
==== Front
Food Chem X
Food Chem X
Food Chemistry: X
2590-1575
Elsevier

S2590-1575(24)00643-6
10.1016/j.fochx.2024.101755
101755
Research Article
Evaluation of physicochemical characteristics, bioactivity, flavor profile and key metabolites in the fermentation of goji juice by Lacticaseibacillus rhamnosus
An Xin ab
Li Tongtong a
Hu Jiaxue a
Li Yaoran a
Liu Huiyan a
Fang Haitian fanght@nxu.edu.cn
a⁎
Wei Xiaobo weixiaobo1992@126.com
a⁎
a School of Food Science and Engineering, Ningxia University, Ningxia Key Laboratory for Food Microbial-Applications Technology and Safety Control, Yinchuan 750021, China
b Institute of Modern Services, Bingtuan Xingxin Vocational and Technical College, Tiemenguan, 841007, China
⁎ Corresponding authors. fanght@nxu.edu.cnweixiaobo1992@126.com
22 8 2024
30 10 2024
22 8 2024
23 10175518 7 2024
8 8 2024
19 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
This study aimed to investigate the changes in physicochemical properties, bioactivities and metabolites of fermented goji juice (FGJ) by Lacticaseibacillus rhamnosus at different fermentation stages. The results showed that Lacticaseibacillus rhamnosus fermentation significantly decreased the content of soluble protein, total phenolic, total flavonoid and total sugar. Meanwhile, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and the inhibition rate of xanthine oxidase (XOD) activity were remarkably enhanced by Lacticaseibacillus rhamnosus fermentation. Flavor profiles analysis indicated that FGJ produced novel volatile compounds such as 4-methylpentanol and 2-butanol, which provide its distinct aroma. The non-targeted metabolomics analysis showed that the differential metabolites in the FGJ28 vs. FGJ0 group were mainly included 1,7-bis (3,4-dihydroxyphenyl) heptan-3-yl acetate, isoplumbagin, triacetylresveratrol, sulochrin, indole-3-acetaldehyde, etc., which might have an effect on the promotion of the bioactivity of goji juice. These findings will contribute to understanding the biotransformation effect of Lacticaseibacillus rhamnosus fermentation on goji juice.

Graphical abstract

Unlabelled Image

Highlights

• Lacticaseibacillus rhamnosus (L. rhamnosus) was used for goji juice fermentation.

• L. rhamnosus fermentation significantly enhanced the bioactivity of goji juice.

• L. rhamnosus fermentation could improve the flavor of goji juice.

• L. rhamnosus fermentation affected the accumulation of metabolites of goji juice.

Keywords

Lacticaseibacillus rhamnosus
Goji berry
Probiotic fermentation
Bioactivity
Metabolomics
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pmc1 Introduction

Goji (Lycium barbarum) fruit contains various active compounds, including polysaccharides, alkaloids, flavonoids, organic acids, lignans, and carotenoids (Miranda et al., 2024). These active compounds have the functions of intestinal barrier repair, anti-inflammatory, antioxidant, and flora regulation (Qiang et al., 2023). Goji juice, as one of the most popular goji processing products currently, suffers from major product homogenization, poor value addition, and low deep processing, resulting in insufficient market competitiveness for goji processing products (Yu et al., 2023). Thus, there is an urgent need to develop processing methods and strategies to increase the competitiveness of goji products.

Currently, the demand for functional beverages with high functional value is increasing. Probiotic lactic acid bacteria fermentation is regarded as a simple and valuable non-alcoholic fermentation process that can maintain and improve nutritional and sensory quality while also extending the shelf life of food (Meng et al., 2022). Many studies have shown that fruit juices can serve as a suitable carrier or medium for probiotics, while fermentation increases the activity of probiotics and increases their antioxidant capacity (Palencia-Argel et al., 2024). Fonseca et al. (2021) found that the number of viable bacteria exceeded 9.0 lgCFU/mL after 18 h of Lactobacillus fermentation of three different fruit juices, while the fermentation also increased the bioactive compounds in the juices, with fermentation using a single Lactobacillus plantarum, CCMA 0743, increasing the flavonoid content of passion fruit juices by approximately 3.0 μg/mL. In addition, fermentation metabolites can synergistically enhance the health benefits of probiotics. Marnpae et al. (2022) revealed that β-carotene content, DPPH radical scavenging activity, iron-reducing antioxidant capacity, and lipid peroxidation inhibition were higher in fermented gac juice than in unfermented gac juice. Thus, Fermentation can significantly alter the key components of fruit juice, leading to changes in its functional activity (Dogan et al., 2021). However, probiotic lactic acid bacteria exhibit strain specificity and different biotransformation abilities in different food matrices (Fonseca et al., 2022). In our previous study, a strain of Lacticaseibacillus rhamnosus (CICC6161) with good probiotic and fermentation properties was identified. However, its potential in the development of fermented goji juice and its effect on the physicochemical properties and the transformation of key active components of goji juice are not yet known.

Non-targeted metabolomics is capable of comprehensively detecting a wide range of metabolites in samples and can be used to characterize the metabolic processes throughout microbial fermentation utilizing substrates (Cao et al., 2022). Zhang et al. (2022) detected a total of 218 metabolites, including 51 differential metabolites, in Lactobacillus fermented chickpea milk using a non-targeted metabolomics, and found that a variety of actives were produced through multiple metabolic pathways during fermentation of Lactobacillus, which ultimately enhanced the functionality and antioxidant properties of the fermented chickpea milk. In addition, Duan et al. (2023) found that 23 differential metabolites were screened from 453 metabolites in Lactobacillus fermented goji juice by non-targeted metabolomics analysis, and the contents of isoquercitrin and m-coumaric acid in fermented goji juice from different Lactobacillus strains differed, which suggests that different Lactobacillus strains have specificity in flavanol and flavonoid biosynthesis and phenylalanine, tyrosine and tryptophan biosynthesis. Therefore, we expect to comprehensively analyze the effects of Lacticaseibacillus rhamnosus fermentation on key metabolites of goji juice through untargeted metabolomics, which can also reveal the role of Lacticaseibacillus rhamnosus biotransformation during the fermentation process.

In this study, goji berry was used as the raw material and inoculated with Lacticaseibacillus rhamnosus for liquid fermentation. The changes in the physicochemical properties and biological activities of fermented goji juice at different fermentation stages were studied. Next, the key volatile substances and non-volatile metabolites in fermented goji juice at different fermentation stages were further studied using gas chromatography-ion mobility spectrometry (GC-IMS) and untargeted metabolomic, respectively, to provide theoretical support for the biotransformation pathway of fermented goji juice by Lacticaseibacillus rhamnosus.

2 Materials and methods

2.1 Material and microorganism preparation

The dried goji berry (Lycium barbarum L. Ningqi 10) was provided by Ningxia Zhongning Wolfberry Industry Innovation Research Institute Co., Ltd. 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), caumars brilliant blue, bovine serum albumin, rutin, vitamin c, gallotannic acid, hydrogen peroxide, salicylate, ferrous sulfate, aluminum nitrate and glucose were all analytical pure and purchased from Aladdin Reagent (Shanghai) Co., Ltd.

