FIELD OF THE INVENTION

The present invention is related to the field of production of purified saponins and saponin extracts from plants, promoting or preventing the formation of sediments of less soluble saponins during the preparation of aqueous extracts. More particularly, the present invention is in the field of preparation of extracts of Yucca schidigera Roezl. containing monodesmosidic and bidesmosidc steroidal saponins. Upon operational adjustment, the disclosed invention allows either the isolation and purification of monodesmosidic saponins for their use as vaccine adjuvants, or the stabilization of the bidesmosidic saponins extracted during the processing of the biomass of yucca, rendering stable aqueous extracts.

DESCRIPTION OF RELATED ART

Saponins comprise a large and diverse family of high molecular weight glycosides, widely distributed in the plant kingdom (Faizal, A., & Geelen, D. 2013. Saponins and their role in biological processes in plants. Phytochemistry Reviews, 12, 877-893; Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press; Sparg, S. G., Light, M. E., & Staden, J. Van. 2004. Biological activities and distribution of plant saponins. Journal of Ethnopharmacology, 94, 219-243; Vincken, J. P., Heng, L., de Groot, A., & Gruppen, H. 2007. Saponins, classification and occurrence in the plant kingdom. Phytochemistry, 68, 275-297). They have a remarkable structural diversity which derives from their sapogenin structures and the sugars attached to them. Saponins are classified regarding the structure of the hydrophobic core known as aglycone or sapogenin, as triterpene or steroid saponins. Regarding the number of hydrophilic carbohydrate chains bonded to the hydrophobic aglycone, saponins containing one, two or three carbohydrate chains bonded to the aglycone are classified as mono-, bi- or tri-desmosides, respectively. Depending on the source of saponins, the sapogenins may contain different functional groups (e.g. methyl, hydroxyl, aldehyde, carboxylic acid, etc.) and spatial configuration. Besides, the composition, conformation, linkage, substitution, and branching patterns of the sugars attached increase enormously the diversity of the saponins (Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press). Although in different grades, most saponins share the property of forming colloidal solutions and lower the surface tension in aqueous media, which distinguishes them from other glycosides. Nonetheless, their structural diversity is reflected by the myriad of biological activities and known uses for this family of compounds (Guglu-Ustundag, 0., & Mazza, G. 2007. Saponins: Properties, applications and processing. Critical Reviews in Food Science and Nutrition, 47, 231-258; Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press), indicating the importance of their study and their specific analysis in commercial products.

Saponins are normally very difficult to separate due to their occurrence in highly complex mixtures of components with subtle structural differences among them, and their polar and amphiphilic properties (Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press). In general, their isolation and purification require substantial efforts by sequential chromatographic steps under normal and/or reversed phase conditions and/or ultra I nano filtration, depending on the target homogeneity, and degree of purity. These processes are time consuming and expensive, and in the case of chromatographic techniques -with or without support- they require the use of organic solvents which might be toxic and harmful to the environment. Therefore, there is need for greener alternatives for saponin isolation in the industry.

Yucca schidigera Roezl. ex Ortgies (yucca) is a plant belonging to the Agavaceae family, native to the southwestern United States and northern Mexico. Yucca extracts are one of the major industrial sources of steroid saponins (Figure 1, Tables 1 and 2), approved in the USA by the Association of American Feed Control Officials (AAFCO) and the Food and Drug Administration (FDA), for their use as additives for the manufacture of beverages, cosmetics, animal food and poultry (Cheeke, P. 2000. Actual and potential applications of Yucca schidigera and Quillaja saponaria saponins in human and animal nutrition. In W. Oleszek & A. Marston (Eds.), Saponins in Food, Feedstuffs and Medicinal Plants, pp. 241-254. Springer Netherlands; Tamura, Y., Miyakoshi, M., & Yamamoto, M. 2012. Application of saponin-containing plants in foods and cosmetics in alternative medicine. In H. Sakagami (Ed), Alternative Medicine, pp. 85-102. InTech. Croatia). Their saponins - particularly monodesmosidic saponins - also have potent antifungal activity against phytopathogenic fungi and yeast strains recognized as food contaminants (Chapagain, B. P., Wiesman, Z., & Tsror, L. 2007. In vitro study of the antifungal activity of saponin-rich extracts against prevalent phytopathogenic fungi. Industrial Crops and Products, 26, 109-1 15; Miyakoshi, M., Tamura, Y., Masuda, H., Mizutani, K., Tanaka, O., & Ikeda, T. 2000. Antiyeast steroidal saponins from Yucca schidigera (Mohave Yucca), a new anti-food-deteriorating agent. Journal of Natural Products, 25, 332-338; Wulff, E. G., Zida, E., Torp, J., & Lund, O. S. (2012). Yucca schidigera extract: A potential biofungicide against seedborne pathogens of sorghum. Plant Pathology, 61 , 331 -338).

