Bacterial spores in spices and dried herbs - Scinapse

Received: 6 July 2020 Revised: 15 November 2020 Accepted: 20 November 2020

DOI: 10.1111/1541-4337.12690

COMPREH ENS IVE REVIEWS IN FOOD SC IENCE AND FOOD SAFETY

Bacterial spores in spices and dried herbs: The risks forprocessed food

Anne Gabrielle Mathot1 Florence Postollec2 Ivan Leguerinel1

1 Université de Brest, LaboratoireUniversitaire de Biodiversité et d’EcologieMicrobienne, Quimper, France2 ADRIA Développement, UMT ACTIA19.03 ALTER’iX, Quimper, Cedex, France

CorrespondenceIvanLeguérinel, LaboratoireUniversitairedeBiodiversité et EcologieMicrobienne,6 ruede l’Université, F-29334Quimper,France.Email: [email protected]

AbstractProduction andworld consumption of spices are constantly increasing. Althoughthe antimicrobial properties of some spices are well documented, their use in theagri-food industry is also responsible for microbial contamination and spoilage.Bacterial spores introduced by spices can withstand different preparation pro-cesses, particularly thermal treatments, leading to food alterations during stor-age. This reviewbrings together data from the literature about the prevalence andconcentrations of spore-forming bacteria in all commercially available spices.The sporeformers found in spices belong mainly to the genera Bacillus andClostridium. Such contaminations are very common and sometimes reach highlevels, as in pepper and turmeric.Bacillus licheniformis andBacillus cereus are themost frequently detected species. Studying the harvesting, processing, and stor-age procedures for spices provides elements to explain why high prevalence andconcentrations are observed. Spices aremostly produced in developing countrieson small farms using traditional production methods. Spices become contami-nated by bacterial spores in two main ways: by contact with soil during harvest-ing or drying, as for pepper, or by cross-contamination during the water-cookingstep, as for turmeric. From these observations, we propose some recommen-dations. Different methods that can be used to eliminate bacterial spores fromspices are presented indicating their efficiency and the limitations of their use.

KEYWORDSbacillus, clostridium, concentrations, herbs, inactivation, prevalence, spice processing, spices,spores

1 INTRODUCTION

Spices and herbs are dry aromatic substances of plant ori-gin used to flavor or color food. Spices are seeds, nuts, flow-ers, rhizomes, or plant bark. Herbs are the green leaves ofdried aromatic plants. Dry aromatic vegetables are asso-ciated with spices and herbs (Peter, 2001). Spice produc-tion is increasing, and these ingredients are now widelyused in the development of new food products. Since 1990,a constant and steady production increase of 2.9% per

year has been recorded (values based on FAO economicdata: FAOSTAT; http://faostat.fao.org/), reaching 49.6 ×106 tons in 2018 (chilies, peppers, ginger, anise, fennel,coriander, mustard seed, cinnamon, cloves, nutmeg,mace,cardamoms, and spices nes)The main spice producing countries are in southern

Asia: India, China, Indonesia, and Bangladesh, all ofwhich can have hot and humid environments. Theconventional classification of spices is based on theirflavor intensity: strong spices (pepper, chili, ginger), mild

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BACTERIAL SPORES IN SPICES AND HERBS. . . 841

spices (paprika, coriander), aromatic spices (cinnamon,turmeric, cloves, cumin, aniseed, celery), dried herbs(basil, bay, dill, marjoram, tarragon, thyme), and aromaticvegetables (onion, garlic) (Peter, 2001). In addition to thesecategories, there are also spice blends and herbal mix-tures. Many spices are grown and, especially, collected inpoor sanitary conditions with most drying processes donedirectly on the soil. All these conditions lead to high level ofmicrobial contamination including mycotoxin-producingmolds, pathogenic microorganisms such as Salmonella,or bacterial spores, as documented in numerous articlesand reviews (e.g., McKee, 1995). In all heat-treated foods,bacterial spores are important microbial contaminantsbecause they can withstand processes used in the foodindustry, such as cooking, pasteurization, sterilization, anddisinfection.The soil is a direct source of bacterial food contamina-

tion. It is considered an important reserve of spores andany prolonged contact with food results in contamina-tion by bacterial spores (Carlin, 2011). Spices in particu-lar can come into contact with the soil, and therefore, beexposed to this contamination, during the threshing or dry-ing stages of traditional methods. Drying processes varygreatly depending on the spices concerned, the regions ofproduction, and the levels of economic development ofthese regions. In developing countries, traditional meth-ods of postharvest treatment of spices are still commonlyused such as drying on the ground (and hence in contactwith the soil). However, various drying techniques couldbe applied to spices and herbs such as solar drying, hot airdrying, and microwave drying (Jin, Mujumdar, Zhang, &Shi, 2017). Other methods, such as cooking by boiling, canalso be critical steps where spices become contaminatedwith bacterial spores.During the manufacture and formulation of food prod-

ucts, the diversity of their microbiota increases (Postollecet al., 2012), which is at least partly associated with theaddition of spices (Hampikyan, Bingol, Colak, & Aydin,2009). Twomain features of thismicrobial diversity need tobe considered. Although the concentrations of added bac-terial spores are low, their diversity is quite large (McKee,1995). Bacterial spores are resistant to heat treatments, sothey can germinate, grow, and even sporulate during man-ufacturing processes and storage. Some species of spore-forming bacteria cause food poisoning (Hariram & Labbé,2015; VanDoren et al., 2013), while others cause alterationsto products resulting in food degradation and significanteconomic losses. Spore-forming bacteria from herbs andspices have, for instance, been implicated in the instabil-ity of canned food (André, Zuber, & Remize, 2013; Wit-skowska et al., 2011). Better knowledge of the level of sporespresent could be helpful for evaluating the risks associatedwith the use of these ingredients (Van Doren et al., 2013).

There exist few decontamination processes for bacte-rial spores in spices and they have many disadvantages.First, ionizing treatments, which are, in fact, very effec-tive, are poorly accepted by consumers and require mar-keting authorizations. Steam-heating treatments alter sen-sory and physicochemical properties of spices. The use offumigation with ethylene oxide can even produce carcino-genic or mutagenic compounds. Alternative methods are,however, being developed.Knowledge about spice contaminations by bacterial

spores is therefore of great interest for foodmanufacturers.The first objective of this review is to consolidate and ana-lyze the published data on the prevalence and levels of bac-terial spores found in herbs and spices. Second, this studywill attempt to identify the potential sources of bacterialspore contamination of spices to improve spice preparationprocesses, and thus, limit the potential contamination.

2 SPORE-FORMING BACTERIA OFCONCERN AND CHARACTERISTICS OFTHE DATA COLLECTED

2.1 Pathogenic spore-forming bacteria

One of the most obvious ways to assess the risk associatedwith a food category is to look at the associated reportsof foodborne disease outbreaks. A shortcoming of thisapproach is that many countries are not able to track food-borne illness, and for some of those that are, the reportingstructure or information collected may be insufficient toattribute outbreaks to spices (e.g., distinction betweenfresh and dry in Europe, geographical scattering of out-breaks caused by pathogens such as Bacillus spp. in theUnited States, lack of microbiological or epidemiologicalevidence linking cause and effect). Consequently, thenumber of worldwide outbreaks associated with the con-sumption of pathogen-contaminated spices is likely under-reported. Ingredient-related outbreaks are especially chal-lenging to investigate because of the many possible foodsthat could have been involved and the potentially complexsupply chains associated with each ingredient. In addition,consumers of contaminated food may not be aware of allingredients in a food, especially minor ingredients suchas spices. The long shelf-life of spices and the ability ofpathogens to persist in them for long periods also createchallenges for outbreak identification because illnessesarising from the consumption of contaminated spices maybe separated in time and space from the contamination.A previous review identified 14 reported outbreaks

attributed to consumption of pathogen-contaminatedspice during the period 1973 to 2010 (Van Doren et al.,2013). Countries reporting outbreaks included Canada,

842 BACTERIAL SPORES IN SPICES AND HERBS. . .

Denmark, England and Wales, France, Germany, NewZealand, Norway, Serbia, and the United States. Together,these outbreaks resulted in 1946 reported human illnesses,128 hospitalizations, and two deaths. Although Salmonellawas identified as the major causative agent, accountingfor 71% of outbreaks, Bacillus spp. was identified as thecausative agent in the remaining 29% of outbreaks. Accord-ing to Mader (2016), from 1973 to 2012, nine outbreaksin Europe can be attributed to spices contaminated withBacillus cereus, which represent 50% of the total outbreaksassociated with spices. This could explain whymany of thedata we found in the literature concern this pathogenicspore-forming bacterium (135 numerical data from 20 arti-cles). Bacillus cereus, a member of the mesophilic aerobicflora, is enumerated on specific media developed due toits pathogenicity. These media are based on its resistanceto polymyxin (Mossel, Mannitol Yolk Polymyxin [MYP],Polymyxin pyruvate Egg yolk mannitol Bromothymol blueAgar [PEMBA], Phenol Red Egg Yolk Agar with PolymixinB [PREYPA], Kim-Goepfert [KG] Agar], presence of alecithinase (on egg yolk), and absence of mannitol degra-dation. They are incubated for 24 to 48 hr, usually at 30 ◦C,but sometimes at 35 ◦C or even 37 ◦C, thus revealing the B.cereus sensu lato group. Confirmation of typical colonies,although recommended, is not always done, which canlead to an overestimation of the presence of B. cereus sensustricto. Indeed, some samples showing high counts of typ-ical colonies may have been slightly contaminated with B.cereus after confirmation (Baxter & Holzapfel, 1982). Oth-erwise, incubation at 37 ◦C may limit the developmentof psychrotrophic species such as Bacillus weihenstepha-nensis (Guinebretière et al., 2008), which can produce anemetic toxin (Thorsen et al., 2006; Thorsen, Budde, Hen-richsen, Martinussen, & Jakobsen, 2009). Another pointis that B. cereus and Bacillus thuringiensis (Bc-Bt) are verysimilar and can only be differentiated by crystal toxin pro-duction in the latter. Although B. cereus/B. thuringiensisfrom onion powder was implicated in a foodborne out-break, only two articles specifically mention B. thuringien-sis (Freire &Offord, 2002; Hariram&Labbé, 2015). The lat-ter authors reported that 12.5% of Bc-Bt isolates from spicesmarketed in the United States are indeed B. thuringiensis.The emetic toxin gene (ces) was absent from all isolates,but percentages of positive isolates forB. thuringiensiswereonly slightly below those of B. cereus isolates for bothnheA and nheB genes (72/82%), hbl genes (hblA and hblD)(67/72%), and the hblC gene (67/71%). As B. thuringien-sis var kurstaki and var aizawai are authorized as pes-ticides against phytophagous caterpillars on spice cropsin Canada, France, and other countries, its spores couldpotentially contaminate the spice and resist subsequentprocessing. Growing interest in the issue of B. thuringiensisfood contamination, the risks it poses and possible coun-

termeasures (EFSA, 2016; Johler et al., 2018) increase theneed for a detailed review of present knowledge.Other aerobic spore-forming bacteria belonging to the

