Isolation and Aflatoxin B1-Degradation Characteristics of a Microbacterium proteolyticum B204 Strain from Bovine Faeces

Aflatoxin B1 (AFB1) is one of the most harmful mycotoxins, raising serious global health and economic problems. Searching for biological approaches for effective and safe AFB1 degradation is imminent. In our study, Microbacterium proteolyticum B204 isolated from bovine faeces degraded 77% of AFB1 after 24 h, becoming the first reported bacteria from the Microbacterium family to possess AFB1 degradation characteristics. Temperature variation showed little effect on its degradation ratio, demonstrating high thermostability of 75% and 79% after boiling and sterilization, respectively. We suppose that the components playing a key role during this process were proteins, considering the decreased degradation rate caused by Proteinase K. Cell viability detection on HepG2 cells indicated that the degradation products were much less toxic than pure AFB1. Furthermore, B204 cell-free culture supernatant also degraded AFB1-contaminated food, such as peanuts, corn and cheese. These results suggested that this strain with AFB1 degradation properties could be a prospective candidate for application in the food and feed industries.


Introduction
Aflatoxins (AFs) are typical mycotoxins ubiquitous in agricultural and sideline commodities that are generated as highly toxic secondary metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus [1,2]. Among the four major isoforms of AFs-B1, B2, G1 and G2-AFB 1 has been classified as a Class IA Danger and a Category I carcinogen by the WHO and IARC's Human Carcinogen Risk Assessment Working Group due to its high toxicity [3][4][5]. AF contamination in tropical and subtropical regions becomes more severe, since fungi grow better under high temperatures and humidity. The harmful and toxic effects of AFs not only occur in food or feed, but also accumulate as food chains extend, which further spreads these effects [6,7]. As a result, AF contamination in food and the environment is an international concern on account of its tremendous hypertoxicity, mutagenicity, teratogenicity and carcinogenicity to both humans and livestock [2,8].
Searching for strategies to detoxify AFB 1 safely and effectively therefore has become a hot topic of scientific studies; there is also an emergent demand for these strategies within national welfare and public health. The application of adsorbent products is one of the most commonly used physical methods to remove AF contamination, including heating and irradiation [9]. Acid, alkali and oxidizing agents (e.g., chlorine dioxide) are mainly used as chemical substances to disinfect toxins [10]. Moreover, active components, including

Isolation, Identification and Characterization of AFB 1 Degradation Bacterial Strains
AFs are a group of bisfuranocoumarin derivatives, and coumarin is the fundamental molecular structure of all AFs [40]. Thus, coumarin solid plates were applied as the sole carbon source for preliminary screening, followed by secondary screening using high-performance liquid chromatography (HPLC). Through two-round screening, Microbacterium proteolyticum strain B204, which showed the highest degradation activity (77.00 ± 1.53%) after 24 h of incubation at 30 • C (Figure 1) was then selected for further investigation. After incubation at 30 • C for 1 h, 19.33 ± 3.79% AFB 1 was removed successfully, as shown in Figure 1. With the incubation time extended, the removal of AFB 1 appeared to increase slowly, and the degradation ratio reached 77.00 ± 1.53% at 24 h. Conversely, the concentration of AFB 1 in the control group remained stable from 0 to 24 h. Based on the 16S rRNA gene sequence blast and phylogenetic evolution analysis, the M. proteolyticum strain B204 showed 100% nucleotide identity with Microbacterium proteolyticum ( Figure 2). Morphological and biochemical analysis also confirmed the typical characteristics of Microbacterium sp., a Gram-positive, creamy yellow bacterium. This strain of Microbacterium proteolyticum was the first species in the Microbacterium genera discovered to possess the ability to degrade AFB1.

