Aqua Extracts of Lyophilized Sea Buckthorn Modify Fermentation and Quality Properties of Set-Type Yogurt

Abstract

Sea buckthorn is a promising ingredient for the food industry because it is a good source of vitamins, polyphenols, phytosterols, etc. In this research, it is the first time that aqueous extracts of lyophilized sea buckthorn (LSB) 0%, 0.5%, 1%, 2%, and 3% w/w were used to enrich set-type yogurts. Therefore, fermentation kinetics, hardness, color, titratable acidity, syneresis, water holding capacity, total phenolic content microstructure, and sensory analysis were investigated. Extracts of lyophilized sea buckthorn shorten the yogurt fermentation time, change the microstructure, reduce syneresis, and increase water-holding capacity compared to plain yogurt. Also, the titratable acidity for all yogurts remained the same but the total phenolic content of yogurts increased as the concentration of extracts from lyophilized sea buckthorn increased. The color parameters of the fortified set-type yogurts were affected by the color of the sea buckthorn extract with increasing a* and b* values according to extract concentrations. Finally, yogurts fortified with 0.5% and 1.0% w/w extracts of LSB have good quality characteristics, increased total phenolic content, and higher scores of being liked compared to the rest of the enriched samples. This study could increase the knowledge of the uses of aqueous extracts of lyophilized sea buckthorn in dairy products.

1. Introduction

In China, sea buckthorn is one of the oldest plants on earth and is well known as the “Holy fruit” and “King of vitamin C” [1]. Sea buckthorn (Hippophae rhamnoides L.) is native to Central Asia and North-Westen Europe and the family includes around 100 species in three genera mostly found in the moderate geographic latitudes of the Northern Hemisphere. Also, sea buckthorn (Hippophae rhamnoides L.) which belongs to the family Elaeagnaceae is cultivated worldwide. It can survive in water-deficient soils, and is resistant to extreme temperatures. The ripe berries of Hippophae rhamnoides L are oval and their color is yellow–orange or red, depending on the variety [2].

The color of sea buckthorn berries comes from pigments and more specific carotenoids such as lycopene and zeaxanthin. Also, they are rich in vitamins C, E, A, and K, polyphenols, flavonoids, phytosterols, unsaturated fatty acids, phenolic acids, glucose, minerals, and other bio-active compounds that offer numerous health advantages [3]. Consequently, sea buckthorn has important functional properties for improving health such as antimicrobial, antioxidant, dermatological, protection against cardiovascular and bowel diseases, and relieving inflammation [4].

Sea buckthorn has emerged as a promising ingredient for food companies due to its health benefits and physicochemical profile and it is already found in many food products [5,6]. Its fruits are not usually consumed due to their high astringency and acidity; therefore, it is used for the preparation of various products. The rich spectrum of bioactive compounds has prompted many researchers to investigate the application of some of the fruit’s parts or its extracts in food. Some of the researchers have developed a soy drink with sea buckthorn syrup for those who are lactose intolerant. In addition, it can also be applied to alcoholic beverages, e.g., sea buckthorn wine. The juice obtained from sea buckthorn has been found to promote the growth of some beneficial gut bacteria possibly due to its prebiotic characteristics [3,5] and many researchers have tried to combine the nutrients from sea buckthorn with probiotic bacteria that help strengthen the gut.

In recent years, dairy producers have been making great efforts to offer products with increased nutritional value and improved organoleptic and quality characteristics that will meet the needs and expectations of the consumer. For this reason, several attempts have been made to produce dairy products enriched with natural additives and extracts such as fruits that are a source of bioactive substances and that will improve their quality characteristics [6]. Dairy products, especially fermented ones, are very widespread because they provide nutrients and bioactive compounds that promote health [7,8]. Not all are equal sources of nutrients and not all have the same impact on human health. However, yoghurt is the most common dairy product consumed by humans as it is considered a good source of vitamins, proteins, and minerals, and its nutritional value is particularly high. Sea buckthorn berries have been added to foods in a few research studies on dairy products such as an ingredient in novel cheese [9] and novel frozen yogurt [10]. Both research studies showed great potential for commercialization and application in the manufacturing of probiotic functional products. In yogurt production, sea buckthorn was added as pulp [11], demonstrating that sea buckthorn may be incorporated in milk as a functional ingredient for improving fermentative properties. In another research study [12], sea buckthorn fruits were added to yogurt in the form of an extract after being crushed in a juice processor and then processed by a microwave digestion system. The addition of sea buckthorn in this form showed that it could selectively enhance probiotic counts. Also, another way of adding sea buckthorn was using syrup, which affected the taste and viscosity of yogurt made with skim milk powder [12].

