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Microbial quality of "Kunun Zaki": a Nigerian indigenous fermented food drink.

Introduction

Fermented foods are essential parts of diets in all parts of the world. Indigenous fermented foods form an important part of an average Nigerian diet contributing important nutrients such as proteins, calories, minerals and some vitamins (Achinewhu, 1983), and they include Ogiri, dawadawa, ugba, kunu, brukutu, nono, etc (Iwuoha and Eke, 1996). Foods are fermented for reasons like preservation, improvement of flavour, texture, aroma, solubility and digestibility (Hessel tine, 1980).

"Kunun--zaki" is a popular indigenous food drink. It is mainly from millet (Sorghum grains). Sorghum is one of the cereals cultivated in the tropical region of Africa in the northern Guinea Savanna areas of Nigeria (Asiedu, 1987). It constitutes a major source of energy and protein for people in Asia and Africa and it serves as a staple food of many of the world's poorest and least privileged people (Hulse et al, 2007). It is traditionally prepared under unhygienic a condition which in turn predisposes it to contamination by pathogenic microbes like Staphycococci sp and E. coli (Onuorah et al, 1987). "Kunun" is also not shelf--stable, readily undergoing microbial induced spoilage within 2-3 days. It has immense social, economic and nutritional benefits to its numerous consumers. Non-alcoholic nature of this drink made it to be readily consumed by both Muslims and Christians alike. The spices used with the cereal during production include: dry sweet potato chips, ginger (Zingiber officinale), malted rice (Oryza sativa) and cloves (Eugenia caryophillata).

This study tends to isolate and identify the microorganisms involved in fermentation and of spoilage of "Kunun--Zaki". This would assist industrial microbiologists in the bids to upgrade the traditional fermentation of the product by using only standardized, known inoculum mixture and to also employ appropriate, preservation methods on this readily spoilt beverage.

Materials and Methods

Sample Collection

The millet (sorghum grains) and the spices used in this study were purchased at military mini market in Ike Nwachukwu army barracks in Obinze, Imo State. Samples were taken to the laboratory immediately after purchase for analysis.

Preparation of Samples for Analysis

Cleaned sorghum grains (80g) were washed and soaked in 250ml of sterile distilled water for a period of 18hours while the steep liquor was being sampled for analysis at a 6hourly interval.

The traditional method for the production of "Kunu" as described by Onuorah et al, (1987) and modified slightly by Egbere (1988) was followed. The viscosity, pH value, sensory quality, total plate count and the microbial load of the fermenting and spoiling "Kunu - Zaki" samples were determined with respect to time.

Fermenting mixture: The pre-gelatinized mixture of sorghum starch with the spices and other ingredients were properly mixed and sampled out for analysis at three hours interval up to the 18th hour of fermentation at room temperature.

Microbial Enumeration and Isolation

The media of choice were nutrient agar, macConkey agar and sabouraud dextrose agar for the enumeration of total heterotrophic bacteria, lactose fermenting bacteria and fungi respectively. The media were prepared according to the manufacture's instruction and allowed to set in petri dishes in the case of the sabouraud dextrose agar, the medium was fortified with chloramphenicol to prevent bacterial growth. Ten fold serial dilutions of the samples were made. 0.1ml aliquot of the appropriate dilution was spread plated in duplicates on surfaces of the appropriate medium (APHA 1985). The plates were then incubated for between 24 and 96 hours at room temperature. Following incubation, the colonies that developed were counted. Colonies were randomly selected based on their colonial characteristics and streaked for purity on nutrient agar and sabouraud dextrose agar, respectively.

Characterization and Identification of the Isolates

The bacterial isolates were examined for colonial morphology as well as for cell micro morphological and biochemical characteristics according to the methods described by Gerhardt et. al., (1981).

Fungal isolates were examined macroscopically and then microscopically using the needle mount technique and identified following the schemes of Alexopoulous and Mims (1981) and Barnett and Hunter (1972).

Determination of the Fermentation Rate

The rate of fermentation of each bacterium was assessed by period testing for pH, TTA and detection of characteristic aroma of the fermenting samples. A control experiment containing no inoculum was also set up to validate results of the experiment.

Determination of pH, Titratable acidity and sensory attributes of the fermenting beverages

The pH values of various samples were determined using a laboratory pH meter (Jenway, 3015, model 10). The total titratable acidity (TTA) calculated as percentage lactic acid was determined following the method described by Pearson, 1973. The viscosity of the samples was determined using the brookefield viscometer. Sensory characteristics (taste, aroma and colour) of the fermenting, finished and stored "Kunu zaki" were assessed periodically to detect when a change in the organoleptic attributes took place.

Qualitative test for production of Ethanol

A simple qualitative test for alcoholic fermentation spoilage by CO2 evolution (effervescence) was determined using the method of Egbere (1988) as follows: 25ml of the fermenting or stored Kunu were put into sterile universal bottles and tightly closed. The samples in the bottles were opened at a stipulated time required with the mouths of the bottles facing a flame on a held match stick. The presence of CO2 evolution was indicated by rapid extinguishing of the flame.

