Abstract
The aim of this study was to assess the gamma-aminobutyric acid (GABA) production in plant-based fermented beverages with kefir cultures (milk and water kefir). Water-soluble extracts of peanut and Brazil nut were evaluated as non-dairy substrates for the development of new bioactive beverages. A total of 12 formulations were developed and evaluated for their chemical composition, physical chemical characterization, and microbiological counts (aerobic mesophilic bacteria, lactobacilli, lactococci and yeasts). Results proved that the composition of kefir culture, the nature of the extract and the addition of glutamic acid affected GABA production. Even peanut extracts presenting higher protein content, Brazil nut extracts was the best substrate for GABA production. In addition, fermented beverages were evaluated for in vitro antihypertensive activity through angiotensin-converting enzyme (ACE) inhibition. The formulations with GABA presented the most efficient ACE-inhibition, wherein beverages based on Brazil nut extract showed the highest results of ACE-inhibition (80%). This study evidenced that kefir fermentation enhanced plant-based beverages bioactivity, indicating their potential as functional beverages. The plant-based extracts and the bioactive compounds produced in this study characterize the beverages in an innovative way, due to the process, composition and simultaneous presence of probiotics and GABA, which had not been reported before.
Keywords: Plant-based fermented beverages, Kefir grain, Gamma-aminobutyric acid, Angiotensin-converting enzyme, Nut milk
Highlights
Peanut and Brazil nut extracts were substrates for non-dairy kefir fermented beverages.
GABA was produced by kefir fermentation of plant-based beverages.
Brazil nut extract was the best substrate for GABA production.
Fermented beverages with GABA presented higher in vitro antihypertensive activity.
First study relating production of GABA in plant-based kefir fermented beverages.
Introduction
The growing concern of consumers with quality of life, well-being and health has resulted in the demand for healthy habits and care with food. Consumers are more aware of the relationship between good nutrition and health and therefore have increased the demand for foods that, in addition to nourishing, provide health benefits. In this context, research to produce functional foods is a field that has been growing considerably in recent years (Dias et al. 2018). Within the context of healthier foods, the plant-based feeding pattern has gained a lot of strength, whose main reasons are the search for more sustainable habits and the evidences revealing the plant-based diet improving health conditions, such as obesity, cardiovascular diseases, diabetes, cancer and anxiety (Euromonitor International 2019). However, a diet based exclusively on plant-based foods is not always able to meet the demands of all necessary nutrients, especially essential minerals (such as iron, calcium, and zinc), due to their low bioaccessibility and bioavailability, in addition to several bioactive compounds (Luz and Pallone 2022). Thus, seeking a balanced diet with health benefits is essential to meet the current consumer demand.
The kefir fermented beverage, traditionally produced from the fermentation of milk, provides a consortia of yeasts and bacteria (lactic acid and acetic acid bacteria) that live in symbiosis and can provides a set of biological functions, such as antioxidant, anti-inflammatory, immunomodulatory, antimicrobial, antihypertensive potential, antidepressant and supply of probiotics (e.g. Lactobacillus acidophilus, Bifidobacterium bifidum, and others) (Araujo Filho et al. 2023). The fermentation of milk by lactic acid bacteria induces the production of a set of bioactive compounds, including the gamma-aminobutyric acid (GABA). GABA is one of the main neurotransmitters of the central nervous system and has several biological activities reported in the literature, being responsible for preventing neurological disorders, having antihypertensive action, antioxidant, and antidiabetic effects, among others (Tang et al. 2018). Microorganisms, including lactic acid bacteria and fungi, have the ability to synthesize GABA during the fermentation process of food matrices, such as milk, fruits, and cereals (Nikmaram et al. 2017). Kefir grains are also capable of fermenting different non-dairy substrates such as fruit juices, and plant extracts (Uruc et al. 2022; Comak Gocer and Koptagel 2023a). Thus, the development of a new bioactive fermented beverage, naturally enriched in GABA, and using plant-based matrices, meets food industry and consumers demands. According to Brazilian food regulation, it is not allowed the addition of GABA to food (Brasil 2018). Food fermentation process is an option for having foods supplemented with GABA.
The aim of this study was to produce fermented beverages based on plant extracts (peanut and Brazil nut), source of GABA, using water and milk kefir grains, and to evaluate the beverages chemical composition, physical chemical parameters, microbiological counts and in vitro antihypertensive activity through angiotensin-converting enzyme (ACE) inhibition. To the best of authors knowledge, no research has been undertaken relating the production of plant-based kefir beverages with GABA production.
Materials and methods
Materials
The milk kefir grains were donated by local users, and water kefir grains were donated by Bioprocess Engineering Laboratory from University of São Paulo. The commercial kefir culture (eXact® Kefir 1 starter culture) was purchased from Chr. Hansen (Hersholm, Denmark). The peanut kernels and Brazil nuts were purchased from local suppliers. Xanthan gum was purchased from Aminna Alimentos (Blumenau, Brazil) and Inulin was purchased from Ingredion (São Paulo, Brazil). Plate count agar (PCA), De Man, Rogosa and Sharpe Agar (MRS agar) and Dicloran Rose-Bengal Chloramphenicol Agar (DRBC agar) were purchased from Neogen (USA). M17 Agar was purchased from Merck (Darmstadt, Germany). ACE inhibition assay fluorescence kit was purchased from Sigma-Aldrich (CS0002, Germany). All other chemicals were used in analytical grade.
