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Food Biotechnology
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Dhiraj A. Vattem a; Kalidas Shetty a a Department of Food Science, Laboratory of Food Biotechnology, University of Massachusetts, Amherst, MA, U.S.A. Online Publication Date: 12 January 2002

To cite this Article Vattem, Dhiraj A. and Shetty, Kalidas(2002)'SOLID-STATE PRODUCTION OF PHENOLIC ANTIOXIDANTS FROM

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FOOD BIOTECHNOLOGY Vol. 16, No. 3, pp. 189–210, 2002

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Dhiraj A. Vattem and Kalidas Shetty* Laboratory of Food Biotechnology, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

Cranberry pomace is a byproduct of the cranberry processing industry that can be targeted for production of value-added phenolic ingredients. Bio-processing of pomace by solid state fermentation (SSF) using food grade fungi provides unique strategies to improve nutraceutical properties and to produce functional phenolic ingredients. Several functional phenolic phytochemicals exist as glycosides or as other conjugated forms with reduced biological activity. We hypothesize that during SSF the fungal glycosidases mobilize some phenolic antioxidants in cranberry pomace and their activity by hydrolysis via b-glucosidase and releasing the aglycone. To develop this strategy we used food grade fungus Rhizopus oligosporus. Our goal was to target the release of simple phenolic aglycones and mobilized diphenyls. SSF of cranberry pomace was done for 16 days with nitrogen sources, ammonium nitrate (NH4NO3) and fish protein hydrolysate (FPH). The two nitrogen treatments increased water extractable phenolics by 15À26% by day 10 in the pomace. Antioxidant protection factor was highest on day 10 for both nitrogen treatments and was 20À25% higher than control for water extracts and 16.5À19.5% for ethanol extracts. The DPPH radical inhibition (DRI) capacity increased by 5% only for the NH4NO3 treatment and gradually decreased for FPH treatment in water extracts.
*Corresponding author. Fax: 1-413-545-1262; E-mail: 189 DOI: 10.1081=FBT-120016667 Copyright # 2002 by Marcel Dekker, Inc. 0890-5436 (Print); 1532-4249 (Online)

1-Diphenyl-2-picrylhydrazl (DPPH). It is rich in fiber and has relatively small amounts of protein and carbohydrates. Once the juice is extracted from the fruit. Phenolic aglycone. Around 520 million tons are produced every year and the projected production is higher for the coming years. Antioxidant activity. However. The antioxidant function has implications for prevention of major oxidation-linked diseases such as cancer and CVD. Pomace is the byproduct of the cranberry juice processing industry with limited applications. Only 5 percent of the annual crop is harvested for fresh fruit and most of it is used for processing.[2. The HPLC profile showed ellagic acid. Pomace is mainly composed of the skin.3] In this investigation we are exploring whether water and ethanol soluble phenolics can be enriched during SSF. We conclude that SSF of cranberry pomace increased the antioxidant activity concurrent with increased b-glucosidase activity. b-carotene oxidation model system. Further. Cranberry juice relished for its taste and ever increasing health benefits is one of the major products produced by the food processing industry. This value-added SSF strategy is an innovative approach to enhance nutraceutically-relevant functional phytochemicals for food and feed applications. The b-glucosidase activity increased by 60-fold for NH4NO3 treatment and by over 100-fold for FPH treatment and correlated well with the increase in the extractable phenolics and antioxidant activity. Previous investigation had explored potential new uses of cranberry pomace using solid state fermentation (SSF).[1] Cranberries are used as ingredients in over 700 products from cereals to salsas. b-glucosidase. however due to its low protein and carbohydrate content it has little nutritive value as an animal feed. Phenolics. Key Words: Cranberry Pomace. we are interested in mobilizing functional diphenyls. due to its relatively low pH it poses significant environmental and ecological problems.190 VATTEM AND SHETTY Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 There was no significant change in DRI of the ethanol extracts. 1. Changes in diphenyl profiles during the solid-state process analyzed using HPLC indicated that ellagic acid increased by 4À5 fold in water extracts for both the nitrogen treatments. This increase was between 15À27% in the ethanol extracts. flesh and seed of the fruit. then enzymes are added to transform some sugars contained within the natural fruit. Rhizopus oligosporus INTRODUCTION Cranberry is an important commercial crop in the United States of America. First. a compound with anti-carcinogenic properties was enriched. the fruit is slightly heated. . Traditionally it has been used as an ingredient in animal feed. Other ways of handling pomace are by its disposal into soil or as a landfill. the remaining product is called cranberry pomace.

