Milk and milk based-products, which are considered a principal part of sensitive age-group meals (infants, children, and elders), can be contaminated with aflatoxins (AFs), which are the most toxic and carcinogenic class of mycotoxins. AFs are produced by several Aspergillus species on tropical and subtropical agricultural commodities. Among AF types, aflatoxin B1 (AFB1), which is the most prevalent and toxic compound found in food and feed, is metabolized by cow-liver enzymes to the 4-hydroxy derivative (AFM1), and secreted in milk (Mohammedi-Ameur et al., Reference Mohammedi-Ameur, Dahmane, Brera, Kardjadj and Ben-Mahdi2020).
Health risks associated with the consumption of food contaminated with AFs are acute and chronic toxicity. AFB1 can cause critical liver injury, liver cirrhosis and tumors, while AFM1 is considered a major etiological factor for hepatocellular carcinoma, mutagenicity and teratogenicity in mammals. Recently, hepatocellular carcinoma associated with AF exposure has led to death (IARC, 2002). Furthermore, Food and Agriculture Organization (FAO) estimated that approximately 25% of feed and food are lost annually worldwide because of fungi and the associated mycotoxins (Moretti et al., Reference Moretti, Logrieco and Susca2017).
Because of the high intake of milk powder by all age groups, the heavy consumption of milk-based infant formulae by sensitive age groups (infants and children) which is necessary for growth, the major toxicity of AFM1 and the major economic losses (Ahmed et al., Reference Ahmed, Awad, Mohamed and El Kutry2020; GadAllah et al., Reference GadAllah, Abou Zied and Fahim2020), many countries have set a maximum permissible levels for AFM1 in milk and dairy products which vary from 0.025 to 0.05 μg kg−1 in Egypt, European Union, and USA (USA/FDA, 2005; EC European Commission Regulation, 1881/2006; ES Egyptian Standard, 7136/2010). Despite these regulatory control measures adopted for AFM1, several studies proved its presence in milk and dairy products and their implication in human intoxication, which is mainly explained by its heat stability during the food processing. Therefore, it is tremendously important to control the quality of the animal feed in order to avoid contamination with AFB1 and consequently bio transformation into AFM1, in addition to adopting innovative methods for reducing or eliminating molds and aflatoxins from food and feed (Ahmed et al., Reference Ahmed, Hafez, Morgan and Awad2015). In this respect, prevention of fungal growth and the subsequent mycotoxin production in foods and feeds, as well as the detoxification of mycotoxins are the main proposed strategies. Chemical and physical methods are among the primary preventative methods. However, several health risks resulted from the extensive use, environmental problems, and lowering the food quality. Therefore, biological control is the safest and effective alternative for control the toxigenic fungi and their mycotoxins (Suresh et al., Reference Suresh, Cabezudo, Pulicharla, Cuprys, Rouissi and Kaur Brar2020).
Probiotics, defined as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host, can be used for this purpose. Lactic acid bacteria (LAB) are on the top of these probiotic microorganisms because of their good safety history in food applications, linked to the production of several bioactive metabolites, the fact that they are generally recognized as safe, and their low production cost. Among the LAB used, Bifidobacterium and different Lactobacilli (L. paracasei, L. plantarum, L. reuteri, L. amylovorus, L. rhamnosus, and L. fermentum), act through the adsorption and binding of mycotoxins by their cell wall that contains polysaccharides, protein, and peptidoglycans (Badr et al., Reference Badr, Ali, Abdel-Razek, Shehata and Albaridi2020; Ren et al., Reference Ren, Zhang, Zhang, Mao and Li2020). Viability of the LAB is affected by the gastrointestinal ecosystem following the consumption of food contained probiotics (Shah, Reference Shah2007). Consequently, encapsulation is considered a functional solution for protection of the LAB under adverse conditions without influencing their antimicrobial effects. Use of whey protein as an encapsulation biomaterial increases the viability of these microorganisms under adverse conditions (Mohammadi et al., Reference Mohammadi, Abbaszadeh, Sharifzadeh, Sepandi, Taghdir, Miri and Parastouei2021). However, only a limited number of studies have estimated the antifungal effects of the encapsulated LAB and its effect on AF production.
