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Fructophilic Lactic Acid Bacteria, a Unique Group of Fructose-

Fermenting Microbes

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Akihito Endo,a Shintaro Maeno,a Yasuhiro Tanizawa,b Wolfgang Kneifel,c Masanori Arita,b,d Leon Dicks,e Seppo Salminenf

a Department of Food, Aroma and Cosmetic Chemistry, Tokyo University of Agriculture, Hokkaido, Japan
b National Institute of Genetics, Shizuoka, Japan
c Department of Food Sciences and Technology, University of Natural Resources and Life Science Vienna,
Vienna, Austria
d RIKEN Center for Sustainable Resource Science, Kanagawa, Japan
e Department of Microbiology, University of Stellenbosch, Stellenbosch, South Africa
f
Functional Foods Forum, University of Turku, Turku, Finland

ABSTRACT Fructophilic lactic acid bacteria (FLAB) are a recently discovered group,
consisting of a few Fructobacillus and Lactobacillus species. Because of their unique
characteristics, including poor growth on glucose and preference of oxygen, they
are regarded as “unconventional” lactic acid bacteria (LAB). Their unusual growth
characteristics are due to an incomplete gene encoding a bifunctional alcohol/acet-
aldehyde dehydrogenase (adhE). This results in the imbalance of NAD/NADH and the
requirement of additional electron acceptors to metabolize glucose. Oxygen, fruc-
tose, and pyruvate are used as electron acceptors. FLAB have significantly fewer
genes for carbohydrate metabolism than other LAB, especially due to the lack of
complete phosphotransferase system (PTS) transporters. They have been isolated
from fructose-rich environments, including flowers, fruits, fermented fruits, and the
guts of insects that feed on plants rich in fructose, and are separated into two
groups on the basis of their habitats. One group is associated with flowers, grapes,
wines, and insects, and the second group is associated with ripe fruits and fruit fer-
mentations. Species associated with insects may play a role in the health of their
host and are regarded as suitable vectors for paratransgenesis in honey bees. Be-
sides their impact on insect health, FLAB may be promising candidates for the pro-
motion of human health. Further studies are required to explore their beneficial
properties in animals and humans and their applications in the food industry.

KEYWORDS fructophilic lactic acid bacteria, Fructobacillus, Lactobacillus, taxonomy,

genomics, microbial ecology

L actic acid bacteria (LAB) are present in a variety of environments, including dairy
products, fermented food, plant surfaces, soil, and the gastrointestinal and vaginal
tracts and oral cavities of humans and animals. Their different habitats, each with
Accepted manuscript posted online 27 July
2018
unique stress conditions and nutrient compositions, forced these bacteria to develop Citation Endo A, Maeno S, Tanizawa Y, Kneifel
specific physiological and biochemical characteristics. Examples are proteolytic and W, Arita M, Dicks L, Salminen S. 2018.
Fructophilic lactic acid bacteria, a unique
lipolytic activities to obtain nutrients from milk (1), tolerance to phytoalexins in plants
group of fructose-fermenting microbes. Appl
(2), or tolerance to bile salts in the gastrointestinal tract (3). In general, such highly Environ Microbiol 84:e01290-18. https://doi
adapted microorganisms are in several instances suitable starter cultures in food .org/10.1128/AEM.01290-18.
fermentations and probiotic candidates for humans and animals (4–6). Editor Harold L. Drake, University of Bayreuth
Fructophilic LAB (FLAB) are a special group of LAB that were described only recently Copyright © 2018 American Society for
Microbiology. All Rights Reserved.
(7). This group is easily differentiated from other LAB on the basis of a few unique
Address correspondence to Akihito Endo,
biochemical characteristics. The optimum substrate for the growth of FLAB is fructose, a3endou@bioindustry.nodai.ac.jp.
and unlike other LAB, growth on glucose is very poor (7). It is thus not surprising to find

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FLAB in fructose-rich environments, such as on flowers and fruits and in fermented food
derived from fruits (7, 8). Recent studies suggested that fructose-feeding insects host
high numbers of FLAB cells in their guts (9, 10). To adapt to these environments, FLAB
had to change on a genomic level (11), which may benefit their use as starter cultures
in food fermentation or as probiotics in their host animals. Since FLAB are a newly
discovered group, their unique characteristics have not been reviewed. This article
summarizes the history, taxonomy, biochemical and genomic characteristics, ecology,

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and practical importance of FLAB.

