The Open Biotechnology Journal




ISSN: 1874-0707 ― Volume 13, 2019
RESEARCH ARTICLE

Molecular Identification of Yeasts and Lactic Acid Bacteria Involved in the Production of Beninese Fermented Food Degue



Angel I. Angelov1, *, Galya Petrova2, Angel D. Angelov1, Petya Stefanova1, Innocent Y. Bokossa3, Célestin K. C. Tchekessi3, Maria L. Marco4, Velitchka Gotcheva1
1 Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria
2 Primoris Bulgaria, 134 Bratia Bakston Str., Plovdiv 4004, Bulgaria
3 Laboratory of Microbiology and Food Technology, Department of Vegetable Biology, Faculty of Sciences and Techniques, University of Abomey-Calavi, 04BP 1107 Cotonou, Benin
4 Department of Food Science & Technology, University of California, Davis, CA, 95616, USA

Abstract

Background:

Traditional Beninese fermented food Degue is widely consumed in Benin and other countries in West Africa. It was originally made from milk and millet flour, but currently other cereals are used as well. Nowadays, Degue production occurs by spontaneous fermentation in individual households and information about the microorganisms involved is currently limited.

Objective:

The microbiota of Degue from Benin has not been studied so far, but its growing production in the country sets a demand for revealing the biodiversity of the microbial population involved in the fermentation process in order to take future steps for development of industrial technology and offer products with improved quality and safety.

Method:

In the present study, yeast and lactic acid bacteria from raw materials for Degue production and from several Degue products were isolated and identified by molecular methods including RFLP and ITS1-5.8S-ITS2 rRNA gene sequence analysis in yeasts, and 16S rRNA gene sequence analysis in lactic acid bacteria.

Results:

Lactic acid bacteria isolates were assigned to eight species within the genera Lactobacillus, Enterococcus, Pediococcus, Streptococcus and Weisella. Four species of yeasts were found in Degue: Cyberlyndnera fabianii, Candida glabrata, Kluyveromyces marxianus, and Meyerozyma caribbica.

Conclusion:

The microbial population revealed is unique to Beninese Degue and needs further characterization for development of defined starter cultures.

Keywords: Degue, Benin, Identification, Yeast, Lactic acid bacteria, Molecular methods.


Article Information


Identifiers and Pagination:

Year: 2017
Volume: 11
First Page: 94
Last Page: 104
Publisher Id: TOBIOTJ-11-94
DOI: 10.2174/1874070701711010094

Article History:

Received Date: 11/04/2017
Revision Received Date: 19/06/2017
Acceptance Date: 09/09/2017
Electronic publication date: 21/09/2017
Collection year: 2017

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© 2017 Angelov et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria, E-mail: angelov@uft-bio.com




1. INTRODUCTION

Fermentation processes are regarded as an important economical form of food production and preservation. Advantages of food fermentation include extended shelf-life of the products, improved palatability, digestibility, nutritive value, texture, taste and aroma resulting from the activity of the microorganisms involved [1Blandino A, Al-Aseeri ME, Pandiella SS, et al. Cereal-based fermented foods and beverages. Food Res Int 2003; 36: 527-43.
[http://dx.doi.org/10.1016/S0963-9969(03)00009-7]
-3Shett PH, Jespersen L. Saccharomyces cerevisiae and lactic acid bacteria as potential mycotoxin decontaminating agents. Trends Food Sci Technol 2006; 17: 48-55.
[http://dx.doi.org/10.1016/j.tifs.2005.10.004]
]. The most common microorganisms found in fermented foods are yeasts and lactic acid bacteria (LAB). These organisms form stable mixed populations and the species composition depends on the raw materials used, geographical factors, preparation methods, production hygiene, etc [4Abriouel H, Ben Omar N, López RL, Martínez-Cañamero M, Keleke S, Gálvez A. Culture-independent analysis of the microbial composition of the African traditional fermented foods poto poto and dégué by using three different DNA extraction methods. Int J Food Microbiol 2006; 111(3): 228-33.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2006.06.006] [PMID: 16842876]
-8Tchekessi CK, Bokossa IY, Azokpota P, et al. Isolation and quantification of lactic acid bacteria from traditional fermented products in Benin. Int J Curr Microbiol Appl Sci 2014; 3: 1-8.]. Africa is regarded to be the continent with the richest variety of lactic acid fermented foods prepared from different raw materials [2Holzapfel WH. Appropriate starter culture technologies for small-scale fermentation in developing countries. Int J Food Microbiol 2002; 75(3): 197-212.
[http://dx.doi.org/10.1016/S0168-1605(01)00707-3] [PMID: 12036143]
, 9Anukam KC, Reid G. African traditional fermented foods and probiotics. J Med Food 2009; 12(6): 1177-84.
[http://dx.doi.org/10.1089/jmf.2008.0163] [PMID: 20041769]
-12Odunfa SA. In: Wood BJB, Ed. African Fermented Foods. Microbiology of Foods. Elsevier, London 1985; 155-191.]. However, yeasts are also involved in these fermentations, but information on the dominant yeast species in African fermented foods is limited [13Chelule PK, Mokoena MP, Gqaleni N. In: Méndez-Vilas A, Ed. Advantages of traditional lactic acid bacteria fermentation of food in Africa. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology 2010; 1160-7.].

