Occurrence of Toxic Biogenic Amines in Various Types of Soft and Hard Cheeses and Their Control by Bacillus polymyxa D05-1

: Egyptian cheeses are considered an important part of the Egyptian diet. This study aimed to examine 60 random samples of different types of commercial cheeses in Egypt, including soft cheeses (Domiati and Tallaga) and hard cheeses (Cheddar and Ras). The samples were subjected to chemical and microbial examination. Biogenic amines (BAs) are nitrogenous compounds found in a variety of foods; their presence is undesirable and related to spoilage, and can result in toxicological effects in humans. Thus, BAs were determined by using a high-performance liquid chromatography (HPLC) analysis. Moreover, the ability of Bacillus polymyxa D05-1 to reduce levels of experimentally added biogenic amines during the manufacturing of Tallaga cheese was investigated. The obtained results revealed variations in the chemical composition between the investigated samples. Furthermore, many cheese samples contained high levels of BAs, including histamine, tyramine and putrescine. Domiati cheese had the highest levels of BAs, followed by Tallaga and Cheddar, whereas Ras cheese had the lowest levels. The existence of yeasts, molds, coliforms and the high levels of BAs in cheese samples indicate the unsanitary conditions in which they were made and stored. Furthermore, addition of B. polymyxa D05-1 during Tallaga cheese manufacturing resulted in a reduction in BA levels.


