Patent Application: US-13760405-A

Abstract:
methods for increasing the amount of resveratrol in a peanut material are provided , comprising the steps of providing a peanut kernel , size - reducing the peanut kernel , abiotically stressing the size - reduced peanut kernel , and incubating the abiotically stressed size - reduced peanut kernel under conditions capable of increasing the amount of resveratrol in the size - reduced peanut kernel . resveratrol - enhanced peanut compositions prepared according to these methods are also provided .

Description:
increases in aoa , total phenolic compounds , and trans - resveratrol in peanut kernels are desirable . accordingly , exposure to post - harvest stress in peanut kernels has been studied to determine the effect of size - reduction methods such as slicing , chopping , or grinding ; to determine the effect of stress application , illustratively by uv light or ultrasound exposure ; and to determine the effect of incubation time on aoa , total phenolic compounds , and trans - resveratrol concentration in peanut kernels . illustratively , the size - reduction takes place by slicing surface - sterilized , fully imbibed peanut kernels into slices of about 1 mm to about 8 . 5 mm in thickness , more illustratively about 2 mm , about 4 mm , about 6 mm , about 7 mm , and about 1 cm , and various thicknesses between these thicknesses . in an illustrative example , slices of about 2 mm in thickness were used . in other work , it has been found that slicing to about 7 mm produces excellent results . however , it is understood that other thickness may be used and other methods of size reduction may be used . illustratively , the stress is applied using uv light or ultrasound . when uv light is used to apply stress , a light of 254 nm for 2 - 25 minutes , illustratively for 10 minutes is used . however , this light frequency and time are illustrative only . when ultrasound is used to apply stress , a variety of power densities for various lengths of time may be used . in one embodiment , ultrasound is applied at a power density of 39 . 2 mw / cm 3 is used for about 2 - 8 minutes at 25 °. in one embodiment , ultrasound is applied at this power density for about 4 minutes . however , it is understood that other times and power densities may be used . a combination of uv light and ultrasound may be used as well . illustratively , incubation takes place subsequent to stress application , illustratively for about 24 to 72 hours , more particularly about 36 to 48 hours , illustratively at about 25 ° c . in one suitable example , surface - sterilized , imbibed peanut kernels are sliced to about 5 mm , stressed , and incubated for about 48 hours . uv light , ultrasound , or a combination of uv light and ultrasound may be used to apply the stress . experimental design . raw peanut kernels were subjected to post - harvest treatments using a 4 × 3 × 4 factorial design . factors studied were size - reduction methods : chopping , slicing , grinding , and no size - reduction treatment ; post size - reduction stress applications : uv light , ultrasound , and no stress application ; and incubation times : 0 , 24 , 36 and 48 h . three replications of the 48 treatments were conducted for at total of 144 samples . peanut samples were analyzed for aoa and total phenolic compound concentration in each duplicate for a total of 288 samples per analyses . duplicate analysis for aoa and total phenolic compounds was conducted generally . however , triplicate analysis was conducted when large variation occurred between results . trans - resveratrol analyses were conducted in triplicate . control samples were prepared from untreated raw peanuts . sample preparation . peanuts used in the analysis were georgia green medium runners ( mccleskey mills inc .) harvested in smithville , ga . in 2002 . the 22 . 68 kg bag ( 50 lb .) had been stored under refrigerated storage in a seed storage room at 7 ° c . for approximately 2 wk prior to the study . approximately 3 . 6 kg of raw peanuts were surface sterilized in 4 l of 20 % hydrogen peroxide ( sigma , st . louis , mo .) for 15 minutes ( 17 ) then rinsed in a sterile colander with 4 l of sterilized deionized water , sterilized by passing through a 0 . 2 μm nylon filter ( millipore corporation , bedford , mass .). the sterilized peanuts were soaked in 4 l of sterile deionized water for 16 h to reach the maximum water holding capacity of the peanut as determined by preliminary studies . the entire process was conducted under yellow light to avoid resveratrol isomerization ( 28 ). it is understood that sterilization using hydrogen peroxide is illustrative , and that other methods of sterilization may be used if necessary , as is appropriate for the particular application . size - reduction . approximately 1 . 2 kg of sterilized fully - imbibed peanut kernels were reduced by one of the four size - reduction methods as follows . peanuts were ground into 1 - 2 mm pieces using a food processor ( model 14181 , sunbeam oskar , china ) for 20 s in 6 batches of 200 g each . kernels were chopped into 0 . 