Use of microbial cell lines to maximize organic acid production

Abstract

Described herein are methods, microbial cell lines, and media used in co-cultivation to enhance propionic acid production using optimized fermentation media, as well as methods for increasing propionic acid yields, for example, by co-cultivating Lactobacillus casei and highly acid-tolerant propionic acid bacteria.

Description

[Technical Field]

[0001] Claim of priority This application claims the benefits of U.S. Provisional Application No. 62 / 977087, filed on 14 February 2020. The entire foregoing is incorporated herein. Technical field of inventions This disclosure relates to methods, microbial cell lines, and media generally used for co-culturing to enhance propionic acid production using optimized fermentation media, as well as methods for increasing propionic acid yield by co-culturing, for example, Lactobacillus casei and highly acid-resistant propionic acid bacteria (Acidipropionibacterium Acidipropionici). [Background technology]

[0002] Organic acids are carbon-containing compounds that possess acidic properties. Examples of organic acids include, among others, acetic acid, citric acid, gluconic acid, lactic acid, and propionic acid. Because they are completely biodegradable, organic acids can be used in the production of biodegradable polymers. They also have other important industrial applications, primarily as preservatives and as food and feed additives.

[0003] Propionic acid (PA) has considerable commercial value and is a carboxylic acid that can be produced by microbial fermentation of sugars from several carbon sources (see, e.g., Gonzalez-Garcia et al., 2017 and Ahmadi et al., 2017). Historically, propionic acid has been commercially produced using “petrochemical” processes because it was considered economically viable. This has changed as the cost of crude oil and petrochemicals has risen over the years, and consumers have begun to seek natural sources of propionic acid for use in food, resulting in increased interest in production methods that utilize microbial fermentation. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Gonzalez-Garcia et al., 2017 [Non-Patent Document 2] Ahmadi et al., 2017 [Overview of the Initiative]

[0005] Given concerns regarding the use of non-renewable base products and their environmental impacts, the microbial biosynthesis of propionic acid using renewable energy sources on a commercial scale is of great interest. This specification describes a method for producing propionic acid in high yield in a microbial fermentation medium containing glucose as a carbon source. These methods can be applied to the commercial production of propionic acid.

[0006] This disclosure provides microbial cell lines suitable for industrial-scale production of organic acids using co-culture, and a method for maximizing organic acid yield using an optimized culture medium. Therefore, what is provided herein is a method for producing organic acids. This method involves culturing (i) acid-resistant organic acid-emitting microorganisms and (ii) acid-resistant lactic acid-producing microorganisms in a medium with a pH of approximately 5.8 to 6.5 under controlled fermentation conditions, thereby yielding a higher yield of organic acids compared to a single microbial cell line used under the same conditions.

[0007] In some aspects, acid-tolerant organic acid-emitting microorganisms include those of the genera Propionibacterium, Anerovibrio, Bacteroides, Clostridium, Fusobacterium, and Megaspha. Genera include Ellaria, Propionispira, Selenomonas, and Veillonella. It is an object. In some aspects, organisms of the genus Propionibacterium are propionic acid bacteria (NFS 2018) On July 10, 2019, it was internationally deposited with the ATCC under the Budapest Convention. )

[0008] In some embodiments, lactic acid-producing microorganisms are cultured or spore-forming lactic acid-producing organisms. In some embodiments, non-spore-forming and acid-resistant lactic acid bacteria include the genera Lactococcus, Pediococcus, Oenococcus, Enterococcus, Leuconostoc, and Bifidobacterium. In some embodiments, spore-forming and acid-resistant lactic acid bacteria include organisms of the genera Bacillus, Lactobacillus, Clostridium, Paenibacillus, or Sporolactobacillus. In some embodiments, Lactobacillus organisms are Lactobacillus casei.

[0009] In some embodiments, the pH of the culture medium is approximately 6.0. In some embodiments, this method involves glucose, yeast extract, MgSO4, NaPO4, and vitamin B 12 The invention provides a starting medium containing KPO4, wherein the starting medium has a pH of approximately 6; an acid-tolerant organic acid-effluxing microorganism is added to the medium; a lactic acid-producing microorganism is added to the medium after 6 to 36 hours, for example after 12 to 24 hours; and the culture is maintained for a sufficient time to produce a desired amount of organic acid.

[0010] In some embodiments, lactic acid-producing microorganisms provide carbon for acid-tolerant organic acid-effluxing microorganisms. In some embodiments, acid-tolerant organic acid-effluxing microorganisms consume glucose from carbon sources derived from whey (a by-product of cheese production), grains (wheat, maize, or starch), or plants (legumes), and / or consume lactic acid produced by one or more cultured or spore-forming lactic acid-producing organisms.

[0011] In some embodiments, the organic acid includes propionic acid; acetic acid; lactic acid; and / or succinic acid. In some embodiments, controlled fermentation conditions during the fermentation process include aerobic conditions, anaerobic conditions, or both.

[0012] This specification also provides an organic acid production method comprising: providing an acid-tolerant organic acid-emitting microorganism; providing an acid-tolerant lactic acid-producing microorganism; providing a growth medium comprising a glucose carbon source and a lactose carbon source and having a pH of about 5.8 to about 6.5; adding the acid-tolerant organic acid-emitting microorganism and the acid-tolerant lactic acid-producing microorganism to the growth medium to produce a culture; and incubating the culture under controlled fermentation conditions to produce organic acids.

[0013] In some embodiments, acid-tolerant organic acid-excreting microorganisms are selected from the group consisting of the genera Propionibacterium, Anerovibrio, Bacteroides, Clostridium, Fusobacterium, Megasphaera, Propionispira, Selenomonas, Veillonella, and combinations thereof. In some embodiments, the Propionibacterium microorganism is propionic acid bacterium (NFS 2018).

[0014] In some embodiments, acid-tolerant organic acid-emitting microorganisms are provided as growth cultures. In some embodiments, acid-tolerant lactic acid-producing microorganisms are selected from the group consisting of the genera Lactococcus, Pediococcus, Oenococcus, Enterococcus, Leuconostoc, Bifidobacterium, Bacillus, Lactobacillus, Clostridium, Paenibacillus, Sporolactobacillus, and combinations thereof.

[0015] In some embodiments, acid-tolerant lactic acid-producing microorganisms are selected from the group consisting of the genera Lactococcus, Pediococcus, Oenococcus, Enterococcus, Leuconostoc, Bifidobacterium, and Bacillus.

[0016] In some embodiments, acid-resistant lactic acid-producing microorganisms are provided as growth cultures. In some embodiments, the acid-tolerant lactic acid-producing microorganism is selected from the group consisting of the genera Bacillus, Lactobacillus, Clostridium, Paenibacillus, Sporolactobacillus, and combinations thereof. In some embodiments, the acid-tolerant lactic acid-producing microorganism is Lactobacillus casei.

[0017] In some embodiments, the acid-tolerant lactic acid-producing microorganism is provided as spores, and optionally, the spores are provided as part of a carbon source. In some embodiments, the pH of the medium is about 6.0.

[0018] In some embodiments, the growth medium further comprises a nitrogen source, vitamins, MgSO4, NaPO4, and KPO4. In some embodiments, the glucose carbon source and the lactose carbon source are the same. In some embodiments, the glucose carbon source and the lactose carbon source are different.

[0019] In some embodiments, the glucose carbon source is a plant-based glucose carbon source. In some embodiments, the plant carbon source is selected from the group consisting of hydrolyzed wheat, hydrolyzed corn, hydrolyzed beans, hydrolyzed starch, and combinations thereof.

[0020] In some embodiments, the lactose carbon source is whey. In some embodiments, the nitrogen source is selected from yeast extract, peptides, ammonium sulfate, ammonium hydroxide, amino acids, and combinations thereof. In some embodiments, the nitrogen source is yeast extract.

[0021] In some embodiments, the vitamin is vitamin B 12 is. In some embodiments, the acid-tolerant lactic acid-producing microorganism is added to the medium 6 to 36 hours, optionally 12 to 24 hours after adding the acid-tolerant organic acid-excreting microorganism to the medium; and the culture is maintained for a time sufficient to produce a desired amount of organic acid.

[0022] In some embodiments, the organic acid is selected from the group consisting of propionic acid, acetic acid, lactic acid, succinic acid, and combinations thereof. The terms "glucose" and "dextrose" are used interchangeably herein.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains. Methods and materials are described herein for use in this invention, but other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to limit the scope of this invention. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are invoked in their entirety. In case of any inconsistency, this specification, including definitions, shall prevail.

