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SOLID-STATE PRODUCTION OF PHENOLIC ANTIOXIDANTS FROMCRANBERRY POMACE BY RHIZOPUS OLIGOSPORUSDhiraj 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 FROMCRANBERRY POMACE BY RHIZOPUS OLIGOSPORUS',Food Biotechnology,16:3,189 — 210

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SOLID-STATE PRODUCTION OF PHENOLIC

ANTIOXIDANTS FROM CRANBERRY POMACEBY RHIZOPUS OLIGOSPORUS

Dhiraj A. Vattem and Kalidas Shetty*

Laboratory of Food Biotechnology, Department of Food Science,

University of Massachusetts, Amherst, MA 01003, USA

ABSTRACT

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 proper-

ties 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 pomacewas 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: [email protected]

189

DOI: 10.1081=FBT-120016667 0890-5436 (Print); 1532-4249 (Online)

Copyright # 2002 by Marcel Dekker, Inc. www.dekker.com

FOOD BIOTECHNOLOGY

Vol. 16, No. 3, pp. 189–210, 2002



There was no significant change in DRI of the ethanol extracts. 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. Changes in diphenylprofiles 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. We conclude that SSF of cranberry pomace increased the

antioxidant activity concurrent with increased b-glucosidase activity. The

HPLC profile showed ellagic acid, a compound with anti-carcinogenic

properties was enriched. The antioxidant function has implications for

prevention of major oxidation-linked diseases such as cancer and CVD.

This value-added SSF strategy is an innovative approach to enhance

nutraceutically-relevant functional phytochemicals for food and feedapplications.

Key Words: Cranberry Pomace; Phenolics; Phenolic aglycone; Anti-

oxidant activity; b-glucosidase; 1,1-Diphenyl-2-picrylhydrazl (DPPH);

b-carotene oxidation model system; Rhizopus oligosporus

INTRODUCTION

Cranberry is an important commercial crop in the United States of America. Around 520 million tons are produced every year and the projected

production is higher for the coming years.[1] Cranberries are used as

ingredients in over 700 products from cereals to salsas. Only 5 percent of the

annual crop is harvested for fresh fruit and most of it is used for processing.

Cranberry juice relished for its taste and ever increasing health benefits is one

of the major products produced by the food processing industry. Pomace

is the byproduct of the cranberry juice processing industry with limited ap-

plications. First, the fruit is slightly heated, then enzymes are added to

transform some sugars contained within the natural fruit. Once the juice is

extracted from the fruit, the remaining product is called cranberry pomace.Pomace is mainly composed of the skin, flesh and seed of the fruit. It is rich in

fiber and has relatively small amounts of protein and carbohydrates.

Traditionally it has been used as an ingredient in animal feed, however due

to its low protein and carbohydrate content it has little nutritive value as an

animal feed. Other ways of handling pomace are by its disposal into soil or as

a landfill. However, due to its relatively low pH it poses significant

environmental and ecological problems. Previous investigation had explored

potential new uses of cranberry pomace using solid state fermentation

(SSF).

[2,3]

In this investigation we are exploring whether water and ethanolsoluble phenolics can be enriched during SSF. Further, we are interested in

mobilizing functional diphenyls.

190 VATTEM AND SHETTY



Phenolic phytochemicals are important secondary metabolites that are

ubiquitous to all plants. They are usually synthesized in plants as a defense

against pathogenic attack or environmental stress such as UV exposure or

hyperhydracity.[4] Because of their important role in plant defense systems

they are often referred to as phytoalexins. Recent research has shown that

these phenolic phytochemicals posses excellent antioxidant properties[5À8] and

thus may have potential beneficiary effect on human health.[9À12] There is also

evidence now that some of these phenolics have antimicrobial activity against

certain bacterial pathogens.[13À19] In addition to their functional properties it

is long known that they contribute to important quality attributes such as

color, taste[20] and flavor in both fresh and processed foods.[21,22]

Phenolics are ubiquitous in plants, but seeds and skins are especially

rich sources of phenolics[12,23À26]

probably because of the role they play inprotecting the fruit and the seed to ensure healthy propagation of the species.

Several phenolics that are found in plants however, exist in conjugated forms

either with sugars (primarily glucose) as glycosides or other moieties. This

conjugation occurs via the hydroxyl groups of the phenolics, this reduces

their ability to function as good antioxidants since, 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. Therefore, if free phenolics are released

from their glycosides or other conjugates then the antioxidant and thushealth functionality of these phytochemicals could be improved.

