The Test of Saccharomyces sp. Potential Filtrate to Inhibit The

Growth of Aspergillus flavus FNCC6109 Broiler Chicken

Concentrate Feed Model

Putu AnggaWiradana

, Ida Bagus Gede Darmayasa

and Ngurah Intan Wiratmini

1Postgraduate, Airlangga University, Surabaya, Indonesia

2Department of Biology, Faculty of Mathematics and Natural Science, Udayana University, Bali, Indonesia

Keywords: Aspergillus flavus, Broiler Chicken, Concentrate Feed, Saccharomyces sp.

Abstract: The test of Saccharomyces sp. culture filtrate potential aims to determine the ability of Saccharomyces sp.

isolates that was obtained on Bali cattle swap saliva by in vitro and in vivo tests on FNCC6109 Aspergillus

flavus in broiler chicken concentrate feed model. The highest inhibitory ability on A. flavus FNCC6109

growth in vitro with experimental method was conducted in Saccharomyces sp. filtrate culture. The in vivo

study used 24 experimental units divided into 8 treatment groups with 3 replicates respectively, i.e. A:

Concentrate without A. flavus FNCC6109 and without Sc.I culture filtrate; B: Concentrate + 15 mL of

sterile water; C: Concentrate + A.flavus FNCC6109; D: Concentrate + A.flavus FNCC6109 + 10% Sc.I; E:

Concentrate + A.flavus FNCC6109 + 20% Sc.I; F: Concentrate + A.flavus FNCC6109 + 30% Sc.I; G:

Concentrate + A.flavus FNCC6109 + 40% Sc.I; H: Concentrate + A.flavus FNCC6109 + 50% Sc.I. with a

15 days of storage period. The quantitative results data was analyzed using ANOVA assay and followed by

Duncan test. The filtrate culture had been incubated for 48 hours at 62.6%, therefore it could be used in in

vivo testing. The addition of Saccharomyces sp.I culture filtrate concentrate by 40% and 50% was able to

inhibit the population of A. flavus FNCC6109 by 97% in broiler chicken concentrate feed model. The results

showed a significant difference (P≤0.05), which means that Saccharomyces sp.I culture filtrate with the

concentration of 40% and 50% in broiler chicken concentrate feed model had the highest inhibition on the

total population of A. flavus FNCC6109.

1 INTRODUCTION

Livestock business in Indonesia is dominated by

local farms with quite large production output

(Subandriyo, 2006). Lack of feed availability can

lead to the decrease of production, decreased health

status and bad effects on livestock reproduction

(Saptahidayat, 2005).

According to Sudarmono and Sugeng (2008), in

general animal feed ingredients are classified into

three types, namely forage feed, concentrate feed

and additional feed. According to Kartadisastra

(1997), concentrate feed is a staple food made from

a mixture of several sources of nutrients such as

energy, protein, vitamins and minerals. Feed quality

is not only determined from the nutrient value

composition of the feed, but it also must be free of

contamination such as aflatoxin that has the potential

to contaminate fodder (Rachmawati, 2005).

Aflatoxin that contaminates the concentrate feed

and its processed ingredients is produced by

Aspergillus flavus. The optimum condition of this

mold in producing aflatoxin is at the temperature of

25-300C with relative humidity 85% and water

content 15-30% (Dwidjoseputro, 1989). According

to Rachmawati (2004), maize is the basic ingredient

of feed and used most up to 50-60% in poultry

rations.

Application of Saccharomyces sp. as a biocontrol

agent is one of the efforts to prevent the pathogen

growth. Further research conducted by El-Sayed and

Eman, (2011) mentioned the use of yeast as a

biocontrol agent in controlling leaf disease in sugar

beet plant with the application of 5 types of yeast

and fungicide significantly reduced leaf infection in

sugar beet plant compared with control.

Effort to suppress the growth of A. flavus

FNCC6109 is still important. Therefore, it is

532

Wiradana, P., Darmayasa, I. and Wiratmin, N.

The Test of Saccharomyces sp. Potential Filtrate to Inhibit The Growth of Aspergillus ﬂavus FNCC6109 Broiler Chicken Concentrate Feed Model.

DOI: 10.5220/0007546405320536

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 532-536

ISBN: 978-989-758-348-3

necessary to study the Saccharomyces sp. culture

filtrate potential to be used in the field of animal

husbandry to control A.flavus contamination in

concentrate feed as an effort to increase livestock

productivity.

