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Knowledge of binary emulsifiers' influence on the formation and stability of emulsion-based products is still limited. The aim of this study was to investigate the emulsifying properties of Quillaja saponin-egg lecithin mixtures at different concentration ratios (r = 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5) with total emulsifier concentration set to 0.5% or 1.0% (w/w). For this, oil-in-water emulsions (10% oil, pH 7) were prepared via high-pressure homogenization. Furthermore, emulsion stability against different environmental stresses was tested. All the binary emulsifier mixtures formed submicron sized emulsions upon homogenization. The most stable emulsions among the mixed emulsifiers were obtained at low Quillaja saponin concentration at r = 1:4 that showed similar physical stability over time to emulsions stabilized by Quillaja saponins and egg lecithin alone. The data suggested that the mixtures of Quillaja saponins and egg lecithins built mixed interfacial layers that were prone to changes over time. Emulsions stabilized by the binary mixtures were in general less stable against changes in pH and ionic strength than the emulsions stabilized by the individual emulsifiers. An exception were the emulsions at r = 1:4 that showed improved stability at pH 2 over the phase separated Quillaja saponin-stabilized emulsions at the same pH. Moreover, all the emulsions were heat stable up to 90 °C. On the other hand, none of the emulsions were stable upon freeze-thawing. These results increase our understanding of technofunctionality of binary emulsifier systems. PRACTICAL APPLICATION: Food-grade and natural emulsifier mixtures composed of Quillaja saponins and egg lecithin may be used in selected emulsion-based food or personal care product applications to replace synthetic surfactants due to issues with consumer acceptance and regulatory restrictions.

Introduction Quillaja brasiliensis (St. A. -Hil. & Tul) Mart (Quillajaceae) is a species native to South America, which is rich in saponins. Saponins are used in different industries, so there is a constant demand for this type of compound. Based on the wide range of applications for the saponins found in this species, notably as immunoadjuvants, we conducted a comprehensive study of this tree and its saponins. Objective The purpose of this work is to complete the characterisation of the immunoadjuvant saponin fraction from Q. brasiliensis leaves and further study the saponin fraction obtained from Q. brasiliensis bark. Methodology Saponin fractions were studied using mass spectrometry in combination with classical methods of monosaccharide and methylation analysis. We performed direct infusion and liquid chromatography/electrospray ionisation ion trap multiple-stage mass spectrometry (DI-ESI-IT-MSn and LC-ESI-IT-MS2 ). Results Seventy-five saponins, 21 from leaves and 54 from bark, were tentatively identified according to their molecular mass, fragmentation pattern and chromatographic behaviour. This work represents the first investigation of saponins from the bark of Q. brasiliensis and some of them presented new structural motifs not previously reported in the genus Quillaja. Conclusion The efficiency and selectivity of the data dependent LC-MS2 method allowed the rapid profiling of saponins from Q. brasiliensis. The results of the monosaccharide and methylation analysis performed in saponins from Q. brasiliensis fractions and Q. saponaria Molina (Quillajaceae) fraction gives further support to the structures proposed according to the mass spectral data, validating the strategy used in the present work.

Natural alternate methods to control the spread of Shiga toxin-producing Escherichia coli (STEC) are important to prevent foodborne outbreaks. Quillaja saponaria aqueous bark extracts (QE), cleared by the U.S. Food and Drug Administration as a natural flavorant, contain bioactive polyphenols, tannins, and tri-terpenoid saponins with anti-inflammatory and antimicrobial activity. The objective of this study was to determine the effects of commercial QE against E. coli O157:H7 and non-O157 strains over 16 h at 37 °C and RT. Overnight cultures of 4 E. coli O157:H7 strains and 6 non-O157 STECs in Tryptic Soy Broth (TSB) were washed and resuspended in phosphate-buffered saline (PBS, pH 7.2), and treated with QE and controls including citric acid (pH 3.75), sodium benzoate (0.1% w/w), acidified sodium benzoate (pH 3.75) or PBS for 6 h or 16 h. Recovered bacteria were enumerated after plating on Tryptic Soy Agar, from duplicate treatments, replicated thrice and the data were statistically analyzed. The 4 QE-treated E. coli O157:H7 strains from initial ∼7.5 log CFU had remaining counts between 6.79 and 3.5 log CFU after 16 h at RT. QE-treated non-O157 STECs showed lower reductions with remaining counts ranging from 6.81 to 4.55 log CFU after 16 h at RT. Incubation at 37 °C caused reduction to nondetectable levels within 1 h, without any significant reduction in controls. Scanning electron microscopy studies revealed damaged cell membranes of treated bacteria after 1 h at 37 °C. QE shows potential to control the spread of STECs, and further research in model food systems is needed.

