Use of paracasei K56 in the preparation of products for improving intestinal motility and promoting defecation

Abstract

The application provides application of Lactobacillus paracasei K56 in preparation of products for improving intestinal peristalsis and promoting defecation, and relates to the technical field of biotechnology. The application comprises application of Lactobacillus paracasei K56 in preparation of products containing at least one efficacy of improving small intestinal propulsion rate of a subject, promoting intestinal peristalsis of the subject and promoting defecation of the subject, wherein the products comprise a fermentation product fermented by using a fermenting agent containing Lactobacillus paracasei K56, and the fermentation substrate contains milk; and the application comprises increasing the proportion of the number of dead Lactobacillus paracasei K56 cells in the fermented product relative to the total number of Lactobacillus paracasei K56 cells. The application alleviates the technical problem that inactivation of bacteria in a fermented product causes the probiotic function to be affected.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of Lactobacillus paracasei K56 in the preparation of products that improve intestinal motility and promote defecation. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Probiotic fermented milk beverages are fermented dairy products produced by combining traditional starter cultures with probiotics, thus combining the health benefits of traditional starter cultures with the health benefits of fermented milk. Probiotics can regulate the composition of the host's intestinal microbiota and its metabolites, alleviate intestinal inflammation, and promote intestinal peristalsis. Probiotic fermented milk beverages also have various beneficial functions such as anti-cholesterolemia, antioxidant, anti-tumor, and immune-enhancing effects. Through fermentation by probiotics, milk breaks down nutrients such as lactose, protein, and fat into various flavorful nutrients such as amino acids, organic acids, polypeptides, and fatty acids, not only retaining the milky aroma of fresh milk but also adding a unique fermented flavor. Probiotic fermented milk beverages are popular with consumers due to their rich nutrition, easy digestibility and absorption, and unique flavor.

[0004] However, many fermented milk beverages currently contain live bacteria, leading to instability, severe post-acidification, and insufficient levels of live lactic acid bacteria to meet industry standards, thus affecting shelf life and limiting sales to regional areas. In response, inactivated fermented milk beverages have emerged, offering longer shelf life, better stability, and eliminating post-acidification issues compared to live lactic acid bacteria beverages. However, since inactivated fermented milk no longer contains live bacteria, it loses the functions associated with live bacteria. For example, probiotics promote intestinal peristalsis and fecal excretion primarily because they continuously produce various organic acids in the intestines, such as lactic acid, acetic acid, and propionic acid. These organic acids lower the pH level in the intestines, acidifying it. When the intestinal pH is low, intestinal peristalsis is enhanced, promoting fecal excretion. Live probiotics continue to ferment dietary fiber and other substances, producing large amounts of gas and organic acids, increasing stool volume. Increased stool volume stimulates intestinal movement, promoting peristalsis and improving digestive problems such as constipation. However, when fermented milk is inactivated, the live bacteria die, and the fermented milk can no longer continuously produce organic acids and fermented dietary fiber in the intestines after consumption. It is evident that while sterilized fermented milk is convenient for storage and transportation, it undeniably loses the function of the live bacteria, especially when the starter culture contains probiotics, whose beneficial functions are affected. Therefore, how to improve this deficiency is a problem that needs to be solved.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of K56 in the preparation of products that improve intestinal motility and promote defecation, so as to alleviate the technical problem that the inactivation of strains in fermented products affects the probiotic function.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] In a first aspect, there is an application of Lactobacillus paracasei subsp. paracasei K56 in the preparation of a product containing at least one of (i) to (iii) effects, said product comprising a fermentation product fermented using a starter containing Lactobacillus paracasei K56, and the fermentation substrate containing milk; said application includes increasing the ratio of the number of dead Lactobacillus paracasei K56 cells to the total number of Lactobacillus paracasei K56 cells in the fermentation product;

[0009] (i) Improve the small intestinal propulsion rate of subjects;

[0010] (ii) Promotes intestinal peristalsis in the subjects;

[0011] (iii) Promote defecation in subjects.

[0012] In a second aspect, a method for preparing a fermented product is provided, the method comprising fermenting a milk-containing fermentation substrate using a fermentation agent containing *Lactobacillus paracasei* K56, and wherein the ratio of the number of dead *Lactobacillus paracasei* K56 cells in the fermented product to the total number of *Lactobacillus paracasei* K56 cells is 0 to 100%, and not 0%.

