Application In Juice Clarification Research Articles

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

A review on pectinase properties, application in juice clarification, and membranes as immobilization support.

Pectic substances cause haziness and high viscosity of fruit juices. Pectinase enzymes are biological compounds that degrade pectic compounds. Nontoxicity and ecofriendly nature make pectinases excellent biocatalysts for juice clarification. However, the poor stability and nonreusability of pectinases trim down the effectiveness of the operation. The immobilization techniques have gained the attention of researchers as it augments the properties of the enzymes. Literature has reported the stability improvement of enzymes like lipase, laccase, hydrogen peroxidase, and cellulase upon immobilization on the membrane. However, only a few research articles divulge pectinase immobilization using a membrane. The catalysis-separation synergy of membrane-reactor has put indelible imprints in industrial applications. Immobilization of pectinase on the membrane can enhance its performance in juice processing. This review delineates the importance of physicochemical and kinematic properties of pectinases relating to the juice processing parameters. It also includes the influence of metal-ion cofactors on enzymes' activity. Considering the support and catalytic-separation facets of the membrane, the prediction of the membrane as support for pectinase immobilization has also been carried out.

Yellow mombin pulp residue valorization for pectinases production by Aspergillus niger IOC 4003 and its application in juice clarification

Pectinases are enzymes responsible for cleaving pectin and other pectic polysaccharides in galacturonic acid monomers. They are widely applied in industrial sectors. In this context, the present study evaluated polygalacturonase (PG), pectin lyase (PMGL) and FPase production by Aspergillus niger IOC 4003 in solid-state fermentation using yellow mombin pulp residue as substrate. Three parameters were investigated for extraction of PG such as temperature, pH stability and ionic strength. The action of PG was assessed under different states of temperature and pH as well in yellow mombin juice clarification. The antibacterial activity of yellow mombin juice was additionally evaluated. Results revealed that the highest yield of PG produced (38.22 ± 0.63 U/g) was achieved at 72 h fermentation using washed yellow mombin pulp residue. Characterization studies indicated stability of PG for 2 h at 30 and 40 °C with pHs between 4 and 6. The PG applied in the yellow mombin was able to reduce turbidity by 41% and viscosity by 52%. Also, it was able to increase color by 36% and antioxidant capacity by 39%. Finally, the yellow mombin juice exhibited antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Klebsiella pneumoniae. Hence, this study demonstrated the feasibility of using tropical fruit residues from industries for pectinases synthesis and showed their application in the food industry.

Production, Characterization, and Industrial Application of Pectinase Enzyme Isolated from Fungal Strains

Pectinases are the group of enzymes that catalyze the degradation of pectic substances. It has wide applications in food industries for the production and clarification of wines and juices. The aim of this study was to isolate, screen and characterize pectinase from fungi isolated from various soil samples and evaluate its application in juice clarification. Fungal strains were isolated and screened primarily using 1% citruspectin incorporated potato dextrose agar (PDA) and secondarily using pectinase screening agar medium (PSAM) for pectinolytic organisms. The enzyme was produced by submerged state fermentation and assayed using the dinitro salicylic acid (DNS) method. From 20 different soil samples, 55 fungal isolates were screened primarily and, among them, only 14 isolates were subjected for secondary screening. Out of 14, only four strains showed the highest pectinolytic activity. Among four strains, Aspergillus spp. Gm showed the highest enzyme production at a 48-h incubation period, 1% substrate concentration, and 30 °C temperature. The thermal stability assessment resulted that the activity of pectinase enzyme declines by 50% within 10 min of heating at 60 °C. The optimum temperature, pH, and substrate concentration for the activity of enzyme was 30 °C (75.4 U/mL), 5.8 (72.3 U/mL), and 0.5% (112.0 U/mL), respectively. Furthermore, the yield of the orange juice, the total soluble solid (TSS), and clarity (% transmittance) was increased as the concentration of the pectinase increased, indicating its potential use in juice processing. Overall, the strain Aspergillus spp. Gm was identified as a potent strain for pectinase production in commercial scale.

