# Probiotic-Based Optimization of Pistachio Paste Production and Detoxification of Aflatoxin B1 UsingBifidobacterium lactis

Authors: Mehdi Pakizeh; Leila Nouri; Mohammad Hossein Azizi
Journal: Journal of Food Quality
 (2022)

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## Abstract

Pistachio paste is very popular for breakfast or supper thanks to its desirable taste, flavor, and texture. One of the hazards that are directly related to agricultural practices, processing, storage, and transportation of pistachios and the byproducts is aflatoxin, which can cause irreversible effects on the consumer. Probiotics are one of the most effective and safe methods to reduce aflatoxins. The variables under study were temperature and time, aflatoxin concentration, and probiotic content. In total, 30 treatments were determined through the rotatable central composite design. This is the first and most comprehensive study to optimize the production of probiotic pistachio paste and investigate the detoxification effects of aflatoxin B1 usingBifidobacterium lactis with six treatments and three replications in the pistachio paste matrix. In simple terms, it is possible to remove a higher percentage of toxins by increasing the number of microorganisms and decreasing the toxin level. The highest aflatoxin B1 reduction was observed in pistachio paste with aflatoxin B1 contamination of (19.7039 ng/g), which was spiked with Bifidobacterium lactis (109 CFU/g) and then stored at 25°C for 26.1853 days (aflatoxin B1: 8.00007 ng/g = 59.4% reduction), which is consistent with the permissible limits of the Iran National Standards Organization and the European Commission Regulation. The results showed a significant reduction in the aflatoxin B1 level in pistachio paste. The probiotics reduced aflatoxin B1 contamination to a permissible level. This is an important, safe, and effective solution, and unlike other methods, it increases the nutritional value of the product.


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## Body

## 1. Introduction

Edible nuts are a significant part of people’s daily food basket [1]. Meanwhile, pistachio (Pistacia vera L.) from the family Anacardiaceae is superior to many foods given its high phosphorus, calcium, iron, potassium, magnesium, and energy level (about 626 kcal/100 g of pistachio kernels) [2]. Pistachio paste containing 100% pistachio is used in food products such as cakes, ice cream, and sweets. Considering its nutritional value and high price, it is of great importance to ensure its health for domestic and international markets. The processes that pistachio goes through after harvesting include transportation, processing, grading, packaging, storage, and finally, transportation to supply centers. These processes affect the quality and health of the final pistachio product [3]. One of the hazards that is directly related to agricultural practices, processing, storage, and transportation of pistachios and their byproducts is aflatoxin, which causes acute to chronic side effects after entering the bodies of humans and animals mainly through the mouth [4]. So far, more than 18 types of aflatoxins have been identified, of which aflatoxin B1 (AFB1) is the most toxic and potent liver carcinogen [5]. In order to prevent aflatoxin concentration in food, good agricultural practices (GAP), processing, storage, and proper transportation are recommended [6]. However, it is not possible to have a 100% control on these processes. The risks of contamination of agricultural products and different methods of reducing the aflatoxins content in food have been studied [7, 8]. The use of many physicochemical methods to remove mycotoxins from contaminated food is limited due to safety issues, the possibility of losing the nutritional quality of the product, low efficiency, and high cost [9]. On the other hand, among the biological detoxification methods, probiotics are considered as the most effective and safest methods [10]. In addition to therapeutic effects such as improving the balance of microbial flora of the gastrointestinal tract v [11] and antioxidant activity it can bind to or degrade aflatoxins in food. The ability to remove aflatoxins is probably due to the physical binding of the toxin to the bacterial cell wall or cell wall components [12]. It seems that probiotics, considering their ability to remove aflatoxin from pistachios, in addition to enriching the product, can improve safety and nutritional value [13].To the best knowledge of the authors, there are limited studies on the antiaflatoxin activity of probiotics in pistachios. This is the first and most comprehensive study to optimize the production of probiotic pistachio paste and investigate the detoxification effect of AFB1 byBifidobacterium lactis with six treatments in three replications in the pistachio paste matrix.


## 2. Materials and Methods

### 2.1. Pistachio Samples Collection

Ripe and dry Ahmad Aghaei pistachio (10 kg) cultivar with closed shell was procured in November 2020 from local orchards in Iran.


### 2.2. Preparation of Pistachio Paste

After breaking the shell, the pistachio kernels were turned into pistachio paste using a butter-making machine (BP30, Bekrdaneh Co., Esfahan, Iran) and immediately used for preparing the treatments and analyzing.


### 2.3. Physicochemical Tests of Pistachio Paste

#### 2.3.1. Moisture Measurement

Pistachio paste moisture was measured according to the International Organization for Standardization (ISO) No. 712 and Iran National Standards Organization (INSO) no. 2705 titled “Cereals and its byproducts-moisture measurement method-Reference method” [14, 15].


#### 2.3.2. Fat Measurement (by Soxhlet Method)

Pistachio paste fat was measured according to ISO No. 659 titled “Oilseeds-Determination of oil content (reference method)” [16].


#### 2.3.3. Protein Measurement

Pistachio paste protein content was measured according to ISO No. 20483 and INSO No. 19052 titled “Cereal grain crude protein and its byproducts” [17, 18].


#### 2.3.4. Measurement of Pistachio Paste Fiber

Pistachio paste fiber was measured according to ISO No. 5498 and INSO No. 3105 titled “Measurement of cereal grain crude protein and its byproducts” [19, 20].


#### 2.3.5. Measurement of Pistachio Paste Ash

Pistachio paste ash was measured according to ISO No. 2171 and INSO No. 2706 titled “Cereals, legumes and byproducts-measurement of ash in the furnace” [21, 22].


#### 2.3.6. Pistachio Paste Acidity Measurement

The pistachio paste acidity was measured according to ISO No. 7305 and INSO No. 3380 [23, 24].


#### 2.3.7. Measurement of Pistachio Paste Peroxide Index

Peroxide content in edible oils and fats was measured According to ISO No. 4179 [25, 26].


### 2.4. Preparation of Aflatoxin B1 Standard

Standard AFB1 solution (Sigma–Aldrich, Germany) was prepared at a concentration of 500 mg/μl in methanol and stored at −20°C. Working solutions at concentrations of 50, 100, and 200 μg/ml were diluted in methanol and stored at −20°C.


### 2.5. Preparation and Activation ofBifidobacterium lactis

A lyophilized culture ofBifidobacterium lactis (Persian Type Culture Collection (PTCC) No: 1736) was procured from the Iranian Research Organization for Science and Technology (IROST). The 24-hour bacterial culture in MRS broth culture medium was prepared through incubation at 37°C under anaerobic conditions.


