Obtaining processes for some metabiotic ingredients with bioactive properties based on bovine colostrum and potentially probiotic lactic acid bacteria belonging to lactiplantibacillus spp

Abstract

In conclusion, considering the biochemical characteristics of the metabiotic ingredients obtained, it is considered that the process 1 offers the beneficial fermentative conditions to obtain a fermented product, that preserved by freeze-drying it is constituted in a multifunctional metabiotic ingredient, with enhanced bioactive properties.

Description

[0001] Obtaining processes for some metabiotic ingredients with bioactive properties based on bovine colostrum and potentially probiotic lactic acid bacteria belonging to Lactiplantibacilhis spp.

[0002] Summary:

[0003] The invention refers to the elaboration of two obtaining processes of some metabiotic ingredients with biologically active properties, by combining the paraprobiotics obtained by inactivation with combined treatments (ohmic heating and thermal treatment) of the probiotic cells of the strain Lactiplantibacilhis plantarum MIUG BL21, respectively the postbiotics obtained by fermentation of a culture medium based on 8% bovine colostrum, with the selected probiotic culture of Lactiplantibacilhis paraplantarum MIUG BL74, with multiple applications in food industry. The difference between the two proposed processes is made by the paraprobiotics supplementation into the substrate before the fermentation with the selected potentially probiotic starter culture, respectively by the supplementation of paraprobiotics into the fermented medium after the fermentation process.

[0004] Metabiotics are obtained by combining the well-known biotics (probiotics and prebiotics) in which paraprobiotics are added, known also as "ghost probiotics”, non-viable microbial cells that offer a benefit for the host when they are administrated in adequate amounts, and postbiotics which are probiotics’ metabolites or cellular components biosynthesized during the fermentation process, respectively.

[0005] Claims: 2

[0006] Figures: 2

[0007] Description of the invention

[0008] The invention refers to the development of two processes to obtain some metabiotic ingredients based on probiotics, prebiotics, postbiotics, and paraprobiotics with high functionality.

[0009] The term paraprobiotic was defined by the International Scientific Association for Probiotics and Prebiotics as compounds resulted by the inactivation of probiotics and / or their components that offer a benefit to consumer ’s health. The probiotics are inactivated by different treatments and they are found in inert form, therefore not being able to colonize the gastrointestinal tract. In the scientific literature from the field there were described some beneficial health effects such as: immunomodulatory effect (Kanauchi et al., 2018; Salas-Lais et al., 2020), diarrhea and colitis symptoms’ improvement (Ueno et al., 2011), antiallergic effect (Nakamura & Mitsunaga, 2018), and improvement of the symptoms related to stress and sleep quality (Nishida et al., 2017).

[0010] Postbiotics are classified by their structure in: peptides, teichoic acid and plasmalogens, carbohydrates, proteins (p40, p75 molecules, and lactocepin), vitamins (B-group), organic acids including short-chain fatty acids (formic, acetic, propionic, butyric etc) and different complex molecules (such as lipoteichoic acids, muropeptides derived from peptidoglycans) (Rafique et al., 2022). Postbiotics can be obtained by fermentation of some formulated culture media. Depending on the fermentation medium’s composition, the probiotics are capable of producing some metabolites with functional role, for which there are scientific proofs referring to the improvement of intestinal microbiome's activity with positive impact on the health estate. It should be mentioned that the human gastrointestinal tract has a significant microbial diversity, considering its biodiversity, as well as its quantitative properties.

[0011] In the scientific literature there was identified a series of patents which valorizes the nutritional and functional potential of the metabiotics, as following:

[0012] WO2015181534A1 WIPO (PCT). Anti-bacterial lysate of probiotic bacteria. The invention refers to probiotic bacteria, specifically but not exclusively, to antibacterial compositions derived from probiotic bacteria. In this patent there are developed different fermented products based on the secondary metabolites synthesized by the probiotic bacteria, and uses thereof. There are taken into consideration especially products containing metabolites synthesized by a strain of Lactobacillus rhamnosus and uses thereof.

[0013] WO2019149940A1 WIPO (PCT), Use of a postbiotic-based composition for the treatment of skin diseases. The invention refers to a product containing a fraction of supernatant resulted after the fermentation of a specific medium with lactic acid bacteria strains, species Lactobacillus casei or Lactobacillus paracasei. that contain in genome at least one DNA sequence identical with one of the sequences of the group SEQ ID No 1-5, involved in the treatment and / or prevention of skin and / or dermatological diseases determined by inflammations, allergic reactions, contact hypersensitivity and intolerance reactions, excessive sebum secretion, exfoliation or dysbiosis.

