Pichia terrestris QJJY1 and application thereof

Abstract

The invention provides a pichia tericorata QJJY1 strain and application thereof, and belongs to the technical field of microorganisms, the pichia tericorata QJJY1 strain is obtained by separating and purifying from a naturally fermented green kumquat sample, and the pichia tericorata QJJY1 strain is used for fermenting green kumquats, so that the pichia tericorata QJJY1 strain is obtained. It is found that the pichia tericorata QJJY1 not only can adapt to the fermentation environment of the whole green kumquat fruits and has high fermentation capacity in the whole green kumquat pulp, but also can remarkably reduce the content of oxalic acid and citric acid when being independently used for fermentation of the whole green kumquat pulp, and the prepared green kumquat enzyme product is controllable in quality and excellent in quality, and has a good market prospect. The content of organic acid in the product is obviously reduced, and the antioxidant activity is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Pichia trichoderma QJJY1 and an application thereof. Background Art

[0002] Green kumquat, a species of the Rutaceae family, is also known as Gongsun orange, green orange, mountain orange, year orange, and green orange. Green kumquats are small in size, with an average of about 15 grams per orange. Green kumquats have a high yield, with an adult green kumquat producing 100 to 200 kilograms per year, and an acre of green kumquats producing 5 to 8 tons per year. Green kumquats not only have a high yield, but also have good nutritional value. Green kumquats are rich in organic acids, vitamin C, flavonoids, phenolic acids, limonin and other functional active ingredients, and have great potential in anti-oxidation, lowering blood lipids and blood sugar.

[0003] At present, green kumquat is mainly used for juicing, which produces a lot of pomace. Since green kumquat pomace contains a lot of limonin, flavonoids and other substances, it has a strong bitter taste and is not suitable for direct consumption. Therefore, green kumquat pomace is basically discarded, which not only causes environmental pollution but also brings great waste of resources.

[0004] At present, the green kumquat products on the market are mainly beverages, and there is a lack of green kumquat enzymes and other related products. Since the total acid content of green kumquat pulp is high and the pH value is low, the pH value of the whole pulp is only 2-3. The high organic acid content not only leads to the poor taste of green kumquat products and the unbalanced sugar-acid ratio, but also has a corrosive effect on consumers' teeth, which limits the development and utilization of green kumquat.

[0005] Therefore, using microbial fermentation methods to reduce the organic acid content and acidity of green kumquat, retain the active ingredients and form a good flavor has become the research focus of green kumquat. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a strain of Pichia tricoli QJJY1 and an application thereof. The present invention separates and purifies a strain of Pichia tricoli QJJY1 from a naturally fermented green kumquat sample, and uses Pichia tricoli QJJY1 to ferment green kumquat to obtain green kumquat enzyme, in which the organic acid content of the enzyme is significantly reduced and the quality is excellent.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a strain of Pichia terricola QJJY1. The Pichia terricola QJJY1 is preserved in Guangdong Province Microbiological Culture Collection Center GDMCC, and the preservation number is GDMCC No: 65006.

[0009] Preferably, the sequence of 26S rRNA of Pichia tricora QJJY1 is as shown in SEQ ID NO.1.

[0010] The present invention provides a microbial agent, which comprises the Pichia tricora QJJY1.

[0011] The present invention also provides the use of the Pichia tricora QJJY1 and the microbial agent in the preparation of plant enzymes.

[0012] Preferably, the raw materials used to prepare the plant enzyme include green kumquat.

[0013] The present invention also provides a method for preparing green kumquat enzyme, comprising the following steps:

[0014] (1) crushing the green kumquat, sieving, and taking the sieved components to obtain green kumquat pulp;

[0015] (2) inoculating the bacteria into the kumquat pulp obtained in step (1) and fermenting it to obtain kumquat enzyme;

[0016] The bacterial species is the Pichia trichoderma QJJY1 or the microbial agent.

