柚苷酶处理、果汁浓缩和低温贮藏对柚子果汁中柠檬苦素的影响

采用高效液相色谱法测定柠檬苦素含量,以柚子果汁为对象研究柚苷酶处理、果汁浓缩和低温贮藏等工序对柑橘果汁中柠檬苦素含量的影响。结果表明,液相色谱方法可使柠檬苦素、普鲁宁、柚皮苷和柚皮素完全分离,消除了柚皮素对柠檬苦素测定的影响;柠檬苦素检出限为0.95μg/m L,定量限为3.17μg/m L,样品回收率为102.6%~104.2%,相对标准偏差为0.34%~1.04%。柚苷酶水解过程中的加热及果汁浓缩和低温贮藏等操作都能促进柠檬苦素的从果汁中结晶析出,综合采用柚苷酶水解、浓缩和低温贮藏等操作,并经过离心分离可去除柚汁中78%的柠檬苦素,使果汁中柠檬苦素含量降低到19μg/m L。     

Anti-inflammatory activity of naringin and the biosynthesised naringenin by naringinase immobilized in microstructured materials in a model of DSS-induced colitis in mice

A great number of antioxidants are naturally present in citrus juice, being responsible for their potentially protective action against inflammation. Naringin, a bitter compound, in citrus, is hydrolysed by naringinase into tasteless naringenin and to rhamnose and glucose. The aim of this study was to evaluate the anti-inflammatory activity of naringin and naringenin in an acute model of induced colitis in mice. Microstructured particles were used for naringinase immobilization. The influence of naringin and naringinase concentration on the bioconversion was evaluated through a central composite design.Colitis was induced by feeding mice 7% dextran sodium sulphate (DSS) dissolved in drinking water for 5 days. Intestine dry/wet weight ratio (indicator of intestine edema), nitrates/nitrites (indicators of inflammatory process), and tissue malondialdehyde levels (indicator of lipid peroxidation) were evaluated. Our results indicate that treatment with both naringin and naringenin significantly reduced the formation of intestine edema, suggesting an anti-inflammatory activity in this model of colitis in mice.     

Immobilization of naringinase from Aspergillus niger CECT 2088 in poly(vinyl alcohol) cryogels for the debittering of juices

Naringinase, induced from Aspergillus niger CECT 2088 cultures, was immobilized into a polymeric matrix consisting of poly(vinyl alcohol) (PVA) hydrogel, cryostructured in liquid nitrogen, to obtain biocatalytically active beads. The effects of matrix concentration, enzyme load and pH on immobilization efficiency were studied. Between 95% and 108% of the added naringinase was actively entrapped in PVA cryogel, depending on the conditions of immobilization used. The optimal conditions were: 8% (w/v) PVA at pH 7 and 1.6–3.7 U ml⁻¹ of enzyme load. The pH/activity profiles revealed no change in terms of shape or optimum pH (4.5) upon immobilization of naringinase. However, the optimum temperature was shifted from 60 °C to 70 °C and the activation energy of reaction, E_a, was decreased from 8.09 kJ mol⁻¹ to 6.36 kJ mol⁻¹ by immobilization. The entrapped naringinase could be reused through six cycles (runs of 24 h at 20 °C), retaining 36% efficacy for the hydrolysis of naringin in simulated juice.     

柚皮苷酶对琯溪蜜柚果汁脱苦效果工艺优化

以琯溪蜜柚果汁为对象,研究在不同条件下柚皮苷酶对蜜柚果汁的脱苦效果。采用HPLC 测定柚皮苷含量,通过单因素试验分别考察加酶量、温度、酶解时间及pH 值对柚皮苷酶酶解反应的影响,在单因素试验基础上以柚皮苷脱除率为指标进行正交试验,确定最佳工艺条件：酶解温度60℃,酶活7.4U/mL 柚汁,酶解时间100min,pH3.6。在此条件下琯溪蜜柚果汁的柚皮苷脱除率达97% 以上。     

Debittering Citrus Juices with β-Cyclodextrin Polymer

A β-cyclodextrin polymer was used to remove the bitter substances limonin and naringin from aqueous solution and from filtered orange and grapefruit juices using batch and continuous flow column treatments. Other components such as naringenin 7β-rutinoside, coumarins and flavonoids were removed, but the total soluble solids (°Brix), total acidity and ascorbic acid content of the juice were unchanged. The polymer could be regenerated by extraction with an organic solvent.     

