PROBIOTIC BEVERAGE FROM BLACK CARROT JUICE

FERMENTED with

3rd International Conference and Exhibition on

Probiotics, Functional and Baby Foods

2014 ItalySelcuk

University

FERMENTED withLactobacillus casei andLactobacillus paracasei

Nihat AKIN, Talha DEMNihat AKIN, Talha DEMNihat AKIN, Talha DEMNihat AKIN, Talha DEMİRCRCRCRCİ, , , , Hale Hale Hale Hale İnci ÖZTÜRKnci ÖZTÜRKnci ÖZTÜRKnci ÖZTÜRK, Kübra AKTA, Kübra AKTA, Kübra AKTA, Kübra AKTAŞ, Enes DERTL, Enes DERTL, Enes DERTL, Enes DERTL

University of Selcuk, Faculty of Agriculture, Department of Food EngineeringTURKEY

BEVERAGE FROM BLACK CARROT JUICE

International Conference and Exhibition on

Probiotics, Functional and Baby Foods,

andparacasei

, Enes DERTL, Enes DERTL, Enes DERTL, Enes DERTLİ

Engineering, Konya

AgendaAgendaAgendaAgenda

1. Introduction

2. Material and Methods

3. Result and Discussion

4. Conclusion4. Conclusion

5. References

TheThe MevlânaMevlânaMevlânaMevlâna Museum, located inMuseum, located in Konya, TurkeyKonya, Turkey

Functional foods are defined as the foodsnutrients and feed on people, in addition tocure the diseases and provide several health(Prado et al 2008).

Probiotics are one of the most popular functional

1. INTRODUCTION1. INTRODUCTION1. INTRODUCTION1. INTRODUCTION

Probiotics are one of the most popular functionalProbiotics are defined as live microbial feedthat beneficially influence the host by enhancinggastrointestinal balance(Fuller, 1989; Yoon, 2006

that providecontribute to

health benefits

functional foods.functional foods.feed supplement

enhancing its2006).

� These microorganisms have many healtheffects such as easing of lactoseprevention of intestinal tract infectionscancer and good industrial characteristicsresistance to acid and bile, attachmentepithelial cells and colonization inepithelial cells and colonization inintestine (Jack,Tagg and Ray, 1995; Prado2008).

� They show antagonistic effect topathogens. One of the most commonlybacteria for commercial probiotic applicationsspecies of Lactobacillus (Sheehan et al., 2007

health-supportingintolerance,and colon

characteristics such asattachment to the

the humanthe humanPrado et al.

food-bornecommonly usedapplications are

2007).

recent years, consumer interest to non-dairyjuices, cereal-based products has increased (probiotic stability in non-fermented foods and beverages

� Probiotics have ordinarily been addedfermented milk products, especiallyyoghurts but there are severaldisadvantages related to their consumptionlike lactose intolerance, the cholesterolcontent and/or allergic to milk proteins(Heenan, Adams, Hosken, & Fleet, 2004Yoon, Woodams, & Hang, 2006; Kunal.2008).

dairy probiotic products such as fruit and(Shah, 2001) and there is a little publishbeverages.

Yoon, Woodams, & Hang, 2006; Kunal.2008).

� However, there is relative little publishedinformation on the survival of probioticsnon-fermented food matrices, whereasstability of probiotics in yoghurts haswidely studied (KailasapathyRybka, 1997).

There are a wide variety of traditional nonaround the world lately. Fruits andappropriate for probiotic products anddairy allergens that might prevent usageDelahunty, 2004). Also they have severalminerals, vitamins, dietary fibers, and antioxidants

In recent years studies about non-dairycabbage, blackcurrant, orange, beetrootperformed in conjuction with differentresults.

non-dairy fermented beverages producedand vegetables have been showed as

fruits and vegetables do not contain anyusage by part of the population (Luckow andseveral functional food components such as

antioxidants (phytochemicals).

dairy probiotic beverages such as tomatobeetroot and carrot juices have beenprobiotic strains and obtained appealing

� Black carrots are a goodanthocyanin pigments

� The anthocyanin contentcarrots was reported1750 mg kg−1 fresh weightMiniati, 1993).

� Black carrots alsoamounts of acylated anthocyanins

RedRedRedRed beetbeetbeetbeet SwedeSwedeSwedeSwedeturnipturnipturnipturnip

amounts of acylated anthocyanins� Moreover, black carrotprovide an excellentstrawberry red shadevalues; therefore, blackcan be a goodcolouring fruit juicessoft drinks, cans,confectionery (Downham2000; Kırca et al.,2006

good source ofpigments.

content of blackreported to be

weight (Mazza &

contain highanthocyanins.

