Production method of phenolic extract from industrial by-product of persimmon with cytoprotective effects against bacteria

Abstract

Process for obtaining extracts enriched in Diospyros kaki Thunb bonded phenolics characterised by use of by-products derived from the production of persimmon juice such as seeds, peels and stalks; tre

Description

Production method of phenolic extract from industrial by-product of persimmon with cytoprotective effects against bacteria TECHNICAL FIELD The present invention belongs to the food industry sector, specifically in the reprocessing of fruit waste. It refers to processing waste from persimmon juice manufacturing to obtain phenolic extract from persimmon. This transformation leads to a modified extract, composed by gallic acid subunits, exhibiting certain physiological activities derived from antibacterial properties to protect human cells against bacteria. STATE OF ART Persimmon (Diospyros kok / Thunb) is a fruit that has been widely consumed in Asian countries, Brazil, and Spain. Spanish production of persimmon has increased exponentially reaching 4.7% of world production (FAO, 2016). The fruit is characterized by its texture and sweetness. The interest for the by-product derived from persimmon industrial juice processing is due to its bioactive content. Persimmon juice industrial by-products tend to be discarded, although it has already been reported that persimmon juice by-product, conformed by seeds, peels and pulps, is rich of bioactive compounds such as polysaccharides, carotenoids and phenolics (Lafka et al., 2007; Russo et al., 2018; Salazar-Bermeo et al., 2021). Among these biocompounds, phenols are popular due to their potential related to effects against reactive oxygen species, anti-obesity, anticarcinogenesis, dermal protection, hypertension, among others (Butt et al., 2015). In persimmon, tannins have been reported as the main phenolics class and have also been proven to exert antimicrobial effects. Recently, the potential of bound phenolic compounds linked to polysaccharide matrix of persimmon by-product have been identified as unexplored sources of phenolics (Burlini &Sacchetti, 2020; Moreno-Chamba et al., 2022; Salazar-Bermeo et al., 2021). Even though the antimicrobial activity of phenolic compounds has been reported, most of its determination has been limited to the study of its inhibitory effect. Derived properties from the extracts, such as the potential cytoprotective effect against pathogenic microorganisms, have not been fully explored (Park et al., 2001; Sengul et al., 2009; Silva et al., 2012). Moreover, it has also been published those phenolic extracts may be able to inhibit multidrugresistant bacterial strains. This establishes them as potential alternatives to conventional antibiotics and other antibacterial molecules that have lost efficacy over time. 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 (Matias et al., 2016; Rodriguez-Daza et al., 2021). Also, the use of antibacterial molecules that may regulate bacterial adhesion, a key virulence factor prior any infection disease such as dermatitis or colitis, is of interest. After adhesion, bacteria can infect other parts of the human body by attachment to non-phagocytic epithelial cells, due to an interaction of proteins and polysaccharides present in the human and bacterial cell membrane. Besides, several treatments that remove bacteria or inhibit their adhesion stimulate the release of toxins from fastidious bacteria like Salmonella enterica or Escherichia coli, causing damages in the gastrointestinal tract and inflammatory symptoms (Barbosa et al., 2021; Guerrant et al., 1999; Yu et al., 2015). Phenolics can also act as potential prebiotic substrates that may regulate the growth and / or activity of gut-health beneficial bacteria (Moreno et al., 2022; Rodriguez-Daza et al., 2021). This effect may be related to an enhancement of the host immune response, protection of the integrity of epithelial barrier, and a selective modulation of bacterial adhesion in oral, respiratory, gastrointestinal tracts and skin, with a cytoprotective effect as a result. The present invention focuses on obtaining an extract rich in phenolics, which are compounds related to the colour and maturation state of persimmon, being the gallic acid the most abundant. This phenolic acid is found in the by-product of the fruit as well as in other fruits and vegetables. The biological activity of this extract in pathogenic and non-pathogenic bacteria as well as human cells, showed the potential of the cytoprotective effect of the persimmon bonded-phenolic extract to be assessed in more complex systems and with different bacteria and human cell lines. The extraction of phenolics from natural sources has been studied extensively, but mainly for gallic acid. 