Exploring the use of NIR and Raman spectroscopy for the prediction of quality traits in PDO cheeses.

The aims of this proof of principle study were to compare two different chemometric approaches using a Bayesian method, Partial Least Square (PLS) and PLS-discriminant analysis (DA), for the prediction of the chemical composition and texture properties of the Grana Padano (GP) and Parmigiano Reggiano (PR) PDO cheeses by using NIR and Raman spectra and quantify their ability to distinguish between the two PDO and among their ripening periods. For each dairy chain consortium, 9 cheese samples from 3 dairy industries were collected for a total of 18 cheese samples. Three seasoning times were chosen for each dairy industry: 12, 20, and 36 months for GP and 12, 24, and 36 months for PR. A portable NIR instrument (spectral range: 950-1,650 nm) was used on 3 selected spots on the paste of each cheese sample, for a total of 54 spectra collected. An Alpha300 R confocal Raman microscope was used to collect 10 individual spectra for each cheese sample in each spot for a total of 540 Raman spectra collected. After the detection of eventual outliers, the spectra were also concatenated together (NIR + Raman). All the cheese samples were assessed in terms of chemical composition and texture properties following the official reference methods. A Bayesian approach and PLS-DA were applied to the NIR, Raman, and fused spectra to predict the PDO type and seasoning time. The PLS-DA reached the best performances, with 100% correctly identified PDO type using Raman only. The fusion of the data improved the results in 60% of the cases with the Bayesian and of 40% with the PLS-DA approach. A Bayesian approach and a PLS procedure were applied to the NIR, Raman, and fused spectra to predict the chemical composition of the cheese samples and their texture properties. In this case, the best performance in validation was reached with the Bayesian method on Raman spectra for fat (R2VAL = 0.74). The fusion of the data was not always helpful in improving the prediction accuracy. Given the limitations associated with our sample set, future studies will expand the sample size and incorporate diverse PDO cheeses.

Exopolysaccharides produced by Bacillus spp. inhibit biofilm formation by Staphylococcus aureus strains associated with bovine mastitis.

Bovine mastitis is a costly disease in the dairy sector worldwide. Here the objective was to identify and characterize anti-biofilm compounds produced by Bacillus spp. against S. aureus associated with bovine mastitis. Results showed that cell-free supernatants of three Bacillus strains (out of 33 analysed) reduced S. aureus biofilm formation by approximately 40 % without affecting bacterial growth. The anti-biofilm activity was associated with exopolysaccharides (EPS) secreted by Bacillus spp. The EPS decreased S. aureus biofilm formation in a dose-dependent manner, inhibiting biofilm formation by 83 % at 1 mg/mL. The EPS also showed some biofilm disruption activity (up to 36.4 %), which may be partially mediated by increased expression of the aur gene. The characterization of EPS produced by Bacillus velezensis 87 and B. velezensis TR47II revealed macromolecules with molecular weights of 31.2 and 33.7 kDa, respectively. These macromolecules were composed mainly of glucose (mean = 218.5 µg/mg) and mannose (mean = 241.5 µg/mg) and had similar functional groups (pyranose ring, beta-type glycosidic linkage, and alkynes) as revealed by FT-IR. In conclusion, this study shows the potential applications of EPS produced by B. velezensis as an anti-biofilm compound that could contribute to the treatment of bovine mastitis caused by S. aureus.

Recovery of Protein from Industrial Hemp Waste (Cannabis sativa, L.) Using High-Pressure Processing and Ultrasound Technologies.

Hemp seeds are currently used mainly for oil extraction, generating waste that could be potentially exploited further as a source of proteins and other bioactives. This study aims to valorise hemp waste (Cannabis sativa, L.) from previous oil extraction as a source of protein by analysing the effect of high-pressure processing (HPP) pre-treatments (0-600 MPa; 4-8 min) combined with conventional or ultrasound-assisted extraction (UAE) methods on protein recovery/purity, amino acid composition, and protein structure. Overall, maximum protein recovery (≈62%) was achieved with HPP (200 MPa, 8 min) with UAE. The highest protein purity (≈76%) was achieved with HPP (200 MPa, 4 min) with UAE. Overall, UAE improved the extraction of all amino acids compared to conventional extraction independently of HPP pre-treatments. Arg/Lys ratios of the protein isolates ranged between 3.78 and 5.34, higher than other vegetable protein sources. SDS-PAGE did not show visible differences amongst the protein isolates. These results seem to indicate the advantages of the use of UAE for protein recovery in the food industry and the need for further studies to optimise HPP/UAE for an accurate estimation of processing costs and their effects on the composition and structure of proteins to contribute further to the circular economy.

