Antibacterial and Sensory Impact of β-carotene and Riboflavin on Pseudomonas Biofilm in Raw Milk

Milk is a highly nutritious yet perishable food that is susceptible to microbial contamination and spoilage, particularly by Pseudomonas species. The present study aimed to assess the antimicrobial and sensory effects of β-carotene and riboflavin as natural additives for preserving raw milk, with a specific emphasis on controlling biofilm-forming Pseudomonas strains. A total of 100 raw milk samples were collected from different markets in Gharbia, Egypt, and tested for Pseudomonas spp. Biofilm formation was assessed in isolates, followed by the detection of the quorum-sensing transcriptional regulator lasR, pellicle formation protein A (polysaccharide biosynthesis protein A) pelA, and polysaccharide synthesis locus protein A pslA genes. An in vitro trial was performed on eight groups of raw milk to evaluate the effects of β-carotene and riboflavin at concentrations of 1 µg/ml and 4 µg/ml, respectively, on Pseudomonas counts and sensory properties. The groups include positive control (G1), raw milk inoculated with Pseudomonas and fortified with β-carotene (G2), raw milk inoculated with Pseudomonas and fortified with riboflavin (G3), raw milk inoculated with Pseudomonas and fortified with a combination of β-carotene and riboflavin (G4), negative control (G5), milk fortified with β-carotene only (G6), milk fortified with riboflavin only (G7) and milk fortified with both β-carotene and riboflavin (G8). Reverse transcription polymerase chain reaction (RT-PCR) was performed to assess the impact of these additives on Pseudomonas, specifically on 16S rRNA and pslA gene expression. Pseudomonas spp. were found in 20% of samples, with 75% forming biofilms. The pslA gene was detected in 83.33% of these samples. β-carotene and riboflavin indicated dose-dependent antibacterial effects, with minimum inhibitory concentration values, and minimum bactericidal concentration values. Milk fortified with these compounds, particularly in G4, demonstrated the most significant reduction in Pseudomonas during a 6-day storage period. Additionally, β-carotene maintained a preferable sensory quality compared to other groups. RT-PCR confirmed the highest pslA gene suppression with the combined treatment. The current findings indicated that β-carotene at a concentration of 1 µg/ml, whether used independently or in combination with riboflavin, can serve as an effective natural preservative in dairy products.