The enzyme b-glucosidase (b-D-glucoside glucohydrolase, E.C. 3.2.1.21)

catalyzes the hydrolysis of glycosidic linkages in alkyl or aryl b-glucosides

as well as glucosides containing only carbohydrate residues.[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 ligno-

cellulosic wastes.[27,31À36] The enzyme is capable of hydrolyzing phenolic

glycosides and releasing extractable free aglycones potentially having high

antioxidant activity, therefore making them very useful for applications in

food and beverage industries. Attempts however, to find a b-glucosidase,which is food grade, has broad substrate specificity, 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.[27,37]

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. It has been successfully grown on

various fruit pomace including cranberry using solid-state system.[2] In

addition we have determined that it produces high amounts of b-glucosidasewhen grown on cranberry pomace during solid-state growth. In this

investigation we have examined the ability of R. oligosporus to release the

PHENOLIC ANTIOXIDANTS FROM CRANBERRY POMACE 191



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.

MATERIALS AND METHODS

Microorganism

Rhizopus oligosporus was isolated from un-pasteurized Tempeh product.

The Tempeh product was kindly provided by Life-Life Foods Co.,

Greenfield, MA. The fungus was maintained on PDA slants and Petri plates

at 4C and sub-cultured monthly. The fungus was revived by transferring

onto PDA plate and cultured at room temperature 10 days before use.

Media and Cultivation

Freshly pressed cranberry pomace was obtained from Veryfine, Inc.,

Westford, MA, and was vacuum-dried and stored in a refrigerator before use.

125 mL Erlenmeyer flasks containing 10 g cranberry pomace 0.5 g of CaCO3,

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. FPH was obtained from

Ocean Crest (Gloucester, MA) as herring waste containing 0.6575 g mL7 1 of

soluble solids. The media contained in flasks were autoclaved at 121

C for20 min and the spores from one PDA plate were inoculated into 8 flasks. The

flasks would be incubated at 28C 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, and

then centrifuged at 15,000 g at 4C for 20 min. The supernatant was then

filtered through a Whatman No. 1 filter paper.

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

then centrifuged at 15,000 g at 4C for 20 min. The supernatant was then

filtered through a Whatman No. 1 filter paper.

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., Houston,




TX) against distilled water at 2C for 24 h. The resultant clear liquid was used

as crude enzyme solution after adjusting the same volume for each respective

culture.

b-Glucosidase Activity Assay

The enzyme activity was measured by a modified procedure based on

the methods of Gunata et al.[38] and Hang and Woodams.[35] A standard

reaction mixture contained 0.1 mL of 9 mM p-nitrophenol b-D glucopyrano-

side (pNPG), 0.8 mL of 200 mM sodium acetate buffer (pH 4.6) and 0.1 mL

of enzyme solution. After 15 min incubation at 50C, the reaction was

stopped by addition of 1 mL of 0.1 M sodium carbonate and released

p-nitrophenol was measured at 400 nm. The standard curve was establishedusing pure p-nitrophenol (Fisher Scientific Co., Fair Lawn, NJ). One unit of

enzyme was defined as the amount of enzyme that releases 1 mmole

p-nitrophenol per min at pH 4.6 at 50C 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, Bio-Rad Laboratory,

Hercules, CA) was diluted 1:4 with distilled water. Five mL of diluted dyereagent was added to 100 mL of the fungus-pomace water extract. After

vortexing and incubating for 5 min, the absorbance was measured at 595 nm

against a 5 mL reagent blank and 100mL buffer using a UV-VIS Genesys

spectrophotometer (Milton Roy, Inc., Rochester, NY).

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. It wasdetermined by the modified method of Sakurai et al.[40] Briefly, the culture

flasks were mixed with 100 mL of distilled water and homogenized in a

Waring blender for 1 min. 1 mL of homogenized sample was transferred into

a test tube to which 2 mL of 5% H2SO4 was added. After standing for 24 h at

25C, it was diluted to 50 mL with distilled water and autoclaved at 120C for

1 h. The hydrolysate was then neutralized with 5 M NaOH to pH 7.0 and

diluted to 100 mL with distilled water, from this 0.5 mL was mixed with

0.5 mL of NaNO2 (5%) and 0.5 mL of KHSO4 (5%) in a centrifuge tube.

