CN104762350A - Biological fermentation wheat bran with antibacterial effect and preparation method and application of biological fermentation wheat bran - Google Patents
Biological fermentation wheat bran with antibacterial effect and preparation method and application of biological fermentation wheat bran Download PDFInfo
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Abstract
The invention discloses biological fermentation wheat bran with an antibacterial effect and a preparation method and application of the biological fermentation wheat bran. The preparation method of the biological fermentation wheat bran with the antibacterial effect comprises the following steps: (1) inoculating Paenibacillus sp. into a wheat bran culture medium and performing aerobic culture to obtain a fermented matter; and (2) drying the fermented matter obtained in the step (1) for preparation. The preparation method disclosed by the invention adopts a solid-state fermentation method, has the advantages of single strain and raw material, simple process and low cost, and is conductive to standardization of fermentation wheat bran quality and cost control of industrial large-scale production. The biological fermentation wheat bran with the antibacterial effect, prepared by the preparation method disclosed by the invention, can effectively relieve and even fundamentally change the present situation of abuse of antibiotics in existing animal cultivation industry after being used in animal feed industry.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of biological fermentation wheat bran with bacteriostatic action and its preparation method and application.
Background technology
Wheat bran is wheat processing Main By product, and have source wide, cost is low, the feature of easily processing, and the primary raw materials being mainly used to feedstuff industry at present uses.Although contain many nutritive ingredients in wheat bran (as starch, oligose, protein, food fibre, VITAMIN etc.), but still there is the macromolecular substance (as robust fibre etc.) being difficult in a large number directly be digested and assimilated by animal, the fact shows, microorganism is utilized to carry out fermentation processing to wheat bran, the decomposition not only contributing to macromolecular substance absorbs, the benefit materials simultaneously relying on microbial metabolism to produce improves the using value of wheat bran, therefore, the raising by greatly promoting cultivation level of biological fermentation wheat bran.
Bacteriocin is some bacteriogenic material with anti-microbial activity, and main component is polypeptide, protein or protein complex, is proposed the earliest by Jacob in nineteen fifty-three.In recent years, the universal negative effect brought of even abusing of Broad spectrum antibiotics becomes increasingly conspicuous, and the research of new antibiotic substitute is extremely urgent.As the bacteriocin of protein substance, owing to being easily degraded by proteases, its security is very high, therefore receives increasing concern.
Can infer, if bacteriocin producing strains can be utilized to ferment to wheat bran, and suppress the fermentation wheat bran of harmful bacteria activity to be applied in animal-feed by obtaining to have, can alleviate, even solve the present situation of current animal cultivation industry abuse of antibiotics to a great extent.But, not yet find the report with the fermentation wheat bran of bacteriostatic activity at present, meanwhile, utilize series bacillus also to belong to space state to the research that wheat bran carries out separately fermentation aspect.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly the present situation for the correlation technique blank at present with bacteriostatic activity biological fermentation wheat bran and series bacillus list bacterium fermentation bran mass, provides a kind of biological fermentation wheat bran with bacteriostatic action and its preparation method and application.
The present situation of current animal cultivation industry abuse of antibiotics, constitute great contradiction with the food safety requirements that current consumer strengthens day by day and conflict, in order to solve this contradiction, contriver is to the preparation method of biological fermentation wheat bran with bacteriostasis property, the particularly training method of bacterial classification, the selection of bran mass formula and preparation, a series of technical parameters such as the temperature of fermentation culture, the time of fermentation culture have carried out conscientious analysis and screening, finally obtain the biological fermentation bran product that technical solutions according to the invention and gained have bacteriostasis property.The source of this biological fermentation wheat bran is natural, its preparation method simple process, and the storage stability of gained biological fermentation wheat bran is splendid, can obtain the antibacterial technique effect that stability is splendid.
The present inventor finds series bacillus (Paenibacillus sp.) CGMCC No.8333, after the fermented product drying that fermentation bran mass obtains makes fermentation wheat bran, still there is strong bacteriostatic activity, by repeatedly verifying, the fungistatic effect of this biological fermentation wheat bran is stablized, thus completes the present invention.
For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: a kind of preparation method with the biological fermentation wheat bran of bacteriostatic action, and described preparation method comprises the following steps:
(1) be inoculated in bran mass by series bacillus (Paenibacillus sp.), aerobic cultivation obtains fermented product;
(2) step (1) gained fermented product is dried and be get final product.
