Lactobacillus reuteri YYS-Pb1 with heavy metal adsorption and fermentation high-yield amino acid function and application thereof

Abstract

This invention relates to the field of microbial technology, providing a *Lactobacillus reuteri* YYS-Pb1 strain with heavy metal adsorption and high amino acid production capabilities during fermentation, and its applications. The preservation number of this *Lactobacillus reuteri* YYS-Pb1 is CGMCC No. 28613. This *Lactobacillus reuteri* YYS-Pb1 exhibits good tolerance in simulated gastric and intestinal fluids, can adsorb heavy metals lead and cadmium, can produce high levels of phenyllactic acid, possesses good hydrophobic interactions and biofilm formation capabilities, can ferment various fruits to remove the sweet potato flavor caused by high temperatures in fruit pulp, and significantly increases the total amino acid content in the fruit pulp. This bacterium can provide a new probiotic source for the development of heavy metal detoxification products, phenyllactic acid-producing products, and high-amino acid fermented fruit pulp products, and has significant social application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a type of Lactobacillus reuteri YYS-Pb1 with heavy metal adsorption and high amino acid production through fermentation, and its applications. Background Technology

[0002] Lead and cadmium are recognized heavy metal pollutants. Lead and cadmium pollution can be generated from gasoline combustion exhaust, lead-containing paints, mining, smelting, casting, batteries, printing and dyeing, electronic waste, and fertilizers. Because lead and cadmium are highly accumulative heavy metals that cannot be degraded, their accumulation in the body poses a significant threat to human health. Lead, in particular, can inhibit the activity of various enzymes, suppress nerve synaptic transmission, compete with calcium ions, and affect vitamin D production, thereby causing corresponding metabolic disorders, interfering with nerve transmission and brain development, and damaging the nervous, immune, respiratory, digestive, urinary and reproductive, cardiovascular, and skeletal systems.

[0003] The main harms of chronic cadmium poisoning to the human body are damage to the kidneys and bones. In the kidneys, cadmium damages the renal tubules, causing proteinuria, hypocalcemia, aminoaciduria, and hyperphosphouria. In the bones, cadmium leads to osteoporosis, chondrogenesis imperfecta, and spontaneous fractures; in severe cases, multiple pathological fractures may occur. Furthermore, chronic cadmium poisoning can cause lung damage, such as pulmonary fibrosis and emphysema, as well as liver damage, such as hepatocellular injury. In addition to kidney and bone damage, long-term exposure to cadmium can also cause neurasthenia, including headaches, dizziness, fatigue, insomnia, mood changes, loss of appetite, as well as stomatitis, tremors, nephritis, and nephrotic syndrome.

[0004] Currently, lead poisoning is mainly treated with laxatives and emetics, which have significant side effects and are unsuitable as a routine daily detoxification method. Therefore, it is necessary to find an effective method for daily detoxification. Acute heavy metal poisoning requires medical treatment, but because lead and cadmium are easily exposed in the modern environment, it is extremely important to adjust diet to facilitate their timely elimination in daily life.

[0005] Phenylated acid is a natural organic acid with broad-spectrum antibacterial and bactericidal effects. It can be used as a food preservative to extend the shelf life of food. It can also reduce the use of antibiotics and is used to treat intestinal and skin infections. Probiotics that produce phenyllactic acid can improve human immunity and resistance by adjusting the intestinal flora, thus preventing disease. Developing high-yield phenyllactic acid probiotics plays a crucial role in ensuring food safety, promoting human health, and reducing antibiotic use.

[0006] Protein is an essential building block for human life, and amino acids, as the basic units of protein, play an indispensable role. Fruit pulp enzymes, as a modern technological beverage, possess excellent flavor, promote gut health, and replenish energy. Developing fruit pulp enzyme products with high amino acid content not only enhances the added value of fruits and other agricultural products but also provides essential nutrients for human health.

[0007] Probiotics are a class of microorganisms that are beneficial to human health. Currently, there are very few probiotics on the market that can adsorb heavy metals, produce phenyl lactic acid, and increase the amino acid content of fruit pulp. Furthermore, there are no publicly reported probiotics of *Lactobacillus reuteri* that can efficiently adsorb heavy metals such as lead / cadmium, produce high levels of phenyl lactic acid, and increase the amino acid content of fruit pulp. How to develop a probiotic that can significantly adsorb heavy metals such as lead / cadmium, produce phenyl lactic acid, and increase the amino acid content of fruit pulp to help human health is precisely the problem that those skilled in the art are dedicated to solving. Summary of the Invention

[0008] To address the shortcomings of the prior art mentioned in the background section, this invention provides a *Lactobacillus reuteri* YYS-Pb1, the technical solution of which is as follows:

[0009] The Lactobacillus reuteri ( Limosilactobacillus reuteri YYS-Pb1 was deposited on October 12, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28613.

[0010] The present invention also provides a composition containing *Lactobacillus reuteri* YYS-Pb1 as described above.