[0014] WO2018024833A1 WIPO (PCT), Postbiotic-based composition for treatment of ocular inflammation. The invention refers to a obtaining method and characterization of a fermented product obtained by fermentation with the lactic acid bacteria strains of Lactobacillus casei or Lactobacillus paracasei, to be used in the treatment of orbital inflammatory syndrome.

[0015] The technical problem solved by the invention refers to the processes particularization and control of the composition for the fermented products according to the invention, by using two selected lactic acid bacteria strains with demonstrated probiotic potential, Lactiplantibacillus paraplantarum MIUG BL74 and Lactiplantibacillus plantarum MIUG BL21, stored by freezing (40% glycerol at -80°C), belonging to the Collection of Microorganisms of "Dunarea de Jos” University of Galati (acronym MIUG). The probiotic potential of the above-mentioned strains was demonstrated by preliminary studies (Cotarlet et al., 2023). The strain Lactiplantibacillus plantarum MIUG BL21 was used to obtain paraprobiotics by a combination of treatments, specific and adapted, applied to the cells' suspension, as following: ohmic heating (20V7cm, 15 min) and thermal treatment (75°C for 15 min). In the first process, the paraprobiotics (10%, v / v) were added at the beginning of the fermentation process ensured by the probiotic starter culture of Lactiplantibacillus paraplantarum MIUG BL74, to ferment a culture medium based on 8% bovine colostrum to produce postbiotics. In the second process, the paraprobiotics were added (10%, v / v) after the fermentation of the culture medium based on 8% bovine colostrum with the probiotic starter strain of Lactiplantibacillus paraplantarum MIUG BL74. The parameters of the fermentation process were similar for both proposed processes. The resulting metabiotic products have a complex composition, combining in unique formulas probiotics, prebiotics, paraprobiotics, and postbiotics with multifunctional properties.

[0016] Parameters of the invention

[0017] 1. The obtainment of paraprobiotics

[0018] The first step of the invention consisted in the paraprobiotics obtainment by combined treatments for inactivation. Therefore, the stock culture of Lactiplantibacilliis plantarum MIUG BL21 was reactivated on De Man. Rogosa and Sharpe agar (MRS) medium, at 37°C for 48 hours. From the reactivated culture, the biomass of a single colony was transferred in MRS broth medium, being further incubated at 37°C for 48 hours in aerobiosis, stationary conditions. The biomass was washed three times with phosphate buffer saline (PBS), pH 7.2, and the suspension was centrifuged at 7,000 rpm, 10 min, 4°C. The biomass was then mixed with PBS, and in the resulting suspension it was dimensioned the cells number (by assessing the optical density at the wavelength of 600 nm, OD600, at a value of 2.0), afterwards being subjected to a combined treatment for cells inactivation. First, the suspension was subjected to ohmic heating treatment following the parameters: 20V / cm, 10 kW, 15 min, followed by thermal treatment at 75°C for 15 min until the complete inactivation of the cells. The viability was evaluated by inoculation on MRS agar medium and incubation at 37°C for 48 hours. A total inactivation degree was considered when the colonies were absent on the culture medium. The paraprobiotics suspension was used, in a concentration of 10% (v / v), for the designing of the two metabiotics obtaining processes.

[0019] 2. The obtainment of postbiotics

[0020] The stock culture of Lactiplantibacilliis paraplantarum MIUG BL74 was reactivated by cultivation on MRS agar medium, at 37°C for 48 hours. From the reactivated culture, the biomass from a single culture was then inoculated in sterile MRS broth medium, the cultivation being carried out at 37°C for 48 hours, in aerobiosis, stationary conditions. The obtained culture was dimensioned regarding the cells number (by assessing the optical density at the wavelength of 600 nm, OD600, at a value of 2.0), being afterwards used as starter for the inoculation of the fermentation medium formulated to obtain the metabiotics.

[0021] 3. The obtainment of metabiotics

[0022] In the obtaining process of the metabiotic ingredients, there were developed two processes:

[0023] Process 1 (Figure 1): It was formulated the fermentation medium, based on 8% bovine colostrum, dissolved in distilled water and sterilized at 105°C for 15 min. The sterile medium was inoculated with 5% (v / v) culture of Lactiplantibacilliis paraplantarum MIUG BL74 (OD6002.0) and 10% (v / v) inactivated suspension of Lactiplantibacillus paraplantarum MIUG BL21. The fermentation was realized at 37°C for 72 hours, in aerobic and stationary conditions. The metabiotic ingredient (MI) (the fermented product) was subjected to freeze-drying at -42°C and 0.1 mbar (product coded MIi). The resulted powders were collected in airtight dark- glass containers stored at 4°C until further analysis. Process 2 (Figure 2): It was formulated the fermentation medium, based on 8% bovine colostrum, dissolved in distilled water and sterilized at 105°C for 15 min. The sterile medium was inoculated with 5% (v / v) culture of Lactiplantibacillus paraplantarum MIUG BL74 (OD6002.0) and the fermentation was realized at 37°C for 72 hours, in aerobic and stationary conditions. At the end of the fermentation step, in the fermented product was added 10% (v / v) inactivated suspension of Lactiplantibacillus paraplantarum MIUG BL21, followed by homogenization for a uniform distribution of paraprobiotics in the fermented product. The metabiotic ingredient was then subjected to freeze-drying at -42°C and 0.1 mbar (product coded MI2). The resulted powders were collected in airtight dark-glass containers stored at 4°C until further analysis.

[0024] 4. The bioactive characteristics of the metabiotic ingredients

[0025] 4.1. Phenolic compounds and organic acids content

[0026] The phenolic and organic acids content was determined by the HPLC method, described by Vlad et al., 2022 for the phenolic compounds (expressed as μg / g DW) and by Santana et al., 2022 for the organic acids (expressed as mg / g DW). The results demonstrated a varied spectrum of phenolic compounds, respectively phenolic acids such as gallic, protocatechuic, vanillic, and ellagic acid, procyanidins and catechin, the last one being the most abundant compound identified. Thus, the metabiotic ingredients presented a total content of phenolics of 8,032 μg / g DW in MI1, and a significantly lower content in Mb of 189 μg / g DW. In the first experimental variant ( MI1), catechin was the major compound (7,693.61±29.72 μg / g DW), whereas in the variant MI2, kaempferol was the major compound (62.06±2.08 μg / g DW). On the other hand, the obtaining process of the metabiotic ingredients determined a different composition regarding the organic acids identified in the analyzed samples, such differences being observed especially for citric acid, determined only in MI2. In MI1the eluted organic acids from the freeze-dried fermented products, such as isovaleric, butyric, and hexanoic acid, with significant impact on the functionality of the formulated ingredients. The two ingredients presented a significantly different content of organic acids, with a concentration of 10.90 mg / g DW in MI1, and 72.49 mg / g DW in MI2. It should be mentioned that the citric acid was quantified only in Mb., whereas the hexanoic acid had the highest concentration in Mb (46.38±0.35 mg / g DW).

[0027] 4.2. Antioxidant activity

[0028] For the antioxidant activity assessment, the ABTS antiradical method was used, described by Robles-Ramirez et al., 2020. The ABTS radical scavenging activity was determined at 56% for the experimental variant MI1and 43.4% for the sample MI2. These significant differences are determined by the high content of phenolic compounds in Mb .

[0029] 4.3. Cells viability

[0030] The viability of the probiotic culture from the fermented samples was determined in aerobic and anaerobic conditions using the indirect counting method (Koch’s method) (Yang et al., 2021 ; Alamanda et al., 2021). Results were expressed as logarithm of colony forming units per gram dry weight analyzed sample (log CFU / g DW) and cells’ surviving rate after freeze-drying using the formula: where: log ;V- log CFU / g DW analyzed sample after freeze-dry ing; log No - log CFU / g DW analyzed sample before freeze-drying.

[0031] Cellular viability for the probiotic culture of Lactiplantibacillus paraplantarum MIUG BL 74 from the fermented products based on bovine colostrum was determined in aerobic and anaerobic conditions (Table 1).

[0032] Table 1. Lactiplantibacillus paraplantarum MIUG BL 74 cells viability from fermented samples based on bovine colostrum supplemented with paraprobiotics

[0033] The surviving rates for the probiotic cells from the fermented products are presented in Table 2.

[0034] Table 2. Surviving rate of the bacterial cells from the fermented products after freeze-drying

[0035] Compared to the initial number of probiotic cells, the sample ML, fermented sample based on bovine colostrum supplemented with paraprobiotic before fermentation, had the highest viability after freeze-drying, respectively 87.42±0.10% by cultivation in aerobic conditions, whereas by cultivation in anaerobic conditions a viability of 84.71±0.05% was calculated.

