Effects of Different Yeasts on the Physicochemical Properties and Aroma Compounds of Fermented Sea Buckthorn Juice

Abstract

Sea buckthorn juice (SBJ) has a sour taste and can lead to the demineralization of tooth enamel when consumed over a long period of time, whereas fermentation reduces the acidity of sea buckthorn juice, improves its taste, and enhances its antioxidant activity. Flavor components are important factors that affect the quality of fermented beverages. Yeast is one of the most important factors affecting the flavor of beverages during the fermentation process, where yeast converts sugars into alcohol and produces flavor substances. Therefore, two commercial yeast strains, Angel RW and Angel RV171, were selected in this study for the single and mixed bacterial fermentation of sea buckthorn juice (FSBJ). Physicochemical analyses showed that RV171-FSBJ had the highest total reducing sugar (0.069 ± 0.02 g/L) and total acid content (1.86 ± 0.03 g/L), as well as the highest fermentation efficiency and free radical scavenging capacity (1,1-diphenyl-2-picrylhydrazyl (DPPH) 98.54 ± 0.03%, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 88.35 ± 0.14%, ·OH 48.61 ± 0.4%). RWRV-FSBJ had the highest content of functional compounds (total flavonoid content (TFC): 176.09 ± 0.44 μg/mL; total phenolic content (TPC): 157.9 ± 1.35 μg/mL; total anthocyanin concentration (TAC): 0.04 ± 0.004 μg/mL) and good color (L* 50.53 ± 0.04, a* 27.98 ± 0.04, b* 173.64 ± 0.34). Among the three FSBJs, a total of 54 volatile compounds were identified, with RV171-FSBJ having the highest content of volatile compounds. OAV analysis showed that 15, 14, and 11 volatile compounds of RW, RV, and RWRV, respectively, were greater than 1. Among them, ethyl hexanoate had the highest OAV, followed by ethyl isovalerate, phenylethyl alcohol, and 3-methylbutyl 3-methylbutanoate, which are characteristic flavor substances common to FSBJ.

1. Introduction

Sea buckthorn (Hippophae rhamnoides L.) is a species of deciduous thorny shrubs or trees from the genus Hippophae [1]. Sea buckthorn berries are rich in nutrients and contain a variety of functional components and bioactive substances such as phenols, flavonoids, sterols, ascorbic acid, tocopherol, fatty acids, carotenoids, and organic acids [2]. Yadav et al. showed that sea buckthorn berries have a high content of vitamin C (VC), almost 4–100 times that of other fruits [3]. In addition, sea buckthorn is not only rich in VC but also contains a large amount of oils and is rich in polyunsaturated fatty acids n − 3, n − 6, and n − 9 [2]. Therefore, as a plant with both medicinal and culinary values, sea buckthorn has significant health benefits and has been traditionally used in traditional oriental medicine systems for the treatment of liver damage, cardiovascular disease, and stomach disorders, among others [4,5]. For example, sea buckthorn extracts can exert antiplatelet effects by inhibiting thrombin-activated platelets, as well as antimicrobial effects by inhibiting adhesion and biofilm formation [6]. Therefore, the nutritional and medicinal value of sea buckthorn has attracted extensive attention from researchers around the world [7].

However, sea buckthorn berries have a high water content (about 70%) and do not tolerate room temperature storage, making them susceptible to mechanical damage and microbial infection. Second, due to their low sugar content and high acidity, they often have a sharp, sour taste when consumed fresh or in juice. Finally, studies have shown that long-term consumption of high-acid beverages may cause demineralization of tooth enamel and even acid erosion [8]. Because of this, products prepared from sea buckthorn are still in the primary stage of development, and the consumer market is limited. In order to expand the industrial distribution of sea buckthorn and to fill the current market demand for sea buckthorn juice (SBJ) products, we try to use fermentation to reduce its acidity and improve its taste. Currently, lactic acid bacteria and yeasts are often used to prepare fermented beverages. Because lactic acid would further increase the level of acid and reduce the pH of SBJ, we choose yeast to ferment SBJ, eliminate or reduce its sour taste and bad smell, and improve its flavor profile.

In the production of yeast-fermented beverages, the quality and volatile compound content are closely related to the yeast used, so the selection of yeast is one of the key factors in determining the quality of fermented beverages [9]. Han et al. used three different yeasts to ferment greengage alcoholic beverage (GAB) and found that although fermented GAB from RV171 and BV818 showed greater antioxidant capacity, GAB fermented by Lalvin 71B showed the highest efficiency, a suitable sugar–acid ratio, and the richest volatile flavor compounds, and so Lalvin 71B was considered to be a more suitable strain for producing fermented GAB [10]. Therefore, different yeasts fermenting SBJ may exhibit different physicochemical qualities and produce different flavor substances. In order to investigate the effect of yeast strains on the physicochemical qualities of fermentation of sea buckthorn juice (FSBJ), a redundancy analysis (RDA) was carried out with different yeast strains as “explanatory variables” and physicochemical indexes as “response variables” to evaluate the “explanatory variables” and “response variables” components. Redundancy analysis (RDA) was conducted to evaluate the linear relationship between the components of “explanatory variables” and “response variables”. In addition, to understand the effect of different yeasts on the volatile compounds in SBJ, principal component analysis (PCA) was used to understand the overall separation trend of volatile compounds among the sample groups, which revealed whether there were any differences in volatile compounds among the sample groups [11].

In the present study, two commercial yeast strains were selected to prepare FSBJ. RV171 and RW are both commonly used commercial yeasts. RV171 is a low-alcohol sweet brewing active dry yeast, suitable for fermentation of high acidity fruits, with a certain conversion ability to malic acid, and at the same time, it can generate more esters and glycerol substances, adding alcohol and ester flavors to the fermentation broth. RW has excellent tannin and pigment extraction power, and it can retain the original color of the fruits, highlighting the fruity aroma of the fermentation broth. Therefore, RW and RV171 are suitable for fermenting most fruits, providing ester and alcohol flavors to the fermentation broth, and fermentation is stable and durable [12]. There have been many studies on the flavor of SBJ, but there have been fewer reports comparing the functional components and aroma profiles of SBJ fermented by monomicrobial complex bacteria. Therefore, the aim of this study was to compare the differences in the physicochemical properties, functional components, antioxidant capacity, and volatile components of different yeast-fermented SBJs to determine the most suitable yeast strain for the fermentation of sea buckthorn juice (FSBJ) and to provide a reference for the subsequent production of sea buckthorn yeast-fermented beverages.

