CN104762350B - Biological fermentation bran with antibacterial effect and preparation method and application thereof - Google Patents
Biological fermentation bran with antibacterial effect and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a biological fermentation bran with an antibacterial effect and a preparation method and application thereof. The preparation method of the biological fermentation bran with the bacteriostatic action comprises the following steps: (1) inoculating Paenibacillus sp in a bran culture medium, and carrying out aerobic culture to obtain a fermentation product; (2) and (2) drying the fermentation product obtained in the step (1). The preparation method adopts a solid state fermentation method, has single strain and raw material, simple process and low cost, and is beneficial to the standardization of the quality of the fermented bran and the cost control of industrial large-scale production. The biological fermentation bran with the antibacterial effect prepared by the invention can effectively relieve even radically change the current situation of antibiotic abuse in the existing animal breeding industry after being used in the animal feed industry.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to biological fermentation bran with an antibacterial effect, and a preparation method and application thereof.
Background
Bran is a main byproduct in wheat processing, has the characteristics of wide source, low cost and easiness in processing, and is mainly used as a primary raw material in the feed industry at present. Although the bran contains a lot of nutrient components (such as starch, oligosaccharide, protein, dietary fiber, vitamins and the like), a large number of macromolecular substances (such as crude fiber and the like) which are difficult to be directly digested and absorbed by animals still exist, and the fact shows that the bran is fermented by using microorganisms, so that the decomposition and absorption of the macromolecular substances are facilitated, and meanwhile, the application value of the bran is improved by virtue of beneficial substances generated by microbial metabolism, and therefore, the improvement of the culture level is greatly promoted by biologically fermenting the bran.
Bacteriocins are substances with antibacterial activity produced by certain bacteria, the main component of which is polypeptides, proteins or protein complexes, and were first proposed by Jacob in 1953. In recent years, the negative effects caused by the popularization and even abuse of broad-spectrum antibiotics are increasingly highlighted, and the research on novel antibiotic substitutes is urgent. Bacteriocins, which are proteinaceous substances, are attracting increasing attention because they are degraded by proteases and are highly safe.
It can be concluded that if bran can be fermented by bacteriocin-producing bacteria and the obtained fermented bran with activity of inhibiting harmful bacteria is applied to animal feed, the current situation of antibiotic abuse in the animal breeding industry can be relieved to a great extent, even solved. However, no report on fermented bran with bacteriostatic activity is found at present, and meanwhile, the research on the fermentation of the bran by using the paenibacillus is blank.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide the biological fermentation bran with the bacteriostatic action and the preparation method and the application thereof aiming at the current situation of the related technical blank of the culture medium of the biological fermentation bran with the bacteriostatic activity and the single-bacterium fermentation bran of the paenibacillus.
At present, the current situation of antibiotic abuse in the animal breeding industry forms great contradiction and conflict with the increasingly strengthened food safety requirements of current consumers, and in order to solve the contradiction, the inventor carries out serious analysis and screening on a series of technical parameters of a preparation method of biological fermentation bran with antibacterial performance, particularly the culture mode of strains, the selection and preparation of a bran culture medium formula, the temperature of fermentation culture, the time of fermentation culture and the like, and finally obtains the technical scheme and the obtained biological fermentation bran product with antibacterial performance. The biological fermentation bran is natural in source, the preparation method is simple and convenient in process, the obtained biological fermentation bran is excellent in storage stability, and an antibacterial technical effect with excellent stability can be obtained.
The inventor finds that the bacillus subtilis sp (CGMCC No. 8333) has strong bacteriostatic activity after a fermented product obtained by fermenting the bran culture medium is dried to prepare the fermented bran, and the biological fermented bran has stable bacteriostatic effect through repeated verification, thereby completing the invention.
In order to solve the technical problems, one of the technical schemes adopted by the invention is as follows: a preparation method of biological fermentation bran with bacteriostatic action comprises the following steps:
(1) inoculating Paenibacillus sp in a bran culture medium, and carrying out aerobic culture to obtain a fermentation product;
(2) and (2) drying the fermentation product obtained in the step (1).
