A complex probiotic capable of synergistically metabolizing pectic polysaccharides and application thereof

Abstract

The present application relates to the field of biotechnology, in particular to a kind of complex probiotic bacteria capable of synergistic metabolism pectin polysaccharide and application thereof, the complex bacterial agent includes plant lactobacillus FMBL L23180CLL and rhamnose lactobacillus FMBL L23121YWT, preservation number is CCTCC NO:M 20241793 and CCTCC NO:M 20241794 respectively;Can be realized in the medium of pectin as carbon source only rapid proliferation, high-efficiency metabolism pectin polysaccharide, promote the growth of strain, biomass is significantly improved, and probiotic index is also significantly improved;In the process of co-culture, a large amount of short-chain fatty acid is produced, and the content of acetic acid and valeric acid produced at the end of fermentation is the highest, more than 90% of total fermentation product, can be used for preparing microcapsule, complex probiotic bacterial agent, fermented milk, fruit and vegetable beverage, plant milk, probiotic milk tablet and the like, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a complex probiotic capable of synergistically metabolizing pectin polysaccharide and application thereof. BACKGROUND

[0002] Pectin is a natural polysaccharide polymer, which is an important component of the cell wall of higher plants, and widely exists in the primary cell wall and middle layer cells of fruits, vegetables, cereals and bran, accounting for about two-thirds of the dry mass of the main cell wall of plants, and is an important component for maintaining the integrity, strength and flexibility of the cell wall. Pectin is a new prebiotic, which has functions of antioxidant, anti-tumor, immune enhancement and the like.

[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 intestine fluid, that is, the human body cannot directly digest and utilize pectin in the stomach and small intestine, and only in the large intestine can pectin be utilized with the help of probiotics. The pectin that is not completely utilized in the intestinal tract may combine with certain minerals (such as calcium, iron, zinc, etc.) to form insoluble complexes, thereby interfering with the absorption of these minerals. Long-term may lead to mineral deficiency in the human body, 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 diseases, diabetes, etc. may be affected by pectin, reducing their therapeutic effect. Therefore, screening of probiotics capable of fermenting pectin has become a technical problem to be solved by those skilled in the art.

[0004] Different genes expressed by different probiotics and metabolites produced by different probiotics have different growth characteristics, antioxidant capacity and antibacterial performance. With further research, researchers have found that single strain has certain limitations in comprehensive probiotic performance and is difficult to produce expected health benefits, and the combination of complex probiotics provides a new idea for breaking the limitations of 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 the study of antagonizing Helicobacter pylori infection that the combination of two strains significantly relieved the symptoms of infection compared with single strain. Therefore, the use of multiple probiotics together is the future direction of probiotic products.

[0005] The inventors accidentally obtained a complex probiotic with synergistic metabolism of pectin polysaccharide and its application during the research process. The complex probiotic comprises Lactiplantibacillus plantarum FMBL L23180 CLL and Lacticaseibacillus rhamnosus FMBL L23121 YWT. The complex probiotic can achieve rapid proliferation in a culture medium with pectin as the only carbon source, efficiently metabolize pectin polysaccharide, promote the growth of the strain, and significantly improve the biomass and the probiotic index. During the co-culture process, the complex strain produces a large amount of short-chain fatty acids, and the contents of acetic acid and valeric acid produced at the end of fermentation are the highest, accounting for more than 90% of the total fermentation products. The amount of SCFA produced by the complex probiotic during the co-culture process is significantly higher than that of Lactiplantibacillus plantarum FMBL L23180 CLL and Lacticaseibacillus rhamnosus FMBL L23121 YWT cultured alone, which is 1.35 and 1.48 times that of the single culture, respectively, especially the increase of valeric acid is the most significant, which is 1.43 and 1.52 times that of the single culture, respectively. It can be used for preparing microcapsules, complex probiotic agents, fermented milk, fruit and vegetable beverages, plant milk, probiotic milk tablets, etc. SUMMARY

[0006] The primary object of the present application is to provide Lacticaseibacillus rhamnosus FMBL L23121 YWT, which was deposited with the China Center for Type Culture Collection on August 15, 2024, and the deposit number is CCTCC NO: M 20241794.

