Isolation Of Lactic Acid Bacteria

Published Date: 02 Nov 2017

Lactic acid bacteria (LAB) were isolated from spontaneously fermented milk (Amasi) purchased from the Bulawayo market. The spontaneously fermented milk samples were aseptically collected and stored at 5 °C in a cooler box and then taken to the microbiology laboratory for analysis.

The samples were prepared for analysis by separately adding 10 g of each sample into 90 ml of 0.1% buffered peptone water (BIOLAB) and homogenized for 2 minutes to make a 10-1 dilution. Serial dilutions were then performed in 0.1% buffered peptone water (BIOLAB) up to 10-7 dilution by transferring 1 ml aliquots from each separate preceding dilution into 9 ml of 0.1% buffered peptone water (BIOLAB) to make a corresponding succeeding dilution. The dilutions (0.1 ml of each dilution) were then plated onto the de Man Rogosa Sharpe (MRS) agar medium (BIOLAB) through the spread plate technique and incubated at 37 °C (Thermo Scientific Incubator) for 48 hours in anaerobic jars. Pure cultures of the LAB isolates were obtained by repeated streaking of separate single colonies from the 10-6 to 10-7 dilutions onto fresh MRS agar (BIOLAB) at least three times. The purity of the isolates was checked by streaking and sub-culturing onto fresh MRS agar plates (BIOLAB), followed by microscopic examinations.

The pure cultures of the LAB isolates were subjected to short-term storage by separately keeping on triplicated MRS agar (BIOLAB) slants at 4 °C and subcultured every four weeks. When the isolates were needed for use, at least two transfers were made in fresh medium before use.

3.1.2 Identification of lactic acid bacteria isolates

3.1.2.1 Phenotypic identification

For initial presumptive characterisation of the lactic acid bacteria isolates, microscopic, physiological and biochemical techniques were used for characterisation based on some of the characteristics of lactic acid bacteria shown in table 3.1.

Table 3.1: Lactic acid bacteria differential characteristics

Genus

Cell morphology

Carbohydrate fermentation

Catalase test

Gram stain test

Lactobacillus

Rods, coccobacilli cells in singles or in chains

Homo- or heterofermentative (facultative heterofermentative)

Negative

Positive

Lactococcus

Spherical or lenticular cells in chains

Homofermentative

Negative

Positive

Leuconostoc

Spherical or lenticular cells in pairs or chains

Heterofermentative

Negative

Positive

Pediococcus

Spherical cells in pairs or tetrads

Homofermentative or facultative heterofermentative

Negative

Positive

Weissella

Spherical, lenticular irregular cells

Heterofermentative

Negative

Positive

Facultatively heterofermentative Pediococcus species: P. pentosaceus, P. acidilactici, P. claussenii (Kandler and Weiss, 1986; Garvie, 1986).

Methylene blue staining

To examine morphologically for cell shape and cell arrangements for the LAB isolates, the colonies for the purified LAB isolates were separately examined microscopically after performing simple staining with methylene blue. This was done by transferring the colonies onto separate microscopic slides, drying and fixing with a flame and then staining with methylene blue. After staining, the smears were washed with tap water and then blotted dry. The slides were then viewed under a light microscope.

Gram staining

For the Gram reaction test, smears of the purified LAB isolates were separately made on glass slides and allowed to air dry. Once dried, the smears were flooded with crystal violet (5-10 drops) and allowed to stand after which they were flushed with Lugol’s iodine for a few minutes. After flushing with Lugol’s iodine, the slides were washed briefly with water after which they were blotted dry. The smears were then decolourised with acetone/ alcohol for a few seconds, washed with water and blotted or air dried. Finally, counterstaining was performed briefly by adding 2 % solution of safranin for a few seconds. The smears were then washed with water briefly and blotted dry after which they were viewed for Gram staining under a light microscope.

