CHOLESTEROL LOWERING PROPERTY OF LACTOBACILLUS PLANTARUM ISOLATED FROM COW MILK

CHOLESTEROL LOWERING PROPERTY OF LACTOBACILLUS PLANTARUM ISOLATED FROM COW MILK

Amutha^* and V. Kokila

Department of Biotechnology, Vels University, Pallavaram, Chennai, Tamil Nadu, India.

ABSTRACT:  The aim of this study is to investigate the cholesterol lowering property of bacterial isolates. Different cultures of the bacteria were isolated from cow milk. Out of the cultured bacterial isolates Lactobacillus species, Micrococcus luteus and Staphylococcus aureus were screened. Three isolates such as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei were identified based on morphological, physiological and biochemical characteristics. Sugar fermentation test was performed to identify Lactobacillus species. The sugars such as D- glucose, galactose, lactose, mannitol and D-fructose were used. The five isolates were inoculated on Nutrient agar medium with cholesterol (1mg/1ml) concentration and incubated for 24 hours to check the cholesterol utilization. Cholesterol lowering activities of these organisms were checked at different concentration of cholesterol, different pH, different temperature and different incubation time. The results of this study, suggests that Lactobacillus plantarum was an effective organism for cholesterol utilization at pH 7, temperature at 35ºC and incubation time was at 24 hrs. Hence Lactobacillus plantarum has been reported as cholesterol lowering agent.

Lactic acid bacteria, Lactobacillus plantarum, Cholesterol, Milk

INTRODUCTION: Lactic acid bacteria (LAB) are the largest group of probiotic bacteria. Among the various LAB, Lactobacillus plays a vital role in both medical and also in food industry ¹. A Lactobacillus species reported to have active benefits on nutrient, improved lactose utilization, anti-carcinogenic, anti-cholesterol and protection against disease ^{2, 3, 4}. The anti oxidative activity was reported in lactic acid bacteria ⁵. Most of the Lactic acid bacteria may show antagonistic activity against pathogenic bacteria ^{6, 7}. Consumption of large amount of milk fermented with a wild Lactobacillus strains and Bifidobacteria showed reduction in serum cholesterol level in human ^{8, 9}. Some Lactobacilli have reported in vivo studies in albino rats that can lower the cholesterol and low density lipoprotein deposition^{10, 11}.

Lactobacillus plantarum 837 significantly decreased the high-density cholesterol level in Ross and Broiler chickens of both sexes ¹². Lactobacillus plantarum ATCC 14917 was investigated for cholesterol assimilation in culture media under stimulated intestinal conditions ¹³. Lactobacillus plantarum MDL 1118, isolated from milk was shown to remove cholesterol from broths of natural hen egg yolk and skimmed milk ¹⁴. Strains of Lactobacillus fermentum such as NCIMB 5221, NCIMB 2797 ¹⁵ and Lactobacillus plantarum ATCC 14917¹⁶ were selected for investigation into cholesterol lowering property.

Probiotic strains could assimilate cholesterol in the range of 14-22 µg/ml ¹⁷ and Bifidobacteriumbifidum MB 109 assimilated 50 µg/ml of cholesterol in MRS broth ¹⁸. According to Ning Xie (2011) ¹⁹ Lactobacillus plantarum 9-41A may play role in fat metabolism in order to showing decreases in the body weight gain, liver and fat pad weight and adipocytes size. The probiotic potential of the strains such as Lactobacillus plantarum was able to assimilate cholesterol by in vitro method ²⁰. Lactobacillus plantarum Lp09 and Lp45 were evaluated on plasma cholesterol in Sprague-Dawley rats ²¹.

Lactobacillus plantarum has antagonistic activity, bile resistance, bile salt deconjugation, β-galactosidase activity, antibiotic resistance activity and production of biogenic amines ²².The purpose of this study was to isolate and identify Lactobacillus plantaruma Lactic acid bacteria from cow milk and to evaluate its action on cholesterol lowering effect.

MATERIALS AND METHODS:

Isolation and Identification of bacteria from cow milk: The cow milk was collected from the North Chennai, Tamilnadu, India and stored at 5 ºC. The sample was pour plated on Nutrient agar medium in order to isolate organisms. It was incubated anaerobically for 24 hours at 37 ºC. Based on the morphological, physiological and biochemical characterization each isolates were identified ²³. Lactobacillus were identified at species level, on the basis of sugar fermentation test²⁴. The following five sugars were used for fermentation test such as D- glucose, galactose, lactose, mannitol and D-fructose. The results of sugar fermentation test were evaluated with the Bergey’s manual to identify the Lactobacilli species. Production of gas from glucose was observed by inoculating culture into inverted Durham tubes and incubating at 37 ºC for 24-48 hours.

Growth of the isolates in the presence of cholesterol: Nutrient agar media with 1mg/1ml of cholesterol concentration was prepared, five isolates were inoculated and incubated anaerobically for 24 hours at 37 ºC. Nutrient media without cholesterol as control was used in this study.

Screening of the isolates for Cholesterol lowering property:

Cholesterol lowering activity at different concentration of cholesterol: 0.2, 0.4, 0.6, 0.8, 1.0 (mg/ml) different concentration of cholesterol in 10 ml broth was used. Three isolates of Lactobacilli, two isolates such as  Micrococcus luteus and Staphylococcus aureus were inoculated and incubated at 37 ºC for 24 hours. Colorimetry reading, Optical density at 650nm were recorded and compared with positive control.