Results

Bacterial species belonging to genera Bacillus, streptoccocus, Lactobacillus and Corynbacterium were isolated (Table 1). Bacillus and Lactobacillus spp had 100% frequency occurrence followed by Streptoccocus and lastly by Corynbacterium with 50% and 44.30% respectively.

Fungal isolates during the fermentation and storage stages were Aspergillus niger and Sacharomyces cerevisiae (yeast) (table 2). S. cerevisiae had 100% frequency of occurrence while A. niger got 55.40%.

The results on the physio-chemical and sensory attributes of "kunun - zaki" (table 3) indicate that spoilt "kunun - zaki" is characterized by high acidity (pronounced sourness), decreased viscosity, effervescence on the corked bottle "kunu" due to ethanol production and fading in the characteristic brown to pale-pink colour of the beverage.

Discussion and Conclusion

The result of this study showed that during the fermentation of "kunun--zaki" production--more bacteria were isolated than fungi. Most of the microorganisms have been isolated by other researchers such as Faparusi et. al., (1973), Kolawole et. al., (1987), Gaffa and Gaffa (2004) and Egbere et al, 2007. Bacillus, Lactobacillus and Sacharomyces spp had 100% frequency occurrence. Bacillus sp is widely distributed bacteria that is commonly found in soils and have been isolated in various countries from a variety of foods.

Aspergillus niger and Sacharomyces cerevisiae persisted in occurrence. They have been reported to be acid tolerant and indeed, strains of A. niger are known to be acid (citric acid) producers (Adam and Moss, 1999). From the study, Lactobacillus sp, Bacillus subtilis and S. cerevisiae are both fermenters and deteriogens, Corynebacterium sp could have been only a fermenter, Egbere et. al., 2007 had the same result during their studies on the microorganisms association with the bacterial and fungal isolates have been known to associated with certain foodborne illnesses, most of which arise from improper handling, preparation and storage (FDA 2005). However good hygiene practices before, during and after food preparation can reduce the chance of contracting an illness. The results of the physico-chemical and sensory attributes of "kunun" indicate that spoilt "kunun-zaki" is characterized by high acidity, sourness, decreased viscosity, effervescent etc. The result of this study have shown that lactobacillus sp was found to be the most effective fermenter followed by Streptococcus sp. Lactobacillus can now be harnessed for the industrial production of "kunu-zaki".

References

[1] Achinewhu S.C (1983). Protein quality of African Oil Bean Seeds (pentaclethia macrophylla) Journal of Science 48:1374-1375.

[2] Adams M.R and Moss M.O (1990). Food Microbiology. The Royal Society of chemistry, Cambridge UK ISBN 085404

[3] Alexopoulous C.J and Nums . C.W (1979). Introductory Mycology 3rd edition, John Wiley and Sons Publishers, Edinburgh.

[4] Asiedu, J.J (1987). Processing Tropical Crops. A technological Approach. 11th Edn Maccmillian Education Ltd, pp 189-222.

[5] APHA (1985). Standard methods for the examination of water and wastewater American Public Health Association, Washington DC.

[6] Barnett, H.L and Hunter, B.B (1972). Illustrated Genera of imperfect fungi 3rd Edition Burgress Publishing Company, Minnesota, lest.

[7] Egbere, O.J. (1988). The Microbiology of "kunun-zaki", sorghum based fermented beverage. B.Sc. Thesis Department of Food Science and Technology, University of Jos, Makurdi Campus Nigeria.

[8] Egbere, O.J., Adik, C., Inyang, C.U., Henry, U.I. and Ogbonna, C.I.C (2007). Microorganisms associated with fermentation and spoilage of kunun zaki. Nig. J. Biotech. 18 (1-2): 49-55

[9] Faparusi, S.I., olofinobi, M.O and Ekundayo, J.A (1975) The Microbiology of Burukutu Beer. Allog Microbiologie, 563-568

[10] FDA (2005). Food borne pathogenic Microorganisms and National Toxins handbook FDA, Rockvile, MD.

[11] Gaffa, T.and Gaffa A.T (2004). Microbial Succession during "kunun-zaki" production with Sorghum (Sorghum bicolor) grains. World Journal of Microbiology 20:499-453.

[12] Gerhardt, P., Murray R.G.E., Costilon, R.N., Wester, E.N., Wood, W.A., Krieg, W.R and Philips, G.B (1981). Manual of methods for General bacteriology. American Society for Microbiology Washington, D.C. 612p.

[13] Hesseltine, C.W (1980). Some unimportant fermented foods in Mid Asia, the Middle east and Africa Journal of American Oil Chemists Society 636: 374637.

[14] Hulse J,H Laing, E.M. and Pearson, O.E (2007). In Sorghum and Millets, their Composition and Nutritive value. Fifth Edition, Academy Press, New York, P.997.

[15] Iwuoha, C.I. and eke, O.S (1996). Nigerian indigenous fermented foods, the traditional process operation inherent problems, improvements and current status. Food Research International 29 (9): 527-540

[16] Kolawole, D.O., Ogundiwin, J.O and Okwuosa E.C. (1987). The microbiology of fermentation of Guinea corn (Sorghum bicolor linn). In the preparation of Sorghum status J. FoodAgric. 1:43-45

[17] Onuorah, S.I. Adesiyun, A.A. and Adekeye, J.O. (1987). Occurrence of Staphylococci and coliforms in "kunun-zaki" and utensils used in its preparation in Samara-Zaria Journal of Food Science and Agriculture 1: 31-34.

