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Isolation and characterization of inhibitory substance producing lactic acid bacteria from Ergo, Ethiopian traditional fermented milk

Esayas Assefa, Fekadu Beyene* and Amutha Santhanam**

Adami Tulu Agricultural Research Center, P.O.Box-35, Ziway, Ethiopia
ifeso2003@yahoo.com
* Hawasa University, College of Agriculture, P.O.Box-05, Awassa, Ethiopia
** Addis Ababa University, Department of Biology, P.O.Box-1176, Ethiopia

Abstract

Isolation and characterization of lactic acid bacteria strains with a potential for starter and production of antimicrobial factor should be the first essential step to produce microbiologically and chemically safe and health promoting food products. Ergo is traditional Ethiopian fermented milk produced by natural fermentation using different utensils and time honored technology adapted to the local environment. Ergo samples have been collected from several farms in Adami Tulu, Lome and Fentale districts of Oromia State, to isolate, and characterize Lactic Acid Bacteria (LAB) and evaluate their potential for antimicrobial activity.

 

Strains of (112) lactic acid bacteria , belonging to genus Lactococcus, Lactobacillus, Entorococcus, Streptococcus, Leuconostoc and Pediococcus were isolated from Ergo and the culture filtrates of the isolates were examined for antimicrobial activity on some test strains (Salmonella thyphi, Shigella flexineri, Staphylococcus aures (ATCC-25923) and Escherchia coli (ATCC-25922)) using disc diffusion assay method. Twelve strains of lactic acid bacteria that produced the greatest antimicrobial substance as indicated by the inhibition zone were screened as positive. Among these three were identified as strains of the Lactococcus lactic spp lactic, two as Lactobacillus acidophilus, two as Lactococcus lactic ssp crimoris, two as Lactobacillus plantarum, one as Leuconostoc lactic, one as Pediococcus pentosaceus and one as Pediococcus spp.

 

This study indicated that Ergo is a rich source of diverse antimicrobial activity, and the isolates are of great potential for use in the production of probiotics and cultured dairy products.

Key words: disc-diffusion assay, Ergo, fermented milk, inhibition, lactic acid bacteria


Introduction

Microbial fermentations have played an important role in food processing for thousands of years. Fermentations provide a way to preserve food products, to enhance nutritive value, to destroy undesirable factors, to make a safe product, to improve the appearance and test of some foods, and to reduce the energy required for cooking (Paredes Lopez1992). These significant changes causing desirable biochemical effects involve in the development of new aroma, flavor, and taste and texture there in increasing the sensory quality, palatability and acceptability of the product. Besides this, fermentation is relatively a low energy preservation that promotes the shelf life of products (Cooke et al 1987).

 

Traditional methods of preparing fermented foods are not complicated and do not require expensive equipment (Ayele Negatu 1992). Fermentation of indigenous food is, therefore, considered by many people to be an effective, inexpensive and nutritionally beneficial household technology for communities with food scarcity and malnutrition (Jay 1994). In traditional lactic acid (LAB) fermented foods, it is very common to use and follow controlled natural fermentation processes.

 

Lactic acid bacteria (LAB) are widespread in nature and predominate in microflora of milk and its products, many species are involved in the daily manufacturing of dairy products (Ayad et al 2004). Lactic acid bacteria (LAB) are widely utilized to produce fermented foods contributing to flavor development as well and safe metabolic activities while growing in foods therein utilizing available sugar for the production of organic acids and other metabolites (Ayele Nigatu 1998). In common fermented products such as yogurt, lactic acid is produced by the starter culture bacteria to prevent the growth of undesirable microorganisms (Daeschel 1993).

 

The rural people in Ethiopia are producing fermented milk by traditional methods. The major fermented milk products produced by smallholder farmers by traditional methods include “Ergo” (fermented sour milk), “Ititu” (Fermented milk curd), “Kibe” (traditional butter), “Neter kibe” (spiced butter), “Ayib” (cottage cheese), “Arerra” (Sour defatted milk), and “Aguat” (whey). “Ergo” is a traditional naturally fermented milk product, which has some resemblance to yogurt. It is thick, smooth and of uniform appearance and usually has a white milk color when prepared carefully. The product is semi solid and has a pleasant odor and taste. It constitutes a primary sour milk product from which other products may be processed. Almaz Gonfa (2001) reported that Ergo can be stored for 15-20 days depending on the storage temperature.

