Litchi enzyme, its production method and use

Abstract

To provide a lychee enzyme, a production method thereof, and use thereof.SOLUTION: Provided are uses of a lychee enzyme in the manufacture of a composition, the composition being used for improving intestinal barrier function, regulating intestinal immunity bi-directionally, improving intestinal inflammation, maintaining intestinal steady state, or improving intestinal health. A method for producing lychee enzyme comprises: (1) performing primary fermentation; (2) extruding and filtering the product after primary fermentation to obtain a primary fermentation filtrate; (3) performing secondary fermentation; and (4) filtering and sterilizing the secondary fermentation liquid to obtain lychee enzyme. The lychee enzyme of the present invention is used for improving intestinal barrier function, regulating intestinal immunity bi-directionally, improving intestinal inflammation, maintaining intestinal steady state, and further improving intestinal health.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention belongs to the field of microbial fermentation, and particularly relates to the use of litchi enzyme in improving intestinal barrier function, bidirectionally regulating intestinal immunity, improving intestinal inflammation, maintaining intestinal steady state, or improving intestinal health. [Background technology]

[0002] Lychee is a common fruit in the south of China, with a sweet taste and mild nature, and has the effects of enhancing the function of the spleen, enhancing the function of the liver, improving the function of qi, enhancing the function of blood, warming the spleen and stomach, relieving pain, replenishing the heart and qi, and stabilizing the mind, etc. Enzymes are products containing specific bioactive ingredients obtained by microbial fermentation using animals, plants, fungi, etc. as raw materials, with or without the addition of auxiliary materials, and the bioactive ingredients include polysaccharides, oligosaccharides, polyphenols, proteins, and polypeptides, amino acids, vitamins, etc. Enzymes have the effects of promoting metabolism, breaking down alcohol, protecting the liver, and whitening the skin, etc.

[0003] The traditional litchi processing process is complicated, has a long production cycle, or requires the introduction of specific equipment, which is disadvantageous for large-scale industrialized production. In the traditional enzyme products, there are few products that can improve the intestinal barrier and intestinal immune function, and there are also phenomena in which the improvement mechanism cannot be clearly explained. The present invention obtains litchi enzyme through a unique fermentation process and a combination of probiotics, and intentionally improves the intestinal barrier function and bidirectionally regulates the intestinal immunity. At the same time, the present invention also clearly explains the improved mechanism. The technical significance of the present invention is not only to extend the function of enzyme products, but also to provide a mechanism explanation for the applicability of the product. Summary of the Invention

[0004] In the conventional enzyme products, there are few products that can improve the intestinal barrier and intestinal immune functions. In view of this, the present inventor has developed a litchi enzyme that can improve the intestinal barrier function, regulate the intestinal immunity in two directions, improve intestinal inflammation, maintain the intestinal steady state, and further improve intestinal health.

[0005] The present invention also provides a use of litchi enzyme in preparing a composition for improving intestinal barrier function, bidirectionally regulating intestinal immunity, improving intestinal inflammation, maintaining intestinal steady state, or improving intestinal health.

[0006] The present invention also provides a litchi enzyme for improving intestinal barrier function, bidirectionally regulating intestinal immunity, improving intestinal inflammation, maintaining intestinal steady state, or improving intestinal health.

[0007] In one embodiment, the litchi enzyme is the litchi enzyme of the present invention.

[0008] The present invention also provides a method for producing a litchi enzyme, the method comprising the steps of: (1) Primary fermentation, including adding a first carbon source to lychee fruit juice and lychee fruit pomace, inoculating a first bacterial agent, which is a mixture of yeast and lactic acid bacteria, and sealing and standing at 20-45°C for 24-96 hours; (2) extruding and filtering the product after primary fermentation to obtain a primary fermentation filtrate; (3) secondary fermentation, which includes adding a second carbon source to the primary fermentation filtrate, inoculating a second bacterial agent, which is acetic acid bacteria, and allowing to stand or aerate at 15-40° C. for 3-15 days to obtain a secondary fermentation broth; and (4) filtering and sterilizing the secondary fermentation liquid to obtain the lychee enzyme.

[0009] In certain embodiments, the method includes a pre-treatment step of washing and squeezing the lychee fruit.

[0010] In some embodiments, the first carbon source is one or more of fructose, sucrose, brown sugar, maltose, and arabinose.

[0011] In one embodiment, the amount of the first carbon source added is 0-5 wt%.

[0012] In some embodiments, the yeast is one or more of Hansenula anomala, sake yeast, Saccharomyces rouxii Boutroux, Saccharomyces cerevisiae, Saccharomyces willianus, Pichia pastoris, or Rhodotorula mucilaginosa. Preferably, the yeast is one or more of Hansenula anomala, sake yeast, or Saccharomyces cerevisiae.

[0013] In certain embodiments, the lactic acid bacteria is one or more of Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus casei, Lactobacillus gasseri, or Lactobacillus paracasei.

[0014] In one embodiment, the weight ratio of yeast:lactic acid bacteria is 50:1-500:1. Preferably, the weight ratio of yeast:lactic acid bacteria is 300:1-500:1.

[0015] In one embodiment, the amount of the first fungicide added is 0.1‰-10‰ of the total mass of the litchi.

[0016] In some embodiments, the second carbon source is one or more of lactose, brown sugar, glucose, molasses, or honey.

[0017] In one embodiment, the amount of the second carbon source added is 1%-20% of the total mass of the primary fermentation filtrate.

[0018] In certain embodiments, the second bacterial agent is one or more of Acetobacter pasteurianus, Acetobacter aceti aceti, Acetobacter ghanensis, Acetobacter rancens, or Acetobacter aceti.

[0019] In one embodiment, the amount of the second fungal agent added is 0.1‰-10‰ of the total mass of the primary fermentation filtrate.