2. Materials and Methods

2.1. Sample Collection

Frozen sea buckthorn berries were purchased in November 2022 from a farmer’s market (Shihezi City, Xinjiang, China). The berries were grown in Korla, Xinjiang, China (without pesticide spraying), and workers harvested the fruits in October 2022 when they were ripe and similar in shape and size, with intact skins, no mechanical damage, no pests or diseases, and no residues on the stalks. Immediately after harvest, the berries were placed in −20 °C cold storage for freezing and preservation. The variety of sea buckthorn berries called Hippophae rhamnoides L. subsp. mongolica “Shenqiuhon” [13]. The berries were stored at −20 °C until use.

The commercial yeast strains (Saccharomyces cerevisiae), Angel RW and Angel RV171, were purchased from ANGEL YEAST Co., Ltd. (Yichang, China).

2.2. Chemical and Reagents

1, 1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS⁺) were supplied by Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Folin–phenol, gallic acid standard, and rutin standard were supplied by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Other chemical reagents (analytical purity) were provided by Tianjin Beifang Tianyi Chemical Reagent Factory (Tianjin, China).

2.3. Beverage Preparation and Fermentation

First, frozen sea buckthorn berries were washed and thawed in ultrapure water. Next, the berries were added to a blender (JYL-Y921 Joyoung, Jiuyang Co., Jinan, China) and blended for 2 min. The SBJ was filtered through a gauze to remove solids. Then, the soluble solids were adjusted to 12 Brix, and the pH was adjusted to 4.0 using white sugar and anhydrous sodium carbonate, respectively. The SBJ was then sterilized and inoculated with 0.3 g/L of pre-activated yeast strains (RW, RV171 [RV], and RW&RV171 (1:1) [RWRV]) to prepare three separate fermentation mixtures (SBJ viable bacteria count > 10⁶ CFU/mL). The mixtures were placed in an incubator at 20 °C and fermented for 20 h. When the alcohol content reached 0.45 ± 0.05% by volume, fermentation was stopped. According to the first method in GB 5009.225–2016 [14], the sample density at 20 °C was measured by the density bottle method, and the comparison table was used to calculate the alcohol content based on the density of the alcohol aqueous solution [15]. The FSBJ was removed, sterilized, and stored at −20 °C for subsequent analysis.

2.4. Determination of Physicochemical Properties

The total content of reducing sugar and acid was measured using previously described methods, namely, the 3,5-dinitrosalicylic acid (DNS) method and titratable acidity method [10,16], respectively. For the latter, the findings were expressed based on the equivalents of tartaric acid (g/L). A colorimeter (WSC-C Sincere Dedication of Science and Technology Innovation, Shanghai, China) was used to measure color parameters (L*, a*, and b*) based on the method described by Hojjatpanah et al. [15].

2.5. Phytochemical Concentration Assay

The total flavonoid content (TFC) in the FSBJ was measured using the aluminum chloride colorimetric method described by Zhao et al, and the TFC was expressed as rutin content in μg/mL [17]. The Folin–Ciocalteu colorimetric method was adopted to determine the total phenolic content (TPC) based on previous me hods described by Zhao et al., and the TPC was expressed as gallic acid content in μg/mL [17]. The total anthocyanin concentration (TAC) was determined using the pH differential method described by Kwaw et al., and the TAC was expressed as microgram equivalent of cyanidin 3-glucoside per milliliter of juice in μg/mL [18].

2.6. Antioxidant Activity Assay

The method of Zhao et al. was referred to for ABTS radical scavenging activity (ABTS-SA) and DPPH radical scavenging activity (DPPH-SA) detection with slight modification [17]. The ABTS stock solution was prepared by taking 0.20 mL of ABTS and adding 0.20 mL of potassium persulfate and reacting under dark conditions for 12 h. The solution was diluted with anhydrous ethanol (approx. 40–50 times) to A₇₃₄ = 0.70 ± 0.02. We took 0.10 mL of sample solution and added 3.00 mL of ABTS reserve solution and shook it well, let it stand at 35 ± 2 °C for 10 min, and then measured the absorbance value (A_s) at 734 nm. Anhydrous ethanol was used to replace the ABTS reserve solution in the control group (A_d), and distilled water was used to replace the sample in the blank group (A₀). A total of 20 μL of sample solution was added to 80 μL of deionized water and 100 μL of 0.20 mM DPPH solution and placed at 37 °C for 45 min, and the absorbance value (A_s) was measured at 517 nm. Methanol was used as a control instead of DPPH solution (A_d), and distilled water was used as a blank instead of sample (A₀). Both ABTS⁺ and DPPH were calculated using Formula (1).

% clearance = (1 − (A₀ − (A_s − A_d)/A₀)) × 100%

(1)

The reducing power capacity (RP-CA) assay was performed by referring to the method of Kwaw et al. with slight modification [18]. The sample solution was diluted 100-fold to 1 mL, and 0.05 mL of HCl (0.01 M), 0.4 mL of potassium ferricyanide (0.02 M), 0.4 mL of 0.02 M FeCl₃, and 0.7 mL of distilled water were added to the sample solution. The mixture was incubated in an incubator at 37 °C in darkness for 30 min, and the absorbance value was measured at 720 nm. The RP-CA of FSBJs is expressed in millimoles of ascorbic acid.

The hydroxyl free radical scavenging capacity (•OH-SA) assay was performed by referring to the method of Wen et al. with slight modification [4]. We took 1 mL of the sample solution, added 1.5 mL of 1.8 mM ethanol–salicylic acid solution, 2 mL of 1.8 mM ferrous sulfate solution, and 0.1 mL of 0.3% hydrogen peroxide, mixed it well, placed it in a water bath at 37 °C for 30 min, and then determined the absorbance value (A_s) at 510 nm. The control group was made by replacing the three reagent solutions with distilled water (A_d), and the blank group was made by replacing the sample with distilled water (A₀). •OH-SA were calculated using Formula (2).

% clearance = (1 − (A_s − A_d)/A₀) × 100%

(2)

The above antioxidant tests were carried out using TAS-964 enzyme marker (Taian Technology Co., Ltd., Taian, China) for absorbance value detection, and the tests on each sample were repeated three times.