� Black carrots (BCs) aregrown in Turkey, Afghanistan,Pakistan and India.

� Ereğli district in Konya is theproduction area for BCs. BCsin Turkey were often processedjuice, concentrate and shalgama traditional lactic acid fermentedanthocyanins.

carrot anthocyaninsexcellent bright

shade at acidic pHblack carrot juice

choice forjuices and nectars,

jellies andDownham & Collins,2006).

a traditional lactic acid fermentedbeverage (Turkyilmaz ve ark.,

The aim ofto determineL. caseiblack carrotblack carrotrefrigeratedand thereforesuitabilityraw materialprobiotic beverages

of the present study wasdetermine the survivability of

and L. paracasei oncarrot juices throughoutcarrot juices throughout

refrigerated storage for 42 daystherefore to assess

of black carrot as amaterial for production

beverages.

RawRawRawRaw MaterialMaterialMaterialMaterial

Black carrots were provided by blackDistrict, Konya as a concentrate, it was

A strain of L. casei NRRL B-442 and

2222. . . . MATERIAL AND METHODSMATERIAL AND METHODSMATERIAL AND METHODSMATERIAL AND METHODS

A strain of L. casei NRRL B-442 andwere obtained from ARS Culture BacterialCulture Collection, United StatesAgriculture, Peoria П,USA)

black carrot producers in Ereğliwas diluted 1:10.

and L. paracasei NRRL B-442and L. paracasei NRRL B-442Bacterial Collection (NRRLStates Department of

Microorganisms,Microorganisms,Microorganisms,Microorganisms, inoculuminoculuminoculuminoculum preparationpreparationpreparationpreparation

Pasteurization were applied to freshly80°C for 20 min. for the purpose of decreasebelow the detection limit.

A strain of L. casei NRRL B-442 andA strain of L. casei NRRL B-442 andstatically activated for 12 h at 37°Ccontaining 100 ml of MRS Broth1960).

� Stock cultures were

preparationpreparationpreparationpreparation andandandand juicejuicejuicejuice fermentationfermentationfermentationfermentation

freshly prepared black carrot juices atdecrease microbial population to

and L. paracasei NRRL B-442 wasand L. paracasei NRRL B-442 wasC in 25 ml erlenmayer flasks(de Man, Rogosa, & Sharpe,

were prepared and stored frozen (-20

� The growth of L. caseithe optical density at 590fresh juice inoculated withfermentation (final absorbance)

� The procedure consistedjuice containing microbialjuice containing microbialagainst water.

� The difference betweenthe growth of the microorganisms

� Growth was expressedusing the calibration curve

solutionsolutionsolutionsolution

L. casei (g/L) = ABS(590 nm

casei and L. paracasei was quantified by measuring590 nm. The absorbance was recorded forwith L.casei (initial absorbance) and after 24

absorbance).

consisted of diluting with distilled water an aliquot ofmicrobial cells and reading the absorbance at 590microbial cells and reading the absorbance at 590

between final and initial absorbance correspondedmicroorganisms during the fermentation.

expressed as dry mass concentration (g/L) calculatedcurve given in Eq. (1), built using L.casei dry cells

L. casei (g/L) = ABS(590 nm ) Eq. 1

Serial dilutions were prepared for microbial countsdiluted samples were inoculated on plates containingAgar, plating on the surface with the aidDrigalsky.

The plates were incubated at 37°C for 72 h. Typicalare round, white creamy with diameters ranging

,3 mm (Vinderola and Reinheimer, 2000).,3 mm (Vinderola and Reinheimer, 2000).

For stability assay, black carrot juice was fermentedfor 48 h. After fermentation the bottles wererefrigeration temperature for 42 days. Each sevenbottle of each sample was analysed (pH, microbialand color).

counts. Thesecontaining MRS

of a Handle

Typical coloniesranging from 0,9 to

fermented at 30°Cstored under

seven days, amicrobial viability

pHpHpHpH AnalysisAnalysisAnalysisAnalysis

pH values of the black carrot juicepH meter (İnolab Ph720, Weilheim, Germany)

ColorColorColorColor AnalysisAnalysisAnalysisAnalysis

Color analysis of samples waschroma meter (Konica Minolta, Incchroma meter was standardized byand measurements were made through(Minolta, 1998). The instrument(L*), redness (a*) and yellowness (b*)

juice was determined by WTWGermany).

determined using CR400Inc., Osaka, Japan). Theby using the illuminant D65

through an 8 mm viewing areainstrument measured lightness

(b*).

dddd.... SugarSugarSugarSugar DeterminationDeterminationDeterminationDetermination

