CN112063565B - Inactivated lactobacillus agent and preparation method and application thereof - Google Patents
Inactivated lactobacillus agent and preparation method and application thereof Download PDFInfo
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- CN112063565B CN112063565B CN202011008511.XA CN202011008511A CN112063565B CN 112063565 B CN112063565 B CN 112063565B CN 202011008511 A CN202011008511 A CN 202011008511A CN 112063565 B CN112063565 B CN 112063565B
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- lactobacillus paracasei
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
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Abstract
The invention provides an inactivated lactobacillus agent and a preparation method and application thereof, belonging to the technical field of microbial preparations. According to the invention, two strains of lactic acid bacteria, namely lactobacillus paracasei HYZQF3DS-2 and lactobacillus plantarum HYZQF1675-63, are obtained through screening and further mutagenesis treatment, are very suitable for heat inactivation treatment, and fermentation process research is further carried out on the lactobacillus paracasei HYZQF3 and lactobacillus plantarum HYZQF1675-63, so that the fermentation performance of the strains is remarkably improved, and meanwhile, tests prove that the microbial inoculum obtained after fermentation culture of the lactobacillus paracasei and the lactobacillus paracasei has good constipation and diarrhea relieving effects; meanwhile, the composition has the effects of improving sleep disorder, anxiety and the like, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical field of microbial preparations, and particularly relates to an inactivated lactobacillus preparation and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
The human body has huge and various microorganisms which jointly form a human body microecological system, normal flora and the human body are in a symbiotic state through a long biological evolution process, and a close relation is established between the normal flora and the human body, so that the human body microecological system plays an important role in promoting the improvement of human body physiological functions, particularly the improvement of immune function, sleep regulation, emotion and intestinal tract peristalsis.
Domestic and foreign researches show that the lactobacillus, the inactivated dead bacteria and cell metabolites thereof play a probiotic role in human intestinal tracts.
The live lactic acid bacteria, which are intended to smoothly enter the intestinal tract to exert a probiotic effect, must first be subjected to tests of saliva, gastric acid, bile and various digestive enzymes, because these substances have a function of killing the active lactic acid bacteria. The living bacteria are sensitive to temperature, high in temperature and high in humidity, can be inactivated easily, are difficult to colonize and propagate in intestinal tracts after being taken orally, and cannot play a probiotic role.
The inactivated lactobacillus is inactivated under specific technological conditions, the inactivated lactobacillus cells are not limited by saliva, gastric acid, bile, various digestive enzymes, high temperature, high humidity, antibiotics and the like, and the inactivated lactobacillus cells can competitively inhibit various pathogenic bacteria only by having high adhesion to intestinal cells, so that the invasion of pathogenic bacteria microorganisms and the invasion of enterotoxins are prevented.
The inactivated lactobacillus has many advantages and disadvantages in use, the currently adopted inactivation methods are all heat inactivation, but the method can cause a large amount of lactobacillus cells to be crushed and cell adhesin to fall off, because the inactivated lactobacillus cells can only be adhered to the intestinal tract to play a role, but the heat-inactivated lactobacillus cells are only adhered to the intestinal tract by 10 percent, so that the effect of the product is influenced, and the production cost is also improved.
The metabolite is produced by lactobacillus in fermentation process, and is original lactobacillus food in intestine. Inositol and vitamin B can promote the rapid proliferation of human intestinal lactic acid bacteria, improve the immunity of human body, improve the intestinal peristalsis, promote the sleep, improve the mood of human and the like.
The metabolites produced by lactic acid bacteria during fermentation are different in composition and yield because the strains, fermentation methods and culture media are different.
Disclosure of Invention
In order to overcome the technical problems, the invention provides an inactivated lactobacillus preparation and a preparation method and application thereof, two strains of lactobacillus are obtained by screening and further carrying out mutagenesis treatment, the inactivated lactobacillus preparation is very suitable for carrying out heat inactivation treatment, and fermentation process research is further carried out aiming at the two strains, so that the fermentation performance of the strains is obviously improved, and a solid foundation is laid for practical industrial production and application.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a lactic acid bacteria preparation, wherein the active ingredients of the lactic acid bacteria preparation at least comprise any one or more of the following components:
1) lactobacillus paracasei (Lactobacillus paracasei) HYZQF3 DS-2;
2) lactobacillus plantarum (Lactobacillus plantarum) HYZQF 1675-63;
3) a metabolite of Lactobacillus paracasei (Lactobacillus paracasei) HYZQF3 DS-2;
4) lactobacillus plantarum (Lactobacillus plantarum) HYZQF 1675-63.
Preferably, the lactobacillus agent comprises the components 1) to 4) and the mass ratio of 1), 2), 3) and 4) is 1-3: 1-3: 0.5-2: 0.5 to 2; preferably, the ratio of 2: 2: 1: 1. more preferably, the 1) and the 2) are inactivated bacteria.
The metabolite of the invention comprises a thallus intracellular metabolite and/or an extracellular metabolite.
The lactobacillus paracasei HYZQF3DS-2 is obtained by separating red wine, performing repeated mutagenesis treatment and screening of UV and chemical mutagenesis, and is preserved in China center for type culture Collection (CCTCC, address: Wuhan city Lojia mountain, Wuhan university) 3 months and 22 days in 2019, wherein the preservation number is CCTCC NO: m2019194.
Lactobacillus plantarum HYZQF1675-63 is separated from jam, is obtained by repeated mutagenesis treatment and screening of UV and chemical mutagenesis, is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan city Wuchang Lojia mountain, Wuhan university) in 2019, 3 months and 22 days, and has the preservation number of CCTCC NO: m2019192.
