International Journal of

Molecular Sciences

Review
Review on Bifidobacterium bifidum BGN4:
Functionality and Nutraceutical Applications as
a Probiotic Microorganism
Seockmo Ku 1,2 , Myeong Soo Park 3 , Geun Eog Ji 1,4, * and Hyun Ju You 1,5, *
1 Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National University,
Seoul 151-742, Korea; [email protected]
2 Laboratory of Renewable Resources Engineering, Department of Agricultural and Biological Engineering,
Purdue University, West Lafayette, IN 47907-2022, USA
3 Department of Hotel Culinary Arts, Yeonsung University, Anyang 430-749, Korea; [email protected]
4 Research Center, BIFIDO Co., Ltd., Hongcheon 250-804, Korea
5 Institute of Health and Environment, Graduate School of Public Health, Seoul National University,
Seoul 151-742, Korea
* Correspondences: [email protected] (G.E.J.); [email protected] (H.J.Y.);
Tel.: +82-2-880-6282 (G.E.J.); +82-2-880-2790 (H.J.Y.); Fax: +82-2-884-0305 (G.E.J. & H.J.Y.)

Academic Editor: Alejandro Cifuentes

Received: 26 July 2016; Accepted: 8 September 2016; Published: 14 September 2016

Abstract: Bifidobacterium bifidum BGN4 is a probiotic strain that has been used as a major ingredient
to produce nutraceutical products and as a dairy starter since 2000. The various bio-functional effects
and potential for industrial application of B. bifidum BGN4 has been characterized and proven by
in vitro (i.e., phytochemical bio-catalysis, cell adhesion and anti-carcinogenic effects on cell lines,
and immunomodulatory effects on immune cells), in vivo (i.e., suppressed allergic responses in
mouse model and anti-inflammatory bowel disease), and clinical studies (eczema in infants and
adults with irritable bowel syndrome). Recently, the investigation of the genome sequencing was
finished and this data potentially clarifies the biochemical characteristics of B. bifidum BGN4 that
possibly illustrate its nutraceutical functionality. However, further systematic research should be
continued to gain insight for academic and industrial applications so that the use of B. bifidum BGN4
could be expanded to result in greater benefit. This review deals with multiple studies on B. bifidum
BGN4 to offer a greater understanding as a probiotic microorganism available in functional food
ingredients. In particular, this work considers the potential for commercial application, physiological
characterization and exploitation of B. bifidum BGN4 as a whole.

Keywords: Bifidobacterium; functional foods; probiotics; nutraceuticals

1. Introduction
The term functional foods and/or nutraceuticals can be defined as certain foods reserving
bioactive compounds that likely have beneficial effects in the body beyond basal nutritional ingredients
(i.e., carbohydrate, protein and fat) [1,2]. According to USA Food and Drug Administration (FDA) [3],
“terms such as functional foods or nutraceuticals are widely used in the marketplace and these are
regulated by FDA under the authority of the Federal Food, Drug, and Cosmetic Act, even though they
are not specifically defined by law”. Probiotics and probiotic foods are included within functional
foods and have a growing market and large economic value [4]. The beneficial effects of probiotics on
hosts beyond normal nutrition have attracted special interest from food industry and academia [5].
Functional properties of probiotic cells may offer various solutions to meet commercial demands
for a variety of functional or conventional food products. Health benefits of probiotic cells coupled

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Int. J. Mol. Sci. 2016, 17, 1544 2 of 23

with consumers’ positive awareness regarding self-care, wellbeing and complementary medicine
have reflected into multiple nutraceuticals as a promising ingredient in food industry [6,7]. Recently,
multiple researchers have displayed interests to applications of food and nutraceutical processing to
develop a novel concept of probiotic food or supplements [8,9].
Among the various probiotic bacteria, Bifidobacterium, is one of the most widely used and studied
probiotic bacteria. According to Soto et al. [10], Lactobacillus and Bifidobacterium spp. accounted for
67.5% and 25.6% of microbial population, respectively, in breast milk (obtained from German and
Austrian women, n = 160). Because the initial bacterial colonization is happening at an early stage
of human life cycle, the primary colonization by breastfeeding or formula feeding has an important
role to the individual health by affecting later host homeostasis during the development of the infant
digestive and immune system [11]. Although, Lactobacillus is the major microbial flora in human milk,
Bifidobacterium is the predominant cell species in fecal samples from breastfeed infants [12].
Naturally occurring microbiota in the intestinal tract of breast-fed infants, Bifidobacterium
accounts for more than 80% of microorganisms within the intestine [13–15]. Among the various
Bifidobacterium spp., B. bifidum, B. breve, B. infantis and B. longum are commonly detected bacteria from
breastfed infants [12], whereas formula-fed infants have a complex ecosystem comprising mostly
of coliform bacteria and Bacteroides, with significantly lower prevalence of Bifidobacterium spp. [16].
For marketing purposes, some food researchers in industry have tried to develop infant formula
that stimulates Bifidobacterium spp. to become the dominant flora by constituting bifidogenic factors
(e.g., non-digestible carbohydrates, and galactooligosaccharides) [17].
Recently reported studies showed that Bifidobacterium bifidum, (B. bifidum) is the second most
prominent species that identified in breast-fed infants (the first was B. breve and the third was
B. longum) [18]. As an early colonizer of the infant gut, B. bifidum is widely present among fecal
microbiota, however, the concentration of overall Bifidobacterium spp. is decreased during the
progression of age while B. adolescentis and B. catenulatum reach greater levels in adult guts [19].
Individual results from multiple studies had a little variation, however, it was clear that B. bifidum
is considered a dominant species of gut population in healthy breast-fed infants. This distinctive
ecological feature of B. bifidum spp. attracted microbiologists’ interests. Multiple experiments were
carried out with clinical and pre-clinical studies, and proved significant health benefits (e.g., reducing
bowel syndrome, diarrhea and pathogen infections) [20–23].
B. bifidum BGN4 (BGN4) obtained from a breast-fed infant’s fecal sample came to
the forefront in 1996 by its distinctive enzymatic representation: β-glucosidase (E.C 3.2.1.21) negative [24].
This microorganism was first used to evaluate the expression of mutagenic activity by
β-glucosidase-producing gut microbiota that produce deglycosyl hydrolases and catalyze carcinogenic
glycosides (i.e., amygdalin, anthrone-6-O-rhamnoside, 8-hydroxyquinoline-β-D-glucoside, neocycasin
A, quercetin-3-O-rutinoside, guercitrin, robinin, and cycasine).
BGN4 has been applied to multiple nutraceutical products and conventional foods in the global
food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore,
Thailand, Turkey, USA, and Vietnam) as a probiotic microorganism because of possible benefits to
consumers [25]. Multiple researchers have proven outstanding bio-functional characteristics of BGN4
by in vitro, in vivo, and clinical experiments. Potential benefits of BGN4 include: (i) notable colon cell
binding properties [26,27]; (ii) improved immune function [28–34]; (iii) anti-tumor effects [27,35,36];
(iv) aid in bioconversion of phytochemicals [37–41]; and (v) production of biogenic metabolites [42,43].
These represent the five main findings that have been discussed with regard to functional benefits
from BGN4 with in-depth research. Optimizing cell culture conditions could increase not only BGN4
cell biomass recovery but also its bioactive metabolites. Recently, BGN4 chromosome sequencing was
completed and will therefore be analyzed to further understand the correlation between genetics and
physicochemical properties [44]. This review will highlight the importance of each of the five areas.
In addition, this review will address prominent prototypes of BGN4 products, distinguished genome
Int. J. Mol. Sci. 2016, 17, 1544 3 of 23

analysis and changed physicochemical attributes of BGN4 and the effect of altered culture conditions
that were studied for the purpose of commercial manipulation.

