*5.4. Determination of Malondialdehyde Levels (Thiobarbituric Acid Assay)*

The level of malondialdehyde (MDA) was measured according to the method described by Weitner et al. [59] with small modifications. Briefly, the tissue supernatant with buthylohydroxytoluene (BHT, Sigma Aldrich, Saint Louis, MO, USA) was deproteinized with 20% TCA (trichloroacetic acid, Avantor, Poland) and centrifuged for 1 h; 100 μl of the supernatant was then combined with thiobarbituric acid (TBA, Sigma Aldrich, Saint Louis, MO, USA) and incubated (1 h at 95◦C). The level of MDA was read from a calibration curve (TBA, MDA Standard, Cayman, Ann Arbor, MI, USA). Absorbance was read in a Perkin Elmer spectrophotometer Lambda 25 at a wavelength of λ= 520 nm (Biocompare, Baltimore, MD, USA). The level of MDA was expressed in picomoles per milligram of whole protein in the tissue supernatant.

### *5.5. Determination of Sulfhydryl Groups (–SH)*

Thiol groups were measured according to the modified Ellman method. Briefly, 1.0 ml of 40 mM Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) solution (Serva, Heidelberg, Germany) was added to the sample (86 mM Tris (Sigma Aldrich, Saint Louis, MO, USA), 90 mM glycine (Avantor, Gliwice, Poland), 4 mM ethylenediaminetetraacetic acid (EDTA) (Avantor, Gliwice, Poland), 8 M urea (Sigma Aldrich, Saint Louis, MO, USA), 0.5% sodium dodecyl sulfate (SDS, Serva, Heidelberg, Germany), 0.2 M Tris-HCl (Sigma Aldrich, Saint Louis, MO, USA)) with pH 8.0. Next, 200 μl of each sample was added to 1.0 ml of DTNB. The samples were incubated at room temperature for 30 min. Cysteine was used (Avantor, Gliwice, Poland) as a standard, and absorbance was measured by a Perkin Elmer spectrophotometer Lambda 25 at a wavelength of λ= 412 nm (Biocompare, Baltimore, MD, USA). The concentration of -SH groups was measured from a calibration curve based on cysteine solution in PBS. The concentration of -SH groups was expressed in micromoles per milligram of whole protein in the ileal supernatant.

### *5.6. Determination of Fructose and Glucose Concentrations*

Fructose level was determined in the ileum by the method described by Messineo and Musarra [60] with some modifications. This method is specific for fructose and similar to sucrose and inulin measurements method without interference from glucose (aldohexoses), aldopentose, and ketopentose. Glucose concentration was evaluated by Trinder's glucose oxidase method modified by Lott and Turner [61], with further modifications. The measurements were conducted spectrophotometrically with glucose oxidase reagent (G7521, Pointe Scientific, Canton, MI, USA) with the appropriate modifications. Fructose and glucose concentrations were expressed in micrograms per milligram of whole protein in the ileal supernatant.

### *5.7. Determination of Proline Concentration*

Proline concentration in ileal Peyer's patches was determined by the modified method for the determination of proline levels in plants [62]. Tissue homogenates in the amount of 500 mL were placed in glass test tubes, and 1.0 mL of 2.5% ninhydrin solution, 1.0 mL of 3% sulfosalicylic acid, and 1.0 mL of glacial acetic acid were added. The samples were incubated (boiling water bath for 15 min.). The glass test tubes were cooled under running water, and 2 mL of toluene was added. The tubes were shaken for 15 min and left to stand until the separation of mixture components. The toluene layer was measured spectrophotometrically at a wavelength of 520 nm. Proline concentration was read from the standard curve, and points on the curve were determined based on the sample preparation method.

### *5.8. Statistical Analysis*

The results were processed in Excel (Microsoft, Redmond, WA, USA) and GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) applications. Mean values and standard error of the mean (SEM) were determined for all groups. Population distributions were evaluated by the Shapiro–Wilk normality test. The results were processed by two-way ANOVA with post hoc Tukey's multiple comparison test. The results were regarded as statistically significant at *p* < 0.05.

**Author Contributions:** The experiments were conceived and designed by K.O. and P.W. The experiments were performed by W.T., K.O. and P.W. Data were analyzed and interpreted by K.O., P.W. and W.T. The manuscript was drafted by K.O., W.T. and P.W. and critically edited by Ł.Z., J.T. and M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the "Healthy Animal—Safe Food" Scientific Consortium of the Leading National Research Centre (KNOW) pursuant to a decision of the Ministry of Science and Higher Education No. 05-1/KNOW2/2015. The project was financially supported by the Minister of Science and Higher 613 Education under the program entitled "Regional Initiative of Excellence" for the years 2019–2022, 614 Project No. 010/RID/2018/19, amount of funding PLN 12,000,000.

**Conflicts of Interest:** The authors declare no conflict of interest.
