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Copyright: © 2018 EndeshawAbatenh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 30 November -0001 | Accepted: 31 January 2018 | Published: 19 February 2018
Keywords: Azo, Bactria, Fermentation, Lactic acid, Gen III Microplate, MRS media, Omnilog.
Azo is a local fermented condiment prepared by fermenting cooked Endod (Phytolaccadodecandra) leaves and sorghum porridge along with different spices in KaftaHumera, North West Tigray regional state. A wide variety of traditional fermented foods and condiments are consumed in Ethiopia The study aimed to identify Lactic acid bacteria (LAB) fromAzo condiment by using OmnilogGenIII identification system. LAB was cultured on DeMan Rogosa Sharpe Agar (MRS agar) media. Pure bacterial cells were suspended in inoculum fluid A at 90-98% turbidity, 100 µ L cell suspension were transferred into 96 wells of GenIII micro Plate(71 carbon source utilization assays + 23 chemical sensitivity assays+ 2 controls) by digital pippetor and incubated at 30+2°C for 48 h, then read by Omnilog Reader, results were recorded and processed for identification by comparing the test organism with the data base of Omni log species library. Omni log acceptable results were above 0.5 similarity index value (SIM).The final result revealed that 3bacterial species were identified. Lactobacillus plantarum (0.795SIM), Lactobacillus delbrueckiiss lactis(0.719 SIM),Streptococcus criceti(0.689SIM).There was little information on LAB species associated with Azo condiments in Ethiopia. Currently, LAB is a focus of intensive international research for their essential role in fermented foods as a starter culture and for their ability to produce various antimicrobial compounds promoting probiotic properties in human health improvement.
Fermented foods, beverges,condiments constitute a major portion of peoples diet in all parts of the world(Ashenafi, 2002).It estimated that 25% of the European diet and 60% of the diet in many developing countries consist of fermented foods (Stiles, 1996; Ogunbanwo et al., 2004). Commonly known like Kefir, Sauerkraut, Kimchi, Cortido,Sourdough, Kvass, Kombucha, Pulque, Kaffir beer, Ogi, Igunaq, Miso, Tepa, Dosa, Surströmming, Wine,Crème fraîche, are consumed by large people all over the world (Stephanie et al.,2015). Ethiopia is rich in cultural diversity where, fermented food products constitute a major portion of people’sdiet(Ashenafi,2006).Among the diversified traditional fermented foods currently mentioned are those of which studied in their microbiological characteristics includes; traditional fermented dairy productsergo (sourmilk), ayib (cottage cheese), qibe (traditional butter), arrera (defatted buttermilk)(Almaz et al., 2001). Some from traditional fermented plant foods includes injerra, kotcho, siljo (Tetemkeand Mogessie, 1995)and awaze, datta (qotchqotcha)are the commonly known condiments (Ahmed et al., 2001). Some of the traditional fermented alcoholic and non-alcoholic beverages aretella (Samueland Berhanu, 1991), tej (Vogel et al., 1983)keribo, ckeka, boka, bordae and shamita (Tetemke and Mogessie, 1995).Fermented condiments areused as taste enhancers in many traditional dishes. They have constituted a significant proportionof the diet of many people in different parts of the world (Achi, 2005).Although, a wide variety of traditional fermented foods,beverage and condiments are consumed in Ethiopia.Azo condiment fermentation process is not widely recognized by many people and fermentermicrobes are not studiedwell compared to other Ethiopian traditional fermented food and beverages. Azo is a traditional fermented semi-solid condiment, which is consumed during Easter fasting season in western Tigray region, particularly in Kafta and Welkait as well as in Gonder. Azo is prepared by fermenting cooked Endod (Phytolacca dodecandra) leaves and sorghum porridge along with different spices (rue, garlic, ginger, korerima, black cumin, black mustards). In recent years, the use of fermented food condiments and flavoring agents are becoming popularin the diets of many nations. Apart from the fact that those condiments improve sensoryproperties of foods and vitamins (Kolapo et al., 2007), some of them contain antioxidants andnutraceuticals that provide health benefits (Omafuvb et al., 2004; Dakwa et al., 2005). The use of microbial fermenters has been instrumental in making a large range of foods, popular around the world. Microbial fermentation has played an important role to preserve food products, to enhance nutritive value, to destroy undesirable factors, to ensure product safety, and to improve the appearance and taste of food products (Paredes-Lopez, 1992). It also results in desirable biochemical changes such as development of aroma and flavor compounds in food products (Cooke et al., 1987). Fermentation plays an essential role in conferring the required stability, safety and sensory properties of the food products. Therefore, there are only four main fermentation processes, which are alcoholic, lactic acid, acetic acid and alkali fermentation (Soni and Sandhu, 1990). Various microorganisms of the genera Enterococcus, Bacillus, Clostridium, Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Trichosporon, Torulopsis, Rhodotrula, Saccaromyces, Kluyveromyceswere dominant in Ethiopian fermented foods, beverages and condiments (Ketema,1998;Moges and Tetemk 1995; Ahmed 2001; Samuel and Birhanu ,1991; Yoneya etal., 1999;Almaz et al., 1999).Currently, LAB are a focus of intensive international research for their essential role in fermented foods as a starter culture and for their ability to produce various antimicrobial compounds promoting probiotic properties (Temmermanet al., 2002),However lactic acid bacteria isolated and identified from many fermented food (Injera,Kotcho,Bulla,Yoghurt,Curdle, Cottage cheese), beverages (Tella,Teje,Bordae,Shameta, Keribo) and condiments (Data, Awaze,Siljo) in Ethiopia using different conventional and biochemical methods, There is little information on Lactic acid bacteria fromAzo condiment. This study was aiming at identification of lactic acid bacteria from traditional Azo condiments using Ominilog GEN IIIfrom kaftaHumera,Tigray regional state.
