Production method of phenolic extract from industrial wine by-product of grape with bacterial antibiofilm properties

Abstract

Process for obtaining extracts enriched in Vitis vinifera phenolics characterised by use of by-products derived from the production of wine such as seeds, skins, stems and pulp, treatment of said bypr

Description

Production method of phenolic extract from industrial wine by-product of grape with bacterial antibiofilm properties TECHNICAL FIELD The present invention belongs to the technical field of food manufacturing, specifically in the processing of fruit raw material waste. It refers to processing waste from wine manufacturing to obtain phenolic extracts from grape. This transformation leads to a modified extract exhibiting certain physiological activities derived from antibacterial properties. STATE OF ART Grape (Vitis vinifera L.) cultivars are cultivated in the regions of Middle and Low Vinalopo due to the economic importance in wine-making industry because of the ideal environmental conditions over the year (Bertolini et al., 2010). Viticulture carries an enormous weight in the economy of several countries. By-products from purple grape varietals, like "Monastrell" or "Tempranillo", are more interesting due to their higher content of polyphenols (Teixeira et al., 2014). Grape by-product is generally conformed by peels and seeds from wine industry. Grape by-products have gained attention in recent years due to its antioxidant activity produced by polyphenols, such as flavonoids like quercetin or resveratrol, as well as ellagitannins and phenolic acids. In recent years, phenolic extracts from fruit by-products have gained attention because they can exhibit a more beneficial or synergistic effect than conventional extracts because of its wide complexity of biocompounds (Kowalska et al., 2017). In plants, phenols are secondary metabolites that participate in plant defence processes, pigmentation, protection, allelopathy, among other functions. Also, it has been mentioned that phenolics are able to manifest a beneficial effect on human health and are related to a lower risk of developing serious diseases such as cancer or diabetes (Tako et al., 2020). Among their reported functional properties, some phenolic compounds have been identified as inhibitors of bacterial growth (Alvarado-Martinez et al., 2020), as well as a control against biofilm formation. Biofilms confer a degree of intrinsic resistance that often infect tissues and / or devices, such as orthopedic devices, admitted to a hospital with S. aureus bacteremia having developed an implant-associated infection. Thus, while there is an urgent need for new antibiotics, there is an equally urgent need to develop therapeutic agents that could be used to limit biofilm formation. While such agents would not necessarily function as antibiotics in and of themselves, they could be used as a prophylactic to limit biofilm formation. Also, producing new antibiotics is expensive and their manufacture is not profitable for the pharmaceutical industry due to the rapid emergence of resistances (Hatfull et al., 2022). Therefore, the use of phenolic extracts prior to the administration of antibiotics may reduce its consumption and, consequently, combat the appearance of antibiotic resistance, which is already considered a new pandemic. Our invention focuses on obtaining an extract rich in phenolics, compounds related to the colour and maturations state of grape, being flavonoids, such as quercetin and resveratrol, the most abundant in the by-product of the fruit, as well as in other fruits and vegetables. The biological activity of this extract against reference bacterial strains, that manifest specific virulence factors related to antibacterial resistance, showed the potential of the anti-virulence effect of the grape phenolic extract to be assessed against another bacterial pathogens. The extraction of phenolics from natural sources has been studied extensively, but mainly for flavonoids of grape. Phenolics oxidize easily due to their antioxidant capacity, and they are also sensitive to light and heat. These characteristics reduce the possible range of extraction methodologies. Various extraction and purification methods can be applied to obtain natural phenolics, such as solvent extraction, supercritical fluid extraction (SFE), distillation, membrane separation, chromatography, or crystallization (Example patents: CN107841268A, CN101703635A, EP2875822A1, KR20110031722A, US2011250344A1, CN112022935A). Among all of them, the extraction with solvents has been the most used in the industry due to its simplicity and its low cost. However, techniques that minimize the use of organic solvents to produce food ingredients are beginning to be used such as vacuum assisted expansion (US2015258225A1, ES2694437A1, ES2537936A1). On