Black Garlic Powder as an Ingredient to Enhance the Functional and Sensorial Properties of Bread and Its Shelf Life

Featured Application

The addition of black garlic powder in bakery products to improve their antioxidant compounds and their shelf life is a method of significant interest that does not decrease their sensory quality or consumer acceptance. Obtaining black garlic powder from bulbs or products that are not directly usable is notable for improving the circular economy; at the same time, it can be introduced onto the market as a new functional product.

Abstract

Black garlic is a processed product prepared via the heat treatment of whole garlic bulbs (Allium sativum L.) at high temperatures and humidity levels for several days, resulting in black cloves with a sweet taste and increased bioactive substances. The purpose of this study was to evaluate the quality, chemical and functional characteristics, and shelf life of bread with different percentages (0.5%, 1%, 2%, and 3%) of Voghiera black garlic powder (BGP). The analysis conducted on the powder showed important changes in composition, and the polyphenol content and antioxidant activity increased when the garlic was processed. The data obtained demonstrated that BGP did not modify the nutritional composition of the bread, while the total polyphenol and total flavonoid content and antioxidant activity progressively increased from 1.40 mg GAE/g, 0.28 mg CE/g, and 0.15 mg TE/g to 1.75 mg GAE/g, 0.56 mg CE/g, and 0.47 mg TE/g, respectively, as the amount of BGP increased from 1 to 3% in comparison with wheat bread. Moreover, BGP improved the shelf life of breads enhanced with 2 and 3% of powder, reducing microorganism growth and water loss; however, on the other hand, the added powder caused an increase in acidity and color intensity. At higher powder percentages, the acceptability and palatability of the bread to the consumers were reduced.

1. Introduction

Black garlic is a highly popular product typical of various Asian cuisines. Recently, it has been studied and appreciated more all over the world, particularly after a study by Badura et al. [1], where the authors described the presence of bulbs of black garlic in medieval cargo shipwrecked in the 15th century near the port of Gdansk (Poland). Black garlic is produced as a result of a thermal process at a temperature of 40–90 °C and a 60–90% humidity for an incubation period of 10–90 days. Due to the applied conditions, white garlic progressively loses its pungent odor and taste, its color grows dark, and its consistency becomes soft and elastic. Black garlic has an extended shelf life and a slight sweet taste [2,3]; the Maillard reaction and a decrease in water activity are responsible for the change in its organoleptic properties [4].

When compared with that of white garlic, the chemical composition of black garlic contains an increased amount of melanoidins, reducing sugars, organic acids, and bioactive substances such as 5-hydroxymethylfurfural and phenolic compounds, while there are reduced amounts of polysaccharides and organosulfur compounds [4]. Several studies have reported that black garlic extract demonstrates antioxidant, anti-allergic, anti-diabetic, anti-inflammation, and anti-carcinogenic effects similar to or greater than those of white garlic due to this new composition [2,3,5].

Garlic (Allium sativum) is an aromatic herbaceous annual spice, and it has been authenticated as one of the oldest and most important herbs, which has been used since ancient times in traditional medicine due to its anti-inflammatory, cardiovascular-protective, hepatoprotective, renal-protective, antibacterial, and antifungal activities [6]. Organic sulfides, such as allicin, alliin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, ajoene, and S-allyl-cysteine, are major bioactive components in garlic [7], but they also have a pungent flavor that limits its application in the development of food products. Aqueous garlic extract [8] and microencapsulated garlic oil nanoemulsions [9] have been added to bread to develop functional and consumer-accepted products with prolonged shelf lives.

Wheat bread and cereal-based food products are widely consumed across the globe. They contribute macronutrients—mainly carbohydrates but also proteins and lipids—and micronutrients to the human diet. The fortification of wheat bread is a historical strategy to improve its dietary value and efficiency and decrease its micronutrient deficiencies. Currently, diabetes, cardiovascular disease, and colon cancer are increasing in prevalence, associated with the high consumption of refined cereal products rich in easily digestible carbohydrates [10].

In recent years, there has been an increase in the tendency to incorporate fruits and vegetables into bread and its substitutes. Cereals and cereal-based products are generally the chosen foodstuffs to enrich the diet with functional compounds such as fiber, polyphenols, and minerals, with the aims of preserving human health and providing new flavors more appreciated by consumers [11].

The added vegetable materials have to be carefully chosen so as to not reduce the acceptability and rheology of a product [12]. Nonetheless, they represent an opportunity for producing newly textured, functional, ready-to-eat cereal-based snacks with a high nutritional value. In this study, black garlic powder (BGP) obtained from white garlic var. Voghiera PDO was used to enrich bread. Garlic var. Voghiera PDO is a typical Italian product cultivated in Voghiera (Ferrara, Italy), and its chemical and genomic profiles have been completely characterized in our previous research [13]. The aim of this study was to incorporate black garlic powder to enhance the functional and sensorial properties and shelf life of bread.