[0020] In one embodiment, the static culture is sealed with eight layers of gauze and statically cultured.

[0021] In one embodiment, the aeration rate for the aerobic culture is 0.1-20 mL / min / mL of fermentation broth.

[0022] In another aspect, there is provided a composition comprising the litchi enzyme of the present invention. The composition may be a medicine. Alternatively, the composition may be a food or functional food, in the form of a drink, beverage, jelly, or gel candy. In some embodiments, the litchi enzyme is in liquid form.

[0023] Advantages of the present invention include: 1. Providing litchi enzymes that improve intestinal barrier function, bidirectionally regulate intestinal immunity, improve intestinal inflammation, maintain intestinal steady state, and further improve intestinal health; and 2. A new lychee enzyme production process is provided, which adopts different bacterial agent combinations and carries out secondary fermentation in stages. [Brief description of the drawings]

[0024] Figure 1 shows the effect of different concentrations of litchi enzyme on the transepithelial electric resistance (TEER) of lipopolysaccharide (LPS)-treated human colon cancer cell Caco-2. When the concentration of litchi enzyme is greater than 0.1 μg / mL, it can significantly improve the transepithelial electric resistance (p<0.05) of intestinal cells, indicating that litchi enzyme can repair the intestinal barrier and has the effect of improving the intestinal barrier function. Note: ### indicates a highly significant difference (p<0.0001) compared with the blank control group; ns indicates no significant difference compared with the model group (LPS group); * indicates a significant difference (p<0.05) compared with the model group (LPS group).

[0025] Figure 2 shows the effect of different concentrations of litchi enzyme on the amount of NO released by macrophages RAW264.7. Different concentrations of litchi enzyme can promote macrophages RAW264.7 to secrete NO, indicating that the enzyme sample can activate macrophages and participate in immune regulation. Note: Different alphabets in the figure indicate significant differences (p<0.05), and containing the same alphabet indicates no significant differences. Significant difference analysis was performed with Duncan's multiple range test in single factor analysis of variance (ONE-WAY ANOVA), and p<0.05 was defined as the significant difference threshold.

[0026] Figure 3 shows the effect of different concentrations of litchi enzyme on the amount of NO released by macrophages RAW264.7 induced by lipopolysaccharide (LPS). Litchi enzyme can inhibit inflammatory cells from releasing NO within a certain concentration range and has anti-inflammatory activity, the concentration range of which is 0.1-8μg / mL. The experimental results of macrophages show that the regulation of immunity by litchi enzyme is bidirectional and helps maintain the steady state of the intestinal tract. Note: #### indicates highly significant difference (p<0.0001) compared with the blank control group; ns indicates no significant difference compared with the model group (LPS group), * indicates significant difference (p<0.05) compared with the model group (LPS group), ** indicates highly significant difference (p<0.01) compared with the model group (LPS group), *** indicates highly significant difference (p<0.001) compared with the model group (LPS group), and **** indicates highly significant difference (p<0.0001) compared with the model group (LPS group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] These and other aspects, features, and advantages of the present invention will be apparent to those skilled in the art upon reading the following detailed description and the appended claims. Any feature of one aspect of the present invention can be used in any other aspect of the present invention. The term "comprises" is intended to be open ended and can be replaced with "consisting of" where appropriate. In this specification, unless otherwise indicated, percentages refer to weight / weight percentages. Except in the examples or more clearly described, all numbers indicating the quality, physical properties, and the like of materials in the specification and claims should be understood to be modified by "about" and can represent said numerical value ±10% or 5%.

[0028] As used herein, "enzyme" refers to a product containing specific bioactive components obtained by microbial fermentation with or without the addition of auxiliary materials, using animals, plants, fungi, etc. as raw materials. The present invention provides litchi enzyme, which refers to a product obtained by microbial fermentation when litchi is used as raw material and a carbon source is added.

[0029] As used herein, "bioactive ingredient" refers to a trace or small amount of a substance produced by fermentation that affects a life phenomenon. Bioactive ingredients can include polysaccharides, oligosaccharides, polyphenols, proteins and polypeptides, amino acids, vitamins, etc.

[0030] The present invention provides a method for preparing litchi enzyme, which includes a two-stage fermentation process. Specifically, the method includes a first fermentation, which includes adding a first carbon source to litchi fruit juice and litchi fruit pomace, inoculating a first fungal agent, and performing sealed static fermentation at a first temperature for a first time period, and the first fungal agent is a mixed fungal agent of yeast and lactic acid bacteria. The yeast can be one or more of Hansenula anomala, sake yeast, Saccharomyces rouxii Boutroux, Saccharomyces cerevisiae, Saccharomyces willianus, Pichia pastoris, or Rhodotorula mucilaginosa. The lactic acid bacteria can be one or more of Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus casei, Lactobacillus gasseri, or Lactobacillus paracasei.

[0031] The first temperature may be in the range of 20-45° C., such as 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or any range therebetween. The first time period may be in the range of 24-96 hours, such as 25, 30, 35, 40, 45, 50, 55, 60, 75, 70, 75, 80, or 90 hours.

[0032] The first carbon source may be one or more of fructose, sucrose, brown sugar, maltose, and arabinose. The amount of the first carbon source added may be 0-5 wt%, for example, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, or 4 wt%. The first carbon source may include two or more sugars, for example, two or more sugars having the same weight percent. In some embodiments, the first carbon source is fructose:brown sugar=1:1. In some embodiments, the first carbon source is sucrose. In some embodiments, the first carbon source is maltose. In some embodiments, the first carbon source is fructose:sucrose:brown sugar=1:1:1. In some embodiments, the first carbon source is sucrose:arabinose=1:1.

[0033] The weight ratio of yeast:lactic acid bacteria may be 50:1-500:1, for example 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1 or 400:1.

