Chickpeas (Shihezi Market, Xinjiang, China) were soaked overnight in water (1:5 bean: water, m/v). The next day, removing moldy and insect-infested chickpeas, one part of the soaked beans was crushed in six parts of water (2) with heat processing for 30 min by mixer grinder (Joyoung, JYL-Y921, China). Then the soy drink was separated from solid residue using sterile gauze, eliminating impurities to make soymilk with fine taste and homogeneous texture. About 250 mL of soymilk sample was prepared by boiling for 10 min to confirm pasteurization.

The commercial strain L. plantarum (LP-56) was obtained from CINOBIO-TEC Co., Ltd. (Shanghai, China), and preserved at −20°C. The lyophilized strain powder was dissolved in sterile distilled water (1:50, w/v) (25), which was used to inoculate (0.07%, w/v) chickpea milk at 10 log CFU/mL (cell count at activation), as determined by spread plate method. The fermentation was performed for 24 h at 37°C (9). Samples were collected in triplicates at 0 and 24 h.

In total, 1 mL of sample was added with 9 mL of saline (0.9%, w/v) and then serially diluted with saline multiple times in the same ratio. 1 mL of respective dilution was plated onto a Petri dish containing about 15 mL of the MRS agar medium. The inoculated Petri dish was incubated at 37°C for 48 h and the bacterial count was estimated as described previously (26).

Where N = the number of microorganisms in the sample, ΣC = microbial total number, n₁ = the number of plates for the first dilution; n₂ = the number of plates for the second dilution; d = dilution factor.

The β-glucosidase activity was determined as described previously with some modifications (27). Took 0.1 mL of fermented soymilk, added 0.2 mL of sodium phosphate buffer (0.1 mol/L, pH 7.0) containing 5 mM p-NPG (p-nitrophenyl-β-D-glucoside) and put it into a 37°C water bath for 30 min. Then 0.5 mL of 1 mol/L NaCO₃ solution (4°C) was added to terminate the reaction, followed by centrifugal treatment (8,000 r/min, 30 min, 4°C), and the supernatant was taken to determine the absorbance value at 400 nm. Control group was inactivated with boiling water for 5 min instead of heating with water bath 37°C for 30 min. One unit of β-glucosidase activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol of ρ-nitrophenol per minute under the assay conditions. Standard curve: y  =  0.1028x  -   0.0078, r² =  0.9999.

The sample color evaluation was performed with a colorimeter to obtain the L*, a*, and b* values.

The total protein and total fat contents were determined spectrophotometrically following the Chinese National Standard GB5009.5-2016 and GB5009.6-2016, respectively. The method details are mentioned in Supplementary Text 1.

The total phenolic content (TPC) was determined using the Folin-Ciocalteu colorimetric method by measuring absorbance at 765 nm (26). This value denotes mg gallic acid equivalent (GAE) per mL of soymilk. The total flavonoid content (TFC) was determined using the aluminum nitrate colorimetric method by measuring absorbance at 508 nm (28). This value denotes mg rutin equivalent (RE) per mL of soymilk. The method details are in Supplementary Text 2.

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical cation (DPPH⁺) scavenging activity of the samples was estimated spectrophotometrically at 517 nm as described previously with some modifications (29). The control group used methanol in place of the DPPH⁺ solution, while the blank samples had deionized water. The calculation was performed as follows:

Where A₂, A₁, and A₀ are the absorbance of experimental, control, and blank samples, respectively.

The ABTS [2,2′-azino-bis(3-ethyl-benzothiazoline)-6-sulphonic acid] radical cation (ABTS⁺) scavenging activity of the samples was estimated by spectrophotometry at 734 nm as reported previously with some modifications (30). In control samples, absolute ethanol replaced the ABTS⁺ solution. The hydroxyl radical cation (OH^–) scavenging activity was analyzed spectrophotometrically at 510 nm (31). In control samples, deionized water replaced the ethanol-salicylic acid, FeSO₄, and H₂O₂ solutions, while the blank samples had deionized water. The calculation was performed as follows:

Where A₂, A₁, and A₀ are the absorbance of experimental, control, and blank samples, respectively.

Following the China National Standard GB5009.210-2016, vitamin B5 content was estimated by high performance liquid chromatography (HPLC) equipped with an ultraviolet spectrophotometric detector (Shimadzu, LC-2010, Japan). The HPLC conditions were as follows: chromatographic column, C18 column (4.6 mm × 250 mm, 5 μm); mobile phase, potassium dihydrogen phosphate solution (0.02 mol/L): acetonitrile (95:5); flow rate, 1 mL/min; column temperature, 30°C; detection wavelength, 210 nm; injection volume, 20 μL. The vitamin B5 standard solutions were 0, 2, 4, 8, 16, and 32 μg/mL (y = 106295x – 13160, r² = 0.998).

