Quality Assurance of Stored Pepper (Piper guineense) Using Controlled Processing Methods

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2 Pakistan Jurnal f Nutritin 3 (4): , 2004 Asian Netwrk fr Scientific Infrmatin, 2004 Quality Assurance f Stred Pepper (Piper guineense) Using Cntrlled Prcessing Methds B.O. Omafuvbe* and D.O. Klawle Department f Micrbilgy, Obafemi Awlw University, Ile-Ife, Osun State, Nigeria bmafuvbe@yah.c.uk Abstract: The micrflra f black and white pepper (P. guineense) during prcessing and strage were enumerated, islated and identified. The fresh untreated pepper samples gave appreciable ttal aerbic mesphilic bacteria (TAMB) cunts f 6.65 lg cfu/g in the white pepper and 7.04 lg cfu/g in the black pepper. Clifrm cunts ranged in number frm 6.23 t 6.80 lg cfu/g while yeast and muld cunts ranged frm 2.00 t 3.74 lg cfu/g sample. The micrflra assciated with the untreated fresh pepper samples included species f Staphylcccus, Micrcccus, Bacillus, Serratia, E. cli, Aspergillus, Fusarium, Itersnilia, Btrydipldia, Penicillium, Mucr, Candida, and Brettanmyces. Pretreatments invlving steeping in biling water fr up t 20mins, surface disinfectin with 2% frmaldehyde slutin and washing reduced the micrbial lad f the pepper samples t zer r less than 1.0 lg cfu/g; this was accmpanied by the disappearance f yeast, clifrms and certain ther species f bacteria. The lw numbers were maintained during the drying perid, whether in the ven r in the sun and later als during strage f the dried pepper samples. Our findings suggest that even in the traditinal setting, simply washing and steeping, fllwed by cntrlled sun drying, culd drastically and effectively decrease the assciated micrbial ppulatins n pepper samples. The pretreatments f pepper samples described in this study kept the micrbial lad within the ICMSF acceptable limits thrughut the strage perid. This prcessing methd has therefre been shwn t be capable f elngating the shelf-life f the prduct and thus ensure the maintenance f gd quality during strage fr at least three mnths. Key wrds: Black pepper, white pepper, micrflra, fresh pepper Intrductin Piper guineense (Schum and Thnn) cmmnly referred t as African black pepper r Ashanti pepper, is very similar t Piper nigrum, which is the true pepper f cmmerce frm which 'black' and 'white' peppers are prcessed (Isawumi, 1984). Piper guineense as a plant has been fully described in literature (Hutchinsn and Dalziel, 1954). Apart frm its culinary uses, Piper guineense has been reprted t have medicinal, csmetic (Dalziel, 1955) and insecticidal (Fasakin and Aberej, 2002) prperties. White and black pepper can be prduced frm P. guineense in much the same way as frm P. nigrum. The fruit f P. guineense, called peppercrn, is the spice. 'Black' pepper is prduced frm unripe fully develped berries while 'white' pepper is frm fully ripe decrticated and dried peppercrns (Purseglve et al., 1981). Spice plants are usually harvested and prcessed prductin f black and white pepper frm P. guineense purchased fresh frm retailers and subjecting them t micrbilgical studies during strage fr a perid f three mnths. Materials and Methds Surce f samples: Fresh fruits f fully develped unripe (fr black pepper) and fully ripe (fr white pepper) P. guineense seeds were purchased frm lcal markets in Ile-Ife, Osun State, Nigeria. Prcessing f samples Black pepper: Fresh unripe berries f P. guineense were remved frm the spikes and divided int three lts. One lt was left untreated, the secnd lt was steeped in biling water fr 15min as described by Parry (1969) while the third lt was steeped in biling water and surface disinfected with 2% frmaldehyde slutin traditinally under variable sanitary cnditins in many fr 2 min as described by Christensen et al. (1967). develping cuntries like Nigeria. This gives rm fr Each f these lts was further divided int tw prtins, high levels f bacteria and fungal cntaminatin. With ne f which was subjected t sun drying while the ther the increasing awareness f hygiene and fd spilage, was dried in the ven at 40 C. The drying cntinued until Micrbilgists have acknwledged that traditinal a misture cntent f -12% was achieved (Purseglve spices and herbs can increase the bacterial levels f et al., 1981). sme fds, which may result in their deteriratin and pssibly cause fd pisning. This paper is a reprt f White pepper: White pepper was prepared frm fresh the use f imprved prcessing methds in the fruits f fully ripe Piper guineense fllwing the water 244

