Microbial, Chemical Composition Evaluation and Development of a Technological Process for the Production of Compound Spices in Nigeria

Article

Microbial, chemical composition evaluation and development of a technological process for the production of compound spices in Nigeria

Olalekan David Adeniyi*, A S Abdulkareem, B M Idris and J O Odigure Chemical Engineering Department Federal University of Technology, Minna, Nigeria *Correspondent author, E-mail : [email protected] volatile oils, which contains terpene, Abstract aldehydes, ketones and alcohol of various phenol based compounds. Spices are vegetable products derived from fruits, seeds, roots Most spices and condiments do not and tree barks. They are important mainly as additives to food because have any reasonable bacteriostatic of the presence of essential oil having anti-microbial and fungicidal effect on the concentration of the properties. These properties have been used to some degree as the basis food material they are used on.1 The for food preservation and as medicinal products for certain types of United States, Food and Drug diseases in Nigeria. This paper takes a look at the microbial load and Administration (FDA) specifies that chemical compositions of some of the compound spices found in Nigeria. paprika, saffron and turmeric are Results reveal that some compound spices are highly contaminated with colours and they must be labelled microorganisms. Isolates from sample B were identified as Salmonella, with common names on food Bacillus and Escherichia coli bacteria while those from sample A labels.1, 2 contained Staphylococcus aureus and Streptococcus. The fungi isolates Normally high were Aspergillus niger, A. flavus and Microsporum caris. The bio-load concentrations of plant extracts are of the spices ranged from 1.64 105 to 2.09 107 cells per gram of spice. × × required before anti-microbial Salmonella species and Bacillus sp. were the most common properties become apparent. It is microorganisms. It is recommended that higher sanitary conditions be well documented that vegetables employed during the processing, handling and storing of the spices. This like, onions, garlic and horse-radish paper proposes a technological process route for the production of more provide anti-microbial extracts.3,4 hygienic spices. For centuries it has been known that certain foods especially spices Introduction seasonings and imparting aroma to possess naturally occurring 1 food. Spices and condiments are preservative qualities but only The definition of spices, usually grown in tropical or semi recently has the technology been herbs and condiments is still the tropical climates and comprise of developed to extract, quantify and focus of much debate over the years. one portion of the plant. Spices are identify these substances. The International Organization for the whole or ground seeds, fruits, Spices vary in their Standardization (ISO) defines spices bark or roots of a plant. effectiveness depending upon the and condiments as the natural Spices have characteristic source, freshness and method of vegetative products or mixture odours or flavours. The chemicals processing and storage. The thereof without extraneous matter responsible for their distinctive inhibitory effect of spices differs that are used for flavourings, tastes and smell are essential oils or with the kind of spice and the

314 Natural Product Radiance Vol 2(6) November-December 2003 Article microorganism being tested for. Pseudomonas aeruginosa were the Methodology Mustard and the volatile oil of most resistant except towards thyme mustard have varying effectiveness oil. Antibacterial and antifungal The material used for the against Saccharomyces cerevisiae effectiveness of the compounds of experiment include spice product but are not effective against most the spice Aframomum melegueta such as garlic (Allium sativum bacteria as cinnamon and cloves.1,5,6 have been carried out by Oloke et Linn.), ginger root (Zingiber Cinnamon and cloves contain al7 and the volatile oil showed officinale Rosc.), allspice [Pimenta cinnamic aldehydes, which makes considerable bactericidal activities dioica (Linn.) Merrill], mansoro, them more bacteriostatic than against Escherichia coli, cloves [Syzygium aromaticum mustard. Thyme, bay leaves, Pseudomonas, etc. and fungicidal (Linn.) Merrill & Perry], gyada marjoram saucy, rosemary, black activities against Candida albicans, muyan, thyme (Thymus vulgaris pepper and others have weak Trichophyton mentagrophytes, etc. Linn.), crayfish, alligator pepper inhibitory power against most These properties have been [Aframomum melegueta (Rosc.) organism. used to some degree as the basis for K. Schum.], white pepper, African Several studies have been food preservation. However, certain hot pepper and locust beans conducted using different spices to spices are known to contain large (Ceratonia siliqua Linn.). The inhibit the growth of pathogenic number of microorganism despite spices were gathered from the local organism. Kivanc and Akgul6 tested their apparent anti-microbial effect. farm and market, cleaned and sun the bacteriostatic and bactericidal Some of these organisms and dried at temperature of 45°C activities of twenty-two essential chemical content may cause maximum, until a constant dried oils from Turkish spices and citrus spoilage or become pathogenic to weight was attained. It was then against Aerobacter aerogenes, man when introduced to food.10 passed through magnetic separator Bacillus subtilis, etc. The results There is little information available to remove the foreign material showed that the essential oils tested on the microflora, volatile oil, manually, sieved to remove the dirt, varied in their antibacterial activity. production techniques and quality of insects and ground to fine particles Anise, celery, coriander and sage local Nigerian spices. This paper of size 0.05 µm. Two samples (A & were inactive or had little activity presents results of some of the B) as proposed by Djmen11 for while corn, mint, cumin, lemon peel analyses carried out on the local mixing by weight were used; the and ziziphora were active against all spices and proposes a technological ratios are given in Table 1. tested bacteria to a variable extent. process for the production of more Staphylococcus aureus and hygienic spices.

