original research article doi: 10.26479/2019.0503.20 ... · polyunsaturated fatty acids (pufas),...

Suja & Paari RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications

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2019 May – June RJLBPCS 5(3) Page No.244

Original Research Article DOI: 10.26479/2019.0503.20

STUDIES ON FERMENTATIVE LACTIC ACID BACTERIA FOR ENHANCED

GROWTH, ANTIOXIDANT ACTIVITY ON ZEBRAFISH (DANIO RERIO)

Suja P, Paari K.A.*

Department of Life sciences, CHRIST (Deemed to be University), Bangalore, India.

ABSTRACT: This study is aimed to find out the survival, growth, antioxidant capacity and

pigmentation of the Danio rerio supplemented with marine seaweed Gracilaria corticata and

probiotic Pediococcus pentosaceus. The marine seaweeds Gracilaria corticata and probiotic

Pediococcus pentosaceus were fed to the fishes for 30days. Diet without the Gracilaria corticata

supplement and probiotic served as control. The experimental study on parameters of proximate

analysis of feed, gradual growth of fishes, antioxidant activity, and pigmentation on skin of the fish

were measured. Varied antioxidases expression was noted in the treatment groups. Survival rate was

better in the probiotic and Gracilaria corticata supplemented samples compared to the control

samples. Fluorescence chromatophores were identified in fish tail under fluorescent microscopy

using FITC dye. Our research concluded that probiotic and seaweed supplementation enhanced the

activity of antioxidant enzymes and could be used as a potential ingredient in aquaculture feed.

KEYWORDS: Probiotic, seaweed, antioxidant capacity, fluorescence chromatophores

Corresponding Author: Dr. Paari K. A.* Ph.D.

Department of Life sciences, CHRIST (Deemed to be University), Bangalore, India.

1. INTRODUCTION

Aquaculture being the innovative part of economy in culturing higher growth of marine organisms

is answering the demands of food production and food security sectors for the growing human

population with enhanced culture methods [1]. Increasing threat from aquaculture diseases and

epizootics to the fisheries community is increasing every year [2]. Lactic acid bacteria commonly

used in household activities are used for its beneficial role in various sectors. Microorganisms

belonging to the batch LAB fabricate various antimicrobial substances that act against different

gram positive and gram negative pathogens. Beneficial properties of probiotic microorganism are

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extended to different host ranging from mice to humans [3]. Immunomodulatory effects of probiotic

strains have been conferred in host organism when supplemented through oral administration [4].

Mode of action of the supplemented probiotic strain might be due to the secretion of excretory

products such as exopolysaccharides, bacteriocins, lactic acid [5]. Most of the organisms belonging

to the group LAB counteract oxidative stress by quenching the expression of free radicals [6].

Identifying a potential novel strains that could control reactive radicals will pave way for

development of novel food supplements which could quench ROS mediated damages in the organ

system [7]. Dietary supplements are effectively converted by the intestinal micro flora by production

of secretory exopolysaccharides which in turn improve the bioavailability of antioxidants [8].

Research is focused towards enhancing the activity of microbiota by supplementing prebiotic

supplements. Supplementation of fermentative lactic acid bacteria along with sea weeds directly

influences the viability of gut microbiota which might have positive influence on the host

metabolism [9]. The ability of the seaweed to enhance the gut microbiome community provides the

microenvironment for the growth of beneficial probiotic bacteria. Red algae Gracilaria which are

found naturally beside the sea coasts was refined and exploited for their biological applications in

food, fodder and therapeutics industries [10]. Bioactive compounds from unexploited seaweeds have

emerged as an attractive element to feed industry. Asian countries particularly Japan, China, and

Korea, have an extensive belief of seaweed utilization while western countries are chiefly involved

in phycocolloids construction such as alginate, agar, and carrageenan from seaweeds[11]. High

quality components of seaweeds which have rich sources of protein, polyphenols, carbohydrates,

polyunsaturated fatty acids (PUFAs), amino acids, antioxidants, minerals, dietary fibers, and

vitamins make it highly demanding element in feed industries [12] Using seaweed supplements

along with fermenting lactic acid bacteria will aid in immune enhancement through up regulation of

antioxidant enzymes. Though various methods such as solvent extraction and chemical digestion

are practiced to extract compounds from seaweeds, product recovery and production cost is

controlling its application in feed industries [14]. Fermentation and breakdown of complex

substances in the seaweed in to simpler available forms are carried out by fermentative bacteria.