3 steeping and retting technique described by Pruthi pepper and 7.04 lg cfu/g in black pepper. Clifrm (1980) with slight mdificatin. Essentially, ripe berries cunts ranged frm 6.23 lg t 6.80 lg cfu/g sample were washed with tap water, packed in muslin bags and while yeast and muld cunts were generally lw, saked in a basin f water fr 7 days with daily changes ranging frm 2.00 t 3.74 lg cfu/g. The misture f the steeping water. Retted berries were de-skinned by cntent f black pepper was 64.20% while that f white rubbing with hands and washed in running water. De- pepper was 55.40%. skinned berries were divided int tw prtins, ne was Pretreatments such as steeping alne r steeping and surface-disinfected befre the lts were dried as surface disinfectin (befre drying) f fresh P. guineense described abve fr black pepper. samples reduced their micrbial lad t <1.0 lg cfu/g r even zer except fr white pepper (Table 1). Steeped Strage f samples: Fr strage, each f the dried white pepper had TAMB cunts f 2.86 lg cfu/g and sample lts was further divided int tw prtins. One clifrms cunt f 1.48 lg cfu/g n the surface, while set was grund int fine pwder using the dry grinding the hmgenate had a TAMB cunt f 3.99 lg cfu/g cmpartment f a Mulinex mixer-blender mill 2 and a clifrm cunt f 3.08 lg cfu/g sample. (sterilized by washing and rinsing with 80%ethanl Pretreatments caused a general increase in the slutin) while the ther set was left as dried whle misture cntent f black pepper (frm 64.20% in the fruits. Equal amunts f the samples were then untreated sample t 66.70% in steeped and surfacedistributed int screw-capped bttles, screwed tight and disinfected sample) while it decreased in white pepper stred fr three mnths at rm temperature. (frm 55.40% in the untreated sample t 49.50% in the steeped and surface-disinfected sample). Micrbilgical analysis: The micrbial lad n the The effect f sun-and ven-drying n the micrbial lad surface f black and white pepper samples cllected and misture cntent f pretreated and untreated P. immediately befre drying, during drying and strage guineense samples is shwn in Table 2. The desired and their respective hmgenates were estimated. Five misture cntent f pepper samples was achieved grams f each sample was aseptically transferred int within 4 t 7 days f ven-drying and 7 t 14 days f sunsterile 45ml f 0.1% peptne water diluent in a cnical drying. In general, the desired misture cntent was flask, the mixture was vigrusly shaken fr 5min and achieved earlier in white pepper than in black pepper the wash-water kept. The washed sample was samples. transferred int a sterile stmacher bag and In the untreated black pepper that was sun-dried fr 14 hmgenized in 45ml f fresh 0.1% peptne water with days, the TAMB cunts drpped frm 7.04 lg cfu/g t the aid f a Clwrth Stmacher (Mdel 400). The wash 5.11 lg cfu/g n the surface and frm 6.78 lg cfu/g water and sample hmgenates were then diluted t 3.54 lg cfu/g in the hmgenate. In cmparisn, serially and apprpriate dilutins plated ut in triplicates. untreated black pepper shwed a drp in TAMB cunts One set was plated ut n Nutrient agar (NA Oxid) and frm 7.04 lg cfu/g t 4.08 lg cfu/g n the surface and incubated at 30 C fr up t 48h fr ttal aerbic frm 6.78 lg cfu/g t 3.32 lg cfu/g in the mesphilic bacteria (TAMB), the secnd n Esin hmgenate after ven-drying fr 7 days (Table 2). The methylene blue agar (EMB), incubated at 37 C fr 