Introduction
Production of healthy food that is free of toxic residues, pesticides, and allergens, as well as pathogenic microbes, is one of the greatest challenges.In this regard, it is very important to make sure that consumed foods are not contaminated by potentially dangerous toxic compounds at any time throughout production, distribution and consumption [1].Food poisoning can be caused by pathogenic microbes, viruses, natural toxins and some bioactive compounds.Biogenic amines (BAs) including histamine, tyramine, cadaverine, putrescine, spermidine or spermine are engaged in a variety of pathogenic syndromes and have deleterious consequences on human health [2].
BAs are organic bases that have biological activity due to the action of decarboxylase enzyme from bacteria (Figure 1), which can remove a carboxyl group from amino acids selectively, and leads to formation of the correspondent amine and CO 2 [3].Enterobacteriaceae and Pseudomonads are known as BA-generator microorganisms, together with lactic acid bacteria, through the production and storage of fermented milk products, especially cheeses [4].Furthermore, the main biogenic amine-producing bacteria can be found: (i) in the raw milk, (ii) as contamination through the entire cheese making process, and (iii) as part of starter or adjunct cultures.Improper storage of food can lead to the formation of biogenic amines [5].
Fermentation 2022, 8, x FOR PEER REVIEW 2 of 13 some bioactive compounds.Biogenic amines (BAs) including histamine, tyramine, cadaverine, putrescine, spermidine or spermine are engaged in a variety of pathogenic syndromes and have deleterious consequences on human health [2].BAs are organic bases that have biological activity due to the action of decarboxylase enzyme from bacteria (Figure 1), which can remove a carboxyl group from amino acids selectively, and leads to formation of the correspondent amine and CO2 [3].Enterobacteriaceae and Pseudomonads are known as BA-generator microorganisms, together with lactic acid bacteria, through the production and storage of fermented milk products, especially cheeses [4].Furthermore, the main biogenic amine-producing bacteria can be found: (i) in the raw milk, (ii) as contamination through the entire cheese making process, and (iii) as part of starter or adjunct cultures.Improper storage of food can lead to the formation of biogenic amines [5].Foods containing high concentrations of BAs may represent a serious threat to public health, as BAs may cause severe toxicological effects.Foods containing BAs are accountable for various food-borne diseases such as tyramine toxicity (cheese-reaction) and histamine poisoning (scombroid poisoning).The detrimental impact of histamine and tyramine increases in the presence of other BAs, such as cadaverine (CAD) and putrescine (PUT).BAs can interact with nitrates and generate carcinogenic nitrosamines [6,7].BAs have been frequently used as quality index and chemical markers in various foods (meat, fish, wines, etc.), showing the degree of spoilage, the use of non-hygienic raw material and poor manufacturing practice [6,8,9].Thus, controlling and monitoring of BAs is critical not only for toxicological and health issues, but also because some individual BAs, such as histamine, tyramine, cadaverine, or a combination of several amines, may be used as quality and/or acceptability indicators in particular foods, and regulating this quality is the way to ensure and guarantee food safety [10,11].Safety, nutrition, availability, convenience, integrity, and freshness are all important aspects of food quality [2].
Cheeses provide a favorable environment for the biosynthesis of amines by bacterial decarboxylation of appropriate amino acids [12].Degradation of casein occurs during cheese ripening, resulting in formation of free amino acids, which could be transformed into BAs.There are many different types of cheeses manufactured all over the world, and their BA content may be influenced by several factors such as free amino acids level, Foods containing high concentrations of BAs may represent a serious threat to public health, as BAs may cause severe toxicological effects.Foods containing BAs are accountable for various food-borne diseases such as tyramine toxicity (cheese-reaction) and histamine poisoning (scombroid poisoning).The detrimental impact of histamine and tyramine increases in the presence of other BAs, such as cadaverine (CAD) and putrescine (PUT).BAs can interact with nitrates and generate carcinogenic nitrosamines [6,7].BAs have been frequently used as quality index and chemical markers in various foods (meat, fish, wines, etc.), showing the degree of spoilage, the use of non-hygienic raw material and poor manufacturing practice [6,8,9].Thus, controlling and monitoring of BAs is critical not only for toxicological and health issues, but also because some individual BAs, such as histamine, tyramine, cadaverine, or a combination of several amines, may be used as quality and/or acceptability indicators in particular foods, and regulating this quality is the way to ensure and guarantee food safety [10,11].Safety, nutrition, availability, convenience, integrity, and freshness are all important aspects of food quality [2].
Cheeses provide a favorable environment for the biosynthesis of amines by bacterial decarboxylation of appropriate amino acids [12].Degradation of casein occurs during cheese ripening, resulting in formation of free amino acids, which could be transformed into BAs.There are many different types of cheeses manufactured all over the world, and their BA content may be influenced by several factors such as free amino acids level, bacterial contamination, pH, salt concentration, storage temperature, water activity, and ripening time [13,14].
BA-mediated intoxications have been linked to a variety of cheese varieties in a number of countries.BAs are found in significant concentrations in a variety of cheeses, particularly in tinned and in well-ripened cheeses.However, intoxicating levels can be Fermentation 2022, 8, 327 3 of 12 found in cheeses that have been subjected to severe proteolysis, such as Cheddar and Swiss cheeses.High levels of histamine and/or tyramine are frequently associated with high levels of putrescine and cadaverine [15].The ability of amines to have a direct or indirect impact on human vascular and neurological systems has sparked interest in amine analysis.The high levels of BAs such as histamine (histamine intoxication) and tyramine (cheese syndrome) can cause several health problems [16].These reactions can be potentiated by other biogenic amines, such as putrescine, cadaverine, spermine and spermidine.Furthermore, diamines, putrescine and cadaverine can form the carcinogenic nitrosamines when they react with nitrate [13].
The permitted limit of biogenic amines is 20 mg/100 g, according to the Egyptian Organization for Standardization [17], and 10 mg/100 g, according to FDA [18].Controlling BAs in fermented foods is thus one of the food industry's current challenges [19].Using bacterial starter cultures producing histamine-decomposing enzyme systems has been reported as a promising important way to decompose histamine and remove BAs in foods; in particular, in fermented foods.Furthermore, encouraging more responsible production by improving hygienic conditions, selecting starters without the potential to synthesize BAs and storing cheese at low temperature would help to prevent BA accumulation and produce healthier cheeses.
Bacillus polymyxa was reported as a potential probiotic species, and has the capacity to produce a variety of secretory proteins, enzymes and antimicrobial substances (Bacteriocins) [20].It has numerous applications in agriculture, chemotherapy and food technology, etc. B. polymyxa D05-1, isolated from Taiwanese salted fish product, has the ability to decrease the levels of histamine and biogenic amine accumulation during salted fish fermentation [21].Furthermore, Shaghayegh et al. [22] found that addition of B. polymyxa starter is an effective method for lowering the biogenic amine content in Mahyaveh fermented fish sauces.
The information about the concentration of biogenic amines and health aspects of manufactured cheese in Egypt is interesting.There is a lack of data needed to conduct relevant risk assessments, which is considered a key action to provide efficient control and to assist decision makers in enacting appropriate legislative and regulatory measures to ensure food safety.Therefore, this study aimed to determine the levels of BAs in different types of soft (Domiati and Tallaga) and hard (Cheddar and Ras) cheeses in Egypt, and investigate, for the first time, the impact of adding B. polymyxa D05-1 on the decomposition of biogenic amines during the manufacturing of selected Tallaga cheese.

