Preparation and application of lactobacillus reuteri HADIG-LR003 metagen rich in organic copper

Abstract

The invention discloses preparation and application of a lactobacillus reuteri HADIG-LR003 metagen rich in organic copper, and belongs to the technical field of metagen preparation. The invention discloses an application of lactobacillus reuteri HADIG-LR003 in preparation of organic copper, and a lactobacillus reuteri culture rich in organic copper is obtained by optimizing a copper enrichment condition of the lactobacillus reuteri HADIG-LR003. According to the invention, the lactobacillus reuteri culture rich in organic copper is prepared into a metagen product, and the growth promoting effect of the organic copper product is verified at the rat level, so that a new idea is provided for the development of novel organic copper products.

Description

Technical Field

[0001] The present invention relates to the technical field of postbiotic preparation, and in particular to the preparation and application of a Lactobacillus reuteri HADIG-LR003 postbiotic rich in organic copper. Background Art

[0002] Copper is an essential trace element for the animal body, playing a crucial nutritional and physiological role in growth, development, and immune regulation. It is not only a component or activator of numerous enzymes involved in metabolism, but also maintains iron metabolism and promotes hemoglobin synthesis and red blood cell maturation. Furthermore, copper is involved in the formation of bone cells, collagen, and elastin, playing a crucial role in skeletal development. The addition of copper sulfate to feed can improve the growth performance of livestock and poultry. Since Barker et al. first discovered in 1955 that adding high copper to pig feed significantly improved feed utilization and growth rate, thereby significantly enhancing pig growth performance, copper sulfate has become increasingly widely used in animal husbandry. Subsequent studies, such as those conducted by Hawbaker et al. in 1961, further confirmed that copper sulfate levels between 125 mg / kg and 250 mg / kg effectively promoted piglet growth, with optimal growth responses achieved at 250 mg / kg. Therefore, the livestock and poultry industry, especially the pig farming industry, began to use high-dose copper sulfate as a feed additive to improve the production performance of weaned piglets and growing and fattening pigs.

[0003] At present, copper sulfate is the main source of inorganic copper in feed. Although it occupies an important position, it has some unfavorable factors that cannot be ignored. These factors include low absorption rate, high emission, strong hygroscopicity and easy caking, which not only affect the quality of feed, but also may damage the vitamins and oils in the feed. In addition, since livestock and poultry cannot fully absorb the copper in the feed, a large amount of copper is excreted from the body with feces. This behavior directly pollutes the surrounding soil, water sources and air, and may indirectly affect the yield and quality of crops, posing a threat to species diversity and aquatic ecological environment. More seriously, high doses of copper may also increase the deposition of trace elements in tissues and organs, thereby posing a potential food safety risk. Therefore, for the use of copper sulfate in feed, it is necessary to actively seek more environmentally friendly and efficient alternatives. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of a Lactobacillus reuteri HADIG-LR003 postbiotic rich in organic copper, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is the use of Lactobacillus reuteri HADIG-LR003 in the production of organic copper from inorganic copper. The Lactobacillus reuteri HADIG-LR003 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 10, 2023, with the deposit number CGMCC No. 27852.

[0007] The second technical solution of the present invention is a method for preparing a Lactobacillus reuteri postbiotic rich in organic copper, comprising the following steps:

[0008] (1) inoculating Lactobacillus reuteri HADIG-LR003 seed solution into a culture medium for cultivation;

[0009] (2) adding copper solution to the culture;

[0010] (3) directly collecting the culture and freeze-drying it to obtain the organic copper-rich Lactobacillus reuteri postbiotic.

[0011] The third technical solution of the present invention is a Lactobacillus reuteri postbiotic rich in organic copper prepared by the preparation method.

[0012] A fourth technical solution of the present invention is the use of the organic copper-rich Lactobacillus reuteri postbiotic in the preparation of organic copper-rich feed.

