Production method of phenolic extract from industrial by-product of pomegranate with bacterial quorum quenching properties

Abstract

Process for obtaining extracts enriched in Punica granatum L. phenolics characterized by use of by-products derived from the production of pomegranate juice such as rind, lobe membranes and seeds, and

Description

Production method of phenolic extract from industrial by-product of pomegranate with bacterial quorum quenching properties. TECHNICAL FIELD The present invention belongs to the food industry technical field, specifically in the processing of fruit waste. It refers to processing waste from pomegranate juice manufacturing to obtain phenolic extracts from pomegranate. This transformation leads to a modified extract exhibiting certain physiological activities derived from antibacterial properties. STATE OF ART Pomegranate (Punica granatum L.) is a deciduous shrub that can reach 5 to 8 m in height. Its fruit is a globular berry with a coriaceous rind, the interior is subdivided into several lobes containing numerous seeds covered with a covering, called sarcotesta, of red and juicy pulp. It opens spontaneously at maturity through fissures that expose the contents of each lobe. The main producing countries are Israel, Egypt, Lebanon, Spain, Tunisia, and Italy. There are different varieties on the market, both for consumption (Fine Tendral, Gabes, Mekens, Mollar de Elche, Provence, Maroc, Gafsa, Seedless, Wonderfull, Sweet, Plentiflora, Flavescnes, Albescent) and for decoration (Flore Pleno, Luteum Plenum, Legrelliae, Maxima Rubra, Nana). Cultivation is mainly by cuttings. Multiplication by seed is also possible but produces very variable specimens. Pomegranate by-products have gained attention in recent years due to its antioxidant activity attributed to polyphenols such as punicalagin. In recent years, phenolic extracts from fruit byproducts have gained attention because they may exhibit higher biological effects than conventional extracts due to their wide range of biocompounds (Kowalska et al., 2017). In the plant, phenols are secondary metabolites that participate in plant defense processes, pigmentation, protection, allelopathy, among other functions. Also, it has been extensively reported 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). Moreover, it has also been published those phenolic extracts may be able to inhibit multidrugresistant bacterial strains (Sharma et al., 2022). This establishes them as a potential support to conventional antibiotics and other antibacterial molecules that have lost efficacy over time (Khare et al., 2021). The production of new antibiotics is not happening at a sufficient rate to stop this issue and, progressively fewer antibiotics are showing efficacy against their target organisms (Aslam et al., 2018). It has been shown that plant phenolic compounds can resensitize some bacteria against antibiotics that were considered obsolete (Khare et al., 2021). 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. The present invention focuses on obtaining an extract rich in phenolics, which are compounds related to the colour and maturations state of pomegranate, being the punicalagin (a and b) the most abundant. This ellagitannin is found 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 pomegranate phenolic extract to be assessed against another bacterial pathogens. The extraction of phenolics from natural sources has been studied extensively, but mainly for punicalagin. 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 WO2004056444A1, CN101481372A, CN108299845A, CN106889506A, CN110357900A, US9526754B1, CN111671781A, CN102849859A). Among of 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 (JP2021145660A, US2018035685A1, CN105123914A, CN108030722A, CN103155953A). But in this case, the extract is obtained from vacuum assisted expansion-treatment of the by-products of pomegranate. The process object of the present invention is based on the use of a process that does not require the use of organic solvents for the extraction and purification of these compounds from the byproducts of pomegranate industry. The process subjects the product to destruction and crushing by bursting the hot material by exposing it suddenly to high vacuum conditions. 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. 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). htlps; / / dpi,prWlQ / OW$inPbiotigs9ip0668 Aslam, B., Wang, W., Arshad, M. I., Khurshid, M., Muzammil, S., Rasool, M. H., . . . Baloch, Z. (2018). Antibiotic resistance: a rundown of a global crisis. Infect Drug Resist, 11, 1645-1658. Hatfull, G. F., Dedrick, R. M., &Schooley, R. T. (2022). Phage Therapy for Antibiotic-Resistant Bacterial Infections. Annual Review of Medicine, 73,197-211. htfes: / / dQ-.Qrg / 10,n • 7 >tit 0 ■■ v? <5 <5 Khare, T., Anand, U., Dey, A., Assaraf, Y. G., Chen, Z. S., Liu, Z., &Kumar, V. (2021). Exploring Phytochemicals for Combating Antibiotic Resistance in Microbial Pathogens. Frontiers in Pharmacology, 12, 720726. https: / / doi.org / 10.3389 / fphar.2021.720726 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. Sharma, S., Barman, P., Joshi, S., Preet, S., &Saini, A. (2022). Multidrug resistance crisis during COVID-19 pandemic: Role of anti-microbial peptides as next-generation therapeutics. Colloids and Surfaces B: Biointerfaces, 211, 112303. https: / / doj,Qrg / 10,1016 / ;.coi^ 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). DETAILED DESCRIPTION OF THE INVENTION The present invention describes the phenolics obtained from the pomegranate, preferably -punicalagin, 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 given virulence factors. The phenolic extract of the present invention is not particularly limited in its composition and, for example, may refer to extracts comprising punicalagin, gallic acid, and / or ellagic acid. The punicalagin of the present invention is not particularly limiting and for example punicalagin-a and punicalagin-b may be included in the invention. In addition, vegetables and whole fruits should be considered as valuable sources of those punicalagin, especially pomegranate (Punica granatum), which production is highly desirable. The extract was produced in the following way: The raw material of pomegranate used to produce phenolic extract comes from the industrialization residue of pomegranate, 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 pomegranate: Use dried products derived from the production of pomegranate juices such as rind, lobe membranes and seeds, 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 liquid fraction is subjected to centrifugation or filtration by resins. Concentration of clarified liquid by evaporation or by spray-drying processes. The process yields an extract with a high total content of phenols, 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) 403.47 ± 16.53 254.75 ± 22.43 1530.63 ± 162.47 13216.001563.80 Table 1. Total phenolic (TPC) and flavonoid content (TFC), and antioxidant activity (AA) present in the extract of pomegranate 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 CLSI standard protocol. After 24 h of incubation, minimal inhibitory concentration, and sub-inhibitory