[21. They are usually synthesized in plants as a defense against pathogenic attack or environmental stress such as UV exposure or hyperhydracity.[27. but seeds and skins are especially rich sources of phenolics[12. oligosporus to release the Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 . 3.21) catalyzes the hydrolysis of glycosidic linkages in alkyl or aryl b-glucosides as well as glucosides containing only carbohydrate residues.22] Phenolics are ubiquitous in plants.[2] In addition we have determined that it produces high amounts of b-glucosidase when grown on cranberry pomace during solid-state growth. E.[4] Because of their important role in plant defense systems they are often referred to as phytoalexins.[27. Therefore.2. to find a b-glucosidase. therefore making them very useful for applications in food and beverage industries.C. has broad substrate specificity.[13À19] In addition to their functional properties it is long known that they contribute to important quality attributes such as color. availability of free hydroxyl groups on the phenolic rings is important for resonance stabilization of free radicals. Lowered antioxidant capacity has direct implications on decreasing health functionality when these phenolics are ingested via food or nutraceuticals.31À36] The enzyme is capable of hydrolyzing phenolic glycosides and releasing extractable free aglycones potentially having high antioxidant activity. if free phenolics are released from their glycosides or other conjugates then the antioxidant and thus health functionality of these phytochemicals could be improved.[27À30] The enzyme is fairly common in all living organism but has been shown to be expressed in high quantities by fungi during solid state fermentation on lignocellulosic wastes.[9À12] There is also evidence now that some of these phenolics have antimicrobial activity against certain bacterial pathogens.1. taste[20] and flavor in both fresh and processed foods. This conjugation occurs via the hydroxyl groups of the phenolics. which is food grade. and that exhibits low-pH and high temperature stability while efficiently hydrolyzing free phenolics for potential applications in juice and wine processing industries have been relatively few. Recent research has shown that these phenolic phytochemicals posses excellent antioxidant properties[5À8] and thus may have potential beneficiary effect on human health. Several phenolics that are found in plants however. It has been successfully grown on various fruit pomace including cranberry using solid-state system. this reduces their ability to function as good antioxidants since.37] Rhizopus oligosporus or Tempeh fungi is a food grade fungi and has been used for thousands of years to make fermented and partially fermented indigenous foods like Soy Tempeh in Asia. Attempts however. exist in conjugated forms either with sugars (primarily glucose) as glycosides or other moieties. The enzyme b-glucosidase (b-D-glucoside glucohydrolase.23À26] probably because of the role they play in protecting the fruit and the seed to ensure healthy propagation of the species. In this investigation we have examined the ability of R.PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE 191 Phenolic phytochemicals are important secondary metabolites that are ubiquitous to all plants.