HISTORY
Fructobacillus fructosus, originally classified as Lactobacillus fructosus, was first described
in the FLAB group in 1955 (12), although the name of the flower from which the strain was
isolated was not mentioned in the original article. Kodama selected a single strain from
approximately 2,000 strains in his culture collection and differentiated the strain from other
LAB on the basis of its poor growth on modified Thompson synthetic medium (13). The
growth of the strain was stimulated by adding tomato juice to glucose-yeast extract-
peptone broth (13). In a follow-up study of this strain, fructose was identified as the growth
factor in tomato juice (14). The taxonomic position of L. fructosus was again evaluated when
a phylogenetic relative, Leuconostoc ficulneum, was isolated from a ripe fig in 2002 (15).
Lactobacillus fructosus was then reclassified as Leuconostoc fructosum on the basis of its
phylogenetic position. Both species grew poorly on glucose. Leuconostoc durionis was
isolated from a Malaysian acid-fermented condiment, tempoyak, in 2005 (16). The
authors reported limited growth of L. durionis in modified MRS broth with glucose,
xylose, ribose, lactose, sucrose, or fructose as the sole carbon source. However, the
latter study showed a fructose preference by the strain (17). Leuconostoc pseudoficul-
neum was isolated from a ripe fig in Portugal, as shown with L. ficulneum in 2006 (18).
These two species share high sequence identities on the basis of their 16S rRNA gene
sequences. The preference for fructose over glucose was not addressed in this article.
Leuconostoc fructosum, L. durionis, L. ficulneum, and L. pseudoficulneum were later
reclassified as species in the novel genus Fructobacillus on the basis of their unique
fructophilic characteristics, phylogenetic positions, and morphological characteristics
(17). The fifth species in the genus, Fructobacillus tropaeoli, was isolated from the flower
Tropaeolum majus and was characterized in depth (19).
In 1998, Lactobacillus kunkeei was isolated from wine that underwent sluggish
alcoholic fermentation (20). At the time, the fructophilic characteristics of the species
were not reported, but the biochemical characteristics of the isolate were similar to
those reported for nine other strains of L. kunkeei isolated from wine, flowers, and fresh
honey (21). This study clearly demonstrated that fructophilic characteristics are well
conserved in the species, regardless of their origins. Lactobacillus florum was described
from flowers as novel FLAB in 2010 (22). This species shares slightly different charac-
teristics with other FLAB, and L. florum was described as the first facultative FLAB (7, 22).
This is discussed in more detail later on. Lactobacillus apinorum was isolated from the
honey stomach of honey bees, together with other novel lactobacilli in 2014 (23). The
unique biochemical characteristics of the species were not discussed in the original
article but were well characterized in a later study (24). As described above, the
fructophilic characteristics of many of the species, now classified as FLAB, were not
described in the original articles, although all of them originated from fructose-rich
environments. Since LAB sometimes possess specific biochemical and physiological
characteristics to survive in unique habitats, a detailed characterization is essential to
understand how they adapt, the roles they play in nature, and their possible symbiosis
with plants, insects, animals, and humans.

TAXONOMY
FLAB species belong to the genus Fructobacillus or Lactobacillus. The genus Fructo-
bacillus was proposed for the reclassification of four Leuconostoc spp. on the basis of
their phylogenetic positions and biochemical and morphological characteristics (17).

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FIG 1 Phylogenetic relationships between FLAB and related LAB species based on the multiple align-
ments of the 235 conserved genes. FLAB species are shown in bold. The values on the branches are
bootstrap support from 1,000 rapid bootstrapping replicates. Lactococcus lactis NBRC 100933T was used
as an out group. This is an update of results presented in a previously published paper (29) and includes
Lactobacillus spp.

On the basis of 16S rRNA gene sequences, species within the genus Fructobacillus are
94.2% to 98.0% identical and 90.4% to 93.1% related to Leuconostoc spp. (17). On the
basis of 16S-23S rRNA gene intergenic spacer region sequences, Fructobacillus spp.
share 81.3% to 92.4% identities and are 69.2% to 80.1% related to Leuconostoc spp. A
phylogenetic analysis based on 16S rRNA gene sequences revealed that Fructobacillus
spp. fall into a single subcluster related to a subcluster of Leuconostoc sensu stricto (17).
On the basis of 16S rRNA gene sequences, Leuconostoc fallax, originally proposed as an
atypical Leuconostoc species (25), is located outside the Fructobacillus-Leuconostoc
sensu stricto group. The phylogenetic position of the species is strongly influenced by
the genes used in the analyses (17). A phylogenetic tree constructed from multiple
alignments of 235 conserved genes placed L. fallax within the Leuconostoc sensu stricto
subcluster (Fig. 1). Fructobacillus spp. form a well-defined subcluster in the single-gene-
based (17), as well as the conserved-gene-based, phylogenetic analyses (Fig. 1).
Species within the genus Lactobacillus are divided into more than 10 phylogenetic
groups. L. kunkeei and L. apinorum are, however, excluded from these phylogenetic
groups (21, 23). The two species share high 16S rRNA gene sequence identities (98.9%)
but are less than 95% similar to other lactobacilli. These fructophilic lactobacilli form a
subcluster in a phylogenetic tree based on multiple alignments of the conserved 235

