Composite probiotics capable of synergistically metabolizing pectic polysaccharides and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a compound probiotic capable of synergistically metabolizing pectic polysaccharides and application of the compound probiotic, the compound probiotic comprises plant lactobacillus FMBL L23180CLL and lactobacillus rhamnosus FMBL L23121YWT, and the preservation numbers of the plant lactobacillus FMBL L23180CLL and the lactobacillus rhamnosus FMBL L23121YWT are CCTCC NO: M 20241793 and CCTCC NO: M 20241794 respectively; rapid proliferation and efficient metabolism of pectic polysaccharides can be realized in the only culture medium taking pectin as a carbon source, the growth of strains is promoted, the biomass is remarkably improved, and the probiotic index is also remarkably improved; in the co-culture process, a large amount of short-chain fatty acid is generated, the content of acetic acid and valeric acid generated at the end of fermentation is the highest, the acetic acid and valeric acid account for 90% or above of the total fermentation product, and the strain can be used for preparing microcapsules, compound probiotic agents, fermented milk, fruit and vegetable beverages, plant milk, probiotic milk tablets and the like and has wide application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a composite probiotic capable of synergistically metabolizing pectin polysaccharide and application thereof. Background Art

[0002] Pectin is a natural polysaccharide polymer and an important component of the cell wall of higher plants. It is widely present in the primary cell walls and middle cells of fruits, vegetables, grains, and bran, accounting for about two-thirds of the dry mass of the main cell walls of plants. It is an important component for maintaining the integrity of the cell wall structure, strength, and flexibility. It is a new type of prebiotic with antioxidant, anti-tumor, and immune-enhancing functions.

[0003] In recent years, pectin and its derivatives have been found to have little change in molecular structure when passing through the stomach and small intestinal fluid, that is, the human body cannot directly digest and utilize pectin in the stomach and small intestine, and can only be utilized in the large intestine, and it needs the help of probiotics to be utilized. Pectin that is not fully utilized may combine with certain minerals (such as calcium, iron, zinc, etc.) in the intestine to form insoluble complexes, thereby interfering with the absorption of these minerals. Long-term human mineral deficiency may occur, especially for those who are prone to mineral deficiency, such as children, pregnant women, the elderly, etc. In some cases, pectin may combine with certain drugs, affecting the absorption and bioavailability of the drugs. For example, some drugs for treating chronic diseases such as cardiovascular disease and diabetes may be affected by pectin, reducing their therapeutic effects. Therefore, screening probiotics that can ferment pectin has become a technical problem that needs to be solved urgently by those skilled in the art.

[0004] Different probiotics express different genes and produce different metabolites, and have different growth characteristics, antioxidant capacity and antibacterial properties. As the research deepens, researchers have found that a single strain has certain limitations in comprehensive probiotic performance and is difficult to produce the expected health benefits. The combination of compound probiotics provides a new idea for breaking the limitations of a single strain and improving application performance. Studies have shown that mixed probiotics are superior to single strains in inhibiting the growth of co-cultured pathogens. For example, MCFARLAND et al. found in a study on antagonizing Helicobacter pylori infection that the combination of two strains significantly alleviated the symptoms of infection compared to a single strain. Therefore, the use of multiple strains of probiotics together is the future direction of probiotic products.

[0005] The inventor unexpectedly obtained a composite probiotic with synergistic metabolism of pectin polysaccharide and its application during the research process, the composite probiotic includes Lactobacillus plantarum FMBLL23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT, the composite probiotic can achieve rapid proliferation in a culture medium with pectin as the only carbon source, efficiently metabolize pectin polysaccharide, promote the growth of strains, significantly improve biomass, and significantly improve the probiotic index. In addition, the composite strain produces a large amount of short-chain fatty acids during the co-cultivation process, and the acetic acid and valeric acid produced at the end of fermentation have the highest content, accounting for more than 90% of the total fermentation products. The amount of SCFA produced by the composite probiotics during co-culture was significantly higher than that of the single-cultured Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT, which were 1.35 and 1.48 times of the single-cultured ones, respectively. In particular, the increase in valeric acid was the most significant, which was 1.43 and 1.52 times of the single-cultured ones, respectively. It can be used to prepare microcapsules, composite probiotic agents, fermented milk, fruit and vegetable beverages, plant milk, probiotic milk tablets, etc. Summary of the invention

[0006] The primary purpose of the present invention is to provide a rhamnosus lactobacillus (Lacticaseibacillusrhamnosus) FMBL L23121 YWT, wherein the rhamnosus lactobacillus FMBL L23121 YWT is deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241794.