Cheese Samples
A total of 60 random samples with a similar use-by date (500 g from each sample) of different commercial cheese types, including soft cheeses (Domiati and Tallaga) and hard cheeses (Cheddar and Ras), 15 samples of each (in block forms), were collected in sterile plastic bags from different local markets in Egypt, then stored at 5

Chemical Analysis of Cheese
Cheese samples were prepared and analyzed for pH, acidity (% of lactic acid) and salt (NaCl) concentration according to AOAC [23] standards 920.124-33.7.14 and 935.43-33.7.10, respectively.

Determination of BAs
Five BAs including histamine (HI), tyramine (TY), putrescine (PU), cadaverine (CA) and tryptamine (TR) were determined in the collected and manufactured cheese samples using HPLC, according to Hwang et al. [24] with some modifications.A representative homogenized cheese sample (5 g) was added to 20 mL of perchloric acid (HClO 4 , 0.4 M), then mixed using a vortex followed by centrifugation at 3000× g for 10 min (at 4 • C).Then, the residue was re-extracted with an equal volume of perchloric acid.The collected supernatants were combined and adjusted to 50 mL with perchloric acid (0.4 M), then filtered and stored at 4 • C ± 1 • C for HPLC (Agilent 1100 series; Agilent, Santa Clara, CA, USA) analysis within a week.Figure 2  and tryptamine (TR) were determined in the collected and manufactured cheese sa using HPLC, according to Hwang et al. [24] with some modifications.A represen homogenized cheese sample (5 g) was added to 20 mL of perchloric acid (HClO4, then mixed using a vortex followed by centrifugation at 3000 ×g for 10 min (at 4 °C) the residue was re-extracted with an equal volume of perchloric acid.The collected natants were combined and adjusted to 50 mL with perchloric acid (0.4 M), then f and stored at 4 ± 1 °C for HPLC (Agilent 1100 series; Agilent, Santa Clara, CA, USA ysis within a week.Figure 2 illustrates a diagram of different BAs detection in the tigated cheese samples by HPLC. Sample preparation and analysis were carried out at the National Research (Mycotoxins Central and Food Safety), Cairo, Egypt.

Activation of B. polymyxa D05-1
B. polymyxa D05-1 was obtained from the Microbiology Resource Center (MIR Faculty of Agriculture, Ain Shams University, Cairo, Egypt.The strain was cultiva brain heart infusion (BHI) broth medium (Fluka, Sigma-Aldrich Chemie GmbH) fo at 37 °C.In peptone water, one milliliter of the cultivated strain was decimally d Accordingly, the viable count of the B. polymyxa D05-1 strain was carried out accord the plate count method (a volume of the culture broth corresponding to approxim × 10 7 cells/mL bacteria was centrifuged at 5000 rpm for 15 min at 5 °C), and the ba pellets were washed twice with sterilized DI water [25].Sample preparation and analysis were carried out at the National Research Center (Mycotoxins Central and Food Safety), Cairo, Egypt.

