INTRODUCTION:

Lactobacilli can exert health benefits in several ways, for example by inhibiting growth of pathogenic bacteria as they produce lactic, propionic, and acetic acids lowering the pH that suppresses proliferation of pathogens in the GIT. Lactobacilli have a significant beneficial effect on host growth, especially by improving body weight and size. There is a growing interest to develop a variety of fermented milk products for other beneficial purposes, particularly for health purposes and preventing of toxins produced by foodborne pathogens and spoilage bacteria that enter human body [1,2,3,4]. The beneficial effects of fermented milk products are produced by a variety of bioactive compounds of LAB [5]. They are recognised for the fermentative ability and thus enhancing food safety and improving organoleptic attributes, enriching nutrients and increasing health benefits.

Products containing lactobacilli dominate the global probiotics market; from a wide range of sources, they are used widely in biotechnology and food preservation, and are being explored as therapeutics [6]. The probiotic microorganisms must be safe, i.e., the probiotic bacteria like lactobacilli, essentially be incompetent to cause hemolysis as well as gelatin liquefaction in host body. In addition, the probiotic LAB be sensitive to antibiotics so as to be inept disseminating the resistance property to other pathogenic bacteria in the same niche, or the antibiotic resistance among them should be innate and non-transferable [7].

MATERIALS AND METHODS:

CHEMICALS:

MRS broth, MRS media, Mueller Hinton Agar, Nutrient broth

COLLECTION OF SAMPLES:

Freshly collected samples of milk, butter, ghee, curd, whey, khao, ice cream, cheese and paneer was commercially purchased for identifying the species of lactic acid bacteria which is actually required for humans for its probiotic potential and it’s utilization in the future.

BACTERIAL STRAINS AND CULTURE CONDITIONS:

Samples were taken and was inoculated in MRS broth (HI media) at 37°C for 24-36 hours and after that it is again inoculated on the MRS media in order to get isolated colonies.

MICROSCOPIC OBSERVATIONS:

Grown colonies were subjected to microscopic identification. Identification of isolated LAB was subjected to species level for sugar carbohydrate fermentation test for Lactobacillus.

ANTIMICROBIAL SENSITIVITY TESTING:

DOSE DIFFUSION METHOD:

Antimicrobial assay of Lactobacillus species was performed by agar well diffusion method in Mueller Hinton Agar (MHA) plates. The test organisms were inoculated in Nutrient broth and incubated overnight at 37°C to adjust the turbidity to 0.5 McFarland standards giving a final inoculum of 1.5 × 108 CFU/ml. MHA plate was lawn cultured with standardized microbial culture broth. 50mg/ml of sample i.e different antibiotics such as Sulfasalazine, metronidazole, ciprofloxacin and ampicillin was tested with four different lactobacillus species. 6 mm of wells were bored in the inoculated media with the help of sterile cork-borer (6mm). Each well was filled with 50μl of samples i.e antibiotics with different dilutions such as Sulfasalazine, Metronidazole, Ciprofloxacin and Ampicillin respectively. It was allowed to diffuse for about 30 minutes at room temperature and incubated for 18-24 hours at 37°C. After incubation, plates were observed for the formation of a clear zone around the well which corresponds to the antimicrobial activity of tested compounds. The zone of inhibition (ZOI) was observed and measured in mm.

RESULT:

Lactobacillus was isolated from diary products such as ghee, milk, butter, cheese, curd, whether, ice cream, paneer on MRS agar plates.

The isolates were characterized based on morphology, staining and antimicrobial activity.

Morphology:

Four different species of Lactobacillus was isolated such as L. lactis, L. bulgaricus, L. acidophilus and L. brevis from all this diary products.

Colonies with entire margins, convex, smooth, glistening, opaque were found. It was again subcultured on nutrient agar and was maintained in refrigerator. Colonies of Lactobacillus with a Gram +ve, Catalase -ve, and non spore formation. It does not produce any pigment.

Table No. 1 Characteristics of Lactobacillus

S. No

Characteristics

Lactobacillus

Cultural

a) Colony morphology

b) Diameter of rods

c) Size

d) Gram reaction

e) Motility

f) Sporulation

g) Strict Anaerobes

Entire margins, convex, smooth, glistening, opaque.

