Grape Juice Clarification Research Articles

Optimization of the conditions for immobilization of crude pectinase on chitin using ultrasound and glutaraldehyde and its application in grape juice clarification

The current research focuses on optimizing the immobilization of crude pectinase, produced by solid-state fermentation, onto chitin using ultrasound and glutaraldehyde as a crosslinking agent for grape juice clarification. A Box-Behnken design was used to model the effects of ultrasound time (5–15 min), ultrasound amplitude (20 – 60 %), and water-to-support ratio for ultrasound application (16−24) on immobilized crude pectinase activity and immobilization efficiency. Optimal conditions, comprising ultrasound time of 10 min, ultrasound amplitude of 20 %, and water-to-support ratio for ultrasound application of 24, yielded an immobilized crude pectinase activity of 2.19 IU/mL and 74 % immobilization efficiency. The immobilized crude pectinase showed improved kinetic properties and retained 69 % activity after three use cycles. Both free and immobilized crude pectinase enhanced juice clarity, reducing sugar, and TSS while lowering viscosity.

Valorisation of Citrus limetta peel for Aspergillus terreus FP6 mediated pectinase fermentation and application in grape juice clarification

ObjectivesThe present investigation highlights the process parameters influencing the pectinase fermentation by a newly isolated Aspergillus terreus using valorised agro-wastes and determination of the efficacy of the pectinase in grape juice clarification. MethodsThe fungal pectinase was produced under submerged fermentation with the optimisation of carbon and nitrogen sources, initial pH of the medium, inoculum load, incubation time and effect of agitation. The pectinase was later used for the clarification of grape juice and improvement of its total phenolic content. Results and ConclusionsMedium supplemented with sweet lime peel and yeast extract (1 % w/v), at initial pH 6.0, when incubated for 72 h under agitated conditions favoured the highest pectinase production by the fungal culture. Following the pectinase action for 1 and 3 h, the grape juice clarification percentages and total phenolic content were 34.21 %, 69.54 %, 3004, and 3484 mg GAE/L, respectively. The results highlight the valorisation of sweet lime peel for the industrial production of fungal pectinase. The study pronounces the cost-effective pectinase fermentation due to the fungi’s non-fastidious nutritional requirement and ubiquitous nature. The increase in the grape juice clarification and total phenolic content following the A. terreus FP6 pectinase treatment was significantly superior to similar commercial enzyme formulations. The result advocates the candidature of the enzyme in the beverage industry, where it can help mediate a greener approach for fruit juice clarification.

Pectinases production using an exotic caatinga passion fruit waste and wheat bran as a substrate for clarification of grape juice

Caatinga passion fruit waste (CPFW):Wheat bran has shown reliable performance for enzyme production. However, the mixture of wheat bran and CPFW may provide all nutrient sources needed by Aspergillus niger for pectinase generation. This work investigates the production of pectinolytic enzymes using a substrate composed of CPFW:Wheat bran using Aspergillus niger FSDE16 via semi-solid-state fermentation (SSSF). A blend of wheat bran:CPFW (1:1, g/g) with 60% moisture content was inoculated with 106 spores/mL for 144 h. Substrates were fermented using the 7 combinations of temperature (25–75 °C) and pH (3–6) dispose in an experimental design. Upon the completion of fermentation, all analyses were conducted including the enzymatic production, optimal activity, and enzymatic thermostability. Afterwards, grape juice clarification was conducted, and the clarified juice was evaluated considering viscosity reduction, yield increase, turbidity reduction, yield increase, colour, and phenolic profile. Results demonstrate that enzyme stability was achieved within the temperature range of 30–40 °C, with optimal activity recorded at 50 °C (42.7 U/g). The application of crude enzymatic extract in grape juice clarification led to an increase of yield (>280%), and a viscosity (>80%) and turbidity (>98%) reduction. The clarification treatment revealed a significant increase in the phenolic compounds evaluated, except epicatechin gallate, Pro-B2, and resveratrol, which reduced their concentrations. The clarification resulted in a colour intensity reduction. This study underscores the potential of using food waste for enzyme production, presenting a sustainable approach with implications for cost reduction and waste management in the food industry.

