Application of lactoferrin in detection of content of lactobacillus acidophilus in combined bacterium fermentation product

Abstract

The invention provides an application of lactoferrin in detecting the content of lactobacillus acidophilus in a combined bacterium fermentation product. The lactoferrin is used for detecting the content of the lactobacillus acidophilus in the combined bacterium fermentation product, a sample with the content of the lactobacillus acidophilus lower than 1.0 E + 05CFU / mL is accurately detected, the detection limit of the lactobacillus acidophilus is reduced, and the detection range of an existing detection method is expanded.

Description

Technical Field

[0001] The invention relates to application of lactoferrin in detecting the content of Lactobacillus acidophilus, in particular to application of lactoferrin in detecting the content of Lactobacillus acidophilus in a combined bacteria fermentation product, and belongs to the technical field of biological detection. Background Art

[0002] The global probiotics market is expected to grow at a compound annual growth rate of 8.3% and may reach US$91 billion in 2026. Among them, the scale of China's probiotics consumption market may grow rapidly at an annual rate of 11-12%, and it is expected that the scale of China's probiotics market will reach 137.7 billion yuan in 2026. In the future, the global probiotics industry will show a trend of more diverse product types, among which the mixed bacteria fermentation products developed through mixed bacteria fermentation process technology are one of the research hotspots in the global probiotics industry.

[0003] Lactobacillus acidophilus is one of the most important species in the probiotic family. Most of the combined fermentation products on the market are obtained by multi-bacteria fermentation of Lactobacillus acidophilus and other species. For example, most probiotic yogurts are obtained by combined fermentation of Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus bulgaricus.

[0004] Because the number of viable bacteria is an important factor affecting the quality of combined fermentation products, it is very necessary to accurately detect the number of viable bacteria in order to control the quality of combined fermentation products. At present, the combination of "MRS medium + antibiotics" is often used to detect the content of Lactobacillus acidophilus in combined fermentation products. However, when using the combination of "MRS medium + antibiotics" for detection, the detection accuracy is related to the concentration of Lactobacillus acidophilus in the test sample. Once the concentration of Lactobacillus acidophilus is too low, for example, less than 1.0E+05CFU / mL, it is basically impossible to accurately detect the content of Lactobacillus acidophilus. Summary of the invention

[0005] The invention provides an application of lactoferrin in detecting the content of Lactobacillus acidophilus in a combined fermentation product. Lactoferrin is applied to detecting the content of Lactobacillus acidophilus in the combined fermentation product, and samples with a content of Lactobacillus acidophilus lower than 1.0E+05CFU / mL can be accurately detected, thereby reducing the detection limit of Lactobacillus acidophilus.

[0006] The present invention also provides a detection medium for the content of Lactobacillus acidophilus in a combined fermentation product. The detection medium includes lactoferrin. The detection medium is used to detect the content of Lactobacillus acidophilus in a combined fermentation product with a content of Lactobacillus acidophilus lower than 1.0E+05 CFU / mL, and the detection result has high accuracy.

[0007] The present invention also provides a method for preparing the above-mentioned detection culture medium. The detection culture medium obtained by the preparation method can be used to accurately detect the content of Lactobacillus acidophilus in a co-fermentation product with a content of Lactobacillus acidophilus lower than 1.0E+05 CFU / mL.

[0008] The present invention also provides a method for detecting the content of Lactobacillus acidophilus. The method uses the above detection culture medium to detect the content of Lactobacillus acidophilus. The detection result is accurate, the detection limit of Lactobacillus acidophilus is reduced, and the detection range of the existing detection method is expanded.

[0009] The invention provides an application of lactoferrin in detecting the content of Lactobacillus acidophilus in a combined bacteria fermentation product.

[0010] As described above, the combined bacterial fermentation product comprises a first bacterial species and a second bacterial species, the first bacterial species is Lactobacillus acidophilus, and the second bacterial species comprises at least one of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, etc.

[0011] In the application as described above, the detecting of the content of Lactobacillus acidophilus in the combined fermentation product specifically comprises: culturing the combined fermentation product using a culture medium comprising the lactoferrin, and determining the content of the Lactobacillus acidophilus according to the colony count in the culture medium.

