comparative effects of food preservatives on the...

Research ArticleComparative Effects of Food Preservatives onthe Production of Staphylococcal Enterotoxin I fromStaphylococcus aureus Isolate

Yanying Zhao Anni Zhu Junni Tang Cheng Tang and Juan Chen

College of life Science and Technology Southwest University for Nationalities Chengdu 610041 China

Correspondence should be addressed to Junni Tang junneytangaliyuncom

Received 6 November 2016 Revised 15 January 2017 Accepted 24 January 2017 Published 16 February 2017

Academic Editor Rosa Perez-Gregorio

Copyright copy 2017 Yanying Zhao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Staphylococcal enterotoxin I (SEI) is associated with staphylococcal food poisoning but little is known about different foodpreservatives on the production of SEI In this study the effect of different food preservatives (sodium nitrite polylysine chitosanand tea catechin) on the bacteria growth sei gene expression and extracellular SEI production of Staphylococcus aureus isolate H4was detected in tryptone soya broth (TSB) culture Our results showed that all of these preservatives depressed S aureusH4 growthand the order of inhibitory effect was 08 gL tea catechin gt 6 gL chitosan gt 025 gL polylysine gt 04 gL tea catechin gt 015 gLsodium nitrite Furthermore 025 gL polylysine or 015 gL sodium nitrite did not significantly alter sei gene transcription while6 gL chitosan obviously increased the relative mRNA level of sei gene expression 04 gL tea catechin remarkably inhibited seigene transcription In addition 015 gL sodium nitrite and 6 gL chitosan significantly enhanced SEI secretion 025 gL polylysineespecially 04 gL tea catechin sharply inhibited the level of SEI secretionThe results indicated that tea catechin not only suppressedStaphylococcus aureus growth but also inhibited SEI production and secretion suggesting that tea catechin may be better thansodium nitrite polylysine or chitosan for keeping the food from the contamination of SEI These investigations would be usefulfor food industry to provide safer food products due to S aureus enterotoxins-related control strategy

1 Introduction

Staphylococcus aureus (S aureus) is a major bacterialpathogen that causes clinical infection and foodborne ill-nesses [1] The organism is robust which permits it to growin many types of foods improperly prepared or stored andto produce staphylococcal enterotoxins (SEs) SEs are thecausative agents of staphylococcal food poisoning (SFP) [2]Even though the bacteria can be killed throughheat treatmentof the food the SEs are heat-resistant Thus although thebacteria are eliminated the toxins will remain in food andsubsequently cause SFP [3]

To date more than twenty-four SEs and staphylococcalenterotoxin-like proteins (SEls) have been identified anddesignated SEA to SElY [4ndash6] These SEsSEls have beentraditionally subdivided into classical (SEA to SEE) and newtype (SEG to SElY) It is supposed that SEs are the toxinsthat elicit emesis the related SEls either lack emetic activity

or have not yet been examined [4] In recent years severalstudies indicate that SEG and SEI may be responsible forcases of SFP in humans [7ndash9] However commercial kitsfor immunological detection of SEI protein are not availablecurrently [10 11] Thus there is very limited data on theproduction of SEI

Food preservatives are widely used to reduce the riskof food poisoning Conventional preservatives are syntheticchemical substances such as sodiumnitrite sodiumbenzoateand potassium sorbate Because of side effects the use of arti-ficial preservatives is being reconsidered [12] With growingconsumer demand for natural preservatives to replace chem-ical compounds new antimicrobial products of various ori-gins are being developed including animal-derived products(lysozymes lactoperoxidase chitosan antimicrobial peptideand others) plant-derived products (polyphenolics essentialoils plant antimicrobial peptides and others) and microbialmetabolites (nisin natamycin 120576-polylysine organic acid and

HindawiJournal of Food QualityVolume 2017 Article ID 9495314 5 pageshttpsdoiorg10115520179495314

2 Journal of Food Quality

others)These products must be investigated for the potentialto control foodborne pathogens in foods