Ethanol Extraction One hundred milliliters of 95% ethanol was added to fungus-pomace flask and the culture was homogenized for 1 min using Waring blender. and was vacuum-dried and stored in a refrigerator before use. Houston. MATERIALS AND METHODS Microorganism Rhizopus oligosporus was isolated from un-pasteurized Tempeh product.. MA) as herring waste containing 0. MA. FPH was obtained from Ocean Crest (Gloucester. 1 filter paper.5 g of CaCO3. The supernatant was then filtered through a Whatman No. Media and Cultivation Freshly pressed cranberry pomace was obtained from Veryfine. The Tempeh product was kindly provided by Life-Life Foods Co. The media contained in flasks were autoclaved at 121 C for 20 min and the spores from one PDA plate were inoculated into 8 flasks.. Inc.000 g at 4 C for 20 min. 20 mL water and 0.5 g of NH4NO3 or 2 mL fish protein hydrolysate (FPH) as the supplemental nitrogen source were used for SSF. MA. The supernatant was then filtered through a Whatman No. The flasks would be incubated at 28 C for 16 days. Water Extraction One hundred milliliters of distilled water was added to fungus-pomace flask and the culture was homogenized for 1 min using Waring blender. Westford. and then centrifuged at 15.. 1 filter paper. The fungus was maintained on PDA slants and Petri plates at 4 C and sub-cultured monthly.000 g at 4 C for 20 min. and then centrifuged at 15.192 VATTEM AND SHETTY phenolic antioxidants from cranberry pomace via its high b-glucosidase activity and further if the released phenolic aglycones potentially could be remobilized for synthesis of functional diphenyls. Greenfield. The fungus was revived by transferring onto PDA plate and cultured at room temperature 10 days before use. Crude Enzyme Extraction A 5 mL portion of the filtrate from water extract was dialyzed using a Spectro=Pro membrane tubing (Spectral Medical Industries Inc. Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 . 125 mL Erlenmeyer flasks containing 10 g cranberry pomace 0.6575 g mL 7 1 of soluble solids.

[38] and Hang and Woodams. Bio-Rad Laboratory.2 mL of 3-methyl-2-benzothiazolinone Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 . After 15 min incubation at 50 C. 0. it was diluted to 50 mL with distilled water and autoclaved at 120 C for 1 h. After vortexing and incubating for 5 min. the culture flasks were mixed with 100 mL of distilled water and homogenized in a Waring blender for 1 min. Fair Lawn.6 at 50 C under the assay conditions.. Protein Assay Protein content was measured by the method of Bradford assay.[39] The dye reagent concentrate (Bio-Rad protein assay kit II. 0.8 mL of 200 mM sodium acetate buffer (pH 4.6) and 0.1 mL of enzyme solution. CA) was diluted 1:4 with distilled water.PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE 193 TX) against distilled water at 2 C for 24 h. 1 mL of homogenized sample was transferred into a test tube to which 2 mL of 5% H2SO4 was added. the absorbance was measured at 595 nm against a 5 mL reagent blank and 100 mL buffer using a UV-VIS Genesys spectrophotometer (Milton Roy. Rochester.1 mL of 9 mM p-nitrophenol b-D glucopyranoside (pNPG). The standard curve was established using pure p-nitrophenol (Fisher Scientific Co. The hydrolysate was then neutralized with 5 M NaOH to pH 7. Five mL of diluted dye reagent was added to 100 mL of the fungus-pomace water extract. Glucosamine Assay The glucosamine content of the fermented culture mixture containing fungal mycelia and cranberry pomace was used to estimate fungal biomass during the SSF as the growth indicator of Rhizopus oligosporus. One unit of enzyme was defined as the amount of enzyme that releases 1 mmole p-nitrophenol per min at pH 4. After standing for 24 h at 25 C. the reaction was stopped by addition of 1 mL of 0.0 and diluted to 100 mL with distilled water.[40] Briefly. it was centrifuged at 1500 g for 3 min 0.. b-Glucosidase Activity Assay The enzyme activity was measured by a modified procedure based on the methods of Gunata et al.2 mL of NH4SO3NH2 (12.1 M sodium carbonate and released p-nitrophenol was measured at 400 nm. It was determined by the modified method of Sakurai et al. After shaking occasionally for 15 min. Hercules.5 mL of KHSO4 (5%) in a centrifuge tube. NJ). from this 0.5 mL was mixed with 0. Inc.5 mL of NaNO2 (5%) and 0.[35] A standard reaction mixture contained 0. To the mixture.5%) and shaken for 3 min. The resultant clear liquid was used as crude enzyme solution after adjusting the same volume for each respective culture.6 mL of supernatant was mixed with 0. NY).