[0012] The present invention also provides a detection medium for the content of Lactobacillus acidophilus in a combined bacteria fermentation product, wherein the detection medium comprises lactoferrin.

[0013] The detection medium as described above, wherein the mass percentage of the lactoferrin in the detection medium is 0.01%-0.12%.

[0014] The detection medium as described above, wherein the mass percentage of the lactoferrin in the detection medium is 0.04%-0.08%.

[0015] The detection culture medium as described above, wherein the detection culture medium is mainly composed of MRS culture medium and also includes antibiotics.

[0016] The detection culture medium as described above, wherein the mass ratio of lactoferrin to antibiotic is (0.1 g / kg-1.2 g / kg): (0.1 mg / kg-10 mg / kg).

[0017] The present invention also provides a method for preparing the detection culture medium described in any one of the above items, comprising the following steps:

[0018] The main body of the detection culture medium and the lactoferrin are sterilized separately and then mixed evenly to obtain the detection culture medium.

[0019] The present invention also provides a method for detecting the content of Lactobacillus acidophilus, which uses any of the above-mentioned detection culture media to detect the content of Lactobacillus acidophilus.

[0020] The present invention provides an application of lactoferrin in detecting the content of Lactobacillus acidophilus in a combined fermented product, because the detection of the content of Lactobacillus acidophilus in the combined fermented product needs to exclude the interference of other strains, and the technology of excluding the interference of other strains will have an adverse effect on the growth activity of Lactobacillus acidophilus, and this adverse effect will affect the accuracy of the test result. At the same time, the magnitude of this adverse effect has a certain corresponding relationship with the concentration of Lactobacillus acidophilus in the combined fermented product, which is specifically manifested as, when the concentration of Lactobacillus acidophilus is too low, for example, when it is lower than 1.0E+05CFU / mL, the detection result of the content of Lactobacillus acidophilus is lower by at least one order of magnitude. The application of lactoferrin provided by the present invention in detecting the content of Lactobacillus acidophilus in the combined fermented product can not only exclude the interference of other strains on Lactobacillus acidophilus during the detection process, but also avoid the adverse effect of the technology of excluding the interference of other strains on Lactobacillus acidophilus, thereby ensuring the accuracy of the test result, especially the accurate detection of samples with a content of Lactobacillus acidophilus lower than 1.0E+05CFU / mL, reducing the detection limit of Lactobacillus acidophilus and expanding the detection range of the existing detection method. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The first aspect of the present invention provides an application of lactoferrin in detecting the content of Lactobacillus acidophilus in a combined bacteria fermentation product.

[0023] The combined bacteria fermentation product of the present invention refers to a product prepared by multi-bacteria combined fermentation using Lactobacillus acidophilus and other strains. When detecting the content of Lactobacillus acidophilus in the combined bacteria fermentation product, it is necessary to exclude the interference of other strains, and the technology of excluding the interference of other strains will have an adverse effect on the growth activity of Lactobacillus acidophilus, and the size of this adverse effect has a certain corresponding relationship with the concentration of Lactobacillus acidophilus. When the concentration of Lactobacillus acidophilus is too low, for example, when it is lower than 1.0E+05CFU / mL, this adverse effect is larger, and the detection result of the content of Lactobacillus acidophilus will be lower by at least one order of magnitude. The application of lactoferrin provided by the present invention in detecting the content of Lactobacillus acidophilus in the combined bacteria fermentation product not only effectively excludes the interference of other strains on the content of Lactobacillus acidophilus, but also avoids the adverse effect caused by the technology of excluding the interference of other strains on Lactobacillus acidophilus, thereby ensuring that the test result is accurate, especially the sample with the content of Lactobacillus acidophilus lower than 1.0E+05CFU / mL can be accurately detected, the detection limit of Lactobacillus acidophilus is reduced, and the detection range of the existing detection method is expanded.

[0024] The present invention does not impose any particular limitation on the type and specification of the lactoferrin, and for example, it may be TATUA lactoferrin powder (protein content ≥ 95%).

[0025] The present invention does not impose any particular limitation on the type of the above-mentioned combined bacteria fermentation product, for example, it may be at least one of a fermented dairy product, a fermented tea product, a fermented cereal product, and a fermented vegetable product.

