Protease-assisted clarification of black currant juice: synergy with other clarifying agents and effects on the phenol content

Conventional clarification with gelatin and silica sol removes a considerable amount of antioxidant phenolics from berry juices. This study examined the clarification and haze-diminishing effects of alternative clarification strategies on black currant juice including centrifugation and addition of acidic protease and pectinolytic enzyme preparations and gallic acid. Centrifugation of freshly pressed juice (10,000 g for 15 min) resulted in a approximately 95% reduction of immediate turbidity and had a decreasing effect on haze development in the juice during cold storage without significantly compromising the total phenols levels. The extent of clarification and haze diminishment varied after individual treatments with five different acidic proteases, but one of the protease preparations, Enzeco, derived from Aspergillus niger, consistently tended to perform best. The individual and interactive effects on juice turbidity, total phenols, and total anthocyanin contents of clarification treatments involving the use of two selected acid proteases (Enzeco and Novozyme 89L), a pectinase (Pectinex BE 3-L), and gallic acid were evaluated in a full factorial 2(4) experimental design. Haze development during cold storage decreased when gallic acid or any of the enzyme preparations were employed individually, but negative interaction effects resulted when the pectinase was employed in combination with any of the proteases. After 28 storage days at 2 degrees C, the lowest levels of haze formation were achieved when the Enzeco protease preparation, added at 0.025 g/L, was added with 0.050 g/L of gallic acid and allowed to react in the juice for 90 min at 50 degrees C. The corresponding anthocyanin reduction was approximately 12% (compared to approximately 30% with gelatin silica sol treatment). The data support the hypothesis that phenol-protein interactions are involved in juice turbidity development during cold storage of berry juices and demonstrate that precentrifugation and protease-assisted clarification show promise as an alternative, phenolics-retaining clarification strategy in black currant juice processing.     

Effect of fining and filtration on the haze formation in bayberry (Myrica rubra Sieb. et Zucc.) juice

Bayberry juice was fined with the methods of xanthan/chitosan (XC) or gelatin/bentonite (GB), and then filtered with diatomaceous earth filtration (DF) or ultrafiltration (UF, MWCO 100 kDa). Their effects on juice haze formation were investigated. The XC fining method was more effective than the GB method in removal of the total monomeric anthocyanin, total phenolics, and protein, with less haze formed in the XC fined juice. The DF reduced 2-5% of the total phenolics and 21-23% of protein, while UF reduced 19-24% of the total phenolics and 34-38% of protein, respectively. The results showed that fining and then UF can reduce but cannot eliminate haze formation in bayberry juice. The storage temperature was a critical factor affecting haze formation, and the juice was more stable when stored at lower temperature (4 degrees C).     

草莓胡萝卜复合澄清汁后浑浊机理研究

为探讨草莓胡萝卜复合澄清汁的后浑浊机理,研究了化学试剂、贮藏温度和顶隙大小对草莓胡萝卜复合澄清汁的澄清度、褐变指数、黄烷醇类多酚含量、可溶性蛋白含量的影响。结果表明：贮藏温度越高,包装顶隙越大,氧气存留越多,随贮藏时间延长,果蔬汁浑浊形成越多,褐变程度越大,黄烷醇类多酚降解程度越大,可溶性蛋白损失越多;果蔬汁中添加尿素和十二烷基磺酸钠(SDS),都能减少果蔬汁中浑浊的产生,抑制产品褐变,减缓黄烷醇类多酚的降解,减少可溶性蛋白的损失,说明氢键和疏水键都是和浑浊有关的化学键。蔗糖的添加对果蔬汁浑浊的形成没有太     

Sulfate--a candidate for the missing essential factor that is required for the formation of protein haze in white wine

