Abstract
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.
Keywords: Lacticaseibacillus rhamnosus, Goji berry, Probiotic
fermentation, Bioactivity, Metabolomics
Graphical abstract
[35]Unlabelled Image
[36]Open in a new tab
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.
1. Introduction
Goji (Lycium barbarum) fruit contains various active compounds,
including polysaccharides, alkaloids, flavonoids, organic acids,
lignans, and carotenoids ([37]Miranda et al., 2024). These active
compounds have the functions of intestinal barrier repair,
anti-inflammatory, antioxidant, and flora regulation ([38]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 ([39]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 ([40]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 ([41]Palencia-Argel
et al., 2024). [42]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. [43]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 ([44]Dogan et al., 2021). However,
probiotic lactic acid bacteria exhibit strain specificity and different
biotransformation abilities in different food matrices ([45]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 ([46]Cao et al., 2022). [47]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, [48]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 [49]Li et al.
(2023). The total phenol content of the samples was determined using
the forintol method outlined by [50]Liu et al. (2023). The total
flavonoid content (TFC) of the samples was measured by the method
described by [51]Luo et al. (2024). The total sugar content of the
samples was determined using the phenol‑sulfuric acid method
([52]Nicolle et al., 2021).
The reducing sugar of the samples was determined using the
3,5-dinitrosalicylic acid colorimetry (DNS) method ([53]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 (R^2 = 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 [54]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. [55](1).
[MATH: DPPH Radical scavenging rate%=1−A1−A2A0×100 :MATH]
(1)
A[1] is the absorbance value of the sample solution, A[2] is the
absorbance value obtained after reacting the sample with anhydrous
ethanol, and A[0] 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 [56]Sun et al. (2022) with slight
modifications. Briefly, 7.4 mmol/L ABTS+ solution and 2.6 mmol/L
K[2]S[2]O[8] 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 A[0]. 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. [57](2).
[MATH: ABTS+Radical scavenging rate%=1−AA0×100 :MATH]
(2)
The hydroxyl radical (OH) scavenging capacity of the samples was
determined by referring to the method from [58]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. [59](3):
[MATH: OHRadical
scavenging rate%=1−A1−A2A0×100 :MATH]
(3)
A[1] indicates the absorbance value of the sample solution; A[2]
indicates the absorbance value of the sample control group; A[0]
indicates the absorbance value of the blank control group.
The xanthine oxidase (XOD) inhibition of goji juice was determined by
high-performance liquid chromatography ([60]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 [61]Wang, Deng, et al. (2023),
[62]Wang, Guan, et al. (2023), [63]Wang, Mi, et al. (2023) and
[64]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 [65]Suo et al. (2024a) and [66]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 [67]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 [68]Sharma et al. (2023).
Meanwhile, organic acids dissolve hydrogen ions in solution, lowering
the pH following fermentation and inhibiting spoilage bacteria
([69]Muhialdin et al., 2020).
Fig. 1.
[70]Fig. 1
[71]Open in a new tab
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.
The survival of lactic acid bacteria is an important prerequisite for
its probiotic effect ([72]Dabbagh Moghaddam et al., 2018). As shown in
[73]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 ([74]Meng et
al., 2022). As shown in [75]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 [76]Lan et al.
(2023), who also found that the soluble solids of the fermented juice
were effectively retained. [77]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 ([78]Li et al., 2023).
As presented in [79]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 [80]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 ([81]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 ([82]Xu et al., 2023).
The change patterns of total and reducing sugar contents in f goji
juice at different fermentation periods are shown in [83]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 ([84]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 [85]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 [86]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 ([87]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 ([88]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
([89]Ge et al., 2021; [90]Wu et al., 2020). [91]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.
[92]Fig. 2
[93]Open in a new tab
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).
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 ([94]Xu et al., 2023). As shown in [95]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 ([96]Deng et al., 2019; [97]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 [98]Fig. 3.
