Mannoproteins Research Articles

Extraction of mannoprotein from Saccharomyces cerevisiae and analysis of its chemical composition and molecular structure

The purpose of this study was to screen the method with the highest yield of mannoprotein (MP) without damaging their functional structure. The recovery rates of the extracts, protein, and mannose were determined along with the mannose/protein ratio, molecular weight and distribution, monosaccharide and amino acid compositions, and secondary structures of the three MP extracts to compare the extraction methods. The MP extracts recovery rate prepared by the thermal method was significantly higher (35.89%) than those obtained using fermentation (31.66%) and SDS treatment (19.77%). Three protein bands with similar molecular weights of 59, 47, and 34 kDa were detected in the MPs obtained via the different extraction methods. The thermally extracted MP has a broader molecular weight distribution. After purification, the proportion of mannose in the polysaccharide parts of the MPs increased from 6–7 to 90.4–91.3%. The essential amino acid content of the hot-extracted MP (170.07 mg/g) was the highest. The thermally extracted MP had similar secondary structural characteristics to that isolated at room temperature, and had a higher protein characteristic peak intensity. In general, the heating method to extract yeast mannoprotein is time-saving and efficient.

Structural characteristics of Saccharomyces cerevisiae mannoproteins: Impact of their polysaccharide part

While they have many properties of interest in enology, the structure-function relationships of mannoproteins and the part played by their polysaccharide moiety are not yet well understood. Mannoproteins (MP) extracted with β-glucanase from a laboratory yeast strain (WT), two of its mutants (Mnn2 with unbranched N-glycosylated chains and Mnn4 without mannosyl-phosphorylation), and an enological strain (Com) were purified and thoroughly characterized. The protein moiety of the four MPs had the same amino acid composition. Glycosyl-linkage and net charge analyses confirmed the expected differences in mutant strain MPs. MP-Com had the highest mannose/glucose ratio followed by MP-WT/MP-Mnn4, and MP-Mnn2 (13.5 > 5.6 ≈ 5.2 > 2.2). The molar mass dependencies of Rg, Rh, and [η], determined through HPSEC-MALLS-QELS-Viscosimetry, revealed specific conformational properties of mannoproteins related to their nature of highly branched copolymers with two branching levels. It also clearly showed structural differences between MP-Com, MP-WT/Mnn4, and MP Mnn2, and differences between two populations within the four mannoproteins.

Mannoproteins interfering wine astringency by modulating the reaction between phenolic fractions and protein in a model wine system

The positive effect of mannoproteins (MPs) on wine astringency is of great interest during winemaking. Phenolic components with different structural characteristics in wine could elicit different intensities of astringency when combined with salivary proteins. Aiming to use commercial MPs to modulate the unbalanced wine astringency, this work selected three commercial MPs and phenolic fractions (phenolic acids, monomeric/oligomeric procyanidins, polymeric procyanidins) with different mDP extracted from wine. The relationship between the structure and concentration of MPs, phenolic fractions, and astringency was investigated by physicochemical techniques (fluorescence quenching, SDS-PAGE, dynamic light scattering, and isothermal titration calorimetry) and sensory evaluation. Results showed that 0.6 g/L of MPs played a role in reducing wine astringency. MPs were more effective in inhibiting the interaction between monomeric/oligomeric or polymeric procyanidins and proteins than that of phenolic acids, through ternary or competitive mechanisms. Moreover, MPs with high protein portion and high molecular weight exhibited a stronger binding capacity to phenolics or protein, thus showing competing effect with phenolics for binding to protein. This work could provide the wine industry with valuable information for optimizing the application of commercial MPs to modulate wine astringency.

Effect of Sonication Treatment and Maceration Time in the Extraction of Polysaccharide Compounds during Red Wine Vinification.