2. Materials and Methods

2.1. Materials

In this study, white garlic var. Voghiera PDO was provided by the Garlic Producers Consortium of Voghiera (Ferrara, Italy), while black garlic and black garlic powder (BGP) were provided by a local firm (Nero Fermento S.r.l., Ravenna, Italy). The black garlic was produced from the white garlic by conducting a thermal process at an elevated temperature between 50 and 70 °C for 55 days, while the powder was obtained via grinding the dried black garlic cloves at 90 °C for 4 h. Wheat flour (9.4% proteins, W 150), extra-virgin olive oil, yeast, and salt were purchased at a local supermarket.

2.2. Bread-Making Procedure

The control bread was prepared using the following formula: 590 g of wheat flour, 25 g of extra-virgin olive oil, 25 g of yeast, 5 g of sugar, 5 g of salt, and 350 g of water. BGP was added at 5, 10, 20, and 30 g/kg of dough. Different breads were made using a commercial bread machine (Panasonic SD-YR2550, Kadoma, Japan) with an electronically controlled kneading oven, capable of carrying out the three main operations of bread production: kneading (15 min, room temperature), leavening (2 h, 37 °C), and baking (1 h, 200 °C). The time required was 3 h and 15 min.

Five different breads were obtained, designated as wheat bread, 0.5 g/100 g, 1 g/100 g, 2 g/100 g, and 3 g/100 g, each of 1 kg and with an increasing content of BGP (Figure 1).

The breads were cooled at room temperature and cut into three parts. One part of each was stored inside a plastic bag designed for food storage at −20 °C for chemical analysis, the second part of each was used for sensory analysis, and the third part of each was stored for 8 days at room temperature for shelf-life evaluation. For each bread type, the bread-making process was repeated in triplicate, and the samples were homogenized and analyzed in triplicate.

2.3. Proximate Composition

Moisture content was determined through drying the samples at 105 °C to a constant weight and expressed as g per 100 g of fresh weight (g/100 g fw) [14]. Total nitrogen compounds were evaluated using 1 g of dry matter and the Kjeldahl method [15]. Ashes were quantified through the incineration of 1 g of dry matter placed in a muffle furnace (VELP Scientifica, Usmate, MB, Italy) at 570 °C for 5 h [16]. Total lipids were assessed via Soxhlet extraction (VELP Scientifica, Usmate, MB, Italy) [17]. Insoluble dietary fiber and soluble dietary fiber were measured according to the instruction protocol provided by the Megazyme Total Dietary Fibre Assay Procedure Kit (Megazyme International, Co., Wicklow, Ireland) [18]. Finally, carbohydrates were determined using the difference in mass. All results were expressed as g/100 g dw.

2.4. Extraction of Free and Bound Phenolic Compounds

Briefly, 5 g of bread or garlic samples and 2 g of BGP were extracted with a 15 mL solution of methanol/water (4:1 v/v). The sample was shaken on a magnetic stirrer at 500 rpm and room temperature, and it was protected from light with silver foil. The sample was extracted three times, and after each step, it was centrifuged (Centrifuge Thermo PK121R, Thermo Fisher Scientific, Waltham, MA, USA) at 2500× g and 4 °C for 5 min, and the supernatants were recovered and combined. The solvent extracts were used to determine free phenols, free flavonoids, and antioxidant activity.

To obtain the bound phenolic compounds, the residues of the previous extraction were suspended in distilled water and digested with 0.5 mL of simulated gastric solution (1 g of pepsin in 25 mL of HCl 0.1N) and 2 mL of simulated intestinal solution (0.1 g of pancreatin and 0.625 g of biliar salt in 25 mL of NaHCO₃ 0.5 N) for 2 h at 37 °C. The samples were centrifuged at 2500× g and 4 °C for 5 min, and the supernatants were collected. The enzymatic extracts were used to determine bound phenolic compounds, bound flavonoids, and antioxidant activity.

The total phenolic compounds, total flavonoids, and total antioxidant activity were obtained by summing the free and bound results.

2.5. Total Phenols, Total Flavonoids, and Antioxidant Capacity Determination

The total phenol content (TPC) in the extracted samples was measured using the Folin–Ciocalteu assay as described by Singleton et al. [19]. The results were expressed as g of gallic acid equivalents per g of dry weight (dw), mg GAE./g dw. According to Chlopicka et al. [20], the total flavonoid content was spectrophotometrically calculated using a method based on the formation of complex flavonoid–aluminum with a maximum absorbability at 510 nm. The results were expressed as g of catechin equivalents per g of dry weight, mg CE./g dw.

The antioxidant activities of the samples were determined through DPPH assay. This was conducted using DPPH (2,2-diphenyl-1-picrylhydrazyl), which could determine the samples’ ability to inhibit free radicals, and a UV–Vis (VWR, UV-633PC) spectrophotometer operating at 515 nm [20]. The results were expressed as mg of Trolox equivalents per g of dry weight, mg TE./dw.

