# Metabolomic Insights into the Potential of Chestnut Biochar as a Functional Feed Ingredient
## 4 Discussion
Specific derivatives, such as methylcumarins, hydroxycumarins, and methoxycumarins, exhibit varying biological effects based on the addition of methyl (-CH\({}_{3}\)), hydroxyl (-OH), and methoxy (-OCH\({}_{3}\)) groups, respectively. Methylation enhances antimicrobial and anti-inflammatory properties, while hydroxyl and methoxy groups contribute to antioxidant activity. Chestnut is also a rich source of bioactive polyphenols, including gallic acid, ellagic acid, quercetin, and catechins, which offer numerous health benefits [31]. However, these polyphenols are thermally unstable and degrade into smaller molecules during pyrolysis at temperatures above 500 \({}^{\circ}\)C. The degradation of plant cells during pyrolysis releases metabolites such as hydroxybenzoic acids, which are known for their antibacterial and antifungal properties. Hydroxybenzoic acid has been shown to disrupt bacterial cell membranes and inhibit essential microbial enzymes, leading to cell death [32]. It also interferes with fungal cell wall synthesis and provides antioxidant and anti-inflammatory effects [33]. The antioxidant potential of the biochar water extract is supported by [PERSON] et al. (2020) [34], who reported the antioxidant activity of chestnut shell extract in water ranging from 50.5 to 88.00 \(\upmu\)g/mL of Trolox equivalent (TE). This finding aligns with the antioxidant compounds found in the chestnut biochar, further confirming its potential for various applications, including animal feed. The extract of chestnut biochar exhibited significant inhibitory activities against _E. coli_, particularly strains F4+ and F18+, which are key pathogens in enterotoxigenic infections in pigs. These infections can lead to severe health issues, especially in young animals. The ability of the biochar extract to inhibit bacterial growth suggests its potential for improving animal health by reducing pathogenic bacteria and limiting their replication in feed. Understanding the molecular mechanisms by which biochar extract interferes with _E. coli_ growth is essential for optimizing its efficacy and developing targeted applications. One important area of focus is the effect of the biochar extract on gene expression in _E. coli_, particularly genes involved in quorum sensing (QS), which regulates bacterial communication and biofilm formation. The formation of _E. coli_ biofilms is controlled by a complex network of genes and metabolic pathways, which enable bacteria to coordinate their activities based on cell density [35; 36]. Among these genes, the FliA gene encodes Sigma 28 (\(\sigma\)28), which plays a key role in regulating the expression of genes involved in flagellar biosynthesis and motility, including the production of flagellin (FliC) [37; 38]. Additionally, sigma factors \(\sigma\)28 and \(\sigma\)70 regulate the expression of the MotA gene, which is crucial for the assembly and function of the bacterial flagellar motor. Another key gene, FtsE, is part of the bacterial cell division machinery. It encodes a membrane protein that, together with FtsX, forms the FtsEX complex, which is essential for regulating the early stages of cell division and septum formation [39]. Disruption of FtsE expression can lead to defects in cell division, cell morphology, and viability, as well as increased susceptibility to antibiotics. The HfIX gene encodes a GTP-binding protein involved in ribosome assembly, protein synthesis, and stress response mechanisms [39; 40; 41]. Down-regulation of HfIX affects bacterial adaptation to environmental stress, including ribosomebiogenesis and cell division. Research has shown that FliA downregulation is associated with incomplete flagellar synthesis, reduced motility, and impaired chemotaxis [42], while disruption of FtsE leads to defects in cell division and morphology [43]. Similarly, loss of HfIX function compromises the ability of bacteria to form stress granules, which are protective structures that help cells survive under stressful conditions [44].