[0013] A fifth technical solution of the present invention is a feed rich in organic copper, comprising the organic copper-rich Lactobacillus reuteri postbiotic.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] This invention discloses the use of Lactobacillus reuteri HADIG-LR003 in the preparation of organic copper. The copper-enriched conditions are optimized to obtain a Lactobacillus reuteri culture rich in organic copper. This invention prepares the organic copper-rich Lactobacillus reuteri culture into a postbiotic product and verifies the growth-promoting effect of the organic copper product in rats, providing new ideas for the development of novel organic copper products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1Figure 2 shows the difference in copper content in Lactobacillus reuteri HADIG-LR003 at different treatment concentrations and treatment times. A represents the amount of copper accumulated by Lactobacillus reuteri HADIG-LR003 at different copper addition time periods; B represents the amount of copper accumulated by Lactobacillus reuteri HADIG-LR003 at different copper addition concentrations.

[0018] Figure 2 Figures 1 and 2 show the distribution of inorganic copper and the copper distribution of the experimental group. Figure A shows the copper distribution after desalting inorganic copper sulfate; Figure B shows the copper distribution after desalting the entire HADIG-LR003 culture after disruption; Figure C shows the copper distribution after desalting the HADIG-LR003 culture after centrifugation, retaining only the supernatant; Figure D shows the copper distribution after desalting the HADIG-LR003 culture after centrifugation, retaining only the bacterial cells; and Figure E is a combined graph of Figures A and B. The data for Figure D corresponds to the right X-axis, while the data for the remaining three figures correspond to the left X-axis.

[0019] Figure 3 It is a purified growth and reproduction feed ingredient.

[0020] Figure 4 The following table represents the bacterial yield and copper content of Lactobacillus reuteri HADIG-LR003. A represents the bacterial yield of Lactobacillus reuteri HADIG-LR003 when different exogenous copper concentrations are added; B represents the copper content of pure cells after freeze-drying at exogenous copper concentrations of 50 mg / kg and 75 mg / kg. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0027] The present invention provides an embodiment of the present invention. The use of Lactobacillus reuteri HADIG-LR003 in the production of organic copper from inorganic copper is provided. The Lactobacillus reuteri HADIG-LR003 was deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection on July 10, 2023, with a deposit number of CGMCC No. 27852. This strain has been disclosed in patent CN116904371A.

[0028] The present invention also provides a method for preparing a Lactobacillus reuteri postbiotic rich in organic copper, comprising the following steps:

[0029] (1) inoculating Lactobacillus reuteri HADIG-LR003 seed solution into a culture medium for cultivation;

[0030] (2) adding copper solution to the culture;

[0031] (3) directly collecting the culture and freeze-drying it to obtain the organic copper-rich Lactobacillus reuteri postbiotic.

[0032] In some specific embodiments, the culture conditions include: static culture at 37°C for 10-12 hours.

[0033] In some specific embodiments, the copper solution is added to a final concentration of 300 mg / L;

[0034] The treatment conditions for adding the copper solution to the culture are: static culture at 37° C. for 20-24 hours.

[0035] The embodiment of the present invention also provides a Lactobacillus reuteri postbiotic rich in organic copper prepared by the preparation method.

[0036] The embodiment of the present invention also provides the use of the organic copper-rich Lactobacillus reuteri postbiotic in preparing organic copper-rich feed.

[0037] An embodiment of the present invention further provides a feed rich in organic copper, comprising the organic copper-rich Lactobacillus reuteri postbiotic.

[0038] In some specific embodiments, the content of Lactobacillus reuteri HADIG-LR003 in the feed is 0.33‰ to 0.67‰.

[0039] The Lactobacillus reuteri HADIG-LR003 used in the present invention was deposited in the General Microbiology Center of the China National Committee for the Preservation of Microorganisms on July 10, 2023, with a deposit number of CGMCC No. 27852. This strain has been disclosed in patent CN116904371A.

[0040] In order to better reflect the copper-enriching effect of the strains screened in this experiment, the present invention purchased a standard strain of Lactobacillus reuteri: ATCC 23272, with a strain collection number of 1.614, from the Guangdong Provincial Microbial Culture Collection Center.

[0041] The MRS culture medium used in the present invention is prepared as follows: accurately weigh 52.4 g of MRS broth powder (Qingdao Haibo) into a 2 L beaker, add 1 L of deionized water, stir thoroughly, transfer to a suitable conical flask or reagent bottle, and sterilize by autoclaving at 118°C for 15 minutes before use.