prepared within 3 days) was added. The absorbance values were converted to total phenolics and were expressed in milligrams equivalents of gallic acid per 10 grams dry weight (dw) of the sample.1-diphenyl-2-picrylhydrazyl Radical (DPPH) Inhibition System[42] To 3 mL of 60 mM DPPH in ethanol.194 VATTEM AND SHETTY hydrazone hydrochloride (MBTH. the decrease in absorbance was monitored at 517 nm until a constant reading was obtained. 50 mL of 50 mM H2O2 was added and shaken vigorously until a uniform emulsion was obtained. 0. To this. After standing it for 30 min. The glucosamine was measured as milligrams per 10 gram of pomace. Determination of Antioxidant Activity 1. Aliquots (5 mL) of this emulsion were transferred to each test tube containing . To each sample 0. Pure glucosamine in distilled water was used to make calibration curve. Chloroform was evaporated using a rotary evaporator under vacuum at 40 C for 5 min. The total phenolics were determined by an assay modified from Shetty et al.5%.5%. 1 mL of 5% Na2CO3 was added to the reaction mixture and allowed to stand for 60 min. the absorbance was measured at 650 nm. Standard curves were established using various concentrations of gallic acid in 95% ethanol.2 mL of FeCl3 (0. The % inhibition was calculated by: % inhibition ¼ fðA517 control À A517 extract Þ=A517 control g  100 b-Carotene Oxidation Model System[9] One milliliter of 200 mg=mL of b-carotene in chloroform was pipetted into a round-bottomed flask. Total Phenolics Assay Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 The water extracts (before dialysis) and ethanol extracts of the fermented pomace were used for the phenolic assay. 500 mL of cranberry pomace SSF extracts were added. The absorbance was read at 725 nm. prepared daily) was added and the mixture was allowed to boil for 3 min in a water bath. After 5 min. The b-carotene adhered to the sides of the flask were scraped and dissolved with 20 mL of purified linoleic acid and 184 mL of Tween 40 emulsifier.[41] One milliliter of supernatant was transferred into a test tube and mixed with 1 mL of 95% ethanol and 5 mL of distilled water. which contained 500 mL of 95% ethanol instead of the extract.5 mL of 50% (v=v) Folin-Ciocalteu reagent was added and mixed. The reaction mixture was immediately brought to room temperature following boiling and 0. The readings were compared with the controls.

resveratrol and ellagic acid (purchased from Sigma Chemical Co. MO) in 100% methanol were used to calibrate the standard curve and retention times. then decreased to 0% for the next 3 min and was maintained for the next 7 min (total run time. absorbance readings were recorded at 417 nm and compared to a control which had 100 mL of ethanol in place of the extract. The fungus grown with FPH as nitrogen source showed 5-fold increase in glucosamine content.d. 2) was typical of a sigmoidal growth curve for both the treatments. Protein content (Fig.PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE 195 100 mL of extract. Palo Alto. 25 min). For the NH4NO3 treatment there was a rapid increase initially until day 6 after which it stabilized and then increased gradually to 13 mg=10 g dw of the pomace. 5 mL of sample was injected using Agilent ALS 1100 autosampler into a Agilent 1100 series HPLC (Agilent Technologies. St. The antioxidant activity was expressed as protection factor (PF) and was calculated as follows: Antioxidant protection factor (APF) ¼ A417 Sample=A417 Control HPLC Analysis of Resveratrol[43. it had increased to over 7-fold to 23 mg=10 g dw of pomace. Subsequently. Louis. CA) equipped with VWD 1100 variable wavelength detector. The final glucosamine concentration was 15 mg=10 g dw and 18 mg=10 g dw for NH4NO3 and FPH treatments. Pure rosmarinic acid.5) and (B) 100% methanol.6 mm i. RESULTS Biomass Biomass was estimated using protein and glucosamine content. The solvents used for gradient were (A) 10 mM phosphoric acid (pH 2. 250 6 4.2 mm filter. The analytical column used was Agilent Zorbax SB-C18. 1) for both the treatments increased gradually until day 12. The glucosamine content (Fig. . In the FPH sample more protein was synthesized by the fungus and by day 12. an increase of over 4-fold from an initial value of 3 mg=10 g dw of pomace. respectively. During each run the chromatogram was recorded at 306 nm and integrated using Agilent Chemstation enhanced integrator. The glucosamine content increased gradually until day 10 before saturating.44] Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 Two mL of cranberry pomace-fungal extracts were filtered through a 0... with packing material of 5 mm particle size at a flow rate of 1 mL=min at ambient temperature. which was one-fold higher than the values obtained when NH4NO3 was used as the nitrogen source. The samples were vortexed for 1 min and incubated at 50 C for 30 min. The methanol concentration was increased to 60% for the first 8 min and to 100% for the 7 min.

