CN104651271B - Lactobacillus casei Lactobacillus casei strain Qian working stock cultures products and its enteron aisle adjust health purpose - Google Patents

Abstract

The invention discloses a working starter product of Lactobacillus casei strain Qian (CCTCC No. M2013514) and its use for intestinal regulation and health care. .0 artificial gastric juice under treatment for 3 hours, the survival rate reached 62.51±7.35%. It can grow slowly under 1.0% concentration of bile salt, and the growth efficiency reaches 7.86±0.45% of the culture without bile salt. The hydrophobicity of Lactobacillus casei strain Qian cells It also reached 51.30±4.28%, and it grows normally in the human intestine; it can slow down the weight loss trend of mice caused by constipation, accelerate the time of black stool, improve the small intestinal propulsion rate, and lower the gene expression level of TRPV1 and NOS in mice , to reduce the defecation volume, the number of defecation particles, and the trend of decreasing the water content of feces, which has a similar effect to constipation drugs, showing a certain effect of preventing constipation.

Description

Lactobacillus casei strain Qian working starter Products and their use for intestinal regulation and health care

technical field

The invention relates to the technical field of microbes, in particular to Lactobacillus casei strain Qian which can regulate intestinal motility and prevent constipation and use thereof.

Background technique

Lactic acid bacteria (LAB) is a general term for bacteria that can produce large amounts of lactic acid from fermentable carbohydrates. These bacteria are widely distributed in nature and have rich species diversity. Lactic acid bacteria can not only improve the nutritional value of food, improve food flavor, improve food preservation and added value. Except for a few, most of them are essential flora in the human body and have important physiological functions, which widely exist in the intestinal tract of the human body. A large number of studies have shown that lactic acid bacteria can regulate the normal flora of the gastrointestinal tract, maintain micro-ecological balance, and improve food digestibility. Lactic acid bacteria are also the main source of probiotic strains. Currently, probiotic strains such as Lactobacillus casei, Lactobacillus plantarum, and Bifidobacterium have been found to have multiple health functions. Such as lowering serum cholesterol, controlling endotoxin, inhibiting the growth and reproduction of spoilage bacteria and production of spoilage products in the intestinal tract, producing nutrients, stimulating tissue development, etc.

Constipation is a very common disease, with a prevalence of up to 27% in the population, and it can affect people of all ages. Habitual constipation is the main culprit in inducing hyperlipidemia, cardiovascular and cerebrovascular diseases, arteriosclerosis, tumors and other diseases and complications in middle-aged and elderly men due to increased abdominal pressure due to exertion, and breath-holding and defecation, which leads to a sharp increase in the mortality rate of cardiovascular and cerebrovascular diseases in the elderly Increase, hemorrhoids, anal fissure and other problems appear, which is very painful. Due to the high incidence of constipation and complicated etiology, patients often have many distresses, and when constipation is severe, it will affect the quality of life. Therefore, it is of great practical significance to find a biological product that can replace drugs to prevent constipation. Microecological therapy solves various problems existing in traditional therapy with a brand-new method.

There are a lot of research reports on the physiological functions of lactic acid bacteria at home and abroad, but there are few reports on the functional research of lactic acid bacteria in preventing constipation. Research on the prevention of constipation by lactic acid bacteria is of great value to many disciplines such as food science, preventive medicine and microecology. Fermented dairy products are an important channel for people to consume lactic acid bacteria. Screening excellent strains of lactic acid bacteria that can prevent constipation and be safe will be of great significance for the development of functional dairy products, enriching the types of existing dairy products, and increasing the added value of dairy products. Therefore, lactic acid bacteria that can prevent constipation have great research and application value.

In the literature and patents or patent applications that have been published so far, for example, CN1796540A discloses bifidobacteria with anti-intestinal pathogenic bacteria and anti-oxidative properties and uses thereof, for preparing food compositions or pharmaceutical compositions, killing or Inhibits Helicobacter pylori or Escherichia coli and treats various diseases caused by them. CN101144064A discloses a Lactobacillus brevis with anti-mutagenic activity and exopolysaccharide production and its application, which is used in fermented food to improve food quality and increase its nutritional function. CN101144065A discloses a hydrogen peroxide-resistant, free-radical-scavenging antioxidant Lactobacillus casei and its application, which is used in fermented food and dairy capsule products to develop functional dairy products, enrich existing dairy products, and increase the additional value of dairy products. value. CN102174450A discloses a Lactobacillus plantarum resistant to Helicobacter pylori infection and its use, which is used in the preparation of pharmaceutical compositions, fermentation agents, animal feed additives and health products to prevent or reduce Helicobacter pylori infection. However, these patents or patent applications do not fully relate to microorganisms that have properties such as regulating intestinal motility and preventing constipation. Therefore, there is also a need for a microorganism that regulates intestinal motility and prevents constipation and food containing them.