2.7. HS-SPME-GC-MS Analysis

Based on Cai et al.’s study [19], the volatile compounds were extracted from FSBJ samples using the head space solid phase microextraction (HS-SPME) method. First, 5 mL of the sample and 1 g of NaCl were placed in a 20 mL headspace vial, and 10 μL of 2-octanol solution (1000 g/L) was added as the internal standard. Next, after equilibration at 45 °C for 15 min, a SPME fiber (50/30 μm, DVB/CAR/PDMS) (Supelco, Bellefonte, PA, USA)was extended into the headspace vial for 30 min to extract volatile compounds from the sample. Then, the extracted compounds were immediately injected into the GC injection port (250 °C for 3 min), and the volatile compounds were desorbed.

GC-MS (Agilent8890-7000D) (Agilent Technologies (China) Co., Beijing, China) was performed using an HP-INNOWax (30 m × 250 μm × 0.25 μm) (Agilent Technologies (China) Co., Beijing, China) capillary column to analyze the volatile compounds. The initial temperature was 40 °C. The temperature was then increased to 85 °C (3 °C/min), followed by increases to 105 °C (1 °C/min), to 180 °C (3 °C/min), and finally to 230 °C (10 °C/min) for 5 min. The volatile compounds were identified using mass spectrometry based on comparisons with the NIST 2017. The content of each volatile compound was calculated using the method described by Cai et al. [20].

2.8. Odor Activity Value (OAV) Calculation

The contribution of volatile compounds is assessed by calculating the odor activity value (OAV), and volatile compounds with OAV ≥ 1 are generally considered to contribute significantly to the overall flavor profile [11], as calculated by Formula (3).

OAV = C_X/T_X

(3)

where C_X is the volatile compounds content/(μg/kg); and T_X is the olfactory sensory threshold of the volatile compounds in water/(μg/kg).

2.9. Statistical Analysis

All the treatments and assays were carried out thrice, and the results are presented as mean ± standard deviation. Analysis of variance (ANOVA) was performed using OriginPro version 2021. Tukey’s test was used to compute significant differences at p < 0.05.

3. Results and Discussion

3.1. Analysis of Physicochemical Properties

3.1.1. The Total Reducing Sugar and Acid Content in FSBJs

The total reducing sugar and acid content in FSBJs is shown in Figure 1. As the initial reducing sugar content and pH were adjusted to the same level before fermentation, there was no significant difference (p > 0.05) in the total acid content of the fermented juices. The total acid content was the highest in RV-FSBJ (1.86 ± 0.03 g/L). Hence, RV-FSBJ was slightly sourer than the other two FSBJs. The total reducing sugar content of RW-FSBJ was significantly lower than that of RV-FSBJ and RWRV-FSBJ (p < 0.05). Compared with RWRV-FSBJ, RV-FSBJ showed a slightly higher total reducing sugar content. Therefore, different yeast strains showed different sugar metabolizing abilities [21], among which the RW strain had higher sugar metabolizing ability and showed higher bioconversion efficiency. Zhao et al. selected 10 commercial yeasts to ferment jujube wine and found that date wine fermented by different yeast strains presented different sugar contents, which was consistent with the results of this paper [15]. As shown in Figure 1, RV consumed the least reducing sugar before the alcohol content of SBJ reached 0.45 ± 0.05% by volume, indicating that RV-FSBJ may have had the highest sugar–alcohol conversion rate. A higher sugar–alcohol conversion not only helps in maintaining an appropriate sugar–acid ratio in the beverage but also saves production costs.

3.1.2. The Color Properties of FSBJs

The effects of different yeasts on the color properties of FSBJ were examined based on the following color features: L*, a*, and b*. The findings (Figure 2) revealed significant differences in L*, a*, and b* among the three kinds of FSBJs. Based on the L*, a*, and b*, we calculated the total color difference (ΔE) as follows: ΔE (RW) = 1.63 ± 0.59; ΔE (RV) = 2.88 ± 0.18; and ΔE (RWRV) = 3.18 ± 0.23. The ΔE value of RWRV-FSBJ was significantly higher (p < 0.05) than that of RW and RV, indicating that different yeasts had significantly different effects on the color of FSBJs. The L* and b* of the FSBJ obtained using RWRV were significantly higher than those of the FSBJs obtained using RW and RV (50.53 ± 0.03 and 173.63 ± 0.33, respectively), whereas its a* is significantly lower but higher than that of RV. This suggested that the FSBJ obtained using RWRV was brighter and more yellow. The bright and yellow color of SBJ is an important parameter affecting its consumer acceptance [22]. Therefore, RWRV-FSBJ had good color properties and was considered to have favorable and pleasing color properties.

3.1.3. The Content of Functional Components in FSBJs

The TFC, TPC, and TAC of the FSBJs are shown in

Table 1. Phytochemical properties of SBJ juice fermented using different yeasts.

Sample	TFC (μg/mL)	TPC (μg/mL)	TAC (μg/mL)
RW	139.60 ± 1.16 b	143.70 ± 1.02 b	0.02 ± 0.01 b
RV	122.73 ± 0.33 c	133.30 ± 1.12 c	0.02 ± 0.002 b
RWRV	176.09 ± 0.44 a	157.90 ± 1.35 a	0.04 ± 0.004 a

^a,b,c Different letters in the same row indicate significant differences (p < 0.05).

. The yeasts were found to significantly change the functional components of SBJ to different extents (p < 0.05). Flavonoids are widely found in plants in nature, and most of them combine with sugars to form glycosides or carbon sugar groups in plants, which have medicinal value and antioxidant effects. The TFC in RW-FSBJ, RV-FSBJ, and RWRV-FSBJ was 139.60 ± 1.16, 122.73 ± 0.329, and 176.09 ± 0.44 μg/mL, respectively. Therefore, the TFC in SBJ fermented using a mixture of yeasts was significantly higher than that in SBJ fermented using a single yeast.

In sea buckthorn berries, phenols are the main contributors to biological characteristics such as antioxidant activity and have extensive pharmacological effects [23]. The TPC in RW-FSBJ, RV-FSBJ, and RWRV-FSBJ was 143.70 ± 1.02, 133.30 ± 1.12, and 157.90 ± 1.35 mg/mL, respectively. This value was significantly higher in SBJ fermented using a mixture of yeasts than in that fermented using a single yeast. Živković et al. also showed that different yeast strains of the same type will cause different effects on the antioxidant activity and phenolic content of the juice, which is consistent with the results of this study [24].