[0042] To prepare a solid culture medium, accurately weigh 52.4g of MRS broth powder and 20g of agar into a 2L beaker. Add 1L of deionized water, stir thoroughly, and transfer to a suitable Erlenmeyer flask or reagent bottle. Autoclave at 118°C for 15 minutes. Remove the autoclaved solid culture medium, cool it to 60°C, and pour approximately 20mL into disposable sterilized plates. Once the culture medium has completely solidified, seal the plates with parafilm and store them upside down in a refrigerator at 4°C until ready for use.

[0043] Example 1

[0044] 1. Test for determining copper treatment time

[0045] (1) Resuscitate Lactobacillus reuteri HADIG-LR003. Use a sterile inoculation loop to pick up the bacterial solution and streak the plate on an MRS solid plate to obtain pure colonies. Place the streaked plate upside down in a sealed anaerobic culture jar, add an anaerobic gas production bag and an anaerobic indicator. Place the sealed anaerobic culture jar in a 37°C constant temperature incubator and culture for 24 hours. Remove the cultured plate, pick a single colony, inoculate it into 5 mL of fresh MRS broth, and culture it at 37°C overnight to prepare the seed solution.

[0046] (2) Inoculate the seed solution into fresh MRS broth at a ratio of 2% (v / v), mix well, and incubate at 37°C for 10-12 hours.

[0047] (3) Gently shake the culture to mix thoroughly, add copper sulfate solution with a final concentration of 75 mg / L, and then divide it into 6 portions. Continue to incubate at 37°C. Remove the corresponding culture at 4, 8, 12, 16, 20, and 24 hours.

[0048] (4) Harvest the bacteria (take 15-40 mL of bacterial solution, transfer to a 50 mL centrifuge tube, centrifuge at 4°C, 4000 rpm, for 10 minutes, and discard the supernatant), wash the bacteria (take 15 mL of DB (Desalting Buffer), resuspend the bacterial pellet with a Pasteur pipette, centrifuge at 4°C, 4000 rpm, for 10 minutes, and discard the supernatant), and disrupt the bacteria using a cell disruptor. Repeat the disruption three times. Take a sample of the fully disrupted bacteria and add 5% (v / v) nitric acid for nitrolysis. Determine the copper content of the bacteria using ICP-MS, and measure each sample three times.

[0049] 2. Experiment on determination of copper treatment concentration

[0050] (1) Resuscitate Lactobacillus reuteri HADIG-LR003. Use a sterile inoculation loop to pick up the bacterial solution and streak the plate on an MRS solid plate to obtain pure colonies. Place the streaked plate upside down in a sealed anaerobic culture jar, add an anaerobic gas production bag and an anaerobic indicator. Place the sealed anaerobic culture jar in a 37°C constant temperature incubator and culture for 24 hours. Remove the cultured plate, pick a single colony, inoculate it into 5 mL of fresh MRS broth, and culture it at 37°C overnight to prepare the seed solution.

[0051] (2) Inoculate the seed solution into fresh MRS broth at a ratio of 2% (v / v), mix well, and incubate at 37°C for 10-12 hours.

[0052] (3) Gently shake the culture to mix thoroughly, divide it into five portions, add copper solution with final concentrations of 25, 50, 75, 150, and 300 mg / L, respectively, and continue to incubate at 37°C for 20-24 hours.

[0053] (4) Collect the bacteria (take 15-40 mL of bacterial solution, transfer to a 50 mL centrifuge tube, centrifuge at 4°C, 4000 rpm, for 10 minutes, and discard the supernatant), wash the bacteria (take 15 mL of DB, resuspend the bacterial pellet with a Pasteur pipette, centrifuge at 4°C, 4000 rpm, for 10 minutes, and discard the supernatant), and disrupt the bacteria using a cell disruptor. Repeat the disruption three times. Take a sample of the fully disrupted bacteria and add 5% (v / v) nitric acid for nitrolysis. Determine the copper content of the bacteria using ICP-MS, and measure each sample three times.

[0054] The experimental results are shown in Figure 1 ,Depend on Figure 1 As shown in Figure A, after 20 hours of copper treatment, the copper content in the bacteria no longer increases. Figure 1 As shown in Figure B, among the copper concentrations selected in this experiment, the higher the copper concentration, the higher the copper content in the bacteria.