Wherein the step (1) is to inoculate Paenibacillus sp in a bran culture medium and carry out aerobic culture to obtain a fermentation product. Wherein the Paenibacillus sp is conventional Paenibacillus in the field, preferably Paenibacillus with the preservation number of CGMCC No. 8333. The Paenibacillus is the prior art, and the preparation method thereof is the conventional preparation method in the field, or is obtained by purchasing from a preservation center. Wherein the bran culture medium is conventional in the art, preferably wheat bran culture medium. The preparation method of the bran culture medium is a conventional preparation method in the field, and the feed-liquid ratio of the bran culture medium is preferably 1: 1-1: 1.5, more preferably 1: 1.2-1: 1.4, wherein the feed-liquid ratio is the mass ratio of the bran to the water in the culture medium.
Wherein the inoculation amount of the paenibacillus is preferably 1-5%, more preferably 2-4%, and preferably 3%, the percentage is volume percentage, the temperature of the aerobic culture is preferably 25-37 ℃, more preferably 30-35 ℃, and preferably 33 ℃, and the time of the aerobic culture is preferably 72-120 hours, more preferably 96-118 hours, and preferably 98 hours.
Before the fermentation method of the invention inoculates the paenibacillus CGMCC No.8333 in the bran culture medium, the fermentation method preferably also comprises the step of activating the paenibacillus CGMCC No.8333 to obtain paenibacillus seeds. The steps preferably include the steps of: inoculating the paenibacillus CGMCC No.8333 in a TYC solid culture medium, and culturing for 18-28 hours at 25-30 ℃ to obtain the paenibacillus CGMCC No.8333 seeds; uniformly dispersing the bacterial colony of the obtained paenibacillus CGMCC No.8333 seed in a TYC liquid culture medium, inoculating the bacterial colony in the TYC liquid culture medium according to the inoculation amount of 2-5% volume percentage, carrying out shake culture at the rotating speed of a shaking table of 100-200 rpm for 18-28 hours, centrifuging the obtained culture, removing supernatant, washing the obtained thallus with sterile distilled water, and suspending with the sterile distilled water of the original culture volume to obtain the paenibacillus seed for fermentation.
Wherein the step (2) is to dry the fermentation product obtained in the step (1). The drying method is a conventional drying method in the field, and the drying temperature is preferably 80-100 ℃, more preferably 85-95 ℃, and most preferably 92 ℃.
In order to solve the above technical problems, the second technical solution adopted by the present invention is: the biological fermented bran with the bacteriostatic action is prepared by the preparation method.
The preferable range of the technical features of each step in the preparation method of the bio-fermented bran with the bacteriostatic action is completely consistent with the corresponding technical content in the preparation method, and for details, reference is made to the technical content described above, and details are not repeated here.
In order to solve the technical problems, the invention adopts the third technical scheme that: the application of the biological fermentation bran with the bacteriostatic action in preparing the functional animal feed.
The functional animal feed is conventional in the field, and refers to an animal feed with specific nutrient components or functions. The functional animal feed is preferably animal feed for enhancing animal immunity or animal feed for preventing animal diseases, and the like.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
1. the invention creatively applies the paenibacillus to the bran fermentation and obtains the biological fermentation bran product with the bacteriostatic effect, and the related technology is not reported.
2. The method adopts a solid state fermentation method, has single strain and raw material, simple process and low cost, and is beneficial to the standardization of the quality of the fermented bran and the cost control of industrial large-scale production.
3. Compared with the existing biological fermentation bran, the biological fermentation bran prepared by the invention has the definite effect of inhibiting the growth and the reproduction of Staphylococcus aureus (Staphylococcus aureus), Micrococcus luteus (Micrococcus luteus) and Listeria monocytogenes (Listeria monocytogenes), so that the biological fermentation bran can be effectively relieved after being applied to animal feed, and even the current situation of antibiotic abuse in the existing animal breeding industry can be fundamentally changed.
4. The biological fermentation bran prepared by the invention shows good antibacterial activity under the conditions of heating, enzymolysis and long-term storage, can be used as a novel preparation method to be applied to the industrial production and related fields of biological fermentation bran with antibacterial action, and has very wide application prospect.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The "room temperature" in the present invention means a temperature between tests, and is generally 15 to 25 ℃. The reagents used in the examples, unless otherwise specified, were all analytical reagents and were purchased from the national pharmaceutical group.