2 MATERIAL AND METHODS

2.1 Preparation of Saccharomyces sp.

Culture Filtrate in Broth Media

The isolated yeast successfully isolated from Bali

cattle (data was not shown) was grown on Yeast

Extract Peptone Dextrose (YEPD) Broth media by

taking 1 dose inoculated on 3 Erlenmeyer containing

25 mL of YEPD Broth media. Each Erlemenyer

containing media and isolates was incubated

consecutively at room temperature for 24 hours; 48

hours and 72 hours

2.2 Inhibitory Test of Saccharomyces

sp. Filtrate Culture on Aspergillus

flavus FNCC6109

Inhibitory test of Saccharomyces sp. filtrate culture

was conducted experimentally by preparing 3 sterile

Petri dishes, each Petri dish was deposited with 1

mL of Saccharomyces sp. culture filtrate that had

been incubated for 24 hours; 48 hours and 72 hours,

after that it was poured with 15 mL of PDA media

and then shaken simultaneously to obtain a

homogeneous mixture. After the culture mixture of

the filtrate and media solidified, then right in the

middle of the Petri dish a piece of A. flavus colony

with a diameter of 0.5 cm was placed. As for the

control, sterile Petri dish filled with 1 mL of sterile

water and 15 mL of PDA media was prepared, as

well as A. flavus with a diameter of 0.5 cm. All the

treated Petri dishes were incubated at room

temperature for 7 days and repeated 5 times

2.3 Effects of Saccharomyces sp.

Filtrate Culture on Aspergillus

flavus FNCC6109 Population in

Broiler Chicken Concentrate Feed

Model

Effects of Saccharomyces sp. culture filtrate on A.

flavus FNCC6109 population in broiler chicken

concentrate feed model was obtained by Completely

Randomized Design (RAL) with 8 treatment types

and 3 replications. Saccharomyces sp. isolates used

in in vivo testing was the ones with the highest

inhibitory ability in the previous test (in vitro).

Before the formulation was done, the feed ingredient

was treated in autoclave first. Treatment to the

concentrate feed model included:

A: Concentrate without A. flavus FNCC6109

and without Sc.I culture filtrate; B: Concentrate + 15

mL of sterile water; C: Concentrate + A.flavus

FNCC6109; D: Concentrate + A.flavus FNCC6109 +

10% Sc.I; E: Concentrate + A.flavus FNCC6109 +

20% Sc.I; F: Concentrate + A.flavus FNCC6109 +

30% Sc.I; G: Concentrate + A.flavus FNCC6109 +

40% Sc.I; H: Concentrate + A.flavus FNCC6109 +

50% Sc.I. After treatment, all of the feed was dried

in an oven with a temperature of 400C for 48 hours.

Concentrate feed was then stored for 15 days at

room temperature. Observation of total A. flavus

FNCC6109 population was determined by using

plating method with dilution series (Nester et al.,

2007).

3 RESULT

3.1 The Saccharomyces sp. Filtrate

Culture Inhibitory Potential to the

Growth of Aspergillus flavus

FNCC6109 in Vitro

From in vitro test, the results obtained was the

percentage of Saccharomyces sp. culture filtrate

inhibitory power where the highest was 63.6 ±

2.07% by Saccharomyces sp.I culture filtrate isolates

with an incubation period of 48 hours. When

compared to Saccharomyces sp.II culture filtrate

isolates, the highest inhibition percentage occurred

at incubation period for 24 hours of 60.8 ± 8.43%.

However, when compared with the control treatment

of A. flavus FNCC6109 diameter that grew on PDA

media and in incubation for 7 days, it reached 4.00

cm (data was not shown).

The data shown in Table 1 shows that the

treatment of Saccharomyces sp.I culture filtrate with

48-hours incubation period used in this study had the

highest inhibitory ability so that it can proceed to the

in vivo testing stage by testing several

concentrations of the Saccharomyces sp.I filtrate

culture added to the broiler chicken feed concentrate

model in inhibiting the growth of A. flavus

FNCC6109.

The Test of Saccharomyces sp. Potential Filtrate to Inhibit The Growth of Aspergillus ﬂavus FNCC6109 Broiler Chicken Concentrate Feed

Model

533

Table 1: Percentage of Saccharomyces sp. filtrate

inhibition at different incubation periods to the growth of

A. flavus FNCC6109.

3.2 Aspergillus flavus FNCC6109

Population in Chicken Broiler

Concentrate Feed Model Added

with Isolate Filtrate Saccharomyces

sp. I

The analysis result of total Aspergillus flavus

FNCC6109 population on broiler chicken feed

concentrate model showed the decrease in the total

population of A. flavus FNCC6109 after given

Saccharomyces sp. culture filtrate I with various

concentration. Differences in A. flavus FNCC6109

population before and after storage for 15 days were

able to maintain the quality of concentrate feed. The

highest population of A. flavus FNCC6109 was

found in concentrate feed which only added A.

flavus FNCC6109 suspension at 29x105 CFU/g

before storage and 66.2x105 CFU/g after storage.

The lowest population of A. flavus FNCC6109 was

found in the concentrate feed model with the

addition of 50% (15mL/25gr) concentration of

Saccharomyces sp.I culture filtrate by 1.4x105

CFU/g.

