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Isolation of Bacteria from Sourdough and the Identification of Lactobacillus Bacteria using Biochemical Tests and Comparative Morphology

INTRODUCTION

Lactobacillus is a gram positive, catalase negative, oxidase negative, endospore forming, rod shaped bacterium (1, 2, 6, 17, 18). It can be found in environments such as; dairy products, the gastrointestinal tract, fermented foods, beer, wine, sourdough bread, plant surfaces and in fruit. A sample taken from sourdough bread was used in this experiment.

The metabolism of Lactobacillus and other microflora in sourdough bread is what makes it different to other breads (11, 16). Lactobacillus undergoes fermentative processes, however being a facultative anaerobe, it can grow in either anaerobic (without oxygen) or aerobic (with oxygen) conditions (6).

Lactobacillus is a homofermentative bacterium. Under anaerobic conditions, it is able to convert glucose entirely into lactate (lactic acid), as opposed to heterofermentative bacteria whose by-products of fermentation are lactate, ethanol and CO2 (11). It uses an endogenous electron acceptor, usually pyruvate. However, in a study by Murphy et al (13) in aerobic conditions, O2 was found to be present as the final exogenous electron acceptor; the study focused on L. plantarum. Many Lactobacillus species break lactate down to acetic acid and under specific conditions H2O2, through the reduction of O2, depending on the level of aeration (5, 13, 19).

Sourdough bread is unique in comparison to regular breads, due to the addition of sourdough starter. This starter typically contains cultures of, on average, 43 species of Lactic acid bacteria, the predominant genus being Lactobacillus and 23 species of yeast, mainly Saccharomyces and Candida (4). These cultures are associated with health benefits (probiotic), superior flavours, and long lasting properties of sourdough; a direct result of the cultures’ metabolism and by-products (11, 16).

Alternative microorganisms other than Lactobacillus and yeast can be found in sourdough bread. They include: Enterococcus, Leuconostoc, Lactococcus, Pediococcus, Weisella, and Streptococcus. Pediococcus (3) is a gram positive, non-motile, catalase and oxidase negative, homofermentative, spherical bacteria and grows in pairs or tetrads. (9, 17, 21) Lactococci are non-spore forming, non motile, gram positive, catalase and oxidase negative and spherical shaped bacteria. (17, 18) Weisella are gram positive, catalase negative, non motile short rods that grow in pairs (10) Leuconostoc are gram positive, catalase negative cocci and grow at 28°C in pairs or chains (14, 17). Streptococcus and Enterococcus are morphologically and metabolically very similar. They are a gram positive, catalase negative, coccus shaped, and lactic acid bacteria that grow in pairs or chains. They are non-motile and do not form spores (6, 17). It was also demonstrated by Hamad (18) that gram negative, catalase positive, rod shaped bacteria are also present but are contaminants from the air.

Throughout the fermentation process a small number of Leuconostoc species are the first to grow. Leuconostoc creates an acidic environment through the slow production of lactic or acetic acid depending on whether conditions are either aerobic or anaerobic. This initiates the growth of a large number of species of Lactobacillus, between the pH range of 4.5 - 6.5. In the final stages of fermentation, a small number of species of Pediococcus starts to grow as they can tolerate highly acidic environments (13, 14).

Corsetti et al demonstrated (1) that by-products produced by Lactobacillus, in particularly, L.sanfrancisco, inhibited the growth of fungi such as Aspergillus niger, Cladosporium herbarum and Penicillium verrucosum in bread. As a result, sourdough breads have a longer shelf life.

Species other than Lactobacillus and yeast can be found in sourdough bread. The majority of these include: Pediococcus, a gram positive, non-motile, catalase and oxidase negative, spherical bacteria that grow in pairs or tetrads (21); Lactococcus, which are non-spore forming, non motile, gram positive, catalase and oxidase negative spherical bacteria (18); and Weisella, which are, gram positive, catalase negative, non motile short rods that grow in pairs (10).

The conditions it is grown in need to be adjusted to promote Lactobacillus growth and inhibit the growth of other micro-organisms. Tomas et al (15) reported that Lactobacillus grows best in temperatures of 30-37°C (15) and in the presence of an inorganic carbon source in the form of CO2 (8). Acidity is also another important consideration, which, for optimal growth, should be maintained in a pH range of 4.5-6.5 (1, 2, 7, 8, 15, 19, 21).

This study was performed with the aim of isolating Lactobacillus from sourdough bread, as a pure culture. Sourdough is a good sample to use as it contains a large number of Lactobacillus species so there is a higher likelihood of isolating it as a pure culture. Also Lactobacillus metabolism alone will assist in inhibiting other bacteria from growing and in this way make it easier to isolateas a pure culture. This was to be achieved by growing micro-organisms in aerobic conditions on nutrient agar and MRS plates. Lactobacillus was then to be isolated by conducting biochemical and morphological tests.

MATERIALS AND METHODS

Preparing the sample in water

A small piece of sourdough bread (not from the crust) about the size of a twenty cent coin was torn into small pieces and placed into a sample tube. Water was added to the tube to about 5mm above the bread. The sample was left to stand for about twenty-four hours at room temperature to allow any microorganisms present to enter the water. Further water was added to ensure the bread did not absorb all the water.

