Trends in Food Science & Technology 34 (2013) 80e95Review* Corresponding author.

0924-2244/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tifs.2013.07.008Hearts of palms

preserves and

botulism in Brazil: An

overview of

outbreaks, causes

and risk management

strategies

Humberto Moreira Hungaroa,

Veronica Ortiz Alvarengab,

Wilmer Edgard Luera Pe~naa andAnderson de Souza SantAnab,*aDepartment of Food Technology, Federal University

of Vicosa, CEP: 36570-000, Vicosa, MG, BrazilbDepartment of Food Science, Faculty of Food

Engineering, University of Campinas, CEP: 13083-862,Campinas, SP, Brazil (Full Postal Address: Rua

Monteiro Lobato, 80, Cidade Universitaria Zeferino

Vaz, CEP: 13083-862, Campinas, SP, Brazil.

Tel.: D55 (19) 3521 2174; e-mail: and@unicamp.br)Botulism is a serious disease that causes paralysis or weakness

of the muscles, including those needed for breathing. It is

caused by a neurotoxin produced by Clostridium botulinum.

This manuscript reviews the association of hearts of palm pre-

serves with botulism outbreaks in Brazil, examines the main

causes and presents a chronology of the preventive and correc-

tive measures taken by public health authorities. The changes

in legislation, processing conditions and controls stipulated to

avoid the re-occurrence of botulism outbreaks were also dis-

cussed. A perspective on the current situation of hearts ofpalm preserve-associated botulism outbreaks highlights the

success of the risk management strategies implemented.IntroductionBotulism, from the Latin botulus (sausage), is a rare andserious neuroparalytic disease caused by a neurotoxin pro-duced under anaerobic conditions by a few species of Clos-tridium. This group, constituted of Gram-positive, rod-shaped, obligate anaerobic and spore-forming bacteriaable to produce the neurotoxin, is called botulinogenicClostridium (Johnson, 2007). The members of the botulino-genic Clostridium group include Clostridium botulinum,Clostridium butyricum, Clostridium argentiniensis andClostridium baratii (Aureli, Franciosa, & Fenicia, 2008;Johnson, 2007). Botulism can affect a variety of mammals,birds and fishes, and six different types are currentlyknown: i) wound botulism, ii) infant botulism, iii) adult in-testinal toxemia, iv) inhalational botulism, v) iatrogenicbotulism and vi) foodborne botulism (Sobel, 2005). Sincethe botulinum toxin is the etiological agent of the disease,the different types of botulism are characterized by thesame clinical symptoms, although the toxin may be pro-duced at different sites (Sobel, 2005). Due to the economic,high social and medical costs involved, foodborne botulismrepresents a major concern for consumers, food industriesand public health authorities throughout the World (CDC,2011; ECDC, 2011; Peck, 2006; Setlow & Johnson,1997). Foodborne botulism is acquired by eating foodscontaminated with the preformed botulinum neurotoxin(Peck, 2006; Setlow & Johnson, 1997).

A variety of foods, industrialized or home-made, ofanimal or vegetable origin, have been associated with food-borne botulism, with several reports existing on the inci-dence of this disease (CDC, 2011; ECDC, 2011; Gelli,Jakabi, & Souza, 2002; Peck, Stringer, & Carter, 2011;Rowlands et al., 2010; Tseng et al., 2009). In Brazil, a totalof 173 suspected cases of botulism were registered between1999 and 2011, of which 68 (40%) were confirmed. Of the68 confirmed cases, 66 were foodborne, one was woundbotulism and one was intestinal botulism (Anonymous,2011a). The majority of the foodborne cases of botulismnotified in Brazil (26 cases) were associated with the con-sumption of industrialized products, with hearts of palmin preserve being the main implicated food (Anonymous,2002a, 2002b, 2007, 2011b; Eduardo et al., 2007).

Delta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnamemailto:and@unicamp.brhttp://dx.doi.org/10.1016/j.tifs.2013.07.008http://dx.doi.org/10.1016/j.tifs.2013.07.008http://dx.doi.org/10.1016/j.tifs.2013.07.008

81H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95Hearts of palm preserves are highly appreciated inBrazil, which is one of the largest World producers andconsumers of this vegetable (IBGE, 2010). Hearts ofpalm in preserve is the product prepared by immersion ofthe edible part of healthy palm trees of the species Jucara(Euterpe edulis mart), Aca (Euterpe oleracea mart) andPupunha (Bactris gasipaes) in acidified brine, followed bypasteurization and hermetic packaging (Anonymous,1999b; Berbari, Prati, & Junqueira, 2008).

The botulism outbreaks associated with the consumptionof hearts of palm preserves occurred because spores of C.botulinum were able to germinate and outgrow in improp-erly processed preserves. Hearts of palms are contaminatedby C. botulinum spores due to direct or indirect contact ofthe palms with soil during field operations (Costa et al.,2010; Johnson, 2007). Although presenting pH values be-tween 5.6 and 6.2 (Chaimsohn, 2002), hearts of palmcannot be submitted to severe heat treatment (>100 C)since their sensory characteristics, principally the colorand texture, would be significantly altered (Resende &Saggin Junior, 2004). Thus, the microbiological stabilityof this product relies on the combination of low pH(

Fig. 1. Phylogenetic dendrogram of Clostridium species based on the 16S rRNA genes, illustrating the genetic diversity within the clostridia (from Hillet al., 2007, with permission). A neighbor-joining tree of 54 sequences reported in the GenBank and 36 sequences representative of the strains fromthis collection are shown. C. botulinum strains cluster into four distinct groups that follow the group I to group IV designation historically based onphysiological characteristics. These groups are interspersed amongst the 27 other clostridial species on the tree. The tree was constructed using analignment of 16S rRNA gene sequences that contained 1329 bases, after removal of any columns containing more than 80% gap characters, and

includes sequences from bivalent, non-proteolytic, and proteolytic toxin-producing strains.

