Process for increasing the bioavailability of bioactive compounds in bee pollen

Abstract

The present invention relates to a process for increasing the bioavailability and efficacy of bioactive compounds in bee-collected pollen, primarily polyphenols and biosilica, with the aim of improving the quality of nutritional supplements, medical devices, and / or cosmetic products based on pollen. The process is characterized by comprising the following steps: semi-solid fermentation of bee-collected pollen, homogenized together with polyfloral honey at 9.5 grams of pollen, at room temperature, for 2 days; mixing 30 grams of semi-solid fermented pollen with 200 ml of distilled water and ultrasonication for 5 minutes at 750 W and a frequency of 30 kHz; adding to the 30 grams of semi-solid fermented pollen, homogenized in 200 ml of water, 8 grams of wheat bran and 4 grams of chaff containing at least 10A8 cfu / g lactobacilli, and fermenting for 3 days at 30 degrees; colloidal grinding of the fermented pollen, bran, and chaff, together with the fermentation medium for 1 minute, ultrasonication for 20 minutes at 750W and a frequency of 30 kHz, followed by cooling the mixture at 4°C for 2 hours and drying by lyophilization.

Description

[0001] PROCESS FOR INCREASING THE BIOAVAILABILITY OF BIOACTIVE COMPOUNDS IN BEE POLLEN

[0002] This invention pertains to a process for enhancing the bioavailability and efficacy of bioactive compounds found in bee-collected pollen, primarily polyphenols and biosilica, aiming to improve the quality of nutritional supplements, medical devices, and / or cosmetic products based on pollen. Various methods for increasing the bioavailability of bioactive compounds in pollen are known. These bioactive compounds include polyphenols (Rzepecka-Stojko et al. 2015, Molecules, 20(12), 21732-21749), especially flavonoids (Algethami et al. 2022, Nutrients, 14(14), 2858), and minerals (Thakur and Nanda, 2020, Trends in Food Science & Technology, 98, 82-106), including soluble silicon (Utoiu et al. 2018, Nutrients, 10(10), 1365). The bioavailability of these compounds from pollen is reduced by over 50% due to the structure of the pollen grain wall (Kostic, et al. 2020. Biomolecules, 10(1), 84). The primary component of the pollen grain cell wall is sporopollenin, a biopolymer that is not biodegradable and has remarkable mechanical resistance (Maruthi & Ramakrishna 2022, International Journal of Biological Macromolecules, 222, 2957-2965).

[0003] Patent application RO135449 A2 relates to a method for producing chewable tablets with pollen and natural vitamin C. The process, according to the invention, involves the steps of pulverizing bee pollen to a fineness of 200...800 pm, preparing dry extracts from rose hips, sea buckthorn, acerola, cranberries, or blackcurrants and standardizing them for ascorbic acid content, respectively, ascorbic acid and beta-carotene, preparing the chewable tablets by mixing 100...500 parts by weight pollen, 45...100 parts standardized extract, 25...50 parts honey as a binding agent, and 75...150 parts glucose, optionally a flavoring agent, wet and dry calibrating the granulate and compressing it, resulting in tablets weighing 0.5...2 g, with a marbled appearance, sweet taste, and improved bioavailability. The patent application RO135449 A2 mentions the increase in bioavailability but does not claim steps that would break the pollen grain wall and increase the bioavailability resulting from this cell wall disruption. Patent RU 2538635 C2 presents a method for processing bee pollen that involves extracting the lipophilic components with supercritical carbon dioxide, followed by enzymatic hydrolysis of the hydrophilic fraction. The ferment is separated into solid and liquid phases, the solid phase is dried, and the liquid phase is filtered and preserved by adding potassium sorbate and sodium benzoate. This method uses chemical preservatives, which are less acceptable to consumers. However, its major flaw is that the extraction and fractionation operations destroy the complexity of the system represented by the bioactive compounds in pollen.