### 2.6. Evaluation of Aflatoxin B1 Detoxification in Pistachio Paste UsingBifidobacterium lactis

Pistachio paste was contaminated with AFB1 at 5, 15, and 25 ng/g concentrations and spiked with 107, 108, and 109 CFU/ml of Bifidobacterium lactis. Then, the treatments were incubated at 4, 25, and 46°C and the aflatoxin content was determined on days 0, 15, and 30 and at 4, 25, and 46°C (Table 1) [27].Table 1
AFB1 reduction in the studied treatments at different temperatures, storage times, and the different probiotic contents.
FactorsTreatments123456789101112131415161718192021222324252627282930Probiotics content (CFU/g)107107107107107107107107108108108108108108108108109109109109109109109109108107108109108108Storage time (day)030030030030015151515151530030030030030151515151515Storage temperature (°C)444446464646252525252525252544444646464625254254625Primary aflatoxin (ng/g)552525552525151515151515151555252555252551515151525Secondry aflatoxin (ng/g)52.82515.2551.952512.25157.47.347.467.537.327.366.252.152513.551.35258.752.28.258.46.36.611.75Reduction %044039061051050.751.150.349.851.250.9258.7057046073065564544585653


### 2.7. Chemical and Reagents

AFB1 standard were purchased from Sigma–Aldrich. AFB1 immunoaffinity columns were obtained from Palo Alto, CA, USA. HPLC grade solvents were purchased from Merck (Darmstadt, Germany).


### 2.8. Analysis of Aflatoxin Content

The method used for the analysis of AFB1 was the method reported by Rastegar et al. with a minor modification. After being prepared with the pistachio paste (50 g), the samples were analyzed in terms of AFB1 levels by high-performance liquid chromatography (HPLC) (Hewlett Packard, Agilent 1100, Palo Alto, CA, USA). For this purpose, AFB1 was extracted by 300 ml methanol-water (80 : 20) and n-hexane (100 ml) applied to immunoaffinity column conditioned with 10 ml of phosphate buffer saline before used. The mobile phase was water: methanol: and acetonitrile (54 : 29 : 17, v/v/v) at a flow rate of 1 ml/min [28].


#### 2.8.1. Calibration Standards

AFB1 stock standard solutions (10μg/ml) were prepared and spiked calibration standards were prepared by addition of 2.5, 5, 10, 100, 200, 300, and 500 μl of mixed standard stock solution, respectively, to 1 g of blank pistachio paste samples in each case.


#### 2.8.2. Recovery Studies

In this regard, spiked pistachio paste samples at concentration levels of 15, 25, 75, 150, 250, 500, and 750μg/g were prepared in triplicates and then treated according to the procedure described in sample preparation. The recoveries were calculated using the spiked calibration curves.


### 2.9. Sensory Testing

Sensory evaluation of probiotic pistachio paste samples (according to the AFB1-free treatments listed in Table1) was performed by a group of 20 trained individuals (10 men and 10 women-aged between 21 and 32 years old) among the students and employees of the food industry department. The pistachio paste samples were evaluated based on acceptance of appearance, color, aroma, texture, taste, and overall acceptance on a 9-point hedonic scale and a 9-point scoring scale ranging from 1 (disliked extremely) to 9 (liked extremely) [29]. A sensory evaluation was conducted in the sensory room with the following properties:(i)
Temperature of evaluation room: room temperature.(ii)
Light of evaluation room: white fluorescent lamp.(iii)
Evaluation time: midmorning, about 4 h after breakfast.(iv)
Process of serving: randomly served in cup.


### 2.10. Statistical Analysis

The statistical analysis was performed in two stages. In the first stage, the experiments were designed and the collected data were analyzed. In the second stage, the formulation optimization operation was performed.


#### 2.10.1. Experimental Design and Statistical Analysis

In order to produce probiotic pistachio paste and also to investigate the effects ofBifidobacterium lactis probiotic on aflatoxin, the response surface methodology (RSM) and a RCCD were used in Design Expert ver. 11.1.1.0 [30]. This method allows optimization of the probiotic pistachio paste formulation with the minimum aflatoxin level and also the maximum overall acceptance in sensory evaluation. In addition, four factors of aflatoxin concentration, probiotic level, temperature, and time were taken into account. Each factor was evaluated at three levels of +1, 0, and −1 (Table 2). After performing the experimental tests, regression analysis was performed based on the experimental data, and a quadratic polynomial model was fitted to each one of the responses using the following equation:(1)Y=β0+∑i=12β0xi+∑i=12βiixi2+∑i=1∑j=i+1βijxixj,where Y is the estimated response and the β0, βi, βii, and βij are the equation constant (intercept), linear effects coefficient, quadratic effects coefficient, and interaction coefficients, respectively; and xi and xj are the levels of independent variables. Finally, significant expressions (p < 0.05) for each response were identified using the analysis of variance (ANOVA) [31]. Test design and statistical data analysis were performed using Design Expert software.Table 2
Effective factors in RCCD.
FactorsNamesUnitsTypesMinMaxCoded lowCoded highMeanStd. dev.AProbioticCFU/gNumeric107109−1↔107+1↔1091080.79BTimeDayNumeric030−1↔0+1↔301511.82CTemperature°CNumeric446−1↔4+1↔462516.54DPrimary AFng/gNumeric525−1↔5+1↔25157.88


#### 2.10.2. Formulation Optimization

The formulation optimization was performed after statistical data analysis. In order to optimize the formulation of probiotic pistachio paste with the minimum aflatoxin level and the maximum overall acceptance, numerical optimization techniques and desirability functions were used [32]. For this purpose, the maximum consumer acceptance response and the minimum aflatoxin level were taken into account.


## 2.1. Pistachio Samples Collection

Ripe and dry Ahmad Aghaei pistachio (10 kg) cultivar with closed shell was procured in November 2020 from local orchards in Iran.


## 2.2. Preparation of Pistachio Paste

After breaking the shell, the pistachio kernels were turned into pistachio paste using a butter-making machine (BP30, Bekrdaneh Co., Esfahan, Iran) and immediately used for preparing the treatments and analyzing.


## 2.3. Physicochemical Tests of Pistachio Paste

### 2.3.1. Moisture Measurement

Pistachio paste moisture was measured according to the International Organization for Standardization (ISO) No. 712 and Iran National Standards Organization (INSO) no. 2705 titled “Cereals and its byproducts-moisture measurement method-Reference method” [14, 15].


### 2.3.2. Fat Measurement (by Soxhlet Method)

Pistachio paste fat was measured according to ISO No. 659 titled “Oilseeds-Determination of oil content (reference method)” [16].


### 2.3.3. Protein Measurement

Pistachio paste protein content was measured according to ISO No. 20483 and INSO No. 19052 titled “Cereal grain crude protein and its byproducts” [17, 18].


### 2.3.4. Measurement of Pistachio Paste Fiber

Pistachio paste fiber was measured according to ISO No. 5498 and INSO No. 3105 titled “Measurement of cereal grain crude protein and its byproducts” [19, 20].


### 2.3.5. Measurement of Pistachio Paste Ash

Pistachio paste ash was measured according to ISO No. 2171 and INSO No. 2706 titled “Cereals, legumes and byproducts-measurement of ash in the furnace” [21, 22].


### 2.3.6. Pistachio Paste Acidity Measurement

The pistachio paste acidity was measured according to ISO No. 7305 and INSO No. 3380 [23, 24].


### 2.3.7. Measurement of Pistachio Paste Peroxide Index

Peroxide content in edible oils and fats was measured According to ISO No. 4179 [25, 26].