Optimization of conditions for production of pectinase in solid-state fermentation with Aspergillus flavus using dried Assam lemon peel powder as substrate

The culture conditions for pectinase production with Aspergillus flavus by solid-state fermentation (SSF) using dried Assam lemon peel were optimized and the pectinase was used for clarification of grape juice. A central composite rotatable design (CCRD) was used for optimization experiments with moisture content (50–90 %), incubation temperature (20–60 ℃), incubation time (96 h–192 h), and urea concentration (0–1 %) as independent variables, and pectinase activity as dependent variable. The optimum conditions for pectinase production obtained were moisture content of 60 %, incubation temperature of 50 ℃, incubation time of 96 h, and urea concentration of 0.30 % with a pectinase activity of 3.68 ± 0.20 U/mL where one unit of pectinase activity corresponds to number of µmole of galacturonic acid released in one minute in 1 % pectin solution at 37 °C and 4.8 pH. The crude pectinase was found to have maximum activity at a temperature of 40 ℃ and pH 4 and had an apparent Vmax and Km of 6.10 U/mL and 6.62 mg/mL respectively. The grape juice clarified using the crude pectinase showed decreased optical density (1.45 ± 0.06 to 1.17 ± 0.07) and viscosity (1.76 ± 0.10 to 1.46 ± 0.07 mPa.s) and increased reducing sugar content (13.90 ± 0.10 to 16.96 ± 0.06 g/100 mL juice), and total soluble solids (17.77 ± 0.16 to 18.50 ± 0.30 °Brix).

Studies on white must clarification using enzyme preparations

The use of pectolytic enzymes to clarify the white grape juice is a simple and economical solution for positively influencing the subsequent technological steps. The positive effects refer to improving the filterability of the final wine, the behavior of the wine during the stabilization treatments, and, most important, wine quality. The experimental study consisted of treating the grape juice with Zymoclaire Pro Ice for the maceration and extraction steps, and Zymoclaire CC Plus for the clarifying step. The evolution of grape juice clarification parameters such as turbidity, oxidation stability, and the dynamics of the alcoholic fermentation were monitored. The study revealed that after 24 h of treatment, a higher volume of clarified grape juice was achieved. The volume of clarified grape juice was 55% in the control samples, while in case of the sulfited grape juice, clarified with maceration and extraction enzymes or with clarification enzymes, reached 72% and 84%, respectively. The optimal turbidity levels of the juice’s enzymatic clarifying stage were reached after 24 hours in case of both enzyme treatments. The comparative analysis of the physicochemical parameters did not reveal significant differences between samples regarding free and total SO2 contents, potential alcoholic concentrations, volatile acidity, and residual sugars. Also, the enzymatically treated samples were the most appreciated from a sensory point of view. In conclusion, the results revealed that the enzyme tested in these experiments showed high efficiency in grape juice clarifying.

Removal of Ochratoxin A from Grape Juice by Clarification: A Response Surface Methodology Study.

This study achieved maximum removal of ochratoxin A (OTA) during the grape juice clarification process with minimal reduction in antioxidant compounds (phenolic acid, flavonoids, and antioxidant capacity by FRAP) by the RSM method. Independent variables included three types of clarifiers—gelatin, bentonite, and diatomite (diatomaceous earth)—at a concentration level of 0.25–0.75% and clarification time of 1–3 h. OTA was measured by high-performance liquid chromatography with fluorescence detection. Clarifying agent concentration and clarification time affected the reduction amount of OTA and antioxidant compounds in grape juice. There was a direct linear correlation between the reduction amounts of OTA and antioxidant compounds and capacity with the concentration of bentonite, gelatin, and diatomite, and the clarification time. The reduction amount of OTA and antioxidant capacity followed the linear mode. However, the decreased phenolic acid and flavonoid values followed the quadratic model. The study results showed that if the concentrations of bentonite, gelatin, and diatomite and clarification time were 0.45, 0.62, 0.25%, and 1 h, respectively, the maximum amount of OTA reduction (41.67%) occurred. Furthermore, the phenolic acid, flavonoid, and antioxidant activity decrease amounts were at their lowest levels, i.e., 23.86, 7.20, and 17.27%, respectively.

