Journal of Food Science & Nutrition Category: Agriculture Type: Research Article
Isolation and Characterization of Lactic Acid Bacteria Strains from Raw Camel Milk for Potential Use in the Production of Yogurt
- Imen Fguiri1*, Manel Ziadi2, Karchoufi Rekaya3, Samira Arroum3, Touhami Khorchani3
- 1 Laboratory Of Livestock And Wild Life, Institute Of Arid Lands (IRA Medenine), Médenine, Tunisia
- 2 Laboratory Of Microbial Ecology And Technology Letmi, National Institute Of Applied Sciences And Technology (INSAT), Tunis, Tunisia
- 3 Laboratory Of Livestock And Wild Life, Institute Of Arid Lands (ira Medenine), Médenine, Tunisia
*Corresponding Author:
Imen FguiriLaboratory Of Livestock And Wild Life, Institute Of Arid Lands (IRA Medenine), Médenine, Tunisia
Tel:+216 23804007,
Fax:+216 75633006
Email:imen.fguiri@yahoo.com
Received Date: Sep 25, 2017 Accepted Date: Oct 13, 2017 Published Date: Oct 27, 2017
Abstract
The objective of this work is to formulate a starter lactic seen application camel milk to prepare fermented product yoghurt. Lactic acid bacteria isolated from camel milk has different characterization tests and selection: morphological study, catalase test, gram stain, use of citrate, acidifying power, lipolytic power and proteolytic power. These tests have to choose the strains: 1, 4, 5, 6, 7, 8, 9 and 10 which has ΔpH ≥ 0.3U after 6h as the most acidifying and EPS producing strains. All the strains showed a proteolytic activity with zone diameters and proteolysis was between 15 and 21mm. In addition, these lactic acid bacteria were considered low lipolytic but all having antimicrobial activity against 11 pathogenic strains. Then freeze-dried lactic acid bacteria were prepared from these strains starters (1, 4, 5, 6 and 9). These were used to inoculate three types of milk after pasteurization, using each time a combination of two strains. These strains were applied to goat, camel and cow’s milk for the preparation of yoghurt. The monitoring of these fermented products shows the combinations of strain nº1 with strain nº6 in the goat milk and cow milk certainly give us the desired product (yogurt). Because of pH, titratable acidity and viscosity seem so similar to those of natural yoghurt
Keywords
INTRODUCTION
In arid regions, camel milk is considered as one of the most important source of dairy products for human diet with potential therapeutic effects. Recent studies showed that camel milk is a natural source for probiotics [1]. The dominant and beneficial microflora in camel milk represented by LAB is a potential source of biological materials to be used in dairy technology [2]. Today, LAB are a focus of intensive international research for their essential role in most fermented food, for their ability to produce various antimicrobial compounds promoting probiotic properties [3], including antitumoral activity [4,5], reduction of serum cholesterol [6,7], alleviation of lactose intolerance [8], stimulation of the immune system [9] and stabilization of gut microflora [10]. LAB strains that produce Exopolysaccharide (ESP) are employed in the manufacture of fermented milk to improve its texture and viscosity [11,12]. Dairy products traditionally made are usually preserved due to spontaneous fermentation. However, modern large-scale production techniques generally make use of starter systems with defined strains so as to guarantee uniformity, safety and quality in the final product [13].
Camel milk has been used fresh or fermented in different regions of the world. Traditional fermented camel’s milk is widely consumed in Africa and in Middle Eastern countries [14].
It is produced by spontaneous souring of camel’s milk. In Tunisia, some practices of camel milk fermentation were known [15]. Development of new fermented functional camel milk in Tunisia can be promised. In fact, during the last decade, the interest of industries and consumers for functional foods has been exponentially increasing. The use of milk with particular nutritional properties such as camel milk, alone or in combination with bacterial strains having probiotic properties and/or producing physiologically active metabolites, represents one of the technology options for manufacturing dairy functional beverages [16]. The traditional method of milk fermentation results in a product with varying taste and flavor and often of poor hygienic quality.