[0013] Thirdly, a fermented product is provided, wherein the fermentation agent of the fermented product contains Lactobacillus paracasei K56, and the fermentation substrate contains milk; the fermented product is sterilized after fermentation.

[0014] Fourthly, a method for storing a fermented product is provided, wherein the fermentation agent of the fermented product contains *Lactobacillus paracasei* K56, and the fermentation substrate contains milk; the storage method includes increasing the ratio of the number of dead *Lactobacillus paracasei* K56 cells in the fermented product to the total number of *Lactobacillus paracasei* K56 cells.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention, through experiments, reveals that the effect of K56 cell death in the fermentation product after milk-based fermentation of *Lactobacillus paracasei* K56 on efficacy differs from that of K56 without milk-based fermentation. The difference in efficacy between live and dead K56 cells after milk-based fermentation is smaller than that between live and dead cells, particularly in promoting intestinal motility and fecal excretion. Live *Lactobacillus paracasei* K56 is significantly superior to dead cells. However, after milk-based fermentation of *Lactobacillus paracasei* K56, sterilized and unsterilized fermented milk show comparable effects in promoting intestinal motility and fecal excretion; in fact, sterilized fermented milk even performs better in small intestinal propulsion tests. In conclusion, sterilized fermentation products of *Lactobacillus paracasei* K56 after milk-based fermentation retain or enhance some of the functions of unsterilized fermentation products. This discovery provides new insights into the preparation and preservation of fermented products.

[0017] Based on this discovery, the present invention provides the application of *Lactobacillus paracasei* K56 in the preparation of products having at least one effect of improving small intestinal propulsion rate, promoting intestinal peristalsis, and promoting defecation. By increasing the ratio of the number of dead *Lactobacillus paracasei* K56 cells in the fermented product to the total number of *Lactobacillus paracasei* K56 cells, the function of the fermented product in improving small intestinal propulsion rate, promoting intestinal peristalsis, and promoting defecation can be enhanced. The storage method of the fermented product provided by the present invention alleviates the defect of partial functional loss due to cell death during the storage of fermented products containing live bacteria; or, based on the above discovery, sterilized fermented products obtained from fermented milk bases containing K56 can also be prepared, which can also reduce the functional loss caused by strain inactivation while possessing the advantages of inactivated fermented products. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The results of blackout time in the model mice of Example 2 are statistical results.

[0020] Figure 2 The results of the fecal pellet count of the model mice in Example 2 after 6 hours;

[0021] Figure 3 The results of fecal water content analysis for model mice in Example 2;

[0022] Figure 4 This is a flowchart of the experiment in Example 4;

[0023] Figure 5 HE staining images of the colon of the blank group, model group, drug group, and lactic acid bacteria beverage-dead bacteria group in Example 2;

[0024] Figure 6 HE staining images of the colon of the K56 live bacteria group, K56 dead bacteria group, K56 fermented milk-live bacteria group, K56 fermented milk-dead bacteria group, compound bacteria fermented milk-live bacteria group and compound bacteria fermented milk-dead bacteria group in Example 2. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In this article, *Lactobacillus paracasei* subsp. *paracasei* K56 was isolated from the intestines of a healthy 2-year-old Chinese infant. *Lactobacillus paracasei* strain K56 was deposited on June 27, 2013, at the German Collection of Microorganisms and Cell Cultures (DSM27447); it was also deposited on December 29, 2017, at the China General Microbiological Culture Collection Center (CGMCC) (CGMCC 15139). In this article, *Lactobacillus paracasei* subsp. *paracasei* K56 is also referred to simply as "Lactobacillus paracasei K56" or "K56".

[0027] In this article, "total number of Lactobacillus paracasei K56 cells" refers to the total number of dead and live Lactobacillus paracasei K56 cells.

[0028] In this document, "fermented products" refers to products made through the metabolic activities of microorganisms, which transform organic matter in raw materials into other substances. "Fermented products" includes fermentation products obtained directly after fermentation and products obtained after further processing of fermentation products. Subsequent processing can be conventional processing methods known in the art for fermentation products, including but not limited to one or more of sterilization, drying, dilution, concentration, and flavoring. Examples of fermented products include, but are not limited to, yogurt, cheese, fermented milk beverages, lactic acid bacteria candies, and starter cultures (e.g., starter cultures obtained by fermenting a K56-containing starter culture in milk, followed by inactivation and drying).