 

Ergo is mainly produced on farm by women who may further process in to more stable products which may be sold in the market, and thus generates income by which other household items purchased (Lemma Fita 2004). As the major fermented dairy product, Ergo is popular and is consumed in all part of the country and by every member of the family. Ergo is considered as a special food which serves as a basis for further processing and it is particularly used as a nutritional support to sick people, children and to pregnant and lactating mothers of the family (O’Connor 1994).

 

Ergo fermentation is usually natural, with no defined starter cultures used to initiate it. This is made possible only through the proliferation of the initial milk flora, with microbial succession determined by chemical changes in the fermenting milk. In most urban homes, no attempt seems to be made to control the fermentation. Raw milk is either left at ambient temperatures or kept in warmer places to ferment. In rural areas, particularly among the pastoralists raw milk is usually kept in a well-smoked container and milk from a previous fermentation serves as inoculum. Lactic acid bacteria also become established on the inner walls of the container and serve as starter cultures. Incubation temperature dose not usually vary significantly and the test of the fermented product may, in general, be more or less uniform (Mogessie Ashenafi 2002).

 

Fermented milk Ergo plays important role in the diet of low income and the majority of people in the rural areas of Ethiopia using traditional utensils. But if “Ergo” is to be produced on large scale some tasks have to be undertaken according to Mogessie Ashenafi (2002), Thus it is logical to start with isolating and characterizing as many lactic acid bacteria as possible from ‘Ergo’ produced in the various ecological zones of the country. These cultures could be identified, selected and various combinations of them could be used for a controlled fermentation of ‘Ergo’. Moreover, isolation and characterization of promising strains of lactic acid bacteria for use as starter culture is the essential first step in the right direction (Fekadu Beyene et al 1998) and use of starter culture with the potential to produce inhibitory factors would result in the improved safety of fermented products. The aim of this study was isolating and identifying inhibitory substance producing lactic acid bacteria from Ergo and collection of strains for further evaluation as starter culture and probiotic preparation.

 

Materials and methods  

The study areas

 

The area of the study was in three districts of East Shewa Zone (Adami Tulu-Jidocombolcha, Lumme and Fentale) of Oromiya Regional State, Ethiopia. The altitude of these areas ranges from 910-2300 meters above sea level (masl) and have arid and semi-arid type of climate with an erratic, unreliable rainfall averaging between 500 and 900 mm per annum (Lemma Fita 2004). The rainfall is bimodal with the short rains from February to May, and long rains from June to September. The predominant production system in the area is mixed crop livestock farming, except for Fentale where pastoral and agro-pastroral production system predominates.

 

Test (indicator) strains

 

Salmonella thyphi (Clinical isolate), Shigella flexineri (Clinical isolate), Staphylococcus aureus (ATCC-25923) and Escherichia coli (ATCC-25922) obtained from Ethiopian Health and Nutrition Research Institute, Addis Ababa, Ethiopia were used as test microorganisms. 

Ergo sample collection  

Samples of ‘Ergo’ (250 ml) were collected from 10-selected farms from each of three districts, and 30 samples were collected using sterilized flasks and kept under refrigeration temperature using an icebox and brought to the dairy laboratory. Samples were then kept in refrigerator until analysis. 

Plating and isolation of lactic acid bacteria (LAB) 

Plating was done within 3-6hrs of arrival of the sample in the laboratory. Enumeration of lactic acid bacteria were done after plating well diluted (10-5) 0.1 ml of sample on de Mann Rogosa Sharp agar (MRS) (HIMIEDIA) and incubated anaerobically in anaerobic jar (BBL, GasPak Plus) at 35oC for 48hrs with replications.

 

Ten colonies were randomly selected from countable MRS agar plate replica. The colonies were purified by successive streaking on appropriate agar media (MRS) before being subjected to characterization. Five colonies with different morphology from each plate were transferred to MRS broth, incubated for about 12 hours and maintained in the refrigerator at 4 oC . The isolates were grouped as lactic acid bacteria after examining for their Gram reaction, cell and colony morphology, catalase reaction and Gas production from glucose fermentation. Those that are characterized as Lactic acid bacteria were kept as a stock culture in the refrigerator at -20 oC in a glycerol solution.  

Antimicrobial activity and bioassay 

The antimicrobial activities of the isolates were quantified by modifying the disc-diffusion method assay procedure of Savadogo et al (2004) and Girum Tadesse et al (2005).

