# Cold-Adapted Fungi: Goldmine of Biomolecules Applicable in Industry
## 4 Biotechnological Application of Psychrophilic Fungi in Food and Agriculture
### Ice-Binding Proteins  
Various organisms, including psychrophilic yeast and filamentous fungi, produce ice-binding proteins (IBPs) [54]. Among these extracellular proteins, some have been demonstrated to inhibit ice crystal growth (antifreeze proteins--AFPs), whereas others have the ability to trigger the formation of new ice crystals (ice-nucleating proteins--INPs) [98,99]. Antifreeze proteins (AFPs) constitute a family of proteins with highly diverse 3D structures. Most AFPs are glycoproteins or lipoproteins with molecular weights ranging from approximately 2 to 50 kDa. These proteins bind to nascent ice crystals and inhibit ice recrystallization inhibitors (IRI) through thermal hysteresis (TH). Both TH and IRI activities depend on the affinity of AFPs for ice. In contrast, ice-nucleating proteins (INPs) are secreted outside the cell as large, multimeric aggregates, which gives them higher activity. These proteins initiate ice crystallization near the melting temperature range (down to about \(-3\)\({}^{\circ}\)C) by interfering with the ordering and stabilization of water molecules in an ice-like structure, thus lowering the activation energy barrier required for freezing and nucleation of ice. To protect the intracellular space of psychrophilic microorganisms, INPs redirect crystal nucleation outside the cell by removing liquid water molecules. Additionally, INPs promote the formation of smaller ice crystals, which exert less deleterious effects on cells. Furthermore, the lethal energy released during the crystallization process prevents further temperature declines in the cellular environment [49,100,101]. Three yeast strains, namely, _Glaciozyna antarctica_ PI12, _Glaciozyna_ sp. AY30, and _Goffeauxzyna gastrica_[102], have been identified as producers of AFP proteins. Analysis of the _Glaciozyn antarctica_ PI12 genome revealed the presence of nine genes encoding AFPs, of which only two (Afp1 and Afp4) had been previously described. Functional characterization demonstrated that AFP1 has a molecular mass of 15 kDa, while AFP4 has a molecular mass of 25.3 kDa, both exhibiting TH activity of 0.1 \({}^{\circ}\)C [54]. _Glaciozyna_ sp. AY30 produced AFP with a molecular weight of 26.8 kDa and showed TH activity of 1.38 \({}^{\circ}\)C [103]. Among filamentous fungi, three producers have been described: _Typhula isthariensis_[104], _Antarctonyces psychrotrophicus_[105], and _Coprius psychromorbidus_[104], which belong to the group of snow molds [98,99]. _T. ishikariensis_ expresses seven isoforms of the AFP protein TrisAFP6 and TrisAFP8, showing noteworthy activity. TrisAFP8 exhibited a TH of 1.9 \({}^{\circ}\)C and IR activity [104]. _A. psychrotrophicus_ produces the 28 kDa AnpAFP protein, which exhibits high thermal hysteresis activity of 0.42 \({}^{\circ}\)C and IR activity at a concentration of 0.05 mg/mL [105]. Additionally, [PERSON] et al. (2020) demonstrated in their investigation of the Antarctic endemic fungus _Antarctonyces pellizariae_ UFMGCB 12416 genome that this strain possesses three distinct isoforms of IBP [106]. Recent research has enabled the identification and characterization of the properties of AFP proteins, thereby expanding their potential applications. The growth of ice crystals presents a significant challenge in a number of fields, including food technology and agriculture (Figure 4). In contrast to some classic cryoprotectants like DMSO or PVA, which are not suitable for food due to their toxicity to humans [107], AFPs offer a safer alternative. In the food industry, it is important to control ice crystal growth during processes like freezing, storage, and transport of food products [98]. The formation of large ice crystals in high quantities and/or irregularly can result in irreversible damage to food products. The TH and IRI activity exhibited by AFP proteins is postulated to underpin the preservation of frozen foods' freshness, flavor, and quality by reducing cell membrane damage, water loss, and protein oxidation [99].