production of fruit wines using novel enzyme …

PRODUCTION OF FRUIT WINES USING NOVEL ENZYME PREPARATIONS

Anastasia VOLCHOK1*, Alexandra ROZHKOVA1, Ivan ZOROV1,2, Sergey SHCHERBAKOV3 and Arkady SINITSYN1,2

1 : The A.N. Bach Institute for Biochemistry, Russ. Acad. Sci., 119071, Leninsky prospect 33/26 Moscow, Russia2 : The Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119991, Leninskie Gory, 1/3,

Moscow, Russia3 : The Russian State Agrarian University, Timiryazev Moscow Agric. Acad., 127550, Timiryazevskaya 49,

Moscow, Russia

*Corresponding author : [email protected]

Aim : This work describes the activities of new-generationenzymatic preparations in fruit-berry substrates engineeredfor use in the fruit-wine industry. The enzymes wereproduced after genetic modification and selection of fungiPenicillium verruculosum, which produce efficientcellulase and pectinase enzymatic complexes.

Methods and results : This paper covers the maincharacteristics of novel multi-enzyme complexes and theresults of in-lab fruit-wine production with addition ofenzymatic preparations, which could be used on anindustrial scale. The juice yield and the content ofsuspended materials in the enzymatically treated sampleswere compared. Experiments included the sensory analysisof produced juices and fruit wines.

Conclusion : Results show a significant increase in juiceyield from the fruit pulp processed with the enzymaticpreparations, without any negative effect on the quality andorganoleptic attributes of the final product.

Significance and impact of the study : The obtained dataclearly show that the use of the new-generation enzymaticpreparations in the fruit-wine industry is effective.

Key words : enzyme preparations, enzymatic treatment,Penicillium verruculosum, fruit wines, multi-enzymecomplex, fruit-wine industry

Objectif : Ce travail décrit les activités de la nouvellegénération de préparations enzymatiques sur substrats defruits/baies développée pour l’application dans l’industriedu vin de fruits. Ces enzymes ont été produites après lamodification génétique et la sélection de champignonsPenicillium verruculosum, source d’efficaces complexesenzymatiques de cellulase et de pectinase.

Méthodes et résultats : Cet article présente les principalescaractéristiques des nouveaux complexes multi-enzymatiques et les résultats, après application depréparations enzymatiques, de la fabrication de vins defruits en laboratoire, production qui pourrait se décliner àune échelle industrielle. Les rendements en jus et lesmatières en suspension contenues dans les échantillonsenzymatiques traités ont été comparés. Les paramètresphysico-chimiques et les caractéristiques d’intensité decouleur de la boisson finale ont été enregistrés.

Conclusion : Les résultats montrent une augmentationsignificative du rendement de jus extrait de la pulpe de fruittraitée avec des préparations enzymatiques sans influencenégative sur les aspects qualitatifs et les propriétésorganoleptiques du produit final.

Signification et impact de l’étude : Les données obtenuesmontrent clairement la haute efficacité de l’utilisation de lanouvelle génération de préparations enzymatiques dansl’industrie du vin de fruits.

Mots clés : préparations d’enzymes, traitementenzymatique, Penicillium verruculosum, vins de fruits,complexe de multi-enzymes, industrie du vin de fruits

Abstract Résumé

manuscript received 4th September 2014 - revised manuscript received 18th August 2015

J. Int. Sci. Vigne Vin, 2015, 49, 205-215©Vigne et Vin Publications Internationales (Bordeaux, France)- 205 -

06_vochok_05b-tomazic 25/09/15 17:41 Page205

- 206 -J. Int. Sci. Vigne Vin, 2015, 49, 205-215©Vigne et Vin Publications Internationales (Bordeaux, France)

Anastasia VOLCHOKet al.

INTRODUCTION

This paper addresses an important technologicalproblem of the fruit-wine industry (CodexAlimentarius : wine made from fruit other thangrapes) : how to increase juice yield from rawmaterial without compromising the quality of thefinal product. Fruit-wine consumption is significantlylower compared to traditional grape wines; however,in countries such as Great Britain, Poland or Russia,this type of beverage is well known and appreciated(Noller and Wilson, 2009; Kiselev et al., 2013). Avast range of raw materials can be used for fruit-wineproduction : apple, pear, pineapple, guava, kiwi(Soufleros et al., 2001), Chinese lychee, orange,cherry, cranberry, mango, passion fruit, papaya,peach, etc. Selection of the raw material is mainlydetermined by traditional recipes existing in thecountry of origin.