 

A well-isolated colony was selected from MRS agar plate culture. The top of the colony was touched with a loop, and the growth is transferred into a tube containing sterile 5ml MRS broth. And the broth culture is incubated at 35oC for about 24hrs. To get the culture filtrate a 24hrs cultures were centrifuged (10,000 rpm for 20min, at 4oC) then was adjusted to pH 7 by 1M NaOH to exclude antimicrobial effect of organic acid (Savadogo et al 2004). Two control test materials also were prepared using un-inoculated MRS broth.

 

An actively growing test (indicator) microorganism in a Tryptone Soya broth (OXOID) of 24hrs culture at 35oC were dipped with a sterile cotton swab which was rotated several times and pressed firmly on the inside wall of the tube above the fluid level to remove excess inoculum from the swab. The dried surface of a Mueller-Hinton agar (HIMIDIA) plates were inoculated by streaking the swab over the entire sterile agar surface. This procedure was repeated by streaking two more times, rotating the plate approximately 60oC each time to ensure an even distribution of inoculum.

 

A sterile approximately 6 mm in diameter discs (Whatman filter paper no. 1) were delivered with culture filtrate of each isolate by using a wire loop of 20 gauge wire having 2mm of diameter, that can deliver 5Ál of the extract to each disc (Lalitha 2005). Four discs (with culture filtrate) were placed on a 100mm plate with in 5 to 15 minutes of striking of the test organisms.

 

After 12 and 24 hours of anaerobic incubation, each plate was examined. The diameter of the zones of complete inhibition was measured, including the diameter of the disc. Zones are measured to the nearest whole number in millimeter, using transparent ruler.

 

Characterization and identification of inhibitory substance producing LAB strains

 

The carbohydrate fermentation profiles of the selected 12 LAB isolates were investigated using API 50 CH strips, and API CHL medium according to manufacturer’s instruction (API system, bio-merieux, France). Overnight cultures of the isolates grown in 10ml MRS broth at 30oC were washed twice with sterile peptone water and the pellets were re-suspended in API 50 CHL medium, using sterile pasture pipettes. With subsequent mixing, homogenized suspensions of the cells in the medium where transferred into each of the 50 wells on the API 50 CH strips. Strips were covered as recommended by the manufacturer instruction and incubated at 30oC. Changes in color were monitored after 24hrs of incubation. The first strip served as a control well and Esculine hydrolysis (well 25) was revealed by change to darker or black colors, others changed to yellow or no change at all. Results were represented by positive sign (+) while a negative sign (-) was designated for no change. 

 

Results  

Isolation of lactic acid bacteria 

Gram positive, catalase negative, cocci, cocco-bacilli or rod shaped isolates with characteristic cell arrangements when grown on MRS growth media were considered as lactic acid bacteria (Savadogo et al 2004). From a total of 150 bacteria isolated from the 30 samples (10 from each district) 112 of them are tentatively considered as lactic acid bacteria (Table.1).


Table 1.  Lactic acid bacteria isolated from Ergo that were collected from three districts (Lumme, Adami Tulu, Fentale).

Site

Number and category of LAB isolated and identified from the different districts

Lactobacillus

Lactococcus

Luconostoc

Entrococcus

Streptococcus

Pediococcus

None LAB

Lumme

10

9

0

5

4

2

20

Adami Tulu

16

20

3

3

2

4

2

Fentale

11

0

5

8

5

5

16

Total

37

29

8

16

11

11

38


Thirty of which are isolated form Lumme, 48 from Adami Tulu and 34 from Fentale districts. The LAB isolated comprised of five genera, Lactobacillus, Lactococcus, Leuconostoc, Entrococcus and Streptococcus. 

Antimicrobial activity of the isolates 

The antimicrobial activity of isolated LAB and degree of inhibition is given in Table 2.


Table 2.   Average diameter of inhibition and antimicrobial activity of the inhibitory culture filtrate, on some indicator strains, using disc diffusion assay technique

No

LAB strain

Test bacteria

Diameter of inhibition, mm (Mean ▒ SD)

1

Mj-622

Salmonella typhi

10 ▒ 0.00

Shigella flexineri

11.8 ▒ 0.35

2

Ad-211

Salmonella typhi

11.8 ▒ 0.35

Shigella flexineri

10 ▒ 0.00

Staphylococcus aureus (ATCC-25923)

9 ▒ 0.00

3

Ad-233

Salmonella typhi

9 ▒ 0.00

Shigella flexineri

10 ▒ 0.00

Staphylococcus aureus (ATCC-25923)

10 ▒ 0.35

4

Ad-355

Salmonella typhi

9.5 ▒ 0.70

Shigella flexineri

9 ▒ 0.00

Staphylococcus aureus (ATCC-2592)