Lacticaseibacillus rhamnosus CICC6161 was obtained from the pre-preservation strain of our laboratory. After the Lacticaseibacillus rhamnosus stored in a − 80 °C refrigerator was thawed at room temperature, it was inserted into 10 mL De Man, Rogosa and Sharpe (MRS) broth medium cooled to room temperature at 2% inoculation rate (v/v) and activated for two generations, cultured for 13 h, centrifuged at 8000 r/min at 4 °C for 10 min, and the bacterial precipitate was washed twice with normal saline. The bacterial concentration was adapted to approximately 7 lg CFU/mL.

2.2 Preparation of fermented goji juice

Dried goji berries of uniform size and without obvious defects were selected and rinsed three times with tap water. After drying at room temperature, the mass ratio of distilled water to dried goji berries was 5:1 (g/g) and 0.05% (g/mL) sodium isoascorbate solution was added for color protection. After 10 h of maceration, the mixture was added with 0.15% pectinase and pulped, and then filtered through 4 layers of sterile gauze to remove the precipitate. Next, the goji juice was adjusted to pH 6.0 by adding citric acid buffer, after which it was pasteurized by holding at 65 °C for 30 min. After cooling to room temperature, Lacticaseibacillus rhamnosus was inoculated into goji juice (5%, v/v) and fermented at 34 °C. The fermented goji juice samples were collected at 0, 6, 12, 14, 18, 24, 28 and 32 h for further analysis.

2.3 Determination of fermentation characteristics

A pH meter (PHSJ-3F, Shanghai Leimagnet Sensor Technology Co., Ltd., China) was used to determine the pH of the samples.

Bacterial counts of Lacticaseibacillus rhamnosus CICC6161 were determined using the standard plate count method. Serial dilutions of fermented goji juice(1, 10−1, 10−2, 10−3, 10−4 10−5 and 10−6) were prepared. Aliquots of 0.1 mL of appropriate dilutions were plated in MRS agar (pH 6.5 ± 0.2) plates using the spread plate method. The plates were incubated at 37 °C for 48 h. Plates containing 20–350 colonies were measured and recorded as colony-forming units (CFU) per mL of solution.

2.4 Determination of nutritional characteristics

The soluble solids content of the samples was determined using a saccharimeter (TD-45, Zhejiang Toppan Yunnong Science and Technology Co., Ltd., China). The soluble protein content of the samples was detected according to published methods described by Li et al. (2023). The total phenol content of the samples was determined using the forintol method outlined by Liu et al. (2023). The total flavonoid content (TFC) of the samples was measured by the method described by Luo et al. (2024). The total sugar content of the samples was determined using the phenol‑sulfuric acid method (Nicolle et al., 2021).

The reducing sugar of the samples was determined using the 3,5-dinitrosalicylic acid colorimetry (DNS) method (Sheng et al., 2022). The goji berry juice was centrifuged at 3000 ×g for 10 min. The resulting supernatant was diluted, mixed with 1 mL of DNS solution (0.2 mg/mL), and boiled for 5 min. A blank was formed using distilled water and DNS solution. The absorbance was measured at 550 nm using a colorimetric method. 80, 100, 120, 140, 180, 200 μg/mL glucose standard solutions were prepared, resulting in a glucose standard curve of y = 3.7304× + 0.0988 (R2 = 0.9908).

2.5 Determination of antioxidant and anti-hyperuricemia activity

The DPPH radical scavenging capacity of the samples was assessed using a method derived from Szydłowska-Czerniak et al. (2010), with minor adjustments. 0.2 mmol/L DPPH ethanol was combined with goji juice samples. The reaction mixture was incubated in the dark at room temperature for 30 min before being measured for absorbance at 517 nm. For each group of samples, the average value was determined from three parallel measurements. The DPPH radical scavenging rate was calculated according to the eq. (1).(1) DPPH Radical scavenging rate%=1−A1−A2A0×100

A1 is the absorbance value of the sample solution, A2 is the absorbance value obtained after reacting the sample with anhydrous ethanol, and A0 is the absorbance value obtained by reacting anhydrous ethanol and DPPH working solution.

ABTS+ radical scavenging capacity of the samples was determined by referring to the method from Sun et al. (2022) with slight modifications. Briefly, 7.4 mmol/L ABTS+ solution and 2.6 mmol/L K2S2O8 solution were combined in equal volumes to create the ABTS+ working solution. The solution was diluted with anhydrous ethanol to 0.7 ± 0.02 absorbance after being left in the dark for 12 h with a homogeneous mixture. The absorbance value of the mixture of ABTS+ dilution and sample was measured at 734 nm, which was recorded as A, and the absorbance value of the sample was measured with anhydrous ethanol instead of the sample, which was recorded as A0. The average value of each group of samples was measured for three parallel measurements, and the formula of the radical scavenging capacity of ABTS+ was calculated as Eq. (2).(2) ABTS+Radical scavenging rate%=1−AA0×100

The hydroxyl radical (OH) scavenging capacity of the samples was determined by referring to the method from Arise et al. (2016). The goji juice (1 mL) was added to 1 mL of salicylic acid solution (9 mmol/L). After mixing, ferrous sulfate solution (9 mmol/L, 1 mL) and 1 mL (30%, v/v) hydrogen peroxide solution were added. The absorbance of the mixture was measured at 510 nm following an incubation for 30 min at 37 °C. The average value of each group of samples was measured for three parallel measurements, and the hydroxyl radical scavenging rate was calculated as Eq. (3):(3) OHRadical scavenging rate%=1−A1−A2A0×100

A1 indicates the absorbance value of the sample solution; A2 indicates the absorbance value of the sample control group; A0 indicates the absorbance value of the blank control group.

The xanthine oxidase (XOD) inhibition of goji juice was determined by high-performance liquid chromatography (Masuda et al., 2019). The enzymatic reaction was initiated by adding 150 μL of xanthine solution (1 mmol/L) after keeping 50 μL of goji juice sample and 50 μL of xanthine oxidase working solution (0.05 U/mL) at 25 °C for 10 min. The reaction was then carried out for 30 min at 25 °C and terminated by the addition of 100 μL of 1 mol/L HCl solution. Subsequently, the reaction solution was mixed well, diluted 10-fold with ultrapure water, filtered through a microporous nylon filter membrane (0.22 μm), and then determined by high-performance liquid chromatography (LC-20 CE, Shimadzu Corporation, Japan). The mobile phase was set as a 0.02 mol/L phosphate buffer solution at pH 7.4 with 1% methanol, isostatically eluted for 10 min at a flow rate of 1.0 mL/min with a detection wavelength of 290 nm and a column temperature of room temperature., and the injection volume was 20 μL. The negative control was phosphate buffer solution substituted for goji juice samples for enzymatic reaction. The XOD inhibitory activity was calculated with the formula as follows: XOD inhibition rate (%) = [(Peak area of uric acid in the negative experiment -Peak area of uric acid in the negative experiment)] / (Peak area of uric acid in the negative experiment) × 100.

