Application of pGFPuv mutant to study Cronobacter sakazakii survival in corn flour during storage

Cronobacter sakazakii is more resistant in dry conditions for more than 2 years. However, study on the behavior of C. sakazakii in dry product using wild type may encounter problem of inability to distinguish target C. sakazakii with naturally occuring C. sakazakii. This research is useful in studying of C.sakazakii pFGPuv in corn flour having different initial moisture content stored in different relative humidity to ensure food safety and sustainability. The corn flour had a water content of 9% and 12 (w.b.) which had been inoculated with C. sakazakii pFGPuv for 16 weeks at 3 different RHs (50%, 70%, and 90%). The survival ability of C. sakazakii of GFPuv was better in corn flour with an Aw of 0.42-0.44 stored at RH 50% compared to RH 70 and 90%. C. sakazakii pGFPuv survived better at RH 50% which had Aw of 0.42-0.44 which was stored at RH 50% compared to RH 70 and 90%. The number of C. sakazakii pGFPuv in corn flour decreased by 0.470-0.489 log cycle/week. Therefore, the number of C. sakazakii in corn flour that will be stored needs to be considered to produce food that is safe to support good health and well-being.


Introduction
The pathogenic bacterium, Cronobacter sakazakii, can cause disease in unhealthy and immunocompromised people where it becomes virulent among neonates, infants and children [1,2].The organism is an opportunistic pathogen that can contaminates a dry foods such as baby formula milk, dry baby food, dry herbs and spices [3].There have been several reports of its survival in dry conditions for more than 2 years [4,5].Products with low water activity conditions and storage temperatures are the preferred conditions for C. sakazakii to survive.In infant formula, the population of these pathogenic bacteria survive better at low water activity 0.25-0.30than high water activity 0.69-0.82during 12 months of storage [6,7].C. sakazakii is still alive in skim milk after spray drying at an inlet temperature of 160 o C and outlet temperature of 82 o C, and survived for 3 months of storage at RH 50%, 70% and 90%.A decrease in the activity of water of skim milk after the drying process and during storage can increase the survival of C. sakazakii Yrc3a during dissolution with water at a temperature of 50 o C [8]. Cronobacter spp.has been isolated from corn and corn products such as corn starch, corn grits, and corn 1302 (2024) 012101 IOP Publishing doi:10.1088/1755-1315/1302/1/012101 2 flour [9,10,11,12].In Indonesia, one positive corn starch sample contained two isolates of Cronobacter spp.The presence of C. sakazakii in cornstarch products is probably caused by contamination from the environment, after the drying process and before the product is packaged [13].
The labeling of Cronobacter sakazakii aims to be a marker to easily differentiate it from other bacteria.With these markers, the behavior of target bacteria is easier to study without interference from other microbes in the environment.Labeling can be done using fluorescent compounds that can covalently bind to identifiable DNA, such as Green Fluorescent Protein (GFP) which does not interfere with the host's growth characteristics [14,15].This GFP-labeled C. sakazakii can grow on media containing ampicillin and shows specific fluorescent green colonies under UV light Several studies have used Cronobacter sakazakii bacteria labeled with GFP to study their characteristics.C. sakazakii pGFPuv in corn kernels could survive at RH 50% for 12 weeks storage in lower Aw conditions, i.e. activity of water was 0.45 [16].The toxic isolate C. sakazakii pGFPuv FWHd16 was more resistant at temperatures of 40 o C, 45 o C, and 50 o C compared to the nontoxic C. sakazakii pGFPuv YRt2a [17].
Therefore, this study used Cronobacter sakazakii which has been labeled with GFP because the use of the wild-type strain has limitations in that it cannot be differentiated from C. sakazakii which may occur naturally in the food product.This research aimed to evaluate C. sakazakii survival during storage of flour with different initial moisture contents of 9% and 12% and different RH (relative humidity) of 50%, 70% and 90% using Cronobacter sakazakii pGFPuv.The results of this research are intended to be used as a basis for determining the initial water content of corn flour and relative humidity when storing corn flour so that food safety and food sustainability are maintained.

Methods 2.2.1.Making corn flour.
The Pioneer P27 Gajah corn variety with a harvest age of 98 days is shelled manually.Dried shelled corn is sorted and ground using a hummer mill.The corn kernels that have been ground are then separated from the body, pericarp and base of the kernels by washing to obtain corn grits.The corn grits are soaked for 2 hours then the corn grits are washed, drained and dried until the moisture content is around 14% bw.The corn grits are floured using a disc mill.The resulting flour is then dried with a cabinet dryer, then the flour is sieved with a 60 mesh sieve to obtain corn flour which has a uniform particle size.