Hydrolysis of seaweed supplements by fermentative lactic acid bacteria enhance antioxidant activity

which can serve the purpose of growth and metabolism enhancement in aquaculture sector. With a

recent huge unease for the usage of antibiotics and the threat of developing antibiotic resistance

among aquaculture pathogens force researchers to identify alternatives for aquaculture sectors. Use

of fermentative bacteria for livestock boost up the growth as well as survival without causing any

side effects [15]. Though vast amount of studies have reported the usage of lactic acid bacteria and

their secretory products for aquatic health, very limited information are available on the role of

utilization of fermentative product of red algae and its effect on the antioxidant enzymes. Feed

formulations and its effects on the metabolism of antioxidant enzymes can be analyzed in animal






models which mimic systems close to human system. Basically this effort was carried to know the

outcome of fermentative lactic acid bacteria on hydrolysis of red algae Gracilaria and its role on the

expression of antioxidant enzymes, growth performance using zebrafish as a model system.

2. MATERIALS AND METHODS

Collection and preparation of the plant material

The seaweed Gracilariacorticata was collected from the coastal areas of Mandapam (9°16'58.8"N

79°07'01.2"E), India. The collected samples were thoroughly washed with fresh water several times,

dried at 40°C for 48 to 72 hrs and then ground to powder using a pulverizer. To acquire methanolic

extract, 30g of the powdered plant material and 100 ml of the methanol was loaded in soxhlet. The

extracts were evaporated to total dryness by vacuum distillation. To make sure maximum extraction

was carried out, the process was repeated twice. The solvent was then evaporated under reduced

pressure using a rotary evaporator to obtain extracts and preserved in refrigerator at 4°C for further

analysis.

DPPH Radical Scavenging Activity

The capability to quench the stable free radical DPPH was recorded according to the procedure of

Blois [16]. Methanolic extracts from the seaweed Gracilaria corticata of 0.1ml were mixed with

0.9ml of 0.041 mM DPPH radicals dissolved in methanol. The mixture was shaken robustly and

was incubated for 60min in the dark. The reduction of the DPPH radical was determined by

measuring the absorption at 517nm.Methanol served as blank. AS is the absorbance of the solution

when the sample extract is been added and ADPPH is the absorbance of the DPPH solution.

% RSA = 𝐴𝐷𝑃𝑃𝐻−𝐴𝑆

𝐴𝐷𝑃𝑃𝐻× 100

Reducing Power

The reducing power of Gracilaria corticata extracts was determined [17]. Briefly, Methanolic

extract of Gracilaria corticata (1 ml) was mixed with 1 ml of 0.2 M phosphate buffer (pH 6.6) and

1 ml of potassium ferricyanide (10 mg/ml) and was incubated at 55 °C for 25 min. Following

incubation, 1 ml of TCA (trichloroacetic acid- 100 mg/ml) was added, and the mixture was

centrifuged at 14000rpm for 4 min. The upper layer (1.0 ml) was separated and to this 1 ml of

deionised water and 0.1 ml of ferric chloride (1.0 mg/ml) was added. Absorbance was measured at

700 nm. Higher the absorbance designates stronger reducing power.

Analysis of Fluorescent Chromatophores

Experimental fishes were identified for the biochrome pigments on their body following the protocol

of Matthias F. Wucherer [18]. Experimental fishes were dissected after exposure to Gracilaria

corticata extracts. Fins or tails were immersed in the physiological saline for an hour and was stained

using fluorescein isothiocyanate (FITC)dye. Stained sample was air dried at room temperature and

was focused using fluorescence microscopy. Wavelengths of excitation was recorded at 490nm and






the emissions was measured at 525nm.

