Prebiotics in Aquaculture:New Strategy for Larviculture

Improvment?

Mahious A.S.

LARVI 05 Fish & Shellfish Larviculture Symposium September 5-8 2005, Belgium

Probiotics Synbiotics? Prebiotics

% survival?

Adhesion?Activity?

Total transfer to the colon

Smallintestine

ColonStimulation of

active endogenousbeneficial bacteria

Stomach

Active exogenous bacteria

Living bacteriain the diet

Oligofructose or inulin in the diet

General concept

Delzenne & Roberfroid, 1994

Why use prebiotics in aquaculture

Exogenous probiotics => Adherence? Activity?

Lactic acid bacteria (LAB) are part of the natural intestinal microflora of healthy fish (Ringo & Gatesoupe, 1998)

LAB have probiotic properties

Prebiotics stimulate growth and activity of probiotic bacteria (LAB) resident in the gut

Prebiotics => food ingredients => Easier to apply in dried feed (stable after feed processing and during storage)

Definition of a prebiotic• Gibson al., 2004

Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon.

There are 3 criteria to be classified as a preboiotic:

Resistance to gastric acidity, hydrolysis by digestive enzymes and gastrointestinal absorption

Fermentation by intestinal flora

Selective stimulation of the growth and/or the activity of intestinal bacteria associated with health

Prebiotics classification

Yes

Yes

Yes

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++

+

+

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++

+

+

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++

(+)

(+)

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Inulin, Oligofructose

Transgalacto-oligosaccharides

Lactulose

Isomalto-oligosaccharides

Lactosucrose

Xylo-oligosaccharides

Soybean oligosaccharides

PrebioticCriterion 3Criterion 2Criterion 1Candidates

Gibson et al., 2004

INULIN SOURCE(by water extraction)

75% water17% inulin8% others

SALAD

«WITLOOF»«BELGIAN ENDIVES»

(2nd growth in the dark)

COFFEE SUBSTITUTE(by roasting)

Inulin and oligofructose from chicory, Cichorium Intybus

Chemical structure of inulin and oligofructose

Oligofructose

Partial hydrolysis

(enzymatic)

DP = 2 - 8

GFn and Fn

Chicory roots

Inulin

> 400.000 T/year15-17% inulin

Extraction

(hot water)

GFn

DP = 3 - 60

Glucose Fructose Reducing fructose

DP = Degree of Polymerisation

ß(2-1) ß(2-1)

Inulin or oligofructose in diet

Bifidobacteria, Lactobacillus

harmfull bacteriaImmunomodulators anticarcinogen

Short chain fatty acid (acetate, butyrate, propionate)

pH, mineral absorption

Mucosal cell proliferation

Inulin and oligofructose activity (Van Loo, 2004)

+

+

+ -

Application of inulin and oligofructose to fish nutrition

Turbot, Psetta maxima

Siberian sturgeon, Acipencer baeri

African catfish, Clarias gariepinus

Turbot, Psetta maxima(Mahious et al.,2005)

• Experiment 1: Early larval stage

Artemia enriched with inulin or oligofructose

• Experiment 2: Weaning stage

Inert feed supplemented with inulin or oligofructose

Turbot, Psetta maxima(Mahious et al.,2005)

• Experiment 1: Early larval stage

Artemia enriched with inulin or oligofructose

• Experiment 2: Weaning stage

Inert feed supplemented with inulin or oligofructose

• Experiment 2: Weaning stage

Inert dietArtemia + Inert dietArtemia naupliiRotifers

39 dph7dph3 dph

Brachionus Artemia 2% Cellulose (control),

2% Raftiline ST (inulin) or

2% Raftilose P95 (oligofructose)

55 dph29 dph

Start End

Control Inulin Oligofructose0,0

0,1

0,2

0,3

0,4

0,5

0,6

a a

b (P<0,01)

Fin

al w

eigh

t (g

)

Growth of turbot larvae weaned with inulin and oligofructose

Intestinal flora of turbot larvae weaned with inulin and oligofructose

Vibrio sp. S12411

Vibrio sp. L2C55

Other Vibrio spp.

Bacillus sp. L171P95

Other bacteria

Control

36% 28%

36%

Inulin

34%22%

44%

Oligofructose

42%16%

25%

14%

3%

0,87

0,88

0,89

0,9

0,91

0,92

0,93

ControlInulinOligofructose

3,33,43,53,63,73,83,9

44,14,2

Siberian sturgeon, Acipenser baeri African catfish, Clarias gariepinus

Specific growth rate (SGR % day-1) of fish fed prebiotics

0%

20%

40%

60%

80%

100%

Control Inulin Ologofructose0123456789

In vitro short chain fatty (SCFA) production with the content of spiral valveof Siberian sturgeon

ControlInulinOligofructose

AcetatePropionateButyrate

Total SCFA (mmol/g)

Lactate(mmol/g)

Bacterial flora of Siberian sturgeon and African catfish under study

Culturable flora => description of microbial biodiversity in the gut

(GTG)5-PCR: clustering and selection

Partial 16S sequencing

API, FAME…

Non-culturable flora => microbial composition of fish fed prebiotics

Bacterial DNA extraction from gut mucus

DGGE: needs to be further improved

Results coming …

ConclusionsIntroduction of prebiotics in inert feed is easy

Oligofructose improves growth in turbot larvae at weaning

Emergence of probiotic bacteria in the gut of turbot weaned with prebiotics

Inulin and oligofructose improve growth of Siberian sturgeon and African catfish

Production of organic acids = indication of specific flora that use inulin and oligofructose as substrate

Impact of prebiotic needs to be tested in several fish species and on long term feeding period

WeaningLarvae

Probiotics

Juveniles, adult

PrebioticsSymbiotics

Is it possible to control bacterial flora by using pro-, pre- and symbiotics at ≠ feeding stages?

??

Isolation of probiotic strains