[0034] The amount of the first fungicide added may be 0.1‰-10‰ of the total mass of the litchi, for example 0.5‰, 0.9‰, 1‰, 2‰, 3‰, 4‰, 5‰, 6‰, 7‰, 8‰ or 9‰.

[0035] In one embodiment, the first fungal agent has a yeast:lactic acid bacteria ratio of 400:1, the yeast is sake yeast, and the lactic acid bacteria is Lactobacillus paracasei:Lactobacillus reuteri:Lactobacillus gasseri ratio of 1:1:1. In one embodiment, the amount of the first fungal agent added is 0.9‰ of the total mass of the lychee.

[0036] In one embodiment, the first fungal agent is a yeast:lactic acid bacteria ratio of 100:1, the yeast is a Saccharomyces cerevisiae:Pichia pastoris:Saccharomyces rouxii Boutroux ratio of 1:1:1, and the lactic acid bacteria is a Lactobacillus plantarum:Lactobacillus casei:Lactobacillus reuteri ratio of 1:1:1. In one embodiment, the amount of the first fungal agent added is 5‰ of the total mass of the lychee.

[0037] In one embodiment, the first fungal agent is a yeast:lactic acid bacteria ratio of 50:1, the yeast is a Saccharomyces willianus:Rhodotorula mucilaginosa:Saccharomyces rouxii Boutroux ratio of 1:1:1, and the lactic acid bacteria is a Lactobacillus sakei:Lactobacillus casei ratio of 1:1. In one embodiment, the amount of the first fungal agent added is 0.1‰ of the total mass of the lychee.

[0038] In one embodiment, the first fungal agent is yeast:lactic acid bacteria at 300:1; the yeast is Hansenula anomala; and the lactic acid bacteria is Lactobacillus gasseri. In one embodiment, the amount of the first fungal agent added is 10‰ of the total mass of the lychee.

[0039] In one embodiment, the first fungal agent is yeast:lactic acid bacteria at a ratio of 500:1, the yeast is Saccharomyces cerevisiae:sake yeast at a ratio of 1:1, and the lactic acid bacteria is Lactobacillus plantarum:Lactobacillus gasseri:Lactobacillus sakei at a ratio of 1:1:1. In one embodiment, the amount of the first fungal agent added is 8‰ of the total mass of the lychee.

[0040] In one embodiment, the first fungal agent is yeast:lactic acid bacteria at 200:1, the yeast is Rhodotorula mucilaginosa, and the lactic acid bacteria is Lactobacillus plantarum:Lactobacillus sakei at 1:1. In one embodiment, the amount of the first fungal agent added is 2‰ of the total mass of the lychee.

[0041] In one embodiment, the first fungal agent is yeast:lactic acid bacteria at 150:1; the yeast is Saccharomyces cerevisiae; and the lactic acid bacteria is Lactobacillus casei. In one embodiment, the amount of the first fungal agent added is 4‰ of the total mass of the lychee.

[0042] The product after the primary fermentation can be extruded and filtered to obtain a primary fermentation filtrate. The process can be carried out in various ways known in the art, for example, by a filter press. The primary fermentation process can be preceded by a pretreatment step of washing and squeezing the lychee fruit.

[0043] The method for producing litchi enzyme further includes a second fermentation process, which includes adding a second carbon source to the first fermentation filtrate, inoculating a second bacterial agent, and statically or aerated culturing at a second temperature for a second period of time to obtain litchi enzyme, and the second bacterial agent is acetic acid bacteria. The second bacterial agent may be one or more of Acetobacter pasteurianus, Acetobacter aceti aceti, Acetobacter ghanensis, Acetobacter rancens, or Acetobacter aceti. The second carbon source may be one or more of lactose, brown sugar, glucose, molasses, or honey. The second carbon source may include two or more sugars, for example, two or more sugars with the same weight percentage. The amount of the second carbon source added is 1%-20% of the total mass of the primary fermentation filtrate, for example 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% or 19%. In some embodiments, the second carbon source is lactose. In some embodiments, the second carbon source is brown sugar. In some embodiments, the second carbon source is molasses:lactose 1:1. In some embodiments, the second carbon source is glucose. In some embodiments, the second carbon source is honey. In some embodiments, the second carbon source is glucose:honey 1:1.

[0044] The second temperature range may be 15-40° C., such as 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., or any range therebetween. The second time period may be in the range of 3-15 days, such as 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0045] The amount of the second bacterial agent added may be 0.1‰-10‰ of the total mass of the primary fermentation filtrate, for example 0.5‰, 1‰, 2‰, 3‰, 4‰, 5‰, 6‰, 7‰, 8‰ or 9‰.

[0046] In one embodiment, the second bacterial agent is Acetobacter aceti aceti: ​​Acetobacter aceti. In one embodiment, the amount of the second bacterial agent added is 1‰ of the total mass of the primary fermentation filtrate.

[0047] In some embodiments, the second bacterial agent is Acetobacter pasteurianus. In some embodiments, the dosage is 5‰ of the total mass of the primary fermentation filtrate.

[0048] In one embodiment, the second bacterial agent is Acetobacter aceti. In one embodiment, the dosage is 9‰ of the total mass of the primary fermentation filtrate.

[0049] In one embodiment, the second bacterial agent is Acetobacter ghanensis:Acetobacter aceti:Acetobacter pasteurianus at 1:1:1. In one embodiment, the amount added is 0.1‰ of the total mass of the primary fermentation filtrate.

[0050] In one embodiment, the second bacterial agent is Acetobacter rancens:Acetobacter aceti aceti:Acetobacter pasteurianus at 1:1:1. In one embodiment, the amount of the second bacterial agent is 0.9‰ of the total mass of the primary fermentation filtrate.

[0051] In one embodiment, the second bacterial agent is Acetobacter rancens. In one embodiment, the dosage is 3‰ of the total mass of the primary fermentation filtrate.

