CN111019847B - Issatchenkia terricola and application thereof in citric acid degradation - Google Patents
Issatchenkia terricola and application thereof in citric acid degradation Download PDFInfo
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- CN111019847B CN111019847B CN201911331670.0A CN201911331670A CN111019847B CN 111019847 B CN111019847 B CN 111019847B CN 201911331670 A CN201911331670 A CN 201911331670A CN 111019847 B CN111019847 B CN 111019847B
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- citric acid
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- acid
- issatchenkia
- issatchenkia terricola
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
- A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
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Abstract
The invention discloses a terrestrial Issatchenkia terricola yeast, namely WJL-G4 and application thereof in citric acid degradation, wherein the terrestrial Issatchenkia terricola yeast is named as terrestrial Issatchenkia terricola yeast WJL-G4, and the classification name of the strain is named as terrestrial Issatchenkia terricola yeast. The strain is preserved in the China general microbiological culture Collection center in 2019, 10 and 21 months, and the preservation number is CGMCC No. 18712. The strain is obtained from fresh red raspberry fruits in a red raspberry orchard, and can grow by taking citric acid as a unique carbon source. The ITS nucleotide sequence of the strain is shown in a sequence table. The invention also discloses application of Issatchenkia terricola WJL-G4 in degrading citric acid and degrading citric acid in raspberry juice. The strain disclosed by the invention has excellent capacity of degrading citric acid, can be applied to degrading citric acid and biologically degrading high-citric-acid fruits, and has important application value in the aspects of improving the quality of processed products of the citric-acid fruits and the like.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to a terrestrial issatchenkia terricola and application thereof in citric acid degradation.
Background
Organic acid is a natural compound existing in fruits and vegetables, and the type and content of the organic acid are important factors influencing the sensory characteristics of the fruits and vegetables; meanwhile, the method is also an important factor influencing the flavor, taste and quality of products such as fruit juice, fruit wine, beverage and the like processed by fruits and vegetables. Organic acid endows the fruits and vegetables with refreshing and pleasant sour feeling, but the fruit and vegetable tastes sour and astringent and unpalatable due to the over high content of the organic acid; the variety and content of organic acids in different kinds of fruits vary widely; for example, the acidity of the red raspberry is high, the content of organic acids is high, the main organic acid is citric acid, the acidity is high, the sugar-acid ratio of the red raspberry is not coordinated, the taste is not good, the formed products such as fruit juice or fruit wine and other beverages need to obtain good taste, the strong acid feeling caused by the high content of the citric acid is overcome, the proper sugar-acid ratio is obtained, sugar is added, the sugar is contradictory to low-sugar diet advocated by the healthy diet concept, the cost of the products is increased by adding a large amount of sugar, and the resource of the sugar is wasted, so the high citric acid is a bottleneck problem in the development and utilization of the products of the red raspberry and the citric acid type fruits similar to the red raspberry, and urgent breakthrough is needed.
Related studies have shown that high concentrations of organic acids and low pH in fruit-based beverages have an erosive effect on human enamel and dentin. Therefore, the method effectively reduces the citric acid content in the citric acid type fruits, improves the pH value, breaks through the processing bottleneck problem caused by high citric acid, simultaneously retains active ingredients and forms good flavor, and becomes a hotspot of research.
Citric acid (critic acid), chemically named 3-hydroxy-1, 3, 5-pentanedioic acid, with molecular formula C6H8O7Is the major intermediate metabolite of the tricarboxylic acid cycle, the tricarboxylic acid. The natural citric acid is widely distributed in nature and mainly exists in fruits such as lemon, orange, pineapple, hawthorn, kiwi fruit, sea buckthorn, green plum, waxberry, blackcurrant, raspberry, indigo honeysuckle and the like.
The effective method is adopted to carry out deacidification treatment on the high-citric acid type fruits, the pH value is increased, so that processed products with excellent quality are produced, and the method has important significance for prolonging the industrial chain of the fruits, forming high-quality products and enriching the market.
The current common acid reduction method mainly comprises the following steps: chemical deacidification method, physical deacidification method and biological deacidification method. The chemical acid-reducing method is to reduce acid by adding a chemical acid-reducing agent, and the common acid-reducing agent comprises the following components: CaCO3、K2CO3、KHCO3、Na2CO3And chemical reagents such as the following. In the processing of fruit juice or fruit wine and other products, the acid reducing effect is obvious after chemical reagents are introduced, the operation is simple, but the outstanding defects are that the raw material components and the color are obviously influenced, and the introduced metal ions not only cause the turbidity and even the precipitation of the fruit juice, but also influence the sensory quality, so the method is not favorable for producing high-quality fruit and vegetable processing productsAnd (5) preparing the product. The commonly used physical acid reduction method comprises an ion exchange resin acid reduction method, an electrodialysis acid reduction method, a low-temperature freezing acid reduction method and the like. For example, the low-temperature freezing deacidification method can utilize a freezing device to cool the fruit wine, so that tartrate in the wine body is crystallized and precipitated, thereby achieving the purpose of deacidification, and is mainly applied to the deacidification of tartaric acid. The ion exchange resin acid reduction method has great influence on products with darker colors such as fruit and vegetable juice, has a certain adsorption effect on sugar and partial pigment substances while adsorbing organic acid, and obviously changes the sensory quality of the products. The biological acid reduction method is characterized in that organic acid is used as a carbon source by microorganisms, and the organic acid is utilized and decomposed, so that the purpose of acid reduction is achieved. One is the malic acid-lactic acid fermentation (MLF), such as Oenococcus (Oenococcus), Leuconostoc (Leuconostoc), Lactobacillus (Lactobacillus) and Pediococcus (Pediococcus), which can convert malic acid into lactic acid and CO more specifically by malic acid-lactic acid fermentation2Thereby achieving the purpose of reducing acid; however, this method is not suitable for citric acid deacidification. The other is fermentation with yeast, so that organic acid is degraded through relevant metabolism.
A patent (publication number: CN101870955A) published by the national intellectual property office discloses a strain of Issatchenkia terrestris which can degrade malic acid and citric acid in fruit wine at the same time, the strain is collected from vineyard soil, can degrade 5g/L of malic acid or citric acid, and the acid reduction rate is more than 80%. However, for most citric acid type fruits, the citric acid content in the fruit juice is far higher than 5g/L, for example, the citric acid content in the raspberry juice is about 20-25 g/L, and varies according to varieties, maturity, picking period and the like, and even is higher than 25 g/L; therefore, from the published data, the Issatchenkia terrestris of the patent is not suitable for deacidification of fruits with high citric acid content.