In Benin, a country in West Africa which is highly dependent on agriculture, fermented foods represent a major part of the daily diet and their production plays an important role in the Beninese domestic economy [14Tchekessi CK, Bokossa A, Agbangla C, et al. Production and microbiological evaluation of three types of “Dèguè”, a local fermented drink made from milk in Benin. Int J Multidisc Cur Res 2014; 2: 714-20., 15Tchekessi CK, Banon J, Seni I, et al. Socio-economic study of a fermented drink “Dèguè” made with milk and cereals in Benin. Int J Multidisc Cur Res 2014; 2: 626-32.]. Most fermented foods are prepared by spontaneous fermentations performed either at individual households or by small-scale companies using the locally available cereals maize, sorghum and millet, sometimes combined with milk [1Blandino A, Al-Aseeri ME, Pandiella SS, et al. Cereal-based fermented foods and beverages. Food Res Int 2003; 36: 527-43.
[http://dx.doi.org/10.1016/S0963-9969(03)00009-7]
, 4Abriouel H, Ben Omar N, López RL, Martínez-Cañamero M, Keleke S, Gálvez A. Culture-independent analysis of the microbial composition of the African traditional fermented foods poto poto and dégué by using three different DNA extraction methods. Int J Food Microbiol 2006; 111(3): 228-33.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2006.06.006] [PMID: 16842876]
, 14Tchekessi CK, Bokossa A, Agbangla C, et al. Production and microbiological evaluation of three types of “Dèguè”, a local fermented drink made from milk in Benin. Int J Multidisc Cur Res 2014; 2: 714-20.-17Byaruhanga YB, Bester BH, Watson TG. Growth and survival of Bacillus cereus in mageu, a sour maize beverage. World J Microbiol Biotechnol 1999; 15: 329-33.
[http://dx.doi.org/10.1023/A:1008967117381]
]. Fermented products of these cereals are commonly consumed as refreshing beverages in the urban areas of Benin, especially appreciated during periods of extreme heat. One of these fermented beverages is Degue (Dèguè), which is also consumed in Burkina Faso and Mali. Degue varieties are produced by varying methods from mixtures of cereals with/or fermented milk [4Abriouel H, Ben Omar N, López RL, Martínez-Cañamero M, Keleke S, Gálvez A. Culture-independent analysis of the microbial composition of the African traditional fermented foods poto poto and dégué by using three different DNA extraction methods. Int J Food Microbiol 2006; 111(3): 228-33.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2006.06.006] [PMID: 16842876]
, 8Tchekessi CK, Bokossa IY, Azokpota P, et al. Isolation and quantification of lactic acid bacteria from traditional fermented products in Benin. Int J Curr Microbiol Appl Sci 2014; 3: 1-8., 14Tchekessi CK, Bokossa A, Agbangla C, et al. Production and microbiological evaluation of three types of “Dèguè”, a local fermented drink made from milk in Benin. Int J Multidisc Cur Res 2014; 2: 714-20., 15Tchekessi CK, Banon J, Seni I, et al. Socio-economic study of a fermented drink “Dèguè” made with milk and cereals in Benin. Int J Multidisc Cur Res 2014; 2: 626-32., 18Hama F, Savadogo A, Outara CA, Traore S. Biochemical, microbial and processing study of Degue- a fermented food (from pearl millet dough). Pak J Nutr 2009; 8: 759-64.
[http://dx.doi.org/10.3923/pjn.2009.759.764]
, 19Soro-Yao AA, Brou K, Amani G, Thonart P, Djè KM. The use of lactic acid bacteria starter cultures during the processing of fermented cereal-based foods in West Africa: A review. Trop Life Sci Res 2014; 25(2): 81-100.
[PMID: 27073601]
]. In Benin, due to the growth of street food consumption, production scale of this beverage has been rapidly increasing in the past few years. However, Degue production still occurs without any control of manufacturing parameters and hygiene practices [8Tchekessi CK, Bokossa IY, Azokpota P, et al. Isolation and quantification of lactic acid bacteria from traditional fermented products in Benin. Int J Curr Microbiol Appl Sci 2014; 3: 1-8., 15Tchekessi CK, Banon J, Seni I, et al. Socio-economic study of a fermented drink “Dèguè” made with milk and cereals in Benin. Int J Multidisc Cur Res 2014; 2: 626-32.]. Information on the microbial species involved in the fermentation process is currently only available for Degue produced in Burkina Faso [4Abriouel H, Ben Omar N, López RL, Martínez-Cañamero M, Keleke S, Gálvez A. Culture-independent analysis of the microbial composition of the African traditional fermented foods poto poto and dégué by using three different DNA extraction methods. Int J Food Microbiol 2006; 111(3): 228-33.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2006.06.006] [PMID: 16842876]
, 18Hama F, Savadogo A, Outara CA, Traore S. Biochemical, microbial and processing study of Degue- a fermented food (from pearl millet dough). Pak J Nutr 2009; 8: 759-64.
[http://dx.doi.org/10.3923/pjn.2009.759.764]
, 20Ouattara CA, Somda MK, Moyen R, Traore AS. Isolation and identification of lactic acid and non-acid lactic bacteria from “dèguè” of Western Africa traditional fermented millet-based food. Afr J Micr Res 2015; 36: 2001-5.], while information related to microbiota of Beninese Degue extends mainly to enumeration of yeast and lactic acid bacteria populations [8Tchekessi CK, Bokossa IY, Azokpota P, et al. Isolation and quantification of lactic acid bacteria from traditional fermented products in Benin. Int J Curr Microbiol Appl Sci 2014; 3: 1-8., 14Tchekessi CK, Bokossa A, Agbangla C, et al. Production and microbiological evaluation of three types of “Dèguè”, a local fermented drink made from milk in Benin. Int J Multidisc Cur Res 2014; 2: 714-20.]. Therefore, an accurate identification of the microbial species involved in the production of different Degue types and understanding of their role in the mixed population is required in order to further develop defined starter cultures and industrial Degue products with improved quality and safety to meet the growing market demand. An industrial technology for the production of Degue may also be of interest to companies in Europe and North America aiming to diversify the food market by the introduction of new fermented products.

