The mass of 100 fresh sea buckthorn fruits was 72 ± 10 g, with 18.1 ± 0.11% dry weight, 8.94 ± 0.15° Brix soluble solids in fruit pulps, and total titratable acidity of 1.7 ± 0.1 g citric acid/100 g fresh weight.
2.1. Characteristics of Wheat Flour and Sea Buckthorn Berry Flour
The physicochemical quality indicators of the wheat flour and the concentrations of polyphenols and the antioxidant activity of the berry flour are represented in
Table 1 and
Table 2, respectively.
In our study, white wheat flour without grain husk (which was separated from flour after milling, in the form of bran) was used. It is known that white wheat flour has very low phenolic content, since the polyphenols are present especially in the removed bran. Phenolic acids and flavonoids are the most common forms of phenolic compounds in whole wheat. They are found mainly in grain husk, as soluble free compounds, soluble conjugates that are esterified to sugars and other low molecular mass compounds, and in the insoluble bound form linked through ester bonds to cell wall structural components such as cellulose, lignin, and proteins [
9].
Concerning the berry flour, the results for total polyphenols revealed 1467 mg gallic acid equivalents (GAE)/100 g, when determined by the Folin-Ciocalteu method and 1311 mg GAE/100 g, when determined by absorbance at 280 nm. Among them, 555 mg GAE/100 g were flavonoids. These differences can be explained by the interferences which occur when employing the Folin-Ciocalteu method, which basically quantifies the reducing potential of a solution and, therefore, overestimates the total polyphenol content [
10].
The main seven individual phenolics identified in the sea buckthorn berry flour were catechin (35.3 mg/100 g), hyperoside (23.6 mg/100 g), chlorogenic acid (11.1 mg/100 g), trans- and cis-resveratrol (10.4 mg/100 g and 10.8 mg/100 g, respectively), ferulic acid (10.3 mg/100 g) and protocatechuic acid (7.0 mg/100 g).
The study of Hajazimi et al. [
11] evaluated and applied a HPLC-CoulArray method (high-performance liquid chromatography with coulometric array detection) for simultaneous determination of flavonols and phenolic acids in several berries including sea buckthorn. The aforementioned authors found quercetin, myricetin, isorhametic, caffeic, ferulic, and
p-coumaric acids in an extract prepared with 50% aqueous methanol containing TBHQ (tert-butylhydroquinone) antioxidant and 1.2 M HCl. But, contrary to our results, they didn’t identify gallic and vanillic acids. The total concentration of polyphenols determined in sea buckthorn berries was 270.5 mg/100 g dry weight [
11], which is significantly lower compared to the amount found in the current research. Ma et al. [
12] found twenty-six flavonol glycosides in wild sea buckthorn varieties from China, Finland and Canada. Quercetin was also one of the major aglycones identified in the respective study, while Arimboor et al. [
13] showed that gallic acid was the predominant phenolic acid in both sea buckthorn berries and leaves, among gallic, protocatechuic,
p-hydroxybenzoic, vanillic, salicylic,
p-coumaric, cinnamic, caffeic and ferulic acids. The present study showed that ferulic acid is the most abundant phenolic acid. Geographical origin, climate, soil, harvest time, genetic and cultivar variability have a great effect on both type and content of the identified polyphenols [
14,
15], thus such differences are not surprising. Ten sea buckthorn populations from different natural habitats located in the Central Albraz Mountains in Iran were collected and evaluated in 2014 and 2015 [
15]. The highest fruit flavonoid content in 2014 and 2015, i.e., 2.40 and 3.19 mg/g was reported for Baladeh populations, while the lowest, i.e., 1.04 and 0.92 mg/g—for the Dehdar population. The same authors have documented that flavonoid content varies significantly among genotypes from Russia, Canada, China, and Finland [
15,
16].
Ma et al. [
12] and Burri et al. [
17] reported that antioxidant capacity and the content of polyphenols depend significantly on the species and the cultivar in question. The method used to determine this parameter can also impact greatly the result, as presented in
Table 2 (total polyphenols).
Kant et al. [
18] have analyzed the content of polyphenols and the antioxidant activity using various methods (among which ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) radical scavenging), of Indian sea buckthorn extracts obtained in various solvents, i.e., methanolic and aqueous. The extracts were able to scavenge different radicals in a concentration dependent manner. The linear regression analysis showed that 100% methanolic extract was better scavenger of ABTS, DPPH (2,2-diphenyl-1-picrylhydrazyl) and hydroxyl radicals. Therefore, both the extraction technique and the solvent influence the results for total polyphenols [
18].
A relatively low amount of carotenoids of 34.93 ± 1.3 mg/100 g was found in sea buckthorn berry flour (
Table 2), while, the comparison of the abovementioned result with those published by Pop et al. [
19] showed slight differences. The authors obtained values comprised between 53 and 97 mg/100 g dry weight in berries of six varieties of Carpathian sea buckthorn (
Hippophae rhamnoides L., ssp.
Carpatica) analyzed using a combination of the HPLC-PAD (high-performance liquid chromatography with photodiode array detector), GC-MS (gas chromatography–mass spectrometry) and UHPLC-PAD-ESI-MS (ultra-high performance liquid chromatography-photodiode array detector electrospray ionization-mass spectrometry) techniques [
19]. Andersson et al. [
20] report contents comprised between 12 mg/100 g dry weight and 142.5 mg/100 g dry weight after analyzing berries from four cultivars of sea buckthorn during ripening in three consecutive years. A study of the carotenoids’ composition in sea buckthorn berry flour is therefore recommended, especially considering that carotenoids also exhibit antibacterial properties [
21,
22].
