1992
JUlI/'Ilal uf Fuod Protenion. \101. 77. No.1 /. 20/4. Pages /992-1997 doi: J 0.43 15/0362-028X.JFP-1 3-483
Copyright ©, International Association for Food Protection
Research Note
Staphylococcal Enterotoxin A Gene-Carrying
Staphylococcus
aureus
Isolated from Foods and Its Control by Crude Alkaloid
f.Jm Papaya Leaves
UTA HANDAYANl,' DIJ)AH ｎｕｒｆｾｉ｜ＮｒｉｄａｈＬＧﾷＲ＠ AND HARSI D. KlJSlJMANINGIWMI2'i<
'Food Science SlUdy Program. DCl'urfmel?! o(Food Science and Technology. Bugor AgriclIlrural [fniversiTY. Cuml'!/.\ IPB Dal"!1wgu. P.O. Box 220. Bogor 16002. Indonesia. and 2Southcu.\{ Asian Food 011(1 Agriwlrurul Science und Teclinologv (SD1FAS"T) Ccnle/", Bogor AgriclIltllral Unil"ersiry. SD1FAST
Center Building, .fl. PI/spa No I. Callipus flJB Damwga. Bogor /6680, Indonesia MS 13-4H3: Received 10 November 20 I 3/Accepted 27 .June 2014
ABSTRACT
Staphylococcus aureus is a known pathogen causing intoxication by producing enterotoxins in food. Staphylococcal enterotoxin A is one of the enterotoxins commonly implicated in staphylococcal food poisoning. The ability of crude alkaloid extract from papaya leaves to inhibit the growth of S. ({[{reus and staphylococcal enterotoxin A synthesis was investigated. Staphylococcal cntcrotoxin A gene-carrying S. aurells was isolated from raw milk and rcady-to-eat food s. Cillde alkaloid was extracted from ground, dried papaya leaves using ultrasonic-assisted extraction, and aMIC of the alkaloid was determincd by the broth macrodilution mctllod. Furthermore, S. (lweus isolate was exposed to the crude alkaloid extract at onc- and twofolclMIC, and the expression of sea was subsequently analyzed using a quantitative reverse transcription real-time PCR. Ten isola tes of S. (wreus were obtained , and nine of those isolates were sea carriers. The yield of crude alkaloid extract was 0.48 to 1.82% per dry weight of papaya leaves . A MIC of cl1lde alkaloid to S. i/li/"eus was 0 .25 mg/ml. After exposure to the alkaloid at 0.25 and 0.5 mg/ ml for 2 h, a significant increasc in cycle threshold values of sea was observed, The sea was cxpressed 29 and 41 times less whcn S. (lurellS was exposed to crude alkaloid at one- and twofold .MTC respectively. This study revealed that crude alkaloid of papaya leaves could control staphylococcal enterotoxin A gene-carrying S. (lureus by suppressing the expression of s('a, in addition to the ability to inhibit the growth of S. aureu,\". The expression of sea was successfully quantified.
Staphylococcus aI/reus is a known pathogen, causing intoxication by producing staphylococcal enterotoxin in food. Staphylococcal enterotoxin A is one of the staphylo-coccal ente'rotoxins commonly impLicated in staphylostaphylo-coccal food poisoning (8, 28) that is found in raw milk (30) and ready-to-eat food (26). Staphylococcal enterotoxin is expressed from the mid-exponential phase of growth, synthesized from the sea gene. Staphylococcal enterotoxin A acts as a superantigen and stimulates the release of inflammatory cytokines, causing symptoms of poisoning, sllch as nausea and vomiting (29).
