Comparative Studies of Ginger (Zingiber Officinale) and West African Black Pepper (Piper Guineense) Extracts at Different Concen

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Nigerian Food Journal Official Journal of Nigerian Institute of Food Science and Techonology www.nifst.org NIFOJ Vol. 30 No. 2, pages 38 – 43, 2012

Comparative Studies of Ginger (Zingiber officinale) and West African Black Pepper (Piper guineense) Extracts at Different Concentrations on the Microbial Quality of Soymilk and Kunun-zaki Udensi, E.A.1, * Odom, T.C.2 and Dike, C.O.

ABSTRACT Extracts of two spices, namely ginger (Zingiber officinale) and black pepper (Piper guineense) were prepared in 0.4%, 1.2%, 2.4% and 3.6% concentrations. Soymilk and kunun-zaki were treated respectively with the different concentrations and stored at ambient temperature for 5 days. The microbial load and identification were determined every day of storage until samples were adjudged spoilt. On the first day, 0.4% ginger extract in soymilk and kunun-zaki had a microbial load of 7.77 x 106b and 5.17 x 106b respectively. 3.6% ginger extract in soymilk and kunun-zaki recorded 3.73 x 106b and 3.30 x 106 each. 0.4% black pepper extract in soymilk had 6.273 x 106b and recorded 4.63 x 106b in kunun-zaki. 3.6% black pepper extract in soymilk and kunun-zaki had a microbial load of 3.20 x 106d and 2.90 x 106c respectively. On the third day the microbial load increased for both ginger and black pepper extract. Ginger extract recorded 9.13 x 106b in soymilk and 5.60 x 106b in kunun-zaki at 0.4% concentration. Black pepper extracts recorded 7.43 x 106b in soymilk and 3.27 x 106b in kunun-zaki also at 0.4% extract. 3.6% black pepper extract recorded 4.10 x 106a in soymilk and 2.20 x 106c in kunun-zaki. There was linear reduction of microbial load as spice concentration increased. Black pepper recorded lower microbial load, thus having more antimicrobial activity and may be preferred to be used as natural antimicrobial preservatives to extend the shelf-life of food. Keywords: Ginger, black pepper, extracts, soymilk, kunun-zaki.

