Preparation of compound probiotic fermented milk and application of compound probiotic fermented milk in relieving constipation

Abstract

The invention discloses preparation of compound probiotic fermented milk and application of the compound probiotic fermented milk in relieving constipation, and belongs to the technical field of food. The preparation method of the compound probiotic fermented milk is as follows: the probiotic combination ratio of lactobacillus paracasei JY062 to lactobacillus gasseri JM1 is 1: 2 (viable count ratio), the fermentation temperature is 7.9 hours, the fermentation time is 39.5 DEG C, and the sugar adding amount is 7.5%. According to the preparation method, the serious post-acidification phenomenon of the lactobacillus paracasei JY062 fermented milk is relieved, and the low viable count phenomenon of the lactobacillus gasseri JM1 fermented milk is improved. The compound probiotic fermented milk can obviously improve the gastric emptying rate, the small intestine propulsion rate and the excrement water content of mice, obviously weaken the tissue damage degree, and improve gastrointestinal regulatory peptides, neurotransmitters and the like. The constipation relieving function of the compound probiotic fermented milk is superior to that of unfermented milk and positive control fermented milk, and the compound probiotic fermented milk has a certain application prospect.

Description

Technical Field

[0001] The invention relates to preparation of composite probiotic fermented milk and application of the composite probiotic fermented milk in alleviating constipation, belonging to the technical field of food. Background Art

[0002] Constipation is a common functional gastrointestinal disease, and its clinical symptoms include difficulty or less defecation, prolonged gastrointestinal emptying time, hard stools, and abdominal discomfort. Epidemiological surveys show that the global incidence of chronic constipation is between 14% and 30%. Constipation can be divided into three categories: normal transit constipation, slow transit constipation, and defecation disorder or rectal defecation disorder. Among them, slow transit constipation is considered to be the main type of disease that leads to reduced colon transit rate, accounting for 30% of the total incidence of constipation. Slow transit constipation is caused by dysfunction of colon smooth muscle or nerve innervation, resulting in abnormal neurocolonic motility, which is closely related to the disorder or defect of colon propulsion movement pattern. Constipation can be relieved by a variety of drugs, such as laxatives and prokinetic drugs. However, the onset of the disease in patients may be caused by a variety of inducements, and there are overlapping effects of multiple factors, which leads to clinical limitations such as low response and frequent recurrence after drug discontinuation in drug therapy. At the same time, drug therapy can also cause side effects of different forms and degrees, such as headache, nausea and diarrhea.

[0003] At present, many studies have confirmed that intestinal function can be regulated and constipation symptoms can be improved through dietary intervention without side effects. Among them, probiotic fermented milk plays an important role in regulating gastrointestinal function. Regular intake of probiotic fermented milk can improve intestinal flora and relieve constipation symptoms. Studies have shown that probiotic fermented milk containing L. plantarum ST-III can regulate the levels of SCFAs and secretory immunoglobulin A in patients' feces and the distribution of intestinal flora, improve human gastrointestinal health, and relieve functional constipation symptoms. At the same time, many studies have found that regular consumption of probiotic fermented milk containing Lactococcus lactis subsp.cremoris FC, probiotic fermented milk containing B. lactis GCL2505, and probiotic fermented milk containing Lactobacillus acidophilus NCFM and Bifidobacterium lactis HN019 can regulate gastrointestinal function and improve constipation symptoms in terms of shortening colon transit time, improving intestinal peristalsis and fecal characteristics, and regulating intestinal flora structure. Probiotic fermented milk plays a pivotal role in relieving gastrointestinal diseases such as constipation, further providing direction for exploring alternative treatments that do not involve prescription drugs.

