Livestock Research for Rural Development 30 (10) 2018 Guide for preparation of papers LRRD Newsletter

Citation of this paper

Production of mannan oligosaccharides (MOS) extracted from fermented palm kernel cake and cassava by-product mixture and its efficacy as prebiotic

Nurhayati1,2, Hartutik3, Osfar Sjofjan3 and Eko Widodo3

1 Department of Animal Science, Lampung State Polytechnic, Lampung-Indonesia
2 Postgraduate Program of Animal Science, University of Brawijaya Malang, East Java-Indonesia
3 Department of Animal Nutrition and Feed, Faculty of Animal Husbandry, University of Brawijaya Malang, East Java-Indonesia
eko.widodo@ub.ac.id

Abstract

The aims of this research were to evaluate production of mannan oligosaccharides (MOS) extracted from fermented palm kernel cake (PKC) and cassava by-product (CB) mixture using Aspergillus niger and to investigate the efficacy of the extracted MOS in reducing pathogenic bacteria (Salmonella sp. and Escherichia coli) and increasing non-pathogenic bacteria ( Lactobacillus sp.) by in vitro test. This research consisted of two trial stages e.i. Trial 1 was to find the best combination between ratio of PKC-CB and length of incubation in producing maximum MOS. Completely Randomised Factorial Design with twenty combinations (4x5) between ratio of PKC-CB (R0 = 100% PKC; R1 = 87.5% PKC : 12.5% CB; R2 = 75% PKC : 25% CB; and R3 = 62.5% PKC : 37.5% CB) and length of incubation (L0 = 0; L1 = 24; L2 = 48; L3 = 72; and L4 = 96 hours) was used in this research. Trial 2 was to evaluate the extracted MOS from the best treatment combination of Trial 1 with dosages of 0 (T0), 1000 (T1), 2000 (T2), 3000 (T3) and 4000 ppm (T4) in reducing pathogenic bacteria and increasing non-pathogenic bacteria by in vitro test. The best dosage of extracted MOS was compared to commercial MOS (T5). Result of this research showed that combination between the ratio of 75% PKC : 25% CB and 72 hours incubation produced the highest MOS. The result also indicated that in vitro test using extracted MOS 4000 ppm was the best in reducing pathogenic bacteria and increasing non-pathogenic bacteria. It can be concluded that MOS extracted from fermented PKC-CB mixture could be an alternative prebiotic. It is suggested that further research should investigate this extracted MOS as poultry feed additive to know its efficacy in improving broiler performance.

Key words: cassava by-product, incubation length, in vitro test, MOS, palm kernel cake


Introduction

Indonesia is known as one of the greatest palm oil producers in the world. Yearly production palm fresh fruit bunches was about 33.5 million ton, 25% of the fraction is in the form of crude palm oil and 2.5-3.0% or about 1 million ton in the form of by-product known as palm kernel cake (Directorate General of Estate Crops 2015). The palm kernel cake is a potential source of mannan oligosaccharide (MOS) because one of the important contents of crude fibre is hemicellulose which contains predominantly mannan (about 58%) (Jaafar and Jarvis, 1992).

In poultry industry, MOS is commonly used as prebiotics feed additive to improve health and production performance. Commercial MOS which is currently available in the market is an imported product. It is usually isolated from cell wall of Saccharomyces cerevisiae. Therefore, elaborative research on the possible utilization of palm kernel cake (PKC) as an alternative source of MOS has been investigated by Tafsin (2007), Syahruddin et al (2008) and Jahromi et al (2016).

There are 4 types of mannan in hemicellulose of PKC namely linear mannan, galactomannan, glucomannan and galactoglucomannan. These polysaccharides can be broken down into simple forms as MOS. Degradation of mannan polysaccharides can be done by physical, chemical and enzymatic methods. Tafsin (2007) extracted mannose from PKC by using chemical and combination of chemical and physical methods. The result showed that chemical and physical combination treatment (NaOH 0.05N and addition of glass particle) resulted in the highest total sugar content (3.2%) with mannose content 68.19% from the total sugar extracted. Whilst Yokomizo (2005) extracted mannose from PKC by using mannanase enzyme resulted in higher mannose (84.59%). Mannan polysaccharides degradation by enzymatic method had a better result of mannose compared to that of physical or chemical method. According to Tafsin (2007) the use of chemical with high concentration (NaOH 0.1 N) could destroy extracted sugar so that decrease mannose content.

