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M. A. Nor Azni and Q. L. YeohJ. Trop. Agric. and Fd. Sc. 26(1)(1998): 79–84

The effects of chemical preservatives on the microbial populationand shelf-life of chilli slurry(Kesan bahan pengawet kimia terhadap populasi mikrob dan tempoh simpan cili bo)

M. A. Nor Azni* and Q. L. Yeoh*

Key words: chilli slurry, cili bo, chemical preservatives, microbiology

AbstrakKesan natrium benzoat, kalium sorbat, natrium laktat dan kalsium propionatterhadap tempoh simpan cili bo pada suhu bilik telah dikaji. Hasil daripada kajianmenunjukkan bahawa penggunaan natrium benzoat dan kalium sorbat dengantambahan asid asetik dapat memanjangkan masa simpan cili bo sehingga 10minggu pada suhu bilik. Dengan penggunaan natrium laktat dan kalsiumpropionat, tempoh simpan cili bo masing-masing hanya 4 minggu dan 2 hari.Semasa penyimpanan, perubahan kandungan mikroorganisma dalam sampel yangmengandungi natrium benzoat + asid asetik dan kalium sorbat + asid asetik jugadiawasi. Jumlah hitungan plat bagi kedua-dua sampel tidak banyak berubah.

AbstractThe effects of sodium benzoate, potassium sorbate, sodium lactate and calciumpropionate on the shelf-life of chilli slurry stored at room temperature wereinvestigated. The results indicated that the use of sodium benzoate and potassiumsorbate with the addition of acetic acid was able to preserve the chilli slurry up to10 weeks at room temperature, while sodium lactate and calcium propionate gavea storage life of 4 weeks and 2 days respectively. The changes in microbiologicalpopulation during the storage period were also monitored for the samples treatedwith benzoate + acetic acid and sorbate + acetic acid. There were no markedchanges in the total plate counts for both treatments.

*Food Technology Centre, MARDI Headquarters, P.O. Box 12301, 50774 Kuala Lumpur, MalaysiaAuthors’ full names: Nor Azni Md. Adnan and Yeoh Quee Lan©Malaysian Agricultural Research and Development Institute 1998

IntroductionChilli slurry, a food ingredient widely usedin Malaysian households and food outlets, isprepared from dried chillies which havebeen soaked in water and ground into a finepaste (Sharifah Norhidayat and Abu Othmann.d.). It is a popular ready-to-use ingredientthat makes cooking less tedious.

According to the Food Regulations1985, “Chilli slurry or commonly known ascili bo shall be the slurry obtained bygrinding the clean, wholesome, fresh ordried chillies with clean potable water. It

shall contain not less than 15% of chilli. Itmay contain salt and permitted preservative.It shall not contain any other addedsubstance” (Regulation 299). The maximumpermitted level of sodium benzoate allowedin cili bo is 750 mg/kg (Regulation 20).

Cili bo has a short shelf-life at normalroom temperature. Its moist nature providesfavourable conditions for growth ofmicroorganisms and thus it is susceptible tomicrobial spoilage. Although its shelf-lifemay be prolonged with preservationtechniques such as refrigeration and

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freezing, these techniques are costly andsometimes not available, especially inremote areas. Another technique forpreserving cili bo is the use of chemicalpreservatives.

Generally, bacteria grow best at pH6.5–7.5 while yeasts and moulds which havea wider pH range, can grow in foods withpH of less than 3.5. One of the effectiveways of controlling microbial growth is,therefore, by pH control. A number oforganic acids can be used as antimicrobialsor acidulants. The main organic acids usedas antimicrobials are benzoic, propionic andsorbic acids, and their salts, besides acetic,lactic, malic, citric and tartaric acids. Allthese acids have “Generally Regarded AsSafe” (GRAS) status if used at levels below1 000 ppm (Giese 1994), and are moreeffective at low pH when they are in theirundissociated forms (Doores 1983).

