Lactobacillus gasseri jm1 fermentation culture additive and use thereof

Abstract

The present application belongs to the field of microbial fermentation and microbial food technology, and particularly relates to a fermentation culture additive of Lactobacillus gasseri JM1 and use thereof. The fermentation culture additive is selected from at least two of polysaccharides of Irpex lacteus, Hericium erinaceus and Inonotus obliquus, or inactivated products of fermentation products of Lactobacillus gasseri JM1. The fermentation culture additive can promote the growth and proliferation of the strain when Lactobacillus gasseri JM1 is fermented. The present application also provides a beverage, and the preparation raw materials include milk powder, polysaccharide products and inactivated Lactobacillus gasseri JM1 blended fermentation products. The polysaccharides in the polysaccharide products are selected from at least two of polysaccharides of Irpex lacteus, Hericium erinaceus and Inonotus obliquus. The inactivated Lactobacillus gasseri JM1 blended fermentation products are added in the fermentation stage of Lactobacillus gasseri JM1, and the polysaccharide products are further added after beverage ingredients are prepared, which is beneficial to improve the viable count and physical stability of the beverage.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and microbial food technology, specifically relating to a fermentation culture additive for Lactobacillus gasseri JM1 and its uses. Background Technology

[0002] Lactobacillus gasseri ( Lactobacillus gasseri Lactobacillus JM1 is a strain of lactobacillus isolated from the feces of healthy infants by our unit (Northeast Agricultural University). This strain and one application have been disclosed in patent document CN202410426416.3 (Fermentation Method and Application of Lactobacillus gasseri JM1). This patent document provides a method for fermenting soybean flour using Lactobacillus gasseri JM1. However, the fermentation growth of Lactobacillus gasseri JM1 is still not ideal. The absorbance (OD600) of the culture medium obtained by culturing at 37℃ for 48 hours using traditional MRS medium (3% inoculum, 2% carbon source) is low (below 0.3). This limits the industrial application of Lactobacillus gasseri JM1.

[0003] Furthermore, patent document CN202410426416.3 discloses the advantages of Lactobacillus gasseri JM1 in fermented soybean flour, including improving the antioxidant activity and calcium content of soybean milk. However, research on other functions of Lactobacillus gasseri JM1, which does not contain plant protein, including its stability, is lacking.

[0004] In view of the above, there is still a need in the field to further improve the fermentation survival rate of Lactobacillus gasseri JM1 and improve the stability of foods containing Lactobacillus gasseri JM1.

[0005] Obviously, the above background information contains technical information that the inventors had previously researched but was not disclosed before the application date. Apart from documents already published before the application date, the above background information does not imply that a person skilled in the art was aware of all the above technical information or the existence of the above technical problems before the application date. Summary of the Invention

[0006] To address the aforementioned technical problems, the purpose of this invention is to provide a fermentation culture additive for Lactobacillus gasseri JM1 and its uses. Therefore, the technical solution provided by this invention is as follows:

[0007] A fermentation culture additive for Lactobacillus gasseri JM1, wherein the fermentation culture additive is selected from at least two of the following: Hericium erinaceus polysaccharide, Hericium erinaceus polysaccharide, and Inonotus obliquus polysaccharide, and wherein the fermentation culture additive contains at least Inonotus obliquus polysaccharide.

[0008] Preferably, the fermentation culture additive is composed of *Clerodendrum trichotomum* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide. More preferably, the weight ratio of *Clerodendrum trichotomum* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide in the fermentation culture additive is (1~2):(1~2):(1~2). Even more preferably, the weight ratio of *Clerodendrum trichotomum* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide in the fermentation culture additive is 1:2:2.

[0009] Another fermentation culture additive for Lactobacillus gasseri JM1 is an inactivated product obtained from the co-fermentation of milk powder, polysaccharide products, and Lactobacillus gasseri JM1. The polysaccharide product is selected from at least two of the following: *Lactobacillus thunbergii* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide, and the polysaccharide product includes at least *Inonotus obliquus* polysaccharide. The difference between this Lactobacillus gasseri JM1 fermentation culture additive and the first Lactobacillus gasseri JM1 fermentation culture additive is that it undergoes further fermentation processing; that is, the inactivated product after fermentation treatment of Lactobacillus gasseri JM1 is used as the additive for Lactobacillus gasseri JM1 fermentation culture.