The commercial yucca products can be divided in three main groups: 1 ) The juice or aqueous extract produced by cold pressing the stem and concentrated by evaporation to approximately 50% w/w solids; 2) the juice absorbed onto inert carriers (clays, etc.); and 3) the dried pulverized yucca trunks comprised of pure botanical material.

Yucca derived products are widely used as an animal feed additive for the control of ammonia and other odorous compounds in the gut and feces (Wallace, R. J., Arthaud, L., & Newbold, C. J. 1994. Influence of Yucca schidigera extract on ruminal ammonia concentrations and ruminal microorganisms. Applied and Environmental Microbiology, 60, 1762-1767). Additionally, the aqueous extract -juice- of yucca is a preferred natural foaming agent for beverages for human consumption and for cosmetics and personal care products (Cheeke, P. 2000. Actual and potential applications of Yucca schidigera and Quillaja saponaria saponins in human and animal nutrition. In W. Oleszek & A. Marston (Eds.), Saponins in Food, Feedstuffs and Medicinal Plants, pp. 241-254. Springer Netherlands}. A further additional application is its use as a food additive due to its antimicrobial activity (Miyakoshi, M., Tamura, Y., Masuda, H., Mizutani, K., Tanaka, O., & Ikeda, T. 2000. Antiyeast steroidal saponins from Yucca schidigera (Mohave Yucca), a new anti-food-deteriorating agent. Journal of Natural Products, 25, 332-338; Suzuki, R., Ohno, H., Murakami, T., Shirataki, Y., 2020. Improving quality control of yucca extracts used as food additives by screening antimicrobial activity using NMR metabolomics. Journal of Natural Medicine. 74, 306-310). Up to now, there are no reports of yucca saponins used as vaccine adjuvants.

The conventional processing of yucca biomass (Figure 2), by the extrusion of the plant botanical material renders an unstable fresh yucca juice, which become unstable short after the cold pressing of the biomass, forming an abundant and insoluble material at the bottom of the receiving tank. In addition to the afore mentioned instability, and also due to the natural variability of the active compounds in plants, some properties of the yucca extracts, such as profiles of saponins and antimicrobial activity become variable across production lots (Sastre, F., Ferreira, F., & Pedreschi, F. 2017. A systematic approach for the chromatographic fractionation and purification of major steroid saponins in commercial extracts of Yucca schidigera Roezl. Journal of Chromatography B, 1046, 235-242; Suzuki, R., Ohno, H., Murakami, T., Shirataki, Y., 2020. Improving quality control of yucca extracts used as food additives by screening antimicrobial activity using NMR metabolomics. Journal of Natural Medicine. 74, 306-310).