Bacillus subtilis group sensu lato, mainly the species sub-tilis, licheniformis, amyloliquefaciens, and pumilus, aremore rarely involved in foodborne outbreaks (and havenever been shown to come from spices). Nevertheless, theyare able to produce toxins and are sometimes not discrim-inated from B. cereus-related outbreaks (Logan, 2012).To our knowledge, only a few outbreaks directly impli-

cate the major anaerobic spore-forming food pathogenClostridium perfringens from spices (five in Europe, allfrom Denmark in 2011 to 2012), but as a major safety con-cern, many publications deal with its presence in herbsand spices (72 numerical data from nine articles). Thesequantitative numerical data were obtained with very dif-ferent methods (for heat treatment, media, and comple-mentary tests) and may not present the same selectivitytowardC. perfringens. Moreover, in 1998, Rodríguez-Romo,Heredia, Labbé, andGarcía-Alvarado reported only a 4.25%presence of the enterotoxin gene (cpe) in the isolatesfrom spices obtained from a retail market in Mexico. Veryrecently, an article about the toxinogenic potential of C.perfringens isolated from spices available in U.S. retail out-lets indicated amuch higher percentage (63%) ofC. perfrin-gens cpe positive isolates, but seven random spice isolatesproduced the enterotoxin at very low levels (4 to 16 ng/mL),compared with outbreak strains (>1,024 ng/mL) (Lee &Labbé, 2018).Few studies have been done on prevalence and levels

of the other anaerobic spore-forming pathogen Clostrid-ium botulinum. Although it has been found in spiced food(Chukwu et al., 2016) and some outbreaks of botulism haveinvolved garlic in oil or pesto (Burke et al., 2016; Peck,2014), no dehydrated spices or herbs have been reported asthe cause of an outbreak. Using enrichment methods, Car-lin, Broussolle, Perelle, Litman, andFach (2004) concludedthat it was absent from 65 samples of spices, herbs, anddehydratedmushrooms, as did Barker,Malakar, Plowman,and Peck (2016) for 36 samples of dried herbs and spices.Because there is known thatmany herbs and spices possessantimicrobial activity that could inhibit spore detection,the procedure for preparing these samples before addingthe enrichment medium was modified in these studies.

2.2 Spoilage-specific spore-formingbacteria

For certain sectors of the food processing industry (e.g.,canning), the contamination of ingredients by highlyheat-resistant bacterial spores is especially troublesome(Table 1). Indeed, in some canned foods, the microbiota


TABLE

1Physiologicalcharacteristicsofspore-form

ingbacteriaspeciesidentified

inspices:Enzym

eproduction,temperatureandpH

grow

thlim

its,and

heatresistance

Hydrolysisof

Temperature:growth/n

ogrow

thpH

:growth

/nogrow

thD

100◦C

Cas

Tri

Lec

Sta

LacA/An

5◦C

10◦C

20◦C

30◦C

35◦C

40◦C

50◦C

55◦C

65◦C

70◦C

75◦C

56

78

910

minutes

B.am

yloliquefaciens

++

–+

±.+/-

––

++

++

+–

–nd

nd+

––

–2.8m-507.2n

B.cereus

++

+–

.+/+

nd±

++

++

––

nd+

+nd

ndnd

0.2j-45.5c

B.circulans

ww

–w

+.+/+

ndnd

nd+

++

+±

ndnd

nd±

++

++

+1.65c-240.1a

B.coagulans

––

+±

.+/+

ndnd

nd+

++

±±

ndnd

nd+

++

++

+0.1g

-8.1l

B.firmus

ww

–+

–.+/+

ndnd

++

++

±–

–+

++

++

170.6c

B.lentus

––

++

.+/-

nd+

++

+nd

––

±+

++

+±

447.6c

B.licheniformis

+–

++

.+/+

––

++

++

+±

nd+

+nd

ndnd

0.9f-7.8n

B.megaterium

+–

++

.+/-

±±

++

nd±

±±

ndnd

ndnd

++

ndnd

nd0.2f -2.35

a

B.pumilus

+–

–+

.+/-

±±

++

++

±–

–±

++

++

–0.2b-10.0i

B.subtilis

+–

++

.+/-

±±

++

++

±±

–nd

++

+nd

nd0.2g-5123.3m

C.Perfrin

gens

–+

±+

.-/+

nd±

++

++

+nd

ndnd

ndnd

++

+nd

nd7.2b-7.4b

G.stearothermophilus±

nd+

nd.+/-

––

––

±+

++

++

±nd

++

+–

–9.6e-1282.6l

L.sphaericus

±+

––

.+/-

–±

++

nd±

––

2.3a-2.9d

P.alvei

–nd

+nd

.+/+

P.macerans

+nd

+nd

.+/+

ndnd

nd+

ndnd

–nd

ndnd

ndnd

nd7.8L-69.9k

P.polymyxa

+nd

+nd

.+/-

ndnd

nd+

ndnd

–nd

ndnd

ndnd

nd0.4l-22.2c

Heatresistancedatareferences:aMikolajcik(1970);bGibrieletal.(1973);cDavies(1975);

dlAndréetal.(2013);Beam

an,Pankratz,andGerhardt(1989);

nBerendsenetal.(2016);

mBerendsen,Zw

ieterin

g,Kuipers,and

Wells-Bennik(2015);hBradshaw,Peeler,andTw

edt(1977);jGonzález,López,Martınez,Bernardo,andGonzález(1999);eHaasetal.(1996);kHuo

etal.(2012);iLeuschner,O’Callaghan,and

Arendt(1998);fNakayam

a,Yano,Kobayashi,Ishikawa,andSakai(1996);gPalop,Raso,Condon,andSala(1996);oSadiqetal.(2016).

Growthandenzymeproductiondataarefrom

Bergey’smanual(DeVosetal.,2009).E

nzym

aticactivity:+

85%positive,±16%to84%positive;<15,15%

positive;w,w

eakreaction;nd,nodata;Cas,hydrolysisofcasein;

Tri,hydrolysisoftributyrine;Lec,hydrolysisoflecithin;Sta,hydrolysisofstarch;Lac,hydrolysisoflactose;GrowthA/An,grow

thinaerobic/anaerobicconditions.


of the major ingredient consists of relatively heat-sensitiveorganisms. However, a minor ingredient, such as natu-ral spice, may introduce highly heat-resistant microorgan-isms (i.e., bacterial spores). In such cases, bacterial sporesbecome the single most important component determin-ing the bacteriological quality of a product. Indeed, tocontrol the quality of the finished product in terms ofspore forming bacteria, it is necessary to control the sporeload of this ingredient (Farkas & Mohácsi-Farkas, 2014).Thermophilic species are more resistant to thermal treat-ments than are other bacteria (Warth, 1978). Despite inter-est from the canning industry, aerobic thermophilic sporeshave rarely been analyzed (Isolation by 15 min at 100 ◦Cand incubation at 55 ◦C on TSA; Oomes et al., 2007). Ofthe bacterial species isolated in this way, only Geobacil-lus stearothermophilus (six numerical data from one arti-cle (Giaccone, Colavita, Torriani, Ciocca, & Augelli, 1996)was identified, although the species Bacillus licheniformis,B. circulans, and Bacillus coagulans, as moderate ther-mophilic species can be connected to this group (Table 1).Few bibliographic data were found on thermophilic anaer-obic spore-forming bacteria either. Moreover, some datashould be treated with caution as thermal selection isomitted before enumeration on clostridial agar at 55 ◦C(Geeta & Kulkarni, 1987). To our knowledge, no publica-tions have reported the prevalence or level of Moorella orThermoanaerobacterium in herbs and spices although theyare major spoilage species in the canned food industrydue to the particularly high heat resistance of their spores(André et al., 2013).Although comprehensive knowledge of species present

can provide important information for evaluating spoilagepotential, few authors have gone as far as the identifica-tion of nonpathogenic species of bacillus or other sporulat-ing genera present in herbs and spices. Identified speciesmainly belong to the B. subtilis sensu lato group: B. sub-tilis, B. licheniformis, Bacillus amyloliquefaciens, and Bacil-lus pumilus. The most frequently mentioned are B. licheni-formis (12 numerical data/three articles) and B. pumilus(14 numerical data/3 articles). Other genera/species men-tioned in the present report are Paenibacillus (Paenibacil-lus macerans and P. polymyxa species) and Lysinibacillus(all formerly Bacillus). The growth limits and main enzy-matic activities of these species are given in Table 1. Theirenzymatic activities indicate significant spoilage potential.

2.3 Overall levels of spore-formingbacteria

Apart from the above-mentioned species, most data giveinformation on “groups” defined by common growth spec-ifications. Indeed, aerobic mesophilic spores, sometimes

called “total spores,” broadly match the cells selected by alimited thermal treatment to destroy vegetative cells with-out affecting spores. This treatment varies from 10 min at70 ◦C (Julseth & Deibel, 1974) up to 5 min at 100 ◦C (Debs-Louka, El Zouki, & Dabboussi, 2013), but most studies arebased on treatments of 5 to 10 min at 80 ◦C. In Freire andOfford (2002), selection of sporeformers is made with aspecific method, using a 5-min 30% H2O2 treatment.After treatment, a nonselective spore culture medium

is used: plate count agar (PCA) in the majority of studies(Julseth & Deibel 1974; Kneifel & Berger 1994; Witkowska,Hickey, Alonso-Gomez, & Wilkinson, 2011; Debs-Loukaet al., 2013), but DTA (Dextrose Tryptone Agar) (Karap-inar & Aktug, 1986) or TSA (Trypticase Soy Agar) (Oomeset al., 2007) are also used. The last of thesemedia lacks dex-trose but has more peptone hydrolysate. All have a neutralpH (6.9 to 7.3). Incubation conditions used are the same asthose for mesophilic aerobic flora, that is, 30 ◦C for 72 hr(Kneifel & Berger, 1994; Witkowska et al., 2011) or occa-sionally 48 hr. Other authors use a temperature of 37 ◦C for72 hr (Oomes et al., 2007) or 48 hr (Debs-Louka et al., 2013).Although Farkas and Mohácsi-Farkas (2014) indicate thatpsychrotrophic bacteria (growth at 7 ◦C) are generally lessnumerous in herbs and spices than mesophilic ones, veryfew data, if any, exist on this category. This lack of inter-est could be due to their lower heat resistance; they are,nevertheless, capable of growth in refrigerated products.Mesophilic aerobic spores therefore encompassmesophilicspecies of Bacillus, Paenibacillus, and Lysinibacillus with asignificant spoilage potential.The spore-forming bacteria found in spices and herbs

