Sequentially fermented dealcoholized apple juice intervenes fatty liver induced by high-fat diets via modulation of intestinal flora and gene pathways

Highlights

• S. cerevisiae and L. plantarum sequentially fermented dealcoholized apple juice has the effect of reducing fat and regulating fatty liver.

• Long-term consumption of low-alcohol cider under high-fat diet can aggravate fatty liver and inflammation of the body.

• S. cerevisiae and L. plantarum sequentially fermented dealcoholized apple juice reduce inflammation by regulating the LPS/LBP/CD14/TLR4 signaling pathway.

• Genes MBOAT7, TMC4, CYP2E1 and CYP7A1 have potential mechanisms for lipid accumulation and fatty liver regulation.

• S. cerevisiae and L. plantarum sequentially fermented dealcoholized apple juice enrich the gut microbial abundance and short-chain fatty acid content.

Abstract

Low-alcohol beverages damage the liver, whereas dealcoholized apple juice sequentially fermented by Saccharomyces cerevisiae and Lactobacillus plantarum is a promising dietary intervention for hyperlipidaemia as a functional non-alcoholic beverage that lowers lipid levels and regulates fatty liver. However, their mechanisms of action have not been identified. In this study, we found that low-alcohol cider exacerbated inflammation in mice on a high-fat diet, up-regulate fatty liver CYP2E1 gene, and inhibit the expression of MBOAT7 and TMC4. Apple juice traditionally fermented by S. cerevisiae and then dealcoholized, followed by sequential fermentation by L. plantarum, can improve obesity and fatty liver, reduce the production of liver cholesterol and fat accumulation, and promote the production of short-chain fatty acids. Our research demonstrates that the lipopolysaccharide/lipopolysaccharide-binding protein/cluster of differentiation 14 protein/Toll-like receptor 4 protein signaling pathway affects the occurrence of obesity and inflammation in mice, and the expression of CYP7A1 inhibits the production of lipids. Further research reveals that L. plantarum sequentially fermented dealcoholized apple juice not only regulate and restore the intestinal flora, but also change the ratio of Firmicutes-to-Bacteroides, and decreased endotoxin-bearing Proteobacteria levels. Together, this functional product may be a potential dietary strategy to interfere with hyperlipidemia and obesity-related metabolic disorders.

Introduction

The excessive intake of calories and fat associated with improvements in people's quality of life and changes in dietary structure causes fat accumulation in the liver and abnormal lipid synthesis, leading to obesity. Obesity often leads to hyperlipidemia, non-alcoholic fatty liver disease (NAFLD), insulin resistance and metabolic syndrome(Younossi, 2019). According to the World Health Organization, obesity is a major disease: the total number of people who die of hyperlipidemia and related diseases worldwide is approximately 17 million annually. It is estimated that by 2030, that number will rise to 23.6 million. The increase in adipose tissue cells caused by obesity is closely related to NAFLD (Cohen et al., 2011, Wree et al., 2013) and threatens human health.

High-fat diets (HFDs) lead to the enlargement and differentiation of fat cells in mice. With the synthesis and release of lipopolysaccharide (LPS) and lipopolysaccharide-binding protein (LBP) (Ceccarelli et al., 2015), LPS in the intestine can enter the blood through the gastrointestinal wall, interacting with LBP in the serum. LBP delivers LPS to cluster of differentiation 14 (CD14) protein on the cell membrane and soluble CD14 in the serum, to form an LPS/LBP/CD14 complex. TLR4 is the receptor or signal transduction molecule of LPS, LPS interacts with the TOLL-like receptor 4 (TLR4) of the cell membrane to transmit the stimulatory signal into the cell, resulting in the expression of a large amount of inflammation factors (interleukin 6 [IL 6], tumor necrosis factor alpha [TNF α], etc.) and chronic systemic inflammation (Ryu et al., 2017, Cremonini et al., 2022). TNF can regulate the expression of cytochrome P450 family 7 subfamily A member 1 (CYP7A1) through TNF receptors and various signaling pathways. Inhibition of CYP7A1 causes an accumulation of metabolic intermediates in the mevalonate pathway, leading to elevated blood lipids(Okin & Medzhitov, 2016), and ultimately causing obesity, insulin resistance and NAFLD(Chambers et al., 2019, Soares et al., 2010). The LPS/LBP/CD14/TLR4 signaling pathway is also key in intestinal flora imbalance, which leads to obesity (Ceccarelli et al., 2015).