Current methods to prevent the afore mentioned defects include the heating of the resultant suspension to 80 - 90 °C (below the boiling point), filtering and recovery of the filtered juice in a second tank. However, the formation of precipitates in the filtered juice is still observed after the above treatment. Prior art analysis regarding process and/or methods for saponins extraction from yucca are showed in US Patent Application 2715122A which discloses a method of extraction of yucca saponins suitable for production at industrial scale. Nevertheless, the evidence described in US2715122A includes a process for saponin extraction using benzene as organic solvent, which is a known human carcinogen, and therefore, a forbidden supply for the production of extracts intended as raw materials for the formulation of vaccine adjuvants in the pharmaceutical industry. In that regard, the regulatory framework of agencies such as FDA or EMA is very tight: The FDA issued an alert in June 2022 to drug manufacturers to avoid the use of benzene in their manufacture process; when the use of benzene become unavoidable to produce a drug product with a significant therapeutic advance, then its levels should be restricted to 2 parts per million (ppm), unless otherwise justified (Food and Drug Administration (2022). “FDA alerts drug manufacturers to the risk of benzene contamination in certain drugs”, https://www.fda.gov/drugs/pharmaceutical-quality- resources/fda-alerts-drug-manufacturers-risk-benzene-contami nation-certain-drugs).

Chinese patent application CN108837775 A discloses a method to obtain steroid saponins from yucca which includes several steps of filtration, concentration and resin cleaning; this document discloses a process which combine several unitary operations in line with processes that are described in the state of art so far. Additionally, using this process it is not possible to obtain different kind of stable products enriched in mono and/or bidesmosidic yucca saponins, according to the client’s and/or researcher’s necessities.

This analysis of prior art shows that despite the extraction processes of yucca saponins have been described, these are composed by several steps, use organic solvents, or does not offer the possibility to obtain stable products enriched in mono and/or bidesmodidic yucca saponins, respectively, which are oriented to different applications according to their composition, such as in the pharmaceutical industry.

SUMMARY OF THE INVENTION

The present invention is related to the field of production of highly purified saponins and stable extracts containing saponins from plants, either to promote, or to prevent the formation of sediments of less soluble saponins during the preparation of aqueous extracts containing steroidal saponins, for their use as immunological adjuvants.

As mentioned above, this technology can be used either to promote or prevent the sediment formation according to the following: a) Promoting the sediment formation, through a controlled conversion of the filtered aqueous extract I juice precursor, allows the purification of a highly purified fraction of monodesmosic saponins having low water solubility, which might be used as ingredients of vaccine adjuvants. The above purification can be done heating up the fresh yucca extract below the boiling point, ideally to a maximum of 88 °C to promote a controlled conversion of the yucca juice precursor, and the corresponding precipitation of monodesmosidic saponins, followed of the removal of water-soluble components, and one or more cycles -preferably 3 cycles- of washing with water the precipitated components, and centrifugation to obtain the final product. The technology disclosed in this application allows the purification of monodesmosidic saponins without using organic solvents, chromatographic techniques, ultra I nano I nor reverse osmosis membranes (Figure 9). b) Preventing the sediment formation: To overcome the above-mentioned difficulties during the preparation of Yucca schidigera Roezl. (yucca) aqueous extracts by boiling the fresh yucca juice coming from the cold press step (Figure 12) during 0.5 - 3.0 h before further processing. In an alternate embodiment, this objective is accomplished by a process which comprises the step of treating the fresh yucca juice coming from the cold press step (Figure 13) with bentonite before further processing. The goal of this object is to preserve the relative amount of bi- and monodesmosidic saponins contained in the extracts, which could also open an opportunity for their functionalization, and hence for the development of new products with different physicochemical and/or biological properties. The technology disclosed in this document also allows the large-scale production of stable yucca extracts containing mono and bidesmosidic saponins. BRIEF DESCRIPTION OF THE DRAWINGS.

Figure 1. Chemical structure of mono- and bidesmosidic saponins previously reported in yucca, and their assignment in the HPLC-HRMS chromatograms.

Figure 2. Flow diagram of the currently employed process for the production of yucca extracts (Prior Art).

Figure 3. HPLC-ELSD chromatogram of the methanolic extract of a fresh botanical sample of yucca.

Figure 4. HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after incubation at 35 °C during 44 h.

Figure 5. HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after boiling during 0.5 h, followed by incubation at 35 °C during 44 h.