Contents of the invention

In order to solve the above problems, the present invention provides a Lactobacillus casei that can regulate intestinal movement and prevent constipation and its application, mainly provides a kind of Lactobacillus casei strain that can regulate intestinal movement and prevent constipation and provides a kind of said Use of Lactobacillus casei in fermented foods.

A strain of Lactobacillus casei strain Qian was screened from naturally fermented yak yoghurt, and the Lactobacillus casei strain Qian was identified as Lactobacillus casei by using microbiological characteristics such as morphological characteristics, cultural characteristics and physiological and biochemical characteristics. The strain was deposited in the China Center for Type Culture Collection (China. Wuhan. Wuhan University) on October 25, 2013, with the preservation number CCTCC NO: M2013514.

The morphological characteristics of the Lactobacillus casei are specifically:

Thale characteristics: Gram-positive, rod-shaped cells, 0.5-1.0 μm wide, 2.5-4 μm long, single, paired or chained, no spores formed, rounded at both ends.

Colony characteristics: Obvious colonies are formed on the MRS medium, with a diameter between 0.5-2.0mm, round, with neat edges, milky white, transparent, moist and smooth surface, and no pigmentation.

The in vivo tolerance characteristics of the Lactobacillus casei are:

The strain of Lactobacillus casei has strong acid resistance, and the survival rate reaches 62.51±7.35% after being treated for 3 hours under the artificial gastric juice of pH 3.0. It can grow slowly under the concentration of 1.0% bile salt, and the growth efficiency reaches 7.86% of the culture without bile salt. ±0.45%, the hydrophobicity of Lactobacillus casei strain Qian cells also reached 51.30±4.28%, and they grow normally in the human intestine.

The Lactobacillus casei is derived from traditional fermented food and belongs to generally recognized as safe (Generally Recognized AsSafe, GRAS) strains.

The realization means of the object of the present invention is:

A working starter product of Lactobacillus casei strain Qian, the preservation number of China Type Culture Collection Center CCTCC NO:M2013514, adopts the following steps to obtain the target object: 1) Inoculate the original strain of Lactobacillus casei above 12 % weight percentage of skim milk, sterilized at 110°C for 10min, cultured at 37°C for 14-16h until curdling, continuous culture and activation for two generations, used as mother starter; The volume percentage is inoculated in sterilized milk, cultivated for 14-16 hours until the curdled milk, the number of viable bacteria in the curdled milk is about 10 ⁹ cfu/mL, and the working starter is obtained; 2) The target working starter obtained in 1) is directly added Into food or used together with symbiotic commercial starters for fermented milk to prepare fermented milk.

The commercial starter is Lactobacillus bulgaricus or Streptococcus thermophilus.

Another object of the present invention is to provide a working starter product of Lactobacillus casei for intestinal regulation and prevention of constipation. The specific means used are:

A working starter product of Lactobacillus casei for intestinal regulation and prevention of constipation. It is used as a starter to add to food or used together with a symbiotic commercial starter for the preparation of fermented milk to prepare fermented milk for the purpose of regulating intestinal motility and preventing Constipation health effects, the optimal dosage is 1.0×10 ⁹ CFU per kilogram of body weight.

The Lactobacillus casei working starter product is used for intestinal regulation, constipation prevention and health care, and the food includes human food, animal feed or health care product.

The edible objects include human beings and farmed animals, and the method is added in daily food so that the edible objects can obtain the health care function of regulating intestinal movement and preventing constipation.

The fermented food is one or more of lactic acid bacteria milk beverage, milk powder, capsule product or fermented milk containing Lactobacillus casei.

