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Isolation and Identification of Lactobacilli from Garris, a Sudanese Fermented Camel’s Milk Product



Abdel Moneim E. Sulieman , Ro Osawa and Roumiana Tsenkova
 
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ABSTRACT

In the present study, microbiological quality of Garris, a traditional fermented camel’s milk product obtained from two production sites in western and central Sudan was investigated. The microbiological analyses showed that Butana Garris was found to contain relatively high counts of lactobacilli (8.22±0.28 log10 cfu mL-1) when compared with the counts found in Kordufan Garris (7.85±0.45 log10 cfu mL-1). However, Kordufan Garris contained relatively higher counts of yeasts (8.42±0.55 log10 cfu mL-1) when compared with that found in Butana Garris which contained 7.65±0.32 log10 cfu mL-1). The coliforms averaged 3.2±0.21 and 3.5±0.14 log10 cfu mL-1) in Kordufan and Butana Garris, respectively. Twenty strains of lactic acid bacteria were isolated from the fermented milk products and identified based on Polymerase Chain Reaction (PCR) method as belonging to the genus lactobacillus. Rapid and reliable two-step multiplex PCR assays were used to identify Garris lactobacilli at species level. A multiplex PCR was used for grouping lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flaking 23S rRNA gene of members of the genus Lactobacillus registered at the GeneBank and DNA Data Bank of Japan. Five lactobacilli isolates from Kordufan Garris were identified as Lactobacillus plantarum, three isolates as Lactobacillus paracasei and two not determined. As for the Butana LAB isolates, five isolates were identified as Lactobacillus paracasei subsp paracasei, two as Lactobacillus plantarum and three were not determined.

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Abdel Moneim E. Sulieman , Ro Osawa and Roumiana Tsenkova , 2007. Isolation and Identification of Lactobacilli from Garris, a Sudanese Fermented Camel’s Milk Product . Research Journal of Microbiology, 2: 125-132.

URL: https://scialert.net/abstract/?doi=jm.2007.125.132

INTRODUCTION

Traditional fermented dairy products have been existence in Sudan from a long period of time. These products are widely popular and consumed by a larger section of human population.

Identification of lactic acid bacteria (LAB) lactic acid bacteria mostly depends on traditional phenotypic analyses, although molecular biology-based methods have become available (Hertel et al., 1993; Pot et al., 1993). Hence, until now modern identification techniques have not been used to a large degree for identification to the species level of lactic acid bacteria from East and Middle African foods.

Garris is a special kind of fermented milk prepared from camel’s milk in Sudan. The product is prepared and consumed primarily, by camel’s boys, roaming pasturelands. The camel boys taking care of these camels prepare Garris on which they live for months as a sole source of nourishment (Dirar, 1993). Garris differs from other kinds of Sudanese fermented milks in that it has substantial amounts of ethanol. The product is thus a member of the acid alcoholic fermented milks, which include kefir, koumiss and bukhsa of central Asia (Kosikowski, 1982).

A semi-continuous fed-batch fermentation process that involves lactobacilli and yeasts, makes Garris. Whenever, part of Garris is withdrawn for consumption, an equal volume of fresh camel’s milk is added. The milk is fermented in two large leather bags covered or imbedded in green grass damped with water. The process of separation of Garris and replacement of milk continues for months (Dirar, 1993).

The nutritive value of Garris is that of fresh camel’s milk as modified by fermentation. Fresh camel milk from the Sudan contains 3.3- 4.7% protein, 2.8-3.6% fat, 4.0- 5.2% lactose, 0.7% ash, 9.2-15.4% solids and to has a pH of 6.0-6.5 (Sulieman et al., 2005). In India, it is used to cure dropsy, jaundice, tuberculosis and anaemia. In Russia, camel’s milk koumiss has been used to treat tuberculosis and other lung ailments. In Sudan, fermented camel milk is used to cure leishmaniasis and protozoal disease of belly beside many other uses. From the documented literature, it appears that few work has been attempted on assessing the microbiological quality of the Sudanese traditionally fermented milk products. Besides, no study has been initiated identify the fermenting lactobacilli of Garris. In this background, the present work aims at isolation and identification of the lactobacilli dominating Garris microflora, which are responsible of its fermentation and bringing about its desirable traits.