2.6. pH and Acidity

pH and acidity were determined according to the method proposed by Fik [21]. In order to measure the pH, which was determined with a pH meter (Velp Scientifica Srl, Usmate Velate, Italy), 10 g of crumbs was homogenized with 50 mL of distilled water for 5 min on a magnetic stirrer (Velp Scientifica Srl, Usmate Velate, Italy). For the determination of acidity, 25 g of crumbs was shaken for 1 h with 250 mL of distilled water on a magnetic stirrer. The homogenate was filtered under vacuum, and 50 mL of it was titrated with NaOH 0.1 M to a pH = 7. The degree of acidity in the bread was expressed as the mL of NaOH 0.1 M required to neutralize 100 g of bread crumbs.

2.7. Water Holding Capacity (WHC)

To measure the WHC, 10 g of powdered dry crumbs was homogenized with 10 mL of distilled water in a pre-weighed centrifuge tube for 30 min. The sample was mixed for 30 min using a magnetic stirrer (Velp Scientifica Srl, Usmate Velate, Italy) at room temperature. Then, the homogenate was centrifuged for 10 min at 3000× g, and the sediments were weighed after completely removing the supernatant. The amount of water in the sediment was calculated as follows:

WHC (g_water/g_dw) = [(W₂ − W₁)/W₀]

where W₀ is the sample weight, W₁ is the sample + centrifuge tube weight, and W₂ is the weight of the tube plus the sediments [22].

2.8. Crumb Color

Bread crumb color was measured using the Colorflex L2 colorimeter (Hunter Associates Laboratory Inc., Reston, VA, USA) and L*a*b*. In this model, the L* value indicates brightness (0: dark color; 100: bright color), +a* values suggest a red color, −a* values reflect a green color, +b* values signal a yellow color, and −b* values represent a blue color [23].

2.9. Microbiological Analyses

For the microbiological analysis of bread with and without black garlic powder (samples stored in plastic bags for different durations of 0 days, 4 days, and 8 days, designated as t₀, t₄, and t₈), 10 g of a crumb sample was dissolved with 90 mL of 0.1% sterilized peptone solution and shaken for 5 min in a vortex (Velp Scientifica Srl, Usmate Velate, Italy). Dilutions were made using the same solution (0.1% peptone). Yeasts and molds were quantified on plates with Sabouraud agar medium (10 mL on each plate) incubated at 25 °C for 5 days [24]. Total aerobic mesophilic bacteria were determined on plates with potato dextrose agar (PDA) medium (10 mL on each plate), also incubated at 25 °C for 5 days.

2.10. Sensory Analysis

The overall appreciation for different sensory attributes was evaluated using 8 untrained panelists (5 women and 3 men) and according to Roncolini et al. [25]’s study on the textural and sensory assessment of a bread with insects. The following attributes were evaluated: aroma, taste, color, texture, and overall acceptability. For each parameter, a five-point hedonic scale was used, where 5, 4, 3, 2, and 1 corresponded to ratings of very good, good, fair, poor, and very poor, respectively. The panelists were asked to evaluate randomly coded 2 cm slices of bread and rinse their mouth with water before and after each bread tasting. Data were expressed as the means.

2.11. Data Analysis

All analyses were carried out in triplicate, and the values of each parameter were expressed as the mean ± standard deviation. A one-way analysis of variance was performed using SPSS for Windows (Version 22.0). Tukey’s test provided the mean comparison, with the level of statistical significance set at p < 0.05.

3. Results and Discussion

3.1. Composition and Functional Properties of White and Black Garlic and Black Garlic Powder

The Voghiera fresh garlic contained 9.03 ± 0.6% proteins, 0.07 ± 0.01% lipids, 1.04 ± 0.06% ash, 5.13 ± 0.89% total fiber and 19.12 ± 1.5% sugar. The white garlic was transformed into black garlic through a thermal process with rising temperatures between 50 and 70 °C and a controlled humidity over a period of 55 days. The black garlic developed a sweet taste and gelatinous texture in contrast to the white garlic. Due to its rubbery consistency, black garlic is difficult to process and add to dough; therefore, it was converted to powder via drying at about 90 °C for 4 days and subsequent grinding (Figure 2).

The proximate compositions of the white and black Voghiera garlic and relative powder (BGP) are shown in

Table 1. Composition of white and black garlic and relative powder dry weight (mean ± SD).