the other hand, as regards to the biological activity of extracts rich in phenolics, studies exist that refer to their role in their antimicrobial effect (ES2532547A1). But in this case, the extract is obtained from vacuum assisted expansion-treatment of the by-products of vine making. The process object of the present invention is based on a hydroalcoholic extraction and purification of these compounds from the by-products of grape vine making industry. The process subjects the waste to destruction and crushing by bursting the hot material by exposing it suddenly to high vacuum conditions. Subsequently, the material is subjected to tangential filtration and extraction by adsorption resins, which manage to isolate and purify the phenolics contained in the original by-product. References Alvarado-Martinez, Z., Bravo, P., Kennedy, N. F., Krishna, M., Hussain, S., Young, A. C., &Biswas, D. (2020). Antimicrobial and Antivirulence Impacts of Phenolics on Salmonella Enterica Serovar Typhimurium. Antibiotics, 9(10). https: / / dss.Qrg / lQ.339p / antjbsQtscsSlpp6S8 Bertolini, E., Garcia, J., Yuste, A., &Olmos, A. (2010). High prevalence of viruses in table grape from Spain detected by real-time RT-PCR. European Journal of Plant Pathology, 128(3), 283-287. Hatfull, G. F., Dedrick, R. M., &Schooley, R. T. (2022). Phage Therapy for Antibiotic-Resistant Bacterial Infections. Annual Review of Medicine, 73,197-211. hitps: / / doi,os>; Kowalska, H., Czajkowska, K., Cichowska, J., &Lenart, A. (2017). What's new in biopotential of fruit and vegetable by-products applied in the food processing industry. In (Vol. 67, pp. ISO-159): Trends in Food Science &Technology. Tako, M., Kerekes, E. B., Zambrano, C., Kotogan, A., Papp, T., Krisch, J., &Vagvblgyi, C. (2020). Plant Phenolics and Phenolic-Enriched Extracts as Antimicrobial Agents against Food-Contaminating Microorganisms. Antioxidants (Basel), 9(2). Teixeira, A., Baenas, N., Dominguez-Perles, R., Barros, A., Rosa, E., Moreno, D. A., &Garcia-Viguera, C. (2014). Natural bioactive compounds from winery by-products as health promoters: a review. International Journal of Molecular Sciences, 15(9), 15638-15678. DETAILED DESCRIPTION OF THE INVENTION The present invention describes the phenolics obtained from the grape, preferably -flavonoid-rich extract, were subjected to studies with bacterial reference strains that manifest several virulence factors related to antimicrobial resistance and were used for a trial to other microorganisms that express said virulence factors. The phenolic extract of the present invention is not particularly limited and, for example, comprises extracts with quercetin, resveratrol, catechin, gallic acid, and ellagic acid. The punicalagin of the present invention is not particularly limited and for example flavonoids such as quercetin or resveratrol may be included. In addition, vegetables and whole fruits should be considered as sources of those flavonoids, and especially, grape (Vitis vinifera), which use in wine making is highly desirable. The extract was produced in the following way: The raw material of grape by-product used to produce phenolic extract comes from the industrialization residue of wine making, composed of seeds, skins, stems, and pulp from wine industry and from the whole fruit. Furthermore, the present invention describes the process of obtaining phenolic extract from grape: Use dried products derived from the production of wine industry such as seeds, skins, stems, and pulp. The use of the raw material influence in the yield and purity of the extract. Adding 50% of ethanol solution from 1:5 to 1:10 (w / v) to the dry substrate at a temperature of 50-90°C. Subject the mixture to cylindrical vacuum sudden expansion system. The liquid and solid fractions are separated, and the liquid fraction is subjected to centrifugation or filtration by resins. Concentration of clarified liquid by evaporation or by spray-drying processes. The process allows to obtain an extract rich in total phenolic content, flavonoids, and antioxidant activity (Table 1). TPC (mg gallic acid equivalent per g) TFC (mg of quercetin equivalent per g) AA by DPPH (mg of Trolox equivalent per g) AA by ABTS (mg of Trolox equivalent per g) 145.57 ± 5.72 101.53 ± 11.48 641.08 ± 7.66 6604 ±639.10 Table 1. Total phenolic (TPC), total flavonoid (TFC) content, and antioxidant activity (AA) present in the extract of grape obtained from vacuum sudden expansion system. For biological assays, the extract was resuspended in sterile water. The biological studies carried out were: An antibacterial activity studied against reference strains: antibiogram by microdilution method according to Clinical and Laboratory Standard Institute (CLSI) standard protocol. After 24 h of incubation, minimal inhibitory concentration (MIC), and sub-inhibitory concentrations (sIC) confirmation by aqueous solution with 0.5% of tetrazolium chloride and visualization of formazan. The optical density of samples and therefore, an inhibitory effect was calculated by a reader plate. The sub-inhibitory concentrations recorded previously served as active concentrations to assess a preventive effect of biofilm formation, inhibitory effect of pre-formed biofilm or destruction of mature biofilm: biomass was determined by crystal violet staining solution. In addition, the anti-biofilm activity of grape phenolic extract was recorded in micrographs by Field Emission Scanning Electron Microscope (FESEM). Given that biofilms are modulated by quorum sensing expression, an anti-quorum sensing effect of grape extract against metabolites responsible for the quorum sensing was determined by quantifying the inhibition of these metabolites by liquidliquid extraction of violacein and pyocyanin by dimethyl sulfoxide and chloroform with acid hydrochloride, respectively. Also, a determination of the inhibition of energy-dependent efflux pumps activity in Pseudomonas aeruginosa PAO1, by real-time monitorization of Red Nile hydrolysis after pump activation with glucose, was measured in fluorescence units in a microplate reader. Simultaneously, a disruption in swarming motility was assessed by modified Luria Bertuni agar plate method with 0.5% of bacteriological agar in the same strain. The effect against the production of p-lactamase was determined by kit of p-lactamase activity of Klebsiella pneumoniae subsp. pneumoniae. Due to the nature of grape extract, the phenolics that conform the extract are of natural origin and soluble in aqueous solutions. The present document also includes an evaluation of the viability of human cells like adenocarcinoma of human colon (Caco-2), determined by crystal violet staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solutions. In the present invention, unless it is specified, all quantities, ratios or percentages will always refer to the dried weight of extract. The period in which the effects of the present invention can be obtained after intake is not particularly limited, but it is desirable that intake be before or at the same time of pathogenic infection. Given the nature of the invention, its intake is not toxic, so it is desirable that the number of days ingested per year be one day or more or 365 days or less, preferably between 1 day or more and 365 days or less and more preferably between 60 days or more. The objects to which the present invention can be used against are not particularly limited, but preferably are microorganisms, especially pathogenic Gram-positive and Gram-negative bacteria that manifest these virulence factors. In the present invention, functional ingredient is understood as that which is consumed as part of a normal diet and that contains biologically active ingredients, which offer health benefits and reduce the risk of suffering from chronic diseases. In the present invention, it is understood as parapharmacy food products, those food products that are not drugs, are consumed and made available to users, in accordance and according to what is established, in specific technical-sanitary regulations of the different categories of products that exist in the market, as well as in the general regulations in force in the field. Throughout the description and the claims, the word "comprises", and its variants are not intended to exclude other technical characteristics, components, or steps. For those skilled in the art, other objects, advantages, and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. EXEMPLARY EMBODIMENTS The following specific examples that are provided in this patent serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and are not to be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting the field of application thereof. The water used in the extraction was distilled while the pure ethanol used in the extraction was potable. The extraction is generally carried out with dried raw material. The raw material of grape used to produce phenolic extract comes from the industrialization residue of wine making, composed of seeds, skins, stems, and pulp from wine industry. The by-product can be dehydrated by lyophilization, vacuum oven or zeodratation treatment that allows drying at room temperature. The by-product can be grounded (milled) and homogenized their particle size between 0.1 to 0.5 mm. After mixture with 30-50% of ethanol solution at 1:5 to 1:10 (w / v), the solution can be kept under agitation between 250 to 800 rpm to increase the efficiency of the process at a temperature between 50-90°C. Once homogenized, the slurry is introduced in a sudden expansion equipment allowing a greater breakdown of the material and increasing the extraction performance of the same. The extraction proceeded