Okechukwu R.I., Ewelike N.C. (2), Okechi R.N. (1), Duru C.M. (3) and Ezejiofor T.I.N. (1)

(1) Department of Biotechnology, (2) Department of Microbiology, (3) Department of Biology,

Federal University of Technology, Owerri, Imo State, Nigeria Corresponding Author E-mail: rositaokechukwu@yahoo.com
Table 1: Bacterial Isolates During Fermentation and Storage Stages

Stage/sample Time Total Bacillus Streptococcus
 During Hr plate sp
fermentation count
 cfu/ml

 Fermented 0 1.0 x [10.sup.1] + +
 slurry 6 1.2 x [10.sup.1] + +
 " " 12 5.3 x [10.sup.1] + +
 " "/ 18 6.4 x [10.sup.1] + +
 Storage 24 7.5 x [10.sup.3] + -
 (Fermented 48 5.3 x [10.sup.3] + -
 slurry) 72 3.9 x [10.sup.3] + -
 96 2.5 x [10.sup.1] + -
 120 1.0 x [10.sup.1] + -
Frequency of 100% 50%
occurrence %

Stage/sample Lactobacillus Corynebacterlum
 During
fermentation

 Fermented + +
 slurry + +
 " " + -
 " "/ + +
 Storage + +
 (Fermented + -
 slurry) + -
 + -
 + -
Frequency of 100% 44.30%
occurrence %

Table 2: Fungal Isolates During Fermentation and Storage Stages

Stage/sample Time Total plate count cfu/
 ml A. nlger S. cerevlslae

During fermentation Hr

Fermented slurry 0 1.0 x [10.sup.1] + +
" " 6 1.2 x [10.sup.1] + +
" " 12 5.3 x [10.sup.1] - +
 18 - +
Storage (Fermented slurry) 24 8.0 x [10.sup.1] - +
 48 5.5 x [10.sup.6] + +
 72 3.0 x [10.sup.5] + +
 96 2.0 x [10.sup.2] + +
 120 1.0 x [10.sup.2] + +
Frequency of occurrence % 55.40% 100%

Key: (+) = present, (-) = absent

Table 3: Changes in Physio-Chemical and Sensory Properties in Sample
of Fermentation and Stored Laboratory Produced 'Kunu'

 Stage/sample Time pH Viscosity Evolution Taste
 During Hr of [O.sub.2]
 fermentation

 Pre Fermented
 slurry 0 7.0 21.00 - Not
 Fermented 6 6.5 16.50 - sour
 Fermented 12 5.50 09.00 - Not
 sour
 Fermented 18 3.95 5.00 - Slight
 sour
 Sweet
 Storage 24 3.10 4.00 - Sweet
 (Fermented slurry) 48 2.50 4.00 + sour
 72 2.40 3.50 + Sweet
 96 2.30 3.30 + sour
 120 2.10 3.00 ++ Very
 sour
 Very
 sour
 Very
 sour

 Stage/sample Colour Remarks
 During
 fermentation

 Pre Fermented
 slurry Brown Raw fermented
 Fermented Brown Raw fermented
 Fermented Brown characteristic
 'kunu'
 Fermented Brown characteristic
 'kunu'

 Storage Brown
 (Fermented slurry) Light " Spoilt 'kunu'
 Pale Spoilt 'kunu'
 pink Spoilt 'kunu'
 Pale Spoilt 'kunu'
 pink
 Pale
 pink

Key: + = effervescence, ++ = profuse effervescence

Table 4: Relative Rate Of Acid Based Fermentation In "Kunun--Zaki" By
Selected Bacterial Isolates.

Time Lactobacillus Streptococcus Bacillus sp
(Hrs) sp sp
 pH TTA
 PH TTA pH TTA
0 7.00 0.006 7.00 0.060.06 7.00 0.006
3 6.60 0.015 6.70 0.015 6.50 0.008
6 6.00 0.016 5.40 0.020 5.80 0.020
9 5.36 0.038 4.46 0.080 5.30 0.038
12 4.00 0.040 4.00 0.042 4.40 0.040
15 3.90 0.049 - - 4.00 0.039
18 - - - - 5.70 0.038

Time Combination of all Control
(Hrs) bacteria
 pH TTA pH TTA

0 7.00 0.060.006 7.00 0.006
3 6.70 0.019 7.00 0.006
6 5.22 0.027 6.49 0.007
9 3.70 0.043 6.38 0.008
12 3.23 0.056 6.30 0.007
15 - - 6.08 0.011
18 - - 6.03 0.013

Key: -= not determined, TTA = % titratable acidity calculated as
lactic acid
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Author:R.I., Okechukwu; N.C., Ewelike; R.N., Okechi; C.M., Duru; T.I.N., Ezejiofor
Publication:International Journal of Biotechnology & Biochemistry
Date:Nov 1, 2011
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