Ph meter

Using a pH meter (Mettler Toledo AG, Zurich, Switzerland), the pH measurements of the supernatant were carried out at each time by first calibrating the pH meter in standard solution (pH 7) and then immersing the probe into the solution/suspensions until the pH reading was stabilized. CS powder and NPs suspensions in 0.2 mg/mL lysozyme solution were the test samples and lysozyme (in PBS solution) was considered as a reference standard.

The model honey was prepared by dissolving fructose (2.24 M), glucose (1.85 M), maltose (0.219 M) and sucrose (0.04 M) (all purchased from Sigma-Aldrich, United Kingdom) in deionized sterile water at 37°C as described previously (Bogdanov, 1997 (link)). Stock solutions of gluconic acid and H₂O₂ (Sigma-Aldrich, United Kingdom) were prepared in deionized sterile water and were added, at the appropriate concentrations, immediately before the start of the assay. The H₂O₂ stock contained stabilizer, which in theory could affect data interpretation, but we expect its effects if any to be small. The compositions of the model honey stocks (A and B) used in this study are presented in Table 1. The pH of model honeys (A; pH 4.1, B; pH 3.8) was measured using a Mettler Toledo pH meter (Mettler-Toledo Ltd., United Kingdom). The effects of model honeys were always assayed following dilution with an equal volume of bacterial cell culture.

Samples of feeds and fecal were analyzed according to the AOAC (1990 ). Nitrogen content was calculated as crude protein (CP) ÷ 6.25. Nitrogen retention was computed from dietary N intake (NI) less total nitrogen output (fecal N [FN] and urinary N [UN] losses) by Katsande et al. (2016 (link)). $$\begin{array}{c}\text{Nitrogen retention}\left({\mathrm{g}}^{\mathrm{d}-1}\right)=\mathrm{NI}–\left(\mathrm{FN}+\mathrm{UN}\right)\\ \text{Apparent digestibility}\left(\%\right)=\mathrm{NI}–\left(\mathrm{FN}+\mathrm{UN}\right)\mathrm{NI}\times 100\end{array}$$
Purine derivatives, such as allantoin, uric acid, xanthine, and hypoxanthine concentrations in urine samples, were measured according to Balcells et al. (1992 (link)). The samples were examined using high-performance liquid chromatography (HPLC) (Agilent 1100 Series HPLC System Agilent Technologies, USA) by two 4.6 mm × 250 mm C-18 reverse-phase column (Spherisorb), and the effluent was monitored at 205 nm.
Determination of volatile fatty acids (VFA) in ruminal fluid was as described previously (Cottyn and Boucque 1968 (link)). The pH of the rumen fluid was assessed with the aid of a Mettler-Toledo pH meter (Mettler-Toledo, Ltd. England). Ammonia nitrogen (NH₃-N) in ruminal fluid was determined in accordance with Parsons et al. (1984 ).