[0011] In one embodiment, the composition comprises a microbial preparation.

[0012] In one embodiment, the number of *Lactobacillus reuteri* YYS-Pb1 in the composition is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g. In one embodiment, the number of *Lactobacillus reuteri* YYS-Pb1 in the composition is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0013] In one embodiment, the composition comprises one or more combinations of uninactivated *Lactobacillus reuteri* YYS-Pb1, inactivated *Lactobacillus reuteri* YYS-Pb1, metabolites of *Lactobacillus reuteri* YYS-Pb1 strain, and lyophilized *Lactobacillus reuteri* YYS-Pb1 strain.

[0014] The present invention also provides a fermentation product obtained by fermentation of *Lactobacillus reuteri* YYS-Pb1 as described above.

[0015] The present invention also provides the use of Lactobacillus reuteri YYS-Pb1 and / or its ferments as described above in the preparation of functional products.

[0016] In one embodiment, the *Lactobacillus reuteri* YYS-Pb1 and / or its ferments in the functional product comprise at least one of the following functions:

[0017] (1) It can adsorb heavy metal lead;

[0018] (2) It can adsorb heavy metal cadmium;

[0019] (3) Produces phenyllactic acid;

[0020] (4) It has hydrophobic properties;

[0021] (5) It has the ability to form biofilms;

[0022] (6) It can be used to ferment a variety of fruits, improve the flavor of the pulp and increase the amino acid content in the pulp.

[0023] In one embodiment, the functional products include heavy metal detoxification products, phenyl lactic acid producing products, and fermented fruit pulp enzyme functional products.

[0024] The present invention also provides the use of Lactobacillus reuteri YYS-Pb1 as described above or the composition described above in the preparation of fermented foods.

[0025] In one embodiment, *Lactobacillus reuteri* YYS-Pb1 is used as a probiotic to ferment fruits and traditional Chinese medicines to prepare fermented foods.

[0026] Based on the above, compared with the prior art, the *Lactobacillus reuteri* YYS-Pb1 provided by the present invention has the following beneficial effects:

[0027] The *Lactobacillus reuteri* YYS-Pb1 provided by this invention exhibits good tolerance in artificial gastric and intestinal fluids, can adsorb heavy metals lead and cadmium, can produce phenyl lactic acid, possesses good hydrophobic interactions and excellent cell membrane formation ability, can ferment various fruits to improve fruit flavor characteristics, and increase the amino acid content of fermented fruit pulp. This bacterium can provide a new probiotic source for the development of detoxification products, phenyl lactic acid-producing products, and fruit pulp fermentation bacteria with high amino acid content, and has significant application value.

[0028] Other features and beneficial effects of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other beneficial effects of the invention can be realized and obtained by means of the structures particularly pointed out in the description, claims and drawings. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the 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 based on these drawings without creative effort. Unless otherwise specified, the positional relationships shown in the drawings in the following description are based on the direction in which the components are drawn in the figure.

[0030] Figure 1 This is a colony morphology diagram of Lactobacillus reuteri YYS-Pb1 provided by the present invention.

[0031] Figure 2 Gram staining morphology of *Lactobacillus reuteri* YYS-Pb1 provided by this invention.

[0032] Figure 3 Scanning electron microscope image of Lactobacillus reuteri YYS-Pb1 provided by the present invention.

[0033] Figure 4 Agarose gel electrophoresis image of the 16S rDNA target fragment amplified from Lactobacillus reuteri YYS-Pb1 provided by this invention.

[0034] Figure 5 Phylogenetic tree diagram of the 16S rDNA gene of Lactobacillus reuteri YYS-Pb1 provided by the present invention.

[0035] Figure 6 A graph showing the phenyllactic acid production data of Lactobacillus reuteri YYS-Pb1 provided for this invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0038] The present invention also provides the following operation examples and embodiments:

[0039] The present invention provides *Lactobacillus reuteri* ( Limosilactobacillus reuteri YYS-Pb1 was obtained from the feces of 9-month-old infants through anaerobic culture in MRS medium.

[0040] An example of the procedure for extracting this bacterium from feces according to the present invention is as follows:

[0041] Example 1: Screening and Isolation of Bacteria

[0042] Take approximately 1g of infant stool sample and dilute it in physiological saline solution for 10 minutes. -2 times, 10 -3 times, 10 -4 Take 0.1 mL of each diluted solution and place it in MRS medium for anaerobic culture. Select several colonies suspected to be lactic acid bacteria, purify them for three generations, and then test the lead adsorption effect. Select the strain with better adsorption effect, preserve it, and name it YYS-Pb1.

[0043] Example 2 Identification of bacterial strains

[0044] 2.1 Morphological observation of YYS-Pb1 bacteria

[0045] The colony morphology of YYS-Pb1 is as follows: Figure 1 As shown, Gram staining and bacterial cell morphology are as follows: Figure 2 and 3 As shown.