Following the China National Standard GB5009.154-2016, vitamin B6 content was also estimated by HPLC equipped with a fluorescence detector (Shimadzu, LC-2010, Japan) under the following conditions: chromatographic column: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol 50 mL, sodium octane sulfonate 2 g, and triethylamine 2.5 mL were dissolved in 1,000 mL ultrapure water of pH 3.0 ± 0.1 adjusted with glacial acetic acid; detection wavelength: excitation 293 nm, emission 395 nm; injection volume: 20 μL. The vitamin B6 standard solutions were 0, 0.1, 0.2, 0.4, 0.6, and 1.0 μg/mL (y = 6000000x – 154981, r² = 0.995).

Analysis of organic acids was performed as follows (32): the sample was centrifuged and the obtained supernatant was filtered using a 0.45 μm membrane filter. 20 μL of the filtered sample was injected into a Waters 2998 HPLC system with a C18 column (4.6 mm × 250 mm, 5 μm) and diode-array detector (DAD). The conditions were as follows: mobile phase, 0.1% phosphoric acid + methanol (97 + 3, v/v); elution, isometric; column temperature, 40°C; detection wavelength, 210 nm. The standard solutions were 0, 4, 10, 20, 40, 100, 200, and 400 μg/mL (lactic acid: y =  243.07x + 163.46, r² =  0.999; citric acid: y = 591.87x + 170.28, r² = 0.999).

In total, 5 mL of the sample was placed in a 20 mL headspace vial and equilibrated on a magnetic stirrer for 20 min at 60°C. An SPME fiber 50/30 μm carboxyene/dimethicone/divinylbenzene (CAR/PDMS/DVB) was extended through the needle and exposed in the vial and adsorbed in the headspace for 30 min. Afterward, the fiber was immediately injected into the GC inlet and desorbed at 250°C for 3 min. The samples were measured by GC-MS (Agilent 8890-7000D, USA) and the analysis of volatile compounds was performed in the splitless injection mode on an Agilent 19091N-133 with an HP-INNO Wax column (30 m × 0.25 mm, 0.25 μm). High-purity helium was the carrier gas at a constant flow rate of 1 mL/min. The GC oven was set to 40°C for 3 min, ramped to 82°C at 2°C/min, then to 103°C at 1°C/min, 124°C at 7°C/min, 138°C at 2°C/min, and finally 220°C at 10°C/min to hold for 3 min. Qualitative analysis of volatile compounds was identified by matching the National Institute of Standards and Technology database (NIST17.L) and retention index (RI).

Analysis of metabolites by UHPLC/MS (Thermo Scientific, Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the previous method with some modifications (33). Sample preparation: 20 mg sample was extracted with 400 μL of acetonitrile: methanol solution (1:1, v:v) containing 0.02 mg/mL of L-2-chlorophenylalanine (internal standard); Frozen tissue was ground for 6 min at −10°C and 50 Hz; Cryogenic ultrasonic extraction was performed for 30 min at 5°C and 40 KHz; The samples were incubated at −20°C for 30 min and then centrifuged for 15 min at 13.000 × g and 4°C, Also, 20 μL of supernatant from each sample was separately mixed with the quality control (QC) sample. A QC sample was run after every 5-15 analytical samples to test the stability of the whole detection process.

Chromatographic conditions were as follows: column: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm i.d., 1.8 μm; Waters, Milford, MA, USA); mobile phase A: 95% water : 5% acetonitrile (containing 0.1% formic acid), mobile phase B: 47.5% acetonitrile : 47.5% isopropanol : 5% water (contains 0.1% formic acid); flow rate: 0.40 mL/min; injection volume: 2 μL; column temperature: 40°C. The gradient elution condition was: 95% A and 5% B, 0.1 min; 75% A and 25% B, 2 min; 100% B, 9 min; 100% B, 13 min; 100% A, 13.1 min; 100%, 16 min.

Mass spectrometry conditions were as follows: Samples were electrosprayed/ionized, and MS signals were collected in the negative ion scanning mode. Scan type (m/z): 70–1,050; Sheath gas flow rate (arb): 40; Aux gas flow rate (arb): 10; Heater temp (°C): 400; Capillary temp (°C): 320; Spray voltage (–) (V): −2,800; S-Lens RF Level: 50; Normalized collision energy (eV): 20, 40, 60; Resolution (Full MS): 70000; Resolution (MS2): 17500.

