Biochemical and physical investigations on detoxification of ginkgo kernel juice using probiotic fermentation with macroporous resin addition

Yuyu Sun; Jiaying Zhao; Sivakumar Manickam; Jingyang He; Dandan Li; Yongbin Han; Xiaosan Jiang; Yang Tao; Yuyu Sun; Jiaying Zhao; Sivakumar Manickam; Jingyang He; Dandan Li; Yongbin Han; Xiaosan Jiang; Yang Tao

doi:10.48130/FIA-2023-0032

The toxicity of ginkgo kernel is a global concern, restricting its consumption as a medicinal food. This study focuses on eliminating the toxic components, specifically ginkgolic acid, from ginkgo kernel juice. The approach used was probiotic fermentation with autochthonous lactic acid bacteria combined with macroporous resin. Compared to using lactic acid fermentation alone, adding macroporous resin during probiotic fermentation significantly enhanced the removal of toxic ginkgolic acid and 4'-O-methylpyridoxine from ginkgo kernel juice. After 48 h of fermentation with macroporous resin, the contents of ginkgolic acid and 4'-O-methylpyridoxine decreased by more than 69% and 61%, respectively. Interestingly, the adsorption of microbial growth inhibitors, such as ginkgolic acid, 4'-O-methylpyridoxine, and phenolics, by the resin did not hinder the growth of lactic acid bacteria or their metabolic activities involving organic acids and monosaccharides. The study further confirmed that microbial adsorption was the primary reason for removing ginkgolic acid during probiotic fermentation. Also, the adsorption mechanism of ginkgolic acid during probiotic fermentation with macroporous resin was explored. From a mass transfer perspective, incorporating macroporous resin during the probiotic fermentation of ginkgo kernel juice reduced the mass transfer resistance for surface diffusion. Consequently, this lowered the contribution of surface diffusion to the overall diffusion process and facilitated the efficient removal of toxic ginkgolic acid. This work can help to understand the physical mechanism regarding detoxification of ginkgo kernel juice by probiotic fermentation, and offer potential strategies to enhance the safety of ginkgo kernel products.

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Materials and methods

Materials

In this study, Ginkgo biloba kernels of the cultivar Dafozhi were provided by Taizhou Xiyang Food Co., Ltd. (China) Autochthonous lactic acid bacteria were isolated from ripe Ginkgo biloba fruits and characterised (Supplementary Table S1 & Supplementary Fig. S2). The isolated lactic acid bacteria were found to be strains of Lactobacillus plantarum and Lactobacillus pentosus. Moreover, four previously isolated microbial strains, namely Lactobacillus plantarum LSJ-TY-HYB-T7 (L. plantarum T7), Lactobacillus plantarum LSJ-TY-HYB-T9 (L. plantarum T9), Lactobacillus fermentum LSJ-TY-HYB-C22 (L. fermentum C22) and Lactobacillus fermentum LSJ-TY-HYB-L16 (L. fermentum L16) from different berry fruits, were also utilised in this study^[20]. To compare their abilities in removing toxins and enriching bioactives in ginkgo kernel juice after 48 h of fermentation, the selected microbial strains underwent evaluation (Supplementary Fig. S3). Based on the results, Lactobacillus plantarum TY-HYB-SYY-Y2 (L. plantarum Y2), Lactobacillus plantarum TY-HYB-SYY-Y4 (L. plantarum Y4), Lactobacillus pentosus TY-HYB-SYY-Y3 (L. pentosus Y3), and Lactobacillus plantarum LSJ-TY-HYB-T7 (L. plantarum T7) were chosen for further investigations. All these LAB strains were stored in 50% glycerol tubes and kept at −20 °C for future use.

Experimental

Pretreatment with macroporous resin
The macroporous resin D101 and DA201 were pretreated following the method described by Wu et al.^[21]. The pretreatment process involves the following steps. The macroporous resin were soaked in 500 mL of 95% aqueous ethanol for 24 h. After soaking, the resin were thoroughly washed with deionised water. Then, 500 mL of 4% HCl was added to the resin for rinsing, which continued for 4 h. The resin were then washed with deionised water until their pH reached 7.0. Subsequently, 500 mL of 5% NaOH was used to rinse the resin for another 4 h. After the alkaline rinsing, the resin were washed with deionised water until their pH returned to 7.0. Finally, the pretreated resin were dried at 30 °C until they reached a constant weight.

