eight 150 g samples of tomato dice were subjected to a range of ultra high pressures for different lengths of time using a high hydrostatic pressure unit . the pressure and time treatments used are listed in table 1 . pressure , up to a maximum of 600 mpa , was provided by water pumped into the base of the unit . water was used as the pressure transferring medium . generally , it took 2 - 3 minutes for the uhp system to reach the chosen pressure . it also took 2 - 3 mins to depressurize after treatment . the pressure and temperature , measured at four points within the unit , were logged continuously by a computer . all experiments were carried out at ambient temperature . a portion of each pressure - treated sample was stored for analysis either by freezing it rapidly using dry ice and storing at - 20 ° c . or by packing it in 150 g of tomato juice in foil pouches , heat sealing the pouches and heat treating for 30 minutes in a waterbath set at 97 ° c . this heat treatment was sufficient to inactivate all enzymes and to achieve pasteurisation . the remainder of each pressure - treated sample was placed in a sachet and incubated for 30 minutes in a waterbath set at either 40 ° c . or 60 ° c . these samples were also stored for analysis , using the method described above . for each stored sample , the activity of the enzymes pectin methyl esterase ( pme ), polygalacturonase ( pg ) , lipoxygenase and peroxidase was determined using methods i to iv described below . the activity of these enzymes was also measured in a sample of the tomato dice which had not undergone the pressure and thermal incubation treatments described above ( this was the control sample ). i ) pme activity was assayed by adding 200 μl of sample to 20 mls 1 % apple pectin in 0 . 15m sodium chloride ( ph 7 . 5 with sodium hydroxide ). activity was calculated from the slope of mls 0 . 025m naoh used to maintain ph 7 . 5 at 30 ° c . against time . ii ) pg activity was measured by the detection of reducing ends , which are generated when the enzyme breaks down polygalacturonic acid , using the calorimetric pahbah assay ( lever m . ( 1972 ) anal biochem 47 : 273 - 279 ). pahbah is an abbreviation of the chemical p - hydroxybenzoic acid hydrazide , which is used for the colour detection of reducing sugars . a high salt extract was prepared by adding low salt buffer ( 25 mm sodium acetate buffer ph 5 . 6 ) to a tomato sample at a ratio of 1 : 2 volume : weight , shaking vigorously then centrifuging at 10 , 000 g for 20 minutes at 4 ° c . after centrifugation , the supernatant was poured away and the pellet resuspended in an equivalent volume of a high salt buffer ( 25 mm sodium acetate buffer ph 5 . 6 containing 1 . 0m sodium chloride ) and left to stand at 4 ° c . for 1 hour . the suspension was then respun at 10 , 000 g , 4 ° c . for 20 minutes and the supernatant ( high salt extract ) stored at - 70 ° c . in 1 ml aliquots . for pg assays , the high salt extract was diluted 1 : 100 in 50 mm sodium acetate buffer ph 4 . 0 containing 0 . 2m nacl ; the activity was assayed using 0 . 2 % polygalacturonic acid in a total assay volume of 500 μl . the assays were incubated at 40 ° c . for 1 hour and terminated by generating colour , which comprised adding 5 mls of pahbah reagent and boiling for 6 minutes . after cooling under running water to room temperature , absorbance was read at 410 nm using quartz cuvettes against a standard curve of galacturonic acid . controls were performed by terminating assays with pahbah reagent as soon as the enzyme extract was added , ie . at zero time , and boiling as above . iii ) lipoxygenase activity was determined at 30 ° c . using the polarographic method of nicolas et al ( 1982 j . sci food agric 33 : 365 - 372 ) using linoleic acid ( 28 mm final concentration ) dispersed in 0 . 1m sodium phosphate buffer ph 7 . 0 containing polyoxyethylenesorbitan monolaurate ( 0 . 8 % final concentration ) in a total volume of 5 ml . iv ) peroxidase activity was determined by the spectrophotometric quantification of o - quinone production in a mixture containing 3 . 0 μl 30 % hydrogen peroxide ( 8 . 8 mm final concentration ), phosphate buffer ph 7 . 0 and enzyme extract , in a total volume of 2 . 925 ml . the reaction was initiated by adding 0 . 