3.2 Cellls Inhibitionn Percentag

ges (CIP)
The ceells inhibitiion percenttages (CIP)) have beeen evaluateed under vvarious con nsidered
parametters (conceentration of the aqueouus extract, incubation temperaturre, number of cells
species present). Similarly, the percentagge inhibition
n cells (CIP
P) make it ppossible to evaluate
the imppact of the aqueous
a extract of theese seeds on
n the abunddance data oof the two bacterial
b
species studied.
At 4 °C
C when one species
s of cell was usedd, the E. colli CIP values varied fromm 55.12 to 99.84%.
Those oof A. hydropphila varied from 13.2 tto 96% (Figure 2). The lowest CIP value was recordedr
at the eextract conccentration 1 g/L for E. coli and A. A hydrophilla. The highhest CIP vaalue was
registerred at the exxtract conceentration 100 and 40 g/LL for E. colii and 15 g/LL for A. hyddrophila.
Under 223 °C incubbation, the E. coli CIP P values varried from 74 4.04 to 99.99% and tho ose of A.
hydrophhila varied from
f 21.2 to
o 97.8%. Foor E. coli, thhe lowest annd the higheest CIP valuues were
recordeed at the extrract concenttration 1g/L
L and 30g/L respectively y. For A. hyddrophila, th
he lowest
CIP vallue was regiistered at 5g g/L whereass the highest was record ded at 15 g//L (Figure 2).
2
When bboth cells sppecies were simultaneoously presen nt and at 4 °C
C, the lowesst (64.12%)) and the
highest (100%) CIP P values forr E. coli werre recorded respectively
r y at the extraact concentrration of
5 g/L aand 40 g/L. For A. hyd drophila, thhe lowest (993.9%) CIP P value wass registered at 1g/L
whereass the highesst (100%) was
w recordedd at 40 g/L. At A 23 °C, th he lowest CIIP values foor E. coli
and A. hhydrophila were 74.48% and 73.88% respectiv vely, all of them at 1g//L. The high hest was
100% fo
for both cellls species (F
Figure 2).

20 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

Figure 22. Cells inhiibition percentages of E

E. coli cells in monospeecies cells inncubation condition
(A) annd bispeciess cells incubbation condiition (B), annd of A. hyd
drophila cellls in monosspecies
cells incuubation condition (C) aand bispeciees cells incu
ubation conddition (D)

21 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

3.3 Relaations Amonng the Conssidered Para

rameters
Spearmman's correlaation test sh
hows that aan increase in incubatio on temperatture is conccomitant
with a significant (P < 0.05) decrease inn E. coli ab bundance when
w it is thhe only celll species
present and when thet concentrrations of seeeds extractts are 5, 10 and 40 g/L (Table 1). When
W A.
hydrophhila cells are
a present alone, the increase in n incubation n temperatuure is significantly
concommitant (P < 0.05)
0 with the
t decreasee in cell abuundance at the concenttration of 10 g/L of
the seedds extract (T
Table 1). When the twoo-cell speciees are present simultaneneously, the increase
in incuubation tem mperature ap ppears to bbe significantly (P < 0.05) conncomitant with w the
decreasse in the ceells abundan nces at conncentrationss at 1 and 30 g/L for E. coli an nd at the
concenttration 1g/LL only, for A.
A hydrophila
la (Table 1)..

Table 11. Spearmann correlation n coefficiennts betweenn the cells abundances

a and the inccubation
temperaature at eachh seeds extrract concenttration, in monospecies
m s and bispeccies cells inccubation
conditioons after 6 hours
h
Cells sspecies conssidered
Seeds extract
e concentration (gg/L)
and inccubation coondition
C
Cells
Cells
incuubation 1 5 10
1 15 20 30 40
species
conndition
Monoospecies
-0.000 -00.909* -0.8864* -0.68 82 -0.000 -0.441 -0.864*
c
cells
E. coli
Bisppecies
-1.000* -00.400 -0.800 -0.60 00 -0.400 -1.000* /
c
cells