[0010] Preferably, the polysaccharide product is composed of *Inonotus obliquus* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide. More preferably, the weight ratio of *Inonotus obliquus* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide in the polysaccharide product is (1~2):(1~2):(1~2). Even more preferably, the weight ratio of *Inonotus obliquus* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide in the polysaccharide product is 1:2:2.

[0011] Preferably, the inactivation method is as follows: first, ultra-high pressure treatment is performed, followed by ultrasonic treatment; the conditions for ultra-high pressure treatment are: treatment pressure 300~400MPa, treatment time 5~10min; the conditions for ultrasonic treatment are: ultrasonic frequency 20~30 kHz, ultrasonic power 200~300w, ultrasonic treatment time 5~10min.

[0012] The present invention also provides the use of the aforementioned Lactobacillus gasseri JM1 fermentation culture additive as a fermentation culture additive to promote the growth of Lactobacillus gasseri JM1. Specifically, it is added to the fermentation culture medium of Lactobacillus gasseri JM1 as an additive to promote the growth and proliferation of Lactobacillus gasseri JM1.

[0013] The present invention further provides a beverage, the preparation method of which includes the following steps:

[0014] Fermentation preparation stage of S1 Lactobacillus gasseri JM1 fermentation product: Dissolve milk powder, add polysaccharide products, inoculate with Lactobacillus gasseri JM1, ferment at 37℃ for 24h, and freeze-dry to obtain Lactobacillus gasseri JM1 fermentation product.

[0015] Preparation stage of fermentation substrate of Lactobacillus gasseri JM1 in step S2: Dissolve milk powder, add fermentation product obtained in step S1, inoculate with Lactobacillus gasseri JM1, ferment at 37°C until pH is below 3.8, and obtain fermentation substrate of Lactobacillus gasseri JM1.

[0016] S3 Beverage Ingredient Preparation Stage: The fermentation base obtained in step S2 is mixed with water and beverage additives, the pH value is adjusted, and homogenization is performed to obtain the beverage.

[0017] The polysaccharide product is composed of at least two of the following: white thorn fungus polysaccharide, hericium erinaceus polysaccharide, and Inonotus obliquus polysaccharide, and the polysaccharide product contains at least Inonotus obliquus polysaccharide;

[0018] The beverage's excipients include at least two of the following: *Inonotus obliquus* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide, with at least *Inonotus obliquus* polysaccharide included.

[0019] Preferably, in step S1, the inactivation method is as follows: first, ultra-high pressure treatment is performed, and then ultrasonic treatment is performed; the conditions for ultra-high pressure treatment are: treatment pressure 300~400MPa, treatment time 5~10min; the conditions for ultrasonic treatment are: ultrasonic frequency 20~30 kHz, ultrasonic power 200~300w, ultrasonic treatment time 5~10min.

[0020] Preferably, in step S3, the pH value is adjusted to 4.0~4.5.

[0021] Preferably, in step S2, the content of milk powder in the milk powder dissolution product is 13g / 100mL; the amount of fermentation product added is 2g / 100mL.

[0022] The "auxiliary materials" mentioned in step S3 refer to additives other than water (water is also an auxiliary material), such as white sugar, monoglycerides (dihydroxypropyl octadecanoate), sucrose esters (sucrose fatty acid esters), carboxymethyl cellulose, propylene glycol alginate, etc.

[0023] The aforementioned polysaccharides from *Inonotus obliquus*, *Hericium erinaceus*, and *Inonotus obliquus* are all commercially available polysaccharides, with a purity exceeding 70% as determined by the phenol-sulfuric acid method. The Latin name of the aforementioned *Inonotus obliquus* is... Irpexlacteus Hericium erinaceus, Latin name: Hericium erinaceus The Latin name of Inonotus obliquus is Inonotus obliquus.