[0029] This invention, through experiments, found that live K56 bacteria are significantly more effective than dead K56 bacteria in promoting intestinal peristalsis and fecal excretion. However, when K56 is fermented on a milk base and then sterilized to kill the K56 bacteria, the sterilized fermented milk (containing dead K56 bacteria) is comparable to the unsterilized fermented milk (containing live K56 bacteria) in promoting intestinal peristalsis and fecal excretion. In fact, the sterilized fermented milk even showed better performance in the small intestinal propulsion rate test.

[0030] In other words, this invention, through experiments, has found that after fermentation with K56 on a milk base, sterilization of the fermented milk to kill the K56 bacteria results in sterilized K56 fermented milk (containing dead K56 bacteria), which can further effectively enhance the effects of promoting intestinal peristalsis and fecal excretion. Based on this finding, the following technical solution is proposed:

[0031] In a first aspect, there is an application of Lactobacillus paracasei subsp. paracasei K56 in the preparation of a product containing at least one of (i) to (iii) effects, said product comprising a fermentation product fermented using a starter containing Lactobacillus paracasei K56, and the fermentation substrate containing milk; said application includes increasing the ratio of the number of dead Lactobacillus paracasei K56 cells to the total number of Lactobacillus paracasei K56 cells in the fermentation product;

[0032] (i) Improve the small intestinal propulsion rate of subjects;

[0033] (ii) Promotes intestinal peristalsis in the subjects;

[0034] (iii) Promote defecation in subjects.

[0035] In this solution, "increasing the ratio of the number of dead Lactobacillus paracasei K56 cells in the fermented product to the total number of Lactobacillus paracasei K56 cells" means making the ratio higher than that obtained by conventional processes known in the art, such as, but not limited to, higher than 0%, i.e., not inactivated after the fermented product is prepared.

[0036] The experimental results show that fermented milk containing dead K56 bacteria after milk-based fermentation exhibits better small intestinal propulsion rate compared to fermented milk containing live K56 bacteria. Simultaneously, it maintains comparable levels to unsterilized fermented milk in terms of first-time black stool expulsion, fecal water content, and fecal pellet number. Therefore, it can be expected that a higher proportion of dead K56 bacteria in the fermented product will be more beneficial in promoting intestinal peristalsis and defecation in the subjects. Based on the aforementioned findings, this invention applies *Lactobacillus paracasei* K56 in a manner that increases the proportion of dead bacteria to prepare products with at least one of the aforementioned effects (i) to (iii), thereby enhancing at least one of the functions of increasing small intestinal propulsion rate, promoting intestinal peristalsis, and promoting defecation in the subjects.

[0037] In an optional embodiment, the application includes making the ratio of the number of dead Lactobacillus paracasei K56 cells in the product to the total number of Lactobacillus paracasei K56 cells 100%.

[0038] In an optional embodiment, the application includes sterilizing the fermentation product to increase the number of dead K56 cells in the fermentation product.

[0039] Secondly, a method for preparing a fermented product is provided, the method comprising fermenting a milk-containing fermentation substrate using a fermentation agent containing *Lactobacillus paracasei* K56, and wherein the ratio of the number of dead *Lactobacillus paracasei* K56 cells in the fermented product to the total number of *Lactobacillus paracasei* K56 cells is 0% to 100%, and not 0%. For example, it can be, but is not limited to, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0040] In an optional embodiment, the preparation method includes ensuring that the ratio of the number of dead Lactobacillus paracasei K56 cells in the fermented product to the total number of Lactobacillus paracasei K56 cells is 100%.

[0041] In an optional embodiment, the fermentation agent contains a single strain, namely Lactobacillus paracasei K56.

[0042] In an optional embodiment, the fermentation agent is a compound agent containing *Lactobacillus paracasei* K56 and other strains optionally used for fermentation in the art.

[0043] In an optional embodiment, the preparation method includes sterilizing the fermented product.

[0044] In an optional embodiment, the fermented product further contains prebiotics.

[0045] In an optional embodiment, the sterilization process includes a first sterilization and a second sterilization; the conditions for the first sterilization include a sterilization temperature of 100-115°C and a sterilization time of 4-10 seconds; the conditions for the second sterilization include a sterilization temperature of 80-100°C and a sterilization time of 15-20 minutes.