Evaluation of Tannin-Delivery Approaches for Gut Microbiota Modulation: Comparison of Pectin-Based Microcapsules and Unencapsulated Extracts.

The aim of this study was to investigate the impact of tannins on gut microbiota composition and activity, and to evaluate the use of pectin-microencapsulation of tannins as a potential mode of tannin delivery. Thus, pectin-tannin microcapsules and unencapsulated tannin extracts were in vitro digested and fermented, and polyphenol content, antioxidant capacity, microbiota modulation, and short-chain fatty acid (SCFA) production were analyzed. Pectin microcapsules were not able to release their tannin content, keeping it trapped after the digestive process, and are therefore not recommended for tannin delivery. Unencapsulated tannin extracts were found to exert a positive effect on the human gut microbiota. The digestion step resulted to be a fundamental requirement in order to maximize tannin bioactive effects, especially with regard to condensed tannins, as the antioxidant capacity exerted and the SCFAs produced were greater when tannins were submitted to digestion prior to fermentation. Moreover, tannins interacted differently with the intestinal microbiota depending on whether they underwent prior digestion or not. Polyphenol content and antioxidant capacity correlated with SCFA production and with the abundance of several bacterial taxa.

Determination of Minerals in Soft and Hard Cheese Varieties by ICP-OES: A Comparison of Digestion Methods.

For sample preparation prior to mineral analysis, microwave digestion (~2 h) is quicker and requires lower acid volume as compared to dry (6-8 h) and wet digestion (4-5 h). However, microwave digestion had not yet been compared systematically with dry and wet digestion for different cheese matrices. In this work, the three digestion methods were compared for measuring major (Ca, K, Mg, Na and P) and trace minerals (Cu, Fe, Mn and Zn) in cheese samples using inductively coupled plasma optical emission spectrometry (ICP-OES). The study involved nine different cheese samples with moisture content varying from 32 to 81% and a standard reference material (skim milk powder). For the standard reference material, the relative standard deviation was lowest for microwave digestion (0.2-3.7%) followed by dry (0.2-6.7%) and wet digestion (0.4-7.6%). Overall, for major minerals in cheese, strong correlation was observed between the microwave and the dry and wet digestion methods (R2 = 0.971-0.999), and Bland-Altman plots showed best method agreement (lowest bias), indicating the comparability of all three digestion methods. A lower correlation coefficient, higher limits of agreement and higher bias of minor minerals indicate possibilities of measurement error.

Nanostructural changes in Polysaccharide-Casein Gel-Like structures upon in vitro gastrointestinal digestion.

This work reports on the nanostructural changes taking place during the in vitro gastrointestinal digestion of polysaccharide-casein gel-like structures through the use of small angle X-ray scattering (SAXS). The results indicated that during the gastric phase, the hydrolysis of casein led to a swelling of the micellar structure, yielding peptide clusters. The presence of sulphated polysaccharides such as agar and κ-carrageenan was seen to limit the hydrolysis of casein during the gastric phase, hence decreasing the size of the formed clusters. After the intestinal phase, the produced peptidic fragments appeared to interact with the bile salts present in the digestion medium, yielding a mixture of bile salt lamellae/micelles and vesicular structures. However, in the presence of polysaccharides, which can interact with bile salts, the formation of vesicular structures was limited. Interestingly, the inclusion of casein within hybrid gel-like structures led to the formation of strong polysaccharide-protein interactions, especially in the case of κ-carrageenan. As a result, in some of the formulations, polysaccharide-peptide complexes were released towards the liquid medium, which formed larger vesicular structures. This was related to the greater protective effect of these particular gel-like structures. Furthermore, κ-carrageenan hindered the formation of bile salt lamellae/micelles. These results are of high relevance to understand the intestinal transport mechanism of the digestion products from protein-based ingredients and will allow a rational design of novel products with optimum nutritional and functional properties.

The Protein Composition and In Vitro Digestive Characteristics of Animal- versus Plant-Based Infant Nutritional Products.