Table 1 : Mixing ratio by weight (wt.) for spices mixture (samples)

Sample A Sample B 10 % wt. Garlic 5%wt.Gyanda muyan 15 % wt. Garlic 10 % wt. Thyme 10 % wt. Ginger 10 % wt. Thyme 15 % wt. Ginger 10 % wt. Crayfish 5 % wt. Allspice 5 % wt. Crayfish 5 % wt. Allspice 10% wt.White pepper 10%wt. African 10% wt. Alligator 15%wt. African 10% wt. Alligator hot pepper pepper hot pepper pepper 5 % wt. Mansoro 20% wt.White pepper 5 % wt. Mansoro 10 % wt. Cloves 5 % wt. Cloves

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estimating the difference in fresh expelling the contents of the pipette. and dry weight. This pipette was also discarded and the second dilution tube was labell- Determination of protein ed 10-2. Taking fresh sterile pipettes and carbohydrate in each case other specimen were -3 -4 -5 Protein content was prepared for 10 , 10 and 10 . measured by Kjedahl method and Plate preparation procedure the total carbohydrate was Taking a fresh sterile pipette determined by using estimation the content of the final dilution tube method.8 If the total of protein, lipid (10-3) was mixed by sucking up and acid content was subtracted from down five times. One ml of the organic matter, the remainder dilution was withdrawn by touching accounts for carbohydrate and the tip of the pipette against the side nucleic acid. Thus, % carbohydrate Ceratonia siliqua of the tube to remove excesses = 100 - (% moisture + % ash + % adhering to the outside and the protein + % lipid + % fibre) contents was transferred to a sterile contents. petri dish. Allowing about 3 minutes Microbiological analysis of to elapse the tip of the pipette was samples A and B touched against the dish away from the previous inoculum and the Dilute Solution Preparation remaining drops gently blown away. Procedure This same pipette was used to Syzygium aromaticum A sterile 1 ml blowout transfer 1 ml from the 10-2 dilution pipette was held vertically and the to a sterile petri dish but before pipette tip was introduced to the taking the samples it was raised and surface of samples A and B and lowered for about three times in sucked up and down five times to order to rinse the sides of the pipette the 1 ml mark. One ml of samples and also to give the dilution a final A and B was withdrawn. The pipette mixing. The same procedures were content was transferred to the first repeated for the 10-1 dilution. tube of dilution solution series with To each plate 15 to 20 ml of the tip touching the side above the molten compound spices agar level of the diluents. This pipette (bacterial) and PDA medium at 45°C was then discarded and the first was added and immediately the dilution tube was labelled 10-1. medium and inoculum was mixed Taking a fresh sterile pipette, by a combination of to and fro and Pimenta dioica the content of the first dilution tube shaking for about 5-10 seconds. The Determination of moisture was mixed by sucking up and down plates were allowed to set, then to the 1 ml mark five times. Then inverted and incubated and ash content 1ml of the first dilution was at the appropriate temperature of The moisture and ash withdrawn and transferred to a 25-30°C for fungi and 37°C for contents were determined by second tube of sterile dilution bacteria.1, 8, 9, 12, 13, 14

316 Natural Product Radiance Vol 2(6) November-December 2003 Article Fungi analysis Preparation of nutrient agar thoroughly cooked. The suspension was filtered through cheese cloth Twenty-eight grams of and water added to make up to 1 ml. Preparation of compound spices compound spices agar powder was Dry ingredient was also added and broth weighed and dissolved in 100 ml of agar was dissolved with heat. distilled water. It was boiled, stirred Sterilization was conducted in an Thirteen grams of and autoclaved at 121°C for 15 autoclave at 121°C for 15 minutes. compound spices broth powders minutes. It was then cooled at 45°C was weighed and dissolved in 1000 and powdered into sterile petri dish ml of distilled water. It was boiled, and allowed to set. Results and Discussion stirred, bottled and autoclaved at Preparation of potato dextrose 121°C for 15 minutes. The bottle The results obtained from agar tubes were kept in the refrigerator the various analysis carried out are at 4°C until required. Potato tube was boiled in presented in Tables 2-5. 500 ml of distilled water until

