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Full Research Article

Evaluation of Lactobacillus rhamnosus as a Protective Agent against Bacillus cereus Contamination in Legume-based Infant Foods

A
Ashraf Albrakati1,*
1Department of Human Anatomy, College of Medicine, Taif University, Saudi Arabia.

  • Submitted04-12-2025|

  • Accepted14-04-2026|

  • First Online 22-04-2026|

  • doi 10.18805/LRF-918

ABSTRACT

Background: Bacillus cereus poses a critical food safety challenge in developing countries, emphasising the need to find ways to combat its toxicity. Commercially produced baby food is authorized as a main source of nourishment for infants; thus, it should include a high concentration of nutrients and be free from any potential hazards.

Methods: 300 random food samples were obtained from supermarkets and pharmacies in Taif and Makkah cities, Saudi Arabia. Microbiological examinations, molecular techniques and anatomical examinations were applied to confirm B. cereus pathogenicity. Probiotic bacteria were tested for their antimicrobial activity.

Result: The bacteria were observed in almost all samples with mean total viable counts of 1.586 to 3.543 log10 cfu/g. Furthermore, 196 Bacillus colonies were identified and 27% of them were B. cereus. Finally, L. rhamnosus is an anti-B. cereus agent was used for detoxification and to reduce the pathogenicity of this pathogen. After mixing L. rhamnosus with B. cereus and/or its emetic toxins and feeding it to mice, the toxicity of B. cereus and its enterotoxins was reduced by 37 to 62%, paving the way for using this strain to protect infant meals from hazardous microbial elements. Anatomical examination showed that mice fed with B. cereus exhibited severe gastrointestinal abnormalities, whereas those treated with combinations of B. cereus and Lactobacillus rhamnosus displayed normal anatomical features. This study concluded that Lactobacillus rhamnosus may have significantly mitigated the toxicity of Bacillus cereus in infant meals, highlighting its potential as a protective agent in food safety.

INTRODUCTION

Bacillus cereus plays a significant role as a foodborne pathogen in developing countries, where the combination of environmental conditions and limited public health infrastructure heightens the risk of outbreaks (Woh and Ng, 2024). In these regions, food safety is often compromised by inadequate refrigeration, poor sanitation and lack of awareness about proper food handling practices. The toxins produced by B. cereus can cause severe gastrointestinal illnesses characterised by vomiting and diarrhoea (Duan et al., 2023).
       
The challenge is exacerbated by the bacteria’s ability to form durable spores, allowing it to survive. Strategies targeting hygiene, storage and cooking practices remain relevant in reducing the impact of B. cereus.
       
Because infants rely heavily on limited food sources during early development, any microbial contamination poses a direct health risk due to their immature immune systems. For this reason, the hygienic quality of commercially prepared infant foods is critical, particularly during the complementary feeding period. Foodborne diseases remain a major global health concern, especially where bacterial spores can persist through processing and storage (Wang et al., 2022). Among these pathogens, Bacillus cereus has been repeatedly identified as one of the most common contaminants in pasteurized and ready-to-eat infant foods, emphasizing the need for strict safety guidelines and targeted hygienic control measures to prevent exposure in this vulnerable age group (Radmehr et al., 2020).
       
B. cereus is a psychrotrophic, facultative anaerobic, spore-forming pathogen associated with food-borne outbreaks (Zhao et al., 2020). It can grow at low temperatures (6-10oC) and even under refrigeration, producing emetic and diarrheal toxins (Granum and Lund, 1997). Diarrheal illness results from vegetative cells producing enterotoxins when ingested at levels exceeding 105 cfu/g (Warda, 2016). In contrast, several Lactobacillus species and lactic acid bacteria produce antimicrobial substances that can inhibit pathogens and help prevent food spoilage (Jørgensen et al., 2017; Mori et al., 2026; Timothy et al., 2021).
       
Despite previous studies addressing Bacillus cereus contamination in food products, limited research has specifically evaluated the detoxifying role of probiotic strains such as Lactobacillus rhamnosus against B. cereus and its toxins in legume-based infant foods (Abdel-Rauf et al., 2025; Granum and Lund, 1997; Zhao et al., 2020). Therefore, this study aimed to (i) evaluate the microbiological quality of infant foods, (ii) determine the prevalence and toxigenic potential of Bacillus cereus and (iii) investigate the antimicrobial and detoxifying effects of Lactobacillus rhamnosus against B. cereus and its enterotoxins.

MATERIALS AND METHODS

Infant food sample collection and preparation
 
A total of 300 samples of ready-to-use baby milk-based powders were obtained randomly from diverse supermarkets and pharmacies in Taif and Makkah cities, Saudi Arabia during the period from 2024 to 2025, as shown in Table 1.

Table 1: Type, numbers and locations of the collected infant foods.


 
Probiotic strain growth and preservation
 
Lacticaseibacillus rhamnosus B-445 and Lactobacillus dulbrueckii subsp. bulgaricus Lb-12 DRI-VAC strains were delivered from the Northern Regional Research Laboratory (NRRL), Illinois, USA: Hansen’s Lab, Denmark. Lactobacillus cremoris was obtained from the Department of Dairy, National Research Centre, Egypt. They were proven to be probiotics (Adebayo-Tayo and Fashogbon, 2020) by the possession of their characteristics.
 
Bacteriological analysis of infant foods
 
For all collected samples, 25 g of each sample in 225 mL of sterile peptone water was mixed well by homogenizer for 2 min. Total bacterial counts were tested using tryptic soya agar and plate count agar media (Darwesh et al., 2019). In the case of psychrotrophic spore-forming bacterial counts, the procedure followed (Sadek et al., 2018).
 
Isolation and identification of psychrotrophic Bacillus spp.
 
Three to five isolates from each plate of typical colonies of the total psychrotrophic spore-forming plates were picked randomly. Each colony was purified on PEMBA medium, characterized as psychrotrophic Bacillus spp. and then inoculated into 10 mL of tryptic soya broth at 37oC for 24 h and then subjected to several biochemical examinations and API 50 CHB (Biomerieux, France) for identification.

