CN111743158B - Probiotic tablet with function of enhancing immunity and preparation method thereof - Google Patents
Probiotic tablet with function of enhancing immunity and preparation method thereof Download PDFInfo
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- CN111743158B CN111743158B CN202010515851.5A CN202010515851A CN111743158B CN 111743158 B CN111743158 B CN 111743158B CN 202010515851 A CN202010515851 A CN 202010515851A CN 111743158 B CN111743158 B CN 111743158B
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- lactobacillus helveticus
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- tablet
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Abstract
The invention relates to the field of food, and discloses a probiotic tablet with an immune enhancing function and a preparation method thereof. The tablet comprises Lactobacillus helveticus freeze-dried powder, acerola cherry powder, direct compression type auxiliary materials and magnesium stearate, can improve in-vitro spleen lymphocyte proliferation and interleukin IL-12 secretion of spleen lymphocytes and promote interleukin IL-8 secretion of human colon cancer epithelial cells HT-29, and can be used for enhancing immunity. The selected Lactobacillus helveticus WHH2580 strain has excellent acid resistance, cholate resistance and adhesion performance; after the strain is inactivated, the organism immunity can be improved by improving the organ index, nonspecific immunity and specific immune response of the organism, so that the product stability and the quality guarantee period of the strain are longer.
Description
Technical Field
The invention relates to the field of food, in particular to a probiotic tablet with an immune enhancing function and a preparation method thereof.
Background
With the accelerated urbanization process and the prevalence of unhealthy life styles of residents, various diseases and sub-health people caused by low immunity in China are increased rapidly. The body can not normally play a protective role due to low immunity, and is very easy to be infected by bacteria, viruses, fungi and the like, and the most direct manifestation is that the body is easy to get ill. The frequent illness aggravates the consumption of the organism, and the manifestations of hypoimmunity such as weak constitution, malnutrition, listlessness, fatigue, weakness, appetite reduction, sleep disorder and the like appear in different degrees, so that the sick people become the frequent meals of the family by injection and medication. Recovery from each illness can take a long time and often attacks repeatedly. This causes poor development of the body and intelligence and is also likely to induce serious diseases. Therefore, enhancing immunity and preventing and treating low immunity are important means for preventing diseases.
At present, a lot of products with immunity enhancement are declared on the market, and clinically common immunity enhancement medicines comprise chemical synthesis medicines of levamisole, isoprinosine and the like, human or animal immune products of thymosin, transfer factor, interferon, interleukin and the like, bacillus calmette guerin from microorganisms, biological polysaccharide from other sources, traditional Chinese medicine active ingredients and the like. The action mechanisms of these drugs for regulating immune function are: (1) enhancing the activity of macrophage and natural killer cell, and improving nonspecific immunity; (2) promoting division, proliferation, maturation and differentiation of T lymphocytes, and enhancing cellular immune function; (3) improving humoral immunity function; (4) inducing the production of interferon and some cell factors, and activating immune cells to play a role. However, these drugs have some adverse side effects to different degrees, such as nausea, vomiting, abdominal pain and other gastrointestinal reactions caused by levamisole; BCG vaccine has more adverse reactions, and can cause skin redness, swelling, induration and ulcer at local part of injection, and occasional anaphylactic shock and even death. Interferon can cause flu-like symptoms, and some patients can also have symptoms such as bone marrow suppression, arrhythmia, confusion, hallucinations and the like.
The probiotics can play a physiological function in the intestinal tract of a human body to help healthy beneficial microorganisms, and after being taken by a host, the probiotics adhere to epithelial cells of the intestinal mucosa and colonize to form stable flora. A large number of researches prove that some lactobacillus species can generate beneficial effects on hosts, which not only is beneficial to maintaining the balance of intestinal microecology and absorbing nutrient substances, but also can adjust the immune function of the hosts, promote the barrier function of intestinal mucosa to be perfect and enhance the anti-infection capability.
The intestinal tract is the largest immune organ of a human body, more than 50% of lymphatic tissues of the human body are distributed in intestinal mucosa, and intestinal microorganisms play an important role in regulating the health of the human body. Starting with intestinal microorganisms, a new method for regulating immunity is tried to be found so as to overcome the defects of side effects of the existing therapeutic drugs and therapeutic methods. Probiotics may balance local microbiota by inhibiting the growth of pathogenic microorganisms, or stimulate the metabolism of intestinal mucosa and intestinal epithelial cells by secreting various enzymes and acids, thereby enhancing local and systemic immune responses. The probiotics can regulate the immunity of organisms in various ways and signal ways, the main ways comprise TLRs signal way, NF-KB, MAPKs, STAT ways and the like, and the intestinal immunity can be stimulated through the regulation of the probiotics, and meanwhile, the whole immune system of the organisms is influenced. In addition, the lactobacillus can regulate body inflammation by regulating immune system, cell factor, intestinal canal penetration, intestinal canal flora balance, bacterial colonization and the like. Therefore, the development of the probiotic product with the function of enhancing the immunity has great economic and social benefits.
Disclosure of Invention
In order to solve the technical problems, the invention provides a probiotic tablet with an immunity enhancing function and a preparation method thereof, the invention compounds lactobacillus helveticus freeze-dried powder, acerola cherry powder, a direct compression type auxiliary material and magnesium stearate, and by using the direct compression type auxiliary material and adopting a direct compression technology, the process flows of granulation and the like can be reduced, the energy consumption is reduced, the productivity is improved, and the tabletting performance can be obviously improved; the obtained tablet can enhance immunity and prevent hypoimmunity. The preparation method of the product has simple steps and low cost, and is beneficial to realizing large-scale industrial production.
The specific technical scheme of the invention is as follows: a probiotic tablet with immunity enhancing effect comprises Lactobacillus helveticus lyophilized powder, acerola cherry powder, direct compression type adjuvant and magnesium stearate.
The lactobacillus helveticus freeze-dried powder is prepared from lactobacillus helveticus and/or mutants thereof; the lactobacillus helveticus is named as WHH2580 and is obtained by screening in yoghourt collected in the Hongshan ditch of Xinjiang Nalat. Has been preserved in China general microbiological culture Collection center (CGMCC) in 2019, 10 and 23 months, the preservation number of the microorganism is CGMCC No.18730, and the microorganism is classified and named as Lactobacillus helveticus; the Lactobacillus helveticus has a 16s rRNA gene sequence shown in SEQ ID NO. 1. The mutant is obtained by carrying out mutagenesis, domestication, gene recombination or natural mutation on the lactobacillus helveticus.
