Effectiveness of Acid/Chlorine rinses at Eliminating E. coli O157:H7 from Alfalfa Seeds Prior to Sprouting
Megan Lang, Steve Ingham, Barbara Ingham; University of Wisconsin-Madison

Scientists at the University of Wisconsin-Madison have studied the effectiveness of two-step acid/hypochlorite treatments as alternatives to 20,000 ppm active chlorine for eliminating E. coli O157:H7 from alfalfa seeds prior to sprouting. Commercially available alfalfa seeds were dipped in a mixture of E. coli O157:H7 bacteria and then dried to attain roughly 1,000,000 bacteria per gram of seeds. These treated seeds were then soaked using one of several treatments in an attempt to remove the pathogenic bacteria.

Introduction
Acetic acid and lactic acid rinses were tested at various strengths and at various temperatures, either alone or followed by various concentrations of hypochlorite. Acetic acid is commonly found in vinegar (5% acid) and lactic acid is a common food ingredient, produced in the manufacture of cheese and other fermented foods.

Initially, 33 combinations of acid/hypochlorite rinse were evaluated based on seed viability and germination. The 5 treatments that allowed for greatest viability/germination (>90%) were selected for further study. Those 5 treatments were:

1) 5% (v/v) lactic acid for 10 min at 42°C
2) 5% acetic acid (v/v) for 10 min at 42°C
3) 2.5% lactic acid for 10 min at 42°C followed by 2,000 ppm active chlorine (from calcium hypochlorite) for 15 min at 25°C
4) 5% lactic acid for 10 min at 42°C followed by 2000 ppm active chlorine for 15 min at 25°C
5) 20,000 ppm active chlorine for 15 min at 25°C.

Results
Each rinse treatment reduced the number of pathogenic bacteria inoculated onto the seeds by about 1,000,000 bacteria per gram. A second series of laboratory tests indicated that at least half of the bacteria were injured (not killed) by the rinse treatment. A combination of both lactic acid AND active chlorine was more effective at removing bacteria than either acetic acid or lactic acid alone. Treatment with 20,000 ppm active chlorine appeared to kill all the contaminating bacteria on the seeds. But, regardless of the rinse treatment given to the seeds, E. coli O157:H7 increased to at least 10,000,000 bacteria per gram during sprouting – even after those treatments where all contaminating bacteria appeared to be destroyed before sprouting. Results of this study show that:

1. a) bacteria may be injured by rinse treatments and these injured cells may repair during sprouting. Testing the effectiveness of any rinse treatment must take into account these injured cells. Failure to account for the injured cells may lead to a false sense of security regarding the effectiveness of a seed rinse treatments.

   b) an acid wash can reduce bacteria by 100 to 10000 per gram; without using 20,000 ppm active chlorine

   c) rinsing the seeds with both lactic acid and hypochlorite destroyed more bacteria than using just acid

   d) none of the rinse treatments tested can prevent re-growth of surviving E. coli O157:H7 during sprouting. This is true for the acid rinses, the acid/chlorine rinses, and the 20,000 ppm active chlorine rinse.