Effect of Oral Administration of Weissella confusa on Fecal and Plasma Ethanol Concentrations, Lipids and Glucose Metabolism in Wistar Rats Fed High Fructose and Fat Diet

Figures & data

Table 1 Composition of the Diets Used

Diet Contents	I. (Ssniff NR)	II. LFru-LF	III. MFru-MF	IV. HFru-HF
Product code*	V1444-0	S8318-E001	Mix of II+IV	S8318-E003
Crude nutrients (g nutrient/100 g diet)
Protein (casein; N x 6.25) [%]	26.1	17.6	17.6	17.6
Starch [%]	26.9	53.8	26.9	–
Fructose [%]	–	–	28.0	56.0
Cellulose powder [%]	3.7	15.0	9.0	3.0
Porc lard [%]	–	–	6.0	12.0
Other fat [%]	5.8		4.0	4.0
Vitamin + mineral mix [%]	5.0	5.0	5.0	5.0
Metabolizable energy [MJ ME/kg diet]
Total ME [MJ/kg]	14.3	13.8	16.2	18.7
Thereof from carbohydrates [%]	45	67.4	59.9	51.7
Lipids [%]	14	11.3	21.9	32.5
Protein [%]	41	21.3	18.6	15.8

Note: *(Product code) Given by the diet provider (Ssniff, Soest, Germany).

Abbreviations: Ssniff NR, maintenance diet for nude rats; LFru-LF, low fructose-low fat diet; MFru-MF, medium fructose-medium fat diet; HFru-HF, high fructose-high fat diet; N, measured nitrogen content of the diet; MJ, megajoule; ME, metabolizable energy.

Figure 1 Feeding scheme.

Table 2 Effects of WC NRRL-B-14171^† and of a Medium or High Fat and Fructose Content in the Diet on Body Weight, Parameters of Host Metabolism and Microbial Metabolites in Wistar Rats

Parameter	Control Group (Without WC)			WC Group (+ WC)			WC vs Control	Impact of Fruct + Fat
	nc	LSm	SE	nc	LSm	SE	pvc^#	pd ^##
A) Body parameters
BW [g]	43	390.6	5.1	45	388.9	4.9	0.811	0.000**
BW (week 15) ^††[g]	13	462.4	11.3	14	450.0	6.9	0.277	n.m.^‡‡
B) Liver parameter
Hepatic index [%]	13	3.008	0.092	14	3.293	0.089	0.036*	n.m.
AST [U/L]	26	95.02	11.87	25	83.15	12.10	0.487	0.629
ALT [U/L]	26	40.58	2.968	25	41.10	3.027	0.903	0.205
C) Blood parameters (p = sodium fluoride plasma; s = serum; b = blood)
pGlucose [mg/dL]	14	358.9	17.67	15	358.2	17.07	0.980	n.m.
sTG [mg/dL]	27	263.9	29.25	25	335.5	30.40	0.097^§	0.002**
sCholesterol [mg/dL]	13	83.95	3.414	14	99.39	3.290	0.003**	n.m.
bEthanol [µg/mL]	13	0.115	16.28	14	75.07	15.69	0.003**	n.m.
D) Fecal (F) parameters (1 mL in the concentration units correspondents to 0.1 g fresh feces)
fEthanol [µg/mL]	43	53.19	20.62	43	155.2	20.62	0.001**	0.023*
fADH [U/L]	45	3.233	0.056	45	3.994	0.056	0.000**	0.000**
fAcetate [µg/mL]	43	668.7	83.82	44	1268.7	82.87	0.000**	0.000**
fPropionate [µg/mL]	43	292.1	42.26	44	539.6	41.78	0.000**	0.025*
fButyrate [µg/mL]	43	192.3	43.20	44	400.8	42.71	0.001**	0.000**
fLactate [µg/mL]	43	18.43	10.38	44	39.62	10.26	0.151	0.144
E) Detection of Weissella in rat feces
Week 15 (Ssniff NR)	–
Week 6 (MFru-MF)	–			+
Week 15 (HFru-HF)	+			+

Notes: ^†Addition of 10.56 ± 0.20 log CFU per g diet of lyophilized WC. ^††Weight (week 15) is the (arithmetic) mean weight at the end of the trial. *Significant effect; **Highly significant effect; ^§Significance on one-sided statistical testing. ^#P-value of the comparison of the WC vs control group, p-values below 0.05 are considered significant. ^##P-value of the dose effect of fructose and fat, ie medium versus high fructose and fat diet, p-values below 0.05 are considered significant. Ssniff NR (Product name) given by the provider (Ssniff, Soest, Germany).