Preparation of ginkgo kernel juice
The method outlined by Wang et al. was followed to prepare ginkgo kernel juice^[12]. Fifty g of fresh ginkgo biloba kernels were taken and thoroughly washed. The washed kernels were ground in 250 mL of distilled water using a fruit juicer (MJ-BL25B22, Midea Holding Co., Ltd, China). For starch hydrolysis, 0.25 g of α-amylase (with an activity of 4,000 U/g) and 0.015 g of glucoamylase (with an activity of 100,000 U/g) were added to the ginkgo kernel juice. The mixture underwent enzymolysis at 60 °C for 2 h. After enzymolysis, the mixture was centrifuged at 5,000 rpm for 20 min to remove any insoluble precipitates. The ginkgo kernel juice was pasteurised at 90 °C for 20 min in a water bath (HH-S, Changzhou Wandasheng Experimental Instrument Co., Ltd, Changzhou, China) to ensure its safety and preservation. To facilitate the measurements of ginkgolic acids using the HPLC method, standard ginkgolic acid C15:1 and ginkgolic acid C13:0 were added to the pasteurised ginkgo kernel juice, making their contents 2.70 and 2.20 mg/L, respectively.

Fermentation of ginkgo kernel juice
Before fermentation, the pre-stored Lactobacillus strains were activated in 50 mL of MRS broth and then incubated at 30°C for 24 h in thermostatic incubator (PYX-DHS, Shanghai Yuejin Medical Equipment Co., Ltd, Shanghai, China). At the same time, the activated resin were sterilised at 121 °C for 20 min.

The sterilised ginkgo kernel juices were divided into two groups for the fermentation experiment. Activated Lactobacillus inoculum, 2.0% (v/v), was added directly to the juice in one group. In the other group, 5 g of sterilised resin were first added to the 250 mL juice samples, and then the 2.0% (v/v) inoculum was added. The initial cell counts in all the ginkgo kernel juice samples were approximately 7.34 ± 0.05 Log CFU/mL. Subsequently, the inoculated samples were cultured in a thermostatic oscillation incubator (SPH-100B Shiping Test Equipment Co., Ltd, China) at 30 °C and 200 rpm for 48 h. Samples were collected at different fermentation periods (0, 24, 48 h) for further analysis.

Comparative experiments investigating the variations in ginkgolic acid content in broths containing active and inactive Lactobacillus strains
The pre-stored Lactobacillus plantarum Y2 strains were activated in 50 mL of MRS broth and incubated at 30 °C for 16 h, allowing the strains to reach the stationary phase. After activation, the broth samples were divided into two groups. In one group, the samples were sterilised at 121 °C for 20 min, while the microbial strains remained alive in the other group. Then, 2 mg of standard ginkgolic acid C15:1 was added to 25 mL of the aforementioned MRS broth in both groups. The samples were further incubated at 30 °C and 200 rpm for 8 h in an orbital shaker to test the influence of both active and inactivated strains on the retention of ginkgolic acid.

After the incubation, the microbial strains were separated from the broths by centrifugation at 10,000 rpm for 10 min. The collected strains were mixed with methanol at 30 °C and 200 rpm for 1 h to investigate whether ginkgolic acid was desorbed from the microbial strains.

Adsorption studies
L. plantarum Y2 and macroporous resin D101 were chosen as the representative adsorbents for the investigation of the bio-adsorption mechanism.

Adsorption kinetics of ginkgolic acid
L. plantarum Y2 was first cultivated until it reached the stationary phase, and then it was inactivated following the procedure described above. The resulting microbial debris was collected by centrifugation and washed three times with deionised water. Subsequently, the microbial debris was dried at 30 °C to reach a constant weight and then milled into powders (Supplementary Fig. S4).