concentrations 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 disruption effect of quorum sensing: 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 pomegranate 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 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 tis 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 pomegranate used to produce phenolic extract comes from the residue of pomegranate industrialization, composed of rind, lobe membranes and seeds, to produce juice and from the whole fruit. The by-product or the whole fruit can be dehydrated by lyophilization, vacuum oven or zeodratation treatment that allows drying at room temperature. The by-product may be grounded (milled) and homogenized their particle size between 0.1 to 0.5 mm. After mixture with distilled water at 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 suspension is filtered by centrifugation or filtration by resins. The obtained liquid can be concentrated by evaporation of by spray-drying processes. Composition of the pomegranate phenolic extract obtained by solid-liquid extraction For HPLC analysis of the extract, the extract was homogenized in a vortex until completely dissolved and filtered through a 0.22 pm 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 pomegranate phenolic extract (Table 2) by HPLC-DAD, showed that it contained mostly punicalagin a and punicalagin b, ellagic acid isomer and gallic acid, among other compounds. n^ Compound Retention time [min] Concentration [mg / g] 1 Gallic acid* 7.2 2.76 ±0.13 2 Galloyl glucose** 8.1 — 3 Galloyl-hexahydroxydiphenoyl- 8.2 — hexose** 4 Granatin** 9.1 — 5 Gallocatechin** 9.3 — 6 Punicalagin a* 10.6 168.55 ± 18.56 7 Peduncalagin** 10.9 — 8 Phellatin** 11.0 — 9 Ellagic acid glucoside** 11.4 30.45 ± 4.99 10 Punicalagin p* 11.6 213.66 ±40.84 11 Prunin** 12.2 — 12 Ellagic acid pentoside** 13.1 — 13 Ellagic acid isomer * 13.4 — * Based on authentic standards, ** tentative identification Table 2. Phenolic compounds present in pomegranate 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 pomegranate extract in virulence factors. At the same time, positive controls with authentic antibiotics were included for comparative efficacy. The doses of the aqueous pomegranate 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 pomegranate 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 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 coculture with bacterial pathogens. Example 1 Antibacterial activity of pomegranate extract against reference bacterial strains (Figure 1). The antibiogram was performed according to the Clinical and Laboratory Standards Institute (CLSI) instructions for microdilution assays. The data indicate that the pomegranate phenolic 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 0.23 and 1.88 mg dry extract per mL, with Pseudomonas putida being the most sensitive bacterium to this extract. These results show the potential application of pomegranate extract as an antibacterial, given that at low doses, an inhibitory effect on pathogenic bacteria can be obtained, increasing the application options of the extract. Example 2 Anti-quorum sensing activity of pomegranate phenolic extract against quorum sensing of Chromobacterium violaceum and Pseudomonas aeruginosa (Figure 2). The disc diffusion method showed a halo inhibition of pyocyanin (Figure 2A) and violacein (Figure 2B) after incubation with pomegranate extract, as well as streptomycin (Figure 2C and D). Violacein production (Figure 2E) was reduced by more than 70% after 24 hours of incubation at l / 128xMIC while pyocyanin production (Figure 2F) was reduced by almost 50% at l / 32xMIC, when compared to the negative control (NC). The results indicate that the pomegranate extract obtained, using 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 pomegranate extract has a potential to modulate bacterial quorum sensing-dependent virulence factors. Example 3 Antibiofilm effect of pomegranate phenolic extract on Staphylococcus aureus biofilm (Figure 3). Pomegranate phenolic extract destroyed mature S. aureus biofilm after 24 hours of exposure by more than 50% at an antibiofilm concentration (AbC) of 2xMIC. This result indicates that the extract, obtained by sudden expansion and ultrasound, is particularly active against Grampositive bacterial biomasses. Its inhibitory effect may facilitate the destruction of cutaneous S. aureus biofilms, as well as in foods susceptible to S. aureus contamination. Example 4 Effect of pomegranate phenolic extract on efflux pump activation in Pseudomonas aeruginosa (Figure 4). Pomegranate phenolic extract blocked the activation of P. aeruginosa energydependent efflux pumps from l / 4xMIC (*p<0.05) to IxMIC (****p<0.0001), even after addition of glucose, compared to bacterial cells with glucose (Control). The glucose-free control (Cw / glucose) showed that P. aeruginosa efflux pumps are energy-dependent. These results reinforce the potential application of pomegranate extract, obtained by a process of sudden expansion and ultrasound, to be able to block the activation of these transport pumps. This may facilitate the application of pomegranate extract as an antibiotic adjuvant, preventing pathogenic bacteria from being able to expel the compound out of their system. Example 5 Inhibition of p-lactamase activity (mU / mL) of Klebsiella pneumoniae (Figure 5). Sub-inhibitory concentrations of the pomegranate phenolic extract (l / 2x to l / 16x the minimum inhibitory concentration) were compared to the untreated control. At l / 2xMIC of the pomegranate phenolic extract, the lowest amount of p-lactamases was produced (****p<0.0001). This result indicates that the pomegranate 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, and can resensitise bacteria to p-lactams that have lost their efficacy. Example 6 Viability of Caco-2 cells exposed to different concentrations (15 to 0.03 mg / mL) of pomegranate phenolic extract by crystal violet staining and MTT reduction assays (Figure 6). A loss of viability was observed by the crystal violet staining assay at higher concentrations (***p<0.001). The results showed that the pomegranate phenolic extract was not toxic to Caco-2 cells according to the MTT assay. The crystal violet assay showed that the extract inhibited cell adhesion at higher concentrations. The results show that the pomegranate extract, obtained by sudden expansion and ultrasound, is safe for human consumption at the doses tested and can be incorporated into food, nutraceuticals, or pharmaceuticals for commercialisation. Example 7 Mode of antibacterial action of pomegranate phenolic extract (Figure 7). Fluorescence leakage of 5(6)-carboxyfluorescein succindimyl ester (CFSE) out of bacterial cells due to membrane damage after exposure to pomegranate phenolic extract. The extract generated significantly more membrane damage in the bacterial cells than the kanamycin positive control. The results show that the pomegranate extract, obtained by sudden expansion and ultrasound, exhibits antibacterial activity on bacterial cells and can damage their cell membranes. The phenolic composition of the extract, in which punicalagin predominates, allows this result to be obtained.