075 ml of guaiacol ( 5 mm final concentration ) and measuring the change in absorbance at 400 nm using a spectrophotometer with a temperature controlled cuvette holder at 30 ° c . the results of these measurements for each sample after pressure - treatment alone are shown in table 1 below . table 1______________________________________ % activity remaining after treatmenttreatment pme pg lipoxygenase peroxidase______________________________________300 mpa , 5 min 100 100 -- -- 300 mpa , 15 min 100 99 -- -- 400 mpa , 10 min 100 100 74 96400 mpa , 20 min 100 62 51 79500 mpa , 10 min 100 3 1 77500 mpa , 20 min 100 0 0 71600 mpa , 10 min 100 0 0 5600 mpa , 20 min 100 0 0 0______________________________________ the results of these measurements for samples after pressure - treatment alone and after both pressure - treatment and thermal incubation are shown in table 2 below , together with the results for the control sample . table 2______________________________________ % activity remaining after treatmenttreatment pme pg lipoxygenase peroxidase______________________________________no treatment 100 100 100 100 ( control ) 400 mpa 10 mins 100 100 74 96400 mpa 10 mins + 90 76 33 10040 ° c . 30 mins400 mpa 10 mins + 58 45 0 10060 ° c . 30 mins600 mpa 20 mins 100 0 0 0600 mpa 20 mins + 86 0 0 040 ° c . 30 mins600 mpa 20 mins + 72 0 0 060 ° c . 30 mins______________________________________ the results in table 1 show that pme is the most barostable enzyme . its activity is not affected after a pressure treatment of 600 mpa for 20 minutes . peroxidase is the next most stable enzyme , followed by pg and lipoxygenase , which have similar stabilities . after a pressure treatment of 500 mpa for 20 mins , both pme and peroxidase are active whilst pg and lipoxygenase have been inactivated ; the presence of peroxidase is favourable to flavour and aroma . from table 2 , it is clear that there are pressures which inactivate pg , lipoxygenase and peroxidase but do not inactivate pme . subsequent incubation allows the active pme to promote firming in the absence of pg - promoted texture degradation , as evidenced by example 2 below . in accordance with the methods of example 1 , tomato juice samples were treated with uhp ( either 400 mpa for 10 mins which is not sufficient for pg inactivation or 600 mpa for 20 mins which is sufficient for pg inactivation ) and incubated at either 40 ° or 60 ° c . for 30 mins . samples were then pasteurized by filling into foil pouches , heat sealing and placing in a waterbath at 95 ° c . for 30 minutes . all enzyme activities were inactivated after this pasteurising heat treatment . the effects of the pressure and incubation treatments on the consistency of the tomato juice were measured by determining the serum to pellet ratio and the bostwick value of the treated tomato juice . the serum to pellet ratio and bostwick value were also determined for non - treated tomato juice , as a control . serum to pellet ratio is a measure of the volume occupancy of the insoluble solids component of tomato juice and was determined by centrifuging the juice ( 3000 g , 10 minutes ) and then weighing the resultant serum ( solubles ) and pellet ( insolubles ) fractions to determine the ratio . the higher the level of insolubles ( ie the lower the serum to pellet ratio ), the thicker the sample and the higher its consistency . bostwick is the standard industry measurement for tomato product viscosity . to obtain juice bostwick values , the serum was removed from the pellet by centrifugation as above and then added back to 70 % of the original sample weight . the measurements were made in a standard bostwick viscometer . the sample was placed in the levelled viscometer , the shutter opened and a timing clock started . the distance ( in centimetres ) at 20 ° c . over which the sample flowed in 30 s was recorded . the lower the bostwick value , the thicker the sample and the higher its consistency . the serum to pellet ratios and the bostwick values determined are shown in table 3 . table 3______________________________________ bostwicktreatment serum : pellet ( cm ) ______________________________________no treatment ( control ) 3 . 2 8 . 1400 mpa 10 mins + 40 ° c . 2 . 7 10 . 130 mins400 mpa 10 mins + 60 ° c . 2 . 8 10 . 330 minsno treatment ( control ) 2 . 8 8 . 8600 mpa 20 mins + 40 ° c . 1 . 4 6 . 130 mins600 mpa 20 mins + 60 ° c . 1 . 2 5 . 