CHANGES IN BACTERIAL FLORA DURING STORAGE OF SELECTED AQUACULTURE

FEED AND FEED INGREDIENTS

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

MASTER OF FISHERIES SCIENCE (MARICULTURE)

OF THECENTRAL INSTITUTE OF FISHERIES EDUCATION

(DEEMED UNIVERSITY)MUMBAI-400 061

BY

RAJANNA, M. R.(MC-65)

■ IVI 1JT V-J q-I C A R

CENTRAL MARINE FISHERIES RESEARCH INSTITUTE INDIAN COUNCIL O F AGRICULTURAL RESEARCH

C O C H IN - 682 014, K E R A LA INDIA.

JULY 2002

DEDICATED TO

THE ALMIGHTY

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CENTRAL AlARINE FISH ER IES RESEARCH IN ST IT U IE POST BOX No. 1603, ERNAKULAM, COCHIN- 682 014

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CERTIFICATE

Certified that the thesis entitled “ CHANGES IN B A C TE R IA L FLO R A DURING

STORAGE O F SELECTED AQUACULTURE FEED A N D FEED INGREDIENTS”

is a record o f independent bonafide research work carried out by S h rL Rajanna,

M. R., during the period of study from September 2000 to August 2002 under our

supervision and guidance for the degree of M a s te r o f F ish e rie s Science

(M aricu lture) and that the thesis has not previously formed the basis fo r the

award of any degree, diploma, associateship, fe llowship o r any other title.

Principal Scientist Head, PNP D iv is ion & GIG, PGPM CMFRl, Kochi-14

M a jo r A d v is o r/ C ha irm a n

Dr. (M rs .) IMELDA JO S E P HScientist PNPD CMFRl, Kochi-14

Advisory Committee

D r (M rs.) SOMY KU R IAKO SEScientist, FRAD CMFRl, Kochi-14

DECLARATION

1 hereby declare tha t the thesis entitled "C H AN G ES IN

BACTERIAL FLO R A DURING STORAGE OF SELEC TED AQ U AC ULTUR E

FEED AND FEED INGREDIENTS” is an authentic record of the w ork done by

me and no part thereof has been presented fo r the award o f a n y degree,

diploma, associateship, fellowship, o r any similar title .

( '

Date: July 2002 (RAJANNA, M.R)

Place: Kochi M .F .Sc. Student

C entra l Marine

F isheries Research Institute

ACKNOWLEDGEMENTS1 express my gratitude and indebtedness to my Major Adviser, Dr.

(Mrs.) Imelda Joseph, Scientist, PNP Division, CMFRI, Kochi, for her valuable advice,

timely help and support throughout my work. It is indeed my pleasure to express my

gratefulness to my co-guide, Dr. R. Paulraj, Principal Scientist, Head, PNP Division

and OIC, PGPM, CMFRI, Kochi, who has been a perennial source of encouragement

throughout my post-graduate programme at CMFRI. I express my sincere thanks to

my co-guide, Dr. (Mrs.) Somy Kuriakose, Scientist, FRAD, CMFRI, Cochin.

I express my extreme sense of gratitude to Prof. Dr. Mohan Joseph

Modayil, Director, CMFRI, Kochi, fo r providing me with the facilities to carryout the

research work in the institute and for his advice and encouragement.

My sincere gratitude is due to Dr. (Mrs.) K.S. Sobhana, Scientist (SS)

and Dr. K. C. George, Principal Scientist, PNP Division for their timely help. I am

thankful to Shri. S. Nandakumar Rao and Shri. T. G. Thippesw/amy and Shri. K. K.

Surendran, Technical Assistants, PNPD, CMFRI, Kochi, for their cooperation and

help during my research v/ork.

I express my persona) gratitude to Shri. Ramalinga, Ph.D. Scholar,

CMFRI, Kochi, for his valuable support throughout my research work. My sincere

thanks are due to Shri. Susanth Kumar Behera, Bhavani, C.N.,Divya,P.R., Anand, C..

Govindaraju, G. S., Vikas Kumar., Edwin, S., Smitha and Seema (all my classmates)

for being strength to me during my study at CMFRI. I also express my personal

thanks to Shri. Joice Abraham, Susanth Kumar Patra, Chandrashekar, A. (Ph.D.

Scholars) and Honnananda, B.R. (I M. F. Sc. Student) for their help in typing the

manuscript.

I am greatly indebted to CIFE, Mumbai and ICAR, New Delhi for

providing me with the institutional fellowship for M.F.Sc.

I am extremely grateful to my parents for being constant support to me

throughout my studies.

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ABSTRACT

The bacteriological changes in two shrim p feeds and th ree protein rich ingredients on

short-term storage fo r 60 d wias studied. Feed 1 was a commercial sh rim p 'grower'

diet and Feed (I w as a cost effective d ie t for shrimp fornnulated at CMFRI, Kochi. The

protein-rich feed ingredients used were fishmeal, g roundnut oil cake and soybean

flour. Sealed sam ples were stored under identical conditions at room temperature

and were analysed fo r bacterial population at day 0. day 30 and day 60. The total

plate count of feeds and feed ingredients was well w ith in the reported non-degrading

limit. Gram-positive cocci (42.8- 73% ) were dominant in all the five sam ples. During

storage gram -positive cocci showed slight reduction, while, gram -negative rods

showed marginal increase. Five hundred isolates from feed and feed ingredients

were used for fu rthe r characterization and identification. The major genera identified

were Micrococcus spp., Arthrobacter spp. and Bacillus spp. Besides enum eration of

indicator bacteria such as Vibrio spp., faecal streptococci, Escherichia coli and

Staphylococcus aureus were quantified in the feeds and feed ingredients. Feed II

was devoid o f all the above Indicator forms, except Vibrio spp., while, Feed I showed

all the above groups in vary/ng numbers. Short-term sto rage (up to 60 d ) d id not have

much effect on bacteria l load in feeds and feed Ingredients.

CONTENTSCERTIFICATE

DECLARATION

ACKNOWLEDGEMENTS

ABSTRACT (IN HINDI)

ABSTRACT (IN ENGLISH)

1 INTRODUCTION 1

2. REVIEW OF LITERATURE 4

2.1. Feed And Feed Management In Aquaculture 4

2.1.2. Quantitative and Qualitative Occurrence of

Bacteria in Feed 5

2.1.3. Bacterial Diseases of Shrimp and

Seafood-borne Human Health Problems 8

3. MATERIALS AND METHODS 10

3.1. Quantitative Analysis of Bacteria In Feeds

and Feed Ingredients 10

3.2. Qualitative Analysis of Bacteria in Feeds

and Feed Ingredients 21

4. RESULTS 25

4.1. Quantitative Variation of Bacterial Population

in Feeds and Feed Ingredients on Short­

term Storage 25

4.2. Qualitative Analysis of Bacterial Genera

in Feeds and Feed Ingredients 26

5. DISCUSSION 41

5.1. Qualitative Study of Bacteria Present in Feeds

and Feed Ingredients 41

5.1.1. Qualitative Study of Bacteria Present in Feeds

and Feed Ingredients 44

6. SUMMARY 47

7. REFERENCES 49

LIST OF TABLES

1. Variation of Bacterial Populations in

Feed I 28

2. Variation of Bacterial Populations in

Feed II 29

3. Variation of Bacterial Populations in

Fish Meal 30

4. Variation of Bacterial Populations in

Groundnut Oil Cake 31

5. Variation of Bacterial Populations in

Soybean Flour 32

LIST OF FIGURES

1. Percentage Occurrence of Different

Bacterial Genera in Feed 1 33

2. Percentage Occurrence of Different

Bacterial Genera in Feed II 34

3. Percentage Occurrence of Different

Bacterial Genera in Fish Meal 35

4. Percentage Occurrence of Different

Bacterial Genera In Groundnut Oil Cake 36

5. Percentage Occurrence of Different

Bacterial Genera in Soybean Flour 37

1. Total Plate Count (TPC) in Zobell Marine

Agar in Fish Meal 38

2. Total Plate Count (TPC) in Zobell Marine

Agar in Groundnut Oil Cake 38

3. Staphylococcus aureus, Gram-Positive Cocci

in Soybean Flour 39

4. Staphylococcus aureus, Gram-Positive Cocci

in Feed I 39

5. Gram-Positive Cocci in Fish Meal 40

6. Gram-Negative Rods in Fish Meal 40

LIST OF PLATES

INTRODUCTION

1. INTRODUCTION

C apture fisheries yie lded a steady increase in tonnage fo r human

consumption from 1960s to tlie iate 1980s, but production has rem ained relatively

constant at approximately GO m illion metric tons (m m t) since then (FAO , 1995).