Molecular detection of toxicogenic-related genes and identification of their producing pathogens
 
Extraction of bacterial DNA of the 22 B. cereus isolates was obtained by the lysozyme (20 mg/mL) and proteinase K (1 mg/mL) method, which was detailed previously (18). Three virulence genes, hbla, nhec and cytk, were amplified as enterotoxin genes using extracted DNA and specific primers as shown in Table 2 (19-23). PCR amplification was carried out starting with denaturation at 95oC for 5 min, followed by 30 cycles of 95oC for 1 min, 58oC for 1 min, 72oC for 1 min and a final elongation step at 72oC for 10 min, with a final hold at 4oC. PCR products were analysed in 1% (w/v) TAE agarose gel. All PCR experiments for each strain were carried out twice (Barakat et al., 2015). One isolate of B. cereus had the three tested virulence genes. Thus, it was identified using molecular biology techniques according to Adesetan et al., (2022).

Table 2: Primer sequences and amplicon sizes used in PCR analysis.


 
Antibacterial and antagonistic activities of the probiotic strains
 
The antimicrobial activity of the four probiotic strains against B. cereus was evaluated using cell-free supernatants obtained after centrifugation of overnight MRS cultures. The inhibitory effect was assessed by the agar well diffusion test and the inhibition zones were measured (Khalid et al., 2021).
       
The antagonistic activity of Lact. rhamnosus B-445 against B. cereus was further examined in broth co-culture. Standardized dilutions of both strains were mixed in tryptic soya broth and bacterial counts were determined at selected time intervals. B. cereus was enumerated on PEMBA medium (Finlay et al., 1999), while Lact. rhamnosus was counted on MRS agar under anaerobic conditions.
 
Detoxification and reduced pathogenicity of B. cereus after Lact. rhamnosus treatment
 
Evaluation of the pathogenicity and production of toxins by the B. cereus isolate was carried out using the mouse toxicity technique. Additionally, the effect of Lact. rhamnosus on this pathogenicity was evaluated. B. cereus was cultured in 10% skim milk medium to produce enterotoxin as a supernatant. The pellet was re-suspended in a suitable quantity of phosphate buffer to attain a bacterial suspension equivalent to 0.5 McFarland standard (Darwesh et al., 2020). In another case, Lact. rhamnosus strain was cultured in 1% (w/v) of MRS. After bacterial cultures and B. cereus crude toxins were prepared, an equal volume of Lact. rhamnosus culture was mixed with B. cereus culture or its emetic toxin and incubated at 37oC for 6 h.
       
To evaluate the pathogenicity of B. cereus and study the role of Lact. rhamnosus in attenuating the toxicity of B. cereus, 30 adult male mice (Mus musculus domesticus) with an average weight of 100 g were obtained from the animal house at Taif University. The mice were divided into six groups containing five mice each. The animals were fed a basal diet containing 50% corn starch, 20% casein, 10% sugar cane, 10% corn oil, 5% cellulose, 4% salt mixture and 1% vitamin mixture (Darwesh et al., 2023). A total of 0.5 mL of each treatment was given orally to the mice by intragastric gavage for three days. The trials (6 groups) were as follows: The control group was fed phosphate-buffered saline and their regular food, the 2nd group was fed B. cereus and the emetic toxin, the 3rd group was fed B. cereus culture, the 4th group was fed a mixture of B. cereus, the emetic toxin and Lact. rhamnosus culture, the 5th group was fed a mixture of B. cereus and Lact. rhamnosus cultures and the 6th group was fed Lact. rhamnosus culture. The results were recorded for the mice after three days and the animals were thoroughly observed for any toxic signs through this period. Convulsions, motor activity, tremors, sedation, aggressiveness, relaxation of muscle, analgesia, hypnosis, ptosis, paralysis, lacrimation, skin colour and diarrhoea were noted during the experiment. The observed death was also recorded in each group.
 
Statistical analyses
 
All results were expressed as the mean±standard deviation (SD). Statistical significance was evaluated using analysis of variance (ANOVA, SAS software) followed by determining the least significant difference at 0.05.
 
Ethical approval
 
All experiments followed Taif University’s Ethics Committee’s guidelines (Approval No. TU 20-050, Taif, Saudi Arabia).

RESULTS AND DISCUSSION

Spreading of microbial communities in infant foods
 
Three hundred infant food samples were collected randomly from pharmacies and supermarkets in Makkah and Taif cities. The collected samples were subjected to micro-biological analyses. The results showed that the total bacterial counts (TBCs) were detected in almost all examined samples, with mean total viable counts ranging from 1.586 to 3.543 log10 cfu/g. The results revealed that the total psychrotrophic spore bacteria were noticed in 40 and 60% of the whole samples of milk-based infant food with vegetables and fruits, respectively.
 
Distribution and identification of Bacillus spp
 
The samples were examined to determine their content of Bacillus spp. by isolation of different morphological aerobic spore-forming colonies and then characterized (morphologically, biochemically and API CHB50 tests) using acceptable protocols of Bacillus spp. identification. The distribution of diverse isolated Bacillus species is illustrated in Fig 1. The results revealed that B. subtilis was the most common species compared with other Bacillus spp. with an incidence of 36%, followed by B. cereus (27%), B. circulans (20%), B. licheniformis (12%) and B. coagulans (5%). Vegetables and fruit milk-based infant foods had the highest numbers of Bacillus spp. From the obtained Bacillus isolates, B. subtilis and B. cereus, due to their hydrolytic activities, are essential for food hygiene.

Fig 1: Distribution of different total psychrotrophic Bacillus species in the obtained samples.


 
Distribution and identification of Bacillus cereus
 
B. cereus bacteria calculated on PEMBA medium were detected in almost all tested samples with the highest percentages in milk-based infant food with fruits and vegetables, which reached 62.2 and 26.6%, respectively (Fig 2).

Fig 2: Distribution of B. cereus in the obtained samples.