24h fr micrbial lad f the black pepper samples pretreated clifrm, while the third set was plated ut n Malt by steeping alne r steeping and surface disinfectin Extract agar (cntaining 50µg streptmycin per ml) and increased slightly during ven and sun drying. On the incubated at 28 C fr up t 7 days fr yeast and muld. ther hand, the micrbial lad f white pepper drpped The bacterial islates were identified accrding t the slightly r was maintained at the lw r negligible levels schemes f Harrigan and McCance (1976) and (<1.0 lg r zer cfu/g) during drying. Buchanan and Gibbns (1974). Yeast and mulds were The micrbial lads f all dried pretreated samples identified as described by Cllins and Lyne (1970), stred as whle fruits r in pwdered frm remained Barnett and Hunter (1972) and Ldder (1971). either at zer r negligible levels thrughut the 3 mnths f strage. Untreated sun-dried whle black Misture cntent determinatin: The misture cntent pepper samples hwever still had appreciable numbers f the Samples was determined at each sampling time f micrrganisms ranging frm 4.00 t 4.48 lg cfu/g in accrdance with AOAC (1990). while the ven-dried prtins gave apprximately 3.88 lg cfu/g. Pwdered samples gave cmparable Results The micrbial lad and misture cntent f Piper guineense samples befre drying are shwn in Table 1. The ttal aerbic mesphilic bacteria cunts f bth fresh untreated pepper sample surface and their numbers. The bacteria assciated with black and white peppers during prcessing and strage are shwn in Table 3.They included Staphylcccus, Micrcccus, Bacillus, Serratia, and Escherichia. Of these bacteria species, respective hmgenates were 6.65 lg cfu/g in white nly Micrcccus and Bacillus were detected by the end 245

4 Table 1: The micrbial lad* and misture cntent f untreated and treated black and white pepper samples befre drying Treatment/analysis Pepper type Black White Surface Hmgenate Surface Hmgenate Untreated Samples: ** Misture Bacteria (TAMB) Clifrm Yeast Muld Steeped Samples: Misture a Bacteria (TAMB) Clifrm Yeast Muld Steeped and surface disinfected: Misture Bacteria (TAMB) Clifrm Yeast Muld Values are means f three determinatins. *Micrbial lad expressed as lg f clny frming unit (cfu) / g sample. **Misture cntent a f pepper fruits expressed as percentage. P. guineense fruits were retted in water in additin t steeping befre misture cntent determinatin. TAMB - Ttal aerbic mesphilic bacteria. Table 2: The micrbial lad* and misture cntent f untreated and treated black and white pepper samples during drying Pepper Pretreatment Drying Sun-drying Oven-drying Type perid (Days) Micrbial lad Micrbial lad Mc Surface Hm- Mc Surface Hmgenate genate Black Untreated ND ND ND Steeped nly <1.48 < <1.48 <1.48 ND ND ND Steeped & surface disinfected <1.48 < <1.00 < < ND ND ND b White Steeped nly 7(4) <1.00 <1.00 Steeped & surface disinfected 7(4) < <1.00 <1.00 Values are means f three determinatins. *Micrbial lad expressed as lg f cfu/g sample and represents ttal aerbic mesphilic bacteria cunt nly. Clifrm, muld and yeast were either nt detected r their cunts were nt significant. Mc - Misture cntent b expressed as percentage. ND - Nt determined since desired misture cntent was already achieved within 7 days. The desired misture cntent f white pepper was achieved within 4 and 7 days fr ven and sun drying respectively. f the drying perid and during strage f the pepper Penicillium and yeast species such as Brettanmyces samples. and Candida. Mst f the yeast and muld species The fungi islated frm white and black pepper during disappeared during the drying perid and were hence prcessing included muld species namely Aspergillus, absent frm the stred samples. Of significant nte is Fusarium, Itersnilia, Btrydipldia, Mucr and the absence f Aspergillus flavus, Fusarium slani and 246

