Bioprocess based on probiotic bacteria for the simultaneous production of acetate, propionate and butyrate short-chain fatty acids for the production of food products and neutraceuticals
A bioprocess combining Levilactobacillus brevis KEK-4A and Lactobacillus paracasei KEF-5A2 bacteria in a two-step fermentation with MRSMS medium and sodium acetate optimizes SCFA production, addressing inefficiencies in current methods and providing a high-concentration, safe dietary supplement.
Patent Information
- Application Number
- PCT/CL2024/050091
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-08-12
- Publication Date
- 2026-02-19
AI Technical Summary
Current methods for producing short-chain fatty acids (SCFAs) are inefficient, requiring separate production stages and purification processes, and their concentration in probiotic foods is low, limiting their health benefits and industrial applications.
A bioprocess using a specific combination of Levilactobacillus brevis KEK-4A and Lactobacillus paracasei KEF-5A2 bacteria, optimized in a two-step fermentation process with MRSMS medium and sodium acetate, to produce acetate, propionate, and butyrate simultaneously, followed by drying with maltodextrin to create a powdered supplement.
The process significantly enhances SCFA production, achieving high concentrations suitable for dietary supplements and nutraceuticals, with demonstrated safety for human consumption and potential health benefits.
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Abstract
Description
[0001] Bioprocess based on probiotic bacteria for the simultaneous production of short-chain fatty acids of the acetate, propionate and butyrate type, for the production of food and nutraceutical products
[0002] DESCRIPTIVE MEMORANDUM
[0003] Background of the invention
[0004] Non-communicable chronic diseases (NCDs) are the leading cause of death worldwide, resulting in healthcare costs of US$11.2 billion. Among the main risk factors for developing these diseases is diet, where an unbalanced diet high in saturated fats, sodium, and refined sugars, and low in dietary fiber, contributes to the pathophysiology of NCDs. Likewise, one of the main strategies for their prevention is the implementation of a balanced and healthy diet. In this context, functional foods and dietary supplements have emerged in recent decades as an alternative for NCD prevention.Among the compounds of interest for health are flavonoids, catechins, anthocyanins, lycopene, and short-chain fatty acids (SCFAs). The latter are naturally produced by the microorganisms of the gut microbiota and probiotic microorganisms. While these compounds are normally produced by the gut microbiota, the dysbiosis associated with various non-communicable diseases (NCDs) causes an alteration in SCFA synthesis, contributing to their pathophysiology.
[0005] Short-chain fatty acids (SCFAs) comprise three main representatives: acetic acid, propionic acid, and butyric acid. As previously mentioned, they are naturally produced by the metabolism of bacteria that are part of the de novo, redundant, or recycling metabolism of bacteria that make up the gut microbiota. SCFAs regulate physiological and metabolic processes such as blood pressure, intestinal permeability, cholesterol, immune system regulation, and glucose metabolism, among others. In this sense, an imbalance of SCFAs is associated with the development of chronic diseases such as diabetes, dyslipidemia, and hypertension. In addition to being produced by the gut microbiota, SCFAs are present in probiotic foods; however, their concentration in these foods is low, fluctuating between 0.57 and 6.49 pg / L. This necessitates the consumption of large quantities of these foods to reach the desired levels.Additionally, the acidic characteristics of these compounds limit their consumption due to the flavor they impart to foods containing them. Therefore, the development of functional ingredients based on short-chain fatty acids (SCFAs), which allow for their incorporation into the diet, is of particular interest for contributing to a balanced and healthy diet and, consequently, to the prevention of chronic non-communicable diseases.
[0006] Additionally, short-chain fatty acids such as acetic acid, propionic acid, and butyric acid are used in various industries, including cosmetics, pharmaceuticals, animal feed, and human food. In the case of human food, these compounds are primarily used as food additives and flavorings. Currently, these compounds are produced through both chemical synthesis and bioprocesses; however, the demand for new, environmentally friendly production processes using renewable resources necessitates an increase in the bioprocesses available for their production. In this context, this proposal aims to develop a dietary supplement based on short-chain fatty acids of microbial origin and a bioprocess using a group of microorganisms for its production.