196 VATTEM AND SHETTY Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 Figure 1. . Figure 2. Protein content of cranberry pomace during SSF. Glucosamine content of cranberry pomace during SSF.

3). When solid-state growth was carried out with NH4NO3 the enzyme showed a longer lag phase until day 8 after which the relative activity increased 60-fold by day 16. Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 Total Phenolics The water extractable phenolics for both the nitrogen sources showed a similar trend (Fig. b-glucosidase activity of cranberry pomace during SSF. Figure 3. The increase was however. When FPH was used as a nitrogen source the enzyme showed a lag phase of 4 days with little activity and then increased exponentially by 100-fold until day 10 before stabilizing. higher for the FPH supplemented pomace which increased by 26% to 120 mg=10 g dw of pomace compared to over 15% increase to 110 mg=10 g dw of pomace observed when NH4NO3 was used as nitrogen source.PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE 197 b-Glucosidase Activity There was a marked difference in the changes in the relative activity of b-glucosidase between the two treatments over the course of fermentation (Fig. . 4). The amounts of free phenolics for both nitrogen sources were constant until day 8 before sharply increasing by day 10 and then gradually decreasing for the remaining days of the fermentation.

they decreased gradually towards the end of the growth period. in the water extracts the DRI capacity increased 5% by day 8 compared to the initial value before rapidly decreasing. When changes in phenolic content were measured in the ethanol extracts (Fig. DPPH Radical Inhibition (DRI) The ability of phenolics to inhibit the DPPH radical formation was measured both in water and ethanol extracts.198 VATTEM AND SHETTY Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 Figure 4. 5). There was no significant increase in the DRI capacity when FPH was used as a nitrogen source. When NH4NO3 was used as a nitrogen source. 7). but on day 10 the DRI capacity decreased below the initial values (Fig. the NH4NO3 supplemented extract showed a gradual increase in total phenolics until day 10 after which it rapidly fell. fell rapidly after day 10. Total phenolic content of water extracts of cranberry pomace during SSF. In the ethanol extract the there was no significant inhibition of the DPPH radical formation for both the nitrogen sources until day 10 (Fig. 8). however. 6). b-Carotene Antioxidant Protection Factor (APF) The APF of water extract for both the nitrogen sources showed similar trends (Fig. The DRI capacity of the extracts however. When FPH was used as a nitrogen source the ethanol extractable phenolics did not show any significant increase. From an initial value of 1 the APF gradually increased until .

PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE 199 Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 Figure 5. SSF. Figure 6. Total phenolic content of ethanol extracts of cranberry pomace during SSF. DPPH radical inhibition capacity of water extracts of cranberry pomace during .

