8172019 Fermented Guava by L Plantarum

httpslidepdfcomreaderfullfermented-guava-by-l-plantarum 13

NOTE

Lactobacillus plantarum mediated fermentation of Psidium guajava L fruit extract

Ravish Bhat Lakshminarayana Chikkanayakanahalli Suryanarayana Karunakara Alageri ChandrashekaraPadma Krishnan Anil Kush and Puja Ravikumar

Vittal Mallya Scienti 1047297c Research Foundation 943 amp 945 23rd Cross 29th Main BTM II Stage Bangalore 560076 India

Received 18 February 2014 accepted 5 September 2014

Available online xxx

Sixteen hour fermentation of the white 1047298esh raw guava Lucknow 49 cultivar using Lactobacillus plantarum NCIM 2912

was taken up for enhancing the antioxidant potential The fermented guava product with high antioxidant potentialtotal phenolic content and short and medium chain fatty acids can be used as functional food

2014 The Society for Biotechnology Japan All rights reserved

[Key words Psidium guajava L Phenolic content Antioxidant activity Fermented guava product Fermentation Functional foods]

Guava (Psidium guajava L) family Myrtaceae is a popular

commercial fruit from tropical and semitropical regions of world

with India accounting for 45 of total production followed by

Pakistan Mexico and Brazil Based on pigments produced in pulp

guava fruits can be classi1047297ed as white and pink cultivars Nutri-

tionally guava fruit contains high amounts of fermentable sugars

such as fructose glucose sucrose pectin minerals (calcium and

phosphorus) antioxidants (vitamins A B C E niacin lycopene

carotinoids polyphenols) and high dietary 1047297bers Jimeacutenez-Escrig

et al (1) reported that guava pulp and peel are good sources of antioxidant dietary 1047297ber with 779 total extractible phenols

Vitamin C E along with phenolics and carotinoids are potent free

radical scavengers Raw guava fruits though rich in antioxidants

are hard (due to high pectin content) less sweet and not preferred

for direct consumption The antioxidant content in fruit declines as

they ripen (2) and the berries have a very limited shelf life Guava

fruits are highly prone to chilling and mechanical injury followed

by fungal decay Several post harvest techniques exist to preserve

guava fruits for retaining the nutritional properties and enhancing

the shelf life Various products like guava pulp concentrates syrup

paste puree and yeast fermented wine are available Sevda and

Rodrigues (3) reported that Saccharomyces cerevisiae NCIM 3095

strain was best suited for guava must fermentation for wine pro-

duction Increased consumer awareness about the bene1047297ts of pre-ventive health is a major contributor in increasing the demand of

functional foods However the perceptible off 1047298avors caused by

microbes pose a challenge to acceptability of these products Flavor

masking technique helps address this problem by incorporating

attractive fruit 1047298avors (45) We took up fermentation of raw guava

fruit extract using Lactobacillus plantarum (NCIM 2912) and the

product was analyzed for antioxidant content total phenolics and

production of short and mediumchain fatty acids To the best of our

knowledge this work is the 1047297rst attempt to improve nutritional

value of guava fruit extract by fermentation using lactic acid

bacteria

Raw guava fruits (Lucknow49 variety) procured from Lalbagh

Horticulture Department Bangalore were used in this study Fruits

were round to oval in shape green-pale yellow with white pulp

(each fruit weighing about 011 kg) L plantarum NCIM 2912

(source National Collection of Industrial Microorganisms Pune

India) was used for all the experiments The cultures were revived

from glycerol stocksin de Man Rogosa Sharpe (MRS) broth and MRS

agar (Himedia Mumbai India) at 37

C for 48 h A single isolatedcolony was sub cultured in MRS broth and used for shake 1047298ask

fermentation Methyl ester derivatives of butyric caproic caprylic

capric and lauric acids220-diphenyl-1-picryl hydrazyl (DPPH) 22-

azinobis 3-ethylbenzothiazolin 6-sulfonic acid (ABTS) butylated

hydroxy toluene (BHT) 35-dinitrosalicylic acid (DNS) were pro-

cured from Sigma Chemical Company (Mumbai India) 246-

tripyridyl-s-triazine (TPTZ) was obtained from Alfa Aesar (Hyder-

abad India) HPLC grade solvents and analytical reagent grade

chemicals were obtained from Merck (Mumbai India)