2. Cell Adhesive Property

Foodborne illness (e.g., salmonellosis, listeriosis and shigellosis) occurs when food consumers eat
a contaminated food with Salmonella, Listeria, and Shigella spp. [45]. Intestinal mucus and epithelial
cells are predominantly susceptible to the attachment of pathogenic microorganisms resulting in active
proliferation and colonization. This microbial adhesion is critical for the beginning of pathogen and
epithelial cell interaction [46]. Consequently, avoiding bacterial adhesion onto the gastrointestinal
mucosa is regarded as an efficient approach for decreasing the risk of foodborne disease [47,48].
Recently, Serafini et al. [49] observed inhibitory properties of B. bifidum PRL2010 against pathogenic
bacteria (i.e., Escherichia coli and Cronobacter sakazakii) regarding enteric adaptation properties using
epithelial intestinal cell monolayers (i.e., Caco-2 and HT-29).
Probiotic microorganisms are defined as “live microorganisms which when administered in
adequate amounts confer a health benefit on the host” by WHO and FAO. Among the many
probiotic strains, Lactobacillus and Bifidobacterium spp. are known as autochthonous microbiota
in the human intestinal tract. These microorganisms have been used in various functional foods
for centuries. The major functional effects that are provided by probiotics are: (i) production of
anti-microbial peptides (i.e., bacteriocins) [50–52]; (ii) assimilation of dietary fibers [53]; (iii) regulation
of fat storage [54,55]; (iv) modulation of mucosal immunity [56]; and (v) regulation of gut flora via
competitive exclusion of pathogenic bacteria resulting in decreased pathogen colonization [57–59].
Among the five key functional effects of probiotics, attachment of probiotic bacteria onto the mucosal
surface of the gastrointestinal tract is regarded as essential for the competitive exclusion of pathogens
and must occur before effective regulation of immune activities, resulting in protective function against
intestinal pathogens [60,61]. The cell adhesion stage of probiotics onto colon cells is essential for the
successful microbial colonization inside of the host’s intestinal tract. This cell adhesion ability has been
regarded as one of the critical screening standards for active probiotic strains [62], since adhesion is
necessary to actively proliferate and provide a resistance to excretion from the intestinal tract as waste
by peristalsis.
Mechanisms of bacterial adhesion onto epithelial cell can be divided into: (i) non-specific
adhesion when regarding physicochemical factors of outer cell surfaces; or (ii) specific adhesion
when considering the expression of specific molecules onto the microbial membranes that directly
attach to the binding sites of epithelial cell mucosal surfaces [60–62]. This adhesion ability is a
function of hydrophobic properties, level of ions, pH, and physical morphology [63]. These factors
considerably affect microbial adhesion onto intestinal tissues of the host, demonstrating the complexity
of preliminary microbial adhesion onto the mucosal surface.
According to Krasowska and Sigler [64], microbial hydrophobicity plays a key role in the initial
interaction with the mucosal surface and epithelial cells of the intestinal lining due to the chemical
composition of the bacterial surfaces. The physicochemical characteristics of the microbial outer
membrane are generally estimated by analysis of cell surface hydrophobicity. It has been proven
that microorganisms that express higher hydrophobicity more effectively attach onto the colon cells
compared to hydrophilic microbial strains [61,62]. High cost and complexity of in vivo models
encouraged attention into the use of an in vitro system for the initial selection and screening of
potentially adherent probiotic microorganisms. Microorganisms that express high adhesive activity
to inanimate surfaces (e.g., hydrocarbon surface) or non-polar solvents are considered hydrophobic,
and cells that express lower adhesive activity are considered hydrophilic [62,64]. Pelletier et al. [65]
reported that the existence of proteinaceous components on the microbial outer layer cause higher
hydrophobicity, while hydrophilic properties are related to the existence of polysaccharides in the cell
wall structure.
Int. J. Mol. Sci. 2016, 17, 1544 4 of 23

The hydrophobicity of strain BGN4 showed greater affinity towards xylene in similar studies
(Table 1). Abdulla et al. [66] reported six different Lactobacillus strains with the hydrophobicity ranging
from 29.5% to 77.4%. The three strains of Lactobacillus (i.e., L. acidophilus, L. gasseri, and L. jensenii) used
by Boris et al. [67] showed about 80% surface hydrophobicity. B. lactis Bb12 and L. acidophilus LA5
showed surface hydrophobicity with values between 61% and 75% [68]. B. pseudolongum CIDCA 531
expressed 85% surface hydrophobicity [69]. Recently, Pan et al. [70] reported significant correlation
between microbial adhesion property and cell hydrophobicity using 5 different Bifidobacterium strains
(i.e., B. longums P-3, B. animalis H-9, B. animalis P-4, B. asteroids H-10 and B. pseudocatenulatum I-6) and
Caco-2 with in vitro model. These results suggest that BGN4 may possess high cell adhesion properties
and potent colonization abilities.

Table 1. Microbial hydrophobicity of the cellular surface (CHS) among reference strains.

No. Cell CHS (%) No. Cell CHS (%)

1 B. bifidum BGN4 93 20 B. longum ATCC 15707 <5
2 Bifidobacterium KJ 90 21 B. longums P-3 18.5
3 Bifidobacterium HJ-30 90 22 B. animalis H-9 37.13
4 B. adolescentis ATCC 15703 90 23 B. animalis P-4 17.4
5 B. animalis ATCC 2552 86 24 B. asteroids H-10 49.5
6 B. animalis M6 85 25 B. pseudocatenulatum I-6 47.3
7 B. animalis Rd60 69.6 26 B. pseudolongum CIDCA 85
8 B. animalis SI 66.3 27 B. lactis Bb12 75
9 B. animalis CN2 21 28 L. acidophilus LA5 75.1
10 B. bifidum ATCC 2952 12 29 L. paracasei (lac 1) 80
11 B. bifidum RD54 7 30 L. acidophilus (lac 2) 65
12 B. bifidum MS1 6 31 L. acidophilus (lac 3) 60
13 B. bifidum SH5 6 32 L. acidophilus (lac 4) 30
14 B. bifidum E15 5 33 L. fermentum (lac 5) 45
15 B. bifidum E2-18 <5 34 L. fermentum (lac 6) 65
16 B. bifidum JS9 <5 35 L. acidophilus 80
17 B. bifidum SH2 <5 36 L. gasseri 80
18 B. bifidum SJ32 <5 37 L. jensenii 80
19 B. infantis ATCC 15697 <5 - - -
The data number 1 to 20, 21 to 25, 26, 27 to 28, 29 to 34 and 35 to 37 were adapted from Kim et al. [26],
Pan et al. [70], Pérez et al. [69], Shakirova et al. [68], Abdulla et al. [66] and Boris et al. [67], respectively.
The level of CHS was evaluated by the cell adhesive method into xylene.