The lactic acid bacteria milk drink containing Lactobacillus casei is prepared according to the following steps:

The original strain of Lactobacillus casei is preserved in the form of a 30% (weight) glycerin suspension at a temperature of -75°C, or in the form of freeze-dried bacteria powder at a temperature of 4°C for subsequent use;

Utilize the original bacterial classification of described Lactobacillus casei to prepare Lactobacillus casei working starter;

Raw milk is heat-sterilized at 95°C for 20 minutes or at 140°C for 2 seconds, cooled to 4°C, added to the working starter of Lactobacillus casei to make the concentration above 10 ⁶ cfu/mL, and stored in cold storage at 4°C .

The milk powder containing Lactobacillus casei is prepared according to the following steps:

The original strain of Lactobacillus casei is preserved in the form of a 30% (weight) glycerin suspension at a temperature of -75°C, or in the form of freeze-dried bacteria powder at a temperature of 4°C for subsequent use;

Utilize the original strain of Lactobacillus casei to prepare Lactobacillus casei working starter, raw milk is heat sterilized at 95°C for 20min or high temperature heat sterilization at 140°C for 2s, then cooled to 37°C, and then 4% of the volume of raw milk % inoculation amount inoculates the described Lactobacillus casei working starter, ferments at 37 DEG C for 16h to obtain Lactobacillus casei fermented milk; said Lactobacillus casei fermented milk and raw milk after sterilization are according to 1:3 (V:V ) in a proportioning manner, homogenized, concentrated in vacuo, and spray-dried to obtain milk powder containing Lactobacillus casei.

The capsule product containing Lactobacillus casei is prepared according to the following steps:

The original strain of Lactobacillus casei is preserved in the form of a 30% (weight) glycerin suspension at a temperature of -75°C, or in the form of freeze-dried bacteria powder at a temperature of 4°C for subsequent use;

Utilize the original bacterial classification of described Lactobacillus casei to prepare Lactobacillus casei working starter;

Heat the raw milk at 140°C for 2 seconds, then cool it down to 37°C, inoculate the working starter of Lactobacillus casei with 4% of the volume of the raw milk, and ferment at 37°C for 16 hours to obtain Lactobacillus casei fermented milk ; Add the Lactobacillus casei fermented milk and the sterilized raw milk according to the ratio of 1:3 (V:V), homogenize, vacuum concentrate, and spray dry to obtain milk powder, and will obtain cheese milk containing Bacillus capsules.

The fermented milk containing Lactobacillus casei is prepared according to the following steps:

The original strain of Lactobacillus casei is preserved in the form of a 30% (weight) glycerin suspension at a temperature of -75°C, or in the form of freeze-dried bacteria powder at a temperature of 4°C for subsequent use;

Utilize the original strain of Lactobacillus casei to prepare Lactobacillus casei working starter; raw milk is sterilized at 95°C for 20min or at 140°C for 2s at high temperature, then cooled to 37°C, and then 3-5% ( volume) add Lactobacillus casei working starter, then add 3-5% (volume) symbiotic starter for the preparation of fermented milk products, mix and ferment mixed bacteria at 37°C until the titrated acidity is 0.6- 0.7%, then cooled to 4°C, and then refrigerated to obtain fermented milk containing Lactobacillus casei.

Described commercial starter is preferably Lactobacillus bulgaricus or Streptococcus thermophilus.

In the sense of the present invention, the raw material milk is one or more raw material milks selected from skimmed milk, fresh milk and reconstituted milk.

The beneficial effects brought by the technical solutions of the embodiments of the present invention are as follows:

The present invention relates to Lactobacillus casei which is isolated from naturally fermented yak yoghurt at the herdsman's home in Mozhugongka County, Tibet Autonomous Region, which can regulate intestinal movement and prevent constipation, and the strain is used as a starter or added fermented milk, health food and animal health care Application in products, Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) strains have strong acid resistance, and the survival rate reached 62.51 ± 7.35% after being treated for 3 hours under the artificial gastric juice of pH 3.0, and can slowly Growth, the growth efficiency reached 7.86±0.45% of that cultured without bile salts, and the hydrophobicity of Lactobacillus casei strainQian cells also reached 51.30±4.28%, and they grew normally in the human intestine. Experiments in mice also showed that Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) working starter product and its downstream products can slow down the weight loss trend of mice caused by constipation, accelerate the time of black stool discharge, improve the propulsion rate of the small intestine, and increase in different degrees Serum levels of MTL, Gas, ET, AchE, SP, and VIP factors, decreased SS factors, increased food intake and water intake, decreased defecation volume, the number of defecation particles, and the trend of decreasing stool water content, which has a similar effect to constipation drugs , showing a certain preventive effect on constipation.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings that need to be used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For Those of ordinary skill in the art can also obtain other drawings based on these drawings without making creative efforts.