In a second aspect of the present invention, there is provided a process for producing the above-mentioned lactic acid bacterium agent, which comprises a fermentation process of the above-mentioned lactobacillus paracasei HYZQF3DS-2 and lactobacillus plantarum HYZQF 1675-63; taking a fermentation method of lactobacillus paracasei HYZQF3DS-2 as an example:
specifically, the fermentation method comprises the following steps: carrying out expanded strain culture, primary seed culture, seed tank culture and fermentation culture on the lactobacillus paracasei HYZQF3DS-2 to obtain lactobacillus plantarum fermentation liquor;
and centrifuging the lactobacillus paracasei fermented liquid to respectively collect lactobacillus paracasei bacterial sludge and centrifuged fermentation liquid, and freeze-drying and crushing the lactobacillus paracasei bacterial sludge and the centrifuged fermentation liquid to obtain lactobacillus paracasei bacterial powder and lactobacillus paracasei metabolite powder.
By optimizing fermentation process parameters and conditions, the fermentation period of strains obtained by screening and optimizing in the invention is obviously shortened, the operation is easier, metabolites are comprehensive and rich, the inactivated extracellular polysaccharide of lactic acid bacteria is improved to 3.1% (dry weight) from 0.6% of the conventional method, the total dry weight of metabolites is improved to 4.9% from 1.8% of the conventional method, inositol is improved to 1.9% (dry weight) from 0.7% of the conventional method, the intestinal adhesion rate of inactivated bacteria is improved to 55% from 10% of the conventional method, the fermentation time is reduced to 15 hours from 24 hours of the conventional method, the equipment utilization rate is improved, and the production cost is reduced.
The third aspect of the invention provides the application of the microbial inoculum in the preparation of health-care food or special medical food.
In a fourth aspect of the invention, a health food is provided, which comprises the above fungicide, and can also comprise auxiliary ingredients allowed to be added in the field of conventional foods.
The health food includes but is not limited to solid food, liquid food; such solid food products include, but are not limited to, baked goods (e.g., cookies, breads, cakes, etc.), candies, solid beverages, and the like; the liquid food includes, but is not limited to, liquid beverages (such as lactic acid bacteria beverages) and the like.
In a fifth aspect of the invention, a special medical food is provided, which comprises the above fungicide, and can also comprise other nutrient components or auxiliary material components allowed to be added in the field of special medical foods.
In a sixth aspect of the invention, the application of the microbial inoculum, the health food and the special medical food in any one or more of the following items is provided:
(1) relieving constipation;
(2) relieving diarrhea;
(3) improving sleep disorders;
(4) improve anxiety.
The beneficial technical effects of one or more technical schemes are as follows:
in the technical scheme, two strains of lactic acid bacteria, namely lactobacillus paracasei HYZQF3DS-2 and lactobacillus plantarum HYZQF1675-63, are obtained through screening optimization, and the fermentation performance of the two strains is remarkably improved through further optimizing a fermentation culture method; meanwhile, tests prove that the microbial inoculum obtained by fermenting and culturing the two has good effects of relieving constipation and diarrhea; meanwhile, the composition has the effects of improving sleep disorder, anxiety and the like, thereby having good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph showing the results of adhesion test in parallel experiment 1 in the example of the present invention;
FIG. 2 is a graph showing the results of adhesion test parallel experiment 2 in the example of the present invention;
FIG. 3 is a graph showing the results of adhesion test in parallel experiment 3 according to the example of the present invention;
FIG. 4 is a graph showing the comparison of the ratio of mixed lactic acid bacteria cells adhering to the intestinal tract after inactivation according to the embodiment of the present invention;
FIG. 5 is a graph of a glucose standard curve according to an embodiment of the present invention;
FIG. 6 is a glucose detection chart of a sample to be detected in the embodiment of the present invention;
FIG. 7 is a graph of a standard inositol curve in an example of the present invention;
FIG. 8 is a diagram showing the detection of inositol in a sample to be tested in the example of the present invention;
FIG. 9 is a graph of the genetic stability of a strain of Lactobacillus paracasei after mutagenesis in an example of the invention;
FIG. 10 is a diagram showing the fermentation cycle of a Lactobacillus paracasei strain after mutagenesis in an example of the present invention;
FIG. 11 is a graph showing the genetic stability of Lactobacillus plantarum after mutagenesis in an example of the present invention;
FIG. 12 is a diagram showing the fermentation cycle of the Lactobacillus plantarum strain after mutagenesis in the example of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
In one embodiment of the present invention, a lactobacillus preparation is provided, wherein the lactobacillus preparation comprises at least one or more of the following active ingredients:
1) lactobacillus paracasei (Lactobacillus paracasei) HYZQF3 DS-2;
2) lactobacillus plantarum (Lactobacillus plantarum) HYZQF 1675-63;
3) a metabolite of Lactobacillus paracasei (Lactobacillus paracasei) HYZQF3 DS-2;
4) lactobacillus plantarum (Lactobacillus plantarum) HYZQF 1675-63.
In still another embodiment of the present invention, the lactobacillus preparation comprises the above 1) to 4), and the mass ratio of 1), 2), 3), 4) is 1 to 3: 1-3: 0.5-2: 0.5 to 2; preferably, the ratio of 2: 2: 1: 1.
in another embodiment of the present invention, the metabolites of the present invention comprise intracellular metabolites and/or extracellular metabolites of bacteria.
In another embodiment of the present invention, the lactobacillus paracasei HYZQF3DS-2 is separated from red wine, and obtained by repeated mutagenesis treatment and screening of UV and chemical mutagenesis, which has been preserved in the china typical culture collection (CCTCC, address: wuhan city, marchan university) 3 month and 22 days in 2019, and the preservation number is CCTCC NO: m2019194.
In another embodiment of the present invention, lactobacillus plantarum HYZQF1675-63 is obtained by separating from fruit jam and screening through repeated mutagenesis treatment of UV and chemical mutagenesis, and has been preserved in China center for type culture Collection (CCTCC, address: Wuhan city Wuchang Loa Jia mountain, Wuhan university) in 2019, 3.22.20, with the preservation number of CCTCC NO: m2019192.
