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Postharvest Biology and Technology91 (2014) 72–77

Contents lists available at ScienceDirect

Postharvest Biology and Technology

 j ournal homepage : www.elsevier .com/ locate /postharvbio

Chitosan applications pre- or postharvest prolong raspberry shelf-lifequality

 Jaqueline Visioni Tezotto-Uliana∗, Gabriela Possati Fargoni, Gabriela Maria Geerdink,Ricardo Alfredo Kluge

Universidade de SãoPaulo,Escola Superior de Agricultura “Luiz de Queiroz”,13418-900 Piracicaba, SP, Brazil

a r t i c l e i n f o

 Article history:Received17 September 2013

Accepted31 December2013

Keywords:

Rubus idaeus

Edible coating

Postharvest life

Decay

Ethylene

Anthocyanins

a b s t r a c t

Raspberries are fruit with high metabolism that makes them very perishable, impairing their storageand shelf-life. Chitosan coatings have the potential to improve their postharvest life by reducing water

loss, respiration rate and decay incidence. The purpose of this work was to study the effect of differentconcentrations of  chitosan, applied pre- or postharvest, on the retention of quality attributes of  freshraspberries. The chitosan concentrations tested were 0 (control), 0.5, 1.0 or 2.0%. The postharvest treat-

ment was applied immediately after harvest, dipping the fruit in the solutions for 5min. The pre-harvesttreatment was done with one hand-spray application per week for three weeks, starting when the fruit

were just turning pink. In both experiments the fruit were stored at 0 ◦C and 90% RH. Pre- or postharvestuse of chitosan at 1 or 2% was effective in maintaining titratable acidity and retarding respiration and

ethylene production, weight loss and decay incidence. Application by both means resulted in the highestchitosanconcentrationsacceleratinga reduction of ascorbic acid contents. Firmnesswasmaintainedonly

when the fruit were treated pre-harvest at 2%. Thus, application of chitosan at 1 or 2% postharvest and2% pre-harvest was able to retain key raspberry quality attributes for 15 and 12 days, respectively.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Recent research on the characteristics of cultivation, marketingand nutritional properties of berries have attracted the inter-est of growers, researchers and consumers, especially because of 

their high value and benefits for human health when consumedfresh. However, a common problem with berries is their high per-ishability which results in a fast ripening period and senescence,hampering storage and marketing (El Ghaouth et al., 1991; Han

et al., 2004). The postharvest life of berries is generallydeterminedby their susceptibility to water loss, softening,mechanical injuries,andespecially to thepresence of pathogens such as Botrytis cinereaand Rhizopus sp (Mass, 1981; Reddy et al., 2000).

Various studies have proposed techniques to control pathogenswhile preserving the qualityof these fruit, such as modified atmo-spheres with high CO2, cooling, heat and osmotic treatments,irradiation and edible coatings (Mass, 1981; Brown, 1922; Vicente

et al., 2005; Fan et al., 2009; Castello et al., 2010; Velickova et al.,2013). Edible coatings have been of increasing interest because of their capacity to reduce respiration and transpiration rates, andincrease storage periods, firmness retention and decay control

(Debeaufort et al., 1998; Vu et al., 2011; Velickova et al., 2013).

∗ Correspondingauthor. Tel.: +55 19 34294136; fax: +55 19 34294100.

E-mail addresses: jaqueline.tezotto@usp.br, jaque vt@hotmail.com

(J.V. Tezotto-Uliana).

Among thevarious coatings, chitosan hasbeen tested in berries.It isa cationicpolysaccharide,with highmolecularweight,obtainedfrom alkaline deacetylation of chitin, and is found abundantly in

nature, constituting theexoskeleton of crustaceans and insectsandthe structure of the fungi (El Ghaouth et al., 1991). These charac-teristics allow chitosan to be considereda non-toxic,biodegradableand biocompatible product (Azevedo et al., 2007). The advantages

of chitosan also include the relative low cost and that its use asa food additive has been approved by the United States Food andDrug Administration (USFDA) (Knorr, 1986).