Maintenance of kefir cultures
The water kefir grains were cultivated in an aqueous solution (filtered water) of sucrose (brown sugar) with soluble solids (SS) concentrations of 5 g/100 g. Fermentations were carried out at 25 ± 1 °C in controlled incubator (Tecnal, Brazil), without agitation. The grains were kept active and viable for fermentation, with continuous change of whole milk and sucrose solution, every 24 h.
The commercial kefir (eXact® Kefir 1 starter culture), containing 6 microorganisms in its composition (Debaryomyces hansenii, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar. diacetylactis, Leuconostoc and Streptococcus thermophilus), was activated according to the manufacturer’s recommendations (Chr. Hansen), inoculating the entire contents of the package (100 units) in one liter of whole cow milk. Fermentation was carried out at 25 ± 1 °C, in controlled incubator, without agitation. After 24 h of fermentation, the grains were fractionated, and one part continued to be fermented with substrate change every 24 h to keep the viability of the culture, and the other stored at -20 °C until use. The milk kefir grains were cultivated in whole cow milk, as described to commercial kefir.
Production of water-soluble plant extracts
The water-soluble extracts of peanut and Brazil nut were produced according to the method described by from Lopes (Lopes 2012). For the production of one liter of extract, 150 g of the raw material was added of water at 50 °C in a ratio of 1:8 (w/v), the mixture was crushed in a homogenizer (Blender High Rotation, Marchesoni, Brazil) during 4 min and then filtered in a 60-mesh sieve. Both water-soluble plant extracts were pasteurized (90 °C/3 min), packed and stored in a freezer (-20 °C) for later use.
Fermentation of vegetable drinks
The beverages were prepared by inoculating 5% (w/v) of kefir grains in 50 g of water-soluble plant extracts. The Erlenmeyer was covered with sterile gauze, allowing gas exchange. Fermentation was carried out at 25 ± 1 °C for 24 h. Then, the grains were filtered and the beverages were stored in 50 mL sterile tubes at -20 °C for further analysis. A total of 8 fermented formulations were developed with kefir grains (Table 1), with addition of 0.26% xanthan gum, 0.3% inulin, which concentrations were based on previous literature (Alves et al. 2021). Formulations with only water-soluble plant extract and 0.26% xanthan gum were named as “standard formulation”. With the commercial kefir culture, 4 formulations were developed with 0.26% xanthan gum and 3% inulin, two of which was added 0.5% glutamic acid, based on the literature previous results (Kantachote et al. 2017). Table 1 describes the formulations and their respective coding.
Table 1.
Formulations of fermented plant extracts kefir beverages
| Formulation | Water-soluble extract | Kefir grain | Inulin (%) | Glutamic acid (%) |
|---|---|---|---|---|
| WSP-MK | Peanut | Milk kefir grain | - | - |
| WSP-MKI | Milk kefir grain | 3 | - | |
| WSP-WK | Water Kefir grain | - | - | |
| WSP-WKI | Water Kefir grain | 3 | - | |
| WSBN-MK | Brazil nut | Milk kefir grain | - | - |
| WSBN-MKI | Milk kefir grain | 3 | - | |
| WSBN-WK | Water Kefir grain | - | - | |
| WSBN-WKI | Water Kefir grain | 3 | - | |
| WSP-CK | Peanut | Commercial kefir culture | 3 | - |
| WSP-CKGA | 3 | 0.5 | ||
| WSBN-CK | Brazil nut | Commercial kefir culture | 3 | - |
| WSBN-CKGA | 3 | 0.5 |
WSP-MK (water-soluble extract of peanut + milk kefir); WSP-MKI (water-soluble extract of peanut + milk kefir + 3% of inulin); WSP-WK (water-soluble extract of peanut + water kefir); WSP-WKI (water-soluble extract of peanut + water kefir + 3% of inulin); WSBN-MK (water-soluble extract of Brazil nut + milk kefir); WSBN-MKI (water-soluble extract of Brazil nut + milk kefir + 3% of inulin); WSBN-WK (water-soluble extract of Brazil nut + water kefir); WSBN-MKI (water-soluble extract of Brazil nut + water kefir + 3% of inulin); WSP-CK (water-soluble extract of peanut + commercial kefir); WSP-CKGA (water-soluble extract of peanut + commercial kefir + glutamic acid); WSBN-CK (water-soluble extract of Brazil nut + commercial kefir); WSBN-CKGA (water-soluble extract of Brazil nut + commercial kefir + glutamic acid)
Physical chemical analysis
The chemical composition of the water-soluble plant extracts was determined according to the Association of Official Analytical Chemists (AOAC 1995). Moisture and fat content were determined gravimetrically. Lipids in the samples were extracted following the method of Bligh and Dyer, the total protein was determined by Kjeldahl nitrogen determination (using the factor 5.46 to protein conversion). Ash content was determined by mineralization of the samples at 450 °C. The content of carbohydrate was calculated based on the difference (100—moisture—lipidis—protein—ash). The pH and Soluble Solids values were determined using a digital potentiometer (Tecnopon, Brazil) and digital refractometer (Milwaukee, USA), respectively.