[0017] Preferably, the mesh size of the sieving is 20 to 70 meshes.

[0018] Preferably, the kumquat pulp is sterilized before inoculating the bacteria, the sterilization temperature is 110-120° C., and the sterilization time is 10-20 min.

[0019] Preferably, the inoculation amount of the strain is 1% to 3% of the mass of the green kumquat pulp, and the live bacteria concentration of the strain is 1×10 7 ~5×10 7 CFU / mL.

[0020] Preferably, the fermentation temperature is 28-32° C., and the fermentation time is 10-25 days.

[0021] Compared with the prior art, the present invention has the following beneficial effects: the present invention obtains a strain of Pichia tricoli QJJY1 by isolating and purifying from a naturally fermented green kumquat sample, and ferments green kumquat with Pichia tricoli QJJY1. It is found that Pichia tricoli QJJY1 can not only adapt to the fermentation environment of the whole green kumquat fruit, but also has a strong fermentation ability in the whole green kumquat pulp, and when used alone for the fermentation of the whole green kumquat pulp, it can significantly reduce the content of oxalic acid and citric acid, and the prepared green kumquat enzyme product has controllable quality and excellent quality, the organic acid content in the product is significantly reduced, and the antioxidant activity is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the colony morphology of Pichia trichoderma QJJY1;

[0023] Figure 2 This is the bacterial morphology of Pichia trichoderma QJJY1 under a microscope (400 times), where the scale bar is 20 μm;

[0024] Figure 3 This is the bacterial morphology of Pichia trichoderma QJJY1 under transmission electron microscope;

[0025] Figure 4 Phylogenetic tree constructed for the sequencing alignment results of Pichia trichoderma QJJY1;

[0026] Figure 5 This is the chromatogram of the organic acid extract of the green kumquat pulp of the control group;

[0027] Figure 6 This is the chromatogram of the organic acid extract of Pichia trichoderma QJJY1.

[0028] Collection Instructions

[0029] Pichia terricola QJJY1, Latin for Pichia terricola, is deposited in Guangdong Microbiological Culture Collection Center, with the address at Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The date of deposit is August 14, 2024, and the deposit number is GDMCCNo: 65006. DETAILED DESCRIPTION

[0030] The invention provides a strain of Pichia terricola QJJY1. The Pichia terricola QJJY1 is preserved in Guangdong Province Microbiological Culture Collection Center GDMCC, and the preservation number is GDMCC No: 65006.

[0031] In the present invention, the sequence of 26S rRNA of Pichia tricora QJJY1 is shown in SEQ ID NO.1, which is as follows:

[0032] ; Said Y represents C or T.

[0033] The present invention provides a microbial agent, which comprises the Pichia tricora QJJY1.

[0034] The present invention also provides the use of the Pichia tricora QJJY1 and the microbial agent in the preparation of plant enzymes.

[0035] In the present invention, the raw materials used for preparing the plant enzyme include green kumquat.

[0036] The present invention also provides a method for preparing green kumquat enzyme, comprising the following steps:

[0037] (1) crushing the green kumquat, sieving, and taking the sieved components to obtain green kumquat pulp;

[0038] (2) inoculating the bacterial strain into the kumquat pulp obtained in step (1), and performing a fermentation treatment to obtain kumquat enzyme;

[0039] The bacterial species is the Pichia trichoderma QJJY1 or the microbial agent.

[0040] In the present invention, the green kumquat is crushed, sieved, and the sieved components are taken to obtain green kumquat pulp. The present invention does not specifically limit the crushing method, and a conventional crushing method can be used. Fresh green kumquats with slightly yellow or yellow skin, unique green kumquat fragrance, no cracks, and no mold are washed and dried, crushed, sieved through a 20-70 mesh sieve, and the sieved components are taken to obtain green kumquat pulp; the mesh number of the sieve is preferably 30-60 mesh, and more preferably 50 mesh.

