Effects of pH, temperature and NaCl concentration on the growth kinetics, proteolytic activity and...

International Journal of Food Microbiology 64 (2001) 105–117www.elsevier.nl / locate / ijfoodmicro

Effects of pH, temperature and NaCl concentration on the growthkinetics, proteolytic activity and biogenic amine production of

Enterococcus faecalis

a , b b b*Fausto Gardini , Maria Martuscelli , Marisa Carmela Caruso , Fernanda Galgano ,b b a bMaria Antonietta Crudele , Fabio Favati , Maria Elisabetta Guerzoni , Giovanna Suzzi

a `Dipartimento di Protezione e Valorizzazione Agroalimentare, Universita degli Studi di Bologna, Via San Giacomo 7, 40126 Bologna,Italy

b `Dipartimento di Biologia, Difesa e Biotecnologie Agroforestali, Universita della Basilicata, Via Nazario Sauro 85, 85100 Potenza,Italy

Received 18 October 1999; received in revised form 23 June 2000; accepted 29 September 2000

AbstractIn this work, the combined effects of temperature, pH and NaCl concentration on the growth dynamics of Enterococcus

faecalis EF37, its proteolytic activity and its production of biogenic amines have been studied. The effects of the selectedvariables have been analysed using a Central Composite Design. The production of biogenic amines, under the adoptedconditions, was found to be mainly dependent on the extent of growth of E. faecalis. Its proteolytic activity was not alimiting factor for the final amine production, because in the system studied (skim milk) an excess of precursors wasguaranteed. Quantitatively, the most important biogenic amine produced was 2-phenylethylamine but substantial amounts oftyramine were detected in all the samples. This work confirms that the main biological feature influencing the biogenicamine formation is the extent of growth of microorganisms, like E. faecalis, characterised by decarboxylase activity. In the

7traditional and artisanal cheeses produced using raw milk, enterococci usually reach levels of 10 cells /g. With thisperspective, it is important that the presence of biogenic amines due to the activities of these microorganisms is maintainedwithin safe levels, without affecting the positive effects of enterococci on the final organoleptic characteristics of the cheese. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Biogenic amine; Enterococcus faecalis; pH; Temperature; NaCl

1. Introduction 1994). Milk is an ideal source for the growth of theseorganisms. Enterococci may be present in substantial

Enterococci commonly occur in raw milk and numbers in cheese curd and ripened cheeses (Clarkdairy products (Garg and Mital, 1991; Giraffa et al., and Reinbold, 1966; Mucchetti et al., 1982; Fontecha

et al., 1990; Poullet et al., 1991; Litopoulou-Tzanetaki et al., 1992; Freitas et al., 1996; Centeno*Corresponding author. Tel.: 139-051-2099-783; fax: 139-et al., 1996; Cogan et al., 1997). Many researchers051-2099-782.

E-mail address: [email protected] (F. Gardini). have also reported their presence in pasteurised and

0168-1605/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0168-1605( 00 )00445-1