[0004] Fermented milk is a good carrier for probiotics, providing sufficient nutrients for the growth and metabolism of strains. The good fermentation characteristics of probiotics are the basis for their application in milk fermentation. Probiotic fermented milk contains a large number of active probiotics, which is conducive to maintaining and improving the safety and nutritional value of fermented dairy products. When sufficient probiotics (more than 10 6 CFU / mL), the fermented milk can promote the health of the body and produce probiotic functions. In addition, probiotic fermented milk also contains functional ingredients such as high-quality protein, amino acids, minerals, niacin, vitamin B12 and riboflavin, which play a certain functional role. Summary of the invention

[0005] The present invention prepares a composite probiotic fermented milk containing Lactobacillus paracasei JY062 and Lactobacillus gasseri JM1, and studies its application in relieving constipation. The present invention provides a probiotic combination with independent intellectual property rights for the fermented milk industry, and provides a reasonable and powerful basis for the development of new functional products and intervention strategies for regulating constipation.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A method for preparing composite probiotic fermented milk comprises the following steps:

[0008] Step 1: Activation and preservation of strains. L. paracasei JY062 and L. gasseri JM1 were inoculated at a 5% (v / v) inoculation rate into MRS broth medium, and cultured anaerobically at 37°C for 20 hours to activate the two strains. Use an inoculation loop to dip the strain culture solution into MRS agar plates, cultured anaerobically at 37°C for 48 hours, pick single colonies and cultured anaerobically at 37°C in sterile MRS broth medium for 20 hours, and use part of the bacterial solution for strain identification. The remaining bacterial solution was mixed with 80% (v / v) glycerol at a ratio of 3:1 and frozen for preservation. After the identification results were correct, it was used for subsequent experiments. The method for preparing bacterial powder is as follows: (1) Strain activation: The frozen bacterial solution was inoculated into MRS broth medium, and the activation method was the same as above to obtain the first-generation bacterial solution. (2) Strain propagation: Inoculate the first-generation bacterial solution at a 5% (v / v) inoculation volume into MRS broth medium, and culture anaerobically at 37°C for 20 hours to obtain the second-generation bacterial solution. (3) Expansion culture: Inoculate the second-generation bacterial solution at a 5% (v / v) inoculation volume into 12% (w / w) skim milk powder medium, and culture anaerobically at 37°C for 16 hours. (4) Collect bacteria: Centrifuge at 5000g for 5 minutes to harvest the bacteria. (5) Pre-freeze and freeze-dry, collect freeze-dried powder for subsequent fermentation tests.

[0009] Step 2: Processing of fermentation raw materials. Commercially available fresh milk was filtered and standardized at 15-25 MPa, sterilized at 85-95°C for 4-7 min, and rapidly cooled to 35-37°C. The number of viable bacteria inoculated was 1×10 7 CFU / mL.

[0010] Step 3: Optimize fermentation conditions. The acidity and sensory properties of fermented milk are affected by three main factors: fermentation time, fermentation temperature, and sugar addition. Through single-factor experiments, the strain ratio of compound fermented milk was determined to be L. paracasei JY062: L. gasseri JM1 = 1:2, fermentation time 7.9h, fermentation temperature 39.5℃, and sugar addition 7.5%.

[0011] Step 4: Preparation of fermented milk: The fermentation raw materials obtained in step 2 are fermented according to the fermentation conditions obtained in step 3 to obtain compound probiotic fermented milk.

[0012] The beneficial effects achieved by the present invention are:

[0013] 1. The compound fermentation combination of Lactobacillus paracasei JY062 and Lactobacillus gasseri JM1 alleviated the serious post-acidification phenomenon of Lactobacillus paracasei JY062 fermented milk (FMJY), and improved the low viable count phenomenon of Lactobacillus gasseri JM1 fermented milk (FMJM).

