Tryptone soya broth tsb

BEO was purchased from Sinergy Flavors Italy S.p.A. (Trieste, Italy). BEO was extracted by hydro distillation as reported in the technical sheet of the product. CH (molar mass 3.7 × 10⁴ g/mol, 91% N-deacetylation) was a gift from prof. R.A.A. Muzzarelli (University of Ancona, Italy) and characterized as previously described [18 (link)]. All further chemicals and solvents were analytical grade and are cited with supplier and code in bracket. Gram-positive and Gram-negative bacteria reported in Table 1 were from the culture collections of the Department of Agricultural Sciences, University of Naples Federico II. All of them were previously identified at genome level by 16S rRNA gene sequencing. Microbial strains were routinely grown in tryptone soya broth (TSB, Thermo Fisher Scientific, Rodano, Italy) for 24 h.

3-Hydroxytyramine hydrochloride (98%), NH₄OH (28–30%), indocyanine green (ICG), potassium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide, and agar were purchased from Sigma-Aldrich (Shanghai, China). Tryptone Soya Broth (TSB; OXOID, Basingstoke, UK), RPMI-1640 medium, Fetal Bovine Serum, penicillin, streptomycin, trypsin-EDTA, SYTO^®9 and Cell Counting Kit-8 (CCK-8) were obtained from Thermo Fisher Scientific, Inc. (Beijing, China). Ethanol, hydrochloric acid, dimethyl sulfoxide and tetrahydrofuran were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All aqueous solutions were prepared in 18.2 MΩ cm purified Milli-Q water (Millipore, Bedford, MA, USA). All chemicals were of analytical grade and used without further purification.

The sterility testing was performed following the method described in the fifth Edition of the International Pharmacopoeia (http://apps.who.int/phint/pdf/b/Jb.7.3.2.pdf). Briefly, before and after first using all dispensers (placebo and treatment groups), 1 ml content was added to 10 ml of Soybean-Casein Digest sterilized Medium (Tryptone Soya Broth (TSB), Oxoid, Thermo scientific, UK) and 1 ml to 10 ml of Nutrient broth (Nutrient Broth, Difco, BD, USA). Dispenser 1 for both placebo and active treatment was also tested at day 119 to evaluate the sterility of its content overtime.
TSB was incubated at 22.5 °C and Nutrient Broth at 37 °C for 14 days. Cultures were assessed daily. In case of increased turbidity due to growth of potential contaminants such as fungal, yeast, aerobic and anaerobic bacteria, further identification occurred.