[0024] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 shows the production of organic acids by propionic acid bacteria (NFS-2018) in a bioreactor using a fermentation sugar medium, with a 5% inoculum in a 30 L working volume, maintained at a temperature of 30°C, and pH 6 (NaOH as the neutralizing base). [Figure 2] Figure 2 shows the production of organic acids by Propionic acid bacteria (NFS-2018) and Lactobacillus casei in a bioreactor using a fermentation sugar medium, with a 5% inoculum in a 1 L working volume, maintained at a temperature of 30°C, and pH 6 (NaOH, neutralizing base). [Figure 3] Figure 3 shows the production of organic acids by Propionic acid bacteria (NFS-2018) and Lactobacillus casei in a bioreactor using a fermentation sugar medium, with a 5% inoculum in a 30 L working volume, maintained at a temperature of 30°C, and pH 6 (NaOH, neutralizing base). [Modes for carrying out the invention]

[0026] Propionic acid (PA) is used as an antifungal agent to control the growth of yeast and mold in food products, including bakery foods, dairy products, meat, poultry, and pet food. Propionic acid is also widely used as an intermediate in the production of various final chemical products used in herbicides, flavorings, and other products.

[0027] Propionic acid production is generally achieved by fermenting a carbon source and nitrogen source-containing medium with a suitable microbial cell line. Typically, organisms of the genus Propionibacterium are used to convert the carbon source in the medium into propionic acid and other metabolites. The growth rate of Propionibacterium cells in the fermentation medium is usually limited due to the inhibitory effect of propionic acid accumulation and the availability of nutrients over the fermentation time. Methods have been developed to improve propionic acid production by developing acid-tolerant Propionibacterium strains and co-culturing the microorganisms. However, production yields have remained limited due to nutrient imbalances for the microorganisms. This specification describes a method for increasing the propionic acid production yield by fed-batching a carbon source and adding Lactobacillus casei in optimized time steps (co-fermentation).

[0028] While other bacteria can also produce propionic acid, the genus Propionibacterium is the most commonly used and widely studied bacterium that excretes propionic acid (Gonzalez-Garcia et al., 2017). Advances in culture medium formulations, strain development, and optimization of fermentation parameters have resulted in propionic acid production yields of 25–50 g / L in broth (Gonzalez-Garcia et al., 2017; Stower et al., 2014; Wang et al., 2014; 2014; Liu et al., 2011; Kagliwal et al., 2013). Although there have been reports of propionic acid production reaching 100+ g / L during fermentation, these values ​​were achieved by extending fermentation for several weeks to several months and were not suitable for commercial scale-up (Zhang and Yang, 2009; Wang et al., 2014; and Jiang et al., 2015).

[0029] In addition to improvements using the strategies described above, efforts have also been made to increase the productivity of propionic acid by using mixed cultures. For example, the co-culture method has been used to produce PA using whey as the feedstock (WO85 / 04901;EP0141642 A1). WO85 / 04901 describes the conversion of lactic acid to propionic acid via a two-step fermentation process using Lactobacillus casei subspecies rhamnosus in the presence of Veillonella crisetii. In the first step, carbohydrates are converted to lactic acid by Lactobacillus casei; in the second step, the lactic acid is fermented to PA by Veillonella crisetii. (The genera Lactobacillus and Veillonella both belong to the phylum Firmicutes, while the genus Propionibacterium belongs to the phylum Actinobacteria.) EP0141642 also describes the use of a mixed culture of lactic acid-producing bacteria (Lactobacillus casei) and PA-producing bacteria (Propionibacterium shamaenii) to maximize fermentation yield. The co-culture systems in WO85 / 04901 and EP0141642 have been reported to be highly productive in terms of PA production from lactose, with final yields of 20-100 g / L. However, such co-culture systems are quite closely related to process parameters. For example, they have the problem of lacking control over the growth and metabolic activity of each organism in the system, which may result in failure of growth of one of the organisms or the inability of one of the organisms to contribute to the formation of the desired product. Lack of reproducibility is common in co-culture systems. A study by Border et al., 1987, showed that adding Lactobacillus strains to fermentation increased propionic acid production from 20 g / L to 30 g / L. Another process using mixed culture showed that propionic acid in the broth increased to 65 g / L when mixed culture was used, but this process was performed using whey only (EP0141642A1).

[0030] This disclosure describes a process for maximizing propionic acid production, using a simple culture medium and glucose from multiple carbon sources such as wheat flour and starch, and simultaneously producing other organic acids during the process. This process can be used for all propionic acid and lactic acid-producing bacteria, and fermentation can be carried out with or without the addition of nitrogen gas to make the medium anaerobic during mixed culture fermentation. As described herein, when propionic acid bacteria (e.g., NFS-2018) were fermented with glucose without the addition of lactic acid bacteria, the propionic acid yield within 168 hours was approximately 5.3%. When lactic acid bacteria were added to the fermenter, propionic acid production increased by 1.4% using a co-culture method, and the final propionic acid yield was 6.7%.

[0031] This specification describes a method using co-culture of microbial cell lines to increase organic acid production. In this method, an organic acid-producing microbial cell line (e.g., Acidipropionibacterium) is used for 24 hours in a pH-controlled medium supplemented with a carbon source, and another microbial cell line (e.g., Lactobacillus) is added to further enhance organic acid production. In some embodiments, the bioreactor is maintained at a temperature of 30°C throughout the fermentation period. In some embodiments, the pH is maintained at 6 under these culture conditions. In some embodiments, the microbial cell lines can be cultured for the same period in all fermenters and bioreactors. In some embodiments, the microbial cell lines are inoculated simultaneously into the fermenters and bioreactors.

[0032] In some embodiments, propionic acid can be produced by co-culturing Lactobacillus casei with the highly acid-resistant propionic acid bacterium NFS-2018. As shown herein, the propionic acid yield was improved from 50 g / L in monoculture to 65 g / L using the co-culture fermentation method described herein. Lactobacillus casei was added to the microbial fermentation in optimized time steps to maximize the propionic acid yield. In addition to propionic acid, the fermentation broth from the co-culture fermentation contained 7-8 g / L of acetic acid and 9-10 g / L of succinic acid as co-metabolites. Organic acid excreting microorganisms The method of the present invention involves the use of organic acid-producing bacteria, such as the genus Propionibacterium. The genus Propionibacterium produces PA (and vitamin B 12It is the most commonly used microorganism in the production of (and Swiss cheese). The genus Propionibacterium is a gram-positive, non-motile, non-spore-forming, rod-shaped, anaerobic bacterial genus that includes species such as Propionibacterium freudenleisii, P. acidifaciens, P. cyclohexanicum, P. australiense, Propionibacterium jensenii, P. thoenii, P. microaerophilum, P. olivae, P. damnosum, P. propionicum, Propionibacterium acnes, Propionibacterium avidum, Propionibacterium granulosum, P. humerusii, and P. lymphophilum. In industrial PA production, the most commonly used strain is Propionibacterium propionicum. (A proposal has been made to reclassify species within the genus Propionibacterium into three new genera: Acidipropionibacterium, Cutibacterium, and Pseudopropionibacterium (Scholz & Kilian, 2016)). However, Propionibacterium acidipropionici and Acidipropionibacterium Acidipropionici are still used somewhat interchangeably. The optimal pH and temperature for Propionibacterium cell proliferation are approximately 6.0–7.0°C and 30–37°C, respectively (Ahmadi et al., 2017). Fermenters started at neutral pH can reach pH 4.4, but cell proliferation is inhibited at pH below approximately 5.0 (Rehberger and Glatz, 1998). Ahmadi et al., as reported in the literature (Ahmadi et al., 2017), provide an overview of PA production by various species of the genus Propionibacterium on several carbon sources, which are incorporated herein by reference. PA can also be produced by other anaerobic bacteria, such as certain species of the genera Anerovibrio, Bacteroides, Clostridium, Fusobacterium, Megasphaera, Propionispira, Selenomonas, and Veillonella.

[0033] In a preferred embodiment, the organic acid-producing microorganism is acid-tolerant and can grow at a pH of, for example, 5–7.5, and produces propionic acid. In some embodiments, the acid-producing microorganism is a highly acid-tolerant propionic acid bacterium (NFS-2018) as described in WO2019 / 245985, deposited with the ATCC as PTA-125895. In some embodiments, the acid-producing microorganism is not Acidipropionibacterium shamaenii (ATCC 39393) (see EP0141642A1).

[0034] In some embodiments, the organic acid-producing microorganisms are wild-type. In some embodiments, the organic acid-producing microorganisms are manipulated. Lactic acid-producing microorganisms This method involves the use of lactic acid-producing microorganisms, such as lactic acid bacteria (LABs). In some embodiments, the microorganisms are derived from the genus Lactobacillus, such as Lactobacillus casei.