can also include anaerobic bacteria. High numbers ofanaerobic spore-forming bacteria can also cause rapidspoilage of anaerobic food products. Studies rarely men-tion the total anaerobic spores, mainly because facultativeanaerobic spore-forming bacteria also grow on nonselec-tive media in anaerobiosis (Table 1). This second group istherefore likened to Clostridia. Clostridia are sometimesenumerated at 37 ◦C directly on clostridial agar (Geeta& Kulkarni, 1987) or on Gifu anaerobic medium agar(GAM agar)) with confirmation on Clostridium welchiiagar (CW agar) (Fujisawa, Aikawa, Takahashi, Yamai, &Ueda, 2001). Sulfate-reducing Clostridia (SRC) can alsobe found (Cosano et al., 2009), corresponding to blackcolonies on sulfite polymixin sulfadiazine agar anaerobi-cally incubated for 72 hr at 37 ◦C after a heat treatmentof 5 min at 80 ◦C. Among the anaerobes, the only speciesthat has been identified is C. perfringens. Proteolytic andsaccharolytic Clostridia can also be differentiated, as wasdone on dehydrated garlic samples (Kłębukowska, Zader-nowska, & Chajęcka-Wierzchowska, 2015), leading to thedetection of these types in 0.01 g of spice for 84.6% and80.8% of the samples, respectively. Saccharolytic species


may be responsible for defects in food products associatedwith the accumulation of butyric acid fermentation prod-ucts (CO2 and H2).

2.4 Data characteristics

A total of 680 prevalence and concentration data were col-lected from 38 articles (published between 1971 and 2018),yielding results on 21 bacterial species or groups and 55kinds of spice.The studied articles include data on 55 spices or herbs.

The most commonly represented spices are chili (69numerical data from 22 articles), black pepper (67 numeri-cal data from 22 articles), turmeric (34 numerical data from13 articles), ginger (31 numerical data from 12 articles), andalso white pepper, cinnamon, red pepper, and cumin. Fewdata are available on contamination of herbs by bacterialspores, resulting in one to seven data depending on thetype of herb.The origins of the analyzed spices in these articles are

very miscellaneous. Although they originate from all thecontinents in the world, most of the studied spices wereproduced in India (151 out of 680 numerical data). Manyanalyses were also conducted on spices obtained fromretail outlets, but in packaging that did not specify theircountry of production (189 out of 680 numerical data). Forthe 28 countries of origin listed, the number of samplesper country was equal or less than 26, with a median valueof 2.5.

3 PREVALENCE ANDCONCENTRATIONS OF BACTERIALSPORES IN SPICES IN RELATION TOCROP-GROWING AND POSTHARVESTPROCESSES

Spices are classified according the conventional classifi-cation depending on the intensity of their taste. For eachspice and bacterial species pair, Table 2 gives themaximumpercentages of contaminated batches reported in the arti-cles. These percentages were calculated based on 1 to 2,090samples per batchwith amedian value of 22 samples for allthe articles.Prevalence datawere obtained using culture techniques.

In hot spices, chili, pepper, and ginger mainly containedthe pathogenic species B. cereus and C. perfringens. Molec-ular biology tools using the real-time PCR-based biochipassay commercially known as the GeneDisc R© Plate (PallGeneSystems, Bruz, France) should now help us to selectand more quickly identify target spore forming bacte-rial species that may be present in various spices in

order to obtain additional prevalence data (Postollec et al.,2012).In addition to assessing prevalence, many studies have

examined the bacterial spore concentrations in spices anddehydrated herbs. Tables 2 and 3 present spore prevalenceand concentrations, respectively, for each spice or herb.In Table 3, the data are summarized for each bacterium-spice pair, indicating the maximum or average concen-trations when available. An overall analysis of this tablereveals that studies have mainly focused on the overallspore flora of mesophilic or thermophilic aerobic or morespecifically on the pathogenic species B. cereus and C. per-fringens. Spoilage species studies are less frequent. OnlyB. subtilis, B. licheniformis, B. pumilus, B. thuringiensis,P. macerans, Paenibacillus alvei, and Lysinibacillus sphaer-icuswere quantified in some studies concerning black andwhite pepper (Freire & Offord 2002; Giaccone et al., 1996;Palumbo, Rivenburgh, Smith, & Kissinger, 1975). Surpris-ingly, no study has quantified G. stearothermophilus inspices.

3.1 Black and white peppers

Pepper is the fruit of the Piper nigrum creeper. On theplant, the stems carrying bunches of berries are harvestedmanually according to berry ripeness. Harvest is followedby a threshing stage. The traditional threshing techniqueis to trample the bunches on the ground and is thereforean unhygienic operation because soil particles come intocontact with the berries (Jayashree, 2011). Mechanicalbeaters can be used to limit contamination related tobacterial spores, but their use is limited to large farms andcooperatives (Pushpadaas & Korikanthimath, 2003). Thesecond stage of bacteriological risk during pepper posthar-vest processing is the blanching step. The immersion inboiling water for 1 min unsure cleaning and black coloringand facilitates the drying step (Risfaheri & Nurdjannah,2000; Zachariah, 2000). However, as in canning factories,the blanching step leads to recontamination by bacterialspores (Durand et al., 2015). These contaminations arelinked to successive splatter and fouling on the equip-ment surfaces allowing the development of thermophilicbacteria and their sporulation. The third stage that posescontamination risks is drying, which is done in the sun.The pepper berries are spread on different types ofmatting,a cement floor or polyethylene sheeting, with the lattermethod offering the best subsequent microbiological qual-ity. Solar dryers or mechanical dryers heated with woodcan also be used to carry out this step. After threshing anddrying, many impurities such as stems, soil, and stonesare present among the peppercorns and they then requirecleaning by winnowing or manual sorting. The different


TABLE

2Prevalenceofspore-form

ingbacteriaspeciesindifferentspicesandherbs(expressedasthepercentageofcontam

inated

samplesam

ongthesamplesstudied)

Bacillu

sam

y-lolique-

faciens

Bacillu

scereus

Bacillu

scirculans

Bacillu

scoagu-

lans

Bacillu

sfirmus

Bacillu

slentus

Bacillu

slichen-

iform

is

Bacillu

smega-

terium

Bacillu

spumilu

sBa

cillu

ssubtilis

Clost-

ridia

Clost-

ridium

perfrin-

gens

Geoacillu

sstearothe-

rmophilus

Lysinib-

acillus

sphaericus

Paenib-

acillus

alvei

Paeniba-

cillu

spolymyxa

Blackpepper

185to82

028

04

8to59

44to36

18to28

00to11

274

016

Whitepepper

53to8

5621

210

0to25

23Chili&red

pepper

33to24

820

04

16to58

164to38

9to20

020

260

840

Ginger

12to25

456

48

812

2016

1212

24Mustard

00

Coriander

130

0Cinnamon

100

0to28

Clove

750

0Turm

eric

600

440

012

00

120

80

32Cum

in8to40

0to38

Nutmeg

110

446

170

5022

Caraway

0Curry

100

0to62

12to33

Spicemix

50to17

455

350

30Basil

100

50Bayleaves

02to12

Dill

00

Oregano

0to53

Rosemary

330

Saffron

0to4

650to47

Sesame

0Thym

e100

050

Garlic

0to84

Datareferences:SeenappaandKem

pton

(1981);K

arapinarandAktug

(1986);teGiffeletal.(1996);Giacconeetal.,1996);Rodríguez-Romoetal.(1998);Fujisawaetal.(2001);Littleetal.(2003);Aguileraetal.(2005);

Stankovicetal.(2006);Cosanoetal.(2009);Ham

pikyan

etal.(2009);Sagooetal.(2009);Witkow

skaetal.(2011);Fogeleetal.(2018).


TABLE

3Sporeconcentrationsofbacterialspeciesindifferentspicesandherbs(expressedasthedecimallogarithm

ofsporeconcentrationsinthesamplesstudied,

*correspondstoa

meanvalue)

Aerobic

mesophilic

spores

Aerobic

ther-

mophilic

spores

Bacillu

scereus

Bacillu

slichen-

iform

isBa

cillu

spumilu

sBa

cillu

ssubtilis

Bacillu

sthurin-

gensis

clostridium

Clostridium

perfringens

Lysinib-

acillus

sphaericus

Paenib-

acillus

alvei

Paenib-

acillus

macerans

Blackpepper

4.9to8.9

4.3

0.0to6.0

6.0

4.6

4.1to7.3

0.0to4.5

0.9to5.0

4.8

Whitepepper

3.0to6.8

2.5to6.0

6.0

1.91.1

1.6to5.0

1.01.2

Chillies&red

pepper

4.7to6.7

4.2

0.0to5.3

6.0

6.2

3.5

0.0to3.0

Paprika

0.0to6.4

3.5to4.0

3.4to7.5

0.6to4.0

Ginger

5.5to6.9

3.9

3.0to5.7

7.2

3.7

3.0

Mustard

0.0to3.9

0.0to5.0

0.0

2.7

0.0to2.7

Coriander

3.6to5.6

6.1

3.0to5.1

0.0to3.0

Cinnamon

2.0*to5.3

2.6to5.3

5.0*

0.0to4.0

Cardamom

1.8to6.0*

4.0to5.0

0.0to2.4

Clove

3.2*to4.4*

0.0to4.0

3.3*

0.0to2.0

Turm

eric

4.8to8.8

6.0

0.0to6.0

00.0to3.0

Nutmeg

3.3*to5.5*

4.0

6.0

5.0*

3.7

1.0to3.0

Mace

0.0to6.7

3.0to6.0

2.0*

3.7

4.0

Cum

in1.6

to5.2

3.6

1.5to6.0

00.0to2.0

Aniseed

2.7to5.6*

5.0

0.6to2.0

Caraway

2.6to4.6*

0.0

2.0

Ajmud

seeds

4.7*

3.0

4.5

0.0to1.0

Fennelseeds

2.8to4.1

0.0to1.0

Fenugreekseeds

3.6

5.0

0.0to2.0

Curry

3.4to6.3

2.8*to4.4

0.6to2.4

China

spice

6.7

Sambarpow

der

5.6

1.7Mixed

spices

3.7to7.0

1.7to5.0

6.0

6.7*

0.0to3.0

(Continues)


TABLE

3(Continued)