Transformation of camel’s milk into traditional Tunisian Yoghurt is achieved in addition of the yogurt starters Streptococcus thermophilus and Lactobacillus Bulgaricus. Unfortunately, some of strains of Lactobacillus delbrueckii subsp. Bulgaricus and Streptococcus thermophilus did not produce EPS or produce only low yields of EPS, which may affect the end products quality [17,18]. Therefore, screening LAB from natural sources has been one of the powerful means to obtain strains for the food industry. Thus, the objectives of this work are to isolate and characterize Lactic Acid Bacteria (LAB) from raw camel milk and to study their potential use in the production of fermented Tunisian dairy products like yoghurt.
MATERIALS AND METHODS
Sampling
Milk composition
Physicochemical analysis:
The complex viscosity (in Pa s) was determined by applying a shear stress of 0.1Pa at an oscillation frequency of 1Hz for 1min with a Brookfield type viscometer (model DV-E, MA, USA).
Dry matter expressed in grams per litter milk is calculated after weighing the sample at 105ºC for 24h of its dry residue. The sample is 5g, Ash content, expressed in g/l of milk was determined after drying at 505ºC [19].
The fat content was measured by an acid-butyrometric method using a "Neusol solution" cited by Farah. This method is a direct reading on a butyrometer the amount of fat contained in 12ml of sample after centrifugation in the presence of amyl alcohol. The direct reading of graduations determines the amount of fat in g/l.
Microbiological analysis:
Milk samples (1ml) were diluted in buffered peptone saline (10-1 to 10-3), mixed in stomacher bag. In order to quantify the various microbial groups, appropriate dilutions were surface plated:
Aerobic Total Plate Count (ATPC) (Sharlau Chemie S.A) was carried out on Plate Count Agar (PCA), incubated at 32ºC for 72h [21]. Yeast and moulds on Sabouraud Chloramphenicol (Pronadisa Micro & Molecular Biology) and incubated at 25ºC for 3 to 5 days. Total coliform were grown in Violet Red Bile Agar (VRBA) (AppliChem - Biochemica.Chemica Services) in double layer. After solidifying of the agar, the plates were incubated at 30ºC for 22h [22]. The lactic acid bacteria on MRS [23] are shown on the surface and then incubated 30ºC for 48h.
Isolation and identification of strains
Strains conservation
Technological characterization
Proteolytic activity:
Lipolytic activity:
Biomass production:
Exopolysaccharides production:
Antibacterial effect:
Lyophilization survival rate
Survival rate (%) = Ln N / Ln N0 * 100
With:
N: Number of viable cells after concentration,
N0: number of viable cells before concentration.
Yogurt preparation
Sensory evaluation
Statistical analysis
RESULT AND DISCUSSION
Milk composition
Parameter |
Cow Milk |
Goat Milk |
Camel Milk |
pH |
6.77±0.00 |
6.63±0.06 |
6.736±0.005 |
Acidity (ºD) |
17.1±0.9 |
16.2±0.9 |
13.8±0.5 |
Density |
1.032±0.000 |
1.028±0.000 |
1.027±0.000 |
Viscosity |
3.03±0.10 |
3.84±0.06 |
3.3±0.04 |
Ash (g/l) |
8.43±0 .72 |
8.73±0.15 |
9.1±0.72 |
Matter fat (g/l) |
20.33±4.61 |
60.33±7.50 |
23.66±11.01 |
Dry matter (g/l) |
108.1±17 |
162.2±5.5 |
133.7±1.2 |
|
Aerobic Total Plate Count (UFC/ml) |
Total Coliform (UFC/ml) |
Yeast and Molds (UFC/ml) |
Lactic Acid Bacteria (UFC/ml) |
Camel milk |
4.7×104 |
0 |
0 |
8×102 |
Cow milk |
2.09×106 |
2.15×105 |
8.3×104 |
6.5×103 |
Goat milk |
2.39×105 |
2.67×104 |
3×102 |
1.15×103 |
Table 2: Microbiological characteristics of the 3 types of milk (cow, goat and camel).
Isolation and identification of strains
The ten strains encoded SCC1-2, SCC1-6, SCC1-7, SCC1-8, SCC1-13, SCC1-15, SCC1-24, SCC1-33, SLch6 and SLch14 are characterized by their ability to grow at different temperatures (10, 39 and 45ºC) at different salt concentrations (4, 6.5) while growth of these strains at 8% of salts concentration were not observed. All strains grow only at pH = 9.6 (Table 3).
