Research Article Effects of Processing and Storage on … · 2019. 7. 31. · (v/v) glycerol...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 680767 8 pageshttpdxdoiorg1011552013680767

Research ArticleEffects of Processing and Storage on Pediococcus pentosaceusSB83 in Vaginal Formulations Lyophilized Powder and Tablets

Sandra Borges1 Paulo Costa2 Joana Silva1 and Paula Teixeira1

1 Centro de Biotecnologia e Quımica Fina (CBQF) Laboratorio Associado Escola Superior de Biotecnologia Universidade CatolicaPortuguesaPorto Rua Dr Antonio Bernardino Almeida 4200-072 Porto Portugal

2 Departamento de Ciencias do Medicamento Laboratorio de Tecnologia Farmaceutica Faculdade de Farmacia Universidade doPorto Rua de Jorge Viterbo Ferreira 228 4050-313 Porto Portugal

Correspondence should be addressed to Paula Teixeira pcteixeiraportoucppt

Received 5 April 2013 Accepted 4 June 2013

Academic Editor Vicky Kett

Copyright copy 2013 Sandra Borges 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

Vaginal probiotics have an important role in preventing the colonization of the vagina by pathogensThis study aimed to investigatedifferent formulations with Pediococcus pentosaceus SB83 (lyophilized powder and tablets with and without retarding polymer)in order to verify its stability and antilisterial activity after manufacture and during storage The bacteriocinogenic activity of Ppentosaceus SB83 against Listeria monocytogenes was evaluated in simulated vaginal fluid Suspension of Pediococcus pentosaceusSB83 reduced the pathogen only after 2 h and the lyophilized bacteria after 24 h of contact and in the tablets P pentosaceus SB83lost the antimicrobial activity The pH of simulated vaginal fluid decreased for all the tested conditions As lyophilized powderdemonstrated better results concerning antimicrobial activity this formulation was selected to evaluate the antilisterial activityduring the 12 months of storage During storage at room temperature lyophilized bacteria totally inhibited the pathogen only untilonemonth of storageAt 4∘CP pentosaceus SB83 showed antimicrobial activity during all the time of storage investigatedThereforethe better formulation of P pentosaceus SB83 is the lyophilized powder stored at 4∘C which may be administered intravaginally asa washing solution

1 Introduction

The dominant microorganisms in vaginal microbiota arelactobacilli with a concentration of 107 to 108 CFUmLof vaginal fluid in healthy premenopausal women [1 2]However other lactic acid bacteria (LAB) genera have beenfound in the vaginal tract such as Pediococcus spp Weisellaspp Streptococcus spp and Leuconostoc spp [3]Moreover itis acknowledged that the composition of the microbiota canvary from day to day even in women without an indicationof infections [4]

The normal vaginal pH of a healthy woman is acidic(4ndash45) with variations from 66 (+minus03) to 42 (+minus02)between day 2 and day 14 of the menstrual cycle [5] althoughvaginal pH can rise with the diminishing of LAB existingin the vaginal microbiota since glucose is not converted tolactic acid High pH values promote growth of pathogenicorganisms particularly colonization by enteric bacteria [2]

The microorganisms that are normally present in thevaginal environment play a major role in preventing illnessesof the host including bacterial vaginosis (BV) urinary tractinfections (UTI) yeast vaginitis and sexually transmitteddiseases including human immunodeficiency virus (HIV)[6] The vaginal LAB are also able to reduce the incidenceof ascending infections of the uterus and the subsequentproduction of proinflammatory molecules [7] During preg-nancy several genital microorganisms such as Escherichiacoli Listeriamonocytogenes and viridans streptococcimay beinvolved in chorioamnionitis [8]

Antimicrobial treatment of urogenital infections is notalways effective and problems remain due to bacterial andyeast resistance To minimize recurrent infections as well asside effects it is important to develop and produce alternativetreatments or drugs [9]

Due to the beneficial characteristics of LAB there hasbeen a growing interest in the potential use of LAB as

2 BioMed Research International

probiotics for maintaining normal urogenital health [10]TheWorld Health Organization and the Food and AgricultureOrganization of theUnitedNations have defined probiotics asldquolivemicroorganisms which when administered in adequateamounts confer a health benefit on the hostrdquo [11]

A probiotic may act indirectly through treating andpreventing recurrent BV or directly by secreting substances(eg hydrogen peroxide bacteriocins and lactic acid) thatinhibit pathogens [12]

Vaginal dosage forms available include creams gelstablets capsules pessaries foams ointments films tamponsrings and douches While the majority of vaginal drugs sofar have been in the form of gels there is a growing interestin alternative dosage forms such as rings tablets and filmsThemajority of issues with product development and scaleupare similar to other pharmaceutical products although thereare some unique challenges because of the site of deliveryprophylactic nature of product application and diversity insex and hygiene practices across the developing world [13]

The purpose of this study was to develop a vaginalformulation containing viable cells of a LAB isolate whichcan be used by women to prevent vaginal colonization ofmicroorganisms (eg Lmonocytogenes) thatmay be harmfulto the fetusneonate In this study the probiotic properties(survival and antagonistic activity) of formulations wereevaluated after manufacture and during long-term storage ofthe product at different temperatures

2 Material and Methods

21 Microorganisms and Culture Conditions Pediococcuspentosaceus SB83 (Escola Superior de Biotecnologia CultureCollection) was selected because previous studies demon-strated that this strain is able to inhibit the growth of clinicalisolates of L monocytogenes (serotype 12a 12b and 4b) byproduction of a bacteriocin [14 15] This strain has beenshown to survive in simulated vaginal fluid at normal vaginalpH (pH of 42) and P pentosaceus SB83 does not producevirulence factors such as gelatinase lipase and DNase andhemolytic activity nor does it possess virulence genes (genesesp agg gelE efaAfm efaAfs cylA cylB and cylM) [15]

Pediococcus pentosaceus SB83 was grown on de ManRogosa and Sharpe (MRS) agar (Lab M Bury United King-dom) and was subcultured twice in MRS broth (Lab M) at37∘C for 24 h before use in all tests