82 H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95proteases present in the human gut (Lund & Peck, 2000).The resulting structure is a double-chained molecule whichis more toxic than its predecessor (DasGupta, 1989; Peck,2009; Sugiyama, 1980). C. botulinum neurotoxins can beeven more toxic when complexed with other proteins thatincrease their stability during passage through the intestinaltract (Johnson & Bradshaw, 2001; Peck, 2009). Estimatesmade using data obtained from epidemiologicalinvestigations and animal experiments, suggest that as littleas 30e100 ng of botulinum neurotoxins may be enough tocause botulism (Lund & Peck, 2000; Peck, 2009). The le-thal dosis for A and B of group I and B and E of groupII toxins range from 5 103 to 5 105 mouse LD50 and1 105 to 5 105 mouse LD50 (Hauschild, 1990).

The symptoms of botulism are mostly neurological, suchas blurry vision, dry mouth and difficulty in swallowing and

Table 1. Characteristics of the six physiologically and phylogenetically distinct clostridia that produce the botulinum neurotoxin (from Peck,2009, with permission).

NeurotoxigenicClostridia

Group I(proteolyticC. botulinum)

Group II (non-proteolyticC. botulinum)

C. botulinumgroup III

C. botulinumgroup IV(C. argentinense)

C. baratii C. butyricum

Neurotoxins formed A, B, Fa B, E, F C, D G F ENon-neurotoxigenicequivalent clostridia

C. sporogenes No species name given C. novyi C. subterminale All typicalC. baratiistrains

All typicalC. butyricumstrains

Ferment glucose b e Ferment fructose / / e Ferment maltose / / e Ferment sucrose e e e Optimum growthtemperature

37 C 25 C 40 C 37 C 30e45 C 30e37 C

Minimum growthtemperature

10e12 C 2.5e3.0 C 15 C 10e15 C 12 C

Minimum pH forgrowth

4.6 5.0 5.1 4.8

NaCl concentrationpreventing growth

10% 5% 6.5%

Minimum wateractivity for growth,humectants: NaCl/glycerol

0.94/0.93 0.97/0.94

Spore heat resistancec D121 C 0.21 min

D82.2 C 2.4/231 mind D104 C 0.9 min

D104 C 1.1 min D100 C < 0.1 min

a In dual-toxin strains, more than one toxin is formed.b , all strains positive, /, some strains are positive and others negative, e, all strains negative.c Spore heat resistance determined in phosphate buffer, pH 7.0.d D-value without/with lysozime during recovery.

83H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95talking. These symptoms are followed by a descendingbilateral flaccid paralysis, which in severe cases can evolveinto flaccid paralysis of the respiratory and heart muscles,and death (Hatheway, 1988). Gastrointestinal symptomsFig. 2. (A) Crystal structure of botulinum neurotoxin A (BoNT/A). The three fuare no contacts between the catalytic and binding domains. The binding dominal sub-domain comprises a jelly roll motif while the C-terminal sub-domab-trefoil fold. The central translocation domain has two unique structural feamotif, and a long loop (translocation belt) that wraps around the catalytic dcatalytic zinc atom and the conserved HExxH motif characteristic of zinc-d(B) Structure of the botulinum neurotoxin molecule. The native molecule is

and the molecule is surrounded by protective proteins (such as nausea, vomit, diarrhea or constipation may alsobe observed (Rowlands et al., 2010; Villar, Elliot, &Davenport, 2006). The symptoms of botulism typicallyappear within 18e36 h after consumption of neurotoxinnctional domains are structurally distinct and oriented such that theremain is made up of two sub-domains of roughly equal size: the N-ter-in (which is believed to contain the ganglioside-binding motif) adopts atures e a pair of long helices that are reminiscent of a viral coiled-coilomain and occludes the active site. The catalytic domain houses theependent endopeptidases (from Turton et al., 2002, with permission).cleaved to form a light and heavy chain, joined by a disulfide bond,from Dutton and Fowler, 2007, with permission).

84 H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95contaminated food. However, the incubation period mayvary from a few hours to up to 10 days depending on theamounts of neurotoxin ingested (Rowlands et al., 2010;Villar et al., 2006). Due to the seriousness and potentialmagnitude of foodborne botulism, one case of this diseaseis treated as a public health emergency, and thereforeconsidered as an outbreak (Chaudhry et al., 1998).

All the symptoms of botulism are credited to extracel-lular binding of the C. botulinum neurotoxins to glycopro-tein structures on cholinergic nerve terminals, followed byblockage of acetylcholine release as illustrated in Fig. 3(Rowland, 2002; Sobel, 2005). Since several review articlesare available on this subject, no further details on the modeof action of C. botulinum neurotoxin have been given in thepresent paper. Detailed information is easily available inTurton et al. (2002), Sobel (2005), Dickerson and Jand(2006) and Villar et al. (2006). Since the damage causedto the pre-synaptic membrane by the C. botulinum neuro-toxins is permanent, clinical recovery requires a long perioduntil new nerve endings are formed (Sobel, 2005; Turtonet al., 2002; Villar et al., 2006). Data from botulism out-breaks have shown that in some cases, up to 2e3 yearswere necessary for a full recovery, demonstrating the grav-ity of this disease (Colebatch et al., 1989; Eduardo, 2002).

