Streptomyces psammoticus and methods of using the same for vanillin production

Abstract

The present invention relates to a bacterial strain, Streptomyces psammoticus OMK-4, and a method for vanillin production by fermentation of the bacterial strain. The method of the fermentation includes the following steps: activating the strain, culturing the seed, fermenting, extracting and so on. This method using ferulic acid as one of the raw materials to produce vanillin through microbial fermentation is safe and environmentally friendly.

Claims

1. A method for producing vanillin using Streptomyces psammoticus strain OMK-4 having preservation number CCTCC M 2015329, wherein the method comprises the steps of: 1) strain activation; 2) seed culture; and 3) fermentation of ferulic acid to produce vanillin wherein strain activation step 1 comprises: under aseptic conditions, a full inoculation loop of strain OMK-4 from a glycerin stock tube is spread evenly over an agar slant and cultured in a biochemical incubator at 26-30 C. for 24-48 hours, and wherein by percentage of weight, the agar slant comprises the following components: soluble starch 1.0-3.5%, KH.sub.2PO.sub.4 0.1-1.0%, NaCl 0.05-0.3% and yeast extract powder 0.1-1.0%, and wherein seed culture step 2 comprises: under aseptic conditions, a full inoculation loop of well-grown OMK-4 cells from the ager slant in step 1 is inoculated into a seed culture medium having an initial pH value of 5-8 and cultured at 28-35 C. with 200-500 rpm shaking until exponential growth phase, wherein the said seed culture medium by weight percentage comprises the following components: soluble starch 1.0-3.5%, KH.sub.2PO.sub.4 0.1-0.5%, urea 0.1-0.3%, MgSO.sub.4 0.05-0.1%, CaCO.sub.3 0.1-0.3%, yeast extract powder 0.1-1.0%, corn syrup 0.1-1.0%, (NH.sub.4).sub.2SO.sub.4 0.1-0.6% and ferulic acid 0.1-0.3%, and wherein fermentation step 3 comprises: the OMK-4 cells from step 2 in exponential growth phase are inoculated into a fermentation medium with the volume ratio of 5-15% under aseptic conditions wherein the initial pH of the fermentation medium is 7.2 to 7.8 at 30-40 C. and 200-500 rpm shaking and 1:0.5 ventilation, to ferment the cells for 70-120 hours, wherein the fermentation medium by weight percentage comprises the following components: soluble starch 2.0-5.0%, KH.sub.2PO.sub.4 0.1-0.3%, urea 0.1-0.5%, MgSO.sub.4 0.05-0.1%, CaCO.sub.3 0.5-2.0%, yeast extract powder 0.1-1.0%, (NH.sub.4).sub.2SO.sub.4 0.1-0.5% and ferulic acid 0.1-3.0%.

2. The method for producing vanillin according to claim 1, further comprising the steps of: 4) extracting the vanillin produced by fermentation at step 3 by pasteurizing the fermentation liquid at 80 C., filtration through a ceramic membrane followed by ultrafiltration and treatment of the ultrafiltrate by reverse osmosis (RO) for 5 to 10 times to concentrate the vanillin crude product, and crystallizing the vanillin crude product by adjusting the pH of the concentrated liquid to pH 5-6 and cooling.

3. The method for producing vanillin according to claim 2, wherein filtration through a ceramic membrane in step 4 is to remove the OMK-4 cells and large molecular weight proteins.

4. The method for producing vanillin according to claim 2, wherein the ultrafiltration in step 4 is to remove small molecular weight proteins and pigments.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1A HPLC diagram of ferulic acid standards;

(2) FIG. 1B HPLC diagram of vanillin standards;

(3) FIG. 1C HPLC diagram of vanillin fermentation broth

(4) The Figures shows that the resulting product is vanillin.

PREFERRED EMBODIMENTS OF THE INVENTION

(5) 1, The Strain

(6) The Streptomyces psammoticus OMK-4 capable of producing natural vanillin was isolated from the soil of a cinnamon plantation forest. The said strain has been preserved at the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei Province on May 27, 2015, with the preservation number of CCTCC M 2015329.

(7) Streptomyces psammoticus OMK-4 is a gram-positive bacterium. Streptomyces psammoticus OMK-4 grows less well in cica culture. Streptomyces psammoticus OMK-4 has straight spore chain when observed under the light microscope. The spores of Streptomyces psammoticus OMK-4 are phalange shape. Streptomyces psammoticus OMK-4 is able to use glycogen and dextrin as energy sources, but Streptomyces psammoticus OMK-4 is not able to use carbon sources such as d-fructose, d-fiber diose, etc. as energy sources. The results of the 16S rRNA gene of Streptomyces psammoticus OMK-4 were identical to that of Streptomyces psammoticus, so it was identified as a strain of Streptomyces psammoticus.

(8) 1) Strain Activation

(9) Under aseptic conditions, a full inoculation loop of bacteria from the glycerol stock tube is spread evenly over an agar slant wherein the said bacteria are Streptomyces psammoticus OMK-4. The agar slant containing Streptomyces psammoticus OMK-4 is cultured in a biochemical incubator at 28-30 C. for 24 to 48 hours.

(10) By the percentage of weight, the agar slant comprises the following components: soluble starch 1.0-3.5%, KH.sub.2PO.sub.4 0.1-1.0%, NaCl 0.05-0.3% and yeast extract powder 0.1-1.0%.

(11) 2) Seed Culture:

(12) Under aseptic conditions, Streptomyces psammoticus OMK-4 that have grown well in the ager are inoculated into a sterile seed culture medium with a shovel. The initial pH of the seed culture medium is 5-8. Under the conditions of 28-35 C. cultivating temperature and 150-500 rpm shaking speed, Streptomyces psammoticus OMK-4 are cultured to the exponential growth phase.