The pathogenic bacteria L monocytogenes 2092 4b (iso-lated from blood Escola Superior de Biotecnologia CultureCollection) was grown on Tryptone Soya agar with YeastExtract 06 (wv) (TSA-YE Lab M) and subcultured inTryptone Soya broth with Yeast Extract (TSB-YE Lab M) at37∘C for 24 h

All strains were stored at minus80∘C in the presence of 30(vv) glycerol (Panreac Barcelona Spain)

22 Preparation of Freeze-Dried Cells After growth of Ppentosaceus SB83 in MRS broth cultures were harvestedby centrifugation at 8877timesg for 10min at 4∘C and washedtwice in sterile Ringerrsquos solution (Oxoid Hampshire United

Kingdom) The harvested cells were then suspended to theoriginal volume in 11 (wv) of skim milk (Oxoid) Thecultures were frozen at minus80∘C for 24 h and subsequentlyfreeze-dried (Martin Christ Osteradam Harz Germany) for2-3 days

23 Preparation of Vaginal Tablets The formulations of vagi-nal tablets constituted different excipients 33 talc (Aco-farma Barcelona Spain) 30 magnesium stearate (JMVPereira Portugal) 02 colloidal silicon dioxide (Acofarma)75 hydroxypropyl methylcelluloses (HPMC Acofarma)as retarding polymer and 860 of lyophilized bacteriaSimultaneously tablets without the retarding polymer werealso prepared

The HPMC are known to show bioadhesive propertiesbut they are also able to hydrate and gel very slowly dueto their high molecular weights and viscosities for thisreason the tablet dissolves over a longer time and providesa prolonged release of the active ingredient embedded in thepolymeric matrix [16] The powder contained an amount ofmagnesium stearate as lubricant and talc as antiadherent [17]

All compounds were mixed in a Turbula apparatus for15min Then tablets were prepared by direct compressionusing a single-punch tablet press (Specac Ltd Kent UnitedKingdom) A quantity of themixture (approximately 600mg)was filled into a die of 11mm diameter and under a pressureof 98 kN and tablets with a plane surface were formed

The possible adverse effects of pressing on bacterialviability were investigated by comparing the viability of cellsin tablets with those in formulated powders

24 Release Study of Vaginal Tablets The determinationof the release of P pentosaceus SB83 from vaginal tabletswas done using simulated vaginal fluid (SVF) Simulatedvaginal fluid was prepared according to Owen and Katz[18] 35 gL NaCl (Panreac) 14 gL KOH (Pronalab LisbonPortugal) 02 gL Ca(OH)

2(Merck Darmstadt Germany)

002 gL bovine serum albumin (Sigma-Aldrich SteinheimGermany) 20 gL lactic acid (Fluka Steinheim Germany)10 gL acetic acid (Panreac) 02 gL glycerol 04 gL urea(Sigma-Aldrich) 50 gL glucose (Pronalab) Once thesecompounds were combined in solution the mixture wasadjusted to a pH of 42 55 and 65 using HCl (Pronalab) orNaOH (Pronalab) These pH values were used to reproducethe vaginal pH in normal conditions (pH 42) and in the caseof infection (pH 55 and 65) As control Ringerrsquos solutionwasused

Tablets with and without retarding polymer were placedin ten millilitres of each solution at 37∘C since the dailyproduction of vaginal fluid ranges between 189ndash1100mLday[18] At appropriate time intervals aliquots were withdrawnfor further enumeration of viable bacteria Two independentreplicates of these assayswere performedThe enumeration ofP pentosaceus SB83 was performed by serial 10-fold dilutionsin Ringerrsquos solution and 20 120583L was spread-plated in duplicateon MRS agar Colony forming units per gram (CFUg) wasdetermined after 48 h of incubation at 37∘C

BioMed Research International 3

25 Viability of Pediococcus pentosaceus SB83 in Tablets andLyophilized Powder Freeze-dried powder and pharmaceu-tical preparations (freeze-dried powder with excipients andtablets) were stored in plastic containers at room temperatureand at 4∘C for 12 months At several time intervals (0 13 6 9 and 12 months) the viability of P pentosaceus SB83was determined by plate method using MRS agar medium(described above) The detection limit of the enumerationtechnique was 14 log CFUg

26 Evaluation of Antimicrobial Activity of Pediococcus pen-tosaceus SB83 in Simulated Vaginal Fluid Pediococcus pen-tosaceus SB83 produce a bacteriocin against Lmonocytogenesthat is resistant to the components and pH of vaginal fluid[14]

The bacterial suspension lyophilized powder and tabletswere used in these experiments to evaluate the antimicrobialactivity of P pentosaceus SB83 in SVF To obtain the bacterialsuspension the microorganism was subcultured twice (24 hat 37∘C) in 10mL MRS broth using a 1 vv inoculumThen cultures were harvested by centrifugation at 8877timesg for10min at 4∘C washed twice in sterile Ringerrsquos solution andthe pellet of bacteria was used The lyophilized powder andtablets were prepared as described above

Simulated vaginal fluid at pH of 65 was inoculated withan overnight culture of L monocytogenes 2092 4b suspendedinRingerrsquos solutionwith a final concentration of 105CFUmLThen P pentosaceus SB83 (bacterial suspension lyophilizedpowder or tablet) was added

During 24 h aliquots were withdrawn for further enu-meration (P pentosaceus SB83 and L monocytogenes 2092 4b)and for pH measurement The enumeration of P pentosaceusSB83 was performed on MRS agar and the enumerationof L monocytogenes on PALCAM agar (Merck) after 48 hincubation at 37∘C

The experimental conditions were (1) SVF inoculatedwith P pentosaceus SB83 (bacterial suspension lyophilizedpowder tablet with retarding polymer and tablet withoutretarding polymer) and L monocytogenes 2092 4b (2) SVFwith 1mgmLof trypsin (Sigma-Aldrich) and inoculatedwithP pentosaceus SB83 and L monocytogenes 2092 4b (3) SVFinoculated with a nonbacteriocinogenic LAB strain ESB67(Escola Superior de Biotecnologia Culture Collection) andL monocytogenes 2092 4b (4) SVF inoculated with P pen-tosaceus SB83 and (5) SVF inoculated with L monocytogenes2092 4b Each trial was performed in duplicate