In this study comparative effects of several food preser-vatives sodium nitrite polylysine chitosan and tea catechinon bacteria growth sei gene expression and extracellularSEI secretion from a penicillin-resistant S aureus isolate H4associated with food poisoning were detected to assess theseproducts for use as antimicrobial agents in food preservation

2 Materials and Methods

21 Bacterial Plate Counting S aureus strain H4 (penicillin-resistant) was isolated from vomitus of hospitalized patientassociated with food poisoning in Chongqing China [13]2 times 105 CFU S aureus H4 was inoculated in 250mL tryp-tone soya broth (TSB) medium supplemented with differentfood preservatives at maximum permissible concentrations(015 gL sodiumnitrite Sichuan Jinshan Pharmaceutical CoLtd China 025 gL polylysine Zhengzhou Tianfeng FoodScience and Technology Co Ltd China 6 gL chitosanZhengzhou Tianhe Biological Science and Technology CoLtd China and 08 gL tea catechin Zhengzhou GreenBanko Trade Ltd China) according to Food Safety Law ofthe Peoplersquos Republic of China (GB2760-2014) In addition0075 gL (12x) and 00375 gL (14x) sodiumnitrite 0125 gL(12x) and 00625 gL (14x) polylysine 3 gL (12x) and15 gL (14x) chitosan and 04 gL (12x) and 02 gL (14x)tea catechin were also used After incubation at 37∘C withshaking at 150 revolutions per minute (rpm) for 24 hours1mL cell suspension was collected for viable plate countingand the best doses of the food preservatives were chosenThen 2 times 105 CFU S aureus H4 was inoculated in 250mLTSB medium containing the food preservative at the bestconcentration for 12 24 and 48 hours respectively 1mL cellsuspension was also harvested for viable plate counting

22 Real Time Quantitative PCR Real time quantitative PCRwas employed to detect relativemRNA level of sei in S aureusH4 treated with food preservatives After incubation for 24hours 200mL cell suspension was collected and centrifugedat 10000119892 for 15min The supernatant was harvested forthe following SEI protein quantification and the cells werelysed by TE buffer (10mmolL Tris-HCl 1mmolL EDTApH = 80) supplemented with 3mgmL lysozyme TotalRNA was extracted with TRIzol reagent (Tiangen BeijingChina) according to the product protocol The concentrationof RNA was detected in a spectrophotometer (Biowave IIBiochrom UK) and 2 120583g RNA was used to synthesize cDNAby reverse transcription with a PrimeScript RT reagent Kit(Fermentas Life Science Hanover MD US) as described inthe manufacturerrsquos instructions

Real time fluorescence-based quantitative PCR was per-formed in a fluorescence temperature iCycler (Bio-RadHercules CA USA)The specific primers for sei gene that wepreviously cloned (GenBank accession number KT8530461)were sei-qF 51015840-TGCCTTTACCAGTGTTATT-31015840 and sei-qR 51015840-AGGACAATACTTAAATTCTGCT-31015840 These primerswere designed with Primer Premier 50 software and thespecificity of the primers was assayed by PCR Conditions for

PCR amplification were 120 s at 95∘C followed by 39 cyclesof 10 s at 95∘C 30 s at 55∘C and 30 s at 72∘C The thresholdcycle (CT) was analyzed with a 2minusΔΔCt method [14] FtsZ(primers FtsZ-F 51015840-TGAAGATGCAATCCAAGGTG-31015840 andFtsZ-R 51015840-GTTAATGCGCCCATTTCTTT-31015840) was used as aloading control for normalization

23 Double-Antibody Sandwich ELISA Double-antibodysandwich ELISA was developed for detecting SEI secretionfrom S aureus H4 with food preservative administration aspreviously described [13] In brief 289120583gmL monoclonalantibody against SEI in PBS was coated into microtiter plateovernight at 4∘C the plate was blocked with 5 skimmedmilk in PBS at 37∘C for 60min 01mL supernatant of Saureus H4 collected at the 24th hour was added to each wellat 37∘C for 60min washed extensively with PBS contain-ing 005 Tween-20 followed by incubation with 01mLpolyclonal antibody against SEI (dilution 1 1000) at 37∘Cfor 60min and washed again Then 01mL goat anti-rabbitIgG-horseradish peroxidase (dilution 1 6000 Santa CruzUSA) was added to each well at 37∘C for 60min and washedagain Thereafter 01mL of substrate tetramethylbenzidine(TMB) solution was used and finally H2SO4 was employedto terminate reaction The measurement was carried out at450 nm photometrically