[0046] The main morphological characteristics of YYS-Pb1 are as follows: the colonies are round, milky white, raised, smooth and opaque on MRS medium; Gram-positive, the cells are mainly short rods, with a long division phase, 0.8-1.1 μm wide and 1.2-5 μm long.

[0047] 2.2 Physiological and biochemical analysis of YYS-Pb1 bacteria

[0048] The biochemical experiments of lactic acid bacteria were conducted according to the standard method of GB4789.35. Specifically, a lactic acid bacteria basal culture medium containing cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% (w / v) sodium hippurate was prepared, and YYS-Pb1 was inoculated at 1% (w / v).

[0049] In addition, for 1% (w / v) sodium hippurate, 0.2 mL of ninhydrin solution (2 mL for 1% sodium hippurate) should be slowly added along the wall of the test tube after the culture is completed. Do not shake. Place the tube in a water bath at 36℃±1℃ for 10 min and then interpret the results. See Table 1 for the interpretation results.

[0050] Table 1. Glycohydrates and major biochemical reactions of YYS-PB1

[0051]

[0052] Note: "+" indicates a positive test result, and "-" indicates a negative test result.

[0053] Conclusion: According to the results in Table 1, YYS-Pb1 can utilize carbohydrates such as cellobiose, maltose, sucrose, raffinose, and lactose, but cannot utilize mannitol, salicin, sorbitol, inulin, or sodium hippurate.

[0054] 2.3 Molecular biological identification of YYS-Pb1

[0055] ① Extraction of YYS-Pb1 bacterial genomic DNA: Extraction was performed using a bacterial genomic DNA extraction kit from TIANGEN.

[0056] ②PCR amplification of 16S rDNA sequence: The primers used to amplify the 16S rDNA gene sequence were F9-27: 5'-GAGTTT GAT CCT GGC TCA G-3'; R1525-1542: 5'-AGA AAG GAG GTG ATC CAG CC-3';

[0057] PCR reaction system: 12.5 μL of 2×Mix, 1 μL each of primers and DNA, and 9.5 μL of ddH2O.

[0058] PCR amplification program: 93℃ pre-denaturation for 4 min. Then denature at 94℃ for 30 s, 55℃ (16S rDNA), 72℃ extension for 90 s, for a total of 30 cycles, and finally extend at 72℃ for 10 min, and store at 4℃.

[0059] ③ PCR product detection and sequencing analysis: 5 μL of PCR product was separated and examined by gel electrophoresis in 1.0% agarose gel containing EB (staining solution). The amplified 16S rDNA target fragment was 1491 bp in length (see agarose gel electrophoresis image of 16S rDNA target fragment amplification). Figure 4 );

[0060] ④ Phylogenetic analysis: Blast alignment analysis was performed on each 16S rRNA sequence in NCBI data to obtain the sequence correlation with *Lactobacillus reuteri* (…). Limosilactobacillus reuteri) The sequences showed greater than 99% homology, and a phylogenetic tree was constructed using the Neighbor-joining method in MEGA4 (results are shown in [link to results]). Figure 5 The 16S rDNA sequence of YYS-Pb1 is as follows:

[0061]

[0062] Conclusion: Based on morphological observation, biochemical identification of lactic acid bacteria, and homology analysis in the DNA phylogenetic tree, YYS-Pb1 was identified as *Lactobacillus reuteri*. Limosilactobacillus reuteri ) bacterial strains.

[0063] The performance characterization of Lactobacillus reuteri YYS-Pb1 provided by this invention is as follows:

[0064] Example 3: Adsorption capacity of Lactobacillus reuteri YYS-Pb1 for lead acetate

[0065] The mother liquor of *Lactobacillus reuteri* YYS-Pb1 was inoculated into MRS medium at a rate of 1% (w / v) and cultured anaerobically at 37°C for 24-48 hours to obtain the fermentation broth of *Lactobacillus reuteri* YYS-Pb1.

[0066] Take the fermentation broth of Lactobacillus reuteri YYS-Pb1 after 48 hours of fermentation, add lead acetate to the initial lead concentration of about 10 mg / kg, and use uninoculated MRS medium as a control. YYS-Pb1 is inoculated and fermented at 37℃ for 24 hours. The lead content of the fermentation broth is measured by centrifugation. These are recorded as the treatment group and the control group after 24 hours of MRS fermentation broth treatment.

[0067] Further, the fermentation broth of *Lactobacillus reuteri* fermented for 24 h was collected by centrifugation at 4500 r / min for 10 min, and the cells were counted using flow cytometry after adding PBS buffer. The concentration of the bacterial suspension was adjusted to (1.0±0.1)*10⁻⁶. 9 The concentrations were 100 cfu / mL, with PBS buffer without bacterial suspension as a control. Lead acetate was added to a final concentration of approximately 2.0 and 5.0 mg / kg. The mixtures were incubated at 37°C and mixed every 30 minutes. After 4 hours, the mixtures were centrifuged and the supernatant was collected for lead content determination. These were designated as the treatment group and control group after 4 hours of PBS treatment.