After this experiment the formulation of P pentosaceusSB83 that demonstrated the better results of antilisterialactivity was selected the formulation was then stored atroom temperature and 4∘C for 12 months At 1 3 6 9and 12 months the evaluation of antimicrobial activity of Ppentosaceus SB83 was performed as previously described

27 Statistical Analysis An analysis of variance (ANOVA)was carried out to assess (1) the effect of pH in the releaseof P pentosaceus SB83 in tablets with and without retardingpolymer (2) the effect of time and temperature of storageon viability of P pentosaceus SB83 in tablets and lyophilized

powder (3) the effect of different forms of P pentosaceusSB83 (bacterial suspension lyophilized powder and tablets)for antilisterial activity and reduction of pH of SVF and (4)the effect of storage on antilisterial activity and reduction ofpH of SVF All calculations were carried out using the soft-ware Kaleidagraph (version 404 Synergy Software ReadingUSA)

3 Results

31 Enumeration of Pediococcus pentosaceus SB83 in Lyo-philized Powder and Tablets The number of P pentosaceusSB83 in lyophilized powder and in tablets (with and withoutretarding polymer) was 1010 CFUg

32 Release Study of Vaginal Tablets The release of Ppentosaceus SB83 from tablets with and without retardingpolymer is shown in Figure 1

In tablets with retarding polymer P pentosaceus SB83 wasnot completely released in all media tested In SVF at pH 42a lower release was observed (approximately 50 log CFUg)than the release in the other media (SVF at pH 55 SVF atpH 65 and Ringerrsquos solution) that was approximately 80 logCFUg

In tablets without retarding polymer P pentosaceus SB83was completely released after 48 h in SVF at pH 55 SVF at pH65 and Ringerrsquos solution The release of bacteria was lowerin SVF at pH 42 with values being approximately 80 logCFUg

Therefore in both tablets (with and without retardingpolymer) the bacteria release characteristics were found to bedifferent when comparing the release in SVF at pH 42 withthe other media (119875 lt 00001)

In either of the tablets there was a large release of bacteriain the early hours of contact

33 Viability of Pediococcus pentosaceus SB83 in Tabletsand Lyophilized Powder Figure 2 shows the viability of Ppentosaceus SB83 in lyophilized powder and pharmaceuticalformulations (lyophilized powderwith excipients and tablets)during storage at room temperature and 4∘C

At room temperature in conditions with retarding poly-mer (Figure 2(a)) the survival was similar during 9 monthsof storage (119875 = 009) However after 12 months of storagethe tablets showed a greater decrease of viable cells (reductionof 95 log CFUg) than that of lyophilized powder andlyophilized powder with excipients In conditions withoutretarding polymer (Figure 2(b)) no significant differenceswere found between lyophilized powder lyophilized powderwith excipients and tablets (119875 = 018) during storage

At 4∘C in both conditions (with and without retardingpolymer) a higher survival of P pentosaceus SB83 wasrecorded during storage than at room temperature (119875 lt005)

34 Evaluation of Antimicrobial Activity of Pediococcuspentosaceus SB83 in Simulated Vaginal Fluid Figure 3 showsthe antimicrobial activity of P pentosaceus SB83 (in bacterial


0

2

4

6

8

10

12

0 2 4 6 8 10Time (hours)

log

(CFU

mL)

(a)

0

2

4

6

8

10

12

0 2 4 6 8 10Time (hours)

log

(CFU

mL)

(b)

Figure 1 Release of Pediococcus pentosaceus SB83 from (a) tablets with retarding polymer and (b) tablets without retarding polymer Therelease of bacteria was tested in SVF at pH = 42 (◼) pH = 55 (998771) pH = 65 () and in Ringerrsquos solution (⧫) All points are means plusmn standarddeviations

0 2 4 6 8 10 12

Time (months)minus12

minus10

minus8

minus6

minus4

minus2

0

2

log

(N

)N

0

(a)

0 2 4 6 8 10 12


minus10

minus8

minus6

minus4

minus2

0

2

log

(N

)N

0

(b)

Figure 2 Viability of Pediococcus pentosaceus SB83 in preparations with (a) retarding polymer and (b) without retarding polymer duringstorageThe viability was examined in freeze-dried powder (⧫) freeze-dried powder with excipients (◼) and tablets (998771) at room temperature(black lines) and at 4∘C (grey lines) All points are means plusmn standard deviations

suspension lyophilized powder and tablets with and withoutretarding polymer) against L monocytogenes 2092 4b in SVFat pH = 65

The bacterial suspension of P pentosaceus SB83 reducedL monocytogenes 2092 4b below the detection limit after only2 h The lyophilized bacteria inhibited Listeria populationmainly over 8 h of contact however total inhibition (up to thelimit of detection) was recorded after 24 hThe tablets did notreduce L monocytogenes 2092 4b during 24 h of experiment(119875 = 011) (Figure 3(a))

The viable counts of P pentosaceus SB83 were maintainedthroughout the 24 h in all conditions tested (data not shown)

All conditions tested demonstrated the ability to decreasethe pH of SVF (Figure 3(b)) However the highest reductionof pH was observed for bacterial suspension followed by thelyophilized cells The lowest reduction of pH was observedwhen tablets formulations were used No significant differ-ence was achieved for tablets with and without retardingpolymer (119875 = 094)

After obtaining these results lyophilized bacteria wereselected to evaluate the antilisterial activity during storage atroom temperature and at 4∘C for 12 months (Figure 4)

The lyophilized bacteria when stored at room temper-ature totally reduced L monocytogenes 2092 4b (up to thelimit of detection) until onemonth of storage After 3monthsa decrease in antimicrobial capability was observed From 3

to 12 months of storage there was a similar behaviour (119875 gt005) and the pathogen was reduced in the early hours ofcontact with the LAB isolate though there was an increaseof L monocytogenes 2092 4b after 24 h (Figure 4(a))