Figure 6. Shows a HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after boiling during 3.0 h, followed by incubation at 35 °C during 44 h.

Figure 7. HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after bentonite treatment, followed by incubation at 35 °C during 44 h.

Figure 8. HPLC-ELSD chromatogram of the stabilized yucca extract (50 °Bx) prepared by the laboratory scale process described in the Example 4.

Figure 9. Flow diagram of the disclosed process for the production of highly purified monodesmosidic saponins.

Figure 10. HPLC-ELSD chromatogram of a highly purified fraction (91.1 % purity HPLC- ELSD) containing monodesmosidic saponins obtained by centrifugation and washing with water -three cycles- the yucca juice (50 °Bx) sediment.

Figure 11. Relative titers of total IgG against OVA (expressed as titers relative to the control without adjuvant) in female BALB/c mice immunized with PBS buffer containing ovalbumin (OVA, 50 pg) (Control) and the following adjuvants (25 pg per dose): Freund's Complete Adjuvant (FCA), purified quillaja saponin QS-21 (>95% purity UHPLC-UV), and monodesmosidic yucca saponins (91.1% purity HPLC-ELSD). On weekly basis, four doses of antigen/adjuvant were applied to each group of mice (immunized intraperitoneally; seven mice per condition). Three days after the fourth immunization, blood samples were recovered from each mouse for determination of the immunoglobulin titer by ELISA.

Figure 12. Flow diagram of the disclosed process to produce yucca extracts including the step of boiling of the fresh yucca juice after cold pressing step.

Figure 13. Flow diagram of the disclosed process to produce yucca extracts including the step of bentonite treatment of the fresh yucca juice after cold pressing step.

Figures 14. Comparison of the lot of yucca juice prepared by the standard process (Figure 2) (bottle 2) and the lot prepared according to the flow diagram shown in the Figure 12 (bottle 1 ), including the boiling step for 3 h after cold pressing step.

Figure 15. Shows a HPLC-ELSD chromatogram of the yucca extract prepared by the standard process described in the Figure 2.

Figure 16. Shows a HPLC-ELSD chromatogram of the yucca extract prepared by the disclosed process described in the Figure 12.

DETAILED DESCRIPTION OF THE INVENTION.

As described earlier, the present application relates to a method to produce either: a) a highly purified monodesmosidic saponins as vaccine adjuvants, and b) stable of extracts of Yucca schidigera Roezl. (yucca) containing monodesmosidic and bidesmosidic saponins found in the biomass of the said plant.

The term “stable” as used herein refers to extracts of yucca free of precipitation upon incubation at room temperature.

The term “yucca plant” as used herein refers to plants of the genus Yucca comprising, including, but not limited to: Yucca schidigera Roezl. , Yucca brevifolia Engelm., Yucca baccata T orr. The term “monodesmosidic saponin” as used herein refers to steroidal glycosides of yucca substituted with one chain of oligosaccharides as shown in Tables 1 and 2 and Figure 1.

The term “bidesmosidic saponin” as used herein refers to steroidal glycosides of yucca substituted with two chains of oligosaccharides as shown in the Tables 1 and 2 and Figure 1.

The term “fresh yucca biomass” as used herein refers to the roots, stems, and leaves of the yucca plant.

The term “hydrated dried biomass” as used herein refers to hydrated dried roots, stems, and leaves of the yucca plant.

The term “vaccine adjuvant” as used herein refers to as any immunomodulatory component(s) formulated with antigens in a vaccine to promote and/or enhancing the host defense mechanism.

The present invention provides a method to produce stable yucca juice precursor having a Brix grade between 15 - 25 °Bx, preferably 19° Bx and the process for the obtention of concentrated yucca extracts having a Brix grade between 45 - 55 °Bx, preferably 50 °Bx, comprising:

- harvesting fresh or hydrated dried yucca biomass,

- cutting the harvested fresh or hydrated yucca biomass in fragments having an appropriate size to be cold pressed,

- conventionally cold pressing the fragmented harvested fresh or hydrated yucca biomass,

- boiling the yucca extract resulting from cold pressing,

- filtering the boiled yucca extract, and

- concentrating the filtered yucca extract to a desired concentration.