Aquaculture production has doubled since 1990 (Aiken and Sinclair, 1995), and this

remarkable trend will have to continue to meet expected future seafood demand.

A iken and Sinclair (1995) predict tha t by the year 2025, there will be a shortfa ll of 55

m m t between seafood harvest o f 60 mmt and dem and for seafood o f 115mmt.

Csavas (1994) calculated that aquaculture production w ould have to increase by 3.5

tim es over current levels to meet the expected shortfa ll. It is in this context, the

technical advances achieved in aquaculture practices came up as a boom to

aquaculture industry. Shrimp fanning witnessed spectacular growth during the 1980s.

But, majority o f shrim p farming countries in Asia had suffered heavy production

losses due to outbreak of diseases, starting with the collapse of Ta iw anese shrimp

industry in 1988. India too became a victim of viru lent viral diseases since 1994

onwards. It is believed that unscientific and unscrupulous uses of resources have led

to such a collapse. Thus, the only so lution for the p resen t problem encountered by

entrepreneurs all over the country Is to adopt sound management at all steps of

aquaculture.

Nutritional cost in shrim p culture am ounts to about 40 to 60% of the

operational cost. It is by now w ell established that success of sh rim p farming

depends upon sc ien tific and sound management at all facets of aquaculture. This is

especially im portant as the focus is getting shifted from extensive to modified-

extensive and sem i-intensive culture systems. The shrim p feed industry has to shift

its focus to cost effective and eco-friendly formulations. As in the case o f any other

manufacturing enterprise, a number o f management interventions are im portant in

aquafeed industry also. Ingrediont quality is of prime im portance in feed manufacture.

Besides, feed should supply all the essentia l nutrients a t required level to sustain the

best growth and feed conversion. Feed derived waste a lso should be m inim um , since

it can cause deleterious changes in w ater quality and lead to eutrophication of the

system, which in turn would enable proliferation of pathogenic m icroorganism s or

create a stressed environmani, reEiulting in poor growth o f the cultured organism s.

Prepared aquafoods are perishable products, which require utmost

care during storage and supply. P roper storage o f feed ingredients and feed is

important in m aintaining tfie quality. Feed processors attempt to fo rm ula te and

manufacture aquafeeds aimed at extending their she lf life. During storage, in many

w ays feed quality can be dim in ished, including physical wastage, chemical

deterioration and microbial degradation.

S ince feed is biological in nature with nutrients, it is equa lly capable of

supporting the growth of contaminating bacteria. Two types of bacterial food borne

diseases are recognized; intoxication and infection. Feed borne bacterial intoxication

is caused by the Ingestion of food containing bacterial toxin resulting from bacterial

growth in the feed. On the other hand, food borne infection is caused by ingestion of

food containing viab le bacteria, w hich then grow and establish in the hos t resulting in

diseases. Even though certain bacteria are ubiquitous in nature, o thers enter the

system through contaminated feed. Th is principle is applicab le to aquafeed as well as

to human food (seafood items). Jansen (1970) has reported that fish m ay be more

important vector o f human diseases than has been realized. Uncontro lled and

im proper feed management could upset the natural flora in the cu ltu ra l systems,

leading to heavy loss. Even though she lf life of aquafeed is not well established, it is

being presumed that a well-processed feed could be stored for s ix months by

follow ing proper storage measures, w ithout any nutrient loss. Small fa rm ers store left

over feed for subsequent crops In case of a crop fa ilu re or when it is bought in

excess. Does th is practice have any implication on the bacteria! flora in the feed?

W hat are the sources of certain bacterial contam inants in feed as well as

environment? P resent investigation is being carried out in this line fo r a valuable

conclusion. There is hardly any w ork being conducted on these aspects o f aquafeed

and thus it will be o f use to farmers as well as nutritionists in designing proper feed

management practices.

The m ain ob jec tives of this work are:

1. T o study the bacteria l profile during short-term storage of feed and

feed ing red ien ts .

2. T o isolate and iden tify bacterial s tra ins from the fee d and feed

ingred ients.

2. REVIEW OF LITERATURE

2.1. Feed and Feed Management in Aquaculture

Like any other organism, shrimps also need nutrients tha t w ill provide

fo r growth, m aintain ing life and resistance to diseases (Pascual, 1983). Extensive

studies have been carried out on shrim p nutrition during the past tw o decades as

m ay be seen from the reviews of New (1976, 1987), Pascual (1983), Akiyam a and

Dominy (1989) Paulra j (1990). In the natural environm ent, shrimp derive nutrients

from a variety o f food sources, both o f plant and anim al origin, like a lgae, diatoms,

sm all crustaceans, molluscs, polychaetes and fishes tha t are found at the bottom of

the aquatic system s (Gopalkrishnan, 1952; Pascual. 1983; Baily-Brock and Moss,

1992). They a lso consume significant amounts o f de tritus aggregates, w h ich serve

as important carrie rs of attached microorganisms that constitute important

components in shrim p diet. There are, however, considerable differences o f opinion

on the food va lue o f bacteria, w hich are ingested by shrimp along w ith detritus

(Chong and Sesekum ar, 1981). C erta in bacteria like Purp le Sulphur Bacteria (PSB)

and Rhodobacter capsulatus have been employed in the artificial feed o f fish larvae

(Bhosle, 1997).

Paralle l to intensillcation of shrimp farm ing industry, nutritionally

balanced and h igh quality compounded diets were developed and commercialized

(New, 1976,1987,1990; Shigueno, 1984; Akiyama and Dominy, 1989; Tacon, 1993;

A li, 1997). Besides commercial aquafeeds, many fa rm ers manufacture the ir own

feeds to reduce the cost of farming to considerable extent.

Se lection of proper ingredients is decisive to ensure nutritionally- rich,

cost-effective and environment-friendly shrimp feeds. Many have reviewed the

conventional feed ingredients and noted fish meal, squid meal, shrimp meal, other

crustacean meals, soybean mtsal, cerea ls and yeast as the ones extensively used in

the manufacture o f commercial shrim p feeds in Asian countries (Raveen and Walker

(1980), New (1987), Tacon, (1993), Akiyama (1991). Paulraj (1993, 1998) and FDS

(1994).

The best feed produced in the world proves to be worthless if it is not

properly managed. Feed mannoement is a sequential process com pris ing feed

selection, handling and storage, feeding regimes and adjustments to feed ing rates

(New, 1990; De- la-Cruz, 1989; Tacon, 1993; Cruz. 1994; Akiyama and Chwang,

1995; Jory, 1995). Poor storage and handling o f feeds results in product

deterioration, reduced feed atlractability and palatability, nutritional defic iencies and

disease out b reaks (Jory, 1996). The effects o f am bien t tem peratures on the

chemical characteristics or nutritional value of shrim p feeds during storage was

reported by De la Cruz et al. (1989). Divakaran e t a i (1994) have demonstrated

effects of long te rm storage.

2.1.2. Quantitative and Qualitative Occurrence of Bacteria In Feed

S tud ies on fish feeds have shown that commercial feeds do contain a

mixed m icroflora including bacterial species potentially harmful to m an and fish

(Trust, 1975). An im al feed is a recognized vector in the transmission o f pathogens to

live stock (Me C apes et al., 1009; H inton and Mead, 1992). The ingredients used in

feed formulation partly determine the nature of its m icroflora (Trust and W ood, 1973).

F ish feeds support grow th of a wide varie ty of bacteria (Trust and

Money, 1972). Feeds are known to be pathogenic to fish when the m oistu re content

exceeds 25% (Ja in, 1998). The enum eration of aerobic hetereotrophic bacteria l load

o f feed (cow-dung and grass) used at AIuu, Nigeria, indicated that, it had higher

count of 5.6 ± 0.4 ><10® cfu/g compared to that of sedim ent 8.4 ± 1.6 x 10^ c fu / g and

w ater 4.7 ± 1.0 xIO ^ cfu/ml (O pokwasili and Alapiki, 1990). Similar va lues for 9

indigenous and 16 imported shrimp feed have been reported from India (Kalaimani et

al., 1998) and fo r tilap ia feed from N igeria (Ogbodeminu e t al., 1991).

