Mrs broth

B. bifidum E3 used in the current study was provided by Northeast Agricultural University (Harbin, China) and inoculated (3% v/v) into the MRS broth (Hopebio Company, Qingdao, China, HB0384-5) supplemented with 2% v/v L-cysteine hydrochloride (Biotopped Technology Co., Ltd., Beijing, China) at 37 °C for 24 h under the anaerobic condition. The strain was subcultured twice and then centrifuged (5000× g) for 10 min. The experimental supernatant was CFS. The precipitates from centrifugation were washed with PBS buffer three times, and B. bifidum E3 was resuspended with PBS (pH 7.4) buffer to achieve a final concentration of 1 × 10⁹ CFU/mL. The precipitates of the strains were the IC of the experiment. CFE was extracted by ultrasonic crushing of the IC. The treatment was performed under ultrasonic conditions at a power of 800 W for 3 s, stopped for 5 s, and continued for 15 min. All experimental samples were directly used for follow-up experiments.

The frozen strains F17 and H52 were removed from a –80 °C ultra-low temperature freezer (DW-HL668, Zhongke MeiLing Cryogenics Company, Ltd., Hefei, China) and thawed at room temperature, and then cultured in Man–Rogosa–Sharpe (MRS) broth (Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) and incubated at 37 °C for 24 h. The bacteria were transferred to fresh MRS broth and incubated at 37 °C for 24 h, then the bacteria count of both F17 and H52 broths was adjusted to 1 × 10⁸ colony forming units (CFU)/mL using a UV/vis double beam U-2910 spectrophotometer (Hitachi, Tokyo, Japan). After that, the fermentation supernatant was separated from the bacteria by centrifugation at 8000× g for 15 min at 4 °C with a cryogenic high-speed Allegra 64R centrifuge (Beckman Coulter, Brea, CA, USA). The bacterial precipitate was discarded and the supernatants containing the products of strains F17 and H52 were stored at 4 °C until use.

Sx YCC3 and Lp MSZ2 were isolated from traditional Chinese fermented foods, identified by 16S rDNA sequencing, and stored at the Meat Laboratory Center of Shanxi Agricultural University of China. Sx YCC3 was cultured in Mannitol Salt Broth at 37 °C for 48 h and Lp MSZ2 was cultured in de Man, Rogosa and Sharpe (MRS) Broth (Qingdao Hope Bio-Technology Co. Ltd., Qingdao, China) at 37 °C for 48 h. The Mannitol Salt Broth was formulated according to the method that was reported by Chen et al. [20 (link)]. The bacterial cells were collected by centrifuging at 1000× g for 5 min and then the bacterial cells were washed twice with 0.85% saline water and stored at 4 °C for subsequent use.

Defatted Antarctic krill powder was provided by Zhejiang Hailisheng Biotechnology Co., Ltd. (Zhoushan, China), with a proximate composition as follows: moisture (8.29%), protein (78.33%), fat (1.17%), sugar (0.84%), and ash (4.62%). Neutrase (5 × 10⁴ U/g), Alcalase (2 × 10⁵ U/g), Papain (1 × 10⁵ U/g), Trypsin (2.5 × 10⁵ U/g), Protamex (1 × 10⁵ U/g), the BCA protein assay kit, the β-GAL assay kit, and the LDH assay kit were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). L. rhamnosus ATCC7469 was provided by Shanghai Luwei Microbial Sci. &Tech. Co., Ltd. (Shanghai, China). MRS broth and MRS agar were purchased from Qingdao Hope Bio-Technology Co., Ltd. (Tsingtao, China). The other reagents and chemicals were of analytical grade.

Bifidobacterium animalis KV9 (KV9) and Lactobacillus vaginalis FN3 (FN3) were stored in the Functional Dairy and Probiotic Engineering Laboratory of Ocean University of China. The strains were cultured successively twice in de Man, Rogosa, and Sharpe (MRS) broth (Qingdao Hope Bio-Technology Co., Ltd., China) before use, then incubated anaerobically at 37°C for 24 h. The bacteria were washed twice by sterile phosphate-buffered saline (PBS) with pH 7.2 and collected by centrifugation and resuspended in PBS to give a concentration of 5×10⁸ colony-forming units (CFU)/ml.