106 F. Gardini et al. / International Journal of Food Microbiology 64 (2001) 105 –117

UHT milk and investigated the survival upon milk and produce pressor amines in cheeses (Garg andprocessing (Garg and Mital, 1991). With respect to Mital, 1991; Giraffa et al., 1997).growth temperature, enterococci grow from 08C up An important role in amine production is playedto 508C and survive a treatment of 608C for 30 min by the proteolytic activity in the medium, whichin a neutral medium (Shattock, 1963). In addition, provides the substrates for the decarboxylase activitytheir capacity to develop in the presence of 6.5% (Joosten, 1988). Tham et al. (1990) investigated theNaCl and in broth at pH 9.6 is commonly used as histamine producing ability of enterococci isolatedphysiological criteria for distinguishing enterococci from artisanal goat cheeses and found that En-from other streptococci (Sherman, 1937). The terococcus faecalis did not produce histamine,prolific growth of enterococci in many foods, leading whereas E. faecium produces only small amounts ofto formation of significant levels of biogenic amines, this amine in cheeses.has been observed (Garg and Mital, 1991; Giraffa et It is not clear which factors are most important inal., 1995). These compounds, which are produced accounting for the occasional high amounts offrom free amino acids by decarboxylase activity of biogenic amines in cheeses. There are many en-microorganisms, have various toxicological implica- vironmental factors that can affect the amine forma-tions. Large quantities of tyramine and histamine can tion by enterococci, such as temperature, pH, andevoke symptoms such hypertension, hypotension, salt concentration. Many studies have investigatedheadache, urticaria, nausea, and vomiting (Edwards the single effects of these factors, but little in-and Sandine, 1981). Putrescine and cadaverine can formation exists on their combined or interactivealso react with nitrite to form carcinogenic nitro- effects.samines (Askar and Treptow, 1996). The toxicologi- Tyramine and 2-phenylethylamine positive strainscal levels of amines is very difficult to establish of enterococci from artisanal goat cheese werebecause it depends on individual characteristics and isolated from Suzzi et al. (2000). They found thatthe presence of other amine. A legal upper limit of proteolytic strains of E. faecalis always produced100 mg of histamine /kg of food and 2 mg/ l of significant levels of biogenic amines. The aims ofethanol have been suggested. Values of 100–800 this work were to investigate the combined effects ofmg/kg for tyramine and 30 mg/kg for phenylethyl- temperature, pH and NaCl concentration on theamine have been reported as toxic doses in food growth dynamics of a strain of E. faecalis, its(Taylor, 1985; Silla Santos, 1996). proteolytic activity and the production of biogenic

A variety of amines (i.e. histamine, tyramine, amine. With these purposes, an amine positivecadaverine, putrescine and 2-phenylethylamine) have enterococcal strain (E. faecalis 37), isolated frombeen found in many different kinds of cheese (Rice goat cheese, was grown at different levels of theet al., 1976; Maga, 1978; Silla Santos, 1996). Several three considered variables, according to a threeoutbreaks of biogenic amine poisoning from dairy variables and five levels Central Composite Designproducts occurred because of the consumption of (CCD) (Box et al., 1978).Gouda, Cheddar and Swiss cheese containing highconcentrations of histamine (Doeglas et al., 1967;Taylor and Woychit, 1982). However, the dataconcerning the histamine intoxication are underesti-mated, because the illness is easily misdiagnosed as 2. Materials and methodsan allergic reaction (Silla Santos, 1996). The onlyrelevant decarboxylase activity from enterococci 2.1. Cultureisolated from dairy products concerned tyrosine. Themicroorganisms known to possess significant E. faecalis EF37, belonging to the Collection oftyrosine decarboxylase activity in cheeses are Department of Biologia, Difesa e Biotecnologiecoliforms and enterococci. Since raw milk is general- Agroforestali (University of Basilicata), was storedly pasteurised during the cheese-making process, the at 2 808C and pre-cultivated in M17 broth (Oxoid,coliforms are inactivated and enterococci, being UK). In skim milk, the strain was characterised bymore heat and acid resistant, can possibly survive high proteolytic activity and produced 2-phenylethyl-

F. Gardini et al. / International Journal of Food Microbiology 64 (2001) 105 –117 107

amine, tyramine, cadaverine, spermidine and sper- is the constant, B , B and B are coefficients of thei ii ij

mine (Suzzi et al., 2000) model, and x and x are the independent variables.i j

The variables with a significance lower than 95%(P.0.05) were not included in the final models.2.2. Experimental design

The three-dimensional surface plots were drawn toillustrate the effects of the independent variables onThe E. faecalis EF37 activities were monitored inthe dependent ones. These graphs were drawn im-M17 broth and in skim milk in relation to threeposing a constant value (i.e. the central points of thedifferent variables: the incubation temperature [8C]interval taken into consideration) to one of the(ranging from 16 to 448C), the percentage (w/v) ofindependent variables.sodium chloride added to the medium [NaCl] (from

2 to 6%) and the environmental pH [pH] (from 5.42.3. Measurements of the growth parametersto 7).