Continuous clarification of grape juice using a packed bed bioreactor including pectinase enzyme immobilized on glass beads

A continuous clarification of grape juice was undertaken in a packed bed reactor (PBR) using pectinase enzymes immobilized on glass beads. The enzyme immobilization process decreased the specific activity and the volumetric productivity from 3 ± 0.2 to 2.7 ± 0.1 units mg−1 and from ~0.085 to ~0.070 mg mL−1 min−1, respectively. Also, it increased the optimum pH from 5.0 to 5.5, but did not influence the optimum temperature (50 °C). The best conditions for clarification of the grape juice were obtained at a flow rate of 0.5 mL min−1 and a treatment time of 42 min. After clarification, pH, total soluble solids, turbidity, total phenolic content, antiradical activity and viscosity of grape juice were decreased, and on the other hand, total titratable acidity and the color parameter L were increased. Generally, this study showed that the continuous operation in a PBR including glass bead immobilized enzyme can be a potential alternative for the batch system used in juice industries.

Evaluation of antiproliferative and anti-inflammatory effects of non-pomace sediment of red grape juices (Vitis labrusca L.) in healthy and cancer cells after in vitro gastrointestinal simulation

Grape polyphenols are studied for their bioactive activities and health benefits. Grape juice clarification produces a colored non-pomace solid residue that is rich in polyphenols and may have potential for nutraceutical applications. This study therefore firstly investigated antiproliferative and anti-inflammatory properties of the non-pomace sediment of Bordo and Isabel grape juices before and after the gastrointestinal simulation, and evaluated the effects of the main individual phenolic compounds found in these sediments in regards to their antiproliferative action in healthy and cancer cells. The non-pomace sediments inhibited significantly the proliferation of colorectal adenocarcinoma cells (Caco-2) cells, whereas their digested fractions inhibited proliferation of hepatocellular carcinoma (HepG2) cells. However, the exposure to treatments caused no cell toxicity to healthy cells. When tested individually, the phenolic compounds quercetin and myricetin showed antiproliferative activity and led to cell viability of 56.47 and 51.07 % in Caco-2 cells and of 33.96 and 27.03 % in HepG2 cells, respectively. The non-pomace sediments and its digested fractions exhibited anti-inflammatory action by inhibiting the release of tumor necrosis factor alpha (TNF-α) in murine macrophages. These encouraging data suggests the potential use of this residue as dietary supplement or as a promising chemotherapy for antitumor therapies.

Efficient hydrolysis of wine and grape juice anthocyanins by Malbranchea pulchella β-glucosidase immobilized on MANAE-agarose and ConA-Sepharose supports

β-glucosidases (BGLs) hydrolyze short-chain cellulooligosaccharides. Some BGLs can hydrolyze anthocyanins and be applied in the clarification process of food industries, especially grape juice and wine. Enzyme immobilization is a valuable tool to increase enzyme stabilization. In this work, Malbranchea pulchella BGL was immobilized on Monoaminoethyl-N-ethyl-agarose ionic support, MANAE-agarose, and Concanavalin A-Sepharose affinity support, Con-A-Sepharose. The formed biocatalysts, denominated BLG-MANAE and BLG-ConA, were applied in the grape juice and red wine clarification. BGL-MANAE and BGL-ConA hyperactivated M. pulchella BGL 10- and 3-fold, respectively. Both biocatalysts showed at least 70% activity at pH range 2–11, until 24 h incubation. BGL-MANAE and BGL-ConA showed activity of 60% and 100%, respectively, at 50 °C, up to 24 h. Both biocatalysts were efficiently reused 20-fold. They were stable in the presence of up to 0.1 M glucose for 24 h incubation, and with 5%, 10% and 15% ethanol kept up to 70% activity. BGL-MANAE biocatalyst was 11% and 25% more efficient than BGL-ConA in clarification of concentrate and diluted wines, respectively. Likewise, BGL-MANAE biocatalysts were 14% and 33% more efficient than the BGL-ConA in clarification of diluted and concentrated juices, respectively. Therefore, the BGL-MANAE biocatalyst was especially effective in red wine and grape juice clarification.

