Isolation and Identification of Lactic Acid Bacteria Isolated from Traditional Drinking Yoghurt in Khartoum State, Sudan
Asmahan Azhari Ali
The present study was conducted to evaluate the technological characteristics of lactic acid bacteria used as lactic acid starter in the manufacturing of fermented dairy products and which are suitable to local conditions. Morphological, cultural, physiological and biochemical characteristics were employed to identify Lactic Acid Bacteria (LAB), isolated from drinking yoghurt in different areas in Khartoum state, Sudan. The purification of isolates was done by transferring Gram +ve rods and cocci shaped bacteria to the plates of selective media MRS and M-17, respectively. These isolates were further sub cultured until pure isolates were obtained. From 18 drinking yoghurt samples a total of 303 LAB positives were determined, in which 47 (17.38%) and 256 (82.62%) were identified as lactic acid cocci and lactic acid bacilli, respectively. Additionally, our biochemical tests showed the occurrence of 22 (44.44%) Lactococcus lactis subsp. cremoris and 25 (55.56%) Leuconostoc mesenteroides subsp. cremoris among lactic acid cocci. While, in the case of lactic acid bacilli, Lactobacillus helveticus 35 (15.3%); Lactobacillus plantarum 74 (22.3%); Lactobacillus brevis 17 (21%); Lactobacillus casei subsp. casei 36 (15.5%) and Lactobacillus delbrueckii subsp. bulgaricus 94 (25.9%) was found. Among lactic acid cocci and bacilli, Leuconostoc mesenteroides subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus were found to be the more dominant species, respectively. The current study constitutes the first step in the designing process of LAB starter cultures, in order to protect the typical organoleptic characteristics of traditional drinking yoghurt. However, in the future we can consider genetical characterization and selection of the most desirable strains and assess their potential as starter cultures for commercial use.
August 03, 2010; Accepted: September 28, 2010;
Published: March 25, 2011
Fermented milks are products prepared by controlled fermentation of milk to
produce acidity and flavour to desired level. Fermented milks are the most common
products from which other products are also made (Thapa, 2000).
Starter culture organisms used in fermentations belongs to a family of bacteria
collectively known as the Lactic Acid Bacteria (LAB). These LABS are united
by a constellation of morphological, metabolic and physiological characteristics.
There are several factors, which influence the quality of yoghurt. These include
type of milk, processing conditions, storage conditions etc., however, quality
of starter culture is the most important factor that influence the development
of quality yoghurt. Lactic Acid Bacteria (LAB) are widely distributed in nature
and occur naturally as indigenous microflora in raw milk, drinking yoghurt,
etc. They are gram positive bacteria that play an important role in many food
fermentation processes. Some species of the genus Lactobacillus (Lb.),
Lactococcus (Lc.) and Leuconostoc (Ln.) are included
in this group. The lactic acid fermentation has long been known and applied
by humans for making different food stuffs. For many centuries, LAB have been
an effective form of natural preservation. In addition, they strongly determine
the flavour, texture and frequently, the nutritional value of food and feed
products. However, the application of well-studied starter cultures has been
established for decades (Lee, 1996; Tserovska
et al., 2002). The dairy industry has developed considerably, thanks
to the use of selective lactic acid bacteria, the choice being based on their
production of lactic acid, aromatic compounds, bacteriocins and their resistance
to the phages (Herrero et al., 1996). Industrialization
of the biological transformation of foodstuffs has increased the economic importance
of lactic acid bacteria, because they play crucial role in the sensorial and
safety aspects of fermented products. It is well recognized that technological
properties of yoghurt, such as acidification, flavour production and viscosity
in great extent are strain dependent (Accolas and Auclair,
1977). Lactic acid is used today by food industry as acidulent and preservative
for the production of sour curd cheese and yoghurt (Linkater
and Griffin, 1971) but Lipinsky (1981) has emphasized
on the potential importance of biotechnologically produced lactic acid as chemical
feedstock via lactonitrile and lactides. Lactococci are the major mesophilic
bacteria used for acid production in dairy fermentations and used as starter
cultures in the manufacture of a vast range of dairy foods including fermented
milks, lactic butter, cheese and lactic casein (Ward et
al., 2002). The aim of the present study was isolation and identification
of a large number of lactic acid bacteria from drinking yoghurt in order to
constitute an original collection of Khartoum state LAB strains and to study
their technological characteristics in order to select strains of lactic acid
bacteria used as lactic acid starter in the manufacturing of fermented dairy
products and which are suitable to local conditions.
MATERIALS AND METHODS
Drinking yoghurt samples: The present study was conducted during the period from January to April 2010, a total of 18 drinking-yoghurt samples were collected from the households of three geographical regions of Khartoum state. The samples were collected in sterile bottles and kept cool until they could be taken to the laboratory, where they were kept at 4°C for further use.
