Enhancing the properties of soy protein isolate and dialdehyde starch films for food packaging applications through tannic acid crosslinking.

The utilization of naturally derived biodegradable polymers, including proteins, polysaccharides, and polyphenols, holds significant promise in addressing environmental concerns and reducing reliance on nonrenewable resources. This study aimed to develop films with enhanced UV resistance and antibacterial capabilities by covalently cross-linking soy protein isolate (SPI) with dialdehyde starch (DAS) through the incorporation of tannic acid (TA). The covalent crosslinking of TA with DAS and SPI was shown to establish a stable chemical cross-linking network. The tensile strength of the resulting SPI/DAS/15TA film exhibited a remarkable increase of 208.27 % compared to SPI alone and 52.99 % compared to SPI/DAS film. Notably, the UV absorption range of SPI/DAS/10TA films extended from 200 nm to 389 nm. This augmentation can be attributed to the oxidation of TA's phenolic hydroxyl groups to quinone under alkaline conditions, which then facilitated cross-linking with the SPI chain via Michael addition and Schiff base reactions. Furthermore, the film demonstrated robust antibacterial properties due to the incorporation of TA. Collectively, the observed properties highlight the significant potential of the SPI/DAS/10TA film for applications in food packaging, where its enhanced mechanical strength, UV resistance, and antibacterial characteristics can contribute to improved product preservation and safety.

Non-extractable polyphenols from blue honeysuckle fruit pomace with strong antioxidant capacity: Extraction, characterization, and their antioxidant capacity.

The aim of this study was to investigate a more practical method for obtaining non-extractable polyphenols (NEPPs) from blue honeysuckle fruit pomace. Three methods, namely acid, alkaline, and enzymatic hydrolysis, were utilized to extract NEPPs. The findings indicated that alkaline hydrolysis was the most effective method for releasing NEPPs, which demonstrated higher levels of total flavonoid content (TFC) and total phenolic content (TPC) from blue honeysuckle fruit pomace. Additionally, higher TPC and TFC levels were related to a stronger antioxidant capacity. Qualitative and quantitative analysis using HPLC-HR-TOF-MS/MS revealed that acid hydrolysis resulted in a greater concentration of certain phenolic acids, while alkaline hydrolysis yielded a higher concentration of flavonoids, and enzymatic hydrolysis produced a wider range of phenolic compositions. Despite the fact that enzymatic hydrolysis is considered a gentler method, the researchers concluded that alkaline hydrolysis was the most appropriate method for obtaining NEPPs from blue honeysuckle fruit pomace.

Comprehensive structural analysis of polyphenols and their enzymatic inhibition activities and antioxidant capacity of black mulberry (Morus nigra L.).

In this paper, the structures of polyphenols and their bioactivity of black mulberry (Morus nigra L.) cv. 'Heisang No. 1' were comprehensively analyzed. The 11 anthocyanins and 20 non-anthocyanin phenolic compounds were identified and quantified by liquid chromatography high-resolution time-of-flight mass spectrometry (LC-HR-TOF/MS2). The cyanidin-3-glucoside and cyanidin-3-rutinoside were the major anthocyanins in the black mulberry. In addition, the black mulberry showed potent antioxidant capacity as assessed by DPPH, ABTS, and FRAP assays. Black mulberry anthocyanins exhibited stronger inhibition activities against α-amylase, α-glucosidase, and lipase compared to non-anthocyanin polyphenols, with IC50 values of 1.10, 4.36, and 9.18 mg/mL, respectively. The total anthocyanin content of black mulberry crude extracts and anthocyanins was 570.10 ± 77.09 and 1278.23 ± 117.60 mg C3GE/100 g DW, respectively. Black mulberry may be a rich source of polyphenols, natural antioxidants, and effective antidiabetic substances with great potential in the food industry.

Polyphenols in twenty cultivars of blue honeysuckle (Lonicera caerulea L.): Profiling, antioxidant capacity, and α-amylase inhibitory activity.

