A strain of Lactobacillus paracasei YYS-EN2 producing cellulase, pectinase, tanninase and phytase and its applications

Abstract

This invention belongs to the field of microbial technology, specifically relating to *Lactobacillus paracasei* YYS-EN2, which produces cellulase, pectinase, tanninase, and phytase, and its applications. This *Lactobacillus paracasei* YYS-EN2 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28531. The *Lactobacillus paracasei* YYS-EN2 provided by this invention possesses the ability to produce cellulase, pectinase, tanninase, and phytase, and can degrade plant cellulose and pectin, decompose tannins and phytic acid. When applied to fermented products, it can reduce bitterness and improve product clarity. It can be widely used in the production of fermented products, such as fermented plant-derived nutritional powders and fermented plant-derived polypeptide powders, and can also be used in probiotic products and functional foods. It colonizes the human intestinal tract, improving gut microbiota structure and promoting digestion and absorption.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Lactobacillus paracasei YYS-EN2 that produces cellulase, pectinase, tanninase and phytase, and its applications. Background Technology

[0002] Grains, bran, fruits, and vegetables are rich in crude fiber substances such as cellulose, pectin, and tannins, which are generally difficult to hydrolyze. Their rigid crystalline structure makes production difficult, increasing processing steps, reducing yield, and even damaging nutritional value. Physical and chemical degradation methods are extreme, time-consuming, and prone to secondary pollution. Therefore, microbial degradation is the most effective, healthy, and environmentally friendly method. Cellulase and pectinase hydrolyze plant crude fiber, facilitating the release of starch and protein, improving nutrient absorption, and increasing product value. They are widely used in fruit and vegetable extraction and clarification, as well as tea and coffee fermentation. Tanninase and phytase degrade tannins and phytic acid, improving the pressing and color of fruit juices and vegetables, and enhancing the flavor and color of wine.

[0003] In conclusion, cellulase, pectinase, tanninase, and phytase have a broader application prospect in plant-based fermented beverages, fermented plant-derived nutritional powders, fermented plant-derived polypeptide powders, and fermented plant-derived protein powders.

[0004] Chinese invention patent application number CN202110864302.3, published on 20230203, discloses a method for preparing cellulase fermentation broth from a Streptomyces strain GZUIRF-Y1, and its application. The cellulase activity in the fermentation broth of this strain reaches 111.0 U·mL. -1 In this technology, the strain that degrades cellulose is a mold.

[0005] Chinese invention patent application number CN202111637611.3, published on April 12, 2022, discloses a strain of Aspergillus oryzae and its application in high-salt, high-nitrogen fermented foods. The strain has a cellulase activity of 18.6 U·g. -1 The acid pectinase activity was 33.1 U·g. -1 In this technology, the strain that degrades cellulose is a mold.

[0006] The aforementioned molds are capable of producing cellulase and / or pectinase, but do not simultaneously produce cellulase, pectinase, tanninase, or phytase. Furthermore, currently, most strains that degrade cellulose, pectin, tannins, and phytic acid are molds or Bacillus species, which contain few beneficial bacteria and therefore cannot be used in food fermentation.

[0007] In summary, how to develop natural probiotics that produce cellulase, pectinase, tanninase, and phytase is precisely the technical problem that this field is committed to solving. Summary of the Invention

[0008] To address the shortcomings of the prior art mentioned in the background section, this invention provides a Lactobacillus paracasei YYS-EN2 strain. This Lactobacillus paracasei YYS-EN2 is a natural strain that produces cellulase, pectinase, tanninase, and phytase. It can degrade plant cellulose and pectin, decompose tannins and phytic acid, reduce bitterness, and improve product clarity. It is widely used in the production of fermented products, such as fermented plant-derived nutrient powder and fermented plant-derived polypeptide powder.

[0009] The Lactobacillus paracasei YYS-EN2 provided by this invention has the following Latin scientific name: Lactobacillus paracasei It was deposited on September 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28531.

[0010] The Lactobacillus paracasei YYS-EN2 strain was isolated from the oral saliva of a healthy adult. After sequencing analysis, the strain was found to be highly homologous to Lactobacillus paracasei by Blast sequence comparison, and it was named Lactobacillus paracasei YYS-EN2.

[0011] Among them, Lactobacillus paracasei YYS-EN2 has the ability to produce cellulase, pectinase, tanninase and phytase, and it has the ability to degrade cellulose, pectin, tannin and phytic acid.

[0012] This invention also provides the application of *Lactobacillus paracasei* YYS-EN2, as described above, in the preparation of fermented products. The application of *Lactobacillus paracasei* YYS-EN2 in the preparation of fermented products can significantly reduce the cellulose and pectin content in the products.

[0013] In some embodiments, the fermented products include, but are not limited to, fermented nutrient powder, fermented polypeptide powder, fermented protein powder, enzymes, and silage.

[0014] In some embodiments, the fermented nutrient powder includes fermented plant-derived nutrient powder; the fermented polypeptide powder includes fermented plant-derived polypeptide powder; and the fermented protein powder includes fermented plant-derived protein powder.

[0015] In some embodiments, the fermented plant-derived nutrient powder includes fermented corn nutrient powder. Lactobacillus paracasei YYS-EN2 is used in corn nutrient powder products, which can ferment corn husks and germ, break down cellulose and pectin, and reduce tannins and phytic acid, thereby enhancing the flavor and texture of the corn nutrient powder.

[0016] The present invention also provides a method for producing cellulase, pectinase, tanninase and phytase: Lactobacillus paracasei YYS-EN2 as described above is inoculated into an enzyme-producing liquid culture medium and cultured at (30-40)℃ for 48-72h; the enzyme-producing liquid culture medium after fermentation is separated into solid and liquid components to obtain a supernatant; the supernatant is an enzyme solution containing cellulase and / or pectinase and / or tanninase and / or phytase.

