*2.4. In Vitro Pro-Health Potency and Antioxidant Capacity* Antioxidant Capacity

The analysis of the antioxidant activity of the examined black garlic varieties showed that the type of pretreatment, drying method, and conditions significantly (*p* < 0.05) affected the antioxidant capacity of the tested samples. The antioxidant properties of black garlic varied widely. The highest antioxidant potential measured by the ORAC method was shown by the black garlic dried by VMD500W (7.90 mmol Trolox/100 g dm), while the lowest one by the black garlic obtained through PEF + H2O (4.38 mmol Trolox/100 g dm). Similar trends, but slightly lower results, were obtained for the ABTS, and FRAP methods. In these cases, the highest antioxidant potential was recorded for the VMD500W—6.05, and 3.73 mmol Trolox/100 g dm (ABTS, FRAP, respectively), while the lowest value was observed for the PEF + H2O process—2.03, and 1.09 mmol Trolox/100 g dm (Table 4).

The conducted study showed that the antioxidant activity positively correlated with the content of bioactive compounds, especially monomers and dimmers, flavan-3-ols, and procyanidin polymers. Other authors confirm this relationship. Their studies indicated that the antioxidant activity depends not only on their amount but also on the structure of the compound and the proportion of individual fractions, i.e., anthocyanins, phenolic acids, flavan-3-ols, and flavonols, in the tested material [58,59].


**Table 4.** Antioxidant, and antidiabetic potential of black garlic dried by different methods.

\* Values followed by the same letter, within the same column, were not significantly different (*p* > 0.05), according to Tukey's HSD test.

### *2.5. α-Amylase, and α-Glucosidase Inhibitory Effect*

According to the estimates by the World Health Organization (WHO), chronic noncommunicable diseases, including diabetes are currently the main cause of death worldwide. Type 2 diabetes is characterized by hyperglycemia with impaired carbohydrate, lipid, and protein metabolism resulting from defects in insulin secretion, insulin action, or both. A rapid postprandial increase in glycemia is due to starch degradation by pancreatic amylase, followed by the blocking of the resultant glucose by intestinal α-glucosidase. Therefore, it is suggested that the inhibition of these enzymes is an important strategy for the management of type 2 diabetes. In addition, several studies have investigated the antidiabetic potential of black garlic. It has been shown that black garlic exhibited ameliorative action on glycometabolic biomarkers in diabetic rats, as well as the ability to decrease blood glucose, glycated hemoglobin, and markedly increase serum insulin [15]. Thomson et al. [7] showed garlic extract significantly attenuated the elevation of serum triglyceride, and lowered lipid peroxidation in the kidney, and liver tissues. Therefore, in the present study, black garlic after various treatments was tested for its ability to inhibit α-amylase, and α-glucosidase (Table 4).

The IC<sup>50</sup> values of the black garlic for inhibiting α-amylase activity ranged from 61.73 mg/mL to 229.46 mg/mL, and the inhibition effect was the highest for black garlic dried by VMD500W, while samples with CD70 ◦C-9h/VD60 exhibited the lowest inhibition effect (Table 4). In turn, the ability to inhibit α-glucosidase ranged from 51.21 mg/mL (CD70 ◦C-3h/125W) to 231.85 mg/mL in garlic treated by MF + H2O. Nevertheless, no recurring pattern was observed in terms of the creation of anti-α-amylase, and anti-αglucosidase potential by type of fraction, the content of polyphenolic compounds, or drying method. In this case, it is worth emphasizing the high efficiency of black garlic after PEF treatment to inhibit both enzymes, especially since this sample was characterized by both low contents of polyphenolics and low antioxidant activity. This may be due to the higher amount of specific amino acids, which are abundantly described in the literature as molecules with a significant antidiabetic potential [60]. Numerous studies describe the key role of amino acids in shaping the health potential of black garlic [15,57]. The CEF + H2O treatment may be an effective tool to increase the availability of amino acids in plant materials and thus result in their greater effectiveness in the area of antidiabetic properties, but confirmation of this statement requires detailed studies, which are planned in the future.