[0014] 2. In the study on the effect of compound probiotic fermented milk (FMMIX) of Lactobacillus paracasei JY062 and Lactobacillus gasseri JM1 on relieving constipation, the constipation-relieving function of FMMIX was better than that of unfermented milk (UFM) and positive control fermented milk, and it has certain application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The pH changes during the fermentation of single strains and composite strains with different proportions;

[0016] Figure 2 The number of live bacteria in fermented milk at the end point of fermentation and during storage of single strain and composite strains in different proportions;

[0017] Figure 3 The acidification of fermented milk with single strain and different proportions of compound probiotics after storage;

[0018] Figure 4 The effect of compound probiotic fermented milk on gastric emptying rate in mice;

[0019] Figure 5 The effect of compound probiotic fermented milk on the small intestinal propulsion rate of mice;

[0020] Figure 6 To study the effect of compound probiotic fermented milk on the water content of mouse feces;

[0021] Figure 7 HE staining of mouse gastric antrum, small intestine and colon tissue (200×);

[0022] Figure 8 is the serum MTL content of mice;

[0023] Fig. 9 is the serum GAS content of mice;

[0024] Fig.10 PYY content in mouse serum

[0025] Fig.11 is the serum VIP content of mice;

[0026] Fig.12 is the serum 5-HT content of mice;

[0027] Fig.13 is the serum NO content of mice;

[0028] Fig.14 Effect of compound probiotic fermented milk on ICC distribution, a) ICC distribution (200×), b) optical density value of c-kit protein;

[0029] Fig.15 Expression levels of c-kit and SCF proteins in mice, a) c-kit and SCF band images, b) c-kit and SCF protein expression levels; DETAILED DESCRIPTION

[0030] Combine the following Figure 1-Figure 15 The present invention is further described in the following examples, which are only used to more clearly illustrate the technical solution of the present invention and are not intended to limit the protection scope of the present invention.

[0031] Step 1: Activation and preservation of strains. L. paracasei JY062 and L. gasseri JM1 were inoculated at a 5% (v / v) inoculation rate into MRS broth medium, and cultured anaerobically at 37°C for 20 hours to activate the two strains. Use an inoculation loop to dip the strain culture solution into MRS agar plates, cultured anaerobically at 37°C for 48 hours, pick single colonies and cultured anaerobically at 37°C in sterile MRS broth medium for 20 hours, and use part of the bacterial solution for strain identification. The remaining bacterial solution was mixed with 80% (v / v) glycerol at a ratio of 3:1 and frozen for preservation. After the identification results were correct, it was used for subsequent experiments. The method for preparing bacterial powder is as follows: (1) Strain activation: The frozen bacterial solution was inoculated into MRS broth medium, and the activation method was the same as above to obtain the first-generation bacterial solution. (2) Strain propagation: Inoculate the first-generation bacterial solution at a 5% (v / v) inoculation volume into MRS broth medium, and culture anaerobically at 37°C for 20 hours to obtain the second-generation bacterial solution. (3) Expansion culture: Inoculate the second-generation bacterial solution at a 5% (v / v) inoculation volume into 12% (w / w) skim milk powder medium, and culture anaerobically at 37°C for 16 hours. (4) Collect bacteria: Centrifuge at 5000g for 5 minutes to harvest the bacteria. (5) Pre-freeze and freeze-dry, collect freeze-dried powder for subsequent fermentation tests.

[0032] Step 2: Processing of fermentation raw materials. Commercially available fresh milk was filtered and standardized at 15-25 MPa, sterilized at 85-95°C for 4-7 min, and rapidly cooled to 35-37°C. The number of viable bacteria inoculated was 1×10 7 CFU / mL.

[0033] Step 3: Optimize fermentation conditions. The acidity and sensory properties of fermented milk are affected by three main factors: fermentation time, fermentation temperature, and sugar addition. Through single-factor experiments, the strain ratio of compound fermented milk was determined to be L. paracasei JY062: L. gasseri JM1 = 1:2, fermentation time 7.9h, fermentation temperature 39.5℃, and sugar addition 7.5%.

[0034] Step 4: Preparation of fermented milk: The fermentation raw materials obtained in step 2 are fermented according to the fermentation conditions obtained in step 3 to obtain compound probiotic fermented milk.