[0036] 4.4. Antimicrobial activity

[0037] The assessment of the antimicrobial activity for the metabiotic ingredients was carried out by agar diffusion method (Mihalcea et al., 2021). The microbial pathogenic strains used to test the antimicrobial activity were: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Listeria monocytogenes Scott A (indicator microorganisms). The results related to the antimicrobial effect of the two metabiotic ingredients, determined against the above- mentioned three bacterial strains with pathogenic potential, respectively two Gram-positive strains (Listeria monocytogenes and Staphylococcus aureus) and one Gram-negative strain (Escherichia coli), are presented in Table 3.

[0038] The results presented in Table 3 represent the diameters for the inhibition zones formed by the diffusion of the antimicrobial compounds from the metabiotic ingredients (freeze-dried powders) in the culture medium inoculated with the tested potentially pathogenic bacteria. As such, it was observed that the antimicrobial effect against all the pathogenic strains tested was obtained for the metabiotic ingredients made by applying the proposed process 1, respectively Conclusions

[0039] In conclusion, considering the biochemical characteristics of the metabiotic ingredients obtained, it is considered that the process 1 offers the beneficial fermentative conditions to obtain a fermented product, that preserved by freeze-drying it is constituted in a multifunctional metabiotic ingredient, with enhanced bioactive properties.

[0040] the supplementation of the fermentation medium based on 8% bovine colostrum, when the probiotic starter culture is inoculated (before fermentation).

[0041] Table 3. The antimicrobial potential of the metabiotic ingredients

[0042] Moreover, the ingredient obtained by applying process 2 wasn't characterized by inhibitory effect against the analyzed pathogenic bacteria. Thus, it can be appreciated that the metabiotic ingredients obtained by fermentation of the medium based on 8% bovine colostrum with the selected probiotic strain Lactiplantibacillus paraplantarum MIUG BL74, by supplementation, before fermentation, with 10% (vw) paraprobiotics originated from the probiotic culture of Lactiplantibacillus plantarum MIUG BL 21, contain antimicrobial compounds with demonstrated activity against bacteria with pathogenic potential Escherichia coli. Staphylococcus aureus, and Listeria monocytogenes. The metabolites with antimicrobial role can be compounds with low molecular weight (carbon dioxide, diacetylene, hydrogen peroxide, organic acids), and with higher molecular weight (bacteriocins, bioactive peptides) (de Almeida Junior et al., 2015).

[0043] 4.5. a-amylase inhibitory activity of the metabiotic ingredients

[0044] The freeze-dried fermented products (in a concentration of 0.1 g%) were solubilized in distilled water, and incubated overnight at room temperature, 250 rpm, and then centrifuged at 4°C, 8,000 rpm, for 10 min. The resulted supernatants were analyzed for their potential to inhibit a- amylase, according to the method described by Costamagna et al., 2016. The extracts' inhibitory effect was expressed as percent, suggesting a superior inhibition degree of the experimental variant MIi, of approximately 25%, compared to 12.4% calculated for Mb. This aspect is correlated with a superior content of phenolic compounds for the experimental variant MIi.

[0045] 4.6. Bioactive peptides content

[0046] To determine the bioactive peptides content of the metabiotic ingredients, an extraction in distilled water was performed, followed by a fractionation using membrane ultrafiltration units (AmiconUltracel YM-10, Millipore, Germany), with molecular weight of 30 kDa (Cotarlet et al., 2020). The obtained supernatants were tested for antioxidant activity (ABTS), a-amylase inhibitory activity, and antipathogenic potential. For the antioxidant activity, it can be estimated a higher content of peptides in MI1, which allowed an antiradical activity of 53%, compared to only 44% for Mh. The bioactive peptides showed, as well, a-amylase inhibitory potential, with a percent of 24% for MI1, and 14.5% for MI2. The peptides with 30 kDa molecular weight extracted from the metabiotic ingredients did not show antibacterial activity against the tested pathogenic bacteria. References:

[0047] Cotarlet. M.. Pihurov. M.. Pacularu-Burada. B.. Vasile. A.M.. Bahrim. G.E.. Grigore-Gurgu. I . (2023). Selection of new lactobacilli strains with potentially probiotic properties. The Annals of the University Dunarea de Jos of Galati Fascicle VI Food Technology. 47 ( 1 ), in press.

[0048] Cotarlej. M., Vasile, A.M., Gaspar-Pintiliescu, A., Oancea, A., Bahrim, G.E. (2020). Tribiotication strategy for the functionalization of bovine colostrum through the biochemical activities of artisanal and selected starter cultures. CyTA-Journal of Food. 18. 274-280.