Protein haze formation in white wine is dependent on the presence of both wine protein and other unknown wine components, termed factor(s) X. The ability to reconstitute protein haze upon heating artificial model wine solutions (500 mg/L thaumatin, 12% ethanol, 4 g/L tartaric acid) to which candidate components were added was employed to identify factor(s) X. No protein haze was formed in the absence of additives. The individual or combined addition of caffeic acid, caftaric acid, epicatechin, epigallocatechin-O-gallate, gallic acid, or ferulic acid at typical white wine concentrations did not generate protein haze. However, PVPP fining of commercial wines resulted in a reduction in protein haze, suggesting that phenolic compounds may play a modulating role in haze formation. To elucidate the nature of the unknown factor(s) wine was fractionated and fractions were back-added to model wine and tested for their essentiality. Wine fractions were generated by ultrafiltration, reverse-phase chromatography, and mixed-mode anion-exchange and reverse-phase chromatography. The only purified fraction containing the essential component(s) was free of phenolic compounds, and analysis by mass spectrometry identified sulfate anion as the dominant component. Reconstitution with KHSO4 using either commercially available thaumatin or wine proteins confirmed the role of sulfate in wine protein haze formation. The two main wine proteins, thaumatin-like protein and chitinase, differed in their haze response in model wines containing sulfate. Other common wine anions, acetate, chloride, citrate, phosphate, and tartrate, and wine cations, Fe(2+/3+) and Cu(+/2+), when added at typical white wine concentrations were not found to be essential for protein haze formation.     

Influence of prefermentary clarification on the composition of apple musts

The polyphenol contents and colors of cider apple juices were compared before (NCM, not clarified must) and after five clarification treatments: enzymatic depectinization by pectinases followed by (i) sedimentation (depectinized and decanted juice), (ii) tangential microfiltration (microfiltered juice) or (iii) fining using gelatin (gelatin-treated juice); (iv) enzymatic gelification of pectin by pectin methylesterase followed by natural keeving by a cider manufacturer (producer keeved juice), or (v) flotation (floated with nitrogen gas juice). The pressing of the apples led to the highly selective extraction of the flavan-3-ols with the lowest molecular weights: In the apples, the number average degree of polymerization of the flavanols was 14.7, and it dropped to 2.2 in the NCM. Keeving had the highest impact on the reduction of both flavanol content and number average degree of polymerization. The flavanol concentrations were decreased in the permeate by fining (30%) much more than by depectinization. The clarification step led to a further decrease of the number average degree of polymerization. Hydroxycinnamic acids were less affected by the extraction process (with extraction yields >50%) and not affected by clarification. The color evolved with all treatments: L*, a*, b*, and chromaticity distance index measures indicated a reduction of orange-yellow saturation except after sedimentation.     

Chemical composition of clarified bayberry (Myrica rubra Sieb. et Zucc.) juice sediment

Clarified bayberry juice turned hazy upon storage at 25 degrees C for 6 months, and the chemical composition of centrifugally separated sediment was analyzed. Bayberry juice haze was mainly protein-tannin haze. The lyophilized sediment contained 20.4 +/- 4.3% of protein, 70.2 +/- 2.6% of total polyphenols, 7.2% of monosaccharides, and 6.7 +/- 0.6% of ash. Amino acid analyses and molecular weight distribution estimation indicated that bayberry proteins were haze-active proteins with a molecular weight less than 8 kDa. Gallic acid, quercetin hexoside, quercetin deoxyhexoside, and quercetin were found in the methanol-dissolved sample, while gallic acid, protocatechuic acid, cyanidin, ellagic acid, and quercetin were detected in the acid-hydrolyzed sample. Ellagic acid was the dominant individual phenolic (9.9 +/- 0.19 g/100 g dry weight, 55.3% of the total amount) in the sediment. Monosaccharides of rhamnose, arabinose, mannose, glucose, and galactose in the sediment were most probably the glycoside moieties of the anthocyanins, flavonols, and elllagitannins. Metal ions of calcium, magnesium, potassium, iron, and copper also indicated the heterogeneous characteristics of the sediment.     

Preheating and incubation of cane juice prior to liming: a comparison of intermediate and cold lime clarification.

