Co-fermentation method for promoting proliferation of lactobacillus rhamnosus based on pre-degradation of pectin by bacteroides multiforme

Abstract

The invention discloses a co-fermentation method for promoting proliferation of lactobacillus rhamnosus based on pre-degradation of pectin by bacteroides multiforme, and belongs to the technical field of food processing. The co-fermentation method specifically comprises the following steps: by taking pectin with the molecular weight of 10kDa as a substrate, pre-inoculating bacteroides multiforme for fermentation so as to degrade the pectin into oligosaccharide fragments, and then inoculating lactobacillus rhamnosus for co-culture. The utilization capacity of the lactobacillus rhamnosus on the pectin is remarkably improved by oligosaccharide fragments generated by pre-degrading the pectin through the bacteroides polymorpha, so that the efficient proliferation of the lactobacillus rhamnosus is promoted. Compared with traditional single fermentation, the biomass of lactobacillus rhamnosus can be increased by 40% or above. The method simulates the natural process of synergistically metabolizing pectin by intestinal flora, is beneficial to development of fermentation preparations closer to intestinal microecology, can be widely applied to production of feed probiotic preparations, and provides a new technical support for improving the probiotic effect of pectin and developing intestinal microecology directional regulation and control products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on Bacteroides polymorphonuclear pre-degradation of pectin. Background Technology

[0002] Pectin is a complex polysaccharide widely found in plant cell walls and has long been a part of the human diet. Currently, pectin has been shown to have various physiological benefits, and its probiotic effects are mainly achieved through fermentation by gut microbiota. Compared to other polysaccharides with simpler structures, such as xylan and fructan, pectin exhibits extremely complex characteristics in terms of monosaccharide composition, linkage sequence, glycosidic bond type, and non-glycosidic substituents. Therefore, pectin degradation usually requires the synergistic action of multiple gut bacteria. Different species of gut bacteria have different pectin utilization capabilities and thus occupy different ecological niches in pectin degradation. Competition, synergy, or cross-feeding relationships may exist among them, all of which affect the efficiency and extent of pectin degradation and the type of final metabolites.

[0003] Bacteroides thetaiotaomicron and Lactobacillus rhamnosus are common bacteria in the human gut. The former is one of the most important carbohydrate-degrading bacteria in the gut, encoding a wide range of carbohydrate hydrolases to degrade and utilize polysaccharides. The latter is a typical probiotic and an important target for dietary intervention; both play crucial roles in maintaining gut microbiota balance. In particular, Lactobacillus rhamnosus has limited ability to degrade pectin, typically acting as a secondary pectin-degrading bacterium, primarily utilizing partially degraded pectin fragments in the gut. This cross-feeding of pectin among the bacteria can compensate for the lack of a complete pectin degradation system in most gut bacteria, thus effectively realizing the probiotic effects of pectin. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0006] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on Bacteroides polymorpha pre-degradation pectin.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions, including:

[0008] Strain activation: Bacteroides polymorpha and Lactobacillus rhamnosus were inoculated into activation medium and cultured at 37°C under anaerobic conditions.

[0009] Pre-fermentation: Bacteroides polymorpha was transferred to a fermentation medium with pectin as the sole carbon source and anaerobic fermentation was carried out at 37°C to obtain a pre-fermentation broth;

[0010] Secondary co-culture: Using the pre-fermentation broth as a secondary culture medium, Lactobacillus rhamnosus was inoculated for mixed culture fermentation;

[0011] Fermentation termination: Fermentation is terminated when the number of viable Lactobacillus rhamnosus cells reaches the stationary phase. The cells are collected by centrifugation and freeze-dried to obtain the cell fermentation agent.

[0012] As a preferred embodiment of the co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha according to the present invention, wherein: the Bacteroides polymorpha includes strain-78496.

[0013] As a preferred embodiment of the co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha according to the present invention, wherein: the OD of the activated Bacteroides polymorpha... 600 ≥1.2, OD of activated Lactobacillus rhamnosus 600 ≥0.8.