In another embodiment of the present invention, there is provided a method for preparing the above-mentioned lactobacillus preparation, said method comprising the fermentation method of lactobacillus paracasei HYZQF3DS-2 and lactobacillus plantarum HYZQF1675-63 as described above; taking a fermentation method of lactobacillus paracasei HYZQF3DS-2 as an example:
in yet another embodiment of the present invention, the fermentation process comprises: carrying out expanded strain culture, primary seed culture, seed tank culture and fermentation culture on the lactobacillus paracasei HYZQF3DS-2 to obtain lactobacillus plantarum zymocyte liquid;
in another embodiment of the present invention, the lactobacillus paracasei fermented bacterial liquid is centrifuged to collect lactobacillus paracasei bacterial sludge and centrifuged fermented liquid, and the lactobacillus paracasei bacterial sludge and the centrifuged fermented liquid are freeze-dried and pulverized to obtain lactobacillus paracasei bacterial powder and lactobacillus paracasei metabolite powder.
In another embodiment of the present invention, the medium formula for the expanded strain culture is: 32g of oligoisomaltose, 7g of beef extract powder, 1g of sodium acetate, 800.5 ml of tween-tween, 1g of monopotassium phosphate and 1000ml of water, and the pH is adjusted to 7.
In another embodiment of the present invention, the culture conditions for expanding the culture of the bacterial species comprise: culturing in the above culture medium at 35-40 deg.C (preferably 37 deg.C) for 10-14 hr (preferably 12 hr).
In another embodiment of the present invention, the formula of the culture medium for the first-stage seed culture is: 10 g of isomaltooligosaccharide, 5 g of peptone, 4 g of beef extract powder, 1g of corn steep liquor, 1g of sodium acetate, 0.5 g of tyrosine, 801 ml of tween-801 ml and 1000ml of tap water, and the pH value is natural.
In another embodiment of the present invention, the primary seed culture is performed under conditions comprising: culturing in the above culture medium at 35-40 deg.C (preferably 37 deg.C) for 10-14 hr (preferably 12 hr); the inoculation amount is controlled to be 0.1-1% (preferably 0.5%);
in yet another embodiment of the present invention, the media formulation selected for the seeding tank culture is as follows (w/w): 5% of isomaltose hypgather, 1% of peptone, 0.2% of dipotassium phosphate, 1% of glycine, 0.2% of sodium acetate, 800.1% of tween-800, 0.1% of polyether defoamer and 92.4% of tap water, and the pH value is natural; the inoculation amount is controlled to be 0.5-2% (preferably 1%);
in another embodiment of the present invention, the formula of the culture medium selected for fermentation culture is as follows (w/w): 5% of isomaltose hypgather, 1% of peptone, 4% of fructo-oligosaccharide, 3% of water-soluble corn starch, 15% of yam starch, 1% of sodium acetate, tween-800.2%, 0.2% of glycine, 0.3% of methionine, 1% of disodium hydrogen phosphate, 0.1% of polyether defoamer and 69.2% of tap water; the inoculation amount is controlled to be 1-5% (preferably 2%);
in another embodiment of the present invention, the culture conditions of the fermentation culture include adjusting the culture temperature based on different culture time periods, specifically: the culture temperature is 33 ℃ in 0-5 hours at the initial stage; the fermentation temperature is 30 ℃ within 6-10 hours; the fermentation temperature is 37 ℃ after 11 to 13 hours; the fermentation temperature is 44 ℃ for 14-15 hours.
In another embodiment of the present invention, the stirring speed is maintained at 50-70 rpm, preferably 60 rpm, and the pH is maintained at weak acidity, preferably 6.0-7.0, and more preferably 6.5 during the fermentation process.
In another embodiment of the invention, the fermentation method of lactobacillus plantarum HYZQF1675-63 is the same as the fermentation method of lactobacillus paracasei HYZQF3 DS-2.
By optimizing fermentation process parameters and conditions, the fermentation period of strains obtained by screening and optimizing in the invention is obviously shortened, the operation is easier, metabolites are comprehensive and rich, the inactivated extracellular polysaccharide of lactic acid bacteria is improved to 3.1% (dry weight) from 0.6% of the conventional method, the total dry weight of metabolites is improved to 4.9% from 1.8% of the conventional method, inositol is improved to 1.9% (dry weight) from 0.7% of the conventional method, the intestinal adhesion rate of inactivated bacteria is improved to 55% from 10% of the conventional method, the fermentation time is reduced to 15 hours from 24 hours of the conventional method, the equipment utilization rate is improved, and the production cost is reduced.
In another embodiment of the invention, the application of the microbial inoculum in preparing health-care food or special medical food is provided.
In another embodiment of the invention, the health food comprises the microbial inoculum and can also comprise auxiliary ingredients (such as diluents, coloring agents, sweeteners, antioxidants and the like) which are allowed to be added in the conventional food field.
The health food includes but is not limited to solid food, liquid food; such solid food products include, but are not limited to, baked goods (e.g., cookies, breads, cakes, etc.), candies, solid beverages, and the like; the liquid food includes, but is not limited to, liquid beverages (such as lactic acid bacteria beverages) and the like.
In another embodiment of the invention, the special medical food comprises the microbial inoculum and can also comprise other nutrient components or auxiliary material components which are allowed to be added in the field of special medical foods.
In another embodiment of the present invention, the application of the above microbial inoculum, health food and special medical food in any one or more of the following applications is provided:
(1) relieving constipation;
(2) relieving diarrhea;
(3) improving sleep disorders;
(4) improve anxiety.
The technical solution of the present invention will be described below with specific examples. The starting materials used in the following examples are commercially available and the equipment used is conventional.
Examples
The culture method of lactobacillus paracasei HYZQF3DS-2 and lactobacillus plantarum HYZQF1675-63 is as follows:
one, expanding strain culture
The strain is stored in a refrigerator at a low temperature of-80 ℃ and is subjected to amplification culture, and a culture medium is prepared: 32g of isomaltooligosaccharide, 7g of beef extract powder, 1g of sodium acetate, 800.5 ml of tween-phosphate, 1g of monopotassium phosphate and 1000ml of tap water, and the pH is adjusted to 7 (adjusted by alkaline solution).
