Evaluation of Market Curd for Sanitary Quality and Bacteriocin-Producing Lactic acid Bacteria for Potential Application as a Natural, Healthy Food Preservative

 

Shalini Singh*, Sujata Das

School of Biotechnology and Biosciences, Lovely Professional University, Punjab, India-144411.

 

ABSTRACT:

The quality of curd in local market varies as there are no well described standards for milk products, making evaluation of their microbiological quality, important.  Curd is a great source of lactic acid bacteria, important producers of bacteriocins (proteinaceous antimicrobials). The study evaluates curd samples, collected from local market of Jalandhar, for sanitary quality and presence of  bacteriocin-producing lactic acid bacteria (LAB). Of the total 21 curd samples tested, only 14% showed confluent microbial growth and categorized as ‘poor quality’ in terms of microbial load. Sample 6 exhibited best sanitary quality. 3 isolates (LABI, II, and III), of the total of 42 bacterial colonies tested, were promising bacteriocin producers against Vibrio parahemolyticus and E.coli with, LABIII (17357 U mL ^-1) as the best bacteriocin producer, exhibiting 13.1% higher activity than standard reference, Lactococcous lactis subspecies lactis MTCC440, against E.coli. LABI and II showed similar bacteriocin activity against V. parahemolyticus while, against E.coli, bacteriocin activity was 7539 U mL ^-1 and 10995 U mL ^-1 for LABI and II, respectively. The future work will explore efficiency of LABIII as bio-preservative in different food materials in cost effective manner. This would help to provide economic solutions for food preservation with distinct and well proved health benefits for the consumers.

 

 

 

INTRODUCTION:

A variety of pathogenic organisms may reach milk and milk products like, curd and contribute towards a variety of food borne illnesses. Milk and milk products may carry toxic metabolites of different organisms growing in it. Ingestion of such products, contaminated with these metabolites, cause food poisoning. On the other hand, the ingestion of microbial pathogens along with the food product leads to food borne infection¹. The quality of curd in local market varies from shop to shop and poor quality milk, unhygienic practices associated with the process involved, and the use of wild type of starter cultures, give rise to poor grade curd².

 

Evaluation of the microbiological quality of milk products is thus, an important task and numerous attempts have been made by researchers worldwide to check milk and milk products including, curd for their microbiological quality^3-4. Apart from the presence of pathogenic microorgansisms, milk and milk products have also been characterized for the presence of beneficial bacteria as well. The lactic acid bacteria (LAB), commonly found in milk and milk products, exhibit antimicrobial activity against a number of microorganisms, including food spoilage organisms and pathogens^3,5 and hence, are highly beneficial to us. Bacteriocins are proteinaceous compounds lethal to bacteria other than the producing strain. As a group, bacteriocins are heterogeneous, and they are classified largely based on their molecular weight differences. Some examples of bacteria that produce bacteriocins are: Lactococci (L. delbrueckii L. lactis), Lactobacilli (L. casei, L. delbrueckii)^1,6,7.  The antimicrobial nature of the bacteriocins has highlighted their usage in food preservation to a great extent. The bacteriocins produced by LAB are suitable for food preservation as they are generally recognised as safe substances, they are not active and nontoxic on eukaryotic cells, they become inactivated by digestive proteases, having little influence on the gut microbiota, they are usually pH and heat-tolerant, they have a relatively broad antimicrobial spectrum, against many food-borne pathogenic and spoilage bacteria, they show a bactericidal mode of action, usually acting on the bacterial cytoplasmic membrane: no cross resistance with antibiotics, and their genetic determinants are usually plasmid-encoded, facilitating genetic manipulation⁸.

 

To date nisin is the only bacteriocin that has found many applications in industrially processed foods. Nisin has been found to be suitable for use in a wide variety of foods^9-10. Thus, extensive research needs to be done to explore the bacteriocins produced by lactic acid bacteria in the hope of obtaining better, efficient antimicrobial agents that can be utilized for preserving food. Keeping in mind the significance of strict compliance of quality of curd, and the useful properties bacteriocins exhibit, this study was carried to check the microbiological quality of curd and to evaluate curd samples for the presence of bacteriocin producing Lactic acid bacteria and to check the antimicrobial activity of bacteriocin against specific test bacterial strains for evaluating their potential to be used as a source of natural antimicrobials that can play an important role in safe, environment friendly food preservation strategies.