The application of high-power ultrasounds (US) at 28 kHz to the crushed grapes and the use of different pomace contact times caused changes in the content and composition of monosaccharides and polysaccharides in the musts and wines. These differences were maintained from the moment of pressing (end of maceration) until the end of the alcoholic fermentation. The US increased the content of monosaccharides and polysaccharides in the musts by facilitating their extraction from the solid parts during maceration. The application of medium maceration time (3 days) to sonicated grapes led to an extraction of polysaccharides rich in arabinose and galactose, rhamnogalacturonan type II (RG-II) and mannoproteins (MP), similar to that observed in the control wines made with an extended maceration of 7 days (968.21 vs. 1029.45; 895.04 vs. 1700.50; 356.81 vs. 343.95, respectively). This fact was attributed to a higher extraction in the must during the sonication process and to an important release of pectic polysaccharides during the pressing of the sonicated pomace, which is reported here for the first time. Therefore, the US technology could be useful for increasing the polysaccharide content in the wines or for reducing the maceration time needed to achieve certain levels of wine polysaccharides.

Anthocyanin and Phenolic Acids Contents Influence the Color Stability and Antioxidant Capacity of Wine Treated With Mannoprotein.

Wine is consumed by humans worldwide, but the functional components are lost and the color changes during its production. Here, we studied the effects of mannoprotein (MP) addition (0, 0.1, and 0.3 g/L) upon crushing and storage. We measured anthocyanins, phenolic acids profiles, color characteristics, and antioxidant activities of wine. The results showed that the addition of MP before fermentation significantly increased the total phenolic content (TPC), total anthocyanin content, total tannin content (TTC), total flavonoid content, and total flavanol content in wine, whereas the addition of MP during storage had the opposite effect. The addition of MP before alcohol fermentation significantly increased the amount of individual anthocyanins and individual phenolic acids, maintained the color, and increased the antioxidant capacity of wine. In addition, the addition of 0.3 g/L MP during storage increased the content of individual phenolic acids and TPC of wine. However, the addition of 0.1 g/L MP during storage significantly reduced the TPC, TAC, TTC, and individual anthocyanin content (except for malvidin-3-glucoside and malvidin-3-acetly-glucoside); meanwhile, the treatment attenuated the color stability and antioxidant capacity of wine. The results demonstrated that the addition of MP before alcohol fermentation could increase the functional components and improve the color stability and antioxidant capacity of wine.

In-silico Studies on Virulence Factors of Cryptococcus Species: Phylogenetic Analysis and B-cell Epitope Prediction

Virulence proteins ensure the survival of Cryptococcus in its host. The epitopes present in these virulence factors can modulate the host's immune system and contribute tocryptococcosis's pathobiology significantly. The amino acid sequences of virulence factors (glucuronoxylomannan (GXM), superoxide dismutase (SOD), mannoprotein (MP), urease, CAP binding protein, galactoxylomannan (GalXM), phospholipase-B, and laccase) of C. neoformans, C. n. grubii, and C. gattii were retrieved from NCBI. Analyses of the phylogenetic relationship between virulence factors were performed by using PhyML software and JMP 13.1 software. Further, ABCpred, BCPred, BcePred web servers were employed for the prediction of linear B-cell epitopes in amino acid sequences of said virulence factors. In all the three Cryptococcus species, laccase, CAP binding protein, and mannoprotein were highly conserved compared to GalXM, GXM, and SOD virulence factors. Superoxide dismutase (SOD) with the lowest gamma distribution value is considered to be highly adaptable. Further, the maximum number of B-cell epitopes was observed on the urease of C. n. grubii. In due course of time, Cu, Zn Superoxide dismutase (SOD) might play the main role in Cryptococcus species' pathogenicity due to its highly variable nature. Additionally, urease could be used to design epitope-based anti-cryptococcal drugs. Nonetheless, the results of this in-silico study need wet lab validation.

Ultrasound treatment of crushed grapes: Effect on the must and red wine polysaccharide composition

This paper studied the effect on the molecular weight and polysaccharide composition of musts and wines of the application of high-power ultrasound (US) at 20 and 28 kHz on crushed grapes. Two different pomace maceration times (short and mid) were tested for sonicated and control vinifications. A long pomace maceration time was also tested for non-treated wines. In must samples, US significantly increased the content of monosaccharides and polysaccharides rich in arabinose and galactose (PRAG), and the average molecular weight of smaller PRAG, mannoproteins (MP) or mannans. The 28 kHz had a major effect on most wine monosaccharides and grape polysaccharides. The wine obtained from sonicated grapes at 28 kHz and with mid maceration had higher rhamnogalacturonans type II and PRAG content than its control, and closer polysaccharide and monosaccharide content to long maceration control wines. No significant differences were obtained in the MP content between sonicated and control wines.