[0007] The second object of the present application is to provide a complex bacterial agent capable of synergistically metabolizing pectin polysaccharide, which comprises Lactiplantibacillus plantarum FMBL L23180 CLL and the Lacticaseibacillus rhamnosus FMBL L23121 YWT. The Lactiplantibacillus plantarum FMBL L23180 CLL was deposited with the China Center for Type Culture Collection on August 15, 2024, and the deposit number is CCTCC NO: M 20241793.

[0008] The third object of the present application is to provide the use of the Lacticaseibacillus rhamnosus FMBL L23121 YWT or the complex bacterial agent in the preparation of functional products, which have one or more of the following effects:

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

[0010] (2) produces short-chain fatty acids by utilizing pectin.

[0011] Preferably, the functional product is one or more of a food, a food additive, a dietary supplement, a health product, a pharmaceutical product, a feed or a feed additive.

[0012] Preferably, the food is a fermented food, a fruit and vegetable beverage, a milk tablet, etc.

[0013] The pharmaceutical product can be prepared as a microcapsule, a tablet, a powder, a sustained-release preparation, etc.

[0014] A fourth object of the present application is to provide the use of the Lactobacillus rhamnosus FMBL L23121 YWT or the complex bacterial agent in the preparation of a fermented dairy product, a bean product or a fruit and vegetable product.

[0015] A fifth object of the present application is to provide a synbiotic containing the Lactobacillus rhamnosus FMBL L23121 YWT or the complex bacterial agent.

[0016] Preferably, the synbiotic further comprises one or more of pectin, pectin oligosaccharide, resistant starch, D-trehalose, soybean oligosaccharide, galactooligosaccharide, raffinose, malt dextrin, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose and inulin.

[0017] The present application has the following beneficial effects: (1) the present application provides a complex probiotic with synergistic metabolism of pectin polysaccharide and its application, the complex probiotic comprises Lactiplantibacillus plantarum FMBL L23180 CLL and Lacticaseibacillus rhamnosus FMBL L23121 YWT;

[0018] (2) the complex probiotic can be rapidly proliferated in a culture medium with pectin as the sole carbon source, efficiently metabolize pectin polysaccharide, promote the growth of the strain, and significantly improve the biomass and the probiotic index.

[0019] (3) during the co-culture process, a large amount of short-chain fatty acids are produced, and the contents of acetic acid and valeric acid produced at the end of fermentation are the highest, accounting for more than 90% of the total fermentation products. The amount of SCFA produced by the complex probiotic during the co-culture process is significantly higher than that of Lactiplantibacillus plantarum FMBL L23180 CLL and Lacticaseibacillus rhamnosus FMBL L23121 YWT cultured alone, which is 1.35 and 1.48 times that of the single culture, respectively, and the increase in valeric acid is the most significant, which is 1.43 and 1.52 times that of the single culture, respectively.

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

[0021] (5) The present invention also provides a synbiotic, wherein the synbiotic contains the Lactobacillus rhamnosus FMBL L23121YWT or the compound bacterial agent, and the synbiotic further comprises one or more of pectin, pectin oligosaccharide, resistant starch, D-trehalose, soybean oligosaccharide, galactooligosaccharide, raffinose, maltodextrin, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose, and inulin. Attached Figure Description

[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 cultured alone in a pectin-based carbon source medium.

[0023] Figure 3 Growth curves of different combinations of probiotics in a pectin-based carbon source medium.

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

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

[0026] Figure 6 Compound probiotic microcapsule form Detailed Implementation

[0027] The present invention will be further illustrated in detail below through several embodiments, but this does not limit the scope of the claims of the present invention in any way.

[0028] The apple pectin in the following implementation case was purchased from Maclean's reagents.

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

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

[0031] MRS solid medium: proteose 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: proteose 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: proteose peptone 5.0 g, beef extract 3.0 g, sodium chloride 5.0 g, agar powder 20.0 g, deionized water 1000 mL.

[0034] PYG medium: proteose 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: tryptone 15.0 g, soybean peptone 5.0 g, sodium chloride 5.0 g, agar powder 20.0 g, deionized water 1000 mL;

[0036] Example 1, isolation and identification of Lactobacillus plantarum FMBL L23180 CLL and Lacticaseibacillus rhamnosus FMBL L23121 YWT

[0037] 1. Strain isolation and purification

[0038] Fresh fecal samples of Kazakh children in Yining Prefecture of Xinjiang and fresh fecal samples of healthy adults in Wuwei, Gansu were collected, gradient dilution plate coating method was used, and the strains were cultured under anaerobic conditions (80% nitrogen, 10% hydrogen, 10% carbon dioxide) and isolated.

