Evaluation of the Antimicrobial Potential of Astragalus fasciculifolius Gum Extract Against Clostridium perfringens in Meatball Formulations Using Response Surface Methodology
الموضوعات : Food and Health
Najmeh Khademi Pour
1
(Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran)
Anousheh Sharifan
2
(Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran)
Hossein Bakhoda
3
(Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran)
الکلمات المفتاحية: Astragalus fasciculifolius Boiss, / Bioactive Compounds, /Response Surface Methodology, / Meatball,
ملخص المقالة :
Astragalus fasciculifolius Boiss is one of the native medicinal plants of Iran that has a special place in Iranian medicine. We investigated the phenolic compounds profile of ethanolic gum extracts, antimicrobial activity (MIC and MBC), and modeling and optimization of Clostridium perfringens growth dynamics in meat matrices. The results showed that the highest phenolic composition in the ethanolic extract was hesperidin (17.61%). Ethanolic A. fasciculifolius gum extract had antimicrobial activity. The MIC and MBC of Clostridium perfringens were reported as 156 and 78 (mg/g extract). The ethanolic gum extract caused shrinkage and changes in bacterial membranes. Dynamic modeling of bacterial growth in the meat matrix in the presence of the ethanolic A. fasciculifolius gum extract was performed as a quadratic equation. It was found that the lowest number of bacteria would be observed at 7200.8 ppm of extract, a storage time of 14.29 hours, and a storage temperature of 4.00 °C. This study showed that A. fasciculifolius gum has important active ingredients that can be used in the food, cosmetics, and drug industries.
Evaluation of phenolic compounds profile, antimicrobial activity of Astragalus fasciculifolius Boiss, and growth dynamic modeling of Clostridium perfringens in Meatball in the presence of ethanolic extract gum by response surface methodology
Abstract
Astragalus fasciculifolius Boiss, is one of the native medicinal plants of Iran that has a special place in Iranian medicine. We investigated the phenolic compounds profile of ethanolic gum extracts, antimicrobial activity (MIC and MBC); and modeling and optimization of Clostridium perfringens growth dynamics in meat matrices. The results showed that the highest phenolic composition in the ethanolic extract was hesperidin (17.61%); Ethanolic A. fasciculifolius gum extract had antimicrobial activity. The MIC and MBC of Clostridium perfringens reported as 156 and 78 (mg/g extract). The ethanolic gum extract caused shrinkage and changes in bacterial membranes. Dynamic modeling of bacterial growth in the meat matrix in the presence of the ethanolic A. fasciculifolius gum extract was performed as a quadratic equation. It was found that the lowest number of bacteria would be observed at 7200.8 ppm of extract, a storage time of 14.29 hours, and a storage temperature of 4.00 °C. This study showed that A. fasciculifolius gum has important active ingredients that can be used in the food, cosmetics, and drug industries.
Keywords: Astragalus fasciculifolius Boiss, Bioactive compounds, Response surface methodology, Meatball.
1- Introduction
Maintaining food safety and quality throughout life has attracted the attention of food industry experts and national health officials, and neglect or insufficient attention to this issue could lead to irreparable harm to society (1). Diseases caused by eating contaminated food are a significant problem worldwide. Therefore, the need to reduce or eliminate food pathogens using new methods is quite noticeable (2). Meat is a food with high perishability. Various factors, such as storage temperature, the amount of oxygen in the atmosphere, internal enzymes, light, moisture, and especially microorganisms, are involved in its spoilage (2). Predictive microbiology models the growth, survival, and death responses of important microbes in food by considering the primary and influential controlling factors. If mathematical models for a wide range of foods are developed, the need for specific and case-specific microbiological tests for new foods will be significantly reduced (3). Therefore, modeling the growth response of pathogenic bacteria in the food matrix under the influence of natural compounds can be substantial; consumers welcome natural preservatives, including natural compounds that can be used as preservatives in food, plant extracts, and essential oils. These compounds have antibacterial, antifungal, antioxidant, and anti-cancer properties and can control the growth of pathogens and the production of toxins by microorganisms (4). According to the World Health Organization, nearly 80% of people use herbal medicines for primary health care and preventive care (5). In addition, more than 50% of newly approved drugs are derived directly from modified herbs or their active ingredient (6). Astragalus fasciculifolius Boiss belongs to the legume family, which is also used in traditional Iranian medicine (7). This plant has anti-inflammatory, anti-viral, anti-diabetic, anti-cancer, and anti-poisoning effects due to its compounds such as flavonoids; phthalides are another group of effective compounds of this plant, which is considered a dietary supplement and a chemical preventive agent against cancer and gastric wounds and protects the liver (8).