However, we should point out that the hydrophobic surface characteristics of probiotic bacteria
do not consistently bind to epithelial colon cells [71,72]. The distinguished physicochemical surface of
probiotics do not guarantee binding to epithelial colon cells. The adhesion property of microorganisms
is significantly inconsistent and heterogeneous among cell strains [73]. Specifically, some probiotic
strains show effective cell adhesion ability although they express significant hydrophilic properties on
their cell surface [74]. This shows that other aspects that affect cell adhesion should also be considered.
To overcome the limitation of cell hydrophobicity that often accompanies adhesion ability, microbial
adhesion experiments using in vitro models with intestinal epithelial cells have been extensively
investigated [26,27,44,62,73]. The number of microorganisms attached to culture tissues directly shows
the cell adhesion property. Among the various intestinal epithelial cell lines, the enterocyte-like
Caco-2 cells obtained from a human colon have been routinely used to examine microbial adhesion
mechanisms because of their distinctive physicochemical characteristics (i.e., active proliferation
and differentiation under normal enrichment conditions, similar biological characteristics to normal
enterocytes) [75].
BGN4 was compared to twenty different strains of Bifidobacterium spp. (i.e., B. bifidum, B. animalis,
B. adolescentis, B. infantis and B. longum) separated from human fecal samples to evaluate cell adhesion
properties [26]. According to Crociani et al. [76], cell adhesive properties of Bifidobacterium spp. are
highly variable between strains of the identical genus. Kim et al. [26] clearly illustrated that binding
between BGN4 whole cells and well-defined brush border microvilli on Caco-2 using scanning electron
microscope (SEM). Among the various strains of Bifidobacterium, BGN4 showed the largest number
of cells bound to the Caco-2 cells with highest cell surface hydrophobicity (93%) (Figure 1). Recently,
 Int. J. Mol. Sci. 2016, 17, 1544 5 of 22

illustrated
Int. J. Mol. Sci. 2016, 17, that
1544 binding between BGN4 whole cells and well-defined brush border microvilli on 5 of 23
Caco-2 using scanning electron microscope (SEM). Among the various strains of Bifidobacterium,
BGN4 showed the largest number of cells bound to the Caco-2 cells with highest cell surface
hydrophobicity (93%) (Figure 1). Recently, 2.2 Mb of the BGN4 genome sequence was completely
2.2 Mb of the BGN4 genome sequence was completely decrypted [44]. The comparative genomic
decrypted [44]. The comparative genomic analysis clearly elucidated the existence of a homolog
analysis clearly elucidated
(BBB_0596) thebifidum
of the B. existence
MIMBb75of a homolog (BBB_0596)
outer protein (BopA) that the B.
of aids in bifidum MIMBb75
the sticking of outer
protein (BopA) that aidsonto
microorganisms in the sticking
a Caco-2 of microorganisms
cell layer [44]. onto a Caco-2 cell layer [44].

Figure 1. Adhesion of B. bifidum BGN4 onto the epithelial Caco-2 cell observed by: (a) optical
Figure 1. Adhesion of B. bifidum BGN4 onto the epithelial Caco-2 cell observed by: (a) optical
(magnification of 1000×); and (b) scanning electron microscopy (magnification of 20,000×, interaction
(magnification of 1000×
with microvilli of);Caco-2
and (b)
andscanning electron
B. bifidum BGN4). microscopy
Microbial adherence(magnification of 20,000
in (a) was observed ×, interaction
after simple
with microvilli ofwith
staining Caco-2
crystaland B. bifidum
violet. Panel (b) BGN4). Microbial
was adapted from Kimadherence
et al. [26]. in (a) was observed after simple
staining with crystal violet. Panel (b) was adapted from Kim et al. [26].
Despite this, very little research was done on specific adhesion related to the molecular
mechanisms which possibly affects the strong adhesion properties, overall these findings evidently
Despite this, very
demonstrated thelittle research
notable was done
cell adhesive ability on specific
of BGN4 ontoadhesion
epithelial related
cell with to
its the
highmolecular
mechanisms hydrophobicity under in
which possibly vitro conditions,
affects the strong andadhesion
thus could properties,
represent better ability these
overall to colonize in the evidently
findings
gastrointestinal tract with protracted transit. More detailed understanding of the: (i) adhesive
demonstrated the notable cell adhesive ability of BGN4 onto epithelial cell with its high hydrophobicity
mechanisms of BGN4 under the molecular level; (ii) fecal samples; and (iii) intestinal lining by
under in vitro conditions,
biopsies could allowand
us to thus
know could represent
the significance better ability
of adhesive ability ofto colonize
BGN4 and its in the gastrointestinal
applications to
tract with functional
protracted transit. More detailed understanding of the: (i) adhesive mechanisms of BGN4
foods.
under the molecular level; (ii) fecal samples; and (iii) intestinal lining by biopsies could allow us to
3. Immune-Modulatory Effects of B. bifidum BGN4
know the significance of adhesive ability of BGN4 and its applications to functional foods.
Multiple probiotic strains have shown significant bio-functional properties concerning boosted
host immune functions.
3. Immune-Modulatory Effects OneofofB.the important
bifidum BGN4roles of probiotic bacteria is immune-modulatory
activities for the prevention and regulation of multiple enteric diseases in the host [77]. According to
Multiple probiotic
Galdeano strains
[78], orally have shown
consumed significant
fluorescent-labeled bio-functional
probiotic properties
cells were identified concerning
in the immune boosted
system (i.e., Payer´s patches and lamina propria mucosa) in the small
host immune functions. One of the important roles of probiotic bacteria is immune-modulatory intestine and lymphoid tissues
(i.e., lymph nodules and colonic crypts). This report provides convincing evidence of a direct
activities for the prevention and regulation of multiple enteric diseases in the host [77]. According to
interplay between probiotic microorganisms and immune cells in the host’s intestinal lining.
Galdeano [78],Among orallythe consumed fluorescent-labeled
various immune cells, phagocytic probiotic cells
cells (i.e., were identified
neutrophils, monocytesin and the immune
macrophages)
system (i.e., Payer´s patchesin the intestinal mucosa
and lamina play anmucosa)
propria essential role in both
in the smallstimulation
intestine of and
inflammatory
lymphoid tissues
(i.e., lymphresponses
nodulesagainst potential crypts).
and colonic enteric pathogens and tolerance
This report provides of normal colonicevidence
convincing luminal nutrients and interplay
of a direct
microbes as an innate immune system [79]. When macrophages are under an inflammatory stimuli,
between probiotic microorganisms and immune cells in the host’s intestinal lining.
they generate cytokines, including Interlukin (IL)-1, IL-6, IL-8, IL-12, and tumor necrosis factor
Among the various
(TNF), which recruits immune othercells, phagocytic
inflammatory cells
cells. (i.e., neutrophils,
Phagocytic monocytes
cells are attracted towardand macrophages)
specific
infection sites to engulf the opsonized targets using phagocytosis. They recognize
in the intestinal mucosa play an essential role in both stimulation of inflammatory responses against pathogens using
chemotaxis stimuli and/or straight physical connections [80–82]. Multiple reports have shown a
potential enteric pathogens and tolerance of normal colonic luminal nutrients and microbes as an
significantly promoted phagocytic capacity of phagocytes by probiotic supplementation as an
innate immune system
immunomodulator [79].
[83].When macrophages
Therefore, the evaluationare under
of the ancytokines
level of inflammatory stimuli,
and macrophage they generate
activity
cytokines, including Interlukin (IL)-1, IL-6, IL-8, IL-12, and tumor necrosis factor (TNF), which recruits
other inflammatory cells. Phagocytic cells are attracted toward specific infection sites to engulf the
opsonized targets using phagocytosis. They recognize pathogens using chemotaxis stimuli and/or
straight physical connections [80–82]. Multiple reports have shown a significantly promoted phagocytic
capacity of phagocytes by probiotic supplementation as an immunomodulator [83]. Therefore, the
evaluation of the level of cytokines and macrophage activity using an in vitro assay is considered an
indirect way of analyzing bio-functional effects of probiotic cells.
Lee et al. [28] reported the significant immunoregulatory capacities of whole-cell and cell-free
extracts derived from BGN4. In this work, when macrophages were exposed to BGN4, active cell
division, greater cytokine production and active phagocytic property were observed. Since then,
various studies have focused on the interactions between outer cell wall and immune cells, however,
Int. J. Mol. Sci. 2016, 17, 1544 6 of 23