Anthocyanins are widely found in the cell sap of flowers, fruits, stems, leaves, and roots, giving these structures different colors. However, because anthocyanins are not very stable, they often change with changes in pH and temperature during food processing. The TAC of RW-FSBJ, RV-FSBJ, and RWRV-FSBJ was 0.02 ± 0.01, 0.02 ± 0.002, and 0.04 ± 0.004 μg/mL, respectively. The TAC of RWRV-FSBJ was significantly higher than that of RW-FSBJ and RV-FSBJ. This indicates that fermentation using mixed yeasts can increase the anthocyanin content in SBJ.

Overall, the findings showed that the TFC, TPC, and TAC of RWRV-FSBJ were significantly higher than those of RW-FSBJ and RV-FSBJ. The reason for the RW-FSBJ being higher than the RV-FSBJ may be that the RW is widely used in the fermentation of fruit wines such as grapes, blueberries, and strawberries; therefore, it has better fermentation properties [25]. The differences in TFC, TPC, and TAC among the three FSBJs may result from differences in the individual adaptability of yeasts and their abilities to produce hydrolase [18]. Based on the above results, it appears that the use of RWRV for fermentation may increase the levels of functional compounds in SBJ. However, the specific underlying mechanism remains to be clarified.

3.1.4. The Antiradical Properties of FSBJs

SBJ is rich in phenols, flavonoids, anthocyanins, and tannins, which have strong antioxidant effects, reducing the risk of chronic diseases and cancer as naturally occurring bioactive components [26]. Therefore, we determined the antioxidant capacity of FSBJs (Figure 3) based on DPPH-SA, ABTS-SA, ·OH-SA, and FRAP these factors. The antioxidant capacity of FSBJs was significantly different (p < 0.05) among those obtained using RW, RV, and RWRV. Compared with RW-FSBJ, RWRV-FSBJ had significantly higher values of ABTS-SA, ·OH-SA, and RP-CA; thus, RWRV-FSBJ had a higher antioxidant capacity. Meanwhile, although the reducing ability of RV-FSBJ was lower than that of RWRV-FSBJ, its ability to scavenge the three free radicals was significantly higher than that of the other two SBJs. Therefore, RV-FSBJ is also highly resistant to oxidation. Han et al. fermented greengage to produce an alcoholic beverage using RV171 and found that it had strong antioxidant and free radical scavenging abilities, consistent with our findings [10].

3.1.5. Correlation Analysis

We studied the correlation between phytochemical properties, color characteristics, and antioxidant activity indices of FSBJ (Figure 4). The results revealed no correlation between the total acid content of FSBJs and other physical and chemical factors. The reason for this is hypothesized to be that yeast is a parthenogenetic anaerobic bacterium whose metabolites are mainly alcohol and carbon dioxide, which do not have a significant effect on the acid content of the beverage [27]. Therefore, total acid cannot be considered an important factor affecting FSBJs quality. Second, total reducing sugar content was positively correlated (p < 0.05) with (ΔE), which, in turn, was significantly positively correlated (p < 0.05) with DPPH-SA, ABTS-SA, and RP-CA values; therefore, the total reducing sugar content and color may be related to the antioxidant capacity of the beverage. Anthocyanins are considered primary natural antioxidants and have many beneficial physicochemical and biological characteristics [19], while proanthocyanins can produce anthocyanins when heated in an acidic medium. In this study, the TAC of FSBJs was found to be positively correlated to the TPC, consistent with the findings of Kwaw et al. [18].

3.2. HS-SPME-GC-MS Analysis of FSBJs

This study identified a total of 54 volatile compounds in FSBJs, comprising 7 alcohols (A1 to A7), 4 acids (B1 to B4), 29 esters (C1 to C29), 2 ketones (D1 to D2), 1 aldehyde (E1), 7 hydrocarbons (F1 to F7), and 4 other compounds (G1 to G4), with their respective levels quantified (

Table 2. Volatile aroma compounds of fermented sea buckthorn juices.