Preparation of sample in nutrient broth

Two tubes containing nutrient broth were prepared according to Microbiological Methods (20). A small amount of bread (not from the crust) about the size of a five cent piece was torn into small pieces. Using sterile techniques, the same amount of bread pieces were placed in each tube. This promotes the growth of large amounts of different bacteria due to the nutritional content. The lids were loosely placed on the tubes and the samples were incubated at room temperature for two days.

Initial sub-culture of sample, growth conditions and media

From the original bread and water suspension, four nutrient agar plates were subcultured. From the sample tube containing the bread in water, sterile methods were used to streak out onto four nutrient agar plates, prepared as stated in Microbiological Methods (20).
Using aseptic technique, each plate was inoculated with a bacteriological loop full (approximately 10mL) of original suspension, by smearing a small section of the plate. To establish pure culture, single colonies were picked up with the bacteriological loop, and sub cultured onto nutrient agar (NA) and MRS (de Man, Rogosa, Sharpe) plates. Two nutrient agar plates and twoMRS plates were used. These were labelled 1-4.

Subculture of nutrient broth

The nutrient broths were streaked as follows. The jars were shaken and then streaked onto two nutrient agar plates. The plates were incubated at room temperature for two days. Only one plate returned any useful colonies to test. So half of each colony was streaked onto a nutrient agar plate and the other half streaked onto a MRS plate. Four nutrient agar plates and four MRS plates were used. Microorganisms only grew on the nutrient agar plates and these were labelled A-D.

Isolation of pure colonies and inoculation into broth

For each pure colony found on the plates (A-D), a colony was inoculated into a nutrient broth. Next, without burning the loop, a nutrient or MRS agar plate was streaked. In this study, four nutrient agar plates, four MRS agar plates and 4 nutrient broths were used. These plates were then incubated at room temperature for two days.

Gram stain

A gram stain (20) was conducted on each pure culture dish that had white bacteria growing. Yellow cultures were disregarded as they were identified to be yeast (11). Since yeast is a fungus, the biochemical tests performed would not be appropriate. There were a total of four pure cultures.

This stain is used to determine the make up of the cell membrane of bacteria. Gram positive bacteria stain a deep purple indicating they have a thick outer layer of peptidoglycan. Gram negative bacteria stain pink/red indicating they have a thin inner layer of peptidoglycan. This stain is also useful in determining morphology.

Catalase test

From each pure culture of white bacteria, a catalase test was conducted (20). This biochemical test detects the presence of the enzyme catalase, which is present in aerotolerant aerobic bacteria. An end product of aerobic respiration is the toxic compound hydrogen peroxide. The enzyme catalase reduces hydrogen peroxide to water and oxygen, and may be represented by the following balanced equation:

equation

Catalase positive bacteria will produce bubbles of O2 when H2O2 is applied. Lactic acid bacteria do not contain catalase, they contain peroxidase. Catalase negative bacteria have a delayed reaction or no reaction as peroxidase reduces H2O2 to H2O.

Oxidase test

Using one colony from each plate that contains a pure culture of white bacteria, an oxidase test was conducted (20). This biochemical test identifies if cytochrome c, a haem-containing protein required for respiratory metabolism, is present in a bacteria in its oxidised form (20). The iron (Fe3+) present in the haem-group causes the oxidation of the cytochrome c. Due to the presence of Fe, a platinum loop was used, otherwise the Fe in the loop reacts with the tetramethyl–p-phenylene diamine (TMPD) giving a false result. When the bacteria is applied to the TMPD, if it is oxidase positive a blue colour will be produced. If it is oxidase negative, no colour change will occur or there is a delayed colour change of over one minute. A green/brown colour will result if an iron loop is used and not a platinum loop.

Subculture of the control and biochemical tests

To obtain a control bacterial sample that is known to be Lactobacillus, a pure colony was streaked onto one nutrient agar plate and one MRS agar plate and incubated at room temperature for two days. The pure colony was used to conduct a gram stain, catalase test and oxidase test, Hugh- Leifson and spore stain.

Hugh- Leifson (oxidation-fermentation) test

The Hugh-Leifson test indicates the metabolic process undertaken by bacteria; oxidation, fermentation or peptone utilising. If the original green media turns yellow all the way through, fermentation is occurring as it can occur in aerobic (top of the tube) and anaerobic (bottom of the tube) conditions. If the top quarter of the tube turns yellow, oxidation is occurring as O2 is available at the top, but it is anaerobic at the bottom. If the media turns blue, this means neither oxidation, or fermentation of carbohydrates is taking place; however, the bacteria are able to ferment the peptones in the media. A positive result for Lactobacillus would be if the media turns yellow all the way through the tube as it can ferment carbohydrates in aerobic and anaerobic conditions.