MATERIALS AND METHODS

Materials
Twenty samples of the Sudanese traditionally fermented camel’s milk product, Garris were collected from local households in two different sources Kordofan (western Sudan) and Butana (Eastern Sudan) during November 2004. The age of Garris samples ranged from 1 to 4 days. The samples were transported in a cooling box to the Department of Food Science and Technology, University of K hartoum, Khartoum, Sudan where the microbiological analyses were performed. Other samples were kept at 4°C, transported by air to the Department of Microbiology, University of Kobe, Japan and kept at low temperature (using a cooling box) temperature pending the isolation and identification analyses.

Viable Microbial Counts
Lactobacilli counts were determined on MRS agar (Merck, Darmstadt, Germany) with glucose a source of energy. Appropriate dilutions were plated on MRS agar (Difco) and incubated anaerobically at 30°C for 48 h using anaerobic jars and gas generating kits (gas pack). Yeasts and moulds were enumerated by surface plating on Potato Dextrose Agar (PDA) (Oxoid) and incubated aerobically at 25 C for 3 days. Coliforms were enumerated by pour plating on MacConkey agar (Oxoid). Characteristic colonies appearing on the respective selective agar media were counted, multiplied by the dilution factor and expressed as colony forming units per milliliter (cfu mL-1).

Isolation of Lactobacilli and Preliminary Tests of the Isolates
MRS was used for isolation of twenty strains of lactobacilli from Garris samples, 10 from Kordofan Garris and 10 from Botana Garris. The isolated strains were purified by random selection of colonies from the MRS agar plates and transformed to tubes containing MRS broth (Oxoid) and incubated again for 24 h at 37 °C. Then, broth cultures were streaked onto MRS agar. The procedure was repeated until purity of the culture was established based on colony appearance.

Preliminary tests were employed on the isolates to ensure that they belong to LAB. These tests included microscopic examination, Gram staining and catalase reaction according to the methods described by Harrigan and McCane (1976). Growth at 15 and 45°C was tested in MRS broth incubated in a Memmert incubator (854 Schwabch, West Germany) and a Salvis water-bath, respectively.

Identification of Lactobacilli at the Genus Level
Identification of Lactobacilli at the genus level was accomplished by a PCR-based method as described by Dubernet et al (2002). The reference strains are shown in Table 1

DNA Isolation
For subsequent genotypic analyses, completely genomic DNA from each isolate was prepared following the method described by Marmur (1961). The purity and the amount of DNA in each preparation was estimated colourimetrically and stored at 4°C until use.

Identification of Lactobacilli at the Species Level
The lactobacilli species were identified by PCR assays using group- and species- specific primers derived from 16S-23S rRNA intergenic spacer region and its flaking 23S rRNA as described by Song et al. (2000), as follows:

Table 1: Reference strains used for identification of lactobacilli at genus level
Image for - Isolation and Identification of Lactobacilli from Garris, a Sudanese Fermented Camel’s Milk Product
CND, not determined, CIPa, Collection de I’institute Pasteur, Paris, France; CNRZ, Centre National de Recherches Zootechniques, Jouy-en-josas, France; DSMZ, Deutsche Sammlung von Mikroorganismen und Zelkultren, Braunschweig, Germany. bHylogenetic group: a, L. delbruekii group; b, L. casei Pediococcus group; Leuconostoc group. CFermentative group: 1, obligatory homofermentative; 11, facultatively heterofermentative; 111, obligatory heterofermentative

Table 2: Reference strains and the nucleotide sequence accession numbers for the 16S 23S rRNA and its flanking 23S rRNA gene of these strains
Image for - Isolation and Identification of Lactobacilli from Garris, a Sudanese Fermented Camel’s Milk Product

Bacterial Strains and Culture Conditions
Twenty two strains of Lactobacillus species or subspecies and 84 lactobacilli isolated from Japanese stool specimens, which were identified to species level by DNA-DNA hybridization method in a previous study by Song et al. (1999), were used as reference strains for identification of lactobacilli at species level (Table 2). All strains were cultured on MRS (Becton Dickinson) at 37°C in an aerobic chamber (Hirasawa, Tokyo, Japan) with an atmosphere consisting of 82%N2, 10%CO2 and 8% H2.