[0017] The present invention also provides a freeze-dried product whose components include Lactobacillus paracasei YYS-EN2 as described above.

[0018] In some embodiments, the viable count of *Lactobacillus paracasei* YYS-EN2 in the freeze-dried product is (2–8) × 10⁻⁶. 11 CFU·g -1 .

[0019] This invention also provides a method for preparing a freeze-dried product, comprising the following preparation steps:

[0020] 1) Preparation of Lactobacillus paracasei YYS-EN2 seed culture;

[0021] 2) Seed culture expansion;

[0022] 3) Seed culture fermentation to obtain fermentation broth;

[0023] 4) Centrifuge and discard the supernatant to obtain mycelial sludge;

[0024] 5) After the bacterial sludge and freeze-drying protectant are mixed evenly, they are emulsified and embedded to obtain an emulsion;

[0025] 6) Freeze-dry the emulsion and pulverize it to obtain freeze-dried powder of Lactobacillus paracasei YYS-EN2.

[0026] The present invention also provides a microbial agent, wherein the components of the microbial agent comprise the lyophilized powder as described above, and / or, the components of the microbial agent comprise Lactobacillus paracasei YYS-EN2 as described above.

[0027] In some embodiments, the microbial agent further includes other ingredients, which are existing ingredients suitable for microbial agents, such as at least one of prebiotics, fillers, acidulants, solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, stabilizers, flow aids, flavoring agents, preservatives, coating materials, fragrances, anti-adhesion agents, binding agents, thickeners, and inclusion agents.

[0028] The present invention also provides the application of Lactobacillus paracasei YYS-EN2 as described above in the preparation of probiotic products.

[0029] This invention also provides the application of Lactobacillus paracasei YYS-EN2 as described above in the preparation of functional products, said functional products comprising at least one of the following functions:

[0030] (1) It has the ability to break down cellulose;

[0031] (2) It has the ability to decompose pectin;

[0032] (3) It has the ability to decompose tannins;

[0033] (4) It has the ability to decompose phytic acid.

[0034] In some embodiments, the functional product includes food or health products; Lactobacillus paracasei YYS-EN2 can be used in the preparation of food or health products.

[0035] Based on the above characteristics, the Lactobacillus paracasei YYS-EN2 provided by the present invention has the following beneficial effects:

[0036] The Lactobacillus paracasei YYS-EN2 provided by this invention has the ability to produce cellulase, pectinase, tanninase and phytase, and can degrade plant cellulose, pectin, tannin and phytic acid.

[0037] The Lactobacillus paracasei YYS-EN2 provided by this invention has the ability to produce cellulase, which can degrade cellulose and can be applied to the development of fermented plant-derived nutrient powder and silage products.

[0038] It has the ability to degrade pectinase and can be widely used in the production of fruit juice, vegetables, wine, enzymes and other products. It can significantly reduce the pectin content in raw materials and improve the clarity and yield of products.

[0039] It has the ability to produce tannins and can be widely used in the production of wine, enzymes and other products. It can reduce the tannin content in wine and improve the taste of the product.

[0040] It has the ability to produce phytase, which can be widely used in flour and soybean food processing to improve the degradation rate of phytic acid and enhance its nutritional and commercial value.

[0041] In summary, the Lactobacillus paracasei YYS-EN2 provided by this invention produces cellulase, pectinase, tanninase and phytase, and can be applied to various probiotic fermentation products, laying the foundation for providing green, healthy, high-nutritional-value and high-utilization plant-derived agricultural products. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 This is a colony diagram of Lactobacillus paracasei YYS-EN2 on MRS agar plates.

[0044] Figure 2 A graph showing the glucose standard curve;

[0045] Figure 3 The standard curve for D-galacturonic acid is shown in the figure.

[0046] Figure 4 The standard curve for gallic acid is shown in the figure.

[0047] Figure 5 This is a graph showing the standard curve for inorganic phosphorus. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] The solution of the present invention:

[0050] This invention provides a Lactobacillus paracasei ( Lactobacillus paracasei YYS-EN2:

[0051] Lactobacillus paracasei ( Lactobacillus paracasei YYS-EN2 was deposited at the China General Microbiological Culture Collection Center on September 25, 2023, with accession number CGMCC No. 28531;

[0052] Source: The Lactobacillus paracasei was isolated from the oral saliva of healthy adults. After sequencing analysis, the strain was found to be highly homologous to Lactobacillus paracasei by Blast sequence comparison, and was named Lactobacillus paracasei YYS-EN2.

[0053] Colony morphology: In MRS solid medium, colonies are milky white, round, and have smooth and neat edges.

[0054] Functions: It has the ability to produce cellulase and pectinase, which can degrade cellulose and pectin; it also has the ability to produce tanninase and phytase, which can decompose tannins and phytic acid.

[0055] The present invention also provides an operational example of a method for producing cellulase and / or pectinase and / or tanninase and / or phytase:

[0056] Lactobacillus paracasei YYS-EN2 as described above was inoculated into an enzyme-producing liquid culture medium and cultured at (30-40) °C for 48-72 h. The enzyme-producing liquid culture medium after fermentation was separated into solid and liquid components to obtain a supernatant. The supernatant was an enzyme solution containing cellulase and / or pectinase and / or tanninase and / or phytase.

