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E L S E V I E R

Process iochemistry Vol . 33, No. 3 , pp. 323-3 29, 1998© 1998 P ub l i s he d by E l s e v ie r S c i e nc e L t d

A l l r i gh t s r e se r ve d . P r i n t e d i n G r e a t B r i t a in0032-9592/98 19.00 + 0.00

P I I : S 0 0 3 2 - 9 5 9 2 9 7 ) 0 0 0 8 6 - 1

ranberry process ing waste for sol id s tatefungal inoculant product ion

Zuo xing Zh eng Ka lidas Shetty*

De par tm ent o f Food Science, Universi ty of M assachuset ts , Am herst , MA 01003, USA

(Received 7 July 1997; revised version received 28 Augu st 1997; accepted 1 Septem ber 1997)

bstract

Cran berry pom ace is a primary by-product of the trad it ional cranberry juice processing industry and i tsdisposal presents economic and environmental problems. Microbial conversion of cranberry pomace intovarious value-added products is a practical approach for solving such disposal problems. The presentresearch was undertaken to test the growth of several agriculturally and industrial ly important fungi oncranberry pomace substrate through solid-state fermentation. Fungi, such as Trichoderma viride If-26, Trichoderma harzianum ATC C 24274 , and Trichoderma pseudokoningii ATCC 26801, a novel polymeric dyedecolorizing PeniciUium isolate, and a food-grade Rhizopus st rain i solated f rom Tempeh, that produceindustrially imp ortan t extracellular enzym es were grown on a cranb erry pomace-based me dium a t 25°C for4 days. The glucosamine content o f the h eterogeneous ferme nted m ixture was a good indicator of fungalgrowth. Th e maximum growth o f al l fungi was establi shed on cranberry poma ce su pplemen ted wi th 0 .05 go f C a C O 3 , 2 0 ml o f water, and 0 05 g of N H 4 N O 3 o r 0 2 ml o f fish protein hydrolysate pe r gram of pomace.It was concluded that bioconversion of cranberry processing waste by industrial ly beneficial fungi throughsolid-state ferm enta tion was feasible. This po tential can be coup led with the uti lization of fish processingwaste as an organ ic nitrogen source to develop m utually com plem entary produ cts benefi t ing bo th the fisheryand c ranbe rry processing industries. © 1998 Elsevier Science Ltd

Keywords: cranb erry waste, fungal inoculants, glucosamine co ntent, solid-state ferm entation .

I n t r oduc t i on

A p p r o x i m a t e l y 4 2 0 m i l l i o n p o u n d s o f c r a n b e r r i e s a r e

p r o d u c e d a n n u a l l y i n t h e U S A , a b o u t 9 0 % o f w h i c h

a r e u s e d f o r p r o c e s s i n g p u r p o s e s [ 1 ]. T h e p r i m a r y

b y - p r o d u c t o f t r a d i t i o n a l c r a n b e r r y j u i c e p r o c e s s i n g i s

c r a n b e r r y p o m a c e . I t c o n si s ts o f t h e p r o c e s s e d s k i n s ,

s e e d s a n d s t e m s , a n d c o n s t i t u t e s a b o u t 5 % o f t h e w e t

w e i g h t o f t h e o r i g i n a l f r u it . F r e s h l y p r e s s e d c r a n b e r r y

p o m a c e c o n t a i n s a l a r g e a m o u n t o f i n s o l u b l e c a r b o -

h y d r a t e s w i t h s m a l l a m o u n t s o f p r o t e i n , m i n e r a l s a n d

s o m e r e m a i n i n g j u ic e w i t h s u g a r s a n d o t h e r s o l u bl e

s u b s t a n c e s . O w i n g t o i t s h i g h m o i s t u r e c o n t e n t , f r e s h l y

p r e s s e d c r a n b e r r y p o m a c e i s s u s c e p t i b l e t o r a p i d

m i c r o b i a l g r o w t h . L i k e o t h e r f r u i t p r o c e s s i n g w a s t e s ,

s u c h a s a p p l e p o m a c e [ 2 ] , g r a p e p o m a c e [ 3 ] , t o m a t o

pomace [4 ] , c i t ru s was t e [5 ] , p i neapp l e was t e [6 ] ,

*To whom correspondence should be addressed. Tel . : (+1)413 545 10 22 ; fa x : (+1) 413 545 12 62 ; e-mail :kalidas@foodsci .umass.edu