Other studies have shown that the post harvest use of chitosan

helps to control diseases in fresh raspberries (R. idaeus L.) (Zhangand Quantick, 1998; Han et al., 2004), but there has been no inves-tigation into the pre-harvest use of this product on the raspberry

quality. Furthermore, there is a need for studies on the most effec-tiveconcentration.This kind of information is extremelyimportantfor growers to obtain the best results with chitosan. Therefore, thepurpose of this workwas to study theeffect ofdifferent concentra-tions of chitosan, applied pre- or post harvest, on the retention of 

key quality attributes of raspberry.

2. Materials and methods

 2.1. Fruit material

‘Autumn Bliss’ raspberries (R. idaeus L.) were obtained from a

commercial orchard in Ibiúna,SP, Brazil, from three year oldplants

0925-5214/$ – seefrontmatter © 2014 Elsevier B.V. All rightsreserved.

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cultivatedon an espalier system.The experiments were performed

in the 2011/2012 growing season during the summer.For both pre- and postharvest treatments, the fruit were hand-

pickedwhen they hadreached full maturity (pinkish surface color).Harvested fruit were selected for uniform color, shape and size

of the fruit, and absence of mechanical or pathogen injuries. Theharvested fruit were directly placed in commercial plastic contain-ers (perforated polyethylene terephthalate, with 120g fruit andnomodified atmosphere) which were set in polystyrene boxes con-

taining ice sheets, and immediately transported to the laboratoryat São Paulo University (USP), Piracicaba, SP, Brazil.

 2.2. Preparation of chitosan solutions

The irradiated chitosan powder (‘Opcão Fenix’ pharmaceuticalindustry, Brazil) extracted from crab and shrimp shells, was char-acterized as a food-grade, odorless and tasteless powder with low

deacylation.The concentrations tested in this study were 0, 0.5, 1.0 and 2.0%

(w/v). Thechitosan waspreviously dissolvedusing 30m/L of aceticacid (5%) per gram of chitosan, and then the volume was com-

pleted with deionized water. For homogenization, the solutionswere heated and stirred for 3 h, then neutralized with 1.0N NaOH

to pH 5.5 and filtered.

 2.3. Treatments

The postharvest treatment was done in the laboratory imme-diately after harvest. The raspberries were randomly divided into

fourgroupsof 4.2kg (35 plastic containers) each and dipped in10L of thechitosan solution for 5min. The fruit were allowed to dry for40min onabsorbentpaper at room temperature,placed back in theplastic containers andthen storedat 0±1 ◦Cand90±5%RH, for 15

days.The pre-harvest treatment was applied once a week for three

weeks between when the fruit were turning pink and the har-vest point. For each concentration, in each weekly application,

20L of chitosan solution was used, hand-sprayed in rows with 30plants until complete coverage, totaling 13.33L m−2 (consideringone plant per 0.15m−2). The fruit were harvested the day follow-ing the last application, and after transportation, were stored at

0±1 ◦C and 90±5% RH, for 12 days. The following assessmentswere conducted with four replicates of 120g each.

 2.4. Respiration rate and ethylene production

Therespiratoryrate andethyleneproductionanalyseswere per-formed on the harvest day, the following day and thereafter everytwodays.Forthe analysis,eight raspberries were placed into 80mL 

glass flasks andhermetically sealed for30minat the same temper-ature andrelative humidity conditions of thecold storage. A 0.5mL 

sampleofinternalatmospherewascollectedthroughasiliconesep-tum fitted in each flask lid and measured by flame ionization gas

chromatographyusinga gaschromatograph(modelTraceGCUltra,ThermoElectron Corporation, MA, USA), which wasequipped withtwoflame ionization detectors (FID), twoinjectors, twoPorapack N

columns and one methanator set to 350◦C. The results were deter-mined considering the chromatographic values, fruit mass, flaskvolume and the time it remained closed. The respiratory rate wasexpressed in mg kg−1 h−1 of CO2   and ethylene production in L 

kg−1 h−1 of C2H4.