H ow ever, the viable m icrobia l counts in the range of 3.7x10^ to 1.4x10“*

/g of diet (Yoshim izhu, 1980) and total heterotrophic o f 2.2 x lo^/g d ie t (Sera and

Kimata, 1971) have been reported. On the contrary R ingo and Strom (1994) recorded

the total viable counts as low as 3.7 x io ^ /g of roe diet and 6.7 xiQ ^/g o f commercial

feed. Trust and w ood (1973) reported 10^ to 10' aerobic bacteria per gram o ff is h diet.

There are even instances where the diet for aquarium fishes was found to be sterile

(Trust and Money, 1972). Bissetts, et al. (1998), studied the characteristics of

microbes of a form ulated abalone (Haliotis laevigata) diet. Bacterial num bers on

unimmersed d ie t and diet immersed in sterile seawater for 2 and 4 days were

negligible. Bacteria proliferated after 2 days immersion in seawater w ith abalone (1.2

X 10^ cells/mm^) and without abalone (5.7 x 10* cells/mm^). Murago e t al. (1987)

have reported the total bacterial counts o f 2.1 x 10^ c fu /g for live d iets (ro tifer and

brine shrimps) and 1.2 x lO'^cfu/g fo r artificial diets. The same author a lso reported

differences in the bacterial counts o f these diets on tw o different media. The mean

bacterial counts w ere 2.1 x 10'' c fii/g w et weight and 2.2x10® cfu/g wet w e igh t for live

diets and 1.2 x io '* cfu/g and x io cfu/g for artificial diets respectively in Zobell

agar and Bromothymol blue agar.

T rust (1971) examined 47 samples o f fish diets belonging to nine

different production batches from tw o commercial hatcheries. The mesophilic,

psychrophilic and thermophilic bacterial counts were found to be in the range of 10^

tolO^/g., <10^ to 10^ and 10^ to lO^g respectively. M any o f the m esophilic bacteria

(10^ to10®/g) w ere able to grow on the media, which contained 3% (w /v) fish diets.

Aerobic and anaerob ic spore forming bacteria (<10^ to lO'^/g) were also present in the

feeds analysed and the faocal Streptococci count ranged between 10^ and 10^/g

feed.

T rust and Money (1972) have studied bacterial populations of 25

commercial diets formulated and offered to aquarium fishes. The to ta l count of

aerobic mesophilic bacteria in 24 sam ples ranged from 9.0 x lO ^to 1.3 x lO ^ /g of diet

and Enterobacteriaceae were detected in 23 of the feeds analyzed. The ir Most

Probable Number estimates ranged from 40 to 1100/ 10 gram of diet w ith a mean of

1023/ lOg. V iable heterotropliio bateria ranged from 10® to 10® cfu/ m l o f Artemia

naupli homogenate (Lopez-Torres and Lizarraga-Partida, 2001). The sam e authors

also isolated bacteria on TCBS m edium with hatched Artem ia cysts o f commercial

brands. The TCBS population has show n an inverse correlation (r^= 0 .5775,a= 0.05)

w ith cyst age, va lues comprising betw een 10^ and 10^ cfu/m l. The standard two-day-

old A.franciscana had 24000 ± 10700 cfu (mean plus o r minus STD) pe r anim al with

presumptive Vibrio spp. and h tiem olylic bacteria constituting 58% and 10% of the

total respectively (O lsen, etai , 2000).

G reater variations in the dominant bacterial flora associated v^itii the

feeds have been reported (Ogbondeminu et al., 1991). The predom inant bacterial

genera associated w ith feed (rice bran) were Pseudom onas with the incidence of

25% along with 25% gram positive bacteria, representd by Streptococcus faecalls

(12%) and l^ ic rococcus (15%) and Staphylococcus spp (10%). They cou ld identify

80% of feed m icroflora in culture w ater as well. Yoshimizhu (1980) reported

diminance of Pseudomonas, Coryneform s and M icrococcus with the percentage

incidence ranging from 13-20, 44-79 and 4-50 respectively for the diet, w hich was

used to feed the fry and fingorlings o f salmons. R ingo and Strom (1994) reported

dominance of Pseudomonas spp. (31.6% ) in capelin roe d ie t and Enterobacteriaceae

(26.9%) in a com m ercia l diol. Dom inance of gram-positive bacteria has been noticed

in the isolates o f feed (cow dung and grass) with B acillus to the extent o f 56.25%.

O ther bacteria! species' such as Streptococcus, Staphylococcus, A erom onas and

Escherichia spp. constituted 6.25% each (Opokwasili and Alapiki, 1990). However,

Hamid et al. (1978) reported the presence of 100% Bacillus in mullet d ie ts . Among

the bacteria isola ted from live diets (brine shrimp and Rotifer), Pseudom onas was the

m ost predominant bacterial genus, constituting 48,3% and Vibrio, Cytophaga, and

Moraxelia constituting 11.4%, 10.2% and 8.1% respective ly (Muroga e t al., 1987).

The same authors reported that gram-positive bacteria (48.4% ) dominated in artificial

commercial diets.

On TC BS medium, Lopez-Torres and Lizarraga-Partida (2000) isolated

bacteria associated with hatched A rtem ia cyst of com m ercial brands. O f the 617

isolates, 94% w ere gram posillve and only 6% were gram-negative, but w ere oxidase

negative.

2.1.3. Bacterial Disoases of Shrimp and Seafood-borne Human

Health Problems

Increasing occurrence o f shrimp diseases in aquaculture system s and

public health risks have been a serious problem In sh rim p farming countries of the

world (Jory, 1996; Karunasagar et al., 1998). Though a number of bacteria l genera

have been found to constitute llie norm al microflora o f the cultured and farmed

animals, members o f only 10 genera, namely >Aero/T?onas, Alcaligenes, Alteromonas,

Flavobacterium, Flexibacter, Leucothrix, Moraxella, Mycobacterium, Pseudomonas

and Vibrio have so fa r been reported to be pathogenic in penaeid shrim p (Fulks and

Main, 1992; Shariff and Subhaslnghe, 1992; Lightner, 1993; Otta et al., 1998). Among

the genera, Vibrio are numerically the most abundant and com m only m et with

bacterial pathogens in cultured shrim p (Karunasagar and Indrani, 1995; O tta et a!.,

1998)

H um an health hazards associated w ith consumption o f fish and

shellfish contam inated with bacterial and pathogenic m icroorganisms are well

recognized (Shewan, 1961; Liston, 1980; Austin and Austin, 1987; Inglis et a i,

1993a&b). Many outbreaks of hum an disease have been associated w ith marine

fishery products (Reilly, 1098), especia lly those from wild stocks, and similar

problems can resu lt from aquaculture due to poor m anagem ent (GESAMP, 1991). A

number of pathogenic or potentially pathogenic bacteria such a V.parahaemolyticus,

V.vulnificus, V .cholerae and Salmonella sp., which are often implicated w ith food

poisoning or ou tb reaks like gastroenteritis, typhoid, cho le ra etc. in hum an beings,

have been encountered in significant levels in cultured shrim ps and their environment

(Fonseka, 1990; Karunasagar o/ al., 1991; Reilly and Tw iddy, 1992; Nayyarahamad

ef al., 1995; Surendran al., 1995). Consumption o f raw or partly cooked shrimp

from affected areas is likely to cause diseases due to pathogens or tox ins (Shuval,

1986). Consumption o f raw shrimp w as identified as the main mode o f transmission

in the explosive outbreak of cholara witnessed in Philippines in 1961 (GESAMP,

1991) and in m any other countries in subsequent years (Reilly, 1998). Karunasagar

et al. (1990) reported that V.parahaemolyticus is the commonest am ong the

potentially pathogenic Vibrio spp. encountered in Indian seafood. Though the

ingestion of a sm all number of V.parahaemolyticus is apparently harmless, in as little

as two hours they can multiply resu lting in a population capable of causing food

poisoning. Food poisoning duo lo V. parahaemofyticus has been reported from many

parts of the world (Aoki and Chun. 1967; Quadri and Zuberi, 1977).

As reviewed above, m any have w orked on various aspects of

aquafeed, but a lacuna exists In lha she lf -life studies o f aquafeed, especia lly on the

bacteriological changes during storage of shrimp feeds in tropical countries. An

attempt has been m ade in the present investigation to study the changes in bacterial

flora during short-term storago of w ide ly used shrimp feed and feed ingredients.