	White Garlic	Black Garlic	BGP
Moisture (g/100 g fw)	65.61 ± 0.58 a	31.73 ± 0.30 b	5.83 ± 0.13 c
Proteins (g/100 g dw)	26.26 ± 1.76 a	26.07 ± 1.13 a	28.48 ± 0.14 a
Lipids (g/100 g dw)	0.19 ± 0.01 a	0.10 ± 0.00 b	0.10 ± 0.01 b
Ash (g/100 g dw)	3.04 ± 0.16 a	3.92 ± 0.11 b	4.74 ± 0.30 c
Total Fiber (g/100 g dw):	14.91 ± 2.58 a	39.02 ± 2.47 b	46.36 ± 2.02 c
Insoluble fiber (g/100 g dw)	7.75 ± 1.56 a	33.70 ± 2.25 b	45.95 ± 2.14 c
Soluble fiber (g/100 g dw)	7.15 ± 0.43 a	5.31 ± 0.17 b	0.40 ± 0.22 c
Sugars (g/100 g dw)	55.61 ± 4.51 a	30.89 ± 1.44 b	20.32 ± 2.17 c
Total phenols (mg GAE./g dw)	0.62 ± 0.04 a	3.95 ± 0.02 b	10.97 ± 0.17 c
Total flavonoids (mg CE./g dw)	0.02 ± 0.00 a	2.12 ± 0.09 b	2.64 ± 0.11 c
DPPH (mg TE./g dw)	0.05 ± 0.00 a	1.09 ± 0.01 b	3.45 ± 0.01 c

Rows with different letters indicate significant differences (p < 0.05). BGP: black garlic powder.

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During the thermal process, the samples lost a major amount of water, the content of which decreased from 65.61 ± 0.58% in the white garlic to 31.73 ± 0.30% in the black garlic and 5.83 ± 0.13% in the powder. The protein, lipid, and ash content slightly increased in the final product as compared with the starting white garlic. In the fatty acids composition, an increase of saturated and monounsaturated and a decrease of polyunsaturated were observed (Supplementary Material, Table S1). The BGP showed a high total fiber content, represented above all by insoluble fiber, while sugars and soluble fiber were significantly reduced after the thermal process.

The effects of temperature, relative humidity, and incubation time on garlic’s composition, especially regarding carbohydrates, have been widely reported [26,27]. Garlic carbohydrates include fructan, sucrose, fructose, and glucose [3]. In the intermediate stage of the Maillard reaction, in which sugars are fragmented, Yuan et al. [28] demonstrated that the content of fructan in black garlic decreased remarkably during thermal processing, while the level of fructose drastically increased. In contrast, the content of glucose and sucrose was found to be almost equivalent to that in fresh garlic. Glucose and fructose derived from fructan can further react with amino acids, increasing the concentrations of the Amadori and Heyns compounds [29]. The Maillard reaction explains the reduction in carbohydrates and soluble fiber between the white and black garlic dry matter and between the black garlic and black garlic powder observed in our samples.

Čeryová et al. [30] analyzed seven garlic cultivars and determined an average content of total phenol compounds and flavonoids of 954.17 ± 124.30 mg GAE./Kg and 21.02 ± 4.04 mg CE./kg of dry matter, respectively. In the Voghiera PDO garlic (Table 1), the total phenolic compounds and total flavonoids were 0.62 ± 0.04 mg GAE./g and 0.02 ± 0.00 mg CE./g, respectively. Phenolic compounds and flavonoids have strong antioxidant properties, and the white garlic showed an antioxidant capacity of 0.05 mg TE./g. At the end of the process, the total phenols and total flavonoids were 3.95 and 10.97 mg GAE./g dw and 2.12 and 2.11 mg CE./g dw in the black garlic and BGP, respectively, while the antioxidant capacity increased to 1.09 mg TE./g dw in the black garlic and 3.45 mg TE./g dw in the BGP.

According to the literature, black garlic’s total phenol content, total flavonoid content, and antioxidant capacity are higher than those of white garlic [3,27]. Recently, Sari and Najman [31,32] demonstrated an increase in total phenols, total flavonoids, and antioxidant capacity in black garlic powder in comparison with black garlic. Navajas-Porras et al. [33] demonstrated that furosine, hydroxymethyl furfural, and furfural produced by the Maillard reaction contributed to the value of these effects. The increase in antioxidant activity in the black garlic and BGP samples may be attributed in part to the drying process, which releases phenolic compounds, and in part to the Maillard reaction, which produces phenol-like compounds. This could be an reason for the increase in phenols and antioxidant capacity, but not in total flavonoids, observed in the BGP.

3.2. Enriched Breads’ Nutritional and Functional Composition

Table 2. Nutritional value of wheat bread and breads enriched with black garlic powder (mean ± SD).