Currently, benzoic acid is the onlychemical preservative permitted for use incili bo under the Food Regulations. Benzoicacid is generally more effective as anantimycotic agent and it is only effective inits undissociated form. Thus, benzoic acidand sodium benzoate are more suitable forfoods with pH 4.5 and below (Chipley1983). They are effective against yeasts andmoulds at levels of 500–1 000 ppm butconcentrations greater than 2 000 ppm arerequired to be effective against spoilagebacteria (Chipley 1983).

Like benzoic acid, sorbic acid is notvery soluble in water at room temperature,and the sodium, potassium or calcium saltsare more commonly used as preservatives.They are more effective against yeasts andfungi, and have a selective action againstcertain bacteria. The pH-dependant sorbateswhich are effective up to pH 6.5 (Sofos andBusta 1983), have been used at 500–3 000ppm to prevent microbial growth in differenttypes of food (Giese 1994).

The antimicrobial action of propionatesis mainly directed towards fungi though theyalso retard the growth of certain bacteria,especially those causing rope spoilage in

bakery products. Generally, they have anupper effective pH limit of 5.5. Levels usedin foods range from 1 000 ppm to more than3 000 ppm (Doores 1983). Currently underthe Food Regulations 1985, propionic acidand its salts are only allowed in bread.

Lactic acid has been well accepted as anaturally occurring preservative, especiallyin fermented foods such as pickles andyoghurts. It lowers pH and displaysinhibitory properties against spore-formingbacteria at pH 5.0 and against psychrotrophicgram negative bacteria, especially in meat(Daeschel 1989). In recent years, there hasbeen increased interest in the use of sodiumlactate as a preservative. It is reported to beeffective against various Bacillus spp., andcan reduce or delay the growth of aerobes,psychrotrophs and coliforms in variousproducts stored at refrigeration temperatures.It is normally used as a 60% solution at2–3% (Schefel 1994).

Acetic acid has been found to be moreeffective against yeast and bacterial growththan mould growth (Doores 1983). Althoughit has a strong odour and taste, acetic acid iscommonly used as an acidulant as it isinexpensive.

This paper compares the effectivenessof some GRAS chemical preservatives forextending the shelf-life of cili bo at roomtemperature.

Materials and methodsPreparation of cili boDried chillies were used in the preparationof the cili bo (Figure 1). It was preparedaccording to the procedure outlined bySharifah and Abu Othman (n.d.).

TreatmentsFour food grade chemical preservatives,namely sodium benzoate, calciumpropionate, sodium lactate and potassiumsorbate, were evaluated for the preservationof cili bo. The preservatives were usedindividually and with addition of glacialacetic acid to reduce the pH to 4.1–4.3. Thepreservatives were added at 750 mg/kg. The

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Specifications of Food (ICMSF 1978a). Tengram sample of cili bo was added to 90 mLquarter-strength Ringer’s solution, whichgave a 10-1 dilution. The mixture was thenblended using the Stomacher Lab-blender400 for 2 min. Further 10-fold serialdilutions, ranging from 10-2 to 10-6 wereprepared from the 10-1 homogenate using9 mL quarter-strength Ringer’s solution asdiluent. Total plate, spore-former and mouldcounts were determined according to themethods described by the ICMSF (1978a)and Speck (1976).

pH determinationThe pH of cili bo was determined using theOrion pH meter.

Water activity determinationThe water activity (aw) of cili bo wasdetermined using the Rotronic water activitymeter.

Titratable acidityTitratable acidity was determined accordingto Speck’s method (1976).

Results and discussionGas production and slime formation in thesamples were taken as indicators ofspoilage. Visual observation showed that thecontrol sample, which did not contain anypreservatives and had an average pH of5.67, spoiled after 24 h of storage at roomtemperature. The pH of cili bo samples withthe addition of only benzoate, sorbate,propionate and lactate was 5.18–5.93(Figure 2). All samples spoiled after 48 h atroom temperature. The rapid spoilage wasprobably due to the high pH values of thecili bo samples which permitted microbialgrowth that consequently caused spoilage.Spoilage was mainly due to bacteria as noyeast or mould growth was detected in allsamples analysed.