Parameter |
Cow Milk |
Goat Milk |
Camel Milk |
pH |
6.77±0.00 |
6.63±0.06 |
6.736±0.005 |
Acidity (ºD) |
17.1±0.9 |
16.2±0.9 |
13.8±0.5 |
Density |
1.032±0.000 |
1.028±0.000 |
1.027±0.000 |
Viscosity |
3.03±0.10 |
3.84±0.06 |
3.3±0.04 |
Ash (g/l) |
8.43±0 .72 |
8.73±0.15 |
9.1±0.72 |
Matter fat (g/l) |
20.33±4.61 |
60.33±7.50 |
23.66±11.01 |
Dry matter (g/l) |
108.1±17 |
162.2±5.5 |
133.7±1.2 |
Table 3: Biochemical criteria of presumptive lactic species isolated from raw camel milk.
Technological properties of LAB isolates
Acidifying activity

The difference observed from one lactic acid bacteria species to another were explained by Badis et al. [26]. In fact, the acidifying activity of each strain is related to its specific capacity to break down the substances in the medium and render the capability of assimilation. On occasion, differences are also due to the presence or absence of nutrient transport systems [45].
Proteolytic activity
The proteolytic activity of dairy lactic acid bacteria is essential for the bacterial growth in milk and involved in the development of organoleptic properties of different fermented milk products [46,47]. The production of high quality fermented dairy products is dependent on proteolytic systems of starter bacteria, since peptidase and amino acids formed have a direct impact on flavor or serve as flavor precursors in these products.
Lipolytic activity:
Strains |
Lipolytic Diameter Zone (mm) |
Proteolytic Activity |
||
1% tween 80 |
3% tween 80 |
5% tween 80 |
Diameter Zone mm |
|
SCC1-2 |
11,375 |
9 |
9 |
15±1.4 |
SCC1-6 |
8,5 |
9 |
9,125 |
15±0.0 |
SCC1-7 |
9,5 |
10 |
9,875 |
18±1.41 |
SCC1-8 |
9,25 |
9 |
9,125 |
16±0.0 |
SCC1-13 |
13,5 |
9,75 |
9,5 |
21±0.0 |
SCC1-15 |
9,625 |
10,5 |
11,5 |
16.5±3.53 |
SCC1-24 |
8,875 |
9,5 |
10,25 |
16.5±3.53 |
SCC1-33 |
9,5 |
8,75 |
9,5 |
16.5±3.53 |
SLch6 |
8,625 |
9,5 |
11 |
19±0.0 |
SLch14 |
11,125 |
9 |
9,5 |
18±0.0 |
Table 4: Proteolytic and lipolytic activity of lactic acid bacteria.
Biomass production and growth rate:
The fermentation broth was centrifuged and the pellet was dried in order to determine biomass. The difference between the initial Optical Density (OD600) and the OD600 at which cells were collected (ΔOD600) as well as the dry weight of strains were used to reflect the growth amount (Table 5). Based on the biomass, cultures were divided into 3 groups: major yields when biomass ≥ 1.30mg/L, an average yield when the formed biomass ranged from 0.6 to 1.29mg/L, poor performance when the biomass was <0.6mg/L [35]. Strains SCC1-6, SCC1-15, SCC1-33 and SLCch14 were characterized by a high value of ΔOD600 and an important growth rate. The strains SCC1-24, SCC1-2, SCC1-13 and SCC1-13 presented a weak biomass and growth rate.
