Process for producing single-cell protein and malic acid

Abstract

The invention discloses a process for producing single-cell protein and malic acid. The process comprises the following steps: pretreating apple pomace, fermenting by using an apple pomace substrate, inoculating a strain into the apple pomace substrate, regulating nutrients required by microorganisms, ensuring rapid growth of thalli, producing single-cell protein, and co-producing malic acid with a certain concentration in fermentation liquor. The process realizes resource utilization of agricultural wastes, and has remarkable economic and environmental benefits.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a process for producing single cell protein and malic acid. Background Art

[0002] my country's apple planting scale and total output account for about one-third of the world's total production level, and a large amount of apple pomace is produced every year. As a by-product of apple juice production, apple pomace has a high water content and is rich in soluble nutrients, but it is often abandoned because it is easy to rot and stink, causing serious environmental pollution and waste of resources.

[0003] The bacterial protein feed produced by fermenting apple pomace is green, pollution-free, rich in protein, complete in vitamin composition and coexisting with biologically active substances. In addition, the fermentation process is easy to control, the raw material sources are wide and the production time is short. Therefore, apple pomace fermented protein feed has received widespread attention. The nitrogen-free extract content of apple pomace is high, and its main components are soluble carbohydrates, pectin, organic acids other than fatty acids and hemicellulose. By adding a suitable nitrogen source, it can be converted into single-cell protein feed through microbial fermentation. This is an important way to solve the problem of waste pomace. It is of great significance to solve the lack of protein feed resources in my country, improve the benefits of fruit planting and processing industries, and reduce environmental pollution. Summary of the invention

[0004] The present invention provides a process for producing single cell protein and malic acid, the process comprising the following steps:

[0005] 1) treating the apple pomace with acid or alkali, and filtering to obtain a homogeneous suspension of the apple pomace;

[0006] 2) preparing a homogeneous suspension of apple pomace into a fermentation liquid, adding an auxiliary culture medium, sterilizing at high temperature, and then inoculating the bacterial strain after cooling;

[0007] 3) fermenting the homogeneous suspension of apple pomace inoculated with the bacterial strain, and centrifuging the fermentation liquid after the fermentation to precipitate a single cell protein product, wherein the supernatant is rich in malic acid.

[0008] In some specific embodiments, the methods of the present invention include:

[0009] In step 1), the alkali treatment is performed by adding alkali solution to the apple pomace, adjusting the pH to 11-12, such as 11, 11.2, 11.4, 11.6, 11.8, 12, etc., and reacting for 2-4 hours, such as 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, etc.;

[0010] Preferably, the alkali solution is an aqueous solution of alkali, wherein the alkali is selected from at least one of sodium hydroxide, potassium hydroxide and ammonia water;

[0011] Preferably, the concentration of the alkali solution is 0.3-0.5 mol / L, for example, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L, etc.

[0012] In step 1), the acid treatment is performed by adding acid solution to the apple pomace, adjusting the pH to 1-2.5, such as 1, 1.5, 2, 2.5, etc., and reacting for 2-4 hours, such as 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, etc.;

[0013] Preferably, the acid solution is an aqueous acid solution, wherein the acid is selected from at least one of sulfuric acid, hydrochloric acid and nitric acid;

[0014] Preferably, the acid solution concentration is 0.01-0.05 mol / L, for example, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, etc.

[0015] The present invention has no requirement on the order of acid and alkali treatment. The acid treatment may be performed first and then the alkali treatment, or the alkali treatment may be performed first and then the acid treatment. Preferably, the alkali treatment is performed first and then the acid treatment.

[0016] The apple pomace of the present invention is treated with acid and alkali to facilitate the decomposition of cell walls and cellulose therein under acid / alkaline conditions, which is more conducive to the metabolism and absorption of microorganisms.

[0017] In step 1), the filtration is carried out by using a membrane filtration method;

[0018] Preferably, the pore size of the filter membrane is 0.2-0.6 μm, for example, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm, etc.