Another patent (publication number: CN108676736A) published by the national intellectual Property office separates and obtains a strain of pichia pastoris from citrus garden soil, the pichia pastoris can tolerate 17.5g/L of citric acid, and the pichia pastoris can reduce the kiwi fruit juice with the initial citric acid content of 12.85g/L to 10.89g/L through fermentation; biological acid reduction tests of the blueberry fruit wine show that the citric acid content can be reduced from 0.81g/L to 0.52 g/L. Therefore, the pichia pastoris is not suitable for reducing the acidity of fruits with high citric acid content.
In conclusion, the physical deacidification method and the chemical deacidification method have the problem that the sensory quality of the processed product is obviously influenced for the deacidification of the high-citric acid type fruit product, while the biological deacidification method is used for the deacidification of malic acid, and the method is not suitable for the deacidification of the citric acid, so that the strain which is screened and obtained to efficiently degrade the citric acid is the main breakthrough point of the biological deacidification of the citric acid type fruit.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a terrestrial issatchenkia strain and application thereof in citric acid degradation.
Thereby solving the problems of less biodegradation amount, low degradation rate and poor effect of the existing microorganism on the high-content citric acid.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention discloses a terrestrial Issatchenkia yeast, which is named as terrestrial Issatchenkia terricola WJL-G4, wherein the terrestrial Issatchenkia terricola WJL-G4 is preserved in China general microbiological culture Collection center, and is called as follows: CGMCC with the preservation number of: CGMCC No. 18712; the preservation date is as follows: 21/10/2019, the preservation address is as follows: china, Beijing, China academy of sciences and microbiology research institute strain preservation center.
The nucleotide sequence of ITS of the WJL-G4 of the Issatchenkia terricola (Issatchenkia terricola) is shown in a sequence table.
The invention relates to a screening method of Issatchenkia terricola WJL-G4, which comprises the following steps: in a culture medium with citric acid as a unique carbon source, the strain for degrading the citric acid on fresh red raspberry fruits picked in a red raspberry orchard is obtained by separation, screening and identification.
Specifically, with flora on fresh red raspberry fruits picked in a red raspberry orchard as a screening target, adding a proper amount of fresh red raspberry fruits into a 250mL conical flask containing 100mL of sterile normal saline, adding 8-12 sterile glass beads, oscillating at room temperature at 150r/min for 20min, and standing to obtain a supernatant; taking 5mL of supernatant, inoculating the supernatant into a citric acid liquid fermentation culture medium with citric acid concentration of 2g/L and citric acid as a unique carbon source, and performing shake culture for 24h in a conical flask with liquid loading capacity of 100mL/250mL and at 28 ℃ and 120 r/min. Sequentially transferring the cultured bacteria liquid under the same culture conditions, gradually increasing the concentration of citric acid in the citric acid liquid fermentation medium to 20g/L according to the concentration of 2g/L, 4g/L, 8g/L, 12g/L and 16g/L, and continuously transferring and culturing for 5 times; selecting strains with better acid reducing effect on citric acid through primary screening and secondary screening, carrying out streak pure culture on a citric acid solid fermentation culture medium, selecting a single colony repeat plate, streaking on the citric acid solid fermentation culture medium, and numbering respectively after the pure culture.
Respectively picking and inoculating the purified and numbered bacterial colonies into a citric acid liquid culture medium, respectively carrying out shake culture for 24h under the conditions of liquid loading of 100mL/250mL conical flask, 28 ℃ and 120r/min, respectively obtaining seed liquids corresponding to the numbered bacterial strains, respectively inoculating the seed liquids into a citric acid liquid fermentation culture medium with the inoculation amount of 4% (v/v), respectively carrying out culture for 3d under the conditions of liquid loading of 50mL/250mL conical flask, 28 ℃ and 120r/min, and respectively measuring the change of the citric acid content, wherein the result is shown in figure 1. The strain WJL-G4 is the strain with the strongest acid-reducing ability, the seed solution with the strain WJL-G4 is inoculated into red raspberry juice at the inoculation amount of 4% (v/v), the red raspberry juice is cultured under the conditions that the liquid loading amount is 50mL/250mL conical flask, the temperature is 28 ℃, and the temperature is 120r/min, and the change of the total acid and the pH is measured, and the result is shown in figure 2. In order to verify the acid reducing capability of the strain WJL-G4, samples were taken every 12h, and the types and content changes of organic acids were detected by liquid chromatography, and the results are shown in FIGS. 3 and 4.
The molecular identification of the strain WJL-G4 is completed by Shanghai biological engineering company Limited; and (3) performing homology comparison on the sequencing result and known sequences in the NCBI gene sequence library by using a Blast program, finding out a strain with the highest similarity, determining the biological classification status of the strain, and making a phylogenetic tree, wherein the phylogenetic tree is shown in figure 5. According to the analysis and identification results of the strain WJL-G4 (figure 6 and figure 7), physiological and biochemical characteristics (table 2) and ITS sequences (shown in a sequence table), the strain WJL-G4 is identified as the terrestrial Issatchenkia terricola yeast (Issatchenkia terricola) named as the terrestrial Issatchenkia terricola WJL-G4.
Specifically, the citric acid liquid fermentation medium is prepared by dissolving 2.0g of ammonium sulfate, 2.5g of potassium dihydrogen phosphate, 0.1g of ferrous sulfate heptahydrate, 0.5g of yeast extract and 2-20 g of citric acid in 1L of distilled water. When preparing a citric acid solid fermentation culture medium, dissolving substances except citric acid in the citric acid liquid fermentation culture medium in 800mL of distilled water, and adding 25g of agar powder; 2-20 g of citric acid is dissolved in 200mL of distilled water, sterilized respectively, cooled to 50-60 ℃, mixed uniformly and poured onto a flat plate or a test tube inclined plane.
The citric acid liquid fermentation medium has other components fixed except the concentration of citric acid changed according to the experimental requirements. If not specifically stated, the citric acid liquid fermentation medium or the citric acid solid fermentation medium has a citric acid concentration of 20 g/L.
The Issatchenkia terricola WJL-G4 provided by the invention grows faster on a wort solid culture medium, and after the culture is carried out for 2-3 days, the colony: the diameter is 3 ~ 5mm, is milk white, and circular bacterial colony has certain thickness, and the centre has a little arch, and the texture is even, and the surface is smooth, and is sticky moist, and is opaque, picks up easily, and the edge is more neat. The colony morphology is shown in FIG. 6.
And (4) microscopic observation: the cell morphology is shown in FIG. 7, the cells are round or oval, and the propagation mode is budding. Culturing in yeast ascospore-producing medium at 25 deg.C for 3d, and observing under microscope: 1-4 smooth spherical ascospores are generated in the ascomycetes. The cell morphology is shown in FIG. 7. Culturing at 28 deg.C in wort liquid culture medium for 2 days to form pellicle on the surface of the culture medium.
The culture medium for WJL-G4 preservation of the terrestrial Issatchenkia terricola comprises the following components: 10g of yeast extract powder, 20g of peptone, 20g of glucose and 25g of agar powder, metering the volume to 1L, and sterilizing. Making into slant test tube culture medium, culturing for 24 hr, and storing at 4 deg.C.
