苹果类产品中甲醇含量的控制

Fruit Processing Technical Service13/06/2000 -1-Methanol in apple products§Origin and production of methanolMethyl alcohol or methanol CH3OH is present in wood and plant pectin. Fruit pectinα-D-1,4-polygalacturonic acid backbone is partially esterified with methanol at C6 position. Methyl esterification degree is specific of fruit species. It is expressed in percentage (number of methanol groups for 100 galacturonic residues (see the table).FRUIT PECTIN (% wet weight)METHYLATION %Apple0.5 - 1.680 - 92 Blackcurrant 1.0 - 1.250 - 80Grape0.2 - 1.050 - 65Orange 2.3 - 3.065 - 70Pear0.5 - 0.850 - 70 Pineapple0.04 - 0.122 - 40Strawberry0.5 - 0.720 - 60Methanol releasing enzyme pectinmethylesterase (E.C. 3.1.1.11)has been widely described in plants and micro-organisms. The methylation degree tends to decrease during fruit ripening, released by the fruit endogenous pectinmethylesterase. Then it is released by exogenous fungal pectinmethylesterase when this enzyme is added at different stages during juice processing together with polygalacturonase for pectin hydrolysis.Pectin quantity and methylation degree of fruit species both determine the “potentially releasable methanol”.§Toxicity of methanol, daily intake and recommended maximum limitFruit pectin is de-esterified during digestion in the human intestine, not by human pectinases, but by symbiotic bacteria from the intestine flora which have a pectinmethylesterase, releasing about fifty per cent of methanol from ingested pectin as toxic compound. Calculation of the daily intake takes into account the total methanol content ingested since both forms free and bound to pectin are absorbable. Then methanol is slowly oxidised in formic acid in the blood increasing the acidity that can leser the optic nerve above 5 ml ingestion. Ingestion or inhalation of pure methanol can induce from headache, nausea, to partial or total blindness. Environmental Protection Agency (EPA) in the USA indicates a NAEL (No Adverse Effect Level) of 500 mg/kg/day for rats (J. Applied Toxicology,1994,14,pp351-353). Death from ingestion of less than 30 ml has been reported. Usual fatal dose is 100-250 ml.There is no methanol limit value defined by IFU or AFNOR in fruit juices.Average methanol content and standard in different beverages are detailed in the following table.Fruit Processing Technical Service13/06/2000 -2-§Methanol content and standard in beverages (1)Free methanol ppm(released by PME)Total potential methanol ppm in fruit Fruit juicesAppleClarified with pectinasesLiquefaction process10-5020-50450-55060-140Apple cloudy juice20-90Cherry140220White grape30-4525-100Red grape30-110120-190BlackcurrantClarified with pectinases150-200550120Orange1050-75Tomato160300Carrot50260Kiwi15200Fruit nectarsApricot30-50130-300Pear35310Peach 35300-325§Effect of raw material, pectinesterase and processing on methanol contentØApple pectin compositionMethanol is volatile and released by specific enzyme pectinmethylesterase from esterified pectin.Apple pectin has the highest methylation degree around 90% at the harvest time decreasing slowly not below 80% along post-harvest storage due to apple pectinesterase. Except for Citrus fruit, apple pectin content is also the highest. This is why methanol potentially released is more a concern in apple processing industry than in berry fruit industry.ØPectinmethylesterasePectinmethylesterase (PME) is naturally present in fruit and vegetable. This enzyme plays an important role in different technologies like juice clarification or fruit firming. PME activity in fruit is very low. When its action is desired, it can be increased by the addition of exogenous PME coming from molds like Aspergillus sp. Apple PME mode de-esterification has been described as “pectin mono-chain mechanism” w ith block-wise attack (1). Aspergillus PME mode of de-esterification has been described as “pectin multi-chain mechanism” with random attack (2). However, a change in amino-acid sequence could change the mode of action from one to the other, according to Danisco patent (3). PME releases methanol and free carboxylic acid functions are revealed, that can later bind with cations like calcium,making a strong insoluble gel of calcium pectate.ØFruit juice process§Eating an appleExample of one 100g apple:apple pectin content 0.4% (pectin content given in the table take into account the weight of neutral sugar side chains linked to the polygalacturonic backbone)methylation degree = 80% methanol MW = 32 galacturonic acid MW = 194total potential methanol absorbable from the apple = 80 x 32 / 100 x 194 = 0.131 x 0.4 = 53 mgFruit Processing Technical Service13/06/2000 -3-but in fact only about half of it is absorbed and the rest is eliminated 53 / 2 =27 mgIt is obvious that methanol content in fruit juices is directly related with the pectin content in final product, this latter depending on the process. Methanol has been assayed from 10 to 600 mg/L in fruit juices.