Joint effect of temperature and insect chitosan on the heat resistance of Bacillus cereus spores in rice derivatives

The heat resistance of Bacillus cereus spores inoculated in a rice substrate supplemented with insect chitosan as an alternative antimicrobial was studied. Two concentrations of insect chitosan were considered in order to assess the role of the insect chitosan concentration during the heat process. Results of the study indicated that the DT values were higher in the substrate without chitosan than in the substrate containing chitosan thus indicating a greater heat resistance to heat treatment of the microorganism inoculated in the substrate without chitosan. This behaviour was also evidenced in the survival curves. There were no great differences between either of the insect chitosan concentrations tested regarding the DT values. The z values were 9.8°C on rice substrate and8.9°C on rice substrate supplemented with insect chitosan at 150 μg/mL and 10.7°C on rice substrate supplemented with 250 μg/mL of insect chitosan. The chitosan concentration appears to affect the z value of the microorganism. Our results indicate that the combination of heat with insect chitosan as an antimicrobial on foodstuffs subjected to cooking is feasible and can improve the safety of rice derivatives.


Introduction
Bacillus cereus is present in many foods due to its ubiquitous nature. This microorganism is one of the top ten pathogens responsible for many foodborne diseases in humans [1]. According to the latest EFSA and ECDC report [2] there is strong evidence for B. cereus was involvement in 38 outbreaks and weak evidence of involvement in 117 outbreaks out of a total of 155 outbreaks reported in 2019. Some recent outbreaks in non-EU countries have also been associated with this pathogen; 45 people were affected in an outbreak in a restaurant in Canberra (Australia) in 2018 [3] and 200 students in an outbreak in a school in China in 2018 [4].
Bacillus cereus causes two types of food poisoning one of an emetic nature and the other of a diarrheal nature [5]. On the one hand diarrheal syndrome is caused by a gastrointestinal disorder due to the ingestion of B. cereus spores present in food and at a dose given, an appreciable probability that cells cross the stomach barrier and implanting themselves in the small intestine is possible. Once they germinate in the small intestine they produce enterotoxins that cause disease. On the other hand emetic syndrome is associated with the production of cereulide toxin in the food contaminated with spores that germinate and produce the toxin resulting in foodborne poisoning [6]. In general, this microorganism is associated with complex food products that may include rice as a component; however, other rice-based products and farinaceous foods such as pasta and noodles are also frequently contaminated and involved in cases of B. cereus poisoning [7].
The ability of B. cereus to form spores and biofilms enables its persistence in various ecological niches and food products resulting in its presence in processed foods such as cooked rice [8]. Furthermore, it is the bacteria most commonly present in rice and rice-based products [9].
Rice is a basic cereal in many diets and is widely consumed by the general population given its ample supply of nutrients and its relatively low cost. This cereal is one of the most important staple crops feeding almost half of the world's population [10]. Starch is the most abundant component of a rice grain constituting about 80% of the dry weight of a brown rice grain and approximately 90% of a milled rice grain [11]. Rice also provides an important variety of micronutrients including vitamins such as niacin, thiamine, pyridoxine or vitamin E, and minerals such as potassium, phosphorus, magnesium and calcium [12]. These conditions provide a very good substrate for B. cereus growth and subsequent toxin production.
This cereal is habitually contaminated by B. cereus spores throughout all production stages from cultivation to the later stages of processing and consumption. It is believed that the primary habitat of emetic strains could be related to roots tubers and mycorrhizae of some plants such as rice which could explain the generally higher prevalence of these strains in carbohydrate-rich foods. In fact, starch has been shown to promote B. cereus growth and emetic toxin production. This would explain why most outbreaks of emetic disease are associated with starch-rich farinaceous foods [13].
Some works pointed out that the current cooking processes for rice and rice derivatives do not inactivate B. cereus spores and consequently they can germinate and grow in food if it is not stored properly [1]. Different control measures have been proposed to control Bacillus cereus in foods. As an additional strategy, heat treatment can be combined with other control measures (hurdle technology). In this respect, chitosan from different sources (crustacean or fungi) has received attention as an antimicrobial. It is a polysaccharide with a well-documented antibacterial activity towards vegetative cells which has already been effectively applied as edible chitosan films [14] and in food packaging applications [15,16]. According to Van Huis et al. [17] rearing insects is a sustainable activity more friendly with the environment than fishing or traditional farming. Besides, as indicated by Mohan et al. [18], the extraction of chitin and chitosan from insects is more advantageous in terms of extraction methods, chemical consumption, time and yield compared to existing sources. Existing chitin resources have some natural challenges, including insufficient supplies, seasonal availability, and environmental pollution. As an alternative, insects could be utilized as unconventional but feasible sources of chitin and chitosan. According to previous in vitro studies [19], insect chitosan could be used as antimicrobial instead of chitosan from other sources. Based on those results it could be also applied as an additional control measure during heat processing of rice thus favouring the destruction of B. cereus spores by affecting their heat resistance. Currently there are no data on the joint effect of insect chitosan and heat on the heat resistance of B. cereus spores since chitosan from other sources is used as a natural antimicrobial in the preservation processes.
The purpose of this study is to determine how B. cereus spore inactivation is affected by the presence of insect chitosan during the heat treatment. This knowledge can pave the way to a better control of B. cereus during and after the cooking processes of rice and its derivatives.