Immobilization of enological pectinase in calcium alginate hydrogels: A potential biocatalyst for winemaking

A biocatalyst was obtained by immobilizing an enological commercial pectinase within calcium alginate hydrogels using an entrapment technique, and its catalytic activity was evaluated during different storage conditions. Hydrogel beads were stored at 4 °C in three different ways: (i) wet, in citrate buffer solution (pH 3.8); (ii) dehydrated by using a vacuum stove; and (iii) freeze-dried. Biocatalyst surface and their internal morphology were characterized by Scanning Electron Microscopy and a good enzyme distribution throughout alginate matrix was observed. Fourier Transform Infrared Spectroscopy results confirmed the presence of absorption bands associated with amino groups present in enzymes. Immobilization procedure did not modify the optimal pH and temperature (pH = 4.0 and 20 °C) for pectinase activity, comparing to free enzyme. Entrapped pectinase showed activity until six reaction cycles with 40% residual activity. Storage stability studies demonstrated that wet entrapped pectinase retained its initial enzymatic activity up to 11 weeks, whereas that lyophilized hydrogels retained its original activity after 8 months of storage. These results suggest that immobilized pectinase may be successfully exploited in various industrial applications, with special concern in grape juice clarification process. Thus, the turbidity of grape must decreased significantly using the immobilized pectinase during 150 min at 20 °C. This biocatalyst could be easily removed after clarification process and it can be reused, minimizing production economic costs in wine industry.

Magnetic biocatalysts of pectinase and cellulase: Synthesis and characterization of two preparations for application in grape juice clarification

In the present study, we prepared two different magnetic biocatalysts of pectinase and cellulase: carrier-free magnetic CLEAs (CLEA-MP*) and immobilization on glutaraldehyde-activated magnetite (Enz-Glu-MP*). The biocatalysts were compared to their magnetic properties, immobilization parameters, stability and grape juice clarification. Enz-Glu-MP* presented higher magnetic properties than CLEA-MP*, whereas this presented higher surface area and pore volume. The KM of the enzyme immobilized on Enz-Glu-MP* was 25.65mM, lower in comparison to the CLEA-MP* (33.83mM). On the other hand, CLEA-MP* was the most active and stable biocatalyst, presenting higher recovered activity (33.4% of cellulase), higher thermal stability (2.39 stabilization factor) and improved reusability (8cycles). The integration of magnetic technology with enzymatic immobilization emerges as a possibility to increase the recover and reuse of biocatalysts for application in juice technology.

Effect of feather meal as proteic feeder on combi-CLEAs preparation for grape juice clarification

This work describes the preparation of combi-CLEAs of pectinases using feather meal as novel proteic feeder. Initially, four solvents were tested as precipitant agents, and ethanol was chosen as the best solvent. Subsequently, a 23 factorial design was carried out to define the optimal conditions for combi-CLEAs preparation, which were set as 110 mM of glutaraldehyde, 0.66 mg protein mL−1 and 5 h of reaction time. Then, the effect of BSA or feather meal (FM) as proteic feeders was evaluated. Combi-CLEAs, combi-CLEAs-BSA and combi-CLEAs-FM were characterized and evaluated by their activity recovery, optimal pH and temperature, operational and thermal stabilities and e effectiveness on juice clarification. The addition of feather meal increased two-fold the activity recovery compared to BSA. The thermal stability was similar for all combi-CLEAs, providing stabilization factors of 1.12, 1.48 and 1.53, respectively, for the combi-CLEA, combi-CLEA-FM and combi-CLEA-BSA, compared to free enzyme. CLEAs presented turbidity reduction near to 50%, 2.5-fold than soluble enzyme. Feather meal was used for the first time as proteic feeder for CLEA preparations and appears to be a good, and low price, alternative.

