Periodic Reporting for period 1 - LACALAB4wine (Characterisation of lactic acid bacterial enzymes and their biotechnological potential in wines)
Periodo di rendicontazione: 2021-09-01 al 2023-08-31
Wine has become a daily consuming-product and the EU is the world’s leading wine producer (60% of world production). To guarantee food quality and safety control for consumers, winemakers normally use physical and chemical methods to reduce toxins and off-flavours in wines, which will be replaced with a biological method using laccases enzymes from lactic acid bacteria (LAB) to avoid environmental and health problems.
This project is aimed at applying laccases to eliminate undesirable substances in wines such as toxic compounds (BA and ochratoxin A-OTA) and off-flavors (volatile phenols - VP) produced from yeasts, fungi and bacteria during the winemaking process. The ultimate objective is to explore the structure and characteristics of LAB laccases as well as their biotechnological and practical applications to the oenological industry, and activity on BA, OTA and VP in wines in order to improve the wine quality.
As conclusions of this action, the knowledge and results generated from this project should be transferred to the wine/food industry with possible great benefits in terms of wine quality and safety.
This project is aimed at applying laccases to eliminate undesirable substances in wines such as toxic compounds (BA and ochratoxin A-OTA) and off-flavors (volatile phenols - VP) produced from yeasts, fungi and bacteria during the winemaking process. The ultimate objective is to explore the structure and characteristics of LAB laccases as well as their biotechnological and practical applications to the oenological industry, and activity on BA, OTA and VP in wines in order to improve the wine quality.
As conclusions of this action, the knowledge and results generated from this project should be transferred to the wine/food industry with possible great benefits in terms of wine quality and safety.
Specific objectives of this project were divided in workpackages and the obtained results are next summarized:
In the first workpackage (WP1), the project was launched and then managed through continuous meetings with my supervisor. Also, we developed a career development plan.
In WP2, I cloned the specific laccase genes of four lactic acid bacteria (LAB) from Companilactobacillus bobalius, Pediococcus parvulus, Lactobacillus paracasei and Lactococcus lactis using pET28a vector into E. coli DH5α due to its easiness to be cloned and manipulated. After that, vector+laccase genes were extracted and cloned into E. coli BL21 as an expression strain given that possessed a second plasmid called pGRO7 which codifies to chaperones GROES-EL that facilitate the protein folding. In addition, some chaperone genes from LAB species were amplified for using these proteins owned by LAB instead of using E. coli ones. In this context. CopZ and GROEL genes from P. parvulus and C. bobalius were amplified. Nevertheless, the only chaperone gene that was successfully cloned into E. coli was CopZ- C bobalius.
In the third workpackage, laccases were produced and purified in Ni+2-NTA agarose and biochemically characterized. The most acidophilus laccases were from C. bobalius and P. parvulus. The optimum temperature for all laccases was 28ºC, except for C. bobalius which turned out the most termophilic laccase (45ºC). On the other hand, the most susceptible laccase to inhibitor compounds was P. parvulus laccase.
In the fourth workpakage, I analyzed all the laccases in buffer reaction systems using either ABTS or epicatechin as mediators, extracts from polyphenolic compounds from both red and white wines, as well as real wines to evaluate their degrading capacity on BA, OTA and volatile phenols. It was evidenced that some laccases could degrade histamine, tyramine and putrescine in around 25% in red wine, and mainly tyramine and phenyethylamine at 15% in white wine. The highest OTA degradation in red wine was around 20%, and only 4-ethylguaiacol was degraded by bacterial laccases in acetate buffer without any mediator. In this sense, L. paracasei laccase was able to degrade it at 75%.
In the fifth workpackage, we could not crystalize any of the four laccases given the low protein concentration obtained for each one and their purity, which could not allow obtaining crystals. Nevertheless, C. bobalius laccase possess two different laccases. Therefore, the second laccase, described as bilirubin oxidase, was crystallized and their structure was resolved. Also, the rest of laccases were modeled.
I was trained since the beginning of the project by colleagues working in the lab and secondments labs about the different protocols and equipment that I used. I also took the courses Data protection and new rights, Know-how protection and intellectual property in research and university, From manuscript to publication in JCR journals academic writing workshop, and Work risks prevention at UV.
Concerning my leadership activities, I taught Microbiology practices and I supervised one trainee for his final degree work. In addition, I supported daily activities of Master degree students in lab, I wrote my proposal for the Spanish call “Ramón y Cajal”, and I prepared my CV for new opportunities in the oenological and biotechnological industry.
Finally, I disseminated my work in next congresses: Gienol, VIII national congress of Industrial microbiology and microbial biotechnology (CMIBM) and FEMS congress in 2022, and the 16th Meeting of the Spanish Network on lactic acid bacteria and Oeno Macrowine in 2023. Soon I will communicate my last results in the II International congress on grapevine and wine sciences in November 2023.
In addition, I communicated the project summary through a video recorded for the European Researchers’ night, celebrated at UV, and I set up a wine tasting during the event. Moreover, I participated in Expociencia 2022 and 2023, the biggest public engagement event organized by the UV to inspire scientific vocation in children.