All samples were tested in triplicates. Significant differences between data were identified by ANOVA Tukey’s test. Origin 9.8.5 was used for plotting the bar charts, stacked histograms, etc. Heatmaps were plotted by R Version 3.6.3 to analyze the changes. Adobe Illustrator 2020 was used to map the metabolic pathways.

Food color is a critical indicator of food quality (34). The mean L*, a*, and b* values of different samples are shown in Figure 1A. Compared with RJ (raw juice), the change in b* and a* values indicated a significant color change of LPFJ (L. plantarum fermented juice), however, the L* value did not change much (p < 0.05). The LAB fermentation changed the color of chickpea milk to more red and yellow colors.

Also, after fermentation, both total protein and total fat contents showed a downward trend (Figure 1B, P < 0.05). The decrease in total protein content may be because LAB promoted protein degradation during fermentation to meet the bacterial needs for nitrogen-containing substances (35). The lactic acid bacteria-degraded proteins are easily absorbed by the body; the formed ACE (angiotensin-converting enzyme) inhibiting peptides have anti-cardiovascular disease and blood pressure-lowering activities (36). Regarding the total fat content, its decrease may be due to the content of linoleic and linolenic acid decreased (37). The linolenic acid in soymilk positively correlates with the content of hexanal, which produces beany flavor (38). Therefore, LAB fermentation should reduce the beany flavor of chickpea milk. Hence, a decrease in total protein and total fat contents improved human digestion/absorption and the flavor of LPFJ.

The post-fermentation change in microorganism number is significantly increased (p < 0.05). The results indicated that LAB had good growth in chickpea milk and rapidly accumulated after 24 h of fermentation, reaching 8.87 log₁₀CFU/mL. Consistently, past research has also demonstrated that LABs grew better in soymilk with microbiological counts ranging from 5.7 to 10.4 log₁₀CFU/mL (39). Overall, chickpea milk is a suitable fermentation substrate with enough nutrients for the good growth of LAB.

As shown in Figures 1C,D, LAB fermentation significantly enhanced the TPC and DPPH+, ABTS+, and OH– scavenging capacity of LPFJ (p < 0.05), indicating a positive effect. However, TFC did not change much (p ≥ 0.05). Increased phenolic content can be attributed to the production of β-galactosidase during LAB fermentation, which catalyzes the release of phenolic compounds from bound sugars increasing antioxidant activity (40). Also, there could be other microbial transformations and depolymerization of compounds (41). Next, a small increase in TFC may be because LAB promote the release of flavonoids by producing enzymes and acids. Lactobacillus plantarum has been shown to increase TFC during fermentation (42).

Hydrolysis of glucosides through enzymatic processes using β-glucosidase to increase their bioavailability in soy-based products, and probiotic micro-organisms have been found to possess β-glucosidase (43, 44). The results showed a significant increase in β-glucosidase activity measured after fermentation (p < 0.05), reaching 28.13 mU/mL. Remarkably, the higher β-glucosidase activity in fermented soymilk indicates that the strain converts glycosidic soy isoflavones to glycosidic soy isoflavones faster, and the antioxidant capacity is enhanced (27, 44, 45). Subsequently, we utilized three different methods to assess the antioxidant properties of LPFJ (Figure 1D). The enhancement of antioxidant capacity indicates the accumulation of antioxidant compounds, such as phenolic compounds, flavonoids, and superoxide dismutase (SOD) (46). The lactic acid bacteria-fermented fruit and vegetable juices also show an increase in free radical scavenging and antioxidant activity (47, 48). Compared with RJ, LPFJ showed a significant increase in antioxidant activity, which may promote its health benefits in daily use (49).

Besides having abundant proteins, chickpeas are also rich in vitamins and minerals, especially vitamins B5 and B6 (50). Here, we found that the content of vitamin B5 decreased and the content of vitamin B6 increased significantly in LPFJ after LAB fermentation (p < 0.05; Figure 1E). Consistently, past research has also indicated that LAB fermentation can increase the contents of vitamin B6 in soymilk (51). The lactic acid bacterias are considered the biological factories for vitamin B production, and utilization (52). During LAB fermentation, lactic acid, a major organic acid in fermented beverages, increases (53). Meanwhile, the content of citric acid, which is an intermediate metabolite of the tricarboxylic acid cycle (TCA cycle), shows a dynamic change. The TCA cycle is the pivot for the liaison and transformation of key substances, such as sugar, lipid, protein, and even nucleic acids. Therefore, we measured the change in contents of lactic and citric acids (Figure 1F). Lactic acid production is not limited to sugars and citric acid as it may also originate from the conversion of amino acids (54). Here too, the lactic acid content increased and the citric acid content decreased in LPFJ.