Microorganisms and sporulation procedure
The Bacillus cereus CECT 148 strain used in this study was obtained from the Spanish Type Culture Collection (CECT), (Valencia, Spain). The strain was reactivated in nutrient broth by shaking for 24 hours at 32˚C and subsequently 0.5 mL of the B. cereus culture was inoculated in 20 Roux flasks (Fisher Scientific SL, Madrid, Spain) with Fortified Nutritive Agar (Scharlab. Barcelona, Spain) and incubated at 30˚C. When the sporulation level reached approximately 90% the spores were collected.
Spore harvesting was performed using a modified metal Digralsky loop (Deltalab, Barcelona, Spain) gently sweeping the agar surface and washing it with double distilled water. The collected solution was centrifuged at 2500g for 15 minutes at 5˚C the supernatant was removed suspended again in 5mL of double distilled water and was centrifuged under the same previously described conditions this process was repeated 4 times. Finally, the spores from the pellet were stored at 4˚C in distilled water.

Substrate preparation
The rice solutions from cooked and lyophilized rice supplied for a local company were prepared by dissolving 0.4 g in 19 mL H 2 O. All solutions were heat sterilized. After sterilizing the rice solution, 1 mL of the spore solution was added and homogenous distribution was guaranteed by a vortex.
Two solutions of rice with insect chitosan from Tenebrio molitor (150 and 250 μg/mL chitosan) (ecoProten, Cordoba, Spain) were used for the heat resistance studies on the food matrix. Those concentrations were chosen because in previous studies carried out by Valdez et al. [20] 250 μg/mL showed a higher antimicrobial effect than the 150 μg/mL concentration. The pH was adjusted to between 6.8 and 6.9 by using NaOH. Finally, 1 mL of the spore suspension was added and homogenous distribution was guaranteed by a vortex. The resulting 20 mL of solution containing spores and chitosan were poured into a 50 mL sterile beaker.
In all cases the spore concentration in the resulting rice solution was 10 8 spores/mL.

Capillary filling and heat treatment
The capillary tubes with one end closed were supplied by Vitrex, reference 217913 (1.50 x 2.00 x 100 mm). For the heat resistance study capillaries were filled using a drying chamber with a vacuum pump. Once the vacuum was achieved, it was broken and the rice solution rose through the capillaries, which were filled to a volume of 2/3 of their capacity. After that, the solution column was centred in the capillaries they were removed from the chamber and the open end was closed with a quick-drying silicone. Before the heat resistance study spores were heat activated in order to create the conditions for them to germinate and grow in the culture medium. For the activation of B. cereus spores the capillaries were placed in hooked racks designed for this type of study. The racks with the capillaries were immersed in a water bath (HAAKE N3) at 80˚C ± 0.5 for 10 minutes.
Both the rice solution alone and the rice solution containing chitosan were heat treated at 90 95 100 and 105˚C for different exposure times. A silicone oil bath (HAAKE DC5) was used for this treatment. For time zero (0) and for each treatment temperature a capillary rack was removed after spore activation and was not heat-treated thus considered as control. The rest of the racks were withdrawn from the activation bath and immediately immersed in the oil bath at the selected temperature. A rack was removed at each time interval and immersed in ice water to stop the treatment.
Before the solution was plated the capillaries were cleaned with 96% ethanol and using forceps the ends were split to extract the solution. The content of eight capillaries was deposited into sterile Eppendorf tubes. With the solution recovered from the capillaries two series of serial decimal dilutions (series A and B) were made up to 10 −6 in duplicate. From each decimal solution 100 μL was plated in duplicate on nutrient agar (Scharlab, Barcelona, Spain) enriched with 1g/L starch (Scharlab, Barcelona, Spain) and incubated for 18-20 hours at 30˚C. After the incubation time, a manual count of B. cereus colonies was carried out. Spore aggregation was prevented by vigorous shaking with glass beads before taking each sample for plating.