       
In contrast, the B. cereus count was not high in the ready-to-use infant food with fruits and vegetables. Generally, 40 out of 300 samples (approximately 26.67%) of the examined infant foods were positive for B. cereus counts.
       
Due to Bacillus cereus isolate No. 8 pathogenicity and production of the 3 virulent harmful toxin-related genes, B. cereus was identified using molecular biology techniques to confirm the biochemical and API CHB50 tests. The obtained sequence was compared with available sequences in the GenBank database and the similarity percentage of this isolate accounted for 98% with Bacillus cereus strains. Additionally, a phylogenetic tree was constructed and it showed that this strain was very close to the type strains of B. cereus deposited in the culture collection centre of the National Center for Biotechnology Information (Fig 3).

Fig 3: Phylogenetic tree of B. cereus and related species in the gene bank.


 
Molecular detection of toxicogenic-related genes
 
Three virulence genes (hbla, nhec, cytk) were screened in 25 B. cereus isolates using PCR. Only two isolates (8 and 9) carried all three genes. The hbla gene (1200 bp) appeared in five isolates and was mainly detected in samples from milk-based infant foods with fruit, rice, wheat and honey but with variable frequencies. The nhec gene showed a wider distribution across isolates from infant foods containing fruit, vegetables, honey and wheat, while it was absent in rice-based samples. In contrast, the cytk gene exhibited the highest prevalence, being detected in 92% of isolates collected from all infant food types examined. Overall, isolate 8-obtained from milk-based infant food with fruit-showed the strongest hbla band intensity among the positive samples (Fig 4).

Fig 4: Agarose gel pictures for the PCR product of hbla (A), cytk (B) and nhec (C) genes in B. cereus isolates compared with the marker (1 kb).


 
Detoxification and reduced pathogenicity of B. cereus using Lact. rhamnosus
 
The cell-free supernatant containing bioactive metabolites of probiotic strains was obtained and employed against the B. cereus strain using an agar well diffusion assay. The data from the pictures illustrated in Fig 5 show that the strain Lact. rhamnosus B-445 produced high antibacterial activity against the B. cereus strain, with a 21 mm inhibition zone diameter, followed by Lb. dulbrueckii and Lb. cremoris, but Leuconostoc mesenteroides appeared to have no activity. The antagonistic activity of Lact. rhamnosus against B. cereus was assessed in the broth culture media system and the results are illustrated in Fig 5. From that, we can clearly show the antagonism between the two tested strains. The growth of the B. cereus strain was inhibited under mixed culture with Lact. rhamnosus starting from 6 h of incubation. Lact. rhamnosus exposed a high inhibitory activity against the B. cereus strain, resulting in a decrease in the total counts from 5.3 to 3.3 Log10 cfu/mL after 24 h with a reduction of 37.7% and reached 2.0 Log10 cfu/mL with a reduction of 62.2% after 48 h of incubation.

Fig 5: Anti-Bacillus cereus activity of some probiotic strains.


 
Toxicology studies of B. cereus cells and crude toxins
 
The probiotic strain Lact. rhamnosus was mixed with B. cereus and/or its emetic toxins and added to the diets of the experimental mice. After three days of the experiment, the groups treated with B. cereus cells and its emetic toxin, groups two and three, showed some clinical signs, such as increased mortality and decreased weight compared with the other groups. The control group did not show any abnormal weight changes (ranging between 100 and 103 g) after three days. While mice fed with bacterial cells of B. cereus showed loss of their body weight and slower movement; their weights ranged from 72 to 75 g and two of five mice died. Additionally, four out of five mice in the group fed emetic toxins died and the remaining mice had a clear decrease in their weight to 60 g and slower movement. However, the group fed a mixture of B. cereus cells and Lact. rhamnosus exhibited normal appearance and movement with weights reaching 93-94 g. Moreover, the mouse group provided a mix of crude toxins and Lact. rhamnosus culture displayed normal appearance, motion and weight, with weights ranging between 98 and 101 g. The group was fed with a diet containing Lact. rhamnosus culture appeared to exhibit normal behaviour and their weight reached 100-105 g. Anatomical examination of the gastrointestinal tract of dissected mice revealed swelling of the intestinal tract due to fluid accumulation in the intestine’s lumen and inflammation of the intestines and stomach, particularly when mice were fed with the B. cereus. Additionally, ulcers and dark red spots were observed in the liver of groups fed with B. cereus, indicating severe vascular effects of the toxin. In contrast, no significant changes were noted in the stomach and intestines of mice treated with crude emetic enterotoxin. However, the group fed a mixture of B. cereus and Lactobacillus rhamnosus showed a normal stomach, intestine and liver compared to the control group. Similarly, the group fed a mixture of B. cereus emetic enterotoxin and Lactobacillus rhamnosus B-445 exhibited normal stomach, intestine and liver conditions. Furthermore, the group fed the diet with Lactobacillus rhamnosus B-445 appeared normal in all examined body contents (Fig 6).

Fig 6: Anatomical examination of mice under various treatments like control (a), bacterial cells of B. cereus (b), B. cereus the emetic toxins (c), mixture of B. cereus toxins with L. rhamnosus B-445 culture (d), mixture from bacterial cells of B. cereus and L. rhamnosus B-445 culture (e) and L. rhamnosus B-445 culture (f). The long arrow indicates the stomach and the short arrow indicates the intestines.


       
Infant foods are certified as a primary nutrition source for babies from seven months to 2 years old. Thus, they should represent a rich source of nutrients from several origins. However, germs in babies’ food can cause severe infection due to their immune systems not being completely developed (Martin et al., 2010). This study evaluated the hygiene of infant foods and tried to improve the quality of these foods. To achieve this objective, three hundred infant food samples were collected randomly from pharmacies and supermarkets in Makkah and Taif cities, Saudi Arabia. The collected samples were subjected to microbiological analyses. The results showed that the total bacterial counts (TBCs) were detected in almost all samples, with mean complete viable counts ranging from 1.586 to 3.543 log10 cfu/g. A similar observation was reported by other researchers (Gautam, 2015; Luby et al., 2011).
       