Papaya plants (Carica papaya) are widely cultivated in tropical cOllntTies. In addtion to the usefulness of papaya fruit, papaya leaves are also consumed as vegetables, used to improve the tenderness of meat, and used as traditional medicine (l, 19). To improve meat lenderness , the meat is usually wrapped in papaya leaves or marinated with pastes made from papaya (I. 25 }.Furthermore, different solvent extracts of papaya leaves have been reported to have . antimicrobial activity against both positive and gram-negative bacteria, such as Escherichia coli. S aureus, Bacillus cereus. Klebsiella pneumolliae. anel Pseudomonas
" Author for correspondence. Tel: +62 251 8626725; Fax: -1- 62 251 8626725; E-mail: [email protected].
aerllginosa (3). The antimicrobial activity of plant extracts are usually associated with the phytochemical content, i.e., t1avonoids (13) , tannins, terpenoids. and alkaloids (9!. Tannins and terpenoids can ･ｬｩ ｾｲ ｵｰｴ＠ cell membranes (3. 9), whereas alkaloiels have been shown LO intercalate into DNA and inhibit DNA synthesis (6). Because papaya leaves have been reported to contain alkaloids, namely. carpai ne. pseudocarpaine, and dehydrocarpaine I anel II (/9), there is a potential action of papaya leaf extract to inhibit staphylococcal enterotoxin A production through alkaloid activity .
1. ,Fooel Prot., VoL 77, No. II SEA GENE-CARRYING S AUREUS CONTROL t3Y CRUDE ALKALOID 1993
TABLE I. Nucleotide sequences of' primers used in PCR ana/vsis (20)
Gene Primer Sequence (5' - ? 3') PCR product
sea
16S rRNA
SEAl SEA2 16sF 16sR3
TTGGAJil\CGG'I'TAAJ-'\,,4CGlo,.A Gl\Z\CCTTCCCI\ ｔｃｉｬｉｾａａｬ｜ｃＷ｜＠
CCGCCTGGGGAGTACG ]\"AGGGTTGCGCTCGTTC;C
120 bp
240 bp
leaves to inhibit the growth of S. {lUrClfS and staphylococcal
enterotoxin A synthesis. Staphylococcal enterotoxin A synthesis was evaluated by relative quantification of sea expression by the qRT-PCR technique.
MATERIALS AND METHODS
Isolation and identification of S. aureus from food samples. S. aI/reus was isolated from raw cow' s milk and ready-to-eat foods, i.e., egg dishes, sauteed chicken cuts. and chicken satay, purchased from a livestock and street food vendor in Bogor, Indonesia. The isolation proccdure was according to the modified
Bacteriological Analytical lvlanual (4) A sample (25 g or 25 ml)
was added to 225 ml of n01111al salinc and homogenized in a stomacher (BagMixer 400P, Interscience, Saint Nom, France). 'fhe homogenate was serially di luted and spread onto Baird··Parker agar (Oxoid Ltd., Hampshire . UK) supplemcntcd with egg yolk tellurite. Plates were incubated at 3TC for 18 to 24 h. and then presumptive colonies were picked and streaked on mannitol salt agar (Ox()id Ltd.). Typical colonies Oil mannitol salt agar were then tested f(Jr the production of catalase using Staphylase test kit (Oxoid Ltd.) and identified using /\P[ Staph (bioMcrieux, Inc .. Durham, NC), according to the manufacturer's instl1lctions.
Detection of 168 rRNA and staphylococcal enterotoxin A encoding gene. The DNA of S. aurcus ATCC 25923 and S. aureus
isolates from foods was isolated using a method adopted from Mason et al. (23) , with minor modification, i.c., thc use of 5 pI of lysostaphin (10 rng/rnl) was substituted with 100 pI of lysozyme (Bio Basic Canada Inc., Markham, Ontario, Canada) solution (10 lng/ml). The DNA pellet was then dried and resuspended in 30 pi of sterile water. The amplification of the gene encoding 16S rRNA and staphylococcal enterotoxin A was performed us ing the Thermal Cycler 2nO (Applied Biosystems, Foster City, CAl. peR master mix consistcd of 12.5 ftl of DreamTaq Green master mix (Thermo Fisher Scientific, Waltham , MA), 1 pI of each primer (10 pM; Table ll, 2 pI of DNA template, and S.S fd of nuclease-free water (Thermo Fisher Scientific). Cycling parameters were one denatur-ation cycle fo r .5 min at 95 "C and 30 amplification cycles for denaturation (1 min at 95'C), anncaling (1 min at 55' ), extension
(1 min at 72'C), and termination for 5 min at 72 "C. adopted from Lee et a1. (20). The amplification product was analyzed by 1.5% agarose gel electrophoresis at 120 V for 35 min.