[0019] In step 2), the high temperature sterilization is performed at a temperature of 110-125°C, such as 110°C, 115°C, 120°C, 125°C, etc., and the time is 10-30 min, such as 10 min, 15 min, 20 min, 25 min, 30 min, etc.

[0020] Preferably, the auxiliary culture medium, based on the volume of the apple pomace homogeneous suspension, comprises components in the following concentrations: yeast powder 5-20 g / L, casein peptone 5-25 g / L, sodium acetate 10-35 g / L, ammonium sulfate 5-15 g / L, riboflavin 0.45-0.8 g / L, magnesium sulfate 8-15 g / L, and ammonium sulfate hydrochloride 2-10 g / L.

[0021] Preferably, the homogeneous suspension of apple pomace after high temperature sterilization is cooled to room temperature, and then the pH is adjusted to 6.5-7.5, such as 6.5, 7, 7.5, etc., with an acid or a base; preferably, at least one acid selected from pure acetic acid, formic acid, phosphoric acid, and hydrochloric acid or at least one base selected from ammonia water, sodium hydroxide, and potassium hydroxide is used to adjust the pH value;

[0022] In an optional solution, a trace element solution is added to the homogeneous suspension of apple pomace, and the trace element solution is added after high-temperature sterilization operation;

[0023] The trace element solution uses water as solvent and includes the following components in concentrations: 5-6 g / L manganese sulfate, 0.1-0.2 g / L sodium molybdate, 0.04-0.08 g / L boric acid, 0.6-0.8 g / L cobalt chloride, and 18-21 g / L zinc chloride, and is used after sterilization and filtration;

[0024] Preferably, the trace element is added in an amount of 0.2-0.5% of the volume of the apple pomace homogeneous suspension, such as 0.2%, 0.3%, 0.4%, 0.5%, etc.

[0025] In a preferred embodiment, step 2) specifically includes: adding auxiliary culture medium to the apple pomace homogeneous suspension, sterilizing at high temperature, cooling to room temperature, and then inoculating bacteria.

[0026] In a preferred embodiment, step 2) specifically includes: adding auxiliary culture medium to the apple pomace homogeneous suspension, sterilizing at high temperature, cooling to room temperature, adjusting the pH with acid or alkali, and then inoculating the bacteria.

[0027] In a preferred embodiment, step 2) specifically includes: adding auxiliary culture medium to the apple pomace homogeneous suspension, sterilizing at high temperature, cooling to room temperature, adding trace element solution, and then inoculating bacteria.

[0028] In a preferred embodiment, step 2) specifically includes: adding auxiliary culture medium to the apple pomace homogeneous suspension, sterilizing at high temperature, cooling to room temperature, adjusting pH with acid or alkali, adding trace element solution, and then inoculating bacteria.

[0029] In step 2), the bacterial species is selected from yeast, preferably at least one of Yarrowia lipolytica and Saccharomyces cerevisiae;

[0030] Preferably, the inoculation amount of the strain is 5-10% by volume, such as 5%, 6%, 7%, 8%, 9%, 10%, etc. It should be noted that in this application, the term "inoculation amount" refers to the proportion of the volume of the strain in the total volume of the culture system after inoculation. Exemplarily, the inoculation amount is 5%, that is, the volume percentage of the strain in the apple pomace homogeneous suspension (including the strain, auxiliary culture medium and optional trace element solution) is 5%.

[0031] In step 3), the fermentation temperature is controlled at 28-32° C., the pH is 6.5-7.5, the fermentation process is accompanied by stirring, the initial speed is 150-200 rpm, such as 150 rpm, 170 rpm, 190 rpm, 200 rpm, etc., and the initial ventilation is 0.5-1 vvm, such as 0.5 vvm, 0.7 vvm, 0.9 vvm, 1 vvm, etc.;

[0032] As fermentation proceeds, dissolved oxygen continues to decrease. Preferably, during the fermentation process, the dissolved oxygen is controlled at 10-20%, for example, 10%, 12%, 14%, 16%, 18%, 20%, etc., by increasing the rotation speed and ventilation volume.