All the above research indicates the necessity of investigation of functional yogurts with sea buckthorn. In the current research, it is the first time that sea buckthorn extracts have been incorporated into yogurt in the form of lyophilized powder. This method could be useful for the food industry as it is the most widely used technique for producing powdered stable foods and consequently, rendering ingredients easier and more stable to handle [13]. In this study, we aim to investigate the influence of aqua extracts of lyophilized sea buckthorn (LSB) on fermentation kinetics and the quality characteristics of the yogurts derived. For the first time, the aqua extracts of lyophilized sea buckthorn are added directly to milk before fermentation and the fermentation kinetics, color, titratable acidity, water retention, syneresis, total phenolic content, hardness, microstructure, and sensory analysis of set-type yogurt were investigated to understand the effect of the aqua extracts of lyophilized sea buckthorn on the yogurt casein network, and consequently on the production and quality of fortified yogurts.

2. Materials and Methods

2.1. Preparation of Lyophilized Sed Buckthorn Powder (LSB) and Aqua Extracts of LSB

Frozen sea buckthorn berries were milled in a blender and then lyophilized sea buckthorn (LSB) powder was obtained through the process of lyophilization using a freeze-dryer ZIRBUS (Bad Grund, Germany) with brand stainless trays. Cooling of sea buckthorn was carried out at −35 °C, while the creation of a vacuum was achieved with a pressure of 0.5 mbar for 18 h with a gradual increase in temperature up to 35 °C. Aqua extracts of lyophilized sea buckthorn were derived from the lyophilized powder by adding it at appropriate concentrations in distilled water at a temperature of 100 °C (boiling temperature) for 15 min, as described previously by other research [14] with minor modifications. Then, the samples were filtered (Whatman filter paper, Sigma-Aldrich, St. Louis, MO, USA) and the extracts were used for the preparation of yogurt.

2.2. Preparation of Set-Yogurt

Yoghurt was produced with extracts of LSB (0.5%, 1%, 2%, and 3% w/v) and the experimental procedure was followed as described in similar research studies with minor modifications [14,15,16]. The commercial skim milk powder was homogenized with distilled water, pasteurized at 90 °C for 15 min, and cooled at 40 °C. Then, the aqueous sea buckthorn extract was added, and the milk was inoculated with the commercial starter culture YC-380 Thermophilic Yoghurt Culture-Yo Flex of the company CHr. Hansen (Hørsholm, Denmark), which contained Streptococcus salivarius ssp. Thermophilus and Lactobacillus delbrueckii, ssp. Bulgaricus. The inoculated samples were put into 40 mL plastic cylindrical cups and incubated at 37 °C until they reached a pH value of 4.6. At the end of fermentation, the samples were cooled and stored at 4ᵒC for further analysis. Yogurt without extract of LSB was defined as a control sample.

2.3. Determination of Fermentation Kinetics

During milk fermentation, the pH values of samples were measured with a digital pH meter LAB-860 at regular intervals in each treatment until the pH reached a value of 4.6 [16]. The kinetic parameters of acidification estimated were as follows: [I] the maximum acidification rate (Vmax), where it was defined as the time-dependent pH variation (dpH/dt) expressed in 10⁻³ pH/min units; [II] the time to reach the maximum acidification rate (T_vmax) (min); [III] the pH to reach the maximum acidification rate (pH_vmax); [IV] the time to reach a pH of 5.0 (T_pH5.0) (min); and [V] the time required to complete the fermentation (t_pH4.5) (min). The whole process was repeated 3 times for all yogurt samples [17,18].