323

o r a n g e w a s t e [ 7 ] , s u g a r - c a n e p r e s s m u d [ 8 ] , a n d k i w i -

f r u i t p e e l w a s t e [ 9] , c r a n b e r r y p r o c e s s i n g w a s t e i s c o m -

m o n l y u s e d a s a n i m a l f e e d o r f e r t i l i z e r . H o w e v e r , i t s

v a l u e a s a n i m a l f e e d i s v e r y l i m i t e d b e c a u s e o f i t s l o w

p r o t e i n c o n t e n t a n d i t s u s e a s f e r t i l i z e r m a y n o t b e

e c o n o m i c a l l y c o m p e t i ti v e . F u r t h e r , d i r e c t d is p o s a l o f

pomace was t e t o so i l o r i n a l and f i l l poses s i gn i f i can t

e n v i r o n m e n t a l p r o b l e m s . T h u s , t h e e x p l o r a t i o n o f

n o v e l u s e s f o r c r a n b e r r y w a s t e i s n e e d e d .

B i o l o g i c a l c o n v e r s i o n o f f r u i t p r o c e s s i n g w a s t e s i n t o

v a r i o u s v a l u e - a d d e d p r o d u c t s t h r o u g h s o l i d - s t a t e f e r -

m e n t a t i o n ( S S F ) h a s b e e n o f m a j o r i n t e re s t t o m a n y

l a b o r a t o r i e s a r o u n d t h e w o r l d . S S F d e a l s w i t h t h e u t i l i -

z a t i o n o f w a t e r - i n s o l u b l e m a t e r i a l s f o r m i c r o b i a l

g r o w t h a n d m e t a b o l i s m , a n d i t is u s u a ll y c a r r i e d o u t i n

s o l id o r s e m i - s o l i d s y s te m s i n t h e n e a r a b s e n c e o f f r e e

w a t e r o r r e d u c e d w a t e r c o n t e n t c o m p a r e d w i t h s u b -

m e r g e d f e r m e n t a t i o n [ 1 0 ] . M a n y o f t h e p o t e n t i a l

p r o d u c t s f r o m f r u i t p o m a c e w a s t e s h a v e b e e nd e v e l o p e d u s i n g t h e S S F t e c h n i q u e , a n d s u c h p r o d u c t s

i nc l ude e t hano l [11 ] , met hane [7 ] , l ac t i c ac i d [8 ] , c i t r i c



324 Bioconversion of cranberry processing waste

acid [6], protein [5], mushrooms [12], enzyme [13], and

fo o d i n g re d i en t [ 3 ] . H o w e v e r , n o r e p o r t h a s b e e n

foun d dea l ing wi th the u t i l iza t ion o f c ranberry p ro -

cess ing was te .

Trichoderma i s no tab ly capab le o f p roducing var ious

p o l y s a c c h a r i d e -d e g ra d i n g e n z y m e s w h i c h e n a b l e i t t o

g ro w o n c r a n b e r ry p o m a c e s u b s t r a te . I t h a s b e e n

re p o r t e d t h a t s o m e Trichoderma spec ies a re wide ly

used to p roduce var ious indus t r i a l ly impor tan t enzymes

[14-16] . Trichoderma spec ies can a l so be e f fec t ive as

b io log ica l con t ro l agen ts aga ins t pa thogen ic o rgan isms

which usua l ly cause many p lan t roo t d i seases [17 ] . Tr / -

c h o d e r ma h a r z i a n u m a l so has the ab i l i ty to degrade

organoch lo r ine pes t i c ides , such as DDT, d ie ld r in ,

e n d o s u l f a n , p e n t a c h l o ro n i t ro b e n z e n e , a n d p e n t a c h l o r -

opheno l , and hence has po ten t ia l app l ica t ions fo r b io -

remed ia t ion [18 ] .