[0035] In some embodiments, lactic acid-producing microorganisms are selected from lactic acid bacteria (LAB), Bacillus species, Escherichia coli, Corynebacterium glutamicum, and combinations thereof. See, for example, Abdel-Rahman et al., “Recent Advances in Lactic Acid Production by Microbial Fermentation Processes,” Biotechnology Advances 31:877-902 (2013).

[0036] In some embodiments, LAB is homofermentable. In some embodiments, LAB is heterofermentable. See, for example, Eitman and Ramalingam, “Microbial Production of Lactic Acid,” Biotechnol. Lett. 37:955-72 (2015).

[0037] In some embodiments, the method involves the use of a mixture of LABs. In some embodiments, the mixture includes both homofermentable and heterofermentable LABs. In some embodiments, the lactic acid-producing microorganisms are wild-type. In some embodiments, the lactic acid-producing microorganisms are manipulated. Carbon source Several carbon sources, including glucose, fructose, maltose, sucrose, xylose, lactose, glycerol, lactate, wheat flour hydrolysate, molasses, whey, and combinations thereof, have been used for microbial PA production.

[0038] In some embodiments, hydrolyzed wheat flour is used as a carbon source. Methods for hydrolyzing wheat flour are known in the art and can include the use of enzymatic hydrolysis, for example, as described in Kagliwal et al., 2013. In some embodiments, enzymatic hydrolysis includes the use of bacterial α-amylase at 90°C / pH 6.0, followed by glucoamylase and protease at 60°C / pH 7.0.

[0039] In some embodiments, corn or other starches are used as the carbon source. In some embodiments, dairy carbon sources, such as whey from cheese production, are used. In some embodiments, a mixture of sources is used as a carbon source. In some embodiments, the mixture of carbon sources includes a glucose carbon source and a lactose carbon source.

[0040] In some embodiments, the glucose carbon source is derived from a plant carbon source, such as hydrolyzed cereals and / or legumes. In some embodiments, the lactose carbon source is derived from a dairy source, such as whey from cheese production. Nitrogen source Several nitrogen sources are used for PA production by microorganisms. In some embodiments, the nitrogen source is yeast extract. In some embodiments, the nitrogen source is selected from yeast extract, peptides, ammonium sulfate, ammonium hydroxide, amino acids, and combinations thereof. Culture medium and growth conditions Typical simple culture media used in this method include glucose, yeast extract, MgSO4, NaPO4, and vitamin B 12 The starting medium may contain KPO4 and has a pH of approximately 6.

[0041] In some embodiments, the culture medium has a pH of approximately 5.5 to approximately 6.9. In some embodiments, the culture medium is approximately 5.5 to 6.8, 5.5 to 6.7, 5.5 to 6.6, 5.5 to 6.5, 5.5 to 6.4, 5.5 to 6.3, 5.5 to 6.2, 5.5 to 6.1, 5.5 to 6.0, 5.5 to 5.9, 5.5 to 5.8, 5.5 to 5.7, 5.5 to 5.6, 5.6 to 6.9, 5.6 to 6.8, 5.6 to 6.7, 5.6 to 6.6, 5.6 to 6.5, 5.6 to 6.4, 5.6 to 6.3, 5.6 to 6.2, 5.6 to 6.1, and 5. 6~approx. 6.0, approx. 5.6~approx. 5.9, approx. 5.6~approx. 5.8, approx. 5.6~approx. 5.7, approx. 5.7~approx. 6.9, 5.7~approx. 6.8, approx. 5.7~approx. 6.7, approx. 5.7~approx. 6.6, approx. 5.7~approx. 6.5, approx. 5.7~approx. 6.4, approx. 5.7~approx. 6.3, approx. 5.7~approx. 6.2, approx. 5.7~approx. 6.1, approx. 5.7~approx. 6.0, approx. 5.7~approx. 5.9, approx. 5.7~approx. 5.8, approx. 5.8~approx. 6.9, 5.8~approx. 6.8, approx. 5.8~approx. 6.7, approx. 5.8~approx. 6.6, approx. 5.8~approx. 6.5, approx. 5.8~approx. 6.4, approx. 5.8~approx. 6.3, approx. 5.8~approx. 6.2, approx. 5.8 to approximately 6.1, approximately 5.8 to approximately 6.0, approximately 5.8 to approximately 5.9, approximately 5.9 to approximately 6.9, 5.9 to approximately 6.8, Approximately 5.9-6.7, approximately 5.9-6.6, approximately 5.9-6.5, approximately 5.9-6.4, approximately 5.9-6.3, approximately 5.9-6.2, approximately 5.9-6.1, approximately 5.9-6.0, approximately 6.0-6.9, 6.0-6.8, approximately 6.0-6.7, approximately 6.0-6.6, approximately 6.0-6.5, approximately 6.0-6.4, approximately 6.0-6.3, approximately 6.0-6.2, approximately 6.0-6.1, approximately 6.1-6.9, 6.1-6.8, approximately 6.1-6.7, approximately 6.1-6.6, approximately 6.1-6.5, approximately 6.1- Approximately 6.4, approximately 6.1-6.3, approximately 6.1-6.2, approximately 6.2-6.9, 6.2-6.8, approximately 6.2-6.7, approximately 6.2-6.6, approximately 6.2-6.5, approximately 6.2-6.4, approximately 6.2-6.3, approximately 6.3-6.9, 6.3-6.8, approximately 6.3-6.7, approximately 6.3-6.6, approximately 6.3-6.5, approximately 6.3-6.4, approximately 6.4-6.9, 6.4-6.8, approximately 6.4-6.7, approximately 6.4-6.6, approximately 6.4-6.5, approximately 6.5-6.9, 6.5-6.8, approximately 6.It has a pH of approximately 5 to 6.7, approximately 6.5 to 6.6, 6.6 to 6.9, 6.6 to 6.8, approximately 6.6 to 6.7, approximately 6.7 to 6.9, 6.7 to 6.8, or approximately 6.8 to 6.9.

[0042] In some embodiments, the culture medium contains about 1 to about 10% by weight / volume of a carbon source, such as glucose. In some embodiments, the culture medium is approximately 1 to approximately 10, approximately 1 to approximately 9.5, approximately 1 to approximately 9, approximately 1 to approximately 8.5, approximately 1 to approximately 8, approximately 1 to approximately 7.5, approximately 1 to approximately 7, approximately 1 to approximately 6.5, approximately 1 to approximately 6, approximately 1 to approximately 5.5, approximately 1 to approximately 5, approximately 1 to approximately 4.5, approximately 1 to approximately 4, approximately 1 to approximately 3.5, approximately 1 to approximately 3, approximately 1 to approximately 2.5, approximately 1 to approximately 2, approximately 1 to approximately 1.5, approximately 1.5 to approximately 10, approximately 1.5 to approximately 9.5, approximately 1.5 to approximately 9, approximately 1.5 to approximately 8.5, approximately 1.5 to approximately 8, approximately 1.5 to approximately 7.5, approximately 1.5 to approximately 7, approximately 1.5 to approximately 6.5, approximately 1.5 to approximately 6.0, approximately 1.5 to approximately 5.5, approximately 1.5~approx. 5, approx. 1.5~approx. 4.5, approx. 1.5~approx. 4, approx. 1.5~approx. 3.5, approx. 1.5~approx. 3, approx. 1.5~approx. 2, approx. 2~approx. 10, approx. 2~approx. 9.5, approx. 2~approx. 9, approx. 2~approx. 8.5, approx. 2~approx. 8, approx. 2~approx. 7.5, approx. 2~approx. 7, approx. 2~approx. 6.5, approx. 2~approx. 6, approx. 2~approx. 5.5, approx. 2~approx. 5, approx. 2~approx. 4.5, approx. 2~approx. 4, approx. 2~approx. 3.5, approx. 2~approx. 3, approx. 2~approx. 2.5, approx. 2.5~approx. 10, approx. 2.5~approx. 9.5, approx. 2.5~approx. 9, approx. 2.5~approx. 8.5, approx. 2.5~approx. 8, approx. 2.5~approx. 7.5, approx. 2.5~approx. 7, approximately 2.5~6.5, approximately 2.5~6, approximately 2.5~5.5, approximately 2.5~5, approximately 2.5~4.5, approximately 2.5~4, approximately 2.5~3.5, approximately 2.5~3, approximately 3~10, approximately 3~9.5, approximately 3~9, approximately 3~8.5, approximately 3~8, approximately 3~7.5, approximately 3~7, approximately 3~6.5, approximately 3~6, ​​approximately 3~5.5, approximately 3~5.0, approximately 3~4.5, approximately 3~4, approximately 3~3.5, approximately 3.5~10, approximately 3.5~9.5, approximately 3.5~9, approximately 2.5~8.5, approximately 3.5~8, approximately 3.5~7.5, approximately 3.5~ Approximately 7, approximately 3.5-6.5, approximately 3.5-6, approximately 3.5-5.5, approximately 3.5-5, approximately 3.5-4.5, approximately 3.5-4, approximately 4-10, approximately 4-9.5, approximately 4-9, approximately 4-8.5, approximately 4-8, approximately 4-7.5, approximately 4-7, approximately 4-6.5, approximately 4-6, approximately 4-5.5, approximately 4-5, approximately 4-4.5, approximately 4.5-10, approximately 4.5-9.5, approximately 4.5-9, approximately 4.5-8.5, approximately 4.5-8, approximately 4.5-7.5, approximately 4.5-7, approximately 4.5-6.5, approximately 4.5-6, approximately 4.5-5.5, approximately 4.5 to approximately 5, approximately 5 to approximately 10, approximately 5 to approximately 9.5, approximately 5 to approximately 9, approximately 5 to approximately 8.5, approximately 5 to approximately 8, approximately 5 to approximately 7.5, approximately 5 to approximately 7, approximately 5 to approximately 6.5, approximately 5 to approximately 6, approximately 5 to approximately 10, approximately 5 to approximately 9.5, approximately 5 to approximately 9, approximately 5 to approximately 8.5, approximately 5 to approximately 8, approximately 5 to approximately 7.5, approximately 5 to approximately 7, approximately 5 to approximately 6.5, approximately 5 to approximately 6, approximately 5 ~5.5, 5.5~10, 5.5~9.5, 5.5~9, 5.5~8.5, 5.5~8, 5.5~7.5, 5.5~7, 5.5~6.5, 5.5~6, 6~10, 6~9.5, 6~9, 6~8.5, 6~8, 6~7.5, 6~ Approximately 7, approximately 6-6.5, approximately 6.5-10, approximately 6.5-9.5, approximately 6.5-9, approximately 6.5-8.5, approximately 6.5-8, approximately 6.5-7.5, approximately 6.5-7, approximately 7-10, approximately 7-9.5, approximately 7-9, approximately 7-8.5, approximately 7-8, approximately 7-7.5, approximately 7.5-10, approximately 7.5-9 Includes a carbon source, such as glucose, in the following weight / volume percentages: 0.5, approximately 7.5-9, approximately 7.5-8.5, approximately 7.5-8, approximately 8-10, approximately 8-9.5, approximately 8-9, approximately 8-8.5, approximately 8.5-10, approximately 8.5-9.5, approximately 8.5-9, approximately 9-10, approximately 9-9.5, or approximately 9.5-10 wt / volume.