On this basis, the aim of the present study was to identify the naturally occurring yeast and LAB in cereal raw materials and milk used for the production of Degue, as well as in several types of Degue products.

2. MATERIALS AND METHODS

2.1. Samples for Isolation of LAB and Yeast

LAB and yeast strains were isolated from 26 samples of raw materials, combinations of them and Degue products, provided by the Laboratory of Microbiology and Food Technology, University of Abomey-Calavi (UAC), Benin (Table 1).

Table 1
Raw materials and product samples for the isolation of LAB and yeasts.


The cereal raw materials were maize (Zea mays L.), millet (Pennisetum glaucum) and sorghum (Sorghum bicolor) from the local market in the city of Abomey-Calavi, Benin. “Yogurt nature” is a starter mix of Lactobacillus bulgaricus and Streptococcus thermophilus (label information), which has been bought at a local pharmacy in Abomey-Calavi. Cow’s milk has also been purchased at the local market. Milk powder (LACSTAR, Ireland) has been bought from Cotonou. The mixed products and the several types of Degue were prepared at the Laboratory of Microbiology and Food Technology, UAC, Benin.

2.2. Isolation of Yeast and Bacteria

For the isolation of yeasts, a decimal dilution method was employed. Diluted samples were plated on Wort agar (Sigma-Aldrich, USA) and incubated at 30oC for 3 days.

Bacteria, and specifically LAB were isolated using the reference ISO 15214:1998 method [21ISO 15214:1998 “Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of mesophilic lactic acid bacteria – Colony count technique at 30°C.” ] with minor modifications. A total of 10 g of each sample was homogenized with 90 ml sterile peptone water, serially diluted and plated on the surface of MRS agar (Oxoid, UK). The plates were incubated anaerobically at 30oC for 48 to 72h prior to colony enumeration.

Single yeast and bacterial colonies with different morphologies were individually selected for purification on Malt Extract and MRS plates, respectively. Yeast isolates were maintained on Malt Extract agar (Sigma Aldrich, USA) at 4oC, while bacterial isolates were maintained in MRS at 4oC (sub-cultured once a month) and in 20% glycerol for long-term storage at -20oC.

2.3. Phenotypic and Biochemical Characteristics of Yeast and Bacterial Isolates

The screening for yeast and LAB strains was based on cultural characteristics, cell morphology and biochemical tests. Cell morphology was examined by microscope (Laboval 4, Carlzeiss, Jena, Germany). Each bacterial isolate was Gram-stained, and only the Gram-positive ones were further tested for catalase production by placing a drop of 3% hydrogen peroxide solution on bacterial biomass. Based on the results, 8 yeast isolates and 30 Gram-positive and catalase-negative putative LAB isolates were further subjected to molecular identification.

2.4. Yeast Identification by Sequencing of ITS1-5.8S-ITS2 Region

Amplification of the ITS1-5.8S-ITS2 region was carried out by transferring biomass from a fresh colony with the tip of a sterile toothpick into a PCR tube containing 10 μl sterile deionized water. Then, 40 μl PCR reaction mix was added to the cell suspension. The PCR reaction mix contained 1 μM of ITS4 (5-TCCTC-CGCTTATTGATATGC-3) and 1 μM of ITS5 (5-GGAAGTAAAAGTG- CTAACAAGG-3) primers (Metabion, Germany), 1 mM dNTPs (Thermo Scientific, USA), 0.8 U Taq polymerase and 1 x PCR buffer (Thermo Scientific, USA) [22Alvarez-Martin P, Florez AB, Lopez-Diaz TM, Mayo B. Phenotypic and molecular identification of yeast species associated with Spanish blue-veined Cabrales cheese. Int Dairy J 2007; 17: 961-7.
[http://dx.doi.org/10.1016/j.idairyj.2006.11.005]
]. The amplification was carried out in a PCR 2720 Thermal Cycler (Biosystems, Germany) using the following program: initial denaturation at 95оС for 10 min, followed by 35 cycles of denaturing at 94оС for 30 s, annealing at 55оС for 30 s, extention at 72оС for 1 min, and final extention at 72оС for 7 min. PCR products were visualized in 2% agarose gel stained with SafeView (NBS Biologicals, UK) at 100 V for 50 min using VWR Mini Electrophoreis System (VWR, Germany) and MiniBis Pro (DNR Bio-Imaging Systems, Israel) for gel visualization. GeneRuler 1kb plus (Thermo Scientific, USA) was used as the molecular marker. Fragment sizes were calculated using image editing software ImageJ64 (freeware).