MATERIALS AND METHODS

3. MATERIALS AND METHODS

The change in the bacteria l population during short-term (0- 60d)

storage of two shrim p feeds and three protein rich feed ingredients w as studied. Of

the two feeds, Feed 1 was a com m ercial grower shrimp feed collected from a farmer

and Feed II, a cos t effective formulated diet from Central Marine Fisheries Research

Institute. The three feed ingredionts w ere fishmeal, g roundnut oil cake and soybean

flour.

One kg each of feed and feed ingredients was used fo r the present

study. The feed ingredients were purchased from the local market. Dry fish (anchovy)

and GNOC were dried in hot hair oven at 60°C overnight and ground in a domestic

grinder to make fine particles of <1 m m size. Soybean flou r was used as such since

the particle size w as <1 mm.

The feed and feed ingredients were stored at room temperature

aseptically in sealed black polythene bags to avoid any aerial contamination.

Sampling was done on day 0, day 30 and day 60.

3.1. Quantitative Analysis of Bacteria in Feeds and Feed

Ingredients

3.1.1. Quantitative analysis

The quantitative analysis includes Total P late Count, Staphylococcus

aureus count. F a eca l streptococci count, Escherichia co li count, Vibrio spp. Count.

3.1.1.1. Preparation of sample

Ten gram s of the sam ple (feed and feed ingredients) w as aseptically

weighed into a sterile mortar and ground with pestle (sterilized by burning with

alcohol and flam ing). Ninety ml saline w as added into m orta r and mixed uniform ly by

10

agitating and a llow ed for 15 niln. Th is becomes 10 tim es dilution. Using a sterile

pipette, 1 ml o f the supernatant was aseptically transferred into 9 ml o f norm al saline.

This becomes 10^ dilutions. Sim ilarly, further dilutions w ere made w henever required

for the inoculation.

3.1.1.2. Total plate counts (TPC)

O ne ml each of the appropriate d ilu tions was pipetted to sterile

petridishes taken in duplicate for each dilution. About 15-18 ml molten and cooled

(45°C) Zobell m arine agar was poured to each plate, m ixed well with the inoculum

and allowed to se t fo r 30 min. The plates were incubated at 37° C fo r 48 h in an

inverted position. A fte r the Incubation, the individual colonies were counted. The

average count o f the duplicates w as calculated. The TPC per gram o f the sample

w as calculated as follows:

TPC /g o f the sample = {Average count x dilution factor)/ W e igh t of the

sample (g)

3.1.1.3. Staphylococcus aureus count

Appropria te dilutions (0.1m l each) were pipetted to pre-set and dried

Baired Parker a ga r plates, taken In duplicate for each dilution. The inoculum was

spread well ove r the surface using the sterile bent g lass rod. The p lates were

incubated at 37° C in an inverted position for 48 hr. B lack colonies w ith thin white

margin and a zone o f clearance around colonies w ere counted as Staphylococcus

aureus. The average count of the duplicates was calculated. The Staphylococcus

aureus count per g sample was ca lcu la ted using the re la tion.

Staphylococcus aureus count/g sample = (Average count x 10 x

dilution factor)/ sample weight

11

Appropria te dilutions (0.1 ml each) were pipetted to pre-set and dried

Tergitol -7 taken in duplicate for each dilution. Thie inocu lum s were spread well over

the surface using a sterile bent glass rod. The plates w ere incubated at 37"^ C for 18 -

24 hr in an inverted position.

Non mucoids not raised, line yellow co lon ies with or w ithout occasional

rets brown at the centre and yellow zones around w ere counted as Escherich ia coH

.The average o f the duplicates w as calculated. E .co li per gram sam ple was

calculated using the relation:

Escherichia coli count per gram of the sam ple =Average count x10 x

dilution factor)/ sam ple weight.

However, to confirm them as E.coli the following procedure was also fo llow ed.

3.1.1.4a. Streak on Eosine- Methylene Blue (EMB) agar

EM B agar was melted and cooled to 50°C poured into petri d ishes and

allowed to set. T he set plates were dried and cooled to room temperature. Typical

yellow colonies from T -7plale(> were picked with a sterile platinum loop and streaked

onto EMB agar plates, by streak d ilu tion method, incubated at 37°C fo r 18-24 hr.

Well-isolated co lon ies, 2-3miTi d iam eter with a greenish metallic sheen by reflected

and dark purple centre by transmitted light were picked up and sub cu ltu red on to

Zobell agar slants and incubated at 37°C for 18- 24 hr.

3.1.1.5. Faecal streptococc\ count

O ne ml each of the appropriate dilutions w as pipetted to appropriately

marked sterile petridishes taken in duplicate for each dilution. About 15-18 ml of

molten Kenner Faecal Streptococci A ga r medium cooled to 45°C was poured on to

each plate. The m edium was mixed w ell with the inoculum and allowed to set for 30

min. The plates w ere incubated at 37°C for 48 hr. A ll surface and subsurface light

3.1.1.4. Escherichia coii count

12

pink to deep red co lour colonies w ere counted as Faeca l streptococci. The average

of the duplicates w as counted. Faeca l sfrep/ococci count per gram sam ple was

calculated as fo llows:

F aeca l streptococci count /gram sample = (Average count x dilution

factor)/sample weight.

3.1.1.6. Vibrio spp. count

Thiosulphate Citrate B ile Salt Sucrose (TC BS) Agar was m elted and

cooled to 45- 50*^C and poured to petridishes and dried. 0.1m l sample o f appropriate

dilution was inoculated on to preset TC BS plates. Inoculum was spread w ell over the

surface using a sterile bent glass rod. Plates were incubated at 37°C fo r 48 hr in an

inverted position.

Y e llow flat smooth colonies with opaque centres and transparent

peripheries, 2-3 m m diameter were taken as Vibrio spp. and were transferred to

Kligier Iron Agar and incubated at 37°C for 18 hr. V.cholerae will give acid butt in

alkaline slant.

V ibrio spp.counl/yrarn o f sample= (Average countxdilution factor

x10)/sample w e igh t weight.

3.1.2. Media used

The media and chem icals used for m icrobial study were the supply of

Hi Media Laboratories Ltd., Mumbai.

The list of media/ broth, staining solution and test reagents used are given below.

3.1.2,1. Alkaline peptone water

Peptone

Sodium chloride

DW

lO g

5g1000 ml

13

3.1.2.2. Baired Parker Medium

Basal medium

Tryptone 10 g

Beef extract : 5 g

Yeast extract : 1g

Sodium pyruvate : 12 g

Glycine 12 g

Lithium chloride : 5 g

Distilled water ; 1000 ml

Heated with agitation to obtain complete solution, cooled to 50°-60°C and

adjusted to pH 7.0 ± 0.2. Dispensed in 100ml quantities and sterilised at 15lbs for 15

min.

To the melted basal m edium of temperature 45-50°C follow ing solutions

were added asceptically.

1 % filte r sterilized solution of potassium te llurite : 1 ml

Egg yo lk emulsion : 5 ml

Mixed well and poured immediately in 15-ml quantities into petriplates.

3.1.2.3. Ec broth

Tryptone

Lactose

Bile sa lt

K2H PO 4

KH2 PO 4

NaCI

Distilled water

2 g

5 g

1 5 g

4 g

1 5 g

5g

100 ml

14

D isso lve d , adjusted to pH 6.9 ± 0.1 and dispensed in 5 ml quantities in

test tubes (w ith in ve rted Durhanm’s tube). Sterilised a t 10 lbs for 20 min.

3.1.2.4. EM B agar

15 lbs.

P ep ton e

L a c to se

K 2H P O 4

E o s in e Y

M e thy lene blue

A g a r

pH 7.1+0.1

H ea ted to dissolve the medium com pletely and sterilised in autoclave at

lO g

lO g

2 g

0.4 g

0 .065 g

1 5 g

3.1.2.5. Kenner Faecal Streptococcus Agar

P ep ton e ; 10 g

Y e a s t extract : 10 g

S o d iu m chloride : 5 g

S o d iu m glycerophosphate : 10 g

M a lto s e : 20 g

L a c to se : 1 g

S o d iu m azide ; 4 g

B rom ocreso l purple : 0.015 g

A g a r : 1 5 g

D W : 1000 ml

pH 7.2

H e a ted with agitation till all ingredients ge t dissolved and coo led to 45-

50°C and pH is adjusted to 7.2. Dispensed in 100 ml quantities and sterilised in

autoclave at 15 lbs fo r 15 min. Before pouring into p la tes, to melted and cooled to

(45-50°c) m ed ium , 1ml of 1% TTC solution (dissolved 1g triphenyl tetrazolium

chloride in 1 00m l sterilized dislllled w a te r by steaming fo r 1 hr) was added.