Effects of Incubation pH and Temperature on AFB1 Degradation by M. proteolyticum B204
The influence of pH and temperature on AFB1 degradation by the M. proteolyticum strain B204 is presented in Figure 3. As we can see from Figure 3A, the strain M. proteolyticum B204 shows a broad working range of pH gradient between five and eight, and the degradation rate reaches up to 55.86 ± 2.73% at pH 7, while the ratios are similar at pH 5 and 6 (35.28 ± 5.90% and 38.16 ± 3.30%, respectively). Nevertheless, the minimum degradation efficiency fell to 12.77 ± 3.42% at pH 4. Based on the 16S rRNA gene sequence blast and phylogenetic evolution analysis, the M. proteolyticum strain B204 showed 100% nucleotide identity with Microbacterium proteolyticum (Figure 2). Morphological and biochemical analysis also confirmed the typical characteristics of Microbacterium sp., a Gram-positive, creamy yellow bacterium. This strain of Microbacterium proteolyticum was the first species in the Microbacterium genera discovered to possess the ability to degrade AFB 1 . Based on the 16S rRNA gene sequence blast and phylogenetic evolution analysis, the M. proteolyticum strain B204 showed 100% nucleotide identity with Microbacterium proteolyticum ( Figure 2). Morphological and biochemical analysis also confirmed the typical characteristics of Microbacterium sp., a Gram-positive, creamy yellow bacterium. This strain of Microbacterium proteolyticum was the first species in the Microbacterium genera discovered to possess the ability to degrade AFB1.

Effects of Incubation pH and Temperature on AFB1 Degradation by M. proteolyticum B204
The influence of pH and temperature on AFB1 degradation by the M. proteolyticum strain B204 is presented in Figure 3. As we can see from Figure 3A, the strain M. proteolyticum B204 shows a broad working range of pH gradient between five and eight, and the degradation rate reaches up to 55.86 ± 2.73% at pH 7, while the ratios are similar at pH 5 and 6 (35.28 ± 5.90% and 38.16 ± 3.30%, respectively). Nevertheless, the minimum degradation efficiency fell to 12.77 ± 3.42% at pH 4.

Effects of Incubation pH and Temperature on AFB 1 Degradation by M. proteolyticum B204
The influence of pH and temperature on AFB 1 degradation by the M. proteolyticum strain B204 is presented in Figure 3. As we can see from Figure 3A, the strain M. proteolyticum B204 shows a broad working range of pH gradient between five and eight, and the degradation rate reaches up to 55.86 ± 2.73% at pH 7, while the ratios are similar at pH 5 and 6 (35.28 ± 5.90% and 38.16 ± 3.30%, respectively). Nevertheless, the minimum degradation efficiency fell to 12.77 ± 3.42% at pH 4. It should be noted that the degradation of the AFB1 by strain M. proteolyticum was barely affected by temperature ( Figure 3B). The degradation rate remained a stable at 15.78 ± 0.37%-20.31 ± 0.58% with increasing temperature up to 30 °C. The lent thermostability suggests that strain 204 has terrific potential practical applicati

AFB1 Degradation by Cell-Free Culture Supernatant and Cell Extracts of M. proteolyt B204
As Figure 1 shows, the AFB1 degradation ratio reaches its peak after 24 h incub The cell-free culture supernatant of M. proteolyticum B204 exhibited the highest deg tion ratio of 80.09 ± 1.29%, making it much more effective than cell extracts and which degraded AFB1 at a ratio of 16.13 ± 2.95%, 35.28 ± 1.55%, respectively (Figu This implies that the removal of AFB1 using M. proteolyticum B204 mainly depends biodegradation process, rather than the bio-adsorption to the bacterial cell wall. A ingly, cell-free culture supernatant was then used in the follow-up experiments.  It should be noted that the degradation of the AFB 1 by strain M. proteolyticum B204 was barely affected by temperature ( Figure 3B). The degradation rate remained almost stable at 15.78 ± 0.37%-20.31 ± 0.58% with increasing temperature up to 30 • C. The excellent thermostability suggests that strain 204 has terrific potential practical application.

AFB 1 Degradation by Cell-Free Culture Supernatant and Cell Extracts of M. proteolyticum B204
As Figure 1 shows, the AFB 1 degradation ratio reaches its peak after 24 h incubation. The cell-free culture supernatant of M. proteolyticum B204 exhibited the highest degradation ratio of 80.09 ± 1.29%, making it much more effective than cell extracts and cells, which degraded AFB 1 at a ratio of 16.13 ± 2.95%, 35.28 ± 1.55%, respectively ( Figure 4). This implies that the removal of AFB 1 using M. proteolyticum B204 mainly depends on the biodegradation process, rather than the bio-adsorption to the bacterial cell wall. Accordingly, cell-free culture supernatant was then used in the follow-up experiments.  It should be noted that the degradation of the AFB1 by strain M. proteolyticum B204 was barely affected by temperature ( Figure 3B). The degradation rate remained almost stable at 15.78 ± 0.37%-20.31 ± 0.58% with increasing temperature up to 30 °C. The excellent thermostability suggests that strain 204 has terrific potential practical application.