In the formula of the invention, lactobacillus helveticus freeze-dried powder is used as a main functional substance, and acerola powder, a direct compression type auxiliary material and magnesium stearate are used as auxiliary materials. The lactobacillus helveticus WHH2580 for preparing the lactobacillus helveticus freeze-dried powder is a strain obtained by screening yoghourt collected from Hongshan ditch of Xinjiang nalat, and animal experiments prove that the lactobacillus helveticus WHH2580 has good tolerance and adhesiveness, can smoothly reach the intestinal tract, is adhered to intestinal tract epithelial cells to play a probiotic effect, can improve the in-vitro spleen lymphocyte proliferation and the secretion of interleukin IL-12 by spleen lymphocytes and can promote the secretion of interleukin IL-8 by human colon cancer epithelial cells HT-29; in an animal model with low immunity, the strain can also obviously improve the organ index of a mouse, promote the proliferation of spleen lymphocytes and improve the activity of NK cells, and has good effect of enhancing immunity; moreover, it also has the function of enhancing immunity after extinguishing fire. In addition, in the animal experiment process, the adverse states of death, lassitude, inappetence and the like of the experimental animals are not found, which indicates that the strain has higher safety.
Specifically, the lactobacillus helveticus WHH2580 has the following advantages:
(1) The acid-resistant and cholate-resistant composite material has the following characteristics: under the environment of pH =3.0, the survival of the strain is not obviously affected after 2h of incubation, and the number of viable bacteria is reduced by 0.9 order of magnitude after 3 h; the pepsin has no obvious influence on the viability of the bacillus within 2 hours, and the viable count is reduced by 0.7 orders of magnitude within 3 hours; 3.6 orders of magnitude reduction in 0.5% bile salt system over 3 h. The food has good acid resistance and bile salt resistance, so that the food can better pass through the stomach and duodenum and smoothly reach the intestinal tract, thereby better playing the probiotic role; meanwhile, the good acid resistance enables the composition to adapt to a wider pH range in the process of preparing freeze-dried powder and the composition, particularly in the fermentation process of preparing the freeze-dried powder, so that the composition based on the lactobacillus helveticus has higher viable bacteria content;
(2) The cell adhesive has good adhesion characteristics (living body and non-living body), shows very good adhesion capability in HT-29 cell model test, and has viable single cell adhesion bacteria number as high as 15.69 +/-5.94 which is 6-8 times that of a contrast commercial strain; the number of the single cell adhesion bacteria of the inactivated thallus is as high as 11.59 +/-4.02, which is far beyond the adhesion capability of commercial control live bacteria strains;
(3) The compound preparation has good inhibition effect on staphylococcus aureus, salmonella paratyphi B, escherichia coli, shigella flexneri and micrococcus luteus, so that pathogenic bacteria in intestinal tracts can be well inhibited in the intestinal tracts, intestinal flora is regulated, and a probiotic effect is exerted;
(4) In an in vitro splenic lymphocyte proliferation assay, splenic lymphocyte proliferation fold was higher than that of two commercial strains with and without ConA induction;
(5) Can improve the in vitro spleen lymphocyte proliferation and the secretion of interleukin IL-12 by the spleen lymphocytes, promote the secretion of interleukin IL-8 by the epithelial cells HT-29 of human colon cancer, and has better performance than that of a commercial control strain;
(6) The administration concentration of the viable bacteria strain is 2X 108CFU/d, and the administration concentration of the fire extinguishing strain is 2X 108CFU/d, so that the visceral organ index of the mouse can be obviously improved, the spleen lymphocyte proliferation is promoted, and the NK cell activity is improved.
In addition, the acerola cherry powder contains abundant natural vitamin C, the vitamin C has multiple functions in body metabolism, such as the functions of protecting blood vessels, muscles, teeth and bones, promoting wound healing, promoting the absorption of iron and calcium and the like, and meanwhile, modern scientific research shows that the vitamin C can activate the immune response function, so that the immune function is enhanced. Vitamin C is a necessary nutrient for human body, and the vitamin C can not be synthesized by the human body and can only be supplemented by external intake. In case of vitamin C deficiency, the fighting ability of the white blood cells is weakened, and the human body is susceptible to diseases.
Magnesium stearate is one of the most common auxiliary materials in the production process of tablets in the modern food industry, can change the binding force among material particles, and has the functions of retention and lubrication.
Preferably, the straight compression type auxiliary material comprises one or more of straight compression type maltitol, straight compression type sorbitol, straight compression type lactose, straight compression type starch sugar and straight compression type microcrystalline cellulose.
Preferably, the probiotic tablet comprises 1-10 parts of lactobacillus helveticus freeze-dried powder, 3-15 parts of acerola powder, 50-90 parts of direct compression type auxiliary materials and 0.2-2 parts of magnesium stearate by weight.
Preferably, the number of viable bacteria in the lactobacillus helveticus freeze-dried powder is 1 × 10 8 CFU/g-2×10 12 CFU/g。
The invention also provides a preparation method of the probiotic tablet, which comprises the following steps:
(1) Weighing the raw materials for later use;
(2) Uniformly mixing all the raw materials except the magnesium stearate to obtain a primary mixture;
(3) Uniformly mixing the primary mixture obtained in the step (2) with magnesium stearate to obtain a total mixed semi-finished product;
(4) Tabletting the total mixed semi-finished product obtained in the step (3) by using a tabletting machine to obtain tablets;
(5) And (5) packaging the tablets obtained in the step (4) by using a packaging bottle to obtain the probiotic tablets.
The invention adopts a powder direct compression technology, is a method for directly compressing main components and proper auxiliary materials into tablets after being uniformly mixed without granulation technologies such as wet granulation, dry granulation, one-step granulation and the like, and has the characteristics of simple process, high efficiency and low cost. The direct-compression type auxiliary material is the core of the powder direct-compression technology, and must have good fluidity, compressibility, lubricity, and wide adaptability and reproducibility. The auxiliary materials are directly related to the performance of the dosage form in the tabletting process such as tabletting pressure, tabletting efficiency and the like and the characteristics of the tablet such as hardness, friability and the like.
Preferably, in the step (2), the mixing rotation speed is controlled to 10 to 35rpm, and the mixing time is controlled to 10 to 20min.
Preferably, in step (3), the mixing speed is controlled to 10 to 25rpm, and the mixing time is controlled to 5 to 20min.
Preferably, in the step (4), the tablet pressing pressure is controlled to be between 5KN and 20KN during the tablet pressing.
Preferably, in the step (5), the packaging bottles used for packaging adopt high-density polyethylene packaging bottles with built-in drying agents.
Preferably, all steps (1) to (5) are carried out in a GMP workshop in a constant temperature and humidity environment at a temperature of 18 to 26 ℃ and a humidity of 25 to 40%.
Preferably, in the step (5), the obtained probiotic tablet has a water content of 1.5-7% and a water activity of 0.1-0.6aW.
Preferably, the preparation method of the lactobacillus helveticus freeze-dried powder comprises the following steps:
1) Preparing a culture medium;
2) Preparing a strain protective agent;
3) Inoculating lactobacillus helveticus and/or a mutant thereof in a fermentation substrate in an inoculation amount of 2% -10% for fermentation culture;
4) Taking a fermentation product after fermentation is finished, and centrifuging;
5) Mixing the centrifugal product with a strain protective agent;
6) Freeze-drying;
7) And crushing and sieving the freeze-dried product to obtain the lactobacillus helveticus freeze-dried powder.