At the pH range of the above samples,none of the preservatives tested waseffective in preserving cili bo at roomtemperature. This was because the pH values

products were packed into plastic cups withlids in 80 g lots, similar to those used forcommercial sale, and stored at roomtemperature. The experiments werereplicated and the results are the meanaverage of duplicate samples.

SamplingAll the samples for each experiment wereprepared as one lot. As such, it was assumedthat the chilli samples were homogenous andthe microbial load was uniform among thesamples. Thus, less samples are needed thanfor non-homogeneous samples (ICMSF1978a). At each storage interval of oneweek, the samples to be examined wererandomly selected and the contentsthoroughly mixed aseptically before the sub-samples for examination were asepticallytaken. One of the ways to ensure that themicrobiological analyses would moreaccurately estimate the true microbiologicalstatus of the products is to homogenise alarger sample prior to taking sub-samples forexamination (ICMSF 1978b; Brown andBaird-Parker 1982). This method was,therefore, used to ensure that the samplesused were representative.

Microbiological analysesThe samples for analysis were preparedbased on the methods recommended by theInternational Commission on Microbiological

Dried chillies

Sort and remove stem

Weigh and wash

Soak in hot water for 10 min

Drain and grind with a little water till fine

Add more water, acetic acid, preservative and salt

Mix well and pack into containers

Cili bo

Figure 1. Production of cili bo

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Effects of chemical preservatives on the shelf-life of chilli slurry

The incorporation of acetic acidtogether with the chemical preservativesshowed a positive effect on extending theshelf-life of cili bo at room temperature.Samples treated with benzoate + acetic acidand sorbate + acetic acid lasted for morethan 10 weeks. While for lactate andpropionate with the addition of acetic acid,the sample shelf-life was 4 weeks and 2days respectively. With the addition of aceticacid, the pH of samples containing benzoateor sorbate were reduced to 4.12–4.17(Figure 2). At this pH range, the benzoateand sorbate were found to be effective inpreserving the cili bo. However, this was notso for samples with lactate and propionatewhich had a higher pH range of 4.22–4.32(Figure 2). Furthermore, much higher levelsof both propionate and lactate are required

Figure 2. Water activity, titratable acidity and pH of cili bo samples treated with various preservativesand acetic acid

were not within the effective pH range formaximum activity of the preservativestested. These preservatives are effective atlower pH values when they are in theirundissociated forms (Chipley 1983; Doores1983; Sofos and Busta 1983; Daeschel1989).

The addition of acetic acid alone toreduce the pH to below pH 4.5 (about4.1–4.2) was effective in giving a slightlylonger shelf-life at room temperature (2–3days) as this lowered the pH range to belowthat for the optimum growth for bacteria,thus delaying spoilage. However, furtheraddition of acid to lower the pH gave aproduct that was too sour and was notacceptable to the taste panel. Thus, the useof acetic acid alone is not suitable forprolonging the shelf-life of cili bo.

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Figure 3. Changes in totalviable counts and spore-formercounts during the storage ofcili bo samples treated withbenzoate + acetic acid andsorbate + acetic acid

to be effective against the spoilageorganisms (Giese 1994).

The microbiological analysis of the cilibo samples treated with benzoate + aceticacid as well as sorbate + acetic acid isshown in Figure 3. There was no distinctchange in the total plate count during the10-week storage period. At zero day, thetotal plate count was found to be 1.9 x 106

cfu/g (colony forming units/g) of sample.After 10 weeks of storage, the countincreased slightly to 3.0 x 106 cfu/g. In thecase of spore-formers, there was a slightdecrease in counts with an initial count of7.5 x 105 cfu/g decreasing to 2.5 x 105 cfu/gat the end of the 10-week storage period.Thus, the effect is more bacteriostatic thanbactericidal. For the mould counts, nogrowth was detected during the 10-weekstorage period. A similar trend was observedin the samples treated with sorbate + aceticacid. There was no distinct change in thetotal plate count, which had an initial countof 1.2 x 106 cfu/g and a final count of 2.2 x106 cfu/g. For the spore-formers, there was aslight decrease in the count from 6.2 x105 to2.7 x 105 cfu/g (Figure 3). Moulds were alsonot detected during the storage period. Assorbate is effective against selected bacteria,the preservative action against bacterialspoilage is probably due to the combined

effect of the preservative and the pHlowering effect.