In the U.S., cold lime clarification remains the clarification process of choice in raw sugar manufacturing. A comparative study of cold vs intermediate lime clarification was undertaken at a factory that operated intermediate liming (approximately 30% mixed juice (MJ) of pH 5.2 +/- 0.3 was preheated to 87-93 degrees C to help maintain clean limed juice heaters, incubated at approximately 54 degrees C, and then limed) but still had the pipes to revert to cold liming (MJ incubated and limed at approximately 40 degrees C) for this study. Hourly samples were collected over a 6 h sampling period across cold and intermediate clarification processes on two consecutive days, respectively, and this was repeated three times across the 1999 grinding season. A total of 1.57% less sucrose was lost to inversion reactions across intermediate rather than cold liming. In intermediate liming, which required approximately 4.6% less lime, preheating of only 30% of the MJ markedly removed color (-29%), dextran (-10%), and starch (-24%) and caused large flocs to form that settled faster in the clarifiers. Faster settling led to an impressive 4.6% (season average) more turbidity removal across the clarifiers in intermediate rather than cold liming. Intermediate clarified juice (CJ) turbidity (season average 2028 ICU +/- 675) was approximately half of cold CJ turbidity (average 3952 ICU +/- 1450) with over 2-fold more CJ turbidity control. Subsequent turbidity values and control were significantly improved in the final evaporator syrup samples too. For both processes, juice incubation caused approximately 10% color removal, but this was offset by color formation on liming, because of the alkaline degradation of invert; however, overall, more color was removed than formed in intermediate liming. Starch was reduced in the incubator tank, for both processes, because added filtrate reduced the acidity enabling natural diastase from the cane to degrade starch. Some dextran occasionally formed in the incubator tank, in both processes. Summed across measured parameters, intermediate liming appears to offer several advantages over cold liming.     

Effect of mash maceration on the polyphenolic content and visual quality attributes of cloudy apple juice

The effects of enzymatic mash treatments on yield, turbidity, color, and polyphenolic content of cloudy apple juice were studied. Using HPLC-ESI-MS, cryptochlorogenic acid was identified in cv. Brettacher cloudy apple juice for the first time. Commercial pectolytic enzyme preparations with different levels of secondary protease activity were tested under both oxidative and nonoxidative conditions. Without the addition of ascorbic acid, oxidation substantially decreased chlorogenic acid, epicatechin, and procyanidin B2 contents due to enzymatic browning. The content of chlorogenic acid as the major polyphenolic compound was also influenced by the composition of pectolytic enzyme preparations because the presence of secondary protease activity resulted in a rise of chlorogenic acid. The latter effect was probably due to the inhibited protein-polyphenol interactions, which prevented binding of polyphenolic compounds to the matrix, thus increasing their antioxidative potential. The results obtained clearly demonstrate the advantage of the nonoxidative mash maceration for the production of cloud-stable apple juice with a high polyphenolic content, particularly in a premature processing campaign.     

Characterization of haze-active proteins in apple juice

The nature of the haze-active protein (HAP) in apple juice was investigated. Heat treatment removed protein indiscriminately while polyvinylpolypyrrolidone (PVPP) treatment was fairly specific for proteins of 15 and 28 kDa. Presumably, the PVPP bound to polyphenols, which in turn were complexed with protein. Three candidate apple HAPs were isolated. Two were extracted from juice with acetone and fractionated by hydrophobic interaction chromatography and solid phase extraction with C18 (HAP I) or SAX (HAP II) material. Hydroxyproline-rich protein was extracted from apple tissue (HAP III). The order of haze formation with tannic acid was gliadin > HAP III > HAP II > HAP I > bovine serum albumin, which shows increasing haze formation with increasing proline content. The sizes of HAP I, II, and III were 28, 15, and 12 kDa; the first two corresponded to the sizes of proteins removed by PVPP treatment and are involved in juice haze formation.     