Example 1 preparation of biologically fermented bran and detection of bacteriostatic activity
1. Materials and methods
Preparation of seeds (fermentation strain): dissolving lyophilized powder of Paenibacillus (Paenibacillus sp.) (the preservation number of the Paenibacillus is CGMCC No.8333, the source of the strain is shown in Chinese patent with the publication number of CN 103740618A) with a small amount of sterile distilled water, taking a ring by using an inoculating ring, streaking the ring on a TYC solid culture medium (purchased from OXOID Co., UK), taking out the strain after aerobic culture at 30 ℃ for 48h, picking a single colony by using the inoculating ring, putting the single colony into 10mL of TYC liquid culture medium (purchased from OXOID Co., UK), uniformly dispersing the colony in the liquid culture medium by using a vortex oscillator, taking out the colony by shaking culture at 30 ℃ and 180rpm for 24h, inoculating the colony in the TYC liquid culture medium (purchased from OXOID Co., UK) with the inoculation amount of 2% (volume percent) into the TYC liquid culture medium, shaking culture at 30 ℃ and 180rpm for 24h, centrifuging the culture at 15,000rpm for 10min, discarding supernatant, washing the thallus for 2 times by using sterile distilled water with the original culture volume, obtaining seeds for fermentation.
Wheat bran is purchased from yanto lotus (Taobao.com/item. htm.
Indicator strain: staphylococcus aureus (Staphylococcus aureus) CGMCC 1.879, Micrococcus luteus (CGMCC 1.1848), Listeria monocytogenes (Listeria monocytogenes) CGMCC1.9136, all of which are purchased from CGMCC.
Preparing an indication bacterial liquid: dissolving freeze-dried powder of Staphylococcus aureus (Staphylococcus aureus), Micrococcus luteus (Micrococcus luteus) and Listeria monocytogenes (Listeria monocytogenes) with a small amount of sterile distilled water, taking a ring by using an inoculating ring, marking on an LB solid culture medium (purchased from OXOID Co., UK), aerobically culturing for 48h at 30 ℃, taking out a single colony by using the inoculating ring, putting the single colony into 10mL of LB liquid culture medium (purchased from OXOID Co., UK), uniformly dispersing the colony in the liquid culture medium by using a vortex oscillator, performing shake culture at 30 ℃ and 180rpm for 24h, taking out, inoculating the single colony into the LB liquid culture medium (purchased from OXOID Co., UK) at 2 percent of inoculation amount by volume, performing shake culture at 30 ℃ and 180rpm for 20h, and obtaining the corresponding indication bacterial liquid.
Preparation of indicator plates: diluting the indicator bacterium liquid prepared by the method to ensure that the bacterium concentration is about 107cfu/mL, and mixing the indicator bacterium liquid with the indicator bacterium liquid in a volume ratio of 1: and (3) sucking diluted indicator bacterium liquid according to the proportion of 150, injecting the indicator bacterium liquid into a 45-DEG C sterile LB solid culture medium, fully and uniformly mixing, quickly pouring the indicator bacterium liquid into a flat plate, and after the flat plate is solidified and the surface moisture is completely evaporated.
The detection method of the antibacterial activity comprises the following steps: dropping 20 μ L of sample to be tested on the indication plate by a dibbling method, culturing at 30 ℃ for 20h, and measuring and recording the diameter of the inhibition zone.
Preparation of a fermentation medium: mixing wheat bran and distilled water at a required ratio, sterilizing at 118 deg.C for 15min, and cooling to room temperature to obtain sterile fermentation culture medium.
2. Preparation of biological fermented bran
Inoculating a Paenibacillus (Paenibacillus sp.) CGMCC No.8333 strain into a sterile bran culture medium in an inoculation amount of 5% (volume percentage), carrying out aerobic culture at 25 ℃ for 72h to obtain a fermented product, and drying the fermented product at 100 ℃ to obtain the biological fermentation bran A. Wherein the feed-liquid ratio of the bran culture medium is 1: 1.5, wherein the feed-liquid ratio is the mass ratio of the bran to the water.