## 2.3.1. Moisture Measurement

Pistachio paste moisture was measured according to the International Organization for Standardization (ISO) No. 712 and Iran National Standards Organization (INSO) no. 2705 titled “Cereals and its byproducts-moisture measurement method-Reference method” [14, 15].


## 2.3.2. Fat Measurement (by Soxhlet Method)

Pistachio paste fat was measured according to ISO No. 659 titled “Oilseeds-Determination of oil content (reference method)” [16].


## 2.3.3. Protein Measurement

Pistachio paste protein content was measured according to ISO No. 20483 and INSO No. 19052 titled “Cereal grain crude protein and its byproducts” [17, 18].


## 2.3.4. Measurement of Pistachio Paste Fiber

Pistachio paste fiber was measured according to ISO No. 5498 and INSO No. 3105 titled “Measurement of cereal grain crude protein and its byproducts” [19, 20].


## 2.3.5. Measurement of Pistachio Paste Ash

Pistachio paste ash was measured according to ISO No. 2171 and INSO No. 2706 titled “Cereals, legumes and byproducts-measurement of ash in the furnace” [21, 22].


## 2.3.6. Pistachio Paste Acidity Measurement

The pistachio paste acidity was measured according to ISO No. 7305 and INSO No. 3380 [23, 24].


## 2.3.7. Measurement of Pistachio Paste Peroxide Index

Peroxide content in edible oils and fats was measured According to ISO No. 4179 [25, 26].


## 2.4. Preparation of Aflatoxin B1 Standard

Standard AFB1 solution (Sigma–Aldrich, Germany) was prepared at a concentration of 500 mg/μl in methanol and stored at −20°C. Working solutions at concentrations of 50, 100, and 200 μg/ml were diluted in methanol and stored at −20°C.


## 2.5. Preparation and Activation ofBifidobacterium lactis

A lyophilized culture ofBifidobacterium lactis (Persian Type Culture Collection (PTCC) No: 1736) was procured from the Iranian Research Organization for Science and Technology (IROST). The 24-hour bacterial culture in MRS broth culture medium was prepared through incubation at 37°C under anaerobic conditions.


## 2.6. Evaluation of Aflatoxin B1 Detoxification in Pistachio Paste UsingBifidobacterium lactis

Pistachio paste was contaminated with AFB1 at 5, 15, and 25 ng/g concentrations and spiked with 107, 108, and 109 CFU/ml of Bifidobacterium lactis. Then, the treatments were incubated at 4, 25, and 46°C and the aflatoxin content was determined on days 0, 15, and 30 and at 4, 25, and 46°C (Table 1) [27].Table 1
AFB1 reduction in the studied treatments at different temperatures, storage times, and the different probiotic contents.
FactorsTreatments123456789101112131415161718192021222324252627282930Probiotics content (CFU/g)107107107107107107107107108108108108108108108108109109109109109109109109108107108109108108Storage time (day)030030030030015151515151530030030030030151515151515Storage temperature (°C)444446464646252525252525252544444646464625254254625Primary aflatoxin (ng/g)552525552525151515151515151555252555252551515151525Secondry aflatoxin (ng/g)52.82515.2551.952512.25157.47.347.467.537.327.366.252.152513.551.35258.752.28.258.46.36.611.75Reduction %044039061051050.751.150.349.851.250.9258.7057046073065564544585653


## 2.7. Chemical and Reagents

AFB1 standard were purchased from Sigma–Aldrich. AFB1 immunoaffinity columns were obtained from Palo Alto, CA, USA. HPLC grade solvents were purchased from Merck (Darmstadt, Germany).


## 2.8. Analysis of Aflatoxin Content

The method used for the analysis of AFB1 was the method reported by Rastegar et al. with a minor modification. After being prepared with the pistachio paste (50 g), the samples were analyzed in terms of AFB1 levels by high-performance liquid chromatography (HPLC) (Hewlett Packard, Agilent 1100, Palo Alto, CA, USA). For this purpose, AFB1 was extracted by 300 ml methanol-water (80 : 20) and n-hexane (100 ml) applied to immunoaffinity column conditioned with 10 ml of phosphate buffer saline before used. The mobile phase was water: methanol: and acetonitrile (54 : 29 : 17, v/v/v) at a flow rate of 1 ml/min [28].


### 2.8.1. Calibration Standards

AFB1 stock standard solutions (10μg/ml) were prepared and spiked calibration standards were prepared by addition of 2.5, 5, 10, 100, 200, 300, and 500 μl of mixed standard stock solution, respectively, to 1 g of blank pistachio paste samples in each case.


### 2.8.2. Recovery Studies

In this regard, spiked pistachio paste samples at concentration levels of 15, 25, 75, 150, 250, 500, and 750μg/g were prepared in triplicates and then treated according to the procedure described in sample preparation. The recoveries were calculated using the spiked calibration curves.


## 2.8.1. Calibration Standards

AFB1 stock standard solutions (10μg/ml) were prepared and spiked calibration standards were prepared by addition of 2.5, 5, 10, 100, 200, 300, and 500 μl of mixed standard stock solution, respectively, to 1 g of blank pistachio paste samples in each case.


## 2.8.2. Recovery Studies

In this regard, spiked pistachio paste samples at concentration levels of 15, 25, 75, 150, 250, 500, and 750μg/g were prepared in triplicates and then treated according to the procedure described in sample preparation. The recoveries were calculated using the spiked calibration curves.


## 2.9. Sensory Testing

Sensory evaluation of probiotic pistachio paste samples (according to the AFB1-free treatments listed in Table1) was performed by a group of 20 trained individuals (10 men and 10 women-aged between 21 and 32 years old) among the students and employees of the food industry department. The pistachio paste samples were evaluated based on acceptance of appearance, color, aroma, texture, taste, and overall acceptance on a 9-point hedonic scale and a 9-point scoring scale ranging from 1 (disliked extremely) to 9 (liked extremely) [29]. A sensory evaluation was conducted in the sensory room with the following properties:(i)
Temperature of evaluation room: room temperature.(ii)
Light of evaluation room: white fluorescent lamp.(iii)
Evaluation time: midmorning, about 4 h after breakfast.(iv)
Process of serving: randomly served in cup.


## 2.10. Statistical Analysis

The statistical analysis was performed in two stages. In the first stage, the experiments were designed and the collected data were analyzed. In the second stage, the formulation optimization operation was performed.


