High-solids enzymatic saccharification of starch-rich raw herbal biomass residues for producing high titers of glucose.

The bioresource utilization of herbal biomass residues (HBRs) has been receiving more attention. Herein, three different HBRs from Isatidis Radix (IR) and Sophorae Flavescentis Radix (SFR) and Ginseng Radix (GR) were subjected to batch and fed-batch enzymatic hydrolysis to produce high-concentration glucose. Compositional analysis showed the three HBRs had substantial starch content (26.36-63.29%) and relatively low cellulose contents (7.85-21.02%). Due to their high starch content, the combined action of cellulolytic and amylolytic enzymes resulted in greater release of glucose from the raw HBRs compared to using the individual enzyme alone. Batch enzymatic hydrolysis of 10% (w/v) raw HBRs with low loadings of cellulase (≤ 10 FPU/g substrate) and amylolytic enzymes (≤ 5.0 mg/g substrate) led to a high glucan conversion of ≥ 70%. The addition of PEG 6000 and Tween 20 did not contribute to glucose production. Furthermore, to achieve higher glucose concentrations, fed-batch enzymatic hydrolysis was conducted using a total solid loading of 30% (w/v). After 48-h of hydrolysis, glucose concentrations of 125 g/L and 92 g/L were obtained for IR and SFR residues, respectively. GR residue yielded an 83 g/L glucose concentration after 96 h of digestion. The high glucose concentrations produced from these raw HBRs indicate their potential as ideal substrate for a profitable biorefinery. Notably, the obvious advantage of using these HBRs is the elimination of the pretreatment step, which is typically required for agricultural and woody biomass in similar studies.

Effects of sodium selenite on the growth, biochemical composition and selenium biotransformation of the filamentous microalga Tribonema minus.

This study aimed to investigate the physiological and biochemical responses of filamentous microalga Tribonema minus to different Na2SeO3 concentrations and its selenium absorption and metabolism to evaluate the potential in treating selenium-containing wastewater. The results showed that low Na2SeO3 concentrations promoted growth by increasing chlorophyll content and antioxidant capacity, whereas high concentrations caused oxidative damage. Although Na2SeO3 exposure reduced lipid accumulation compared with the control, it significantly increased carbohydrate, soluble sugar, and protein contents, with the highest carbohydrate productivity of 117.97 mg/L/d at 0.5 mg/L Na2SeO3. Furthermore, this alga effectively absorbed Na2SeO3 in the growth medium and converted most of it into volatile selenium and a small part into organic selenium (predominantly as selenocysteine), showing strong selenite removal efficacy. This is the first report on the potential of T. minus to produce valuable biomass while removing selenite, providing new insights into the economic feasibility of bioremediation of selenium-containing wastewater.

Highly efficient glucose production from raw non-pretreated Chinese medicinal herbal residues via the synergism of cellulase and amylolytic enzymes.

Available literature on Chinese medicinal herbal residues (CMHRs) bioconversion highlights pretreatment prior to saccharification with cellulase without considering the presence of starch constituent. Herein, four commonly found CMHRs were tested for starch content, and it was found they all contained starch with content ranging from 4.74% to 16.78%. Hydrolysis of raw CMHRs with combined cellulase and amylolytic enzymes yielded increments of 16.85% to 26.51% in 48-h glucan conversion compared to cellulase alone. Further study showed 48-h glucan conversion of raw CMHRs outperformed that pretreated by water-ethanol successive extraction, ultrasound and acid, but underperformed alkali-pretreated CMHRs. Although increasing 48-h glucan conversion in the range of 7.40% to 24.10% compared to raw CMHRs, alkaline pretreatment demonstrated low glucose recovery and incurred additional cost, making it unfavorable. Saccharification of the four raw CMHRs with combined enzymes seems like a preferred option considering the elimination of high-cost pretreatment step.

Production of Cardamine violifolia selenium-enriched peptide using immobilized Alcalase on Fe3O4 modified by tannic acid and polyethyleneimine.