5 cm pieces in two batches of 600 g each using a commercial food cutter ( model 84142 , hobart , troy , ohio ) at 1725 rpm for 30 s . peanuts were sliced into 2 mm thick pieces , by hand , with a razor blade , a ruler was used as a guide for size . whole peanut kernels were also collected to represent samples with no size - reduction treatment . each of the size - reduction batches were collected and mixed by hand with a spoon . all equipment used was sterilized prior to use with 20 % hydrogen peroxide . post size - reduction stress application . peanuts from each of the size - reduction treatments were divided into 3 batches of 400 g each then stressed with uv light , ultrasound , or had no further stress application . peanuts arranged 1 cm in depth on a plastic tray were exposed to uv light for 10 min ( 20 , 21 ) using a germicidal lamp ( 254 nm , 30 w , model uvsl - 58 , ultra violet products , inc ., san gabriel , calif .) placed 40 cm ( 21 ) above the surface of the peanuts . peanuts were stirred after 5 minutes to ensure exposure of uv light to all sides of the kernels . ultrasound treatment was performed in a sonic cleaner ( model fs60120v , 29 . 5 l × 15 . 5 w × 14 . 5 d cm , 260 w , 2 . 2 a , 50 / 60 hz ; fisher scientific , fair lawn , n . j .) with a power density of 39 . 2 mw / cm 3 . approximately 400 g of peanuts were placed into a 1 l glass beaker containing 800 ml of sterile deionized water . the beaker was placed into the sonic cleaner filled with enough water to completely surround the beaker and sonicated for 4 min at room temperature ( 22 ). after the sample was sonicated the water was drained from the peanuts by placing them into a sterile colander for 5 min . approximately 100 g of sample was stored in half - pint glass mason jars ( ball corporation , muncie , ind .) with two piece lids ( alltrista corporation , muncie , ind .). the mason jars were wrapped with aluminum foil to protect samples from light exposure . samples receiving none of the stress treatments were immediately placed in jars and incubated . stress application was done in dim light to prevent trans - resveratrol isomerization ( 28 ). sample incubation . mason jars were incubated at 25 ° c . ( environmental growth chamber , chargrin falls , ohio ) for 24 , 36 and 48 h . after incubation , the samples were stored at − 23 ° c . until analysis for approximately 1 month . extraction of antioxidants and total phenolic compounds . each peanut sample was extracted for antioxidants and total phenolic compounds in duplicate . ten g of each peanut sample was ground and 5 g was placed in a 250 ml centrifuge tube ( nalgene , rochester , n . y .) with 50 ml of methanol ( fisher scientific , fair lawn , n . j .). tubes were shaken for 12 hrs with a wrist action shaker ( model 75 , burrell corporation , pittsburgh , pa .) at ambient temperature , 25 ° c . the extracts were filtered ( whatman no . 42 , england ) into a round bottom flask and the residue was extracted again under the same conditions . the combined filtrate in round bottom flask was placed in a water bath ( buchi 461 water bath , switzerland ) at 40 ° c . and evaporated under vacuum in a rotary evaporator ( brinkmann re 111 , buchi , switzerland ), approximately 30 min , to a final volume of 5 ml . analysis of aoa . the aoa of the 144 peanut extracts was determined according to the thiocyanate method of osawa and namiki ( 29 ) for eucalyptus leaves as adapted by duh and yen ( 10 ). aoa was calculated as percent inhibition of oxidation of linoleic acid relative to the control after a 60 min incubation according to emmons et al . ( 31 ). analysis of total phenolic compounds . the concentration of total phenolic compounds present in the peanut sample was determined using a spectrophotometric method with folin - denis reagent ( 32 ) established by yen and duh ( 9 ). the blue color was measured at 726 nm ( 33 ) using a spectrophotometer ( model 8451a , diode array spectrophotometer , hewlett packard , palo alto , calif .). absorbance was measured in triplicate for all peanut samples and standard solutions . analysis of trans - resveratrol . trans - resveratrol was analyzed using an hplc method . trans - resveratrol was extracted following a procedure established by sanders et al . ( 34 ) modified by rudolf et al . ( 35 ) using phenolphthalein as the internal standard . approximately 35 g of peanut sample was prepared as described by rudolf et al . ( 35 ). the entire procedure was conducted under yellow light to prevent resveratrol isomerization ( 28 ). the dried peanut residue was prepared for hplc analysis by adding 0 . 