Isolation of bacteriocin - producing lactic acid bacteria from 'Ugba' and 'Okpiye', two locally fermented nigerian food condiments

FOOD/FEED SCIENCE AND TECHNOLOGY

Isolation of bacteriocin - producing lactic acid bacteria from 'Ugba' and 'Okpiye', two locally fermented nigerian food condiments

Charles Ogugua Nwuche^*

Department of Microbiology; University of Nigeria; Nsukka-Enugu State; Nigeria

ABSTRACT

In this work, 100 samples each of 'ugba' and 'okpiye' were evaluated for the presence of bacteriocin producing lactic acid bacteria. Thirty strains showing antibacterial activity against at least one of the indicator organisms were selected from a total of 752 colonies isolated from the condiments. Out of the 30, only five strains retained activity after the pH of the broth supernatant was adjusted to 6.5. When evaluated by the agar-well diffusion assay, the spectra of inhibitory activity showed that Staphylococcus aureus was the most sensitive indicator organism tested, while Listeria monocytogenes was the most resistant. One strain (UG 2) was active against Escherichia coli. The assays using the cell-free supernatant of the cultures showed that the bacteriocins were completely inactivated by the proteolyses as well as by the chloroform treatment. In ethanol, the activity of the compounds was only partially modified. When incubated in a water bath at 80°C for 30 min, no significant activity loss was recorded. The antimicrobial activity of the bacteriocins produced by the lactic acid bacteria has potential for use in biopreservation of condiments against the spoilage and food - borne pathogens.

Key words: 'Ugba', 'Okpiye', Lactic acid bacteria, Bacteriocins, Inhibitory activity, Condiments, Cell free supernatant

INTRODUCTION

Ugba' and 'Okpiye' (okpei) are two popular traditionally fermented food condiments in Nigeria. They are protein based and are used to complement the nutrient content of soups and sauces. They are sometimes substituted for the meat or fish. 'Ugba' is obtained from the seeds of the African oil bean (Pentaclethra macrophylla) while 'Okpiye' is prepared from the mesquite seeds (Prosopis africana). Both are products of natural fermentation. As a result, diverse groups of microorganisms including Bacillus, Micrococcus, Leuconostoc, Staphylococcus, Enterobacteriaceae and lactic acid bacteria (LAB) have been reported to play active roles in the process (Obeta 1983; Achi 1992; Njoku and Okemmadu 1998; Enujiugha 2009). They produce antimicrobial substances such as organic acids, diacetyl, hydrogen peroxide and bacteriocins (Ogunbanwo et al. 2003) which are suspected to be associated with the preservation of many fermented food condiments in Nigeria. The preservation and safety are presently the two major challenges of the food industry because, huge economic losses are sustained yearly due to food spoilage while numerous consumers have been reported to develop adverse sensitivity reactions to chemical based preservatives. The LAB which are used widely as starter cultures for food fermentation are thought of as having the potential to bridge this gap (Budde et al. 2003; Delves-Broughton 2005). They produce antimicrobial compounds that show antagonism toward the closely related species. The LAB in 'ugba' and 'okpiye' enhance fermentation and also improve the product storage, quality and safety by preventing the spoilage microflora from deteriorating the food condiments and at the same time keeping off the pathogens such as L. monocytogenes, S. aureus and E.coli (Ogunshe and Olasugba 2008).

Bacteriocins are extracellularly produced primary compounds of bacterial ribosomal synthesis which have a relatively narrow spectrum of bactericidal activity (Caplice and Fitzgerald 1999). They are active against other bacteria despite varying greatly in the chemical nature and mode of action. Bacteriocins have important advantage over the classical antibiotics in being easily degraded by the digestive enzymes without the risk of disruption of normal tract ecology (Caplice and Fitzgerald 1999). Bacteriocin producing LAB have the 'generally recognized as safe' (GRAS) status and have been shown to strengthen the barrier function of the gut microflora as well as promote the non-specific enhancement of the immune system of man and animals (Tome et al. 2008).