The protein composition and digestive characteristics of four commercially available infant formulae (IF) manufactured using bovine (B-IF), caprine (C-IF), soy (S-IF), and rice (R-IF) as a protein source were examined in this study. Plant-based formulae had significantly higher crude protein and non-protein nitrogen (NPN) concentrations. Static in vitro gastrointestinal digestion of these formulae, and subsequent analysis of their digestates, revealed significantly higher proteolysis of B-IF at the end of gastrointestinal digestion compared to the other formulae, as indicated by the significantly higher concentration of free amine groups. Furthermore, differences in structure formation during the gastric phase of digestion were observed, with formation of a more continuous, firmer coagulum by C-IF, while R-IF demonstrated no curd formation likely due to the extensive hydrolysis of these proteins during manufacture. Differences in digestive characteristics between formulae manufactured from these different protein sources may influence the bio-accessibility and bioavailability of nutrients, warranting additional study.

Tremella fuciform Polysaccharides: Extraction, Physicochemical, and Emulsion Properties at Different pHs.

The chemical composition, macromolecular characteristics, and structure of four types of Tremella fuciform polysaccharides (TPS) were analyzed, including one TPS that was extracted in the laboratory (L-TPS) and three commercial TPS. The effects of pH on the properties of TPS emulsions were investigated by analyzing their zeta potential, particle size, apparent viscosity, and stability. The results showed that L-TPS presented a higher percentage content of protein (2.33%) than commercial TPS (0.73-0.87%), and a lower molecular mass (17.54 × 106 g/mol). Thus, L-TPS exhibited the best emulsifying activity but gave poor emulsion stability. The droplet sizes and apparent viscosity of commercial TPS-stabilized emulsions were larger or higher in acidic environments. At pH 2, the apparent viscosity was the lowest for L-TPS. Commercial TPS emulsions were most stable at pH 6, while the L-TPS-stabilized emulsion was most stable at pH 2. The obtained results revealed that the emulsifying properties of TPS varied and the effects of pH on emulsion characteristics differed, as determined from the molecular mass, macromolecular characteristics, and structure. This research is useful for expanding the application of TPS as a novel food ingredient in emulsions.

A Review on the Effect of Calcium Sequestering Salts on Casein Micelles: From Model Milk Protein Systems to Processed Cheese.

Phosphates and citrates are calcium sequestering salts (CSS) most commonly used in the manufacture of processed cheese, either singly or in mixtures. Caseins are the main structure forming elements in processed cheese. Calcium sequestering salts decrease the concentration of free calcium ions by sequestering calcium from the aqueous phase and dissociates the casein micelles into small clusters by altering the calcium equilibrium, thereby resulting in enhanced hydration and voluminosity of the micelles. Several researchers have studied milk protein systems such as rennet casein, milk protein concentrate, skim milk powder, and micellar casein concentrate to elucidate the influence of calcium sequestering salts on (para-)casein micelles. This review paper provides an overview of the effects of calcium sequestering salts on the properties of casein micelles and consequently the physico-chemical, textural, functional, and sensorial attributes of processed cheese. A lack of proper understanding of the mechanisms underlying the action of calcium sequestering salts on the processed cheese characteristics increases the risk of failed production, leading to the waste of resources and unacceptable sensorial, appearance, and textural attributes, which adversely affect the financial side of processors and customer expectations.

Fructose-induced topographical changes in fructophilic, pseudofructophilic and non-fructophilic lactic acid bacterial strains with genomic comparison.

Fructophilic Lactic Acid Bacteria (FLAB), Fructobacillus fructosus DPC7238 and pseudofructophilic Leuconostoc mesenteroides DPC7261 and non-FLAB Limosilactobacillus reuteri DSM20016 strains were studied for their growth and morphological evolution as a function of increased fructose concentrations (0, 25, and 50% w/v) in the media. A comparison of the genomics of these strains was carried out to relate observed changes and understand fructose-rich adaptations. The viability of FLAB strains were reduced by approx. 50% at a 50% fructose concentration, while the Limosilactobacillus reuteri strain was reduced to approx. 98%. Electron microscopy demonstrated that FLAB strain, Fructobacillus. fructosus and pseudofructophilic Leuc. mesenteroides, were intact but expanded in the presence of high fructose in the medium. Limosilactobacillus reuteri, on the other hand, ruptured as a result of excessive elongation, resulting in the formation of cell debris when the medium contained more than 25% (w/v) fructose. This was entirely and quantitatively corroborated by three-dimensional data obtained by scanning several single cells using an atomic force microscope. The damage caused the bacterial envelope to elongate lengthwise, thus increasing width size and lower height. The cell surface became comparatively smoother at 25% fructose while rougher at 50% fructose, irrespective of the strains. Although Fructobacillus fructosus was highly fructose tolerant and maintained topological integrity, it had a comparatively smaller genome than pseudofructophilic Leuc. mesenteroides. Further, COG analysis identified lower but effective numbers of genes in fructose metabolism and transport of Fructobacillus fructosus, essentially needed for adaptability in fructose-rich niches.