Monoospecies
-0.091 -00.455 -0.8
864* -0.37
75 -0.088 -0.530 -0.727
A. c
cells
hydrophhila Bisppecies
-1.000* -00.400 -0.800 -0.94
49 -0.600 -0.400 /
c
cells
*: P ≤ 00.05; df (deggree of freed
dom) = 5 (nnumber of observations
o s)

The Taable 2 shoows that att all incubbation temp peratures, an

a increasee in seeds extract
concenttration signiificantly deecrease (P < 0.05) the abundance
a of
o E. coli annd A. hydro
ophila in
the soluutions whenn cells are of
o monospeecies. When n cells belon
ng to the tw
wo-bacteriall species
consideered, the sam
me observaation was m made except the abundaance of E. ccoli when solutions
s
were inncubated at 4 °C (Table 2).

22 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

Table 22. Spearmann correlation

n coefficientts between the
t cells abu undances annd the seeds extract
concenttrations at each incubattion temperaature, after 6 hours in monospecies
m s and bispeccies cells
incubation conditioons
Cells sspecies connsidered andd incubationn condition In
ncubation te
temperature
Cells sppecies
Cells in
ncubation coondition 4 °C
° 23 °C
C
consideered
Mo onospecies ccells -0.61
14** -0.547**
E. coli
Bispecies
B cellls -0.4
402 -0.484
4*

Mo
onospecies ccells -0.71
10** -0.744**
A. hydrrophila
Bispecies
B cellls -0.86
67** -0.894**
*: P ≤ 00.05; **: P ≤ 0.01; df (d
degree of freeedom) =20
0 (number of
o observatioons)

A compparison betw ween the ceells abundannces record

ded at 4 °C with thosee recorded at a 23 °C
shows that, in thhe monospeecies cellullar conditio on, the abuundances oof E. coli differed
significcantly (P ≥ 0.05) from each otherr at each off the concen
ntrations off seeds extract used
(Table 33).

Table 3. The “P” values

v indicating the deegrees of significance of the differ
erence relateed to the
comparrison of the cells abundances regiistered betw ween 4 °C and
a 23 °C incubation, at each
seeds eextract conccentration after
a 6 houurs in mon nospecies and bispeciees cells inccubation
conditioons
Cells sspecies conssidered
Seeds ex
xtract conceentrations (gg/L)
and inccubation conndition
Cells C
Cells
species incuubation 1 5 10
0 15 20 30 40
consideered condition
Monoospecies
0.043*
0 0.5513* 0.51
13* 0.043* 0.043* 0.043* 0.043*
cells
E. coli
Bisppecies
0.439
0 0. 121 1.00
00* 0.439
9 0.121 0.439 1.000*
cells

Monoospecies
A. hyddro- 0.043*
0 0. 589 0.513 0.487* 0.043* 0.500 0.043*
cells
phila
Bisppecies
0.121
0 0.4439 0,12
21 0.221
1 0.121 1.000* 1.000*
cells
*: P ≥ 00.05; df (deggree of freed
dom) = 40 ((number of observation
ns).

The abuundances off A. hydrop phila at bothh incubationn temperatu

ures significcantly differred (P ≥
0.05) frrom each otther only at concentratiions 1, 15, 20 and 40 g/L
g of seedss extract. When
W the

23 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

two baacterial speccies were present sim multaneouslly, the abu

undances off the E. co oli cells
recordeed at 4 °C C significanntly differeed (P≥0.05
5) from those recordded at 23 °C and
concenttrations 100 and 40 g/L of seeeds extractt. The A. hydrophilaa cells abu undance
significcantly differred (P≥0.05
5) between the two temmperatures only at conncentrationss 30 and
40 g/L oof extract (TTable 3).
It was also noted that the reecorded abuundances off E. coli siignificantly differed frrom one
extract concentration to anoth
her (P<0.05)), at the incuubation tem
mperature off 4 ° C as well
w as at
23 ° C, when the cells
c presen
nt belongs tto a single species.
s The same obsservation was made
for A. hydrophilaa (Table 4)). On the other hand d, when th he two baccterial speccies are
simultanneously present, the abundancess of the reecorded cells do not differ significantly
(P>0.055) from one extract con
ncentration tto another (Table
( 4).