Activation of B. polymyxa D05-1
B. polymyxa D05-1 was obtained from the Microbiology Resource Center (MIRCEN), Faculty of Agriculture, Ain Shams University, Cairo, Egypt.The strain was cultivated in brain heart infusion (BHI) broth medium (Fluka, Sigma-Aldrich Chemie GmbH) for 24 h at 37 • C. In peptone water, one milliliter of the cultivated strain was decimally diluted.Accordingly, the viable count of the B. polymyxa D05-1 strain was carried out according to the plate count method (a volume of the culture broth corresponding to approximately 1 × 10 7 cells/mL bacteria was centrifuged at 5000 rpm for 15 min at 5 • C), and the bacterial pellets were washed twice with sterilized DI water [25].Tallaga cheese was manufactured as described by El-Kholy et al. [26], with some modifications.The bacterial pellets of B. polymyxa D05-1 (1 × 107) and 40 mg of histamine or tyramine (Sigma-Aldrich; St. Louis, MO, USA) were suspended in 1 kg of milk mixture directly before the addition of rennet enzyme (Chr-Hansen's laboratories, Denmark).The manufactured cheese was divided into three groups: Group 1 (contaminated cheese with histamine or tyramine; kept as control) was prepared by suspending 40 mg/kg of histamine or tyramine in 1 kg of milk mixture, directly before the addition of rennet enzyme; Group 2 was prepared by inoculating the milk mixture with B. polymyxa D05-1 and 40 mg/kg of histamine (T 1 ); Group 3 was prepared by inoculating the milk mixture with B. polymyxa D05-1 and 40 mg/kg of tyramine (T 2 ).The samples were examined at 0, 12, and 24 h after processing to measure the content of histamine and tyramine using HPLC analysis.

Microbiological Analysis of the Investigated Cheese Samples
Total aerobic count (TAC), coliform, yeasts and molds were determined in order to characterize the microbiological properties of the investigated cheese samples.Plate count agar (PCA, Oxoid) for total aerobic bacteria, violet red bile agar (VRB, Difco) for coliform bacteria, and PDA (potato dextrose agar) for yeasts and molds were used.The conditions were: PCA for 48 h at 30 • C; VRB at 37 • C for 24-48 h; and PDA at 25 • C for 5 days [27].

Statistical Analysis
The obtained data were expressed as mean ± S.E of triplicates.Significant differences between the samples were detected using analysis of variance (ANOVA) and Duncan's multiple range test.The level of p < 0.05 was used to define the significant differences.The SPSS program was used to conduct all of the analyses (Version 20; SPSS Inc., Chicago, IL, USA).Data were visualized by RStudio (R version 4.0.2) software using ggplot2 package.