[0033] Preferably, the homogeneous suspension of apple pomace is added to a fermentation tank for fermentation, and the volume proportion of the homogeneous suspension of apple pomace in the fermentation tank is 60-70%, for example, 60%, 62%, 64%, 66%, 68%, 70%, etc., wherein the added auxiliary culture medium and / or trace elements are calculated based on the total volume after addition.

[0034] In step 3), sampling is performed during the fermentation process to monitor the bacterial OD and malic acid concentration of the fermentation liquid. When the OD of the fermentation liquid does not increase for 2 consecutive hours, the tank can be stopped, the fermentation is terminated, and the fermentation liquid is centrifuged and dried;

[0035] The invention can prepare a single cell protein product with a crude protein quality content of ≥68% and an L-malic acid concentration of ≥120 g / L, and has the advantages of stable and simple process, low cost and the like.

[0036] The beneficial effects of the present invention are that the cell walls and cellulose of apple pomace are destroyed by acid-base treatment to release nutrients. The process provided by the present invention can not only effectively utilize apple pomace, an agricultural waste, to reduce environmental pollution, but also produce single-cell protein and malic acid with high added value, thus having important economic and environmental benefits. In addition, the process is simple and easy to implement and is suitable for large-scale industrial production. DETAILED DESCRIPTION

[0037] In order to facilitate the understanding of the present invention, the present invention will be further described below in conjunction with examples. It should be understood that the following examples are only for a better understanding of the present invention and do not mean that the present invention is limited to the following examples.

[0038] 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 the present invention belongs. The term "and / or" as may be used herein includes any and all combinations of one or more of the related listed items. The top, bottom, etc. mentioned or may be mentioned in this specification are defined relative to the structures shown in each scheme. They are relative concepts and may therefore be changed accordingly according to their different locations and different usage states.

[0039] (1) OD detection

[0040] In the present invention, the term "OD value" refers to the OD value of the fermentation broth, which is obtained by ultraviolet spectrophotometry, specifically: using an ultraviolet spectrophotometer (Alpha-1106, Shanghai PUYUAN Instrument Co., Ltd.) to detect the OD value of the fermentation broth at 600nm.

[0041] (2) Determination of L-malic acid

[0042] Determination of L-malic acid: Use 2,7-naphthalene diol colorimetric method, take 1.0mL of sample solution, add 6.0mL of analytical grade concentrated sulfuric acid, then add 0.1mL of 2,7-naphthalene diol solution, then heat in a 100℃ water bath for 20min, take out and cool to room temperature, then perform colorimetric determination at 385nm, and use distilled water as a control to calibrate the instrument zero point. Use standard samples to make a standard curve, with malic acid content as the horizontal coordinate and the absorption value at 385nm, i.e. OD385, as the vertical coordinate. The corresponding L-malic acid content can be found on the standard curve through the OD value of the unknown sample at 385nm.

[0043] (3) Crude protein content: unit: %

[0044] According to the national standard GB / T 6432-2018 "Determination of crude protein in feed by Kjeldahl method", the crude protein content (mass fraction) is obtained through digestion, ammonia distillation and titration.

[0045] Saccharomyces cerevisiae (Saccharomyces cerevisiae) was purchased from China Industrial Microbiological Culture Collection Center, with the collection number of CICC1388.

[0046] Yarrowia lipolytica (Yarrowia lipolytica) was purchased from China Industrial Microbiological Culture Collection Center, with the collection number: CICC 32291.

[0047] Example 1

[0048] 1) Add 0.3 mol / L sodium hydroxide aqueous solution to apple pomace and stir, adjust the pH to 11 and react for 2 h, then add 0.03 mol / L sulfuric acid aqueous solution and stir, adjust the pH to 1 and react for 4 h, then filter through a filter membrane with a pore size of 0.45 μm to obtain a homogeneous suspension of apple pomace.