[0055] Example 2

[0056] Verification of the conversion of inorganic copper into organic copper by Lactobacillus reuteri culture

[0057] After adding exogenous inorganic copper (copper sulfate) to the Lactobacillus reuteri culture, during the incubation process, inorganic copper ions can be combined with materials such as amino acids, carbohydrates, polypeptides and proteins in the culture to be converted into organic copper. In order to verify the transformation of copper from inorganic to organic, the present embodiment utilizes the molecular sieve principle, with the help of an AKTA protein purification system and a desalting column, to desalinate the Lactobacillus reuteri mixed solution after crushing, separate high-molecular-weight substances from low-molecular-weight substances, collect samples, and respectively measure the copper concentration in the samples, and verify the morphological changes of copper by the change of the copper distribution state in the samples.

[0058] 1. Sample preparation

[0059] (1) First, inorganic copper is dissolved in Desalting Buffer as an inorganic copper control sample. After desalting treatment and copper concentration determination, the inorganic copper distribution state diagram can be obtained.

[0060] (2) The Lactobacillus reuteri HADIG-LR003 seed liquid was inoculated into fresh MRS broth at a ratio of 2% (v / v) and mixed. Incubate at 37°C for 10-12 hours. Add 75 mg / L copper concentration solution and treat for 20 hours. First, divide the culture into two parts. One part is directly crushed with a cell crusher and centrifuged at 4°C, 4000 rpm for ten minutes. The supernatant is collected as test group 1. The other part is centrifuged at 4°C, 4000 rpm for ten minutes, and the supernatant and bacteria are separated. The supernatant is directly collected as test group 2. The bacteria are collected, washed with DB and crushed. After the crushing is completed, it is centrifuged at 4°C, 4000 rpm for ten minutes, and the supernatant is collected as test group 3.

[0061] 2. Prepare the AKTA protein purification system

[0062] (1) Open the software, enter the purification interface, and install the High Trap Desalting Column.

[0063] (2) Wash the pumps: Place the probes of pump A and pump B into the balancing solution DB and the eluent EB respectively. On the software interface, click Manual-pump-pump wash basic in sequence to turn pump A and pump B to the ON state to clean pumps A and B.

[0064] (3) After the pump is cleaned, start cleaning the sample loop and desalting column. Inject 2 mL of deionized water into the sample loop and run the set desalting program to clean it. Rinse 2-3 times before use.

[0065] (4) Inject the sample to be desalted into the sample loop using a 2 mL syringe (up to 2 mL), run the desalting program, and fill the collector with 1.5 mL EP tubes.

[0066] (5) After desalting, collect all EP tubes and temporarily place them on ice. Then, measure the copper concentration manually.

[0067] (6) To prevent cross-contamination between samples, the sample column needs to be cleaned with deionized water after desalting a sample. That is, after each sample is measured, the water sample needs to be washed once.

[0068] Test results:

[0069] After desalination, all EP tubes were collected, a total of 29, and the copper concentration of the liquid in all EP tubes was measured. After the measurement was completed, a copper distribution map in different EP tubes was drawn. The results are shown in Figure 2 .Depend on Figure 2 It can be seen that the peak graphs of each group of copper-containing samples shifted significantly compared with the inorganic copper sulfate control group, and new absorption peaks appeared. The peak elution time in the molecular sieve was shorter, indicating that the copper ions were mainly bound copper and the molecular weight increased. It was preliminarily judged to be organic copper.

[0070] Example 3

[0071] Preparation of Lactobacillus reuteri postbiotics rich in organic copper

[0072] 1 Calculation of Lactobacillus reuteri yield and bacterial copper content

[0073] (1) Lactobacillus reuteri HADIG-LR003 was selected and copper treatment was performed after 10-12 hours of culture. The final concentrations of copper treatment were 0, 50 mg / L and 75 mg / L, and the copper treatment time was 20 hours.

[0074] (2) Collect and wash the bacteria as before to remove the influence of copper ions in the culture medium.

[0075] (3) The washed bacterial pellet was placed in a -80°C ultra-low temperature freezer for pre-freezing overnight, and then freeze-dried using a vacuum freeze dryer. The freeze-dried bacterial cells were weighed using an analytical balance to calculate the yield.

