Periodic Reporting for period 1 - GLYCO-WAY (Glycomics of Winery Antimicrobial Yeast)
Período documentado: 2021-02-15 hasta 2023-02-14
The recent emergence and spread of anti-microbial resistance (AMR) generates serious concerns. AMR related infections result in 25K deaths and €1.5 billion healthcare costs in the EU annually. A promising strategy involves strengthening the intestinal barrier and modulating gut microbiota using prebiotics. Cell walls polysaccharides, mainly from S. cerevisiae, showed great potential for mitigating antibiotic burden. At present, however, our understanding of the mechanism of action underlying these biological effects is significantly lacking, mostly due rudimentary characterization, which hinders identification of key molecules and delays production of effective feed supplements. A key reason for incomplete characterization is the heterogeneous nature of polysaccharides, which have challenged researchers for years. Recent technological breakthroughs now provide analytical toolsets that enable a more comprehensive understanding of the fine structure of large polysaccharides. The overall objective of the project was to use the Glycomics techniques to characterize in-depth the structure of polysaccharides, specifically mannan and glucan due to their reported bioactivities, present in different non-saccharomyces yeasts, to evaluate and compare their potential anti-adherent and gut microbiota modulator functions, and to unravel their structure-function relation to mitigate the use of antimicrobials in humans and livestock production.
According to the preliminary results obtained, we have identified that the cell-walls from the nine yeast species included in the study contain different ratio and structure of monosaccharides, which could play a beneficial role when compared to cell-walls from S. cerevisiae that only contains glucose and mannose units. Also, since there is a high amount of commercial yeast cell-walls product, mainly for livestock, and with poor structural characterization, this study also included the analysis of these products from supplier based on the USA and in the European Union, and we have concluded that some of the products have important difference among batches, and the purity claimed on the label is not always reliable.
According to the preliminary results obtained, we have identified that the cell-walls from the nine yeast species included in the study contain different ratio and structure of monosaccharides, which could play a beneficial role when compared to cell-walls from S. cerevisiae that only contains glucose and mannose units. Also, since there is a high amount of commercial yeast cell-walls product, mainly for livestock, and with poor structural characterization, this study also included the analysis of these products from supplier based on the USA and in the European Union, and we have concluded that some of the products have important difference among batches, and the purity claimed on the label is not always reliable.
The following work-packages were achieved during the duration of the project:
Work Package 1. Biomass production. In this case, we increased to 20 the number of yeast strains to be analyzed. These strains were activated and growth in nutritional media and then the biomass obtained was fractionated for further analysis.
Work Package 2. Purification and fractionation of cell wall polysaccharides. Once the biomass was washed with deionized water, the cell-walls were isolated by lysing the cells and then fractionated by using alkali and ethanolic precipitation.
Work Package 3. Glycomic study of yeast cell wall polysaccharides. The fraction obtained were first analyzed using LC-QqQ to identify the monosaccharide composition and their relative abundance. From this, 9 strains were selected based on the difference monosaccharide structure and abundance. Then, the samples were permethylated and hydrolyzed to determine their glycosidic linkages, as well as, hydrolyzed using a patented method based on Fenton Reaction to study, more in detail, their structures by using high-resolution mass spectrometry.
In this package we also include the commercial samples of S. cerevisiae cell-walls. Since these products were already available for consumption, the samples were not fractionated, as analyzed them as it is.
In general, the results show a wide range of structural differences of the cell-wall of the yeast species studied. This implies that we could find new source of bioactive polysaccharides with different beneficial properties when compared to S. cerevisiae cell walls products.
On the other hand, some of the most used commercial S. cerevisiae cell-wall products showed some differences between batches and purity, as well as a misused of the nomenclature to identify the carbohydrates contained in such as products, this is despite the high amount of research concluding the bioactive potential of these products. This highlights the importance of a comprehensive structural characterization of these products. This issue has been raised and explained by us in a recent edition of the Magazine The World of Food Ingredients (Jan-Feb 2022).
The results obtained are still being interpreted, and we can foresee a potential to exploit them as a new generation of bioactive carbohydrates from non-saccharomyces yeast strains. However, this will depend on the results obtained after in vitro and in vivo experiments with these products.
Work Package 1. Biomass production. In this case, we increased to 20 the number of yeast strains to be analyzed. These strains were activated and growth in nutritional media and then the biomass obtained was fractionated for further analysis.
Work Package 2. Purification and fractionation of cell wall polysaccharides. Once the biomass was washed with deionized water, the cell-walls were isolated by lysing the cells and then fractionated by using alkali and ethanolic precipitation.
Work Package 3. Glycomic study of yeast cell wall polysaccharides. The fraction obtained were first analyzed using LC-QqQ to identify the monosaccharide composition and their relative abundance. From this, 9 strains were selected based on the difference monosaccharide structure and abundance. Then, the samples were permethylated and hydrolyzed to determine their glycosidic linkages, as well as, hydrolyzed using a patented method based on Fenton Reaction to study, more in detail, their structures by using high-resolution mass spectrometry.
In this package we also include the commercial samples of S. cerevisiae cell-walls. Since these products were already available for consumption, the samples were not fractionated, as analyzed them as it is.
In general, the results show a wide range of structural differences of the cell-wall of the yeast species studied. This implies that we could find new source of bioactive polysaccharides with different beneficial properties when compared to S. cerevisiae cell walls products.
On the other hand, some of the most used commercial S. cerevisiae cell-wall products showed some differences between batches and purity, as well as a misused of the nomenclature to identify the carbohydrates contained in such as products, this is despite the high amount of research concluding the bioactive potential of these products. This highlights the importance of a comprehensive structural characterization of these products. This issue has been raised and explained by us in a recent edition of the Magazine The World of Food Ingredients (Jan-Feb 2022).
The results obtained are still being interpreted, and we can foresee a potential to exploit them as a new generation of bioactive carbohydrates from non-saccharomyces yeast strains. However, this will depend on the results obtained after in vitro and in vivo experiments with these products.
Despite that the characterized cell-walls from non-saccharomyces strains have not been yet subjected to functional studies, we identified monosaccharides residues that can have an important effect on immunological and gut microbiota modulation that could have also an impact in decreasing the use of antibiotics. Not only from livestock but also for human consumption.
However, more studies and research must be conducted to have a real impact from a social point of view.
This project was terminated earlier (12 months out of 36) because the main researcher was offered a new position as Scientist in a Food Company to pursue the challenge of finding novel ways of producing more sustainable milk replacers.
However, more studies and research must be conducted to have a real impact from a social point of view.
This project was terminated earlier (12 months out of 36) because the main researcher was offered a new position as Scientist in a Food Company to pursue the challenge of finding novel ways of producing more sustainable milk replacers.