Food Technology and Biotechnology

Microorganisms and materials

The microorganisms were deposited in the China General Microbiological Culture Collection Center (CGMCC) in Beijing. The CGMCC accession number of Lactobacillus plantarum TK9 is 11891, and that of L. paracasei TK1501 is 13130.

The Tartary buckwheat samples used in this study were grown in Kunming, Yunnan province, PR China, and collected in October 2016. The grains were cleaned and stored in the dark in polyethylene containers at room temperature for less than 3 months.

Preparation of microbiological cultures

Lactobacillus plantarum TK9 and L. paracasei TK1501 were activated in 10 mL of De Man, Rogosa and Sharpe (MRS) broth (Oxoid, Basingstoke, UK) at 37 °C for 18 h, using 1% inocula. The A_{600 nm} of the resulting culture of L. plantarum TK9 was between 1.0 and 1.2, with viable counts of (3.5±0.4)·10⁹ CFU/mL; and the A_{600 nm} of L. paracasei TK1501 was between 1.4 and 1.6, with viable counts of (5.5±0.4)·10⁹ CFU/mL. The cultures were centrifuged (GL20A, Xiangyi, Hunan, PR China) at 5000×g for 10 min, the supernatants were discarded, and the bacterial cells resuspended in sterile saline solution and adjusted to 10⁹ CFU/mL. Thus obtained suspensions were applied as inocula for SSF.

Optimization of fermentation conditions using orthogonal experimental design

Table 1 shows the influence factors and level values selected in this study. The orthogonal design helped to analyze the performance of the fermented Tartary buckwheat and determine the level of influence of factors (water ratio, inoculum size, time) affecting the total viable counts of the probiotic bacteria.

Tartary buckwheat grain substrate (40 g) and sterile distilled water were mixed in conical flasks (250 mL; Deschem, Changshu, PR China) in mass per volume ratios as shown in Table 2 and Table 3, autoclaved (SX-500; Tomy, Fukushima, Japan) at 121 °C for 20 min, and cooled to 37 °C before the addition of the required amounts of L. plantarum TK9 or L. paracasei TK1501 starter cultures. Fermentation of the inoculated substrates occurred at 37 °C in an incubator (SHKE6000-1CE; Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, samples from the optimal combinations were freeze-dried using a DW3 freeze dryer (Heto-Holten A/S; Allerød, Denmark) and stored at –20 °C for further analysis. The native unfermented samples (inoculated with the same volume of sterile saline) collected at 0 h were used as the negative control. SSF was performed in triplicate. SPSS software v. 22.0 (17) was used to perform the statistical analysis.

Determination of the total viable counts

Total viable counts of bacterial cells were assessed according to the method described by Zhang et al. (18). The fermented Tartary buckwheat (10 g) was homogenized for 15 s in 90 mL of sterilized physiological saline (0.85%) in a blender (JT-C; Jintian, Luohe, PR China). Total viable counts of L. plantarum TK9 and L. paracasei TK1501 were made using a pour plate method and MRS agar (Oxoid) after serial dilution in maximum recovery diluents. Serial dilutions were prepared in sterilized physiological saline and 1 mL of the appropriate dilution was poured on plates in triplicate. The poured plates of L. plantarum TK9 were incubated at 37 °C for (48±2) h. The cultures of L. paracasei TK1501 were incubated at 37 °C for (60±2) h. The colonies were then counted, and the viable counts were expressed as colony forming units per gram (CFU/g) of the sample.

Preparation of extracts

The freeze-dried samples from the optimal combination in the orthogonal experiment and the negative control were ground to a powder using an M20 universal mill (IKA, Staufen, Germany). Then, 10 g of the freeze-dried SSF powder were extracted with 200 mL of 70% (by volume) ethanol for 2 h in an ultrasonic extractor (KH-600TDV; Hechuang, Kunshan, PR China). Afterwards, the samples were centrifuged (Heraeus^TM, Multifuge^TM X1R; Thermo Fisher Scientific) at 25 155×g and 4 °C for 10 min, and the supernatants were collected. The residue was then suspended in 100 mL of 70% (by volume) ethanol, ultrasonicated and centrifuged under the same conditions. The supernatants were combined, filtered through Whatman no. 1 paper (GE Healthcare, Chicago, IL, USA) and freeze-dried. An aliquot comprising 5 mg of the freeze-dried sample was stored at –20 °C and dissolved in 1 mL of phosphate buffer (0.1 M, pH=6.8; Sinopharm Chemical Reagent Co., Ltd, Shanghai, PR China) immediately before analysis.