In an alternate description, the production of stable yucca juice precursor having a Brix grade between 15 - 25 °Bx, preferably 19 °Bx, is achieved by the method comprising:

- harvesting fresh or hydrated dried yucca biomass. - cutting the harvested fresh or hydrated dried yucca biomass in fragments as mentioned above.

- conventionally cold pressing the fragmented harvested fresh or hydrated yucca biomass as mentioned above.

- treating of the yucca extract resulting from cold pressing with bentonite, preferably, with an amount of 0 - 100 g/L bentonite suspension.

- filtering the bentonite-treated yucca extract.

- concentrating the filtered yucca extract to a desired concentration as mentioned above.

Specifically, the present method allows to control the content of mono and bidesmosidic saponins in yucca extracts, suitable for isolation of steroidal saponins with immunostimulant properties, comprising: a) crushing and extruding fragmented harvested fresh or hydrated yucca biomass to obtain a first yucca juice extract (first precursor) having 15 - 25 °Bx, preferably, 19 °Bx, such first yucca juice extract then is submitted to: a.1 ) heating up below the boiling point and at a maximum temperature of 88 °C by steps, in 1 -10 steps, but preferably in 2-3 steps to promote a controlled conversion and a precipitation of monodesmosidic saponins; or a.2) a one-step boiling at 100 °C, or a heating above 100 °C, preferably, by Ultra High Temperature processing (UHT), or a.3) a bentonite treatment, or a.4) performing sequentially step a.2) and step a.3) but cooling after step a.2) or vice versa, b) concentrating such first yucca juice extract obtained from step a) by either, conventional evaporation or membrane concentration techniques or a combination thereof, to obtain a second yucca juice extract (second precursor) having 25 - 35 °Bx, preferably 30 °Bx; c) filtering such second yucca juice extract after adjusted pH with a pH buffer, preferably such pH buffer is selected from H3PO4, HCI, citric acid, ascorbic acid or a mixture of two or more thereof. d) concentrating such filtered yucca juice extract, either by conventional evaporation, membrane concentration techniques or a combination thereof, to obtain a third yucca juice extract having 45 - 55 °Bx, preferably, 50 °Bx; and e) pasteurizing at 88 °C, such third yucca juice extract to obtain a fourth yucca juice extract having 45 - 55 °Bx, preferably, 50 °Bx and a pH between 3.5 - 4.5, preferably, pH 3.9, and then optionally, drying to be stored, wherein after step a.1), the method further comprising submitting such heated first fresh or hydrated dried yucca juice extract to a controlled conversion to promote instability, preferably 30 - 40 °C, more preferably at 35 °C, and, further f) optionally, after step a.1 ) and either after steps b, c), or d), the method further comprises submitting the sediment obtained after filtering to a water washing and centrifugation step in 1 -5 cycles, preferably 2-3 cycles, and purified monodesmosidic saponins are separately recovered.

The present invention is also related to a highly pure yucca monodesmosidic saponins precipitate having between 60-100% purity, preferably 70-99% purity, at HPLC-ELSD, which is useful. as immunological adjuvant.

The advantages of the present invention include, without limitation, the production of purified monodesmosidic saponins useful as vaccine adjuvants, and stable extracts of Yucca schidigera Roezl. containing mono and bidesmosidic saponins as commercial chemical raw material. Thus, the method of the present invention allows the purification of monodesmosidic saponins useful as vaccine adjuvants, and could be applied without using organic solvents, chromatographic techniques, ultra/nano/nor reverse osmosis membranes as described in the state of art so far. Besides, the method of the present invention allows the large-scale production of stable yucca extracts containing mono and bidesmosidic saponins, which are described as follows.