[0024] The inventors' research revealed that,Adding polysaccharides from *Lactobacillus thuringiensis*, *Hericium erinaceus*, and *Inonotus obliquus* to the culture medium can enhance the growth / proliferation rate of *Lactobacillus gasseri* JM1 during fermentation. In particular, complex polysaccharides composed of these three polysaccharides in specific proportions, such as a 1:2:2 weight ratio, show a more significant promoting effect on the growth and proliferation of *Lactobacillus gasseri* JM1. Furthermore, after a single fermentation of *Lactobacillus thuringiensis*, *Hericium erinaceus*, and *Inonotus obliquus* polysaccharides with the mixture, followed by inactivation of the fermentation product, and then repeated fermentation with *Lactobacillus gasseri* JM1, the effect on promoting the growth / proliferation rate of the *Lactobacillus gasseri* JM1 strain is even more pronounced.

[0025] The aforementioned term "polysaccharide product" refers to polysaccharides or combinations thereof (physical mixtures of several polysaccharides); it differs from the aforementioned fermentation products because fermentation products are a more complex system of substances resulting from the fermentation of polysaccharides by Lactobacillus gasseri JM1.

[0026] The aforementioned Lactobacillus gasseri JM1, namely Lactobacillus gasseri JM1 is a type of Lactobacillus isolated from the feces of healthy infants at Northeast Agricultural University. This strain has been disclosed in patent document CN202410426416.3 (Fermentation Method and Application of Lactobacillus gasseri JM1) and the dissertation "Study on the Immunomodulatory Effect and Regulatory Pathway of a Strain of Lactobacillus gasseri" (Sun Linlin, Northeast Agricultural University, 2020 thesis). This strain is deposited by the Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, and its 16S rDNA sequence has been determined and uploaded to the National Center for Biotechnology Information (NCBI) in the United States. Furthermore, in patent document CN202410426416.3 (Fermentation Method and Application of Lactobacillus gasseri JM1), the depository institution has provided a certificate / declaration for this strain to be distributed free of charge to the public.

[0027] The beneficial effects of this invention are:

[0028] (1) This invention, through research on the fermentation growth of Lactobacillus gasseri JM1, obtained a polysaccharide and its composition or fermentation product that can significantly promote the fermentation growth and proliferation of Lactobacillus gasseri JM1. This improves the productivity of Lactobacillus gasseri JM1, thus providing a good foundation for its large-scale industrial application.

[0029] (2) Through research on the inactivation methods of Lactobacillus gasseri JM1, this invention further obtained an inactivation method for an additive fermentation product that can promote the fermentation growth and proliferation of Lactobacillus gasseri JM1. The fermentation product prepared using this inactivation method, when used for Lactobacillus gasseri JM1 fermentation, can improve the fermentation growth and proliferation of Lactobacillus gasseri JM1.

[0030] (3) By investigating different preparation methods of fermented milk beverages, this invention further obtained a fermented milk beverage prepared using polysaccharide products and their fermentation products, which has a high number of live bacteria and stability.

[0031] In summary, this invention, through the investigation of polysaccharides from *Lactobacillus gasseri* JM1, *Hericium erinaceus*, and *Inonotus obliquus*, has yielded a novel fermentation method for *Lactobacillus gasseri* JM1 and a fermented milk beverage. This fills a technological gap in the production technology of *Lactobacillus gasseri* JM1 and the production technology of fermented milk beverages, providing a solid foundation for its industrial production and application in the food industry. Detailed Implementation

[0032] To clearly demonstrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with embodiments.

[0033] In the following examples, Lactobacillus gasseri JM1, namely Lactobacillus gasseri JM1 is a type of Lactobacillus isolated from the feces of healthy infants at Northeast Agricultural University. This strain has been disclosed in patent document CN202410426416.3 (Fermentation Method and Application of Lactobacillus gasseri JM1) and the dissertation "Study on the Immunomodulatory Effect and Regulatory Pathway of a Strain of Lactobacillus gasseri" (Sun Linlin, Northeast Agricultural University, 2020 thesis). This strain is deposited by the Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, and its 16S rDNA sequence has been determined and uploaded to the National Center for Biotechnology Information (NCBI) in the United States. Furthermore, in patent document CN202410426416.3 (Fermentation Method and Application of Lactobacillus gasseri JM1), the depository institution has provided a certificate and statement that this strain will be made available to the public free of charge.