[0007] The second object of the present invention is to provide a composite bacterial agent that can synergistically metabolize pectin polysaccharides, wherein the composite bacterial agent includes plant lactobacillus FMBL L23180 CLL and the rhamnosus lactobacillus FMBL L23121 YWT, and the plant lactobacillus FMBL L23180 CLL is deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241793.

[0008] The third object of the present invention is to provide the use of the Lactobacillus rhamnosus FMBL L23121 YWT or the composite bacterial agent in the preparation of a functional product, wherein the functional product has one or more of the following effects:

[0009] (1) Have better ability to metabolize pectin;

[0010] (2) Use pectin to produce short-chain fatty acids.

[0011] Preferably, the functional product is one or more of food, food additives, dietary supplements, health products, medicines, feed or feed additives.

[0012] Preferably, the food is fermented food, fruit and vegetable beverage, milk tablets and the like.

[0013] The medicine can be prepared into microcapsules, tablets, powders, sustained-release preparations, etc.

[0014] The fourth object of the present invention is to provide the use of the Lactobacillus rhamnosus FMBL L23121 YWT or the composite bacterial agent in the preparation of fermented dairy products, bean products or fruit and vegetable products.

[0015] The fifth object of the present invention is to provide a synbiotic, wherein the synbiotic contains the Lactobacillus rhamnosus FMBL L23121 YWT or the composite bacterial agent.

[0016] Preferably, the synbiotics also include one or more of pectin, pectin oligosaccharides, resistant starch, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, fructo-oligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose and inulin.

[0017] The beneficial effects of the present invention are as follows: (1) The present invention provides a composite probiotic with synergistic metabolism of pectin polysaccharide and its application, wherein the composite probiotic comprises Lactobacillus plantarum FMBLL23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT;

[0018] (2) The composite probiotics can achieve rapid proliferation in a culture medium with pectin as the only carbon source, efficiently metabolize pectin polysaccharides, promote the growth of the strain, significantly increase the biomass, and significantly improve the probiotic index.

[0019] (3) The composite strain produced a large amount of short-chain fatty acids during the co-culture process, with the highest content of acetic acid and valeric acid produced at the end of fermentation, accounting for more than 90% of the total fermentation products. The amount of SCFA produced by the composite probiotics during the co-culture process was significantly higher than that of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBLL23121YWT cultured alone, which were 1.35 and 1.48 times of those cultured alone, respectively. In particular, the increase in valeric acid was the most significant, which was 1.43 and 1.52 times of those cultured alone, respectively.

[0020] (4) The composite strain can be used to prepare microcapsules, composite probiotic agents, fermented milk, fruit and vegetable beverages, plant milk, probiotic milk tablets, etc.

[0021] (5) The present invention also provides a synbiotic, which contains the Lactobacillus rhamnosus FMBL L23121YWT or the composite bacterial agent, and further contains one or more of pectin, pectin oligosaccharides, resistant starch, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, fructo-oligosaccharides, isomalto-oligosaccharides, xylo-oligosaccharides, stachyose and inulin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Phylogenetic tree of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT Figure 2 Growth curves of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT when cultured alone in medium with pectin as the carbon source.

[0023] Figure 3 Growth curves of different combinations of probiotics in culture medium with pectin as carbon source.

[0024] Figure 4 Copy number changes of strains during co-culture of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT.

[0025] Figure 5 Changes in total sugar content during single and co-fermentation of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT;

[0026] Figure 6 Composite probiotic microcapsule morphology DETAILED DESCRIPTION

[0027] The specific implementation modes of the present invention are described in detail below through several embodiments to further illustrate the present invention, but no limitation is imposed on the claims of the present invention.

[0028] The apple pectin in the following examples was purchased from MacLean's reagent.