Heteroaggregated oil-in-water (O/W) emulsions formed by targeted combination of oppositely charged emulsion droplets were proposed to be used for the modulation of physical properties of food systems, ideally achieving the formation of a particulate 3-dimensional network at comparably low-fat content. In this study, rheological properties of Quillaja saponins (QS), sugar beet pectin (SBP), and whey protein isolate (WPI) stabilized conventional and heteroaggregated O/W emulsions at oil contents of 10% to 60% (w/w) were investigated. Selected systems having an oil content of 30% (w/w) and different particle sizes (d43 ≤ 1.1 or ≥16.7 μm) were additionally subjected to chemical (genipin or glutaraldehyde) and thermal treatments, aiming to increase network stability. Subsequently, their rheological properties and stability were assessed. Yield stresses (τ0) of both conventional and heteroaggregated O/W emulsions were found to depend on emulsifier type, oil content, and initial droplet size. For conventional emulsions, high yield stresses were only observed for SBP-based emulsions (τ0,SBP approximately 157 Pa). Highest yield stresses of heteroaggregates were observed when using small droplets stabilized by SBP/WPI (approximately 15.4 Pa), being higher than those of QS/WPI (approximately 1.6 Pa). Subsequent treatments led to significant alterations in rheological properties for SBP/WPI systems, with yield stresses increasing 29-fold (glutaraldehyde) and 2-fold (thermal treatment) compared to untreated heteroaggregates, thereby surpassing yield stresses of similarly treated conventional SBP emulsions. Genipin-driven treatments proved to be ineffective. Results should be of interest to food manufacturers wishing to design viscoelastic food emulsion based systems at lower oil droplet contents.

The miscibility behavior of mixed surfactant systems and the influence of extrinsic parameters are crucial for their application as emulsifiers. Therefore, the objective of this study was to evaluate the miscibility behavior of mixed systems composed of commercial Quillaja saponin and a co-surfactant, namely sodium caseinate, pea protein, rapeseed lecithin, or egg lecithin. These mixtures were evaluated macro- and microscopically at different concentration ratios (maximum concentration 5% w/v) at pH 3, 5, and 7 at 25 °C. The individual ingredients were also assessed for their charge properties and surface hydrophobicity. The results showed that Quillaja saponin-caseinate mixtures were miscible only at pH 7, and showed aggregation and precipitation at lower pH due to increasing electrostatic attraction forces. Rheological measurements showed that Quillaja saponin-pea protein mixtures formed gelled structures at all tested pH values mainly via association of hydrophobic patches. Quillaja saponins mixed with rapeseed lecithin were miscible at all tested pH values due to electrostatic repulsion. Quillaja saponin-egg lecithin mixtures aggregated independent of pH and concentration ratio. The microscopic analysis revealed that the lower the pH and the higher the Quillaja saponin ratio, the denser were the formed Quillaja saponin-egg lecithin aggregates. The results are summarized in ternary phase diagrams that provide a useful tool in selecting a surfactant system for food applications.