[0039] Take 1 g of fecal sample dissolved in 9 mL of MRS liquid medium, mix well, dilute the fecal sample to 10 -3 、10 -4 、10 -5 , and spread on MRS agar medium, incubate at 37°C for 24-48 hours. According to the colony characteristics, cell morphology, pick the suspected lactic acid bacteria colonies and purify 3 times, then store the isolated strains in MRS liquid medium with 30% glycerol and store at -20°C. Two strains were isolated and named FMBL L23180 CLL and FMBL L23121 YWT.

[0040] 2. Strain identification

[0041] The DNA of the strains was extracted using a kit, and the groEL gene of the strains was amplified by PCR. The amplification and reaction conditions are shown in Table 1. After the PCR reaction, the products were subjected to 1.2% agarose gel electrophoresis to observe the molecular size, and the appropriate PCR products were sent to the company for sequencing. The returned sequencing results were uploaded to the NCBI database for BLAST comparison. After comparison, the corresponding genus sequences were obtained from the database, and a 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 shown in Figure 1 The strain FMBL L23180 CLL was identified as Lactiplantibacillus plantarum, with the Latin name Lactiplantibacillus plantarum. It was named Lactiplantibacillus plantarum FMBL L23180 CLL and preserved in the China Center for Type Culture Collection on August 15, 2024, with the preservation number CCTCC NO: M 20241793 and the preservation address Wuhan University, Wuhan, China, telephone (027)-68752319.

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

[0046] 3. Carbohydrate utilization ability of Lacticaseibacillus rhamnosus FMBL L23121 YWT

[0047] Activated Lacticaseibacillus rhamnosus FMBL L23121 YWT was inoculated into modified MRS liquid medium with prebiotic: resistant starch, chitosan, D-trehalose, soybean oligosaccharide, galactooligosaccharide, raffinose, maltodextrin, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose, inulin as the only carbon source, with glucose as the positive control, and no carbon source as the negative control, at an inoculation amount of 2% (v / v) (OD 600 : 1.0 ± 0.05), and the absorbance of 0h was OD 600 , the absorbance of 37℃ culture for 24h was OD 600 , the final OD 600 = OD1- OD2, and the experiment was repeated three times to take the average value.

[0048] Prebiotics are not 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 show that the Lactobacillus plantarum FMBL L23180 CLL strain can effectively utilize 10 kinds of carbon sources including resistant starch, D-trehalose, soybean oligosaccharide, galactooligosaccharide, raffinose, maltodextrin, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose, and inulin, and the utilization ability of xylooligosaccharide is slightly poor, and chitosan cannot be effectively utilized.

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

[0050] soybean oligosaccharides 1.156 resistant starch 0.758 maltodextrin 1.375 raffinose 1.509 xylooligosaccharides 0.465 galactooligosaccharides 1.559 fructooligosaccharides 1.427 stachyose 1.507 isomaltooligosaccharides 1.491 inulin 0.692 chitooligosaccharides 0.185 D-fucosylated oligosaccharides 1.515 positive control 1.674 negative control 0.018

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

[0052] Example II, Effect of pectin on the growth of Lactiplantibacillus plantarum FMBL L23180 CLL and Lacticaseibacillus rhamnosus FMBL L23121 YWT

[0053] Strains to be tested: Lactiplantibacillus plantarum FMBL L23180 CLL, Lacticaseibacillus rhamnosus FMBL L23121 YWT, and Lactobacillus rhamnosus strain LGG.

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

[0055] Lacticaseibacillus rhamnosus FMBL L23121 YWT was deposited with the China Center for Type Culture Collection on August 15, 2024, and the deposit number is CCTCC NO: M 20241794.

[0056] After activation, Lactiplantibacillus plantarum FMBL L23180 CLL, Lacticaseibacillus rhamnosus FMBL L23121 YWT, and control strain Lactobacillus rhamnosus LGG were centrifuged to remove the supernatant, and the bacterial slurry was washed twice with sterile physiological saline and resuspended to prepare a bacterial solution with a concentration of 1.0 x 10 7 cfu / mL, inoculated into MRS medium with 2% pectin as the carbon source at an inoculation amount of 1%, and fermented at 37°C for 24h. The OD600 value was measured every 2h, and the growth curves of the bacteria were plotted.