15

3.1.2.6. K ligler Iron agar

Peptone : 20 g

Yeast extract 3 g

Beef extract 3 g

NaCI : 5 g

Lactose 10 g

Glucose 1 g

Ferric citrate 0 g

Sodium thiosulphate ; 0 .3 g

Phenol red 0 .05 g

Agar 15 g

DW 1000 ml

pH 7.4+0.2

D issolved and dispensed in test tubes (15m l), plugged and sterilized at

121 °C, 15 lbs fo r 15 min. Kept In slanting position, so as to get about o f 2 .5 cm dept

3.1.2.7. Potassium tellurite

1% potassium leliurile provided by Hi M edia Laboratories Ltd., Mumbai,

w as sterilized by membrane filter techn ique and was taken in sterilised con ica l flask

kept in refrigerator a t 5-8°C. Whenever, it was required it w as taken and used.

3.1.2.8. Tergitol-7 agar medium

Peptone

Yeast extract

Beef extract

Lactose

Tergitol-7

Brom othym ol blue

lO g

6g

5 g

20 g

0.1 g

0 .05 g

16

Agar

DW

pH

1 5 g

1000 ml

7 .2±0.2

H eated to dissolvo m ed ium completely, cooled to 50°C and pH was

adjusted to 7.2 ± 0 .2 . Dispensed in 100 ml quantities and sterilised in au toc lave at 15

lbs fo r 15 min. Be fore pouring into sterile Petri p lates 0.4 ml of 15 2, 3. 5 TTC

previously sterilised by steaming, w as added.

3.1.2.9. Thiosulphate Citrate Bile Salts Sucrose Agar (TCBS)

Peptone 1 0 g

Yeast extract 5 g

Sodium thiosulphate 1 0 g

Sodium citrate lO g

Sodium cholale 3 g

Oxgall 5 g

Sucrose 20 g

Sodium chloride lO g

Ferric citrate 1 g

Brom othym ol blue 0.04 g

Agar 1 5 g

Boiled to dissolve Ihe m edium completely. Not autoclaved. Cooled to

5 0 °C and poured in to plates.

3.1.2.10. Tryptone broth

Trypone

NaCI

DW

pH 7.1±0.1

D ispensed in 5 ml quan tities in test tubes and sterilised a t 15 lbs for 15

I .O g

0 .5 g

100 ml

m in .

17

C om m ercially available Zobell marine B ro th was taken in appropriate

quantities(40.25grams per litre d is tilled water), into w hich 2% agar w as added.

Heated to dissolve the medium com p le te ly and sterilized a t 15 lbs for 15 m in.

3.1.2.11a. Zobell Slant proparation

Zobell marine broth in appropriate quantity w as taken and into which

Agar was added in appropriate proportion and heated to dissolve medium

completely. M edium w as transferred to test tubes and kept in slanting position and

allowed till it got solid ified.

3.1.3. Staining Solutions

3.1.2.11. Zobell marine agar

3.1.3.1. Gram’s crystal violet

Crystal violet

Ethyl alcohol

Am m onium oxalalo

DW

G ram ’s Iodine

Potassium iodide

Iodine crystal

D istilled water

2 g

20 ml

0 .8 g

80 ml

2 g

1 g

300 ml

3.1.3.2. Gram’s saffranine

Saffranine

E lhyl alcohol

D istilled water

1 g40 ml

360m!

Com m erciliy available G ram s staining kit w as used (HIMEDIA).

3.1.4. Indicator Solutions

3.1.4.1. Andrade’s indicator (for sugar fermentation test)

Acid Fuschsin

D istilled water

IN NaOH

0.5 g

100 ml

16ml

3.1.4.2. Bromothymol blue indicator

Sodium hydroxide : 40 mg

Bromothymol blue : 250 mg

DW 100 ml

40 m g NaOH was dissolved in 100ml d istilled water. Bromothymol blue

was added and dissolved.

3.1.4.3. Methyl red indicator

Methyl red

Ethyl alcohol

D istilled water

50 mg

150 ml

100 ml

M ethyl red was dissolved in alcohol and w as diluted with d istilled water.

19

Test: To a tube of 48-h old bacterial cu ltu re in iVlRVP m edium , 0.25ml of

MR indicator w as added. Developm ent o f red colour w as taken as positive.

3.1.4.4. Kovac's Indole reagent

P-dim ethyl amino benzaldehyde : 5 g

N- butyl alcohol or am yl alcohol : 75 ml

D issloved and kept in am ber coloured bo ttle overnight before test.

Test: To a tube o f 48h old tryptone broth culture. 0.2m l o f indole reagent w as added,

shaken and allowed to stand. Developm ent of pink o r red colour at the top indicated

a positive test fo r indole.

3.1.4.5. Vogue’s Parker test reagents

Solution A

a-N aphthol : 0 .25 g

Ethyl alcohol 5 ml

Solution B

KOH 2 g

D istilled water : 5 ml

Test: In a small test tube, 1 ml o f 48h old bacterial cu lture grown in

MRVP medium w as taken and 0.6 ml solution A and 0.2 ml solution B w as added,

shaken well, sm all crystals of creatine w as added, shaken and allowed to stand for 4

hours. Eosin p ink co lour indicated positive VP test.

20

Five chicken eggs w ere washed free o f d irt w ith vim and w ater, wiped,

dried and kept im m ersed in rectified sp irit in a 1L beaker fo r 2 hours. The alcohol was

drained into a bo ttle , eggs wore (aken out (one at a tim e), small opening w as made at

one end and all the egg while was poured out carefully. The egg yolk w as transferred

into a sterile con ica l flask, each egg provided around 15 ml of yolk. Equal volume of

sterile normal sa line was added, ag ita ted well and a llow ed to stand. 5 m l each of the

yolk saline was p ipetted into sterile test tubes plugged w ith sterile cotton and kept in

refrigerator (5 to 8° C).

3.1.5. Prepartion of Sterile Egg-yolk (50%) fo r Baired Parker Agar

3.2. Qualitative Analysis of Bacteria in Feeds and Feed

Ingredients

3.2.1. Isolation and identification of colonies

3.2.1.1. Isolation

A bou t 20-30 cultures from each feed g iving preferably a coun t o f 30- 60

well separated colonies on Zobell marine agar w ere randomly se lected and

transferred into peptone water tubes. The tubes were incubated at 37°C fo r 18-24 hr.

The broth cultures w ere purified on Zobell marine agar by streak dilution method.

3.2.1.2. Streak dilution method for purification of cultures

Pre se t and dried Zobell p lates were prepared. A loopful o f the culture

grown in peptone w ate r broth was streaked across the firs t quarter on the surface of

Zobell plates. T he loop was sterilized, continued as above to the third quarter and

similarly to the fo u rth quarter. The p la tes were incubated at 37°C for 18-24 hr. A well

isolated colony w as picked to a zobell slant, labeled and incubated a t 37°C for

21

18-24hr. The p u re culture thus o b ta in ed was m aintained at room tem p eratu re and

used for identification.

3.2.1.3. Identification

T h e cultures were initially differentiated in to gram positive and gram-

negative bacteria. G ram -positive b a c te ria w ere further identified upto the g en u s level

by following the identification s ch e m e o f Surendran and G opakum ar (1 9 8 1 ). The

identification s ch e m e Includes gram reac tio n and m icroscopy, catalase a n d motility.

3.2.1.4. Gram reaction and microscopy

P rep ara tio n of smear: O n ly young cultures (1 8 -2 4 h old) w e re used for

staining. On to a dust free m icroscopic glass slide a sp eck of young culture was

emulsified with a drop of sterile physio logical normal sa lin e into middle and spread

uniformly. Dried in the air. Heat fixed by passing the s lide three to four tim es through

the blue flam e o f B unsen burner.