When storage was performed at 4∘C the antimicrobialactivity of P pentosaceus SB83 was maintained during storage(119875 = 066) showing only a slight decrease in antimicrobialpotential after 12 months (Figure 4(d))

As previously observed lyophilized P pentosaceus SB83were more stable at 4∘C than room temperature (Figures 4(b)and 4(e))

During storage of the lyophilized bacteria at room tem-perature the ability of P pentosaceus SB83 to reduce the pHof SVF decreased over time (119875 lt 0001) (Figure 4(c)) In con-trast during storage at 4∘C the ability to reduce the pHof SVFthroughout the time of storage was observed This capacityonly decreased slightly with time (119875 = 066 Figure 4(f))

4 Discussion

Theintravaginal route is normally used for the administrationof drugs such as antimicrobials labour-inducing agentsspermicidal agents prostaglandins and steroids [19] Accord-ingly the vaginal route can be used to prevent vaginal colo-nization by pathogenic microorganisms in pregnant womenand thereby is a method of protection of the fetusneonate


0 5 10 15 20 250

2

4

6

8

10

Time (hours)

log

(CFU

mL)

(a)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(b)

Figure 3 Antimicrobial activity of Pediococcus pentosaceus SB83 against Listeria monocytogenes 2092 4b in simulated vaginal fluid at pH =65 (a) Counts of viable cells of Listeria monocytogenes 2092 4b and (b) pH of simulated vaginal fluid during 24 h of contact are representedThe tested conditions were SVF inoculated with bacterial suspension of P pentosaceus SB83 and L monocytogenes 2092 4b (⧫) lyophilized ofP pentosaceus SB83 and L monocytogenes 2092 4b (◼) tablet of P pentosaceus SB83 with retarding polymer and L monocytogenes 2092 4b(998771) tablet of P pentosaceus SB83 without retarding polymer and L monocytogenes 2092 4b () Controls were SVF with 1mgmL of trypsinand inoculated with P pentosaceus SB83 and L monocytogenes 2092 4b (sdot sdot sdot ) SVF inoculated with a non-bacteriocinogenic LAB strain ESB67and L monocytogenes 2092 4b (ndash ndash) and SVF inoculated with P pentosaceus SB83 (mdash) SVF inoculated with L monocytogenes 2092 4b (ndash sdot)All points are means plusmn standard deviations

Based on this principle different pharmaceutical formu-lations with P pentosaceus SB83 a bacteriocin-producingLAB [14] were performed for subsequent vaginal adminis-tration

In this study freeze-dried cells of P pentosaceus SB83were used containing 1010 CFUg which remained after theproduction of tablets so tabletting had no adverse effecton the viability of bacteria Generally the quantity used forvaginal probiotics is between 108 and 1010 CFU in the product[16 20ndash22]

The normal vaginal pH in premenopausal women isacidic however vaginal pH can be increased due to a deple-tion of vaginal lactobacilli [23] menstruation unprotectedsexual intercourse with the deposition of semen [24] andvaginal medications Therefore we tested the release of Ppentosaceus SB83 in SVF at different values of pH (42 55 and65) The pH influenced the release of bacteria from tabletsTablets with retarding polymer as well as tablets withoutretarding polymer released fewer cells of P pentosaceus SB83at pH 42

The presence of retarding polymer affected the totalnumbers of bacteria released The polymer used (HPMC)retained P pentosaceus SB83 inside the tablets releasinga maximum of 108 CFUg However this release occurredin the early hours of contact with SVF such as in tabletswithout the retarding polymer Fazeli et al [25] performedan assay with tablets with HPMC and concluded that a highinitial bacterial load release occurs during the first 15minin deionized sterile water however the tablets showed acontinuous bacterial release during 575 h

The tablets and lyophilized powder were stored during12 months in order to determine their stability There wasan effect of temperature on viability of cells during storageP pentosaceus SB83 showed a higher survival rate at 4∘Cthan at room temperature According to other studies freeze-dried bacteria are more stable at low temperatures than at

room temperature [17 26] It was reported thatmilk promotessurvival at low temperature by stabilizing the cell membraneconstituents and forming a protective coating on the cell wallproteins (see review [27])

At 4∘C the viability of P pentosaceus SB83 wasmaintainedat a high level in all formulations tested for at least 12 monthsFazeli et al [25] also demonstrated that slow-release tabletswere stable at 4∘C during 6 months of storage Maggi et al[16] tested the viability of 10 lactobacilli strains in freeze-driedpowder and tablets and have shown that the stability dependson the bacterial strain and also on the polymer used as aretarding compound In their study four strains of lactobacillimaintained a high viability for 18 months at 4∘C

While observing an elevated survival of bacteria duringstorage it is important to confirm that the antimicrobial char-acteristics are alsomaintained In previous studies our groupcharacterized the bacteriocin produced by P pentosaceusSB83 in MRS medium [14] and tested the antimicrobialactivity of P pentosaceus SB83 after exposure to SVF at pH42 after 24 h [15] However it is relevant to evaluate theproduction and effectiveness of bacteriocin production bythe LAB after storage in SVF in case of vaginal colonizationby pathogens (elevated pH) The pathogen selected to assessthese parameters was L monocytogenes This bacteriumcan be acquired via vertical transmission from mother tofetusneonate through the placenta or birth canal [28 29] andcan lead to preterm labour chorioamnionitis spontaneousabortion stillbirth and neonatal infection [30]

In experiments to determine the inhibitory activity ofstored cells of P pentosaceus SB83 SVF at pH of 65 wasused because it was demonstrated previously that clinicalstrains of L monocytogenes are inhibited by the normalvaginal pH for 48 h butmay proliferatewhen the pH increases[31]

The bacterial suspension of P pentosaceus SB83 reducedL monocytogenes 2092 4b below the detection limit after only


0 5 10 15 20 250

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)0 5 10 15 20 25

0

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(a)

(b)

(c)

(d)

(e)

(f)

log

(CFU

mL)

log

(CFU

mL)

log

(CFU

g)

log

(CFU

g)