2.6 Volatile component analysis

The volatile components of the samples were determined by reference to identified using the method from Wang, Deng, et al. (2023), Wang, Guan, et al. (2023), Wang, Mi, et al. (2023) and Wang, Yang, et al. (2023). 3 mL of goji juice was transferred to a headspace glass sampling vial. 500 μL of each sample was headspace injected into the gas chromatography-ion mobility spectrometry (GC-IMS) instrument (FlavourSpec®, G. A.S. Company, Germany) after 15 min of incubation at 50 °C, with three sets of parallels determined for each sample. The inlet temperature was 280 °C, and the chromatographic column temperature was 60 °C. The carrier gas was high-purity nitrogen gas (purity ≥99.999%), with a flow rate of 2 mL/min at the initial flow rate for 2 min, linearly increasing to 10 mL/min within 8 min, 100.0 mL/min within 10 min, and 130.0 mL/min within 20 min. The flavor analysis was performed by using the Vocal analytical spectrum, and the volatile compounds were analyzed qualitatively by comparing the NIST database and IMS database, in combination with retention time and ion migration time.

2.7 Untargeted metabolite analysis

The metabolites extraction of goji juice was conducted by the method described by Suo et al. (2024a) and Suo et al. (2024b). Briefly, 100 μL goji juice was mixed with 400 μL methanol, sonicated on ice for 20 min, then stored at −20 °C for 1 h. After centrifugation at 16000g and 4 °C for 20 min to separate the precipitate, the supernatant was collected and evaporated in a high-speed vacuum concentration centrifuge. Post-addition of 100 μL methanol-water (1:1, v/v) solution for dissolution, the mixture was centrifuged at 20,000g and 4 °C for 15 min before analysis by injecting the supernatant into the sample.

Metabolomics profiling of goji juice was analyzed using a UPLC-ESI-Q-Orbitrap-MS system (UHPLC, Shimadzu Nexera X2 LC-30 CE, Shimadzu, Japan) coupled with Q-Exactive Plus (Thermo Scientific, San Jose, USA). The injection volume was 4 μL, the column temperature was 40 °C, and the flow rate was 0.3 mL/min. The chromatographic mobile phases were 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the chromatographic gradient elution program was as follows: 0–2 min, 0 %B; 2–6 min, the B varied linearly from 0% to 48%; 6–10 min, the B varied linearly from 48% to 100%; 10–12 min, the B was maintained at 100%; 12–12.1 min, the B was maintained at 100%; 12–12.1 min, the B was maintained at 100%. From 12 to 12.1 min, B varied linearly from 100% to 0%, and from 12.1 to 15 min, B was maintained at 0%.

The electrospray ionization (ESI) with positive mode and negative mode were applied for MS data acquisition separately. The HESI power conditions were set as follows: spray voltages of 3.8 kv (positive) and 3.2 kv (negative); capillary temperature of 320 °C; sheath gas (nitrogen) flow rate of 30 arb (arbitrary units); auxiliary gas flow rate of 5 arb; probe heater temperature of 350 °C; and S-lens RF levels of 50. The instrument was set to acquire over the m/z range 70–1050 Da for full MS. The full MS scans were acquired at a resolution of 70,000 at m/z 200, and 17,500 at m/z 200 for MS/MS scan. The raw MS data were processed using MS-DIAL for peak alignment, retention time correction and peak area extraction. The metabolites were identified by accuracy mass (mass tolerance <10 ppm) and MS/MS data (mass tolerance <0.02 Da) which were matched with massbank and the self-built metabolite standard library of Shanghai Bioprofile Co., Ltd.

2.8 Statistical analysis

Each experimental result was reported three times with the means and standard deviations given as M ± SD. The data were analyzed statistically using SPSS 27 (IBM Company, USA) and plotted using OriginPro 2024 (OriginLab Company, USA). Significant and highly significant differences between treatments were determined using one-way of variance (ANOVA), followed by Duncan's test at P < 0.05 or P < 0.01.

3 Results and discussion

3.1 Changes of physicochemical properties during fermentation of goji juice by Lacticaseibacillus rhamnosus

As shown in Fig. 1A, the pH of the fermented goji juice gradually decreased with the fermentation process. The pH in FGJ32 was significantly reduced by 28.3% to 4.3 compared with UFGJ, probably owing to the generation of organic acids such as acetic acid and lactic acid during the fermentation process, and resulting in a pH decrease, which is in agreement with the results of Sharma et al. (2023). Meanwhile, organic acids dissolve hydrogen ions in solution, lowering the pH following fermentation and inhibiting spoilage bacteria (Muhialdin et al., 2020).Fig. 1 Changes in pH value (A), bacterial count (B), soluble solid content (C), soluble protein content (D), total phenol content (E), total flavonoid content (F), total sugar content (G) and reducing sugar content (H) in the goji juice. Different letters indicate significant differences (P < 0.05). UFGJ, unfermented goji juice; FGJ0, fermented goji juice at 0 h; FGJ6, fermented goji juice at 6 h; FGJ12, fermented goji juice at 12 h; FGJ18, fermented goji juice at 18 h; FGJ24, fermented goji juice at 24 h; FGJ28, fermented goji juice at 28 h.

Fig. 1

The survival of lactic acid bacteria is an important prerequisite for its probiotic effect (Dabbagh Moghaddam et al., 2018). As shown in Fig. 1B, the number of viable bacteria in FGJ12 stabilized after 12 h of fermentation. At 32 h, the viable bacterial number reached 9.5 lgCFU/mL, indicating that the nutrients in goji berry juice can meet the basic growth requirements of Lacticaseibacillus rhamnosus.

The nutrient species and content of the juice are influenced by the metabolic activity of microorganisms during fermentation (Meng et al., 2022). As shown in Fig. 1C, there is no significant difference in the change of soluble solids content between pre-fermentation and post-fermentation, which is similar to the findings of Lan et al. (2023), who also found that the soluble solids of the fermented juice were effectively retained. Fig. 1D shows that the soluble protein content of goji juice reduced dramatically as fermentation progressed, probably due to the strains consuming proteins in the samples to meet their survival demands during fermentation. Besides, another explanation for the decreased soluble protein level in goji juice might be the degradation of complex plant components into simpler molecules by certain hydrolytic enzymes generated by microbes, resulting in protein depletion and consequently lower content (Li et al., 2023). As presented in Fig. 1E and F, the total phenol and flavonoid contents of fermented goji juice were significantly lower than those in UFGJ. This finding is consistent with the results of Zhou et al. (2020), which shows that probiotic bacteria employ nutrient resources during fermentation to generate new metabolites, altering the overall phenol and flavonoid content of the fermented lychee juice. The decrease in total phenolic content of fermented goji juice could be caused by phenolic compounds binding or adsorbing to proteins (Kwaw et al., 2018). In addition, the degradation of phenolic acids, especially hydroxycinnamic acid, induced by the microbial fermentation process may also be responsible for the decrease in total phenolic content (Xu et al., 2023).

The change patterns of total and reducing sugar contents in f goji juice at different fermentation periods are shown in Fig. 1G and H. The total sugar content (except at 6 h and 32 h) and reducing sugar content (except at 12 h and 18 h) of goji juice did not significantly change throughout fermentation. At 6 h, the total sugar content in fermented goji juice was significantly higher than that at other fermentation periods, potentially because of extracellular polysaccharide generation by Lacticaseibacillus rhamnosus (Monteiro et al., 2021). At 12 h and 18 h, the reduced sugar content of fermented goji juice was significantly lower than that at the other fermentation periods, which can be attributed to the increase in the number of surviving bacteria during the growth of Lacticaseibacillus rhamnosus and the resulting increase in the amount of reducing sugars consumed.