The functional properties of paraprobiotics (non-viable cells or the cellular extract) in AF control have been reported by many researchers who have recorded the higher binding efficiency of the nonviable cells compared to the viable cells because of a greater number of binding sites exhibited for AFs and the absence of undesirable fermentative changes in milk that are caused by viable cells (Zhang et al., Reference Zhang, Liu, Zhao, Wang, Jiang, Xin and Zhang2019). Variation in the stability of this form of binding suggested the use of a postbiotic as a promising alternative method for fungal growth inhibition and AF degradation (biological detoxification). These are defined as the metabolic byproducts secreted by live bacteria or released after bacterial lysis that have beneficial effects on the host. They include organic acids, short chain fatty acids, carbon dioxide, hydrogen peroxide, phenyllactic acid, and bioactive low molecular weight peptides, reuterin, diacetyl and bacteriocins and bacteriocin-like inhibitory substances. This innovative method is characterized by broad spectrum of target mycotoxins, low cost, minimal side effects regarding nutrients and the suitability for a wide range of liquid and solid foods (Moradi et al., Reference Moradi, Molaei and Guimarães2021).
Owing to the importance of the continued AFM1 monitoring in milk products, especially those intended for infants, as well as the scarcity of reports covering the reduction control of AFM1 using the encapsulated probiotics and acid treated cells (parabiotics) together with the absence of studies covering AFM1 detoxification by the postbiotics, the present study was designed to evaluate AFM1 levels in infant milk formulae and milk powder retailed in the Egyptian markets with assessing the degree of compatibility with the different standard regulations. As a practical solution, the study proposed the use of the bioactive compounds; postbiotic (cell free supernatant), parabiotic (acid treated probiotic) and the encapsulated cells of L. plantarum RM1 and L. paracasei KC39 for the reduction of AFM1 & AFB1 and the assessment of their antifungal effect against toxigenic mold strains.
Materials and methods
Collection of samples
A total number of fifty random samples of infant cow's milk-based formulae (n = 25) and full-fat milk powder (n = 17 unpacked and 8 packed) were collected from local markets in Cairo and Giza governorates, Egypt.
Determination of AFM1 content
Prevalence of AFM1 in the examined samples was carried out using indirect enzyme-linked immune-sorbent assay (ELISA) test kit, BioFront Technologies, Commonwealth Blvd., Tallahassee, USA according to (Sani et al., Reference Sani, Khezri and Moradnia2012).
Reduction of the AFs using the bioactive compounds of Lactobacillus plantarum RM1 and Lactobacillus paracasei KC39
Preparation of the probiotic cell pellets
The new strains of Lactobacillus plantarum RM1 and Lactobacillus paracasei KC39, isolated from the fermented Rayeb and Karesh cheese, respectively by (Shehata et al., Reference Shehata, Badr and El Sohaimy2018, Reference Shehata, Badr, El Sohaimy, Asker and Awad2019) were activated on MRS broth media at 37°C/24 h, then, transferred to Lab-fermenter (Jupiter stirred mini-fermenter 4L, Solaris Biotech., Porto Mantovano, Italy) containing MRS-broth and incubated at 37°C/24 h and the yield of each strain was separately centrifuged at 4100 g/5°C/30 min to obtain the cell-pellets.
Preparation of RM1 and KC39 postbiotics
The cell-free supernatant (CFS) was prepared according to Shehata et al. (Reference Shehata, Badr and El Sohaimy2018, Reference Shehata, Badr, El Sohaimy, Asker and Awad2019). The solution derived from L. plantarum RM1 and L. paracasei KC39 bacterial-pellet centrifugations were known as postbiotics. They were collected, purified and sterilized by an individual sterile-membrane (0.22 μm), then lyophilized by a Dura-Dry MP freeze-dryer (FTS System, USA) to yield dry-pure powder.
Preparation of RM1 and KC39 parabiotic
The treated probiotic cell-pellets were prepared according to El-Nezami et al. (Reference El-Nezami, Kankaanpaa, Salminen and Ahokas1998) with modification. Hydrochloric acid (1 m) was used for acidification of the media that made the bacterial cells to die. The bacterial concentrations were 2.1 × 1011 and 1.7 × 1011 CFU ml−1 media for RM1 and KC39, respectively.
