CN111676117A - Method for immobilized fermentation of vinegar - Google Patents

Abstract

The invention discloses a method for immobilized fermentation of vinegar, which comprises the following steps: (1) constructing a fixed reaction system: an immobilization bracket is arranged in the reactor, and an immobilization carrier is fixed on the immobilization bracket; (2) strain immobilization and immobilized fermentation: adding strain seed liquid and wine liquid into the fixed reaction system constructed in the step (1), and simultaneously carrying out strain immobilization and immobilized fermentation. The method can increase the adsorption area of the strain, improve the fermentation rate, reduce the flow resistance of the fermentation liquid, and is beneficial to gas-liquid-solid three-phase contact in the fermentation process; immobilization and fermentation are carried out simultaneously, so that the fermentation time is shortened; the immobilized strain can be repeatedly utilized, the utilization rate of the strain is improved, and the utilization rate of a fermentation substrate is improved.

Description

Method for immobilized fermentation of vinegar

Technical Field

The invention relates to a method for immobilized fermentation of vinegar, in particular to a method for immobilized fermentation of vinegar with high fermentation efficiency and high substrate utilization rate.

Background

The vinegar is essential seasoning in human life. The fermentation process for preparing vinegar mainly comprises solid fermentation and liquid fermentation. The vinegar produced by liquid fermentation has the advantages of short production period, stable quality of fermentation products, high degree of mechanization of production equipment, small occupied area and the like. At present, the white vinegar, the rice vinegar and the fruit vinegar are mainly produced by liquid fermentation. Liquid vinegar is gradually accepted and favored by consumers due to its unique flavor and function. The white vinegar is suitable for seasoning western dishes and light dishes, and has the advantages of refreshing air, sterilizing, cleaning, decontaminating and the like.

The liquid vinegar mainly uses ethanol as a substrate to produce acetic acid. During the fermentation process, the high concentration of ethanol and acetic acid has serious inhibition effect on the growth and metabolism of acetic acid bacteria. In the method for producing the white vinegar by adopting the divided material feeding fermentation method, a lag phase occurs in the growth and fermentation of the acetic acid bacteria after each material feeding, the whole fermentation time is prolonged due to the lag phase, and the production efficiency of the liquid fermentation is directly influenced. The immobilized cell fermentation technology has the characteristics of improving cell tolerance, reducing substrate inhibition effect in fermentation and the like, and is widely applied to the field of fermentation. At present, the immobilized cell vinegar brewing technology mainly utilizes an embedding method to embed cells in materials such as polyacrylamide gel, sodium alginate, calcium alginate, agarose, gel and the like for fermentation. However, high concentrations of ethanol and acetic acid in the fermentation process have some degradation effect on the immobilized material, resulting in contamination of the fermentation product. In addition, cells embedded in the immobilized material are aged, decayed and the like after being fermented for many times, so that the later fermentation efficiency is directly reduced, and the utilization rate of the immobilized cells is low.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a method for immobilized fermentation of vinegar, which has high fermentation efficiency and high substrate utilization rate.

The technical scheme is as follows: the method for immobilized fermentation of vinegar comprises the following steps:

(1) constructing a fixed reaction system: an immobilization bracket is arranged in the reactor, and an immobilization carrier is fixed on the immobilization bracket;

(2) strain immobilization and immobilized fermentation: adding strain seed liquid and wine liquid into the fixed reaction system constructed in the step (1), and simultaneously carrying out strain immobilization and immobilized fermentation.

Preferably, the immobilization support in the step (1) is in a spiral barrel shape, and a grid-shaped hollow structure is arranged on the barrel; further preferably, the immobilization support in step (1) is in a double helix barrel shape.

The design of the immobilized bracket can increase the number of inoculated strains in unit volume and improve the adsorption area of the strains, thereby improving the fermentation speed; in addition, the structure of the immobilized bracket does not influence the flow of gas and fermentation liquor, so that fermentation substrates are fully fermented, and the fermentation quality is improved.

Preferably, the immobilized carrier in the step (1) is a fiber material; further preferably, the fiber material is one or more of bamboo shoot skin, reed leaves, palm fiber and water chestnut shell.

The selected fiber material has rich pore structure, and is not easy to soften and deform in long-term soaking.

Preferably, the strain in the step (2) is European coltform bacillus HS-K1, and the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 16345.