Strains |
ΔOD600* |
Biomass (g/l) |
μmax (h-1) |
EPS |
OD600 Supernatant |
SCC 1-2 |
0.44 |
0.53 |
0.05 |
- |
0.003 |
SCC1-6 |
1.13 |
0.81 |
0.13 |
- |
0.008 |
SCC1-7 |
0.67 |
0.69 |
0.07 |
- |
0.01 |
SCC1-8 |
0.86 |
0.20 |
0.08 |
+ |
0.10 |
SCC1-13 |
0.73 |
0.06 |
0.07 |
+ |
0.11 |
SCC1-15 |
1.32 |
0.79 |
0.12 |
+ |
0.02 |
SCC1-24 |
0.73 |
0,06 |
0,07 |
+ |
0.04 |
SCC1-33 |
1 .32 |
0.79 |
0,12 |
+ |
0.05 |
SLch6 |
0.68 |
0.887 |
0.14 |
+ |
0.06 |
SLCch14 |
1.93 |
0.98 |
0.13 |
+ |
0.02 |
Exopolysaccharide production:
Antagonism Effect:

Stability of lyophilized cultures:
Strains
|
Survival Rate Before Lyophilization |
Survival Rate After Lyophilization |
||
CFU/ml |
% |
CFU/ml |
% |
|
SCC1-2 |
8 108 |
100 |
2.3 108 |
28.75 |
SCC1-6 |
7.6 108 |
100 |
5.6 108 |
73.68 |
SCC1-7 |
6.5 108 |
100 |
5.3 108 |
81.53 |
SCC1-8 |
8.9 107 |
100 |
6.7 106 |
75.28 |
SCC1-13 |
5.7 108 |
100 |
5.9 108 |
103.5 |
SCC1-15 |
9.6 107 |
100 |
5.3 108 |
55.21 |
SCC1-24 |
8 .1 108 |
100 |
5.1 106 |
62.96 |
SCC1-33 |
9.2 107 |
100 |
5.2 106 |
56.52 |
SLch6 |
7.2 108 |
100 |
6.3 108 |
87.50 |
SLch14 |
5.6 108 |
100 |
7.2 106 |
128.5 |
Application of strains in the preparation of yoghurt from goat, cow and camel milk:
Camel, cow and goat milk were inoculated with these selected strains to prepare the pre-culture. After preparing dairy product, pH, acidity, and microbial count were measured. The results were shown in figure 3, 4 and table 7.


|
Day 1 |
Day 2 |
||
(1+6) |
(5+9) |
(1+6) |
(5+9) |
|
Camel milk |
172×107 |
8×1010 |
4×107 |
4.1×1010 |
Cow milk |
3.16×109 |
5.36×109 |
>300×109 |
>300×109 |
Goat milk |
>300×107 |
>300×107 |
>300×107 |
>300×107 |
The fermentation profiles obtained for the three types of milk (camel, cow and goat) are similar. The acidity increases rapidly over time in different dairy products; this is explained by the importance the inoculum (2106) and its adaptation to the fermentation because the pre-culture is carried out on the same type of milk. Indeed, the fermentation of lactose into lactic acid lowers the pH and promotes the proteolysis of proteins [57]. The increase in acidity is accompanied by a decrease in pH to a final value 4.45, 4.83 and 5.04 respectively for camel, goat and cow milk in day 1 and this decrease continue after 24h (Figure 3 and 4).
Thus, it is observed that the pH of the fermented milks for the combination of strain 1 with strain 6 appear closest to being included in the pH range of plain yogurt (4.2 to 4.3) cited by Maurice M [58].
In some countries the yoghurt-equivalent product may contain others organisms beside Lb. bulgarius and S. thermophilus. For example, in India, other bacteria are used (Lactobacilus plantarum and Lactococcus lactis subsp lactis; Marshall) [59], while Chander et al., [60] have used only mixed cultures comprising Lac.lactis biovar diacetylactis and cremoris. Lactobacilus plantarum is a heterofermentative lactobacillus producing acetate in addition to lactate, and the lactococci produce only diacetyl and no acetataldehyde.
Sensory evaluation

CONCLUSION
The present study described the technological potential of combination of 2 selected strains of Lactic Acid bacteria isolated from camel milk and their use as starters for yoghurt preparation.
Based on the overall evaluation of the obtained results, the strains selected have high acidifying activity, high proteolytic activity and sensitive reaction to antibiotics. Among 10 strains, only 2 which have high acidification rate and high yield of biomass at the end of fermentation were applied to prepare dairy product and these dairy product present lowest pH values, highest acidity and lowest microbial cell count. This work may have important implication to put in the market yoghurt based of camel, cow and goat milk using a combination of lactic acid bacteria other than Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus. After sensory evaluation, these implied that our own authority starter could produce the yogurt with similar or better quality compared with the commercial starters.
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Citation:Fguiri I, Ziadi M, Rekaya K, Arroum S, Khorchani T (2017) Isolation and Characterization of Lactic Acid Bacteria Strains from Raw Camel Milk for Potential Use in the Production of Yogurt. J Food Sci Nut 3: 026.
Copyright: © 2017 Imen Fguiri, 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.