A standard aqueous solution of ginkgolic acid C15:1 at 10 mg/L was prepared. Separate mixtures of 100 mg microbial debris powders, 12.5 mg resin, and a combination of 100 mg microbial debris powders with 12.5 mg resin were added to 25 mL of the ginkgolic acid C15:1 solutions. The adsorption processes were carried out in a thermostatic oscillation incubator at 30 °C and 200 rpm. The concentration of ginkgolic acid C15:1 was measured at regular intervals until the adsorption equilibrium was achieved. The absorption capacity of each adsorbent is expressed in the following Eq (1):

$ {q}_{s,e}=\dfrac{\left({C}_{0}-{C}_{t}\right)}{m}V $ (1)

where, q_s,e is mass of ginkgolic acid adsorbed by the adsorbent at equilibrium (i.e. microbial strains, macroporous resin, and their mixture) (mg/g); C₀ is ginkgolic acid concentration in the solution at 0 min (mg/L); C_t is ginkgolic acid concentration in the bulk solution at t min (mg/L); m is weight of adsorbent (i.e. microbial strains, macroporous resin, and their mixture) (g); V is the volume of bulk solution of ginkgolic acid (mL).

Adsorption isotherm of ginkgolic acid
Standard aqueous solutions of ginkgolic acid C15:1 were prepared at concentrations of 5, 10, 12, and 15 mg/L. The microbial debris, the resin, and mixtures were added for each solution, following the procedures described previously. All adsorption processes were allowed to reach equilibrium. Afterwards, the concentration of ginkgolic acid C15:1 at equilibrium (C_e) and the adsorption capacity (q_e) were determined. The obtained data were then fitted to the Langmuir isotherm Eq (2):

$ {q}_{s}=\dfrac{{q}_{m}b{C}_{e}}{1+b{C}_{e}} $ (2)

where, q_s is mass of ginkgolic acid adsorbed by the adsorbent (i.e. microbial strains, macroporous resin, and their mixture) (mg/g); q_m is the maximum adsorption capacity of the adsorbent (i.e. microbial strains, macroporous resin, and their mixture) predicted by the Langmuir model (mg/g); b is langmuir constant (L/mg); C_e is ginkgolic acid concentration in the solution at equilibrium (mg/L).

Physicochemical analysis during fermentation

pH
The pH of ginkgo kernel juice was measured using a digital pH meter (PHS-3C, Shanghai INESA Scientific Instrument Co., Ltd, Shanghai, China).

Measurement of viable cell count
The viable cell counts were determined using the standard plate counting method after performing serial dilutions^[22]. The number of viable bacteria is expressed as Log CFU/mL.

Quantification of organic acid content
The analysis of individual organic acid contents in the ginkgo kernel juice was conducted using an HPLC system (LC-2010A, Shimadzu Tokyo, Japan) with separation achieved on the Agilent TC-C18 column (4.6 mm × 25 mm, 5.0 μm). The specific chromatographic method employed in this study can be referred to in our previous work^[23]. The concentrations of different organic acids are expressed as mg of each standard/L.

Quantification of monosaccharide content
The analysis of different monosaccharide contents in the ginkgo kernel juice followed the methodology described in our previous work^[24]. Calibration curves were established using various monosaccharide standards. The concentrations of different monosaccharides are expressed as mg of each standard/L.

Quantification of ginkgolic acid content
In this study, the types of ginkgolic acids present in the ginkgo kernel juice were initially identified using HPLC-MS, leading to the tentative identification of three ginkgolic acids (Supplementary Text 1 & Supplementary Table S2). Subsequently, the contents of two representative ginkgolic acids, namely ginkgolic acid C15:1 and ginkgolic acid C13:0, were quantified in the ginkgo kernel juice using a Waters 1525 HPLC system (Mirford, USA) connected to a YMC Carotenoid column (4.6 mm × 150 mm, 3.0 μm). The chromatographic conditions were adopted from a previous study by Wang et al.^[12]. Specifically, the mobile phase consisted of 3% glacial acetic acid in deionised water:methanol (8:92, v/v), and the elution was carried out at a flow rate of 0.8 mL/min. The detection wavelength and column temperature were set at 310 nm and 30 °C, respectively. An injection volume of 20 μL was used for the analysis. The content of each ginkgolic acid in the ginkgo kernel juice is expressed as mg of each standard/L.