The E. faecalis strain was grown at differentThe evaluation of enterococci growth was per-values of the three variables considered according to

formed by measuring the optical density (OD) at 590a Central Composite Design (CCD). Table 1 reportsnm of M17 cultural broth conditioned according tothe values of pH, temperature and NaCl concen-

6the CCD. The broth was inoculated with 10 cells /tration in the 17 runs of the CCD (a 5 2). Theml. The OD was monitored by means of a Cary 1Eresults obtained in each run of the CCD for theUV–visible Spectrophotometer (Varian, Australia) atdifferent dependent variables considered (growthregular intervals (2 h). The OD data obtained overparameters, proteolytic activity and biogenic aminetime were modeled according to the Gompertzproduction) were fitted by a second-order model toequation as modified by Zwietering et al. (1990):the independent variables using a quadratic polyno-

mial equation: m emax]]y 5 A ? exp 2 exp ? (l 2 t) 1 1H FS D GJA2y 5 B 1 SB x 1 SB x 1 SB x x0 i i ii i ij i j

where y is the OD at time t, A represents themaximum OD reached, m is the maximum ODwhere y is the dependent variable to be modelled, B max0

21increase rate (as DOD h ) and l is the lag time (h)for OD increase.

Table 1Central Composite Design (three factors, five levels) used for the

2.4. Proteolytic activityevaluation of the effects of pH, temperature and NaCl con-centration on the growth parameters, proteolytic activity andbiogenic amine production by E. faecalis EF37 Proteolytic activity (expressed as free amino acids

in the medium) was evaluated in skim milk after 72 hRuns pH Temperature (8C) NaCl concentration (%)

of incubation. Free amino acids (as mg of leucine /1 5.8 23 3100 ml) were determined at 507 nm after reaction2 6.6 23 3

3 5.8 37 3 with Cd-ninhydrin (Folkertsma and Fox, 1992) with4 6.6 37 3 a Cary 1E UV–visible Spectrophotometer (Varian,5 5.8 23 5 Australia).6 6.6 23 57 5.8 37 5

2.5. Biogenic amine determination8 6.6 37 59 6.2 30 4

10 6.2 30 4 After 72 h of incubation, the inoculated skim milk11 5.4 30 4 was tested for the presence of tryptamine, 2-phenyl-12 7.0 30 4

ethylamine, putrescine, cadaverine, histamine,13 6.2 16 4tyramine, spermidine and spermine. Total biogenic14 6.2 44 4

15 6.2 30 2 amines are defined as the sum of the individual16 6.2 30 6 amines. Biogenic amines produced were determined17 6.2 30 4 by HPLC analysis using a Varian Model 9012 liquid


chromatograph (Varian, Australia). Amine derivatisa- E. faecalis EF37, in relation to pH, incubationtion was performed according to Eerola et al. (1993), temperature and NaCl concentration, are reported.while the chromatographic analysis was conducted as Moreover, the equation relative to the OD reacheddescribed by Moret et al. (1992). after 72 h of incubation (the time after which the

proteolytic activity and the biogenic amine product-2.6. Statistical treatment of data ion have been monitored) is reported.

The maximum optical density reached (A) wasAll the data were elaborated by means of the affected only by the interactive terms [Eq. (1), Table

statistical package Statistica for Windows (Statsoft 3]. However, pH was found to be the most importantInc. USA). variable influencing this growth parameter. In fact, as

shown by the Fig. 1a,b, relative to the interactions[8C]3[pH] and [pH]3[NaCl], the increase of OD

3. Results mostly depended on the pH increase. Fig. 1c, relativeto the interaction [NaCl]3[8C], shows that increas-

3.1. Growth parameters ing salt concentrations had positive effects on E.faecalis EF37 cell yield when the incubation tem-

Table 2 reports, for each run of the CCD, the perature is lower than 248C.response measured for the different dependent vari- By contrast, the m [Eq. (2), Table 3] was moremax

able considered (growth parameters, proteolytic ac- susceptible to salt concentration, as showed by Fig.tivity and biogenic amine production). In Table 3, 2b,c, relative to the interactions [pH]3[NaCl] andthe surface response equations describing the be- [8C]3[NaCl], in which a strong reduction of mmax

haviour of the growth parameters (A, m and l) of was observed when NaCl concentration increased upmax

Table 2Measured responses of the dependent variables in the different runs of the CCD

aRun Growth parameters Proteolytic Biogenic aminesactivity

bA m l A (ppm of free 2-phenylethylamine Tyramine Totalmax 72

(OD) (OD/h) (h) (OD) amino acids) (ppm) (ppm) (ppm)