The 16S rRNA gcne sequence of S. aurells isolates was obtained by sequencing and analyzed by nucleotide BLAST program. Phylogenetic analysis was conducted between 16S rRNA sequence of S. alll'ellS isolates and 16S rRNA sequence of S. aurcus
ATCC 25923 that was obtained from the GcnBank database. S.
aurcils isolates that had the closest relat ionship with S. aureus '
ATCC 25923 would be used in the next stage of this study .
Preparation of crude alkaloid. Healthy. disease free, and mature fresh leavcs of Cal ina papaya (TPB9) wcrc collected from University Fa11n, Bogar Agricultural University (Bugor, Indone-sia). The fresh leaves were rinsed thoroughly two to three times
under running tap water and once with sterile water and dried at 55"C for 22 h Llsing a vacuum oven (VWR AI43 A- 143. Sheldon Manufacturing. Inc. , Cornelius, OR) (7) . Dried leaves were ground into a fine tcxture using an electric blender. Tbe dried leaves wcrc stared in sealed and labeled containers for latcr use. Moisture content of fresh and d ried leaves was detennined llsing an oven method, according to the /\OAC International (2).
The crude alkaloid was extracted by adopting the extraction method, described by Dji lani et al. (10) Ten grams of dried papaya leaves was suspended in 400 ml of sodium dodecylsulfate (Merck & Co., Kenilworth, NJ) solution (2%. mass/vol) in an Erlenmeyer llas k and sonicatcd for 2.5 h at a temperature of 25 to 35' C in an ultrasonic bath (Bransonic Ultrason ic Cleaner mode l S510E MTH . Branson Ultrasonic Corporation, Danbury, CT). The extract was separated by Whatrnan no. I filter paper (Sigma-Aldrich. St. Louis, MO), and the residual materials were washed w ith 20 rnl of pure water The solution of combined filtrates was acidified with sulfuric acid (Merck & Co.) solution (2(k, vol/vol) to a pH of 3 to 4, and the alkaloids were precipitated with 15 ml of Mayer's reagent. The precipitate was then dissolved in sodium carbonate (Merck & Co.) solution (5%, mass/vol) and extracted by CHCI, (,1.'1'. Baker. Center Valley, PAl. The organic layer was washed by water to neutral pH, dried with Na2S(}, (Merck & Co.) to remove the rem aining water, and concentrated to dryness using a rotary evaporator (Butchi Rotavapor R-nO , BCTCHT Labortcchnik AG, Flawil. Switzerland) and N2 gas to obtain alkaloids. The extracts were collected from several extraction processes and combined into one solution using dimethyl su]f()xidc (Merck & Co.) as the so lvent.
Preparation of baderial inoculum. The 18- to 24-11-0Id staphylococcal enterotoxin A gene-carrying S. (lllreliS isolate culture in tryptic soy broth (TSB; Oxoid Ltd.) , incubated at 37' C. was centrifuged at 9,SOO x g for 10 rn in (Hennle Z3S3K. Hemlle Labortec hnik GmbH, Wehingen, Germany). The sllpematant was discarded. and the pclIet was resuspended in normal sa line solution. The ccll suspension was adjusted to an optica l density at 625 mil of OJJ8 that ,vas equivalent to the McFarland 0 .5 turbidi ty standard (lOx CFU/ml).