Statistical analysis
All statistical analyses including the one step nonlinear regression were performed using Statgraphics Centurion XVI Software (Addinsoft SARL. New York. NY. USA). Non-linear regression is a powerful technique for standardizing data analysis [21], it allows obtaining the D and z values from survival curves at once

Results
In the present work, the heat resistance of Bacillus cereus was studied in a rice substrate without insect chitosan and with insect chitosan at two concentrations.
The survival curves at each temperature tested in the study can be seen in Fig 1A-1D.
In general, at all temperatures studied B. cereus spore's inactivation in the rice substrate was lower than in the substrate without chitosan. Regarding chitosan concentrations, we also observed that for all temperatures the heat resistance of B. cereus spores was quite similar, so the chitosan concentration in the heating medium did not affect the survival of these spores.
The parameters defining the spore's inactivation were derived by a non-linear one-step fitting of the survival data. Nonlinear models often capture the relationships in a data set better than linear models. Perrin [22] described the disadvantages of the usual linear least squares analysis of first-and second-order kinetic data and nonlinear least squares fitting was recommended as an alternative. In our study the value of the studentized residuals was in all cases two or less than two in any case three as absolute value this means that in no case the residuals exceed two standard deviations. Table 1 shows the estimation of the parameters that define the heat resistance of B. cereus spores D T for each of the substrates and temperatures studied. Table 2 shows the z value for each of the studied substrate.
The value of the parameter D T estimated by the model is clearly higher in the substrate without chitosan than in the substrate containing chitosan which is related with the lower spore inactivation as previously shown by the survival curves. Regarding the value of the parameter, D T estimated by the model when chitosan is present little difference was found between the two chitosan concentrations. It seems that the effect of chitosan on the inactivation of B. cereus spores does not depend on the concentration of chitosan between 150 and 250 μg/mL during heating. With respect to the value of the z parameter estimated by the model varied between 8.9 and 10.7, those are quite common values for this microorganism [23,24].

Discussion
Bacillus cereus is a ubiquitous microorganism that can cause serious food safety issues especially in rice products and their derivatives. Proper characterization of its thermal resistance is essential for the design and development of suitable cooking processes. Likewise the prospect of using combined processes in this case with natural antimicrobials can pave the way to improving the safety of these widely consumed products around the world. Currently there is information on the effect of temperature and on the effect of chitosan separately on B. cereus spores.
Several works have reported the variation in the D T and z values of the microorganism in different heating substrates. Pendurka and Kulkarni [25] studied the heat resistance of the spores of five Bacillus species including B. cereus in distilled water and pasteurized skim milk. The authors found that in all cases the spores survived the cooking conditions applied to the rice. At 100˚C a D T value of 19 min was shown by B. cereus in distilled water while B. cereus spores were completely inactivated in skim milk at the same temperature (100˚C). This result indicates low levels of heat resistance. In the present work at 100˚C a D T value of 1.82 min was recorded when the spores were heated in a rice solution. However, the great variability that exists between B. cereus spores in relation to heat resistance is well known. Fernandez et al. [23] studied the heat resistance of two Bacillus cereus strains isolated from cooked chilled foods containing vegetables and found D T values between 0.22 and 2.5 min at 100˚C. More recently Salwa Abu El-Nour Ali Hammad [26] found D 85 -values of B. cereus spores ranging from 24.9 to 35.2 min. D 90 -values ranging from 7.6 to 11.6 min. whereas D 95 -values ranged from 2.4 to 4.7 min. depending on the type of substrate. The values obtained in the present work are slightly higher probably due to the strain and substrate differences.
Regarding the z value Fernandez et al. [23] reported values of 8.1 and 8.4˚C depending on the strain considered obtained on a reference substrate. Salwa Abu El-Nour Ali Hammad [26] reported z values of B. cereus spores suspended in different media ranging from 9.81 to 11.24˚C. In the present work, the z value ranged from 8.9˚C to 10.7˚C depending on the substrate used. The z values obtained in the present work are in accordance with previously reported results; therefore, these results can be considered a suitable reference to develop suitable cooking conditions for rice.
Today, chitosan is extensively studied given the multiple applications that it can have in both the food and the pharmaceutical industries. One of these applications is its use as a natural antimicrobial in food preservation. Ke et al. [27] indicated that the broad-spectrum antimicrobial activity of chitosan offers great commercial potential for this product. Some studies have been published in which the effectiveness of chitosan against B. cereus has been demonstrated. Fernandes et al. [28] found a relationship between the molecular weight of chitosan and its antimicrobial activity for both vegetative cells and spores of B. cereus. Mellegård et al. [29] studied the inhibition of B. cereus spore outgrowth and multiplication by chitosan; they found chitosan exerts antimicrobial activity that appears to be concentration-dependent and related to the average molecular weight and fraction of acetylation of the chitosan used as antimicrobial.
Currently the industry is looking into combined treatments in which the different control measures are administered with lower intensities than when applied individually. In this way, pathogenic microorganisms are inactivated in a way that improves both the nutritional and sensory quality of food. In some cases, this combination is interesting because it can provide greater inactivation by heat than when heat is administered alone. There are no studies in the literature reporting the combination of heat treatment and chitosan to achieve control and inactivation of B. cereus in rice-based substrates. However, the effect of combining heat treatment or other control measures with natural antimicrobials has been reported in the scientific literature. Ueckert et al. [30] reported that exposure to heat and nisin caused synergistic reductions of Lactobacillus plantarum viability. Huertas et al. [31] studied the combined effect of natural antimicrobials (nisin. citral and limonene) and thermal treatments on Alicyclobacillus acidoterrestris spores. Authors concluded that the antimicrobial agents tested did not affect the heat resistance of the spores; however, the antimicrobials were effective in controlling the growth of the microorganisms after the heat treatment. Kamdem et al. [32] studied the effect of mild heat treatments on the antimicrobial activity of some essential oils. Authors indicated that the combination of temperature and those essential oils reduced the treatment time needed to inactivate 7 log cfu/mL of Salmonella enteritidis. In the present work a joint effect of heat and chitosan on B. cereus spore's inactivation was found, D T values were in general lower on samples containing chitosan than in the sample without chitosan. The decrease in the D T values may be due to a reduction in the spores' resistance caused by the joint effect of the thermal treatment and the chitosan, as it has been observed in vegetative cells of E. coli, L. mocytogenes and S. Typhimurium [19]. Probably, the additive effect during heat treatment depends on the type of microorganism or the type of antimicrobial. It is also possible that the reduction in D T could be also due to chitosan is blocking outgrowth of Bacillus spores that have been damaged by wet heat. In any case, the presence of chitosan increases the inactivation or prevents the development of spores of B. cereus, significantly improving the food safety of the food. Besides, in the present work, we found that the effect on D T values was not dependent on chitosan concentration. It is possible that at this level the chitosan concentration does not play an important role but rather it is the molecular structure of the chitosan that facilitates the action of heat on the bacterial spores thus reducing the number of spores capable of germinating and growing.

Conclusions
This study investigated the nature of the inactivation of Bacillus cereus spores by combining insect chitosan with heat treatment. The results indicated that the presence of chitosan regardless of its concentration produced reductions in the D T value of B. cereus spores in a rice substrate. These findings pave the way to a better control of B. cereus during and after the cooking processes of rice and its derivatives making the combination of chitosan with heat treatment feasible in order to improve the safety of these types of products. These results also indicate that insect chitosan could be also used as chitosan from other sources, in combination with heat treatment as an additional control measure.