Overview of botulism in Brazil and the role of heartsof palms preserves as vehicles of the disease

Records on the occurrence of foodborne botulism inBrazil have been hardly found in the literature before2001, when the notification of the disease became obliga-tory (Anonymous, 2001, p. 35). Nonetheless, a fewFig. 3. Mode of action of botulinum neurotoxin. Acetylcholine in nerve termthose of the nerve terminals, releasing the transmitter into the synaptic cleft.SNARE proteins. Botulinum toxin, taken up into vesicles, cleaves the SNARE

the release of acetylcholine (from Roregisters of the occurrence of foodborne botulism in Brazilbefore 2001 can be found in the literature. The report byJeffman (1960) comprises the oldest data available on theoccurrence of foodborne botulism in Brazil. According tothe author, this outbreak of botulism involved the consump-tion of a home-made fish preserve containing type A toxin.A total of nine people affected and seven deaths in the Stateof Rio Grande do Sul, Brazil (Jeffman, 1960). Anotheroutbreak occurred in 1982 in Rio de Janeiro affecting twopeople, with one death, due to the consumption of a cannedchicken pate (Serrano, 1982). In 1985 seven people, from asingle family in Minas Gerais, Brazil, were intoxicated bytype A botulism toxin, and two died, possibly due to theingestion of home-made canned pork (Ferreira et al.,1987). In 1997 the ingestion of a home-made pequi pre-serve contaminated with type A botulism toxin was respon-sible for a botulism outbreak in Goias, Brazil, affecting fourpeople all of whom died (Eduardo & Sikusawa, 2003).

Using various official research sources, Eduardo andSikusawa (2002) carried out a survey with the objectiveof estimating and making available information concerningthe occurrence of botulism in Brazil from 1979 to 2001,particularly in the State of S~ao Paulo. They used reportsfrom the epidemiological and sanitary surveillance system,laboratory data of diseases registered in the public healthlaboratories, registers of the supply of anti-botulism toxin,data of hospital internments, data from information servicesconcerning mortalities and various databases. The data ob-tained indicated a total of 26 isolated cases of botulism inthe State of S~ao Paulo, Brazil from 1979 to 2001, withsix deaths. As the notification of botulism was still notinals is packaged in vesicles. Normally, vesicle membranes fuse withThe process is mediated by a series of proteins collectively called theproteins, preventing assembly of the fusion complex and thus blockingwland, 2002, with permission).

Year

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

Nu

mb

er o

f cas

es

0

5

10

15

20

25

30

35

Confirmed

Suspected

Fig. 4. Number of suspected and confirmed cases of botulism in Brazil(1999e2011) (adapted from Anonymous, 2011a, 2011b).

85H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95compulsory in this period and because the epidemiologicalinvestigations were not conclusive, it was not possible tomake the association with foods in the majority of cases.By taking into account the percentage of sub-notification(81%), they estimated that 55 cases of botulism (2.4 casesyear) occurred in the State of S~ao Paulo, Brazil in theperiod from 1979 to 2001 (Eduardo & Sikusawa, 2002).However, taking into consideration the results of a labora-tory investigation of biological samples and of foods ob-tained from botulism outbreaks in Brazil in this period,only three foodborne cases of botulism, of the eight sus-pected, were confirmed. In three of the eight suspectedcases, type A toxin was detected in a home-made preserveand in the leftovers of hearts of palm preserves. In four ofthe cases/outbreaks, type A botulism toxin was detected inthe serum or blood clot and in one case it was not possibleto typify the toxin in the serum or feces (Gelli et al., 2002).Further details on some of these cases/outbreaks can befound in Gelli et al. (2002).

From the year 2001, when notification of botulismbecame compulsory in Brazil (Anonymous, 2001, p. 35),and probably also because of improvements in the epidemi-ological surveillance of the disease, a diversification of theTable 2. Cases of botulism related to the consumption of hearts of palm i

Year State Cases Orig

1997 S~ao Paulo 1 Indu1998 S~ao Paulo 1 Indu1999 S~ao Paulo 1 Indu2002 Minas Gerais 1 Hom2005 Rio Grande do Sul 2 Indu2007 S~ao Paulo 1 Indu2007/2008 Minas Gerais 1 Indu

a Consumption of hearts of palm as an ingredient in the preparation of ob Type of toxin not identified and case confirmed by the clinical-epidemfoods associated with the disease could be observed. Fig. 4shows the incidence of foodborne botulism cases/outbreaksas reported to the Brazilian Ministry of Health from 1999 to2011. The 66 cases of foodborne botulism confirmed in thisperiod gave a mean annual incidence of 0.04 cases permillion inhabitants, with a mortality rate of 29% (20deaths) (Anonymous, 2011a). Between 1999 and 2011,the foods most implicated in botulism outbreaks in Brazilwere pork products (15 cases, 23%), canned sardines andItalian sausage (11 cases, 17%), hearts of palm (6 cases,9%), chicken pie (9 cases, 14%), tofu (4 cases, 6%), others(5 cases, 7%) and non-determined (16 cases, 24%)(Anonymous, 2011a). The majority of cases (62.5%) wererelated to type A botulism toxin, whereas the AB toxinswere identified in 7 cases and in 14 cases the type of toxininvolved could not be elucidated (Anonymous, 2011a). Thehigh incidence of type A toxin is cause for great concern,since this is the most potent of the botulism toxins. In addi-tion, this toxin is capable of causing the severest and mostlong-lasting form of the disease (Arnon et al., 2001). Moredetailed information on most of these cases can be found inRowlands et al. (2010).