[0014] As a preferred embodiment of the co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha according to the present invention, wherein: the pre-fermentation comprises: an inoculum of Bacteroides polymorpha of 1-3% (v / v); a fermentation medium comprising: 1.0-3.0 g / L soybean peptone, 3.0-5.0 g / L yeast extract, 2.0-4.0 g / L beef meal, 0.5-2.0 g / L potassium dihydrogen phosphate, 1.0-3.0 g / L sodium chloride, 0.1-0.3 g / L L-cysteine, 0.1-0.3 g / L sodium thioglycolate, 0.5-1 ml 0.1% vitamin K1, 2.5-5 mg / L heme chloride, and 5.0-8.0 g / L pectin; and a fermentation time of 10-14 h.

[0015] As a preferred embodiment of the co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degraded pectin of Bacteroides polymorpha according to the present invention, the pectin has the following structural characteristics: molecular weight ≤12kDa, degree of esterification ≤50%, and galacturonic acid content ≤75%.

[0016] As a preferred embodiment of the co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degraded pectin of Bacteroides polymorpha according to the present invention, wherein: in the secondary co-culture, the inoculum amount of Lactobacillus rhamnosus is 2-6% (v / v), and the fermentation time is 16-24h.

[0017] As a preferred embodiment of the co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha according to the present invention, wherein: oligosaccharide fragments are produced by pre-fermentation of pectin by Bacteroides polymorpha, and after co-culture, the biomass of Lactobacillus rhamnosus reaches more than 10 LogCFU / mL during the stable period.

[0018] As a preferred embodiment of the co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha according to the present invention, wherein: during the termination of fermentation, the stabilization period is ±5% of the cell count fluctuation.

[0019] Another objective of this invention is to overcome the shortcomings of existing technologies and provide a cell fermentation primordial prepared by a co-fermentation method based on Bacteroides polymorpha pre-degrading pectin to promote the proliferation of Lactobacillus rhamnosus. The Lactobacillus rhamnosus biomass in the cell fermentation primordial reaches 10... 10 CFU / mg or higher, which increases the yield by 40% to 65% compared to single-strain fermentation.

[0020] The third objective of this invention is to overcome the shortcomings of the prior art and provide an application of microbial fermentation agents in animal feed additives.

[0021] Beneficial effects of this invention:

[0022] This invention significantly enhances the pectin utilization capacity of *Lactobacillus rhamnosus* by pre-degrading oligosaccharide fragments from pectin using *Bacteroides polymorpha*, thereby promoting its efficient proliferation. Compared to traditional single fermentation, the biomass of *Lactobacillus rhamnosus* can be increased by more than 40%. This method cleverly utilizes the complementary ecological niches of the two strains. *Bacteroides polymorpha*, as the primary degrader, is responsible for breaking down the complex pectin structure, while *Lactobacillus rhamnosus* relies on its metabolic capacity to further utilize the degradation products, forming an efficient cross-feeding network and optimizing pectin utilization efficiency. This co-culture model overcomes the limitations of single-strain pectin utilization and also simulates the natural process of intestinal flora co-metabolizing pectin, which is helpful for developing fermentation preparations that are closer to the intestinal microecology. This technology can be widely applied to the production of probiotic feed preparations, providing new technical support for improving the probiotic effect of pectin and developing products for targeted regulation of the intestinal microecology. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0024] Figure 1 The effects of different fermentation methods (Examples 1 and 2, Comparative Examples 1 and 3) on the proliferation of Lactobacillus rhamnosus were investigated.

[0025] Figure 2 The growth curves of Bacteroides polymorpha and Lactobacillus rhamnosus under the fermentation method used in Comparative Example 3 are shown.

[0026] Figure 3 This describes the utilization of pectin of different molecular weights by Bacteroides polymorpha. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0030] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0031] The accession number for Bacteroides thetaiotaomicron-78496 is ATCC29148; the accession number for Lactobacillus rhamnosus-22825 is CICC S22825.