1 1.40 0.08 19.17 1.40 121.75 7.52 0.30 10.152 1.90 0.19 13.97 1.90 155.78 13.12 5.25 25.053 1.76 0.20 3.40 1.75 131.01 8.97 3.92 16.714 2.03 0.59 4.17 2.03 174.55 14.14 7.93 24.235 1.53 0.05 25.32 1.44 34.33 0.05 1.60 1.786 1.80 0.10 17.67 1.80 76.53 0.71 2.31 5.547 1.45 0.13 5.77 1.45 79.58 0.05 1.60 1.788 1.84 0.28 5.01 1.83 130.80 3.68 3.06 7.029 1.71 0.20 6.96 1.17 187.74 3.71 2.93 6.70

10 1.76 0.19 7.10 1.76 163.29 3.49 2.49 6.1411 1.08 0.05 12.40 1.08 93.63 0.10 1.95 1.9512 1.96 0.20 5.70 1.96 182.80 7.59 3.56 11.3213 1.66 0.07 38.83 1.55 7.02 0.58 1.71 2.8814 1.45 0.27 4.93 1.45 88.43 1.62 2.15 4.9715 1.96 0.35 5.36 1.96 121.10 11.92 4.64 18.9416 1.69 1.14 12.24 1.69 20.32 0.10 2.20 3.7917 1.66 0.21 6.80 1.66 193.34 2.80 2.39 6.50

a A, maximum OD; m , maximum growth rate; l, lag phase length; and A (OD reached after 72 h) are obtained by fitting withmax 72

Gompertz equation the OD data over time.b Total biogenic amines is the sum of tryptamine, 2-phenylethylamine putrescine, cadaverine, histamine, tyramine, spermidine and

spermine.


Table 3Models obtained for the growth parameter calculated by means of the Gompertz equation as modified by Zwietering et al. (1990)

b cEquation Growth Model R Faparameter

(1) A (OD) 0.00651[NaCl][pH]2 0.997 857.540.00151[NaCl][8C]10.0101[pH][8C]

(2) m (OD/h) 0.00463[pH][8C]20.00017[8C][NaCl]20.0158[8C] 0.975 93.39max2(3) l (h) 370.26265.70[pH]28.90[8C]10.006[NaCl] 2 0.992 155.37

2 23.58[pH] 10.077[8C] 10.57[pH][8C]2

0.01[NaCl][8C](4) Growth after 0.327[pH]20.0088[NaCl] 0.996 1053.41

72 h (OD)a Growth parameters calculated by fitting the OD data over time with the Gompertz equation as modified by Zwietering et al. (1990).b Regression coefficient.c Fisher test value. All the model were characterised by high significance (P,0.0001).

to 6%. A positive effect on m was observed when The proteolytic activity [Eq. (1), Table 4] wasmax

pH and temperature increased (within the ranges found to be affected by all the variables considered.considered for these variables), as shown by Fig. 2a. In particular, the presence of the quadratic terms of

The most important factor influencing the lag NaCl concentration and temperature (with negativephase length (l) was temperature [Eq. (3), Table 3], signs) indicates the presence of a maximum of thiswhich minimised this growth parameter at values of physiological activity in relation to these variables.about 388C, as shown by Fig. 3a,c, relative to the This is showed in Fig. 4, which suggests that theinteractions [8C]3[pH] and [8C]3[NaCl]. The ef- maximum proteolysis was reached at an incubationfects of the other two variables were less pro- temperature of about 32–348C and at NaCl con-nounced, even if it is possible to underline a linear centration of about 3.2%. The effect of pH on thisincrease of l with the increase of NaCl concentration activity was rather weak (at least within the intervaland a slight increase of lag phase due to the decrease of values considered in this investigation), even if anof pH (Fig. 3b). increase of proteolysis can be observed with the

Eq. (4) of Table 3 reports the model relative to the increase of pH.OD reached after 72 h of incubation. In this case,differently from the model relative to A, temperaturedid not significantly affect this value, which linearly 3.3. Biogenic amine productionincreased with the increase of pH and the decrease ofNaCl concentration. The same medium (skim milk) in which the

proteolytic activity has been determined was used for3.2. Proteolytic activity the determination of biogenic amines produced by E.

faecalis EF37. After 72 h of incubation, the mediumMany authors (Chang et al., 1985; Joosten, 1988; was tested for the presence of tryptamine, 2-phenyl-