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, fermentation, or enzymatic treatments (Example patents: KR101854120B1, KR101289726B1, CN106539848A, KR20150051438A, CN101294048A, KR102084950B1, KR20030067082A, CN106008616A). 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). A disadvantage of these methods is that they are usually focused on the easily extractable phenolics, discarding bonded phenols in polysaccharide matrices. The hydrolysis of these saccharide matrices to obtain bonded phenolics may contribute to the complete use of the by-product, leaving the polysaccharides for other purposes with 0% of polyphenolic residues. 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 (KR20180055303A, KR20180055306A, KR101162581B1, KR20140122588A). But in this case, the extract of bonded phenolics is obtained from the hydrolysis of vacuum assisted expansion-treated by-products of persimmon. References Barbosa, P. d. P. M., Ruviaro, A. R., Martins, I. M., Macedo, J. A., LaPointe, G., &Macedo, G. A. (2021). Enzyme-assisted extraction of flavanones from citrus pomace: Obtention of natural compounds with anti-virulence and anti-adhesive effect against Salmonella enterica subsp. enterica serovar Typhimurium. Food Control, 120, 107525. Burlini, I., &Sacchetti, G. (2020). Secondary Bioactive Metabolites from Plant-Derived Food Byproducts through Ecopharmacognostic Approaches: A Bound Phenolic Case Study. Plants, 9(9), 1060. Butt, M. S., Sultan, M. T., Aziz, M., Naz, A., Ahmed, W., Kumar, N., &Imran, M. (2015). Persimmon (Diospyros kaki) fruit: hidden phytochemicals and health claims. EXCLI J, 14, 542-561. FAO. (2016). FAO. Food and Agricultural Organization of the United Nations (2016). Retrieved from http; / / ww^ Guerrant, R. L., Steiner, T. S., Lima, A. A. M., &Bobak, D. A. (1999). How Intestinal Bacteria Cause Disease. The Journal of Infectious Diseases, 179(Supplement_2), S331-S337. Lafka, T.-L, Sinanoglou, V., &Lazos, E. S. (2007). On the extraction and antioxidant activity of phenolic compounds from winery wastes. Food chemistry, 104(3), 1206-1214. Matias, A. A., Rosado-Ramos, R., Nunes, S. L., Figueira, 1., Serra, A. T., Bronze, M. R.,. .. Duarte, C. M. (2016). Protective effect of a (poly) phenol-rich extract derived from sweet cherries culls against oxidative cell damage. Molecules, 21(4), 406. Moreno-Chamba, B., Salazar-Bermeo, J., Martinez-Madrid, M. C., Lizama, V., Marti'n-Bermudo, F., Berna, G., . . . Valero, M. (2022). Bound galloylated compounds in persimmon upcycled dietary fiber modulate microbial strains associated to human health after in vitro digestion. In (Vol. 156): Lebensmittel-Wissenschaft und-Technologie. Park, E.-S., Moon, W.-s., Song, M.-J., Kim, M.-N., Chung, k.-H., &Jin-San, Y. (2001). Antimicrobial activity of phenol and benzoic acid derivatives. International Biodeterioration &Biodegradation, 47(4), 209-214. https: / ZdoLprg / https: / / ^^ Rodriguez-Daza, M. C., Pulido-Mateos, E. C., Lupien-Meilleur, J., Guyonnet, D., Desjardins, Y., &Roy, D. (2021). Polyphenol-mediated gut microbiota modulation: Toward prebiotics and further. Frontiers in Nutrition, 8, 689456. Salazar-Bermeo, J., Moreno-Chamba, B., Martinez-Madrid, M. C., Saura, D., Valero, M., &Marti, N. (2021). Potential of Persimmon Dietary Fiber Obtained from Byproducts as Antioxidant, Prebiotic and Modulating Agent of the Intestinal Epithelial Barrier Function. Antioxidants, 10(11), 1668. https: / / doi.org / 10.3390 / antioxl0111668 Sengul, M., Yildiz, H., Gungor, N., Cetin, B., Eser, Z., &Ercisli, S. (2009). Total phenolic content, antioxidant and antimicrobial activities of some medicinal plants. Pakistan Journal of Pharmaceutical Sciences, 22(1). Silva, J. C., Rodrigues, S., Feas, X., &M. Estevinho, L. (2012). Antimicrobial activity, phenolic profile and role in the inflammation of propolis. Food and Chemical Toxicology, 50(5), 1790-1795. https: / / doi.Org / https: / / doi.org / 10.1016 / j.fct.2Q12.Q2.097 Yu, Q., Yuan, L., Deng, J., &Yang, Q. (2015). Lactobacillus protects the integrity of intestinal epithelial barrier damaged by pathogenic bacteria [Original Research], Frontiers in Cellular and Infection Microbiology, 5. http sj / / do mb,2015,QQQ26 DETAILED DESCRIPTION OF THE INVENTION The process object of the present invention is based on a process that does not require the use of organic solvents for the extraction and purification of these compounds from the by-products of persimmon industry. The process subjects the by-product to destruction and crushing by bursting the hot material by exposing it suddenly to high vacuum conditions. The solid fraction is subjected to hydrolysis process to release bonded