Catalase test

In carrying out the catalase test, colonies from the purified LAB isolates were separately placed on glass slides using sterilised loops after which a few drops of 0.1-0.3% H2O2 (hydrogen peroxide) were added. A positive test was indicated by gas bubbles (effervescence) whilst absence of effervescence indicated a negative test. Lactic acid bacteria are the only microbes which can grow aerobically but lack the enzyme catalase (Holzapfel et al., 2001; Carr et al., 2002; Gadaga et al., 2007).

Fermentation test: hot-loop test… prep of MRS broth with glucose

The hot-loop test was carried out to determine if the purified LAB isolates were homofermentative or heterofermentative. This test is based on CO2 production, a by-product of sugar (e.g., glucose) fermentation which is a major end-product of heterofermentation. The purified isolates were separately grown in MRS broth medium (BIOLAB) containing glucose in which the CO2 gas is highly soluble at high pH and will stay in solution. After incubation at 37 °C (Thermo Scientific Incubator) for 48 hours, a wire loop (inoculating loop) was heated to redness and plunged into the broth culture causing the liquid around the loop to heat up. For heterofermentative organisms, CO2 bubbles evolved close to the loop. This is because if the temperature of the solution is increased, CO2 will become insoluble and will be released in the gaseous form.

Based on the phenotypic characteristics in reference to cell morphology and cell grouping, only the catalase-negative and Gram-positive isolates were selected. By also referring to the fermentation test properties, the genera of the purified LAB isolates were identified. Furthermore, for the identification of the LAB isolates to species level, API 50 CHL biochemical kits (carbohydrate fermentation strips at 30 °C) were used according to the manufacturer’s instructions for the API identification profile (API system, Bio-Mérieux, France). After the investigation of the LAB isolates for biopreservative activity as highlighted in the next sections, those that showed properties of interest were also characterised through molecular techniques as detailed in section 3.6 to give a more accurate identity.

3.2 Bacteriocins assay

3.2.1 Isolation of crude bacteriocins from the lactic acid bacteria isolates

The LAB isolates were separately propagated in liquid medium for maximum bacteriocin production i.e. 10 ml of MRS broth (BIOLAB). After incubation at 30 °C (Thermo Scientific Incubator) for 48 hours, the cells were removed from the growth medium by centrifugation at 5,000 × g for 10 min, 4 °C (centrifuge) to obtain the supernatants which were then separately filtered through 0.45 μm pore size filters to ensure they were sterile and cell free.

The cell-free supernatants were adjusted to pH 6.2 (Mettler-Toledo) using 1 mol/L NaOH (40g of NaOH in 1L distilled water) to exclude/eliminate the antimicrobial effect of the organic acids present in the supernatants. The antimicrobial activity of the crude bacteriocin extracts was then investigated as detailed in section 3.2.2.

3.2.2 Crude bacteriocins antimicrobial activity assay

The antimicrobial effect of the cell-free supernatants (crude bacteriocin extract) from the different purified LAB isolates and for the controls was separately investigated against gram-positive and gram-negative pathogenic or spoilage micro-organisms (indicator bacteria) through the agar-well diffusion assay. The test/indicator microbes that were used in the antimicrobial assay are; Escherichia coli, Staphylococcus aureus, Salmonellae typhimurium, Pseudomonas aeruginosa, Enterococcus faecalis, Bacillus cereus, Bacillus subtilis, Klebsiella pneumoniae, Clostridium perfringens and Listeria monocytogenes. These pathogens are vital in food spoilage and in food-borne illnesses.

The test bacteria were separately propagated in 10 ml nutrient broth at 37 °C (Thermo Scientific Incubator) for 24 hours in sets corresponding to each LAB isolate to be investigated. Petri dishes were then separately inoculated with 0.1 ml aliquots of the different 24 hour nutrient broth cultures after which 20 ml of the molten mueller-hinton agar (OXOID) was poured into each petri dish and thoroughly mixed with the aliquots. The plates were then allowed to solidify after which, four agar wells were made in each of the plates seeded with the indicator bacteria. The seeded plates were prepared in sets that corresponded to the purified LAB isolates whose bacteriocins were being investigated for antimicrobial activity. The cell-free supernatants (100 µl) were added into the agar wells after which the plates were then placed in the refrigerator at 4 °C for 20 minutes to enhance diffusion of the cell-free supernatant. The plates were then incubated at 37°C (Thermo Scientific Incubator) for 48 hours and then examined for zones of growth inhibition exerted on the test bacteria by the crude bacteriocins from the LAB isolates.