2.4. Determination of Titratable Acidity, Water Holding Capacity and Syneresis

The titratable acidity in yogurt was expressed as lactic acid %w/w. Specifically, 25 g of yogurt sample was mixed with 25 mL of distilled water and titrated with 0.1 M NaOH (sodium hydroxide) solution, using phenolphthalein as indicator until the appearance of a pale pink color [16,17,18]. This process was applied on all yogurt treatments after one day of storage at 4 °C. Titratable acidity was expressed in % (w/w) lactic acid and calculated using Equation (1), where V is the volume (mL) of NaOH used in the titration, A the molar mass, 0.009 for lactic acid, and D is the factor of dilution.

$$\mathrm{Titratable} \mathrm{acidity} (\% \mathrm{lactic} \mathrm{acid})={\displaystyle \frac{\mathrm{V}\ast \mathrm{N}\mathrm{a}\mathrm{O}\mathrm{H} \mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\ast \mathrm{A}\ast \mathrm{D}}{\mathrm{V}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}\ast 100$$

(1)

Syneresis and water holding capacity (WHC) of yogurt were also determined [19], after one day of storage at 4 °C. In brief, 10 g samples of fermented yogurt were transferred to centrifugal tubes and centrifuged at 5000× g for 10 min at 4 °C using the high-speed refrigerated Hitachi Model CR22N (Koki Co., Ltd., Tokyo, Japan). Then the supernatants (whey) and pellets were weighed [20]. Syneresis and WHC were calculated with the following Equations (2) and (3) [21].

$$\mathrm{Syneresis} (\%)={\displaystyle \frac{\mathrm{W}\mathrm{h}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{w}\mathrm{h}\mathrm{e}\mathrm{y} \left(\mathrm{g}\right)}{\mathrm{W}\mathrm{h}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{y}\mathrm{o}\mathrm{g}\mathrm{u}\mathrm{r}\mathrm{t} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \left(\mathrm{g}\right)}}\ast 100$$

(2)

$$\mathrm{WHC} (\%)={\displaystyle \frac{\mathrm{W}\mathrm{h}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{t}\mathrm{a}\mathrm{t}\mathrm{e} \left(\mathrm{g}\right)}{\mathrm{W}\mathrm{h}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{y}\mathrm{o}\mathrm{g}\mathrm{u}\mathrm{r}\mathrm{t} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \left(\mathrm{g}\right)}}\ast 100$$

(3)

All measurements were performed three times.

2.5. Total Phenolic Content

Five grams of each yogurt was treated with thirty mL of methanol (100%) at room temperature and methanol extracts were stored at −18 °C until analysis. The total phenolic content was determined using the Folin–Ciocalteu assay, as reported by previous research [22]. Then, the reaction took place and yogurt samples were centrifuged at 4 °C, 3000× g, and for 25 min. Then, at 760 nm, the absorbance of the supernatant was measured by using a UV–visible spectrophotometer (UV-1800 Shimadzu, Kyoto, Japan). The results were expressed as mg gallic acid equivalents (GAE) per 100 g of sample. All measurements were repeated three times.

2.6. Determination of Color

The color of the yoghurt sample was evaluated after one day of storage at 4 °C according to the system defined by the Commission International de l’Éclairage (CIE) L*A*b*, using the colorimeter (Hunter Lab Miniscan XE Plus, Reston, VA, USA) [16]. The color was expressed as L*: darkness–lightness (0–100), a*: greenness–redness (−60–+60) and b*: blueness–yellowness (−60–+60) [15,16]. The mean values of these parameters were obtained from three samples for every treatment. From L*, a* and b* parameters chroma (C*) or chroma saturation index and hue* angle were calculated using the following equations:

Chroma* = √(a² + b²),

(4)

Hue angle (h*) = arctangent (b*/a*),

(5)

All measurements were performed three times.

2.7. Texture Measurements

Hardness was measured using an eXpert 5601 texture analyzer (ADMET, Norwood, MA, USA), with a cylindrical probe of 20 mm in diameter [16,21]. The height of the samples was 30 mm and the diameter 40 mm. The determination of the structure was evaluated as an analysis of the structure profile (TPA) with 75% compression at a rate of 1 mm/s one day after storage at 4 °C. For each treatment, three replications were analyzed and averaged.