High-level expression and characterization of a novel aspartic protease from Talaromyces leycettanus JCM12802 and its potential application in juice clarification

Aspartic proteases are promising fining agents used in the production of fruit juices. In this study, a novel aspartic protease gene (Tlap) was identified in Talaromyces leycettanus JCM12802 and heterologously expressed in Pichia pastoris. Using casein as the substrate, purified recombinant TlAP showed optimal activities at pH 3.0 and 55 °C with a specific activity of 1795.4 ± 62.8 U/mg, and remained stable over a pH range of 3.0–6.0 and at temperatures of 45 °C and below. Moreover, the enzyme was highly resistant to most metal ions and chemical reagents except for Fe3+ and β-mercaptoethanol. When added to apple, orange, grape and kiwi fruit juice, it showed excellent proteolytic activity against haze-forming proteins, decreasing the turbidity by up to 49.9 nephelometry turbidity units (NTU). These favorable enzymatic properties make TlAP attractive for potential use in the juice industry.

A low-temperature polygalacturonase from P. occitanis: characterization and application in juice clarification

An extracellular endo-polygalacturonase (PGase) was purified, after a single purification step, from the constitutive and hyperpectinolytic CT1 mutant of Penicillium occitanis. This enzyme named PG2 has a molecular weight of 42kDa. It was optimally active at 35°C and pH6 with more than 85% of activity at pH7 in contrast to the majority of fungal PGase, generally acting at 50°C and pH5. The specific activity obtained was among the highest ones, 31397.26U/mg. The PGase activity increased with the decrease of the degree of methylation (DM) of pectin, but it was also able to degrade the highly methyl-esterified substrates, 70% (DM) and 90% (DM), with almost 80% and 40% of residual activity respectively. Interestingly, PG2 is completely inhibited by DEPC, suggesting the implication of a Histidine residue in the active site. The sequencing of P. occitanis whole genome allowed us to identify the pga2 gene encoding PG2 and to localize the His residue, target of DEPC, while it was absent in the PG1 that resisted to DEPC. Besides that, the potentialities of PG2 have been put in use in juice clarification of pear, banana and citrus juice.

Stabilization by multipoint covalent attachment of a biocatalyst with polygalacturonase activity used for juice clarification

Derivatized-agarose supports are suitable for enzyme immobilization by different methods, taking advantage of different physical, chemical and biological conditions of the protein and the support. In this study, agarose particles were modified with MANAE, PEI and glyoxyl groups and evaluated to stabilize polygalacturonase from Streptomyces halstedii ATCC 10897. A new immobilized biocatalyst was developed using glyoxyl-agarose as support; it exhibited high performance in degrading polygalacturonic acid and releasing oligogalacturonides. Maximal enzyme activity was detected at 5h of reaction using 0.05g/mL of immobilized biocatalyst, which released 3mg/mL of reducing sugars and allowed the highest product yield conversion and increased stability. These results are very favorable for pectin degradation with reusability up to 18 successive reactions (90h) and application in juice clarification. Plum (4.7°Bx) and grape (10.6°Bx) juices were successfully clarified, increasing reducing sugars content and markedly decreasing turbidity and viscosity.

