Trace Minerals

Saccharomyces cerevisiae strain AXT3K (△ena1::HIS3::△ena4, △nha1::LEU2, and △nhx1::KanMX4), which is deficient in the main endogenous Na⁺ transporters, is a derivative of W303 (MAT ura3–1 leu2–3,112 his3–11,15 trp1–1 ade2–1 can1–100) [10 (link)]. To assess the functionality of the SpSOS1 and SpAHA1 proteins, p416-SpSOS1, p414-SpAHA1 and p416-SpSOS1 and p414-SpAHA1 were transformed into AXT3K. The wild-type yeast strain W303 was used as a positive control [18 (link)]. W303, AXT3K, and AXT3K transformed with p416-SpSOS1, AXT3K transformed with p414-SpAHA1, and AXT3K co-transformed with p416-SpSOS1 and p414-SpAHA1 were grown in AP medium (10 mM arginine, 8 mM phosphoric acid, 2% glucose, 2 mM MgSO₄, 1 mM KCl, 0.2 mM CaCl₂, and trace minerals and vitamins) [19 (link)]. When these precultures grew to saturation, they were diluted 10-fold, 100-fold, 1,000-fold, and 10,000-fold, and then 10 μl of each serial dilution was spotted onto AP plates with the indicated concentration of NaCl. After incubation for 3–5 days at 30°C, growth was imaged and analyzed.

The cloning of lpmo9A gene from P. anserina strain S mat⁺, encoding PaLPMO9A (protein ID CAP73254), was described by Bey et al. [14 (link)] Genes encoding PaLPMOC (protein ID CAP68173), PaLPMO9D (protein ID CAP66744), PaLPMO9E (protein ID CAP67740), PaLPMO9F (protein ID CAP71839), PaLPMO9G (protein ID CAP73072), and PaLPMO9H (protein ID CAP61476) were codon optimized for P. pastoris (GenScript, Piscataway, USA) and further inserted into the vector pPICZαA (Invitrogen, Cergy-Pontoise, France) using XhoI and XbaI restriction sites in frame with the (His)₆ tag (located at the C terminus of recombinant proteins). P. pastoris strain X33 and the pPICZαA vector are components of the P. pastoris Easy Select Expression System (Invitrogen). All media and protocols are described in the Pichia expression manual (Invitrogen). Recombinant expression plasmids were sequenced to check the integrity of the corresponding sequences. Transformation of competent P. pastoris X33 was performed by electroporation with SacI-linearized pPICZαA recombinant plasmids as described in [45 (link)]. Zeocin-resistant P. pastoris transformants were then screened for protein production. The best-producing transformant was grown in a 1 l of BMGY containing 1 ml.l⁻¹ of Pichia trace minerals 4 (PTM₄) salts (2 g.l⁻¹ CuSO₄.5H₂O, 3 g.l⁻¹ MnSO₄.H₂O, 0.2 g.l⁻¹ Na₂MoO₄.2H₂O, 0.02 g.l⁻¹ H₃BO₃, 0.5 g.l⁻¹ CaSO₄.2H₂O, 0.5 g.l⁻¹ CaCl₂, 12.5 g.l⁻¹ ZnSO₄.7H₂O, 22 g.l⁻¹ FeSO₄.7H₂O, biotin 0.2 g.l⁻¹, H₂SO₄ 1 ml.l⁻¹) in shaken flasks at 28 °C in an orbital shaker (200 rpm) for 16 h to an OD₆₀₀ of 2–6. Expression was induced by transferring cells into 200 ml of BMMY containing 1 ml.l⁻¹ of PTM₄ salts at 20 °C in an orbital shaker (200 rpm) for another 3 days. Each day, the medium was supplemented with 3 % (v/v) methanol. Bioreactor production of the best-producing transformant was carried out in a 2-l bioreactor Tryton (Pierre Guerin, Mauze, France) according to the P. pastoris fermentation process guidelines (Invitrogen).

Fiber addition to meat products has changed their overall composition, which has led to the development of new fiber sources and opened up exciting prospects for their application across several industries. The use of dried pumpkin pulp reduced the patties’ moisture content and raised their ash content. Raw and cooked patties both had a higher pH after incorporation. The capacity to retain moisture improved, as the amount of dry pumpkin pulp added rose. The cooking yield and diameter change, when pumpkin pulp was added were not significantly different (Serdaroğlu et al., 2018 (link)). This increase in ash content of meat product was probably due to higher mineral content of pumpkin matrix. As meat ash content rises, it could help end specific mineral deficiency. Trace minerals (iron, zinc, selenium, iodine, copper, chromium, manganese and molybdenum) perform vital functions within the body including thyroid metabolism, antioxidant activity and immune function (Vural et al., 2020 (link)). For example, zinc (Zn) is the second most abundant trace element in human, which can’t be stored in the body, thus regular dietary intake is required Zn microelement is very essential for male fertility. It could be considered as a nutrient marker with many potentials in prevention, diagnosis, and treatment of male infertility (Fallah, Mohammad-Hasani & Colagar, 2018 (link)). In one study, protein content, mineral content, and crude fiber content were increased by the incorporation of white cauliflower by-product flour in beef sausage; pH was found to be increased by the incorporation of flour made from the upper stem of white cauliflower. The cooking yield was also increased gradually with the incorporation of white cauliflower stem powder, because of its water yielding property (Abul-Fadl, 2012 ). The quantity of protein and fat in emulsified pork meatballs reduced as rice bran was added. In contrast, the amount of carbohydrates in the meatballs grew dramatically as rice bran was added (Choi et al., 2011 (link)). The protein level of frankfurter beef sausages was raised by one percentage point, and the ash content was raised dramatically when 7% of the residue was added. The benefits of increased ash content has already been discussed in previous example. Furthermore, overall lipid levels dropped, and this reduction in lipid provided longer shelf life by cutting down the chance of lipid oxidation. The question of what sort of quality this residue adds to the protein remains unanswered (Savadkoohi et al., 2014 (link)). A few other examples are showing the changes in different characteristics of meat products by incorporating various plant-based materials, especially waste, have been listed in Table 1.

The prevalent levels of saturated fat in meat, especially red meat, give it a bad reputation regarding people’s health. Therefore, it is recommended to limit one’s consumption of meat, particularly red meat, in order to reduce an individual’s chance of developing certain disorders and diseases including cardiovascular (CV) diseases (Giromini & Givens, 2022 (link)). However, this perspective takes into account the fact that meat is a rich source of several micronutrients, including vitamins and trace minerals that are either absent from meals derived from plants or have a low bioavailability in those foods (Biesalski, 2005 (link)). In addition, because it is high in protein and low in carbohydrates, meat has a lesser glycemic index (GI), which is thought to be advantageous in preventing obesity, diabetes, and cancer. Meat is a product that is rich in protein and low in carbohydrates (Biesalski, 2005 (link)).