Sausage Fermentation and Greening – a Laboratory Exercise


Based on Experiment 12 in the Laboratory Manual for the Food Microbiology Laboratory (1998 edition, edited by John L. and published at the University) which was used in Bacteriology/Food Science 324 at the University of Wisconsin – Madison.

 Lab procedure as given in the manual is presented herein.


The fermentation step in sausage production involves the action of a lactic acid bacterium on the sugar added to ground meat. This organism, generally a strain of Pediococcus cerevisiae added as a starter culture, ferments the sugar to lactic acid. The acid causes the meat to develop the characteristic texture associated with sausage. The flavor of sausage is due to the lactic acid, other byproducts of bacterial fermentation, salt, seasonings and nitrite. Salting also affords protection against many spoilage organisms.

Most sausage formulas include sodium nitrite (NaNO2) as a curing agent. The addition of nitrite to a meat product inhibits the growth of the pathogen Clostridium botulinum. Also, nitrite is responsible for the pink color associated with sausage and other cured meats, such as bologna and ham. Third, a nitrite cure adds a particular flavor to the sausage.

In rare instances, sodium nitrate (NaNO3) is added during manufacture rather than sodium nitrite. One then relies on bacteria in the meat to reduce the nitrate to nitrite. It is more difficult to control the concentration of nitrite in a sausage by the use of this method. On occasion, excessive nitrate reduction will take place, resulting in overly high concentrations of nitrite in the product. The excessive amount of nitrite, in the presence of the lactic acid, will cause severe oxidation of the meat pigment (myoglobin), producing a green pigment (oxymyoglobin). This form of greening of cured meats is referred to as nitrite burn.

A second form of greening results from the accumulation of hydrogen peroxide on meats. Lactics on the surfaces of meats will, in the presence of atmospheric oxygen, produce hydrogen peroxide if they are allowed to grow. Meat must therefore be handled appropriately, keeping initial lactic counts low and avoiding temperature abuse to preclude growth of these organisms and subsequent greening of the meat. Lactobacillus viridescens is commonly associated with peroxide greening of meats.

In this exercise, sausage will be prepared, using both a straight nitrite cure and also an excessive nitrate cure to induce nitrite burn. In the latter case, a nitrate-reducing strain of Staphylococcus will be added. Sausage prepared with the use of a starter culture (Pediococcus) will be compared with that prepared with a "wild fermentation." In addition, the effects of greening by nitrite and hydrogen peroxide on sliced, packaged bologna will be demonstrated.

This experiment is done most economically and instructively with groups of four students.

PERIOD 1

Cultures

  • Pediococcus cerevisiae grown at 37°C in APT Broth containing 5% NaCl
  • Staphylococcus sp. strain N9A grown at 37°C in BHI Broth

Materials

  • Fresh ground beef (500 g)
  • Four 100 ml beakers
  • One 800 ml beaker
  • One thick glass stirring rod
  • Parafilm
  • Balances and paper for weighing (For the dry chemicals, approximate amounts may be achieved by the use of measuring spoons as indicated in the procedure below.)
  • Glucose, NaCl and sodium nitrate (NaNO3)
  • One tube of 2.5% sodium nitrite (NaNO2), 3 ml
  • Pipettors and sterile tips

Procedure

1.  Weigh 250 grams of ground beef and place in an 800 ml beaker. Add 7.5 g of NaCl (1 level teaspoon), 3.8 g of glucose (1 heaping teaspoon) and 1 ml of the sodium nitrite solution. Mix thoroughly and withdraw half of the mixture and pack it into a 100 ml beaker, excluding as much air as possible in the packing process. This serves as the control mixture. To the remaining mixture, add one ml of P. cerevisiae and mix thoroughly. Withdraw and pack into a second 100 ml beaker, again excluding as much air as possible in the process. Label both 100 ml beakers.

2.  Thoroughly clean the 800 ml beaker and add another 250 grams of ground beef. Add 7.5 g of NaCl (1 level teaspoon), 3.8 g of glucose (1 heaping teaspoon), 2.5 g of sodium nitrate (1/2 level teaspoon) and 1 ml of the sodium nitrite solution. Mix well and pack half into a 100 ml beaker; again this will serve as a control sample. To the remaining mixture, add one ml of the Staphylococcus culture. Mix and pack into the remaining 100 ml beaker. Label appropriately.

3.  Cover all beakers with parafilm to prevent excessive dehydration during incubation. Incubate the beakers at 37°C for 2-3 days, taking care to place (at least) a double layer of paper towels under the beakers.

PERIOD 2

Procedure

1.  With a spatula, carefully remove each meat mixture from its beaker onto a paper towel. Compare the textures and colors. Be able to explain what has happened in each situation to cause the results observed. (Note the significance of the controls!) Caution: Do not taste or eat the sausage! (This procedure is reserved only for sauerkraut and yogurt!)

2.  Discard the meat into the plastic bag provided when you have completed your observations.



SUMMARY

In fermented food products – sausage, for example – note how one or more of the following listed items can directly or indirectly affect fermentation by desired organisms (starter and/or wild), preservation qualities (retardation of spoilage organisms), texture, taste and sometimes color:

  • SUGAR PRESENT
  • ACID PRODUCED
  • ADDITION OF NaCl
  • ADDITION OF NITRITE (if any)

In this experiment, remember that our four batches of "beaker sausage" have the following in common: fresh ground beef, an indigenous population of lactic acid bacteria (which often rival the starter culture in performance), glucose, NaCl and nitrite (as NaNO2). To some batches we added one or more of the following: starter culture (Pediococcus cerevisiae), nitrate (as NaNO3) and an "overachieving" nitrate reducer (Staphylococcus sp. strain N9A). Relevant activities are summarized on the following table:

ACTIVITIES OF STARTER
CULTURE AND/OR
DESIRED WILD
FERMENTATION
AFFECT
ON / OF
NITRITE
ACTION
ON MEAT
PIGMENT
 NO3
(nitrate)
(if added) 
glucose bacterial
nitrate
reduction
*
 
fermentation by
lactic acid
bacteria
NO2
(nitrite)
 
lactic acid 
 HNO2
(nitrous acid)
 
 spontaneous
reaction in
H2O
myoglobin
(purple-red
pigment)
 NO
(nitric oxide)
 cured meat pigment
(pink; fully developed
with heat)
* Reduction by strains of Micrococcus
and occasionally Staphylococcus
excess NO
 oxidized porphyrins
(green)

Here is a photo of two samples of beaker sausage, vintage 1997. The sample on the left has the desired cohesiveness and color. The sample on the right was made with an excess of nitrate and the addition of the nitrate-reducer Staphylococcus sp. strain N9A. (We still haven't gotten around to speciating that strain after all these dozens of years!) Besides the green color, note how the second sample has expanded due to gas production. What gas did that, and how was it produced?



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