[0046] In an optional embodiment, the preparation method includes cooling the sterilized milk, adding an inoculum containing Lactobacillus paracasei K56 at 33-37°C, fermenting for more than 72 hours, cooling to obtain a fermented product containing live Lactobacillus paracasei K56, and then sterilizing it.

[0047] Thirdly, a fermented product is provided, wherein the fermentation agent of the fermented product contains Lactobacillus paracasei K56, and the fermentation substrate contains milk; the fermented product is sterilized after fermentation.

[0048] In an optional embodiment, the fermentation agent contains a single strain, namely Lactobacillus paracasei K56.

[0049] In an optional embodiment, the fermentation agent is a compound agent containing *Lactobacillus paracasei* K56 and other strains optionally used for fermentation in the art.

[0050] In an optional embodiment, the fermented product further contains prebiotics.

[0051] In an optional embodiment, the fermented product is prepared by the following method: the sterilized milk is cooled, and a bacterial agent containing Lactobacillus paracasei K56 is added at 33-37°C. The mixture is fermented for more than 72 hours, cooled to obtain a fermented product containing live Lactobacillus paracasei K56, and then sterilized.

[0052] In an optional embodiment, the sterilization process includes a first sterilization and a second sterilization; the conditions for the first sterilization include a sterilization temperature of 100-115°C and a sterilization time of 4-10 seconds; the conditions for the second sterilization include a sterilization temperature of 80-100°C and a sterilization time of 15-20 minutes.

[0053] Fourthly, a method for storing a fermented product is provided, wherein the fermentation agent of the fermented product contains *Lactobacillus paracasei* K56, and the fermentation substrate contains milk; the storage method includes increasing the ratio of the number of dead *Lactobacillus paracasei* K56 cells in the fermented product to the total number of *Lactobacillus paracasei* K56 cells.

[0054] Based on the aforementioned findings, in K56 fermentation products containing milk as the fermentation substrate, the death of K56 does not reduce the function of K56 when it is a live bacterium; in fact, it may even show better performance in promoting intestinal motility. Therefore, this storage method differs from existing technologies that aim to maintain the number of live bacteria when storing fermentation products containing live bacteria. Instead, it does not prevent the death of live K56 bacteria to maintain or enhance the function of the fermentation product. In this scheme, "increasing the ratio of dead to live *Lactobacillus paracasei* K56 cells in the fermentation product" means that the ratio of dead to live K56 cells is greater than the ratio at the end of the fermentation product's preparation period.

[0055] It should be noted that this invention does not limit the content of dead K56 cells in the fermented product after preparation. In an optional embodiment, the fermented product can be a non-sterilized finished product, meaning that K56 and other optional strains are all live bacteria, and the bacteria die during storage, increasing the ratio of dead K56 cells to live K56 cells. Alternatively, the fermented product can also be a mixture of non-sterilized and sterilized fermented milk, resulting in a finished product containing some dead K56 cells, which continue to die during storage, further increasing the ratio of dead K56 cells to live K56 cells.

[0056] In an optional embodiment, the storage method includes increasing the ratio of the number of dead K56 cells to the number of live K56 cells in the fermentation product over a prolonged storage period.

[0057] In an optional embodiment, the storage method includes placing the fermented product at room temperature, for example, at 15–35°C, to increase the ratio of the number of dead K56 cells to the number of live K56 cells.

[0058] In an optional embodiment, the storage method includes heating the fermented product in an environment above room temperature, such as heating it at 35 to 100°C. This embodiment allows people who are not suitable for eating cold food to consume the fermented product without losing its function.

[0059] In an optional embodiment, the storage method includes using a sterilization method known in the art, to kill some or all of the K56 cells in the fermentation product, thereby increasing the ratio of the number of dead K56 cells to the number of live K56 cells.

[0060] In an optional embodiment, the fermentation agent of the fermented product contains a single strain, namely Lactobacillus paracasei K56.

[0061] In an optional embodiment, the fermented milk also contains prebiotics.

[0062] In optional embodiments, the fermentation substrate of the fermented product in any of the above embodiments contains milk, including but not limited to raw milk or modified milk. The milk can be from conventional mammals known in the art, including but not limited to one or more of cattle, sheep, and camels.