Rearing conditions and Experimental design

The treatment slots in our research were categorized into four batches (1) fish supplemented with

diet containing 6LogCFU of Pediococcus pentosaceus, (2) fish fed a diet supplemented with 5%

Gracilaria corticata (3). Fishsupplemented with Pediococcus pentosaceus (6LogCFU)and

Gracilaria corticata (5%) (4) Control group was fed with diet devoid of Pediococcus pentosaceus

and Gracilaria corticata. Zebrafish (Danio rerio) of same weights were purchased from the

aquafarm, Bangalore, India and was maintained in in-house aquaria with proper freshwater

maintenance at 28◦C for a period of 30 days. All aquaria were given proper aeration and water was

circulated and changed daily in a routine basis. Four batches consisting of 10 individuals per batch

were grouped as Control, probiotic minus Gracilaria corticata, probiotics with Gracilaria corticata,

and only Gracilaria corticata

Feed formulation

Feed was prepared using the raw materials such as fishmeal (150gm/kg), wheat flour (140gm/kg),

soya flour (160gm/kg), corn flour (140gm/kg), tapioca flour (180gm/kg), groundnut oil cake

(180gm/kg). The formulations were mixed with 5% of Gracilaria corticata. Prepared ingredients

were collected, shade dried, powered and was sieved by 0.5mm extruder. Distilled water was used

to make dough with feed ingredients. The extruded feed was air dried for 24 hours to prevent the

degradation of vitamin, mineral mix and marine seaweeds. The feed was then trampled, sieved to

get a pellet size ranging 0.2– 0.5 mm, and kept at −20°C to evade oxidation of the marine seaweed.

Proximate composition analysis in feed

Protein content was estimated by the procedure of Lowry [18] by means of Bovine Serum Albumin

(BSA) as standard. Carbohydrate content was established according to the procedure of Dubois [19]

using D-Glucose as standard. Lipid content was estimated following the procedure of Folch[20].

Moisture content in fish was calculated by the method of Rajendran [21]. The ash content was

determined based on the methods mentioned in AOAC [22]

Determination of nutritional effects and survival:

At every 5 days of interval, fishes were taken for wet weight measurement. Indicators of growth

such as feed conversion ratio (FCR), feed efficiency (FE), specific growth rate (SGR), weight gain

(WG) and length gain (LG) percentage were calculated as follows;

Feed Conversion Ratio

Dry feed intake (g)

FCR (g) =

Wet body weight gain (g)






Feed Efficiency

Wet weight gain (g)

FE (g) =

Dry feed consumed (g)

Percentage Of Survival

Final no. of fish

P(S) = x 100

Initial no. of fish

Specific Growth Rate

Wf – Wi

SGR = x 100

T

Wf = Final average weight of fish

Wi = Initial average weight of fish

T = Duration of feeding trials in days

Weight Gain Percentage

Final weight- Initial weight

WG (%) = × 100

Initial weight

Length Gain Percentage

Final length - Initial length

LG (%) = ×100

Initial length

Antioxidant enzyme activity

Superoxide Dismutase Activity (SOD)

Superoxide Dismutase (SOD) activity was determined based on the capacity of the enzyme to slow

down the autoxidation of pyrogallol [23]. 100µl of sample was added in the clean glass tube. To this,

0.15ml of chloroform was added and mixed well for 15min. After centrifugation, at 13000rpm for

15mins, 2ml of Tris- EDTA buffer and 0.5ml of pyrogallol was added to the supernatant and the

absorbance was read at 470nm against blank. 0.5ml of pyrogallol and 3.5ml of distilled water was

used as blank.

Catalase Activity (CAT)

The CAT activity assay was performed using spectropohotometric determination of hydrogen

peroxide (H2 O2) which form stable complex with ammonium molybdate that absorbs at 405nm [24].

50µl of sample and 1ml of H2O2 were added into the clean glass tube. It was incubated at 37°C for

60 seconds. Following incubation, 1 ml of ammonium molybdate was added and the absorbance






was read at 405 nm against blank. 1050µl of phosphate buffer and 1000µl of ammonium molybdate

served as blank.

