A novel polyphenolic prebiotic and probiotic formulation have synergistic effects on the gut microbiota influencing Drosophila melanogaster physiology

Figures & data

Table 1. Primer sequences for immunological genes in Drosophila.

Gene name	Sequence 5′–3′	Annealing temp.
Duox	F: GCTGCACGCCAACCACAAGAGACT	54 °C
	R: CACGCGCAGCAGGATGTAAGGTTT
IMD	F: TCGAATGCCAATAATCTGCA	52 °C
	R: CGCGATGCTGGGACTCCCAC
Drosocin	F: GCACAATGAAGTTCACCATCGT	56 °C
	R: CCACACCCATGGCAAAAAC
PIMS	F: CCATCTACGCCAGCAAGGA	59 °C
	R: CGATTACTTGCAGTTGCC
Rp49	F: AGATCGTGAAGAAGCGCACCAAG	52 °C
	R: CACCAGGAACTTCTTGAATCCGG

Table 2. Primers for quantification of Drosophila microbiota.

Gene name	Sequence 5′–3′	Annealing temp. (°C)
27F	AGAGTTTGATCMTGGCTCAG	48
1429R	CGGTTACCTTGTTACGACTT
Universal primers (340F/804R)	F: ACCATGCACCACCTGTC	52
	R: GGACTACCAGGGTATCTA
L. plantarum	F: TCCATGTCCCCGAAGGGAACG	57
	R: TGGATGGTCCCGCGGCGTAT
L. breve	F: ACGTAGCCGACCTGAGAGGGT	52
	R: AGCTTAGCCTCACGACTTCGCA
A. malorum	F: CTGGTTTGAGGCTTGAGTGATATG	51
	R: CCATGCGACTAAGTGACACGTCT
A. tropicalis	F: AGGGCTTGTATGGGTAGGCT	50
	R: CAGAGTGCAATCCGAACTGA
A. aceti	F: TGGAGCATGTGGTTTAATTCGA	50
	R: GCGGGAAATATCCATCTCTGAA
A. pasteurianus	F: TCAAGTCCTCATGGCCCTTATG	51
	R: TCGAGTTGCAGAGTGCAATCC
A. pomorum	F: TGTTTCCCGCAAGGGACCTCT	58
	R: AGAGTGCCCAGCCCAACCTGA
A. cerevisiae	F: CTGGTTTGAGGCTTGAGTGATATG	50
	R: CCATGCCACTAAGTGACACTTCC
Enterococcus spp.	F: AGCGCAGGCGGTTTCTTAA	48
	R: CTCGTTGTACTTCCCATTGT
Gluconobacter spp.	F: ATGATGACGGTACCCGTAGA	50
	R: CTAGCCCCCTTCGTATTACC

Table 3. Primers for quantification of human microbiota.

Gene name	Sequence 5′–3′	Annealing temp. (°C)
Firmicutes	F: TGAAACTCAAAGGAATTGACG	48
	R: ACCATGCACCACCTGTC
Bacteriodetes	F: GCACGGGTGMGTAACRCGTAT	52
	R: GTRTCTCAGTDCCARTGTGGG
Prevotella	F: CCAGCCAAGTAGCGTGCA	52
	R: TGGACCTTCCGTATTACCGC
Enterococcus	F: CCCTTATTGTTAGTTGCCATCATT	49
	R: ACTCGTTGTACTTCCCATTGT
Lactobacillus	F: AGCAGTAGGGAATCTTCCA	48
	R: CACCGCTACACATGGAG
Bifidobacteria	F: CTCCTGGAAACGGGTGG	49
	R: GGTGTTCTTCCCGATATCTACA
E. coli	F: CATGCCGCGTGTATGAAGAA	51
	R: CGGGTAACGTCAATGAGCAAA
Ruminococcus	F: GGCGGCYTRCTGGGCTTT	53
	R: CCAGGTGGATWACTTATTGTGTTAA
C. coccoides–E. rectale group	F: CGGTACCTGACTAAGAAGC	45
	R: AGTTTYATTCTTGCGAACG
Enterobacteriaceae	F: ATGGCTGTCGTCAGCTCGT	55
	R: CCTACTTCTTTTGCAACCCACTC
Staphylococcus	F: GGCCGTGTTGAACGTGGTCAAATCA	50
	R: TDACCATTTCAGTACCTTCTGGTAA
Actinobacteria	F: TACGGCCGCAAGGCTA	50
	R: TRCTCCCCACCTTCCTCCG