9.25 ▒ 0.35

5

Ad-411

Salmonella typhi

10 ▒ 0.00

6

Ad-522

Shigella flexineri

10.5 ▒ 0.00

Staphylococcus aureus(ATCC-25923)

9.25 ▒ 0.35

7

Ad-855

Salmonella typhi

11.8 ▒ 0.35

Shigella flexineri

11.3 ▒ 0.35

Escherichia coli (ATCC-25922)

10 ▒ 0.00

8

Fn-133

Salmonella typhi

10 ▒ 0.00

Escherichia coli (ATCC-25922)

9 ▒ 0.00

9

Fn-144

Salmonella typhi

9.25 ▒ 0.35

10

Fn-233

Salmonella typhi

9.25 ▒ 0.35

11

Fn-533

Shigella flexneri

9 ▒ 0.00

12

Fn-555

Shigella flexneri

9 ▒ 0.00


From a total of 112 lactic acid bacteria isolates that were subjected to antimicrobial activity test, using disc diffusion method, the extracts of 12 strains of lactic acid bacteria gave zone of inhibition on to indicator strain tested. The diameters of inhibition ranged from 7mm to 12mm.

 

The biggest diameters of 12mm were obtained from the extracts of strains Mj-622 on Shigella flexinery, Ad-211 on Salmonella typhi and Ad-855 on Salmonella typhi and Shigella flexineri (Photo.1).



Photo 1.
  Inhibition Zone on test strains formed by lactic acid bacteria isolated from Ergo


In contrast, Escherichia coli (ATCC-25922) was the most resistant strain it was only inhibited by two strain (Ad-855 and Fn-133) by a diameter of 10mm and 9mm respectively, followed by Staphylococcus aures (ATCC-25923) that was inhibited by four strains with low diameter of inhibition, Strain Ad-211 by the diameter of inhibition 9mm, Ad-233 by 10mm, Ad-411 by 10mm and Ad-522 by 10mm exhibited inhibitory activity against indicator organisms.

 

Identification of the inhibitory substance producing strains

 

The morphological and physiological characteristics (Table3.) and the carbohydrate fermentation profile of the inhibitory substance producing LAB isolates were used to tentatively designate to species according to Bergey’s Manual of Determinative Bacteriology (Holt et al 1994) in addition to identification table in API 50 CH user manual.


Table 3.  Morphological, cultural and physiological characteristics of inhibitory substance producing strains of lactic acid bacteria isolated from  isolated from Ergo

Characteristics

Inhibitory substance producing lactic acid bacteria isolates

Mj-622

Ad-211

Ad-233

Ad-355

Ad-411

Ad-522

Ad-855

Fn-133

Fn-144

Fn-233

Fn-533

Fn-555

Cell morphology

Coc

Coc

Coc-baci

Coc

Coc

Rod

Coc

Coc

Coc

Rod

Coc

Coc

Cell arrangement

Pr

Pr,S. ch

Pa,S.ch

Pair

Pr & ch

Pr&ch

Pr&ch

S.ch

Pr

Pr

Pr&ch

Pair

Gram stain reaction

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

Spore formation 

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

Colony morphology

Ft & Irg

Ra & cir

Ra& cir

Ra& cir

Ra &cir

Ra&cir

Ra&cir

Ra&cir

Ra&cir

Ra&cir

Ra&cir

Ra&cir

Catalase activity

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

Glucose fermentation.

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

Growth at

Temperature, oC

10

+ve

+ve

-ve

ND

+ve

-ve

+ve

-ve

-ve

-ve

-ve

+ve

45

-ve

+ve

-ve

-ve

+ve

ND

-ve

ND

+ve

ND

-ve

+ve

Growth in medium

NaCl, %

4

-ve

+ve

-ve

+ve

+ve

-ve

-ve

-ve

+ve

-ve

-ve

+ve

Milk curdling

-ve

+ve

-ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

-ve

+ve

pH optimum

5.20

5.11

4.43

4.41

4.13

4.13

4.41

4.14

4.20

4.13

4.50

4.22

Legend: Positive reaction (+), Negative reaction (-ve), Not Determined (ND), Flat (Ft),Raised (Ra), Circular (Cir), Irregular ( Irg), Pair (Pr), Short chain (S.ch), Chain (Ch) 


Twelve strains among totally isolated (one hundred and twelve) lactic acid bacteria isolated from “Ergo” were selected according to their maximum antimicrobial activity against indicator strains. Two of these isolates were identified as Lactobacillus plantarum 2 (Ad-522 and Ad-233), two as Lactococcus lactic ssp cremoris of different strain 1 and 2 (Mj-622 and Ad-855 respectively) three as Lactococcus lactic ssp lactic of different strains (Fn-555 and Ad-411 as strain 1 and Ad-211 as strain 2), two as Lactobacillus acidophyilus of different strain 1 and 3 (Fn-133 and Fn-233 respectively), one as Leuconostoc lactic (Fn-533), one as Pediococcus pentosace (Fn-144) and one as Pediococcus sp (Ad-355).