The operation is as follows: adding isomaltooligosaccharide and other raw materials into 1000ml of water in the formula before and after the raw materials are not separated, stirring for 10 minutes to dissolve the raw materials, then subpackaging the raw materials into test tubes, 5ml of each test tube, sealing rubber plug kraft paper, sterilizing for 20 minutes at 0.1-0.12 MPa in a medical sterilizer, and preparing the culture medium.
Inoculation: opening a low-temperature refrigerator at minus 80 ℃ under aseptic conditions (a clean bench), preserving two bacteria in a freezing tube, melting the freezing tube at room temperature, taking 1 sterilized test tube culture medium, adding 0.5ml of each of the two dissolved bacteria into the test tube, sealing the tube opening with rubber plug kraft paper, shaking up by hand gently, and placing in a constant-temperature incubator at 37 ℃ for standing culture for 12 hours, wherein the culture medium is called as an expanded culture medium.
Second and first order seed culture
The first-level seed culture formula comprises: 10 g of isomaltooligosaccharide, 5 g of peptone, 4 g of beef extract powder, 1g of corn steep liquor, 1g of sodium acetate, 0.5 g of tyrosine, 801 ml of tween-801 ml and 1000ml of tap water, and the pH value is natural.
The operation is as follows: the above-mentioned raw materials were added to 1000ml of tap water before and after the addition, and the mixture was stirred for 10 minutes to dissolve the whole of the raw materials, and this was referred to as a primary seed culture solution.
Adding the stirred and dissolved first-stage seed culture solution into a triangular flask according to the using amount, then wrapping a bottle mouth with 6 layers of gauze and a layer of kraft paper, putting the bottle mouth into a sterilizer for sterilization, firstly opening an exhaust valve on the sterilizer before sterilization and closing the exhaust valve when a small amount of steam is exhausted, opening the exhaust valve on the sterilizer for exhausting 6-8 minutes when the pressure on the sterilizer reaches 0.05 Mpa, then closing the exhaust valve, continuing heating, starting timing when the steam pressure in the sterilizer reaches 0.1-0.12 MPa, sterilizing for 20 minutes, leaving a heat source after the sterilization is finished, naturally cooling the heat source, opening the sterilizer when the pressure gauge on the sterilizer returns to zero, taking out the sterilized culture medium, putting the sterilized culture medium into an ultra-clean workbench for naturally cooling, and transferring the cultured expanded strain solution into the sterilized culture solution in the triangular flask when the temperature is reduced to 37 ℃, the inoculum size is 0.5%, namely 100ml of first-stage seed culture solution, 0.5ml of expanded bacterial solution is inoculated, after the inoculation is finished, the bottle mouth is sealed by using original gauze and kraft paper, the inoculated strain and the culture solution are uniformly mixed by slightly shaking by hand, and then the mixture is placed in a thermostat and cultured for 12 hours at 37 ℃, so that the first-stage strain solution is obtained.
Sterilization of fermentation equipment, pipelines and sterile filtration systems:
the operation is as follows: firstly, opening valves of each inlet and outlet pipeline and a sterile air pipeline, introducing steam of 0.12-0.14 MPa, communicating the steam with the pipeline valves and discharging a small amount of steam, ventilating for 40 minutes, and then closing valves of each inlet and outlet pipeline for later use.
Third, seeding tank culture
And (3) empty tank sterilization of the seed tank and the fermentation tank:
closing each valve, opening a drain valve at the bottom of the tank and a drain valve in an interlayer of the tank, opening a direct steam valve, introducing steam of 0.12-0.14 MPa into the pipe, starting timing when the temperature in the tank reaches 121 ℃, wherein the sterilization time is 40 minutes, then closing the drain valve at the bottom of the tank and the interlayer drain valve, and naturally reducing the temperature in the tank to 37 ℃ for later use.
The seeding tank culture formula comprises: 5 percent of isomaltose hypgather, 1 percent of peptone, 0.2 percent of dipotassium phosphate, 1 percent of glycine, 0.2 percent of sodium acetate, 800.1 percent of tween-1, 0.1 percent of polyether defoamer, 92.4 percent of tap water and natural pH value.
The method comprises the following specific operations: firstly, putting tap water used in a formula into a seeding tank sterilized in an empty tank, starting a stirrer, respectively adding raw materials (the adding sequence is not divided), then continuously stirring and introducing steam for heating, firstly heating to 95 ℃ by using tank interlayer steam, then directly heating by using the steam, keeping the temperature in the tank for 20 minutes when the temperature reaches 121 ℃ to achieve a sterilization effect, then closing the steam, starting to cool tap water passing through the tank interlayer, and keeping for later use when the temperature in the tank reaches 37 ℃, wherein the seed culture medium is called as a seed culture medium. Then inoculate the seed tank with above cultured first-order bacterial culture solution under aseptic condition go, the inoculum size is 2% of culture medium in the jar respectively, 100 kg seed tank culture medium respectively access first-order fungus liquid 2 kg can, then the inoculation cap on the lid jar begins the stirred culture, the culture condition: if the tank body is lowered, sterile air can be introduced to maintain the tank pressure, and the over-high tank pressure can be regulated by a gas release valve at the top of the tank, which is called seeding tank strain.
Fourthly, fermentation culture
The formula is as follows: 5% of isomaltose hypgather, 1% of peptone, 4% of fructo-oligosaccharide, 3% of water-soluble corn starch, 15% of yam starch, 1% of sodium acetate, tween-800.2%, 0.2% of glycine, 0.3% of methionine, 1% of disodium hydrogen phosphate, 0.1% of polyether defoamer and 69.2% of tap water.