The TBC falls within standard regulations; therefore, it is less critical than spore-forming and pathogenic bacterial counts. High TBC values generally indicate poor hygienic practices during handling, packaging, or the use of low-quality ingredients (Mokgaotsi, 2019). For this reason, total psychrotrophic spore-forming bacteria were also evaluated and they were detected in 40% and 60% of milk-based infant foods with vegetables and fruits, respectively.
       
At this stage, psychrotrophic spore bacteria in infant milk meals were reported at 2.9 × 10² cfu/g (Ahmed et al., 2008) and another study showed that 40% of vegetable-based baby foods tested positive (Sadek et al., 2018). Bacillus spp. were isolated and identified following standard morphological, biochemical and API CHB50 protocols, with B. subtilis being the most common species in milk-based vegetable and fruit foods. Controlling B. subtilis and B. cereus is essential due to their hydrolytic activities, toxin production and ability to grow at refrigeration temperatures. These findings agree with previous reports detecting B. subtilis (28%), B. licheniformis (20%) and B. cereus (14%) in infant formulae (Mostafa et al., 2002). Although less prevalent, B. cereus remains the most harmful due to its pathogenicity and toxin production. FDA regulations require B. cereus levels to remain below 100 cfu/g. In this study, B. cereus was detected in all tested samples, especially milk-based infant foods with fruits and vegetables, while absent in ready-to-use products. Other studies similarly reported B. cereus contamination in powdered milk and a variety of infant foods (Kim et al., 2011). Differences in prevalence are likely related to sample type, testing method and product formulation. Previous research has suggested applying stable, nontoxic bioactive compounds and food-grade natural materials to reduce B. cereus in infant foods (Sadek et al., 2018).
       
The pathogenicity of B. cereus was assessed by detecting three key enterotoxin-related genes hbla, nhec and cytk using PCR. Two isolates (8 and 9) carried all three virulence genes. Toxigenic B. cereus strains have been previously reported in a wide range of starchy and dairy-based foods (Jovanovic et al., 2021; Mohammadi, 2023), emphasizing the need to evaluate the safety of infant foods commonly marketed in Saudi Arabia. PCR detection methods are increasingly applied for identifying these genes (Kim et al., 2000). In this study, isolate 8 originating from milk-based infant food with fruit was positive for the hbla gene, consistent with earlier reports detecting hbla in dairy and child-related foods (Rahimi et al., 2013). Gene distribution patterns varied according to food type, season and collection conditions. The nhec gene was widespread among isolates from fruit-, vegetable-, honey- and wheat-based infant foods but absent in rice-based samples, matching previous findings where nhec occurred in 80% of infant-food isolates (Rahimi et al., 2013). The cytk gene showed the highest prevalence, appearing across all food categories examined. As cytk is strongly associated with severe food-poisoning outbreaks, isolate 8 was selected as the pathogenic reference strain. Its genomic DNA was extracted and the 16S rRNA gene sequence showed 98% similarity to known B. cereus strains. Phylogenetic analysis confirmed its close relationship to established B. cereus type strains in the NCBI database.
       
The antimicrobial activity of the probiotic strains was assessed using cell-free supernatants in an agar well diffusion assay, where Lact. rhamnosus B-445 showed the strongest inhibition against B. cereus (21 mm), followed by Lb. dulbrueckii, Lb. cremoris and Leuconostoc mesenteroides. This variation reflects differences in the production of bioactive compounds such as organic acids, polysaccharides and bacteriocins (Liu et al., 2023; Moniri et al., 2017). LAB strains, including Lact. rhamnosus B-445 and Lb. dulbrueckii, are known for their antibacterial activity against Bacillus spp. (Ahmad et al., 2022; Zeinab et al., 2015). In broth co-culture, Lact. rhamnosus markedly suppressed B. cereus growth, reducing counts from 5.3 to 3.3 Log10 cfu/mL at 24 h and to 2.0 Log10 cfu/mL at 48 h. This inhibition is attributed to bacteriocins and related metabolites with applications in food biopreservation (Fuochi et al., 2019).
     
Infant foods require high hygiene standards because B. cereus is a pathogenic bacterium that poses significant health risks. The bacterium is particularly dangerous due to its spore-forming ability, which makes it resistant to thermal treatments and its toxins remain stable even under hygienic controls. To investigate a safe protective agent, the probiotic strain Lact. rhamnosus was mixed with B. cereus cells and/or emetic toxins and incorporated into mice diets. Mice fed only B. cereus cells or toxin alone experienced severe clinical signs including significant weight loss (60-75 g), reduced mobility and high mortality with 4 out of 5 mice in the toxin-treated group dying. However, mice fed B. cereus (cells or toxins) combined with Lact. rhamnosus maintained normal weight (93-105 g) and exhibited normal behavior and movement. Pathological examination revealed that mice exposed to B. cereus alone developed swollen intestinal tracts, ulcers and dark coloration of the liver. In contrast, groups treated with B. cereus mixed with Lact. rhamnosus showed normal stomach, intestine and liver tissues. These findings demonstrate that Lact. rhamnosus B-445 could serve as an effective and environmentally safe protective agent against B. cereus in infant foods. Probiotic supple-mentation has been widely applied to improve microbial balance and health performance in food-related biological systems, as reported in previous publications (Barde et al., 2025).

CONCLUSION

The findings demonstrate that Bacillus cereus contamination in infant foods represents a significant health risk due to its toxigenic potential and ability to persist under storage conditions. Moreover, Lactobacillus rhamnosus effectively reduced bacterial pathogenicity and toxin-associated effects, supporting its potential application as a natural bioprotective agent to enhance the safety of infant food products.

ACKNOWLEDGEMENT

The authors would like to acknowledge the Deanship of Graduate Studies and Scientific Research at Taif University for funding this work.
 
Author contributions
 
The principal author completed all research work items in this study.
 
Data availability statement
 
Data is contained within the article.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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    Evaluation of Lactobacillus rhamnosus as a Protective Agent against Bacillus cereus Contamination in Legume-based Infant Foods

    A
    Ashraf Albrakati1,*
    1Department of Human Anatomy, College of Medicine, Taif University, Saudi Arabia.