[0029] Lactobacillus rhamnosus LGG was purchased from China General Microbiological Culture Collection Center; Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT were isolated from the intestines of school-age children in Yining County by the Food Biotechnology Center of the School of Food Science and Technology of Shihezi University and preserved in the laboratory.

[0030] It should be noted that in the following examples, the culture medium formula used is as follows:

[0031] MRS solid medium: peptone 10.0 g; beef extract 10.0 g; yeast extract 5.0 g; glucose or pectin 20.0 g; Tween 80 1.0 mL; K2HPO4 2.0 g; sodium acetate 5.0 g; diammonium hydrogen citrate 2.0 g; MgSO4·7H2O 0.58 g; MnSO4·4H2O 0.25 g, agar powder 20.0 g, deionized water 1000 mL;

[0032] MRS liquid medium: peptone 10.0 g; beef extract 10.0 g; yeast extract 5.0 g; glucose or pectin 20.0 g; Tween 80 1.0 mL; K2HPO4 2.0 g; sodium acetate 5.0 g; diammonium hydrogen citrate 2.0 g; MgSO4·7H2O 0.58 g; MnSO4·4H2O 0.25 g, deionized water 1000 mL;

[0033] Nutrient agar medium: 5.0 g peptone, 3.0 g beef extract, 5.0 g sodium chloride, 20.0 g agar powder, and 1000 mL deionized water.

[0034] PYG medium: peptone 10.0 g, yeast extract 5.0 g, glucose 1.0 g, agar powder 20.0 g, deionized water 1000 mL;

[0035] TSA medium: 15.0 g tryptone, 5.0 g soy peptone, 5.0 g sodium chloride, 20.0 g agar powder, 1000 mL deionized water;

[0036] Example 1: Isolation and identification of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT

[0037] 1. Strain Isolation and Purification

[0038] Fresh stool samples from Kazakh children in Yili Prefecture, Xinjiang and healthy adults in Wuwei, Gansu were collected and cultured under anaerobic conditions (80% nitrogen, 10% hydrogen, 10% carbon dioxide) using the gradient dilution plate method to isolate strains.

[0039] Take 1g of fecal sample and dissolve it in 9mL MRS liquid culture medium. Mix thoroughly and dilute the fecal sample to 10 -3 , 10 -4 , 10 -5 , and spread on MRS agar medium and cultured at 37℃ for 24-48 hours. According to the characteristics of colonies and cell morphology, the suspected lactic acid bacteria colonies were picked and purified three times, and the isolated strains were preserved in MRS liquid medium supplemented with 30% glycerol and stored at -20℃. Two strains were isolated and named FMBL L23180 CLL and FMBL L23121 YWT.

[0040] 2. Strain Identification

[0041] The DNA of the strain was extracted using a kit, and the groEL gene was amplified by PCR. The amplification factors and reaction conditions are shown in Table 1. After the PCR reaction, the product was subjected to 1.2% agarose gel electrophoresis to observe the molecular size, and the appropriate PCR product was selected and sent to the company for sequencing. The returned sequencing results were uploaded to the NCBI database for BLAST comparison. After the comparison, the sequence of the corresponding genus was obtained from the database, and the phylogenetic tree was established using MEGA11.0.

[0042] Table 1 PCR amplification of groEL gene

[0043]

[0044] The phylogenetic tree of the strains FMBL L23180 CLL and FMBL L23121 YWT is as follows Figure 1 The strain FMBL L23180 CLL was identified as Lactobacillus plantarum, with a Latin name of Lactiplantibacillus plantarum. It was named Lactobacillus plantarum FMBL L23180 CLL, and was deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241793. The deposit address is Wuhan University, Wuhan, China, and the telephone number is (027)-68752319.

[0045] The strain FMBL L23121 YWT is Lactobacillus rhamnosus, with a Latin name of Lactobacillus rhamnosus. It is named Lactobacillus rhamnosus FMBL L23121YWT, and was deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M20241794. The deposit address is Wuhan University, Wuhan, China, and the telephone number is (027)-68752319.