� The sugars were analysed by high performanceShimadzu HPLC equipped with LC-1010ACvp column oven and DGU-14Aminex by HPX-87C carbohydrate columnvolume was 20 μL and flow velocitycarried out according to Vebericvolume was and flow velocitycarried out according to Vebericmodification.

dddd.... StatisticalStatisticalStatisticalStatistical AnalysisAnalysisAnalysisAnalysis

� JMP 5.0 (SAS Institute Inc., Cary, NC,the statistical analysis according toMeans that were statistically differentusing Student’s t comparison tests at

performance liquid chromatography in a10ADvp pump, RID 10A dedector, CTO-14A degasser. Seperation was appliedcolumn (300*7.8mm) at 80°C. Injection

velocity 0,6 ml/min. Sample preparation wasVeberic and Stampar (2005) with somevelocity 0,6 ml/min. Sample preparation wasVeberic and Stampar (2005) with some

NC, USA) software was used to performone-way analysis of variance (ANOVA).

different from each other were compared byat %5 confidence interval.

NameNameNameName FermentationFermentationFermentationFermentation andandandandStorageStorageStorageStorage Time Time Time Time

(day(day(day(dayssss))))

pH pH pH pH valuesvaluesvaluesvalues

0 3,78a

1 3,75b

NameNameNameName FermentationFermentationFermentationFermentationandandandand

Storage Storage Storage Storage Time Time Time Time (day(day(day(dayssss))))

pH pH pH pH valuesvaluesvaluesvalues

LLLL

0 3,78a

1 3,74b

3333. . . . RESULT AND DISCUSSION

LactobacillusLactobacillusLactobacillusLactobacillusparacaseiparacaseiparacaseiparacasei

1 3,75b

2 3,74a

7 3,74b

14 3,72c

21 3,71cd

28 3,69d

35 3,69d

42 3,69d

LactobacillusLactobacillusLactobacillusLactobacilluscaseicaseicaseicasei

1 3,74b

2 3,74b

7 3,67c

14 3,64d

21 3,63d

28 3,64d

35 3,64d

42 3,65d

valuesvaluesvaluesvaluesLLLL****

valuesvaluesvaluesvaluesaaaa**** valuesvaluesvaluesvalues bbbb**** valuesvaluesvaluesvalues

a 7,88c 21,43cd -6,45d

b 7,95c 15,25f -6,73d

LLLL**** valuesvaluesvaluesvalues aaaa**** valuesvaluesvaluesvalues bbbb**** valuesvaluesvaluesvalues

8,10a 23,37ab -5,78a

8,98a 22,18ab -4,81ab 7,95c 15,25f -6,73d

a 10,56a 28,78a -2,45a

b 8,37c 16,20ef -6,35d

c 9,94ab 26,64ab -3,11ab

cd 10,02ab 19,14de -5,51cd

d 9,62b 23,06c -4,34bc

d 9,49b 23,86bc -4,21abc

d 8,16c 17,54ef -5,83cd

8,98a 22,18ab -4,81a

9,43a 25,22ab -4,21a

8,33a 18,77b -5,97a

9,19a 27,53a -4,78a

9,16a 19,60b -5,67a

9,44a 22,45ab -5,13a

9,38a 24,22ab -3,59a

8,70a 20,89ab -4,50a

3333. . . . RESULT AND DISCUSSION

a.a.a.a. pHpHpHpH AnalysisAnalysisAnalysisAnalysis

� No pH adjustment was donethe fermentation.

� At the beginning of the fermentationfor L. casei and L. paracasei

� At the beginning of the fermentationfor L. casei and L. paracasei3,78. It was measured as 3,65and L. paracasei , respectively

� Statistically significant reductiondetermined, after 21st dayparacasei and 14th day of storage

done at the beginning of

fermentation, pH valuesparacasei were measured as

fermentation, pH valuesparacasei were measured as

65 and 3,69 for L. caseirespectively, after the storage.

reduction was notday of storage for L.storage for L. casei .

pH reduction which would affect toand L. paracasei too much was not

In a study about fermented cashewexplained that initial pH value wasand it decreased to about 3,8and it decreased to about 3,8storage, and viable cell populationremained around 8,5 log CFU/mlconditions after the storage.

In our study, reduction of pHunfavourably viability as well, afterremained approximately 7,5 log CFU/ml

to viability of L. caseinot observed.

cashew apple juice, it waswas approximately 4,3

at the end of theat the end of thepopulation of L. caseiCFU/ml in these pH

pH did not affectafter 42 days viabilityCFU/ml.