No.	Lable	RT	CAS	Concentrations (mg/L)
	Alcohols			RW	RV	RWRV
A1	Trans-2-dodecen-1-ol	22.775	69064-37-5	-	-	0.68 ± 0.05
A2	Phenylethyl alcohol	50.276	60-12-8	178.07 ± 19.30 b	257.55 ± 2.99 a	174.34 ± 24.97 b
A3	Linalool	28.211	78-70-6	-	-	0.77 ± 0.08
A4	D(+)-2-Octanol	20.342	6169-06-8	2.35 ± 0.25 a	1.91 ± 0.19 b	1.88 ± 0.28 b
A5	1-Octanol-2, 7-dimethyl-	18.854	15250-22-3	-	0.32 ± 0.06	-
A6	1-Dodecanol	24.264	112-53-8	0.79 ± 0.16	-	-
A7	3-Methyl-1-butanol	11.537	123-51-3	42.48 ± 2.09	61.93 ± 6.68	-
	Total concentrations			223.69 ± 20.74 b	321.71 ± 6.37 a	177.67 ± 24.76 b
	Acids
B1	Octanoic acid	56.435	124-07-2	48.59 ± 2.53 b	71.14 ± 6.03 a	47.68 ± 5.23 b
B2	Nonanoic acid	59.94	112-05-0	0.87 ± 0.13	-	0.66 ± 0.13
B3	Hexanoic acid	48.131	142-62-1	26.96 ± 1.91 a	28.67 ± 2.57 a	21.02 ± 0.45 b
B4	Heptanoic acid	20.065	111-14-8	1.95 ± 0.20 a	1.72 ± 0.26 ab	1.35 ± 0.03 b
	Total concentrations			78.37 ± 0.90 b	101.53 ± 7.80 a	70.71 ± 5.26 b
	Esters
C1	Ethyl caprylate	20.983	106-32-1	44.92 ± 4.10 b	75.68 ± 5.92 a	39.57 ± 2.48 b
C2	Ethyl nonanoate	26.959	123-29-5	1.13 ± 0.19 b	1.58 ± 0.17 a	0.75 ± 0.03 c
C3	Hexanoic acid, propyl ester	15.454	626-77-7	-	0.65 ± 0.12	-
C4	Ethyl hexanoate	12.178	123-66-0	146.14 ± 12.50 b	197.42 ± 14.46 a	143.56 ± 7.44 b
C5	Ethyl heptanoate	16.181	106-30-9	3.07 ± 0.36 b	4.7 ± 0.38 a	2.85 ± 0.28 b
C6	Ethyl trans-4-decenoate	36.66	76649-16-6	13.27 ± 0.78 a	10.68 ± 0.51 b	9.11 ± 0.88 b
C7	Ethyl 3-hydroxy-3-methylbutanoate	19.739	18267-36-2	16.36 ± 1.65 a	16.51 ± 0.37 a	11.77 ± 1.23 b
C8	Ethyl laurate	47.307	106-33-2	6.56 ± 0.83	-	-
C9	Diphosphoric acid, diisooctyl ester	28.728	72101-07-6	4.69 ± 0.50 a	3.98 ± 0.38 a	0.04 ± 0.01 b
C10	Benzyl isovalerate	9.227	103-38-8	2.42 ± 0.24 b	3.12 ± 0.32 a	2.02 ± 0.28 b
C11	Ethyl isovalerate	6.356	108-64-5	49.43 ± 10.89 a	52.98 ± 4.32 a	50.42 ± 2.39 a
C12	3-Methylbutyl 3-methylbutanoate	14.487	659-70-1	60.6 ± 7.10 ab	77.2 ± 11.05 a	55.36 ± 7.05 b
C13	Ethyl 2-methylbutyrate	5.962	7452-79-1	-	17.12 ± 2.97	-
C14	Butanoic acid, 2-methyl-3-methylbutyl ester	13.64	27625-35-0	-	4.27 ± 0.68	-
C15	Boronic acid, ethyl-dimethyl ester	15.154	7318-82-3	4.01 ± 0.75	-	2.94 ± 0.46
C16	Borinic acid, diethyl-methyl ester	13.46	7397-46-8	0.98 ± 0.10 b	1.7 ± 0.18 a	0.07 ± 0.00 c
C17	Benzoyl isothiocyanate	25.767	532-55-8	3.05 ± 0.32 b	3.56 ± 0.2 ab	4.24 ± 0.45 a
C18	Ethyl benzoate	36.102	93-89-0	90.83 ± 11.76 a	114.55 ± 8.05 a	89.36 ± 9.66 a
C19	Ethyl phenylacetate	44.233	101-97-3	13.11 ± 1.56	18.44 ± 1.02	-
C20	Ethyl trans-4-octenoate	22.952	78989-37-4	15.68 ± 1.34	-	-
C21	Ethyl (Z)-oct-4-enoate	23.086	34495-71-1	4.84 ± 0.37 b	18.99 ± 2.03 a	15.53 ± 1.83 a
C22	2-Tetrahydrofurfuryl isothiocyanate	36.301	36810-87-4	-	-	2.56 ± 0.09
C23	Ethyl trans-2-octenoate	27.847	2351-90-8	0.39 ± 0.02 b	0.56 ± 0.08 a	0.56 ± 0.07 a
C24	Ethyl hex-2-enoate	16.65	1552-67-6	0.83 ± 0.16	0.83 ± 0.04	-
C25	Ethyl 3,3-dimethylacrylate	11.807	638-10-8	5.16 ± 0.55	-	-
C26	Ethyl 2,4-hexadienate	23.641	110318-09-7	-	-	0.35 ± 0.04
C27	Isoamyl benzoate	49.923	94-46-2	76.69 ± 10.5 ab	100.99 ± 12.94 a	64.08 ± 5.17 b
C28	Isoamyl acetate	8.148	123-92-2	-	-	0.9 ± 0.07
C29	Diisobutyl phthalate	69.458	84-69-5	-	-	0.46 ± 0.05
	Total concentrations			564.17 ± 30.85 b	725.53 ± 9.14 a	496.5 ± 20.01 b
	Ketones
D1	2-Cyclohexyl-1-tetrazol-2-yl-ethanone	38.602	74897-66-8	-	0.51 ± 0.09	-
D2	2, 4-Pentanedioneion (1-)lithium	9.936	18115-70-3	0.36 ± 0.02	-	-
	Total concentrations			0.36 ± 0.02	0.51 ± 0.09	-
	Aldehydes
E1	Hexanal, 3-(hydroxymethyl)-4-methyl-	18.633	56805-30-2	0.92 ± 0.11	-	-
	Total concentrations			0.92 ± 0.11	-	-
	Hydrocarbons
F1	Cyclohexane, 1-butenylidene-	9.981	36144-40-8	10.23 ± 1.02 a	8.97 ± 0.48 ab	7.79 ± 0.58 b
F2	Cyclohexane, ethenylidene-	40.99	5664-20-0	2.83 ± 0.19 b	3.83 ± 0.28 a	2.34 ± 0.27 b
F3	Butane, 1-chloro-3-methyl-	11.331	107-84-6	-	-	43.54 ± 3.41
F4	Borane, dimethoxy-	15.184	4542-61-4	-	5.06 ± 0.51	-
F5	Boranic acid dimethyl ester	67.82	10468-64-1	0.63 ± 0.09 b	1.15 ± 0.13 a	0.69 ± 0.04 b
F6	2, 3-Heptadien-5-yne-2-4-dimethyl-	16.02	41898-89-9	0.7 ± 0.07	-	0.3 ± 0.04
F7	1-Silacyclo-2-4-hexadiene	22.772	52023-17-3	0.46 ± 0.06 b	0.43 ± 0.05 b	0.64 ± 0.04 a
	Total concentrations			14.85 ± 0.84 b	19.45 ± 0.63 b	55.3 ± 4.15 a
	Others
G1	Morpholine, 4-methyl-4-oxide	15.649	7529-22-8	-	-	0.5 ± 0.04
G2	2,5-Dimethylbenzaldehyde	44.758	5779-94-2	39.62 ± 2.52 b	48.48 ± 5.54 ab	49.5 ± 2.57 a
G3	3, 5-Methanocyclopentapyrazole-3a-6a-hexahydro-3a-4-trimethyl-	11.218	87143-58-6	0.06 ± 0.00	-	-
G4	1H-1, 2, 3, 4-Tetrazol-5-ylmethanamine	22.213	31602-63-8	13.76 ± 1.39	16.24 ± 0.63	-
	Total concentrations			53.44 ± 2.38 b	64.72 ± 5.43 a	50.00 ± 2.57 b

Results represent the mean ± standard for three independent experiments. Values in the same column with different superscripted alphabets are significantly different at p < 0.05.

and Figure 5). Esters, alcohols, and acids were found to be the volatile compounds with the highest levels in FSBJs.