The fruit-wine technology is characterized by thespecificity of the raw materials, which vary in theirchemical content and requirements for differentprocessing conditions. The production of such typesof wine is often confronted with numerous problemssuch as low juice yield, difficulties with pressing,slow juice clarification, clouding and color changesin the final product (Volchok et al., 2013).

Currently, preprocessing of fruits and berries withvarious enzymes prior to pressing and filtration isconsidered to be the most effective solution to thesetechnological problems (Jayani et al., 2005 ; LiewAbdullah et al., 2007), ensuring better fibermaceration and juice clarification, prevention ofcolloidal hazes, and achievement of balanced anddiverse flavors (Ageeva and Markosov, 2013).Selection of enzymes is based on their activitiesrequired for a particular fruit or berry.

The Enzyme Biotechnology Laboratory of the BachInstitute for Biochemistry, Russ. Acad. Sci. (INBIRAS) is developing new superior enzymes andenzymatic complexes with several activities at ratiosallowing the efficient processing of various rawmaterials. This article describes the processing ofseveral fruit substrates, containing cellulose andhemicellulose, with the new multi-enzymatic

complexes BI_3-227.7 and BI_3-227.4, followed bythe lab-scale production of fruit wines. Both enzymepreparations were derived from recombinant strainsof Penicillium verruculosum. These complexes wereselected in relation to earlier experiments processingfresh viburnum and strawberry juice yield from thepulp and larger content of reducing sugarspreliminaryprocessing experiments. The main criteria were yieldincreasing of juice and larger content of reducingsugars (Volchok et al., 2013). The results of theorganoleptic analysis and the comparisons ofenzyme-processed juices and wines against the non-processed samples are presented to acknowledge theefficiency of the proposed method and multi-enzymatic complexes.

MATERIALS AND METHODS

1. Substrates

Ash berry, plum and black currant provided by theRussian State Agrarian University named after K.A.Timiryazev were used as raw fruit and berry material.Table 1 shows the dates of harvest and sampling.

2. Enzyme preparations

Multi-enzyme preparations were obtained bycotransformation of the auxotrophic host strain P.verruculosum 537. Expression plasmid encoding P.canescens pectin lyase (PELA) and Aspergillus nigerβ-glucosidase (BG), and transforming plasmid pSTA10 were used in transformation experiments. Detailsof the developed process for the recombinant strainsand enzyme preparations are described in Bushina etal. (2012). Preparations are in the form of a lightbrown powder (easily soluble in water) obtained bylyophilization of culture filtrates (micro-filtrated andconcentrated by ultrafiltration method) afterfermentation of recombinant P. verruculosum strains,and they show stable, high enzymatic activity in therange of 25-50ºС for temperature and 4.0-5.0 for pH.

Enzyme complexes contained pectin lyase A,cellobiohydrolase, endo-1,4-glucanase and β-glycosidase. Earlier activity of enzymes was tested onapple, citrus and beet pectin (Morozova et al., 2010;Bushina et al., 2012). Their main enzymatic activitiesare presented in Table 2.

Table 1 - Harvest and sampling dates of raw fruit and berry material

Materials Harvest dates Sampling dates

Ash berry 25/09/14 27/09/14

Yellow plum 20/08/14 23/08/14

Black currant 30/07/14 02/08/14


Enzymatic activities towards polysaccharidesubstrates were determined from the initial rates offormation of reducing sugars by the Somogyi–Nelsonmethod (Nelson, 1944 ; Somogyi, 1952). Activitiesagainst p-NP-derived substrates were determined atpH 5.0 and 40ºC by measuring p-nitrophenol release,as described elsewhere (Gusakov et al., 2005). Allactivities are expressed as international units per mgprotein (U/mg) (one unit corresponds to thehydrolysis of 1 μmol of glycoside bonds from thesubstrate per minute). Methods of determination ofenzymatic activities are described in detail in Bushinaet al. (2012).

3. Scheme for producing wines

Fruit-wine materials were produced in the lab usingthe methodology presented in Table 3.

4. Analytical methods

During the experiments, juice yield, viscosity andsuspension content in fermented samples werecompared.

Characteristics of fruit juices

Suspension content of fruit must was evaluatedgravimetrically by centrifugation. 10-cm3 sampleswere put in pre-measured sedimentation tubes andcentrifuged for 10 min at 3,000 rpm. Supernatant wasremoved, leaving the tubes upside down for 1 min.Sediment content was calculated by the equation:

С= (m2-m1)*100/V, where

m2 – mass of sedimentation tube with sediment, g;m1 – mass of empty sedimentation tube, g; andV – sample volume, cm3.