3.2 Bioactivities of Lacticaseibacillus rhamnosus fermented goji juice

The probiotic fermentation process has an important impact on the alteration of juice bioactivity. As shown in Fig. 2A, the DPPH free radical scavenging capacity of fermented goji juice increased substantially during the fermentation process, and the scavenging capacity of FGJ32 increased by 195.7% (P < 0.05) compared with UFGJ, which is consistent with the findings of Li et al. (2018). This may be because fermentation can better facilitate the dissolution of the active ingredients in the juice, which in turn improves the scavenging capacity of the juice for DPPH free radicals (Drużyńska et al., 2021). However, the ABTS+ radical and hydroxyl radical scavenging capacity of FGJ32 were significantly decreased by 9.9% and 7.5% compared with UFGJ (Fig. 2B and C), respectively. Many studies have shown that probiotic fermentation improves ABTS+ radical scavenging activity possibly due to changes in the type and content of phenolics (Ge et al., 2021; Wu et al., 2020). Wu et al. (2021) reported that reducing the content of anthocyanins with strong antioxidant capacity and increasing the content of phenolic acids with weak antioxidant capacity in fermented blueberry and blackberry juices reduced the scavenging capacity of ABTS+ radicals. The decrease in total phenolic content of goji juice during fermentation might explain the decrease in ABTS+ and hydroxyl radical scavenging activity.Fig. 2 Changes in bioactivities of DPPH (A), ABTS+ (B), OH (C) and XOD inhibition (D) in goji juice during fermentation. Different letters indicate significant differences (P < 0.05).

Fig. 2

Xanthine oxidase (XOD) is a key enzyme in the regulation of uric acid production, and increased levels of uric acid in the blood can lead to hyperuricemia (Xu et al., 2023). As shown in Fig. 2D, the inhibition of xanthine oxidase in fermented goji juice increased slowly and then rapidly as fermentation progressed. After 28 h of fermentation, the XOD inhibition of fermented goji juice significantly increased (P < 0.05) with a maximum value of 79.1%, which was 8.25-fold higher than that in UFGJ. This may be because probiotic fermentation promotes the transformation and production of relevant substances in the substrate matrix, decreasing or increasing the content of the original substances or producing new compounds. It has been shown that probiotic fermentation promoting the production of phenolic acids such as ferulic acid and caffeic acid could significantly inhibit XOD activity (Deng et al., 2019; Nile et al., 2016). Thus, further investigation is required to validate the elevated xanthine oxidase inhibition induced by fermented goji juice. These together results indicated that Lacticaseibacillus rhamnosus fermentation could dramatically increase the antioxidant activity and possible uric acid-lowering effect of goji juice.

3.3 Flavor substance composition of Lacticaseibacillus rhamnosus fermented goji juice

GC-IMS can preserve the “smell” of the samples to the maximum extent, and the results are closer to the real state. The mapping of flavor fingerprints of goji juice fermented by Lacticaseibacillus rhamnosus at different fermentation stages was determined by GC-IMS in Fig. 3. The top view of the 2D topography of the GC-IMS is shown in Fig. 3A, and the horizontal and vertical coordinates represent the drift time and retention time relative to the reactive ion peak (RIP) for the separation of the substances in LBP juice, respectively. The vertical line at 1.0 of the horizontal coordinates is the RIP peak, and the spots on both sides of the RIP peak on either side of it represent the substances, and the presence or absence of the spots and the darkness of the color indicate the degree of accumulation and decomposition of the substances, and the color ranges from white to red to indicate that the concentration ranges from low to high (Xia et al., 2021). Based on the variation of spots, it can be seen that these goji juices were different in the position, number, intensity and time of the ion peaks. These results indicate that these goji juices have significant differences in volatile substance composition and that Lacticaseibacillus rhamnosus fermentation has a significant effect on the production of flavor substances in goji juice.Fig. 3 Compositional profiles of volatiles in goji juice at different fermentation stages. Plotting of peak areas of selected signals in fermented goji juice (A), fingerprints of fermented goji juice (B).

Fig. 3

As presented in Fig. 3B and Table 1, a total of 24 volatiles were identified in all the samples, including ketones (7 types), alcohols (3 types), aldehydes (7 types), esters (2 types), pyrazines (2 types), and others (3 types). A comparison of the two-dimensional spectra of goji juice before and after fermentation showed that there were significant differences in the types and concentrations of volatile flavor substances in unfermented and fermented goji juice. Region a in Fig. 3B shows the characteristic volatiles of the pre-fermentation goji juice, which decreased as fermentation proceeds, including ehy1 E-2-hexenoate D, n-pentanal D, hexanal D, ehy1 E-2-hexenoate, 6-methy1–5-hepten-2-one, n-pentanal, 2,3-pentanedione, hexanal, ac. acetic ethy1 ester D, heptanal, 2,3-pentanedione D, ac. acetic ethy1 ester, 2-methy1–2-propenal, 2,3-butaneddiol. Region b in Fig. 3B presents the characteristic volatiles of the post-fermentation goji juice, which increased as fermentation proceeds, including 2-butanone, 4-methy1–1-pentanol, butanal, 3-hydroxybutan-2-one, 1,3-dioxolane, 2,4-dimethy1, 2-pentyl furan, 2,3-butanddioe, 2,3-butaneddione, 2-pentyl furan D. Meanwhile, fermented goji berry juice produced the new volatile substances, such as 4-methylpentanol, 2-butanol, compared with UFRJ. Alcohols are generally produced by lactic acid bacteria through the metabolism of amino acids (Chen et al., 2022), and the increase of alcohols can provide the unique fermentation flavor of goji juice. In addition, the ketones content of fermented goji juice significantly increased compared with UFRJ, while the aldehydes content significantly decreased. It has been shown that high concentrations of aldehydes and ketones bring about off-flavors and are unstable compounds under microbial action (Zhang et al., 2023). Therefore, Lacticaseibacillus rhamnosus fermentation could balance the content of volatile compounds and play an important role in improving the flavor of goji juice.Table 1 Volatile components of UFGJ, FGJ0, FGJ6, FGJ12, FGJ18, FGJ24, FGJ28 and FGJ32 samples.