Table 2 : Moisture, Ash, Protein, Lipid and Carbohydrate contents of the spice samples A and B Spice sample Moisture % Ash % Protein % Lipid % Carbohydrate %

A 7.00 21.40 7.50 19.70 44.47 B 10.80 15.00 8.25 19.78 46.17

Table 3: Bacterial count of the compound spices sample A and B Sample Dilution factor Average Bacterial Count

10-1 10-2 10-3 A 1.64 × 105 1.40 × 106 9.80 × 106 1.33 × 106 B 2.75 × 105 1.92 × 106 1.60 × 106 2.09 × 107

Table 4: Fungi count of the compound spices sample A and B Sample Dilution factor Average Fungi Count

10-1 10-2 10-3 A 7.80 × 103 5.60 × 104 3.60 × 106 5.67 × 106 B 1.20 × 105 9.80 × 104 7.60 × 106 9.80 × 106

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Table 5 : Morphological characteristics and identification of fungi isolated from samples A and B Serial No. Sample Description of species of fungi Fungi species type

1 A Upper side: blackish Aspergillus niger Under side: yellowish Upper side: yellowish green Aspergillus flavus Under side: brownish Upper side: light brown Microsporus caris

2 B Upper side: greenish Aspergillus parasiticus Under side: yellowish Upper side: grey white Trichophyton verrucosum sp. Under side: brown Upper side: pink Fusarium sp. Under side: deep red Upper side: yellowish green Rhizopus sp. Under side: grey black Upper side: blackish Aspergillus niger Under side: yellowish Upper side: yellowish green Aspergillus flavus Under side: brownish Upper side: white grey Microsporum caris Under side: brownish

The laboratory tests were spices with A having 19.70% and B The level of contamination conducted to determine the physical with 19.78%. The protein and depended on the type of spices. The composition and the microbial load carbohydrate contents for sample A microfloras were heterogeneous and of the compound spices and the were lower than that of B (Table 2). these data appear to agree with those fungi growth on these spices. The The percentage protein values for A described elsewhere for spices microbial analysis showed that the and B are 7.5% and 8.25%, prepared for marketing.13 The flora spices carried a fairly heavy load of respectively while the carbohydrate of these spices was mainly Gram microorganisms (Table 3). Table 2 was 44.47% and 46.17%, negative rods, although Gram gives the percentage estimation of respectively. positive spore formers and spool the moisture and ash contents of the Table 3 showed that the were present. In this study, the spices spices under investigation. The compound spices sample B seem to examined contained Salmonella, moisture content of spices sample have more bacterial load than Bacillus cereus, Staphylococcus A (7.0%) is lower than that of B sample A. The total viable count of aureus and Pseudomonas (10.8%). The reverse was the case the spices ranged from 1.64 × 105 to aeruginosa. Gilbert and Roberts,3 in the ash contents; that of A has 2.09 × 107 cells per gram of spice. and Laidley et al15 examined 21.4% while that of B was 15.0%. The highest average counts were samples of certain spices from These discrepancies are attributed to obtained from sample B with a value military bases and reported that only the differences in the composition of 2.09 × 107, due to the higher a small proportion contained of the spices mixture. Table 2 gives moisture, content of B (crayfish, organisms of public health the percentage lipid content of the garlic, ginger and mansoro).15, 16 significance. Samples of herbs and

318 Natural Product Radiance Vol 2(6) November-December 2003 Article spices examined by several authors plant consists of a magnetic radiate heat transfer between the sun in some other countries have been separator, sterilization tank, milling and the materials; this acts as a solar shown to contain pathogenic machines, drying machines, mixers collectors in the warehouse. In this organisms.13, 14 Their microbial and other ancillary equipment. case the convective air acts only to populations have been shown to be carry away the removed vapour. 7 Spices raw Raw materials very high often exceeding 10 cells materials bulk tank per gram. In this study the microbial Magnetic centrifuge/ 5 count ranged from 1.5 × 10 to Magnetic sterilizer 9.0 × 107 (Tables 3 and 4). The separator presence of E. coli in some of the This comprises heating spices suggests the need for high Sterilization cylindrical container, with electro- sanitary procedure particularly tank magnetic properties capable of during handling and storage. removing metallic material present The fungal species Milling/ in the raw material as it goes associated with the spices examined Grinding through it. The spices are then were Aspergillus niger, A. flavus, sterilized at a temperature of about A. parasiticus, Microsporum caris, Drying 160°C with a retention time of 120 Fusarium sp. and Rhizopus sp. minutes or 170°C for 69 minutes. (Table 5). These fungi are often During this process the material associated with mycotoxin Mixing loses about 2% of the moisture production particularly the content.9 The heat penetration time Aspergillus. It is however not known depends on the nature and volume Packaging if the isolate obtained from these of the spices. The contamination and spices is toxigenic. Sanitary Fig. 1 : Flow diagram for the production infection of microbial load are measures should therefore be of compound spices reduced by 90% during this stage. introduced early in the storage of the The sterilized material is then passed spices to minimize excessive fungal Raw material offloads on to the milling and grinding unit. growth. It is recommended that The spices raw materials are immediately after harvest, the spices Milling/grinding offloaded from the tank into the should be cleaned by washing with conveyor, where they are passed on The mills consist of a potable water and kept dry during to the raw material storage specially designed rotors mounted the handling process. Some form of warehouse. vertically in a housing which acts as packaging would be very useful to a stator and is equipped with a ensure reduced microbial growth. Raw material storage warehouse removable corrugated inner casing. Development of process This consists of large A fan disc is attached to each of the cabinets with transparent cover and technology rotor shaft. The spices are fed into solar collector for pre-drying and the machine through an inlet in the In order to minimize the conditioning. The spice materials wall of the housing at the level of contamination of the produced are exposed to sunlight through the lowest grinding disc, being compound spices the following transparent cover and the required drawn onto the grinding zone by the technological process was energy for drying purpose is air stream provided there. The developed as shown in Fig. 1. The achieved as provided essentially by rotation of the rotors causes strong