Enzymatic synthesis of selenium (Se)-enriched peptides is vital for their application in supplementing organic Se. However, the poor stability and reusability of the free enzyme impedes the reaction. In this work, a highly stable immobilized Alcalase was synthesized by immobilizing Alcalase on tannic acid (TA) and polyethyleneimine (PEI) modified Fe3O4 nanoparticles (NPs). The optimal immobilization conditions for immobilized Alcalase were found at a TA/PEI (v/v) ratio of 1 : 1, pH of 10, and temperature of 40 °C, and the results from scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier Transform Infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) characterization confirmed the successful immobilization of Alcalase. The results of an enzyme property test showed that immobilized Alcalase had higher thermal and pH stability than free Alcalase, and retained 61.0% of the initial enzyme activity after 10 repetitions. Furthermore, the organic Se content of Se-enriched peptide prepared through enzymatic hydrolysis of Cardamine violifolia (CV) protein with immobilized Alcalase was 2914 mg kg-1, and the molecular weight was mainly concentrated in 924.4 Da with complete amino acid components. Therefore, this study proposes the feasibility of immobilized enzymes for the production of Se-enriched peptides.

Formation of protein-anthocyanin complex induced by grape skin extracts interacting with wheat gliadins: Multi-spectroscopy and molecular docking analysis.

In order to broaden the application of grape skin anthocyanin extract (GSAE) in flour products, the interaction of gliadin (Gli) and GSAE were investigated. Seven anthocyanin components from GSAE were identified by HPLC-MS2, which could form complexes with Gli having different binding rates based on UV, FTIR and HPLC analysis. The fluorescence quenching experiment showed that GSAE was capable of efficiently quenching the intrinsic fluorescence of Gli through hydrophobic interactions, and the binding parameters were near to one unit at the given temperatures. Additionally, GSAE binding changed the conformational properties of Gli, increasing α-helix and ß-turn content, but decreasing ß-sheet and irregular coil content. The molecular docking suggested that Gli possessed various binding sites bound with different anthocyanin monomers, mainly depending on hydrogen bonds and hydrophobic interactions. These findings further proved the formation of Gli-GSAE complex, indicating the potential of anthocyanins as natural colorant.

Structure and stability analysis of antibacterial substance produced by selenium enriched Bacillus cereus BC1.

Microorganisms can produce many antibiotics against bacteria and fungi, which have been used as a potential choice of new antibiotics. In this paper, we studied the characteristics of antibacterial substances by Bacillus cereus BC1. The results showed that the acid-precipitated substance played the main role in antibacterial activity, and further characterization indicated that the antibacterial substance might be a lipopeptide substance. Then the antibacterial spectrum suggested that the antibacterial substance had an inhibitory effect on Gram-positive bacteria and fungi, while selenium-riched antibacterial substance of Bacillus cereus BC1 could significantly enhance the inhibition. Then the morphological effects of the antibacterial substance to indicator bacteria were determined. The effects of different treatment methods on the stability of antibacterial substances were studied and the results showed that the antibacterial substance was stable to heat, ultrasonic, and ultraviolet treatment, and their antibacterial activity would not be greatly affected. However, they were sensitive to pepsin. The optimum pH range of antibacterial activity was 3-5. This study may contribute to reusing the fermentation supernatant often discarded in the previous fermentation process. At the same time, the lipopeptide antibacterial substance extracted from the fermentation broth of selenium-enriched Bacillus cereus BC1 can be used in the development of antibiotics and biopesticides, and open up a new way for the control of plant diseases.

Effects of selenate on Se, flavonoid, and glucosinolate in broccoli florets by combined transcriptome and metabolome analyses.

Broccoli is a nutritious vegetable popular all over the world. This study investigated the effects of different concentrations of selenate (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mmol/L) on the selenium (Se), glucosinolate, and flavonoid contents of broccoli florets. Results showed that the total Se, selenomethionine, and methyl selenocysteine contents increased following selenate dosage. Interestingly, selenate treatment of 0.4 mmol/L decreased the flavonoid but increased the glucosinolate content. Metabolome analysis revealed changes in the individual contents of glucosinolates and flavonoids. Conjoint analysis of transcriptome and metabolome showed that the glucosinolate and flavonoid compounds were potentially regulated by two sulfate transporter genes (Sultr3;1 and Sultr4;2) and several cytochrome P450 genes (e.g., CYP71B21, CYP72C1, and CYP81F1). These new findings indicated that Se treatment may influence glucosinolate and flavonoid accumulation by regulating the expression of these genes. The results of this study provide some novel insights into the effects of Se on glucosinolates and flavonoids in broccoli florets and deepen our understanding of the regulatory network between some specific genes and these compounds.

