Lacticaseibacillus rhamnosus GG and Lactobacillus casei Shirota Growth on a Medium Enriched with Rye Protein, and Assessment of DPP-IV Inhibitory Activity ^†

Abstract

Due to climate change and the development of sustainable foods, protein vegetable sources are being considered as promising food commodities. Fermentation is an ancient tool for obtaining bioactive compounds, and has been exploited for bioactive peptide production from different sources. Thus, this work aimed to evaluate growth and the antidiabetic peptides released from a rye-enriched medium fermented by probiotics. The culture was made with 7.5% rye protein isolate and 1% glucose, with buffering at pH = 6.8. Fermentation began with 1% inoculum addition and was performed for 24 h. The proposed medium allowed the growth of L. rhamnosus GG and L. casei Shirota to reach concentrations of 9.72 and 10.52 log cfu/mL, respectively, superior to those recommended to obtain beneficial effects on humans. In addition, the nitrogen demands of each strain tested produced peptides capable of inhibiting the DPP-IV enzyme at percentages between 20 and 27%, which converted the hydrolysates into an interesting tool for glycemic control. Finally, rye fermentation by probiotics is a promising process for developing plant-based products with functional properties.

1. Introduction

Since ancient times, fermentation has been considered a powerful tool for obtaining value-added products. During this process, the metabolism of microorganisms is exploited to generate foods, biofuels, or medicines [1]. In this context, bioactive compounds, such as peptides with biological functions, can be produced from protein hydrolysis by the lactic acid bacteria proteolytic system, where the Lactobacillus genera are the leading bacterial group applied [2,3]. Bioactive peptides have mainly been made from animal sources such as milk, egg, meat, or fish [4,5]; however, novel trends due to climate change and animal protein allergies suggest that the use of vegetable sources of proteins can offer higher sustainability and cover particular issues in a specific population [6,7]. Cereals and seeds are essential and common vegetable protein sources worldwide. They contain variable crude protein contents, ranging from 7 to 20%, and lactic acid bacteria can hydrolyze them according to their nitrogen auxotrophy [8,9]. They also release amino acid sequences with positive health benefits, in particular for chronic diseases, such as cancer, hypertension, and diabetes [10,11]. Antioxidant peptides have been produced from wheat, maize, rice, oat, and quinoa fermentation [3,12]. On the other hand, antihypertensive peptides have been obtained through the lactic fermentation of soy, wheat, oats, lupin, quinoa, or amaranth [13]. Meanwhile, starter cultures have generated antidiabetic peptides from sorghum, lupin, quinoa, wheat, or lentil protein hydrolysis [14,15,16]. Concerning the last peptide type, in recent years, several studies have suggested that foods fermented by lactic acid bacteria have a beneficial impact on the control and prevention of type 2 diabetes, which is considered a pandemic health problem because the incidence has increased by fourfold from 1980 to 2019, passing from 108 million to 463 million cases [17]. Rye is not a sufficiently appreciated grain, although it can resist climatological adversities and contains bioactive compounds such as phenolic acids, flavonoids, or arabinoxylans; it has been exploited poorly to benefit human health [18,19]. Also, rye maintains 5.8–18% protein, which could be hydrolyzed for bioactive peptide production. Indeed, in recent years, the enzymatic hydrolysis of rye protein has shown antioxidant, antihypertensive, and antidiabetic properties [20,21]. Thus, this work aimed to evaluate the growth of two probiotics in a medium enriched with rye protein and to explore the release of DPP-IV inhibitory peptides from lactic acid bacteria proteolysis.

2. Materials and Methods

Chemical reagents: The MRS broth used was GranuCult from Merck Millipore (Darmstadt, Germany). Bacteriological agar, picryl sulfonic acid solution (5%, w/v), Tris (hydroxy) aminomethane, and Dipeptidyl Peptidase IV (DPP-IV) recombinant enzyme from humans (>1 unit/vial) came from Sigma-Aldrich (Saint Louis, MO, USA). Glucose, HCl, KH₂PO₄, K₂HPO₄, and K₂CO₃ came from JT Baker Thermo Fisher Scientific (CDMX, Mexico). Gly-Pro-p-nitroanilide came from Santa Cruz Biotechnology (Dallas, TX, USA). All chemicals were ACS grade, and Milli-Q deionized water at 18.2 MΩ·cm (Merck Millipore; Darmstadt, Germany) was used for dissolutions.

Instrumentation: Vertical autoclave (Evar, Guadalajara, Mexico). Convection incubator (Benchmark, Guadalajara, Mexico). Refrigerated microcentrifuge Sorvall Fresco and UV–vis spectrophotometer (Thermo Fisher Scientific, CDMX, Mexico). Dry-Bath (Barnstead Thermolyne; Dubuque, IA, USA).