PCR Amplification of the 16S-23S rRNA ISR and its Flaking 23S rRNA
A primer pair, 16 and 23-10C (Table 3) (Berthier and Ehrlich, 1998; Gutler and Stanisich, 1996), corresponding to positions of 1526-1543 of the 16S rRNA and positions of 456-474 of the 23S rDNA of Escherichia coli, was used to amplify the 16S-23S rRNA ISRs and its flanking 23S rRNA genes of 17 reference strains of the genus Lactobacillus (see strains with the accession numbers in Table 2), which represent Lactobacillus commonly found in human intestinal microflora and their closely related species. PCR amplification was performed as previously described by Kato et al 1991). Briefly, one or two colonies of bacterial strains on an agar plate were suspended in 50 μL of Tris-HCl-EDTA-saline (pH 8.0). The bacterial suspension was incubated for 10 min at 95°C and centrifuged at 186000x g for 2 min to obtain the DNA sample as the PCR template. PCR amplification was performed with a perkin-Elmer DNA thermal cycle 480 (Perkin-Elmer Corporation, Norway, CT, USA) programmed for 35 cycles comprising 95°C for 20 s for denaturation and 55°C for 2 min for annealing and extension.

Sequencing of the PCR-Amplified 16S-23S rRNA Isr and its Flanking 23s rRNA
A major PCR product, an amplicon of about 600-700 bp in size, was excised from a 1% agarose gel after electrophoresis and purified using the Pre-A-Gene purification matrix kit (Bio-Rad, Richmond, CA, USA) and was sequenced directly with ABI PRISM Dye Terminator Cycle Sequencing Really Reaction Kit (PE Applied Biosystems, Foster City, CA, USA) and ABI Prism Genetic Analyzer (PE Applied Biosystems). Cycle sequencing was carried out as recommended by the manufacturer. The analysis of alignment and homology and the construction of a phylogenetic tree for the nucleotide sequences obtained in this study were carried out by DNASIS software ver. 3.6 (Hitachi Software Engineering, Yokohama, Japan).

Table 3: Oligonucleotide primers used in the study
Image for - Isolation and Identification of Lactobacilli from Garris, a Sudanese Fermented Camel’s Milk Product

Primers for PCR were designed by OLIGO software ver. 4.0 (Hitachi Software Engineering).

Identification of Lactobacilli by Two-step Multiplex PCR Assays
Based on nucleotide sequences of the 16S-23S rRNA ISR of lactobacilli which were determined in this study, lactobacilli were first grouped by multiplex PCR designated multiplex PCR-G) and then identified to species level by four multiplex PCR assay (named multiplex PCR I I-1, multiplex PCR II-2, multiplex PCR III and multiplex PCR IV). Primers used for the grouping and species identification and the aim of each multiplex PCR are given in Table 3.

The DNA sample was extracted as described above. Thirty microliters of a reaction solution for PCR amplification was composed of 0.15 U Taq DNA polymerase (Promega Corporation, Madison, WI, USA), 26.6 μL of a reaction buffer (Promega) supplemented with 200 μM each of dCTP, dATP, dGTP and dTTP (Pharmacia Biotech, Uppsala, Sweden), 10 pmol primer mix comprising one portion of each primer and 0.3 μL of template DNA. PCR was carried out for 35 cycles. Each cycle consisted of 95°C for 20 s for denaturation; annealing and extension was performed for 2 min at 55°C for multiplex PCR-G, 68°C for multiplex PCR II-1, 65°C for multiplex PCR II-2, 62°C for multiplex final extension. Amplicons were analyzed by electrophoresis on a 5% polyacrylamide gel followed by ethidium bromide.

RESULTS AND DISCUSSION

The microbial viable counts of Garris samples obtained from the two different production sites are presented in Table 4. Butana Garris was found to contain relatively high counts of lactobacilli (8.22±0.28 log10 cfu mL-1) when compared the counts found in B utana Garris (7.85±0.45 log10 cfu mL-1). On the other hand, Kordufan Garris contained relatively higher counts of yeasts (8.42±0.55 log10 cfu mL-1)) when compared with that found in Butana Garris which contained 7.65±0.32 log10 cfu mL-1). Presence of yeast in such a high counts suggests that these microorganisms play a role in Garris fermentation.