Insights to the clarification of sugar cane juice expressed from sugar cane stalk and trash

Processing of juice expressed from green sugar cane containing all the trash (i.e., tops and leaves, the nonstalk component) of the sugar cane plant during sugar manufacture has been reported to lead to poor clarified juice (CJ) quality. Studies of different liming techniques have been conducted to identify which liming technique gives the best clarification performance from juice expressed from green cane containing half of all trash extracted (GE). Results have shown that lime saccharate addition to juice at 76 °C either continuous or batchwise gives satisfactory settling rates of calcium phosphate flocs (50-70 cm/min) and CJ with low turbidity and minimal amounts of mineral constituents. Surprisingly, the addition of phosphoric acid (≤ 300 mg/kg as P?O?), prior to liming to reduce juice turbidity (≤ 80%), increased the Mg (≤ 101%) and Si (≤ 148%) contents particularly for clarified GE juices. The increase was not proportional with increasing phosphoric acid dose. The nature of the flocs formed, including the zeta potential of the particles by the different liming techniques, has been used to account for the differences in clarification performance. Differences between the qualities of the CJ obtained with GE juice and that of burnt cane juices with all trash extracted (BE) have been discussed to provide further insights into GE processing.     

Roles of grape thaumatin-like protein and chitinase in white wine haze formation

Grape chitinase was found to be the primary cause of heat-induced haze formation in white wines. Chitinase was the dominant protein in a haze induced by treating Sauvignon blanc wine at 30 °C for 22 h. In artificial wines and real wines, chitinase concentration was directly correlated to the turbidity of heat-induced haze formation (50 °C for 3 h). Sulfate was confirmed to have a role in haze formation, likely by converting soluble aggregates into larger visible haze particles. Thaumatin-like protein was detected in the insoluble fraction by SDS-PAGE analysis but had no measurable impact on turbidity. Differential scanning calorimetry demonstrated that the complex mixture of molecules in wine plays a role in thermal instability of wine proteins and contributes additional complexity to the wine haze phenomenon.     

Flocculation and Haze Removal from Crude Beer Using In-House Produced Laccase from Trametes versicolor Cultured on Brewer's Spent Grain

The potential of brewer's spent grain (BSG), a common waste from the brewing industry, as a support-substrate for laccase production by the well-known laccase producer Trametes versicolor ATCC 20869 under solid-state fermentation conditions was assessed. An attempt was made to improve the laccase production by T. versicolor through supplementing the cultures with inducers, such as 2,2-azino bis(3-ethylbenzthiazoline-6-sulfonic acid), copper sulfate, ethanol, gallic acid, veratryl alcohol, and phenol. A higher laccase activity of 13506.2 ± 138.2 IU/gds (gram dry substrate) was obtained with a phenol concentration of 10 mg/kg substrate in a tray bioreactor after 12 days of incubation time. The flocculation properties of the laccase treated crude beer samples have been studied by using various parameters, such as viscosity, turbidity, ζ potential, total polyphenols, and total protein content. The present results indicated that laccase (25 IU/L) showed promising results as a good flocculating agent. The laccase treatment showed better flocculation capacity compared to the industrial flocculation process using stabifix as a flocculant. The laccase treatments (25 IU/L) at 4 ± 1 °C and room temperature have shown almost similar flocculation properties without much variability. The study demonstrated the potential of in-house produced laccase using brewer's spent grain for the clarification and flocculation of crude beer as a sustainable alternative to traditional flocculants, such as stabifix and bentonite.     

Effect of pectolytic enzyme preparations on the phenolic composition and antioxidant activity of asparagus juice

Commercial pectolytic enzymes were investigated for their influence on phenolics and antioxidant activities of asparagus juice. The antioxidant activity of asparagus juice was analyzed according to 2,2'-diphenyl-l-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) methods. The enzymes, with the exception of pectinase from Rhizopus sp., contained rutinase, which hydrolyzed rutin to quercetin. Asparagus juice treated with Viscozyme had the highest quercetin content without exhibiting a significant increase in the antioxidant activity. For a pectinase from Aspergillus niger, the antioxidant activity of asparagus juice was markedly reduced. Caution should be paid in the selection of pectolytic enzyme preparations for production of antioxidant activity-rich juice.     

Free and conjugated polyamines and phenols in raw and alkaline-clarified sugarcane juices.