[0041] In the present invention, the green kumquat pulp obtained in step (1) is inoculated with bacterial strains for fermentation to obtain green kumquat enzyme; the green kumquat pulp of the present invention is preferably sterilized before inoculation with bacterial strains; the sterilization temperature is 110-120° C., preferably 112-118° C., and more preferably 115° C.; the sterilization time is 10-20 min, preferably 12-18 min, and more preferably 15 min; after the sterilization, the present invention inoculates bacterial strains, the inoculation amount of the bacterial strains is 1%-3% of the mass of the green kumquat pulp, preferably 2%-2.8%, and more preferably 2.5%, and the live bacteria concentration of the bacterial strains is 1×10 7 ~5×10 7 CFU / mL; the fermentation temperature is 28-32°C, preferably 29-31°C, and more preferably 30°C; the fermentation time is 10-25d, preferably 15-22d, and more preferably 20d.

[0042] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1 Isolation and screening of Pichia trichoderma QJJY1

[0044] (1) Cultivation: Pour the prepared YPD medium (purchased from Guangdong Huankai Microbiological Technology Co., Ltd.) into the plate and wait for the plate to solidify. Dilute the naturally fermented green kumquat sample 10 times in series, select the 3rd, 4th, and 5th dilutions, take 100 μL of the gradient dilution solution and spread it on the YPD plate. After culturing at 28°C for 3 days, observe the growth of the colonies on the plate.

[0045] (2) Isolation and purification: Yeast colonies of different morphologies were selected and the isolated strains were continuously purified by the plate streak method using YPD medium.

[0046] (3) Screening: A film-forming yeast that grows well in whole fruit pulp of kumquat was obtained by preliminary screening, and its bacterial morphology was observed under a microscope. The yeast colony morphology and bacterial morphology were as follows: Figure 1 , 2 , as shown in Figure 3.

[0047] Depend on Figures 1 to 3 It can be seen that the vegetative cells of this strain are elliptical or oval, and the asexual reproduction mode is budding, without forming pseudohyphae; sexual reproduction produces asci, each asci contains 1 to 3 ascospores. After culturing on MA (malt extract agar medium, purchased from Guangdong Huankai Microbiological Technology Co., Ltd.) plates at 28°C for 5 days, the colonies are milky white, convex, rough on the surface, and irregular on the edges.

[0048] The bacteria were transferred to YPD medium slant tubes for storage and used in subsequent experiments.

[0049] (4) Cultivation: Perform secondary cultivation on the strain stored in the slant tube to collect a sufficient amount of bacteria and store them for later use.

[0050] (5) Strain identification and preservation: The isolated strain was sent to the Guangdong Microbiological Culture Collection Center for sequencing and identification. Its 26S rRNA gene sequence is shown in SEQ ID NO: 1, and the identification result is Pichia terricola. It was deposited in the Guangdong Microbiological Culture Collection Center on August 14, 2024 and named Pichia terricola QJJY1, with a preservation number of GDMCC No: 65006.

[0051] SEQ ID NO: 1:

[0052] .

[0053] (6) Morphological analysis of QJJY1: The vegetative cells of QJJY1 are elliptical or oval, and the asexual reproduction mode is budding without forming pseudohyphae; sexual reproduction produces asci, each of which contains 1 to 3 ascospores. After culturing on MA (malt extract agar) plates at 28°C for 5 days, the colonies are milky white, convex, with a rough surface and irregular edges.

[0054] (7) Phylogenetic tree construction: The strain identification sequencing results were imported into the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) for nucleic acid sequence blasting. Strains with a similarity of more than 97% were selected based on the comparison results, and their fasta format sequence files were downloaded. The downloaded sequence files were imported into Mega 11 software for sequence comparison, and a phylogenetic tree was constructed based on the comparison results. The results are shown in Figure 2. Figure 4 shown.

[0055] (8) The physiological and biochemical characteristics of yeast QJJY1 are shown in Table 1.