5 Table 3: The ccurrence f bacteria islated frm white and black pepper during prcessing and strage Bacteria Islates Pepper type/treatment Black pepper White pepper Prcessing Strage Prcessing Strage Staphylcccus aureus S. epidermidis Micrcccus rseus M. luteus M. varians Bacillus cereus Bacillus subtilis B. firmus Serratia marcescens Escherichia cli Present - Absent Table 4: The ccurrence f fungi islated frm white and black pepper during prcessing and strage Fungi islates Pepper type /Treatment Black pepper White pepper Prcessing Strage Prcessing Strage Mulds Aspergillus niger A. flavus A. fumigatus A. chraceus Fusarium slani Itersnilia sp Btrydipldia thebrmae Mucr hiemalis Penicillium verrucsum Yeasts Brettanmyces claussenii B. intermedius Candida trpicalis C. guilliermndii C. lusitaniae Present - Absent Btrydipldia thebrmae in the dried stred pepper generally accmpanied with the disappearance f the samples (Table 4). clifrms and the yeasts leaving Micrcccus, Staphylcccus and Bacillus species and a variety f Discussin The micrbial cunts f fresh untreated black and white pepper in this study appear t cnfrm with earlier reprts n black and white pepper prepared frm P. nigrum in ther parts f the wrld (Krishnaswamy et al., 1971; Beckmann et al., 1996; Garcia et al., 2001). The grwing and harvesting cnditins f the pepper samples and pst-prcessing envirnmental expsure may have cntributed t their high micrbial lad. The micrbial lad f the untreated pepper samples drpped mulds dminated by Aspergillus sp. Hence the aerbic plate cunts n the dried samples still shwed the presence f a few hundred rganisms per gram f each f the samples and these were carried int strage whether as whle berries r grund spices. Pretreatments such as steeping in biling water alne r steeping and surface disinfectin f fresh pepper fruits befre drying drastically reduced their micrbial lad. This was reflected in the eliminatin f clifrms, yeasts, Staphylcccus species and Serratia sp. The with drying whether in the sun r in the ven and was lw numbers resulting frm the pretreatment 247

6 manipulatins were sustained by bth drying methds. This trend agrees bradly with earlier reprts (Christensen et al., 1967). These investigatrs reprted the reductin in micrbial lad f spices t negligible numbers fllwing treatment invlving steeping in biling water fr up t 20mins, surface disinfectin with 2% frmaldehyde and drying. Als, Andress et al. (2001) reprted a significant reductin in the aerbic micrflra f spices when preliminary treatments such as washing with water and dipping in chlrine were emplyed. These pretreatments caused a general increase in the misture cntent f black pepper but a decrease in that f white pepper samples. The black pepper samples may have absrbed misture during the steeping treatment while the decrease in white pepper may be a result f the retting and de-skinning prcess. This study shwed that drying in the ven generally achieved the required misture cntent earlier and mre unifrmly than sun-drying. This may be related t the fact that ven drying is free frm the vagaries f the weather which culd lead t a re-wetting f the samples as is the case with sun-drying. Sun-drying culd be affected by annual seasnal changes, fr example effective drying culd be achieved in nly a few days in the dry seasn while it culd take weeks during the raining seasn. In the latter case, the samples als stand the risk f further recntaminatin befre they are dry enugh t withstand micrbial grwth (Ayres et al., 1980). Once the required misture cntent f -12% was achieved hwever, bth drying methds effectively kept dwn the numbers f micrrganisms, since the level f misture essential fr micrbial grwth was nt available (Purseglve et al., 1981). The predminant micrrganisms islated during prcessing and strage included Micrcccus sp, and the spre frmers Bacillus, Aspergillus, and Penicillium species all f which are able t survive dry cnditins. These micrrganisms have been reprted in dried spices frm ther parts f the wrld (Mckee, 1995; Garcia et al., 2001; Banerjee and Sarkar, 2003). It is interesting t nte, hwever, that