[0007] PREVIOUS ART
[0008] The benefits of short-chain fatty acids for gut health and overall health are well-established. This is demonstrated, for example, in the review by Xiong, Ruo-Gu, et al. ("Health benefits and side effects of short-chain fatty acids." Foods 11.18 (2022): 2863). Currently, the use of probiotics and fiber is recommended to increase the production of these compounds in our own intestines. Additionally, their presence or production by probiotic microorganisms and their production in bacterial fermentations have been studied; see, for example, the previous publication by the inventors Ibacache-Quiroga, C., González-Pizarro, K., Charifeh, M., Canales, C., Díaz-Viciedo, R., Schmachtenberg, O., & Dinamarca, M.A. (2022). Metagenomic and functional characterization of two Chilean kefir beverages reveals a dairy beverage containing active enzymes, short-chain fatty acids, microbial 0-amyloids, and bio-film inhibitors. Foods, 11(7), 900.
[0009] Furthermore, due to their diverse applications, the current production of short-chain fatty acids (SCFAs) from microorganisms is carried out independently for each required SCFA. This involves not only a production stage but also subsequent purification. In this regard, the present initiative proposes the development of a unique bioprocess for the combined production of short-chain fatty acids based on a fermentation process using bacteria isolated from probiotic foods.
[0010] The use of bacteria isolated from probiotic foods will allow for the implementation of a bioprocess that yields short-chain fatty acids suitable for human consumption. Additionally, during microbial metabolism, the microorganisms produce other molecules of great interest to human health, such as B vitamins and vitamin D precursors, among others, giving the product of the invention an advantage.
[0011] DESCRIPTION OF THE FIGURES
[0012] Figure 1. Graph showing the concentration of short-chain fatty acids of the acetate, propionate and butyrate type in MRSMS medium with and without pre-culture acetate (sterile) and in the supernatant after the production process of the invention.
[0013] Figure 2. Graph showing the toxicity of the dry product of the invention in human fibroblast cultures. The variation in cell viability was evaluated when the culture medium was supplemented with different amounts of the food supplement of the invention.
[0014] Human fibroblasts were grown in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Different concentrations of the food supplement of the invention were added (31.25, 15.625, 7.1825, and 3.90625 mg of supplement per mL of culture medium). The results are shown after 48 h of incubation at 37°C in a humidified atmosphere with 5% CO2.
[0015] DESCRIPTION OF THE INVENTION
[0016] The invention relates to a method based on probiotic bacteria for the simultaneous production of short-chain fatty acids of the acetate, propionate, and butyrate type, for the production of cell-free food ingredients, supplements, and nutraceuticals.
[0017] Specifically, the invention relates to a probiotic bacteria composition for the simultaneous production of short-chain fatty acids, acetate, propionate, and butyrate, comprising the Levilactobacillus brevis strain KEK-4A (Bacteria A, Deposit RGM 3602) and the Lactobacillus paracasei strain KEF-5A2 (Bacteria B, Deposit RGM 3603) in a ratio of between 100:1 and 10:1 and a carrier.
[0018] Where the inventors have developed a specific method employing this composition for the optimized and simultaneous production of short-chain fatty acids, acetate, propionate, and butyrate, comprising the following steps: a) obtaining a culture of Levilactobacillus brevis KEK-4A (Bacteria A, Deposit RGM 3602) and separately a culture of Lactobacillus paracasei KEF-5A2 (Bacteria B, Deposit RGM 3603); b) cultivating Levilactobacillus brevis KEK-4A (Bacteria A, Deposit RGM 3602) in a culture medium for lactic acid bacteria supplemented with between 0.1 and 2% sodium acetate, at a temperature between 36-38°C for 24 to 72 h, with agitation; c) incorporate Lactobacillus paracasei KEF-5A2 (Bacteria B, RGM Depot 3603) into the previous culture from point b) in a proportion between 1:10 and 1:100 and incubate for 12-96 h with shaking; d) separate the cell-free supernatant, which contains the short-chain fatty acids acetate, propionate and butyrate.