Figure 4 Antimicrobial activity of lyophilized of Pediococcus pentosaceus SB83 against Listeria monocytogenes 2092 4b in simulated vaginalfluid at pH = 65 during storage at room temperature (black lines) and at 4∘C (grey lines) ((a) and (d)) Counts of viable cells of Listeriamonocytogenes 2092 4b ((b) and (e)) counts of viable cells of Pediococcus pentosaceus SB83 ((c) and (f)) pH of simulated vaginal fluidovertime are represented These parameters were evaluated at storage time of 0 (⧫) 1 (◼) 3 (998771) 6 (times) 9 (5) and 12 () months All pointsare means plusmn standard deviations

2 h (reduction of approximately 40 log) The pH of SVF wasdecreased to normal vaginal pH (42) in 24 h

The lyophilized bacteria showed a total inhibition of thepathogen after 24 h of contact The pH of SVF decreased tovalues of 45 in 24 h

The tablets did not reduce counts of L monocytogenes2092 4b although the bacteria in the tablets were able todecrease the pH to values of 55 in 24 hThese results showedthat the activity of the bacteriocin SB83 was affected bythe excipients used in production of tablets probably by itsadsorption to the excipients It has been reported that somecompounds commonly used as pharmaceutical excipientsandor starter culture cryoprotectants can alter or completelyinhibit the activity of bacteriocins [32] The reduction inpH was rather less by cells in tablets than by lyophilized

cells this also suggests that the tabletted cells are rather lessmetabolically active than lyophilized cells

In the controls used (SVF with 1mgmL of trypsin andinoculated with P pentosaceus SB83 and L monocytogenes2092 4b SVF inoculated with a nonbacteriocinogenic LABstrain ESB67 and L monocytogenes 2092 4b) there was noinhibition of pathogen while there was a decrease of pHThissuggests that the antimicrobial activity observed in bacterialsuspension and lyophilized in SVF was due to the productionof bacteriocin SB83

The studies performed on the production of bacteriocinsin the vaginal environment are very few According tothe study by Tomas and Nader-Macıas [33] Lactobacillussalivarius CRL 1328 was able to grow in SVF at 425 but thatbacteriocin production was minimal only 40AUmL


The lyophilized powder demonstrated antimicrobialactivity therefore this formulation was selected to evaluatethe antilisterial activity during the 12 months of storage Inpharmaceutical products it is important to verify the stabilityof the bioactive compounds and the maintenance of thebiological activity during long periods of storage

When stored at room temperature lyophilized cellsinhibited totally the pathogen (up to the limit of detection) upto one month of storage After this time there was a decreasein antimicrobial potential that may be caused by diminishingnumbers of viable cells of P pentosaceus SB83 during storageat room temperature This decrease in the number of cellscould also have influenced the reduction of pH of SVF theproduction of acid is lower with storage time of P pentosaceusSB83 cells

When lyophilized cells were stored at 4∘C P pentosaceusSB83 maintained their viability and consequently the anti-listerial activity and the capacity to produce acid wereobserved throughout the time of storage The bacteriocinSB83 remained active during at least 12 months in spite of adecrease that occurred in antimicrobial activity at the twelfthmonth of storage

Zarate and Nader-Macias [32] evaluated the bacterio-cinogenic activity of L salivarius CRL 1328 after lyophiliza-tion (with different protective substances such as lactoseskim milk ascorbic acid and combinations of them) duringstorage at 5∘C It was demonstrated that bacteriocin synthesiswas not affected by the lyophilization but a slight decrease ofactivity was observed during storage in cells lyophilized withlactose and completely abolished in cells lyophilized withascorbic acid

Pingitore et al [34] tested the activity of a lyophilizedbacteriocin (salivaricin) during storage at minus25∘C 4∘C and25∘C for 18 months The antimicrobial activity of salivaricinwas dependent mainly on temperature and also on the timeof storage and protectant used to lyophilize The stability ofsalivaricin was higher at temperatures lower than 25∘C anddecreased during the time of storage

To our knowledge this is the first study where thebacteriocin production of lyophilized bacteria was testedagainst L monocytogenes under the conditions of vaginalfluid and throughout 12 months of storage

The assays for antimicrobial activity were performedagainst L monocytogenes nevertheless bacteriocin SB83 alsoinhibits the growth of Enterococcus spp [14] thus thelyophilized cells may also be used to prevent UTI caused bythis pathogen

Pediococcus pentosaceus SB83 showed the ability to reducethe pH of SVF which is relevant because in addition toinhibiting those pathogens previously mentioned this LABhas the capability of preventing colonization by other vagi-nal pathogens by reduction of vaginal pH acid one studyreported the inhibition of group B Streptococcus in SVF atpH 42 [35] Moreover the maintenance of the acidic vaginalpH will allow the proliferation of the vaginal Lactobacillusspp belonging to the normal microbiota of the vaginaltract Hemalatha et al [36] demonstrated that a probioticlactobacilli tablet was found to be better than a pH loweringvaginal tablet in preventing BV in healthy subjects

5 Conclusions

The better formulation of P pentosaceus SB83 is thelyophilized powder whichmay be administered vaginally as awashing solution Based on the tests carried out the productmust be stored at 4∘C and can be used for a period of timeof 9 months If storage is performed until this time thecharacteristics of cells are maintained that is the number ofviable cells the production of an active bacteriocin and theability to reduce vaginal pH

Further assays need to be performed including adhesionto vaginal epithelial cells and clinical trials to determinethe frequency of administration and the effectiveness ofin vivo of the lyophilized powder of P pentosaceus SB83Other formulations can be tested using lyophilized bacteriaincluding tablets with suitable excipients ovules and gels

Conflict of Interests

The authors have no conflict of interests to disclose

Acknowledgments

This work was supported by National Funds from (FCT)Fundacao para a Ciencia e a Tecnologia through project PEst-OEEQBLA00162011 Financial support for author S Borgeswas provided by a PhD fellowship SFRHBD454962008(Fundacao para a Ciencia e a Tecnologia) (FCT) Editing ofthis paper by Dr P A Gibbs is gratefully acknowledged