[0046] 3. Carbohydrate Utilization Capacity of Lactobacillus rhamnosus FMBL L23121 YWT

[0047] Activated Lactobacillus rhamnosus FMBL L23121 YWT was added at 2% (v / v) (OD 600 : 1.0 ± 0.05) was inoculated into the modified MRS liquid medium with prebiotics: resistant starch, chitosan, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, fructo-oligosaccharides, isomalto-oligosaccharides, xylo-oligosaccharides, stachyose, and inulin as the sole carbon source, respectively. Glucose was used as the positive control, and no carbon source was added as the negative control. The OD at 0 h was measured. 600 The absorbance of OD1 was determined by culturing at 37℃ for 24h. 600 The absorbance is OD2, and the final OD 600 =OD1-OD2, the experiment was repeated three times and the average value was taken.

[0048] Prebiotics are substances that cannot be digested and absorbed by the human body, but can stimulate or promote the proliferation of probiotics. As shown in Table 2, the results of the carbohydrate metabolism experiment showed that the plant lactobacillus FMBL L23180 CLL strain could effectively utilize 10 carbon sources, including resistant starch, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, oligofructose, oligomalto-oligosaccharides, oligoxylose, stachyose, and inulin, but had a slightly poor ability to utilize oligoxylose, and could not effectively utilize chitosan.

[0049] Table 2 Carbohydrate utilization ability of Lactobacillus rhamnosus FMBL L23121 YWT

[0050] soy oligosaccharides 1.156 Resistant Starch 0.758 Maltodextrin 1.375 Raffinose 1.509 Xylo-oligosaccharide 0.465 Galacto-oligosaccharides 1.559 Fructo-oligosaccharides 1.427 Stachyose 1.507 Isomaltooligosaccharide 1.491 Inulin 0.692 Chitosan Oligosaccharide 0.185 D-Trehalose 1.515 Positive Control 1.674 Negative control 0.018

[0051] In the following examples, Lactobacillus plantarum FMBL L23180CLL is abbreviated as Lactobacillus plantarum FMBL L23180 CLL, and Lactobacillus rhamnosus FMBL L23121 YWT is abbreviated as Lactobacillus rhamnosus FMBL L23121 YWT.

[0052] Example 2 Effect of pectin on the growth of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121YWT

[0053] The strains to be tested were: Lactobacillus plantarum FMBL L23180 CLL, Lactobacillus rhamnosus FMBL L23121 YWT, and Lactobacillus rhamnosus LGG.

[0054] Lactobacillus rhamnosus FMBL L23121 YWT was deposited in China Center for Type Culture Collection on August 15, 2024, with the deposit number CCTCC NO:M 20241793;

[0055] The Lactobacillus rhamnosus FMBL L23121 YWT was deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241794.

[0056] After activation, Lactobacillus plantarum FMBL L23180 CLL, Lactobacillus rhamnosus FMBL L23121 YWT and the control strain Lactobacillus rhamnosus LGG were centrifuged to remove the supernatant, and the bacterial sludge was washed twice with sterile saline and resuspended to prepare a concentration of 1.0×10 7 cfu / mL bacterial liquid was inoculated into MRS medium with 2% pectin as carbon source at an inoculum size of 1%, fermented at 37°C for 24 h, OD600 values ​​were measured every 2 h, and growth curves of each bacteria were drawn.

[0057] Results: By Figure 2 It can be seen that Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT entered the logarithmic growth phase after 4 hours of fermentation, entered the stable phase after 12 hours, and the OD value was about 1.3 at the end of fermentation; while the OD value of Lactobacillus rhamnosus LGG was less than 0.35 at 24 hours of fermentation. The results showed that Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT can better utilize pectin and can achieve rapid proliferation in a culture medium with pectin as the only carbon source.

[0058] Example 3: Synergistic Metabolic Effect of Composite Probiotics on Pectin

[0059] (1) Screening of compound probiotic combinations

[0060] The strains to be tested are: Lactobacillus plantarum FMBL L23180 CLL, Lactobacillus plantarum FMBL L23184 CLL, Lactobacillus rhamnosus FMBL L23136 YWT, Lactobacillus rhamnosus FMBL L23121 YWT, Lactobacillus rhamnosus FMBLL23137 YWT and Lactobacillus rhamnosus LGG.