Materials And Methods
The study was conducted at KaftaHumera, AddiHirdiwhich is found in part of the West Tigray Zone. The study area is located 984 km (611 mi) northwest of the capital Addis Ababa, 515 km (320 mi) west of Mek'ele, and 267 km (166 mi) east of Shire.with 14° 00' 0.00" N latitude and 37° 00' 0.00" E longitude at an altitude of 1094 meter above sea level (masl).KaftaHumera is bordered on the South by Tsegede, on the west by Sudan, then by the Tekezé River which separates KaftaHumera from Eritrea on the north and SemienMi'irabawi Zone on the east, and on the southeast by Wolqayt. Towns in KaftaHumera include AddiHirdi. Town of Humera was part of KaftaHumera district.
Sample Collection and Transportation
During Azo condiment sample collection all necessary equipment were disinfected with ethanol (70%). 50mL azo condiment sample were collected aseptically from each house hold using sterile screw bottles and kept in icebox. The sample immediately transported to Ethiopian biodiversity institute microbial laboratory and kept at +40C until processed (Figure.1)
A total of 31 bottle Azo condiment samples were collected purposively from different household where azo food preparation is frequent and highly consumed in KaftaHumera district, AdiHerdi,West TigrayZoneEthiopia.
Isolation of microorganisms
From each Azocondiment samples approximately about 0.1 mL transferred by swab and streaked by nichrom loop on MRS media (Proteose peptone 10g, Beef extract 10.g, Yeast extract 5.g, Dextrose 20.g, Polysorbate 80 1.000mL, Ammonium citrate 2.g, Sodium acetate 5.g, Magnesium sulphate 0.1g, Manganese sulphate 0.050g, Dipotassium phosphate 2.g Agar, 12.g/1L, final pH at 25°C 6.5±0.2). The primary cultures wereincubated anaerobically for 35°C+2 in digital incubator for 48 h. Isolates were subculture twice until pure colony obtained for morphological identification and further Omnilog identification.
Identification of colonial morphology
The bacterial morphology grown on MRS agar media at 35°C+2 for 48h were examined and its colony morphology such as form, size, texture,elevation, margin/edge, appearance, optical property, andcolor/pigmentation of colony were observed using hand lens and recorded.
Identification of LABusing OmniLog ID System
After a pure bacteriumcolonygrown MRS agar media and incubated at 35+2˚C for 48h. Pure bacterial colony suspension were prepared in inoculation fluid (IF A) and adjusted to 90-98T using biologturbidiameter.100µ-L of inoculum was dispensed using digital pipettor to each of 96 wells of GEN III micro pate and incubated at 30˚C 48h. The GEN III micro plate is tagged with 96 carbon source. An isolate ability to metabolize each carbon source is measured in the presence or absence of purple hue in the wells. Tetrazolium violet a redox dye forms a purple color when oxidized by cellular respiration of microorganisms. The GEN III micro plate measures both metabolic reactions as well as turbidity growth to produce identifications.The Biolog GEN III MicroPlate analyzes a microorganism in 94 phenotypic tests: 71 carbon source utilization assays and 23 chemical sensitivity assays.GEN III micro plate was read by the Omnilog identification system reader at 48 h.Using all Biolog databases, over 2650 species of bacteria, yeast and filamentous fungi can be identified.The OmniLog® GEN III Data Collection Software, Retrospect 2.0 compared the results obtained with the test strain to the database and provided identification based on distance value of match and separation score produces similarity index value and probability for species identification. Above 0.5 similarities index value result is acceptable species identification (Biolg1993).