[0004] In general, the disadvantage of such processes aimed at fractionating and purifying the bioactive components of pollen is that they destroy complex systems in the attempt to increase their bioavailability. A complex product is more than the sum of its parts, due to emergent characteristics, that is, "unexpected behaviors that result from the interaction between the components of an application and their environment" (Johnson, Reliability Engineering & System Safety, 91(12), 1475-1481). One of the most well-known emergent properties in complex systems is synergy, which is the mutual enhancement of the biological activities of various biologically active ingredients. Generally, biological compounds are more active in their natural combinations than the extracted compounds.

[0005] A solution for increasing the bioavailability of bioactive compounds in pollen is fermentation with microorganisms. This solution is biomimetic because it mirrors the way in which the bioavailability of bioactive components in bee bread - the fermented pollen by bees - is? increased (Aylanc et al., Trends in Food Science & Technology, 109, 464- 481).

[0006] Patent application W02020016770 A1 describes a biotechnological process for producing fermented pollen that includes inoculating the pollen with at least one lactic bacteria from the species Lactobacillus kunkeei selected among the strains L. kunkeei PF12 (DSM 32843), PF13 (DSM 32845), and / or PL13 (DSM 32844) and fermenting the inoculated pollen with the lactic bacteria. The obtained fermented pollen has nutritional and organoleptic properties similar to naturally produced bee bread inside the hive's honeycomb and is applicable in the food and nutraceutical field. The patented process includes the strains of L. kunkeei PF12 (DSM 32843), PF13 (DSM 32845), and / or PL13 (DSM 32844) as such and compositions comprising these strains.

[0007] Patent application CN 115537344 A reveals a process for improving the content and activity of antioxidant substances in pine pollen through fermentation with Rhizopus oryzae. The process includes the following steps: preparing a liquid culture medium of pine pollen, inoculation with R. oryzae, and fermenting to obtain a fermentation product. According to the method described in the invention, the content of polyphenols, the content of flavones, and the antioxidant activity of pine pollen are enhanced through fermentation.

[0008] The disadvantage of such processes, where only fermentation procedures are applied, is the exclusive biochemical action on the cell wall. In the case of bee bread, there is also a mechanical disaggregation process of the cell wall, due to the chewing of pollen grains by worker bees (Kieliszek et al., Trends in Food Science & Technology, 71 , 170-180).

[0009] Patent application CN 112956668 A describes a process involving the fine grinding of pollen, followed by suspension in distilled water at a material-to-liquid ratio of 1:10-1:30 and pH adjustment to 6 ±0.5, ultrasonic treatment at 800 W and a frequency of 80 kHz at 30 degrees Celsius for 15 minutes, and sequential enzymatic hydrolysis. Sequential enzymatic hydrolysis involves adding 5% pectinase and performing enzymatic hydrolysis at 45°C, pH 5, for 6 hours, adding 10% papain and conducting enzymatic hydrolysis at 55°C, pH 5, for 6 hours, and adding 8% cellulase and carrying out enzymatic hydrolysis at 55 degrees Celsius, pH 5, for 6 hours.

[0010] Patent application DE102010022994 A1 claims a process that involves homogenizing pollen in a piston homogenizer at 100 MPa, followed by fermentation with Streptococcus. The fermented liquid is either used to produce beverages or is sterile filtered and dried by lyophilization. The disadvantage of these processes is that they apply these steps of mechanical disaggregation induced by shear forces in liquids before biochemical (enzymatic, microbiological) processes, and not during or after them. Pollen fermentation not only increases the bioavailability of bioactive ingredients from pollen but also leads to the formation of postbiotic compounds that enhance the biological value of the resulting products (U|oiu et al 2018, Nutrients, 10(10), 1365). Interleaving steps of mechanical disaggregation and fermentation enhances the formation of post-biotic compounds, which potentiate the bioactive compounds in bee-collected pollen, increasing their efficacy.

[0011] The technical problem solved by the invention is to achieve a combined process, through which the processes of fermentation and mechanical disaggregation by shear forces induced in liquids mutually enhance each other to increase the bioavailability and to enhance the efficacy of bioactive compounds from pollen grains, due to the breaking of cell walls and combining the action of bioactive components from pollen with those produced in fermentation.