Twenty one cases (31%) of the foodborne cases of botu-lism in Brazil is related to the consumption of home-madefoods such as cooked chopped meat filled into vessels andsealed with pork fat. However, the majority of cases arerelated to industrialized products (26 cases, 40%)(Anonymous, 2011a). Of these, hearts of palm preservescontaminated with type A botulinum toxin were involvedin various outbreaks in Brazil between 1990 and 2000(Table 2).

The botulism outbreaks occurring between 1997 and1999 involving hearts of palm preserves in the State ofS~ao Paulo, were widely divulged by the Brazilian pressand caused great social commotion. In these outbreaks,different brands of industrialized products, both nationaland imported, were confirmed as the causers of the out-breaks, affecting three people with no deaths (Table 2)(Anonymous, 2001, p. 35, 2011a). Only one outbreak,occurring in the State of Minas Gerais in 2002 (Table 2),appears to have been caused by the consumption ofhome-made hearts of palm preserve. The fact that the ma-jority of the botulism outbreaks associated with then Brazil.

in Type of toxin Reference

strialized A Gelli et al. (2002)strialized A Gelli et al. (2002)strialized A Gelli et al. (2002)e-made A Anonymous (2007)strialized b Anonymous (2005)strializeda A and B Eduardo et al. (2007)strializeda b Anonymous (2011b)ther types of food.iological criteria.

Fig. 5. Sequence of events that probably led to the occurrence of outbreaks of botulism associated with the consumption of hearts of palm preservesin Brazil. Where: (1) C. botulinum and its spores are widely spread in the environment, with soil and sediments being the main source. (2 and 3)Handling in the field and during early stages of industrial processing may result in contamination of hearts of palms by spores of C. botulinum. Sen-sory properties of hearts of palms are destroyed at severe heat treatments (>100 C). Thus, the microbiological safety of hearts of palms preservesrelies on acidification (pH

Fig. 6. Processing steps of hearts of palm in preserve (adapted from Resende and Saggin Junior, 2004; Berbari et al., 2008).

87H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95plants with diameters between 7 and 10 cm are used, cut ata height 80 cm from the neck (Pimentel, Souza, & Cabral,1999, pp. 1e3). The crude palm is pared to remove theouter layers just leaving the two or three internal layers(Chaimsohn, 2000, 121 p.). During this step one mustalso be careful not to cut the tip of the crude palm exces-sively, since this procedure can increase the risk of micro-biological contamination and rotting of the material(Chaimsohn, 2000, 121 p.) (Fig. 6).

Transport of the palms should be as quick as possibleand at times of the day when the temperatures are lower.Processing is carried out as soon as possible to avoidweight losses, browning and rotting. However when thisis not possible, the palms can be stored in a cold chamberat 5e10 C for up to 24 h. During storage, the relative hu-midity should be from 85 to 90% so as to preserve the phys-ical, chemical and sensory characteristics of the finalproduct (Chaimsohn, 2000, 121 p.; Raupp, 2001).

The palm is subsequently washed with potable water,before and after removal of the external layers, to avoid mi-crobial contamination (Resende & Saggin Junior, 2004).Guaranteeing the use of potable water is highly importantto avoid the possibility of this being a source of productcontamination, including by sporulated microorganisms(Fig. 6). After washing and separation of the edible portionof the palm, it is cut into three parts: namely the foliar,

88 H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95basal or caudal and apical parts of the heart of palm. Thefoliar parts are normally 9 cm long and are classified ac-cording to their diameter into fine (up to 3.0 cm), medium(3.1e4 cm) and thick (above 4.1 cm) (Raupp, 2001;Resende & Saggin Junior, 2004) (Fig. 6).

Soon after removing the external layers and cutting thehearts of palm, they are immersed in brine which normallycontains 5% NaCl and 1% citric acid. This procedure iscarried out to minimize the oxidation process, browningand development of deteriorative microorganisms (Raupp,2001). Hearts of palm are normally packed into glass con-tainers and acidified saline added so as to reach an equilib-rium pH value 4.5 (Fig. 6). The acidified brine isnormally prepared with citric acid (Raupp, 2001; Resende& Saggin Junior, 2004). The equilibrium pH is reachedwhen the ground hearts of palm, macerated with brine inthe same proportions present in the container, show thesame pH value.

After filling, the containers are steam exhausted in orderto produce a vacuum and contribute to a more efficient seal-ing of the product. Also exhaustion serves to restrict theoxidative reactions which mainly affect the color and flavorof the hearts of palm (Camargo et al., 1986, 298 p.; Medina,1979, 277p.). After exhaustion, the containers are closed ata temperature not inferior to 85 C so as to avoid reducingthe vacuum on the inside of the final product (Resende &Saggin Junior, 2004). The heat treatment of the hearts ofpalm is carried out at the temperature of boiling water(100 C) for a time of 25e60 min, which varies accordingto the size of the container and texture of the product(Resende & Saggin Junior, 2004) (Fig. 6).

