NL2028177B1 - Process for producing acetoin by utilizing wheat b-starch - Google Patents

Process for producing acetoin by utilizing wheat b-starch Download PDF

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NL2028177B1
NL2028177B1 NL2028177A NL2028177A NL2028177B1 NL 2028177 B1 NL2028177 B1 NL 2028177B1 NL 2028177 A NL2028177 A NL 2028177A NL 2028177 A NL2028177 A NL 2028177A NL 2028177 B1 NL2028177 B1 NL 2028177B1
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starch
acetoin
wheat
fermentation
slurry
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Liu Liping
Liu Jianjun
ZHANG Jiaxiang
Tian Yanjun
Han Mo
Zhao Xiangying
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Shandong Food Ferment Industry Res & Design Institute
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    • C12P7/00—Preparation of oxygen-containing organic compounds
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    • C07C45/81—Separation; Purification; Stabilisation; Use of additives by change in the physical state, e.g. crystallisation
    • C07C45/82—Separation; Purification; Stabilisation; Use of additives by change in the physical state, e.g. crystallisation by distillation
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    • C07C45/83—Separation; Purification; Stabilisation; Use of additives by change in the physical state, e.g. crystallisation by distillation by extractive distillation
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    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/04—Saturated compounds containing keto groups bound to acyclic carbon atoms
    • C07C49/17—Saturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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Abstract

The present invention provides a process for producing acetoin by utilizing wheat B-starch, and belongs to the technical field of deep processing of agricultural products. The specific process includes: saccharifying the wheat B-starch to obtain starch sugar; preparing a fermentation medium from the starch sugar as a raw material; and inoculating acetoin producing strain into the fermentation medium to produce acetoin by fermentation. By adopting the aforementioned process, the production cost of acetoin can be effectively reduced, and meanwhile the added value of the wheat B-starch can be effectively increased, so the process has a good practical application value.

Description

i PROCESS FOR PRODUCING ACETOIN BY UTILIZING WHEAT B-STARCH Field of the Invention The present invention belongs to the technical field of deep processing of agricultural products, and particularly relates to a process for producing acetoin by utilizing wheat B-starch.
Background of the Invention The information disclosed in this background section is only for the purpose of increasing understanding of the general background of the present invention, and is not necessarily regarded as an acknowledgement or any form of suggestion that this information constitutes the prior art already known to those of ordinary skills in the art.
A acetoin is a widely used food flavor and an important C4 platform compound.
It is widely used in fields of food, medicine, chemical industry, tobacco and the like.
At present, acetoin products in the market are mainly produced by chemical synthesis.
For the chemical production process, sources of raw materials are limited and many by-products are produced, also the products do not conform to the natural, green and safe consumption concept.
Acetoin is a metabolite of various microorganisms, and can be produced by microbial transformation from glucose as the raw material.
The acetoin produced by biotransformation has advantages such as abundant sources of raw materials, high product purity and high product safety.
The inventor has been developing biotechnology to produce acetoin for many years, and screened out a strain of Bacillus subtilis which has a high yield of acetoin and a good industrial application potential (Zhao Xiangying ef al, BACILLUS SUBTILIS CAPABLE OF PRODUCING HIGH PURITY acetoin, Chinese patent No.
ZL2007100134025, published as CN101016530B, 2007; Zhao Xiangying et al., BACILLUS SUBTILIS PRODUCING acetoin AND USE THEREOF, Chinese patent No.
ZL201310289934.7, 2013; and Fan Yixiao et al, The Screening of Acetoin-producing Strain and Identification of Metabolite, Food and Fermentation Industries, 2012, 38(11): 42-46.). At present, the reported biotransformation technologies for producing acetoin basically use glucose as the raw material.
In addition, it also needs to add organic nitrogen sources such as yeast extract and corn steep liquor, inorganic nitrogen sources such as ammonium sulfate and urea, and some other inorganic salts into the fermentation medium.
The cost of the raw material accounts for about 60-70% of the total production cost.
Wheat gluten production is one of the main deep processing methods of wheat.
Wheat contains about 12-13% of proteins and 65-70% of starch.
Therefore, a large amount of wheat starch will be co-produced in the process of wheat gluten production.
Wheat starch can be divided into A starch and B starch according to the particle size.
The A starch has a larger particle size, is relatively easier to separate by centrifugation, and can be further refined and sold as commercial starch.
The B starch is a kind of starch with smaller particle size, together with non-starch components such as pentosan and residual proteins in wheat starch slurry.