[0012] The process according to the invention consists of the following steps: Semi-solid fermentation of bee-collected pollen, homogenized together with 0.5 grams of polyfloral honey to 9.5 grams of pollen, at room temperature, for 2 days;

[0013] Mixing 30 grams of semi-solid fermented pollen with 200 ml of distilled water and ultrasonication for 5 minutes at 750 W and a frequency of 30 kHz;

[0014] Adding to the 30 grams of semi-solid fermented pollen, homogenized in 200 ml of water, 8 grams of wheat bran and 4 grams of chaff containing at least 10A8 cfu / g lactobacilli, and fermenting for 3 days at 30 degrees;

[0015] Colloidal grinding of the fermented pollen, brans, and chaff, along with the fermentation medium for 1 minute, ultrasonication for 20 minutes at 750W and a frequency of 30 kHz, followed by cooling the mixture at 4°C for 2 hours and drying by lyophilization.

[0016] The process presents the following advantages: It releases significant quantities of bioactive compounds, polyphenols, and soluble silicon from pollen grains, due to the combined action of fermentation and mechanical disaggregation processes.

[0017] It combines the fermentative action of fructophilic lactic bacteria from pollen and lactic bacteria from traditional fermented wheat bran beverage (bors) to release bioactive compounds from the pollen grains. It releases additional quantities of hydroxycinnamic acids and silicon from the bran, further potentiating the action of bioactive compounds from pollen. It forms para-probiotic compounds as a result of inactivation and breaking of the cell walls of lactic bacteria under the action of ultrasonication. It combines the action of bioactive compounds from pollen with the action of pre- and post-biotic compounds formed as a result of the development of specific lactic bacteria from bors. It maintains the bioactivity of thermolabile compounds and increases shelf life due to lyophilization drying.

[0018] The following are examples of embodiments that illustrate the invention without limiting it. Example 1 : 38 grams of polyfloral bee-collected pollen are homogenized with 2 grams of polyfloral honey. Pollenological analysis of the bee-collected pollen shows a significant major composition of 40% Helianthus annuus pollen, and a significant minor composition, of 5-10%, of Robinia pseudoacacia, Zea mays, Tilia spp., Fagus sylvatica. It is left at room temperature, in a sterilized 400 ml jar, for 2 days. 30 grams of semi-solid fermented pollen, on which the specific fructophilic lactic bacteria of pollen and honey have developed, are transferred into another glass jar, sterilized by autoclaving (10 min, 121°C), together with 200 ml distilled water. The sonotrode of an ultrasonication equipment (Sonics VCX-750 Vibracell Ultrasonic, Sonics, Newtown, CT, USA) is introduced, and the mixture is ultrasonicated for 10 minutes at 750 W and a frequency of 30 kHz. To the 30 grams of pollen homogenized in 200 ml of water, 8 grams of wheat bran and 4 grams of chaff are added. The wheat bran has a content of 68.8 g / 100 g dw carbohydrates, 2.3 g / 100 g dw proteins, 3.9 g / 100 g dw lipids, and 25.1 g / 100 g dw fibers. The chaff, coming from the sediment of a traditional fermented wheat bran beverage culture, contains at least 10A8 cfu / g lactobacilli. The jar is covered with sterile gauze and kept at 30°C for 3 days.

[0019] After 3 days, the fermented pollen, bran, and chaff, along with the fermentation medium, are passed through a colloid mill with stator-rotor (magic LAB®, IKA, Staufen, Germany) for 1 minute. The resulting suspension is collected in the 400 ml jar where fermentation was conducted and is ultrasonicated with ultrasonication equipment (Sonics VCX-750 Vibracell Ultrasonic, Sonics, Newtown) for 20 minutes at 750W and a frequency of 30 kHz. The resulting mixture is cooled in the refrigerator at4°C for 2 hours. It is then placed in the chamber of a lyophilizer (CoolSafe 4-15 L, LaboGene, Lilerod, Denmark) and dried. In the sample of fermented pollen, total polyphenols, total flavonoids, total hydroxycinnamic acids, antioxidant activity (FRAP), soluble silicon, antimicrobial activity, and cytocompatibility are determined. Analyses were performed comparatively on pollen maintained in water (30 grams in 200 ml water), traditional fermented wheat bran beverage (bors), and fermented pollen according to this Example 1.