Two acidic, thermophilic GH28 polygalacturonases from Talaromyces leycettanus JCM 12802 with application potentials for grape juice clarification

Efficient hydrolysis of pectic materials to sugars requires the synergistic action of endo- and exo-polygalacturonases. Two novel polygalacturonases (exo-TePG28a and endo-TePG28b) were identified in Talaromyces leycettanus JCM12802, overexpressed in Pichia pastoris, and characterized in this report. The specific activities of TePG28a and TePG28b towards polygalacturonic acid were 280±9 and 25,900±502U/mg, respectively. Both enzymes exhibited optimal activities at pH 3.5 and retained highly stable over a broad pH range of 2.0–7.0. Distinct from most fungal polygalacturonases that have low temperature optima, TePG28a and TePG28b were optimally active at 70°C. When treated the grape juice with the enzyme combination (the unit ratio of TePG28a:TePG28b was 1:4), higher pectin-degrading efficiency (up to 140%) was achieved, and light transmittance was improved from 14% to 82%. These favorable enzymatic properties make TePG28a and TePG28b attractive for the applications in the juice industry.

Effect of Juice Turbidity and Yeast Lees Content on Brandy Base Wine and Unmatured Pot-still Brandy Quality

The aims of this project were to identify a suitable grape juice clarification technique for the attainment of the optimal brandy base wine turbidity, to determine the importance of chemical components (volatile components and long-chain fatty acids) in brandy base wine and unmatured pot-still brandy quality, and to study the effect of yeast lees content on quality. Although common industry practice is to use the grape cultivars Colombar(d) and Chenin blanc for the production of brandy base wine, the optimal conditions for Chenin blanc have been defined in this study. The juice clarification treatments applied included no settling, cold settling, whisk, large- and small-scale centrifugation and bentonite. Yeast strain 228 was compared with VIN13, large-scale (L) distillation was compared with small-scale (s) distillation, and the use of no enzyme was compared with the use of pectolytic enzyme. The data for four vintages were compiled and evaluated. Settling with or without pectolytic enzyme, bentonite, small-scale centrifugation and whisk treatments gave clearer Chenin blanc juice, higher concentrations of certain volatile components and long-chain fatty acids, and higher quality brandy base wine and unmatured pot-still brandy. No settling and large-scale centrifugation yielded the most turbid and lowest quality products. There is a definite relationship between treatments, turbidity, concentrations of esters, higher alcohols and acids, and overall brandy base wine and unmatured pot-still brandy quality. The use of yeast strain VIN13 (as opposed to strain 228), in conjunction with an increased yeast lees content of 1.5x that is normally found in brandy base wine, yielded the best quality unmatured pot-still brandy. Based on the results of this study, it is possible to recommend the best juice clarification method(s) for optimal turbidity as well as optimal levels of yeast lees addition, and to identify chemical compounds that positively relate to quality.

Preparation and characterization of a Combi-CLEAs from pectinases and cellulases: a potential biocatalyst for grape juice clarification

Combi-CLEAs of pectinases and cellulases were prepared for grape juice clarification.

Production, characterization of tannase from Penicillium montanense URM 6286 under SSF using agroindustrial wastes, and application in the clarification of grape juice (Vitis vinifera L.).

