and the B-HBA concentration was recorded. Data were interpreted using 208 determinations in the
two aforementioned sampling time points.

2.7. *Antioxidant Status, Immune Response, and Milk Fatty Acid Profile*

Eight (n = 8) ewes of each group with comparable weights (Control: 60.2 ± 5.11 kg; ActiSaf: 60.3 ± 4.88 kg), ages (Control: 1.84 ± 0.16 kg; ActiSaf: 1.85 ± 0.18 kg), milk performance (Fat corrected milk 6% (FCM<sub>6%</sub>) data used up 14th day in milk (DIM), Control: 2.3 ± 0.15 kg; ActiSaf: 2.3 ± 0.21 kg), prolificacy (Control: 2; ActiSaf: 2), and same lactation stage (up to 3 days deviation between animals) were selected for determining the antioxidant status of both milk and blood, for immune system gene response, and for milk fatty acid profile. Milk samples were collected (as mentioned above) in the 3rd and 6th week postpartum and stored at -80 °C. Blood samples were also collected, before the morning feeding (0700 h), at the same time points in heparin contained tubes for cell extraction and plasma isolation.

2.8. *Enzyme Assays, Oxidative Stress Biomarkers, and Total Antioxidant Capacity*

The enzyme activities, oxidative stress biomarkers, and the total antioxidant capacity were
measured spectrophotometrically (Helios alpha, UNICAM, Cambridge, UK) as previously described
by Tsiplakou et al. [22]. Briefly, Glutathione transferase (GST) activity in blood plasma was measured
according to the method described by Labrou et al. [23] by measuring the conjunction of reduced
glutathione to 1-chloro-2,4-dinitrobenzene at 340 nm. Catalase (CAT) activity in blood plasma and
milk were assessed using a continuous spectrophotometric rate for the determination of H₂O₂ at
520 nm, according to the Sigma-Aldrich Catalase Assay Kit (CAT100). Glutathione peroxidase (GSH-Px)
activity in blood plasma was measured according to the method of Paglia and Valentine [24] at 340 nm.
Glutathione reductase (GR) activities in both blood plasma and milk were measured according to
the method of Mavis and Stellwagen [25] by measuring the reduction in oxidized glutathione at
340 nm. Superoxide dismutase (SOD) activities in both blood plasma and milk were assayed using the
method of McCord and Fridovich [26] by measuring the inhibition of cytochrome c oxidation at 550 nm.
Lactoperoxidase (LPO) activity in milk was performed according to the methods of Keesey [27] by
measuring the oxidation of ABTS present in hydrogen peroxide at 340 nm. Malondialdehyde (MDA)
was determined according to the method of Nielsen et al. [28] with some modifications. More specifically,
100 µL blood plasma was added to 700 µL ortho-phosphoric acid (Panreac ITW Companies) and
200 µL aquarius thiobarbituric acid (TBA, Sigma-Aldrich CO USA) and then the samples were heated
at 100 °C for 60 min. In milk samples, 1 mL of raw milk was added to 7 mL ortho-phosphoric acid
(Panreac ITW Companies) and 2 mL of aquarius TBA (thiobarbituric acid, Sigma-Aldrich CO USA)
and then incubated at 100 °C for 60 min. After that, absorbance was recorded at 532 nm. The protein
carbonyl (PC) content was determined according to the method of Patsoukis et al. [29] by measuring the
conjunction of 2,4-dinitrophenylhydrazine (DNPH) on protein carbonyls at 375 nm. The 2,2'-Azino-bis
(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging assay was based on the published
methods [30,31]. Ferric reducing ability of plasma (FRAP) assay was used to measure total antioxidant
potential according to the method described by Benzie and Strain [32].

2.9. *Milk Fatty Acid Profile*

Milk fatty acid profile was determined using Gas Chromatography (Agilent 6890 N GC, Agilent
7683 B autosampler injector), equipped with an HP-88 capillary column (60 m × 0.25 mm i. d. with
0.20 µm film thickness, Agilent Technologies, USA) and a flame ionization detector (FID) as previously
described by Mavrommatis and Tsiplakou [33].

2.10. *Monocytes and Neutrophils Immune Genes Expression*

Blood monocytes and neutrophils were isolated and then total RNA was extracted as previously
described by Tsiplakou et al. [34]. Pure RNA (500 ng) from 64 individual (monocytes (32) and
neutrophils (32)) samples was reverse transcribed with the PrimeScript First Strand cDNA Synthesis Kit (Takara, Japan) according to the manufacturer's instructions using a mix of random hexamers and oligo-dT primers. A pair of primers specific for each target gene was designed using Geneious software (Biomatters, New Zealand) according to the respective *Ovis aries* gene coding sequences (CDSs in GenBank) (Table S7). The specificity of each pair of primers was tested against genomic DNA (positive control) to confirm that a single amplicon would emerge after quantitative real-time PCR. In addition, dissociation curves were generated, and the amplification products were subjected to agarose gel electrophoresis to confirm the production of a single amplicon per reaction. The relative expression levels of the target genes were calculated as $(1 + E)^{-\Delta Ct}$, where $\Delta Ct$ is the difference between the geometric mean of the two housekeeping genes' Cts and the Ct of the target gene, and the primer efficiency is the mean of each amplicon's efficiency per primer, which was calculated by employing the linear regression method on the log (fluorescence) per cycle number ($\Delta Rn$) using the LinRegPCR software [35]. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) and Tyrosine 3-monoxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) were used as housekeeping genes to normalize the cDNA template concentrations; the RT-PCR protocols are described in Tsiplakou et al. [18].

## 2.11. Statistical Analysis

Experimental data were analyzed using the SPSS.IBM statistical package (version 20.0) and results are presented as mean ± mean standard error (SEM). Dietary treatment effects were determined using a general linear model (GLM) for a repeated measures analysis of variance (ANOVA). With the dietary treatments (D = Control and ActiSaf) used as the fixed factor and the sampling time (S) as the repeated measure, while including their interactions (D*S) to evaluate differences over time, according to the model:

$$Yijkl = \mu + Di + Sj + Ak + (D \times S)ij + eijkl$$

where $\Upsilon$ijk is the dependent variable, $\mu$ the overall mean, Di the effect of dietary treatment (i = 2; Control and ActiSaf), Sj the effect of sampling time (j = 6 for milk performance, 2 for B-HBA concentration, fatty acids profile, antioxidant and immune system), Ak the animal's random effect, (D*S)ij the interaction between dietary treatments and sampling time, and eijk the residual error. Posthoc analysis was performed when appropriate using a Tukey's multiple range test [36]. For all tests, the significance level was set at $p = 0.05$. In order to simplify the visualization of the results, GraphPad Prism 6.0 (2012) was used for interleaved bars while error bars represent the mean standard error (SEM).

Moreover, discriminant analysis was also applied to pooled data to establish those variables capable of distinguishing and classifying samples among the two dietary treatments. Wilk's lambda ($\lambda$) criterion was used for selecting discriminant variables [37]. Forty variables were entered to develop a model to discriminate the thirty-two samples of each case. Specifically, five variables were used for grouped fatty acids in milk, ten and 10 for immune system genes' relative expression in monocytes and neutrophils, respectively, and seven and eight in antioxidant indices in milk and blood, respectively.

# 3. Results

## 3.1. Animal Performance

Dietary supplementation with LY ActiSaf significantly reduced the B-HBA concentrations in ewes' blood by 27% (0.86 ± 0.07 vs. 0.63 ± 0.06 mmol/L, $p = 0.018$) in the prepartum period, and by 17% (0.67 ± 0.04 vs. 0.56 ± 0.03 mmol/L, $p = 0.028$) in the postpartum period. Overall, the B-HBA was reduced by 24% (0.77 vs. 0.59 mmol/L, SEM = 0.04, $p = 0.003$) in the whole experimental period (Figure 1; Tables S1 and S2). Mean BW did not differ between the dietary treatments in the whole experimental period (Tables S3 and S4). However, ewes' BW recovered between lambing and 6th week
of lactation tended to increase by 75% (2.00 ± 0.31 vs. 3.44 ± 0.28 kg, *p* = 0.092) in the ActiSaf compared with the Control group (Figure 2; Table S5).

<figure><img src="image_27.png" /><figcaption>**Figure 1.** Graphical representation of (A) β-hydroxybutyric acid (B-HBA) in ewes' blood prepartum (mean ± SE), (B) β-hydroxybutyric acid in blood postpartum (mean ± SE), and (C) β-hydroxybutyric acid in blood of ewes in Control (black, *n* = 51 ewes) and ActiSaf (grey, *n* = 53 ewes) groups in experimental period of 12 weeks (mean ± mean standard error (SEM)).</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>**Figure 2.** Graphical representation of body weight gain (recovery) between lambing and 6th week of lactation of ewes in Control (black, *n* = 51 ewes) and ActiSaf (grey, *n* = 53 ewes) groups (mean ± SE).</figcaption></figure>

Milk, fat corrected milk 6% (FCM<sub>6%</sub>), and energy corrected milk yield (ECM) were numerically increased by 7.6 (2.50 vs. 2.69 kg/day, SEM = 0.159, *p* = 0.395), 12 (2.07 vs. 2.32 kg/day, SEM = 0.126, *p* = 0.161), and 10% (1.93 vs. 2.13 kg/day, SEM = 0.116, *p* = 0.231), respectively, in the ActiSaf compared to the Control group (Figure 3; Table S6; Figure S1). Concerning milk chemical composition, fat and protein contents were slightly decreased by 1.2 (*p* = 0.740) and 3% (*p* = 0.381), respectively, in the ActiSaf group, due to higher daily milk yield. However, both daily milk fat (114 vs. 131 g/day, SEM = 7.188, *p* = 0.104) and milk protein production (133 vs. 143 g/day, SEM = 8.288, *p* = 0.434) were increased by 15 and 7.5%, respectively, in the ActiSaf group (Figure 3; Supplementation Table S6). The milk yield in the treated ewes showed a moderate increase after the third week in lactation, and a peak in the fourth week, indicating a more intense milk persistence.
<figure><img src="image_29.png" /><figcaption>**Figure 3.** Graphical representation of milk yield and chemical composition of ewes in Control (black, *n* = 51 ewes) and ActiSaf (grey, *n* = 53 ewes) groups (mean ± SEM). FCM: Fat corrected milk in 6% according to the equation Y6% = (0.28 + 0.12F) M, where F = fat% and M = milk yield in kg. ECM: Energy corrected milk = milk yield × (0.071 × fat (%) + 0.043 × protein (%) + 0.2224) [38].</figcaption></figure>
## 3.2. Milk Fatty Acid Profile

Milk fatty acid profile was not altered among dietary supplementation except for certain minor differences. Specifically, pentadecanoic acid (C₁₅:₀), palmitoleic acid (C₁₆:₁), trans linoleic acid (C₁₈:₂n6t), and linolenic acid (C₁₈:₃n₃) were increased in ActiSaf milk by 15 (0.82 vs. 0.95%, SEM = 0.045, *p* = 0.042), 13 (0.29 vs. 0.33%, SEM = 0.014, *p* = 0.033), 9 (0.19 vs. 0.22%, SEM = 0.008, *p* = 0.049), and 20% (0.40 vs. 0.48%, SEM = 0.027, *p* = 0.075), respectively, while stearic acid (C₁₈:₀) decreased by 5% (8.91 vs. 8.44%, SEM = 0.490, *p* = 0.029) (Table 2).

<table><caption>**Table 2.** The mean individual fatty acids (FAs) (% of total FA), FA groups and Saturated Fatty Acids (SFAs)/Unsaturated Fatty Acids (UFAs) of milk from ewes fed Control (n = 8 ewes) and ActiSaf (n = 8 ewes) diet throughout the experimental period (21 and 42 experimental days).</caption><thead><tr><th rowspan="2"></th><th colspan="3">Diets (D)</th><th colspan="3">Sampling Time (T) in Weeks</th><th colspan="3">Effect *</th></tr><tr><th>Control</th><th>ActiSaf</th><th>SEM †</th><th>3</th><th>6</th><th>SEM †</th><th>D</th><th>T</th><th>DxT</th></tr></thead><tbody><tr><td>C<sub>4:0</sub></td><td>4.81</td><td>4.59</td><td>0.288</td><td>4.79</td><td>4.61</td><td>0.205</td><td>0.522</td><td>0.230</td><td>0.989</td></tr><tr><td>C<sub>6:0</sub></td><td>3.75</td><td>3.60</td><td>0.191</td><td>3.42</td><td>3.93</td><td>0.181</td><td>0.788</td><td>0.005</td><td>0.589</td></tr><tr><td>C<sub>8:0</sub></td><td>3.17</td><td>3.12</td><td>0.171</td><td>2.90</td><td>3.89</td><td>0.125</td><td>0.922</td><td>0.010</td><td>0.489</td></tr><tr><td>C<sub>10:0</sub></td><td>9.08</td><td>9.14</td><td>0.639</td><td>8.05</td><td>10.17</td><td>0.451</td><td>0.789</td><td>0.001</td><td>0.367</td></tr><tr><td>C<sub>12:0</sub></td><td>4.88</td><td>5.01</td><td>0.395</td><td>4.33</td><td>5.56</td><td>0.285</td><td>0.422</td><td>0.000</td><td>0.148</td></tr><tr><td>C<sub>14:0</sub></td><td>10.84</td><td>11.16</td><td>0.325</td><td>10.41</td><td>11.58</td><td>0.251</td><td>0.398</td><td>0.000</td><td>0.257</td></tr><tr><td>C<sub>14:1</sub></td><td>0.24</td><td>0.30</td><td>0.030</td><td>0.26</td><td>0.28</td><td>0.028</td><td>0.152</td><td>0.890</td><td>0.789</td></tr><tr><td>C<sub>15:0</sub></td><td>0.82</td><td>0.95</td><td>0.045</td><td>0.83</td><td>0.94</td><td>0.037</td><td>0.042</td><td>0.004</td><td>0.174</td></tr><tr><td>C<sub>15:1</sub></td><td>0.24</td><td>0.29</td><td>0.032</td><td>0.28</td><td>0.25</td><td>0.027</td><td>0.259</td><td>0.520</td><td>0.258</td></tr><tr><td>C<sub>16:0</sub></td><td>23.94</td><td>24.90</td><td>0.596</td><td>24.10</td><td>24.42</td><td>0.428</td><td>0.189</td><td>0.890</td><td>0.478</td></tr><tr><td>C<sub>16:1</sub></td><td>0.29</td><td>0.33</td><td>0.014</td><td>0.309</td><td>0.314</td><td>0.011</td><td>0.033</td><td>0.621</td><td>0.585</td></tr><tr><td>C<sub>17:0</sub></td><td>0.60</td><td>0.65</td><td>0.031</td><td>0.701</td><td>0.544</td><td>0.020</td><td>0.698</td><td>0.003</td><td>0.984</td></tr><tr><td>C<sub>17:1</sub></td><td>0.31</td><td>0.34</td><td>0.030</td><td>0.40</td><td>0.25</td><td>0.022</td><td>0.980</td><td>0.000</td><td>0.970</td></tr><tr><td>C<sub>18:0</sub></td><td>8.91</td><td>8.44</td><td>0.490</td><td>8.86</td><td>8.48</td><td>0.352</td><td>0.029</td><td>0.045</td><td>0.368</td></tr><tr><td>Σ<sub>trans</sub></td><td>1.07</td><td>1.01</td><td>0.110</td><td>1.05</td><td>1.02</td><td>0.088</td><td>0.893</td><td>0.880</td><td>0.489</td></tr><tr><td>‡<sub>trans-11</sub></td><td>1.26</td><td>1.16</td><td>0.158</td><td>1.22</td><td>1.20</td><td>0.125</td><td>0.358</td><td>0.499</td><td>0.984</td></tr><tr><td>C<sub>18:1</sub></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>cis<sub>9</sub> C<sub>18:1</sub></td><td>20.99</td><td>20.05</td><td>1.260</td><td>23.09</td><td>17.97</td><td>0.785</td><td>0.639</td><td>0.002</td><td>0.874</td></tr><tr><td>C<sub>18:2n6t</sub></td><td>0.19</td><td>0.22</td><td>0.008</td><td>0.19</td><td>0.22</td><td>0.008</td><td>0.049</td><td>0.019</td><td>0.321</td></tr><tr><td>C<sub>18:2n6c</sub></td><td>3.02</td><td>3.09</td><td>0.213</td><td>2.84</td><td>3.26</td><td>0.168</td><td>0.890</td><td>0.009</td><td>0.284</td></tr><tr><td>C<sub>20:0</sub></td><td>0.12</td><td>0.12</td><td>0.004</td><td>0.11</td><td>0.13</td><td>0.006</td><td>0.980</td><td>0.033</td><td>0.678</td></tr><tr><td>C<sub>18:3n3</sub></td><td>0.40</td><td>0.48</td><td>0.027</td><td>0.45</td><td>0.43</td><td>0.020</td><td>0.075</td><td>0.459</td><td>0.574</td></tr><tr><td>C<sub>20:3n3</sub> + C<sub>22:1</sub></td><td>0.31</td><td>0.28</td><td>0.015</td><td>0.34</td><td>0.25</td><td>0.012</td><td>0.784</td><td>0.000</td><td>0.348</td></tr><tr><td>trans-11<sup>†</sup></td><td>0.76</td><td>0.77</td><td>0.110</td><td>0.74</td><td>0.80</td><td>0.081</td><td>0.899</td><td>0.269</td><td>0.635</td></tr><tr><td>cis<sub>9</sub> C<sub>18:2</sub></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>§§SCFA</td><td>20.81</td><td>20.45</td><td>0.896</td><td>19.16</td><td>22.10</td><td>0.678</td><td>0.678</td><td>0.002</td><td>0.354</td></tr><tr><td>¶MCFA</td><td>41.07</td><td>42.07</td><td>0.858</td><td>40.69</td><td>43.04</td><td>0.689</td><td>0.201</td><td>0.008</td><td>0.528</td></tr><tr><td>††LCFA</td><td>9.03</td><td>8.56</td><td>0.489</td><td>8.97</td><td>8.62</td><td>0.361</td><td>0.302</td><td>0.099</td><td>0.598</td></tr><tr><td>‡‡MUFA</td><td>24.40</td><td>23.48</td><td>1.140</td><td>26.61</td><td>21.28</td><td>0.651</td><td>0.522</td><td>0.008</td><td>0.789</td></tr><tr><td>§§§PUFA</td><td>4.68</td><td>4.84</td><td>0.308</td><td>4.57</td><td>4.95</td><td>0.212</td><td>0.622</td><td>0.028</td><td>0.654</td></tr><tr><td>¶¶SFA</td><td>70.91</td><td>71.67</td><td>1.046</td><td>68.82</td><td>73.75</td><td>0.721</td><td>0.589</td><td>0.007</td><td>0.354</td></tr><tr><td>†††UFA</td><td>29.09</td><td>28.33</td><td>1.046</td><td>31.18</td><td>26.25</td><td>0.712</td><td>0.453</td><td>0.007</td><td>0.352</td></tr><tr><td>‡‡‡SFA/UFA</td><td>2.43</td><td>2.53</td><td>0.143</td><td>2.21</td><td>2.80</td><td>0.086</td><td>0.870</td><td>0.006</td><td>0.123</td></tr><tr><td>§§§§AI</td><td>2.56</td><td>2.68</td><td>0.133</td><td>2.33</td><td>2.92</td><td>0.099</td><td>0.256</td><td>0.002</td><td>0.099</td></tr></tbody></table>