The European foal bacillus HS-K1 is described in detail in the Chinese patent application CN 201811337264.0.

Preferably, the culture medium of the strain seed liquid in the step (2) is 2-4g/L of glucose, 2-4g/L of peptone, 1-3g/L of yeast extract, 20-40ml/L of acetic acid and 10-30ml/L of ethanol; the culture conditions of the seed liquid are 28-32 ℃, 150-.

Preferably, the strain immobilization method in the step (2) is to add the strain seed liquid and the wine liquid which are cultured to the logarithmic phase into the reactor according to the volume ratio of 1:3-1:5, and the fermentation time is 12-48 h.

Preferably, the wine liquid in the step (2) is grain wine or fruit wine, wherein the grain wine is one or more of rice wine, edible alcohol, sorghum wine, wheat wine, potato wine and sweet potato wine, and the fruit wine is one or more of cider, grape wine, lychee wine, pineapple wine, green plum wine, blueberry wine and strawberry wine.

Preferably, the immobilized fermentation method in the step (2) is repeated batch fermentation or continuous fermentation, the fermentation temperature is 28-30 ℃, the rotation speed is 120-150r/min, and the ventilation volume is 0.1-0.2 vvm.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:

(1) the immobilization and the fermentation are carried out simultaneously, so that the immobilization culture time and a culture medium in the fermentation process are saved, the fermentation time is shortened, and the production benefit is improved; meanwhile, the cells adapt to the fermentation environment earlier, so that the method has the function of domesticating the cells and shortens the lag phase in repeated batch fermentation;

(2) compared with a vertical barrel-shaped immobilized frame structure, the spiral barrel-shaped carrier frame structure provides a larger surface area for thallus adsorption, so that thallus participating in fermentation reaction is increased, and the fermentation speed is increased; compared with a three-clamping-plate type immobilized frame structure parallel to the cross section of the reactor, the spiral barrel-shaped carrier frame structure not only increases the adsorption surface of thalli, but also can reduce the flowing resistance of fermentation liquor, and is beneficial to the contact of gas-liquid-solid phases in fermentation;

(3) the natural materials adopted by the method have low cost and no toxicity, have no toxic action on the acetic acid bacteria, and do not influence the growth and metabolism of the acetic acid bacteria; the material is not easy to be decomposed by microorganisms, the service cycle is long, and harmful and toxic substances are not introduced or generated in the fermentation process; the material as an immobilized carrier has the characteristics of high void ratio and specific surface area, so that the mass transfer efficiency and the oxygen transfer efficiency of the material are obviously higher than those of the traditional immobilization mode;

(4) cells in the immobilized reactor are adsorbed on the surface of a fiber material to form a biological membrane, so that the cell density is improved to a great extent, and the formation of the biological membrane is used as a natural barrier of acetic acid bacteria, so that the ethanol and acetic acid tolerance of the immobilized cells is greatly enhanced compared with free cells; the biological membrane immobilization mode formed by the acetic acid bacteria automatically avoids physical and chemical damages to cells in the process of immobilizing other cells;

(5) in the process of repeated batch fermentation, the immobilized thalli in the immobilization reactor can be directly used as seeds for each batch of fermentation, so that the use efficiency of the strains is improved; the quantity of bacteria with a certain base number in the reactor greatly shortens the lag phase among fermentation batches and shortens the fermentation time; the substrate for strain proliferation is reduced in each batch of fermentation, more ethanol is used for synthesizing acetic acid, and the utilization rate of the substrate is improved;

(6) the immobilized acetic acid bacteria fermentation has the advantages of strong cell environment tolerance, high substrate utilization rate, high fermentation yield and the like, is used for producing the high-acidity white vinegar, further improves the acidity of the white vinegar, and improves the fermentation process of the high-acidity white vinegar.

Drawings

FIG. 1 is a schematic view of the structure of an immobilization support according to the present invention;

FIG. 2 is a schematic diagram of the immobilization reaction system of the present invention;

FIG. 3 is a schematic diagram of another immobilization reaction system according to the present invention.

Detailed Description

The technical solution of the present invention is further illustrated by the following examples.