200 VATTEM AND SHETTY Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 Figure 7. Figs. The individual peaks were confirmed using authentic pure standards under the same analytical conditions. after which they rapidly decreased until day 8 before increasing again by 19% and 11% compared to the initial values (Fig. 9). 10a and 10b illustrate the chromatographic profiles of diphenyls in standard mixtures and in SSF extracts. HPLC analysis showed that ellagic acid was the major diphenyl in the water and ethanol extract for both the nitrogen sources with low levels . The APF peaked on day 10 by when it had increased by 25% for FPH and 20% for NH4NO3 compared to the initial values. The individual retention times for each of the diphenyls (rosmarinic acid. On day 4 the APF increased by 13% and 16% for NH4NO3 and FPH nitrogen sources respectively. The changes in the APF for ethanol extracts were similar for both the nitrogen sources. DPPH radical inhibition capacity of ethanol extracts of cranberry pomace during day 4 before decreasing by day 8. SSF. HPLC of Diphenyls Possible mobilization of functionally relevant diphenyls during SSF were investigated using HPLC. ellagic acid and resveratrol) tested are given in Table 1. Both showed two peaks of increase in APF during the course of growth before stabilizing by day 12.

Antioxidant protection factor of water extracts of cranberry pomace during SSF. SSF. Figure 9. Antioxidant protection factor of ethanol extracts of cranberry pomace during .PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE 201 Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 Figure 8.

of rosmarnic acid and resveratrol. (a) HPLC profiles of standard diphenyls. 11) when NH4NO3 was used as a nitrogen source.202 VATTEM AND SHETTY Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 Figure 10. the ellagic acid content increased linearly 5-fold by day 12 to a concentration of 375 mg=g dw of pomace. . In the water extracts when FPH was used as nitrogen source the ellagic acid showed two sharp peaks of increase. In both the treatments after an initial increase there was a sharp decrease in ellagic acid content. which remained unchanged for the duration of growth. (b) HPLC profiles of diphenyls present in extracts of cranberry pomace during SSF. After day 12 the ellagic acid content decreased during the remaining days of growth period. In the water extract (Fig. By day 2 the ellagic acid increased over 3-fold to 275 mg=g dw of pomace and then increased to over 4-fold to 325 mg=g dw of pomace by day 12 compared to initial values.

15 203 Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 The ellagic acid content in ethanol extracts behaved similarly for both the nitrogen sources (Fig.26 12. 12). Peak 1 2 3 Retention Times of Standard Diphenyls Diphenyl Ellagic acid Resveratrol Rosmarnic acid Retention Time (min) 11.09 12.02 Æ 0.PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE Table 1. DISCUSSION Biomass Glucosamine is a complex carbohydrate that is found in the shells of many crustaceans and is abundant in the cell wall of fungi. In this study Figure 11.5 Æ 0. Ellagic acid content of water extracts of cranberry pomace during SSF. . When FPH was used as a source the ellagic acid initially increased by 15% by day 4 before rapidly decreasing and again increasing to the same value by day 12. When NH4NO3 used as a nitrogen source the ellagic acid increased gradually until day 8 by 25% and then decreased rapidly before increasing by 27% by day 14 to a value of 330 mg=g dw of pomace.6 Æ 0.

FPH is a hydrolysate of fish protein containing many small peptides and amino acids. These peptides are easily assimilated by the fungus as amino acids and into proteins for growth.204 VATTEM AND SHETTY Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 Figure 12. The amounts of total phenolics in both water and ethanol extracts were comparable to the phenolics in various varieties of fresh cranberry and their juices.[45] The increased activity of the enzyme correlated well with the . This may explain the better growth of fungus on cranberry pomace when FPH was used as nitrogen source instead of NH4NO3. glucosamine content was used as an indicator of biomass to monitor the growth of fungus during the solid state fermentation. When NH4NO3 is used as a nitrogen source the fungus spends extra energy to first assimilate nitrogen and then synthesize amino acids. An increase in glucosamine content was observed and this resembled a typical fungal growth curve. Production of Free Phenolics and b-Glucosidase b-glucosidase activity increased by 60À100 fold for both the nitrogen sources. The fungus showed enhanced growth when FPH was used as nitrogen source and this was further confirmed by the 3-fold increase in protein content that was synthesized by the fungus. Ellagic acid content of ethanol extracts of cranberry pomace during SSF. These treatments had an important effect on the production of total free phenolics from cranberry pomace during solid-state growth.