Effect of different yeast mannoproteins on the interaction between wine flavanols and salivary proteins

Unbalanced wine astringency, caused by a gap between phenolic and technological grape maturities, is one of the consequences of the global climate change in the vitiviniculture. To resolve it, potential strategies are being currently used, like the addition of commercial yeast mannoproteins (MPs) to wines. In this work, the main interactions responsible for the wine astringent sensation, namely, interactions between human salivary proteins and wine flavanols have been studied by Dynamic Light Scattering (DLS) and liquid chromatography coupled to DAD and MS detectors (HPLC–DAD–MS), in presence or absence of two MPs with different saccharide/protein ratio. The results indicate that there are differences on the substrate specificity for each mannoprotein and that its action mechanism could change not only depending on the mannoprotein composition but also on the flavanol structure. MPs with elevated carbohydrate content could act thought the stabilization of soluble aggregates with human salivary proteins and flavanols, mainly non-galloylated flavanol oligomers, whereas MPs with higher protein percentage mostly could precipitate flavanols (mainly non-galloylated ones with low degree of polymerization) which partially prevents the formation of insoluble flavanol–salivary protein aggregates.

A Predicted Mannoprotein Cmp1 Regulates Fungal Virulence in Cryptococcus neoformans.

The capsule of the fungal pathogen Cryptococcus neoformans consists of glucuronoxylomannan (GXM), glucuronoxylomannogalactan (GXMGal), and mannoproteins (MPs). MPs are a kind of glycoproteins with low content but high immunogenicity, which can stimulate the immune protection of the host. However, there is not much information about the role of mannoproteins in virulence of the human fungal pathogen C. neoformans. In this study, we reported the identification and functional analysis of a predicted mannoprotein Cmp1 that regulates fungal virulence in C. neoformans. Gene expression pattern analysis indicates that the CMP1 gene was ubiquitously expressed at all stages of cryptococcal development. Subcellular localization analysis indicated that Cmp1 was localized in the cytoplasm of cryptococcal cells. Disruption or overexpression of CMP1 results in impairing capsule formation in Cryptococcus, but it does not affect the melanin production and sensitivity under various stress conditions, nor does it affect the sexual reproduction process of Cryptococcus. Survival assay showed that the pathogenicity of the cmp1Δ mutant or the CMP1 overexpression strain was significantly attenuated in a murine inhalation model of cryptococcosis. In conclusion, our findings implied that the mannoprotein Cmp1 is required for the virulence of C. neoformans.

Coronary Vasculitis Induced in Mice by the Cell Wall Mannoprotein of <i>Candida krusei</i>

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances, such as the hot water extract of C. albicans (CADS) and Candida water-soluble fraction (CAWS), induced coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the hot water extract of C. krusei, inherently resistant to fluconazole, induces vasculitis in mice. Three strains of C. krusei, NBRC1395, NBRC1162, and NBRC10737, were cultured in natural (Y) and chemically defined (C) media and cell wall mannoprotein (MN) fractions were prepared by autoclaving cells (CKY1395MN, CKC1395MN, CKY1162MN, CKC1162MN, CKY10737MN, and CKC10737MN). All MN fractions reacted strongly with Concanavalin A (Con A) and dectin-2 and induced anaphylactoid shock in ICR mice. MNs induced severe coronary vasculitis in DBA/2 mice, resulting in cardiac hypertrophy. MNs also induced coronary vasculitis in C57Bl/6 mice. These results suggest that the MNs of non-albicans Candida, such as C. krusei, induce similar toxicity to those of C. albicans.

Influence of the mannoproteins of different strains of <em>Starmerella bacillaris</em> used in single and sequential fermentations on foamability, tartaric and protein stabilities of wines

Aim: In this work, seven strains of Starmerella bacillaris were analysed for their ability to release polysaccharides during alcoholic fermentation (AF), both in single-strain and in sequential AF together with Saccharomyces cerevisiae.Methods and Results: A synthetic polysaccharide-free must was used to characterise the mannoproteins (MPs) released. The MPs were quantified, characterised in terms of carbohydrate composition, and tested to assess their ability to reduce protein and tartrate instabilities and their ability to affect the foaming properties of wine.Conclusions: All the tested strains in sequential AF increased the total MPs production. Moreover, the strains affected the MPs properties in different ways regarding tartaric and protein stabilities. The MPs released in sequential AF by some S. bacillaris strains showed a significant effect on protein stabilisation and tartaric stability. An effect on the foamability was found for MPs obtained in single-strain AFs of S. bacillaris.Significance and impact of the study: An improvement in wine stability can be achieved using the sequential AF.