 2.5. Physicochemical and biochemical quality

Qualitative analyses were carried outatharvestday (day 0), and

thereafter every three days. Decay and weight loss were measured

in the same four packages, each containing 35 raspberries, from

the beginning to the end of the experiment. Decay incidence wasvisually inspected, counting the number of fruit that showed signsof fungal growth and mealiness (a condition of extreme softnessand oozing) and the results were expressed in %. To determine

weight loss, the raspberries were weighed at the harvest (day 0)and thereafter each day, the results being expressed as percentageloss of the initial total weight. For the following analyses, the fruitwere removed from cold storage and held at room temperature

(25 ◦C, 65% RH)24h beforeanalysis to simulate shelf-life. Firmnesswas determinedby a horizontal flattening technique, representinga ratio between applied force and measured flattened area of thefruit, this measured according to the smaller and larger diameters

of the presumed elliptical figure formed and the result expressedin newtons (Calbo and Nery, 1995). The pectin solubilization per-centage was determined by washing the samples with ethanol,complexing with EDTA, pH adjustmentandpectinase, according to

McCready and McCoomb (1952), and the determination was per-formed colorimetrically using the method described by Bitter andMuir (1962), with alterations, reducing the fruit mass from 5 to 2gin each sample, and the aliquot used for the quantification, from

1 to 0.2mL. The ascorbic acid content was determined by titrationwith DCFIand results express inmg 100g−1 of fruit (Carvalho et al.,

1990). Thetitratableacidity wasdetermined from 10g pooledjuicesample diluted with 90mL distilled water, titrated with 1N NaOH

to pH 8.1 and expressed in % citric acid. The anthocyanin contentswere determined according to Lees and Francis (1972) and wasexpressedin g 100g−1 of fruit. Thepulp masseswere reduced from100 to 10g and homogenized in a 10mL ethanol and HCl solution.

The color index (CI) to express the intensity of the red color wasdetermined using a colorimeter (model CR-400, Konica MinoltaInc., Japan), calculated as CI= 100×a/(L×b). The CI ranged from35 to 75,witha greater CI value indicating a more intense red color

of the fruit.

 2.6. Statistical analysis

Data analyses were performed by GLM (general linear model)using SAS statistical software 9.2 (SAS Institute, Cary, NC, USA).Multiple comparisons among the treatments with significant dif-ferences tested were conducted by using Tukey’ test (P<0.05).

3. Results

 3.1. Respiration rate and ethylene production

We hypothesized that the use of chitosan as coating could reduce respiration

and ethylene production fresh raspberries and thus extend their postharvest life.

The datarevealed thatthe postharvestuseof chitosanat 2% reduced therespiration

of the fruit when compared to the control from the first day after harvest (Fig. 1a).

This behavior also occurred with thefruit treated with chitosan at 1% starting from

the ninth day. When the application was made pre-harvest, the fruit treated with1 and 2% had lower respiratory rates from the harvest day, and during the storage

period the three chitosan concentrations reduced the respiratory rate (Fig.1b).Regardingethylene, we found that the higher the chitosan concentration used,

the lower was the ethylene production. In the postharvest treatments, the three

concentrations resutled in lower production comparedto thecontrol from thefirst

day after harvest (Fig. 1c), while in thepre-harvest treatments, trhis only occurred

with the 1 and 2% treatments (Fig. 1d). We also observed that between harvest

and the first day of storage, the respiratory rate and ethylene production were

respectively reduced more than 80 and 65% in both pre- and postharvest treat-

ments, but then showed an increase through the evaluation period, related to the

cold temperature during storage. Thus, the data revealed that the application of 1or 2% chitosanpre-or postharvest waseffectivein slowing respiration andethylene

production.

 3.2. Decay incidence and weight loss

One of the main benefits from the chitosan application was decay control,

whichwas observedin both the pre-and postharvest applications.The postharvest

concentrations of 0.5, 1 and 2% reduced decay incidence by 75.5, 80.9 and 88.9%,

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0

10

20

30

40

50

60

70

80

151311975310

   R  e  s  p   i  r  a   t   i  o  n  r  a   t  e   (  m  g   C   O

   2   k  g  -   1   h  -   1   )

Postharvest

0

10

20

30

40

50

60

70

80

11975310

Pre-harvest

0

2

4

6

8

10

12

14

151311975310   E   t   h  y   l  e  n  e

  p  r  o   d  u  c   t   i  o  n   (  µ   L   C

   2   H

   4

   k  g  -   1   h  -   1   )

Days after harvest

0% 0.5% 1% 2%

0

2

4

6

8

10

12

14

11975310

Days after harvest

a   b

c  d

Fig. 1. Respiration and ethylene production of ‘Autumn Bliss’ raspberriestreatedwith chitosan postharvest (a and c) or pre-harvest (b andd) and cold storedat 0 ◦C and 90%

of RH. Vertical bars represent thestandard error (n=4).