[0034] In the following examples, the polysaccharides of *Hericium erinaceus*, *Hericium erinaceus*, and *Inonotus obliquus* were all purchased from Shaanxi Yuzhou Biotechnology Co., Ltd., and their purity was above 70% as determined by the phenol-sulfuric acid method.

[0035] Prior to the experiments described in the following embodiments, the inventors conducted preliminary exploratory experiments, including comparisons with polysaccharides from Bacillus amyloliquefaciens JM033, Lactobacillus rhamnosus JM039, and other monosaccharides or oligosaccharides. Clearly, the following embodiments are only for explaining the present invention and are not intended to limit the scope of protection of the present invention or to represent that those skilled in the art can obtain the solutions of the present invention without creative effort.

[0036] Example 1: Effects of different polysaccharides and compositions on the fermentation and proliferation of Lactobacillus gasseri JM1

[0037] A 10% (10g / 100mL) whole milk powder culture medium (excluding glucose and other carbon sources) was prepared, with 2% sugar (2g / 100mL; see Table 1 for the types and ratios of sugars added), sterilized, and inoculated with Lactobacillus gasseri JM1 at an inoculation amount of 3%; cultured at 37℃ for 24h, and the fermentation broth was collected for viable cell counting after fermentation was completed.

[0038] Table 1. Effects of polysaccharide addition types and proportions (by weight) on viable cell count in fermentation broth

[0039]

[0040] Note: The viable cell count data in Table 1 are the mean values ​​calculated from the measurements of three batches of samples. The letters following the values ​​are significance markers. Different letters indicate significant differences between groups; the same letter indicates no significant differences between groups. For example, the letter 'j' after the viable cell count data for groups A, B, and C indicates no significant differences among multiple groups within these three groups; the letter 'i' after the viable cell count data for groups B, C, and D indicates no significant differences among multiple groups within these three groups; and the letter 'h' after the viable cell count data for groups D and E indicates no significant differences between these two groups.

[0041] As shown in Table 1, compared with whole milk powder medium containing glucose (2%) but without polysaccharides, the whole milk powder medium with added polysaccharides, at the same addition amount (2%), showed at least a 1.5-fold increase in the viable count of Lactobacillus gasseri JM1 in the fermentation product after fermentation (Group B). vs Group A). ​​However, except for Inonotus obliquus polysaccharide, there were no significant differences between the polysaccharides of Inonotus alba, Hericium erinaceus, and glucose groups. When single polysaccharides were added, the order of their effects on promoting the proliferation of Lactobacillus gasseri JM1 was: Inonotus obliquus polysaccharide (Group D) > Hericium erinaceus polysaccharide (Group C) > Inonotus alba polysaccharide (Group B).

[0042] When two polysaccharide combinations were added, the order of their effects on promoting the proliferation of *Lactobacillus gasseri* JM1 was: Hericium erinaceus polysaccharide + Inonotus obliquus polysaccharide (Group G, weight ratio of the two polysaccharides 1:1) > *Inonotus alba* polysaccharide + Inonotus obliquus polysaccharide (Group F, weight ratio of the two polysaccharides 1:1) > *Inonotus alba* polysaccharide + Hericium erinaceus polysaccharide (Group E, weight ratio of the two polysaccharides 1:1). Furthermore, the effect of adding polysaccharides in pairs on promoting the proliferation of *Lactobacillus gasseri* JM1 was stronger than that of adding a single polysaccharide. However, the effect of the *Inonotus alba* polysaccharide + Hericium erinaceus polysaccharide combination (Group E) was not significantly different from that of the *Inonotus obliquus* polysaccharide group (Group D).

[0043] When the three polysaccharide combinations were added, the weight ratio of *Lactobacillus thuringiensis* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide was 1:2:2, which significantly promoted the proliferation of *Lactobacillus gasseri* JM1 than other combinations of the three polysaccharides. Moreover, when the three polysaccharide combinations were added, the promotion effect on the proliferation of *Lactobacillus gasseri* JM1 was significantly higher than that of the two polysaccharide combinations.