Determination of the total phenolic content

The total phenolic content (TPC) in the extracts was determined by a modified Folin-Ciocalteu method (19). Briefly, 100 μL of each diluted extract were mixed with 500 μL of Folin-Ciocalteu reagent (Sangon Biotech, Shanghai, PR China) and 6 mL of distilled water, and shaken for 1 min. Afterwards, 2 mL of a 15% (by mass per volume) Na₂CO₃ (Sinopharm Chemical Reagent Co., Ltd, Shanghai, PR China) solution were added to the mixture, shaken once again for 2 min, and the volume of the solution was adjusted to 10 mL with distilled water. Finally, the mixture was incubated in the dark for 2 h at room temperature. The absorbance at 750 nm was recorded on an InfiniteM200 PRO multifunctional microplate reader (Tecan, Männedorf, Switzerland) against a solution without sample as blank (100 μL of 70% ethanol instead of the test samples). A standard curve was prepared using gallic acid (Sigma-Aldrich, St. Louis, MO, USA):A=0.4789c±0.0303 /1/where A is the absorbance at 750 nm and c is the concentration of gallic acid (c=0.2–1 mg/mL, R²=0.9978).

Samples were independently analyzed in triplicate and the TPC was expressed as milligrams of gallic acid equivalents (GAE) per gram of extract.

Determination of total flavonoid content

Determination of total flavonoid content (TFC) followed a published colorimetric method (20) with slight modifications as follows: the mixture contained 0.3 mL NaNO₂ (Damao Chemical Reagent Factory, Tianjin, PR China) solution (5%, by mass per volume), 0.6 mL AlCl₃ (Sinopharm Chemical Reagent Co., Ltd) solution (10%, by mass per volume), 2 mL NaOH (Sinopharm Chemical Reagent Co., Ltd) solution (1 M) and 1 mL diluted sample solution. The volume of the mixture was adjusted to 10 mL with distilled water. After 5 min of incubation at room temperature, the absorbance at 507 nm was recorded with multifunctional microplate reader (InfiniteM200 PRO; Tecan) against the mixture without sample solution as a blank. The TFC was expressed as rutin (Yuanye Biological Technology, Shanghai, PR China) equivalents per g of sample using the calibration curve of rutin:A=0.636c±0.0388 /2/where A is the absorbance at 507 nm and c is the concentration of rutin (R²=0.9974).

Determination of α-glucosidase inhibition

The α-glucosidase inhibition by the samples was assessed according to the method described by Zeng et al. (12) with slight modifications. Briefly, the reaction mixture contained 25 μL of 10 mM p-nitrophenyl-α-d-glucopyranoside (PNPG; Sigma-Aldrich) and 25 μL of the sample preincubated at 37 °C for 10 min. The reaction was initiated by the addition of 50 μL α-glucosidase solution (0.16 U/mL; Sigma-Aldrich) diluted with 0.1 M phosphate buffer, and incubated at 37 °C for 30 min. The reaction was terminated by adding 100 μL of 0.1 M Na₂CO₃ (Sigma-Aldrich). The enzymatic activity was quantified based on the measurements of the absorbance of the samples at 405 nm on a Multiskan MK3 plate reader (Thermo Fisher Scientific). Each test sample was analyzed in technical triplicate, and the absorbance values were corrected against sample blanks in which α-glucosidase was replaced with phosphate buffer. The positive control (α-glucosidase activity with no inhibitor) and negative control (no α-glucosidase activity) were prepared by using phosphate buffer instead of the sample or instead of the sample and the α-glucosidase solution, respectively. The α-glucosidase inhibition rate was calculated as follows:

Determination of DPP-IV inhibition

Dipeptidyl-peptidase IV (DPP-IV) inhibitory activity was assessed according to the method described by Zeng et al. (12) with some modifications. Briefly, 25 μL Gly-Pro-p-nitroanilide (6 mM; Sigma-Aldrich) and 25 μL Tartary buckwheat sample, or 25 μL phosphate buffer saline (PBS; Sigma-Aldrich) as a control, were mixed and preincubated at 37 °C for 10 min. The reaction was initiated by adding 50 μL DPP-IV from porcine kidney (3·10^–4 U/L, ≥10 U/mg protein; Sigma-Aldrich) and the mixture was incubated at 37 °C for 60 min. The reaction was terminated by adding 100 μL of 1 M sodium acetate buffer (pH=4.0; Sinopharm Chemical Reagent Co., Ltd), and the absorbance of the samples at 405 nm was measured on a Multiskan MK3 plate reader (Thermo Fisher Scientific). Each sample was analyzed in technical triplicate, and the absorbance values were normalized to sample blanks in which DPP--IV was replaced with Tris-HCl buffer (0.1 M, pH=8.0; Solarbio, Beijing, PR China). The negative control (no DPP-IV activity) and positive control (DPP-IV activity with no inhibitor) were prepared by using Tris-HCl buffer (100 mM, pH=8.0; Solarbio) instead of the sample or instead of the DPP-IV solution and the sample, respectively. Diprotin A (Sigma-Aldrich) was used as a standard inhibitor. The DPP-IV inhibition rate was calculated using Eq. 3.

Statistical analysis of the results obtained using orthogonal experimental design

The most important factors for consideration in the development of functional food are the bioactive components. In this study, we developed a probiotic product in a solid-state fermentation on Tartary buckwheat, taking into consideration the final viable probiotic cell count. Thus, the fermentation parameters were optimized in order to obtain a high growth rate of the lactic acid bacteria (LAB) in Tartary buckwheat. We designed an orthogonal experiment with three factors and four levels (Table 1). The results served to find the optimal fermentation conditions and analyze the relationship between the factors and systemic performance of the fermentation (Table 2 and Table 3). K_i represents the sum of the corresponding test results when the level number is i for any factor (Table 3), and k_i=K_i/4 (4 is the number of levels). The larger the R value (R=K_max–K_min), the greater the influence of this factor on the viable count. We found that the R values of the water ratio (A), inoculum size (B) and time of fermentation (C) for TK9 and TK1501 were R=0.345, 0.305, 0.330 and R=0.6575, 0.4375, 0.5025, respectively. The effect of the factors on the number of viable bacteria was in the order: A>C>B. Thus, the optimal fermentation conditions were a 1:1.5 water ratio, a 24-hour fermentation period and 10⁷ CFU/g inoculum size (A1C2B2). Subsequently, analysis of variance (ANOVA) determined the significance of the model (Table 4 and Table 5). The p-values served as a tool to check the significance of each coefficient; the lower the p-value, the higher the significance of the corresponding coefficient. The corresponding p<0.05 suggested that the water ratio, inoculum size and time of fermentation are significant terms. Therefore, a small variation of their values will notably alter the total viable count. Under the optimal conditions, the highest viable counts of Tartary buckwheat fermented by L. plantarum TK9 and L. paracasei TK1501 were (2.3±0.7)·10⁹ CFU/g and (3.3±0.4)·10⁹ CFU/g, respectively.