40 ml of 10 % ethanol to a vial mixed and filtered into a 300 μl polypropylene plastic insert ( national scientific co ., lawrenceville , ga . ), to minimize sample volume , which was placed in a 2 ml hplc amber vial ( national scientific co ., lawrenceville , ga .). the vial was sealed with a screw cap that was fitted with a teflon / silicone septum ( national scientific co ., lawrenceville , ga .). hplc analyses were performed using a waters ( waters corporation , milford , mass .) system with a w717 sample injector , w2695 separations module , and w996 photodiode array detector ( pda ) set to monitor the uv spectrum from 240 to 400 nm . an econosphere ( alltech associates , inc ., deerfield , ill .) c 18 reversed - phase column ( 250 l × 4 . 6 i . d . mm , 5 μm particle size ) preceded by an econosphere c18 ( alltech associates , inc ., deerfield , ill .) guard column ( 7 . 5 l × 4 . 6 i . d . mm , 5 μm particle size ) was used for analysis . trans - resveratrol was analyzed using a reversed - phase hplc method developed by rudolf et al . ( 35 ). the mobile phase was 0 . 1 % acetic acid ( j . t . baker , phillipsburg , n . j .) in double deionized water that was filtered through a 0 . 2 μm nylon filter by vacuum , as solvent a , and 100 % acetonitrile ( hplc grade , aldrich , milwaukee , wis .) as solvent b , with a flow - rate of 1 . 5 ml / min and the column temperature maintained at 25 ° c ., ambient . the gradient elution increased acetonitrile linearly from 5 to 41 . 8 % over 23 min then increased to 77 % over 5 min and finally returned to 5 % over 1 min and held for an additional 5 min . waters millenium 32 software , version 3 . 05 was used to control the hplc auto sampler , gradient conditions , pda and data acquisition . peanut samples were injected at a volume of 80 μl . peak areas of trans - resveratrol and phenolphthalein were quantified at 307 nm ( 27 ) and 254 nm ( 35 ), respectively . ratios of trans - resveratrol and phenolphthalein peak areas from analysis of peanut samples and standards were used to calculate the concentration of trans - resveratrol using the following equation ( 35 ): equation ⁢ ⁢ 2 ⁢ : ⁢ ⁢ μg ⁢ ⁢ of ⁢ ⁢ i ⁢ ⁢ in ⁢ ⁢ sample = [ ( μg ⁢ ⁢ of ⁢ ⁢ i ⁢ ⁢ in ⁢ ⁢ standard pa ⁢ ⁢ of ⁢ ⁢ i ⁢ ⁢ in ⁢ ⁢ standard ) × pa ⁢ ⁢ of ⁢ ⁢ i ⁢ ⁢ in ⁢ ⁢ sample ( μg ⁢ ⁢ of ⁢ ⁢ is ⁢ ⁢ in ⁢ ⁢ standard pa ⁢ ⁢ of ⁢ ⁢ is ⁢ ⁢ in ⁢ ⁢ standard ) × pa ⁢ ⁢ of ⁢ ⁢ is ⁢ ⁢ in ⁢ ⁢ sample ] × μg ⁢ ⁢ of ⁢ ⁢ is ⁢ ⁢ in ⁢ ⁢ sample where i is trans - resveratrol , is is phenolphthalein ( internal standard ) and pa is the peak area . the five levels of standards , 5 , 3 . 125 , 1 . 250 , 0 . 625 and 0 . 375 ppm ( μg / ml ), for trans - resveratrol and phenolphthalein were analyzed at the beginning of each hplc sample set . trans - resveratrol concentrations were reported as μg / g of peanut on a dry weight basis . statistical analysis . data was analyzed using sas ( 37 ) statistical software version 8 . regression analysis ( proc reg ) was used to relate total phenolic compound concentrations as the dependant variable with peak area as the independent variable . the general linear model ( proc glm ) was used to detect significant differences in stress treatments for aoa , total phenolic compounds and trans - resveratrol concentration . fishers least significant difference ( lsd ) test was used to compare means of stress treatments on aoa , total phenolic compounds and trans - resveratrol concentration . pearson &# 39 ; s product correlation coefficients ( proc corr ) were calculated between mean aoa , total phenolic compounds and trans - resveratrol concentration . aoa . results from analysis of variance are presented in table 1 . incubation time was the only significant factor ( p & lt ; 0 . 01 ) that affected aoa . size - reduction and post size - reduction stress did not significantly affect aoa . means and their significant differences of aoa are shown in table 2 . aoa of untreated peanut kernels was 39 . 34 ± 0 . 87 %, which is similar to results from stressed peanuts not exposed to post size - reduction stress at 0 h of incubation , 39 . 93 - 42 . 30 %, regardless of size - reduction ( table 2 ). no literature comparison could be made due to the lack of documentation on peanut kernels . however , results are lower than those reported for peanut hulls , ranging from 94 . 8 to 93 . 9 % in hulls at varied levels of maturity , 74 to 144 d after planting ( 9 ), and 99 . 7 % in untreated hulls ( 24 ). peanut hulls contain large concentrations of phenolic compounds ( 9 , 10 ), such as the three flavoniods 5 , 7 - dihydroxychromone , eriodictyol and luteolin ( 38 ), which function as antioxidants ( 1 ). therefore , hulls are expected to have higher levels of aoa than peanut kernels . aoa of chopped , sliced and whole peanuts not exposed to post size - reduction stress did not change over incubation from 0 to 48 h . however ground peanuts not exposed to post size - reduction stress had a significant decrease ( α & lt ; 0 . 05 ) in aoa after 24 h of incubation , then remained stable until 48 h ( table 2 ). all peanut samples exposed to uv light had a significant decrease ( α & lt ; 0 . 