Sugarcane juice contains a lot of sucrose associated with several monosaccharides, defined as low molecular mass carbohydrates (LMMC), as well as some polysaccharides and glycoproteins, which are defined as mid and high molecular mass carbohydrates (MMMC and HMMC, respectively). These three categories of carbohydrates can be separated by size-exclusion chromatography through Sephadex G-10 and Sephadex G-50 columns, but elution profiles change drastically after juice clarification performed by adjusting the pH value of the juice to 8.0. In addition, polyamines and some phenolics are currently associated with carbohydrate preparations, and the distribution pattern of these conjugates also changes after clarification. Polyamine levels generally decrease after juice clarification. Cadaverine is completely removed from the different carbohydrate preparations, whereas spermidine is the main polyamine occurring in association with sugarcane carbohydrates, as free or acid-soluble form in LMMC preparation or as acid-soluble and -insoluble forms in both MMMC and HMMC preparations. Polyamines, presumably spermidine, conjugate to p-hydroxybenzoic acid in LMMC, mostly to caffeic acid in MMMC, and to syringic acid in HMMC preparations. HMMC-associated polyamines appear in both acid-soluble and -insoluble fractions. Syringic acid also occurs in the LMMC preparation, but juice clarification changes it from acid-soluble to free form, and it coelutes with sucrose.     

Improved sugar cane juice clarification by understanding calcium oxide-phosphate-sucrose systems

It is accepted that the efficiency of sugar cane clarification is closely linked with sugar juice composition (including suspended or insoluble impurities), the inorganic phosphate content, the liming condition and type, and the interactions between the juice components. These interactions are not well understood, particularly those between calcium, phosphate, and sucrose in sugar cane juice. Studies have been conducted on calcium oxide (CaO)/phosphate/sucrose systems in both synthetic and factory juices to provide further information on the defecation process (i.e., simple liming to effect impurity removal) and to identify an effective clarification process that would result in reduced scaling of sugar factory evaporators, pans, and centrifugals. Results have shown that a two-stage process involving the addition of lime saccharate to a set juice pH followed by the addition of sodium hydroxide to a final juice pH or a similar two-stage process where the order of addition of the alkalis is reversed prior to clarification reduces the impurity loading of the clarified juice compared to that of the clarified juice obtained by the conventional defecation process. The treatment process showed reductions in CaO (27% to 50%) and MgO (up to 20%) in clarified juices with no apparent loss in juice clarity or increase in residence time of the mud particles compared to those in the conventional process. There was also a reduction in the SiO2 content. However, the disadvantage of this process is the significant increase in the Na2O content.     

Enzyme-assisted extraction of antioxidative phenols from black currant juice press residues (Ribes nigrum).

Enzymatic release of phenolic compounds from pomace remaining from black currant (Ribes nigrum) juice production was examined. Treatment with each of the commercial pectinolytic enzyme preparations Grindamyl pectinase, Macer8 FJ, Macer8 R, and Pectinex BE, as well as treatment with Novozym 89 protease, significantly increased plant cell wall breakdown of the pomace. Each of the tested enzyme preparations except Grindamyl pectinase also significantly enhanced the amount of phenols extracted from the pomace. Macer8 FJ and Macer8 R decreased the extraction yields of anthocyanins, whereas Pectinex BE and Novozym 89 protease showed no effect. A decrease in pomace particle sizes from 500-1000 microm to <125 microm increased the phenol yields 1.6-5 times. Black currant pomace devoid of seeds gave significantly higher yields of phenols than pomace with seeds and seedless wine pomace. Four selected black currant pomace extracts all exerted a pronounced antioxidant activity against human LDL oxidation in vitro when tested at equimolar phenol concentrations of 7.5-10 microM.     

Wheat gluten used as a clarifying agent of red wines.

Bovine spongiform encephalopathy caused a situation of crisis leading the public and winemakers to lose their confidence in the use of gelatin as a fining agent and to reject animal proteins in general. Therefore, we started the search for a substitute for gelatin and egg protein by comparing gluten with these fining treatments currently used. This study concerned the fining of a Burgundy red wine (Rully, Controlled Appellation). For 6 g/hL, enzymatically hydrolyzed glutens (EHG) gave better efficiencies than deamidated glutens. The efficiency of the egg proteins treatment was situated between those of the hydrolyzed glutens and deamidated glutens. For 12 and 18 g/hL, turbidities of the wine treated by five glutens were 67 to 86% less than that of the control wine. Better results were obtained with egg proteins for short kinetics particularly. Wine fining with gluten was always better than gelatin treatments. The differences between the five glutens became very small when the dose incorporated in the wine increased. The volumes of lees generated by fining with gluten are situated between the values obtained with egg proteins and gelatin. After fining, immunodetection with gluten polyclonal antibodies failed to detect residual deamidated gluten.     