The containers are then cooled in potable water at roomtemperature in order to interrupt the cooking process andcontribute to maintenance of the desired sensory character-istics. Cooling is dimensioned to reduce the temperature to40 C in approximately 15 min (Fig. 6). The product is thenmaintained in quarantine for a minimum of 15 days, and in-spected regularly for undesirable changes such as cloudi-ness of the saline, bulging of the cans or top (whenpacked into cans) and/or leakage (Resende & SagginJunior, 2004).

The steps of obtaining the raw material, its reception,storage and removal of the outer layers are important con-trol points to avoid contamination of the hearts of palm byC. botulinum spores. Cooling after heat treatment also con-stitutes an important control point with respect to microbialhazards due to the possibility of re-contaminating it viaopening/leakage of the container. Thus, as a preventativemeasure, the use of chlorinated water with a minimum of2 ppm residual free chlorine is recommended for the cool-ing procedure by immersion. After this procedure, thehearts of palm packs are stored at room temperature in adry place sheltered from the light (Resende & SagginJunior, 2004). Complying with the requirements of GoodManufacturing Practices constitutes a way of reducing thelikelihood of the hazard related to these steps. Theacidification and heat treatment are the critical steps forthe microbiological safety of hearts of palm in preserveand therefore demand a special focus. The heat treatmentconstitutes a critical step for the safety of hearts of palmin preserve since it is responsible for inactivation ofcontaminating microorganisms, mainly non-spore-formingbacteria. Inadequate heat treatments can allow the survivalof spoilage microorganisms which can raise the pH of thepreserve and allow for growth of C. botulinum (Huhtanen,Naghski, Custer, & Russell, 1976). The control of thetime/temperature binomial and maintenance of records ofthe heat treatment are fundamental in reducing the risksto consumer health. In addition to the heat treatment, acid-ification has a fundamental role in guaranteeing the micro-biological safety of the hearts of palm in preserve, since itis the reduction of the pH to a value below 4.5 that effec-tively impedes the germination, outgrowth and productionof the neurotoxin by C. botulinum (Derossi et al., 2011;FDA, 2009; Johnson, 2007). For this reason actions relatedto the acidification of the saline used in processing hearts ofpalm and the guarantee of an equilibrium pH value below4.5 will be discussed in detail in Section 5.1.

The responses of authorities to hearts of palmspreserves-associated botulism outbreaks

The investigations carried out as a result of outbreaksof botulism found processing failures in the production ofa large number of clandestine products, showing a lackof criteria for quality and safety control (Anonymous,1999c, p. 10). The main irregularities found were the super-imposition of labels of products from different origins andthe lack of important information such as the manufacturer,batch, date of manufacture, expiry date and registers of thecontrol of the productive process (Anonymous, 1999c, p.10; Costa et al., 2010).

Table 3 shows the chronology of the measures and ac-tions implemented aimed at controlling the outbreaks ofbotulism related to the consumption of hearts in palm inpreserve and avoid their re-occurrence. A group made upof the interested parts (representatives from industry, con-sumer defense, academia, and epidemiological and sanitarysurveillance) was first formed to discuss, create and aid thesanitary surveillance authorities implement the appropriatemeasures. Thus, ANVISA divulged alerts to the populationindicating that the products from suspect brands (clandes-tine or those associated with the cases of botulism) shouldnot be consumed. In addition they alerted the populationthat even the products from trustworthy brands shouldonly be consumed after boiling for 15 min in water or inthe brine in which they were packed. In addition, the integ-rity of the container and appearance of the liquid should beverified before consumption (Inmetro, 1999). Finally, as apreventative measure, ANVISA determined, by way of de-cree n 304 of April 8th 1999 that the following warningphrase be stuck on the label of all hearts of palm preservescommercialized in Brazil: For your safety, this product

Table 3. Measures adopted by ANVISA to reduce the outbreaks of botulism associated with the consumption of hearts of palm preserves.

Measures Objectives Reference

Label with warning to consumer suggesting heatingof the hearts of palm preserve before consumption.

Inactivation of the botulinum toxin and reduction in the riskof new botulism outbreaks.

Anonymous (1999a), p. 12

Approval of the technical regulation for the identityand quality of hearts of palm preserve,

Standardization of the product. Definition of the minimum quality requirements.

Anonymous (1999b), p. 10

Implantation of the Good Manufacturing Practices,HACCP and quality control in industries producinghearts of palm preserves.

Improvement in the hygienic-sanitary conditions. Establishment of the critical control points. Greater control of the processing steps. Maintenance of registers.

Anonymous (1999c), p. 10

Registers of the industries in IBAMA and ANVISA. Guarantee the legality of the raw material and the origin ofthe product.

Allow inspection.

Anonymous (1999c), p. 10

Making the labeling adequate. Traceability of the product. Guarantee information for the consumer about quality and

safety.

Anonymous (1999b, p. 10,1999c), p. 10

Inspection of the industries, canceling of registersand removal of irregular products from the market.

Guarantee of complying to the legal production demands. Guarantee food safety.

Anonymous (1999c), p. 10

Elaboration, implantation and maintenance of theStandard Operating Procedures for the critical stepsof the processing of hearts of palm.

Avoid failures in the acidification and heat treatment steps ofhearts of palm.

Establish corrective measures for non-conformists. Maintain registers of the processing steps. Reduce the risks of products not conforming and the devel-

opment of C. botulinum.

