Mulberry is regarded as multipurpose plant due to recognition of its role in environmental safety approach, as a medicinal plant and its industrial uses in various sectors [1
]. The broad ecological adaptability of mulberry to different environmental conditions such as temperature, water, and soil, and enables it to have multiple ecological protective functions in water and soil conservation, wind resistance and sand consolidation, water source preservation and air refreshment [2
]. The health-promoting effect of white mulberry (Morus alba
L.) results from the presence of many compounds with biological activity. The literature indicates the presence of polyphenolic compounds, such as, quercetin, kaempferol, rutin, isoquercetin, astragalin, myricetin derivatives and other glucosides [3
]. Mulberry preparations are also a source of tannins and coumarin compounds (scopolin and skimin). The presence of numerous phenolic acids (chlorogenic acid, caffeic acid, hydroxybenzoic or ferulic acid) was determined in mulberry leaves and fruits [4
]. Furthermore, mulberry was shown to contain terpenes such as citral, linalol acetate, linalol or cis-3-hexen-1-ol, and steroids such as β-sitosterol [5
]. From the pharmacological point of view, the important active ingredients of mulberry are alkaloids which may affect carbohydrate metabolism: 1,5-dideoxy-1,5-imino-D-sorbitol (DNJ) and its derivatives. The variety and specificity of the compounds contained in the leaves, roots, fruits and bark of mulberry has an effect on the antioxidant and health-promoting activity in model and biological systems, which has been repeatedly demonstrated in the available literature [11
Cereal products, including bread, provide components necessary for the proper functioning of the body such as proteins, saccharides, minerals and biologically active substances (e.g., dietary fiber and vitamins). Fresh bread is a short-life product, so freezing the dough is a common practice in industrial production due to its many economic advantages. Freezing of bread saves time and labor during production and extends the shelf-life of the product, which facilitates product distribution [12
]. However, the process of freezing and storage affects the structure and quality of the dough, mainly due to the formation of ice crystals, recrystallization and redistribution of water in the product [13
]. These processes negatively affect the quality of the dough and lead to an increase and destruction of the crumb pores, weakening of the gluten network and deformation of the starch grains, thus there is a search for ways to reduce the adverse changes associated with the cooling processes [14
Bread fortification is a common practice used to prevent malnutrition and nutritional deficiencies in populations and adding functional properties, but additional ingredients can also influence other parameters, such as appearance, flavor and taste [15
] Presence of various ingredients in bread, such as plants’ components and extracts, can also influence the physicochemical stability and quality parameters during storage [17
]. In studies in which bread was enriched with Garcinia mangostana
fruit extract, it was shown that such an addition increases the attractiveness of the bread in terms of its functionality, while reducing the bread volume [19
]. Sun-Waterhouse et al., by adding extracts of dried apples, kiwi and blackcurrants to bread, showed that they can be a beneficial functional addition [20
]. Peng et al. proved that the antioxidant properties of bread can be improved by adding grape seed extract rich in catechins and epicatechins [21
]. In another publication, it was shown that Cistus
extract contributes to the improvement of the microbiological quality of bread [22
]. The addition of Cistus
extract influenced the content of polyphenols by increasing it from 8.88 (wheat bread) to 78.71 mg/100 g (bread with 7.5% cistus extract). The literature also indicates other raw materials that can be used to enrich bread, such as hops, rice, blueberries, etc. However, there is no data in the literature on the impact of refrigerated storage on baking or ready-made bread on the functional properties resulting from the addition of white mulberry. Using mulberry leaves to enrich bread is consistent with the principle of sustainable development due to the use of raw materials that are a good source of compounds contributing to the improvement of the well-being of the population. Mulberry leaves are currently not a component of many foods for humans and are available in many locations around the world. Bread enriched with ingredients derived from white mulberry and the good quality of such bread after storage in a frozen state may indicate new directions in reducing food waste by extending the shelf life of bread that maintains high sensory and microbial quality and functional properties. The aim of the study was to investigate the effect of additives in the form of mulberry leaves and fruit extract on the quality and properties of fresh and frozen bread. Enriched and control bread products were compared in terms of sensory characteristics and polyphenol content, as well as antioxidant and microbiological activity.