Figure 1. Total phenolic, flavonoid and tannin constitution in Triphala extracts. Triphala extracts were made using water, water:HCl, methanol (MeOH), MeOH:HCl, ethanol (EtOH) or EtOH:HCl solutions as extract media. Total (a) phenolic, (b) flavonoid, (c) condensed tannin and (d) hydrolysable tannins were assessed using various colourimetric assays. Each group contained n = 3 independent trials while significant differences are indicated by *p < .05 and **p < .01 between groups.

Figure 2. Characterization of the prebiotic activity of Triphala compared to known controls in both aerobic and anaerobic species. The prebiotic activity of Triphala was assessed in a variety of (a) aerobic and (b) anaerobic species using in vitro isolated cultures. Each bacterial strain was incubated in mMRS supplemented with either glucose (dark grey), inulin (light grey), scFOS (medium grey) or Triphala (black) as the only fermentable source of energy. Each group contained n = 5 independent samples and significance is indicated as *p < .05 and **p < .01 between groups.

Figure 3. Toxicity of Triphala in Drosophila melanogaster. After eclosion, Drosophila were placed on media supplemented with different concentrations (0–2% w/v) of Triphala water extracts. A variety of physiological parameters were assessed including (a) average body weight, (b) motility through the negative geotaxis test and (c) longevity. All groups contained n = 5 independent samples and significance is indicated as *p < .05 and **p < .01.

Figure 4. Triphala induces variations in Drosophila gut microbiota populations. After 14 days exposure to the various concentrations (0–2% w/v) of Triphala-treated media, quantification of the Drosophila gutx microbiota was assessed using real-time PCR. Each group contains n = 5 independent samples and significance is marked as *p < .05 relative to the 0% Triphala control.

Figure 5. Triphala is not toxic to variations in weight in Drosophila melanogaster. After eclosion, Drosophila were exposed to media containing a dose-curve of the probiotic bacteria (a) L. plantarum NCIMB 8826 (Lp8826), (b) L. fermentum NCIMB 5221 (Lf5221) and (c) B. longum subsp. infantis NCIMB 702255 (Bi702255) and variations in the Drosophila’s total weight was recorded weekly from Day 0 to Day 56. Each group contained n = 5 independent samples and significance is marked as *p < .05 and **p < .01. Stars indicate significance relative to the normal-media control where bars indicate variations between groups.

Table 4. Motility of Drosophila over time after exposure to various probiotic bacteria.