Table 44. The “P” values

v indiccating the ddegree of sig
gnificance of
o the differrence relateed to the
comparrison of the cells abund dances regisstered amon ng the seedss extract conncentration
ns after 6
hours, iin monospeccies and bisspecies cellss incubation
n conditionss, at 4 °C annd 23 °C
Cells sppecies consiidered and incubation
i ccondition Incubation tem
mperatures
Cells sppecies
Cells inccubation coondition 4 °C
C 23 °C
C
consideered
Mon nospecies ceells 0.0044* 0.004
4*
E. coli
Biispecies cellls 0,06
60 0.067
7

A. hydro
rophila Mon
nospecies ceells 0.0055* 0.004
4*
Biispecies cellls 0.07
70 0.070
0
*: P ≤ 00.05; df (deggree of freed
dom) =19 (nnumber of observation
o ns).

3.4 Disccussion
The baccteria decayy curves obtained show w that the Moringa
M oleeifera extracct can be used
u as a
natural,, green alteernative forr effectivenness water treatment. The antibaacterial acttivity of
Moringga oleifera seed
s extract would be ddue to the presence
p of a short, catiionic protein within
the seedd. This prottein, comm monly knownn as the Mo oringa oleiffera cationiic protein (MMOCP),
has beeen shown too cause bactterial cell daamage throu ugh rapid flocculation
f and fusionn of their
inner annd outer meembranes (S Shebek et all., 2015). Th
his protein would inhibbit bacteriall growth
and thaat stronger concentrattions faciliitate higherr bactericid dal propertiies. Howev ver, this
activityy is dependdent on thee bacterial load, and increased bacterial
b cooncentration n would
require a larger dosse or a stron
nger concenntration of th
he seed extrract.
The M M. oleifera extract inh hibitory effe
fect against bacterial cell wouldd be linked d to the
phytochhemicals. Working
W on
n the structuure-functionn characterization andd optimizatiion of a
plant-deerived antibbacterial peptide, Suarrez et al. (2008) noted that one oof the seed peptides
p
mediatees both the sedimentati
s on of suspeended bacterrial cells an nd a direct bbactericidal activity,
raising the possibillity that the two activitiies might bee related. In
n addition, SShebek et all. (2015)