Figure 1 Lactobacillus casei (Lactobacillus casei strain Qian) colony morphology;

The thalline morphology (l000x) of Fig. 2 lactobacillus casei (Lactobacillus casei strain Qian);

Fig. 3 change of mouse body weight with experimental time;

The time required for the first row of black stools in different groups of mice;

The defecation patterns of different groups of mice in Fig. 5;

The total length of small intestine of different groups of mice in Fig. 6;

Figure 7 c-Kit, SCF, GAPDH gene expression in different groups of mice

Figure 8 TRPV1, GDNF, NOS, GAPDH gene expression in different groups of mice

The Lactobacillus casei strain Qian of the present invention was preserved in the China Center for Type Culture Collection on October 25, 2013, and the number is CCTCC NO: M2013514.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on this The embodiments in the invention, and all other embodiments obtained by persons of ordinary skill in the art without creative effort, all belong to the scope of protection of the present invention.

Example 1: Isolation, purification and preliminary identification of Lactobacillus casei strain Qian.

This embodiment is carried out according to the following steps:

(1) Separation and purification of Lactobacillus casei strain Qian.

Aseptically draw 500 μL of traditional fermented yak yogurt sample and add it to 5 mL sterilized saline to make a uniform dilution of 1:10, and continue to make a certain proportion of dilution. Select an appropriate gradient dilution solution, pipette 100 μL each into the MRS solid plate medium with a sterile pipette tip, and incubate at 30°C for 48-72 hours. Observe and record the colony morphology as shown in Figure 1.

Use an inoculation loop (or a sterilized toothpick) to pick different colonies from the surface and interior of the plate and inoculate them in the MRS liquid medium, and culture them on a shaker at 30°C and 300r/min for 24-48h; repeat the above steps for continuous activation for 2 generations before coating Microscopic examination of Gram staining is shown in Figure 2.

It was determined to be the continued activation of G+ bacteria until pure colonies were obtained (microscopic examination without miscellaneous bacteria), and a catalase test was carried out. The G+ and catalase-negative non-bacillus, cocci and streptococci are tentatively designated as lactic acid bacteria and stored. For specific operations, refer to Microbiology Experimental Techniques.

The experimental results showed that more than 58 strains of lactic acid bacteria were isolated from ten samples in Mozhugongka County, and the average number of live lactic acid bacteria in the samples was on the order of 10 ⁸ cfu/mL. Among them, Lactobacillus casei strain Qian is one of the strains.

(2) Preliminary identification of Lactobacillus casei strain Qian.

Gram staining: take typical colony smears, perform Gram staining, and observe the shape and arrangement of bacteria under the oil lens of a microbial microscope. Pick the strains with clear vision and typical shape and take pictures. Catalase test: Pick an inoculation loop from a colony on the solid medium, place it in a clean test tube, add 2 mL of 3% hydrogen peroxide solution dropwise, and observe the results (those with bubbles within half a minute are positive, those without bubbles) is negative).

The results showed that 58 strains of lactic acid bacteria were isolated and purified from the samples. These strains could all grow on the MRS plate medium containing 5‰CaCO ₃ , and formed calcium-dissolving circles around the colonies, Gram staining was positive, catalase The test was negative.

Example 2: In vitro screening of Lactobacillus casei strain Qian.

(1) Screening of probiotics resistant to pH 3.0 artificial gastric juice.

Preparation of artificial gastric juice: 0.2% NaCl, 0.35% pepsin, adjust the pH value to 3.0 with 1M HCl, filter and sterilize with a vacuum pump in an aseptic operating table for later use.

Determination of the tolerance of probiotics to artificial gastric juice: take 5mL of the activated strain culture solution, pour it into a sterilized 10mL centrifuge tube in a sterile operating table, collect the bacteria by centrifugation at 3000r/min for 10min, add 5mL of sterilized Mix physiological saline to make bacterial suspension, take 1mL bacterial suspension and 9mL artificial gastric juice with pH3. Samples were taken, poured into MRS agar medium and cultured at 37°C for 48h. The number of viable bacteria was determined by plate counting method, and the survival rate (%) was calculated: survival rate (%)=number of viable bacteria in 3 h/number of viable bacteria in 0 h×100%.