Introduction availability (Nsofor and Osuji 1997). It is a non- The use of soybean for various food products has alcoholic and non-fermented beverage. However, become popular and has acquired a confirmed soymilk is susceptible to spoilage, it has poor shelf health food status by the United States Food and stability during ambient temperature (Nsofor and Drug Administration (USFDA) of USA (Enwere, Anyanwu 1992) and pasteurization treatment is 1998; Ohr 2004). One main product of soybean not sufficient to inactivate the bacterial spores in is soymilk which contributes to the provision it. Sterilization by heat causes denaturation of soy for the much needed cheap protein source that protein thus resulting in coagulation (Nsofor and would alleviate the problems of declining protein Osuji, 1997). Kunun-zaki is a traditionally fermented non- 1 Department of Food Science and Technology, Abia State Uni- alcoholic beverage mostly consumed in Northern versity Uturu. 2 Department of Food Science and Technology, Abia State Nigeria. It is usually produced from millet (Pennisetum Polytechnic Aba. typhoidum), sorghum (Sorghum bicolor) or maize (Zea * corresponding author: [email protected] mays). Graffa et al. (2002) reported that 73% of Comparative Studies of Ginger and West African Black Pepper Extracts at Different Concentrations ... Udensi, Odom and Dike 39 the population in Nigeria consumes Kunun-zaki Production of soymilk daily and 26% occasionally. Kunun-zaki is low in Soymilk was processed by the method described viscosity and has a sweet sour taste, with creamy by Iwe (2003). Soybeans were sorted, and 250 g appearance. of sample steeped in water for 12 h. The soaked According to Odunta (1985) numerous different soybeans were hand dehulled by rubbing and hulls fermented products have the advantage of prolonged removed by floatation. The steeped beans were shelf life due to the organic acid produced during grounded to fine paste using locally fabricated fermentation which is fatal to diverse spoilage attrition mill. 2.4 litres of water was added to the microorganisms. This could be the reason why paste. The resulting paste was sieved using muslin Kunun-zaki preserved longer than soymilk under cloth and the filtrate boiled for 1 h. the same condition as observed in this work. Production of kunun-zaki However, Kunun-zaki does not store long at ambient The millet grains were sorted to remove extraneous temperature though it is a fermented product. It materials. It was weighed, steeped in water for 18 h has a shelf life of 24 h at ambient temperature. at ambient temperature, washed, wet milled with (Adeyemi and Umar, 1994). attrition mill and sieved. The filtrate was mixed with Efforts are being made in order to maintain the water and divided into two unequal portions in the nutritional components of food using various ratio 1: 2. The greater portion was boiled until it means such as reduction in temperature or the use gelatinized and was allowed to cool. The cooked of local spices as a preservative. The use of local and uncooked portions were mixed together and spices to control the activities of microorganisms in allowed to ferment for 12 h to give kunun-zaki. food has been reported (Akpomedaye and Ejechi, Extraction of spices 1998; Nwafor, 2003). The antimicrobial activity Ginger and black pepper extracts were prepared varies depending on the type of spice or herb. The by the method described by Zia-ur-Rehman et need to which a spice material is used as a spice is al. (2003). Samples were grounded into powder dictated primarily by its essential oils or oleoresins form and extracted with hot sterile water (80oC at (Dziezak, 1989).The volatile oils are responsible for 5 g/100 ml for 72 h). Extracts were filtered using the aroma and taste of most spices. These volatile filter papers. The spice extracts were concentrated oils contain terpenes, sesquiterpenes, alcohols, by exposure to 100oC for 20 min in a water bath. esters, aldehydes, ketones, and phenols, etc. (Juliana Different concentrations of the spices were et al. 2004). prepared according to the method developed by The application of extracts of spice could possibly Akpomedaaye (1998) and Ogiehor et al. (1998) control the microbial activities associated with food (10.4%, 1.2%, 2.4% and 3.6% w/v) respectively. samples while retaining the nutritive and economic 50 mls each of soymilk and kunun-zaki were placed quality. Hence, in this work, two spices, ginger in five separate sterile containers respectively and (Zingiber officinale) and black pepper (Piper guineense) treated with the different concentration of the were used to enhance the shelf life of soymilk and spices. kunun-zaki at ambient temperature. Group A: Ginger extract (0.4%, 1.2% 2.4%, 3.6% Materials and Methods w/v) Soybeans, millet, ginger and black pepper were all Group B: Black pepper extract (0.4%, 1.2%, 2.4%, obtained from a local market (Ahaiohuru) in Abia 3.6% w/v) State. Group C: Control (No treatment). 40 Nigerian Food Journal Vol. 30 No. 2, 2012 ... Research Note

After the various treatments the samples were isolates were obtained on the nutrient agar and stored at ambient temperature until they were sabouraud dextrose agar respectively. adjudged spoilt. Characterization and identification of isolates Microbiological analysis Bacteria isolates were characterized and identified One ml from each of the samples was separately by initially examining colonies macroscopically on homogenized in 9.0 ml of sterile peptone water. their cultural properties followed by physiological The dilution was serially made until 10-5 level of and biochemical tests (Motility test, citrate test, dilution was obtained. Isolation and identification coagulase test, indole test, starch fermentation test, was done according to the method of Ogbulie gram stain, spore stain catalase test and oxidase et al. (2005) and ICMMSF (1978). For bacterial test, etc). The fungal isolates were characterized isolation, nutrient agar, MacConkey agar were by their cultural properties stained with cotton- used, while sabouraud dextrose agar was used for blue lactophenol solution and observed under low fungi isolation. Total viable counts of bacteria were power objective lens (Chessbrough, 1984; Kovac determined by enumerating the colony forming 1956; ICMMSF, 1978; Ogbulie et al. 2005). units (cfu) by pour plating 1.0 ml of 10-5 diluent Results and Discussion incubated at 37oc for 48 h. Total fungi counts were Results of the effect of spice concentration on determined by pour plating also and incubated at storage time of 3 to 4 days on the microbial quality 37oc for 3 days. The experiments were carried out of soymilk and kunun-zaki is presented in Tables 1 in triplicates. Pure cultures of bacteria and fungal and 2.