[0076] (4) Take the freeze-dried bacteria and dissolve them with an appropriate amount of deionized water. Then, measure the copper concentration manually and calculate the copper content of the bacteria.

[0077] 2. Determination of viable counts in Lactobacillus reuteri culture

[0078] (1) The frozen bacteria were revived and seed solution was prepared using the method of Example 1.

[0079] (2) Inoculate the seed solution into fresh MRS broth at a ratio of 2% (v / v), mix well, and incubate at 37°C for 10-12 hours.

[0080] (3) Measure and record OD 600 The bacterial solution was diluted with physiological saline in a gradient manner, with the dilution multiples of 10 -5 , 10 -6 , 10 -7 Place 100 μL of the mixed dilution onto an MRS solid plate and spread it with a sterile spreader to evenly cover the surface of the plate. Repeat three times for each dilution. After standing upright in a clean bench for ten minutes, place all plates in an anaerobic culture tank, along with an anaerobic gas bag and anaerobic indicator. Place the anaerobic culture tank in a 37°C incubator for 24 hours.

[0081] (4) Take out the plates and observe the number of colonies on the plates with different dilutions. Select the plates corresponding to the dilutions with a colony count of 30-300 and count them to calculate the CFU / mL.

[0082] 3 Preparation of Lactobacillus reuteri rich in organic copper

[0083] (1) Lactobacillus reuteri HADIG-LR003 was selected and copper was added to the culture at 10-12 hours. The final concentration of copper addition was 300 mg / L and the copper addition time was 20 hours. After the culture was completed, the front sample was collected by two methods.

[0084] (2) The first method is to directly collect the culture. (Then prepare it into lactic acid bacteria copper)

[0085] (3) The second method is to collect the culture in a collection bottle and separate the bacterial precipitate and supernatant using a floor centrifuge. The centrifugation conditions are 4°C, 4000 rpm, and 10 minutes. If necessary, the centrifugation time can be appropriately extended. (This is then prepared as lactic acid bacteria fermented copper)

[0086] (4) The two pre-samples collected were placed in suitable containers and freeze-dried using a vacuum freeze dryer.

[0087] 4. Use of vacuum freeze dryer

[0088] (1) Seal the collected samples with plastic wrap and place them in a -80°C ultra-low temperature freezer for pre-freezing overnight.

[0089] (2) Check the status of the vacuum freeze dryer, turn on the power, and check the status of the exhaust port and the freezing tube.

[0090] (3) Turn on the freezer switch and wait for 30 minutes until the freezer compartment temperature is below -40°C.

[0091] (4) Take out the pre-frozen sample from the refrigerator, use a tool to poke several small holes in the plastic wrap, place the sample on the sample rack, and cover it with a plexiglass cover.

[0092] (5) Select the warm-up vacuum pump on the instrument to preheat the oil pump. Wait for 10-15 minutes until the system pressure drops below 0.1 mbar.

[0093] (6) Select Main Drying on the instrument to enter the main drying mode. Adjust the drying time according to the thickness of the sample. Generally, it takes 24-72 hours to dry.

[0094] (7) After the sample freeze-drying is completed, select "standby" on the instrument to end the drying process and turn off the oil pump.

[0095] (8) Find the vent valve of the freeze dryer and rotate the micro-vent valve very slowly. When you hear a slight venting sound, stop and wait for gas to enter. Observe the vacuum value displayed on the controller. When the vacuum value is above 200 mbar, slightly increase the venting volume until it is balanced with the external atmospheric pressure. Then open all valves. This process takes about 20 minutes.

[0096] (9) After the deflation is completed, close the micro-vent valve, turn off the main unit, remove the organic glass cover, and take out the sample. Finally, the condensation chamber must be defrosted, and the moisture in the cavity must be wiped with a clean cloth to keep the cavity clean; and the defrost water must be drained and the drain valve closed.

[0097] 5. Preparation of feed rich in organic copper

[0098] The prepared Lactobacillus reuteri dry powder was used as an additive and a purified growth and reproduction feed (AIN-93G) was used as the base to prepare a high-copper feed. Copper in the purified growth and reproduction feed was added in the form of copper carbonate at a concentration of 6 mg / kg. The preparation of the high-copper feed was commissioned to Synergy Biotechnology (Nanjing, Jiangsu). The ingredients of the purified growth and reproduction feed are shown in the table below. Figure 3 .