in a nitrogen-rich environment to inhibit the potential oxidation processes that may occur during the extraction of some of the active ingredients. The obtained suspension is filtered by centrifugation or filtration by resins. The obtained liquid can be concentrated by evaporation or by spray-drying processes. Composition of the grape phenolic extract obtained by solid-liquid extraction For the HPLC analysis of the extract, solutions of x g / L were prepared. The extract was homogenized in a vortex until completely dissolved and filtered through a 0.22 mm filter. The composition of the extracts was analysed by liquid chromatography RRLC 1200 series (Agilent Technologies, 40 CA) coupled to diode array detector in a range of the spectrum between 120 and 950 nm. A Poroshell column 120 SB-C18, 2.7 um, 4.6 x 150 mm was used. Phenolic compounds were analysed with a flow rate elution of 0.7 mL / min. The mobile phases used were acetic acid and ultrapure water (0.5:99.5, v / v) as solvent A, and acetonitrile as solvent B. The composition of grape phenolic extract (Table 2) by HPLC, showed that it contained gallic acid as well as several flavonoids and ellagitannins n^ Compound Retention time [min] Concentration [mg / g] 1 Gallic acid* 7.2 3.00±0.71 2 Protocatechuic acid** 9.2 — 3 Vanillic acid** 9.4 — 4 Caffeic acid** 9.6 — 5 Syringic acid** 9.9 — 6 o-Coumaric acid** 10.4 — 7 p-Coumaric acid** 10.8 — 8 Catechin** 11.5 — 9 Epicatechin** 11.9 — 10 Quercetin* 21.2 0.2010.02 * Based on authentic standards, ** tentative identification Table 2. Phenolic compounds present in grape by-product extract obtained from vacuum sudden expansion system, identified by HPLC-DAD EXAMPLES In all the examples carried out, the procedure was similar. Freshly culture of bacteria was prepared 24 h prior any assay. Then, the bacterial suspension was normalized to 0.5 McFarland optical density in the corresponding culture media for each tested microorganism. For each assay, a control of untreated bacterial cells was included to quantify the effect of grape extract in virulence factors. At the same time, positive controls with authentic antibiotics were included for comparative efficacy. The doses of the aqueous grape extract were from 15.00 to 0.03 mg of dried extract per mL, prepared in 96-well plates by serially 2-fold dilutions. Each result represents the mean of three independent experiments carried out separately. All the incubation steps were performed at 37°C in darkness and with no agitation unless it is indicated otherwise. The anti-virulence effect of grape extract against reference strains was performed with sub-inhibitory concentrations of the extract, which are concentrations below minimal inhibitory concentrations that do not inhibit bacterial viability but interfere with their pathogenic actions such as bacterial communication (quorum sensing), bacterial biofilms, efflux pumps, swarming motility disruption, and anti-p-lactamase activity. The viability human Caco-2 cells exposed to grape phenolic extract was assessed by crystal violet staining and MTT solution. Example 1 Antibacterial activity of grape extract against reference strains (Figure 1). The antibiogram was performed according to Clinical Laboratory Standard Institute (CLSI) instructions for microdilution assays. The data indicated that the phenolic grape extract inhibited the growth of bacterial strains, most of them pathogenic, after 24 h of incubation. The inhibitory effect was observed if the minimum inhibitory concentrations (MIC) were higher than 50% inhibition. Sub-inhibitory concentrations (sIC) were concentrations below the minimum inhibitory concentration. All minimum inhibitory concentrations against bacteria were between 1.87 and 15.00 mg dry extract per mL, with B. cereus being the most sensitive bacterium to this extract. These results show the potential application of grape extract as a broad-spectrum antibacterial at low doses. Example 2 Anti-quorum sensing activity of grape phenolic extract against quorum sensing of Chromobacterium violaceum and Pseudomonas aeruginosa (Figure 2). Violacein production (Figure 2A) was reduced by more than 60% after 24 hours of incubation at l / 8xMIC (p<0.05) while pyocyanin production (Figure 2B) was reduced by 50% at l / 64xMIC (p<0.05), when compared to the negative control (NC). The results indicate that the grape extract, obtained by the procedure detailed in this patent, inhibits horizontal bacterial communication of C. violaceum and P. aeruginosa by reducing the production of their metabolites. Low concentrations of the extract show quorum quenching activity, indicating that the grape extract has a potential to modulate bacterial quorum sensing-dependent virulence factors. Example 3 Antibiofilm activity of grape phenolic extract. Prevention of biofilm formation (Figure 3A), inhibition of