24 Statistical Analysis Data were presented as mean plusmn SEMand the differences between food preservatives treatmentsand controls were statistically analyzed using SPSS130 (SPSSChicago IL USA)

3 Results

31 Bacterial Plate Counting The number of S aureus H4with different food preservative treatments was determinedby viable plate counting as shown in Figure 1 All of thesepreservatives depressed S aureus H4 growth especially atmaximum permissible concentrations (Figure 1(a)) It isnoteworthy to mention that 08 gL tea catechin absolutelydisrupted bacteria after 24-hour incubation thus the con-centration of tea catechin was adjusted to 04 gL Thereafterthe best dose of the food preservative for the followingstudy or comparison was 015 gL sodium nitrite 025 gLpolylysine 6 gL chitosan and 04 gL tea catechinThe orderof inhibitory effect was 6 gL chitosan gt 025 gL polylysinegt 04 gL tea catechin gt 015 gL sodium nitrite from 12- to48-hour incubation (Figure 1(b)) Therefore it seemed thatnatural preservatives chitosan polylysine and tea catechinwere more efficient than chemical compound sodium nitritein inhibiting S aureus growth

32 Effect of Food Preservatives on sei Gene TranscriptionFurthermore relative mRNA level of sei gene expression inS aureusH4 was detected by real time quantitative PCRTheresults indicated that 025 gL polylysine (091 plusmn 012 versus103 plusmn 03 119901 = 0207) or 015 gL sodium nitrite (073 plusmn 016versus 103 plusmn 03 119901 = 056) did not significantly alter seigene transcription while 6 gL chitosan (229 plusmn 023 versus103plusmn03119901 = 00046) obviously increased the relativemRNA

Journal of Food Quality 3

The concentrations of food preservatives

Num

ber o

f bac

teria

(times108)

1x

12

x1

4x 0

1x

12

x1

4x 0

1x

12

x1

4x 0

1x

12

x1

4x 0

015 gL sodium nitrite025 gL polylysine

6 gL chitosan08 gL tea catechin

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowastlowast lowastlowastlowast0

10

20

30

40

(a)

0

10

20

30

40

0 10 20 30 40

ControlSodium nitritePolylysine

ChitosanTea catechin

Time (h)

Num

ber o

f bac

teria

(lowast108

CFU

)

(b)

Figure 1 Viable plate count of penicillin-resistant S aureus strain H4 treated with different food preservatives (a) 2 times 105 CFU S aureusH4was inoculated in 250mL TSB medium supplemented with 015 gL (1x) 0075 gL (12x) or 00375 gL (14x) sodium nitrite 025 gL (1x)0125 gL (12x) or 00625 gL (14x) polylysine 6 gL (1x) 3 gL (12x) or 15 gL (14x) chitosan and 08 gL (1x) 04 gL (12x) or 02 gL(14x) tea catechin respectively After incubation at 37∘C for 24 hours 1mL cell suspensionwas collected for viable plate count (b) 2times 105 CFUS aureusH4was inoculated in 250mL TSBmedium supplemented with 015 gL sodium nitrite 025 gL polylysine 6 gL chitosan or 04 gLtea catechin for 12 24 and 48 hours respectively Data were the mean plusmn SEM of three independent experiments (lowastlowastlowast119901 lt 0001)

lowast

lowastlowast

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

0

1

2

Relat

ive m

RNA

leve

l of S

EI

Figure 2 Effect of different food preservatives on sei gene tran-scription S aureus H4 was treated with sodium nitrite polylysinechitosan or tea catechin respectively as described above At the24th-hour incubation S aureus were lysed by lysozyme and themRNA was detected by real time quantitative PCR Data were themean plusmn SEM of three independent experiments (lowast119901 lt 005 lowastlowast119901 lt001)

level of sei gene expression 04 gL tea catechin remarkablyinhibited sei gene transcription (048 plusmn 011 versus 103 plusmn 03119901 = 0043) (Figure 2) Thus only tea catechin prevented seitranscription