<footnote>* Effect: The dietary treatment (D), time (T), and the interaction between dietary treatment × time (DxT) effects were analyzed by analysis of variance (ANOVA) using a general linear model (GLM) for repeated measures and posthoc analysis was performed when appropriate using Tukey's multiple range test. †SEM = Standard error of the mean. ‡<sub>trans-11</sub> C<sub>18:1</sub> = these values are not included in the Σ<sub>trans</sub> C<sub>18:1</sub> content. §SCFAs: Short-Chain Saturated Fatty Acids = C<sub>6:0</sub> + C<sub>8:0</sub> + C<sub>10:0</sub> + C<sub>11:0</sub>; ¶MCFAs: Medium-Chain Saturated Fatty Acids = C<sub>12:0</sub> + C<sub>13:0</sub> + C<sub>14:0</sub> + C<sub>15:0</sub> + C<sub>16:0</sub> + C<sub>17:0</sub>. ††LCFAs: Long-Chain Saturated Fatty Acids = C<sub>18:0</sub> + C<sub>20:0</sub>. ‡‡MUFA: Mono-Unsaturated Fatty Acids = C<sub>14:1</sub> + C<sub>15:1</sub> + C<sub>16:1</sub> + C<sub>17:1</sub> + C<sub>18:1</sub> cis-9 + <sub>trans-11</sub> C<sub>18:1</sub> + <sub>trans</sub> C<sub>18:1</sub>; §§PUFAs: Poly-Unsaturated Fatty Acids = <sub>cis-9</sub>, <sub>trans-11</sub> C<sub>18:2</sub> (CLA) + C<sub>18:2n-6c</sub> + C<sub>18:2n-6t</sub> + C<sub>18:3n-3</sub> + C<sub>18:3n-6</sub> + C<sub>20:3n-3</sub>; ¶¶SFA: Saturated Fatty Acids = SCFA + MCFA + LCFA; †††UFAs: Unsaturated Fatty Acids = PUFA + MUFA; ‡‡‡S/U: Saturated/Unsaturated = (SCFA + MCFA + LCFA)/(PUFA + MUFA), and §§§AI: Atherogenicity index = (C<sub>12:0</sub> + 4 * C<sub>14:0</sub> + C<sub>16:0</sub>)/(PUFA + MUFA).</footnote>
### 3.3. Oxidative Status

Amongst the dietary treatments, we did not report any significant differences in both blood and milk antioxidant enzymes. However, Glutathione Reductase (GR) in blood plasma was significantly increased by 13% (0.067 vs. 0.076 units/mL, SEM = 0.002, *p* = 0.004) in ActiSaf fed ewes. A numerical increase in lactoperoxidase (LPO) and catalase (CAT) activities by 20 and 10%, respectively, in milk of ActiSaf fed ewes was observed. The total antioxidant capacity measured by ABTS assay was significantly higher by 16.7% (37.563 vs. 43.850% inhibition, SEM = 3.564, *p* = 0.001) in the milk of ActiSaf fed ewes (Table 3). A negative correlation between the total antioxidant capacity determined by FRAP assay and the proportions of MUFA and oleic acid (C<sub>18:1 cis-9</sub>) in milk was found. The same trend was reported between Glutathione Peroxidase (GPx) activity in blood plasma and the aforementioned fatty acids of milk. The correlation between blood malondialdehyde (MDA) content and the proportions of milk’s PUFA was also negative. On the other hand, the correlations between blood MDA content and the proportions of MUFA and oleic acid, respectively, were positive (Figure 4).

<table><caption>**Table 3.** Enzymes activities (Units/mL), total antioxidant capacity, and oxidative status biomarkers in blood plasma and milk of ewes fed the two diets (Control, *n* = 8 and ActiSaf, *n* = 8) at two sampling times.</caption><thead><tr><th rowspan="2"></th><th colspan="3">Diets (D)</th><th colspan="3">Sampling Time (T) in Weeks</th><th colspan="3">Effect *</th></tr><tr><th>Control</th><th>ActiSaf</th><th>SEM <sup>†</sup></th><th>3</th><th>6</th><th>SEM <sup>†</sup></th><th>D</th><th>T</th><th>DxT</th></tr></thead><tbody><tr><td colspan="10" style="text-align: center; font-weight: bold;">Blood plasma</td></tr><tr><td>GST</td><td>0.150</td><td>0.132</td><td>0.011</td><td>0.148</td><td>0.134</td><td>0.011</td><td>0.282</td><td>0.336</td><td>0.903</td></tr><tr><td>GR</td><td>0.067</td><td>0.076</td><td>0.002</td><td>0.067</td><td>0.076</td><td>0.002</td><td>0.004</td><td>0.003</td><td>0.458</td></tr><tr><td>GSH-Px</td><td>0.398</td><td>0.423</td><td>0.025</td><td>0.353</td><td>0.468</td><td>0.021</td><td>0.476</td><td>0.000</td><td>0.847</td></tr><tr><td>SOD</td><td>18.544</td><td>20.304</td><td>0.987</td><td>18.409</td><td>20.440</td><td>1.170</td><td>0.227</td><td>0.301</td><td>0.427</td></tr><tr><td>CAT</td><td>4.969</td><td>5.008</td><td>0.146</td><td>4.893</td><td>5.084</td><td>0.131</td><td>0.855</td><td>0.255</td><td>0.477</td></tr><tr><td>FRAP</td><td>0.641</td><td>0.764</td><td>0.045</td><td>0.675</td><td>0.730</td><td>0.036</td><td>0.099</td><td>0.230</td><td>0.409</td></tr><tr><td>ABTS</td><td>36.888</td><td>33.045</td><td>0.497</td><td>35.577</td><td>34.356</td><td>0.617</td><td>0.000</td><td>0.246</td><td>0.195</td></tr><tr><td>MDA</td><td>0.601</td><td>0.566</td><td>0.067</td><td>0.604</td><td>0.562</td><td>0.055</td><td>0.729</td><td>0.497</td><td>0.304</td></tr><tr><td>PC</td><td>4.065</td><td>4.015</td><td>0.085</td><td>3.886</td><td>4.193</td><td>0.110</td><td>0.685</td><td>0.114</td><td>0.981</td></tr><tr><td colspan="10" style="text-align: center; font-weight: bold;">Milk</td></tr><tr><td>LPO</td><td>0.504</td><td>0.602</td><td>0.059</td><td>0.628</td><td>0.478</td><td>0.047</td><td>0.265</td><td>0.006</td><td>0.467</td></tr><tr><td>SOD</td><td>3.677</td><td>3.569</td><td>0.173</td><td>3.577</td><td>3.669</td><td>0.157</td><td>0.664</td><td>0.660</td><td>0.859</td></tr><tr><td>CAT</td><td>29.284</td><td>32.359</td><td>3.564</td><td>33.558</td><td>28.085</td><td>2.836</td><td>0.552</td><td>0.054</td><td>0.750</td></tr><tr><td>FRAP</td><td>3.013</td><td>3.090</td><td>0.179</td><td>2.855</td><td>3.247</td><td>0.158</td><td>0.766</td><td>0.078</td><td>0.445</td></tr><tr><td>ABTS</td><td>37.563</td><td>43.85</td><td>1.014</td><td>40.254</td><td>41.158</td><td>1.057</td><td>0.001</td><td>0.572</td><td>0.726</td></tr><tr><td>MDA</td><td>0.363</td><td>0.369</td><td>0.018</td><td>0.363</td><td>0.369</td><td>0.015</td><td>0.843</td><td>0.693</td><td>0.687</td></tr><tr><td>PC</td><td>2.916</td><td>2.877</td><td>0.106</td><td>2.925</td><td>2.867</td><td>0.108</td><td>0.801</td><td>0.718</td><td>0.541</td></tr></tbody></table>

<footnote>† SEM = Standard error of the mean. GST: Glutathione transferase. GR: Glutathione reductase. SOD: Superoxide dismutase. GSH-Px: Glutathione peroxidase. CAT: Catalase. ABTS: 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (inhibition%). FRAP: Ferric Reducing Ability of Plasma (µM ascorbic acid). MDA: Malondialdehyde (µM MDA). PCs: Protein carbonyls (nmol/mL). LPO: Lactoperoxidase. Significance level below 0.05 indicates significant difference. * Effect: The dietary treatment (D), time (T), and the interaction between dietary treatment × time (DxT) effects were analyzed by ANOVA using a general linear model (GLM) for repeated measures and post hoc analysis was performed when appropriate using Tukey's multiple range test.</footnote>
<figure><img src="image_30.png" /><figcaption>**Figure 4.** Heat-map represents a Pearson correlation of milk fatty acids, immune system gene expression in monocytes and neutrophils, antioxidant enzymes activities, total antioxidant capacity, and oxidative indices in both blood plasma and milk of ewes. In immune system genes, *M* = monocytes and *N* = neutrophils, while in antioxidants *B* = blood plasma and *M* = milk. CCL5: C-X-C motif chemokine 5, CXCL16: C-X-C motif chemokine ligand 16, INFG: Interferon γ, IL1B: Interleukin-1 beta, IL2: Interleukin-2, IL6: Interleukin-6, IL8: Interleukin-8, IL10: Interleukin-10, TNF: Tumor Necrosis Factor, NFKB: Nuclear Factor kappa B, GST: Glutathione transferase, GR: Glutathione reductase, SOD: Superoxide dismutase, GPx: Glutathione peroxidase, CAT: Catalase, ABTS: 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), FRAP: Ferric Reducing Ability of Plasma, MDA: Malondialdehyde, PCs: Protein carbonyls, LPO: Lactoperoxidase, VA: Vaccenic acid, CLA: Conjugated linoleic acid, SCFAs: Short-Chain saturated fatty acids, MCFAs: Medium-Chain saturated fatty acids, LCFAs: Long-Chain saturated fatty acids, MUFAs: Mono-unsaturated fatty acids, and PUFAs: Poly-unsaturated fatty acids.</figcaption></figure>

3.4. *Immune Status*

The relative transcript levels of both CCL5 and CXCL16 in monocytes of ActiSaf fed ewes were significantly suppressed by 30% (0.053 vs. 0.037, SEM = 0.003, *p* = 0.007 and 0.042 vs. 0.029, SEM = 0.008, *p* = 0.008, respectively) (Table 4). Amongst cytokines, Interleukin 8 (IL8) relative transcript levels were significantly decreased by 80% (0.0020 vs. 0.0004, SEM = 0.0000, *p* = 0.031), while Interleukin 1β (IL1B) showed a tendency to decrease by 43% (0.007 vs. 0.004, SEM = 0.001, *p* = 0.087) in ActiSaf fed ewes (Table 4). A significant downregulation in the relative expression of Interleukin 10 (IL10)
by 30% (0.014 vs. 0.010, SEM = 0.002, *p* = 0.047) in the neutrophils of the treated ewes was observed (Table 4). In addition, the relative transcript levels of *IL1B* in monocytes were positively correlated with the proportions of both MUFA and oleic acid in milk. The same trend was found between the relative expression of the Interleukin 2 gene and the proportion of palmitic acid in milk (Figure 4). Monocytes and neutrophil relative transcript levels of *IL1B* were negatively correlated with GR and Glutathione Transferase (GST) activities in blood plasma, respectively (Figure 4).

<table><caption>**Table 4.** Relative transcript levels of several genes in blood monocytes and neutrophils of ewes fed the two diets (Control, *n* = 8 and ActiSaf, *n* = 8) at two sampling times.</caption><thead><tr><th rowspan="2"></th><th colspan="3">Diets (D)</th><th colspan="3">Sampling Time (T) in Weeks</th><th colspan="3">Effect *</th></tr><tr><th>Control</th><th>ActiSaf</th><th>SEM<sup>†</sup></th><th>3</th><th>6</th><th>SEM<sup>†</sup></th><th>D</th><th>T</th><th>DxT</th></tr></thead><tbody><tr><td colspan="10" style="text-align: center; font-weight: bold;">Monocytes</td></tr><tr><td>CCL5</td><td>0.053</td><td>0.037</td><td>0.003</td><td>0.047</td><td>0.043</td><td>0.004</td><td>0.007</td><td>0.621</td><td>0.030</td></tr><tr><td>CXCL16</td><td>0.042</td><td>0.029</td><td>0.008</td><td>0.037</td><td>0.034</td><td>0.004</td><td>0.008</td><td>0.599</td><td>0.032</td></tr><tr><td>IFNG</td><td>0.009</td><td>0.008</td><td>0.001</td><td>0.006</td><td>0.010</td><td>0.001</td><td>0.301</td><td>0.034</td><td>0.856</td></tr><tr><td>IL1B</td><td>0.007</td><td>0.004</td><td>0.001</td><td>0.007</td><td>0.004</td><td>0.001</td><td>0.087</td><td>0.010</td><td>0.016</td></tr><tr><td>IL2</td><td>0.005</td><td>0.006</td><td>0.001</td><td>0.004</td><td>0.007</td><td>0.001</td><td>0.484</td><td>0.147</td><td>0.837</td></tr><tr><td>IL6</td><td>0.002</td><td>0.002</td><td>0.000</td><td>0.001</td><td>0.002</td><td>0.000</td><td>0.896</td><td>0.061</td><td>0.786</td></tr><tr><td>IL8</td><td>0.0020</td><td>0.0004</td><td>0.0000</td><td>0.0020</td><td>0.0004</td><td>0.0000</td><td>0.031</td><td>0.007</td><td>0.007</td></tr><tr><td>IL10</td><td>0.019</td><td>0.018</td><td>0.002</td><td>0.020</td><td>0.017</td><td>0.002</td><td>0.727</td><td>0.126</td><td>0.383</td></tr><tr><td>TNF</td><td>0.053</td><td>0.048</td><td>0.003</td><td>0.040</td><td>0.061</td><td>0.003</td><td>0.266</td><td>0.000</td><td>0.886</td></tr><tr><td>NFKB</td><td>0.251</td><td>0.245</td><td>0.022</td><td>0.254</td><td>0.242</td><td>0.024</td><td>0.842</td><td>0.727</td><td>0.708</td></tr><tr><td colspan="10" style="text-align: center; font-weight: bold;">Neutrophils</td></tr><tr><td>CCL5</td><td>0.083</td><td>0.079</td><td>0.014</td><td>0.067</td><td>0.095</td><td>0.014</td><td>0.854</td><td>0.179</td><td>0.356</td></tr><tr><td>CXCL16</td><td>0.069</td><td>0.065</td><td>0.012</td><td>0.055</td><td>0.079</td><td>0.012</td><td>0.856</td><td>0.190</td><td>0.355</td></tr><tr><td>IFNG</td><td>0.026</td><td>0.015</td><td>0.007</td><td>0.018</td><td>0.023</td><td>0.005</td><td>0.289</td><td>0.424</td><td>0.719</td></tr><tr><td>IL1B</td><td>0.014</td><td>0.010</td><td>0.002</td><td>0.017</td><td>0.008</td><td>0.002</td><td>0.171</td><td>0.003</td><td>0.022</td></tr><tr><td>IL2</td><td>0.023</td><td>0.021</td><td>0.004</td><td>0.011</td><td>0.033</td><td>0.003</td><td>0.703</td><td>0.003</td><td>0.975</td></tr><tr><td>IL6</td><td>0.007</td><td>0.006</td><td>0.002</td><td>0.001</td><td>0.012</td><td>0.002</td><td>0.821</td><td>0.005</td><td>0.642</td></tr><tr><td>IL8</td><td>0.003</td><td>0.004</td><td>0.001</td><td>0.004</td><td>0.003</td><td>0.001</td><td>0.781</td><td>0.142</td><td>0.119</td></tr><tr><td>IL10</td><td>0.014</td><td>0.010</td><td>0.002</td><td>0.016</td><td>0.008</td><td>0.002</td><td>0.047</td><td>0.007</td><td>0.032</td></tr><tr><td>TNF</td><td>0.298</td><td>0.442</td><td>0.052</td><td>0.262</td><td>0.478</td><td>0.062</td><td>0.075</td><td>0.061</td><td>0.578</td></tr><tr><td>NFKB</td><td>0.182</td><td>0.185</td><td>0.028</td><td>0.239</td><td>0.0128</td><td>0.025</td><td>0.935</td><td>0.018</td><td>0.359</td></tr></tbody></table>

Significance level below 0.05 indicates significant difference. † SEM = Standard error of the mean. CCL5: C-X-C motif chemokine 5. CXCL16: C-X-C motif chemokine ligand 16. INFG: Interferon γ. IL1B: Interleukin-1 beta. IL2: Interleukin-2. IL6: Interleukin-6. IL8: Interleukin-8. IL10: Interleukin-10. TNF: Tumor Necrosis Factor. NFKB: Nuclear Factor kappa B. * Effect: The dietary treatment (D), time (T), and the interaction between dietary treatment × time (DxT) effects were analyzed by ANOVA using a general linear model (GLM) for repeated measures and posthoc analysis was performed when appropriate using Tukey's multiple range test.

### 3.5. Holistic Statistics

Discriminant analysis was applied to pooled data of two sampling times (3rd and 6th week postpartum) according to fatty acids in milk, immune gene expression in both monocytes and neutrophils, and antioxidant indices in blood plasma and milk (Figure 5) to investigate if the samples can be distinguished according to the type of the diet (Control and ActiSaf). The percentages of the samples that were classified into the correct group, according to the dietary treatment, were 100%. Wilks' lambda was observed at 0.001 for Function 1 (*p* = 0.159), while the relative transcript levels of CCL5, CXCL16, and IL6 in monocytes', IL10, IL6, and IL8 in neutrophils, and the GR activity in blood plasma were the variables that contributed the most.
<figure><img src="image_31.png" /><figcaption>**Figure 5.** Discriminant plots separating the Control and ActiSaf fed ewes according to their fatty acid grouped values, immune gene expression in both monocytes and neutrophils, and antioxidant indices in blood and milk.</figcaption></figure>

**4. Discussion**

Blood B-HBA concentrations reflect the magnitude of negative energy balance (NEB) and lipid mobilization and are a diagnostic marker for subclinical (SCK) and clinical ketosis (CK) in ruminants. The B-HBA content in the blood of sheep with SCK ranges from 0.5 to 1.6 mmol/L [39–42], while in those with CK from 1.6 to 7 mmol/L [39,40,43]. However, in the case of healthy pregnant sheep these values could be around 0.8–0.9 mmol/L [44,45]. Nonetheless, the values of the Control group, first and foremost during the prepartum period, may indicate a moderate NEB (0.86 mmol/L). On the other hand, results regarding the B-HBA concentration in the blood of ActiSaf fed ewes indicate an improvement in the energetic status of the animals. It should be underlined here that the prolificacy between the two groups was the same (around 2.35), which means that although the number of fetuses affects B-HBA content [46], it had minimum impact in our trial. The same levels of B-HBA content in blood of healthy ewes of the same breed in early lactation have been previously reported [18,38]. An increase in the host energy availability might be due to a better rumen function and microbiome homeostasis. During the peripartum period, the energy and nutrient demands increase exponentially while the dry matter intake decreases. Thus, the optimal rumen function and the balance between VFA for a maximum feed efficiency are momentous in the transition period. The mechanism underling LY contribution in rumen may be down to yeast’s oxygen scavenging properties (anaerobiosis). Specifically, the improvement of anaerobiosis in rumen increases the bacteria viability and thus, microbial protein synthesis and fiber digestibility [6]. Energy balance might be improved as a result of the dry matter (DM) and NDF digestion enhancement as have been reported by Plata et al. [47]. Furthermore, Panda et al. [48] also found that crude protein (CP) digestibility was also increased by 4.8% with dietary supplementation of yeast culture on male calves. In agreement with our findings, ActiSaf dietary inclusion (5g/day) in early lactating dairy cows, significantly decreased serum B-HBA and non-esterified fatty acids (NEFA) concentrations [9], while in mid-lactating cows, 4 g/day of LY supplementation did not affect B-HBA concentration since animals were not prone to NEB [49]. In addition, ewes in the ActiSaf group recovered their body weight from lambing until the sixth week postpartum in a more efficient manner, considering the increased available energy, as concluded by B-HBA concentration.
In compliance with our findings (12% FCM<sub>6%</sub>, *p* = 0.161), Stella et al. [12], reported a significant increase in goats' milk yield by 14% when their diet was supplemented with *S. cerevisiae*. It is worth mentioning that, in the same study, treated goats showed an upward trend in milk yield after the fourth week postpartum which decreased slower compared to the control group, showing a persistence in milk similar to our study. The dietary inclusion of *S. cerevisiae* enhanced cows' milk yield in early [13] mid- [14] and late [7] lactation. However, Dehghan-Banadaky et al. [49], showed that the milk yield was not affected in *S. cerevisiae* supplemented cows after the 145 DIM, possibly due to the absence of NEB. The results from 22 studies with more than 9039 lactating dairy animals showed an increase in their milk production by 7.3% (ranging from 2 to 30%) when their diets were supplemented with Yea-Sacc®1026 yeast [50].