Cashew apple

� In a study of Yoon et al. (2004that probiotic cultures, includingmaintained their viability intomato juice’s pH after 72 hourat 30 OCat 30 C

2004), they expressedincluding L. casei,in low pH such ashour of fermentation

b. b. b. b. ColorColorColorColor analysisanalysisanalysisanalysis “L”, “a”, “b”“L”, “a”, “b”“L”, “a”, “b”“L”, “a”, “b”

� “L”“L”“L”“L” (lightness) value increasedfluctuated during storage afterwas determined that this valuestorage compared to the beginningstorage compared to the beginningresearch about viability of L.was determined that “L”“L”“L”“L” value, authors associated this reductionduring the storage.

increased during fermentation, it hadafter the fermentation, however, itvalue declined by the end of thebeginning of the storage. In abeginning of the storage. In a

casei in cashew apple juice, itvalue decreased during the storagereduction with increase of biomass

� “a” (redness) value had fluctuateddetermined last of the storage comparedstorage. In the study about viabilitywas explained that “a” value decreased

�Reduction of a value of L. caseisignificant but reduction of “a”significant but reduction of “a”observed statistically significant. However,açai, acerola, pomegranate and applesamples decreased any without probiotic

�It was observed that “b” (yellownessparacasei, more decreased than at

� Negative values was observed inwas more dominant than yellow in black

fluctuated, too. But a decrease wascompared to at the beginning of the

of L. casei in cashew apple juice, itdecreased during the storage, as well.

casei were observed no statistically“a” value of L. paracasei were“a” value of L. paracasei wereHowever, in a study about storage of

apple, was reported that “a” values ofprobiotic culture.

yellowness) value for both L. casei and L.the beginning of the storage.

in “b” value.This showed that Blueblack carrot juice.

Again, it was found significant for L.paracaseistatistically important for L.casei.

As it known to all, black carrot is one of the widely

Even if black carrot juice colour diluted 1\10 rateEven if black carrot juice colour diluted 1\10 ratedecreases of “a” and “b” values in blackabsolutely distinctive as visual during storage.

From this point of view (during fermantation andcomfortly that adding L.casei and L.paracasei

carrot juice characteristic colour.

paracasei whereas it was not

widely used natural colorant.

rate , it was so intensive,rate , it was so intensive,black carrot juice were not

and storage ) it could beparacasei did not change black

c. c. c. c. ViabilityViabilityViabilityViability

0 1 2 7 14 21 28 35 42

Time (days)

CFU/ml by the end of the end of the storage atnumbers belonging to both two bacteria were observedremained in the range of 7,5 log CFU/ml after 42sd day.

L. paracasei

� Count of L. casei which was 6,5CFU/ml, increased to about 8CFU/ml after 48 hourfermentation. At the same time,paracasei was 7.4 log CFU/ml atbeginning of the fermentation whilewas 8,4 log CFU/ml afterL. paracasei

L. casei

was 8,4 log CFU/ml afterfermentation.

� Raises of both L. casei andparacasei’s numbers which were 1log CFU/ml after fermentation, wereexpected increases. After theseraises, it was anticipated that numberof probiotic bacteria which reached8,5 log CFU/ml remained upper than

at 4OC. And it occured as it expected to be. Fallsobserved after 21st day, however, number of bacteria

.

� In a study conducted by Yoon etinoculated into beet juice and theirfermentation and storage time (28acidophilus was falling to 16x104

remained approximately 9x106 CFU/mlreported that L. casei and L. paracasei7,2x107 and 7,7x107 CFU/ml, respectivelyreported that L. casei and L. paracasei7,2x107 and 7,7x107 CFU/ml, respectively

� In another study about tomato(2004), it was notified that L. caseiCFU/ml after the storage for 4 weeksresearch about probiotic cashew, initialCFU/ml in the fermentation and thenafter the fermentation. It increaseddeclined after that day, and remainedday.

et al. (2005), 4 type of bacteria weretheir viabilities were examined during

28 days). While number of L.CFU/ml, number of L. delbrueckii

CFU/ml. On the other hand, it wasparacasei stayed alive such high rate asrespectively.paracasei stayed alive such high rate asrespectively.

tomato juice performed by Yoon et al.casei kept its viability about 1,7x108

weeks. When it comes to anotherinitial number of L. casei was 7,5 logthen it rose up around 8,5 log CFU/ml

increased by the time 21st day and hadremained about 8,6 log CFU/ml on 42sd

d. d. d. d. SugarSugarSugarSugar analysisanalysisanalysisanalysis

Exchange of Exchange of Exchange of Exchange of SucroseSucroseSucroseSucrose withwithwithwithTimeTimeTimeTime