This study aimed to investigate the antimicrobial properties of the ethanolic gum extract of A. fasciculifolius. Also the growth dynamics of Clostridium perfringens bacteria in meatballs in the presence of the ethanolic extract of Astragalus fasciculifolius were modeled and optimized using Design-Expert software.
2- Materials and Methods
Astragalus fasciculifolius was collected from the cultivation pastures of this plant in the north of Hormozgan-Iran (25° 24′ 28°.53″N 52° 44′ 59.14″E), and a Botanist approved the genus and species of this plant of Hormozgan University. Plants were harvested on 18 July 2020, and the root, aerial parts, and gum of the plant were separated; after that, the A. fasciculifolius were cleaned and dried in the shade at room temperature and proper airflow for 72 h and were grounded into an adequate powder particles size using an industrial mill. The plants were stored in the refrigerator at 4 to 6 °C.
2-1 Preparation and extraction of A. fasciculifolius extract
For ethanolic extraction, the ethanol was used as solvent at a rate of 1:20 (solids: solvent) for 24 hours by the maceration method, and the remaining organic solvents were evaporated using an evaporator (9). The extracts were labeled and kept at 2-8 °C until needed.
2-2 Identification of the phenolic profile in the ethanolic extract
The resulting extract was diluted with hexane and injected into a gas chromatograph (GC) model (Hewlett Packard-Sunnyvale, USA-GC 5890 Series II). The most suitable thermal programming was performed to separate the components of the extract. The chromatography device was equipped with a BPX5 column (column length: 30 m, internal diameter: 0.33, and stationary phase thickness: 0.25 μm). The extract's constituents were identified by comparing the standard compounds' mass spectra and inhibition indices using the Wiley 275. L database in the GC/MS device. The relative percentage of each extract constituent was obtained according to their sub-curved surface in the relevant chromatogram (10)
2-3 The Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)
The dilution broth method was used to determine the MIC. Twelve test tubes were used to determine the MIC for ethanolic A. fasciculifolius gum extract. From 1 mg/ml to 625 mg/ml, 5 l of the prepared bacterial suspension was added to each ethanolic extract concentration, from 1mg/ml to 625 mg/Ml. The tubes were incubated at 37°C for 24 hours. After 24 hours, the tubes were examined for turbidity due to the growth of inoculated bacteria. In order to estimate, the first concentration in which microbial growth did not fall out and was not seen recorded for MIC. All the cultures that showed no visible growth were cultured on media and incubated at 37 °C for 24 h. The MBC was taken as the concentration that kills all bacterial cells cultured and prevents the growth of any bacteria colony on media (3).
2-4 Preparation of meatball samples and microbial counting
The meat and fat were ground with 1.5% salt using a meat grinder with a blade diameter of 3 ml. For 1000 g of beef, 200 g of fat, 15 g of salt, and 50 g of bread were mixed and ground again (11). Then they were heat-treated with 80 ° C water vapor for two hours until the center temperature reached 72 ° C. 1010 CFU Clostridium perfringens (activated according to the manufacturer's instructions) were inoculated separately into meatballs. Ethanolic Astragalus fasciculifolius extract was injected into the meatball samples at concentrations of 0 to 8000 ppm, and the samples were stored at 4 to 12 °C and examined for 1 to 96 hours. Table 4 presents the proposed tests of Design-Expert software with the Central Composite method (file version: 12.0.3.0).
Table 1. The input factors for modeling and optimization
2-8-2- Clostridium perfringens bacteria Count in meatballs Sulfite polymyx sulfadiazine agar was the media for the growth of Clostridium perfringens. The plates were incubated for 37 h at 37 °C, and the colony counter was used for counting (12).
2-9- Data analysis
The phenolic compounds analysis: All experiments were triplicated. Average results were reported as the mean and standard error, and SPSS (Version 18.0, SPSS Inc., Chicago, USA) statistical software, one-way ANOVA followed by Duncan's multiple were used. Optimization and modeling of the microbial population of Clostridium perfringens with the extract concentration, time, and storage temperature were evaluated by Design-Expert software for RSM design and the Central Composite method. Analysis of variance was performed at a 95% confidence level.
1. Results and Discussion
3-1. Investigation of the total phenol, total phenol, and flavonoid content and the profile of Enzrot gum extract phenolic compounds.
The content of total phenols and total flavonoids in methanolic, ethanolic, and aqueous extracts are shown in Table 2.
Table 2- Total phenol and total flavonoid content in ethanolic extract.