[0057] Results: As can be seen from Figure 2 Lactiplantibacillus plantarum FMBL L23180 CLL and Lacticaseibacillus rhamnosus FMBL L23121 YWT entered the logarithmic growth phase at 4h of fermentation, and entered the stationary phase after 12h. The OD value at the end of fermentation was about 1.3. Lactobacillus rhamnosus LGG had an OD value of less than 0.35 at 24h of fermentation. The results showed that Lactiplantibacillus plantarum FMBL L23180 CLL and Lacticaseibacillus rhamnosus FMBL L23121 YWT could better utilize pectin and could achieve rapid proliferation in a culture medium with pectin as the sole carbon source.

[0058] Example 3: Synergistic metabolism of pectin by complex probiotics

[0059] (1) Screening of complex probiotic combination

[0060] Strains to be tested: Lactobacillus plantarum FMBL L23180 CLL, Lactobacillus plantarum FMBL L23184 CLL, Lactobacillus rhamnosus FMBL L23136 YWT, Lactobacillus rhamnosus FMBL L23121 YWT, Lactobacillus rhamnosus FMBL L23137 YWT, and Lactobacillus rhamnosus LGG.

[0061] After activation, the supernatant was removed by centrifugation, and the bacterial slurry was washed twice with sterile physiological saline and resuspended to prepare a bacterial solution with a concentration of 1.0 x 10 7 cfu / mL. The bacterial solution was inoculated into MRS medium with 2% pectin as the carbon source at a ratio of 1:1 and at an inoculation amount of 1%. The mixture was incubated at 37°C for 24 h, and the OD600 value was measured every 2 h. The growth curve of each combination was plotted, and the optimal probiotic combination was selected.

[0062] The results, as shown in Table 5, showed that after 24 h 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 when they were cultured alone. The growth of other combinations was not significantly different from that when they were cultured alone, and the OD value was about 1.25, which was significantly lower than that of the combination of Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT. Therefore, this combination was selected for subsequent experiments. Figure 3 (2) Synergistic metabolism of pectin by Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT

[0063] After activation, the supernatant was removed by centrifugation, and the bacterial slurry was washed twice with sterile physiological saline and resuspended to prepare a bacterial solution with a concentration of 1.0 x 10 7 cfu / mL. The bacterial solution was inoculated into MRS medium with 2% pectin as the carbon source at a ratio of 1:1 and at an inoculation amount of 1%. The mixture was incubated at 37°C for 24 h, and the OD600 value was measured every 2 h. The growth curve of each combination was plotted, and the optimal probiotic combination was selected.

[0064] Table 3 Real-time fluorescence quantitative PCR

[0065]

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

[0067]

[0068] Standard curve preparation, configure different concentrations of plantaricin FMBL L23180 CLL and rhamnolactic FMBL L23121 YWT, extract strain DNA according to the bacterial DNA extraction kit instructions, and determine the OD value of different gradient DNA by micro nucleic acid instrument 260 , and the extracted DNA is subjected to QPCR to establish the standard curve relationship between Ct value and bacterial liquid concentration, and draw the standard curve (Table 5). After fermentation, the DNA of the fermentation liquid sample is extracted, and the real-time fluorescence quantitative PCR reaction is carried out according to the reflection system and reflection condition of the standard curve. The obtained Ct value is substituted into the corresponding standard curve, and the concentration of the corresponding lactobacillus in the fermentation liquid is calculated, and the result is expressed as CFU / mL.

[0069] Table 5 Standard curve of fluorescence real-time quantitative PCR amplification

[0070]

[0071] Results: The results of real-time fluorescence quantitative PCR are shown in Figure 3 Compared with single culture, the biomass of plantaricin FMBL L23220 CLL and rhamnolactic FMBL L23121 YWT is significantly increased during co-culture, and the viable count of plantaricin FMBL L23180 CLL reaches 9.58×10 9 CFU / mL, which is 19.32% higher than that of single culture, and the viable count of rhamnolactic FMBL L23121 YWT is 3.83×10 9 CFU / mL, which is 17.79% higher than that of single culture. The above results show that the biomass of plantaricin FMBL L23180 CLL and rhamnolactic FMBL L23121 YWT is significantly improved during co-culture, which further shows that plantaricin FMBL L23180 CLL and rhamnolactic FMBL L23121 YWT can synergistically utilize pectin in the medium with pectin as carbon source, thereby promoting the growth of each other.