The method comprises the following specific operations: firstly, putting tap water used in the formula into a fermentation tank which is sterilized in an empty tank, starting a stirrer, putting raw materials used in the formula into the fermentation tank (before and after the addition sequence is not divided), and then introducing steam for heating. Firstly heating to 95 ℃ by using interlayer steam, then heating by using direct steam, timing when the temperature in the tank reaches 121 ℃, keeping for 30 minutes to achieve a sterilization effect, then cooling and cooling by using interlayer tap water, starting inoculating strains when the temperature in the tank is reduced to 37 ℃, firstly increasing the tank pressure of a seeding tank to 0.1 MPa and the tank pressure of a fermentation tank to 0.05 MPa before inoculation, then opening an inoculation pipeline valve, conveying the seeds cultured in the seeding tank to the fermentation tank by using a pressure difference mode, and then closing an inoculation channel valve.
The inoculation amount is2 kilograms of strains cultured in a seeding tank by inoculating every 100 kilograms of culture medium in a fermentation tank, and the culture conditions are as follows: the initial culture temperature is 33 ℃ in 0-5 hours, the stirring speed is 60 rpm, the fermentation temperature is 30 ℃ in 6-10 hours, the stirring speed is constant, the fermentation temperature is 37 ℃ in 11-13 hours, the stirring speed is constant, the fermentation temperature is 44 ℃ in 14-15 hours, the stirring speed is constant, the tank pressure is kept at 0.05 MPa, an automatic acid and alkali regulation and control system is used for keeping the pH value at 6.5 in the fermentation process, if the tank pressure is reduced, sterile air can be introduced to keep the tank pressure until the fermentation is finished, after the fermentation is finished, sterile glycerol with the content of 98 percent of 1 percent of the volume of the fermentation liquid is added into the fermentation tank, the mixture is stirred for 30 minutes, then the temperature is reduced to 20 ℃ and is kept for 20 minutes, then the temperature is increased to 75 ℃ and the inactivation is kept for 40 minutes, and the fermentation liquid is obtained.
Fifthly, centrifuging the fermentation liquor, and collecting inactivated bacteria mud
Pumping the fermentation liquor into a storage tank for centrifugation, and operating: starting the centrifugal machine, adjusting the rotating speed to 10000 r/min, centrifuging at the feeding speed of 3 kg/min, collecting wet bacterial sludge after centrifugation, carrying out freeze drying and vacuum drying on the centrifugally collected wet bacterial sludge to obtain inactivated lactic acid bacteria powder, and using the centrifuged fermentation liquor (inactivated wet bacterial sludge metabolite) to prepare the inactivated bacteria metabolite freeze-dried powder.
Sixthly, freeze drying of bacterial sludge metabolite
Preparing a suspension liquid: and adding 3000 ml of sterile normal saline, 500 g of maltodextrin and 1000 g of anhydrous glucose into 1000 g of the inactivated wet bacterial sludge metabolite.
The operation is as follows: firstly, 2000ml of normal saline in the formula is taken, maltodextrin is added, the mixture is stirred and melted, 1000 g of inactivated wet bacterial sludge metabolite is added, the rest normal saline and anhydrous glucose in the formula are added, the mixture is fully and uniformly stirred to obtain suspension, the suspension is frozen and dried, and finally, the water content is controlled to be 4%, so that the inactivated bacterial metabolite freeze-dried powder is obtained.
Taking powder of lactobacillus paracasei HYZQF3DS-2 and metabolite powder thereof, and powder of lactobacillus plantarum HYZQF1675-63 and metabolite powder thereof according to the ratio of 2: 1: 2: 1 to prepare the microbial inoculum.
Effect verification
The method comprises the following steps of performing fermentation culture on lactobacillus paracasei HYZQF3DS-2 and lactobacillus plantarum HYZQF1675-63 by adopting a conventional fermentation method, wherein the conventional fermentation method is the same as the fermentation method, and the difference is that a culture medium used in the conventional fermentation is as follows: 0.9 percent of peptone, 0.72 percent of beef extract, 0.36 percent of yeast powder, 1.81 percent of glucose, 0.18 percent of sodium acetate, 0.18 percent of triammonium citrate, 800.45 percent of Tween, 0.01 percent of magnesium sulfate, 0.003 percent of manganese sulfate, 0.09 percent of dipotassium hydrogen phosphate, 5 percent of molasses and the balance of distilled water.
The content of extracellular polysaccharide, metabolite and inositol is detected by gas chromatography.
1. Intestinal adhesion rate of bacteria:
the method comprises the following steps:
bacterial culture
Mixing plant milkRespectively inoculating Bacillus (viable), Lactobacillus paracasei (inactivated) and Lactobacillus plantarum (inactivated) in MRS culture medium, culturing at 37 deg.C in anaerobic box for 48 hr, collecting thallus, and adjusting bacterial suspension to 1 × 10 with MRS culture medium8Each/ml, divided into live bacteria group and inactivated bacteria group.
Adhesion test of probiotics in two biological states and intestinal mucosal epithelial cells
Inoculating human intestinal mucosa epithelial cell Lovo strain in 1640 culture solution containing 10% calf serum and no antibiotics, placing 6-well plate of clean cover glass, anaerobically culturing at 37 deg.C until cell density reaches 80%, washing with D2Hanks solution for 3 times, adding live bacteria and inactivated bacteria respectively, and making final concentration be 1 × 108Adding no antibiotic 1640 culture solution of lactobacillus plantarum (viable bacteria), lactobacillus paracasei (killed bacteria) and lactobacillus plantarum (killed bacteria) per ml, carrying out symbiotic culture in an anaerobic environment at 37 ℃, stopping culture for 1h and 3h respectively, taking out a slide, washing for 3 times by using D2Hanks solution, and removing non-adhered probiotics. Methanol fixation for 15min, gram staining, random selection of 60 cells under oil microscope, calculation of visible cell surface probiotics, three replicates of adhesion test, and statistical calculation of the average adhesion index, which is the number of adhered cells/cell number × 100%.