Interestingly, in a meta-analysis study, Dehghan-Banadaky [49] concluded that an enhancement in milk yield was accompanied by an increase in feed intake in supplemented animals with yeast products. Moreover, yeast administration in prepartum cows' diets improved DMI [51]. Additionally, Habeeb et al. [52] reported that an enhancement of animal performance by the inclusion of yeast in their diet was mainly attributed to an increase in feed intake rather than feed digestibility improvement.

The milk fatty acid's profile was not holistically modified; however, certain interesting results, related to the biohydrogenation process (BH), were unveiled. Julien et al. [53] first observed the impact of LY administration on ruminal biohydrogenation processes. Specifically, LY promotes growth and activity of rumen lactate-utilizing bacteria, such as *Megasphaera elsdenii* or *Selenomonas ruminantium*, *Actinobacteria*, including *Propionibacterium acnes* as well as fibrolytic bacteria. Consequently, LY could be involved at different stages of BH; firstly, by altering biohydrogenating microorganisms, i.e., improving growth of either t11 or t10 isomer producing bacteria, and secondly by modulating the ruminal biotope, i.e., by stabilizing ruminal pH or favoring stronger reducing conditions. In addition, Julien et al. [53] reported that LY supplementation increased the accumulation of trans C<sub>18:1</sub> in vitro and decreased the proportion of C<sub>18:0</sub>, suggesting an inhibition of the last step of BH of C<sub>9c12</sub>-C<sub>18:2</sub> fatty acids. Thus, in our study it could be hypothesized that the improved rumen conditions by LY administration may favor the isomerisation of C<sub>9c12</sub>-C<sub>18:2</sub> and consequently increased the production of intermediate fatty acids in the rumen, which induced an inhibition or a saturation of the enzyme activity of bacteria involved in the second reduction step [54].

During the peripartum period, animals' augmented requirement for energy and nutrient results in lipid mobilization and blood hyperketonemia which induce oxidative stress [55]. Optimizing nutrition requirements by improving rumen efficiency may suppress the concentration of such trigger metabolites and improve the oxidative status. Glutathione reductase (GR) has a central role in the antioxidant defense system since it catalyzes the conversion of oxidized glutathione disulfide to the reduced form of glutathione, which is a critical molecule in resting oxidative stress [56]. It is known that glutathione is extremely important since it acts as substrate or co-substrate in enzymatic reactions (e.g., the glutathione-S-transferase or glutathione-shuttle enzymes), reacts directly with free radicals and lipid peroxides, and protects cells [57]. The mechanism under which GR increased its activity remains unclear, thus our assumptions are oriented toward a prudent liver function where glutathione is de novo synthesized due to the lower B-HBA concentration. Another possible mechanism that can increase GR activity might be the Flavin Adenine Dinucleotides' (FADs) co-substrate. Specifically, yeasts are sources of B-complex vitamins that act as precursors of the essential co-enzymes NAD and FAD that are responsible for biological oxidation [58]. In addition, high genetic merit dairy animals often burden their metabolism since they require increased levels of energy in order to meet their demands, leading to ROS production and later to the annihilation of the milk oxidative stability [59]. Hence, total antioxidant capacity enhancement in the milk of ActiSaf fed ewes may be important for the dairy industry.

Concerning chemokines, CCL-5 is involved in the activation of T cells, macrophages, eosinophils and basophils, and its enhancement is related to an inflammation response [60]. On the other hand, CXCL-16, a transmembrane protein, is detached from the membrane by metalloproteinase
ADAM10 induced chemotaxis [61]. In this study, the downregulation in the relative transcript levels of both CCL5 and CXCL16 in monocytes of ActiSaf treated ewes, indicates a lower inflammatory response during the first 6 weeks of lactation. The IL1B which was suppressed in our study, regulates B-cell maturation and proliferation, activates the Natural Killer (NK) cells and is generally related to the acute manifestation of inflammation in immune cells [62]. Interleukin-8, on the other hand, has a chemotaxis-inducer effect mainly in neutrophils. Pro-inflammatory chemokines and cytokine downregulations is directly attributed to B-HBA mitigation. Specifically, blood ketones derived from ketogenesis through acetyl-CoA metabolization have been shown to act as stimulants in chemokines and cytokines in cows' mammary epithelia cells [63].

**5. Conclusions**

In conclusion, supplementing dairy sheep diets with 2 g of the ActiSaf live yeast/day/ewe during the transition and early lactation periods have a beneficial impact on animals' performance whilst simultaneously portraying an improvement on pro-inflammatory responses attributed to a lower lipomobilization. This overall stress suppression during this turning point for the ruminants' period may unveil the potential of live yeasts as health modulators towards the collective effort of reducing antibiotic dependence at the farm scale. However, further research is needed to deeply understand the mechanism under the enhancement of energy supply in small ruminants.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2309-608X/6/4/334/s1, Table S1. Analysis of variance in blood β-hydroxybutyric acid in prepartum and postpartum period; Table S2. Repeated measure analysis of variance in blood β-hydroxybutyric acid in overall experimental period using prepartum and postpartum sampling time as repeated factor; Table S3. Analysis of variance in body weight at the start of the experiment, at lambing, and at the end; Table S4. Repeated measure analysis of variance in body weight in overall experimental period using the weighing at the start of the experiment, at lambing and at the end, as repeated factor.; Table S5. Analysis of variance in body weight recovery from lambing to 6 weeks postpartum. Table S6. Milk yield and milk chemical composition of ewes fed the Control and ActiSaf diets in the six sampling times; Table S7. Sequences of primers for target genes used in real-time qPCR; Figure S1. Graphical representation of milk chemical composition of the Control and ActiSaf groups.

**Author Contributions:** Conceptualization, G.Z. and V.N.; methodology, A.M., C.M. and C.C.; software, A.M.; validation, A.M., C.M., C.C., V.N. and E.T.; formal analysis, A.M.; investigation, G.Z. and E.T.; resources, V.N.; data curation, A.M. and D.K.; writing—original draft preparation, A.M.; writing—review and editing, E.T., C.C. and G.Z.; visualization, A.M.; supervision, E.T.; project administration, G.Z.; funding acquisition, G.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was partially funded by Phileo Lesaffre Animal Health, Lille, France.

**Acknowledgments:** Authors would like to thank Flessas Dairy Farm for their collaboration.

**Conflicts of Interest:** The authors declare no conflict of interest.

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Article

# Assimilation of Cholesterol by *Monascus purpureus*

Theresa P. T. Nguyen* ◎, Margaret A. Garrahan †, Sabrina A. Nance †, Catherine E. Seeger †
and Christian Wong †

Department of Chemistry & Biochemistry, Loyola University Maryland, Baltimore, MD 21210, USA;
magarrahan@loyola.edu (M.A.G.); sanance@loyola.edu (S.A.N.); ceseeger@loyola.edu (C.E.S.);
ckwong@loyola.edu (C.W.)

* Correspondence: tptnguyen@loyola.edu; Tel.: +1-410-617-2862

† These authors contributed equally to this work.

Received: 31 October 2020; Accepted: 8 December 2020; Published: 9 December 2020

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updates

**Abstract:** *Monascus purpureus*, a filamentous fungus known for its fermentation of red yeast rice, produces the metabolite monacolin K used in statin drugs to inhibit cholesterol biosynthesis. In this study, we show that active cultures of *M. purpureus* CBS 109.07, independent of secondary metabolites, use the mechanism of cholesterol assimilation to lower cholesterol in vitro. We describe collection, extraction, and gas chromatography-flame ionized detection (GC-FID) methods to quantify the levels of cholesterol remaining after incubation of *M. purpureus* CBS 109.07 with exogenous cholesterol. Our findings demonstrate that active growing *M. purpureus* CBS 109.07 can assimilate cholesterol, removing 36.38% of cholesterol after 48 h of incubation at 37 °C. The removal of cholesterol by resting or dead *M. purpureus* CBS 109.07 was not significant, with cholesterol reduction ranging from 2.75–9.27% throughout a 72 h incubation. Cholesterol was also not shown to be catabolized as a carbon source. Resting cultures transferred from buffer to growth media were able to reactivate, and increases in cholesterol assimilation and growth were observed. In growing and resting phases at 24 and 72 h, the production of the mycotoxin citrinin was quantified via high-performance liquid chromatography-ultraviolet (HPLC-UV) and found to be below the limit of detection. The results indicate that *M. purpureus* CBS 109.07 can reduce cholesterol content in vitro and may have a potential application in probiotics.

**Keywords:** *M. purpureus*; red yeast rice; filamentous fungi; cholesterol reduction; probiotic potential

## 1. Introduction

*Monascus purpureus* is a filamentous fungus that produces a variety of secondary metabolites, including pigments, lipids, and monacolins. *M. purpureus* is most widely known for the fermentation of white rice to produce a deep red rice known as angkak or beni koji [1–3]. More commonly, the fermented product is called "red yeast rice", though *Monascus* species are more accurately molds. *M. purpureus* fermented rice is used in food preparation for flavoring, coloring, and preservation, and is also consumed in traditional Chinese medicine to improve ailments of circulation and heart health [2,4–7]. Modern research explored the health claims, and found a plausible cause: *M. purpureus* can synthesize monacolins, naturally occurring compounds capable of decreasing cholesterol levels by inhibiting HMG-CoA reductase, the rate-limiting step in cholesterol biosynthesis [7,8]. The most potent of the *Monascus* monacolins, monacolin K, was isolated and patented as lovastatin and is widely prescribed to treat hypercholesterolemia [8]. Statins are effective treatments for high cholesterol; however, side effects, low tolerance, and the cost of the drug have led patients to pursue alternative options to lower their cholesterol levels [9]. As lyophilized red yeast rice (RYR) supplements emerged as a naturopathic alternative in the U.S., the Food and Drug Administration (FDA) restricted the
market, determining that without standardization and quality control for the amount of monacolin K, RYR was not a dietary supplement, but rather an unauthorized new drug [5,10]. As a result, current U.S. supplements marketed as RYR may contain, at most, only trace amounts of monacolin K [5,10–12]. Nevertheless, several clinical studies report that RYR supplements may be an effective treatment option for hypercholesterolemia as significant reductions in low-density lipoprotein (LDL) cholesterol and total cholesterol levels were observed in patients taking RYR [4,10–14].

Two-thirds of cholesterol required for cell membranes and biosynthesis of steroid hormones and bile acids is endogenously synthesized; however, an excess of cholesterol is a major risk factor for cardiovascular disease [15–17]. The World Health Organization (WHO) projects that by 2030, nearly 23.6 million people will die from cardiovascular disease, which includes heart disease and stroke as the leading causes of death [18]. Research into the efficacy of therapies such as RYR supplements in the treatment of elevated cholesterol levels is ongoing and critical for treating cardiovascular disease.

*M. purpureus* strains administered via RYR supplements in clinical trials often have unspecified viability [19,20]. This led us to ask if living *M. purpureus*, and not simply its secondary metabolites, have cholesterol-lowering properties. Several studies have demonstrated that active probiotic microorganisms introduced into intestinal microbiota can improve the overall lipid content in human blood serum [15,21–25]. Probiotics are defined as live microorganisms that, when administered in adequate amounts, can confer a health benefit on the host [26]. The fermented product of *M. purpureus* can be labeled as “contains live and active cultures,” but clinical studies on the safety, viability, and dosage of live *M. purpureus* strains are still necessary before characterizing strains of *Monascus* as probiotic [26–28]. Additionally, factors like mycotoxins should considered; *Monascus* species, like *Aspergillus* and *Penicillium*, naturally synthesize the cytotoxic citrinin at low levels [29,30]. In the European Union (EU) and US, *Monascus* pigments are prohibited from use in food industries [3,31,32]. Still, many *Monascus* strains are generally regarded as safe (GRAS) in many Asian countries and the commercial interest in fermentation has led to easy access of RYR outside of Asia [6,11,33].

While the cholesterol-lowering benefits of probiotics have been highlighted in in vivo studies, the mechanisms of probiotics remain not fully understood [34–36]. Potential mechanisms have been proposed in vitro, such as the removal of cholesterol from media by the assimilation or uptake of cholesterol by probiotic strains of *Lactobacilli* and *Bifidobacteria* [35,37–41]. The ability of cholesterol removal appears to be growth- and strain-specific, with nearly all research focused on bacterial strains [35–41]. We anticipate that as beneficial fungi are characterized, greater attention will be drawn to the roles of fungi in health, nutrition, and the mycobiome. Currently, *Saccharomyces boulardii* is the only fungus with a strain commercially labeled in the U.S. as a probiotic, as it can survive the passage through the gastrointestinal tract, present good growth at 37 °C, complement treatment of gastrointestinal diseases, and also assimilate cholesterol [37,42–46].

In this study, we evaluated the ability of the fungus *M. purpureus* CBS 109.07 to assimilate cholesterol in vitro. We developed new sample collection methods and used gas chromatography to quantify the levels of cholesterol remaining after incubation with *M. purpureus* CBS 109.07. Our data indicate that this strain of *M. purpureus* is capable of cholesterol assimilation, a cholesterol-lowering mechanism separate from its ability to produce monacolins and which has not been previously reported. The present study does not establish the safety and efficacy of CBS 109.07 as a therapeutic agent, however, these results lay the groundwork for the possibility that active *M. purpureus* CBS 109.07 may have probiotic potential based on its ability to assimilate cholesterol.

## 2. Materials and Methods

### 2.1. Strains and Media Conditions

*M. purpureus* Went teleomorphic type strain CBS 109.07 (ATCC 16365) was obtained from the American Type Culture Collection (ATCC) strain bank and chosen for its ability to grow at 30 °C. *M. purpureus* CBS 109.07 has been used in food-grade studies for human and animal food
applications [6,47,48]. *M. purpureus* was grown in a malt extract media (MEA) containing: 2% soluble Bactomalt extract (BD Bioscience), 2% glucose, 1% peptone, and pH adjusted to pH 7. Plate media contained 2% agar. Phosphate-buffered saline (PBS) solution contained 8.0 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄ for every 1.0 L solution and was pH adjusted to pH 7. Where indicated, PBS was supplemented with 6.72 g/L of yeast nitrogen base without amino acids (BD Difco) or 5 g/L ammonium sulfate, and pH was adjusted to 7 before sterilization. Bile salt supplemented media contained 0.3% (w/v) oxgall (BD Difco).

## 2.2. Submerged Culture Preparation

A sterilized 5 mm cork-borer was used to remove an agar plug of *M. purpureus* CBS 109.07 grown on MEA plate media. The agar plug was subcultured into 4 mL of liquid MEA media and incubated at 30 °C. After four day incubation at 30 °C at 150 rpm, a colorless, spherical fungal pellet was formed. The pellet was transferred into new media with 0.3% oxgall at 37 °C and 60 rpm for growth curve, cholesterol assimilation, and citrinin production experiments.

## 2.3. Cholesterol Assimilation

### 2.3.1. Cholesterol Reagents

A stock solution of cholesterol (Lipids Cholesterol Rich from adult bovine serum; Sigma-Aldrich, St. Louis, MO, USA) at 10 mg/mL was used to prepare cholesterol assimilation assays and to prepare a 6-point calibration curve as described in Section 2.3.6 [37]. A stock solution of 5-α-cholestane (Sigma-Aldrich, St. Louis, MO, USA) at 2.5 mg/mL was used as an internal standard in the lipid extractions.

### 2.3.2. Culture Preparation for Growing, Resting, Dead, and Control Conditions

Growing, resting, and *M. purpureus* control conditions contained *M. purpureus* CBS 109.07 pellets that were homogenized using a sterilized glass duncer in a sterile 50 mL conical tube and divided into replicates. Dead culture conditions contained *M. purpureus* CBS 109.07 pellets that were autoclaved at 121 °C for 20 min under 15 psi pressure and transferred to fresh media. Growing and dead cultures contained 10 mL MEA media in sterile 50 mL borosilicate tubes; resting cultures contained 10 mL of PBS in sterile 50 mL borosilicate tubes; resting cultures supplemented with nitrogen sources contained 10 mL of PBS with ammonium sulfate or 10 mL of PBS with yeast nitrogen base. Cholesterol assimilation and dry weight experiments were supplemented with 0.3% oxgall and incubated at 37 °C and 60 rpm. With the exception of the *M. purpureus* control, all conditions were incubated with 120 µg/mL cholesterol. Media control without *M. purpureus* contained 120 µg/mL cholesterol and 0.3% (w/v) oxgall in 10 mL MEA.

### 2.3.3. Cholesterol Assimilation and Dry Weight Growth Curve

Cholesterol assimilation and dry weight experiments for each growth condition were prepared in triplicate, where three independent sets of culture were homogenized and divided into six sterile 50 mL borosilicate tubes to account for six timepoints (0, 24, 36, 48, 60, and 72 h). At designated time point, a 1.0 mL aliquot of culture supernatant was collected, centrifuged at 2000× g for 15 min, and stored in a 50 mL borosilicate glass tube with a PTFE-lined cap at -20 °C. The remaining 9.0 mL of the culture was then harvested and filtered via vacuum flask and a pre-weighed Whatman filter #1. The contents were allowed to air dry for five days and dry weight was measured on an analytical balance.

### 2.3.4. *M. Purpureus* Dormancy Experiment

Cholesterol assimilation and dry weight experiments were prepared in triplicate, where three independent sets of cultures were homogenized and divided into five sterile 50 mL borosilicate tubes to account for five timepoints (0, 24, 48, 72, and 96 h). *M. purpureus* was incubated in PBS buffer, pH 7
with 0.3% oxgall and 120 µg/mL cholesterol. At designated time points, a 1.0 mL aliquot of culture supernatant was collected, centrifuged at 2000× g for 15 min, and stored in a 50 mL borosilicate glass tube with a PTFE-lined cap at -20 °C. *M. purpureus* samples were then washed twice under sterile conditions with 10 mL of PBS + 0.3% oxgall, and transferred to 10 mL MEA media with 0.3% oxgall and 120 µg/mL cholesterol. A 1.0 mL aliquot of culture supernatant was collected at the starting point (t = 0 h), and also at day 4 and day 7 of incubation in MEA media. At day 7, the culture was harvested and filtered via vacuum flask and a pre-weighed Whatman filter #1. The contents were allowed to air dry for five days and dry weight was measured on an analytical balance.