References

[1] S Boris andC Barbes ldquoRole played by lactobacilli in controllingthe population of vaginal pathogensrdquo Microbes and Infectionvol 2 no 5 pp 543ndash546 2000

[2] M A Farage K W Miller and J D Sobel ldquoDynamics of thevaginal ecosystem-hormonal influencesrdquo Infectious Diseasesvol 3 pp 1ndash15 2010

[3] L Jin L Tao S I Pavlova et al ldquoSpecies diversity and relativeabundance of vaginal lactic acid bacteria from women inUganda and Koreardquo Journal of Applied Microbiology vol 102no 4 pp 1107ndash1115 2007

[4] J R Schwebke ldquoRole of vaginal flora as a barrier to HIVacquisitionrdquo Current Infectious Disease Reports vol 3 no 2 pp152ndash155 2001

[5] C Charlier M Cretenet S Even and Y le Loir ldquoInteractionsbetween Staphylococcus aureus and lactic acid bacteria an oldstory with new perspectivesrdquo International Journal of FoodMicrobiology vol 131 no 1 pp 30ndash39 2009

[6] G Reid andA Bocking ldquoThe potential for probiotics to preventbacterial vaginosis and preterm laborrdquo American Journal ofObstetrics and Gynecology vol 189 no 4 pp 1202ndash1208 2003

[7] MWilks RWiggins AWhiley et al ldquoIdentification andH2

O2

production of vaginal lactobacilli from pregnant women at highrisk of preterm birth and relation with outcomerdquo Journal ofClinical Microbiology vol 42 no 2 pp 713ndash717 2004

[8] L Donati A di Vico M Nucci et al ldquoVaginal microbialflora and outcome of pregnancyrdquo Archives of Gynecology andObstetrics vol 281 no 4 pp 589ndash600 2010


[9] S Cribby M Taylor and G Reid ldquoVaginal microbiota andthe use of probioticsrdquo Interdisciplinary Perspectives on InfectiousDiseases vol 2008 9 pages 2008

[10] K C Anukam ldquoThe potential role of probiotics in reducingpoverty-associated infections in developing countriesrdquo Journalof Infection in Developing Countries vol 1 no 2 pp 81ndash83 2007

[11] T Iannitti and B Palmieri ldquoTherapeutical use of probioticformulations in clinical practicerdquo Clinical Nutrition vol 29 no6 pp 701ndash725 2010

[12] M Bolton A van der Straten and C R Cohen ldquoProbioticspotential to prevent HIV and sexually transmitted infections inwomenrdquo Sexually Transmitted Diseases vol 35 no 3 pp 214ndash225 2008

[13] S Garg D Goldman M Krumme L C Rohan S Smootand D R Friend ldquoAdvances in development scale-up andmanufacturing of microbicide gels films and tabletsrdquo AntiviralResearch vol 88 supplement pp S19ndashS29 2010

[14] S Borges J Barbosa J Silva and P Teixeira ldquoCharacterizationof a bacteriocin of Pediococcus pentosaceus SB83 and its poten-tial for vaginal applicationrdquo Anti-Infective Agents vol 11 no 22013

[15] S Borges J Barbosa J Silva and P Teixeira ldquoEvaluationof characteristics of Pediococcus spp to be used as a vaginalprobioticrdquo Journal of Applied Microbiology 2013

[16] L Maggi P Mastromarino S Macchia et al ldquoTechnologicaland biological evaluation of tablets containing different strainsof lactobacilli for vaginal administrationrdquo European Journal ofPharmaceutics andBiopharmaceutics vol 50 no 3 pp 389ndash3952000

[17] S Klayraung H Viernstein and S Okonogi ldquoDevelopment oftablets containing probiotics effects of formulation and pro-cessing parameters on bacterial viabilityrdquo International Journalof Pharmaceutics vol 370 no 1-2 pp 54ndash60 2009

[18] D H Owen and D F Katz ldquoA vaginal fluid simulantrdquo Contra-ception vol 59 no 2 pp 91ndash95 1999

[19] E Baloglu Z A Senyigit S Y Karavana and A Bernkop-Schnurch ldquoStrategies to prolong the intravaginal residence timeof drug delivery systemsrdquo Journal of Pharmacy and Pharmaceu-tical Sciences vol 12 no 3 pp 312ndash336 2009

[20] P Mastromarino P Brigidi S Macchia et al ldquoCharacterizationand selection of vaginal Lactobacillus strains for the preparationof vaginal tabletsrdquo Journal of Applied Microbiology vol 93 no5 pp 884ndash893 2002

[21] P-G Larsson B Stray-Pedersen K R Ryttig and S LarsenldquoHuman lactobacilli as supplementation of clindamycin topatients with bacterial vaginosis reduce the recurrence rate a6-month double-blind randomized placebo-controlled studyrdquoBMCWomenrsquos Health vol 8 article 3 2008

[22] S Ehrstrom K Daroczy E Rylander et al ldquoLactic acidbacteria colonization and clinical outcome after probiotic sup-plementation in conventionally treated bacterial vaginosis andvulvovaginal candidiasisrdquoMicrobes and Infection vol 12 no 10pp 691ndash699 2010

[23] G G G Donders T Caeyers P Tydhof I Riphagen T van denBosch and G Bellen ldquoComparison of two types of dipsticksto measure vaginal pH in clinical practicerdquo European Journalof Obstetrics Gynecology and Reproductive Biology vol 134 no2 pp 220ndash224 2007

[24] C Tevi-Benissan L Belec M Levy et al ldquoIn vivo semen-associated pHneutralization of cervicovaginal secretionsrdquoClin-ical and Diagnostic Laboratory Immunology vol 4 no 3 pp367ndash374 1997

[25] M R Fazeli T Toliyat N Samadi S Hajjaran andH JamalifarldquoViability of Lactobacillus acidophilus in various vaginal tabletformulationsrdquo Daru vol 14 no 4 pp 172ndash177 2006

[26] S Kaewnopparat and N Kaewnopparat ldquoFormulation andevaluation of vaginal suppositories containing LactobacillusrdquoWorld Academy of Science Engineering and Technology vol 55pp 25ndash28 2009