Purification and characterisation of Aspergillus sojae naringinase: The production of prunin exhibiting markedly enhanced solubility with in vitro inhibition of HMG-CoA reductase

Aspergillus sojae isolated from a traditional Korean fermented soybean product exhibited strong naringinase activity. The naringinase enzyme was purified and had a molecular weight of 70 kDa. The α-l-rhamnosidase activity of this enzyme was optimal at pH 6.0 and stable in the pH range of 5.5–8.0. The purified enzyme also had β-d-glucosidase activity, but the activity was relatively weak compared to the activity of the naringinase from Penicilliumdecumbens. The enzymatic bioconversion by A. sojae naringinase of naringin to prunin was efficiently performed with a 91% yield and a negligible amount of naringenin. The bioconversion was achieved by repetitive batch reactions with enzyme recycling. Prunin exhibited a markedly enhanced solubility compared to naringenin and naringin while maintaining the in vitro inhibition of HMG-CoA reductase. The results reported in this paper show that the naringinase produced by A. sojae will be useful in enhancing the potential bioavailability of naringin by efficiently converting it to prunin as a food component in citrus.     

黑曲霉DB056柚苷酶制剂对琯溪蜜柚果汁的脱苦效果

采用高效液相色谱法测定琯溪蜜柚果汁中柚皮苷及其降解产物的变化,研究黑曲霉DB056柚苷酶制剂脱苦琯溪蜜柚果汁的优化条件;并且在此优化条件下,使用该柚苷酶制剂对琯溪蜜柚鲜柚汁、浑浊浓缩柚汁和澄清浓缩柚汁进行脱苦。结果表明：黑曲霉DB056柚苷酶制剂催化琯溪蜜柚果汁中柚皮苷水解成普鲁宁和柚皮素;该柚苷酶制剂在40～55℃、果汁自然pH值条件下对琯溪蜜柚鲜柚汁进行脱苦,酶用量90U/mL、处理时间80min,柚皮苷脱除率达到89%;处理不同浓缩程度的浑浊浓缩柚汁,脱苦率约为80%左右。结果说明黑曲霉DB056柚苷酶制剂对琯溪蜜柚果汁具有良好的脱苦作用。     

Reduction of bitterness and acidity in grapefruit juice by adsorptive processes

By choosing an appropriate adsorbent, the bitter principles (naringin and limonin) and titratable acids could be removed from grapefruit juice in varying combinations. For example, Amberlite XAD-7 removed about 63% naringin, 85% limonin and 3% titratable acids and Deacidite-FFIP removed about 20% naringin, 8% limonin and 23% titratable acids when contacted for 1 h with grapefruit juice at the rate of 20 g (dry wt) of adsorbent per litre of juice. Such treatments, applied consecutively, would eliminate excessive bitterness and acidity from juices containing up to 2 g naringin and 36 mg limonin litre^?1 and with a total soluble solids:titratable acid ratio of < 6. The adsorbents are readily available, may be used as supplied and can be reactivated simply and economically; some of them have already been approved for use with foods, and such approval seems possible for the others.     

Limonoids and Flavonoids in Juices of Oroblanco and Melogold Grapefruit Hybrids

Oroblanco and Melogold are hybrids obtained from pummelo and grapefruit. Limonoids and flavonoids in both juices were analyzed. Oroblanco and Melogold juices contained low concentrations of limonoid glucosides, an average of 99 and 59 ppm, respectively. However, they contained relatively high concentrations of bitter limonoid aglycones, limonin and nomilin, at levels above the limonin bitterness threshold. For comparison, limonoid glucosides in juices of grapefruit, another pummelo hybrid, were also analyzed. Limonin glucoside was the major limonoid glucoside in all juices analyzed. Nomilin glucoside and nomilinic acid glucosides were also present. Oroblanco and Melogold juices contained bitter flavonoids normally found in grapefruit and pummelo including naringin, neohesperidin and poncerin in total amount of 440 ppm in Oroblanco juice and 495 ppm in Melogold juice. They also contained several other nonbitter flavonoids found in grapefruit.     

黑曲霉TC-01产柚苷酶对柚皮苷酶解作用的研究

柚皮苷是一类主要的类黄酮物质,它是引起柚类和葡萄果实苦味的主要来源,去除柚皮苷会使果汁苦味减轻,而柚皮苷酶能在不影响柑橘果汁品质的前提下较好地去除苦味。但是由于不同来源的柚苷酶的结构不同,其催化特性也不尽相同,本文通过利用前期筛选的黑曲霉(Aspergillus.niger TC-01)产柚苷酶对柚皮苷酶解作用进行研究,实验采用高效液相色谱法测定柚皮苷、普鲁宁和柚皮素的三者之间含量变化,确定酶解最适温度、时间、pH及酶用量,并测定TC-01产柚苷酶的米氏常数以确定酶解反应的最佳条件。结果表明,TC-01产柚苷酶酶解最适温度为60℃、酶解最适时间为30-40min,最适pH4.0,酶最佳用量为24u/mL。米氏方程为Y=0.0018X-3.0×10-6,其中Km值为598.8ug/mL,Vmax=2.06g/(min.mL)。此研究结果为柚苷酶的应用提供了理论依据。     