[0032] The materials obtained in the embodiments of the present invention were subjected to performance testing according to the following method:

[0033] Biomass counting of Lactobacillus rhamnosus

[0034] Take 1 mg of the prepared bacterial fermentation spawn and suspend it in 10 mL of physiological saline (0.9%, m / v). Then, perform a 10-fold serial dilution. Spread 100 μL of the diluted bacterial suspension onto MRS agar plates and incubate at 37°C under anaerobic conditions for 48 h. Record the total number of colonies formed. The biomass of *Lactobacillus rhamnosus* in the bacterial fermentation spawn is calculated as: total colony count * 10^6. n+2 The concentration is CFU / mg, where n is the dilution factor. The dilution factor used should ensure that the final colony count is within the range of 30–300 CFU. (Because MRS medium lacks essential nutrients for the growth of Bacteroides polymorpha, such as vitamin K1 and ferric heme, this bacterium cannot grow normally on MRS agar plates, and therefore will not interfere with the biomass determination of Lactobacillus rhamnosus.)

[0035] Example 1

[0036] This embodiment provides a co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on Bacteroides pre-degrading pectin, specifically as follows:

[0037] (1) Activation of Bacteroides polymorpha and Lactobacillus rhamnosus: Take 1.5 mL of Bacteroides polymorpha-78496 and Lactobacillus rhamnosus-22825 frozen at -80℃ respectively, and inoculate them into 5 mL of commercial GAM medium and MRS medium respectively, and incubate at 37℃ under anaerobic conditions for 24 h.

[0038] (2) Pre-fermentation of pectin substrate: 500 μL (2%, v / v) of activated Bacteroides polymorpha-78496 was inoculated into 25 mL of fermentation medium with pectin as the sole carbon source and fermented at 37℃ under anaerobic conditions for 12 h. The fermentation medium consisted of: soybean peptone 2.0 g / L, yeast extract 4.0 g / L, beef meal 3.0 g / L, potassium dihydrogen phosphate 1.5 g / L, sodium chloride 2.0 g / L, L-cysteine ​​0.2 g / L, sodium thioglycolate 0.2 g / L, vitamin K1 0.0008 g / L, heme chloride 3.5 mg / L, and pectin 6.0 g / L. The pectin structure was characterized by a molecular weight of 10 kDa, a degree of esterification of 30%, and a galacturonic acid content of 65%.

[0039] (3) Secondary co-culture: 25 mL of the pre-fermentation broth after 12 h of fermentation was used as the secondary culture medium. 1 mL (4%, v / v) of activated Lactobacillus rhamnosus-22825 was inoculated and cultured at 37 °C under anaerobic conditions for 18 h.

[0040] (4) The culture medium that has undergone secondary fermentation is centrifuged at 10,000 rpm for 10 minutes at 4°C. After collecting the cells, they are washed with 10 mL of physiological saline. This process is repeated 3 times. The cells obtained are then freeze-dried to obtain the prepared cell fermentation agent.

[0041] Example 2

[0042] The difference between this embodiment and embodiment 1 is that the pre-fermentation time of the pectin substrate in step (2) is adjusted to 10 hours, while other parameters and conditions are the same as in embodiment 1, and a bacterial fermentation agent is obtained.

[0043] Example 3

[0044] The difference between this embodiment and embodiment 1 is that the pre-fermentation time of the pectin substrate in step (2) is adjusted to 14 hours, while other parameters and conditions are the same as in embodiment 1, and a bacterial fermentation agent is obtained.

[0045] Example 4

[0046] The difference between this embodiment and Example 1 is that the inoculation amount of Lactobacillus rhamnosus in the secondary co-culture in step (3) is adjusted to 0.5 mL (2%, v / v), while other parameters and conditions are the same as in Example 1, and the bacterial fermentation agent is obtained.

[0047] Example 5

[0048] The difference between this embodiment and Example 1 is that the inoculation amount of Lactobacillus rhamnosus in the secondary co-culture in step (3) is adjusted to 1.5 mL (6%, v / v), while other parameters and conditions are the same as in Example 1, and the bacterial fermentation agent is obtained.

[0049] Comparative Example 1

[0050] The difference between this comparative example and Example 1 is that the pre-fermentation treatment in step (2) is omitted. The activated Lactobacillus rhamnosus is directly inoculated into a fermentation medium with pectin as the sole carbon source at an inoculation amount of 1 mL (4%, v / v) for single-cell fermentation. Other parameters and conditions are the same as in Example 1 to obtain the cell fermentation agent.