[0099] Test results:

[0100] After calculation, the yield of Lactobacillus reuteri HADIG-LR003 was 2.06±0.36g / L. When the exogenous copper concentration was 50mg / L, the bacterial copper content was 1.83±0.02mg / g; when the exogenous copper concentration was 75mg / L, the bacterial copper content was 3.17±0.04mg / g. Figure 4 The results showed that the bacterial yield per liter of fermentation broth was only about 2g / L, and the copper content per gram of bacterial cells was also limited. The present invention has demonstrated that the copper in the bacterial cells and fermentation broth exists primarily in the form of organically bound copper. Therefore, considering the cost implications of practical applications, subsequent preparations will involve freeze-drying the entire culture, rather than just the bacterial cells.

[0101] In the test of viable bacterial count, 10 -5 The dilution corresponding to the number of colonies on the plate is too large (>1000), 10 -7 The number of colonies on the plate corresponding to the dilution is too small (<20), 10 -6 The number of colonies on the plate corresponding to the dilution was moderate. After calculation, the CFU / mL of Lactobacillus reuteri HADIG-LR003 was 3.24×10 8 ±1.42×10 7 .

[0102] Lactobacillus reuteri HADIG-LR003 was used to successfully produce lactobacillus copper and lactobacillus fermentation copper postbiotics, producing a high-copper feed. In addition to the two postbiotics produced in this experiment, the inventors also used inorganic copper (CuSO4·5H2O) and copper glycinate as additives to produce high-copper feed. Subsequent animal experiments were conducted to verify and compare the effects of different copper sources.

[0103] The high-copper feed ingredients were categorized into four groups: copper sulfate (CuSO₄·5H₂O), copper glycinate (CuGly), lactic acid bacteria copper (Lactobacillus reuteri HADIG-LR003) culture supernatant (Cu-treated Lactobacillus reuteri HADIG-LR003), and lactic acid bacteria copper (Lactobacillus copper) culture supernatant (Cu-treated Lactobacillus reuteri HADIG-LR003). Two copper concentrations were set for each copper source: 50 mg / kg and 100 mg / kg. The lactic acid bacteria copper feed at 50 mg / kg contained approximately 0.33‰ of lactic acid bacteria, while the lactic acid bacteria copper feed at 100 mg / kg contained approximately 0.67‰ of lactic acid bacteria.

[0104] Example 4

[0105] Effects of organic copper-rich Lactobacillus reuteri postbiotics on growth performance in rats

[0106] SPF-grade Sprague-Dawley male rats were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Animal husbandry experiments were conducted in the SPF animal facility at Wenzhou Medical University. All rats were housed in a controlled environment at 20–25°C, 50%–60% humidity, and a 12-h light-dark cycle. They had free access to feed and deionized water.

[0107] The basal diet used in the experiment was a purified growth and reproduction feed formulated according to the American Institute of Nutrition standard AIN-93G. Aside from variations in the form and concentration of copper, all other components of the diet remained identical. Feed production was contracted to Synergy Biological (Nanjing, Jiangsu).

[0108] 1 Experimental design

[0109] (1) This study used a single-factor experimental design. Forty-five male, three-week-old, SPF-grade SD rats of similar body weight were randomly divided into nine treatment groups, with five replicates in each treatment group and one rat in each replicate.

[0110] (2) This experiment can be divided into three stages. The first stage is the rat adaptation period, which takes 4 days and is fed with ordinary growth and reproduction feed.

[0111] (3) The second stage is the establishment of the copper-deficient animal model, which takes 10 days and is fed with a purified growth and reproduction feed (i.e., basic copper-deficient feed). Before the modeling period, the animals were fasted for 14 hours, with free access to water during this period, and then weighed and blood was collected from the orbital cavity. After the modeling period, the animals were fasted for 14 hours, with free access to water during this period, and then weighed and blood was collected from the orbital cavity.

[0112] (4) The third stage was the copper supplementation period, which lasted for 10 days. One control group was fed a basic copper-deficient diet, and the copper sources and copper supplementation concentrations of the remaining eight groups are shown in Table 1.