24 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

indicateed that a caationic proteein isolatedd from the seeds

s of thee Moringa ooleifera treee has an
importaant antibacteerial activity
y. Its dominnant mechannism of antimmicrobial acctivity is meembrane
fusion. Its activity includes addsorption, sttalk formation, and fusiion betweenn membranees.
Temperrature is onne of the explanatory
e parameterss of changees in bacter
erial abundaances. It
indirecttly influencees bacterial productivitty by modiffying the ph
hysical and cchemical prroperties
of their environment. In this study, tempeerature appeears as an immportant facctor involveed in the
cellularr inhibition.. The incubbation tempperature inccreases the effectiveneess of the aqueous
extract of Moringga oleifera seeds, the inhibition being conssiderable att the psych hrophilic
temperaature (Maugguin et al., 2004).
2
It is obsserved that an
a increase in the conccentration off seeds extraacts significcantly impro oved the
seeds exxtract inhibiitory activitty (Table 2).. The seeds have been indicated as containing calcium,
magnessium, phospphorus, copp per, vitaminns (A, B and d E), and are also rich iin organic elements
e
(Hans & Bindandaa, 2003). Th hese differennt secondarry metabolittes in excesss could acccumulate
in the bbacterial ceell walls an nd become toxic. Bactteria inhibittion could also be du ue to the
presencce of α-L-rhhamnosyloxy benzyl isoothiocyanatte molecules which aree found in th he seeds
and whoose antibactterial and an ntifongic prroperties haave been desscribed (Cac
aceres, 1991). These
molecules are solubble and are positively
p ccharged. Theey can easily cross the bbacterial meembrane
to bind to cation prroteins negaatively charrged on the cells memb brane surfacce and supp port their
inhibitioon (Thevisssen et al., 19996).
Jahn (19988) reporteed that wateer disinfectioon by Morin nga seeds reequires relat
atively high doses of
200 g/L
L of extract to t have a geermicidal efffect. In thiss study, from
m 1 g/L to 400 g/L of exttract, the
bacteriaal inhibitionns varying frrom 55.12% % to 99.9% forf E. coli, and
a from 13 .2% to 97.8% for A.
hydrophhila. This suggest
s thatt the enviroonment, as well as thee genetic chharacteristiccs of the
bacteriaa or other abiotic
a propperties of thhe water used, could affect
a the acctivity of th
he seeds
constituuents and otther parts off the plant ((roots and fllowers). It has
h also beeen indicated d that the
content of chemicaal componen nts varies w
with Moring ga species (JJahn, 1988)..
The anttimicrobial activity off Moringa eextracts wass previously y attributedd to plant-p
produced
benzyl isothiocynaate derivativ
ves (Eilert eet al., 1981). Suarez et al. (2003) sshowed thatt at least
part off the antimmicrobial activity
a of Moringa seedss extraact may sttem from Flo-like
polypepptides. Accoording to Zaasloff (20022), they act by forming g essential eenzymes, leading to
cell deaaths.
Chuangg et al., (20007) has stuudied the m mode of attaack of Moringa oleiferra seeds ex xtract on
fungus. The resultts showed th hat the cytooplasmic membrane
m of the fungaal cell was ruptured
r
and thee intercellullar compon nents were sseriously damaged aftter treatmennt with M. oleifera
seed crrude extracct. Howeveer, the interrcellular co omponents did not leeak out. Based on
previouus studies of
o cell lysiss pathways of antimicrobial peptides on baccteria (Chaan et al.,
1998; CChen et al.,, 2003), this indicated that extraccted compounds interaacted with the t lipid
bilayerss in membrranes leadin ng to the seeparation of
o the two membranes
m s (outer and d inner).
Subsequuently, wateer dips in to
o the cell, w
which causess cell to sweell more andd leads to deeath.
It has bbeen noted that
t incubattion temperrature relativ
vely impactts the seedss extract acttivity. In
bispeciees cells incuubation con
ndition, the high percen ntages of in
nhibition obbserved at 4 °C and