little work that employs intercellular ingredients has been reported before. Therefore, they also
extracted four different BGN4 cell fractions (i.e., whole-cell, cell free extracts, purified cell wall and
supernatant) and treated cell lines to evaluate the level of cytokine produced by macrophages. As a
result, each fraction showed different patterns of immune reactions. The whole cell fraction represented
the strongest TNF-α expression. The cell-free extracts of BGN4 induced the highest IL-6 production.
This work was confirmed and further explained by Kim and Ji [29]. They made an attempt to
determine the significance of type of BGN4 cell fractions with special focus on location within the
host immune system. All BGN4 cell fractions (i.e., cell free extracts, whole cell fractions and cell wall
fractions) significantly stimulated the production of IL-10 and IL-6. Cell free extracts of the BGN4
were able to induce greater morphological modification of macrophages with increased phagocytosis
properties compared to macrophages treated with other BGN4 fractions (i.e., whole cell and cell wall
fractions). The use of an in vitro assay clearly showed the immunomodulatory properties of BGN4
that activate differentiation of macrophages.
Another experiment was performed to examine the immune responses of intragastrically
administrated BGN4 in a murine model of peanut allergy to provide further support function of
BGN4 based on in vivo experiments [30]. They concluded that BGN4 treatment in an animal model
showed anti-allergic and immunomodulatory effects by decreased levels of peanut-specific IgE and
IL-4 and increased levels of IL-12 and the ratio of Interferon (IFN)-γ/IL-4. Kim et al. [31] also reported
clinical properties of BGN4 to inflammatory bowel disease using a mouse model. The BGN4-fed
group showed minimal signs of thickened wall and inflammatory cell infiltration, in a clinical sense,
such as: (i) thickened wall; (ii) crypt elongation; (iii) reduction of goblet cells; and (iv) maintaining
the level of cluster of differentiation (CD) 69, IFN-γ, TNF-α and MCP-1 in the mouse intestine than
its counter group. The in vivo approaches employed in this work clearly suggest further functional
characterization of BGN4 on the control of the aberrant intestinal immunity.
However, an additional question is: does BGN4 show potent immune stimulating effects within
clinical experiments? This answer is critical to prove practical benefits of BGN4. Despite various
in vitro, in vivo data, the precise mechanism of action of BGN4 was not fully demonstrated and could
be multifactorial in clinical research.
According to Hong et al. [32], a probiotics mixture containing 2 × 1010 of lyophilized cells
(i.e., BGN4, B. lactis AD011, L. acidophilus AD031and L. casei IBS041) was effective to relieve irritable
bowel syndrome. They randomly divided two groups (n = 36 and 34; age: 19–75 years; sex: male and
female; symptoms: presence of previous gastrointestinal disease) as probiotics and placebo groups,
respectively. Their work clearly demonstrated that probiotics treatment was statistically significant in
the reduction of abdominal pain and defecation discomfort after eight weeks of probiotics treatment
compared to placebo groups (n = 70, −31.9 vs. −17.7, p = 0.045), and concluded “composite probiotics
containing BGN4, L. acidophilus AD031, and other species are safe and effective, especially in patients
who excrete normal or loose stools”.
Kim et al. [33] used different strategies to characterize functional effect of BGN4 for their role in
eczema. Through the randomized, double-blind and placebo-controlled experimental design (n = 112,
screened and randomized pregnant women having family history of allergic diseases), they evaluated
the preventive function of BGN4 against progress of eczema. They concluded that the prevalence
of eczema can be statistically significantly decreased by BGN4 treatment compared to its counter
placebo group (completed sample number: n = 68, p = 0.048, BGN4 group: 18.2% vs. placebo: 40.0%).
Interactions among gut microbiota, intestinal epithelial cells and mucosal dendritic cells in the lamina
propria, and their impact in innate immunity has been the focus of multiple researchers in recent
decades [84]. Specifically, Kim et al. [34] discussed function of BGN4 treatment into the dendritic cells.
With comparison of cell culture conditions (i.e., single culture of dendritic cells or co-culture of dendritic
cells and mouse epithelial cell monolayers), multiple conditions for exerting immune-modulatory
reactions were evaluated. The authors concluded that BGN4 significantly upregulated the expression
of I-Ad and cluster differentiation (i.e., CD86 and CD40) (p < 0.05) with increased secretion levels
Int. J. Mol. Sci. 2016, 17, 1544 7 of 23

of pro-inflammatory cytokines (i.e., IL-6 and TNF-α). These results indicate that BGN4 potentially
stimulates immune modulation via interaction of dendritic cells in the gut homeostasis.
As noted above, there are multiple experiments that provide evidence that BGN4 potentially
affects the host immune systems and exerts protective actions from allergens through in vitro and
in vivo studies, and show promising advances in the application of nutraceutical fields. Accumulating
results indicate that some symptoms that are triggered by artificially treated allergens or antigens
can be relieved by BGN4. Although physiologic outcomes have suggested possible benefits of
BGN4, clinical efficacy of BGN4 has not been clearly established using single type of cell ingredient.
Therefore, available evidence is not enough to demonstrate whether BGN4 may be more effective
for bio-functionality in the human body than other microorganisms. Interpretation of the functional
evidence of BGN4 is hampered by the presence of numerous other microorganisms. Further physiologic
investigations are necessary to design formulations and to understand the basic mechanisms and
bioavailability for studies of physiologic actions using single type of BGN4.

4. Anticancer Effects of B. bifidum BGN4

Colorectal cancer is a global health issue; in particular, South Korea showed the highest number
of colon cancer cases in the world. As the third leading type of cancer in the world, approximately
1.4 million cases were identified in 2012 [85]. According to the American Cancer Society [86], colon
cancer is the second leading cause of cancer mortalities with an expected 49,190 deaths in 2016.
The international incidence and mortality rates of colorectal cancer are rapidly increasing in multiple
countries as eating habits have been altered to a more occidental manner (i.e., low-dietary fibers,
high-fat and high-protein) [87].
Various studies have reported that fermented food products can significantly prevent tumor
growth by decreasing the risk of long-standing inflammatory responses in colon cancer. Probiotic
microorganisms normally contained in fermented food products are known to offer functional effects
on mucosal damages, specifically preventing the effects of cancer on the digestive tract [88]. Probiotics
have multiple therapeutic advantages, playing significant roles in decreasing the mutagenicity of
the epithelial layer, as reported in various experimental models of colorectal cancer [56]. In-depth
investigations have shown that the relationship between colorectal cancer and probiotics seems to be
primarily dependent on bioactive metabolites of probiotic bacteria, which lead to the generation of
therapeutic anti-carcinogenic compounds [89].
Certain probiotic microorganisms and their extractions demonstrate growth inhibitory activities
on adenocarcinoma cell lines. In particular, fractions from Bifidobacterium and Lactobacillus spp.
containing high levels of microbial carbohydrates (i.e., extracellular glycoproteins, peptidoglycan,
and polysaccharide) displayed profound tumor-suppressing activities [90–92]. These studies have
demonstrated on evaluating the properties of probiotic fractions and extractions with regard to the
decrease of viability or size of cell lines. However, proving the selective inhibition on cancer cells
by probiotics treatment is critical with regard to the screening and selection of anti-carcinogenic
substances. Moreover, anti-carcinogenic properties of probiotic microorganisms on colorectal cancer
significantly differ from strain to strain, making it necessary to screen novel probiotic strains for tumor
inhibitory effects [93]. Therefore, researchers should attempt to study the selectivity, sensitivity and
specificity on noncancerous as well as cancerous cell lines using multiple probiotics.
Microbial polysaccharides are produced by probiotic bacteria with various health-promoting
effectiveness. Their chemical structures, complexity and molecular weights differ among the probiotic
species, resulting in expression of different physicochemical characteristics in both in vitro and
in vivo systems. The antagonistic properties of probiotic bacteria against gastrointestinal illneses
have been the subject of many clinical investigations, demonstrating varied functional properties.
According to Nagaoka et al. [94], probiotic polysaccharides extracted from B. breve YIT4014 and
4043, and B. bifidum YIT4007 have shown anti-ulcer activities both directly (i.e., epidermal and
fibroblast growth factor) and indirectly (e.g., immune system stimulating: increased production
Int. J. Mol. Sci. 2016, 17, 1544 8 of 23

