Effects of Ligustrum robustum on gut microbes and obesity in rats

Table 1 Culture media and incubation conditions

Bacterial group	Bacteria preparation		MIC
	Medium	Incubation conditions	Medium	Incubation conditions
Bacteroides	GAM1 agar	37 °C anaerobic, 48 h	BHI2 (10% HS4)	37 °C Anaerobic, 48 h
Bifidobacteria	BBL2 agar	37 °C anaerobic, 48 h	RC2	37 °C Anaerobic, 48 h
Lactobacilli	LBS2 agar	37 °C aerobic with 5% CO2 48 h	RC2	37 °C Anaerobic, 48 h
C. perfringens	SPS2 agar	37 °C anaerobic, 48 h	RC2	37 °C Anaerobic, 48 h
Aerobic E. coli	EMB2 agar	37 °C aerobic, 18 h	MH2	37 °C Aerobic, 18 h
Enterococci	BEA2 agar	37 °C aerobic, 18 h	MH2	37 °C Aerobic, 18 h
Candida albicans	Salouraud2 agar	37 °C aerobic, 18 h	Salouraud2	37 °C Aerobic, 18 h
Staphylococcus aureus	blood plate3	37 °C aerobic, 18 h	MH2	37 °C Aerobic, 18 h
Other bacteria	LB1 agar	37 °C aerobic, 18 h	MH2	37 °C Aerobic, 18 h

¹Prepared in the laboratory according to methods defined by the Chinese Ministry of Health[8];

²Purchased from Beijing Land Bridge Technology Co., Ltd., China;

³Purchased from Chengdu RICH Technology Co., Ltd., China;

⁴Purchased from Hyclone. MIC: Minimum inhibitory concentration; GAM: Gifu anaerobic medium; BBL: Bifidobacterium culture; LBS: Lactobacillus selection; SPS: Sulfite polymyxin-sulfadiazine; EMB: Eosin methylene blue; BEA: Bile esculin azide; LB: Luria-Bertani; BHI (10% HS): Brain heart infusion with 10% horse serum; RC: Reinforced Clostridium medium; MH: Mueller-Hinton medium.

Table 2 Primers for target gut bacteria

Target bacteria (amplicon size, bp)	Sequence (5’ to 3’)
Bifidobacteria sp. (243)	F: TCGCGTC(C/T)GGTGTGAAAG
	R: CCACATCCAGC(A/G)TCCAC
Lactobacilli sp. (341)	F: AGCAGTAGGGAATCTTCCA
	R: CACCGCTACACATGGAG
Clostridium perfringens groups (120)	F: ATGCAAGTCGAGCGA(G/T)G
	R: TATGCGGTATTAATCT(C/T)CCTTT
Enterobacteriaceae (195)	F: CATTGACGTTACCCGCAGAAGAAGC
	R: CTCTACGAGACTCAAGCTTGC
Enterococci sp. (144)	F: CCCTTATTGTTAGTTGCCATCATT
	R: ACTCGTTGTACTTCCCATTGT
Bacteroidetes (126)	F: GGARCATGTGGTTTAATTCGATGAT
	R: AGCTGACGACAACCATGCAG
Firmicutes (126)	F: GGAGYATGTGGTTTAATTCGAAGCA
	R: AGCTGACGACAACCATGCAC

F: Leading strand; R: Lagging strand.

Table 3 Richness, Shannon-Weiner diversity indices, and evenness

Group	n	Richness				Shannon-Weiner				Evenness
		Hae III-FAM	Hae III-HEX	Hha I-FAM	Hha I-HEX	Hae III-FAM	Hae III-HEX	Hha I-FAM	Hha I-HEX	Hae III-FAM	Hae III-HEX	Hha I-FAM	Hha I-HEX
LR-L	10	44.80 ± 8.64ac	32.50 ± 5.20ac	44.90 ± 14.90	24.00 ± 7.77	3.16 ± 0.31ac	2.76 ± 0.22ac	3.23 ± 0.20a	2.36 ± 0.16	0.84 ± 0.07	0.80 ± 0.05	0.86 ± 0.03a	0.76 ± 0.05
LR-M	10	31.70 ± 8.08	26.00 ± 5.68	39.40 ± 5.32	25.00 ± 4.99	2.93 ± 0.25	2.54 ± 0.18c	3.19 ± 0.11a	2.34 ± 0.15	0.85 ± 0.04	0.79 ± 0.05	0.87 ± 0.03a	0.73 ± 0.05
LR-H	10	31.80 ± 11.79	24.50 ± 4.58	37.00 ± 7.56	26.40 ± 6.11	2.88 ± 0.38	2.50 ± 0.25c	3.04 ± 0.18	2.51 ± 0.22	0.85 ± 0.04	0.79 ± 0.06	0.85 ± 0.04	0.77 ± 0.07
Normal	9	29.11 ± 10.40	21.56 ± 8.17	35.89 ± 7.03	21.44 ± 4.50	2.66 ± 0.25	2.28 ± 0.23a	3.09 ± 0.22	2.35 ± 0.16	0.81 ± 0.08	0.77 ± 0.09	0.87 ± 0.04a	0.77 ± 0.06
HFD	9	33.56 ± 11.20	27.11 ± 5.80	38.67 ± 12.75	24.78 ± 6.36	2.80 ± 0.47	2.50 ± 0.20c	2.95 ± 0.39	2.38 ± 0.23	0.81 ± 0.06	0.76 ± 0.06	0.82 ± 0.04c	0.75 ± 0.08

S (Richness) = Σpi; H (Shannon-Weiner diversity index) = -ΣpiLnpi; D (Simpson index) = 1 - Σpi²; E (Evenness) = H/Hmax (Hmax = Ln S).

^aP < 0.05 vs HFD group;

^cP < 0.05 vs Normal group. Normal: Normal chow diet-fed mice; HFD: High-fat diet-fed control mice; LR-L/M/H: Low-, medium- and high-dose of Ligustrum robustum aqueous extract-treated HFD rats.