[0113] Table 1

[0114]

[0115] (5) During the experiment, the rat bedding was changed regularly and deionized water was replenished regularly.

[0116] 2. Determination of production performance indicators

[0117] (1) Daily weight gain (ADG): The rats were weighed before and after each period, and fasted for 14 h before weighing.

[0118] (2) Feed intake (ADFI): Calculated based on feed consumption. Feed is replenished regularly and feed consumption is measured.

[0119] (3) Feed-to-gain ratio (F / G): calculated based on feed intake and daily weight gain.

[0120] 3. Determination of blood copper content

[0121] (1) Serum copper content was determined using the Nanjing Jiancheng kit.

[0122] (2) Principle of determination: Under acidic conditions, Cu 2+ Ascorbic acid dissociates Cu from ceruloplasmin and albumin. 2+ Reduction to Cu + , Cu + It reacts with the complexing agent 3,5-dibromo-PAESA to produce a blue complex. The absorbance of the blue complex is measured at a wavelength of 600 nm to calculate the Cu 2+ concentration.

[0123] (3) Operation method: Take out the reagent kit from the 4°C refrigerator and allow the pure copper ion to equilibrate at room temperature for about 20 minutes. At the same time, prepare the sample and deionized water.

[0124] (4) Add 10 μL of deionized water, standard, and sample to be tested into a 96-well plate.

[0125] (5) Add 150 μL of Reagent 1 and gently shake to mix, avoiding bubbles. Incubate in a 37°C incubator for 5 minutes, then remove the sample and set the wavelength of the microplate reader to 700 nm. Read the absorbance A1.

[0126] (6) Remove the 96-well plate from the microplate reader and add 50 μL of Reagent 2. Mix gently to avoid bubbles. Incubate in a 37°C incubator for 5 minutes, then remove the plate and set the wavelength of the microplate reader to 600 nm. Read the absorbance A2, ΔA = A2 - A1.

[0127] (7) Calculate the copper concentration in the sample using the formula: Cu (μM) = [ΔA measured value - ΔA blank value) / (ΔA standard value - ΔA blank value)] * standard concentration.

[0128] 4. Determination of serum ceruloplasmin (CP) activity

[0129] (1) The activity of serum ceruloplasmin was determined using the Nanjing Jiancheng kit.

[0130] (2) Principle of Assay: Ceruloplasmin (CP) catalyzes the conversion of dianisidine into a light yellow-brown product. After adding a terminator, a purple-red solution is formed and the absorbance is measured at 540 nm. The enzyme activity can be calculated based on the absorbance coefficient of the product.

[0131] (3) Take out the reagent kit, prepare deionized water and samples, and set the water bath temperature to 37°C.

[0132] (4) Add deionized water and 10 μL of sample to a 1.5 mL EP tube.

[0133] (5) Add 160 μL of buffer and 40 μL of matrix solution.

[0134] (6) Vortex mix and incubate in a water bath at 37°C for 20 minutes.

[0135] (7) Add 400 μL of the terminator. Vortex to mix thoroughly, let stand at room temperature for 5 minutes, and then measure the absorbance at 540 nm. Adjust the absorbance to zero with deionized water. The measurement can be performed using a spectrophotometer or an enzyme-linked microplate reader. The two use different measurement formulas, so the appropriate formula should be used for calculation.

[0136] (8) Determination formula (spectrophotometer): [(measured OD - control OD) / absorbance coefficient (9.46)] * (1 / 20) * (610 / 10) * 1000. If using a microplate reader, multiply the end of the formula by 10 / 6. The unit is U / L.

[0137] Test results:

[0138] 1 The growth performance indicators of rats in each group during the adaptation period are shown in Table 2. As shown in Table 2, there was no difference in the growth performance indicators of rats among the groups during the adaptation period.

[0139] Table 2 Growth performance indicators of rats in each group during the adaptation period

[0140]

[0141]

[0142] 2 The growth performance indicators of rats in each group during the modeling period are shown in Table 3. As shown in Table 3, there was no difference in the growth performance indicators of rats among the groups during the modeling period.