The volatile compounds in the three types of FSBJs were compared and visualized using stacked histograms (Figure 5a) and heatmaps (Figure 5b). Significantly different concentrations of volatile compounds among the three FSBJs (p < 0.05). In total, 38 volatile compounds were detected in RW-FSBJ (935.80 ± 28.12 mg/L), and 34 were detected in RV-FSBJ (1233.44 ± 16.51 mg/L) and RWRV-FSBJ (850.19 ± 41.84 mg/L). Although RW-FSBJ had the most types of volatile compounds, the content of volatile compounds was significantly higher in RV-FSBJ (p < 0.05) than in RW-FSBJ and RWRV-FSBJ. This indicated that different yeasts had different aroma-generating abilities during the fermentation of SBJ [21]. In addition, compared with single-yeast fermentation, mixed fermentation provided a lower concentration of volatile compounds, likely owing to metabolism differences between RV and RW strains. These specific metabolic mechanisms need to be explored further.

3.2.1. Esters

Esters are among the largest and most important volatile compounds in fermented beverages, and their synthesis is associated with the metabolism of lipids and acetyl-CoA (coenzyme A) [27]. In this study, we found that esters are the most volatile compounds in FSBJs, and these esters give them a fruity and floral flavor. A total of 29 esters were detected in FSBJs generated from different yeast strains, with 22 in RW and 21 in RV and RWRV each. The main esters present in FSBJs were fatty acid esters, including high levels of ethyl caprylate (fruity, floral, and sweet), ethyl hexanoate (fruity), ethyl benzoate (similar to wintergreen and ylang oil aromas), ethyl phenylacetate (strong, sweet honey aroma), and 3-methylbutyl 3-methylbutanoate (apple, mango, fruity aromas) [22]. The contents of ethyl caprylate, ethyl hexanoate, ethyl benzoate, ethyl phenylacetate, and 3-methylbutyl 3-methylbutanoate were significantly higher in RV-FSBJ than in RW-FSBJ and RWRV-FSBJ. Among them, ethyl hexanoate was the most abundant ethyl ester in FSBJs. Interestingly, it is also the most abundant and important ester in sea buckthorn berries [22]. Different yeasts have distinct effects on the ester content due to their specific physiological activities and metabolic processes. RV-FSBJ had a higher content of volatile compounds that contribute positively to sea buckthorn flavor compared to RW-FSBJ and RWRV-FSBJ, resulting in a stronger fruity aroma.

3.2.2. Alcohols

Alcohols are produced by yeasts during sugar or amino acid metabolism, promoting aroma coordination and imparting an ester aroma. FSBJs contained seven types of alcohols, with significantly higher content in RV-FSBJ (321.71 ± 6.37 mg/L) than in RW-FSBJ and RWRV-FSBJ. The alcohols were divided into two groups: higher alcohols and phenylethyl substances, with higher levels of higher alcohols, which are the largest volatile compounds in alcoholic beverages and a secondary metabolite of yeast alcohol fermentation. The higher alcohol content in FSBJs varied considerably with different yeast strain; RV-FSBJ had the highest, followed by RW-FSBJ and RWRV-FSBJ. The ester-to-higher-alcohol ratio affects the sensory quality of samples, with the highest ratio observed in RV and lowest in mixed strains. RV-FSBJ had high levels of phenethyl alcohol and 3-methyl-1-butanol, contributing positively to its fruity and floral aroma. 3-methyl-1-butanol has a calvados aroma and spicy taste, while phenethyl alcohol has a rose and honey flavor. Vitova et al. detected 26 alcohols in FSBJ, with 1-dodecanol and 3-methyl-1-butanol also being found in FSBJ [22]. Phenethyl compounds give FSBJ a floral and fruity fragrance, with phenylethyl alcohol being the main compound, giving a rose-like sweetness and fragrance. RV-FSBJ had the highest 3-methyl-1-butanol content (61.93 ± 6.68 mg/L) and a significantly higher phenylethyl alcohol content compared to RW-FSBJ and RWRV-FSBJ. Differences in yeast glycosidase and cell metabolism lead to significant differences in alcohol type and levels in the three FSBJ types.

3.2.3. Acids, Ketones, Aldehydes, Hydrocarbons, and Other Compounds

Fatty acids react with alcohols to form esters, balancing the aroma of FSBJs. RV-FSBJ had significantly higher fatty acid content (101.53 ± 7.80 mg/L) compared to RW-FSBJ and RWRV-FSBJ (p < 0.05). Octanoic, hexanoic, heptanoic, and nonanoic acids had high content in all FSBJs. While octanoic acid has an undesirable oily and mildew aroma, it contributes to pleasant cheese and fruit flavors at low concentrations. Overall, RV-FSBJ had the highest fatty acid content, while RWRV-FSBJ had the lowest.

Ketones, aldehydes, hydrocarbons, and other compounds were also detected in FSBJs at low levels (Figure 5). A total of two ketones, one aldehyde, seven hydrocarbons, and four other compounds were detected in the FSBJs. In general, volatile aldehydes contribute to aroma, including apple, citrus, and nutty flavor notes.

A large number of research papers and reviews have shown that yeast fermentation for aroma production is closely related to certain key yeast genes in the fermentation process. In addition, many fruit and vegetable juices often do not have the ability to convert volatile compounds’ precursors into aroma compounds on their own, but certain yeast strains possess the necessary enzymes to facilitate the conversion of precursors to release an aroma [21]. This may therefore be the reason why two different yeasts of the same strain produce different volatile compounds.

3.2.4. PCA Analysis of the FSBJs

In order to understand the influence of different yeasts on volatile compounds present in SBJ, principal component analysis (PCA) of the compounds in FSBJs produced using different yeast strains was conducted (Figure 6). Principal components 1 and 2 (PC1 and PC2) together explained 86.3% of the total variance (50.30% and 36.00% individually, respectively). Figure 6 shows that PC1 can clearly separate the three kinds of FSBJs, indicating that there was a significant difference between single-strain fermentation and mixed-strain fermentation. A large number of esters and a small number of alcohols and acids were observed near the left side of PCA chart.