    • Submitted04-12-2025|

    • Accepted14-04-2026|

    • First Online 22-04-2026|

    • doi 10.18805/LRF-918

    ABSTRACT

    Background: Bacillus cereus poses a critical food safety challenge in developing countries, emphasising the need to find ways to combat its toxicity. Commercially produced baby food is authorized as a main source of nourishment for infants; thus, it should include a high concentration of nutrients and be free from any potential hazards.

    Methods: 300 random food samples were obtained from supermarkets and pharmacies in Taif and Makkah cities, Saudi Arabia. Microbiological examinations, molecular techniques and anatomical examinations were applied to confirm B. cereus pathogenicity. Probiotic bacteria were tested for their antimicrobial activity.

    Result: The bacteria were observed in almost all samples with mean total viable counts of 1.586 to 3.543 log10 cfu/g. Furthermore, 196 Bacillus colonies were identified and 27% of them were B. cereus. Finally, L. rhamnosus is an anti-B. cereus agent was used for detoxification and to reduce the pathogenicity of this pathogen. After mixing L. rhamnosus with B. cereus and/or its emetic toxins and feeding it to mice, the toxicity of B. cereus and its enterotoxins was reduced by 37 to 62%, paving the way for using this strain to protect infant meals from hazardous microbial elements. Anatomical examination showed that mice fed with B. cereus exhibited severe gastrointestinal abnormalities, whereas those treated with combinations of B. cereus and Lactobacillus rhamnosus displayed normal anatomical features. This study concluded that Lactobacillus rhamnosus may have significantly mitigated the toxicity of Bacillus cereus in infant meals, highlighting its potential as a protective agent in food safety.

    INTRODUCTION

    Bacillus cereus plays a significant role as a foodborne pathogen in developing countries, where the combination of environmental conditions and limited public health infrastructure heightens the risk of outbreaks (Woh and Ng, 2024). In these regions, food safety is often compromised by inadequate refrigeration, poor sanitation and lack of awareness about proper food handling practices. The toxins produced by B. cereus can cause severe gastrointestinal illnesses characterised by vomiting and diarrhoea (Duan et al., 2023).
           
    The challenge is exacerbated by the bacteria’s ability to form durable spores, allowing it to survive. Strategies targeting hygiene, storage and cooking practices remain relevant in reducing the impact of B. cereus.
           
    Because infants     rely heavily on limited food sources during early development, any microbial contamination poses a direct health risk due to their immature immune systems. For this reason, the hygienic quality of commercially prepared infant foods is critical, particularly during the complementary feeding period. Foodborne diseases remain a major global health concern, especially where bacterial spores can persist through processing and storage (Wang et al., 2022). Among these pathogens, Bacillus cereus has been repeatedly identified as one of the most common contaminants in pasteurized and ready-to-eat infant foods, emphasizing the need for strict safety guidelines and targeted hygienic control measures to prevent exposure in this vulnerable age group (Radmehr et al., 2020).
           
    B. cereus     is a psychrotrophic, facultative anaerobic, spore-forming pathogen associated with food-borne outbreaks (Zhao et al., 2020). It can grow at low temperatures (6-10oC) and even under refrigeration, producing emetic and diarrheal toxins (Granum and Lund, 1997). Diarrheal illness results from vegetative cells producing enterotoxins when ingested at levels exceeding 105 cfu/g (Warda, 2016). In contrast, several Lactobacillus species and lactic acid bacteria produce antimicrobial substances that can inhibit pathogens and help prevent food spoilage (Jørgensen et al., 2017; Mori et al., 2026; Timothy et al., 2021).
           
    Despite previous studies addressing Bacillus cereus contamination in food products, limited research has specifically evaluated the detoxifying role of probiotic strains such as Lactobacillus rhamnosus against B. cereus and its toxins in legume-based infant foods (Abdel-Rauf et al., 2025; Granum and Lund, 1997; Zhao et al., 2020). Therefore, this study aimed to (i) evaluate the microbiological quality of infant foods, (ii) determine the prevalence and toxigenic potential of Bacillus cereus and (iii) investigate the antimicrobial and detoxifying effects of Lactobacillus rhamnosus against B. cereus and its enterotoxins.

    MATERIALS AND METHODS

    Infant food sample collection and preparation
     
    A total of 300 samples of ready-to-use baby milk-based powders were obtained randomly from diverse supermarkets and pharmacies in Taif and Makkah cities, Saudi Arabia during the period from 2024 to 2025, as shown in Table 1.

    Table 1: Type, numbers and locations of the collected infant foods.


     
    Probiotic strain growth and preservation
     
    Lacticaseibacillus rhamnosus B-445 and Lactobacillus dulbrueckii subsp. bulgaricus Lb-12 DRI-VAC strains were delivered from the Northern Regional Research Laboratory (NRRL), Illinois, USA: Hansen’s Lab, Denmark. Lactobacillus cremoris was obtained from the Department of Dairy, National Research Centre, Egypt. They were proven to be probiotics (Adebayo-Tayo and Fashogbon, 2020) by the possession of their characteristics.
     
    Bacteriological analysis of infant foods
     
    For all collected samples, 25 g of each sample in 225 mL of sterile peptone water was mixed well by homogenizer for 2 min. Total bacterial counts were tested using tryptic soya agar and plate count agar media (Darwesh et al., 2019). In the case of psychrotrophic spore-forming bacterial counts, the procedure followed (Sadek et al., 2018).
     
    Isolation and identification of psychrotrophic Bacillus spp.
     
    Three to five isolates from each plate of typical colonies of the total psychrotrophic spore-forming plates were picked randomly. Each colony was purified on PEMBA medium, characterized as psychrotrophic Bacillus spp. and then inoculated into 10 mL of tryptic soya broth at 37oC for 24 h and then subjected to several biochemical examinations and API 50 CHB (Biomerieux, France) for identification.