### 2.3.5. Resting *M. purpureus* Supplemented with Nitrogen Sources Experiment

Cholesterol assimilation and dry weight experiments were prepared in duplicate, where two independent sets of cultures were homogenized and divided into three sterile 50 mL borosilicate tubes to account for three timepoints (0, 24, and 72 h). *M. purpureus* was incubated in PBS buffer supplemented with either ammonium sulfate or yeast nitrogen base without amino acids, pH 7 with 0.3% oxgall and 120 µg/mL cholesterol. At designated time points, a 1.0 mL aliquot of culture supernatant was collected, centrifuged at 2000× g for 15 min, and stored in a 50 mL borosilicate glass tube with PTFE lined cap at -20 °C. *M. purpureus* samples were then washed twice under sterile conditions with 10 mL of PBS + 0.3% oxgall, and transferred to 10 mL MEA media with 0.3% oxgall. At day 4, the culture was harvested and filtered via vacuum flask and a pre-weighed Whatman filter #1. The contents were allowed to air dry for five days and dry weight was measured on an analytical balance.

### 2.3.6. Cholesterol Extraction

Cholesterol assimilation samples were thawed and a stock solution of cholesterol (10 mg/mL) was used to prepare cholesterol standards in a range from 10-150 µg/mL in a borosilicate glass tube. To each sample and standard, 20 µL of 2.5 mg/mL internal standard 5-α-cholestane was added. Direct saponification was carried out on all samples and standards based on the method described by Fletouris et al. [49]. Four millimeters of methanolic 0.5 M KOH solution was added to each tube which was then capped and vortexed for 15 s. The samples and standards were heated for a total of 15 min in an 80 °C water bath, and removed every 5 min to vortex for 10 s. After cooling to room temperature, 4 mL of hexane was added for lipid extraction and vortexed for 1 min. After incubating at room temp for 10 min to permit phase separation, the entire hexane layer of each sample was transferred to a clean test tube. The hexane layer was evaporated using speed vacuum at -109 °C. Dried samples and standards were resuspended in 0.6 mL of hexane and transferred to autosampler vials for gas chromatography (GC) analysis.

### 2.3.7. Gas Chromatography Methods

Cholesterol was determined using gas chromatography (Shimadzu GC-2014, Kyoto, Japan) with a flame ionized detector (FID) and an autosampler [49]. The separation was completed using an SPB-1 column (15 m × 0.32 mm i.d.; film thickness 1.0 mm) (Supelco Inc., Bellefonte, PA, USA) using helium as a carrier gas at a flow rate of 2 mL/min. The oven temperature was set at 285 °C, injection port temperature at 300 °C, and flame ionization detector temperature at 300 °C. The injection volume was 1 µL with a split ratio of 20:1. Matrix effects were addressed by the addition of an internal standard of 5-α-cholestane to all samples and standards. In addition, extracting the standards for each set of experiments using the same process as for the samples helped account for any errors in the preparation process. This allowed for the determination of the limits of detection (LOD) and quantitation (LOQ) for the experimental conditions of 8.31 µg/mL and 27.71 µg/mL, respectively.

### 2.3.8. Calculations for Cholesterol Assimilation

The integrated peak areas for cholesterol and the internal standard 5-α-cholestane were used to determine a 6-point calibration curve for cholesterol and used to extrapolate cholesterol recovered.
The experimental calibration curve to determine LOD and LOQ were created by combining the calibration curves from five experiments to generate a linear calibration curve with R² = 0.9832. Cholesterol assimilated and % cholesterol assimilated were calculated as follows, where Cholesterolᵢ represents cholesterol content recovered at t = 0 and Cholesterolᵣ represents cholesterol content recovered at given time point.

$$ \text{Cholesterol assimilated} = \text{Cholesterol}_i - \text{Cholesterol}_f \qquad (1) $$

$$ \% \text{ Cholesterol assimilated} = \frac{\text{Cholesterol assimilated}}{\text{Cholesterol}_i} \times 100\% \qquad (2) $$

## 2.4. Citrinin Production

### 2.4.1. Citrinin Reagents

Citrinin, high-performance liquid chromatography (HPLC)-grade, was purchased from Sigma-Aldrich. A stock solution of citrinin at 100 µg/mL was prepared in HPLC-grade methanol (J.T. Baker) and used to construct a 7-point calibration curve as described in Section 2.4.3. Acetonitrile and water used in chromatography were HPLC grade (J.T. Baker), and trifluoroacetic acid (TFA) (Sigma-Aldrich, St. Louis, MO, USA) was analytical grade.

### 2.4.2. Culture Preparation and Extraction for Citrinin Production

A 5 mm agar plug of *M. purpureus* was pre-cultured at 150 rpm and 30 °C for 4 days, and transferred to 10 mL of MEA + 0.3% or PBS + 0.3% oxgall and grown at 60 rpm and 37 °C, as described in culture preparations for cholesterol assimilation assays Section 2.3.2. At 24 h, 72 h, and 14 days, cultures were extracted for citrinin as described in Liu and Xu, with some modifications [50]. Briefly, 10 mL cultures were dounced and extracted with 10 mL of ethanol (1:1). Samples were then vortexed for 5 min and sonicated for 20 min. Samples were spun down at 4200× g for 10 min. Supernatant was collected, dried down, and resuspended in 1 mL HPLC-grade methanol. Extraction method was validated with recovery controls, where *M. purpureus* grown in MEA + 0.3% oxgall at 24 h and 72 h were spiked with citrinin at 10 µg/mL and extracted as described previously.

### 2.4.3. High-Performance Liquid Chromatography Methods

Citrinin was determined using high-performance liquid chromatography, HPLC (Agilent 1100 liquid chromatograph) with a diode array detector (DAD) and an autosampler. The separation was completed using a Discovery C18 column (5 µm, 150 × 4.6 mm column) (Supelco Inc., Bellefonte, PA, USA) and an isocratic elution. The mobile phase consisted of acetonitrile:water containing 0.05% TFA and the volume ratio was 35:65 [50,51]. All samples, standards, and solvents were filtered through 0.22 µm membrane filters prior to HPLC analysis. The flow rate was 1 mL/min, and 20 µL sample was injected. The UV-DAD detection was monitored at 254 nm and 334 nm. The integrated peak areas at 334 nm for standard citrinin were used to determine a 7-point calibration curve and used to extrapolate citrinin recovered. The LOD and LOQ for the experimental conditions were 1.11 µg/mL and 3.70 µg/mL, respectively. Recovery of citrinin was determined by dividing citrinin concentration recovered by known citrinin concentration injected.

## 2.5. Statistical Analysis

For cholesterol assimilation and growth curves, growing, resting, and dead *M. purpureus* cultures and controls were conducted in triplicate for each time point. For citrinin assays, growing and resting *M. purpureus* cultures were conducted in duplicate for each time point. All GC-FID and high-performance liquid chromatography-ultraviolet (HPLC-UV) samples were measured in duplicate. Two-way ANOVA was carried out to examine the effect of *M. purpureus* × incubation time interaction
on growing, resting, or dead conditions. Tukey's test was used to compare means. Significance was defined at *p* < 0.05 or *p* < 0.01. Standard deviation is calculated as either absolute error, or percent error through propagation of uncertainty from Equation (2) of Section 2.3.8. All statistical analyses were carried out using GraphPad Prism 8.0.

# 3. Results

## 3.1. Cholesterol Assimilation

The in vitro removal of cholesterol by the filamentous fungi *M. purpureus* CBS 109.07 (hereon referred to in the Results section as *M. purpureus*) was analyzed at 37 °C in media containing 0.3% (*w/v*) oxgall and 120 µg/mL cholesterol. Three growth phases were assessed: growing, where culture is active in MEA media; resting, where culture is dormant in PBS buffer; and dead, where culture has been heat-killed and incubated in MEA media. At indicated time points, an aliquot of spent media was collected from three independent replicates.

After 36 h, *M. purpureus* removed 18.69 µg/mL or 18.78% of the cholesterol in spent media, which is a significant decrease from the initial concentration (Table 1, Figure 1, *p* < 0.01). The rate of cholesterol removal was most dramatic from 36 to 60 h, and cholesterol removed increased from 18.78% to 50.27% (*p* < 0.05). At 72 h, 69.65% cholesterol was removed.

<table><caption>**Table 1.** Cholesterol assimilated in *M. purpureus* CBS 109.07 at different growth phases. All cultures were incubated at 37 °C with 120 µg/mL cholesterol and 0.3% (*w/v*) oxgall bile salts. Cholesterol assimilated was calculated from initial cholesterol, and determined from three independent trials conducted for each growth phase at each time point, and measured in duplicate via gas chromatography-flame ionized detection (GC-FID). Standard deviation calculated is absolute error (µg/mL) and percent error (%).</caption><thead><tr><th rowspan="3">Time (h)</th><th colspan="6">Cholesterol Assimilated</th></tr><tr><th colspan="2">Growing</th><th colspan="2">Resting</th><th colspan="2">Dead</th></tr><tr><th>M. purpureus<br>(µg/mL)</th><th>M. purpureus<br>(%)</th><th>M. purpureus<br>(µg/mL)</th><th>M. purpureus<br>(%)</th><th>M. purpureus<br>(µg/mL)</th><th>M. purpureus<br>(%)</th></tr></thead><tbody><tr><td>0</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>24</td><td>10.89 ± 5.62 <sup>+</sup></td><td>10.91 ± 7.47</td><td>6.78 ± 2.79</td><td>6.07 ± 6.97</td><td>5.68 ± 4.40</td><td>5.59 ± 4.95</td></tr><tr><td>36</td><td>18.69 ± 4.53 <sup>a,+</sup></td><td>18.78 ± 7.03 <sup>a,+</sup></td><td>7.20 ± 3.33 <sup>b</sup></td><td>6.40 ± 6.02 <sup>b</sup></td><td>6.04 ± 4.54 <sup>b</sup></td><td>5.94 ± 5.02 <sup>b</sup></td></tr><tr><td>48</td><td>36.18 ± 3.84 <sup>a,+</sup></td><td>36.38 ± 5.38 <sup>a,+</sup></td><td>10.53 ± 7.94 <sup>b,+</sup></td><td>9.29 ± 7.25 <sup>b</sup></td><td>2.88 ± 4.45 <sup>c</sup></td><td>2.75 ± 4.69 <sup>b</sup></td></tr><tr><td>60</td><td>49.86 ± 5.30 <sup>a,+</sup></td><td>50.27 ± 13.5 <sup>a,+</sup></td><td>4.54 ± 2.48 <sup>b</sup></td><td>4.03 ± 6.03 <sup>b</sup></td><td>4.21 ± 4.63 <sup>b</sup></td><td>4.08 ± 4.71 <sup>b</sup></td></tr><tr><td>72</td><td>69.13 ± 3.95 <sup>a,+</sup></td><td>69.65 ± 12.5 <sup>a,+</sup></td><td>6.70 ± 6.33 <sup>b</sup></td><td>5.91 ± 7.23 <sup>b</sup></td><td>4.44 ± 4.67 <sup>b</sup></td><td>4.33 ± 5.16 <sup>b</sup></td></tr></tbody></table>

In contrast, *M. purpureus* cultures dormant in PBS for resting phase or heat-killed in dead phase removed a negligible amount of cholesterol after 72 h (Table 1) (*p* > 0.05). Non-growing *M. purpureus* conditions removed less than 10% of cholesterol, with a high percent error accounting for the propagation of the standard deviation. The media control containing MEA or PBS incubated without *M. purpureus* similarly showed no significant change in cholesterol content or cholesterol assimilation over 72 h (Table S1). We note that cholesterol content of resting and dead *M. purpureus* and media controls did not change significantly from the initial concentration of cholesterol within each trial (*p* < 0.01).

<footnote><sup>a, b, c</sup> Means within a row are significantly different (*p* < 0.01). <sup>+</sup> Means significantly different from the initial value at *t* = 0 (*p* < 0.01).</footnote>
<figure><img src="image_35.png" /><figcaption>**Figure 1.** Cholesterol assimilated (%) by *M. purpureus* CBS 109.07 at different growth phases. Growing, resting, and dead *M. purpureus* cultures were incubated with 120 µg/mL cholesterol and 0.3% (*w/v*) oxgall bile salts at 37 °C. Malt extract media (MEA) control contained 120 µg/mL cholesterol and 0.3% (*w/v*) oxgall bile salts incubated without *M. purpureus*. Three independent trials were conducted for each condition at each time point, and cholesterol in samples were measured in duplicate via GC-FID. Standard deviation in cholesterol assimilated is percent error.</figcaption></figure>

## 3.2. Growth of *M. purpureus*

The morphology of *M. purpureus* in submerged media is a compact, smooth, and spherical pellet consisting of intertwined hyphae [52–54]. Consistent with studies on other filamentous fungi, we found that dry weight measurement is the most reproducible method of quantifying *M. purpureus* growth [55–61].

After an aliquot of spent media was collected from three independent replicates, the remaining culture was harvested to measure dry weight (Table 2, Figure 2). Growth phases growing, resting, and dead were assessed and conditions included 0.3% oxgall and 120 µg/mL cholesterol. *M. purpureus* control was grown in MEA media without cholesterol. The dry weight of growing *M. purpureus* was significantly different from that of resting or dead *M. purpureus* (*p* < 0.05). The presence of cholesterol did not significantly enhance or inhibit the growth of *M. purpureus* [62] (*p* > 0.05). In both resting and dead conditions, *M. purpureus* had no significant growth (*p* > 0.05).

<table><caption>**Table 2.** Dry weight of *M. purpureus* CBS 109.07. Growing, resting, or dead *M. purpureus* cultures were incubated in 120 µg/mL cholesterol and 0.3% (*w/v*) oxgall bile salts at 37 °C. *M. purpureus* incubated without cholesterol served as a control. Three independent trials, corresponding to samples used for cholesterol assimilation, were conducted for each growth phase at each time point.</caption><thead><tr><th rowspan="2">Time (h)</th><th colspan="4">Dry Weight (mg)</th></tr><tr><th>Growing<br><i>M. purpureus</i></th><th>Resting<br><i>M. purpureus</i></th><th>Dead<br><i>M. purpureus</i></th><th><i>M. purpureus</i><br>without Cholesterol</th></tr></thead><tbody><tr><td>0</td><td>17.54 ± 2.5</td><td>18.40 ± 1.5</td><td>18.60 ± 1.3</td><td>19.91 ± 2.9</td></tr><tr><td>24</td><td>30.77 ± 4.9 <sup>a,+</sup></td><td>24.17 ± 2.0 <sup>a</sup></td><td>21.17 ± 1.5 <sup>b</sup></td><td>37.05 ± 0.5 <sup>c,+</sup></td></tr><tr><td>36</td><td>40.77 ± 0.9 <sup>a,+</sup></td><td>21.07 ± 3.5<sup>b</sup></td><td>16.93 ± 5.6 <sup>b</sup></td><td>40.45 ± 4.6 <sup>a,+</sup></td></tr><tr><td>48</td><td>45.40 ± 4.2 <sup>a,+</sup></td><td>22.93 ± 0.5 <sup>b</sup></td><td>25.00 ± 3.6 <sup>b,+</sup></td><td>43.72 ± 2.5 <sup>a,+</sup></td></tr><tr><td>60</td><td>45.17 ± 2.7 <sup>a,+</sup></td><td>24.60 ± 3.3 <sup>b</sup></td><td>24.17 ± 1.3 <sup>b</sup></td><td>50.87 ± 5.6 <sup>a,+</sup></td></tr><tr><td>72</td><td>48.20 ± 3.9 <sup>a,+</sup></td><td>22.27 ± 1.7 <sup>b</sup></td><td>21.50 ± 2.2 <sup>b</sup></td><td>54.63 ± 1.8 <sup>a,+</sup></td></tr></tbody></table>

<footnote><sup>a, b, c</sup> Means within a row are significantly different (*p* < 0.05). <sup>+</sup> Means significantly different from the initial value at *t* = 0 (*p* < 0.05).</footnote>
<figure><img src="image_36.png" /><figcaption>**Figure 2.** Dry weight of *M. purpureus* CBS 109.07. Growing, resting, or dead *M. purpureus* cultures were incubated in 120 µg/mL cholesterol and 0.3% (*w/v*) oxgall bile salts at 37 °C. *M. purpureus* incubated without cholesterol served as a control. Three independent trials were conducted for each growth phase at each time point. Standard deviation is represented by error bars.</figcaption></figure>

### 3.3. Reactivating Dormant *M. purpureus*

*M. purpureus* incubated in PBS buffer, pH 7 with 0.3% oxgall and 120 µg/mL cholesterol does not assimilate cholesterol during a 96 h incubation (Table 3). To examine if resting conditions correspond to a dormant *M. purpureus*, cultures incubated in PBS were washed and transferred to MEA media with 0.3% oxgall and 120 µg/mL cholesterol. After four days of incubation in MEA, cholesterol assimilation was measured, but was not significantly different than MEA media control (*p* > 0.05). After seven days of incubation, cholesterol assimilation was initiated and cholesterol content is comparable to growing phase *M. purpureus* (Table S1). Dry weight of rescued *M. purpureus* was increased from resting *M. purpureus* (Table 2). The length of time incubated in PBS before the transfer to MEA did not have a significant effect on the ability to assimilate cholesterol (*p* < 0.05).

<table><caption>**Table 3.** Cholesterol content (µg/mL) and dry weight (mg) of *M. purpureus* CBS 109.07 incubated in phosphate-buffered saline (PBS) and rescued in MEA. All cultures were incubated at 37 °C with 120 µg/mL cholesterol and 0.3% (*w/v*) oxgall bile salts. Resting *M. purpureus* was washed in PBS + 0.3% oxgall and then transferred to MEA with 0.3% oxgall and 120 µg/mL cholesterol, and sample collected after 4 and 7 days. Data were determined from three independent trials conducted at each time point. Cholesterol content was measured in duplicate via GC-FID. Standard deviation in cholesterol content is absolute error.</caption><thead><tr><th colspan="2">PBS <sup>1</sup></th><th colspan="2">Day 4 Incubation in MEA <sup>1</sup></th><th colspan="2">Day 7 Incubation in MEA <sup>1</sup></th></tr><tr><th>Time (h)</th><th>Cholesterol Content (µg/mL)</th><th>Time <sup>2</sup> (h)</th><th>Cholesterol Content (µg/mL)</th><th>Cholesterol Content (µg/mL)</th><th>Dry Weight (mg)</th></tr></thead><tbody><tr><td>24</td><td>114.46 ± 3.73 <sup>a</sup></td><td>24</td><td>97.73 ± 2.66 <sup>b</sup></td><td>38.83 ± 9.95 <sup>c,+</sup></td><td>32.87 ± 2.9 <sup>++</sup></td></tr><tr><td>48</td><td>118.30 ± 3.12 <sup>a</sup></td><td>48</td><td>97.12 ± 4.40 <sup>b</sup></td><td>40.88 ± 9.51 <sup>c,+</sup></td><td>31.70 ± 2.7 <sup>++</sup></td></tr><tr><td>72</td><td>117.36 ± 6.84 <sup>a</sup></td><td>72</td><td>98.36 ± 2.10 <sup>b</sup></td><td>37.24 ± 11.47 <sup>c,+</sup></td><td>31.60 ± 4.9 <sup>++</sup></td></tr><tr><td>96</td><td>114.81 ± 3.86 <sup>a</sup></td><td>96</td><td>98.63 ± 8.19 <sup>b</sup></td><td>52.23 ± 5.30 <sup>c</sup></td><td>25.33 ± 2.0</td></tr></tbody></table>

<footnote n="1">¹ Media contained 0.3% oxgall and 120 µg/mL cholesterol. ² Time corresponds to duration incubated in PBS prior to rescue in MEA media. a, b, c Cholesterol content means within a row are significantly different (*p* < 0.05). + Means significantly different from the MEA media control Table S1 (*p* < 0.05). ++ Means significantly different from the resting *M. purpureus* dry weight Table 2 (*p* < 0.05).</footnote>
To determine if cholesterol is catabolized by *M. purpureus* as a carbon source, resting phase cultures in PBS were incubated with a nitrogen source, either ammonium sulfate or yeast nitrogen base without amino acids, at concentrations found in minimal media. We observed that resting *M. purpureus* in PBS supplemented with nitrogen sources had no significant cholesterol assimilation throughout a 24 and 72 h incubation (Table 4, *p* > 0.05).