The starch component in the dry matter of this component accounts for about 80%, which has high viscosity and strong water holding capacity and is difficult to be processed for adding value.
Summary of the Invention In order to increase the processing value of wheat B starch, the present invention provides a process for producing acetoin by utilizing wheat B-starch.
Particularly, the wheat B-starch slurry separated from the process of wheat gluten production is saccharified and then used for the fermentation production of acetoin.
By adopting the aforementioned process, the production cost of acetoin can be effectively reduced, and meanwhile the added value of the wheat B-starch can be effectively increased, so the process has a good practical application value.
In order to achieve the aforementioned technical objective, the technical solution of the present invention is as follows.
In a first aspect of the present invention, it is provided a use of wheat B-starch in fermentation production of acetoin.
In a second aspect of the present invention, it is provided a process for producing acetoin by utilizing wheat B-starch, which includes: saccharifying the wheat B-starch to obtain a starch syrup; preparing a fermentation medium by using the starch syrup as a raw material; and inoculating an acetoin producing strain into the fermentation medium, and performing a fermentation to produce acetoin.
In some embodiments, there is no limitation on the method for saccharifying the wheat B-starch, and a commonly used "double-enzymatic" saccharifying process can be used.
In some embodiments, a starch syrup is obtained by adding water into the wheat B-starch to prepare a starch slurry, adjusting the concentration of the starch slurry to be 10-15 Bé, and then subjecting the slurry the slurry to a "double-enzymatic" saccharifying process.
In some embodiments, in the fermentation medium, the initial concentration of glucose is controlled to be 120-160 g/L; In some embodiments, the fermentation medium further includes an inorganic nitrogen source; and preferably, the inorganic nitrogen source can be urea.
It has been found in the present invention that, for acetoin fermentation when B starch sugar slurry is used as the raw material, it does not need addition of yeast extract, and the yield and conversion rate of acetoin are obviously higher than those used glucose as the raw material.
In a third aspect of the present invention, it is provided acetoin prepared by the aforementioned process for producing acetoin by utilizing wheat B-starch.
Beneficial technical effects of the aforementioned one or more technical solutions are mentioned as below: 1) In the B starch portion separated in the production process of wheat gluten, the dry matter accounts for about 40%. Starch accounts for about 80% of the dry matter and the other components include proteins, fats, crude fibers, ash and the like.
It is found that when B starch 1s used as the raw material for fermentation for producing acetoin, it only needs to add a small amount of inorganic nitrogen source, and does not need to add expensive organic nitrogen sources such as the yeast extract.
Meanwhile, the cost of B starch is much lower than that of glucose, so the raw material cost of acetoin production can be greatly reduced by using the wheat B-starch. 2) Acetoin is azeotroped with water, the acetoin can be collected from the fermentation broth by vacuum distillation or multi-effect concentration, and other unfermented components such as pentosan brought by the B starch raw materials are enriched in the bottoms fraction.
Therefore, the extraction cost of acetoin will not increase compared with glucose as the raw material.
Meanwhile, the bottoms fraction can be further process to be a feed. 3) The B starch separated from the wheat starch is used for producing acetoin with a high added value, which realizes high added value utilization of the wheat B-starch and thus has a good practical application value.
Detailed Description of Embodiments It should be noted that, the following detailed description is exemplary and is intended to provide further description of the present invention.
Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skills in the art to which the present invention belongs.
It should be noted that the terms used here are only for describing specific implementations, and are not intended to limit exemplary implementations according to the present invention.
As used herein, the singular form is also intended to include the plural form, unless the context clearly indicates otherwise.
In addition, it should also be understood that when the terms “comprising/comprise” and/or “including/include” are used in this specification, it indicates the presence of a feature, a step, an operation, a device, an assembly, and/or a combination thereof. It should be appreciated that the protection scope of the present invention is not limited to the following specific embodiments. It should also be appreciated that the terms used in the embodiments of the present invention are to describe the specific embodiments, not to limit the protection scope of the present invention. In a specific embodiment of the present invention, it is provided a use of wheat B-starch in fermentation production of acetoin. The present invention reports for the first time that a starch syrup of the wheat B-starch is used as a component of a culture medium for producing acetoin by fermentation, and as verified by experiments, the fermentation production of acetoin can be realized only by further adding some inorganic nitrogen source into the culture medium, and the yield and conversion rate of acetoin are both high.
In another specific embodiment of the present invention, it is provided a process for producing acetoin by utilizing wheat B-starch, which includes: saccharifying the wheat B-starch to obtain a starch syrup; preparing a fermentation medium by using the starch syrup as a raw material; and inoculating acetoin producing strain into the fermentation medium to produce acetoin by fermentation.