2.8. Microstructure

The preferment mixture of extract of LSB and milk—(0.5 mL)—was transferred to a microscopic slide, incubated at 37 °C until the pH reached 4.6., and then sealed and stored at 4 °C. One day after the preparation of the yogurts, the microstructure was determined [16]. The observation of the microstructure was performed using a ZEISS Primovert Inverted microscope equipped with an Axiocam 105 color (Zeiss, Oberkochen, Germany). Images were captured at a magnification of 10×. The light goes through the slide in the areas where loose or no yogurt gel is formed, whereas the darker zones in the pictures represent dense compact yogurt gel.

2.9. Sensory Analysis

In this research, 12 untrained panelists utilized sensory analysis to assess variations among the yogurt samples, employing a 7-point hedonic scale [23]. The panelists’ ages were within the range of 25 to 55 years old. Yogurts with different concentrations of extracts of LSB of 0%, 0.5%, 1%, 2%, and 3% w/w were evaluated using a 7-point hedonic scale. Panelists were asked to rate the samples according to the 1–7 scale: 1—very much, 2—dislike moderately, 3—dislike slightly, 4—neither like nor dislike, 5—like slightly, 6—like moderately, 7—like very much. Color, flavor, texture, sourness, overall taste, and overall acceptance attributes were measured to identify the ideal formulation of yogurt enriched with extracts of LSB. Samples were tagged with three-digit random numbers, ensuring a balanced serving order. Sensory measurements are presented as the mean ± standard error.

2.10. Statistical Analysis

Data from triplicate determinations of samples from each yogurt were expressed as mean ± standard deviation (SD). The statistical package IBM SPSS STATISTICS 26.0 was used for the analysis with the non-parametric test of one-way Kruskal–Wallis analysis with a 95% confidence level. Tukey’s post hoc test was applied for means comparison and statistical significance was considered when p-value < 0.05.

3. Results and Discussion

3.1. Kinetic Measurements

The commercial fermentation time and the strength of the formed casein network are very important quality factors in yogurt preparation. In this research, the kinetics of acidification of yogurt samples by lactic acid bacteria were studied as enriched with extract of LSB of varied concentrations (0% w/w, 0.5% w/w, 1% w/w, 2% w/w, and 3% w/w). The addition of aqua extracts of LSB differentiates milk’s initial pH, as shown in

Table 1. Measurement of the initial pH, V_max: the maximum acidification rate; pH_Vmax the pH at the maximum acidification rate; T_pH5, the time when pH value is equal to 5; T_pH4.6, the time when pH value is equal to 4.6, in yogurts enriched with extracts of lyophilized sea buckthorn (0%, 0.5%, 1%, 2%, 3% w/w).

Extracts of Lyophilized Sea Buckthorn (% w/w)	pH initial	Vmax (10−3 upH/min)	pHVmax	TpH5 (min)	TpH4.6 (min)
0	6.6 ± 0.21 a	8.9 ± 1.17 b	6.15 ± 0.15 a	308.00 ± 50.48 a	421.00 ± 1.22 a
0.5	6.29 ± 0.18 b	11.22 ± 0.39 a	5.90 ± 0.17 a	227.00 ± 12.29 b	337.22 ± 2.56 b
1	6.02 ± 0.11 c	6.6 ± 2.01 c	5.64 ± 0.25 b	266.00 ± 21.07 b	338.04 ± 1.67 b
2	5.82 ± 0.09 d	8.00 ± 0.33 b	5.23 ± 0.02 c	175.00 ± 13.88 c	243.00 ± 2.08 c
3	5.58 ± 0.13 e	7.00 ± 0.00 b c	5.13 ± 0.14 c	165.00 ± 3.22 c	241.39 ± 1.59 c

^a–e values with different superscript letters indicate statistical differences among treatments (p < 0.05). All measurements were conducted in triplicate.