Pre-harvestPostharvest

0

5

10

15

20

25

30

35

40

15129630

   D  e  c  a  y  r  a   t  e   (   %   )

0

5

10

15

20

25

30

35

40

129630

a   b

0

1

2

3

4

5

6

15129630

   W  e   i  g   h   t   l  o  s  s

   (   %   )

Days after harvest

0% 0.5% 1% 2%

0

1

2

3

4

5

6

129630

Days after harvest

c 

d

Fig. 2. Decayand weight loss of ‘Autumn Bliss’raspberries treated with chitosanpostharvest(a and c) or pre-harvest (band d),and cold storedat 0 ◦C and90% of RH. Vertical

bars representthe standard error (n=4).

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0.3

0.4

0.5

0.6

0.7

0.8

0.9

0  3  6  9  12  15

   F   i  r  m  n  e  s  s   (   N   )

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

0 3 6 9  12

5

10

15

20

25

30

35

0 

3 

6 

9 

12 

15

   P  e  c   t   i  n   S  o   l  u   b   i   l   i  z  a   t   i  o  n   (   %   )

20

40

60

80

100

0 3 6 9 

12

12

14

16

18

20

22

24

26

28

30

32

0 3 6 9  12  15

   A  s  c  o  r   b   i  c  a  c   i   d   (  m  g   1   0   0  g  -   1   )

Days after harvest

0% 0.5%  1%  2%

18

20

22

24

26

28

30

32

0 3 6  9  12

Days after harvest

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

0 3 6  9  12  15

   T   i   t  r  a   t  a   b  e   l  a  c   i   d   (   %   )

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

0 3 6 9  12

Postharvest   Pre-harvesta b

c  d

e  f 

g 

h

Fig. 3. Physicochemical analyses of ‘Autumn Bliss’ raspberries treated with chitosan postharvest (a, c, e and g) or pre-harvest (b, d f andg), andcold storedat 0 ◦C and 90%of RH. Fruit were removed from the cold storage and held at room temperature (25 ◦C, 65% RH) 24h before the analysis to simulate shelf-life. Vertical bars represent the

standarderror (n=4).

respectively, at the 12th day, although the treatments extended the postharvest

life until the 15th day after harvest (Fig. 2a). In the pre-harvest application, the 1

and2% concentrationsreduceddecayincidenceby 13.98% and27.96% respectively,

extending thepostharvestlife until the12th day (Fig. 2b).

We found that the weight loss was slightly reduced by all pre- and posthar-vest treatments, butwerenot statistically differentcomparedto the control (Fig.2c

and d).

 3.3. Firmness, titratable acidity and ascorbic acid

Firmnessmaintenance is oneof themostimportant physicalattributes in main-

taining quality of raspberries. The postharvest application of chitosan was not

effectivein thisaspect, andall treatments sufferedan averagelossof45%in firmness

(Fig. 3a). However, when the treatment was applied pre-harvest, the fruit treated

with 2%chitosan remained firmer than the untreated and0.5% treated fruit at from

day 0 t o 9 (Fig.3b).

Pectin solubilization analysis was performed in order to confirm the firmness

results.In thepostharvestapplicationthe control fruit hadhigher solubilized pectinpercentagesthan theother treatments in the lasttwo daysof analysis. However,the

differentconcentrationsof chitosan showed thesame behavior, with an average of 

26.3%ofthepectinsolubilizedbythe15th day(Fig.3c).Inthepre-harvestapplication,

the fruit treated with 2%chitosan differed from the others at the harvest and ninth

days, although throughout the experimental period, fruittreated with 0.5% had the

lowest solubilization (Fig. 3d). Thus, we observed that the postharvest application

of chitosan did not prevent the loss of firmness in raspberries, but pre-harvest use

at 2% was able to slow this loss.