Severa l fungal spec ies be long ing to the genus Rhi -

z o p u s have been used in SSF fo r severa l cen tu r ies ,e s p e c i a l l y i n A s i a fo r p r e p a r i n g m a n y f e rm e n t e d fo o d -

stuffs . R h i z o p u s no t on ly enhances the d iges t ib i l i ty and

pro te in c on ten t o f foods tu f fs , bu t a l so p reven ts the

fo rmat ion o f tox ic subs tances such as a f la tox in B1 .

S o m e R h i z o p u s spec ies can a l so p roduce an t i -carc ino -

genic substances and ant ibiot ics [19,20]. A s train of

Rhizopus oligosporus, w h i c h w a s i s o l a t e d f ro m c o m m e r -

c ia l Tempeh in ou r l abora to ry , was used in th i s s tudy in

o rd e r t o d e v e l o p a p o t e n t i a l p ro t e i n - e n h a n c e d v a l u e -

a d d e d p ro d u c t f r o m c r a n b e r ry p o m a c e fo r u s e a s

an imal feed .

An o the r fun gus used in th i s s tudy is a novel Pen-icillium i so la te wh ich i s capab le o f deco lo r iz ing the

po lymer ic dyes Po ly R-478 and Po ly S-119 in l iqu id

med ia [21 ] . Th is i so la te has po ten t ia l app l ica t ions in

b i o r e m e d i a t i o n o f a ro m a t i c p o l l u t a n t s s i n ce i t c o u ld b e

u s e d t o r e m o v e s o m e d y e s t u ff s f r o m d y e -c o n t a m i n a t e d

water or soi l .

F i sh o f fa l i s a majo r f i shery by -p roduct wh ich i s

u s u a l l y d i s p o s e d o f o n l a n d o r o f f s h o re a s w a s t e e v e ry

year. Since i t has a h igh ni t rogen content [22], i ts acid

h y d ro l y s a t e c o u l d b e s u p p l e m e n t e d t o c r a n b e r ry

p o m a c e m e d i u m t o e n r i c h t h e o rg a n i c n i t r o g e n fo r

fungal g rowth . The ob jec t ive o f th is research w as to

d e v e l o p n o v e l a p p ro a c h e s t o u t i l i z e c r a n b e r ry p o m a c e ,coup led wi th u t i l i za t ion o f f i shery was te , to genera te

va lue-added p roduct s , l ike microb ia l inocu lan t s , u s ing

t h e b e n e f i c i a l f u n g i m e n t i o n e d a b o v e . C ra n b e r ry

p o m a c e c o u l d n o t o n l y s e rv e a s a n e x c e l l e n t c a rb o n

source , bu t in add i t ion i t cou ld be used as an o rgan ic

car r ie r fo r fungal inocu lan t s fo r food , ag r icu l tu ra l and

env i ronmen ta l app l ica t ions .

a t e r ia l s a n d m e t h o d s

Microorgan isms

T. viride IF-26, T. h a r z i a n u m ATCC 24274 , and T. pseu-

dokoningii A TC C 2 6 8 0 1 w e re o b t a i n e d f ro m t h e

A m e r i c a n Ty p e C u l t u r e C o l l e c t i o n (R o c k v i l l e , M D ) ; a

Penicillium sp . ATCC 74414 tha t deco lo r ized po lymer ic

dyes was i so la ted in ou r l a bora to ry [21] ; a s tra in o f

Rhizopus oligosporus w a s i s o l a t e d f ro m u n p a s t e u r i z e d

Te m p e h p ro d u c t . Th e Te m p e h p ro d u c t w a s k i n d l y p ro -

v i d e d b y L i f e -L i f e F o o d s C o . , G re e n f i e ld , M A .