[0019] The supernatant obtained is conveniently mixed with a thickener such as maltodextrin and subjected to drying to obtain the product of the invention, a powdered food / nutraceutical composition with a high content of SCFAs, which can be consumed directly with food or in capsules, to improve the microbiota and health of the people who consume them.
[0020] To develop the invention process, the applicants evaluated various probiotic strains, selecting those with the best production rates of these metabolites. Simultaneously, they reviewed various culture media for lactic acid bacteria, finding that, surprisingly, one particular culture medium yielded the best results: the MRSMS medium, which uses sucrose as the carbon source sugar.
[0021] To further improve SCFA production, the inventors noticed that incorporating sodium acetate into the medium also had a promoting effect on this production, both in the production of acetate itself and in the production of propionate and butyrate. Based on this background, the inventors developed the SCFA production method of the invention, which requires selected strains, in a specific ratio of between 100:1 and 10:1, of Levilactobacillus brevis KEK-4A (Bacteria A, RGM Depot 3602) and Lactobacillus paracasei KEF-5A2 (Bacteria B, RGM Depot 3603). In the first stage, each strain is cultivated separately, and then they are mixed to continue a combined culture in the second stage.
[0022] Once the combined culture or fermentation is complete, the supernatant, which contains the produced SCFAs, is separated. Conveniently, this supernatant is combined with a stabilizing thickener, such as maltodextrin, and subjected to drying to obtain the product of the invention: a dry powder rich in SCFAs, which can be consumed directly, for example, mixed with foods such as milk, yogurt, or juices, or can be consumed as a capsule.
[0023] In a specific embodiment, in the method of the invention the proportion in which both bacteria are mixed is determined by quantifying the optical density of the microbial cell suspensions at a wavelength of 600 nm, so as to establish the relative concentrations of the bacteria in the medium.
[0024] The preferred culture medium of the invention is MRSMS, where the sugar is sucrose, the exact composition of the medium is
[0025] Peptone 2%
[0026] Yeast extract (0.6%)
[0027] Tween 80 (0.1%)
[0028] Sodium acetate (0.1-1%)
[0029] Magnesium sulfate (0.01%)
[0030] Manganese sulfate (0.005%)
[0031] Dipotassium phosphate (0.2%)
[0032] Sucrose (0.5-5%) In a preferred embodiment, the method for the simultaneous production of short-chain fatty acids of the acetate, propionate, and butyrate type comprises the following steps: a) obtaining a culture of Levilactobacillus brevis KEK-4A (Bacteria A, RGM Deposit 3602); b) obtaining a culture of Lactobacillus paracasei KEF-5A2 (Bacteria B, RGM Deposit 3603); c) cultivating the bacteria obtained from step a), in a proportion between 0.1-10% v / v, between 12 and 96 h at a temperature between 30°C and 37°C, with agitation between 5-300 rpm; d) incorporating the culture from point b) into the previous culture in a proportion between 0.1-10% v / v, thus obtaining the composition of the invention, and incubating for 12-96 h with agitation between 5-300 rpm; e) recover the supernatant from point d). Preferably the separation is carried out by centrifugation, at between 3000 and 10000 rpm for between 10 and 60 minutes at a temperature between 4°C and 20°C.
[0033] It should be noted that in this case the inoculum ratio used in stages C and D of the process can be expressed in either v / v or w / v / p, as they are very similar. It is convenient to work with volume.
[0034] In the method of the invention, the ratio of the bacteria Levilactobacillus brevis KEK-4A (Bacteria A, Deposit RGM 3602) and Lactobacillus paracasei KEF-5A2 in step c) (Bacteria B, Deposit RGM 3603) in step d) is between 100:1 and 10:1.
[0035] The culture medium used is a lactic acid bacteria medium containing (0.1-1%) sodium acetate and (0.5-5%) sucrose. Preferably, the culture medium is MRSMS, the composition of which was described previously.
