Total dietary fiber assay kit

The moisture content of cookies was determined according to AACC method 44-15.02 in triplicate. Total dietary fiber (TDF) of dried and defatted samples was determined according to AACC method 32-05.01 and AOAC Method 985.29 (if fat content is >10%) in duplicate using Total Dietary Fiber Assay Kit (Megazyme, Bray, Ireland). The results are expressed se mean value in grams per dry weight of cookie.

The dog food samples were analyzed without pre-drying. To determine dry matter (DM) content, the samples were dried at 103 °C for 16 h, and then placed in a desiccator to cool before weighing³⁶ . For ash determination, samples were incinerated at 550 °C for three hours and then cooled in a desiccator before weighing. The Kjeldahl method³⁷ was applied to determine nitrogen content, using a 2020 digester and a 2400 Kjeltec analyzer (FOSS Analytical A/S, Hilleröd, Denmark). Crude protein was then calculated as N × 6.25. Crude fat was analyzed in accordance with Commission Directive EC/152/2009³⁸ , on a Soxtec extraction unit (FOSS Analytical A/S, Hilleröd, Denmark). Crude fiber was analyzed as previously described³⁹ . Gross energy (GE) was measured on a Parr isoperobol Bomb Calorimeter 6300 (Parr Instrument Company, Moline, Illinois, USA). Total dietary fiber (TDF) as well as soluble and insoluble dietary fiber were analyzed in accordance with AOAC 991.43 using a Total Dietary Fiber Assay Kit (Megazyme, Bray, Ireland), while resistant and non-resistant starch were analyzed according to AOAC 2002.02 using a Resistant Starch Assay Kit (Megazyme, Bray, Ireland).

The chemical composition of samples was determined according to AOAC methods [57 ]. Total protein content was estimated by determination of elemental nitrogen content by thermal conductivity using a TruSpec 630-200-200 CNHS analyzer from LECO (St. Joseph, MI, USA), multiplied by the conversion factor of 5, which is specific for seaweeds [58 (link)]. Ash content was determined by incineration in a muffle furnace at 550 °C for 6 h and gravimetric quantification. Crude fat was obtained by soxhlet extraction with light petroleum for 8 h, followed by filtration through a 0.2 µm nylon filter, solvent removal in a rotary evaporator and drying, followed by gravimetric quantification. Total carbohydrates were calculated by difference. Dietary fiber contents (soluble, insoluble and total) were estimated according to the enzymatic gravimetric method AOAC 991.43, using the Total Dietary Fiber Assay kit from Megazyme (Bray, UK). Calorific content was determined according to AOAC [57 ]. The free sugars content was measured according to Dubois et al. [59 (link)] with slight modifications. In particular, 50 µL of 4% phenol and 250 µL of 96% sulfuric acid was added to 100 µL of sample/standard. The absorbance of the mixture was read at 490 nm after 10 min of incubation.

The same methods were applied as described in a previous study [41 (link)]. Briefly, seaweeds were freeze-dried and powdered (BFP660, Breville, Sydney, Australia). Moisture and ash contents were measured by a thermal gravimetric analysis using method no. 950.46 AOAC (Association of Official Analytical Chemists) 2008 and method no. 938.08 AOAC 2008, respectively. Protein content was obtained by the Kjeldahl method (AOAC 2008, no. 988.05) using a protein factor of 5 [87 (link)], and lipid content was obtained by the Bligh and Dyer method [88 (link)]. Total carbohydrate content was determined by difference [21 (link)], and fiber content was determined with a total dietary fiber assay kit (Megazyme, Bray, Ireland) using method no. 985.29 AOAC 2008. Calcium, iron, magnesium, potassium, and sodium contents were quantified using a flame atomic absorption spectrophotometer (220FS, Varian, CA, USA) with AOAC 2008 method no. 968.08, and iodine content was assessed with an iodine ion-selective electrode (Thermo Scientific 258508, Waltham, MA, USA).

Insoluble (IDF), soluble (SDF), and total (TDF) dietary fiber content was determined, using the Total Dietary Fiber Assay Kit (Megazyme International Ireland, Ireland) according to the manufacturer’s instructions, and based on the enzymatic–gravimetric method. All measurements were performed in triplicates. Results are expressed as a percentage (%).

The following reagents, consumables and special materials were used in this study: ethanol (96%, 70%) (Chempur, Piekary Śląskie, Poland), petroleum ether ACS grade (Chempur, Piekary Śląskie, Poland), Total Dietary Fiber Assay Kit (Megazyme, Bray, Ireland), Tashiro indicator (Chempur, Piekary Śląskie, Poland), hydrochloric acid 35–38% (Chempur, Piekary Śląskie, Poland), sodium hydroxide 0.1 M (Chempur, Piekary Śląskie, Poland), MES-2-(N-morpholio)ethanesulfonic acid (Chempur, Piekary Śląskie, Poland), tris(hydroxymethyl)aminomethane 99% (Chempur, Piekary Śląskie, Poland), acetone ACS grade (Chempur, Piekary Śląskie, Poland), NaOH ACS grade (Chempur, Piekary Śląskie, Poland), chloroform ACS grade, BHT (butylated hydroxytoluene) (Chempur, Piekary Śląskie, Poland), KOH ACS grade (Chempur, Piekary Śląskie, Poland), methanol, HPLC grade (Chempur, Piekary Śląskie, Poland), boron trifluoride 14% in methanol (Merck, Darmstadt, Germany), n-hexane HPLC 99% (Chempur, Piekary Śląskie, Poland), capillary column of dimensions 15 m × 0.10 mm, 0.10 μm (Supelcowax™ 10 Capillary GC Column, Supelco, Bellefonte, PA, USA), internal standard heneicosanoic acid (Merck, Darmstadt, Germany).