25 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

23 °C iin the preseent study may

m be expllained by th he fact that these tempperatures acccelerate
bacteriaal metabolissm, with to
oxic produccts being metabolized.
m Lessard annd Sieburth
h (1983)
suggestted that low
w temperaturres can leadd to better survival by the
t fact thatt the metabolism of
mesophhilic bacteriia is very slow,
s the tooxic produccts present at the samme time as the
t high
concenttrations of nutrients are
a only veery slowly metabolized. The bioochemical reactions
r
underlyying cellulaar metabolism depennd on the activities of the enzzymes, wh hich are
themsellves largelyy influenced
d by the envvironmental temperature (Regnaultt, 2002; Maauguin et
al., 2004).
In monoospecies ceell incubatio on conditionn as well ass in bispeciees, a slight cells regrow wth was
sometimme noted. Itt is known thatt bacteriaal cells are made up off a variety oof moleculees, some
of whicch may be nutritious
n (H
Holt et al., 22000; Main nil, 2005; McInerney
M ett al., 2008).. During
seeds exxtract activiity, the cell inhibition ffollowed by y the degraddation of som
ome cells coould lead
to the rrelease intoo the mediu um of cellullar compou unds. Surviv ving cells wwould probaably use
some oof these eneergetic consstituents of these comp pounds for the time oof their surv vival. In
bispeciees cell incuubation con ndition, theese slight cells
c regrow
wth could aalso be duee to the
metabolism of the cells of botth species. T This phenomenon is usually know wn as a synthrophy.
In this fform of association, th he catabolism m products of one wou uld become a source of carbon
and energy for thhe other (M McInerney et al., 200 08). This form
f of mu
mutualism hash been
mentionned betweeen Enterobaacteriaceae bacteria an nd lactic accid bacteriaa by other authors
(Jorgennsen et al., 2004).
2 In addition,
a somme molecu ules from thhe extracts can be a so ource of
nutrientts and alloww the cell reegrowth, ass it was indicated that a seed of M M. oleifera contains
organic compoundds (Ferreira & Janick, 11996). The doublingd off the bacteriial populatio
on could
contribuute to a rapiid depletionn of nutrientt moleculess and an acccumulation oof metaboliic waste.
This woould lead too the slight aspect
a of thee cells regro
owth noted.
4. Concclusion
This stuudy showedd a significan
nt effect of bacterial in
nhibition of the
t aqueouss extract of seeds of
Moringga oleifera on
o the two bacteria sppecies studiied. Cell ab bundances ddecreases gradually
g
with ann increased seeds extraact concentrration. How wever, a maarked regrow
owth of E. coli
c was
sometimmes noted. When
W cells belonging tto one species were used, E. coli ccells abundances as
well as those of A.
A hydrophilla were som metimes relatively low wer at 4 °C compared to those
recordeed at 23 °CC. The seed ds extract cconcentratio ons also plays an impportant rolee in this
inhibitioon.
Acknow
wledgemen
nt
We exteend our thaanks to the authorities
a oof the Labooratory of Microbiology
M gy and Envirronment
of Centtre Pasteur of Cameroo on, the Univversity of Yaounde1
Y (C
Cameroon) and the Un niversity
Clermoont Auvergnne (France), for their loggistic and material
m con
ntributions.
nces
Referen
Adrianaa, B., Almoodovar, A. N.
N M., Pereeira, C. T., & Mariangla, T. A. (20007). Antim microbial
efficacyy of Curcuma zedoa aria extracct assessedd by linearr regressioon compareed with
commerrcial mouthh rinses. Bra
azilian J. M
Microbiol, 38
8, 440-445. https://doi..org/10.1590/S1517
-8382200070003000011