[0063] In optional embodiments, the starter culture of the fermented product in any of the above embodiments, in addition to Lactobacillus paracasei K56, may also contain strains known in the art, optionally used for fermenting milk bases, including but not limited to: one or more of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactococcus lactis, Bifidobacterium, Lactobacillus casei, and Lactobacillus plantarum.

[0064] In optional embodiments, the sterilization method in any of the above embodiments includes, but is not limited to, pasteurization, browning sterilization or UHT sterilization.

[0065] In an optional embodiment, the sterilization process in any of the above embodiments includes a first sterilization and a second sterilization; the conditions for the first sterilization include a sterilization temperature of 100-115°C and a sterilization time of 4-10 seconds; the conditions for the second sterilization include a sterilization temperature of 80-100°C and a sterilization time of 15-20 minutes.

[0066] In optional embodiments, the prebiotic in any of the above embodiments may be selected from prebiotics known in the art, including but not limited to one or more of fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), resistant starch, and polydextrose.

[0067] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0068] Example 1

[0069] Sample preparation:

[0070] (1) Live bacteria K56: *Lactobacillus paracasei* K56 was cultured overnight at 37°C in MRS broth. Prior to the experiment, K56 was stored at -80°C. The cultured *Lactobacillus paracasei* K56 was centrifuged at 4500×g for 10 minutes, followed by washing three times with sterile water. Bacterial counts were determined by plate counting, and the bacterial concentration was adjusted to 1.3×10⁻⁶ using sterile water. 9 CFU / mL.

[0071] (2) K56 dead bacteria: The K56 live bacteria suspension prepared in (1) was inactivated at 121°C for 10 minutes. The inactivated bacterial solution is heat-inactivated bacteria.

[0072] (3) K56 Fermented Milk - Live Bacteria: Heat a portion of the water for the ingredients to 35-55℃, add milk powder and stir for 10-30 minutes, then let stand for 10-30 minutes. Then homogenize (pressure 150-200 bar) and brown sterilize (94-99℃, 1-3 hours). Cool the sterilized reconstituted milk, add K56 bacteria at 33-37℃, ferment for more than 72 hours, and cool to obtain K56 fermented milk live bacteria or compound bacteria fermented milk live bacteria.

[0073] (4) K56 fermented milk - dead bacteria: The fermented milk containing live bacteria prepared in step (3) above is homogenized (pressure of 100-150 bar), and then sterilized at 110-115℃ for 4-10 seconds; then sterilized again at 90℃ for 15-20 minutes to obtain K56 fermented milk - dead bacteria sample.

[0074] (5) Compound fermented milk - live bacteria: Prepared according to step (3), the only difference from step (3) is that the reconstituted milk is cooled and the compound fermentation agent is added at 33-37℃ and fermented for more than 72 hours.

[0075] The compound starter culture contains the following strains: Lactobacillus paracasei K56 and commercially available Lactobacillus paracasei (Chr. Hansen).

[0076] (6) Compound bacteria fermented milk - dead bacteria: The fermented milk containing live bacteria prepared in step (5) above is homogenized (pressure of 100-150 bar) and sterilized at 100-115℃ for 4-10 seconds to obtain compound bacteria fermented milk - dead bacteria sample.

[0077] (7) Lactic acid bacteria beverage - dead bacteria: Heat a portion of the ingredients to 35-55℃, add milk powder and stir for 10-30 minutes, then let stand for 10-30 minutes. Then homogenize (pressure 150-200 bar) and browning sterilize (94-99℃, 1-3 hours). Cool the sterilized reconstituted milk, add compound bacteria at 33-37℃ and ferment for more than 72 hours, homogenize a second time (pressure 100-150 bar), and cool to obtain fermented milk. Heat a portion of the ingredients to 50-70℃, add prebiotics (including polydextrose and soybean polysaccharide), white sugar, stabilizer and sweetener, mix well, then add fermented milk and mix well. Perform ultra-high temperature sterilization at 100-115℃ for 4-10 seconds, cool and perform secondary sterilization to obtain lactic acid bacteria beverage.

