Combined acid hydrolysis and fermentation improves bioactivity of citrus flavonoids in vitro and in vivo

Phytochemicals, particularly citrus flavonoids like naringin and hesperidin, hold promise for improving human health due to their anti-inflammatory and antioxidant properties. However, their bioavailability and bioactivity are limited by conjugation with sugar moieties (neohesperidose or rutinose). These sugars hinder efficient absorption in the small intestine, reducing their effectiveness. This necessitates strategies to improve their bioavailability and bioactivity. One approach is deglycosylation, which increases aglycone levels. While enzymatic deglycosylation using α-L-rhamnosidase and β-D-glucosidase is effective, it is not cost-effective for large-scale applications. Fermentation offers a potentially sustainable and economical alternative. Lactic acid bacteria (LAB), especially *Lactobacillus* species like *Lactiplantibacillus plantarum*, are known for their ability to catabolize flavonoids and are generally recognized as safe. Previous studies have shown that LAB can cleave flavanones, but the conversion process was slow and the aglycone yield was moderate. This research aimed to accelerate the conversion of flavonoid glycosides to aglycones using a combined acid hydrolysis and LAB fermentation approach. The goal was to develop a biotransformation method suitable for food and feed industries, and assess the impact of this biotransformation on the bioactivity of citrus extracts using in vitro and in vivo models. The in vitro models included human intestinal epithelial Caco-2 cells for studying flavonoid transport and uptake; Caco-2 and porcine intestinal epithelial IPEC-J2 cells for investigating oxidative stress and cell migration; and human monocytic THP-1 cells for assessing anti-inflammatory activity. The *Drosophila melanogaster* model was used for in vivo evaluation of intestinal barrier integrity and antioxidant properties.

Numerous studies highlight the health-promoting effects of phytochemicals, especially citrus flavonoids. These flavonoids, including naringin and hesperidin, are naturally found in their glycoside form, where aglycones are attached to sugar moieties. These sugars reduce bioavailability. Previous research demonstrated that enzymatic removal of these sugars improves bioavailability and shifts absorption from the colon to the small intestine. The traditional enzymatic approach utilizes α-L-rhamnosidase and β-D-glucosidase, however this method can be expensive. Fermentation using lactic acid bacteria (LAB) presents a cost-effective and sustainable alternative. Several *Lactobacillus* species have demonstrated the capacity to cleave flavanones; however, the reported conversion times were long and yields were moderate. Existing literature establishes the use of Caco-2, IPEC-J2, and THP-1 cells as in vitro models for studying intestinal processes and inflammation, and *Drosophila melanogaster* as a suitable in vivo model for assessing intestinal barrier integrity and antioxidant properties.

The study involved several key steps. First, the researchers prepared two types of citrus extracts: an aqueous extract (AQE) and a citric acid hydrolyzed extract (CAE). The CAE underwent hydrolysis with citric acid at varying concentrations (0.25 M, 0.5 M, and 1.0 M) at 90°C for four hours to cleave the L-rhamnose group from naringin, neohesperidin, hesperidin, and narirutin. Next, four different LAB strains (*L. plantarum*, *L. rhamnosus*, *L. brevis*, and *L. paracasei*) were screened for their ability to convert naringenin-7-O-glucoside and hesperetin-7-O-glucoside into their respective aglycones using modified MRS broth at 37°C. The effects of glucose addition on bacterial growth and β-glucosidase activity were also evaluated. *L. plantarum* demonstrated the highest aglycone yield after 24 hours of fermentation with CAE. The optimum citric acid concentration for hydrolysis was determined to be 0.25 M. Four final extracts were prepared: AQE, FermAQE (fermented AQE), CAE, and FermCAE (fermented CAE). The flavonoid profiles of these extracts were analyzed by HPLC-UV. Cellular uptake and transport of flavonoids were studied in differentiated Caco-2 cells using AQE and FermCAE. Oxidative stress and cell migration were evaluated in Caco-2 and IPEC-J2 cells using AAPH and tBHP stressors, respectively. The anti-inflammatory activities of the extracts were examined in LPS-stimulated THP-1 cells using cytokine arrays and a multiplex immunoassay. In vivo studies were conducted using *Drosophila melanogaster*. Female fruit flies were subjected to dextran sulfate sodium (DSS)-induced intestinal barrier damage, and the effects of AQE and FermCAE on mortality and intestinal barrier integrity were assessed. Another group of flies underwent oxidative stress induced by ferrous iron supplementation, with ROS levels, metabolic activity, mortality, and climbing performance being measured. HPLC-UV was used extensively for flavonoid quantification throughout the experiments, and various statistical methods were employed to analyze the data.

The combination of 0.25 M citric acid hydrolysis and 24-hour fermentation with *L. plantarum* yielded the highest aglycone levels (a 4.5-fold increase in naringenin and a 3.1-fold increase in hesperetin compared to AQE). Caco-2 cells showed significantly higher uptake and transport of naringenin and hesperetin from FermCAE compared to AQE, indicating improved bioavailability. In vitro studies demonstrated that the aglycones naringenin and hesperetin exhibited stronger antioxidant properties in Caco-2 and IPEC-J2 cells under oxidative stress than their glycosylated counterparts. FermCAE showed the most significant reduction in ROS levels in both cell lines compared to other extracts. In IPEC-J2 cells, FermCAE significantly enhanced cell migration under tBHP stress. The anti-inflammatory effects of FermCAE were also evident in LPS-stimulated THP-1 macrophages, where it reduced the production of various pro-inflammatory cytokines more effectively than AQE. In vivo studies using *Drosophila melanogaster* revealed that FermCAE significantly reduced mortality and intestinal barrier damage induced by DSS and reduced ROS levels and improved climbing performance under ferrous iron-induced oxidative stress, with better protective effects compared to AQE.

The results demonstrate that the combined acid hydrolysis and fermentation method significantly enhances the bioactivity of citrus flavonoids by increasing the concentration of bioavailable aglycones. The superior in vitro and in vivo activities of the biotransformed extracts are attributed to the increased levels of naringenin and hesperetin. These aglycones show better cellular uptake and transport, resulting in enhanced antioxidant and anti-inflammatory effects. The findings are consistent with previous research highlighting the superior bioactivity of aglycones over glycosides. However, it's crucial to note that the impact of glycosylation on flavonoid bioactivity can vary depending on the specific compound and its biological target. The use of citric acid hydrolysis offers a sustainable and cost-effective alternative to enzymatic deglycosylation, making this biotransformation method suitable for industrial applications in food and feed production. The Drosophila model provided valuable in vivo data, supplementing the in vitro findings.

This study successfully demonstrates a novel biotransformation approach for improving the bioactivity of citrus flavonoids. The combined acid hydrolysis and fermentation method significantly increased the aglycone content, leading to enhanced bioavailability and superior antioxidant, anti-inflammatory, and intestinal barrier-protective effects both in vitro and in vivo. This cost-effective and sustainable approach holds significant promise for the development of functional foods and feed supplements. Future research could explore the optimization of this method for other plant-based sources and investigate the long-term effects of these biotransformed extracts on human health.

The study primarily focused on a specific citrus flavonoid complex and a limited number of LAB strains. The in vitro studies used simplified models that may not fully replicate the complexities of the human gastrointestinal tract. While the *Drosophila melanogaster* model provided valuable in vivo data, extrapolation of these findings directly to humans requires further investigation. The HPLC analysis for transport and uptake studies was conducted in HBSS rather than a more realistic gastrointestinal fluid simulant due to limitations in detecting analyte concentrations with purification of interfering compounds.