Anonymous (2003)

Compulsory notification of outbreaks of botulism. Improve the accompanying of botulism outbreaks. Investigate the factors related to the intoxication. Obtain reliable epidemiological data.

Anonymous (2001, 2010),p. 50

89H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95should be consumed after boiling in the preserving liquid orin water for 15 minutes (Anonymous, 1999a, p. 12). Thewarning stickers had to be placed on the primary containerin a legible form and in a place visible to the consumer,within a maximum of ten days. This measure was basedon the impossibility of impeding the commercializationof the product in the country, since not all the industriespresented irregularities in their production system. Theplacing of these warning stickers was based on the factthat the botulinum neurotoxin is heat labile and can be de-stroyed by heating at 80 C/20 min or 85 C/5 min (Siegel,1993). Thus, the objective of ANVISA was to prevent theoccurrence of new botulism cases associated with the con-sumption of hearts of palm until the planned measures wereeffectively implemented (Anonymous, 2000). The warningsticker would be used on the products until ANVISA in-spected the manufacturers, and those approved would sub-sequently be dispensed from its use (Anonymous, 2002a,2002b). In addition, the manufacturers themselves tookthe initiative to create an association of hearts of palm man-ufacturers with its own quality seal. The quality seal wouldbe conceded after approval by a third party in inspectionaudits of the production by the various industries(Inmetro, 1999).

In July 1999 ANVISA approved a technical regulationreferring to the standard of identity and quality of heartsof palm in preserve (Anonymous, 1999b, p. 10). Thisregulation was created with the objective of establishingthe minimum quality characteristics for the hearts ofpalm preserves commercialized in Brazil. The minimumquality characteristics established in this regulationwere: presentation form (sticks, slices or cubes), composi-tion and essential quality factors (maximum pH < 4.5,minimum vacuum and filling), processing hygiene as-pects, labeling (including the registration number inIbama), and the analytical methods used in the qualitycontrol (Anonymous, 1999b, p. 10). This regulation stillapplies in Brazil, although it was subsequently alteredwith respect to the minimum vacuum for the containersof hearts of palm preserves with varied capacities andtypes of closure (Anonymous, 2003, p. 64).

Also in 1999, regulation n 18 of November 19th 1999was published, which led to a series of changes in the pro-cessing and quality management of hearts of palm pro-ducers (Anonymous, 1999c, p. 10). This regulation madeit compulsory to implement the Good Manufacturing Prac-tices (GMP) and the Hazard Analysis and Critical ControlPoints (HACCP) and Quality Control and Guaranteesystems in all hearts of palm processing industries(Anonymous, 1999c, p. 10). In addition the companieswere obliged to contract a technical manager who shouldreceive specific training on GMP and HACCP as directedto the processing of hearts of palm, in institutions recog-nized by ANVISA.

Regulation n 18 of November 19th 1999 also made itcompulsory to present their registers to both IBAMA andANVISA (Anonymous, 1999c, p. 10). In practice this regu-lation signified the canceling of their previous registers, andin order to renew them they had up to 90 days to present thefollowing documents: i) copy of the register in IBAMA, ii)

90 H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95copy of the report of a sanitary inspection of the factoryexpedited by a sanitary authority, iii) layout of the factoryand a detailed production flow diagram, iv) GMP manualand HACCP and quality control and guarantee plan, v)technical term of responsibility, vi) training certificate ofthe technical manager, vii) copy of the social contract ofthe company, viii) copy of the contract of the technicalmanager and his working hours, ix) proof that the productsare being commercialized according to the specificationsapproved in the register in the Ministry of Health, x)form requesting registration/re-registration. In the case ofdistributors of hearts of palm preserves (importers, forexample), the following documents became necessary torenew the register: i) form requesting registration/re-registration, ii) copies of the contracts with the factoriessupplying the hearts of palm, approved by the sanitary in-spection and duly registered in ANVS/MS, iii) report ofthe sanitary inspection and License to Function of thedistributor, iv) copy of the social contract of the company,v) list of the commercial establishments who buy the prod-uct (Anonymous, 1999c, p. 10).

The obligation to obtain a register in IBAMA aimed toreduce the chances of commercializing clandestine prod-ucts, whose processes do not follow the basic principlesof hygienic production and food safety. The register inANVISA, for its part, aimed to guarantee that only pro-ducers inspected and qualified with respect to the regulato-ry aspects were allowed to industrialize hearts of palm. Inaddition, the commercial establishments, supermarkets, res-taurants and others that use hearts of palm in preserve or innatura, were also obliged to present legal proof of theorigin of the product (Anonymous, 1999c, p. 10).

Regulation n 18 of November 19th 1999 also estab-lished the implantation of a National Program for Food In-spection e Hearts of Palm in preserve (Anonymous, 1999c,p. 10), with the following activities: i) the re-registration ofthe hearts of palm factories and their distributors aftercomplying to the demands established by this regulation,ii) sanitary inspection of the factories and distributors ofhearts of palm by technicians from the municipal or statesanitary surveillance or by consultants and technicians indi-cated by ANVISA, iii) sanitary inspection of the commer-cial establishments, transporters and storage units. Tocarry out the inspections, a routine list for inspection wasmade available by the National Program for Food Inspec-tion (Hearts of Palm in preserve), and is presented asSupplementary Material. The registers of the hearts ofpalm in preserve processing industries that did not adequatethemselves to the demands of Regulation n 18 ofNovember 19th 1999 were canceled (Anonymous, 2002b).