<table><caption>**Table 4.** Cholesterol content (µg/mL) of *M. purpureus* CBS 109.07 incubated in PBS with nitrogen sources. All cultures were incubated at 37 °C with 120 µg/mL cholesterol and 0.3% (*w/v*) oxgall bile salts. Cholesterol content was determined from two independent trials conducted for each growth phase at each time point, and measured in duplicate via GC-FID. All means were not significantly different from the initial value at t = 0 (*p* < 0.05). Standard deviation in cholesterol content is absolute error.</caption><thead><tr><th colspan="3">Cholesterol Content of <i>M. purpureus</i> Incubated in PBS with 0.3% Oxgall (µg/mL)</th></tr><tr><th>Time (h)</th><th>With Yeast Nitrogen Base without Amino Acids</th><th>With Ammonium Sulfate</th></tr></thead><tbody><tr><td>0</td><td>105.82 ± 2.20</td><td>105.46 ± 4.59</td></tr><tr><td>24</td><td>97.91 ± 2.03</td><td>89.64 ± 2.59</td></tr><tr><td>72</td><td>97.57 ± 1.60</td><td>93.45 ± 8.03</td></tr></tbody></table>

Fungi samples were then washed twice and transferred to MEA + 0.3% oxgall. The dry weight at day 4 in MEA of *M. purpureus* previously incubated in yeast nitrogen base was a significant increase from the initial weight before rescue (Table 5, *p* < 0.05), supporting the observation that dormant *M. purpureus* can be reactivated.

<table><caption>**Table 5.** Dry weight (mg) of *M. purpureus* CBS 109.07 after incubation in PBS with 120 µg/mL cholesterol and 0.3% (*w/v*) oxgall bile salts and rescued in MEA with 0.3% (*w/v*) oxgall bile salts. *M. purpureus* is washed in PBS + 0.3% oxgall and then transferred to MEA with 0.3% oxgall, and sample collected after 4 days. Two independent trials, corresponding to samples used in PBS cholesterol assimilation, were conducted for each growth phase at each time point. Standard deviation in cholesterol content is absolute error.</caption><thead><tr><th colspan="5">Dry Weight of <i>M. purpureus</i> (mg)</th></tr><tr><th rowspan="2">Time <sup>1</sup> (h)</th><th colspan="2">PBS + 0.3% Oxgall</th><th colspan="2">Day 4 Incubation in MEA + 0.3% Oxgall</th></tr><tr><th>With Yeast Nitrogen Base</th><th>With Ammonium Sulfate</th><th>With Yeast Nitrogen Base</th><th>With Ammonium Sulfate</th></tr></thead><tbody><tr><td>0</td><td>19.4 ± 3.1 *</td><td>18.8 ± 3.1 *</td><td></td><td></td></tr><tr><td>24</td><td></td><td></td><td>37.5 ± 2.8 <sup>+</sup></td><td>34.7 ± 3.3 <sup>+</sup></td></tr><tr><td>72</td><td>25.8 ± 5.3</td><td>23.3 ± 3.2</td><td>39.3 ± 0.4 <sup>+</sup></td><td>30.8 ± 1.8 <sup>+</sup></td></tr></tbody></table>

### 3.4. Citrinin Production in *M. purpureus*

The production of the mycotoxin citrinin was measured in *M. purpureus* grown under conditions used in cholesterol assimilation assays, where a 5 mm agar plug of *M. purpureus* was precultured in MEA at 150 rpm and 30 °C and then transferred to fresh media with oxgall and incubated at 60 rpm and 37 °C (Table 6). Samples collected at 24 h and 72 h did not have citrinin production above the limit of detection. The culture broth in these samples were also colorless. After 14 days, *M. purpureus* grown in MEA + 0.3% oxgall began to produce red pigment and culture broth turned reddish. *M. purpureus* in resting phase did not become pigmented. Though 14 days is outside the incubation period for this study's cholesterol assimilation experiments, we extracted the red cultures, and measured 6.77 ± 1.02 µg citrinin per mL of culture broth. To validate extraction methods and show

<footnote n="1">¹ Time corresponds to duration incubated in PBS prior to rescue in MEA media. * Initial weight at t = 0.</footnote>

<footnote>⁺ Means significantly different from the initial weight at t = 0 (*p* < 0.05).</footnote>
effectiveness, a set of *M. purpureus* cultures grown in MEA + 0.3% oxgall at 24 h and 72 h were spiked with citrinin at 10 µg/mL and extracted. Recovery of citrinin was 72.9% ± 3.8.

<table><caption>**Table 6.** Citrinin production under experimental conditions. Citrinin concentration (µg/mL) of *M. purpureus* CBS 109.07 under experimental conditions of growing and resting phases was measured at 24 and 72 h via HPLC-UV. Two independent trials were conducted for each time point. Standard deviation in cholesterol content is absolute error.</caption><thead><tr><th colspan="4">Citrinin Production of <i>M. purpureus</i> (µg Citrinin/mL of Culture Broth)</th></tr><tr><th>Media Conditions</th><th>After 24 h <sup>1</sup></th><th>After 72 h <sup>1</sup></th><th>After 14 Days</th></tr></thead><tbody><tr><td>MEA + 0.3% oxgall</td><td>ND</td><td>ND</td><td>6.77 ± 1.02</td></tr><tr><td>PBS + 0.3% oxgall</td><td>ND</td><td>ND</td><td></td></tr></tbody></table>

## 4. Discussion

As a natural source for monacolins, fermentation products of *Monascus purpureus* are widely used as alternative treatments for hypercholesterolemia. To the best of our knowledge, our data is the first to show that a strain of *M. purpureus* is capable of a cholesterol-lowering mechanism separate from its ability to produce monacolins and other secondary metabolites. We observed that active growing *M. purpureus* CBS 109.07 can assimilate cholesterol in vitro, and after 48 h incubation at 37 °C and high bile salt conditions, 36.38% of cholesterol content was removed. The removal of cholesterol by resting or dead *M. purpureus* CBS 109.07 was not statistically significant, and cholesterol was not catabolized as a carbon source. When resting cultures were washed and transferred to MEA media, *M. purpureus* CBS 109.07 became active and cholesterol assimilation and growth were observed. Citrinin production of *M. purpureus* CBS 109.07 incubated in growing or resting phase conditions at 24 h and 72 h was lower than the limit of detection, and we note that CBS 109.07 produced citrinin under our experimental conditions at day 14 when red pigment production was observed.

The ability of microorganisms to remove cholesterol in vitro from growth media is an indicator of probiotic potential, and the range of reduction percentage is wide and dependent on strain. We note that as with any therapeutic dosage, the concentration of microorganisms present will play a major role in cholesterol assimilation percentage. Miremadi et al. tested strains of *Lactobacilli* and *Bifidobacteria* and found 14 strains capable of removing cholesterol with a range of 34–65% assimilation after 24 h [41]. Eukaryotes capable of lowering cholesterol include strains of *S. boulardii*, *S. cerevisiae*, and *I. orientalis*, which after 48 h of incubation was observed to assimilate 90.6%, 96.8%, and 88.1% of cholesterol, respectively [37]. Strains of *P. kudriazevii*, *Galactomyces* sp., and *Y. lipolytica* were observed to assimilate 45.7%, 36.3%, and 30.9% of cholesterol, respectively, after 48 h of incubation in Chen et al. [45]. In the same study, the commercially available yeast probiotic *S. boulardii* lowered cholesterol by 36.5% at 48 h, and 41.5% cholesterol at 72 h. In this study, active growing *M. purpureus* CBS 109.07 was comparable to *S. boulardii* and was able to lower cholesterol from the media by 36.38% at 48 h, and 69.65% cholesterol at 72 h (Table 1). We note that at higher aeration and agitation, *M. purpureus* CBS 109.07 was able to assimilate a higher percentage of cholesterol (Table S2). When *M. purpureus* CBS 109.07 is resting or dead, cholesterol removal is not significant (Figure 1) and ranged from 2.75–9.29% removal after 72 h of incubation (Table 1). Other studies observed similar trends where resting and heat-killed cultures did not significantly reduce cholesterol [41,63–65], suggesting a mechanism where actively growing strains are more efficient at removing cholesterol.

To eliminate the possibility that cholesterol assimilation by *M. purpureus* was an artifact of starvation and the uptake of available carbon sources, we allowed cultures to incubate undisturbed until the entire culture was collected at the designated time point. This procedure differed from other cholesterol assimilation studies, where one-tenth of the culture volume was removed at each time point and, thus, could significantly impact nutrient availability [38,39,41,63,65–67]. We note that in our

<footnote n="1">¹ ND is not detected with a peak below the limit of detection (LOD) of 1.11 µg/mL.</footnote>
methods, the presence of cholesterol at 120 µg/mL did not enhance or inhibit the growth of *M. purpureus* CBS 109.07 as measured by dry weight (Figure 2).

We investigated the ability of *M. purpureus* CBS 109.07 to transition out of microbial dormancy after one to four days of incubation in PBS. Resting phase cultures incubated in PBS with 120 µg/mL cholesterol did not show significant cholesterol assimilation (Table 3). When washed and transferred to MEA media with 120 µg/mL cholesterol, previously resting phase *M. purpureus* CBS 109.07 cultures were able to restore cholesterol assimilation and growth at day 7 of rescue (Table 3) at levels comparable to growing cultures (Table S1, Table 2). There was no significant difference in reactivation of cholesterol assimilation between cultures that were incubated for one day or four days in PBS. We also observed that *M. purpureus* CBS 109.07 incubated in PBS with cholesterol and supplemented with nitrogen sources showed insignificant cholesterol assimilation between 24 to 72 h (Table 4), and were able to grow after transfer into MEA media for four days (Table 5). These results on resting phase reveal that *M. purpureus* CBS 109.07 incubated in PBS is indeed dormant and that cholesterol is not significantly taken up as a carbon source during dormancy. Additionally, the absence of cholesterol assimilation in PBS with nitrogen sources supports the assertion that *M. purpureus* CBS 109.07 does not metabolize cholesterol (Table 4). We posit that such an absence of cholesterol removal may reflect a mechanism where growing *M. purpureus* CBS 109.07 is more efficient at assimilating cholesterol.

Microorganisms can utilize the cholesterol-lowering mechanisms of active assimilation and passive adhesion to decrease host absorption of intestinal cholesterol [21,36,38]. Our results suggest that *M. purpureus* CBS 109.07 is capable of an in vitro active assimilation mechanism by growing cells. The dense pellet morphology of *M. purpureus* CBS 109.07 has made it difficult to measure the cholesterol content of the cell membrane, as similarly noted in biosorbent studies on other filamentous fungi such as *Aspergillus niger* and *Penicillium* sp. L1 strains [55-57]. Follow-up experiments will be conducted to lyse the *Monascus* membrane and examine membrane cholesterol content. Other cholesterol-lowering mechanisms by probiotic microorganisms include modulation of lipid metabolism and deconjugation of bile salts [36]. *M. purpureus* is capable of directly modulating lipid metabolism, as it synthesizes monacolins that directly inhibit HMG-CoA reductase, the committed step of cholesterol biosynthesis in the liver [7,8]. In future studies, we will assay *M. purpureus* CBS 109.07 for bile salt hydrolase (BSH) activity, the enzyme responsible for the deconjugation of bile salts found in many probiotic strains [68,69].

Like many strains within *Monascus*, *Aspergillus*, and *Penicillium* genera, *M. purpureus* CBS 109.07 can biosynthesize citrinin, with levels highly dependent on the growth conditions and amount of microorganisms used. In this study, the citrinin production in *M. purpureus* CBS 109.07 under growing and resting phase conditions replicated from our cholesterol assimilation experiments was below our limit of detection of 1.11 µg/mL (Table 6). Our results at 24 h and 72 h are consistent with published studies on other *M. purpureus* strains which measured citrinin production in different growth conditions over time. These studies observed delays in citrinin production, with detection of citrinin beginning as early as day 5 or late as day 10 [70-72]. Notably, the commencement of citrinin production corresponded to the commencement of red pigment production, and increases in agitation and aeration increased citrinin production [72,73]. *M. purpureus* CBS 109.07 studies in particular did not measure citrinin at early time points. However using thin-layer chromatography (TLC), they reported citrinin level to be 5 µg/mL after 14 day incubation in glucose media and unspecified agitation [30], and 65 µg/mL after 7 day incubation in ethanol media and 220 rpm agitation [74,75]. We used HPLC-UV to quantify citrinin production after 14 day incubation in MEA + 0.3% oxgall and 60 rpm. At day 14 under our conditions, the culture broth began to turn reddish, and we measured a citrinin concentration of 6.77 µg/mL [30,74-76]. The differences in citrinin production between CBS 109.07 studies highlight how critical growth conditions are to the control of citrinin levels in *Monascus* strains [77,78]. In future studies, we can target the citrinin issue as many studies have successfully eliminated or reduced the levels of citrinin by disrupting the citrinin biosynthetic genes *pksCT* or *ctnA* in *M. purpureus* [70,79-81].
To be beneficial for human health, probiotic microorganisms must be capable of surviving transit through the human gastrointestinal tract. Absorption of dietary cholesterol into the bloodstream occurs predominantly in the duodenum of the small intestine, where the pH varies from pH 6 to 7 and bile salts excreted from the bile duct assist in solubilizing cholesterol [82,83]. *M. purpureus* CBS 109.07 was cultured in media at physiological temperature and pH, and with a high bile salt concentration and low aeration and agitation. However we recognize the limitations of an in vitro study in reproducing gastrointestinal conditions. Additionally, the clinical safety of *M. purpureus* CBS 109.07 needs to be established—a potentially complicated issue if the restrictive regulations on *Monascus* pigments and red yeast rice supplements by the FDA and European Food Safety Authority are any indication [4,5,31,84]. In the current study, we are only beginning to raise the possibility of an application for *M. purpureus* CBS 109.07 in probiotics; we recognize that additional safety and gastrointestinal survival experiments are required, and that such advancement in understanding *Monascus* biochemistry may improve the restrictions on their usage in the US and EU [85]. We also recognize that other candidate strains of *M. purpureus* or other *Monascus* species may be found [86], and that CBS 109.07 may not be unique or exemplary. However, we note that the human consumption of *M. purpureus* CBS 109.07 has precedents, as several food-grade studies have considered CBS 109.07 an edible filamentous fungus and used it as the representative *Monascus* strain in human and animal food applications of mycoprotein [6,47,48].

**5. Conclusions**

Our findings demonstrate that *M. purpureus* CBS 109.07, which can biosynthesize statin-like monacolins, can also reduce cholesterol content in vitro via a mechanism of cholesterol assimilation at 37 °C with a high concentration of bile salts. The most effective removal of cholesterol occurred in growing *M. purpureus* CBS 109.07 cultures, while non-growing *M. purpureus* CBS 109.07 minimally adhered to cholesterol and did not metabolize cholesterol. Dormant cultures, once transferred from buffer to nutrient rich media, were able to be resume cholesterol assimilation at levels observed in active cultures. Citrinin production under our experimental conditions was not detected. Our results show that it is valuable to continue examining the cholesterol-lowering potential of active *M. purpureus* CBS 109.07 cultures, as further research may provide a possible insight in the treatment of hypercholesterolemia and will draw attention to the significance of filamentous fungi in human health and nutrition.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2309-608X/6/4/352/s1, Table S1: Cholesterol content of *M. purpureus* CBS 109.07, Table S2. Cholesterol content, cholesterol assimilated, and dry weight of growing *M. purpureus* CBS 109.07.

**Author Contributions:** Conceptualization, T.P.T.N.; methodology, T.P.T.N.; validation, T.P.T.N.; M.A.G.; S.A.N.; C.E.S.; C.W.; formal analysis, T.P.T.N.; investigation, T.P.T.N.; M.A.G.; S.A.N.; C.E.S.; C.W.; resources, T.P.T.N.; data curation, T.P.T.N.; M.A.G.; S.A.N.; C.E.S.; C.W.; writing—original draft preparation, T.P.T.N.; writing—review and editing, T.P.T.N.; M.A.G.; S.A.N.; C.E.S.; C.W.; visualization, T.P.T.N.; supervision, T.P.T.N.; project administration, T.P.T.N.; funding acquisition, T.P.T.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We thank the members of the Chemistry & Biochemistry Department and the Natural & Applied Sciences College at Loyola University Maryland for support to carry out this project. We are especially grateful to Elizabeth E. Dahl for assistance with GC-FID and quantitative and statistical analyses, Courtney J. Hastings for assistance with HPLC-UV, Heather R. Schmidt for assistance with materials, and Timothy J. McNeese for helpful discussions.

**Conflicts of Interest:** The authors declare no conflict of interest.

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Article

# Exploring the Antibacterial Activity of *Pestalotiopsis* spp. under Different Culture Conditions and Their Chemical Diversity Using LC-ESI-Q-TOF-MS

Madelaine M. Aguilar-Pérez <fnref n="1" />,†, Daniel Torres-Mendoza <fnref n="1" />,<fnref n="2" />,†✉, Roger Vásquez <fnref n="1" />, Nivia Rios <fnref n="3" /> and Luis Cubilla-Rios <fnref n="1" />,*✉

Received: 16 July 2020; Accepted: 17 August 2020; Published: 19 August 2020

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**Abstract:** As a result of the capability of fungi to respond to culture conditions, we aimed to explore and compare the antibacterial activity and chemical diversity of two endophytic fungi isolated from *Hyptis dilatata* and cultured under different conditions by the addition of chemical elicitors, changes in the pH, and different incubation temperatures. Seventeen extracts were obtained from both *Pestalotiopsis mangiferae* (*man*-1 to *man*-17) and *Pestalotiopsis microspora* (*mic*-1 to *mic*-17) and were tested against a panel of pathogenic bacteria. Seven extracts from *P. mangiferae* and four extracts from *P. microspora* showed antibacterial activity; while some of these extracts displayed a high-level of selectivity and a broad-spectrum of activity, *Pseudomonas aeruginosa* was the most inhibited microorganism and was selected to determine the minimal inhibitory concentration (MIC). The MIC was determined for extracts *man*-6 (0.11 µg/mL) and *mic*-9 (0.56 µg/mL). Three active extracts obtained from *P. mangiferae* were analyzed by Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry (LC-ESI-Q-TOF-MS) to explore the chemical diversity and the variations in the composition. This allows us to propose structures for some of the determined molecular formulas, including the previously reported mangiferaelactone (1), an antibacterial compound.