 

Discussion  

The lactic acid bacteria isolated from fermented milk includes in the Lactobacillus, Lactococcus, Pediococcus, Entrococcus and Streptococcus genera. These results showed the heterogeneousness of fermented milk (‘Ergo’) at different location or agro climatic zones either in its chemical or microbial composition and quality attributes. These finding was inline with the observations of others who worked on fermented milk, Almaz Gonfa et al (1999) in Ethiopia, Savadogo et al (2004) in fermented milk of Burkina Faso, Ayad et al (2004) in Egypt.

 

The variation in the type and number of the lactic acid bacteria present as well as the traditional practices (like smoking) would influence the hygienic quality and organoleptic property of the fermented milk. Traditionally different herbs and pepper are used in the consumption of fermented milk as beverage, or side dish, perhaps to get acceptable flavor. The variations in the characteristics, quality and acceptability of traditional fermented milk products are the result of unregulated nature of natural fermentation. Indeed some of these organisms could perhaps have beneficial effect on health of the consumers.

 

All the lactic acid bacteria were subjected to inhibitory activity test using a disc diffusion method. Only twelve isolates (three strains of Lactococcus lactic ssp lactic, two Lactobacillus acidophilus, two Lactococcus lactic ssp crimoris, two Lactobacillus plantarum, one Leuconostoc lactic, one Pediococcus pentosaceus and one as Pediococcus sp. ) showed inhibition zone on the test bacteria, Salmonella thyphi (Clinical isolate), Shigella flexineri (Clinical isolate), Staphylococcus aureus (ATCC-25923) and Escherchia coli (ATCC-25922), to varying degree. The spectrum of antimicrobial activity for the species suggested that the inhibitory components were different (Larsen et al 1993 and Schillinger and Lucke 1989). Similarly Girum Tadesse et al (2005) and Cadirici and Citak (2005) observed varying degree of inhibition of various food born pathogens by the culture filtrate of lactic acid bacteria, although these inhibitory substances produced by the lactic acid bacteria strains acts differently on the pathogenic reference indicator strains, inhibitive substances produced by the lactic acid bacteria can be generally protein (Vandenberg 1993). Inhibition caused by hydrogen peroxide and organic acids was ruled out as the producer strains were cultured anaerobically and the culture supernatant was neutralized before assaying the antimicrobial activity. However, the importance of the inhibition effect varies according to serotypes (Savadogo et al 2004).

 

Escherchia  coli strains were the least sensitive to inhibitory substance produced by the lactic acid bacteria as compared to the other indicator strains. The resistance of Gram negative bacteria is attributed to the particular nature of their cellular envelop, the mechanisms of action described for bacteriocin bringing in phenomenon of adsorption. According to Bhunia et al (1991) the pediocin (bacteriocin produced by Pediococcus acidilactic) interact with lipoteichoic acids that are absent in Gram-negative bacteria. Also all the Lactococcus lactic ssp lactis isolates (Fn-555, Ad-411 and Ad-211) were not shown inhibitory effect on Escherchia coli ATCC-25922. This could be due to the fact that these isolates are nisin producers (Soomro et al 2002) and the primary target of nisin’s antimicrobial action is the cell membrane. Nisin has an inhibitory effect against a wide variety of Gram-positive food-born pathogens and spoilage microorganisms (Rodriguez 1996). In this study wider inhibition zones were observed by the species of Lactococcus (Mj. 622, Ad.855 and Ad.211) on the test strains. Similarly (Kelly et al 1996) reported that several lactococci produced a nisin-like activity, and showed a broad inhibitory spectrum against the indicator strains tested.

 

Conclusions  

                 

Acknowledgement  

The authors want to thank Hawassa University-NORAD Food Safety Project for providing all research facilities and resources.

 

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Received 22 October 2007; Accepted 26 December 2007; Published 1 March 2008

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