Isolation of lactic acid bacteria: The samples were aseptically weighed
and homogenized. From each sample, a 1:10 dilution was subsequently made using
peptone water followed by making a 10 fold serial dilution. The 0.1 mL from
each dilution was then sub cultured, in duplicate, into the M 17 and MRS agars
(Merck, Germany) used for isolating LAB (Badis et al.,
2004a; Guessas and Kihal, 2004). To prevent the
growing of yeasts, the media were then supplemented with 100 mgL-1
of cycloheximide before being incubated at the appropriate temperatures (42,
35 and 30°C) for 2-3 days (Beukes et al., 2001;
Kalavrouzioti et al., 2005). The MRS agar plates
were incubated anaerobically using the Gas Pack system (Merck Anaerocult type
A) at 42, 35 and 30°C for 3 days, in order to provide an optimal temperature
for growing Thermophilic lactobacilli, mesophilic lactobacilli and Leuconostoc,
respectively. M17 agar plates were also incubated aerobically at 30°C for
2 days, in order to set up an optimal temperature for growing lactococci. To
perform the total counts, the higher dilutions were used. Colonies were randomly
selected and streak plating was then used to purify the strains which were subsequently
kept in two different conditions including at 4°C for MRS and M17 plates
and at -20°C for M17 and MRS broths supplemented by 20% glycerol for further
use (Mathara et al., 2004).
Identification of the bacterial strains: All strains were initially
tested for gram reaction, catalase production and spore formation (Harrigan
and McCance, 1976). Colonies were characterized on MRS and M 17 agar. Strains
with gram positive and catalase negative reactions were finally used for further
identification (Sharpe, 1979). Growth at different temperatures
(10, 15, 37, 40 and 45°C) for 5 days, resistance to 60°C for 30 min
(Sherman test), growth in the presence of 2, 3, 4 and 6.5% NaCl and different
pHs (4.5 and 6.5) were considered to identify the strains. Hydrolysis of arginine
and asculin, utilization of citrate, production of acetone, gas formation from
glucose and dextran production from sucrose were also determined (Samelis
et al., 1994). All strains were also tested for fermentation of L-arabinose,
D-xylose, galactose, D-fructose, sorbitol, lactose, melibiose, saccharose, D-raffinose,
melezitose, mannose and glucose (Tserovska et al.,
2002). The growth of bacterial strains at 10, 15, 37, 40 and 45°C was
visually confirmed by the changes in turbidity of MRS or M17 broth after 24,
48 and 72 h of incubation. The tolerance of microorganisms to the different
levels of salt, pH and heat (60°C) was also visually evaluated (Harrigan
and McCance, 1976). Arginine dihydrolase agar and asculin azid agar (Merck,
Germany) were employed to perform the hydrolysis tests. For evaluation of citrate
utilization and acetone production, citrate and MR-VP agars (Merck, Germany)
were used. MRS or M17 broths containing inverted Durham tubes were used for
evaluation of gas production and the production of dextran from sucrose was
done in MRS agar (Mayeux et al., 1962). In order
to assess the fermentation of sugars a medium with the following composition
was employed (gL-1): bovine extract, 10.0; neopepton, 10.0; yeast
extract, 5.0; K2HPO4, 2.0; CH3COONa+3H2O,
5.0; diamonium citrate, 2.0; MgSO4, 0.2; MnSO4, 0.05;
brom-cresol-purple, 0.17; tween 80, 1 mL. Carbon sources were added individually
to this medium as filter sterilized solutions to a final concentration of 1%.
Carbohydrate utilization was assessed at the 24th and 48th h and on the 7th
day of the growth at the corresponding temperature (Tserovska
et al., 2002).
All 303 Gram positive, catalase negative and non spore-forming isolates were further characterized as follows:
Mesophilic homo-fermentative cocci, 22 isolates: This group was represented
by ADH (-) (arginine dihydrolase) (negative arginine hydrolysis), citrate (-)
(negative citrate utilization) and acetoin (-) (negative acetoin production)
isolates, which were identified as Lactococcus lactis subsp. Cremoris
(Table 1). In this group, the microorganisms were spherical
or ovoid in shape, occurring in pairs and short chains with non motile, facultative
anaerobic fermentative metabolism (Holt, 1994).
Mesophilic heterofermentative cocci, 25 isolates: The microorganisms
in this group were closely related to Leuconostoc mesenteroides subsp.
cremoris which represented a reduced fermentative profile, unable to
hydrolyse arginine, producing gas from glucose with citrate and acetoin positive
and dextrane negative reactions (Table 1). These microaerophilic
organisms were also characterized by the fermentation metabolism of lactose,
glucose and Galactose (Server-Busson et al., 1999;
Hemme and Foucaud- Scheunemann, 2004).