These results showed that with theaddition of benzoate + acetic acid or sorbate+ acetic acid, there was no marked increasein the microbial population in the cili bosamples during the 10-week storage periodat room temperature. The pH of the samplesremained relatively constant throughout thestudy. The pH was in the range of 4.11–4.16for samples treated with benzoate + aceticacid and 4.17–4.20 for samples with sorbate+ acetic acid. As mentioned earlier, these pHvalues are within the effective pH range formaximum activity. This probably resulted inthe inhibition of microbial development inthe cili bo although the water activity valuesin both cases were in the range of 0.92–0.94throughout the storage period and werefavourable for growth of microorganisms.Titratable acidity in both cases alsoremained fairly constant throughout thestudy, ranging from 0.60% to 0.66% aceticacid.

ConclusionFrom the experiments conducted, it can beseen that the addition of 750 ppm benzoateor sorbate in the presence of acetic acid (pHrange: 4.1–4.2) has more potential inpreserving cili bo stored at room temperature

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than lactate and propionate or acetic acidalone. However, as sorbate is currently notapproved for use in cili bo under the FoodRegulations, the problem of cili bo spoilagecan be effectively overcome by using aceticacid to lower the pH to the effective rangefor benzoate and adding the permissibleamount of benzoate. In view of the fact thatsorbate is also effective in prolonging theshelf-life of cili bo and is effective to ahigher pH level (pH 6.5 compared to pH4.5), it may be appropriate to petition for itsinclusion in the Food Regulations as analternative to benzoate.

AcknowledgementsThe authors wish to thank Ms JosephineMary, Ms Fatimah Hassan and Ms NorrijahOsman for their assistance.

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microbiological examination of meat. In Meatmicrobiology (Brown, M. H., ed.) p. 423–521.London and New York: Applied SciencePublishers Ltd.

Chipley, J. R. (1983). Sodium benzoate and benzoicacid. In Antimicrobials in foods (Branen, A.L. and Davidson, P. P., ed.) p. 11–35. NewYork: Marcel Dekker Inc.

Daeschel, M. A. (1989). Antimicrobial substancesfrom lactic acid bacteria for use as foodpreservatives. Food Technol. 3(1): 164–7

Doores, S. (1983). Organic acids, p. 75–108. SeeChipley (1983)

Giese, J. (1994). Antimicrobials: Assuring foodsafety. Food Technol. June: 102–10

International Commission on MicrobiologicalSpecifications of Food (1978a).Microorganisms in foods: Their significanceand methods of enumeration (Thatcher, F. S.and Clark, D. S., ed.) 2nd ed., p. 52–63.Toronto: University of Toronto Press

–––– (1978b). Microorganisms in foods 2.Sampling for microbiological analysis:Principles and specific applications 2nd ed.p. 32–64. Toronto: University of TorontoPress

Schefel, L. A. (1994). Antimicrobial effects oflactates: A review. J. Food Protection 57:445–50

Sharifah Norhidayat, A. R. A. and Abu Othman, A.R. (n.d.). Perusahaan memproses cili boh. SiriPanduan Untuk Usahawan No. 22 7 p.Serdang: MARDI

Sofos, J. N. and Busta, F. F. (1983). Sorbates,p. 141–75. See Chipley (1983)

Speck, M. L., ed. (1976). Compendium of methodsfor the microbiological examination of foodsp. 107–130, 223, 225–8, 235–8, 568–73.Washington: American Public HealthAssociation

Accepted for publication on 16 April 1998