[0044] Example 2: Effects of fermentation products obtained by different inactivation methods on the fermentation and proliferation of Lactobacillus gasseri JM1

[0045] The fermentation product from Example 1 was inactivated using different inactivation processes (see Table 2 for treatment methods), and then freeze-dried to prepare the fermentation broth. The fermentation broth was then added at a rate of 2% (2 g / 100 mL) to 10% (10 g / 100 mL) whole milk powder medium (which does not contain glucose or other carbon sources) according to the method in Example 1 (the sugar addition method is the same as in Example 1), and inoculated with 3% *Lactobacillus gasseri* JM1. The mixture was incubated at 37°C for 24 h, and viable cell counts were performed. The effects of different inactivation methods on the fermentation and proliferation of *Lactobacillus gasseri* JM1 were investigated.

[0046] The thermal inactivation conditions are as follows: the inactivation effect is achieved by setting the water bath temperature and time. The temperature is 60~80℃ (70℃ is selected in this embodiment), and the holding time is 10~30min (15min is selected in this embodiment).

[0047] The ultrasonic conditions are as follows: ultrasonic frequency is 20~40kHz (30kHz is selected in this embodiment), power is 200~500w (300w is selected in this embodiment), processing time is 5~15min (10min is selected in this embodiment), and during the processing, there is a 1min pause every 2min of processing;

[0048] The ultra-high pressure conditions are: 300~500 MPa (400 MPa is selected in this embodiment), 5~15 min (10 min is selected in this embodiment);

[0049] Ultra-high pressure + ultrasonic conditions: First, ultra-high pressure treatment is performed, followed by ultrasonic treatment. The ultra-high pressure treatment conditions are 300~400 MPa (350 MPa is selected in this embodiment), 5~10 min (8 min is selected in this embodiment), ultrasonic frequency is 20~30 kHz (25 kHz is selected in this embodiment), power is 200~300 W (250 W is selected in this embodiment), and treatment time is 5~10 min (8 min is selected in this embodiment).

[0050] Table 2. Effects of fermentation products obtained by different inactivation methods on the viable count of Lactobacillus gasseri JM1

[0051]

[0052] Note: The viable cell count data in Table 2 are the mean values ​​calculated from the measurements of three batches of samples. The letters after the values ​​are significance markers. Different letters indicate significant differences between groups; the same letter indicates no significant differences between groups (see "Note" in Table 1 for examples of specific meanings).

[0053] As shown in Table 2, under the same heat inactivation conditions, compared with the fermentation product containing glucose (2%) but no polysaccharide (Group A1), the fermentation products of all polysaccharide groups increased the viable count of *Lactobacillus gasseri* JM1 (Groups B1 to L1). Among them, except for Group B1, the viable count of *Lactobacillus gasseri* JM1 after use was significantly higher in the other groups than in the fermentation product containing glucose (2%) (Group A1). In the fermentation products with added single polysaccharides, except for *Inonotus obliquus* polysaccharide (Group B1), the viable count of *Lactobacillus gasseri* JM1 after use was significantly higher in the fermentation product containing glucose (2%) (Group A1) after use. When two polysaccharides were added, the viable count of *Lactobacillus gasseri* JM1 after use was significantly higher in the fermentation product containing *Inonotus obliquus* polysaccharide than in all groups containing single polysaccharides. That is, the viable count of *Lactobacillus gasseri* JM1 in Groups F1 and G1 was significantly higher than in Groups A, B, C, and D. The viable count of Lactobacillus gasseri JM1 in the fermentation products obtained by adding all three polysaccharides was significantly higher than that in the groups with two polysaccharides or the groups with a single polysaccharide. Specifically, the viable count of Lactobacillus gasseri JM1 in the H1, I1, J1, K1, and L1 groups was significantly higher than that in the A, B, C, D, E, F, and G groups.