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genes, indicating their close genetic relationships (Fig. 1). Maeno et al. (11) reported the
average nucleotide identity (ANI) of 16 strains of L. kunkeei. Contrary to the widely
accepted ANI threshold of 0.95 used to discriminate species (26, 27), the 16 strains of
L. kunkeei showed a broader distribution of intraspecies ANI values, ranging from 0.905
to 1.000 (Table 1). Several strains showed similar threshold values of 0.945 to 0.952,
suggesting that the widely used ANI threshold is not simply applicable to L. kunkeei
(Table 1). Relatively low ANI values (ranging from 0.905 to 0.918) were recorded

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between strain LAdo and the other strains (Table 1), indicating that the taxonomic
position of the strain is unclear. Another fructophilic species, L. florum, belongs to the
L. fructivorans phylogenetic group, together with L. fructivorans, L. homohiochii, L.
lindneri, and L. sanfranciscensis (22). None of the members in this phylogenetic group,
except L. florum, show fructophilic characteristics. All FLAB are classified as heterofer-
mentative LAB on the basis of the profiles of metabolic end products from glucose. The
reason for the absence of FLAB among homofermentative LAB is discussed below.

BIOCHEMICAL CHARACTERISTICS
FLAB prefer fructose over glucose as a growth substrate, and their growth on
glucose is limited or delayed compared to that on fructose (7). Glucose is usually the
best substrate for other LAB, and these characteristics clearly distinguish FLAB from
most LAB. FLAB are separated into two groups, i.e., the obligate FLAB and the
facultative FLAB, on the basis of their growth characteristics. Obligate FLAB grow well
on fructose but poorly on glucose (7). However, growth on glucose is stimulated when
the medium is supplemented with pyruvate or fructose or when the incubation is
under aerobic conditions. This is due to the requirement for an external electron
acceptor when glucose is metabolized. Pyruvate, fructose, and oxygen serve as the
electron acceptors. Unlike other LAB, strains in this group produce pinpoint colonies
(0.1 to 0.2 mm in diameter after 2 days of incubation) when grown on a medium with
glucose as the sole carbon source and incubated under anaerobic conditions. The
colony size increases to 1 to 2 mm in diameter when incubated under aerobic
conditions (7, 19). This would be the reason for the “yet-to-be cultured” status ascribed
to FLAB until recently. All species in the genus Fructobacillus, L. kunkeei, and L. apinorum
are classified as obligate FLAB.
Facultative FLAB, similar to obligate FLAB, grow well on fructose and on glucose in
the presence of electron acceptors (7). Facultative FLAB are able to grow on glucose but
at a delayed rate compared to growth on fructose or on glucose in the presence of
electron acceptors (7). L. florum is the only recognized species in the group of
facultative FLAB (22).
FLAB species are classified as heterofermentative LAB and produce accessory prod-
ucts together with lactate. Unlike other heterofermentative LAB, the accessory products
in obligate FLAB are CO2 and acetate (Fig. 2), while ethanol is hardly produced (7, 17,
21). Almost equal molar ratios of lactate and acetate are produced, while the proportion
of ethanol to lactate is less than 5%. This is due to a deletion of the bifunctional
alcohol/acetaldehyde dehydrogenase gene (adhE) in obligate FLAB (11, 28, 29). Because
of the deletion, they lack alcohol dehydrogenase (ADH) and acetaldehyde dehydroge-
nase (ALDH) activities (11), while L. kunkeei possesses ALDH activity but lacks ADH
activity. This results in a failure to convert acetyl phosphate to ethanol via acetaldehyde.
Acetyl phosphate is instead converted to acetate by acetate kinase (Fig. 2). During the
production process of acetate, heterofermentative LAB generate an additional ATP,
which results in the production of more biomass in these LAB (30). On the other hand,
the ethanol production system is essential to maintain the NAD/NADH balance in the
heterolactic phosphoketolase pathway (30). The lack of ADH and ALDH activities causes
an imbalance of NAD/NADH in obligate FLAB, and they thus require electron acceptors
for glucose metabolism (Fig. 2). Facultative FLAB, such as L. florum, produce lactate,
ethanol, and acetate at a ratio of 1:0.8:0.2 (22) and CO2 from glucose. This is different
from other heterofermentative LAB. Facultative FLAB possess ADH and ALDH activities
(11). Homofermentative LAB use glycolysis for glucose metabolism, which does not

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TABLE 1 ANI values obtained between L. kunkeei strains and L. apinoruma