[0072] (3) Probiotic index PI of compound probiotics

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

[0074] PI = [ODP24 - ODP0 - (ODC24 - ODC0)] x 100 / [ODG24 - ODG0 - (ODC24 - ODC0)]

[0075] In the formula, P represents pectin, C represents no carbon source, and G represents glucose. ODP0 and ODP24 are the OD values at 600 nm of the lactobacillus at 0 h and 24 h, respectively, when the carbon source is pectin, and the other notations are similar.

[0076] Table 6 Probiotic index PI (%)

[0077]

[0078] As shown in Table 6, the PI values of the plantaricin FMBL L23180 CLL and rhamnolactic lactococcus FMBL L23121 YWT are 79.68% and 78.69, which are lower than the probiotic index 92.18% when the two are co-cultured. Therefore, the two are co-cultured to better metabolize pectin.

[0079] Example Four, Change of total sugar in the process of fermentation of pectin by compound probiotic bacteria

[0080] After the plantaricin FMBL L23180 CLL and rhamnolactic lactococcus FMBL L23121 YWT were activated, the supernatant was removed by centrifugation, the bacterial slurry was washed twice with sterile normal saline and resuspended, and a bacterial solution with a concentration of 1.0 x 10 7 cfu / mL was prepared. The two were inoculated into MRS medium with 2% pectin as the carbon source at a ratio of 1:1 and at an inoculation amount of 1%, and co-cultured at 37°C for 24 h. The total sugar content of the fermentation liquid was measured at 0 h, 4 h, 8 h, 12 h, 18 h and 24 h, respectively.

[0081] The total sugar content was measured by anthrone-sulfuric acid method. 0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mL of glucose standard solution were taken into 10 mL test tubes, water was added to 2 mL, 6 mL of anthrone sulfuric acid solution was added, boiling water bath was performed for 15 min, it was taken out and quickly cooled for 15 min, and OD 620 was measured. The standard curve was drawn. The lactobacillus fermentation liquid was diluted 20 times with distilled water, the absorbance of the lactobacillus fermentation liquid was measured according to the above method, and the total sugar content was calculated by substituting the standard curve.

[0082] Results: The change of total sugar is an important indicator to characterize the carbon source utilization ability of microorganisms in the fermentation system. From the results in Table 7, it can be seen that the total sugar content of the fermentation liquid of the two co-cultured lactobacillus is lower than that of the single culture, and the total sugar content of the fermentation liquid of the two co-cultured lactobacillus is lower than that of the single culture. Figure 5It can be seen that during the fermentation process, the total sugar and reducing sugar rapidly decreased within 4-18h, and there was no significant difference in the utilization efficiency of pectin between the Lactobacillus plantarum FMBLL23180 CLL and the Lactobacillus rhamnosus FMBLL23121 YWT cultured alone, and the total sugar consumption rate was about 73%. However, 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 of the compound probiotics increased significantly, and the total sugar consumption rate reached nearly 85%. The above results also showed that there was a synergistic effect between the Lactobacillus plantarum FMBLL23180 CLL and the Lactobacillus rhamnosus FMBLL23121 YWT during the co-culture process, which could promote the growth of each other and the metabolic capacity of pectin.

[0083] Example Five, Production of Short-Chain Fatty Acids by Compound Probiotics

[0084] The production of short-chain fatty acids (SCFA) by the Lactobacillus plantarum FMBLL23180 CLL, the Lactobacillus rhamnosus FMBLL23121 YWT and the compound probiotics during the fermentation process was analyzed by gas chromatography. 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 being loaded. The gas chromatography analysis was performed on an Agilent 7890B gas chromatograph.

[0085] The GC determination conditions were as follows: a DB-WAX analysis column (60m x 250μm x 0.25μm) was used for determination. The injection port temperature was 250℃; the column oven conditions were as follows: the initial temperature was 100℃ for 30s, increased by 8℃ per minute, maintained at 160℃ for 1min, then increased by 20℃ / min, and maintained at 200℃ for 5min; the injection volume was 1μL; the carrier gas conditions were as follows: N2, flow rate was 1.2mL / min, split ratio was 2:1; the FID detector conditions were as follows: temperature was 250℃, tail gas: N2(25mL / min), H2(30mL / min), air(400mL / min).