Feeding of yeast cell wall extracts during a necrotic enteritis challenge enhances cell growth, survival and immune signaling in the jejunum of broiler chickens

Necrotic enteritis (NE) is one of the most common and costly diseases in the modern broiler industry, having an estimated economic impact of $6 billion dollars annually. Increasing incidents of NE have resulted from restrictions on the use of antibiotic feed additives throughout the broiler industry. As such, finding effective antibiotic alternatives has become a priority. In this study, an experimental model of NE was used, comprising a commercial infectious bursal disease virus vaccine and Clostridium perfringens (C. perfringens) inoculation. Yeast cells wall (YCW) components, β-glucan (BG), and mannoproteins (MPTs) were evaluated for their effects on disease development. Chicken-specific immunometabolic kinome peptide arrays were used to measure differential phosphorylation between control (uninfected), challenged (infected), and challenged and treated birds in duodenal, jejunal, and ileal tissues. Treatment groups included crude YCW preparation, BG, MPT, or BG+MPT as feed additives. Data analysis revealed kinome profiles cluster predominantly by tissue, with duodenum showing the greatest relative signaling and jejunum showing the greatest response to treatment. BG, MPT, and BG+MPT cluster together, separate from controls and challenge birds in each tissue. Changes in signaling resulting from the treatments were observed in cell growth and survival responses as well as immune responses. None of the treatments of disease challenge returned the profiles to control-like. This is attributable to immune modulation and metabolic effects of the treatments generating distinct profiles from control. Importantly, all the treatments are distinct from the challenge group despite being challenged themselves. Only BG+MPT treatment had a significant effect on bird weight gain compared with the NE challenge group, and this treatment had the greatest impact on gut tissue signaling in all segments. The signaling changes elicited by BG+MPT during an NE challenge were increased cell growth and survival signaling, reducing cell death, apoptosis and innate inflammatory responses, and generating compensatory signaling to reduce disease severity.

Cell Wall Mannoproteins from Yeast Affect Salivary Protein-Flavanol Interactions through Different Molecular Mechanisms.

It is known that interactions between wine flavanols and salivary proline-rich proteins (PRPs) are one of the main factors responsible for wine astringency. The addition of commercial yeast mannoproteins (MPs) to wines has been pointed to as a possible tool to modulate the excessive astringency due to a lack of phenolic maturity at harvest time that might occur as a consequence of global climate change. The aim of this work was to study by isothermal titration calorimetry and molecular dynamics simulation the molecular mechanisms by which mannoproteins could modulate astringency elicited by tannins and if it can be influenced by mannoprotein composition. Results obtained indicate that the MPs assayed had an important impact on astringency through the formation of ternary aggregates with different solubilities or by preventing the flavanol-PRP interaction by a competitive mechanism, although in a different strength, depending on the size and the compositional characteristic of the mannoprotein.

Mannoproteins from Saccharomyces cerevisiae stimulate angiogenesis by promoting the akt-eNOS signaling pathway in endothelial cells

Mannoproteins (MPs) are a major component of yeast cell walls and consist of high levels of mannose in covalent complexes with proteins. MPs complexly enhance the immune system. We previously isolated a mutant yeast, K48L3, with a higher yield of MP from its cell wall than wild-type Saccharomyces cerevisiae, YPH499. We determined that K48L3 induces the release of nitric oxide in macrophage cells. The present study reports nitric-oxide-mediated angiogenesis by MP from K48L3 and the induction of the Akt/eNOS signal pathway. Western blotting and RT-PCR were used to demonstrate that MP treatment resulted in the upregulation of p-Akt, p-eNOS, and angiogenesis-mediated gene expression. Moreover, the angiogenesis activity of the MPs was demonstrated using three angiogenesis assays, namely, a cell migration assay, a tube-forming assay, and an ex vivo aorta ring assay. Thus, this study demonstrates for the first time that MPs from S. cerevisiae K48L3 induce angiogenesis in HUVECs via the Akt-eNOS-dependent signaling pathway.