After isolation and identification of bacterial species using omnilog ID system, the genetic materials were kept in microbial gen bank using a long term cryopreservation techniques.
Conservation of biological resource is one of the Ethiopian biodiversity institute mandate and Convention of BiologicalDiversity (CBD) treaty to protect genetic resource loss due to several factors, for access and benefit sharing, further characterization in other academic and scientific research, all identified LAB were kept in long term preservation protocol 10% glycerol cry protectant at +4OC and -80OC in microbial Gene Bank of Ethiopian Biodiversity Institute.
All laboratory generated data was analyzed using descriptive statistics like percentage frequency. And omnilog result is analyzed by Bayesian statistics about probability and similarity result.
Screening and isolation
A total of 68 bacterial colonies were grown MRS agar media.Pure colonies having similar morphology were counted and clustered in order to detect percentage frequencies of the microorganisms. Thirty five percent were Lactobacillus plantarum,Lactobacillus delbrueckiisslactis(29%), Streptococcus criceti (17%), Unidentified colony (19%).The following table will summarize (Table 1)
Identified bacterial species
Percentage of Frequency
Lactobacillus plantarum
Lactobacillus delbrueckiisslactis
Streptococcus criceti
Table1. Percentage frequency of LAB on MRS media.
Colony morphology characteristics of LAB
Their shape, size, elevation, reverse side, mycelium color, margin and color of the colony were observed on MRS agar. Macro morphological results were noted down in summarized table 2.
Identified bacterial species
Table.3. Identified bacteria species by GNE III omnilogID system
Lactobacillus plantarum
Streptococcus criceti
Lactobacillus delbrueckiisslactis
Table.2. Bacterial colony morphology
Omnilog identification result
GEN III micro Plate “Metabolic finger print” when read against a BiologMicroLog database showed a similarity index calculated based on the reaction profilesof 96 carbon sources. (Biolog 2016). By comparing the test organisms with the bacteria databaseof omnilog species library. Result revealed that three LAB were identified and two bacteria were unidentified. The result summarized in table 3
KaftaHumera /AddiHirdi/
Lactobacillus plantarum
KaftaHumera /AddiHirdi/
Streptococcus criceti
KaftaHumera / AddiHirdi/
Lactobacillus delbrueckiisslactis
Fermented foods and the microorganisms that contribute to the fermentation process have beenassociated with many beneficial effects on human health.They have long been a part of the human diet, and with further supplementation of probiotic microbes in some cases, they offer nutritional and health attributes worthy of recommendation of regular consumption. Active fermenting microorganisms, such as lactic acid bacteria or Enterobacteriaceae, which are usually important in the fermentation of various traditional Ethiopian food products (Berhanuet al., 1982; Berhanu 1985, 1987; Ashenafi 1994). Many LAB species find wide industrial applications, mainly as starter or complementary cultures, in several food fermentations. The most commonly food-associated genera belong to Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Oenococcus, and Streptococcus (Pot, 2008). Various microorganisms, the genera Enterococcus, Bacillus, Clostridium, Lactoccus, Lactobacillus, Leuconostoc, Streptoccus, Trichosporon, Torulopsis, Rhodotrula, Saccaromyces, Kluyveromyces were dominant in Ethiopian fermented foods, beverages and condiments (Ketema,1998;Moges and Tetemk, 1995; Ahmed, 2001; Samuel and Birhanu,1991;Yoneya etal., 1999;Almaz et al., 1999). In this study three fermenter bacteriaLactobacillus plantarum Lactobacillus delbrueckiisslactis, Streptococcus cricetiwere identified from Azo condiment using omnilog ID system(Table3). Fermented condiment is used to make the food more tasty and enjoyable (Hesseltine,1983).Although in most part of Africa, protein rich food ingredientare often fermented to make condiments, low protein ingredients are also fermented into condiments in Ethiopia. Siljo, Datta, Awaze are traditional condiments of Ethiopia.Teeemk and Mogese 1995 reported Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus delbruekiiintiated fermentation and later the member of the genera Enterococcus, Bacillus, Lactoccus, Lactobacillus and yeast were the dominant microorganism fermenting Siljo (SenaitZewdie et al 1995). According to Ahmede 2001 Bacillus and Lactic acid bacteria were involved in Datta and Awaze fermentation.However Lactobacillus plantarum isolated from Azo condiment in this study also corresponds with research done in