930 mins______________________________________ from this example and example 1 , the following conclusions can be drawn . tomato juice given a uhp treatment of 400 mpa for 10 minutes still has 100 % of its pg activity remaining ( table 1 ). even after a thermal incubation of 60 ° c . for 30 mins there is still 45 % pg activity remaining ( table 2 ). during the mild thermal incubation this remaining activity is able to depolymerize pectin in the juice which is reflected in the higher bostwick values and serum to pellet ratios ( ie lower consistencies ) of the treated samples . tomato juice given a uhp treatment of 600 mpa for 20 minutes has no pg activity remaining after treatment ( table 1 ). however pme is unaffected by this treatment and during the mild thermal incubation pme activity is stimulated ( pme has a temperature optimum close to 60 ° c .) to catalyse the removal of methyl ester groups from pectin molecules resulting in the pectin molecules being able to associate with each other via cation cross - links to provide a decrease in bostwick values and serum to pellet ratios , ie . an increase in consistency . the juice colours of the final samples of example 2 were measured using an ultrascan xe ™; this is a machine for measuring colour available from hunterlab , usa . the measurements are shown in table 4 below ; the values given are the ratios of the hunterlab &# 34 ; a &# 34 ; and &# 34 ; b &# 34 ; scores ; these scores are used by hunterlab to describe food colour ; &# 34 ; a &# 34 ; is a red to green scale ; &# 34 ; b &# 34 ; is a yellow to blue scale . table 4______________________________________treatment a / b ratio______________________________________no treatment ( control ) 1 . 89400 mpa 10 mins + 40 ° c . 1 . 9630 mins400 mpa 10 mins + 60 ° c . 1 . 8530 mins600 mpa 20 mins + 40 ° c . 2 . 0030 mins600 mpa 20 mins + 60 ° c . 1 . 9330 mins______________________________________ from these results it can be concluded that uhp treatment in combination with mild thermal incubation has very little effect on tomato colour . this is beneficial as it is desirable to retain as much of the original fresh tomato red colour as possible in the final product . in contrast , the prior art heat processes lead to the generation of brown colours due to the maillard reaction . a sample of 150 g of tomato dice was subjected to a pressure of 500 mpa for 15 mins , incubated in a water bath for 30 mins at 42 ° c . and pasteurised , all in accordance with the method of example 1 . qualitative volatile analysis was then carried out on the sample using a tekmar ™ dynamic headspace ( purge & amp ; trap ) apparatus ( model lsc2000 available from interscience ). four grams of the sample was purged for 10 minutes with helium at 45 ° c . the volatiles were trapped on tenax ™ absorbing material and cryofocussed before injection into a gas chromatograph . qualitative information was obtained by means of a dual column system , using chromatography columns of differing polarities ( namely models cpsil05 and cpsil13 available from chrompack ), and a reference database . semi - quantitative information was obtained by comparing peak heights and areas . this analysis was also carried out on a sample of untreated tomato dice pasteurised in tomato juice , as a control . relative peak heights for selected volatiles in the samples are shown in table 5 . 3 - methylbutanal , hexanal , cis - 3 - hexenal and 6 - methyl - 5 - hepten - 2 - one are important for both fresh and processed tomato aroma ( buttery rg , 1993 , in flavour science : sensible principles and techniques , acs , 259 - 286 ). cis - 3 - hexen - 1 - ol is important for fresh tomato aroma . table 5______________________________________ peak height relative to untreated control uhp 500 mpa 15 mins + volatile untreated ( control ) 42 ° 30 mins______________________________________3 - methylbutanal 100 1352 - methylbutan - 1 - ol 100 100hexanal / cis - 3 - 100 350hexenalcis - 3 - hexen - 1 - ol 100 1251 - hexanol 100 1256 - methyl - 5 - hepten - 100 1502 - one______________________________________ this shows that the uhp treatment resulted in an increase in 3 - methylbutanal , hexanal , cis - 3 - hexenal , cis - 3 - hexen - 1 - ol , 1 - hexanol and 6 - methyl - 5 - hepten - 2 - one . it can be concluded from table 1 that a uhp treatment of 500 mpa for 15 minutes is sufficient to inactivate pg , but not sufficient to inactivate peroxidase or pme . without wishing to be bound by theory , the inventors believe that the presence of peroxidase during the incubation step is responsible for the generation of free radicals which may be involved in the oxidation of lipids and carotenoids , to generate most of the volatiles listed in table 5 . the results of this experiment show that uhp treatment can be used to produce tomato dice with altered aroma volatile profiles .
a process for providing a tomato - based product comprising the following steps : a ) applying ultra high pressure to a tomato piece , such that polygalacturonase is inactivated and pectinmethylesterase and preferably peroxidase are not inactivated ; b ) incubating the tomato piece with endogenous pectinmethylesterase to achieve a desired consistency and preferably with peroxidase to achieve a fresh aroma and / or flavour profile ; and c ) inactivating the pectinmethylesterase and any peroxidase .