[0036] The method of the invention may further comprise the following steps: f) adding to the supernatant obtained in step e) a food-grade thickener at a concentration of 1-15%. Preferably the thickener is maltodextrin. g) drying this composition. Preferably, the drying is carried out by spray drying at a temperature between 160-180°C. Q C and a flow rate of between 1-10 mL / min
[0037] This process yields a powdered food supplement containing short-chain fatty acids of the acetate, propionate, and butyrate types. Specifically, for the two-step method of the invention, a culture of each of the strains, Levilactobacillus brevis KEK-4A (Bacteria A, RGM Deposit 3602) and Lactobacillus paracasei KEF-5A2 (Bacteria B, RGM Deposit 3603), is first obtained. In these primary cultures, a concentration of between 10 7 10 9CFU / mL for each strain. From this first culture, an aliquot of strain RGM 3602 is taken and diluted to 5% in fresh MRSMS medium, for example, 5 mL of inoculum in a final 100 mL of culture medium. This strain is cultured for 48 h, reaching a concentration between 10 and 100 times its initial concentration. Subsequently, 5 mL of culture of strain RGM3603 are added to this culture, and this mixture is cultured for an additional 48 h. After this period, the supernatant containing the SCFAs is separated. The method of the invention may be better understood through the illustrative examples included below.
[0038] EXAMPLES
[0039] Example 1. Obtaining the strains
[0040] The strains presented in the present invention were obtained from probiotic dairy ferments belonging to the Micro-Bioinnovation Center (University of Valparaíso, Valparaíso, Chile).
[0041] The strains of the invention were isolated in selective media for lactic acid bacteria (LAB), specifically, the samples were plated on MRS agar and incubated under anaerobic conditions at 37 Q C.
[0042] Five strains that grew well under these conditions were analyzed. The isolated microorganisms were characterized using molecular and microbiological techniques, including 16S rRNA amplification and sequencing, biochemical characterization using the API20 NE system, and the production of acetate, butyrate, and propionate. The production of short-chain fatty acids was evaluated by high-performance liquid chromatography (HPLC), a factor that allowed for the selection of the strains for the invention.
[0043] The inventors evaluated the production of short-chain fatty acids for each of these 5 strains in 4 different culture media for BAL.
[0044] The results are shown in Table 1 below, and the culture media used are described in Table 2. Table 1
[0045] The results show that of the 5 strains evaluated, only one was able to produce butyrate, and surprisingly, the MRSMS medium consistently showed the best results. Table 2. Composition of the culture media evaluated in examples 1 and 2.
[0046] From these results, the inventors determined that the best strains for the intended purpose were strains KEK-4A and KEF-5A2. These were molecularly characterized as Levilactobacillus brevis KEK-4A (Bacteria A, Deposit RGM 3602) and Lactobacillus paracasei KEF-5A2 (Bacteria B, Deposit RGM 3603). Both strains were deposited in the Chilean Collection of Microbial Genetic Resources (Av. Vicente Méndez 515, Chillán, Chile) on July 4, 2024, where they received registration numbers RGM 3602 for the bacterium Levilactobacillus brevis KEK-4A and RGM 3603 for the bacterium Lactobacillus paracasei KEF-5A2. Example 2. Culture conditions
[0047] The inventors verified the importance of supplementing the culture medium with acetate through the following experiment. MRSMS culture medium and acetate-free MRSMS were prepared as shown in Table 2.
[0048] Table 3 shows the production of SCFAs using a production process based on the two selected microorganisms (Levilactobacillus brevis KEK-4A and Lactobacillus paracasei KEF-5A2), in MRSMS media with and without acetate. For this, the KEK-4A strain was inoculated in a 1:20 ratio in each of the culture media and incubated for 48 h. Q C and 300 rpm. At 48 h, this culture was inoculated with KEF-5A2 of the Lactobacillus paracasei strain and incubated at 37°C and 300 RPM for 48 h. The concentration of SCFAs was evaluated in the culture medium before and after production.
[0049] The results show that incorporating acetate into the culture medium leads to a significant increase in SCFA production, notably inducing the production of propionate and butyrate, which are not present in the unsupplemented medium. This demonstrates that the initial presence of acetate stimulates SCFA production, including the production of acetate itself.