Immobilization of Commercial Pectinase (Polygalacturonase) on Celite and Its Application in Juice Clarification

The commercial pectinase (polygalacturonase) was immobilized on celite through adsorption. There was 2.5% concentration of glutaraldehyde used as cross-linking agent and a binding time of 4 h was found to be the best time for its proper binding. Binding efficiency of enzyme to the matrix was 43.18%. The immobilized enzyme retained almost 50% of activity after third cycle of reuse. Free enzyme showed maximum activity with polygalacturonic acid (PGA, 0.9%) as substrate at 40C temperature, pH 5.5 and incubation time of 20 min while immobilized enzyme showed maximum activity with PGA (0.9%) as substrate at temperature of 45C, pH 5.5 and incubation time of 15 min. Both free and immobilized enzyme were inhibited by metal ions, thiols, phenolics and protein inhibitors. For clarification of 1.0 mL of pineapple juice, 20 mg of immobilized enzyme per mL of juice at 45C temperature and 1 h of holding time was optimized. Practical Applications Pectinases find commercial application in fruit juice, wine, oil, tea, coffee, textile and paper-making industries using a wide variety of carriers and methods. One of the vital applications is the clarification and depectinization of fruit juices. The raw fruit juice obtained after pressing is very turbid viscous and contains a significant amount of colloidal compounds, mainly pectin that causes cloudiness; therefore, clarification of fruit juices involves the removal of juice haze by enzyme hydrolysis with pectolytic enzymes. Although pectinases enhance the clarification of juices, immobilization of these enzymes proves to be beneficial for industrial use. Immobilization of pectinase on celite through adsorption is a simple, cheap and effective method. For the clarification of pineapple juice, excellent results were observed using immobilized polygalacturonase in comparison with free enzyme.

Inactivation ofEscherichia coliand Quality Changes in Black Mulberry Juice Under Pulsed Sonication and Continuous Thermosonication Treatments

Effects of pulsed sonication and continuous thermosonication treatments on Escherichia coli ATCC 25922 inactivation in black mulberry juice were investigated at different acoustic power densities and different temperatures. The inactivation data were evaluated with five selected kinetic models with regard to adjusted correlation coefficient and root mean square error. The inactivation data of all treatments were well fitted with Weibull model followed by biphasic linear and first-order models. Treatment time for 5 log reduction of the E. coli was estimated in 14.10 min for pulsed sonication at 25C and 1.63 W/mL and 10.45 min for continuous thermosonication at 50C. Additionally, some physicochemical properties of black mulberry juices were tested for the treatments. Color and turbidity values increased, whereas monomeric anthocyanin contents decreased by the treatments. However, remarkable changes were not observed in titratable acidity, pH, percent polymeric color and antioxidant activity of black mulberry juice. Practical Applications This study demonstrates that the Weibull was the best model for estimation of ultrasonic pasteurization parameters of the black mulberry juice. Black mulberry juice could be pasteurized by the sonication treatments even at low temperatures. Because it takes a long time at low temperature, the treatment could be assisted by heat treatment at mild temperature to decrease pasteurization time. The ultrasonic treatment which led to increase in turbidity of the juice can be a potential application in juice clarification to reduce the risk of post-turbidity during storage.

Cloning, expression and characterization of an acidic endo-polygalacturonase from Bispora sp. MEY-1 and its potential application in juice clarification

An endo-polygalacturonase gene, pga1, was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. The 1455-bp full-length complementary DNA of pga1 encoded a 485-amino acid polypeptide (endo-PGA1) including a putative 21-residue signal peptide and a catalytic domain belonging to glycoside hydrolase family 28. Purified recombinant endo-PGA1 exhibited activity towards polygalacturonic acid and pectin was optimally active at pH 3.5 and 50 °C, and showed good stability at pH 2.0–7.0. When tested against pectin, endo-PGA1 exhibited 40% of maximum activity at pH 2.0 and over 50% maximum activity at pH 2.5–4.5. The K m,app and V max,app values for polygalacturonic acid were 1.25 mg/ml and 2526 μmol/min/mg, respectively. When treated apple juice at the concentration of 10 U/ml, endo-PGA1 reduced the intrinsic viscosity (7.7% vs. 8.0%) and increased the light transmittance (84% vs. 86%) almost on the same level as the commercial compound pectinase did. These properties make endo-PGA1 an interesting biocatalyst for acidic industrial processes, especially in the juice clarification.