For determination of relative viscosity, samples werecentrifuged for 10 min at 8,000 rpm. Then 5 cm3 ofliquid was incubated in an Ostwald viscometer for5 min at 20ºC (Ashapkin et al., 2005). Relativeviscosity was calculated by the equation:

η=Тi/Т0, where

Тi – flow time of selected sample, sec; andТ0 – flow time of water, sec.

5. Sensory analysis of fruit juices and wines

Ten people were recruited for participating in thesensory analysis of produced juices and fruit wines.The range of descriptors and reference termsallowing the complete organoleptic description of thejuices and wines was selected previously (Baxter etal., 2005). 10-scale evaluation maps were developedusing the following key terms : “weak”, “little” or“absent” for the left anchors and “strong” or “much”for the right anchorskey words. During the week (5days), panel members participated in trainingsessions to ensure an homogeneous interpretation ofthe terms and correct filling of the score cards(Laboissiere et al., 2007).

3 terms (2 for wine) were selected for color andturbidity, 5 for aroma, and 6 for flavor and aftertaste.Samples were served in 100-mL transparent plasticglasses coded with three-digit codes. Water and

- 207 -J. Int. Sci. Vigne Vin, 2015, 49, 205-215

©Vigne et Vin Publications Internationales (Bordeaux, France)

Table 2 - Characteristics of multi-enzymatic preparations

BI_3-227.4 BI_3-227.7Protein content, mg/g of preparation 854±39,77 503±33,6%RSD 1,873 2,684Cellulase (CMCase) activity, U/g of preparation 3346±388,84 3194±310,6%RSD 4,673 3,911Cellulase (Avicelase) activity, U/g of preparation 170±19,92 174±14,9%RSD 4,736 3,448!-glycosidase activity, U/g of preparation 3999±388,11 395±25,49%RSD 3,93 2,58Pectinlyase activity, U/g of preparation 2694±388,38 1164±113,2%RSD 5,86 3,925Xylanase activity, U/g of preparation 3490±288,28 5310±334,9%RSD 3,321 2,522

!"#$%$&'()Enzyme preparation

* - Sum of cellulase activities is equated to 1.




Mat

eria

lTe

chno

logi

cal s

chem

e w

ithou

t enz

ymat

ic tr

eatm

ent o

f fru

it pu

lpTe

chno

logi

cal s

chem

e w

ith e

nzym

atic

trea

tmen

t of

frui

t pul

p

4. M

acer

atio

n of

frui

t pul

p ca

rrie

d ou

t in

durin

g 24

h a

t 50

º!4.

Mac

erat

ion

of fr

uit p

ulp

carr

ied

out i

n pr

esen

ce o

f enz

yme

prep

arat

ion

#3-

227.

7 (0

,03%

from

pul

p m

ass)

dur

ing

24 h

at 2

5 º!

4. M

acer

atio

n of

frui

t pul

p ca

rrie

d ou

t in

durin

g 24

h a

t 50

º!4.

Mac

erat

ion

of fr

uit p

ulp

carr

ied

out i

n pr

esen

ce o

f enz

yme

prep

arat

ion

#3-

227.

4 (0

,03%

from

pul

p m

ass)

dur

ing

24 h

at 2

5 º!

1. W

ashi

ng a

nd so

rting

of r

aw m

ater

ial

2. C

rush

ing

pulp

, int

rodu

ctio

n of

pul

p w

ater

to o

btai

n tit

rate

d ac

id c

onte

nt 5

g/1

000

mL

3. S

ulfit

atio

n of

frui

t pul

p (K

2S2O

5, «M

egah

im»,

RU

) up

to 8

0 m

g/10

00 m

L salt

5. P

ress

ing

pulp

(lab

orat

ory

mec

hani

cal p

ress

cap

acity

150

0 m

L)A

sh b

erry

dr

y w

ine

6. In

trodu

ctio

n su

gar (

suga

r syr

up 8

0%) u

p to

22

g/10

0 m

L co

ncen

tratio

n, su

lfita

tion

of fr

uit m

ust u

p to

120

mg/

1000

mL

conc

entra

tion

7. S

edim

enta

tion

(24

h, 4

, 4

ºC),

sepa

ratio

n8.