phenolics. Subsequently, the product is subjected to tangential filtration and extraction by adsorption resins, which manage to isolate and purify the phenolics contained in the product. The phenolics thus obtained from the persimmon by-product, preferably -gallic acid, were subjected to studies with bacterial reference strains that adhere to human cells with results related to antimicrobial resistance and were used for a trial with other microorganisms that express other virulence factors. The phenolic extract of the present invention is not particularly limited in its composition and, for example, may refer to extracts comprising gallic acid, ellagic acid, salicylic acid, cyanidin, and / or ferulic acid. The gallic acid of the present invention is not particularly limiting and, for example, other phenolic acids may be included in the invention. In addition, other vegetables and whole fruits may be considered as valuable sources of those phenolics, especially persimmon (Diospyros kaki Thunb), which production is highly desirable. The extract was produced in the following way: The raw material of persimmon used to produce phenolic extract comes from the industrialization residue of persimmon, composed of rind, lobe membranes and seeds, to produce juice and from the whole fruit. Furthermore, the present invention describes the process of obtaining phenolic extract from persimmon: Use dried products derived from the production of persimmon juices such as peel, seeds, and stalk, as well as whole fruit. The use of the raw material influence in the yield and purity of the extract. Adding water 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 solid fraction is subjected to alkaline / acidic hydrolysis. The liquid fraction from hydrolysis process is concentrated by evaporation or by spraydrying processes. The present process allows to obtain an extract rich in bonded phenolic compounds to polysaccharides from persimmon polysaccharide byproduct, as well as a high 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) 46.92 ±4.02 2.14 ±0.18 331.02 ±8.98 1330 ± 186.70 Table 1. Total phenolic (TPC) and flavonoid content (TFC), and antioxidant activity (AA) present in the dried extract of persimmon 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 several virulence factors related to human cell colonization: disk diffusion assay as initial approach in the inhibition of violacein and pyocyanin, metabolites responsible for the quorum sensing. Quantification of the inhibition of these metabolites by liquid-liquid 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. At the same time, the effect against the production of p-lactamase was determined with a kit of p-lactamase activity of Klebsiella pneumoniae subsp. pneumoniae. Due to the nature of persimmon extract, the phenolics that conform the extract are of natural origin and soluble in aqueous solutions and their use may be to protect human cells. 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. The protective effect of the extract against bacterial adhesion was assessed as well as an enhancement of probiotic effect of Lactococcus lactis subsp. lactis in co-culture with Caco-2 cells. The mode of antibacterial action was determined by the probe carboxyfluorescein diacetate succinimidyl ester and detection of the fluorescence of the carboxyfluorescein succinimidyl ester was measured after antibacterial effect. 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 the virulence factors herein assessed. 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 provided in this patent application 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 scope of protection thereof. The water used in the extraction was distilled. The extraction is generally carried out with dried raw material. The raw material of persimmon used to produce phenolic extract comes from the residue of persimmon industrialization, composed of seeds, peel and stalk, to produce juice and from the whole fruit. The by-product or the whole fruit can be subjected to dehydration by lyophilization, vacuum oven or zeodratation treatment that allows drying at room temperature. The by-product may be grounded (milled) and homogenized with a particle size between 0.1 to 0.5 mm. After mixture with distilled water from 1:5 to 1:10 (w / v), the solution may 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 solid fraction is subjected to alkaline / acidic hydrolysis, by adjusting the pH of a solution of solid fraction and water with sodium hydroxide between 3-6 M until reaching a pH near to 12 and immediately, the pH of the solution was lowered with citric acid between 3-6 M until reaching a pH close to 2. The process is performed under room temperature. The obtained liquid from hydrolysis is