[0051] Comparative Example 2

[0052] The difference between this comparative example and Example 1 is that the activated Bacteroides polymorpha and Lactobacillus rhamnosus were simultaneously inoculated into a fermentation medium with pectin as the sole carbon source at inoculation amounts of 0.5 mL (2% v / v) and 1 mL (4% v / v), respectively. All other parameters and conditions were the same as in Example 1 to prepare the bacterial fermentation agent.

[0053] Comparative Example 3

[0054] The difference between this comparative example and Example 1 is that the pre-fermentation time of the pectin substrate was adjusted to 24 hours, while other parameters and conditions were the same as in Example 1, and a bacterial fermentation agent was obtained.

[0055] Comparative Example 4

[0056] The difference between this comparative example and Example 1 is that the molecular weight of the pectin used is 15kDa, while the other parameters and conditions are the same as in Example 1, and the bacterial fermentation agent is obtained.

[0057] The biomass of Lactobacillus rhamnosus in the bacterial fermentation broths prepared in each embodiment and comparative example is shown in Table 1.

[0058] Table 1

[0059] Lactobacillus rhamnosus biomass (CFU / mg) Example 1 <![CDATA[6.20*10 10 ]]> Example 2 <![CDATA[5.40*10 10 ]]> Example 3 <![CDATA[5.77*10 10 ]]> Example 4 <![CDATA[5.66*10 10 ]]> Example 5 <![CDATA[5.75*10 10 ]]> Comparative Example 1 <![CDATA[3.79*10 10 ]]> Comparative Example 2 <![CDATA[3.91*10 10 <!-- 4 -->]]> Comparative Example 3 <![CDATA[4.12*10 10 ]]> Comparative Example 4 <![CDATA[4.06*10 10 ]]>

[0060] As shown in the table above, the biomass of *Lactobacillus rhamnosus* in Comparative Example 1 was much lower than that in Examples 1-5, indicating that the pectin substrate without pre-fermentation cannot be directly utilized by *Lactobacillus rhamnosus*. Similarly, when *Lactobacillus rhamnosus* and *Bacteroides polymorpha* were inoculated simultaneously (Comparative Example 2), the biomass of *Lactobacillus rhamnosus* also failed to increase effectively. This is because the rapid proliferation and acid production of *Lactobacillus rhamnosus* inhibited the growth of *Bacteroides polymorpha*. Figure 2 This resulted in pectin not being pre-degraded by *Bacteroides multiforme* during mixed-culture fermentation. These results indicate that using a stepwise inoculation method to ensure that *Bacteroides multiforme* pre-degrades pectin into oligosaccharide fragments is a prerequisite for subsequently promoting the proliferation of *Lactobacillus rhamnosus*. Furthermore, in Comparative Example 3, extending the pre-fermentation time of *Bacteroides multiforme* to 24 hours led to a significant decrease in *Lactobacillus rhamnosus* biomass, attributed to excessive pre-fermentation consuming other nutrients in the culture medium. In Comparative Example 4, pectin with a molecular weight of 15 kDa was used as the substrate, but it failed to effectively increase the final *Lactobacillus rhamnosus* biomass. Figure 3 The result is that Bacteroides polymorpha failed to effectively degrade pectin with a molecular weight greater than 15 kDa.