Preparation of the encapsulated bacterial cells
Bacterial strains were encapsulated by wall material consists of maltodextrin and whey protein (1:2), according to the method designated by Abdel-Razek et al. (Reference Abdel-Razek, Badr, El-Messery, El-Said and Hussein2018) with bacterial cell concentrations of 2.1 × 1011 and 1.7 × 1011 CFU ml−1 for RM1 and KC39, respectively.
Determination of the antifungal activity of bacterial treatments
The antifungal activities of the postbiotics, parabiotics, and encapsulated probiotics were assessed against four toxigenic fungal strains (A. flavus ITEM 698, A. parasiticus ITEM 11, Fusarium moniliforme KF 488, and Penicillium chrysogenum ATCC 10106), which were obtained from ISPA, Bari, Italy using the agar well diffusion method according to Badr et al. (Reference Badr, Ali, Abdel-Razek, Shehata and Albaridi2020).
Estimation of the effect of postbiotics, parabiotics and the encapsulated probiotics on AFB1 secretion in a liquid media
Impact of the bacterial treatments on AFB1 reduction in a liquid media was done as described by Shehata et al. (Reference Shehata, Badr, El Sohaimy, Asker and Awad2019) with modifications. The spores of Aspergillus flavus ITEM 698 were inoculated into yeast extract sucrose (YES) broth media at a concentration of 105 ml−1 and incubated for 18 h. Postbiotics, parabiotics, and the encapsulated cell pellets were inoculated separately (1 mg ml−1 media), then re-incubated (96 h/30°C). AFB1 secreted in the liquid media was determined using ELISA method according to Sani et al. (Reference Sani, Khezri and Moradnia2012). The reduction in fungal mycelial weight was compared to the control one and the antifungal efficacy (AE) was calculated as a percentage by the following equation:
$${\rm AE\% }\,\,{\rm} = [ {( {{\rm MFWc}-{\rm MFWt}} ) {\rm /\ MFWc}} ] \,\,\times \,100$$Where AE: is the antifungal efficacy of the treatment, MFWc: is the dried mycelia-weight of control fungal growth, MFWt: is the dried mycelia-weight of treated fungal growth.
The effect of postbiotics, parabiotics, and encapsulated probiotics on AFM1 reduction
The reduction in AFM1 concentration owing to each treatment was determined in a model of reconstituted milk powder according to Negera and Washe (Reference Negera and Washe2019) with some modification. AFM1 residue was determined using ELISA according to Sani et al. (Reference Sani, Khezri and Moradnia2012). Toxin inhibition was calculated as a ratio of inhibition by the following equation:
$${\rm \% TRR}\,{\rm} = [ {{\rm Toc \ndash Tot\ /Toc}} ] \times 100$$Where TRR: is the toxin reduction ratio achieved by the treated components, Toc: is the toxin concentration in the control buffer solution, Tot: is the toxin concentration in a buffer solution with the different treatments.
Statistical analysis
The data were statistically analyzed using SPSS Version 17.0 software. An independent sample t-test was conducted to compare the AFM1 levels in infant milk and milk powder, Mann–Whitney U test was used when data were not normally distributed. Pearson's correlation coefficient (r) was used for assessing the impact of using postbiotics, parabiotics, and the encapsulated cells on the reduction of AFM1 and AFB1. Significance was set at P-value <0.05.
Results
The samples of milk powder and infant formulae were positive for AFM1 contamination, with no statistically significant differences (P > 0.05) between them, although AFM1-concentration in the packed milk powder sample (46.58 ± 7.69 ng kg−1) was numerically lower than unpacked milk powder (88.78 ± 33.31 ng kg−1) and infant formulae (78.83 ± 19.31 ng kg−1) (Table 1).
Table 1. Prevalence of AFM1 (ng/kg) in samples of dairy products
Aflatoxin M1 was determined in nano-gram per kg using ELISA technique.
