Millex syringe driven filter unit

One hundred mg of caecum contents from each were homogenized in MQ water (100 mg/mL) by vortex for 30 s, and then centrifuged at 8000 rpm for 10 min at room temperature to get supernatant. A mixture of 100 μL of the caecum supernatant and 200 μL of 0.25 mM 2-ethylbutyric acid as internal standard were labelled with 2-nitrophenyl hydrazide (2-NPH) using a short- and long-chain fatty acid analysis kit (YMC Co., Ltd., Kyoto, Japan) according to the manufacturer’s manual. 2-NPH labelled caecum supernatant was filtered through a 0.45 μm syringe filter (Millex Syringe Driven Filter Unit, MERCK, Darmstadt, Germany), and then injected into a high-performance liquid chromatography (HPLC) system (JASCO Corporation, Tokyo, Japan) with a YMC-Pack FA column (250 × 4.5 mm; YMC Co., Ltd. Tokyo, Japan). Mobile phase was acetonitrile-methanol-water (30:16:54, v/v/v), which adjusted to pH 4.5 by 0.01 M of hydrochloric acid. The injection volume was 20 μL and flow rate was kept at 1 mL/min and column oven temperature was set as 50 °C.

All procedures relevant to animals used in this study were carried out in accordance with the UK Animals (Scientific Procedure) Act 1986 and were approved by the University of Hull Ethical Review Process (No. PPL 70/7966). Cardiac, skeletal, liver and kidney tissues from male Sprague-Dawley rats were washed for excess blood, freeze clamped with Wollenberger tong cooled in liquid nitrogen and stored at −80 °C. Tissues were subsequently weighed and powdered in liquid nitrogen according to Seymour et al. [40 (link)]. At time of tissue harvesting, blood samples were taken, and a portion centrifuged at 4 °C. The serum was obtained and whole blood was snap frozen in liquid nitrogen and stored at −80 °C for future glutathione analysis.
Powdered tissues, whole blood and serum were subjected to 6% PCA extraction [51 (link)] and neutralised with 6 M potassium hydroxide (KOH). The resultant supernatant was filtered through 0.45 µm Millex syringe-driven Filter unit (Merck KGaA, Darmstadt, Germany) for glutathione analysis.

Standardized lyophilized plant powder of the root of horseradish (Armoracia rusticana Radix, batch number PN19869) that is used in pharmacological remedy was provided by Repha GmbH (Langenhagen, Germany). Plant powder extraction was done as described before [10 (link)]. Briefly, one gram of plant powder was mixed with 10 mL double-distilled water or DMSO and directly incubated at 37°C for 30 min at 100 rpm. For the water extract, only glassware like vials, fennels, and flasks was used. The extract was strained through gauze and sterile filtered using a Millex syringe driven filter unit, 0.2 μm (Merck Millipore, Darmstadt, Germany), and 6 serial dilutions were prepared in a 1 : 3 ratio. The initial concentration of the extracts was 33.33 mg/mL. 1 mL PBMC (1 × 10⁶ cells) suspension, prepared in a 24-well plate, was treated with 10 μL of water extract. For the samples exposed to DMSO extract, 1 μL was used in 1 mL cell suspension. The final concentration of DMSO in the cell suspension did not exceed 0.1%.

To produce LAB-fermented both, a pre-grown culture of each LAB isolate was inoculated into fresh MRS broth to reach an initial density of 10⁵ cells ml⁻¹. The liquid portion of each culture took 10% of the volume inside the container and each culture was incubated at 200 r.p.m. at 28 °C for 24 h. The final density of each fermented broth was approximately 10⁹ cells ml⁻¹. As a control treatment, the MRS broth was not inoculated with LAB.
To collect the cell-free fermented broth of each isolate, each whole fermented broth was centrifuged, and the supernatant was filter-sterilized through a 0.22-µm Millex® syringe-driven filter unit (Merck Millipore Ltd., Co. Cork, Ireland) and then stored at − 20 °C before use.

To enable accurate chemical compound extractions, powders of the cheese samples were prepared. Therefore, cheese core and rind samples were frozen using liquid nitrogen (Air Liquide, Paris, France) and subsequently milled into a fine powder with a coffee grinder (De’Longhi KG49, Treviso, Italy). These cheese powders were subjected to two types of extractions. To assess organic acids, free amino acids, and biogenic amines, an aqueous extraction was performed, as described previously (Le Boucher et al., 2016 (link); Zhang et al., 2019 (link)). Briefly, 1.0 g of cheese powder was mixed with 9.0 mL of ultrapure water (Milli-Q; Merck) on a rotating wheel at 30 rpm for 30 min at room temperature, followed by centrifugation at 1,000 × g for 5 min. Extracts were stored at −25°C until further analysis. To assess volatile organic compounds, ethyl acetate extracts were prepared by mixing 0.5 g of cheese powder with 9.5 mL of ethyl acetate (SupraSolv^® grade; Merck) and supplemented with 100 μg/L of toluene-D8 (Sigma-Aldrich) as internal standard (IS). Ethyl acetate extracts were filtered with a Millex Syringe Driven Filter Unit (Millex; Merck) and immediately used for further analysis.

Size‐exclusion chromatography (SEC) measurements and fractionations were performed using a HLC8220 GPC system (Tosoh, Tokyo, Japan) equipped with a refractive index detector (RID). Separation was carried out using two TSKgel multipore H_XL‐M columns (7.8 mm × 300 mm) connected in series following a multipore Hxl guard column. CHCl₃ was used as the mobile phase at a flow rate of 1 mL min^–1. EVA samples were dissolved in CHCl₃ at 2 mg mL^–1, filtered using Millex syringe‐driven filter units (Merck Millipore, Carrigtwohill, Ireland) and 200 μL of the so‐formed solution were injected for the SEC elutions. Fractionation of the polymeric samples was done by collecting aliquots of 0.5 mL (30 s) into vials directly after the RID, further allowed to air dry for a couple of hours and submitted to mass analysis. Following this re‐concentration procedure, the sensitivity of the mass spectrometer allowed satisfactory mass spectra to be recorded from one single analytical SEC run only. The fractions of interest in the present study were collected after 18'30''–19'00'' (fraction #1), 19'00''–19'30'' (fraction #2) and 19'30''–20'00'' (fraction #3) of elution. Fractions of higher molecular weights were also collected (16'30''–18'30'') and mass‐analyzed (see Supporting Information).