As shown in Figure 6, RV-FSBJ had the most volatile compounds, and these were related to the properties of RV-FSBJ. RV is suitable for the fermentation of most fruit and can also provide fruit raw materials with characteristics such as high acidity and early maturity. Further, more ester and glycerol substances can be simultaneously generated during fermentation; the product has an obvious ester, the mellow fragrance is obvious, and the fermentation is stable.

3.2.5. OAV Analysis of the FSBJs

Although GC-MS analysis can characterize and quantify the volatile compounds in the samples, the aroma thresholds of each volatile compound are different; therefore, the contribution of each volatile compounds to the overall aroma of the samples cannot be accurately determined by relying solely on GC-MS detection analysis. Further determination of the extent of the contribution of each volatile compounds to the overall aroma of the sample requires an odor activity value (OAV) analysis.

The OAV is an indicator of the contribution of an aroma to the overall flavor of a sample. The OAV values of volatile compounds in the three groups of the samples are presented in

Table 3. Odor thresholds and odor activity values of key aroma-active compounds in FSBJs.

No.	Odorants	Threshold (mg/kg)	OAVs			Odor Description
	Alcohols		RW	RV	RWRV
A2	Phenylethyl alcohol	0.045 m	3957	5723	3874	Honey, spice, rose, lilac *
A3	Linalool	0.0015 m	/	/	513	Flower, lavender *
A6	1-Dodecanol	0.066 m	12	/	/	Fat, wax *
A7	3-Methyl-1-butanol	0.25 m	169.9	247.72	/	Alcoholic, banana, fruity, malt, whiskey ※
	Acids
B1	Octanoic acid	101 m	<1	<1	<1	Sweat, cheese *
B2	Nonanoic acid	1.5 m	<1	/	<1	Green, fat *
B3	Hexanoic acid	80 m	<1	<1	<1	Sweat *
B4	Heptanoic acid	10 m	<1	<1	<1	Apricot, Floral, Sour ★
	Esters
C1	Ethyl caprylate	10 m	4.5	7.6	4	Apricot, banana, brandy, fat, fruit, pear, sweet ※
C2	Ethyl nonanoate	1.2 m	<1	1.32	<1	Fruity, natural, rose, waxy, wine ※
C4	Ethyl hexanoate	0.0005 m	292,280	394,840	287,120	Apple, peel, fruity *
C5	Ethyl heptanoate	0.17 m	18.06	27.65	16.75	Pineapple, cognac, rummy, winey ※
C8	Ethyl laurate	0.33 m	19.88	/	/	clean, floral, leaf ※
C10	Benzyl isovalerate	0.1 m	24.2	31.2	20.2	Sweet, fruity, apple, pineapple, herbal ★
C11	Ethyl isovalerate	0.0002 m	247,150	264,900	252,100	Apple, fruit, pineapple, sweet ※
C12	3-Methylbutyl 3-methylbutanoate	0.046 n	1317.4	1678.3	1203.5	Apple, mango, fruity, jammy ※
C13	Ethyl 2-methylbutyrate	0.00015 m	/	114,133	/	Apple, delicious *
C14	Butanoic acid, 2-methyl-3-methylbutyl ester	0.14 n	/	30.5313	/	Apple, blueberry, cherry, citrus ※
C18	Ethyl benzoate	300 m	<1	<1	<1	Camomile, flower, celery, fruit *
C19	Ethyl phenylacetate	0.1 m	131.1	184.4	/	Anise, balsam, bitter, chocolate *
C20	Ethyl trans-4-octenoate	0.05 n	313.6	/	/	Fruity, pear, citrus ★
C25	Ethyl 3,3-dimethylacrylate	0.025 n	20.64	/	/	/
C27	Isoamyl benzoate	0.25 n	306.76	403.96	256.32	Balsamic, green, sweet ※
C28	Isoamyl acetate	0.003 m	/	/	300	Banana, bitter, fruity ※
	others
G2	2,5-Dimethylbenzaldehyde	0.2	198.1	242.4	247.5	/

^m Odor thresholds are from the Compendium of Compound Odor Thresholds [28]. ⁿ Odor thresholds are taken from the Compilations of Odour Threshold Values in Air, Water and Other Media [29]. * Odor descriptions from the website www.flavornet.org/flavornet.html (accessed on 5 May 2024). ^※ Odor descriptions from the website https://foodb.ca/compounds (accessed on 6 May 2024). ^★ Odor descriptions from the website http://www.thegoodscentscompany.com (accessed on 6 May 2024)).

. Of the volatile compounds, 15, 14, and 11 in the RW, RV, and RWRV, respectively, had OAVs > 1. Aroma constituents with an OAV > 1 can be considered to contribute to the odor profile of a food or beverage, and constituents with an OAV > 10 can be considered to be important contributors to overall flavor [11].

Among the volatile compounds from the three groups of fermentation juices, all volatile compounds had OAV values > 10 except for ethyl caprylate (1 < OAV < 10), which had apricot, banana, and brandy flavors. Previously reported studies indicated that ethyl caprylate was also present in SBJ, so it was presumed that ethyl caprylate was the volatile compound contained in SBJ itself that contributed prominently to the overall aroma [30,31,32]. Referring to the sea buckthorn-related literature, it was found that the volatile compounds (OAV > 10) common to the three groups in this experiment were detected in SBJ, so it was hypothesized that these volatile compounds were the characteristic flavor notes contained in SBJ itself [12,30,31], which mainly provide a fruity, sweet flavor to SBJ. This shows that yeast fermentation can retain the characteristic flavor substances of SBJ while metabolizing and producing a large number of new complex characteristic flavor substances, thus improving the sensory quality of the product. Among them, ethyl hexanoate had the highest OAV, followed by ethyl isovalerate, phenylethyl alcohol, and 3-methylbutyl 3-methylbutanoate, ranked 3 and 4. Ethyl hexanoate was mainly synthesized by the enzyme catalyzed synthesis of caproic acid contained in SBJ itself and ethanol produced by yeast fermentation. Ethyl isovalerate is produced by yeast fermentation under the action of acetyl coenzyme A. Ethanol is oxidized to acetaldehyde, which is oxidized to acetic acid and then dehydrogenated to isoamyl alcohol, which is oxidized to isovaleric acid; and finally, isovaleric acid and ethanol are catalyzed by enzymes to produce ethyl isovalerate. Phenylethyl alcohol mainly provides the rich honey flavor and floral aroma for SBJ, and its synthesis pathway is as follows: yeast synthesizes phenylethyl alcohol by three major biochemical reaction pathways, namely, the Embden–Meyerhof–Paras pathway (EMP pathway), the pentose phosphate pathway (PPP pathway), and the Shikimate pathway, with glucose as the substrate [33,34,35]. 3-Methylbutyl 3-methylbutanoate is produced through a series of reactions between isoamyl alcohol and isovaleric acid produced during the generation of ethyl isovalerate.