26 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

Allison, S. D., Wallenstein,

W M. D., & Bradford, M. A. (20
010). Soil ccarbon resp
ponse to
warminng dependdent on microbial
m physiology
y. Nature Geosciennce, 3, 336-340.
3
https://ddoi.org/10.11038/ngeo84
46
APHA (American Public Heaalth Associaation). (2012 2). Standard
d methods ffor the exam
mination
of waterr and wastee water (22tth ed.). Wasshington DC
C.
Bennettt, R. N., Mellon,
M F. A.,
A Foidl, N.., Pratt, J. H.,
H Dupont, M. S., & Perkins, L. (2003)
Profilinng glucosinnolates and
d phenolicss in vegettative and reproductiv ive tissues of the
multi-puurpose treees Moringa oleifera L
L. (horserad dish tree) an
nd Moringaa stenopeta ala L. J.
Agric. FFood Chem,, 51(12), 35
546-3553. hhttps://doi.orrg/10.1021/jjf0211480
Caceress, A. (1991)). Pharmaco ological prooperties of Moringa olleifera: Effeect of seed extracts
in the trreatment off experimenttal pydermiia. Fitoterap
pia, 62, 449-454
Chan, S. C., Yauu, W. L., Wang,W W., Smith, D.,, Sheu, F. S., & Cheen, H. M. (1998).
Microsccopic observations off the diffeerent morph hological changes
c byy the anti-b bacterial
peptides on Klebsiella pneumo oniae and H
HL-60 leukeemia cells. J.
J Peptide SScience, 4, 413-425.
4
https://ddoi.org/10.11002/(SICI))1099-1387((199811)4:77%3C413::A AID-PSC1660%3E3.0.C CO;2-W
Chen, X X.-L., Zhang, Y.-Z., Gao, P.-J.,, & Luan, X.-W. (20 003). Two different proteases
p
produceed by a deep-sea psycchrotrophicc bacterial strain,
s Pseu
udoaltermonnas SM991
13. Mar.
Biol, 1443, 989-993. https://doii.org/10.10007/s00227-0
003-1128-2
Chuangg, P.-H. Chi--Wei, L., Jiaa-Ying, C., Murugan, M.,
M Bor-Jinn n, S., & Hueeih-Min, C.. (2007).
Anti-funngal activitty of crude extracts andd essential oil
o of Morin nga oleiferaa Lam. Biorresource
Technollogy, 98, 2332-236. https://doi.org/110.1016/j.biiortech.2005.11.003
Devêvre, O. C., & Horwáth, W. W R. (20000). Decompoosition of riice straw annd microbiaal carbon
ficiency undder differen
use effi nt soil tem
mperatures and
a moisturres. Soil B Biol. Biocheemy, 32,
1773-17785. https:///doi.org/10.1016/S00388-0717(00)0
00096-1
Edima, H. Tatsadjiieu, L., Mb bofung, C., & Etoa, F. X. (2010). Anti-bacterrial profile of some
beers annd their effeect on somee selected paathogens. Affr. J. Biotecchnol, 5938--5945.
Eilert, U
U., Wolters, B., & Nah
hrstedt, A. ((1981). Thee antibiotic principle off Moringa Oleifera
ant Medica, 42, 55-61. https://doi.o
and Mooringa Stenoopetala. Pla org/10.10555/s-2007-9771546
Ferreiraa, J. F. S., & Janick, J.
J (1996). D
Distribution
n of Artemissinin in Arrtemisia ann
nua (pp.
579-5844). In New Crops
C Arlin
ngton, VA.
Ferreiraa, P. M. P., Farias, D. F., Oliveirra, J. T. A.,, & Carvalh ho, A. F. U
U. (2008). Moringa
M
oleiferaa: bioactive compound ds and nutriitional poten ntial. Rev. Nutr.
N Camppinas, 21, 431-437.
4
https://ddoi.org/10.11590/S1415-527320080000400007
Fuglie, L. J. (20011). The Mira
acle Tree: M
Moringa olleifera -Natural Nutritiion for the Tropics.
Church World Servvice, Dakar,, Senegal
Hans, M
M., & Binddanda, M. (22003). Troppical Natura
al Medicinee: How to H
Heal With Tropical
Plants. Schafweidee, Winnendeen.

27 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

Holt, J.. N. Krieg, P., Sneath,, J., & Stalley, W. S. (2000).