[0054] When comparing different inactivation methods, the comparison from group L1 to group L4 (all groups using *Lactobacillus oryzae* polysaccharide: *Hericium erinaceus* polysaccharide: *Inonotus obliquus* polysaccharide = 1:2:2) showed that the fermentation product of the ultra-high pressure + ultrasonic inactivation group (group L4) significantly increased the viable count of *Lactobacillus gasseri* JM1 (group L4). vs Groups L1, L2, and L3). Specifically, the use of ultrasonic disruption and ultra-high pressure mixing significantly improved the proliferative capacity of the fermentation complex for *Lactobacillus gasseri* JM1, achieving a viable count of 1.15 × 10⁻⁶. 9 CFU / mL. High pressure disrupts the cell membrane and cell wall, enhancing the inactivation effect; while the cavitation and mechanical effects of ultrasound further damage the cell structure. Therefore, ultrasound treatment after high-pressure inactivation can further destroy the remaining bacterial cells, thereby causing the fermentation complex to release more substances that are beneficial to the proliferation of Lactobacillus gasseri JM1.

[0055] Example 3: Different preparation methods of fermented milk beverages and their effects on the viable count and physical properties of fermented milk beverages

[0056] The L-group polysaccharides (see Example 1, i.e., *Lactobacillus thuringiensis* polysaccharide: *Hericium erinaceus* polysaccharide: *Inonotus obliquus* polysaccharide = 1:2:2) and their inactivated fermentation products (see Example 2, L4 group fermentation products, inactivated using the same method as L4 group) were applied to the preparation of *Lactobacillus gasseri* JM1 fermented milk beverages. The effects of the complex polysaccharides and fermentation products on the fermented milk beverages were investigated.

[0057] The preparation steps for different fermented milk beverages are as follows (the culture medium and additives must be routinely sterilized before inoculation with Lactobacillus gasseri JM1):

[0058] (1) Preparation of different fermentation substrates

[0059] Dissolve whole milk powder in water (w / v concentration of whole milk powder is 13%, i.e., 13g / 100mL), and sterilize at 95℃ for 3 hours. Then, add L-group polysaccharide under aseptic conditions (for comparative examples 1 and 2, add equal amounts of inulin or glucose respectively). The amount of sugar added is 2% (w / v, i.e., 2g / 100mL). Heat to 45℃ and stir for 10 minutes, then hydrate for 30 minutes. Cool to 37℃, and then inoculate with Lactobacillus gasseri JM1 (inoculation amount is 1×10⁻⁶). 6 The fermentation substrate (CFU / mL) was placed in a 37℃ constant temperature incubator for fermentation. Once the fermentation endpoint was reached (pH below 3.80 is acceptable; pH 3.6~3.8 was selected here), fermentation was stopped to obtain the fermentation substrates for each group (i.e., using sugar or polysaccharide fermentation products directly as fermentation substrates without secondary fermentation).

[0060] In addition, the fermentation substrate was prepared using the fermentation product of group L4 in Example 2 (ultra-high pressure + ultrasonic inactivation, method as in Example 2) as follows (i.e., the primary fermentation product of polysaccharide was inactivated and freeze-dried, and then fermented again with Lactobacillus gasseri JM1, and the resulting product was used as the fermentation substrate): Whole milk powder was dissolved in water (the mass-volume concentration of whole milk powder was 13%, i.e., 13g / 100mL), and after sterilization at 95℃ for 3 h, the fermentation product of group L4 in Example 2 (ultra-high pressure + ultrasonic inactivation, method and conditions as in Example 2 L4 group) was added under aseptic conditions at a concentration of 2% (w / v, i.e., 2g / 100mL); the mixture was heated to 45℃ and stirred for 10 min, then hydrated for 30 min, cooled to 37℃, and then inoculated with Lactobacillus gasseri JM1 (inoculation amount 1×10⁻⁶). 6 The fermentation material (CFU / mL) was placed in a 37℃ constant temperature incubator for fermentation. After reaching the fermentation endpoint (pH below 3.80 is acceptable; pH 3.6~3.8 was selected here), the fermentation was stopped to obtain the fermentation substrate prepared by secondary fermentation of L4 fermentation material.

[0061] (2) Preparation of basic ingredients

[0062] Using different fermentation substrates prepared in step (1), the basic ingredients were prepared according to the ingredient list in Table 3, resulting in four basic ingredients, which were named as the basic ingredients of Experimental Example 1, the basic ingredients of Experimental Example 2, the basic ingredients of Comparative Example 1, and the basic ingredients of Comparative Example 2, respectively.