2.2. Effects of Sea Buckthorn Berry Flour Adition on Wheat Bread’s Properties
The results of sensory analysis of the wheat bread samples with added sea buckthorn berry flour are presented in
Table 3.
The results of the sensory analysis have shown that the addition of 1% sea buckthorn concentration influenced favorably the organoleptic index of the sample obtained. This sample had a smooth, glossy, golden crust, elastic core, dry taste, well-developed porosity, pleasant taste and aroma. On the other hand, samples with 3% and 5% sea buckthorn berry flour (SBBF) had dark crust, dry crumb, poorly developed porosity, and a specific sea buckthorn berry flavor and odor. Nevertheless, the total scores on the organoleptic analysis of the samples containing 3% and 5% SBBF are within the 24.1–30.0 interval, which implies that the products are of very good quality [
23].
The physicochemical quality indicators of the products obtained with the addition of sea buckthorn berry flour and control are presented in
Table 4.
The analysis of the results presented in
Table 4 has shown that the addition of sea buckthorn berry flour influenced the moisture content of the bread core. Therefore, sea buckthorn slows down the aging process due to the ability of its components (cellulose, hemicellulose, pectin) to bind and retain water in the product. The slow migration of moisture during the storage of the bakery product contributes to maintaining the freshness of the bread core [
24].
The acidity of the bakery products increased with the concentration of the added sea buckthorn berry flour. This phenomenon can be explained by the presence of organic acids and sugars from sea buckthorn berry flour, which accelerated dough fermentation. Consequently, the acidity of the samples with sea buckthorn increased by 100–291.7% compared to control.
Porosity plays a leading role in the digestibility of bakery products. The higher the porosity of the bread core, the easier to digest it by the consumer’s body [
25]. According to our research, the porosity of the core in the sample with the addition of 1% SBBF increased to 72.7%. Vitamins and simple carbohydrates of sea buckthorn stimulate the fermentative activity of bakery yeast, influencing the porosity of the bread core. The porosity decreased by 5.7% and 17.4% in the samples with 3% and 5% sea buckthorn, respectively, compared to control. This can be explained by the fact that the obtained dough had a low extensibility, contributing to the lowering of the gas retention capacity during fermentation. As a result, the specific volume of the respective samples was decreased by 17.7% and 23.6% compared to control. In the case of 1% SBBF, the increase of specific volume was by 1% compared to control.
The influence of the addition of sea buckthorn on the microbiological safety of wheat bread was investigated (
Table 5). The analysis of the results showed that even 1% of added sea buckthorn berry flour reduced the risk of rope spoilage in wheat bread. The spoilage appeared after 96 h for 1% SBBF, after 120 h for 3% SBBF and after 144 h for 5% SBBF in bread, compared to the control, for which spoilage appeared after 72h. The shelf life of the fortified products was extended by 24, 48 and, 72 h, respectively.
The increasing time of storage in the presence of berry flour may be connected to the significant content of phenolics that we noticed, as many studies document the antimicrobial effect of the polyphenols and recommend the commercial use of polyphenol rich extracts in food processing [
26,
27,
28,
29]. Sea buckthorn berries contain a series of biologically active substances, such as polyphenols and other natural antioxidants, which inhibit the development of microorganisms and allow the stabilization of the food matrix [
30].
It is well known that adding rich sources of antioxidants of plant origin influences not only the microbiological stability of food, but also the antioxidant activity [
31,
32].
The control sample had a negative value of the DPPH antioxidant activity, i.e., −15.26 ± 0.36% (
Figure 1). This can be explained by the fact that in the experimental conditions of gastric digestion simulation (acid pH), the starch of wheat bread breaks down into glucose. According to Pilar de Torre et al. (2019), glucose exhibits a prooxidant effect [
33].
The antioxidant activity was improved by a slightly increase, but remained negative (−8.65 ± 0.62%), when 1% sea buckthorn berry flour was added. Adding 3% SBBF provided a positive value (13.96 ± 0.45). For samples with 5% SBBF, an appreciable antiradical activity (20.05 ± 0.51%) was noticed. The explanation is that in the SBBF 1% bread, even if antioxidant properties are improved compared to control, the prooxidant effect of the glucose still prevails, while at higher concentrations of added sea buckthorn berry flour (SBBF 3% and SBBF 5%), the antioxidant effects of the bioactive compounds originated from sea buckthorn exceeds the mentioned prooxidant ones, and consequently, the antioxidant activity has positive values and increases when the SBBF percent in bread increases (
Figure 1).
Thus, the use of sea buckthorn berry flour at low concentration (1%) in wheat flour products positively influences the structural-mechanical, physicochemical, organoleptic, antioxidant properties and microbiological stability of the finished products.
Figure 2 summarizes the influences exhibited by the addition of various amounts of sea buckthorn berry flour on the physicochemical indicators (moisture content, acidity, porosity, bread specific volume), the total score of the organoleptic indices, the in vitro DPPH antioxidant activity and the development of rope spoilage in wheat bread, i.e., the appearance of the first signs of disease. The values of the mutual influence are presented on the arrows.
It was shown that the added amounts of sea buckthorn berry flour influence greatly (0.856 bits) DPPH antioxidant activity. On a decreasing scale, the next influence is on the development of rope spoilage in bread, 0.755 bits. The third influence in size is on the specific volume of bread, with mutual information of 0.726 bits and the smallest influence has been exhibited on the porosity of bread, 0.612 bits.