Amelioration of Alcoholic Hepatic Steatosis in a Rat Model via Consumption of Poly-γ-Glutamic Acid-Enriched Fermented Protaetia brevitarsis Larvae Using Bacillus subtilis

Abstract

Alcoholic hepatic steatosis (AHS) is a common early-stage symptom of liver disease caused by alcohol consumption. Accordingly, several aspects of AHS have been studied as potential preventive and therapeutic targets. In this study, a novel strategy was employed to inhibit fatty liver accumulation and counteract AHS through the consumption of microorganism-fermented Protaetia brevitarsis larvae (FPBs). By using an AHS rat model, we assessed the efficacy of FPB by examining the lipid profile of liver/serum and liver function tests to evaluate lipid metabolism modulation. After FPB administration, the lipid profile-including high-density lipoprotein, total cholesterol, and total triglycerides-and histopathological characteristics exhibited improvement in the animal model. Interestingly, AHS amelioration via FPBs administration was potentially associated with poly-γ-glutamic acid (PγG), which is produced by Bacillus species during fermentation. These findings support the formulation of novel natural remedies for AHS through non-clinical animal studies, suggesting that PγG-enriched FPBs are a potentially valuable ingredient for functional foods, providing an ameliorative effect on AHS.

Keywords: Bacillus subtilis; Protaetia brevitarsis larvae; alcoholic hepatic steatosis; fermentation; lipid metabolism modulation; poly-γ-glutamic acid.

Figure 1

Biological properties of fermented Protaetia brevitarsis larvae (PbsLs) fermented for 3 d using different microbial species: total phenolic compound (a) and flavonoid contents (b). Values are presented as the mean ± standard error (n = 3). Means with the same letter are not significantly different (α = 0.05), as determined using one-way analyses of variance followed by Duncan’s new multiple-range tests. Abbreviations include N, non-fermented; F3, PbsLs fermented by Lactobacillus plantarum JBMI F3; F5, PbsLs fermented by L. plantarum JBMI F5; Ba9, PbsLs fermented by L. gaseri Ba9 F3; Ak, PbsLs fermented by Aspergillus kawachii KCCM 32819; Sc, PbsLs fermented by Saccharomyces cerevisiae KACC 93023; and Bs, PbsLs fermented by Bacillus subtilis KACC 91157.

Figure 2

Effect of fermented Protaetia brevitarsis larvae (PbsLs) on the serum lipid profiles of alcohol-exposed rats: total cholesterol (a), HDL cholesterol (b), LDL cholesterol (c), total lipids (d), free fatty acids (e), triglycerides (f), and atherogenic index (g). Values are presented as the mean ± standard error (n = 6). Means with the same letter are not significantly different (α = 0.05), as determined using one-way analyses of variance followed by Duncan’s new multiple-range tests. Treatments include N, normal; C, control (alcohol only); PC, positive control (silymarin); NPB, non-fermented PbsLs combined with Bacillus subtilis KACC 91157; and FPB100 and FPB400, 100 and 400 mg/kg/d, respectively, of PbsLs fermented by B. subtilis for 3 d.

Figure 3

Effect of fermented Protaetia brevitarsis larvae (PbsLs) on the serum-based liver function indicators of alcohol-exposed rats: alanine aminotransferase (ALT; (a)), aspartate aminotransferase (AST; (b)), alkaline phosphatase (ALP; (c)), lactate dehydrogenase (LDH; (d)), gamma-glutamyl transferase (GGT; (e)), total protein (f), albumin (g), globulin (h), and the albumin/globulin (A/G) ratio (i). Values are presented as the mean ± standard error (n = 6). Means with the same letter are not significantly different (α = 0.05), as determined using one-way analyses of variance followed by Duncan’s new multiple-range tests. Treatments include N, normal; C, control (alcohol only); PC, positive control (silymarin); NPB, non-fermented PbsLs combined with Bacillus subtilis KACC 91157; and FPB100 and FPB400, 100 and 400 mg/kg/d, respectively, of PbsLs fermented by B. subtilis for 3 d.

Figure 4

Effect of fermented Protaetia brevitarsis larvae (PbsLs) on the alcohol (a), acetaldehyde (b), alcohol dehydrogenase (ADH) (c), and aldehyde dehydrogenase (ALDH) (d) and glutathione concentrations in the liver (e) and serum (f) of alcohol-exposed rats. Values are presented as the mean ± standard error (n = 6). Means with the same letter are not significantly different (α = 0.05), as determined using one-way analyses of variance followed by Duncan’s new multiple-range tests. Treatments include N, normal; C, control (alcohol only); PC, positive control (silymarin); NPB, non-fermented PbsLs combined with Bacillus subtilis KACC 91157; and FPB100 and FPB400, 100 and 400 mg/kg/d, respectively, of PbsLs fermented by B. subtilis for 3 d.

Figure 5

Effect of fermented Protaetia brevitarsis larvae (PbsLs) on the malondialdehyde (MDA) content in the liver (a), serum (b), mitochondria (c), and microsome (d) of alcohol-exposed rats. Values are presented as the mean ± standard error (n = 6). Means with the same letter are not significantly different (α = 0.05), as determined using one-way analyses of variance followed by Duncan’s new multiple-range tests. Treatments include N, normal; C, control (alcohol only); PC, positive control (silymarin); NPB, non-fermented PbsLs combined with Bacillus subtilis KACC 91157; and FPB100 and FPB400, 100 and 400 mg/kg/d, respectively, of PbsLs fermented by B. subtilis for 3 d.

Figure 6

Effect of fermented Protaetia brevitarsis larvae (PbsLs) on the triglyceride content (a) of alcohol-exposed rats, along with histopathological images of their hepatic tissue (b) and images of their livers (c). Values presented in (a) are the mean ± standard error (n = 6). Means with the same letter are not significantly different (α = 0.05), as determined using one-way analyses of variance followed by Duncan’s new multiple-range tests. Treatments include N, normal; C, control (alcohol only); PC, positive control (silymarin); NPB, non-fermented PbsLs combined with Bacillus subtilis KACC 91157; and FPB100 and FPB400, 100 and 400 mg/kg/d, respectively, of PbsLs fermented by B. subtilis for 3 d.