Limonin and Naringin Removal from Grapefruit Juice with Naringinase Entrapped in Cellulose Triacetate Fibers

Naringin and limonin are two bitter components of some citrus products such as grapefruit juice. Naringin could be removed by hydrolysis with immobilized naringinase and limonin might be removed by adsorption with cellulose monoacetate gel beads. Cellulose triacetate fibers show a similar limonin adsorption capacity as cellulose monoacetate gel beads. Thus when naringinase from Penicillium sp. was entrapped in such fibers, an enzyme column was made which could remove both bitter components simultaneously. When grapefruit juice was debittered with this enzyme column, sugar components, total organic acids and turbidity were not affected. The enzyme column could be regenerated by washing with warm water. In addition, operational stability of the enzyme column was satisfactory. Such enzyme column could be considered for industrial use.     

Treatment of Grapefruit Juice for Bitterness Removal by Amberlite IR 120 and Amberlite IR 400 and Alginate Entrapped Naringinase Enzyme

ABSTRACT: Treatment of grapefruit ( Citrus aurantium ) juice with Amberlite IR 400 resulted in 69.23% naringin removal after 5 min exposure with significant clarification and 89.41% tartness (acidity) reduction. Amberlite IR 120 removed only 9% naringin in 1 min with increased shelf life of the juice without any change in clarity, quality, and naringin content. Alginate entrapped naringinase treatment of the juice resulted in 83.84% naringin hydrolysis at 55°C and 220 rpm in 180 min with 1.98 enzyme units/mL of juice. The soluble enzyme hydrolyzed only 65.53% naringin under similar conditions. Kinetic constant values of the immobilized and soluble enzymes were found to be 9.75 mM and 20 mM, respectively.     

Immobilization of naringinase on asymmetric organic membranes: Application for debittering of grapefruit juice

An enzymatic membrane reactor (EMR) is assembled for the immobilization of naringinase on a polyethersulfone ultrafiltration membrane, based on fouling-induced method. The effects of molecular weight cut-off, membrane configuration, applied pressure, enzyme concentration and pH are studied in terms of permeate rate, immobilization efficiency, and biocatalytic conversion. The 10 kDa membrane operating in reverse mode, 0.2 MPa, 0.3 gL⁻¹ of enzyme in acetate buffer at pH 5 and cross-linking with 0.25% glutaraldehyde showed the highest naringin conversion (73%). It was determined that the intermediate pore blocking model was the predominant fouling mechanism for the enzymatic immobilization. The EMR was applied for debittering of grapefruit juice, achieving a conversion of naringin below bitterness threshold and maintaining the antioxidant capacity of the juice. Furthermore, the biocatalytic activity of immobilized enzyme was retained at a high level at least during three consecutive reaction runs, with overnight storage at 4 °C after each run.Industrial relevanceThe potential of membrane technologies in the juice industries is widely recognized today. The development of EMR with naringinase activity is an attractive option for reducing bitterness that could replace current techniques, due to its high specificity and effectiveness, possibility of repeated and continuous use, and in order to retain the properties of juice as much as possible. The research therefore represents an advance in the application of biocatalytic membranes as technological alternative for juice debittering.     

Evaluation of the Properties of Polystyrene Divinylbenzene Adsorbents for Debittering Grapefruit Juice

Various physical properties of polystyrene divinylbenzene adsorbents involved in the removal of the bitter compounds naringin and limonin from grapefruit juice were investigated in a series of batch reaction processes. The removal of naringin from model systems and, naringin and limonin from grapefruit juice increased with the degree of cross-linkage and surface area of the adsorbents which had a solid density higher than 1.2 g/mL (dry) to ensure complete submersion. The specific surface area appeared to be the major factor in the debittering process. Regeneration with 95% ethanol was found to be more efficient than warm water. Sensory evaluation tests showed that more than 65% of the panelists preferred the less bitter treated juice over the control.     

Naringinase Immobilization in Packaging Films for Reducing Naringin Concentration in Grapefruit Juice

Naringin, a bitter compound in citrus, may be converted to a nonbitter form by enzymic hydrolysis. Our objective was to determine the feasibility of immobilizing naringinase in a food contact approved packaging film. Naringinase from Penicillium sp. was immobilized in cellulose acetate films with up to 23% efficiency at 7°C. Kinetic studies showed that the free enzyme had an optimum pH=3.5 and the immobilized enzyme pH=4.0. Activation energy decreased upon immobilization (from 14.2 to 11.0 Kcal/mol), thus providing an increased catalytic efficiency for immobilized naringinase. The Michaelis constant for immobilized naringinase (K_m=2.1 mM) was lower than for free enzyme (K_mm=3.6 mM). Keeping films under dry storage for 1 mo at room temperature did not cause decreased enzyme activity. A film area/volume ratio (cm²/mL of 10° Brix grapefruit juice) of 7.2 hydrolyzed 60% of the naringin in 15 days at 7°C.     