the radical inhibition capacity was highest on day 8 and day 10 in the water extracts in NH4NO3 and FPH treatments respectively. This suggests that since the substrate depletion in the FPH sample was not very high. . in both the FPH and NH4NO3 treatments there was no significant correlation between the protein synthesized by fungus and phenolics enrichment for the total duration of growth. In the b-carotene system. These differences may be due to reduced nitrogen depletion stress response in the presence of FPH vs. which corresponded to the stage when the b-glucosidase enzyme activity was at its highest.37] The Folin-Ciocalteu assay may have contribution from the aromatic amino acids from proteins from the substrate and from the fungus. NH4NO3 during the course of growth. An insignificant increase in the DRI capacity when FPH was used as nitrogen source suggests the difference in the chemical nature of phenolics released in response to the two nitrogen sources may have affected their ability to respond to this particular assay. for both the nitrogen sources the APF was highest on day 10 in the water extracts. The good correlation in both the water and ethanol extracts suggests that the enzyme is likely to be involved in the release of free phenolics from its glycosides in cranberry pomace.[46À48. The contribution from the intrinsic phenolics in the fungus was determined in an earlier investigation and found to be insignificant (Zheng and Shetty. the antioxidants that may have been released by the b-glucosidase were either being rearranged or were being converted into dimers or trimers which due to their higher lipid solubility had higher antioxidant activity at the interface. This mechanism may be similar to the release of flavors occurring during fermentation of wine. The increase in APF was higher in FPH containing medium than in the treatment with NH4NO3. In the DPPH system. A significant difference was not observed in the ethanol extracts indicating that most of these antioxidants having radical quenching capacity were water extractable. The b-carotene system potentially quantifies the ability of the antioxidant to function at a lipid water interface and therefore. When NH4NO3 was used as the nitrogen source the DRI capacity peaked on day 8 which may have been because of rapid synthesis and release of antioxidants in response to the stress on the fungus as a result of nutrient depletion in the substrate. This difference was further substantiated by the APF values measured by the b-carotene assay. however. unpublished results). the antioxidant has to be a partially hydrophobic in nature. Antioxidant Activity Is Correlated to b-Glucosidase Activity Antioxidant activity was measured by two methods.PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE 205 Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 increase in the total phenolic content of the extracts. The antioxidant protection factor (APF) directly measures the ability of the antioxidant=extract to prevent the H2O2 catalyzed oxidation of b-carotene.

The second peak of increase observed for both the FPH and NH4NO3 treatments could be due to dimerization and trimerization of simple phenolic monomers during the later stages of growth. Therefore. The comparable concentrations of ellagic acid in both water and ethanol extracts could possibly be due to the non-covalent interactions of ellagic acid with soluble proteins and peptides in the pomace matrix increasing their solublity and therefore extractable with water. are known to be rich in flavanoids and its derivatives[49] but are likely to be extracted with the juice. This process resulted in enrichment of phenolics to a level found usually in fresh cranberry and it juice. Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 HPLC Determination of Diphenyls The phenolics in the pomace was determined in an earlier investigation[3] and were found to contain some simple phenolics such as gallic acid.206 VATTEM AND SHETTY The two peaks of high APF in the ethanol extracts could be due to the rearrangements of the hydroxyl groups carried out by the fungal growth when the antioxidants were being released during the initial stages of growth. These deletions and rearrangements of hydroxyl groups on the phenolic ring may have transiently increased their solubility and antioxidant activity in ethanol. the presence and enrichment of functionally mobilized diphenyls in cranberry fruit waste (pomace) was monitored in this investigation. Concentrations in the water extract ranged from 325 mg=g dw of pomace for NH4NO3 and 375 mg=g dw of pomace for FPH treatment.[50À54]. and p-coumaric acid. CONCLUSIONS The investigation was carried out to enrich the cranberry pomace with phenolic antioxidants via solid-state growth using food grade fungus Rhizopus oligosporus. This enrichment of the cranberry pomace may have been occurring due to the hydrolysis of ellagotannins and ellagic acid esters in the cranberry pomace during the initial stages of fermentation by enzymes such as tannin-acyl-hydrolase and dimerization of the simple phenolics such as PABA during the later stages. Ellagic acid was found in water and ethanol extracts for both NH4NO3 and FPH nitrogen treatments. Cranberries like other fruits of the Vaccinium Spp. p-hydroxybenzoic acid. Ellagic acid is well documented to have anti-carcinogenic effect. HPLC of the extracts showed a 3À5 fold enrichment of the diphenyl ellagic acid in the cranberry pomace. Such high concentrations of ellagic acid have not been previously reported in cranberry or its pomace.[44] The role of . This process may lead to initiation of a polymerization by fungal growth in order to form tannins or similar polymers arising due to nutrient depletions. chlorogenic acid.