Sample	Moisture (g/100 g fw)	Proteins (g/100 g dw)	Lipids (g/100 g dw)	Ash (g/100 g dw)	Total Fiber (g/100 g dw)	Sugars (g/100 g dw)
Wheat bread	41.85 ± 0.69 a	12.56 ± 0.27 a	2.40 ± 0.06 a	1.08 ± 0.08 a	9.62 ± 0.31 a	75.35 ± 0.02 a
0.5 g/100 g	40.92 ± 0.36 a	12.69 ± 0.09 a	2.40 ± 0.02 a	1.18 ± 0.06 a	9.10 ± 0.01 a	75.14 ± 0.06 a
1 g/100 g	39.57 ± 0.73 a	12.64 ± 0.23 a	2.37 ± 0.05 a	1.13 ± 0.03 a	9.27 ± 0.17 a	74.79 ± 0.04 ab
2 g/100 g	38.24 ± 0.69 a	12.63 ± 0.23 a	2.33 ± 0.04 a	1.24 ± 0.01 a	10.15 ± 0.17 b	74.05 ± 0.37 ab
3 g/100 g	38.41 ± 0.06 a	12.60 ± 0.28 a	2.34 ± 0.01 a	1.29 ± 0.02 a	10.59 ± 0.10 b	72.88 ± 0.19 b

Columns with different letters indicate significant differences (p < 0.05), and 0.5 g/100 g, 1 g/100 g, 2 g/100 g, and 3 g/100 g correspond to wheat bread with 5, 10, 20, 30 g/kg of black garlic powder added, respectively.

shows the composition of the wheat bread and breads enriched with black garlic powder. The wheat bread, used as a control, contained 12.56 g/100 g of proteins, 2.40 g/100 g of lipids, 1.08 g/100 g of ash, 9.62 g/100 g of total fiber, and 75.35 g/100 g of sugars. The incorporation of black garlic powder at 0.5, 1, 2, and 3 g/100 g produced differences in the nutritional composition of the breads only at the major concentrations. When adding 0.5 and 1 g/100 g of BGP, there were no significant influences on the composition of the bread, while adding 2 and 3 g/100 g produced a slight increase in the total fiber content and slight decrease in sugars. Overall, the added quantities did not change the nutritional values of the breads. These amounts were decided based on the results reported by Liu et al. [34], who incorporated black garlic into rye flour noodles to develop enhanced healthy noodles. They demonstrated that the addition of less than 5.0% BGP had no significant influence on the textural properties of rye flour noodles, with satisfactory consumer acceptability.

Free and bound forms of phenolic compounds are present in vegetable foods.

Table 3. Free, bound, and total phenolic and flavonoid content and antioxidant activity in bread samples (mean ± SD) and black garlic powder (BGP).

Sample	Free Phenols (mg GAE./g dw)	Bound Phenols (mg GAE./g dw)	Total Phenols (mg GAE./g dw)
Wheat bread	0.14 ± 0.00 a	1.26 ± 0.08 a	1.40 ± 0.08 a
0.5 g/100 g	0.15 ± 0.01 a	1.26 ± 0.06 a	1.41 ± 0.104 b
1 g/100 g	0.17 ± 0.01 ab	1.38 ± 0.02 a	1.55 ± 0.102 c
2 g/100 g	0.24 ± 0.03 bc	1.36 ± 0.09 a	1.60 ± 0.112 d
3 g/100 g	0.30 ± 0.02 bc	1.45 ± 0.09 a	1.75 ± 0.07 e
BGP	2.17 ± 0.12	8.79 ± 0.05	10.97 ± 0.17
	Free flavonoids (mg CE./g dw)	Bound flavonoids (mg CE./g dw)	Total flavonoids (mg CE./g dw)
Wheat bread	0.02 ± 0.01 a	0.26 ± 0.02 a	0.28 ± 0.03 a
0.5 g/100 g	0.01 ± 0.01 a	0.30 ± 0.02 ab	0.30 ± 0.03 a
1 g/100 g	0.01 ± 0.01 a	0.36 ± 0.02 bc	0.37 ± 0.03 ab
2 g/100 g	0.05 ± 0.00 a	0.41 ± 0.01 c	0.46 ± 0.01 bc
3 g/100 g	0.12 ± 0.01 b	0.44 ± 0.03 c	0.56 ± 0.02 c
BGP	0.45 ± 0.04	2.19 ± 0.18	2.64 ± 0.11
	DPPH solvent extract (mg TE./g dw)	DPPH enzymatic extract (mg TE./g dw)	DPPH total (mg TE./g dw)
Wheat bread	0.10 ± 0.01 a	0.05 ± 0.00 a	0.15 ± 0.02 a
0.5 g/100 g	0.10 ± 0.00 a	0.09 ± 0.01 b	0.20 ± 0.02 b
1 g/100 g	0.11 ± 0.00 a	0.15 ± 0.01 c	0.25 ± 0.01 c
2 g/100 g	0.14 ± 0.00 b	0.18 ± 0.01 d	0.32 ± 0.01 d
3 g/100 g	0.17 ± 0.00 c	0.30 ± 0.01 e	0.47 ± 0.00 e
BGP	1.09 ± 0.01	2.36 ± 0.02	3.45 ± 0.01

Columns with different letters indicate significant differences (p < 0.05), and 0.5 g/100 g, 1 g/100 g, 2 g/100 g, and 3 g/100 g correspond to wheat bread with 5, 10, 20, and 30 g/kg of black garlic powder added, respectively.