[0043] In some embodiments, the culture medium contains a nitrogen source, such as yeast extract, in an amount of about 1 to about 2.5% by weight / volume. In some embodiments, the culture medium is approximately 1 to 2.4, approximately 1 to 2.3, approximately 1 to 2.2, approximately 1 to 2.1, approximately 1 to 2, approximately 1 to 1.9, approximately 1 to 1.8, approximately 1 to 1.7, approximately 1 to 1.6, approximately 1 to 1.5, approximately 1 to 1.4, approximately 1 to 1.3, approximately 1 to 1.2, approximately 1 to 1.1, approximately 1.1 to 2.5, approximately 1.1 to 2.4, approximately 1.1 to 2.3, approximately 1.1 to 2.2, approximately 1.1 to 2.1, approximately 1.1 to 2, approximately 1.1 to 1.9, approximately 1.1 to 1.8, approximately 1.1 to 1.7, approximately 1.1 to 1.6, approximately 1.1 to 1.5, approximately 1.1 to 1 0.4, approximately 1.1-1.3, approximately 1.1-1.2, approximately 1.2-2.5, approximately 1.2-2.4, approximately 1.2-2.3, approximately 1.2-2.2, approximately 1.2-2, approximately 1.2-1.9, approximately 1.2-1.8, approximately 1.2-1.7, approximately 1.2-1.6, approximately 1.2-1.5, approximately 1.2-1.4, approximately 1.2-1.3, approximately 1.3-2.5, approximately 1.3-2.4, approximately 1.3-2.3, approximately 1.3-2.2, approximately 1.3-2.1, approximately 1.3-2, approximately 1.3-1.9, approximately 1.3-1.8, approximately 1.3-1.7, approximately 1.3-1.6, Approximately 1.3-1.5, approximately 1.3-1.4, approximately 1.4-2.5, approximately 1.4-2.4, approximately 1.4-2.3, approximately 1.4-2.2, approximately 1.4-2.1, approximately 1.4-2, approximately 1.4-1.9, approximately 1.4-1.8, approximately 1.4-1.7, approximately 1.4-1.6, approximately 1.4-1.5, approximately 1.5-2.5, approximately 1.5-2.4, approximately 1.5-2.3, approximately 1.5-2.2, approximately 1.5-2.1, approximately 1.5-2, approximately 1.5-1.9, approximately 1.5-1.8, approximately 1.5-1.7, approximately 1.5-1.6, approximately 1.6-2.5, approximately 1. 6~approx. 2.4, approx. 1.6~approx. 2.3, approx. 1.6~approx. 2.2, approx. 1.6~approx. 2.1, approx. 1.6~approx. 2, approx. 1.6~approx. 1.9, approx. 1.6~approx. 1.8, approx. 1.6~approx. 1.7, approx. 1.7~approx. 2.5, approx. 1.7~approx. 2.4, approx. 1.7~approx. 2.3, approx. 1.7~approx. 2.2, approx. 1.7~approx. 2.1, approx. 1.7~approx. 2, approx. 1.7~approx. 1.9, approx. 1.7~approx. 1.8, approx. 1.8~approx. 2.5, approx. 1.8~approx. 2.4, approx. 1.8~approx. 2.3, approx. 1.8~approx. 2.2, approx. 1.8~approx. 2.1, approx. 1.8~approx. 2, approx. 1.8~approx. 1.9, approx. 1.9~approx. 2.5, approx. 1.9~approx. 2.4. The medium contains a nitrogen source, e.g., yeast extract, in an amount of approximately 1.9–2.3, 1.9–2.2, 1.9–2.1, 1.9–2, 2–2.5, 2–2.4, 2–2.3, 2–2.2, 2–2.1, 2.1–2.5, 2.1–2.4, 2.1–2.3, 2.1–2.2, 2.2–2.5, 2.2–2.4, 2.2–2.3, 2.3–2.5, 2.3–2.4, or approximately 2.4–2.5% by weight / volume. In some embodiments, the medium contains a nitrogen source, e.g., yeast extract, in an amount of approximately 2% by weight / volume.

[0044] In some embodiments, the medium contains about 0.2 to about 0.8 g / L of MgSO4. In some embodiments, the medium contains about 0.2 to about 0.7, about 0.2 to about 0.6, about 0.2 to about 0.5, about 0.2 to about 0.4, about 0.2 to about 0.3, about 0.3 to about 0.8, about 0.3 to about 0.7, about 0.3 to about 0.6, about 0.3 to about 0.5, about 0.3 to about 0.4, about 0.4 to about 0.8, about 0.4 to about 0.7, about 0.4 to about 0.6, about 0.4 to about 0.5, about 0.5 to about 0.8, about 0.5 to about 0.7, about 0.5 to about 0.6, about 0.6 to about 0.8, about 0.6 to about 0.7, or about 0.7 to about 0.8 g / L of MgSO4. In some embodiments, the culture medium contains approximately 0.4 g / L of MgSO4.

[0045] In some embodiments, the culture medium contains about 0.5 to about 3.0 g / L of NaPO4. In some embodiments, the culture medium contains about 0.5 to about 2.5, about 0.5 to about 2, about 0.5 to about 1.5, about 0.5 to about 1, about 1 to about 3, about 1 to about 2.5, about 1 to about 2, about 1 to about 1.5, about 1.5 to about 3, about 1.5 to about 2.5, about 1.5 to about 2, about 2 to about 2.5, or about 2.5 to about 3 g / L of NaPO4. In some embodiments, the culture medium contains about 1 g / L of NaPO4.

[0046] In some embodiments, the culture medium contains a vitamin source of approximately 0.0015 to 0.004 g / L, such as vitamin B12. 12The medium contains a vitamin source in the range of approximately 0.0020 to 0.0040 g / L, such as vitamin B12. 12 Includes.

[0047] In some embodiments, the culture medium contains about 0.5 to about 3.0 g / L of KPO4. In some embodiments, the culture medium contains about 0.5 to about 2.5, about 0.5 to about 2, about 0.5 to about 1.5, about 0.5 to about 1, about 1 to about 3, about 1 to about 2.5, about 1 to about 2, about 1 to about 1.5, about 1.5 to about 3, about 1.5 to about 2.5, about 1.5 to about 2, about 2 to about 2.5, or about 2.5 to about 3 g / L of KPO4. In some embodiments, the culture medium contains about 1 g / L of KPO4.