05 ) in aoa after 24 h of incubation ( table 2 ). at 36 h of incubation aoa remained stable , except for chopped peanut samples , at which time aoa increased to levels not significantly different than 0 h of incubation . after 48 h of incubation , aoa of sliced and whole peanuts increased to the initial level ( 0 h ). aoa of ground and chopped peanuts after 48 h remained at the same level after 36 h of incubation . the decrease in aoa for ground peanuts after 24 h though 48 h in this study are contradictory to results found in the literature ( 24 ) for methanolic extracts of peanut hulls , where uv exposure did not significantly ( p & lt ; 0 . 05 ) affect aoa . ultrasound exposure did not have any effect on aoa of chopped and whole peanuts . however , aoa in sliced peanuts exposed to ultrasound decreased from 0 h of incubation after 24 and 36 h , but increased to levels at 0 to 24 h of incubation after 48 h of incubation ( table 2 ). aoa in ground peanuts did not significantly change after 24 to 48 h of incubation . no literature comparison can be made on the effect of ultrasound exposure to peanuts . total phenolic compounds . results from the analysis of variance of total phenolic compounds are presented in table 3 . time of incubation was the only significant factor ( p & lt ; 0 . 01 ) affecting the total phenolic compound concentration in peanut samples ( table 3 ). size - reduction and post harvest size - reduction stress did not significantly affect total phenolic compound concentration . mean values and significant differences of total phenolic compounds are shown in table 4 . a means were calculated from three replications with duplicate extractions measured for a total of 6 analyses at each incubation time . when large variation in total phenolic compounds occurred between samples , triplicate analysis was conducted . outliers were removed prior to statistical analysis . b for each size reduction method , means for a stress not followed by the same letter are significantly different at α & lt ; 0 . 05 as determined by fisher &# 39 ; s least significant difference mean comparison test . c total phenolic compound concentration determined in peanut kernels using a regression equation , y = [ 44 . 37 ( au ) x + 0 . 019 ( au / mg / g )] where y is total phenolic compound ( mg / g ) and x is peak area , are reported above . untreated control peanutshad 1 . 35 ± 0 . 46 mg / g of total phenolic compounds . d the general linear model ( proc glm ) was used to determine the f - value and probability . untreated peanut samples contained 1 . 35 ± 0 . 46 mg / g of total phenolic compounds . imbibition of peanuts with water has no bearing on total phenolic compounds . low concentration of total phenolic compounds in whole peanuts not exposed to post size - reduction stress may be a result of method variability due to interfering compounds , described previously . results show that the peanut kernels contain lower concentrations of total phenolic compounds than peanut hulls , 7 . 80 mg / g ( 24 ), which is expected as hulls are darker in color , and darker color is associated with increased levels of phenolic compounds ( 38 ). total phenolic compound concentration of ground and sliced peanuts not exposed to post size - reduction stress was not affected over incubation for 0 to 48 h . total phenolic compound concentration in chopped peanuts not receiving post size - reduction stress significantly decreased ( α & lt ; 0 . 05 ) after 36 h of incubation then increased to levels not significantly different than 0 and 24 h after 48 h ( table 4 ). concentration of total phenolic compounds in whole peanuts not exposed to post size - reduction stress significantly increased ( α & lt ; 0 . 05 ) to a maximum after 24 h of incubation then decreased at 36 h and remained constant after 48 h . exposure to uv light had no significant effect on total phenolic compounds , regardless of size reduction or incubation time ( table 4 ). results found in the study are contradictory to the literature ( 24 ), where a significant ( p & lt ; 0 . 05 ) decrease in total phenolic compounds occurred in methanolic extracts of peanut hull powder after exposure to uv light . however hull samples in the literature were exposed to uv light over a longer period of time , 6 d ( 24 ), compared to 10 min in the present study . ultrasound had no significant effect on total phenolic compound concentration in chopped , sliced and whole peanuts ( table 4 ). only ground peanuts exposed to ultrasound had a significant decrease ( α & lt ; 0 . 05 ) in total phenolic compounds after 24 and 36 h of incubation ( table 4 ). however concentration of total phenolic compounds in ground peanuts increased to levels not significantly different ( α & lt ; 0 . 