[0050] Table 3. Comparison of SCFA production in culture medium supplemented or not with acetate. Example 3. Process Development
[0051] Since the best strains and the best culture medium had been determined, the inventors incubated combinations of the best strains selected in the previous stage using the traditional method. In addition to strains KEK-4A and KEF-5A2, they also included the third-best performing strain from Example 1, strain KEK-3. The results of these experiments are shown in Table 4 below.
[0052] The inventors observed that the combined one-step cultures produced an amount of between 0.03 and 0.05 mg of SCFAs per milligram of food supplement, corresponding to the dry supernatant of the culture medium and maltodextrin, as explained below.
[0053] Table 4. One-step bioprocess.
[0054] The results obtained were lower than expected based on the individual cultures of each strain separately, so the inventors designed a new process to improve SCFA production. Different conditions were tested, starting with an inoculum and then supplementing with a second culture in the same flask, always using MRSMS culture medium. The results are shown in Table 5. Table 5. Two-step bioprocess.
[0055] Surprisingly, although the KEK-4A bacterium was included under all conditions, butyrate was not obtained in the supernatant of the process under all of them. Without wishing to be limited by theory, we believe there is a metabolic pathway for butyrate production that, surprisingly, is only achieved with the strains and sequences of the method of the invention. Thus, the inventors determined that the best condition is to start a culture of the KEK-4A strain and then supplement this culture with KEF-5A2 in a ratio of between 10:1 and 100:1. This concentration is achieved by the concentration of KEF-5A2 reached in the culture after 48 h of incubation, which is estimated to correspond to 10 to 100 times the initial inoculum.
[0056] Specifically, to carry out this 2-step method, the inventors first obtained a culture of each of the strains, where a concentration of between 10 7 10 9CFU / mL. From this first culture, the inventors took an aliquot and diluted it to 5% in fresh MRSMS medium, specifically 5 mL of inoculum in a final 100 mL of culture medium. This was cultured for 48 h. Subsequently, 5 mL of the second bacterial culture were added to this culture, and this mixture was cultured for an additional 48 hours.
[0057] In all the above cases, the SCFA concentration was evaluated in the food supplement generated from this production process, which is the final product of the method of the invention. To obtain the powdered food supplement, the supernatant was dried by spray drying under the following conditions (Table 6).
[0058] Table 6. Spray drying conditions for obtaining food supplement
[0059] Example 4. Process and product of the invention
[0060] Based on the results obtained in example 3, the inventors developed an optimized process that allows for the highest production of SCFAs, resulting in a nutraceutical product suitable as a food supplement. The developed example is described below.
[0061] Stage 1.
[0062] 1. Preinoculum A: The strain Levilactobacillus brevis KEK-4A (Bacteria A, Repository RGM 3602) was grown in MRSMS medium at 37 Q C and 250 rpm for 24 h until reaching an optical density (OD) 600 nm >1.
[0063] 2. Starting from step 1, the optical density of the culture was adjusted to an OD=1 with MRSMS. 3. The MRSMS culture medium, previously sterilized, was inoculated with 5% v / v of the Preinoculum A adjusted to an OD = 1 from the previous step.
[0064] 4. It was incubated at 37 Q C and 250 rpm, for 48 h
[0065] Stage 2
[0066] 1. Pre-inoculum B: The strain Lactobacillus paracasei KEF-5A2 (Bacteria B, Deposit RGM 3603) was grown in MRSMS medium at 37 Q C and 250 rpm for 48 h until reaching an optical density (OD) 600 nm >2. After incubation, the OD of the culture was adjusted to an OD=2 with MRSMS.
[0067] 2. The culture obtained from Stage 1 was inoculated with 5% v / v of the Pre-inoculum B from the previous step.
[0068] 3. It was incubated at 37 Q C and 250 rpm, for an additional 48 hours.
[0069] 4. After the incubation period, it was centrifuged at 5714 g for 30 min, at room temperature
[0070] 5. And the supernatant containing the FATCs was recovered
[0071] Stage 3
[0072] Obtaining a powdered product for food or nutraceutical use
[0073] 1. 5 g / L of maltodextrin was added to the cell-free supernatant obtained from stage 2 of the bioprocess
[0074] 2. The composition from the previous step was subjected to drying, using a spray dryer, at an inlet temperature of 180 °C, inlet flow at 10%, Qflow at 40-50 and aspiration at 100%.