:vIle deteTminatiol1. M Ie of crude a lkaloid was determined lIsing the broth macrocJilutiun method (24 J. One hundred microlitcrs of bacterial suspension (106 CFU/rnl) was inoculatcd
into I rn l of TSB containing ciUde alkaloid at 0.125, 0.25, 0.5 and I rngirni. A tube containing inoculum and TSB (0 mg/ml of crude alkaloid) was Llsed as a control. After incubation at 3TC at ISO IVI11 for 24 h, bacterial culture at each cnJcle alkaloid concentration and controJ were diluted and spread on a tryptic soy agar (TSA) plate. The plates were incubated at 3TC for 48 h. and the bacterial cell nuinbcr was counted. ?vllC(J() was determined as the lowest concentration that resulted in inh ibition of 90% of tested isolates compared with the number of the initial inoculum (/2). Each experiment lVas carried out three times.
[image:2.595.88.576.59.133.2]1994 HANDA YANI ET AL
carrying S. aurells suspension (108 CFU/ml) was inoculated into 5 lIll of TSB without crude alka loid (control) and with crude alkaloid at one- and ｴｷｯｪｾＩｬ､＠ MIC. The tubes were inc llbated at
room temperature at 150 rpm for 2 h. The short exposure was designed to give mild exposure to the cells so that the reduction oj' cell number did not occur, but it could still affect the gene expression. Bacterial cell number. before and after the 2-h exposure, was determined using tile spread plate technique on a TSA plate.
RNA extt'action and d)NA synthesis. Two m ill iliters of treated S. au reus clliture with crude alkaloid was centrifuged at 5 ,000 ;< g for 5 min. The S. aI/reus pellet was then sllspended in
LOO ftl of 1;< TE buffer (PEQLAB Bio teehnologie GmbH, Erlangen, Gennany) and 30 [ti of lysozyme (PEQLAB Biotedmo-logie GmbH) solution (10 mg/ml). The RNA was extracted using a peqGOLD Bacterial RNA Kit (PEQLAB Biotechnologie GmbH), according to the manufacturer's instructions. The residual DNA was digcsted llsing a DNascI, RNase frce (Thermo Fisher Scientific) trcatment. RNA concentration and purity (/\260/A2.10 and /\261//\2811 ratios) wcre assessed llsing a UV-1800 Spectropho-tometer (Shimadzu, Kyoto, Japan).
Onc microliter of total RNA was then applicd in a 20-p.l
rcversc transcription to synthcsize cDNA using a RcvertAid F irst Strand eDNA Synthesis Kit (Thermo Fisher Scientific). The reverse transcription reactions were perfomled for I h at 42"C, and the reverse transcriptaseinactivated for 5 min at nrc, as described
in the manufacturer's protocol.
Relative quantification of sea expression. The qPCR analysis was perfoITned lIsing Swift Spectrum Themal Cycler 48 (Esco Healthcare Pte. Ltd., Singapore). PCR master mix consists of I [1.1 of the relevant cON A. 0.8 pI of each primer (10 pM; Table I),
10 ｾｌｉ＠ of KAPA SYBR FAST qPCR Kit Master (K.appa Biosystems, Woburn, MA), and nucleasc-free water, up to 20 pI
of reaction volume . Cycling parameters were one de naturation cycle for 5 min at 95"C and 45 arnp lificatioll cycles for denaturation (I min at 95'C), annealing (I min at 55'C), extension (I min at 72°C), and termination for 5 min at 72'e (20 J.
Fluorescence rcadings were taken after each extension step. During'relative quantification by the qRTPCR techn ique, the changes in gene exp ression in the sample treatcd with crude alkaloid wcre measured relative to the calibrator sample and the reference gene . The C r values of the target gcne were compared dircctly to an internal reference C r, and the resu lt s were expressed as ratios of the target-specific signal to the internal reference (36).