other fermented food and condiments of Ethiopian where also isolated from cottage cheese, InjeraErsho,fermentedkotcho(Ketema ,1998., Moges and Tetemk, 1995., Samuel andBirhanu ,1991).Lactobacillus pluntururn is important in vegetable fermentation (Vaughn, 1985) and can inhibit a variety of food-borne pathogens during tempeh-type fermentations of horse bean and other legumes (Ashenafi 1991; Ashenafi andBusse1991a, 1992) and other food fermentations (Gilliland & Speck 1972).Similar work indicated by Teteemk and Mogese 1995 reportedLactobacillus delbrueckiisslactis also isolated from other condiments like siljo, Data and awaze corresponds to azo LAB identification result. (Yoneya etal., 1999;Almaz et al., 1999) reported that Lactococcusgarviae, Lactococcuslactis sub Lactisstreptoccus, Leconostoc and Lactobacillus were identified from fermented dairy products, Ergo andMicrobacterium,Brevibacteriumspp,Lactobasillusfermenti,andLactobacillusplantarumdominated flora of cottage cheese (Mogess Asshenafi1994a).According to BerhanuAbegazGashe 1985 a complex group of microorganisms was involved during injerafermentation and a dominant microflorawere,Leuconostocmesenteroids,enterococcusfacalispediococcuscerevisieae,Lactobacillusbrevis, Lactobacillus plantarumand Lactobacillus fermentum.BirhanuAbegazGashe 1987astudied and described the microbiology of Kotcho fermentation, Leuconostocmesenteroids, Lactobacillus plantarum, Lactobacillus coryneformis,Clostridiumbutyricum, Clostridium beijerinckii, Clostridium sticklandi, Bacillus subtilis,Bacillus megaterrium,Bacillus licheniformis, Bacillus cereus, Trichosporon, Torulopsis,Rhodotrulaand Candida sppwere the dominant micro flora. Various groups of microorganisms also involved in the fermentation of beverages indigenous to Ethiopia.In Tella, Tej, Shameta,Bordae, beverages, Saccaromycescerevisieae, Lactobacillusspp, Micrococci, staphylococci,Bacillus,coliforms,Enterobacteriaceae,kluyveromycesbulgaricus, Debromycesphaffi, kluyveromycesveronaewere reported (Ketema ,1998., Moges and Tetemk 1995., Samuel andBirhanu,1991). In this study Streptococcus criceti species from Azo condiment might benew fermenter not yet identified from other fermented food,beverage and condiments of Ethiopia.This might be from Endod(Phytolaccadodecandra).LAB are considered to have several beneficial physiological effects, such as enhancing of immune potency (Kullisaaret al., 2002) and prevention of cancer and lower serum cholesterol levels (Kauret al., 2002). LAB also have beneficial influence on nutritional and sensory characteristic as well as on the standardization of end product (Gawad et al., 2010). Furthermore, they play a vital role in antagonistic activities of human pathogens directly through production of secondary metabolites such as organic acids, bacteriocin and bacteriocin-liked inhibitory substances (GirumTadasse et al., 2005), which is good solution to address the current serious problem of drug resistant (Cuetoet al., 2007). Thus, recently receive higher attention of many researchers to achieve nutritional and heath problem in the world (Temmermanet al., 2002).
In the present study, Lactobacillus plantarum, Lactobacillus delbrueckiisslactis, and Streptococcus cricetiwere identified from Azo Condiments using selective MRS media and andOmnilogGenIII identification system. However Lactobacillus plantarum, Lactobacillus delbrueckiisslactisSpecies are common fermenter in other fermented food, beverage and condiments of Ethiopia, Streptococcus cricetinewly identified LAB from Azocondiments. These three LAB species are very important to prepare starter culture, Probiotics, and other nutricitical and pharmaceutical application in the era of Biotechnology.
Based on the above concluding remarks further research should be continued on Azo condiments on yeast fermenter and bacteria other than LAB using molecular identification because this is not studied well condiments of Ethiopia. These identified organisms should further evaluated and characterizedfor fermentative ability test and probiotics potential for human health.
Conflict of Interests
The authors have not declared any conflict of interest.
Authors would like to acknowledge DerjeHailufor his unreserved guidance and encouragement and support in providing and facilitating the necessary equipment. Finally goes to Ethiopian biodiversity institute, microbial directorate for every budget grant to carry out this study and its research team for their unreserved support at laboratory work. Lastly, author’s acknowledgeWoyenshetLule and RomaneNega for her kindly support especially laboratory chemicals facilitation.
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