Since the cases of botulism registered between 1997 and1999 and associated with hearts of palm occurred in theState of S~ao Paulo (Gelli et al., 2002), notification of thedisease already became compulsory in this state in 1999(Anonymous, 1999d). In addition, a reference center forbotulism was created in the same year with the followingobjectives: i) reorganize and integrate the actions of theepidemiological and sanitary surveillance to investigatesuspected cases of botulism, ii) reorganize the laboratoriesto give the necessary support to the actions of the epidemi-ological and sanitary surveillance, iii) allow the rapidrelease of anti-botulinum serum to attend cases of botulism,iv) orientate, divulge and provide knowledge to the profes-sionals of the epidemiological and sanitary surveillance or-gans, doctors and the population by producing technicaland educational materials, v) improvement of the systemof notification, consultation and register of the disease(Anonymous, 1999d, 2009).

Due to the constant need for strengthening hearts ofpalms in preserve legislation, in 2003 ANVISA publishedthe regulations concerning the elaboration, implantationand maintenance of the Standard Operating Procedures(SOPs) for the production of these foods. The SOPs werespecifically focused on the most critical steps to ensurethe production of safe hearts of palms in preserve: acidifi-cation and heat treatment (Anonymous, 2003, p. 64). Theproper implementation of Standard Operating Procedureswas evaluated in the routines inspections of these heartsof palms in preserve industries (Anonymous, 2003, p. 64).

In 2001 the notification of outbreaks of botulism becameobligatory at a national level in Brazil. This action had theobjective to allow proper record of outbreaks of botulismoccurring in the country and to investigate the factorsrelated to the intoxication such as the types of toxin andfood involved, number of people, age and sex, and the pro-cessing and storage conditions of the foods (Anonymous,2001, p. 35).

An overview of the acidification of the brine as acritical step in the safety of hearts of palm preservesand of the actions taken to ensure safe products

The regulations of ANVISA passed in 1999 had a directimpact on the processing of hearts of palm preserves(Anonymous, 1999a, p. 12, 1999b, p. 10, 1999c, p. 10).These regulations took special care with the acidificationstep since it is considered to be a critical step in the pro-cessing. In addition, epidemiological investigations indi-cated failures in the acidification of the brine and a lackof knowledge concerning its importance in avoidingmultiplication of the C. botulinum as the probable causesof the outbreaks of botulism between 1997 and 1999(Anonymous, 1999a, p. 12, 1999b, p. 10, 1999c, p. 10).

Acidification of the brine is carried out with the objec-tive of impeding the development of C. botulinum afterpasteurization, without significantly affecting the flavor ofthe product. This procedure is based on an acidificationcurve, and the amount of acid to be added to the salinevaries according to the acid added and the batch of heartsof palm to be processed (Bernhardt, 1989; Zapata &Quast, 1975). Thus the amount of acid to be added to thebrine should not be standardized, since there are variationsin the pH of different batches of hearts of palm. The

91H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95standardization of the amount of acid without taking the pHof each batch into account can result in a great risk to pub-lic health, since it may not always be sufficient to reducethe pH of the product to below 4.5. Thus some batches ofthe hearts of palm in preserve might have a pH > 4.5, acondition that favors multiplication of C. botulinum andthe production of neurotoxin (Costa et al., 2010; Derossiet al., 2011; Johnson, 2007) (Fig. 7).

The acidification curve allows one to calculate theamount of acid, generally citric acid, to be added to100 g of fresh hearts of palm with the objective of reducingthe equilibrium pH to values below 4.5 (Fig. 7). AlthoughpH 4.5 marks the limit below which C. botulinum doesnot multiply (Johnson, 2007; Peck, 2009), a lower valueis used to provide a good margin of safety due to naturalvariations in the pH that can occur, or to some variabilitybetween strains of C. botulinum in relation to the minimumpH value for multiplication (Chea, Chen, Montville, &Schaffner, 2000; Raatjes & Smelt, 1979). Thus a pH valueof 4.3 has been used to allow for an adequate penetration ofthe acidity to the center of the hearts of palm sticks, withouttoo much effect on product flavor (Berbari et al., 2008;Bernhardt, 1989; Zapata & Quast, 1975) (Fig. 7).

Some factors such as age, weight and length of thehearts of palm, humidity of the soil at the time of harvest,quantity and purity of the acid added to the brine and theamount of brine in the container can influence penetrationof the acid into the sticks, and consequently affect obtain-ing an equilibrium pH value below 4.5 (Chaimsohn,2002; Derossi et al., 2011; Quast, Zapata, & Bernhardt,1975). The diameter of the sticks is of great importancein the microbiological safety of the product, since thethinner they are, the quicker the acidified brine penetratesto the center (Quast, Ruiz, Schmdit, & Vitali, 2010). Asg of anhydrous citric acid per 100 g of heart of palm

0.0

00

0.0

75

0.1

50

0.2

25

0.3

00

0.3

75

0.4

50

0.5

25

0.6

00

0.6

75

0.7

50

0.8

25

0.9

00

0.9

75

1.0

50

pH

3.8

4.0

4.2

4.4

4.6

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

6.4

6.6

"Real" heart of palm

Fig. 7. Hypothetic acidification curve of Real hearts of palm (Arch-ontophoenix alexandrae and A. cunninghamiana) (adaptation pre-pared based on Berbari et al., 2008). An arrow indicates the amount

of anhydrous citric acid needed to reach the target pH 4.3.the acidification process is not instantaneous, dependingon the diameter of the hearts of palm sticks, it can takeup to 6 days for the pH at the center of the stick to reacha value below 4.5 (Quast et al., 2010). Such a time wouldbe sufficient for multiplication and production of neuro-toxin by C. botulinum, if spores of the organism were pre-sent on the inside of the hearts of palm sticks (Quast et al.,2010).