[0068] The lead content was determined according to the graphite furnace atomic absorption spectrometry method in the national standard GB5009.12-2017, using a PinAAcle900Z atomic absorption spectrometer. The formula for calculating the lead adsorption rate is:

[0069] Lead adsorption rate / % = (lead content) 对照 - Lead content 处理组 ) / Lead content 对照 *100.

[0070] Table 2. Adsorption capacity analysis of YYS-Pb1 for lead acetate

[0071]

[0072] The results show that:

[0073] Both the YYS-Pb1 fermentation broth and the bacterial suspension showed good adsorption of lead. After treatment, the lead content in the fermentation broth decreased from 8.55 mg / kg in the control to 1.49 mg / kg, with an adsorption rate of 83.180%.

[0074] In PBS medium, after treatment, the lead content decreased from 1.390 mg / kg and 3.37 mg / kg in the control group to 0.075 mg / kg and 0.052 mg / kg, respectively, with adsorption rates as high as 94.640% and 98.457%, indicating that YYS-Pb1 has excellent adsorption capacity for heavy metal lead.

[0075] Example 4: Test of cadmium adsorption capacity of Lactobacillus reuteri YYS-Pb1

[0076] After centrifuging the fermentation broth of *Lactobacillus reuteri* YYS-Pb1 for 24 hours, the bacterial cells were collected. PBS was added, and the bacterial suspension was adjusted by flow cytometry to achieve a total particle count of (1±0.1)*102. 9 Cfu / mL was added to cadmium sulfate to an initial concentration of approximately 10 mg / kg. The mixture was incubated at 37℃ for 4 hours for adsorption. PBS containing the same concentration of cadmium sulfate without bacterial suspension was used as a control. The supernatant was collected by centrifugation. The Cd adsorption rate was determined according to the national standard GB 5009.268-2016, National Food Safety Standard, Determination of Multiple Elements in Food, Method I: Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The formula for calculating the Cd adsorption rate is as follows:

[0077] Cadmium adsorption rate (%) = (Cadmium content) 对照 -Cadmium content 处理组 ) / Cadmium content 对照 *100.

[0078] The adsorption rate of heavy metal cadmium by YYS-Pb1 bacterial suspension was calculated to be 67.02±0.50%, indicating that YYS-Pb1 has a good adsorption effect on Cd.

[0079] Example 5: Phenolic lactic acid production capacity of Lactobacillus reuteri YYS-Pb1

[0080] YYS-Pb1 yeast broth was inoculated into different MRS media (where MRS+0: indicates that no phenylalanine was added to the MRS medium, and MRS+0.5: indicates that exogenous phenylalanine was added to the MRS medium until the final concentration of phenylalanine was approximately 0.5 g / L). The fermentation was carried out at 37℃ for 96 h to obtain YYS-Pb1 fermentation broth. The supernatant of the fermentation broth was centrifuged and filtered through a 0.22 μm microporous membrane. The phenyllactic acid content was determined by high-performance liquid chromatography using a Waters Alliance e1695 to obtain the yield data of phenyllactic acid.

[0081] The HPLC detection conditions were as follows: column: SunFire C18 column (4.6 × 250 mm, 5 μm); column temperature: 30℃; injection volume: 10 μL; flow rate: 0.8 mL / min; mobile phase: phase A was 0.55% trichloroacetic acid methanol solution, phase B was 0.05% trichloroacetic acid solution; elution program: 0–20 min, A:B linearly changed from 10% to 100%; 20–23 min, 100% phase A; 23–27 min, maintained A:B at 10%; detection wavelength: 210 nm. The peak time for phenyl lactic acid was between 12.1 and 13.4 min.

[0082] YYS-Pb1 utilizes its ability to produce phenyllactic acid. Figure 6 The data shows that:

[0083] After 96 hours of fermentation, the yields of phenyllactic acid in the fermentation broth were 194.97 and 440.13 mg / L, respectively, indicating that YYS-Pb1 has extremely high phenyllactic acid production capacity, and the addition of phenylalanine can significantly promote its phenyllactic acid production.

[0084] Example 6 Hydrophobic interaction test of Lactobacillus reuteri YYS-Pb1

[0085] Take *Lactobacillus reuteri* YYS-Pb1 cultured for 48 h, centrifuge to remove supernatant, wash twice with PBS, and then add PBS to adjust OD. 600nm The value was 0.6 ± 0.05. YYS-Pb1 bacterial suspension was obtained, and the actual OD value was measured. 600nm The value is calculated as A0. Take 3 mL of YYS-Pb1 bacterial suspension, add 3 mL of xylene and n-hexane respectively, mix thoroughly for 5 min, let stand for 1 h, take the lower aqueous phase, and measure the OD. 600nm The value is denoted as A. T The hydrophobic interaction forces of the bacteria were calculated, and the results are detailed in Table 3. The calculation formula is as follows:

[0086] Hydrophobic interaction force of bacteria / % = (A0 - A T ) / A0*100.