[0026] The above-mentioned combined bacterial fermentation product includes a first bacterial species and a second bacterial species, the first bacterial species is Lactobacillus acidophilus, and the second bacterial species includes at least one of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, etc.

[0027] In a specific embodiment, the combined bacteria fermented product is a fermented dairy product, specifically a triple bacteria fermented dairy product, wherein the first bacterial species is Lactobacillus acidophilus, and the second bacterial species includes Lactobacillus bulgaricus and Streptococcus thermophilus.

[0028] Of course, the present invention does not specifically limit the preparation method of the combined bacteria fermentation product, especially does not limit the order of adding each strain. Taking the above-mentioned triple bacteria fermented dairy product as an example, in a specific embodiment, the preparation method includes the following steps:

[0029] 1) heating a first raw material including raw milk to 50-70° C. to obtain a first liquid;

[0030] 2) homogenizing the first liquid at 50-65° C. and 15-30 MPa to obtain a second liquid;

[0031] 3) sterilizing the second liquid at 93-97° C. for 5-10 min to obtain a third liquid;

[0032] 4) cooling the third feed liquid to an inoculation temperature of 37° C.-42° C., inoculating a mixed fermentation strain of Lactobacillus bulgaricus and Streptococcus thermophilus, and stirring evenly to obtain a fourth feed liquid;

[0033] 5) inoculating Lactobacillus acidophilus into the fourth liquid, stirring evenly, and then fermenting. When the acidity reaches 70-80°T or the pH is less than 4.5, the fermentation is terminated to obtain a triple bacteria fermented dairy product.

[0034] It is understandable that fermentation can be terminated when one of the above acidity index and pH index reaches the requirement. At this time, the fermentation time is generally 4 hours, and during this period of time, in the presence of Lactobacillus bulgaricus and Streptococcus thermophilus, Lactobacillus acidophilus is still in the delayed phase and has not yet entered the logarithmic phase, so it can be considered that the viable count of Lactobacillus acidophilus in the above triple-bacteria fermented dairy product is the same as the inoculum size.

[0035] The raw milk mentioned above is at least one of fresh milk, reconstituted milk, whole milk, partially skimmed low-fat milk, fully skimmed milk, and milk powder that meet my country's raw milk purchase standards. The present invention does not limit the mass percentage of the total dry matter of the raw milk in the raw materials of the above-mentioned fermented dairy products, and the conventional amount used in the field can be used, for example, 11%-14%, more preferably 12%-14%. In addition, when skimmed milk is used, the mass percentage of the dry matter of the skimmed milk in the raw materials of the above-mentioned fermented dairy products is at least 8.5%.

[0036] The first raw material also includes at least one of a sweetener, a stabilizer, an edible flavor, a conventional auxiliary material and a protein supplement.

[0037] The above-mentioned sweet substances include at least one of monosaccharides, sucrose, lactose and maltose.

[0038] The monosaccharide includes at least one of glucose, fructose, galactose and fructose-glucose syrup, and the mass percentage of the monosaccharide in the fermented dairy product raw material is 2%-5%.

[0039] The stabilizer includes at least one of pectin, CMC, gellan gum, propylene glycol alginate, modified starch and gelatin.

[0040] The above-mentioned edible flavors include at least one of artificial synthetic flavors, natural flavors and nature-equivalent flavors. The present invention does not limit the types and addition amounts of edible flavors used in the above-mentioned fermented milk, and conventional types and addition amounts of edible flavors in the art can be used.

[0041] The conventional auxiliary materials include at least one of cream, butter, anhydrous butter, evaporated milk, and sweetened condensed milk. It is understood that in order to control the sweetness of the fermented dairy product, when adding sugar-containing auxiliary materials, the amount of the sweetener should be reduced accordingly.

[0042] The protein supplement comprises at least one of concentrated whey protein, concentrated milk protein and soy protein.

[0043] In addition, the color of the sample will not affect the detection result of Lactobacillus acidophilus, for example, the fermented dairy product can be a white fermented dairy product or a brown fermented dairy product. It is understandable that the white fermented dairy product is a product prepared by conventional fermentation technology in the art; the brown fermented dairy product is a product obtained by browning the white fermented dairy product.