. As the added concentration % w/w of extracts of LSB increased, the pH value of the initial milk mixture at fermentation time 0 min decreased probably due to the organic acids which are contained in the extract. In addition, the extract of LSB affected the initial acidity of the samples, but, further changes in the pH values of yogurt samples took place during fermentation and these changes represent the effects of lactic acid bacteria [24,25] and the studied fermentation kinetic parameters, as shown Table 1. In yogurt with 0.5% w/w extract of LSB, the maximum acidification rate was observed as 11.22 × 10⁻³ ± 0.39 × 10⁻³ upH/min which was significantly different compared to the other concentrations (p < 0.05). Sea buckthorn contains organic acids, phenolic acids, etc., which even in the small concentration of 0.5% extract of LBG could be responsible for accelerating the growth of LAB and causing a rapid drop in pH. Samples with 0% and 2% w/w extract of LSB showed similar V_max values, 8.9 × 10⁻³ ± 0.00 × 10⁻³ upH/min and 8.0 × 10⁻³ ± 0.33 × 10⁻³ upH/min, respectively. The results are in contrast to previous research on guarana aqua extracts added to yogurt [16] where the yogurt with 0.5% w/w was similar to the control sample and the peak of V_max was noticed at 1% w/w guarana extract followed by a drop at concentration increase to 2% w/w guarana extract. Also, in another research study, moringa extract added at varied concentrations to yogurt increased V_max according to the concentration increase [26]. Probably aqua extracts of LSB contain fibers, organic acids, and phenolics which could be responsible for these different effects on V_max.

The required time for yogurt samples to reach a pH of 5 was also determined in Table 1. The highest time required for yogurt with 0% w/w extract of LSB to reach a pH of 5 was T_pH5 308 ± 50.48 min and the smallest for the yogurts with the highest concentrations of extract of LSB 2% w/w (175 ± 13.88 min) and 3% w/w (165 ± 3.22 min). The presence of bioactive compounds and indigestible polysaccharides in LSB extracts could influence acid lactic bacteria growth. Similar results were found in other studies, in yogurts enriched with extracts such as Pleurotous ostreatus [27], guarana aqua [16], and green tea [28].

Acidification of milk influences yogurt’s physicochemical stability as the way that the pH lowers casein micelles makes them lose their negative charge and enhances the attraction of proteins. When the pH reaches the value of 4.6, the agglomeration of caseins takes place [16]. In this study, the fermentation completion time was the largest for the sample with 0% w/w LSB extract 421.00 ± 1.22 min (p < 0.05) and the yogurt samples with 2% and 3% w/w extracts of LSB were the fastest ones with T_pH4.6 243.00 ± 2.08 and 241.39 ± 1.59 min, respectively. As mentioned above, the initial pH of samples at fermentation time T = 0 min was decreased as the concentration of LSB extracts increased. This could be explaining the smallest needed fermentation time for the yogurts with 2% and 3% w/w LSB extracts. Lactic acid bacteria are also influenced by the presence of extracts of LSB in milk as this extract could provide bacteria with many carbon sources such as phytosterols, phenolic acids, glucose, etc. [29], necessary for the acceleration of fermentation time due to the increase in lactic acid bacterial growth. Other researchers have found analogous results when they incorporated red ginseng extract in yogurt [30], and green tea powder [28]. Also, this research work agrees with our previous study where guarana aqua extracts reduced the fermentation time and influenced starter bacteria [16].

3.2. Titratable Acidity, Syneresis and Water Holding Capacity (WHC) Measurements

Lactic acid is the main component of yogurt that imparts a characteristic flavor and is produced by the action of the starter bacteria during the formation of the casein network and storage of yogurt at low temperatures [31,32]. In this research, titratable acidity (TA) of yogurt samples enriched with extracts of LSB 0%, 0.5%, 1%, 2%, and 3% w/w is expressed in mg/L of lactic acid and presented in Figure 1. All samples had a similar TA, for example, the yogurt with 0% w/w LBS had 1.00 ± 0.03%, that with 1% w/w LBS had 1.01 ± 0.02%, and that with 3% w/w LSB had 1.01 ± 0.04% and no significant differences were found between the samples (p > 0.05) So, the extracts of LSB did not influence lactic acid production.

This is in contrast with other research where the enrichment of yogurt with aqua guarana extracts showed a differentiation of TA values of the lowest concentration of guarana extract compared to the rest of the samples [16] and for yogurt with flaxseed powder and pineapple peel powder [33,34]. On the other hand, our results agree with the TA values of yogurt with 0.5% w/w SB in the form of mousse which had no significant differences with the control sample [35].