In conclusion, four recombinant bacterial laccases were cloned, produced and biochemically characterized. These enzymes turned out acidophilus and mesophilic with different degradation potential of BA, OTA and VP either using canonical substrates or by their own. These laccases revealed more degradation of BA in red wines than in white wines. OTA degradation in both red and white wine was also accomplished.
In the first workpackage (WP1), the project was launched and then managed through continuous meetings with my supervisor. Also, we developed a career development plan.
In WP2, I cloned the specific laccase genes of four lactic acid bacteria (LAB) from Companilactobacillus bobalius, Pediococcus parvulus, Lactobacillus paracasei and Lactococcus lactis using pET28a vector into E. coli DH5α due to its easiness to be cloned and manipulated. After that, vector+laccase genes were extracted and cloned into E. coli BL21 as an expression strain given that possessed a second plasmid called pGRO7 which codifies to chaperones GROES-EL that facilitate the protein folding. In addition, some chaperone genes from LAB species were amplified for using these proteins owned by LAB instead of using E. coli ones. In this context. CopZ and GROEL genes from P. parvulus and C. bobalius were amplified. Nevertheless, the only chaperone gene that was successfully cloned into E. coli was CopZ- C bobalius.
In the third workpackage, laccases were produced and purified in Ni+2-NTA agarose and biochemically characterized. The most acidophilus laccases were from C. bobalius and P. parvulus. The optimum temperature for all laccases was 28ºC, except for C. bobalius which turned out the most termophilic laccase (45ºC). On the other hand, the most susceptible laccase to inhibitor compounds was P. parvulus laccase.
In the fourth workpakage, I analyzed all the laccases in buffer reaction systems using either ABTS or epicatechin as mediators, extracts from polyphenolic compounds from both red and white wines, as well as real wines to evaluate their degrading capacity on BA, OTA and volatile phenols. It was evidenced that some laccases could degrade histamine, tyramine and putrescine in around 25% in red wine, and mainly tyramine and phenyethylamine at 15% in white wine. The highest OTA degradation in red wine was around 20%, and only 4-ethylguaiacol was degraded by bacterial laccases in acetate buffer without any mediator. In this sense, L. paracasei laccase was able to degrade it at 75%.
In the fifth workpackage, we could not crystalize any of the four laccases given the low protein concentration obtained for each one and their purity, which could not allow obtaining crystals. Nevertheless, C. bobalius laccase possess two different laccases. Therefore, the second laccase, described as bilirubin oxidase, was crystallized and their structure was resolved. Also, the rest of laccases were modeled.
I was trained since the beginning of the project by colleagues working in the lab and secondments labs about the different protocols and equipment that I used. I also took the courses Data protection and new rights, Know-how protection and intellectual property in research and university, From manuscript to publication in JCR journals academic writing workshop, and Work risks prevention at UV.
Concerning my leadership activities, I taught Microbiology practices and I supervised one trainee for his final degree work. In addition, I supported daily activities of Master degree students in lab, I wrote my proposal for the Spanish call “Ramón y Cajal”, and I prepared my CV for new opportunities in the oenological and biotechnological industry.
Finally, I disseminated my work in next congresses: Gienol, VIII national congress of Industrial microbiology and microbial biotechnology (CMIBM) and FEMS congress in 2022, and the 16th Meeting of the Spanish Network on lactic acid bacteria and Oeno Macrowine in 2023. Soon I will communicate my last results in the II International congress on grapevine and wine sciences in November 2023.
In addition, I communicated the project summary through a video recorded for the European Researchers’ night, celebrated at UV, and I set up a wine tasting during the event. Moreover, I participated in Expociencia 2022 and 2023, the biggest public engagement event organized by the UV to inspire scientific vocation in children.
In conclusion, four recombinant bacterial laccases were cloned, produced and biochemically characterized. These enzymes turned out acidophilus and mesophilic with different degradation potential of BA, OTA and VP either using canonical substrates or by their own. These laccases revealed more degradation of BA in red wines than in white wines. OTA degradation in both red and white wine was also accomplished.
Preliminary results about biogenic amines degradation by bacterial laccases had been reported in acetate buffer, not in wine. In addition, reported results about OTA and other mycotoxins degradation refer to the use of lactic acid bacteria cells or the use of fungal laccases, but not bacterial laccases, and also their application had not been tried directly on wine.
Therefore, the four recombinant bacterial laccases that we have obtained should be further explored in order to analyze and evaluate their direct application to wines in terms of the real degradation percentage of toxic compounds and the application time during the winemaking process, as well as the generated subproducts after their possible reactions with other polyphenolic compounds integrated in wine, which has not been yet analyzed.
The exploration of the application of these recombinant bacterial laccases on wine could have not only a scientific importance, but also a positive socio-economic impact at enhancing the wine quality and healthiness and considering that wine is one of the most consumed alcoholic beverage worldwide according to the OIV.
Therefore, the four recombinant bacterial laccases that we have obtained should be further explored in order to analyze and evaluate their direct application to wines in terms of the real degradation percentage of toxic compounds and the application time during the winemaking process, as well as the generated subproducts after their possible reactions with other polyphenolic compounds integrated in wine, which has not been yet analyzed.
The exploration of the application of these recombinant bacterial laccases on wine could have not only a scientific importance, but also a positive socio-economic impact at enhancing the wine quality and healthiness and considering that wine is one of the most consumed alcoholic beverage worldwide according to the OIV.