Natural Product Radiance Vol 2(6) November-December 2003 319 Article eddies of air to form in the chamber, after harvesting, higher sanitary and antifungal activities of which together with the impact of conditions should be employed certain components of the particles on the walls of the during the processing, handling Aframomum melegueta fruits, grinding tools and the stator casing, and storing of the spices. The Fitoterapia, 1988, 59,384-388. grind the materials. The finer proposed technological process 8. Flick A S, Microbiology of particles leave the mill as finished will help in reducing the microbial foods, (W H Freeman ground spices and are collected after growth of the produced compound Publication, San Francisco), passing through a sieve. spices. 1976, 249-259. 9. Macrae S, Joshyn MA and Drying References Webster J, Methods in food A vertical dryer is fitted with 1. Frazier WC and Westhoff DC, Analysis, 2nd edition, (Academic three heating elements arrayed in Food Microbiology, 3rd edition, Press, New York), 1997, 7-24. form of triangle on the dryer (McGraw-Hill, New York), 10.Macrae S and Robinson J, sidewalls. The dryer contains some 1978, 1,191,273, 313-316. Influence of the activity of water drying trays. Here the grains are 2. Idris BM, Evaluation of on spoilage of foodstuffs, J Food gradually heated to a maximum technological processes for the Tech, 1997, 3,131-142. temperature of about 50°C. The production of compound spices, 11. Djmen K, Quality and uses of spices are transferred into various M. Eng Thesis, Federal Nigerian spices, Home bins and passed unto the mixer. University of Technology, economics report, Dept. of Agr., Minna, Nigeria, 2002, 1-96. Niger State, Nigeria,1997, 3-8. Mixing/packaging 3. Gilbert RJ and Roberts D, Food 12. Christopher O, The microbial The spices now in form of poisoning risks associated with content of some Nigerian spices, flour are mixed in desired food other than meat and PhD Thesis, University of Port proportions. Here complete poultry-outbreak and Harcourt, 1986. homogenization takes place and the surveillance studies, Health 13. Christensen G M, Fanse H A , required consistency of spices is Hyg, 1979, 3, 33-40. Nelson G H and Bates F, produced. The blended spices are 4. Purseglove JW and Brown EG, Microflora of black and red packaged into smaller units of 20g Spices, (Longman Publication, pepper, Appl Microbiology, nylon bags and are ready for London), Vol 1, 1981, 524-528, 1967, 15, 626-662. marketing. 5. Bullerman LB, Lieu FY and 14. Kaferstein F K, The microflora Seier Sally A, Inhibition of of parsley, J Milk Food Tech, Conclusions growth and aflatoxin production 1976, 39, 837-840. The formulated compound by cinnamon and clove oils, 15. Laidley R, Handzel S, Sever D spices under study were fairly cinnamic aldehyde and eugenol, and Butler R, Salmonella contaminated with some J Food Sci, 1977, 42, 1107- weltevreden outbreak associated microorganism and were not 1109. with contaminated pepper, E suitable for consumption. The type 6. Kivanc M and Akgul A, Bull, 1974, 18, 62. of the spices composition also Antibacterial activities of 16.Oyebiodun F, The effect of contributed to the level of essential oils from Turkish extracts of some Nigerian spices contamination. The level of bio-load spices and citrus, Flavour Fragr on biodegradation of okro by on these spices was greatly J, 1986, 1,175-179. fungi, PhD Thesis, Delta State dependent on the level of moisture 7. Oloke JK, Kolawole DO and University, Abraka, Nigeria, content. It is recommended that Erhun WO, The antibacterial 1982.

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