Media and cul t ivat ion condit ion

Th e m i c ro o rg a n i s m s w e re m a i n t a i n e d o n p o t a t o d e x -

t rose agar (PDA) s lan t s and pe t r i p la tes a t 4 °C and

subcu l tu red mon th ly . Al l fung i were cu l tu red a t room

t e m p e ra t u r e fo r 7 d a y s b e fo re u s e . 1 2 5 m l E r l e n m e y e r

f lasks con ta in ing 10 g o f c ranbe rry pom ace , 0 .5 g o f

CaCO 3, 20 ml water , and 0 5 g o f NH aNO 3 o r 2 ml f ish

p ro t e i n h y d ro l y s a t e (F P H ) a s t h e s u p p l e m e n t a l

n i t rogen source were used fo r SSF. The f resh ly p ressed

cranberry pomace was ob ta ined f rom Very f ine , Inc . ,

W e s t fo rd , M A , a n d w a s d r i e d a n d g ro u n d a n d s t o r e din a re f r igera to r b efo r e use . The wate r con ten t o f c ran -

berry pomace used in the exper imen t was 5 .8% (w/w,

w e t b a s i s ) . F P H w a s o b t a i n e d f ro m O c e a n C re s t

(Glouces te r , MA) as her r ing was te con ta in ing

0 .6575 g m1-1 o f so lub le so l ids . The spores f rom one

PDA p la te were inocu la ted in to abou t 20 f l asks . The

f lasks were incubated a t 25°C fo r 4 days . The cu l tiva-

t ion o f a l l fung i was a l so ex t rapo la ted fo r 100 g o f

c r a n b e r ry p o m a c e w i t h p ro p o r t i o n a l a d d i t i on o f o t h e r

supp lem en ts ca lcu la ted f ro m the 10 g l eve l.

Protein assay

100 ml o f d i s t il l ed wa ter was ad ded in to the f ung us -

pomace-con ta in ing f l asks and the cu l tu re was homoge-

n ized us ing a War ing b lender , then cen t r i fuged a t

1500g fo r 15 min . The supern a tan t was used fo r p ro te in

assay . So lub le p ro te in was de te rmined us ing a commer-

c ia l as say k i t (B io -R ad Pro te in Assay Ki t I I , B io -Ra d

La b o ra t o ry , H e rc u l e s , C A ) w i t h b o v i n e s e ru m a l b u m i n

a s s t a n d a rd , a c c o rd i n g t o t h e p ro c e d u re d e s c r i b e d b y

Bra dfo rd [23] . The so lub le p ro te in p rod uce d by fungal

s t ra i n s i n t h e c r a n b e r ry p o m a c e m e d i u m w a s e x p re s se d

as mi l l ig rams per g ra m o f pom ace (o r ig ina l d ry

weigh t ) .

Mo isture content M C) and wa ter act ivi ty determination

T h e M C o f cr a n b e rr y p o m a c e m e d i u m w a s d e t e r m i n e d

b y m e a s u r in g b o t h t h e w e t w e i g h t a n d d ry w e i g h t o f

the sample . Af te r measur ing the wet weigh t , the

samp le was d r ied in an ov en a t 105°C fo r 2 days , o r

un t i l the weigh t was cons tan t , befo re reco rd ing the d ry

w e i g h t. Th e w a t e r a c ti v it y a w o f th e c r a n b e r ry p o m a c e

m e d i u m w a s d e t e rm i n e d a c c o rd i n g t o t h e m e t h o d

d e s c r i b e d b y M c C u n e et al. [24 ] . A re ference mater ia l

(c i rc le o f f i l t e r paper) o f known so rp t ion i so therm wasob ta in ed by equ i l ib ra t ing fo r 24 h to each o f s ix sa lt

s lu shes and was equ i l ib ra ted fo r 24 h to the sample ; the



Z. Zheng and K. Shetty 325

water activity of the equilibrated filter paper was cal-

culated using the linear equation of the isotherm curve

from its MC, which was determined by measuring the

weight gain of the filter paper during equilibration. The

water activity of the sample was equal to that of the

equilibrated filter paper. The relationship between MC

(wet basis) and aw of the sample was expressed by the

equation

MC = A + B log (1 - a w

where, at 25°C,A = 0.0033 and B = -0.1155.

