Phase 4

Development of the simulated practical component of the learning strategy

Course:

Development of new products

Presented by:

Angela Fernanda Pérez Abril

Code: 1057591006

Group:

216005_4

Tutor

Andrea Vasquez

National Open and Distance University

2020
 SIMULATION IN COMBASE

I. Registration and admission to the simulator ComBase

Registration and admission to the ComBase simulator is performed following the

instructions given by the practical guide where the user and password are

generated

II. Review of the ComBase tools

A general review of the ComBase software browser was carried out, which could verify

that it is composed of microbial growth and survival curves

Check that the ComBase Predictor is a cake which has a collection of tools to predict the

growth or inactivation of microorganisms. It was also observed that the software has

predictive models

III. Preliminary Simulation. To use the tool ComBase Predictor,

specifically the growth model

ComBase Predictor is used as a highly effective tool for the design, comparison and

validation of predictive models. I know

Perform the step by step, selecting the growth model, temperature, time and others,

including the micro-organism recommended by the example in the guide.

IV. ComBase Growth Model: Make a prediction

Below is the simulation of make a prediction where the Growth model was selected as the

category in temperature the static mode is selected, in aqueous activity NaCl is selected as

the microorganism Brochothrix thermosphacta microorganism responsible for the

deterioration of meat and fishery products was taken into account. Regarding the physical

state, the table was left empty, as for the temperature, it was included at 20 ° C, pH 7, in

the NaCl table (% ), the figure of 0.6 is left last the time which is included at 25 hours.

Results in Table:
Analysis of the results

Making use of the data provided by the guide we can observe the 3 phases of the bacterial

growth curve, where in the time of 0 to 4 hours the analyzed microorganism is in the

latency phase, also at a neutral pH of 7 and at a temperature of 20 ° C it will reach the

exponential phase of Brochothrix thermosphacta microbial growth from 4 hours of

exposure under the conditions exposed in the simulation reaching its peak of exponential

growth up to 20 hours. Finally, the latency phase is evident from 20 to 25 hours of

exposure.

Making a comparison with the analysis of the same microorganism this time with the

growth model with an Aw of 0.997 instead of NaCl at a time of 60 hours also has a

similar behavior where the latency phase is from 0 to 6 hours a slightly longer than that

exposed to NaCl but the exponential phase was observed from 7 to 19 hours and

observing a fairly long stationary phase from 19 hours extending to 60 hours of analysis.
V. Simulation for the pathogenic microorganism involved in the deterioration of the

new food product

Estimated simulation of useful life

Predictive microbiology simulation will be performed using the ComBase microbiology

simulation program, which is available online. First, it is chosen from a list of

microorganisms, in this case E. coli since it is the problem microorganism and has an

incidence in dairy products, then working conditions such as sodium concentration

(NaCl), temperatures, are determined. and hydrogen potential (pH), from there a graph of

the growth of the microorganism under these conditions will be generated.

Enterobacteriaceae are naturally present widely in nature, for example in contaminated

and flooded water. Coliforms are part of this family and are natural inhabitants of the

intestinal flora of animals and humans. They include Escherichia Coli, a gram negative

bacterium that is facultative anaeobia and ferments lactose, some strains of this battery are

enteropathogenic and among them is E. Coli O157: H7 which can cause bleeding.

Also within this family is Salmonella and Shigella which can cause intestinal disorders.

Gram-positive spore-forming bacteria belong to the Bacillacea family. The most

important are Bacillus and Colstridium. (Walstra, Wouters & Geurts, 2006, p.).

The lactose-free honey yogurt with oatmeal, rice and quinoa it is a product of animal

origin and rich in proteins, with plant components, due to its composition, it can promote

the growth of pathogenic microorganisms in case of contamination.

The microorganism Escherichia coli is chosen because it is one of the main indicators of

contamination by bad GMP, it ferments lactose at 44.5 ° C with acid and gas production.

Resolution 2310 of 1986 requests the absence of this microorganism in the product, and
 since the product is lactose-free and that the enzyme B-galactosidase is included - a

potential trigger for the fermentation of lactose by pathogenic microorganisms - its

analysis will allow determine not only the quality of the product but its shelf life. Table 1

shows the behavior of the E. coli microorganism as soon as the milk has been finished.

Table1. Initial condition (finished milk)

Source: own elaboration based on ComBase

E. coli is a producer of the Shiga toxin and can grow at temperatures between 7 ° C and 50

° C with the optimum growth temperature being 37 ° C. Some can proliferate in acidic

foods, up to a pH of 4.4 and in foods with a minimum Aw of 0.95. (OMS, 2018, para.8)

In Table 2, the simulations are based on changing the temperature to the same water

activity, incubation temperature, and pH. The initial condition of the milk is represented

after the thermization process that happens at 37 ° C, where the fat of the milk melts.

Aw 0.997, 37 ° C, pH 4.5 and 24 hrs.

Table2. Thermization process that occurs at 37 ° C

Source: own elaboration based on ComBase

Aw 0.997 37 ° C, pH 4.5 and 48 hrs

During the first 10 hours of incubation until 30 hours, the exponential phase of the

microbial growth represented in Table 3 develops.

Table3. Microbial exponential growth

Source: own elaboration based on ComBase

Aw 0.97 48 hrs

Table 4 shows the change in microbial growth under different conditions of water activity

and how it changes with respect to time; It is observed how when decreasing the aqueous
activity, the microorganism goes into lethargy and only until 20 hours the growth in

exponential phase begins to be observed.

Table4. Microorganism lethargy phase changing AW conditions and time.

Source: own elaboration based on ComBase

NaCl 1.2% 24 hrs

The growth of the microorganism is observed by varying the concentration of NaCl to

1.2%, where exponential growth begins at five hours, which is evidenced in Table5.

Table5. Microorganism varying the concentration of NaCl to 1.2%

Source: own elaboration based on ComBase

NaCl 2.4% 24 hrs

Table 6 doubles the previous concentration of NaCl, and it can be seen that the E. coli

microorganism takes longer to start its exponential phase, which begins to increase at eight

hours.

Table6. Microorganism doubling the previous concentration of NaCl to 1.2%

Source: own elaboration based on ComBase

NaCl 3.6% 24 hrs

By tripling the initial concentration, it is concluded that this increase contributes to

retarding microbial growth, as can be seen in Table 7, the trend is almost flat and only until

15 hours the amount of CFU begins to increase.

Table7. Microorganism tripling the previous concentration of NaCl to 1.2%

Source: own elaboration based on ComBase

pH 4.5

In Table 8 the exponential phase develops from five hours to 30 hours, and obtains from 8

to 9 CFU / g maximum. Since this pH is typical of dairy products, it is important to note

that it develops at 37 ° C, whose effect at 4 ° C (refrigeration temperature) will change.

Table8. pH 4.5
Source: own elaboration based on ComBase

pH 6.00

In this phase development begins from two hours to 10 hours, therefore it is clear that a

semi-neutral pH favors the growth of the microorganism, this occurs with raw milk and

before termination (see Table 9).

Table9. pH 6

Source: own elaboration based on ComBase

pH 7

In the same way, the same behavior is observed, and it is validated that the neutral pH

favors growth in less time (see Table 10).

Table10. pH 7
Source: own elaboration based on ComBase

In the previous tables it is observed that at higher water activity (Aw), alkaline pH and low

NaCl concentrations, bacteria tend to develop in a shorter time, that is, the proliferation of

microorganisms is faster, so that the food is would deteriorate easier under these

conditions.

Since Yogurt is an acidified product, the parameters would be with a pH 4.5, an incubation

temperature of 37 ° C, Aw = 0.98 (see Table 11).

Table11. pH 4.5 incubation temperature 37 ° C Aw = 0.98

Source: own elaboration based on ComBase

In this case, under the established conditions, it was required to increase the prediction

time, where it is observed that at hour 100 8.68 CFU / g are obtained.

According to the hours of the latency phase, it is compared against Table 12 obtaining:

Table 12. Data comparison

Source: own elaboration based on ComBase

The stationary phase, compared to the latency phase, doubles the concentration of

microorganisms, but requires seven times more, to reach the stationary point (see Table

13).

Table13. Stationary phase vs. latency

Source: own elaboration based on ComBase

The latency phase decreases as the temperature decreases, therefore, the microorganism is

thermophilic in nature. Hence the importance of keeping the product in refrigerated

conditions and maintaining the cold chain.

This analysis compared to the results of other accelerated useful life studies, for example,
that of Vásquez-Villalobos et al. (2015), found that Yogurt at higher temperatures (at 4 °

C) has sensory rejection of 25% at 49 and 69 hours, which shows, as in this ComBase

predictive study, that when exceeding the variables of control and especially the cold

chain, the product changes physicochemically and microbiologically, so a dairy product

must remain in refrigeration throughout its production and consumption chain.

a. Under what parameters of aw - NaCl - pH; Does microbial kinetics take

the microbial growth curve in its 4 phases: initial (Lag) - Exponential -

Stationary and death?

Answer: At the aw parameters of 0.997 - pH 4.5 and at a temperature of 37 ° C

the microbial kinetics takes the microbial growth curve in its 4 phases: initial-

Exponential - Stationary and death

b. What is the microbial growth reached at the end of the time established for

the simulation?

Answer: Taking into account that in neutral or alkaline pH, it favors

microbiological growth in less time, making use of the data in normal yogurt

conditions where its pH is acidic, with a temperature of 4.5 incubation 37 ° C

Aw = 0.98 in a 100 hour prediction time, 8.68 CFU / g are obtained.

c. In what time range, is the stationary phase of the microbial growth curve

presented and what is the microbial count expressed in Conc (Log10 cells / g)?

Answer: Under temperature conditions of 32 ° C at a pH of 4.5 and an Aw of

0.98 in a 180-hour time prediction of exposure under the above conditions, the

stationary phase is approximately at the 80th hour, maintaining time with 8, 68

CFU / g.
References

Walstra, P., Wouters, J., & Geurts, T. (2006). Dairy Science Technology. Chapter 22. Boca
Raton, United States: CRC Press.

Vásquez, V., Aredo, V., Velásquez, L, & Lázaro, M. (2015). Physicochemical properties
and Sensory acceptability of fruity skim milk yogurt with mango and banana in
accelerated tests. Scientia Agropecuaria, 6 (3), 177-189.

OMS. (2018). E. coli. Obtained from https://www.who.int/es/news-room/fact-

sheets/detail/e-coli

ComBase. (2020). ComBase. Retrieved on 06/03/2020, from ComBase:

https://browser.combase.cc/membership/Login.aspx?ReturnUrl=%2f