The raw fruits were washed with double distilled water several

times followed by surface disinfection with 95 ethanol Grated

guava was mixed with equal amount of sterile water (11 ratio

wv) and pulverized in to slurry in an electric juicer blender

(Kenwood JE 720) To this pulp 025 (ww) pectinase enzyme(Tris enzymes Mysore India) was added and the mixture was

incubated at 38

C with shaking at 200 revolutions per min for 2 h

to obtain clear fruit extract The slurry was 1047297ltered and extract was

diluted with sterile water (11 ratio) and supplemented with

following media components (Merck) (gl) K2HPO4 60 KH2PO4

140 MgSO4 7 H2O 04 NaCl 40 (NH4)2SO4 20 Glycine 30

Sucrose 500 and the pH was adjusted to 705 005 followed by

sterilization by autoclaving

The fermentation experiments were carried out in triplicate as

separate 1047298asks and uninoculated media was maintained as unfer-

mented control Fermentation was carried out at 37

C for 16 h with

mild shaking at 120 rpm and sampling was done at an interval of

every 4 h starting from 0 h for measuring antioxidant activity and Corresponding author Tel thorn91 80 26 68 72 23 fax thorn91 80 26 68 71 70

E-mail address pujavmsrforg (P Ravikumar)

wwwelseviercomlocatejbiosc

Journal of Bioscience and BioengineeringVOL xx No xx 1e3 2014

1389-1723$ e see front matter 2014 The Society for Biotechnology Japan All rights reservedhttpdxdoiorg101016jjbiosc201409007

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8172019 Fermented Guava by L Plantarum

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pH The viability of L plantarum during fermentation was checked

by plating on MRS agar plates

The antioxidant potential of the fermented product was evalu-

ated by determining the DPPH scavenging activity (6) ABTS radical

scavenging activity (7) and ferric reducing antioxidant power

(FRAP) assay (8) BHT was used to plot the standard graph and the

FRAP activity was expressed in terms of BHT equivalents (mgmL)

The concentration of total phenolics in fermented product was

determined using colorimetric method as described by Singleton

et al (9) The concentration of phenolics calculated from the stan-

dard graph using gallic acid and the phenolic content in fermented

product was expressed in terms of gallic acid equivalents (mg GAE

mL) Reducing sugars were estimated in the fermentation medium

to quantify the amount of unutilized sugars and the estimation was

done by DNS method (10) D-Glucose (Himedia) was used to plot

standard graph (02e

10 mgmL) Detection and quanti1047297cation of short chain fatty acids (SCFAs) such as butyric acid (40) and me-

dium chain fatty acids (MCFAs) like caproic acid (60) caprylic acid

(80) capric acid (100) and lauric acid (C120) was done by one

step liquideliquid extraction of fermented sample using chlor-

oformmethanol (21) solvent system conversion of free fatty acids

to fatty acid methyl esters followed by gas chromatographic anal-

ysis was done as described by Christie (11)

The mean values of percent free radical scavenging activity

FRAP activity total phenolics and short and medium chain fatty

acids in fermented product were obtained from triplicate 1047298asks of

two fermentation experiments and represented as mean SE The

correlation analysis was performed between total phenolic content

and antioxidant activities of fermented product using Pearson

correlation analysis in XLSTAT The signi1047297cance of correlation wasdone at P lt 0001 and the differences at P lt 0001 were considered

as statistically signi1047297cant

In our study guava extract fermentation using L plantarum led

to increase in antioxidant activity total phenolics and short and

medium chain fatty acids The increase in antioxidant potential is

observed because L plantarum degrades some poly phenolic com-

pounds into simple forms due to its wide portfolio of enzymes (ieb-glucosidase p-coumaric acid decarboxylase and decarboxylase)

The number of viable L plantarum cells increased exponentially

during 4e8 h of fermentation and reached stationary growth phase

between 12 h and 16 h The pH gradually dropped between 0 h and

8 h (pH 705e656) rapidly at 12 h (pH 565) reached 483 at 16 h

Approximately 67 of available soluble sugars from fruit material

were utilized by L plantarum by 12 h and consumption declined

thereafter as the microbial growth declined (Fig 1) After 16 h of

fermentation the liquid supernatant of guava fermented broth was

brown colored aqueous solution with fruity odor and the lyophi-

lized product was a hygroscopic brown crystalline powder withfruity fermented odor and freely miscible in water