By using gas chromatography, 20 phenolic compounds were found. A one-way analysis of variance and a Duncan analysis were used to determine how the type of solvent affected these 20 compounds.
Table 3shows that the change in solvent type led to a change in the number of phenolic compounds. However, the highest phenolic compounds identified were related to methanolic A. fasciculifolius gum extract. In the ethanol solvent, hesperidin had the highest amounts.
3-2 Investigation of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
Researchers looked into the effect of ethanolic A. fasciculifolius gum extract on the number of bacteria that could grow and the number of bacteria that could die. The results are shown in Table 4.
Table 4. Evaluation of the MIC and MBC of A. fasciculifolius gum extract on Clostridium perfringens
According to Table 4, Clostridium perfringens was more sensitive to A. fasciculifolius gum extract than Pseudomonas aeruginous and had lower MIC and MBC.
3-3 Modeling bacterial growth dynamics in the meatballs formulated with ethanolic A. fasciculifolius gum extract by response surface methodologyThe independent effects of extract concentration, storage time, and storage temperature on the microbial growth of Clostridium perfringens were evaluated in 20 tests, shown in Table 5.
Table 5- Designed tests with test results
3-3-1-Evaluation of coefficients affecting the growth rate of Clostridium perfringens and Pseudomonas aeruginosa
The logarithm of the number of Clostridium perfringens and Pseudomonas aeruginosa was analyzed by a quadratic model. An ANOVA analysis was performed to estimate the significance of the independent variables and their interaction. Multivariate correlation analysis was performed to obtain the coefficients of the final equation. The variables that did not significantly affect the response were removed to obtain the final model and the final equation was estimated. The model's performance was judged by the lack of fit values, the R2 and adj R2 coefficients, and the model's P-value.
3-3-1-1-Growth dynamics of Clostridium perfringens
Table 6 - Results of analysis of variance of dynamic growth of Clostridium perfringens bacteria
The results in Table 6 show that the concentration of ethanolic extract of A. fasciculifolius gum, storage time, and storage temperature of meatballs significantly affected the final growth dynamic model of Clostridium perfringens bacteria (P≤0.05). Also, the interaction of two variables of A. fasciculifolius gum ethanolic extract concentration and storage time (AB), as well as A. fasciculifolius gum ethanolic extract concentration and storage temperature of meatballs (AC), and the quadratic power of A. fasciculifolius gum ethanolic extract concentration (A2), had a significant effect on the model ( P≤0.05).
Fig. 1. Effect of ethanolic extract concentration of A. fasciculifolius gum (A), storage time (B), and storage temperature (C) on the growth of Clostridium perfringens
As shown in Fig. 1, with increasing the concentration of ethanolic extract of A. fasciculifolius gum, the logarithm of microbial growth of Clostridium perfringens decreased, which, according to Table 6, was significant (p≤0.05). The lowest growth logarithm of Clostridium perfringens was observed at 6000 ppm and above concentrations. It is also known that with increasing the storage time of meatball samples, the microbial growth logarithm of Clostridium perfringens has increased, which has also been significant according to the reports in Table 6 (p≤0.05). The highest logarithm of the number of Clostridium perfringens was observed at 96 h. Fig. 5 shows that Clostridium perfringens microbial growth increased with increasing storage temperature. The highest logarithm of the number of Clostridium perfringens was observed at 96 h. Figure 5 shows that Clostridium perfringens microbial growth increased with increasing storage temperature, which was also significant (p≤0.05). According to the , the lowest logarithm of the number of Clostridium perfringens was observed at 4 ° C. By removing the ineffective variables on the model, the final model was expressed as a quadratic equation.
4.47- 0.009A+0.034B+0.47C -0.00005AB-0.0001AC+8.32 A2
According to the fitted coefficients (Table 6), the proposed model has high adequacy and can be used to predict the logarithm of the number of Clostridium perfringens bacteria.