§Apple ciderClassically, the french cider clarification occurred thanks to the natural apple PME. Addition of calcium chloride resulted in the formation of strong calcium pectate or brown coagulum, entrapping proteins and floating at the top of the juice surface due to CO2 bubbles released during alcoholic fermentation. This clarification was slow and not standardised because of variations of the PME activity of raw material. DSM has developed with INRA Le Rheu (F) a purified PME from Aspergillus niger, for fastening coagulum formation and cider clarification. The addition of exogenous PME increases apple PME activity and standardise the process. Because the demethylation is not complete and bound methanol discarded with coagulum (few hours process compared to few days in the classical process with almost complete pectin demethylation), methanol content in the clarified juice is lower. Then, methanol is partially removed during distillation for Calvados production.§Apple cloudy and clear juices and concentrateCloudy juice still contains pectin and theoretically more methanol than a clarified juice. Clear apple juice is processed with pectinases among which PME for juice clarification. Free methanol should be thus higher compared to cloudy juice. Apple pulp maceration process (pectinases 1h at 20°C) or liquefaction process (pectinases + cellulases 3h at 50°C) release more methanol because pectin demethylation is more complete. Methanol content range is 20-40 mg/L without maceration, 100-250 mg/L with maceration and 300-500 mg/L in liquefaction process. However, in every process, the juice is pasteurised just after pressing and part of methanol is removed. Apple juice is later concentrated under vacuum and methanol is also removed at this stage. Methanol content is concentrate is normally in standard range.§Grape mustGrape PME and pectinases added for must clarification play a role on methanol release. We can see an average increase of 20% of methanol content in grape must processed with pectinases with PME, + 30% in press juice compared to free run juice, however wine processed with pectinases never exceed the standard value after alcoholic fermentation.§Orange juiceCitrus fruit have a high natural PME activity, but juice is pasteurised as fast as possible after squeezing to destroy the PME and prevent clarification. Major part of methanol is removed at this stage.§Fruit firmingRapidase FP Super is a pure PME. Its application on whole fruit or fruit pieces results in demethylation of fruit pectin in situ. The demethylated pectin gelifies at acidic pH with calcium present in the fruit creating a strong pectate network, increasing the viscosity and consistency of fruit pieces or fruit sauce. Fruit identity and firmness are improved in fruit preparation added to dairy products like yoghurt or ice cream.Example of one 120 g yoghurt with 20% strawberry preparation at 50% fruit (10% fruit)Strawberry pectin content 0.5% (60 mg pectin in the yoghurt) with methylation degree = 50%Total theoritical demethylation is from 50% down to 20% = -30%methanol MW = 32 - galacturonic acid MW = 194free methanol released in fruit prep in the yoghurt = 30 x 32 / 100 x 194 = 0.05 x 0.06 =3 mgFruit Processing Technical Service13/06/2000 -4-§Methods of determinationMethanol assay is performed by Gas Chromatography (5).§DSM recommendationsIn addition to methanol potentially present then released in fruit by the natural PME, process conditions including the use of exogenous PME (next to other pectinases PL and PG) increase with about + 25% the final methanol content but this is difficult to predict. The example of blackcurrant is significant since starting from same pectin content and methylation percentage as apple, the methanol content in the juice is very much higher, because of the much longer depectinisation time (ambiant temperature process).The limitation of methanol content requires:§Fruit heating for denaturation of endogenous PME (i.e. for orange)§Maceration duration must be controlled for a limited release of soluble pectin§Depectinisation duration must be controlled as well§Use of a low PME pectinase for berry cold process§Metyhanol content is not an issue in the case of apple concentrate processing since methanol is removed in the vacuum concentration equipment.§Literature(all papers are available upon request)(1) Wucherpfennig K., Dietrich H. and Bechtel J.,1983,Flüssiges Obst,8(2) Denes J.M., PhD thesis,1999,University of Rennes,France.(3) Baron et al.,1980,Lebensm.Wiss.und Technol.,13,pp330-333.(4) Danisco WO 00/15830 patent. Pectin enzymatic modification(5) GC methanol assay。