Aerobic

mesophilic

spores

Aerobic

ther-

mophilic

spores

Bacillu

scereus

Bacillu

slichen-

iform

isBa

cillu

spumilu

sBa

cillu

ssubtilis

Bacillu

sthurin-

gensis

clostridium

Clostridium

perfringens

Lysinib-

acillus

sphaericus

Paenib-

acillus

alvei

Paenib-

acillus

macerans

Rasampowder

5.5

1.0to1.4

Tandoorispices

5.4

Basil

3.6*to5.0*

4.4*

3.0*

Bayleaves

4.0*

2.4

2.7

Dill

2.7to3.1*

3.0

Mint

2.8to3.1

4.0

4.0

Oregano

3.2*to4.9

3.8*to4.5*

3.6to5.0

3.7

3.5toto4.0

Parsley

2.6

3.0

0.0

Rosemary

2.4*to5.8

2.0*

5.2

Saffron

2.0

2.0

Sage

3.2*to3.6*

3.0

Savory

3.4*

2.8*

3.0

Tarragon

4.0*

3.2*

Thym

e2.6to5.1

3.6*

2.2*to4.0

3.6

Garlic

2.7*to5.1*

0.0to4.0

0.0to3.0

Onion

2.3*to4.5

5.0

0.0to1.2

Provenceherbs

4.6*

Mixed

herbs

4.0

4.0

Datareferences:Krishnaswam

yetal.(1974);JulsethandDeibel(1974);Palumbo

etal.(1975);Powersetal.(1976);Baxterand

Holzapfel(1982);D

eBoeretal.(1985);KarapinarandAktug

(1986);Pafum

i(1986);Geetaand

Kulkarni(1987);Antai(1988);M

unasirietal.(1987);Salmeron

etal.(1987);Sharmaetal.(1989);Te

Giffeletal.(1996);Kneifeland

Berger(1994);G

iacconeetal.(1996);Oom

esetal.(2007);Aksuetal.(2000);Freireand

Offord(2002);BanerjeeandSarkar(2003);A

guileraetal.,2005);Cosanoetal.(2009);Ham

pikyan

etal.(2009);Littleetal.(2003);Wójcik-Stopczyńskaetal.(2009);Witkow

skaetal.(2011);Debs-Loukaetal.(2013);

Kłębukowskaetal.(2015);Leeetal.(2018);Fogeleetal.(2018).


stages of harvesting and postharvest processing, especiallyusing traditional methods, include several bacterial riskstages that explain the high concentration of bacterialspores found in pepper: 8.9 log aerobic mesophilic sporesper gram (Geeta & Kulkarni, 1987). Black pepper is thespice most heavily contaminated with bacterial spores(Table 3), while white peppers appear comparatively lesscontaminated. This difference can be explained by thepostharvest treatment of the grains. To obtain white pep-per, the outer shell of the berry is softening by soaking upfor to aweek in slow runningwater, which limitsmicrobialgrowth. Then, the berries are trampled to remove the outershell. After peeling, the pepper berries are then washedand dried in the same way as black pepper (Azam, 2007).The soaking and washing steps remove soil, dust, andspores from the outer shell of the berries. White pepper istherefore less contaminatedwith spores than black pepper,showing an aerobicmesophilic spore concentration from 0to 6.8 log spores per gram (Debs-Louka et al., 2013; Kneifel& Berger, 1994). Overall, the prevalence appears lower inwhite than in black pepper, which can be explained by itsdifferent production processes. Black pepper is harvestedbefore maturity, dried in the sun for several days, andthen packaged, which explains its high contamination.White pepper is harvested at maturity. The fine bark ofthe peppercorns is removed manually or mechanically.Following brining, the peppercorns are stored away fromsunlight. This process combining hulling, brining, andrinsing may account for the lower contamination.Among the spices classified as strong, the prevalence

of pepper contaminants has been studied extensively andmany data are available. In black peppers, B. cereus alsohas high prevalence: (% of samples where contaminationwas detected) 85% of 11 samples (Fogele, Granta, Valcin, a,& Berzin, š, 2018), 40% of 25 samples (Seenappa & Kemp-ton, 1981), and 11.6% of 60 samples for peppers from India(Hampikyan et al., 2009). In this latter study, Hampikyanet al. (2009) also observed a lower B. cereus prevalencein white peppers (8.3% of 60 samples). B. licheniformispresents a maximum prevalence of 59.1% of 22 samples ofblack pepper and 56.4% for 39 samples of white pepper(Giaccone et al., 1996). In black peppers, spores of otherBacillus species can be found, with a prevalence above36% for B. pumilus, 28% for B. subtilis, 28% for B. coag-ulans, and 27% for G. stearothermophilus. In white pep-pers, prevalences are lower: 21% for B.3 pumilus, 21% forB. subtilis, and 23% for G. stearothermophilus (Giacconeet al., 1996; Seenappa & Kempton, 1981). The presence ofBacillus amyloliquefians in white pepper (5.1% for 39 sam-ples) and in black pepper (18.2% for 22 samples) has alsobeen noted (Giaccone et al., 1996). Concerning Clostridiaspecies, Stankovic, Comic, and Kocic (2006) did not detecttheir presence in white or black peppers in a limited num-

ber of samples (six to seven). However, Rodríguez-Romo,Heredia, Labbé, and García-Alvarado (1998) detected 11%Clostridium sporogenes prevalence in 60 samples of blackpepper obtained from retail outlets in Mexico, and Fuji-sawa et al. (2001) detected 25% ofC. sporogenes in four sam-ples of white pepper from retail outlets in Japan. Strongspices show the highest spore concentrations, with, forexample, a total spore concentration of 7.7 log spores pergram in black pepper (Julseth&Deibel, 1974). Several stud-ies have quantified mesophilic aerobic spores, with maxi-mum concentrations varying from 8.9 log spores per gramquantified in an Indian black pepper sample (Geeta &Kulkarni, 1987) to 4.9 log spores per gram (Debs-Loukaet al., 2013), and 4.3 log spores per gram of thermophilicspores (Oomes et al., 2007). B. cereus has been the sub-ject of numerous studies on black peppers of various ori-gins (Aksu, Bostan, & Ergün, 2000; Antai, 1988; Baner-jee & Sarkar, 2003; Baxter & Holzapfel, 1982; Giacconeet al., 1996; Pafumi, 1986; Salmeron, Jordano, Ros, & Pozo-lora, 1987). In these studies, the maximum concentra-tions detected ranged from 6 log spores per gram to totalabsence. B. subtilis was detected at very high concentra-tions, with an average of 7.3 log spores per gram (Palumboet al., 1975). Freire and Offord (2002) also detected thisspecies, but at lower concentrations, in Brazilian blackpepper (4.1 log spores per gram). B. licheniformis is a ubiq-uitous microorganism and its spoilages have been fre-quently observed.Maximumconcentrations of 6 log sporesper gram were detected by Giaccone et al. (1996) in blackpepper. In this spice, Freire and Offord (2002) quantifiedthe concentrations of other species such as B. pumilus (4.6log spores per gram) and P. alvei (4.8 log spores per gram).In black pepper, Julseth andDeibel (1974) quantified 4.5 logspores per gram of Clostridia. The concentration of C. per-fringens varied among studies from 0.9 to 5 log spores pergram (Banerjee & Sarkar, 2003; Pafumi, 1986; Lee & Labbé,2018; Salmeron et al., 1987).The lower concentrations of bacterial spores in white

pepper than in black pepper could be explained by theelimination of the bark of the peppercorns and the brin-ing. Debs-Louka et al. (2013) quantified maximum con-centrations of mesophilic aerobic spores of 6.8 log sporesper gram, while several authors have found maximumconcentrations ranging from 6 to 3 log spores per gram(De Boer, Spiegelenberg, & Janssen, 1985; Giaccone et al.,1996; Pafumi, 1986; Salmeron et al., 1987). The maximumconcentration of B. cereus ranged from 6 log spores pergram (Giaccone et al., 1996) to 2.5 log spores per gramwhen quantified in 60 white pepper samples from Turkey(Hampikyan et al., 2009).High concentrations ofB. licheni-formis (6 log spores per gram) were found in white as wellas black pepper (Giaccone et al., 1996). Freire and Offord(2002) identified and quantified other species of the genus


Bacillus or species from similar genera, such as P. mac-erans, B. pumilus, L. sphaericus, and B. thuringiensis, buttheir concentrations were below 2 log spores per gram. Inthe Clostridia, C. perfringens was found at lower concen-trations than Bacillus, with observed concentrations from1.6 log spores per gram (Lee & Labbé, 2018) to 5 log sporesper gram (Pafumi, 1986).

3.2 Chili, cayenne pepper, red pepper,and paprika

Chili, cayenne pepper, red pepper, and paprika are allspices produced from Capsicum spp. fruits. The morepiquant species such as Capsicum frutescens produce thehottest tasting spices: cayenne pepper, red pepper, andchili. For a milder taste, Capsicum annuum, known as“pepper,” bell pepper, or sweet pepper is dried and pow-dered to produce paprika.The peppers (Capsicum spp) are harvested from the

plants when the fruits are mature and red. The peppersare dried and can then be ground to produce red pepperor paprika. Drying is the stage where there is a risk ofcontamination by bacterial spores. The traditional methodof drying consists in spreading the peppers on dry soil inthe sun (Topuz, Feng, & Kushave, 2009; Jayashree, 2011).This soil contact explains the high concentrations of bac-terial spores (6.3 log) observed in chili powder produced inIndia (Munasiri et al., 1987). Such traditional techniquescan be improved to limit these contaminations by using acement floor or polyethylene films. The use of mechanicalor solar dryers (Thirupathi, Balakrishnan, & Visvanathan,2013) could also reduce bacterial spore contaminations.Concerning red pepper, Hampikyan et al. (2009) founda high prevalence of B. cereus, with 18.3% of 60 samplescontaminated. For the same spice, Seenappa and Kemp-ton (1981) found a B. cereus prevalence close to 24% in 25samples. However, Giaccone et al. (1996) detected a lowerprevalence of 2.9% in 34 samples of red chili imported intoItaly.Red and chili peppers are contaminated by various bac-

terial spores of species such as B. licheniformis (58% of 34samples, Giaccone et al., 1996; 16% in 25 Indian samples,Seenappa & Kempton, 1981), B. pumilus (38.2% in 34 sam-ples),G. stearothermophilus (26.5% in 34 samples Giacconeet al., 1996), Paenibacillus polymixa (40% in 25 samples), B.subtilis (20% in 25 samples), B. coagulans (20% in 25 sam-ples), Bacillus megaterium (16% in 25 samples), and P. alvei(8% in 25 samples) (Seenappa & Kempton, 1981). Powers,Lawyer, and Masuoka (1975) detected the presence of C.perfringens in Cayenne pepper (20% in 15 samples). To ourknowledge, no identification of bacterial spore species inpaprika has been reported in the literature.