4- Discussion
4-1-Investigation of total phenol and flavonoid and phenolic compounds profile of A. fasciculifolius gum extract
The ethanolic extract had a significant total phenol and flavonoid content. In similar research, Hadzri et al. (2014) showed that extraction of P.niruri by Soxhlet using methanol as solvent had the highest yield, followed by ethanol, ethyl acetate, and hexane (13). Do et al. (2014) stated that the amounts of phenolic compounds in the total aqueous extract were significantly lower than in other solvents. They also reported that the extraction of total phenol compounds in the extracts decreased with increasing water content in the solvent, except for the methanol system. Increasing water content in solutions may be due to recycling more non-polar compounds, such as carbohydrates and terpenes, in the aqueous extract. They may also be due to the possible complex formation of some phenolic compounds in the extracts dissolved in methanol, acetone, and ethanol (14). The study of the phenolic compound profile found that p-coumaric acid in methanolic extract, hesperidin in ethanolic extract, and salicylic acid in aqueous extract had the most phenolic compounds. Identifying phenolic compounds in plant extracts to purify the effective compounds is essential for health, treatment, and nutritional purposes. Lekmine et al. (2021) identified the phenolic compound profiles of ethanolic extract of Astragalus gombiformis aerial parts and stated that 17 phenolic compounds were identified with the highest concentrations of crasiliol, silymarin, quercetin, and kaempferol (15).
4-2-Evaluation of antimicrobial activity of A. fasciculifolius gum extract.
The antimicrobial activity of medicinal plants is typically associated with phenolic compounds having a (-0H) group. The hydroxyl group in phenolic compounds binds to the active part of enzymes and inhibits their metabolism (16). Another possible mechanism is that phenolic compounds bind to phospholipids in cell membranes, thereby reducing selective permeability and increasing membrane permeability. In this case, the substance of which the cell is composed is removed from the cell and the energy metabolism is damaged, as well as the absorption of nutrients by the microbial cell, the transfer of electrons into the cell and the synthesis of genetic material. (16).
4-2-1-Investigation of Minimum Inhibitory concentration (MIC) and the Minimum Bactericidal Concentration (MBC)
It was found that Clostridium perfringens was more sensitive to A. fasciculifolius gum extract than Pseudomonas aeruginous and had lower levels of minimum inhibitory and lethality. Gram-positive bacteria do not have an outer membrane, so phenolic compounds enter the cell more easily than gram-positive bacteria. In other words, the outer membrane of Gram-negative bacteria forms a barrier against excess fatty acids, and this may represent the difference between resistance to Gram-negative and positive bacteria. (17). Khan et al. (2018) investigated the antimicrobial properties of a methanolic extract of Astragalus eremophilus. They reported that the highest inhibitory halos were against Salmonella Typhimurium, Enterococcus faecalis, Klebsellesa pneumonia, and Staphylococcus aureus, respectively, and had the lowest inhibitory concentration of 7.5 mg/mL. This study states that the antimicrobial properties of Astragalus eremophilus extract are due to the presence of compounds such as phenolic compounds, tannins, saponins, and flavonoids (18).
Conclusion
Due to the importance of new plant resources with biostatic properties, in this study, according to the background of traditional Iranian medicine, the plant Anazrut, which is native to Iran and West and South Asia, was studied. First, the phenolic compounds in Anazrut gum extract extracted with methanol, ethanol, and water were analyzed by gas-mass chromatography, and it was reported that the highest content of phenolic compounds was in the extract extracted with methanol solvent. The P-coumaric acid compounds were the most extracted phenolics with methanol solvent. The MIC and MBC of Anazrut gum extract against Clostridium perfringens were calculated, and it was found that the minimum inhibitory and lethal concentrations for Clostridium perfringens were 78 and 156 mg/ml, respectively, indicating that Clostridium perfringens was sensitive to Anazrut gum extract.