Table 1: Bacteria load of spice treated soymilk during storage at ambient temperature Spice Storage time concentration (Days) % 1 2 3 4 0.4 7.77 x 106b 10.01 x 106b 9.13 x 106b 9.00 x 106b 1.2 5.63 x 106c 8.80 x 106c 7.80 x 106b 7.20 x 106b 2.4 4.40 x 106d 6.57 x 106d 5.80 x 106b 5.20 x 106b 3.6 3.73 x 106d 6.13 x 106d 4.70 x 106b 4.10 x 106b 0.4 6.27 x 106c 8.67 x 106d 7.43 x 106b 7.10 x 106b 1.2 5.03 x 106d 6.43 x 106d 5.53 x 106b 5.25 x 106b 2.4 3.93 x 106d 5.37 x 106d 4.47 x 106b 4.23 x 106b 3.6 3.20 x 106d 5.27 x 106d 4.10 x 106a 3.90 x 106b Control 10.90 x 106a 12.40 x 106a 12.20 x 106a 11.90 x 106a Means with the same superscripts on the same row are not significantly different (p < 0.05). Comparative Studies of Ginger and West African Black Pepper Extracts at Different Concentrations ... Udensi, Odom and Dike 41

Table 2: Bacteria load of spiced treated kunun-zaki during storage at ambient temperature Spice Storage time Concentration (%) Days 1 2 3 4 0.4 5.17 x 106b 4.33 x 106b 5.60 x 106b 2.83 x 106b 1.2 4.90 x 106b 3.7.3 x 106b 3.33 x 106b 2.60 x 106b 2.4 3.83 x 106c 3.33 x 106bc 3.07 x 106bc 2.47 x 106b 3.6 3.30 x 106c 3.03 x 106bc 2.53 x 106bc 2.17 x 106b 0.4 4.63 x 106b 4.10 x 106b 3.27 x 106b 2.87 x 106b 1.2 3.83 x 106c 3.43 x 106bc 3.03 x 106bc 2.60 x 106b 2.4 3.37 x 106c 2.63 x 106cd 2.40 x 106c 2.20 x 106b 3.6 2.90 x 106c 2.40 x 106cd 2.20 x 106c 2.00 x 106b Control 8.44 x 106a 10.31 x 106a 9.23 x 106 8.0 x 106b Means with the same superscript on the same row are not significantly different (p < 0.05)

The microbial count on the day of production was (Powers et al., 1995). The chemical composition generally low, 1.30 x 106 and 9.7 x 105 in both samples varies in spices, hence black pepper kept the shelf respectively, until the second day of storage when life of the soymilk and kunun-zaki longer than there was a spontaneous increase in the bacterial ginger as found in this test. load in both soymilk and kunun-zaki ranging from However, the microbial load started to decline 7.77 x 106b at a concentration of 0.4% and 3.73 x by the third day. This could be attributed to the 106d at a concentration of 3.6% for ginger extract possibility of increase in acidity resulting from and 6.27 x 106c at a concentration of 0.4% and microbial fermentation of the samples and the 3.20 x 106d at a concentration of 3.6% using black depletion of nutrients available and the release of pepper extract; 5.17 x 106b at 0.4% and 3.30 at 3.6% toxins by the microorganisms. ginger extract; 4.63 x 106b at 0.4% and 2.90 x 106d at 3.6% black pepper extract for kunun-zaki. Tables 3 and 4 present a summary of the microorganisms identified. At lower concentration of It was observed that there was a linear reduction the spice extract, five species of bacteria were in the bacteria load with increase in concentration isolated including staphylococcus aureus bacillus cereus, of spice. This indicates the antimicrobial activity pseudomonas, proteus and lactobacillus species. At higher of ginger and black pepper extract. In the ginger concentration of spices two species were identified. treated samples, there was a moderate reduction This indicates that at higher concentration of these in microbial load when compared with those of spices, some types of bacteria are killed. black pepper treated samples, showing that black pepper spice has more antimicrobial activity than Comparing Tables 1 and 2, it was observed that the ginger. The active component of spices at low microbial load in soymilk is generally higher than concentration might have interacted synergistically the kunun-zaki sample, also kunun-zaki was able with other factors to increase preservative effect. to preserve longer than soymilk. Kunun-zaki is a fermented product. According to Odunta (1985), The preservative effect of these spices is due to their different fermented products have the advantage chemical composition especially the essential oil of prolonged shelf life because of the organic fractions which are inhibitory to microbial growth 42 Nigerian Food Journal Vol. 30 No. 2, 2012 ... Research Note acid produced during fermentation which is fatal fermentation helps to increase the shelf life and to certain microorganisms. This also agreed with digestibility of foods. the work done by Achi (1999) who observed that

Table 3: Bacteria flora of the spice treated soymilk at ambient temperature

Organisms Staphylococcus Bacillus Pseodomonas Proteous Lactobacillus Spice Conc % 0.4 + + + + + 1.2 + + + + + 2.4 + + – + +

Ginger extract 3.6 + + – – + 0.4 + + + + + 1.2 + + – + + 2.4 + + – – + 3.6 – + – – + Black pepper