[0075] This production process yielded 70 g of powdered product, or food / nutraceutical product of the invention, per liter of supernatant with a concentration of 4.7% w / w acetate, 2.8% w / w propionate, and 0.36% w / w butyrate in the dry product. Example 5. Toxicity test
[0076] Since the study involved probiotic bacteria, it is known that these bacteria and their products are not toxic to humans. However, for added safety, a toxicity study of the product obtained in the previous example was conducted on human fibroblast cultures. For this purpose, human fibroblast cultures, grown in DMEM medium (10% fetal bovine serum) supplemented with 1% penicillin / streptomycin antibiotics, were exposed to different concentrations of the dietary supplement obtained in Example 3 and incubated at 37°C for 48 h in a humidified atmosphere with 5% CO2, after which cell viability was assessed.
[0077] The results are shown in Figure 1, where it can be seen that at concentrations of 7.18 mg / mL of the supplement in the culture medium, or lower, no toxicity to fibroblasts is observed. This concentration is quite high, thus demonstrating that there is no risk of toxicity from consuming the supplement of the invention.
[0078] The estimated daily dose is 3.5 g of supplement / day (to achieve an intake of 275 mg of short chain fatty acids / day).
Claims
CLAIMS 1. Probiotic bacteria composition for the simultaneous production of short-chain fatty acids, acetate, propionate and butyrate, CHARACTERIZED in that it comprises Levilactobacillus brevis KEK-4A (Bacteria A, Deposit RGM 3602) and Lactobacillus paracasei KEF-5A2 (Bacteria B, Deposit RGM 3603) in a ratio of between 100:1 and 10:1 and a carrier.
2. A method for the simultaneous production of short-chain fatty acids, acetate, propionate, and butyrate, CHARACTERIZED in that it comprises the following steps: a) obtaining a culture of Levilactobacillus brevis KEK-4A (Bacteria A, RGM Depot 3602); b) obtaining a culture of Lactobacillus paracasei KEF-5A2 (Bacteria B, RGM Depot 3603); c) cultivating the bacteria from step a) in a proportion of 0.1-10% v / v for 12-96 hours at a temperature between 30°C and 37°C, with agitation between 5-300 rpm; d) incorporating the culture from point b) into the previous culture in a proportion of 0.1-10% v / v and incubating for 12-96 hours with agitation between 5-300 rpm; e) recovering the supernatant from step d).
3. Production method according to claim 2, CHARACTERIZED in that the ratio of the bacteria Levilactobacillus brevis KEK-4A (Bacteria A, Deposit RGM 3602) in step c) and Lactobacillus paracasei KEF-5A2 (Bacteria B, Deposit RGM 3603) in step d) is between 100:1 and 10:
1.
4. Production method according to claim 2, CHARACTERIZED in that the culture medium employed is a medium for lactic acid bacteria containing carbon and energy sources that induce fatty acid metabolism and biomass production: (0.1-1%) sodium acetate and (0.5-5%) sucrose.
5. Production method according to claim 4, CHARACTERIZED in that the culture medium has the following composition: Peptone 2% Yeast extract (0.6%) Tween 80 (0.1%) Sodium acetate (0.1-1%) Magnesium sulfate (0.01%) Manganese sulfate (0.005%) Dipotassium phosphate (0.2%) Sucrose (0.5-5%) 6. Production method according to claim 2, CHARACTERIZED in that in step e) the separation is carried out by centrifugation, at between 3000 and 10000 rpm for between 10 and 60 minutes between 4°C and 20°C.
7. Method according to claim 2, CHARACTERIZED in that it further comprises the following steps: f) adding to the supernatant obtained in step e) a food-grade thickener product in a concentration of 1-15%; g) drying this composition; and obtaining a food supplement containing short-chain fatty acids of the acetate, propionate, and butyrate type.
8. Production method according to claim 7, CHARACTERIZED in that the thickener is maltodextrin.
9. Production method according to claim 7, CHARACTERIZED in that the drying is carried out by spraying.
Citation Information
Patent Citations
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