Mann–Whitney test was performed for comparing AFM1 content in infant formulae and milk powder and between the packed and unpacked milk powder samples, showing non-significant differences between the different groups.
According to the critical limit of AFM1 depicted in different legislations, 96.0, 29.41, and 25.0% of the examined infant formulae, unpacked, and packed milk powder exceeded the Egyptian (ES Egyptian Standard, 7136/2010) and European regulations (EC European Commission Regulation, 1881/2006) which states a maximum limit of 25 and 50 ng kg−1 for infant formula and milk powder, respectively. All samples were acceptable within the USA/FDA regulation, which specifies a fixed maximum limit of 500 ng kg−1 for milk products and milk-based formulae (Table 2).
Table 2. Acceptability of the samples in relation to AFM1 content with the various regulation standards
EC: European Regulations (EC European Commission Regulation, 1881/2006), ES: Egyptian Standard (ES Egyptian Standard 7136/2010).
Data presented in Fig. 1 show that all mycotoxigenic fungi strains were sensitive to the different treatments of probiotics, with the highest sensitivity for Fusarium strain with L. paracasei KC39 compared to the other genera. Moreover, postbiotics exhibited the best antifungal activity against F. moniliforme (16.7 ± 1.3 and 23.7 ± 1.5 mm) for L. plantarum RM1 and L. paracasei KC39, respectively.
Fig. 1. Antifungal impacts of probiotic components against four toxigenic mold strains. *Antifungal impacts were represented as diameter of the inhibition zone (millimeter) of fungal growth regarding each component treatment.
Results of the bioactive compound application in yeast extract sucrose media containing a high producer strain of AFB1 are shown in Table 3. Growth of A. flavus in the control and its secreted AFB1 content were assessed as a reference used for comparing the different treatments. The obtained results demonstrated fungal-growth inhibition, with AFB1 reduction ratio of 56.40, 50.27 and 38.60% for L. plantarum postbiotics, encapsulated cells and parabiotics, respectively. In relation to L. paracasei, the parabiotic KC39 achieved the highest reduction ratio (60.56%) of AFB1 secretion followed by the encapsulated KC39 (52.73%), whilst postbiotic KC39 yielded a reduction percentage of 42.94%. These results potentially give a safe solution for AFB1 contamination issues in milk and milk products.
Table 3. Impact of the bioactive components of two probiotic strains on AFB1 secretion in the liquid media
Results are expressed as means ± se. AE: the antifungal efficacy of the treatment. Mycelia weight represents the mycelia weight of A. flavus ITEM 698 in the broth media.
The three RM1-bioactive compounds gave lower activities compared to those of the KC39-bacterial strain. It is important to refer to the postbiotics of KC39-strain as being the most effective, capable of a reduction of 89.8% from the applied models (Fig. 2).
Fig. 2. Reduction control of AFM1 in reconstituted milk powder using the bioactive components of the two probiotic strains. *The data expressed as means of AFM1 concentration.
The study revealed a very strong positive and significant correlation between both probiotic strains against -AFB1 -AFM1 in liquid media for CFBE and postbiotic (r = 0.99, P < 0.05). In contrast, only a weak correlation was found for encapsulated RM1-AFB1 & -AFM1 (r = 0.34). Besides, the capsuled probiotic KC39 showed a moderate significant correlation with AFB1 & -AFM1 (r = 0.58, P < 0.05) and postbiotic of KC39-AFB1 & -AFM1 (r = 0.58, P < 0.05). The positive and highly significant relationship between these effective compounds with detoxification effect indicated that bioactive compounds play a major role in the detoxification effect of probiotic strains.
Discussion
Mycotoxins pose a serious health threat to humans and animals, and AFM1 is the main mycotoxin found in milk and dairy products. This includes milk powder, which is used in the manufacture of many other milk products such as ice cream, cheese, evaporated milk and condensed milk, in addition to its use as an ingredient in many bakery products, processed meats, and soups (Ahmed et al., Reference Ahmed, Hafez, Morgan and Awad2015). Therefore, its contamination with AFM1 means contamination of all downstream products. The problem is exacerbated since neither heat treatment nor mild acidic conditions have been reported to cause any substantial reduction in AFM1 content. Moreover, infant milk formulae constitute the main meal for some newborns and infants during the first months of life, so it should be AFM1 free (Meucci et al., Reference Meucci, Razzuoli, Soldani and Massart2009).