pre-formed biofilm (Figure 3B) and destruction of mature biofilm (Figure 3C) of Pseudomonas putida, Bacillus cereus and Staphylococcus aureus. Inhibition of biofilms above 50% was considered significant, being antibiofilm concentrations (Abe). Grape phenolic extract prevented biofilm formation of P. putida (2xMIC, IxMIC and l / 2xMIC), B. cereus (2xMIC and IxMIC) and S. aureus (2xMIC); grape phenolic extract inhibited the pre-formed biofilm of P. putida (2xMIC), B. cereus (2xMIC) and S. aureus (2xMIC and IxMIC); grape phenolic extract destroyed mature biofilm of P. putida (2xMIC), B. cereus (2xMIC) and S. aureus (2xMIC and IxMIC); all by more than 50%. These results indicate that the extract, obtained by sudden expansion and ultrasound, is especially active against bacterial biomasses by preventing biofilm formation, inhibiting pre-formed biofilms, and eliminating mature biofilms. These results also show the potential of grape extract as an agent to treat surfaces with biofilms and to eliminate them. Example 4 FESEM micrographs of untreated Pseudomonas putida biofilm exposed to grape phenolic extract during its initial formation and pre-formed biofilm (Figure 4). Biofilm prevention by grape phenolic extract occurred at l / 2xMIC. Inhibition of pre-formed biofilm by grape phenolic extract occurred at 2xMIC. Mature P. putida biofilm was destroyed by grape phenolic extract at 2xMIC by crystal violet staining assay. The figure demonstrates the antibiofilm effect of the grape extract, which can be used as an antibiofilm agent in different food, parapharmaceutical, nutraceutical and pharmaceutical formulations. Example 5 Effect of grape phenolic extract on Pseudomonas aeruginosa swarming motility (Figure 5A). Motility was altered compared to the untreated control (Figure 5B). The P. aeruginosa inoculation site is represented by a red cross. The dashed line in Figure 5A represents the motility contour of the bacteria. Grape phenolic extract disrupted P. aeruginosa swarming motility at IxMIC, l / 2xMIC and l / 4xMIC. The inhibition is observed as an obstacle for the bacteria to move when interacting with the extract-impregnated disc, avoiding contact with the disk, and migrating through the agar in areas without extract. The grape extract, obtained by sudden expansion and ultrasound, can disrupt the motility of P. aeruginosa, preventing it from migrating to specific areas, which can prevent future colonisation of surfaces and future infections. This can prevent the formation of cell aggregates and the formation of biofilms. Example 6 Effect of grape phenolic extract on efflux pump activation in Pseudomonas aeruginosa (Figure 6). Grape phenolic extract blocked the activation of P. aeruginosa energy-dependent efflux pumps from l / 8xMIC to IxMIC, even after addition of glucose, compared to bacterial cells with glucose (Control) (p<0.0001). The glucose-free control (Cw / glucose) showed that P. aeruginosa efflux pumps are energy-dependent. These results reinforce the potential application of grape extract, obtained by sudden expansion process and ultrasound, to block the activation of transport pumps. This may facilitate the application of grape extract as an antibiotic adjuvant, preventing pathogenic bacteria from being able to expel the compound out of their system. Example 7 Inhibition of p-lactamase activity (mU / mL) of Klebsiella pneumoniae (Figure 8). Sub-inhibitory concentrations of grape phenolic extract (l / 2x to l / 4xMIC) were compared to the untreated control. At l / 2xMIC of grape phenolic extract, the lowest amount of p-lactamases was produced (p<0.001). This result indicates that the grape extract, obtained by sudden expansion and ultrasound, can be used as an adjuvant for p-lactam antibiotics against bacteria that produce enzymes that modify these antibiotics, being able to resensitise bacteria to p-lactams that have lost their efficacy. Example 8 Viability of human colon adenocarcinoma cells (Caco-2) after 24 h incubation with grape phenolic extract (Figure 8). Grape phenolic extract showed non-toxic effects on Caco-2 cells according to the MTT assay (Figure 8A) compared to untreated cells (p<0.05), indicating that grape phenolic extract does not interfere with the metabolic processes of Caco-2 cells. According to the crystal violet staining assay (Figure 8B), the loss of viability of Caco-2 cells at higher concentrations of grape phenolic extracts is related to impaired cell adhesion (p<0.001). These results indicate that grape phenolic extract is not toxic for human application / consumption. The results indicate that the grape extract, obtained by sudden expansion and ultrasound in a hydroalcoholic solution, is safe for human consumption at the doses evaluated and can be incorporated into food, nutraceuticals, parapharmaceuticals or pharmaceuticals for commercialisation.