The PCR products were further subjected to RFLP analysis by digestion with restriction enzymes Hae III, Cfo I and Hinf I (Thermo Scientific, USA) at 37оС for 4h. Restriction fragments were visualized on 2% agarose gel stained with SafeView at 100 V for 55 min, and fragment sizes were calculated using ImageJ64. The obtained RLFP profiles were used for confirmation of yeast identification, which was carried out by DNA sequencing (MacroGen Europe, Inc.). The nucleotide sequences were compared to the GenBank database using the BLAST software [23 Basic local alignment search tool. Available from: http://www.mbio.ncsu.edu/BioEdit/bioedit.html].

2.5. Extraction of Bacterial DNA

Total genomic DNA from putative LAB strains was extracted from overnight cultures, grown in MRS by using a previously optimized Chelex-100 method. Briefly, 18 h-culture of each isolate was centrifuged at 10 000 x g for 10 min (4ºС), washed twice with DNAse-free deionized water and re-suspended in 100 μl 6% Chelex (Bio-Rad Laboratories, Foster City, CA). The suspension was incubated at 56ºС for 20 min, vortexed and subjected to boiling and freezing cycles for 8 min and 5 min, respectively. After centrifugation at 14 000 х g for 5 min (4ºС), 80 μl of the supernatant was incubated with 20 μl Proteinase К (20 mg/mL) (Thermo Scientific, USA) at 65ºС for 60 min. Cell debris was then collected by centrifugation at 14 000 х g for 5 min (4ºС), and the supernatant was stored at -20ºС. Concentration and purity of the extracted DNA (A260/A280) was measured by a spectrophotometer (Shimadzu UV-1800, Japan).

2.6. Molecular Identification of Bacteria by Sequencing of 16S rRNA Gene

PCR amplification of 16S rRNA gene for sequence analysis was performed in thermal cycler (Bio-Rad Laboratories Inc., Hercules, USA). Each PCR contained 10µM of each 27F – AGAGTTTGATCMTGGCTCAG and 1492R – TACCTTGTTACGACTT primer [24Lane DJ. 16S/23S rRNA Sequencing. Nucleic Acid Techniques in Bacterial Systematics 1991; 17: 115-75.], 200 ng DNA, 2.5 mM of each dNTP, and 1.25 U Тakara Ex taqTM DNA polymerase (Takara Biotechnology Co. Ltd., China). Cycling conditions were set at an initial denaturation at 95oC for 3 min followed by30 cycles of denaturation at 94oC for 30 s, annealing at 50oC for 30 s, elongation at 72oC for 90 s, and final elongation at 72oC for 5 min. PCR products were then visualized in 1% agarose gel stained with SYBR Safe DNA gel stain (Invitrogen, USA) and 1 kb DNA ladder (Invitrogen, USA) was used as molecular marker. PCR products were purified with Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and sequenced with universal primer 27F. The nucleotide sequences were analyzed by BLAST, ClustalW and BioEdit software packages [25 Biological sequence alignment editor for Win95/98/NT/2000/XP/7. Available from: http://www.mbio.ncsu.edu/BioEdit/bioedit.html].

3. RESULTS AND DISCUSSION

A total of 42 yeast and bacterial isolates were obtained from cereal and milk samples used for the preparation of Beninese Degue, as well as from several product types. Isolates selection was based on colony morphology, microscopic cell observation and biochemical tests aiming to obtain different yeast and lactic acid bacteria. Eight yeast isolates were selected based on differences of cultural and morphological characteristics, and thirty isolates were tentatively assigned as LAB based on colony and cell characterization, Gram-positive and catalase-negative reactions. The yeast and LAB isolates were further subjected to molecular identification to the species level.

Molecular identification of the yeast isolates was carried out by PCR amplification of the ITS1-5.8S-ITS2 rRNA region [22Alvarez-Martin P, Florez AB, Lopez-Diaz TM, Mayo B. Phenotypic and molecular identification of yeast species associated with Spanish blue-veined Cabrales cheese. Int Dairy J 2007; 17: 961-7.
[http://dx.doi.org/10.1016/j.idairyj.2006.11.005]
]. The amplicons obtained were between 660 to 940 bp. Because of the differences in analytical conditions, none of the yeast isolates could be identified by RFLP profile comparison with previously published patterns of common yeast species [22Alvarez-Martin P, Florez AB, Lopez-Diaz TM, Mayo B. Phenotypic and molecular identification of yeast species associated with Spanish blue-veined Cabrales cheese. Int Dairy J 2007; 17: 961-7.
[http://dx.doi.org/10.1016/j.idairyj.2006.11.005]
, 26Esteve-Zarzoso B, Belloch C, Uruburu F, Querol A. Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Int J Syst Bacteriol 1999; 49(Pt 1): 329-37.
[http://dx.doi.org/10.1099/00207713-49-1-329] [PMID: 10028278]
]. Strain identification based on RFLP profiles alone would require a pattern database obtained with a unified analytical method, as well as more detailed digestion with additional restriction endonucleases, such as AluI, DdeI, ScrFI, and TaqI as strain related pattern variability could occur within a species [26Esteve-Zarzoso B, Belloch C, Uruburu F, Querol A. Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Int J Syst Bacteriol 1999; 49(Pt 1): 329-37.
[http://dx.doi.org/10.1099/00207713-49-1-329] [PMID: 10028278]
].