AFB1 Degradation by Cell-Free Culture Supernatant and Cell Extracts of M. proteolyticum B204
As Figure 1 shows, the AFB1 degradation ratio reaches its peak after 24 h incubation. The cell-free culture supernatant of M. proteolyticum B204 exhibited the highest degradation ratio of 80.09 ± 1.29%, making it much more effective than cell extracts and cells, which degraded AFB1 at a ratio of 16.13 ± 2.95%, 35.28 ± 1.55%, respectively ( Figure 4). This implies that the removal of AFB1 using M. proteolyticum B204 mainly depends on the biodegradation process, rather than the bio-adsorption to the bacterial cell wall. Accordingly, cell-free culture supernatant was then used in the follow-up experiments.   The degradation rate remained stable even after boiling and sterilization at 75.33 ± 0.58% and 79.33 ± 1.53%, respectively, compared to the control group with the addition of culture broth without other treatments, demonstrating excellent thermostability. The detoxification efficiency (79.07 ± 1.94%) suffered little impact from the addition of EDTA, while this value was markedly affected by Proteinase K and Proteinase K plus SDS in the cell-free supernatant of strain M. proteolyticum, falling to 44.93 ± 3.68% and 31.21 ± 4.66%, respectively ( Figure 5).

Effects of Heat Treatment, SDS, Proteinase K and EDTA on AFB1 Degradation by M. proteolyticum B204 Cell Culture Supernatant
The degradation rate remained stable even after boiling and sterilization at 75.33 ± 0.58% and 79.33 ± 1.53%, respectively, compared to the control group with the addition of culture broth without other treatments, demonstrating excellent thermostability. The detoxification efficiency (79.07 ± 1.94%) suffered little impact from the addition of EDTA, while this value was markedly affected by Proteinase K and Proteinase K plus SDS in the cell-free supernatant of strain M. proteolyticum, falling to 44.93 ± 3.68% and 31.21 ± 4.66%, respectively ( Figure 5).

Cytotoxicity Analysis of M. proteolyticum B204
The potential toxicity and mutagenicity had to be considered, as some degradable products of toxins could still be toxic, similarly to their parent compounds. Concerns as to whether AFB1 degradation products formed by the strain M. proteolyticum B204 cellfree culture supernatant were more, less or not toxic are not unreasonable. Therefore, it is imperative to detect and analyze the toxicity of degradation products in comparison with AFB1 (the parent compound). The cytotoxicity of AFB1 degradation extracts was performed on human hepatocellular liver carcinoma (HepG2) cells by MTT assay in this study. The cell viability was relatively stable at around 37% with 1, 5 and 10 μg/mL AFB1, until the concentration of AFB1 was raised to 20 μg/mL, which then decreased to 16.16% ± 2.42% ( Figure 6). Delightfully, with the addition of cell-free culture supernatants of strain M. proteolyticum B204, cell mortality underwent an obvious loss. The cell viability was not less than 75% in every experimental group, which was much higher than in the control group, with the help of M. proteolyticum B204′s degradation of AFB1 ( Figure 6). The cell viability was 86.99%, 79.59%, 75.82% and 75.36% with the addition of AFB1 at 1, 5, 10 and 20 μg/mL, respectively. These results indicate that the AFB1 degradation metabolites produced by strain M. proteolyticum B204 could help reduce the cytotoxicity AFB1 caused, and the degradation products themselves were harmless to cells as well.