Chemical Analysis of Cheese Samples
Temperature, pH, acidity and salt (NaCl content) are the key environmental elements that influence the microbial activity in foods.These factors are responsible for the metabolism of the living cell that secretes the decarboxylase, and they also affect the activity of this enzyme [28].The pH is a significant factor affecting the formation of amines, because amino acid decarboxylase activity is much higher in acidic conditions, i.e., at pH 4.0-5.5 [29].The mean values of pH for the investigated cheese samples were 5.02, 5.27, 5.36 and 5.35 for Domiati, Tallaga, Cheddar and Ras cheese, respectively (Figure 3A).Kanotor et al. [30] reported that the cheese making process represents an ideal condition for BA formation.The development of acidity is the most important process control factor in terms of cheese quality and safety.As seen in Figure 3B, the mean values of acidity (as lactic acid) were 1.39%, 1.14%, 0.87% and 1.80% for Domiati, Tallaga, Cheddar and Ras cheese, respectively.Relative variations in the acidity percentage of cheese samples may be attributed to variation in the manufacturing process, ripening periods, temperature of storage and/or age of the cheese samples [31].During the manufacturing and ripening processes, acidity has an important impact on the coagulant's activity.Furthermore, acidity encourages syneresis (the expulsion of fluid from the cheese curd), and hence has a significant impact on the cheese's composition, notably its moisture content.Lactic acid also affects the enzyme activity during ripening and, as a result, cheese flavor and quality.Finally, acidity aids in the management or prevention of spoilage and pathogenic microbe growth [32].
Salt concentration has a role in controlling the microbial growth and the different enzyme activities in cheese, and in reducing cheese moisture content and the formation of BAs [33].The results in Figure 3C revealed that values of salts (NaCl) were 4.85%, 2.43%, 1.65% and 3.49% for Domiati, Tallaga, Cheddar and Ras cheese, respectively.Mean values of salt in Cheddar and Ras cheese are similar to that obtained by Farag [34].On the other hand, the obtained result of salt content in Domiati cheese was lower than that obtained by Salama et al. [35]; they reported that the mean value of sodium chloride content was 5.10%.Salt concentration has a role in controlling the microbial growth and the different enzyme activities in cheese, and in reducing cheese moisture content and the formation of BAs [33].The results in Figure 3C revealed that values of salts (NaCl) were 4.85%, 2.43%, 1.65% and 3.49% for Domiati, Tallaga, Cheddar and Ras cheese, respectively.Mean values of salt in Cheddar and Ras cheese are similar to that obtained by Farag [34].On the other hand, the obtained result of salt content in Domiati cheese was lower than that obtained by Salama et al. [35]; they reported that the mean value of sodium chloride content was 5.10%.

Occurrence and Levels of BAs in the Collected Cheese Samples
Biogenic amines are valuable for judging the hygienic quality of cheese; therefore, they can be used as indicators of food spoilage, and can cause toxicological effects to consumers at high levels.In this study, 15 samples of each cheese type-Domiati, Tallaga,

Occurrence and Levels of BAs in the Collected Cheese Samples
Biogenic amines are valuable for judging the hygienic quality of cheese; therefore, they can be used as indicators of food spoilage, and can cause toxicological effects to consumers at high levels.In this study, 15 samples of each cheese type-Domiati, Tallaga, Cheddar and Ras-were examined for the occurrence of BAs including HI, TY, PU, TR and CA.The data in Table 1 illustrates the percentage of positive samples containing BAs, and the levels of the different BAs (mg/100 g) that existed in these positive samples.A total of 73.33% of Domiati cheese samples contained HI, and the levels of HI varied from 2.1 to 28.9 mg/100 g.The levels of HI in Domiati cheese were the highest among all the tested cheese samples.Ras cheese had 46.67% positive samples to HI occurrence, with levels of 2.3 to 21.0 mg/100 g.The most common dangerous type of food poisoning is histamine poisoning.Scombroid poisoning, commonly known as histamine intoxication, is a serious foodborne illness that affect consumers all over the word [36].Tyramine (TY) causes the so-called "cheese-reaction" (intoxication caused by the intake of cheese containing high levels of histamine and tyramine); TY was the most abundant biogenic amine in Domiati cheese, followed by HI and CA.All the Domiati cheese samples contained TY (100%), with levels ranging from 3.8 to 29.5 mg/100 g (Table 1).The positive samples of Tallaga cheese represented 86.67% of the total collected samples, and the levels of TY ranged from 2.9 to 33.2 mg/100 g.Conversely, Ras cheese recorded the lowest levels of TY, which ranged from 1.3 to 25.7 mg/100 g, with a mean value of 9.57 mg/100 g.The presence of TY is usually related to the contamination of the milk that is used for cheese making with Levilactobacillus brevis and Enterococcus faecalis [37].
Domiati and Tallaga cheese gave the highest levels of PU (1.6-23.1 and 1.2-21.8mg/100 g, respectively), whereas Cheddar and Ras cheeses recorded lower levels of PU (1-20.1 and 1-16.7 mg/100 g, respectively) (Table 1).On the other hand, all Domiati cheese samples contained CA, and the levels ranged from 1.3 to 20.6 mg/100 g, whereas Ras cheese had four samples positive to the occurrence of CA, and recorded the lowest levels of CA which ranged from 1 to 9.4 mg/100 g.According to Marino et al. [38], Enterobacteriaceae are associated with the formation of cadaverine, putrescine and histamine primarily when raw materials or end products deteriorate.
Tryptamine was not detected in all Ras cheese samples, whereas cheddar cheese recorded lower levels of TR (1-1.2 mg/100 g) (Table 1).The presence of TR in foods can cause hypertension, headache, fever and sometimes vomiting and sweating [39].
In general, HI, TY and PU were detected in high levels in some of the tested cheese samples in comparison with the other types of Bas (CA and TR).Domiati and Tallaga cheese samples recorded the highest levels of the tested BAs in comparison with other cheese samples.Domiati and Tallaga cheese are classified as soft cheeses; this means that the moisture content of Domiati and Tallaga cheeses is higher than Cheddar and Ras cheese, which are classified as hard cheeses.Gardini et al. [28] reported that the rate of BAs accumulation in fermented sausage decreased with the decrease of a w due to the water losses.
Some of the cheese samples exceeded the allowable threshold of HI, TY and PU, according to the Egyptian Organization for Standardization [17] (20 mg/100 g) and the FDA [18] (10 mg/100 g).For CA, only Domiati cheese exceeded the permissible level (20 mg/100 g).The levels of TR in the investigated cheese samples did not exceed the permissible level (20 mg/100 g) compared with the other BAs.Therefore, the presence of high concentrations of BAs can be attributed to the insufficient microbiological quality of raw materials, unsuitable storage of the products, and uncontrolled fermentation [40,41].Conclusively, the mean concentration of all examined BAs did not exceed the permitted level of the Egyptian Organization for Standardization [17].

