Freeze dryer

The stem and leaves of H. dulcis were obtained from a certified company (Hambakjae Bio Farm Co., Ltd., Jeju Island, South Korea). The identity of H. dulcis was confirmed by a taxonomist (Dr. ZI-Eum Im) and voucher specimens (Voucher number KU-FST-010) were deposited at the Department of Food Science and Technology, Keimyung University, South Korea. The dried and crushed parts of the stem and leaves of H. dulcis were macerated with water (100 g in 1000 ml of water) for 8 h at 100 °C. A novel functional test product (FHDE) was prepared by co-fermenting 50 ml of the concentrated H. dulcis extract (HDE) with probiotics. Briefly, the extract was mixed with glucose (3%) and monosodium L-glutamic acid (5%), and autoclaved at 121 °C for 15 min. Then, Bacillus subtilis HA (KCCM 10775P) starter culture was inoculated and incubated at 42 °C for 3 days. Then, the product was mixed with skim milk (1%, v/v) and glucose (1.5%, v/v) solution. Lactobacillus plantarum EJ2014 (KCCM 11545P) was inoculated and incubated at 30 °C for 7 days for secondary fermentation. Finally, the fermented product was lyophilized via Freeze Dryer at − 70 °C for 3 days (Freeze Dryer, Ilshin BioBase Ltd., Ede, Netherlands; Pilot LP20).

The IBC extract was prepared according to a previously described method [16 (link)]: 300 g of IBC was heated to 105 °C in 3 L of distilled water for 3 h. After cooling at −20 °C for 30 min, the mixture was filtered once through a filter paper (HA-030, Hyundai Micro, Seoul, Korea) and then lyophilized in a freeze dryer (Ilshin BioBase, Gyeonggi-do, Korea) to obtain IBC dry extract. The extract yield was calculated and then re-dissolved in phosphate-buffered saline (PBS) to the desired concentration. It was stored at −20 °C until use.

LPE powder was prepared as follows: Leaves of Potentilla rugulosa Nakai were collected from Yeongwol-gun, Gangwon-do, Republic of Korea, in July 2016 (NIBRGR0000597649) and were identified by Dr. Jong Seok Lee at the National Institute of Biological Resources (Incheon, Korea). Coarse, dried, and grounded samples (100 g) were extracted for 24 h with 1000 mL 70% ethanol for the extraction of both fat-soluble and water-soluble compounds. The extracted material was filtered (Whatman, No. 3, Maidstone, Kent, UK) and concentrated with a rotary evaporator (EYELA, N-3000, Tokyo, Japan). Finally, the extracted material was subsequently dried using a freeze dryer (Ilshin Biobase Co., LTD, Yangju, Korea).

The Chinese yam (D. batatas) was purchased from Taesan-nongjang (Andong, Korea). The Chinese yam tubers were peeled off, and then the flesh and the peel were separated. The peel was washed with water. Then, both the flesh and the peel were cut into slices and dried with a freeze-dryer (Ilshinbiobase, Dongducheon, Korea).

Lentil cultivars used in this research were LG, FG, and LR, which were purchased from Asia Seed (Seoul, Republic of Korea). Seeds were soaked in tap water for 2 h. After removing seed coats, thirty seeds were placed in a plant culture dish (ø 90 mm × h 40 mm) containing four layers of sterilized gauze with 3 mL of distilled water. The seeds were grown for 6 days under different light conditions in a cultivation room with constant temperature and humidity at 26 ± 2 °C and 70 ± 5%, respectively. At 2, 4, and 6 days after germination (DAGs), ten individuals were collected from each dish for a total of four dishes per treatment. All of the lentil seeds and sprouts, including shoot, root, and cotyledon, were lyophilized at −80 °C in a freeze dryer (IlshinBioBase, Dong-ducheon, Republic of Korea).

Curcumin was conjugated with Poly (D, L-lactic-co-glycolic acid) (PLGA) via an ester linkage. We prepared conjugate using N, N’-Dicyclohexylcarbodiimide (DCC) and 4-(Di-methylamino) pyridine (DMAP) as activating agents in dichloromethane (DCM). In the resulting suspension, curcumin was added and stirred for 2 h at room temperature. The reaction mixture was lyophilized in freeze dryer (ILSHIN BIOBASE, Korea) and re-crystallized with DCM to obtain curcumin-PLGA conjugate. The resulting conjugate was characterized by Fourier transform infrared spectroscopy (FT-IR) using FT-IR-2000A, ABB Spectrophotometer (ZrCl₂). For further confirmation of the conjugation, ¹H NMR spectra of PLGA, native curcumin and curcumin-PLGA conjugate were recorded on Bruker, Avance II (500MHz) Bru spectrometer. Chemical shifts were reported as δ (ppm) relative to Tetramethylsilane (TMS) as a standard. Various shifts in the peaks were interpreted for different groups present in the conjugated system.