Table 4: Bacteria flora of the spice treated Kunun-zaki at ambient temperature Organisms Staphylococcus Bacillus Pseodomonas Proteous Lactobacillus Spice Conc % 0.4 + + + + + 1.2 + + + + + 2.4 + + – – + Ginger extract 3.6 – + – – + 0.4 + + + + + 1.2 + + + – + 2.4 + + – – +

Black pepper 3.6 – + – – +

+ = Present – = Absent Comparative Studies of Ginger and West African Black Pepper Extracts at Different Concentrations ... Udensi, Odom and Dike 43

Conclusion ICMMSF: International Commission of Microbiology It is evident from this study that ginger and black Specifications of Foods (1978). Microbial Ecology of Food Vol. 2 Food Commodities. Academic Press, London. pepper extracts possess antimicrobial properties, since they can extend the shelf life of soymilk Iwe, M.O. (2003) The science of technology of soybean, pp. 64 – 93. and kunun-zaki from 12 and 24 h respectively to a maximum of 4 days at ambient temperature. The Juliani, H.R., Zygadlo, J.A., Scrivanti, R., Sofa dela, E. and Simo, J.E. (2004) The essential oil of anemia tomentosa (savigny) Sar microbial activity of the spices varies especially at Var. anthriscifolia schrad. Michel Flavour Frag. J. 19: 541 – 543. concentration from 0.4% and above. Black pepper Kovac, N. (1956) Identification of pseudomonas pyocyanea by spice possesses more antimicrobial properties than oxide the reaction nature, London, 178 – 703 Reaction nature, ginger and may be preferred for use as natural anti- London 178 – 703. microbial preservatives to extend the shelf life of Nwafor, O.E. and Ogiehor, I.S. (2003) Medical stability of food. meat preserved by Hurdle technology, Prog Niger. Inst. Food Science, pp. 74 – 76. References Nsofor, L.M. and Anyanwu, K.B. (1992) Development and Achi, O.K. (1999): The potential for upgrading traditionally evaluation of concentrated soymilk beverage. J. Fd Sc Technol fermented food through biotechnology. African Journal of 29 (5): 331 – 332. Biotechnology 5: 376 – 378. Nsofor, C.M. and Osuji, C.M. (1997) Storage stability and Adeyemi, I.A. and Umar, S. (1994) Effect of method of chemical properties of soymilk from sprouted soybeans J. Food manufacture on quality characteristics of kunun-zaki a millet Sc. Techn. 54 (7): 527 – 530. based beverage. Nigerian Food Journal 12: 34 – 41. Odunta, S.A. (1985) Microbiological and toxicology aspect Akpomedaye, D.C. and Ejechi, B.O. (1998) The hurdle effect of fermentation of castor oil seeds for original production. of mild heat and two tropical spices extract on growth of three Journal of Food Science 50: 1758 – 1759. fungi in fruit juices. Res. Int. 31 (58): 339 – 341. Ogbulie, T.N., Uwaezuoke, T.C. and Ogiehor, S.I. (2005) Chessbrough, M. (1984) Medical Laboratory Manual for Tropical Introductory Microbiology Practicals. Springfield Pub., Owerri Countries Vol. II Microbiology. 1st Edn., English Language Nigeria, pp. 67 – 82. Book Society, London. Ogiehor, I.S., Owhe Ureghe, U.B. and Momodu, I.O. (1998) Dziekak, J.D. (1989) Innovation food trends. Spices J. Food Microbial, chemical and organepltic quality of palm-wine Technol 43 (1): 102 – 116. preserved by combination of preservatives. Nigerian J. Microbial Enwere, M.J. (1998) Foods of Plant Origin Processing and Utilization 1.12 (1 – 2): 63 – 68. with Recipes and Technology Profiles. Afro Orbis Publication Ltd., Ohr, M.C. (2004) Neutraceuticals and functional foods. Food Nsukka, pp. 24 – 228. Technol 58 (8): 71 – 72. Graffia, T., Jidean, I.A. and Nkama, L. (2002) Nutrient and Powers, E.M., Layer, R. and Masuoka, Y. (1995) Microbiology sensory qualities of Kunun-zaki from different saccharifying of processed spices J. Mild Food Technol. 35: 683 – 689. agents. Int. J. Food Sci. Nut. 53: 109 – 115. Zia-ur-Rehman, Salariya and Habib, F. (2003) Antioxidant activity of ginger extract in sunflower oil. J. Sc. Food Agric; 83: 624 – 639.