Cholesterol lowering activity at different pH level: 10 ml of Nutrient broth at different pH such as 2,4,5,7 and 8 were used. Each isolates were inoculated and incubated at 37 ºC for 24 hours. Colorimetry reading, Optical density at 650nm were recorded.

Cholesterol lowering activity at different temperatures: 10 ml of Nutrient broth at different temperature such as 20 ºC, 25 ºC, 30 ºC, 35 ºC and 45 ºC were used. Each isolates were inoculated and incubated for 24 hours. Colorimetry reading, was taken Optical density at 650nm.

Cholesterol lowering activity at different incubation time: 10 ml of Nutrient broth at different incubation time such as 1hr, 6hr, 12hr, 24hr and 48hr were used. Each isolates were inoculated and incubated at 37 ºC. Colorimetry reading, was taken Optical density at 650nm.

RESULTS: Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Micrococcus luteus and Staphylococcus aureus were isolated and identified from cow milk were tabulated in Table 1. Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei were subjected to sugar fermentation test for further identifications. Gas and acid producing positive Lactobacillus species were identified and listed out in Table 2 and Figure1. These five isolates were assessed for cholesterol lowering property. Nutrient agar with cholesterol at 1mg/1ml concentration plates when inoculated with five isolates, cholesterol utilizing organism grown very well. Out of five isolates Lactobacillus plantarum, Lactobacillus fermentum and Micrococcus luteus were shown maximum growth compared to Lactobacillus casei and Staphylococcus aureus were shown in Figure 2.

Lactobacillus plantarum grown maximum at (1g/1ml) concentration of cholesterol as shown in Figure 3. At pH 7 maximum absorbance was observed in Lactobacillus plantarum as shown in Figure 4. At 35ºC for 24 hours of incubation period Lactobacillus plantarum gave maximum absorbance at 650 nm that shows maximum growth of the organism (Figure 5 & 6). The results of present study suggest that the Lactobacillus plantarum was able to utilize cholesterol at 1g/1ml concentration of cholesterol at pH7 temperature at 35ºC and incubation period was at 24 hours.

TABLE 1: MORPHOLOGICAL, PHYSIOLOGICAL CHARACTERIZATION OF THE ISOLATES

(+) Positive, (–) Negative

TABLE 2: CARBOHYDRATE FERMENTATION PATTERN

(+) Positive reaction, (A) acid, (G) gas

FIGURE1: CARBOHYDRATE FERMENTATION TEST FOR SPECIES IDENTIFICATION

C- Control plate containing medium without cholesterol C1- Lactobacillus plantarum , C2- Lactobacillus fermentum , C3- Micrococcus luteus, C4- Staphylococcus aureus , and C5- Lactobacillus casei;

T- Test plate containing medium with cholesterol T1- Lactobacillus plantarum , T2- Lactobacillus fermentum , T3- Micrococcus luteus, T4- Staphylococcus aureus , and T5- Lactobacillus casei;

FIGURE 2: GROWTH OF ISOLATESON THE NUTRIENT MEDIUM CONTAINING WITH AND WITHOUT THE PRESENCE (CONTROL) OF CHOLESTEROL

FIGURE 3: CHOLESTEROL LOWERING ACTIVITY AT DIFFERENT CONCENTRATION OF CHOLESTEROL

FIGURE 4: CHOLESTEROL LOWERING ACTIVITY AT DIFFERENT PH LEVEL

FIGURE 5: CHOLESTEROL LOWERING ACTIVITY AT DIFFERENT TEMPERATURES

FIGURE 6: CHOLESTEROL LOWERING ACTIVITY AT DIFFERENT INCUBATION TIME

DISCUSSION: Five isolates such as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Micrococcus luteus and Staphylococcus aureus were isolated from cow milk. Bergey’s Manual was used to identify five isolates. Dora et al. (2002) ²⁵ reported cholesterol assimilation of lactic acid bacteria. Nguyen et al. (2007) ²⁶ evaluated Lactobacillus plantarum as potential probiotic with cholesterol lowering effect. Sasithorn Sirilun et al. (2010) ²⁷ selected four strains of Lactobacillus plantarum showing probiotic potential in cholesterol lowering property.  Kuda et al. (2013) ²⁸ reported Lactobacillus plantarum can be used as a profitable starter organism and probiotic. In my present study Lactobacillus plantarum shown higher utilization of cholesterol from the medium. At different concentration of cholesterol at different pH, temperature, incubation time Lactobacillus plantarum proved its stability in utilization of cholesterol. 1g/1ml concentration of cholesterol was observed by Lactobacillus plantarum for its optimal growth at pH7, temperature 35 ºC and incubation time at 24 hours.

CONCLUSIONS: Lactobacillus plantarum was able to utilize cholesterol from the culture medium at optimum pH, temperature, incubation time and different cholesterol concentration. Lactobacillus plantarum has optimum absorbance of cholesterol was about 1g/1ml hence this has been reported as cholesterol lowering agent.

ACKNOWLEDGEMENT: This research was supported by Vels University Research grant.

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    How to cite this article:

    Amutha K and Kokila V: Cholesterol Lowering Property of Lactobacillus Plantarum Isolated from Cow Milk. Int J Pharm Sci Res 2015; 6(3): 1184-89.doi: 10.13040/IJPSR.0975-8232.6(3).1184-89.

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