The current study records a high rate of occurrence of AFM1 in the tested samples of infant formulae and milk powder, whether packed or unpacked. AFM1 is a metabolite, not a regular secretion by fungi, which might explain the lack of packed/unpacked difference. These outcomes are similar to those reported by Murshed (Reference Murshed2020) and may relate to lack of awareness, insufficient regulatory infrastructure, and/or the absence of proper storage conditions for the animal feed, which resulted in contamination with AFB1 that bio transformed into AFM1 and was secreted in milk (Mohammedi-Ameur et al., Reference Mohammedi-Ameur, Dahmane, Brera, Kardjadj and Ben-Mahdi2020).
The risk posed by AFM1 may potentially extend to increased cancer incidence. For instance, AFM1 increases risk of liver cirrhosis which in turn is a risk factor for hepatocellular hepatoma. Morsy et al. (Reference Morsy, Saif-Al-Islam and Ibrahim2018) reported a doubling of hepatocellular carcinoma (HCC) over the past decade in Egypt but did not attempt to relate this to AFM1. Nevertheless, the confirmed toxic and carcinogenic impacts of AFM1 lead the international agency for research on cancer (IARC) to change its classification from possibly carcinogenic to carcinogenic to humans (IARC, 2002). Carcinogenicity of AFM1 is influenced by the duration and level of exposure, which may be increased by frequent milk consumption (Ahlberg et al., Reference Ahlberg, Grace, Kiarie, Kirino and Lindahl2018). Accordingly, many countries have issued strict regulations concerning the maximum permissible AFM1 levels in milk and dairy foods to protect the consumers from their public health effects. The elimination of risk sources represents a major challenge not only in relation to contaminated consumer products, but also to animal feeds which ultimately constitute the main source of AFM1 in milk and milk products. Consequently, the main way for avoiding AFM1 presence in milk is to prevent cattle from feeding contaminated rations, through adopting good feeding practices, having good storage conditions and applying good manufacturing practices.
Where contamination of dairy products is concerned, investigators search to solve this problem using natural components, including probiotic bacterial strains, which have been suggested to bind and inactivate aflatoxins. The binding ratio is affected by heat or acid treatment of the bacterial cells (El-Nezami et al., Reference El-Nezami, Mykkanen, Kankaanpaa, Suomalainen, Salminen and Ahokas2000; Badr et al., Reference Badr, Ali, Abdel-Razek, Shehata and Albaridi2020). Recently, researchers referred to the bioactive components of the probiotic strains such as L. plantarum RM1 and L. paracasei KC39 basically as a new type of bacteriocin which could have a significant role in aflatoxin degradation (Shehata et al., Reference Shehata, Badr and El Sohaimy2018, Reference Shehata, Badr, El Sohaimy, Asker and Awad2019).
An alternative approach is to inhibit the growth of mycotoxin-producing fungi (Ahmed et al., Reference Ahmed, Nehal, Abdel-Salam and Fahim2021). Lactic acid bacteria (LAB) have been shown by several studies to be a suitable solution for preventing the fungal growth and prolonging the shelf-life of food owing to the produced antifungal compounds, such as organic acids, diacetyl, fatty acids, bioactive antimycotic peptides, bacteriocins, carboxylic acids, lactones, hydrogen peroxide, reuterin and alcohols (Faizan et al., Reference Faizan, Bowen, Fengwei, Jianxin, Hao and Wei2019). However, there are only a few reports concerning the antifungal effect of the bacterial bioactives, postbiotics, parabiotics and encapsulated LAB. By comparing the antifungal impact of these compounds we were able to show that, in general, parabiotics derived from LAB are the most effective treatment in exhibiting fungal growth suppression, followed by the encapsulated cells, then the postbiotics. The encapsulated cells of L. paracasei KC39 (isolated from the Egyptian traditional fermented milk ‘Laban rayeb’ and Karesh cheese by Shehata et al., Reference Shehata, Badr and El Sohaimy2018, Reference Shehata, Badr, El Sohaimy, Asker and Awad2019) were particularly effective at inhibiting the mycelia of the four toxigenic mold strains that we tested. Moreover, Fusarium moniliforme was the most sensitive strain for the tested bioactives.