Cronobacter sakazakii pGFPuv isolate confirmation.
Frozen stocks of Cronobacter sakazakii pGFPuv mutant isolates were incubated in BHI medium + 100 µg/ml ampicillin at 37 o C for 24 hours.Cronobacter sakazakii pGFPuv was confirmed by growing it on TSAA media by scratching one loop onto the surface of the media, then incubating at 37 o C for 24 hours using an incubator (Heraeus, Germany) and observing under UV light (Desaga, Heidelberg, Germany) with a wavelength of 366 nm.C. sakazakii colonies will fluoresce (glow green) if observed under UV light [17].

Preparation of Cronobacter sakazakii pGFPuv as inoculum.
Fluorescent colonies growing on TSAA media were suspended with BPW (Buffered Peptone Water) diluent solution and centrifuged for 10 minutes at 4 o C at a speed of 3500 rpm (2,600 x g).The supernatant was discarded and centrifugation was carried out again by adding 10 ml BPW to the pellet.The number of cells was standardized by the Optical Density (OD) value at λ=590 nm using spectrophotometer (Shimadzu UV-2450).Dilution was carried out until it reached an OD value of 0.4 which was equivalent to a colony number of 10 8 CFU/mL [17], and then this mutant suspension was used as an inoculum.

C. sakazakii pGFPuv inoculation.
The liquid isolate of C. sakazakii pGFPuv was inoculated into corn flour in a sterile plastic bag and then mixed thoroughly so that the initial amount of C. sakazakii pGFPuv in corn flour was 10 6 CFU/g with an initial moisture content (MC) of corn flour of 9% and 12%.

Analysis of moisture content and water activity during storage of corn flour.
Moisture content and water activity analysis was carried out on the control group every two weeks during storage.The water content of corn flour was analyzed using the oven method at a temperature of 105 o C for a minimum of 6 hours [21].Water acitivity measurements were carried out using a calibrated Aw meter (Ro-Tronic) [22].

Analysis of the number of C. sakazakii pGFPuv during storage of corn flour.
A total of 10 g of sample was dissolved in 90 ml of BPW, homogenized by shaking in sterile plastic, and a dilution series was carried out to be fertilized into TSAA media using the spread method.After incubation for 24 hours at 37 o C, colonies were counted under a UV lamp with a wavelength of 366 nm, according to Standard Plate Count [23].Then the number of fluorescent C. sakazakii colonies was converted into log units.The curve for the number of C. sakazakii during storage was created by plotting the number of C. sakazakii colonies (log) on the Y axis and the time interval (weeks) of storage on the X axis.

Analysis of total plate number, number of molds and yeast during storage of corn flour.
Analysis of total plate number and total mold and yeast was carried out every two weeks during storage by dissolving 10 g of sample in 90 mL BPW, homogenizing by shaking in sterile plastic, and carrying out serial dilutions.Analysis of total plate numbers with PCA media and incubation at 35 o C for 48 ± 2 hours [23] and analysis of mold and yeast numbers with APDA media and incubation at 25 o C for 5 days [24].The plates included in the count for TPC are 25-250 colonies per petri plate, while the number of molds and yeasts is 10-150 colonies per petri plate.The number of colonies was calculated using the Standard Plate Count formula.

Survival of C. sakazakii during storage of corn flour.
The survival of C. sakazakii pGFPuv isolates was created by creating a curve by plotting the logarithm of the number of colonies surviving at a certain time (Nt) divided by the initial number of colonies (No) on the Y axis and the time interval (weeks) of storage on the X axis. the amount (log/week) during storage is shown by the slope of the linear equation.

Statistical analysis.
The treatment was carried out in two replications.Data on the number of C. sakazakii pGFPuv that survived during storage were tested for normality using the Shapiro Wilk method.The effect of initial MC on the number of C. sakazakii pGFPuv during storage was analyzed using the paired t test if the data were normally distributed or using the Mann-Whitney test if the data were not normally distributed.The effect of RH in the storage room on the number of C. sakazakii pGFPuv during storage was analyzed using One Way Anova with Duncan's follow-up test if the data was normally distributed or the Friedman test with Wilcoxon's follow-up test if the data was not normally distributed.

Changes in moisture content and water activity of corn flour during storage
Corn flour with an initial MC of 9% stored at RH 50% and corn flour with an initial MC of 12% stored at RH 70% reach equilibrium moisture content at the beginning of storage, but the other moisture content of corn flour under other storage conditions reached equilibrium after two weeks of storage (Figure 1).The moisture content of corn flour during storage at RH 50%, 70%, and 90% respectively was 9.0-9.3%;12.2-12.4%;15.1-15.8% at initial MC of 9% and 9.2-9.3%;12.2-12.4%;15.4-16% at an initial MC of 12%.The water activity value of corn flour reached equilibrium after two weeks of storage (Figure 2).The higher the storage RH, the corn flour will reach a high equilibrium Aw, and conversely, the lower the storage RH, the corn flour will also reach a low equilibrium Aw.Corn flour stored at RH 90% has a higher equilibrium Aw compared to corn flour stored at RH 50% and 70%.The equilibrium water activity of corn flour stored at RH 50%, 70%, and 90% respectively was 0.42-0.44;0.67-0.69;0.8-0.86 at initial MC of 9% (initial Aw 0.38) and 0.42-0.44;0.65-0.69;0.84-0.87 at initial MC of 12% (initial Aw 0.62).The moisture content and water activity of corn flour is influenced by storage RH.Corn flour stored at RH 90% has a higher equilibrium moisture content and water activity than that stored at RH 50% and 70%.When corn flour is stored at a higher relative humidity (RH 90%) it will absorb water vapor from the environment until its equilibrium moisture content and water activity are reached, and conversely, when it is stored at a lower relative humidity it will release some of the water into the environment until the equilibrium moisture content is reached.

3.2.Changes in the number of C. sakazakii pGFPuv in corn flour during storage
The initial number of Cronobacter sakazakii pGFPuv is around 6-6.6 log CFU/g at the begining of storage.During storage, the decrease in the number of Cronobacter sakazakii pGFPuv in corn flour with initial MC of 9% and 12% at RH 50% was slower than RH 70% and 90% (Figure 3).The number of C. sakazakii pGFPuv in corn flour at 16 weeks of storage with an initial MC of 9% and 12% was 1.4 ± 0.26 and 2.2 ± 0.21 log CFU/g respectively, whereas at RH 70% and 90% there were no Cronobacter sakazakii pGFPuv surviving.The number of C. sakazakii pGFPuv colonies can be easily counted under UV light on media containing ampicillin marked with fluorescent colonies so that wild type strains of C. sakazakii that may be present are not counted (Figure 4).The number of C. sakazakii pGFPuv that survived on corn flour was influenced by storage RH (p<0.05).The decrease in C. sakazakii pGFPuv in corn flour with an initial MC of 9% and 12% stored at 50% RH was slower than at 70% RH and 90% RH.The amount of decrease C. sakazakii pGFPuv at 50% RH was 4.4-4.7 log CFU/g for 16 weeks, while at 70% RH of 6.59 log CFU/g for 14 weeks and at 90% RH of 6.59 log CFU/g for 12 weeks.A similar thing happened with skim milk, the decrease in the number of C. sakazakii during 12 weeks of storage was greatest at RH 90% compared to RH 50% and 70% [8].The slow decrease in the number of C. sakazakii pGFPuv at 50% RH was also related to the decrease in its Aw during storage.The Aw of corn flour stored at RH 50% is lower than RH 70% and RH 90%.C. sakazakii survived better in infant formula and cereal with low Aw during 12 months of storage [7].

Changes in total plate number of corn flour during storage
Total plate numbers at RH 50%, 70%, and 90% experienced a slight decrease in corn flour during 16 weeks of storage (Figure 5).In corn flour with an initial MC of 9%, the total plate number after storage at RH 50%, 70%, 90% respectively was 7.2 ± 0.06 log CFU/g; 7.2±0.08log CFU/g, and 7.1±0.06log CFU/g from the initial amount of 7.6±0.05log CFU/g.Meanwhile, in corn flour with an initial MC of 12%, the total plate number after storage at RH 50%, 70%, 90% was respectively 7.3 ± 0.04 log CFU/g, 7.3 ± 0.02 log CFU/g, and 7.3 ±0.11 log CFU/g from the initial amount of 7.6±0.02log CFU/g.The reduction in total plate number in corn flour with a MC of 9% was 0.4; 0.4; 0.5 log CFU/g respectively at RH 50%, 70%, and 90%, while for corn flour with a MC of 12% it is 0.3; 0.3; 0.3 log CFU/g respectively at RH 50%, 70%, and 90%.A high total plate count in corn flour can be caused by microbes that are already present in the shelled corn, milling machines, milling methods, and the water used when making the flour [25].The decrease in total plate number was caused by the water content of shelled corn and corn flour which was not suitable for growth, namely at a water content of 14-18% [26].The number of molds and yeasts in corn flour with an initial MC of 9% after being stored for 16 weeks at RH 50%, 70%, and 90% respectively was 3.2 ± 0.04 log CFU/g, 3.2 ± 0.02 log CFU/g, and 7.2 ±0.56 log CFU/g from the initial amount of 3.5±0.10log CFU/g.The number of molds and yeasts in corn flour with an initial MC of 12% after being stored for 16 weeks at RH 50%, 70%, and 90% respectively was 3.2 ± 0.01 log CFU/g, 3.2 ± 0.01 log CFU/g, and 7.2 ±0.03 log CFU/g from the initial amount of 3.2±0.21log CFU/g.The number of molds and yeasts in corn flour with an initial MC of 9% at the end of storage decreased by 0.3 log CFU/g at RH 50% and RH 70%.The number of molds and yeasts in corn flour with an initial MC of 12% at the end of storage remained at RH 50% and 70%.However, at 90% RH the number of molds and yeasts for 16 weeks increased by 3.7 log CFU/g in corn flour with an initial MC of 9% and by 4 log CFU/g in corn flour with an initial MC of 12% (Figure 6).

Survival of C. sakazakii during storage of corn flour
The rate of reduction in the number of C. sakazakii pGFPuv isolates in corn flour with an initial MC of 9% and 12% stored at RH 50% was slower than at RH 70% and 90% (Figure 7).The rate of decline in the number of C. sakazakii pGFPuv in corn flour with an initial water content of 9% and 12 % at 50% RH is relatively the same.This is indicated by the slope value of the linear equation for corn flour with an initial MC of 9% of 0.489 log cycles/week and corn flour with an initial MC of 12% of 0.470 log cycles/week.The number of C. sakazakii pGFpuv that survived on corn flour during storage at 50% RH with different initial water contents (9% and 12%) was not significantly different (p>0.05) or had no significant effect.Thus, it can be said that the number of C. sakazakii pGFPuv is not greatly influenced by the initial water content of corn flour because the initial water content is in equilibrium with the RH of the storage room.The survival of C. sakazakii in dry conditions is due to its ability to accumulate trehalose which will bind in hydrogen bonds with phospholipid membranes so that it can increase surface tension with intracellular fluid and thus prevent autolysis [27].Furthermore, there is an accumulation of proteins that play structural and protective roles such as heat shock proteins (Hsp), cold shock proteins (Cspc), protection and repair proteins (Dps), and DNA binding proteins (Hns) [28].In addition, corn flour with an initial MC of 9% and 12% stored at RH 50% and 70% was still good for 16 weeks, whereas at RH 90% it had already experienced damage, namely the corn flour had solidified and was growing by mold (Figure 8).Therefore, it is best not to store corn flour at high RH (90%) because both physical quality quickly decreases.Research done by [16] showed that the number of molds and yeasts in shelled corn during 12 weeks of storage with an initial MC of 12% stored at RH 50 and RH 70% decreased by 1.1 Log CFU/g, whereas at RH 90% experienced an increase of 3.1 Log CFU/g.

Conclusion
The moisture water and water activity of corn flour is influenced by the relative humidity factor in the storage room compared to the initial water content factor during 16 weeks of storage and the moisture water and water activity value influences the survival of C. sakazakii pGFPuv in corn flour.Cronobacter sakazakii pGFPuv survived better at RH 50% compared to RH 70 and 90% which is related to its ability to survive at lower aw conditions.Cronobacter sakazakii pGFPuv can survive on corn flour with Aw of 0.42-0.44stored at 50% RH for 16 weeks.The rate of decrease in the number of C. sakazakii pGFPuv in corn flour with an initial MC of 9% and 12% at 50% RH was relatively the same, namely 0.489 log cycles/week and 0.470 log cycles/week, respectively.Therefore, reducing the number of C. sakazakii in dry food products such as corn flour must be a consideration to produce safe food products, maintain food sustainability, and contributes to the Sustainable Development Goals (SDG) especially goal number 3 about good health and well-being.