05 ) than 0 and 24 h after 48 h ( table 4 ). a comparison with findings in the literature can not be made due to the lack of reports on this effect . a large variation occurred in total phenolic compound concentration in chopped and whole peanuts not exposed to post size - reduction stress and ground peanuts exposed to ultrasound ( table 4 ). since no trend in total phenolic compound concentration can be identified in these samples , variation could be attributed to method variability . trans - resveratrol . evaluation of the uv spectrum for the peak corresponding to trans - resveratrol in peanut extracts revealed an irregular shape compared to that in standards . however , previous analysis of peanut extracts by gc - ms confirmed that the compound identified was trans - resveratrol . the compound identified had an identical retention time and similar fragmentation pattern to that of trans - resveratrol standard and published data ( 34 , 39 ). results from analysis of variance of trans - resveratrol concentration are presented in table 5 . size - reduction , post size - reduction stress , incubation time , and the interaction between size - reduction and incubation time were the factors that significantly ( p & lt ; 0 . 01 ) affected trans - resveratrol concentration in peanut kernels ( table 5 ). significant differences in mean values of trans - resveratrol concentrations are shown in table 6 and fig1 . the highest amount of trans - resveratrol was 3 . 96 ± 0 . 96 μg / g , found in sliced peanuts exposed to ultrasound after 36 h of incubation . untreated peanut kernels contained 0 . 48 ± 0 . 08 μg / g of trans - resveratrol where as approximately half of that amount ( 0 . 18 ± 0 . 21 to 0 . 25 ± 87 μg / g ) was found in stressed peanuts not exposed to post size - reduction stress at 0 h of incubation . lower concentrations of trans - resveratrol in the stressed peanut kernels may be a result of soaking the peanuts in water for 16 h prior to size reduction treatment . however results for trans - resveratrol concentration of untreated peanuts are similar to those reported in the literature ( 34 ), where peanuts in storage for up to 3 months contained 0 . 02 - 1 . 79 μg / g . trans - resveratrol concentration in all peanut samples not receiving post size - reduction stress significantly increased ( α & lt ; 0 . 05 ) from 0 to 24 h of incubation ( table 6 ; fig1 ). findings are consistent with those found by cooksey et al . ( 17 ) where resveratrol concentration increased to a range of 4 . 3 to 23 . 8 μg / g in eleven different cultivars of peanuts sliced 2 mm thick and incubated for 24 h . in all size - reduced peanuts , the trans - resveratrol concentration remained constant or decreased in concentration from 24 to 36 h . trans - resveratrol concentration increased in whole kernels from 0 to 48 h of incubation . after 48 h of incubation , trans - resveratrol concentration increased from levels at 0 h of incubation . however , from 36 to 48 h of incubation the concentration remained equal or decreased , except for sliced kernels which increased in resveratrol content . peanuts that were exposed to size - reduction increased in trans - resveratrol concentration as particle size increased from ground to sliced ( fig1 ). slicing with 48 h of incubation produced the largest amount of trans - resveratrol ( 2 . 15 ± 0 . 63 μg / g ) in peanuts not exposed to post size reduction stress . in subsequent work , it has been noted that excellent results are obtained with slicing to about 7 mm . also , slicing to 5 mm results in a good increase in trans - resveratrol concentration . exposure of uv light to peanut kernels resulted in a significant increase ( α & lt ; 0 . 05 ) in trans - resveratrol concentration as incubation period increased from 0 to 36 h ( table 6 ). trans - resveratrol concentration increased from 0 to 24 h of incubation . then , from 24 to 36 h concentration increased even further . results are consistent with other studies where uv light exposure to grapes leaves ( 16 ) and grapes ( 19 ) increased synthesis of resveratrol after incubation for 24 and 23 h , respectively . trans - resveratrol remained level or increased significantly ( α & lt ; 0 . 05 ) from 36 to 48 h in size reduced kernels , but not in whole kernels ( table 6 ). as particle size increased trans - resveratrol concentration increased , but not in whole kernels ( fig1 ). the highest trans - resveratrol concentration ( 3 . 42 ± 0 . 95 μg / g ) in peanuts exposed to uv light was found in sliced peanuts after 48 h of incubation . creasy and coffee ( 19 ) also found that uv light exposure to grapes and incubation for 48 h increased trans - resveratrol concentration . fritzemeier and kindl ( 40 ) found that leaves of vitaceae exposed to uv light stimulated the production of stilbene synthase and catalyzed the reaction of 4 - hydroxycinnamoyl - coa and malonyl - coa to produce resveratrol . ultrasound exposure caused a significant increase ( α & lt ; 0 . 