Fructophilic Lactic Acid Bacteria, a Unique Group of Fructose-Fermenting Microbes

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).

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/acetaldehyde dehydrogenase (adhE). This results in the imbalance of NAD/NADH and the requirement of additional electron acceptors to metabolize glucose. Oxygen, fructose, 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 fermentations. 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. Besides their impact on insect health, FLAB may be promising candidates for the promotion of human health. Further studies are required to explore their beneficial properties in animals and humans and their applications in the food industry.

INTRODUCTION

Lactic 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 unique stress conditions and nutrient compositions, forced these bacteria to develop specific physiological and biochemical characteristics. Examples are proteolytic and lipolytic activities to obtain nutrients from milk (1), tolerance to phytoalexins in plants (2), or tolerance to bile salts in the gastrointestinal tract (3). In general, such highly adapted microorganisms are in several instances suitable starter cultures in food fermentations and probiotic candidates for humans and animals (4–6).

Fructophilic LAB (FLAB) are a special group of LAB that were described only recently (7). This group is easily differentiated from other LAB on the basis of a few unique biochemical characteristics. The optimum substrate for the growth of FLAB is fructose, and unlike other LAB, growth on glucose is very poor (7). It is thus not surprising to find 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, 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 pseudoficulneum 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 characteristics 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 Fructobacillus was proposed for the reclassification of four Leuconostoc spp. on the basis of their phylogenetic positions and biochemical and morphological characteristics (17). 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).

FIG 1.

Phylogenetic relationships between FLAB and related LAB species based on the multiple alignments 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 100933^T was used as an out group. This is an update of results presented in a previously published paper (29) and includes Lactobacillus spp.

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 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 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 heterofermentative 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.

TABLE 1.

ANI values obtained between L. kunkeei strains and L. apinorum^a

Species	Strain	L. kunkeei																L. apinorum
		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
	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.

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 products 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 dehydrogenase (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 include an ethanol production system, and the NAD/NADH balance is well kept. Thus, homofermentative LAB do not have fructophilic characteristics.

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).

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 carbohydrates (7, 21); hence, carbohydrates metabolized in the API50CHL test are usually two to four. These poor carbohydrate utilization properties are due to their genetic characteristics, 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 heterofermentative 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 activities, suggesting possible beneficial roles of FLAB in food fermentation.

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).

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.

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 Leuconostoc (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 carbohydrate 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 carbohydrate 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, recombination, and repair, and (iv) intracellular trafficking, secretion, and vesicular transport are comparable between Fructobacillus spp. and Leuconostoc spp. (29). The conservation of genes in these classes against genome reduction may indicate that their functions are essential for the reproduction of the organisms.

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.

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 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 Leuconostoc 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 Fructobacillus, as described elsewhere.

TABLE 2.

Discriminative pathways between FLAB and related LAB groups^a

Pathway definition (KEGG no.)	No. of genes (mean [SD]) for groupb:
	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 biosynthesis (map00130)	1 (0)	2 (0.5)	2	0	8 (1.0)	2 (3.4)	0 (0)
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.

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 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 phylogenetic 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. sanfranciscensis and L. florum (22, 44). Certain species are strongly linked with specific environments, e.g., L. sanfranciscensis in sourdough (44), L. lindneri in beer (41), L. homohiochii 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. Heterofermentative 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).

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.

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 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.

FIG 6.

Currently known habitats of FLAB.

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 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 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, paratransgenesis is an interesting application tool for FLAB in honey bees. This approach uses genetically transformed symbiotic microorganisms as vectors to control certain diseases 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 consumption 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 fermentation, 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 investigated 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 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 characteristics 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.