( Berrgey Manuaal of Determ
minative
Bacteriology (9th ed.).
e Lipponncott William ms and Willkins, Philad
delphia.
Jahn & Al, A. S. (1988).
( Usinng moringa seeds as cooagulant in developingg countries. Journal
AWWA, 6, 43-50. https://doi.o
h rg/10.1002//j.1551-8833.1988.tb033052.x
Jorgenssen, P. R., Larry, D. M., & Kisstrup, J. (20 004). Aquifer Vulneraability to Pesticide
P
Migratiion Throughh Till Aquittards. Grounnd Water, 5,
5 841-855. https://doi.oorg/10.1111
1/j.1745-
6584.20004.t01-3-.xx
Kuete, V
V. (2017). Medicinal
M sp
pices and vvegetables from
f Africa. Therapeuttic potentiall against
metabolic, inflamm
matory, infecctious and ssystemic disseases. Acad
demic Presss, London.
Lacassee, D. (2004)). Introductiion to food m
microbiolog
gy. Québec..
Lessardd, E., & Sieebuth, J. (19
983). Survivval of naturral sewage populations
p s of enteric bacteria
in diffuusion and bath
b chamb bers in thee marine en nvironment. Applied aand Enviro onmental
Microbiiology, 45, 950-959
9
Mahmoood, K. T., Mugal,
M T., & Ul Haq, I . (2010). Moringa
M oleiffera: a natuural gift-a reeview. J.
Pharm. Sci. Res, 2, 775-781
Mainil, J. (2005).. Génétiquee et régulaation de la virulence bactériennee: Vers la version
moléculaire des poostulats de Koch.
K Annalles de Médeecine Vétériinaire, 149, 24-32
Marchaal, N., Bourrdon, J. L.,, & Richarrd, C. (1991). Milieux de culturee pour isoleement et
identificcation des bactéries.
b Paris.
Martin, D., & Segalen, P. (1966). C
Carte pédollogique du Camerounn oriental: Notice
explicattive. Yaoundde.
Mauguiin, G., Dellion, N., & Corsin, P P. (2004). La tempéraature de l’’eau, un paaramètre
importaant pour la production
p d’eau destiinée à la co
onsommatio
on humaine.. L’eau, l’In
ndustrie,
les Nuissances, 27, 23-29.
M'Biandoun, M., Guibert,
G H.., & Olina, J.-P. (20022). Caractérrisation du climat dan
ns quatre
terroirs en région soudano-sah
s hélienne duu Nord-Cam meroun et coonséquencess pour l'agrriculture.
Actes ddu colloque sur «Savannes africainnes: des esp
paces en mutation, dess acteurs faace à de
nouveauux défis», mai
m 2002, Maroua,
M Cam
meroun.
Mbikayy, M. (2012). Theraapeutic pootential of Moringa oleifera lleaves in chronic
hypergllycemia andd dyslipidem
mia: A revieew. Front. Pharmacol,
P 3, 24. httpss://doi.org/1
10.3389/
fphar.20012.00024
McInernn, M. J., Struchtemey
S yer, C. G., Sieber, J., Mouttaki, H., Stams, A. J., Schhink, B.,
Rohlin, L., & Guunsalus, R. P. (2008). Physiology y, ecology, phylogenyy, and geno
omics of
microorrganisms caapable of syntrophic
s metabolism
m. Annals of o the New
w York Acad demy of
Sciencees, 1125, 58--72. https:///doi.org/10. 1196/annalss.1419.005
Prabhu,, K., Muruggan, K., Naareshkumarr, A., Ramaasubramaniaan, N., & B
Bragadeesw
waran, S.