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Table 1. The input factors for modeling and optimization
Factor | Name | Units | Type | Minimum | Maximum | Coded Low | Coded High |
A | Cons. Extract | ppm | Numeric | 0.0000 | 8000.00 | -1 ↔ 0.00 | +1 ↔ 8000.00 |
B | Time | hr | Numeric | 12.00 | 96.00 | -1 ↔ 12.00 | +1 ↔ 96.00 |
C | Temperature | degree centigrade | Numeric | 4.00 | 12.00 | -1 ↔ 4.00 | +1 ↔ 12.00 |
Table 2- Total phenol and total flavonoid content in ethanolic extracts
Total flavenoeid(mg RUE/g) | Total phenol(mg GAE/g)
| Solvent |
15.12±0.8 | 24.39±1.2 | Ethanol |
Table 3- Comparison of phenolic compounds identified in ethanolic extracts
Ethanol |
| |
8.781±0.009 |
| |
1.380±0.01 |
| |
4.333±0.009 |
| |
2.428±0.005 |
| |
1.857±0.020 |
| |
2.122±0.002 |
| |
1.311±0.001 |
| |
6.026±0.005 |
| |
3.212±0.006 |
| |
11.953±0.005 |
| |
8.333±0.006 |
| |
2.665±0.110 |
| |
17.615±0.004 |
| |
2.428±0.020 |
| |
3.834±0.007 |
| |
2.354±0.577 |
| |
0.00±0.00 |
| |
0.00±0.00 | Kaempferol | |
0.00±0.00 |
| |
7.202±0.015 |
|
Table 4. Evaluation of the MIC and MBC of A. fasciculifolius gum extract on Clostridium perfringens
MBC(mg/ml) | MIC(mg/ml) | Bacteria |
156 | 78 | Clostridium perfringens |
Table 5- Designed tests with test results
|
| Factor 1 | Factor 2 | Factor 3 | Response 2 |
Std | Run | A: Cons. Extract | B: Time | C: Temperature | Cl. Perfrigenes |
|
| ppm |
|
|
|
16 | 1 | 4000 | 54 | 8 | 5 |
20 | 2 | 4000 | 54 | 8 | 5.6 |
19 | 3 | 4000 | 54 | 8 | 5.3 |
15 | 4 | 4000 | 54 | 8 | 5.1 |
12 | 5 | 4000 | 96 | 8 | 5.6 |
13 | 6 | 4000 | 54 | 4 | 4.8 |
5 | 7 | 0 | 12 | 12 | 7.2 |
6 | 8 | 8000 | 12 | 12 | 3.5 |
10 | 9 | 8000 | 54 | 8 | 4.2 |
3 | 10 | 0 | 96 | 4 | 8.9 |
9 | 11 | 0 | 54 | 8 | 9.1 |
17 | 12 | 4000 | 54 | 8 | 5.1 |
11 | 13 | 4000 | 12 | 8 | 4.8 |
7 | 14 | 0 | 96 | 12 | 10.5 |
2 | 15 | 8000 | 12 | 4 | 3.5 |
1 | 16 | 0 | 12 | 4 | 6.3 |
8 | 17 | 8000 | 96 | 12 | 4.3 |
18 | 18 | 4000 | 54 | 8 | 4.8 |
4 | 19 | 8000 | 96 | 4 | 4.1 |
14 | 20 | 4000 | 54 | 12 | 5.1 |
Table 6 - Results of analysis of variance of dynamic growth of Clostridium perfringens bacteria
Source | Sum of Squares | df | Mean Square | F-value | p-value |
|
Model: Quadratic | 66.92 | 9 | 7.44 | 68.45 | < 0.0001 | significant |
A-Cons. Extract | 50.18 | 1 | 50.18 | 461.88 | < 0.0001 |
|
B-Time | 6.56 | 1 | 6.56 | 60.40 | < 0.0001 |
|
C-Temperature | 0.9000 | 1 | 0.9000 | 8.28 | 0.0164 |
|
AB | 2.53 | 1 | 2.53 | 23.30 | 0.0007 |
|
AC | 0.6613 | 1 | 0.6613 | 6.09 | 0.0333 |
|
BC | 0.1013 | 1 | 0.1013 | 0.9320 | 0.3571 |
|
A² | 4.88 | 1 | 4.88 | 44.90 | < 0.0001 |
|
B² | 0.0384 | 1 | 0.0384 | 0.3536 | 0.5653 |
|
C² | 0.3728 | 1 | 0.3728 | 3.43 | 0.0937 |
|
Residual | 1.09 | 10 | 0.1086 |
|
|
|
Lack of Fit | 0.7113 | 5 | 0.1423 | 1.90 | 0.2496 | not significant |
Pure Error | 0.3750 | 5 | 0.0750 |
|
|
|
Cor Total | 68.01 | 19 |
|
|
|
|
Std. Dev. | 0.329 |
| R² | 0.9840 |
|
|
Mean | 5.46 |
| Adjusted R² | 0.9696 |
|
|
CV % | 5.84 |
| Predicted R² | 0.9095 |
|
|
|
|
| Adeq Precision | 29.6061 |
|
|
|
|
Fig. 1. Effect of ethanolic extract concentration of A. fasciculifolius gum (A), storage time (B), and storage temperature (C) on the growth of Clostridium perfringens
Highlights files
Solvent polarity is one of the most critical factors affecting the extraction of phenolic
compounds from the Astragalus fasciculifolius Boiss. gum
2. The Astragalus fasciculifolius Boiss gum extract has Significant amounts of phenolic
compounds and antimicrobial activities.
3. The Astragalus fasciculifolius Boiss gum extract causes shrinkage and changes in
Clostridium perfringens bacterial membranes.
4. Dynamic modeling of bacterial growth in the meat matrix in the presence of the ethanolic
A.fasciculifolius gum extract showed the quadratic equation could explain the decrease
in Clostridium perfringens growth.s