As a control, non-inoculated MRS broth (BIOLAB) was subjected to the same conditions and treatments as the MRS broth which was inoculated with the LAB isolates for bacteriocin production. It was incubated, centrifuged, filtered through a 0.45 μm pore size filter and corrected to pH 6.2 and used as a control for the bacteriocins assay for antimicrobial activity.

After 48 hours of incubation, the diameter of the zones of growth inhibition around the agar-wells was measured with vernier callipers in millimetres. A zone of growth inhibition is defined as the area with turbidity < 50% of the turbidity of the control culture grown without the antimicrobial under investigation (Jack et al., 1995; Mufandaedza et al., 2006). The antimicrobial activities of the cell-free fractions were classified as; – for 0 mm (no activity); + for inhibition zone < 6.5 mm; ++ for inhibition zone 6.5 – 9 mm; +++ for inhibition zone > 9 mm (Cleveland et al., 2001; Todorov et al., 2007). Each assay for antimicrobial activity was performed in duplicate.

The LAB isolates whose cell-free supernatants exhibited superior antimicrobial activity against the test bacteria were selected for partial purification of the bacteriocins for subsequent characterisation in terms of biopreservation and physico-chemical properties.

3.3 Biopreservative activity

3.3.1 Bacteriocins partial purification (bioassay guided fractionation)

The LAB isolates whose cell-free supernatants exhibited superior antimicrobial activity were separately propagated in 10 ml of MRS broth (BIOLAB) for bacteriocin production in preparation for the partial purification of the bacteriocins. After incubation at 30 ° C (Thermo Scientific Incubator) for 48 hours in MRS broth (BIOLAB), the cells were removed from the growth medium by centrifugation at 5,000 × g for 10 min, 4° C (centrifuge) to obtain the supernatants which were then separately filtered through 0.45 μm pore size filters to ensure they were cell free and sterile. The resultant supernatants were used as the crude bacteriocin extracts.

For the partial purification of the bacteriocins, a set of 9 tubes, each containing 2 ml of the cell-free supernatant (crude bacteriocin extract) corresponding to each LAB isolate were inoculated with equal volumes (2 ml) of saturated ammonium sulphate (NH4)2SO4) at concentrations of 40-80% at 5% intervals. Most bacteriocins are precipitable with (NH4)2SO4 of concentration of 40–80% saturation (Ennahar et al., 2000; Cleveland et al., 2001). After stirring on a magnetic stirrer, the different mixtures were kept undisturbed at 4 °C overnight. The precipitates that formed were collected through centrifugation at 10 000 ×g for 10 min (centrifuge) by separating and discarding the supernatant. The precipitates were then redissolved in one-tenth volume of 20 mmol/L sodium phosphate or 25 mmol/L ammonium acetate buffer at pH = 6.0 (Mettler-Toledo) after which they were desalted by dialysis against distilled water using 1 KDa cut-off dialysis membranes…. More info on dialysis

The different precipitated fractions of the bacteriocins (redissolved/reconstituted) that corresponded to the selected LAB isolates were then investigated for antimicrobial activity test indicator bacteria to allow for bioassay follow up on the bacteriocin containing fractions. This was done through the agar-well diffusion assay as highlighted in subsection 3.2.2. The zones of growth inhibition elicited by the different precipitated fractions were recorded in comparison with those of the crude bacteriocin for each LAB isolate investigated. The fractions with comparable or higher antimicrobial activity were considered to be containing the bacteriocins in a partially purified form and were subjected to further characterisation. Based on their antimicrobial activity, the selected fractions of bacteriocins also pointed out on the concentrations of saturated ammonium sulphate suitable for the subsequent precipitation of the respective bacteriocins.