[0049] 2) Add auxiliary culture medium to the homogeneous suspension of apple pomace, shake well, sterilize at 110° C. for 30 minutes, cool to room temperature, adjust pH to 6.5 with pure acetic acid or ammonia water, add 0.2% by volume of trace elements, and then inoculate Yarrowia lipolytica with an inoculation volume of 5%;

[0050] The amount of auxiliary culture medium added, based on the volume of the apple pomace homogeneous suspension, was: yeast powder 5 g / L, casein peptone 5 g / L, sodium acetate 10 g / L, ammonium sulfate 5 g / L, riboflavin 0.45 g / L, magnesium sulfate 8 g / L, and ammonium sulfate hydrochloride 2 g / L;

[0051] Trace elements, using water as solvent, include components of the following concentrations: manganese sulfate 5g / L, sodium molybdate 0.1g / L, boric acid 0.04g / L, cobalt chloride 0.6g / L, zinc chloride 18g / L, and are used after sterilization and filtration.

[0052] 3) The homogeneous suspension of apple pomace inoculated with the bacteria was inoculated into a fermentation tank (60% by volume) for fermentation, with the temperature controlled at 28° C., pH 6.5, initial speed at 150 rpm, initial ventilation at 0.5 vvm, and the dissolved oxygen was controlled between 10-20% by increasing the speed and ventilation during the fermentation process;

[0053] During the fermentation process, samples were taken every hour to test the OD of the fermentation liquid. When the OD of the fermentation liquid did not increase for two consecutive hours, the tank was stopped and the fermentation liquid was centrifuged and dried.

[0054] 4) After testing, the fermentation OD was 240 when the tank was stopped, the crude protein content of the bacteria was 68wt%, and the L-malic acid concentration was 120g / L.

[0055] Example 2

[0056] 1) Add 0.4 mol / L sodium hydroxide aqueous solution to apple pomace and stir, adjust the pH to 11.5 and react for 3 h, then add 0.05 mol / L sulfuric acid aqueous solution and stir, adjust the pH to 1.5 and react for 3 h, then filter through a filter membrane with a pore size of 0.45 μm to obtain an apple pomace homogeneous suspension.

[0057] 2) Add auxiliary culture medium to the homogeneous suspension of apple pomace, shake well, sterilize at 110° C. for 30 minutes, cool to room temperature, adjust pH to 7 with pure acetic acid or ammonia water, add 0.5% by volume of trace elements, and then inoculate Yarrowia lipolytica with an inoculation volume of 10%;

[0058] The amount of auxiliary culture medium added, based on the volume of the apple pomace homogeneous suspension, was: yeast powder 10 g / L, casein peptone 15 g / L, sodium acetate 20 g / L, ammonium sulfate 10 g / L, riboflavin 0.6 g / L, magnesium sulfate 10 g / L, and ammonium sulfate hydrochloride 5 g / L;

[0059] Trace elements, using water as solvent, include components of the following concentrations: manganese sulfate 5.5 g / L, sodium molybdate 0.15 g / L, boric acid 0.05 g / L, cobalt chloride 0.7 g / L, zinc chloride 20 g / L, and are used after sterilization and filtration.

[0060] 3) The homogeneous suspension of apple pomace inoculated with the bacteria is inoculated into a fermentation tank (60% by volume) for fermentation, the temperature is controlled at 30° C., the pH is 7, the initial rotation speed is 170 rpm, the initial ventilation is 0.8 vvm, and the dissolved oxygen is controlled between 10-20% by increasing the rotation speed and the ventilation volume during the fermentation process; the OD of the fermentation liquid is sampled and tested every hour during the fermentation process, and the tank is stopped when the OD of the fermentation liquid does not increase for 2 consecutive hours, and the fermentation liquid is centrifuged and dried.

[0061] 4) After testing, the fermentation OD was 242 when the tank was stopped, the crude protein content of the bacteria was 70%, and the L-malic acid concentration was 124 g / L.

[0062] Example 3

[0063] 1) Add 0.5 mol / L sodium hydroxide aqueous solution to apple pomace and stir, adjust the pH to 12 and react for 4 h, then add 0.05 mol / L sulfuric acid aqueous solution and stir, adjust the pH to 2.5 and react for 2 h, then filter through a filter membrane with a pore size of 0.45 μm to obtain an apple pomace homogeneous suspension.