[0048] Methods for preparing culture media are known in the art and are provided herein. In some embodiments, the culture medium for organisms does not contain one or more, for example all, of the following: dipotassium hydrogen phosphate; potassium dihydrogen phosphate; ferrous sulfate; 5,6-diaminobenzimidazole; cobalt chloride; manganese sulfate; enzymes; proteases; or calcium carbonate.

[0049] In some embodiments, the culture medium is inoculated with a culture of organic acid-producing microorganisms, such as propionic acid-producing microorganisms, such as propionic acid bacteria, such as propionic acid bacteria (NFS-2018), in an amount of about 1 to about 10 volumes / volt%. In some embodiments, the culture medium is inoculated with about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 5, about 2 to about 4, about 2 to about 4, about 2 to about 3, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, Inoculate cultures of organic acid-producing microorganisms, such as propionic acid-producing microorganisms, such as propionic acid bacteria, such as propionic acid bacteria (NFS-2018), in amounts of approximately 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 volumes / vol.

[0050] In some embodiments, cultures of organic acid-producing microorganisms, such as propionic acid-producing microorganisms, such as propionic acid bacteria, such as propionic acid bacteria (NFS-2018), contain cells with an optical density of approximately 5 to approximately 7 OD / mL. In some embodiments, cultures of organic acid-producing microorganisms, such as propionic acid-producing microorganisms, such as propionic acid bacteria, such as propionic acid bacteria (NFS-2018), contain cells with an optical density of approximately 5 to approximately 6.8, approximately 5 to approximately 6.6, approximately 5 to approximately 6.4, approximately 5 to approximately 6.2, approximately 5 to approximately 6, approximately 5 to approximately 5.8, approximately 5 to approximately 5.6, approximately 5 to approximately 5.4, approximately 5 to approximately 5.2, approximately 5.2 to approximately 7, and approximately 5.2 to approximately 6 0.8, approximately 5.2~6.6, approximately 5.2~6.4, approximately 5.2~6.2, approximately 5.2~6, approximately 5.2~5.8, approximately 5.2~5.6, approximately 5.2~5.4, approximately 5.4~7, approximately 5.4~6.8, approximately 5.4~6.6, approximately 5.4~6.4, approximately 5.4~6.2, approximately 5.4~6, approximately 5.4~5.8, approximately 5.4~5.6, approximately 5.6~7, approximately 5 0.6~approx. 6.8, approx. 5.6~approx. 6.6, approx. 5.6~approx. 6.4, approx. 5.6~approx. 6.2, approx. 5.6~approx. 6, approx. 5.6~approx. 5.8, approx. 5.8~approx. 7, approx. 5.8~approx. 6.8, approx. 5.8~approx. 6.6, approx. 5.8~approx. 6.4, approx. 5.8~approx. 6, approx. 6~approx. 7, approx. 6~approx. 6.8, approx. 6~approx. 6.6, approx. 6~approx. 6.4, approx. 6~approx. 6.2, approx. 6.2~approx. 7 This includes organic acid-producing microorganisms with OD / mL (optical concentration) of approximately 6.2 to 6.8, 6.2 to 6.6, 6.2 to 6.4, 6.4 to 7, 6.4 to 6.8, 6.4 to 6.6, 6.6 to 7, 6.6 to 6.8, or 6.8 to 7, such as propionic acid-producing microorganisms, such as propionic acid bacteria, such as propionic acid bacteria (NFS-2018).

[0051] In some embodiments, the culture medium is inoculated with a culture of lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, at a volume of about 1 to about 10 / vol. In some embodiments, the culture medium is inoculated with about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 4 to about 10, about 4 Inoculate with lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, in amounts of approximately 9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or approximately 9-10 volumes / volt%.

[0052] In some embodiments, a culture of lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, contains lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, at an optical concentration of about 5 to about 7 OD / mL. In some embodiments, a culture of lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, contains about 5 to about 6.8, about 5 to about 6.6, about 5 to about 6.4, about 5 to about 6.2, about 5 to about 6, about 5 to about 5.8, about 5 to about 5.6, about 5 to about 5.4, about 5 to about 5.2, about 5.2 to about 7, about 5.2 to about 6.8, about 5.2 to about 6.6, Approximately 5.2-6.4, approximately 5.2-6.2, approximately 5.2-6, approximately 5.2-5.8, approximately 5.2-5.6, approximately 5.2-5.4, approximately 5.4-7, approximately 5.4-6.8, approximately 5.4-6.6, approximately 5.4-6.4, approximately 5.4-6.2, approximately 5.4-6, approximately 5.4-5.8, approximately 5.4-5.6, approximately 5.6-7, approximately 5 0.6~approx. 6.8, approx. 5.6~approx. 6.6, approx. 5.6~approx. 6.4, approx. 5.6~approx. 6.2, approx. 5.6~approx. 6, approx. 5.6~approx. 5.8, approx. 5.8~approx. 7, approx. 5.8~approx. 6.8, approx. 5.8~approx. 6.6, approx. 5.8~approx. 6.4, approx. 5.8~approx. 6.2, approx. 5.8~approx. 6, approx. 6~approx. 7, approx. 6~approx. 6.8, approx. 6~approx. 6.6, approx. 6~approx. 6.4, approx. 6 This includes lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, with OD / mL (optical concentration) of approximately 6.2, 6.2-7, 6.2-6.8, 6.2-6.6, 6.2-6.4, 6.4-7, 6.4-6.8, 6.4-6.6, 6.6-7, 6.6-6.8, or 6.8-7.

[0053] In some embodiments, lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, are inoculated into the culture medium simultaneously with organic acid-producing microorganisms, such as propionic acid-producing microorganisms, such as propionic acid bacteria, such as propionic acid bacteria (NFS-2018). In some embodiments, lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, are inoculated into the culture medium after organic acid-producing microorganisms, such as propionic acid-producing microorganisms, such as propionic acid bacteria, such as propionic acid bacteria (NFS-2018). In some embodiments, lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, are inoculated into the culture medium approximately 6 to 36 hours after organic acid-producing microorganisms, such as propionic acid-producing microorganisms, such as propionic acid bacteria, such as propionic acid bacteria (NFS-2018).

[0054] In some embodiments, lactic acid-producing microorganisms, such as lactic acid bacteria, such as Lactobacillus casei, are inoculated into the culture medium about 6 to 36, 6 to 30, 6 to 24, 6 to 18, 6 to 12, 12 to 36, 12 to 30, 12 to 24, 12 to 18, 18 to 24, 24 to 36, 24 to 30, or 30 to 36 hours after inoculation of organic acid-producing microorganisms, such as propionic acid-producing microorganisms, such as propionic acid bacteria, such as propionic acid bacteria (NFS-2018).

[0055] In some embodiments, fermentation is carried out at approximately 30 to 37°C. In some embodiments, fermentation is carried out at approximately 30-36°C, 30-35°C, 30-34°C, 30-33°C, 30-32°C, 30-31°C, 31-37°C, 31-36°C, 31-35°C, 31-34°C, 31-33°C, 31-32°C, 32-37°C, 32-36°C, 32-35°C, 32-34°C, 32-33°C, 33-37°C, 33-37°C, 33-36°C, 33-35°C, 33-34°C, 34-37°C, 34-36°C, 34-35°C, 35-37°C, 35-36°C, or 36-37°C.