Thus, stable yucca extracts can be obtained by boiling the fresh or hydrated dried yucca juice or by bentonite treatment which remains stable even after controlled conversion (at 35 °C for 44 h). Boiling the fresh yucca juice (during 0.5 and 3.0 h) or treating it with bentonite after the extraction process, allowed a lower conversion rate of the bidesmosidic saponins into monodesmosic ones, and an equilibrated relative amount (conversion rate of 0 to 95%) of bidesmosidic and monodesmosic saponins contained in the extract, which is stable even after its controlled conversion (at 35 °C for 44 h). On the other hand, after a controlled conversion of the untreated fresh extract (at 35 °C for 44 h), bidesmosidic saponins are almost completely converted into monodesmosidic ones (groups 1 - 3, see Table 2 below), being the dominant components of the sediment, particularly saponins belonging to group 1 , see Table 2 below. For the untreated fresh extract, a conversion rate of the bidesmosidic saponins into monodesmosidic ones of 99% was achieved at 35 °C for 44 h. On the other hand, a conversion rate of 35, 49 and 84% was achieved by boiling (0.5 and 3.0 h) or by a bentonite treatment after the extraction/extrusion process (see Tables 1 and 2, and Figures 1 , 3 - 7).

A highly purified fraction containing monodesmosidic saponins can be obtained by centrifugation and washing of a yucca juice (50 °Bx) sediment with water. Such highly purified fraction containing monodesmosidic saponins (91.1 % purity HPLC-ELSD, yield 4.2% dry weight) (Figures 9 and 10). After tested sixteen different yucca juice lots, purities (HPLC-ELSD) and yields (dry weight) of the purified monodesmosidic saponins of 78 - 99% and 1 - 14%, respectively, were achieved.

The invention disclosed in this document may be further understood by reference based on the following Examples, which are not intended to be limiting of the scope of the invention.

Example 1. HPLC-ELSD analysis of steroidal saponins in extracts of yucca.

The analysis of the samples was performed by Reversed Phase High Performance Liquid Chromatography (RP-HPLC) according to Sastre, F., Ferreira, F., & Pedreschi, F. 2016, “MALDI-TOF mass spectrometry and reversed-phase HPLC-ELSD chromatography for structural and quantitative studies of major steroid saponins in commercial extracts of Yucca schidigera Roezl”. Journal of Pharmaceutical and Biomedical Analysis, 120, 270- 282. The samples were separated on an octadecylsilane end-capped column (250 x 4.6 mm, 5 |im particle size) maintained at 40 °C, eluted with a linear gradient from 68% MeOH in H2O to 88% MeOH in 30 min and a flow rate of 1 mL/min. The detection was performed on an evaporative light scattering detector (ELSD), set at 50 °C and an air set at 2.5 bar was used as nebulizer gas.

For quantification purposes, a calibration curve was determined by injection of standards of purified yucca saponins. The method allowed the determination of total yucca saponins, and the relative quantitation of bi- and monodesmosidic components by HPLC- ELSD. Example 2. HPLC-HRMS analysis of steroidal saponins in extracts of yucca.

The analysis of the samples was performed by RP-HPLC, employing the same chromatographic conditions described in the Example 1. The HPLC chromatograph was coupled to a High-Resolution Electrospray Ionization Mass Spectrometer (HRMS) Thermo Fisher Scientific Exactive Plus Orbitrap (Bremen, Germany) in the mass range 100 - 1500 Da (negative mode). The sheath, auxiliary and sweep nitrogen flow rate were set at 9, 2 and the capillary temperature at 320 °C. The S lens RF was set at 50 and 1 arbitrary units, respectively. Spray voltage was set at -5 kV and the heater temperature at 150 °C. Extracted compound chromatograms were processed employing Xcalibur™ Software.