The lactobacillus freeze-dried powder is prepared by adopting a bioengineering constant pH high-density fermentation technology, a freeze-drying technology and a crushing and sieving process, and the obtained freeze-dried powder has the characteristics of high viable count, low water content and low water activity, can ensure the stable activity of lactobacillus plantarum in the freeze-dried powder, and has the characteristics of reasonable particle size distribution, good mixing performance with other materials and the like.
Preferably, in step 1), the culture medium comprises the following components: 15-50g of desalted whey powder, 2-15g of casein hydrolysate, 3-15g of tryptone, 7-20g of soybean peptone, 3-6g of yeast powder, 5-15g of sodium citrate, 1-5g of dipotassium phosphate, 0.4-0.8g of magnesium sulfate, 0.2-0.5g of manganese sulfate monohydrate, 5-30mL of tomato juice and 1000mL of water; the pH value is adjusted to 6.5+0.2.
Preferably, in step 2), the strain protective agent comprises the following components: skim milk 20-150g/L, sucrose 30-100g/L, and glycerol 5-45g/L.
Preferably, in the step 3), the fermentation temperature is 34-45 ℃, the fermentation time is 6-18h, and the pH value is controlled to be 4.0-6.0 in the fermentation process.
Preferably, in the step 4), the centrifugal speed is 4000-10000rpm, and the centrifugal time is 3-15min.
Preferably, in the step 5), the mixing weight ratio of the centrifugal product to the strain protecting agent is 1: 1-3.
Preferably, in step 7), the pulverized product is sieved, and the sieve is selected from a standard sieve of 10 to 80 meshes.
Compared with the prior art, the invention has the beneficial effects that:
(1) The lactobacillus helveticus freeze-dried powder, the acerola cherry powder, the direct compression type auxiliary material and the magnesium stearate are compounded. Besides the function of enhancing the immunity of lactobacillus helveticus WHH2580, the vitamin C rich in the acerola cherry powder can play a good auxiliary role; the direct-compression auxiliary material can endow the composition with good compressibility; magnesium stearate can remarkably improve the flowability and compressibility of the material.
(2) The direct compression type auxiliary material adopted by the invention can endow the composition with good compressibility, so that a direct compression technology can be adopted, the process flows of granulation and the like can be reduced, the energy consumption is reduced, and the productivity is improved. The preparation method has simple steps and low cost, and is beneficial to realizing large-scale industrial production.
(3) The lactobacillus helveticus freeze-dried powder is obtained by adopting a bioengineering constant pH high-density fermentation technology, a freeze-drying technology and a crushing and sieving process, and has the characteristics of high viable count, low water content and low water activity, reasonable particle size distribution, good mixing performance with other materials and the like.
(4) In the process of preparing the lactobacillus helveticus freeze-dried powder, the constant pH high-density fermentation technology is adopted to regulate and control the fermentation pH, and when the pH is controlled to be 4.0-6.0 in the fermentation process, the viable count of the fermentation liquid reaches 3.0 multiplied by 10 9 CFU/mL, 1.82 times higher than without pH control. Then, by matching with a strain protective agent and a freeze drying process adopted by the invention, the number of the obtained lactobacillus helveticus WHH2580 living bacteria reaches 2.75 multiplied by 10 11 CFU/g, water content up to 3.5%, water activity up to 0.24aW.
(5) The preparation process of the tablet is completely carried out in a constant-temperature and constant-humidity environment in a GMP workshop, and meanwhile, the finished tablet is packaged by adopting a high-density polyethylene material with a built-in drying agent, so that the low-moisture and low-water-activity environment of the tablet can be well kept, and the stability of the activity of Lactobacillus helveticus WHH2580 is favorably kept; in addition, the tablet manufacturing process is carried out in a GMP workshop, so that the possibility of microbial pollution of finished products can be obviously reduced.
(6) The Lactobacillus helveticus WHH2580 adopted in the tablet has excellent acid resistance, cholate resistance and adhesion performance, can improve the in-vitro spleen lymphocyte proliferation and the secretion of interleukin IL-12 by the spleen lymphocytes, and promotes the secretion of interleukin IL-8 by the human colon cancer epithelial cells HT-29; in addition, in an animal model with low immunity, the strain can also obviously improve the organ index of a mouse, promote the proliferation of splenic lymphocytes and improve the activity of NK cells, and has good immune enhancement function; in addition, the inactivated strain also has the function of enhancing immunity, so that the strain is richer in product form, wider in application range and longer in product stability and quality guarantee period.
Drawings
FIG. 1 shows the morphology of Lactobacillus helveticus WHH2580 observed under a microscope;
FIG. 2 is a graph showing the effect of cell adhesion between viable, nonviable and commercial strains of Lactobacillus helveticus WHH2580, LGG and Lcs, and HT-29;
FIG. 3 is a graph showing the body weight change of BALB/c mice in each group in the animal test of examples;
FIG. 4 is a graph showing organ ratios of BALB/c mice in each group in the animal test of examples;
FIG. 5 is a graph showing spleen lymphocyte transformation numbers of groups of BALB/c mice in the animal experiments of examples;
FIG. 6 is a graph showing NK cell activities of groups of BALB/c mice in examples.
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
A probiotic tablet with an immunity enhancing function comprises 1-10 parts of Lactobacillus helveticus WHH2580 freeze-dried powder, 3-15 parts of acerola cherry powder, 50-90 parts of direct compression type auxiliary materials and 0.2-2 parts of magnesium stearate.
The straight pressing type auxiliary materials comprise single or multiple compounds of straight pressing type auxiliary materials with common straight pressing performance, such as straight pressing type maltitol, straight pressing type sorbitol, straight pressing type lactose, straight pressing type starch sugar, straight pressing type microcrystalline cellulose and the like.
The lactobacillus helveticus freeze-dried powder is prepared from lactobacillus helveticus and/or mutants thereof; the lactobacillus helveticus is named as WHH2580 and is obtained by screening in yoghourt collected in the Hongshan ditch of Xinjiang Nalat. Has been preserved in China general microbiological culture Collection center (CGMCC) in 2019, 10 months and 23 days, the preservation number of the microorganism is CGMCC No.18730, and the microorganism is named Lactobacillus helveticus by classification; the Lactobacillus helveticus has a 16s rRNA gene sequence shown in SEQ ID NO. 1. The mutant is obtained by carrying out mutagenesis, domestication, gene recombination or natural mutation on the lactobacillus helveticus.
Preparation of lactobacillus helveticus WHH2580 freeze-dried powder:
1) Preparing a culture medium; the formula of the culture medium comprises 15-50g of desalted whey powder, 2-15g of casein hydrolysate, 3-15g of tryptone, 7-20g of soybean peptone, 3-6g of yeast powder, 5-15g of sodium citrate, 1-5g of dipotassium hydrogen phosphate, 0.4-0.8g of magnesium sulfate, 0.2-0.5g of manganese sulfate monohydrate, 5-30mL of tomato juice and 1000mL of water; the pH was adjusted to 6.5. + -. 0.2.
2) Preparing a strain protective agent; the formula of the strain protective agent is skim milk 20-150g/L, sucrose 30-100g/L and glycerol 5-45g/L.