The invention also aims to provide an Issatchenkia terricola strain WJL-G4 for application in citric acid degradation. The degradation of citric acid in a medium in which citric acid is the sole carbon source and the degradation of citric acid in raspberry juice are exemplified.
The purpose of the invention can be realized by the following technical scheme:
1. the method comprises the following steps of screening Issatchenkia terricola WJL-G4 deacidification culture medium and application of citric acid degradation in the culture medium with citric acid as a unique carbon source:
(1) issatchenkia terricola (Issatchenkia terricola) WJL-G4 seed culture: selecting WJL-G4 single colony of Issatchenkia terricola (Issatchenkia terricola) on citric acid solid fermentation culture medium, inoculating into citric acid liquid fermentation culture medium, culturing in conical flask with liquid content of 100mL/250mL at 28 deg.C and 120r/min for 24 hr to obtain viable bacteria count of 8 × 107CFU/mL of Issatchenkia terricola WJL-G4 seed liquid.
(2) Optimization study of culture medium for acid reduction of Issatchenkia terricola WJL-G4
Taking citric acid liquid fermentation culture medium as reference, determining the maximum tolerance range of Issatchenkia terricola WJL-G4 to citric acid, and setting the viable count at 8 × 107CFU/mL Issatchenkia terricola WJL-G4 seed solution was inoculated into citric acid liquid fermentation medium and citric acid-tolerant liquid medium with citric acid mass concentration of 20G/L, 40G/L, 60G/L, 80G/L, 100G/L, 120G/L, 140G/L, respectively, to obtain OD600The maximum citric acid tolerance concentration of Issatchenkia terricola WJL-G4 was determined for the growth index, and the results are shown in FIG. 9. As can be seen from FIG. 9, Issatchenkia terricola WJL-G4 was able to tolerate a maximum citric acid concentration of 60G/L, good growth was achieved at a concentration of 60G/L or less, and growth was affected at a concentration of 60G/L or more.
The citric acid tolerant liquid culture medium is prepared by dissolving 2.0g of ammonium sulfate, 2.5g of monopotassium phosphate, 0.1g of ferrous sulfate heptahydrate, 0.5g of yeast extract and 20-140 g of citric acid in 1L of distilled water and sterilizing.
The concentration gradients of the citric acid adopted in the citric acid-tolerant liquid culture medium in the test are sequentially increased by 20g/L, 40g/L, 60g/L, 80g/L, 100g/L, 120g/L and 140 g/L.
Taking a basic culture medium without a nitrogen source as a blank, selecting yeast extract powder, beef extract, peptone, ammonium sulfate, urea and potassium nitrate to replace the nitrogen source in the basic culture medium to prepare the culture medium, enabling the nitrogen content to be 1g/L, and screening the most suitable nitrogen source by taking the deacidification rate and the growth amount as judgment indexes, wherein the culture conditions are as follows: the inoculum size is 1% (v/v), shaking culture is carried out at 28 ℃ and 120r/min for 48h, and the liquid loading amount is 100mL/250mL conical flask. As shown in FIG. 10, the optimum nitrogen source was yeast extract powder.
The results of screening the optimum addition amount of the optimum nitrogen source yeast extract powder by using the screened optimum addition amounts of the nitrogen source yeast extract powder as 1g/L, 5g/L, 10g/L, 15g/L and 20g/L and the deacidification rate and the growth amount as judgment indexes are shown in FIG. 11, and it can be seen from the figure that the optimum addition amount of the yeast extract powder is not less than 5g/L, and the optimum addition amount of the yeast extract powder is 5-10 g/L from the economical point of view.
The basic culture medium comprises the following components in percentage by weight: 10g of yeast extract powder, 1g of magnesium sulfate and 20g of citric acid are dissolved in 1000mL of distilled water.
Taking a basic culture medium without inorganic salt as a blank (without salt), selecting magnesium sulfate, zinc sulfate, potassium dihydrogen phosphate, manganese sulfate, sodium chloride and ferric sulfate to replace inorganic salt (magnesium sulfate) in the basic culture medium to prepare the culture medium, wherein the salt content is 0.01mol/L, the optimum inorganic salt is screened by taking the deacidification rate and the growth amount as judgment indexes, and the culture conditions are as follows: carrying out shake culture at 28 ℃ for 48h at the inoculum size of 1% (v/v) and 120r/min in a conical flask with the liquid loading amount of 100mL/250 mL; the results are shown in FIG. 12, where the most suitable inorganic salt species is magnesium sulfate.
The optimum addition amount of magnesium sulfate as inorganic salt is 0.001 mol/L, 0.005 mol/L, 0.01mol/L, 0.015 mol/L, 0.02 mol/L, and the culture conditions are as follows: carrying out shake culture at 28 ℃ for 48h at the inoculum size of 1% (v/v) and 120r/min in a conical flask with the liquid loading amount of 100mL/250 mL; the optimum amount of magnesium sulfate was selected using the deacidification rate and the growth amount as judgment indexes, and the results are shown in FIG. 13. As can be seen from FIG. 13, the influence of different amounts of magnesium sulfate on the deacidification rate and the growth rate was small, and 0.001 mol/L (0.12g/L) or more was selected as the optimum amount of magnesium sulfate, and from the economical viewpoint, 0.12 to 0.6g/L was selected.
The optimal formula of the acid reducing culture medium screened by the experiment is 5g of yeast extract powder, 0.122g of magnesium sulfate and 20g of citric acid, and the components are dissolved in 1000mL of distilled water.
(3) Issatchenkia terricola WJL-G4 Deacidification conditions and growth characteristics
Taking viable count as 8 × 107CFU/mL Issatchenkia terricola WJL-G4 seed solution with 1% (v/v) inoculation amount and 100mL/250mL conical flask in deacidification medium, respectively culturing at 25 deg.C, 28 deg.C, 30 deg.C, 33 deg.C, 35 deg.C for 48h at 120r/min, and determining deacidification rate and OD600The optimum temperature was determined and the results are shown in FIG. 14. As can be seen from FIG. 14, the optimum deacidification temperature is determined by integrating the deacidification rate and the growth amount at 28 ℃, and the optimum deacidification temperature is selected from 27 to 29 ℃ in consideration of the temperature change of the actual situation.
Taking viable count as 8 × 107CFU/mL Issatchenkia terricola WJL-G4 seed solution was inoculated in a 1% (v/v) inoculum size in 20, 40, 60, 80, 100mL/250mL Erlenmeyer flask in a deacidification medium, shake-cultured at 28 deg.C for 48h at 120r/min, and then the deacidification rate and OD were measured600The optimum liquid loading was determined and the results are shown in FIG. 15. As can be seen from FIG. 15, the difference in the acid-reducing rate is not so great when the liquid contents are 20mL/250mL and 40mL/250mL, but it is more preferable to select 40mL/250mL from an economical point of view, and in this case, the volume ratio is 16% (v/v), and it is most preferable to select 12% to 20% (v/v) in practice.
