Expert view: Validation of Baking as a Kill Step
Scientists from VH Berlin e.V. Research Institute for Baker’s yeast are conducting research into the effectiveness of baking killing the microorganisms that naturally appear in dough. They conducted different experiments on dough and obtained interesting results.
By Dr. Michael Quantz; Ann-Maria Dinse; Christine Stadelmeyer
Located at the Institute for Fermentation Industries and Biotechnology (IfGB), VH Berlin is a non-profit association (e.V.), registered in Germany that provides services and applied research in the field of yeast technology to its 40 international members who hail from Europe and further afield. 
Grains and milled products are raw agricultural commodities, therefore, a variety of microorganisms, including moulds, yeasts, coliform and others can be present at low levels. The risk of bacteria occurring happens naturally. In the case of the industrial production of baked goods, microorganisms, such as Salmonella spp., can lead to consumer infection and the risk of this occurring is also increased by the rapid proliferation of microorganisms during the proofing of the dough. In the manufacturing of baked goods, baking is an important process step in destroying pathogens that may be present.
Therefore it is necessary to further confirm that baking represents a kill step, as per the report prepared by the Institute of Food Technologists for the U.S. FDA entitled “Evaluation and Definition of Potentially Hazardous Food – Chapter 4. Analysis of Microbial Hazards Related to Time/Temperature Control Foods for Safety”, in particular, Section 5.3 which examines the effects of processing related to cereal grains and related products.
Choosing the bacteria
The objective of this study was to validate the efficiency of baking as a kill step. For this purpose, the Research Institute for Baker’s yeast (VH Berlin e.V.) used two different dough methods, i.e., “no time dough” and “sponge & dough”. Those two different recipes were chosen to examine the potential effect of proofing. The doughs were spiked with Salmonella enterica subspecies enterica, strain ident. no. DSM 11320. Salmonella was selected as a model germ because of its resistance to drying and its relevance as a pathogen in order to obtain a representative candidate for microbial contamination of dough.
Materials and methods
Salmonella enterica subspecies enterica, strain ident no. DSM 113200, was supplied by “DSMZ – German Collection of Microorganisms and Cell Cultures” in freeze dried form. The pellet was resuspended in 5 ml of sterile peptone-water and grown overnight at 37 °C to a total of 1.8 x 108 CFU/ml, as detected using RIDA Count® Salmonella/Enterobacteriaceae dry plates from Co. R-biopharm. On the day of analyses, serial dilutions to a concentration of 5,000 CFU/ml (“no time dough”) and of 3,100 CFU/ml (“sponge & dough”) were prepared.
The bread-making procedure was according to the “no time dough” and “sponge & dough” recipes (Table 1). In order to achieve comparable dough texture and viscosity, all ingredients were mixed rapidly in a Stephan mixer, 1.5 kW model, for 20 s at speed 2, with a short break for scraping the dough, and for 40 s at speed 2 (total 1 minute).
Proofing the dough
The “no time dough“ was split by hand into four dough pieces of 400 g, rounded into cylindrical bread loaf shapes and put into baking pans. All doughs were proofed for 1 h at 44 °C (112 °F) and spiked with the Salmonella solution using a Hamilton syringe. Figure 1 shows the proofed spiked dough from “no time dough”, after proofing for 60 minutes at 44 °C (112 °F). The dough pieces of 400 g were inoculated with 5 x 0.2 ml Salmonella solution (c= 5,000 CFU/ml) at five different positions from the top – once in the center and four times at a 3-cm distance to the baking pan wall. The spiked sponge (70 % of total flour) was inoculated with 1 ml of Salmonella solution (3.100 CFU/ml) and then proofed for three hours at 30 °C (86 °F), mixed with the residual flour, split into four loaves as before and finally proofed for another hour at 44 °C (112 °F). During the four hours of proofing, growth of the injected Salmonella was estimated. From experience, the final CFU concentration in the dough was assumed to go up to ~8.500 CFU per 400g of dough.
Analysing the baked product
Two doughs of each series were baked for 17 minutes. The baking oven, Miwe model “Concepto”, was preheated to “level 3”, two hours before the baking test, to achieve upper and lower heat: 225 °C (440 °F).
Figure 2 shows the injection of methylene blue dye, to illustrate the distribution in the baked bread after baking a “no-time dough” for 17 minutes. The injection was prepared with a Hamilton syringe. The depth of injection was chosen to spot the dye into the center of the dough. This method was also used for spotting the Salmonella solution.
The Figures 3 and 4 show breads prepared from “no time dough” (bread core temperatures after baking varied between 86 °C and 90 °C[187 °F – 194 °F]) and “sponge & dough” (bread core temperature after baking was 87.2 °C [189 °F]) after baking for 17 minutes at 225 °C (440 °F).
After cooling down, one bread (“no time dough”) was packed as a whole (375 g), whereas the other bread was sliced into five slices of about 74 g (+/- 7 g) and then packed, each slice separately, into sterile bags (whirl-Paks) for dissolving, enrichment and analysis according to the technical instructions of the detection test. The two breads (“sponge & dough”) were packed as a whole (375 g) into sterile bags.
The contaminants in spiked dough samples before and after baking were analyzed using specific colony plate methods, after proper enrichment, according to “ASU Amtliche Sammlung Untersuchungsmethoden” (German Food Stuff analyses SOP collection) no. ASU L 00.00-20.
Results
The results show that the baked breads and slices of bread from the “no time dough” (Table 2) and “sponge & dough” (Table 3) recipes contain no Salmonella in contrast to the raw doughs.