(2) Determination of tolerance of probiotics to different concentrations of bile salts.

Select the strains whose survival rate is above 10% in pH3.0 artificial gastric juice to do the growth test under different bile salt concentrations. Inoculate 5 mL of the activated strain into 0.0% ox bile salt (i.e. blank), 0.3% ox bile salt, 0.5% ox bile salt, 1.0% Bovine bile salt (W/V) MRS-THIO medium (0.2% sodium thioglycolate added to MRS medium). After culturing in a constant temperature shaker at 37°C for 24 hours, the OD values of the above-mentioned medium with different concentrations were measured with the blank medium as the control (uninoculated MRS-THIO medium), and the tolerance of the strain to bile salts was calculated. Bile salt tolerance = OD value of bile salt-containing medium/OD value of blank medium × 100%.

(3) Determination of hydrophobicity.

Select bacteria with a survival rate of more than 10% in artificial gastric juice at pH 3.0 and strong tolerance to bile salts. After microscopic examination, it is observed that the growth of the bacteria is in good shape, and then the test is carried out. 5 mL of the activated bacterial culture is placed in a sterile Pour it into a sterilized 10mL centrifuge tube in the operating table, and centrifuge at 3000r/min for 10min to collect the bacteria. The cells were washed with 5 mL of PBS (50 mM, pH 6.5) buffer, centrifuged at 3000 r/min for 10 min, and washed twice. Using PBS buffer as a blank control, adjust the concentration of the tested strains with buffer so that the A0 value at 560nm wavelength is about 1.00.

Take 4mL of the bacteria solution with adjusted turbidity in a sterilized 10mL centrifuge tube, add 0.8mL xylene, the control group does not add xylene, shake for 30s, pause for 10s, then shake for 30s, and put it on the test tube stand for 5~ After 10 minutes of separation, the lower aqueous phase was removed, and the PBS buffer was used as a blank control, and the A value was measured at 560 nm and recorded. Hydrophobic rate H%=[(A0-A)/A0]×100 where A0 and A are the A values measured at 560nm before and after mixing with xylene, respectively.

Lactic acid bacteria are part of the normal intestinal flora. As one of the first conditions for probiotics, they can survive in the stomach and the front of the small intestine. The strains to be screened should have sufficient acid resistance and growth and development characteristics in an acidic environment.

The screening results of Lactobacillus casei strain Qian are shown in Table 1 below:

Note: LC-Qian: Lactobacillus casei strain Qian LB: Lactobacillus bulgaricus

Table 1

The screening results showed that the survival rate of Lactobacillus casei strain Qian reached 62.51±7.35% in gastric juice at pH3.0.

The surviving bacteria after passing through the stomach will be in contact with bile salts in the small intestine. In this experiment, the resistance of lactic acid bacteria to bile salts was used as a selection criterion for potential probiotics. The result of Lactobacillus casei strain Qian with better acid resistance tolerance to different concentrations of bile salts is that it has a certain tolerance to 0.3% bile salts. When the concentration of bile salts increases to 0.5% and 1.0%, Lactobacillus casei strain Qian Demonstrated good tolerance to bile salts.

In addition to having a certain resistance to bile salts in the small intestine, lactic acid bacteria also need to have good adhesion on the small intestinal mucosa, so the hydrophobicity of lactic acid bacteria is used as another selection criterion. It can be seen from the table that Lactobacillus caseistrain Qian The hydrophobicity reached 51.30±4.28%.

Through the above experiments, we screened out Lactobacillus casei strain Qian with good acid resistance, bile salt resistance and hydrophobicity.

Example 3: Activated carbon induced constipation experiment.