[0035] 55 male KM mice (6-7 weeks old) were selected and placed in cages at room temperature of 22±2℃ and humidity of 55±5%, and light / dark cycle was performed every 12h. In the first week, the mice were fed adaptively and had free access to food and water. After 7 days, the mice were divided into NC group, M group, UFM group, FMMIX group and positive control fermented milk (PCFM) group, with 11 mice in each group.

[0036] Table 1 Grouping and gavage conditions of animal experiments

[0037]

[0038] The NC group was continuously gavaged with an equal amount of PBS. After the modeling was completed, three mice were randomly selected from each group, fasted for 24 hours, gavaged with 0.5 mL of ink, and killed 30 minutes later. The defecation of the mice was observed, and the gastric emptying rate, small intestinal propulsion rate and fecal water content were measured. The significant difference between each modeling group and the NC group indicated that the modeling was successful.

[0039] Example 1: The NC group was continuously gavaged with an equal amount of PBS.

[0040] Example 2: Group M was intragastrically administered with 5 mg / (kg·d) loperamide for 7 days, and then intragastrically administered with 10 mL / (kg·d) PBS for 14 days.

[0041] Example 3: The UFM group was intragastrically administered with 5 mg / (kg·d) loperamide for 7 days, and then intragastrically administered with 10 mL / (kg·d) UFM for 14 days.

[0042] Example 4: The FMMIX group was intragastrically administered with 5 mg / (kg·d) loperamide for 7 days, and then intragastrically administered with 10 mL / (kg·d) FMMIX for 14 days.

[0043] Example 5: The PCFM group was intragastrically administered with 5 mg / (kg·d) loperamide for 7 days, and then intragastrically administered with 10 mL / (kg·d) PCFM for 14 days.

[0044] The above five groups of mice were tested for indicators

[0045] Determination of biochemical indicators in mice

[0046] The mice were fasted for 24 hours before being killed, and then gavaged with 0.5 mL of ink and killed 30 minutes later. The specific measurement method is as follows: (1) Weigh the ink weight (a1) and the total weight of the stomach (a2). Wash the stomach contents, dry them and weigh the net weight of the stomach (a3). (2) Use a ruler to determine the distance from the pylorus to the front end of the ink (L1) and the distance from the pylorus to the ileocecal area (L2). (3) Use low-temperature freeze-drying method to determine the moisture content of feces. Weigh the weight of the plate (b1), the total weight of the plate and feces before freeze-drying (b2), and the total weight of the plate and feces after freeze-drying (b3).

[0047]

[0048] Hematoxylin-eosin staining

[0049] Fresh mouse gastric antrum, small intestine and colon tissues were collected, and 0.5×0.5×0.5cm3 of animal tissues were fixed in 10% neutral formaldehyde solution. After fixation, the fixative was washed away, dehydrated with different concentrations of ethanol, and the alcohol was replaced by the transparent agent xylene, and then embedded in paraffin and sliced. After removing the paraffin, washing with different concentrations of ethanol and distilled water, and staining with hematoxylin-eosin (HE), the histological characteristics of the mice were analyzed using an optical microscope.

[0050] Mouse serum parameter determination

[0051] The levels of MTL, GAS, PYY, VIP and 5-HT in mouse serum were detected by ELISA kits, and the NO content in mouse serum was detected by NO content detection kit.

[0052] Immunohistochemistry

[0053] Colon tissue sections were dewaxed in xylene, antigens were recovered with citric acid antigen recovery solution, cooled to room temperature, blocked with goat serum for 1 hour, incubated with c-kit antibody at 4°C overnight, and labeled with biotin secondary antibody, incubated at room temperature for 1 hour, and incubated with horseradish enzyme-labeled avidin at 37°C for 30 minutes. After diaminobenzidine staining, photos were collected in a 200x field of view. Image-ProPlus 6.0 software was used to select the same brown-yellow color as the unified standard for judging positivity, analyze the positive cumulative optical density value (IOD) and tissue pixel area (AREA) of the photos, and calculate the mean optical density value IOD / AREA (Mean Density).