### 2.10.1. Experimental Design and Statistical Analysis

In order to produce probiotic pistachio paste and also to investigate the effects ofBifidobacterium lactis probiotic on aflatoxin, the response surface methodology (RSM) and a RCCD were used in Design Expert ver. 11.1.1.0 [30]. This method allows optimization of the probiotic pistachio paste formulation with the minimum aflatoxin level and also the maximum overall acceptance in sensory evaluation. In addition, four factors of aflatoxin concentration, probiotic level, temperature, and time were taken into account. Each factor was evaluated at three levels of +1, 0, and −1 (Table 2). After performing the experimental tests, regression analysis was performed based on the experimental data, and a quadratic polynomial model was fitted to each one of the responses using the following equation:(1)Y=β0+∑i=12β0xi+∑i=12βiixi2+∑i=1∑j=i+1βijxixj,where Y is the estimated response and the β0, βi, βii, and βij are the equation constant (intercept), linear effects coefficient, quadratic effects coefficient, and interaction coefficients, respectively; and xi and xj are the levels of independent variables. Finally, significant expressions (p < 0.05) for each response were identified using the analysis of variance (ANOVA) [31]. Test design and statistical data analysis were performed using Design Expert software.Table 2
Effective factors in RCCD.
FactorsNamesUnitsTypesMinMaxCoded lowCoded highMeanStd. dev.AProbioticCFU/gNumeric107109−1↔107+1↔1091080.79BTimeDayNumeric030−1↔0+1↔301511.82CTemperature°CNumeric446−1↔4+1↔462516.54DPrimary AFng/gNumeric525−1↔5+1↔25157.88


### 2.10.2. Formulation Optimization

The formulation optimization was performed after statistical data analysis. In order to optimize the formulation of probiotic pistachio paste with the minimum aflatoxin level and the maximum overall acceptance, numerical optimization techniques and desirability functions were used [32]. For this purpose, the maximum consumer acceptance response and the minimum aflatoxin level were taken into account.


## 2.10.1. Experimental Design and Statistical Analysis

In order to produce probiotic pistachio paste and also to investigate the effects ofBifidobacterium lactis probiotic on aflatoxin, the response surface methodology (RSM) and a RCCD were used in Design Expert ver. 11.1.1.0 [30]. This method allows optimization of the probiotic pistachio paste formulation with the minimum aflatoxin level and also the maximum overall acceptance in sensory evaluation. In addition, four factors of aflatoxin concentration, probiotic level, temperature, and time were taken into account. Each factor was evaluated at three levels of +1, 0, and −1 (Table 2). After performing the experimental tests, regression analysis was performed based on the experimental data, and a quadratic polynomial model was fitted to each one of the responses using the following equation:(1)Y=β0+∑i=12β0xi+∑i=12βiixi2+∑i=1∑j=i+1βijxixj,where Y is the estimated response and the β0, βi, βii, and βij are the equation constant (intercept), linear effects coefficient, quadratic effects coefficient, and interaction coefficients, respectively; and xi and xj are the levels of independent variables. Finally, significant expressions (p < 0.05) for each response were identified using the analysis of variance (ANOVA) [31]. Test design and statistical data analysis were performed using Design Expert software.Table 2
Effective factors in RCCD.
FactorsNamesUnitsTypesMinMaxCoded lowCoded highMeanStd. dev.AProbioticCFU/gNumeric107109−1↔107+1↔1091080.79BTimeDayNumeric030−1↔0+1↔301511.82CTemperature°CNumeric446−1↔4+1↔462516.54DPrimary AFng/gNumeric525−1↔5+1↔25157.88


## 2.10.2. Formulation Optimization

The formulation optimization was performed after statistical data analysis. In order to optimize the formulation of probiotic pistachio paste with the minimum aflatoxin level and the maximum overall acceptance, numerical optimization techniques and desirability functions were used [32]. For this purpose, the maximum consumer acceptance response and the minimum aflatoxin level were taken into account.


## 3. Results

### 3.1. Physicochemical Tests of Pistachio Paste

The physicochemical properties of the pistachio paste are listed in Table3.Table 3
Chemical properties of pistachio paste.
ProductMoisture (%)Fat (%)Protein (%)Fiber (%)Ash (%)AcidityPeroxide indexPistachio paste75817.511.72.60.549 mg KOH/g2.63 meq/kg


### 3.2. Statistical Models

#### 3.2.1. Summary of Statistical Model

A model was proposed according toR2 and PRESS indices. Low standard deviation (0.0043), high R2 (0.9982), and low PRESS indicate (0.0014) that the quadratic model is the best model.


#### 3.2.2. Sequential Sum of Squares

The software also suggested a quadratic model with sum of squares: 0.0036, df: 4, mean square: 0.0009, F-value: 48.83, and p value: <0.0001). For each model, the probability value (prob > F) was investigated in terms of significance (>0.05) to cover a larger part of the data.


#### 3.2.3. Lack of Fit Test

In this test, the significance of the models was investigated and since the proposed model had a low significance, a quadratic model was proposed (sum of squares: 0.0003, df: 10, mean square: 0.0000, F-value: 26.08, and p value: 0.0011).


#### 3.2.4. Analysis of Variance Test

The important point regarding the results of ANOVA is that thep value of the model was significant. The test was evaluated at p value >0.05 and data with this p value were entered into the equation. The Adeq precision value indicates the difference between the predicted response value of the model and the average prediction error value. If this ratio is higher than 4, it indicates an appropriate model of discrimination. Thus, the 89.9929 ratio proves the quadratic model.


#### 3.2.5. Coefficient Estimate

Impact factors and the type of their effect on responses were determined based on the coefficient estimate index. Positive index means direct and negative index means reverse impact and the higher the value, the greater the impact. The results showed that the probiotic factors, i.e. temperature and time, had a reverse impact on the response, i.e. the higher the value of these factors, the lower the response. Moreover, the aflatoxin factor had a direct effect.


#### 3.2.6. Final Response Formula Based on (Coded and Real) Factors

In the equation section, the response is between 1- and +1 in terms of coded factors and the final equation is shown in terms of real factors.


### 3.3. Three-Dimensional Diagrams

A three-dimensional (3D) diagram was used to highlight the effects of the two parameters on the response. Thus, the response changes were displayed according to the two desired parameters in the three-dimensional diagram. The three-dimensional diagrams showed the interactions of factors AB, AC, AD, BC, BD, and CD against aflatoxin residues. Where. A: probiotic content (CFU/g), B: time (day), C: temperature (°C), D: primary AF (Spiked AF: ng/gr) (Figures1–6). In addition, the effect of factors A, B, and C against overall acceptance are shown in (Figures 7–9).Figure 1
Interaction of storage temperature (25°C) and the inoculated aflatoxin level (15 ng/g) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)Figure 2
Interaction of time (15 days) and the inoculated aflatoxin level (15 ng/g) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)(e)Figure 3
Interaction of time (15 days) and the storage temperature (25°C) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)Figure 4
Interaction of probiotic content (108 CFU/g) and the inoculated aflatoxin level (15 ng/g) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)Figure 5
Interaction of probiotic content (108 CFU/g) and the storage temperature (25°C) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)Figure 6
Interaction of probiotic content (108 CFU/g) and the storage time (15 days) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)(e)Figure 7
The effect of storage temperature (25°C) on the overall acceptance of pistachio paste.
(a)(b)(c)(d)(e)Figure 8
The effect of storage time (15 days) on the overall acceptance of pistachio paste.
(a)(b)(c)(d)(e)Figure 9
The effect of probiotic content (108 CFU/g) on the overall acceptance of pistachio paste.
(a)(b)(c)(d)


### 3.4. Quality Assurance

The average recoveries were 107.2%. (97.8–111.8%). Limit of detection (LOD) and limit of quantification (LOQ) for AFB1 were 0.1 and 0.3 ng/g, respectively.