[27] A S Carvalho J Silva P Ho P Teixeira F X Malcata andP Gibbs ldquoRelevant factors for the preparation of freeze-driedlactic acid bacteriardquo International Dairy Journal vol 14 no 10pp 835ndash847 2004

[28] A R Delgado ldquoListeriosis in pregnancyrdquo Journal of Midwiferyand Womenrsquos Health vol 53 no 3 pp 255ndash259 2008

[29] K M Posfay-Barbe and E R Wald ldquoListeriosisrdquo Seminars inFetal and Neonatal Medicine vol 14 no 4 pp 228ndash233 2009

[30] H DiMaio ldquoListeria infection in womenrdquo Primary Care Updatefor ObGyns vol 7 no 1 pp 40ndash45 2000

[31] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[32] G Zarate and M E Nader-Macias ldquoInfluence of probioticvaginal lactobacilli on in vitro adhesion of urogenital pathogensto vaginal epithelial cellsrdquo Letters in Applied Microbiology vol43 no 2 pp 174ndash180 2006

[33] M S J Tomas and M E Nader-Macıas ldquoEffect of a mediumsimulating vaginal fluid on the growth and expression ofbeneficial characteristics of potentially probiotic lactobacillirdquoCommunicating Current Research and Educational Topics andTrends in Applied Microbiology vol 2 pp 732ndash739 2007

[34] E V Pingitore E Bru and M E Nader-Macıas ldquoEffectof lyophilization and storage temperature on the activity ofsalivaricin CRL 1328 a potential bioactive ingredient of aurogenital probiotic productrdquo Journal of General and AppliedMicrobiology vol 58 no 2 pp 71ndash81 2012

[35] S Borges J Silva and P Teixeira ldquoSurvival and biofilmformation by Group B streptococci in simulated vaginal fluidat different pHsrdquo Antonie van Leeuwenhoek vol 101 no 3 pp677ndash682 2012

[36] R Hemalatha P Mastromarino B A Ramalaxmi N VBalakrishna and S Sesikeran ldquoEffectiveness of vaginal tabletscontaining lactobacilli versus pH tablets on vaginal health andinflammatory cytokines a randomized double-blind studyrdquoEuropean Journal of Clinical Microbiology and Infectious Dis-eases vol 31 no 11 pp 3097ndash3105 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


probiotics for maintaining normal urogenital health [10]TheWorld Health Organization and the Food and AgricultureOrganization of theUnitedNations have defined probiotics asldquolivemicroorganisms which when administered in adequateamounts confer a health benefit on the hostrdquo [11]

A probiotic may act indirectly through treating andpreventing recurrent BV or directly by secreting substances(eg hydrogen peroxide bacteriocins and lactic acid) thatinhibit pathogens [12]

Vaginal dosage forms available include creams gelstablets capsules pessaries foams ointments films tamponsrings and douches While the majority of vaginal drugs sofar have been in the form of gels there is a growing interestin alternative dosage forms such as rings tablets and filmsThemajority of issues with product development and scaleupare similar to other pharmaceutical products although thereare some unique challenges because of the site of deliveryprophylactic nature of product application and diversity insex and hygiene practices across the developing world [13]

The purpose of this study was to develop a vaginalformulation containing viable cells of a LAB isolate whichcan be used by women to prevent vaginal colonization ofmicroorganisms (eg Lmonocytogenes) thatmay be harmfulto the fetusneonate In this study the probiotic properties(survival and antagonistic activity) of formulations wereevaluated after manufacture and during long-term storage ofthe product at different temperatures

2 Material and Methods

21 Microorganisms and Culture Conditions Pediococcuspentosaceus SB83 (Escola Superior de Biotecnologia CultureCollection) was selected because previous studies demon-strated that this strain is able to inhibit the growth of clinicalisolates of L monocytogenes (serotype 12a 12b and 4b) byproduction of a bacteriocin [14 15] This strain has beenshown to survive in simulated vaginal fluid at normal vaginalpH (pH of 42) and P pentosaceus SB83 does not producevirulence factors such as gelatinase lipase and DNase andhemolytic activity nor does it possess virulence genes (genesesp agg gelE efaAfm efaAfs cylA cylB and cylM) [15]

Pediococcus pentosaceus SB83 was grown on de ManRogosa and Sharpe (MRS) agar (Lab M Bury United King-dom) and was subcultured twice in MRS broth (Lab M) at37∘C for 24 h before use in all tests

The pathogenic bacteria L monocytogenes 2092 4b (iso-lated from blood Escola Superior de Biotecnologia CultureCollection) was grown on Tryptone Soya agar with YeastExtract 06 (wv) (TSA-YE Lab M) and subcultured inTryptone Soya broth with Yeast Extract (TSB-YE Lab M) at37∘C for 24 h

All strains were stored at minus80∘C in the presence of 30(vv) glycerol (Panreac Barcelona Spain)

22 Preparation of Freeze-Dried Cells After growth of Ppentosaceus SB83 in MRS broth cultures were harvestedby centrifugation at 8877timesg for 10min at 4∘C and washedtwice in sterile Ringerrsquos solution (Oxoid Hampshire United

Kingdom) The harvested cells were then suspended to theoriginal volume in 11 (wv) of skim milk (Oxoid) Thecultures were frozen at minus80∘C for 24 h and subsequentlyfreeze-dried (Martin Christ Osteradam Harz Germany) for2-3 days

23 Preparation of Vaginal Tablets The formulations of vagi-nal tablets constituted different excipients 33 talc (Aco-farma Barcelona Spain) 30 magnesium stearate (JMVPereira Portugal) 02 colloidal silicon dioxide (Acofarma)75 hydroxypropyl methylcelluloses (HPMC Acofarma)as retarding polymer and 860 of lyophilized bacteriaSimultaneously tablets without the retarding polymer werealso prepared