[0052] In some embodiments, the second bacterial agent is Acetobacter ghanensis. In some embodiments, the dosage is 10‰ of the total mass of the primary fermentation filtrate.

[0053] In one embodiment, the yeast content for inoculation is at least 1.0×10 6 CFU / g, e.g. 1.5×10 6CFU / g, 2×10 6 CFU / g, 2.5×10 6 CFU / g, 3×10 6 CFU / g, 3.5×10 6 CFU / g, 4×10 6 CFU / g, 4.5×10 6 CFU / g, 5×10 6 CFU / g, 5.5×10 6 CFU / g, 6×10 6 CFU / g, 6.5×10 6 CFU / g, 7×10 6 CFU / g, 7.5×10 6 CFU / g, 8×10 6 CFU / g, 8.5×10 6 CFU / g, 9×10 6 CFU / g or 9.5 x 10 6 CFU / g.

[0054] In one embodiment, the lactic acid bacteria content for inoculum is at least 1.0×10 5 CFU / g, e.g. 1.5×10 6 CFU / g, 2×10 6 CFU / g, 2.5×10 6 CFU / g, 3×10 6 CFU / g, 3.5×10 6 CFU / g, 4×10 6 CFU / g, 4.5×10 6 CFU / g, 5×10 6 CFU / g, 5.5×10 6 CFU / g, 6×10 6 CFU / g, 6.5×10 6 CFU / g, 7×10 6 CFU / g, 7.5×10 6 CFU / g, 8×10 6 CFU / g, 8.5×10 6 CFU / g, 9×10 6 CFU / g or 9.5 x 10 6 CFU / g.

[0055] In one embodiment, the protozoal content of the acetic acid bacteria for inoculation is at least 1.5×10 6CFU / g, 2×10 6 CFU / g, 2.5×10 6 CFU / g, 3×10 6 CFU / g, 3.5×10 6 CFU / g, 4×10 6 CFU / g, 4.5×10 6 CFU / g, 5×10 6 CFU / g, 5.5×10 6 CFU / g, 6×10 6 CFU / g, 6.5×10 6 CFU / g, 7×10 6 CFU / g, 7.5×10 6 CFU / g, 8×10 6 CFU / g, 8.5×10 6 CFU / g, 9×10 6 CFU / g or 9.5 x 10 6 CFU / g.

[0056] In the present specification, the static culture may be any type of static culture known to those skilled in the art, for example, static culture sealed with eight layers of gauze. Also, the aerated culture may be any type of aerated culture known to those skilled in the art. The aeration rate may be 0.1-20 mL / min / mL fermentation liquid, for example, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 mL / min / mL fermentation liquid.

[0057] The litchi enzyme produced by the present method can be added to a product as an additive ingredient, for example, added to a food or functional food, or used as a food or functional food. The form of the litchi enzyme is not particularly limited. For example, the litchi enzyme may be liquid. The litchi enzyme of the present invention is used to improve intestinal barrier function, bidirectionally regulate immunity (e.g., intestinal immunity), improve intestinal inflammation, maintain intestinal steady state, and further improve intestinal health. The litchi enzyme of the present invention can achieve these effects by regulating NO secretion from macrophages. In addition, the litchi enzyme of the present invention can activate macrophages to produce NO and exert non-specific immune effects. In addition, the litchi enzyme of the present invention can inhibit the release of NO from inflammatory cells induced by LPS, and has anti-inflammatory activity.

[0058] Although several embodiments of the present invention have been described above, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0059] The present application will be described in more detail below with reference to examples. However, those skilled in the art should understand that these embodiments are provided for illustrative purposes only and are not intended to limit the present application.

[0060] Working Example The embodiments of the present application will be described in detail below with reference to the examples. Those skilled in the art should understand that the following examples are merely for the purpose of illustrating the present application and are not to be considered as limiting the scope of the present application. In the examples, the specific conditions are not specified, and are carried out according to normal conditions or conditions suggested by the manufacturer. The reagents or equipment used are not specified by the manufacturer, and are conventional products that can be obtained commercially. Unless otherwise specified, all amounts listed are described based on the weight percentage of the total weight. The present application should not be interpreted as being limited to the specific examples described.

[0061] The fungicides used in the examples are as follows: Sake yeast: Saccharomyces sake CCTCC CY 20081270 (purchased from the typical culture depository center); Saccharomyces cerevisiae: Saccharomyces sp. GDMCC 2.115 (purchased from the Guangdong Microbial Culture Collection Center); Pichia anomala: Pichia anomala CICC 1859 (purchased from the China Center of Industrial Culture Collection); Saccharomyces rouxii Boutroux: Saccharomyces rouxii Boutroux GDMCC 2.55 (purchased from the Guangdong Microbial Culture Collection Center); Saccharomyces willianus: Saccharomyces willianus Saccardo GDMCC 2.60 (purchased from the Guangdong Microbial Culture Collection Center); Rhodotorula mucilaginosa: Rhodotorula mucilaginosa CICC 13237 (purchased from China Industrial Microbial Seed Deposit Center); Hansenula anomala: Hansenula anomala var. Anomala CICC 1641 (purchased from China Industrial Microbial Seed Deposit Center).

[0062] Lactobacillus paracasei: Lactobacillus paracasei CGMCC 1.9089 (purchased from China General Microbiological Culture Collection Center); Lactobacillus plantarum: Lactobacillus plantarum CGMCC 1.572 (purchased from China General Microbiological Culture Collection Center); Lactobacillus casei: Lactobacillus casei CGMCC 1.575 (purchased from China General Microbiological Culture Collection Center); Lactobacillus reuteri: Lactobacillus reuteri CICC 6121 (purchased from China Industrial Microbiological Culture Collection Center); Lactobacillus sakei: Lactobacillus sakei subsp. sakei CICC 21858 (purchased from China Industrial Microbiological Culture Collection Center); Lactobacillus gasseri: Lactobacillus gasseri FRI 2008082 (NING BO TESTOBIO LIMITED).