Among obese people, a large proportion drink alcohol, and the number of such individuals has increased greatly. An HFD can cause metabolic disorders and metabolic impairment(Despres & Lemieux, 2006). According to the World Health Organization, alcohol is a carcinogen and consumption thereof is the main risk factor for many diseases(Wood, Kaptoge, & Butterworth, 2018). Alcohol decreases gray matter in the brain(Topiwala, Ebmeier, Maullin-Sapey, & Nichols, 2021). Experiments in mice have shown that excessive drinking for 2–3 weeks damages the body's immune system and can cause alcoholic fatty liver disease(Y. Y. Li & Zhou, 2017); this disease manifests as steatosis and inflammatory infiltration, and develops into fibrosis or cirrhosis with continued drinking(Armstrong & Guo, 2019). Studies have found that two genes, membrane-bound O-acyltransferase domain-containing 7 (MBOAT7) and transmembrane channel-like 4 (TMC4), are associated with alcoholic cirrhosis, NAFLD, non-alcoholic steatohepatitis and liver fibrosis in vivo (Buch et al., 2015, Helsley et al., 2019, Luukkonen et al., 2016, Mancina et al., 2016, Viitasalo et al., 2016). Other research has demonstrated that cytochrome P450 family 2 subfamily E member 1 (CYP2E1) plays a critical role in NAFLD, participates in lipid metabolism and causes various forms of liver cell damage and cell death (K. Y. Wang et al., 2021, Leung and Nieto, 2013). Therefore, suppressing the overexpression of CYP2E1 can be effective for treating NAFLD.

Apples can lower blood lipids (Koutsos et al., 2020); our previous research found that alcohol-free apple juice fermented by Saccharomyces cerevisiae can be used as a substrate for fermentation by Lactobacillus plantarum. Moreover, sequential fermentation of alcohol-free apple juice with mixed bacteria increases the polyphenol content and improves antioxidant activity(H. C. Li et al., 2021), where the γ–aminobutyric acid and short-chain fatty acids (SCFAs) produced by fermentation have a lipid-lowering effect(Feng, Zhang, Mujumdar, & Gao, 2017). Studies have found that the SCFAs obtained by fermentation with L. plantarum can upregulate genes related to lipid oxidation and thermogenesis, reducing the likelihood of metabolic diseases caused by HFD(Cani et al., 2008) and thus preventing obesity (Den Besten et al., 2015; X. Wang et al., 2022). The abundance of intestinal flora is correlated with serum cholesterol and blood sugar levels (Dao et al., 2016), and intestinal microbes can promote intestinal peristalsis and inhibit the growth of pathogenic bacteria. Therefore, maintaining the intestinal microecological balance is critical to human health(Fetissov, 2017). However, it is not clear that the product obtained by fermenting apple juice with S. cerevisiae, and then removing ethanol and sequentially fermenting with L. plantarum, has a lipid-lowering effect in people with an HFD, and its effect on intestinal microflora still needs to be explored.

No research has addressed the effect of long-term consumption of low-alcohol fruit wine on people with HFDs, or the mechanisms by which “S. cerevisiae and L. plantarum sequentially fermented non-alcoholic apple juice” (SLSFAJ) lowers lipids or improve the fatty livers and intestinal flora of people with HFDs. Therefore, in this study, we focused on the mechanism underlying the harm caused by long-term consumption of low-alcohol fruit wine by people with a HFD. We investigated the effect of SLSFAJ on the blood lipid-lowering mechanism, SCFAs and intestinal microbial population in C57BL/6 mice fed an HFD, and studied the regulation of the LPS/LBP/CD14/TLR4 signaling pathway in a hyperlipidemia model, as well as the effects of genes MBOAT7, TMC4, CYP2E1 and CYP7A1 on NAFLD and alcoholic fatty liver disease. This study aimed to provide an economical, safe and effective dietary intervention (SLSFAJ) as an adjuvant treatment for HFD-induced obesity and NAFLD.