[0057] This invention also provides a method for preparing a freeze-dried product, comprising the following preparation steps:

[0058] 1) Preparation of Lactobacillus paracasei YYS-EN2 seed culture;

[0059] 2) Seed culture expansion;

[0060] 3) Seed culture fermentation to obtain fermentation broth;

[0061] 4) Centrifuge the fermentation broth to obtain bacterial sludge;

[0062] 5) After mixing the bacterial sludge with the freeze-drying protectant, emulsify and embed it to obtain an emulsion;

[0063] 6) The emulsion is freeze-dried and pulverized to obtain Lactobacillus paracasei YYS-EN2 freeze-dried powder.

[0064] This invention also provides an operational example of its application method in the production of corn nutrient powder:

[0065] Corn germ and corn husk are crushed, then treated with lipase and amylase, sterilized at high temperature, and then Lactobacillus paracasei YYS-EN2 bacterial powder and / or freeze-dried powder are added, mixed and fermented together. After fermentation is completed, the mixture is sterilized at high temperature and then dried and stored to obtain corn nutrient powder.

[0066] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1: Isolation and identification of Lactobacillus paracasei YYS-EN2

[0068] Separation:

[0069] Aseptic sampling was performed on saliva samples from healthy adults using the plate spread method. A sterile cotton swab was used to collect the saliva sample, which was then placed in a sterile homogenizing bag. 45 mL of 0.9% physiological saline was added, and the mixture was homogenized to obtain the sample. 100 μL of the sample was then serially diluted 10-fold. -2 10 -3 10 -4 100 μL of the sample was spread onto MRS solid agar plates containing 2.5% CaCO3 and incubated upside down at 37°C for 24 h. Colonies with good growth and large calcium dissolution zones were selected and repeatedly isolated and purified by streak plating until single colonies were obtained. The isolated bacteria were screened to obtain strains that produced high levels of cellulase, pectinase, tanninase, and phytase. This isolated strain was named YYS-EN2 and stored in a bacterial culture library at -80°C with added glycerol.

[0070] The colony morphology of the isolated and purified Lactobacillus paracasei YYS-EN2 was as follows: in MRS solid medium, the colonies were milky white, round, and had smooth and regular edges.

[0071] The process for screening bacteria that produce high levels of cellulase, pectinase, tanninase, and phytase is as follows:

[0072] Screening of cellulase-producing strains:

[0073] 1. Screening medium (g / L): Sodium carboxymethyl cellulose (CMC-Na) 20g, peptone 10g, yeast extract 5g, sodium chloride 5g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.3g; Congo red 0.4g;

[0074] 2. Strain screening: The bacteria were inoculated into MRS liquid medium from a -80℃ freezer and cultured at 37℃ for 24h; then, 100 μL of culture medium was placed on a screening medium plate using the Oxford cup method and cultured at 37℃ for 48h. The diameter of the clear zone was observed, and strains with high cellulase production were selected based on the diameter of the clear zone. Cellulase activity was measured to screen out strains with high cellulase production.

[0075] Similarly, the screening of bacteria that produce high levels of pectinase, tanninase, and phytase was carried out sequentially. The specific process is as follows:

[0076] Screening of pectinase-producing strains:

[0077] 1. Screening medium (gL-1): pectin 10g, Congo red 0.2g, peptone 1.5g, dipotassium hydrogen phosphate 4g, potassium dihydrogen phosphate 2g, ammonium sulfate 4g, magnesium sulfate heptahydrate 0.5g.

[0078] 2. Strain screening: The bacteria were inoculated into MRS liquid medium from a -80℃ freezer and cultured at 37℃ for 24h; then, 100μL of culture medium was placed on a screening medium plate using the Oxford cup method and cultured at 37℃ for 48h. The diameter of the clear zone was observed, and strains with high pectinase production were selected based on the diameter of the clear zone. Pectinase activity was measured to screen out strains with high pectinase production.

[0079] Screening of tannin-producing strains

[0080] 1. Screening medium (gL-1): Sodium nitrate 3 g, dipotassium hydrogen phosphate 1 g, potassium chloride 0.5 g, magnesium sulfate 0.5 g; bromophenol blue 0.04 g, tannic acid 10 g, sterilized separately.

[0081] 2. Strain Screening: Bacteria were inoculated into MRS liquid medium from a -80℃ freezer and incubated at 37℃ for 24 h. Then, using the Oxford cup method, 100 μL of the culture was placed on screening medium plates and incubated at 37℃ for 48 h. The screening plates contained tannic acid and bromophenol blue indicator. If the strain produced tanninase, it would hydrolyze the tannic acid in the medium to produce gallic acid, causing the bromophenol blue indicator around the colony to change from blue-purple to yellow, thus forming a distinct color-changing zone around the colony. High-tanninase-producing strains were selected based on the intensity and diameter of the color-changing zone, and tanninase activity was measured to screen for high-tanninase-producing strains.

[0082] Screening of phytase-producing strains

[0083] 1. Screening medium (g / L) -1 Ingredients: 15g glucose, 5.0g ammonium nitrate, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.3g ferrous sulfate, 0.3g manganese sulfate, 2g calcium phytate.

[0084] 2. Strain Screening: The bacteria were inoculated into MRS liquid medium from a -80℃ freezer and cultured at 37℃ for 24 hours. Then, using the Oxford cup method, 100 μL of the culture solution was placed on a screening medium plate and cultured at 37℃ for 48 hours. Phytase-producing strains were able to hydrolyze calcium phytate, forming a clear zone around the colony. High-yielding phytase-producing strains were selected based on the diameter of the clear zone, and phytase activity was measured to further screen for high-yielding phytase-producing strains.

[0085] The formula for MRS liquid medium is as follows: 10.0g beef extract, 20.0g glucose, 10.0g tryptone, 5.0g yeast extract, 1.0mL Tween 80, 2.0g dipotassium hydrogen phosphate, 2.0g ammonium citrate, 5.0g anhydrous sodium acetate, 0.5g magnesium sulfate, 0.25g manganese sulfate monohydrate, 1.0L deionized water, pH 6.5 (adding 2% agar makes it MRS solid medium).

