FERMENTATION PROCESS

Abstract

The present invention is related to sustainable fermentation processes with increased efficiency and less environmental impact. Particularly, the present invention is related to a process wherein in one fermentation process two or more fermentation products can be produced and isolated, i.e. a “primary” fermentation product and a “secondary” fermentation product, particularly wherein one is a water soluble organic compound and one is a fat-soluble organic compound particularly a fat-soluble vitamin, preferably vitamin K2.

Claims

1. Process for co-fermentation of at least two fermentation products in a suitable host cell capable of co-production of a primary fermentation product and a secondary fermentation product, wherein one fermentation product is water-soluble and one fermentation product is fat-soluble organic compound, and wherein the fat-soluble fermentation product is preferably a fat-soluble vitamin, more preferably vitamin K, most preferably vitamin K2.

2. Process according to claim 1, wherein the primary fermentation product is water-soluble and the secondary fermentation product is fat-soluble.

3. Process according to claim 1, wherein the primary and the secondary fermentation products are selected from vitamins.

4. Process according to claim 1, wherein the primary fermentation product is selected from the group consisting of vitamin B2, vitamin B5, vitamin B6, vitamin B12, vitamin B3, vitamin B1, vitamin B7, preferably vitamin B2, B5, B6, B12, more preferably vitamin B2.

5. Process according to claim 1, wherein the secondary fermentation product is extracted from the biomass generated during the fermentative production of the primary fermentation product.

6. Process according to claim 1, wherein the secondary fermentation product is vitamin K2, preferably vitamin K2 with a percentage of at least about 80% (wt/wt) of MK-7.

7. Process according to claim 1, wherein the secondary fermentation product is vitamin K2 with a percentage of about 10 ng or less DNA per g of fermentation product, preferably per g of vitamin K2.

8. The process according to claim 1, wherein the secondary fermentation product is vitamin K2 with a percentage of less than about 1 colony forming unit (CFU) production strain per 1 g of fermentation product, preferably per g of vitamin K2.

9. The process according to claim 1, in a host cell selected from the group consisting of Lactococcus, Lactobacillus, Enterococcus, Leuconostoc, Streptococcus, Bacillus, Corynebacterium, Pseudomonas, Flavobacterium, Elizabethingia and Escherichia, preferably selected from B. amyloliquefaciens, B. subtilis, B. licheniformis, B. subtilis natto, B. polymyxa, B. firmus, B. megaterium, B. cereus, B. thuringiensis, Flavobacterium sp., more preferably Flavobacterium meningosepticum, Elizabethingia meningoseptica, E. coli, Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremonis, Flavobacterium meningosepticum or C. glutamicum, most preferably from Bacillus subtilis.

10. Process according to claim 1 comprising the steps of: (a) cultivation of the host cell under suitable conditions and in a suitable culture medium suitable for production of the primary fermentation product, preferably water-soluble vitamin, (b) isolation of the primary fermentation product from step (a) from the production stream, (c) recovery of the secondary fermentation product, preferably vitamin K2 from the biomass generated during step (a), and optionally (d) purification of the primary and/or secondary fermentation product.

11. Process for production of bio-based vitamin K2 according to claim 1 comprising the step of recovery/extraction from genetically modified biomass, wherein the recovered vitamin K2 has a content of MK-7 in the range of at least about 80% (wt/wt) and maximal content of about 10 ng DNA, preferably recombinant DNA, per g vitamin K2 as measured by PCR.

12. Process according to claim 1 comprising extraction of vitamin K2 as secondary fermentation product in a hexane-free solvent, preferably in an aqueous solution with at least about 80% (wt/wt) ethanol.

13. Process according to claim 1 further comprising production of a nutritional product comprising mixing the bio-based fermentation product(s) with one or more ingredients selected from the group consisting of encapsulation agents, organic solvent(s), oils, supercritical fluids, antioxidant(s), sugar, co-crystallizing agent(s), coacervate(s), and combinations thereof.

14. Bio-based vitamin K2 with a content of about 1 CFU or less production strain per g vitamin K2 obtainable by a process according to claim 1.

Description

FIGURES

[0057] FIG. 1: Co-extraction of vitamin K2 and riboflavin in B. subtilis. Dotted box indicates vitamin K2 recovery, solid box indicates vitamin B2 recovery.

[0058] The following examples are illustrative only and are not intended to limit the scope of the invention in any way. The contents of all references, patent applications, patents, and published patent applications, cited throughout this application, particularly EP405370, EP1186664, WO2010052319, WO2004113510, WO2005014594, WO2017036903, WO2007051552, WO20050145949, WO2013124351, EP1814987, CN101422446, WO2007045488, WO2015169816 are hereby incorporated by reference.

EXAMPLES

Example 1: General Methods

[0059] Unless otherwise mentioned, all media and general methods are disclosed in Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or according to WO2017036903.