clarified by centrifugation or filtration by resins. The obtained liquid can be concentrated by evaporation by spray-drying processes, rotavapor or zeodratation. Composition of the persimmon phenolic extract obtained by hydrolysis 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 persimmon phenolic extract (Table 2) by HPLC showed that it contained mostly gallic acid. n^ Compound Retention time [min] Concentration [mg / g] 1 Gallic acid* 7.2 6.35 ±0.60 2 3,5-Dicaffeoylquinic acid** 12.1 — 3 Ellagic acid* 15.3 0.09 ± 0.01 4 Salicylic acid** 18.6 — 5 Spinacetin** 21.3 — 6 Galloyl-hexoside 1** 22.6 — 7 Galloyl-hexoside II** 22.8 — 8 Cyanidin** 23.2 — 0 Resveratrol-glucoside 1** 26.1 — 10 7-Hydroxy-4'-methoxyisoflavone** 29.4 — 11 5,4'-Dihydroxy-6,7-dimethoxyflavone** 29.5 — 12 Ferulic acid** 30.1 — 13 p-Coumaronyl tartaric acid** 30.6 — * Based on authentic standards, ** tentative identification Table 2. Bonded-phenolic compounds present in persimmon 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 persimmon extract in virulence factors. At the same time, positive controls with authentic antibiotics were included for comparative efficacy. The doses of the aqueous persimmon 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 otherwise indicated. The anti-virulence effect of persimmon extract against reference strains and in co-culture with human adenocarcinoma colon cells 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 adhesion, efflux pumps, and anti-p-lactamase activity. To determine the antibacterial effect of the extract, an evaluation of damages in bacterial membrane was also performed. The cytoprotective effect of the extract in human Caco-2 cells as well as a promotion of probiotic effect of L. lactis was also assessed in co-culture with bacterial pathogens. The Caco-2 cell monolayer barrier function was monitored for 48 hours challenged with lipopolysaccharide from Escherichia coli and treated with persimmon phenolic extract. Example 1 Antibacterial activity of persimmon extract against reference strains (Figure 1). The antibiogram was performed according to the Clinical and Laboratory Standards Institute (CLSI) instructions for microdilution assays. The data indicated that the phenolic persimmon extract inhibited the growth of the bacterial strains, most of them being pathogenic, while Lactococcus lactis, a non-pathogenic bacterium, was more resistant to the extract, 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 MICs against bacteria were between 0.94 and 3.75 mg of dry extract per mL and inhibited at least 60% of the bacterial population, with Bacillus cereus being the most sensitive bacterium to this extract. Results are expressed as mean inhibition (n=3) ± standard deviation (SD). SD <0.01 are not shown. These results show the potential application of persimmon extract as an antibacterial with an inhibitory effect on pathogenic bacteria of major importance. Example 2 Viability of Caco-2 cells exposed to different doses (15.00 to 0.03 mg / mL) of phenolic persimmon extract by crystal violet staining and MTT methods (Figure 2). Compared to untreated cells, the viability recorded by MTT indicates that the persimmon phenolic extract did not cause loss of viability (p>0.05) relative to the extract and did not interfere with the metabolic activity of human cells. Using the crystal violet staining method, the loss of viability of Caco-2 cells at doses above 7.50 mg / mL indicated that phenolic extracts interfere with Caco-2 adhesion (***p<0.001). Results are expressed as mean viability (n=3) ± standard deviation (SD). SD <0.01 are not represented. The results show that the persimmon extract, obtained by sudden expansion, ultrasound, and alkaline / acid hydrolysis, is safe for human consumption at the doses evaluated and can be incorporated into food, nutraceuticals, or pharmaceuticals for commercialisation. Example 3 Adhesion of Salmonella enterica and Klebsiella pneumoniae to Caco-2 cell monolayers treated with phenolic extract of persimmon at l / 2x or l / 4xMIC (Figure 3). Overall, persimmon phenolic extract reduced bacterial adhesion to monolayers compared to untreated (C-) cells (****p>0.0001). Furthermore, at l / 2xMIC the extract showed a similar effect as antibiotic-treated cells (C+) against K. pneumoniae adhesion (p>0.05) and a greater reduction of S. enterica adhesion to Caco-2 cells (++p>0.01). The results indicate that persimmon extract, obtained by sudden expansion, ultrasound, and alkaline / acid hydrolysis, inhibits bacterial adhesion of pathogens on human intestinal cells, with a better effect than penicillin / streptomycin. Persimmon extract can be incorporated into food, nutraceutical, parapharmaceutic, and drug formulations as a cytoprotective