[0056] In some embodiments, fermentation is carried out in a volume of approximately 1 to 189,000 liters. In some aspects, fermentation occurs in approximately 1 to 180,000, approximately 1 to 170,000, approximately 1 to 160,000, approximately 1 to 150,000, approximately 1 to 140,000, approximately 1 to 130,000, approximately 1 to 120,000, approximately 1 to 110,000, approximately 1 to 100,000, approximately 1 to 90,000, approximately 1 to 80,000, approximately 1 to 70,000, approximately 1 to 60,000, approximately 1 to 50,000, approximately 1 to 40,000, approximately 1 to 30,000, approximately 1 to 20,000, approximately 1 to 10,000, approximately 10,000 to 189,000, approximately 10,000 to approximately 180,000, approximately 10,000 to approximately 170,000, approximately 10,000 to approximately 160,000, approximately 10,000 to approximately 150,000, approximately 10,000 to approximately 140,000, approximately 10,000 to approximately 130,000, approximately 10,000 to approximately 120,000, approximately 10,000 to approximately 110,000, approximately 10,000 to approximately 100,000, approximately 10,000 to approximately 90,000, approximately 10,000 to approximately 80,000, approximately 10,000 to approximately 70,000, approximately 10,000 to approximately 60,000, approximately 10,000 to approximately 50,000, approximately 10,000 ~40,000, 10,000~30,000, 10,000~20,000, 20,000~189,000, 20,000~180,000, 20,000~170,000, 20,000~160,000, 20,000~150,000, 20,000~140,000, 20,000~130,000, 20,000~120,000, 20,000~110,000, 20,000~100,000, 20,000~90,000, 20,000~80,000 00, approximately 20,000~approximately 70,000, approximately 20,000~approximately 60,000, approximately 20,000~approximately 50,000, approximately 20,000~approximately 40,000, approximately 20,000~approximately 30,000, approximately 30,000~approximately 189,000, approximately 30,000~approximately 180,000, approximately 30,000~approximately 170,000, approximately 30,000~approximately 160,000, approximately 30,000~approximately 150,000, approximately 30,000~approximately 140,000, approximately 30,000~approximately 130,000, approximately 30,000~approximately 120,000, approximately 30,000~approximately 110,000, approximately 30,000~approx. 100,000, approx. 30,000~approx. 90,000, approx. 30,000~approx. 80,000, approx. 30,000~approx. 70,000, approx. 30,000~approx. 60,000, approx. 30,000~approx. 50,000, approx. 30,000~approx. 40,000, approx. 40,000~approx. 189,000 0, approximately 40,000 to approximately 180,000, approximately 40,000 to approximately 170,000, approximately 40,000 to approximately 160,000, approximately 50,000 to approximately 160,000, approximately 50,000 to approximately 150,000, approximately 50,000 to approximately 140,000, approximately 50,000 to approximately 130,000, approximately 50, 000~approx. 120,000, approx. 50,000~approx. 110,000, approx. 50,000~approx. 100,000, approx. 50,000~approx. 90,000, approx. 50,000~approx. 80,000, approx. 50,000~approx. 70,000, approx. 50,000~approx. 60,000, approx. 60,000~approx. 189, 000, approximately 60,000-180,000, approximately 60,000-170,000, approximately 60,000-160,000, approximately 60,000-150,000, approximately 60,000-140,000, approximately 60,000-130,000, approximately 60,000-120,000, approximately 6 0,000 to approximately 110,000, approximately 60,000 to approximately 100,000, approximately 60,000 to approximately 90,000, approximately 60,000 to approximately 80,000, approximately 60,000 to approximately 70,000, approximately 70,000 to approximately 189,000, approximately 70,000 to approximately 180,000, approximately 70,000 to approximately 17 0,000, approximately 70,000-160,000, approximately 70,000-150,000, approximately 70,000-140,000, approximately 70,000-130,000, approximately 70,000-120,000, approximately 70,000-110,000, approximately 70,000-100,000 Approximately 70,000 to 90,000, approximately 70,000 to 80,000, approximately 80,000 to 189,000, approximately 80,000 to 180,000, approximately 80,000 to 170,000, approximately 80,000 to 160,000, approximately 80,000 to 150,000, approximately 80,000 0 to approximately 140,000, approximately 80,000 to approximately 130,000, approximately 80,000 to approximately 120,000, approximately 80,000 to approximately 110,000, approximately 80,000 to approximately 100,000, approximately 80,000 to approximately 90,000, approximately 90,000 to approximately 189,000, approximately 90,000 to approximately 180,000, approximately 90,000-170,000, approximately 90,000-160,000, approximately 90,000-150,000, approximately 90,000-140,000, approximately 90,000-130,000, approximately 90,000-120,000, approximately 90,000-110,000, approximately 90,000-100,000, approximately 100,000-189,000, approximately 100,000-180,000, approximately 100,000-170,000, approximately 100,000-160,000, approximately 100,000-150,000, approximately 100,000- 140,000, approximately 100,000-130,000, approximately 100,000-120,000, approximately 100,000-110,000, approximately 110,000-189,000, approximately 110,000-180,000, approximately 110,000-170,000, approximately 110,000-160,000, approximately 110,000-150,000, approximately 110,000-140,000, approximately 110,000-130,000, approximately 110,000-120,000, approximately 120,000-189,000, approximately 120,000-180,000 , approximately 120,000~approximately 170,000, approximately 120,000~approximately 160,000, approximately 120,000~approximately 150,000, approximately 120,000~approximately 140,000, approximately 120,000~approximately 130,000, approximately 130,000~approximately 189,000, approximately 130,000~approximately 180,000, approximately 130,000~approximately 170,000, approximately 130,000~approximately 160,000, approximately 130,000~approximately 150,000, approximately 130,000~approximately 140,000, approximately 140,000~approximately 189,000, approximately 140,000~approximately 180,000, approximately 140, The project will be implemented with capacities of approximately 170,000 to 140,000 to 160,000, 140,000 to 150,000, 150,000 to 189,000, 150,000 to 180,000, 150,000 to 170,000, 150,000 to 160,000, 160,000 to 189,000, 160,000 to 180,000, 160,000 to 170,000, 170,000 to 189,000, 170,000 to 180,000, or approximately 180,000 to 189,000 L.

[0057] The following table shows general parameters for culture media and cultures that can be used with this method.

[0058] [Table 1]

[0059] Fermentation approach Several different fermentation approaches are known in the art and can be used in the methods described herein.

[0060] In some embodiments, fermentation is batch, fed-batch, sequential batch, repeated batch, or continuous batch fermentation. In some embodiments, fermentation is carried out in a bioreactor. In some embodiments, the bioreactor is a PEI-Poraver bioreactor. In some embodiments, the bioreactor is a fiberbed bioreactor. In some embodiments, the fiberbed bioreactor is a multi-point fiberbed bioreactor. In some embodiments, the fiberbed bioreactor is a plant fiberbed bioreactor.

[0061] In some aspects, fermentation is immobilized cell fermentation. In some embodiments, fermentation involves adding additional culture medium and / or culture medium components during the reaction process.

[0062] In some embodiments, one or more of the following are added to the culture medium: a carbon source, a nitrogen source, a vitamin source, magnesium sulfate, or sodium diphosphate, as described herein. process The methods described herein may include the treatment and / or purification of organic acids produced during the fermentation described herein, such as propionic acid. In some embodiments, the fermentation is terminated and the organic acids are extracted from the culture. In some embodiments, a portion of the culture is removed for treatment before the fermentation is terminated. In some embodiments, the fermentation is terminated within the portion of the culture removed for treatment.

[0063] In some embodiments, fermentation is terminated by heat sterilization. In some embodiments, fermentation is terminated by separating the cells from the culture medium, for example, by filtration or other physical means. [Examples]

[0064] method The growth medium was sterilized at 121°C for 1 hour, then allowed to stand and cool to 30°C. After sterilization, Vitamin B 12 The following steps were taken: the headspace was replaced with nitrogen gas for 2 hours when using a 3L bioreactor, or nitrogen gas was sprayed for 48 hours when using a 30L fermenter. Propionic acid bacteria, which had been grown for 48 hours in advance, were inoculated into the fermentation sugar medium at hour 0. If used, lactic acid bacteria were added to the fermenter 12-24 hours later. After 168 hours of fermentation, the cells were heat-sterilized for 1 hour for further downstream processing. This process was also used for monoculture fermentation of NFS-2018 without the addition of lactic acid bacteria. Example 1. PA production by Propionic acid bacteria (NFS-2018) - 30L reaction Propionic acid bacteria (NFS-2018) were cultured from frozen glycerol stocks in M24 medium supplemented with 2% glucose under anaerobic conditions at 30°C. Cells were started in 10 mL of fresh M24 medium and then subcultured every 48 hours in 50 mL (2-3 OD / mL) to be used as seed cells in a 1 L bioreactor container.

[0065] Fermentation was carried out in a 42L fermenter with a working volume of 30L. To a fermenter containing 25L of ddH2O, the following were added while stirring: 1.8kg of dextrose (6%), 250g (1%) of yeast extract + 20g of MgSO4 (0.08%), 0.15g of MnSO4 (0.0015%), 75g of NaPO4 (dibasic: 0.3%), and 25g of KPO4 (dibasic: 0.1%). After complete mixing, the mixture was autoclaved at 121°C for 1 hour. The temperature was reduced to 30°C, and the pH was lowered to 6 using 15M NaOH. Vitamin B 12The solution was added to the fermenter at a final concentration of 2 mg / L. The fermenter was inoculated with 1.5 L (5 - 7 OD) of Propionic acid bacteria (NFS - 2018) that had been grown in advance for 48 hours. Samples were taken every 12 - 24 hours using a YSI analyzer and HPLC respectively for glucose and organic acid analysis. During the entire fermentation test period, 2 - 3% glucose was added as appropriate. After 168 hours, the fermentation was terminated by heat sterilization at 80 °C for 1 hour. The broth was collected for further downstream processing.