[0056] Table 1 Physiological and biochemical characteristics of Pichia trichoderma QJJY1

[0057]

[0058]

[0059] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction.

[0060] Example 2 Application of Pichia trichoderma QJJY1 in green kumquat enzyme

[0061] The Pichia trichoderma QJJY1 isolated in Example 1 of the present invention was used to prepare the green kumquat enzyme, and the specific experimental method was as follows:

[0062] (1) Crushing and sieving: fresh kumquats with slightly yellow or yellow skin, characteristic kumquat fragrance, no cracks and no mold are selected, washed and dried, and air-dried until there is no moisture on the surface of the kumquats, and the whole fruit is crushed in a crusher, and the crushed fruit is passed through a 50-mesh sieve. The purpose of sieving is to ensure that the crushed particles are uniform, and the sieved matter is taken to obtain the whole kumquat pulp (kumquat pulp).

[0063] (2) Sterilization: sterilize the whole fruit pulp of the kumquat at 115° C. for 15 min to obtain sterilized whole fruit pulp of the kumquat.

[0064] (3) Feeding: Pichia trichoderma QJJY1 was inoculated into YPD liquid medium and cultured for 48 h. Centrifugation was performed at 4°C and 1800 g to collect the bacterial precipitate, which was washed twice with saline and diluted with saline to a concentration of 1×10 7 CFU·mL -1 Pichia Tricora QJJY1 seed liquid. Add 2.5% of the weight of Pichia Tricora QJJY1 yeast seed liquid into the sterilized green kumquat whole pulp.

[0065] (4) Fermentation: Ferment at 30°C for 20 days to obtain green kumquat enzyme.

[0066] The physicochemical indexes of the green kumquat whole pulp and green kumquat enzyme obtained above were measured. 0.2 g of the freeze-dried and ground sample was placed in a 15 mL centrifuge tube, 10 mL of 70% methanol was added for soaking, and the sample was placed in an ultrasonic instrument, and ultrasonicated at 360W and 50°C for 30 minutes. The sample extract was filtered with a 0.45 μm pore size filter membrane and placed in a 4°C refrigerator for use.

[0067] Determination of total phenol content (calculated as gallic acid): The above sample extract was taken and determined using the Folin phenol colorimetric method. The result was expressed as mg gallic acid equivalent (GAE) / 100 g freeze-dried sample.

[0068] Determination of total flavonoid content (in terms of rutin): The above sample extract was taken and determined using the sodium nitrite-aluminum nitrate colorimetric method. The result was expressed as mg rutin equivalent (RE) / 100 g freeze-dried sample.

[0069] DPPH free radical scavenging ability determination: 60 μL sample extracts (diluted to obtain the above 0.2g / 10mL sample extracts) with mass concentrations of 4 mg / mL, 8 mg / mL, 12 mg / mL, 16 mg / mL, and 20 mg / mL were dispersed in a 96-well plate and mixed with 40 μL 1mmol / L DPPH solution (DPPH dissolved in methanol), and then 150 μL methanol solution was added. The reaction was carried out in the dark at room temperature for 30 minutes, and the absorbance value was measured at a wavelength of 517 nm.

[0070] ABTS free radical scavenging ability determination: 7mmol / L ABTS solution (prepared with water) and 2.4mmol / L potassium persulfate solution were mixed in equal volumes, reacted in the dark under room temperature conditions for 12 to 16 hours to prepare a fresh ABTS free radical solution. Before use, the freshly prepared ABTS solution was diluted with methanol to an absorbance of 0.7±0.02 at a wavelength of 734nm. 150μL ABTS dilution was mixed with 30μL sample extract (mass concentrations were 2mg / mL, 4mg / mL, 6mg / mL, 8mg / mL, 10mg / mL, respectively), reacted at room temperature in the dark for 10min, and the absorbance was measured at a wavelength of 734nm.