Example 1: apple vinegar is produced by taking apple wine as a raw material and adopting immobilized acetic acid bacteria to perform repeated batch fermentation

As shown in fig. 1, 2cm × 2cm diamond shell 2 is uniformly fixed on 10cm long spiral barrel-shaped immobilization support 1; as shown in figure 2, a section of iron wire 3 is welded on the spiral barrel-shaped fixed support 1, and the other end of the iron wire 3 is bent and hooked on the mouth of the triangular flask 4, so that the spiral barrel-shaped fixed support 1 is vertical to the bottom of the triangular flask 4. Then 0.3L of seed liquid and 0.6L of cider with alcohol concentration of 9% vol (obtained by fermenting cider juice and containing 0.6g/L of nutrient salt for fermentation and 1.4g/L of glucose) are added into the bottle, and the cell immobilization and fermentation are carried out at the temperature of 28-30 ℃ and the rotation speed of 120-150 r/min. When the concentration of ethanol in the fermentation liquid is lower than 1g/L, pouring out the fermentation liquid in the fermentation tank in a super clean bench, and pouring 1L of cider with the alcohol concentration of 9% vol. 10 batches of fermentation were performed in sequence.

And (3) from the beginning of the 3 rd batch, the fermentation time of each batch is shortened to 12.5h, the shortest fermentation time only needs 11h, and the total acid of the fermentation liquor reaches 8.6g/100 ml. Compared with the traditional free fermentation, the average fermentation time of all batches in the whole fermentation process is shortened by 2.6 hours compared with the free fermentation, and the average total acid yield of all batches is improved by 3.3 percent. The fermentation remained in steady state until batch 10.

Example 2: taking rice wine as raw material, adopting immobilized acetic acid bacteria to carry out repeated batch fermentation to produce rice vinegar

The bamboo shoot skin with the size of 4 multiplied by 4cm is evenly fixed on a spiral barrel-shaped bracket of a fermentation tank. As shown in FIG. 3, the spiral barrel-shaped immobilization support 1 is placed in a 50L liquid fermentation tank 5, 25L of rice wine with the alcoholic strength of 5% vol is added, and the seed solution in a shake flask is inoculated according to the inoculation amount of 20% (v/v), wherein the seed culture medium is as follows: 2g/L glucose, 2g/L peptone, 1g/L yeast extract, 20ml/L acetic acid, 10ml/L ethanol. Adjusting ventilation amount to 0.1-0.2vvm, stirring speed to 120-. When the ethanol concentration of the fermentation liquor is less than 0.5g/L, 1/2 volumes of the fermentation liquor are removed, 1/2 volumes of 10% vol rice wine are supplemented for continuous fermentation, and the fermentation is sequentially circulated for 8 batches of fermentation.

And after the 2 nd divided feeding, the fermentation enters a stable stage, and the divided feeding process is 2 times less than the process of free fermentation entering a stable stage. After the fermentation is stable, the total acid concentration of the rice vinegar reaches 9.02g/100ml, which is 0.24g/100ml higher than that of the free segmentation fed-batch fermentation, and the fermentation time is shortened by 1.7 h.

Example 3: the high acidity white vinegar is produced by using edible alcohol as a raw material and adopting immobilized acetic acid bacteria to perform repeated batch fermentation

The fermenter and immobilization support were the same as in example 2. Uniformly fixing reed leaves with the size of 4 multiplied by 4cm on a spiral barrel-shaped bracket of a fermentation tank, adding 25L of edible alcohol solution with the alcoholic strength of 5 percent vol, containing 0.9g/L of nutrient salt and 2.5g/L of glucose, inoculating the edible alcohol solution into seed liquid in a shake flask according to the inoculation amount of 30 percent (v/v), wherein the seed culture medium is as follows: glucose 4g/L, peptone 4g/L, yeast extract 3g/L, acetic acid 40ml/L, ethanol 30 ml/L. Adjusting ventilation amount to 0.1-0.2vvm, stirring speed to 120-. And when the ethanol concentration of the fermentation liquor is less than 1g/L, removing 12.5L of fermentation liquor, supplementing 12.5L of 20% vol edible alcohol solution in volume to continue fermentation, circulating, removing 12.5L of fermentation liquor after fermentation of each batch is finished, and adding 12.5L of 20% vol edible alcohol solution in volume.

After the fermentation is stable, the fermentation time of each batch is 16-18h, the total acid of the white vinegar can reach more than 18.7g/100ml, and the concentration of the total acid is improved by 5.2% compared with the concentration of the free fermentation total acid under the same condition.

