Two techniques for investigation of proteins and short-chain fatty acids of Lactobacillus plantarum in presence of galactooligosaccharides
(2014) MOBN18 20132Degree Projects in Molecular Biology
- Abstract
- Popular science summary:
This thesis is a starting point of a bigger project, aimed to develop synbiotic food supplements. The role of probiotics and prebiotics in human health is getting increasingly recognised as offering beneficial impact to the host. WHO defines probiotics as “live microorganisms which, when administered in adequate amounts, confer a health benefit to the host.” Prebiotics are defined as “selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastro-intestinal microbiota thus conferring benefit(s) upon host’s health.” Synbiotics are compositions of probiotics and prebiotics, in which the prebiotic selectively supports the growth of the probiotic.
Delivering... (More) - Popular science summary:
This thesis is a starting point of a bigger project, aimed to develop synbiotic food supplements. The role of probiotics and prebiotics in human health is getting increasingly recognised as offering beneficial impact to the host. WHO defines probiotics as “live microorganisms which, when administered in adequate amounts, confer a health benefit to the host.” Prebiotics are defined as “selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastro-intestinal microbiota thus conferring benefit(s) upon host’s health.” Synbiotics are compositions of probiotics and prebiotics, in which the prebiotic selectively supports the growth of the probiotic.
Delivering probiotics to the site of their action, i.e. the colon, poses a great challenge. They have to pass the natural barriers in the human body – mainly the acidic conditions in the stomach and digestive processes in the small intestine. Since prebiotics are per definition non-digestible by humans, they can assist in the probiotic’s survival and promote its growth. Prebiotics offer another benefit to human health. When a prebiotic is fermented by a bacterium, short-chained fatty acids (SCFAs) are produced and released in the environment. These molecules (mainly acetic, propionic and butyric acid) are taken up by the cells in the colon and utilised in important cell processes.
In this study, a selected probiotic strain, Lactobacillus plantarum F44, was grown with a prebiotic galactooligosaccharide (GOS). To determine how efficient the bacteria are at fermentation of the GOS, the concentration of SCFAs produced should be measured. With this information, the appropriate synbiotic preparations can be designed. The analyses of SCFAs were done by high performance liquid chromatography (HPLC). In order to remove substances which can interfere with the chromatography from the bacterial supernatant, sample preparation and SCFA isolation are performed before the HPLC analysis. Filtration of the sample and liquid/liquid extraction were tested. However, these were not sufficient methods as certain impurities remained in the sample. Time was limited, so other methods were not performed in practice. Suggestions for the future experiments in this project include solid-phase extraction of the SCFAs from the medium. Utilising the volatile nature of the SCFAs by using liquid-gas-liquid extraction with hollow fibres to purify and concentrate the SCFAs from aqueous samples is another possible solution.
It is possible that growth in the presence of a prebiotic causes changes in gene expression in bacteria. Proteins were isolated from two parallel L. plantarum F44 cultures that were grown with and without GOS in the media. Protein patterns were investigated and compared. No major difference was observed between these two cultures. It would be interesting to compare protein patterns when the bacteria grow with other available prebiotics as well. This can later be combined with methods to identify bacterial proteins, especially those that play a role in the bacterial interaction with the human host. That way it may be possible to determine how the prebiotic affects and improves bacterial ability to colonise human gastrointestinal tract. The adaptation of these techniques for investigations of bacterial behaviour in the presence or absence of prebiotics is important to facilitate preparation and optimisation of synbiotic food supplements.