Lactobacilli bacteria, 256 isolates: We have divided the Lactobacilli
group into three subgroups according to Stiles and Holzapfel
(1997), as follows (Table 1): (1) Mesophilic facultative
heterofermentative Lactobacilli (110 isolates), which included Lb. plantarum
(74 isolates, 22.3%) and Lb. casei subsp. casei (36 isolates,
15.5%). (2) Thermophilic obligate homo-fermentative Lactobacilli (129 isolates),
including Lb. helveticus (35 isolates, 15.3%) and Lb. delbrueckii
subsp. bulgaricus (94 isolates, 25.9%) which had a narrow fermentation
profile and was able to ferment lactose and fructose and thus, would likely
belong to Lactobacillus delbrueckii subsp. bulgaricus (Samelis
et al., 1994; Guessas and Kihal, 2004; Ammor
et al., 2005) and (3) mesophilic obligate hetero-fermentative Lactobacilli
(17 isolates). Lb. brevis (17 isolates, 21%) was included in this group.
|| Physiological and biochemical characteristics of isolated
|1: Lactobacillus helveticus, 2: Lactobacillus plantarum,
3: Lactobacillus brevis, 4: Lactobacillus casei subsp.
casei, 5: Lactobacillus delbrueckii subsp. Bulgaricus,
6: Lactococcus lactis subsp. cremoris, 7: Leuconostoc mesenteroides
It was noted that the mesophilic facultative hetero-fermentative lactobacilli
group was represented by two species; 74 isolates were identified as Lb.
plantarum and 36 isolates as Lb. casei subsp. casei according
to Collins et al. (1989). The last species together
with Lb. helveticus is included in starter cultures during the production
of the cheese Gruyere, Gorgonzola and Mozzarella (Tserovska
et al., 2002). The above-mentioned results are in accordance with
other research groups, in raw goat milk (Guessas and Kihal,
2004). Lb. plantarum was also the major lactobacillus species found
in kule naoto, the Maasai traditional fermented milk (Mathara
et al., 2004). For group two, 35 and 94 isolates were identified
as Lb. helveticus and Lb. delbrueckii subsp. bulgaricus,
respectively. Moreover, in the last group, a supplementation test for mannose
and melezitose fermentation permitted the identification of 17 isolates as Lb.
brevis (Samelis et al., 1994). Olarte
et al. (2000) noted that the presence of Lb. plantarum in
the cheese (Cameros) from goat milk decreased the number of the enterobacteriacae
and fecal coliforms in the final product. Lactobacilli isolated from household
bushera belonged to Lb. plantarum, Lb. brevis and Lb. delbrueckii
subsp. bulgaricus (Muyanja et al., 2003).
In the characterization of microflora of Homemade semi hard white zlatar cheese,
Lactobacillus brevis was found as one of the main groups (Terzic-Vidojevic
et al., 2007). In the cocci group, 25 isolates were identified as
Leuconostoc mesenteroides subsp. cremoris and 22 isolates as Lactococcus
lactis subsp. cremoris. The lower number of lactic acid cocci is
probably due to their inability to compete with lactic acid bacilli in mixed
cultures (Teuber and Geis, 1981; Togo
et al., 2002). As starter cultures, LAB are omnipresent in dairy
manufacturing. Specific fermentation processes have been developed in order
to encourage the growth of the desired species, some of which are fastidious
organisms such as Lb. delbrueckii subsp. bulgaricus and Lb.
helveticus (Bottazzi, 1988). Isolates belonging
to the Lb. plantarum group were shown to be the predominant members of
the LAB flora of acid-fermented condiment (Tempoyak). In addition, isolates
belonging to the Lb. brevis group and Ln. mesenteroides were
also observed (Leisner et al., 2001). Beukes
et al. (2001) found Lb. plantarum, Lb. delbrueckii,
Ln. mesenteroides and Lc. lactis as dominant microorganisms of
South African traditional fermented milks. The most abundant isolated species
from raw goats milk of four Algerian races were Lb. helveticus,
Lb. plantarum, Lb. delbrueckii subsp. bulgaricus, Lb.
brevis and Lc. lactis subsp. lactis (Badis
et al., 2004b). Leisner et al. (1999)
identified Lactic Acid Bacteria (LAB) of Chili Bo and found Lb. plantarum
to be the most important predominant organism. Isolation and identification
of Sudanese traditional drinking yoghurt has been conducted for the first time.
There is no record in the literature to demonstrate the isolation and identification
of the Sudanese traditional drinking yoghurt, so far. There is, however, a big
economic loss due to the import of yoghurt starters, annually. Because of increased
demands for traditional fermented products, the results of the present study
might be able to launch a considerable native achievement in the production
of drinking yoghurt. The identified isolates are used to establish the production
of volatile compounds and to assess their potential as starter cultures for
their commercial uses.
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