[0049] Costamagna. M.S.. Zampini, I.C.. Alberto. M.R., Cuello. S., Torres. S., Perez. J.. Quispe. C.. Schmeda-Hirschmann. G., Isla, M.L (2016). Polyphenols rich fraction from Gepffroea decorticans fruits flour affects key enzymes involved in metabolic syndrome, oxidative stress and inflammatory process. Food Chemistry, 190. 392-402. de Almeida J.W. L. G., I. D. S. Ferrari, J. V. de Souza, C. D. A. da Silva, M. M. da Costa and F. S. Dias. (2015). Characterization and evaluation of lactic acid bacteria isolated from goat milk. Food Control, 53, 96-103.

[0050] Kanauchi. O., Andoh, A., AbuBakar, S., & Yamamoto, N. (2018). Probiotics and paraprobiotics in viral infection: Clinical application and effects on the innate and acquired immune systems. Current Pharmaceutical Design, 24(6), 710-717.

[0051] Mehmood, N.. Hussain, T., Hameed, A., Yaseen, A., Ahmed, A., Hussain. M., Al Jbawi, E. (2023). Prospective effect of vitamin E and selenium supplementation on colostrum quality in Beetal goats. Cogent Food & Agriculture, 9( 1 ), 2197711.

[0052] Mihalcea, L., Turturica, M., Cucolea, E.L, Danila, G.-M., Dumitrascu, L., Coman, G., Constantin, O.E., Grigore-Gurgu, L.. Stanciuc, N. (2021). CO? Supercritical Fluid Extraction of Oleoresins from Sea Buckthorn Pomace: Evidence of Advanced Bioactive Profile and Selected Functionality. Antioxidants, 10, 1681.

[0053] Nakamura, S., & Mitsunaga, F. (2018). Anti-allergic effect of para-probiotics from non-viable acetic acid bacteria in ovalbumin-sensitized mice. Food and Nutrition Sciences, 09(12), 1376- 1385.

[0054] Nishida, S. M. K.., Sawada, D., Kawai, T., Kuwano, Y., Fujiwara, S., & Rokutan, K. (2017). Para- psychobiotic Lactobacillus gasseri CP2305 ameliorates stress-related symptoms and sleep quality. Journal of Applied Microbiology, 123, 1561-1570.

[0055] Raftque, N„ Jan, S.Y., Dar, A.H., Dash, K.K., Sarkar, A., Shams, R., Pandey, V.K., Khan, S.A., Amin, Q.A., Hussain, S.Z. (2023). Promising bioactivities of postbiotics: A comprehensive review. Journal of Agriculture and Food Research, doi: https: doi.org 10.1016, j.jafr.2023.100708. Salas-Lais. A. G., Robles-Contreras. A., Balderas-Lopez, J. A., & Bautista-de Lucio, V. M. (2020). Immunobiotic and paraprohiotic potential effect of Lactobacillus casei in a systemic toxoplasmosis murine model. Microorganisms, 8. 1 13.

[0056] Santana. A., J. K., Chagas Barros. R. G., Pereira. U. C.. Nogueira, J. P., Gualberto, N. C., Santos de Oliveira. C., Shanmugam, S., Narain. N. (2022). Bioaccessibility of bioactive compounds after in vitro gastrointestinal digestion and probiotics fermentation of Brazilian fruits residues with antioxidant and antidiabetic potential. LILT Food Science and Technology, 153, 112469.

[0057] Ueno, N.. Fujiya. XL. Segawa. S.. Nata. T.. Moriichi. K... Tanabe. H Kohgo, Y. (2011 ). Heat- killed body of Lactobacillus brevis SBC8803 ameliorates intestinal injury in a murine model of colitis by enhancing the intestinal barrier function. Inflammatory Bowel Diseases, 17(11 ). 2235- 2250.

[0058] Vlad, C.C., Pacularu-Burada, B., Vasile, A.M., Milea, S.A., Bahrim, G.E., Rapeanu, G., Stanciuc, N. (2022). Upgrading the Functional Potential of Apple Pomace in Value-Added Ingredients with Probiotics. Antioxidants. 11(10).

Claims

Claims1. A process to obtain metabiotic ingredients in freeze-dried estate (Process 1). comprising paraprobiotics obtained by inactivation of the cells from Lactiplantibacillus plantarum MIUG BL21. and postbiotics obtained by fermentation of a medium based on 8% bovine colostrum with Lactiplantibacillus paraplantarum MIUG BL74. with improved bioactive properties.

2. A process to obtain metabiotic ingredients in freeze-dried estate (Process 2), comprising paraprobiotics obtained by inactivation of the cells from Lactiplantibacillus plantarum MIUG BL21, added after the fermentation of a medium based on 8% bovine colostrum with Lactiplantibacillus paraplantarum MIUG BL74.

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