The taxonomy and distribution of the genus Quillaja (Quillajaceae) is examined and two species, Q. brasiliensis and Q. saponaria, are recognised and keyed out. Quillaja bra- siliensis is distributed in southern Brazil, northern Uruguay, northeastern Argentina and eastern Paraguay. The presence of Q. brasiliensis in Peru, indicated in some sources, is not confirmed with herbarium specimens. Quillaja saponaria is distributed in central Chile, besides one doubtful collection from Andean Bolivia. The mention of its presence in Peru is likewise unjustified. A distribution map of the species is provided and two names are lectotypified here. Se examina la taxonomia y distribucion del genero Quillaja (Quillajaceae), donde dos especies, Q. brasiliensis y Q. saponaria, se reconocen y diferencian mediante una clave. Quillaja brasiliensis se distribuye en el sur de Brasil, norte de Uruguay, noreste de Argentina y este de Paraguay. La presencia de Q. brasiliensis en Peru, indicada en algunas fuentes, no se ve respaldada por ejemplares de herbario. Quillaja saponaria se distrbuye en Chile central, mas una coleccion dudosa proveniente de los Andes de Bolivia. La mencion de Q. saponaria para Peru tampoco se justifica. Se presenta un mapa de distribucion de las especies y se lectotipifican dos nombres. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Five saponins purified from the leaves of three Ilex species (saponins 1 and 2 from I. dumosa; saponin 3 from I. argentina; saponin 4 from I. paraguariensis) and from Passiflora alata (saponin 5) were evaluated for their in vitro haemolytic activity and in vivo immunostimulatory ability in a mouse model using tetanus toxoid (TT) as a model antigen. The assayed saponins showed very weak or no haemolytic activity over the tested concentration range. Mice were immunized twice with TT formulated with pure saponins 1-5, or with a mixture of saponins from Quillaja saponaria, aluminum hydroxide gel or saline, which were used as controls. The elicited humoral response was evaluated by means of the time course of specific serum antibody levels up to day 131 post-priming (total IgG and isotypes); the cellular response was tested through a delayed-type hypersensitivity (DTH) assay. The assayed saponins, in particular saponins 3 and 5, showed an adjuvant effect similar to that of alum for all tested parameters. The immunostimulating potential of these compounds deserves further investigation, especially taking into account that some Ilex spp. and Passiflora alata are native crops of widespread use and economical importance in Latin America.

Thirty-eight saponins in two chromatographic fractions (QH-B and QH-C) from Quillaja saponaria Molina have been separated by a two-step high-performance liquid chromatography (HPLC) procedure and investigated by electrospray ionisation ion trap multiple-stage mass spectrometry (ESI-ITMS(n)) in positive ion mode. MS(2) and MS(3) spectra of the compounds were investigated by principal component analysis (PCA) and could be classified by partial least squares - discriminant analysis (PLS-DA) according to the structures of the oligosaccharides at C-3 and C-28 of the saponins. Four minor components with novel structures were found in a previously non-investigated fraction of QH-C. The structures of two of these components, J1 and J1a, were predicted by PLS-DA whereas the structures of the two others, J2 and J3, were only partly predicted. The structures of J1 and J1a were composed of structural elements found in the 34 known saponins whereas a new acyl substituent, not included in the training set used for calibration of the PLS-DA models, was found in J2 and J3, making these two components outliers. The complete structures of the four components were confirmed by monosaccharide analysis, MS(n) data and (1)H NMR spectroscopy.

Akara, a deep-fat-fried cowpea paste product, is a popular snack food in West Africa. Foaming capacity of the cowpea paste is important for achieving a spongy light-textured fried product. The effect of two saponins, Yucca and Quillaja, on the foaming capacity of cowpea paste was investigated. Results showed that an increase in the amount of saponin added (1, 2, 4%) resulted in a decrease in specific gravity and apparent viscosity of the cowpea paste. Addition of saponin also significantly reduced the firmness and increased the cohesiveness of the akara compared with the control. Akara made using the Quillaja saponin was less firm and more cohesive than akara made using the Yucca saponin. An increase in the amount of saponin added to the cowpea paste resulted in a darker (lower L*) and browner colour of akara. However, akara containing 1% Yucca saponin had similar colour attributes to the control but was less firm and more cohesive.