[0071] Copper ion reducing ability determination: In a 96-well ELISA plate, add 25 μL sample extract, 50 μL CuCl 2 10mmol / L, 50μL 7.5mmol / L new cuprous reagent solution, 50μL 1mol / L ammonium acetate buffer (pH 7), and finally 30μL water were added, reacted for 30min under room temperature conditions, 70% methanol by volume was used as blank, and the absorbance value was measured at a wavelength of 450nm. The results are expressed as μgGAE / g sample.

[0072] Determination of α-glucosidase inhibition rate: 10 μL sample extract and 50 μL α-glucosidase (1.0 U / mL dissolved in 0.1 mol / L pH 6.8 phosphate buffer) were added to a 96-well plate, shaken evenly, and incubated at 37°C for 15 min. Then 50 μL p-nitrophenylα-D-glucopyranoside (pNPG) (5 mmol / L dissolved in 0.1 mol / L pH 6.8 phosphate buffer) was added to the 96-well plate, shaken evenly, and continued to incubate at 37°C for 15 min. 150 μL 0.5 mmol / L sodium carbonate solution was added to terminate the reaction, and the absorbance was measured at a wavelength of 415 nm. α-glucosidase activity is expressed as p-nitrophenol released from pNPG.

[0073] The results are shown in Table 2. As shown in Table 2, compared with the unfermented green kumquat whole fruit pulp, the pH of the green kumquat enzyme obtained by fermentation with Pichia trichoderma QJJY1 was higher than that of the green kumquat whole fruit pulp, the organic acid content was significantly reduced, the soluble total phenol, total flavonoids content and antioxidant activity were increased, and the ability to inhibit α-glucosidase activity was enhanced.

[0074] Table 2 Physicochemical indicators of Pichia trichoderma QJJY1 applied to green kumquat enzyme

[0075]

[0076]

[0077] Comparative Example

[0078] The green kumquat enzyme was prepared in the same manner as in Example 2, except that Hanseniaspora thailandica QJJY15 (deposited in Guangdong Microbial Culture Collection Center GDMCC, deposit number: GDMCC No: 65007) was used to replace Pichia trichoderma QJJY1.

[0079] Experimental Example 1

[0080] The key phenolic substances of the green kumquat enzyme obtained in Example 2 and the comparative example were determined, and the phenolic substances were determined using HPLC. The specific test condition parameters are as follows: the fermented enzyme product was freeze-dried, 1.00 g of the freeze-dried sample was accurately weighed and placed in a 100 mL centrifuge tube, 50.0 mL of distilled water was accurately added, 320 W, 50 ° C ultrasonic for 30 min, 5000 r / min centrifugation for 10 min, the supernatant was collected, and 4 ° C was stored for standby use. Each sample was tested 3 times.

[0081] Preparation of standard curve: accurately weigh oxalic acid, citric acid, succinic acid, tartaric acid, DL-malic acid, lactic acid, and acetic acid, and accurately prepare mixed standard solutions with concentrations of 1, 2, 5, 10, 20, 50, 100, and 200 μg / mL, of which the concentrations of citric acid are 3, 6, 15, 30, 60, 150, 300, and 600 μg / mL, and store at 4°C for future use. Quantification was performed using the external standard method, and the standard curve and linear regression equation were drawn with the mass concentration of the standard solution as the abscissa and the peak area as the ordinate.

[0082] Chromatographic conditions: HPLC method was used for quantitative determination. The mobile phase was selected to be methanol: 0.01 mol potassium dihydrogen phosphate (v:v = 2:98, pH 2.85) as the best. Chromatographic column: Thermo Fisher Acclaim 120Cis chromatographic column (4.6x250mm, 5mm); column temperature: 30℃; injection volume: 10μL; flow rate: 0.8mL / min; UV detector wavelength: 210nm; elution mode was isocratic elution.

[0083] The results are shown in Table 3. Figure 5 , 6 shown.