Microbiological analysis
The microbiological counts of kefir beverages were performed after 24 h of fermentation, according to enumeration method described before (Garofalo et al. 2015). An aliquot of 10 mL of each kefir beverage sample were homogenized in 90 mL of sterile 0.1% peptone solution. Serial decimal dilutions were prepared in the same diluent and inoculated in duplicate by deep (lactobacilli and lactococci) or surface (total aerobic mesophilic and yeasts) plating on specific solid media. Total aerobic mesophilic counts were performed on PCA, after aerobic incubation at 25 °C for 72 h. Lactobacilli and lactococci were counted using MRS and M17 agar, respectively, after anaerobic incubation at 35 °C for 72 h. PCA, MRS and M17 media were supplemented with 0.4 mg/ml of nystatin to avoid yeast growth. Yeasts counts were analyzed in DRBC Agar, after aerobically incubation at 30 °C for 5 days. Results were expressed as means of log colony-forming units (CFU) per ml of sample (log CFU/mL).
Qualitative determination of gamma-aminobutyric acid
The determination of the presence of GABA was carried out qualitatively, using the paper chromatography (PC) method, as previously described, with modifications (Li et al. 2009). PC was developed at 30 °C with n-butanol, acetic acid, and water (5:2:3) containing 1% ninhydrin (w/v) by the ascending technique. After development, the paper was dried directly for color yield in an air-circulating oven (Tecnal, Brazil) at 70 °C for 80 min. After drying, pictures were taken of the chromatograms.
Evaluation of antihypertensive activity in vitro
The antihypertensive activity of vegetable kefir beverages was evaluated by the ACE inhibition assay – Fluorescence Kit, following the manufacturer’s instructions. The assay was performed with control (buffer solution and enzyme solution), blank (buffer solution without enzyme), sample (kefir beverages) and captopril solution at 50 mg/ml, the main drug inhibitor of angiotensin-converting enzyme (ACE). ACE inhibition (%) was calculated based on Eq. 1, where AAm: sample activity and ACTL: reaction control activity.
 |
1 |
Statistical analysis
The mean values of the physical chemical characterization were obtained in triplicate and the other analyses in duplicate. Microbial counts were analyzed by analysis of variance (ANOVA) with the aid of the R software (version 4.0) followed by the Scott-Knott test (p ≤ 0.05).
Results and discussion
Physical chemical characterization
Table 2 illustrates the proximate composition of the standard formulations of vegetable kefir beverages after fermentation by homemade kefir grains.
Table 2.
Chemical composition (mean ± standard deviation) of plan-based kefir beverages after 24 h fermentation
| Components (g/100 g) | WSP-MK | WSP-WK | WSBN-MK | WSBN-WK |
|---|---|---|---|---|
| Moisture | 94.58 ± 0.08 | 93.55 ± 0.12 | 94.42 ± 0.07 | 93.46 ± 0.06 |
| Ash | 0.08 ± 0.05 | 0.14 ± 0.12 | 0.17 ± 0.05 | 0.30 ± 0.08 |
| Protein | 1.09 ± 0.03 | 1.46 ± 0.07 | 0.77 ± 0.01 | 0.86 ± 0.02 |
| Lipids | 4.79 ± 0.03 | 4.74 ± 0.04 | 3.54 ± 0.12 | 3.97 ± 0.16 |
| Carbohydrates | - | 0.20 ± 0.25 | 1.09 ± 0.12 | 1.41 ± 0.22 |
WSP-MK (water-soluble extract of peanut + milk kefir); WSP-WK (water-soluble extract of peanut + water kefir); WSBN-MK (water-soluble extract of Brazil nut + milk kefir); WSBN-WK (water-soluble extract of Brazil nut + water kefir)
The vegetable kefir beverages presented high moisture content as expected, ranging from 93.46 to 94.58% (Atalar 2019). The four formulations analyzed had ash contents below 0.20%. According to Perfeito et al. (2017), foods with high water content tend to have a low amount of incinerated waste. However, there is still a higher concentration of ash in Brazil nut-based beverages, due to the composition of the raw material (Perfeito et al. 2017). Deziderio (2019), developed a fermented beverage with lactic acid bacteria in a water-soluble Brazil nut extract and found 0.10% ash (Deziderio 2019). Comak Gocer and Koptagel (2023b), found in their study a content of 0.24% ash in a fermented beverage with a commercial kefir starter culture in water-soluble peanut extract, a content slightly higher than the observed in the formulations of water-soluble extract of peanut (WSP) and water-soluble extract of Brazil nut (WSBN) (Comak Gocer and Koptagel 2023b). The protein content ranged from 0.77 to 1.46%. It was observed that WSP beverages had higher protein content, corroborating the fact that among the raw materials, fresh peanuts have 27% protein and Brazil nuts 14% (TACO 2011). The lipid content ranged from 3.54 to 4.79%. Rios (2021) developed two fermented beverages with homemade milk kefir grains based on water-soluble Brazil nut and coconut extract. The authors reported lipid levels ranging of 7.97 and 5.32%, respectively (Rios 2021). The lipid content reported in the literature is higher than that observed in all formulations evaluated in this study, which could be attributed to the plant of peanut crop or chestnut used in the production of the extracts and to the process of the extracts production.
Microbial metabolism and fermentation results in the degradation of carbohydrates and the release of less complex compounds, as a source of energy during their development and multiplication. WSP-WK formulation had the lowest carbohydrate content among the formulations (0.20%). Thus, the variation in the content of moisture, ash, proteins, lipids, and carbohydrates in vegetable fermented beverages can be attributed to the matrix, water-soluble extracts and fermentation processes.