S ta in ing: The slides w e re placed on a sta in ing bridge. T h e s m e a r was

flooded with g ram crystal violet for o n e m inute. W ashed w ith water. G ram iodine was

flooded for one m inute . W ashed w ith w ater. Gram's decolouriser was a d d e d for 30

seconds. W ash ed w ith water. C o u n ter stained with saffran in for one m inute . W ashed

and dried.

M icroscopy: Using oil im m ersion objective, slides were o b served under

microscope. C ells s ta ined violet, bluish violet or bluish purple were ta k en as gram

positive. Cells s ta in e d red w ere considered gram negative. S hape, s ize and

arrangem ent of ce lls w ere also noted . In case of gram -positive obsen/ation o f spore

formation was done.

22

3.2.1.5. M o tility o f bacteria

H a n g in g drop m ethod w a s adopted to o b s e rv e motility of b acteria under

microscope using h igh power o b jec tive . (40x, 45x, 5 0 x ). Sm all drop o f distilled water

was placed on th e m iddle of a c o v e r slip; a speck o f young culture from a g a r slant

was emulsified w ith it. A cavity s lide w a s taken; the m arg in o f the cavity w a s smeared

with paraffin je lly . T h e slide w as in v e rte d on the c o ver slip in such a w a y that the

cover slip gets a tta c h e d to the s lid e and on turning upside down, th e culture drop

hangs in to the cav ity . O bserved u n d e r a high power objective.

3.2.1.6. Catalase test

A s p e c k of young cu ltu re w as placed on a d e a n glass slide and flooded

with two drops o f 3 0 % of hydrogen peroxide. Evolution of gas from the culture

indicates positive tes t for catalase.

3.2.1.7. Oxidase test

O n e percent N: N: N: N- T e tram eth y l- p- phenylene diamine

dihydrochloride w a s freshly p repared and used for th e test. A sm ear o f tes t culture

was prepared an d streaked on to a filter paper im pregnated with Kovac's cytochrome

oxidase reagent us ing a sterile p la tin u m loop. D eve lo p m en t of blue co lour indicates

positive reaction.

23

RESULTS

4. RESULTS

4.1 Quantitative Variation of Bacterial Population in Feeds and

Feed Ingredients on Short Term Storage

T h e bacterial loads determ ined in two shrimp feeds and three protein

rich feed ingredients during 60 d storage are shown in Tables 1 to 5. The total plate

counts (TPC) of bacteria in feeds and feea ingredients showed variations during the

storage period o f 60 days.

In the commercial shrim p feed (Feed I), the initial TPC was 2 .7 6 5 x 10

cfu/g, which increased to 8.8 x 10' cfu/ g in 30 days and then reduced to 2 .73 x 10

cfu/g. The num ber of colony forming units (cfu) of Vibrio spp. almost doubled from 3.0

to 6.4 X 10 cfu/g on the 60"' day. The faecal Streptococci count also showed a

marginal increase. The Esclwrichia coli count remained constant throughout ie. 3

x io ’ cfu/g. Staphylococcus aureus appeared only on 30 days of storing (2 .2 x 10

cfu/ g), while it w as absent in the 6 0 ‘ day sampling (Tab le 1).

In Feed II, which is a cost effective formulated feed of C M F R I, the TPC

showed a decline after 60 days. T h e total count of Vibrio spp., however, increased

from 2 x 1 0 ’ to 4 x 10 ’ cfu/ g in 30 days and remained the same on day 60. Faecal

Streptococci,' E. coli and S. aureus were absent in this feed throughout the period of

observation (T ab le 2).

In all the three feed ingredients there w as variation in bacterial count

during storage. The total plate count o f fishmeal which was 5.12 x 10^ cfu/g on day 0

showed a decline to 2.7 x 10^ cfu/g in 60 days. The counts of Vibrio spp., E. coli (2 x

10 ’ cfu/gj and S. aureus (3 x 1 0 ^ cfu/gm ) did not show any significant variation along

with F. streptococci (6.5 x 10^ cfu/gm and 5.1 x 10^ cfu/gm). F. Streptococci and S.

aureus were absent in fishmeal on day 60 (Table 3).

The total plale count o f groundnut oil cake showed an increase from 3.3

X 10^ cfu/g to 5.1 X 10^ cfu/g in 30 days and then a reduction to 2.5 x 10^ cfu/g. Vibrio

25

spp. count rem ained almost the sam e (3 x 10' cfu/g) with an increase in 3 0 days (1.2

X 10^cfu/gm). Faecal streptococci and E.coli count showed little or no variation.

S.aureus though occurred in day 0. in the remaining samplings it was absent (Table

4).

Total plate count of soybean flour showed a decline from 3 .15 x 10

cfu/g to 2.75 x 10^ cfu/g on day 60. Vibrio spp. did not show much quantitative

variation and rem ained at 2 x 10’ cfu/g. Faecal streptococci and E.coli w ere absent in

the soyflour. S.aureus count remained a 3.5 x 10^cfu/gm in 30 d sample (Tab le 5).

4.2. Qualitative Analysis of Bacterial Genera in Feeds and Feed

Ingredients

For qualitative study of the bacterial isolates, Zobell M arine Agar was

used. For this purpose 500 isolates were m aintained. The tests done for

characterization include gram staining, motility, catalase, oxidase, nitrate reduction.

Micrococci were gram positive, aerobic cocci. The cells non-motile, and

were showing oxidative respiration. Arthrobacter w ere gram positive, aerobic,

asperogenus, coccoid bacteria, which were non-fermentative and nitrate reducing

forms. Bacillus spp. were facultatively fermentative rods, gram positive, motile and

showed catalase positive and were nitrate reducing.

Vibrio spp., Faecal Streptococci. E.coli and Staphylococcus aureus

were plated in their respective selective media. Vibrio spp. from TCBS m edia were

gram-negative rods, oxidase and catalase positive with round entire, glossy and

yellow or green colonies. Faecal streptococci plated on Kenner Faecal A gar medium

was gram positive, facultatively anaerobic, non-motile. catalase and oxidase negative

cocci. E, coli plated on Tergitol-7agar was gram-negative, motile, and oxidative. The

positive indole test was used as confirmative test. Staphylococcus aureus plated on

Baired Parker A gar medium is cocci form, gram positive, facultatively anaerobic, and

non-motile showing catalase test positive.

26

T h e percentage frequencies of occurrence of respective bacterial

colonies in feed and feed ingredients are shown in Figures 1-5. The commonest

genera was Micrococcus spp. in all the samples. O ther genera were Arthrobacter

spp and Bacillus spp. The occurrence of these genera did not show much variation

on storage. Gram-negative rods increased from 13.6% to 23.8% in 60 days in Feed I

(Fig.1).

In Feed II, the occurrence of Micrococcus spp. reduced from 73% to

53.27% in 60 days, while, the percentage of Arthrobacter (11.5% to 14.7% ), Bacillus

spp. {4.6%- 10 .74% ) and gram-negative rods (10.6 - 10.74%), increased during

storage (Fig. 2 ).

in all the triree leea ingreo:ent samples, Micrococcus was the dominant

group. In fishmeal, Micrococcus spp. decreased from 73% to 65.17% in 60 days.

Arthrobacter (6 - 7 .74% ) and Bacillus spp. (2.0 • 5 .7 4 % ) showed slight increase,

whereas, gram negative rods showed slight increase in 30 days (22% ) and then a

reduction on the 60'^ day (19,04 - 17% ) (Fig.3). In groundnut oil cake, Micrococcus

reduced from 73% to 53.33%, while, Arthtrobacter spp., Bacillus spp. and gram

negative rods w ere 6-13.3%, 5.3- 13.33% , 15.7 to 20% respectively on the 60*^ day

(Fig.4).

In soybean flour Micrococcus spp, was less than 50% (42 - 4 8 % ) and

the percentage occurrence increased on storage (42 .8 - 51.72) while, Arthrobacter

occurrence increased from 17.8 to 28 .5% in 30days and then reduced to 23 .9% on

the 60'" day. Grarh-negative rods showed a gradual reduction in percentage

occurrence (28 .5 to 24.3). Bacillus also showed an increasing trend in 30 d (28.5%)

and then reduced to 20% (Fig. 5).

