Effectiveness of Acid Chlorine Rinses At Eliminating E Coli From Alfalfa Seeds Prior to Sprouting

Effectiveness of Acid/Chlorine rinses atEliminating 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 effectivenessof two-step acid/hypochlorite treatments as alternatives to 20,000 ppm activechlorine 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:H7bacteria 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 anattempt to remove the pathogenic bacteria.

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

Initially, 33 combinations of acid/hypochlorite rinse were evaluated based onseed viability and germination. The 5 treatments that allowed for greatestviability/germination (>90%) were selected for further study. Those 5treatments 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 for15 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 ontothe seeds by about 1,000,000 bacteria per gram. A second series of laboratorytests indicated that at least half of the bacteria were injured (not killed) bythe rinse treatment. A combination of both lactic acid AND active chlorine wasmore effective at removing bacteria than either acetic acid or lactic acidalone. Treatment with 20,000 ppm active chlorine appeared to kill all thecontaminating bacteria on the seeds. But, regardless of the rinse treatmentgiven to the seeds, E. coli O157:H7 increased to at least 10,000,000 bacteriaper gram during sprouting - even after those treatments where all contaminatingbacteria appeared to be destroyed before sprouting. Results of this study showthat:

a)bacteria may be injured by rinse treatments and these injured cells may repairduring sprouting. Testing the effectiveness of any rinse treatment must takeinto account these injured cells. Failure to account for the injured cells maylead to a false sense of security regarding the effectiveness of a seed rinsetreatments.

b) an acid wash can reduce bacteria by 100 to 10000 per gram; without using20,000 ppm active chlorine

c) rinsing the seeds with both lactic acid and hypochlorite destroyed morebacteria 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, theacid/chlorine rinses, and the 20,000 ppm active chlorine rinse.

Summary
It is important to test the effectiveness of any rinse treatment on both theseeds and the resulting sprouts. The conditions for sprouting are ideal forbacterial growth, and may promote the repair of injured cells. Alternatives mayexist to hypochlorite, depending on the level of destruction required to ensuresafety. In other words, is it sufficient to reduce bacterial counts by 1000 pergram, or is a kill of 100000 required? This study used seeds artificiallyinoculated with high levels of pathogenic E. coli 0157:H7. No conclusions can bedrawn as to how effectively this methodology parallels a situation where seedsare naturally contaminated with low levels of pathogens.