Tannase is an enzyme that hydrolyzes esters and lateral bonds of tannins, such as tannic acid, releasing glucose and gallic acid and stands out in the clarification of wines and juices. Fungi of the genera Aspergillus and Penicillium are excellent producers of this enzyme. The search for fungi that produce high levels of tannase as well as new substrates for the enzyme production by the SSF is required. The objectives of this study were to evaluate the production of tannase by Aspergillus and Penicillium species through SSF using leaves and agroindustrial waste barbados cherry and mangaba fruit as substrate, select the best producer, optimize production, characterize the crude enzyme extract, and apply it the clarification of grape juice. Selecting the best producer was performed by planning Placket-Burman and RSM. P. montanense showed highest activity with 41.64 U/mL after 72 h of fermentation residue using barbados cherry, with 3.5% tannic acid and 70% moisture. The enzyme showed the highest activity at pH 9.0 and 50°C. The tannase of P. montanense was stable over a wide pH range and temperature and, when applied to grape juice, showed higher efficiency by reducing 46% of the tannin content after incubation 120 m.

Effect of the addition of inert cellulose substrates to grape must on Saccharomyces cerevisiae diversity and the evolution of alcoholic fermentation

To study the addition of cellulose-based adjuvant as a resource to offset the negative effects produced by grape juice clarification during alcoholic fermentations. The effect of the addition of two kinds of inert cellulose substrates in white wine vinification was investigated in two different musts. In one of these musts, stuck fermentations were detected. One of the types of cellulose examined had a fining effect, which caused a decrease in the number of viable yeasts in the medium and altered the distribution and frequency of the clones, which performed the fermentation. The other cellulose substrate made the medium cloudier but did not alter the distribution of yeasts in comparison with the control. The behaviour of the inert cellulose substrates on vinification depends on its physical characteristics and its capacity for making the must cloudy. The addition of inert cellulose substrates in white wine vinification improves the fermentation process and the quality of wines obtained. This effect is more noticeable in difficult fermentations. One variety of cellulose showed an inhibitory effect on Torulaspora delbrueckii yeasts.

The Influence of Enological Practices on the Selection of Wild Yeast Strains in Spontaneous Fermentation

The aim of this work is to establish the influence of some enological treatments: grape juice clarification (by settling and by vacuum filtering), fermentation temperature (14°C and 18°C) and nitrogen addition (DAP 30 g/ hL and commercial activator 15.5 g/hL), on the natural selection of wild <i>Saccharomyces cerevisiae</i> strains during the spontaneous fermentation of white grape juice, of the Viura variety from La Rioja (Spain). Mitochondrial DNA analysis is applied to the study of 240 isolates from two consecutive harvests. The results reveal one new effect of the clarification treatment: the selection effect on the wild yeast strain of <i>Saccharomyces cerevisiae</i> in spontaneous fermentation. However, the strain distribution is independent of fermentation temperature and nitrogen addition.

The use of commercial enzymes in white grape juice clarification

Abstract A commercial pectinase from Aspergillus niger containing various polysaccharases clarified the white grape juice to an extent of 98–99% and also degraded the grape mash by 25–30%. This was achieved by optimising the grape mash treatment with 0.048% of enzyme at 27–30°C for 30 min without changing the mash pH. After pectinolytic juice clarification, both juice viscosity and total phenols were reduced by 25% and 32% respectively.

Effects of Some Treatments of Grape Juice on the Population and Growth of Yeast Species During Fermentation

The effects of grape juice clarification on the initial population and growth of wild yeasts were studied. Two musts were clarified using different treatments. One of them was treated with pectolytic enzyme, and then a single cold treatment at 3°C to 5°C was performed. The other must was submitted to an additional cold settling treatment combined with bentonite, gelatine, and coloidal silica. Despite the different occurrences of yeast species in the musts, the effects of the treatments were similar. The population of almost all yeasts decreased during the clarification, yet at the end of the treatment the levels reached were similar to those existing at the beginning of the process. The extent of removal of yeasts depended on each particular species. The most affected yeasts were <i>Issatchenkia terricola, Hansenula anomala</i>, and <i>Saccharomyces cerevisiae</i>. An unexpected proliferation of <i>Pichia membranaefaciens</i> in the early stages of the fermentation was observed. This fact was a consequence of the changes in the relative populations of the yeast species during clarification of the must.