Glucosamine assay

The glucosamine content of the fermented culture

mixture consisting of fungal mycelia and cranberry

pomace medium was used to estimate fungal biomass

during the SSF as the growth indicator of Trichoderma,

Rhizopus , and Penicillium strains. It was determined bythe modified method of Sakurai et al. [25]. After culti-

vation, the culture mixture in each flask was mixed with

100ml of distilled water. The mixture was then

homogenized using a Waring blender. A 1 ml suspen-

sion of homogen ized sample was mixed with 2 ml of

H2504 (98 ) in a test tube. After standing for 24 h at

25°C, it was diluted with 47 ml of water and autoclaved

at 120°C for 1 h. The hydrolysate was then neutra lized

with NaOH to pH7-0 and diluted to 100ml, from

which 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 at1500g for 2 min. 0.6 ml of supernatant was then mixed

with 0 2 ml of NHaSO3NH2 (12 5%) and shaken for

3 min. To the mixture, 0.2 ml of 3-methyl-2-benzothia-

zolinone hydrazone hydrochloride (MBTH, 0.5%, pre-

pared daily) was added and then the mixture was

boiled for 3 min. The reaction mixture was immediately

cooled to room temperature following boiling and

0.2 ml of FeCI3 (0 5%, prepared within 3 days) was

added. After standing for 30min, the absorbance at

650nm was measured spectrophotometrically. The

glucosamine content was calculated as milligrams per

gram of pomace (original dry weight) according to the

standard curve.

e s u l t s a n d d i s c u s s i o n

The effect of Ca C0 3 supplementation on the growth of

selected fungi

Since the cranberry pomace contains organic acids, the

pH of cranberry-pomace-based medium was relatively

low (approximately 3.0 to 3-2) and hence inhibited the

growth of fungi. In order to obtain the maximal

growth, it was necessary, therefore, to neutralize the

medium before inoculation. In general, most fungi areable to grow in a pH range of neutral to slightly acidic.

Considering the desired pH range, CaCO3 was con-

8 .

0

i i i i i i i

1 2 3 4 5 6 7 8 9 1

CaC03 g/ lOOg pomace)

Fig. l. The effect of CaCO3addition on the growth of Tricho-derma, Rhizopus and Penicillium strains on cranberry pomace.

sidered as an ideal neutralizer because it can increase

the pH of cranberry pomace medium to 5.8-7.0 and it

is inexpensive for large-scale use. Again, Trichoderma

viride , Rhizopus isolate, and Penicillium G-1 strains

were used to examine the effect of CaCO3 addition in

cranberry pomace on fungal growth using 2 mlg

water and 0 .05 g NH4NO3. It was demonstra ted that

cranberry pomace medium supplemented with

0.04-0.05 g of CaCO3 per gram of pomace would be

satisfactory for the growth of all three selected fungi

(Fig. 1).

The effect o f water addit ion in cranberry pom ace on the

growth of se lected fungi

The moisture level of the medium is a critical factor

influencing the growth of fungi in SSF. In general, a

higher moisture level results in decreased porosity or

intracellular spaces, lower oxygen diffusion and gas

exchange and enhanced formation of aerial mycelium.

