# Fermented Cashew Nut Cheese Alternative Supplemented with _Chondrus crispus_ and _Porphyra_ sp.
## 2 Materials and Methods  
### Preparation of FCNCAs Supplemented with S seaweeds and Sampling Strategies  
The experiments utilized dehydrated red seaweeds from the division Rhodophyta: Chondrus crispus ([PERSON], 1797), commonly known as Irish Moss (order Gigartianales, family Gigartinaceae), and _Porphyna_ sp. ([PERSON], 1824), known as Nori (order Banglades, family Bangiaceae). These seaweeds were harvested from Atlantic coastal waters and industrially air-dried (ALGAplus, Ltd., Ilhavo, Portugal). They were mechanically ground into small pieces using a grinder (Orbegozo BV 9600, Murcia, Spain), resulting in particle sizes of 2.87 \(\pm\) 0.99 mm for _C. crispus_ and 1.47 \(\pm\) 0.49 mm for _Porphyna_ sp. Then, they underwent UV irradiation (DNA/RNA UV-Cleaner Box, UVC/T-AR, Biosan, Riga, Latvia) for 48 h to prevent contamination, were vacuum-sealed (Sammic SU-316G, Azkoitia, Spain), and stored at room temperature for three days before being used to prepare FCNCAs, as detailed below. Cashew nuts (Alesto(r), Neckarsulm, Germany) were soaked in water for approximately 12 h at 6-7 \({}^{\circ}\)C. After soaking, they were boiled in water (-88 \({}^{\circ}\)C) for about 2 min, then drained and processed in a Thermomix(r) TM5 (Vorwerk, Wuppertal, Germany) at speed 8 during 3 min until a thick paste was formed. The paste was then mixed with 3% nutritional yeast flakes enriched with vitamin B12 (Marigold Health Foods(r) Engevita, London, UK), 3% unpasteurized white miso paste (Clearspring(r), London, UK), and mineral water (Luso(r), Vacarica, Portugal) and mixed until a thick paste was achieved. Then, 0.1% of Advanced Acidophilus Plus (Solgar(r), Lisboa, Portugal), containing Lactobacillus acidophilus LA-5(r) (250 million organisms) and Bifidobacterium lactis BB-12(r) (250 million organisms), was added. The resulting mixture was transferred to a cheesecloth and pressed to remove excess water and/or fat. Two percent of the selected seaweed (_Chondrus crispus_ or _Porphyna_ sp.) was incorporated into the paste. This mixture was then shaped using steel molds (\(\O\)10 cm and 6-6.5 cm in height), placed in a hermetically sealed container, and stored at 6-7 \({}^{\circ}\)C. After two days, the product was removed from the molds, and the entire surface was sprinkled with 2% refined salt (Saldomar(r), Olhao, Portugal). The fermented cashew nut cheese alternatives were then dried in an oven at 40 \({}^{\circ}\)C for 30 h (TS 9135, Termaks AS(r), Bergen, Norway). Finally, the FCNCAs, uncovered, were ripened in a refrigerator (7.25 \(\pm\) 1.50 \({}^{\circ}\)C at 59.17 \(\pm\) 14.52% RH) (Thermo Meter, Max-Min Thermo Hygro TA318, KT)(r), Shenzhen, China), with daily flipping for 15 days. Three different types of FCNCAs were manufactured: fermented cashew nut cheese alternative control (FCNCA-C), fermented cashew nut cheese alternative with _Chondrus crispus_ (FCNCA-CC), and fermented cashew nut cheese alternative with _Porphyna_ sp. (FCNCA-P) (Figure 1). After manufacturing, FCNCAs were stored according to the requirements of subsequent analyses. Some were vacuum-packed (Sammic SU-316, Azkoitia, Spain) in labeled polypropylene bags (90 \(\upmu\)m, 180 mm \(\times\) 300 mm, PA/PE, Sammic, Azkoitia, Spain); part of these were refrigerated (4-6 \({}^{\circ}\)C) and another part frozen (\(-\)18 \({}^{\circ}\)C) until further analysis. The remaining FCNCAs were freeze-dried in a laboratory freeze-dryer (ScanVac Cool Safe 4 L, LaboGene, Lillered, Denmark), operating at a working temperature of \(-\)50 \({}^{\circ}\)C under a pressure of 0.0005-0.002 mBar, for 48 h. Subsequently, these were also vacuum-packed in labeled polypropylene bags and stored at \(-\)18 \({}^{\circ}\)C until further analysis. The experimental design and general sampling strategy of the FCNCAs are summarized in Figure 2.  
Figure 1: Representative images of the fermented cashew nut cheese alternatives (FCNCAs) after 15 days of ripening.