[0061] After the strain to be tested was activated, the supernatant was removed by centrifugation, and the bacterial sludge was washed twice with sterile saline and resuspended to prepare a concentration of 1.0×10 7 cfu / mL bacterial liquid was inoculated into MRS medium with 2% pectin as the carbon source at a ratio of 1:1 with an inoculum size of 1% for co-cultivation. The culture was carried out at 37°C for 24 hours. The OD600 value was measured every 2 hours, and the growth curves of each combination were drawn to screen the optimal probiotic combination.

[0062] The results are as follows Figure 3 As shown in the figure, after 24 hours of fermentation, the OD value of the co-culture system of Lactobacillus plantarum FMBL L23220 CLL and Lactobacillus rhamnosus FMBL L23121 YWT reached 1.41, which was significantly higher than the OD value of the single culture; the growth of other combinations was not significantly different from that of the single culture, and the OD values ​​were all around 1.25, which was significantly lower than the combination of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT. Therefore, this combination was selected for subsequent experiments. (2) Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT synergistically metabolize pectin

[0063] After activation, Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT were centrifuged to remove the supernatant, and the bacterial sludge was washed twice with sterile saline and resuspended to prepare a concentration of 1.0×10 7 cfu / mL bacterial solution, inoculated into MRS medium with 2% pectin as carbon source at a ratio of 1:1 with an inoculum of 1% for co-cultivation, and fermented at 37°C for 24h, with plant lactobacillus FMBL L23180 CLL and rhamnosus lactobacillus FMBLL23121YWT inoculated separately as controls. DNA was extracted, and the concentrations of the two bacteria were determined by real-time fluorescence quantitative PCR at 24h of fermentation. The reaction conditions and primer selection of real-time fluorescence quantitative PCR are shown in Tables 3 and 4.

[0064] Table 3 Real-time fluorescence quantitative PCR

[0065]

[0066] Table 4 Specific primers for different lactic acid bacteria

[0067]

[0068] Standard curve preparation: different concentrations of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YW bacterial suspension were prepared, and the strain DNA was extracted according to the instructions of the bacterial DNA extraction kit. The OD of different gradient DNA was measured by micro-nuclease analyzer. 260 The extracted DNA was put into QPCR machine to establish the standard curve relationship between Ct value and bacterial liquid concentration, and the standard curve was drawn (Table 5). After the fermentation was completed, the DNA of the fermentation broth sample was extracted, and the real-time fluorescence quantitative PCR reaction was performed according to the reflection system and reaction conditions of the standard curve. The obtained Ct value was substituted into the corresponding standard curve, and the corresponding lactobacillus concentration in the fermentation broth was calculated, and the result was expressed as CFU / mL.

[0069] Table 5 Fluorescence real-time quantitative PCR amplification standard curve

[0070]

[0071] Results: Real-time fluorescence quantitative PCR results were as follows: Figure 3 As shown in the figure, compared with single culture, the biomass of Lactobacillus plantarum FMBL L23220 CLL and Lactobacillus rhamnosus FMBL L23121 YWT increased significantly during co-culture, and the number of viable cells of Lactobacillus plantarum FMBL L23180 CLL reached 9.58×10 9 CFU / mL, which was 19.32% higher than that when cultured alone, and the viable count of Lactobacillus rhamnosus FMBL L23121 YWT was 3.83×10 9 The above results show that the biomass of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT is significantly increased during co-culture, which further shows that Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT can synergistically utilize pectin in a culture medium with pectin as a carbon source, thereby promoting each other's growth.

[0072] (3) Probiotic Index PI of Compound Probiotics

[0073] Probiotic index (PI) was calculated according to the following formula:

[0074] PI=[ODP24–ODP0–(ODC24–ODC0)]×100 / [ODG24–ODG0–(ODC24–ODC0)]

[0075] Wherein: P represents pectin, C represents no carbon source; G represents glucose, ODP0 and ODP24 are the OD values ​​at 600 nm when the carbon source is pectin at 0 h and 24 h of lactobacillus culture, respectively, and the others are similar.

[0076] Table 6 Probiotic Index PI (%)

[0077]

[0078] It can be seen from Table 6 that the PI values ​​of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT are 79.68% and 78.69, which are lower than the probiotic index of 92.18% when the two are co-cultured. Therefore, the co-culture of the two can better metabolize pectin.