L. kunkeei L. apinorum
Species Strain YH-15T FF30-6 AR114 MP2 EFB6 Fhon2 LAan LAce LAdo LAfl LAko LAla LAni LAnu LMbe LMbo Fhon13T
L. kunkeei YH-15T 0.936 0.938 0.951 0.949 0.976 0.940 0.930 0.905 0.931 0.933 0.931 0.939 0.932 0.980 0.943 0.799
FF30-6 0.937 0.973 0.949 0.954 0.935 0.949 0.976 0.915 0.976 0.952 0.964 0.948 0.951 0.938 0.947 0.800
AR114 0.938 0.973 0.954 0.951 0.939 0.948 0.976 0.915 0.977 0.951 0.959 0.946 0.948 0.945 0.947 0.800
MP2 0.950 0.949 0.954 0.968 0.953 0.963 0.946 0.913 0.950 0.943 0.944 0.961 0.944 0.953 0.944 0.800
EFB6 0.948 0.954 0.952 0.968 0.944 0.968 0.949 0.913 0.950 0.948 0.948 0.966 0.947 0.945 0.949 0.799

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Fhon2 0.977 0.936 0.940 0.953 0.945 0.939 0.930 0.907 0.930 0.932 0.930 0.937 0.930 0.978 0.942 0.799
LAan 0.940 0.949 0.948 0.963 0.967 0.938 0.949 0.917 0.951 0.952 0.948 0.979 0.951 0.941 0.947 0.800
LAce 0.931 0.976 0.977 0.946 0.950 0.930 0.951 0.916 0.951 0.954 0.964 0.949 0.952 0.934 0.944 0.799
LAdo 0.905 0.915 0.914 0.913 0.913 0.905 0.916 0.916 0.916 0.918 0.916 0.915 0.918 0.907 0.913 0.801
LAfl 0.931 0.976 0.977 0.950 0.950 0.930 0.951 0.951 0.916 0.954 0.964 0.949 0.952 0.934 0.944 0.800
LAko 0.933 0.950 0.951 0.943 0.946 0.931 0.952 0.953 0.918 0.954 0.951 0.950 0.972 0.934 0.950 0.800
LAla 0.930 0.963 0.958 0.943 0.947 0.928 0.948 0.963 0.916 0.964 0.951 0.947 0.948 0.932 0.942 0.799
LAni 0.939 0.947 0.947 0.960 0.966 0.935 0.980 0.949 0.915 0.949 0.951 0.948 0.951 0.939 0.946 0.799
LAnu 0.931 0.949 0.948 0.943 0.946 0.928 0.951 0.951 0.918 0.952 0.971 0.949 0.950 0.932 0.949 0.800
LMbe 0.980 0.938 0.945 0.954 0.945 0.978 0.941 0.933 0.907 0.934 0.934 0.932 0.939 0.932 0.945 0.800
Lmbo 0.942 0.946 0.946 0.944 0.947 0.940 0.947 0.943 0.913 0.944 0.950 0.941 0.944 0.949 0.944 0.799
L. apinorum Fhon13T 0.798 0.800 0.799 0.800 0.799 0.798 0.800 0.800 0.800 0.800 0.800 0.799 0.799 0.799 0.799 0.800
aCompiled from data published in reference 11.

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FIG 2 Pathways and electron flows for metabolism of fructose and glucose in typical heterofermentative LAB and obligate FLAB. Dotted lines represent electron
flow. xpk, phosphoketolase; ack, acetate kinase; adhE, bifunctional alcohol/acetaldehyde dehydrogenase; ldh, lactate dehydrogenase. Compiled from data
previously presented (29).

include an ethanol production system, and the NAD/NADH balance is well kept. Thus,
homofermentative LAB do not have fructophilic characteristics.
FLAB produce mannitol from the metabolism of fructose, suggesting that fructose
is used as a substrate and an electron acceptor (30). This has been reported for other
heterofermentative LAB (31, 32). FLAB metabolize only a limited number of carbohy-
drates (7, 21); hence, carbohydrates metabolized in the API50CHL test are usually two
to four. These poor carbohydrate utilization properties are due to their genetic char-
acteristics, which are described later. In the carbohydrate metabolism test, fructose is
always metabolized within 24 h. Glucose metabolism is delayed and takes 2 to 4 days
(33). Mannitol metabolism is usually recorded for FLAB but only after 5 to 6 days. The
pentoses arabinose, ribose, and xylose, which are usually metabolized by heterofer-
mentative LAB, are not utilized by FLAB. Filannino et al. (34) reported interesting
findings on phenolic acid metabolism in L. kunkeei and F. fructosus strains that were
isolated from honey bees. All strains tested had the potential to metabolize p-coumaric
acid to phloretic acid or p-vinylphenol. p-Coumaric acid is the main phenolic acid in
pollens which are linked with the habitats of FLAB. The conversion of caffeic acid to
dihydrocaffeic acid was also commonly seen in the FLAB tested. The end products from
the metabolism of phenolic acids possess aroma characteristics and antioxidant activ-
ities, suggesting possible beneficial roles of FLAB in food fermentation.