[0063] Acetobacter aceti : Acetobacter aceti ATCC 15973 (from American Type Culture Collection); Acetobacter aceti : Acetobacter aceti BNCC 159639 (purchased from BeNa Culture Collection (BNCC)); Acetobacter pasteurianus : Acetobacter pasteurianus GDMCC 1.67 (purchased from Guangdong Provincial Microbial Seed Depository Center); Acetobacter ghanensis : Acetobacter ghanensis CICC 7002 (purchased from China Industrial Microbial Seed Depository Center); Acetobacter rancens : Acetobacter rancens BNCC 183824 (purchased from BeNa Culture Collection (BNCC)). The above strains were purchased as bacterial powder, and the yeast protobacterium content was 1.0 × 10 6 CFU / g, and the protozoan content of lactic acid bacteria was 1.0×10 5 CFU / g, and the protozoan content of acetic acid bacteria was 1.0 × 10 5 CFU / g. The strains were added as powder during the experiment.

[0064] Example 1: Preparation of litchi enzyme (1) Pre-treatment: 5 tons of lychee fruit are washed and pressed, (2) Primary fermentation: Add 1 liter of sugar to the mixture of fruit juice and fruit pomace, inoculate with 1 liter of fungal agent, seal and leave at 30°C for 72 hours to ferment. (3) Filtration: pressing with a filter press to obtain a primary fermentation filtrate, and the mass of the primary fermentation filtrate is greater than 60% of the total mass of the lychee fruit; (4) Secondary fermentation: Sugar 2 is added to the primary fermentation filtrate, and bacterial agent 2 is inoculated and cultured at 15°C for 5 days with aeration. (5) Filter and sterilize.

[0065] In this embodiment, sugar 1 was fructose:brown sugar = 1:1; the amount of sugar 1 added was 2% (w / w); bacterial agent 1 was yeast:lactic acid bacteria = 400:1; the yeast was sake yeast; the lactic acid bacteria was Lactobacillus paracasei:Lactobacillus reuteri:Lactobacillus gasseri = 1:1:1; and the amount of bacterial agent 1 added was 0.9‰ of the total mass of lychee.

[0066] In this embodiment, sugar 2 is lactose; the amount of sugar 2 added is 15% of the total mass of the primary fermentation filtrate; bacterial agent 2 is Acetobacter acetate aceti:Acetobacter acetate at a ratio of 1:1; the amount of bacterial agent 2 added is 1‰ of the total mass of the primary fermentation filtrate; and the culture method is aeration culture with an aeration volume of 1mL / min / mL fermentation liquid.

[0067] The litchi enzyme was prepared by the above method.

[0068] Example 2: Preparation of litchi enzyme (1) Pre-treatment: 5 tons of lychee fruit are washed and pressed, (2) Primary fermentation: Add 1 part of sugar to the mixture of fruit juice and fruit pomace, inoculate with 1 part of fungal agent, and leave to ferment in a sealed container at 21°C for 96 hours. (3) Filtration: pressing with a filter press to obtain a primary fermentation filtrate, and the mass of the primary fermentation filtrate is greater than 60% of the total mass of the lychee fruit; (4) Secondary fermentation: Sugar 2 is added to the primary fermentation filtrate, and bacterial agent 2 is inoculated and cultured at 37°C for 7 days with aeration. (5) Filter and sterilize.

[0069] In this embodiment, sugar 1 is sucrose; the amount of sugar 1 added is 5% (w / w); bacterial agent 1 is yeast:lactic acid bacteria at 100:1; yeast is Saccharomyces cerevisiae:Pichia pastoris:Saccharomyces rouxii Boutroux at 1:1:1; lactic acid bacteria is Lactobacillus plantarum:Lactobacillus casei:Lactobacillus reuteri at 1:1:1; and the amount of bacterial agent 1 added is 5‰ of the total mass of lychee.

[0070] In this embodiment, sugar 2 is brown sugar; the amount of sugar 2 added is 1% of the total mass of the primary fermentation filtrate; bacterial agent 2 is Acetobacter pasteurianus; the amount of bacterial agent 2 added is 5‰ of the total mass of the primary fermentation filtrate; and the culture method is aeration culture of the fermentation liquid with an aeration amount of 15mL / min / mL.

[0071] Example 3: Preparation of litchi enzyme (1) Pre-treatment: 5 tons of lychee fruit are washed and pressed, (2) Primary fermentation: Add 1 part of sugar to the mixture of fruit juice and fruit pomace, inoculate with 1 part of fungal agent, seal and leave at 45°C for 24 hours to ferment. (3) Filtration: pressing with a filter press to obtain a primary fermentation filtrate, and the mass of the primary fermentation filtrate is greater than 60% of the total mass of the lychee fruit; (4) Secondary fermentation: Sugar 2 is added to the primary fermentation filtrate, and bacterial agent 2 is inoculated and cultured at 30°C for 3 days with aeration. (5) Filter and sterilize.

[0072] In this embodiment, sugar 1 is maltose:arabinose at 1:1; the amount of sugar 1 added is 4% (w / w); bacterial agent 1 is yeast:lactic acid bacteria at 50:1; the yeast is Saccharomyces willianus:Rhodotorula mucilaginosa:Saccharomyces rouxii Boutroux at 1:1:1; the lactic acid bacteria is Lactobacillus sakei:Lactobacillus casei at 1:1; and the amount of bacterial agent 1 added is 0.1‰ of the total mass of lychee.

[0073] In this embodiment, sugar 2 is molasses:lactose at 1:1; the amount of sugar 2 added is 5% of the total mass of the primary fermentation filtrate; bacterial agent 2 is Acetobacter aceti; the amount of bacterial agent 2 added is 9‰ of the total mass of the primary fermentation filtrate; and the culture method is aeration culture with an aeration volume of 20mL / min / mL fermentation liquid.