Selenium yeast promoted the Se accumulation, nutrient quality and antioxidant system of cabbage (Brassica oleracea var. capitata L.).

The application of Se yeast as a Se source to cultivate Se-rich cabbage has a significant effect on cabbage growth and quality indices. Results showed that total plant weight, head weight, and head size in cabbage were notably increased by 48.4%, 88.3%, and 25.4% under 16 mg/kg Se yeast treatment, respectively. Compare with the control, a high proportion of 3874% of Se accumulation in cabbage head was also detected in 16 mg/kg Se yeast treatment. Selenocystine (SeCys2) and Methyl-selenocysteine (MeSeCys) were the main Se speciations in the cabbage head. Application of 8 mg/kg Se yeast improved cabbage quality and antioxidant system indices, including free amino acid, soluble sugar, ascorbic acid, phenolic acid, glucosinolates, and SOD activity, which had 81.6%, 46.5%, 34.9%, 12.3%, 44.8%, 25.2% higher than that of the control, respectively. In summary, considering 8 mg/kg Se yeast as the appropriate level of Se enrichment during cabbage cultivation. These findings enhanced our understanding of the effects of Se yeast on the growth and quality of cabbage and provided new insights into Se-enrichment vegetable cultivation.

Complexation of maltodextrin-based inulin and green tea polyphenols via different ultrasonic pretreatment.

Ultrasound has been applied in food processing for various purpose, showing potential to advance the physical and chemical modification of natural compounds. In order to explore the effect of ultrasonic pretreatment on the complexation of inulin and tea polyphenols (TPP), different frequencies (25, 40, 80 kHz) and output power (40, 80, 120 W) were carried out. According to the comparison in particle size distribution and phenolic content of different inulin-TPP complexes, it was indicated that high-intensity ultrasonic (HIU) treatment (25 kHz, 40 W, 10 min) could accelerate the interaction of polysaccharides and polyphenols. Moreover, a series of spectral analysis including UV-Vis, FT-IR and NMR jointly evidenced the formation of hydrogen bond between saccharides and phenols. However, the primary structure of inulin and the polysaccharide skeleton were not altered by the combination. Referring to field emission scanning electron microscopy (FESEM), the morphology of ultrasound treated-complex presented a slight agglomeration in the form of bent sheets, compared to non-treated sample. The inulin-TPP complex also revealed better stability based on thermogravimetric analysis (TGA). Thus, it can be speculated from the identifications that proper ultrasonic treatment is promising to promote the complexation of some food components during processing.

Ultrasound as a Promising Tool for the Green Extraction of Specialized Metabolites from Some Culinary Spices.

Spices are a popular food of plant origin, rich in various phytochemicals and recognized for their numerous properties. The aim of the study was to evaluate the antioxidant and antimicrobial activity, as well as the content of specialized metabolites, of aqueous extracts of three spice species--garlic (Allium sativum L.), ginger (Zingiber officinalle L.) and turmeric (Curcuma longa L.)--prepared by green extraction methods. Ultrasound treatment increased the chromaticity parameter b value of turmeric and ginger extracts, thus indicating a higher yellow color predominantly due to curcuminoids characteristic of these species. Ultrasound-assisted extraction significantly increased the content of total soluble solids, phenolic compounds, total carotenoids and vitamin C. The temperature of the system was also an important factor, with the highest (70 °C) conditions in ultrasound-assisted extraction having a positive effect on thermolabile compounds (vitamin C, phenolics, total carotenoids). For example, turmeric extract treated with ultrasound at 70 °C had up to a 67% higher vitamin C content and a 69.4% higher total carotenoid content compared to samples treated conventionally at the same temperature, while ginger extracts had up to 40% higher total phenols. All different concentrations of spice extracts were not sufficient for complete inhibition of pathogenic bacterial strains of Salmonella, L. monocytogenes and S. aureus; however, only garlic extracts had an effect on slowing down the growth and number of L. monocytogenes colonies. Spice extracts obtained by ultrasonic treatment contained a significantly higher level of bioactive compounds and antioxidant capacity, suggesting that the extracts obtained have significant nutritional potential and thus a significant possibility for phytotherapeutic uses.

