Systematic evaluation of antimicrobial food preservatives on glucose metabolism and gut microbiota in healthy mice

Food additives, including antimicrobial preservatives (APs), are increasingly used to enhance food quality and shelf life. While some additives have been shown to negatively impact gut microbiota and host health, the effects of a broader range of APs, and the comparative safety of biogenic versus synthetic APs, remain largely unknown. This study aimed to systematically evaluate the impact of chronic exposure to eleven commonly used APs (five synthetic and six biogenic) on glucose metabolism and gut microbiota in healthy wild-type mice. This approach is crucial because it assesses the effects on normal hosts, providing more relevant information than studies using disease models. The gut microbiota's vital role in metabolic homeostasis suggests that AP-induced dysbiosis could further disrupt host metabolism, warranting investigation into the impact on glucose metabolism alongside gut microbiota changes.

Previous research has hinted at the potential for APs to disrupt gut microbiota. Studies have shown that certain APs, such as sodium bisulfite, sodium sulfite, and potassium sorbate, inhibited the growth of beneficial gut bacteria *in vitro* or altered microbiota composition in animal models like zebrafish. Studies on rodents have also indicated that mixtures of APs can cause dysbiosis. However, a comprehensive, systematic study on the impact of a diverse range of APs on the gut microbiota and host metabolism was lacking, particularly comparing synthetic and biogenic options. The role of biogenic APs as potentially safer alternatives to synthetic ones also required further investigation.

Wild-type male C57BL/6J mice (8–10 weeks old) were randomly assigned to control and various AP-treated groups (n=9 per group). The APs were added to the standard diet at three times the acceptable daily intake (ADI) level for 8 weeks. Glucose tolerance tests (GTT) were performed at weeks 2 and 8 to assess the time-dependent effects of AP consumption on glucose homeostasis. Fecal samples were collected at week 8 for 16S rDNA sequencing to analyze gut microbiota composition and diversity. Targeted metabolomics analysis of fecal samples was performed using UPLC-MS/MS to identify potential metabolic biomarkers altered by nisin, the AP showing the most significant effects. Finally, qRT-PCR was used to measure the expression of genes related to GLP-1 production in colonic samples, and ELISAs were performed to measure serum glucagon and insulin levels.

Chronic exposure to various APs, irrespective of their synthetic or biogenic origin, led to glucose intolerance in the mice. Five synthetic APs (sodium benzoate, potassium sorbate, ethylparaben, sodium nitrate, and sodium propionate) and the biogenic AP nisin significantly increased blood glucose levels after two weeks of treatment. After eight weeks, sodium benzoate, sodium propionate, and all biogenic APs significantly impacted blood glucose. The 16S rDNA sequencing analysis revealed that AP treatment altered the gut microbiota composition and diversity. Three synthetic and three biogenic APs significantly impacted the gut microbiota's beta diversity. Nisin exhibited the most significant effects on gut microbiota, increasing alpha diversity while significantly reducing *Bifidobacterium*, *Coriobacteriaceae*, and *Allobaculum*, and increasing *Oscillospira*, S24-7 family, *Clostridiales*, *Ruminococcaceae*, and *Lactobacillus*. Targeted metabolomics analysis of the nisin-treated group revealed significant alterations in fecal metabolites, with a substantial decrease in amino acids. Correlation analysis showed relationships between these altered metabolites and the abundance of specific gut genera. Furthermore, nisin treatment significantly reduced the expression of *Pcsk1* (involved in GLP-1 production) and increased serum glucagon levels, suggesting a possible mechanism for the observed glucose intolerance.

This study demonstrated that chronic consumption of both synthetic and biogenic APs, including nisin, can induce glucose intolerance and alter gut microbiota composition in healthy mice. The fact that nisin, a biogenic AP often considered a safer alternative to synthetic preservatives, caused the most pronounced effects challenges this assumption. The observed reduction in amino acids due to nisin treatment may have impacted GLP-1 release and subsequent glucoregulatory hormone imbalances, contributing to glucose intolerance. The altered gut microbiota profile likely plays a role in these metabolic changes, though further studies such as fecal microbiota transplantation are needed to confirm causality. The current study utilized a high dose of APs (three times the ADI), a limitation considering that most consumers are unlikely to ingest such high quantities. However, it demonstrates the potential for adverse effects even with a significant safety margin.

This study provides strong evidence that commonly used antimicrobial food preservatives, regardless of whether they are synthetic or biogenic, can induce glucose intolerance and disrupt the gut microbiota. The biogenic preservative nisin was found to have the most pronounced effect and warrants further investigation. Future research should focus on elucidating the specific mechanisms underlying these effects and identifying potential strategies to mitigate the negative impacts of APs on gut health and metabolism. Longitudinal studies with more diverse populations and lower AP dosages are necessary to establish the clinical significance of these findings.

The study used a high dose of APs (three times the ADI), which may not accurately reflect typical human consumption levels. Furthermore, while correlations between microbiota changes and metabolic alterations were identified, the causal relationship requires further investigation, possibly through fecal microbiota transplantation experiments. The study focused primarily on nisin's mechanism of action; more research is needed to fully understand the mechanisms by which other APs influence glucose metabolism and gut health.