3) Inoculating lactobacillus helveticus WHH2580 into a fermentation substrate in an inoculation amount of 2% -10% for fermentation culture; the fermentation temperature is 34-45 deg.C, the fermentation time is 6-18h, and the pH is controlled at 4.0-6.0 during fermentation.
4) Taking a fermentation product after fermentation, and centrifuging; the centrifugal speed is 4,000-10,000rpm, and the centrifugal time is 3-15min.
5) Mixing the centrifugal product with a strain protective agent; the mixing weight ratio of the centrifugal product to the strain protective agent is 1: 1-3.
6) Freeze-drying;
7) And (3) crushing and sieving the freeze-dried product, and selecting a standard sieve with a sieve of 10-80 meshes to obtain lactobacillus helveticus WHH2580.
Preparation of probiotic tablets with enhanced immune function:
(1) Weighing the raw materials according to the weight part ratio for later use.
(2) Mixing all adjuvants except magnesium stearate uniformly to obtain primary mixture; the mixing speed is controlled at 10-35 rpm, and the mixing time is controlled at 10-20 min.
(3) Uniformly mixing the primary mixture obtained in the step (2) with magnesium stearate to obtain a total mixed semi-finished product; the mixing speed is controlled at 10-25 rpm, and the mixing time is controlled at 5-20 min.
(4) Tabletting the total mixed semi-finished product obtained in the step 3) by using a tabletting machine to obtain tablets; the tablet pressing pressure is controlled between 5KN and 20KN during the tablet pressing process.
(5) Packaging the tablets obtained in the step (4) by using a packaging bottle to obtain a finished product; the packaging bottle used for packaging adopts a High Density Polyethylene (HDPE) packaging bottle with a built-in drying agent. The water content and water activity of the obtained finished product need to be controlled, the water content is controlled to be 1.5-7%, and the water activity is controlled to be 0.1-0.6aW.
All the steps (1) to (5) are carried out in a constant-temperature and constant-humidity environment in a hundred thousand-grade GMP workshop, the temperature is controlled to be 18-26 ℃, and the humidity is controlled to be 25-40%.
Example 1
A probiotic tablet with an immunity enhancing function comprises 10 parts of Lactobacillus helveticus freeze-dried powder, 5 parts of acerola powder, 84.5 parts of direct compression sorbitol and 0.5 part of magnesium stearate. The number of viable bacteria in the Lactobacillus helveticus freeze-dried powder is 3.5 multiplied by 10 10 CFU/g。
The preparation method of the lactobacillus helveticus freeze-dried powder comprises the following steps:
1) Preparing a culture medium; the formula of the culture medium comprises 15g of desalted whey powder, 13g of casein hydrolysate, 3g of tryptone, 20g of soybean peptone, 6g of yeast powder, 5g of sodium citrate, 3g of dipotassium hydrogen phosphate, 0.4g of magnesium sulfate, 0.3g of manganese sulfate monohydrate, 15mL of tomato juice and 1000mL of water; the pH was adjusted to 6.5.
2) Preparing a strain protective agent; the formula of the strain protective agent is 90g/L of skim milk, 80g/L of cane sugar and 30g/L of glycerol.
Inoculating lactobacillus helveticus WHH2580 into a fermentation substrate in an inoculation amount of 2% for fermentation culture; the fermentation temperature is 35 ℃, 3) the fermentation time is 18h, and the pH value is controlled to be 4.8 in the fermentation process.
4) Taking a fermentation product after fermentation, and centrifuging; the centrifugation speed is 4,000rpm, and the centrifugation time is 15min.
5) Mixing the centrifugal product with a strain protective agent; the mixing weight ratio of the centrifugal product to the strain protective agent is 1: 2.
6) Freeze-drying;
7) And (4) crushing and sieving the freeze-dried product, and selecting a 40-mesh standard sieve by using a sieve to obtain the lactobacillus helveticus freeze-dried powder.
Preparation of probiotic tablets:
(1) Weighing the raw materials according to the weight part ratio for later use.
(2) Uniformly mixing 10 parts of lactobacillus helveticus freeze-dried powder, 5 parts of acerola powder and 84.5 parts of direct-compression sorbitol to obtain a primary mixture; the mixing speed was controlled at 35rpm and the mixing time was controlled at 20min.
(3) Uniformly mixing the primary mixture obtained in the step (2) with 0.5 part of magnesium stearate to obtain a total mixed semi-finished product; the mixing speed was controlled at 25rpm and the mixing time was controlled at 15min.
(4) Tabletting the total mixed semi-finished product obtained in the step (3) by using a tabletting machine to obtain tablets; the tablet pressing pressure was controlled at 10KN during the tablet pressing.
(5) Packaging the tablets obtained in the step (4) by using a packaging bottle to obtain a finished product; the packaging bottle used for packaging adopts a High Density Polyethylene (HDPE) packaging bottle with a built-in drying agent. The water content and water activity of the obtained finished product need to be controlled, the water content is controlled to be 5 percent, and the water activity is controlled to be 0.25aW.
All the steps (1) to (5) are carried out in a constant-temperature and constant-humidity environment in a hundred thousand-grade GMP workshop, the temperature is controlled at 26 ℃, and the humidity is controlled at 40%.
And (3) carrying out key index detection on the finished tablet, wherein the detection result is as follows:
example 6
A probiotic tablet with an immunity enhancing function comprises 3 parts of Lactobacillus helveticus freeze-dried powder, 10 parts of acerola powder, 86 parts of direct compression maltitol and 1 part of magnesium stearate. The number of viable bacteria in the Lactobacillus helveticus freeze-dried powder is 2.7 multiplied by 10 11 CFU/g。
Preparation of lactobacillus helveticus freeze-dried powder:
1) Preparing a culture medium; the formula of the culture medium comprises 40g of desalted whey powder, 10g of casein hydrolysate, 8g of tryptone, 15g of soybean peptone, 5g of yeast powder, 8g of sodium citrate, 2g of dipotassium phosphate, 0.6g of magnesium sulfate, 0.4g of manganese sulfate monohydrate, 20mL of tomato juice and 1000mL of water; the pH was adjusted to 6.5.
2) Preparing a strain protective agent; the formula of the strain protective agent comprises 110g/L skim milk, 100g/L sucrose and 60g/L glycerin.
Inoculating lactobacillus helveticus WHH2580 into a fermentation substrate in an inoculation amount of 8% for fermentation culture; the fermentation temperature was 37 ℃. 3) The fermentation time is 14h, and the pH value is controlled at 5.4 in the fermentation process.
4) Taking a fermentation product after fermentation is finished, and centrifuging; the centrifugation speed is 7,000rpm, and the centrifugation time is 10min.
Mixing the centrifugal product with a strain protective agent; the mixing weight ratio of the centrifugal product to the strain protective agent is 1: 2.
6) Freeze-drying;
7) And (3) crushing and sieving the freeze-dried product, and selecting a 20-mesh standard sieve by using a sieve to obtain the lactobacillus helveticus freeze-dried powder.