[0054] Western Blot Analysis

[0055] The expression of c-kit and SCF proteins in colon tissue was determined by WB. First, the total protein was extracted, and the tissue blocks rinsed with pre-cooled PBS buffer were cut into small pieces and placed in a homogenizer. 1 mL of tissue protein lysis buffer (protease inhibitors were added within a few minutes before use) was added to obtain a homogenate in an ice bath. The homogenate was transferred to a centrifuge tube and oscillated. After an ice bath for 30 minutes, centrifuged at 13000g for 5 minutes at 4°C, and the supernatant was collected, which was the total protein solution. The protein concentration was determined using a BCA protein assay kit. The protein sample (40 μg) was separated using 10% SDS-PAGE. The concentrated gel (50V) and the separation gel (120V) were prepared in an electrophoresis tank, and electrophoresis buffer was added. The sample was added to the spotting well and constant voltage electrophoresis was performed until bromophenol blue reached the bottom edge of the gel plate. The transferred membrane was blocked with TBST (5% skim milk and 0.1% Tween-20 in Tris buffer solution) at room temperature for 1 hour. GAPDH was used as an internal reference. The membrane was incubated overnight at 4°C with the primary antibody (1:1000). After washing 3 times with TBST, the secondary antibody was added at 1:5000, incubated at room temperature for 30 minutes, and then washed 4 times with TBST. Freshly prepared ECL mixed solution was added dropwise to the protein side of the membrane and exposed in a dark room. The optical density value of the target band was analyzed using the Image-pro Plus6.0 software processing system.

[0056] Effects of FMMIX on Physiological Indexes of Mice

[0057] The results of the dietary intervention test were as follows Figure 2 As shown in the figure, compared with the NC group, the gastric emptying rate, small intestinal propulsion rate and fecal water content of mice in the M group were significantly reduced (P<0.05), indicating that the modeling effect of loperamide-induced constipation mice was significant. Compared with the M group, the above three indicators of mice intervened by UFM, FMMIX and PCFM were significantly increased (P<0.05), among which there was no significant difference between the two groups of mice intervened by FMMIX and PCFM (P>0.05), but the improvement effect of FMMIX was the most obvious. At the same time, the fecal water content of mice in the FMMIX group can reach the level of the NC group, and there is no significant difference between the two groups (P>0.05).

[0058] Effects of FMMIX on histopathological analysis in mice

[0059] HE staining showed that ( Figure 3), the gastric antrum tissue structure of mice in the M group was disordered, epithelial cells eroded and fell off (red arrows), a large number of closely arranged tubular glands were visible in the lamina propria, the glands were arranged in a disordered manner (black arrows), and a small amount of inflammatory cell infiltration was visible (blue arrows); the small intestinal villi were loosely arranged, shortened in length, and disordered in structure. Some epithelial cells in the mucosal layer fell off, the lamina propria was exposed, and the crypt structure of the mucosal layer partially disappeared, accompanied by a large number of inflammatory cell infiltrations; the crypt structure of the colon mucosal layer partially disappeared, accompanied by focal infiltration of inflammatory cells, and there were visible goblet cells (green arrows). After intervention, UFM, FMMIX, and PCFM could alleviate the degree of tissue damage in the gastric antrum, small intestine, and colon of mice. In addition, there was still visible cell necrosis in the small intestine of the UFM group (yellow arrows), but it was alleviated after the intervention of FMMIX and PCFM. In addition, the degree of tissue damage in mice in the FMMIX and PCFM groups was significantly weakened, and the degree of relief was better than that in the UFM group.