[0044] The above-mentioned browning treatment is a conventional technique in the art, for example, the first raw material including monosaccharides is placed in a water bath at 90° C. for 2 h-3 h or in a water bath at 115° C. for 5 min. It is understood that when the first raw material does not include monosaccharides, in order to perform the browning treatment, lactase can be used to hydrolyze lactose in the raw milk to obtain monosaccharides, and the monosaccharides obtained after hydrolysis can be used for the browning treatment.

[0045] It is understandable that the form of the fermented dairy product, such as solidified, stirred, or flowing, does not affect the detection result of Lactobacillus acidophilus.

[0046] In a specific embodiment, the use of the above-mentioned lactoferrin in detecting the content of Lactobacillus acidophilus in a combined fermentation product specifically includes: culturing the combined fermentation product using a culture medium including lactoferrin, and determining the content of Lactobacillus acidophilus based on the colony count in the culture medium.

[0047] Specifically, the principle of using the culture medium containing lactoferrin to culture the combined fermentation product and determining the content of Lactobacillus acidophilus according to the number of colonies in the culture medium is the same as the principle of the pour plate method. The Lactobacillus acidophilus in the combined fermentation product is fully dispersed and evenly distributed in the culture medium in the plate. After anaerobic culture, a single Lactobacillus acidophilus grows and multiplies, gradually forming a colony visible to the naked eye. The number of colonies is counted to determine the content of Lactobacillus acidophilus in the combined fermentation product.

[0048] The present invention also provides a detection medium for the content of Lactobacillus acidophilus in a combined fermentation product, and the detection medium includes lactoferrin. Using the detection medium to detect the content of Lactobacillus acidophilus in the combined fermentation product can not only exclude the influence of other strains on the content of Lactobacillus acidophilus during the detection process, but also effectively avoid the adverse effects of the technology of excluding other strains on Lactobacillus acidophilus, thereby ensuring that the content of Lactobacillus acidophilus is accurately detected.

[0049] In the specific implementation process of the present invention, the mass content of lactoferrin in the detection medium is generally controlled to be more than 0.01%. Reasonably controlling the addition amount of lactoferrin in the detection medium is conducive to further improving the accuracy of the detection result of the content of Lactobacillus acidophilus. The inventor has found that as the amount of lactoferrin increases within a certain range, the accuracy of the detection result of the content of Lactobacillus acidophilus first increases, and then remains basically unchanged. Therefore, for economic considerations, the mass content of lactoferrin in the detection medium is generally controlled to be 0.01% to 0.12%.

[0050] Of course, the use of different specifications of lactoferrin in the culture medium, or even different culture medium bodies, will have an impact on the detection accuracy. Therefore, generally speaking, for the above different bodies and different lactoferrin specifications, when the mass content of lactoferrin in the detection culture medium is controlled at 0.04% to 0.08%, the detection accuracy of Lactobacillus acidophilus content can be basically optimized.

[0051] The present invention does not limit the composition of the detection medium, as long as it contains lactoferrin. Generally, a common bacterial culture medium can be used as the main body, and then lactoferrin is added thereto. Further, based on considerations of cost and detection accuracy, the mass percentage of lactoferrin in the detection medium can be controlled to meet the above definition.

[0052] In one embodiment, the main component of the detection medium is MRS medium, and in addition, it also includes antibiotics.

[0053] The present invention does not limit the MRS culture medium, and the MRS culture medium used in conventional technical means in the art can be used.

[0054] The role of the above-mentioned antibiotics is to eliminate the interference of other bacterial species in the sample, thereby ensuring that the colonies cultured in the detection medium are all Lactobacillus acidophilus. The present invention does not limit the types of the above-mentioned antibiotics, and the types of antibiotics used in conventional technical means in the art can be used. For example, the antibiotics include fluoroquinolone antibiotics and lincosamide antibiotics. In a specific embodiment, the above-mentioned fluoroquinolone antibiotics are chloracrylamide hydrochloride, and the above-mentioned lincosamide antibiotics are ciprofloxacin.

[0055] The present invention does not impose any particular limitation on the amount of antibiotics added, and any amount added in the conventional manner in the art may be used.