Wherein step (1) is for be inoculated in bran mass by series bacillus (Paenibacillus sp.), and aerobic cultivation obtains fermented product.The series bacillus that wherein said series bacillus (Paenibacillus sp.) is this area routine is preferably series bacillus that deposit number is CGMCC No.8333.Described series bacillus is prior art, and its preparation method is this area customary preparation methods, or obtains by buying from preservation center.Wherein said bran mass is the bran mass of this area routine, is preferably Testa Tritici substratum.The preparation method of described bran mass is the preparation method of this area routine, and the solid-liquid ratio of described bran mass is preferably 1:1 ~ 1:1.5, and be more preferably 1:1.2 ~ 1:1.4, described solid-liquid ratio is the mass ratio of wheat bran and water in substratum.
The inoculum size of wherein said series bacillus is preferably 1 ~ 5%, be more preferably 2 ~ 4%, be preferably 3%, described per-cent is volume percent, and the temperature of described aerobic cultivation is preferably 25 DEG C ~ 37 DEG C, is more preferably 30 DEG C ~ 35 DEG C, be preferably 33 DEG C, the time of described aerobic cultivation is preferably 72 hours ~ 120 hours, is more preferably 96 hours ~ 118 hours, is preferably 98 hours.
Before described series bacillus CGMCC No.8333 is inoculated in bran mass by fermentation process of the present invention, preferably also comprises this series bacillus CGMCC No.8333 and activate the step obtaining series bacillus seed.Described step preferably comprises the following steps: be seeded in TYC solid medium by series bacillus CGMCC No.8333 of the present invention, and namely 25 ~ 30 DEG C of cultivations obtain series bacillus CGMCC No.8333 seed for 18 ~ 28 hours; The bacterium colony of gained series bacillus CGMCC No.8333 seed is dispersed in TYC liquid nutrient medium, be inoculated in concussion in TYC liquid nutrient medium by the inoculum size of 2% ~ 5% volume percent again to cultivate, shaking speed is 100 ~ 200rpm, cultivate 18-28 hour, by centrifugal for gained culture supernatant discarded, gained thalline, with after sterile distilled water washing, suspends with the sterile distilled water of former volume of culture, the seed of the series bacillus that must ferment.
Wherein step (2) is for dry step (1) gained fermented product and get final product.The method of described oven dry is the furnace drying method of this area routine, and the temperature of described oven dry is preferably 80 DEG C ~ 100 DEG C, is more preferably 85 DEG C ~ 95 DEG C, is 92 DEG C best.
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: a kind of preparation method's gained as described in the present invention has the biological fermentation wheat bran of bacteriostatic action.
The technology contents that the preferable range with technical characteristic described in each step in the preparation method of the biological fermentation wheat bran of bacteriostatic action of the present invention is corresponding to preparation method mentioned above is completely the same, specifically refer to technology contents mentioned above, repeat no more herein.
For solving the problems of the technologies described above, three of the technical scheme that the present invention takes is: the biological fermentation wheat bran as described in the present invention with bacteriostatic action is preparing the application in performance animal feed.
Performance animal feed of the present invention is the performance animal feed of this area routine, refers to the animal-feed with specific nutrition composition or function.Performance animal feed of the present invention is preferably for the animal-feed or prevention animal that strengthen animal immunizing power produce the animal-feed etc. of disease.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
1, series bacillus is creatively applied to wheat bran fermentation by the present invention, and obtains the biological fermentation wheat bran product with fungistatic effect, and correlation technique not yet had report.
2, the preparation of existing biological fermentation wheat bran adopts the mixed fermentation of bacterium enzyme mostly, raw material is also in variation, make the cost of preparation very high, and the streaking one of product between each batch, the inventive method adopt process for solid state fermentation, bacterial classification and raw material single, technique is simple, with low cost, be conducive to the ferment stdn of wheat bran quality and the cost control of industrial mass production.
3, compared with existing biological fermentation wheat bran, biological fermentation wheat bran prepared by the present invention has clear and definite suppression streptococcus aureus (Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), singly increases the effect of listeria spp (Listeria monocytogenes) growth and breeding, therefore, effectively can alleviate after being applied to animal-feed, even fundamentally can change the present situation of existing animal cultivation industry abuse of antibiotics.