[0084] Table 3 Analysis of organic acid substances in green kumquat enzyme (unit: mg / g)

[0085]

[0086] As shown in Table 3, fermentation of green kumquat by Pichia trichoderma QJJY1 can reduce the total amount of organic acids, and significantly reduce the content of oxalic acid and citric acid. Compared with Hanseniaspora thailandica QJJY15, it was found that Pichia trichoderma QJJY1 not only reduced the content of citric acid, but also had lower production of tartaric acid, L-malic acid, and D-malic acid, resulting in a lower total amount of organic acids.

[0087] Experimental Example 2

[0088] The green kumquat enzyme obtained in Example 2 was subjected to sensory evaluation, and the sensory evaluation table is shown in Table 4. Sensory evaluation method: The evaluation panel consisted of 10 members, and the color, flavor, taste and tissue state of the green kumquat enzyme were scored independently according to the scoring criteria in Table 4, and the average value of the cumulative total score was used as the sensory score of the product. The results are shown in Table 5.

[0089] Table 4 Sensory evaluation table

[0090]

[0091]

[0092] Table 5 Sensory evaluation results

[0093]

[0094] From the sensory evaluation experimental results in Table 5, it can be seen that fermenting green kumquat with Pichia trichoderma QJJY1 can enrich the aroma of green kumquat enzyme, reduce acidity, and improve flavor and taste scores. The preparation method of green kumquat enzyme provided by the present invention retains the typical aroma and taste of green kumquat to the greatest extent, improves the total phenol, total flavonoid content, antioxidant activity and α-glucosidase inhibition rate, reduces the total amount of organic acid, and improves the sensory score.

[0095] From the results of the above embodiments and experimental examples, it can be seen that the Pichia tricora QJJY1 provided by the present invention has a strong fermentation ability in the whole fruit pulp of kumquat, and when used alone for the fermentation of the whole fruit pulp of kumquat, it can significantly reduce the total amount of organic acids, improve the total polyphenols, total flavonoids, DPPH free radical scavenging rate, ABTS free radical scavenging rate, copper ion reducing ability and α-glucosidase inhibition rate, enrich the aroma of kumquat enzymes, and improve the sensory score.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Pichia terricola QJJY1, characterized in that: The Pichia tricora QJJY1 is deposited in Guangdong Microbial Culture Collection Center GDMCC, with the deposit number: GDMCC No: 65006.

2. The Pichia tricora QJJY1 according to claim 1, characterized in that The sequence of the 26S rRNA of Pichia tricora QJJY1 is shown in SEQ ID NO.

1.

3. A microbial agent, characterized in that: The microbial agent includes the Pichia trichoderma QJJY1 described in claim 1.

4. Use of the Pichia tricora QJJY1 described in claim 1 or 2 and the microbial agent described in claim 3 in the preparation of plant enzymes.

5. The use according to claim 4, characterized in that: The raw materials used for preparing the plant enzyme include green kumquat.

6. A method for preparing green kumquat enzyme, characterized in that: The following steps are involved: (1) crushing the green kumquat, sieving, and taking the sieved components to obtain green kumquat pulp; (2) inoculating the bacteria into the kumquat pulp obtained in step (1) and fermenting it to obtain kumquat enzyme; The bacterial species is the Pichia tricora QJJY1 described in claim 1 or the microbial agent described in claim 3.

7. The preparation method according to claim 6, characterized in that: The mesh number of the sieving is 20 to 70 meshes.

8. The preparation method according to claim 6, characterized in that: The green kumquat pulp is sterilized before inoculating the bacteria, the sterilization temperature is 110-120° C., and the sterilization time is 10-20 minutes.

9. The preparation method according to claim 6, characterized in that: The inoculation amount of the strain is 1% to 3% of the mass of the green kumquat pulp, and the live bacteria concentration of the strain is 1×10 7 ~5×10 7 CFU / mL.

10. The preparation method according to claim 6, characterized in that: The fermentation temperature is 28-32° C., and the fermentation time is 10-25 days.

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