Industrial extract from mulberry leaves was obtained by extraction of leaves by a counter-flow method at a temperature of 80–90°C, with water in counter-flow in a quantity 10 times higher than the raw material. Vacuum concentration of the extract was performed at 70–80°C in 7.5–10 h at 0.6–0.8 atm. The concentration was completed when the extract reached 25–30% of dry substance. Then spray drying of the concentrated extract was carried out using a Niro Atomizer SR16 type spray dryer equipped with a disc as a spraying element. The capacity of the dryer was about 16 kg of evaporated water per hour. The drying was carried out with hot air in counter-flow. Inlet air temperature at 175–185℃and outlet air temperature at 85–95℃ were controlled. As a result of drying, a fine powder of dry herbal mixture extracts was obtained. The obtained extract in the form of powder was stored at room temperature without access to light.
3.2. Bread Recipe and Baking Process
The bread was baked using the direct, single-phase method with ingredients listed in Table 1
. The dough was obtained by mixing the ingredients in a laboratory mixer GM-2 (ZBPP, Bydgoszcz). Fermentation at 37℃ at 80% relative humidity lasted 1 h (with punching after 30 min). The dough was fermented in the same way as wheat dough, without using leaven. Pieces of dough weighing 250 g were placed in the fermentation chamber until optimal growth (37℃, relative humidity 80%). The baking was carried out for 30 minutes at 220℃ in a convection oven. After baking, breads were allowed to cool down to room temperature for 3 h. Subsequently, the breads were sliced (slices about 1.5 cm thick), and kept frozen (−20°C) until analysis. Breads for analysis were thawed in the air at room temperature until they reached the temperature of 20°C. Bread with mulberry (Morus alba
fruit) was determined as PM1, analyzed on the day of baking, and PM30 after 30 days of freezing storage, and bread without additives as control bread PK1 analyzed on the day of baking and PK30 after the storage.
3.3. Qualitative and Quantitative Analysis of Phenolic Acids and Flavonoids
The qualitative and quantitative analysis of phenolic acids, flavonoids and ascorbic acid was carried out using a high-performance liquid chromatograph Agilent Infinity (Agilent Technologies, USA) according to the methodology of Kobus et al. 2019 [23
]. The NovaPack C18 column (5 mm, 150 × 3.9 mm) was used to separate phenolic acids. The separation using a UV detector was monitored at 250 nm for p-hydroxybenzoic, protocatechuic, gallic and vanillic acids and at 320 nm for caffeic, chlorogenic, p-coumaric, ferulic and sinapic acids. In the analysis of flavonoid content, the separation was monitored at a wavelength of 370 nm. Myricetin, quercetin, rutin, hyperoside and isoquercetin, as well as kaempferol and astragalin were determined in the examined extracts. Phenolic acids and flavonoids were identified and quantified by comparing their retention times and peak areas with those of their standards.
3.4. Antioxidant Activity in Model Systems with DPPH and ABTS
The determination of activity with respect to DPPH radicals was performed based on the method described earlier by Kobus et al. [3
]. The principle of the method was based on spectrophotometric measurement (Metertek SP-830, Taiwan) of the color of the reaction mixture, in which, depending on the antioxidative capacity of the extract under investigation, the free radicals of DPPH (1,1-diphenyl-2-pyrylhydrazil) were scavenged. Measurement of absorbance was performed at a wavelength of 517 nm. The antiradical activity was expressed in mmol Trolox per 1 g of extract dry matter and in quercetin equivalents (mmol/1 g of extract dry matter). The results were also expressed as EC50 (mg extract/ml) and AE coefficient (AE = 1/EC50). The determination of activity against ABTS cationic radicals was performed based on the method described by Re et al. (1999). Antiradical activity was expressed in Trolox and quercetin equivalents (mmol/1 g of extract dry matter), EC50 (mg extract/ml) and AE coefficient (AE = 1/EC50) were determined.
3.5. Chelating and Reducing Properties
Ferrous ion-chelating effects and reducing power of samples were estimated according to the method of Kobus-Cisowska et al. by colorimetric measurement [24
]. For this colorimetric assay, 1 mL of sample, 0.1 mL of 2 mM FeCl2
and 0.2 mL of the ferrozine reagent were added to each tube. The mixture was vortexed for ~60 s and left for 20 min at room temperature. Absorbance values were recorded (λ = 700 nm) using the Meterek SP 830 apparatus (Taipei, Taiwan). Deionized water was used as the control, and ferrozine was used as a reference.