[0078] Example 2

[0079] (1) Mouse model construction:

[0080] One hundred and twenty six-week-old male C57 mice were housed in the SPF-grade experimental animal facility of China Agricultural University. Humidity was strictly controlled at 22±2℃ and 45±10%, with a 12-hour day-night cycle. Mice were fed standard feed but allowed free access to food and water. After the acclimatization period, subsequent experiments were conducted. The specific modeling and intervention protocols are as follows:

[0081] During the modeling period, the control group was administered 0.1 mL of physiological saline by gavage daily, while the other groups were administered 0.1 mL of loperamide hydrochloride (15 mg / kg bw) by gavage once daily for 10 consecutive days. During the intervention period, the control group was administered 0.1 mL of physiological saline by gavage for 1 hour followed by 0.2 mL of ink by gavage. The model group was administered 0.1 mL of loperamide hydrochloride by gavage for 1 hour followed by 0.2 mL of ink by gavage. The other groups were administered 0.1 mL of loperamide hydrochloride by gavage for 1 hour followed by 0.2 mL of the intervention group containing ink, respectively. This gavage was performed once daily for 14 consecutive days. From the start of ink administration, the time of the first black feces excreted by each mouse, the number and weight of black feces excreted within 6 hours, and the water content of the feces were recorded. After the intervention, blood was collected from the eyeballs of all mice, and they were euthanized by cervical dislocation.

[0082] Preparation of loperamide hydrochloride suspension: Take 20 loperamide hydrochloride tablets (dosage: 2mg / tablet) and add physiological saline to 100mL to prepare a 0.04% solution. Prepare immediately before use.

[0083] Preparation of ink: Accurately weigh 100g of gum arabic, add 800mL of water, and boil until the solution is clear. Weigh 50g of activated carbon (powder) and add it to the above solution, then boil three times. After the solution cools, add physiological saline to make up to 1000mL. Store in a refrigerator at 4℃ and shake well before use.

[0084] Mouse model construction and evaluation results are as follows Figures 1-3 As shown. The results show:

[0085] The average time for the first black particle to be expelled in the blank group was 61.88 min, while the average time for the first black particle to be expelled in the model group was 141.2 min. Compared with the blank group, the time for the first black particle to be expelled in the model group was significantly longer, by 2.28 times.

[0086] The average number of fecal particles in the control group after 6 hours was 68, while the average number of fecal particles in the model group after 6 hours was 48. The number of fecal particles in the control group after 6 hours was 1.42 times that of the model group.

[0087] The fecal moisture content of the control group was 69.19%, and the average number of fecal particles in the model group after 6 hours was 48.30%. The number of fecal particles in the control group after 6 hours was 1.42 times that of the model group.

[0088] The above results show that the model group differed significantly from the control group in terms of the first black fecal pellet and the number of fecal pellets. The above modeling method can successfully construct a mouse constipation model.

[0089] (2) The performance of the samples prepared in Example 1 was tested. The experimental procedure is as follows: Figure 4 As shown in Table 1, the experimental groups were divided according to the guidelines. Mice were acclimatized for 7 days after purchase and then administered loperamide (15 mg / kg / day) by gavage to induce functional constipation. The control group received an equal volume of physiological saline. After 10 consecutive days of administration, the intervention group was administered the drugs according to Table 1, receiving 200 μL daily by gavage for 2 weeks (14 days). Samples were collected for analysis after the intervention. From the start of the ink administration, the time of blackening, the number of fecal pellets per 6 hours, and the fecal water content were recorded daily. After the intervention, colons were collected for HE staining to observe intestinal inflammation and to measure the thickness of the colonic muscle layer.

[0090] Table 1

[0091]

[0092] The experimental method for small intestinal propulsion rate is as follows: The methods for testing the time of blackening of mice, the number of fecal pellets after 6 hours, and the water content of feces are the same as described above. Small intestinal propulsion rate: Mice were euthanized by cervical dislocation immediately after being given ink for 25 minutes. The abdominal cavity was opened and the mesentery was separated. The intestinal segment from the upper end to the pylorus to the lower end to the ileocecal junction was cut off and placed on a tray. The small intestine was gently pulled into a straight line, and the length of the intestinal segment was measured as the "total length of the small intestine". The length from the pylorus to the leading edge of the ink was measured as the "ink propulsion length".

[0093]

[0094] (3) The test results of small intestinal propulsion rate, first black expulsion time, fecal water content and number of fecal particles are shown in Table 2.

[0095] Table 2

[0096]

[0097] According to Table 2 above, the test results for small intestinal propulsion rate, fecal water content, and fecal particle number of the K56 live bacteria group (without fermentation) were better than those of the K56 dead bacteria group. Although the test results for the first black pellet expulsion time were slightly better in the K56 dead bacteria group, the results were similar between the two groups. Therefore, it can be concluded that when K56 is not fermented on a milk base, the K56 live bacteria group is more effective than the K56 dead bacteria group in promoting intestinal peristalsis and fecal expulsion.