 

MATERIALS AND METHODS:

Microorganisms:

Lactic acid bacterial isolates were obtained from curd samples collected from Jalandhar region of Punjab, India and screened for their ability to secrete bacteriocin. Lactococcus lactis subspecies lactis MTCC 440 and Lactobacillus plantarum MTCC 1407 procured from IMTECH Chandigarh, India were taken as reference LAB strains for comparison of antimicrobial activity of the lactic acid bacterial isolates obtained in the present study. Vibrio parahaemolyticus and E. coli were used as test bacterial strain against which, antimicrobial activity of bacterial isolates was tested

 

Collection of curd samples:

A total of 21 curd samples were collected from dairy outlets located in Jalandhar region of Punjab, India. All samples were collected in sterile container plastic bottles and refrigerated at 4^oC for further analysis.

 

Microbiological quality of curd samples:

For enumeration of bacteria in the curd samples, standard plate count (SPC)^1,11 was done for each of the curd samples. Under aseptic conditions, one ml of each curd sample at appropriate dilutions was inoculated on sterile nutrient agar medium by spread plate method. The plates were incubated at 37°C for 24-48 hours and the number of bacterial colonies that appeared after the incubation period was counted using a colony counter. Similarly, yeast and mould count was also made using by inoculating appropriately diluted curd samples and inoculating them on Sabouraud dextrose agar medium (SDA) by spread plate method. The inoculated plates were incubated at 37^oC and the fungal colonies counted using colony counter¹².

 

Isolation, partial identification and screening of curd microflora (Lactic acid bacteria) for the production of bacteriocin:

The curd samples at, appropriate dilutions, were spread plated on MRS agar medium. The plates were incubated at 37⁰C for 24-36 hours and subsequently observed for colony morphology, Gram staining reaction and catalase test and sugar fermentation test¹³. The lactic acid bacterial isolates were then checked for their ability to produce bacteriocin and the same was compared with those for standard reference strains (Lactococcous lactis subspecies lactis MTCC440 and Lactobacillus plantarum MTCC1407). All the bacterial isolates and the standard reference strains were inoculated in MRS broth and incubated overnight at 37°C. After 24 hours of incubation, the broth cultures were centrifuged at 10,000 rpm for 20 minutes at 4°C. The pH of the supernatant was maintained at 7.0. The supernatant, maintained at pH 7, was then filtered through 0.2 µm pore size cellulose acetate filter. Dialysis of the filtered supernatant was performed for 24 hrs at 4°C using 0.05 M potassium phosphate buffer. The dialyzed samples were subjected to ammonium sulphate precipitation at a concentration of 20% and 40%. Centrifugation was repeated for the precipitated samples at 20,000 rpm for 1hr at 4°C, followed by dialysis of the supernatant received for 18 hours^14-15. The antimicrobial activity of bacteriocin against test pathogenic strains (Vibrio parahaemolyticus and E. coli) was determined by agar well diffusion method. The test pathogenic strains were swabbed on sterile Mueller Hinton agar plates. 25µl of the dialyzed samples was placed in wells cut in plates using a sterile well puncture. The plates were incubated at 37°C and examined for zones of inhibition of growth after 24 hours of incubation¹².      

 

RESULTS AND DISCUSSION:

Microbiological quality of curd samples:

As table 1 indicates, curd samples 3, 4 and 11 showed confluent growth (indicated by standard plate count) and hence, classified as ‘Too Many Too Count, TNTC’ as the enumeration of bacteria present could not be done for the given samples. In terms of bacteriological growth, sample 2 showed the best quality as the bacteroiological count (149x10¹⁰) was the lowest for this particular curd sample. Curd samples 3, 4, and 11 were also found to show confluent growth in reference to Yeast and Mold Count and hence, classified as ‘Too Many Too Count, TNTC’. The best sanitary quality of curd was for sample 6 as indicated by the Yeast and Mold Count with least number of fungal counts reported (105x10⁶), compared to other samples tested. The quality of curd in local market varies from shop to shop as there is no well described standard for these products. Poor quality milk, unhygienic practices associated with the process involved and the use of wild type of starter culture give rise to poor grade curd².