3.3.2 Biopreservative activity of bacteriocins in selected foods

After isolation and partial purification, the bacteriocins (100 µl) were tested in a food system to investigate on their suitability to act as biopreservatives. Fresh milk was used as a testing food system since liquid foods were more feasible for this investigation as compared to the use of solid foods.

Fresh milk samples were separately were spiked with test bacteria and their shelf lives followed over a period of time to determine if the respective bacteriocins were suitable for use as biopreservatives. The fresh milk samples were first pasteurised to establish a common starting ground from a microbiological perspective, after which they were then separately inoculated with the test bacteria; Salmonella, E. coli and S. aureus in sets corresponding to the partially purified bacteriocins from the LAB isolates. The sets of the fresh milk samples were then inoculated with the corresponding partially purified bacteriocins and incubated at 37° C. Positive control samples were only incorporated with the test pathogens while the negative control samples were only incorporated with the bacteriocins. All the samples were incubated at 37° C and the microbial counts of the test bacteria followed at 24 hour intervals i.e. 0, 24, 48, 72 hrs etc until the bacterial counts recorded constant.

The shelf life of the test samples and the controls was followed at 24 hour intervals from the inoculation time by determining the counts of the test bacteria i.e. E. coli, S. aureus and Salmonella through the standard plate count (SPC) method utilizing the spread plate technique. For the isolation and enumeration of E. coli, 1 ml of the milk sample was introduced into 9 ml of saline broth and subsequent serial dilutions made in saline broth up to 10-6 after which the dilutions were plated on violet red bile agar (VRBA) and incubated at 37 °C for 24 hours. The colonies that grew on VRBA were counted and confirmed on eosin methylene blue (EMB) agar. For the isolation of S. aureus, a similar technique was employed but the serial dilutions were plated on Baird-Parker agar whilst the growing colonies were confirmed on mannitol salt agar (MSA). For Salmonella, the sample dilutions were prepared in buffered peptone water and incubated on Xylose lysine deoxycholate (XLD) agar at 37 °C for 18-24 hours after which colonies with black centres were enumerated. To determine the bacterial counts, only the plates with 30-300 colonies were considered and the bacterial counts were calculated as shown in Appendix II.

The colony counts for the respective test bacteria in the test samples and positive control samples were recorded in cfu/ml corresponding to the partially purified bacteriocins from the LAB isolates being investigated. The E. coli, S. aureus and Salmonella counts that were determined in the positive control samples and the test samples were statistically compared through the student’s t-test as independent samples to determine the effectiveness of the bacteriocins as biopreservatives (ability to exert antimicrobial activity in food systems). It is from these results that bacteriocin activity corresponding to a particular LAB isolate could be inferred to be a potential biopreservative or not. The LAB isolates with biopreservative activity of interest were subjected to more accurate identification to species level through genotypic identification techniques as described in section 3.6 and preserved for future use and for reference purposes as highlighted in section 3.5.

3.4 Characterisation of the bacteriocins

3.4.1 Molecular weight estimation of partially purified bacteriocins

The molecular weight (MW) of the partially purified bacteriocins was estimated using SDS-PAGE. One half of the SDS-PAGE gel was run containing the sample and a low molecular-weight marker with sizes ranging from 2.5 to 45 kDa and fixed for 30 min in 5% (v/v) formaldehyde, rinsed with distilled water and stained for 50 minutes with Coomassie. The other half of the gel was run containing the sample only and was fixed for 30 min in 20% (v/v) isopropanol and 10% (v/v) acetic acid, rinsed with sterile water at 4 °C and overlain with nutrient agar (seeded with susceptible indicator bacteria). It was then incubated at 37 °C for 24 hours. The position of the active bacteriocin was visualised by an inhibition zone around the active protein band which was then aligned to the stained band on the other half of the SDS-PAGE gel to estimate the molecular weight (MW) of the bacteriocin.