In contrast, a low moisture level will lead to decreased

substrate swelling and decreased microbial growth. In

this study, partially dried and ground cranberry pomace

with an MC of 5 8% (w/w) was used. Since it had very

low water content, additional water was required toincrease the moisture level of the medium. To deter-

mine the optimal amount of water for maximum

growth, the growth of T. viride, T. pseud okonin gii, T.

harzianum, Penicil l ium G-l, and the Tempeh Rhizopus

isolate were examined using the basal cranberry

pomace medium with inorganic nitrogen as described

in the Material and methods section with varying water

contents. The soluble protein produced by various

fungi in the pomace medium was measured as the indi-

cator of growth (Fig. 2). All fungi tested in the experi-

ment grew very poorly if no additional water was

added to the medium; they exhibited very similargrowth patterns as the amount of water supplemented

to the medium increased up to 4mlg -~ before



326 Bioconve rsion of cranberry processing waste

0

0

¢ / / . / o - *- r . ~,, ,~o,,m

l

100 200 300 400 500

E

g

a 1

H20 ml/100g pomace)

Fig. 2. The effect of water addition in cranberry pomacemedium on the growth of Trichoderma Penicillium and Rhi-zopus strains.

declining slightly at higher water addition

(4.5-5-0 ml g-I). Addition of about 2 ml of water per

gram of cranberry pomace was sufficient for the

maximum growth of all selected fungal strains.

The relat ionship between M C a nd w ater activity o f

cranberry pom ace med ium

A better way of expressing the MC of cranberry

pomace medium is the water activity aw, which indi-

cates the availability of water for the growth of fungi.

The MC and water activity of the cranberry pomace

medium corresponding to each level of water supple-

mented to the medium were determined using the

methods described in the Materials and methods

section. It appeared that the optimal MC in the

pomace for the growth of all selected fungi was about

67 (wet basis), whereas the corresponding optimal

water activity was 0.99 (Table 1).

Bioma ss es t ima t ion

In SSFs, one of the most important problems encoun-

tered is the biomass measurement. Unlike submergedcultivation, fungal mycelia are intimately bound to the

Table 1. The effect of water addition on the MC and aw ofcranberry pomace medium

Water addition

to the medium(ml/g pomace)

MC in the pomace(%, wet basis)

w

0 5 79 0-46431 51 49 0-97472 67 34 0 99043 75-38 0 9938

4 80-05 0-99455 83 50 0-9960

solid matrix and cannot be quantitatively separated

from it; so, direct measurement of fungal biomass is

impossible. Many authors have described methods of

indirect biomass estimation including measuring: (a)

fungal cell constituents, such as ergosterol, nucleic

acids, protein, nitrogen and chitin; (b) primary metabo-

lites, such as CO2, ATP, or enzymatic activity; (3)

nutrient consumption [26, 27].

As glucosamine is an essential and stable compo-

nent in chitin of mycelial cell walls, the glucosamine

content seems to be a useful parameter for the estima-

tion of the total sum of the growing mycelium and its

changes may correspond to the development of the

mycelium, although the values cannot be converted to

mycelial weight quantitatively [25]. Roche et al. [27]

reported that a linear correlation existed between

cumulative biomass and cumulative glucosamine of fila-

mentous fungi during SSF. Desgranges et al. [26] also

suggested that the glucosamine measurement gave agood indication of fungal biomass development, but

the biomass indicator could only be used to compare

different media having the same constituents, even if

the C/N ratios were different.

In our study, two methods for biomass estimation

were compared when additional nitrogen source was

added to the medium. In the case when NH4NO3 was

supplemented as a nitrogen source, as shown in Figs 3

and 4, the measurement of soluble protein content was

consistent with the measurement of glucosamine

content, as both gave a similar growth pattern. Under

such conditions, therefore, either soluble protein or

glucosamine content could be used as the growth indi-

cator of the selected strains in SSF.

The e f fec t o f NH 4N 03 on the growth o f selec ted fung i

An important indicator of nutritional regulation of

growth in SSF is the C/N ratio. The optimal C/N ratio

Eg

J

n

i . - . , - -~

1

0 2 4 6 8 10

NI-14NOa g/100g Ix)mace)

Fig. 3. The effect of NH4NO3 addition on the growth ofTrichoderma Rhizopus and Penicillium strains on cranberrypomace.