Effect of Adding B. polymyxa D05-1 Culture on the Levels of Histamine and Tyramine Experimentally Added to Processed Tallaga Cheese
Tallaga cheese is a soft cheese, and is very popular in the Egyptian market; its consumption is high due to its desirable flavor and texture [26].Our results revealed that all the tested BAs were found in this cheese at high levels.Therefore, a decrease of BAs in this type of cheese is a good way to prevent the health problems related to the occurrence of BAs in cheeses.Data in Table 2 show that the addition of B. polymyxa D05-1 into Tallaga cheese during processing resulted in a decrease in the level of histamine from 40 mg/kg at 0 h to 13.1 mg/kg (67.3% decrease) after 12 h of storage at 5 • C, then decreased after 24 h to reach 6.6 mg/kg (83.5% decrease).Similarly, the tyramine levels decreased from 40 mg/kg at 0 h to 16.8 mg/kg (58.0%decrease), then reached 11.3 mg/kg after 24 h (71.8% decrease).The decrease in the experimentally added BAs during processing of Tallaga cheese is a result of the addition of B. polymyxa D05-1.In contrast, the control treatment did not show any reduction of both histamine and tyramine.Lee et al. [42] suggested two mechanisms for the action of B. polymyxa D05-1 on the reduction of biogenic amines: (1) B. polymyxa D05-1 competes with the present microorganisms which can produce BAs; and (2) B. polymyxa D05-1 has the ability to degrade the BAs.