of 6-keto-prostaglandin F1 α by macrophage). Specifically, polysaccharides containing rhamnose as a

major content (more than 60%) showed greater effectiveness in healing gastric ulcers. The soluble
polysaccharides produced from L. acidophilus 606 also expressed the inhibitory effects on progression
of colorectal cancer cell lines such as HeLa, PANC-1 and HT-29 cells and partially induced apoptosis
into the HT-29 [90]. However, the polysaccharides from L. acidophilus 606 exhibited minimum toxicity
into healthy human embryo fibroblasts.
These findings provided motivation to our group for observation and hypothesis on the correlation
between BGN4 treatments and human colon cancer. Ku et al. [27] and You et al. [36] have demonstrated
the tumor-suppressing activity of whole cell and its fractions of BGN4 on diverse adenocarcinoma cell
lines. They also attempted to clarify whether such properties were human colon cancer cell-specific.
Among the 30 kinds of different strains of Bifidobacterium tested in these studies, BGN4 showed the
greatest anti-proliferative effects on human colon cancer cell lines. In particular, the polysaccharide
fractions comprising chiroinositol, rhamnose, glucose, galactose, and ribose that were extracted from
BGN4 induced significant growth inhibition of cancer cell lines (i.e., HT-29 and HCT-116), but did not
show any growth inhibition of FHC (normal human colon cell) or Caco-2 cells, which are generally
used as control group because of similar physicochemical properties with normal cells [95].
Previously, Shim et al. [35] discussed health benefits of fermented soy milk by BGN4, and
confirmed that dietary BGN4 decreases the size of azoxymethane-induced aberrant crypt foci in rats.
The combination of soymilk and BGN4 showed significant synergistic effects on reducing the number
of aberrant crypt foci. The size and number aberrant crypt focus are commonly used biomarkers for
colorectal cancer in rodents [96] and often regarded as the earliest histopathologic lesion linked to
colon cancers [97].
The simple assessments of a decreased cell proliferation and aberrant crypt foci levels using
in vitro and in vivo experiment are not enough to evaluate anti-cancer and/or anti-tumor properties of
BGN4 due to the complexity of cancer development, which is linked to numerous cellular mechanisms.
However, the anti-proliferation properties of BGN4 onto multiple cancer cell lines could be used as
an example of the interaction between Bifidobacterium spp. and host. The interactions between BGN4
and colon cancer stimulated novel manners of cancer suppression and suggested the treatments of
cell extractions with probiotic substances for the purpose of gaining anticancer properties. Further
mechanistic studies and human epidemiological studies are necessary to elucidate the role of BGN4
and its extractable polysaccharides as a therapeutic option for anticancer or antitumor effects using
animal models to take advantage of clinical properties derived from BGN4.

5. Industrial Application: Biocatalysis

Applications of health benefits from probiotics depend on the production of functional
cell metabolites [5,7,52,56]. The commercial significance of health beneficial metabolites (i.e.,
polyssacchrides, bacteriocine, γ-Aminobutyric acid (GABA), and S-Adenosyl-L-Methionine (SAM))
from probiotic bacteria has stimulated the use of these bacteria as “Biological Factories” of value
added products [98]. Bio-functional metabolites are produced by a variety of probiotic cells, especially
Lactobacillus and Bifidobacterium spp., and have been intensively studied due to their broad spectrum
of bio-functional properties and beneficial roles in human body. Due to the significance of probiotic
cells in the expression properties of functional molecules, probiotic cells have been used in industry
manufacturing for the production of value-added molecules [7,9,21,33,75].
Normally, the claimed functional benefits are likely achieved with high level of probiotic cells,
however, multiple studies showed poor nutraceutical availability of some probiotic microorganisms
in functional food products and found that they often exist at lower concentration in cells than those
claimed on product packages [99]. Due to severe food processing and storage conditions (e.g., heat and
acid treatment, artificial and natural preservatives, freeze and osmotic shock, and oxygen stress) often
applied during the manufacturing step, maintaining cell activity and viability are practical challenge.
Moreover, during the long-term period of circulation, viability of probiotic cells in product does not
Int. J. Mol. Sci. 2016, 17, 1544 9 of 23

meet the criteria at the end of shelf life. Therefore, it is necessary to maintain cell viability and ensure
probiotic effect for consumers’ needs [100]. A simple addition of probiotic cells into foods cannot
guarantee health benefits to consumers. Therefore food industries have also encountered a number
of difficulties when claiming the functional effects on the package of food products. For this reason,
industry researchers and marketers have pursued to explore more applications of probiotics that can
potentially be utilized in industry geared at several different markets [99]. Recently, various studies
have proposed to use probiotic immobilization techniques to maintain microbial functionality and
viability [101].
Extensive attention has been paid to the potential of using whole cell and/or cell fractions to
facilitate the production of functional molecules [7,36,102]. Food and biotechnology industries have
used advances in probiotics and their enzymes to produce value-added plant metabolites and/or
their chemically transformed substances. Recently, bio-functional potentials of traditional herbal
medicines and normal plants have emerged, resulting in notable progress in commercial developments
of functional foods and/or nutraceuticals [41,102–106]. Specifically, to improve the quality of herbal
resources, multiple probiotic cells and their enzymes have been applied for decades with commercial
and domestic purpose in Korea under the concept of “fermented plant medicine” in the development
and launch of nutraceuticals that is conceptually differentiated to other products [9,107].
This trend can be explained by favorable images of probiotics and herbal medicines among
Korean consumers. According to Siró et al. [1], “Consumers need to understand the benefits, not the
science behind the product”. Because of consumers’ limited understanding of functional foods and
their health benefits, use of novel bio-functional materials could generate unnecessary work load and
marketing cost to advertise and inform specific functional effects to consumers [99].
Phytochemicals are biologically and nutraceutically valuable plant metabolites. The isolation
and recovery of target natural products from plants is often available for small quantities, specifically
when small amounts of target molecules are naturally produced by plants and preserved in them [108].
Therefore, artificial pre-treatment (i.e., physical, chemical and enzymatic treatment) of phytochemicals
to modify their chemical structures results in an increased yield of bioactive molecules and has
conventionally been used to overcome limited supply issues. According to Gao et al. [107],
biotransformation is “a chemical reaction that is catalyzed by whole cells (microorganisms, plant
cells, animal cells), or by isolated enzymes due to high stereo- or regioselectivity combined with the
high product purity and high enantiomeric excesses”. Bioavailable plant metabolites, specifically
when they are in a glucoside form, are known to be functionally fortified by a deglycosylating
process [9]. This biotransformation process selectively hydrolyzes target molecules and enables the
structural conversion into valuable products. Biotransformation using biological catalysis can be
carried out under relatively mild operational conditions compared to physical (heat treatment) reaction
and/or chemical (i.e., acid and basic) catalyst counterparts, abridging the multifaceted manufacturing
process [108–110]. Recently, biotransformation utilizing catalytic activity of microbial glycosidases
has been recognized as useful technology in nutraceutical and pharmaceutical industries. Specifically,
biotransformation of phytochemical glycosides using probiotic glycosyl hydrolases has played a great
role in the production of bio-functional phytochemical aglycones with attractive potential for practical
applications [102]. This bio-catalytic process using probiotic enzymes has been studied and applied
as an essential manufacturing tool for enhancing the bio-functional and nutritional values of herbal
medicines [107]. Through the fermentation process, plant glycosides can be catalyzed to aglycone,
which has better bio-functional effects. Probiotic whole cells and their extracts (i.e., purified enzyme,
cell-free extracts and crude homogenates) are increasingly utilized in the nutraceutical industry as
key ingredients [9]. They have also been used as bio-catalytic agents that play a fundamental role in
the bioconversion of herbal glycosides into aglycones induced by microbial enzymes belonging to
different groups of glycosidases.
Panax ginseng, meaning “cure-all”, and its major functional metabolite, ginsenosides, are
one of the widely-researched herbal medicines and phytochemicals [109]. There are multiple
Int. J. Mol. Sci. 2016, 17, 1544 10 of 23