Preparation of probiotic tablets:
(1) Weighing the raw materials according to the weight part ratio for later use.
(2) Uniformly mixing 3 parts of lactobacillus helveticus freeze-dried powder, 10 parts of acerola powder and 86 parts of direct-compression maltitol to obtain a primary mixture; the mixing speed was controlled at 30rpm and the mixing time was controlled at 20min.
(3) Uniformly mixing the primary mixture obtained in the step (2) with 1 part of magnesium stearate to obtain a total mixed semi-finished product; the mixing speed was controlled at 15rpm and the mixing time was controlled at 8min.
(4) Tabletting the total mixed semi-finished product obtained in the step (3) by using a tabletting machine to obtain a tablet; the tablet pressing pressure was controlled at 13KN during the tablet pressing.
(5) Packaging the tablets obtained in the step (4) by using a packaging bottle to obtain a finished product; the packaging bottle used for packaging adopts a High Density Polyethylene (HDPE) packaging bottle with a built-in drying agent. The water content and water activity of the obtained finished product need to be controlled, the water content is controlled at 3 percent, and the water activity is controlled at 0.3aW.
All the steps (1) to (5) are carried out in a constant-temperature constant-humidity environment in a hundred thousand GMP workshop, the temperature is controlled at 20 ℃, and the humidity is controlled at 25%.
And carrying out key index detection on the finished tablet, wherein the detection result is as follows:
example 3
The strain provided by the invention belongs to Lactobacillus helveticus (Lactobacillus helveticus) through identification, is named as WHH2580, is preserved in the China general microbiological culture Collection center at 23/10 th 2019, and has the microbiological preservation number as follows: CGMCC No.18730.
The strain provided by the invention is obtained by screening yogurt collected from the Hongshan ditch of Xinjiang Nalati.
1. Biological Properties:
morphological characteristics: the growth state in MRS agar medium is: the growth form of the bacterial colony in the MRS agar culture medium is light milky colony, and the bacterial colony is semitransparent, round, rough in surface, irregular in edge and flat. Gram staining is typically positive, and cells observed under a microscope are long-rod-shaped, have no flagellum, do not produce spores and do not move.
The culture characteristics are as follows: the optimal growth temperature is 37 ℃, and the culture medium is facultative anaerobic and grows in MRS culture medium.
2. Identification of strains
2.1 biological identification:
and (3) sequencing a 16s rRNA gene sequence, and performing homology comparison analysis on the obtained result and an NCBI GenBank database to show that the strain is Lactobacillus helveticus (Lactobacillus helveticus).
The gene sequence is shown as SEQ ID NO. 1:
2.2 API50CHL system identification of WHH2580 strain:
WHH2580 zymocyte liquid is centrifuged by 1mL, and bacterial mud is resuspended by normal saline. Adjusting OD 600 Until 5mL of the resuspended bacterial suspension had an OD600 of about 0.5. The supernatant was centrifuged off and resuspended in 5mL of API50 medium. Add to API50 plate, 100uL per well. Incubate at 37 degrees for 24h and 48h.
WHH2580 strain can utilize 6 carbon sources: d-glucose, D-fructose, D-mannose, N-acetylglucosamine, D-lactose and D-trehalose.
Example 4
1. Tolerance of strains
Acid and bile salt resistance of the lactobacillus helveticus WHH2580 strain:
1.1 acid resistance: the bacterial solution after the second generation activation of WHH2580 strain is centrifuged at 20 mL/tube, and washed twice with PBS (pH7.2M). The bacterial sludge was placed in 20mL sterile PBS pH3.0, PBS +0.3% peptin pH3.0 and PBS pH7.2, respectively, with two replicates per system. Sampling at 37 deg.C for 0h, 1h, 2h and 3h respectively, and counting viable bacteria.
The acid resistance test results show that: the pH of 3.0 has no obvious influence on the viability of the bacteria within 2h, and the viable count is reduced by 0.9 order of magnitude within 3 h. The pepsin pH3.0 has no obvious influence on the viability of the bacteria within 2h, and the viable count within 3h is reduced by 0.7 order of magnitude. The above results indicate that the bacterium has excellent acid resistance.
1.2 bile salt resistance: the second generation of WHH2580 bacterial strain after activation is centrifuged in 20 mL/tube and washed twice with PBS (pH 7.2PBS). Placing the bacterial sludge in 20mL sterile PBS +0.1%, trypsinPh8.0, PBS +0.3%, BS pH8.0, PBS +0.5%, BS pH8.0 and PBS pH7.2, respectively, and repeating each system for two times. Sampling at 37 deg.C for 0h, 1h, 2h and 3h respectively, and counting viable bacteria.
The results of the bile salt resistance experiment show that: 0.1% by 3h by 0.8 orders of magnitude in Trypsin system, 0.3% by 3h by 2.8 orders of magnitude in BS system, 0.5% by 3h by 3.6 orders of magnitude in BS system. The results show that the bacterium has good bile salt resistance.
2. Adherence of strains
Adhesion of lactobacillus helveticus WHH2580 strain:
the probiotics colonize epithelial cells of a host intestine through adhesion, gradually form a biological membrane and produce various metabolites, so that a layer of natural protective barrier is formed, and various physiological effects are produced on the host. Thus, the adherence and colonization of the host intestinal epithelial cells by probiotics is a prerequisite for their probiotic action.
HT-29 cells are used as cell strains for adhesion tests, and the specific experimental method is as follows:
HT-29 cells were passed to the third generation, centrifuged at 1000rpm for 5min after digestion with 0.25% trypsin, and resuspended in 5mL of DMEM medium containing 10% fetal calf serum (100U/mL penicillin, 100mg/mL streptomycin) until the cells were a single cell suspension. Counting appropriate amount of cells with a blood cell counting plate, diluting the cells with DMEM medium containing 10% fetal calf serum (containing penicillin 100U/mL, streptomycin 100 mg/mL) until the cell suspension concentration is 1X 10 6 cells/mL, 1mL was inoculated into 12-well cell culture plates containing cell slides, and 5% CO was measured at 37% 2 The culture was carried out for 2 days.
After the WHH2580 strain (LGG of lactobacillus rhamnosus and LcS are positive control strains) is activated for the second generation, 10mL of bacterial liquid is taken, 4200g of centrifugal strain is taken at room temperature for 10min to collect the thallus, 10mL of DMEM complete culture medium containing 10% fetal calf serum is used for culture (without double antibody) for heavy suspension, and the bacterial concentration is adjusted to 2 × 10 8 CFU/mL. Get5mL of strain adjustment solution was inactivated at 95 ℃ for 5min. Inoculating 1mL of each of the strain adjusting solution and the inactivated strain adjusting solution into 12-well cell culture plate containing cell slide, 3 of each of the solutions in parallel to CO 2 Incubate in incubator at 37 ℃ for 2h.
After incubation, the culture medium was slowly aspirated, washed 3 times with PBS, and fixed with 100% methanol for 8min. Taking out the cell slide, standing for 20min, gram staining, and sealing with neutral resin. Observation was performed under an optical microscope.
The results of the adhesion experiments are given in the table below.