[0087] Table 3: Hydrophobic interaction test of YYS-Pb1 at different initial concentrations

[0088]

[0089] It can be seen that the hydrophobic interaction forces of YYS-Pb1 in xylene and n-hexane as organic phases are 76.440% and 74.125%, respectively, indicating that YYS-Pb1 exhibits excellent hydrophobic interaction forces in different organic phases.

[0090] Example 7: Biofilm Formation Ability Analysis of Lactobacillus reuteri YYS-Pb1

[0091] MRS medium was prepared by adding galactose at a concentration of 0.5 g / L to obtain MRS and MRS+galactose medium. YYS-Pb1 was inoculated into these mediums at 1% (w / v) and fermented at 37℃ for 24 h. Using the corresponding medium as a dilution, the bacterial count in each fermentation broth was adjusted to (1.0±0.1)*10⁻⁶ using flow cytometry. 8 Take 200 μL of the adjusted fermentation dilution (cfu / mL) and place it in a 96-well plate. Use various culture media without inoculation as controls. Incubate at 37°C for 24 h. After incubation, discard the culture medium, add 0.2 mL of 1% (w / v) crystal violet and incubate for 30 min. Rinse with distilled water, then dissolve in 0.2 mL of 95% ethanol. Measure the absorbance of the resulting colored solution at 590 nm. Repeat the experiment three times and record the average value as OD. X The value, with uninoculated culture medium as a control, is recorded as a replicate and is denoted as OD. C The test results are shown in Table 4.

[0092] Table 4. Analysis of YYS-Pb1 biofilm formation ability

[0093]

[0094] When OD X OD C A ratio >1 indicates that the bacteria have the ability to form biofilms; the higher the ratio, the stronger the ability. OD values ​​for each treatment group... X :OD C The values ​​were all greater than 3, indicating that YYS-Pb1 has a good biofilm formation ability. After adding galactose to MRS, its biofilm formation ability was significantly higher than that of the fermentation group on MRS (P<0.05), indicating that galactose helps YYS-Pb1 to further promote its biofilm formation.

[0095] Example 8: Survival analysis of *Lactobacillus reuteri* YYS-Pb1 in a simulated gastric fluid environment.

[0096] (1) The survival rate test process is as follows:

[0097] Take the YYS-Pb1 bacteria that have been fermented for 24 hours, centrifuge at 12000 r / min for 5 min to collect the bacterial cells, add the same volume of physiological saline (0.85%) and mix well for later use;

[0098] Prepare artificial gastric fluid (125 mM NaCl, 7 mM KCl, 45 mM NaHCO3 and 3 g / L pepsin), adjust the pH to 2.0, 2.5 and 3.0, filter through a 0.22 μM microporous membrane and set aside.

[0099] Take 1 mL of the treated sample and place it in 9 mL of artificial gastric fluid with a pH of 2.75. Incubate at 37℃. Take 0.9 mL of untreated (control) and treated samples for x (1, 2, 3, 5) h each time. Add 0.1 mL of PI and stain at 37℃ for 10 min. Take 0.1 mL of the PI and place it in 0.9 mL of ultrapure water. Detect the total bacterial count P1 / % and the number of dead bacteria P2 / % by flow cytometry. Calculate the survival rate / % at different time points. With the survival rate of untreated samples as 100% control, calculate the gastrointestinal tolerance of each treatment.

[0100] The formula for calculating bacterial survival rate at different treatment times is as follows:

[0101] Survival rate / % = [(P1) 处理组 - P2 处理组 ) / P1 处理组 ] / [ (P1 对照 - P2 对照 ) / P1 对照 ]*100.

[0102] (2) The survival rate of YYS-Pb1 in different gastric fluid environments is shown in Table 5 below:

[0103] Table 5 Survival rate of YYS-Pb1 in simulated gastric fluid environment (survival rate / %)

[0104]

[0105] The data shows that when YYS-Pb1 was treated in a simulated gastric fluid environment with a pH of 2.5 for 1-5 hours, the survival rate was 70.23-90.11%, and when treated in a simulated gastric fluid environment with a pH of 3.0 for 1-5 hours, the survival rate was 88.55-95.28%, indicating that YYS-Pb1 has good tolerance in an artificial simulated gastric fluid environment.

[0106] Example 9: Survival analysis of *Lactobacillus reuteri* YYS-Pb1 in a simulated artificial pancreatic juice environment.