Spore stain (Schoeffer and Fulton)

Using a pure colony of the Lactobacillus control, a spore stain was conducted according to Microbiological Methods (20). The spore stain is used to determine whether the bacteria produce spores or not. When stained with malachite green, then counter stained with safranin, spores will turn a bright green, whereas vegetative cells will stain a red/brown. Since Lactobacillus is a non-spore forming bacteria then a positive result for Lactobacillus would be red/ brown stained cells.

RESULTS

Description of bacterial grown on plates

There were two distinct colonial morphologies: yellow and white. On the original plates, the white microbes were surrounded by the yellow microbes. The white bacteria formed small, smooth looking colonies, whereas the yellow microbes formed larger grainy looking colonies. The white colonies were biochemically tested, but not the yellow ones because yellow microbes indicate yeast and the odour being released was sour, which is characteristic of yeast.

Results from biochemical tests

The biochemical tests that were performed on possible Lactobacillus candidates returned the overall result of negative for Lactobacillus. Refer to Table 1 for a summary of these results. The pure culture plate A was determined to be gram positive, catalase negative and oxidase negative chains of spheres. The pure culture plate B was identified as gram positive, catalase negative and oxidase negative spheres that appeared to grow in groups; possibly pairs or tetrads. Plate C bacteria were gram negative, catalase positive and oxidase negative rods. Finally, the pure bacteria in plate D were gram positive, catalase negative and oxidase negative spheres that formed groups of pairs and tetrads as well as single cells.

Table 1: Results of biochemical tests on cultures A, B, C, D

Pure culture plate Gram stain Morphology Catalase test Oxidase test
A + Chains of spheres - -
B + Spheres in groups (pairs or tetrads+) - -
C - Rods + -
D + Spheres in groups (pairs or tetrads +) - -

Results of the control

Results of the biochemical tests on the control that was known to be Lactobacillus confirmed this fact. The tests and stains showed it to be gram positive, catalase negative, and oxidase negative, fermentative and non-spore forming rods. Refer to Table 2 for a summary of the test results for the Lactobacillus control.

Table 2: Results of Biochemical tests for Lactobacillus Control

Biochemical Test/ stains Result
Gram Stain +
Morphology Rod
Catalase test -
Oxidase test -
Hugh- Leifson Test Yellow colour change though entire tube.
Spore stain -

It is possible the yellow bacteria were yeast (11) as the grainy appearance and the odour being released from the plates in the pure colonies are characteristic of yeast. These conflicting data are explored in the discussion. A hypothesis as to why the yeast was growing around the white bacteria can be formulated if the white bacteria are assumed to be lactic acid bacteria. These data suggest that the yeast grew after the lactic acid bacteria (LAB) because the LAB makes the environment more acidic through the production of lactic acid. This enabled the yeast to grow as it grows best in acidic conditions.

Possible identities of the pure cultures

None of the pure cultures were Lactobacillus (Table 3). However, identification of these pure cultures can be hypothesised. The bacteria in plate A could be Leuconostoc, Streptococcus or Enterococcus. The bacteria in plate C could be contaminants from the air. Also, the bacteria in plate D could be Weisella or Pediococcus. Further tests are needed to determine the genera of the cultured bacteria.

Table 3: Summary of Conclusions

Pure culture plate Possible conclusions
A Leuconostoc, Streptococcus, Enterococcus.
B Leuconostoc, Streptococcus, Enterococcus.
C Contaminants from the air.
D Weisella, Pediococcus

DISCUSSION

The results can only lead to very limited conclusions. This is as a result of the few biochemical tests that were performed and the limited number of pure cultures that they were performed on. This research was unable to identify the genera of bacteria that actually were isolated due to the limited information from the biochemical tests employed.

Lactobacillus was not isolated as a pure culture from sourdough for two possible reasons. The first being that the preparation of the sourdough bread before baking was not done properly, or that Lactobacillus cultures were not used in the sourdough starter. The second is that the method used was not very well planned and that the growth conditions were not right.

One way in which to remedy the first case, which was of the sourdough not being prepared properly, would be to obtain a wider range of sourdough bread samples for testing. These could be from sourdough loaves prepared by different bakeries (about 5) and one prepared by the researcher which would include a Lactobacillus starter.

There are many ways in which to remedy the second case, which was that the method was not appropriate. One way is to make the conditions more favourable to grow Lactobacillus. This would require raising the incubation temperature to 30°C, growing in greater CO2 levels, and growing in anaerobic as well as aerobic conditions (3, 4). Another way would have been to streak out more plates to test as many pure colonies as possible. Another alternative would be to use samples other than sourdough, for example Yakult, leaves and fruit. Also a greater variety of biochemical tests should be performed in order to determine exactly which genera of bacteria each of the pure cultures were and hence more research conducted into the bacteria in the sourdough.

To increase the possibility of actually identifying each pure culture more biochemical tests should have been performed. Some of these tests could include a differential test to determine if the culture is homofermentative or heterofermentative. For example the Gibson and Abd-el-Malek which tests for CO2 production (21). The methyl red test could also be used to estimate the pH of the environment. Another test includes the motility test to determine whether the microbes are motile or not. The Litmus milk test could also be used to determine changes in pH as a result of Lactose fermentation.

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