[0060] Assay of riboflavin production in deep-well microtiter plates (MTP) was performed as follows: an overnight culture was made from a single colony in 3 ml of VY containing selective antibiotics where appropriate. The preculture was incubated at 39° C., 550 rpm, 80% humidity. The next day, 3 ml of RSM were inoculated with the preculture with a starting OD.sub.600 nm˜0.05. Cultures in MTP were made in triplicate, covering the wells with a breath seal. MTP were incubated at 39° C., 550 rpm, 80% humidity for 48 hours. 250 μl of the 48 hour-culture were treated with 20 μl of 4M NaOH solution to solubilize the riboflavin crystals (shaken for 1 min at 300 rpm). 230 μl of a 1M potassium phosphate buffer, pH 6.8 were added (shaken for 1 min at 300 rpm). Riboflavin was assayed by HPLC using an Agilent 1100 series HPLC system with a quaternary pump, an autosampler, a UV detector and Fluorescence detector. The separation was achieved using a Supelcosil LC-8 DB (150 mm×4.6 mm×μum). Optimal column temperature was 20° C. The mobile phase was a gradient from 100% 0.1M acetic acid to 50/50 0.1M acetic acid/methanol at 15 minutes for total of 33 minutes per run. The flow rate was 1.0 ml/min and the injection volume set to 5 μl. The UV signal was monitored and used for detection. Calibration range from 0.1 μg/ml to 500 μg/ml. Additionally, the potential accumulation of glucose in the culture broth was analyzed by a Waters HPLC system using a binary pump, an autosampler, a UV- and a refractive index detector. The separation was achieved on a CAPCELL PAK NH2 UG80 column (4.6 mm×250 mm, 5 μm; Shiseido). The optimal column temperature was 40° C. The mobile phase was a mixture of acetonitrile and deionized water at a ratio of 65:35. The flow rate was 1.0 ml/min and the injection volume set to 5 μl or 10 μl. The refractive index signal was monitored and used for detection. The calibration range for each compound was from 0.3 mg/ml to 3 mg/ml.

[0061] The analysis of K2 vitamers was performed by HPLC after extraction of the analyte from dried biomass. The biomass was freeze dried and the extraction was carried out by using a Precellys homogenizer. Two disruption cycles were applied. The analysis was carried out on an Agilent 1200 series HPLC system (or similar) using a quaternary pump, an auto sampler and a UV- detector. The separation was achieved on a C18 column, 150×4.6 mm, 3 μm. The optimal column temperature was 15° C. The mobile phase was a mixture of methanol and ethanol, in a 60:40 ratio. Gradient elution was applied ranging from 60:40 MeOH:EtOH to 40:60 in 15 minutes. The flow rate was 1.0 ml/min. The UV signal at 270 nm was monitored and used for quantification.

Example 2: Fermentative Production of the Primary Fermentation Product in Strains of Bacillus or Corynebacterium and Recovery of Vitamin K2

[0062] For riboflavin production as being the primary fermentation product, the protocol according to WO2007051552 has been performed. At the end of the fermentation, the broth was pasteurized and the decanted vitamin B2 crystals (Sedicanter®: Flottweg, Germany) further processed (see e.g. WO20050145949).

[0063] For production of pharma-grade riboflavin, crystals were dissolved in concentrated HCL followed by filtration and re-crystallization with water as anti-solvent.

[0064] In case of fermentative production of enzymes as primary fermentation product in Bacillus the protocol according to WO2013124351 can be used.

[0065] In case of fermentative production of amino acids as primary fermentation product in Corynebacterium the protocol according to EP1814987 can be used.

[0066] At the end of the fermentation process, the supernatant (biomass slurry) from the 1.sup.st decantation step with Sedicanter® as described above comprising vitamin K2 was treated with ethanol as single extraction solvent. followed by passing through a microfiltration membrane for removal of most of the EtOH and waste biomass. To remove remaining rDNA, proteins or part of lipids still present in the solution, a further purification step was applied, comprising ultrafiltration and/or nanofiltration, with further removal of waste biomass and EtOH. After further evaporation and cooling crystallization, a further filtration and drying step was performed, resulting in purified vitamin K2 crystals with at least 80% (wt/wt) of MK-7 as compared to the less than 1% MK-7 (wt/wt) at the beginning of the treatment as described above. The purity of the menaquinone fraction (containing especially MK-4, MK-5 and MK-6) was also increased from 87 to 88% at the start of the treatment to 97 to 98. Vitamin K2 crystals were analyzed for their rDNA content, which was measured below 10 ng/g product by PCR.

[0067] The ratio of riboflavin to vitamin K2 (MK-7) obtained with this procedure as described above was in the range of 1000 to 1.

Example 3: Solid Vitamin K2 Formulation

[0068] Vitamin K2 crystals with at least 80% MK-7 (wt/wt) is dissolved in medium chain triglyceride oil at 62° C. Gum acacia and sugar are dissolved in water and the oil-phase comprising vitamin K2 is added at 62° C.

[0069] After homogenization (e.g. in a rotor/stator or high pressure homogenization) the resulting dispersion can be spray-dried including a thin starch coating. Ideally, the final size of the droplets is 100-800 nm.

[0070] Thus generated vitamin K2 microcapsules can further be mixed with dietary minerals (e.g. MgO, CaCO.sub.3) microcrystalline cellulose powder, Mg-stearate or the like in order to produce solid tablets, as exemplified in WO2015169816 (Ex. 1 to 7).