    Molecular detection of toxicogenic-related genes and identification of their producing pathogens
     
    Extraction of bacterial DNA of the 22 B. cereus isolates was obtained by the lysozyme (20 mg/mL) and proteinase K (1 mg/mL) method, which was detailed previously (18). Three virulence genes, hbla, nhec and cytk, were amplified as enterotoxin genes using extracted DNA and specific primers as shown in Table 2 (19-23). PCR amplification was carried out starting with denaturation at 95oC for 5 min, followed by 30 cycles of 95oC for 1 min, 58oC for 1 min, 72oC for 1 min and a final elongation step at 72oC for 10 min, with a final hold at 4oC. PCR products were analysed in 1% (w/v) TAE agarose gel. All PCR experiments for each strain were carried out twice (Barakat et al., 2015). One isolate of B. cereus had the three tested virulence genes. Thus, it was identified using molecular biology techniques according to Adesetan et al., (2022).

    Table 2: Primer sequences and amplicon sizes used in PCR analysis.


     
    Antibacterial and antagonistic activities of the probiotic strains
     
    The antimicrobial activity of the four probiotic strains against B. cereus was evaluated using cell-free supernatants obtained after centrifugation of overnight MRS cultures. The inhibitory effect was assessed by the agar well diffusion test and the inhibition zones were measured (Khalid et al., 2021).
           
    The antagonistic activity of Lact. rhamnosus B-445 against B. cereus was further examined in broth co-culture. Standardized dilutions of both strains were mixed in tryptic soya broth and bacterial counts were determined at selected time intervals. B. cereus was enumerated on PEMBA medium (Finlay et al., 1999), while Lact. rhamnosus was counted on MRS agar under anaerobic conditions.
     
    Detoxification and reduced pathogenicity of B. cereus after Lact. rhamnosus treatment
     
    Evaluation of the pathogenicity and production of toxins by the B. cereus isolate was carried out using the mouse toxicity technique. Additionally, the effect of Lact. rhamnosus on this pathogenicity was evaluated. B. cereus was cultured in 10% skim milk medium to produce enterotoxin as a supernatant. The pellet was re-suspended in a suitable quantity of phosphate buffer to attain a bacterial suspension equivalent to 0.5 McFarland standard (Darwesh et al., 2020). In another case, Lact. rhamnosus strain was cultured in 1% (w/v) of MRS. After bacterial cultures and B. cereus crude toxins were prepared, an equal volume of Lact. rhamnosus culture was mixed with B. cereus culture or its emetic toxin and incubated at 37oC for 6 h.
           
    To evaluate the pathogenicity of B. cereus and study the role of Lact. rhamnosus in attenuating the toxicity of B. cereus, 30 adult male mice (Mus musculus domesticus) with an average weight of 100 g were obtained from the animal house at Taif University. The mice were divided into six groups containing five mice each. The animals were fed a basal diet containing 50% corn starch, 20% casein, 10% sugar cane, 10% corn oil, 5% cellulose, 4% salt mixture and 1% vitamin mixture (Darwesh et al., 2023). A total of 0.5 mL of each treatment was given orally to the mice by intragastric gavage for three days. The trials (6 groups) were as follows: The control group was fed phosphate-buffered saline and their regular food, the 2nd group was fed B. cereus and the emetic toxin, the 3rd group was fed B. cereus culture, the 4th group was fed a mixture of B. cereus, the emetic toxin and Lact. rhamnosus culture, the 5th group was fed a mixture of B. cereus and Lact. rhamnosus cultures and the 6th group was fed Lact. rhamnosus culture. The results were recorded for the mice after three days and the animals were thoroughly observed for any toxic signs through this period. Convulsions, motor activity, tremors, sedation, aggressiveness, relaxation of muscle, analgesia, hypnosis, ptosis, paralysis, lacrimation, skin colour and diarrhoea were noted during the experiment. The observed death was also recorded in each group.
     
    Statistical analyses
     
    All results were expressed as the mean±standard deviation (SD). Statistical significance was evaluated using analysis of variance (ANOVA, SAS software) followed by determining the least significant difference at 0.05.
     
    Ethical approval
     
    All experiments followed Taif University’s Ethics Committee’s guidelines (Approval No. TU 20-050, Taif, Saudi Arabia).

    RESULTS AND DISCUSSION

    Spreading of microbial communities in infant foods
     
    Three hundred infant food samples were collected randomly from pharmacies and supermarkets in Makkah and Taif cities. The collected samples were subjected to micro-biological analyses. The results showed that the total bacterial counts (TBCs) were detected in almost all examined samples, with mean total viable counts ranging from 1.586 to 3.543 log10 cfu/g. The results revealed that the total psychrotrophic spore bacteria were noticed in 40 and 60% of the whole samples of milk-based infant food with vegetables and fruits, respectively.
     
    Distribution and identification of Bacillus spp
     
    The samples were examined to determine their content of Bacillus spp. by isolation of different morphological aerobic spore-forming colonies and then characterized (morphologically, biochemically and API CHB50 tests) using acceptable protocols of Bacillus spp. identification. The distribution of diverse isolated Bacillus species is illustrated in Fig 1. The results revealed that B. subtilis was the most common species compared with other Bacillus spp. with an incidence of 36%, followed by B. cereus (27%), B. circulans (20%), B. licheniformis (12%) and B. coagulans (5%). Vegetables and fruit milk-based infant foods had the highest numbers of Bacillus spp. From the obtained Bacillus isolates, B. subtilis and B. cereus, due to their hydrolytic activities, are essential for food hygiene.

    Fig 1: Distribution of different total psychrotrophic Bacillus species in the obtained samples.


     
    Distribution and identification of Bacillus cereus
     
    B. cereus bacteria calculated on PEMBA medium were detected in almost all tested samples with the highest percentages in milk-based infant food with fruits and vegetables, which reached 62.2 and 26.6%, respectively (Fig 2).

    Fig 2: Distribution of B. cereus in the obtained samples.