Monitoring the effects and side-effects on wine colour and flavonoid composition of the combined post-fermentative additions of seeds and mannoproteins.

One of the main consequences of the advancement of harvest date associated to global climate change is that the phenolic maturity of grapes can be delayed in relation to their technological maturity. As a consequence, wines made from these grapes can be poor in phenolic compounds or possess an unbalanced phenolic composition, affecting their global quality. The combined post-fermentative addition of seeds and an astringency-modulator mannoprotein (MP) might be a potential strategy to solve this problem, since seeds might supply flavanols and improve wine chemical stability and the mannoprotein might modulate the changes induced in astringency by the addition of seeds and improve wine colloidal stability. The present study aimed at monitoring at different moments of winemaking and ageing the effects and side-effects of this combined strategy on the detailed flavanol, flavonol and anthocyanin compositions and on colour of wines made from Syrah grapes. Seeds were obtained from Pedro Ximénez overripe grapes. Flavanol composition and flavonol and anthocyanin compositions were determined by HPLC-MSn-MRM and HPLC-DAD-MSn analyses, respectively. Colour changes caused by these additions were studied from CIELAB parameters as well as the ability of these techniques to protect colour from bleaching agents, such as SO2. In general, the addition of seeds initially increased the levels of flavanols and anthocyanins. However, during bottle ageing a reduction in the levels of flavanols, flavonols and anthocyanins could be observed in seed treated wines, which might be related to the greater formation of flavanol aggregates associated with greater levels of flavanols. This effect was partially solved for most of the flavonoids studied with the additional MP treatment. Treated and control wines showed colour differences that were visible to human eye, although they were reduced over time. At the end of the study, treated wines showed colour parameters corresponding to younger wines than those observed in control wines. A greater resistance against SO2 bleaching was also observed in treated wines, which can be mainly associated to the greater percentages of polymeric pigments caused by seed treatment and to the improvement of the colloidal stability of SO2-resistant pigments caused by the addition of the MP.

Wine polysaccharides modulating astringency through the interference on interaction of flavan-3-ols and BSA in model wine

Polysaccharides and flavan-3-ols from Cabernet Sauvignon wine were isolated and then characterized by high performance liquid chromatography coupled with diode array detector (HPLC-DAD), high performance size exclusion chromatography (HPSEC) and HPLC-MS. The influence of purified polysaccharides on the aggregation of flavan-3-ols-proteins were investigated using fluorescence spectroscopy, circular dichroism (CD) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate the co-effect of polysaccharides on wine astringency. The results indicated that mannoproteins (MPs) and rhamnogalacturonans II (RG II) were the major polysaccharides to significantly alter the interaction of flavan-3-ols with bovine serum albumin (BSA). The interaction of polysaccharides-flavan-3-ols-BSA was enhanced with the concentration (from 0.2 to 0.6 g/L) of the studied polysaccharides. Furthermore, the reaction of BSA-flavan-3-ols was strengthened in the absence of polysaccharides when percentage of galloylation (%G) was higher (11.29). When mean degree of polymerization (mDP) of flavan-3-ols was from 5.14 to 6.88, the secondary structure of proteins was changed from mainly α-helix to random curl and the formation of protein precipitation increased. This work pointed out that the important property of polysaccharides and different structural characteristic flavan-3-ols are able to modulate wine astringency perception.

Characterization of the composition and the techno-functional properties of mannoproteins from Saccharomyces cerevisiae yeast cell walls

Broad molecular weight (MW) distribution and variability of mannan to protein ratio of purified mannoproteins (MP), isolated from yeast cell walls upon the enzymatic treatment, revealed their multiplicity. The main fraction of high-MW Agrimos®-MP1 and YCW-b-MP1′ contained mannoproteins with a mannan to protein ratio of 3.5 and 6.9, respectively. Low-MW YCW-b-MP2′ was mainly comprised of mannan, with a ratio of 181, whereas low-MW Agrimos®-MP2 was characterized by a ratio of 12.2. The solubility of MP1/MP2 was higher than that of MP1′/MP2′. Mannoproteins showed similar or lower solubility than mannan, and they exhibited a Newtonian behaviour. Sonication was the appropriate method for the formation of mannoproteins-based emulsions. Contrary to MP1/MP1′-based emulsions, MP2/MP2′-based ones showed higher affinity towards soybean oil than glyceryl-trioleate. pH affected the emulsifying ability of MP1/MP1′. MP1/MP1′ showed similar or slightly inferior emulsifying properties than lecithin. This study is expected to broaden the applications of mannoproteins as value-added ingredients.