3. Detection of bacteriostatic activity of biologically fermented bran
And adding sterile distilled water into the biological fermented bran A to prepare a fermented bran solution with the concentration of 50mg/mL, uniformly mixing, centrifuging at 15,000rpm for 10min, taking the supernatant, and measuring the bacteriostatic activity of the supernatant. The results are shown in the following table:
TABLE 1 bacteriostatic effect of biofermented bran
As can be seen from table 1, the diameters of inhibition zones generated by the action of the bio-fermented bran a on staphylococcus aureus (staphylococcus aureus), Micrococcus luteus (Micrococcus luteus) and Listeria monocytogenes (Listeria monocytogenes) are 10mm, 13mm and 9mm, respectively, and thus, the bio-fermented bran obtained by the present invention has a significant inhibition effect on the gram-positive bacteria.
Example 2 preparation of biologically fermented bran and detection of bacteriostatic activity
1. The material and the method are as follows: the same as in example 1.
2. Preparation of biological fermented bran
Inoculating a Paenibacillus (Paenibacillus sp.) CGMCC No.8333 strain into a sterile bran culture medium in an inoculation amount of 1% (volume percentage), carrying out aerobic culture at 37 ℃ for 120h to obtain a fermented product, and drying the fermented product at 80 ℃ to obtain the biological fermentation bran B. Wherein the feed-liquid ratio of the bran culture medium is 1: 1, wherein the feed-liquid ratio is the mass ratio of the bran to the water.
3. Detection of bacteriostatic activity of biologically fermented bran
And adding sterile distilled water into the biological fermented bran B to prepare a fermented bran solution with the concentration of 50mg/mL, uniformly mixing, centrifuging at 15,000rpm for 10min, taking the supernatant, and measuring the bacteriostatic activity of the supernatant. The results are shown in the following table:
TABLE 2 bacteriostatic effect of biofermented bran
As can be seen from table 2, the diameters of the inhibition zones generated by the action of the bio-fermented bran B on staphylococcus aureus (staphylococcus aureus), Micrococcus luteus (Micrococcus luteus) and Listeria monocytogenes (Listeria monocytogenes) are 12mm, 15mm and 13mm, respectively, and thus, the bio-fermented bran B obtained by the present invention has a significant inhibition effect on the gram-positive bacteria.
Example 3 preparation of biologically fermented bran and detection of bacteriostatic activity
1. The material and the method are as follows: the same as in example 1.
2. Preparation of biological fermented bran
Inoculating a Paenibacillus (Paenibacillus sp.) CGMCC No.8333 strain into a sterile bran culture medium in an inoculation amount of 2 percent (volume percentage), carrying out aerobic culture at 30 ℃ for 96 hours to obtain a fermented product, and drying the fermented product at 90 ℃ to obtain the biological fermentation bran C. Wherein the feed-liquid ratio of the bran culture medium is 1: 1.3, wherein the feed-liquid ratio is the mass ratio of the bran to the water.
3. Detection of bacteriostatic activity of biologically fermented bran
And adding sterile distilled water into the biological fermented bran C to prepare a fermented bran solution with the concentration of 50mg/mL, uniformly mixing, centrifuging at 15,000rpm for 10min, taking the supernatant, and measuring the bacteriostatic activity of the supernatant. The results are shown in the following table:
TABLE 3 bacteriostatic effect of biofermented bran
As can be seen from table 3, the diameters of the inhibition zones generated by the action of the bio-fermented bran C on staphylococcus aureus (staphylococcus aureus), Micrococcus luteus (Micrococcus luteus) and Listeria monocytogenes (Listeria monocytogenes) are 15mm, 18mm and 15mm, respectively, and thus, the bio-fermented bran obtained by the present invention has a significant inhibition effect on the gram-positive bacteria.
Example 4 preparation of biologically fermented bran and detection of bacteriostatic activity
1. The material and the method are as follows: the same as in example 1.
2. Preparation of biological fermented bran
Inoculating a Paenibacillus (Paenibacillus sp.) CGMCC No.8333 strain in a sterile bran culture medium in an inoculation amount of 4% (volume percentage), carrying out aerobic culture at 35 ℃ for 118h to obtain a fermented product, and drying the fermented product at 85 ℃ to obtain the biological fermentation bran. Wherein the feed-liquid ratio of the bran culture medium is 1: 1.2, wherein the feed-liquid ratio is the mass ratio of the bran to the water.