[0064] 2) Add auxiliary culture medium to the homogeneous suspension of apple pomace, shake well, sterilize at 110° C. for 30 minutes, cool to room temperature, adjust pH to 7.5 with pure acetic acid or ammonia water, add 0.3% by volume of trace elements, and then inoculate Yarrowia lipolytica at an inoculation volume of 8%;

[0065] The amount of auxiliary culture medium added, based on the volume of the apple pomace homogeneous suspension, was: yeast powder 20 g / L, casein peptone 25 g / L, sodium acetate 35 g / L, ammonium sulfate 15 g / L, riboflavin 0.8 g / L, magnesium sulfate 15 g / L, and ammonium sulfate hydrochloride 10 g / L;

[0066] Trace elements, using water as solvent, include components of the following concentrations: manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, and are used after sterilization and filtration.

[0067] 3) The homogeneous suspension of apple pomace inoculated with the bacteria was inoculated into a fermentation tank (60% by volume) for fermentation, the temperature was controlled at 32° C., the pH was 7.5, the initial speed was 190 rpm, the initial ventilation was 1 vvm, and the dissolved oxygen was controlled between 10-20% by increasing the speed and ventilation during the fermentation process;

[0068] During the fermentation process, samples were taken every hour to test the OD of the fermentation liquid. When the OD of the fermentation liquid did not increase for two consecutive hours, the tank was stopped and the fermentation liquid was centrifuged and dried.

[0069] 4) After testing, the fermentation OD was 245 when the tank was stopped, the crude protein content of the bacteria was 70%, and the L-malic acid concentration was 130 g / L.

[0070] Example 4

[0071] Fermentation was carried out according to the steps of Example 1, except that in step 2), the Yarrowia lipolytica strain was replaced by Saccharomyces cerevisiae, and other operations and conditions remained unchanged.

[0072] After testing, the fermentation OD was 240 when the tank was stopped, the crude protein content of the bacteria was 69%, and the L-malic acid concentration was 125g / L.

[0073] Example 5

[0074] Fermentation was carried out according to the steps of Example 1, except that in step 2), no trace elements were added, and other operations and conditions remained unchanged.

[0075] After testing, the fermentation OD was 204 when the tank was stopped, the crude protein content of the bacteria was 61%, and the L-malic acid concentration was 101g / L.

[0076] Fermentation was carried out according to the steps of Example 1, except that in step 1), 0.03 mol / L sulfuric acid was replaced with an equal amount of water, and other operations and conditions remained unchanged.

[0077] After testing, the fermentation OD was 96 when the tank was stopped, the crude protein content of the bacteria was 41%, and the L-malic acid concentration was 35g / L.

[0078] Comparative Example 2

[0079] Fermentation was carried out according to the steps of Example 1, except that in step 1), 0.3 mol / L sodium hydroxide water was replaced with an equal amount of water, and other operations and conditions remained unchanged.

[0080] After testing, the fermentation OD was 83 when the tank was stopped, the crude protein content of the bacteria was 40%, and the L-malic acid concentration was 32g / L.

[0081] Comparative Example 3

[0082] Fermentation was carried out according to the steps of Example 1, except that in step 2), the apple pomace homogeneous suspension was replaced with an equal amount of water (without adding apple pomace matrix, and only using auxiliary culture medium and trace elements for culture), and other operations and conditions remained unchanged.

[0083] After testing, the fermentation OD was 36 when the tank was stopped, the crude protein content of the bacteria was 38%, and the L-malic acid concentration was 22g / L.

[0084] Comparative Example 4

[0085] The fermentation was carried out according to the steps of Example 1, except that in step 2), the auxiliary culture medium was replaced with an equal amount of water, and other operations and conditions remained unchanged.

[0086] After testing, the fermentation OD was 65 when the tank was stopped, the crude protein content of the bacteria was 41%, and the L-malic acid concentration was 38g / L.