Optimization of Bacillus cereus Fermentation Process for Selenium Enrichment as Organic Selenium Source.

Selenium is an essential trace element and micronutrient for human health. Application of organic selenium in plants and microorganisms as trace element supplement is attracting more and more attention. In this study, Bacillus cereus, an important probiotic, was used for selenium enrichment with sodium selenite as selenium source. The growth curve of B. cereus was investigated, and 150 µg/ml was selected as the concentration of selenium for B. cereus fermentation. With application of response surface methodology, the optimal fermentation conditions were obtained as follows: inoculation quantity of 7%, culture temperature of 33°C, and shaking speed of 170 rpm, leading to the maximal selenium conversion ratio of 94.3 ± 0.2%. Field emission scanning electron microscope and energy dispersive spectrometry evidenced that inorganic selenium had been successfully transformed. This study may contribute to get a strain with high Se conversion ratio, so as to extract organic selenium in the form of selenoprotein to be used for further application.

Effect of linear charge density of polysaccharides on interactions with α-amylase: Self-Assembling behavior and application in enzyme immobilization.

The co-existence of polysaccharides and enzymes in the food matrix could form complexes that directly influence the catalytic efficacy of enzymes. This work investigated the self-assembly behaviors of α-amylase and charged polysaccharides and fabricated the α-amylase/polysaccharides complex coacervates. The results showed that the linear charge density of polysaccharides had a critical impact on the complex formation, structure, and enzyme protection under acidic conditions. At low pH, α-amylase formed compact and tight coacervates with the λ-carrageenan. However, α-amylase/pectin coacervates dissociated when the pH was lower than 3.0. The optimized binding ratio of α-amylase/λ-carrageenan was 12:1, and α-amylase/pectin was 4:1. Finally, the α-amylase/λ-carrageenan complex coacervates effectively immobilized the enzyme and almost 70% of enzyme activity remained in coacervates after exposure to pH3.0 for 1 h. This study demonstrates that the change in the linear charge density of polysaccharides could regulate the enzyme-catalyzed process in food processing by a simple and fine-controlled method.

Protective effects of selenium-enriched peptides from Cardamine violifolia against high-fat diet induced obesity and its associated metabolic disorders in mice.

Selenium-enriched peptides from Cardamine violifolia (CSP) have excellent antioxidant functions but little is known about their effects on obesity and associated metabolic disorders in mice fed with a high-fat diet (HFD). In this study, C57BL/6 mice were fed a HFD with or without CSP supplementation (CSPL: 26 µg Se per kg bw per d; CSPH: 104 µg per kg bw per d) for 10 weeks. The results showed that both CSPL and CSPH could ameliorate overweight gain, excess fat accumulation, serum lipid metabolism, and insulin resistance. The potential mechanism might be associated with the increase in thermogenesis, reduced oxidative stress, and inflammation, which regulated the gene expression in lipid and cholesterol metabolism. In addition, CSPL and CSPH also maintained the intestinal integrity and modulated the gut microbiota. Increased Blautia in CSP may be involved in the protective effect against obesity. Furthermore, a distinct increase in Lactobacillus was exclusively found in CSPH, suggesting that a more effective function of CSPH on metabolic disorders might be through the synergism of Blautia and Lactobacillus. Spearman's correlation analysis revealed that these specific genera were significantly correlated with the metabolic improvements. Taken together, CSP supplementation prevented HFD-induced obesity and metabolic disorders, probably by ameliorating oxidative stress and inflammation, regulating metabolic genes, and modulating the gut microbiota compositions.

Molecular characteristics of kappa-selenocarrageenan and application in green synthesis of silver nanoparticles.

Selenium is an essential trace element in human body, and kappa-selenocarrageenan (Se-car) is an organic source of selenium supplement. To further utilize Se-car in food packaging, biotherapy or biosensor, the molecular information of Se-car was characterized here and multi-functional Ag NPs synthesized by Se-car were fabricated. Results of GPC-MALLS, FTIR, potentiometric titration, and intrinsic viscosity showed that Se-car was polymerized by nearly 22 basic units of disaccharide. Sixty-four percentage of sulfated groups (SO42-) in carrageenan was replaced by selenium acid (SeO32-), which belonged to weak acid resulting from a gradually decrease of ζ-potential with acidity process to pH 1.0. Besides, the capacity of biosynthesis silver nanoparticles (Ag NPs) by Se-car was studied and it made a comparison with κ-carrageenan. Results exhibited that Se-car could serve as an efficient reducing and capping agent for Ag NPs fabrication (remarked as Se-car@Ag). The kapp of Se-car@Ag NPs for catalyzing 4-NP degradation was 2.14 × 10-2 s-1. Antibacterial test revealed Se-car@Ag had an ability to inhibit the growth of Escherichia coli and Staphylococcus aureus. To combine the selenium health benefit and functional metal nanoparticles, Se-car@Ag might have potential applications in multiple areas like medicine, disease diagnostic, and drug delivery.