#### **3. Materials and Methods 3. Materials and Methods** *3.1. Material*

*Molecules* **2023**, *28*, x FOR PEER REVIEW 13 of 19

#### *3.1. Material* Reagents for antioxidant and biological activity tests (α-amylase, α-glucosidase,

Reagents for antioxidant and biological activity tests (α-amylase, α-glucosidase, starch, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, TPTZ, p-nitrophenyl-α-D-glucopyranoside) were purchased from Sigma-Aldrich (Steinheim, Germany). Standards for polyphenols were acquired from Extrasynthese (Lyon Nord, France). Acetonitrile, methanol, and formic acid for ultra-performance liquid chromatography (UPLC; gradient grade) were purchased from Merck (Darmstadt, Germany). Black garlic was produced from regular garlic (*Allium sativum* L.) as a result of the aging process. The material used in this study was obtained from the local producer ("PASZKÓW" Farma Tadeusz Kaczmarczyk, Swidnica, Poland). Garlic slices were sliced in half and each sample ´ consisted of 100 g of material of the initial moisture content *Mc* = 0.66 kg/kg db. starch, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, TPTZ, pnitrophenyl-α-D-glucopyranoside) were purchased from Sigma-Aldrich (Steinheim, Germany). Standards for polyphenols were acquired from Extrasynthese (Lyon Nord, France). Acetonitrile, methanol, and formic acid for ultra-performance liquid chromatography (UPLC; gradient grade) were purchased from Merck (Darmstadt, Germany). Black garlic was produced from regular garlic (*Allium sativum L.*) as a result of the aging process. The material used in this study was obtained from the local producer ("PASZKÓ W" Farma Tadeusz Kaczmarczyk, Świdnica, Poland). Garlic slices were sliced in half and each sample consisted of 100 g of material of the initial moisture content *Mc* = 0.66 kg/kg db.

#### *3.2. Pretreatment Methods 3.2. Pretreatment Methods*

A pulsed electric field (PEF) treatment was performed in prototype equipment built at the University of Agriculture in Krakow, Poland (model ERTEC-SU1 with Line Parameters Analyzer type AS3 Mini) [29,61]. The material was immersed in water and then placed in a treatment chamber between the electrodes. Each time, the material was treated with 300 impulses with 10 s break between the impulses. The strength of the electric field was fixed at 5 kV/cm. A pulsed electric field (PEF) treatment was performed in prototype equipment built at the University of Agriculture in Krakow, Poland (model ERTEC-SU1 with Line Parameters Analyzer type AS3 Mini) [29,61]. The material was immersed in water and then placed in a treatment chamber between the electrodes. Each time, the material was treated with 300 impulses with 10 s break between the impulses. The strength of the electric field was fixed at 5 kV/cm.

A constant electric field (CEF) was applied in an apparatus consisting of two flat electrodes placed in a chamber with the material in between [32]. The high voltage pulse generator was set to 9 kV. The treatment consisted of 1272 impulses for 10 s each, with a 5 s break between the impulses. Two variants of CEF treatment were performed: with and without the addition of water to the material. A constant electric field (CEF) was applied in an apparatus consisting of two flat electrodes placed in a chamber with the material in between [32]. The high voltage pulse generator was set to 9 kV. The treatment consisted of 1272 impulses for 10 s each, with a 5 s break between the impulses. Two variants of CEF treatment were performed: with and without the addition of water to the material.

A magnetic field (MF) was used to treat the material alone and with the addition of water using a magnetic field of 100 mT at the frequency of 50 Hz for 2 h using equipment built at the University of Agriculture in Krakow (Kraków, Poland) (Figure 4). A magnetic field (MF) was used to treat the material alone and with the addition of water using a magnetic field of 100 mT at the frequency of 50 Hz for 2 h using equipment built at the University of Agriculture in Krakow (Kraków, Poland) (Figure 4).

**Figure 4.** Diagram of the solenoid for magnetic stimulation of black garlic samples: 1—supply chamber; 2—cooling water outlet; 3—cooling water inlet; 4—power cables; 5—sample containers; 6—carcass; 7—coil. **Figure 4.** Diagram of the solenoid for magnetic stimulation of black garlic samples: 1—supply chamber; 2—cooling water outlet; 3—cooling water inlet; 4—power cables; 5—sample containers; 6—carcass; 7—coil.