Mixing the inactivated bacteria lactobacillus plantarum HYZQF1675-63 and lactobacillus paracasei HYZQF3DS-2 obtained by the fermentation method in the embodiment of the application according to the ratio of 1:1 for performing an adhesion index test; the control group was an adhesion index test in which the inactivated bacteria lactobacillus plantarum HYZQF1675-63 obtained by the conventional method and lactobacillus paracasei HYZQF3DS-2 were mixed at a ratio of 1:1, and the test demonstrated that the intestinal adhesion rate of the inactivated mixed lactobacillus in this example was increased by 55% from 10% in the conventional method.
Ultrastructural observation of interaction of probiotics in two biological states and intestinal mucosa epithelial cells
Live bacteria and inactivated probiotics in two biological states act on intestinal mucosal epithelial cells for 1h, the cover glass is taken out, the intestinal mucosal epithelial cells are fixed for more than 2h at 4 ℃ by using 215% glutaraldehyde, and the adhesion of the probiotics to the intestinal mucosal epithelial cells is observed by using a Hitachi S2520 scanning microscope on a critical dry specimen.
Method for detecting inositol and extracellular polysaccharide
1. Determination of extracellular polysaccharide by anthrone-sulfuric acid colorimetric method
(1) Experiment principle that saccharide can be dehydrated into furfural or hydroxymethyl furfural by concentrated sulfuric acid at higher temperature and then is reacted with anthrone (C)14H10O) dehydration condensation to form furfural derivative in blue-green color. The material has a maximum absorption at 620nm, and the shade of the color is proportional to the content of soluble sugar in the range of 150 mug/mL. The method has high sensitivity, and the sugar content can be measured at about 30 μ g.
(2) Reagent equipment
Anthrone reagent: accurately weighing 0.1g anthrone, adding 80% concentrated H2SO4Dissolve 100mL and shake well.
Preparing and using the composition on the same day;
glucose standard solution: placing anhydrous glucose in phosphorus pentoxide drier, accurately weighing 100mg after 12hr, and diluting with distilled water to 100 ml;
other apparatuses: analytical balance, spectrophotometer, volumetric flask (100ml, 50ml, 10ml), beaker, stoppered test tube, pipette tip, vortex shaker, waste vat, and the like.
(3) Procedure for the preparation of the
Preparation of glucose Standard Curve
Taking 7 supports to plug the test tube, precisely preparing a series of glucose solutions with different concentrations according to the data in the following table, and repeating the concentration for 2-3 times:
| |
0 | 1 | 2 | 3 | 4 | 5 | 6 |
| Standard glucose solution/ |
0 | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 | 1.2 |
| Distilled water/ml | 2.0 | 1.8 | 1.6 | 1.4 | 1.2 | 1.0 | 0.8 |
Adding 6mL of anthrone reagent into each test tube, shaking, mixing, and heating in boiling water bath for 15 min. Taking out, and rapidly soaking in ice water bath for cooling for 15 min. The absorbance of each of the remaining tubes was rapidly measured at a wavelength of 625nm using the 1 st tube as a blank.
Note: a standard curve was plotted with the standard glucose content (. mu.g) as the abscissa and the absorbance value as the ordinate, and the result is shown in FIG. 5.
Determination of samples
Adjusting the sugar concentration of the sample solution to a measuring range, accurately sucking 2mL of the sample solution, placing the sample solution in dry and clean test tubes, immediately adding 6mL of anthrone reagent into each test tube, uniformly mixing the test tubes by oscillation, and placing the test tubes in a boiling water bath for heating for 15min after the tubes are added. Taking out, rapidly soaking in ice water bath, cooling for 15min, and repeating for 2-3 times at each concentration. The absorbance of each tube was rapidly measured at a wavelength of 625 nm. And according to the standard curve of the glucose content, calculating the concentration of the sugar in each sample solution according to the light absorption value of the sample solution, and calculating the sugar content of the sample solution.
| |
0 | 1 | 2 | 3 | 4 | 5 | 6 |
| Standard glucose solution/ |
0 | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 | 1.2 |
| Sample solution sugar/ml | 2.0 | 1.8 | 1.6 | 1.4 | 1.2 | 1.0 | 0.8 |
The standard curve shown in FIG. 6 has good linearity, and the exopolysaccharide obtained according to the curve has high sugar content.
2. Method for assaying inositol
(1) Principle of experiment and pretreatment
The determination method is according to [ determination of inositol in food products of national standard for food safety ] (GB5009.270-2016).
(2) Drawing of standard curve
Water, inositol standard working solutions and inositol assay medium were added in triplicate to the culture tubes in the order listed below.
Preparation of Standard Curve
(3) Preparation of test solution
Water, test solution and inositol assay medium were added in triplicate to the culture tubes as follows.
Preparation of test solution
| |
1 | 2 | 3 | 4 |
| Water/ |
4 | 3 | 2 | 1 |
| Sample extract/ |
1 | 2 | 3 | 4 |
| Culture Medium/ |
5 | 5 | 5 | 5 |
(4) Inoculation of
Fixing the test tube in a shaking incubator, and carrying out shaking culture for 22-24 h at 30 +/-1 ℃ at a shaking speed of about 140 times/min-160 times/min.
(5) Measurement of
Note: visual inspection of each tube should be clear from the culture in the inoculated blank tube S1, and if turbidity occurs, the results are invalid.
1. Taking out the test tube from the shaking incubator, placing the test tube into a sterilization kettle, and keeping the temperature at 100 ℃ for 5min to stop the growth of microorganisms.
2. The inoculated blank tube S1 was blanked, the spectrophotometer transmittance was adjusted to 100% (or absorbance was 0), and the reading of the inoculated blank tube S2 was read. And then, using the inoculated blank test tube S2 as a blank, adjusting the light transmittance to be 100% (or adjusting the absorbance to be 0), and sequentially reading the light transmittance (or absorbance) of each other test tube.