The obtained RLFP profiles of the yeast isolates were used to confirm identification performed by DNA sequencing. Results from the DNA sequencing showed 4 yeast species belonging to 4 genera - Cyberlyndnera, Candida, Kluyveromyces and Meyerozyma (Table 2). Three of the isolates were identified as Cyberlindnera fabianii (formerly Pichia fabianii, Hansenula fabianii, and Lindnera fabianii). These yeast originated from corn and red sorghum pellets and from Degue prepared from millet pellets. Kluyveromyces marxianus was found in the Degue sample prepared from millet, and an isolate obtained from millet flour was identified as Meyerozyma carribica (formerly Pichia carribica).

Table 2
Identification of yeasts from different raw materials and Degue products by RFLP and sequencing of ITS1-5.8S-ITS2 rDNA region.


Candida glabrata isolates were recovered from millet and red sorghum grains and from Degue prepared with millet and cow’s milk. The three isolates had identical PCR product size (940 bp), and the RFLP profile comparison showed identical patterns with all three restriction endonucleases (Figs. 1A, 1B, 1C - lanes 1, 2 & 4). This confirmed that the isolates were of the same species. Fig. (1) shows similar results with regards to C. fabianii isolates (lanes 5, 7 and 8).

Fig. (1)
Restriction fragment length polymorphism of ITS1-5.8S-ITS2 region of rDNA using restriction enzymes HaeIII (A), HinfI (B), CfoI (C). Lanes 1, 2, 4 - C. glabrata, Lane 3 - M. carribica, Lanes 5, 7, 8 - C. fabianii, Lane 6 - K. marxianus.


The yeast species isolated in this study are commonly found in cereal and dairy products, as well as other food matrices from various regions in the world. Kluyveromyces marxianus was found in other traditional cereal-based fermented foods from Benin - the sorghum-based “gowe”, and in “mawe”, prepared from maze. In mawe it was identified along with C. glabrata in the initial fermentation stage of the product [6Greppi A, Rantsiou K, Padonou W, et al. Determination of yeast diversity in ogi, mawè, gowé and tchoukoutou by using culture-dependent and -independent methods. Int J Food Microbiol 2013; 165(2): 84-8.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2013.05.005] [PMID: 23727651]
, 27Greppi A, Rantisou K, Padonou W, et al. Yeast dynamics during spontaneous fermentation of mawè and tchoukoutou, two traditional products from Benin. Int J Food Microbiol 2013; 165(2): 200-7.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2013.05.004] [PMID: 23756236]
]. It is interesting to note that C. glabrata was not recovered from samples of the final products. K. marxianus has been predominant in dairy products such as the Spanish “Cabrales” cheese [22Alvarez-Martin P, Florez AB, Lopez-Diaz TM, Mayo B. Phenotypic and molecular identification of yeast species associated with Spanish blue-veined Cabrales cheese. Int Dairy J 2007; 17: 961-7.
[http://dx.doi.org/10.1016/j.idairyj.2006.11.005]
] and Central Asian “Koumis” [28Mu Z, Yang X, Yuan H. Detection and identification of wild yeast in Koumiss. Food Microbiol 2012; 31(2): 301-8.
[http://dx.doi.org/10.1016/j.fm.2012.04.004] [PMID: 22608237]
]. Lopandic et al. [29Lopandic K, Zelger S, Bánszky LK, Eliskases-Lechner F, Prillinger H. Identification of yeasts associated with milk products using traditional and molecular techniques. Food Microbiol 2006; 23(4): 341-50.
[http://dx.doi.org/10.1016/j.fm.2005.05.001] [PMID: 16943023]
] isolated 71 strains of K. marxianus from dairy products available on the Austrian market. These findings show that K. marxianus is not limited to cereal-based fermented foods only, and same applies to C. glabrata. Padonou et al. [30Wilfrid Padonou S, Nielsen DS, Hounhouigan JD, Thorsen L, Nago MC, Jakobsen M. The microbiota of Lafun, an African traditional cassava food product. Int J Food Microbiol 2009; 133(1-2): 22-30.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2009.04.019] [PMID: 19493582]
] found C. glabrata in the African fermented cassava food “Lafun”, whereas Nyanga et al. [31Nyanga LK, Nout MJ, Gadaga TH, Theelen B, Boekhout T, Zwietering MH. Yeasts and lactic acid bacteria microbiota from masau (Ziziphus mauritiana) fruits and their fermented fruit pulp in Zimbabwe. Int J Food Microbiol 2007; 120(1-2): 159-66.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2007.06.021] [PMID: 17904237]
] identified this yeast species during fermentation of masau fruits in Zimbabwe. C. glabrata was also identified in Bulgarian wheat-based fermented beverage “boza” [5Gotcheva V, Pandiella SS, Angelov A, et al. Microflora identification of the Bulgarian cereal-based fermented beverage boza. Process Biochem 2000; 36: 127-30.
[http://dx.doi.org/10.1016/S0032-9592(00)00192-8]
]. C. fabianii was previously isolated from traditional Indian rice wine starter “hamei” [32Jeyaram K, Singh WM, Capece A, Romano P. Molecular identification of yeast species associated with ‘Hamei’--a traditional starter used for rice wine production in Manipur, India. Int J Food Microbiol 2008; 124(2): 115-25.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2008.02.029] [PMID: 18433905]
], and Mukisa et al. [33Mukisa IM, Porcellato D, Byaruhanga YB, et al. The dominant microbial community associated with fermentation of Obushera (sorghum and millet beverages) determined by culture-dependent and culture-independent methods. Int J Food Microbiol 2012; 160(1): 1-10.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2012.09.023] [PMID: 23141639]
] isolated C. fabianii from Ugandan traditional food “Obushera”, prepared from sorghum and millet.