3. Detection of bacteriostatic activity of biologically fermented bran
The detection method is as described in example 1, and the detection result is as follows: the diameters of inhibition zones generated by staphylococcus aureus, micrococcus luteus and listeria monocytogenes are respectively 13mm, 16mm and 14mm, so that the biological fermentation bran obtained by the invention has a remarkable inhibition effect on the gram-positive bacteria.
Example 5 preparation of biologically fermented bran and detection of bacteriostatic activity
1. The material and the method are as follows: the same as in example 1.
2. Preparation of biological fermented bran
Inoculating a Paenibacillus (Paenibacillus sp.) CGMCC No.8333 strain into a sterile bran culture medium in an inoculation amount of 3 percent (volume percentage), carrying out aerobic culture at 33 ℃ for 98 hours to obtain a fermented product, and drying the fermented product at 92 ℃ to obtain the biological fermentation bran. Wherein the feed-liquid ratio of the bran culture medium is 1: 1.4, wherein the feed-liquid ratio is the mass ratio of the bran to the water.
3. Detection of bacteriostatic activity of biologically fermented bran
The detection method is as described in example 1, and the detection result is as follows: the diameters of inhibition zones generated by staphylococcus aureus, micrococcus luteus and listeria monocytogenes are 14mm, 18mm and 15mm respectively, so that the biological fermentation bran obtained by the invention has a remarkable inhibition effect on the gram-positive bacteria.
Example 6 preparation of biologically fermented bran and detection of bacteriostatic activity
1. The material and the method are as follows: the same as in example 1.
2. Preparation of biological fermented bran
Inoculating a Paenibacillus (Paenibacillus sp.) CGMCC No.8333 strain into a sterile bran culture medium in an inoculation amount of 3 percent (volume percentage), carrying out aerobic culture at 33 ℃ for 96 hours to obtain a fermented product, and drying the fermented product at 95 ℃ to obtain the biological fermentation bran. Wherein the feed-liquid ratio of the bran culture medium is 1: 1.1, wherein the feed-liquid ratio is the mass ratio of the bran to the water.
3. Detection of bacteriostatic activity of biologically fermented bran
The detection method is as described in example 1, and the detection result is as follows: the diameters of inhibition zones generated by staphylococcus aureus, micrococcus luteus and listeria monocytogenes are respectively 15mm, 17mm and 13mm, so that the biological fermentation bran obtained by the invention has a remarkable inhibition effect on the gram-positive bacteria.
Effect example 1 stability of bacteriostatic activity of bio-fermented bran under normal temperature storage condition
The bio-fermented bran A, B and C prepared in examples 1 to 3 were separately packaged in aseptic aluminum foil bags, stored at normal temperature (25 ℃) for 6 months and 12 months, taken out, added with sterile distilled water to prepare a 50mg/mL fermented bran solution, mixed well, centrifuged at 15,000rpm for 10min to take the supernatant, and the bacteriostatic activity was measured, the results are shown in the following table.
TABLE 4 stability of bacteriostatic activity of biofermented bran under normal temperature storage conditions
As can be seen from table 4, all of the tested biofermented brans stably maintained bacteriostatic activity against Staphylococcus aureus (Staphylococcus aureus), Micrococcus luteus (Micrococcus luteus), and Listeria monocytogenes (Listeria monocytogenes) after 12 months of storage at normal temperature.
Effect example 2 stability of bacteriostatic Activity of biofermented bran at different pH conditions
The bio-fermented bran A, B and C prepared in examples 1 to 3 were added to 0.2M phosphate buffer solution with pH 3, 5, 7, and 9, respectively, to prepare 50mg/mL bio-fermented bran solution, after mixing well, the mixture was centrifuged at 15,000rpm for 10min to obtain the supernatant, and samples a-3, a-5, a-7, a-9, B-3, B-5, B-7, B-9, C-3, C-5, C-7, and C-9 to be tested were obtained, and the bacteriostatic activity of each group of samples is shown in the following table:
TABLE 5 bacteriostatic Activity of biofermented bran at different pH
As can be seen from table 5, the bio-fermented bran has very stable bacteriostatic effect under different pH conditions, and thus, the bio-fermented bran prepared by the present invention can still maintain good bacteriostatic activity in the acidic digestive fluid in the animal body.