Candida albicans cell wall mannoprotein synergizes with lipopolysaccharide to affect RAW264.7 proliferation, phagocytosis and apoptosis

With the widespread use of invasive surgery, immunosuppressive therapy and broad-spectrum antibiotics, there has resulted a corresponding increase in severe systemic infections as produced by Candida albicans (C.albicans), as it combines with bacterial infections. Such infections often result in high rates of mortality. In this report, we examined the effects of the C. albicans cell wall mannoprotein (MP) on macrophage immunity. The MTS assay was used to detect cell proliferation activity and neutral red staining to observe cell phagocytosis. The Griess method was used to detect NO secretion in culture supernatants and apoptosis of macrophages were determined with use of FITC-Annexin V and PI staining. mRNA and protein expressions of JAK2, STAT3, IL-1β, IL-6, TNF-α and iNOS in RAW264.7 cells were determined with use of RT-PCR and western blot. MP significantly promoted the proliferation of RAW264.7 cells, inhibited their phagocytic capacity, but exerted no significant effects on apoptosis of macrophages. In addition, MP not only up-regulated the expression of cytokines, but also the expressions of p-stat3 and p-jak2. Interestingly, when MP was combined with lipopolysaccharide (LPS) a markedly accentuated release of inflammatory cytokines was observed. MP promotes macrophage inflammation induced by LPS and participates in the inflammatory response. One of the potential mechanisms of this effect involves MP activation of the JAK2/STAT3 signaling pathway in RAW264.7 cells, which enables macrophages to transform from M0 to M1 and promote the occurrence of inflammation.

Impact of Commercial Oenotannin and Mannoprotein Products on the Chemical and Sensory Properties of Shiraz Wines Made from Sequentially Harvested Fruit.

The tannin and polysaccharide profiles and therefore sensory properties of wine are influenced by fruit maturity at harvest, and practices employed during winemaking. This study investigated the extent to which commercial winemaking supplements (skin and seed tannins, and mannoprotein (MP)) can enhance the mouthfeel properties of red wine, in particular, wine made from grapes harvested before commercial ripeness (early-harvest). Supplements were added to wines made from Shiraz grapes harvested at 20.8 and 24.5 °Brix. The chemical composition and mouthfeel properties of wines were then determined by high performance liquid chromatography and descriptive analysis (DA), respectively. Wines made from riper grapes had higher levels of tannin than wines made from early-harvest grapes, but similar polysaccharide levels were observed. The addition of seed oenotannin yielded higher tannin levels than addition of skin oenotannin, particularly for wines made from early-harvest grapes. The DA panel perceived sensory differences between H1 and H2 wines, but could not perceive any effect of supplementation on wine mouthfeel properties, with the exception of a minor increase in sweetness, attributed to mannoprotein addition to H1 wines, even when MP was added to wines at 2.5 times the level recommended for use in Australia.

Effects on the color, taste, and anthocyanins stability of blueberry wine by different contents of mannoprotein

Blueberry wine is a new fruit wine with good taste and rich nutrition, but color change and anthocyanins (ACNs) content readily decrease during the production process. The effects of different content (0.2 g/L, 0.25 g/L, and 0.3 g/L) of mannoprotein (MP) on the blueberry wine were investigated in this study. The result showed that MP treatment inhibited the decrease in ACN content, reduced the content of total acid, increased the content of alcohol content in blueberry wine, maintained the color and improved the taste of blueberry wine. In addition, the effect was more pronounced as the MP concentration increased, with the optimum effect at 0.3 g/L. However, MP has no significant effect on the total sugar in blueberry wine. The results arising from this study provide new insights into blueberry wine production, by which treatment with MP maintain the color and ACNs contents, and improve the taste of blueberry wine.