33 Influence of Different Food Preservatives on ExtracellularSEI Production In addition the extracellular SEI production

was tested by double-antibody sandwich ELISA As shown inFigure 3(a) 015 gL sodium nitrite (880 plusmn 141 times 10minus9 ngcellversus 354plusmn004times10minus9 ngcell119901 = 0038) and 6 gL chitosan(940 plusmn 042 times 10minus9 ngcell versus 354 plusmn 004 times 10minus9 ngcell119901 = 00034) significantly enhanced SEI secretion SEI proteinincrement by chitosan was consistent with the result of PCRwhile 025 gL polylysine (290 plusmn 014 times 10minus9 ngcell versus354 plusmn 004 times 10minus9 ngcell 119901 = 0043) especially 04 gLtea catechin (170 plusmn 007 times 10minus9 ngcell versus 354 plusmn 004 times10minus9 ngcell 119901 = 00008) sharply inhibited the level of SEIsecretion Among these food preservatives tea catechin notonly downregulated sei gene transcription but also depressedits protein secretion On the other hand except sodiumnitrite all of the three natural food preservatives remarkablydecreased total extracellular concentration of SEI in TSBculture (Figure 3(b))

4 Discussion

Nowadays consumers want more convenient ready-to-eatfood Convenient food offers a suitable growth environmentfor S aureus an important pathogen associated with food-borne diseases including toxic shock syndrome and foodpoisoning by secreting a wide variety of SEsSEls [15] Inorder to produce high-quality microbiologically safe food forconsumers data on SEsSEls production in foodpreservativesenvironments are needed to improve existing understandingabout the growth and survivability of S aureus in liquidcultures

In this study we evaluated the effects of sodium nitritepolylysine chitosan and tea catechin on the growth of Saureus H4 All of tested preservatives inhibited bacterial


lowast

lowast lowastlowast

0

2

4

6

8

lowastlowastlowast

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

Leve

l of S

EI se

cret

ion

(times10minus9

ngc

ell)

(a)

0

5

10

lowastlowastlowastlowastlowastlowastlowastlowastlowastLe

vel o

f SEI

pro

tein

(ng

mL)

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

(b)

Figure 3 Influence of different food preservatives on extracellular SEI secretion S aureus H4 was administrated with sodium nitritepolylysine chitosan or tea catechin respectively as described above At the 24th-hour incubation the level of SEI in supernatant of S aureusH4was tested by double-antibody sandwich ELISA (a)The level of SEI produced by per bacterium (b)The concentration of total extracellularSEI (lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001)

growth and 08 gL tea catechin was lethal to S aureus After24 h incubation the numbers of bacterial cells were as controlgt 015 gL sodium nitrite gt 04 gL tea catechin gt 025 gLpolylysine gt 6 gL chitosan It seemed that the three naturalfood preservatives were more effective in inhibiting bacterialgrowth than the chemical food preservative sodium nitrite atthese concentrations

Furthermore sei expression was investigated by realtime quantitative PCR and ELISA method respectively Thetraditional point was that SEs production was correlatedwith bacterial growth the more bacterial cells and themore toxins Thus the number of bacterial cells was usuallycounted to determine the level of SEs production In ourstudy tea catechin polylysine and chitosan indeed reducedthe total content of SEI which was consistent with thedecrement of bacterial cell numbers Even though sodiumnitrite inhibited S aureus growth it did not decrease SEIproduction which indicated that bacterial growth and SEIproduction was decoupled Chitosan a linear biopolymercomposed of 120573-(1-4)-linked N-acetyl-D-glucosamine is adeacetylated derivative of chitin [16] Chitosan has beenrecommended as a potential candidate for targeting antimi-crobial agents due to a broad spectrum of antimicrobialactivity and biocompatibility [17ndash19] In this study chitosanhad a good inhibitory effect on the bacterial growth and hadthe potential to control bacterial cell number however itincreased the relative mRNA level of sei gene expression andenhanced the SEI secretion which indicated that chitosanmight be not effective to control the staphylococcal entero-toxins production