[0107] (1) Take the YYS-Pb1 bacteria that have been fermented for 24 h, centrifuge at 12000 r / min for 5 min to collect the bacterial cells, add the same volume of physiological saline (0.85%) and mix well for later use;

[0108] Prepare protein pancreatic juice (0.1% secretin w / v, 0.15% bovine bile), adjust the pH to 7.5 and 8.0 respectively, filter through a 0.22 μM microporous membrane and set aside. Take 1 mL of the treated bacterial culture and add it to 9 mL of protein pancreatic juice at different pH values. Incubate at 37℃. Take samples at 3 and 6 (h) after treatment, take 0.9 mL each time, add 0.1 mL of PI dilution, stain at 37℃ for 10 min, and detect the total bacterial count P1 / % and mortality P2 / % by flow cytometry. Calculate the survival rate / % at different time points. Use the untreated survival number as a 100% control to calculate the bacterial survival rate.

[0109] The formula for calculating bacterial survival rate is as follows:

[0110] Survival rate / % = [(P1) 处理组 - P2 处理组 ) / P1 处理组 ] / [ (P1 对照 - P2 对照 ) / P1 对照 ] *100.

[0111] (2) The bacterial survival rate of strain YYS-Pb1 is shown in Table 6:

[0112] Table 6 Survival rate of YYS-Pb1 in artificial simulated pancreatic juice environment (survival rate / %)

[0113]

[0114] According to the data:

[0115] When treated in pancreatic juice at pH 7.5 for 3 and 6 (h), the survival rate of strain YYS-Pb1 was 85.87% and 63.01%, respectively, and the survival rate in pancreatic juice at pH 8.0 was 85.46% and 62.26%, respectively, indicating that it has excellent pancreatic juice tolerance.

[0116] This invention also provides the following application examples of Lactobacillus reuteri YYS-Pb1:

[0117] Example 10: Preparation of fermented food using Lactobacillus reuteri YYS-Pb1

[0118] YYS-Pb1 yeast culture of *Lactobacillus reuteri* was prepared using plums, bayberries, blueberries, oranges, and kiwifruit as fermentation raw materials. The fruits were peeled and prepared into fruit pulp. The pulp, sugar, and water (w:w:v) were added at a ratio of 4:1:5 and mixed well. The mixture was sterilized at 121℃ for 20 min. YYS-Pb1 yeast culture was inoculated at a rate of 2.5% (w / v). After fermentation for 3 days, pH was measured and sensory evaluation was performed.

[0119] It was found that the fruits without YYS-Pb1 inoculation fermentation had a strong sweet potato flavor after autoclaving; however, after fermentation with YYS-Pb1, the sweet potato flavor disappeared. The plums, bayberries, blueberries, and kiwis transformed from their natural sweetness into a richer sweet and sour fermented flavor, exhibiting more unique and pleasant flavor characteristics. Further testing of the pH of the fruit substrate before and after fermentation revealed that the pH of the fruit substrate decreased to a certain extent after fermentation (see Table 7 for details).

[0120] The changes in amino acid content of fermented plums and blueberries with good flavor and texture were analyzed (with uninoculated fruit pulp of YYS-Pb1 as the control group). Amino acid detection was performed using the GB 5009.124-2016 National Food Safety Standard for the Determination of Amino Acids in Food, with an LA8080 amino acid analyzer. The results are shown in Table 8. The calculation method for the amino acid growth rate is as follows:

[0121] Amino acid growth rate % = (fermentation sample - control) / control * 100%.

[0122] Table 7 Effect of YYS-Pb1 fermentation on the pH value of fruit pulp

[0123]

[0124] Table 8 Effects of YYS-Pb1 fermentation on amino acids in fruit pulp

[0125]

[0126] The test data shows that:

[0127] After fermentation with YYS-Pb1 bacteria, the total amino acid content in the plum and blueberry pulp matrices increased from 1.518 (g / kg) and 1.368 (g / kg) to 1.894 (g / kg) and 1.937 (g / kg), respectively, representing increases of 24.78% and 41.61%, indicating that YYS-Pb1 fermentation can significantly increase the amino acid content in the pulp (p≤0.01).

[0128] It should be noted that, based on the above design concept, the Lactobacillus reuteri YYS-Pb1 can also be applied to the fermentation treatment of other fruit fermentation raw materials, including but not limited to plums, bayberries, blueberries, oranges, kiwis, etc.

[0129] Example 11: Preparation of probiotic agent from Lactobacillus reuteri YYS-Pb1

[0130] Lactobacillus reuteri YYS-Pb1 is inoculated into a culture medium, such as MRS medium, and cultured at 0-38°C for more than 15 hours. The bacterial cells are collected by centrifugation and resuspended in, for example, physiological saline or PBS buffer to prepare a liquid bacterial preparation containing Lactobacillus reuteri YYS-Pb1. Optionally, Lactobacillus reuteri YYS-Pb1 bacterial cells are resuspended in a cell protectant and a carrier, and then freeze-dried to obtain a solid bacterial powder preparation containing Lactobacillus reuteri YYS-Pb1.

[0131] Optionally, *Lactobacillus reuteri* YYS-Pb1 can be used as a raw material component in heavy metal lead / cadmium detoxification products, phenyl lactic acid producing products, and fruit pulp fermentation products. *Lactobacillus reuteri* YYS-Pb1 can exist in the products in liquid or solid form.