The adhesion effect of live bacteria, non-live bacteria and commercial strains LGG and Lcs of Lactobacillus helveticus WHH2580 and HT-29 cells is shown in figure 2.
The adhesive capacity of the live bacteria and the non-live bacteria of the lactobacillus helveticus WHH2580 is obviously higher than that of the positive control strain lactobacillus rhamnosus LGG and the field strain LcS. The adhesion rate of non-living bacteria of lactobacillus helveticus WHH2580 is slightly lower than that of living bacteria, but the adhesion effect of the non-living bacteria is better than that of the living bacteria, the thallus among HT-29 cells is less, and most of the non-living bacteria are tightly adhered to the cells.
3. Resistance of strains
Sensitivity of lactobacillus helveticus WHH2580 strain antibiotics:
the sensitivity of probiotics to antibiotics is an important indicator for testing the safety of strains. In order to test the safety of the WHH2580 strain, the following 9 antibiotics are selected for testing, and the sensitivity of the WHH2580 strain to the 9 antibiotics is tested, and the specific experimental design is as follows.
The antibiotic preparation concentration is 0.5ug/mL, 1ug/mL, 2ug/mL, 4ug/mL, 8ug/mL, 16ug/mL, 32ug/mL, 64ug/mL, 128ug/mL, 256ug/mL, and the actual concentration is 0.125ug/mL, 0.25ug/mL, 0.5ug/mL, 1ug/mL, 2ug/mL, 4ug/mL, 8ug/mL, 16ug/mL, 32ug/mL, 64ug/mL.
100 μ L of medium was added to each well of a sterile 96-well polystyrene plate, and columns 2-11 are labeled according to the following table concentrationsAdding 50 μ L antibiotic diluent (ug/mL) into each well, and adding 50 μ L bacterial suspension into each well in 1-11 rows (final concentration of bacterial liquid is 10) 5 -10 6 CFU/mL), sealing, placing in 37 deg.C incubator for 24-48h, and observing and measuring OD with naked eye 625 . Wherein the Lactobacillus rhamnosus LGG and the Tatian strain LcS are positive control strains.
| Name (R) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Penicillin | Sterile solvent | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | Sterile water |
| Ampicillin | Sterile solvent | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | Sterile water |
| Imipenem | Sterile solvent | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | Sterile water |
| Meropenem | Sterile solvent | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | Sterile water |
| Vancomycin | Sterile solvent | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | Sterile water |
| Daptomycin | Sterile solvent | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | Sterile water |
| Erythromycin | Sterile solvent | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | Sterile water |
| Clindamycin | Sterile solvent | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | Sterile water |
| Linezolid | Sterile solvent | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | Sterile water |
The experimental results are shown in the following table, MIC values ug/mL.
According to the criteria for determining the sensitivity of the CLSI M4 strain to antibiotics, the following conclusions are drawn. The WHH2580 strain is resistant to Daptomycin and Clindamycin, neutral to Erythromycin, and sensitive to Penicillin, ampicillin, imipenem, meropenetem, vancomycin, and Linezolid. LGG strains are resistant to Imipenem, meroperem, vancomycin, daptomycin, clindamycin, neutral to Erythromycin, sensitive to Penicillin, ampicillin, and Linezolid. LcS strain is resistant to Vancomycin and Daptomycin, neutral to Imipenem, meroperem and Erythromycin, and sensitive to Penicillin, ampicillin, clindamycin and Linezolid.
4. Bacterial strain for inhibiting pathogenic bacteria
Lactobacillus helveticus WHH2580 strain bacteriostasis experiment:
sterilized 1.5% agar was dispensed in sterile petri dishes. The oxford cup is placed. MRS culture of sterilized 1% agarCulturing in NB medium at 50 deg.C or below, inoculating 10 6 -10 7 Five indicator bacteria (Escherichia coli, staphylococcus aureus, shigella flexneri, salmonella paratyphi B and Micrococcus luteus) with the concentration of CFU/mL are shaken, mixed uniformly and poured on the previously poured vegetarian agar medium with an Oxford cup. And (4) after the culture medium is cooled and solidified, pulling out the Oxford cup. 100uL of the corresponding bacterial suspension (WHH 2580) and positive control (10 g/mL polymyxin B or 1% Nisin) were added to different wells and diffused in a 4 ℃ freezer for 7h. After culturing at 37 ℃ for 16h, observing and recording the size of the inhibition zone.
The experimental results are shown in the following table, and the numerical values in the table are the size em of the inhibition zone.
The experimental result shows that the WHH2580 strain has inhibiting effect on 5 pathogenic bacteria, has no obvious difference on the inhibiting effect on salmonella paratyphi B, escherichia coli and shigella flexneri and 10g/mL polymyxin B, has no obvious difference on the inhibiting effect on staphylococcus aureus and 1 percent Nisin, and is slightly lower than other control strains.
Example 5
1. The strain has the function of enhancing immunity
In vitro and in vivo experiments to enhance immune function:
1.1 splenic lymphocyte proliferation in vitro:
the mice were sacrificed, spleens were aseptically taken, and were minced with an ophthalmic scissors, and then crushed with a sterile glass syringe core, filtered through a 200-mesh metal screen, added with sterile Hank's solution containing 10% fetal bovine serum, and centrifuged at 1000rpm at 4 ℃ for 5min, to thereby harvest the cells. Adding cell lysis solution 5 times the cell volume, lysing for 5min, adding sterile Hank's solution containing 10% fetal calf serum to stop lysing, and centrifuging at 1000rpm at 4 deg.C for 5min to remove supernatant. Washing with RPMI-1640 medium containing 10% fetal bovine serum, and centrifuging at 1000rpm at 4 deg.C for 5min. Washed twice and the pellet resuspended in 5mL RPMI-1640 medium containing 10% fetal bovine serum. Counting cells and adjusting finenessCell concentration of 5X 10 6 cells/mL。
Centrifuging lactobacillus activated bacteria liquid at 4000rpm at room temperature for 10min to remove supernatant, washing with PBS twice, adding appropriate amount of RPMI-1640 medium containing 10% fetal calf serum, adjusting OD 600 To 0.50. + -. 0.10. The cell suspension was added to a 96-well cell culture plate, 3 replicates per sample. A zero-adjustment group (cell culture medium), a blank control group (cell suspension + cell culture medium, inducer group (cell suspension + 10. Mu.g/mL ConA), and lactobacillus group (cell suspension + 10. Mu.g/mL ConA + bacterial suspension) were set, and the cells were incubated at 37 ℃ for 72 hours in a carbon dioxide incubator, 20. Mu.L of MTT solution (2.5 mg/mL) was added to each well, developed at 37 ℃ for 4 hours, and then centrifuged at 1500rpm and 4 ℃ for 10 minutes, the supernatant was aspirated, 100. Mu.L of DMSO was added, and the absorbance was measured at 490nm using a microplate reader.
And (3) data analysis: probiotics that significantly promote splenic lymphocyte proliferation both in the case of ConA induction and in the case of no induction are preferred strains.
