The Euglena gracilis CCAP 1224/5Z powders (autotrophic Euglena as “AE,” heterotrophic Euglena as “HE” afterward) were supplied by Yougetiancheng Biotechnology Co., (Yiwu, China) (15). The nutrients in the Euglena powders, such as paramylon, total proteins, lipids, amino acids, vitamins, and minerals, were determined using standard methods. 2,2-Dipheny1-1-picrylhydrazyl (DPPH) and Pyrogallic acid were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All other chemicals were purchased from commercial suppliers and of analytical grade.

Six probiotics, Lactobacillus hordei (MRS 102), L. kefiri (MRS 103), L. brevis (MRS 104), L. parabuchneri (MRS 107), L. buchneri (MRS 108), and L. fructivorans (MRS 109), were achieved from Liqing Zhao laboratory of Shenzhen University (14). The probiotics were stored in MRS broth containing 25% glycerol at −80°C.

Six probiotics were employed to investigate the effect of Euglena powders on prebiotic activity. The green Euglena powder AE and yellow powder HE were separately added to MRS broth (30 ml) at the final concentrations of 0.1, 0.2, 0.5, 1.0, and 3.0% (w/w). The mixtures were then 5.0% inoculated and cultured overnight culture of a lactic acid bacteria strain and incubated with shaking at 200 rpm at 30°C and sampled in triplicates at different time points (0, 4, 6, 8, 12, and 24 h).

Meanwhile, the sample was diluted and plated onto the full MRS agar plates to determine viable bacterial count (cfu/ml). Based on results from our preliminary experiments, 1.0% Euglena powder additions, 12 and 24 h as logarithmic and platform growth phases were chosen for bacterial growth evaluation. The MRS medium without Euglena powder was used as the control group for metabolomic analysis.

The overnight culture of Lactobacillus was centrifuged at 1,000 × g for 5 min at 4°C to separate the insoluble Euglena powder. Then the resulting supernatant was harvested and centrifuged at 7,000 × g for 15 min at 4°C. Finally, the cell pellet was washed thrice with PBS (pH 7.2). The antioxidant analysis of Lactobacillus was evaluated using DPPH free radical scavenging ability and superoxide anion free radical scavenging ability.

A total of 2 ml cell suspension and 2 ml of 0.2 mmol/L ethanolic solutions of DPPH radical were mixed and incubated at room temperature for 30 min in the dark. After incubation, the reaction mixture was centrifuged at 7,000 × g for 15 min at 4°C, and the absorbance at 517 nm was measured. The mixture of DPPH and sterile distilled water served as the blank sample. The mixture of DPPH and Euglena powder was used as the control group. The scavenging ability was defined as follows:

The first ml of cell suspension was mixed with 3 ml of Tris–HCl buffer (pH 8.2). And the sterile distilled water was used as the blank sample. Then, 1 ml pyrogallol solutions (1.2 mmol/L) were added, and the autoxidation activity was evaluated by measuring the absorbance at 325 nm after incubation for 25 min. The following formula calculated the scavenging ability:

Species MRS 104 showed the most apparent promotion growth with the additions of EA and HE was selected for further metabolomic analysis. The bacterial media during logarithmic growth was collected for metabolic analysis. The metabolites in the sample were extracted and analyzed according to the method mentioned by He et al. (16). Each sample group included four biological replicates. Metabolites were extracted from bacterial solution using the method of Doppler. LC–MS/MS analyses were performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The autosampler temperature was 4°C, and the injection volume was 3 μl. The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur, Thermo). To include as many metabolites as possible, both Electron Spray Ionization (ESI) positive and negative ion modes data were collected. The metabolomic data were log2-transformed and then processed using principal component analysis (PCA) from MetaboAnalyst. It summarizes the contribution of each variable to the model. The metabolites with VIP > 1 and p < 0.05 (Student’s t-test) were significantly changed. In addition, commercial databases, including the KEGG database¹ and MetaboAnalyst², were used for metabolic pathway enrichment analysis. From these analyses, bubble diagrams and metabolic pathways were made.

All the experiments were conducted in triplicate. The experimental results were presented as the mean ± SD. Significant analysis was analyzed by one-way ANOVA. All the tests were performed using SPSS statistical software (version 16.0, SPSS Inc., United States).

As summarized in Table 1, noticeable content differences in AE and HE were observed, especially for paramylon, proteins, and lipids. High content of paramylon (70.1% w/w), low proteins (22.2%), and lipids (3.4% w/w) were detected in HE, while contents of proteins and lipids in AE were much higher, 55.1 and 24.5%, respectively. Similarly, higher contents of Fe, Zinc, linolenic acid, and linoleic acid in AE were also detected in this study. According to amino acids, all have higher ranges in AE than those of HE, almost with two-folds.

As shown in the differentially expressed metabolomic data (Supplementary Table 1, POS groups) at the “positive ion” mode, among top 45 metabolites with > 100 fold increase in AE+ culture medium compared to that of the control 33 (73.3%) are “lipids and lipid-like molecules,” and relatively low number (total 29 metabolites > 100-fold increase) detected in HE+ samples 19 are lipids. They are PC(14:0/14:0), LysoPC(20:4(5Z,8Z,11Z,14Z)), PC(14:0/15:0), LysoPC(20:2(11Z,14Z)), LysoPC(22:4(7Z,10Z,13Z,16Z)), PC(18:3(9Z,12Z,15Z)/18:1(11Z)), PC(20:1(11Z)/14:0), PC(18:1(11Z)/14:0), etc. (Supplementary Table 1), most of them are unsaturated fatty acids.

