Malic acid

All the solvents or reagents used were of HPLC or analytical grade. Ethanol, FeCl₃·6H₂O, HCl, K₂S₂O_8, and acetic acid were purchased from Titan Scientific Co., Ltd. (Shanghai, China). Gallic acid was purchased from Energy-Chemical Co., Ltd. (Shanghai, China). ChCl and xylitol were obtained from Adamas Co., Ltd. (Shanghai, China). Na₂CO₃ was purchased from Sinopharm Chemical Reagent (Shanghai, China). D-sorbitol, glucose, maleic acid, malonic acid, malic acid, sucrose, and Folin–Ciocalteu’s phenol reagent were all obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Citric acid, ethylene glycol (EG), and 1,2-propanediol (PD) were purchased from TCI Shanghai (Shanghai, China). Lactic acid and sodium acetate were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Glycerol was purchased from MP Biomedicals Co., Ltd. (Shanghai, China). 2, 4, 6-Tri(2-pyridyl)-s-triazine (TPTZ), [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Aldrich (St. Louis, MO, USA). Commercial standards of catechins [(−)-EC, (−)-EGC, (−)-ECG, and (−)-EGCG, purity ≥ 98%] were purchased from Chengdu RefMedic Biotech Co., Ltd. (Chengdu, China).

The fresh hybrid Pennisetum was harvested from a commercial plantation base located in Meizhou City, Guangdong Province (115.82°E, 24.52°N). Plants were cut down at a height of 2–2.5 m and chopped into pieces (1–2 cm per segment) via a fully automatic grass shredding machine. The chemical composition of hybrid Pennisetum was determined (Table 1). After moderate drying, the chopped hybrid Pennisetum was divided into five treatments and ensiled with (1) no additive (CK), (2) 1% malic acid (purity ≥ 99.5%; Shanghai Macklin Biochemical Co., Ltd., Shanghai) of fresh matter (MA), (3) 1% glucose (purity ≥ 99.5%; Shanghai Macklin Biochemical) of fresh matter (GL), (4) 100 U/g cellulase (VTR Bio-Tech Co., Ltd., Zhuhai, Guangdong) of fresh matter (CE), and (5) 10⁶ cfu/g Bacillus subtilis (VTR Bio-Tech) of fresh matter (BS). After adequate mixing, approximately 200 g hybrid Pennisetum was packed into a polyethylene bag (20 cm × 30 cm), compacted, and sealed via an automatic vacuum packager. Six replicates were set in a treatment; 30 bags were obtained and preserved at ambient temperature (25–28°C). After a 120-day fermentation, all 30 packages were opened for subsequent experiments, including the determination of fermentation parameters and chemical compositions and the analyses of bacterial community and metabolic profile.