the number f micrrganisms islated during drying and strage falls within the acceptable ICMSF (Internatinal Cmmissin n Micrbilgical Specificatin fr Fds) level (> 6 cfu/g) reprted by Banerjee and Sarkar (2003). Als, the numbers are nt sufficient t cause the spilage f the spices prvided that they are kept at the required dryness. This finding des nt cnfrm t thse in existing literature (King et al., 1981; Seenappa and Kemptn, 1981; Banerjee and Sarkar, 2003). These investigatrs reprted high micrbial cunts n dried spices f exprt quality. This may be due t the preparatin methds and handling f the spices by the prducing cuntries, since prductin practices range frm sanitary t unsanitary. If the aerbic mesphilic cunt is high r allwed t increase, this may accelerate prduct spilage while relatively significant fungal cunts may be a prblem, especially when such spices are used in lng-term strage type prducts. Mst f the micrrganisms assciated with the fresh pepper samples were eliminated by the pretreatments and drying prcess. This finding suggests that even in the traditinal setting, washing and steeping alne fllwed by cntrlled sun-drying, culd g a lng way in prviding spices with minimal micrbial lad and which culd therefre be stred fr lng perids f time withut underging spilage. References Andress, E.L., I.C. Blackman,, E.M. D Sa and M.A. Harrisn, Micrbita f fresh herbs and whle spices used in hme fd preservatin and effectiveness f micrbial interventin methds. Prceedings f the institute f Fd Technlgists Annual Meeting, New Orleans, LA. June 26,88D-27. th A.O.A.C., Official Methds f Analysis. 15 edn. Virginia, Assciatin f Official Analytical Chemists. Ayres, J.C., J.O. Mundt and W.E. Sandine, Micrbilgy f Fds. W.H. Freeman and C. San Francisc. Banerjee, M. and P.K. Sarkar, Micrbilgical quality f sme retail spices in India. Fd Res. Int., 36: Barnett, H.L. and B.B. Hunter, Illustrated genera f rd imperfect fungi 3 edn. Burgess Publ. C. Minneaplis, Minnesta. Beckmann, G., D. Kszegi, B. Snnenschein and R. Leimbeck, On the micrbial status f herb and spices. Fleischwirtschaft, 76: Buchanan, R. and N.E. Gibbns, Bergey`s Manual f Determinative Bacterilgy, Williams and Wilkins, Baltimre. Christensen, C.K., H.A. Fanse, G.H. Nelsn, F. Bates, and C.J. Mircha, Micrflra f black and red pepper. Appl. Micrbil., 15: Cllins, C.H. and P.M. Lyne, Micrbilgical rd Methds 3 ed. University Press, Baltimre. Dalziel, J.M., The useful plants f West Trpical nd Africa 2 printing, Crwn Agents, Lndn. Fasakin, E.A. and B.A. Aberej, Effect f sme pulverized plant materials n the develpmental stages f fish beetle, Dermestes maculates Dageer in smked cat fish (Clarias gariepinus) during strage Biresur Tec., 85: Garcia, S., F. Iracheta, F.Galvan and N. Heredia, Micrbilgical survey f retail herbs and spices in Mexican markets. J. Fd Prt., 64: Harrigan, W.F.and M.E. McCance, Labratry Methds in Fd and Dairy Micrbilgy. Academic Press, New Yrk. 248

7 Hutchinsn, J. and J.M. Dalziel, Flra f West Trpical Africa.Vl. 1. Part 1. Crwn Agents Lndn. Isawumi, M.A., The peppery fruits f Nigeria. Nigerian Field, 49: King, A.D., A.D. Hcking and J.I. Pitt, The mycflra f sme Australian fds. Fd Tec. Aust., 33: Krishnaswamy, M.A., J.D. Patel and N. Parthasarathny, Enumeratin f micrrganisms in spices and spice mixtures. J. Fd Sci. Tec., 8: Ldder, J., The yeasts: A Taxnmic Study. 2 nd edn. Nrth Hlland Publ. C. Amsterdam. McKee, L., Micrbilgical cntaminatin f spices and herbs: A Review. Lebensmittel- Wissenschaft un Technlgie, 28: Parry, J.W., Spices vl1 and 11.New Yrk, Chemical Publ.C. Pruthi, J.S., Spices and Cndiments: Chemistry Micrbilgy Technlgy. Advances in Fd Research Suppl.4. Academic Press. Inc. New Yrk. Purseglve, J.W., E.G. Brwn, C.L. Green and S.R.T. Rbbins, Spices. Vl.1Trpical Agric.Series.Uk, Lngman, Lndn. Seenappa, M. and A.G. Kemptn, A nte n the ccurrence f Bacillus cereus and ther species f Bacillus in Indian spices f exprt quality. J. Appl. Bacteril., 50: