The overall goal of this experiment is to evaluate if thyme essential oil, applied in vapor phase, can serve as effective preservative during meat-drying process. This method can help answer questions in the field of food processing and safety, such as how to effectively apply natural preservatives instead of synthetic ones. The main advantages of this technique is that enables fast and equal distribution of thyme essential oil while the meat dries and that all vapor can be used in lower volumes than in case of liquid oil application.
The use of this method can provide insight into improving the safety and quality of dried meat and also can be applied for treatment of other food products against other unfavorable microorganisms. Generally, individuals new to this method will struggle because the drop plate methods used to count the number of the bacteria requires properly prepared plates and practice. Obtain a short loin of beef from a local butchery, and transfer it to the lab in a sealed bag at ambient temperatures within 20 minutes of purchase.
In a Laminar safety cabinet, sterilize the outer surface of the beef muscle using a 10-second application of 70%ethanol, sprayed on from a squeeze bottle. Then, using sterile technique, cut out the outer surface of the muscle so that no ethanol remains on the preparation. Then pack the muscle into a sealed bag, and freeze it for one day at negative 18 degrees Celsius.
The next day, thaw the frozen muscle at four degrees Celsius over six hours. Then, in a Laminar safety cabinet, use a meat cutter to make 0.5 centimeter thick slices. And further reduce the size of the slices into 5x2.5 square centimeter rectangular samples.
For storage, pack the samples into plastic bags and keep them in the freezer. In a Laminar safety cabinet, prepare a standardized inoculum of E.coli ATCC 25922 to inoculate the meat samples. For the inoculation, arrange at least four samples on an aluminum foil sheet.
Then load just as many samples on a second foil sheet to serve as uninoculated controls. Next, inoculate each sample by applying 400 microliters of inoculum onto one side and gently spreading it over the surface using a sterile cell-spreader. Then, let the samples dry for 10 minutes.
Next, flip the samples over with tweezers, and repeat the procedure for a total of 800 microliters of inoculum per sample. After this, check the inoculation efficiency of two of the inoculated raw samples and two uninoculated controls. And the rest of the samples, wrap them in foil.
Wash the raw samples in peptone water for 10 minutes at room temperature with 140 rpm of shaking. After the wash, on a Microtiter plate, make five 10-fold serial dilutions of the bathwater which now contains bacteria. Next, apply five microliter drops of each dilution by using a multichannel pipette onto plates with MacConkey agar and plate count agar using the 6x6 drop method.
The drops should absorb quickly into the agar. For the cultivation of the dilutions by the 6x6 drop method, ensure that the plate count agar and MacConkey agar plates are appropriately dried so that the drops will absorb quickly into the agar. Then, culture the inoculated plates at 37 degrees Celsius for 24 hours.
Prepare a meat drying oven for the application of thyme essential oil by vapor. Soak a 12x20 centimeter filter paper with the desired volume of TEO containing 79%thymol. Now take the samples left in the foil and transfer them together with the filter paper to the prepared drying oven with TEO to dry for six hours.
In parallel, treat a control set of samples in a meat drying oven without TEO. After the drying, to assess the survival of E.coli on the bacteria-treated samples, repeat the same procedure of washing the samples in peptone water with one altercation. In this case, after the 10 minutes of shaking, transfer the samples in the bath to an incubator at 37 degrees Celsius for a six-hour pre-enrichment incubation.
After the pre-enrichment, make five 10-fold dilutions of the bathwater as before. Then, use the 6x6 drop method to plant the dilutions on the detection plates and incubate the plates for 24 hours. The next day, determine the colony forming units per gram of meat.
Examine the plates for the presence of mesophilic aerobic bacteria which appear as white spots on plate count agar, and count the typical E.coli colonies on the MacConkey agar which appear as red to dark pink. To analyze the microbiological data, convert the counts to the log of the CFU per gram and use an analysis of variants to determine the main affects of treatment. During the drying process, not all E.coli is eliminated.
Some bacteria enter a viable but nonculturable state. So, when meat is stored improperly under high temperature and/or humidity, these bacteria can become viable again and potentially harm consumers. Treatment of the meat with different doses of TEO during drying could completely or partially eliminate E.coli.
The number of mesophilic aerobic bacteria and enteric bacilli on meat samples was determined on PCA and MCA plates. When 1.5 milliliters of TEO was applied, no growth of bacteria was observed even after a six-hour pre-enrichment phase. This dose corresponds to 0.028 milliliter of TEO vapor per liter of air.
After watching this video, you should have a good understanding of how to treat meat with vapor phase of essential oils during drying and evaluate the number of viable bacteria in the final product. In addition to this procedure, other methods like sensory evaluation and GC analysis can be performed in order to answer additional questions about consumer acceptability of the final product and amount of thyme essential oil residue. Don't forget that working with a meat cutter and pathogenic bacteria can be hazardous, and precautions such as wearing appropriate protective equipment and use of a Laminar safety cabinet should always be taken.