agent. Example 4 Effect of l / 2xMIC of persimmon phenolic extract on the ability of Lactococcus lactis to (Figure 4A and D) compete, (Figure 4B and E) exclude or (Figure 4C and F) displace Salmonella enterica or Klebsiella pneumoniae adhesion to Caco-2 cell monolayers. Persimmon stimulated a greater ability of L. lactis to compete with, exclude or displace pathogens than L. lactis by itself (Control) (p>0.001), especially S. enterica. The results obtained indicate that persimmon extract, obtained by sudden expansion, ultrasound, and alkaline / acid hydrolysis, can selectively protect beneficial microorganisms against pathogens in human cell monolayers. In other words, persimmon extract can be used as a cytoprotective ingredient of beneficial bacteria and as a promoter of their probiotic activity against pathogens to favour their competitiveness as a cytoprotective ingredient in food, nutraceuticals, parapharmaceutic, and pharmaceuticals. Example 5 Trans epithelial electrical resistance (TEER) response of Caco-2 cell monolayers exposed to lipopolysaccharide (LPS) during 48 h of incubation with phenolic persimmon extract (Figure 5). An increase in TEER values was observed in monolayers treated with l / 2xMIC of the extract at 24 h (p>0.001). After incubation, TEER values were similar to those of healthy (blank) monolayers (untreated monolayers not exposed to LPS (p>0.05), while a significant loss of TEER was observed in monolayers with LPS (C-) (p>0.001). The persimmon extract, obtained by sudden expansion system, ultrasound, and alkaline / acid hydrolysis, shows a cytoprotective effect on cells exposed to E. coli LPS toxin, and can be used in product formulations aiming to protect cells from endotoxins. Example 6 Anti-quorum sensing activity of persimmon phenolic extract against quorum sensing of Chromobacterium violaceum and Pseudomonas aeruginosa (Figure 6). The disc diffusion method showed an inhibition halo of pyocyanin (Figure 6A) and violacein (Figure 6B) after incubation with persimmon extract at l / 2xMIC and l / 4xMIC, with an inhibition halo like that of streptomycin (Figure 6C and D, respectively). Violacein production (Figure 6E) was reduced by more than 70% after 24 hours incubation at l / 2xMIC (p>0.0001), while pyocyanin production (Figure 6F) was reduced by almost 50% at l / 16xMIC of persimmon phenolic extract (p>0.001). The persimmon extract, obtained by sudden expansion, ultrasound, and alkaline / acid hydrolysis, exhibits quorum sensing disruptive activity, indicating that it has the potential to modulate bacterial horizontal communication-dependent virulence factors. Example 7 Effect of phenolic persimmon extract on efflux pump activation in Pseudomonas aeruginosa (Figure 7). Persimmon phenolic extract blocked the activation of energy-dependent efflux pumps of P. aeruginosa from l / 4xMIC to IxMIC (p>0.0001), even after addition of glucose, compared to bacterial cells not treated with glucose (Control). The glucose-free control (Cw / glucose) confirmed that P. aeruginosa efflux pumps are energy-dependent. The results indicate that the persimmon extract, obtained by sudden expansion, ultrasound, and alkaline / acid hydrolysis, has the potential to block the activation of transport pumps; it could be applied as an antibiotic adjuvant, preventing bacteria from being able to expel the compound out of their system. Example 8 Inhibition of p-lactamase activity (mU / mL) of Klebsiella pneumoniae (Figure 8). Sub-inhibitory concentrations of the phenolic persimmon extract (l / 2x to l / 16x the minimum inhibitory concentration) were compared to the untreated control. At l / 2xMICof the persimmon phenolic extract, 50% of the p-lactamase activity was reduced (p>0.001). The results indicate that the persimmon extract, obtained by sudden expansion, ultrasound, and alkaline / acid hydrolysis, can be used as an adjuvant for p-lactam antibiotics against bacteria that produce enzymes that modify these molecules, and can resensitise bacteria to p-lactams that have lost efficacy. Example 9 Mode of antibacterial action of phenolic persimmon extract (Figure 9). Release of 5(6)-carboxyfluorescein succindimyl ester (CFSE) out of bacterial cells due to membrane damage after exposure to persimmon phenolic extract. The extract generated remarkable membrane damage in bacterial cells, superior even to the 2xMIC kanamycin positive control (p>0.001), after 24 h of incubation. The results show that the persimmon extract, obtained by sudden expansion, ultrasound, and alkaline / acid hydrolysis, exhibits antibacterial activity on bacterial cells and may damage their cell membranes. The phenolic composition of the extract, in which gallic acid predominates, allows this result to be obtained.