Chili, cayenne pepper, and red pepper all contain highconcentrations of bacterial spores. In chili from Pakistan,Kneifel and Berger (1994) observed an average mesophilicspore concentration of 6.7 log spores per gram. The ther-mophilic spore concentration was lower (4.7 log spores pergram) (Oomes et al., 2007). In red peppers of different ori-gins, several studies quantified B. cereus spores, with themaximum concentrations ranging from 0 to 5.3 log sporesper gram (Aksu et al., 2000; Antai, 1988; Banerjee & Sarkar,2003; Baxter & Holzapfel, 1982; Bhat, Geeta, & Kulkarni,1987; Giaccone et al., 1996; Hampikyan et al., 2009; Powerset al., 1975; Powers, Latt, & Brown, 1976; Sharma, Pawal-Desaiand, & Nair, 1989). Several authors did not detectany C. perfringens spores in chili (Banerjee & Sarkar, 2003;Krishnaswamy, Patel, Nair, & Muthu, 1974; Pafumi, 1986;Powers et al., 1975). However, Power et al. (1975) and Leeand Labbé (2018) quantified C. perfringens in cayenne pep-per at concentrations ranging from 0.6 to 2.4 log sporesper gram. Aguilera, Stagnitta, Micalizzi, and de Guzmán(2005) quantified the spores of C. perfringens at a maxi-mum concentration of 3 log spores per gram in red pep-per. A maximum Clostridium spore concentration of 3.5log spores per gram was quantified in Indian chili powder(Bhat et al., 1987).High concentrations of bacterial spores have also been

found in sweet paprika, with a total maximum spore con-centration of 7.5 log spores per gram (Julseth & Deibel,1974). For mesophilic aerobic spores, 5.6 log spores pergramwere quantified (Debs-Louka et al., 2013), withmeanconcentrations from 4.7 to 6.4 log spores per gram (Kneifel& Berger, 1994). B. cereus was quantified at concentrationsof 4 log spores per gram (Pafumi, 1986) and 3.5 log sporesper gram (Baxter&Holzapfel, 1982). For anaerobic bacteriaspores, Clostridia concentrations were significant, with amaximumconcentration of 7.5 log spores per gram (Julseth& Deibel, 1974) and 4 log spores per gram for C. perfringens(Banerjee & Sarkar, 2003) in paprika.

3.3 Ginger

Ginger spice, obtained from the rhizome of Zingiber offici-nale, is classified as a strong spice. The fact that this spicegrows underground in the soil naturally leads to a highinitial bacterial spore contamination level. The first stepin ginger preparation is peeling, which is done to removethe scaly epidermis and facilitate drying. This step can becarried out manually with a bamboo blade or by mechan-ical brushing (Thirupathi et al., 2013). The ginger is thentraditionally dried on the ground for 7 to 10 days andrubbed to remove wrinkles formed during drying. (Bal-akrisnan, 2005; Jayashree, 2011; Jayashree, Visvanathan, &Zachariah, 2014). It can also be powered by grinding. These


postharvest stages can only slightly reduce the contamina-tion of the rhizomes by bacterial spores, which explains thehigh concentrations of aerobicmesophilic spores (5.8 to 6.9log spores per gram) found in this spice (Kneifel & Berger,1994; Oomes et al., 2007; Witkowska et al., 2011)It is not surprising to find awide variety of species of bac-

terial spores in this spice because it is derived from a rhi-zome that grows in the soil. Seenappa and Kempton (1981)detected 11 species of Bacillus in 25 samples of India ginger.For some species, the prevalence is high: B. coagulans 56%,P. polymixa 24%, B. pumilus 20%, B. cereus 12%, B. mega-terium 12%, L. sphaericus 12%, and P. alvei 12%. Hampikyanet al. (2009) quantified a B. cereus prevalence of 25% in 60samples of Turkish ginger.The fact that ginger is a rhizome also explains the

high total spore concentrations (6.7 log spores per gram)observed by Julseth and Deibel (1974). Mesophilic aero-bic spores were quantified, with maximum detected val-ues of 5.5 log spores per gram (Baxter & Holzapfel, 1982;Kneifel & Berger, 1994; Witkowska et al., 2011) and 6.9log spores per gram (Oomes et al., 2007) Aerobic ther-mophilic spores were quantified at 3.9 log spores per gramby Oomes et al. (2007). In ginger, like in other strongspices, many authors have quantified spores of B. cereus,and concentrations range from 5.7 to 3 log spores per gram(Aksu et al., 2000; Banerjee & Sarkar, 2003; De Boer et al.,1985; Hampikyan et al., 2009; Pafumi, 1986). For Clostridia,Julseth and Deibel (1974) reported a bacterial spore con-centration of 3.7 log spores per gram, consistentwithC. per-fringens concentrations, which have been reported rangingfrom 2 log spores per gram (Banerjee & Sarkar, 2003) to 3log spores per gram (De Boer et al., 1985; Pafumi, 1986).

3.4 Mustard

The term “mustard” includes seeds from different speciesof plants of the Cruciferae family: Sinapsis alba (whitemustard), Brassica juncea (brown mustard), and Bras-sica nigra (black mustard) (Vaughan & Hemingway, 1959).Mustards aremultibranched herbaceous plants cultured insouthern Europe, western Asia (black andwhitemustard),and India (brown mustard). Mustard seeds are harvestedby cutting the stems when the seeds are fully grown butnot fully ripe to avoid the bursting pods dispersing theircontents. The stems are then dried and beaten to separateout the seeds. Studies on bacterial spore prevalence inmus-tard are scarce. In a study of 16 mustard samples frommar-kets on U.S. army bases, no Clostridiawere detected (Pow-ers et al., 1975). Compared with other strong spices, con-centrations of bacterial spores in mustard seed are low, forexample, 4.5 log spores per gram (Julseth & Deibel 1974),with concentrations of mesophilic aerobic spores at 3.9

log spores per gram (Krishnaswamy et al., 1974). B. cereusspores ranged from absent (Powers et al., 1976) to 5 logspores per gram in Indian samples (Banerjee & Sarkar,2003). ForC. perfringens, spore concentrations ranged fromabsent (Powers et al., 1975) to 2.7 log spores per gram inIndian samples (Krishnaswamy et al., 1974). These differ-ences could be due to the origin or the harvest period ofthis spice.

3.5 Coriander

Coriander (Coriandrum sativum) is an annual herb usedas a spice. This plant is grown in many parts of the world,including India, Russia, and North and South America(Azam, 2008). Contamination of the seeds of this spice bybacterial spores, quantified at 5.2 log spores per gram byMunasiri et al. (1987), can be related to the traditional sun-drying techniques used in India. Seeds may be dried by thesun or in mechanical dryers (Azam, 2008), which couldreducemicrobial contaminations. In coriander, concentra-tions of mesophilic aerobic spores were detected at levelsfrom 3.3 log spores per gram (Kneifel & Berger, 1994) to 7.0log spores per gram in South African samples (Baxter &Holzapfel, 1982). Concerning thermophilic aerobic spores,Oomes et al. (2007) observed maximum concentrations of6.1 log spores per gram. In 60 Turkish samples, B. cereusspores were detected in 13% (Hampikyan et al., 2009). Forthis bacterial species, 3.0 to 5.5 log spores per gram werequantified in Indian, Turkish, and South African corian-der samples (Banerjee & Sarkar, 2003; Baxter & Holzapfel,1982; Hampikyan et al., 2009). Spores ofC. perfringenswerefound to be absent in one study of coriander seeds (Pafumi,1986), while quantified at concentrations up to 3 log sporesper gram in Indian coriander powder by Banerjee andSarkar (2003).

3.6 Cinnamon

Cinnamon is an aromatic spice obtained from the innerbark of the cinnamon speciesCinnamomum verum orCin-namomum zeylanium, which are small evergreen trees, 2to 3 m in height, and native to Sri Lanka. The bark isharvested from the trees, then cut into sticks, piled up,and pressed, during which a slight fermentation occurs.The bark folds on itself forming sticks, which are thendried in the sun or by using a mechanical dryer. Sulfurtreatments can also be applied for the conservation ofthis spice (Jayashree, 2011). The concentrations of aerobicmesophilic spores found in this spice are highly variable,with concentrations from 2 (Witkowska et al., 2011) to 5.3log spores per gram (Baxter &Holzapfel, 1982; Karapinar &


Aktug, 1986). The absence of soil contact explains the rel-atively low levels of bacterial spore contamination. Cinna-monhas a high prevalence ofB. cereus: 100% for 20 samplesfrom the Turkish retail market (Karapinar & Aktug, 1986)and in seven samples of imported spices in Riga, Latvia(Fogele et al., 2018). In this spice, many studies have specif-ically quantified the spores of B. cereus (Banerjee & Sarkar,2003; Fogele et al., 2018; Giaccone et al., 1996; Karapinar& Aktug, 1986; Pafumi, 1986; Power et al., 1976). The max-imum concentrations detected ranged from 2.6 to 5.3 logspores per gram. High concentrations of B. subtilis (meanof 5 log spores per gram) were observed by Palumbo et al.(1975). C. perfringens was detected in 28% of 18 cinnamonsamples from markets on U.S. army bases (Power et al.,1975). Quantifications of C. perfringens by Pafumi (1986)ranged from absence in cinnamon chips to 4 log spores pergram in ground cinnamon.

3.7 Cardamom

Cardamom is the dried fruit of the plant Elettaria car-damonum, a small evergreen shrub 3 m in height onwhich mature seed capsules stand out easily. Harvestedcardamom capsules are cleaned to remove dust and thenrinsed in clean water. The capsules can be soaked in a2% sodium bicarbonate solution for 10 min so that theykeep their green color (Jayashree, 2011; Korikanthimath,2013). After draining, the capsules are immediately driedat a temperature below 50 ◦C. Traditionally, the capsulesare spread on a concrete floor away from direct sun-light to avoid color loss, although they can be contami-nated by dust from the floor environment (Azam, 2008).Since the use of solar dryers leads to a loss of the greencolor, cardamom capsules can also be dried in mechani-cal wood-heated or electric dryers (Korikanthimath, 2013;Patil, 1987). Artisanal techniques of drying on the groundexplain the high level of contamination by bacterial spores.Kneifel and Berger (1994) showed high concentrations ofaerobic mesophilic spores (6 log spores per gram) in car-damom, which were mainly represented by B. cereus at 4to 5 log spores per gram (Banerjee & Sarkar, 2003). Lee andLabbé (2018) quantified C. perfringens at 2.4 log spores in acardamom sample obtained from a U.S. retail outlet.