Turbidity-based assay for polygalacturonic acid depolymerase activity

A technically simple and inexpensive discontinuous turbidity assay for qualitative and/or quantitative assessments of polygalacturonic acid depolymerase activity is presented. The enzyme reaction is initiated by the addition of enzyme preparation to a reaction mixture containing 0.02% polygalacturonic acid (PGA) in acetate buffer. The progress of the reaction is monitored by terminating aliquots of the reaction mixture (via heat treatment at appropriate times), subsequently adding poly(diallyldimethylammonium chloride) (PDADMAC) for turbidity development (approximately 30 min), and then measuring the turbidity (typically at 420 nm) of the resulting PGA-PDADMAC complex-containing solution. PGA depolymerase activity causes a decrease in the observed turbidity of PGA-PDADMAC-containing solutions because the stability of the interpolyelectrolyte PGA-PDADMAC complex is a function of the degree of polymerization of the PGA. The rate of turbidity change is herein shown to be proportional to a relatively wide range of enzyme concentrations. The assay is demonstrated using a commercial pectinase preparation and tomato fruit extracts.     

Comparative changes in color features and pigment composition of red wines aged in oak and cherry wood casks

The color features and the evolution of both the monomeric and the derived pigments of red wines aged in oak and cherry 225 L barriques have been investigated during a four months period. For cherry wood, the utilization of 1000 L casks was tested as well. The use of cherry casks resulted in a faster evolution of pigments with a rapid decline of monomeric anthocyanins and a quick augmentation formation of derived and polymeric compounds. At the end of the aging, wines stored in oak and cherry barriques lost, respectively, about 20% and 80% of the initial pigment amount, while in the 1000 L cherry casks, the same compounds diminished by about 60%. Ethyl-bridged adducts and vitisins were the main class of derivatives formed, representing up to 25% of the total pigment amount in the cherry aged samples. Color density augmented in both the oak and cherry wood aged samples, but the latter had the highest values of this parameter. Because of the highly oxidative behavior of the cherry barriques, the use of larger casks (e.g., 1000 L) is proposed in the case of prolonged aging times.     

Effect of clarification techniques and rat intestinal extract incubation on phenolic composition and antioxidant activity of black currant juice

This study examined the phenolic composition and the antioxidant potencies of black currant juices that had been experimentally clarified with acidic proteases and pectinases to retain the phenolics and which had been subjected to rat intestinal mucosa extract incubation to mimic gut cell mediated biotransformation of phenolics. When compared at equimolar levels of 2.5 microM gallic acid equivalents, the black currant juice samples prolonged the induction time of human low-density lipoprotein oxidation in vitro by 2.6-3.6 times, and the order of antioxidant potency of differently clarified black currant juices was centrifuged juice > gelatin silica sol clarified juice > enzymatically clarified juice approximately raw juice. No immediate relationship between the, almost similar, phenolic profiles of the juice samples and their relative antioxidant activities could be established. Incubation of juices with a rat small intestine cell extract for 19 h promoted significant decreases in the contents of the anthocyanin 3-O-beta-glucosides (cyanidin 3-O-beta-glucoside and delphinidin 3-O-beta-glucoside), but did not affect the anthocyanin 3-O-beta-rutinosides (cyanidin 3-O-beta-rutinoside and delphinidin 3-O-beta-rutinoside) of the black currant juice. Black currant juice samples subjected to such intestinal cell extract incubation had approximately 30% decreased antioxidant capacity. Incubation of juices with the rat small intestine cell extracts at neutral pH appeared to decrease the levels of delphinidin glucosides more than the levels of cyanidin glucosides. The results provide an explanation for the predominant detection of anthocyanin rutinosides, and not anthocyanin glucosides, in plasma and urine in in vivo studies and provide important clues to better understand the complex mechanisms affecting dietary phenols in the gut.