In another specific embodiment of the present invention, the wheat B-starch can be selected from B starch slurry separated during processing of wheat gluten; or a B starch product prepared by directly drying of B starch.
In another specific embodiment of the present invention, the wheat B-starch can be: an intermediate flowing powder slurry portion obtained by a process of taking fresh wheat starch slurry produced in the production process of wheat gluten, subjecting it to a centrifugation with a centrifuge at a rotating speed of 3000 rpm for 2 seconds, removing the supernatant, and collecting the intermediate flowing powder slurry portion; alternatively, B starch slurry obtained after separating wheat gluten from the intermediate phase separated in the production process of wheat gluten by a three-phase decanter centrifuge; alternatively, commercial B starch (dry powder) produced by wheat starch manufacturers.
In another specific embodiment of the present invention, there is no limitation on the method for saccharifying the wheat B-starch, and a commonly used "double-enzymatic" saccharifying process can be used; In another specific embodiment of the present invention, a starch slurry is obtained by adding water into the wheat B-starch and adjusting the slurry. The concentration of the starch slurry is controlled to be 10-15 Bé, and then the starch syrup is prepared by employing a
"double-enzymatic" saccharifying process.
In another specific embodiment of the present invention, the fermentation medium further includes an inorganic nitrogen source; and still further, the inorganic nitrogen source can be urea. It has been found in the present invention through research that, acetoin fermentation 5 with B starch sugar slurry as the raw material does not need addition of yeast extract, and the yield and conversion rate of acetoin are obviously higher than those of the fermentation with glucose as the raw material.
In another specific embodiment of the present invention, the fermentation medium includes the aforementioned starch syrup and an inorganic nitrogen source, and the inorganic nitrogen IO source is urea.
In another specific embodiment of the present invention, in the fermentation medium, the initial concentration of glucose is controlled to be 120-160 g/L and the concentration of urea is controlled to be 1-2 g/L.
In another specific embodiment of the present invention, it is preferably to use Bacillus for the production of acetoin, especially Bacillus subtilis. In an embodiment of the present invention, Bacillus subtilis BS168D was used to produce acetoin.
In another specific embodiment of the present invention, the process further includes acetoin collection and purification. Particularly, the specific method of collecting and purifying comprises: collecting and fractionating the fermentation broth of acetoin, subjecting the obtained aceton fraction to extraction with an added salt and a rectification separation to obtain an acetoin crystal. The specific purifying method can also be conducted with reference to CN 2013 10426868.3.
In yet another specific embodiment of the present invention, the fractionating specifically includes: collecting the fraction from the fermentation broth of acetoin by vacuum distillation or multi-effect concentration, wherein the temperature of distillation is controlled at 55-75°C; and the fraction collection continues until the collected fraction accounts for 75-85% of the total volume of the fermentation broth. In the present invention, acetoin is azeotroped with water, the acetoin can be collected from the fermentation broth by vacuum distillation or multi-effect concentration, and other unfermented components such as pentosan brought by the B starch raw materials are enriched in the bottoms fraction, which does not increase the extraction cost of acetoin compared with that used glucose as the raw material.
In another specific embodiment of the present invention, it is provided an acetoin prepared by the aforementioned process for producing acetoin by utilizing wheat B-starch.
The present invention is further illustrated by examples hereafter, but the examples do not constitute a limitation of the present invention. It should be understood that, the following examples are only intended to illustrate the present invention, rather than limiting the scope of the present invention. Experimental Materials Experimental strains: Bacillus subtilis BS168D (Bacillus subtilis 168 engineering strain with 2,3-butanediol dehydrogenase gene (bdhA)-deleted) (Junjiao Zhang, Xiangying Zhao, Jiaxiang Zhang, Chen Zhao, Jianjun Liu, Yanjun Tian & Liping Yang (2017) Effect of deletion of 2,3-butanediol dehydrogenase gene (bdhA) on acetoin production of Bacillus subtilis, Preparative Biochemistry and Biotechnology, 47:8, 761-767). The aforementioned strain can be obtained from Shandong Food Ferment Industry Research & Design Institute. Acquisition of B starch: taking fresh wheat starch slurry produced in the production process of wheat gluten, subjecting it to a centrifugation with a centrifuge at a rotating speed of 3000 rpm for 2 seconds, removing the supernatant, and collecting the intermediate flowing powder slurry portion to prepare starch syrup.