Microbiological Quality of Cheese Samples
The shelf life of dairy products is limited, as it represents a suitable growth environment for a large number of microorganisms.As a result, it is critical to control the microbiological quality of dairy products, particularly the total viable count and concentration of coliform bacteria, as these are indicators of the product's hygienic state.The obtained results in Figure 4A revealed that total aerobic counts (TAC) were 4.83, 5.17, 4.62 and 5.75 Log cfu/g for Domiati, Tallaga, Cheddar and Ras cheese, respectively.TACs for Domiati and Tallaga cheese were higher than that obtained by Hassan and Gomaa [43].APHA [44] reported that TAC is considered an index of quality, which gives an idea about the hygienic conditions during the processing of dairy product.Coliform bacteria are commonly found in the intestine of animals, and contamination of ready-to-eat food is attributed to post-processing contamination due to many reasons, such as unhygienic practices during food handling, lack of personal hygiene, and unhygienic utensils, equipment and packaging [45].The data in Figure 4B show that coliform counts in Domiati, Tallaga, Cheddar and Ras cheese were 3.41, 3.47, 2.60 and 3.67 Log cfu/g, respectively.These results are similar to those obtained by Hassan and Gomaa [43].
Occurrence of yeasts and molds in cheeses was variable because they have been associated with the production of flavor compounds as a result of their relatively strong Coliform bacteria are commonly found in the intestine of animals, and contamination of ready-to-eat food is attributed to post-processing contamination due to many reasons, such as unhygienic practices during food handling, lack of personal hygiene, and unhy-gienic utensils, equipment and packaging [45].The data in Figure 4B show that coliform counts in Domiati, Tallaga, Cheddar and Ras cheese were 3.41, 3.47, 2.60 and 3.67 Log cfu/g, respectively.These results are similar to those obtained by Hassan and Gomaa [43].
Occurrence of yeasts and molds in cheeses was variable because they have been associated with the production of flavor compounds as a result of their relatively strong proteolytic and lipolytic activities [46].By examination of Domiati, Tallaga, Cheddar and Ras cheese for yeasts and molds, data revealed that the counts were 3.30, 3.61, 2.72 and 3.66 Log cfu/g, respectively (Figure 4C).These results agree with those obtained by Osama et al. [47].El-Badry and Raslan [48] reported that the presence of yeast and molds in cheese samples indicated the occurrence of unsatisfactory conditions during the manufacturing of cheese.
It has been reported that the occurrence of dangerous concentrations of BAs is connected with a relevant growth (>7 log cfu/g) of decarboxylating microorganisms.For this reason, many authors suggested using BAs content as indirect indicators for the microbial quality of food products [28,49,50].However, in our study, the microbial load did not exceed the relevant growth of decarboxylating microorganisms.The high BAs content in some investigated samples is probably related to poor-quality raw materials, processing and preservation conditions.

Conclusions
The high amounts of biogenic amines in some cheese samples suggest that they were processed and stored in unsanitary conditions.Additionally, the use of B. polymyxa D05-1 reduced the added HI and TY by 83.5% and 71.8%, respectively, after 24 h.It is worth mentioning that there is a necessity for maintaining the sanitary conditions in the raw material supply and manufacturing processes, as well as in the indigenous bacterial population, which are responsible for high BAs production in traditional cheeses.Likewise, using competitive adjunct cultures is advisable to control the formation of the biogenic amine.Another requirement for reducing the occurrence of BAs in dairy products and assessing the efficacy of the measures implemented is the development of robust and effective epidemiological and surveillance programs.Finally, further studies are needed to investigate the safety of using B. polymyxa as a starter in food product production.

Figure 1 .
Figure 1.Biogenic amine (BA) formation and the action of decarboxylase from bacteria.

Figure 1 .
Figure 1.Biogenic amine (BA) formation and the action of decarboxylase from bacteria.
illustrates a diagram of different BAs detection in the investigated cheese samples by HPLC.

Figure 2 .
Figure 2. Diagram of different BAs detection in the investigated cheese samples by a high-p mance liquid chromatography (HPLC).

Figure 2 .
Figure 2. Diagram of different BAs detection in the investigated cheese samples by a highperformance liquid chromatography (HPLC).

Figure 4 .
Figure 4. Bar plot of microbiological quality of the investigated cheese samples (Total aerobic count (A), Coliform count (B) and Yeast & moulds count (C)).Data expressed as mean ± SE (n = 15).

Table 1 .
Occurrence and levels of BAs "mg/100 g sample" in the examined cheese samples (n = 15).
Different superscript letters within the same column are significantly different (p < 0.05).* = refers to the mean ± standard error.

Table 2 .
Effect of adding B. polymyxa D05-1 culture on the levels of BAs (40 mg/kg) experimentally added to Tallaga cheese.