The enzymes which are able to degrade crude fibre and mannan polysaccharides in PKC were cellulase, hemicellulose, xylanase, mannanase, glucosidase and galactosidase (Yokomizo 2005; Purnawan et al 2017; Saenphoom et al 2011) which can be produced by Aspergillus niger. Combination of these enzymes is needed to degrade the complex structure of crude fibre of PKC and to release its sugar content. Releasing of MOS from PKC might also be achieved by fermentation process using bacteria and yeast. Aspergillus niger has been reported to be able to degrade mannan polysaccharides due to its various enzymatic activities produced by the microbe (de Vries et al 2001; Norita 2005; Youssef et al 2006; Ab Rasyid et al 2009; Andersen et al 2012).

In a previous research, Nurhayati et al (2006) reported that fermentation of combination between 75% PKC and 25% cassava by-product (CB) with Aspergillus niger decreased crude fibre (13.7%) more than that of substrate 100% PKC (6.5%). The more decrease of crude fibre of mixture substrate of 75% PKC and 25% CB is due to the role of CB as source of energy which boost the growth and development of Aspergillus niger impacting on maximum enzyme production. The decrease of crude fibre may be followed by degradation process of mannan polysaccharides to become MOS due to enzymes produced by Aspergillus niger. MOS was reported to be able to be agglutinated by pathogenic bacteria such as Escherichia coli and Salmonella sp (Spring et al 2000). This agglutination happened due to pathogen bacteria which have type-1 fimbriae which are sensitive to adhere to MOS. This research aimed to find the best combination between the ratio of PKC-CB and length of incubation if fermented by Aspergillus niger to produce MOS. Finally, the extracted MOS obtained was tested as prebiotic by using in vitro test (agglutination and resistance tests and inhibition test in liquid medium).


Materials and method

This research consisted of two trial stages, Trial 1 and Trial 2. Trial 1 aimed to find the best combination of ratio PKC-CB and length of incubation of fermentation to produce maximum MOS. This Trial was done in the laboratory of Animal Nutrition and Feed, Animal Husbandry Faculty, University of Brawijaya Malang and in laboratory of Biochemistry, Mathematic and Natural Sciences Faculty, University of Brawijaya Malang. Trial 2 was to test dosage use of MOS extracted from fermentation product from Trial 1 in reducing pathogenic bacteria ( Salmonella sp. and Escherichia coli) and increasing non-pathogenic bacteria (Lactobacillus sp.) by using in vitro test. This Trial was done in Central Laboratory of Life Sciences, University of Brawijaya Malang.

Trial 1

Materials used in Trial 1 were PKC, CB, Aspergillus niger, and some minerals (urea, KCl, NPK and ZA). PKC was obtained from palm oil industry of PTPN VII Lampung Province, Indonesia, while CB was obtained from tapioca industry of Bumi Waras East Lampung, Indonesia. Yeast of Aspergillus niger was obtained from the laboratory of Microbiology, Medicine Faculty, University of Brawijaya Malang. The NDF and ADF contents of PKC on Dry Matter (DM) basis were 68.62% and 40.60%, while the respective NDF and ADF contents of CB were 52.80% and 39.96%.

Analysis of fibre components and MOS extraction from fermentation product

The method of research was a laboratory experiment, arranged in completely randomised factorial design of 4x5. There were 2 factors employed, namely ratio PKC-CB and length of incubation. The first factor consisted of ratio of PKC-CB, namely R0 = 100% PKC : 0% CB ; R1 = 87.5% PKC : 12.5% CB; R2 = 75.0% PKC : 25.0% CB and R3 = 62.5% PKC : 37.5% CB. The second factor was length of incubation consisted of L0 = 0 hour, L 1 = 24 hours, L2 = 48 hours, L3 = 72 hours and L4 = 96 hours. Each treatment combination was replicated three times. Twenty combinations of PKC and CB mixtures were then homogenised before being fermented with Aspergillus niger (Nurhayati et al 2006) at certain length of incubation as mentioned in the second factor. Fermentation product of each mixture was dried and grinded, then used for fibre components analysis consisted of neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), cellulose and hemicellulose.