[0056] In addition, the inventors found that when the mass ratio of lactoferrin to antibiotics is (0.1g / kg-1.2g / kg): (0.1mg / kg-10mg / kg), not only can the antibiotics be used to effectively eliminate the interference of other bacterial species, but also the adverse effects of antibiotics on Lactobacillus acidophilus are eliminated, thereby accurately detecting the content of Lactobacillus acidophilus in the co-fermented product.

[0057] The present invention also provides a method for preparing any of the above detection culture media, comprising the following steps:

[0058] The main body of the detection culture medium and the lactoferrin are sterilized separately and then mixed evenly to obtain the detection culture medium.

[0059] The main body of the above-mentioned detection medium refers to the medium conventionally used in the art when culturing Lactobacillus acidophilus, and its components include water, carbon source, nitrogen source, inorganic salt, and growth factor, which can provide necessary nutrients for Lactobacillus acidophilus to enable normal growth and reproduction of Lactobacillus acidophilus. In one embodiment, the main body of the above-mentioned detection medium is MRS medium.

[0060] The present invention does not limit the sterilization technology of the main body of the above-mentioned detection culture medium and lactoferrin. Any sterilization technology that does not damage the main body of the above-mentioned detection culture medium and lactoferrin can be used in the present invention. For example, the main body of the above-mentioned detection culture medium is sterilized by high temperature technology, and the sterilization conditions are 121±1℃20min; lactoferrin is sterilized with a 0.22μm filter.

[0061] The detection culture medium prepared by the above preparation method can be used to accurately detect the content of Lactobacillus acidophilus in samples with a content of Lactobacillus acidophilus lower than 1.0E+05 CFU / mL, which significantly reduces the detection limit of Lactobacillus acidophilus.

[0062] It is understandable that, in order to facilitate the sterilization operation of lactoferrin and to fully mix lactoferrin with the main body of the above-mentioned detection culture medium, lactoferrin can be mixed with sterile distilled water to prepare a lactoferrin aqueous solution. The present invention does not limit the concentration of the lactoferrin aqueous solution. For example, TATUA lactoferrin powder (protein content ≥ 95%) and sterile distilled water are used to prepare a lactoferrin aqueous solution with a mass concentration of 1%, and the above mass concentration is equal to the percentage of the mass of dissolved lactoferrin to the volume of the dissolved lactoferrin aqueous solution.

[0063] It is understandable that when the above-mentioned detection culture medium also includes antibiotics, in order not to destroy the function of the antibiotics, before adding the antibiotics to the main body, a 0.22 μm filter can be used to sterilize the antibiotics.

[0064] The present invention also provides a method for detecting the content of Lactobacillus acidophilus. The method uses any one of the above detection culture media to detect the content of Lactobacillus acidophilus.

[0065] The working principle of the detection method is that each live bacterium can grow a colony on the above detection medium, and the number of live bacteria in the sample is determined by recording the number of colonies on the above detection medium. The detection method has high sensitivity and can accurately detect the content of Lactobacillus acidophilus, especially for samples with a content of Lactobacillus acidophilus lower than 1.0E+05CFU / mL, which significantly reduces the detection limit of Lactobacillus acidophilus and expands the scope of application of the method.

[0066] The detection method can be used to detect the content of Lactobacillus acidophilus in various materials such as food, medicine, water, secretions, excrement, etc.

[0067] The detection method specifically comprises the following steps:

[0068] 1) Pour the test medium into the plate;

[0069] 2) Pour a quantitative sample into a plate containing the above-mentioned detection medium, spread it evenly, and culture it anaerobically at 37°C for 3 days. Calculate the number of viable bacteria in the sample based on the number of colonies cultured.

[0070] It is understandable that when using the above detection method, it should be noted that generally plates with colony counts between 30 and 300 are selected for counting. Too many or too few will be inaccurate.

[0071] In order to make the number of colonies cultured in the plate between 30 and 300, the sample can be diluted in series according to the estimated value of the Lactobacillus acidophilus content in the sample and the quantitative requirement of the sample poured into the plate to obtain a bacterial suspension with a series of gradient concentrations. A certain amount of the bacterial suspension is added to the plate including the above-mentioned detection culture medium, and the suspension is evenly spread and then cultured.