           
    In contrast, the B. cereus count was not high in the ready-to-use infant food with fruits and vegetables. Generally, 40 out of 300 samples (approximately 26.67%) of the examined infant foods were positive for B. cereus counts.
           
    Due to Bacillus cereus isolate No. 8 pathogenicity and production of the 3 virulent harmful toxin-related genes, B. cereus was identified using molecular biology techniques to confirm the biochemical and API CHB50 tests. The obtained sequence was compared with available sequences in the GenBank database and the similarity percentage of this isolate accounted for 98% with Bacillus cereus strains. Additionally, a phylogenetic tree was constructed and it showed that this strain was very close to the type strains of B. cereus deposited in the culture collection centre of the National Center for Biotechnology Information (Fig 3).

    Fig 3: Phylogenetic tree of B. cereus and related species in the gene bank.


     
    Molecular detection of toxicogenic-related genes
     
    Three virulence genes (hbla, nhec, cytk) were screened in 25 B. cereus isolates using PCR. Only two isolates (8 and 9) carried all three genes. The hbla gene (1200 bp) appeared in five isolates and was mainly detected in samples from milk-based infant foods with fruit, rice, wheat and honey but with variable frequencies. The nhec gene showed a wider distribution across isolates from infant foods containing fruit, vegetables, honey and wheat, while it was absent in rice-based samples. In contrast, the cytk gene exhibited the highest prevalence, being detected in 92% of isolates collected from all infant food types examined. Overall, isolate 8-obtained from milk-based infant food with fruit-showed the strongest hbla band intensity among the positive samples (Fig 4).

    Fig 4: Agarose gel pictures for the PCR product of hbla (A), cytk (B) and nhec (C) genes in B. cereus isolates compared with the marker (1 kb).


     
    Detoxification and reduced pathogenicity of B. cereus using Lact. rhamnosus
     
    The cell-free supernatant containing bioactive metabolites of probiotic strains was obtained and employed against the B. cereus strain using an agar well diffusion assay. The data from the pictures illustrated in Fig 5 show that the strain Lact. rhamnosus B-445 produced high antibacterial activity against the B. cereus strain, with a 21 mm inhibition zone diameter, followed by Lb. dulbrueckii and Lb. cremoris, but Leuconostoc mesenteroides appeared to have no activity. The antagonistic activity of Lact. rhamnosus against B. cereus was assessed in the broth culture media system and the results are illustrated in Fig 5. From that, we can clearly show the antagonism between the two tested strains. The growth of the B. cereus strain was inhibited under mixed culture with Lact. rhamnosus starting from 6 h of incubation. Lact. rhamnosus exposed a high inhibitory activity against the B. cereus strain, resulting in a decrease in the total counts from 5.3 to 3.3 Log10 cfu/mL after 24 h with a reduction of 37.7% and reached 2.0 Log10 cfu/mL with a reduction of 62.2% after 48 h of incubation.

    Fig 5: Anti-Bacillus cereus activity of some probiotic strains.


     
    Toxicology studies of B. cereus cells and crude toxins
     
    The probiotic strain Lact. rhamnosus was mixed with B. cereus and/or its emetic toxins and added to the diets of the experimental mice. After three days of the experiment, the groups treated with B. cereus cells and its emetic toxin, groups two and three, showed some clinical signs, such as increased mortality and decreased weight compared with the other groups. The control group did not show any abnormal weight changes (ranging between 100 and 103 g) after three days. While mice fed with bacterial cells of B. cereus showed loss of their body weight and slower movement; their weights ranged from 72 to 75 g and two of five mice died. Additionally, four out of five mice in the group fed emetic toxins died and the remaining mice had a clear decrease in their weight to 60 g and slower movement. However, the group fed a mixture of B. cereus cells and Lact. rhamnosus exhibited normal appearance and movement with weights reaching 93-94 g. Moreover, the mouse group provided a mix of crude toxins and Lact. rhamnosus culture displayed normal appearance, motion and weight, with weights ranging between 98 and 101 g. The group was fed with a diet containing Lact. rhamnosus culture appeared to exhibit normal behaviour and their weight reached 100-105 g. Anatomical examination of the gastrointestinal tract of dissected mice revealed swelling of the intestinal tract due to fluid accumulation in the intestine’s lumen and inflammation of the intestines and stomach, particularly when mice were fed with the B. cereus. Additionally, ulcers and dark red spots were observed in the liver of groups fed with B. cereus, indicating severe vascular effects of the toxin. In contrast, no significant changes were noted in the stomach and intestines of mice treated with crude emetic enterotoxin. However, the group fed a mixture of B. cereus and Lactobacillus rhamnosus showed a normal stomach, intestine and liver compared to the control group. Similarly, the group fed a mixture of B. cereus emetic enterotoxin and Lactobacillus rhamnosus B-445 exhibited normal stomach, intestine and liver conditions. Furthermore, the group fed the diet with Lactobacillus rhamnosus B-445 appeared normal in all examined body contents (Fig 6).

    Fig 6: Anatomical examination of mice under various treatments like control (a), bacterial cells of B. cereus (b), B. cereus the emetic toxins (c), mixture of B. cereus toxins with L. rhamnosus B-445 culture (d), mixture from bacterial cells of B. cereus and L. rhamnosus B-445 culture (e) and L. rhamnosus B-445 culture (f). The long arrow indicates the stomach and the short arrow indicates the intestines.


           
    Infant foods are certified as a primary nutrition source for babies from seven months to 2 years old. Thus, they should represent a rich source of nutrients from several origins. However, germs in babies’ food can cause severe infection due to their immune systems not being completely developed (Martin et al., 2010). This study evaluated the hygiene of infant foods and tried to improve the quality of these foods. To achieve this objective, three hundred infant food samples were collected randomly from pharmacies and supermarkets in Makkah and Taif cities, Saudi Arabia. The collected samples were subjected to microbiological analyses. The results showed that the total bacterial counts (TBCs) were detected in almost all samples, with mean complete viable counts ranging from 1.586 to 3.543 log10 cfu/g. A similar observation was reported by other researchers (Gautam, 2015; Luby et al., 2011).
           