Syneresis and WHC in a milk protein network are critical quality parameters that could affect the physicochemical stability of the set-type yogurt. The liquid phase of serum could not be kept from the yogurt gel network and thus separation of whey proteins takes place giving a negative appearance in the final yogurt product. In this research, both syneresis and WHC were measured and the effects of the extracts of LSB are presented in Figure 2. The highest value of syneresis was found for the sample with 0% w/w LSB and the lowest for the sample of yogurt enriched with 3% w/w extract of LSB with values of 65.03 ± 1.89% and 51.83 ± 1.80%, respectively. The opposite was found for WHC, and specifically the yogurt without LSB had the smallest value and the yogurt with 3% w/w extracts of LSB showed the highest WHC compared to the rest of the samples, 29.25 ± 1.25% and 36.00 ± 0.09%, respectively.

Our results showed that when the extract concentration of LSB increases, the water retention also increases and the yogurt gel network exhibits greater stability concerning serum elimination. Probably the polysaccharides that are present in extracts of LSB stabilize the casein network. Also, the sort fermentation time is a factor that increases syneresis. In this study, the addition of extracts of LSB drives the formation of molecular structures that retain moisture more effectively compared to the control yogurt [20]. Our results differ from our previous research which showed that guarana aqua extracts added to yogurt samples increase syneresis and decrease WHC [16]. The addition of edible rose extract [20] and moringa [26] in yogurt showed similar results on WHC and syneresis with the enriched set-type yogurt with the extracts of LSB.

3.3. Total Phenolic Content (TPC)

According to epidemiological research, the consumption of polyphenols has been linked with health improvements [36]. In addition, the consumption of yogurts enriched with polyphenols provokes potential advantages in human health by reducing low-density lipoprotein (LDL), overweight, high blood pressure, cancer, cardiovascular diseases, etc. [37]. In the current study, the total phenolic content in yogurts with extracts of lyophilized sea buckthorn was measured and the results are presented in Figure 3.

Although polyphenols are commonly found in fruits and vegetables, plain yogurt (0% w/w extract of LSB) has a TPC 9.6 ± 0.17 mg GAE/100 g probably because TPC is naturally present in cows’ milk from the feed of cows and the amino acid catabolism [38]. Yogurt with 3% w/w extract of LSB has the highest TPC (30.1 ± 0.21 mg GAE/100 g) and the sample with 0.5% w/w extract of LSB had the lowest concentration (18.7 ± 0.15 mg GAE/100 g) compared to the rest of the fortified yogurts (p < 0.05). It should be mentioned that the total phenolic content is increasing according to the increased concentration of extract of LSB. The sample with 3% w/w extract of LSB has a level of TPC three times higher compared to plain yogurt. So, the addition of LSB extracts increases the yogurt’s nutritional value. Similar results were found in other research studies, such as in yogurts fortified with peanuts [39] and moringa extracts [40].

3.4. Color Characteristics of Yogurt Samples

Color is an important quality factor of yogurt, which affects the acceptance of the product by the consumer. The presence of milk fat globules and casein micelles, which can scatter light in the visible spectrum [41], is responsible for the white color of yogurt. The addition of plant extracts provokes changes in yogurt samples and influences consumer choices. In

Table 2. Color parameters L*, a*, b*, chroma*, and hue angle of yogurt samples enriched with extracts of lyophilized sea buckthorn.

Lyophilized Sea Buckthorn Extract (% w/w)	L*	a*	b*	Chroma*	Hue Angle (h*)
0	78.99 ± 2.37 c	−1.92 ± 0.02 d	10.26 ± 0.57 e	10.44 ± 0.56 e	178.61 ± 0.01 a
0.5	74.85 ± 0.71 b	1.25 ± 0.18 c	16.32 ± 0.21 d	16.37 ± 0.20 d	1.49 ± 0.01 b
1	74.95 ± 0.23 b	1.28 ± 0.10 c	17.84 ± 0.18 c	17.88 ± 0.18 c	1.50 ± 0.01 b
2	82.26 ± 1.41 a	6.33 ± 0.20 b	30.37 ± 0.49 b	31.03 ± 0.47 b	1.37 ± 0.01 c
3	81.88 ± 0.79 a	12.57 ± 0.27 a	56.19 ± 0.90 a	57.58 ± 0.82 a	1.35 ± 0.01 c

Different letters within the columns indicate significant differences (p < 0.05) among the different treatments. All measurements were conducted in triplicate.