Extraction of MOS from fermentation product was done by using modified method of Tafsin, Jaelani and Krisnan (Tafsin 2007; Jaelani 2007; Krisnan 2010). The extraction process was started by grinding 100 g dried fermentation product and 50 g broken glass mixture using mortar grinder for 15 minutes, and then adding 500 ml aquadest and re-grinding for another 15 minutes. The product was then heated by using autoclave (121oC; 15 minutes), and then let it cool. The next step was filtering the cool product to get the supernatant. Finally, the supernatant was separated from solid by using centrifuge (12000 G; 15 minutes). The supernatant was collected and then concentrated by using rotary evaporator (Yamato RE 50). The result was then used for analysis of reducing sugar and mannose contents.

Variables observed in this research were NDF, ADF, ADL (lignin), cellulose, hemicellulose, reducing sugar and mannose contents. Acid detergent fiber, NDF, and ADL were determined as described by Goering and Van Soest (1970). Cellulose content was calculated as ADF−ADL, whereas hemicellulose content was calculated as NDF−ADF. Reducing sugar content was analysed by using Luff Schoorl method (Sudarmadji et al 1984) and analysis of mannose content was analysed using High Performance Liquid Chromatography (HPLC) (Ramli et al 1994).

Statistical Analysis

Data were subjected to analysis of statistic according to the design which was Completely Randomised Factorial Design by using Agricolae package from R environment (de Menndiburu 2017). Means within each main factor and inter simple factor were then compared by using Duncan’s Multiple Range Test (DMRT) when variance analysis was significant (p<0.05) using the package.

Trial 2

Materials used in this experiment were aquadest, HCl, medium of nutrient Broth, Phosphate Buffer Saline (PBS) 0.05M,Salmonella sp., Escherichia coli, Lactobacillus sp ., NaCl 0.9%, medium of nutrient agar, and discs paper. In addition, MOS extracted from the best fermentation product in Trial 1and commercial MOS were also used in this research. The experimental treatments included T0 = without addition of extracted MOS (MOS 0 ppm), T1= adding extracted MOS of 1000 ppm, T2 = adding extracted MOS of 2000 ppm, T3 = adding extracted MOS of 3000 ppm, T4 = adding extracted MOS of 4000 ppm and T5 = adding commercial MOS. Each treatment was repeated three times. In vitro test consisted of agglutination test, resistance test and inhibition test in liquid medium. The best treatment of extracted MOS for the three test was compared to commercial MOS. Criterion of the best treatment was based on positive result with the more MOS particles were clumping together for agglutination test, positive result with no clear zone formed for resistance test and the lowest pathogenic bacteria number and the highest non-pathogenic bacteria number for inhibition test in liquid medium.

Agglutination test was carried out by using a qualitative method according to Spring et al (2000) modified by Tafsin (2007) which tested MOS in binding pathogenic bacteria (Salmonella sp. and Escherichia coli)and non-pathogenic bacteria ( Lactobacillus sp.). Initially, to standardize the concentration of bacteria, bacterial cultures were grown in nutrient broth medium for 24 hours. Bacteria cells were harvested by centrifugation and suspended in PBS (0.05 M, pH 7.2) to reach an optical density at 660 nm (OD 660) of 1.60. MOS was suspended as well in PBS (OD 660) of 1.60.Agglutination tests were then performed with mixing the suspension of bacteria and MOS on object glass with proportion of 1:1 for 5 minutes. Finally, agglutination result was determined microscopically.

Resistance test was done by using Kirby-Bauer protocol (Hudzicki 2009) which evaluated MOS in resisting bacteria. Initially, bacteria culture (Salmonella sp., Escherichia coli, and Lactobacillus sp.) were planted in petri dish by using medium of nutrient agar. Different levels of MOS as mentioned in the treatments were entered into discs paper around50 μl each and put on the medium of nutrient agar. Furthermore, they were incubated for 24-48 hours to observe the availability of clearing zone in the culture medium. Resistance test is positive if clear zone was not formed around discs paper meaning that MOS is not able to kill bacteria.