[0132] Based on the results of the above embodiments, the *Lactobacillus reuteri* YYS-Pb1 provided by the present invention has the following properties and effects:

[0133] In summary, compared with the prior art, the *Lactobacillus reuteri* YYS-PB1 provided by this invention has the following beneficial effects:

[0134] a. YYS-Pb1 can utilize carbohydrates such as cellobiose, maltose, sucrose, raffinose, and lactose;

[0135] b. It can adsorb heavy metal lead, with an adsorption rate as high as 98.457%;

[0136] c. It can adsorb the heavy metal cadmium, with an adsorption rate of 67.02%;

[0137] d. It can produce phenyllactic acid. After adding 0.5 g / L of phenylalanine to the culture medium, the yield of phenyllactic acid is 440.13 mg / L.

[0138] e. It has good hydrophobic properties, with a hydrophobicity greater than 74% when xylene and n-hexane are organic phases.

[0139] f. It has a good biofilm formation ability, which is 3.263 times that of the control. Adding galactose to the culture environment can increase its biofilm formation.

[0140] g. After treatment in a gastric juice environment with pH 2.5 for 1-5 hours, the survival rate was 70.23-90.11%, and after treatment in an artificial simulated gastric juice environment with pH 3.0 for 1-5 hours, the survival rate was between 88.55-95.28%.

[0141] h. When treated in pancreatic juice environment at pH 7.5 for 3-6 hours, the survival rate was as high as greater than 63%, and when treated in pancreatic juice environment at pH 8.0 for 3 hours and 6 hours, the survival rates were 85.46% and 62.26%, respectively.

[0142] i. It can ferment a variety of fruits such as plums, bayberries, blueberries, oranges and kiwis, and produce good flavor characteristics, improve the sweet potato flavor after fruit pulp sterilization, and significantly increase the amino acid content of fermented plum and blueberry pulp. The total amino acid value-added rate after fermentation is 24.78% and 41.61%, respectively.

[0143] j. YYS-Pb1 was isolated from the feces of healthy infants. It is a natural beneficial bacterium with high safety. It can be used as a detoxification product for heavy metals such as lead / cadmium, a product for producing phenyl lactic acid, and a fermentation strain for fruit pulp in functional products, and has broad application prospects.

[0144] For example:

[0145] (1) Lactobacillus reuteri YYS-Pb1 can be used as a raw material component of the composition to prepare a composition with the above-mentioned functions.

[0146] The bacterial strain present in the composition includes, but is not limited to, one or more combinations of *Lactobacillus reuteri* YYS-Pb1 (non-inactivated), *Lactobacillus reuteri* YYS-Pb1 (inactivated), metabolites of *Lactobacillus reuteri* YYS-Pb1 strain, and lyophilized *Lactobacillus reuteri* YYS-Pb1 strain. Preferably, the number of *Lactobacillus reuteri* YYS-Pb1 in the composition is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g. More preferably, the number of *Lactobacillus reuteri* YYS-Pb1 is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0147] (2) Various plants (such as fruits, Chinese herbal medicines, grains, etc.) can be used as raw materials, combined with various ingredients, and inoculated with Lactobacillus reuteri YYS-Pb1 for fermentation treatment to prepare fermented products. These fermented products can be used to prepare products with functions such as detoxification (based on its ability to adsorb heavy metals lead and cadmium) and improving immunity (based on its ability to produce phenyl lactic acid).

[0148] The fermentation raw materials can be various conventionally used plant fermentation raw materials, and the auxiliary materials can also be existing conventional auxiliary materials, including but not limited to the above-mentioned options. The fermented product is, but not limited to, used for the preparation of fermented foods.

[0149] In summary, *Lactobacillus reuteri* YYS-Pb1 and / or its ferments, based on their characteristics, can be used in functional products that include at least one of the following functions:

[0150] (1) Can adsorb heavy metal lead

[0151] (2) Can adsorb heavy metal cadmium

[0152] (3) High production of phenyllactic acid

[0153] (4) It can be used to ferment a variety of fruits, improve the flavor of fruits, improve the flavor of fruit pulp and increase the amino acid content in berry pulp.

[0154] Among them, products with the above-mentioned (1)-(4) functions include, but are not limited to, heavy metal detoxification products, phenyl lactic acid functional products, fruit pulp fermentation strain products that improve the fermentation effect of fruit pulp, and products with obvious effects such as detoxification, anti-oxidation and improving immunity; or products with other obvious effects based on the functions of (1)-(4).

[0155] In addition, Lactobacillus reuteri YYS-Pb1 also has good hydrophobic interaction and good biofilm formation ability;

[0156] The hydrophobic forces of bacteria refer to the adsorption forces on bacterial surfaces that resist the stickiness of water molecules. These forces are mainly controlled by cell membrane structure, biomolecular surface properties, and chemical composition, and have a significant impact on their vitality. Studies have found that bacterial surface hydrophobicity can promote their reproduction, protect them from environmental pollutants, and enable them to adapt to environmental changes. Therefore, the YYS-Pb1 bacterium possesses excellent hydrophobic forces, which is beneficial for its applications (e.g., as a raw material for detoxification or phenyl lactic acid production, or as a fermentation strain for fruit pulp), as it exhibits good reproductive capacity and environmental adaptability.