[0066] The results showed that when NFS-2018 was cultured alone in the fermenter, the maximum PA reached 53 g / L at 168 hours. See Table 1. Example 2. PA production of propionic acid bacteria (NFS-2018) using co-culture with Lactobacillus casei - 1L reaction Propionic acid bacteria (NFS-2018) and Lactobacillus casei were cultured from frozen glycerol stocks under anaerobic conditions at 30 °C in M24 medium supplemented with 2% glucose. Cells were started in 10 mL of fresh M24 medium and then subcultured at 50 mL (2 - 3 OD / mL) every 48 hours and used as seed for a 1 L bioreactor vessel.

[0067] Fermentation was carried out in a 3 L fermenter at a volume of 1 L. While stirring, the following were added to the fermenter containing 1 L of ddH2O: 70 g of dextrose, 10 g (1%) of yeast extract + 0.4 g of MgSO4 (0.08%), 3 g of NaPO4 (dibasic: 0.3%), and 1 g of KPO4 (dibasic: 0.1%). After thorough mixing, it was autoclaved at 121 °C for 1 hour. The temperature was lowered to 30 °C and the pH was lowered to 6 using 15 M NaOH. Vitamin B 12The solution was added to the fermenter at a final concentration of 2 mg / L. At hour 0, 1.5 L (5-7 OD) of propionic acid bacteria (NFS-2018) that had been grown for 48 hours prior was inoculated into the fermenter. At 24 hours of fermentation, 1.5 L (5-7 OD / mL) of Lactobacillus casei was added to the bioreactor. Samples were taken every 12-24 hours using a YSI analyzer and HPLC, respectively, for glucose and organic acid analysis. 2-3% glucose and a separate 1% yeast extract were added as appropriate throughout the fermentation period. After 168 hours, fermentation was terminated by heat sterilization at 80°C for 1 hour. The broth was collected for further downstream processing.

[0068] The results showed that when NFS-2018 and Lactobacillus casei were co-cultured in a bioreactor, the maximum PA reached 68 g / L in 168 hours. See Table 1. Example 3. PA production by Lactobacillus casei (NFS-2018) in a 30L reaction. Lactobacillus propionic acid bacteria (NFS-2018) and Lactobacillus casei were cultured from frozen glycerol stocks in M24 medium supplemented with 2% glucose under anaerobic conditions at 30°C. Cells were started in 10 mL of fresh M24 medium and then subcultured every 48 hours in 50 mL (2-3 OD / mL) to be used as seed for 1 L bioreactor containers.

[0069] Fermentation was carried out in a 42L fermenter with a volume of 30L. To a fermenter containing 25L of ddH2O, the following were added with stirring: 1.8kg of dextrose (6%), 250g (1%) of yeast extract + 20g (0.08%) of MgSO4, 0.15g of MnSO4 (0.0015%), 75g of NaPO4 (dibasic: 0.3%), and 25g of KPO4 (dibasic: 0.1%). After complete mixing, the mixture was autoclaved at 121°C for 1 hour. The temperature was reduced to 30°C, and the pH was lowered to 6.0 using 15M NaOH. Vitamin B 12The solution was added to the fermenter at a final concentration of 2 mg / L. At hour 0, 1.5 L (5-7 OD) of propionic acid bacteria (NFS-2018) that had been pre-grown for 48 hours was inoculated into the fermenter. After 12 hours, 1.5 L (5-7 OD) of Lactobacillus casei that had been pre-grown for 48 hours was added. Samples were taken every 12-24 hours using a YSI analyzer and HPLC, respectively, for glucose and organic acid analysis. 2-3% glucose was added as needed throughout the fermentation period. After 168 hours, fermentation was terminated by heat sterilization at 80°C for 1 hour. The broth was collected for further downstream processing.

[0070] The results showed that when NFS-2018 and Lactobacillus casei were co-cultured in a bioreactor, the maximum PA reached 63 g / L after 168 hours. See Table 1.

[0071] [Table 2]