In addition, the OAV values of these volatile compounds showed a consistent size arrangement, i.e., RV > RW > RWRV, among the three fermentation groups, indicating that the fermentation of different yeasts had a role in regulating the aroma of SBJ, and the differences in the contents might be related to the metabolic abilities of different strains. The OAV value of volatile compounds in RWRV was the lowest, and the comprehensive aroma description was the worst among the three groups, and it was hypothesized that the two strains might be partially antagonistic during the fermentation process; thus, RW and RV171 strains were not suitable for fermentation of SBJ at the inoculum of 1:1 ratio. The OAV value of the volatile compounds in RV was the highest, and that of the volatile compounds detected by HS-SPME-GC-MS of theRV content was also the highest, while the OAV value of the volatile compounds in RW was in between the two groups, so RW was not the most suitable yeast strain for FSBJ.

1-Dodecanol, ethyl laurate, ethyl trans-4-octenoate, and ethyl 3,3-dimethylacrylate are volatile compounds unique to the RW, providing a floral, fruity, and slightly fatty alcohol flavor; linalool and isoamyl acetate are volatile compounds unique to the RWRV, providing a mild fruity flavor; and ethyl 2-methylbutyrate, butanoic acid, and 2-methyl-3-methylbutyl ester are volatile compounds unique to the RV. Ethyl 2-methylbutyrate has a strong apple skin, pineapple skin, and unripe plum skin aroma; butanoic acid, 2-methyl-3-methylbutyl ester has fruity flavor such as an apple flavor, blueberry flavor, cherry flavor, and citrus flavor. The unique volatile compounds in RV gave the FSBJ an intense sweet and fruity flavor, especially an apple-like flavor, and the slight alcohol and bitterness added to the flavor layers. Therefore, RV171 is the most suitable strain for the yeast fermentation of SBJ. Ma et al. selected six yeasts for fig wine fermentation and compared the quality changes of single-yeast-fermented fig wine during 6 months of aging and found that strain RV171 was the most suitable yeast strain for its fermentation [12]. 1-Dodecanol, octanoic acid, nonanoic acid, benzeneacetic acid, and ethyl ester may be compounds with unwanted sensory attributes because they mostly present fatty, bitter, grassy, etc., flavors and have an OAV < 1; therefore, they do not contribute significantly to the aroma of the FSBJ.

3.3. Redundancy Analysis

Redundancy analysis (RDA) is a ranking method that combines regression analysis with PCA [20]. RDA provides the fitting value matrix of multiple linear regressions between the response and explanatory variables. Using the different yeast strains as explanatory variables, the quality indexes of FSBJ were explored. As shown in Figure 7, the yeasts influenced all the quality indexes of FSBJ. A Monte Carlo permutation test was performed, and the high correlation between the yeasts and the quality indexes was confirmed (p = 0.002 < 0.01).

A clear positive correlation was observed between the use of RV and RWRV for fermentation and the total reducing sugar content, total acid content, DPPH·-SA, ABTS·-SA, ·OH·-SA, RP-CA, L*, b*, a*, ΔE, TPC, TAC, and TFC values. Moreover, a significant correlation was also observed with the levels of most volatile compounds. This indicates that RV and RWRV had an extremely significant influence (p = 0.002 < 0.01) on the quality of FSBJs. RV-FSBJ had the highest content of alcohols and esters, which play an important role in flavor. Hence, the flavor of RV-FSBJ was better than that of RW-FSBJ and RWRV-FSBJ. Although RWRV-FSBJ had a high content of functional components, RV-FSBJ had a stronger free radical scavenging ability than the other two FSBJs. These findings on physicochemical properties, antioxidant capacities, functional components, and volatile compounds revealed that RV is more suitable for FSBJ.

4. Conclusions

The physicochemical properties and volatile compounds of FSBJs prepared using a single inoculation and a 1:1 mixed inoculation of two commercial S. cerevisiae strains (RV171 and RW) were compared in this study. The FSBJ prepared using the mixed strains (RWRV) showed the highest brightness and a pleasing yellow color and also had the highest content of functional components. The FSBJ prepared using RV171, which had a high fermentation efficiency, had a strong antioxidant and free radical scavenging capacity. In terms of volatile compounds, RV171-FSBJ showed the highest content, followed by RW-FSBJ and RWRV-FSBJ. Esters, alcohols, and acids were found to be the main volatile compounds in FSBJs, giving them a fruity and floral fragrance. The content of ester, alcohol, and acid compounds was highest in RV171-FSBJ. In addition, OAV analysis showed that 15, 14, and 11 volatile compounds with OAV values > 1 in RW, RV, and RWRV, respectively, mainly provided sweet and fruity flavors—especially an apple flavor—to the SBJ. Among the volatile compounds shared by the three groups, RV had the largest OAV value; the volatile compounds unique to RV provided its FSBJ with a strong sweet and fruity flavor and slight alcohol and bitterness, which enriched its flavor layers. Hence, RV171 appeared to be more suitable for the fermentation of SBJ. It is expected that the results generated from this study will provide a theoretical basis for developing a new FSBJ, which can lay a foundation for extending the sea buckthorn processing industry chain and developing a sea buckthorn consumption market.

With the natural antioxidant properties, flavor advantages, and high nutritional value of sea buckthorn resources, this product has broad prospects in the field of functional beverages, nutritional supplements, etc. It is especially suitable for the health beverage market and the development of regional characteristic agricultural products. In the future, we can focus on the optimization of fermentation parameters, flavor stability control, the synergistic fermentation effect with other probiotics, and further carry out animal experiments such as cell, nematode, and mouse experiments to evaluate the safety and functionality of the product so as to accelerate the industrialization process in the food industry and to contribute to the high-value utilization of sea buckthorn resources and the development of the whole industrial chain.