**Keywords:** Endophytic fungi; *Hyptis dilatata*; *Pestalotiopsis mangiferae*; *Pestalotiopsis microspora*; chemical elicitors; antibacterial activity; LC-ESI-Q-TOF-MS

## 1. Introduction

The World Health Organization (WHO; Geneva, Switzerland) has established an urgent pathogen list of antibiotic-resistant bacteria to guide the research, discovery, and development of new antibiotics. This list includes carbapenem-resistant *Pseudomonas aeruginosa* and *Enterobacteriaceae* and third generation cephalosporin-resistant bacteria as critical priorities as a result of the continuous and indiscriminate use of antibiotics, not only in the treatment of human diseases, but also in animals [1,2]. This list includes antifungal compounds [3].

Pharmaceutical conglomerates have abandoned this field of research due to the high costs. Despite the efforts made in recent years, e.g., investment in research and development (R&D) as well as in scientific and technological research, the strategies for the search of new antibiotics and antifungals

<footnote n="1">¹ Laboratory of Tropical Bioorganic Chemistry, Faculty of Natural, Exact Sciences and Technology, University of Panama, Panama 0824, Panama; mad25aguilar@gmail.com (M.M.A.-P.); dtorresm.507@gmail.com (D.T.-M.); royi071123@gmail.com (R.V.)</footnote>

<footnote n="2">² Vicerrectoría de Investigación y Postgrado, University of Panama, Panama 0824, Panama</footnote>

<footnote n="3">³ Department of Microbiology, Faculty of Natural, Exact Sciences and Technology, University of Panama, Panama 0824, Panama; toxogondii@gmail.com</footnote>

<footnote>* Correspondence: luis.cubilla@up.ac.pa; Tel.: +507-6676-5824</footnote>

<footnote>† These authors contributed equally to this work.</footnote>
remain uncertain [4,5]. In this context, natural products produced by endophytic fungi provide an
alternative to supply new molecules with antimicrobial activities [6–9].

Endophytic fungi spend a large part of their life cycle inside the tissue of the host organism
without causing apparent damage [10]. In the last 15 years, interest in endophytic fungi has grown
exponentially because of their ability to produce a wide range of secondary metabolites with diverse
and important biological activities. Plant endophytes are considered one of the least studied groups of
microorganisms and have proven to be a source of natural products and therefore provide a way to
discover novel compounds with biological activities [11,12]. Two species of the genus *Pestalotiopsis*,
isolated from *Hyptis dilatata* (Labiatae), a plant that is distributed in the north and east of the Republic
of Panama and that is known for producing abietane and pimarane diterpenes [13] were selected for
these studies.

The genus *Pestalotiopsis* is considered a vast source of natural products from which more than
300 compounds have been isolated and characterized, including terpenoids, polyketides, chromones,
quinones, coumarins, lactones, and nitrogen-containing molecules with a wide range of biological
activities such as antifungal, antibacterial, anticancer, antioxidant, antiparasitic, antihypertensive,
anti-inflammatory, and neuroprotective activities [14–19]. Previously, our group reported the isolation
of a set of eleven compounds (see Figure S1) from the crude extract of *P. mangiferae*, including
a polyhydroxylated macrolide named mangiferaelactone [20]; the crude extract showed growth
inhibition against *Listeria monocytogenes* (29 mm diameter inhibition zone), and showed a minimal
inhibitory concentration (MIC) of 1.69 mg/mL and 0.55 mg/mL against *L. monocytogenes* and *Bacillus
cereus*, respectively. This compound belongs to the nonalide class that is associated with important
biological activities such anticancer, antifungal, antibacterial, and antiviral activities. Its synthesis has
been developed by different research groups [21–23]. Compounds such as taxol, isopestacin, and some
polyketides have been isolated from *P. microspora* [24–27].

The production of secondary metabolites by microorganisms could be impacted by environmental
dynamics, such as growing conditions, which include biotic and abiotic factors [28]. Therefore,
the selective variation of these parameters during the cultivation of fungi [29,30] and/or the induction
of stress through competition with other microorganisms in a co-culture represent interesting ways to
generate greater activity, chemical diversity, and novel active molecules [31–33]. Hence, the opportunity
to modify culture conditions allows for the optimization of secondary metabolite production [34].
Therefore, given the growing interest in enhancing the production of secondary metabolites by
endophytic fungi, the study of the methods and strategies to stimulate the gene clusters responsible
for the biosynthesis of new molecules has been intensified and could include chemical or physical
factors [35,36]. For example, the use of metallic ions, organic and inorganic compounds, pH,
and incubation temperature to optimize the production of enzymes or secondary metabolites have
been described [37–47].

For this study, we focused on modifying the conditions of the culture medium by varying abiotic
parameters and through this, activate fungal silent gene clusters [48–50] in *P. mangiferae* Hd08 and
*P. microspora* Hd18 in order to increase the chemical diversity and to detect new antibacterial activities.

**2. Materials and Methods**

**2.1. *Chemicals and Reagents***

All of the following chemicals were acquired from Sigma-Aldrich® (Sigma-Aldrich, St. Louis, MO, USA): arginine, glutamic acid, CuSO₄, CaCl₂, FeSO₄, tri-sodium citrate dihydrate, dimethyl sulfoxide (DMSO), and formic acid (FA). Ethyl acetate, acetone, and methanol used for extraction were American Chemical Society grade (Tedia®, Tedia Company Inc., Fairfield, OH, USA). The methanol for the liquid chromatography-mass spectrometry (LC-MS) analysis was LC-MS grade (J. T. Baker®, Avantor Performance Materials, Inc., Center Valley, PA, USA).
## 2.2. Isolation and Identification of Fungal Isolates

A healthy specimen of *Hyptis dilatata* (Labiatae) was collected in La Mesa, Veraguas Province in the Republic of Panama, in November 2010. An exsiccate from the plant material was deposited in the Herbarium of the University of Panama (PMA 084861). Mature leaves were surface sterilized as we previously reported in [51], and small fragments were cultured on 2% malt-extract agar (MEA; Difco™, Becton, Dickinson and Co., Sparks, MD, USA) under sterile conditions. Strains Hd08 and Hd18 were further isolated from the collection plate and successively re-plated until pure strains were obtained. Pure fungal strains were stored at -80 °C in a cryoprotectant solution of 10% glycerol and were preserved in the collection of the International Cooperative Biodiversity Group (ICBG) at the University of Panama. The identification of endophytic fungi was carried out as described previously [20]. Briefly, the total genomic DNA of each strain was isolated from fresh mycelium following U'Ren et al. [52]. Polymerase chain reaction (PCR) was used to amplify the nuclear ribosomal internal transcribed spacers and 5.8s gene (ITS rDNA; ca. 600 bp), and the first ca. 600 bp was sequenced bidirectionally [52]. The entire sequences for each strain were compared to the nucleotide database of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tools (BLAST®) Website.

## 2.3. Media Preparation and Cultivation of Fungal Strains

Strains Hd08 and Hd18 were reactivated aseptically on Petri dishes containing potato dextrose agar (PDA; Difco™, Becton, Dickinson and Co., Sparks, MD, USA) and incubated at 26 °C for seven days. Then, the mycelium was removed using a sterile spatula and was placed in sterile water to obtain a homogeneous solution. This solution was poured on the surface of Petri dishes (145 × 90 mm) containing MEA for all of the experimental conditions. After 15 days of incubation, the material was extracted, and the amount of crude organic extract was measured. Sterile controls were established for all of the experiments.

Chemicals as elicitors. MEA was prepared as described above and buffered with a 50 mmol/L tri-sodium citrate dihydrate solution to set values of 4.0, 4.6, and 5.6. The chemical elicitors were CaCl₂ and CuSO₄. The strains then were inoculated, and the plates were incubated at 26 °C for 15 days.

pH as an elicitor. MEA was prepared as described above and buffered with a 50 mmol/L tri-sodium citrate dihydrate solution to set values of 4.0, 4.6, and 5.6. The chemical elicitors were CaCl₂ and CuSO₄. The strains then were inoculated, and the plates were incubated at 26 °C for 15 days.

Incubation temperature as an elicitor. MEA was prepared and buffered at pH = 4.0 by adding CaCl₂ or CuSO₄, as described above. The incubation temperatures were set at 24, 28, and 30 ± 2 °C in the incubation chamber (Sheldon Manufacturing, Inc., Cornelius, OR, USA) for 15 days.

## 2.4. Extraction and Sample Preparation

In all of the experiments, after the incubation time, the mycelium was cut into small pieces and placed into a 1 L beaker and 500 mL of ethyl acetate was added. After 30 min, the mixture was triturated and homogenized using a Polytron® (Brinkmann Instruments, Westbury, NJ, USA) and subsequently filtered through filter paper (Whatman No. 7) using a vacuum. The organic solvent was evaporated under a vacuum at 30 °C using a rotary evaporator. The resulting crude extract was re-dissolved in acetone and transferred to scintillation vials, which were previously labeled and weighed, and then evaporated on a Speed Vac® Plus (Thermo Savant™, Thermo Fisher Scientific, Waltham, MA, USA) for 24 h. Thereafter, the amount of extract was determined.

## 2.5. Antibacterial Activity

Tested microorganisms: Among the microorganisms used for the antimicrobial test, eight strains (Bacillus cereus CECT 5050, Escherichia coli CECT 433, Kocuria rhizophila CECT 241, Legionella pneumophila
CECT 7109, *Listeria monocytogenes* CECT 935, *Pasteurella multocida* CECT 962, *Salmonella enterica* CECT 7161, *Salmonella enterica* CECT 7160, and *Shigella flexneri* CECT 4804) were acquired from the Spanish Types Culture Collection of the University of Valencia, Spain, and eight strains (*Enterobacter cloacae* ATCC 13047, *Enterococcus faecalis* ATCC 19433, *Klebsiella pneumonia* ATCC 13883, *Klebsiella pneumonia* ozaenae ATCC 11296, *Proteus vulgaris* ATCC 9484, *Pseudomonas aeruginosa* ATCC 10145, *Staphylococcus aureus* ATCC 25923, and *Streptococcus oralis* ATCC 35037) were acquired from the American Type Culture Collection.

In vitro bacterial growth inhibition: The antibacterial activity of each extract was determined through the susceptibility test of the British Society for Antimicrobial Chemotherapy (BSAC) [53]. The turbidity standard (0.5 McFarland Turbidity Standard) was a BaCl₂ solution which absorbance (0.08-0.10 at 625 nm) was verified in a spectrophotometer (Spectronic 21, Bausch & Lomb, Rochester, NY, USA). The solution was stored in the dark at 24 ± 2 °C. The bacterial inoculum of the seventeen pathogenic strains were prepared in Trypticase-Soy Agar (TSA; Bacto™, Becton, Dickinson and Co., Sparks, MD, USA) for 24 h. Thereafter, five colonies were picked up and transferred into a tube containing a saline and isotonic solution, then visually compared to the turbidity standards previously reported [54].

Evaluation of the minimal inhibitory concentration (MIC): A broth dilution susceptibility testing method was applied for the determination of the (MIC) [55], using a stock solution prepared by adding 15 mg of the organic extract in 3 mL of Trypticase-Soy Broth (TSB). Serial dilutions of the organic extract (3.33 µg/mL, 1.67 µg/mL, 0.56 µg/mL, 0.18 µg/mL, 0.061 µg/mL, 0.0021 µg/mL, and 0.007 µg/mL) and positive control (gentamycin sulfate: 104.5 µg/mL, 35.0 µg/mL, 11.6 µg/mL, and 3.87 µg/mL) were performed. Each solution was inoculated with 50 µL (0.5 McFarland) of a culture of *Pseudomonas aeruginosa* and incubated at 37 °C for 18 h. The negative control was DMSO. A sterile culture media control was also used. Each assay was performed in duplicate.

## 2.6. Analysis of Organic Extracts by LC-MS

LC-MS analysis was carried out in an Agilent 1290 Infinity LC System (Agilent Technologies, Santa Clara, CA, USA) using a Zorbax® Eclipse Plus (1.8 µm) C₁₈ reverse phase LC column, 100 × 3 mm (Agilent Technologies, Santa Clara, CA, USA). The mass spectrometer was a micrOTOF-QIII (Bruker Daltonics, Billerica, MA, USA) supplied with an electrospray ionization (ESI) source. For the positive mode Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry (ESI+−Q-TOF-MS) analysis, extracts were re-dissolved in methanol and filtered through a 0.45 µm cellulose acetate membrane filter. Solutions of 0.5 µg/mL were prepared, and aliquots of 10 µL were injected. The chromatographic analysis was carried out using a 37 min step gradient (UHPLC) run using mixtures of methanol and acidified water (99.9% H₂O-0.1% FA) as mobile phase, starting from: (a) 5-95% MeOH-H₂O for 2 min; (b) a 25 min gradient from 5:95 methanol:H₂O to 100% methanol; (c) 100% methanol for 8 min. The column was returned to the initial condition for 2 min. Prior to collecting the data, two level of calibration were employed; before the analysis, an external calibration was performed using an Agilent ESI-L Low-Calibration Tuning Mix, and during the evaluation of each sample, we used hexakis (1H, 1H, 2H-difluoroethoxy)-phosphazene (m/z 622.0290 [M + H]⁺; Synquest Laboratories, Alachua, FL, USA) as an internal reference, for the lock mass calibration.

# 3. Results

## 3.1. Culture Conditions

The results obtained from evaluating the changes in culture conditions are listed in Table S1. The addition of chemical elicitors impacted the amount of crude extract obtained. In *P. mangiferae*, the best result was achieved in presence of Fe²⁺ and Ca²⁺ ion (*man*-3, 256.0 mg and *man*-4, 232.0 mg); in *P. microspora*, under the presence of Cu²⁺ and Ca²⁺ ions (*mic*-4, 297.0 mg and *mic*-5, 329.0 mg).
Low pH values increased the amount of crude extract obtained. For both species, the best results were obtained at pH = 4.0 and Cu²⁺ as elicitor: in *P. mangiferae* (*man*-9, 264.3 mg); in *P. microspora* (*mic*-9, 260.0 mg).

The third factor to consider was the incubation temperature. Maintaining constant the pH at 4.0, we found that the highest amount of extract in *P. mangiferae* was at 30 °C for both elicitors (*man*-14, 528.0 mg; *man*-17 448.0 mg); the production of extract using Ca²⁺ was 34-fold higher and 1.7-fold higher using Cu²⁺ compared to the amounts obtained in phase II. For *P. microspora*, the highest amount of extract was obtained at 24 °C using Cu²⁺ as elicitor (*mic*-15, 1194.0 mg) that was 4.6-fold higher than the obtained amount in phase II.

## 3.2. Antibacterial Activity

A total of 34 extracts were assayed against a panel of pathogenic bacteria in a preliminary antibacterial test (disc diffusion method, mm), and only 11 extracts showed growth inhibition: seven from *P. mangiferae* and four from *P. microspora* (Table 1). These extracts were capable of inhibiting the growth of 13 of 17 pathogenic bacterial strains. Larger inhibition zones were observed against *P. aeruginosa* (12.5 mm) and *L. monocytogenes* (11.0 mm). Extract *man-6* displayed the highest broad-spectrum of antimicrobial activity (inhibited seven pathogenic strains), followed by extracts *man-9* and *mic-9* (both inhibited six pathogenic strains). Nevertheless, the culture condition for *man-6* (*CaCl₂*, pH = 4.0 and T = 26 °C) induced one of the lowest amounts of organic extract (15.4 mg). The culture condition for extracts *man-9* and *mic-9* (*CuSO₄*, pH = 4.0, T = 26 °C) were more favorable.

Extracts *man-15* and *mic-11* exhibited selectivity against *P. aeruginosa*, the most sensitive strain. To determine the MIC using *P. aeruginosa*, five of the eleven extracts were selected. In this experiment, only extracts *man-6* (0.11 µg/mL) and *mic-9* (0.56 µg/mL) demonstrated growth inhibition.

Hence, to correlate the chemical profile with the antibacterial activity against *P. aeruginosa*, three samples were selected from *P. mangiferae* to be analyzed by LC-MS: (1) one active, *man-7*; (2) one with broad-spectrum activity, *man-9* (12.5 mm inhibition zone); and (3) one with selective activity, *man-15* (9.5 mm inhibition zone).

To our knowledge, there are only two reports of secondary metabolites from *P. mangiferae* [20,56].