Table 1Count	Classification	Compound	Formula	RI	Rt [sec]	Dt [a.u.]				Peak intensity				
UFGJ	FGJ0	FGJ6	FGJ12	FGJ18	FGJ24	FGJ28	FGJ32	
1	Ketones	2,3-butandione	C4H6O2	573.7	121.911	1.17826	739 ± 1.89	727.1 ± 6.49	846.39 ± 282.11	593.46 ± 15.12	605.95 ± 44.56	676.19 ± 330.01	1117.03 ± 11.7	1114.61 ± 19.69	
2		2,3-Butanedione	C4H6O2	620.3	139.518	1.17519	109.21 ± 5.46	136.3 ± 5.66	111.1 ± 49.24	63.38 ± 6.52	80.46 ± 5.17	87.69 ± 54.66	143.64 ± 8.7	148.86 ± 22.35	
3		2,3-Pentanedione D	C5H8O2	713.4	189.425	1.20396	3364.19 ± 189.99	3653.07 ± 15.06	1904.15 ± 949.11	1819.8 ± 194.8	1984.22 ± 159.82	2114.56 ± 1094.78	3416.31 ± 27.66	3338.89 ± 45.31	
4		2,3-Pentanedione	C5H8O2	746.1	218.236	1.20682	1559.68 ± 91.23	1466.68 ± 32.21	739.14 ± 409.8	613.53 ± 22.48	645.86 ± 37.36	734.9 ± 496.49	1293.57 ± 62.63	1236.08 ± 59.68	
5		2-Butanone	C4H8O	577.3	123.179	1.25082	194.08 ± 20.91	219.58 ± 1.43	428.77 ± 33.34	663.34 ± 138.3	372.23 ± 32.19	1716.71 ± 679.82	2414.98 ± 58.21	2089.35 ± 29.29	
6		3-hydroxybutan-2-one	C4H8O2	737.7	210.465	1.3338	7768.29 ± 510.72	7951.52 ± 74.4	7391.03 ± 1935.68	6873.92 ± 537.09	7970.44 ± 78.89	11,296.32 ± 3509.11	15,864.37 ± 302.26	16,470.26 ± 73.74	
7		5-Hepten-2-one, 6-methyl-	C8H14O	985.9	548.455	1.17459	285.78 ± 12.86	360.37 ± 10.71	316.59 ± 111.07	228.37 ± 40.54	210.4 ± 16.31	236.22 ± 73.85	296.97 ± 31.23	295.58 ± 18.94	
8	Alcohols·	2,3-Butanediol	C4H10O2	782.5	255.624	1.36874	1233.45 ± 25.72	1212.75 ± 4.05	638.47 ± 227.59	494.54 ± 48.29	468.68 ± 22.1	531.37 ± 199.32	721.31 ± 16.14	652.49 ± 13.88	
9		2-Butanol	C4H10O	598.4	130.941	1.14712	147.41 ± 1.02	122.42 ± 6.68	34.73 ± 16.7	48.63 ± 1.67	74.79 ± 4.26	106.84 ± 37.65	126.02 ± 22	142.75 ± 38.48	
10		4-Methyl-1-pentanol	C6H14O	867.5	347.888	1.32556	216.69 ± 12.33	314.38 ± 9.05	817.6 ± 350.67	556.74 ± 49.64	651.52 ± 26.44	757.91 ± 348.52	1062.36 ± 13.62	1022.28 ± 7.72	
11	Aldehydes	2-methyl-2-propenal	C4H6O	559	116.809	1.21512	418.73 ± 21.68	355.38 ± 4.38	261.76 ± 119.93	186.78 ± 25.06	196.37 ± 22.18	164.98 ± 73.6	244.74 ± 18.42	237.21 ± 3.79	
12		Butanal	C4H8O	589.7	127.7	1.29288	749.72 ± 10.03	994.21 ± 21.36	746.61 ± 457.78	639.75 ± 30.33	586.27 ± 59.36	1244.91 ± 727.18	1941.6 ± 67.79	1995.68 ± 71.27	
13		Heptanal	C7H14O	895	384.588	1.33741	743.11 ± 16.15	700.72 ± 14.45	314.53 ± 118.14	170.8 ± 7.23	165 ± 6.13	244.68 ± 111.04	350.42 ± 17.05	331.06 ± 10.04	
14		Hexanal	C6H12O	784	257.244	1.26142	2182.82 ± 7.98	2230.04 ± 31.21	1623.4 ± 537.04	1045.31 ± 77.08	959.23 ± 22.25	1142.71 ± 190.28	1286.62 ± 24.8	1205.94 ± 22.07	
15		Hexanal D	C6H12O	781.8	254.828	1.56235	2032.62 ± 50.42	2369.76 ± 93.69	1285.07 ± 401.42	357.15 ± 4.6	266.37 ± 10.66	361.96 ± 38.22	356 ± 15.41	301.23 ± 28	
16		n-Pentanal	C5H10O	649.8	151.975	1.17887	1401.37 ± 23.08	1120.11 ± 15.04	804.8 ± 562.04	350.87 ± 59.43	349.18 ± 6.62	515.96 ± 190.96	713.06 ± 17.48	812.64 ± 19.67	
17		n-PentanalD	C5H10O	650	152.062	1.40591	2516.67 ± 62.61	1397.05 ± 71.26	991.53 ± 41.03	341.95 ± 21.98	351.28 ± 7.74	332.16 ± 68.61	287.38 ± 2.16	440.35 ± 23.74	
18	Esters	ac. acetic ethyl ester	C4H8O2	604.6	133.335	1.0999	523.73 ± 27.72	437.05 ± 11.4	470.13 ± 179.59	288.47 ± 16.79	196.98 ± 28.41	388.33 ± 73.48	450.79 ± 5.86	441.06 ± 23.14	
19		ac. acetic ethyl ester D	C4H8O2	599	131.186	1.33698	4099.2 ± 269.75	4684.84 ± 107.49	3153.33 ± 1261.17	2341.02 ± 191.63	2856.32 ± 126.1	2091.99 ± 534.95	2677.5 ± 24.35	3167.85 ± 33.46	
20	Pyrazines	ethyl E-2-hexenoate	C8H14O2	1052.3	670.321	1.308	1315.67 ± 214.71	745.94 ± 154.99	349.62 ± 61.98	334.66 ± 59.03	260.79 ± 27.92	222.46 ± 14.97	212.82 ± 13.69	215.95 ± 13.32	
21		ethyl E-2-hexenoate D	C8H14O2	1044.5	655.477	1.78532	439.27 ± 318.55	74.55 ± 14.99	52.74 ± 6.48	49.31 ± 16.72	47.72 ± 5.55	56.41 ± 16.54	63.28 ± 1.9	65.19 ± 11.17	
22	Others	1,3-Dioxolane, 2,4-dimethyl, cis	C5H10O2	726.9	200.846	1.39959	1507.76 ± 60	1820.7 ± 49.49	1678.06 ± 595.22	1553.4 ± 199.04	1884.33 ± 126.99	2147.28 ± 766.97	3040.3 ± 49.24	3259.39 ± 30.49	
23		2-pentyl furan D	C9H14O	988.6	554.256	1.25346	695.59 ± 26.67	518.38 ± 16.5	483.16 ± 184.22	489.53 ± 13.84	448.39 ± 4.41	655.25 ± 242.26	812.77 ± 10.23	902.66 ± 20	
24		2-Pentyl furan	C9H14O	1037.5	642.58	1.26392	269.15 ± 12.37	289.25 ± 11.66	335.31 ± 120.74	147.36 ± 15.79	127.64 ± 7.23	139.95 ± 19.74	157.11 ± 8.8	150.26 ± 10.04	
±: Represents the standard deviation. n = 3. RI: Retention indexes, Rt: Retention time, and Dt: Drift time.