Mus

t fer

men

tatio

n pr

oces

s up

to 1

,5 g

/100

mL

suga

r at 2

0 º!

. Add

ing

com

mer

cial

yea

st «

Fran

ce u

nive

rsal

» (F

ranc

e) 5

00 g

/625

L, i

ntro

duct

ion

in fr

uit m

ust 0

,6 g

/100

0 m

L (N

H4)H

2PO

4 (S

igm

a, U

SA)

6. In

trodu

ctio

n su

gar u

p to

22

g/10

0 m

L (s

ugar

syru

p 80

%),

sulfi

tatio

n of

frui

t jui

ce u

p to

120

mg/

1000

mL

7. S

edim

enta

tion

(24

h, 4

ºC),

sepa

ratio

n8.

Mus

t fer

men

tatio

n pr

oces

s up

to 1

.5 g

/100

mL

suga

r at 2

0 º!

. Add

ing

com

mer

cial

yea

st «

Fran

ce u

nive

rsal

» (F

ranc

e) 5

00 g

/625

L, i

ntro

duct

ion

in fr

uit m

ust 0

,6 g

/100

0 m

L (N

H4)H

2PO

4 (S

igm

a, U

SA)

9. F

erm

enta

tion

in d

urin

g 15

day

s, 20

ºC

9. In

trodu

ctio

n su

gar (

suga

r syr

up 8

0%) u

p to

15

g/10

0 m

L su

gar c

once

ntra

tion,

mas

h fe

rmen

tatio

n ca

rrie

d ou

t in

durin

g 30

day

s, 20

ºC

10. S

edim

enta

tion

(24

h, 4

ºC),

sepa

ratio

n

11. F

ilter

ing

win

e m

ater

ials

(if n

eces

sary

), st

orag

e in

gla

ss b

ottle

s at 4

º!

10. S

edim

enta

tion

(24

h, 4

ºC),

sepa

ratio

n

11. F

ilter

ing

win

e m

ater

ials

(if n

eces

sary

), st

orag

e in

gla

ss b

ottle

s for

4 º!

Yello

w-p

lum

, bl

ack

curr

ant

swee

t win

e

1. W

ashi

ng a

nd so

rting

of r

aw m

ater

ial

2. C

rush

ing

pulp

, int

rodu

ctio

n of

pul

p to

obt

ain

titr t

itret

aed

acid

con

tent

6,8

g/1

000

mL

3. S

ulfit

atio

n of

frui

t pul

p (K

2S2O

5, «M

egah

im»,

RF)

up

to 8

0 m

g/10

00 m

L

5. P

ress

ing

pulp

(lab

orat

ory

mec

hani

cal p

ress

cap

acity

150

0 m

L )

Table 3 - In lab fruit-wine production schemes


unsalted biscuits were provided for clearing thepalate. Spider web plots were made for graphicalrepresentation of the tasting session results (Duarte etal., 2010).

6. Data analyses

Physical characteristics of the juices were measuredin triplicate for each parameter. Identified Rrelativestandard deviation (RSD %) and confidence intervalwere identified.

Student t-test capability on Microsoft Excel 2003 wasused to determine the significance of the differencesin between attributes (Doerffel, 1990). For dataprocessing, a statistical significance level of P = 0.05was used.

RESULTS AND DISCUSSION

The yield of free-run juice in the course of in-labproduction of fruit wine is presented in Figure 1 (dataobtained were recalculated for 1 ton of pulp – rawweight). It is important to note that due to high aciditythe substrate pulp used for the experiment was dilutedwith water. The must yield from untreated pulp wasused as a control.

It is clearly seen from the figure data that the use ofenzymatic preparations significantly increases theyield of high quality free-run juice.

Table 4 shows the results of relative viscosity for themust produced from different raw materials and itssediment content.

The advantages of the enzymatic treatment of rawplant materials compared to non-processed samplesare lower viscosity (lower biopolymer content -cellulose, hemicellulose, pectin - due to enzymaticdestruction) and lower concentration of sediment inthe fermented must.

Thus, enzyme preparation BI_3-227.4 was chosenfor treatment of black currant and plum due to its β-glucosidase and pectin lyase activities leading to therapid rarefaction bioconversion of pectin substrates.Another enzymatic preparation, BI_3-227.7, waschosen for the ash berry treatment due to its cellulaseand hemicellulose activities. These results wereexpected as the preparation formula, either BI_3-227.4 or BI_3-227.7, correlates to the componentcomposition of the cell wall of these plants.