studies available covering various pharmaceutical properties of ginsenosides. In accordance with

molecular mechanism, various pre-clinical and clinical studies have suggested that the regulatory
properties of deglycosylated ginsenosides on diverse cellular mechanisms (i.e., cell cycle regulator
(cyclin-dependent kinase), transcription factor (myc gene), signal protein (vascular permeability factor),
tumor suppressors (cellular tumor antigen p53), cyclin-dependent kinase inhibitor (CDK-interacting
protein 1), negative regulator of the p53 (mouse double minute 2 homolog) and apoptosis regulators
(B-cell lymphoma-extra large, B-cell lymphoma-2 and X-linked inhibitor of apoptosis protein, etc.)) may
have the anti-carcinogenic ability in prevention and management of cancer and chronic diseases [102].
Many studies have reported the phytoestrogenic properties of soy milk and soy isoflavones. Multiple
evidences have shown that soy-derived phytoestrogens play inhibitory roles in osteoporosis, obesity
and diabetes [111–114]. However, the functional effect of ginsenosides and soy isoflavones in
in vivo systems are significantly dependent on enzymes of gut flora [115]. Therefore, merging
probiotics and these phytochemicals may improve beneficial effects associated with intake of this
plant medicine. Daidzein, genistein and glycitein that deglycosylated from of daidzin, genistin and
glycitin have been introduced as chemo-preventive compounds for certain types of cancer (i.e., colon,
breast and prostate) [116] and osteoporosis [117]. Similar to glycosylated ginsenosides, multiple
studies demonstrated that the soy isoflavones in plants should be catalyzed into deglycosylated form
(i.e., daidzein and genistein) for the effective absorption into blood stream across the gastrointestinal
tissue [118]. The higher bioavailability after deglycosylation process has been demonstrated in in vivo
experiments and explained by lower molecular weight and greater hydrophobicity than those of
glycosidic compounds.
Because BGN4 naturally does not produce β-glucosidase during fermentation, soy isoflavones
and ginsenoside glycosides cannot be catalyzed into the more bio-functional aglycones [24]. In this
sense, BGN4 had practical limitation for industry application. Some researchers have manipulated
expression vector to produce the recombinant BGN4 strain by cloning the structural β-glucosidase
gene from naturally β-glucosidase producing Bifidobacterium spp. (i.e., B. lactis AD011, B. lactis SH5
and B. lactis RD68) [37–41]. β-glucosidase of B. lactis AD011 was cloned and overexpressed to apply
ginsenoside conversion by Kim et al. [38]. BGN4 was employed as a sub-cloning and overexpression
host for cloning of β-glucosidase of B. lactis AD011. However, BGN4 transformants (B141 and B893)
could not use to catalyze artificial substrate (pNP-β-D-glucopyranoside) as well as natural substrates
(ginsenosides). To attack this problem, Wang et al. [39] have attempted to highlight the necessity of
powerful promoters for BGN4 in inducing significant expression of cloned genes with special focus
on an exploration of the activity configurations of the promoters in BGN4. They proved that the
activities of promoters were a function of microbial growth rates and that the use of P919 is effective to
express of high-levels of foreign genes as a BGN4 promoter by hydrolyzing pNP-β-D-glucopyranoside
into p-nitrophenyl and glucose. However, it should be considered that microbial β-glucosidases are
quite unspecific catalysts whose specificities and activities possibly depend on a structural diversity
of glycosides.
Recently, Youn et al. [37] highlights further how genetically-transformed BGN4 can be
characterized as a β-glucosidase producer to practically apply for hydrolyzing natural products
in which sugar moieties are linked to functional groups by a glycosidic bond (glycosides). They have
constructed multiple expression vectors systems using bifidobacterial promoters (i.e., pamy, p919
and p572), ORF (i.e., bbg572), terminator (i.e., 572t) and signal sequences (i.e., ssamy) to produce
new recombinant β-glucosidase-positive BGN4. The recombinant β-glucosidase, Bp572bbg572t
was applied to catalyze multiple disaccharides (i.e., cellobiose, sophorose, laminaribiose and
gentiobiose), Isoflavones (i.e., daidzin, genistin, and glycitin), ginsenosdies (i.e., Rb1 and Rb2) and
Quercetins (i.e., isoquercetrin and spiraeoside) and successfully degraded glycosidic linkages between
two molecules. This work would be practically useful for an application in phytochemical and
bioconversion industries due to the understanding the vector systems/enzyme functions relationship.
You et al. [41] also successfully carried out enzymatic catalysis of the isoflavone glycosides (i.e., daidzin,
Int. J. Mol. Sci. 2016, 17, 1544 11 of 23

genistin and glycitin) into isoflavone aglycones (i.e., daidzein, genistein and glycitein) using a novel
recombinant β-glucosidase. The β-Glu gene of B. lactis AD011 consisting of 1.4 kb was cloned and the
recombinant β-glucosidase was overexpressed in BGN4.
As a result, BGN4 notably produced β-glucosidase and could be applied to convert ginsenoside
glycosides and soy isoflavones into deglycosylated forms. The catalysis of plant glycosides using
recombinant BGN4 would be applicable for commercial purposes [111,115]. The combination of this
recombinant BGN4 with plant compounds could be utilized to produce fermented plant medicines
with elevating amount of bioactive forms of ginsenosides and isoflavones. In addition, the synergistic
effects generated by indigenous functional properties of BGN4 and its biogenic metabolites can be
possibly expected. Recently, 110 Nobel Prize winners from diverse domains (i.e., medicine, economics,
physics, chemistry, literature and peace) issued a statement in their support of modern genetic
engineering, such as GMOs [119]. However, resistance to genetically-engineered probiotics from
food consumers may exist in food market, resulting in the search for a new market (i.e., biomedical
and pharmaceutical markets) exploitation beyond food or nutraceuticals, and consumers’ paradigm
shift seems be necessary for successful commercial applications [1,9]. Before extensive utilization
of genetically tailored BGN4 in nutraceutical products, consideration of possible safety issues and
consumers’ prejudice is necessary.

6. Industrial Application: Bioactive Molecules

Among the various functional cell metabolites, GABA, a ubiquitous non-protein amino acid, is
widely present in natural resources including bacteria, plants, and animals [8]. This molecule acts as a
major inhibitory neurotransmitter in the brains of vertebrates and is produced by α-decarboxylation of
glutamate by glutamate decarboxylase [120]. Recently, multiple studies have reported bio-functional
effects of GABA (i.e., hypotensive, energy boosting, tranquilizing, lessens signs of aging, diuretic
effects and anti-diabetes) [120,121]. Microbial glutamate decarboxylase, which is critical in GABA
production, is widely distributed in probiotic cells. Multiple Lactobacillus spp. have expressed an
ability to produce GABA in various levels depending upon the density of glutamates in the cell culture
broth [122,123]. Recent reports have shown that Gastrodia elata, a traditional Asian plant medicine often
applied for the treatment of neurodegenerative diseases and headaches, represses the degradation
of GABA [124] and protects against neuronal damage. As a raw material, Gastrodia elata is regarded
as a useful raw material for the GABA production due to the synergistic anti-hypertensive functions
originated from Gastrodia elata [125].
To develop fermented Gastrodia elata products containing considerable amount of GABA,
Kim et al. [30] applied Lactobacillus brevis GABA 100 and BGN4 simultaneously as a starter culture for
Gastrodia elata fermentation. Previously, Kim et al. [126] reported high GABA-producing properties of
Lactobacillus brevis GABA 100 that is isolated from Korean kimchi. The total GABA productivity was
further increased by the co-culture of L. brevis GABA 100 with BGN4. The level of GABA observed
during the co-culture was higher compared to the culture inoculated only by L. brevis GABA 100
by further decreasing media pH compared to its decrease in the single culture of L. brevis GABA
100. According to Komatsuzaki et al. [127], maintaining low pH (about 5) is necessary for effective
GABA production. However, during the fermentation, normally the pH level of the cell culture media
increased due to the enhanced level of GABA in the media.
As discussed above, due to the bioactive functionality of probiotic bacteria and its value-added
metabolites, interest in mass production and industrial applications of biogenic molecules has been
growing. Specifically, SAM, a commercially available and FDA-approved dietary supplement, has
often been obtained and produced through chemical synthesis and cell fermentation. However,
chemical synthesis has issues with production cost and generation of low purity products with optical
isomers [128]. SAM, which is an amino acid naturally produced in the human body, plays a key role
in transmethylation as a methyl donor. Multiple studies have extensively revealed the functional
effects of SAM. As an important nutraceutical ingredient, SAM showed anti-depressant [129], anti-liver
Int. J. Mol. Sci. 2016, 17, 1544 12 of 23