## 3.3. Evaluation of the Chemical Diversity

Table 2 lists the principal molecular ions present in the analyzed extracts and their determined molecular formulas. Extracts *man-7* and *man-15* showed a similar chemical composition; nevertheless, at least four compounds were only present in extract *man-15*. The molecular ions are linked, mainly to polyoxygenate compounds, but some nitrogenous are present too. In all three extracts (*man-7*, *man-9*, *man-15*), the presence of mangiferaelactone was determined (retention time *t*<sub>R</sub> 21.55–21.59 min; *m/z* 401.2017 [M + H]<sup>+</sup>); none of the other previously isolated compounds from *P. mangiferae* was detected as principal components of the analyzed samples (see Figure S1). The pseudo molecular ion *m/z* 338.341 [M + H]<sup>+</sup>, which appeared at *t*<sub>R</sub> 28.3 min, has been established as a possible molecular formula (calculated for C<sub>22</sub>H<sub>44</sub>NO). This compound appeared in the chromatograms of extracts *man-9* and *man-15*, but it was absent in the *man-7* extract (Figure 1, Table 2), suggesting that it could be responsible for the antibacterial activity against *P. aeruginosa*.
<table><caption>**Table 1.** Antibacterial activity in the disk diffusion test <fnref n="1" /> of organic extracts produced by *Pestalotiopsis* spp.</caption><thead><tr><th>Fungi</th><th>Extract</th><th>E. coli</th><th>P. aeruginosa</th><th>S. tiphy</th><th>S. flexneri</th><th>P. vulgaris</th><th>S. enterica</th><th>L. pneumophila</th><th>E. faecalis</th><th>E. cloacae</th><th>P. multocida</th><th>K. rhizophila</th><th>L. monocytogenes</th><th>B. cereus</th><th>S. oralis</th></tr></thead><tbody><tr><td rowspan="7">P. mangiferae</td><td>man-1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>8.5</td><td></td><td>9.5</td></tr><tr><td>man-2</td><td></td><td>8.5</td><td></td><td></td><td>8.5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>8.0</td><td></td></tr><tr><td>man-3</td><td></td><td>8.5</td><td></td><td>7.5</td><td></td><td></td><td></td><td>9.0</td><td>9.0</td><td></td><td></td><td></td><td>7.5</td><td></td></tr><tr><td>man-6</td><td></td><td>10.0</td><td>8.5</td><td>9.0</td><td></td><td>9.0</td><td></td><td></td><td></td><td>8.0</td><td></td><td></td><td>11.0</td><td>8.0</td></tr><tr><td>man-7</td><td></td><td></td><td></td><td></td><td></td><td></td><td>8.0</td><td>8.5</td><td>8.5</td><td></td><td>9.0</td><td></td><td></td><td>7.5</td></tr><tr><td>man-9</td><td></td><td>12.5</td><td></td><td></td><td>8.5</td><td>8.5</td><td>8.0</td><td></td><td></td><td></td><td>7.5</td><td>7.5</td><td></td><td></td></tr><tr><td>man-15</td><td></td><td>9.5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">P. microspora</td><td>mic-9</td><td></td><td>8.0</td><td>8.5</td><td></td><td>7.5</td><td></td><td></td><td></td><td></td><td></td><td>8.0</td><td>7.5</td><td>8.5</td><td></td></tr><tr><td>mic-10</td><td></td><td>8.0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>8.5</td><td>8.5</td><td></td><td></td></tr><tr><td>mic-11</td><td></td><td></td><td>9.5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>mic-12</td><td></td><td></td><td>9.5</td><td></td><td></td><td>7.5</td><td></td><td></td><td></td><td></td><td></td><td></td><td>7.5</td><td></td></tr><tr><td>Positive control</td><td>Gentamycin sulfate (10 µg/mL)</td><td>18.5</td><td>17.5</td><td>14.5</td><td>13.5</td><td>13.0</td><td>13.0</td><td>12.0</td><td>10.0</td><td>9.5</td><td>9.5</td><td>30.0</td><td>18.0</td><td>17.0</td><td>9.0</td></tr></tbody></table>

<footnote n="1">¹ Results are given in mm of inhibition.</footnote>
<table><caption>**Table 2.** Molecular ions of secondary metabolites present in extracts obtained from *P. mangiferae*.</caption><thead><tr><th>Retention Time<sup>1</sup> (t<sub>R</sub>)</th><th>m/z</th><th>[M + H]<sup>+</sup></th><th>[M + Na]<sup>+</sup></th><th>Dimers</th><th>Molecular Formula</th><th>Extracts</th></tr></thead><tbody><tr><td>19.44–19.50</td><td>588.36567</td><td>589.369</td><td></td><td>1177.720 [2M + H]<sup>+</sup></td><td>C<sub>34</sub>H<sub>52</sub>O<sub>8</sub></td><td>man-7, man-15</td></tr><tr><td>20.14–20.37</td><td>278.18765</td><td>279.194</td><td></td><td></td><td>C<sub>17</sub>H<sub>26</sub>O<sub>3</sub></td><td>man-7</td></tr><tr><td>20.78–20.87</td><td>588.36567</td><td>589.369</td><td></td><td>1177.720 [2M + H]<sup>+</sup></td><td>C<sub>34</sub>H<sub>52</sub>O<sub>8</sub></td><td>man-7, man-15</td></tr><tr><td rowspan="3">21.55–21.59</td><td rowspan="3">282.18524</td><td rowspan="3">283.193</td><td rowspan="3">423.199</td><td>565.375 [2M + H]<sup>+</sup></td><td>C<sub>16</sub>H<sub>26</sub>O<sub>4</sub></td><td>man-7, man-9, man-15</td></tr><tr><td>801.424 [2M + H]<sup>+</sup></td><td></td><td></td></tr><tr><td>823.407 [2M + Na]<sup>+</sup></td><td>C<sub>20</sub>H<sub>32</sub>O<sub>8</sub></td><td>man-7, man-9, man-15</td></tr><tr><td rowspan="4">22.39–22.43</td><td>394.28663</td><td>395.295</td><td></td><td></td><td>C<sub>27</sub>H<sub>38</sub>O<sub>2</sub></td><td>man-7, man-15</td></tr><tr><td>412.29317</td><td>413.303</td><td></td><td></td><td>C<sub>22</sub>H<sub>40</sub>N<sub>2</sub>O<sub>5</sub></td><td>man-7, man-15</td></tr><tr><td>470.33906</td><td>471.347</td><td></td><td></td><td>C<sub>30</sub>H<sub>46</sub>O<sub>4</sub></td><td>man-7, man-15</td></tr><tr><td>488.34560</td><td>489.354</td><td></td><td>995.716 [2M + H<sub>2</sub>O + H]<sup>+</sup></td><td>C<sub>30</sub>H<sub>48</sub>O<sub>5</sub></td><td>man-7, man-15</td></tr><tr><td>23.40–23.45</td><td>314.24516</td><td>315.256</td><td>337.236</td><td></td><td>C<sub>38</sub>H<sub>34</sub>O<sub>4</sub></td><td>man-15</td></tr><tr><td>23.72–23.81</td><td>428.30737</td><td></td><td>451.298</td><td></td><td>C<sub>31</sub>H<sub>40</sub>O</td><td>man-7, man-15</td></tr><tr><td>24.54</td><td>278.22403</td><td>279.231</td><td>301.211</td><td>557.446 [2M + H]<sup>+</sup></td><td>C<sub>24</sub>H<sub>44</sub>O<sub>6</sub></td><td>man-15</td></tr><tr><td>24.60</td><td>452.31324</td><td></td><td>475.304</td><td>927.610 [2M + Na]<sup>+</sup></td><td>C<sub>18</sub>H<sub>30</sub>O<sub>2</sub></td><td>man-7, man-15</td></tr><tr><td>26.86</td><td>281.27132</td><td>282.279</td><td>304.261</td><td></td><td>C<sub>26</sub>H<sub>44</sub>O<sub>6</sub></td><td>man-15</td></tr><tr><td>27.52</td><td>283.28697</td><td>284.294</td><td>306.276</td><td></td><td>C<sub>18</sub>H<sub>35</sub>NO</td><td>man-7, man-15</td></tr><tr><td>27.83–27.87</td><td>390.27646</td><td>391.283</td><td>413.261</td><td>803.536 [2M + Na]<sup>+</sup></td><td>C<sub>18</sub>H<sub>37</sub>NO</td><td>man-7, man-15</td></tr><tr><td>28–33</td><td>337.33392</td><td>338.341</td><td>360.320</td><td>675.670 [2M + H]<sup>+</sup></td><td>C<sub>24</sub>H<sub>38</sub>O<sub>4</sub></td><td>man-7, man-9, man-15</td></tr><tr><td>28.40</td><td>418.28663</td><td>419.308</td><td>441.288</td><td>859.578 [2M + Na]<sup>+</sup></td><td>C<sub>22</sub>H<sub>43</sub>NO</td><td>man-9, man-15</td></tr><tr><td>31.25–31.36</td><td>662.43884</td><td>663.444</td><td>685.40</td><td></td><td>C<sub>29</sub>H<sub>38</sub>O<sub>2</sub></td><td>man-7</td></tr><tr><td></td><td>721.51233</td><td>722.519</td><td></td><td></td><td>C<sub>40</sub>H<sub>36</sub>NO<sub>4</sub></td><td>man-7, man-9, man-15</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>C<sub>41</sub>H<sub>71</sub>NO<sub>9</sub></td><td>man-15</td></tr></tbody></table>

<footnote n="1">¹ Time is given in minutes.</footnote>
<figure><img src="image_40.png" /><figcaption>**Figure 1.** Total ion chromatograms (TICs) of extracts *man-9*, *man-7*, and *man-15*.</figcaption></figure>

Chemical diversity of the three extracts from *P. mangiferae* (*man-7*, *man-9*, and *man-15*) was analyzed by LC-ESI-Q-TOF-MS. The total ion chromatograms for each sample are presented in Figure 1. The peak at *t*<sub>R</sub> 21.5 min is common to all three analyzed samples. The MS spectrum of this peak showed ions [M + H]<sup>+</sup> and [M + Na]<sup>+</sup> at *m/z* 401.217 and 423.199, respectively. Additionally, two ion clusters were detected: M<sub>2</sub>H<sup>+</sup> and M<sub>2</sub>Na<sup>+</sup> at *m/z* 801.424 and 823.407, respectively (Figure 2A), which matched the MS data for mangiferaelactone, a previously characterized compound. This compound had a relatively lower concentration in sample *man-15*. Based on its selectivity against *P. aeruginosa* (9.5 mm inhibition zone), this compound could be proposed as the major component of the extract, for example, the peak at *t*<sub>R</sub> 27.8 min with [M + H]<sup>+</sup>, [M + Na]<sup>+</sup>, and [2M + Na]<sup>+</sup> ions at *m/z* 391.283, 413.261, and 803.536, respectively (Figure 2D). The polar section of *man-15* was the most complex, indicating a higher level of chemical diversity than *man-7* and *man-9*.

Extract *man-7* showed a higher relative concentration among the components of the polar section (peaks at *t*<sub>R</sub> 19.4, 20.2, 20.7, and 21.5 min). Its moderate polarity section included a peak at *t*<sub>R</sub> 28.4 min with [M + H]<sup>+</sup>, [M + Na]<sup>+</sup>, and [2M + Na]<sup>+</sup> ions at *m/z* 419.308, 441.276, and 859.579, respectively (Figure 2B), compared with extracts *man-9* and *man-15* that had a peak at *t*<sub>R</sub> 28.3 min with [M + H]<sup>+</sup>, [M + Na]<sup>+</sup>, and [2M + H]<sup>+</sup> ions at *m/z* 338.337, 360.319, and 675.670, respectively (Figure 2C).
<figure><img src="image_41.png" /><figcaption>**Figure 2.** Mass spectra of selected peaks. (A) Mangiferaelactone at *t*<sub>R</sub> 21.5 min in extracts *man*-7, *man*-9, and *man*-15. (B) Peak in extract *man*-7 at *t*<sub>R</sub> 28.4 min. (C) Peak in extracts *man*-9 and *man*-15 at *t*<sub>R</sub> 28.3 min. (D) Peak in extracts *man*-7 and *man*-15 at *t*<sub>R</sub> 27.8 min.</figcaption></figure>

**4. Discussion**

As we mentioned above, there are only two reports related to the isolation and characterization of secondary metabolites from *P. mangiferae*; early culturing under two different conditions produced antibacterial compounds mangiferaelactone (1) and 4-(2,4,7-trioxa-bicyclo[4.1.0]heptan-3-yl) phenol (see Figure S1) [20,56]. Neither one was among the main components present in the extracts analyzed here by high resolution LC-MS, nor the other compounds with a low molecular weight.

Our results showed that in this initial study, *Pestalotiopsis* showed prolific antibacterial activity. The results of Table 2 indicate that each extract had a different biological activity profile. This means that the chemical composition changes in diversity and concentration.

The changes in the culture conditions played a role in differences in the chemical diversity of *P. mangiferae* and the genus *Pestalotiopsis*. This was confirmed by the wide range of the preliminary antibacterial activities determined for each of the extracts and through the LC-HRMS analysis of the three extracts. According to the revised reviews and recent publications (Table S2) on the secondary metabolites isolated from the genus *Pestalotiopsis* and the antibacterial activity previously determined, most of the molecular formulas for the metabolites reported here did not match with those reported earlier for the genus. Nonetheless, taking into account the previously isolated compounds from the genus *Pestalotiopsis* or from *P. mangiferae* and the molecular formula obtained through high-resolution mass spectrometry, we proposed some molecular structures; also, in most of the cases they are related with a previously isolated compound with antibacterial or antifungal activity.

Five major reviews on the chemistry and bioactivity of the genus *Pestalotiopsis* were published until 2017 [14–18]. An exhaustive exploration of the available compounds’ structures allowed us to establish that the majority of the compounds present in the analyzed extracts of *P. mangiferae* have not been isolated from a species of the genus *Pestalotiopsis*. Nevertheless, it could be proposed that they belong to three of the main classes of compounds isolated from the genus, namely: (a) polyketides/polyols derivatives; (b) terpenoids/triterpenoids; and (c) nitrogen-containing compounds.

The major group of compounds present in the analyzed extracts could belong to polyketide/polyol derivatives. For example, we proposed the hydrolysis of mangiferaelactone (1) (C20H32O8) that will result in the hypothetical trihydroxylactone (1a) (not-yet-detected by MS-analysis), its successive
dehydration and methylation could lead to two lactones (1b) ($C_{16}H_{26}O_4$) and (1d) ($C_{17}H_{26}O_3$), respectively (Figure 3). Compound (1b) has the same molecular formula as koninginins B (1e; $C_{16}H_{26}O_4$) and E (1f; $C_{16}H_{26}O_4$) isolated from the genus *Trichoderma* [57–59], and they probably have the same precursor as compound (1a) ($C_{16}H_{28}O_5$).

<figure><img src="image_42.png" /><figcaption>**Figure 3.** Proposed polyketide/polyol derivatives 1b,d that could be present in extracts man-7, man-9, and man-15, having as a precursor compound 1 and the intermediates 1a,c. Polyketides 1e,f previously isolated from the genus *Trichoderma* with same molecular formula as 1b.</figcaption></figure>

Two molecular formulas could correspond to triterpenoids ($C_{30}H_{46}O_4$/ $C_{30}H_{48}O_5$). From the genus *Pestalotiopsis*, only oleanane- and ursane-type triterpenes have been isolated [14,17,60]. Ursane-type triterpenes have been reported when to the culture medium was added ursolic acid [61]. Nevertheless, oleanane-type were isolated (15α)-15- hydroxysoyasapogenol B (2), (7β, 15α)-7, 15-dihydroxysoyasapogenol B (3) and (7β)-7, 29-dihydroxysoyasapogenol B (4) from *Pestalotiopsis clavispora* [60], together with ursolic acid. These three compounds (2–4) could be synthesized by biological oxidation mechanism derived in one of the triterpenoids 2a, 3a or 4a, respectively; they have the molecular formula $C_{30}H_{46}O_4$ or $C_{30}H_{48}O_5$ established by high resolution MS in extracts man-7 and man-15 (Figure 4).

<figure><img src="image_43.png" /><figcaption>**Figure 4.** *Cont.*</figcaption></figure>
<figure><img src="image_44.png" /><figcaption>**Figure 4.** Proposed structure for compounds **2a**, **3a**, and **4a** based on the molecular formula determined by HRMS in extracts *man-7* and *man-15* and their proposed biosynthetic precursor.</figcaption></figure>

In our results two molecular ions have the same molecular formula $C_{34}H_{52}O_8$, but they eluted at different time and are both present in extracts *man-7* and *man-15*. These isomers could be related to fusapirone (5; $C_{34}H_{54}O_9$) a compound with antifungal activity, previously, isolated from *Fusarium semitectum* [62], it possess multiple chiral centers and can derived into compound (5a; $C_{34}H_{52}O_8$) by dehydration (Figure 5).

<figure><img src="image_45.png" /><figcaption>**Figure 5.** Dehydration of the polyketide derivative fusapirone 5 could produce compound 5a with a molecular formula $C_{34}H_{57}O_8$, determined in extracts *man-7* and *man-15*.</figcaption></figure>

The dehydrogenation of asperacine (6; $C_{40}H_{36}N_6O_4$) results in compound **6a** ($C_{40}H_{36}N_6O_4$) with an imine function (Figure 6), this molecular formula was determined for extracts *man-7*, *man-9*, and *man-15*.
<figure><img src="image_46.png" /><figcaption>**Figure 6.** Nitrogen derivative 6a, that could be present in extracts man-7, man-9, and man-15.</figcaption></figure>

**5. Conclusions**

For this study were selected two strains of *Pestalotiopsis* endophytic fungi (*P. microspora* Hd18 and *P. mangiferae*), that are capable of producing secondary metabolites with relevant biological activities. The strategy developed in this work included variations of the culture conditions, the determination of the antibacterial activity of the obtained extracts, together with the effective analysis of the chemical profile using LC-HRMS. This strategy could improve the discovery of new molecules with a pharmaceutical potential, in this case antibacterial. Hence, this work confirmed changes in the chemical diversity and biological activity of *P. microspora* Hd18 and, principally, *P. mangiferae* Hd08 under varying the culture conditions.

Taking into account the chemical diversity and the preliminary antibacterial activity displayed by *P. mangiferae*, further work will need to establish and confirm the chemical composition of each extract as well as the antibacterial activity of a single compound.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2309-608X/6/3/140/s1, Figure S1: Structure of compounds previously isolated from *P. mangiferae*. Table S1: Culture parameters and amount of organic extract produced by *Pestalotiopsis* spp., Table S2: Molecular ions and formulas of compounds isolated from the genus *Pestalotiopsis*

**Author Contributions:** M.M.A.-P., R.V., and N.R. designed and performed the experiments and analyzed the data. D.T.-M. analyzed the data and reviewed, edited, and wrote the paper. L.C.-R. designed the experiments, analyzed the data, and edited and wrote the paper. All of the authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partially supported by the National Secretariat for Science, Technology and Innovation of Panama (SENACYT, grants COL10-060 and FID11-051), the Projects of Nagoya Protocols´ Application in Panama, and the National Research System of Panama (SNI).

**Acknowledgments:** We gratefully acknowledge the Government of Panama through the Ministerio de Ambiente (MiAMBIENTE) for granting the corresponding permits to collect the samples used in this study.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Article

Interactive Impact of Arbuscular Mycorrhizal Fungi
and Elevated CO₂ on Growth and Functional Food
Value of *Thymus vulgare*

Talaat H. Habeeb <fnref n="1" />, Mohamed Abdel-Mawgoud <fnref n="2" />, Ramy S. Yehia <fnref n="3" />,<fnref n="4" /> ID,

Ahmed Mohamed Ali Khalil 5 ID, Ahmed M. Saleh 4,\* ID and Hamada AbdElgawad 6,\*

<figure><img src="image_50.png" /></figure>

check for
updates

Received: 25 July 2020; Accepted: 4 September 2020; Published: 9 September 2020

**Abstract:** Arbuscular mycorrhizal fungi (AMF) and elevated CO₂ (eCO₂) have been effectively integrated to the agricultural procedures as an ecofriendly approach to support the production and quality of plants. However, less attention has been given to the synchronous application of AMF and eCO₂ and how that could affect the global plant metabolism. This study was conducted to investigate the effects of AMF and eCO₂, individually or in combination, on growth, photosynthesis, metabolism and the functional food value of *Thymus vulgare*. Results revealed that both AMF and eCO₂ treatments improved the photosynthesis and biomass production, however much more positive impact was obtained by their synchronous application. Moreover, the levels of the majority of the detected sugars, organic acids, amino acids, unsaturated fatty acids, volatile compounds, phenolic acids and flavonoids were further improved as a result of the synergistic action of AMF and eCO₂, as compared to the individual treatments. Overall, this study clearly shows that co-application of AMF and eCO₂ induces a synergistic biofertilization impact and enhances the functional food value of *T. vulgare* by affecting its global metabolism.

**Keywords:** mycorrhizae; elevated CO₂; *Thymus vulgare*; growth; photosynthesis; metabolites; biological activity

**1. Introduction**

Herbal plants have been widely used in traditional and folk medicine as an effectual solution to cure many diseases, being a big store for bioactive compounds, especially secondary metabolites [1]. They are known to have various biological activities such as antioxidant, antimicrobial, anti-inflammatory and anticancer properties [2]. Recently, a priority was given to herbal plants in terms of enhancing the production of the economically important phytochemicals through the application of cultivation procedures under stimulated growth conditions [3]. In this aspect, arbuscular mycorrhizal fungi (AMF) have been regarded as one of the most important beneficial microorganisms that are able to associate with almost two thirds of terrestrial plants improving their growth and stress tolerance [4].