In the present study C. fabianii was isolated along with K. marxianus from Degue from millet pellets. Pedersen et al. [34Pedersen LL, Owusu-Kwarteng J, Thorsen L, Jespersen L. Biodiversity and probiotic potential of yeasts isolated from Fura, a West African spontaneously fermented cereal. Int J Food Microbiol 2012; 159(2): 144-51.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2012.08.016] [PMID: 23072700]
] also found both species in the Ghanaian food “Fura”, which, similarly to Degue, was prepared by spontaneous fermentation of millet and milk. M. carribica found in the present study in millet flour has previously been isolated by Leong et al. [35Leong SL, Niba AT, Ny S, Olstorpe M. Microbial populations during maize storage in Cameroon. Afr J Biotechnol 2012; 11: 8692-7.] from maize during storage in Cameroon. This species was also found in a variety of other matrices, such as the Brazilian fermented beverage “cachaça” [36Nova MX, Schuler AR, Brasileiro BT, Morais MA Jr. Yeast species involved in artisanal cachaça fermentation in three stills with different technological levels in Pernambuco, Brazil. Food Microbiol 2009; 26(5): 460-6.
[http://dx.doi.org/10.1016/j.fm.2009.02.005] [PMID: 19465241]
], Ghanaian cocoa bean heap fermentation [37Daniel HM, Vrancken G, Takrama JF, Camu N, De Vos P, De Vuyst L. Yeast diversity of Ghanaian cocoa bean heap fermentations. FEMS Yeast Res 2009; 9(5): 774-83.
[http://dx.doi.org/10.1111/j.1567-1364.2009.00520.x] [PMID: 19473277]
], phylloplane of sugarcane in Thailand [38Limtong S, Kaewwichian R, Yongmanitchai W, Kawasaki H. Diversity of culturable yeasts in phylloplane of sugarcane in Thailand and their capability to produce indole-3-acetic acid. World J Microbiol Biotechnol 2014; 30(6): 1785-96.
[http://dx.doi.org/10.1007/s11274-014-1602-7] [PMID: 24442819]
], and olive fruits, paste, and pomace from Spain [39Romo-Sánchez S, Alves-Baffi M, Arévalo-Villena M, Ubeda-Iranzo J, Briones-Pérez A. Yeast biodiversity from oleic ecosystems: study of their biotechnological properties. Food Microbiol 2010; 27(4): 487-92.
[http://dx.doi.org/10.1016/j.fm.2009.12.009] [PMID: 20417397]
].

It is interesting to note that some yeast species, such as Candida krusei, C. tropicalis and Saccharomyces cerevisiae, commonly found in other cereal-based fermented foods from Benin and other geographical locations countries were not recovered from Degue [5Gotcheva V, Pandiella SS, Angelov A, et al. Microflora identification of the Bulgarian cereal-based fermented beverage boza. Process Biochem 2000; 36: 127-30.
[http://dx.doi.org/10.1016/S0032-9592(00)00192-8]
, 6Greppi A, Rantsiou K, Padonou W, et al. Determination of yeast diversity in ogi, mawè, gowé and tchoukoutou by using culture-dependent and -independent methods. Int J Food Microbiol 2013; 165(2): 84-8.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2013.05.005] [PMID: 23727651]
, 33Mukisa IM, Porcellato D, Byaruhanga YB, et al. The dominant microbial community associated with fermentation of Obushera (sorghum and millet beverages) determined by culture-dependent and culture-independent methods. Int J Food Microbiol 2012; 160(1): 1-10.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2012.09.023] [PMID: 23141639]
, 34Pedersen LL, Owusu-Kwarteng J, Thorsen L, Jespersen L. Biodiversity and probiotic potential of yeasts isolated from Fura, a West African spontaneously fermented cereal. Int J Food Microbiol 2012; 159(2): 144-51.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2012.08.016] [PMID: 23072700]
].

Sequencing of the 16S rRNA gene from each of the selected putative LAB isolates from raw materials and Degue samples revealed the presence of five genera. Specifically, eight species of Lactobacillus, Enterococcus, Pediococcus, Streptococcus and Weisella were identified Table (3). The predominant species was Lactobacillus fermentum (37% of all LAB isolates). It was isolated from the fermented milk powder, red sorghum flour and pellets, and maize flour, as well as in 6 of the 8 analyzed Degue types. An earlier study of Asmahan and Muna [39Romo-Sánchez S, Alves-Baffi M, Arévalo-Villena M, Ubeda-Iranzo J, Briones-Pérez A. Yeast biodiversity from oleic ecosystems: study of their biotechnological properties. Food Microbiol 2010; 27(4): 487-92.
[http://dx.doi.org/10.1016/j.fm.2009.12.009] [PMID: 20417397]
] focused on API 20 CHL identification of LAB involved in the preparation of the sorghum-based fermented food “kisra in Sudan also revealed dominance of Lb. fermentum, and the same applied for African pearl millet slurries [40Asmahan AA, Muna MM. Isolation, characterization and identification of lactic acid bacteria from fermented sorghum dough used in Sudanese Kisra preparation. Pak J Nutr 2009; 8: 1814-8.
[http://dx.doi.org/10.3923/pjn.2009.1814.1818]
].