[0086] Strain identification:

[0087] (1) Observation of fungal morphology

[0088] The screened and purified strain YYS-EN2 was subjected to Gram staining and catalase tests, and its physiological and biochemical indicators were measured. The test results were compared with those in Bergey's Manual of Systematic Bacteriology, 8th Edition, for preliminary identification of the bacterial species. Figure 1 As shown, the test results showed that the selected strain YYS-EN2 stained purple with Gram stain, indicating a positive result. It is rod-shaped, catalase-negative, and does not form spores.

[0089] DNA was extracted from YYS-EN2 according to the instructions of the bacterial DNA extraction kit and then amplified by PCR:

[0090] PCR amplification process:

[0091] The 16S rDNA gene sequence was amplified using primers 27F (5'-AGAGTT TGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction mixture consisted of 1 μL of 27F (10 μM), 1 μL of 1492R (10 μM), and 10X... EasyTag @ R Buffer(2.5 mM) 5μL, dNTPs (2.5 mM) 4μL, DNA template 1μL, EasyTag @ R DNAPolymerase (5 U / L) 0.3 μL, ddH2O 37.7 μL. PCR amplification program: 94℃ 5 min, 94℃ 30 s, 55℃ 30 s, 72℃ 90 s, 72℃ 10 min, cycle from step 2 to step 4 32 times, store at 4℃.

[0092] ③PCR product detection and sequencing analysis: The amplified product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The strain YYS-EN2 was identified as Lactobacillus paracasei.

[0093] The 16S rDNA gene sequence is as follows:

[0094]

[0095] Example 2: Preparation of Lyophilized Lactobacillus paracasei YYS-EN2 Powder

[0096] Activated Lactobacillus paracasei YYS-EN2 was inoculated at a rate of 2% (v / v) into culture medium sterilized at 121℃ for 15 min, and cultured at 37℃ for 24 h. The culture was then centrifuged at 4℃, 6000 r / min for 10 min, and the supernatant was discarded to obtain bacterial sludge. This sludge was then emulsified with a protective agent for 15 min to obtain a bacterial suspension with a concentration of 5 × 10⁻⁶. 10 CFU·mL -1 The emulsion was pre-frozen at -40°C for 4 hours and then freeze-dried at -35°C for 35 hours to obtain a viable bacterial count of 510 billion CFU·g. -1 Lyophilized Lactobacillus paracasei YYS-EN2 active bacterial powder.

[0097] The culture medium used consisted of the following components (by weight percentage): 2.5% glucose, 2.5% soybean peptone, 1% yeast extract, 1% beef extract, 0.6% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.03% manganese sulfate, 0.1% Tween 80, and the balance being water. The protective agent was prepared at 50 g / L. -1 Skim milk powder, 10g·L -1 Sucrose, 10g·L -1 Trehalose and 10g·L -1 The composition of L-glutamate sodium.

[0098] It should be noted that the protective agent can be an existing protective agent component or formulation, including but not limited to the above-described embodiments.

[0099] Example 3: Lactobacillus paracasei YYS-EN2 cellulase activity

[0100] 1. Enzyme-producing medium (g / L) -1 Ingredients: Sodium carboxymethyl cellulose (CMC-Na) 20g, peptone 3g, yeast extract 0.5g, ammonium sulfate 2g, potassium dihydrogen phosphate 4g, magnesium sulfate 0.3g, calcium chloride 0.3g;

[0101] 2. Cellulase Activity Assay

[0102] (1) Definition of enzyme activity:

[0103] Under conditions of 50℃ and pH 4.5, it decomposes at a rate of 10 g·L per minute. -1 The amount of enzyme required to produce 1 mg of glucose from sodium carboxymethyl cellulose (CMC-Na) substrate is defined as one unit of enzyme activity (U·mL). -1 ).

[0104] (2) Reagent preparation:

[0105] 0.1 mol·L -1 Acetic acid-sodium acetate buffer (pH 4.6): Add 0.1 mol·L⁻¹ -1 Sodium acetate solution and 0.1 mol·L -1 The acetic acid solution was mixed and the pH was adjusted to 4.5. The concentration was 0.1 mol·L⁻¹. -1 Sodium acetate solution: Accurately weigh 8.20 g of anhydrous sodium acetate, add distilled water, and dilute to a volumetric flask with distilled water; 0.1 mol·L⁻¹ -1 Acetic acid solution: Accurately weigh 6g of acetic acid, add distilled water, and dilute to 1000mL in a volumetric flask;

[0106] Sodium carboxymethyl cellulose (CMC-Na) solution (10 g·L⁻¹) -1 Accurately weigh 1g of sodium carboxymethyl cellulose (CMC-Na) and add it to 100mL of acetate-sodium acetate buffer.

[0107] (3) Enzyme activity assay: Cellulase activity was determined by the DNS method.

[0108] Preheat a 1% sodium carboxymethyl cellulose (CMC-Na) solution in a 40°C water bath. Add 1 mL of enzyme solution to 1 mL of the sodium carboxymethyl cellulose solution and react at 40°C for 30 min (enzyme time). Then add 1 mL of 0.4 mol·L⁻¹ enzyme solution. -1 The reaction was terminated with NaOH, while the stop solution was added to the blank control group before adding the enzyme solution.

[0109] After the reaction was complete, 1 mL of the reaction solution was mixed with 1 mL of DNS (5-(dimethylamino)-1-naphthalenesulfonic acid hydrate) solution, and the mixture was reacted in a boiling water bath for 5 min to develop color. The reaction solution after color development was diluted 5 times, and the sample absorbance was measured at 540 nm using a spectrophotometer with distilled water as the reference. The enzyme activity was then calculated according to the formula.