Results as shown in the table below, the proliferation fold of splenic lymphocytes of lactobacillus helveticus WHH2580 was higher than that of the two commercial strains with and without Con a induction.
1.2 in vitro spleen lymphocyte cytokines IL-10 and IL-12 assays
The steps of cell material selection, erythrocyte lysis, washing, cell counting and probiotic culture are the same as the steps of the in vitro splenic lymphocyte proliferation. The cell suspension was added to 96-well cell culture plates, and each treatment was repeated 5 times, and divided into a zero-adjustment group (cell culture medium 100. Mu.L) and a blank control group (1X 10) 7 cells/mL 100. Mu.l of cell suspension, cell culture media 100. Mu.1), probiotic group (1X 10) 7 cell suspension 100. Mu.L, 1X 10 of cells/mL 8 cfu/mL bacterial suspension 100. Mu.L), 37 ℃ 5% CO 2 Culturing in an incubator for 24h. Centrifuging at 1500rpm for 10min, sucking supernatant, filtering with 0.22 μm filter membrane, and determining IL-10 and IL-12 content with BD kit.
Strains with high IL-12/IL-10 ratios can be used as immunomodulatory potential strains.
The results are shown in the table below, and the ratio of L.helveticus WHH2580 IL-12/IL-10 is close to that of L.rhamnosus LGG, and is not significantly poor.
1.3 in vitro human Colon cancer cell HT-29 cytokine IL-8 assay
Digesting the human colon cancer cells HT-29 growing and fusing to 70% -80%, and adjusting the cell concentration to 1 x 10 6 And (4) inoculating the cell/hole into a 24-hole plate, culturing at 37 ℃, and changing the solution after 24 hours. The LPS was added at 20ng/mL for 6 hours, followed by 8X 10 8 After the CFU/mL bacterial solution is treated for 18 hours, centrifuging at 3000r/min for 5min to obtain a supernatant, and detecting the content of interleukin IL-8 by using a corresponding ELISA kit.
The strain with high IL-8 secretion can be used as an immune regulation potential strain.
The results are shown in the following table.
| Strain numbering | IL-8 concentration (ng/mL) |
| Lactobacillus rhamnosus LGG | 2981±95 |
| Lactobacillus helveticus WHH2580 | 4021±145 |
Lactobacillus helveticus WHH2580 IL-8 is higher than commercial strain LGG.
Example 6: animal experiments:
a random grouping design is adopted, 192 inbred line BALB/C male mice (6-8 weeks old, 16-20 g) are divided into 8 groups at random, and each group comprises 12 mice, with an adaptation period of 5 days. Keeping the environment temperature of the animal breeding at 21 +/-2 ℃, the humidity of 30-70%, illuminating for 12h alternately, freely drinking water and freely taking the feed. Changing padding every three days, and adding water and feed in time. The grouping is as follows:
control group 1: a blank control group is filled with gastric water;
control group 2: a model control group is subjected to gastric lavage water and intraperitoneal injection of 50mg/kg cyclophosphamide for molding;
control group 3: a positive drug group, 40mg/kg of levamisole hydrochloride by intragastric administration and 50mg/kg of cyclophosphamide by intraperitoneal injection for molding;
experimental group 1: the bacterial suspension of the invention is used for intragastric administration, and the intragastric administration dosage is 2 x 10 8 CFU/d；
Experimental group 2: the bacterial strain suspension of the invention is used for intragastric administration, and the intragastric administration dosage is 2 x 10 8 CFU/d；
Wherein the inactivated bacteria is treated at 80 deg.C for 10min. The existence of no viable bacteria is confirmed by plate culture.
The test is carried out by adopting a model animal with low immune function. The subjects were administered for 38 consecutive days and administration of the immunosuppressant was started after day 32. Body weight was weighed once a week before inhibitor and once a day after inhibitor. The detection is started on the 5 th day after the intraperitoneal injection of the immunosuppressant, and the detection indexes comprise organ ratio, spleen lymphocyte proliferation and NK cell activity.
The experimental results are as follows:
the body weight results are shown in figure 3. The body weight change trend was similar in each experimental group except for the blank control, and the body weight decreased on the first day of inhibitor injection and gradually recovered thereafter.
The results of organ comparison are shown in FIG. 4. The thymus/body weight ratio and spleen/body weight ratio of the model group were significantly lower than those of the control group. The thymus/body weight ratio of the low-dose group and the positive-drug group was significantly higher than that of the model group (P < 0.05), and that of the high-dose group and the dead-bacteria group was significantly higher than that of the model group (P < 0.001). Spleen/body weight ratios were significantly higher in the low dose, medium dose, dead bacteria and positive drug groups than in the model group (P < 0.05).
The proliferation results of splenic lymphocytes of mice are shown in FIG. 5. The OD values of the ultra-low dose group, the medium dose group and the high dose group are obviously higher than those of the inactivated bacteria group of the model group, and are extremely obviously higher than those of the model group. The WHH2580 can obviously promote spleen lymphocyte proliferation of mice with low immunity by virtue of ultralow dose, low dose, medium dose and high dose and inactivated bacteria.
The results of the measurement of NK cell activity of mice are shown in FIG. 6. The activity of NK cells of the medium-dose group is remarkably higher than that of the model group, and the activity of NK cells of the inactivated bacterial group is remarkably higher than that of the model group. The results show that the dose and the inactivated bacteria in the WHH2580 can obviously improve the activity of NK cells of mice with low immunity.
According to the health product function evaluation method, under the establishment condition of an immune function low model, the total number of blood leucocytes, the cellular immune function (a mouse spleen lymphocyte transformation experiment, a delayed type allergic reaction experiment), the humoral immune function (an antibody-producing cell detection, a serum hemolysin determination), the mononuclear-macrophage function (a mouse carbon clearance experiment, a mouse abdominal cavity macrophage phagocytosis fluorescent microsphere experiment) and the NK cell activity are determined to be positive, and the tested sample has the function of enhancing the immunity of a person with low immune function. The test result of the WHH2580 strain is positive in both the cellular immune function and the NK cell activity, and the WHH2580 strain can be judged to have the effect of enhancing the immunity of a person with low immune function. The WHH2580 inactivated strain can also improve the immunity of the organism by improving and regulating the organ index, nonspecific immunity and specific immune response of the organism.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Sequence listing
<110> Hangzhou baby Haha science Co Ltd
Hangzhou Wahaha Group Co.,Ltd.
