Probiotic Potential and Cholesterol-Lowering Capabilities of Bacterial Strains Isolated from Pericarpium Citri Reticulatae 'Chachiensis'

Abstract

Pericarpium Citri Reticulatae 'Chachiensis' (PCR-Chachiensis), the pericarps of Citri Reticulatae Blanco cv. Chachiensis, is a food condiment and traditional medicine in southeast and eastern Asia. Its rich and various bacterial community awaits exploration. The present study is the first report on probiotic screening and characterization of bacteria from PCR-Chachiensis. Based on 64 culturable bacterial isolates, 8 strains were screened out to have great survival in the simulated gastrointestinal stressful condition, being nonhemolytic and without biogenic amine formation. They were identified by 16S rRNA gene sequencing as two Bacillus, three Lactobacillus, and three strains from Bacillales. Their probiotic properties, cholesterol-lowering potential and carbohydrate utilization capability were further investigated. Though these eight strains all displayed distinct cholesterol removal potential, Bacillus licheniformis N17-02 showed both remarkable cholesterol removal capability and presence of bile salt hydrolase gene, as well as possessing most of the desirable probiotic attributes. Thus, it could be a good probiotic candidate with hypocholesterolemic potential. Bacillus megaterium N17-12 displayed the widest carbohydrate utilization profile and the strongest antimicrobial activity. Hence, it was promising to be used as a probiotic in a host and as a fermentation starter in fermented food or feed.

Keywords: Bacillus; Lactobacillus; Pericarpium Citri Reticulatae ‘Chachiensis’; cholesterol-lowering potential; probiotic bacteria.

Figure 1

Phylogenetic tree analysis and primary screening of 65 strains isolated from PCR-Chachiensis. The results of hemolytic, gastrointestinal tolerance and the production of amines were respectively represented by symbols from the inside to the outside as square, circle and star. A grey symbol indicates failure and a red one indicates passing the assay.

Figure 2

Amino acid sequence alignment of two putative BSHs, N15-22a and N15-22b, identified in N15-22 and the reference BSHs from NCBI. The closest reference BSHs were KRM94488.1 and KRM93236.1 from Lactobacillus senioris DSM 24302, while AAV42751.1 and AAV42923.1 were two functional BSHs reported from Lactobacillus acidophilus [45]. The identical amino acids are indicated by asterisks; the conservative and semi conservative amino acids are indicated by two dots and one dot, respectively. N in the LTN-conserved motif and R in the RF-conserved motif are the previously described conserved active-site residues.

Figure 3

Cholesterol removal capability of the probiotic strains. (A). Cholesterol removal capability after incubation in cholesterol medium for 6 h, 12 h, 18 h and 24 h. Different superscripts (a–c) represent significantly different values (p < 0.05). (B). Cholesterol removal capability by Lactobacillus strains N15-22, X16-68, and X16-69 in MRS containing cholesterol (MRS-CHO) with or without bile salt. Values are expressed as mean ± SE (n = 3). The asterisk indicated the significantly different values (p < 0.05) between the mediums with or without bile salt for the same strain.