3.6. Bread Microbiological Quality Evaluation
Microbiological tests were performed to determine the total number of aerobic mesophilic and psychrophilic bacteria, mesophilic yeasts and molds, the presence of Enterobacteriaceae bacteria, Listeria monocytogenes and Salmonella spp., Staphylococcus aureus, the number of aerobic Bacillus spp. and sulfate reducing bacteria. Determination of the total number of mesophilic and psychrophilic microorganisms was performed in accordance with the guidelines of PN-EN ISO 4833:1:2013. Determination of the number of molds and yeasts was in accordance with the guidelines of PN-ISO-21527:2:2009. Detection and determination of Enterobacteriaceae was in accordance with the guidelines of PN-ISO 21528-2:2005.
Determination of the number of microorganisms was performed using the Koch flooding method. The number of microorganisms was expressed as an arithmetic mean in the form of colony forming units in relation to 1 g of product (cfu/g). Microbiological analysis included detection of the presence and number of Listeria monocytogenes according to the guidelines of PN-EN ISO 11290-1:1999/A1:2005 and the presence of Salmonella spp. according to the guidelines of PN-EN ISO 6579:2003/A1:2007. Additionally, the count of indicator microorganisms, anaerobic spore-forming bacteria in Wrzosek’s medium, coliforms in lactose and diamond green medium, Enterococci in sodium azide and bromocresol purple medium, and coagulase-positive staphylococci in Giollitti-Cantoni medium were determined.
3.7. Sensory Evaluation
Sensory evaluation of bread samples was carried out in a sensory laboratory meeting the requirements of the PN-ISO 8589:1998 standard. The method of quantitative descriptive analysis (i.e., sensory profiling) was applied to the detailed sensory characteristics of these samples, which was performed by a 20-person team specially trained for this purpose. A total of 16 individual qualitative characteristics of taste, smell and color selected in the preliminary tests were evaluated. The intensity of each qualitative note was determined by means of a 10 cm unstructured linear scale with appropriate border markings. The obtained results were replaced by numerical values.
3.8. Statistical Analysis
The statistical analysis was carried out with the STATISTICATMPL 13.1 software from StatSoft. Basic descriptive statistics for individual parameters were performed. The results presented in the paper are an arithmetic mean of at least two series performed in three repetitions.
The comparison of mean values of the examined traits was made with the use of variance analysis for factorial systems with different number of observations, and intergroup differences were evaluated with Tukey’s test or Spjotvoll’s test (extended Tukey’s test for unequal number of samples). The fulfillment of the assumptions of this analysis was also checked. Pearson correlation coefficients were calculated to assess the strength of the relationship between the tested samples. The significance of the correlation coefficient was checked with Student’s T-test. Statistical conclusions were made at the significance level of α = 0.05. In order to examine the shape and direction of the relationship between the studied activities, appropriate non-linear regression equations (logarithmic and exponential) were determined. The principal components analysis (PCA) method was used.
Technological processes in which both low and high temperatures are used influence the stability of bioactive compounds. Processing processes, among which drying (high temperature) or freezing is the most effective method of preserving the nutritional value and sensory quality of raw materials. In order to maintain active compounds, the process of freezing most plant materials is preceded by blanching. In the case of bread, it is not necessary because the bread before freezing is usually pre-baked and then finally baked. However, raw materials such as fruit, including mulberry fruit, should be blanched to inactivate the enzymes responsible for the oxidation of natural antioxidants [25
]. Spray drying and freeze drying (freeze drying) are the most commonly used methods used in the food industry to dry thermolabile substances. Dried mulberry fruit and white mulberry spray-dried extract can be a valuable ingredient in functional foods such as bread. However, it is true that during spray drying the temperature reaches 185 degrees, and according to different studies the polyphenol content of the dried extract could possibly be about 20% higher if a carrier such as maltodextrin was used [26
]. The literature on the subject has repeatedly emphasized the beneficial properties of mulberry and the possibilities of its use in food technology. However, there are no reports to date that have determined the influence of mulberry addition on the quality and properties of frozen bread. Earlier studies indicated the possibility of using mulberry fruit as an ingredient in muesli [4
]. It was observed that in the samples with extract addition, the polyphenol content was stable in comparison with the control sample, which showed a statistically significant decrease in total polyphenol content after baking. Therefore, it can be assumed that the addition of mulberry not only increased the content of individual phenolic acids and flavonoles in new bread, but also that these compounds stabilized the presence of other polyphenolic substances. The stability of antioxidant compounds was confirmed in the results of the cited study, during fat stability tests by Rancimat and Oxidograph method. It was shown that heating of oil with mulberry extract at 110°C does not inactivate its antioxidant activity.