Preparation of syrup: adjusting the starch obtained after separation and washing by adding with water to achieve a concentration of the starch slurry of 10-15 Bé, adding an appropriate amount of amylase calculated according to the total amount of starch to conduct double-enzymatic saccharification (the amount of the enzyme preparation is added according to the amount recommended by the manufacturer), and separating by centrifuging after saccharification is completed, and collecting the supernatant for acetoin fermentation.
Seed medium: 50 g/L of glucose, 10 g/L of yeast extract, 10 g/L of corn steep liquor, 5 g/L of sodium chloride, formulated with drinking water, pH 7.0.
Fermentation of acetoin The starch syrup was adjusted to a proper glucose concentration (120-160 g/L), so as to prepare a medium for acetoin fermentation. A control experiment was prepared with oral glucose. The culture medium was sterilized, inoculated with strains, and subjected to fermentation.
Shake-flask fermentation: a 500 ml Erlenmeyer flask with a liquid volume of 50 ml and an inoculation amount of 5%, was incubated under the optimal culture conditions for 3-4 days.
Fermentation in fermenter: the fermentation was carried out in a laboratory-scale (5-50 L)
fermenter, and the appropriate temperature and relative dissolved oxygen concentration are controlled as desired in the fermentation process. The incubation continued until the residual glucose decreased below 0.1 g/L, and the fermentation was ended. Example 1 Effect of medium on fermentation of acetoin Glucose for experimental use was used as a control for acetoin fermentation experiment, and the effect of the addition of a nitrogen source on the fermentation of acetoin from wheat B-starch sugar slurry was investigated. The results were shown in the following table: Raw g/L Nitrogen source g/L Residual Acetoin g/L Yield % material sugar g/L Yeast extract 5 136 Corn steep 10 Glucose 38.0 28.0 liquor Urea 2 Yeast extract 5 Corn steep 10
37.2 26.2 liquor Urea 2 Yeast extract 5 Corn steep 5 0.3
42.0 29.6 142 liquor B starch Urea 2 sugar Yeast extract 2 Corn steep 2 1.5
44.8 32.0 liquor Urea 2
The experiment results showed that, acetoin fermentation with B starch sugar slurry as the raw material did not need addition of yeast extract and corn steep liquor, and the yield and conversion rate of acetoin were obviously higher than those used glucose as the raw material. Example 2 50 L scale fermentation with B starch sugar slurry as raw material Starch sugar slurry was prepared from fresh wheat B-starch slurry by a conventional double-enzymatic saccharifying process. After the saccharification was completed, centrifugal separation was conducted to remove the solid residue. The residue could be washed with equal volume of water, separated by centrifuging, the supernatants of the centrifuging were combined, adjusted to a proper glucose concentration, added into a fermenter, and sterilized. It was further added with 2 g/L. of urea (sterilized separately and added before inoculation). The inoculation was conducted according to the inoculation amount of 5%, the initial sugar concentration after the inoculation was 138 g/L, and the appropriate temperature and relative dissolved oxygen concentration were controlled in the fermentation process. The culture was conducted for 58 hours, and the fermentation was ended when it was determined that the glucose concentration decreased below 0.1 g/L. A total of 35 L fermentation broth was collected, and the content of acetoin was determined to be
53.2 g/L. 10 L of the aforementioned fermentation broth was taken and distilled under reduced pressure at 70-80°C, and 8.3 L of a fraction was collected. The content of acetoin in the fraction was determined to be 55.6 g/L, and the recovery rate of acetoin was 86.7%. It should be noted that the above examples are only used for illustrating the technical solutions of the present invention rather than limiting them. Although the present invention has been described in detail with reference to the given examples, the technical solutions of the present invention can be modified or equivalently substituted by those of ordinary skills in the art as required without departing from the spirit and scope of the technical solutions of the present invention.