3.8 Cloves

Cloves (Eugenia caryophyllus) are dried flower buds. Thesebuds are very moist during their harvest, which raises therisks of fermentation and alteration. To reduce the risk ofmicrobial development, harvesting and drying processesare carried out paying great attention to hygiene. The care

taken explains the lowmesophilic aerobic spore concentra-tions, of 3.2 to 4.7 log spores per gram, observed in this spice(Baxter & Holzapfel, 1982; Kneifel & Berger, 1994). Clovesare known for their antiseptic effects (Arora & Kaur, 1999),but B. cereus spores were detected in 75% of four samples(Fogele et al., 2018) and at a concentration of 4 log sporesper gram (Giaccone et al., 1996; Banerjee & Sarkar, 2003).B. subtilis was quantified at 3.3 log spores per gram byPalumbo et al. (1975). Banerjee and Sarkar (2003) detectedspores of C. perfringens at 2 log spores per gram in sevenIndian clove samples.

3.9 Turmeric

Turmeric is an aromatic spice made from the rhizome ofCurcuma longa. The rhizomes of turmeric undergo severaltreatments after harvest: they are scalded, dried, rubbed,and then colored. The cooking by boiling takes 60 to 90min (Jayashree, 2011) and the rhizomes are then spreadon bamboo mats or on the ground to be dried in the sunfor 10 to 15 days. The boiling step, like the blanching stagein a canning factory, can be a source of spice contami-nation by bacterial spores (Durand et al., 2015). Splatterand fouling allow spores that have resisted the thermaltreatments to germinate,multiply, and sporulate, introduc-ing high concentrations of mesophilic and thermophilicbacterial spores that can recontaminate the turmeric afterthis stage. Munasiri et al. (1987) quantified 5.9 log bacte-rial spores per gram in Indian turmeric samples. Thus,this stage of boiling represents a critical point in turmericpreparation and can explain the high spore concentrationsin this spice. Pressurized steam cooking (Thirupathi et al.,2013) would greatly reduce the risk of high-level bacterialspore contamination of turmeric. Seenappa and Kempton(1981) researched the presence of 12 different species ofBacillus in 25 samples of turmeric. High prevalence lev-els were found for B. cereus (60%), B. coagulans (44%),and Bacillus polymixa (32%). The other species detected,B. licheniformis and B. subtilis, were found at lower preva-lence (12%), as was L. sphaericus (8%). Turmeric may havevery high levels of bacterial spore contamination, with aer-obic mesophilic spores ranging from 4.8 to 8.8 log sporesper gram (Debs-Louka et al., 2013; Geeta & Kulkarni, 1987;Kneifel & Berger, 1994; Krishnaswamy et al., 1974; Oomeset al., 2007;Witkowska et al., 2011). Thermophilic bacterialspores were quantified at 6.0 log spores per gram byOomeset al. (2007). Different studies have found concentrationsof B. cereus spores from total absence up to a maximum of6 log spores per gram (Banerjee & Sarkar, 2003; Pafumi,1986; Sharma et al., 1989). C. perfringens concentrations of3 log spores per gram were quantified by Pafumi (1986)and Krishnaswamy et al. (1974), but Banerjee and Sarkar


(2003) found that the species was absent from 10 studiedsamples.

3.10 Nutmeg and mace

Nutmeg consists of the fruit albumen of the nutmeg tree(Myristica fragrans), a tropical tree of the Myristicaceaefamily, measuring 10 to 15 m. After harvest, the fruitsare transported for processing. The first step is to sepa-rate the mace from the rest of the fruit. The fruits areopened manually and the mace, a thin lace of red fiberssurrounding the core, is removed. The mace is then flat-tened by hand and dried on mats exposed to sunlight for2 or 3 hr. Mace is sold in the form of thin strips of about2.5 cm long and 1 mm thick. Once the mace has beenremoved, the nutmeg in the core is dried. The shells of thecores are then broken, and the nutmeg removed. Duringthe harvesting and drying stages, nutmeg does not comeinto contact with the soil, which explains why the maxi-mum contamination of nutmeg in bacterial spores is rela-tively low: only up to 3.7 log spores per gram in 10 Indiannutmeg samples (Julseth & Deibel, 1974). Giaccone et al.(1996) detected noB. cereus in 18 nutmeg samples, althoughthese same samples showed a high prevalence of B. licheni-formis (44.4%) and G. stearothermophilus (22.2%). Further-more, in three samples of this spice, Stankovic et al. (2006)detected Clostridia species. Among aromatic spices, nut-meg presents average maximum mesophilic aerobic sporeconcentrations from 3.3 log spores per gram (Baxter &Holzapfel, 1982; Debs-Louka et al., 2013; Witkowska et al.,2011) to 5.5 log spores per gram, quantified in imported nut-meg in Austria by Kneifel and Berger (1994). Concentra-tions of 4 log spores per gram B. cereus (Pafumi, 1986), 6log spores per gram B. licheniformis (Giaccone et al., 1996),and 5 log spores per gram B. subtilis (Palumbo et al., 1975)have been recorded by different authors.Clostridiae sporeshave also been detected, with maximum concentrations of3 log spores per gram for C. perfringens in 33 Australianimported nutmeg samples (Pafumi, 1986).Mace has similar or higher contamination spore con-

centrations than nutmeg, for example, 4.9 log sporesper gram quantified by Julseth and Deibel (1974). Bax-ter and Holzapfel (1982) detected higher concentrations ofmesophilic aerobic spores inmace (6.7 log spores per gram)than in nutmeg,withmaximumB. cereus concentrations of6 log spores per gram. These B. cereus concentrations arehigher than the 5 log spores per gram observed by Pafumi(1986) or the 3 log spores per gram quantified by Giacconeet al. (1996). B. subtilis was detected at low levels, 2 logspores per gram, byPalumbo et al. (1975). It should benotedthat the 4.0 log spores per gram concentration of C. per-fringens (Pafumi, 1986) is also higher than that quantified

in nutmeg. The higher spore concentrations in mace rel-ative to nutmeg can be explained by the contamination ofthe tegument, which comes into contactwith the soil whenthe nutmeg fruits fall.

3.11 Cumin

Cumin is an annual herbaceous plant, 30 to 45 cmtall (Cuminum cyminum), grown in sunny countries(Divakara, Sastry, & Anandaraj, 2013). Plants are harvestedwhen the seeds become light brown in color. They arethen dried under direct sunlight and the seeds recoveredby threshing the dry plants. The seeds are then driedon carpets or trays and fanned to remove dirt, dust, andleaves. These cleaning operations can now be carried outby machines equipped with vibrating screens. The cuminseeds can then be crushed. During threshing of wholeplants, rubbing of roots containing soil with the seeds cancause contamination by bacterial spores and explain thelevels reported. B. cereus was detected in 8.3% of 60 sam-ples of Turkish cumin and in 40% of five imported sam-ples into Riga (Leetonia) (Fogele et al., 2018).Cl. sporogeneswas detected in 38% of 60 samples of packaged cumin seedsfrom Mexican retail outlets (Rodríguez-Romo et al., 1998).Cumin can have maximum concentrations of mesophilicaerobic spores from 1.6 to 5.2 log spores per gram (Krish-naswamy et al., 1974; Oomes et al,. 2007; Debs-Louka et al.,2013) or average concentrations of 3.6 log spores per gram(Witkowska et al., 2011). For thermophilic aerobic spores,rates of 3.6 log spores per gramwere determined by Oomeset al. (2007). Among these spores, B. cereus was counted atmaximum concentrations of 1.5 to 6 log spores per gram inbatches of spices from India and Turkey (Aksu et al., 2000;Banerjee& Sarkar, 2003; Bhat et al., 1987;Hampikyan et al.,2009; Pafumi, 1986). C. perfringens has been quantifiedfrom 1.6 log spores per gram (Krishnaswamy et al., 1974)to 2 log spores per gram (Banerjee & Sarkar, 2003), but wasnot detected by Pafumi (1986) in a study on 15 samples.

3.12 Seeds from annual plants

Seeds of anise (Pimpinella anisum), caraway (Carumcarvi), celery (Apium graveolens L.), fennel (Foeniculumvulgare), and fenugreek (Trigonella foenum-graecum) areharvested and then dried on carpets or trays, which keepstheir spore counts relatively low.In celery seeds, Julseth and Deibel (1974) detected max-

imum bacterial spore concentration of 5.9 log spores pergram. These concentrations are consistent with the meanconcentrations of mesophilic aerobic spores quantified byWitkowska et al. (2011) in anise seeds (5.6 log spores per


gram), celery seeds (4.7 log spores per gram), and fen-nel seeds (4.1 log spores per gram). Krishnaswamy et al.(1974) and Debs-Louka et al. (2013) detected lower concen-trations of mesophilic aerobic spores in fenugreek seeds(3.6 log spores per gram), fennel seeds (2.8 log spores pergram), and caraway seeds (2.8 log spores per gram). How-ever, Kneifel and Berger (1994) observed higher concen-trations of mesophilic aerobic spores in caraway seeds,with mean concentrations of 4.6 log spores per gram inseeds and 4 log spores per gram in powders, and a lowerconcentration in aniseed (2.7 log spores per gram). In thegenus Bacillus, only B. cereus was sought and quantifiedin these spices. Concentration of 5 log of B. cereus sporesper gramwas found in anise and fenugreek seeds and 3 logspores per gram in celery seeds (Banerjee & Sarkar, 2003;Pafumi, 1986). In 10 samples of Indian celery seeds, JulsethandDeibel (1974) detectedmaximumClostridia concentra-tions of up to 4.5 log spores per gram. In all seeds fromannual aniseed plants from India or U.S. retail, C. perfrin-gens sporeswere quantified from0.6 to 2 log spore per gram(Banerjee & Sarkar, 2003; Lee & Labbé, 2018)