[0020] The total polyphenol content (TPC) was determined using the Folin-Ciocalteu method (Dimitriu et al., 2022, Antioxidants 11 , 2194). To 10 pL of the sample / standard, 90 pL of bidistilled water was added. After adding 10 pL of Folin-Ciocalteu reagent, the plate was shaken for 5 minutes. Then, 100 pL of 7% Na2CO3 and 40 pL of bidistilled water were pipetted onto the solution. After incubating the plate at room temperature for 60 minutes, absorbance values were recorded at A =765 nm. The calibration curve was constructed starting from a stock solution of 500 pg / mL gallic acid in 70% ethanol, within the concentration range of 0-250 pg / mL (CLARIOstar BMG Labtech, Ortenberg, Germany). For the determination of the total flavonoid content (TFC), 25 pL of sample / standard was mixed with 25 pL of 10% sodium acetate, 30 pL of 2.5% AICI3, and 170 pL of distilled water. After 45 minutes of incubation at room temperature, absorbance was measured at A= 430 nm using a plate reader. The calibration curve was constructed starting from a stock solution of 500 pg / mL quercetin in 70% ethanol, in the concentration range of 0-100 pg / mL (Dimitriu et al., 2022, Antioxidants 11 , 2194).

[0021] The total hydroxycinnamic acids (HCA) content was determined by a method adapted from the European Pharmacopoeia 7.0 (EDQM, 2011). To 25 pL of sample / standard, 50 pL of 0.5 M HCI, 50 pL of a 1% sodium nitrite and 1% sodium molybdate solution, 50 pL of 8.5% NaOH, and 75 pL of bidistilled water were added. Samples were agitated and read spectrophotometrically at A= 524 nm using a plate reader. The calibration curve for the determination of hydroxycinnamic acids content was constructed starting from a stock solution of 1 mg / mL chlorogenic acid, in the concentration range of 0-300 pg / mL (Dimitriu et al., 2022, Antioxidants 11, 2194).

[0022] For the determination of the FRAP antioxidant activity, 3 solutions were prepared: sodium acetate buffer 300 mM, pH=3.6, 10 mM TPTZ in 40 mM HCI, and 20 mM FeCI3 in bidistilled water. The FRAP reagent was prepared by mixing 10 parts of the sodium acetate buffer solution 300 mM, pH=3.6 with one part of the 10 mM TPTZ solution and one part of the 20 mM FeCI3 solution (10:1:1). The FRAP reagent was kept in a water bath at a temperature of 37°C. To 15 pLof sample / standard, 285 pL of FRAP reagent was added, and the solution was incubated for 30 minutes at 37°C in the dark. After incubation, samples were centrifuged at 6000 ref, and absorbance was read at A= 593 nm using a plate reader. The calibration curve was constructed in the concentration range of 0-450 pM Trolox, starting from a stock solution of 10 rnM Trolox in ethanol (Thaipong et al., 2006, Journal of food composition and analysis, 19, 669-675).

[0023] Soluble silicon was determined spectrophotometrically by reading the absorbance at A=410 nm, according to the protocol from the Supelco Silicate Test kit (1.00857.0001 , MQuant®Supelco, Merck, Darmstadt, Germany) for silicon determination (silicic acid). For the determination of soluble silicon content in bors (traditional fermented wheat bran beverage), pollen, and bors with pollen, the MQuant® Colorimetric Test kit (Merck, Darmstadt, Germany) was used. Thus, 3 drops of Si-1 reagent were added to 20 mL of control sample / diluted sample. The mixture was shaken and incubated for 3 minutes at room temperature. Subsequently, 3 drops of Si-2 reagent and 10 drops of Si-3 reagent were added, and the samples were shaken and incubated for 2 minutes at room temperature. The concentration of soluble silicon was determined using the color gradation in the range of 0.01 to 0.25 mg / L Si.