Feed healthy Kunming mice, 6 weeks old, weighing 23±2g, 50 females. After feeding the basal diet for 5 days, the mice were randomly divided into 5 groups according to body weight, namely: normal group, constipation control group, bisacodyl group, Lactobacillus bulgaricus group, and high and low intragastric doses of Lactobacillus casei strain Qian group, 10 in each group. Raise them in stainless steel cages, keep room temperature at 24±2°C, relative humidity at 50±10%, and light-dark rotation for 12 hours (lighting at 8:00-20:00). During the whole experimental period, the normal group and the constipation control group were fed with basic feed, and the mice in the experimental group were fed with probiotics at 1.0×10 ⁹ CFU/kg per day. Two weeks later, at 9:00 every morning, except the normal group, mice in the other 5 groups were gavaged with 10% activated carbon ice water at 2°C, 0.2 mL/mouse, once a day for 3 consecutive days. Until the last day of the experiment, all mice were fasted for 24 hours, and then gavaged with activated carbon and ice water, 0.2 mL/mouse. Five mice in each group were used to measure the body weight change, the quantity of feces excreted and the water content of feces. Mice The other 5 mice were killed and dissected after gavage of activated charcoal for 30 minutes.

The body weight changes of the mice in each group during the experiment period are shown in Figure 3 and Figure 4.

The body weight of the mice in the normal group (Normal) increased with the time of the experiment. The constipation control group (Control) lost weight due to constipation due to gavage of activated charcoal after 6 days. Bisacodyl (bisacodyl) constipation drug group slowed down the weight loss. The Qian group also slowed weight loss, and the high-concentration group lost less weight.

Compared with the normal group, there was a very significant difference (P<0.05) in the time of the first black stool in the constipation control group. The average time for the mice in the high and low intragastric doses of Lactobacillus casei strain Qian group to pass black stool for the first time was lower than that of the constipation control group, and higher than that of the normal group and the constipation treatment drug bisacodyl group, showing a certain preventive effect on constipation .

During the test period, the changes in the amount of defecation, the number of defecated particles and the moisture content of feces in each group of mice are shown in Table 2, and the defecation morphology is shown in Figure 5.

Note: LC-Qian: Lactobacillus casei strain Qian

Table 2

Each group of mice n=10ICR, bisacodyl: 100mg/kg bw, Lactobacillus bulgaricus: 1.0×10 ⁹ CFU/kgb.w., LC-Qian: Lactobacillus casei strain Qian.

The defecation volume, the number of defecated particles and the moisture content of feces were not significantly different among the groups in the first 6 days. After 6 days of constipation induction, there was no significant change in the tablet variables of the normal group. The defecation volume, the number of defecation particles and the moisture content of feces in the control group were significantly lower than those in the normal group. Higher than the control group, the high concentration of Lactobacillus caseistrain Qian's defecation particle number and fecal water content were close to the drug group and the normal group.

Example 4: Small intestine activated carbon propulsion experiment.

The mice were sacrificed by cervical spine dissection, the abdominal cavity was dissected and the mesentery of the mice was separated, and the entire intestinal tube from the pylorus to the ileocecal was cut off, placed on absorbent paper, and the entire small intestine was pulled into a straight line to measure the intestinal tube The length is the "total length of the small intestine", and the "activated carbon propulsion length" is from the pylorus to the front of the activated carbon juice. By measuring the propulsion length of activated carbon in the small intestine, the propulsion rate was calculated according to the following formula. Propulsion rate (%)=(activated carbon propulsion distance)/(total length of small intestine)×100.

The total length of the small intestine and the propulsion rate of the small intestine of the mice in each group during the test period are shown in Figure 6.

The average intestinal propulsion rate of the mice in the constipation treatment drug bisacodyl group was 89.2±6.0%, and the small intestinal propulsion rate of the mice in the constipation control group was 21.3±2.2%. There was a very significant difference (P<0.05). The intestinal propulsion rate of mice in Lactobacillus casei strain Qian group and Lactobacillus bulgaricus group was 65.8±4.8%, 52.4±5.1% and 40.3±4.3%, respectively, higher than that of constipation control group and lower than that of constipation treatment drug bisacodyl group, showing a certain Constipation preventive effect.

Example 5: Determination of the levels of various factors in mouse serum.

Take 0.2mL mouse arterial blood, centrifuge at 4°C, 3000r/min for 10min, and take the supernatant serum. According to motilin (MTL), gastrin (Gas), endothelin (ET), somatostatin (SS), serum acetylcholinesterase (AchE), substance P (SP) and vasoactive intestinal peptide (VIP) The method in the kit instruction manual was used to determine the levels of the above factors in serum.