These results agree with those reported by Russo et al. (Reference Russo, Arena, Fiocco, Capozzi, Drider and Spano2017), whilst lower activity against the fungal growth of A. flavus, A. niger and A. parasiticus by the encapsulated L. casei (LC-01) was recorded by Mohammadi et al. (Reference Mohammadi, Abbaszadeh, Sharifzadeh, Sepandi, Taghdir, Miri and Parastouei2021).
Bacteriocins, organic acids, enzymes, alcohols, and low-molecular-mass substances are the main metabolites responsible for the antimicrobial action of LAB. These bioactive materials were reported to affect the aflatoxigenic fungal growth with subsequent reduction of their AF secretion (Zhao et al., Reference Zhao, Shao, Jiang, Shi, Li, Huang, Rajoka, Yang and Jin2017; Ren et al., Reference Ren, Zhang, Zhang, Mao and Li2020). Other studies demonstrated that lactobacilli inhibited AF production, as well as the growth of Aspergillus spp. (Huang et al., Reference Huang, Duan, Zhao, Gao, Niu, Xu and Li2017). RM1 postbiotics were most effective in reducing AFB1 production, followed by the encapsulated cell then the parabiotics, but for KC39, in contrast, the ordering was parabiotics > encapsulated > postbiotics.
The reductions we obtained are lower than that reported by Mohammadi et al. (Reference Mohammadi, Abbaszadeh, Sharifzadeh, Sepandi, Taghdir, Miri and Parastouei2021), who revealed that the encapsulated L. casei (LC-01) reduced AFB1 almost completely (99.2%). Lactobacillus binding to AFs is the possible mechanism for AFB1 reduction (Hashemi and Amiri, Reference Hashemi and Amiri2020). This was confirmed by Ben Taheur et al. (Reference Ben Taheur, Mansour, Ben Jeddou, Machreki, Kouidhi, Abdulhakim and Chaieb2020) who illustrated the reduction through the binding of AFs to the bacterial cell wall, particularly the gluco-manann component which showed better capability to bind AFs whether bacteria were viable or killed (by acid or heat).
Various studies revealed the higher AF binding efficiency of nonviable cells compared to viable (Elsanhoty et al., Reference Elsanhoty, Salam, Ramadan and Badr2014). Our study revealed that acid dead KC39 (parabiotics) achieved the best AFB1 reduction, which agrees with Haskard et al. (Reference Haskard, El-Nezami, Kankaanpää, Salminen and Ahokas2001) who showed that acid, in particular, and heat treatment have a significant positive impact on the reduction of AFB1 by L. plantarum and L. casei. Azab et al. (Reference Azab, Tawakkol, Hamad, Abou-Elmagd, El-Agrab and Refai2001) observed that AFB1 removal by L. acidophilus, L. casei, L. helveticus and L. bulgaricus was 43.1–87.0% for the acid treatment, which is lower than we observed. These results may perhaps be explained by the effect of acid on the cell wall. Through breaking the glycosidic linkages between polysaccharides or increasing hydrolysis of the proteins into smaller peptides and amino acids, the cell wall thickness is reduced and pore size increased (via decreasing the cross linkages). These changes expose more microbial cell sites for AF binding and inactivation (Haskard et al., Reference Haskard, El-Nezami, Kankaanpää, Salminen and Ahokas2001).