 

Table 1: Microbiological quality of curd samples collected from local market of Jalandhar, Punjab, India

Isolation, partial identification and screening of curd microflora (Lactic acid bacteria) for production of bacteriocin

A total of 42 bacterial colonies were isolated from 21 curd samples. The morphological variations were, pinpointed-creamish-non mucoid, pinpointed-white-non mucoid, low convex-flat-dry, circular-flat-dry, irregular-white-mucoid, low convex-irregular, pin-pointed-creamish-mucoid and pin pointed-creamish-mucoid.

 

Out of the 42 bacterial colonies checked for catalase enzyme, only 3 were found to be catalase negative. All the bacterial isolates were found to be Gram positive but a variation in bacterial cell shape and arrangement was observed for different isolates.

 

The cell shape varied from rods to cocci while the cell arrangement varied from singly occurring rods, chains of rods, pairs of cocci, tetrads of cocci to singly occurring cocci. Bacterial isolates (3 isolates) exhibiting pinpointed, creamish, mucoid colonies and Catalase negative and Gram positive (characteritic features of Lactic acid bacteria) nature, were selected for further study. Thus, LAB were isolated and use further testing for bacteriocin production. The selected isolates were designated as LAB I, LABII and LABIII for future reference.

 

All the 3 isolates selected for testing for bacteriocin activity, were found to be exhibit variable desired activity when tested against different test organisms. LABIII was found to be the best source of bacteriocin against test organism, as compared to the other bacterial isolates. It even exhibited better bacteriocin activity than Lactobacillus plantarum MTCC1407 when tested against E.coli when it showed 13.1% higher bacteriocin activity than that for Lactobacillus plantarum MTCC1407 against the same test organism.

 

This particular bacteriocin activity was in fact, same as that for Lactobacillus plantarum MTCC1407 when tested against Vibrio parahemolyticus. LABI and II showed similar bacteriocin activity against V. parahemolyticus but that against E.coli was different for both the isolates. While LAB isolate II showed similar activity against V. parahemolyticus and E.coli, LABI showed 31.2% times lesser activity against E.coli as compared to that against V. parahemolyticus. Overall, the isolates can be arranged in the decreasing following order of bacteriocin activity against V. parahemolyticus: LABIII > LABI and II. The same can be arranged for bacteriocin activity against E.coli: LABIII > LABII > LABI. Lactococcus lactis CCSULACI has been reported to show broad antimicrobial activity including, activity against both E. coli, Enterobacter sp., among others¹⁶. The LAB isolates especially, LABIII, shows promising bacteriocin activity

 

These strains thus, have the potential to be biopreservatives. Future work will explore LAB strains for cost effective preservation for different kinds of food materials^17-18.

 