Z. Zheng and K. Shetty 327

10

E8

E

(.9/ --X--R/~o/x~

- - - l . -Pe t~

2 4 6 8 10

NH4NOa (g/100g pomace)

Fig. 4. The effect of NH4NO suppleme ntat ion on the glucos-amine product ion by Trichoderma Rhizopus and Penicilliumstrains grown o n cranb erry pomace.

v a r i e s i n a w i d e r a n g e f r o m 1 0 t o 1 0 0 o r h i g h e r i n

v a r i o u s S S F p r o c e s s e s , b u t t h e a v a i l a bi l it y o f C a n d N

c a n b e m o r e i m p o r t a n t t h a n t h e r a t i o . I n m o s t S S F

s y s t e m s , t h e c a r b o n s o u r c e c o m e s f r o m t h e n a t u r a l

s o l u b l e a n d i n s o l u b l e c a r b o h y d r a t e s w h i l e t h e n i t r o g e n

s o u r c e i s a d d e d . C r a n b e r r y p o m a c e c o n t a i n s h i g h e r

c a r b o h y d r a t e s ( ~ 5 0 o n a d r y w e i g h t b a s is ) , s o m e o f

w h i c h a r e f e r m e n t a b l e a n d c o u l d b e u s e d a s a c a r b o n

s o u r c e , b u t a r e l a t i v e l y l o w c o n t e n t o f n i t r o g e n s o u r c e

a v a i l a b le f o r f u n g i b e c a m e t h e l i m i t in g f a c t o r f o r f u n g a lg r o w t h . T h e r e f o r e , i n o r d e r t o o b t a i n t h e o p t i m a l

g r o w t h o f fu n g i o n c r a n b e r r y p o m a c e , s u p p l e m e n t a t i o n

o f n i t r o g e n s o u r c e w a s n e e d e d . T w o k i n d s o f n i t r o g e n

s o u r c e , N H a N O 3 a n d F P H , w e r e u s e d i n t h i s s t u d y .

T h e g r o w t h o f T . v ir ide Rhizopus i so l a t e , and Peni-

cillium G - 1 w a s d e t e r m i n e d b y m e a s u r i n g b o t h t h e

s o l u b l e p r o t e i n c o n t e n t a n d g l u c o s a m i n e c o n t e n t o f

f e r m e n t e d c u l t u r e . T h e e f f e c t o f N H 4 N O 3 o n t h e

g r o w t h o f t h e t h r e e s e l e c t e d f u n g i i s s h o w n i n F ig . 3 ( i n

t e r m s o f s o l u b l e p r o t e i n p r o d u c t i o n ) a n d F i g . 4 ( i n

t e r m s o f g l u c o s a m i n e c o n t e n t ) . A d d i t i o n o f a b o u t

0 - 0 5 g o f N H 4 N O 3 p e r g r a m o f p o m a c e g a v e th e

m a x i m a l g r o w t h o f a l l s e l e c t e d f u n g a l s p e c i e s ( F i g s 3

and 4 ) .

The effect o f FP H on the growth o f selected fung i

F i s h o f f a l i s a n o t h e r i m p o r t a n t f o o d p r o c e s s i n g w a s t e ,

a n d i t s d i s p o s a l h a s n o t y e t b e e n s o l v e d s a t i s f a c t o r i l y

[ 2 2 ] . B y a c o m b i n a t i o n o f p a p a i n a n d a c i d h y d r o l y s i s ,

t h e f i s h er y b y -p r o d u c t w a s c o n v e r t e d i n t o F P H i n

w h i c h a h i g h c o n c e n t r a t i o n o f n u t r i e n t s , s u c h a s

n i t ro g e n , w a s e x p e c t e d t o e n h a n c e t h e f u n g a l g ro w t h

o n c r a n b e r r y p o m a c e m e d i u m . M a r t i n a n d C h i n t a l a p a t i[ 2 2 ] d e m o n s t r a t e d t h a t t h e g r o w t h o f Scytalidium acid-

ophi lum o n a c i d p e a t h y d r o l y s a t e w a s e n h a n c e d w h e n i t

g r e w i n f is h o f f a l - p e a t c o m p o s t . W e p r o p o s e d t h a t

F P H c o u l d b e a n a l t e r n a t i v e n i t r o g e n s o u r c e f o r c r a n -

b e r r y p o m a c e m e d i u m e n r ic h m e n t .