[0110] The crude enzyme solution was obtained as follows: Lactobacillus paracasei YYS-EN2 was inoculated into the enzyme-producing medium and cultured at 37°C for 48 h. The culture medium was centrifuged at 4°C and the supernatant was collected, which was the crude enzyme solution. The crude enzyme solution was diluted to the enzyme solution by a certain factor for enzyme activity determination. The inoculation amount of Lactobacillus paracasei YYS-EN2 was 2% (v / v) of the culture medium.

[0111] Result calculation method:

[0112] The formula for enzyme activity determination is: Enzyme activity = OD 540 ×n×1 / k×1 / 30;

[0113] In the formula, n is the dilution factor for enzyme solution testing (the dilution factor from crude enzyme solution to test enzyme solution), k is the slope of the fitted standard curve, and 30 is the enzyme-catalyzed reaction time.

[0114] The standard curve was plotted using 1 g·L⁻¹. -1 Preparation of standard solutions of different concentration gradients (0 g·L⁻¹) from glucose solution -1 0.2 g·L -1 0.4 g·L -1 0.6 g·L -1 0.8 g·L -1 1.0 g·L -1 Following the enzyme activity assay method, absorbance was measured at 540 nm. A linear standard curve was plotted with glucose concentration on the x-axis and OD value on the y-axis (see [reference]). Figure 2 ).

[0115] The results showed that the cellulase activity of Lactobacillus paracasei YYS-EN2 was 47.34 ± 0.06 U·mL. -1 .

[0116] Example 4: Lactobacillus paracasei YYS-EN2 pectinase activity

[0117] 1. Enzyme-producing medium (g / L) -1 ): Pectin 10g, glucose 10g, peptone 4g, dipotassium hydrogen phosphate 4g, potassium dihydrogen phosphate 2g, magnesium sulfate heptahydrate 0.5g;

[0118] 2. Pectinase activity assay

[0119] (1) Definition of enzyme activity:

[0120] Under conditions of 50°C and pH 5.0, it decomposes at a rate of 10 g·L / min. -1 The amount of enzyme required to generate 1 mg of galacturonic acid from a pectin substrate is defined as one unit of enzyme activity (U·mL). -1 );

[0121] (2) Solution preparation:

[0122] 0.2 mol·L -1 Acetic acid-sodium acetate buffer (pH 4.8): Add 0.2 mol·L⁻¹ -1 Sodium acetate solution and 0.2 mol·L -1 The acetic acid solution was mixed and the pH was adjusted to 5.0. The concentration was 0.2 mol·L⁻¹. -1 Acetic acid solution: Accurately weigh 12g of acetic acid, add distilled water, and dilute to a volumetric flask of 1000mL; 0.2 mol·L⁻¹ -1To prepare a sodium acetate solution, accurately weigh 16.41 g of sodium acetate, add distilled water, and dilute to a volumetric flask with the solution to 1000 mL. Mix the solutions and adjust the pH to 5.0.

[0123] Pectin substrate (10 g·L) -1 ): 1g pectin added to 100mL acetate-sodium acetate buffer

[0124] (3) Enzyme activity assay:

[0125] Preheat the pectin substrate in a 50°C water bath. Add 1 mL of enzyme solution to 1 mL of the preheated pectin substrate, shake well, and react in a 50°C water bath for 30 min. Terminate the reaction by adding 1 mL of 10% trichloroacetic acid solution. For the blank control group, add the stop solution before adding the enzyme solution. After the reaction, mix 1 mL of the reaction solution with 1 mL of DNS solution and react in a boiling water bath for 5 min to develop color. Dilute the color-developed reaction solution 5 times, zero the sample using distilled water as a reference, and measure the absorbance of the sample at 540 nm using a spectrophotometer. Calculate the enzyme activity according to the formula.

[0126] The crude enzyme solution was obtained as follows: Lactobacillus paracasei YYS-EN2 was inoculated into the enzyme-producing medium and cultured at 37°C for 48 hours. The culture medium was centrifuged at 4°C, and the supernatant was collected as the crude enzyme solution. The crude enzyme solution was diluted to the enzyme solution by a certain factor for enzyme activity determination. The inoculation amount of Lactobacillus paracasei YYS-EN2 was 2% (v / v) of the culture medium.

[0127] Result calculation method:

[0128] The formula for enzyme activity determination is: Enzyme activity = OD 540 ×n×1 / k×1 / 30;

[0129] In the formula, n is the dilution factor for enzyme solution testing (the dilution factor from crude enzyme solution to test enzyme solution), k is the slope of the fitted standard curve, and 30 is the enzyme-catalyzed reaction time.

[0130] The standard curve was plotted using 1 g·L⁻¹. -1 Prepare standard solutions of different concentration gradients (0 g·L⁻¹) from D-galacturonic acid solution. -1 0.2 g·L -1 0.4 g·L -1 0.6 g·L -1 0.8 g·L -1 1.0 g·L -1 Following the enzyme activity assay method, absorbance was measured at 540 nm. A linear standard curve was plotted with D-galacturonic acid concentration on the x-axis and OD value on the y-axis (see [link to standard curve]). Figure 3 ).

[0131] The results showed that the pectinase activity of Lactobacillus paracasei YYS-EN2 was 47.81±0.07 U / mL.

[0132] Example 5: Lactobacillus paracasei YYS-EN2 tannin enzyme activity

[0133] 1. Enzyme-producing medium (g / L) -1 Ingredients: 10g glucose, 3g sodium nitrate, 1g dipotassium hydrogen phosphate, 0.5g potassium chloride, 0.5g magnesium sulfate, 20g tannic acid.