### 3.5. Optimization

#### 3.5.1. Optimization of Reduction and Detoxification of Aflatoxin B1 in Pistachio Paste byBifidobacterium lactis

The optimization results were optimized using numerical optimization techniques in Design Expert, and the results related to the optimization of the studied indices are shown in Figure10. Taking into account the minimum aflatoxin residue, the value of the desirability index was estimated to be equal to 0.903.Figure 10
Optimization values of initial levels of aflatoxin, probiotics, temperature, and storage time of pistachio paste on the reduction in AFB1 residue (59.4%).According to the results of the optimization process, the maximum reduction in AFB1 can be found in pistachio paste contaminated with AFB1 (19.7039 ng/g), spiked withBifidobacterium lactis (109 CFU/g), and then stored at 25°C for 26.1853 days (AFB1 residues: 8.00007 ng/g = 59.4% reduction).In addition, the aflatoxin reduction in the studied treatments ranged from 0% in the day-zero treatments to 73% in treatment 22, which is related to pistachio paste with an initial AFB1 contamination of 5 ng/g, spiked withBifidobacterium lactis (109 CFU/g), and stored at 46°C for 30 days (AFB1 residue: 1.35 ng/g = 73% reduction) (Table 1).According to INSO titled “Human Food and Animal Feed-Maximum mycotoxin tolerance” No. 5925 (2020) [33] and the European Commission Regulation No. 2010.165 (26 February 2010) titled “Maximum levels of aflatoxins in food” [34], the permissible aflatoxin level in pistachios and pistachio kernels is 8 ng/g. The treatment that was considered optimal was less than INSO and the European Commission’s requirements:(i)
Initial AFB1 contamination: 19.7039 ng/g.(ii)
Inoculation withBifidobacterium lactis: 109 CFU/g.(iii)
Storage: temperature 25°C for 26.1853 days.(iv)
AFB1 resides: 8.00007 ng/g.(v)
Reduction percentage: 59.4%.


#### 3.5.2. Optimization of Sensory Evaluation of Probiotic Pistachio Paste

The results of the optimization process were analyzed using numerical optimization techniques in Design Expert (Figure11). According to the results and taking into account the maximum overall acceptance in the sensory evaluation, the desirability index value was estimated to be equal to 1.000. Sensory analysis is an important factor in assessing the pistachio paste acceptability. According to the results of the optimization process, the highest overall acceptance in sensory evaluation can be observed in the pistachio paste spiked with Bifidobacterium lactis (109 CFU/g) and then stored at 25°C for 15 days (overall acceptance: 8.25455 = 91.72% of the total acceptance score).Figure 11
Optimization values of probiotic content, temperature, and storage time of pistachio paste on overall acceptance.In general, the sensory evaluation in the studied treatments for texture, color, flavor, odor, and overall acceptance started from 3.9–8.3, 7.1–8.9, 3.8–8.7, 3–8.6, and 4.5–8.6, respectively. Moreover, the percentage of overall acceptance score of the total sensory evaluation score (9) ranged from 50 to 95.6% (Table4).Table 4
Results of sensory evaluation in the studied treatments at different temperatures, storage times, and the different probiotic contents.
FactorsTreatments123456789101112131415161718192021222324252627282930Texture8.37.78.37.78.34.18.34.18.37.37.37.37.37.37.36.98.37.58.37.58.33.98.33.97.37.37.96.95.27.3Color8.98.28.98.28.97.58.97.58.97.97.97.97.97.97.97.28.98.08.98.08.97.18.97.17.97.98.37.28.47.9Tastes8.7758.7758.73.98.73.98.76.86.86.86.86.86.86.18.77.18.77.18.73.88.73.86.86.87.86.55.16.8Odor8.68.18.68.18.63.28.63.28.67.77.77.77.77.77.77.18.67.98.67.98.63.08.63.07.77.78.27.54.37.7Overall acceptance8.6798.6798.64.68.64.68.67.47.47.47.47.47.46.98.67.68.67.68.64.58.64.57.47.48.17.05.87.4Overall acceptance %95.687.895.687.895.651.195.651.195.682.282.282.282.282.282.276.795.684.495.684.495.65095.65082.282.29077.864.482.2


## 3.1. Physicochemical Tests of Pistachio Paste

The physicochemical properties of the pistachio paste are listed in Table3.Table 3
Chemical properties of pistachio paste.
ProductMoisture (%)Fat (%)Protein (%)Fiber (%)Ash (%)AcidityPeroxide indexPistachio paste75817.511.72.60.549 mg KOH/g2.63 meq/kg


## 3.2. Statistical Models

### 3.2.1. Summary of Statistical Model

A model was proposed according toR2 and PRESS indices. Low standard deviation (0.0043), high R2 (0.9982), and low PRESS indicate (0.0014) that the quadratic model is the best model.


### 3.2.2. Sequential Sum of Squares

The software also suggested a quadratic model with sum of squares: 0.0036, df: 4, mean square: 0.0009, F-value: 48.83, and p value: <0.0001). For each model, the probability value (prob > F) was investigated in terms of significance (>0.05) to cover a larger part of the data.


### 3.2.3. Lack of Fit Test

In this test, the significance of the models was investigated and since the proposed model had a low significance, a quadratic model was proposed (sum of squares: 0.0003, df: 10, mean square: 0.0000, F-value: 26.08, and p value: 0.0011).


### 3.2.4. Analysis of Variance Test

The important point regarding the results of ANOVA is that thep value of the model was significant. The test was evaluated at p value >0.05 and data with this p value were entered into the equation. The Adeq precision value indicates the difference between the predicted response value of the model and the average prediction error value. If this ratio is higher than 4, it indicates an appropriate model of discrimination. Thus, the 89.9929 ratio proves the quadratic model.


### 3.2.5. Coefficient Estimate

Impact factors and the type of their effect on responses were determined based on the coefficient estimate index. Positive index means direct and negative index means reverse impact and the higher the value, the greater the impact. The results showed that the probiotic factors, i.e. temperature and time, had a reverse impact on the response, i.e. the higher the value of these factors, the lower the response. Moreover, the aflatoxin factor had a direct effect.


### 3.2.6. Final Response Formula Based on (Coded and Real) Factors

In the equation section, the response is between 1- and +1 in terms of coded factors and the final equation is shown in terms of real factors.


## 3.2.1. Summary of Statistical Model

A model was proposed according toR2 and PRESS indices. Low standard deviation (0.0043), high R2 (0.9982), and low PRESS indicate (0.0014) that the quadratic model is the best model.


## 3.2.2. Sequential Sum of Squares

The software also suggested a quadratic model with sum of squares: 0.0036, df: 4, mean square: 0.0009, F-value: 48.83, and p value: <0.0001). For each model, the probability value (prob > F) was investigated in terms of significance (>0.05) to cover a larger part of the data.


## 3.2.3. Lack of Fit Test

In this test, the significance of the models was investigated and since the proposed model had a low significance, a quadratic model was proposed (sum of squares: 0.0003, df: 10, mean square: 0.0000, F-value: 26.08, and p value: 0.0011).