Quantification of 4'-O-methylpyridoxine (MPN) content
MPN in the ginkgo kernel juice was quantified using the same Shimadzu LC-2010A device coupled with an Agilent TC-C18 column (4.6 mm × 250 mm, 5.0 μm). The chromatographic conditions were adapted from a previous study by Zou et al.^[25]. The mobile phase A used was a 5.0 mmol/L potassium phosphate solution containing 5.0 mmol/L sodium pentane sulfonate (pH 2.5), while mobile phase B consisted of acetonitrile. The gradient program for elution was as follows: 0−10 min, 4%-8% B; 10−15 min, 8%−10% B; 15−20 min, 10%−8% B; 20−40 min, 8−4% B. The flow rate was set at 1 mL/min. The column temperature was maintained at 30 °C, and the detection wavelength was set at 292 nm. An injection volume of 20 μL was used for the analysis. The results are expressed as mg of each standard/L.

Quantification of total phenolics content
The total phenolics content in the ginkgo kernel juice was measured using the Folin-Ciocalteu method using a UV-Vis spectrophotometer (TU-1900, Persee General Instruments Co., Ltd, Shanghai, China), as described in detail in our previous study^[26]. Gallic acid was employed as the standard substance for calibration. The results are reported as mg of gallic acid equivalent /L.

Physicochemical analysis during adsorption

Porosity
The porosity (ε_p) of the adsorbent was estimated using the following Eq (3):^[27]

$ {\varepsilon }_{p}=1-\dfrac{{\rho }_{p}}{{\rho }_{s}} $ (3)

where, ρ_p is apparent density of adsorbent (i.e. microbial strains, macroporous resin, and their mixture) (g/mL); ρ_s is solid density of adsorbent (i.e. microbial strains, macroporous resin, and their mixture) (g/mL).

In this study, the porosity values for the debris powders of L. plantarum Y2, macroporous resin D101, and their mixture were determined as follows: 0.005, 0.238 and 0.057, respectively. The low porosity indicates that microbial debris can be considered solid materials with limited pore space.

Particle size distribution
The particle size distribution of the adsorbents was determined using a laser particle size analyser (Mastersizer 2000, Malvin Instruments Co., Ltd, UK). The average particle sizes for microbial debris, resin, and their mixture were 30.92 μm, 127.10 μm, and 38.70 μm, respectively.

Zeta potential
The Zeta potentials of the dispersions before and after adsorption were determined using the Nano ZS90 Zetasizer (Malvin Instruments Co., Ltd, UK). The temperature was maintained at 25 °C. The dispersant and the number of tests were set to 'water' and 'Automatic' respectively. The measurements were performed in triplicate to ensure accuracy and reliability.

FT-IR
The microbial debris, resin, and their mixture before and after adsorption were collected and dried to remove any residual water. Subsequently, the dried materials were milled into powders. For the FT-IR analysis, 1 mg of each powder was mixed separately with 100 mg of KBr and then pressed into flakes with a height of 2 mm. The resulting flakes were scanned using the Nicolet IR200 FT-IR spectroscopy (Nicolet IR 200, Thermo Electron Corporation, Massachusetts, USA) in the range of 4000 to 400 cm⁻¹. This FT-IR analysis was performed to investigate any changes or interactions in the chemical compositions of the adsorbents before and after the adsorption process.

Mass transfer during adsorption

The pore volume and surface diffusion model (PVSDM) was employed to analyse the concentration decay of ginkgolic acid during physisorption. This model incorporates several phenomena, including external mass transfer of ginkgolic acid from the bulk solution to the adsorbent surface, intraparticle diffusion (surface or pore volume diffusion of ginkgolic acid inside the adsorbent particle), and instantaneous adsorption of ginkgolic acid on the active sites^[28−30]. To simplify the model, the following assumptions were considered^[31,32]:

(1) Microbial debris, resin, and their mixture were assumed to have spherical and isotropic geometry. As a result, the intraparticle diffusion mode of ginkgolic acid was considered to be one-dimensional radial diffusion.