(13) By weight percentage the said seed culture medium comprises the following components: soluble starch 1.0-3.5%, KH.sub.2PO.sub.4 0.1-0.5%, urea 0.1-0.3%, MgSO.sub.4 0.05-0.1%, CaCO.sub.3 0.1-0.3%, yeast extract powder 0.1-1.0%, corn syrup 0.1-1.0%, (NH.sub.4).sub.2SO.sub.4 0.1-0.6% and ferulic acid 0.1-0.3%;

(14) 3) Fermentation:

(15) Streptomyces psammoticus OMK-4 that have been cultured to exponential growth phase in the seed culture medium are added to a fermentation medium with the ratio of 10% V/V in aseptic conditions. The initial pH of the fermentation medium is 7.5. Under conditions of 30-40 C. culture temperature, 200-500 rpm shaking speed and 1:0.5 ventilation, Streptomyces psammoticus OMK-4 are fermented for 70-120 hours. The fermentation medium by weight percentage comprises the following components: soluble starch 2.0-5.0%, KH.sub.2PO.sub.4 0.1-0.3%, urea 0.1-0.5%, MgSO.sub.4 0.05-0.1%, CaCO.sub.3 0.5-2.0%, yeast extract powder 0.1-1.0%, (NH4).sub.2SO.sub.4 0.1-0.5% and ferulic acid 0.1-3.0%.

Embodiment 1: The Production of Natural Vanillin (Flask Shaking Fermentation)

(16) Preparation of the seed medium: the seed medium composition was as follow (g/L): glucose 20, KH.sub.2PO.sub.4 3, urea 5, MgSO.sub.4 0.3, NaCl 1.0, yeast extraction powder 5, corn starch 5, (NH.sub.4).sub.2SO.sub.4 6 and CaCO.sub.3 0.2. Water was used as solvent. The initial pH was 7.2. The seed culture medium was sterilized for 20 minutes at 121 C.

(17) Preparation of the fermentation medium: the medium composition was as follow (g/L): glucose 30, KH.sub.2PO.sub.4 5, urea 5, MgSO.sub.4 0.6, yeast extraction powder 8, (NH.sub.4).sub.2SO.sub.4 3, CaCO.sub.3 1.0 and ferulic acid 20. Water was used as solvent. The initial pH was 7.2. The fermentation medium was sterilized for 20 minutes at 121 C.

(18) Seed preparation: under aseptic conditions, the strain of Streptomyces psammoticus OMK-4 in low temperature glycerol tube was transferred into a fresh sterile medium plate for activation at 30 C. for 2 days. Streptomyces psammoticus OMK-4 colonies were inoculated into a 500 mL flask for seed culture. The volume of the seed culture medium was 50 mL. The flask containing Streptomyces psammoticus OMK-4 and the seed culture medium was incubated at 30 C. with the rotation speed of 180 rpm. Seed liquid was obtained after 16-24 hours of growth at the aforementioned conditions.

(19) Natural vanillin fermentation: 50 mL of the prepared fermentation medium was introduced into a 500 mL sterile triangle flask, after which 7.5 mL of cultured seed liquid was inoculated into the fermentation medium for fermentation. The flask containing the seed culture and the fermentation medium was incubated at 37 C. with 200 rpm shaking speed. After being fermented for 80 hours, the concentration of natural vanillin in the fermentation medium was measured by HPLC method. The final concentration of natural vanillin in the fermentation medium was 10 g/L.

Embodiment 2: Natural Vanillin Production (Stirring Reactor Fermentation)

(20) Seed medium and fermentation medium were prepared in the same way as described in embodiment 1.

(21) Seed preparation: under aseptic conditions, the strain of Streptomyces psammoticus OMK-4 in low temperature glycerol stock tube was transferred into a fresh sterile medium plate for activation at 30 C. for 2 days. Streptomyces psammoticus OMK-4 colonies were inoculated into a 3 L tank equipped with 1.8 L seed medium. The tank containing Streptomyces psammoticus OMK-4 and 1.8 L seed medium was incubated with ventilation at 30 C. and 300 rpm stirring speed. Seed liquid was obtained after 13-18 hours of culture.

(22) The fermentation of natural vanillin: 10.2 L of the prepared fermentation medium was added into a 20 L fermentor. The fermentor with the fermentation medium was sterilized at 121 C. for 20 min, after which 1.8 L of cultured seed liquid was added into the 20 L fermentor for fermentation, with fermentation temperature of 37 C., stirring speed of 400 rpm and ventilation ratio of 1:0.2. The fermentation period was 90 hours. At the end of the fermentation, the concentration of natural vanillin in fermentation medium measured by HPLC method was 15 g/L. (shown in FIG. 1A to FIG. 1C)

Embodiment 3: The Extraction of Natural Vanillin

(23) Twelve liters of the fermented medium of embodiment 2 which contained 15 g/L vanillin were filtrated by ceramic membrane at 80 C. Ten liters of filtrate were obtained, which was further treated by ultrafiltration (UF). The ultrafiltrate was further treated with reverse osmosis (RO). At the end 5 L of concentrate was obtained. The concentrate was adjusted to pH 5-6 and was allowed to cool for crystallization between 0 C. to 4 C. The concentrate was stranded for 4 hours. Raw natural vanillin was obtained from the concentrate by suction filter. One hundred and forty four gram of crude vanillin was obtained after drying. The purity of the crude vanillin was 98%.

INDUSTRIAL APPLICABILITY

(24) The method of the invention is by fermentation at low temperature and low pressure. The merits of the process are that it is relatively safe with less environmental pollution and that the operation is simple.