05 ) in trans - resveratrol concentration in all samples after 24 h of incubation ( table 6 ). from 24 to 36 h of incubation , trans - resveratrol concentration decreased or was not significantly different ( table 6 ). trans - resveratrol concentration in peanuts incubated for 36 to 48 h did not change ( table 6 ). in peanuts exposed to size - reduction , trans - resveratrol concentration increased as particle size increased from ground to sliced ( fig1 ). the highest trans - resveratrol concentration in peanuts exposed to ultrasound occurred in sliced kernels at 24 , 36 or 48 h of incubation . the parameters for using uv light and ultrasound have not yet been optimized . it is expected that uv light at other wavelengths , intensities , distances from the bulb , and durations would result in significant trans - resveratrol production . similarly , ultrasound at other power densities and durations are also expected to be useful . additionally , it is expected that a combination of uv light and ultrasound , simultaneously or sequentially in either order , would likely result in higher trans - resveratrol concentration . relationship between aoa , total phenolic compounds and trans - resveratrol concentration . significant pearson &# 39 ; s product correlation coefficients ( r ) obtained between aoa , total phenolic compounds and trans - resveratrol concentration are shown in table 7 . significant r was found between trans - resveratrol concentration and aoa . however , the magnitude of correlation was low . since trans - resveratrol has been shown to provide antioxidant activity ( 1 ), correlation between the two factors is expected . none of the other combinations tested had significant correlations . these findings are similar to kahkonen ( 41 ), where no significant correlations could be found between the total phenolic content and aoa of plant extracts , berries , fruits , vegetables , cereals , herbs , plant sprouts , seeds and tree leaves and bark . however these results are contradictory to findings by emmons ( 31 ) where total phenolic content was significantly correlated with aoa in oat fractions . emmons et al . ( 31 ) findings suggest that phenolic compounds in the oat might be responsible for a large proportion of the aoa ( 31 ). peanuts were soaked in 12 l of 0 . 075 % hydrogen peroxide ( h 2 o 2 , 20 %, sigma , st . louis , mo ., u . s . a .) solution for 1 min ( 42 ). peanuts were transferred into a sterile strainer to remove the hydrogen peroxide solution and rinsed with filtered ( 0 . 2 μm nylon filter , millipore corporation , bedford , mass ., u . s . a .) deionized water . the three batches of peanuts were combined and placed into a plastic tub ( 53 . 34 l × 33 . 02 w × 20 . 32 d ), sterilized with 5 % h 2 o 2 , containing 20 l of filtered deionized water and soaked for 16 h ( 11 ) for full imbibition . the water was drained from the peanuts and the kernels were divided into 3 batches , containing 3 kg , for each of the 3 stress treatments . one batch of peanut kernels was manually sliced using a razor blade on a sterile cutting board into 0 . 7 cm thick pieces using a template . the remaining two batches were left as whole kernels , ranging from 1 . 5 to 2 cm in length . sliced peanuts and one batch of whole kernels were exposed to ultrasound ( model fs60120v , 29 . 5 l × 15 . 5 w × 14 . 5 d cm , 260 w ; fisher scientific , fair lawn , n . j ., u . s . a . ), with a power density of 39 . 2 mw / cm 3 , by placing 500 g into a 1 l glass beaker filled with 800 ml of filtered dionized water , for a total of 6 batches per treatment . after sonication for 4 min at 25 ° c . peanuts were placed into a sterile strainer for approximately 5 min , to remove excess water , then transferred to a sterilized quart glass mason jar ( ball corporation , munice , ina ., u . s . a .) with two piece lid ( alltrista corporation , muncie , ind ., u . s . a .). each jar was wrapped with aluminum foil ( reynolds 614c , reynolds metals company , richmond , va ., u . s . a .) to protect samples from light exposure . the entire sample preparation procedure was conducted under dim light to prevent trans - resveratrol isomerizition ( 28 ). after incubation ( environmental growth chamber , chagrin falls , ohio , u . s . a . ), 100 g of peanuts were left in the jar and stored at − 23 ° c ., approximately 1 week , until trans - resveratrol analysis was conducted . peanuts used for descriptive analysis were placed onto an aluminum screen ( 29 . 21 × 43 . 18 cm ), not more than 2 . 54 cm thick , and placed into a mechanical convection oven ( 645 freas , precision scientific , winchester , va ., u . s . a .) at 40 ° c . until moisture was reduced to 10 ± 1 % by weight , approximately 24 h . peanut butter preparation . approximately 2 . 5 kg of the three dried stressed peanut samples and untreated peanuts were roasted separately in a gas roaster ( model l5 , probat in ., memphis , term ., u . s . a .) preheated at 177 ° c . then maintained at 135 ° c . while the peanuts roasted . peanuts were visually inspected for color change every minute . peanuts were removed from the heated barrel of the roaster , approximately 3 to 4 min , when the color looked similar to a medium roast and cooled a perforated tray in the lower base of the roaster . once the peanuts were cool , approximately 5 min , the color was determined using a colorimeter ( gardner xl800 , pacific scientific , bethesda , md ., u . s . a .) with a circumferential sensor ( gardner xl845 , pacific scientific , bethesda , md ., u . s . a .). the colorimeter was calibrated using a yellow reference standard tile ( l = 79 . 