presents the free, bound, and total phenolic and flavonoid content and antioxidant activity of the bread samples and BGP. Bound phenols and flavonoids represented major parts of the total content in the bread samples and BGP. In the BGP, the content of free and bound phenolic compounds was 2.17 and 8.79 mg GAE./g, respectively, while that of free and bound flavonoids was 0.35 and 1.76 mg CE./g, respectively. The enzymatic treatment increased the quantity of phenols and flavonoids by 4–5-fold, but the antioxidant capacity increased by only 2-fold. In the bread samples, the bound phenolic compounds present in the control bread were not statistically different from those in the breads enriched with BGP, while the free phenol compounds increased in the enriched breads with respect to the control bread but only with the addition of 2–3%. The same results were observed for free and bound total flavonoids. The antioxidant capacity determined using solvent and enzymatic extracts showed that the presence of BGP influenced the overall antioxidant activity of the enriched bread samples.

The major phenolic compounds in wheat are phenolic acids and flavonoids, and the C-glycosidic-conjugating form of flavones and flavonols is the most common [35]. Phenolic acids exist in both free and bound forms; the latter represent 80–95% of the total amount, are ester-linked to cell wall polymers, and consist mainly of ferulic acid and its oxidatively coupled dimers [36]. Free and bound phenolic compounds are subjected to neo-formation/degradation/hydrolysis reactions during the baking process due to the effects of temperature [37].

In garlic, 28 polyphenolic compounds, mainly anthocyanins, flavones, flavonols, and phenolic acids, have been identified, and among these, the 3-O-glucosides of quercetin and kaempferol and coumaric and caffeic acids are the most concentrated [38]. The concentrations of phenolic acid and flavonoid constituents increase during thermal processing, and enzymatic digestion could release these phenolic compounds, explaining the increment in bound phenols and flavonoids observed in the BGP and enriched bread samples. Further, the Maillard reaction, responsible for the transformation of white garlic into black garlic during the thermal process, contributes to this increment.

Wu et al. [39] proved that melanoidins from black garlic are nearly indigestible. During the simulated in vitro digestion of melanoidins, numerous volatile compounds were eliminated and several polysaccharides were degraded; furthermore, the antioxidant capacity of melanoidins still remained at over 60%. Additionally, Navajas-Porras et al. [33] determined a statistically positive correlation between antioxidant capacity and furosine and furfural content as a function of time and temperature during cooking.

3.3. Bread Shelf-Life Evaluation

Black garlic has been found to have higher antibacterial activity than fresh garlic. It exhibits inhibiting activity against the growth of pathogenic bacteria, including Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, and Pseudomonas aeruginosa [40,41]. In particular, Gram-positive bacteria are more sensitive than Gram-negative bacteria to black garlic, while yeasts, including Saccharomyces cerevisiae, are the most resistant microorganisms [42]. Moreover, black garlic melanoidins and protein extract exhibit excellent in vitro α-amylase and α- and ß-glucosidase inhibition activity, with a dose-dependent relationship between melanoidin and protein extract concentrations and the inhibition rate [43].

According to Wu et al. 2025 [44], our BGP sample showed low solubility and a low WHC (0.86 ± 0.04 g_water/g_dw). Suspensions of 1% in water showed a pH of 4.19 ± 0.06 and acidity of 1.4 ± 0.0. The BGP dissolved in 0.1% peptone solution at the same concentration did not present any microorganism growth in the bread.

For the evaluation of the bread’s shelf life, the samples were stored in micro-perforated plastic bags designed for food storage for 8 days at room temperature. After 0(t₀), 4(t₄), and 8(t₈) days, the pH, acidity, % of water loss, and water-holding capacity were determined. Moreover, microbiological analyses were performed using the wheat and BGP-added breads at the three storage times.

The results obtained are reported in

Table 4. Effect of storage and BDP addition on % water loss, WHC, pH, and acidity in bread samples (mean ± SD).