The levels of each factor in the serum of each group of mice during the experiment period are shown in Table 3.

table 3

Compared with the constipation control group, the Lactobacillus casei strain Qian high and low groups could increase the levels of serum MTL, Gas, ET, AchE, SP and VIP, which are negative correlation factors for constipation, and decrease the levels of SS positive correlation factors for constipation. So Lactobacillus casei strain Qian has a preventive effect on constipation.

Example 6: Determination of the expression level of constipation-related genes

A total RNA extraction kit (Invitrogen, USA) was used to isolate tissues according to the instructions. Purification was performed using RNeasy kit (Qiagen, Germany) according to the instructions. cDNA was synthesized by reverse transcription of 2 μg total RNA incubated at 37°C for 1 h with avian myeloblastosis virus reverse transcriptase (GE, UK). The gene primer sequences used for nucleic acid amplification are shown in Table 4, and the polymerase chain reaction (PCR) was performed in thermocycling (Eppendorf, Germany). PCR products were electrophoresed in 1.0% agarose gel, stained with ethidium bromide and imaged.

The gene primer sequence of table 4 nucleic acid amplification

Interstitial cells of Cajal (ICC) are mesenchymal cells between the intestinal nervous system and smooth muscle, which play an important role in regulating the nerve signal of intestinal smooth muscle cells. Cytoplasmic tyrosine kinase (C-Kit) is a special marker of ICC, which is the key to ICC proliferation. Stem cell growth factor (SCF) is a key protein for ICC growth. Without SCF, ICC cannot grow. Transient receptor potential channel vanillin subtype-1 (TRPV1) is closely related to defecation and absorption, and its high expression can cause intestinal damage. Constipated patients overexpress TRPV1. Glial cell-derived neurotrophic factor (GDNF) is an active protein factor that can control the growth and development of nerve cells, protect and repair damaged nerve fibers. It also has the function of regulating nerve cells. NOS is the key enzyme for producing endogenous NO. It widely exists in the gastrointestinal tract from the esophagus to the anal sphincter, and plays an important role in regulating the motility of the gastrointestinal tract.

Compared with the control group, Lactobacillus casei strain Qian high and low dose groups can increase the mRNA and protein expression levels of c-Kit, SCF, and GDNF in mice, and decrease the gene expression levels of TRPV1 and NOS in mice, as shown in Figure 7 and Figure 8. From the analysis of the results, we can see that the effect is significantly higher than that of Lactobacillus bulgaricus.

Application example 1: Lactobacillus casei strain Qian is used to produce lactic acid bacteria milk beverage.

First, the original strain of Lactobacillus casei strain Qian is stored at -75°C in the form of 30% by weight glycerin suspension, or at 4°C in the form of freeze-dried bacteria powder for future use.

Then, adopt following method to prepare Lactobacillus casei strainQian (Lactobacillus casei strainQian) working starter of the present invention:

The above-mentioned Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) original strains were inoculated into 12% by weight of skim milk sterilized at 110°C for 10 minutes, cultured at 37°C for 14-16h until the curdled milk, and continuously cultured and activated for two generations. As a mother starter; inoculate the mother starter in sterilized milk at 3-5% (volume), and cultivate for 14-16 hours until the curdled milk, at this time, the number of viable bacteria in the curdled milk is about 10 ⁹ cfu/mL , to obtain the working starter, which can be directly added to food, or used together with symbiotic commercial starters for fermented milk, such as Lactobacillus bulgaricus and Streptococcus thermophilus, to prepare fermented milk.

The second method is to inoculate the above-mentioned original strain of Lactobacillus casei strain Qian into the MRS liquid medium, and activate it by culturing it at 37°C for 12-16 hours, and activate it continuously for two generations, and then inoculate the activated culture by Inoculate 2-4% (volume) in MRS medium, culture for 16-18h, centrifuge at 4000r/min for 15min at 4°C, remove the supernatant to obtain a cell pellet, and make the pellet with a certain amount of sterile skim milk Suspension, get the working starter for later use.

Then, the raw milk was heat-sterilized at 95°C for 20 minutes or at 140°C for 2 seconds, then cooled to 4°C, and then the above-mentioned Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) working starter was added to make the concentration reach 10 ⁶ cfu/ml or more, and refrigerated storage at 4°C to obtain a lactic acid bacterium milk drink containing Lactobacillus caseistrain Qian live bacteria.