X-ray computerized microtomography and confocal Raman microscopy as complementary techniques to conventional imaging tools for the microstructural characterization of Cheddar cheese.

This study explored the use of X-ray computerized microtomography (micro-CT) and confocal Raman microscopy to provide complementary information to well-established techniques, such as confocal laser scanning microscopy (CLSM), for the microstructural characterization of cheese. To evaluate the potential of these techniques, 5 commercial Cheddar cheese samples, 3 with different ripening times and 2 with different fat contents, were analyzed. Confocal laser scanning microscopy was particularly useful to describe differences in fat and protein distribution, especially between the 2 samples with different fat contents. The quantitative data obtained through image analysis correlated well with the nutritional information provided in the product labels. Conversely, micro-CT was more advantageous for studying the size and spatial distribution of microcrystals present within the cheese matrix. Two types of microcrystals were identified that differed in size, shape, and X-ray attenuation. The smallest, with a diameter of approximately 10 to 20 µm, were more abundant in the samples and presented a more uniform roundish shape and higher X-ray attenuation. Larger and more heterogeneous crystals with diameters reaching 50 µm were also observed in scarcer numbers and showed lower X-ray attenuation. Confocal Raman microscopy was useful not only for identifying the distribution of all these components but also allowed comparing the presence of micronutrients such as carotenoids in the cheeses and provided compositional information on the crystals detected. Small and large crystals were identified as calcium phosphate and calcium lactate, respectively. Overall, using micro-CT, confocal Raman microscopy, and CLSM in combination generated novel and complementary information for the microstructural and nutritional characterization of Cheddar cheese. These techniques can be used to provide valuable knowledge when studying the effect of milk composition, processing, and maturation on the cheese quality attributes.

Digestion of protein and toxic gluten peptides in wheat bread, pasta and cereal and the effect of a supplemental enzyme mix.

There has been an increasing interest in the relationship between wheat digestibility and potential toxicity to the host. However, there is a lack of understanding about temporal profile of digestion of wheat proteins from different food matrices under physiologically relevant conditions. In this study, digestion of three wheat-based foods (bread, pasta and cereal) was conducted based on the INFOGEST semi-dynamic protocol in the absence and presence of a commercial supplemental enzyme preparation (a Glutalytic® based supplement, which will be marketed as Elevase®). Protein hydrolysis (OPA- ortho-phthalaldehyde - assay), molecular weight distribution (SEC-HPLC) and potential toxicity (R5 antibody-based competitive ELISA), were assessed. Our results demonstrated that under normal conditions, the complexity of the food influenced the temporal profile of protein hydrolysis and gluten breakdown throughout simulated gastric and intestinal digestion. However, treatment with the enzyme supplement significantly and acutely increased protein hydrolysis and gluten degradation in the gastric stage, and this enhanced digestion was maintained into the intestinal environment. These findings highlight the limitations of temporal gastric proteolysis and gluten degradation under normal conditions to different food types. They also show that supplemental enzyme mixes can effectively accelerate the breakdown of protein and hydrolysis of toxic gliadin fractions from the early stages of gastric digestion, thereby reducing intestinal exposure and potentially limiting the sensitization of the host.

The type of gum arabic affects interactions with soluble pea protein in complex coacervation.