[0060] Effects of FMMIX on gastrointestinal regulatory peptides and neurotransmitters in mice

[0061] Excitatory gastrointestinal regulatory peptides (MTL and GAS) and inhibitory gastrointestinal regulatory peptides (PYY and VIP) play an important role in the regulation of gastrointestinal functional diseases. Figure 4 It can be seen that compared with the NC group, the levels of serum MTL and GAS in mice in the M group were significantly reduced (P<0.05). After dietary intervention, the levels of MTL and GAS in mice in the UFM, FMMIX and PCFM groups increased significantly, especially the levels in the FMMIX group were significantly higher than those in the UFM and PCFM groups (P<0.05), and there was no significant difference with the NC group (P>0.05). The levels of serum PYY and VIP in mice in the M group were significantly increased compared with the NC group. After the intervention of FMMIX, the levels of PYY and VIP in mice were significantly reduced (P<0.05). Although there was no significant difference with the PCFM group, the VIP level of mice in the FMMIX group could reach the level of the NC group (P>0.05). At the same time, the PYY level of mice in the UFM group was lower than that of mice in the FMMIX group, but there was no significant difference (P>0.05). In addition, the content of excitatory neurotransmitter 5-HT in mice in the M group was significantly reduced, while the content of inhibitory neurotransmitter NO was significantly increased (P<0.05). After dietary intervention, their contents were improved, and the effect was most obvious in the FMMIX group. Therefore, the intervention of UFM, FMMIX and PCFM all had a certain improvement effect on the above-mentioned gastrointestinal regulatory peptides and neurotransmitters, and the improvement effect after FMMIX intervention was more obvious in general.

[0062] Effect of FMMIX on the distribution of ICC in mice

[0063] The c-kit antibody was used to label the c-kit on the cell membrane of ICCs to observe the distribution of ICCs and semi-quantitatively determine the number of ICCs ( Figure 5 ). Brown-yellow indicates positive labeling, and the darker the color, the higher the degree of positivity. The colon staining of mice in the M group became significantly lighter, while after FMMIX intervention, the colon staining of mice deepened, indicating that c-kit labeling increased significantly, indicating that the number of ICCs in the colon of mice increased. The average optical density value of mice in the M group was significantly lower than that of the NC group, and significantly increased after dietary intervention (P<0.05), especially the FMMIX group. The most significant increase was consistent with the results of staining labeling.

[0064] Effect of FMMIX on the expression of c-kit / SCF protein in mice

[0065] Depend on Figure 6 It can be seen that compared with the NC group (0.91±0.022 and 0.84±0.039), the expression levels of c-kit and SCF proteins in mice in the M group were significantly reduced (P<0.05), 0.11±0.024 and 0.15±0.033, respectively, with specific decrease rates of 87.4% and 82.5%, respectively. Compared with the M group, the expression levels of c-kit and SCF proteins in mice in the dietary intervention group increased significantly (P<0.05), among which the increase in the FMMIX group was the most obvious, 0.56±0.038 and 0.59±0.051, respectively, with growth rates of 390.0% and 305.9%, respectively. At the same time, the c-kit and SCF protein levels of mice in the FMMIX group were significantly higher than those in the PCFM group (P<0.05). However, there was no significant difference in the expression of c-kit protein in mice in the UFM group compared with the M group (P>0.05), which was consistent with the above immunohistochemical results.

[0066] In summary, in the study on the combined probiotics (FMMIX) of Lactobacillus paracasei JY062 and Lactobacillus gasseri JM1 for relieving constipation, the constipation-relieving function of FMMIX was better than that of unfermented milk (UFM) and positive control fermented milk, and it has certain application prospects.

Claims

1. A preparation method of compound probiotic fermented milk, the preparation method of which is to filter and standardize commercially available fresh milk at 15-25 MPa, sterilize at 85-95°C for 4-7 minutes, and quickly cool to 35-37°C, and inoculate the viable bacteria count to 1×10 7 CFU / mL, wherein the probiotic combination ratio is Lactobacillus paracasei JY062: Lactobacillus gasseri JM1=1:2 (ratio of viable bacteria count), fermentation temperature is 7.9h, fermentation time is 39.5℃, and sugar addition amount is 7.5%.

2. The composite probiotic fermented milk obtained by the preparation method according to claim 1 has an application prospect of alleviating constipation.

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