In Brazil the elaboration of the acidification curve forhearts of palm in preserve is established in Resolution eRDC N 17 of November 19th 1999 e ANVISA/MS(Anonymous, 1999b, p. 10). In addition, the elaboration,implementation and maintenance of Standard OperatingProcedures (SOPs) for the steps critical to the safety ofthe product, i.e. acidification and heat treatment, are regu-lated by Resolution RDC n 81 of April 14th 2003(Anonymous, 2003, p. 64). The SOPs referring to acidifica-tion should contain a description of the specification of theraw material processed, type of organic acid and method ofdetermining the amount to be used, formulation of the brineand proportion of the weight of the product to the weight orvolume of saline, and the method and frequency of deter-mining the pH value at the moment of acidification and af-ter the quarantine period of the product. The SOPs referringto the heat treatment should contain the product specifica-tions, size and type of container, minimum initial tempera-ture, type and characteristics of the heat treatment system,and the time and temperature of the heat treatment itself.Implementation of the SOPs should be monitored periodi-cally so as to guarantee the desired end product and adjust-ments should be made where necessary. The correctiveactions should contemplate the final destiny of the product,the restoring of sanitary conditions and the reevaluation ofthe SOPs under conditions of non-conformity. Periodic reg-isters should be made so as to document the execution andmonitoring of the SOPs, and the adoption of any correctivemeasures.

Concluding remarksHearts of palm is an important food potentially associ-

ated with botulism due to its conditions of acquisition, pro-cessing of the raw material and mode of consumption. Thismaterial is handled a lot in the field, and frequently har-vested under conditions that allow for ample contact withthe soil or with material contaminated with soil, which isthe main reservoir and source of contamination by C. bot-ulinum (Johnson, 2007; Peck, 2009). In addition the heattreatment is mild (100 C/up to 60 min) in order to maintainthe sensory characteristics, and food safety with respect tothe growth of C. botulinum is guaranteed by acidification ofthe product (Costa et al., 2010; Derossi et al., 2011; Quastet al., 2010). Due to the mild heat treatment and acidifica-tion, hearts of palm in preserve is considered to be acommercially sterile product and is therefore stored atroom temperature. Thus failures in processing, principallyin acidification, can provide ideal conditions for any C.

92 H.M. Hungaro et al. / Trends in Food Science & Technology 34 (2013) 80e95botulinum spores to germinate, multiply and then producethe potent neurotoxin (Derossi et al., 2011; Quast et al.,2010).

The outbreaks of botulism in Brazil associated with theconsumption of hearts of palm were widely divulgedcausing a great social commotion in the country, and repre-sented the turning point with respect to surveillance and thecontrol of foodborne botulism in the country. During theinvestigation of the outbreaks, the lack of standardizationand the amount of products available on the market withno quality control and safety criteria became evident.Hence the public health and sanitary surveillance author-ities implemented various corrective and preventive mea-sures throughout the productive chain of this food.

The measures taken by the public health and sanitarysurveillance authorities included effective, immediate ac-tions such as the immediate removal of products suspectof contamination from the commerce and the emission ofa warning to the population, up to profound changes inthe legislation and processing of the hearts of palm(Table 3). These interventions led to a standardization ofthe production and labeling of hearts of palm and to agreater control of the quality and safety criteria by bothproducers and consumers (Anonymous, 1999c, p. 10). Inaddition, the changes in the legislation had an adequate sci-entific basis and already included the concepts of riskanalysis.

Due to the improvements in the processing and qualityof hearts of palm preserves resulting from the measures im-planted over the years, ANVISA has revoked Decree n 304of April 8th 1999, which determined the obligatory pres-ence of a warning sticker on the container, indicating thesuggestion to heat the product before consumption (Brasil,2008).

The data of the epidemiological surveillance of food-borne botulism in Brazil demonstrated that the problemrelated to hearts of palm is under control. The recent casesof botulism registered in Brazil were associated with theingestion of foods prepared at home or by artisans, someof which contained hearts of palm as one of the ingredients(Table 2) (Eduardo et al., 2007; Zandonadi, Botelho, Savio,Akutsu, & Araujo, 2007). The participation of these foodsas responsible for foodborne botulism corresponds to anew challenge to the public health authorities in Brazil.

Hearts of palm, originated from small or large-scale in-dustries, should be produced taking into account adequatesanitary conditions for handling and legislation, that whenadequately implemented, assure high quality productssafe for consumption. According to the data and informa-tion reviewed in the present article, the outbreaks of botu-lism associated with hearts of palm preserves providedgreat lessons for processors, legislators, epidemiologicaland public health surveillance organs and consumers. Asa result of all the preventative and corrective actions adop-ted, the consumer market for hearts of palm is growingannually in Brazil, demonstrating the importance andadequacy of the measures adopted by the governmental or-gans in response to the botulism outbreaks. Despite this, thecontinuous training of processors, the acquisition of knowl-edge by consumers and active, constant inspection by thesanitary and public health surveillance organs are of greatimportance to guarantee that the advances obtained bemaintained and continue to protect public health.Supplementary data

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.tifs.2013.07.008.References

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