3.4 Metabolic profiling of goji juice at different fermentation stages

Based on the above findings, the physicochemical properties and bioactivities of goji juice at different fermentation stages varied significantly, especially the uric acid-lowering potential (Fig. S1). Therefore, LC-MS/MS combined with metabolomics was used to elucidate the characteristics of the changes in non-volatile flavor compounds of goji juice at different fermentation stages. The results of principal component analysis (PCA) showed that the two principal components (PC1 and PC2) together explained 54.85% of the variability, and also indicated that there were significant differences between goji juice and fermented goji juice, as well as significant differences in the metabolites of goji juice at different stages of fermentation (Fig. S2). These results indicated that Lacticaseibacillus rhamnosus fermentation significantly changed the metabolic profile of goji juice.

Orthogonal partial least squares discriminant analysis (OPLS-DA) is a supervised statistical method for discriminant analysis, which improves the parsing ability and validity of the model. The OPLS-DA model was established in this experiment, and the model validation parameter R2Y (cum) was close to 1 as seen in the OPLS-DA replacement test plot, and the intersection of Q2 and the vertical axis is negative in each experimental group, indicating that the model was not overfitted. As shown in Fig. 4A, C, E, G, I and K), the distances between the groups were clearly separated, indicating large differences between the different comparison groups.Fig. 4 Score plots (A, C, E, G, I, K) and model validation (B, D, F, H, J, L) of OPLS-DA.

Fig. 4

The screening criteria for this experiment were based on FC ≥ 1.5 or FC ≤ 1/1.5, and metabolites with P < 0.05 and VIP value >1 were identified as differential metabolites. The differential metabolites were further visualized in the form of volcano plots for each comparison group (Fig. S3). As shown in Fig. S3A, a total of 631 significantly different metabolites were detected in the comparison between the FGJ14 vs. FGJ0 group, of which 243 were significantly up-regulated and 388 were significantly down-regulated, which indicated that the metabolite changes in FGJ14 were highly significant compared with FGJ0. The results were similar for the FGJ28 vs. FGJ0 group (Fig. S3E). A total of 728 significantly different metabolites were detected, of which 238 were significantly up-regulated and 490 were significantly down-regulated. As presented in Fig. S3B, a total of 278 significantly different metabolites were detected in the FGJ18 vs. FGJ14 group, 82 of the significantly up-regulated differential metabolites and 196 of the significantly down-regulated metabolites were detected, which could be seen as less metabolite changes in FGJ18 vs. FGJ14 group compared with the FGJ14 vs. FGJ0 group. Fig. S3C shows the results of the comparison in the FGJ28 vs. FGJ18 group, with a total of 484 significantly different metabolites detected, including 192 significantly up-regulated differential metabolites and 292 significantly downregulated differential metabolites. Fig. S3D shows the comparison results of the FGJ32 vs. FGJ28 group, a total of 189 significantly different metabolites were detected, of which 100 significantly up-regulated different metabolites and 89 significantly down-regulated. The difference in metabolite changes between these two groups was minor, and it is probable that Lacticaseibacillus rhamnosus growth stabilized at a later stage of fermentation, resulting in minimal metabolite changes. The findings of hierarchical clustering of comparative groups of top50 differential metabolites are presented using a hierarchical clustering heatmap, as illustrated in Fig. S4. Red coloration indicates high relative expression, whereas blue coloration denotes low relative expression. The visualized heatmaps showed the changes of metabolites in goji juice at different fermentation stages, and their contents were significantly different before, during and after fermentation.

After screening and comparing the two groups according to the screening criteria, differential metabolites with FC value ≥1.5 and VIP value >1 among significantly up-regulated differential metabolites were screened. The differential metabolites in the FGJ14 vs. FGJ0 group were mainly screened to include 39 carboxylic acids and their derivatives, 32 fatty acyls, 22 organic oxides, 13 isoprenoid lipids, 10 indoles and their derivatives, 6 coumarins and their derivatives, 3 quinolines and their derivatives, 2 cinnamic acids and their derivatives, and 2 keto acids and their derivatives. Fig. 5A and Table S1 show the top 20 significantly up-regulated metabolites, mainly including 1,7-bis (3,4-dihydroxyphenyl) heptan-3-yl acetate, triacetylresveratrol, isoplumbagin, indole-3-lactic acid, aucubin, catalpol and diacetone ketogulonic acid, etc., and organic acids and their derivatives were the most differentiated substance species between the FGJ14 vs. FGJ0 group. The most widely known effects of resveratrol and its derivatives are its antioxidant, anti-inflammatory, and antitumor effects. The systemic anti-inflammatory and antioxidant effects of resveratrol may affect different cells and tissues (Barbalho et al., 2020). Wang, Deng, et al. (2023), Wang, Guan, et al. (2023), Wang, Mi, et al. (2023) and Wang, Yang, et al. (2023) conducted in vivo antitumor experiments on tumor-bearing mice and showed that resveratrol modulated the activity of several enzymes in tumor cells, leading to the accumulation of H2O2 in mitochondria. Indole-3-lactic acid is a tryptophan-containing metabolite with antioxidant activity and immunomodulatory effects. Meanwhile, indole-3-lactic acid produced by Lactobacillus plantarum was found to improve intestinal barrier integrity through the AhR/Nrf2/NF-κB axis. Trichosanthesinic acid is a secondary metabolite of lichens and their endophytic fungi with antimicrobial activity (Studzińska-Sroka et al., 2022). Ferulic acid, as a phenolic acid compound, is a free radical scavenger with a variety of physiological functions. Ferulic acid has a favorable inhibitory effect on XOD and may have a modulating effect on hyperuricemia (Lou et al., 2023). Therefore, these above significantly increased active substances may be metabolites produced by Lacticaseibacillus rhamnosus in fermented goji juice, which may play a major role in promoting the bioactivities of the fermented goji juice, especially possibly in uric acid-lowering activity.Fig. 5 The top20 significantly up-regulated metabolites in the comparison group. (A) FGJ14 vs. FGJ0 group, (B) FGJ18 vs. FGJ14 group, (C) FGJ28 vs. FGJ18 group, (D) FGJ32 vs. FGJ28 group, (E) FGJ28 vs. FGJ0 group.

Fig. 5

In addition, Qin et al. (2024) reported that Wickerhamomyces anomalus fermentation contributes to terpenoid release from red raspberries. Meanwhile, He et al. (2024) found that the content of acids, aromatic alcohols, ethers, terpenoids and amino acids in Lactobacillus fermented blueberry juice increased significantly as the fermentation progressed, and these compounds play an important role in determining the characteristic aroma and flavor of the fruit juice (Kelebek & Selli, 2011). In this study, the production of alcohols (catalpol) and amino acids (acetylglycine, 1-carboxyethylphenylalanine, calycanthine, and acetylhomoserine) may affect the formation of the specific flavor of goji juice.