While many scientific reports are available on 'ugba' and 'okpiye', such studies have hitherto addressed mainly the microorganisms associated with the fermentation processes of the seeds as well as systems for optimization of the fermentation conditions (Obeta 1983; Achi 1992; Odibo et al. 1992). Equally, research on the biochemical changes during the fermentation as well as the proximate composition and properties of the seeds have also received modest scientific attention (Sanni and Onilude 1997; Achi and Okereka 1999; Aremu et al. 2007 and Odibo et al. 2008). There is presently, paucity of scientific information on the ecological contribution of the LAB and bacteriocins for the safety and biopreservation of the food condiments. The objective of this study therefore was to isolate and identify bacteriocin producing LAB from 'ugba' and 'okpiye' as well as evaluate the stability and activity of the bacteriocins against the common food pathogens.

MATERIALS AND METHODS

Sampling

One hundred samples each of traditionally fermented 'ugba' and 'okpiye' were purchased from different selling points at Ogige main market, Nsukka - Nigeria. All the samples were stored at 3±1°C for a maximum of 24 h before the analysis.

Isolation of Lactic acid Producing Bacteria (LAB)

Ten gram of each sample was homogenized separately in a porcelain mortar and then were transferred into 250 ml Erlenmeyer flask containing 90 ml of buffered peptone water (BPW, Oxoid) as diluent. The samples were mixed thoroughly by agitation for 2 - 3min before serial 10-fold dilution was made in 0.1 % peptone water. For the isolation of the LAB, 0.1 ml supernatant of appropriate dilution was inoculated into de Mann, Rogosa and Sharpe (MRS) agar plates containing 0.6 % CaCO₃ (May & Baker, Ltd., England) and distributed properly with a sterile glass spreader. The plates were incubated anaerobically at 30 °C for 24 - 48 h. Anaerobiosis was confirmed with a BBL anaerobic indicator. Halos developing around the colonies were regarded as an indication of colony's ability to solubilize CaCO₃ by acid production (Vanos and Cox 1986). The cultures were purified successively on the MRS agar plates and then investigated to determine their Gram staining and catalase status according to the assay methods of Harrigan and McCance (1976). Gram positive and catalase negative isolates were selected and stored on the MRS agar slants at 4 °C for use in further study.

Test Microorganisms

The food-borne pathogens chosen as indicator strains for testing the antibacterial activity of the LAB bacteriocins were Staphylococcus aureus (ATCC 12600), Listeria monocytogenes (ATCC 15313) and Escherichia coli (ATCC 11775). They were obtained from the culture collection of the Microbiology Department of the University of Nigeria, Nsukka. The organisms were subcultured in Tryptone Yeast Extract Glucose broth (TYG, Oxoid Ltd, UK) for 24 h before use.

Bacteriocin Production by the Isolated LAB

Bacteriocin production by the LAB was performed by inoculating the LAB isolates into 6.0 ml MRS medium and incubating at 30 °C. Cell free supernatants (CFS) were collected by centrifugation (5000 g, 15 min at 4 °C) of the overnight broth cultures. The pH of the CFS was adjusted to 6.5 with 4 N NaOH to eliminate the effect of organic acids. Possible inhibition by the hydrogen peroxide was also removed by the addition of catalase (Sigma Aldrich, Germany) at a final concentration of 1.0 mg/ml at 30 °C for 1 h.

Sensitivity of the Bacteriocin-like Compounds to Various Treatments

The CFS were divided into 2.0 ml portions before subjecting them to treatments in organic solvents (chloroform and ethanol), proteolytic action (pepsin and trypsin, Sigma Aldrich, Germany) and heat in order to evaluate the effect of the different factors on the activity and stability of bacteriocin. The solvents and enzymes were filter sterilized through Millex GV 0.22 µm filters (Millipore SA., France) before adding to the CFS. The controls consisted of distilled water and untreated CFS in 0.1 M phosphate buffer. The samples and controls were incubated at 30 °C for 2 h before the remaining bacteriocin activity was determined by the agar-well diffusion (AWD) assay against the indicator strains.