Reduced Glutathione (GSH)

Reduced glutathione (GSH) was calculated according to the method of Beutler [25], using Elmann’s

reagent (DTNB). 50µl of sample was taken in 2ml centrifuge tube. Equal volume of 10% TCA was

added and centrifuged at 12000g for 5 mins. 1ml of Na2 HPO4 and 0.5ml of DTNB was added and

the absorbance was read at 412nm.

Glutathione Peroxide (GPx)

Glutathione Peroxidase activity assay was performed by using spectropohotometric determination

of hydrogen peroxide (H2 O2) [11]. 50 µl of sample was taken in 2 ml centrifuge tube. For blank,

3ml of distilled water was added with 1ml of DTNB. 0.2ml of EDTA and 0.1ml of sodium azide,

0.1ml H2O2, 0.2 ml of Reduced Glutathione and 0.4 ml of phosphate buffer was added and incubated

at 37° C for 10 mins. To this mixture, 20 µl of 10% TCA was added and was centrifuged at 5000

rpm for 5 mins. l ml of Na2 HPO4 and 0.5ml of DTNB was added and the absorbance was read at

412nm.

3. RESULTS AND DISCUSSION

Proximate Composition Analysis In Feed

The formulated feed containing Gracilaria corticata had about 21.45% of protein, 19.88% of

carbohydrate, 13% of lipid content, 35.89% of moisture content and 2.1% of ash content. Almost

similar nutritional composition was obtained in Gracilaria eduli collected from the same sample

point [26]. Minor changes in the proximate composition value might be due to the seasonal changes

and different regions of sample collection. Variation in the composition is attributed due to the

capacity of the seaweeds to hold minerals, nutrient availability, and geographical location [27].

Higher level of protein, lipid and carbohydrate suggest that the sea weed Gracilaria corticata can

be a potential raw material for designing efficient functional feeds.

Antioxidant property of Gracilaria corticata

The absorbance percentage of Gracilaria corticata extract raised slowly from 0.1 – 0.5 µg/ml in the

DPPH radical scavenging assay (Fig.1). Proton radical scavenging potential was gradually getting

higher at increasing concentration of the Gracilaria corticata extract. Thus the reduction ability of

the DPPH radical is determined by the decrease in its absorbance at 517 nm induced by

antioxidants[28].The reducing power of Gracilaria corticata extract was gradually increasing from

1 – 2 mg/ml (Fig. 2). The high reducing power of compound indicates the ability of the compound

as potential electron donors to quench free radicals competently. Increase in reducing power was

evident with the higher concentration of sea weed extracts. Increasing concentration of the

Gracilaria corticata extract are generally related with the occurrence of reductones which breaks

down the free radical chain by donating a hydrogen atom.






Fig.1. Dose dependent potential of DPPH radical quenching ability of Gracilariacorticataextracts

Fig.2. Dose dependent potential of reducing ability of Gracilariacorticataextracts

The experimental fish pigments on the tail region focused under light microscopy showed

enhancement of color pigments (Fig.3). The black pigment spot, blue pigment spot, yellow pigment

spot indicates the excitation of melanophores, iridophores and xanthophores respectively.

Chromatophore in fishes actively regulates the circulation of fluorescent pigments throughout the

skin [29]. The fluorescence could act as a context-dependent signal in some marine fish and

ornamental species. Such pigment excitation was not observed in any of the control and Pediococcus

supplemented samples (Data not shown).

0

5

10

15

20

25

30

35

40

45

0.1 0.2 0.3 0.4 0.5

DP

PH

act

ivit

y

(%)

Extract concentration (µg/ml)

0

10

20

30

40

50

60

70

80

1 1.2 1.4 1.6 1.8 2

Red

uci

ng p

ow

er a

ctiv

ity

(%

)

Extract concentration (mg/ml)






Fig.3. Effect of Gracilaria corticata on the expression of chromatophores (X- xanthophores,

M- melanpohores, IR- iridophore) on tail

Growth parameters

Comparing the FCR value from day 1 to day 30 showed Pediococcus pentosaceus treated samples

exhibiting a gradual sloppy range from 3.34 ±0.01 to 2.24 ±0.02. Batches of Gracilaria corticata

plus Pediococcus pentosaceus and Gracilaria corticata also showed reduced FCR value compared

to control samples(Fig.4). Milinković Tur S [30] analysed FCR values on fingerlings of Indian major

carp close from the reported value in this study with a range from 2.68 ± 0.05 to 1.57 ± 0.09.