3.13 Mixed spices

It is not surprising that among the great diversity of spicemixtures, different levels of contaminant prevalence havebeen detected.In 161 samples of dried mixed spices, Little, Omotoye,

andMitchell (2003) detected 61.5%with aerobicmesophilicspores including 17% with B. cereus spores. Hampikyanet al. (2009) detected 11.6% contamination in 60 samplesof Turkish mixed spice samples. Giaccone et al. (1996),however, did not detect B. cereus in any of 20 spice blendsthey studied. Fogele et al. (2003) detected B. cereus sporesin each of four samples of curry. B. licheniformis is aubiquitous bacterium producing proteolytic and lipolyticenzymes. Its presence in spice mixtures and its devel-opment can lead to significant food spoilage. Giacconeet al. (1996) found a 45% prevalence of this species in 20samples of dried spice mix. In these samples, the preva-lence of other Bacillus species was also assessed: B. amy-loliquefaciens (5%), B. pumilus (5%), and B. subtilis (35%).Other highly heat-resistant spore-forming bacteria capa-ble of food spoilage include G. stearothermophilus, whichhad a prevalence of 30%. Studies have also sought to detectClostridia, particularly C. perfringens, in spice mixtures.Fujisawa et al. (2001) detected Clostridia in 62% of 60 sam-ples of curry roux imported into Japan, including 12% withC. perfringens. These authors quantified a 33% prevalenceof C. perfringens in curry on the Japanese retail market.In contrast, Stankovic et al. (2006) found no Clostridia ina study of seven samples. Bacterial spore concentrations

have also been quantified in Indian spice mixtures suchas curry, tandoori, sambar, and rasam, as well as in unde-fined spice mixtures. Krishnaswamy et al. (1974) observedmesophilic aerobic spore concentrations from 5.5 to 5.8 logspores per gram. Kneifel and Berger (1994) measured 6.3log spores per gram in curry and 5.4 log spores per gramin tandoori. In undefined spice mixtures, very high con-centrations of mesophilic aerobic spores were observed,with maximum concentrations up to 7 log spores per gram(Little et al., 2003) and a mean of 5.2 log spores per gram(Kneifel & Berger, 1994). Maximum concentrations of B.cereus observed in curry ranged from 2.8 log spores pergram (Fogele et al., 2018) to 4.4 log spores per gram (Antai,1988). In other spice mixtures, maximum spore concen-trations of B. cereus ranged from 1.7 log spores per gram(Hampikyan et al., 2009) to 5 log spores per gram (Littleet al., 2003). B. licheniformis was quantified at 6 log sporesper gram (Giaccone et al., 1996) and B. subtilis at 6.7 logspores per gram (Palumbo et al., 1975) and 5 log sporesper gram (te Giffel, Beumer, Leijendekkers, & Rombouts„1996). C. perfringens spores were detected at low concen-trations of 0.6 to 2.4 log spores per gram in curry, 1.4 logspores per gram in rasam, and 1.7 log spores per gram insambar (Krishnaswamy et al., 1974; Lee & Labbé, 2018). Inspice mixtures, Boer et al. (1985) found that C. perfringensspore concentrations of 3 log spores per gram, but Banerjeeand Sarkar (2003) did not detect any spores of this speciesin six samples of Indian mixed spices.

3.14 Dried herbs and aromaticvegetables

Overall, dried aromatic herbs show low bacterial sporeconcentrations, with maximum aerobic mesophilic sporeconcentrations of up to 5.8 log spores per gram. Herbssuch as parsley (Petroselinum crispum), thyme (Thymusvulgaris), sage (Salvia officinalis), and basil (Ocimumbasilicum) are harvested in dry weather by mowingemploying a cutter bar (Fraser & Whish, 1997). Bacterialspore contaminations come from soil, as the leaves, cantrap dust and soil particles. Plants are therefore washedbefore being dried. For all aromatic herbs, the dryingneeds to be done quickly so as to retain the herbs’ colorsby heat inhibition of enzymes. However, to avoid removingthe aromatic compounds, drying temperatures must notexceed 40 ◦C (Deans & Svoboda, 1992; Fraser & Whish,1997; Rocha, Lebert, & Marty-Audouin, 1993). These con-straints lead to the use of mechanical dryers with a heatingsystem to control the drying temperature. This processlimits contamination of dried herbs by bacterial spores.Production of aromatic plants with limited soil contact,a washing stage, and the use of a mechanical dryer all


contribute to explaining the low levels of contamination ofdried herbs by bacterial spores. Prevalence data are scarcefor herbs. In mixtures of dried herbs, Sagoo et al. (2009)detected the presence of B. cereus (34% of 23 samples) andC. perfringens (4.3% of 23 samples) at high concentrations.The presence of C. perfringens was detected in 53% of 15oregano samples, between 2% of 60 (Rodríguez-Romoet al., 1998) and 12% of 16 bay leaf samples (Power et al.,1975), and in 46.8% of 79 saffron samples (Cosano et al.,2009).Dehydrated herbs therefore appear to be less contami-

nated than strong or aromatic spices.However, the concen-trations show great variability among the aromatic herbsstudied: from 2.6 aerobic mesophilic spores per gram inIrish parsley sample (Witkowska et al., 2011) to 5.8 ina Lebanese rosemary sample (Debs-Louka et al., 2013).Wójcik-Stopczyńska, Jakubowska, and Reichelt (2009)quantified thermophilic aerobic spores in different Pol-ish herb samples (Basil, Oregano, Thyme, Tarragon, andSavory). Mean thermophilic aerobic spore concentrationsranged from 2.8 to 4.5 log spores per gram. Mean aer-obic mesophilic spore concentrations quantified in basilsamples ranged from 3.6 to 5 log spores depending onsample origin (Kneifel & Berger, 1994; Witkowska et al.,2011; Wójcik-Stopczyńska et al., 2009) and mean aerobicthermophilic spore concentration was 4.4 log spores pergram. In an imported sample, Fogele et al. (2018) quan-tified B. cereus mean concentration at 3 log spores pergram. In bay leaves, the mean mesophilic aerobic bacte-ria spore concentration was found to be 4 log spores pergram (Witkowska et al., 2011) with B. cereus concentra-tions of 2.4 log spores per gram (Wójcik-Stopczyńska et al.,2009). Powers et al. (1975) detected C. perfringens sporeswith a maximum concentration of 2.7 log spores per gramin 16 samples. In dill, concentrations of mesophilic aero-bic spores were found to be low, with mean concentrationvalues from 2.7 to 3.1 (Kneifel & Berger, 1994; Witkowskaet al., 2011) with a B. cereus concentration of 3 log sporesper gram (Aksu et al., 2000). In Mint, mesophilic aero-bic spore concentrations ranged from 2.8 to 3.1 log sporesper gram (Kneifel & Berger, 1994; Debs-Louka et al., 2013)and B. cereus concentration was 4 log spores per gram(Aksu et al., 2000; Pafumi, 1986). Pafumi also detectedmaximum C. perfringens concentrations of 4 log sporesper gram. Many authors have investigated and quanti-fied bacterial spores in oregano. In this herb, Julseth andDeibel (1974) quantified a total spore concentration of 4.9log spores per gram. Mesophilic aerobic spore concentra-tions ranged from 3.2 to 4.9 log spores per gram depend-ing on the origin of the samples (Debs-Louka et al., 2013;Kneifel & Berger, 1994; Witkowska et al., 2011; Wójcik-Stopczyńska et al., 2010), and thermophilic aerobic sporeconcentrations ranged from 3.8 to 4.5 log spores per gram

(Wójcik-Stopczyńska et al., 2009). B. cereusmaximum con-centrations of 5 log spores per gram and 3.6 log spores pergram were quantified by Pafumi (1986) and Powers et al.(1976), respectively. In oregano, these authors reported thatsignificant concentrations of C. perfringens from 3.5 to 4log spores per gram. These data agree with the Clostridiaconcentration (3.7 log spores per gram) observed for thisdehydrated herb by Julseth and Deibel (1974). In parsley,concentrations of mesophilic aerobic spores are low, withmean values of 2.6 log spores per gram (Witkowska et al.,2011) and a B. cereus concentration of 3 log spores pergram (Aksu et al., 2000; Pafumi, 1986). Pafumi (1986) didnot detect any C. perfringens spores in six Australian sam-ples of imported origin. Rosemary is the dried herb withthe highest concentrations of aerobic mesophilic spores,with concentrations ranging from 2.4 to 5.8 log spores pergram (Baxter & Holzapfel, 1982; Debs-Louka et al., 2013;Kneifel & Berger, 1994; Witkowska et al., 2011). Fogeleet al. (2018) detected low concentrations of B. cereus (2 logspores per gram). Julseth andDeibel (1974) observed a highClostridia concentration of 5.2 log spores per gram. In saf-fron, Kneifel and Berger (1994) quantified concentrationsof mesophilic aerobic spores at 3.9 log spores per gram.Cosano et al. (2009) quantified maximum B. cereus con-centration at 2 log spores per gram in 79 samples and C.perfringens spores in 47% of these, with one sample reach-ing 2 log spores per gram. In savory and sage, mean con-centrations of mesophilic aerobic spores were quantifiedfrom 3.2 to 3.6 log spores per gram (Witkowska et al., 2011;Wójcik-Stopczyńska et al., 2009).In Savory, mean concentrations of 2.8 log thermophilic

spores were quantified by Wójcik-Stopczyńska et al.(2009). In savory and sage, De Boer et al. (1985) and Aguil-era et al. (2005) quantified concentrations of C. perfringensat 3 log spores per gram. In tarragon, the concentrationof mesophilic aerobic bacteria was 4 log spores per gramand the concentration of thermophilic aerobic bacteriawas3.2 log spores per gram (Wójcik-Stopczyńska et al., 2009).Thyme contains significant concentrations of mesophilicaerobic spores, from 2.6 to 5.1 log spores per gram (Bax-ter & Holzapfel, 1982; Debs-Louka et al., 2013; Kneifel &Berger, 1994; Witkowska et al., 2011; Wójcik-Stopczyńskaet al., 2009), and a thermophilic aerobic spore concen-tration of 3.6 log spores per gram (Wójcik-Stopczyńskaet al., 2009). B. cereus concentrations ranged from 2.2 to4 log spores per gram (Antai, 1988; Fogele et al., 2018).Lee and Labbé (2018) detected 3.6 log spores per gramC. perfringens in one U.S. retail market sample. As aro-matic vegetables, garlic and onions can also be consideredas spices. These aromatic plants contain spore concentra-tions similar to aromatic herbs. In garlic, mesophilic aero-bic spore concentrations have been found to range from 2.7to 5.1 spores per gram (Kłębukowska et al., 2015; Kneifel &


Berger, 1994; Witkowska et al., 2011), with B. cereus sporesranging from absence to 4 log spores per gram (Baner-jee & Sarkar, 2003; Pafumi, 1986; Powers et al., 1976). Leeand Labbé (2018) and Pafumi (1986) detected C. perfrin-gens concentrations from 1 to 3 log spores per gram in garlicpowder. However, Banerjee and Sarkar (2003) and Powers(1975) did not detect this species in 29 studied samples. Inonion powders, mesophilic aerobic spore concentrationsvaried among studies, ranging from 2.3 log spores per gram(Witkowska et al., 2011) to 4.5 log spores per gram (Bax-ter & Holzapfel, 1982). B. cereus spores were quantified atconcentrations of 5 log spores per gram (Pafumi, 1986). Leeand Labbé (2018) quantified up to 1.2 log spores of C. per-fringens in U.S. retail market samples, but Pafumi (1986)did not detect this species in 13 studied samples. In dehy-drated herbmixtures andmixed “Provence” herbs, aerobicmesophilic spores were quantified at mean concentrationsof 4.6 log spores per gram (Kneifel & Berger, 1994) and B.cereus and C. perfringens at 4 log spores per gram (Pafumi,1986).