disease [130], and anti-headache effects [131]. According to Kim et al. [43], BGN4 produced a higher
level of SAM compared to other microorganism. They used 25 kinds of different lactic acid bacteria
(i.e., Bifidobacterium, Enterococcus, Lactobacillus, Lactococcus, Pediococcus, Streptococcus and Weissella spp.)
and evaluated the productivity of SAM in culture media. The SAM productivity of BGN4 was at least
two times higher than other bacterial strains. They also applied BGN4 to develop SAM-reinforced
yogurt and reported favorable sensory value for commercial purposes [42]. However, little work
was done to elucidate how productivity of above mentioned metabolites may be influenced by
environmental factors including media ingredients, temperature and presence of other bacteria for
commercial purposes. Nonetheless, above mentioned experimental data supports that BGN4 can be
used in nutraceutical products as a microbial ingredient due to the benefits for human health.

7. Increase Biomass Productivity

For the commercialization, estimating productivities of cell and/or biogenic metabolites is
necessary for cost-effectiveness of product manufacturing. In this sense, determination of appropriate
media ingredients and formula design are important to enhance total cell-biomass productivity, as both
biochemically and physiologically affect probiotic cultures [132]. When considering production costs
of cell or biogenic molecules for commercial application, microbial enrichment for biomass production
can be halted at the time of maximum productivity and some are left to run for longer, depending
on the culture condition [133]. However, there are several pragmatic obstacles in mass production
Bifidobacterium spp. and its metabolites for commercialization due to: (i) lower cell productivity after
enrichment; and (ii) higher production cost compared to other aerobic or facultative anaerobic cells.
Kwon et al. [134] worked to develop a strategy to obtain high BGN4 biomass with greater
metabolic products by combination of crossflow filter and cell reactor. Specifically, they submerged
hollow fiber membrane (0.4 µm cut off, polyvinylidene fluoride, surface area of 25 m2 ) bioreactor with
suction and gas sparging to maintain anaerobic conditions. By using this method, they were able to
observe higher BGN4 viability and lower microbial harms generated by shear stress during crossflow
filtration processes compared to conventional membrane reactor culturing. About 5 and 7 folds greater
productivity of BGN4 cell biomass and viable cell counts (i.e., 12.0 g/L of biomass productivity and
2.2 × 1010 CFU/mL of maximum cell count) were observed when submerged hollow fiber membrane
bioreactor was applied for BGN4 enrichment compared to the microorganism levels achieved through
conventional batch culture (i.e., 4.5 g/L of biomass productivity and 3.0 × 109 CFU/mL of maximum
cell count).
Recently, Ku et al. [27] and Ji et al. [135] have reported more systematic approaches to increase
BGN4 biomass productivity. They examined multiple factors, including: (i) media ingredients; (ii) types
of acid; and (iii) incubation time when they utilized a two-step culture method that significantly affects
the total recovery of BGN4 biomass and its bioactive metabolites. They reported that the phytic acid in
culture media plays important role in improving the productivity and economic of BGN4 cell biomass
and its biogenic molecules by changing microbial morphology and increasing size of the cell, although
additional phytic acid treatments constitute a small portion from the overall costs of culture media
formulation. These results agreed with various results showing that microbial shape and morphologies
increase in the group in which media was treated with supplemented acids compared with those of
control group [136–139]. It seems that the putative morphological modification effects of organic and
mineral acids are likely due to their ability to act as cation chelators to induce response of the microbial
mesosome control [140].
Even though multiple researchers have observed the putative role of acids as major inducer for the
morphological modification of various bacteria, the molecular mechanisms underlying the producing
properties of cell biomass are still largely unknown. Because chromosome sequencing data is available
for BGN4, this information can be potentially utilized for designing better conditions of the biomass
recovery than is currently possible. Moreover, transcription profiling over the fermentation procedures
will provide information for stress and acid tolerance genes for BGN4. Additional work is necessary to
Int. J. Mol. Sci. 2016, 17, 1544 13 of 23

better understand how genetic characteristics of BGN4 affect the recovery and production of BGN4
and its bioactive molecules using post-genomic approaches.

8. From Comparative Genomics to Functionality of BGN4

The Bifidobacterium genus is currently comprised of 47 recognized taxa, which have been isolated
from six different ecological environments including the gut and oral cavity of human and animals
insect hindgut, sewage and fermented foods [141–143]. The Bifidobacterium taxa can be clustered into
six different phylogenetic taxa: B. adolescentis, B. longum, B. pseudolongum, B. boum, B. pullorum, and
B. asteroidsgroups [144]. Although B. bifidum species have been represented one of the dominant bacteria
from the gastrointestinal tract of breast milk-fed infants, B. bifidum is not included in six phylogenetic
groups mentioned above, suggesting its unique and specific genomic composition [19,145].
The publicly available genome sequences to date contain three complete genomes of B. bifidum
strains obtained from infant stool samples including BGN4 and 12 draft genome sequences
(NCBI source). Among the genus Bifidobacterium, 23 complete bifidobacterial genome sequences
are available. The size of B. bifidum genome is approximately 2.2 Mb (range, 2.14–2.28 Mb) and GC
content is about 62% (Table 2).

Table 2. Publicly available genome datasets of three different B. bifidum strains.

Strain Name B. bifidum BGN4 B. bifidum PRL2010 B. bifidum S17

Accession NC_017999.1 NC_014638.1 NC_014616.1
Sequencing Status Complete Complete Complete
Genome Size (bp) 2,223,664 2,214,656 2,186,882
G + C ratio (%) 62.65 62.67 62.76
Number of Chromosones 1 1 1
Number of Contigs 1 1 1
Number of ORFs 1834 1706 1783
Number of rRNA Genes 9 9 9
Number of tRNA Genes 52 52 53