Significant reduction was noted in all the groups with the growth of zebrafish. This mathematical

calculation provides information regards to amount of feed converted to weight of the fish. With

the increase in the treatment time, decline of FCR value was noted, indicating a better weight gain

in the fish samples. Pediococcus pentosaceus treated sampleshaving less FCR value is due to the

better nutrient absorbtion by the gut microbiota. Gain in body weight due to probiotic

supplementation has been reported in broilers [31]. Significant differences were noted in the feed

conversion among the treatment groups compared to control samples which could be attributed due

to the presence of probiotics Pediococcus pentosaceus and prebiotic Gracilaria corticata causing

improved digestion and nutrient absorption in zebrafish.

X M

I






Fig.4. Feed Conversion Ratio in the control and in the formulated diet supplemented groups

Feed efficiency designates the correlation between the intake of feed and the body weight gain

[32].Feed efficiency is better in the slots treated with formulated diet compared to control slot(Fig.5).

Though during initial days of treatment, significant differences were not noted, with the progression

of the treatment time, Pediococcus pentosaceus treated batchshowed a gradual raise of feed

efficiency ratio from 0.33 ±0.01g to 0.52 ±0.01g. Gracilaria corticata supplemented

groupsexhibited a range from0.32 ±0.01g to 0.49 ±0.02g, Control groups was in the range between

0.315 ±0.015g and 0.46 ±0.01g. Batch treated with combination of probioticPediococcus

pentosaceus and Gracilaria corticata exhibited a feed efficiency range from 0.31 ±0.01g to 0.45

±0.02g. Complementary activity of both Pediococcus pentosaceus and Gracilaria corticata was

visualizedduring the growth of zebrafish.

Fig.5. Feed Efficiency Ratio in the control and in the formulated diet supplemented groups

0

0.5

1

1.5

2

2.5

3

3.5

4

DAYS 5 10 15 20 25 30

Fee

d c

on

ver

sio

n r

ati

o (

g)

Pediococcus pentosaceus

Control

Pediococcus pentosaceus with Gracilaria corticata

Gracilaria corticata

0

0.1

0.2

0.3

0.4

0.5

0.6

DAYS 5 10 15 20 25 30

Fee

d e

ffec

iency

(g)

Control









Results of SGR also supported the influence of fermentative bacteria on the growth rate of zebrafish.

Pediococcus pentosaceus had a boost up in steady growth to0.47 ±0.01, followed by Gracilaria

corticatasupplemented trial(0.44 ±0.01)(Fig.6).

Fig.6. Effect of formulated diet on the Specific Growth Rate

Pediococcus pentosaceus with Gracilaria corticata also exhibited values close to Gracilaria

corticata supplemented group (0 - 0.44 ±0.01).Lowest value was observed in the control slots (0 -

0.31 ±0.01). Results of SGR was in correlation with the results of weight gain and length gain of

the treatment groups. Pediococcus pentosaceus supplemented diet showed enhanced weight and

length gain of about 26.67 ±0.01 and 4.142 ±0.01 respectively at the end of the study period(Fig.7

and Fig.8). Although presence of Gracilaria corticata does not influence the length of the fish,

control had the least weight and length gain compared to other treatment groups.

Fig.7. Effect of formulated diet on the Percentage weight gain in the control and treatment groups

0

0.1

0.2

0.3

0.4

0.5

0.6

DAYS 5 10 15 20 25 30

Sp

ecif

ic gro

wth

rate

( %

)

Control




0

5

10

15

20

25

30

DAYS 5 10 15 20 25 30

Weig

ht

ga

in p

erc

en

tage (

%)




Control






Fig.8. Effect of formulated diet on the length gain in the control and treatment groups

The survival rate was high in Pediococcus pentosaceus supplied batch compared to other samples