Table 2: Total population of Aspergillus flavus FNCC6109

in broiler chicken feed concentrate model added by

Saccharomyces sp.I filtrate before and after storage

period.

Table 2 shows the effect of the addition of

Saccharomyces sp.I culture filtrate to the total

population of A. flavus FNCC6109 after concentrate

feed model was stored for 15 days. The total

population of A. flavus FNCC6109 prior to storage

period had shown a decrease in some concentrate

feed models that had been added Saccharomyces

sp.I culture filtrate. The results of statistical analysis

using Duncan Multiple Range Test (DMRT) showed

a significantly different mean value (P≤0.05)

between controls (A and C) with the concentration

of each treatment (D, E, F, G, and H).

In the concentrate feed model without the

addition of A. flavus FNCC6109 suspension and

Saccharomyces sp.I culture filtrate before and after

storage period, the growth of A. flavus FNCC6109

after analysis with dilution method was not found.

This could be due to the sterilization process on the

concentrate feed model that was running well so that

there was no contamination from other

microorganisms. The population of A. flavus

FNCC6109 contained in the concentrate feed model

was 66.2x105 CFU/g with rate of increase reached

56%. While the lowest population of A. flavus

FNCC6109 was found in the concentrate feed model

that was added with Saccharomyces sp.I culture

filtrate with 50% concentration of 1.4x105 CFU/g

with the increase only 28%.

4 DISCUSSION

The small diameter size of A. flavus that was tested

in vitro by Saccharomyces sp. culture filtrate proved

the effect of an enzyme or other compound excreted

by Saccharomyces sp. culture. According to the

research conducted by Chan and Tian (2005) in

vitro, by using modification method on

Saccharomyces sp. ability in lysing the cell wall of

A. parasiticus, there was a direct interaction of

Saccharomyces sp. cells on the hyphae of A.

parasiticus. It was allegedly due to β-gluconase

enzyme activity produced by Saccharomyces sp.

Furthermore, Albers et al. (1996) mentioned that

yeast culture filtrate is capable to produce several

types of enzymes and organic acids such as ethanol,

glycerol, acetic acid, pyruvic acid, succinic acid, á-

ketoglutarate and fumaric acid. In addition to the

inhibitory ability possessed by yeast isolates, the

role of lactic acid bacteria such as Lactobacillus

plantarum is able to inhibit spore germination from

A. flavus due to pH changes in fermentation media

and nutrient competition (Xu et al., 2002).

ICPS 2018 - 2nd International Conference Postgraduate School

534

The ability of Saccharomyces sp.I culture filtrate

to inhibit the growth of A. flavus FNCC6109 in the

concentrate feed model was suspected to occur due

to the nutrient competition and culture ability in

producing primary metabolite. A research from

Dharmaputra et al. (2003) mentioned that mold has a

faster growth ability compared with A.flavus that has

the potential to control A. flavus attack on peanut

seeds. Based on these results, the percentage of

inhibition to the growth of A. flavus FNCC6109

from the addition of Saccharomyces sp.I culture

filtrate with concentration of 40% and 50% during

storage period had percentage of inhibition equal to

97%. The results were consistent with a study

conducted by Darmayasa (2015) stating that the

administration of Trichoderma asperellum TKD

filtrate with a concentration of 9g/100g could inhibit

the growth of A. flavus FNCC6109 in the

concentrate feed model of 74.93% with 30 days of

storage period. Raharjanti (2006) also mentioned

that the culture filtrate of M. rouxii and

Saccharomyces sp. was able to inhibit the growth

and affected the morphological structure of A.

parasiticus. However, if compared with the M.

rouxii culture filtrate, the inhibitory ability of

Saccharomyces sp. culture filtrate was much higher

as it reached 98.1%.

5 CONCLUSIONS

Based on the research results, it can be concluded

that between 2 isolates obtained from swap saliva of

Bali cattle, the ability of Saccharomyces sp.I culture

filtrate used in this study generally has positive

correlation between in vitro and in vivo testing in

inhibiting the growth of A. flavus FNCC6109.

The Saccharomyces sp.I culture filtrate potential

in inhibiting the growth of A.flavus FNCC6109 in

the concentrate feed model provides an effect in

decreasing the number of A. flavus FNCC6109 after

15 days of storage.

ACKNOWLEDGEMENTS

Head of Bali Cattle Breeding Technical

Implementation Unit, Sobangan Mengwi, Badung

regarding sample taking permission, Mr. I Made

Mudita, S.Pt. M.P on permission to use flotation

machine. Mrs. Dr. Dra. RetnoKawuri, M.Phil, Dr.

IrianiSetyawati, S.Si., M.Si, and Dr. Ir. Ni

LuhSuriani, S.Si., M.Si for all the input, criticism

and suggestion.

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