[0079] Example 4: Changes in total sugar during compound probiotic fermentation of pectin

[0080] After activation, Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT were centrifuged to remove the supernatant, and the bacterial sludge was washed twice with sterile saline and resuspended to prepare a concentration of 1.0×10 7 The cfu / mL bacterial liquid was inoculated into MRS medium with 2% pectin as the carbon source at a ratio of 1:1 with an inoculum size of 1% for co-cultivation. The culture was carried out at 37°C for 24 h, and the total sugar content of the fermentation liquid was measured at 0 h, 4 h, 8 h, 12 h, 18 h, and 24 h.

[0081] The total sugar content was determined by the anthrone-sulfuric acid method. Pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of glucose standard solution into a 10 mL test tube, add water to 2 mL, add 6 mL of anthrone sulfate solution, boil in water for 15 min, take out, cool rapidly for 15 min, and measure OD 620 , draw a standard curve. Dilute the lactobacillus fermentation liquid 20 times with distilled water, determine the absorbance value of the lactobacillus fermentation liquid according to the above method, and substitute it into the standard curve to calculate the content of total sugar.

[0082] Results: The change of total sugar is an important indicator to characterize the ability of microorganisms to utilize carbon sources in the fermentation system. Figure 5It can be seen that during the fermentation process, total sugar and reducing sugar both decreased rapidly within 4-18 hours. There was no significant difference in the utilization efficiency of pectin between Lactobacillus plantarum FMBLL23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT cultured alone, and the total sugar consumption rate was about 73%; during the co-culture process, the reduction rate of total sugar and reducing sugar was significantly higher than that of the strains cultured alone, and the utilization efficiency of pectin by the composite probiotics was significantly increased, and the total sugar consumption rate reached nearly 85%; the above results also showed that there was a synergistic effect in the co-culture process of the composite strains of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT, which could promote each other's growth and promote the metabolic capacity of pectin.

[0083] Example 5: Composite probiotics produce short-chain fatty acids

[0084] Gas chromatography was used to analyze the production of short-chain fatty acids (SCFA) by Lactobacillus plantarum FMBL L23180 CLL, Lactobacillus rhamnosus FMBL L23121 YWT and their composite probiotics using pectin during fermentation. The fermentation broth was centrifuged at 10000r / min for 15min to remove cells and impurities, and then filtered through a 0.22μm filter membrane before loading. Gas chromatography analysis was performed on an Agilent 7890B gas chromatograph.

[0085] GC measurement conditions: DB-WAX analytical column (60m×250μm×0.25μm) was used for measurement. Inlet temperature: 250℃; Column box conditions: starting temperature 100℃ for 30s, increasing by 8℃ per minute, maintaining at 160℃ for 1min, then increasing the temperature at 20℃ / min, and maintaining at 200℃ for 5min; injection volume 1μL; carrier gas conditions: N2, flow rate 1.2mL / min, split ratio 2:1; FID detector conditions: temperature 250℃, tail gas: N2 (25mL / min), H2 (30mL / min), air (400mL / min).

[0086] Table 7 SCFA content produced during the fermentation of pectin by compound probiotics

[0087]

[0088] Results: As shown in Table 7, a large amount of short-chain fatty acids (SCFA) were produced during the fermentation of pectin by Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT. SCFA is an important substance that inhibits pathogenic microorganisms in the intestine, and is very important for regulating the ecological balance of intestinal microorganisms in the body and promoting intestinal health. The results showed that the content of acetic acid and valeric acid produced at the end of fermentation was the highest, accounting for more than 90% of the total fermentation products. The amount of SCFA produced by the composite probiotics fermentation of pectin during the co-culture process was significantly higher than that of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT cultured alone, which were 1.35 and 1.48 times of those cultured alone, respectively, especially the increase of valeric acid was the most significant, which was 1.43 and 1.52 times of those cultured alone, respectively.