[0134] 2. Tannin enzyme activity assay

[0135] (1) Definition of enzyme activity:

[0136] At 30°C and pH 5.0, the substrate decomposes at a rate of 0.01 mol·L⁻¹ per minute. -1 The amount of enzyme required to produce 1 mg of gallic acid from propyl gallate is defined as one unit of enzyme activity (U·mL). -1 );

[0137] (2) Solution preparation:

[0138] 0.1 mol·L -1 Citrate-sodium citrate buffer (pH 5.0): Add 0.1 mol·L⁻¹ -1 Citric acid solution and 0.1 mol·L -1 The sodium citrate solution was mixed and the pH was adjusted to 5.0. The solution contained 0.1 mol·L⁻¹ -1 Citric acid solution: Accurately weigh 19.21 g of citric acid and dilute to a volumetric flask with distilled water to a final volume of 1000 mL; 0.1 mol·L⁻¹ -1 Sodium citrate solution: Accurately weigh 29.41g of sodium citrate, dissolve it in distilled water, and dilute to 1000 mL in a volumetric flask.

[0139] propyl gallate solution (0.01 mol·L⁻¹) -1 Accurately weigh 0.21 g of propyl gallate and dilute to 100 mL in a volumetric flask with citric acid buffer solution.

[0140] Methanol-Rhodanine Solution: Accurately weigh 0.667 g of rhodanine and dilute to 100 mL in a volumetric flask with methanol.

[0141] Gallic acid standard solution (5 mg·L) -1 Accurately weigh 0.5 g of gallic acid and dilute to 100 mL in a volumetric flask with methanol.

[0142] (3) Enzyme activity assay:

[0143] Preheat the propyl gallate solution in a 30°C water bath. Add 0.5 mL of enzyme solution to 0.5 mL of the preheated propyl gallate solution, shake well, and react in a 30°C water bath for 5 min. Add 0.6 mL of methanol rhodanine, and react in a 30°C water bath for 5 min. Add 0.8 mL of KOH, and react in a 30°C water bath for 5 min. Add 7.6 mL of distilled water, and react in a 30°C water bath for 10 min. Zero the sample using distilled water as a reference, and measure the absorbance at 520 nm using a spectrophotometer. Calculate the enzyme activity according to the formula. For the blank control group, add the stop solution KOH before adding the enzyme solution.

[0144] The crude enzyme solution was obtained as follows: Lactobacillus paracasei YYS-EN2 was inoculated into the enzyme-producing medium and cultured at 37°C for 48 h. The culture medium was centrifuged at 4°C and the supernatant was collected, which was the crude enzyme solution. The crude enzyme solution was diluted with the test enzyme solution at a certain ratio for enzyme activity determination. The inoculation amount of Lactobacillus paracasei YYS-EN2 was 2% (v / v) of the culture medium.

[0145] Result calculation method:

[0146] The formula for enzyme activity determination is: Enzyme activity = OD 520 ×n×1 / k×1 / 5×1 / 0.5;

[0147] In the formula, n is the dilution factor for enzyme solution testing (the dilution factor from crude enzyme solution to test enzyme solution or the dilution factor of reaction solution after color development), k is the slope of the fitted standard curve, 5 is the enzyme-catalyzed reaction time, and 1 / 0.5 is converted into 1 mL of enzyme solution.

[0148] The standard curve was plotted using 5 mg·L⁻¹. -1 Standard solutions of different concentration gradients (0 mg·L⁻¹) were prepared from gallic acid solution. -1 1 mg·L -1 2 mg·L -1 3 mg·L -1 4 mg·L -1 5 mg·L -1 Following the enzyme activity assay method, absorbance was measured at 520 nm. A linear standard curve was plotted with gallic acid concentration on the x-axis and OD value on the y-axis (see [reference needed]). Figure 4 )

[0149] The results showed that the tannin activity of Lactobacillus paracasei YYS-EN2 was 29.85 ± 0.03 U·mL. -1 .

[0150] Example 6: Phytase activity of Lactobacillus paracasei YYS-EN2

[0151] 1. Enzyme-producing medium (g / L)-1 ): Peptone 3g, glucose 15g, magnesium sulfate 0.5g, manganese sulfate 0.03g, ferrous sulfate 0.03g.

[0152] 2. Phytase activity assay

[0153] (1) Definition of enzyme activity:

[0154] 1 ml of liquid enzyme decomposes 50 mmol·L⁻¹ per minute at 37°C and pH 5.5. -1 Sodium phytate substrate produces 1 μmol of inorganic phosphorus, which is equivalent to one unit of phytase activity, expressed in U·mL. -1 .

[0155] (2) Reagent preparation:

[0156] 0.2 mol·L -1 Acetic acid-sodium acetate buffer (pH 5.5): Add 0.2 mol·L⁻¹ -1 Acetic acid solution and 0.2 mol·L -1 Sodium acetate solution was mixed and the pH was adjusted to 5.5. The solution contained 0.2 mol·L⁻¹. -1 Acetic acid solution: 12g of acetic acid was diluted to a volumetric flask with distilled water to a final volume of 1000mL; 0.2mol·L⁻¹ -1 16.406 g of sodium acetate was dissolved in distilled water and diluted to a volumetric flask of 1000 mL.

[0157] Sodium phytate solution (75 mmol·L) -1 Accurately weigh 0.69 g of sodium phytate, dissolve it in buffer solution, and dilute to a volumetric flask of 100 mL.

[0158] Nitric acid solution: Nitric acid: Water (v / v) = 1:2

[0159] Ammonium molybdate solution (100 g / L): Accurately weigh 10 g of ammonium molybdate, add 1 mL of 25% (v / v) ammonia solution, and dilute to 100 mL with distilled water in a volumetric flask.