After shaking occasionally for 15 min, it was centrifuged at 1500 g for 3 min0.6 mL of supernatant was mixed with 0.2 mL of NH4SO3NH2 (12.5%) and

shaken for 3 min. To the mixture, 0.2 mL of 3-methyl-2-benzothiazolinone




hydrazone hydrochloride (MBTH, 0.5%, prepared daily) was added and the

mixture was allowed to boil for 3 min in a water bath. The reaction mixture

was immediately brought to room temperature following boiling and 0.2 mL

of FeCl3 (0.5%, prepared within 3 days) was added. After standing it for

30 min, the absorbance was measured at 650 nm. The glucosamine was

measured as milligrams per 10 gram of pomace. Pure glucosamine in distilled

water was used to make calibration curve.

Total Phenolics Assay

The water extracts (before dialysis) and ethanol extracts of the

fermented pomace were used for the phenolic assay. The total phenolics

were determined by an assay modified from Shetty et al.[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. To each sample 0.5 mL of 50% (v=v)

Folin-Ciocalteu reagent was added and mixed. After 5 min, 1 mL of 5%

Na2CO3 was added to the reaction mixture and allowed to stand for 60 min.

The absorbance was read at 725 nm. 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. Standard curves were established

using various concentrations of gallic acid in 95% ethanol.

Determination of Antioxidant Activity

1,1-diphenyl-2-picrylhydrazyl Radical (DPPH) Inhibition System[42]

To 3 mL of 60mM DPPH in ethanol, 500 mL of cranberry pomace SSF

extracts were added, the decrease in absorbance was monitored at 517 nm

until a constant reading was obtained. The readings were compared with the

controls, which contained 500 mL of 95% ethanol instead of the extract. The

% inhibition was calculated by:

% inhibition ¼ fðA517control À A517extract Þ=A517controlg Â 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. Chloroform was evaporated using a rotary

evaporator under vacuum at 40C for 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. To this, 50 mL of 50 mM H2O2 wasadded and shaken vigorously until a uniform emulsion was obtained.

Aliquots (5 mL) of this emulsion were transferred to each test tube containing




100 mL of extract. The samples were vortexed for 1 min and incubated at

50C for 30 min. Subsequently, absorbance readings were recorded at 417 nm

and compared to a control which had 100 mL of ethanol in place of the

extract. 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[43,44]

Two mL of cranberry pomace-fungal extracts were filtered through a

0.2 mm filter. 5 mL of sample was injected using Agilent ALS 1100

autosampler into a Agilent 1100 series HPLC (Agilent Technologies, PaloAlto, CA) equipped with VWD 1100 variable wavelength detector. The

solvents used for gradient were (A) 10 mM phosphoric acid (pH 2.5) and (B)

100% methanol. The methanol concentration was increased to 60% for the

first 8 min and to 100% for the 7 min, then decreased to 0% for the next 3 min

and was maintained for the next 7 min (total run time, 25 min). The analytical

column used was Agilent Zorbax SB-C18, 2506 4.6 mm i.d., with packing

material of 5mm particle size at a flow rate of 1 mL=min at ambient

temperature. During each run the chromatogram was recorded at 306 nm

and integrated using Agilent Chemstation enhanced integrator. Pure

rosmarinic acid, resveratrol and ellagic acid (purchased from Sigma ChemicalCo., St. Louis, MO) in 100% methanol were used to calibrate the standard

curve and retention times.

RESULTS

Biomass

Biomass was estimated using protein and glucosamine content. Protein

content (Fig. 1) for both the treatments increased gradually until day 12. For

the NH4NO3 treatment there was a rapid increase initially until day 6 afterwhich it stabilized and then increased gradually to 13 mg=10gdw of the

pomace, an increase of over 4-fold from an initial value of 3 mg=10 g dw of

pomace. In the FPH sample more protein was synthesized by the fungus and

by day 12, it had increased to over 7-fold to 23 mg=10 g dw of pomace.

The glucosamine content (Fig. 2) was typical of a sigmoidal growth

curve for both the treatments. The glucosamine content increased gradually

until day 10 before saturating. The fungus grown with FPH as nitrogen

source showed 5-fold increase in glucosamine content, which was one-fold

higher than the values obtained when NH4NO3 was used as the nitrogensource. The final glucosamine concentration was 15 mg=10 g dw and

18mg=10 g dw for NH4NO3 and FPH treatments, respectively.




Figure 1. Protein content of cranberry pomace during SSF.

Figure 2. Glucosamine content of cranberry pomace during SSF.




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. 3). 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. 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.

Total Phenolics

The water extractable phenolics for both the nitrogen sources showed a

similar trend (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. The

increase was however, 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.

Figure 3. b-glucosidase activity of cranberry pomace during SSF.