(2) The PVSDM model depicting the adsorption of ginkgolic acid by various adsorbents, based on the assumptions of insignificant convective mass transfer inside the pores and constant pore volume diffusion coefficient (D_ep) and surface diffusion coefficient (D_s), is expressed as follows (Eq 4):

$ {\varepsilon }_{p}\dfrac{\partial {C}_{s}}{\partial t}+{\rho }_{p}\dfrac{\partial {q}_{s}}{\partial t}=\dfrac{1}{{r}^{2}}\dfrac{\partial }{\partial r}\left({r}^{2}\left({D}_{ep}\dfrac{\partial {C}_{s}}{\partial r}+{D}_{s}{\rho }_{p}\dfrac{\partial {q}_{s}}{\partial r}\right)\right) $

(4)

where, ε_p is porosity of adsorbent (i.e. microbial strains, macroporous resin, and their mixture); C_s is ginkgolic acid concentration in the solution within the porous space of adsorbent (i.e. macroporous resin alone or the mixture of resin and microbial strains) (mg/L); t is adsorption time (min); D_ep is pore volume diffusion coefficient of ginkgolic acid (m²/s); D_s is surface diffusion coefficient of ginkgolic acid (m²/s).

The initial conditions are defined as follows (Eq 5):

$ t=0\quad {C}_{s} =0\quad {q}_{s} =0 \quad 0\leq r \leq R $

(5)

where, R is radius of adsorbent (i.e. microbial strains, macroporous resin and their mixture) (cm)

The boundary conditions are described as follows (Eqs 6 & 7):

$ {\left.\dfrac{\partial {C}_{s}}{\partial r}\right|}_{r=0}=0 $

(6)

$ {D}_{ep}{\left.\dfrac{\partial {C}_{s}}{\partial \mathrm{r}}\right|}_{r=R}+{D}_{s}{\rho }_{p}{\left.\dfrac{\partial {q}_{s}}{\partial \mathrm{r}}\right|}_{r=R}={k}_{L}({C}_{L}-{C}_{s}) $

(7)

where, k_L is external mass transfer coefficient of ginkgolic acid (m/s); C_L is total ginkgolic acid concentration in the solution (mg/L).

Since the adsorption on active sites occurred much faster than intraparticle diffusion, it was assumed that the local adsorption equilibrium on the solid-liquid interface could be achieved instantaneously^[33]. Consequently, the term Cs in the PVSDM model can be replaced by q_s using the Langmuir relationship (Eq 2). Subsequently, Eqs (4, 6, & 7) can be transformed into Eqs (8, 9, & 10), respectively as follows:

$ \left({\varepsilon }_{p}\dfrac{{q}_{m}b}{{({q}_{m}b-b{q}_{s})}^{2}}+{\rho }_{p}\right)\dfrac{\partial {q}_{s}}{\partial t}=\dfrac{1}{{r}^{2}}\dfrac{\partial }{\partial r}\left({r}^{2}\left({D}_{ep}\dfrac{{q}_{m}b}{{({q}_{m}b-b{q}_{s})}^{2}}+{D}_{s}{\rho }_{p}\right)\dfrac{\partial {q}_{s}}{\partial r}\right) $

(8)

$ \dfrac{{q}_{m}b}{{({q}_{m}b-b{q}_{s})}^{2}}{\left.\dfrac{\partial {q}_{s}}{\partial r}\right|}_{r=R}=0 $

(9)

$ {D}_{ep}\dfrac{{q}_{m}b}{{({q}_{m}b-b{q}_{s})}^{2}}{\left.\dfrac{\partial {q}_{s}}{\partial r}\right|}_{r=R}+{D}_{s}{\rho }_{p}{\left.\dfrac{\partial {q}_{s}}{\partial \mathrm{r}}\right|}_{r=0}={k}_{L}({C}_{L}-{C}_{s}) $

(10)

To solve the PVSDM model, the two key parameters, D_ep and k_L, need to be calculated. D_ep is closely related to the features of the adsorbent, adsorption temperature, and adsorbate solvent^[34]. It can be estimated using the following Eq (11)^[31,35]:

$ {D}_{ep}=\dfrac{{\varepsilon }_{p}}{\tau }{D}_{AB} $

(11)

where, D_AB is molecular diffusion coefficient of ginkgolic acid at infinite dilution (m²/s); τ is tortuosity factor of adsorbent (i.e. microbial strains, macroporous resin, and their mixture). The values of τ and D_AB are calculated using Eqs (12 & 13), respectively^[36,37].