3.4.2 Physico-chemical properties of the bacteriocins

The physico-chemical properties of the bacteriocins were determined to give insight on the type of foods in which they can be used and on their stability to food processing conditions/ parameters. This was done to give information on the suitability of use of the bacteriocins in food production systems or if they could only be incorporated into foods after processing.

Heat stability

Corresponding to each LAB isolate under investigation, 8 test tubes were incorporated with volumes of 5 ml of partially purified bacteriocins and overlain with paraffin oil to prevent evaporation during heating. The tubes were separately exposed to different temperatures i.e. each tube to a unique specific temperature of either 10 °C, 25 °C, 30 °C, 40°C, 50°C, 80°C, 100 °C for 20-30 minutes or 121 °C for 15 minutes under pressure. Aliquot volumes of the heat-treated bacteriocin samples were then assayed for antimicrobial activity through the agar-well diffusion assay against indicator bacteria. This was done to determine if the bacteriocins retained their activity after heat treatment at a specific temperature and hence their heat stability.

Effect of pH

Corresponding to each LAB isolate under investigation, the sensitivity of the partially purified bacteriocins to pH was investigated. Volumes of 5 ml of the partially purified bacteriocin were taken in test tubes and their pH values separately adjusted to pH 2, 4, 6, 8, 10, 11 using diluted 1 M NaOH and 1 M HCl solution such that each tube was adjusted to a specific pH under study. After incubating the samples for 2-4 hours at room temperature, the bacteriocins were assayed for antimicrobial activity through the agar-well diffusion assay against indicator bacteria. This was done to determine if the bacteriocins retained their activity after specific pH treatment and hence their pH range of activity.

Effect of enzymes

To establish and ascertain the nature of the bacteriocins purified from each isolate, their sensitivity to different enzymes was investigated. Corresponding to each LAB isolate, 5 ml volumes of the partially purified bacteriocins were taken in test tubes and separately treated with amylase, lipase, papain, pepsin, catalase and trypsin (100 TU) 1 mg/mL at pH = 7. The samples were incubated separately with each enzyme for 24 hours at 37 °C to determine the effect of the enzyme on the bacteriocins after which the samples were heated for 3-5 minutes at 50-60 °C to denature the enzymes. After enzyme treatment, the bacteriocins were assayed for antimicrobial activity through the agar-well diffusion assay to determine if the bacteriocins retained their activity and hence their susceptibility to a particular enzymes which inferred their nature. Control samples separately containing the enzymes only and some control samples with the bacteriocins only, were exposed to the same conditions for the same duration as the test samples and assayed for antimicrobial activity.

3.5 Long term preservation of valuable lactic acid bacteria isolates

Purified LAB isolates of interest were preserved at −20 °C in MRS broth with 15% (v/v) glycerol added or in 30 % glycerol stocks. The desirable LAB isolates are those whose bacteriocins exhibited superior antimicrobial and biopreservative activity which was of interest for commercialisation or for further studies and research.

3.6 Genotypic identification

The LAB isolates which were perceived to be valuable were also identified to species level through genotypic identification to give a more accurate identity. The lactic acid bacteria isolates were identified through the amplification and sequencing of part of their genome using PCR primers that targeted the 16S/23S rRNA spacer region. The primers are illustrated below:

Table 3.2: Sequences of the primers used for PCR amplification and sequencing

Primer

Location

Oligonucleotide sequence (5’-3’)

Specificity

16

16S rRNA gene, 3’ end, forward

GCTGGATCACCTCCTTTC

16S rRNA gene

23

23S rRNA gene, 5’ end, reversed

AGTGCCAAGGCATCCACC

23S rRNA gene

(Berthier and Ehrlich, 1998)

3.6.1 Genomic DNA isolation

The LAB isolates were picked from each pure plate and separately transferred to 10 ml MRS broth and cultured overnight at 37 °C in preparation for genomic DNA isolation. DNA was extracted from each isolate and analysed separately.