Without pigment

0.5- 1.6

2-5mm

Gram positive

Uncommon reverse peritrichous flagella (non motile)

Non sporing

-ve

Biochemical test

a. Oxidase

b. Catalase

c. Nitrate reduction

-ve

Figure No 1. Lactobacillus acidophilus on MRS plate

Figure no. 2 Antagonistic Effects of Lactobacillus against Various Bacteria:

ANTIBIOTIC SUSCEPTIVITY:

Four different species of Lactobacillus was isolated such as L. lactis, L. bulgaricus, L.acidophilus and L. brevis from all the diary products mentioned above.

50µl of sample such as Sufasalazine, Metronidazole, Ciprofloxacin and Ampicillin was tested with four different lactobacillus species.

The agar well diffusion assay revealed that all strains of LAB before co-cultured were inhibitory to the different antibiotics

Lactic acid bacteria had some changes in resistance to antibiotics with different organisms in different species. At the concentration of 50mg/ml, when tested with Sufasalazine, L.lactis shows the lowest inhibition of about 1.25mm and L.brevis on ampicilin shows highest inhibition of 3.0mm and incase of metronidazole on L.bulgaricus it shows minimum inhibition of2. 0mm. When tested with L.acidophilus with the same concentration it shows the lowest inhibition of about 1.9mm

Figure No. 3 Antibacterial effect of different antibiotics by Dose diffusion method at 50mg/ml

Figure No.4 Antibacterial effect of Metronidazole by Dose diffusion method at 50mg/ml

Table 2: Zone of inhibition of concentration 50mg/ml of different antibiotics by dose diffusion method

S. No	Organism Used	Amount of Sample Added	Antibiotics used	Zone of Inhibition	% of Inhibition efficiency
1	Lactobacillus lactis	50µl	Sufasalazine	1.25 mm± 1.20	25%
2	Lactobacillus bulgaricus	50µl	Metronidazole	2.0 mm± 0.43	20%
3	Lactobacillus acidophilus	50µl	Ciprofloxacin	1.9 mm± 0.82	34%
4	Lactobacillus brevis	50µl	Ampicillin	3.0 mm± 1.32	30%

CONCLUSION:

Continuous usage of antibiotics in the human body will have the chance of damaging the normal flora. The consumption of antagonism of antibiotics using lactic acid bacteria has been well documented and the antibacterial activity of such friendly microorganisms which is really useful for human has been considered as an important attribute for the maintenance of healthy microbial balance in the gut. It is better and good to consume large amount of milk products for humans when using antibiotics.

REFERENCE:

1. 1.P. S. Panesar, “Fermented Dairy Products: Starter Cultures and Potential Nutritional Benefits,” Food and Nutrition Sciences, Vol. 2, No. 1, 2011, pp. 47-51.

http://dx.doi.org/10.4236/fns.2011.21006

2. R. Sharma, B. S. Sanodiya, D. Bagrodia, M. Pandey, A. Sharma and P. S. Bisen, “Efficacy and Potential of Lactic Acid Bacteria Modulating Human Health,” International Journal of Pharma and Bio Sciences, Vol. 3, No. 4, 2012, pp. 935-948.

3. N. P. Shah, “Functional Cultures and Health Benefits,” International Dairy Journal, Vol. 17, No. 11, 2007, pp.1262-1277.

4. A. A. Ali, “Beneficial Role of Lactic Acid Bacteria in Food Preservation and Human Health,” Research Journal of Microbiology, Vol. 5, No. 12, 2010, pp. 1213-1221.

5. M. W. Griffiths and A. M. Tellez, “Lactobacillus helve- ticus: The Proteolytic System,” Frontiers in Microbiology, Vol. 4, 2013, pp. 1-9.

http://dx.doi.org/10.3389/fmicb.2013.00030

6. Sun, Z.; Harris, H.M.B.; McCann, A.; Guo, C.; Argimon, S.; Zhang, W.; Yang, X.; Jeffery, I.B.; Cooney, J.C.; Kagawa, T.F.; et al. Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera. Nat. Commun. 2015, 6, 1–13. [CrossRef] [PubMed]

7. Jose, N.M.; Bunt, C.R.; Hussain, M.A. Comparison of microbiological and probiotic characteristics of lactobacilli isolates from dairy food products and animal rumen contents. Microorganisms 2015, 3, 198–212. [CrossRef] [PubMed]

Received on 27.09.2019 Modified on 30.11.2019

Research J. Pharm. and Tech. 2020; 13(8):3915-3917.

DOI: 10.5958/0974-360X.2020.00693.9