Lactic acid bacteria propagation: The strains Lacticaseibacillus rhamnosus GG and Lactobacillus casei Shirota were activated from samples stored in glycerol. Samples were grown at 10% inoculum on MRS broth for activation at 37 °C for 48 h. Then, activated cultures were inoculated at 10% into MRS broth and incubated for 24 h at 37 °C. Finally, cultures grown in MRS broth for 24 h were inoculated at 10% into the rye fermentation medium and incubated at 37 °C and 130 rpm for 24 h to allow the bacteria to adapt to the enriched rye medium before fermentation. MRS cultures were sterilized at 121 °C for 15 min, while rye medium was thermally treated at 90 °C for 10 min in autoclave before the inoculation.

Rye-enriched culture preparation and fermentation conditions: A medium enriched with rye protein was made on sterilized phosphate buffer (0.1 M, pH = 6.8), dissolving glucose at 1% (w/v) and dispersing at 7.5% (w/v) with a rye protein isolate with a protein content of 51.06 ± 1.15%, which was obtained using the solubilization–precipitation method [21]. The culture was thermally treated at 90 °C for 10 min and inoculated at 1% with L. rhamnosus GG or L. casei Shirota strains from the previously adapted culture. Fermentation was performed for 24 h at 37 °C and 130 rpm.

Following bacterial growth and proteolysis level: Bacterial concentration at 0 and 24 h was determined by viable count in MRS plates using the drop technique [22]. Decimal dilutions in sterile peptone thinner were made. Then, 5 µL of dilutions was inoculated on MRS plates and incubated for 48 h. The colony count was realized after the incubation time, and the concentration was reported as log CFU/mL. A sample fermentation at 0 and 24 h was centrifuged at 10,000 rpm and 4 °C for 10 min to measure the proteolysis level. Supernatants were submitted for free amino group analysis using the Adler–Nilsen method [23]. In brief, 0.25 mL was mixed with 2 mL of phosphate buffer (0.21 M, pH = 8.2) and 2 mL of pycrylsulfonic acid solution at 0.1% dissolved in buffer. The mixture was incubated at 50 °C in light-absence conditions for 1 h. The reaction was stopped by adding 4 mL of 0.1 N HCl, and the absorbance was recorded at 340 nm. The free amino groups concentration was determined from a calibration curve of glycine at 0–200 mg/L.

Antidiabetic determination by DPP-IV inhibition test: The antidiabetic test was performed with supernatants obtained at 0 and 24 h of fermentation. The in vitro determination was realized according to Nongonierma et al. [24], with some modifications reported in Islas-Martínez et al. [21]. Three systems were evaluated; the first was denominated as the sample system (A_s), containing fermentation supernatants, Gly-Pro-p-nitroanilide at 1.6 mM as a substrate, and DPP-IV enzyme at 0.01 U/mL. The second system was a positive control (A₁₀₀), where the sample was substituted with buffer Tris-HCl (0.1 M, pH = 8). The last systems, were incubated at 37 °C for 60 min and arrested by 0.1 M K₂CO₃. The third system was a sample control (A_sc) to discard absorbance from fermentation centrifugates. It was made from a blending sample, potassium carbonate, and a buffer was used as a substitute for the substrate and enzyme. Finally, the absorbance of all systems was performed at 405 nm, and the DPP-IV inhibition percentage was calculated from the following equation.

$$\mathbf{D}\mathbf{P}\mathbf{P}-\mathbf{I}\mathbf{V}\mathbf{i}\mathbf{n}\mathbf{h}\mathbf{i}\mathbf{b}\mathbf{i}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n}\left(\mathbf{\%}\right)={\displaystyle \frac{{\mathbf{A}}_{\mathbf{100}}-\left({\mathbf{A}}_{\mathbf{s}}-{\mathbf{A}}_{\mathbf{s}\mathbf{c}}\right)}{{\mathbf{A}}_{\mathbf{100}}}}\times \mathbf{100}$$

Statistical analysis: Minitab 18 software (State College, PA, USA) was employed to carry out a one-way ANOVA. Tukey’s contrast was performed to determine differences at a confidence level α = 0.05.

3. Results and Discussion

Table 1. Following growth, hydrolysis, and antidiabetic activity due to rye protein fermentation in lactic acid bacteria.