Taking viable count as 8 × 107CFU/mL Issatchenkia terricola WJL-G4 seed solution 1%, 2%, 3%, 4%, 5% (v/v), respectively, was inoculated into 100mL/250mL Erlenmeyer flask deacidification medium, shake-cultured at 28 deg.C and 120r/min for 48h, and the deacidification rate and OD were measured600To determineThe results of the inoculation are shown in FIG. 16. As can be seen from FIG. 16, the optimum inoculum size was selected from 1% to 2% (v/v).
Taking viable count as 8 × 107Inoculating CFU/mL Issatchenkia terricola WJL-G4 seed solution at an inoculation amount of 1% (v/v) in 100mL/250mL conical flask, placing in shaking culture box at rotation speed of 80r/min, 120r/min, 160r/min, 200r/min, and 240r/min, culturing at 28 deg.C for 48h, and determining acid reduction rate and OD600The optimum rotation speed is determined as shown in fig. 17. As shown in FIG. 17, the acid reduction rate is the highest when the rotation speed is 240r/min, but the difference between the acid reduction rate and 240r/min is not large when the rotation speed is 200r/min, and 160-240 r/min is selected as the optimal rotation speed in consideration of comprehensive factors such as the use safety of the instrument.
(4) Efficient citric acid degrading bacteria Issatchenkia terricola WJL-G4 degrading citric acid of different concentrations under optimal citric acid degrading condition
And (3) taking the deacidification culture medium as a control, and changing the concentration of citric acid in the deacidification culture medium into: 5g/L, 10g/L, 20g/L, 40g/L, 80g/L, 120g/L, 160g/L and 200g/L respectively, and the number of the accessed viable bacteria is 8 multiplied by 107CFU/mL of Issatchenkia terricola WJL-G4 seed liquid, and the degrading capability of the strain Issatchenkia terricola WJL-G4 to different concentrations of citric acid under the optimal acid reducing condition is studied, wherein the culture condition is as follows: the inoculation amount is 2 percent (v/v), shaking culture is carried out for 48 hours at 28 ℃ at 200r/min, and the liquid loading amount is 40mL/250mL conical flask; the results are shown in Table 1, using the acid-reducing rate and the amount of growth as indices.
TABLE 1 acid reducing Effect of Issatchenkia terricola WJL-G4 on different concentrations of citric acid
At a citric acid concentration of 5g/L in Table 1, the citric acid degradation rate in 24 hours has reached 91.62%; at the citric acid concentration of 10g/L, the citric acid degradation rate reaches 92.28% in 24 hours; when the concentration of the citric acid is 20g/L, the degradation rate of the citric acid reaches 85.37% in 24 hours, so that the deacidification time can be selected from 24-48 hours according to actual conditions.
According to the comprehensive experiment results, the optimal deacidification culture medium formula of WJL-G4 of Issatchenkia terricola (Issatchenkia terricola) is obtained by taking citric acid as the only carbon source of the culture medium, wherein the citric acid concentration is 5-40G/L, the yeast extract powder is 5-10G/L, and the magnesium sulfate concentration is 0.12-0.6G/L; the optimal deacidification culture condition is that the temperature is 27-29 ℃, the liquid loading amount is 12-20% (v/v), the inoculation amount is 1-2% (v/v), the rotating speed is 160-240 r/min, and the time is 24-48 h, and the deacidification rate reaches 85.37-99.90 percent.
2. The application of Issatchenkia terricola WJL-G4 in degrading citric acid in red raspberry juice comprises the following steps:
(1) issatchenkia terricola (Issatchenkia terricola) WJL-G4 seed culture: selecting WJL-G4 single colony of Issatchenkia terricola (Issatchenkia terricola) on citric acid solid fermentation culture medium, inoculating into citric acid liquid fermentation culture medium, culturing in conical flask with liquid content of 100mL/250mL at 28 deg.C and 120r/min for 24 hr to obtain viable bacteria count of 8 × 107CFU/mL of Issatchenkia terricola WJL-G4 seed liquid.
(2) Issatchenkia terricola (Issatchenkia terricola) WJL-G4 was tested for its ability to degrade organic acids in red raspberry juice: inoculating 4% (v/v) viable bacteria in 25% (v/v) of raspberry juice to obtain a mixture with viable bacteria count of 8 × 107CFU/mL of Issatchenkia terricola WJL-G4 seed liquid, cultured at 28 ℃ for 72h with shaking at 120r/min, and sampled every 12h, to determine the degradation capability and change of Issatchenkia terricola WJL-G4 for organic acid of raspberry juice, the results are shown in FIG. 4.
(3) Effect of fermentation temperature on degradation of citric acid by indigenous Issatchenkia terricola WJL-G4: inoculating 4% (v/v) viable bacteria in red raspberry juice with the number of 8 × 107CFU/mL of Issatchenkia terricola WJL-G4 seed solution, performing shake fermentation for 48h at 25 deg.C, 28 deg.C, 30 deg.C, 33 deg.C, 35 deg.C, 25% (v/v) liquid loading amount and rotation speed of 120r/min, measuring acid reduction rate and pH in raspberry juice, and examining the fermentation temperature for Issatchenkia terricolaThe effect of Saccharomyces cerevisiae (Issatchenkia terricola) WJL-G4 deacidification is shown in FIG. 18. As can be seen from FIG. 18, the optimum temperature for degrading the red raspberry juice by Issatchenkia terricola WJL-G4 is 28 ℃, and the temperature can be 27-29 ℃ in practical operation.
(4) Effect of fermentation time on degradation of citric acid by indigenous Issatchenkia terricola (lsatchenkia terricola) WJL-G4: inoculating 4% (v/v) viable bacteria in red raspberry juice with the number of 8 × 107CFU/mL of WJL-G4 seed liquid of Issatchenkia terricola (Issatchenkia terricola) was subjected to shaking fermentation for 12h, 24h, 36h, 48h, 60h and 72h at a temperature of 28 ℃, a liquid loading amount of 25% (v/v) and a rotation speed of 120r/min, the acid reduction rate and pH value of the raspberry juice were measured, and the influence of the fermentation time on the WJL-G4 acid reduction of Issatchenkia terricola (Issatchenkia terricola) was examined, and the result is shown in FIG. 19. The acid reducing speed is the highest in 24-36 h, the acid reducing rate is close to 80% in 36h, and in practice, 36-48 h can be adopted.