[0024] The antimicrobial activity was determined against Staphylococcus aureus ATCC 25923, by the semi-quantitative diffusion method. Biocompatibility tests were conducted on gingival fibroblasts (HGF-1 , ATCC). The analysis of cell viability and proliferation was carried out 24 hours after treatment by combining the LIVE / DEAD assay, which uses calcein-AM (acetoxymethyl) and ethidium homodimer (EthD-1) to differentiate viable cells (green fluorescence) from dead cells (red fluorescence), with the CCK-8 assay, which quantifies the number of metabolically active viable cells. Calcein AM reacts with the intracellular esterases of viable cells and emits green fluorescence at A=494 / 517 nm. The ethidium homodimer penetrates the nucleus of cells with damaged plasma membrane, intercalates between the nucleic acid bases, and emits red fluorescence at A=528 / 617 nm. The CCK-8 assay is based on the reduction of a tetrazolium salt, WST-8, by cellular dehydrogenases, to a yellow-colored formazan, soluble in the culture medium, which is evaluated spectrophotometrically at A= 450 nm. The LIVE / DEAD assay contains 2 compounds that act simultaneously: calcein acetoxymethyl ester (AM) and ethidium homodimer (EthD-1).

[0025] Thus, for the microscopic examination of cells 24h after being in contact with the bioproducts, the culture was visualized using the CelenaX High Content Imaging System, with image acquisition done using the CelenaX Explorer software. The results are presented in Table 1.

[0026] Table 1. Characteristics of fermented pollen according to Example 1 compared with pollen maintained in water and bors.

[0027] SEE TABLE

[0028] The results demonstrate a significant increase in the biological activity of the fermented pollen according to Example 1 , compared with pollen maintained in water and bors. This increase is due to the solubilization of bioactive compounds in pollen and their enhancement through preand post-biotic compounds produced during fermentation. Notably, there's a significant increase of about 10% in the number of metabolically active viable cells in the sample with fermented pollen.

[0029] Example 2. The procedure is the same as in Example 1 , with the only difference being the use of bee pollen, which palynological analysis shows a significant major composition of 40% Tillia spp. pollen and a significant minor composition of 5-10% of Allium spp., Helianthus annuus, Asteraceae spp. The test results are similar to those obtained in the sample from Example 1 .

[0030] Example 3. The procedure is as in Example 1 , with the only difference being the use of bee pollen, which palynological analysis shows a significant major composition of 40% Robinia pseudoacacia pollen, and a significant minor composition of 5-10%, of Cucumber spp., Castaneae spp., Rosaceae spp. The test results are similar to those obtained in the sample from Example 1 .

[0031] Example 4. The procedure is as in Example 1 , with the only difference being the use of bee pollen, which palynological analysis shows a significant major composition of 40% Zea mays pollen, and a significant minor composition of 5-10%, of Trifolium spp., Quercus spp., Prunus spp. The test results are similar to those obtained in the sample from Example 1 .

[0032] Example 5. The procedure is as in Example 1 , with the only difference being the use of bee pollen, which palynological analysis shows a significant major composition of 40% Robinia pseudoacacia pollen, and a significant minor composition of 5-10%, of Salicaceae spp., Sophora spp., Poaceae spp. The test results are similar to those obtained in the sample from Example 1 .

Claims

CLAIMSThe process according to the invention is characterized by comprising the following steps: semisolid fermentation of bee-collected pollen, homogenized together with 0.5 grams of polyfloral honey to 9.5 grams of pollen, at room temperature, for 2 days; mixing 30 grams of semi-solid fermented pollen with 200 ml of distilled water and ultrasonication for 5 minutes at 750 W and a frequency of 30 kHz; adding to the 30 grams of semi-solid fermented pollen, homogenized in 200 ml of water, 8 grams of wheat bran and 4 grams of chaff containing at least 10A8 cfu / g lactobacilli, and fermenting for 3 days at 30 degrees; colloidal grinding of the fermented pollen, bran, and chaff, together with the fermentation medium for 1 minute, ultrasonication for 20 minutes at 750W and a frequency of 30 kHz, followed by cooling the mixture at 4°C for 2 hours and drying by lyophilization.

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