Evaluation of Microbial Safety of Dairies using Bacterial Proteomic Profiling via MALDI Approach

We developed a protocol to prepare and isolate microbial communities from various daily products, such as milk, cream, butter, and cheese. Once isolated, this microorganism can be rapidly and accurately identified using MALDI-TOF MS.Optimizing culture conditions remains a key challenge, along with database gaps and the demanding extraction of gram-positive and spore-forming bacteria. To begin, obtain a representative sample of the dairy product and mix thoroughly to ensure uniformity.

Aseptically, transfer one milliliter or one gram of solid samples into a 15-milliliter conical centrifuge tube containing nine milliliters of sterile liquid medium. Vortex the tube vigorously for 30 to 60 seconds to homogenize the sample. Next, prepare a series of tenfold dilutions by transferring one milliliter of the homogenized suspension into successive tubes, each containing nine milliliters of sterile solution.

Mix each dilution thoroughly by vortexing or shaking. Then, take 0.1 milliliters of the original sample, or one of the prepared dilutions, and transfer it onto the surface of a prepared Petri dish containing the appropriate agar medium. Using a sterile spreader, evenly distribute the inoculum over the surface of the agar plate.

For aerobic incubation, inoculate APT, M17, CBL, MPCA, and TSA agar plates with the prepared samples. Incubate the plates under aerobic conditions at 37 degrees Celsius and 30 degrees Celsius for 24 hours. For anaerobic incubation, place the inoculated MRS agar plates in an anaerobic chamber and incubate at 37 degrees Celsius and 30 degrees Celsius for 24 to 72 hours.

Remove the plates from the incubator and examine them daily to monitor microbial growth. Using a one-microliter microbial loop, streak the selected colonies onto the same agar medium to subculture them. Incubate under the same conditions used previously.

For mass spectrometry analysis, begin by preparing the MALDI matrix solution. Prepare the solvent mixture using 50%acetonitrile, 47.5%HPLC-grade water, and 2.5%trichloroacetic acid. Dissolve the matrix compound into the solvent mixture to a final concentration of approximately 10 milligrams per milliliter.

For MS identification of bacteria using the direct colony transfer method, use a sterile loop to transfer one microliter of a grown bacterial colony onto a spot on the MALDI target plate. Spread the bacterial material to create a thin, even layer on the plate. Once dried, add one microliter of HCCA matrix solution on top and allow it to air-dry at room temperature.

For the on-target formic acid extraction method, use a disposable microbial loop or sterile toothpick to pick a small amount of bacterial biomass from a single colony. Smear the material directly onto a spot on the MALDI target plate to create a thin, even layer. Add one microliter of 70%formic acid solution over the smeared area.

Once the spot has dried, overlay it with one microliter of HCCA matrix solution and allow it to air-dry at room temperature. For the in-tube protein extraction method, transfer one to three complete colonies from an agar culture plate into a 1.5-milliliter microcentrifuge tube containing 300 microliters of sterile water. Add 900 microliters of absolute ethanol to achieve a final ethanol concentration of approximately 75%After mixing the content briefly, centrifuge the tube for two minutes at 15, 000 g at room temperature.

Afterward, carefully discard the supernatant without disturbing the cell pellet. Then, add 20 to 50 microliters of 70%formic acid to the dried cell pellet. Mix the contents by pipetting or vortexing until the pellet is fully resuspended.

Now, add an equal volume of acetonitrile to the formic acid suspension and mix thoroughly. Centrifuge the tube at 15, 000 g for two minutes. Transfer one microliter of the resulting supernatant to a designated spot on a MALDI target plate and allow it to dry.

Then, overlay the dried spot with one microliter of HCCA matrix solution. Pipette one microliter of either the bacterial test standard containing an extract of Escherichia coli DH5 alpha or the microbiology calibrator containing E.coli ATCC 25922 protein extract, ribonuclease, and myoglobin. For spectra acquisition and microbial identification, prepare calibration spots on the MALDI target plate.

Load the MALDI target plate with both the prepared sample spots and the calibrant spots into the mass spectrometry instrument. Run the calibration analysis in calibration mode to calibrate the system. Operate the mass spectrometer in linear positive ion mode for general spectra acquisition.

Then, set the mass detection range of the mass spectrometer to cover from 2, 000 to 20, 000 mass-to-charge ratio. Acquire spectra in duplicate for each spot to generate at least four technical replicates per sample. Now, apply the Savitzky-Golay method as a smoothing algorithm.

Use the TopHat algorithm for baseline correction to remove background noise. Then, detect peaks using centroid mode. Upload the processed mass spectra of unknown bacterial isolates to the microbial identification platform within the MALDI software and run the identification.

Evaluate the identification scores according to the manufacturer's threshold standards. The mass spectrometry spectra displayed species-specific protein profiles, enabling the identification at the genus or species level. The dendrogram revealed high similarity observed among strains of the same species and clear differentiation between species.

A shorter incubation time of 12 hours for Bacillus licheniformis produced a higher ID score value of 2.38, while a longer incubation of 18 to 24 hours led to a lower ID score value of 1.85 due to the dominance of spore signals. Among the sample preparation methods, in-tube protein extraction gave the highest ID score value of 2.23, followed by on-target formic acid extraction at 1.97, and direct colony transfer, which resulted in the lowest score of 1.61. By combining culturomics with tailored sample preparation, MALDI enables more precise and faster microbiome analysis at lower cost, producing results comparable to the reference 16S rRNA methods.

This protocol enables routine, high-throughput monitoring of microorganisms in dairy products, improving detection speed and accuracy, ultimately strengthening food quality and safety systems. Expanding MALDI databases and automating workflows in industry labs will enhance microbial identification, increase throughput, reduce errors, and speed microbiota monitoring.