Detection of Antimicrobial Activity of the Bacteriocin

A modification of the agar-well diffusion (AWD) method was employed in this assay. Firstly, loopful of the indicator strains - S. aureus, L. monocytogenes and E. coli were inoculated separately into 6.0 ml TYG broth and incubated at 37 °C for 24 h. Then, TYG agar (100 ml) were prepared in six 250 ml Erlenmeyer flasks and just before the setting, 1.0 ml each of the indicator strains at a concentration of 10⁸ CFU/ml were separately added into each medium, swirled gently to mix and then dispensed into sterile petri-dishes. The plates were left to solidify under a laminar airflow. Thereafter, the wells (8 mm diameter) were made in each of the plates using a sterile cork borer. A 0.1 ml of the CFS was introduced into the different wells and left for 1 h in a refrigerator to allow the diffusion of the metabolite into the agar before incubating at 30 °C. The plates were examined for the development of translucent halos in the bacterial lawn surrounding the wells after 24 h incubation. A direct comparison was made between the diameters of the halos (zones of clearing) produced by the different strains. The percentage bacteriocin activity was calculated from the diameter of zones of clearing measured after each treatment relative to the halos produced from the positive control (untreated CFS) against each target organism.

RESULTS

Strains and Cultures

A total of 752 colonies was isolated from both the condiments - 472 from 'ugba' and 280 from 'okpiye'. The colonies were successively screened for the antibacterial activity against the indicator strains. Thirty colonies which showed reasonable inhibitory action against at least one of the indicator organisms were selected. The CFS of only five colonies composed of Lactobacillus spp. (Table 1) retained their activity after the pH was adjusted to 6.5. All the five isolates showed good activity against S. aureus and L. monocytogenes (Tables 3 and 4). However, only one isolate (UG 2) was active against E. coli (Table 2).

Effect of Different Treatments on Antimicrobial Activity of CFS

The sensitivity of the bacteriocin-like compounds to various treatments is shown in Tables 2 - 4. The activity of the CFS was completely lost by the treatment with chloroform, pepsin or trypsin when they were challenged with the Gram positive indicators. A considerable loss of activity was recorded after the treatment with ethanol. Strain OK 3 had the lowest level of activity loss (71 %) while UG 1 had the highest (89 %) when the CFS was challenged with S. aureus. On the other hand, the CFS from strain UG2 was least affected by the ethanol treatment (70 %), while OK 1 and UG 1 were the most compromised when challenged with L. monocytogenes. The antibacterial activity of the CFS remained stable even after exposure to a temperature as high as 80 °C. Results showed that the activity loss was negligible. When the indicator strains were challenged with the heat treated CFS, the highest recorded activity loss as determined in the assays involving S. aureus varied between 7 - 15% while a corresponding range of 10 - 17% was recorded when L. monocytogenes was used as the test organism. In the tests conducted against E. coli, the percentage loss of activity of the CFS was 17% from strain UG 2.

DISCUSSION

Out of the 752 colonies that were isolated, only five that were confirmed as bacteriocin producing were selected and used in the study. Several factors might have influenced such low number of isolation. According to Sezer and Guven (2009), the culture medium, incubation conditions, target microorganisms as well as the sensitivity of methods used in determining the antimicrobial activity are important factors. The antibacterial activity of the centrifuged culture supernatants are thought to be due to the presence of bacteriocins since the potency of the culture metabolite was neither lost by the addition of catalase nor diminished by the pH adjustment. Thus, the recorded inhibition levels were not due to interference by hydrogen peroxide nor lactic acid produced by the culture in the supernatant fluid. The bacteriocins produced were completely inactivated by the pepsin and trypsin as well as by the chloroform treatment. Such susceptibility to proteolytic degradation is a confirmatory characteristic of proteinaceous compounds. According to Tatsajieu et al. (2009), the LAB are well-known for their production of protein with antimicrobial properties, known as bacteriocins. Fricourt et al. (1994) and Piard and Desmazeand, (1992) reported that many of the bactericidal agents synthesized by the LAB were bacteriocins with a proteinaceous active moiety while others were non - protein agents. The loss of activity of the bacteriocins due to the treatment in organic solvents suggested that the antimicrobial agent might have a lipid moiety in their chemical composition. Studies by Saad and Nadra (1993), and lately by Bromberg et al. (2004) corroborated this view.