Saponin-rich extracts of the Chilean indigenous tree Quillaja saponaria Molina are widely used as natural foaming agents in foods and beverages, food emulsifiers, photographic emulsions, vaccine adjuvants, etc. However, with the exception of the vaccine industry, saponin concentration is not adequately quantified. Normally, the quality and price of the extracts are determined using simple foam tests. This may not be adequate, since similar foam levels can be obtained by blending quillaja extracts with other low-cost saponin sources (eg Yucca shidigera extracts). Also, many products are diluted with high amounts of carriers, reducing significantly their saponin concentration. To overcome these problems and standardise the quality of commercial extracts, the use of reverse phase HPLC techniques is explored. It is shown that RP-HPLC yields consistent and repetitive results and can be easily implemented to control the quality of quillaja extracts. Commercial non-refined extracts contain 190–200 g saponins kg−1 solids, while semi-refined extracts contain 750–800 g saponins kg−1 solids. Also, extracts derived from quillaja bark (traditional raw material) and whole quillaja wood (novel ecological production method) have similar saponin composition and concentration. © 2000 Society of Chemical Industry

The immune responses to immunostimulating complexes (iscoms) containing recombinant Epstein-Barr virus (EBV) gp340 envelope protein was evaluated in BALB/c (H-2(d)) and CBA (H-2(k)) mice. Gp340-iscoms were used either with a low content of Quillaja triterpenoid adjuvant (L-iscoms) or supplemented with additional Quillaja adjuvant in the form of iscomatrix (S-iscoms). Class and subclass distribution of anti-gp340 antibodies, EBV-neutralizing antibodies, antigen-specific T cell proliferation and cytokine production were determined and these results compared to those obtained by immunization with non-adjuvated gp340. The H-2(d) and H-2(k) mice were characterized as low or high responders in respect to the level of specific anti-gp340 antibodies, secretion of IgG2a isotype, antigen-specific lymphoproliferative capacity, interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) production in the basic immunizations with gp340. While presentation of the antigen in iscom formulations with low levels of Quillaja triterpenoids induces a moderate enhancement of the immune responses in the low responder H-2(d) mice, supplementation with high levels of iscomatrix immunomodulator was required to enhance the immune responses in the high responder H-2(k) mice. In both mouse strains subcutaneous immunization with S-iscoms resulted in a significant increase of IgG1- and IgG2a-specific antibodies, as well as in strong antigen-specific proliferative response confirmed by the simultaneous cytokine production. The enhanced antigen-specific secretion of IL-2 and IFN-gamma together with the abrogation of IL-10 and the absence of IL-4 indicates that the responses were driven towards a Th1-type rather than Th2-type immune response. The S-iscom formulations minimized the differences in immune responses between the two mouse strains, but the capacity of immune sera to neutralize EBV transformation in vitro remained completely strain-dependent. These data indicate that immune responses generated by iscoms can be manipulated by altering the triterpenoid composition of the iscoms and that the levels of triterpenoids can determine whether or not a Th1-type response is made.

The rate of fermentation of hay was not affected by purified tannins from Quercus incana and Dichostachys cinerea at 0.13 mg ml -1 of the in vitro medium whereas a decrease of 4% in the rate was observed for Acioa barteri tannins at this level. At 0.23 mg and 0.47 mg of tannins per ml, the decrease in rate varied from 4 to 13% and 13 to 20%, respectively. The decrease in in vitro true digestibility of dry matter was 3, 6 and 7% for Q. incana, D. cinerea and A. barteri, respectively, at a tannin concentration of 0.47 mg ml -1 , and 17, 21 and 27%, respectively, at 0.93 mg ml -1 . There was no change in the potential extent of digestion up to 0.47 mg ml -1 for any of the tannins studied. These results suggested that the rate of digestion is affected to a greater extent than the potential extent of digestion or the in vitro true digestibility and that different tannins even at the same level have different degrees of effect. Tannin-saponin interactions were studied using tannic acid (TA), quebracho tannin (QT) and quillaja saponin (S) at different concentrations (0.2, 0.4 and 0.6 mg ml -1 ) alone and in combination with each other. The decrease in the rate of digestion and true digestibility was additive when both the TA and the S were present. Similar results were obtained when TA was replaced by QT except that the effects with QT were more marked as compared to TA at the same concentration. Saponins had little effect on the potential extent of digestion and true digestibility. These tannins also decreased the production of short-chain fatty acid (SCFA) and their molar proportions (acetate decreased whereas propionate increased). Efficiency of microbial protein synthesis, expressed as the ratio of 15 N incorporation per unit of SCFA production, was higher with both S and the tannins studied. A substantial amount (71-93%) of tannins soluble in aqueous acetone was released from leaves of some trees and shrubs on incubation in the in vitro medium for 48 h. The rumen liquor was not capable of degrading oligomeric condensed tannins.