The top view of the 2D topography of the GC-IMS is shown in [99]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 ([100]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.
[101]Fig. 3
[102]Open in a new tab
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).
As presented in [103]Fig. 3B and [104]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 [105]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
[106]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 ([107]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 ([108]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.
Count Classification Compound Formula RI Rt [sec] Dt [a.u.]
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
Peak intensity
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
UFGJ FGJ0 FGJ6 FGJ12 FGJ18 FGJ24 FGJ28 FGJ32
1 Ketones 2,3-butandione C[4]H[6]O[2] 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 C[4]H[6]O[2] 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 C[5]H[8]O[2] 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 C[5]H[8]O[2] 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 C[4]H[8]O 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 C[4]H[8]O2 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- C[8]H[14]O 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 C[4]H[10]O[2] 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 C[4]H[10]O 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 C[6]H[14]O 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 C[4]H[6]O 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 C[4]H[8]O 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 C[7]H[14]O 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 C[6]H[12]O 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 C[6]H[12]O 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 C[5]H[10]O 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 C[5]H[10]O 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 C[4]H[8]O[2] 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 C[4]H[8]O[2] 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 C[8]H[14]O[2] 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 C[8]H[14]O[2] 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 C[5]H[10]O[2] 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 C[9]H[14]O 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 C[9]H[14]O 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
[109]Open in a new tab
±: 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 R^2Y
(cum) was close to 1 as seen in the OPLS-DA replacement test plot, and
the intersection of Q^2 and the vertical axis is negative in each
experimental group, indicating that the model was not overfitted. As
shown in [110]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.
[111]Fig. 4
[112]Open in a new tab
Score plots (A, C, E, G, I, K) and model validation (B, D, F, H, J, L)
of OPLS-DA.
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. [113]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 ([114]Barbalho et al., 2020).
[115]Wang, Deng, et al. (2023), [116]Wang, Guan, et al. (2023),
[117]Wang, Mi, et al. (2023) and [118]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 H[2]O[2] 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 ([119]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 ([120]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.
[121]Fig. 5
[122]Open in a new tab
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.
In addition, [123]Qin et al. (2024) reported that Wickerhamomyces
anomalus fermentation contributes to terpenoid release from red
raspberries. Meanwhile, [124]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
([125]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 [126]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 ([127]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 [128]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 ([129]Ivanova-Petropulos et al., 2018).
Additionally, [130]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 [131]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 ([132]Kumar et
al., 2023). Ferulic acid, a product in the phenylalanine metabolic
pathway, has good XOD inhibitory activity and antioxidant activity
([133]Wang, Deng, et al., 2023; [134]Wang, Guan, et al., 2023;
[135]Wang, Mi, et al., 2023; [136]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
([137]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 [138]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 ([139]Chang et al., 2023). Isoplumbagin as a
natural quinone is a natural substance with anti-inflammatory,
antimicrobial and anticancer activities ([140]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 ([141]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
[142]Fig. 6. The metabolites including (E,2S,3R,4R,5S)-2-acetamido
3,4,5,14-tetrahydroxyeicos-6-enoic acid ([143]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.
[144]Fig. 6
[145]Open in a new tab
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.
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 ([146]Fig.
7A), a total of 146 metabolic pathways were enriched and the top 30
metabolic pathways are shown in the FGJ18 vs. FGJ14 group ([147]Fig.
7B), 207 metabolic pathways were enriched and the top 30 metabolic
pathways are shown in the FGJ28 vs. FGJ18 group ([148]Fig. 7C), 146
metabolic pathways were enriched and the top 30 metabolic pathways are
shown in the FGJ32 vs. FGJ28 group ([149]Fig. 7D) and 212 metabolic
pathways were enriched and the top 30 metabolic pathways are shown in
the FGJ28 vs. FGJ0 group ([150]Fig. 7E). As shown in [151]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 ([152]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 ([153]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.
[154]Fig. 7
[155]Open in a new tab
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.
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 [156]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.
Acknowledgments