[0089] Application Example 1: Preparation of microcapsules and capsule products containing the composite bacterial agent of the present invention

[0090] The plant lactobacillus FMBL L23180 CLL and the rhamnosus lactobacillus FMBL L23121 YWT of the present invention were cultured in MRS medium for 24 hours, centrifuged at 4°C and 8000 r / min for 10 minutes, the cells were collected and washed twice with sterile saline, and the cells were resuspended with sterile saline to a final cell concentration of 1×10 10 CFU / mL. After the two bacterial suspensions are mixed, an equal volume of 1.5% (w / v) galacto-oligosaccharide solution and 5 times the volume of 3.0% (w / v) pectin solution are added in sequence, and the mixture is stirred and mixed thoroughly. The mixed solution is dripped into a solidifying liquid of 0.8 mol / L calcium chloride by extrusion to form micelles. After solidification for 30 minutes, the micelles are added into a chitosan solution of pH 5.0 and a concentration of 0.5% (w / v) to solidify for 20 minutes. The micelles are collected by filtration, and the collected micelles are freeze-dried to obtain microcapsules containing the composite probiotics of FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT of the present invention ( Figure 6 ), microcapsule particle size 1.93-2.11 mm, embedding rate ≥ 92.83%, strain survival rate ≥ 89%, with the characteristics of tolerance to simulated gastrointestinal fluid and heat stress. The prepared microcapsules are loaded into the medicinal capsules currently sold on the market to obtain the capsule product.

[0091] Application Example 2: Preparation of composite probiotics

[0092] Preparation of culture medium: Use water and culture medium raw materials to prepare a medium containing 20 g / L of glucose, 8 g / L of beef extract, 10 g / L of yeast extract, 9 g / L of NaCl, 1 mL / L of Tween 80, 2 g / L of K2HPO4, 5 g / L of sodium acetate, 2 g of diammonium hydrogen citrate, 0.58 g of MgSO4·7H2O, and 0.25 g of MnSO4·4H2O, and adjust the pH to 6.8 to obtain the culture medium.

[0093] Preparation of protective agent: Use water and protective agent raw materials to prepare a protective agent containing 120 g / L skim milk powder, 20 mL / L glycerol, 22 g / L maltodextrin, 60 g / L trehalose, and 22 g / L galacto-oligosaccharide.

[0094] Plant lactobacillus FMBL L23180 CLL and rhamnosus lactobacillus FMBL L23121 YWT were inoculated in a ratio of 1:1 with a 2% inoculum into the above-mentioned culture medium sterilized at 115°C for 15 minutes; the culture medium contained 20 g / L glucose, 8 g / L beef extract, 10 g / L yeast extract, 9 g / L NaCl, 1 mL / L Tween 80, 2 g / L K2HPO4, 5 g / L sodium acetate, 2 g diammonium hydrogen citrate, 0.58 g MgSO4·7H2O, and 0.25 g MnSO4·4H2O, and the pH was adjusted to 6.8. The culture was cultured at 37°C for 24 hours, washed twice with pH 7.2 PBS buffer, and then resuspended with a protective agent to a concentration of 10 11 CFU / mL. The protective agent contains 120g / L skim milk powder, 20mL / L glycerol, 22g / L maltodextrin, 60g / L trehalose, and 22g / L galacto-oligosaccharide. Then, the suspension is pre-cultured at a temperature of 37°C for 60min, and then freeze-dried to obtain the composite probiotic agent.

[0095] Application Example 3: Preparation of fermented milk using the composite probiotic agent of the present invention

[0096] After sugar is dissolved in fresh milk, it is homogenized at 60°C and 20MPa, and then sterilized at 90-95°C for 5-8min. When the temperature drops to 35°C, a mixed bacteria consisting of the composite probiotic agent of the present invention, commercial dry powder fermentation agent Lactobacillus bulgaricus and thermophilic streptococcus is added, and the mass ratio of the mixed bacteria is 1:1:1. The inoculation amount of the mixed bacteria is 0.03-2.0% of the weight of the fresh milk. The mixture is mixed, and the mixture is fermented at 37°C. After the milk is coagulated, the mixture is refrigerated at 4°C for 16h to obtain the fermented milk.

[0097] Application Example 4: Preparation of Fruit and Vegetable Beverages Containing Composite Probiotics

[0098] Fresh fruits and vegetables are selected and washed, and then the juice is squeezed, and then the juice is sterilized at high temperature, and then the temperature is immediately cooled to about 37°C, and the composite probiotic agent prepared by the present invention is added to make the concentration reach 10 6 CFU / mL or more, and refrigerated storage at a temperature of 4°C to obtain a fruit and vegetable beverage containing the live bacteria of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT of the present invention.