The technical feasibility of separating cholesterol from butteroil by contacting it with aqueous solutions of Quillaja saponaria saponins followed by separation of the cholesterol-saponin complex was studied. Alternative processes and controls compared were (1) Water Control (W): butteroil was contacted with pure water; (2) Reaction (R): butteroil was contacted with aqueous solution of saponins; (3) Adsorption Control (A): butteroil was contacted with water, diatomaceous earth was then added; (4) Reaction + Adsorption (R+A): diatomaceous earth was added to the Reaction medium (from 2); (5) Reaction + Washing (R+W): the aqueous phase resulting from Reaction (2) was discarded and pure water contacted with the fat. the last step in all the above processes and controls was the separation of the fat phase where cholesterol was determined. Statistical analysis indicated that the Adsorption Control did not yield any significant cholesterol reduction over the Water Control, while all other processes gave significant reduction. the order of cholesterol separation observed was R+A > R+W > R. the levels of cholesterol reduction were significantly affected by pH of quillaja solution and amount of adsorbent used; the time of contact with quillaja solution had no significant effect over the range investigated. These results indicate the technical feasibility of reducing the cholesterol levels of butteroil using food grade quillaja saponins.

The separation of cholesterol from butteroil was achieved by various processes based on contacting it with aqueous solutions of Quillaja saponaria saponins followed by separation of the cholesterol-saponin complex. Alternative processes and controls studied were (1) Water Control (W): butteroil was contacted with pure water; (2) Reaction (R): butteroil was contacted with aqueous solution of saponins; (3) Adsorption Control (A): butteroil was contacted with water, diatomaceous earth was then added; (4) Reaction + Adsorption (R+A): diatomaceous earth was added to the Reaction medium (from 2); (5) Reaction + Washing (R+W): the aqueous phase resulting from Reaction (2) was discarded and pure water contacted with the fat. the last step in all the above processes and controls was the separation of the fat phase, after which analyses were made. ANOVA indicated that the Adsorption Control process did not yield any significant cholesterol reduction over the Water Control; all other processes resulted in significant cholesterol separation in the order R+A > R+W > R. the levels of cholesterol reduction achieved by the R, R+W, and R+A processes were significantly affected by the concentration of quillaja solution and temperature; the level of agitation had no significant effect over the range investigated. Quillaja powder residue was present in the fat phase after Reaction (2) but both adsorption and washing procedures significantly reduced it.

The possibility that certain saponins produce growth depression by complexing fat soluble vitamins with a mechanism similar to saponin-cholesterol complexation was studied. An in vitro procedure was developed to quantitate the formation of addition complexes in aqueous solutions. Saponins studied included digitonin, ammoniated glycyrrhizin, quillaja bark saponin, and saponin-containing extracts from alfalfa leaves and roots and soybeans. Vitamins tested included provitamin D3 and vitamins E, A and D3. The study demonstrated that the saponins did not complex fat soluble vitamins in a manner similar to the complexation of cholesterol.

The possibility that certain saponins produce growth depression by complexing minerals was studied. Saponins tested included digitonin, ammoniated glycyrrhizin, quillaja saponin and saponin-containing extracts from alfalfa leaves and roots. Minerals studied were zinc, iron and magnesium. Alfalfa root saponins and ammoniated glycyrrhizin, but not digitonin, quillaja and alfalfa leaf saponins, complexed zinc and iron, but not magnesium. It is proposed that one mechanism of action whereby saponins suppress growth is by the formation of insoluble saponin-mineral complexes. Such complexes could reduce the availability of minerals necessary for growth.

The leafy material from Atriplex polycarpa contains 1.15% saponin. Comparative inhibition of Trichoderma viride mycelium indicated that saponin from A. polycarpa was either much more potent than commercially available saponin from the bark of Quillaja, or that other water-soluble inhibitors were also present. Distilled water, dripping through A. polycarpa plants to simulate rain, also removed inhibitory substances from A. polycarpa.