Complex coacervation is an encapsulation process involving two oppositely charged biopolymers. Since different compositions of gum arabic may affect its interaction with protein, we studied the complex coacervation of two types of gum arabic (GA) (Acacia senegal-GA1 and Acacia seyal-GA2) with soluble pea protein (SPP) through Zeta potential, turbidity, morphology, the secondary structure of SPP, UV/vis absorbance and thermodynamic parameters. The maximum formation of coacervates occurred at SPP:GA 3:1 (w/w) and pH 3.5-4.0 with changes in the secondary structure of SPP. GA1 combination resulted in higher binding constant, implying a stronger affinity between SPP and GA1. Entropy of 0.7 and 0.5 kJ/mol.K, and enthalpy of -151 and -95.5 kJ/mol were obtained for SPP:GA1 and SPP:GA2. The complex coacervation was spontaneous as proved by the negative values of the Gibbs free energy. GA1 resulted in stronger interactions with SPP, offering new alternatives for encapsulation of bioactive compounds.

Tannin-rich extracts improve the performance of amidated pectin as an alternative microencapsulation matrix to alginate.

Microencapsulation of tannin extracts through extrusion-gelation method was performed comparing two alternative encapsulation matrices: alginate and amidated pectin. The microstructure of the generated microbeads was studied, as well as their microencapsulation efficiency and release properties. Overall, pectin-based beads performed better than their alginate-based counterparts. This, combined with a greater incorporation of tannins in the feed formulations led to a higher tannin load in the final beads. The best microencapsulation efficiency was given by pectin microbeads loaded with 10% tannin extract (w/w), but the final tannin content could be further increased by adding a 20% (w/w) concentration of the extracts. During a 14-days storage, only a marginal loss of tannins was recorded for pectin-based microbeads. The results reveal that great potential exists in producing pectin-based microbeads in presence of tannins, which allow better loading capacities and improving structural properties, thanks to the interactions between the tannins and the amidated polysaccharide.

Microstructural Analysis of Whey/Soy Protein Isolate Mixed Gels Using Confocal Raman Microscopy.

This work explores the potential of confocal Raman microscopy to investigate the microstructure of mixed protein gel systems. Heat-set protein gels were prepared using whey protein isolate (WPI), soy protein isolate (SPI), and mixtures thereof, with a total of five different whey-to-soy protein ratios (100, 75, 50, 25, and 0%). These were analysed using confocal Raman microscopy, and different data analysis approaches were used to maximize the amount of structural and compositional information extracted from the spectral datasets generated, including both univariate and multivariate analysis methods. Small spectral differences were found between pure WPI and SPI gels, mainly attributed to conformational differences (amide bands), but SPI exhibited considerably greater auto-fluorescence than WPI. The univariate analysis method allowed for a rapid microstructural analysis, successfully mapping the distribution of protein and water in the gels. The greater fluorescence of the capsule-like structures found in the mixed gels, compared to other regions rich in proteins, suggested that these may be enriched in soy proteins. Further analysis, using a multivariate approach, allowed us to distinguish proteins with different levels of hydration within the gels and to detect non-proteinaceous compounds. Raman microscopy proved to be particularly useful to detect the presence of residual lipids in protein gels.

Characterization and gelling properties of a bioactive extract from Ascophyllum nodosum obtained using a chemical-free approach.

The bioactivity and gelling properties of a carbohydrate-rich algal extract obtained from locally harvested Ascophyllum nodosum seaweed using a chemical-free approach were investigated for its potential interest in food applications. Physicochemical characterisation and compositional analysis of the extract, using FTIR, biochemical methods and monosaccharide analysis, confirmed the presence of alginates and fucoidans, although the main polysaccharide present in it was laminarin. Significant amounts of phenolic compounds (~9 â€‹mg phloroglucinol/100 â€‹mg sample) were also detected. As a result, the extract exhibited good antioxidant activity. It also showed promising prebiotic potential, promoting the growth of beneficial Lactobacillus sp. and Bifidobacteria sp. when compared with commercial prebiotics, but not that of pathogenic bacteria such as E. coli or P. aeruginosa. The gelling properties of the raw extract were explored to optimize hydrogel bead formation by external gelation in CaCl2 solutions. This was enhanced at neutral to alkaline pHs and high extract and CaCl2 concentrations. The mechanical strength, nano- and microstructure of the hydrogel beads prepared under optimised conditions were determined using compression tests, synchrotron small- and wide-angle X-ray scattering (SAXS/WAXS) and scanning electron microscopy (SEM). It was concluded that the raw algal extract at neutral pH had potential for use as a gelling agent, although further enrichment with alginate improved the mechanical properties of the obtained gels. The advantages and disadvantages of applying the non-purified algal extract in comparison with purified carbohydrates are discussed.