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

[0087]

[0088] Results: Table 7 shows that *Lactobacillus plantarum* FMBL L23180 CLL and *Lactobacillus rhamnosus* FMBLL23121 YWT produced a large amount of short-chain fatty acids (SCFAs) during pectin fermentation. SCFAs are important substances in the gut that inhibit pathogenic microorganisms and are crucial for maintaining the balance of gut microbiota and promoting gut health in conditioned organisms. The results indicate that acetic acid and valeric acid were produced at the end of fermentation, accounting for more than 90% of the total fermentation products. The amount of SCFA produced by the co-cultured probiotics fermenting pectin was significantly higher than that of the individually cultured *Lactobacillus plantarum* FMBL L23180 CLL and *Lactobacillus rhamnosus* FMBL L23121 YWT, respectively, being 1.35 and 1.48 times higher, respectively. In particular, the increase in valeric acid was the most significant, being 1.43 and 1.52 times higher, respectively, than that of the individually cultured strains.

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

[0090] The *Lactobacillus plantarum* FMBL L23180 CLL and *Lactobacillus rhamnosus* FMBL L23121 YWT of this invention were cultured in MRS medium for 24 h, centrifuged at 4 °C and 8000 r / min for 10 min, and the bacterial cells were collected. After washing twice with sterile physiological saline, the bacterial cells were resuspended in sterile physiological saline to achieve a final bacterial concentration of 1 × 10⁻⁶. 10 CFU / mL. After mixing the two bacterial suspensions, equal volumes of 1.5% (w / v) galactooligosaccharide solution and 5 times the volume of 3.0% (w / v) pectin solution were added sequentially, and the mixture was stirred thoroughly. The above mixture was then extruded and dripped into a 0.8 mol / L calcium chloride curing solution to form gel particles. After curing for 30 min, the gel particles were added again to a 0.5% (w / v) chitosan solution at pH 5.0 and cured for 20 min. The gel particles were collected by filtration and freeze-dried to obtain microcapsules containing the FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT complex probiotics of this invention. Figure 6 The microcapsules have a particle size of 1.93-2.11 mm, an encapsulation efficiency of ≥92.83%, and a bacterial survival rate of ≥89%. They exhibit tolerance to simulated gastrointestinal fluid and heat stress. The prepared microcapsules are then filled into commercially available pharmaceutical capsules to obtain the aforementioned capsule product.

[0091] Application Example 2: Preparation of Compound Probiotic Agents

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

[0093] Protectant preparation: using water and protectant raw materials to prepare a protectant containing skimmed milk powder 120 g / L, glycerol 20 mL / L, malt dextrin 22 g / L, trehalose 60 g / L, and galactooligosaccharide 22 g / L.

[0094] Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT were inoculated in the above-mentioned medium sterilized at 115°C for 15 min at a ratio of 1:1 and an inoculation amount of 2%, wherein the medium contains glucose 20 g / L, beef extract 8 g / L, yeast extract 10 g / L, NaCl 9 g / L, Tween 80 1 mL / L, K2HPO4 2 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g, MgSO4·7H2O 0.58 g, and MnSO4·4H2O 0.25 g, and the pH was adjusted to 6.8. After being cultured at 37°C for 24 h, the bacteria were washed twice with PBS buffer with pH 7.2 and resuspended in a protectant to reach a concentration of 10 11 CFU / mL. The protectant contains skimmed milk powder 120 g / L, glycerol 20 mL / L, malt dextrin 22 g / L, trehalose 60 g / L, and galactooligosaccharide 22 g / L. Then, the suspension was pre-cultured at 37°C for 60 min, and freeze-dried to obtain the compound probiotic bacteria agent.

[0095] Example 3: preparation of fermented milk using the compound probiotic bacteria agent of the present application

[0096] After the fresh milk was dissolved with sugar, homogenization was performed at 60°C and 20 MPa, and then sterilization was performed at 90-95°C for 5-8 min. After the temperature dropped to 35°C, the compound probiotic bacteria agent of the present application, commercial dry powder starter Lactobacillus bulgaricus and Streptococcus thermophilus were added, and the mass ratio of the three was 1:1:1. The inoculation amount of the mixed bacteria was 0.03-2.0% of the weight of the fresh milk, and then mixed and fermented at 37°C. After coagulation, the fermented milk was stored at 4°C for 16 h to obtain the fermented milk.