When changes in phenolic content were measured in the ethanol

extracts (Fig. 5), the NH4NO3 supplemented extract showed a gradualincrease in total phenolics until day 10 after which it rapidly fell. When FPH

was used as a nitrogen source the ethanol extractable phenolics did not show

any significant increase, however, they decreased gradually towards the end

of the growth period.

DPPH Radical Inhibition (DRI)

The ability of phenolics to inhibit the DPPH radical formation was

measured both in water and ethanol extracts.

When NH4NO3 was used as a nitrogen source, in the water extracts theDRI capacity increased 5% by day 8 compared to the initial value before

rapidly decreasing. There was no significant increase in the DRI capacity

when FPH was used as a nitrogen source, but on day 10 the DRI capacity

decreased below the initial values (Fig. 6).

In the ethanol extract the there was no significant inhibition of the

DPPH radical formation for both the nitrogen sources until day 10 (Fig. 7).

The DRI capacity of the extracts however, fell rapidly after day 10.

b-Carotene Antioxidant Protection Factor (APF)

The APF of water extract for both the nitrogen sources showed similar

trends (Fig. 8). From an initial value of 1 the APF gradually increased until

Figure 4. Total phenolic content of water extracts of cranberry pomace during SSF.




Figure 5. Total phenolic content of ethanol extracts of cranberry pomace during SSF.

Figure 6. DPPH radical inhibition capacity of water extracts of cranberry pomace during

SSF.




day 4 before decreasing by day 8. 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 changes in the APF for ethanol extracts were similar for both the

nitrogen sources. Both showed two peaks of increase in APF during the

course of growth before stabilizing by day 12. On day 4 the APF increased by

13% and 16% for NH4NO3 and FPH nitrogen sources respectively, after

which they rapidly decreased until day 8 before increasing again by 19% and

11% compared to the initial values (Fig. 9).

HPLC of Diphenyls

Possible mobilization of functionally relevant diphenyls during SSF

were investigated using HPLC, Figs. 10a and 10b illustrate the chromato-

graphic profiles of diphenyls in standard mixtures and in SSF extracts. The

individual retention times for each of the diphenyls (rosmarinic acid, ellagic

acid and resveratrol) tested are given in Table 1. The individual peaks were

confirmed using authentic pure standards under the same analyticalconditions. HPLC analysis showed that ellagic acid was the major diphenyl

in the water and ethanol extract for both the nitrogen sources with low levels

Figure 7. DPPH radical inhibition capacity of ethanol extracts of cranberry pomace during

SSF.




Figure 9. Antioxidant protection factor of ethanol extracts of cranberry pomace during

SSF.

Figure 8. Antioxidant protection factor of water extracts of cranberry pomace during SSF.




of rosmarnic acid and resveratrol, which remained unchanged for the

duration of growth. In the water extract (Fig. 11) when NH4NO3 was used as

a nitrogen source, the ellagic acid content increased linearly 5-fold by day 12

to a concentration of 375 mg=g dw of pomace. After day 12 the ellagic acid

content decreased during the remaining days of growth period. In the water

extracts when FPH was used as nitrogen source the ellagic acid showed two

sharp peaks of increase. 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. In both the treatments after an

initial increase there was a sharp decrease in ellagic acid content.

Figure 10. (a) HPLC profiles of standard diphenyls; (b) HPLC profiles of diphenyls present

in extracts of cranberry pomace during SSF.




The ellagic acid content in ethanol extracts behaved similarly for both

the nitrogen sources (Fig. 12). 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 thendecreased rapidly before increasing by 27% by day 14 to a value of 330 mg=g

dw of pomace.

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

Table 1. Retention Times of Standard Diphenyls

Peak Diphenyl Retention Time (min)

1 Ellagic acid 11.6 Æ 0.26

2 Resveratrol 12.02 Æ 0.09

3 Rosmarnic acid 12.5 Æ 0.15

Figure 11. Ellagic acid content of water extracts of cranberry pomace during SSF.




glucosamine content was used as an indicator of biomass to monitor thegrowth of fungus during the solid state fermentation. An increase in

glucosamine content was observed and this resembled a typical fungal growth

curve. 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. 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.

When NH4NO3 is used as a nitrogen source the fungus spends extra energy

to first assimilate nitrogen and then synthesize amino acids. This may explain

the better growth of fungus on cranberry pomace when FPH was used asnitrogen source instead of NH4NO3.