The bacteriocins produced in the CFS were remarkable by its quality of heat stability. After 30 min at 80 °C, about 83 - 93% of their antimicrobial potency was retained. The bacteriocin produced by the UG 2 was considered the most heat stable as only 7% loss of activity was recorded. Several reports on the ability of bacteriocins to withstand the high temperature conditions have been published (Nettles and Barefoot, 1993; Deraz et al. 2005 and Campos et al. 2006). At temperatures as high as 121 °C, bacteriocins have been shown to retain activity and stability (Ogunbanwo et al. 2003). This property of high temperature tolerance may be important in the food industry where some food preparation procedures involve a heating step (Ogunbanwo et al. 2003). The bacteriocin produced by the UG 2 had some interesting characteristic that could justify its further study. The most striking being its activity against a Gram negative bacterium. Bacteriocins are known to harbor the narrow antibacterial spectra of activity (Caplice and Fitzgerald 1999) although some have been reported to show broad activity spectrum.

Studies on the activity of bacteriocins against the target strains usually take place in laboratory media not in the foods. As a result, several intrinsic factors such as water activity could bring about reduction in the activity of bacteriocins. Further insight into this using a system that effectively replicates a food microenvironment (probably solid-state culture) could be imperative in order to study why high in-vitro bacteriocin activity did not always corresponded to high activity in in-vivo environment. That way the use of bacteriocin producing microorganisms to preserve the food products would become easily optimized and readily adaptable.