Giraffa et al., 1995) reported that the production of ethylamine, putrescine, cadaverine, histamine,biogenic amines in cheese could be limited by the tyramine, spermidine and spermine.amount of their precursor, i.e. the free amino acids in Among these, only 2-phenylethylamine andthe medium. In consideration of this, the influence of tyramine were detected in all the runs of the CCD.the three variables considered on the proteolytic For this reason, only for these two amines, togetheractivity of E. faecalis EF37 has been investigated. with the total biogenic amine production, a responseThe proteolysis of the enterococcal strain was mea- surface equation has been obtained.sured (as mg of free amino acids in 100 ml) in skim In particular, 2-phenylethylamine was found to bemilk conditioned according to the CCD after 72 h of the most important quantitative biogenic amine,fermentation. accounting for more than half of the total biogenic


Fig. 1. Three-dimensional surfaces relative to the A values (expressed as OD) calculated according to the modified Gompertz equation(Zwietering et al., 1990). The figures represent the effects on this variable of the interactions [pH]3[T ] (a), [NaCl]3[pH] (b) and[NaCl]3[T ] (c).

amines in almost all the runs of the CCD. The total amine production after 72 h of incubation inequation obtained for 2-phenylethylamine, tyramine relation to pH and NaCl concentration are reported.and total biogenic amines produced are reported in In general, the biogenic amine production was veryTable 4 (Eqs. (2)–(4)). low at the higher NaCl concentrations and this trend

In all the models, the production of biogenic was enhanced by the lower pH. The increase of pHamines was independent of the incubation tempera- resulted in a rapid increase in biogenic amineture. Similarly, the OD (i.e. the cell yield) predicted production. It is interesting to observe that theafter 72 h of incubation was independent on this maximum values of biogenic amines were reached atfactor. values of pH and NaCl concentration determining

In Fig. 5 the 2-phenylethylamine, tyramine and also the higher growth parameters after 72 h.


Fig. 2. Three-dimensional surfaces relative to the m values (expressed as OD/h) calculated according to the modified Gompertz equationmax

(Zwietering et al., 1990). The figures represent the effects on this variable of the interactions [pH]3[T ] (a), [NaCl]3[pH] (b) and[NaCl]3[T ] (c).

4. Discussion 1946). Others reports have supported this theory(Sinell, 1978; Chander et al., 1988). In particular,

The pH level is a key factor influencing the Teodorovic et al. (1994) showed that the amino acidinteractions amino acid decarboxylase activity. Over decarboxylase activities were higher when pH ranged60 years ago, Koessler et al. (1928) suggested that between 4 and 5.5. However, amine formation wasamine formation by bacteria was a physiological found to depend on the growth activity of bacteriamechanism to counteract acid environment. Compat- rather than on the growth conditions per seible with this suggestion, bacterial amino acid de- (Yoshinaga and Frank, 1982). Maijala et al. (1993)carboxylases exhibit an acid pH optimum (Gale, and Maijala (1994) reported that the acidification of


Fig. 3. Three-dimensional surfaces relative to the l values (expressed as h) calculated according to the modified Gompertz equation(Zwietering et al., 1990). The figures represent the effects on this variable of the interactions [pH]3[T ] (a), [NaCl]3[pH] (b) and[NaCl]3[T ] (c).

fortified MRS broth by adding glucono-6-lactone or in turn, is mainly affected by pH, under the adoptedlactic acid decreased the cell yield of an amine conditions. The influence of temperature on the finalpositive Lactobacillus strain and, consequently, the cell yield is low but, above all, its lack of signifi-production of histamine and tyramine. According to cance on the extent of growth after 72 h of incuba-the latter authors, E. faecalis EF37 biogenic amine tion seems to be the reason for which biogenic amineproduction significantly decreased with decreasing amounts were not influenced by temperature in thispH. study. Taking into consideration that the cell con-

However, the role of temperature should not be centration was found to be the most important factorunderestimated. In fact, this variable considerably in biogenic amine production, a significant delay ininfluenced the lag phase of E. faecalis EF37. In this the growth kinetics of decarboxylase positive micro-study, the single and total biogenic amine production organisms due to low temperatures can reduce theseem to be dependent of the extent of growth, which, health risks associated with the presence of these