Table 4: Microbial viable counts log10 cfu mL-1) and pH of raw milk and Garris
Image for - Isolation and Identification of Lactobacilli from Garris, a Sudanese Fermented Camel’s Milk Product

Yeast was reported by many investigators as being aroma producer in certain dairy products. Coliforms were found in appreciable amounts in most of the samples. The coliforms averaged 3.2±0.21 and 3.5±0.14 log10 cfu mL-1) in Kordufan and Butana Garris, respectively. The pH averaged 4.52±0.12 and 4.35±0.13 for Kordufan Garris and Butana Garris, respectively.

The pH and microbial counts of raw camel’s milk used Garris fermentation in both production sites were almost similar with exception to coliform counts which were higher in raw milk used for preparation of Kordufan Garris (6.7±0.33 log10 cfu mL-1) when compared with that of Butana Garris (5.8±0.38 log10 cfu mL-1). The counts of Lactobacilli and yeasts in raw milk were very high which may lead to the quick spoilage of raw milk in these areas and this puts emphasis on the importance of Garris fermentation as a method for cow’s milk preservation. However, presence of coliforms and yeasts in such high counts might be due to the improper hygienic conditions during milk handling. However, carry over of large numbers of these microorganisms in Garris is a critical health problem because those coliforms might contain pathogenic E. coli.

Twenty lactobacillus strains were isolated from Garris samples obtained from two different production sites namely Kordofan and Botana. All isolates were Gram-positive, catalse-negative, rods producing no gas from glucose. The strains were identified based on the above characteristics in addition to growth behavior at 15 and 45°C as belonging to the lactic acid bacteria (LAB). On the other hand, PCR-based method revealed that these LAB as belonging to the genus Lactobacillus.

On the present study, the authors used a system to identify Lactobacilli by two-step multiplex PCR for grouping of lactobacilli followed by a multiplex assays for each group to identify lactobacilli at the species level. This is because of the limited variation of the nucleotide sequence targeted and the complexity of multiplex PCR such as selection of specific primers that generate a distinctive PCR product for each species under the same PCR condition.

Among Lactobacilli grouped by multiplex PCR Lactobacillus plantarum and Lactobacillus paracasei subsp paracasei were successfully identified at the species level by using one of the four second-step multiplex PCR assays (Table 5). Five lactobacilli isolates from Kordufan Garris were identified as Lactobacillus plantarum, three isolates as Lactobacillus paracasei and two not determined (Table 5). As for the Butana LAB isolates, five isolates were identified as Lactobacillus paracasei subsp paracasei, two as Lactobacillus plantarum and three were not determined. The presence of Lactobacillus plantarum was also reported for many African dairy products. Of the identified 21 isolates from naturally fermented milk in Zimbabwe, three were identified as Lactobacillus plantarum. From 100 isolates from fermented milk in Northern Tanzania, Isono et al. (1994) identified four as Lactobacillus plantarum. From cultured milk in Cameroon, Jiwona and Millier (1990) identified 47 out of 426 isolates as Lactobacillus plantarum. Sulieman et al. (2004) isolated few strains of Lactobacillus plantarum from the Sudanese sour milk Robe. On the other hand, presence of Lactobacillus paracasei subsp paracasei is not known in African fermented dairy products (Steinkraus, 1996), but it has been isolated from plant materials and fermented foods (Winter et al., 1998; Paludan-Muller et al., 1999).

Microbial counts of Garris samples obtained from different production sites demonstrated the presence of high counts of lactobacilli, yeasts and coliforms. The study demonstrates the high potential for isolating new species of LAB from Sudanese foods. Further investigations into taxonomic classification of the new taxa demonstrated in this study are in progress. In addition, there is a need for investigating the technological characteristics of Garris dominant microorganisms to select the most appropriate strains as starter culture for a controlled fermentation process.

Table 5: Strains and results of Lactobacillus genus-specific or species-species PCR assays. Identification results based on PCR-based assays
Image for - Isolation and Identification of Lactobacilli from Garris, a Sudanese Fermented Camel’s Milk Product
ND: not tested, *2 Group III includes Lactobacillus paracasei subsp. paracasei, L. paracasei subsp.casei, L. paracasei subsp. tolerans and L. rhamnosus. *3 Group IV includes Lactobacillus plantarum, L. reuteri, L. salivarius and L. fermentum

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