The identification of 12 saponins in the samples was performed by spiking with reference standards and confirmed with highly accurate mass measurements (compounds N°: 9 - 12, 14 - 17, and 21 - 24, Tables 1 and 2, and Figure 2). This information, along with reference data from previous literature (Kowalczyk, M., Pecio, L, Stochmal, A., & Oleszek, W. 2011 , “Qualitative and quantitative analysis of steroidal saponins in crude extract and bark powder of Yucca schidigera Roezl”. Journal of Agricultural and Food Chemistry, 59(15), 8058-8064; Sastre, F., Ferreira, F., & Pedreschi, F. 2016, “MALDI- TOF mass spectrometry and reversed-phase HPLC-ELSD chromatography for structural and quantitative studies of major steroid saponins in commercial extracts of Yucca schidigera Roezl”. Journal of Pharmaceutical and Biomedical Analysis, 120, 270-282 Sastre, F., Ferreira, F., & Pedreschi, F. 2017, A systematic approach for the chromatographic fractionation and purification of major steroid saponins in commercial extracts of Yucca schidigera Roezl”. Journal of Chromatography B, 1046, 235-242} allowed the tentative identification of other 14 components, previously reported in yucca (compounds N°: 1 - 8, 13, 18 - 20, 25 and 26; Tables 1, and 2 and Figure 1).

Table 1. Compounds identified in yucca by High-Pressure Liquid Chromatography coupled to High Resolution Electrospray Ionization Mass Spectrometry (HPLC-HRMS).

¹ 0leszek, W., Sitek, M., Stochmal, A., Piacente, S., Pizza, C., & Cheeke, P. (2001 ). Steroidal saponins of Yucca schidigera Roezl. Journal of Agricultural and Food Chemistry, 49, 4392-4396.

² Kowalczyk, M., Pecio, L., Stochmal, A., & Oleszek, W. (2011 ). Qualitative and quantitative analysis of steroidal saponins in crude extract and bark powder of Yucca schidigera Roezl. Journal of Agricultural and Food Chemistry, 59(15), 8058-8064,

Table 2. Structural diversity of yucca saponins. monomosidic saponins identified (groups 1, 2 and 3), previously reported in yucca bidesmosidic saponins previously reported in yucca, tentatively identified by HPLC-HRMS Example 3. Testing of the stability of yucca extracts obtained by aqueous extraction yucca biomass at laboratory scale.

We tested the stability of yucca extracts obtained under controlled laboratory conditions, by boiling the fresh yucca juice (during 0.5 and 3.0 h), or by bentonite treatment, and subjecting these samples to a controlled conversion (at 35 °C for 44 h) in parallel with the untreated fresh sample. As shown in Figures 2, 3 and 4, after a controlled conversion of the untreated fresh extract (at 35 °C for 44 h), bidesmosidic saponins are completely converted into monodesmosidic ones (groups 1 - 3), being the latter also the dominant components of the sediment, particularly saponins belonging to group 1 . Boiling the fresh yucca juice (during 0.5 and 3.0 h), (Figures 5 and 6) or by treating it with bentonite (Volclay® KWK Food Grade, low dust granular sodium bentonite with an average particle size between 20 and 70 mesh) (Figure 7) short after the extraction process, allowed to lower the conversion rate of the bidesmosidic saponins into monodesmosic ones. These processes disclosed (boiling or bentonite treatment), allows to obtain an equilibrated relative amount (conversion rate of 0 to 95%) of bidesmosidic and monodesmosic saponins contained in the extract, which is stable even after its controlled conversion (at 35 °C for 44 h), suggesting that the amount of sediment can be controlled and mitigated. In particular, the conversion rate of the bidesmosidic saponins into monodesmosidic ones for the analyzed intact untreated fresh sample after a controlled conversion at 35 °C for 44 h was 99%. For the same sample, the conversion rate of the bidesmosidic saponins into monodesmosidic ones by boiling it during 0.5 and 3.0 h, or by treating it with bentonite short after the extraction/extrusion process, was about 35, 49 and 84% compared to the no treated samples (Tables 1 and 2, and Figures 1 , 3 - 7).