Table 5 shows the results of the organoleptic analysisof the juices obtained with enzymes and the juicesobtained by pressing after maceration. Participants inthe sensory analysis especially noted more attractivecolor characteristics in the case of the enzyme-processed plum juice compared to the referencesample.

Figure 2 shows significant differences in aroma andappearance characteristics between the enzyme-processed samples and control.

Juices that have undergone enzymatic treatment arecharacterized by lower amount of suspendedparticles and lower turbidity, which facilitates thesubsequent clarification and filtration processes.Figure 3 depicts the differences in taste attributes of



Figure 1 - Volume of free-run juice from 1 ton of pulp, L


the juices. From this spider web plot illustrating theflavor attributes of the compared juices, we can drawthe conclusion that the enzyme used had a minorinfluence on flavor consistency.

Juices produced by traditional maceration arecharacterized by a stronger aroma. It happens asmaceration done without multi-enzyme complexestakes significantly more time compared to enzymatictreatment. At the same time, the richness of the juiceproduced with enzymatic complexes can be correctedby adjusting the fermentation time.

The organoleptic study of the wines produced fromanalyzed juice samples included the determination offruity and floral notes in aroma and main flavorparameters (Table 6).

Provided data show that the use of multi-enzymecomplexes for fruit-wine production has a positiveeffect on appearance and aroma characteristics(especially noticeable in the case of plum juice)without affecting other organoleptic attributes.

Figure 4 depicts the results of the sensory analysis ofproduced fruit wines. In the case of yellow plum,significant positive difference can be observed. Thesample produced with the developed enzymecomplex is characterized by noticeably lower cookedodor and better clarity compared to the reference.

Comparing flavor parameters of enzyme-processedwine and control showed no significant differences(Figure 5).



Relative viscosity %RSD Mass fraction of suspended, g/100 mL %RSD

Enzyme preparation

BI_3-227.7

control 1,855±0,015 0,329 0,935±0,012 2,529

Enzyme preparation

BI_3-227.4

control 2,240±0,01 0,18 1,356±0,028 0,735

Enzyme preparation

BI_3-227.4

control 1,816±0,013 0,303 1,436±0,037 1,85

1,143±0,075 5,094

Tested samples, fresh must

Ash berry1,715±0,013 0,321

0,44Yellow-plum

1,641±0,01 0,254 1,318±0,017

0,726±0,037 0,704

Black currant2,079±0,01 0,194

Table 4 - Physical attributes of fruit must

Figure 2 - Appearance and aroma attributes for simples of juices




Ash

ber

ry

(enz

.pre

p.)

Ash

ber

ry

(con

trol)

Bla

ck c

urra

nt

(enz

.pre

p.)

Bla

ck c

urra

nt

(con

trol)

Yello

w p

lum

(e

nz.p

rep.

)Ye

llow

plu

m

(con

trol)

Appearance

Littl

e: 0

-1M

uch:

8-1

0A

bsen

t: 0-

1M

uch:

9-1

0

Abs

ent:

0-2

Muc

h: 9

-10

Aroma

Wea

k: 0

-1St

rong

: 8-1

0W

eak:

0-3

Stro

ng: 8

-10

Wea

k: 0

-3St

rong

: 8-1

0A

bsen

t: 0-

2St

rong

: 9-1

0A

bsen

t: 0

Stro

ng: 1

0Flavor

Abs

ent:

0-1

Stro

ng: 8

-10

Wea

k: 0

-3St

rong

: 8-1

0W

eak:

0-3

Stro

ng: 8

-10

Abs

ent:

0-2

Stro

ng: 9

-10

Abs

ent:

0St

rong

: 10

Wea

k: 0

-4St

rong

: 7-1

0Consistency

Wea

k: 0

-3St

rong

: 8-1

0

Attr

ibut

esD

efin

ition

sR

efer

ence

sTh

e re

sults

of t

he e

valu

atio

n sa

mpl

es o

f jui

ces (

aver

age)

9,7

9,85

9,53 *

8,63 *

Cha

ract

eris

tic ju

ice

colo

rC

olor

cha

ract

eris

tic o

f jui

ce (f

or a

sh b

erry

– in

tens

e pi

nk, f

or

blac

k cu

rran

t – d

ark

purp

le, f

or p

lum

– li

ght y

ello

w)