In the present invention, the MRS liquid culture medium is well known to those skilled in the art, and is a culture medium for Lactobacillus sold by Suleibao Company.

The heat sterilization is carried out, for example, using a TW10D1000 tubular sterilizer sold by Shanghai Wodi Automation Equipment Co., Ltd.

The high-temperature heat sterilization is carried out, for example, by using a YC-104 plate-type ultra-high temperature sterilizer sold by Beijing Yongchuang Jiaye Machinery Equipment Co., Ltd.

Application example 2: using Lactobacillus casei strain Qian to produce milk powder.

According to the preparation method of the above-mentioned lactic acid bacteria milk beverage, only the raw milk is heat-sterilized at 95°C for 20 minutes or at 140°C for 2 seconds, then cooled to 37°C, and then inoculated with 4% of the volume of the raw milk. said Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) working starter, then fermented at 37°C for 16h to obtain Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) fermented milk; then said Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) fermented The milk was added to the above-mentioned sterilized raw milk according to the ratio of 1:3 (V:V), homogenized, vacuum concentrated, and spray-dried to obtain milk powder containing Lactobacillus casei strain Qian.

The homogenization is, for example, carried out using a GJB500-40 small homogenizer sold by Changzhou Homogenization Machinery Co., Ltd.

The concentration is, for example, carried out using a vacuum concentration pot sold by Shanghai Weizhou Light Industry Machinery Co., Ltd.

The spray drying is carried out, for example, by using an experimental spray dryer sold by Shanghai Wodi Technology Co., Ltd.

Application example 3: utilize Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) to manufacture capsule product.

Heat the raw milk at 140°C for 2 seconds, then cool it to 37°C, inoculate the working starter of Lactobacillus casei strain Qian of the present invention with 4% of the volume of the raw milk, and ferment at 37°C for 16 hours , to obtain Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) fermented milk. Add Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) fermented milk to the sterilized raw milk at a ratio of 1:3 (V:V) to homogenize, then vacuum concentrate and spray dry to obtain milk powder, and pack the obtained milk powder into into capsules to make capsule products.

Application example 4: using Lactobacillus casei strain Qian to prepare fermented milk.

Prepare according to the preparation method of the above-mentioned lactic acid bacteria milk beverage, except that the raw milk is heat-sterilized at 95°C for 20 minutes or at 140°C for 2 seconds, then cooled to 37°C, and then added with 3-5% (volume) of the above-mentioned Lactobacillus casei strain Qian (Lactobacillus casei strain Qian) working starter, then add 3-5% (volume) symbiosis to prepare fermented milk commercial starter, after mixing, mix the bacteria and ferment at 37°C until the titrated acidity is 0.6- 0.7%, then cooled to 4°C, and then refrigerated to obtain the fermented milk.

Described commercial starter is preferably Lactobacillus bulgaricus or Streptococcus thermophilus.

In the sense of the present invention, the raw material milk is one or more raw material milks selected from skimmed milk, fresh milk and reconstituted milk.

The above is only a specific embodiment of the present invention, but the features of the present invention are not limited thereto, and any changes or modifications that can be easily imagined by anyone familiar with the technology in the field of the present invention should be covered by the following In the patent scope of the present invention.

Claims (2)

1. a kind of Lactobacillus casei Lactobacillus casei strain Qian working stock culture products, Chinese Typical Representative training Support object collection deposit number CCTCC NO:Object is made using following step in M2013514：

1) original strain of above-mentioned Lactobacillus casei is inoculated in the skimmed milk of 12% weight percent, 110 DEG C of sterilizings 10min cultivates 14-16h to curdled milk under the conditions of 37 DEG C, in continuous culture two generations of activation, is used as mother culture；By female fermentation Agent is inoculated in by 3-5% percents by volume in sterile milk, and 14-16h is to curdled milk for culture, until the viable count in the curdled milk is about 10⁹CFU/mL obtains working stock culture product；

2) be directly appended in food by 1) gained target operation leavening product or with can symbiosis the quotient for preparing acidified milk Industry leavening, which is used together, prepares fermented milk products or food.

2. Lactobacillus casei Lactobacillus casei strain Qian working stock cultures according to claim 1 Product, which is characterized in that the commercial fermentation agent is lactobacillus bulgaricus or streptococcus thermophilus.