Table 4Models obtained for the proteolytic activity and biogenic amine production in skim milk

a bEquation Biochemical Model R Factivity

2(1) Proteolytic activity 2108.83[pH]123.64[8C]20.58[8C] 2 0.992 117.462(mg/100 ml of free 22.22[NaCl] 12.17[pH][8C]123.94[pH][NaCl]

amino acids)2 2(2) 2-phenylethyl- 0.882[pH] 10.871[NaCl] 21.776[pH][NaCl] 0.963 59.37

amine (ppm)2(3) Tyramine (ppm) 0.171[pH] 20.146[pH][NaCl] 0.928 46.20

2 2(4) Total biogenic 1.445 [pH] 11.561[NaCl] 22.942[pH][NaCl] 0.958 52.56amines (ppm)

a Regression coefficient.b Fisher test value. All the model were characterised by high significance (P,0.001).

Fig. 4. Three-dimensional surfaces relative to the proteolytic activity (expressed as free amino acids /100 ml of skim milk). The figuresrepresent the effects on this variable of the interactions [pH]3[T ] (a), [NaCl]3[pH] (b) and [NaCl]3[T ] (c).


Fig. 5. Three-dimensional surfaces relative to the biogenic amine production (expressed as ppm) in skim milk. The figures represent theeffect the interaction [T ]3[pH] on 2-phenylethanolamine (a), tyramine (b) and total biogenic amines (c).

compounds in foods. It is well known that tempera- its effects on the activity of proteolytic and de-ture has a marked effect on the formation of biogenic carboxylase enzymes and the relationships betweenamines in the fishing industry and in cheese. Several the microbial populations (Joosten and van Boeckel,authors report that amine content depends on tem- 1988; Maijala et al., 1995) play an important role inperature, and increases with time and storage tem- relation to the total amount of amine produced.perature (Klausen and Lund, 1986; Diaz-Cinco et al., Moreover, NaCl concentration strongly influenced

`1992; Halasz et al., 1994). This is not in contradic- the amine production in the medium after 72 h oftion with the results obtained in this work. In fact, incubation. The lower values of this variable allowedtemperature had different influences on phenomena the maximum amine production. A 5% NaCl con-endowed with the biogenic amine production, such centration minimises the presence of amine, but thisas the growth kinetics and the cell yield. Moreover, value can be lower with a decrease in pH. Sumner et


al. (1990) and Joosten (1988) obtained similar 5. Conclusionsresults. Chander et al. (1989) found that the rate ofamine production by a Lactobacillus bulgaricus The production of biogenic amines is an extremelystrain was considerably reduced when salt concen- complex phenomenon, dependent of several vari-tration in the medium increased from 0 to 6%. This ables, such as the growth kinetics of the micro-negative influence can be attributed either to reduced organisms, their proteolytic and decarboxylase ac-cell yield obtained in the presence of high NaCl tivities, which interact with each other. Furthermore,concentrations and/or to a progressive disturbance of there is not a univocal rule linking these variablesthe membrane located microbial decarboxylase en- with the different metabolic mechanisms necessaryzymes (Sumner et al., 1990). for the formation of biogenic amines. This work

One of the most important factors influencing the suggests that the main biological feature influencingformation of biogenic amines in cheese, is the the biogenic amines formation is the extent ofpresence of the precursor free amino acids (Eitenmil- growth of microorganisms, like E. faecalis, char-ler et al., 1978). These amine precursors arise via acterised by decarboxylase activity. In the traditionalproteolysis. Primary (Grappin et al., 1985) and and artisanal cheeses produced using raw milk,

7secondary (Rank et al., 1985) proteolysis have been enterococci usually reach levels of 10 cells /gextensively studied in cheese. The secondary (Wessels et al., 1990). With this perspective, it isproteolysis produces a complex mixture of large, important that the presence of biogenic amines duemedium, and small peptides and amino acids. to the activities of these microorganisms is heldSumner et al. (1990) found that histamine formation within safe levels, without affecting the positiveseems to be dependent of the degree of proteolysis. effects of enterococci on the final organolepticThe proteolytic activity of E. faecalis EF37 (i.e. the characteristics of the cheese.availability of amine precursors), under the adoptedconditions, seems not to be a limiting factor for thefinal amine production. A study on tyramine and

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