The Impact of Formulation on Lutein, Zeaxanthin, and meso-Zeaxanthin Bioavailability: A Randomised Double-Blind Placebo-Controlled Study.

Lutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ) have been the focus of research and commercial interest for their applications in human health. Research into formulations to enhance their bioavailability is merited. This 6 month randomised placebo-controlled trial involving 81 healthy volunteers compared the bioavailability of different formulations of free L, Z, and MZ in sunflower or omega-3 oil versus L, Z, and MZ diacetates (Ld, Zd, and MZd) in a micromicellar formulation. Fasting serum carotenoids, macular pigment, and skin carotenoid score were analysed at baseline and 6 months. Serum L, Z, and MZ concentrations increased in all active interventions compared to placebo (p < 0.001 to p = 0.008). The diacetate micromicelle formulation exhibited a significantly higher mean response in serum concentrations of Z and MZ compared to the other active interventions (p = 0.002 to 0.019). A micromicellar formulation with solubilised Z and MZ diacetates is a promising technology advancement that enhances the bioavailability of these carotenoids when compared to traditional carotenoid formulations (ISRCTN clinical trial registration number: ISRCTN18206561).

Encapsulation of Plant-derived Bioactive Ingredients through Electrospraying for Nutraceuticals and Functional Foods Applications.

The electrospraying technique, which consists of electrohydrodynamic atomization of polymeric fluids, can be used to generate dry nano- and microparticles by subjecting a polymer solution, suspension or melt to a high voltage (typically in the range of 7-20 kV) electric field. This potential can be exploited for developing nano- and microencapsulation structures under mild temperature conditions. Thus, it constitutes a promising alternative to conventional microencapsulation techniques for sensitive ingredients, like most plant-derived bioactive compounds, especially for their application in the food sector. Given the importance of plants as one of the major sources of dietary bioactive compounds, significant attention has been recently paid to research the encapsulation of phytochemicals through novel techniques such as electrospraying, aiming to provide new tools for the development of innovative functional food products and nutraceuticals. In this review, the latest advances in the application of electrospraying for nano- and microencapsulation of phytochemicals are discussed, with a focus on their potential use in the food sector.

Improved antioxidant activity and oxidative stability of spray dried European eel (Anguilla anguilla) oil microcapsules: Effect of emulsification process and eel protein isolate concentration.

The objective of this study was to prepare European eel oil (EO) microcapsules using European eel protein isolate (EPI) as a wall material and investigate its oxidative stability. The EPI emulsions were obtained at different EO: EPI ratios (1:1, 1:2 and 1:4, w/w) and using two emulsification procedures: Homogenization (H) and homogenization followed by ultrasonication (HU) treatments. The microcapsules prepared by combining the two emulsification processes (HU) and at core and wall ratio of was 1:4 presented the smallest particles size and the greatest encapsulation efficiency (68.50%) and oxidative stability. Scanning electron microscopy (SEM) images proved the spherical shape of all microcapsules without fissure on the surface. The capsules exhibited an interesting antioxidant activity depending on the EO:EPI ratio, especially for the metal chelating potential. Thus, the effect of ultrasonication process and the EPI concentration on the characteristic, the stability and the antioxidant activity of the encapsulated EO has been proved.

Self-assembled gelatin-ι-carrageenan encapsulation structures for intestinal-targeted release applications.

In this work, natural biopolymeric encapsulation structures were developed through the self-assembly of gelatin and ι-carrageenan in aqueous solutions. The interactions of this binary system and of a ternary system containing a polyphenol-rich extract were deeply explored for the development of intestinal delivery systems. The processing of the structures (extrusion vs. freeze-drying) greatly influenced release properties, explained by the specific interactions between gelatin and polyphenols, thus allowing for tuning the processing conditions depending on the desired target application. Release was further controlled by incorporating a divalent salt, giving raise to extract-loaded ι-carrageenan/gelatin capsules with adequate release profiles for intestinal targeted delivery. These results demonstrate the potential of exploiting biopolymer interactions for designing bioactive delivery systems using environmentally friendly processes which do not involve the use of toxic or harsh solvents or cross-linkers.