[0074] Example 4: Preparation of litchi enzyme (1) Pre-treatment: 5 tons of lychee fruit are washed and pressed, (2) Primary fermentation: Add 1 part of sugar to the mixture of fruit juice and fruit pomace, inoculate with 1 part of fungal agent, and leave to ferment in a sealed container at 39°C for 25 hours. (3) Filtration: pressing with a filter press to obtain a primary fermentation filtrate, and the mass of the primary fermentation filtrate is greater than 60% of the total mass of the lychee fruit; (4) Secondary fermentation: Sugar 2 is added to the primary fermentation filtrate, and bacterial agent 2 is inoculated and cultured at 25°C for 10 days with aeration. (5) Filter and sterilize.

[0075] In this embodiment, sugar 1 is maltose; the amount of sugar 1 added is 1% (w / w); bacterial agent 1 is yeast:lactic acid bacteria at 300:1; the yeast is Hansenula anomala; the lactic acid bacteria is Lactobacillus gasseri; and the amount of bacterial agent 1 added is 10‰ of the total mass of lychee.

[0076] In this embodiment, sugar 2 is glucose; the amount of sugar 2 added is 18% of the total mass of the primary fermentation filtrate; bacterial agent 2 is Acetobacter ghanensis:Acetobacter aceti:Acetobacter pasteurianus at 1:1:1; the amount of bacterial agent 2 added is 0.1‰ of the total mass of the primary fermentation filtrate; and the culture method is aeration culture with an aeration volume of 3mL / min / mL.

[0077] Example 5: Preparation of litchi enzyme (1) Pre-treatment: 5 tons of lychee fruit are washed and pressed, (2) Primary fermentation: Add 1 liter of sugar to the mixture of fruit juice and fruit pomace, inoculate with 1 liter of fungal agent, seal and leave at 25°C for 48 hours to ferment. (3) Filtration: pressing with a filter press to obtain a primary fermentation filtrate, and the mass of the primary fermentation filtrate is greater than 60% of the total mass of the lychee fruit; (4) Secondary fermentation: Sugar 2 is added to the primary fermentation filtrate, and bacterial agent 2 is inoculated and cultured at 40°C for 9 days with aeration. (5) Filter and sterilize.

[0078] In this embodiment, sugar 1 is fructose:sucrose:brown sugar at a ratio of 1:1:1; the amount of sugar 1 added is 3% (w / w); bacterial agent 1 is yeast:lactic acid bacteria at a ratio of 500:1; the yeast is Saccharomyces cerevisiae:sake yeast at a ratio of 1:1; the lactic acid bacteria is Lactobacillus plantarum:Lactobacillus gasseri:Lactobacillus sakei at a ratio of 1:1:1; and the amount of bacterial agent 1 added is 8‰ of the total mass of lychee.

[0079] In this embodiment, sugar 2 is honey; the amount of sugar 2 added is 20% of the total mass of the primary fermentation filtrate; the bacterial agent 2 is Acetobacter rancens: Acetobacter acetate aceti: ​​Acetobacter pasteurianus in a ratio of 1:1:1; the amount of bacterial agent 2 added is 0.9‰ of the total mass of the primary fermentation filtrate; and the culture method is aeration culture of the fermentation liquid with an aeration amount of 5mL / min / mL.

[0080] Example 6: Preparation of litchi enzyme (1) Pre-treatment: 5 tons of lychee fruit are washed and pressed, (2) Primary fermentation: Add 1 liter of sugar to the mixture of fruit juice and fruit pomace, inoculate with 1 liter of fungal agent, seal and leave at 32°C for 86 hours to ferment. (3) Filtration: pressing with a filter press to obtain a primary fermentation filtrate, and the mass of the primary fermentation filtrate is greater than 60% of the total mass of the lychee fruit; (4) Secondary fermentation: Sugar 2 is added to the primary fermentation filtrate, and bacterial agent 2 is inoculated and cultured at 35°C for 15 days with aeration. (5) Filter and sterilize.

[0081] In this embodiment, the amount of sugar 1 added is 0%; the ratio of yeast to lactic acid bacteria in the bacterial agent 1 is 200:1; the yeast is Rhodotorula mucilaginosa; the lactic acid bacteria is Lactobacillus plantarum:Lactobacillus sakei at 1:1; and the amount of bacterial agent 1 added is 2‰ of the total mass of the lychee.

[0082] In this embodiment, sugar 2 is glucose:honey at 1:1 ratio; the amount of sugar 2 added is 10% of the total mass of the primary fermentation filtrate; bacterial agent 2 is Acetobacter rancens; the amount of bacterial agent 2 added is 3‰ of the total mass of the primary fermentation filtrate; and the culture method is aeration culture with an aeration volume of 0.1 mL / min / mL fermentation liquid.

[0083] Example 7: Preparation of litchi enzyme (1) Pre-treatment: 5 tons of lychee fruit are washed and pressed, (2) Primary fermentation: Add 1 part of sugar to the mixture of fruit juice and fruit pomace, inoculate with 1 part of fungal agent, and ferment in a sealed container at 28°C for 65 hours. (3) Filtration: pressing with a filter press to obtain a primary fermentation filtrate, and the mass of the primary fermentation filtrate is greater than 60% of the total mass of the lychee fruit; (4) Secondary fermentation: Sugar 2 is added to the primary fermentation filtrate, and bacterial agent 2 is inoculated and incubated at 28°C for 13 days. (5) Filter and sterilize.

[0084] In this embodiment, sugar 1 is sucrose:arabinose at 1:1; the amount of sugar 1 added is 2.5% (w / w); bacterial agent 1 is yeast:lactic acid bacteria at 150:1; the yeast is Saccharomyces cerevisiae; the lactic acid bacteria is Lactobacillus casei; and the amount of bacterial agent 1 added is 4‰ of the total mass of lychee.