On the other hand polyphenols in tea include catechinsflavonoids tannins and theaflavins These compounds pro-vide potential health benefits [20] Catechins are composed of

epigallocatechin-3-gallate (EGCG) epicatechin epicatechin-3-gallate and epigallocatechin The main active ingredientEGCG accounts for approximately 59 of the total catechins[21] A report suggested that negatively charged EGCGexerts its antibactericidal activity by binding to the positivelycharged lipids of the bacterial cell membrane leading todamage to the lipid layer [22 23] Another report indicatedthat tea catechin EGCG inhibited SEB production in doseand time dependent manner [24] Further work is neededto determine the effects of tea catechin EGCG on differ-ent SE production and whether EGCG has neutralizationproperties against other staphylococcal superantigens Inour experiment tea catechin not only sharply inhibited SaureusH4 growth but also depressed seimRNA and proteinproduction which was consistent with Hisanorsquos report

Taken together we detected and compared the effects offood preservatives sodium nitrite polylysine chitosan andtea catechin on penicillin-resistant S aureus H4 growth andSEI production Our results indicated that natural preser-vatives exerts higher antibacterial activity than chemicalcompound and tea catechin was the most efficient amongthe four kinds of food preservatives in inhibiting S aureusgrowth and SEI production The investigation was useful forfood industry to provide safer food products due to S aureusenterotoxins-related control strategy

Competing Interests

All the authors declare that they have no conflict of interestsNone of the authors has a financial or personal relationshipwith other individuals or organizations that could inappro-priately influence or bias the content of this paper


Acknowledgments

This work was jointly supported by the National Natural Sci-ence Foundation of China (31371781 31400794) the AppliedBasic Research Programs of Sichuan Province (2014JY0253)and the NewCentury Excellent Talents in University (NCET-11-0847)

References

[1] R Rasooly andM Friedman ldquoPlant compounds inhibit Staphy-lococcus aureus bacteria and the toxicity of StaphylococcusEnterotoxin A (SEA) associated with atopic dermatitisrdquo inAtopic Dermatitis-Disease Etiology and Clinical Management JEsparza-Gordillo and I Dekio Eds pp 387ndash404 InTech-OpenAccess Publishers Rijeka Croatia 2012

[2] N Balaban and A Rasooly ldquoStaphylococcal enterotoxinsrdquoInternational Journal of Food Microbiology vol 61 no 1 pp 1ndash10 2000

[3] D-L Hu and A Nakane ldquoMechanisms of staphylococcalenterotoxin-induced emesisrdquo European Journal of Pharmacol-ogy vol 722 no 1 pp 95ndash107 2014

[4] M A Argudın M C Mendoza and M R Rodicio ldquoFoodpoisoning and Staphylococcus aureus enterotoxinsrdquo Toxins vol2 no 7 pp 1751ndash1773 2010

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] H K Ono Y Satorsquoo K Narita et al ldquoIdentification andcharacterization of a novel staphylococcal emetic toxinrdquoAppliedand Environmental Microbiology vol 81 no 20 pp 7034ndash70402015

[7] A Kerouanton J A Hennekinne C Letertre et al ldquoCharac-terization of Staphylococcus aureus strains associated with foodpoisoning outbreaks in Francerdquo International Journal of FoodMicrobiology vol 115 no 3 pp 369ndash375 2007

[8] J Tang C Tang J Chen et al ldquoPhenotypic characterizationand prevalence of enterotoxin genes in Staphylococcus aureusisolates from outbreaks of illness in Chengdu cityrdquo FoodbornePathogens and Disease vol 8 no 12 pp 1317ndash1320 2011