27

Table 1. Variation of bacterial populations in Feed I

Days TPC Vibrio sppF.streptococci E. coli S. aureus

0 2.765 X 10^ 3 x1 0 ’ 3.6 X 10’ 3 x 10’ NIL

30 8.8X10'* 5.3 X 10’ 2.9 x 10’ 3 X 10’ 2.2x10^

60 2.73 X 10'* 6.4x10’ 4.5 x 10 3 x 10’ NIL

28

Table 2. Variation of bacterial populations in Feed II

Days TPC Vibrio spp F.streptococci

E. coll S. aureus

0 5 .7 5 X 10^ 2x10 ' NIL NIL NIL

30 5 . 5 X 1 0 “ 4 X 10’ NIL NIL NIL

60 3 .6 5 X 1 0 “* 4x 10' NIL NIL NIL

29

Table 3. Variation of bacterial populations in Fishmeal

Days TPC Vibrio sppF.streptococci E. coli S. aureus

0 5 . 1 2 X 1 0 ^ 2 x 1 0 ^ 6 . 5 X 1 0 ^ 2 x 1 0 ’ NIL

3 0 3 . 8 7 X 1 0 ^ 3 X 1 0 ^ 5 . 1 x 1 0 ^ 2 X 1 0 ’ 2 . 2 x 1 0 2

6 0 2 . 7 X 1 0 ^ 2 X 1 0 ’i 'I

NIL 2 x 1 0 ’ NIL

30

Table 4. Variation of bacterial populations in Groundnut oil cake

Days TPC Vibrio sppF.streptococci E. coli S. aureus

0 3.3 X 10^ 3x10 ’ 6 x 10’ 3 X 10’ 2.3x10^

30 5.1 x10^ 1.2 X 10 5 X 10' 3 X 10^ NIL

60 2.51 X 10^ 3x10 ' 4.9 X 10’ 3 x 10^ NIL

31

Table S.Variation of bacterial population in Soybean flour

Days TPC Vibrio sppF.streptococci E. coli S. aureus

0 3.15X 10^ 2 x1 0 ’ NIL NIL 3.5 x 10^

30 2.075 X 10^ 10x10’ NIL NIL 3.1 x 10^

60 2.75 X 10^ 2 x1 0 ’ NIL NIL NIL

32

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1. Total Plate Count in Zobel marine agar medium in Fish meal

2. Total Plate Count in Zobel marine agar medium in Ground nut oil cake

38

3. Staphylococcus aureus gram positive cocci in soybean flour

4. Staphylococcus aureus gram positive cocci in Feed I

t w * * • . . . , v » > - •

* * *s ^ w ** •

I» a__________- ______ __________ « » - f *

39

5. Bacillus spp in Fish meal

t f. « • I \*

r

■ \t

K ' '

A*.

■*

' /

- 4 r -

>

f •t .

f — t

6. Gram negative rods in Fish meal

40

DISCUSSION

5. DISCUSSION

In addition to enumerating the bacterial load of two shrimp feeds and

three feed ingredients, the present study also attempted to evaluate the ability of

these samples to support bacterial growth on short-term storage. The rationale for

the choice of these bacteria for testing in samples has been based on their role in

sea food- borne epidemics, in feed decomposition and causing diseases in cultured

aquatic organisms. Also, feeds and feed ingredients being sources o f nutrients

required for bacterial growth and contain a significant inoculum in it. Bacteria being

able to produce extracellular enzym es could affect the quality of a diet or play a role

in pathogenicity to fish or man, as were spore-forming bacteria that resist

pasteurization.

5.1. Qualitative Study of Bacteria Present in Feeds and Feed

Ingredients

5.1.1. Total Plate Count

W hen bacterial count is taken into account it was observed that either

of the two shrimp feeds or (he feed ingredients were sterile in the present study. TPC

of Feed I was in the range of 2.7 x 10^ cfu/g to 8,8 x 10'* cfu/g and that of Feed II was

in the range of 5 .75 * 10 cfu/g to 5 .5 x 10“* cfu/g, Sera and Kimata (1971 ) observed

2.2 X 10 cfu/g T P C in fish die!. The mesophilic bacterial counts in fish diets were

reported as 10^ to lO ’ cfu/g by Trust (1971) and Trust and Wood (1973). Muroga et

at. (1994), have reported difference in counts when Zobel! agar and Bromothymol

blue medium w ere used. They could obtain 1.2 x 10^ cfu/g in artificial fish diet using

Zobell agar m edium . The present observations also show a similar trend within the

range as in the above studies when Zobell agar was used.

For feed ingredients, T P C of fish meal w as between 2.7 x 10^ cfu/g to

5.12 X 10^ cfu/g, of ground nut oil cake 3 .3 x 10 cfu/g to 5.1 x 10 cfu/g and soybean

flour 2.075 5< 10^ cfu/g to 3,15 x 10^ cfu/g. There are no studies pertaining to change

41

of bacterial flora in feed ingredients on storage. Thus, the present observations form

a baseline in Ihe area.

Feeds are considered unsatisfactory w hen they contain a large

population of bacteria, even if they are not pathogenic or have not altered the

material. The T P C in the range of 10® to 10® /g feed results in decomposition of feed

(Trust, 1971). In the present study, T P C of feeds as well as feed ingredients were

well within this range and there w as no drastic variation in the count during 60 d

storage period.

Vibr/o spp, Faecal streptococci, Escherichia coli and Staphylococcus

aureus etc are indicators of contamination as well as potential pathogens. T h e feed

containing these bacteria would form an inoculum in the culture systems either

through the uneaten feed or through faeces. The bacterial population would result in

oxygen depletion and increase in am m onia level, thus affecting animal health. The

presence of indicators or pathogens or any other bacteria beyond a limit in feed is,

therefore,dangerous to the culture system s (Trust, 1971).

5.1.2. Vibrio spp.

Vibrio spp. count in Feed I was between 3 x 1 0 cfu/g and 6 .2 x 10 cfu/g.

during 60 d of storage. In Feed II, it ranged from 2 x 1 0 cfu/g to 4 x10 cfu/g. Reilly

and Twiddy (1 9 9 2 ) in their attempt to identify the source of V.cholerae in shrimp

feeds have concluded that commercial shrimp feed is a potential source o f the

pathogen. Vibrio parahaemolyiicus is another potentially pathogenic species reported

to be dangerous to cultured shrimp and seafood (Karunasagar et al. 1990). Muroga

et al. (1987) reported 10% Vibrio spp. from live diets, Artemia and rotifers. In

fishmeal. Vibrio spp. count was between 2-3 x 10 cfu/g, while, in groundnut oil cake it

was between 3 x 1 0 cfu/g and 1.2 x 10^ cfu/g and in soybean flour between 10- 20

cfu/g.

42

5.1.3. Faecal streptococci

Faecal streptococci was present in the Feed I, a commercial shrimp

feed, in the range o f 2 .9 -4.5 x 10 cfu/g, while it was absent in the Feed il, which was

formulated at the Centra! Marine Fisheries Research Institute. In fishmeal, the count

ranged from 5.1 x 10^ to 6.5 x 10 cfu/g and in groundnut oil cake it ranged from 4 .9 x

10 to 6 X 10 cfu/g.whereas in soybean flour it was absent. Storage didn't have much

effect in the count of Faecal streptococci.

5.1.4. Escherichia coil

Feed I has shown E.coli count in the range o f 3 x 10 cfu/g throughout

the period of observation, whereas, Feed II had no E.coli in the sample. Trust and

M oney (1972) observed that Enterobacteriaceae in feeds could indicate a potential

for contamination with enteric pathogens.

5.1.5. Staphylococcus aureus

S. aureus count in Feed I w as found to be in the range of 0 to 2 .2 x 10^

cfu/g, whereas it w as nil throughout the storage period in Feed II. It was observed

that S. aureus appeared in 30 days of storing, while, it w as absent in the 6 0 ’'' day

sampling. Inhibition o f Salmonella enteritldis isolated from fishm eal on 60-d storing is

reported (Pelagic et a i. 1998), Probably similar may be the case here. The

processing method employed during feed preparation, especially heat treatm ent,

seem s to destroy the bacteria. That might be the reason for the absence o f Faecal

streptococci, E.coli and S. aureus in Feed II, which was formulated at the Nutrition

laboratory of the Central Marine Fisheries Research Institute. Feed I, a commercial

shrimp feed, had occasional to regular contamination with the indicator bacteria. This

m ay be attributed to unhygienic handling by the farmer a fter opening of the feedbag

or storage. Ramalinga (2000) has also reported presence of faecal streptococci in

aquafeeds.