[0085] In this embodiment, sugar 2 is brown sugar; the amount of sugar 2 added is 3% of the total mass of the primary fermentation filtrate; bacterial agent 2 is Acetobacter ghanensis; the amount of bacterial agent 2 added is 10‰ of the total mass of the primary fermentation filtrate; and static culture is static culture sealed with 8 layers of gauze.

[0086] Example 8: Efficacy test method Litchi enzyme experimental processing method: 1 mg of litchi enzyme sample was weighed and dissolved in 1 mL of PBS to prepare a mother solution of 1 mg / mL, which was then diluted 31.25, 62.5, 125, 250, 500, and 1000 times to prepare sample solutions with corresponding concentrations of 32, 16, 8, 4, 2, and 1 μg / mL.

[0087] Human colon cancer cell Caco-2 intestinal barrier function test experiment Establishment of cell culture and cell monolayer barrier model: Caco-2 cells stored in liquid nitrogen were resuscitated, and when the cell wall adhesion fusion rate reached 85%, the cells were digested for about 5 min at 37 °C and then subcultured in a complete medium containing 1% 100 μg / mL streptomycin, 100 U / mL penicillin, and 10% fetal bovine serum.

[0088] Before establishing the monolayer barrier model, wet the upper chamber of the migration cup Transwell with pre-chilled PBS solution and then inoculate 1x10 cells. 4 The cells were seeded in the upper chamber of the Transwell plate at a density of cells / well, and the cells were starved for 24 h in serum-free medium, which was then replaced with complete medium. After 18–21 days of continuous growth, the cells were able to form a continuous, dense cell monolayer in the upper chamber of the Transwell plate, at which point subsequent experimental treatments could be performed.

[0089] The whole experiment was divided into three groups: blank group, lipopolysaccharide (LPS) modeling group, and enzyme treatment group. The experiment selected a stepwise incubation model, the blank group represented the complete medium only added, without any experimental treatment; the modeling group represented the complete medium added with LPS at a final concentration of 5 μg / mL, incubated with cells for 24 h, and then washed with PBS; the enzyme treatment group represented the LPS and Caco-2 cells incubated together for 24 h, and then washed with PBS, at which time different concentrations of litchi enzyme solution (Example 5) and Caco-2 cells were incubated together for 6 h.

[0090] Detection of intestinal cell transmembrane resistance (TEER) Caco-2 cells were seeded into 24-well Transwell cells. After the cells were continuously cultured for 18–21 days, the TEER values ​​were between 300 and 1000 Ω cm 2 When the resistance reached 100%, it indicated the formation of a dense cell monolayer. After that, LPS with a final concentration of 5 μg / mL was added to the lower chamber and allowed to interact with the upper layer cells for 24 h, after which the cell TEER value was measured, and modeling was considered successful when the resistance value exceeded 20%. Then, litchi enzyme was further added and incubated with the cells for a total of 6 h, and the TEER value was measured again. Finally, the relative change value of intestinal cell TEER was calculated.

[0091] The TEER value of Caco-2 epithelial cell monolayer can reflect the barrier function of intestinal epithelium. After adopting LPS modeling, the intestinal barrier function was damaged, and the intervention of litchi enzyme could increase the TEER value of epithelial cell monolayer, which showed the effect of restoring the damaged intestinal barrier.

[0092] As can be seen from FIG. 1, when the concentration of litchi enzyme was greater than 0.1 μg / mL, it had the effect of significantly improving the transepithelial electrical resistance of intestinal cells (p<0.05), demonstrating that litchi enzyme can restore the intestinal barrier function.

[0093] Macrophage RAW264.7 immune activity test experiment Measurement of nitric oxide (NO) secretion in macrophages RAW264.7: Nitric oxide (NO) is the main effector molecule produced by macrophages, and after macrophages are activated, the NO produced exerts the main cytotoxic effect of non-specific immunity. If litchi enzyme can affect the secretion of NO, it would indicate that litchi enzyme can regulate immunity. This experiment measured the effect of litchi enzyme on the amount of NO secreted by macrophages.

[0094] 5 x 10 logarithmic growth phase cells were added to each well. 5 The cells were added to a 24-well plate according to the number of cells, and stimulated for 24 hours by adding 1, 2, 4, 8, 16, and 32 μg / mL of litchi enzyme at various concentrations. The supernatant was aspirated and added to a 96-well plate at 50 μL / well according to the number of cells, and a blank control was set up at the same time; 50 μL of room temperature Griess I was added to each well and mixed gently to homogenize; 50 μl of room temperature Griess II was added to each well and mixed gently to homogenize; and the absorbance at 540 nm was measured using a microplate reader.

[0095] As can be seen from FIG. 2, different concentrations of litchi enzyme (Example 5) can stimulate macrophages to secrete NO, indicating that the enzyme samples can activate macrophages and participate in immune regulation.

[0096] Inflammation model experiment using macrophages RAW264.7 Macrophages RAW264.7 were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and the conditions of the carbon dioxide incubator were 37°C and 5% CO2. When the cells grew to 80% of the culture dish, they were subcultured. During subculture, the medium aspirated with a pipette was repeatedly aspirated to detach the cells and form a single cell suspension, which was then centrifuged at 200g for 3 minutes, the supernatant was discarded, and fresh medium was added and sprayed evenly before inoculation into the culture dish. Macrophages RAW264.7 were cultured in each well at 5x10 5The cells were seeded in a six-well plate at a density of 100 μg / mL, with three wells for each treatment. The treatment was started when the cells had attached walls and grown to 70% of the hole area, and the cells were treated with special DMEM medium containing different concentrations (0.01, 0.1, 1, 2, 4, 8, 16 μg / mL) of litchi enzyme (Example 5) and LPS (0.1 μg / mL) for 24 h. After completion, the culture supernatant was collected, and 50 μL of each sample was added to a 96-well plate for detection, and 50 μL of Griess reagent I was added to each well, and then 50 μL of Griess reagent II was added, and the absorbance value was detected at 540 nm after the color reaction occurred.