(5) Effect of inoculum size on citric acid degradation by isaschenkia terricola WJL-G4: inoculating 0.5%, 1%, 2%, 3%, 4%, 5%, 6% (v/v) viable bacteria of 8 × 10 into red raspberry juice7CFU/mL Issatchenkia terricola WJL-G4 seed liquid, shaking and fermenting at 28 deg.C and 120r/min for 48h, measuring acid reduction rate and pH of red raspberry juice, and examining the effect of inoculation amount on acid reduction of Issatchenkia terricola WJL-G4, the result is shown in FIG. 20. The optimum inoculum size was 4%.
(6) Effect of liquid loading on citric acid degradation by Issatchenkia terricola WJL-G4: inoculating 4% (v/v) viable bacteria in red raspberry juice with the number of 8 × 107CFU/mL of Issatchenkia terricola WJL-G4 seed solution was subjected to shake fermentation at 28 ℃ at a rotation speed of 120r/min for 48 hours with liquid contents of 15%, 20%, 25%, 30%, 35%, and 40% (v/v), and then the acid reduction rate and pH in raspberry juice were measured, and the effect of the liquid contents on the acid reduction of Issatchenkia terricola WJL-G4 was examined, and the results are shown in FIG. 21. When the liquid loading amount is 15-25%, the acid reduction rate is over 85%.
(7) Effect of rotational speed on citric acid degradation by isasseki terricola WJL-G4: inoculating 4% (v/v) viable bacteria in red raspberry juice with the number of 8 × 107CFU/mL of Issatchenkia terricola WJL-G4 seed liquid was subjected to shake fermentation at 28 deg.C, 25% (v/v) liquid loading, 100r/min, 120r/min, 140r/min, 160r/min and 180r/min for 48h, and then the acid reduction rate and pH in raspberry juice were measured, and the influence of the rotation speed on the acid reduction of Issatchenkia terricola WJL-G4 was examined, and the results are shown in FIG. 22. As can be seen from the figure, when the rotating speed is more than 120r/min, the acid reduction rate is not changed greatly and is maintained at more than 80 percent. The rotating speed is 120 r/min-180 r/min.
(8) According to the invention, fresh raspberry fruits are used as separation materials, a yeast capable of efficiently degrading citric acid, namely Issatchenkia terricola WJL-G4, is separated and purified, and is applied to degrading citric acid.
Compared with the existing biological deacidification technology, the WJL-G4 strain of Issatchenkia terricola has high citric acid degradation rate and high speed, can efficiently degrade high-concentration citric acid, the citric acid degradation rate in a 20G/L citric acid liquid fermentation culture medium reaches over 90 percent after fermentation for 48 hours, and the total acid degradation rate reaches 85.58 percent and the citric acid degradation rate reaches 99.10 percent for red raspberry juice with the total acid content of 25.15G/L and the citric acid content of 22.87G/L, so that the biological deacidification technology is suitable for being used in high-concentration citric acid juice, such as biological deacidification of citric acid fruits and application of degrading the high-concentration citric acid, and has important practical significance for improving the quality of processed products of the citric acid fruits, saving the use of saccharides in the citric acid type fruit processing, enriching the types of the processed products of the fruits and the like in various aspects.
Measurement method used in the present invention
OD600: measuring the bacteria liquid in-wave by spectrophotometryThe absorbance at 600nm indicates the amount of growth of the strain.
Total acid and pH determination: the total acid is measured by citric acid according to a potentiometric titration method in GB/T12456-; the pH was measured with a pH meter.
And (3) acid reduction rate determination:
in the formula, A0The mass concentration of total acid or citric acid in the sample before acid reduction; a. the1The mass concentration of total acid or citric acid after acid reduction.
Organic acid component determination:
the liquid chromatography conditions for organic acid determination are specifically as follows: agilent ZORBAX extended-C18 (250 mm. times.4.6 mm), mobile phase A to mobile phase B volume ratio of 97:3, mobile phase A: 0.5% KH at pH 2.52PO4Solution, mobile phase B: methanol, liquid phase conditions: isocratic elution for 10min, flow rate: 0.7mL/min, sample size: 10 μ L, column temperature: 35 ℃, detection wavelength: 210 nm. And measuring the mixed standard and the sample in turn. And (4) making a standard curve by using the concentration of the standard sample to the peak area, and calculating the content of the organic acid in the sample solution by adopting an external standard method.
Standard curve: respectively dissolving with purified water to prepare mixed standard solutions of L-malic acid, alpha-ketoglutaric acid, citric acid and succinic acid with different concentrations, filtering with a 0.22 mu m microporous filter membrane, and performing HPLC analysis on the solution on a computer to obtain a regression equation and a correlation coefficient of a peak area (x) and an organic acid mass concentration (y).
Sample treatment: centrifuging the sample (4000r/min, 15min), sucking the supernatant into a volumetric flask, fixing the volume with distilled water, mixing uniformly, and diluting to a proper concentration. The samples were filtered through a 0.22 μm filter and tested on the machine, and the test was repeated 3 times for each sample.
Drawings
FIG. 1 is a schematic diagram showing the variation of citric acid in a citric acid liquid fermentation medium of 8 types of acid-reducing bacteria screened by the method.
FIG. 2 is a schematic diagram showing the change of total acid and pH of Issatchenkia terricola WJL-G4 in fermenting red raspberry juice.
FIG. 3 is a high performance liquid chromatogram of the organic acid assay of the red raspberry juice fermentation at 0d and 3d of the WJL-G4 of Issatchenkia terricola (Issatchenkia terricola) of the present invention.
FIG. 4 is a schematic diagram showing the change of organic acids in WJL-G4 of Issatchenkia terricola (Issatchenkia terricola) in fermenting raspberry juice.
FIG. 5 is a phylogenetic tree of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 6 is a photograph showing the colony morphology of the plate of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 7 is a photograph showing the morphological observation of 1000 times of Meilan stained cells of Issatchenkia terricola WJL-G4 according to the present invention under an optical microscope.
FIG. 8 is a agarose gel electrophoresis of the PCR product WJL-G4 of Issatchenkia terricola (Issatchenkia terricola) of the present invention, with the ITS band of WJL-G4 of Issatchenkia terricola (Issatchenkia terricola) marked WJL-G4.
FIG. 9 shows the tolerance of Issatchenkia terricola WJL-G4 to different citric acid concentrations.
FIG. 10 shows the effect of different nitrogen source species on the acid reduction rate and growth of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 11 shows the effect of the addition amount of yeast extract powder on the acid reduction rate and growth of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 12 shows the effect of different inorganic salts on the acid reduction rate and growth of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 13 shows the effect of magnesium sulfate addition on the acid reduction rate and growth of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 14 shows the effect of different temperatures on the acid reduction rate and growth of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 15 shows the effect of different liquid contents on the acid reduction rate and growth of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 16 shows the effect of different inoculation amounts on the acid reduction rate and growth amount of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 17 shows the effect of different rotation speeds on the acid reduction rate and growth of Issatchenkia terricola WJL-G4 according to the present invention.
FIG. 18 is a graph showing the effect of the Issatchenkia terricola WJL-G4 in degrading citric acid in raspberry juice under different temperature conditions.