Cytotoxicity Analysis of M. proteolyticum B204
The potential toxicity and mutagenicity had to be considered, as some degradable products of toxins could still be toxic, similarly to their parent compounds. Concerns as to whether AFB 1 degradation products formed by the strain M. proteolyticum B204 cell-free culture supernatant were more, less or not toxic are not unreasonable. Therefore, it is imperative to detect and analyze the toxicity of degradation products in comparison with AFB 1 (the parent compound). The cytotoxicity of AFB 1 degradation extracts was performed on human hepatocellular liver carcinoma (HepG2) cells by MTT assay in this study. The cell viability was relatively stable at around 37% with 1, 5 and 10 µg/mL AFB 1, until the concentration of AFB 1 was raised to 20 µg/mL, which then decreased to 16.16% ± 2.42% ( Figure 6). Delightfully, with the addition of cell-free culture supernatants of strain M. proteolyticum B204, cell mortality underwent an obvious loss. The cell viability was not less than 75% in every experimental group, which was much higher than in the control group, with the help of M. proteolyticum B204's degradation of AFB 1 (Figure 6). The cell viability was 86.99%, 79.59%, 75.82% and 75.36% with the addition of AFB 1 at 1, 5, 10 and 20 µg/mL, respectively. These results indicate that the AFB 1 degradation metabolites produced by strain M. proteolyticum B204 could help reduce the cytotoxicity AFB 1 caused, and the degradation products themselves were harmless to cells as well.  Figure 7 shows that 78.00 ± 1.91%, 83.30 ± 1.35% and 58.69 ± 0.51% of AFB1 degradation occurred in peanuts, corn and cheese after incubation with strain M. proteolyticum B204 at 30 °C for 24 h. The most effective detoxification occurred in corn, while the worst was in cheese.   Figure 7 shows that 78.00 ± 1.91%, 83.30 ± 1.35% and 58.69 ± 0.51% of AFB 1 degradation occurred in peanuts, corn and cheese after incubation with strain M. proteolyticum B204 at 30 • C for 24 h. The most effective detoxification occurred in corn, while the worst was in cheese.  Figure 7 shows that 78.00 ± 1.91%, 83.30 ± 1.35% and 58.69 ± 0.51% of AFB1 degradation occurred in peanuts, corn and cheese after incubation with strain M. proteolyticum B204 at 30 °C for 24 h. The most effective detoxification occurred in corn, while the worst was in cheese.

Discussion
Plenty of bacterial species have been revealed to be able to degrade AFB 1 based on current research, including Actinobacteria, Bacillus and α or β-proteobacteria [1,41]. Microbacterium proteolyticum B204 isolated in our study is the first species ever reported in Microbacterium that exhibits AFB 1 degradation ability, the degradation ratio of which was relatively high, reaching nearly 80% after 12 h treatment. However, this value did not exceed 90% in most bacteria. Among the bacteria with the highest degradation rate ever reported, Escherichia coli CG1061 eliminated 93.7% of AFB 1 after 72 h incubation [42]. Bacillus velezensis DY3108 strains degraded up to 94.7% of AFB 1 at 72 h, but only 30% after 12 h treatment. The use of M. proteolyticum B204 would greatly reduce time and cost and make this process more effective. Furthermore, the degradation process in our research was conducted with the incubation of bacteria and AFB 1 solution together instead of the direct addition of substantial amounts of M. proteolyticum B204, which might have affected the degradation efficiency. There is currently no research that suggests that any species of Microbacteriaceae could degrade or adsorb AFB 1 . Streptomyces roseolus is the closest bacterial species to M. proteolyticum B204 that can remove AFB 1 ( Figure 2); it also shows strong inhibitory effects on aflatoxin production [43], suggesting that we could continue to study whether M. proteolyticum B204 has similar functions.
The degradation of AFB 1 by cell-free supernatant after 24 h treatment was approximately 80% (Figure 3), almost equivalent to the effects induced by whole bacteria (Figure 1), but much higher than that of bacterial cell extracts (16.13%, Figure 3), indicating that the AFB 1 -degrading enzyme is mainly located in bacterial secretions. Similarly, it was also found that the cell-free supernatant of Flavobacterium aurantiacum could degrade 74.5% of AFB 1 after 24 h incubation [44]; this AFB 1 -detoxification process primarily took place in the cell-free extracts of Stenotrophomonas Maltophilia 35-3 with a 78.7% degradation ratio after 72 h incubation with its culture supernatant [40]. Furthermore, the cell-free supernatant of M. proteolyticum B204 still possessed a AFB 1 degradation capacity of more than 70% after treatment in boiling water at 100 • C for 20 min (Figure 5), implying its excellent thermal stability. We should also note that Proteinase K reduced almost 50% of the degradation activity and around 40% degradation still occurred. The reason why degradation kept proceeding mainly includes two aspects: firstly, there may have been more than one kind of enzyme that could degrade AFB 1 ; secondly, the digestion of Proteinase K was not very specific, and partial resistance to Proteinase K might have also occurred.
As shown in Figure 6, AFB 1 was cytotoxic to the growth of HepG2 cells, which has also been proved by previous studies [2,36,40,42,45]. Therefore, the AFB 1 degradation ability of M. proteolyticum B204 was investigated by evaluating the remaining AFB 1 s effects on cell proliferation and cell cytotoxicity by MTT assay. Based on the 86.99% cell viability in the 1 µg/mL AFB 1 group instead of 100%, we presumed that the degradation ratio of B204 cell-free supernatant to AFB 1 was about 80%, and it was the undegraded AFB 1 that affected cell viability. As the concentration of AFB 1 increased, the cell viability decreased, which might have been caused by the accumulation of residual AFB 1 that had not been degraded.