8,84

8,93

2,95

**3,

65**

4,35

3 *7,

353 *

Pres

ence

of

susp

ende

d pa

rticl

esPr

esen

ce o

f par

ticle

s fro

m fr

uit p

ulp

4,83

*5,

58*

2,55

**3,

44**

5,75

3 *8,

453 *

Turb

idity

Non

-lim

pid

aspe

ct re

late

d to

the

diffi

culty

of l

ight

pas

sing

th

roug

h ju

ice

4,6*

5,3*

98,

957,

553 *

7,33 *

Nat

ural

Cha

ract

eris

tic a

rom

a fr

om n

atur

al ju

ice

8,69

8,84

7,55

**8,

2**

6,15

5,8

Aci

dA

rom

a re

late

d to

the

pres

ence

of c

hara

cter

istic

org

anic

aci

ds

from

frui

t6,

356,

4

5,12

4,95

4,2

4,05

Swee

tA

rom

a du

e to

the

pres

ence

of s

ucro

se a

nd o

ther

suga

rs fr

om

frui

t 5,

255,

25

0,1

0,1

2,53

*3,

453 *

Coo

ked

Cha

ract

eris

tic a

rom

a fr

om fr

uit s

ubm

itted

to th

erm

al p

roce

ssin

g0,

30,

3

0,5

0,6

11

Ferm

ente

dC

hara

cter

istic

aro

ma

from

frui

t sho

win

g si

gns o

f ear

ly

dete

riora

tion

0,5

0,5

8,5

8,7

7,8

7,6

Nat

ural

Cha

ract

eris

tic fl

avor

from

nat

ural

juic

e9

9,05

8,7

8,95

7,13

*7,

653 *

Aci

dFl

avor

stim

ulat

ed b

y th

e pr

esen

ce o

f cha

ract

eris

tic o

rgan

ic

acid

s fro

m fr

uit

8,22

8,35

5,65

5,3

5,75

5,4

Swee

tFl

avor

stim

ulat

ed b

y th

e pr

esen

ce o

f suc

rose

and

oth

er su

gars

fr

om fr

uit

6,61

6,31

0,4

0,4

2,8

3,8

Coo

ked

Cha

ract

eris

tic fl

avor

from

frui

t sub

mitt

ed to

ther

mal

pro

cess

ing

0,1

0,2

0,1

0,5

11

Ferm

ente

dC

hara

cter

istic

flav

or fr

om fr

uit s

how

ing

sign

s of e

arly

de

terio

ratio

n0,

30,

5

5,3

5,5

3,65

3,25

Ast

ringe

ncy

Har

sh se

nsat

ion

perc

eive

d in

mou

th a

nd to

ngue

cha

ract

eris

tic

of fr

uit

7,32

7,72

9,28

9,3

7,7

7,4

Con

sist

ency

Perc

eptio

n in

mou

th o

f jui

ce d

ilutio

n or

con

cent

ratio

n 7,

758,

15

Table 5 - Fruit juices sensory attributes mean values

*paired parameters a

sh berry ju

ices (*

* for b

lack currant, 3* for y

ellow plum) w

ith t-te

st above the sig

nific

ance level P


In earlier research held in the Enzyme BiotechnologyLaboratory INBI RAS, the organoleptic parametersof the red wine processed with the help of monitoredenzymatic complexes were compared to the wineproduced according to traditional technology.Cabernet Sauvignon and the local grape varietyTsimlyansky Black (provided by the « SARKEL »

department of the « Tsimlyansky wines » company),a local grape variety, served as raw material for thewine. Results obtained during the tasting session aredescribed in Volchok et al. (2014). These data showthat the developed multi-enzymes applied to thegrape-wine industry allow to achieve well balancedwines with rich fruity aromas.



Figure 3 - Flavor and consistency attributes for simples of juices

Figure 4 - Appearance and aroma attributes for simples of fruit-wines


CONCLUSIONS

Samples of fruit wines were produced in-lab,including a maceration stage with the use of newenzymatic preparations consisting of the targetactivities of cellulase, β-glucosidase and pectin lyase.Different ratios of the target activities allowed toapply specific enzymatic agents to a particular type ofraw material. The juice yield was higher compared tothe reference sample. And produced fruit-winesmaterial was were not inferior to the reference samplein quality attributes, showing lower viscosity, lowersediment content and higher color intensity. Lowersediment content was observed in the must treated byenzymatic preparations. Organoleptic analyses ofjuices and fruit wines showed a positive effect ofmulti-enzyme complexes on the sensorycharacteristics of products.

Obtained data clearly show the high efficiency of thenew-generation enzymatic preparations in the fruit-wine industry.