The polyphenols extracted from 20 blue honeysuckle cultivars were comprehensively characterized and quantified by HPLC-DAD and HPLC-ESI-QTOF-MS2 analyses and evaluated for antioxidant capacity (ABTS, DPPH, FRAP) and α-amylase inhibitory activity. The 17 anthocyanins and 59 non-anthocyanin phenolics were characterized. Among them, cyanidin-3-glucoside, quercetin-3-galactoside, myricetin-3-galactoside, and 3-caffeoylquinic acid were the major polyphenols. These polyphenols not only contributed to the antioxidant capacity, but were also good α-amylase inhibitors. 'Lanjingling' showed the strongest antioxidant capacity evaluated by FRAP, while 'CBS-2' and '14-13-1' showed the strongest antioxidant capacity evaluated by ABTS and DPPH. All the twenty cultivars showed α-amylase inhibitory activity, and the IC50 values ranged from 0.12 ± 0.01 to 0.69 ± 0.02 mg/mL. 'Lanjingling' showed the most potent α-amylase inhibitory activity. Additionally, principal component analysis indicated that Lonicera. caerulea subsp. emkuyedao bred in Japan differed markedly in phenolics and bioactivity compared to the other four subspecies bred in China and Russia.

Further evaluation on structural and antioxidant capacities of soy protein isolate under multiple freeze-thaw cycles.

Multiple freeze-thaw (F-T) treatments could change a protein structure and affect its physicochemical activities. In this work, soy protein isolate (SPI) was subjected to multiple F-T treatments, and the changes in its physicochemical and functional properties were investigated. The three-dimensional fluorescence spectroscopy indicated that F-T treatments changed the structure of SPI, including an increase in surface hydrophobicity. Fourier transform infrared spectroscopy showed that SPI underwent denaturation, unfolding and aggregation due to the interchange of sulfhydryl-disulfide bonds and the exposure of hydrophobic groups. Correspondingly, the particle size of SPI increased significantly and the protein precipitation rate also increased from 16.69%/25.33% to 52.52%/55.79% after nine F-T treatments. The F-T treated SPI had a higher antioxidant capacity. Results indicate that F-T treatments may be used as a strategy to ameliorate preparation methods and improve functional characteristics of SPI, and suggest that multiple F-T treatment is an alternative way to recover soy proteins.

Fabrication and characterization of ß-carotene emulsions stabilized by soy oleosin and lecithin mixtures with a composition mimicking natural soy oleosomes.

Natural soy oleosomes are known to have a remarkable stability, given the advantage of their sophisticated membrane. The aim of the present study is to examine the concept of fabricating a ß-carotene emulsion stabilized by soy oleosin (OLE) and lecithin (LEC) mixtures mimicking the membrane composition of soy oleosomes while providing preferable stability and bioaccessibility. For this, the fabricated emulsion was characterized in terms of droplet size distribution, and emulsion structure, stability and digestion (release and absorption of lipophilic ß-carotene). Compared to SPI/LEC (10 : 1) stabilized emulsions, the OLE/LEC (10 : 1) mixture stabilized emulsion exhibited the highest emulsifying activity index (EAI) and emulsifying stability index (ESI) values, and higher encapsulation efficiency. Results show that the ß-carotene emulsion stabilized by OLE and LEC mixtures at the ratio of 10 : 1 (w/w) has the most uniform droplet distribution and highest stability. The in vitro gastrointestinal digestion test revealed that the ß-carotene emulsion stabilized by OLE and LEC mixtures was digested more rapidly than the emulsion stabilized by soy protein isolate (SPI) and LEC mixtures. In turn, the bioaccessibility and cellular uptake of ß-carotene were enhanced, resulting in a higher absorption, a desirable feature of nutrition delivery systems. Our results demonstrated a promising way to fabricate emulsions mimicking natural soy oleosomes.

Wheat germ-derived peptide ADWGGPLPH abolishes high glucose-induced oxidative stress via modulation of the PKCζ/AMPK/NOX4 pathway.

This study explores the antioxidative effect of a specific wheat germ-derived peptide on high glucose-induced oxidative stress in vascular smooth muscle cells (VSMCs) and the underlying mechanisms. The peptide ADWGGPLPH was identified by LC-MS/MS. The effects of this peptide on the production of ROS and the expression of oxidative stress signaling proteins in VSMCs were determined. STZ-induced mice were utilized to confirm the anti-oxidative and anti-diabetic cardiovascular disease effects of this peptide in vivo. The results showed that ADWGGPLPH significantly prevented high glucose-induced cell proliferation, decreased intracellular ROS generation, stimulated AMPK activity, inhibited the PKCζ, AKT and Erk1/2 phosphorylation, and suppressed NOX4 protein expression. In addition, ADWGGPLPH enhanced the antioxidant abilities and attenuated inflammatory cytokine generation in STZ-induced diabetic mice. Therefore, ADWGGPLPH prevents high glucose-induced oxidative stress in VSMCs by modulating the PKCζ/AMPK/NOX4 pathway.