	CNT	Lp8826			Lf5221			Bi702255
CFU/ml	0	5.0 × 10^8	2.5 × 10^9	7.5 × 10^9	5.0 × 10^8	2.5 × 10^9	7.5 × 10^9	5.0 × 10^8	2.5 × 10^9	7.5 × 10^9
Day 0	4.1 ± 0.3	4.8 ± 0.4	4.4 ± 0.2	4.5 ± 0.3	4.6 ± 0.2	4.6 ± 0.4	4.5 ± 0.2	5.0 ± 0.3	4.8 ± 0.4	4.9 ± 0.3
Day 7	4.9 ± 0.3	5.3 ± 0.5	5.4 ± 0.5	5.1 ± 0.3	5.1 ± 0.2	5.4 ± 0.5	4.9 ± 0.3	4.5 ± 0.2	6.4 ± 0.5	6.3 ± 0.6
Day 14	6.2 ± 0.6	7.0 ± 0.5	7.0 ± 0.6	7.0 ± 0.5	4.9 ± 0.3	6.3 ± 0.6	4.8 ± 0.4	4.9 ± 0.3	6.3 ± 0.3	5.8 ± 0.6
Day 21	8.6 ± 0.5	6.4 ± 0.5	5.0 ± 0.3*	7.2 ± 0.5	7.2 ± 0.4	6.3 ± 0.5*	5.8 ± 0.4*	6.4 ± 0.5	7.5 ± 0.6*	6.0 ± 0.6
Day 28	11.2 ± 0.6	11.0 ± 0.6	11.0 ± 0.4	11.0 ± 0.6	9.6 ± 0.5	7.0 ± 0.7*	6.2 ± 0.6*	7.5 ± 0.6*	7.4 ± 0.5*	6.2 ± 0.6*
Day 35	13.0 ± 0.7	13.0 ± 0.7	8.0 ± 0.3*	9.0 ± 0.3*	10.8 ± 0.6*	8.2 ± 0.6*	7.7 ± 0.4*	7.8 ± 0.7*	7.8 ± 0.7*	9.2 ± 0.4*
Day 42	16.0 ± 0.3	14.8 ± 0.6	9.0 ± 0.7*	9.6 ± 0.8*	10.6 ± 0.5*	10.0 ± 0.5*	10.4 ± 0.9*	11.0 ± 0.7*	10.1 ± 0.7*	9.0 ± 0.3*
Day 49	–	14.8 ± 0.6	10.8 ± 0.5*	12.6 ± 0.5*	11.4 ± 0.5*	10.8 ± 0.8*	10.6 ± 0.5*	14.2 ± 0.4	10.4 ± 0.8*	11.0 ± 0.7*

Significance is indicated as *p < .05 relative to the Day 0 control in the same treatment group.

Table 5. Longevity of Drosophila fed various probiotic bacteria.

CFU/ml	Lp8826		Lf5221		Bi702255
Mortality	50%	100%	50%	100%	50%	100%
Control	31 ± 1.3	44 ± 1.5	31 ± 2.1	44 ± 1.8	35 ± 1.4	47 ± 2.0
1.0 × 10^8	31 ± 1.7	40 ± 2.1	24 ± 2.2	48 ± 3.1	29 ± 2.1	47 ± 2.1
1.0 × 10^9	29 ± 2.4	45 ± 3.2	30 ± 3.1	55 ± 2.1*	27 ± 3.1	53 ± 2.5
2.5 × 10^9	37 ± 3.4*	52 ± 2.8*	46 ± 3.4*	61 ± 4.2*	44 ± 4.1*	59 ± 3.5*
7.5 × 10^9	40 ± 2.8*	49 ± 2.5*	43 ± 2.5*	63 ± 3.4*	42 ± 4.1*	57 ± 3.2*
1.0 × 10^10	43 ± 3.5*	51 ± 3.4*	42 ± 3.1*	65 ± 4.5*	42 ± 4.3*	58 ± 4.0*

The longevity of Drosophila raised on media containing a dose-curve of probiotic bacteria from 1.0 × 10⁸ to 1.0 × 10¹⁰ CFU/ml was assessed by counting the survivability of flies daily until each group had total mortality. The number of days it took for a population of 10 flies to reach both 50% and 100% mortality is outlined.

Each group represents n = 5 independent tests and significance is indicated as *p < .05 relative to Drosophila kept on control media.

Figure 6. Probiotics are not recognized as invading pathogens in Drosophila melanogaster. To assess the immunoreactivity of Drosophila after immediate exposure to the probiotic bacteria, 5-day old Drosophila were transferred to media inoculated with various concentrations of Lf5221. After 1, 3, 6 and 12 h, samples of Drosophila were taken and the expression of the immunological genes (a) immune deficiency (IMD), (b) PGRP-LC-interacting inhibitor of IMD signalling (PIMS), (c) dual oxidase (Duox) and (d) defensin were assessed using real-time PCR. Each group contained n = 5 independent samples and significance is marked as *p < .05 and **p < .01. Stars indicate significance relative to the normal-media control where bars indicate variations between groups.

Table 6. Motility of Drosophila subjected to probiotics and/or prebiotic combinations.

