Innovation and technology are allowing the human population to grow in large numbers and have increased longevity. Farms have become more industrialized and many preservation methods are used to preserve food and prevent spoilage when transporting over long distances. By the early 20th century, techniques such as pasteurizing, canning and vacuum sealing were used to extend the life of food products until they reached their destination. However, these preservation methods have certain drawbacks and promote food-borne illnesses that are caused by microbial contaminants comprising a wide range of bacterial, fungal, parasitic and viral agents.
Today, rigorous government standards are followed and continuous microbial testing is performed to ensure food safety. These tests comprise enzyme linked assays, various forms of microscopy (or spectroscopy), selective media culturing, and PCR (or genetic) screenings of food samples before being introduced into the market. However, in spite of these various detection assays, food-borne illnesses continue to pose a public health risk.
Apart from viruses, food-borne microbial agents share a common life process; they eat, grow and create waste. Although all living things do this using a process known as respiration, there are certain microbes which can do this in the absence of oxygen, known as anaerobic respiration. In aerobic respiration, energy is released from complex organic molecules in the presence of oxygen. When this process occurs, water vapor and carbon dioxide are produced as by-products and the organism loses weight or mass.
Using the loss of mass that are experienced by all forms of life during respiration, it is hypothetically possible to determine bioactivity in the form of weight loss. Since most human pathogens exhibit an optimal growth temperature at 37°C, it is feasible to incubate a contaminated food sample at this temperature and calculate the rate of loss. This can be done using traditional incubation techniques, wherein a sample is weighed on a regular basis so as to track the rate of loss as compared to a sterile control sample but without contamination.
In order to test this theory, a loss-on-drying moisture analyzer such as the Computrac MAX 4000XL equipped with a humidity air pump was used to maintain the samples at a controlled humidity. This analyzer maintains a constant temperature, enables downloadable graphs and data, and offers precise weight loss readings over an extended period of time.
For model organism, Arizona Instrument LLC selected a common Baker’s Yeast, Saccharomyces cerevisiae. Commonly used as a leavening agent in bread and pastries, S. cerevisiae is a fast growing unicellular yeast which is harmless in nature. In this study, an in vitro approach was used to demonstrate the proof-of-concept by determining the weight loss on potato dextrose agar plates inoculated by yeast and later testing the ‘real’ sliced bread using similar techniques.
The result of this experiment will reveal a new loss-on-drying method for bioactivity measurement. This approach will further improve the quality assurance of food safety in food industries who wish to expand their scope of microbial testing. Since Baker’s Yeast was utilized in this experiment, this technique may prove useful in determining the feasibility of certain yeast cultures for bread and brewing industries where conventional spectroscopy driven methods may only reveal the population density of yeast, but not their carbon emission capacity.
Methods - Media Preparation and Yeast Culturing
Potato Dextrose Agar
Potato dextrose agar (PDA) is a semi-solid media used for growing fungi in the lab. Agar is a seaweed extract and contains cellulose instead of proteins. It should be noted that the agar is not consumed when microbes are grown on it, it is only scaffolding that retains the liquid broth mixed into it.
Potato Dextrose Broth
Potato dextrose broth (PDB) is produced by stewing potatoes and adding dextrose to produce a nutrient broth that can be consumed by fungi, including S. cerevisiae. This liquid broth is a liquid culture in which unicellular yeasts can grow.
For this study, aluminum deep-well plates were utilized and sterilized at 121°C for about 30 minutes. Then, the molten PDA mixture from the oven was allowed to cool within a 500mL Erlenmeyer flask until it reached ~50°C. At this temperature, ~30mL of molten PDA was poured into the sterile empty plates within a sterile hood, with each plate covered with a heat sterilized aluminum ‘lid’ to prevent contamination. After the PDA solidifies, the plates and lids were closed with Parafilm strips to prevent moisture loss and stored in a refrigerator set at 4°C.
Liquid Culture of S. Cerevisiae
About ~3.5g of freeze dried yeast and 100mL of PDB was added to a 500mL beaker and mixed with a sterile loop. After agitating the culture for 2 hours, it is stored in a refrigerator set at 4°C for subsequent cell culturing.
Inoculation of PDA Plates with S. Cerevisiae
After creating a strong culture of yeast, a flamed loop was immersed into the liquid culture and spread uniformly over each plate. Three ‘loop fulls’ were utilized on each plate to supply an adequate amount of yeast. All inoculated plates were then placed upside down to prevent condensation from the lid (Figure 1).
Figure 1. Inoculating bread slices with S. Cerevisiae.
The bread slices were stored in a refrigerator set at 4°C and then taken out one hour before the experiment so that the entire loaf is warmed up to room temperature. For the control, a bread slice was placed onto a waffle pan and five drops of sterile PDB was placed onto the bread slice (Figure 2). For the experiment, the same drop placement was carried out on a bread slice, but with a uniform mixture of liquid yeast culture held in PDB.
Figure 2. Drops of sterile PDB placed onto the bread slice.
Programming and Procedure of Computrac MAX 4000XL
In order to track the weight loss in real-time, twelve 2-hour tests had to be combined to give a precise and continuous demonstration of weight loss over a period of 24 hours. Since S. cerevisae is not a true human pathogen, 37°C is too warm for optimal yeast growth and therefore the temperature setting is reduced to 30°C. The moisture analyzer serves as an incubator to grow the yeast at optimal temperature and helps determine the weight loss over a fixed amount of time.
Since each test will automatically read as an individual ‘% Moisture’ result, a custom equation was produced to track the entire percent of weight loss. The first 2-hour link does not need this custom equation; however the following links will need it to give a precise real-time reading of weight loss.
MAX 4000XL Testing
Each test was initiated when the first link in the 12 linked series was allowed to reach 30°C. As soon as the temperature was achieved, the instrument tares the pan and prompts the analyst to add the sample within the specified sample window i.e. 10 to 40g. For the PDA samples, pans were uncovered and inverted onto the waffle pan on the 4KXL. The change in sample weight, which was within the acceptable weight limits, was recognized by the instrument. Once the lid was closed, the test continued for 24 hours. Figure 3 shows the humidified MAX 4000XL connected by Tygon tubing.
Figure 3. MAX 4000XL connected by Tygon tubing.
The data was stored on the MAX 4000XL analyzer and was simultaneously reported to the network. The instrument reports the weight and rate of the sample every ~30 seconds. However, in case a new test begins, the analyzer does not instantly recognize that it is a link test and therefore % rate and weight loss drops back to zero.
Figure 4. PDS weight loss (+/- Std. Dev.)
Figure 5. Bread weight loss (+/- Std. Dev.)
The graphs (Figures 4 and 5) were produced using a ‘Scatter Plot’ graph in Excel, taking the averaged ‘total % weight loss’ from three separate runs and plotted against time (in hours). The four dependent variables observed were: PDA (not inoculated) – control; PDA w/ yeast inoculation; bread slice with 5 drops sterile PDB – control; and 4) bread slice with 5 drops liquid culture.
The error bars denoted in each graph matches with the standard deviation taken at two-hour interval among the three run of each variable.
Results and Discussion
The first issue in this analysis was to support the hypothesis that bioactivity can be determined by weight loss. It is understood that any food product placed in the chamber would lose weight from just evaporation alone. Hence, a baseline evaporation curve was needed for the PDA plates, particularly made as an optimal media for S. cerevisiae. The ‘blue’ line of the graph corresponds to three separate PDA plates without any S. cerivisae.
Once the baseline rate of evaporation i.e. the change in total % weight loss was measured, it was projected that if a living organism consumes the dextrose in the media, water vapor and carbon dioxide would be created and the agar plate would start to lose weight at a faster rate when compared to the control plate. Although water vapor or carbon dioxide may not contribute to this loss, it can still be shown that significant weight loss occurred at ~8 hrs into the incubation as compared to the control.
Using incubation-based assays to determine the presence of microbial growth on food products is not a novel method in food safety screening. In truth, spectroscopy methods and selective media culturing often depend on growing potential forms of contamination in large number so as to be detected or visibly perceived using light spectroscopy. However, using loss-of-weight to determine the microbial contamination on food is an innovative technique, which requires a baseline evaporation curve of each product being studied and eliminates the use of specialized vessels or expensive selective media for optical density.
This information has been sourced, reviewed and adapted from materials provided by AMETEK Brookfield Arizona
For more information on this source, please visit AMETEK Brookfield Arizona