[0087] It is easy to understand that the above embodiments are only examples for clear explanation and do not mean that the present invention is limited thereto. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the protection scope of the present invention.

Claims

1. A process for single cell protein and malic acid, characterized in that: The process comprises the following steps: a. subjecting the apple pomace to acid and alkali treatment and filtering to obtain a homogeneous suspension of the apple pomace; b. The apple pomace homogeneous suspension is prepared into a fermentation broth, an auxiliary culture medium is added, and the bacteria are sterilized at high temperature and cooled; c. The homogeneous suspension of apple pomace inoculated with the bacteria is fermented, and after the fermentation is completed, the fermentation liquid is centrifuged to precipitate a single-cell protein product, and the supernatant is rich in malic acid.

2. The process according to claim 1, characterized in that In step a, the apple pomace is added with 0.3-0.5 mol / L alkali solution, the pH is adjusted to 11-12, and the alkali treatment is performed for 2-4 hours; and / or: in step a, the apple pomace is added with 0.01-0.05 mol / L acid solution, the pH is adjusted to 1-2.5, and the acid treatment is performed for 2-4 hours; preferably, the apple pomace after the acid-base treatment is filtered through a 0.2-0.6 μm filter membrane to obtain an apple pomace homogeneous suspension.

3. The process according to claim 1, characterized in that In step b, the high temperature sterilization temperature is 110-125° C. and the time is 10-30 min.

4. The process according to claim 1, characterized in that In step b, an auxiliary culture medium is added to the homogeneous suspension of apple pomace. The auxiliary culture medium comprises the following components based on the volume of the homogeneous suspension of apple pomace: 5-20 g / L yeast powder, 5-25 g / L casein peptone, 10-35 g / L sodium acetate, 5-15 g / L ammonium sulfate, 0.45-0.8 g / L riboflavin, 8-15 g / L magnesium sulfate, and 2-10 g / L ammonium sulfate hydrochloride.

5. The process according to any one of claims 1 to 4, characterized in that In step b, the homogeneous suspension of apple pomace is sterilized at high temperature, cooled, and then adjusted to pH 6.5-7.5 with acid or alkali, and then a trace element solution is added; preferably, the trace elements use water as a solvent and include components with the following concentrations: manganese sulfate 5-6 g / L, sodium molybdate 0.1-0.2 g / L, boric acid 0.04-0.08 g / L, cobalt chloride 0.6-0.8 g / L, zinc chloride 18-21 g / L, and are used after sterilization and filtration; preferably, the trace element solution is added in an amount of 0.2-0.5% of the volume of the homogeneous suspension of apple pomace.

6. The process according to claim 5, characterized in that The acid used to adjust the pH with an acid or a base is selected from at least one of acetic acid, formic acid, phosphoric acid, and hydrochloric acid; and / or the base is selected from at least one of ammonia water, sodium hydroxide, and potassium hydroxide.

7. The process according to any one of claims 1 to 6, characterized in that In step b, the bacterial strain is selected from yeast, preferably at least one of Yarrowia lipolytica and Saccharomyces cerevisiae; preferably, the apple pomace homogeneous suspension is inoculated with the bacterial strain at a volume ratio of 5-10%.

8. The process according to claim 1, characterized in that In step c, the homogeneous suspension of apple pomace is added into a fermentation tank for fermentation, and the volume proportion of the homogeneous suspension of apple pomace is 60-70%.

9. The process according to claim 1 or 8, characterized in that In step c, the fermentation temperature is controlled at 28-32° C., the pH is 6.5-7.5, the initial rotation speed is 150-200 rpm, and the initial ventilation is 0.5-1 vvm; preferably, the dissolved oxygen is controlled at 10-20% during the fermentation process.

10. The process according to any one of claims 1 to 9, characterized in that At the end of fermentation, the OD of the fermentation liquid is ≥240, the crude protein content of the bacteria is ≥68%, and the L-malic acid concentration is ≥120 g / L.