For hum an consumption permitted E.coli count is <10/g, Faecal

conforms nil, and Faecal streptococci <20 /g (Trust, 1971). Such restriction should be

imparted for aqua feed also, because fish have been shown to be vectors of bacterial

43

disease to humans (Jansen, 1970), It is, therefore, important that such bacteria are

not present in the diets of shrimp being raised for human consumption.

When consideration is given for feed preparation and handling under

hygienic conditions and proper heat treatm ent, the number o f bacteria present in the

feed ingredients could be reduced as seen in the case of Feed II in the present study.

The possible source o f contamination o f F eed 1 would be during handling and storage

by the farmer.

5.2. Qualitative Study of Bacteria Present in Feeds and Feed

Ingredients

For qualitative study of bacteria present in feeds and feed ingredients.

500 isolates were m aintained In Zobell M arine Agar slants. O nly gram-positive strains

were identified upto genus lovol, w hereas gram-negative isolates were taken as

such. As shown in Figs. 1-5, gram-positive cocci were the dom inant group in both the

feeds as well as the ingredients. Micrococcus was followed by Arthrobacter spp. and

Bacillus spp. A sim ilar observation was m ad e by Muroga et al. (1994) in commercial

fish diets. Hamid et qI. (1978 ) reported only Bacillus sp. from grey mullet diet. In the

present study, the percentage occurrence of gram negative bacteria in both the feeds

gradually increased on storage. While in feed ingredients, there was no noticeable

trend observed. T h e re was gradual increase in percentage composition of

Arthrobacter spp. in Feed I, II, fishm eal, and groundnut oil cake. In soy flour,

Arthrobacter spp and Bacillus spp were higher in 30 d storage period,

Ringo and Strom (1 9 9 4 ) have reported predominance of

Enterobacteriaceae in commercial feeds and Flavobacterium spp in capelin roe diet.

According to Surendran and Gopakum ar (1981), the gram-positive cocci, usually

found in association with fish and fishery products or aquatic environments are either

of three genera, viz., Staphylococcus. Streptococcus o r Micrococcus. Since

Staphylococcus and Streptococci were absent in feed II, it can be inferred that

Micrococcaceae observed in Feed II belonged to the genus Micrococcus.

Many factors like nature o f microflora of the ingredients used in feed

formulation, type o f heat treatment that the feed has undergone and the final

44

moisture content would have an influence on the final bacterial load of diet (Trust and

W ood, 1973; Blank et al., 1996)

Christian (1980) has observed a shift from gram negative to gram -

posltive flora in proteinaceous foods w hen the water activity dropped from 0 .98 to

0 .96 . As a result of this, the major bacterial flora of such feed could be gram

positives. In the present study, in both the feeds gram positive bacteria were

dominant throughout the period of study, though it gradually reduced, yet, overall,

remained as the dom inant group, Sugita et al. (1986, 1987) studied the microflora in

pellet diets and found them to be com posed of Bacillus spp. (4.8 x 10^/g) and

Clostridium (6 x 10^/g). In Ihe present study, i! was observed that Bacillus spp.

formed 4.5*10% in feeds and 2.0-20% in feed ingredients. It is also a well-established

fact that feed consumed by cultured aquatic organisms does have a definite influence

on the gut microbial flora (DeJ-Rio-Rodriguez et al., 1997). Ogbondeminu et a l (1991)

have reported that 80% of the feed microflora were identified in the water and of the

bacterial population in feed 25% was Pseudomonas spp.. Staphylococcus spp., 10% ,

Streptococcus as 12% , Micrococcus 15% and Aeromonas 8% .

The fact that Staphylococcus spp., Sfrepfococcus spp {faecalis) etc are

facultative pathogens or agents of food poisoning is of importance. Although the

presence of these bacteria is not often associated with fish diseases or enteric

diseases in human beings, the health implications, on the introduction o f these

organisms into natural water via the unconsumed feed and faeces in shrimp culture

waste water cannot be ignored.

Feed quality, generally perceived as the responsibility of the feed

manufacturer, is affected by factors outside the plant such as handling, storage, and

use. Thus, the m aintenance of feed quality becomes partly the responsibility of the

farm er too. Every shrim p farmer must be familiar with the nature and occurrence of

major feed quality problems and be able to prevent and control them by practicing

proper storage m easures.

Based on the results of the present study, it is concluded that the

bacterial microflora of selected feeds and feed ingredients reflects their handling.

45

storage and processing temperature. The commensal bacteria included a diversity of

potenlial pathogens and indicators. It is suggested that feed manufacturers and

farmers should be more aware of the bacterial load of fish/ shrimp diets, and a

minimum standard might need to be imposed in the processing methods. Strict

hygiene and sanitary procedures in aqua feed production and handling would be of

immense help in avoiding problems during later stages of culture operations. Further

studies in this line could help in imposing proper standards for feed ingredients as

well as feeds used in aquaculture.

46

SUMMARY

6. SUMMARY

1. Two fish feeds, one a commercial shrimp ‘grower’ feed (Feed I) and another a

cost effective shrimp feed form ulated at CMFRf, Kochi'(Feed II) and three

protein rich feed ingredients (fish m eal, ground nut oil cake and soybean flour)

were stored at room tem perature and were analysed quantitatively and

qualitatively fo r their bacteriological profile, on 0, 30 and 6 0 days.

2. TPC of both the feeds were in the range of 2.7x10^ to 8.8x10^ cfu/g, w hereas,

TPC of f/sh m ea! was 2 ,7 to 5 .1 2 x 10^ cfu/g, groundnut oil cake 3 .3 to 5.1 x

10 cfu/g and soybean flour 2 .0 7 5 to 3.15 x 10^ cfu/g during 60 d storage

period.

Vibrio spp., count in both the feed s was in the range of 2 x 10’ to 6 .2 x 10''

cfu/g. Fish m eal showed Vibrio spp., count in the order 2-3 x 10 cfu/g, ground

nut oil cake had V/dr/o spp. count in the range 3 x 10^ cfu/g. to1.2 x 10^ cfu/g

and soybean flour in the range o f 10 X 10' 2.0 x 10''

4. Faecal strep iococci count was in the range of 2 .9 x 10' to 4,5 x 10^ cfu/g In

Feed I, w hile, it w as absent in Feed II. In fishmeal, it ranged from 5.1 x 10^ to

6.5 xIO^ cfu/g in groundnut oil cake it was found to be between 4,9 x 1 0 ' to 6

x 10^while it w as absent in soybean flour during the storage period.

5. Esherichia co li count was 3 x 1 0 ’ cfu/g in the Feed I, whereas, it was absent in

Feed II. F ishm eal has shown E .co li count of 2.0 x 1 0 ' cfu/g, groundnut oil cake

3 X 10’ cfu/g and soy bean flour nil during storage.

6. Staphylococcus aureus count ranged from 0 to 2.2x10^ cfu/g in Feed I,

whereas, it w as absent in Feed II. Fishmeal, groundnut oil cake and soybean

flour had Staphylococcus aureus within 10 cfu/g during the 60 d storage

period.

47

7. Qualitatively, in both the feeds, Micrococcus was predominant, followed by

Arthrobacter and Bacillus, with marginal decrease in Micrococcus and

proportionate increase in Bacillus in 60 d.

8. In fishmeal. Micrococcus spp. (65-73% ) was predominant, followed by

Arthrobacter spp., (6-7.4%) and Bacillus spp,, (5 .3-5.74% ). Gram-negative

bacteria ranged from 15.7% to 22 .0% .

9. In groundnut oil cake Micrococcus spp., (48 to 7 3% ) was dominant, followed

by Arthrobater spp (6-13.3%) and Bacillus spp., (2-13.3% ). Gram-negative

bacteria varied between 19.04 - 37 .8% .

10. In soybean flour, Micrococcus sp was dominant in the range of 42.8- 51 .72% ,

followed by Arthrobacter spp. showing variation between13,79- 28 ,55% and

Bacillus spp. in the range of 10 .7 - 28,5%, Gram-negative bacteria showed

slight reduction during storage (2 8 .5 7 - 24.13%).

11. Overall, short-term storage of 60 days had no drastic effect on the bacterial

flora of feeds and feed ingredients. Yet, the effect on long-term storage cannot

be overruled, especially of qualitative changes. So, there is a wide scope in

this area for further studies.

48

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