Lipase catalysis of α-linolenic acid-rich medium- and long-chain triacylglycerols from perilla oil and medium-chain triacylglycerols with reduced by-products.

BACKGROUND: Medium- and long- chain triacylglycerols (MLCTs) are functional structural lipids that can provide the human body with essential fatty acids and a faster energy supply. This study aimed to prepare MLCTs rich in α-linolenic by enzymatic interesterification of perilla oil and medium-chain triacylglycerols (MCTs), catalyzed by Lipozyme RM IM, Lipozyme TL IM, Lipozyme 435, and Novozyme 435 respectively. RESULTS: The effects of lipase loading, concentration of MCTs, reaction temperature, and reaction time on the yield of MLCTs were investigated. It was found that the reaction achieved more than a 70% yield of MLCTs in triacylglycerols under the conditions of 400 g kg-1 MCTs and 60 g kg-1 lipase loading after equilibrium. A novel two-stage deodorization was also applied to purify the interesterification products. The triacylglycerols reach over 97% purity in the products with significant removal (P < 0.05) of the free fatty acids, and the trans fatty acids were strictly controlled at below 1%. There was more than 40% α-linolenic in the purified products, with long-chain fatty acids mostly occupying the desired sn-2 position in acylglycerols, which are more active in hydrolysis. CONCLUSION: A series of novel α-linolenic acid-rich medium- and long-chain triacylglycerols was prepared. Under appropriate reaction conditions, the yield of MLCTs in triacylglycerols was above 70%. A novel two-stage deodorization can be used to promote the elimination of free fatty acids and limit the generation of trans fatty acids. © 2020 Society of Chemical Industry.

Soybean-derived miRNAs specifically inhibit proliferation and stimulate apoptosis of human colonic Caco-2 cancer cells but not normal mucosal cells in culture.

MicroRNAs (miRNAs) are important regulators of gene expression in eukaryotes. Studies have shown that plant-derived miRNAs can be absorbed through diets and regulate gene expression in mammals. Although soybean-derived miRNAs have been reported, their biological functions are still unclear. In this study, we found that soybean-derived small RNAs (sRNAs) significantly inhibited the proliferation and stimulated the apoptosis of Caco-2 cells. Bioinformatics analysis indicated that the target gene set of soybean miRNAs was extensively enriched in cancer pathways. Besides, we obtained 8 target genes, including Transcription factor 7 (TCF7), associated with colon cancer through prediction. Further studies showed that gma-miR159a inhibited the proliferation of Caco-2 cells and played an important role in the inhibitory effect of sRNAs by inhibiting TCF7 protein, which are upregulated in colon cancer cells but not normal mucosal cells in culture. These findings provide a novel molecular mechanism of soybean-derived miRNAs for potential application in tumor prevention.

Purification and Characterization of Antioxidant Peptides from Alcalase-Hydrolyzed Soybean ( Glycine max L.) Hydrolysate and Their Cytoprotective Effects in Human Intestinal Caco-2 Cells.

This study aimed to purify and identify antioxidant peptides from the low-molecular-weight fraction (SPH-I, MW < 3 kDa) of Alcalase-hydrolyzed soybean ( Glycine max L.) hydrolysate and further evaluate the cytoprotective effects of synthesized peptides against oxidative stress in human intestinal Caco-2 cells. After purification by gel filtration chromatography and reversed-phase HPLC, four major peptides were sequenced by nano-LC-ESI-MS/MS as VVFVDRL (847 Da, SPH-IA), VIYVVDLR (976 Da, SPH-IB), IYVVDLR (877 Da, SPH-IC), and IYVFVR (795 Da, SPH-ID). The antioxidant peptides were synthesized and displayed desirable DPPH radical-scavenging activity (from 16.5 ± 0.5 to 20.3 ± 1.0 µM Trolox equivalent (TE)/µM), ABTS•+ radical-scavenging activity (from 3.42 ± 0.2 to 4.24 ± 0.4 mM TE/µM), ORAC (from 143 ± 2.1 to 171 ± 4.8 µM TE/µM), and FRAP (from 54.7 ± 1.2 to 79.0 ± 0.6 mM Fe2+/µM). Moreover, the synthesized peptides protected Caco-2 cells against H2O2-induced oxidative damage via significantly downregulating intracellular ROS generation and lipid peroxidation ( p < 0.05). Additionally, SPH-IC and SPH-ID statistically upregulated total reduced glutathione synthesis, enhanced activities of catalase and glutathione reductase, and suppressed ROS-mediated inflammatory responses via inhibiting interleukin-8 secretion ( p < 0.05).