4, the biological fermentation wheat bran that prepared by the present invention all shows good bacteriostatic activity under heating, enzymolysis and long-term storage conditions, it can be used as in the suitability for industrialized production and association area that a kind of novel preparation method is applied to the biological fermentation wheat bran with bacteriostatic action, and application prospect is very wide.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue." room temperature " described in the present invention refers to the temperature of carrying out the operation room tested, and is generally 15 ~ 25 DEG C.If the reagent used in embodiment does not add explanation, be analytical reagent, buy from traditional Chinese medicines group.
The preparation of embodiment 1 biological fermentation wheat bran and the detection of bacteriostatic activity
1, materials and methods
The preparation of seed (fermented bacterium): (deposit number of described series bacillus is CGMCC No.8333 by series bacillus (Paenibacillus sp.), the source of this bacterial strain refers to the Chinese patent that publication number is CN103740618A) lyophilized powder dissolve with a small amount of sterile distilled water, get a ring with transfering loop to line TYC solid medium and (buy from OXOID Co., Britain), 30 DEG C of aerobic cultivation 48h take out, putting into 10mL TYC liquid nutrient medium with transfering loop picking list bacterium colony (buys from OXOID Co., Britain), vortex oscillator is used to be dispersed in liquid nutrient medium by bacterium colony, 30 DEG C, 180rpm concussion is cultivated 24h and is taken out, being inoculated in TYC liquid nutrient medium with 2% (volume percent) inoculum size (buys from OXOID Co., Britain), 30 DEG C, after 24h is cultivated in 180rpm concussion, culture 15, centrifugal 10 minutes of 000rpm, supernatant discarded, after thalline sterile distilled water washs 2 times, suspend with the sterile distilled water of former volume of culture, obtain the seed fermented.
Testa Tritici buy from Heze, Shandong (Taobao's trade name: beach, the Yellow River peasant household other, http://item.taobao.com/item.htm? spm=a230r.1.14.34.6aY7qR & id=23590856555 & ns=1 & abbucket=5#detail).
Indicator strain: streptococcus aureus (Staphylococcus aureus) CGMCC 1.879, micrococcus luteus (Micrococcus luteus) CGMCC 1.1848, singly increase listeria spp (Listeriamonocytogenes) CGMCC 1.9136, above indicator strain is all purchased from CGMCC.
The preparation of indicator liquid: by streptococcus aureus (Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), the lyophilized powder singly increasing listeria spp (Listeria monocytogenes) dissolves with a small amount of sterile distilled water, get a ring with transfering loop to line LB solid medium and (buy from OXOID Co., Britain), 30 DEG C of aerobic cultivation 48h take out, putting into 10mL LB liquid nutrient medium with transfering loop picking list bacterium colony (buys from OXOID Co., Britain), vortex oscillator is used to be dispersed in liquid nutrient medium by bacterium colony, 30 DEG C, 180rpm concussion is cultivated 24h and is taken out, being inoculated in LB liquid nutrient medium with 2% (volume percent) inoculum size (buys from OXOID Co., Britain), 30 DEG C, after 20h is cultivated in 180rpm concussion, obtain corresponding indicator liquid.
The preparation that instruction is dull and stereotyped: the indicator liquid prepared by aforesaid method dilutes, its bacterial concentration is made to be about 107cfu/mL, the indicator liquid drawing dilution with the ratio of volume ratio 1:150 injects 45 DEG C of aseptic LB solid mediums, abundant mixing is down flat plate rapidly afterwards, after flat board solidifies and surface-moisture evaporation is complete.
The detection method of bacteriostatic activity: drip 20 μ L testing samples with dibbling method on instruction flat board, measures after being placed in 30 DEG C of cultivation 20h and records antibacterial circle diameter.
The preparation of fermention medium: mixed in required ratio with distilled water by Testa Tritici, 118 DEG C of sterilizing 15min, are cooled to room temperature, obtain aseptic fermention medium.
2, the preparation of biological fermentation wheat bran
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic bran mass with 5% (volume percent) inoculum size, 25 DEG C of aerobic cultivation 72h obtain fermented product, by above-mentioned fermented product in 100 DEG C of oven dry, obtain biological fermentation wheat bran A.Wherein, the solid-liquid ratio of bran mass is 1:1.5, and described solid-liquid ratio is the mass ratio of wheat bran and water.