Conclusions
Our study firstly reports that a strain of Microbacterium proteolyticum B204 from bovine faeces could degrade AFB 1 effectively in common culture mediums and contaminated peanuts and corn with a ratio of approximately 77% after 24 h incubation. The toxicity evaluation of byproducts using the MTT assay on HepG2 cells showed a significant decrease in AFB 1 after biodegradation, indicating its biosafety potential for actual use. These findings may offer a novel path for reducing AFB 1 toxicity in practical use. Moreover, the effects of degradation products on human and animal metabolism should be further investigated, and the efficacy of isolated strains for detoxifying AFB 1 may become of great interest for applications in food and feed.

Two-Round Screening and Isolation of AFB 1 Degradation Bacterium
Preliminary Screening: One gram of bovine faeces was dissolved in 10 mL sterilized distilled water after a continuous vortex for 3 min; it was then serially diluted to the final concentration of 10 −4 , 10 −5 and 10 −6 . Aliquots of each diluent (100 µL) were homogeneously spread on coumarin medium plates and incubated at 30 • C for 21 days until visible colonies appeared. Single colonies growing well were picked and then inoculated to fresh new plates, the purification process of which was performed at least three times to obtain the pure isolates.
Secondary Screening: Each of the purified strains was further inoculated in 15 mL fresh LB liquid medium at 30 • C under continuous shaking of 180 rpm/min for 12-24 h. Fifty microliters of activated bacterial solution derived from the previous step were transferred to 9.95 mL fresh LB medium mixed with AFB 1 solution to acquire the desired concentration of 10 µg/µL, then cultured in a rotary shaker incubator at 30 • C for 12 h away from light. The supernatant was collected after centrifugation at 8000 rpm for 20 min, followed by concentration and redissolution in 1 mL acetonitrile (HPLC grade), then filtered through 0.22 µm filter and preserved at 4 • C for the further detection of residual AFB 1 . AFB 1 standard solution mixed with sterile LB medium (final concentration 10 µg/µL) was used as negative control.

Analysis of AFB 1 Degradation Products Using HPLC
The prepared filtered samples were loaded on Waters C18 Column (0.5 µm, 4.6 × 250 mm) equipped with a fluorescence detector according to Guan et al., with some modifications [40]. Millipore water was used as mobile phase A, and a mixture with methanol and acetonitrile (v/v, 1:1) was employed as mobile phase B, with a flow rate of 0.6 mL/min. The concentration of residual AFB 1 was quantitatively determined by fluorescence, the excitation wavelength and detection wavelength of which was set as 350 nm and 450 nm, respectively. AFB 1 standard solution mixed with sterile LB medium instead of cell-culture supernatant was used as negative control. The AFB 1 degradation ratio was evaluated using the following formula: AFB 1 degradation ration % = (AFB 1 peak area in control group − AFB 1 peak area in experimental group)/AFB 1 peak area in control group × 100%

Exploration of Culture Conditions by M. proteolyticum B204 on AFB 1 Degradation
The influences of different culture conditions on the biodegradation of AFB 1 by M. proteolyticum B204 were achieved independently as follows: incubation time 12 h, initial culture pH 4, 5, 6, 7 and 8, incubation temperature 4, 10, 20, 25, 30, 40, 45 and 50 • C, with other conditions kept constant. The final concentration of AFB 1 was consistent (10 µg/µL) and the addition of M. proteolyticum B204 was also maintained in each experimental group. The detection of AFB 1 degradation efficiency was performed as described in Section 5.3 without modification.