Inhibition test in liquid medium used Tafsin method (Tafsin 2007) which evaluated the ability of MOS in inhibiting the development of bacteria. Initially, a series of 104, 105 and 10 6 CFU/ml bacteria was added to the media of nutrient broth, and then incubated for 24 hours with temperature of 37°C to be observed the colony formed. Colony counting was done with dilution using NaCl 0.9% and followed by growing culture in a petri dish using medium of nutrient agar. Colony counting was done after incubation for 48 hours with temperature 37°C. All the result of in vitro test were analysed using a descriptive method.


Results and discussion

Trial 1

The summary result of trial 1 about effect of PKC-CB ratio and length of incubation is presented in Table 1 and Table 2.

Table 1. Combination effects between ratio of PKC-CB (R) and length of incubation (L) on NDF, ADF, lignin, cellulose and hemicellulose contents

Treatment combination

NDF
(%)

ADF
(%)

Lignin
(%)

Cellulose
(%)

Hemicellulose
(%)

R

L

R0

L0

68.62b

40.60f

11.78g

27.91de

28.02a

R0

L1

72.05a

46.11ab

14.87c

30.36c

25.94b

R0

L2

70.82a

47.26a

13.96d

32.45a

23.56d

R0

L3

61.50f

41.08f

14.29d

26.07gh

20.42ef

R0

L4

63.55de

46.15ab

15.91a

25.65hi

17.39h

R1

L0

66.16c

40.18f

11.32h

27.27ef

25.98b

R1

L1

66.58c

45.89bc

13.19ef

31.50b

20.69e

R1

L2

54.24h

40.78f

12.92f

25.18i

13.45jk

R1

L3

51.57i

36.40h

14.23d

20.52l

15.17i

R1

L4

52.15i

38.52g

15.47b

19.52mn

13.63j

R2

L0

65.00cd

40.49f

10.25i

26.74fg

24.51c

R2

L1

68.75b

44.29d

11.90g

28.50d

20.46ef

R2

L2

56.62g

43.57de

15,96a

21.66k

13.06k

R2

L3

53.20hi

41.33f

15.41b

20.19lm

11.87m

R2

L4

51.21i

40.83f

16.21a

18.30o

10.37n

R3

L0

62.53ef

40.46f

10.36i

23.92j

20.06fg

R3

L1

64.11de

44.37d

11.71g

25.27i

19.73g

R3

L2

53.21hi

41.17f

11.08h

19.11n

12.04lm

R3

L3

51.53i

42.57e

14.06d

17.29p

8.96o

R3

L4

57.23g

44.75cd

13.40e

18.01op

12.49l

Pooled SEM

0.92

0.37

0.25

0.60

0.74

Main effects

Length of incubation

L0

65.58b

40.43d

10.93e

26.46b

24.64a

L1

67.87a

45.17a

12.92d

28.91a

21.70b

L2

58.72c

43.19b

13.48c

24.60c

15.52c

L3

54.45e

40.35d

14.50b

21.02d

14.10d

L4

56.04d

42.56c

15.25a

20.37e

13.47e

Ratio of PKC-CB

R0

67.31a

44.24a

14.16a

28.49a

23.07a

R1

58.14c

40.36d

13.42c

24.80b

17.79b

R2

58.95b

42.10c

13.95b

23.08c

16.05c

R3

57.72c

42.67b

12.12d

20.72d

14.66d

Probability

L

<0.001

<0.001

<0.001

<0.001

<0.001

R

<0.001

<0.001

<0.001

<0.001

<0.001

LxR

<0.001

<0.001

<0.001

<0.001

<0.001

Note: R0=100% PKC:0% CB, R1 =87.5% PKC:12.5% CB, R2=75.0% PKC:25.0% CB, R3=62.5% PKC:37.5% CB; L0 = 0 hour, L1 = 24 hours, L2 = 48 hours, L3 = 72 hours, L4 = 96 hours. Different superscript of little or capital letter within the same column indicated significant differences (p<0.05).