[0160] Ammonium metavanadate solution (2.35 g / L): Accurately weigh 0.235 g of ammonium metavanadate, dissolve it in distilled water, add 2 mL of nitric acid solution, and dilute to 100 mL in a volumetric flask with distilled water. Store in the dark and prepare fresh before use.

[0161] Colorimetric solution (stop solution): Nitric acid solution: Ammonium molybdate solution: Ammonium metavanadate solution (v / v) = 2:1:1. Store protected from light and prepare fresh before use.

[0162] (3) Enzyme activity assay:

[0163] First, mix 1.8 mL of acetate buffer with 0.2 mL of the reaction solution (i.e., enzyme solution), preheat the mixture to 37°C for 5 min, then add 4 mL of sodium phytate and mix well. Incubate the mixture at 37°C for 30 min, and finally add 4 mL of colorimetric solution (stop solution) and mix well. Zero the sample using distilled water as a reference, and measure the absorbance at 415 nm using a spectrophotometer. Calculate the enzyme activity according to the formula. For the blank control group, add the stop solution before adding the enzyme solution.

[0164] The crude enzyme solution was obtained as follows: Lactobacillus paracasei YYS-EN2 was inoculated into the enzyme-producing medium and cultured at 37°C for 48 h. The culture medium was centrifuged at 4°C and the supernatant was collected as the crude enzyme solution. The crude enzyme solution was diluted to the enzyme solution by a certain factor for enzyme activity determination. The inoculation amount of Lactobacillus paracasei YYS-EN2 was 2% (v / v) of the culture medium.

[0165] Result calculation method:

[0166] The formula for enzyme activity determination is: Enzyme activity = OD 415 ×n×1 / k×1 / 30×1 / 0.2×10 3 ;

[0167] In the formula, n is the dilution factor for enzyme solution testing (the dilution factor from crude enzyme solution to test enzyme solution), k is the slope of the fitted standard curve, 30 is the enzyme-catalyzed reaction time, 1 / 0.2 is converted to 1 mL of enzyme solution, and 10 3 This is a conversion factor for the concentration of inorganic phosphate ions.

[0168] The standard curve was prepared by using potassium dihydrogen phosphate stock solution to prepare inorganic phosphorus standard solutions of different concentrations (0 mmol·L⁻¹). -1 10 mmol·L -1 20 mmol·L -1 30 mmol·L -1 40 mmol·L -1 50 mmol·L -1 OD was measured according to the enzyme activity assay method. 415 A linear standard curve was plotted with absorbance values, inorganic phosphate ion concentration on the x-axis and OD values ​​on the y-axis (see [reference]). Figure 5 ).

[0169] The results showed that the phytase activity of Lactobacillus paracasei YYS-EN2 was 18.67±0.04 U / mL.

[0170] Example 7: Application of Lactobacillus paracasei YYS-EN2 in the preparation of fermented corn nutrient powder

[0171] Corn germ and corn bran were crushed, then treated with lipase and amylase, sterilized at high temperature, and then 3% (by weight) of *Lactobacillus paracasei* YYS-EN2 bacterial powder and / or freeze-dried powder were added. After mixing thoroughly, the mixture was fermented at 37℃ for 72 hours. After fermentation, it was sterilized at high temperature and dried at 60℃ to produce corn nutrient powder. *Lactobacillus paracasei* YYS-EN2 fermentation of corn bran and corn germ decomposes cellulose and pectin and reduces tannins and phytic acid, enhancing the flavor and texture of the corn nutrient powder. Specific flavor results before and after fermentation are detailed in Table 1 below:

[0172] Table 1

[0173]

[0174] Example 8: Application of Lactobacillus paracasei YYS-EN2 in the preparation of fermented traditional Chinese medicine

[0175] Polygonatum sibiricum, ginseng (artificially cultivated), wolfberry, mulberry, and red dates were pulverized and added to the mixture at mass percentages of 0.5%, 0.3%, 1%, 1.5%, and 2%, respectively. The mixture was thoroughly mixed, boiled, and kept at a gentle simmer for 1 hour. After cooling, 0.03% of Lactobacillus paracasei YYS-EN2 bacterial powder and / or freeze-dried powder and 0.02% of Lactobacillus plantarum BXM2 bacterial powder were added, mixed thoroughly, and fermented at 37℃ for 48 hours. After fermentation, the mixture was sterilized at high temperature. Lactobacillus paracasei YYS-EN2 fermentation of traditional Chinese medicine decomposes cellulose and pectin, reduces tannins and phytic acid, decreases bitterness, and enhances taste.

[0176] The traditional Chinese medicine mentioned is not limited to the specific traditional Chinese medicines described in the examples, but includes those listed in the catalog of medicinal and edible herbs. The probiotic combination includes, but is not limited to, Lactobacillus paracasei YYS-EN2 powder and Lactobacillus plantarum BXM2 powder, and can also be used alone or in combination with other probiotic powders.

[0177] Among them, Lactobacillus plantarum ( Lactobacillus plantarum BMX2 was deposited on September 6, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.16436.

[0178] Based on the results of the above embodiments, the Lactobacillus paracasei YYS-EN2 provided by the present invention has the following properties and effects:

[0179] 1. It has the ability to produce cellulase, with a cellulase activity of 47.34 ± 0.06 U·mL. -1 It can decompose plant cellulose and can be widely used in the development of silage products.

[0180] 2. It possesses pectinase activity, with a pectinase activity of 47.81 ± 0.07 U·mL. -1 It can break down pectin in fruits and vegetables, and can be widely used in the development of products such as fruit juice, vegetables, wine, and enzymes.