Received: June 28, 2011

Revised: March 06, 2012

Accepted: September 25, 2012

• Achi OK. Microorganisms associated with natural fermentation of Prosopis africana seeds for the production of okpiye. Plant Foods for Hum Nutr, 1992; 42: 297-304.
• Achi OK, Okereka EG.Proximate composition and functional properties of Prosopis africana seed flour. J Manage Technol, 1999; 1: 7-13.
• Aremu MO, Olonisakin A, Atolaye BO, Ogbu CF. Some nutritional composition and functional properties of Prosopis africana. Bangledesh J Sci Indust Res. 2007; 42: 269-280.
• Bromberg R, Moreno I, Zaganini CL, Delboni, RR, Oliveria J. Isolation of bacteriocin - producing lactic acid bacteria from meat and meat products and its spectrum of inhibitory activity. Braz J Microbiol, 2004; 35: 137-144.
• Budde BB, Hornbaek T, Jacobsen T, Barkholt V, Koch AG. Leuconostoc carnosum 4010 has the potential for use as a protective culture for vaccum - packed meats: culture isolation, bacteriocin identification and mean application experiments. Int J Food Microbiol. 2003; 83: 171-184.
• Campos CA, Rodriquez O, Calo - Mata P, Prado M, Barros - Velazquez J. Preliminary characterization of bacteriocins from Lactococcus lactis, Enterococcus faecium and Enterococcus mundtii strains isolated from turbot. Food Res Int. 2006; 39: 356-364.
• Caplice E, Fitzgerald GF. Food fermentations: role of microorganisms in food production and preservation. Int J Food Microbiol. 1999; 50: 131-149.
• Delves - Broughton J. Nisin as a food preservative. Food Australia. 2005; 57: 525-527.
• Deraz SF, Karlsson EN, Hedstrom M, Anderson MM. Purification and characterization of acidocin D20079, a bacteriocin produced by Lactobacillus acidophilus DSM 20079. J Biotechnol. 2005; 117: 343-354.
• Enujiugha VN. Major fermentative organisms in some Nigerian soup condiments. Pak J Nutr. 2009; 8: 279-283.
• Fricourt BV,Barefoot SF, Testin RF, Hayasaka SS. Detection and activity of plantaricin F, an antibacterial substance from Lactobacillus plantarum BF001 isolated from processed channel catfish. J Food Protec. 1994; 57: 698-702.
• Harrigan WF, McCance ME. Basic methods. In: Harrigan WF, McCance ME (eds.,), Laboratory methods in food and dairy microbiology. Academic Press, London, 1976. Pp.1-115.
• Nettles CG,Barefoot, SF.Biochemical and genetic characteristics of bacteriocins of food associated lactic acid bacteria. J Food Protect. 1993; 6: 338-356.
• Njoku HO, Okemadu CP. Biochemical changes during the natural fermentation of the African oil bean for the production of 'ugba.' J Sci Food Agric. 1998; 49: 457-465.
• Obeta JAN. A note on microorganisms associated with the fermentation of seeds of African oil bean seed (Pentaclethra macrophylla). J Appl Bacteriol. 1983; 54: 433-435.
• Odibo FJC, Ugwu DA, Ekeoha DC. Microorganisms associated with the fermentation of Prosopis seeds for 'ogiri-okpei' production. J Food Sci Technol. 1992; 29: 306-307.
• Odibo FJC, Ezeaku EO, Ogbo FC. Biochemical changes during the fermentation of Prosopis africana seeds for 'ogiri-okpei' production. J Indust Microbiol Biotechnol. 2008; 35: 947-952.
• Ogunbanwo ST, Sanni AI, Onilude AA. Characterization of bacteriocin produced by Lactobacillus plantarum FI and Lactobacillus brevis OFI. Afr J Biotechnol. 2003; 2: 219-227.
• Ogunshe AAO, Olasugba KO. Microbial loads and incidence of food - borne indicator bacteria in most popular indigenous fermented food condiments from middle - belt and south western Nigeria. Afr J Microbiol Res 2008; 2: 332-339.
• Piard JC, Desmazeaud M. Inhibiting factors produced by lactic acid bacteria. Part 2. bacteriocins and other antibacterial substances. Lait. 1992; 72: 113-142.
• Saad AMS, Nadra MCM. Characterization of bacteriocin produced by Pediococcus pentosaceus from wine. J Appl Bacteriol 1993; 74: 406-410.
• Sanni AI, Onilude AA. Proximate and functional properties of Prosopis africana seeds for ogiri - okpei production. J Indust Microbiol Biotechnol. 1997; 35: 947-952.
• Sezer C, Guven A. Investigation of bacteriocin production capability of lactic acid bacteria isolated from foods. Kafkas Vniv. Vet. Fak Derg, 2009; 15: 45-50.
• Tatsadjieu NL, Njintang VN, Sonfack TK, Daoudou B, Mvofung CMF. Characterization of lactic acid bacteria producing bacteriocins against chicken Salmonella enterica and Escherichia coli. Afr J Microbiol Res., 2009; 3: 220-227.
• Tome E, Pereira VL, Lopes CI, Gibbs PA, Teixera C. 'In vitro' tests of suitability of bacteriocin - producing lactic acid bacteria, as potential biopreservation cultures in vacuum - packaged cold - smoked Salmon. Food Control. 2008; 19: 535-543.
• Vanos, V, Cox L. Rapid routine method for the detection of lactic acid bacteria among competitive flora. Food Microbiol. 1986; 3: 223-234.

Publication Dates

• Publication in this collection
  09 Apr 2013
• Date of issue
  Feb 2013

History

• Received
  28 June 2011
• Accepted
  25 Sept 2012
• Reviewed
  06 Mar 2012