Lactic Acid Bacteria Strain	Bacterial Concentration (Log CFU/mL)		Free Amino Groups Concentration (mg/L)		DPP-IV Inhibition (%)
	0 h	24 h	0 h	24 h	0 h	24 h
L. rhamnosus GG	7.58 ± 0.02 b	9.72 ± 0.10 a	163.33 ± 6.97 a	167.50 ± 1.54 a	5.72 ± 0.14 b	20.32 ± 0.95 a
L. casei Shirota	8.47 ± 0.07 b	10.52 ± 0.07 a	11.80 ± 0.00 b	891.78 ± 48.92 a	10.37 ± 1.04 b	27.04 ± 1.57 a

Different lowercase letters show significant differences between the evaluation times; n = 3 for analysis of viable count and free amino group determination, while n = 2 for antidiabetic capacity.

shows the results obtained for bacterial growth, free amino groups released by strain nitrogen demands, and the corresponding antidiabetic capacity.

The viable count showed that both strains could grow in the rye protein-enriched media, with an increment of two logarithmic cycles according to the initial bacterial concentration. Kocková et al. [25] found similar growth during the fermentation of rye flour, rye grain, and other cereals with L. rhamnosus GG, where the initial concentration for lactic acid bacteria was 5.05–6.20, and it increased to 7.78–8.57 log CFU/g. In the same context, Matejčeková et al. [26] observed initial and final concentrations for L. rhamnosus GG of 5.9–6.7 and 8.4–9 log CFU/mL, respectively, when it was co-cultured with other commercial starter cultures for the fermentation of buckwheat. Also, Němečková et al. [27] carried out the fermentation of rice, barley, and corn flours, supplemented with 1% of glucose, reaching concentrations of 7–8 log cfu/mL after 16 h for different lactic acid bacteria inoculated, representing an increment of two or three logarithmic cycles according to the initial cell concentration. On the other hand, it is highly remarkable that at the end of the fermentation, L. rhamnosus GG as L. casei Shirota reached superior recommended concentrations to obtain beneficial effects on human health from probiotics, considered as between 10⁷ and 10⁹ cells [28,29].

The free amino groups test revealed higher proteolytic activity of L. casei Shirota after 24 h during the fermentation of the rye-enriched medium. Previous studies on the lactic fermentation of cereals have demonstrated that the proteolysis level depends on the lactic acid bacteria strain and cereal substrate. Němečková et al. [27] measured the proteolytic capacity through the ability to reduce protein and phenylalanine content. They found few changes during the fermentation of rice and corn by some strains of L. casei, L. paracasei, and L. helveticus. Similar results were reported by Moiseenko et al. [30] when rice and oat flour were fermented by L. paracasei, L. delbrueckii, L. helveticus, S. thermophilus, and L. lactis. In addition, these strains were also inoculated on wheat flour, but the proteolysis was minimal or null. On the other hand, bacteria inoculated on a rye medium have been applied in dairy fermentation, with similar proteolysis patterns found to those in the present work, which confirms that the main factor in hydrolysis produced by lactic acid bacteria is the nitrogen demand. Lactobacillus casei Shirota has been a highly proteolytic bacteria on milk [31,32], and L. rhamnosus GG produced few proteolytic changes in bovine whey fermentation [33,34].

Fermentation with probiotics increased the antidiabetogenic activity similarly despite differences in proteolytic activity. According to the bioactivity found at the beginning of fermentation, L. rhamnosus GG produced an increment of 14.6 percentual units, while the increase for L. casei Shirota was 16.7. Feng et al. [35] reported increases in percentual units from 10 to 60 in a concentration-dependent manner when strains of L. plantarum and L. paracasei were used to ferment buckwheat.

Rye fermentation with probiotics generated a DPP-IV inhibition of around 20–27%, which was comparable with the findings reported by Garzón et al. [36], who found antidiabetic activities of around 30% in a media composed of sorghum and skim milk and fermented by lactic acid bacteria. In the same way, Feng et al. [35] found DPP-IV inhibition between 30 and 40% at concentrations of 1–2 mg/mL for ethanol extracts obtained from solid-state fermentation, while Wu et al. [37] showed inhibitions of around 30% at the same concentrations for water and ethanol extracts from the fermentation of rice beans by Bacillus amyloliquefaciens. Thus, rye fermentation by probiotic lactic bacteria is very promising compared to similar studies.

4. Conclusions

The culture media proposed with the enrichment of rye isolate protein allowed the efficient growth of lactic acid bacteria tested at levels recommended for obtaining human health benefits from probiotic bacteria. Also, each strain’s proteolytic activity produced bioactive peptides with antidiabetic activity, similar to other vegetable sources. Thus, rye fermentation is a promising method for the obtention of plant-based beverages and functional foods.