, color parameters L*, a*, b*, chroma, and hue of all samples were presented one day after the fermentation and storage at 4 °C.

Yogurt with 0% w/w lyophilized sea buckthorn extract had the lowest a* value of around −1.92 ± 0.02 and the lowest b* value of about 10.2 ± 0.57, (p < 0.05). With the addition of extracts of LSB to the samples, the a* and b* values increased while the L* values fluctuated according to the increase in LSB extract concentrations. However, the samples containing extracts of LSB at a concentration of 2% and 3% w/w presented a higher L* value than the rest of the extract concentrations and the control with values of 82.26 ± 1.41 and 81.88 ± 0.79, respectively (p > 0.05). The more the concentration of extract of LSB increased to 0.5%, 1%, 2%, and 3% w/w, the more the values of a* and b* increased. More specifically, yogurt with 3% w/w lyophilized sea buckthorn, which was the highest concentration added, had the highest a* and b* values of 12.5 ± 0.27 and 57.1 ± 0.90, respectively. These samples have more redness and yellowness due to the yellow–orange color of the sea buckthorn and samples with a higher proportion of sea buckthorn extract had a more intense yellow and red color compared to the control sample. The high content of carotenoids in sea buckthorn is undoubtedly responsible for the yellow color. Hue and chroma measurements were also affected by the addition of different concentrations of extracts of LSB. In general, the color of extracts of LSB is responsible for color measurements on the enriched yogurts. For the concentrations of 0.5% and 1% w/w extracts of LSB, an increase in the b* values compared to control samples and a decrease in their L* values (74.85 ± 0.71 and 74.95 ± 0.23) was noticed, which is due to phenolic degradation and the formation of more brown pigments. Similar results were found in the yogurt samples fortified with extracts of argel leaf [15], chia seed extract [42], and Pleurotous ostreatus [27].

3.5. Texture Measurements

The texture of yogurt is an important quality characteristic for assessing molecular arrangements giving information on the protein network and on the stability of yogurt colloidal emulsions. Many factors could affect the casein network including the yogurt hardness, type of milk, fat concentration, emulsion composition, lactic acid bacteria, and other factors. The force needed to achieve a particular deformation is considered a measure of the hardness of yogurt [7].

As shown in Figure 4, the hardness of the set-type yogurt samples was significantly affected by the addition of the lyophilized sea buckthorn extract. Specifically, the addition of extracts of LSB led to decreased hardness values for all the treatments. The highest hardness values (N) were recorded for the control sample (1.33 ± 0.06 N) and were significantly different (p < 0.05) from the other treatments. In contrast, the lowest hardness values (N) were observed for the yogurt samples containing 3% w/w extract of LSB (0.87 ± 0.06 N). No differences were noticed among the hardness values of the samples containing different amounts of extracts LSB. It should be noticed that although yogurts with the higher extract concentrations of LSB (2% and 3% w/w) have a smaller fermentation duration compared to the rest of the fortified samples, these yogurts did not show weaker structures. In our previous article, similar concentrations of guarana extract affected the hardness of set-type yogurt differently, and more specifically, 3% w/w guarana extract had similar values with the control [16]. Generally, a different type of extract has different bioactive compounds which could affect different yogurt textural properties. Also, the hardness of the yogurt network depends on many parameters such as the type of starter, processing, and milk types [43,44].

3.6. Microstructure of Yogurt

The microstructure of set-type yogurts fortified with sea buckthorn extracts with concentrations of 0%, 0.5%, 1%, 2%, and 3% w/w are presented in Figure 5. Although textural analysis showed no significant differences among the yogurts with extracts of LSB, each sample has its own microstructure, and differences were observed. As the LSB extract concentration increases, areas with a yellow color are also increasing. Probably the high content of carotenoids in sea buckthorn gives the yellow color. Also, as the concentration of LSB extract in yogurt sample increases, the spherical droplets have larger sizes and different shapes, covering a larger part of the sample microstructure.