Effect example 3 stability of bacteriostatic Activity of enzyme-treated biofermented bran
The present invention also employs trypsin (e.c.3.4.21.4, purchased from Sigma), pepsin (e.c.3.4.23.1, purchased from Sigma), lipase (e.c.3.1.1.3, purchased from Sigma), proteinase K (e.c.3.4.21.64, purchased from Sigma) treatment of biofermented bran to simulate in vitro the effects of different enzymes in the animal on the bacteriostatic activity of the biofermented bran as follows:
preparing 0.2M phosphate buffer solution with corresponding pH according to the optimum reaction pH of different enzymes, adding the 0.2M phosphate buffer solution with different pH into the biological fermentation bran A, B and C prepared in examples 1-3 to prepare 50mg/mL fermentation bran solution, mixing uniformly, centrifuging at 15,000rpm for 10min to take supernatant, dissolving corresponding enzyme into the supernatant with different pH to make the final concentration of the enzyme be 10mg/mL, keeping the mixed solution in water bath at 37 ℃ for 2h, adjusting the pH to be neutral, measuring the bacteriostatic activity of each sample group, and comparing the bacteriostatic activity with the bacteriostatic activity before enzyme treatment, wherein the results are shown in the following table:
TABLE 6 comparison of bacteriostatic Activity of biologically fermented bran after enzyme treatment
As can be seen from table 6, after the bio-fermented bran is treated by different enzymes, the bacteriostatic effect is still stable, and therefore, the bio-fermented bran prepared by the invention can maintain good bacteriostatic activity in digestive enzymes in animals.
Comparative example 1
The inoculum size, culture temperature, fermentation time, culture medium liquid ratio and drying temperature in example 3 were adjusted one by one to obtain a set of biofermented bran prepared by different methods as follows. The bacteriostatic effect of each group of bio-fermented bran is shown in the table below.
TABLE 7 bacteriostatic effect of biofermented bran prepared by different methods
As can be seen from the results shown in table 7, when the inoculation amount, the culture temperature, the fermentation time, the culture medium-to-liquid ratio, and the drying temperature in the preparation method of the bio-fermented bran are adjusted to be outside the present invention, the bacteriostatic effect of the obtained bio-fermented bran is significantly reduced.
Comparative example 2
The bacteriostatic effects of biofermented brans prepared from Paenibacillus (Paenibacillus sp.) cgmccno.8333, lactobacillus plantarum (l.plantarum) ATCC 14917 (purchased from ATCC) and lactobacillus casei (l.casei) ATCC 393 (purchased from ATCC) were compared as described in reference to example 3, and were performed as follows:
1. materials and methods
Preparation of seeds (fermentation strain):
the seed of Paenibacillus CGMCC No.8333 was prepared as in example 1.
Preparation of lactobacillus plantarum and lactobacillus casei seeds: dissolving freeze-dried powder of lactobacillus plantarum ATCC 14917 and lactobacillus casei ATCC 393 in a small amount of sterile distilled water, taking a ring by using an inoculating ring, scribing on an MRS solid culture medium (purchased from Merck Co. Germany), carrying out anaerobic culture at 37 ℃ for 24h, taking a single colony by using the inoculating ring, putting the single colony into 1mL of MRS liquid (purchased from Merck Co. Germany), uniformly dispersing the colony in the liquid culture medium by using a vortex shaker, carrying out anaerobic culture at 37 ℃ for 24h, taking out, inoculating the single colony into 50mL of MRS liquid by using an inoculation amount of 2 percent (volume percent), carrying out culture at 37 ℃ for 24h, centrifuging the culture at 9,000rpm for 10min, discarding the supernatant, washing thalli for 2 times by using sterile distilled water, and suspending by using the sterile distilled water with the original culture volume to obtain seeds for fermentation.
The preparation of the indicator strain, the indicator bacterial liquid, the fermentation medium, the indicator plate and the detection method of the bacteriostatic activity are the same as in example 1.