The HPMC are known to show bioadhesive propertiesbut they are also able to hydrate and gel very slowly dueto their high molecular weights and viscosities for thisreason the tablet dissolves over a longer time and providesa prolonged release of the active ingredient embedded in thepolymeric matrix [16] The powder contained an amount ofmagnesium stearate as lubricant and talc as antiadherent [17]

All compounds were mixed in a Turbula apparatus for15min Then tablets were prepared by direct compressionusing a single-punch tablet press (Specac Ltd Kent UnitedKingdom) A quantity of themixture (approximately 600mg)was filled into a die of 11mm diameter and under a pressureof 98 kN and tablets with a plane surface were formed

The possible adverse effects of pressing on bacterialviability were investigated by comparing the viability of cellsin tablets with those in formulated powders

24 Release Study of Vaginal Tablets The determinationof the release of P pentosaceus SB83 from vaginal tabletswas done using simulated vaginal fluid (SVF) Simulatedvaginal fluid was prepared according to Owen and Katz[18] 35 gL NaCl (Panreac) 14 gL KOH (Pronalab LisbonPortugal) 02 gL Ca(OH)

2(Merck Darmstadt Germany)

002 gL bovine serum albumin (Sigma-Aldrich SteinheimGermany) 20 gL lactic acid (Fluka Steinheim Germany)10 gL acetic acid (Panreac) 02 gL glycerol 04 gL urea(Sigma-Aldrich) 50 gL glucose (Pronalab) Once thesecompounds were combined in solution the mixture wasadjusted to a pH of 42 55 and 65 using HCl (Pronalab) orNaOH (Pronalab) These pH values were used to reproducethe vaginal pH in normal conditions (pH 42) and in the caseof infection (pH 55 and 65) As control Ringerrsquos solutionwasused

Tablets with and without retarding polymer were placedin ten millilitres of each solution at 37∘C since the dailyproduction of vaginal fluid ranges between 189ndash1100mLday[18] At appropriate time intervals aliquots were withdrawnfor further enumeration of viable bacteria Two independentreplicates of these assayswere performedThe enumeration ofP pentosaceus SB83 was performed by serial 10-fold dilutionsin Ringerrsquos solution and 20 120583L was spread-plated in duplicateon MRS agar Colony forming units per gram (CFUg) wasdetermined after 48 h of incubation at 37∘C











3 Results












0

2

4

6

8

10

12

0 2 4 6 8 10Time (hours)

log

(CFU

mL)

(a)

0

2

4

6

8

10

12

0 2 4 6 8 10Time (hours)

log

(CFU

mL)

(b)


0 2 4 6 8 10 12


minus10

minus8

minus6

minus4

minus2

0

2

log

(N

)N

0

(a)

0 2 4 6 8 10 12


minus10

minus8

minus6

minus4

minus2

0

2

log

(N

)N

0

(b)












4 Discussion



0 5 10 15 20 250

2

4

6

8

10

Time (hours)

log

(CFU

mL)

(a)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(b)













0 5 10 15 20 250

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)0 5 10 15 20 25

0

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(a)

(b)

(c)

(d)

(e)

(f)

log

(CFU

mL)

log

(CFU

mL)

log

(CFU

g)

log

(CFU

g)

















5 Conclusions





Acknowledgments


References








O2





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results












0

2

4

6

8

10

12

0 2 4 6 8 10Time (hours)

log

(CFU

mL)

(a)

0

2

4

6

8

10

12

0 2 4 6 8 10Time (hours)

log

(CFU

mL)

(b)


0 2 4 6 8 10 12


minus10

minus8

minus6

minus4

minus2

0

2

log

(N

)N

0

(a)

0 2 4 6 8 10 12


minus10

minus8

minus6

minus4

minus2

0

2

log

(N

)N

0

(b)












4 Discussion



0 5 10 15 20 250

2

4

6

8

10

Time (hours)

log

(CFU

mL)

(a)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(b)













0 5 10 15 20 250

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)0 5 10 15 20 25

0

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(a)

(b)

(c)

(d)

(e)

(f)

log

(CFU

mL)

log

(CFU

mL)

log

(CFU

g)

log

(CFU

g)

















5 Conclusions





Acknowledgments


References








O2





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

12

0 2 4 6 8 10Time (hours)

log

(CFU

mL)

(a)

0

2

4

6

8

10

12

0 2 4 6 8 10Time (hours)

log

(CFU

mL)

(b)


0 2 4 6 8 10 12


minus10

minus8

minus6

minus4

minus2

0

2

log

(N

)N

0

(a)

0 2 4 6 8 10 12


minus10

minus8

minus6

minus4

minus2

0

2

log

(N

)N

0

(b)












4 Discussion



0 5 10 15 20 250

2

4

6

8

10

Time (hours)

log

(CFU

mL)

(a)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(b)













0 5 10 15 20 250

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)0 5 10 15 20 25

0

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(a)

(b)

(c)

(d)

(e)

(f)

log

(CFU

mL)

log

(CFU

mL)

log

(CFU

g)

log

(CFU

g)

















5 Conclusions





Acknowledgments


References








O2





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 5 10 15 20 250

2

4

6

8

10

Time (hours)

log

(CFU

mL)

(a)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(b)













0 5 10 15 20 250

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)0 5 10 15 20 25

0

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(a)

(b)

(c)

(d)

(e)

(f)

log

(CFU

mL)

log

(CFU

mL)

log

(CFU

g)

log

(CFU

g)

















5 Conclusions





Acknowledgments


References








O2





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 5 10 15 20 250

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)0 5 10 15 20 25

0

2

4

6

8

10

Time (hours)

0 5 10 15 20 250

2

4

6

8

10

Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

4

45

5

55

6

65

7

pH

0 5 10 15 20 25Time (hours)

(a)

(b)

(c)

(d)

(e)

(f)

log

(CFU

mL)

log

(CFU

mL)

log

(CFU

g)

log

(CFU

g)

















5 Conclusions





Acknowledgments


References








O2





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























5 Conclusions





Acknowledgments


References








O2





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Effects of Processing and Storage on … · 2019. 7. 31. · (v/v) glycerol...

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Transcript of Research Article Effects of Processing and Storage on … · 2019. 7. 31. · (v/v) glycerol...