4 DECONTAMINATIONMETHODSFOR SPICES ANDHERBS

As mentioned earlier, the use and consumption of spicesis constantly increasing. The food industry uses them toimprove the sensory characteristics of dishes and sauces.The presence of bacterial spores in the spices makes thema source of contamination, which can lead to the deteriora-tion of foods, thus reducing their shelf life. Bacterial sporescan resist thermal processes applied to them such as pas-teurization and, in the case of thermophilic species, steril-ization treatments (André et al., 2013). It therefore appearsnecessary to remove these bacterial spores or to reducetheir concentrations in the spices before their use. Treat-ments applied for such decontamination use mainly phys-ical methods such as irradiation, steam, microwave, andchemical fumigation methods (Brodowska, Śmigielski, &Nowak, 2014; Fine & Gervais, 2007).

4.1 Irradiation

Irradiation is a decontamination method that can beapplied effectively to all spices such as black and whitepepper, cinnamon, nutmeg, cumin seeds, and aromaticherbs such as parsley and basil oregano sage onion pow-der (Calucci et al., 2003; Duncan et al., 2017; Emam, Farag,& Aziz, 1995; Sádecká, 2007). This technique causes littlesensory modification because few volatile compounds areeliminated. From a nutritional point of view though, cer-tain compounds such as ascorbate and carotenoids are oxi-

dized (Calucci et al., 2003). Irradiation is a method thatuses gamma rays, X-rays, or electron fluxes to alter theRNA or DNA of microorganisms, inhibiting their replica-tions and increasing food storage time (Farkas &Mohácsi-Farkas, 2011). This cold method was recognized as safeby the FAO/WHO Codex Alimentarius Commission in1980 (Farkas & Mohácsi-Farkas, 2011; Sádecká, 2007) andby 2005 spices and dry herbs accounted for 46% of allirradiated products worldwide (Kume, Furuta, Todoriki,Uenoyama, & Kobayashi, 2009). In Europe, the maximumallowed dose of irradiation to treat spices is 10 kGy (EC1999). However, for dry products such as spices, as dosesabove 10 kGy do not affect their sensory qualities, treat-ments up to 30 kGy can be applied. This level is used forpepper and is the FDAmaximum limit (Morehouse, 2002).The resistance of microorganisms to ionizing treatments isquantified by the parameter D10, which is the dose lead-ing to a decimal reduction of the population (Van Ger-wen, Rombouts, Van’t Riet, & Zwietering, 1999). This valuedepends on thewater content of the cytoplasm and the sizeof the chromosomal DNA (Grecz, Al-Harithy, & Jaw, 1986).In 1999, Van Gerwen et al. published a data analysis of D10values for bacterial vegetative cells and spores under dif-ferent conditions. For vegetative cells of Salmonella, Lis-teria, and E. coli, resistances are better at low water activ-ity levels, as one finds in spices. The average D10 value isclose to 0.6 kGy. Therefore, a dose of 10 kGy would cause16 decimal reductions, which would totally eliminate thevegetative cells from the spice. However, bacterial sporeshave D10 values from 2 kGy up to 5 kGy for species such asG. stearothermophilus and C. sporogenes. Grecz et al. (1986)determined D10 values from 1.8 to 2.1 kGy for spores in anumber of different spices: black pepper, thyme, and seedsof anise, cumin, and cardamom. These values thereforeindicate that a treatment of 10 kGy would result in twoto five decimal reductions of the population of such bac-terial spores in spices. Although this technique of inactiva-tion seems effective for dry products such as spices, ionizedproducts are not well accepted by consumers (Eustice &Bruhn, 2013). According to European regulations, the pres-ence of ionized spices in a food must be indicated on thelabeling by a logo. French manufacturers therefore do notuse ionized spices but steam-decontaminated ones instead.

4.2 Steam treatment

Steam treatment is a technique widely used in Europe forthe decontamination of all spices, but the process is lessusable for herbs because it strongly diminishes aromasand essential oils (Schweiggert, Carle, & Schieber, 2007).The use of steam in treatments of long duration also affectsthe sensory characteristics of spices. It blackens pepper


and greatly reduces the concentration of piperine presentafter processing and storage (Waje, Kim, Kim, Todoriki,& Kwon, 2008). In red pepper, Rico et al. (2010) did notobserve a decrease in concentration of capsium, butobserved a reduction in color. These authors determinedthat the destruction of bacterial spores by heat treatment(100 ◦C for 16 min) is less effective than irradiation. Othermethods using steam have also been developed. Thevacuum-steam-vacuum (VSV) process consists in treatingdry products and spices by injecting steam from 120 to 140◦C for short durations from 10 to 20 s. This method resultsin four decimal reductions of bacterial spores (B. subtilis)without affecting pepper quality (Lilie, Hein, Wilhelm, &Mueller, 2007). This technique is now used industrially(Napasol TM) to decontaminate spices and appears to bean interesting alternative to the use of irradiation.Another technological process that effectively inhibits

bacterial cells is steam combinedwith CO2 under pressure.However, for bacterial spores, the addition of CO does notimprove the effectiveness of steam treatment (Brodowskaet al., 2014; Fine & Gervais, 2007).

4.3 Microwaves

Microwaves or high frequencies are new decontamina-tion methods for inactivating bacterial spores in spicessuch as black pepper, white pepper, chili pepper, cin-namon, cloves, fenugreek, ginger, turmeric, and oregano(Dababneh, 2013). This last author had studied the effectof microwaves (2450 MHz at 900 W) on the decontamina-tion of spices and herbs. The values of the decimal reduc-tion times determined in spices varied from 10 to 20 s formesophilic and thermophilic bacterial spores, resulting ina 60 s treatment of six to three decimal reductions. Emamet al. (1995) also observed good inhibition of spores of B.subtilis, B. megaterium, and Clostridium sp. in black pep-per powder. However, at the industrial scale, microwaveheating treatments are very heterogeneous as a result ofthe variable penetration of microwaves and low humidityof bacterial spores (Fine & Gervais, 2007). Moreover, thisprocess allows large amounts of spice essential oils to belost (Brodowska et al., 2014).

4.4 Fumigation

Fumigation with ethylene oxide, propyl oxide, and methylbromide has long been used to decontaminate all kinds ofspices. However, since the 1980s, its use has been ques-tioned due to the persistence of mutagenic and carcino-genic compounds in the spices treated. This process is nowtotally prohibited in Europe (European Spice Association,

2015). In addition, its effectiveness in inactivating bacterialspores is limited (Russel, 1990) and it affects the sensoryqualities of spices. Furthermore, the effect of ozone fumi-gation against bacterial spores is not significant for a wateractivity less than or equal to 0.5, which corresponds to thewater activity of spices at 10% humidity (Ishizaki, Shinriki,& Matsuyama, 1986).

4.5 Alternative methods

Cold plasma treatment is a novel technology for decon-tamination of foods such as spices (Bhatt, Prasad, Joshi, &Sagarika, 2018). The microbial decontamination effect of“microwave-powered cold plasma treatment” (WCPT) hasbeen studied on black pepper, oregano, paprika powder,red pepper, and onion powder (Hertwig et al., 2015; Kim,Lee, & Min, 2014; Kim, Oh, Won, Lee, & Min, 2017). Coldplasma technology generates a plasma by excitation of agas with ultraviolet production, photons, electron radicals,reactive oxygen species, and atoms at a temperature below50 ◦C. It inhibits microorganisms by destroying the DNAof vegetative cells or spores (Moisan et al., 2002). However,the use of CPT to inhibit bacterial spores in spices is limitedby the nonuniformity of the treatment and the geometricstructure of the powder, which protects spores from radia-tion (Kim et al., 2014).Although pulsed light is effective for removing bacte-

rial spores on surfaces, this technology is unsuitable fortreating bacterial spores in powders such as spices becausethey would not be completely exposed to the radiation dur-ing treatment. The same observation was made for theinfrared treatments. The radiation does not penetrate toreach the inner spice layers, so this process is limited tosurface treatment (Erdogdu, Eliasson, Erdogdu, Isaksson,& Ahrné, 2015).High pressure is another alternative method for remov-

ing microorganisms, but is totally ineffective at inactivat-ing bacterial spores in powders or spices (Brodowska et al.,2014).It seems that few methods can be used industrially to

inactivate bacterial spores in spices, and only irradiationand the VSV technique appear useful and can be appliedin this context.

5 CONCLUSION

The high level of spice contamination by bacterial sporesis an important problem for the food industry. The stud-ies cited in this review show high prevalence of bacte-rial spores, especially in spices such as black pepper andspice blends. High spore concentrations are observed in


highly used spices such as pepper and turmeric. It appearsthat traditional methods, which may lead to contamina-tion, are still widely used in small farms in the main spiceproducing countries of the developing world. Methods ofspore decontamination suitable for spices are very limiteddue to their low applicability or the altering effects theyhave on spice quality. It is therefore important to knowabout bacterial spore contamination in spices and reducepossible contamination during harvesting and posthar-vest treatment and processes by limiting contact with thesoil, using dryers, and respecting the rules of hygieneand cleaning. The adoption of these steps will involve thedevelopment and dissemination of simple and low-costtechnologies to replace traditional methods and improvebasic hygiene practice in producer countries. This controlover the microbiological quality of spices before their salewould limit health risks, costs, and losses associated withalterations to food during storage, while lengthening foodshelf life. This review highlights how the data are insuffi-cient for the application of predictivemicrobiology tools orrisk assessments for pathogenic of spoilage-related spore-forming bacteria. The published data are patchy, particu-larly those concerning spore-forming bacteria responsiblefor spoilage. Through this study, it appears that there areno studies describing the complete spore-forming bacteriamicrobiota present in spices.

AUTHOR CONTRIBUTIONS

Anne Gabrielle Mathot: bibliographical research, dataanalysis, and writing of the manuscript.Florence Postollec: bibliographical research and writing

of the manuscript.Ivan Leguerinel: bibliographical research, data analysis,

and writing of the manuscript.

CONFL ICT OF INTERESTThe authors declare no conflict of interest

ORCIDIvanLeguerinel https://orcid.org/0000-0002-6525-9270

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How to cite this article: Mathot AG, Postollec F,Leguerinel I. Bacterial spores in spices and driedherbs: the risks for processed food. Compr Rev FoodSci Food Saf. 2021;20:840–862.https://doi.org/10.1111/1541-4337.12690

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