[0143] Table 3 Growth performance indicators of rats in each group during the modeling period

[0144]

[0145] To verify the success of the model, we collected blood from the rats' orbits before and after the modeling period and measured serum copper levels and ceruloplasmin activity. The results are shown in Tables 4-6. As shown in Tables 4-6, serum copper levels and ceruloplasmin activity decreased significantly before and after the modeling period, indicating that the copper-deficient rat model was successfully established.

[0146] Table 4 Serum copper content and ceruloplasmin activity of rats in each group before modeling

[0147]

[0148]

[0149] Table 5 Serum copper content and ceruloplasmin activity of rats in each group after modeling

[0150]

[0151] Table 6 Comparison of serum copper content and ceruloplasmin activity in rats before and after modeling

[0152]

[0153] 4 After the modeling period, the copper supplementation period began, and different types and concentrations of copper sources were added to the feed. After the copper supplementation period, the growth performance indicators of rats fed with different copper sources were tested. The results are shown in Table 7. As shown in Table 7, adding 50 mg / kg of lactic acid bacteria copper (lactic acid bacteria content is about 0.33‰) to the feed can significantly increase the ADG of rats; adding 50 mg / kg of glycinate copper, 100 mg / kg of lactic acid bacteria fermented copper, 50 mg / kg of lactic acid bacteria copper and 100 mg / kg of lactic acid bacteria copper (lactic acid bacteria content is about 0.67‰) can all significantly increase the ADFI of rats, and 50 mg / kg of lactic acid bacteria copper has the best effect. A multivariate analysis of variance was performed on the results, and it was found that different copper sources had a significant effect on the ADG and ADFI of rats, and different copper concentrations and copper source*copper concentration had no significant effect on the growth performance of rats.

[0154] Table 7 Growth performance indicators of rats under different copper source treatments during the copper supplementation period

[0155]

[0156] Note: 1) The copper content of the basal diet was 6 mg / kg; 2) In the same column, * indicates p < 0.05; ** indicates p < 0.01.

[0157] In summary, the present invention successfully improved the copper-rich level of copper-rich Lactobacillus reuteri HADIG-LR003 by optimizing culture conditions and adjusting the added concentration of exogenous copper. The present invention also designed an experiment to verify its ability to convert inorganic copper into organic copper. Finally, this organic copper-rich Lactobacillus reuteri culture was made into a postbiotic product and added to the feed of rats. Through experiments on rats, the effects of the new organic copper product and traditional inorganic copper products (copper sulfate) and commercial organic copper products (copper glycinate) on the growth performance of rats were compared. The results of the study found that the new organic copper product produced by the present invention can significantly improve the growth performance of rats, and the effect is better than traditional inorganic copper (copper sulfate), and it also has the potential to replace commercial organic copper products (copper glycinate).

[0158] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of Lactobacillus reuteri HADIG-LR003 in producing organic copper from inorganic copper, characterized in that: The Lactobacillus reuteri HADIG-LR003 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 10, 2023, with the deposit number CGMCC No. 27852.

2. A method for preparing a Lactobacillus reuteri postbiotic rich in organic copper, characterized in that: The following steps are involved: (1) inoculating Lactobacillus reuteri HADIG-LR003 seed solution into a culture medium for cultivation; (2) adding copper solution to the culture; (3) directly collecting the culture and freeze-drying it to obtain the organic copper-rich Lactobacillus reuteri postbiotic.

3. The preparation method according to claim 2, characterized in that The culture conditions include: static culture at 37° C. for 10-12 hours.

4. The preparation method according to claim 2, characterized in that The final concentration of the copper solution added is 300 mg / L; The treatment conditions for adding the copper solution to the culture are: static culture at 37° C. for 20-24 hours.

5. The organic copper-rich Lactobacillus reuteri postbiotic prepared by the preparation method according to any one of claims 2 to 4.

6. Use of the organic copper-rich Lactobacillus reuteri postbiotic according to claim 5 in preparing an organic copper-rich feed.

7. A feed rich in organic copper, characterized in that: The invention comprises the organic copper-rich Lactobacillus reuteri postbiotic according to claim 5.

8. The feed according to claim 7, characterized in that The content of Lactobacillus reuteri HADIG-LR003 in the feed is 0.33‰ to 0.67‰.

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