Claims (12)

Conclusies:Conclusions: 1. Gebruik van tarwe B-zetmeel bij de fermentatieproductie van acetoine.1. Use of wheat B starch in the fermentation production of acetoin. 2. Werkwijze voor de productie van acetoine door gebruik te maken van tarwe B-zetmeel, waarbij de werkwijze omvat: het versuikeren van het tarwe B-zetmeel om een zetmeelsiroop te verkrijgen; het bereiden van een fermentatiemedium door de zetmeelsiroop als grondstof te gebruiken; en het inoculeren van een acetoïne producerende stam 1n het fermentatiemedium, en het uitvoeren van een fermentatie om acetoine te produceren.A method for producing acetoin using wheat B starch, the method comprising: saccharifying the wheat B starch to obtain a starch syrup; preparing a fermentation medium by using the starch syrup as a raw material; and inoculating an acetoin-producing strain into the fermentation medium, and performing a fermentation to produce acetoin. 3. Werkwijze volgens conclusie 2, waarbij het tarwe-B-zetmeel is gekozen uit B-zetmeelslurry die is afgescheiden tijdens de verwerking van tarwegluten, of een B-zetmeelproduct bereid door direct drogen van B-zetmeel.The method of claim 2, wherein the wheat B starch is selected from B starch slurry separated during the processing of wheat gluten, or a B starch product prepared by direct drying of B starch. 4. Werkwijze volgens conclusie 2 of 3, waarbij de zetmeelsiroop wordt verkregen door water toe te voegen aan het tarwe B-zetmeel om een zetmeelbrij te bereiden, de concentratie van de zetmeelbrij aan te passen op 10-15 Bé en vervolgens de brij te gebruiken om zetmeelsuiker te bereiden door een "dubbel-enzymatisch" versuikingsproces. A method according to claim 2 or 3, wherein the starch syrup is obtained by adding water to the wheat B starch to prepare a starch slurry, adjusting the concentration of the starch slurry to 10-15 Bé and then using the slurry to prepare starch sugar by a "double-enzymatic" swaging process. 5 Werkwijze volgens conclusie 2, 3 of 4, waarbij het fermentatiemedium verder een anorganische stikstofbron omvat.The method of claim 2, 3 or 4, wherein the fermentation medium further comprises an inorganic nitrogen source. 6. Werkwijze volgens conclusie 5 waarbij de anorganische stikstofbron ureum is.The method of claim 5 wherein the inorganic nitrogen source is urea. 7. Werkwijze volgens conclusie 6, waarbij het fermentatiemedium de zetmeelsuiker en de anorganische stikstofbron omvat; de anorganische stikstofbron ureum is; en in het fermentatiemedium de beginconcentratie glucose geregeld wordt op 120-160 g /1 en de concentratie ureum gecontroleerd wordt op 1-2 g/1.The method of claim 6, wherein the fermentation medium comprises the starch sugar and the inorganic nitrogen source; the inorganic nitrogen source is urea; and in the fermentation medium the initial concentration of glucose is controlled at 120-160 g/l and the concentration of urea is controlled at 1-2 g/l. 8. Werkwijze volgens een der voorgaande conclusies 2 tot en met 7, waarbij de acetoïne producerende stam Bacillus is.A method according to any one of claims 2 to 7, wherein the acetoin producing strain is Bacillus. 9. Werkwijze volgens conclusie 8, waarbij de acetoine producerende stam Bacillus subtili is. The method of claim 8, wherein the acetoin producing strain is Bacillus subtili. 10, Werkwijze volgens een der voorgaande conclusies 2 tot en met 9, verder omvattende het verzamelen en zuiveren van acetoine; waarbij de specifieke methode voor het verzamelen en zuiveren omvat: het verzamelen en fractioneren van de fermentatiebouillon van acetoine, het onderwerpen van de verkregen acetoïnefractie aan extractie met een toegevoegd zout en een rectificatiescheiding om acetoïne te verkrijgen.The method of any one of claims 2 to 9, further comprising collecting and purifying acetoin; wherein the specific method for collecting and purifying comprises collecting and fractionating the fermentation broth of acetoin, subjecting the obtained acetoin fraction to extraction with an added salt and rectification separation to obtain acetoin. 11. Werkwijze volgens conclusie 10, waarbij de specifieke methode van fractionering omvat: het verzamelen van de fractie uit de fermentatiebouillon van acetoïne door vacuümdestillatie of multi-effectconcentratie, waarbij de destillatietemperatuur wordt geregeld op 55-75 © C; en doorzetten van het verzamelen van de fracties totdat de verzamelde fractie 75-85% van het totale volume van de fermentatiebouillon uitmaakt.The method of claim 10, wherein the specific method of fractionation comprises: collecting the fraction from the fermentation broth of acetoin by vacuum distillation or multi-effect concentration, wherein the distillation temperature is controlled at 55-75°C; and continuing to collect the fractions until the collected fraction constitutes 75-85% of the total volume of the fermentation broth. 12. Acetoine bereid met de werkwijze voor het produceren van acetoïne door gebruik te maken van tarwe B-zetmeel volgens een van de conclusies 2-11.Acetoin prepared by the method of producing acetoin using wheat B starch according to any one of claims 2-11.
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