[0061] Compared to Example 1, the final Lactobacillus rhamnosus biomass decreased in Examples 2 and 3 after shortening or extending the pre-fermentation time of Bacteroides multiforme by 2 hours. This is because insufficient pre-fermentation time may hinder the sufficient growth of Bacteroides multiforme to pre-degrade pectin, while excessive pre-fermentation time may lead to the loss of pectin and other nutrients. Therefore, the optimal technical effect can be obtained when the pre-fermentation time of the pectin substrate is 12 hours in this invention. In Example 4, reducing the initial inoculum size of Lactobacillus rhamnosus prevents it from quickly entering the logarithmic growth phase, thus affecting the final biomass. Conversely, increasing the initial inoculum size can promote the rapid proliferation of Lactobacillus rhamnosus, but may interfere with the pectin degradation efficiency of Bacteroides multiforme due to competitive inhibition. In summary, this invention significantly improves the utilization capacity of Lactobacillus rhamnosus for pectin by using oligosaccharide fragments produced by the pre-degradation of pectin by Bacteroides multiforme, thereby promoting its efficient proliferation. Compared with traditional single fermentation, the biomass of Lactobacillus rhamnosus can be increased by 40% to 65%. This invention promotes the proliferation of *Lactobacillus rhamnosus* and significantly increases its biomass during the stationary phase through stepwise inoculation and co-fermentation. This method cleverly utilizes the complementary ecological niches of the two bacterial species: *Bacteroides polymorpha* acts as a primary degrader responsible for breaking down the complex pectin structure, while *Lactobacillus rhamnosus* further utilizes the degradation products through its metabolic capacity, forming a highly efficient cross-feeding network and optimizing pectin utilization efficiency. This co-culture model overcomes the limitations of single-species pectin utilization and simulates the natural process of intestinal flora co-metabolizing pectin, contributing to the development of fermentation preparations that more closely resemble the intestinal microecology. This technology can be widely applied to the production of probiotic feed preparations, providing new technical support for improving the probiotic effects of pectin and developing products that target and regulate the intestinal microecology.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on Bacteroides polymorphonuclear pluripotentiol pre-degradation pectin, characterized in that: include, Strain activation: Bacteroides polymorpha and Lactobacillus rhamnosus were inoculated into activation medium and cultured at 37°C under anaerobic conditions. Pre-fermentation: Bacteroides polymorpha was transferred to a fermentation medium with pectin as the sole carbon source and anaerobic fermentation was carried out at 37°C to obtain a pre-fermentation broth; Secondary co-culture: Using the pre-fermentation broth as a secondary culture medium, Lactobacillus rhamnosus was inoculated for mixed culture fermentation; Fermentation termination: Fermentation is terminated when the number of viable Lactobacillus rhamnosus reaches the stationary phase. The cells are collected by centrifugation and freeze-dried to obtain the cell fermentation agent.

2. The co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha as described in claim 1, characterized in that: The Bacteroides polymorpha includes strain-78496.

3. The co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha as described in claim 1, characterized in that: OD after activation of Bacteroides polymorpha 600 ≥1.2, OD of activated Lactobacillus rhamnosus 600 ≥0.

8.

4. The co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha as described in claim 1, characterized in that: The pre-fermentation process involves the following components: 1-3% (v / v) of Bacteroides polymorpha inoculum; fermentation medium composition: 1.0-3.0 g / L soybean peptone, 3.0-5.0 g / L yeast extract, 2.0-4.0 g / L beef meal, 0.5-2.0 g / L potassium dihydrogen phosphate, 1.0-3.0 g / L sodium chloride, 0.1-0.3 g / L L-cysteine, 0.1-0.3 g / L sodium thioglycolate, 0.0005-0.001 g / L vitamin K1, 2.5-5 mg / L heme chloride, and 5.0-8.0 g / L pectin; fermentation time: 10-14 h.

5. The co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha as described in claim 4, characterized in that: The pectin has the following structural characteristics: molecular weight ≤12kDa, degree of esterification ≤50%, and galacturonic acid content ≤75%.

6. The co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha as described in claim 1, characterized in that: The secondary co-culture involves an inoculum of Lactobacillus rhamnosus of 2–6% (v / v) and a fermentation time of 16–24 h.

7. The co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha as described in claim 1, characterized in that: Oligosaccharide fragments were produced by pre-fermenting pectin with Bacteroides polymorpha, and after co-culture, the biomass of Lactobacillus rhamnosus reached more than 10 LogCFU / mL during the stationary phase.

8. The co-fermentation method for promoting the proliferation of Lactobacillus rhamnosus based on pre-degradation of pectin by Bacteroides polymorpha as described in claim 1, characterized in that: During the termination of fermentation, the stabilization period is characterized by a fluctuation of ±5% in the cell count.

9. A bacterial starter culture prepared by the co-fermentation method according to any one of claims 1 to 8, based on the pre-degradation of pectin by Bacteroides polymorpha to promote the proliferation of Lactobacillus rhamnosus, characterized in that: The biomass of *Lactobacillus rhamnosus* in the bacterial fermentation ant is 10. 10 CFU / mg or higher.

10. The application of the microbial fermentation agent as described in claim 9 in animal feed additives.

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