Strycharz. showing that the enzyme may play an important role in the release of phenolic aglycones from cranberry pomace and therefore increase the antioxidant capacity. 37 (8). 805À812.S. 4... . This approach has led to process development concepts to produce plant-based nutraceutical such as ellagic acid that is GRAS and permits an alternative use of the cranberry pomace as a functional ingredient for diverse food and feed applications. Antioxidant Activity of Durum Wheat Bran. 2001. Food Agric. E. S. Agric. REFERENCES 1. Onyeneho. The solid-state growth using food grade fungus resulted in the valueaddition of the cranberry pomace. The investigation showed that antioxidant capacity of the pomace can be improved through a solid-state process.C. Both total phenolics and antioxidant capacity correlated well with the increase in the b-glucosidase activity and peaked in a similar manner. Phenolics: Prooxidants or Antioxidants? Nutr. K. Shetty. Food Chem. Z Strain and Polydye R-478 and Implications for Hyperhydricity Prevention in Tissue Culture. Solid-state Bioconversion of Phenolics from Cranberry Pomace and Role of Lentinus edodes Beta-Glucosidase. 1997. 323À329. 895À900.. Hettiarachchy. Z. Process Biochem.. National Agricultural Statistics Service (NASS). 55.N. Zheng. 3. 2. USDA.S. ‘‘Cranberries’’ Annual Reports.. 7. N. 40. J. 5. Fr Nt 4. Rev. 33. K. Hettiarachchy.[50À54] The fungus Rhizopus oligosporus and other ingredients used in this solid-state growth process are food grade and are generally recognized as safe (GRAS). Onyeneho. Zheng. J. We observed that during the course of solidstate growth there was an increase in the total extractable phenolic content. Food Chem. N. Washington D. Shetty. 1998. 1992. Antioxidant Activity. J.A. 345À350. Process Biochemistry 2002. Response of Oregano (Origanum vulgare) Clonal Lines to Pseudomonas sp. Z. 1496À1500. S. In addition. 1993. Sci. Shetty.PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE 207 Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 05:25 13 September 2008 b-glucosidase in the enrichment of phenolic antioxidants by hydrolyzing the glycosides was also investigated. Cranberry Processing Waste for Solid-state Fungal Inoculant Production. Decker. S. The process resulted in enrichment of the pomace with ellagic acid.N. Agric. Agricultural Statistics board. 62. Fatty Acids and Phenolic Acids Compositions of Potato Peels. an important phytochemical well documented to have anti-carcinogenic and cardioprotective properties. 396À407. HPLC analysis indicated that the cranberry pomace was enriched with ellagic acid to a level of 375 mg=g dw of pomace. Antioxidant activity measured by both APF and DRI increased over the course of growth. 6. 48. 2000. K.

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