FIG. 19 is a graph showing the effect of Issatchenkia terricola WJL-G4 in degrading citric acid in red raspberry juice under different time conditions.
FIG. 20 is a graph showing the effect of Issatchenkia terricola WJL-G4 in degrading citric acid in raspberry juice under different inoculation amounts.
FIG. 21 is a graph showing the effect of WJL-G4 of Issatchenkia terricola on degrading citric acid in raspberry juice at different liquid loading levels.
FIG. 22 is a graph showing the effect of Issatchenkia terricola WJL-G4 in degrading citric acid in raspberry juice at different rotation speeds.
Biological preservation Instructions
The classification strain of the terrestrial Issatchenkia terricola WJL-G4 is named as terrestrial Issatchenkia terricola, and is preserved in the general microbiological culture collection center of China Committee for culture Collection of microorganisms in 2019 and 21 days 10 and 2019, and the name is as follows: CGMCC with the preservation number of: CGMCC No. 18712; the preservation address is as follows: china, Beijing, China academy of sciences and microbiology research institute strain preservation center.
Detailed Description
The technical solutions in the embodiments of the present invention will be described more clearly and completely with reference to the following descriptions of specific embodiments of the present invention and the accompanying drawings, but the embodiments are a part of all the embodiments. All other embodiments, which do not exceed the scope of the innovative inventions of the present invention, are within the scope of protection of the present invention.
The experimental procedures used are described in detail below with reference to specific examples, which are conventional unless otherwise specified.
Example 1
Screening, separating and purifying Issatchenkia terricola WJL-G4 as efficient citric acid degrading bacteria
Collecting fresh fruits in a red raspberry orchard as a sample, adding a proper amount of the fresh red raspberry fruits into a 250mL conical flask containing 100mL of sterile normal saline, adding 8-12 sterile glass beads, oscillating at room temperature at 150r/min for 20min, and standing to obtain a supernatant. Taking 5mL of supernatant, adding the supernatant into a citric acid liquid fermentation culture medium containing citric acid as a unique carbon source for continuous transfer culture, gradually increasing the concentration of the citric acid in the citric acid liquid fermentation culture medium from 2g/L, 4g/L, 8g/L, 12g/L and 16g/L to 20g/L, continuously transferring and culturing strains with the citric acid utilization capacity, diluting and coating the cultured bacterial liquid on a citric acid solid fermentation culture medium containing 20g/L of citric acid, culturing at 28 ℃ for 2-3 days to obtain single colonies of 8 strains, selecting the single colonies to be repeatedly streaked and purified and cultured on a citric acid solid fermentation culture medium plate, and numbering the 8 strains respectively; finally, respectively inoculating the purified single colonies into a citric acid liquid fermentation culture medium, respectively oscillating for 24 hours at 28 ℃ and 120r/min in a conical flask with the liquid containing amount of 100mL/250mL to respectively obtain seed liquids of 8 numbered bacteria, respectively inoculating the seed liquids of 8 numbered bacteria into the citric acid liquid fermentation culture medium with the inoculation amount of 4% (v/v), carrying out oscillation culture for 3d at 28 ℃ and 120r/min in the conical flask with the liquid containing amount of 50mL/250mL, and determining the change of citric acid; the results are shown in FIG. 1. Among 8 numbered strains, the strain WJL-G4 has the strongest acid reduction capability and can degrade more than 90% of citric acid, so the strain WJL-G4 is selected for subsequent acid reduction research.
The strain WJL-G4 seed solution was inoculated into raspberry juice at an inoculum size of 4% (v/v), cultured in 50mL/250mL Erlenmeyer flask at 28 deg.C under shaking at 120r/min for 3d, and the total acid and pH changes during fermentation were measured, and the results are shown in FIG. 2. Compared with the red raspberry juice without inoculation under the same condition, the strain WJL-G4 has strong acid reducing capacity in the red raspberry juice, so that the total acid content can be greatly reduced, the acid reducing rate of the total acid reaches 94.66% at the 3 rd day, the pH is greatly improved, and the pH is increased to 4.89 from 3.08.
In order to verify the degradation capability of the strain WJL-G4 on citric acid, the type and content of organic acid in the raspberry fermented juice are detected by high performance liquid chromatography. Sampling every 12h, centrifuging the sample at 4000r/min for 15min, sucking the supernatant into a volumetric flask, diluting by 20 times, fixing the volume with ultrapure water, and uniformly mixing. The assay was performed after filtration through a 0.22 μm filter. The high performance liquid chromatogram is shown in FIG. 3, the retention position of citric acid is marked, and the peak area at 3d is close to 0, which indicates that the strain WJL-G4 has strong capacity of degrading citric acid.
As shown in FIG. 4, it can be seen from FIG. 4 that citric acid is the main component of the raspberry organic acid, and the citric acid content in the raspberry juice is 22.87 g/L. The contents of citric acid and malic acid in the fruit juice inoculated with the strain WJL-G4 are reduced as a whole along with the extension of the fermentation time. After 3d fermentation, compared with unfermented raspberry juice, the citric acid content is reduced by 99.37%, and the malic acid content is reduced by 47.96%; the contents of alpha-ketoglutaric acid and succinic acid are in a trend of increasing firstly and then decreasing secondly, the content of succinic acid is decreased by 57.54%, and the content of alpha-ketoglutaric acid is decreased by 47.50%, which shows that the strain WJL-G4 not only has the capacity of efficiently degrading citric acid, but also has better degradation effect on other organic acids such as malic acid, alpha-ketoglutaric acid and succinic acid.
The citric acid liquid fermentation medium is prepared by dissolving 2.0g of ammonium sulfate, 2.5g of monopotassium phosphate, 0.1g of ferrous sulfate heptahydrate, 0.5g of yeast extract and 2-20 g of citric acid in 1L of distilled water.
When a citric acid solid fermentation culture medium is prepared, 2.0g of ammonium sulfate, 2.5g of monopotassium phosphate, 0.1g of ferrous sulfate heptahydrate, 0.5g of yeast extract and 25g of agar powder are dissolved in 800mL of distilled water; adding 2-20 g of citric acid into 200mL of distilled water, sterilizing at 121 ℃ for 20min, cooling to 50-60 ℃, mixing uniformly, and pouring the mixture onto a flat plate or a test tube inclined plane.
If not specifically stated or emphasized, the citric acid content in the citric acid liquid fermentation medium or the citric acid solid fermentation medium is 20 g/L.
Example 2
Physiological and biochemical identification of citric acid efficient degradation bacterium Issatchenkia terricola WJL-G4
And performing physiological and biochemical identification on the separated and purified strain WJL-G4, wherein the physiological and biochemical identification comprises colony morphology observation, Meilan dyeing observation, sugar fermentation, carbon source assimilation and nitrogen source assimilation experiments and other physiological and biochemical experiments, and the experiment method adopts a conventional experiment method in the field.