[0099] Application Example 5: Plant milk prepared using the composite probiotics of the present invention

[0100] Soybeans, almonds, and other raw materials are soaked at 80°C for 2 hours, peeled, and the soaking water is drained, and the pulp is ground with boiling water and kept at 80-85°C for 12-15 minutes. The obtained slurry is filtered with a 200-mesh screen to remove the residue and then boiled. The supernatant obtained after the solids are removed by centrifugation is cooled to about 37°C, and the composite probiotic agent of the present invention is added to make its concentration reach 10 6 CFU / mL or more, and stored in a refrigerator at a temperature of 4°C to obtain the plant milk containing the live bacteria of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT of the present invention.

[0101] Application Example 6: Preparation of probiotic milk tablets using the composite probiotics of the present invention

[0102] 5.96 parts by weight of the composite probiotic agent of the present invention prepared by a freeze-drying method, 65.0 parts by weight of whole milk powder, 7.92 parts by weight of white granulated sugar, 2.5 parts by weight of magnesium stearate, and 18.0 parts by weight of skim milk powder were respectively weighed, mixed with 1.0 part by weight of water, prepared into wet granules by a conventional method, then tabletted using a tablet press, dried in a dryer, and packaged to obtain the probiotic milk tablets of the present invention.

[0103] In summary, the present invention provides a composite probiotic with synergistic metabolism of pectin polysaccharide and its application, wherein the composite probiotic comprises Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT; the composite probiotic can realize rapid proliferation in a culture medium with pectin as the only carbon source, efficiently metabolize pectin polysaccharide, promote the growth of strains, significantly increase biomass, and significantly improve the probiotic index. During the co-cultivation process, the composite strain produces a large amount of short-chain fatty acids, and the acetic acid and valeric acid produced at the end of fermentation have the highest content, accounting for more than 90% of the total fermentation products. The amount of SCFA produced by the composite probiotics during co-culture is significantly higher than that of plant lactobacillus FMBLL23180 CLL and rhamnosus lactobacillus FMBL L23121 YWT cultured alone, which are 1.35 and 1.48 times of those cultured alone, respectively, and the increase in valeric acid is particularly significant, which is 1.43 and 1.52 times of those cultured alone. The composite strain can be used to prepare microcapsules, composite probiotic agents, fermented milk, fruit and vegetable beverages, plant milk, probiotic milk tablets, etc. The present invention also provides a synbiotic, which contains the rhamnosus lactobacillus FMBL L23121 YWT or the composite agent, and the synbiotic also includes one or more of pectin, pectin oligosaccharides, resistant starch, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, fructo-oligosaccharides, isomalto-oligosaccharides, xylo-oligosaccharides, stachyose, and inulin.

Claims

1. A Lactobacillus rhamnosus FMBL L23121 YWT, characterized in that: The Lactobacillus rhamnosus FMBL L23121 YWT was deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241794.

2. A composite bacterial agent capable of synergistically metabolizing pectin polysaccharides, characterized in that: The composite bacterial agent comprises Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT according to claim 1, and the Lactobacillus plantarum FMBL L23180 CLL was deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241793.

3. The use of Lactobacillus rhamnosus FMBL L23121 YWT as claimed in claim 1 or the composite bacterial agent as claimed in claim 2 in the preparation of functional products, characterized in that: The functional product has one or more of the following functions: (1) Have better ability to metabolize pectin; (2) Use pectin to produce short-chain fatty acids.

4. The use according to claim 3, characterized in that The functional product is one or more of food, food additives, dietary supplements, health products, medicines, feed or feed additives.

5. Use of the Lactobacillus rhamnosus FMBL L23121 YWT according to claim 1 or the composite bacterial agent according to claim 2 in the preparation of fermented dairy products, bean products or fruit and vegetable products.

6. A synbiotic, characterized in that: The synbiotic contains the Lactobacillus rhamnosus FMBL L23121 YWT according to claim 1 or the composite bacterial agent according to claim 2.

7. The synbiotic according to claim 6, characterized in that The synbiotics also include one or more of pectin, pectin oligosaccharides, resistant starch, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, fructo-oligosaccharides, isomalto-oligosaccharides, xylooligosaccharides, stachyose and inulin.

Images