The main differential metabolites (FC value ≥1.5 and VIP value >1) screened in the FGJ18 vs. FGJ14 group were 11 carboxylic acids and their derivatives, 11 fatty acyls, 6 isoprenoid lipids, 5 glycerol lipids, 5 indoles and their derivatives, 3 organic oxides, 1 coumarin and its derivatives, 1 quinoline and its derivatives, and 1 keto acid and its derivatives. The top 20 significantly up-regulated metabolites in FGJ18 vs. FGJ14 group are presented in Fig. 5B and Table S2, and primarily consisted of N-fructosyl isoleucine, 1,7-bis (3,4-dihydroxyphenyl) heptan-3-yl acetate, 6′-o-vanillylpaeoniflorin, caperatic acid, indole-3-lactic acid and 8, 12-octadecadienoic acid, etc. From these metabolites mentioned above, it can be seen that more carboxylic acids are generated in Lacticaseibacillus rhamnosus fermented goji juice at the middle stage of fermentation. In addition to their antibacterial and digestive qualities, fumaric acid and citric acid serve as antioxidants and acidity regulators, preventing the production of free radicals and oxidative cell damage (Li et al., 2022).

The main differential metabolites (FC value ≥1.5 and VIP value >1) screened in the FGJ28 vs. FGJ18 group were 44 carboxylic acids and their derivatives, 36 fatty acyl groups, 8 isoprenoid lipids, 8 organic oxygen compounds, 7 indoles and their derivatives, 5 glycerol sugars, 3 endolipids, 1 cinnamic acid and its derivatives, 1 quinoline and its derivatives, and 1 ketoacid and its derivatives. As shown in Fig. 5C and Table S3, the top 20 significantly up-regulated metabolites in the FGJ28 vs. FGJ18 group were N-fructosyl isoleucylglutamate, methionylleucine, 8, 12-octadecadienoic acid, glycylleucine, vernolic acid and alpha-eleostearic acid, etc. In contrast to FGJ18, FGJ28 produced significantly more malic acid, coumaric acid, and succinic acid throughout the middle and late phases of fermentation. As byproducts of the citric acid cycle, these naturally occurring organic acids exhibit potent antioxidant activity in addition to imparting flavor, color, and aroma (Ivanova-Petropulos et al., 2018). Additionally, Zhao et al. (2014) reported that p-coumaric and ferulic acid-rich brown Coix lacryma extracts have strong antioxidant and xanthine oxidase inhibitory properties, which served to lower uric acid levels in hyperuricemia rats.

The main differential metabolites (FC value ≥1.5 and VIP value >1) screened in the FGJ32 vs. FGJ28 group were 26 carboxylic acids and their derivatives, 26 fatty acyls, 4 isoprenoid lipids, 3 organic oxygen compounds, 7 indoles and their derivatives, 3 glycerol glycolipids, 3 endolipids, 2 keto acids and their derivatives, 1 cinnamic acid and its derivatives, 1 quinoline and its derivatives, and 1 flavonoid and their derivatives. As presented in Fig. 5D and Table S4, the top 20 significantly up-regulated metabolites in the FGJ32 vs. FGJ28 group were ferulic acid, N-lactoyl-tyrosine, avenoleic acid and vernolic acid, etc. Phenylpropanes are synthesized from amino acid (phenylalanine and tyrosine) analogs as precursors (Kumar et al., 2023). Ferulic acid, a product in the phenylalanine metabolic pathway, has good XOD inhibitory activity and antioxidant activity (Wang, Deng, et al., 2023; Wang, Guan, et al., 2023; Wang, Mi, et al., 2023; Wang, Yang, et al., 2023). In addition, tyrosine produced from the catabolism of phenylalanine can be involved in the synthesis of thyroid hormones and can also be catabolized to fumaric acid and acetoacetic acid. Avenoleic acid and linoleic acid are unsaturated fatty acids with rich nutritional value (Capouchová et al., 2021). Compared with FGJ28, FGJ32 showed a significant increase in the content of these above-mentioned substances.

In our present study, the bioactivities (DPPH radical scavenging capacity and xanthine oxidase inhibition) of goji juice reached a maximum value at the fermentation time of 28 h. Therefore, the key differential metabolites (FC value ≥1.5 and VIP value >1) in the FGJ28 vs. FGJ0 group were analyzed. As shown in Fig. 5E and Table S5, the top 20 significantly up-regulated metabolites in the FGJ28 vs. FGJ0 group were 1,7-bis (3,4-dihydroxyphenyl) heptan-3-yl acetate, isoplumbagin, triacetylresveratrol, sulochrin, indole-3-acetaldehyde, indole-3-lactic acid, dictyoquinazol C, 1,4-dihydro-3H-2-benzopyran-3-one, aucubin and deoxycarnitine, etc. Interestingly, 1,7-bis (3,4-dihydroxyphenyl) heptan-3-yl acetate, isoplumbagin, triacetylresveratrol were not detected in FGJ0 but highly significantly increased in the FGJ28 group. 1,7-Bis (3,4-dihydroxyphenyl) heptan-3-yl acetate belongs to the class of diarylheptanes and possesses significant anticancer, antioxidant, and antimicrobial activities (Chang et al., 2023). Isoplumbagin as a natural quinone is a natural substance with anti-inflammatory, antimicrobial and anticancer activities (Tsao et al., 2020). Among the key differential metabolites of the FGJ14 vs. FGJ0 group in this study, it was also found that the contents of 1,7-bis (3,4-dihydroxyphenyl) heptan-3-yl acetate, isoplumbagin, triacetylresveratrol, indole-3-lactic acid and aucubin were also significantly increased in the goji juice after 14 h of fermentation.

In addition, we further analyzed differential metabolites (VIP value >1 and P < 0.05) in multiple groups (FGJ0_FGJ14_FGJ18_FGJ28_FGJ32) comparisons. As shown in Fig. S5, a total of 284 significantly different metabolites were detected. Next, k-value cluster analysis was used to analyze the trends of key metabolites during the fermentation of goji juice. As shown in Fig. S6A, each line indicates the trend of key metabolites in goji juice in different clusters as the fermentation proceeded, and the black line indicates the trend of the average value of the content of differential metabolites in different clusters. Cluster 2 shows that a total of 67 metabolites were accumulated in goji juice as fermentation progressed. The general categorization information of 67 metabolites is shown in Fig. S6B. Based on the information of metabolite categorization, the top 20 of significantly up-regulated differential metabolites with VIP values >1 is presented in Table S6, and mainly included N-lactoyl-tryptophan, o-succinylhomoserine, dodecatrienoic acid, tartaric acid, fumaric acid and citric acid, etc. TCA cycle is the hub connecting sugar, amino acid and lipid metabolism. Organic acids including tartaric acid and fumaric acid enter the TCA cycle via acetyl coenzyme A to form citric acid during the fermentation process (Xie et al., 2023). In our present study, the bioactivities (DPPH radical scavenging capacity and xanthine oxidase inhibition) of goji juice increased with fermentation. The correlation analysis between the differential metabolites of the top 20 and the biological activity of fermented goji juice is shown in Fig. 6. The metabolites including (E,2S,3R,4R,5S)-2-acetamido 3,4,5,14-tetrahydroxyeicos-6-enoic acid (POS11294), N-lactoyl-tryptophan (NEG3533), octadeca-9,12-dienal (POS7072), O-succinylhomoserine (NEG2480), 6,10,14-trimethyl-5,9,13-pentadecatrien-2-one (POS7011), dodecatrienoic acid (POS4631) and citric acid (NEG1934) were significant positive correlation with DPPH free radical scavenging activity and XOD inhibitory activity in goji juice. Thus, these results suggest that the enhancement of DPPH free radical scavenging capacity and XOD activity inhibition may be related to the increase in the content of these metabolites during the fermentation of goji juice, and further research is required to confirm this hypothesis.Fig. 6 Heatmap of correlation of DPPH and XOD with significantly different metabolites in comparison group of FGJ0_FGJ14_FGJ18_FGJ28_FGJ32. * and ** indicate significantly different at P < 0.05 and P < 0.01, respectively.