[0181] 3. It possesses tanninase activity, with a tanninase activity of 29.85±0.03 U·mL. -1 It can reduce the tannin content in wine and can be widely used in the development of wine and enzyme products to improve the taste of the products.

[0182] 4. It possesses phytase activity, with a phytase activity of 18.67 ± 0.04 U·mL. -1 It can effectively decompose phytic acid and can be widely used in flour and soybean food processing to improve their nutritional and commercial value.

[0183] 5. It can be used in the production of corn nutrient powder to degrade cellulose and phytic acid in corn husks and corn germ, thereby improving the nutritional value of corn nutrient powder and enhancing its taste.

[0184] 6. The strain is derived from the saliva of healthy adults and is a natural probiotic that can be used as a fermenting agent in the preparation of feed, wine, enzymes, etc.

[0185] In summary, compared with the prior art, the Lactobacillus paracasei YYS-EN2 provided by the present invention has the following beneficial effects:

[0186] The *Lactobacillus paracasei* YYS-EN2 provided by this invention can be used in probiotic products and functional foods. It colonizes the human gut, improving gut microbiota structure and promoting digestion and absorption. It produces cellulase, pectinase, tanninase, and phytase, which can be used in probiotic fermented products (including but not limited to beverages, foods, and feeds). These fermented products include, but are not limited to, the preparation of fermented plant-derived nutrient powders, fermented plant-derived polypeptide powders, fermented plant-derived protein powders, enzymes, and silage.

[0187] The Lactobacillus paracasei YYS-EN2 strain provided by this invention produces cellulase and pectinase, which can degrade plant cellulose and pectin, and can be applied to the development of fermented plant-derived nutrient powders, fruit juices, vegetable feeds and other products. At the same time, this strain has a high capacity for pectinase and tanninase, and can be widely used in the production of fermented products, such as wines and enzymes, and can significantly reduce the tannin content in the products.

[0188] In summary, it can be specifically applied to functional products that include at least one of the following functions:

[0189] (1) It has the ability to degrade cellulose;

[0190] (2) It has the ability to degrade pectin;

[0191] (3) It has the ability to degrade tannins;

[0192] (4) It has the ability to degrade phytic acid;

[0193] Among them, products with the above (1)-(4) functions include, but are not limited to, functions such as decomposing plant fibers and pectin, improving product clarity, etc., and can also be products with other obvious effects based on the functions of decomposing plant fibers and pectin, degrading tannins or phytic acid.

[0194] It should be noted that:

[0195] (1) Definition:

[0196] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders, tablets, and gels, and also to disperse in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0197] (2) The relevant prior art means or prior art terms involved in this application:

[0198] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through an analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. X "OD" is the optical density value measured when the wavelength is set to X nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. The method for measuring the "OD" value is existing technology, and its principle and method will not be described here.

[0199] This article uses the Oxford cup method as an existing technique, and its process will not be described in detail. (3) Application of strains:

[0200] The example illustrates that Lactobacillus paracasei YYS-EN2 can be applied to corn nutrient powder products. According to the above design concept, this strain can be applied to various products containing cellulose, pectin, tannin and phytic acid, and can be applied to the preparation of fermented products including fermented plant-derived nutrient powder, fermented plant-derived polypeptide powder, fermented plant-derived protein powder, enzymes, silage, etc., including but not limited to the corn nutrient powder in the example.

[0201] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Lactobacillus paracasei ( Lactobacillus paracasei YYS-EN2, characterized in that, Its accession number is CGMCC No. 28531.

2. The *Lactobacillus paracasei* YYS-EN2 according to claim 1, characterized in that: It has the ability to produce cellulase, pectinase, tanninase and phytase.

3. The *Lactobacillus paracasei* YYS-EN2 according to claim 1, characterized in that: It has the ability to degrade cellulose, pectin, tannins and phytic acid.

4. A method for producing cellulase, pectinase, tanninase, and phytase, characterized in that: Lactobacillus paracasei YYS-EN2 was inoculated into enzyme-producing liquid culture medium and cultured at 30-40°C for 48-72 hours. The enzyme-producing liquid culture medium after fermentation was separated into solid and liquid components to obtain the supernatant. The supernatant was an enzyme solution containing cellulase, pectinase, tanninase and phytase. Wherein, the Lactobacillus paracasei YYS-EN2 is the Lactobacillus paracasei YYS-EN2 as described in any one of claims 1-3.

5. The application of Lactobacillus paracasei YYS-EN2 in the preparation of fermented products, characterized by: The Lactobacillus paracasei YYS-EN2 is the Lactobacillus paracasei YYS-EN2 as described in any one of claims 1-3; the fermentation product includes fermented nutrient powder or silage. The fermented nutrient powder includes fermented plant-derived nutrient powder.

6. A freeze-dried powder, characterized in that: Its components include Lactobacillus paracasei YYS-EN2 as described in any one of claims 1-3.

7. A microbial agent, characterized in that: The microbial agent comprises the lyophilized powder as described in claim 6; or the microbial agent comprises Lactobacillus paracasei YYS-EN2 as described in any one of claims 1-3.

8. The application of Lactobacillus paracasei YYS-EN2 in the preparation of probiotic products, characterized by: The Lactobacillus paracasei YYS-EN2 is the Lactobacillus paracasei YYS-EN2 as described in any one of claims 1-3.

9. The application of Lactobacillus paracasei YYS-EN2 in the preparation of functional products, characterized by: The functional product includes at least one of the following functions: (1) It has the ability to degrade cellulose; (2) It has the ability to degrade pectin; (3) It has the ability to degrade tannins; (4) It has the ability to degrade phytic acid; Wherein, the Lactobacillus paracasei YYS-EN2 is the Lactobacillus paracasei YYS-EN2 as described in any one of claims 1-3.

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