## 3.2.4. Analysis of Variance Test

The important point regarding the results of ANOVA is that thep value of the model was significant. The test was evaluated at p value >0.05 and data with this p value were entered into the equation. The Adeq precision value indicates the difference between the predicted response value of the model and the average prediction error value. If this ratio is higher than 4, it indicates an appropriate model of discrimination. Thus, the 89.9929 ratio proves the quadratic model.


## 3.2.5. Coefficient Estimate

Impact factors and the type of their effect on responses were determined based on the coefficient estimate index. Positive index means direct and negative index means reverse impact and the higher the value, the greater the impact. The results showed that the probiotic factors, i.e. temperature and time, had a reverse impact on the response, i.e. the higher the value of these factors, the lower the response. Moreover, the aflatoxin factor had a direct effect.


## 3.2.6. Final Response Formula Based on (Coded and Real) Factors

In the equation section, the response is between 1- and +1 in terms of coded factors and the final equation is shown in terms of real factors.


## 3.3. Three-Dimensional Diagrams

A three-dimensional (3D) diagram was used to highlight the effects of the two parameters on the response. Thus, the response changes were displayed according to the two desired parameters in the three-dimensional diagram. The three-dimensional diagrams showed the interactions of factors AB, AC, AD, BC, BD, and CD against aflatoxin residues. Where. A: probiotic content (CFU/g), B: time (day), C: temperature (°C), D: primary AF (Spiked AF: ng/gr) (Figures1–6). In addition, the effect of factors A, B, and C against overall acceptance are shown in (Figures 7–9).Figure 1
Interaction of storage temperature (25°C) and the inoculated aflatoxin level (15 ng/g) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)Figure 2
Interaction of time (15 days) and the inoculated aflatoxin level (15 ng/g) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)(e)Figure 3
Interaction of time (15 days) and the storage temperature (25°C) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)Figure 4
Interaction of probiotic content (108 CFU/g) and the inoculated aflatoxin level (15 ng/g) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)Figure 5
Interaction of probiotic content (108 CFU/g) and the storage temperature (25°C) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)Figure 6
Interaction of probiotic content (108 CFU/g) and the storage time (15 days) on the aflatoxin residue in pistachio paste.
(a)(b)(c)(d)(e)Figure 7
The effect of storage temperature (25°C) on the overall acceptance of pistachio paste.
(a)(b)(c)(d)(e)Figure 8
The effect of storage time (15 days) on the overall acceptance of pistachio paste.
(a)(b)(c)(d)(e)Figure 9
The effect of probiotic content (108 CFU/g) on the overall acceptance of pistachio paste.
(a)(b)(c)(d)


## 3.4. Quality Assurance

The average recoveries were 107.2%. (97.8–111.8%). Limit of detection (LOD) and limit of quantification (LOQ) for AFB1 were 0.1 and 0.3 ng/g, respectively.


## 3.5. Optimization

### 3.5.1. Optimization of Reduction and Detoxification of Aflatoxin B1 in Pistachio Paste byBifidobacterium lactis

The optimization results were optimized using numerical optimization techniques in Design Expert, and the results related to the optimization of the studied indices are shown in Figure10. Taking into account the minimum aflatoxin residue, the value of the desirability index was estimated to be equal to 0.903.Figure 10
Optimization values of initial levels of aflatoxin, probiotics, temperature, and storage time of pistachio paste on the reduction in AFB1 residue (59.4%).According to the results of the optimization process, the maximum reduction in AFB1 can be found in pistachio paste contaminated with AFB1 (19.7039 ng/g), spiked withBifidobacterium lactis (109 CFU/g), and then stored at 25°C for 26.1853 days (AFB1 residues: 8.00007 ng/g = 59.4% reduction).In addition, the aflatoxin reduction in the studied treatments ranged from 0% in the day-zero treatments to 73% in treatment 22, which is related to pistachio paste with an initial AFB1 contamination of 5 ng/g, spiked withBifidobacterium lactis (109 CFU/g), and stored at 46°C for 30 days (AFB1 residue: 1.35 ng/g = 73% reduction) (Table 1).According to INSO titled “Human Food and Animal Feed-Maximum mycotoxin tolerance” No. 5925 (2020) [33] and the European Commission Regulation No. 2010.165 (26 February 2010) titled “Maximum levels of aflatoxins in food” [34], the permissible aflatoxin level in pistachios and pistachio kernels is 8 ng/g. The treatment that was considered optimal was less than INSO and the European Commission’s requirements:(i)
Initial AFB1 contamination: 19.7039 ng/g.(ii)
Inoculation withBifidobacterium lactis: 109 CFU/g.(iii)
Storage: temperature 25°C for 26.1853 days.(iv)
AFB1 resides: 8.00007 ng/g.(v)
Reduction percentage: 59.4%.


### 3.5.2. Optimization of Sensory Evaluation of Probiotic Pistachio Paste

The results of the optimization process were analyzed using numerical optimization techniques in Design Expert (Figure11). According to the results and taking into account the maximum overall acceptance in the sensory evaluation, the desirability index value was estimated to be equal to 1.000. Sensory analysis is an important factor in assessing the pistachio paste acceptability. According to the results of the optimization process, the highest overall acceptance in sensory evaluation can be observed in the pistachio paste spiked with Bifidobacterium lactis (109 CFU/g) and then stored at 25°C for 15 days (overall acceptance: 8.25455 = 91.72% of the total acceptance score).Figure 11
Optimization values of probiotic content, temperature, and storage time of pistachio paste on overall acceptance.In general, the sensory evaluation in the studied treatments for texture, color, flavor, odor, and overall acceptance started from 3.9–8.3, 7.1–8.9, 3.8–8.7, 3–8.6, and 4.5–8.6, respectively. Moreover, the percentage of overall acceptance score of the total sensory evaluation score (9) ranged from 50 to 95.6% (Table4).Table 4
Results of sensory evaluation in the studied treatments at different temperatures, storage times, and the different probiotic contents.
FactorsTreatments123456789101112131415161718192021222324252627282930Texture8.37.78.37.78.34.18.34.18.37.37.37.37.37.37.36.98.37.58.37.58.33.98.33.97.37.37.96.95.27.3Color8.98.28.98.28.97.58.97.58.97.97.97.97.97.97.97.28.98.08.98.08.97.18.97.17.97.98.37.28.47.9Tastes8.7758.7758.73.98.73.98.76.86.86.86.86.86.86.18.77.18.77.18.73.88.73.86.86.87.86.55.16.8Odor8.68.18.68.18.63.28.63.28.67.77.77.77.77.77.77.18.67.98.67.98.63.08.63.07.77.78.27.54.37.7Overall acceptance8.6798.6798.64.68.64.68.67.47.47.47.47.47.46.98.67.68.67.68.64.58.64.57.47.48.17.05.87.4Overall acceptance %95.687.895.687.895.651.195.651.195.682.282.282.282.282.282.276.795.684.495.684.495.65095.65082.282.29077.864.482.2