Covalent conjugates of anthocyanins to soy protein: Unravelling their structure features and in vitro gastrointestinal digestion fate.

Seeking for healthier dietary foods without compromising their quality has become the first choice for consumers. Various components in foods are able to interact with each other and form conjugates to improve their functionality and nutritional value. The present study investigated on the formation and structural properties of covalently linked conjugates using soy protein isolate (SPI) and black rice anthocyanins at different ratios. The in vitro digestion fate of the conjugates under simulated gastrointestinal conditions was also evaluated. Fourier transform infrared spectroscopy analysis indicated that the secondary structures of SPI was changed upon conjugation with anthocyanins as there was a decrease in α-helix and ß-sheet content. Three-dimensional fluorescence also revealed that the tertiary structure of SPI was less compact after conjugation with anthocyanins as a result of the unfolding of polypeptide chains. The conjugates were shown to have higher degree of hydrolysis than SPI. However, transepithelial transport of peptides across Caco-2 cell monolayer was decreased in SPI after conjugation with anthocyanins. The results of this study suggest that incorporation of anthocyanins in SPI is a way to reconcile consumer demand for healthier foods with better quality and functionality.

Physicochemical and oxidative stability of a soybean oleosome-based emulsion and its in vitro digestive fate as affected by (-)-epigallocatechin-3-gallate.

Oleosomes, which are pre-emulsified oil bodies found naturally in plants, have excellent stability, therefore making their use more popular in the food industries. However, the mechanism of EGCG in regulating the physicochemical and oxidative stability, and digestion of oleosome emulsions is not yet clear. In this study, the effect of EGCG on the properties of soybean oleosome emulsions (SOE) was examined at different pH values (5.0, 7.0, and 9.0). EGCG was significantly more effective in maintaining the stability of SOE at pH 5.0 and 7.0 over the 14 days of storage, but less effective at pH 9.0. Furthermore, lipid oxidation of SOE at pH 7.0 was successfully retarded by incorporating EGCG, but not at pH 5.0 and 9.0. The in vitro gastrointestinal results suggested that EGCG retarded the digestion rate of SOE based on a 20% reduction in free fatty acid release. The results of this study will help food technologists to design slow-digestive oleosome-based products that will satisfy health-conscious consumers' demand for healthier food choices.

Antioxidant activity and protective effects of Alcalase-hydrolyzed soybean hydrolysate in human intestinal epithelial Caco-2 cells.

Soybeans are known as a promising source of bioactive peptides. However, knowledge on the antioxidant behaviors of soybean protein hydrolysate (SPH) in the human intestinal epithelium is limited. In this study, SPH was prepared with Alcalase and subsequently ultrafiltered into four peptide fractions as SPH-I (<3 kDa), SPH-II (3-5 kDa), SPH-III (5-10 kDa) and SPH-IV (>10 kDa). The antioxidant properties of SPH and membrane fractions were investigated using different chemical assays and their protective effects against oxidative stress were evaluated using H2O2-stressed human intestinal Caco-2 cells. Results showed that SPH-I exhibited the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (IC50 = 2.56 mg/mL) and reducing capacity while SPH-III had the best metal ion-chelating activity (IC50 = 0.29 mg/mL). Both SPH and the peptide fractions dose-dependently suppressed intracellular reactive oxygen species (ROS) accumulation induced by H2O2 in Caco-2 cells, but the strongest inhibitory effect was observed for SPH-I. Amino acid (AA) results revealed that SPH-I was rich in hydrophobic and antioxidant AAs, which could contribute to its stronger antioxidant properties. Additionally, SPH-I protected Caco-2 cells from H2O2-induced oxidative stress via inhibiting lipid peroxidation and stimulating antioxidant enzyme activities. These results suggest that SPH-I and constitutive peptides can be beneficial ingredients with antioxidant properties and protective effects against ROS-mediated intestinal injury.