[0072] The technical solution of the present invention is described in more detail below through specific embodiments.

[0073] The specific sources of some raw materials in the following preparation method are:

[0074] Lactobacillus acidophilus powder: NCFM Danisco (China) Co., Ltd.

[0075] Mixed fermentation strains of Lactobacillus bulgaricus and Streptococcus thermophilus: premium 1.0 Chr. Hansen;

[0076] Lactoferrin: TATUA lactoferrin, Fonterra, New Zealand;

[0077] MRS culture medium: Beijing Luqiao Technology Co., Ltd.

[0078] Example 1

[0079] The method for detecting the content of Lactobacillus acidophilus of this embodiment comprises:

[0080] 1. Preparation of the detection medium, the preparation method comprises the following steps:

[0081] 1) Sterilize the MRS medium at 121±1℃ for 20min;

[0082] 2) Prepare a 1% lactoferrin aqueous solution using TATUA lactoferrin powder (protein content ≥ 95%) and sterile distilled water, and then sterilize with a 0.22 μm filter;

[0083] 3) Use sterile distilled water to prepare a 0.2 mg / ml chloracrylamide hydrochloride solution and a 2 mg / ml ciprofloxacin solution, and then sterilize with a 0.22 μm filter;

[0084] 4) Add the sterilized lactoferrin aqueous solution, chloramphenicol hydrochloride solution and ciprofloxacin solution into the sterilized MRS medium, mix well, and obtain a detection medium; wherein, the MRS medium is 100 g, lactoferrin is 0.06 g, chloramphenicol hydrochloride is 0.01 mg, and ciprofloxacin is 0.1 mg.

[0085] 2. Using the above detection medium to detect the content of Lactobacillus acidophilus, comprising the following steps:

[0086] 1) Pour the test culture medium into a plate and cool it to solidify;

[0087] 2) Add the triple bacteria fermented dairy product to the above plate and spread it evenly;

[0088] 3) The mixture was cultured anaerobically at 37° C. for 3 days, and the number of colonies cultured on the culture medium in the plate was recorded. The content of Lactobacillus acidophilus in the triple-bacteria fermented dairy product was obtained by conversion. The specific results are shown in Table 1.

[0089] The preparation method of the triple bacteria fermentation product comprises the following steps:

[0090] 1) heating the milk to 55° C., adding granulated sugar, wherein the mass ratio of granulated sugar to milk is 60 g:1 kg, and stirring in a cycle for 20-30 min to obtain a first liquid;

[0091] 2) homogenizing the first liquid at 50-65° C. and 15-30 MPa to obtain a second liquid;

[0092] 3) sterilizing the second liquid at 95° C. for 10 min to obtain a third liquid;

[0093] 4) Cooling the third liquid to an inoculation temperature of 42°C, inoculating a mixed fermentation strain of Lactobacillus bulgaricus and Streptococcus thermophilus, the inoculation amount of the mixed fermentation strain being 10 6cfu / ml, stirring evenly to obtain the fourth feed liquid;

[0094] 5) Inoculate Lactobacillus acidophilus into the fourth feed liquid, the inoculation amount of Lactobacillus acidophilus is 10 6 cfu / ml, and then fermentation is carried out. When the acidity reaches pH < 4.5, the fermentation is terminated to obtain a triple bacteria fermented dairy product.

[0095] Example 2

[0096] This embodiment is basically the same as embodiment 1, except that the inoculation amount of the mixed fermentation bacteria and Lactobacillus acidophilus in the triple-bacteria fermented dairy product is 10 5 cfu / ml, see Table 1 for specific results.

[0097] Example 3

[0098] This embodiment is basically the same as embodiment 1, except that in the preparation method of triple-bacteria fermented dairy products, the inoculation amount of the mixed fermentation bacteria and Lactobacillus acidophilus is 10 4 cfu / ml, see Table 1 for specific results.

[0099] Example 4

[0100] This embodiment is basically the same as embodiment 1, except that in the preparation method of triple-bacteria fermented dairy products, the inoculation amount of the mixed fermentation bacteria and Lactobacillus acidophilus is 10 3 cfu / ml, see Table 1 for specific results.