The result shows that the bacterial colony is milky white and circular after being cultured in a wort solid culture medium for 2-3 days, the diameter of the bacterial colony is 3-5 mm, the middle of the bacterial colony is slightly convex, the texture is uniform, the surface is smooth, moist and sticky, the bacterial colony is easy to pick up, and the edge is neat and opaque. As shown in fig. 6.
Microscopic morphological characteristics of the strain WJL-G4 after the staining of methylene blue are observed by a microscope, cells are from circular to oval, and the propagation mode is budding. As shown in fig. 7. Culturing the seeds in a yeast ascospore-producing culture medium for 3 days at 25 ℃, and observing by a microscope to generate 1-4 smooth spherical ascospores in the ascospores. Culturing at 28 deg.C in wort liquid culture medium for 2 days to form pellicle on the surface of the culture medium.
The results of physiological and biochemical tests of the strain WJL-G4 are shown in Table 2.
TABLE 2 physiological and biochemical characteristics of strain WJL-G4
The strain WJL-G4 can utilize glucose, cannot utilize other sugar sources such as lactose, galactose, sucrose and the like, can assimilate ammonium sulfate and cannot assimilate nitrate.
Example 3
Molecular identification of citric acid efficient degradation bacterium Issatchenkia terricola WJL-G4
The specific steps are as follows, extracting the genome DNA of the strain WJL-G4 according to the genome DNA extraction kit of the Shanghai's fungi, and using the genome DNA as a template and using the universal primer of the ITS zone to amplify the ITS zone gene of the primer. Amplification conditions for amplification of ITS sequences: pre-denaturation at 94 ℃ for 4.0 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1.0min, and 30 cycles; extension at 72 ℃ for 10.0 min. Sequencing was performed by Shanghai Bioengineering, Inc. The agarose gel electrophoresis pattern of the PCR product is shown in FIG. 8.
And logging in an http:// blast.ncbi.nlm.nih.gov/blast.cgi website after sequencing, and comparing and analyzing the obtained ITS sequence with a known sequence in Genbank by using a Blast program. In the NCBI (national Center for Biotechnology information) database, the ITS sequence of the strain was aligned analytically with the known sequences in Genbank by the online BLAST (basic Local Alignment Search tool) program to create a phylogenetic tree of the strain WJL-G4, the results are shown in FIG. 5. The strains containing the gene segments with higher homology are Issatchenkia terricola, and the sequence homology can reach more than 99 percent. The ITS nucleotide sequence of the strain WJL-G4 is shown in a sequence table.
According to morphological characteristics (figure 6 and figure 7), physiological and biochemical characteristics (table 2) and ITS sequence analysis and identification of the strain WJL-G4, the strain WJL-G4 is identified as Issatchenkia terricola, namely Issatchenkia terricola, and named as Issatchenkia terricola WJL-G4.
Example 4
Citric acid tolerance test of Issatchenkia terricola WJL-G4
To explore the citric acid tolerance concentration of Issatchenkia terricola WJL-G4, Issatchenkia terricola WJL-G4 was cultured in a citric acid tolerant liquid medium with citric acid as the sole carbon source, and the results are shown in FIG. 9.
Specifically, Issatchenkia terricola WJL-G4 seed solution was inoculated into a citric acid-tolerant liquid medium at an inoculum size of 4% (v/v), and the citric acid-tolerant liquid medium used in the test was gradually increased in concentration by a gradient of 20G/L, 40G/L, 60G/L, 80G/L, 100G/L, 120G/L and 140G/L. The amount of growth was measured by shaking culture at 28 ℃ and 120r/min for 48 hours in a 40mL/250mL Erlenmeyer flask, and the change in growth of Issatchenkia terricola WJL-G4 was measured as the absorbance at 600nm, and the results are shown in FIG. 9. The pH of the citrate-tolerant liquid medium for each concentration gradient is shown in table 3. Compared with the concentration of citric acid of 20g/L, when the concentration of citric acid is less than 80g/L, the strain growth amount is better and is not inhibited basically. When the citric acid concentration was 80g/L, the growth amount showed a significant (P <0.05) decrease. The concentration of citric acid is more than or equal to 80G/L, the strain number shows a remarkable descending trend, so that the maximum concentration of citric acid which can be tolerated by Issatchenkia terricola WJL-G4 is 60G/L, and the concentration of citric acid is less than or equal to 60G/L, and the Issatchenkia terricola WJL-G4 can normally grow and is not influenced by the concentration change of citric acid. As shown in Table 3, the maximum pH tolerance of the strain is 2.41, and the growth is affected when the pH of the strain growth environment is less than 2.41. Therefore, Issatchenkia terricola WJL-G4 is tolerant to citric acid at a maximum mass concentration of 60G/L, corresponding to a pH of 2.41.
The citric acid tolerant liquid culture medium is prepared by dissolving 2.0g of ammonium sulfate, 2.5g of potassium dihydrogen phosphate, 0.1g of ferrous sulfate heptahydrate, 0.5g of yeast extract and 20-140 g of citric acid in 1L of distilled water.
The concentrations of citric acid used in the above-described citric acid-tolerant liquid medium in the test were gradually increased in the order of 20g/L, 40g/L, 60g/L, 80g/L, 100g/L, 120g/L and 140 g/L.
The citric acid liquid fermentation culture medium has a citric acid concentration of 2-20 g/L, and the citric acid tolerance liquid culture medium has a citric acid concentration of 20-140 g/L.
TABLE 3 pH corresponding to different citric acid concentrations in citric acid-tolerant liquid medium
Example 5
Efficient citric acid degrading bacteria Issatchenkia terricola WJL-G4 degrading citric acid of different concentrations under optimal citric acid degrading condition
The optimal deacidification liquid culture medium formula of WJL-G4 of Issatchenkia terricola (Issatchenkia terricola) is obtained by taking citric acid as a unique carbon source of the culture medium, wherein the citric acid concentration is 5-40G/L, the yeast extract powder is 3-6G/L, and the magnesium sulfate concentration is 0.12-0.6G/L; the best culture condition is that the temperature is 27-29 ℃, the liquid loading amount is 15-25% (v/v), the inoculation amount is 1-2% (v/v), the rotating speed is 160-240 r/min, and the time is 24-48 h, and the acid reduction rate reaches 85.37-99.90 percent.
When the formula and the culture conditions of the deacidification liquid culture medium are as follows: 5g/L of yeast extract powder, and the concentration of magnesium sulfate is 0.12 g/L; the culture temperature was 28 ℃, the liquid loading was 16% (v/v), the inoculation amount was 1% (v/v), the rotation speed was 160r/min, and the time was 48 hours, the culture medium was started with citric acid of different concentrations, and after 48 hours of deacidification culture, the growth amount and the deacidification rate of the strain Issatchenkia terricola WJL-G4 were measured, and the results are shown in Table 1.