Under the “negative ion” mode, 18 of 19 TOP metabolites with > 100-fold increase in the AE+ culture medium are “lipids and lipid-like molecules,” such as adrenic acid, hypogeic acid, ginsenoside B2, sciadonic acid, leukotriene B4, 15S-hydroxy-5Z,8Z,11Z,13E, 17Z-eicosapentaenoic acid, and hepoxilin B3 in AE+ sample; 13/13 metabolites with > 100-fold increase are “lipids and lipid-like molecules” in HE-treated culture medium, namely, adrenic acid, eicosadienoic acid, sciadonic acid, ginsenoside B2, arachidonic acid (ARA), leukotriene B4, docosapentaenoic acid (22n-3) (DPA), hypogeic acid, myristoleic acid, alpha-tocopherol, hepoxilin B3, and eicosapentaenoic acid (EPA) (Supplementary Table 1, NEG groups).

The contents of lactic acid, isobutyric acid, and dodecanoic acid in different samples were studied (Supplementary Table 2). Similar levels of lactic acid, the relative abundance of 349.2 and 337.85 based on metabolomics data, were also detected in both AE and HE powder controls (AE+MRS, HE+MRS), lower than those in bacterial only (MRS103, MRS104). When AE and HE were added to MRS103, lactic acid in the co-cultured medium was reduced if combined lactic acid contents (as [AE+MRS103] vs. [AE+MRS] + [MRS103]) were considered. To MRS104, the addition of AE showed no significant change, while HE increased lactic acid content significantly.

Autotrophic Euglena (AE) and HE have similar contents of isobutyric acid with a little higher level than that in MRS103 or MRS104 only. The addition of AE or HE into MRS103 and MRS104 caused an obvious reduction of isobutyric acid (as [AE+MRS103] vs. [AE+MRS] + [MRS103]) (Supplementary Table 2). The final level was higher in AE added bacteria (AE+MRS103 and AE+MRS104) than in HE addition (HE+MRS103 and HE+MRS104). The comparative contents of dodecanoic acid showed low contents (i.e., 0.06) in bacteria (MRS103 and MRS104) and high levels in AE and HE (1.89 and 2.53). Thus, AE and HE provided a high level of dodecanoic acid in the coculture medium. Only HE addition resulted in a significant combination decrease in the coculture medium (Supplementary Table 2). Other fatty acids showed similar differential changes in the treatments.

The bacterial growth was first investigated with normal OD600 absorption, however, both 0.2 and 1% addition of Euglena powders showed obvious absorption in the culture medium (Supplementary Figure 1). We used a dilution plate on the full MRS agar plates to determine the viable bacterial count (cfu/ml) in each sample. The counting of viable bacteria during logarithmic growth and platform stages of six selected Lactobacillus on Euglena powders (AE and HE) was shown in Figures 1A,B, respectively. Different Lactobacillus showed different growth patterns, MRS102 with low cell numbers at both growth stages, while MRS104 and 105 reached high cell numbers at the log phase and MRS103 increased cell numbers until the platform stage. All selected Lactobacillus grew well in the presence of both Euglena powders. Compared with controls, in the presence of Euglena powders, especially AE, the numbers (by up to three-fold) of Lactobacillus were significantly increased. Also, the growth-promoting effect of AE is superior to that of HE. At the platform stage, while some Lactobacillus cell viability decreased, the addition of AE or HE promoted bacterial growth at an early stage.

The antioxidant potential of Lactobacillus plays a vital role in protecting the host microflora from the attack by free radicals. The six selected Lactobacillus revealed solid antioxidant activity against free radicals, as shown in Figure 2. In the presence of Euglena powder, the antioxidant activity of Lactobacillus was significantly improved. And the effect of green Euglena powder is superior to the yellow one. The results demonstrated that the Euglena powder could improve significant antioxidant activity, and the AE is better than HE.

After data processing, the total metabolic features were obtained in positive and negative modes. PCA was conducted to see significant metabolomic differences before and after adding Euglena powder. The result is shown in Figure 3. Each group in both models showed good separation, and all samples were nearly within a 95% CI.

This study detected a total of 1,011 and 387 metabolites in our bacterial culture media under positive and negative ion modes. According to the selection criteria (i.e., VIP score > 1 and p values < 0.05 in Student’s t-test), 218 and 196 matched differential metabolites were identified in positive and negative modes between HE and the control group. A total of 399 and 191 matched differential metabolites were identified between AE and the control (Table 2). The addition of Euglena, either AE or HE, provided more metabolites (shown as upregulated metabolites, Table 2), especially in HE (negative ion mode) with 187 upregulated metabolites, including pyruvate, succinate, L-aspartate, L-glutamate, nicotinate, and biotin, with only nine metabolites downregulated when compared with controls.

After the KEGG database annotated these metabolites, critical metabolic pathways were screened according to their position and role in the relevant metabolic pathways (Supplementary Table 2). A total of six pathways were selected and regarded as the key ones with the addition of AE and HE, namely, metabolic pathways, biosynthesis of secondary metabolites, ABC transporters, purine metabolism, cysteine and methionine metabolism, and biosynthesis of amino acids (Supplementary Table 2). According to the bubble chart, critical metabolic pathways related to HE addition are nicotinate and nicotinamide metabolism, cysteine, and methionine metabolism (positive mode, Figure 4A) and nitrogen metabolism, and alanine, aspartate, and glutamate metabolism (negative mode) (Figure 4B). In contrast, several key metabolic pathways are relevant to AE: biotin metabolism and nicotinate and nicotinamide metabolism in the positive mode (Figure 4C); and biotin metabolism in the negative mode (Figure 4D).