Example 4: Laboratory scale preparation of a stabilized yucca extract (50 °Bx) having an equilibrated relative amount of bidesmosidic and monodesmosidic saponins.

Y. schidigera Roezl. (yucca) powdered biomass (50 g) was extracted in deionized water (500 mL) for 15 min at room temperature using a magnetic stirrer. Then, the extract was centrifuged at 3220 RCF for 10 min (20 °C), and the supernatant was transferred to a bottle (1 L). The supernatant was then adjusted to pH = 3.8 with diluted HCI and clarified with bentonite (Volclay KWK Food Grade, 3 g/L) for 1 h using a magnetic stirrer. The extract was then filtered through paper (qualitative grade 2) and concentrated by evaporation in an oven set at 88 °C. After this process, a stabilized yucca juice (30 g) at 50 °Bx, 5.2% total yucca saponins, pH = 3.2, having a conversion rate of the bidesmosidic saponins into monodesmosidic ones of about 66% compared to the intact sample, was obtained (Figures 8 and 13).

Example 5. Preparation of a highly purified fraction containing monodesmosidic saponins obtained by centrifugation and washing of a yucca juice (50 °Bx) sediment with water.

An aliquot of a yucca juice (12 mL, 50 °Bx) was centrifuged at 3100 RCF for 30 min at 20 °C. The sediment was washed 3 times with mili-Q water (12 mL x 3) and centrifuged as previously described. After this step, the sediment was dried in an oven for 24 h at 50 °C, weighted, and analyzed by HPLC-ELSD and HPLC-HRMS as previously described in the Examples 1 and 2, respectively. This process allowed to obtain a highly purified fraction containing monodesmosidic saponins (91 .1% purity HPLC-ELSD, yield 4.2% dry weight) (Figures 9 and 10). This process was tested in sixteen different yucca juice lots and the purities (HPLC-ELSD) and yields (dry weight) of the purified monodesmosidic saponins ranged from 78 - 99% and 1 - 14%, respectively.

Example 6. Pilot production testing of yucca extracts.

The results obtained after the preparation of an experimental lot of yucca juice by adding a boiling step for 3 h short after the extrusion process, as described in Figure 12, were consistent with the laboratory study (Example 3). Both the amount of sediment observed and the conversion rate of bidesmosidic and monodesmosidic was reduced by about 10% compared to two lots prepared by the standard process (Figures 2, 3, 4, 12, 14 - 16).

Based on the experimental results at laboratory (Examples 3 and 4) and pilot plant scale (Example 6), it can be concluded that the disclosed processes (boiling or bentonite treatment) allow to obtain an equilibrated relative amount of bidesmosidic and monodesmosidic saponins, mitigating the formation of sediments -unwanted in yucca extracts-. The same process could also be applied in other sources of steroidal glycosides, such as -but not limited to- Yucca brevifolia Engelm., Yucca baccata Torr., etc.

Example 7. Yucca monodesmosidic saponins as immunological adjuvant.

To determine the immunostimulant activity of the highly purified fraction containing monodesmosidic saponins obtained in the Example 5, female BALB/c mice were immunized with PBS buffer containing ovalbumin (OVA, 50 .g) (Control) and the following adjuvants (25 jig per dose): Freund's Complete Adjuvant (FCA), purified quillaja saponin QS-21 (>95% purity UHPLC-UV), and monodesmosidic yucca saponins (91.1% purity HPLC-ELSD). On weekly basis, four doses of antigen/adjuvant were applied to each group of mice (immunized intraperitoneally; seven mice per condition). Three days after the fourth immunization, blood samples were recovered from each mouse, and the titers of total IgG against ovalbumin (OVA) were determined by ELISA. As shown in Figure 11 , the relative titers of total IgG against OVA (expressed as titers relative to the control without adjuvant) induced by yucca monodesmosidic saponins was comparable at the same dose with the corresponding quillaja saponin QS-21 , a potent immunostimulant compound employed as adjuvant in vaccine formulations (p < 0.0001 ).