In accordance with the analysis of other bifidobacterial taxa, enzymes in charge of the transport
and metabolism of carbohydrates (Clusters of Orthologous Genes, COG category, G) were identified
from the genome of BGN4 (Table 3), such as glycosyl hydrolases whose substrates are various oligo-
and polysaccharides including human milk oligosaccharides (HMOs) and intestinal mucin [146,147].
Genome analysis of bifidobacterial species has revealed that the genus B. bifidum have adapted
to ecological niches where there is a limited source of other nutrients except carbohydrates [148].
The B. bifidum pan-genome consisted of 2970 COGs and the core-genome was represented by 1295 genes.
Those genes were dedicated to housekeeping functions of bacterial cells such as DNA replication,
transcription and translation transport and metabolism of carbohydrates and amino acids, as well as
host-interacting components of bacteria including sortase-dependent pili, tight adherence (tad) locus
and murein lytic enzyme (TgaA). The pili structures reported to be crucial for interacting with host and
other gut microbiota [141].
Duranti et al. [141] analyzed the average abundance of bifidobacterial DNA from 11 metagenomic
datasets of the gut microbiome from infants and found that the relative abundance of B. bifidum DNA
was 12.42% in breast milk-fed infants compared with the 0.24% in formula milk-fed infants. This
result was consistent with the genomic analysis representing specialized catabolic ability of B. bifidum
to utilize host glycans in an aspect of adapting to infant gut. Turroni et al. [146] also showed the
specific ability of B. bifidum PRL2010 metabolizing host-derived glycans, especially HMOs and mucin.
A comparative genomics study based on 15 genomes of B. bifidum strains helped to elucidate the
evolutionary force for successful adaptation of this species to specific ecological niches (i.e., infant gut)
by assessing genomic variability and complexity [141,142,147]. The genetic variability of B. bifidum was
13.7% of the total genomic pool of B. bifidum, and this was relatively lower than 15.3% of mobilome
value in the genus Bifidobacterium [141,149].
Int. J. Mol. Sci. 2016, 17, 1544 14 of 23

Table 3. Summary of genome analysis comparing COGs of three B. bifidum strains (analyzed by the authors based on NCBI datasets).

B. bifidum BGN4 B. bifidum PRL2010 B. bifidum S17

COG Description
Number of Genes % Number of Genes % Number of Genes %
J Translation, ribosomal structure and biogenesis 136 10.56% 135 10.39% 135 10.48%
K Transcription 95 7.38% 95 7.31% 93 7.22%
L Replication, recombination and repair 102 7.92% 107 8.24% 100 7.76%
D Cell cycle control, cell division, chromosome partitioning 24 1.86% 22 1.69% 23 1.79%
O Posttranslational modification, protein turnover, chaperones 50 3.88% 50 3.85% 50 3.88%
M Cell wall/membrane/envelope biogenesis 75 5.82% 81 6.24% 79 6.13%
N Cell motility 6 0.47% 6 0.46% 5 0.39%
P Inorganic ion transport and metabolism 50 3.88% 49 3.77% 49 3.80%
T Signal transduction mechanisms 47 3.65% 50 3.85% 47 3.65%
C Energy production and conversion 50 3.88% 50 3.85% 51 3.96%
G Carbohydrate transport and metabolism 118 9.16% 117 9.01% 118 9.16%
E Amino acid transport and metabolism 135 10.48% 137 10.55% 136 10.56%
F Nucleotide transport and metabolism 56 4.35% 55 4.23% 56 4.35%
H Coenzyme transport and metabolism 45 3.49% 44 3.39% 44 3.42%
I Lipid transport and metabolism 35 2.72% 36 2.77% 36 2.80%
Q Secondary metabolites biosynthesis, transport and catabolism 6 0.47% 7 0.54% 6 0.47%
R General function prediction only 150 11.65% 148 11.39% 153 11.88%
S Function unknown 108 8.39% 110 8.47% 107 8.31%
Total 1288 100% 1299 100% 1288 100%
Int. J. Mol. Sci. 2016, 17, 1544 15 of 23

In case of glycobiomes, B. bifidum showed a relatively small size of genes and especially reduced
capabilities to catabolize high-molecular plant polysaccharides. However, it should be noted that
B. bifidum contain enriched gene-sets undertaking the metabolism of host-derived glycans and
health-beneficial glyco-conjugated phytochemicals. The comparative genomic analysis of BGN4 strain
with other B. bifidum strains and whole bifidobacterial taxa has not been reported yet. Further study
focusing on identification of unique BGN4 genes, which are capable of encoding specific colonizing
factors, key enzymes catalyzing HMO and glycones, and immunomodulatory molecules expressed
and secreted by BGN4, should be helpful to reinforce the multiple functionality of BGN4.

9. Conclusions
This systematic review summarizes bio-functionality of BGN4 assessed by in vitro, in vivo and
clinical studies and potential of BGN4 for industrial applications and explains what is known and
unknown based on available data (Figure 2). To demonstrate a precise mechanism of exploitation
of BGN4 for human body, multifactorial clinical research and well-controlled molecular level work
Int. J. Mol. Sci. 2016, 17, 1544 15 of 22
should be further pursued. However, potential functional value of BGN4 was clearly established
through multiple
practically in vitro and
in commercial in vivo
products andaclinical
with experiments.Summarized
mass production. Moreover, BGN4 has been
information onapplied
BGN4
practically in commercial products with a mass production. Summarized information
would be valuable to guide design with insight of future experiments to know mechanisms on BGN4 wouldof
be valuable to guide design with insight of future
functionality, clinical trials and commercial applications. experiments to know mechanisms of functionality,
clinical trials and commercial applications.

Figure 2. Schematic representation of biofunctional properties: Biotransformation of phytochemicals

Figure 2. Schematic representation of biofunctional properties: Biotransformation of phytochemicals (a);
(a); high cell adhesion property with high surface hydrophobicity (b); and direct (c); and indirect
high cell adhesion property with high surface hydrophobicity (b); and direct (c); and indirect
immunomodulatory effects (activation of macrophages (d); and dendritic cells (e)) of B. bifidum BGN4
immunomodulatory effects (activation of macrophages (d); and dendritic cells (e)) of B. bifidum
to host.
BGN4 to host.
Acknowledgments: This work was carried out with the support of “Cooperative Research Program for
Agriculture Science &
Acknowledgments: Technology
This work wasDevelopment
carried out(Project No. support
with the PJ01123001 and PJ01123002)”,
of “Cooperative Rural Development
Research Program for
Administration
Agriculture of “the
Science PromotingDevelopment
& Technology Regional specialized
(ProjectIndustry (Projectand
No. PJ01123001 No.PJ01123002)”,
R0004140)”, the Ministry
Rural of Trade,
Development
Administration of “the Promoting Regional specialized Industry (Project No. R0004140)”, the Ministry of Trade,
Industry and Energy (MOTIE) and Korea Institute for Advancement of Technology (KIAT), and “Research
program of SGER (Project No. NRF-2015R1D1A1A02062267)”, National Research Foundation of Korea. The
authors wish to thank Raymond RedCorn and Emily R. Coleman at Purdue University, and Jaycey Hardenstein
of Eli Lily for their review and feedback of this paper. The authors would also like to thank Michael R. Ladisch
and Eduardo Ximenes at Purdue University for their supports to Seockmo Ku.
Int. J. Mol. Sci. 2016, 17, 1544 16 of 23

Industry and Energy (MOTIE) and Korea Institute for Advancement of Technology (KIAT), and “Research program
of SGER (Project No. NRF-2015R1D1A1A02062267)”, National Research Foundation of Korea. The authors wish to
thank Raymond RedCorn and Emily R. Coleman at Purdue University, and Jaycey Hardenstein of Eli Lily for their
review and feedback of this paper. The authors would also like to thank Michael R. Ladisch and Eduardo Ximenes
at Purdue University for their supports to Seockmo Ku.
Author Contributions: Seockmo Ku initiated this work in partial fulfillment of his degree at Seoul National
University under the supervision of Geun Eog Ji, and the mentorship of Hyun Ju You and Myeong Soo Park.
Seockmo Ku performed the literature search and was the primary author of the review. Seockmo Ku wrote
Sections 1–7, and Section 9. Hyun Ju You wrote Section 8. Myeong Soo Park and Geun Eog Ji edited and revised
the review. Geun Eog Ji and Hyun Ju You designed the review template. All authors discussed drafts and
approved the final manuscript for publication.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

BGN4 Bifidobacterium bifidum BGN4

CD cluster of differentiation:
FDA USA Food and Drug Administration
GABA γ-Aminobutyric acid
IFN Interferon
IL Interleukin
SAM S-Adenosyl-L-Methionine
SEM scanning electron microscope
TNF tumor necrosis factor

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