#### *3.3. Drying Methods 3.3. Drying Methods*

In the first stage of the experiment, black garlic cloves were subjected to convective drying (CD) performed using the convective dryer designed and constructed at the Institute of Agricultural Engineering (Wrocław, Poland) [62] at 60 and 70 °C and the air velocity of 0.5 m/s for 9 h (Figure 5). Then, pre-dried black garlic was moved to a vacuum dryer (VD) and dried at 60 °C under 100 Pa (SPT-200, ZEAMiL, Horyzont, Kraków, Poland) until the moisture content of the material was below 13%. Vacuum-microwave drying (VMD) was performed using SM200 dryer (Plazmatronica, Wrocław, Poland) [63] at 125, In the first stage of the experiment, black garlic cloves were subjected to convective drying (CD) performed using the convective dryer designed and constructed at the Institute of Agricultural Engineering (Wrocław, Poland) [62] at 60 and 70 ◦C and the air velocity of 0.5 m/s for 9 h (Figure 5). Then, pre-dried black garlic was moved to a vacuum dryer (VD) and dried at 60 ◦C under 100 Pa (SPT-200, ZEAMiL, Horyzont, Kraków, Poland) until the moisture content of the material was below 13%. Vacuum-microwave drying (VMD) was performed using SM200 dryer (Plazmatronica, Wrocław, Poland) [63] at 125, 250, and 500 W power of magnetrons under the pressure in the range of 50–70 hPa. Combined CD-VMD

consisted of 3 h or 6 h of convective pre-drying at 70 ◦C followed by vacuum-microwave finishing drying at 125 W (CD3h-VMD or CD6h-VMD, respectively). i50 (Flir Systems AB, Stockholm, Sweden). Experiments were carried out in two repetitions.

250, and 500 W power of magnetrons under the pressure in the range of 50–70 hPa. Combined CD-VMD consisted of 3 h or 6 h of convective pre-drying at 70 °C followed by vacuum-microwave finishing drying at 125 W (CD3h-VMD or CD6h-VMD, respectively).

In the second stage of this study, black garlic pretreated using a PEF, a CEF, and a MF was subjected to convective pre-drying at 70 °C for 3 h and then vacuum-microwave

The surface temperature of all the samples was measured using an infrared camera

*Molecules* **2023**, *28*, x FOR PEER REVIEW 14 of 19

**Figure 5.** Schematic representation of the two stages of experiments performed in this study and analyses performed in this study. CD—convective drying; VMD—vacuum-microwave drying; VD—vacuum drying; PEF—pulsed electric field; CEF—constant electric field; MF—magnetic field. **Figure 5.** Schematic representation of the two stages of experiments performed in this study and analyses performed in this study. CD—convective drying; VMD—vacuum-microwave drying; VD—vacuum drying; PEF—pulsed electric field; CEF—constant electric field; MF—magnetic field.

*3.4. Physical Properties* 3.4.1. The Moisture Content In the second stage of this study, black garlic pretreated using a PEF, a CEF, and a MF was subjected to convective pre-drying at 70 ◦C for 3 h and then vacuum-microwave finishing drying at 125 W.

The moisture content (Mc) of the samples was measured using vacuum-drying (SPT-200, ZEAMiL, Horyzont, Kraków, Poland) at 100 Pa and 80 °C for 48 h. Measurements The surface temperature of all the samples was measured using an infrared camera i50 (Flir Systems AB, Stockholm, Sweden). Experiments were carried out in two repetitions.

#### *3.4. Physical Properties*

finishing drying at 125 W.

#### 3.4.2. Water Activity 3.4.1. The Moisture Content

were performed in duplicate.

The water activity of the samples both before and after treatments was measured using AquaLab Dew Point 4TE (Decagon Devices Inc., Pullman, WA, USA) water activity meter. Water activity was measured at 25 ± 0.5 °C in triplicate. The moisture content (Mc) of the samples was measured using vacuum-drying (SPT-200, ZEAMiL, Horyzont, Kraków, Poland) at 100 Pa and 80 ◦C for 48 h. Measurements were performed in duplicate.

#### 3.4.2. Water Activity

3.4.3. Color Color measurement was performed with the black garlic powder obtained after grinding the material using Profi Cook grinder (PC-KSW 1021) after each drying treat-The water activity of the samples both before and after treatments was measured using AquaLab Dew Point 4TE (Decagon Devices Inc., Pullman, WA, USA) water activity meter. Water activity was measured at 25 ± 0.5 ◦C in triplicate.

#### ment. Then, the sample was placed in a vessel placed on a Chroma Meter CR-400 colorimeter (Minolta Co., Ltd., Osaka, Japan). The color was expressed within CIE *L\*a\*b\** color 3.4.3. Color

space meaning lightness (*L\**)*,* hues from red to green according to the values of *a\** coordinate, and from yellow to blue for *b\** coordinate. The browning index (*BI*) was calculated based on the equations (Equations (2) and (3)) provided by Subhashree et al. [64]: = <sup>∗</sup> + 1.75 ⋅ ∗ 5.645 ⋅ <sup>∗</sup> + − 3.012 ⋅ ∗ 100 ⋅ ( − 0.31) Color measurement was performed with the black garlic powder obtained after grinding the material using Profi Cook grinder (PC-KSW 1021) after each drying treatment. Then, the sample was placed in a vessel placed on a Chroma Meter CR-400 colorimeter (Minolta Co., Ltd., Osaka, Japan). The color was expressed within CIE *L\*a\*b\** color space meaning lightness (*L\**)*,* hues from red to green according to the values of *a\** coordinate, and from yellow to blue for *b\** coordinate. The browning index (*BI*) was calculated based on the equations (Equations (2) and (3)) provided by Subhashree et al. [64]:

$$X = \frac{a^\* + 1.75 \cdot L^\*}{5.645 \cdot L^\* + a - 3.012 \cdot b^\*} \tag{2}$$

$$BI = \frac{100 \cdot (X - 0.31)}{0.17} \tag{3}$$

(2)

(3)

#### *3.5. Chemical Analysis*

3.5.1. Determination of Phenolic Compounds, including Polymeric Proanthocyanidins by UPLC

Determination of phenolics in black garlic and black garlic after different treatments was performed as described by Nowicka et al. [65] using an Acquity UPLC system (Waters, Milford, MA, USA) with a photodiode and a fluorescence detector with the mass detector G2 Qtof mass spectrometer (Waters, Manchester, UK). The absorbance values of flavan-3-ols and phenolic acids were read at 280 nm, and 320 nm, respectively. The content of polymeric procyanidins was analyzed by the phloroglucinol method [66]. All samples were measured in triplicate, and the results were expressed as mg per 100 g dry mass.

#### 3.5.2. Analysis of Health-Promoting Properties by In Vitro Methods

To analyze the antioxidant activity, the ORAC (oxygen radical absorbance capacity), FRAP (ferric reducing antioxidant power), and ABTS (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) methods were used as described earlier by Ou et al. [67], Benzie et al. [68], and Re at al. [69], respectively. The obtained results were presented as mmol Trolox per 100 g dry matter (dm).

The α-amylase and α-glucosidase inhibitory effects (antidiabetic activity) of the black garlic were determined according to the procedure described by Nowicka et al. [70]. In the α-amylase activity determination, the primary sample consisted of black garlic extracts, to which the starch solution, as well as α-amylase solution, were added. The reaction of these components was carried out at 37 ◦C for 15 min, and then it was stopped with 0.4 M HCl followed by the addition of potassium iodide with iodine.

In the case of α-glucosidase activity analysis, the basic sample including back garlic extracts and enzymes, was incubated at 37 ◦C for 10 min, then the β-D-glucosidase substrate was added, and incubated as before. Acarbose was included as a positive control for αamylase and α-glucosidase assay. The absorbance measurement was done at 600 nm and 405 nm for α-amylase and α-glucosidase activity, respectively, using a Synergy H1 spectrophotometer (BioTek, Winooski, VT, USA). Both tests were performed in triplicate, and the results were expressed as IC<sup>50</sup> values.

#### *3.6. Statistical Analysis*

Statistica 13.3 software (StatSoft, Krakow, Poland) was used for all statistical analyses. The results were presented as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed in this study. HSD Tukey's least significance test (*p* < 0.05) was used to determine homogenous groups. Table Curve 2D v. 5.0 (Systat Software, Inc., San Jose, CA, USA) was used to fit mathematical models to the experimental data based on the lowest values of the root mean square error (RMSE) and the highest values of the coefficient of determination (R<sup>2</sup> ).

#### **4. Conclusions**

The impact of convective, vacuum-microwave, and combined drying methods was investigated in this study together with the effect of nonthermal pretreatment including a pulsed electric field, a constant electric field, and a magnetic field on the quality of black garlic. This study found that the application of different pretreatment methods significantly reduced the overall time of combined drying as well as leading to the lowest values of the final moisture content among considered treatment variants. Drying kinetics were described by the Weibull model, which presented a very good fit. Moreover, pulsed electric field and magnetic field treatments proved to be effective in maintaining the health-promoting properties of black garlic, especially in terms of antidiabetic potential. However, vacuummicrowave drying positively affected the phenolic content and antioxidant capacity of black garlic due to the thermal effect that led to the cleaving of bound forms of polyphenols and consequently an increase in bioactive compounds. This study showed that nonthermal pretreatments considerably affect the drying process as well as the quality of dried materials; however, future studies are needed to find the optimal process parameters.

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

**Funding:** This research was funded by the Program of the Minister MNiSW "Inkubator Innowacyjno´sci 4.0" (Poland) based on the agreement number MNiSW/2020/334/DIR, 28.09.2020.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author upon reasonable request.

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

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**


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