Fig. 6

3.5 KEGG pathway annotation and enrichment analysis of differential metabolites in goji berry juice at different fermentation stages

Fermentation is a very complex metabolic process involving the formation of bioactive components and flavor substances, and pathway analysis of key differential metabolites can reveal mechanisms of substance regulation at the metabolic level. Here, we explored the pathway enrichment analysis associated with differential metabolites among the comparison groups in this study. By comparison of KEGG databases, a total of 214 metabolic pathways were enriched and the top 30 metabolic pathways are shown in the FGJ14 vs. FGJ0 group (Fig. 7A), a total of 146 metabolic pathways were enriched and the top 30 metabolic pathways are shown in the FGJ18 vs. FGJ14 group (Fig. 7B), 207 metabolic pathways were enriched and the top 30 metabolic pathways are shown in the FGJ28 vs. FGJ18 group (Fig. 7C), 146 metabolic pathways were enriched and the top 30 metabolic pathways are shown in the FGJ32 vs. FGJ28 group (Fig. 7D) and 212 metabolic pathways were enriched and the top 30 metabolic pathways are shown in the FGJ28 vs. FGJ0 group (Fig. 7E). As shown in Fig. 7C, the key metabolic pathways in FGJ18 vs. FGJ14 group were ABC transporters, protein digestion and absorption, central carbon metabolism, aminoacyl-tRNA biosynthesis and mineral absorption. For the FGJ28 vs. FGJ18 group (Fig. 7D), the key metabolic pathways were ABC transporters, central carbon metabolism, protein digestion and absorption, aminoacyl-tRNA biosynthesis and biosynthesis of amino acids. During fermentation, bacterial proliferation is influenced by changes in metabolites that can impact various metabolic pathways, leading to increased or decreased metabolite levels (Zhan et al., 2023). Notably, among the KEGG pathways in the FGJ28 vs. FGJ0 group, the fermentation of Lacticaseibacillus rhamnosus led to significant changes in both the tricarboxylic acid (TCA) cycle and amino acid related metabolism (biosynthesis of amino acids, alanine, aspartate and glutamate metabolism, tyrosine metabolism, beta-alanine metabolism and cysteine and methionine metabolism), which may be enhance the flavor of goji juice by producing organic acids and amino acids substances. Besides, phenylalanine metabolism and phenylpropanoid biosynthesis were significantly regulated by the fermentation of Lacticaseibacillus rhamnosus, which may contribute to the production of polyphenolic substances to enhance the biological activity of goji juice.Fig. 7 KEGG enrichment pathways of the differential metabolites among different comparison groups. (A) FGJ14 vs. FGJ0, (B) FGJ18 vs. FGJ14, (C) FGJ28 vs. FGJ18, (D) FGJ32 vs. FGJ28, (E) FGJ28 vs. FGJ0 and (E) Comparison group of FGJ0_FGJ14_FGJ18_FGJ28_FGJ32.

Fig. 7

In addition, 193 metabolic pathways were enriched in the multi-group comparison, of which 84 had a significant effect in metabolite formation (P < 0.05). The top 30 KEGG pathways are shown in Fig. 7F, and the major metabolic pathways were ABC transporters, central carbon metabolism, alcoholism, protein digestion and absorption, biosynthesis of amino acids and aminoacyl-tRNA biosynthesis, which had significant effects on metabolites formation in the different stages of Lacticaseibacillus rhamnosus fermented goji juice. These results indicated that the key metabolic pathways including ABC transporters, aminoacyl-tRNA biosynthesis, biosynthesis of amino acids and protein digestion play important roles for the generation of key metabolites in the pre-, mid-, and post-fermentation phases.

4 Conclusion

In conclusion, Lacticaseibacillus rhamnosus fermentation significantly alters the physicochemical properties, bioactivities, and key characteristics of volatile and non-volatile metabolites of goji juice. The soluble protein, total phenols, and total flavonoids contents decreased while total sugar content increased as Lacticaseibacillus rhamnosus fermented. The ability of fermented goji juice to scavenge DPPH free radicals and inhibit xanthine oxidase was enhanced during the fermentation process. The volatile components undergo significant variations at different stages, and the aroma of fermented goji juice is distinctly influenced by the formation of alcohol substances during fermentation. The primary non-volatile differential metabolites in goji juice at different fermentation stages were amino acids, organic acids, and phenylpropanoids, particularly carboxylic acids and their derivatives, which were significantly enhanced during the process and may contribute to the boost in antioxidant activity and uric acid-lowering capacity. These results may provide a theoretical basis for the biotransformation of bioactive components in fermented goji juice.

CRediT authorship contribution statement

Xin An: Writing – original draft, Investigation, Data curation. Tongtong Li: Investigation, Data curation. Jiaxue Hu: Investigation, Data curation. Yaoran Li: Methodology, Data curation. Huiyan Liu: Investigation, Conceptualization. Haitian Fang: Supervision, Methodology, Data curation, Conceptualization. Xiaobo Wei: Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

Supplementary material: Fig. S1 Changes in the inhibition rate of xanthine oxidase in goji berry juice at different fermentation stages.Fig. S2 PCA of non-fermented and Lacticaseibacillus rhamnosus fermented goji juice.Fig. S3 Volcano plots of metabolite species distribution. A, B, C, D, and E represent volcano plots of significantly different metabolite species in the FGJ14 vs. FGJ0 group, FGJ18 vs. FGJ14 group, FGJ28 vs. FGJ18 group, FGJ32 vs. FGJ28 group and FGJ28 vs. FGJ0 group, respectively.Fig. S4. The significantly different metabolite heat maps of comparison groups. (A) FGJ14 vs. FGJ0 group, (B) FGJ18 vs. FGJ14 group, (C) FGJ28 vs. FGJ18 group, (D) FGJ32 vs. FGJ28 group, (E) FGJ28 vs. FGJ0 and (F) FGJ0_FGJ14_FGJ18_FGJ28_FGJ32 comparison group.Fig. S5 The significantly different metabolites in multiple comparison group (FGJ0_FGJ14_FGJ18_FGJ28_FGJ32).Fig. S6. Trend plot of K-mean clustering (A) and ring plot of differential metabolite classification (B) for comparison groups of FGJ0_FGJ14_FGJ18_FGJ28_FGJ32.

Image 1

Data availability

Data will be made available on request.

Acknowledgments

This work was financially supported by Ningxia Natural Science Foundation (2023AAC03135 ) and Scientific Research Project for Higher Education Institutions of Ningxia (NYG2022009 ).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2024.101755.
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