Table 3
Identification of LAB from from different raw materials and Degue products by sequencing of 16S rRNA gene.


The other species found in Degue products from Benin were Lb. plantarum, Lb. pentosus, Lb. rhamnosus, P. acidilactici and W. paramesenteroides. It is interesting to note that Lb. fermentum was the only species found in common with the bacterial microbiota of millet Degue from Burkina Faso [4Abriouel H, Ben Omar N, López RL, Martínez-Cañamero M, Keleke S, Gálvez A. Culture-independent analysis of the microbial composition of the African traditional fermented foods poto poto and dégué by using three different DNA extraction methods. Int J Food Microbiol 2006; 111(3): 228-33.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2006.06.006] [PMID: 16842876]
]. This shows that despite the similarity in raw materials and production methods, the geography-related factors play a key role for the microbiota formation in Degue.

Similar microbial population of LAB was identified by conventional phenotypic and biochemical methods in other spontaneously fermented cereal-based foods from Africa [41Turpin W, Humblot C, Guyot JP. Genetic screening of functional properties of lactic acid bacteria in a fermented pearl millet slurry and in the metagenome of fermented starchy foods. Appl Environ Microbiol 2011; 77(24): 8722-34.
[http://dx.doi.org/10.1128/AEM.05988-11] [PMID: 22003019]
]. These results also partly confirmed the findings of Adimpong et al. [42Adimpong DB, Nielsen DS, Sørensen KI, Derkx PM, Jespersen L. Genotypic characterization and safety assessment of lactic acid bacteria from indigenous African fermented food products. BMC Microbiol 2012; 12: 75.
[http://dx.doi.org/10.1186/1471-2180-12-75] [PMID: 22594449]
] and Ouoba et al. [43Ouoba LI, Nyanga-Koumou CA, Parkouda C, et al. Genotypic diversity of lactic acid bacteria isolated from African traditional alkaline-fermented foods. J Appl Microbiol 2010; 108(6): 2019-29.
[PMID: 19895650]
] studying indigenous African cereal-based fermented foods. Similar LAB species have been identified by biochemical tests in combination with AFLP analysis in South African sourdoughs [44Kunene NF, Geornaras I, von Holy A, Hastings JW. Characterization and determination of origin of lactic acid bacteria from a sorghum-based fermented weaning food by analysis of soluble proteins and amplified fragment length polymorphism fingerprinting. Appl Environ Microbiol 2000; 66(3): 1084-92.
[http://dx.doi.org/10.1128/AEM.66.3.1084-1092.2000] [PMID: 10698775]
]. Members of Lactobacillus and Weisella have also been found in Zambian fermented products based on maize, millet and/or sorghum by amplification of the V1 to V4 hypervariable regions on the 16S rRNA gene [7Schoustra SE, Kasase C, Toarta C, Kassen R, Poulain AJ. Microbial community structure of three traditional zambian fermented products: mabisi, chibwantu and munkoyo. PLoS One 2013; 8(5): e63948.
[http://dx.doi.org/10.1371/journal.pone.0063948] [PMID: 23691123]
]. Owusu-Kwarteng et al. [45Owusu-Kwarteng J, Akabanda F, Nielsen DS, Tano-Debrah K, Glover RL, Jespersen L. Identification of lactic acid bacteria isolated during traditional fura processing in Ghana. Food Microbiol 2012; 32(1): 72-8.
[http://dx.doi.org/10.1016/j.fm.2012.04.010] [PMID: 22850376]
] and Soro-Yao et al. [19Soro-Yao AA, Brou K, Amani G, Thonart P, Djè KM. The use of lactic acid bacteria starter cultures during the processing of fermented cereal-based foods in West Africa: A review. Trop Life Sci Res 2014; 25(2): 81-100.
[PMID: 27073601]
] reported predominance of Lb. fermentum, Lb. plantraum Lb. reuteri, Lb. salivarius, P. acidilactici, W. confusa and W. cibaria in millet-based spontaneously fermented products in West Africa (Ghana and Cote d’Ivoire). By applying a combination of phenotypic and genotypic methods (API CHL kits) Nwachukwu et al. [46Nwachukwu E, Achi OK, Ijeoma IO. Lactic acid bacteria in fermentation of cereals for the production of indigenous Nigerian foods. Afr J Food Sci Tech 2010; 1: 21-6.] and Oyedeji et al. [47Oyedeji O, Ogunbanwo ST, Onilude AA. Predominant lactic acid bacteria involved in the traditional fermentation of Fufu and Ogi, two Nigerian fermented food products. Food Nutr Sci 2013; 4: 40-6.
[http://dx.doi.org/10.4236/fns.2013.411A006]
] identified Lb. plantarum, Lb. pentosus, Lb. cellobiosus, Ln. mesenteroides, and P. pentosaceus in maize, millet and cassava used for the production of indigenous Nigerian cereal-based fermented foods “fufu and “ogi, while lactic acid populations in sorghum-based drinks from Zimbabwe (“chibuku”, a sorghum beer) and Uganda (“bushera”) were found to consist of Lb. plantarum, Lb. paracasei subsp. paracasei, Lb. fermentum, Lb. brevis and Lb. delbrueckii subsp. delbrueckii, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Lc. raffinolactis, Ln. mesenteroides subsp. mesenteroides, Ln. mesenteroides subsp. dextranicum and W. confusa [48Chamunorwa AT, Feresu SB, Mutukumira AN. Identification of lactic acid bacteria isolated from Opaque beer (Chibuku) for potential use as a starter culture. J Food Technol Afr 2002; 7: 93-7., 49Muyanja CM, Narvhus JA, Treimo J, Langsrud T. Isolation, characterisation and identification of lactic acid bacteria from bushera: a Ugandan traditional fermented beverage. Int J Food Microbiol 2003; 80(3): 201-10.
[http://dx.doi.org/10.1016/S0168-1605(02)00148-4] [PMID: 12494920]
].