[0157] Bacterium YYS-Pb1 has excellent biofilm formation ability, specifically with the following functions:

[0158] Biofilms act as a barrier: water-soluble substances on both sides of the membrane cannot pass freely, preventing antibiotics, antibodies, complement, immune cells, and other substances from entering the membrane, thus protecting bacteria from attack.

[0159] Biofilms play a role in nutrient acquisition: biofilms are semi-permeable, allowing certain small molecules to pass through selectively, which helps bacteria obtain nutrients.

[0160] Biomembrane substance transport function: Biomembranes have a substance transport function, enabling the exchange of substances between cells and the surrounding environment. For example, the cell membrane can achieve the exchange of substances between cells and the surrounding environment through its rotational movement function.

[0161] Biofilms protect bacteria from drug attack: When bacteria form a biofilm, their surface area increases, making it difficult for drugs to penetrate into the biofilm, thus protecting the bacteria from drug attack.

[0162] Therefore, the excellent biofilm-forming ability of YYS-Pb1 is beneficial for its application (e.g., as a raw material for detoxification or phenyl lactic acid production, or as a fruit pulp fermentation strain for fruit fermentation), as it has good reproductive capacity, survival capacity, and environmental adaptability.

[0163] It should be noted that:

[0164] (1) Definition:

[0165] The term "food" as used herein is used in a broad sense, encompassing human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders and tablets, as well as to disperse in liquids to prepare liquid dosage forms and other dosage forms suitable for oral administration to humans, including but not limited to powders.

[0166] The composition includes, but is not limited to, microbial preparations, and the composition containing *Lactobacillus reuteri* YYS-Pb1 can be used in other forms of products.

[0167] The *Lactobacillus reuteri* YYS-Pb1 present in the composition may be in the form of, but is not limited to, non-inactivated bacteria, inactivated bacteria, metabolites, lyophilized strains, etc. It is anticipated that the *Lactobacillus reuteri* YYS-Pb1 may also exist in the composition in other forms.

[0168] (2) The relevant prior art means or prior art terms involved in this application:

[0169] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through an analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. x "OD" is the optical density value measured when the wavelength is set to X nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. The method for measuring the "OD" value is existing technology, and its principle and method will not be described here.

[0170] The determination of total bacterial count P1 and dead bacterial count P2 using flow cytometry is an existing technology, and its principles and methods will not be elaborated here.

[0171] Carbon source utilization analysis was performed using biochemical identification strips for lactic acid bacteria. This is an existing technology, and its principles and methods will not be elaborated here.

[0172] (3) The formulations of the culture media used in the examples are as follows:

[0173] MRS medium (g / L): Casein peptone 10, beef extract 10, yeast extract 5, glucose 5, sodium acetate 5, K2HPO4 2, diammonium citrate 2, MgSO4·7H2O 0.2, MnSO4·H2O 0.05, Tween 80 1; pH 6.2. For solid medium, add 2% agar to the above and sterilize at 121℃ for 15 min.

[0174] Unless otherwise specified, the experimental procedures involved in the embodiments of the present invention are conventional experimental procedures in the art, and the reagents or instruments involved can be obtained from legitimate channels.

[0175] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each embodiment or technical solution of the present invention can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or the background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. A type of *Lactobacillus reuteri* ( Limosilactobacillus reuteri YYS-Pb1, characterized in that: Its accession number is CGMCC No.28613.

2. The composition, characterized in that: Its components include *Lactobacillus reuteri* YYS-Pb1 as described in claim 1.

3. The composition according to claim 2, characterized in that: The composition includes a microbial preparation.

4. The composition according to claim 2, characterized in that: The composition contains one or more of the following: uninactivated Lactobacillus reuteri YYS-Pb1 strain and freeze-dried Lactobacillus reuteri YYS-Pb1 strain.

5. Fermentation broth, characterized in that: It was obtained by fermentation of *Lactobacillus reuteri* YYS-Pb1 as described in claim 1.

6. The application of *Lactobacillus reuteri* YYS-Pb1 as described in claim 1 or the fermentation broth as described in claim 5 in the preparation of any one of the following functional products, characterized in that: (1) Functional products used to adsorb heavy metal lead; (2) Functional products for adsorbing heavy metal cadmium; (3) Functional products used in the production of phenyllactic acid; (4) Functional products used to improve the flavor of fruit pulp and increase the amino acid content in fruit pulp.

7. The use of *Lactobacillus reuteri* YYS-Pb1 as described in claim 1 or the composition as described in any one of claims 2-4 in the preparation of fermented foods, characterized in that... The fermented food includes fermented fruit pulp.

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