[0063] Table 3. Ingredients List for Basic Ingredients

[0064]

[0065] (3) The addition of basic ingredients, i.e. the preparation of fermented milk beverages

[0066] L-group polysaccharides (see Example 1) or their fermentation products (see Example 2, preparation and inactivation of L4-group fermentation products) were added to the basic ingredients of Experimental Example 1, Experimental Example 2, Comparative Example 1, and Comparative Example 2, respectively, to obtain eight kinds of fermented milk beverages (see Table 4). That is, after the basic ingredients were prepared, polysaccharides or their fermentation products were added. The amount of L-group polysaccharides or their fermentation products added was 1% (w / v, i.e., 1g / 100mL). After addition, the pH value was adjusted to 4.0~4.5. Homogenization (5 MPa~10 MPa, temperature 10°C~15°C; the homogenization conditions were the same for different milk beverages) was performed to obtain fermented milk beverages.

[0067] Samples of fermented milk beverages were taken separately and their viable cell count, viscosity, and centrifugal sedimentation rate were determined.

[0068] Viable bacteria count: The count was performed using the plate count method, referring to the national standard "National Food Safety Standard for Microbiological Examination of Food - Examination of Lactic Acid Bacteria" (GB4789.35-2023).

[0069] Viscosity: The viscosity of the milk beverage was measured using a viscometer with a rotor range of 100 mPa·s, a rotation speed of 60 rpm, and an equilibration time of 2 min.

[0070] Centrifugation sedimentation rate: Take 10g of the prepared fermented milk beverage into a centrifuge tube, the mass of the centrifuge tube is m0, centrifuge at 3500rpm for 10min, pour out the supernatant and invert for 10min, weigh the obtained precipitate and the total mass of the centrifuge tube as m1, centrifugation sedimentation rate = (m1-m0) / 10.

[0071] Table 4 compares the ingredients at each stage of preparation for different fermented milk beverages.

[0072] Table 4 Comparison of ingredients at different stages of preparation of different fermented milk beverages

[0073]

[0074] Table 5 shows the test results for different fermented milk beverages.

[0075] Table 5. Results of determination of different fermented milk beverages

[0076]

[0077] Note: The viable cell count, viscosity, and centrifugal sedimentation rate data in Table 5 are the average values ​​calculated from the measurements of three batches of samples. The letters after the values ​​are significance markers. Different letters indicate significant differences between groups; the same letter indicates no significant differences between groups (see "Note" in Table 1 for examples of specific meanings).

[0078] Analysis of the viable count of *Lactobacillus gasseri* JM1 showed no significant differences among comparative groups 1A, 2A, 1B, and 2B. While there were no significant differences between experimental examples 1A and 1B, or between 2A and 2B, significant differences were observed between experimental examples 1A and 2A, and between 1B and 2B. The fermented milk beverage in example 2A had the highest viable count of *Lactobacillus gasseri* JM1. This indicates that further addition of L-group or L4-group polysaccharide fermentation products to the base ingredients had no significant effect on the viable count of *Lactobacillus gasseri* JM1 in the fermented milk beverage. However, during the preparation of the fermentation base, the use of L4-group polysaccharide fermentation products significantly increased the viable count compared to the use of L-group polysaccharides.

[0079] Viscosity analysis showed that the viscosities of Experimental Examples 1A and 2A were significantly higher than those of the other groups, with Experimental Example 2A exhibiting a significantly higher viscosity than Experimental Example 1A. This indicates that the addition of L-group polysaccharides to the base ingredients is beneficial for increasing the viscosity of fermented milk beverages, and that the product viscosity is even better when combined with the use of L4-group polysaccharide fermentation products in the fermentation base preparation stage.

[0080] Analysis of centrifugal sedimentation rates showed that the sedimentation rates of Experimental Examples 1A and 2A were significantly lower than those of the other groups, and the sedimentation rate of Experimental Example 2A was significantly lower than that of Experimental Example 1A. This indicates that the further addition of L-group polysaccharides to the basic ingredients is beneficial to improving the stability of fermented milk beverages, and the product stability is even better when combined with the use of L4-group polysaccharide fermentation products in the fermentation base preparation stage.