<footnote n="1">¹ Biology Department, Faculty of Science at Yanbu, Taibah University, King Khalid Rd., Al Amoedi, Yanbu El-Bahr 46423, Saudi Arabia; thabeeb@yahoo.com</footnote>

<footnote n="2">² Department of Medicinal and Aromatic Plants, Desert Research Centre, Cairo 11753, Egypt; Mohamed_drc@yahoo.com</footnote>

<footnote n="3">³ Department of Biological Sciences, College of Science, King Faisal University, Al-Ahsa 31982, Saudi Arabia; drramy4@hotmail.com</footnote>

<footnote n="4">⁴ Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt</footnote>

<footnote n="5">5 Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo 13759, Egypt; khalilahmed_1980@hotmail.com</footnote>

<footnote n="6">⁶ Botany and Microbiology Department, Faculty of Science, Beni-Suef University, Beni-Suef 62511, Egypt</footnote>

<footnote>* Correspondence: asaleh@sci.cu.edu.eg (A.M.S.); hamada.abdelgawad@uantwerpen.be (H.A.)</footnote>
In some cases, mycorrhizal symbiosis is essential as the host plant cannot grow normally and/or survive without it [5]. The beneficial effects of AMF symbiotic association with plants include enhanced levels of mineral nutrients and accumulation of primary and secondary metabolites [6]. From environmental point of view, AMF can keep the balance of soil aggregates, hence, able to fight erosion [7]. Accordingly, AMF represent a promising trend that has found its way in the sustainable agricultural productivity [8]. For instance, utilization of AMF for enhancing the production and quality of aromatic plants have been reported [8]. In this regard, several medicinal aromatic plants, such as pennyroyal and parsley, showed enhanced levels of bioactive metabolites when associated with AMF [6].

In another aspect, the exposure of plants to elevated CO₂ (eCO₂) has been regarded as an effective approach to improve the nutritional and medicinal values of herbal plants [9]. eCO₂ can increase plant growth and productivity either directly by enhancing photosynthesis [10] or indirectly by stimulating plant water use efficiency [11]. On the other hand, the effect of eCO₂ on belowground communities, including AMF, are still not fully understood [12]. What is well known is that the higher the photosynthetic activity, under eCO₂, the more the photosynthate transfer to plant roots and the higher release to the associated microbial communities [13]. Furthermore, as being dependent on their host plant for carbon, AMF may be sensitive to global climatic changes that influence their host [14]. Therefore, such triple effect resulting from interactions among plant, AMF and eCO₂ is expected to have beneficial roles in increasing the productivity and quality of crops and medicinal plants.

One of the well-known plants for both culinary and medicinal purposes is *Thymus vulgaris* L., a member of the family Lamiaceae, being widely used in folk medicine for treatment of several diseases like bedwetting, diarrhea, stomach ache, arthritis, sore throat, cough, bronchitis and chest congestion [15]. The biological activities of *T. vulgaris* are mainly ascribed to its content of secondary metabolites, particularly essential oils that have been extensively studied for antioxidant, antimicrobial and antitumor activities [16]. Thus, improving the accumulation of these phytochemicals in *T. vulgaris* could support its nutritional, medicinal and pharmacological properties. In this regard, previous studies have reported the positive impacts of both AMF and eCO₂ on the growth and quality of herbal plant [17,18], however, the complete picture on how AMF-eCO₂ combination affect primary and secondary metabolomes is not fully drawn [19,20]. In addition, metabolic profiling of the host plant is essential to understand the mechanisms behind the changes occurring in response to the individual and/or combined effect of AMF and eCO₂. So far, the detailed metabolic implications induced by the synchronous application of eCO₂ and AMF on plants are not investigated. Thus, the current study was conducted to explore, in details, the individual and combined impacts of eCO₂ and AMF on *T. vulgaris*, as a model herbal plant. We have assessed the changes in mycorrhizal colonization, plant biomass production, photosynthesis, respiration and levels of individual primary (sugars, amino acids, fatty acids and organic acids), secondary (phenolic acids and flavonoids) metabolites and volatile oils. Further, the associated changes in nutritional and medicinal values of *T. vulgaris* were investigated.

## 2. Material and Method

### 2.1. *Experimental Setup, Plant Materials and Growth Conditions*

Soil potting was mixed with sterilized sand (1:3) and inculcated with a pure commercial inoculum of *Rhizophagus irregularis* (MUCL 41,833 obtained from Glomeromycota in vitro collection (GINCO)) at a concentration of 50 spores per soil in a pot (25 × 15 cm). The control treatments were represented by non-inoculated soil. The seeds of *T. vulgaris* were disinfected then sown in both treated and non-treated soils. Plants were grown in a controlled greenhouse at 21/18 °C, 16/8 h day/night, and 60% humidity, they were regularly watered. The pots inn each of the control and AMF-inoculated groups were equally subdivided into two sub-groups, one subjected to 410 ppm CO₂ (ambient CO₂; aCO₂) and the other subjected to 620 ppm CO₂ (elevated CO₂; eCO₂,) through the time course of the experiment. The plants were harvested after 6 weeks, then the aerial parts were immediately frozen in liquid nitrogen and
stored at –20 °C to be used in different plant analyses. For determination of dry matter and mineral elements, plant shoots were washed with distilled water and dried at 75 °C for 72 h.

## 2.2. Mycorrhizal Parameters

Mycorrhizal colonization was demonstrated following Phillips and Hayman [21]. About 0.5 g of fresh roots were clarified with potassium hydroxide (10% *w/v*) and potassium hydroxide (10%) + hydrogen peroxide (10% *v/v*) in a ratio of 1:1 (*v/v*), then stained with 0.05% trypan blue in lactoglycerol. A stereomicroscope (40×) was used to show the stained roots, while the colonization rate was calculated by using gridline intersect method [22].

## 2.3. Photosynthesis Parameters

The light-saturated photosynthetic rates (µmol CO₂ m⁻² s⁻¹) of mature leaves were measured (LI-COR LI-6400, LI-COR Inc., Lincoln, NE, USA), according to AbdElgawad et al. [23]. Dark respiration was determined as the absolute CO₂ exchange rate determined at photosynthetic photon flux density (µmolm⁻² s⁻¹).

## 2.4. Metabolic Profiling

For extraction of sugars, plant tissues were homogenized in 50% (*v/v*) acetonitrile. The method described by Hamad et al. [24] was applied to identify the individual sugars in the plant extract by using high-performance liquid chromatography (HPLC), then comparing their retention time with those of a standard mixture. Quantification of the sugar samples was achieved based on peak area comparison with a calibration curve of the corresponding standards. Organic acids were extracted in phosphoric acid (0.1% *v/v*) supplemented with butylated hydroxyanisole (3 g/L) and then analyzed using HPLC with a SUPELCOGEL C-610H column coupled to a UV detection system set at 210 nm (LaChromL-7455 diode array, LaChrom, Tokyo, Japan). The concentration of each organic acid was calculated by using a calibration curve [24]. For extraction of amino acids, a known weight of plant tissues was vigorously homogenized in 80% aqueous ethanol. Amino acids were measured using a Waters Acquity UPLC-tqd system (Milford, Worcester County, MA, USA) equipped with a BEH amide 2.1 × 50 column. The lipophilic fraction of plant samples was obtained by extraction in chloroform/methanol (2:1, *v/v*). Thereafter, fatty acids were detected, according to Hassan et al. [25], by using GC/MS analysis (Hewlett Packard, Palo Alto, CA, USA) with an HP-5 MS column (30 m × 0.25 mm × 0.25 mm). Fatty acids were quantified using NIST 05 database and Golm Metabolome Database (http://gmd.mpimp-golm.mpg.de). Phenolic acids and flavonoids were extracted in acetone-water solution (4:1 *v/v*) for 24 h. The method outlined in Hamad et al. [24] was followed up for determination of Phenolic acids and flavonoids using an HPLC system (SCL-10A vp, Shimadzu Corporation, Kyoto, Japan). The concentration of each compound was calculated with a calibration curve of the corresponding standard. For extraction of volatile oils, two hundred gm of fresh plant material were subjected to steam distillation with about 500 mL of water, where the volatiles were collected [26]. The levels of volatiles were determined using gas chromatography-mass spectrometry (GC-MS) according to the method outlined by El Hattab et al. [27].

## 2.5. Determination of Biological Activities

Several methods were used to determine the total antioxidant capacities of the plant extract, including the ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), inhibition of LDL (low density lipoprotein) oxidation (TBARS and conjugated dienes) and inhibition of hemolysis assays [23,24]. For LDL oxidation, dialyzed LDL (100 µg protein/mL) was diluted in 10 mM PBS (phosphate buffered saline containing 0.01 M phosphate-buffer and 0.15 M NaCl, pH 7.4) and incubated at 37 °C in presence or absence of 10 µM CuSO₄. Oxidation was performed with or without the sample solution of colostrum proteins. After incubation, lipid peroxidation of the LDL was measured. Thiobarbituric acid reactive substances (TBARS) was determined at 532 nm/600 nm,
using 1,1,3,3-Tetramethoxypropane as standard for calibration curve, while conjugated diene formation was measured at 232 nm of LDL solution (100 µg protein/mL) in PBS incubated with CuSO₄ (10 µM) in the absence or presence of various concentrations of bovine colostrums protein [28].

## 2.6. Statistical Analysis

Experiments were carried out following a randomized complete block design. Data normality and the homogeneity of variances were checked using the Kolmogorov-Smirnov test and Levene's test, respectively. Each experiment was done in five replicates (n = 5). All the data was subjected to one-way analysis of variance (ANOVA). Student's t-test at probability levels of 0.05, 0.01 or 0.001 was used to test the difference between the treatment and control or between AMF alone and the combined AMF+eCO₂ treatment. All statistical tests were performed using the computer program PASW statistics 18.0 (SPSS Inc., Chicago, IL, USA).

# 3. Results and Discussion

## 3.1. AMF Colonization and Hyphal Growth

It is known that AMF are largely dependent on their host plant for carbon, so they are sensitive to climatic changes that affect their host plant [14]. In this sense, eCO₂ could have an indirect effect on mycorrhizal colonization by promoting carbon assimilation and allocation to roots [29]. Since AMF are attached to plant roots, they are lucky to receive higher amount of photosynthates under eCO₂ before other soil microbes [30]. Herein, the mycorrhizal growth was significantly enhanced in *T. vulgare* by AMF treatments (Table 1). Such mycorrhizal proliferation was much more stimulated under eCO₂ conditions. Several studies have demonstrated some positive effects for eCO₂ on AMF-plant association such as increased mycorrhizal root length [14] and increased extra-radical hyphae [31]. However, other studies did not show any beneficial effects for eCO₂ on AMF growth in host plants [32,33]. Therefore, the impact of eCO₂ levels on mycorrhizal growth seems to be dependent on plant species, AMF species and soil type [34]. In fact, as being a member of *Gomeraceae*, the ratio of *R. irregularis* has been reported to be more positively influenced by eCO₂ than others, e.g., *Gigasporaceae* [35].

<table><caption>**Table 1.** Mycorrhizal colonization and growth parameters in roots of *Thymus vulgare* grown under normal conditions (control) or the effect of eCO₂ (620 ppm), arbuscular mycorrhizal fungi (AMF) or their combination (AMF-eCO₂). Values are mean ± standard error of five independent replicates. Asterisks indicate significant changes (*** *p* < 0.001) between AMF alone and the combined AMF+eCO₂ treatment.</caption><thead><tr><th>Metabolite</th><th>Control</th><th>eCO<sub>2</sub></th><th>AMF</th><th>AMF + eCO<sub>2</sub></th></tr></thead><tbody><tr><td>Colonization (% root)</td><td>nd</td><td>nd</td><td>33.06 ± 2.39</td><td>54.04 ± 1.11 ***</td></tr><tr><td>Hyphal length (cm g<sup>-1</sup> soil)</td><td>nd</td><td>nd</td><td>12.94 ± 5.82</td><td>19.10 ± 9.74 b ***</td></tr><tr><td>Number of arbuscules (no. cm<sup>-1</sup> root)</td><td>nd</td><td>nd</td><td>4.78±0.27</td><td>5.03 ± 0.18</td></tr></tbody></table>

nd = not detected.

## 3.2. AMF and eCO₂ Acts Synergistically to Improve Photosynthetic Capacity and Biomass Production

It has been known that the photosynthetic rate, and consequently biomass production, could be improved under the effect of AMF inoculation, as a result of the expected increased nutrients uptake [36], and also under eCO₂ atmosphere due to the enhancement of the carboxylation reaction of rubisco [37]. Supporting this hypothesis, the current results revealed that eCO₂ and AMF independently, and to more extent in combination, promoted photosynthetic rate and biomass production in *T. vulgare* (Figure 1). Such increments were much more induced by the interaction between both treatments.
<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /><figcaption>**Figure 1.** Fresh mass (A), dry mass (B), and rates of photosynthesis (C) and respiration (D) in *Thymus vulgare* grown under normal conditions (control) or the effect of eCO₂ (620 ppm), AMF or their combination (AMF-eCO₂). Values are mean ± standard error of five independent replicates. Asterisks indicate significant changes (* p* < 0.05; ** p* < 0.01) compared to control, as revealed by the Student's t-test. Lowercase letters indicate significant differences (a p* < 0.05; b p* < 0.01) between AMF alone and the combined AMF+eCO₂ treatment.</figcaption></figure>

Similar to our results, the positive effects of eCO₂ on biomass of *T. vulgaris* and some other medicinal plants, *Ocimum basilicum*, *Origanum vulgare*, *Mentha piperita* and *Mentha spicata*, have been previously investigated [38]. Moreover, the increased biomass production in plants inoculated with AMF was reported [39]. Regarding the interaction between eCO₂ and AMF, It is well known that eCO₂ stimulates the photosynthetic rate and plant growth [40], which in turn, affects the allocation of photosynthates to AMF, consequently makes more C available to AMF colonizing the roots [13],
thus, increasing sink strength in mycorrhizal plants, and eventually this leads to increased C storage in soils [41]. Such effect is hypothesized to create a balance between carbon cost and nutrient benefits, besides reducing the negative effects of down regulation of photosynthesis caused by acclimation of plants to long-term exposure to eCO₂ [42]. In this regard, it has been found that both eCO₂ and AMF, when applied individually or in combination, improved biomass production of *Pisum sativum* and lettuce [32,43]. In addition, it has been indicated that mycorrhizal plants have higher photosynthetic rate [33] when grown under high CO₂ levels. However, such an effect might differ among variable cultivars [44]. In contrast, it was supposed that eCO₂ may impair the beneficial effects of AMF on plant biomass, especially when the fungal community is dominated by *Glomus* species [45]. This might be due to the difference among AMF taxa in their exchange of carbon and nutrients [46].

### 3.3. Application of AMF and eCO₂ Improves the Nutritional Value of *T. vulgare*

It was assumed that the nutritive value of plants is highly related to its content of primary metabolites, e.g., sugars, proteins and lipids [9]. In this regard, sugars and organic acids are related to taste and flavor [47], essential amino acids are involved in some biological processes, such as protein synthesis [48] and a lower saturated/unsaturated fatty acids (SFA/USFA) ratio is linked to cardio-protective effects [49]. It has been reported that the higher the CO₂ levels, the higher the rate of photosynthetic activity, which is linked to the enhancement of the carboxylation reaction of rubisco, the enzyme responsible for CO₂ fixation [37]. As a consequence of photosynthesis improvement, sugars could be accumulated and also broken down via dark respiration, resulting in production of the precursors necessary for synthesis of different classes of primary and secondary metabolites [50]. Supporting such a concept, the individual AMF and eCO₂ treatments induced significant increases in the content of total soluble sugar of *T. vulgare*, about 1.6 folds, however, starch was significantly accumulated under eCO₂ only (Table 2, Figure 2). Further, the synchronous application of AMF and eCO₂ caused a significant accumulation in the levels of the majority of the measured sugars relative to AMF alone treatment. Similarly, CO₂ enrichment enhanced the accumulation of sucrose and starch in oil palm [51], and increased the accumulation of total soluble carbohydrates and starch in ginger varieties [52]. AMF treatments were found to induce the accumulation of total soluble sugars in lettuce [53]. Further, the interaction between AMF and CO₂ improved forage quality of alfalfa plants by increasing the levels of glucose, fructose and hemicellulose and decreasing that of lignin [54].

<table><caption>**Table 2.** Levels of primary metabolites (mg g⁻¹ dry weight) in *Thymus vulgare* grown under normal conditions (control) or the effect of eCO₂ (620 ppm), AMF or their combination (AMF-eCO₂). Values are mean ± standard error of five independent replicates. Asterisks indicate significant changes (* *p* < 0.05; ** *p* < 0.01) compared to control, as revealed by the Student's t-test. Lowercase letters indicate significant differences (ª *p* < 0.05; ᵇ *p* < 0.01) between AMF alone and the combined AMF+eCO₂ treatment.</caption><thead><tr><th></th><th>Control</th><th>eCO<sub>2</sub></th><th>AMF</th><th>AMF + eCO<sub>2</sub></th></tr></thead><tbody><tr><td><strong>Sugars</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Glucose</td><td>1.34 ± 0.07</td><td>2.18 ± 0.12 **</td><td>1.32 ± 0.07</td><td>2.14 ± 0.01 <sup>b</sup></td></tr><tr><td>Fructose</td><td>0.34 ± 0.05</td><td>0.45 ± 0.02</td><td>0.4 ± 0.02</td><td>0.76 ± 0.06 <sup>b</sup></td></tr><tr><td>Sucrose</td><td>1.67 ± 0.15</td><td>2.07 ± 0.15</td><td>2.19 ± 0.04 *</td><td>2.85 ± 0.1 <sup>b</sup></td></tr><tr><td>Soluble sugars</td><td>6.09 ± 0.31</td><td>10.29 ± 0.3 **</td><td>9.46 ± 0.35 **</td><td>11.44 ± 0.8</td></tr><tr><td>Starch</td><td>62.06 ± 1.89</td><td>79.26 ± 4.63 *</td><td>60.1 ± 1.7</td><td>85.22 ± 1.68 <sup>b</sup></td></tr><tr><td>Total carbohydrates</td><td>105.38 ± 3.08</td><td>137.1 ± 6.75 *</td><td>105.62 ± 6.31</td><td>147.79 ± 2.01 <sup>b</sup></td></tr><tr><td><strong>Organic acids</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Oxalic</td><td>3.84 ± 0.33</td><td>2.97 ± 0.06</td><td>5.7 ± 0.34 *</td><td>7.75 ± 0.29 <sup>b</sup></td></tr><tr><td>Malic</td><td>6.88 ± 0.3</td><td>6.25 ± 0.34</td><td>6.36 ± 0.29</td><td>7.38 ± 0.36</td></tr><tr><td>Isobutyric</td><td>3.46 ± 0.33</td><td>2.89 ± 0.46</td><td>6.34 ± 0.67 *</td><td>7.67 ± 0.53</td></tr><tr><td>Fumaric</td><td>0.93 ± 0.01</td><td>0.96 ± 0.08</td><td>0.67 ± 0.04 **</td><td>1.14 ± 0.07 <sup>b</sup></td></tr><tr><td>Succinic</td><td>3.07 ± 0.33</td><td>4.21 ± 0.29</td><td>5.08 ± 0.39 *</td><td>6.31 ± 0.58</td></tr><tr><td>Citric</td><td>2.88 ± 0.33</td><td>5.32 ± 0.28 **</td><td>4.17 ± 0.54</td><td>5.9 ± 0.2 <sup>a</sup></td></tr></tbody></table>