	Control	Lf5221	TFLA	Probiotic	Synbiotic
Day 0	4.1 ± 0.3	4.6 ± 0.2	4.7 ± 0.3	4.4 ± 0.2	4.3 ± 0.2
Day 7	4.9 ± 0.3	5.5 ± 0.4	5.1 ± 0.4	5.5 ± 0.2	4.9 ± 0.5
Day 14	6.2 ± 0.6	6.2 ± 0.5	5.3 ± 0.5	5.4 ± 0.3	4.6 ± 0.4
Day 21	8.6 ± 0.5	6.7 ± 0.3*	6.1 ± 0.4*	5.6 ± 0.4*	5.1 ± 0.5
Day 28	11.2 ± 0.6	7.2 ± 0.5*	6.5 ± 0.6	6.1 ± 0.5**	5.8 ± 0.6**
Day 35	13.0 ± 0.7	8.5 ± 0.5**	7.9 ± 0.4**	6.9 ± 0.4**^,τ	6.5 ± 0.7**
Day 42	16.0 ± 0.3	10.3 ± 0.6**	10.3 ± 0.3**	8.9 ± 0.7 **^,τ	7.1 ± 0.8**^,τ
Day 49	–	10.7 ± 0.4**	11.0 ± 0.4**	9.1 ± 0.5**^,τ	8.7 ± 0.6**^,τ

Newly eclosed Drosophila were placed on media inoculated with Lf5221 (3.0 × 10⁹ CFU/ml), TFLA (0.5%) or their combination and the motility was measured using the negative geotaxis test as the flies aged. The times shown represent the time it took for five flies to reach the 10 cm mark at each time-point.

Each group represents n = 3 independent tests and significance is indicated as *p < .05 and **p < .01 relative to Drosophila kept on control media, and ^τp < .05 as relative to the Day 0 controls in the same treatment group.

Table 7. Variations in the human gut microbiota following prebiotic and/or probiotic treatment.