Claims

1. An additive used as a carbon source in the fermentation culture of Lactobacillus gasseri JM1, characterized in that, The additives used as carbon sources in the fermentation culture consist of *Tetranychus sylvestris* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide; the weight ratio of *Tetranychus sylvestris* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide in the fermentation culture is (1~2):(1~2):(1~2).

2. The additive used as a carbon source in the fermentation culture of Lactobacillus gasseri JM1 according to claim 1, characterized in that, In the fermentation culture, the weight ratio of the polysaccharides of *Hericium erinaceus*, *Hericium erinaceus*, and *Inonotus obliquus* is 1:2:2, which are used as carbon sources.

3. A deep-processed Lactobacillus gasseri JM1 fermentation culture additive, characterized in that, The deep-processed Lactobacillus gasseri JM1 fermentation culture additive is an inactivated product obtained from the co-fermentation of milk powder, polysaccharide products, and Lactobacillus gasseri JM1; the polysaccharide products are composed of white thorn polysaccharide, Hericium erinaceus polysaccharide, and Inonotus obliquus polysaccharide; in the polysaccharide products, the weight ratio of white thorn polysaccharide, Hericium erinaceus polysaccharide, and Inonotus obliquus polysaccharide is (1~2):(1~2):(1~2).

4. The deep-processed Lactobacillus gasseri JM1 fermentation culture additive according to claim 3, characterized in that, In the polysaccharide product, the weight ratio of *Bacillus thunbergii* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide is 1:2:

2.

5. The deep-processed Lactobacillus gasseri JM1 fermentation culture additive according to claim 3, characterized in that, The inactivation method is as follows: first, ultra-high pressure treatment is performed, followed by ultrasonic treatment; the conditions for ultra-high pressure treatment are: treatment pressure 300~400MPa, treatment time 5~10min; the conditions for ultrasonic treatment are: ultrasonic frequency 20~30kHz, ultrasonic power 200~300w, ultrasonic treatment time 5~10min.

6. The use of the additive as a carbon source in the fermentation culture of Lactobacillus gasseri JM1 as described in claim 1 or 2, or the use of the deep-processed Lactobacillus gasseri JM1 fermentation culture additive as described in any one of claims 3 to 5, as a fermentation culture additive to promote the growth of Lactobacillus gasseri JM1.

7. A beverage, characterized in that, The preparation method of the beverage includes the following steps: Fermentation preparation stage of S1 Lactobacillus gasseri JM1 fermentation product: Dissolve milk powder, add polysaccharide products, inoculate with Lactobacillus gasseri JM1, ferment at 37℃ for 24h, and freeze-dry to obtain Lactobacillus gasseri JM1 fermentation product. Preparation stage of fermentation substrate of Lactobacillus gasseri JM1 in step S2: Dissolve milk powder, add fermentation product obtained in step S1, inoculate with Lactobacillus gasseri JM1, ferment at 37°C until pH is below 3.8, and obtain fermentation substrate of Lactobacillus gasseri JM1. S3 Beverage Ingredient Preparation Stage: The fermentation base obtained in step S2 is mixed with water and beverage additives, the pH value is adjusted, and homogenization is performed to obtain the beverage. The polysaccharide product is composed of *Tetranychus sylvestris* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide; the weight ratio of *Tetranychus sylvestris* polysaccharide, *Hericium erinaceus* polysaccharide, and *Inonotus obliquus* polysaccharide in the polysaccharide product is (1~2):(1~2):(1~2).

8. A beverage according to claim 7, characterized in that, In step S1, the inactivation method is as follows: first, ultra-high pressure treatment is performed, followed by ultrasonic treatment; the conditions for ultra-high pressure treatment are: treatment pressure 300~400MPa, treatment time 5~10min; the conditions for ultrasonic treatment are: ultrasonic frequency 20~30 kHz, ultrasonic power 200~300w, ultrasonic treatment time 5~10min.

9. A beverage according to claim 7, characterized in that, In step S3, the pH value is adjusted to 4.0~4.

5.

10. A beverage according to claim 7, characterized in that, In step S2, the milk powder content in the milk powder dissolution product is 13g / 100mL; the amount of fermentation material added is 2g / 100mL.

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