Claims

1. Process for obtaining extracts enriched in persimmon bonded phenolics (Diospyros kaki Thunb) characterized by:Use of by-products derived from the production of persimmon juice such as seeds, peel, and stalks.Treatment with a system of sudden expansion of said by-products previously crushed and sieved.Obtention of bonded phenolic extracts from hydrolysation of treated by-product of persimmon.

2. Process according to claim 1, characterized in that the by-products originate from the waste of the commercialization of fresh persimmon (Diospyros kaki Thunb).

3. Process according to claim 1 or 2, characterized in that the obtention of bonded-phenolic extracts takes place in aqueous solution.

4. Composition based on persimmon obtained by the process according to claims 1 to 3, characterized by having a composition of phenolics in the following range: a minimum percentage of phenolics between 4 to 5%, which 10-20% of it is gallic acid.

5. A cytoprotective antibacterial agent that produces the following biological effects: inhibition of pathogenic bacteria, quorum quenching of bacterial communication in C. violaceum and P. aeruginosa, blocking of efflux pumps in P. aeruginosa, inhibition of b-lactamase activity of K. pneumoniae, inhibition of S. enterica and K. pneumoniae adhesion to Caco-2 cell monolayers, promotion of the probiotic effect of L. lactis to compete, exclude and displace pathogens, increment stimuli of trans epithelial electric resistance of Caco-2 monolayers exposed to the toxin lipopolysaccharide after 24 h of incubation, and a damage of bacterial membrane as result of antibacterial effect after 24 hours of incubation.

6. A cytoprotective agent according to claim 5 and that contains phenolics.

7. A cytoprotective agent according to claim 6 that contains gallic acid.

8. A cytoprotective agent according to claim 7 that is aqueous.

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

10. Use of the composition according to claim4 in the manufacture of functional foods.

11. Use of the composition according to claim 4 in the manufacture of food supplements.

12. Use of the composition according to claims 4 in the manufacture of parapharmacy food products.

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

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