[0098] However, the inventors unexpectedly discovered that when K56 was fermented on a milk base, the death of K56 in the fermentation product did not significantly reduce the effect of the fermentation product in promoting intestinal peristalsis and promoting fecal excretion. On the contrary, it performed better in some tests.

[0099] Regarding the intestinal propulsion rate test results, the K56 fermented milk-dead bacteria group performed better than the K56 fermented milk-live bacteria group, with the intestinal propulsion rate of the K56 fermented milk-dead bacteria group increasing by approximately 7% compared to the K56 fermented milk-live bacteria group. However, when K56 was not fermented on a milk base, the intestinal propulsion rate of the K56 dead bacteria group was approximately 10% lower than that of the K56 live bacteria group. Similarly, regarding the fecal particle count test results, when K56 was not fermented on a milk base, the number of fecal particles in the K56 dead bacteria group was approximately 10 fewer than that in the K56 live bacteria group. Even after K56 underwent milk base fermentation, the number of fecal particles in the K56 fermented milk-dead bacteria group was still slightly higher than that in the K56 fermented milk-live bacteria group. Although the test results for the first blackening time and fecal moisture content of the K56 fermented milk-dead bacteria group were still inferior to those of the K56 fermented milk-live bacteria group, it can be seen that the difference between the two groups is narrowing compared to the differences between the K56 live bacteria group and the K56 dead bacteria group. Therefore, it can be concluded that when K56 is fermented on a milk base, the sterilized fermented milk has a better ability to promote intestinal peristalsis and promote fecal excretion, which is at least no less than that of fermented milk containing live K56 bacteria.

[0100] Furthermore, comparing the live bacteria group and the dead bacteria group of the compound fermented milk, it can be seen that after sterilization, the fecal water content and fecal particle number of the compound fermented milk group were higher than those of the live bacteria group. Although the small intestinal propulsion rate decreased and the time to first black excretion increased, the increased fecal water content and fecal particle number compensated to some extent for the loss of defecation ability caused by the death of strains in the fermented milk. This may be because the use of compound bacteria for fermentation reduces the proportion of K56 in the entire fermentation bacteria. The commercially available Lactobacillus paracasei in the compound bacteria agent does not have the ability to promote intestinal peristalsis and fecal excretion after milk-based fermentation and sterilization. Only the dead K56 bacteria have an improving effect. This also shows that even among Lactobacillus paracasei, the ability to promote intestinal peristalsis and fecal excretion after milk-based fermentation and sterilization is not present in any Lactobacillus paracasei. K56 has an unexpected technical effect. In summary, as long as the K56 strain is added to fermented milk, the dead K56 bacteria after sterilization can, to some extent, compensate for the weakening effect of the dead bacteria in fermented milk on promoting intestinal peristalsis and promoting fecal excretion.

[0101] Furthermore, the experimental results of the lactic acid bacteria beverage-dead bacteria group show that the addition of prebiotics can increase the small intestinal propulsion rate, fecal water content, and fecal pellet number, while shortening the time for the first black pellet to be expelled. Therefore, it can be expected that when fermented milk is fermented with a bacterial agent containing K56 and prebiotics are added, the resulting fermented milk, whether used in live or sterilized form, can promote intestinal peristalsis and fecal excretion. Moreover, the fermented milk will further enhance its ability to promote intestinal peristalsis and fecal excretion as the bacterial strains die.

[0102] (4) HE staining results of colon in each experimental group are as follows Figure 5 and Figure 6 As shown in Table 3, the results of the colonic basal layer thickness test in mice are shown in Table 3.