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FIG 3 Genome sizes (left) and number of CDSs (right) in Fructobacillus spp. (n ⫽ 5), L. kunkeei (n ⫽ 16), L. apinorum
(n ⫽ 1), L. florum (n ⫽ 1), Leuconostoc spp. (n ⫽ 9), vaginal lactobacilli (n ⫽ 5) and “Firm-5” (n ⫽ 5). Fructobacillus
spp. include F. fructosus, F. durionis, F. ficulneus, F. pseudoficulneus, and F. tropaeoli. Leuconostoc spp. include L.
mesenteroides, L. carnosum, L. citreum, L. fallax, L. gelidum, L. inhae, L. kimchii, L. lactis, and L. pseudomesenteroides.
Vaginal lactobacilli include L. crispatus, L. gasseri, L. iners, L. jensenii, and L. vaginalis. Firm-5 includes Lactobacillus
apis, L. helsingborgensis, L. kimbladii, L. kullabergensis, and L. melliventris. The figure was compiled on the basis of
data published in references 11, 24, and 29.

GENOMIC CHARACTERISTICS
Previous genomic studies of FLAB revealed that FLAB have adapted to their habitats at
the genome level (11, 24, 29). Comparative genomics between Fructobacillus spp. (n ⫽ 5)
and Leuconostoc spp. (n ⫽ 9) showed that Fructobacillus spp. possess significantly fewer
protein coding sequences ([CDSs] median ⫾ standard deviation [SD], 1,387 ⫾ 132 versus
1,980 ⫾ 323, respectively; P ⬍ 0.001) in their small genomes (median ⫾ SD, 1.49 ⫾ 0.30
versus 1.94 ⫾ 0.21 Mbp, respectively; P ⬍ 0.001) (Fig. 3). A quality assessment of genomic
data conducted by CheckM suggested that the genomes of Fructobacillus spp. lack multiple
Leuconostocaceae-specific gene markers (29).
A comparison of the number of genes in the cluster of orthologous groups (COG)
identified for Fructobacillus and Leuconostoc clearly demonstrated that Fructobacillus
has a dramatically reduced number of genes (average ⫾ SD, 65.6 ⫾ 5.7 versus 141.6 ⫾
29.7, respectively) involved in carbohydrate transport and metabolism (29). Compared
to the total number of genes in all COGs, the carbohydrate transport and metabolism
genes are represented at 5.1% ⫾ 0.22% in Fructobacillus and 8.8% ⫾ 1.1% in Leucono-
stoc (P ⬍ 0.001) (Fig. 4). Of the 21 COG functional classes, the number of genes
categorized in carbohydrate transport and metabolism ranks 3rd in Leuconostoc but
only 9th in Fructobacillus. LAB usually possess a large number of genes in the carbo-
hydrate transport and metabolism class, since they colonize a wide variety of habitats.
Available carbohydrates vary considerably among habitats. Honey bee lactobacilli,
so-called “Firm-5,” possess 134 to 304 genes of this class per strain (35). The carbohy-
drate transport and metabolism class ranks 1st in 21 COG classes in Firm-5 (Fig. 4). The
numbers of genes classified in the classes (i) cell cycle, cell division, and chromosome
partitioning, (ii) translation, ribosomal structure, and biogenesis, (iii) replication, recom-
bination, and repair, and (iv) intracellular trafficking, secretion, and vesicular transport
are comparable between Fructobacillus spp. and Leuconostoc spp. (29). The conserva-
tion of genes in these classes against genome reduction may indicate that their
functions are essential for the reproduction of the organisms.
Ortholog assignment and pathway mapping indicated that Fructobacillus spp. lack
most genes used for respiration (genes mapped in the tricarboxylic acid [TCA] cycle and
ubiquinone and other terpenoid-quinone biosynthesis) (Table 2), whereas oxygen
enhances their growth, implicating that they do not respire and use oxygen as an

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FIG 4 Top 10 COG classes in Fructobacillus spp. (n ⫽ 5), Leuconostoc spp. (n ⫽ 9), L. kunkeei (n ⫽ 16), L. apinorum (n ⫽ 1), L. florum (n ⫽ 1),
Leuconostoc spp. (n ⫽ 9), vaginal lactobacilli (n ⫽ 5), and “Firm-5” (n ⫽ 5). Species in each group correspond to those in Fig. 3. The figure was
compiled on the basis of data published in references 11, 24, and 29.