Optimization of Spray-Drying Process of Jerusalem artichoke Extract for Inulin Production.

Jerusalem artichoke is an important natural matrix for inulin production. In this experiment, response surface methodology (RSM) was employed to optimize the spray-drying parameters in order to determine the maximal inulin yield. For this study, three independent variables (heating temperature (Tª, 110-120 °C), creep speed (V, 18-22 rpm) and pressure (P, 0.02-0.04 MPa)) were used in the experimental design. Using the Box-Behnken design, the optimal parameters obtained were: drying temperature 114.6 °C, creep speed 20.02 rpm, and pressure: 0.03 MPa. The inulin yield, water content and particle size of inulin obtained by spray-drying and freeze-drying were compared. In this regard, the spray-dried inulin consisted of a white powder having a fine particle size, and the freeze-dried inulin had a pale-yellow fluffy floc. On the other hand, the drying methods had a great influence on the appearance and internal structure of inulin powder, since the spray-dried inulin had a complete and uniform shape and size, whereas the freeze-dried inulin had a flocculated sheet structure. The analysis showed that the spray-drying led to a higher inulin yield, lower water content and better surface structure than freeze-drying.

Transport of Flavanolic Monomers and Procyanidin Dimer A2 across Human Adenocarcinoma Stomach Cells (MKN-28).

It has been proven that A-type procyanidins, containing an additional ether bond, compared to B-type procyanidins are also bioavailable in vitro and in vivo. However, their bioavailability and absorption in the gastrointestinal tract remain uncertain. In this study, a model of the human adenocarcinoma stomach cell line (MKN-28) was established to explore the cellular transport of flavanolic monomers and procyanidin dimer A2, which were isolated from the litchi pericarp extract. After the integrity and permeability of the cell monolayer were ensured by measurement of the transepithelial electrical resistance and the apparent permeability coefficient for Lucifer yellow, the transportation of procyanidins A2 and B2, (-)-epicatechin (EC), and (+)-catechin (CC) was studied at pH 3.0, 5.0, or 7.0 in the apical side, with compound concentrations of 0.05 and 0.1 mg/mL based on the cytotoxicity test. High-performance liquid chromatography and liquid chromatography-mass spectrometry analyses indicated that EC, CC, and A2 were transported in the MKN-28 cell line from 30 to 180 min, while B2 showed no transport. The maximal transport efficiencies of EC, CC, and A2 were 23 ± 0.81, 13.16 ± 1.53, and 16.41 ± 1.36%, respectively, existing at 120, 180, and 120 min of transportation. Laser scanning confocal microscopy analysis presented the dynamic transmission of EC, in accordance with the result of concentration determination, suggesting that the A-type procyanidins are possibly absorbed through the stomach barrier, which is pH- and time-dependent.

Effects of Pulsed Electric Field Treatment on Compression Properties and Solutes Diffusion Behaviors of Jerusalem artichoke.

Jerusalem artichoke is widely used as raw material for industrial production of inulin. Pressing (compression) and diffusion are two effective technologies for bio-compounds' recovery from plants. In this work, pulsed electric field (PEF) treatment at 400, 600, and 800 V/cm during 100 ms was applied to facilitate juice and solutes recovery from Jerusalem artichoke. The application of PEF led to electroporation of cell membranes and enhanced the tissue compression/juice expression and solutes diffusion. The consolidation coefficient (calculated by application of semi-empirical model) of PEF treated sample at 800 V/cm was 6.50 × 10-7 m²/s, which is significantly higher than that of untreated sample (5.02 × 10-9 m²/s) and close to that of freeze-thawed sample. Diffusion experiments with PEF treated samples were carried out at 25, 50, and 75 °C. A PEF treatment of Jerusalem artichoke at 800 V/cm led to a similar diffusion behavior at 25 °C, compared to diffusion behavior obtained from untreated sample at 75 °C.