Production of Free Phenolics and b-Glucosidase

b-glucosidase activity increased by 60À100 fold for both the nitrogen

sources. These treatments had an important effect on the production of total

free phenolics from cranberry pomace during solid-state growth. The

amounts of total phenolics in both water and ethanol extracts werecomparable to the phenolics in various varieties of fresh cranberry and their

juices.[45] The increased activity of the enzyme correlated well with the

Figure 12. Ellagic acid content of ethanol extracts of cranberry pomace during SSF.




increase in the total phenolic content of the 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. This mechanism may be similar to the release of flavors occurring

during fermentation of wine.[46À48,37] The Folin-Ciocalteu assay may have

contribution from the aromatic amino acids from proteins from the substrate

and from the fungus; however, 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. The

contribution from the intrinsic phenolics in the fungus was determined in an

earlier investigation and found to be insignificant (Zheng and Shetty;

unpublished results).

Antioxidant Activity Is Correlated to b-Glucosidase Activity

Antioxidant activity was measured by two methods. In the DPPH

system, the radical inhibition capacity was highest on day 8 and day 10 in the

water extracts in NH4NO3 and FPH treatments respectively. 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.

An insignificant increase in the DRI capacity when FPH was used as nitrogensource 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. This difference was further substantiated by

the APF values measured by the b-carotene assay. These differences may be

due to reduced nitrogen depletion stress response in the presence of FPH vs.

NH4NO3 during the course of growth. 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 anti-

oxidant to function at a lipid water interface and therefore, the antioxidanthas to be a partially hydrophobic in nature. The antioxidant protection

factor (APF) directly measures the ability of the antioxidant=extract to

prevent the H2O2 catalyzed oxidation of b-carotene. In the b-carotene

system, for both the nitrogen sources the APF was highest on day 10 in the

water extracts, which corresponded to the stage when the b-glucosidase

enzyme activity was at its highest. The increase in APF was higher in FPH

containing medium than in the treatment with NH4NO3. This suggests that

since the substrate depletion in the FPH sample was not very high, the

antioxidants that may have been released by the b-glucosidase were eitherbeing rearranged or were being converted into dimers or trimers which due to

their higher lipid solubility had higher antioxidant activity at the interface.




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 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. 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.

HPLC Determination of Diphenyls

The phenolics in the pomace was determined in an earlier investiga-

tion[3] and were found to contain some simple phenolics such as gallic acid,

chlorogenic acid, p-hydroxybenzoic acid, and p-coumaric acid. Cranberries

like other fruits of the Vaccinium Spp. are known to be rich in flavanoids and

its derivatives[49] but are likely to be extracted with the juice. Therefore, the

presence and enrichment of functionally mobilized diphenyls in cranberry

fruit waste (pomace) was monitored in this investigation. HPLC of the

extracts showed a 3À5 fold enrichment of the diphenyl ellagic acid in the

cranberry pomace. Ellagic acid is well documented to have anti-carcinogeniceffect.[50À54]. Ellagic acid was found in water and ethanol extracts for both

NH4NO3 and FPH nitrogen treatments. Concentrations in the water extract

ranged from 325mg=g dw of pomace for NH4NO3 and 375mg=g d w o f

pomace for FPH treatment. Such high concentrations of ellagic acid have not

been previously reported in cranberry or its pomace. This enrichment of the

cranberry pomace may have been occurring due to the hydrolysis of ellago-

tannins 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.

The comparable concentrations of ellagic acid in both water and ethanolextracts 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.

CONCLUSIONS

The investigation was carried out to enrich the cranberry pomace with

phenolic antioxidants via solid-state growth using food grade fungusRhizopus oligosporus. This process resulted in enrichment of phenolics

to a level found usually in fresh cranberry and it juice.[44] The role of




b-glucosidase in the enrichment of phenolic antioxidants by hydrolyzing the

glycosides was also investigated. We observed that during the course of solid-

state growth there was an increase in the total extractable phenolic content.

Antioxidant activity measured by both APF and DRI increased over the

course of growth. Both total phenolics and antioxidant capacity correlated

well with the increase in the b-glucosidase activity and peaked in a similar

manner, showing that the enzyme may play an important role in the release

of phenolic aglycones from cranberry pomace and therefore increase the

antioxidant capacity. In addition, HPLC analysis indicated that the

cranberry pomace was enriched with ellagic acid to a level of 375mg=g dw

of pomace.

The solid-state growth using food grade fungus resulted in the value-

addition of the cranberry pomace. The investigation showed that antioxidantcapacity of the pomace can be improved through a solid-state process. The

process resulted in enrichment of the pomace with ellagic acid, an important

phytochemical well documented to have anti-carcinogenic and cardio-

protective properties.[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). 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.

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