Evaluation of Bacillus spp. isolated from stingless bee (Heterotrigona itama cockerell) honey as a potential probiotic

In Malaysia, stingless bees are well known for producing special honey called “madu kelulut” that is high in antioxidant and antimicrobial activity. Due to its higher moisture content, it provides favourable condition for microbial growth. However, little is known about the types of microbes that re...

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Main Author:	Zulkhairi Amin, Fatin Aina
Format:	Thesis
Language:	English
Published:	2020
Subjects:	Probiotics Bacillus (Bacteria) Stingless bees
Online Access:	http://psasir.upm.edu.my/id/eprint/99096/1/IB%202021%209%20%20IR.pdf
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id	my-upm-ir.99096
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spelling	my-upm-ir.990962023-02-15T02:08:27Z Evaluation of Bacillus spp. isolated from stingless bee (Heterotrigona itama cockerell) honey as a potential probiotic 2020-12 Zulkhairi Amin, Fatin Aina In Malaysia, stingless bees are well known for producing special honey called “madu kelulut” that is high in antioxidant and antimicrobial activity. Due to its higher moisture content, it provides favourable condition for microbial growth. However, little is known about the types of microbes that resides in this honey. Probiotics are live microbes, which when administered in adequate amounts confer health benefits to the host. This study aimed to find new strains of spore-forming probiotic bacteria from stingless bee (Heterotrigona itama Cockerell) honey. Therefore, Bacillus spp. were isolated from honey produced by 5 different H. itama colonies which were then further identified via 16S rRNA sequencing. Out of 53 isolates, only 23 isolates that are Gram positive, catalase positive and can tolerate 7% NaCl were selected for further molecular identification. Gene sequence analysis revealed that the dominant Bacillus species were Bacillus altitudinis (34%) and Bacillus pumilus (33%) followed by Bacillus megaterium (13%), Bacillus amyloliquefaciens and Bacillus aryabatthai (8%), followed by Bacillus subtilis (4%). Antibacterial activity against pathogenic bacteria assessed using agar well diffusion method showed that B. amyloliquefaciens HTI-19 and B. subtilis HTI-23 have comparable inhibitory activity with commercial probiotic strain Lactobacillus rhamnosus GG. Based on their antibacterial activity, these two strains were evaluated for probiotic properties including acid and bile salt (0.3%) tolerance, hydrophobicity and autoaggregation ability. Both isolates exhibited high tolerance with more than 85% viability in simulated in vitro gastrointestinal (GIT) conditions. Adhesion activity assessed by hydrophobicity and autoaggregation test revealed that B. amyloliquefaciens HTI-19 has the highest autoaggregation ability (84.13%) but lowest hydrophobicity (53.64%) among tested isolates. However, it was not significantly different from L. rhamnosus GG. Both Bacillus spp. were susceptible to all antibiotics with different mode of action. Bacillus amyloliquefaciens HTI-19 was α-hemolytic while B. subtilis HTI-23 exhibited γ-hemolytic activity on blood agar plate. Acute and subacute oral toxicity tests were performed in Sprague-Dawley rats to assess the safety of newly isolated B. amyloliquefaciens HTI-19. The probiotic feeding in acute and sub-acute toxicity study showed no mortality or significant abnormalities in rats throughout the 14-day and 28- ii day experimental period. The rats body weights were not affected by daily administration of probiotic B. amyloliquefaciens HTI-19 except for week 2 of acute study, when the body weight showed significant increase (P<0.05). By gross and microscopic examination of organs, no obvious and significant changes were observed in the morphology of organs from the treated rats. Serum biochemical tests and blood hematology test also revealed no treatment-related changes. Overall, these data indicated that oral administration of B. amyloliquefaciens HTI-19 culture up to 1 x109 CFU/ml for acute and subacute test were safe. The results of this study showed that Bacillus isolates from stingless bee honey, B. amyloliquefaciens HTI-19 and B. subtilis HTI-23, due to its spore forming ability, have remarkable probiotic properties. Probiotics Bacillus (Bacteria) Stingless bees 2020-12 Thesis http://psasir.upm.edu.my/id/eprint/99096/ http://psasir.upm.edu.my/id/eprint/99096/1/IB%202021%209%20%20IR.pdf text en public masters Universiti Putra Malaysia Probiotics Bacillus (Bacteria) Stingless bees Zawawi, Norhasnida
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
advisor	Zawawi, Norhasnida
topic	Probiotics Bacillus (Bacteria) Stingless bees
spellingShingle	Probiotics Bacillus (Bacteria) Stingless bees Zulkhairi Amin, Fatin Aina Evaluation of Bacillus spp. isolated from stingless bee (Heterotrigona itama cockerell) honey as a potential probiotic
description	In Malaysia, stingless bees are well known for producing special honey called “madu kelulut” that is high in antioxidant and antimicrobial activity. Due to its higher moisture content, it provides favourable condition for microbial growth. However, little is known about the types of microbes that resides in this honey. Probiotics are live microbes, which when administered in adequate amounts confer health benefits to the host. This study aimed to find new strains of spore-forming probiotic bacteria from stingless bee (Heterotrigona itama Cockerell) honey. Therefore, Bacillus spp. were isolated from honey produced by 5 different H. itama colonies which were then further identified via 16S rRNA sequencing. Out of 53 isolates, only 23 isolates that are Gram positive, catalase positive and can tolerate 7% NaCl were selected for further molecular identification. Gene sequence analysis revealed that the dominant Bacillus species were Bacillus altitudinis (34%) and Bacillus pumilus (33%) followed by Bacillus megaterium (13%), Bacillus amyloliquefaciens and Bacillus aryabatthai (8%), followed by Bacillus subtilis (4%). Antibacterial activity against pathogenic bacteria assessed using agar well diffusion method showed that B. amyloliquefaciens HTI-19 and B. subtilis HTI-23 have comparable inhibitory activity with commercial probiotic strain Lactobacillus rhamnosus GG. Based on their antibacterial activity, these two strains were evaluated for probiotic properties including acid and bile salt (0.3%) tolerance, hydrophobicity and autoaggregation ability. Both isolates exhibited high tolerance with more than 85% viability in simulated in vitro gastrointestinal (GIT) conditions. Adhesion activity assessed by hydrophobicity and autoaggregation test revealed that B. amyloliquefaciens HTI-19 has the highest autoaggregation ability (84.13%) but lowest hydrophobicity (53.64%) among tested isolates. However, it was not significantly different from L. rhamnosus GG. Both Bacillus spp. were susceptible to all antibiotics with different mode of action. Bacillus amyloliquefaciens HTI-19 was α-hemolytic while B. subtilis HTI-23 exhibited γ-hemolytic activity on blood agar plate. Acute and subacute oral toxicity tests were performed in Sprague-Dawley rats to assess the safety of newly isolated B. amyloliquefaciens HTI-19. The probiotic feeding in acute and sub-acute toxicity study showed no mortality or significant abnormalities in rats throughout the 14-day and 28- ii day experimental period. The rats body weights were not affected by daily administration of probiotic B. amyloliquefaciens HTI-19 except for week 2 of acute study, when the body weight showed significant increase (P<0.05). By gross and microscopic examination of organs, no obvious and significant changes were observed in the morphology of organs from the treated rats. Serum biochemical tests and blood hematology test also revealed no treatment-related changes. Overall, these data indicated that oral administration of B. amyloliquefaciens HTI-19 culture up to 1 x109 CFU/ml for acute and subacute test were safe. The results of this study showed that Bacillus isolates from stingless bee honey, B. amyloliquefaciens HTI-19 and B. subtilis HTI-23, due to its spore forming ability, have remarkable probiotic properties.
format	Thesis
qualification_level	Master's degree
author	Zulkhairi Amin, Fatin Aina
author_facet	Zulkhairi Amin, Fatin Aina
author_sort	Zulkhairi Amin, Fatin Aina
title	Evaluation of Bacillus spp. isolated from stingless bee (Heterotrigona itama cockerell) honey as a potential probiotic
title_short	Evaluation of Bacillus spp. isolated from stingless bee (Heterotrigona itama cockerell) honey as a potential probiotic
title_full	Evaluation of Bacillus spp. isolated from stingless bee (Heterotrigona itama cockerell) honey as a potential probiotic
title_fullStr	Evaluation of Bacillus spp. isolated from stingless bee (Heterotrigona itama cockerell) honey as a potential probiotic
title_full_unstemmed	Evaluation of Bacillus spp. isolated from stingless bee (Heterotrigona itama cockerell) honey as a potential probiotic
title_sort	evaluation of bacillus spp. isolated from stingless bee (heterotrigona itama cockerell) honey as a potential probiotic
granting_institution	Universiti Putra Malaysia
publishDate	2020
url	http://psasir.upm.edu.my/id/eprint/99096/1/IB%202021%209%20%20IR.pdf
_version_	1776100291804921856

Evaluation of Bacillus spp. isolated from stingless bee (Heterotrigona itama cockerell) honey as a potential probiotic

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