The pH and soluble solids values of each formulation after 24 h of fermentation are presented in Table 3. The pH values differed between beverages fermented with different kefir grains (milk or water kefir grains). However, there was no statistical difference between different matrixes. Also, the addition of inulin does not seem to influence the pH drop during fermentation. Inulin is a prebiotic applied to increase lactic acid bacteria viability in foods. It was expected that milk kefir cultures would benefit from this prebiotic, since the plant extracts do not contain the lactose, an important substrate for these bacteria. Gocer and Koptagel (2023a) produced kefir based on water-soluble peanut extract with commercial culture and found a final pH value of 4.97, corroborating the values observed in the beverages developed in this study (Comak Gocer and Koptagel 2023a). Rios (2021) evaluated the pH over the 20-day refrigerated storage period of five fermented beverages with milk kefir grains based on vegetable extract of rice, brown rice, coconut, cashew nuts, and Brazil nuts (Rios 2021). The author concluded that the Brazil nut-based beverage did not decrease pH during this period, which may be explained to the composition of the plant extract (lipid content, type of fatty acid, as well as mineral salts), which may have directly influenced the metabolism of the microorganisms. The soluble solids (SS) presented no statistical differences between the formulations (Table 3). The addition of inulin resulted in higher soluble solids levels. The developed beverage formulations presented physical chemical contents close to those reported in previous literature, even though the beverages were fermented with two different types of kefir grains.
Table 3.
pH and soluble solids (mean ± standard deviation) of plan-based kefir beverages after 24 h fermentation
| Formulation | pH | Soluble solids (°Brix) |
|---|---|---|
| WSP-MK | 4.29 ± 0.11b | 0.95 ± 0.05 b |
| WSP-MKI | 4.17 ± 0.00 b | 3.50 ± 0.00 a |
| WSP-WK | 5.24 ± 0.18 a | 0.60 ± 0.10 b |
| WSP-WKI | 4.83 ± 0.02 b | 2.95 ± 0.05 a |
| WSBN-MK | 4.20 ± 0.01b | 0.85 ± 0.05 b |
| WSBN-MKI | 4.25 ± 0.25 b | 3.05 ± 0.05 a |
| WSBN-WK | 5.01 ± 0.06 a | 0.85 ± 0.05 b |
| WSBN-WKI | 4.83 ± 0.04 a | 3.05 ± 0.05 a |
a, b: Different letters in the same row demonstrates statistically difference between samples (p ≤ 0.05) by the Scott-Knott test
WSP-MK (water-soluble extract of peanut + milk kefir); WSP-MKI (water-soluble extract of peanut + milk kefir + 3% of inulin); WSP-WK (water-soluble extract of peanut + water kefir); WSP-MKI (water-soluble extract of peanut + water kefir + 3% of inulin); WSBN-MK (water-soluble extract of Brazil nut + milk kefir); WSBN-MKI (water-soluble extract of Brazil nut + milk kefir + 3% of inulin); WSBN-WK (water-soluble extract of Brazil nut + water kefir); WSBN-MKI (water-soluble extract of Brazil nut + water kefir + 3% of inulin)
Microbial counts of plant-based kefir beverages
Table 4 shows the logarithmic counts (Log10 CFU/mL) of total aerobic mesophilic bacteria counts, lactobacilli, lactococci and yeasts determined after 24 h of fermentation of plant-based kefir beverages. Only lactobacilli count significantly differed between formulations studied (p ≤ 0.05). It is possible to note that the beverages produced by different water-soluble extracts presented different counts, indicating that the composition of the substrates may result in different responses for this group of bacteria.
Table 4.
Viable cell counts (Log10 CFU/mL) in plant-based kefir beverages after 24 h of fermentation
| Formulation | Total aerobic mesophilic bacteria | Lactobacilli | Lactococci | Yeasts |
|---|---|---|---|---|
| WSP-MK | 8.67 ± 0.84 | 8.02 ± 0.83 c | 8.68 ± 0.89 a | 4.55 ± 0.59 a |
| WSP-MKI | 8.63 ± 0.89 a | 8.52 ± 0.88a | 8.48 ± 0.89 a | 5.23 ± 0.69 a |
| WSP-WK | 8.51 ± 0.90 a | 7.09 ± 0.81 e | 9.10 ± 0.94 a | 6.28 ± 0.77 a |
| WSP-WKI | 8.80 ± 0.93 a | 8.02 ± 0.86 c | 8.85 ± 0.92 a | 5.60 ± 0.62 a |
| WSBN-MK | 8.26 ± 0.84 a | 7.86 ± 0.87 d | 9.15 ± 0.94 a | 4.88 ± 0.56 a |
| WSBN-MKI | 8.31 ± 0.89 a | 7.92 ± 0.86 d | 8.41 ± 0.89 a | 5.15 ± 0.62 a |
| WSBN-WK | 8.47 ± 0.84 a | 8.13 ± 0.80 b | 9.02 ± 0.89 a | 4.46 ± 0.59 a |
| WSBN-WKI | 8.64 ± 0.85 a | 8.00 ± 0.86 c | 8.91 ± 0.91 a | 4.89 ± 0.55 a |
a, b: Different letters in the same row demonstrates statistically difference between samples (p ≤ 0.05) by the Scott-Knott test
WSP-MK (water-soluble extract of peanut + milk kefir); WSP-MKI (water-soluble extract of peanut + milk kefir + 3% of inulin); WSP-WK (water-soluble extract of peanut + water kefir); WSP-MKI (water-soluble extract of peanut + water kefir + 3% of inulin); WSBN-MK (water-soluble extract of Brazil nut + milk kefir); WSBN-MKI (water-soluble extract of Brazil nut + milk kefir + 3% of inulin); WSBN-WK (water-soluble extract of Brazil nut + water kefir); WSBN-MKI (water-soluble extract of Brazil nut + water kefir + 3% of inulin)
Also, the addition of inulin increased the growth of this group of microorganisms. On the other hand, beverages fermented with water grains, without the addition of inulin, had the highest count.