$ \tau =\dfrac{(2-{\varepsilon }_{p})^{-1/3}}{\varepsilon_p}$

(12)

$ {D}_{AB}=7.4\times {10}^{-8}\dfrac{T{\left(\varphi {M}_{B}\right)}^{0.5}}{{n}_{B}{V}_{A}^{0.6}} $

(13)

where, T is adsorption temperature (°C or K); φ is association factor of water; V_A is molecular volume of ginkgolic acid (cm³/mol); M_B is molecular weight of water (mol/g); n_B is the viscosity of water at 30 °C (cp).

The required and calculated values of the indicated parameters are listed in Supplementary Table S3.

The external mass transfer coefficient (k_L) can be obtained based on the concentration decay curve of ginkgolic acid in the solution during the first two sampling periods^[38,39]. It is calculated using the following Eq (14):

$ {\left(\dfrac{d\left(\dfrac{{C}_{L}}{{C}_{0}}\right)}{dt}\right)}_{t\to 0}=-\dfrac{mS{k}_{L}}{V} $

(14)

$ S=\dfrac{3}{R{\rho }_{p}} $

(15)

where, S is the surface area of adsorbent (i.e. microbial strains, macroporous resin, and their mixture) (cm²/g).

The PVSDM model was solved using the pdepe function in Matlab R2009a (The MathWorks, Inc., MA, USA). To obtain the surface diffusion coefficient (D_s), it was initially assigned a value and then iteratively adjusted to minimise the differences between the experimental (q_i,e) and predicted values (q_i,p) of ginkgolic acid adsorption. During this optimisation process, the AAD (Eq 16) and R² (Eq 17) values were calculated to assess the model's predictive accuracy.

$ AAD{\text%}=\left(\dfrac{{\displaystyle\sum }_{i=1}^{n}\left(\left|{q}_{i,e}-{q}_{i,p}\right|/{q}_{i,e}\right)}{n}\right)\times 100 $

(16)

$ {R}^{2}=1-\dfrac{{\displaystyle\sum }_{i=1}^{n}{\left({q}_{i,e}-{q}_{i,p}\right)}^{2}}{{\displaystyle\sum }_{i=1}^{n}{\left({q}_{e,mean}-{q}_{i,p}\right)}^{2}} $

(17)

where, AAD is absolute average deviation (%); q_i,e is the experimentally measured mass of ginkgolic acid adsorbed by the adsorbent (i.e. microbial strains, macroporous resin, and their mixture) (mg/g); q_i,p is the predicted mass of ginkgolic acid adsorbed by the adsorbent (i.e. microbial strains, macroporous resin, and their mixture) (mg/g); n is the number of experimental data; R² is coefficient of determination; q_e,mean is the mean value of adsorption capacity for adsorbent (i.e. microbial strains, macroporous resin, and their mixture) (mg/g).

Based on the numerical results, the mass flux due to the pore volume diffusion (N_AP) and surface diffusion (N_AS) inside the adsorbent were estimated using Eqs (18 & 19), respectively^[40]:

$ {\mathit{N}}_{AP}=-{D}_{ep}\dfrac{\partial {C}_{s}}{\partial r} $

(18)

$ {\mathit{N}}_{AS}=-{D}_{s}{\rho }_{p}\dfrac{\partial {q}_{s}}{\partial \mathrm{r}} $

(19)

Finally, the percentage of surface diffusion contribution (SDCP%) is determined using the following Eq (20)^[34]:

$ S DCP{\text%}=\dfrac{\|{\mathit{N}}_{AS}\|}{\|{\mathit{N}}_{AS}\|+\|{\mathit{N}}_{AP}\|}\times 100 $

(20)

Statistical analysis

All experimental data were collected in triplicate and expressed as mean ± standard deviation. A one-way variance analysis (ANOVA) was performed using SPSS Statistics 20.0 (IBM Corp., NY, USA) to compare the mean values. Statistical significance was considered at the p < 0.05 probability level. Principal component analysis (PCA) was conducted using Origin 2022 (Origin, Inc., USA) for multivariate analysis. Mass transfer investigations were conducted using Matlab R2009 (The MathWorks, Inc., MA, USA).