Table 2. Combination effects between ratio of PKC-CB (R) and length of incubation (L) on reducing sugar and mannose contents

Treatment combination

Reducing
sugar (%)

Mannose
(mg)

R

L

R0

L0

1.93kl

354.51l

R0

L1

1.40n

309.66n

R0

L2

1.68m

387.50j

R0

L3

3.01h

527.21e

R0

L4

5.99e

566.91d

R1

L0

1.78lm

343.88lm

R1

L1

2.74i

424.41i

R1

L2

2.94hi

368.45k

R1

L3

5.02f

526.37e

R1

L4

6.84d

501.33f

R2

L0

1.74lm

344.07lm

R2

L1

2.37j

445.89h

R2

L2

4.18g

479.53g

R2

L3

10.05a

753.64a

R2

L4

6.91d

674.13b

R3

L0

2.07k

333.04m

R3

L1

4.32g

535.71e

R3

L2

7.50c

580.26cd

R3

L3

9.82b

583.00c

R3

L4

5.00f

370.29k

Pooled SEM

0.34

15.64

Main effects

Length of incubation

L0

1.88e

343.88e

L1

2.71d

428.92d

L2

4.08c

453.93c

L3

6.98a

597.56a

L4

6.18b

528.16b

Ratio of PKC-CB

R0

2.80d

429.15c

R1

3.86c

432.89c

R2

5.05b

539.45a

R3

5.74a

480.46b

Probability

L

<0.001

<0.001

R

<0.001

<0.001

LxR

<0.001

<0.001

Note: R0=100% PKC:0% CB, R1 =87.5% PKC:12.5% CB, R2=75.0% PKC:25.0% CB, R3=62.5% PKC:37.5% CB; L0 = 0 hour, L1 = 24 hours, L2 = 48 hours, L3 = 72 hours, L4 = 96 hours. Different superscript of little or capital letter within the same column indicated significant differences (p<0.05).

Result of variance analysis showed that there was interaction (p<0.01) between ratio of PKC-CB and length of incubation on NDF, ADF, lignin, cellulose, hemicellulose, reducing sugar and mannose contents. Based on the MOS production which is indicated by the content of reducing sugar and mannose, the best combination treatment in this research was R2 (75% PKC, 25% CB) and L3 (72 hours). This combination treatment produced the highest content of reducing sugar (10.05%) and mannose (753.64 mg) (Table 2). The highest reducing sugar and mannose content of R2 might be due to the highest degradation of hemicellulose and cellulose. Mannose and glucose from hemicellulose and cellulose degradation might correlate with reducing sugar content.

Table 1 and Table 2 showed that content of NDF, cellulose and hemicellulose tended to decrease with the length of incubation and with the increase of CB proportion in the substrate, but reducing sugar and mannose content tended to increase. While ADF and lignin content tended to increase with the length of incubation, but it decreased with increasing proportion of CB in the substrate. These happened may be because of the availability of starch content in CB that function as energy source for Aspergillus niger so that growth and development of Aspergillus niger could be boosted by the addition of CB. The good growth and development of Aspergillus niger influenced the yeast to produce enzymes such as cellulase, mannanase and ligninase that could degrade the fibre of PKC in maximum capacity. In consequence, addition of CB in the substrate could decrease the content of cellulose and hemicellulose, and finally decreased the content of NDF, ADF and lignin, and increased reducing sugar and mannose content of the substrate of PKC-CB mixture.

Cellulose content of PKC-CB mixture substrate started to decrease at 48 hours to 96 hours incubation. This decrease was caused by cellulose degradation by cellulase enzyme produced by Aspergillus niger to glucose (Subowo 2010). Sharply decrease of cellulose content that started from 48 hours to 72 hours of fermentation process indicated that at this time Aspergillus niger was in logarithmic growth phase with cell proliferation, cell activity and enzyme production increase so that in this phase Aspergillus niger need more energy which can be fulfilled by CB. The result of current study is similar to previous study reporting that cellulase enzyme produced by Aspergillus nigeris in maximum at 3-6 days of fermentation (Kassim, 1982; Ariyani et al 2014; Dos Santos et al 2016). Mrudula and Murugammal, (2011) reported that the optimum time to produce maximum cellulase enzyme is 72 hours on solid state fermentation (SSF) and 96 hours on submerge fermentation (SmF).

Hemicellulose content decreased with the length of incubation, which started from 24 hours to 96 hours. This decrease was faster 24 hours than cellulose, it may be because hemicellulose was more soluble compared to cellulose. The decrease of hemicellulose content was caused by the activity of mannanase enzyme produced by yeast Aspergillus niger in degrading hemicellulose (mannan) to MOS or mannose. Previous study reported that Aspergillus niger produce manannase enzyme with enzyme activity 0,3489 U/ml higher thanAspergillus flavus (0,1830 U/ml) and Aspergillus tamari (0,1546 U/ml) (Agu et al 2014). Yopi et al (2006) reported that Aspergillus niger can produce mannanase enzyme with enzyme activity 0,102 U/ml higher than Eupenicillum javanicum (0,088 U/ml),Streptomyces lipmanii (0,032 U/ml) and lower than Saccharopolyspora flava (0,133 U/ml) in PKC substrate.

Lignin content tended to decrease with addition of CB proportion in the substrate, but tended to increase with the length of incubation. The decrease of lignin content with the addition of CB proportion in the substrate is caused by ligninase enzyme activity produced by yeast Aspergillus niger in degrading lignin. Actually, Aspergillus niger is one of yeast that produce small amount of ligninase enzyme (Hu et al 2011; Subowo 2015), However, the good growth and development of Aspergillus niger as CB addition in this research, this might impact on the increase of ligninase production causing lignin degradation and finally decrease lignin content of the substrate. While the increase of lignin content with the length of incubation might be caused by lignocellulose degradation by lignocellulase enzyme activity produced by Aspergillus niger. The high broken lignin bond with cellulose and hemicellulose by the enzyme lignocellulase during fermentation by 96 hours and not offset with high lignin degradation by ligninase enzyme impacted on lignin accumulation in the substrate.

Reducing sugar content of PKC-CB mixture substrate increased in line with the length of incubation and addition of CB in the substrate. This is because of cellulase and mannanase and other enzymes activities that break down complex carbohydrate to simple sugar components such as disaccharides and monosaccharides. Cassava by-product is an energy source for yeast Aspergillus niger for growing and developing, so that this yeast can produce enzymes maximally and break down carbohydrate to simple sugar which is measured in reducing sugar content. Pangesti et al (2012) reported that addition of carbon source such as molasses in fermentation medium can influence the growth of fungi, enzyme activity and the length of incubation. Addition of molasses 5% in fermentation medium can increase DM of mycelia cell of Aspergillus niger which is the highest (0.0104 g). Mojsov (2010) also reported that addition of glucose in fermentation medium resulted in the highest DM of Aspergillus niger cell (4.5 g/l) compared to other carbon source such as fructose, galactose, lactose and apple dregs. The increase of Aspergillus niger cell DM indicated that the growth and development of the yeast increase meaning enzyme production also increase.

Like reducing sugar, mannose content of PKC-CB mixture substrate increased in line with the addition of CB in the substrate and length of incubation. Cassava by-product is starch source that is useful to supply energy to boost the growth and development of yeast Aspergillus niger, so that mannanase enzyme is produced in maximum capacity to degrade mannan to mannose. This result fitted with the result of Ab Rasyid et al (2009) reported that addition of energy source is useful to boost the growth yeast Aspergillus niger. Addition of carbon source of molasses 4% resulted in the highest activities of mannanase enzyme (411,09 U/g substrate) followed by other source of carbon i.e starch (394,16 U/g substrate), glucose (389,14 U/g substrate), sucrose (382,87 U/g substrate), maltose (365,31 U/g substrate) and lactose (313,25 U/g substrate).

Trial 2
Agglutination test

The summary result on agglutination test is presented in Table 3. Agglutination test indicated that adding either extracted or commercial MOS showed positive results meaning enable to form clumping when tested by using Escherichia coli and Salmonella sp. However, agglutination test showed negative results if tested against Lactobacillus sp. Clumping was formed because of adhering between mannose component and bacteria receptor. Fimbriae type 1 of bacteria is a receptor containing lectin which is sensitive to mannose (Spring et al 2000).These results are in agreement with Spring et al (2000) who reported the same agglutination test results of MOS extracted from Saccharomyces cerevisiae on Escherichia coli and Salmonella sp. The other author also reported similar results on agglutination test by using MOS extracted from PKC on Salmonella sp. (Tafsin 2007).

Table 3. Result of agglutination test on Escherichia coli, Salmonella sp. and Lactobacillus sp.

Treatments

Bacteria

Escherichia coli

Salmonella sp.

Lactobacillus sp.

T0 (Without MOS or MOS 0 ppm)

-*

-

-

T1 (Added extracted MOS 1000 ppm)

+**

+

-

T2 (Added extracted MOS 2000 ppm)

+

+

-

T3 (Added extracted MOS 3000 ppm)

+

+

-

T4 (Added extracted MOS 4000 ppm)

++***

+

-

T5 (Added commercial MOS)

++

+

-

*(-) No MOS particles were clumping together, **(+) MOS particles were clumping together,
***(++) More MOS particles were clumping together

Resistance test

The summary result on resistance test is presented in Table 4. Resistance test using both extracted and commercial MOS showed positive results on pathogenic (Escherichia coli and Salmonella sp.) and non-pathogenic bacteria ( Lactobacillus sp.) (Table 4). The positive result of resistance test indicated that the use of both commercial and extracted MOS did not kill the bacteria so that formation of clear zone is not formed. This result agrees with previous results (Tafsin 2007; Syahruddin, et al 2008).

Table 4. Result of resistance test onEscherichia coli, Salmonella sp. and Lactobacillus sp.

Treatments

Bacteria

Escherichia coli

Salmonella sp.

Lactobacillus sp.

T0 (Without MOS or MOS 0 ppm)

+

+

+

T1 (Added extracted MOS 1000 ppm)

+*

+

+

T2 (Added extracted MOS 2000 ppm)

+

+

+

T3 (Added extracted MOS 3000 ppm)

+

+

+

T4 (Added extracted MOS 4000 ppm)

+

+

+

T5 (Added commercial MOS)

+

+

+

*(+) There was no clear zone around discs paper

Inhibition test in liquid medium

The results of inhibition test in liquid medium by using either extracted or commercial MOS (T1 to T5) on Escherichia coli, Salmonella sp. and Lactobacillus sp. can be seen in Figure 1. Escherichia coli and Salmonella sp. tended to decrease with increasing extracted MOS dosage. The number of Escherichia coli was lower than that of Salmonella sp. The decrease of Escherichia coli in T4 was 0.68x104 to 0.57x104 cfu/g (equal to 16.18%) as compared to T0. Furthermore, the decrease of Salmonella sp. in T4 was 1.12x10 4 to 0.96x104 cfu/g (equal to 14.29%) as compared to T0. Whilst the decrease of Escherichia coli and Salmonella sp. in T5 was 17.65% (from 0.68x104 to 0.56x104 cfu/g) and 13.39% (from 1.12x104 to 0.97x104cfu/g), respectively, compared to T0. The decrease of Escherichia coli and Salmonella sp. might be caused by clumping effect of bacteria, so that during counting colony of bacteria in agar medium the number was counted less.

Figure 1. The result of inhibition test in liquid medium of using extraction MOS (T0-T4) and
commercial MOS (T5) on Escherichia coli, Salmonella sp. and Lactobacillus sp.

The result of inhibition test in liquid medium of using extracted MOS on Lactobacillus sp. showed that colony number increase with addition of MOS dosage. Similarly, the number of Lactobacillus sp. also increased when added commercial MOS. The increase of colony number of Lactobacillus sp. in T4 and T5 was 5% (from 1.20x104 to 1.26x104 cfu/g) and 6.7% (from 1.2 to 1.28x104 cfu/g), respectively, compared to T0. The increase of colony number of Lactobacillus sp. indicated that extracted MOS could function as prebiotic which agree with the result of Chen et al (2015) who reported that oligosaccharide extracted from palm kernel expeller was able to support the growth of Lactobacillus sp. strains and a potential source of prebiotic.


Conclusion


References

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Received 5 March 2018; Accepted 1 September 2018; Published 1 October 2018

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