The expression of sea was caleulated relative to the calibrator sample and an internal control Llsing a comparative C r mcth od (2 -MCT ) (33) . In th is study. the 16S rRNA gcne was Llsed as the reference gene (internal contro l), whereas the sample without the crude alkaloid addition was used as the calibrator sample. The experimen t was performed three times .
Data analysis. Data were analyzed by SPSS vers ion 16.0 (Ch icago, IL) using one-way analysis of variance. Significance was defi.ncd at P < 0.05 by using Duncan's multiple range test. The results were expressed as means
±
standard deviations.RESULTS AND DISCUSSION
Presence of sea carrie r S. aureus. All food sam ples were found to be contaminated with S. aureus. There were a total of 78 presum pti ve S. (fUJ'CliS isolates on Baird-Parker agar, but on ly 65 isolatcs showcd a typical colony of S.
J. Food Prot. , Yo!. 77, No. II
TABLE 2. Tile prescllce 165 rRNA and sea gene ill S. aurells iso/ares ({lid S. aureus ATCC 25923"
S. aureus i\ TCC 25923
S. allrcus Sl
S. ilurCllS S4
S. ourCII.\' S to S. <1l1reus TB I
S. aurC/lS TB 10
S. ill/rellS UAI
S. aurells UA2 S. (II/reus U i\ 13 S. allreus SJ 1 S. (/urCIIS S.l4
" +,
present; '-, absent.Fnoc! source
Raw milk Raw milk Raw milk Egg dishes Egg dishes
Sauteed chicken Cllts
Sautecd chicken Cllts Sauteed chicken Cllts Chicken satay Chicken salay
16SrRNA sea
+
+
+
+
+
' T
+
..l-
+
aurclIs on mannitol salt agar, i.e., could change the color of agar to yellow . All 65 tested isolates were coagulase positive , anc! from 13 isolates tested using API Staph, J 0 isolates (76.9%) were positive for S. aureus, followed by Staphylococcus lel/tlls (15.4(k;) and Staphylococcl/s xvlos/ls
(7 .7%) .
Furthermore, all
or
the obtained S' . aureus isolates exhibited 240 bp of the 16S rRNA gene PCR product after ampli fication using l6sF and 16sR3 primers, the same as the S. aurcus ATCC 25923 as a positive control (Table 2). N inc of lOS. alirCIIS isolates showed 120 bp of sea PCR product that indicated the ability to produce staphylococcal enterolOxin A. On the other hanc!, S. (lureus TB 10 from egg dishes, such as S. (lurCIIS ATCC 25923, did not have sea,confirming that this strain did not produce staphylococcal enterolOxin A (Table 2). The results of the 16S rRNA gene sequence anal ys is using BLAST showed that. the J () isolates were S. aI/reus (data not shown) . 'fllis case confirmed the identi fication results. using API Staph . Phylogenetic analysis rcsults showcd that S. {lUrellS SJl had the closest relationship with S. ollreus A'T'CC 25923 (data not shown). Primers SEA I and SEA2 were also used by Rall et a1.
(32) to dctect the presence of sea in S. allreus isolates from raw and pasteurized milk using PCR. Among the genes that cncode the class ic enleroloxins (A through I:), sea was the most commonly foun d (41 fo llowed by .ICC (20.Y'k), sed (3.7%), seb (7.7%), and sec (::;.1
[image:3.595.341.582.74.230.2]r·
J. Fooel Prot., Voi. 77, No. I j SEA GENE-CARRYING S ill/REUS CONTROL BY CRUDE ALKALOID 199510 9 8
;.. =-
7...