Acknowledgements : This work was supported by FASIE,Russia (grant no. 346 GU1/2013).

REFERENCES

Ageeva N.M., Markosov V.A., 2013. Influence of drugs onthe composition of aromaforming components in thered table wines. Wine-making and Viticulture(Russian), 3, 19-22.

Ashapkin V.V., Kutueva L.I., Zaharova M.G., et al., 2005.Quality Control by Physico-Chemical Methods. 4.Wine and Wine Materials (Russian). DeLi print.

Baxter I.A., Easton K., Schneebeli K., Whitfield F.B.,2005. High pressure processing of Australian navelorange juices : sensory analysis and volatile flavorprofiling. Innovative Food Science and EmergingTechnologies, 6, 372-387.

Bushina E.V., Rozhkova A.M, Zorov I.N., SatrutdinovA.D., Bekkarevich A.O., Koshelev A.V., OkunevO.N., Sinitsyn A.P., 2012. Development of complexenzymatic preparations of pectinases and cellulasesfor processing of sugar beet pulp. AppliedBiochemistry and Microbiology (Russian), 48, 543-549.

Doerffel K., 1990. Statistik in der analytischen Chemie(German). Leipzig, pp. 36-39, 114-125.

Duarte W.F., Dias D.R., Oliveira J.M., Vilanova M.,Teixeira J.A., Almeida у Silva J.B., Schwan R.F.,2010. Raspberry (Rubus idaeus L.) wine : yeastselection, sensory evaluation and instrumentalanalysis of volatile and other compounds. FoodResearch International, 43, 2303-2314.

Gusakov A.V., Sinitsyn A.P., Salanovich T.N.,Bukhtojarov F.E., Markov A.V., Ustinov B.B., vanZeijl C., Punt P., Burlingame A., 2005. Purification,cloning and characterisation of two forms ofthermostable and highly active cellobiohydrolase I(Cel7A) produced by the industrial strain ofChrysosporium lucknowense. Enzyme and MicrobialTechnology, 36, 57-69.



Figure 5 - Flavor and consistency attributes for simples of fruit-wines




Ash

ber

ry

(enz

.pre

p.)

Ash

ber

ry

(con

trol)

Bla

ck c

urra

nt

(enz

.pre

p.)

Bla

ck c

urra

nt

(con

trol)

Yello

w p

lum

(e

nz.p

rep.

)Ye

llow

plu

m

(con

trol)

Appearance

Littl

e: 0

-1M

uch:

8-1

0A

bsen

t: 0-

2M

uch:

9-1

0Aroma

Wea

k: 0

-1St

rong

: 8-1

0W

eak:

0-3

Stro

ng: 8

-10

Wea

k: 0

-3St

rong

: 8-1

0A

bsen

t: 0-

2St

rong

: 9-1

0A

bsen

t: 0-

1St

rong

: 10

Flavor

Abs

ent:

0-1

Stro

ng: 8

-10

Wea

k: 0

-3St

rong

: 8-1

0Fl

avor

stim

ulat

ed b

y th

e W

eak:

0-3

pres

ence

of s

ucro

se a

nd o

ther

suga

rs fr

om fr

uit

Stro

ng: 8

-10

Abs

ent:

0-2

Stro

ng: 9

-10

Abs

ent:

0St

rong

: 10

Wea

k: 0

-4St

rong

: 7-1

0Consistency

Wea

k: 0

-3St

rong

: 8-1

0

Attr

ibut

esD

efin

ition

sR

efer

ence

sTh

e re

sults

of t

he e

valu

atio

n sa

mpl

es o

f fru

it-w

ines

(ave

rage

)

9,5

9,8

93 *7,

93 *C

hara

cter

istic

col

orC

olor

cha

ract

eris

tic o

f win

e (f

or a

sh b

erry

– in

tens

e pi

nk, f

or

blac

k cu

rran

t – d

ark

purp

le, f

or p

lum

– li

ght y

ello

w)

