Study on the Antibacterial Rule in Fermented Feed with Different Amounts of CaCO₃ by Quantitative Real-Time Polymerase Chain Reaction

Fermented feed is needed to maintain the vitality of probiotics and cannot be sterilized. Fermented feeds, especially those with added CaCO₃, have a high risk of contamination with pathogens. <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, and <i>Shigella flexneri</i> are the main pathogenic bacteria threatening animal breeding. This study developed a new microbial quantitative real-time PCR analysis method to investigate the antibacterial rule in fermented feed with different amounts of CaCO₃. Moreover, using the qPCR method, we found that the feed pH decreased slowly with the increase of CaCO₃ addition. In the early stage of fermentation, CaCO₃ addition promoted three pathogenic bacteria growth. In the middle and late fermentation, CaCO₃ addition inhibited the growth of <i>Escherichia coli</i> and <i>Shigella flexneri</i>, and the greater the CaCO₃ addition, the stronger the inhibitory effect. The CaCO₃ addition reduced the growth inhibition of <i>Staphylococcus aureus</i>, and the inhibition effect was weaker with the increase of CaCO₃ addition. From the inhibitory effect on intestinal pathogenic bacteria such as <i>Escherichia coli</i> and <i>Shigella flexneri</i>, the optimal addition amount of CaCO₃ was 12%. At this level of addition, the number of <i>Lactiplantibacillus plantarum</i> subsp. <i>plantarum</i>, <i>Lacticaseibacillus rhamnosus</i>, and <i>Bacillus subtilis</i> were also the highest, and the content of organic acids with antibacterial effects was also the highest. The addition of CaCO₃ had an inhibitory effect on the growth of pathogenic bacteria, mainly attributed to the promotion of the growth of <i>Lactiplantibacillus plantarum</i> subsp. <i>plantarum</i> and <i>Lacticaseibacillus rhamnosus</i>, and the organic acid of its fermentation product had an inhibitory effect on pathogenic bacteria. This study provides theoretical guidance for the antibacterial rule of high-pH fermented feed with different amounts of CaCO₃.