[0072] Non-limitingly, the present invention includes the following embodiments. [Aspect 1] A method for producing organic acids, Provide acid-tolerant organic acid-emitting microorganisms; Provide acid-resistant lactic acid-producing microorganisms; The invention provides a growth medium containing a glucose carbon source and a lactose carbon source, having a pH of approximately 5.8 to 6.5; Acid-tolerant organic acid-extracting microorganisms and acid-tolerant lactic acid-producing microorganisms are added to the growth medium to produce a culture; and The culture is incubated under controlled fermentation conditions to produce organic acids; The method, including the act of [Aspect 2] The method according to embodiment 1, wherein the acid-tolerant organic acid-emitting microorganism is selected from the group consisting of the genera Propionibacterium, Anerovibrio, Bacteroides, Clostridium, Fusobacterium, Megasphaera, Propionispira, Selenomonas, Veillonella, and combinations thereof. [Aspect 3] The method according to embodiment 2, wherein the microorganism of the genus Propionibacterium is propionic acid bacterium (NFS 2018). [Aspect 4] The method according to any one of embodiments 1 to 3, wherein an acid-tolerant organic acid-emitting microorganism is provided as a growth culture. [Aspect 5] The method according to any one of embodiments 1 to 4, wherein the acid-resistant lactic acid-producing microorganism is selected from the group consisting of the genera Lactococcus, Pediococcus, Oenococcus, Enterococcus, Leuconostoc, Bifidobacterium, Bacillus, Lactobacillus, Clostridium, Paenibacillus, Sporolactobacillus, and combinations thereof. [Aspect 6] The method according to embodiment 5, wherein the acid-resistant lactic acid-producing microorganism is selected from the group consisting of the genera Lactococcus, Pediococcus, Oenococcus, Enterococcus, Leuconostoc, Bifidobacterium, and Bacillus. [Aspect 7] The method according to embodiment 6, wherein acid-resistant lactic acid-producing microorganisms are provided as a culture. [Aspect 8] The method according to embodiment 5, wherein the acid-resistant lactic acid-producing microorganism is selected from the group consisting of the genera Bacillus, Lactobacillus, Clostridium, Paenibacillus, Sporolactobacillus, and combinations thereof. [Aspect 9] The method according to embodiment 8, wherein the acid-resistant lactic acid-producing microorganism is Lactobacillus casei. [Aspect 10] The method according to embodiment 8 or 9, wherein an acid-resistant lactic acid-producing microorganism is provided as spores, and the spores are provided as part of a carbon source. [Aspect 11] The method according to any one of embodiments 1 to 10, wherein the pH of the culture medium is approximately 6.0. [Aspect 12] The method according to any one of embodiments 1 to 11, wherein the growth medium further comprises a nitrogen source, vitamins, MgSO4, NaPO4, and KPO4. [Aspect 13] The method according to any one of embodiments 1 to 12, wherein the glucose carbon source and the lactose carbon source are the same. [Aspect 14] The method according to any one of embodiments 1 to 12, wherein the glucose carbon source and the lactose carbon source are different. [Aspect 15] The method according to any one of embodiments 1 to 14, wherein the glucose carbon source is a plant-based glucose carbon source. [Aspect 16] The method according to embodiment 15, wherein the plant carbon source is selected from the group consisting of hydrolyzed wheat, hydrolyzed maize, hydrolyzed beans, hydrolyzed starch, and combinations thereof. [Aspect 17] The method according to any one of embodiments 1 to 16, wherein the lactose carbon source is whey. [Aspect 18] The method according to any one of embodiments 12 to 17, wherein the nitrogen source is selected from yeast extract, peptides, ammonium sulfate, ammonium hydroxide, amino acids, and combinations thereof. [Aspect 19] The method according to embodiment 18, wherein the nitrogen source is yeast extract. [Aspect 20] Vitamin B 12 The method according to any one of embodiments 12 to 19. [Aspect 21] Acid-tolerant lactic acid-producing microorganisms are added to the culture medium 6 to 36 hours after the addition of acid-tolerant organic acid-excreting microorganisms, and optionally 12 to 24 hours afterwards; and, Maintain the culture for a sufficient amount of time to produce the desired amount of organic acid; The method described in Embodiment 1. [Aspect 22] The method according to any one of embodiments 1 to 21, wherein the organic acid is selected from the group consisting of propionic acid, acetic acid, lactic acid, succinic acid, and combinations thereof. References 1. Wang Z, Jin Y, Yang ST. 2015. High cell density propionic acid fermentation with an acid tolerant strain of Propionibacterium acidipropionici. Biotechnol Bioeng 112:502-511. 2. Liu Y, Zhang YG, Zhang RB, Zhang F, Zhu J. 2011. Glycerol / glucose co-fermentation: one more proficient process to produce propionic acid by Propionibacterium acidipropionici. Curr Microbiol 62:152-158. 3. Wang Z, Ammar EM, Zhang A, Wang L, Lin M, Yang ST. 2015. Engineering Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: effects of overexpressing propionyl-CoA:Succinate CoA transferase. Metab Eng 27:46-56. 4. Jiang L, Cui H, Zhu L, Hu Y, Xu X, Li S, Huang H. 2015. Enhanced propionic acid production from whey lactose with immobilized Propionibacterium acidipropionici and the role of trehalose synthesis in acid tolerance. Green Chemistry 17:250-259. 5. Border PM, Kierstan MPJ, Plastow GS. 1987. Production of propionic acid by mixed bacterial fermentation. Biotechnology Letters 9:843-848. 6. Zhang A, Yang S-T. 2009. Propionic acid production from glycerol by metabolically engineered Propionibacterium acidipropionici. Process Biochemistry 44:1346-1351. 7. Wang Z, Yang S-T. 2013. Propionic acid production in glycerol / glucose co-fermentation by Propionibacterium freudenreichii subsp. shermanii. Bioresource Technology 137:116-123. 8. Stowers CC, Cox BM, Rodriguez BA. 2014. Development of an industrializable fermentation process for propionic acid production. Journal of Industrial Microbiology & Biotechnology 41:837-852. 9. Gonzalez-Garcia RA, McCubbin T, Navone L, Stowers C, Nielsen LK, Marcellin E. 2017. Microbial Propionic Acid Production. Fermentation 3:21. 10. Kagliwal LD, Survase SA, Singhal RS, Granstrom T. 2013. Wheat flour based propionic acid fermentation: An economic approach. Bioresource Technology 129:694-699. 11. Ahmadi N, Khosravi-Darani K, Mortazavian AM. 2017. An overview of biotechnological production of propionic acid: From upstream to downstream processes. Electronic Journal of Biotechnology 28:67-75. 12. Woskow S.A., B.A. Glatz. 1991. Propionic acid production by a propionic acid-tolerant strain of Propionibacterium acidipropionici in batch and semicontinuous fermentation. Applied and Environmental Microbiology 57:2821-2828. 13. Zhu Y., J. Li, M. Tan, L. Liu, L. Jiang, J. Sun, P. Lee, G. Du, J. Chen. 2010. Optimization and scale-up of propionic acid production by propionic acid-tolerant Propionibacterium acidipropionici with glycerol as the carbon source. Bioresource Technology 101:8902-8906. 14. Zhuge X., L. Liu, H.-d. Shin, J. Li, G. Du, J. Chen. 2014. Improved propionic acid production from glycerol with metabolically engineered Propionibacterium jensenii by integrating fed-batch culture with a pH-shift control strategy. Bioresource Technology 152:519-525. 15. Zhuge X., J. Li, H.-d. Shin, L. Liu, G. Du, J. Chen. 2015. Improved propionic acid production with metabolically engineered Propionibacterium jenseniiby an oxidoreduction potential-shift control strategy. Bioresource Technology 175:606-612. 16. Coral J. 2008. Propionic acid production by Propionibacterium sp. using low-cost carbon sources in submerged fermentation. Dissertation. Federal University of Parana. 17. Zhang A., J. Sun, Z. Wang, S.-T. Yang, H. Zhou. 2015. Effects of carbon dioxide on cell growth and propionic acid production from glycerol and glucose by Propionibacterium acidipropionici. Bioresource Technology 175:374-381. 18. Wang Z., M. Lin, L. Wang, E. M. Ammar, S.-T. Yang. 2015. Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: Effects of overexpressing three biotin-dependent carboxylases. Process Biochemistry 50:194-204. 19. Suwannakham S., Y. Huang, S.-T. Yang. 2006. Construction and characterization of ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation. Biotechnology and Bioengineering 94:383-95. 20. Suwannakham S., S.-T. Yang. 2005. Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous‐bed bioreactor. Biotechnology and Bioengineering 91:325-337. 21. Suwannakham S. 2005. Metabolic engineering for enhanced propionic acid fermentation by Propionibacterium acidipropionici. Dissertation. Ohio State University. 22. Tufvesson P., A. Ekman, R. R. R. Sardari, K. Engdahl, L. Tufvesson. 2013. Economic and environmental assessment of propionic acid production by fermentation using different renewable raw materials. Bioresource Technology 149:556-564. 23. Thierry A., S.-M. Deutsch, H. Falentin, M. Dalmasso, F. J. Cousin, G. Jan. 2011. New insights into physiology and metabolism of Propionibacterium freudenreichii. International Journal of Food Microbiology 149:19-27. 24. Scholz C.F.P., M. Kilian. 2016. The natural history of cutaneous propionibacteria, and reclassification of selected species within the genus Propionibacteriumto the proposed novel genera Acidipropionibacteriumgen. nov., Cutibacterium gen. nov. and Pseudopropionibacterium gen. nov. International Journal of Systematic and Evolutionary Microbiology 66:4422-4432. 25. Rehberger J.L., B.A. Glatz. 1998. Response of cultures of Propionibacterium to acid and low pH: tolerance and inhibition. Journal of Food Production 61:211-216. 26. Guan N., L. Liu, X. Zhug, Q. Xu, J. Li, G. Du, J. Chen. 2012. Genome-shuffling improves acid tolerance of Propionibacterium acidipropionici and propionic acid production. Advances in Chemistry Research 15:143-152. 27. Guan N., H. Shin, R. R. Chen, J. Li, L. Liu, G. Du, J. Chen. 2014. Under standing of how Propionibacterium acidipropionici respond to propionic acid stress at the level of proteomics. Scientific Reports 4:6951. 28. Guan N., H.D. Shin, G. Du, J. Chen, L. Liu. 2016. Metabolic engineering of acid resistance elements to improve acid resistance and propionic acid production of Propionibacterium jensenii. Biotechnology and Bioengineering 113:1294-304. 29. EP 0141642 30. WO 85 / 04901 31. US 2011 / 0151529 A1 32. WO 2012 / 064883 A2 33. WO 2017 / 055932 A2

Claims

1. A method for producing propionic acid, Provides propionic acid-excreting microorganisms of the genus Propionibacterium; Provides lactic acid-producing microorganisms of the genus Lactobacillus; The invention provides a growth medium containing a glucose carbon source and a lactose carbon source, having a pH of 5.8 to 6.5; Propionic acid-extracting microorganisms and lactic acid-producing microorganisms are added to the growth medium, and the lactic acid-producing microorganisms are added to the medium 12 to 24 hours after the propionic acid-extracting microorganisms have been added, thereby producing a co-culture; and The co-culture is incubated under controlled fermentation conditions to produce propionic acid; and, Maintain the co-culture for a sufficient amount of time to produce the desired amount of propionic acid. The method, including the act of

2. The method according to claim 1, wherein the microorganism of the genus Propionibacterium is Propionibacterium NFS 2018.

3. The method according to claim 1 or 2, wherein propionic acid-extracting microorganisms are provided as a culture.

4. The method according to claim 1, wherein lactic acid-producing microorganisms are provided as a culture.

5. The method according to claim 1, wherein the lactic acid-producing Lactobacillus microorganism is Lactobacillus casei.

6. The method according to claim 5, wherein Lactobacillus casei is provided as spores, and the spores are provided as part of a carbon source.

7. The method according to any one of claims 1 to 6, wherein the pH of the growth medium is 6.

0.

8. The growth medium contains a nitrogen source, vitamins, and MgSO4. ４ NaPO ４ , and KPO ４ The method according to any one of claims 1 to 7, further comprising:

9. The method according to any one of claims 1 to 8, wherein the glucose carbon source and the lactose carbon source are the same.

10. The method according to any one of claims 1 to 8, wherein the glucose carbon source and the lactose carbon source are different.

11. The method according to any one of claims 1 to 10, wherein the glucose carbon source is a plant-based glucose carbon source.

12. The method according to claim 11, wherein the plant-based glucose carbon source is selected from the group consisting of hydrolyzed wheat, hydrolyzed maize, hydrolyzed beans, hydrolyzed starch, and combinations thereof.

13. The method according to any one of claims 1 to 12, wherein the lactose carbon source is whey.

14. The method according to any one of claims 8 to 13, wherein the nitrogen source is selected from yeast extract, peptides, ammonium sulfate, ammonium hydroxide, amino acids, and combinations thereof.

15. The method according to claim 14, wherein the nitrogen source is yeast extract.

16. Vitamin B １２ The method according to any one of claims 8 to 15.

17. A lactic acid-producing microorganism is added to the growth medium 12 to 24 hours after the propionic acid-excreting microorganism is added to the growth medium; and, Maintain the co-culture for a sufficient amount of time to produce the desired amount of propionic acid; The method according to claim 1.

Images