The addition of inulin did not increase lactobacilli counts in the beverages developed by Montanuci et al. (2012). The authors produced kefir in whole milk with the addition of inulin, powdered milk, using kefir grains and starter culture and justify that the growth and viability of bacteria in the presence of inulin vary according to their degree of polymerization, thus the addition of powdered milk, may contribute to the lack of the effect of inulin on the viability of lactic acid bacteria (Montanuci et al. 2012).
Beverages fermented with water kefir grains didn’t have counts higher of yeast counts than those fermented with milk kefir grains, which was expected, since yeasts are usually in higher quantities in water kefir grain. This may have occurred due to the plant extracts, in addition to the fermentation conditions, since these factors may influence the microbiota composition of kefir grains (Lynch et al. 2021). Beverages fermented with the commercial culture were not evaluated for microbial counts, however, according to the manufacturer, the culture was composed of Debaryomyces hansenii, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar. diacetylactis, Leuconostoc and Streptococcus thermophilus.
Evaluation of gamma-aminobutyric acid (GABA) production
The presence of GABA was qualitatively evaluated only in formulations fermented with commercial kefir culture, since the other kefir grains did not present potential for GABA production (results not shown). The addition of glutamic acid in the formulations was carried out aiming of enhance the production of GABA via fermentation by bacteria, since glutamic acid, as well as monosodium glutamate, is one of the main substrates to produce GABA (Yogeswara et al. 2020) Results are illustrated in Fig. 1.
Fig. 1.
Chromatogram of fermented plant-based kefir beverages with commercial kefir culture
Glutamic acid: 0.5% glutamic acid solution; GABA: 0.05% gamma-aminobutyric acid solution; 1: formulation WSP-CK (water-soluble extract of peanut + commercial kefir); 2: formulation WSP-CKGA (water-soluble extract of peanut + commercial kefir + glutamic acid); 3: formulation WSBN-CK (water-soluble extract of Brazil nut + commercial kefir); 4: formulation WSBN-CKGA (water-soluble extract of Brazil nut + commercial kefir + glutamic acid)
The chromatogram clearly showed the presence of GABA in the WSP-CKGA and WSBN-CKGA formulations, which commonly have the addition of glutamic acid in the formulation. Comparing WSP-CK and WSBN-CK (both without glutamic acid addition), the presence of GABA is more evident in the peanut-based formulation than in Brazil nut-based formulation. Apparently, peanut is better substrate for GABA production than Brazil nut, although the fresh Brazil nut has a higher glutamic acid content than raw peanuts (17.7% in Brazil nut against 5,39% in peanut) (Hosseini Taheri et al. 2024).
The identification of the presence of GABA in the WSP-CKGA and WSBN-CKGA formulations suggests that the microbial culture of commercial kefir has the capacity to produce GABA and that the addition of glutamic acid (the main substrate for GABA production) provided favorable conditions to produce this metabolic byproduct during the fermentation of the beverages. The addition of 0.5% glutamic acid to vegetable kefir formulations resulted in higher GABA production than formulations fermented with the commercial culture without the addition of glutamic acid (Fig. 1). As this is a qualitative determination, the concentration of glutamic acid was not calculated, however, studies suggest that GABA production may increase due to the presence of glutamic acid, which can be observed in the chromatogram above. Fan et al. (2023) developed GABA-enriched yogurt by fermenting a mixture of strains (Levilactobacillus brevis CGMCC1.5954, Streptococcus thermophilus ABT-T, and Lactobacillus delbrueckii ssp. bulgaricus BNCC 336,436) in a 3:1:1 ratio with the addition of 0.1% monosodium glutamate. The product had 147.36 mg/100 mL of GABA, a content about 317% higher than that found in the control drink (produced only with the starter culture) (Fan et al. 2023). The incorporation of monosodium glutamate also influenced the GABA content in yogurt developed by Chen et al. (2018), the authors varied the glutamate concentrations between 6 and 12 g/L and identified that the addition of 12 g/L reflected in higher GABA content, in addition, factors such as temperature and fermentation time also influenced the result found (Chen et al. 2018). Plant matrix is also a viable substrate for GABA production through fermentation. A vegetable extract of fermented chickpeas enriched with GABA was obtained using Lactobacillus plantarum M-6 strains and optimized fermentation conditions (Feng et al. 2023). According to the same study, the fermented beverage showed a neuroprotective effect.