electron acceptor. NADH oxidase is usually an indicator for oxygen consumption in LAB
(36–38). In fact, FLAB possess strong NADH oxidase activities (11). Furthermore, genes
for pyruvate dehydrogenase complex subunits are not seen in the genomes of L.
kunkeei strains (39), suggesting that pyruvate generated from sugar metabolism is not
processed further to the TCA cycle but metabolized to lactate. Fructobacillus spp. also
lack the metabolic systems for most carbohydrates (29). In particular, they possess only
one gene for the phosphotransferase system (PTS) (Table 2), whereas the PTS is a major
carbohydrate transport system in LAB. In Leuconostoc spp., the average gene number
for the PTS is 13 (SD ⫽ 3.13). A recent article reported that Lactobacillus kullabergensis
and Lactobacillus kimbladii, classified as Firm-5 honey bee lactobacilli, possess 87 and
88 genes for the PTS, encoding an estimated 41 and 42 complete PTS transporters,
respectively (35). The number of genes for the ATP-binding cassette transporter (ABC
transporter) is significantly reduced in Fructobacillus spp. compared to that in Leucono-
stoc spp. (median ⫾ SD, 33.8 ⫾ 3.1 versus 50.6 ⫾ 8.3, respectively; P ⫽ 0.003). These
findings are consistent with the poor carbohydrate metabolic properties in Fructoba-
cillus, as described elsewhere.
Two species of obligate fructophilic lactobacilli, i.e., L. kunkeei and L. apinorum,
possess genomic characteristics similar to those of Fructobacillus spp. The two species
have genome sizes ranging from 1.41 to 1.58 Mbp and CDS numbers from 1,271 to

TABLE 2 Discriminative pathways between FLAB and related LAB groupsa

No. of genes (mean [SD]) for groupb:
Pathway definition (KEGG no.) 1 2 3 4 5 6 7
Citrate cycle (TCA cycle) (map00020) 0 (0)c 2 (0) 1 1 4 (0.8) 3 (1.5) 2 (0.5)
Ubiquinone and other terpenoid-quinone 1 (0) 2 (0.5) 2 0 8 (1.0) 2 (3.4) 0 (0)
biosynthesis (map00130)
ABC transporter (map02010) 34 (3.1) 26 (1.6) 30 29 51 (8.3) 35 (5.0) 42 (3.5)
Phosphotransferase system (map02060) 1 (0.5) 0 (0) 0 0 13 (3.1) 14 (6.6) 36 (2.2)
aCompiled from references 11, 24, and 29.
bGroups: 1, Fructobacillus spp. (n ⫽ 5); 2, L. kunkeei (n ⫽ 16); 3, L. apinorum (n ⫽ 1); 4, L. florum (n ⫽ 1); 5,
Leuconostoc spp. (n ⫽ 9); 6, vaginal lactobacilli (n ⫽ 5); 7, “Firm-5” (n ⫽ 5). Species in each group
correspond to those in Fig. 4.
cValues indicate means and standard deviations of the number of genes used for the pathways.

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FIG 5 Primary structures of the AdhE protein in FLAB and heterofermentative LAB. The figure was compiled from reference
11 by inclusion of data published in references 24 and 29.

1,457, similar to those reported for Fructobacillus spp. (Fig. 3). However, these sizes and
numbers are significantly smaller than those of other lactobacilli (P ⬍ 0.001) (11). The
two species also have a significantly reduced number of genes in the class for
carbohydrate transport and metabolism compared to those of other lactobacilli (11)
(Fig. 4). Similar to Fructobacillus spp., the two species also lack most of the genes
involved in respiration, and the PTS is completely missing (Table 2). Vaginal lactobacilli,
including Lactobacillus crispatus, L. gasseri, L. iners, and L. jensenii, have significantly
smaller genomes than nonvaginal lactobacilli (40). However, specific gene losses in the
PTS and the class for carbohydrate transport and metabolism have not been observed
among vaginal lactobacilli (11) (Fig. 4; Table 2). This would imply that each small-
genome Lactobacillus sp. has traced a different course of reductive evolution during its
adaptation process. Species in the L. fructivorans phylogenetic group, including L.
florum, showed similar genomic characteristics to those of obligate FLAB (11), meaning
that species of this phylogenetic group have a small-size genome and have specific
gene reductions in carbohydrate transport and metabolism. Species in this phyloge-
netic group have unique growth characteristics, which are slow growth in L. lindneri and
L. fructivorans (41–43) and the preference for a specific growth substrate in L. sanfran-
ciscensis and L. florum (22, 44). Certain species are strongly linked with specific envi-
ronments, e.g., L. sanfranciscensis in sourdough (44), L. lindneri in beer (41), L. homo-
hiochii in sake (45), and L. florum in flowers (22). These fastidious characteristics may be
due to their genomic characteristics.
Fructobacillus spp. are found as the first heterofermentative LAB that lack the adhE
gene (Fig. 5). This observation was originally reported on the basis of data obtained
from PCR with gene-specific primers and Southern blot hybridization (28) and was
confirmed on the basis of genomic information (29). This unique genomic characteristic
is shared with obligate fructophilic lactobacilli, i.e., L. kunkeei and L. apinorum. Hetero-
fermentative LAB usually have a single adhE gene coding for amino acids ranging from
864 to 900 residues, containing a C-terminal ADH domain and an N-terminal ALDH
domain (11). In L. kunkeei, the adhE gene is only partially present, excluding the ADH
domain (Fig. 5). L. apinorum lacks the adhE gene (Fig. 5). Obligate FLAB are thus
characterized by an incomplete adhE gene. On the other hand, facultative FLAB, such
as L. florum, have a normal-size adhE gene and ADH and ALDH activities (11).
Concluded from the genomic data, obligate FLAB share similar genomic characteristics
but are clearly different from their phylogenetic relatives. This suggests that fructose-rich
niches have induced similar gene reductions in phylogenetically distant LAB.