The DPPH free radical scavenging activity of fermented product

increased signi1047297cantly and exhibited highest value of 8251 at 8 h

Gradual decrease in antioxidant activity was observed in subse-

quent fermentation hours and it dropped to 7729 at 16 h ABTS

radical scavenging and FRAP activities were in consensus and

reached the highest levels by 16th h of fermentation (Table 1

Fig S1) The mechanism of antioxidant effect of LAB has been

studied and explained using intact cells and intracellular extracts of

LAB where these strains signi1047297cantly inhibited the FethornthornH2O2

induced peroxidation of lipids and the intracellular extracts of LAB

possess metal chelating ability reactive oxygen species (ROS)

scavenging and reduction activity properties It has also been

illustrated that intracellular cell free extracts of differentL plantarum strains show strong hydroxyl radical scavenging ac-

tivity probably due to their ability to chelate Fethornthorn metal ions

FIG 1 Microbial population pH of the medium and reducing sugar utilization during fermentation of guava fruit extract

TABLE 1 Antioxidant activity and total phenolic content in fermented guava product during fermentation The uninoculated medium is used as control

Fermentation

hours

Antioxidant activity Total phenolic content (mgmL

GAE)DPPH activity ABTS activity FRAP activity

Control FGP Control FGP Control FGP Control FGP

0 3658 056 3701 060 1543 045 1552 028 081 004 080 002 271 006 283 006

4 3833 079 6049 064 1683 057 5357 208 080 002 093 006 285 005 433 009

8 3525 085 8251 139 1828 062 6290 255 075 002 120 003 289 006 782 011

12 3308 043 8081 073 1734 053 9626 049 073 002 160 006 295 007 1026 008

16 364 122 7729 084 1795 053 9916 021 071 001 220 008 285 007 1118 015

Each value in the table is the mean standard error of two fermentations DPPH and ABTS radical scavenging activity expressed in percentage FRAP activity expressed in mg

mL butylated hydroxy toluene (BHT) equivalents GAE gallic acid equivalent

2 BHAT ET AL J BIOSCI BIOENG

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L plantarum mediated fermentation of guava increased the total

phenolics in fermented product to 6648 (1118 mg GAEmL) as

compared to control that contains only 285 mg GAEmL after 16 h

(Table 1) The total phenolic content in guava pulp and peel is

268 mg GAEg and 587 mg GAEg respectively as estimated and

reported in a previous study by Jimeacutenez-Escrig et al (1) We have

used whole fruit extract in our experiment and the total phenolic

content in the unfermented control is 285 mg GAEmL of medium

is comparable to the values reported earlier This increase in the

total phenolic content can be attributed to the enzymatic degra-dation of the polyphenol complexes by the fermenting microor-

ganism Duenas et al (12) also studied the effect of L plantarum

fermentation on total phenolic compounds in Vigna sinensis L 1047298our

and reported that fermentation enhances the phenolic content

because it hydrolyzes complexes of polyphenols to other simpler

and biologically active ones

A considerable increase in butyrate caproate caprylate caprate

and laurate were observed in fermented product vis-a-vis unfer-

mented control (Table 2 Fig S2) The sugars in guava extract that

constitute the digestible carbohydrates of the fruit are converted to

SCFAs Henningsson et al (13) reported that a certain combination

of carbohydrate substrates may have an in1047298uence on SCFA syn-

thesis and these SCFAs and MCFAs (associated with gut health) are

produced by bacterial fermentation of indigestible carbohydrates incolon Vong and Stewart (14) reported that dietary fruit 1047297ber fer-

ments to form SCFAs and butyric acid (SCFA) has been implicated in

preventing and treating colon diseases like ulcerative colitis (1516)

The fermented guava product (FGP) with high antioxidant potential

and important fatty acids can be a ready to blend functional food

with multiple health bene1047297ts In our fermentation experiments we

observed that antioxidant potential is highest at 16th h of

fermentation as assessed by FRAP assay DPPH assay and total

phenolic content We optimized the process for harvesting at

16th h based on the antioxidant activity and fatty acid content of

product as well as the sugar utilization by microbes

In conclusion this study describes value addition to a common

tropical fruit guava using a probiotic strain L plantarum The fer-

mented product is rich in antioxidant content total phenolics short

and medium chain fatty acids The liquid concentrate of fermented

guava product is amenable for blending in a wide range of food

commodities like beverages candies bars ice creams etc Further

optimization in the fermentation process using other LABs can help

in developing a ready to blend fermented guava product for forti-

1047297cation of a range of food items Developing a value added fer-

mented guava product will help circumvent the losses because of

highly perishable nature of fruits

Supplementary data related to this article can be found at http

dxdoiorg101016jjbiosc201409007

Authors are thankful to the Department of Biotechnology Government of India

(Grant Id BTPR-11099FNS203882008) for 1047297nancial support Authors declare no

con1047298ict of interest This article does not contain any studies with human or animal

subjects

References

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(Psidium guajava L) as a new source of antioxidant dietary 1047297ber J Agric Food