Total phenolic content during Tartary buckwheat fermentation

During the fermentation of Tartary buckwheat by LAB, the composition of the buckwheat changes under the influence of bacterial metabolism. Hence, fermentation affected the bio- active constituents (Table 6). In Tartary buckwheat extracts, TPC (expressed as GAE) increased from (243.0±14.0) mg/g in the dry extract of native unfermented sample to (251.8±10.4) mg/g in the extract of Tartary buckwheat fermented by L. plantarum TK9. The TPC of Tartary buckwheat fermented by L. paracasei TK1501 was (241.5±5.4) mg/g dry extract, without significant changes compared to the native unfermented material. Zhu (5) and Ran and Ma (21) reported a similar TPC in Tartary buckwheat. The possible explanation for these results is that the metabolic activity of microbes can modify the levels of bio- active compounds during fermentation (22). Some reports stated that fermentation by L. plantarum is an efficient process for increasing the concentration of phenolic compounds in fermented cowpea flour (23, 24). The β-glucosidase enzyme produced during fermentation is thought to catalyze the hydrolysis of complex polyphenols to yield simpler and biologically more active compounds, leading to an increase of the total phenolic content (25). In addition, some reports suggested that the higher antioxidant activity might be due to the presence of phenolic components (26).

Total flavonoid content during Tartary buckwheat fermentation

The TFC of the different samples is shown in Table 6. The TFC of the native unfermented Tartary buckwheat sample, as well as the samples fermented by L. plantarum TK9 and L. para- casei TK1501 was (25.5±1.1), (25.6±1.4) and (25.7±0.4) mg/g, respectively. No significant differences in the TFC were observable between the native unfermented Tartary buckwheat and the preparations fermented by L. plantarum TK9 and L. paracasei TK1501. Importantly, this also means that there was no loss of TFC during fermentation. Moreover, recent reports stated that the TFC of Tartary buckwheat is much higher than that of common buckwheat (12, 27).

Inhibition of α-glucosidase activity

Diabetes is a chronic metabolic disorder mainly characterized by high blood glucose levels. Therefore, treatment of diabetes mainly focuses on reducing the fluctuations of blood glucose in order to reduce the subsequent complications. This has led to the development of α-glucosidase inhibitors as oral anti-diabetic agents, widely used in treatment of diabetes, that can postpone the digestion and absorption of carbohydrates, and thus reduce postprandial hyperglycaemia (28). In this study, the extracts of Tartary buckwheat fermented using the two different strains were able to inhibit α-glucosidase in a concentration-dependent manner (Fig. 1). All tested Tartary buckwheat extracts at a concentration of 1 mg/mL showed α-glucosidase inhibitory activities ranging from 29.25 to 75.82%, with the higher value obtained using Tartary buckwheat fermented by L. plantarum TK9. This extract at 0.008, 0.04, 0.1, 0.2, 0.4, 0.8 and 1.0 mg/mL displayed α-glucosidase inhibitory activities of (0.03±0.02), (0.3±0.2), (2.3±0.4), (20.2±1.6), (42.8±0.3), (68.91±0.05) and (75.8±0.2) %, respectively, with an IC₅₀ value of 0.51 mg/mL. By contrast, the extract of native Tartary buckwheat at the same concentrations had inhibitory activities of (1.5±7.8), (4.9±1.6), (5.7±4.0), (17.8±2.2), (22.6±1.5), (44.1±1.3) and (60.5±0.6) %, respectively, with an IC₅₀ value 0.87 mg/mL. The extract fermented with L. paracasei TK1501 was even less effective, with respective inhibitory activities of (2.7±5.9), (5.7±3.7), (7.2±1.6), (7.1±1.6), (10.4±5.7), (17.1±7.0) and (29.2±0.5) %, with an IC₅₀ value >1 mg/mL. The IC₅₀ value of the positive control (acarbose) for α-glucosidase inhibition was 0.85 mg/mL (26). Earlier studies have reported that Tartary buckwheat inhibits α-glucosidase (29). Based on the relationship between bioactive components and α-glucosidase inhibition, TFC and TPC are considered to play an important role in the inhibitory activity of Tartary buckwheat (21). Some reports have suggested that many flavonoids can inhibit α-glucosidase activity (30). Moreover, quercetin has a higher inhibitory activity on α-glucosidase than rutin (4). Therefore, we suspected that the L. plantarum TK9 fermentation enhanced the quercetin content. It has already been confirmed that certain strains of Lactobacillus have inhibitory potential against a range of α-glucosidases and at least one β-glucosidase (31). It was found that a product fermented by L. plantarum TK9 has a significantly higher inhibitory effect on α-glucosidase than that by L. paracasei TK1501. The inhibition of intestinal α-glucosidase is a known strategy to regulate blood glucose (32). Although synthetic inhibitors of intestinal α-glucosidase have found wide application in the clinic, there are considerations concerning cost and side effects. Consequently, there is a demand for alternatives from natural plant, animal and probiotic sources (33–35), and many bacteria have been shown to have inhibitory activity. Cell-free supernatants of six strains of L. plantarum showed inhibitory activity ranging from 24.96 to 41.81% (36). Notably, the strain TK9 investigated in this study was even more active than these six strains. Its comparatively high inhibitory activity suggests that Tartary buckwheat fermented by L. plantarum TK9 may have dual beneficial effects on glycaemia regulation by reducing the intestinal absorption of carbohydrates. Similarly, reports have shown that L. plantarum NCU116 and carrot juice fermented by it had the potential to regulate blood glucose levels (37). The Tartary buckwheat fermented by L. plantarum TK9 had very satisfactory α-glucosidase inhibitory activity, and it merits commercial exploration as an efficient agent for the management of glucose metabolism.