ECOLOGY
FLAB are isolated from fructose-rich environments, including flowers, fruit surfaces,
fermented fruits, and the guts of insects. In most cases, the habitat for each species is
unique. L. kunkeei was originally isolated from spoiled wine in the United States (20) but

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FIG 6 Currently known habitats of FLAB.

was also isolated from wines and wine grapes in other wine-producing regions (46, 47).
L. florum was also isolated from grapes and wines, and these strains contain genes
coding for the production of malolactic enzyme, phenolic acid decarboxylase, and
citrate lyase (48). These properties might have a positive impact on the taste and aroma
of wines. In Fructobacillus spp., F. fructosus is the only species found in wine (47). L.
kunkeei was the major FLAB species present in flowers (7). L. florum, F. fructosus, and F.
tropaeoli have also been isolated from flowers (7, 13, 19, 22), while F. durionis, F.
ficulneus, and F. pseudoficulneus have not been reported as present in flowers. The three
Fructobacillus species could be linked with ripe and fermented fruits. F. ficulneus and F.
pseudoficulneus were originally isolated from figs (15, 18) and F. pseudoficulneus from
bananas (7). F. durionis is one of the dominant members in tempoyak (16, 49), a
fermented condiment made from durian. No other FLAB species have been isolated
from this source. This species was also found in fermented palm wine (50). Cocoa bean
fermentation is a rich source of Fructobacillus spp., and F. ficulneus, F. pseudoficulneus,
F. durionis, and F. tropaeoli that have been recovered from these fermentations in
several countries (51–53). L. kunkeei, L. apinorum, and F. fructosus have not been
identified in ripe or fermented fruits (excluding grapes and wines), although all three
species have been isolated from bee microbiota (9, 23). F. fructosus was also detected
in the guts of giant ants and tropical fruit flies (54, 55). Concluded from the isolation
histories, FLAB may be separated into two groups. One group is associated with flowers,
grapes, wines, and insects. L. kunkeei and F. fructosus are representatives of this group
(Fig. 6), and L. florum and L. apinorum are partially affiliated. The second group is
associated with ripe fruits and fruit fermentations (except grapes and wines), and this
group is represented by F. ficulneus, F. pseudoficulneus, and F. durionis (Fig. 6). F.
tropaeoli, found in flowers, fruits, and fruit fermentations, can be placed between the
two groups.
As described above, recent studies revealed that certain FLAB species, especially L.
kunkeei and F. fructosus, are microbial components in the digestive tracts of honey bees
(9, 10, 34). As these two species are flower inhabitants (7), it is suggested that bees
share their gut microorganisms with their diet sources. Flowers provide fructose-rich
diets to bees and to bee commensal microbes. In honey bee larvae, the two FLAB
species account for the major proportions of cultured microbiota in the latter larval
instars (56). This might be due to a royal jelly resistance associated with these species
(57). In adult honey bees, L. kunkeei is mainly found in the crop and hindgut (58, 59).
L. kunkeei and F. fructosus are shared at the strain level in hives, including among adult
bees, larvae, pollen, and fresh honey (9). L. kunkeei is the major microbial component
in other bee species, such as bumblebees, sweat bees, and stingless bees (39, 60, 61).
Lactobacillus apinorum, another bee-relevant FLAB, is a relatively new FLAB species, and
there is only one report about its isolation and detection in bees (23). On the basis of
16S rRNA gene, this species shares high sequence identity (98.9%) with L. kunkeei,
implicating that, in previous metagenomics studies, the species might have been

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hidden within L. kunkeei. In fact, clones that would be regarded as L. apinorum on the
basis of 16 rRNA gene sequence identities were described as L. kunkeei (59).