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Postharvest

35

40

45

50

55

60

65

70

75

0  3  6  9  12  15

   I  n   d  e  x  c  o   l  o  r

Postharvest

35

40

45

50

55

60

65

70

75

0  3  6  9  12

Pre-harvest

5

10

15

20

25

30

35

40

45

0 3 6 9  12  15

   A  n   t   h

  o  c  y  a  n   i  n  s  c  o  n   t  e  n   t   (  g   1   0   0  g  -   1   )

Days after  harvest

0% 0.5%  1%  2%

a   b

c  d

10

15

20

25

30

35

40

45

0 3 6 9  12

Days after  harvest

Fig. 4. Index color and anthocyanin content of ‘Autumn Bliss’ raspberries treated with chitosan postharvest (a and c) or pre-harvest (b and d), and cold stored at 0 ◦C and90% of RH. Fruit were removed from thecold storage and held at room temperature (25◦C, 65% RH) 24h before theanalysis to simulate shelf-life. Vertical bars representthe

standarderror (n=4).

Therewas a decrease in titratable acidity until the sixth dayin the postharvest

application, regardless of the chitosan concentration (Fig. 3e). From this day, the

fruit treatedwith 2% chitosan retainedconstant acidity. With the other treatments,

aciditykeptdecreasing,but the1% treatment hadshowedthe lowest decreasein the

12th and 15th days. In thepre-harvesttreatment, we observed a decrease in acidity

during the entire period, but the fruit treated with 2% chitosan had the highest

acidity percentage comparedto theother treatments (Fig. 3f). Thus, the use of1 and

2% chitosanpostharvest, and2% pre-harvestwerethe mosteffective in maintainingthe acidity of raspberries.

The ascorbic acid contents of all fruit with postharvest applications were

reduced from day 0 to 3 in direct correlation with the chitosan concentration

(Fig. 3g). Thereafter, the control fruit and those which received 0.5% maintained

constantascorbic acidcontents, whereasfruit treatedat1 and2% continuedto show

a decline, presenting66.7and 48.2% of theinitialvalue, respectivelyat the15th day.

In thepre-harvestexperiment,we alsoobserveda decreasein ascorbicacid content;

however, untreated fruit showed the smallest reduction (Fig. 3 h). Thus, we found

that theuse of chitosan accelerated thereduction of theascorbic acid content.

 3.4. Color index and anthocyanin contents

Chitosan interacts with the main pigment of raspberry (Massa and Miniati,

1993), changing its color,so we studied theCI andanthocyanin contents.Our results showedthat itsuse in the1 and2% postharvest treatments resulted

in the smallest CI, indicating less darkening which is linked to better maintenance

of freshness (Fig. 4a). However, pre-harvest use had no influence on the CI, which

increasedby an average of 61.7%during theexperimental period (Fig.4b).

Theanthocyanincontentsfolloweda similar pattern,with the1 and2% posthar-

vest application resulting in the lowest content (Fig. 4c), and the pre-harvest

application having no affect the pigment content (Fig. 4d). Thus, we conclude thatchitosan maintains the color and anthocyanin contents of the raspberries when

applied postharvestat 1 or 2%.

4. Discussion

The use of chitosan slowed respiration rates and ethylene

production of raspberries regardless of the application form. Sev-

eral studies have shown that chitosan has excellent selective

permeability to the respiratory gases, actingas a barrier to thepas-

sageof O2 (Elsabee andAbdou, 2013). This control of gas exchangebetweenthe fruit andenvironment reduces the respiration andthe

action of ACC oxidase and synthase enzymes, which besides beingkey enzymes of ethylene biosynthesis, are greatly influenced by

the presence of O2   (Noh, 2005). In this study we found that theapplication of c1 and 2% chitosan, both pre and postharvest, mighthave formeda barrier aroundthe fruit andreduced respiration andethylene production, which is directly correlated to the retention

of other fruit quality attributes.Weight loss reduction from the use of chitosan is also related

to the formation of a selective barrier around the surface of thefruit, which reduces moisture loss to the environment and reduces

respiration, the main metabolic processes that lead to water loss(Han et al., 2004; Hong et al., 2012).