[0101] Example 5

[0102] This example is basically the same as Example 3, except that the amount of lactoferrin is 0.01 g and the amount of MRS culture medium is 100 g. Specific results are shown in Table 2.

[0103] Example 6

[0104] This example is substantially the same as Example 3, except that the amount of lactoferrin is 0.02 g and the amount of MRS culture medium is 100 g. Specific results are shown in Table 2.

[0105] Example 7

[0106] This example is substantially the same as Example 3, except that the amount of lactoferrin is 0.04 g and the amount of MRS culture medium is 100 g. Specific results are shown in Table 2.

[0107] Example 8

[0108] This example is substantially the same as Example 3, except that the amount of lactoferrin is 0.08 g, and the amount of MRS culture medium is 100 g. Specific results are shown in Table 2.

[0109] Example 9

[0110] This example is basically the same as Example 3, except that the amount of lactoferrin is 0.1 g and the amount of MRS culture medium is 100 g. Specific results are shown in Table 2.

[0111] Example 10

[0112] This example is substantially the same as Example 3, except that the amount of lactoferrin is 0.12 g and the amount of MRS culture medium is 100 g. Specific results are shown in Table 2.

[0113] Comparative Example 1

[0114] This comparative example is basically the same as Example 1, except that lactoferrin is not included in the detection culture medium. Specific results are shown in Table 1.

[0115] Comparative Example 2

[0116] This comparative example is basically the same as Example 2, except that lactoferrin is not included in the detection culture medium. Specific results are shown in Table 1.

[0117] Comparative Example 3

[0118] This comparative example is basically the same as Example 3, except that lactoferrin is not included in the detection culture medium. The specific results are shown in Table 1.

[0119] Comparative Example 4

[0120] This comparative example is basically the same as Example 4, except that lactoferrin is not included in the detection culture medium. The specific results are shown in Table 1.

[0121] Comparative Example 5

[0122] 1. Prepare a Lactobacillus acidophilus solution, comprising the following steps:

[0123] 1) sterilizing the milk at 121±1°C for 20 min, and obtaining sterile milk after cooling;

[0124] 2) Take Lactobacillus acidophilus powder and dissolve it in sterile milk to prepare a lactobacillus acidophilus content of 10 7 cfu / ml bacterial solution;

[0125] 3) Prepare 10 7 The bacterial solution was diluted 10 times to obtain a solution with a Lactobacillus acidophilus content of 10 cfu / ml. 6 cfu / ml bacterial solution A;

[0126] 4) Dilute the bacterial solution A 10 times with sterile milk to obtain a solution with a Lactobacillus acidophilus content of 10 5 cfu / ml bacterial solution B;

[0127] 5) Dilute the bacterial solution B 10 times with sterile milk to obtain a solution with a Lactobacillus acidophilus content of 10 4 cfu / ml bacterial solution C;

[0128] 6) Dilute the bacterial solution C 10 times with sterile milk to prepare a solution with a Lactobacillus acidophilus content of 10 3 cfu / ml bacterial solution D.

[0129] 2. A method for detecting the content of Lactobacillus acidophilus using sterilized MRS culture medium, comprising the following steps:

[0130] 1) Pour the sterilized MRS medium into a plate and cool it to solidify;

[0131] 2) Take four sets of plates containing the above-mentioned MRS medium, add bacterial solution A, bacterial solution B, bacterial solution C, and bacterial solution D respectively, and spread them evenly;

[0132] 3) The samples were cultured anaerobically at 37° C. for 3 days, and the number of colonies cultured on the culture medium in the plate was used to calculate the content of Lactobacillus acidophilus in bacterial solution A, bacterial solution B, bacterial solution C, and bacterial solution D. The specific results are shown in Table 1.