Supervisor: Bengt Danielsson, Acromed Invest AB
Master Degree Project 45 credits in Molecular Biology: Microbiology, 2014
Department of Biology, Lund University (Less) - Abstract
- Abstract
The long-term aim of this project was to develop synbiotic drinks presenting food supplements with beneficial effects on human health. A synbiotic consists of a combination of probiotic bacteria and one or more prebiotics. For the part of the project that was covered by this Master thesis, a probiotic strain, Lactobacillus plantarum F44 and a prebiotic galactooligosaccharide (GOS) were tested in vitro. The probiotic L. plantarum F44 was selected due to previous systematic studies of the strain characteristics. The selected probiotic strain was cultured with GOS, but also grown without GOS (control), to compare growth efficiency, gene expression, and fermentation products. By monitoring the cell density, it was shown that GOS in... (More) - Abstract
The long-term aim of this project was to develop synbiotic drinks presenting food supplements with beneficial effects on human health. A synbiotic consists of a combination of probiotic bacteria and one or more prebiotics. For the part of the project that was covered by this Master thesis, a probiotic strain, Lactobacillus plantarum F44 and a prebiotic galactooligosaccharide (GOS) were tested in vitro. The probiotic L. plantarum F44 was selected due to previous systematic studies of the strain characteristics. The selected probiotic strain was cultured with GOS, but also grown without GOS (control), to compare growth efficiency, gene expression, and fermentation products. By monitoring the cell density, it was shown that GOS in a moderate concentration (2 mg/mL) increased the bacterial growth rate. Bacterial proteins play a major role in interaction with the cells in the human gastrointestinal tract. Investigation of these proteins can help understand and predict how probiotics affect the human host. Methods for protein extraction of the L. plantarum F44 showed that the strain had a high resistance to osmotic stress but not to lysozyme treatment. Two-dimensional protein maps were obtained of the extracted proteins. However, no distinct differences were noted of the protein patterns of bacteria grown in the presence and absence of GOS. A prospect for the future is to analyse whether there are differences when using other prebiotics, such as pectin and resistant starches, and with other probiotic strains. The concentrations of short-chain fatty acids (SCFAs) in the culture, mainly butyric and propionic acids, produced as a result of bacterial fermentation of the GOS, were determined by high performance liquid chromatography (HPLC). The conditions for the chromatographic separation were carefully evaluated and clear chromatograms of the standard solutions of butyric and propionic acids established. However, a different sample preparation will be required for accurate measurement of the SCFAs produced by the bacteria. The attempted liquid/liquid back extraction was not sufficient since too many impurities remained in the sample, interfering with the chromatography. Further purification steps are needed. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/4499348
- author
- Bratus, Marusa
- supervisor
- organization
- course
- MOBN18 20132
- year
- 2014
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 4499348
- date added to LUP
- 2014-06-23 15:27:56
- date last changed
- 2014-06-23 15:27:56
@misc{4499348,
abstract = {{Abstract
The long-term aim of this project was to develop synbiotic drinks presenting food supplements with beneficial effects on human health. A synbiotic consists of a combination of probiotic bacteria and one or more prebiotics. For the part of the project that was covered by this Master thesis, a probiotic strain, Lactobacillus plantarum F44 and a prebiotic galactooligosaccharide (GOS) were tested in vitro. The probiotic L. plantarum F44 was selected due to previous systematic studies of the strain characteristics. The selected probiotic strain was cultured with GOS, but also grown without GOS (control), to compare growth efficiency, gene expression, and fermentation products. By monitoring the cell density, it was shown that GOS in a moderate concentration (2 mg/mL) increased the bacterial growth rate. Bacterial proteins play a major role in interaction with the cells in the human gastrointestinal tract. Investigation of these proteins can help understand and predict how probiotics affect the human host. Methods for protein extraction of the L. plantarum F44 showed that the strain had a high resistance to osmotic stress but not to lysozyme treatment. Two-dimensional protein maps were obtained of the extracted proteins. However, no distinct differences were noted of the protein patterns of bacteria grown in the presence and absence of GOS. A prospect for the future is to analyse whether there are differences when using other prebiotics, such as pectin and resistant starches, and with other probiotic strains. The concentrations of short-chain fatty acids (SCFAs) in the culture, mainly butyric and propionic acids, produced as a result of bacterial fermentation of the GOS, were determined by high performance liquid chromatography (HPLC). The conditions for the chromatographic separation were carefully evaluated and clear chromatograms of the standard solutions of butyric and propionic acids established. However, a different sample preparation will be required for accurate measurement of the SCFAs produced by the bacteria. The attempted liquid/liquid back extraction was not sufficient since too many impurities remained in the sample, interfering with the chromatography. Further purification steps are needed.}},
author = {{Bratus, Marusa}},
language = {{eng}},
note = {{Student Paper}},
title = {{Two techniques for investigation of proteins and short-chain fatty acids of Lactobacillus plantarum in presence of galactooligosaccharides}},
year = {{2014}},
}