[0097] Evaluation criteria: When macrophages are stimulated by LPS or other substances, inducible nitric oxide synthase is significantly increased, which produces a large amount of NO to carry out an immune response. NO has a certain degree of cytotoxicity and further damages the living body, thereby promoting exudation and edema at the inflammatory site. The degree of inhibition of NO production is a direct indicator for detecting anti-inflammatory activity. If the production of NO can be inhibited by adding litchi enzyme sample, it will be explained that litchi enzyme has good anti-inflammatory activity.

[0098] As can be seen from Figure 3, different concentrations of litchi enzyme can inhibit the NO release of inflammatory cells induced by LPS, indicating that litchi enzyme has anti-inflammatory activity. The concentration range with anti-inflammatory activity was 0.1-8μg / mL. The experimental results in Figures 2 and 3 reveal that the immune regulation of litchi enzyme is bidirectional and helps maintain intestinal steady state.

[0099] Comparison of the effects of litchi enzymes in Examples 1-7 Using the litchi enzymes of Examples 1-7, the above-mentioned human colon cancer cell Caco-2 intestinal barrier function test experiment, macrophage RAW264.7 immune activity test experiment, and macrophage RAW264.7 inflammation model experiment were carried out at a sample concentration of 4 μg / mL, and the effects of each litchi enzyme were compared. The effects of the litchi enzymes are shown in Table 1.

[0100] Table 1: Effects of different embodiments JPEG0007610644000001.jpg87134Note: Transepithelial electrical resistance recovery rate = Transepithelial electrical resistance value of enzyme intervention group / Initial transepithelial electrical resistance value * 100% Anti-inflammatory efficiency = (NO release amount in model group - NO release amount in sample group) / NO release amount in model group * 100%

[0101] As can be seen from Table 1, the effect of the litchi enzyme obtained in Example 7 is the lowest. In Examples 1-5, the first carbon source was added and aeration culture was used, while in Example 6, the first carbon source was not added and the aeration amount in the secondary fermentation process was 0.1mL / min / mL, and in Example 7, the first carbon source was added and static culture was used in the secondary fermentation process, which showed that the addition of the first carbon source and aeration culture in the secondary fermentation process produced favorable changes in the fermentation process, and further enhanced the beneficial effect of the litchi enzyme on intestinal health. The litchi enzyme obtained in Example 5 was used to obtain the optimal effect on the transepithelial electrical resistance recovery rate, the amount of nitric oxide (NO) released in the immune activity test, the amount of nitric oxide (NO) released in the inflammation model, and anti-inflammatory efficiency. By adjusting the strains and culture parameters in long-term studies, the inventors have found that other culture strains or conditions (e.g., Examples 1-4 and 6) than Example 5 cannot achieve such high anti-inflammatory efficiency; and the sum of the effects obtained by the experiments carried out using a single bacterium in Example 5 is much lower than the effect obtained by combining the bacteria in Example 5 of the present invention.

[0102] The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention are equivalent replacement methods, and all fall within the protection scope of the present invention.

Claims

1. A method for producing a litchi enzyme, comprising the steps of: (1) Primary fermentation, which includes adding a first carbon source to lychee fruit juice and lychee fruit residue, inoculating a first bacterial agent, which is a mixed bacterial agent of yeast and lactic acid bacteria, and sealing and standing at 20-45°C for 24-96 hours; (2) extruding and filtering the product after the primary fermentation to obtain a primary fermentation filtrate; (3) secondary fermentation, which includes adding a second carbon source to the primary fermentation filtrate, inoculating a second bacterial agent which is an acetic acid bacterium, and allowing to stand or aerate at 15-40° C. for 3-15 days to obtain a secondary fermentation broth; and (4) filtering and sterilizing the secondary fermentation liquor to obtain the lychee enzyme.

2. 2. The method of claim 1, comprising a pre-treatment step of washing and squeezing the lychee fruit.

3. the first carbon source is one or more of fructose, sucrose, brown sugar, maltose, and arabinose; 2. The method of claim 1, wherein the amount of the first carbon source added is 0-5 wt %.

4. The yeast is one or more of Hansenula anomala, sake yeast, Saccharomyces rouxii Boutroux, Saccharomyces cerevisiae, Saccharomyces willianus, Pichia pastoris, or Rhodotorula mucilaginosa; The lactic acid bacteria is one or more of Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus casei, Lactobacillus gasseri, or Lactobacillus paracasei; The weight ratio of yeast:lactic acid bacteria is 50:1-500:1; The method according to claim 1, wherein the amount of the first fungicide added is 0.1‰-10‰ of the total mass of the litchi.

5. the second carbon source being one or more of lactose, brown sugar, glucose, molasses, or honey; 2. The method of claim 1, wherein the amount of the second carbon source added is 1%-20% of the total mass of the primary fermentation filtrate.

6. the second bacterial agent is one or more of Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter rancens, or Acetobacter aceti; The amount of the second fungal agent added is 0.1‰-10‰ of the total mass of the primary fermentation filtrate; Static culture was performed by sealing with eight layers of gauze and then static culture; The method according to claim 1, wherein the aeration rate of the aeration culture is 0.1-20 mL / min / mL of fermentation liquid.

7. A litchi enzyme produced by the method according to any one of claims 1 to 6.

8. The litchi enzyme according to claim 7 is contained therein. A composition used for improving intestinal barrier function, bidirectionally regulating intestinal immunity, improving intestinal inflammation, maintaining intestinal steady state, or improving intestinal health.

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