[0097] Example 4: preparation of fruit and vegetable beverage containing compound probiotic bacteria

[0098] The fresh fruits and vegetables are washed and juiced, then high-temperature instant sterilization is performed, and immediately after cooling to a temperature of about 37℃, the composite probiotic agent prepared by the application is added to make the concentration reach 10 6 CFU / mL or more, and stored in a refrigerator at a temperature of 4℃, to obtain a fruit and vegetable beverage containing live Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT of the application.

[0099] Example 5: Preparation of plant milk using the composite probiotic agent of the application

[0100] The soybeans, almonds, apricots and other raw materials are soaked at a temperature of 80℃ for 2h, peeled and then the soaking water is drained, boiling water is added to grind the pulp, and the temperature is maintained at 80-85℃ for 12-15min. The obtained pulp is filtered to remove residues using a 200-mesh screen, then boiled, and the supernatant obtained after removing the solids by centrifugation is cooled to a temperature of about 37℃, and the composite probiotic agent of the application is added to make the concentration reach 10 6 CFU / mL or more, and stored in a refrigerator at a temperature of 4℃, to obtain a fruit and vegetable beverage containing live Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus FMBL L23121 YWT of the application.

[0101] Example 6: Preparation of probiotic milk tablets using the composite probiotic agent of the application

[0102] 5.96 parts by weight of the composite probiotic agent of the application prepared by freeze-drying method, 65.0 parts by weight of full-fat milk powder, 7.92 parts by weight of white granulated sugar, 2.5 parts by weight of magnesium stearate, 18.0 parts by weight of skim milk powder, and 1.0 part by weight of water are weighed respectively, mixed, and wet granulated by a conventional method, then tableted using a tablet press, dried in a drying machine, packaged, and the probiotic milk tablets of the application are obtained.

[0103] In summary, the present application provides a kind of complex probiotics with synergistic metabolism pectin polysaccharide and its application, the complex probiotics include Lactiplantibacillus plantarum (Lactiplantibacillus plantarum) FMBL L23180 CLL and Lacticaseibacillus rhamnosus (Lacticaseibacillus rhamnosus) FMBL L23121 YWT;The complex probiotics can be rapidly proliferated in the medium with pectin as the only carbon source, efficiently metabolize pectin polysaccharide, promote the growth of strains, and the biomass is significantly improved, and the probiotic index is also significantly improved.The complex strains produce a large amount of short-chain fatty acids during co-culture, and the content of acetic acid and valeric acid produced at the end of fermentation is the highest, accounting for more than 90% of the total fermentation products.The amount of SCFA produced by co-cultured complex probiotics during pectin fermentation is significantly higher than that of Lactiplantibacillus plantarum FMBL L23180 CLL and Lacticaseibacillus rhamnosus FMBL L23121 YWT cultured alone, which is 1.35 and 1.48 times that of single culture, respectively, especially the increase of valeric acid is the most significant, which is 1.43 and 1.52 times that of single culture, respectively.The complex strains can be used for preparing microcapsules, complex probiotic agents, fermented milk, fruit and vegetable drinks, plant milk, probiotic milk tablets, etc.The present application also provides a kind of synbiotic, the synbiotic contains the Lacticaseibacillus rhamnosus FMBL L23121 YWT or the complex bacterial agent, and the synbiotic also includes one or more of pectin, pectin oligosaccharide, resistant starch, D-trehalose, soybean oligosaccharide, galactooligosaccharide, raffinose, malt dextrin, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose and inulin.

Claims

1. A complex microbial inoculant capable of synergistically metabolizing pectic polysaccharides, characterized in that, The compound microbial agent includes *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The *Lactobacillus plantarum* FMBL L23121 YWT and *Lactobacillus rhamnosus* FMBL L23180CLL were deposited at the China Center for Type Culture Collection (CCTCC) on August 15, 2024, with accession number CCTCC NO: M20241793. The *Lactobacillus rhamnosus* FMBL L23121 YWT was also deposited at the same collection on August 15, 2024, with accession number CCTCC NO: M 20241794.

2. The use of the complex microbial agent of claim 1 in the preparation of food, food additive, dietary supplement or feed additive.

3. The use of the complex microbial agent of claim 1 in the preparation of fermented dairy products, bean products or fruit and vegetable products.

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