2. Preparation of biological fermentation bran antibacterial activity
Inoculating each strain into a sterile bran culture medium according to the inoculation amount of 2 percent (volume percentage), respectively culturing (anaerobic culture of lactobacillus plantarum and lactobacillus casei at 37 ℃, aerobic culture of paenibacillus at 30 ℃) for 96 hours to obtain a fermented product, and drying the fermented product at 90 ℃ to obtain the biological fermented bran produced by different strains. Wherein the feed-liquid ratio of the bran culture medium is 1: 1.3, wherein the feed-liquid ratio is the mass ratio of the bran to the water.
3. Detection of bacteriostatic activity of biologically fermented bran
Adding sterile distilled water into the biological fermented bran to prepare a fermented bran solution with the concentration of 50mg/mL, uniformly mixing, centrifuging at 15,000rpm for 10min, taking the supernatant, and measuring the antibacterial activity of the supernatant. The results are shown in the following table:
TABLE 8 bacteriostatic effect of biofermented bran prepared with different strains
As can be seen from table 8, the bacteriostatic effect of the bio-fermented bran prepared by paenibacillus is significantly higher than that of other strains, and therefore, fermenting bran with paenibacillus is a preferred method for preparing bio-fermented bran with bacteriostatic effect.
Comparative example 3
Adding 0.2M phosphate buffer solution with pH values of 3, 5, 7 and 9 into the biological fermentation bran C with the bacteriostatic action prepared in the embodiment 3 to prepare 50mg/mL biological fermentation bran solution, uniformly mixing, centrifuging at 15,000rpm for 10min, and taking supernatant to obtain sample groups S-3, S-5, S-7 and S-9 to be detected.
Meanwhile, the conventional bacteriocin product Nisapilin (Nisaplin, purchased from Densitx) in the market is selected as a control, and is dissolved in 0.2M phosphate buffer solution with pH values of 3, 5, 7 and 9 to obtain control groups of N-3, N-5, N-7 and N-9 with the concentration of 50mg/mL, and the bacteriostatic effect of each group of samples is shown in the following table.
TABLE 9 comparison of the bacteriostatic effect with conventional bacteriocin products
Table 9 shows the bacteriostatic effect of the conventional bacteriocin product and the bio-fermented bran prepared according to the present invention under different pH conditions, and the obtained data indicate that the bacteriostatic activity of nisapline starts to be lost under the neutral or above-neutral pH condition, but the bio-fermented bran prepared according to the present invention still maintains stable bacteriostatic ability under the same condition, so that the bio-fermented bran prepared according to the present invention has more excellent bacteriostatic stability, which overcomes the limitation that the conventional bacteriocin product can only be applied to the food processing field under the meta-acid environment, and therefore, the bio-fermented bran prepared according to the present invention has a wider application range and higher application value in the application of feed.
It should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.
Claims (4)
1. A preparation method of biological fermentation bran with bacteriostatic action is characterized by comprising the following steps:
(1) inoculating Paenibacillus sp in a bran culture medium, and carrying out aerobic culture to obtain a fermentation product; the preservation number of the paenibacillus is CGMCC No. 8333; the feed-liquid ratio of the bran culture medium is 1: 1-1: 1.3, wherein the feed-liquid ratio is the mass ratio of the bran to the water; the inoculation amount of the paenibacillus is 2-4%, and the percentage is volume percentage; the temperature of the aerobic culture is 30-35 ℃, and the time of the aerobic culture is 96-118 hours;
(2) drying the fermentation product obtained in the step (1), wherein the drying temperature is 85-95 ℃.
2. The method according to claim 1, wherein the paenibacillus strain of step (1) is inoculated in an amount of 3%, the percentage being volume percentage, the aerobic culture temperature being 33 ℃, and the aerobic culture time being 98 hours.
3. Biologically fermented bran with bacteriostatic action obtained by the method of claim 1 or 2.
4. Use of the bacteriostatic biologically fermented bran according to claim 3 for the preparation of a functional animal feed.
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| Bacillus amyloliquefaciens ES-2-4 抗菌脂肽固态发酵工艺的研究;朱筱玉等;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20120615(第06期);第B018-97页 * |
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Inventor after: Han Jin Inventor after: Guo Benheng Inventor after: Wu Zhengjun Inventor after: Gao Caixia Inventor after: Liu Zhenmin Inventor before: Han Jin Inventor before: Guo Benheng Inventor before: Wu Zhengjun Inventor before: Gao Caixia Inventor before: Liu Zhenmin |