...｡ ｾＶ＠
:: ｾ＠ 5
= u
ｾ＠ ｾｉＮＧ＠ 4U63
2
o
o
0125 0.25 0.5 [image:4.599.348.577.45.190.2] [image:4.599.90.325.45.204.2]C rude alka loid concentra tion ( m g/ml )
FIGURE 1. Abiliry of crude (/Ikaloid o/,papaya lem'es al \'(/riou,\
cOllcellTrations, by macrodilllfioll resr afier 24-h incubation, to
inhibit and or reduce rhe cell number of'S. aurcus Sf I. l1'irh an
inirial level o/' 4 .65 log CPU/mi. Values are means
0/
rhreereplicares, and error bars represent s[(1ndard del·i(/rions.
(31), i,e" in a concentration of 1.25 rng/ml. This crude alkaloid MIC was also lower than the crude alkaloid extract from Trihulus terresrris leaves against S. ({ureus that was in a concentration of 0.625 mg/ml (16). On the other hand, a MIC of papaya leaf crude alkaloid extract was higher than that of the alka loid extract from AconitunI elwesii and Sida aeuta Bunn. f. leaves, i.e., 0.094 and 0.078 mg/ml, respectively (15, 34).
Effect of crude alkaloid to the cell number of S,
aurellS
and expression ofsea.
Exposure of crude alkaloidto S. allreus SJJ at one- and twofold MIC for 2 h resulted in a lower bacterial number in comparison to the control (Fig. 2), In contrast with the results of MIC detenl1ination in which the cell number without alkaloid exposure increased 4.56 log CFU/ml, the cell numbcr or the control (no alkaloid extract) increased only 1.09 log CFU/ml after 2-h incubation (Figs. 1 and 2), On the other hand, the bacterial cell number at 0,25 anti 0,5 mg/ml decreased 0 .98 and 2,77 log CFU/ml, respectively, after the 24-h incubation with MIC detenni-nation, whereas the cell number slightly increased with 0.42 and 0.21 log CFU/ml, respectively, after 2-h incubation, However, these increases were not significantly different (Fig. 2). This shOlt incubation (2 h) gave mild exposure, so the a lkaloid did not inactivate and decrease the cells but still affected the expression of sea. 'rhe increase of bacterial cell number at the early exposure time with the antimicrobial compound was also round in Hermsen el al. (14). Methicilin-susceptible S. aureus cell number increased until the fourth hour of exposure time and decrcased about 3 log CFU/rnl at the 16th hour when it was exposed to the cefazolin antibiotic in cation-adjusted Mueller-Hinton broth media at pH 7.4.
In comparison to the growth of control, crude alkaloiq at 0,25 mg/ml reeluced 61 % of the cell numbers and crude alkaloid at 0.5 mg/ml reduced 81 % of the cell numbers. These findings suppOlted the results of other researches that found decreasing
S.
ourem numbers after treatment with alkaloids (18.21). Although the exposure to crude alkaloid up to 0.5 rng/ml for 2 h resulted in a slight increase of the10
8
,-.,
t
E
.Q -..
6
::
;.,.;J=
ｾ＠c: U
eli 4
...