3D confocal Raman imaging of oil-rich emulsion from enzyme-assisted aqueous extraction of extruded soybean powder.

The understanding of the structure morphology of oil-rich emulsion from enzyme-assisted extraction processing (EAEP) was a critical step to break the oil-rich emulsion structure in order to recover oil. Albeit EAEP method has been applied as an alternative way to conventional solvent extraction method, the structure morphology of oil-rich emulsion was still unclear. The current study aimed to investigate the structure morphology of oil-rich emulsion from EAEP using 3D confocal Raman imaging technique. With increasing the enzymatic hydrolysis duration from 1 to 3 h, the stability of oil-rich emulsion was decreased as visualized in the 3D confocal Raman images that the protein and oil were mixed together. The subsequent Raman spectrum analysis further revealed that the decreased stability of oil-rich emulsion was due to the protein aggregations via SS bonds or protein-lipid interactions. The conformational transfer in protein indicated the formation of a compact structure.

Deciphering the characteristics of soybean oleosome-associated protein in maintaining the stability of oleosomes as affected by pH.

The understanding of the behavior of natural oleosomes is very important for leading to advancements in liposome manufacturing. Thus, the aims of this work was to evaluate the stable behaviors of oleosomes with regards to a wide pH range (2.0-11.0) using soybean oleosomes. The conformation changes and surface hydrophobicity of soybean oleosome-associated protein, and as well as ζ-potential and particle size distribution of oleosome were investigated. The particle size and ζ-potential of oleosomes, and surface hydrophobicity of oleosome-associated proteins were found to be readily affected by pHs. The secondary structure of oleosome-associated proteins was more susceptible to alkaline treatment than acidic treatment. The most stable oleosomes suspension was occurred at pH9.0. The electrostatic repulsion and hydrophobic interaction simultaneously affected the stability of oleosome. At pH2.0 to 6.0, acidic treatment did not significantly change the secondary structure of oleosome-associated proteins. With an increased treatment pH of 8.0-11.0, the contents of α-helix and random coil structures of oleosome-associated proteins decreased, while the amount of ß-sheet structures increased. Results highlighted the interesting fact that the aggregation of oleosomes occurring at different pHs was attributed to the synergistic effects between conformation changes and interaction properties of oleosome-associated proteins.

Secondary Structure and Subunit Composition of Soy Protein In Vitro Digested by Pepsin and Its Relation with Digestibility.

In the present study, in vitro digestibility and structure of soybean protein isolates (SPIs) prepared from five soybean varieties were investigated in simulated gastric fluid (SGF), using FT-IR microspectroscopy and SDS-PAGE. The result indicated that ß-conformations were prone to be hydrolyzed by pepsin preferentially and transformed to unordered structure during in vitro digestion, followed by the digestion of α-helix and unordered structure. A negative linear correlation coefficient was found between the ß-conformation contents of five SPIs and their in vitro digestibility values. The intensities of the protein bands corresponding to 7S and 11S fractions were decreased and many peptide bands appeared at 11~15 kDa during enzymatic hydrolysis. ß-conglycinin was poorly hydrolyzed with pepsin, especially the ß-7S subunit. On the other hand, basic polypeptides of glycinin degraded slower than acidic polypeptides and represented a large proportion of the residual protein after digestion. 11S-A3 of all SPIs disappeared after 1 h digestion. Moreover, a significant negative linear correlation coefficient (r = -0.89) was found between the ß-7S contents of five SPIs and their in vitro digestibility values. These results are useful for further studies of the functional properties and bioactive properties of these varieties and laid theoretical foundations for the development of the specific functional soy protein isolate.

Changes in the color, chemical stability and antioxidant capacity of thermally treated anthocyanin aqueous solution over storage.