(Fig.9). Results of survival rate symbolize the immunity conferred to zebrafish due to the influence

of Pediococcus pentosaceus. Probiotic organismscould have colonized through the outer layer of

the fish and in addition they might secrete products such as exopolysaccharides, lactic acid,

bacteriocin like substances that prevent colonization of pathogenic bacterium [33]

Fig.9. Survival rate in the control and in the formulated diet supplemented groups

Antioxidant profile of zebrafish treated withGracilaria corticataandPediococcus pentosaceus

Expressions of antioxidant enzymes are synergistically controlled by the nutrition and the balance

of pro-oxidant and antioxidants factors. Oxidative stress causes damage to host due to the reduced

expression of antioxidant enzymes [30]. Expression of antioxidant enzymes is directly proportional

0

1

2

3

4

5

DAYS 5 10 15 20 25 30

Len

gth

gain

per

cen

tage

( %

)

CONTROL




0

20

40

60

80

100

120

DAYS 5 10 15 20 25 30

Per

cen

tage

of

surv

ival

( %

)

Control









to the antioxidant content in the feed [11] stated that SOD dismutates superoxide anion to H2O2.

Pediococcus pentosaceus and Gracilaria corticata simultaneously possessed equal capacity for

SOD activity in the fish samples. Wu [28] reported increased SOD activity in brown algae

supplemented samples. With time, CAT activity deceased gradually, but remained higher than that

of the control category. Takano [36] stated that the increase in CAT activity in muscle suggests

higher level of oxidative stress possibly due to the ROS-derived lophocladine detoxification.

Catalase is the most vital enzyme in detoxifying H2O2. Glutathione peroxidase (GPx) catalyzes the

reduction hydrogen peroxide and lipid peroxides and is denoted as a competent defending enzyme

against lipid peroxidation [46]. GPx exists in muscle and involves in the response of removal of

H2O2 and is documented as one of the most significant antioxidant that barricades against oxygen

toxicity in organisms [43]. Wu [28] confirmed that GSH plays an innermost role in the detoxification

of ROS, which can be generated as by-products throughout the biotransformation of a mixture of

endogenous and exogenous substances. The GSH and GPx antioxidant activity is significantly high

in Pediococcus pentosaceusalone than the other samples such as Gracilaria corticata, Pediococcus

pentosaceus with Gracilaria corticata and control treated fishes. Gracilaria has been reported for

its antioxidant nature both in vitro and in vivo[37].Effective antioxidant activity in the sea weed

treated group could be due the presence of phenolic compounds and polysaccharides. Differential

expression of antioxidant enzymes among the treatment groups is due to the difference in the

antioxidant nutrients in the feeds [38]. Similar trend in enzyme activity was not noted among the

treatment groups as antioxidant enzymes react synergistically with each other which has an impact

on radical quenching [39]. Differential expression of the antioxidant activity is attributed to the age,

susceptibility to environmental stress and pathogenic stress [11]. Though the ability to restrict the

oxidation by probiotic organism is not clearly understood, symptoms of probiotics elevating the

antitoxidases activity in the host is reported [32]. Cultures of lactic acid bacteria comprising the

intestinal microbiota have exerted radical quenching property [40]. Better antioxidant enzyme

system were recorded in probiotics species which are capable to deliver antioxidases such as

catalases [41], SOD [43], GSH [29]. Supplementation of probiotics have been reported for

augmentation of antioxidant metabolites [15].






Fig.10.Effect of formulated diet on the expression of antioxidant enzymes in the

control and treatment groups

4. CONCLUSION

This study has demonstrated that probiotic Pediococcus pentosaceus has a momentous influence on

the survival rate, feed conversion ratio as well as on the final mean weight of the zebrafish.

Fermentative bacteria plays a crucial role in the breakdown of metabolites and increases the

bioavailability of the nutrients which is a crucial factor for aquaculture feed designing. Bioactive

compounds from this less studied seaweeds have emerged as an appealing attribute in designing

functional feeds.

ACKNOWLEDGEMENT

The authors wish to acknowledge Fr.Jobi for his support in providing infrastructure facilities to

perform experiments.

CONFLICT OF INTERST

Authors have no conflict of interest.

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original research article doi: 10.26479/2019.0503.20 ... · polyunsaturated fatty acids (pufas),...

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