j
U Ｎ ｾ＠2
0
0 0.25 0.5
Crud e alkaloid concentration (mg /ml)
FIGURE 2. Cdl //lIl1/her o/' S. ClllreliS SJI hefim' (D) and aller (ffil) nposllre with cmde alkaloid o(paJ!ilva leal'es (/1 zero- ,
o//e-and twoj()ld !'vI/Cji)!' 2 lz. Vailles are means ofrizrec replicales, and
error bars repreSl'1/f siandard del'ialiofls . iV/eans with diflerent
letters are significantly ditJercn( (P
<
G.(5)cell numbers, the decline of sea relative expression was observed. The results indicated that exposure to the papaya leaf crude alkaloid caused a significant increase in the CT value of sea, but the C, values of 16S rRNA gene did not significantly increase (Table 3). The C, value was inversely proportional to the initial number copies of DNA: the higher the C
r
value, the lower the initial amount of DNA, Because the Cr values of the 16S rRNA gene were relatively constant (increased insignificantly) indicated that the cell numbers were similar in all samples. The 16S rRNA gene is a housekeeping gene that is always expressed because it codes for 16S ribosomal RNA and is involved in protein metabolism, This gene is used as an internal standard (control) in qPCR and can serve as a marker for the cell numbers in a sample (35).Fulthermore, amplification of sea without exposure to erude alkaloid (0 rng/ml) required 17.3R cycles until the fluorescence signal could pass through the threshold. After exposure to 0,25 and 0.5 mg/ml of crude alkaloid ror 2 h, the amplirication of sea required more cyclcs to pass through the thre shold, indicating a decrease of the initial amount of
sea
cDNA, which meant less mRNA was synthesized from sea. Lee et al. (20) comprehensively studied the expression of enterotoxin genes in S. aureus by a reverse transcription qPCR that facilitates detection on an RNA level. Various levels of expression were found, depending on the species and enterotoxin gene. Duquenne et al. (II) used qRT-PCR to study the expression of sea andsed
during cheese manufacture. Expression levels or sea remained essentially unchanged during the first 72 h or cheese manufacture, whereas sed expression decreased.Relative expression value of sea was represented by the value of 2 - L\/lCT ,
11C
r was obtained from the difference betweenC
T of sea and 16S rRNA gene. The1111C
7 could be obtained from the difference between11C
r of the exposed scunple (0.25 and 0.5 mg/ml of crude alkaloid) and11C
r of the calibrator samplc. Then, the1111C
r value was entered into the 2-!l!lCr equation to obtain the relative expression value (Table 3). Thesea
was expressed 29 times less inS.
1996 HANDA YANI ET AL. 1. Food Prot. , Vol. 77, No . II
TABLE 3. Relative e.\jJressiol1
of
sea (JlS. aurellS SJt exposed to crude alkaloid of papa va lall'csj()f' 2 II, calcllimcd using a compararil'('Cr method
Crude alkaloid
concn (mg/mll CT 16S rRNA geneO L\CT
o
0.25 0.5
17.38
±
0.28 Il 22.58±
1.07 A23.15 ·t 0.62 A
15.26
±
0.41 c15.63
±
0.17 c] 5.69
±
0.58 c2.12 6.95 7.47
0 1.000
4.83 0.035
5.35 0.025
a Values are means J: stand,u'd deviations of three repl icates. Means with different letters are significarlriy different (P
<
0.(5).alkaloid at 0 .5 mg/ml, compared with S. aureus SJ I \vithoLlt crude alkaloid exposure. This comparative CT method (2 -L\,\CT) was also fruitfully used to calculate the relative expression of target genes in human peripheral blood mononuclear cells by Kharaji and Haghparast (17) and target genes in the human glioblastoma cell line by Malvandi et al. (22).
This study highlighted that the crude alkaloid of papaya leaves possessed antibacterial activity against S. OllrellS that not only inhibited bacte rial growth but also inhibited the expression of sea. In pmticular, the prescnt study successfully examined the expression of sea quantilatively. Considering that papaya leaves are ofLen applied to improve tenderness of meat in Asian and African countrics, this practice will give an additional benefit to improve the safety
or
mcat. During the wrapping of meat with papaya leaves or marinating with papaya leaf pastes , the active compou nd, including alkaloid, will be in contact with bacteria and might result in the inhibition of the growth and f0111ling of enterotoxins.ACKNOWLEDGMENT
This work was funded by National Strategic Competitive Research Grant 2013 from Directorate General of Higher Education, Ministry of Education and Culture of Indonesia.
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ｧｯｶＯｆｯｯ､Ｏｆｯｯ､ｓ｣ｩ･ｮ｣･ｒ･ｳ･｡ｲ｣ｨＯｌ｡｢ｯｲ｡ｴｯｲｹｍ･ｴｨｯ､ｳＯｵｾ［ｾｉｴＩＧ＿＠ ｩＴｩＹｾｨｴｭＮ＠
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