Table 2: Morphological and Biochemical properties of isolated bacterial isolates

Samples

Iso-lates

Morphological Features

Catalase test

Gram staining reaction

Sugar Fermentation

A

B

C

D

E

a

b

a

b

a

b

a

b

a

b

1

I

Pin-Pointed, creamish, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

II

Pin-Pointed, white, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

2

I

Irregular, white, mucoid

+ve

+ve long rods in chains

-

-

+

+

-

-

+

+

+

+

II

Low convex, irregular

+ve

+ve cocci in tetrads

+

+

-

-

+

+

-

-

-

-

3

I

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

II

Pin-Pointed, white, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

4

I

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

II

Pin-Pointed, creamish, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

5

I

Irregular, white, mucoid

+ve

+ve long rods in chains

-

-

+

+

-

-

+

+

+

+

II

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

6

I

Pin-Pointed, white, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

II

Low convex, irregular

+ve

+ve cocci in tetrads

+

+

-

-

+

+

-

-

-

-

7

I

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

II

Irregular, white, mucoid

+ve

+ve long rods in chains

-

-

+

+

-

-

+

+

+

+

8

I

Low convex, irregular

+ve

+ve cocci in tetrads

+

+

-

-

+

+

-

-

-

-

II

Pin-Pointed, white, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

9

I

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

II

Low convex, irregular

+ve

+ve cocci in tetrads

+

+

-

-

+

+

-

-

-

-

10

I

Pin-Pointed, creamish, mucoid

-ve

+ve cocci in pairs

-

-

+

+

+

+

+

+

+

+

II

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

11

I

Pin-Pointed, creamish, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

II

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

12

I

Low convex, irregular

+ve

+ve cocci in tetrads

+

+

-

-

+

+

-

-

-

-

II

Irregular, white, mucoid

+ve

+ve cocci in pairs

-

-

+

+

-

-

+

+

+

+

13

I

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

II

Low convex, irregular

+ve

+ve cocci in tetrads

+

+

-

-

+

+

-

-

-

-

14

I

Pin-Pointed, creamish, mucoid

-ve

+ve cocci in pairs

-

-

+

+

+

+

+

+

+

+

II

Irregular, white, mucoid

+ve

+ve cocci in pairs

-

-

+

+

-

-

+

+

+

+

15

I

Pin-Pointed, white, non-mucoid

+ve

+ve cocci

+

-

-

-

+

-

+

-

+

+

II

Irregular, white, mucoid

+ve

+ve cocci

-

-

+

+

-

-

+

+

+

+

16

I

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

II

Pin-Pointed, white, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

17

I

Pin-Pointed, white, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

II

Pin-Pointed, creamish, mucoid

-ve

+ve cocci in pairs

-

-

+

+

+

+

+

+

+

+

18

I

Pin-Pointed, white, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

II

Pin-Pointed, creamish, non-mucoid

+ve

+ve cocci in pairs

+

-

-

-

+

-

+

-

+

+

19

I

Pin-Pointed, white, non-mucoid

+ve

+ve cocci

+

-

-

-

+

-

+

-

+

+

II

Circular, flat, dry

+ve

+ve cocci in tetrads

-

-

+

-

+

-

+

-

+

+

20

I

Pin-Pointed, creamish, non-mucoid

+ve

+ve short rods

+

-

-

-

+

-

+

-

+

+

II

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

21

I

Circular, flat, dry

+ve

+ve cocci in pairs

-

-

+

-

+

-

+

-

+

+

II

Irregular, white, mucoid

+ve

+ve cocci in pairs

-

-

+

+

-

-

+

+

+

+

*A: Ribose, B: Trehalose, C: Lactose, D: Arabinose, E: Fructose # a: Acid production, b : Gas production

 

Table 3: Antimicrobial activity of selected lactic acid bacterial strains

Lactic acid bacteria	Vibrio parahemolyticus		E.coli
	Zone of inhibition, cm	Bacteriocin activity, U mL -1	Zone of inhibition, cm	Bacteriocin activity, U mL -1
Lactococcous lactis subspecies lactis MTCC440	2	31101	1.4	15079
Lactobacillus plantarum MTCC1407	1.5	17357	2	31101
LABI	1.2	10995 (-64.6%)a (-36.6%)b	1	7539  (-50.0%) a (75.7)b
LABII	1.2	10995 (-64.6%)a (-36.6%)b	1.2	10995 (-27.0%) a (-64.6%) b
LABIII	1.4	15079 (-51.5%)a (-13.1%)b	1.5	17357 (+13.1%) (-44.1%) b

^a % increase/decrease in bacteriocin activity from standard reference bacterial strains-Lactococcous lactis subsp. lactis MTCC440

^b %increase/decrease in bacteriocin activity from standard reference bacterial strains-Lactobacillus plantarum MTCC1407

 

 

 

CONCLUSION:

The study highlights the importance of exploiting bacteriocins for natural food preservation. The bacterial isolates in the investigation, especially LABIII shows promising bacteriocin activity, even better than the reference standard strains of lactic acid bacteria tested. Thus, the isolates obtained can be explored in preservation of different food materials as whole cells or isolated proteins.

 

ACKNOWLEDGEMENT:

The authors are thankful to School of Biotechnology and Biosciences, Lovely Professional University, Punjab, India for providing facilities for carrying out the study.

 

CONFLICT OF INTEREST:

All the authors certify that there is no conflict of interest in relation to this article

 

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Accepted on 29.03.2017           © RJPT All right reserved