Psychrophiles in Commercially Produced Sauces, Glazes, and Marinades

 These are plates from the 2nd dilution of 13 different sauces on week 4 of our study.  We plated on July 3rd and did the 2nd dilution so that we would not have a potential of too many to count after being incubated for 48 hrs at 37C.  The plates were pulled out on July 5th and placed into the refrigerator at 4C.  I did not view the plates until July 9th and observed yellow, bacterial colonies growing.  These photos were taken on July 10th.

The colonies are small, circular, opaque yellow, entire, raised to convex, and glistening.  Perhaps tomorrow I can stop by the lab to streak for singles and the next day do a Gram stain.  From there we will decide what metabolic media to use in identifying this organism.

What is interesting about this growth is that it did not occur the first two weeks of tests.  Those plates had 1 or 2 yeast colonies growing on a few of the sauces.  For the most part the plates have been clean.  After seeing this growth, we looked at the plates from week 1 and week 2.  Week one showed a single bacterial colony growing on one streak for 2 different sauces.  Week 2 showed these yellow colonies on ALL of our plates.  It appears this organism is a psychrophile.

In preparing for this long-term shelf-life study, we aliquoted 25 ml of 13 different sauces into 50 ml conical tubes.  One set remains in the refrigerator for future testing if we see a need to do so.  The other sets were stored at room temp at 30C and 2 sets were placed in the incubator at 37C for week 1 and week 2.  So for week 1 and week 2, we ran a set of tests of the sauces stored at 30C and at 37C.  Based on previous results of these same sauces in a similar study, we did not plan for additional weeks at 37C.  Last week we completed week 4, which was only for the sauces stored at 30C.


From Suite101.com:

Psychrophiles – Properties and Characteristics of Psychrophilic Bacteria

The best earliest review on psychrophiles is that of Ingraham and Stokes who describe these features of psychrophiles:

• are typically Gram-negative bacteria and include members of the genera Pseudomonas, Flavobacterium, Psychrobacter, Colwellia and others.
• often produce green, yellow, orange and purple-violet pigments at reduced temperatures
• can tolerate elevated levels of salt
• can grow well and sometimes optimally at temperatures up to about 20 degrees Centigrade
• can grow at temperatures as low as -3 to -7 degrees Centigrade
• produce extracellular enzymes that can degrade meats, milk, cheeses and other food products during cold conditions

I found a genus of bacteria that may fit but only testing will determine.  Chryseobacterium spp. (Flavobacterium) This genus is a Gram-negative, rod bacterium with yellow pigment.  It is non-motile.  C. indologenes tests positive for oxidase, negative for catalase, cannot ferment Mannitol or Urease, does not grow on MacConkey.  It weakly ferments glucose as seen on TSI slants.  They can grow at 4C but optimum growth is at 25C.  They are also heat stable and have a salt tolerance.  They are commonly found in soil, plants, foodstuffs, and water sources.


Like I said, we have to streak for singles and do a Gram stain first to determine next steps in identifying this organism. 








 

Multivariate Study on Antimicrobial Properties of Raw Honey, Stevia, and HFCS

Today we begin our next experiment to test the antimicrobial properties of raw honey, stevia, and high fructose corn syrup.  We will begin with extracting different groups of phytochemicals from the stevia plant and the stevia flower.  For the raw honey and the HFCS, we will make solutions of different concentrations.  We will be using disk diffusion to look for zone of inhibition against E. coli and S. aureus.

A couple of weeks ago we tested xylitol and HFCS.  We used the following concentrations:  0, 5, 20, and 25%.  There was no inhibition on the disk diffusion against E. coli and S. aureus.  But we did observe that the HFCS encouraged growth.

The other test we did with these two sweeteners was to see if E. coli and S. aureus could metabolize xylitol or HFCS.  We aliquoted 5 mL of PBS into 6 test tubes.  For 2 we added nothing else.  For another 2, we added 1.25 grams of xylitol and for the other 2, we added 1.25 mL of HFCS.  We inoculated 3 tubes with 100 microliters of E. coli and the other 3 with 100 microliters of S. aureus.  The results were interesting.  After incubating at 37 C for 48 hours, we observed growth of both organisms in the HFCS.  The xylitol also showed growth but not as much.  I plated 10 microliters onto a nutrient agar plate of each tube.  I did not do any dilutions.  After 24 hrs at 37 C, there was too many to count growth on the plate. Plus, either by my error or from contaminated PBS, there was also growth of yeast present on the plates.

Two weeks later, the PBS shows no growth for either organism.  The xylitol shows small growth and the HFCS shows moderate growth. We plan to plate for growth but this time will probably take it to the 2nd dilution.

I read some interesting peer-reviewed research papers on the antimicrobial properties of stevia so I am excited about testing this.  I will also be learning how to do protein gel electrophoresis and will be doing chromatography.

ASLT Continues

I am in my second semester of research in food microbiology.  The Spring semester of 2012 was dedicated to accelerated shelf-life testing of numerous sauces for a local company that manufactures these sauces for big-named customers.  Our first assay showed mixed results.  Our second assay of 5 sauces that were fresh off the line and sealed performed much better.  The attached photo shows growth of yeast in small numbers.  We did not have any bacterial growth.  All 5 sauces held up at the 37 C temp with no changes to color or odor.

Since we did not repeat results from the first assay to the second, we discovered that the sauces given to us in the first one were significantly old and were not hermetically sealed.  Additionally, I cultured one of the plates with bacterial growth and did a Gram stain.  We found Gram + and a Gram - organisms with a diplococci morphology.  We did some tests to try and identify the bacteria.  Motility, TSI, citrate and some others I am not recalling at the moment.  The results did not lead to any common bacteria that we, as budding microbiologists, were familiar with.  Dr. Bates, our supervising instructor, concluded it was environmental as in dirt.  We did take a pic of the Gram stain and I need to ask for a copy so I can post.

Right now we are repeating the first assay with the same 19 sauces.  This time the sauces are fresh off the line and hermetically sealed.  They are performing just as well as the sauces did in our second assay.  No bacteria and low yeast growth with some no growth at all.  No significant odor changes and a few of the sauces showing color change.  This is the start of a long-term study requested by the company.  Although we gave them our ASLT results, they tell us their customer wants real-time data so this study is set up to take place over the course of 12 months.

The next post will be about the scientific experiments we designed that have nothing to do with the shelf-life studies.  My research partner and I created a spreadsheet of all the ingredients in all of the sauces so we could analyze the results to see which ingredient we want to isolate and experiment with.  Right now we are getting ready to test the antimicrobial properties of sweeteners.  Raw honey, xylitol, high fructose corn syrup, and stevia.  More to come.