15

Exchange of Exchange of Exchange of Exchange of GlucoseGlucoseGlucoseGlucoseTimeTimeTimeTime

24 s

48 s0

5

10

mg/

ml

mg/

ml

mg/

ml

mg/

ml

GlucoseGlucoseGlucoseGlucose withwithwithwithTimeTimeTimeTime

10

Exchange of Exchange of Exchange of Exchange of FructoseFructoseFructoseFructoseTimeTimeTimeTime

24 s

48 s 0

2

4

6

8

10

mg/

ml

mg/

ml

mg/

ml

mg/

ml

� In our study, it is seemed that the amountof the fermentation and this falling wasGlucose and fructose contents rose upafter fermentation, too. This increaseReduction of sucrose and incrementexpected result. It indicated that probioticmaintain their activity.maintain their activity.

� Costa et all. (2013) performed sugaradded with L. casei. Sucrose decreasedglucose and fructose were increasing.sucrose reduced from 45 g/L to 32 g/Lglucose and fructose contents which wereg/L.

amount of sucrose decreased at the endwas found to be more for L. paracasei.up due to decomposition of sucrose

increase was still more for L. paracasei.increment of glucose and fructose was an

probiotic bacteria used sugar resources to

sugar analysis in pineapple which wasdecreased during the fermentation while

As a conclusion, they observed thatg/L during the fermentation. In additionwere approximately 4 g/L rose up to 6,5

eeee.... BiomassBiomassBiomassBiomass determinationdeterminationdeterminationdetermination

�Biomass analysis was performed only

0.75

0.8

0.85

Bio

mas

s (g

/L)

0.65

0.7

0.75

0 1 2

Bio

mas

s (g

/L)

Time ( Fermentation days)

L. paraceseii

L. caseii

� It increased as expected during the fermentationenriched L.casei was higher than that of Lwith increase of viable count and decreaseviable count in black carrot juice fermentedL.paracasei. Also decrease of pH was determinedthan that of L.paracasei.

only for fermentation.

L. paraceseii

L. caseii

fermentation. Biomass increase of black carrot juiceL. paracasei. This situation showed parallelism

decrease of pH. Because augmentation of increasefermented with L.casei was high compared to that

determined high in black carrot juice with L.casei

4444. CONCLUSION. CONCLUSION. CONCLUSION. CONCLUSION

5555. REFERENCES. REFERENCES. REFERENCES. REFERENCES

�Kyung Young Yoon, Edward E. Woodams, Yong D. Hangbacteria, Lebensm.-Wiss. u.-Technol. 38 (2005) 73–75

�Clarice Maria de Arau´ jo Chagas Vergara a,Rodrigues, Prebiotic effect of fermented cashew appleand Technology, LWT - Food Science and Technology

�Sawaminee Nualkaekul a, Ivan Salmeron b, Dimitris Charalampopoulosthe survival of Bifidobacterium longum in model acidic

�Mayra Garcia Maia Costa, Thatyane Vidal Fonteles,�Mayra Garcia Maia Costa, Thatyane Vidal Fonteles,pineapple juice as substrate for L. casei cultivation forand product stability, Food Chemistry, 139 (2013) 261

�Kyung Young Yoon, Edward E. Woodams, Yong D. Hangbacteria, Bioresource Technology 97 (2006) 1427–1430

�Fla´vera C. Prado a, Jose L. Parada a, Ashok Pandeybeverages, Food Research International 41 (2008) 111

�Ana Lúcia F. Pereira, Tatiane C. Maciel, Sueli Rodrigueswith Lactobacillus casei, Food Research International,

�Kyung Young Yoon, Edward E. Woodams1 and YongBacteria, 2004, The Journal of Microbiology, December

Hang*, Fermentation of beet juice by beneficial lactic acid75.

Talita Lopes Honorato b, Geraldo Arraes Maia, Sueliapple (Anacardium occidentale L) juice, LWT - Food Science

Technology 43 (2010) 141–145.

Charalampopoulos, Investigation of the factors influencingacidic solutions and fruit juices, 129 (2011) 1037–1044.

Ana Laura Tibério de Jesus, Sueli Rodrigues, SonicatedAna Laura Tibério de Jesus, Sueli Rodrigues, Sonicatedfor probiotic beverage development: Process optimisation

261–266.

Hang, Production of probiotic cabbage juice by lactic acid1430,

Pandey b, Carlos R. Soccol, Trends in non-dairy probiotic111–123

Rodrigues, Probiotic beverage from cashew apple juice fermented44 (2011) 1276–1283

Yong D Hang, Probiotication of Tomato Juice by Lactic AcidDecember 2004, p.315-318