Pectinase and naringinase help to improve juice production and quality from pummelo (Citrus grandis) fruit

Pectinase and naringinase were investigated for improving the production of pummelo juice by increasing the juice yield and eliminating juice bitterness. Compared to a control, the enzymatic treatment significantly (p<0.05) increased the juice yield, soluble pectin and total soluble solid (TSS) contents, and the clarity, while decreasing the concentrations of the bitter chemicals naringin, limonin, and nomilin. A combined processing treatment of peeling and enzymatic hydrolyses using 5U/g of pectinase and 0.4 U/g of naringinase at 50oC for 60 min resulted in a juice yield of 42.3%, a TSS content of 11.4oBx, a titrable acidity (TA) of 0.96%, a vitamin C concentration of 38.4 mg/100 mL, and concentrations of naringin, limonin, and nomilin of 42.4 μg/mL, 33.5 μg/mL, and 13.3 μg/mL, respectively. The enzymatic method was effective and practical for production of high quantity pummelo juice.     

Effect of enzymatic debittering on antioxidant capacity and protective role against oxidative stress of grapefruit juice in comparison with adsorption on exchange resin

Antioxidant capacity, radical scavenging activity, as well as protective effect on lipoperoxidation, glutathione oxidation and DNA damage, were evaluated in grapefruit juice subjected to bitterness removal by naringinase or by physical adsorption with Amberlite®IRA-400. The results showed a reduction in naringin content for the naringinase-treated juice (N-PJ) and those processed with the exchange resin (R-PJ), which made both juices acceptable to consumers. Total antioxidant capacity, measured by ABTS and FRAP assays, was lower in R-PJ samples. The highest superoxide and hydroxyl radical scavenger activity was observed in N-PJ. With regard to inhibitory effect of juice samples on lipoperoxidation, N-PJ also provided the greatest effectiveness. In addition, R-PJ showed the lowest levels of GSH. The results showed a protective effect on DNA oxidative damage for all juice samples tested. In summary, enzymatic technology was more effective than physical adsorption in preserving the antioxidant and biomolecule protection capacity of fresh grapefruit juice.     

Stability of black carrot anthocyanins in various fruit juices and nectars

Fruit juices (apple, grape, orange, grapefruit, tangerine and lemon) and nectars (apricot, peach and pineapple) were coloured with black carrot juice concentrate and stability of black carrot anthocyanins in these matrices was studied during heating at 70–90 °C and storage at 4–37 °C. Anthocyanin degradation, in all coloured juices and nectars, followed first-order reaction kinetics. During heating, black carrot anthocyanins in apple and grape juices showed higher stability than those in citrus juices at 70 and 80 °C. High stability was also obtained for the anthocyanins in peach and apricot nectars at these temperatures. Black carrot anthocyanins were the least stable in orange juice during both heating and storage. During storage, degradation of anthocyanins was very fast at 37 °C, especially in pineapple nectar. Refrigerated storage (4 °C) markedly increased the stability in all samples. Activation energies for the degradation of black carrot anthocyanins in coloured juices and nectars ranged from 42.1 to 75.8 kJ mol⁻¹ at 70–90 °C and 65.9–94.7 kJ mol⁻¹ at 4–37 °C.     

Naringinase Immobilization in Polymeric Films Intended for Food Packaging Applications

ABSTRACT: We present a method of obtaining a food-grade active film able to reduce the naringin content of grapefruit juices during storage by a direct interaction with the product. The active film consists of a crosslinked matrix in which naringinase was completely immobilized. The proposed method was tested by first assessing the efficacy of the immobilization procedure and then evaluating the capability of the active film to reduce the naringin concentration in standard solutions. Results indicate that the developed active film has commercial potential to improve the sensory properties of grapefruit juices.     

Reduction of Bitterness and Tartness in Grapefruit Juice with Florisil

Treatment of commercial grapefruit juice with Florisil (activated magnesium silicate) simultaneously reduced the content of citric acid and the bitter compounds limonin and naringin. Ascorbic acid concentration and °Brix (total soluble solids) were not altered by the Florisil treatment. Experienced taste panelists were able to differentiate between the nontreated and Florisil-treated juice on the basis of bitterness and tartness (acidity). The panelists indicated a preference for the Florisil-treated juice.