AFB 1 Degradation by Cell-Free Culture Supernatant, Cell-Free Extracts and Cells
The degradation of AFB 1 by different components of M. proteolyticum B204 was investigated as described below, referring to Wang et al. with minor modifications [2]. Cell-free culture supernatant was collected after centrifugation at 8000 rpm for 20 min at 4 • C, followed by filtration through 0.22 µm filters and preserved on ice for a further AFB 1 degradation assay. The precipitation of bacteria was thoroughly washed by sterilized MilliQ water and re-dissolved in PBS buffer (Biological Industries, Kibbutz Beit Haemek, Israel). The resuspending cells were then disrupted by ultrasonic homogenizer (Sonics, Wallingford, CT, USA) at 28% power with a 4 s pulse on and 6 s pulse off repetition cycle on ice. Cell-free extracts were obtained after centrifugation at 8000 rpm for 20 min at 4 • C and filtration through 0.22 µm filters. AFB 1 standard solution was then introduced into cell-free culture supernatant, cell-free extracts and cells with a final concentration of 10 µg/µL, respectively. LB culture medium + PBS buffer with equivalent AFB 1 was set as a negative control. After incubation at 30 • C for 24 h, the residual AFB 1 was detected by HPLC as described in Section 5.3.

Effects of Heat Treatment, SDS, Proteinase and EDTA on AFB 1 Degradation by Cell-Free Culture Supernatant
The effects of heat treatment, SDS, proteinase and EDTA on AFB 1 degradation by M. proteolyticum B204 cell-free culture supernatant were studied according to Farzaneh et al., with slight adjustments [45]. The impact of heat treatment on AFB 1 degradation efficiency was investigated by boiling for 20 min. Moreover, autoclave sterilization (121 • C, 20 min) was even adopted to evaluate the detoxification of AFB 1 . Sodium dodecyl sulfate (SDS) and Proteinase K treatments were also performed to evaluate the degradation process. One-milliliter of cell-free culture supernatant was mixed with 0.1 mL 10 mg/mL Proteinase K, 0.1 mL 10% SDS buffer and 0.1 mL 10 mg/mL Proteinase K plus 0.1 mL 10% SDS buffer, respectively. Moreover, EDTA with a final concentration of 0.1 mol/L was also added to 1 mL cell-free broth to analyze the degradation efficiency of AFB 1 . All the degradation reactions with the addition of SDS, Proteinase K and EDTA were incubated at 30 • C for 1 h in the dark. The degradation products of AFB 1 were determined by HPLC as described in Section 5.3.

Cytotoxicity Analysis by MTT Assay
The effects of the AFB 1 and B204 cell-free supernatant-induced degradation of AFB 1 were detected on HepG2 cells via evaluating cell cytotoxicity using the MTT assay referenced in Wang et al., with some modifications [2]. The final concentrations of AFB 1 were set as 0, 1, 5, 10 and 20 µg/mL, respectively. The only difference between experimental groups and control groups was the addition of M. proteolyticum B204 cell-free culture supernatant. After degradation for 24 h with the help of B204 cell-free culture supernatant, using equivalent LB culture medium as controls, the mixture was then incubated with HepG2 cells (1 × 10 4 cells per well) in a 96-well multi-plate at 37 • C under 5% CO 2 for 24 h. Then, 20 µL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added and co-incubated for another 4 h. Finally, 150 µL DMSO was introduced to every well, making the cell crystals sufficiently dissolved. Cell viability was assessed according to the absorption value at 560 nm.

Application of AFB 1 Detoxification by M. proteolyticum B204 to Food Matrices
The application to food matrices of M. proteolyticum B204 against AFB 1 contamination was conducted on peanuts, corn and cheese. One gram of each sample mentioned above was sprayed with 100 µL AFB 1 with a concentration of 100 µg/µL. After incubation with 450 µL of M. proteolyticum B204 at 30 • C for 16 h, the residual AFB 1 was then detected by HPLC, as described above.

Statistical Analysis
All experiments were independently repeated in triplicate. The statistical analysis was performed by one-way analysis of variance (ANOVA) within the 95% confidence interval followed by the Student's t test or Duncan's test using SPSS software (SPSS Inc., Chicago, IL, USA).