Claims

1. Process for obtaining extracts enriched in pomegranate phenolics (Punica granatum L.) characterized by:Use of by-products derived from the production of pomegranate juice such as rind, lobe membranes and seeds.Treatment with a system of sudden expansion of said by-products previously crushed and sieved.

2. Process according to claim 1, characterized in that the by-products originate from the waste of the commercialization of fresh pomegranate (Punica granatum L.).

3. Process according to claim 1, characterized in the obtention of aqueous phenolic extract.

4. Composition based on pomegranate 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 40 to 50%, which 20-30% of it is punicalagin.

5. An antibacterial agent that produces the following biological effects: inhibition of pathogenic bacteria, quorum quenching of bacterial communication in C. violaceum and P. aeruginosa, destruction of S. aureus mature biofilm, blocking of efflux pumps in P. aeruginosa, inhibition of b-lactamase activity of K. pneumoniae, no toxic effect on human Caco-2 cells, a damage of bacterial membrane as result of antibacterial effect after 24 hours of exposure.

6. An antibacterial agent according to claim 5 and that contains phenolics.

7. An antibacterial agent according to claim 6 that contains especially punicalagin.

8. An antibacterial 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 claim 4 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 claim 4 in the manufacture of parapharmacy food products.

13. Use of the composition according to claim 4 in the manufacture of antibacterial agents against resistant bacteria.

Images