<120> a probiotic tablet with function of enhancing immunity and a preparation method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1425
<212> DNA
<213> Lactobacillus helveticus (Lactobacillus helveticus)
<400> 1
ccttcccgaa ggttaggcca ccggctttgg gcattgcaga cttccatggt gtgacgggcg 60
gtgtgtacaa ggcccgggaa cgtattcacc gcggcgttct gatccgcgat tactagcgat 120
tccagcttcg tgcagtcgag ttgcagactg cagtccgaac tgagaacagc tttcagagat 180
tcgcttgcct tcgcaggctc gcttctcgtt gtactgtcca ttgtagcacg tgtgtagccc 240
aggtcataag gggcatgatg acttgacgtc atccccacct tcctccggtt tatcaccggc 300
agtctcatta gagtgcccaa cttaatgctg gcaactaata acaagggttg cgctcgttgc 360
gggacttaac ccaacatctc acgacacgag ctgacgacag ccatgcacca cctgtcttag 420
cgtccccgaa gggaactcct aatctcttag gatggcacta gatgtcaaga cctggtaagg 480
ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat 540
tcctttgagt ttcaaccttg cggtcgtact ccccaggcgg agtgcttaat gcgttagctg 600
cagcactgag aggcggaaac ctcccaacac ttagcactca tcgtttacgg catggactac 660
cagggtatct aatcctgttc gctacccatg ctttcgagcc tcagcgtcag ttgcagacca 720
gagagtcgcc ttcgccactg gtgttcttcc atatatctac gcattccacc gctacacatg 780
gagttccact ctcctcttct gcactcaaga aaaacagttt ccgatgcagt tcctcggtta 840
agccgagggc tttcacatca gacttattct tccgcctgcg ctcgctttac gcccaataaa 900
tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt tagccgtgac 960
tttctggttg attaccgtca aataaaggcc agttactacc tctatccttc ttcaccaaca 1020
acagagcttt acgatccgaa aaccttcttc actcacgcgg cgttgctcca tcagacttgc 1080
gtccattgtg gaagattccc tactgctgcc tcccgtagga gtttgggccg tgtctcagtc 1140
ccaatgtggc cgatcagtct ctcaactcgg ctatgcatca ttgccttggt aagccgttac 1200
cttaccaact agctaatgca ccgcggggcc atcccatagc gacagcttac gccgcctttt 1260
ataagctgat catgcgatct gctttcttat ccggtattag cacctgtttc caagtggtat 1320
cccagactat ggggcaggtt ccccacgtgt tactcaccca tccgccgctc gcgtccccag 1380
cgtcattacc gaagtaaatc tgctggttct gctcgctcga cttgc 1425
Claims (10)
1. The application of the lactobacillus helveticus in preparing the probiotic tablet with the function of enhancing the immunity is characterized in that the lactobacillus helveticus is named as WHH2580, has been preserved in China general microbiological culture Collection center (CGMCC) in 2019, 10 and 23 months, and has the microbiological preservation number of CGMCC No.18730 and the microbiological classification name of lactobacillus helveticusLactobacillus helveticus(ii) a The Lactobacillus helveticus has a 16s rRNA gene sequence shown in SEQ ID NO. 1.
2. A probiotic tablet with an immunity enhancing function is characterized by comprising 1-10 parts of Lactobacillus helveticus freeze-dried powder, 3-15 parts of acerola cherry powder, 50-90 parts of direct compression type auxiliary materials and 0.2-2 parts of magnesium stearate by weight;
the lactobacillus helveticus freeze-dried powder is prepared from lactobacillus helveticus; the Lactobacillus helveticus is named as WHH2580 and has been cultured in China general microbiological culture Collection Committee (China Committee for culture Collection of microorganisms) in 2019, 10 months and 23 daysThe center of the culture has the preservation number of CGMCC No.18730, and the classification name of the microorganism is Lactobacillus helveticusLactobacillus helveticus(ii) a The Lactobacillus helveticus has a 16s rRNA gene sequence shown in SEQ ID NO. 1.
3. The probiotic tablet according to claim 2, characterized in that the excipients of the direct compression type comprise one or more of maltitol of the direct compression type, sorbitol of the direct compression type, lactose of the direct compression type, starch sugar of the direct compression type and microcrystalline cellulose of the direct compression type.
4. The probiotic tablet according to claim 2, characterized in that the number of viable bacteria in the freeze-dried powder of lactobacillus helveticus is 1 x 10 8 CFU/g-2×10 12 CFU/g。
5. Method for the preparation of probiotic tablets according to any of claims 2 to 4, characterized in that it comprises the following steps:
(1) Weighing the raw materials for later use;
(2) Uniformly mixing all the raw materials except the magnesium stearate to obtain a primary mixture;
(3) Uniformly mixing the primary mixture obtained in the step (2) with magnesium stearate to obtain a total mixed semi-finished product;
(4) Tabletting the total mixed semi-finished product obtained in the step (3) by using a tabletting machine to obtain a tablet;
(5) And (5) packaging the tablets obtained in the step (4) by using a packaging bottle to obtain the probiotic tablets.
6. The method of claim 5, wherein:
in the step (2), the mixing rotation speed is controlled to be 10 to 35rpm, and the mixing time is controlled to be 10 to 20min; and/or
In the step (3), the mixing rotation speed is controlled to be 10 to 25rpm, and the mixing time is controlled to be 5 to 20min; and/or
In the step (4), the tablet pressing pressure is controlled to be between 5kN and 20kN in the tablet pressing process; and/or
In the step (5), a packaging bottle used for packaging adopts a high-density polyethylene packaging bottle with a built-in drying agent; and/or
And (5) performing the steps (1) to (5) in a constant-temperature and constant-humidity environment in a GMP workshop, wherein the temperature is 18-26 ℃, and the humidity is 25-40%.
7. The method of claim 5, wherein: in the step (5), the water content of the obtained probiotic tablet is 1.5-7%, and the water activity is 0.1-0.6.
8. The method of claim 5, wherein: the preparation method of the lactobacillus helveticus freeze-dried powder comprises the following steps:
1) Preparing a culture medium;
2) Preparing a strain protective agent;
3) Inoculating lactobacillus helveticus in a fermentation substrate in an inoculation amount of 2% -10% for fermentation culture;
4) Taking a fermentation product after fermentation, and centrifuging;
5) Mixing the centrifugal product with a strain protective agent;
6) Freeze-drying;
7) And crushing and sieving the freeze-dried product to obtain the lactobacillus helveticus freeze-dried powder.
9. The method of claim 8, wherein:
in step 1), the culture medium comprises the following components: 15-50g of desalted whey powder, 2-15g of casein hydrolysate, 3-15g of tryptone, 7-20g of soyabean peptone, 3-6g of yeast powder, 5-15g of sodium citrate, 1-5g of dipotassium phosphate, 0.4-0.8g of magnesium sulfate, 0.2-0.5g of manganese sulfate monohydrate, 5-30mL of tomato juice and 1000mL of water; the pH was adjusted to 6.5. + -. 0.2.
10. The method of claim 8, wherein:
in the step 2), the strain protective agent comprises the following components: 20-150g/L skim milk, 30-100g/L sucrose and 5-45g/L glycerol; and/or
In the step 3), the fermentation temperature is 34-45 ℃, the fermentation time is 6-18h, and the pH value is controlled to be 4.0-6.0 in the fermentation process; and/or
In the step 4), the centrifugal speed is 4000-10000rpm, and the centrifugal time is 3-15min; and/or
In the step 5), the mixing weight ratio of the centrifugal product to the strain protective agent is 1:1-3; and/or
In step 7), the crushed product is screened, and a standard sieve with 10-80 meshes is selected as a screen.
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