Author contributions

The authors confirm contribution to the paper as follows: study conception: Tao Y, Han Y; investigation: Sun Y, Zhao J, He J, Tao Y; methodology: Sun Y, Zhao J, He J, Li D; validation: Sun Y, Tao Y; draft manuscript preparation & Formal analysis: Sun Y; data curation: Tao Y; manuscript review & editing: Manickam S, Li D, Han Y, Jiang X, Tao Y; supervision: Han Y, Jiang X; funding acquisition: Tao Y. All authors reviewed the results and approved the final version of the manuscript.

Supplemental Fig. S1 Implementation of probiotic fermentation technology for the industrial processing of ginkgo kernel juices by the research group from Nanjing Agricultural University.
Supplemental Fig. S2 The phylogenetic tree of Lactobacillus plantarum and Lactobacillus pentosus strains isolated from Ginkgo Biloba fruits.
Supplemental Fig. S3 Comparison of the abilities of isolated microbial strains to remove toxins and enhance bioactives in ginkgo kernel juice after 48 h of fermentation. a: ginkgolic acid C15:1; b: ginkgolic acid C13:0; c: 4'-O-methylpyridoxine; d: total phenolics.
Supplemental Fig. S4 The images of active L. plantarum Y2 (a), inactive L. plantarum Y2 (b) and dried L. plantarum Y2 powders (c).
Supplemental Fig. S5 Changes in ginkgolic acid C15:1 content in the dispersions during absorption by L. plantarum Y2, macroporous resin D101, and their mixture as predicted by the PVSDM modeling.
Supplemental Fig. S6 Changes in the mass fluxes (NAP and NAs) within the adsorbent during the adsorption of ginkgolic acid C15:1 by L. plantarum Y2, macroporous resin D101, and their mixture. a and b: mass fluxes at the surface (a) and center (b) of L. plantarum Y2 adsorbent; c and d: mass fluxes at the surface (c) and center (d) of resin D101 adsorbent, respectively; e and f: mass fluxes at the surface (e) and center (f) of the adsorbent composed of L. plantarum and resin, where the mixture was considered as a unified entity.
Supplemental Table S1 Physiological and biochemical identification of autochthonous bacteria isolated from ginkgo biloba fruits.
Supplemental Table S2 Mass information of the identified ginkgolic acids and 4'-O-methylpyridoxine in the studied ginkgo kernels using mass spectrometry.
Supplemental Table S3 The parameters used to calculate the D_ep value.
Supplemental Table S4 The changes in individual organic acid contents (mg/L) in ginkgo kernel juice during lactic acid fermentation with the addition of macroporous resin.
Supplemental Table S5 The changes in individual monosaccharide contents (mg/L) in ginkgo kernel juice during lactic acid fermentation with the addition of macroporous resin.
Supplemental Table S6 The constants and accuracy of the Langmuir isotherm model for the adsorptions of ginkgolic acid by L. plantarum Y2, macroporous resin D101, and their mixture.
Supplemental Text 1 The MS conditions used to identify ginkgolic acids in the studied ginkgo kernels.

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Adsorbent	k_L (m/s)	D_AB (m²/s)	D_ep (m²/s)	D_s (m²/s)	AAD (%)	R²
L. plantarum Y2	6.70 × 10⁻⁹	5.57 × 10⁻¹⁰	n.s.	2.51 × 10⁻¹⁴	8.78	0.975
Macroporous resin D101	6.74 × 10⁻⁶	5.57 × 10⁻¹⁰	8.22 × 10⁻¹¹	1.67 × 10⁻¹²	10.16	0.947
Mixture of L. plantarum and resin	6.97 × 10⁻⁷	5.57 × 10⁻¹⁰	1.23 × 10⁻¹¹	5.03 × 10⁻¹⁴	13.27	0.933
n.s. refers to no pore volume diffusion.

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Biochemical and physical investigations on detoxification of ginkgo kernel juice using probiotic fermentation with macroporous resin addition

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