The GABA content in fermented beverages varies depending on the substrate and the species of lactic acid bacteria used. The beverages developed in this study with homemade milk and water kefir grains were not able to produce GABA (data not shown). Probably due to commercial kefir microbiota (Debaryomyces hansenii, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar. diacetylactis, Leuconostoc and Streptococcus thermophilus) efficiency in the production of GABA when compared to the microbiota of milk and water kefir. The microbial load of the homemade kefir grains and the commercial culture used in the fermentation of the beverages were very different about 5.02 Log10 CFU/g for the milk kefir grains, 6.74 Log10 CFU/g for water kefir grains and 8.96 Log10 CFU/g for the commercial kefir culture. An alternative to produce GABA by homemade kefir grains would be to optimize fermentation conditions. It was evidenced that kefir fermentation of plant extracts (water-soluble peanut extract and water-soluble Brazil nut extract) with the addition of glutamic acid allowed the production of GABA in sufficient quantities for identification using the commercial kefir culture.
Evaluation of the in vitro antihypertensive effect of vegetable kefir beverages
The in vitro antihypertensive potential of vegetable kefir beverages was evaluated by inhibition of angiotensin-converting enzyme (ACE). ACE triggers a catalytic reaction that converts angiotensin I to angiotensin II, a potent vasoconstrictor (Ruviaro et al. 2020). Thus, inhibiting the action of ACE results in a potential reduction in blood pressure. Results obtained of angiotensin-converting enzyme (ACE) inhibitory activity of kefir beverages are presented in Fig. 2. Between milk and water kefir, brazil nut beverages showed higher inhibitory activities of ACE than peanut beverages (Fig. 2). The difference observed in the inhibitory activity between the beverages was probably due to the protein and amino acid composition of the vegetable substrate used. Although peanut extract had the highest protein content (1.78%), ACE inhibition did not exceed 20% in formulations based on this plant extract. For beverages based on Brazil nut extract, inhibition of up to 80% of ACE was achieved. Thus, the amino acid profile present in the plant extracts may interfere in ACE inhibition. Aromatic amino acids, such as phenylalanine, or positively charged amino acids, such as arginine, as well as hydrophobic amino acid residues such as tryptophan or proline, are associated with high affinity for ACE, promoting its inhibition (Uruc et al. 2022). Captopril, an inhibitor of angiotensin-converting enzyme, was evaluated as positive control. Captopril results showed inhibition activity below than the observed for WSBN-MK and WSBN-WK formulations, at the tested concentration. These results demonstrated the great potential of this plant-based kefir fermented beverages to ACE inhibition.
Fig. 2.
Angiotensin-converting enzyme (ACE) inhibitory activity of plant-based kefir beverages, fermented with milk, water and commercial kefir grains
WSP-MK (water-soluble extract of peanut + milk kefir); WSP-WK (water-soluble extract of peanut + water kefir); WSP-CK (water-soluble extract of peanut + commercial kefir); WSP-CKGA (water-soluble extract of peanut + commercial kefir + glutamic acid); WSBN-MK (water-soluble extract of Brazil nut + milk kefir); WSBN-WK (water-soluble extract of Brazil nut + water kefir); WSBN-CK (water-soluble extract of Brazil nut + commercial kefir); WSBN-CKGA (water-soluble extract of Brazil nut + commercial kefir + glutamic acid)
The ACE inhibitory activities by beverages fermented by commercial kefir with positive production of GABA were greatly superior than those observed by beverages obtained through fermentation with homemade grains (Fig. 2). Beverages based on Brazil nut extract showed the highest results of ACE inhibition, with 67% in the formulation without added glutamic acid (WSBN-CK) and 80% for the formulation with Brazil nuts and added glutamic acid (WSBN-CKGA). In peanut extract-based beverages fermented by commercial kefir, ACE inhibition was between 19 and 22% for formulations with glutamic acid and glutamic acid-free (WSP-CKGA and WSP-CK), respectively. The inhibitory activity of the GABA-rich formulation based on Brazil nuts and added glutamic acid (WSBN-CKGA) was about 17.7% higher than that observed in the formulation that showed the highest ACE inhibition among the beverages that did not produced GABA (WSBN-CKGA). Thus, we observed by in vitro assays that plant-based kefir beverages with the presence of GABA presented an increased ACE inhibition and consequently the hypotensive effect of the beverages.
Uruc et al., (2022) developed kefir with apricot seed and after 21 days of storage, the beverages were responsible for 85.48% of ACE inhibition, compared to 55.15% of inhibition by traditional beverages produced with cow’s milk (Uruc et al. 2022). Kefir made with soy milk also showed significant ACE inhibitory activity, with IC50 equal to 3.55 μg/ml (Gamba et al. 2020). These results demonstrate the relevance of ACE inhibitor peptides derived from plant proteins.
In vitro and in vivo studies have already demonstrated that GABA can attenuate hypertension, however, the mechanism of action of this postbiotic is different from that observed by peptides with ACE inhibitory activity (Garavand et al. 2022). Researchers suggest that the antihypertensive effect of GABA occurs through the inhibition of the release of noradrenaline by peripheral sympathetic nerves, via presynaptic GABA B receptors, thus promoting the inhibition of perivascular nerve stimulation, mediating the hypotensive effect (Hayakawa et al. 2002). Thus, to explore the synergistic effect of GABA observed in relation to the antihypertensive action of the beverages developed here, it would be necessary to evaluate in vivo the effect of the vegetable kefir beverages developed in relation to the antihypertensive action.