98,

9

9,5

9,2

8,73 *

7,13 *

clar

ityTh

e de

gree

of t

rans

pare

ncy

9*8,

1*

9,5

98,

237,

45Fr

uity

Cha

ract

eris

tic a

rom

a fr

om fr

uit

8,8

8,71

7,7

7,83

5,4

5,5

Sour

Aro

ma

rela

ted

to th

e pr

esen

ce o

f cha

ract

eris

tic o

rgan

ic a

cids

fr

om fr

uit

7,4

7

6,8

7,2

5,13 *

4,33 *

Flor

alFr

esh

flora

l not

es in

the

arom

a6,

15,

6

0,2

0,2

23 *3,

33 *C

ooke

dC

hara

cter

istic

aro

ma

from

frui

t sub

mitt

ed to

ther

mal

pr

oces

sing

0,5

0,5

11

1,5

1,5

Ferm

enta

tion

The

arom

a of

the

pres

ence

of y

east

0,5

0,5

8,75

8,7

7,83 *

6,93 *

Nat

ural

Cha

ract

eris

tic fl

avor

from

nat

ural

frui

t9,

59,

3

5,5

5,6

Aci

dFl

avor

stim

ulat

ed b

y th

e pr

esen

ce o

f cha

ract

eris

tic o

rgan

ic

acid

s fro

m fr

uit

7,8

7,7

swee

t6,

66,

256,

1

9,1

8,8

5,9

6,2

6,5

cook

edC

hara

cter

istic

flav

or fr

om fr

uit s

ubm

itted

to th

erm

al

proc

essi

ng0,

30,

20,

50,

53,

13 *4,

23 *

ferm

enta

tion

The

arom

a of

the

pres

ence

of y

east

0,3

0,3

0,2

0,2

0,5

0,5

4,5

4,3

2,1

1,8

astri

ngen

cyH

arsh

sens

atio

n pe

rcei

ved

in m

outh

and

tong

ue c

hara

cter

istic

of

frui

t6,

26,

4

99,

17,

16,

96co

nsis

tenc

yPe

rcep

tion

in m

outh

of f

ruit

win

e di

lutio

n or

con

cent

ratio

n 8,

88,

6

Table 6 - Fruit wines sensory attributes mean values

*paired parameters a

sh berry wines (*

* for b

lack currant, 3* for y

ellow plum) w

ith t-te

st above the sig

nific

ance level P


Jayani R.S., Saxena S., Gupta R., 2005. Microbialpectinolytic enzymes : a review. ProcessBiochemistry, 40, 2931-2944.

Kiselev V.M., Gorelikova G.A., Adayeva A.A., 2013.Status and prospects of the Russian market of fruitwines. Wine-making and Viticulture (Russian), 1, 7-11.

Laboissiere L.H.E.S., Deliza R., Barros-Marcellini A.M.,Rosenthal A., Camargo L.M.A.Q., Junqueira R.G.,2007. Effects of high hydrostatic pressure (HHP) onsensory characteristics of yellow passion fruit juice.Innovative Food Science and EmergingTechnologies, 8, 469-477.

Liew Abdullah A.G., Sulaiman N.M., Aroua M.K., MegatMohd Noor M.J., 2007. Response surfaceoptimization of conditions for clarification ofcarambola fruit juice using a commercial enzyme.Journal of Food Engineering, 81, 65-71.

Morozova V.V., Gusakov A.V., Andrianov R.M.,Pravilnikov A.G., Osipov D.O., Sinitsyn A.P., 2010.Cellulases of Penicillium verruculosum.Biotechnology Journal, 5, 871-880.

Nelson N., 1944. A photometric adaptation of the Somogyimethod for the determination of glucose. Journal ofBiological Chemistry, 153, 375-379.

Noller J., Wilson B., 2009. Markets for Tropical FruitWine Products. RIRDC Publication No. 09/033,Australian Government - Rural Industries Researchand Development Corporation.

Somogyi M., 1952. Notes on sugar determination. Journalof Biological Chemistry, 195, 19-23.

Soufleros E.H., Pissa I., Petridis D., Lygerakis M.,Mermelas K., Boukouvalas G., Tsimitakis E., 2001.Instrumental analysis of volatile and othercompounds of Greek kiwi wine; sensory evaluationand optimisation of its composition. Food Chemistry,75, 487-500.

Volchok A.A., Bushina E.V., Rozhkova A.M., Zorov I.N.,Shcherbakov S.S., Sinitsyn A.P., 2013. Newgeneration enzyme complexes for juice production.Russian Journal of Biotechnology (Russian), 5, 78-89.

Volchok A.A., Rozhkova A.M., Zorov I.N., Sinitsin A.P.,Bushina E.V., Cherbakov S.S., 2014. Pretreatment ofgrape pulp by enzymes of new generation in the tablewines manufacturing. Wine-making and Viticulture(Russian), 1, 36-39.




production of fruit wines using novel enzyme …

Documents