Moreover, the components of mulberry leaf extract showed antioxidant activity measured by the protective factor. The temperature during bread baking does not exceed 100°C inside the loaf, so most of the mulberry compounds are probably not thermally inactivated. The stability of phenolic acids and flavonols in model systems was also confirmed in other studies, where high stability was demonstrated in a wide temperature range for ferulic acid, the content of which was constant in the temperature range from 25°C to 100°C, and the antioxidant effect of caffeic and sinapic acid was higher at 90°C than 22°C [27
]. Mulberry fruits have a high antioxidant potential and the ability to chelate metals [29
]. The presence of most flavonols and acids increased the antiradical activity and reducing properties. Other authors pointed out that the activity measured by reducing power and ABTS-+ is influenced by the same compounds [30
]. The antioxidant effect of mulberry polyphenols was documented many times before, which may have determined the effect in bread samples [6
]. The bread consisted mainly of wheat flour (38%) and rye flour (26%). The presence of polyphenols in grains was found mainly in the fruit and seed coat, aleurone cells, husk and rarely in the germ. The basic raw material for bread production was whole wheat flour, in which the main polyphenolic compound is tricin (5,7,4’-trihydroxy-3’,5’-dimethoxy flavone). This compound and others may have interacted with mulberry polyphenols.
The baking process allows for high purity and, consequently, microbiological stability of the resulting product. However, certain groups of yeasts and molds, acidifying type bacteria as well as endospore-forming bacteria can survive the temperature of 95 °C, which is achieved during baking in the crumb. The source of contamination may be plant raw materials (cereals) and the environment of the production process (hygienic conditions of the plant), the most common are molds such as Aspergillus, Penicillium and Fusarium and bacteria of the genera Rhizopus, Penicillium and Bacillus [31
]. It was found that the breads were characterized by high microbiological purity, which is indicated by the results of analyses performed for the count of indicator microorganisms and the absence of pathogens. Precise determination of the components of sensory quality is a subject of interest for food producers due to the possibility of manufacturing a competitive product with repeatable parameters, meeting growing consumer requirements. The addition of the so-called non-bread flours increases taste and smell qualities of bread, prolongs its freshness and modifies its texture characteristics [27
]. The addition of milk and milk products or unusual cereals and cereal products is also frequently used, and literature reports the addition of plant extracts such as grape, rosemary extracts or soya and barley sprouts to bread and cereal products [21
]. All these ingredients must of course have a positive effect on the sensory quality of the food. Data evaluating an effect of rosemary extract on the sensory quality of bread are available in the literature. Costa de Conto et al. indicated that the additive above 2.5 g was negatively perceived by consumers [33
]. The bread was characterized by a typical taste identified as rosemary and had a bitter aftertaste. Therefore, other authors suggested that ground leaves of raw materials that are a source of polyphenols could be used instead of extracts. Polyphenols are responsible for the bitterness of the leaves and extracts made from them, tannins form complexes mainly with polysaccharides and proteins, giving bitterness and astringency. Ferulic acid and vanillic acid, which are found in extracts from mulberry leaves and fruit, are responsible for the bitterness of the taste. These acids also give the wrong bitter bean-like taste to protein isolates from oil plants. Fresh mulberry leaves contain flavonol glycosides, mainly 3-O-di- and 3-O-triglucosides of quercetin and kaempferol, as well as C-glycosides of quercetin. These glucosides may affect the bitter but also the sour taste. However, the appropriately selected composition of the raw materials of the bread being tested, despite the raw materials which may have a negative influence on sensory qualities, allowed to obtain an attractive functional bread with mulberry.