W e t e s t e d t h e e f f e c t o f s u p p l e m e n t a t i o n o f h e r r i n g

F P H o n t h e g r o w t h o f Trichoderma viride Rhizopus

i so l a t e , and Penicillium G - 1 s t r a i n s o n c r a n b e r r y

p o m a c e m e d i u m . F u n g a l g r o w t h w a s e x p e c t e d t o b e

e x p r e s s e d i n t e r m s o f b o t h s o l u b l e p r o t e i n a n d g l u c o s -

a m i n e c o n t e n t s . H o w e v e r , i t w a s p r o b l e m a t i c t o u s e

s o l u b l e p r o t e i n c o n t e n t a s t h e g r o w t h i n d i c a t o r b e c a u s e

t h e p a r t i a l l y h y d r o l y z e d p r o d u c t s o f f is h p r o t e i n , s u c h

a s p o l y p e p t i d e s i n h e r r i n g F P H s u p p l e m e n t e d i n t h e

m e d i u m , c o n t r i b u t e d t o t h e t o t a l s o l u b l e p r o t e i n

c o n t e n t o f t h e f e r m e n t e d m e d i u m . T h i s w o u l d e x p l a i n

t h e m i s l e a d i n g a n d c o n f u s i n g r e s u l t t h a t t h e s o l u b l e

p r o t e i n w a s c o n s t a n t l y i n c r e a s i n g a s t h e l e v e l o f F P H

s u p p l e m e n t e d t o t h e m e d i u m i n c r e a s e d ( d a t a n o t

s h o w n ) . I n t h i s c a s e , t h e r e f o r e , s o l u b l e p r o t e i n c o n t e n t

w a s n o t a r e l i a b l e f u n g a l g r o w t h i n d i c a t o r a s i t t e n d e dt o g i ve a n e r r o n e o u s r e s u l t. I t c o u l d b e u s e d a s a f u n g a l

g r o w t h i n d i c a t o r o n l y if t h e m e d i u m c o n t a i n e d a n i g n-

o r a b l e a m o u n t o f s o l u b l e p r o t e i n s . I n s t e a d , i t w a s m o r e

r e l i a b l e t o u s e t h e g l u c o s a m i n e c o n t e n t a s t h e g r o w t h

i n d i c a t o r . I t i s s h o w n i n F i g . 5 t h a t t h e a d d i t i o n o f

a b o u t 0 . 2 - 0 . 3 m l o f F P H p e r g r a m o f cr a n b e rr y

p o m a c e r e s u l t e d i n t h e o p t i m a l g r o w t h o f a l l s e l e c t e d

fung i .

The growth o f all tested fungi o n a n optimized cranberry

p o m a c e m e d i u m

W i t h t h e g o a l o f o b t a i n i n g a g e n e r a l c r a n b e r r y - b a s e d

m e d i u m f o r g r o w t h o f s e l e c t e d f u n g i , a n o p t i m i z e d

m e d i u m f o r m u l a w a s d e v e l o p e d a s d e s c r ib e d i n t h e

M a t e r i a l a n d m e t h o d s s e c t io n . T h e o p t i m i z a t i o n o f t h e

m e d i u m w a s b a s e d o n t h e h y p o t h e s i s t h a t a l l t h e f u n g i

s e l e c t e d h a d s i m i l a r g r o w t h p a t t e r n s i n r e s p o n s e t o

E 6g

E

o 2J . X ~

I0 20 30 40 50

FPH ml/100g pomace)

Fig. 5 . The effect of FPH addi t ion on glucosamine produc-tion by Trichoderma Rhizopus and Penicillium strains o n cran-berry pomace.



Z Zheng and K Shetty 329

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producción hongos en pomaza de cranberry

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