In Figure 5, the microstructure of the yogurt sample with the 3% w/w extract of LSB is presented with the largest darker yellow areas compared to the rest of the samples. Also, in the same figure, there are only small empty spaces as the extract seems to occupy big areas consisting only of LSB and areas consisting only of the yogurt protein network. More clearly, the microstructure of 3% w/w extract of LSB presents micro-phase separation and spoils the uniform microstructure of the yogurt compared to 0.5% or 1% w/w where the dots of lyophilized sea buckthorn are very small and more uniform.

3.7. Sensory Analysis

Sensory analysis has been used for centuries to evaluate food characteristics through the senses. Today, this analysis is one of the very important methods of quality control that also ensures consumers will accept the final products. In this research, the yogurt samples were evaluated by 12 untrained panelists who gave their degree of liking for color, flavor, texture, sourness, overall taste, and overall acceptance (

Table 3. Specific indicators of sensory analysis for yogurt samples enriched with extracts of lyophilized sea buckthorn (0%, 0.5%, 1%, 2%, and 3% w/w).

Scheme	Extracts of Lyophilized Sea Buckthorn % w/w
	0	0.5	1	2	3
Color	5.83 ± 0.84 a	6.00 ± 0.74 b	5.83 ± 0.94 a	4.83 ± 0.94 c	4.25 ± 1.22 d
Flavor	6.00 ± 0.85 a	5.85 ± 1.31 b	4.92 ± 0.79 c	4.08 ± 0.79 d	3.58 ± 0.52 e
Texture	6.08 ± 0.79 a	5.58 ± 0.79 b	5.50 ± 0.67 c	4.75 ± 0.75 e	5.08 ± 0.67 d
Sourness	5.33 ± 0.65 a	4.67 ± 0.65 b	4.58 ± 0.79 c	4.58 ± 0.52 c	4.58 ± 0.52 c
Overall Taste	5.58 ± 0.52 a	5.42 ± 0.67 b	5.08 ± 0.79 c	3.83 ± 0.72 d	3.83 ± 0.72 d
Acceptance	5.75 ± 0.62 a	5.33 ± 0.65 b	5.08 ± 0.79 c	4.17 ± 0.58 d	4.17 ± 0.58 d

Different letters within the columns indicate significant differences (p < 0.05) among the different treatments.

).

The incorporation of the highest extract concentrations of LSB 2% and 3% w/w led to a decrease in preference for color, flavor, texture, overall taste, and acceptance compared to the rest of the samples (p < 0.05). Further investigation is needed as to whether the addition of sugars and additives could help in improving the overall taste and acceptance of the above samples. Probably the higher extract concentration of LSB influences the taste and the aroma of the high concentration of enriched yogurts. Also, yogurts with LSB extract 0,5% w/w and 1% w/w were preferable by the panelists for color, texture, overall taste, and overall acceptance compared to the rest of the enriched yogurts (p < 0.05). In addition, based on the sensory evaluation and panelist preferences, the above concentrations of extracts of lyophilized sea buckthorn could be incorporated in yogurt for the production of novel products and at the same time increase the nutritional value without notable negative sensory attributes.

4. Conclusions

Yogurts with lyophilized sea buckthorn extracts were successfully produced. As the amount of extract of LSB increases, the enriched yogurts had the same titratable acidity and texture properties, increased total phenolic content, but different microstructure. The addition of 3% w/w extract of LSB disturbs the yogurt’s casein microstructure without any difference in hardness compared to the rest of the enriched samples. Also, the yogurt with the above extract concentration of LSB is the sample with the one of the smallest fermentation times and at the same time, the highest water-holding capacity. Yogurts with small extract concentrations of LSB 0.5% and 1% w/w also need less time to complete fermentation compared to the plain yogurt and at the same time were more preferable samples according to the sensory analysis. These fortified yogurts also have a high nutritional value and good quality characteristics. This research could be interesting in industrial research and development as aqua extracts of lyophilized sea buckthorn could be new ingredients that could fortify foods with their high phenolic content and at the same time improve their quality properties.