## 3.5.1. Optimization of Reduction and Detoxification of Aflatoxin B1 in Pistachio Paste byBifidobacterium lactis

The optimization results were optimized using numerical optimization techniques in Design Expert, and the results related to the optimization of the studied indices are shown in Figure10. Taking into account the minimum aflatoxin residue, the value of the desirability index was estimated to be equal to 0.903.Figure 10
Optimization values of initial levels of aflatoxin, probiotics, temperature, and storage time of pistachio paste on the reduction in AFB1 residue (59.4%).According to the results of the optimization process, the maximum reduction in AFB1 can be found in pistachio paste contaminated with AFB1 (19.7039 ng/g), spiked withBifidobacterium lactis (109 CFU/g), and then stored at 25°C for 26.1853 days (AFB1 residues: 8.00007 ng/g = 59.4% reduction).In addition, the aflatoxin reduction in the studied treatments ranged from 0% in the day-zero treatments to 73% in treatment 22, which is related to pistachio paste with an initial AFB1 contamination of 5 ng/g, spiked withBifidobacterium lactis (109 CFU/g), and stored at 46°C for 30 days (AFB1 residue: 1.35 ng/g = 73% reduction) (Table 1).According to INSO titled “Human Food and Animal Feed-Maximum mycotoxin tolerance” No. 5925 (2020) [33] and the European Commission Regulation No. 2010.165 (26 February 2010) titled “Maximum levels of aflatoxins in food” [34], the permissible aflatoxin level in pistachios and pistachio kernels is 8 ng/g. The treatment that was considered optimal was less than INSO and the European Commission’s requirements:(i)
Initial AFB1 contamination: 19.7039 ng/g.(ii)
Inoculation withBifidobacterium lactis: 109 CFU/g.(iii)
Storage: temperature 25°C for 26.1853 days.(iv)
AFB1 resides: 8.00007 ng/g.(v)
Reduction percentage: 59.4%.


## 3.5.2. Optimization of Sensory Evaluation of Probiotic Pistachio Paste

The results of the optimization process were analyzed using numerical optimization techniques in Design Expert (Figure11). According to the results and taking into account the maximum overall acceptance in the sensory evaluation, the desirability index value was estimated to be equal to 1.000. Sensory analysis is an important factor in assessing the pistachio paste acceptability. According to the results of the optimization process, the highest overall acceptance in sensory evaluation can be observed in the pistachio paste spiked with Bifidobacterium lactis (109 CFU/g) and then stored at 25°C for 15 days (overall acceptance: 8.25455 = 91.72% of the total acceptance score).Figure 11
Optimization values of probiotic content, temperature, and storage time of pistachio paste on overall acceptance.In general, the sensory evaluation in the studied treatments for texture, color, flavor, odor, and overall acceptance started from 3.9–8.3, 7.1–8.9, 3.8–8.7, 3–8.6, and 4.5–8.6, respectively. Moreover, the percentage of overall acceptance score of the total sensory evaluation score (9) ranged from 50 to 95.6% (Table4).Table 4
Results of sensory evaluation in the studied treatments at different temperatures, storage times, and the different probiotic contents.
FactorsTreatments123456789101112131415161718192021222324252627282930Texture8.37.78.37.78.34.18.34.18.37.37.37.37.37.37.36.98.37.58.37.58.33.98.33.97.37.37.96.95.27.3Color8.98.28.98.28.97.58.97.58.97.97.97.97.97.97.97.28.98.08.98.08.97.18.97.17.97.98.37.28.47.9Tastes8.7758.7758.73.98.73.98.76.86.86.86.86.86.86.18.77.18.77.18.73.88.73.86.86.87.86.55.16.8Odor8.68.18.68.18.63.28.63.28.67.77.77.77.77.77.77.18.67.98.67.98.63.08.63.07.77.78.27.54.37.7Overall acceptance8.6798.6798.64.68.64.68.67.47.47.47.47.47.46.98.67.68.67.68.64.58.64.57.47.48.17.05.87.4Overall acceptance %95.687.895.687.895.651.195.651.195.682.282.282.282.282.282.276.795.684.495.684.495.65095.65082.282.29077.864.482.2