    The TBC falls within standard regulations; therefore, it is less critical than spore-forming and pathogenic bacterial counts. High TBC values generally indicate poor hygienic practices during handling, packaging, or the use of low-quality ingredients (Mokgaotsi, 2019). For this reason, total psychrotrophic spore-forming bacteria were also evaluated and they were detected in 40% and 60% of milk-based infant foods with vegetables and fruits, respectively.
           
    At this stage, psychrotrophic spore bacteria in infant milk meals were reported at 2.9 × 10² cfu/g (Ahmed et al., 2008) and another study showed that 40% of vegetable-based baby foods tested positive (Sadek et al., 2018). Bacillus spp. were isolated and identified following standard morphological, biochemical and API CHB50 protocols, with B. subtilis being the most common species in milk-based vegetable and fruit foods. Controlling B. subtilis and B. cereus is essential due to their hydrolytic activities, toxin production and ability to grow at refrigeration temperatures. These findings agree with previous reports detecting B. subtilis (28%), B. licheniformis (20%) and B. cereus (14%) in infant formulae (Mostafa et al., 2002). Although less prevalent, B. cereus remains the most harmful due to its pathogenicity and toxin production. FDA regulations require B. cereus levels to remain below 100 cfu/g. In this study, B. cereus was detected in all tested samples, especially milk-based infant foods with fruits and vegetables, while absent in ready-to-use products. Other studies similarly reported B. cereus contamination in powdered milk and a variety of infant foods (Kim et al., 2011). Differences in prevalence are likely related to sample type, testing method and product formulation. Previous research has suggested applying stable, nontoxic bioactive compounds and food-grade natural materials to reduce B. cereus in infant foods (Sadek et al., 2018).
           
    The pathogenicity of B. cereus was assessed by detecting three key enterotoxin-related genes hbla, nhec and cytk using PCR. Two isolates (8 and 9) carried all three virulence genes. Toxigenic B. cereus strains have been previously reported in a wide range of starchy and dairy-based foods (Jovanovic et al., 2021; Mohammadi, 2023), emphasizing the need to evaluate the safety of infant foods commonly marketed in Saudi Arabia. PCR detection methods are increasingly applied for identifying these genes (Kim et al., 2000). In this study, isolate 8 originating from milk-based infant food with fruit was positive for the hbla gene, consistent with earlier reports detecting hbla in dairy and child-related foods (Rahimi et al., 2013). Gene distribution patterns varied according to food type, season and collection conditions. The nhec gene was widespread among isolates from fruit-, vegetable-, honey- and wheat-based infant foods but absent in rice-based samples, matching previous findings where nhec occurred in 80% of infant-food isolates (Rahimi et al., 2013). The cytk gene showed the highest prevalence, appearing across all food categories examined. As cytk is strongly associated with severe food-poisoning outbreaks, isolate 8 was selected as the pathogenic reference strain. Its genomic DNA was extracted and the 16S rRNA gene sequence showed 98% similarity to known B. cereus strains. Phylogenetic analysis confirmed its close relationship to established B. cereus type strains in the NCBI database.
           
    The antimicrobial activity of the probiotic strains was assessed using cell-free supernatants in an agar well diffusion assay, where Lact. rhamnosus B-445 showed the strongest inhibition against B. cereus (21 mm), followed by Lb. dulbrueckii, Lb. cremoris and Leuconostoc mesenteroides. This variation reflects differences in the production of bioactive compounds such as organic acids, polysaccharides and bacteriocins (Liu et al., 2023; Moniri et al., 2017). LAB strains, including Lact. rhamnosus B-445 and Lb. dulbrueckii, are known for their antibacterial activity against Bacillus spp. (Ahmad et al., 2022; Zeinab et al., 2015). In broth co-culture, Lact. rhamnosus markedly suppressed B. cereus growth, reducing counts from 5.3 to 3.3 Log10 cfu/mL at 24 h and to 2.0 Log10 cfu/mL at 48 h. This inhibition is attributed to bacteriocins and related metabolites with applications in food biopreservation (Fuochi et al., 2019).
         
    Infant foods require high hygiene standards because B. cereus is a pathogenic bacterium that poses significant health risks. The bacterium is particularly dangerous due to its spore-forming ability, which makes it resistant to thermal treatments and its toxins remain stable even under hygienic controls. To investigate a safe protective agent, the probiotic strain Lact. rhamnosus was mixed with B. cereus cells and/or emetic toxins and incorporated into mice diets. Mice fed only B. cereus cells or toxin alone experienced severe clinical signs including significant weight loss (60-75 g), reduced mobility and high mortality with 4 out of 5 mice in the toxin-treated group dying. However, mice fed B. cereus (cells or toxins) combined with Lact. rhamnosus maintained normal weight (93-105 g) and exhibited normal behavior and movement. Pathological examination revealed that mice exposed to B. cereus alone developed swollen intestinal tracts, ulcers and dark coloration of the liver. In contrast, groups treated with B. cereus mixed with Lact. rhamnosus showed normal stomach, intestine and liver tissues. These findings demonstrate that Lact. rhamnosus B-445 could serve as an effective and environmentally safe protective agent against B. cereus in infant foods. Probiotic supple-mentation has been widely applied to improve microbial balance and health performance in food-related biological systems, as reported in previous publications (Barde et al., 2025).

    CONCLUSION

    The findings demonstrate that Bacillus cereus contamination in infant foods represents a significant health risk due to its toxigenic potential and ability to persist under storage conditions. Moreover, Lactobacillus rhamnosus effectively reduced bacterial pathogenicity and toxin-associated effects, supporting its potential application as a natural bioprotective agent to enhance the safety of infant food products.

    ACKNOWLEDGEMENT

    The authors would like to acknowledge the Deanship of Graduate Studies and Scientific Research at Taif University for funding this work.
     
    Author contributions
     
    The principal author completed all research work items in this study.
     
    Data availability statement
     
    Data is contained within the article.

    CONFLICT OF INTEREST

    The authors declare no conflict of interest.

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