Genomic DNA was separately extracted from MRS broth overnight cultures of the LAB isolates as follows; the cells were harvested by spin down of 5 ml overnight culture at 4 °C at a speed of 6000 × g centrifugation for 10 min in eppendorf tubes. The cell pellets were washed twice in sterile ultrapure water or in phosphate-buffered saline (PBS) pH 7.2 after which they were re-suspended in 0.5 ml (500 µl) ice cold lysis buffer pH 8.0 (Tris-HCl 25 mM, EDTA 10 mM, 50mM glucose; with 50 µl lysozyme (50 mg/mL) and then incubated on ice for 10 minutes. After incubation, 50 µl of 10% SDS buffer was added, and the mixture incubated at 37 °C for 5-10 minutes until clear and viscous.

After incubation, 550 µl of phenol (freshly equilibrated with equal volume of 0.3 M Na Acetate) was added and mixed gently by inversion. The mixture was then centrifuged at 4 °C for 15 minutes. This step can be repeated to improve purity of DNA. The final top layer was transferred to an eppendorf tube into which one-tenth of 3 M NaOAc was added. Chromosomal DNA was then precipitated by the addition of 2 volumes of 100% ethanol and mixed by inverting. The sample was cooled at -80 °C for 5 minutes and centrifuged at 4 °C for 15 minutes. The supernatant was then removed and discarded. The pellet was then dried and resuspended in 50-100 µl of distilled water or TE buffer (10 mM Tris–HCl, 1 mM EDTA pH 8.0).

The quantity and purity of the DNA preparations was assessed by 1.5% TBE agarose gel electrophoresis. The DNA quality was also determined through absorbance ratios (A260/A280) which should be in the range of 1.80 - 1.90 using spectrophotometer readings. The DNA preparations and their corresponding 1/20 and 1/50 dilutions were stored at -20 °C until use.

3.6.2 Genomic DNA amplification

One microliter of appropriate diluted extracted genomic DNA (corresponding to 5–10 ng DNA) was used as template for PCR reactions. The amplification of the 16S/23S rRNA spacer region was performed on the template DNA using primers in table 3.2. The amplified products length are between 300-500 bp.

The PCR-mediated amplifications were performed at least in duplicate for each sample and carried out by using an eppendorf in a total volume of 15 to 50 µl containing 5 to 10 ng of template DNA, 1X PCR buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L of each dNTP and appropriate concentration of each primer and Taq DNA polymerase. The following thermal cycles were used; 95 °C for 3 minutes initially; 40 cycles of: 95 °C for 30 s (denaturation), 55 °C for 55 s (annealing), and 72 °C for 60 s (extension); and one cycle final primer extension at 72 °C for 10 min.

The amplified DNA i.e. fifteen microliters of the each PCR product was run on gel together with an appropriate DNA ladder of MW markers. The PCR products were checked by electrophoresis on a 1% agarose gel (agarose gel electrophoresis) stained with 3.1 mg/mL ethidium bromide, visualized using UV-Transilluminator for specific bands and photographed. Electrophoresis was performed in pH 8.0, 1X Tris-acetate-EDTA/ 0.5X Tris-borate-EDTA buffer at constant voltage of 80-100 volts for 30 min. The DNA bands were identified according to their size by comparing against the molecular weight markers (0.2–2 kb DNA ladder) loaded in a separate a lane. After identification of the bands, the DNA was excised from the gel and purified using a gel extraction and PCR purification kit. The DNA was then eluted into 30 µl of sterile water for use in DNA sequencing.

The purified amplification products were sent to a commercial facility for sequencing. After sequencing, the 16S/23S rRNA spacer region were aligned with sequences in GenBank by using the National Centre for Biotechnology Information (NCBI) Basic Local Alignment Search Tools, nucleotide (BLASTn) program available online. The identity of the LAB species was then given from the hits obtained.

3.7 Aseptic techniques

Sampling and sample analysis

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