3. After each test tube is fully mixed by a vortex oscillator (or a drop of antifoaming agent is added), the culture solution is immediately transferred into a cuvette for measurement, the wavelength is 540 nm-660 nm, the light transmittance is read after the reading is stable for 30s, and the stability time of each test tube is the same. The inositol standard, the concentration of the standard series as the abscissa and the light transmittance as the ordinate are used as a standard curve.
4. And according to the light transmittance of the liquid to be detected, obtaining the concentration of inositol in the liquid to be detected from the standard curve, and then calculating the content of the inositol in the sample according to the dilution factor and the sample weighing. And (4) discarding the sample tube with the light transmittance exceeding the range of 3-10 of the standard curve tube.
5. For each test tube of the test solution with the number, the light transmittance of each test tube is used for calculating the concentration of inositol in the test solution with the number per milliliter, and the average value of the concentration of the inositol in the test solution with the number is calculated, wherein the concentration measured in each test tube does not exceed +/-15% of the average value, and the test tubes with the number exceeding the average value are discarded. If the measurement result of 1 tube does not meet the requirement, discarding the measurement result of the tube and recalculating the average value; if the measurement results of 2 tubes do not meet the above requirements, the test is performed again.
Note: and drawing a standard curve, and reading the light transmittance and the absorbance.
The standard curve obtained in FIG. 8, which is linear, gives a high inositol content.
Third, evaluation of genetic stability and fermentation performance of lactobacillus paracasei and lactobacillus plantarum
1. The lactobacillus paracasei strain obtained after mutagenesis has good genetic stability:
as shown in FIG. 9, the extracellular polysaccharide and inositol of the Lactobacillus paracasei strain HYZQF3DS-2 obtained after mutagenesis reach peak values at 18 generations, and then are stable and unchanged, and the genetic stability is better.
2. The lactobacillus paracasei strain obtained after mutagenesis has short fermentation period:
as shown in FIG. 10, the extracellular polysaccharide and inositol of Lactobacillus paracasei HYZQF3DS-2 strain obtained after mutagenesis reach peak values in 15 hours, while the original starting strain needs 24 hours, which shows that the fermentation period of the strain after mutagenesis is shortened, thereby improving the utilization rate of equipment and reducing the production cost. The lactobacillus plantarum strain obtained after mutagenesis has good genetic stability as follows:
as shown in FIG. 11, the extracellular polysaccharide and inositol of Lactobacillus plantarum HYZQF1675-63 obtained after mutagenesis reach peak values at 18 generations, and then are stable and unchanged, and the genetic stability is better. The embodiment that the lactobacillus plantarum strain obtained after mutagenesis has short fermentation period is as follows:
as shown in FIG. 12, the extracellular polysaccharide and inositol of Lactobacillus plantarum HYZQF1675-63 obtained after mutagenesis reach peak values in 15 hours, while the original starting strain needs 24 hours, which indicates that the fermentation period of the mutagenized strain is shortened, thereby improving the utilization rate of equipment and reducing the production cost. Fourth, clinical effect test of inactivated lactobacillus agent
Table one:
description of the invention
Male: after the medicine is taken for 7 days, 35 people can relieve constipation and diarrhea; sleep and anxiety are improved obviously.
After the medicine is taken for 14 days, 58 people with constipation and diarrhea can be obviously relieved; the sleep is improved obviously, the feeling of mood is smooth, and the attention is concentrated.
After the medicine is taken for 20 days, 75 people with constipation and diarrhea are relieved; the sleep disorder and the anxiety patients are obviously improved; the product is not effective in 115 people taking the product in 20 days.
Female: after the medicine is taken for 7 days, 20 people can relieve constipation and diarrhea; sleep and anxiety are improved obviously.
After the medicine is taken for 14 days, 45 people with constipation and diarrhea can be obviously relieved; the sleep is improved obviously, the feeling of mood is smooth, and the attention is concentrated.
After the medicine is taken for 20 days, 60 people with constipation and diarrhea are relieved; the sleep disorder and the anxiety patients are obviously improved; 140 people take the product during the 20-day period, and the product has no effect.
Table two:
description of the invention
Male: after being taken for 7 days, 50 people can relieve constipation and diarrhea; sleep and anxiety are improved obviously.
After the medicine is taken for 14 days, 75 people with constipation and diarrhea can be obviously relieved; the sleep is improved obviously, the feeling of mood is smooth, and the attention is concentrated.
After the medicine is taken for 20 days, the constipation and diarrhea of 90 people are relieved; the sleep disorder and the anxiety patients are obviously improved; the product is not effective in 110 people after taking the product for 20 days.
Female: after the medicine is taken for 7 days, 35 people can relieve constipation and diarrhea; sleep and anxiety are improved obviously.
After the medicine is taken for 14 days, 50 people with constipation and diarrhea can be obviously relieved; the sleep is improved obviously, the feeling of mood is smooth, and the attention is concentrated.
After the medicine is taken for 20 days, 80 people can relieve constipation and diarrhea; the sleep disorder and the anxiety patients are obviously improved; the product is not effective after 120 people take the product in 20 days.
Table three:
description of the invention
Male: after being taken for 7 days, 90 people can relieve constipation and diarrhea; sleep and anxiety are improved obviously.
After the medicine is taken for 14 days, the constipation and diarrhea of 128 people are obviously relieved; the sleep is improved obviously, the feeling of mood is smooth, and the attention is concentrated.
After the medicine is taken for 20 days, 191 people with constipation and diarrhea are relieved; the sleep disorder and the anxiety patients are obviously improved; the product is not effective in 9 people after 20 days.
Female: after being taken for 7 days, 82 people can relieve constipation and diarrhea; sleep and anxiety are improved obviously.
After the medicine is taken for 14 days, the constipation and diarrhea of 131 people are obviously relieved; the sleep is improved obviously, the feeling of mood is smooth, and the attention is concentrated.
After the medicine is taken for 20 days, 187 people with constipation and diarrhea are relieved; the sleep disorder and the anxiety patients are obviously improved; 13 people have no effect after taking the product in 20 days.