56 , a =− 2 . 17 , b = 22 . 98 ) prior to sample analysis . peanut kernels were placed evenly on the bottom of the colorimeter sample cup and four sets of readings were obtained per sample by rotating the cup a quarter of a turn each time ( muego - gnanasekharan and resurreccion , 1992 ) the average of these reading were used in the analysis . peanuts having a hunter l - value of 50 ± 1 were not further roasted . roasted samples were dry blanched ( model ex , ashton food machinery co ., inc ., newark , n . j ., u . s . a .) to crack and remove the skins and testas . peanut kernels with remaining testa were put through the blancher an additional time then manually inspected . all peanuts were put through a seed cleaner ( almaco seed cleaner , allan machine company , ames , iowa , u . s . a .) to remove remaining testa . it is understood that , while the samples are blanched and testas are removed subsequent to size reduction and incubation , the samples could be blanched and testas could be removed prior to size reduction , or at any other step . also , depending on the particular embodiment , blanching and removal of the testa may not be necessary . after roasting , each of the peanut samples were coarsely ground using a colloid mill ( morehouse industries , los angeles , calif ., u . s . a .) set with a stone clearance of 0 . 25 mm ( 10 notches ). the ground sample was manually mixed in a bowl with a wooden spoon with 1 % salt ( morton international inc ., chicago , ill ., u . s . a .) and 6 % corn syrup solids ( star - dri 42f , a . e . staley manufacturing company , decatur , ill ., u . s . a .) added by weight ( gills and resurreccion 2000 ). the entire contents of the bowl were placed into metal pans ( 30 . 48 l × 20 . 32 cm ) and placed into a mechanical oven ( model # m01440sc , lindberg / blue m , asheville , n . c ., u . s . a .) at 60 ° c . for 30 min . samples were removed from the oven and 1 . 5 % stabilizer ( fix - x , procter & amp ; gamble , cincinnati , ohio , u . s . a .) was added , by weight , then the sample was manually mixed with a wooden spoon in the same pan to distribute the stabilizer throughout the peanut butter . samples were passed through a colloid mill with a stone clearance of 0 . 1 mm ( 5 notches ), to get a finer grind , and a hot water jacket maintained at 75 ° c ., to disperse the stabilizer . the peanut butter was placed into quart mason jars with two piece lids and stored at 4 ° c . approximately 1 d until sensory testing was conducted . the mill was cleaned between grinding of each of the different peanut samples . sensory evaluation . subsequent sensory evaluation of the peanut butter revealed that size - reduction and post size - reduction stress exposure do not significantly affect spreadability , texture , and roasted peanut intensity . however , ultrasound exposure did negatively affect overall acceptance , aroma , and flavor . while the addition of flavoring agents and / or dilution with untreated peanut products may be used in any composition herein , the addition of flavoring agents and / or dilution with untreated peanut butter is particularly well suited to improve aroma and flavor for compositions using peanut kernel material that has been exposed to ultrasound as the post size - reduction stress . it has been noted that the effect on flavor is most noticeable when thinner slices were used . thus , for products where aroma and flavor are more important , such as with peanut butter , size reduction to larger slices ( illustratively about 7 to 8 . 5 mm ) may be desirable , whereas for peanut flour or other products for which flavor and aroma are less important , size reduction to smaller sizes ( illustratively about 5 mm ) to obtain a higher trans - resveratrol concentration may be desirable . trans - resveratrol concentration . evaluation of the uv spectrum , using the pda , for the peak corresponding to trans - resveratrol in peanut extracts revealed a similar shape to that in the standard solutions and the literature ( 28 ). the compound identified in the peanut extracts also had an identical retention time ( 16 min ) to the standard solutions therefore we concluded that the compound identified was trans - resveratrol . results from analysis of variance of trans - resveratrol are shown in table 8 . size - reduction , added moisture and time of incubation were significant factors affecting trans - resveratrol concentration in peanut kernels ( table 9 ). replication , ultrasound exposure and skin did not significant affect trans - resveratrol concentration in peanut kernels ( table 8 ). means of trans - resveratrol concentration for the peanut samples are presented in fig2 , table 9 . after incubation all stressed peanuts had significantly higher trans - resveratrol than untreated control peanuts regardless of drying and roasting . skin did not significantly affect the trans - resveratrol concentration of peanut samples ( table 8 ). therefore , removing the skins prior to processing peanut butter would not affect the trans - resveratrol concentration . 