The present study showed that the predominant microbiota in the fermented milk samples from Benin was presented by Lb. plantarum, Lb. fermentum, Lb. rhamnosus and S. thermophilus. These results partly comply with the findings of Nyambane et al. [50Nyambane B, Thari WM, Wangoh J, Njage PM. Lactic acid bacteria and yeasts involved in the fermentation ofamabere amaruranu, a Kenyan fermented milk. Food Sci Nutr 2014; 2(6): 692-9.
[http://dx.doi.org/10.1002/fsn3.162] [PMID: 25493187]
] who identified mainly S. thermophilus, Lb. plantarum and Ln. mesenteroides in traditional Kenyan fermented milk “amabere amaruranu”. A larger species diversity was revealed by Mohammed and Ijah [51Mohammed SS, Ijah UJ. Isolation and screening of lactic acid bacteria from fermented milk products for bacteriocin production. Annals. Food Sci Tech 2013; 14: 122-8.] using biochemical and phenotypic methods for identification of LAB in yogurt and milk products in Nigeria. They reported the presence of Lb. bulgaricus, Lc. lactis, Lb. acidophilus, S. thermophilus, S. cremoris, Lc. lactis, P. halophilus and P. cerevisiae. Similar results were obtained by Savadogo et al. [52Savadogo A, Ouattara CA, Savadogo PW, et al. Identification of exopolysaccharides-producing lactic acid bacteria from Burkina Faso fermented milk samples. Afr J Biotechnol 2004; 3: 189-94.] for identification of LAB from Burkina Faso fermented milks by using spacer region between 16S and 23S rRNA genes.

The comparison of LAB microbiota found in raw materials and Degue products from Benin with those of other similar products show that although individual species may be found in common, the combination of species in Beninese Degue is unique for this product.

CONCLUSION

The present study is the first to explore the yeast and lactic acid bacteria diversity in Degue - a traditional cereal-based fermented beverage produced in Benin. Four yeast species were found in the samples of raw materials and various types of Degue - C. fabianii, C. glabrata, K. marxianus and M. caribbica, and identification of the LAB isolates revealed 8 species: Lb. fermentum, Lb. plantarum, Lb. pentosus, Lb. rhamnosus, E. mundtii, P. acidilactici, S. thermophilus and W. paramesenteroides. The differences of species diversity in comparison to other similar fermented foods suggest that microbial community structure in Degue is not a simple consequence of the nature of the raw materials used, but it is a result from a variety of abiotic and biotic factors affecting the natural selection of specific microbial populations – origin of the raw materials, climatic and geographical factors, preparation method and work environment, etc. Therefore, further studies on the microbiota of Degue products from various locations in Benin on the microbial dynamics throughout the various stages of Degue preparation and on the role of each species in the mixed population involved in Degue fermentation will form the basis for future development of defined starter cultures and for establishment of an industrial technology for Degue production to meet the growing consumption in Benin by offering products with improved quality, safety and nutritional value.

LIST OF ABBREVIATIONS

AFLP  = Amplified Fragment Length Polymorphism
C.  = Candida
DNA  = Deoxyribonucleic Acid
dNTP  = Deoxynucleotide Triphosphate
E.  = Enterococcus
K.  = Kluyveromyces
LAB  = Lactic Acid Bacteria
Lb.  = Lactobacillus
Ln.  = Leuconostoc
M.  =  Meyerozyma
MRS  = de Man, Rogosa and Sharpe
P.  =  Pediococcus
PCR  = Polymerase Chain Reaction
RFLP  = Restriction Fragment Length Polymorphism
RNA  = Ribonucleic Acid
rRNA  = Ribosomal Ribonucleic Acid
S.  = Streptococcus
W.  = Weisella

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLDEGEMENTS

The present study was supported by Project BG051PO001-3.3.05-0001 “Science and Business” of the Bulgarian Ministry of Education and Project 3/14 of Fund “Scientific Research” of the University of Food Technologies, Bulgaria.

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[40] Asmahan AA, Muna MM. Isolation, characterization and identification of lactic acid bacteria from fermented sorghum dough used in Sudanese Kisra preparation. Pak J Nutr 2009; 8: 1814-8.
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[41] Turpin W, Humblot C, Guyot JP. Genetic screening of functional properties of lactic acid bacteria in a fermented pearl millet slurry and in the metagenome of fermented starchy foods. Appl Environ Microbiol 2011; 77(24): 8722-34.
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[42] Adimpong DB, Nielsen DS, Sørensen KI, Derkx PM, Jespersen L. Genotypic characterization and safety assessment of lactic acid bacteria from indigenous African fermented food products. BMC Microbiol 2012; 12: 75.
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Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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