It is also possible that bioactive components in the bacterial growth media could convert AFs to less toxic material. Such bacterial bioactive metabolites, or postbiotics, have been categorized into four distinct groups comprising micro-molecular organics, amino acids, antibiotics, and enzymes (Ren et al., Reference Ren, Zhang, Zhang, Mao and Li2020). The reduction percentage of AFB1 by RM1 postbiotics was nearly similar to cell free supernatants of B. subtilis that provided 60% AFB1 degradation in the study conducted by Suresh et al. (Reference Suresh, Cabezudo, Pulicharla, Cuprys, Rouissi and Kaur Brar2020), who explained the degradation mechanism via enzymes, or other bioactive components expressed by B. subtilis rather than the toxin binding. Regarding AFB1 reduction in the broth media by RM1 postbiotic, presence of protein with a new molecular weight within the RM1 postbiotics previously reported by Shehata et al. (Reference Shehata, Badr and El Sohaimy2018) might be the main cause. This was in accordance with the theory provided by Ren et al. (Reference Ren, Zhang, Zhang, Mao and Li2020). The inhibition recorded by KC39 postbiotic could be related to the presence of organic acids (lactic, phenyl lactic, hydroxyl-phenyl lactic, and indole lactic acid), previously identified by GC-MS, in addition to the purified bacteriocin which is a completely novel active peptide identified as bacteriocin KC39 by Shehata et al. (Reference Shehata, Badr, El Sohaimy, Asker and Awad2019).
Regarding AFM1 degradation, we observed that KC39 bioactives are more effective than their corresponding RM1 bioactive. KC39 postbiotics were the most effective bioactive at reducing AFM1, followed by the encapsulated-KC39 then KC39-parabiotics. These results are in accordance with those recorded by Russo et al. (Reference Russo, Arena, Fiocco, Capozzi, Drider and Spano2017) who referred to the bacterial cell-free supernatant components as a main reason for aflatoxin degradation. The effective role of KC39 and RM1 postbiotics could be explained by the same theory illustrated before with AFB1 (Ren et al., Reference Ren, Zhang, Zhang, Mao and Li2020).
The results of KC39 parabiotics on AFM1 are higher than that recorded by Assaf et al. (Reference Assaf, Atoui, Khoury, Chokr and Louka2017) who reported a reduction of 63% by L. rhamnosus GG in PBS after heat treatment, and Muaz and Riaz (Reference Muaz and Riaz2021) who reported that acid treated L. paracasei (108 CFU g−1) successfully reduced AFM1 in milk spiked with 0.2 μg l−1 to 47, and 62% AFM1 removal against 109 CFU g−1. The ability of the dead cells to remove AFs has been suggested through the formation of a non-covalent complex by the components of the bacterial cell wall (Shetty et al., Reference Shetty, Hald and Jespersen2007). Moreover, the protein denaturation by the acid and heat treatments results in formation of hydrophobic surfaces which further act as binding sites for aflatoxins (Elsanhoty et al., Reference Elsanhoty, Salam, Ramadan and Badr2014).
Finally, postbiotics which are defined as soluble metabolites released by food-grade microorganisms during the growth and fermentation are rich in high and low molecular weight biologically active metabolites. There are still gaps concerning these substances. Postbiotics are suggested as superior to probiotics because of their defined chemical composition, safety, ease of use and storage, stability in a broad range of temperature and pH and their broad-spectrum antimicrobial activity. Moreover, they are a rich source of bacteriocin and bacteriocin-like inhibitory substances with antagonistic activity on major foodborne pathogens (Moradi et al., Reference Moradi, Molaei and Guimarães2021). Furthermore, they will provide a safely practical application through the manufacturing of infant formulae and milk powder via re-regulating the AF levels to be in the acceptable range with an increment of safety properties.
In conclusion, aflatoxins are a major hazard that could threaten food safety and dairy production. Degradation of AFs is vital to maintain the safety of foods and feeds. Contamination of infant formulae and milk powder with AFM1 poses a health risk to specific groups (infants and the elderly, and our samples did exhibit a high incidence of AFM1 contamination. As a novel solution, postbiotics, parabiotics, and the encapsulated L. plantarum RM1 and L. paracasei KC39 were evaluated. They had good antifungal impact against four toxigenic fungal strains. Moreover, these bacterial products were able to reduce the AFB1 level, as well as AFM1 in a simulated milk powder model. KC39 was more effective for AFM1 reduction than RM1. We propose that bacterial bioactives could be applied as a solution to limit aflatoxin contamination in dairy products, particularly those directed to the sensitive age groups.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S002202992100090X