[0133] Test results

[0134] Table 1 Lactobacillus acidophilus content test results of Examples 1-4 and Comparative Examples 1-5

[0135]

[0136]

[0137] The results of the detection of Lactobacillus acidophilus content in Table 1 of Examples 1-4 and Comparative Examples 1-5 show that: first, the detection amounts of the four single Lactobacillus acidophilus bacterial solutions in Comparative Example 5 are higher than those in Examples 1-4 and Comparative Example 1-4, and are closer to the actual amount, that is, compared with the single bacterial species solution, the detection amount of Lactobacillus acidophilus in the triple-bacteria fermented milk is significantly lower, indicating that the combination of clindamycin hydrochloride and ciprofloxacin not only inhibits the growth of Lactobacillus bulgaricus and Streptococcus thermophilus, but also has a certain effect on the growth activity of Lactobacillus acidophilus. Secondly, the data of Examples 1-4 are all close to the inoculum size of the corresponding Lactobacillus acidophilus, while Comparative Examples 1-4 are all lower than the inoculum size of the corresponding Lactobacillus acidophilus, and even Lactobacillus acidophilus is not detected in Comparative Example 4; that is, the number of viable bacteria detected in the culture medium containing lactoferrin is significantly higher than the number of viable bacteria detected in the culture medium without lactoferrin, and is closer to the actual amount, indicating that lactoferrin effectively reduces the adverse effect of the combination of clindamycin hydrochloride and ciprofloxacin on the activity of Lactobacillus acidophilus; thereby improving the detection limit of Lactobacillus acidophilus and expanding the scope of use of the method.

[0138] Table 2 Lactobacillus acidophilus content test results of Example 3 and Example 5-10

[0139]

[0140] The lactobacillus acidophilus content detection result of table 2 embodiment 3 and embodiment 5-10 shows: the different contents of lactoferrin will have an impact on the detection result of lactobacillus acidophilus content, specifically, the higher the lactoferrin content, the more obvious the effect of the adverse effect of the combination of clindamycin hydrochloride and ciprofloxacin on the activity of lactobacillus acidophilus, making the number of detectable viable bacteria more.When lactoferrin content reaches 0.04%, the detected value of lactobacillus acidophilus content is close to the actual value, along with the increase of lactoferrin content, the viable bacteria detection result of lactobacillus acidophilus will increase, and when lactoferrin content ≥ 0.08%, along with the increase of lactoferrin content, the viable bacteria detection result of lactobacillus acidophilus increases not significantly.It is understandable that, when lactoferrin content ≥ 1.2%, at this moment, the detection result is close to the implementation value, and continuing to increase lactoferrin content will increase cost, which is not very significant for practical application, so lactoferrin content ≥ 1.2% is no longer explored.

[0141] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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. An application of lactoferrin in detecting the content of Lactobacillus acidophilus in a mixed bacteria fermentation product.

2. The use according to claim 1, characterized in that: The combined bacterial fermentation product comprises a first bacterial species and a second bacterial species, wherein the first bacterial species is Lactobacillus acidophilus, and the second bacterial species comprises at least one of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus casei, and Lactobacillus rhamnosus.

3. The use according to claim 1 or 2, characterized in that: The detecting of the content of Lactobacillus acidophilus in the combined fermentation product specifically comprises: culturing the combined fermentation product using a culture medium comprising the lactoferrin, and determining the content of the Lactobacillus acidophilus according to the colony count in the culture medium.

4. A medium for detecting the content of Lactobacillus acidophilus in a combined bacterial fermentation product, characterized in that: The assay medium includes lactoferrin.

5. The detection medium according to claim 4, characterized in that The mass percentage of the lactoferrin in the detection culture medium is 0.01%-0.12%.

6. The detection medium according to claim 5, characterized in that The mass percentage of the lactoferrin in the detection culture medium is 0.04%-0.08%.

7. The detection medium according to any one of claims 4 to 6, characterized in that The detection culture medium is mainly composed of MRS culture medium and also includes antibiotics.

8. The detection medium according to claim 7, characterized in that The mass ratio of lactoferrin to antibiotic is (0.1 g / kg-1.2 g / kg): (0.1 mg / kg-10 mg / kg).

9. A method for preparing the detection medium according to any one of claims 4 to 8, characterized in that: The following steps are involved: The main body of the detection culture medium and the lactoferrin are sterilized separately and then mixed evenly to obtain the detection culture medium.

10. A method for detecting the content of Lactobacillus acidophilus, characterized in that: The content of Lactobacillus acidophilus is detected using the detection culture medium described in any one of items 4-9.

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