[0103] Table 3. Coronary basal layer thickness in mice

[0104]

[0105] The experimental results show that in the model group, loperamide induced a decrease in colonic muscle layer thickness and goblet cell count, and inflammatory infiltration occurred. All drug intervention groups restored colonic muscle layer thickness, increased goblet cell count, and improved inflammatory infiltration. The colonic basal layer thickness in the K56 live bacteria group was significantly higher than that in the K56 dead bacteria group, with a mean value approximately 36 μm greater. Although the colonic muscle layer thickness in both the K56 fermented milk-live bacteria group and the compound fermented milk-live bacteria group was higher than that in the corresponding dead bacteria group, it can be seen that after milk-based fermentation with K56 or K56-containing agents, the difference in the ability of live and dead bacteria to restore colonic muscle layer thickness was shortened. The mean colonic basal layer thickness in the K56 fermented milk-live bacteria group was approximately 16.5 μm greater than that in the K56 fermented milk-dead bacteria group, and the mean colonic basal layer thickness in the compound fermented milk-live bacteria group was approximately 27 μm greater than that in the K56 fermented milk-dead bacteria group, both significantly smaller than the differences between the K56 live bacteria group and the K56 dead bacteria group without milk-based fermentation.

[0106] In summary, the effect of K56 cell death on efficacy after milk-based fermentation of *Lactobacillus paracasei* K56 differs from that of K56 without milk-based fermentation. The difference in efficacy between dead and live K56 cells after milk-based fermentation is smaller than that between live and unfermented K56 cells. Furthermore, in some aspects, such as in small intestinal propulsion tests, the ability of dead K56 cells after milk-based fermentation to promote small intestinal propulsion is actually superior to that of live K56 cells after milk-based fermentation. The fact that sterilized fermentation products of *Lactobacillus paracasei* K56 after milk-based fermentation retain or enhance most of the functions of unsterilized fermentation products provides new insights into the preparation and preservation of probiotics.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of Lactobacillus paracasei subsp. paracasei K56 in the preparation of a product containing at least one of the effects (i) to (iii), said product comprising a fermentation product fermented using a starter containing Lactobacillus paracasei K56, and the fermentation substrate containing milk; said application includes increasing the ratio of the number of dead Lactobacillus paracasei K56 cells to the total number of Lactobacillus paracasei K56 cells in the fermentation product; (i) Improve the small intestinal propulsion rate of subjects; (ii) Promotes intestinal peristalsis in the subjects; (iii) Promote defecation in subjects.

2. The application according to claim 1, characterized in that, The application includes ensuring that the ratio of the number of dead *Lactobacillus paracasei* K56 cells in the product to the total number of *Lactobacillus paracasei* K56 cells is 100%.

3. A method for preparing a fermented product, characterized in that, The preparation method includes fermenting a milk-containing fermentation substrate using a fermentation agent containing Lactobacillus paracasei K56, and ensuring that the ratio of the number of dead Lactobacillus paracasei K56 cells in the fermentation product to the total number of Lactobacillus paracasei K56 cells is 0-100%, and not 0%.

4. The preparation method according to claim 3, characterized in that, The preparation method includes ensuring that the ratio of the number of dead Lactobacillus paracasei K56 cells in the fermented product to the total number of Lactobacillus paracasei K56 cells is 100%.

5. The preparation method according to claim 3 or 4, characterized in that, The fermentation agent for the fermented product is Lactobacillus paracasei K56.

6. The preparation method according to claim 5, characterized in that, The preparation method includes sterilizing the fermented product; Optionally, the sterilization process includes a first sterilization and a second sterilization; the conditions for the first sterilization include a sterilization temperature of 100-115°C and a sterilization time of 4-10 seconds; the conditions for the second sterilization include a sterilization temperature of 80-100°C and a sterilization time of 15-20 minutes. Optionally, the preparation method includes cooling the sterilized milk, adding an inoculum containing Lactobacillus paracasei K56 at 33-37°C, fermenting for more than 72 hours, cooling to obtain a fermented product containing live Lactobacillus paracasei K56, and then sterilizing it.

7. A fermented product, characterized in that, The fermentation agent of the fermented product contains Lactobacillus paracasei K56, and the fermentation substrate contains milk; the fermented product is sterilized after fermentation.

8. The fermented product according to claim 7, characterized in that, The fermentation agent is a compound fermentation agent; And / or, the fermented product also contains prebiotics.

9. The fermented product according to claim 7 or 8, characterized in that, The fermented product is prepared using the preparation method described in claim 6.

10. A method for storing fermented products, characterized in that, The fermentation agent of the fermented product contains *Lactobacillus paracasei* K56, and the fermentation substrate contains milk; the storage method includes increasing the ratio of the number of dead *Lactobacillus paracasei* K56 cells in the fermented product to the total number of *Lactobacillus paracasei* K56 cells.

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