Claims

1. Process for obtaining extracts enriched in grape phenolics (Vitis vinifera) characterized by:Use of by-products derived from the production of wine such as seeds, skins, stems, and pulp.Treatment with a system of sudden expansion of said by-products previously crushed and sieved and made into a slurry.

2. Process according to claim 1, characterized in that the derivative by-product originates from the waste of the wine industry of grape (Vitis vinifera).

3. Process according to claim 1, characterized in that the obtained product is an aqueous phenolic extract.

4. Composition based on grape obtained by the process described in any of claims 1 to 3, characterized by having a composition of phenolics in the following range: a minimum percentage of phenolics between 10 to 15%.

5. An antibiofilm agent that produces the following biological effects: inhibition of pathogenic bacteria, quorum quenching of bacterial communication in C. violaceum and P. aeruginosa, prevention of biofilm formation, inhibition of pre-formed biofilm, destruction of mature biofilm, blocking of efflux pumps in P. aeruginosa as well as a disruption on swarming motility of P. aeruginosa, inhibition of b-lactamase activity of K. pneumoniae.

6. An antibiofilm agent according to claim 5 that contains phenolics.

7. An antibiofilm agent according to claim 6 that within the phenolics contains flavonoids such as quercetin and resveratrol.

8. An antibiofilm agent according to claim 5 and is water-soluble and which contains flavonoids.

9. Use of the preparation according to claim 4 in the manufacture of healthy foods and beverages.

10. Use of the composition according to claim 4 in the manufacture of functional foods.

11. Use of the composition according to claim4 in the manufacture of food supplements.

12. Use of the composition according to claim4 in the manufacture of parapharmacy food products.

13. Use of the composition according to claim4 in the manufacture of antibacterial agents to be used in devices as well as on surfaces in contact with humans.

14. Use of the composition according to claim4 in the manufacture of antibacterial agents against resistant bacteria.

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