Conclusion
This study demonstrated that it is possible to use plant extracts of peanuts and Brazil nuts to produce kefir beverages with bioactive activity. The type of kefir grain and vegetable substrate played a fundamental role in determining which compounds were metabolized and produced during fermentation. Only commercial kefir produced GABA after plant-based beverages fermentation. Among the beverages containing GABA, ACE inhibition reached the highest percentage with WSBN-CKGA formulation (80%). Optimizing the fermentation of these beverages are important to increase GABA production. Also, in vivo studies are needed to evaluate the effects of the presence of probiotics and GABA, as well as sensory acceptance studies of beverages. To the best of authors knowledge, no research has been undertaken relating the production of plant-based kefir beverages with GABA production.
Acknowledgements
The authors would like to thank the National Council for Scientific and Technological Development (CNPq) for the master’s scholarship. The present study was carried out with the support of the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001.
Abbreviations
- WSBN-CK
Water-soluble extract of Brazil nut + commercial kefir
- WSBN-CKGA
Water-soluble extract of Brazil nut + commercial kefir + glutamic acid
- WSBN-MK
Water-soluble extract of Brazil nut + milk kefir
- WSBN-MKI
Water-soluble extract of Brazil nut + milk kefir + 3% of inulin
- WSBN-WK
Water-soluble extract of Brazil nut + water kefir
- WSP-CK
Water-soluble extract of peanut + commercial kefir
- WSP-CKGA
Water-soluble extract of peanut + commercial kefir + glutamic acid
- WSP-MK
Water-soluble extract of peanut + milk kefir
- WSP-MKI
Water-soluble extract of peanut + milk kefir + 3% of inulin
- WSP-WK
Water-soluble extract of peanut + water kefir
- WSP-WKI
Water-soluble extract of peanut + water kefir + 3% of inulin
Author contributions
CS: methodology, formal analysis, investigation, data curation, writing original MS; CLSC: methodology; ESK: methodology; JAM: methodology; PPMB: methodology, writing - review & edition; GAM: conceptualization, methodology, supervision, writing - review & edition, Funding.
Funding
Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001.
Data availability
All data generated or analyzed during this study are included in this published article.
Declarations
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Consent to participate
All the authors contributed and agreed to participate in the manuscript.
Consent for publication
All the authors have seen the manuscript and approved to submit to this Journal.
Competing interests
The authors declare that they have no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- Alves V, Scapini T, Camargo AF et al (2021) Development of fermented beverage with water kefir in water-soluble coconut extract (Cocos nucifera L.) with inulin addition. LWT 145:111364. 10.1016/j.lwt.2021.111364 [Google Scholar]
- AOAC (1995) Official methods of analysis of AOAC International, 16th edn. AOAC International, Arlington [Google Scholar]
- Araujo Filho AAL, de Sousa PHM, Vieira IGP et al (2023) Kombucha and kefir fermentation dynamics on cashew nut beverage (Anacardium occidentale L). Int J Gastron Food Sci 33:100778. 10.1016/j.ijgfs.2023.100778 [Google Scholar]
- Atalar I (2019) Functional kefir production from high pressure homogenized hazelnut milk. LWT 107:256–263. 10.1016/j.lwt.2019.03.013 [Google Scholar]
- Brasil, DE 26 DE JULHO DE (2018) INSTRUÇÃO NORMATIVA - IN Nº 28, 2018 - Estabelece as listas de constituintes, de limites de uso, de alegações e de rotulagem complementar dos suplementos alimentares. Brazil: DOU no 144, de 27 de julho de 2018
- Chen L, Alcazar J, Yang T et al (2018) Optimized cultural conditions of functional yogurt for γ-aminobutyric acid augmentation using response surface methodology. J Dairy Sci 101:10685–10693. 10.3168/jds.2018-15391 [DOI] [PubMed] [Google Scholar]
- Comak Gocer EM, Koptagel E (2023a) Production and evaluation of microbiological & rheological characteristics of kefir beverages made from nuts. Food Biosci 52:102367. 10.1016/j.fbio.2023.102367 [Google Scholar]
- Comak Gocer EM, Koptagel E (2023b) Production of milks and kefir beverages from nuts and certain physicochemical analysis. Food Chem 402:134252. 10.1016/j.foodchem.2022.134252 [DOI] [PubMed] [Google Scholar]
- Deziderio MA (2019) Desenvolvimento de bebida fermentada funcional de origem vegetal. Universidade de São Paulo
- Dias CO, dos Santos Opuski J, Pinto SS et al (2018) Development and physico-chemical characterization of microencapsulated bifidobacteria in passion fruit juice: a functional non-dairy product for probiotic delivery. Food Biosci 24:26–36. 10.1016/j.fbio.2018.05.006 [Google Scholar]
- Euromonitor International (2019) Conscious consumers drive sales of vegan products. In: https://www.euromonitor.com/article/consumidores-conscientes-impulsionam-vendas-de-produtos-veganos