28 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

(2011). Larvicidall and repellent potenntial of Mo oringa oleif

ifera againsst malarial vector,
Anopheeles stephennsi Liston (Insecta:
( D
Diptera: Cullicidae). Asian Pac. J. Trop. Bioomed, 1,
124-1299. https://dooi.org/10.10
016/S2221-11691(11)600
009-9
Regnauult, J. (2002)). Eléments de microbioologie et d’’immunolog
gie (p. 601).
Rodier, J. (2009). L’analyse
L de
d l’eau (9thh ed.). Duno
od, Paris.
Sairam,, T. V. (19999). Home Remedies, vol II: A Handbook
H of Herbal C
Cures for Commons
Ailmentts. Penguin,, New Delhii, India.
Shebek, K., Schanntz, A. B., Sines, I., L Lauser, K., Velegol, S.,
S & Kumaar, M. (201
15). The
flocculaating cationic polypeetide from Moringa oleifera seeds damaages bacterrial cell
membraanes by causingg membrrane fussion. Lanngmuir, 31, 449
96-4502.
https://ddoi.org/10.11021/acs.lan
ngmuir.5b000015
Steinweeg, J. M., Pllante, A. F.,, Conant, R
R. T., Paul, E.
E A., & Tan naka, D. L. (2008). Pattterns of
substratte utilizatioon during long-term incubation ns at differrent tempeeratures. Sooil Biol.
Biochemm, 40, 27222-2728. https://doi.org/110.1016/j.so oilbio.2008..07.002
Stohs, S
S. J., & Harrtman, M. J. (2015). Reeview of thee safety and
d efficacy oof Moringa oleifera.
o
Phytothher. Res, 29,, 796-804. https://doi.o
h org/10.1002//ptr.5325
Suarez, M., Entenzza, J. M., Doerries,
D C., Meyer, E., Bourquin, L., Sutherlaand, J., Marrison, I.,
Moreilllon, P., & Mermod, N. N (2003). Expression of a plaant-derived peptide haarboring
water-cleaning and a antim
microbial activities. Biotechn nol. Bioenng, 81, 13-20.
https://ddoi.org/10.11002/bit.105
550
Suarez, M., Haennni, M., Canaarelli, S., FFisch, F., Ch hodanowskii, P., Servis , C., Michielin, O.,
Freitag,, R., Moreiillon, P., & Mermod, N. (2008). Structure--function chharacterizattion and
optimizzation of a plant-derive
p d antibacterrial peptide. Antimicrob
bial Agents and Chemo otherapy,
49, 38447-3857. httpps://doi.org
g/10.1128/AAAC.49.9.38 847-3857.20005
Sunda, M., Rosilloon, F., & Tab ba, K. (20008). Contribution à l’étu
ude de la déésinfection de l’eau
par phootosensibilissation avec des extraitss de plantes.. European J. Water Quuality, 39, 199-209.
1
https://ddoi.org/10.11051/water/2008006
Tamsa, A. A. Nolaa, M., Lontsi, D. C., N Nandjou, N. R. V., Nou ugang, M. EE., Bricheux x, G., &
Sime-N Ngando, T. (2013).
( Commparison off the inhibitiion of comm
mensally annd enteropatthogenic
E. coli strains in thhe presence of Eucalypptus microcorys leaves extract in aaquatic miccrocosm.
Int. J. C
Curr. Microbbiol. Appl. Sci.,
S 2, 80-996.
Thevisssen, K. A., Ghazi,
G G. W.,
W De Samb mblanx, C., Brown,
B R. W.,
W & Brockkaert, W. F.. (1996).
M Funggal membraane responsees induced by plant deefensins and
d thionins. JJ. Biol. Cheem, 217,
15018-115025. https://doi.org/1
10.1074/jbcc.271.25.150
018
UN/WW WD. (2006)). Facts off water Ressources. Reepport 2 UN
N/WWD, A Summary
y of the
united N
Nations World Water Developmen
D nt, UN, Neww York.
Weathers, P. J., & Reed, K. (2014). Whoole plant ap
pproaches to
o therapeutitic use of Artemisia

29 http://jab.macrrothink.org
 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

annua LL. (Asteracceae). In T. Aftab, J. F

F. S. Ferreirra, M. M. A.
A Khan, & M. Naeem m (Eds.),
Artemissia annua. Pharmaco ology and Biotechnollogy. Sprin nger, Londoon. https:///doi.org/
10.10077/978-3-6422-41027-7_4 4
WHO. (2017a). Water
W quality
y and healtth - Review w of turbidiity: informaation for reegulators
and water suuppliers. Retrieved from
www.who.innt/water_san
http://w nitation_heaalth/publicaations/turbid
dity-technicaal-brief/en/
WHO, (2017b). Guidelines
G fo
or drinking--water quallity. Fourth edition inccorporating the first
addenduum. Geeneva: World Health Organizaation. R
Retrieved from
http://w
www.who.innt/water_san
nitation_heaalth/water-q
quality/guideelines/en/
WHO. (2002). Organisation
O n Mondialee de la Santé (OMS
S) Rapportt sur la medicine
m
traditionnnelle: Besooins et poteentiel. N°- 44.6P
WHO. ((2011). Guiidelines for drinking waater qualityy, 4th edition
n. WHO, Geeneva, Swissserland.
Zasloff,
f, M. (2002)). Antimicro
obial peptiddes of multiccellular organisms. Naature, 415, 389-395.
3
https://ddoi.org/10.11038/415389a

Copyriight Disclaiimer
Copyrigght reservedd by the auth
hor(s).
This arrticle is ann open-acceess article ddistributed under the terms andd conditionss of the
Creative Commonss Attribution n license (hhttp://creativ
vecommonss.org/licensees/by/3.0/).

30 http://jab.macrrothink.org