PRACTICAL IMPORTANCE AND FUTURE APPLICATIONS

Because of the dominance and adaptation to the environment, certain FLAB species
have been discussed regarding their role as probiotics for bees. The administration of
a honey bee LAB cocktail including L. kunkeei to honey bee larvae infected by the

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European foulbrood pathogen, Melissococcus plutonius, resulted in a 20% reduction in
the proportion of dead larvae (62). Certain strains of L. kunkeei possess antibacterial
activity against M. plutonius (9). Another study revealed that a mixture of four strains of
L. kunkeei decreased the mortality associated with Paenibacillus larvae (a pathogen of
American foulbrood) infection in larvae and the counts of Nosema ceranae (a causative
agent of nosemosis type C) spores from adult honey bees (63). Similar findings have
been reported by Al-Ghamdi et al. (64). Metabolites of F. fructosus were reported to
support the growth of specific honey bee lactobacilli (65). This would have an impact
on microbiota dynamics in honey bees. All these findings suggest that FLAB are
promising probiotic candidates for honey bees. Since honey bees are one of the most
important pollinators in nature, this observation can be tremendously important for
humans as well as for our environment. The disappearance of bees will result in
severely decreased yields in crops, vegetables, and fruits. In this context, paratransgen-
esis is an interesting application tool for FLAB in honey bees. This approach uses
genetically transformed symbiotic microorganisms as vectors to control certain dis-
eases in host insects (66). The transformed bacteria usually express bioactive proteins
or peptides for the purposes. L. kunkeei and F. fructosus can be genetically modified and
express certain proteins (67, 68). The genetically modified strains did not induce
adverse effects in honey bees (67). This is a promising option to maintain the health of
honey bees, although further research is needed.
Certain FLAB strains originating from honey bees or bee hives were examined for
beneficial effects on human health. For example, the intake of a heat-killed form of an
L. kunkeei strain by healthy adults significantly enhanced the levels of secretory
immunoglobulin A (SIgA) in saliva compared with baseline concentrations (69) and also
resulted in decreased levels of Bacteroides fragilis group (70). Certain strains of B. fragilis
are enterotoxigenic, and a decreased number of the species in humans is considered a
beneficial marker (71). In a separate study, the administration of the heat-killed strain
reduced the symptoms of murine influenza pneumoniae by enhancing SIgA production
in mice infected with influenza virus (72). Biofilm-forming L. kunkeei strains possessed
strong biofilm formation properties, and they showed antibiofilm formation properties
against a virulent Pseudomonas aeruginosa strain in vitro and in an insect infection
model (73). So far, no studies have applied viable FLAB strains in human intervention
studies. This is due to safety concerns of the strains (74), although FLAB have been
reported to be present in certain food fermentations. The history of human consump-
tion of FLAB with viable forms might be still not be enough to sufficiently assess their
safety for human consumption.
Regarding spontaneous food fermentation, the exact role and impact of FLAB have
not been well characterized. Certain species of FLAB seem to dominate the fermenta-
tion, especially of tempoyak and cocoa beans (49, 52), suggesting their relevance in the
fermentation process. FLAB produce large amounts of acetate together with lactate (7),
and acetate is well known to possess strong antibacterial activities against several
foodborne pathogens and food spoilage bacteria (75–77). Thus, FLAB may contribute to
biopreservation in spontaneous food fermentation. An F. fructosus strain was investi-
gated for its starter culture function in wheat bran fermentation (78). The strain reduced
the viable cell numbers of yeasts and molds as well as of Enterobacteriaceae during
fermentation. The fermented bran and breads prepared with this bran displayed
medium acidity and chemical profiles, underlining the low sensory preference. FLAB
species are known to produce large amounts of mannitol from the metabolism of
fructose. Mannitol is a low-calorie sweetener and can be used for the replacement of

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sugar, in particular for people who suffer from diabetes. An F. tropaeoli strain isolated
from ripe figs produced large amounts of mannitol (81 g/liter) with a high yield (77%)
and was able to grow under high osmotic pressure (79). The strain had pronounced
mannitol 2-dehydrogenase activities. These results suggest that the strain may be
considered a promising candidate for industrial mannitol production. Besides mannitol
formation, certain FLAB strains also produce trace amounts of erythritol (15, 80), which
is a calorie-free polyol that is effective in managing oral health (81).
CONCLUDING REMARKS
FLAB are a rather newly discovered group of bacteria. They possess several charac-
teristics unusual for hitherto known LAB. FLAB seem to have well adapted their
metabolic functions at the genomic level according to the environmental niches they
occupy. Meanwhile, a few studies report on their potential for industrial application.
There are several indicators for their beneficial and health-promoting properties in
animals and humans; however, further studies will be needed to explore this more in
detail.
ACKNOWLEDGMENTS
The present study was supported by JSPS KAKENHI (grants 26850054, 17K19248,
and 16H06279), the MEXT Program for the Strategic Research Foundation at Private
Universities 2013 to 2017 (S1311017), and NIG-JOINT (2016 to 2018).
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