- Fan X, Yu L, Shi Z et al (2023) Characterization of a novel flavored yogurt enriched in γ-aminobutyric acid fermented by levilactobacillus brevis CGMCC1.5954. J Dairy Sci 106:852–867. 10.3168/jds.2022-22590 [DOI] [PubMed] [Google Scholar]
- Feng L, Wu Y, Wang J et al (2023) Neuroprotective effects of a novel tetrapeptide SGGY from walnut against H2O2-stimulated oxidative stress in SH-SY5Y cells: possible involved JNK, p38 and Nrf2 signaling pathways. Foods 12:1490. 10.3390/foods12071490 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gamba RR, Yamamoto S, Abdel-Hamid M et al (2020) Chemical, microbiological, and functional characterization of Kefir produced from cow’s milk and soy milk. Int J Microbiol 2020:1–11. 10.1155/2020/7019286 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garavand F, Daly DFM, Gómez-Mascaraque G L (2022) Biofunctional, structural, and tribological attributes of GABA-enriched probiotic yoghurts containing lacticaseibacillus paracasei alone or in combination with prebiotics. Int Dairy J 129:105348. 10.1016/j.idairyj.2022.105348 [Google Scholar]
- Garofalo C, Osimani A, Milanović V et al (2015) Bacteria and yeast microbiota in milk kefir grains from different Italian regions. Food Microbiol 49:123–133. 10.1016/j.fm.2015.01.017 [DOI] [PubMed] [Google Scholar]
- Hayakawa K, Kimura M, Kamata K (2002) Mechanism underlying γ-aminobutyric acid-induced antihypertensive effect in spontaneously hypertensive rats. Eur J Pharmacol 438:107–113. 10.1016/S0014-2999(02)01294-3 [DOI] [PubMed] [Google Scholar]
- Hosseini Taheri SE, Bazargan M, Rahnama Vosough P, Sadeghian A (2024) A comprehensive insight into peanut: chemical structure of compositions, oxidation process, and storage conditions. J Food Compos Anal 125:105770. 10.1016/j.jfca.2023.105770 [Google Scholar]
- Kantachote D, Ratanaburee A, Hayisama-ae W et al (2017) The use of potential probiotic Lactobacillus plantarum DW12 for producing a novel functional beverage from mature coconut water. J Funct Foods 32:401–408. 10.1016/j.jff.2017.03.018 [Google Scholar]
- Li H, Qiu T, Cao Y et al (2009) Pre-staining paper chromatography method for quantification of γ-aminobutyric acid. J Chromatogr A 1216:5057–5060. 10.1016/j.chroma.2009.04.044 [DOI] [PubMed] [Google Scholar]
- Lopes GAZ (2012) Caracterização química, física E Sensorial De Produtos à base de amendoim. Universidade Estadual Paulista (UNESP)
- Luz GM, Pallone JAL (2022) Alimentos à base de plantas: Dietas, tendências de mercado, composição nutricional, e ensaios in vitro de bioacessibilidade e biodisponibilidade de minerais. Avanços em Ciência E Tecnologia De Alimentos - volume 6. Editora Científica Digital, pp 12–29
- Lynch KM, Wilkinson S, Daenen L, Arendt EK (2021) An update on water kefir: microbiology, composition and production. Int J Food Microbiol 345:109128. 10.1016/j.ijfoodmicro.2021.109128 [DOI] [PubMed] [Google Scholar]
- Montanuci FD, Pimentel TC, Garcia S, Prudencio SH (2012) Effect of starter culture and inulin addition on microbial viability, texture, and chemical characteristics of whole or skim milk kefir. Food Sci Technol 32:580–865. 10.1590/S0101-20612012005000119 [Google Scholar]
- Nikmaram N, Dar B, Roohinejad S et al (2017) Recent advances in γ -aminobutyric acid (< scp > GABA) properties in pulses: an overview. J Sci Food Agric 97:2681–2689. 10.1002/jsfa.8283 [DOI] [PubMed] [Google Scholar]
- Perfeito DGA, Corrêa IM, Peixoto N (2017) Elaboração De Bebida com extrato hidrossolúvel de soja saborizada com frutos do Cerrado. Revista De Agricultura Neotropical 4:21–27 [Google Scholar]
- Rios DA da Silva (2021) Extratos Vegetais Fermentados Por Kefir: desenvolvimento, caracterização e potencial antimicrobiano. Universidade Federal do Ceará
- Ruviaro AR, Barbosa P, de Alexandre PM EC, et al (2020) Aglycone-rich extracts from citrus by-products induced endothelium-independent relaxation in isolated arteries. Biocatal Agric Biotechnol 23:101481. 10.1016/j.bcab.2019.101481 [Google Scholar]
- TACO (2011) Tabela Brasileira Da composição de alimentos, 4th edn. Universidade Estadual de Campinas – UNICAMP, Campinas [Google Scholar]
- Tang X, Yu R, Zhou Q et al (2018) Protective effects of γ-aminobutyric acid against H2O2-induced oxidative stress in RIN-m5F pancreatic cells. Nutr Metab (Lond) 15:60. 10.1186/s12986-018-0299-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uruc K, Tekin A, Sahingil D, Hayaloglu AA (2022) An alternative plant-based fermented milk with kefir culture using apricot (Prunus armeniaca L.) seed extract: changes in texture, volatiles and bioactivity during storage. Innovative Food Sci Emerg Technol 82:103189. 10.1016/j.ifset.2022.103189 [Google Scholar]
- Yogeswara IBA, Maneerat S, Haltrich D (2020) Glutamate decarboxylase from lactic acid bacteria—a key enzyme in GABA synthesis. Microorganisms 8:1923. 10.3390/microorganisms8121923 [DOI] [PMC free article] [PubMed]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data generated or analyzed during this study are included in this published article.