Fig. 1

α-Glucosidase inhibition activity by Tartary buckwheat (TBW) ethanol extracts obtained in solid-state fermentation. Each value corresponds to the mean of three independent replicates with error bars indicating the standard deviations

Inhibition of DPP-IV activity

Dipeptidyl peptidase IV (DPP-IV) is a highly specialized aminopeptidase that appears to be a major physiological modulator of a number of regulatory peptides, neuropeptides and chemokines (38, 39). GLP-1 is a gastrointestinal hormone that can reduce appetite, suppress glucagon secretion, stimulate insulin secretion and reduce gastric emptying (13). Reports show that GLP-1 levels reduced after a mixed meal and an oral glucose load in patients with diabetes type 2 (40). The enzyme DPP-IV rapidly metabolizes GLP-1 (41). Preliminary clinical data have shown the potential of DPP-IV inhibitors in treating diabetes type 2 (42). The need to develop safe DPP-IV inhibitors has led to increased attention to natural sources. Recently, natural sources as diverse as dietary proteins, medicinal plants, and marine life have been confirmed to have DPP-IV inhibitory effects (43). All extracts used in our research showed DPP-IV inhibitory activity in a concentration-dependent manner. The Tartary buckwheat extracts (4 mg/mL) showed DPP-IV inhibitory potential, among which the extract of Tartary buckwheat fermented by L. paracasei TK1501 displayed the greatest inhibition (77.2±1.9) %, followed by L. plantarum TK9 (57.3±2.1) % and native non-fermented Tartary buckwheat extract (39.9±1.4) %. Fig. 2 shows the inhibitory effects of the three different Tartary buckwheat samples (native, fermented by L. plantarum TK9 and L. paracasei TK1501) at different concentrations (0.2–5 mg/mL), with IC₅₀>5, 3.45 and 2.47 mg/mL, respectively. Unlike the results of α-glucosidase inhibition, the extract of Tartary buckwheat fermented by L. paracasei TK1501 showed the best inhibition of DPP-IV activity. The positive control diprotin A had an IC₅₀ of 1.15 mg/mL, and behaved as a competitive inhibitor, which was in agreement with the literature (12, 21). Our work represents the first report on the inhibition of DPP-IV activity by Tartary buckwheat. We speculated that effective inhibitory components might not be related to TPC or TFC in view of our previous results. Similarly, some reports suggest that the strains themselves were the most likely producer of the inhibitory compounds (1, 12, 21, 44). The results of this study suggest that fermented Tartary buckwheat has antihyperglycaemic properties, and hence might provide a new dietotherapy food for the control of diabetes.

Fig. 2

Dipeptidyl peptidase IV (DPP-IV) inhibition activity by Tartary buckwheat (TBW) ethanol extracts obtained in solid-state fermentation (SSF). Each value corresponds to the mean of three independent replicates with error bars indicating the standard deviations