Group	Section	TFLA	Lf5221	Probiotic	Synbiotic
Bacteriodetes to firmicutes ratio	Ascending	2.1 ± 0.2*	2.4 ± 0.2*	4.6 ± 0.6*	4.3 ± 0.2**
	Transverse	3.6 ± 0.2*	1.8 ± 0.3*	3.2 ± 0.2*	6.9 ± 0.5**
	Descending	2.6 ± 0.1*	1.7 ± 0.2*	3.0 ± 0.4*	5.5 ± 0.4**
Actinobacteria spp.	Ascending	2.6 ± 0.2*	1.7 ± 0.2*	2.0 ± 0.2*	1.1 ± 0.09
	Transverse	1.1 ± 0.1*	0.56 ± 0.1	3.3 ± 0.3*	5.2 ± 0.7**
	Descending	2.3 ± 0.2*	1.0 ± 0.09	6.2 ± 0.5*	3.2 ± 0.6*
Bifidobacteria spp.	Ascending	3.5 ± 0.3*	1.8 ± 0.2*	3.8 ± 0.4**	2.2 ± 0.1*
	Transverse	5.9 ± 0.4**	1.9 ± 0.3*	2.4 ± 0.5*	3.2 ± 0.2*
	Descending	1.8 ± 0.2*	1.1 ± 0.4	2.8 ± 0.3*	6.1 ± 0.3**
Lactobacillus spp.	Ascending	1.8 ± 0.1*	2.7 ± 0.3*	3.6 ± 0.4**	9.6 ± 0.7**
	Transverse	1.4 ± 0.1	1.9 ± 0.1*	14.9 ± 0.4**	33.7 ± 0.1**
	Descending	4.3 ± 0.3*	2.5 ± 0.5*	4.4 ± 0.3**	29.2 ± 0.4**
Prevotella spp.	Ascending	4.6 ± 0.3*	0.7 ± 0.1	1.7 ± 0.3	3.9 ± 0.3*
	Transverse	5.7 ± 0.2*	0.43 ± 0.08*	1.5 ± 0.2	4.4 ± 0.2*
	Descending	4.6 ± 0.1*	0.86 ± 0.09	5.7 ± 0.5*	2.0 ± 0.1*
Ruminococcus spp.	Ascending	1.9 ± 0.1*	1.5 ± 0.2	0.70 ± 0.01	2.5 ± 0.5*
	Transverse	2.3 ± 0.1 *	1.3 ± 0.2	3.4 ± 0.2*	7.4 ± 0.5**
	Descending	2.3 ± 0.2*	3.3 ± 0.3*	4.5 ± 0.6*	2.3 ± 0.1*
Clostridium cluster XIVa	Ascending	2.0 ± 0.1*	1.0 ± 0.3	1.8 ± 0.5	3.4 ± 0.5**
	Transverse	3.2 ± 0.2*	3.1 ± 0.5*	1.8 ± 0.1	3.3 ± 0.5**
	Descending	1.7 ± 0.3	1.7 ± 0.4	1.8 ± 0.3	5.7 ± 0.6**
Enterococcus spp.	Ascending	0.18 ± 0.02**	0.97 ± 0.1	1.1 ± 0.08	0.61 ± 0.2**
	Transverse	0.32 ± 0.04**	1.84 ± 0.3*	0.49 ± 0.03**	0.16 ± 0.07**
	Descending	0.16 ± 0.3**	3.08 ± 0.4**	0.44 ± 0.07**	0.56 ± 0.1**
Staphylococcus spp.	Ascending	0.79 ± 0.09	0.89 ± 0.1	0.24 ± 0.04**	0.19 ± 0.04**
	Transverse	0.39 ± 0.02**	0.91 ± 0.09	0.96 ± 0.07	0.52 ± 0.07**
	Descending	0.12 ± 0.05**	0.89 ± 0.1	0.29 ± 0.04**	0.46 ± 0.09**
Escherichia coli	Ascending	1.2 ± 0.1	0.22 ± 0.06**	0.12 ± 0.06**	0.67 ± 0.2**
	Transverse	0.6 ± 0.1*	0.31 ± 0.07**	0.27 ± 0.02**	1.1 ± 0.1
	Descending	0.1 ± 0.07**	0.34 ± 0.06**	0.27 ± 0.04**	0.31 ± 0.1**

Using an in vitro model of the human GIT (SHIME), the variations in the gut microbiota were evaluated after treatment with either Lf5221 (3.0 × 10⁹ CFU/ml), TFLA (0.5%) or the probiotic or synbiotic formulations over the course of 3 weeks. Quantification of the gut microbiota is represented as the ratio of relative gene expression between Day 21 and Day 0.

Each group contains n = 3 independent samples and significance is marked as *p < .05 and **p < .01.

Figure 7. The probiotic and/or prebiotics alone or in combination alter the composition of the Drosophila microbiota. Variations in the aging Drosophila gut microbiota were assessed in flies inoculated with each of the individual probiotics (Lf5221, Lp8826 or Bi702255), TFLA or the probiotic or synbiotic formulations. Variations in the gut microbiota are represented by the ratio of relative gene expression from Day 0 to Day 30. Bacterial populations examined include (a) A. malorum, (b) A. pasteurianus, (c) A. aceti, (d) A. tropocalis, (e) A. cerevisiae, (f) L. breve, (g) L. plantarum and (h) Gluconobacter spp. Each group contained n = 5 independent samples and significance is marked as *p < .05 and **p < .01. Stars indicate significance relative to the normal-media control where bars indicate variations between groups.

Figure 8. The probiotic and synbiotic formulations alter SCFA production in a humanized model of the GIT. Supernatant from the humanized in vitro model of the GIT was isolated and the levels of key SCFAs (a) acetate, (b) propionate and (c) butyrate were assessed using isocratic HPLC analysis in the ascending, transverse and descending colon compartments. Quantification is represented as the change in SCFA production from Day 0 to Day 21. Each group contains n = 5 independent samples and significance is marked as *p < .05 and **p < .01. Stars indicate significance relative to the normal-media control where bars indicate variations between groups.