[9] X Yan B Wang X Tao et al ldquoCharacterization of Staphylococ-cus aureus strains associated with food poisoning in ShenzhenChinardquoApplied and Environmental Microbiology vol 78 no 18pp 6637ndash6642 2012

[10] K Omoe M Ishikawa Y Shimoda D-L Hu S Ueda and KShinagawa ldquoDetection of seg seh and seigenes in Staphylococ-cus aureusisolates and determination of the enterotoxin pro-ductivities of S aureus isolates harboring seg seh or seigenesrdquoJournal of ClinicalMicrobiology vol 40 no 3 pp 857ndash862 2002

[11] J L Aguilar A K Varshney X Wang L Stanford M Scharffand B C Fries ldquoDetection and measurement of staphylococcalenterotoxin-like K (SEl-K) secretion by Staphylococcus aureusclinical isolatesrdquo Journal of Clinical Microbiology vol 52 no 7pp 2536ndash2543 2014

[12] S Sharma ldquoFood preservatives and their harmful effectsrdquoScientific Research Publishing vol 5 pp 1ndash2 2015

[13] Y Zhao A Zhu J Tang C Tang J Chen and J Liu ldquoIden-tification and measurement of staphylococcal enterotoxin-likeprotein I (SEll) secretion from Staphylococcus aureus clinicalisolaterdquo Journal of Applied Microbiology vol 121 no 2 pp 539ndash546 2016

[14] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862119879 methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[15] M S Bergdoll ldquoStaphylococcal intoxicationsrdquo in FoodborneInfections and Intoxications H Reimann and F L Bryan Edspp 443ndash494 Academic Press New York NY USA 1979

[16] F Shahidi J K V Arachchi and Y-J Jeon ldquoFood applicationsof chitin and chitosansrdquo Trends in Food Science and Technologyvol 10 no 2 pp 37ndash51 1999

[17] Y-C Chung H-L Wang Y-M Chen and S-L Li ldquoEffect ofabiotic factors on the antibacterial activity of chitosan againstwaterborne pathogensrdquo Bioresource Technology vol 88 no 3pp 179ndash184 2003

[18] E I Rabea M E-T Badawy C V Stevens G Smagghe andWSteurbaut ldquoChitosan as antimicrobial agent applications andmode of actionrdquo Biomacromolecules vol 4 no 6 pp 1457ndash14652003

[19] H Liu Y M Du X H Wang and L P Sun ldquoChitosan killsbacteria through cell membrane damagerdquo International Journalof Food Microbiology vol 95 no 2 pp 147ndash155 2004

[20] N Khan and H Mukhtar ldquoTea and health studies in humansrdquoCurrent Pharmaceutical Design vol 19 no 34 pp 6141ndash61472013

[21] D L McKay and J B Blumberg ldquoThe role of tea in humanhealth an updaterdquo Journal of the American College of Nutritionvol 21 no 1 pp 1ndash13 2002

[22] S Kumazawa K Kajiya A Naito et al ldquoDirect evidence ofinteraction of a green tea polyphenol epigallocatechin gallatewith lipid bilayers by solid-state nuclear magnetic resonancerdquoBioscience Biotechnology and Biochemistry vol 68 no 8 pp1743ndash1747 2004

[23] Y Cui Y J Oh J Lim et al ldquoAFM study of the dif-ferential inhibitory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive andGram-negative bacteriardquo Food Microbiology vol 29 no 1 pp80ndash87 2012

[24] M Hisano K Yamaguchi Y Inoue et al ldquoInhibitory effect ofcatechin against the superantigen staphylococcal enterotoxin B(SEB)rdquo Archives of Dermatological Research vol 295 no 5 pp183ndash189 2003

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Enzyme Research



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others)These products must be investigated for the potentialto control foodborne pathogens in foods

In this study comparative effects of several food preser-vatives sodium nitrite polylysine chitosan and tea catechinon bacteria growth sei gene expression and extracellularSEI secretion from a penicillin-resistant S aureus isolate H4associated with food poisoning were detected to assess theseproducts for use as antimicrobial agents in food preservation