Decay was influenced by both the chitosan concentration andtimeof treatment. According to recent studies, the chemical struc-

ture of chitosan inhibits the growth of fungi and bacteria throughelectrostatic forces between the protonated amino groups (NH2)of the chitosan, and the negative charges or phosphoryl groupspresent on the cell surface of the microorganisms (Elsabee and

Abdou, 2013). Another reason for the beneficial effects of chitosanmay be thereductionof polygalacturonaseproductionby thefungi,limiting its ability to colonize the fruit tissue (El Ghaouth et al.,1997). In our study, the postharvest application possibly left a

greater amount of residue on the surface of the raspberries thanthepre-harvest, andin bothapplications, thehighest chitosan con-centrationprobablyprovidedthe greater thenumberof NH2groupsand consequently had lower decay incidence.

The postharvest use of chitosan did not prevent firmness lossof raspberries unlike that observed in most of the fruit treatedwith pre-harvest applications, such as strawberry (Reddy et al.,

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2000), guava(Hongetal.,2012) andcherry(Martinez-Romeroet al.,

2006). According to these authors, firmness maintenance is due torespiratory rate reduction which decreased release of free radicalsand intensified pectinase activity, factors of cell wall degradationandfruit softening (KonandSchwimmer, 1977). In the postharvest

treatment, this process has probably not occurred due to persis-tent humidity around the fruit after application of the chitosan.The difficulty of drying the raspberries completely after dippedin chitosan solutions was also reported by Han et al. (2004), who

suggested that spraying the chitosan solution might be the bestalternative.

The use of higher concentrations of chitosan minimized acid-ity loss in both experiments. This result is consistent with those

observed by Zhang and Quantick (1998) and Han et al. (2004) andthe other results from this study, because the higher percentageof citric acid in the fruit treated with chitosan is related to lowerrespiration ratesandless fungalinfestation,whichresults in longer

postharvest life.The results observed for ascorbic acid content were not

expected. We predicted that the ascorbate contents of raspberrieswould decrease through the experimental period, and that the use

of chitosan would minimize the loss rather than increase it. Sincechitosan might reduce the amount of O2   available for oxidative

reactions, one possible reason for this result is that the ascorbicacid has reacted with other oxidizing agents present in the fruit

or with metallic ions that perhaps were in the chitosan solutions,or has been involved in cell division during the ripening process(Smirnoff, 1996). Therefore, more research is necessary to affirmthe real reasons for the ascorbate reduction in raspberries due to

the use of chitosan.Thepostharvestapplication ofhigherconcentrationsof chitosan

was effective in reducing darkening and maintaining the antho-cyanin contents, attributes directly related to freshness. The color

change in the raspberries is due to enzyme activity, especiallypolyphenol oxidase and peroxidase, and the synthesis of antho-cyanins. Activity of these enzymes is influenced by the presenceof O2   inside the fruit, so when chitosan form a barrier around it,

enzyme activity will be reduced and consequently the darkeningof the fruit (Kang et al., 2005). It is possible that anthocyanin syn-thesis was reduced by the reduction in gas metabolism resultingfrom the chitosan barrier. In addition, the positive charges present

in the coating may have stabilized the anthocyanins, helping tomaintain the raspberry color (Han et al., 2004). In the pre-harvesttreatmentwe didnotobserve thisbehavior, andthis maybe relatedto the fruit metabolism of this fruit which might have not been

reduced enough to modify enzyme activity and the anthocyaninsynthesis.

In conclusion, we have observed that the postharvest appli-cation of 1 or 2% chitosan and 2% in the pre-harvest treatment

are able to retain key raspberry quality attributes for 15and 12 days, respectively. Future studies should focus on the

properties of the chitosan coating, so that its use can be recom-mended.

 Acknowledgement

The authors would like to thank the ‘São Paulo Research

Foundation (FAPESP)’ for granting the scholarships. Grant n◦

2010/02601-3.

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