As can be seen from the results in the table 1 and the formula and the screening effect of the citric acid liquid fermentation medium combined with the specification, Issatchenkia terricola WJL-G4 can grow well under the conditions of different media and can achieve the ideal acid reduction effect, and the acid reduction rate of 48h fermentation is 98.51% when the citric acid concentration reaches 40G/L in the medium taking citric acid as the only carbon source; and when the concentration of the citric acid is as high as 120G/L, the acid reduction rate can still reach more than 50 percent, which shows that the Issatchenkia terricola WJL-G4 strain can tolerate the citric acid with higher concentration and has extremely strong citric acid degradation capability.
TABLE 1 acid reducing Effect of Issatchenkia terricola WJL-G4 on different concentrations of citric acid
Example 6
Application of Issatchenkia terricola WJL-G4 (Issatchenkia terricola) as efficient citric acid degradation bacterium in citric acid degradation of red raspberry juice
Selecting WJL-G4 single colony of Issatchenkia terricola (Issatchenkia terricola) on citric acid solid fermentation culture medium, inoculating into 100mL/250mL citric acid liquid fermentation culture medium in conical flask, culturing at 28 deg.C for 24 hr at 120r/min to obtain viable count of 8 × 107CFU/mL of Issatchenkia terricola WJL-G4 seed liquid.
As shown in fig. 18, the highest deacidification rate of the rubus parvifolius (Issatchenkia terricola) WJL-G4 at 28 ℃ is 85.72%, after the temperature exceeds 28 ℃, the total deacidification rate is obviously inhibited and is in a descending trend along with the temperature rise, the pH change is similar to the total deacidification trend, and when the temperature is 28 ℃, the temperature is increased from 3.08 to 4.22, which shows that the deacidification temperature at 28 ℃ is the optimum deacidification temperature of the WJL-G4 of the Issatchenkia terricola (Issatchenkia terricola) WJL-G4, so the fermentation deacidification temperature is selected to be 28 ℃.
As can be seen from fig. 19, the acid reduction rate of Issatchenkia terricola (Issatchenkia terricola) WJL-G4 on raspberry juice increases rapidly within 24-36 h, then the slow rising trend appears, the pH rises correspondingly, the acid reduction rate is 94.43% at 60h, and the pH rises from 3.08 to 4.90, which indicates that when the fermentation time is longer than 60h, Issatchenkia terricola (Issatchenkia terricola) WJL-G4 has already reduced most of citric acid in the juice, but in practical application, the lower the citric acid is the better, considering the practical situation of application, the too low the citric acid can affect the flavor and color of the raspberry juice, and comprehensively considering that the fermentation acid reduction time is preferably 36-48 h.
As can be seen from FIG. 20, when the inoculation amount is 0.5% to 4% (v/v), the deacidification rate of Issatchenkia terricola WJL-G4 increases with the increase of the inoculation amount, when the inoculation amount is 4% (v/v), the deacidification rate reaches the highest value and reaches 86.00%, and when the inoculation amount is more than 4% (v/v), the deacidification rate slightly decreases and the pH difference is not obvious, which indicates that the inoculation amount is 4% (v/v), which is the optimum inoculation amount of Issatchenkia terricola WJL-G4 deacidification, so that the deacidification inoculation amount is selected to be 4% (v/v).
As can be seen from fig. 21, the acid reduction rate decreased with the increase in the liquid content of the raspberry juice, indicating that Issatchenkia terricola WJL-G4 was an aerobic bacterium, and the smaller the liquid content, the more the strain contacted the air, and contributed to the improvement in the acid reduction rate. The difference between the liquid loading amounts of 15 percent to 25 percent (v/v) is not obvious, which shows that the liquid loading amount between 15 percent to 25 percent (v/v) is the optimal liquid loading amount for reducing the acid of Issatchenkia terricola WJL-G4, the liquid loading amount is selected to be 25 percent (v/v) by comprehensively considering the practical situation and the acid reducing effect of the application, the acid reducing rate is 86.86 percent at the time, and the pH is increased from 3.08 to 4.32.
As can be seen from FIG. 22, after the rotation speed of the shaker reached 120r/min, the rotation speed had no influence on the acid reduction rate, the acid reduction rate could be maintained at 80% or more, and when the rotation speed was 120r/min, the pH was increased from 3.08 to 5.07, indicating that the rotation speed was 120-180 r/min, which is the optimum rotation speed for acid reduction of Issatchenkia terricola WJL-G4, and the rotation speed was selected to be 120r/min in consideration of the actual application.
The above examples are part of the present invention, and in addition to the above examples, the present invention also includes other embodiments within the range of fermentation culture parameters, and any modifications made without departing from the spirit and principle of the present invention are equivalent or equivalent substitutions, and thus the technical solutions formed are still within the protection scope of the present invention.
Sequence listing
<110> Heilongjiang Tong Sheng food science and technology Co., Ltd
<120> Issatchenkia terricola and application thereof in citric acid degradation
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<170> SIPOSequenceListing 1.0
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tctcgcatcg atgaagagcg cagcgaaatg cgatacctag tgtgaattgc agccatcgtg 180
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Claims (5)
1. A strain of terrestrial Issatchenkia, which is characterized in that the classified name of the terrestrial Issatchenkia is terrestrial Issatchenkia (Issatchenkia terricola) Named Issatchenkia terricola (Issatchenkia terricola) WJL-G4 has been preserved in China general microbiological culture Collection center in 2019, 10.21.20.CGMCC No. 18712.
2. The Issatchenkia terricola strain according to claim 1, characterized in that ITS ITS nucleotide sequence is shown as SEQ ID No.1 in the sequence list.
3. Use of the terrestrial issatchenkia yeast according to claim 1 or 2 for the degradation of citric acid.
4. Use according to claim 3, characterized in that said Issatchenkia terricola is used (A)Issatchenkia terricola) WJL-G4 ferments and degrades citric acid in the culture medium, and the concentration of the citric acid is 5-40, 80, 120, 160 and 200G/L.
5. Use according to claim 3, characterized in that said Issatchenkia terricola is used (A)Issatchenkia terricola) WJL-G4 degrades citric acid in red raspberry juice.
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| Gene cloning,expression,and characterization of a novel acetaldehyde dehydrogenase from Issatchenkia terricola strain XJ-2.;Zhengying Yao et al.;《Appl Microbiol Biotechnol》;20120331;第93卷(第5期);第1999-2009页 * |
| 东方伊萨酵母高密度培养的研究;郑苗等;《中国食品学报》;20160430;第16卷(第4期);第96-103页 * |
| 陆生伊萨酵母生物降酸酿造树莓干型酒工艺研究;隋韶奕等;《食品研究与开发》;20140531;第35卷(第10期);第51页第2栏第1.1节 * |
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