1 peanuts were stressed by slicing into 0 . 7 cm or left whole , 1 . 0 cm , and exposed to ultrasound for 4 min at 25 ° c . then incubated for 44 h at 25 ° c . and analyzed for trans - resveratrol before and after drying approximately 24 h at 40 ° c . 2 means in each row , for each incubation time , not followed by the same letter are significantly different as determined by fisher &# 39 ; s least significant difference ( lsd ) at α & lt ; 0 . 05 mean separation test . 3 trans - resveratrol concentration was determined in samples by reverse - phase high performance liquid chromatography and reported on a dry weight basis . 4 peanuts were fully imbibed with water then stressed by slicing ( 0 . 7 cm ) or left whole ( 1 . 0 cm ), exposed to ultrasound for 4 min at 25 ° c . then incubated for 44 h at 25 ° c . 6 after incubation peanut samples were dried approximately 24 h at 40 ° c . then roasted at 135 ° c . for 3 - 4 min . after 44 h of incubation peanuts that were sliced and exposed to ultrasound had the highest amount of trans - resveratrol concentration , regardless of drying and roasting ( fig8 ). although trans - resveratrol concentration appears to be higher in peanut samples with skins there was no significant difference between samples with or without skins . slicing and ultrasound exposure increased trans - resveratrol concentration to 2 . 73 ± 0 . 7 μg / g and 6 . 80 ± 0 . 37 - 7 . 15 ± 1 . 27 μg / g in peanuts before and after drying , respectively . an unexpected large increase in trans - resveratrol concentration occurred in peanuts with or without skins after the drying period , 24 h at 40 ° c . the highest increase in trans - resveratrol was found in sliced peanuts treated with ultrasound . whereas trans - resveratrol concentration was lower in whole stressed peanuts exposed or not exposed to ultrasound and the lowest in untreated control peanuts . a previous study ( 17 ) showed that resveratrol increased from 4 . 3 - 23 . 8 to 21 . 2 - 42 . 2 μg / g , in eleven different peanut cultivars , as time of incubation increased from 24 to 48 h , respectively , at 25 ° c . therefore the increase in trans - resveratrol after the drying period maybe a result of increased incubation time at conditions that promote trans - resveratrol synthesis . results for sliced peanuts exposed to ultrasound are higher than findings in a previous study ( 43 ) where concentrations increased from 0 . 48 ± 0 . 08 μg / g , in untreated peanuts , to 3 . 96 μg / g in peanuts that were sliced ( 2 mm ), exposed to ultrasound ( 4 min at 25 ° c .) and incubated for 48 h at 25 ° c . in addition , concentrations in this example are much higher than the recent study ( 43 ) where trans - resveratrol increased from 0 . 29 μg / g , in untreated peanuts , to 1 . 38 μg / g in peanuts that were sliced 0 . 7 cm and incubated for 48 h at 25 ° c . increased amount of synthesis of trans - resveratrol in the current example may have been a result of shorter storage conditions of the peanuts , approximately 1 mo compared to 1 yr in the later ( 43 ). arora and strange ( 11 ) reported a decrease in the ability of cotyledons to synthesize phytoalexins , in response to slicing 1 - 2 mm and incubating 48 h at 25 ° c ., after storage for 9 mo . in addition the peanuts used in the current example were harvested in a geographic area similar to that in the previous study by rudolf ( 43 ). mcmurtrey and others ( 26 ) also found that harvest location affects the synthesis of resveratrol in grapes and wine . whole peanuts not exposed to ultrasound had significantly higher trans - resveratrol concentration than whole peanuts exposed to ultrasound after 44 h of incubation . however after drying and roasting the trans - resveratrol concentration in whole stressed peanuts was not significantly different . untreated control peanuts contained 0 . 45 ± 0 . 05 μg / g of trans - resveratrol . results are similar to findings in the previous study by rudolf ( 43 ) for raw peanut kernels , 0 . 48 ± 0 . 08 μg / g . after peanuts were fully - imbibed and exposed to stress , but not incubated , the sliced peanuts contained 0 . 40 ± 0 . 03 μg / g , whole peanuts exposed to ultrasound contained 0 . 57 ± 0 . 16 μg / g and whole peanuts not exposed to ultrasound contained 0 . 74 ± 0 . 10 μg / g of trans - resveratrol . it is understood that the resveratrol - enhanced peanut material may be processed into various forms , including but not limited to sliced or chopped peanuts , peanut butter , and peanut flour , each of which may be used in various product . 1 . stojanovic , s . ; 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[ msc thesis ]. athens , ga . : university of georgia . p . available from : university of georgia library , athens , ga . although the invention has been described in detail with reference to preferred embodiments , variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims .