Effect of Tempeh Supplementation on Gut Microbiota and Immunoglobulin A Profiles in Sprague Dawley Rats
EFFECT OF TEMPEH SUPPLEMENTATION ON
GUT MICROBIOTA AND IMMUNOGLOBULIN A PROFILES
IN SPRAGUE DAWLEY RATS
SUSAN SOKA
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
DECLARATION OF THE SOURCE OF THIS DISSERTATION
I declare that this dissertation, entitled Effect of Tempeh Supplementation on
Gut Microbiota and Immunoglobulin A Profiles in Sprague Dawley Rats is
entirely my own work, assisted by a supervisory committee and has not been
submitted in any form for another degree or diploma to any university or other
tertiary institution of education. Where this dissertation draws on existing
publications, those sources are cited in the text and listed in the references section.
Bogor, November 2014
Susan Soka
G361110041
RINGKASAN
SUSAN SOKA. Pengaruh Suplementasi Tempe terhadap Profil Mikrobiota Usus
dan Imunoglobulin A pada Tikus Sprague Dawley. Dibimbing oleh ANTONIUS
SUWANTO, DONDIN SAJUTHI dan IMAN RUSMANA.
Penelitian nutrigenomika saat ini telah menunjukkan bahwa interaksi gen
dan makanan mempunyai peranan penting dalam menjaga kesehatan yang optimal.
Selain itu, studi nutrigenomika juga telah menunjukkan bahwa triliunan bakteri
yang ada pada saluran pencernaan manusia, atau yang lebih dikenal dengan
mikrobiota usus, dapat mempengaruhi penyerapan nutrisi dan regulasi energi. Hal
ini menunjukkan pentingnya peranan mikrobiota usus pada kesehatan manusia
dalam perkembangan sistem kekebalan tubuh dan penggunaan energi.
Tempe merupakan makanan fermentasi tradisional Indonesia yang terbuat
dari kacang kedelai (Glycine max) dan telah menjadi makanan yang populer di
dunia terutama bagi kelompok vegetarian. Tempe dihasilkan melalui proses
fermentasi kacang kedelai yang melibatkan sejumlah mikroorganisme seperti
cendawan, khamir, bakteri asam laktat dan juga beberapa kelompok bakteri Gram
negatif. Selama proses fermentasi, terjadi biosintesis vitamin, senyawa fitokimia
dan komponen bioaktif lainnya yang dapat berpengaruh terhadap kesehatan.
Selain itu, pada tempe masih dapat ditemukan komponen oligosakarida dan
polisakarida yang bernilai positif terhadap kesehatan.
Mikroorganisme yang ada di dalam tempe akan turut masuk ke dalam
saluran pencernaan jika tempe dimakan. Beberapa penelitian telah menunjukkan
bahwa aktivitas probiotik dari mikroorganisme mati seringkali menunjukkan
pengaruh yang sama dibandingkan dengan mikroorganisme hidup. Kandungan
mikroorganisme yang ada di dalam tempe dapat menjadi salah satu alternatif
suplemen diet yang mengandung probiotik dan memicu stimulan kekebalan tubuh
pada manusia. Imunoglobulin A (IgA) merupakan imunoglobulin utama yang
terdapat di saluran pencernaan dan mempunyai peranan dalam melindungi tubuh
dari komponen dan juga mikrob asing.
Penelitian ini bertujuan menentukan dinamika populasi mikrobiota dan
IgA dalam usus tikus Sprague-Dawley (SD). Sebanyak 30 ekor tikus SD betina
diberi suplementasi pakan standar yang ditambah dengan kedelai yang belum
diberi laru atau tempe (mentah atau masak) selama 28 hari. Teknik real-time
polymerase chain reaction (RT-PCR) dengan primer spesifik terhadap sekuens
gen penyandi 16S rRNA digunakan untuk mengkuantifikasi kelompok bakteri
spesifik dari sampel feses tikus. Analisis ekspresi gen IgA dilakukan
menggunakan metode quantitative RT-PCR, sedangkan protein IgA dianalisis
dengan metode enzyme-linked immunosorbent assay dan pewarnaan
imunohistokimia.
Hasil penelitian ini menunjukkan bahwa tempe dapat memodulasi
komposisi mikrobiota usus, namun jenis tempe yang berbeda dapat memberikan
perubahan komposisi yang berbeda pula. Hal ini disebabkan oleh keberadaan
mikroorganisme yang unik pada setiap tempe.
Mikroorganisme yang mati pada tempe masak dan mikroorganisme yang
hidup pada tempe mentah terbukti dapat meningkatkan ekspresi gen IgA dan
menstimulasi sekresi protein IgA. Proses pemasakan tempe sangat dianjurkan
dengan tujuan untuk menginaktivasi mikroorganisme patogen yang tidak
diinginkan selama proses fermentasi.
Kata kunci: tempe, mikrobiota usus, imunitas mucosal, immunoglobulin A
SUMMARY
SUSAN SOKA. Effect of Tempeh Supplementation on Gut Microbiota and
Immunoglobulin A Profiles in Sprague Dawley Rats. Supervised by ANTONIUS
SUWANTO, DONDIN SAJUTHI and IMAN RUSMANA.
Recent nutrigenomics studies have showed that the interplay between genes
and diet plays an important role in the development of certain diseases and in the
maintenance of optimal metabolism. In addition, nutrigenomics showed that
trillions of bacteria that normally reside within the human gastrointestinal tract,
also referred as the gut microbiota, has been showed to affect nutrient acquisition
and energy regulation. These findings showed the important role of gut microbiota
in human health through its effect on the gut defense barrier, immune
development, and nutrient utilization.
Tempeh is a well-known Indonesian fermented food made from soybean
(Glycine max), and is now becoming popular in the world, especially in the diets
of vegetarians. A diverse group of microorganisms including moulds, yeasts,
lactic acid bacteria and different Gram-negative bacteria plays an important role
during the fermentation process. Many studies have also reported that tempeh is a
good source of protein, vitamin B12, phytochemicals and other bioactive
substances. Stachyose and raffinose are non-digestible-galactooligosaccharides in
soybean which can still be found in tempeh. There are many health benefits
associated with this dietary fiber component, not least the effect on satiety and
fecal bulking.
Tempeh is consumed in the form of cooked tempeh and consuming cooked
tempeh may further expose gastrointestinal tract to many non-viable
microorganisms. Despite the common definition that probiotics are live
microorganisms, various biological responses have been reported after
administering dead, frequently heat-killed, probiotics to various mammalian and
avian species. Direct interactions between the host and non-viable bacterial cells
are based on the capacity of human cells to recognize specific bacterial
components or products, giving rise to responses that commonly involve the
mucosa-associated lymphoid tissue and, therefore, the immune system. Secretory
immunoglobulin A (IgA), as the most abundant intestinal immunoglobulin on the
surface of the mucosa, has further the combined task of protecting against foreign
substances and microbes
The objective of this study was to evaluate the impact of Empang and
Warung Jambu tempeh on gut microbiota composition and mucosal IgA in
Sprague-Dawley rats. In this study, thirty female Sprague-Dawley (SD) rats were
fed a standard diet, supplemented with either non-fermented soybeans or tempeh
(raw or cooked), for 28 days. The specific bacterial groups in fecal samples were
quantified using real-time PCR with 16S rRNA gene-targeted, group-specific
primers. The gene expression of intestinal IgA was analyzed using the semi
quantitative real-time PCR, and intestinal IgA was further quantified from the
ileum wash using the ELISA and immunohistochemistry methods.
The result of this study showed that tempeh might modulate the
composition of gut microbiota. However, different types of tempeh may offer
variable health benefits due to its unique microorganism composition that plays an
important role during the fermentation process.
The result of IgA analysis indicated that both live and dead microorganisms
can confer health benefits by stimulating IgA secretion in gut mucosal tissue.
Consuming cooked tempeh may offer a further health benefit, as the cooking
process inactivates pathogenic microorganisms that may occur in tempeh during
the fermentation processes.
Keywords: tempeh, gut microbiota, mucosal immunity, immunoglobulin A
©Copyright Bogor Agricultural University, 2014
This copyright is protected by law
Any unauthorized quotation of part or this entire dissertation without referencing
the author and institution is prohibited. Reproduction of this material is only
permitted for research and education purposes and must not undermine the rights
of Bogor Agricultural University
It is forbidden to publish or copy this dissertation in any form without prior
permission from Bogor Agricultural University
EFFECT OF TEMPEH SUPPLEMENTATION ON
GUT MICROBIOTA AND IMMUNOGLOBULIN A PROFILES
IN SPRAGUE DAWLEY RATS
SUSAN SOKA
Dissertation
as one of the requirements of the
PhD program in the
Major Microbiology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
Examiners of Closed Examination
: Prof. Dr. Lilis Nuraida, M.Sc.
Raymond Tjandrawinata, Ph.D.
Examiners of Opened Examination : Prof. Dr. Made Astawan
Achmad Dinoto, Ph.D.
Title of Dissertation : Effect of Tempeh Supplementation on Gut Microbiota and
Immunoglobulin A Profiles in Sprague Dawley Rats
Name
: Susan Soka
NIM
: G36111041
Approved by
Supervisory Committee
Prof. Dr. Antonius Suwanto, M.Sc
Head of Committee
Prof. drh. Dondin Sajuthi, MST, Ph.D.
Member of Committee
Dr. Iman Rusmana, M.Sc.
Member of Committee
Authorized by
Head of Major Microbiology
Dean of Graduate School
Prof. Dr. Anja Meryandini
Dr Ir Dahrul Syah, M.Sc.Agr.
Date of Opened Examination:
27 November 2014
Date of Graduation:
ACKNOWLEDGEMENTS
It would not have been possible to complete this dissertation without the
support, patience and guidance of the following people. It is to them that I owe my
deepest gratitude.
My promotor, Prof. Dr. Antonius Suwanto, M.Sc., for his endless
support, enthusiasm, knowledge and patience during my graduate study
at Bogor Agricultural University.
My co-promotor, Prof. drh. Dondin Sajuthi, MST, Ph.D. for his
guidance and knowledge in Immunology and animal study.
My co-promotor, Dr. Iman Rusmana for his support, guidance and input
throughout finalizing my dissertation.
Indonesian Minister of Education for the doctorate scholarship and
research funding.
Dr. Ir. Gayuh Rahayu, MS and Prof. Dr. Anja Meryandini as former
Head and Head of Graduate Programe Microbiology in Bogor
Agricultural University for their support during my study, respectively.
Dean of Faculty of Biotechnology at Atma Jaya Catholic University of
Indonesia, Dr. Diana E. Waturangi, M.Si., for giving me the opportunity
to finish my doctorate study and also for providing the laboratory
facilities.
Dr. Diah Iskandriati, MS for the provision of the animal laboratory
facility in Primate Research Center at the Bogor Agricultural University.
My fellow postgraduate students and friends both at Bogor Agricultural
University and PT. Bimana Indomedical in Bogor for their support and
friendships.
Finally, and most importantly, I take this opportunity to express my
gratitude to my family for their love, unfailing encouragement and support. I
would like to thank my husband Erwin for his patience during the ups and
downs of my study. I would like to thank my sons, Darrence and Jeremy, for
their loves. I thank my parents for their love and faith in me. And also, I thank
Erwin’s parents, for providing me with unending encouragement and support.
Bogor, November 2014
Susan Soka
CONTENTS
LIST OF TABLES
xv
LIST OF FIGURES
xv
INTRODUCTION
Background
Hypothesis
Objectives
Novelty
1
1
2
2
3
LITERATURE REVIEW
Tempeh
Carbohydrates in Soybean
Oligosaccharides in Soybean Tempeh
Gut Microbiota and Immune System
The Paraprobiotic Concept
Real Time-Polymerase Chain Reaction
3
3
4
4
6
8
8
MATERIALS AND METHODS
9
Tempeh Preparation
9
Protein Quantification in Tempeh
9
Animal Study
9
Total Fecal Bacterial DNA Extraction
10
Construction of Standard Curve
10
Quantification of Bacterial Specific-Group from Feces
11
Extraction of the Ileum and the Colon
11
ELISA IgA Quantification
11
Assesment of mRNA Gene Expression Using Real-Time Polymerase Chain
Reaction (PCR)
12
Immunohistochemistry of sIgA in the Gut
13
Statistical Analysis
13
RESULTS AND DISCUSSIONS
Empang- and Warung Jambu Tempeh
Impact of Tempeh Supplementation on Gut Microbiota Composition in
Sprague-Dawley Rats
Impact of Tempeh Supplementation on Mucosal Immunoglobulin A in
Sprague-Dawley Rats
13
13
CONCLUSION
32
REFERENCES
33
APPENDIX
40
CURRICULUM VITAE
42
15
21
LIST OF TABLES
1
2
3
4
5
6
7
8
16S rRNA gene-targeted, group specific primers
Primer sequences used for qRT-PCR
qRT-PCR conditions
The protein content and total added non-fermented soybean and tempeh
in animal feed.
Impact of Empang (EMP) tempeh on gut microbiota composition
Impact of Warung Jambu (WJB) tempeh on gut microbiota composition
The ratio of Firmicutes/Bacteroidetes in the three groups following
supplementation with Empang tempeh for 28 days
The ratio of Firmicutes/Bacteroidetes in the three groups following
supplementation with Warung Jambu tempeh for 28 days.
11
12
13
15
17
18
20
20
LIST OF FIGURES
1 The difference of total fecal bacteria before and after Empang tempeh
supplementation for 28 days
2 The difference of total fecal bacteria before and after Warung Jambu
tempeh supplementation for 28 days
3 Effect of tempeh supplementation on IgA gene expression
4 Effect of tempeh supplementation on IgA concentration in ileum
washes
5 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with cooked Empang tempeh
6 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with cooked Empang tempeh
7 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with non-fermented Empang soybean
8 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with cooked Warung Jambu tempeh
9 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with raw Warung Jambu tempeh
10 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with non-fermented Warung Jambu soybean
19
19
22
23
25
26
27
28
29
30
INTRODUCTION
Background
Recent nutrigenomics studies have showed that the interplay between
genes and diet plays an important role in the development of certain diseases and
in the maintenance of optimal metabolism. In addition, nutrigenomics showed that
trillions of bacteria that normally reside within the human gastrointestinal tract
(referred to as the gut microbiota) can affect nutrient acquisition and energy
regulation. This suggests that obese and lean people have different gut microbiota
(Ley et al. 2005). These findings showed the important role of gut microbiota in
human health through their effect on the gut defense barrier, immune
development, and nutrient utilization. Comparative studies using 16S rRNA
sequencing have shown that the intestinal microbiota in mice and humans is very
similar in composition at the division level (i.e., ≥80% of both mouse and human
microbiota is dominated by two phyla, the Firmicutes and the Bacteroidetes)
(Sekirov et al. 2010). However, composition of a microbial community is host
specific. It evolves throughout an individual’s lifetime and is susceptible to both
exogenous and endogenous modifications. The composition of human gut
microbiota is mainly influenced by maternal colonization but is further influenced
by diet, environmental exposures, and antimicrobial therapies. Diverse disorders,
such as antibiotic-associated diarrhea, Crohn’s disease, ulcerative colitis, and
obesity have been correlated with large-scale imbalances in the gastrointestinal
microbiota, demonstrating the importance of commensal microorganisms in
maintaining gastrointestinal balance.
Tempeh is a well-known Indonesian fermented food made from soybean
(Glycine max). It is gaining popularity in the world, especially in the diets of
vegetarians. A diverse group of microorganisms including molds, yeasts, lactic
acid-producing bacteria, and some Gram-negative bacteria plays an important role
during the fermentation process (Steinkraus et al. 1983). Many studies also have
reported that tempeh is a good source of protein (Astuti et al. 2000), vitamin B12
(Okada 1989), phytochemicals (Murakami et al. 1984), and other bioactive
substances. Stachyose and raffinose are non-digestible galactooligosaccharides in
soybean found in tempeh, although it has been reported that the concentrations of
oligosaccharides in soybeans are reduced during the fermentation process (RuizTerán and Owens 1999; Egounlety and Aworh 2003). Since these dietary
components are undigested by human enzymes, the human colonic bacteria play
important roles in the digestion of dietary fiber through the fermentation process
(Scott et al. 2008). This fermentation process will further lead to the formation of
some acidic compounds, such as acetate, lactate, butyrate, and propionate. These
short-chain fatty acids will be released and cause further decrease in pH value
(Scott et al. 2008; Binns 2013). The benefits of a lower pH in the gut intestine
include enhanced multiplication and survival of organisms that prefer acidic
conditions and general inhibition of some pathogens. There are many health
benefits associated with dietary fiber intake, not including effects on satiety
2
(Samra and Anderson, 2007) and fecal bulking. Therefore, many countries are
attempting to increase the recommended dietary fiber intake in an effort to prevent
some of the diseases afflicting modern society.
Moreover, tempeh is consumed in the form of cooked tempeh and
consuming cooked tempeh may further expose gastrointestinal tract (GIT) to
many non-viable microorganisms. Most scientific studies define probiotics
according to the FAO/WHO definition, which describes them as dietary
supplements containing viable nonpathogenic microorganisms that, when
administered in adequate amounts, confer a health benefit on the host. However,
recent studies have shown that the different amounts of non-viable
microorganisms found in probiotic products might contribute to the variation in
response that is often seen with live probiotics (Kataria et al. 2009). Direct
interactions between the host and non-viable bacterial cells are based on the
capacity of human cells to recognize specific bacterial components or products,
giving rise to responses that commonly involve the mucosa-associated lymphoid
tissue (MALT) and, therefore, the immune system (Adams 2010). Various
microbial components such as cell homogenates, β-glucans, teichoic and
lipoteichoic acids, peptidoglycans (PGNs), and lipopolysaccharides (LPSs) have
been proven to have an immunomodulatory effect by stimulating innate immune
systems (Adams 2010; Taverniti and Guglielmetti 2011). Secretory
immunoglobulin A (sIgA) is the most abundant intestinal immunoglobulin on the
surface of the mucosa, which has the combined task of protecting against foreign
substances and microbes (Harris et al. 2006).
Hypothesis
Many microorganisms are involved during the making of tempeh. Whether
tempeh is consumed in the cooked or raw form, these microorganisms may serve
as non-viable probiotics for the host and therefore increasing the immune system.
Moreover, non-soluble polysaccharides presented mainly by soybean and
Rhizopus sp., can serve as prebiotics. Consuming tempeh that might play a role as
probiotics and prebiotics might shift the bacterial composition of gut microbiota.
The following microbial shifting is expected toward a healthier gut microbiota
composition.
Objectives
The objective of this study was to evaluate the impact of tempeh
supplementation on gut microbiota composition and mucosal immunity in
Sprague-Dawley (SD) rats. In this study, intestinal IgA was quantified at both the
gene- and protein-expression levels in groups of rats that were fed with either raw
or cooked tempeh, as well as in control rats that were fed with non-fermented
soybeans.
3
Novelty
The impact of tempeh supplementation on gut microbiota composition and
mucosal immunity in Sprague-Dawley rats has not been studied. This research
will study how tempeh, which can be considered as a potential source of nonviable probiotics and prebiotics, may become a possible strategy to stimulate the
immune system and manage the complex gut microbial community towards a
health-promoting composition. Therefore, the result of this nutrigenomic studies
will further give tempeh an added nutritional value.
LITERATURE REVIEW
Tempeh
Tempeh is a traditional Indonesian fermented food in which soybeans are
hydrated and acidified, dehulled, cooked and fermented with Rhizopus spp. There
is no standard process for tempeh making, therefore many variations can be found
from one region and one producer from another. During the fermentation process
of tempeh, there are many valuable changes not only in the increase of nutritional
values of some nutrients in soybean, but also in the development of vitamins,
phytochemicals and other bioactive compounds (Astuti et al. 2000). Enzymes
produced during fermentation affect protein, fat and carbohydrates contents in
soybean. Soybean oligosaccharides such as stachyose and raffinose, which cause
flatulence, are broken down into digestible sugars. Moreover, the fungus produces
the enzyme phytase that mobilize phytic acid improving bioavailability of
minerals (Nout and Kiers 2005).
Most of microbiological studies on tempeh production have been focused
on the fungi as starter, however, the association of bacteria and yeast during the
fermentation process has been reported (Nout and Kiers 2005; Barus et al. 2008;
Seumahu et al. 2012). Klebsiella pneumoniae and Citrobacter freundii found in
fresh tempeh implicated their roles in the production of vitamin B12 during the
fermentation (Mulyowidarso et al. 1990; Keuth and Bisping 1994). Increased
protein content in tempeh has been reported due to the cooking and fermentation
steps during tempeh processing, which favor losses of soluble substances from
soybean (Vaidehi and Rathnamani 1990; Bavia et al. 2012). On the other hand,
the oil content observed in soybean is higher than in tempeh (Bavia et al. 2012).
Valeric, propionic, formic and acetic are the main acids in soybean. Their
concentrations at the end of soaking step were decreased progressively
(Mulyowidarso et al. 1991a). At the end of soaking, lactic and malic were the
main acids in soybean. These acids were produced by microbial metabolism of
soybean sugars leached into the water and contribute to the decrease in pH of the
soak-water and soybeans. The main microorganisms responsible for producing
lactic acid are Lactobacillus casei, Staphylococcus epidermidis, Streptococcus
4
faecium and for producing malic acid is L. casei (Mulyowidarso et al. 1991a). The
presence of bacteria producing organic acids during the soaking process showed
their important roles in the inhibition of various pathogenic and spoilage
microorganisms in tempeh (Nout and Kiers 2005).
Carbohydrates in Soybean
Soybean (Glycine max) contains ~35% carbohydrates which consist
mainly of non-starch polysaccharides and oligosaccharides (Dixit et al. 2011).
Polysaccharides are composed mainly of insoluble dietary fiber, such as cellulose
and pectins. Soybean oligosaccharide is a group of soluble low molecular weight
oligosaccharides in soybean seeds, which include sucrose (1%), stachyose (4%)
and raffinose (1.1%) (Choct et al. 2010). Stachyose is a tetraose with a structure
of α-D-galactopyranosyl-[1→6] – α – D – galactopyranosyl – [1→ 6] – α – D –
glucopyranosyl – [1 → 2] – β–D–fructofuranoside, while raffinose is a triose with
a structure of α–D–galactopyranosyl–[1→6]–α–D-glucopyranosyl–[1→2]–β–D–
fructofuranoside. Stachyose and raffinose are defined as non-digestible
galactooligosaccharides (GOS). GOS content of soybean has been shown to vary
with the degree of maturity. Immature green seeds contain fewer amounts of GOS
than fully matured yellow soybean (Espinosa-Martos and Rupérez 2006).
Humans do not posses α-galactosidase necessary for hydrolyzing the
linkage present in oligosaccharides, so that they cannot be digested when
consumed. When these intact oligosacharides reach the colon, they will be
preferentially fermented by colonic bacteria that posses the enzyme (Liu 1999).
Anaerobic bacterial fermentation of these substrates results in the production of
gases which cause uncomfortable flatulence. Many studies have been carried out
to reduce the oligosaccharides content in soybean products by processing
techniques such as soaking, cooking, fermentation and enzyme treatment
(Egounlety and Aworh 2003; Gote et al. 2004). However, many clinical
researches have suggested that oligosaccharides, with approximately 30 to 50%
caloric value of sucrose, are interesting because of their prebiotic activity and
associated health benefit (Roberfroid and Slavin 2000). Along with the progress
of oligosaccharides researches, it was found that soybean oligosaccharides are not
the direct causes of flatulence (Cui 2001).
Oligosaccharides in Soybean Tempeh
Stachyose and raffinose are non-digestible-galactooligosaccharides in
soybean which can still be found in tempeh although it has been reported that the
concentrations of oligosaccharides in soybeans are reduced during the
fermentation process. However, there are considerable differences in the extent of
the reductions reported.
Soaking in water is an essential preliminary step in the use of soybean for
the preparation of tempeh (Steinkraus et al. 1983). Mulyowidarso et al. (1991b)
reported that the concentration of stachyose and raffinose in soybean decreased by
5
65% and 50% respectively after soaking at 30°C for 24h. Meanwhile, when a
mixture of antibiotics was added in the soak-water in order to completely inhibit
the growth of microorganisms, the decrease of stachyose concentrations was
reduced. Thus, at the end of 24h soaking, the soybean contained approximately
1.6% stachyose, compared with approximately 1% stachyose for soaking in the
absence of antibiotics (Mulyowidarso et al. 1991b). The decreased concentration
of raffinose was approximately the same as that which occurred during soaking in
the absence of antibiotics.
The changes of these oligosaccharide concentrations within the beans
during soaking could be attributed to endogenous enzyme metabolism, diffusion
into the external environment of the soak-water and microorganisms activities that
grow in the soak-water (Mulyowidarso et al. 1991b). Total microbial populations
of 1010 CFU mL-1were developed during the first 18h of soaking at 30°C with the
main species are L. casei, S. epidermidis, S. faecium and Streptococcus
dysgalactiae (Mulyowidarso et al. 1989). α-galactosidases produced by these
microbial species in the soak water could diffuse into the soybean and accelerate
the hydrolysis of soybean oligosaccharides. Moreover, microbial fermentation of
glucose and reduction in pH of the soak-water and soybean to 5.0 could affect the
activity of soybean α-galactosidase. Since the pH optimum of this enzyme has
been reported to be 5.0 (Harpaz et al. 1977), microbial fermentation is likely to
stimulate the enzyme activity. Recent studies have also reported the role of some
bacteria during the fermentation process in tempeh production (Barus et al. 2008;
Seumahu et al. 2012). In conclusion, the decrease of the oligosaccharides
concentration within the soybean will depend on the microbial species that
develop and predominate in the soak-water (Mulyowidarso et al. 1991b).
Furthermore, Ruiz-Terán and Owens (1999) studied the fate of
oligosaccharides, stachyose and raffinose, during production of soybean tempeh.
Stachyose and raffinose were monitored during the preparation of bacteria-free
tempeh made with R. oligosporus NRRL 2710. The essential losses of
oligosaccharides were due to leaching during hydration, washing and cooking of
the soybean. Meanwhile, the concentrations of these oligosaccharides did not
change during the fermentation. The result of this study was in agreement with
observations that, in vitro, tempeh moulds did not use the oligosaccharides as sole
sources of carbon and energy (Graffham et al. 1995). Therefore, it is possible to
control the concentration of oligosaccharides by making choice of conditions for
hydrating and cooking the beans. This control might be facilitated by the fact that
the oligosaccharides are not utilized in tempeh by the moulds.
On the other hand, a recent study on the effect of fermentation with
Rhizopus oligosporus on the oligosaccharides of soybean, cowpea (Vigna
unguiculata) and groundbean (Macrotyloma geocarpa) was done by Egounlety
and Aworh (2003). Stachyose and raffinose decreased during 48h fermentation
with a reduction of 83.9% and 55.4%; 91.5% and 53.8%, and 85.5% and 54.0%
respectively for soybean, cowpea and groundbean. This study concluded that α-D
galactosidase and α-D glucosidase produced by Rhizopus species during
fermentation degraded soybean oligosaccharides which resulted in the production
of galactose. This suggested that the α-D galactosidase acts on the
oligosaccharides at the galactose moety (Egounlety 1994).
6
The conflicting reports in the literature on the production of
oligosaccharidases and the utilization of oligosaccharides during tempeh
fermentation are possibly a consequence of the presence of bacteria, or, in some
cases, could be due to the use of different mould strains (Ruiz-Terán and Owens
1999). However, it is still possible to control the concentration of oligosaccharides
by making choice of conditions for hydrating and cooking the beans and selecting
the microorganisms used during fermentation. Therefore, the possible prebiotic
effect of soybean can still be found in tempeh.
Gut Microbiota and Immune System
Recent studies suggested that the trillions of bacteria that normally reside
within the human gastrointestinal tract, also referred as the gut microbiota, affect
nutrient acquisition and energy regulation; it suggests further that obese and lean
people have different gut microbiota. The composition of this microbial
community is host specific, evolving throughout an individual's lifetime and
susceptible to both exogenous and endogenous modifications (Sekirov et al.
2010). Firmicutes and Bacteroidetes are closely related to body fat in humans and
mice, which are the two dominant bacterial divisions of gut microbiota in
mammals. Comparisons of the distal gut microbiota of obese and lean mice, and
those of obese and lean humans revealed that a statistically significant reduction
in the relative abundance of Bacteriodetes and a significantly greater proportion of
Firmicutes were in obese animals than in lean controls (Ley et al. 2005).
In healthy adults, 80% of the identified fecal microbiota can be classified
into three dominant phyla: Bacteroidetes, Firmicutes, and Actinobacteria (Lay et
al. 2005). The Firmicutes and Bacteroidetes ratio is regarded to be of significant
relevance in human gut microbiota composition. However, the fecal microbiota is
a highly complex and diverse bacterial ecosystem. Within this ecosystems exists a
hierarchy of dominant (>109 CFU g-1) anaerobic bacteria, represented by the
genera Bacteroides, Eubacterium, Bifidobacterium, Peptostreptococcus,
Ruminococcus, Clostridium and Propionibacterium, and sub-dominant (
GUT MICROBIOTA AND IMMUNOGLOBULIN A PROFILES
IN SPRAGUE DAWLEY RATS
SUSAN SOKA
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
DECLARATION OF THE SOURCE OF THIS DISSERTATION
I declare that this dissertation, entitled Effect of Tempeh Supplementation on
Gut Microbiota and Immunoglobulin A Profiles in Sprague Dawley Rats is
entirely my own work, assisted by a supervisory committee and has not been
submitted in any form for another degree or diploma to any university or other
tertiary institution of education. Where this dissertation draws on existing
publications, those sources are cited in the text and listed in the references section.
Bogor, November 2014
Susan Soka
G361110041
RINGKASAN
SUSAN SOKA. Pengaruh Suplementasi Tempe terhadap Profil Mikrobiota Usus
dan Imunoglobulin A pada Tikus Sprague Dawley. Dibimbing oleh ANTONIUS
SUWANTO, DONDIN SAJUTHI dan IMAN RUSMANA.
Penelitian nutrigenomika saat ini telah menunjukkan bahwa interaksi gen
dan makanan mempunyai peranan penting dalam menjaga kesehatan yang optimal.
Selain itu, studi nutrigenomika juga telah menunjukkan bahwa triliunan bakteri
yang ada pada saluran pencernaan manusia, atau yang lebih dikenal dengan
mikrobiota usus, dapat mempengaruhi penyerapan nutrisi dan regulasi energi. Hal
ini menunjukkan pentingnya peranan mikrobiota usus pada kesehatan manusia
dalam perkembangan sistem kekebalan tubuh dan penggunaan energi.
Tempe merupakan makanan fermentasi tradisional Indonesia yang terbuat
dari kacang kedelai (Glycine max) dan telah menjadi makanan yang populer di
dunia terutama bagi kelompok vegetarian. Tempe dihasilkan melalui proses
fermentasi kacang kedelai yang melibatkan sejumlah mikroorganisme seperti
cendawan, khamir, bakteri asam laktat dan juga beberapa kelompok bakteri Gram
negatif. Selama proses fermentasi, terjadi biosintesis vitamin, senyawa fitokimia
dan komponen bioaktif lainnya yang dapat berpengaruh terhadap kesehatan.
Selain itu, pada tempe masih dapat ditemukan komponen oligosakarida dan
polisakarida yang bernilai positif terhadap kesehatan.
Mikroorganisme yang ada di dalam tempe akan turut masuk ke dalam
saluran pencernaan jika tempe dimakan. Beberapa penelitian telah menunjukkan
bahwa aktivitas probiotik dari mikroorganisme mati seringkali menunjukkan
pengaruh yang sama dibandingkan dengan mikroorganisme hidup. Kandungan
mikroorganisme yang ada di dalam tempe dapat menjadi salah satu alternatif
suplemen diet yang mengandung probiotik dan memicu stimulan kekebalan tubuh
pada manusia. Imunoglobulin A (IgA) merupakan imunoglobulin utama yang
terdapat di saluran pencernaan dan mempunyai peranan dalam melindungi tubuh
dari komponen dan juga mikrob asing.
Penelitian ini bertujuan menentukan dinamika populasi mikrobiota dan
IgA dalam usus tikus Sprague-Dawley (SD). Sebanyak 30 ekor tikus SD betina
diberi suplementasi pakan standar yang ditambah dengan kedelai yang belum
diberi laru atau tempe (mentah atau masak) selama 28 hari. Teknik real-time
polymerase chain reaction (RT-PCR) dengan primer spesifik terhadap sekuens
gen penyandi 16S rRNA digunakan untuk mengkuantifikasi kelompok bakteri
spesifik dari sampel feses tikus. Analisis ekspresi gen IgA dilakukan
menggunakan metode quantitative RT-PCR, sedangkan protein IgA dianalisis
dengan metode enzyme-linked immunosorbent assay dan pewarnaan
imunohistokimia.
Hasil penelitian ini menunjukkan bahwa tempe dapat memodulasi
komposisi mikrobiota usus, namun jenis tempe yang berbeda dapat memberikan
perubahan komposisi yang berbeda pula. Hal ini disebabkan oleh keberadaan
mikroorganisme yang unik pada setiap tempe.
Mikroorganisme yang mati pada tempe masak dan mikroorganisme yang
hidup pada tempe mentah terbukti dapat meningkatkan ekspresi gen IgA dan
menstimulasi sekresi protein IgA. Proses pemasakan tempe sangat dianjurkan
dengan tujuan untuk menginaktivasi mikroorganisme patogen yang tidak
diinginkan selama proses fermentasi.
Kata kunci: tempe, mikrobiota usus, imunitas mucosal, immunoglobulin A
SUMMARY
SUSAN SOKA. Effect of Tempeh Supplementation on Gut Microbiota and
Immunoglobulin A Profiles in Sprague Dawley Rats. Supervised by ANTONIUS
SUWANTO, DONDIN SAJUTHI and IMAN RUSMANA.
Recent nutrigenomics studies have showed that the interplay between genes
and diet plays an important role in the development of certain diseases and in the
maintenance of optimal metabolism. In addition, nutrigenomics showed that
trillions of bacteria that normally reside within the human gastrointestinal tract,
also referred as the gut microbiota, has been showed to affect nutrient acquisition
and energy regulation. These findings showed the important role of gut microbiota
in human health through its effect on the gut defense barrier, immune
development, and nutrient utilization.
Tempeh is a well-known Indonesian fermented food made from soybean
(Glycine max), and is now becoming popular in the world, especially in the diets
of vegetarians. A diverse group of microorganisms including moulds, yeasts,
lactic acid bacteria and different Gram-negative bacteria plays an important role
during the fermentation process. Many studies have also reported that tempeh is a
good source of protein, vitamin B12, phytochemicals and other bioactive
substances. Stachyose and raffinose are non-digestible-galactooligosaccharides in
soybean which can still be found in tempeh. There are many health benefits
associated with this dietary fiber component, not least the effect on satiety and
fecal bulking.
Tempeh is consumed in the form of cooked tempeh and consuming cooked
tempeh may further expose gastrointestinal tract to many non-viable
microorganisms. Despite the common definition that probiotics are live
microorganisms, various biological responses have been reported after
administering dead, frequently heat-killed, probiotics to various mammalian and
avian species. Direct interactions between the host and non-viable bacterial cells
are based on the capacity of human cells to recognize specific bacterial
components or products, giving rise to responses that commonly involve the
mucosa-associated lymphoid tissue and, therefore, the immune system. Secretory
immunoglobulin A (IgA), as the most abundant intestinal immunoglobulin on the
surface of the mucosa, has further the combined task of protecting against foreign
substances and microbes
The objective of this study was to evaluate the impact of Empang and
Warung Jambu tempeh on gut microbiota composition and mucosal IgA in
Sprague-Dawley rats. In this study, thirty female Sprague-Dawley (SD) rats were
fed a standard diet, supplemented with either non-fermented soybeans or tempeh
(raw or cooked), for 28 days. The specific bacterial groups in fecal samples were
quantified using real-time PCR with 16S rRNA gene-targeted, group-specific
primers. The gene expression of intestinal IgA was analyzed using the semi
quantitative real-time PCR, and intestinal IgA was further quantified from the
ileum wash using the ELISA and immunohistochemistry methods.
The result of this study showed that tempeh might modulate the
composition of gut microbiota. However, different types of tempeh may offer
variable health benefits due to its unique microorganism composition that plays an
important role during the fermentation process.
The result of IgA analysis indicated that both live and dead microorganisms
can confer health benefits by stimulating IgA secretion in gut mucosal tissue.
Consuming cooked tempeh may offer a further health benefit, as the cooking
process inactivates pathogenic microorganisms that may occur in tempeh during
the fermentation processes.
Keywords: tempeh, gut microbiota, mucosal immunity, immunoglobulin A
©Copyright Bogor Agricultural University, 2014
This copyright is protected by law
Any unauthorized quotation of part or this entire dissertation without referencing
the author and institution is prohibited. Reproduction of this material is only
permitted for research and education purposes and must not undermine the rights
of Bogor Agricultural University
It is forbidden to publish or copy this dissertation in any form without prior
permission from Bogor Agricultural University
EFFECT OF TEMPEH SUPPLEMENTATION ON
GUT MICROBIOTA AND IMMUNOGLOBULIN A PROFILES
IN SPRAGUE DAWLEY RATS
SUSAN SOKA
Dissertation
as one of the requirements of the
PhD program in the
Major Microbiology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
Examiners of Closed Examination
: Prof. Dr. Lilis Nuraida, M.Sc.
Raymond Tjandrawinata, Ph.D.
Examiners of Opened Examination : Prof. Dr. Made Astawan
Achmad Dinoto, Ph.D.
Title of Dissertation : Effect of Tempeh Supplementation on Gut Microbiota and
Immunoglobulin A Profiles in Sprague Dawley Rats
Name
: Susan Soka
NIM
: G36111041
Approved by
Supervisory Committee
Prof. Dr. Antonius Suwanto, M.Sc
Head of Committee
Prof. drh. Dondin Sajuthi, MST, Ph.D.
Member of Committee
Dr. Iman Rusmana, M.Sc.
Member of Committee
Authorized by
Head of Major Microbiology
Dean of Graduate School
Prof. Dr. Anja Meryandini
Dr Ir Dahrul Syah, M.Sc.Agr.
Date of Opened Examination:
27 November 2014
Date of Graduation:
ACKNOWLEDGEMENTS
It would not have been possible to complete this dissertation without the
support, patience and guidance of the following people. It is to them that I owe my
deepest gratitude.
My promotor, Prof. Dr. Antonius Suwanto, M.Sc., for his endless
support, enthusiasm, knowledge and patience during my graduate study
at Bogor Agricultural University.
My co-promotor, Prof. drh. Dondin Sajuthi, MST, Ph.D. for his
guidance and knowledge in Immunology and animal study.
My co-promotor, Dr. Iman Rusmana for his support, guidance and input
throughout finalizing my dissertation.
Indonesian Minister of Education for the doctorate scholarship and
research funding.
Dr. Ir. Gayuh Rahayu, MS and Prof. Dr. Anja Meryandini as former
Head and Head of Graduate Programe Microbiology in Bogor
Agricultural University for their support during my study, respectively.
Dean of Faculty of Biotechnology at Atma Jaya Catholic University of
Indonesia, Dr. Diana E. Waturangi, M.Si., for giving me the opportunity
to finish my doctorate study and also for providing the laboratory
facilities.
Dr. Diah Iskandriati, MS for the provision of the animal laboratory
facility in Primate Research Center at the Bogor Agricultural University.
My fellow postgraduate students and friends both at Bogor Agricultural
University and PT. Bimana Indomedical in Bogor for their support and
friendships.
Finally, and most importantly, I take this opportunity to express my
gratitude to my family for their love, unfailing encouragement and support. I
would like to thank my husband Erwin for his patience during the ups and
downs of my study. I would like to thank my sons, Darrence and Jeremy, for
their loves. I thank my parents for their love and faith in me. And also, I thank
Erwin’s parents, for providing me with unending encouragement and support.
Bogor, November 2014
Susan Soka
CONTENTS
LIST OF TABLES
xv
LIST OF FIGURES
xv
INTRODUCTION
Background
Hypothesis
Objectives
Novelty
1
1
2
2
3
LITERATURE REVIEW
Tempeh
Carbohydrates in Soybean
Oligosaccharides in Soybean Tempeh
Gut Microbiota and Immune System
The Paraprobiotic Concept
Real Time-Polymerase Chain Reaction
3
3
4
4
6
8
8
MATERIALS AND METHODS
9
Tempeh Preparation
9
Protein Quantification in Tempeh
9
Animal Study
9
Total Fecal Bacterial DNA Extraction
10
Construction of Standard Curve
10
Quantification of Bacterial Specific-Group from Feces
11
Extraction of the Ileum and the Colon
11
ELISA IgA Quantification
11
Assesment of mRNA Gene Expression Using Real-Time Polymerase Chain
Reaction (PCR)
12
Immunohistochemistry of sIgA in the Gut
13
Statistical Analysis
13
RESULTS AND DISCUSSIONS
Empang- and Warung Jambu Tempeh
Impact of Tempeh Supplementation on Gut Microbiota Composition in
Sprague-Dawley Rats
Impact of Tempeh Supplementation on Mucosal Immunoglobulin A in
Sprague-Dawley Rats
13
13
CONCLUSION
32
REFERENCES
33
APPENDIX
40
CURRICULUM VITAE
42
15
21
LIST OF TABLES
1
2
3
4
5
6
7
8
16S rRNA gene-targeted, group specific primers
Primer sequences used for qRT-PCR
qRT-PCR conditions
The protein content and total added non-fermented soybean and tempeh
in animal feed.
Impact of Empang (EMP) tempeh on gut microbiota composition
Impact of Warung Jambu (WJB) tempeh on gut microbiota composition
The ratio of Firmicutes/Bacteroidetes in the three groups following
supplementation with Empang tempeh for 28 days
The ratio of Firmicutes/Bacteroidetes in the three groups following
supplementation with Warung Jambu tempeh for 28 days.
11
12
13
15
17
18
20
20
LIST OF FIGURES
1 The difference of total fecal bacteria before and after Empang tempeh
supplementation for 28 days
2 The difference of total fecal bacteria before and after Warung Jambu
tempeh supplementation for 28 days
3 Effect of tempeh supplementation on IgA gene expression
4 Effect of tempeh supplementation on IgA concentration in ileum
washes
5 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with cooked Empang tempeh
6 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with cooked Empang tempeh
7 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with non-fermented Empang soybean
8 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with cooked Warung Jambu tempeh
9 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with raw Warung Jambu tempeh
10 Immunohistochemical detection of IgA in the ileum section of rats
supplemented with non-fermented Warung Jambu soybean
19
19
22
23
25
26
27
28
29
30
INTRODUCTION
Background
Recent nutrigenomics studies have showed that the interplay between
genes and diet plays an important role in the development of certain diseases and
in the maintenance of optimal metabolism. In addition, nutrigenomics showed that
trillions of bacteria that normally reside within the human gastrointestinal tract
(referred to as the gut microbiota) can affect nutrient acquisition and energy
regulation. This suggests that obese and lean people have different gut microbiota
(Ley et al. 2005). These findings showed the important role of gut microbiota in
human health through their effect on the gut defense barrier, immune
development, and nutrient utilization. Comparative studies using 16S rRNA
sequencing have shown that the intestinal microbiota in mice and humans is very
similar in composition at the division level (i.e., ≥80% of both mouse and human
microbiota is dominated by two phyla, the Firmicutes and the Bacteroidetes)
(Sekirov et al. 2010). However, composition of a microbial community is host
specific. It evolves throughout an individual’s lifetime and is susceptible to both
exogenous and endogenous modifications. The composition of human gut
microbiota is mainly influenced by maternal colonization but is further influenced
by diet, environmental exposures, and antimicrobial therapies. Diverse disorders,
such as antibiotic-associated diarrhea, Crohn’s disease, ulcerative colitis, and
obesity have been correlated with large-scale imbalances in the gastrointestinal
microbiota, demonstrating the importance of commensal microorganisms in
maintaining gastrointestinal balance.
Tempeh is a well-known Indonesian fermented food made from soybean
(Glycine max). It is gaining popularity in the world, especially in the diets of
vegetarians. A diverse group of microorganisms including molds, yeasts, lactic
acid-producing bacteria, and some Gram-negative bacteria plays an important role
during the fermentation process (Steinkraus et al. 1983). Many studies also have
reported that tempeh is a good source of protein (Astuti et al. 2000), vitamin B12
(Okada 1989), phytochemicals (Murakami et al. 1984), and other bioactive
substances. Stachyose and raffinose are non-digestible galactooligosaccharides in
soybean found in tempeh, although it has been reported that the concentrations of
oligosaccharides in soybeans are reduced during the fermentation process (RuizTerán and Owens 1999; Egounlety and Aworh 2003). Since these dietary
components are undigested by human enzymes, the human colonic bacteria play
important roles in the digestion of dietary fiber through the fermentation process
(Scott et al. 2008). This fermentation process will further lead to the formation of
some acidic compounds, such as acetate, lactate, butyrate, and propionate. These
short-chain fatty acids will be released and cause further decrease in pH value
(Scott et al. 2008; Binns 2013). The benefits of a lower pH in the gut intestine
include enhanced multiplication and survival of organisms that prefer acidic
conditions and general inhibition of some pathogens. There are many health
benefits associated with dietary fiber intake, not including effects on satiety
2
(Samra and Anderson, 2007) and fecal bulking. Therefore, many countries are
attempting to increase the recommended dietary fiber intake in an effort to prevent
some of the diseases afflicting modern society.
Moreover, tempeh is consumed in the form of cooked tempeh and
consuming cooked tempeh may further expose gastrointestinal tract (GIT) to
many non-viable microorganisms. Most scientific studies define probiotics
according to the FAO/WHO definition, which describes them as dietary
supplements containing viable nonpathogenic microorganisms that, when
administered in adequate amounts, confer a health benefit on the host. However,
recent studies have shown that the different amounts of non-viable
microorganisms found in probiotic products might contribute to the variation in
response that is often seen with live probiotics (Kataria et al. 2009). Direct
interactions between the host and non-viable bacterial cells are based on the
capacity of human cells to recognize specific bacterial components or products,
giving rise to responses that commonly involve the mucosa-associated lymphoid
tissue (MALT) and, therefore, the immune system (Adams 2010). Various
microbial components such as cell homogenates, β-glucans, teichoic and
lipoteichoic acids, peptidoglycans (PGNs), and lipopolysaccharides (LPSs) have
been proven to have an immunomodulatory effect by stimulating innate immune
systems (Adams 2010; Taverniti and Guglielmetti 2011). Secretory
immunoglobulin A (sIgA) is the most abundant intestinal immunoglobulin on the
surface of the mucosa, which has the combined task of protecting against foreign
substances and microbes (Harris et al. 2006).
Hypothesis
Many microorganisms are involved during the making of tempeh. Whether
tempeh is consumed in the cooked or raw form, these microorganisms may serve
as non-viable probiotics for the host and therefore increasing the immune system.
Moreover, non-soluble polysaccharides presented mainly by soybean and
Rhizopus sp., can serve as prebiotics. Consuming tempeh that might play a role as
probiotics and prebiotics might shift the bacterial composition of gut microbiota.
The following microbial shifting is expected toward a healthier gut microbiota
composition.
Objectives
The objective of this study was to evaluate the impact of tempeh
supplementation on gut microbiota composition and mucosal immunity in
Sprague-Dawley (SD) rats. In this study, intestinal IgA was quantified at both the
gene- and protein-expression levels in groups of rats that were fed with either raw
or cooked tempeh, as well as in control rats that were fed with non-fermented
soybeans.
3
Novelty
The impact of tempeh supplementation on gut microbiota composition and
mucosal immunity in Sprague-Dawley rats has not been studied. This research
will study how tempeh, which can be considered as a potential source of nonviable probiotics and prebiotics, may become a possible strategy to stimulate the
immune system and manage the complex gut microbial community towards a
health-promoting composition. Therefore, the result of this nutrigenomic studies
will further give tempeh an added nutritional value.
LITERATURE REVIEW
Tempeh
Tempeh is a traditional Indonesian fermented food in which soybeans are
hydrated and acidified, dehulled, cooked and fermented with Rhizopus spp. There
is no standard process for tempeh making, therefore many variations can be found
from one region and one producer from another. During the fermentation process
of tempeh, there are many valuable changes not only in the increase of nutritional
values of some nutrients in soybean, but also in the development of vitamins,
phytochemicals and other bioactive compounds (Astuti et al. 2000). Enzymes
produced during fermentation affect protein, fat and carbohydrates contents in
soybean. Soybean oligosaccharides such as stachyose and raffinose, which cause
flatulence, are broken down into digestible sugars. Moreover, the fungus produces
the enzyme phytase that mobilize phytic acid improving bioavailability of
minerals (Nout and Kiers 2005).
Most of microbiological studies on tempeh production have been focused
on the fungi as starter, however, the association of bacteria and yeast during the
fermentation process has been reported (Nout and Kiers 2005; Barus et al. 2008;
Seumahu et al. 2012). Klebsiella pneumoniae and Citrobacter freundii found in
fresh tempeh implicated their roles in the production of vitamin B12 during the
fermentation (Mulyowidarso et al. 1990; Keuth and Bisping 1994). Increased
protein content in tempeh has been reported due to the cooking and fermentation
steps during tempeh processing, which favor losses of soluble substances from
soybean (Vaidehi and Rathnamani 1990; Bavia et al. 2012). On the other hand,
the oil content observed in soybean is higher than in tempeh (Bavia et al. 2012).
Valeric, propionic, formic and acetic are the main acids in soybean. Their
concentrations at the end of soaking step were decreased progressively
(Mulyowidarso et al. 1991a). At the end of soaking, lactic and malic were the
main acids in soybean. These acids were produced by microbial metabolism of
soybean sugars leached into the water and contribute to the decrease in pH of the
soak-water and soybeans. The main microorganisms responsible for producing
lactic acid are Lactobacillus casei, Staphylococcus epidermidis, Streptococcus
4
faecium and for producing malic acid is L. casei (Mulyowidarso et al. 1991a). The
presence of bacteria producing organic acids during the soaking process showed
their important roles in the inhibition of various pathogenic and spoilage
microorganisms in tempeh (Nout and Kiers 2005).
Carbohydrates in Soybean
Soybean (Glycine max) contains ~35% carbohydrates which consist
mainly of non-starch polysaccharides and oligosaccharides (Dixit et al. 2011).
Polysaccharides are composed mainly of insoluble dietary fiber, such as cellulose
and pectins. Soybean oligosaccharide is a group of soluble low molecular weight
oligosaccharides in soybean seeds, which include sucrose (1%), stachyose (4%)
and raffinose (1.1%) (Choct et al. 2010). Stachyose is a tetraose with a structure
of α-D-galactopyranosyl-[1→6] – α – D – galactopyranosyl – [1→ 6] – α – D –
glucopyranosyl – [1 → 2] – β–D–fructofuranoside, while raffinose is a triose with
a structure of α–D–galactopyranosyl–[1→6]–α–D-glucopyranosyl–[1→2]–β–D–
fructofuranoside. Stachyose and raffinose are defined as non-digestible
galactooligosaccharides (GOS). GOS content of soybean has been shown to vary
with the degree of maturity. Immature green seeds contain fewer amounts of GOS
than fully matured yellow soybean (Espinosa-Martos and Rupérez 2006).
Humans do not posses α-galactosidase necessary for hydrolyzing the
linkage present in oligosaccharides, so that they cannot be digested when
consumed. When these intact oligosacharides reach the colon, they will be
preferentially fermented by colonic bacteria that posses the enzyme (Liu 1999).
Anaerobic bacterial fermentation of these substrates results in the production of
gases which cause uncomfortable flatulence. Many studies have been carried out
to reduce the oligosaccharides content in soybean products by processing
techniques such as soaking, cooking, fermentation and enzyme treatment
(Egounlety and Aworh 2003; Gote et al. 2004). However, many clinical
researches have suggested that oligosaccharides, with approximately 30 to 50%
caloric value of sucrose, are interesting because of their prebiotic activity and
associated health benefit (Roberfroid and Slavin 2000). Along with the progress
of oligosaccharides researches, it was found that soybean oligosaccharides are not
the direct causes of flatulence (Cui 2001).
Oligosaccharides in Soybean Tempeh
Stachyose and raffinose are non-digestible-galactooligosaccharides in
soybean which can still be found in tempeh although it has been reported that the
concentrations of oligosaccharides in soybeans are reduced during the
fermentation process. However, there are considerable differences in the extent of
the reductions reported.
Soaking in water is an essential preliminary step in the use of soybean for
the preparation of tempeh (Steinkraus et al. 1983). Mulyowidarso et al. (1991b)
reported that the concentration of stachyose and raffinose in soybean decreased by
5
65% and 50% respectively after soaking at 30°C for 24h. Meanwhile, when a
mixture of antibiotics was added in the soak-water in order to completely inhibit
the growth of microorganisms, the decrease of stachyose concentrations was
reduced. Thus, at the end of 24h soaking, the soybean contained approximately
1.6% stachyose, compared with approximately 1% stachyose for soaking in the
absence of antibiotics (Mulyowidarso et al. 1991b). The decreased concentration
of raffinose was approximately the same as that which occurred during soaking in
the absence of antibiotics.
The changes of these oligosaccharide concentrations within the beans
during soaking could be attributed to endogenous enzyme metabolism, diffusion
into the external environment of the soak-water and microorganisms activities that
grow in the soak-water (Mulyowidarso et al. 1991b). Total microbial populations
of 1010 CFU mL-1were developed during the first 18h of soaking at 30°C with the
main species are L. casei, S. epidermidis, S. faecium and Streptococcus
dysgalactiae (Mulyowidarso et al. 1989). α-galactosidases produced by these
microbial species in the soak water could diffuse into the soybean and accelerate
the hydrolysis of soybean oligosaccharides. Moreover, microbial fermentation of
glucose and reduction in pH of the soak-water and soybean to 5.0 could affect the
activity of soybean α-galactosidase. Since the pH optimum of this enzyme has
been reported to be 5.0 (Harpaz et al. 1977), microbial fermentation is likely to
stimulate the enzyme activity. Recent studies have also reported the role of some
bacteria during the fermentation process in tempeh production (Barus et al. 2008;
Seumahu et al. 2012). In conclusion, the decrease of the oligosaccharides
concentration within the soybean will depend on the microbial species that
develop and predominate in the soak-water (Mulyowidarso et al. 1991b).
Furthermore, Ruiz-Terán and Owens (1999) studied the fate of
oligosaccharides, stachyose and raffinose, during production of soybean tempeh.
Stachyose and raffinose were monitored during the preparation of bacteria-free
tempeh made with R. oligosporus NRRL 2710. The essential losses of
oligosaccharides were due to leaching during hydration, washing and cooking of
the soybean. Meanwhile, the concentrations of these oligosaccharides did not
change during the fermentation. The result of this study was in agreement with
observations that, in vitro, tempeh moulds did not use the oligosaccharides as sole
sources of carbon and energy (Graffham et al. 1995). Therefore, it is possible to
control the concentration of oligosaccharides by making choice of conditions for
hydrating and cooking the beans. This control might be facilitated by the fact that
the oligosaccharides are not utilized in tempeh by the moulds.
On the other hand, a recent study on the effect of fermentation with
Rhizopus oligosporus on the oligosaccharides of soybean, cowpea (Vigna
unguiculata) and groundbean (Macrotyloma geocarpa) was done by Egounlety
and Aworh (2003). Stachyose and raffinose decreased during 48h fermentation
with a reduction of 83.9% and 55.4%; 91.5% and 53.8%, and 85.5% and 54.0%
respectively for soybean, cowpea and groundbean. This study concluded that α-D
galactosidase and α-D glucosidase produced by Rhizopus species during
fermentation degraded soybean oligosaccharides which resulted in the production
of galactose. This suggested that the α-D galactosidase acts on the
oligosaccharides at the galactose moety (Egounlety 1994).
6
The conflicting reports in the literature on the production of
oligosaccharidases and the utilization of oligosaccharides during tempeh
fermentation are possibly a consequence of the presence of bacteria, or, in some
cases, could be due to the use of different mould strains (Ruiz-Terán and Owens
1999). However, it is still possible to control the concentration of oligosaccharides
by making choice of conditions for hydrating and cooking the beans and selecting
the microorganisms used during fermentation. Therefore, the possible prebiotic
effect of soybean can still be found in tempeh.
Gut Microbiota and Immune System
Recent studies suggested that the trillions of bacteria that normally reside
within the human gastrointestinal tract, also referred as the gut microbiota, affect
nutrient acquisition and energy regulation; it suggests further that obese and lean
people have different gut microbiota. The composition of this microbial
community is host specific, evolving throughout an individual's lifetime and
susceptible to both exogenous and endogenous modifications (Sekirov et al.
2010). Firmicutes and Bacteroidetes are closely related to body fat in humans and
mice, which are the two dominant bacterial divisions of gut microbiota in
mammals. Comparisons of the distal gut microbiota of obese and lean mice, and
those of obese and lean humans revealed that a statistically significant reduction
in the relative abundance of Bacteriodetes and a significantly greater proportion of
Firmicutes were in obese animals than in lean controls (Ley et al. 2005).
In healthy adults, 80% of the identified fecal microbiota can be classified
into three dominant phyla: Bacteroidetes, Firmicutes, and Actinobacteria (Lay et
al. 2005). The Firmicutes and Bacteroidetes ratio is regarded to be of significant
relevance in human gut microbiota composition. However, the fecal microbiota is
a highly complex and diverse bacterial ecosystem. Within this ecosystems exists a
hierarchy of dominant (>109 CFU g-1) anaerobic bacteria, represented by the
genera Bacteroides, Eubacterium, Bifidobacterium, Peptostreptococcus,
Ruminococcus, Clostridium and Propionibacterium, and sub-dominant (