Many anthocyanin-containing foods are thermally processed to ensure their safety, and stored for some time before being consumed. However, the combination of thermal processing and subsequent storage has a significant impact on anthocyanins. This study aimed to investigate the color, chemical stability, and antioxidant capacity of thermally treated anthocyanin aqueous solutions during storage at 4, 25, 45, and 65 °C, respectively. Anthocyanin aqueous solutions were thermally treated before storage. Results showed that the degradation rate of anthocyanins in aqueous solutions was much faster than those in real food. The color of the anthocyanin aqueous solutions changed dramatically during storage. The anthocyanin aqueous solutions stored at 4 °C showed the best chemical stability. Interestingly, the antioxidant capacity of the anthocyanin aqueous solutions stored at lower temperatures remained the same; however, the antioxidant capacity of those thermally treated at 120 or 140 °C and stored at 45 or 65 °C significantly decreased.

Combined effect of pH and high temperature on the stability and antioxidant capacity of two anthocyanins in aqueous solution.

The stability of two cyanidin-based anthocyanins from black rice in an aqueous system containing them with a pH range of 2.2-6.0 was investigated at temperatures ranging from 100 to 165°C. Within these pH and temperature ranges, the stability of the two anthocyanins was found to decrease gradually with increasing pH. The lowest (8.99×10(-4)s(-1)) and highest (0.120s(-1)) degradation rate constants for cyanidin-3-glucoside were obtained at pH 2.2 & 100°C and pH 6.0 & 165°C, respectively, whereas those for cyanidin-3-rutinoside were 5.33×10(-4)s(-1) at pH 2.2 & 100°C and 7.39×10(-2)s(-1) at pH 5.0 & 165°C, respectively. Antioxidant capacity analysis was conducted on thermally processed anthocyanin solutions to further evaluate the effects of pH and thermal treatment on the anthocyanins. The total antioxidant capacity of samples after thermal treatments under various pHs was found to remain at a similar level.

Blending of soybean oil with selected vegetable oils: impact on oxidative stability and radical scavenging activity.

BACKGROUND: Soybean oil may protect against cancer of the breast and prostate. It may also exert beneficial influence in combination with other oils. Here, blends (20%, v/v) of sea buckthorn oil (SEBO), camellia oil (CAO), rice bran oil (RBO), sesame oil (SEO) and peanut oil (PEO) with soybean oil (SBO) were formulated. MATERIALS AND METHODS: Oxidative stability (OS) and radical scavenging activity (RSA) of SBO and blends stored under oxidative conditions (60°C) for 24 days were studied. By blending with different kinds oils, levels of polyunsaturated fatty acids (PUFA) decreased, while monounsaturated fatty acid (MUFA) content increased. Progression of oxidation was followed by measuring peroxide value (PV), p-anisidine (PAV), conjugated dienes (CD) and conjugated trienes (CT). RESULTS: Inverse relationships were noted between PV and OS at termination of storage. Levels of CD and CT in SBO, and blends, increased with increase in time. The impact of SEO as additives on SBO oxidation was the strongest followed by RBO, CAO, SEBO and PNO. CONCLUSIONS: Oxidative stability of oil blends was better than SBO, most likely as a consequence of changes in fatty acids and tocopherols' profile, and minor bioactive lipids found in selected oils. The results suggest that these oil blends could contribute as sources of important antioxidant related to the prevention of chronic diseases associated to oxidative stress, such as in cancer and coronary artery disease.

Monte Carlo modelling of non-isothermal degradation of two cyanidin-based anthocyanins in aqueous system at high temperatures and its impact on antioxidant capacities.

The non-isothermal degradation of two cyanidin-based anthocyanins, cyanidin-3-glucoside and cyanidin-3-rutinoside, was investigated in aqueous system within the temperature range from 100 to 165 °C, and the degradation kinetics was modelled using Monte Carlo simulation. The two anthocyanins showed different stability, with cyanidin-3-glucoside exhibiting a higher degradation rate than cyanidin-3-rutinoside. The derived degradation rate at the reference temperature of 132.5 °C and activation energy of cyanidin-3-glucoside and cyanidin-3-rutinoside were 0.0047 and 0.0023 s(-1), and 87 and 104 kJ/mol, respectively. The antioxidant capacity of the thermally processed anthocyanins solutions was measured by using ABTS and DPPH assays. Results showed that the antioxidant capacity of the samples remained at the same level during the thermal treatment process under various conditions, i.e., there was no significant difference (P>0.05) in the antioxidant capacity amongst the samples despite their significantly different contents of the two anthocyanins.