Prebiotic effect of enzymatically treated citrus polyphenols

Agroindustrial wastes have become a value-added product, due to their rich content of bioactive compounds. Citrus juice by-products (CJB) are source of important molecules, and it is considered an inexpensive source of polyphenols. Usually, phenolic compounds are obtained by chemical synthesis or extraction. An alternative to obtain these molecules is enzymatic treatments, which can increase polyphenol extraction. Biological effects of polyphenols have been widely described, such as their antimicrobial activity. However, new evidence has shown that polyphenols can also have prebiotic effects, as these nutrients are resistant to the digestion process and can improve the proliferation and survival of probiotic and commensal bacteria in the gut. The aim of this study was to investigate the effect of enzymatic treatment on the profile of polyphenols in CJB and its effect on the growth of Lactobacillus acidophilus LA-5.
The enzymatic treatment consisted of β-glucosidase (Sigma-Aldrich) and tannase from Paecilomyces variotii (10 U each), per gram of CJB. The reaction occurred in acetate buffer, at 40 ºC, 120 rpm, for 24 h, along with a non-enzymatic control treatment without enzymes. The main flavanones present in CJB extracts were quantified by HPLC-DAD. The effect of L. acidophilus growth was assayed with an initial inoculum of 105 CFU mL-1, using MRS broth without glucose, at 37 ºC, for 48 h, in anaerobic conditions. The amount of glucose originally present in MRS composition was replaced by citrus extracts (20 mg mL-1) and their effect was measured by plate counting on MRS agar. Glucose (20 mg mL-1), inulin (20 mg mL-1) and the main flavanones present in the extracts (1 mg mL-1) were used as positive controls.
According to HPLC analysis, CJB non-enzymatic (CJB-NE) contained mainly the glycosylated flavanones hesperidin (24.60 µg mg-1) and narirutin (5.64 µg mg-1). In enzymatically-treated CJB (CJB-E), the major compounds were hesperidin (6.58 µg mg-1) and the aglycones, hesperetin (19.53 µg mg-1) and naringenin (2.53 µg mg-1). Thus, these results demonstrate the ability of these enzymes to promote citrus polyphenols deglycosylation, resulting in extracts with modifed composition. Both extracts presented significant stimulatory effect on L. acidophilus La5 survival and proliferation. After 48 h of growth, La5 reached 7.42 log with CJB-NE; 7.64 log with CJB-E; 7.10 log with glucose; 6.94 log with inulin; and 3.29 log in the control (without glucose). The pure flavanones also demonstrated a positive effect on L. acidophilus growth, which reached 5.66 log with naringin; 6.63 log with naringenin; 3.93 log with hesperidin; and 6.71 log with hesperetin. These results show the ability of La5 to metabolize these aglycones, which can occur by C-ring cleavage or their O-methyl hydrolysis of flavonones. Previous publications show that Blautia sp. and Eubacterium ramulus can metabolize naringenin and hesperetin in the gut. However, L. acidophilus ability to metabolize polyphenols was reported only for rutin, anthocyanins, vanillic and tannic acid. These results support the prebiotic effect of citrus polyphenols (mainly aglycones), on this strain of probiotic bacteria, but further research into the metabolism pathway of flavanones is needed.

Financial support: CNPq, Fapesp (grant number 2015/04555-2), Canada Global Affairs