		Wheat Bread	0.5 g/100 g	1 g/100 g	2 g/100 g	3 g/100 g
pH	t0	5.65 ± 0.06 a	5.56 ± 0.07 a	5.25 ± 0.02 b	5.15 ± 0.03 b	5.12 ± 0.04 b
	t4	5.57 ± 0.11 a	5.31 ± 0.05 ab	5.18 ± 0.10 b	4.91 ± 0.10 c	4.71 ± 0.07 c
	t8	5.50 ± 0.09 a	5.25 ± 0.07 b	5.07 ± 0.07 bc	4.82 ± 0.10 cd	4.62 ± 0.08 d
Degree of acidity	t0	1.6 ± 0.0 a	1.9 ± 0.2 a	2.4 ± 0.0 b	2.8 ± 0.0 c	3.2 ± 0.0 d
	t4	1.2 ± 0.0 a	2.0 ± 0.0 b	2.4 ± 0.0 c	2.9 ± 0.2 d	3.9 ± 0.2 e
	t8	1.2 ± 0.0 a	1.7 ± 0.2 b	2.1 ± 0.2 c	2.8 ± 0.0 d	4.0 ± 0.0 e
% water loss	t0	0.0 ± 0.0 a	0.0 ± 0.0 a	0.0 ± 0.0 a	0.0 ± 0.0 a	0.0 ± 0.0 a
	t4	3.5 ± 0.14 a	3.7 ± 0.14 a	3.8 ± 0.07 a	2.4 ± 0.14 b	1.5 ± 0.14 c
	t8	20.4 ± 1.06 a	12.4 ± 1.13 b	10.4 ± 0.92 c	3.5 ± 0.14 d	3.4 ± 0.14 d
WHC	t0	2.55 ± 0.06 ab	2.51 ± 0.06 ad	2.24 ± 0.08 bc	2.25 ± 0.04 c	2.72 ± 0.02 d
gwater/gdw	t4	2.14 ± 0.05 a	1.83 ± 0.08 a	2.07 ± 0.17 a	1.89 ± 0.05 a	2.09 ± 0.11 a
	t8	2.59 ± 0.02 a	2.56 ± 0.17 b	2.59 ± 0.02 a	2.62 ± 0.05 a	2.75 ± 0.06 b

Rows with different letters indicate significant differences (p < 0.05), and t₀, t₄, and t₈ correspond to storage times.

. Before the analysis, the breads were weighed, and the weight decrease was understood as water loss. The percentage of water loss decreased as a function of the quantity of BGP added, and this trend was particularly evident at the time t₈, when the wheat bread lost 20.4% of its water and the bread enriched with 3 g/100 g of BGP lost only 3.4% of its water.

The ability of proteins to absorb and retain water against gravity is expressed by the WHC parameter. This parameter plays an important role in the textural quality of baked products. During the storage period, the WHC decreased from t₀ to t₄ and returned to the initial value at t₈ for each sample. Among different breads, the WHC value slightly decreased from the wheat bread to the bread with 2% BGP added, and there was an increment in the bread with 3% of BGP product. Although the latter product demonstrated a minor loss in water content, its textural acceptability was lower because it was judged as too dry). Liu et al. [45] studied the WHC of black garlic protein extract and observed that enzymatic hydrolysis significantly improved the WHC. During hydrolysis, proteins were cleaved into smaller peptide fragments, exposing more hydrophilic groups.

The pH of the breads decreased as a function of the quantity of BGP added and showed a slight tendency to decrease over the storage period. The percentage of BGP had a great influence on sensory evaluation; in fact, the addition of 3 g/100 g of BGP reduced the overall acceptability, and the taste was judged as having an acidic aftertaste.

According to the pH, the acidity of the bread increased according to the % of BGP added, while no significant change was observed during the storage period, with the exception of the bread prepared with 3 g of BGP, for which the acidity increased from t₀ to t₈.

A microbiological analysis (

Table 5. Effect of storage (0, 4, and 8 days of storage in a plastic bag) and BGP added at different concentrations on the growth of yeasts and molds (PDA medium) and mesophilic bacteria (Sabouraud medium).

After 5 Days of Incubation		Wheat Bread	0.5 g/100 g	1 g/100 g	2 g/100 g	3 g/100 g	BGP
PDA medium	t0	<10	<10	<10	<10	<10	<10
	t4	<850	<850	<850	<800	<500	<10
	t8	>5000	>5000	<1500	<200	<100	<10
Sabouraud medium	t0	<10	<10	<10	<10	<10	<10
	t4	>1000	<100	<100	<100	<10	<10
	t8	>5000	>5000	<1500	<500	<100	<10

) of the wheat bread and bread with black garlic powder prior to storage showed that the initial total microorganism levels were below 10 cfu/g (t₀). At this time, the wheat bread and bread with different powder concentrations added did not contain any microorganisms, and throughout storage at 20 °C for 5 days, no microbiological modifications were observed.

Conversely, after 4 and 8 days of storage in the micro-perforated plastic bags, microorganism growth was detected.

After 4 days of storage, the presence of more than 1000 cfu/g of yeasts and molds in the wheat bread was detected, while less than 100 cfu/g was found in the bread with 0.5, 1, and 2% of black powder added, and in the bread with an additional 3 g/100 g, a notable microbial count of less than 10 cfu was observed. The content of mesophilic bacteria in agar medium was less than 850 cfu/g in the wheat bread and bread with 0.5, 1, and 2% black powder added, and it was less than 500 cfu/g in the 3 g/100 g bread. After 8 days of storage, there was more microorganism growth, with more than 5000 cfu/g in the wheat bread in both media, while the 3 g/100 g bread showed a very low incidence of yeasts, molds, and bacteria, with less than 100 cfu/g. Although the microbial load was quite high, it decreased significantly when 1 and 2% of black garlic powder was added, reducing to a value <200 cfu and <100 cfu, respectively, in the PDA medium and Sabouraud medium.

The BGP analyzed in the same conditions did not present any microorganism growth during the considered storage period.