Example 4: rice vinegar is produced by taking rice wine as raw material and adopting immobilized acetic acid bacteria to carry out continuous fermentation

50L of acetic acid bacteria seed culture medium (glucose 3g/L, peptone 3g/L, yeast extract 2g/L, acetic acid 30ml/L, ethanol 30ml/L) was charged into a 100L fermenter, and inoculated with activated acetic acid bacteria in a shake flask for seed liquid propagation. Selecting 500L liquid fermentation tank, uniformly fixing 4cm palm silk on the net-shaped support of the fermentation tank, and pumping into 50L seed liquid and 200L 5% vol rice wine. Adjusting ventilation amount to 0.1-0.2vvm, stirring speed to 120-.

After the fermentation is stable, the total acid content of the fermentation liquor can reach 9.7g/100ml, the acid production rate is 0.83g/100ml/h, the total acid concentration is improved by 3.5 percent compared with the continuous free fermentation, and the acid production rate is improved by 12 percent.

Example 5: survival rate of cells in different fermentation modes

Experimental groups: carrying out fermentation experiments of 5 batches by adopting the method of example 2, and sampling and detecting the cell decay rate when the fermentation of each batch is finished;

control group: fermentation is carried out in a free mode, namely: the same fermentation equipment as that of example 2 was not supplemented with a fixed carrier, and other culture methods and conditions were the same as those of example 2, and samples were taken after the end of each fermentation batch to examine the cell death. The specific test results are shown in Table 1.

TABLE 1 survival of cells in different fermentation regimes

Under the same batch, in the immobilized fermentation method, the survival rate of the cells after the cells are fermented to the third batch is obviously higher than that of the cells in the free fermentation mode; in the immobilized fermentation method, the survival rate of the cells after the cells are fermented to the fifth batch still reaches more than 80 percent and is still obviously higher than that of the cells in a free fermentation mode. Therefore, the immobilized fermentation method improves the survival rate of cells and is beneficial to fermentation.

Claims (10)

1. The method for immobilizing fermented vinegar is characterized by comprising the following steps of:

(1) constructing a fixed reaction system: an immobilization bracket is arranged in the reactor, and an immobilization carrier is fixed on the immobilization bracket;

(2) strain immobilization and immobilized fermentation: adding strain seed liquid and wine liquid into the fixed reaction system constructed in the step (1), and simultaneously carrying out strain immobilization and immobilized fermentation.

2. The method for immobilizing fermented vinegar according to claim 1, wherein the immobilizing scaffold in step (1) has a spiral barrel shape, and the barrel has a grid-like hollow structure.

3. The method for immobilizing fermented vinegar according to claim 2, wherein the immobilizing scaffold in step (1) has a double helix barrel shape.

4. The method for immobilizing fermented vinegar according to claim 1, wherein the immobilizing carrier is a fibrous material in step (1).

5. The method for immobilized fermented vinegar of claim 4, wherein the fiber material is one or more of bamboo shoot skin, reed leaves, palm fiber and water chestnut shell.

6. The method for immobilized fermentation of vinegar according to claim 1, wherein the strain in step (2) is Colletobacter colatoides HS-K1, which is deposited in the China general microbiological culture Collection center with the preservation number of CGMCC No. 16345.

7. The method for immobilized fermented vinegar according to claim 1, wherein the culture medium of the seed liquid of the strain in step (2) is glucose 2-4g/L, peptone 2-4g/L, yeast extract 1-3g/L, acetic acid 20-40ml/L and ethanol 10-30 ml/L; the culture conditions of the seed liquid are 28-32 ℃, 150-.

8. The method for immobilizing fermented vinegar according to claim 1, wherein the strain is immobilized in step (2) by adding seed liquid and wine liquid of the strain cultured to logarithmic growth phase into a reactor at a volume ratio of 1:3-1:5, and fermenting for 12-48 h.

9. The method for immobilized fermentation of edible vinegar according to claim 1, wherein the wine liquid in step (2) is grain wine or fruit wine, wherein the grain wine is one or more of rice wine, edible alcohol, sorghum wine, wheat wine, potato wine and sweet potato wine, and the fruit wine is one or more of cider, grape wine, lychee wine, pineapple wine, green plum wine, blueberry wine and strawberry wine.

10. The method for immobilized fermentation of vinegar as claimed in claim 1, wherein the immobilized fermentation in step (2) is repeated batch fermentation or continuous fermentation, the fermentation temperature is 28-30 ℃, the rotation speed is 120-.

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