Degradation of anthocyanins and polymeric color formation during heat treatment of purple sweet potato extract at different pH.

Purple sweet potato anthocyanins are common natural pigments widely used in food industry, while they are often thermally processed in application. Degradation of anthocyanins, formation of polymers and color changes of purple sweet potato extract (PSPE) were investigated at 90 °C in the range of pH 3.0-pH 7.0. Data analysis indicated a first-order reaction for anthocyanins degradation in solutions with pH 3.0, 5.0 and 7.0 have half-lives of 10.27, 12.42 and 4.66 h, respectively. The polymeric color formation followed zero-order kinetics, progressively increasing with pH values. The color of PSPE were changed with heating time and pH value through visual observation and colorimetric characterization. Analysis by UV-Vis spectrophotometry and HPLC indicated that anthocyanins in solution with pH 3.0 changed from monomeric anthocyanin into new polymers during heat treatment. Degradation of anthocyanins was accompanied by an increase in polymeric color index, due to the formation of melanoidin pigments and condensation reactions.

Anti-hyperuricemic and nephroprotective effects of extracts from Chaenomeles sinensis (Thouin) Koehne in hyperuricemic mice.

Clinically, Chaenomeles sinensis (Thouin) Koehne (C. sinensis) has been used to treat hyperuricemia and gout. However, the exact mechanism of action is still unknown. In the present study, the ethyl acetate fraction of C. sinensis fruit extract (CSF-E) was separated. Potassium oxonate (PO)-induced hyperuricemic mice and normal mice were administered with CSF-E at 60, 120 and 180 mg kg-1, respectively for 7 days. Serum uric acid, creatinine and BUN levels, liver oxidative damage, and serum and hepatic XOD activities were primarily measured using assay kits. The evaluation of its nephroprotective effects was carried out by renal histopathological analysis. Simultaneously, renal protein levels of organic anion transporters, such as mURAT1 and mOAT1, were detected using western blotting to elucidate the possible mechanisms. The results showed that CSF-E could significantly inhibit XOD activities in both serum and liver (p < 0.05), decreasing uric acid, creatinine and BUN levels in serum, and increasing levels in the excretion of uric acid by down-regulated of mURAT1 and up-regulated mOAT1 protein expression of kidney in hyperuricemic mice. Moreover, PO-induced alterations in the levels of MDA, hepatic SOD and GSH-Px activities and renal inflammation damage in hyperuricemic mice were effectively recovered by CSF-E at 120 mg kg-1. CSF-E possessed anti-hyperuricemic and nephroprotective effects by suppressing XOD activity, improving renal function and regulating renal mURAT1 and mOAT1 protein expression, which resulted in beneficial effects on hyperuricemia and gout prevention.

Increasing Yield and Antioxidative Performance of Litchi Pericarp Procyanidins in Baked Food by Ultrasound-Assisted Extraction Coupled with Enzymatic Treatment.

Extraction with organic solvents is a traditional method to isolate bioactive compounds, which is energy-wasting and time-consuming. Therefore, enzyme and ultrasound treatments were combined to assist the extraction of oligomeric procyanidins from litchi pericarp (LPOPC), as an innovative approach to replace conventional extraction methods. Under optimum conditions (enzyme concentration 0.12 mg/mL, ultrasonic power 300 W, ultrasonic time 80 min, and liquid/solid ratio 10 mL/g), the yield of LPOPC could be improved up to 13.5%. HPLC analysis indicated that the oligomeric procyanidins (OPC) content of LPOPC from proposed extraction was up to 89.6%, mainly including (-)-epicatechin, procyanidin A1, A2, and A-type procyanidin trimer. Moreover, LPOPC powder was added in baked food to inhibit the lipid peroxidation. It was found that 0.2% (w/w) of LPOPC could maintain the quality of cookies in the first 7 days, by decreasing the peroxide values. The procyanidin dimers and trimers in LPOPC played more important roles as antioxidants compared to monomers during storage. The results also showed that the combined extraction process can be considered as a useful and efficient method for the extraction of functional components from other plant sources.