## 4. Discussion

Numerous studies have been performed to reduce the aflatoxin level in pistachios. The majorities of these studies have investigated the effect of different irradiations on the aflatoxin level and have indicated a reduction in high percentage of aflatoxin in pistachios due to irradiation. However, they lacked good desirability in terms of sensory evaluation and overall acceptance by consumers.Hashemi et al. showed that aflatoxin levels reduced by 38.84, 48.79, 53.50, and 77.17% in pistachios treated at doses of 1, 3, 5, and 7 kGy of electron irradiation on different storage days, respectively [35]. Jalili et al. also reported aflatoxin and ochratoxin levels in pistachios under microwave irradiation decreased by 72.5 ± 2.6% [36]. Makari et al. showed gamma irradiation at doses of 4 and 6 kGy led to 73.26 and 83.36 AFB1 degradation in pistachios, respectively [37]. Mehdizadeh and Amini found that pistachio AFB1 in natural uranium boilers decreased from 134.85 to 72.28 ng/g [38]. Mazaheri found that ultraviolet irradiation can reduce the AFB1 concentration in pistachios. This effect on the high AFB1 concentration is greater than the low AFB1 concentration [39], which is consistent and almost in the same line compared to the present study, which showed a 73% reduction.In meta-analysis studies, it was concluded that ultraviolet, ozone-ultraviolet, and citric acid methods were the most effective methods for reducing aflatoxins in cheeses [40], cereals, and nuts, respectively [41].The effects of the pistachio roasting process on the aflatoxin residues were examined. Better results were obtained both in terms of aflatoxin reduction and overall acceptance in cases where the roasting process included adding flavorings.Jalili et al. reported that the roasting process led to 85.78 ± 2.5% reduction in aflatoxin and ochratoxin of pistachios [36]. Rastegar et al. also concluded that the roasting process with lemon juice and citric acid can be used as a useful and safe degradation method for pistachios [28]. Yazdanpanah et al. also stated in a similar study that roasting pistachios at 150°C for 120 minutes destroyed more than 95% of AFB1; however, the resulting product was not edible [42]. The results of studies on the effects of roasting pistachios on the AFB1 reduction have shown the superiority of the biological method used in the present study, which in addition to reducing a high percentage of AFB1 in pistachio paste (73%), leads to an overall acceptance rate of 8.25455 (91.72%).Studies have managed to reduce aflatoxins using oral acids. For example, Esmailzadeh et al. investigated the effects of dehydroacetic acid (DHA) and ozonated water (OW) on pistachio AFB1 and reported the lowest AFB1 levels in pistachios treated with dehydroacetic acid (0.21–1.5μg/kg) and DHA + OW (0.21–1.59 μg/kg) [43]. In this regard, a review study, Babaei et al. reported that acids were more effective than other detoxification methods such as ozone, UV-C, and so on. in terms of degrading aflatoxins in pistachio nuts [44].Microbial populations coexist in the ecosystem and form a complex microbial community [45]. Among these, soil as a natural habitat of Aspergillus flavus has a high degree of complexity and heterogeneity and makes it difficult to analyze the ecological functions of secondary metabolites such as aflatoxins in soil [46]. Thus, research has become an important tool for controlling or reducing specific contaminants in cereals, feed, and the environment [47]. The toxins persist for a long time after soil contamination and this is important as planting in contaminated land transfers toxins from the soil to the cultivated seed and then to the grown forage. As a new insight at this point, if the beneficial microorganisms can multiply in the contaminated soil, the toxin level is greatly reduced. In addition to the mechanisms such as inhibition of aflatoxin production and uptake by microorganisms, another mechanism is aflatoxin degradation by microorganisms that produce specific substances. It alters the basic structure of mycotoxins and turns them into low-toxic or even completely nontoxic substances [48].Various microbiological techniques have emerged in recent decades for removing or reducing mycotoxins. Microorganisms exert detoxification effects on aflatoxins [49]. Studies have shown the effects of the biological activity of microorganisms on gene mutation or activation of silent gene clusters, and thus, reducing the aflatoxin production [50]. In particular, microorganisms reduce the toxin concentration to an undetectable level through interrupting the aflatoxin biosynthesis pathway by Aspergillus flavus in the early stages and before the synthesis of norsolorinic acid [51].The use of microorganisms as a biological method, in addition to reducing and degrading aflatoxins in pistachio paste can bring the health properties of these organisms to the body of consumers, which is one of the strengths and advantages compared to other methods.The results of the present study showed the highest reduction of AFB1 in pistachio paste with initial AFB1 contamination at 19.7039 ng/g which was spiked with 109 CFU/g of Bifidobacterium lactis, and then, stored at 25°C for 26.1853 days. The AFB1 residues in the above pistachio paste, finally, decreased by 59.4% to 8.00007 ng/g, which was within the standard limits recommended by INSO and ISO. The results indicated that high levels of the probiotic Bifidobacterium lactis (109 CFU/g) play an important role in reducing aflatoxin levels. The abovementioned treatment is the optimal treatment, and it is possible to operate it in the industry considering the possibility of storing pistachio paste at room temperature (25°C) for 26.0181 days.The aflatoxin reduction in the studied treatments ranged from 0% for day-zero treatments to 73% for treatment 22, with an initial AFB1 contamination of 5 ng/g, spiked with 109 CFU/g of Bifidobacterium lactis, and stored at 46°C for 30 days. Therefore, in addition to the high level of probiotic Bifidobacterium lactis (109 CFU/g), high temperature (46°C) and long-term storage (30 days) play a more important role in reducing aflatoxin in pistachio paste (73%). However, as to the practical use of the results in industry, it is not possible to store pistachio paste at 46°C for 30 days due to its low desirability for the consumer (50% of total overall acceptance score). The low desirability was probably due to microbial and chemical spoilage of the product.In a study of the effect ofSaccharomyces cerevisiae on AFB1 in pistachios, Rahaei et al. stated that AFB1 removal was related to the toxin concentration and yeast survival played no important role in the AFB1 removal [52]. Abdolshahi et al. stated that mannan extracted from the cell wall of Saccharomyces cerevisiae could bind to aflatoxin and the highest percentage of aflatoxin binding was observed at higher mannan concentrations and reduced pistachio aflatoxin [53], which is consistent with the results of the present study that suggested a high level of probiotic Bifidobacterium lactis had a significant effect on the aflatoxin removal.Farzaneh et al. concluded thatBacillus subtilis can significantly remove AFB1 from culture media and pistachio by 85.66 and 95%, respectively [54]. Failure to completely eliminate AFB1 seems to be a reversible process [55]. Pizzolitto et al. stated that this may be due to the saturated bond between the microorganism and AFB1 [56]. In a simple term, it is possible to remove a higher percentage of toxins by increasing the number of microorganisms and reducing the level of toxins, as the high level of the probiotic Bifidobacterium lactis led to the highest percentage of aflatoxin removal in the present study (73%).Some researchers have suggested that aflatoxin inhibition is due to the presence of lactic acid or other metabolites produced by lactic acid bacteria [57]. On the other hand, other researchers have found no significant relationship between the number of metabolites produced by these bacteria and the reduction of aflatoxin levels [58], and it is more likely that glucose oxidase exerts its effect as a catalyst glucose on the oxidation process [59]. In this regard, Raksha et al. investigated the biological detoxification of AFB1 by Bacillus licheniformis CFR1 and concluded that more than 94.7% of AFB1 reduction was observed in liquid culture medium and cell-free supernatant was able to degrade AFB1 more effectively than the cell [60]. Gonzalez et al. isolated and identified aflatoxin-degrading Bacillus strains. They stated that 10 strains and eight cell-free supernatants were able to significantly reduce AFB1. The tested strains could degrade AFB1 by extracellular and intracellular enzymes. If safe, they can be used to detoxify aflatoxins in contaminated food or feed [61]. This emphasizes the role of metabolites produced by microorganisms in reducing aflatoxins.Rahaei et al. reported that the two strains ofSaccharomyces cerevisiae and Lactobacillus rhamnosus bind to aflatoxins in significant numbers. The results of the study showed that the binding process did not have a significant effect on the pistachio color, texture, and peroxide value, which are very important in the desirability of nuts [62]. In the present study, the overall acceptance score in sensory evaluation at 4°C (refrigerator temperature) and 25°C (room temperature), which are practical and suitable storage temperatures for pistachio paste, ranged from 84.4% to 95.6% and 76.7% to 95.6%, respectively (Table 4).Ansari et al. reported that pretreated kefir grains at 70°C caused a significant reduction in AFB1 levels in pistachio samples [27]. Siahmoshteh et al. showed that Bacillus species can be considered as a potential biocontrol agent to counteract the growth of toxic fungi and subsequent aflatoxin contamination of pistachios and agricultural products [63].Overall, it seems that the use of biological methods as well as microorganisms in the detoxification and reduction of aflatoxin not only leads to a significant reduction in the aflatoxin content of the food matrix, but also increases the consumer’s acceptance. It also benefits the consumer thanks to the valuable benefits of microorganisms and especially probiotics.


## 5. Conclusions

Probiotics can bind to carcinogens and mutagens and their use as an effective, environmentally friendly, inexpensive, and safe strategy to eliminate chemical contaminants in food and feed has been widely examined. The results showed that probiotics have a special potential to remove aflatoxin or reduce its concentration to a safe level in pistachio paste. The effects of five independent variables including aflatoxin concentration, probioticBifidobacterium lactis, storage time, and temperature on the reduction of AFB1 were investigated using an optimization strategy by a RCCD. To improve food and feed safety, it is necessary to use these microorganisms and remove mycotoxins from contaminated food and feed. However, it seems necessary to carry out further studies to investigate the mechanisms involved in the toxin removal process by probiotics and reveal the relevant mechanism, dose, and timing of detoxification. The optimal detoxification conditions obtained in the present study can be proposed for the common and practical detoxification of pistachio paste AFB1 in the food industry. However, since aflatoxin production is a biological process, future in vivo studies are needed to confirm the effectiveness of probiotics in the contaminated pistachio paste.



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*Source: 2504482-2022-12-01.xml*