And (4) conclusion:
the results show that the lactobacillus paracasei, the inactivated lactobacillus plantarum and the metabolite produce synergistic effect on relieving constipation, diarrhea, sleep and anxiety when being taken simultaneously, and the effect of the lactobacillus preparation prepared by the invention is good.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention is described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents of the embodiments described above can be made, or some equivalents thereof can be substituted. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (12)
1. The lactobacillus agent is characterized in that the active ingredients of the lactobacillus agent at least comprise one or more of the following components:
1) lactobacillus paracasei (Lactobacillus paracasei) HYZQF3 DS-2;
2) lactobacillus plantarum (Lactobacillus plantarum) HYZQF 1675-63;
the lactobacillus paracasei HYZQF3DS-2 is already preserved in the China center for type culture Collection in 2019, 4 and 9 months, and the preservation number is CCTCC NO: m2019194;
lactobacillus plantarum HYZQF1675-63 is preserved in China center for type culture Collection in 2019, 4 months and 9 days, and the preservation number is CCTCC NO: m2019192.
2. The method for producing a lactic acid bacterium agent according to claim 1, which comprises a fermentation method of Lactobacillus paracasei HYZQF3DS-2 and Lactobacillus plantarum HYZQF 1675-63; the fermentation method of the lactobacillus paracasei HYZQF3DS-2 comprises the following steps: carrying out expanded strain culture, primary seed culture, seed tank culture and fermentation culture on the lactobacillus paracasei HYZQF3DS-2 to obtain lactobacillus paracasei zymocyte liquid;
and centrifuging the lactobacillus paracasei fermented liquid to respectively collect lactobacillus paracasei bacterial sludge and centrifuged fermentation liquid, and freeze-drying and crushing the lactobacillus paracasei bacterial sludge and the centrifuged fermentation liquid to obtain lactobacillus paracasei bacterial powder and lactobacillus paracasei metabolite powder.
3. The method according to claim 2, wherein the medium used for the expanded strain culture comprises: 32g of isomaltooligosaccharide, 7g of beef extract powder, 1g of sodium acetate, 800.5 ml of tween-phosphate, 1g of monopotassium phosphate and 1000ml of water, and adjusting the pH to 7;
the culture conditions for expanding the strain culture comprise: culturing in culture medium selected for enlarged strain culture at 35-40 deg.C for 10-14 hr;
the formula of the culture medium for first-stage seed culture is as follows: 10 g of isomaltooligosaccharide, 5 g of peptone, 4 g of beef extract powder, 1g of corn steep liquor, 1g of sodium acetate, 0.5 g of tyrosine, 801 ml of tween-801 ml and 1000ml of tap water, and the pH value is natural;
the culture conditions for the primary seed culture include: culturing in the culture medium selected for the first-stage seed culture at 35-40 deg.C for 10-14 hr; the inoculation amount is controlled to be 0.1-1%;
the formula of the culture medium selected in the seeding tank culture is as follows, w/w: 5% of isomaltose hypgather, 1% of peptone, 0.2% of dipotassium phosphate, 1% of glycine, 0.2% of sodium acetate, 800.1% of tween-800, 0.1% of polyether defoamer and 92.4% of tap water, and the pH value is natural; the inoculation amount is controlled to be 0.5-2%;
the formula of the culture medium selected for fermentation culture is as follows, w/w: 5% of isomaltose hypgather, 1% of peptone, 4% of fructo-oligosaccharide, 3% of water-soluble corn starch, 15% of yam starch, 1% of sodium acetate, tween-800.2%, 0.2% of glycine, 0.3% of methionine, 1% of disodium hydrogen phosphate, 0.1% of polyether defoamer and 69.2% of tap water; the inoculation amount is controlled to be 1-5%;
the culture conditions of the fermentation culture include adjusting culture temperature based on different culture time periods, and specifically include: the culture temperature is 33 ℃ in 0-5 hours at the initial stage; the fermentation temperature is 30 ℃ within 6-10 hours; the fermentation temperature is 37 ℃ after 11 to 13 hours; the fermentation temperature is 44 ℃ for 14-15 hours;
and in the fermentation culture process, the stirring speed is maintained at 50-70 r/min, and the pH is maintained at 6.0-7.0.
4. The method according to claim 3, wherein the conditions for culturing the expanded strain include: culturing in the culture medium selected for the expanded strain culture at 37 ℃ for 12 hours;
the culture conditions for the primary seed culture include: culturing in the culture medium selected for the primary seed culture at 37 ℃ for 12 hours; the inoculation amount is controlled to be 0.5 percent;
the inoculation amount is controlled to be 1% when the seeding tank is used for culture;
the inoculation amount is controlled to be 2% during fermentation culture;
the stirring speed is maintained at 60 r/min and the pH is maintained at 6.5 during the fermentation culture process.
5. The process according to any one of claims 2 to 4, wherein the fermentation process of Lactobacillus plantarum HYZQF1675-63 is the same as the fermentation process of Lactobacillus paracasei HYZQF3 DS-2.
6. Use of the lactic acid bacterial agent of claim 1 for the preparation of health food or specialty medicine food.
7. A health food comprising the lactic acid bacterial agent according to claim 1.
8. The health food of claim 7, further comprising an auxiliary ingredient which is allowable in the field of conventional foods.
9. The health food of claim 7, wherein the health food comprises a solid food, a liquid food; the solid food comprises baked food, candy and solid beverage; the liquid food includes a liquid beverage.
10. A special medical food, which comprises the lactic acid bacterial agent according to claim 1.
11. The special medical food according to claim 10, further comprising other nutritional components or adjuvant ingredients that are allowed to be added in the field of special medical foods.
12. Use of a lactic acid bacterial preparation according to claim 1, a health food according to any one of claims 7 to 9 or a specialist food according to claim 10 or 11 in any one or more of:
(1) relieving constipation;
(2) relieving diarrhea;
(3) improving sleep disorders;
(4) improve anxiety.
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