3.4. Color Parameters and Sensory Analysis

Color is one of the most important psychological properties of food products that influence consumers’ perceptions. The BGP exhibited color parameters of 1.52 ± 0.21 L, 0.05 ± 0.03 a*, and 0.26 ± 0.06 b*, which were similar to the results reported by Choi et al. [46]. The color of the bread crumbs changed to dark brown as a function of the % BGP added (

Table 6. Color parameters in bread crumb samples (mean ± SD) and black garlic powder (BGP).

Samples	L	a*	b*
Wheat bread	62.55 ± 1.03 a	−4.08 ± 0.05 a	16.89 ± 1.08 a
0.5 g/100 g	46.36 ± 1.00 b	−1.77 ± 0.22 b	15.40 ± 1.01 ab
1 g/100 g	33.34 ± 3.07 c	−1.47 ± 0.09 b	13.51 ± 1.51 b
2 g/100 g	37.33 ± 1.37 d	−1.07 ± 0.13 c	13.33 ± 0.66 b
3 g/100 g	24.72 ± 1.40 d	0.24 ± 0.13 d	12.83 ± 1.20 b
BGP	1.52 ± 0.21	0.05 ± 0.03	0.26 ± 0.06

Columns with different letters indicate significant differences (p < 0.05), and 0.5 g/100 g, 1 g/100 g, 2 g/100 g, and 3 g/100 g correspond to wheat bread with 5, 10, 20, and 30 g/kg of black garlic powder added, respectively.

). The degree of lightness progressively decreased, the redness (a* value) increased, and the yellowness (b* value) decreased from the wheat bread to the bread with 3% BGP. These results are according to a sensory evaluation (Table 6), in which the color intensity due to the gradual addition of BGP, particularly 3 g/100 g, was judged as too dark.

The effects of the addition of BGP on the sensory characteristics of the bread are reported in

Table 7. Sensory evaluation of the wheat bread and breads enriched with black garlic powder on a 5-point hedonic scale (5 = very good; 4 = good; 3 = fair; 2 = poor; 1 = very poor).

Sample	Color	Texture	Taste	Smell	Overall Acceptability
Wheat bread	4.8 a	4.3 a	4.4 a	4.2 a	4.3 a
0.5 g/100 g	4.3 ab	4.1 a	3.9 ab	3.3 b	3.9 ab
1 g/100 g	4.1 ab	3.7 ab	3.6 ab	3.0 bc	3.6 bc
2 g/100 g	3.3 b	3.3 b	3.3 b	2.7 c	3.2 c
3 g/100 g	1.9 c	2.3 c	1.8 c	2.0 d	2.3 d

Columns with different letters indicate significant differences (p < 0.05), and 0.5 g/100 g, 1 g/100 g, 2 g/100 g, and 3 g/100 g correspond to wheat bread with 5, 10, 20, and 30 g/kg of black garlic powder added, respectively.

. Preferences for color, texture, taste, and smell and overall acceptability were evaluated using a hedonic scale from 1 to 5. The addition of BGP progressively increased the color intensity and changed the taste and smell of the bread, reducing the overall acceptability. The addition of 3 g/100 g BGP significantly reduced the overall acceptability due to a stronger smell, a darker color, and drier crumbs than the control. The addition of 0.5 g /100 g of BGP did not significantly change the sensory characteristics of the bread and its overall acceptability. Finally, the addition of 1–2 g/100 g modified the sensorial attributes, but the product remained acceptable with a characteristic flavor.

4. Conclusions

Significant differences were determined between the white and black var. Voghiera PDO garlic and between the black garlic and its powder with regard to their proximate compositions and bioactive compounds. These changes could be related to the temperature, humidity, and time parameters used in their production. The BGP showed an increase in ash and fiber, which corresponded to a decrease in carbohydrates, and also a large increase in phenol compounds and antioxidant activity related to the black garlic. This powder was used to fortify wheat bread. The nutritional composition of the breads did not change with the addition of 0.5 and 1 g/100 g of BGP, while adding 2 and 3 g/100 g of black garlic powder, a slight increase in the total fiber content and slight decrease in sugars were observed. The phenol compounds, flavonoids, and antioxidant activity increased as a function of % BGP added.

The addition of BGP improved the shelf life of the breads in terms of microbial development and water loss but caused an increase in acidity and a dark color intensity. The color, acidity, and dry texture progressively reduced the overall acceptability of the products.

Comprehensively considering all the determined properties, the 1.0% and 2.0% BGP-fortified breads were the most acceptable products among all the prepared samples.

Therefore, black garlic powder can be used as an ingredient to enhance the functional and sensorial properties and shelf life of bread.

The supplementation of wheat bread and functional, ready-to-eat cereal-based snacks with black garlic or black garlic powder could be a notable future trend to achieve products with different flavors and potential health benefits to consumers, including young people.

This study and its future applications could play a significant role in emphasizing the value of products containing black garlic, which is still not widely used today.