Abbreviations: WC, Weissella confusa NRRL-B-14171; fruct, fructose; n_c, number of measured cases; LSm, least-squares means; SE, standard error; BW, body weight; n.m., not measured/measurable; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TG, triglycerides; U/L, units per liter; ADH, alcohol dehydrogenase; Ssniff NR, maintenance diet for nude rats; MFru-MF, medium fructose-medium fat diet; HFru-HF, high fructose-high fat diet; vs, versus.

Figure 2 Effect of WC on ethanol concentrations in blood (A) and feces (B), on alcohol dehydrogenase activity (C) and on fecal acetate (D) in Wistar rats after six weeks of feeding a medium fructose and medium fat diet (MFru-MF) and after further nine weeks on a high fructose-high fat diet (HFru-HF). In this evaluation, only the ten rats were included, of which blood samples were available from both period and for all parameters. The figure shows mean values and standard deviations. The statistical comparison of the control and Weissella group and of the MFru-MF and HFru-HF periods was carried out by paired t-tests, separately for each period, or the control and Weissella group at the different periods, respectively.

Notes: *Denotes statistically significant (P < 0.05) differences from the corresponding values in rats fed a diet without WC. ^#Denotes statistically significant (P < 0.05) differences from the corresponding values of the MFru-MF period.
Abbreviations: ADH, alcohol dehydrogenase; EtOH, ethanol; M/HFru-M/HF, medium (week 6) or high (week 15) fructose and medium (week 6) or high (week 15) fat diet; MFru-MF, medium fructose and medium fat diet; WC, Weissella confusa.

Figure 3 Amplification by PCR of Weissella confusae-specific lepA gene (225 bp) in male rats. (A) Rats were fed for 15 weeks (105 days) standard rat food (Ssniff NR*). Lane 1: Positive control (2.3 ng WC genomic DNA), Lane 2–6: feces samples from rats at day 0, Lane 7–11: feces samples from rats at day 40, Lane 12–16: feces samples from rats at day 106, Lane 17: negative control, L: GeneRuler TM DNA Ladder Mix (Thermo Scientific, Fisher Scientific Co., Pittsburgh, USA). (B) Rats were fed for six weeks (42 days) the MFru-MF diet (28% fructose, 10% fat) and thereafter for nine weeks the HFru-HF diet (56% fructose, 16% fat) according to a total intervention time of 15 weeks (105 days). Lane 1: Positive control (2.3 ng WC genomic DNA), Lane 2–7: feces samples were collected from rats at day 0, Lane 8–13: feces samples were collected from rats at the end of the MFru-MF diet feeding period (day 40), Lane 14–19: feces samples were collected from rats after the HFru-HF diet feeding period (day 106), Lane 20: negative control, L: GeneRulerTM DNA Ladder Mix (Thermo Scientific, Fisher Scientific Co., Pittsburgh, USA). (C) Rats were fed for six weeks (42 days) MFru-MF diet (28% fructose, 10% fat) mixed with WC. Thereafter, rats were accustomed to HFru-HF diet (56% fructose and 16% fat) mixed with WC for eight weeks according to a total intervention time of 15 weeks (105 days). Lane 1: Positive control (2.3 ng WC genomic DNA), Lane 2–7: feces samples were collected from rats at day 0, Lane 8–13: feces samples were collected from rats at the end of the MFru-MF diet feeding period (day 40), Lane 14–17 and lane 19–20: fecal samples were collected from rats after the HFru-HF diet feeding period (day 106), Lane 18: negative control, L: GeneRulerTM DNA Ladder Mix (ThermoScientific, Fisher Scientific Co., Pittsburgh, USA).

Note: *Product name (Ssniff NR) given by the provider (Ssniff, Soest, Germany).
Abbreviations: HFru-HF, high fructose-high fat; MFru-MF, medium fructose-medium fat; PCR, polymerase chain reaction; WC, Weissella confusa.