Chem 49 5489e5493 (2001)

2 Gull J Sultana B Anwar F Naseer R Ashraf M and Ashrafuzzaman MVariation in antioxidant attributes at three ripening stages of guava (Psidium

guajava L) fruit from different geographical regions of Pakistan Molecules 17

3165e3180 (2012)

3 Sevda S and Rodrigues L Fermentative behavior of Saccharomyces strainsduring guava (Psidium guajava L) must fermentation and optimization of guava

wine production J Food Process Technol 2 118 (2011)

4 Luckow T Sheehan V Fitzgerald G and Delahunty C Exposure health

information and 1047298avour-masking strategies for improving the sensory quality

of probiotic juice Appetite 47 315e323 (2006)

5 Siroacute I Kaacutepolna E Kaacutepolna B and Lugasi A Functional food Product

development marketing and consumer acceptance e a review Appetite 51

456e467 (2008)

6 Aoshima H Tsunoue H Koda H and Kiso Y Aging of whiskey increases

11-diphenyl-2-picrylhydrazyl radical scavenging activity J Agric Food Chem

52 5240e5244 (2004)

7 Re R Pellegrini N Proteggente A Pannala A Yang M and Rice-Evans C Antioxidant activity applying an improved ABTS radical cation

decolorization assay Free Radic Biol Med 26 1231e1237 (1999)

8 Benzie I F and Strain J J The ferric reducing ability of plasma (FRAP) as a

measure of ldquoantioxidant powerrdquo the FRAP assay Anal Biochem 239 70e76

(1996)9 Singleton V L Orthofer R and Lamuela-Raventoacutes R M Analysis of total

phenols and other oxidation substrates and antioxidants by means of Folin-

ciocalteursquos reagent Methods Enzymol 299C 152e178 (1999)

10 Scherz H and Bonn G Analytical chemistry of carbohydrates Georg Thieme

Verlag Stuttgart New York (1998)

11 Christie W W Advances in lipid methodology pp 69e111 Oily Press Dun-

dee (1993)

12 Duenas M Fernandez D Hernandez T Estrella I and Munoz RBioactive phenolic compounds of cowpeas (Vigna sinensis L) Modi1047297cations by

fermentation with natural micro1047298ora and with Lactobacillus plantarum ATCC

14917 J Sci Food Agric 85 297e304 (2005)

13 Henningsson Aring Bjoumlrck I and Nyman M Short-chain fatty acid formation

at fermentation of indigestible carbohydrates Food Nutr Res 45 165e168

(2001)

14 Vong M H and Stewart M L In vitro bacterial fermentation of tropical fruit

1047297bres Benef Microbes 4 291e295 (2013)

15 Cummings J H Short-chain fatty acid enemas in the treatment of distal ul-

cerative colitis Eur J Gastroenterol Hepatol 9 149e

153 (1997)16 Zentek J Buchheit-Renko S Ferrara F Vahjen W Van Kessel A G and

Pieper R Nutritional and physiological role of medium-chain triglycerides

and medium-chain fatty acids in piglets Anim Health Res Rev 12 83e93

(2011)

TABLE 2 Short (SCFAs) and medium chain fatty acids (MCFAs) content in fermented

guava product (FGP)

Components (ng per 100 mL) Unfermented control Fermented guava product

Butyrate (C-4) 130 039 1785 068

Caproate (C-6) 100 034 6203 055

Caprylate (C-8) 240 031 3493 062

Caprate (C-10) 158 027 697 052

Laurate (C-12) 967

036 1797

051The values are in ng100 mL of fermented product uninoculated medium is used as

control Each value in the table is the mean standard error of two fermentations

V OL xx 2014 NOTE 3

Please cite this article in press as Bhat R et al Lactobacillus plantarum mediated fermentation of Psidium guajava L fruit extract J BiosciBioeng (2014) httpdxdoiorg101016jjbiosc201409007