2 Materials and Methods

21 Bacterial Plate Counting S aureus strain H4 (penicillin-resistant) was isolated from vomitus of hospitalized patientassociated with food poisoning in Chongqing China [13]2 times 105 CFU S aureus H4 was inoculated in 250mL tryp-tone soya broth (TSB) medium supplemented with differentfood preservatives at maximum permissible concentrations(015 gL sodiumnitrite Sichuan Jinshan Pharmaceutical CoLtd China 025 gL polylysine Zhengzhou Tianfeng FoodScience and Technology Co Ltd China 6 gL chitosanZhengzhou Tianhe Biological Science and Technology CoLtd China and 08 gL tea catechin Zhengzhou GreenBanko Trade Ltd China) according to Food Safety Law ofthe Peoplersquos Republic of China (GB2760-2014) In addition0075 gL (12x) and 00375 gL (14x) sodiumnitrite 0125 gL(12x) and 00625 gL (14x) polylysine 3 gL (12x) and15 gL (14x) chitosan and 04 gL (12x) and 02 gL (14x)tea catechin were also used After incubation at 37∘C withshaking at 150 revolutions per minute (rpm) for 24 hours1mL cell suspension was collected for viable plate countingand the best doses of the food preservatives were chosenThen 2 times 105 CFU S aureus H4 was inoculated in 250mLTSB medium containing the food preservative at the bestconcentration for 12 24 and 48 hours respectively 1mL cellsuspension was also harvested for viable plate counting

22 Real Time Quantitative PCR Real time quantitative PCRwas employed to detect relativemRNA level of sei in S aureusH4 treated with food preservatives After incubation for 24hours 200mL cell suspension was collected and centrifugedat 10000119892 for 15min The supernatant was harvested forthe following SEI protein quantification and the cells werelysed by TE buffer (10mmolL Tris-HCl 1mmolL EDTApH = 80) supplemented with 3mgmL lysozyme TotalRNA was extracted with TRIzol reagent (Tiangen BeijingChina) according to the product protocol The concentrationof RNA was detected in a spectrophotometer (Biowave IIBiochrom UK) and 2 120583g RNA was used to synthesize cDNAby reverse transcription with a PrimeScript RT reagent Kit(Fermentas Life Science Hanover MD US) as described inthe manufacturerrsquos instructions

Real time fluorescence-based quantitative PCR was per-formed in a fluorescence temperature iCycler (Bio-RadHercules CA USA)The specific primers for sei gene that wepreviously cloned (GenBank accession number KT8530461)were sei-qF 51015840-TGCCTTTACCAGTGTTATT-31015840 and sei-qR 51015840-AGGACAATACTTAAATTCTGCT-31015840 These primerswere designed with Primer Premier 50 software and thespecificity of the primers was assayed by PCR Conditions for

PCR amplification were 120 s at 95∘C followed by 39 cyclesof 10 s at 95∘C 30 s at 55∘C and 30 s at 72∘C The thresholdcycle (CT) was analyzed with a 2minusΔΔCt method [14] FtsZ(primers FtsZ-F 51015840-TGAAGATGCAATCCAAGGTG-31015840 andFtsZ-R 51015840-GTTAATGCGCCCATTTCTTT-31015840) was used as aloading control for normalization

23 Double-Antibody Sandwich ELISA Double-antibodysandwich ELISA was developed for detecting SEI secretionfrom S aureus H4 with food preservative administration aspreviously described [13] In brief 289120583gmL monoclonalantibody against SEI in PBS was coated into microtiter plateovernight at 4∘C the plate was blocked with 5 skimmedmilk in PBS at 37∘C for 60min 01mL supernatant of Saureus H4 collected at the 24th hour was added to each wellat 37∘C for 60min washed extensively with PBS contain-ing 005 Tween-20 followed by incubation with 01mLpolyclonal antibody against SEI (dilution 1 1000) at 37∘Cfor 60min and washed again Then 01mL goat anti-rabbitIgG-horseradish peroxidase (dilution 1 6000 Santa CruzUSA) was added to each well at 37∘C for 60min and washedagain Thereafter 01mL of substrate tetramethylbenzidine(TMB) solution was used and finally H2SO4 was employedto terminate reaction The measurement was carried out at450 nm photometrically

24 Statistical Analysis Data were presented as mean plusmn SEMand the differences between food preservatives treatmentsand controls were statistically analyzed using SPSS130 (SPSSChicago IL USA)

3 Results

31 Bacterial Plate Counting The number of S aureus H4with different food preservative treatments was determinedby viable plate counting as shown in Figure 1 All of thesepreservatives depressed S aureus H4 growth especially atmaximum permissible concentrations (Figure 1(a)) It isnoteworthy to mention that 08 gL tea catechin absolutelydisrupted bacteria after 24-hour incubation thus the con-centration of tea catechin was adjusted to 04 gL Thereafterthe best dose of the food preservative for the followingstudy or comparison was 015 gL sodium nitrite 025 gLpolylysine 6 gL chitosan and 04 gL tea catechinThe orderof inhibitory effect was 6 gL chitosan gt 025 gL polylysinegt 04 gL tea catechin gt 015 gL sodium nitrite from 12- to48-hour incubation (Figure 1(b)) Therefore it seemed thatnatural preservatives chitosan polylysine and tea catechinwere more efficient than chemical compound sodium nitritein inhibiting S aureus growth

32 Effect of Food Preservatives on sei Gene TranscriptionFurthermore relative mRNA level of sei gene expression inS aureusH4 was detected by real time quantitative PCRTheresults indicated that 025 gL polylysine (091 plusmn 012 versus103 plusmn 03 119901 = 0207) or 015 gL sodium nitrite (073 plusmn 016versus 103 plusmn 03 119901 = 056) did not significantly alter seigene transcription while 6 gL chitosan (229 plusmn 023 versus103plusmn03119901 = 00046) obviously increased the relativemRNA



Num

ber o

f bac

teria

(times108)

1x

12

x1

4x 0

1x

12

x1

4x 0

1x

12

x1

4x 0

1x

12

x1

4x 0



lowastlowastlowast


lowastlowastlowast

lowastlowastlowast




10

20

30

40

(a)

0

10

20

30

40

0 10 20 30 40



Time (h)

Num

ber o

f bac

teria

(lowast108

CFU

)

(b)


lowast

lowastlowast

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

0

1

2

Relat

ive m

RNA

leve

l of S

EI





4 Discussion




lowast

lowast lowastlowast

0

2

4

6

8

lowastlowastlowast

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

Leve

l of S

EI se

cret

ion

(times10minus9

ngc

ell)

(a)

0

5

10


vel o

f SEI

pro

tein

(ng

mL)

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

(b)







Competing Interests



Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Num

ber o

f bac

teria

(times108)

1x

12

x1

4x 0

1x

12

x1

4x 0

1x

12

x1

4x 0

1x

12

x1

4x 0



lowastlowastlowast


lowastlowastlowast

lowastlowastlowast




10

20

30

40

(a)

0

10

20

30

40

0 10 20 30 40



Time (h)

Num

ber o

f bac

teria

(lowast108

CFU

)

(b)


lowast

lowastlowast

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

0

1

2

Relat

ive m

RNA

leve

l of S

EI





4 Discussion




lowast

lowast lowastlowast

0

2

4

6

8

lowastlowastlowast

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

Leve

l of S

EI se

cret

ion

(times10minus9

ngc

ell)

(a)

0

5

10


vel o

f SEI

pro

tein

(ng

mL)

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

(b)







Competing Interests



Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowast

lowast lowastlowast

0

2

4

6

8

lowastlowastlowast

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

Leve

l of S

EI se

cret

ion

(times10minus9

ngc

ell)

(a)

0

5

10


vel o

f SEI

pro

tein

(ng

mL)

Con

trol

Sodi

um n

itrite

Poly

lysin

e

Chito

san

Tea c

atec

hin

(b)







Competing Interests



Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

comparative effects of food preservatives on the...

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