3, the detection of biological fermentation wheat bran bacteriostatic activity
Add to biological fermentation wheat bran A the fermentation wheat bran solution that sterile distilled water is mixed with 50mg/mL, after mixing, 15,000rpm is centrifugal, and 10min gets supernatant, measures its bacteriostatic activity.Result is as shown in the table:
The fungistatic effect of table 1 biological fermentation wheat bran
As can be seen from Table 1, biological fermentation wheat bran A acts on streptococcus aureus (Staphylococcusaureus), micrococcus luteus (Micrococcus luteus), singly increase the antibacterial circle diameter that listeria spp (Listeriamonocytogenes) produces is respectively 10mm, 13mm and 9mm, as can be seen here, gained biological fermentation wheat bran of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The preparation of embodiment 2 biological fermentation wheat bran and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of biological fermentation wheat bran
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic bran mass with 1% (volume percent) inoculum size, 37 DEG C of aerobic cultivation 120h obtain fermented product, by above-mentioned fermented product in 80 DEG C of oven dry, obtain biological fermentation wheat bran B.Wherein, the solid-liquid ratio of bran mass is 1:1, and described solid-liquid ratio is the mass ratio of wheat bran and water.
3, the detection of biological fermentation wheat bran bacteriostatic activity
Add to biological fermentation wheat bran B the fermentation wheat bran solution that sterile distilled water is mixed with 50mg/mL, after mixing, 15,000rpm is centrifugal, and 10min gets supernatant, measures its bacteriostatic activity.Result is as shown in the table:
The fungistatic effect of table 2 biological fermentation wheat bran
As can be seen from Table 2, biological fermentation wheat bran B acts on streptococcus aureus (Staphylococcusaureus), micrococcus luteus (Micrococcus luteus), singly increase the antibacterial circle diameter that listeria spp (Listeriamonocytogenes) produces is respectively 12mm, 15mm and 13mm, as can be seen here, gained biological fermentation wheat bran B of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The preparation of embodiment 3 biological fermentation wheat bran and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of biological fermentation wheat bran
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic bran mass with 2% (volume percent) inoculum size, 30 DEG C of aerobic cultivation 96h obtain fermented product, by above-mentioned fermented product in 90 DEG C of oven dry, obtain biological fermentation wheat bran C.Wherein, the solid-liquid ratio of bran mass is 1:1.3, and described solid-liquid ratio is the mass ratio of wheat bran and water.
3, the detection of biological fermentation wheat bran bacteriostatic activity
Add to biological fermentation wheat bran C the fermentation wheat bran solution that sterile distilled water is mixed with 50mg/mL, after mixing, 15,000rpm is centrifugal, and 10min gets supernatant, measures its bacteriostatic activity.Result is as shown in the table:
The fungistatic effect of table 3 biological fermentation wheat bran
As can be seen from Table 3, biological fermentation wheat bran C acts on streptococcus aureus (Staphylococcusaureus), micrococcus luteus (Micrococcus luteus), singly increase the antibacterial circle diameter that listeria spp (Listeriamonocytogenes) produces is respectively 15mm, 18mm and 15mm, as can be seen here, gained biological fermentation wheat bran of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The preparation of embodiment 4 biological fermentation wheat bran and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of biological fermentation wheat bran
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic bran mass with 4% (volume percent) inoculum size, 35 DEG C of aerobic cultivation 118h obtain fermented product, by above-mentioned fermented product in 85 DEG C of oven dry, obtain biological fermentation wheat bran.Wherein, the solid-liquid ratio of bran mass is 1:1.2, and described solid-liquid ratio is the mass ratio of wheat bran and water.
3, the detection of biological fermentation wheat bran bacteriostatic activity
Detection method as described in Example 1, detected result is: be respectively 13mm, 16mm and 14mm to streptococcus aureus, micrococcus luteus and single antibacterial circle diameter increasing listeria spp generation, as can be seen here, the biological fermentation wheat bran of gained of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The preparation of embodiment 5 biological fermentation wheat bran and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of biological fermentation wheat bran
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic bran mass with 3% (volume percent) inoculum size, 33 DEG C of aerobic cultivation 98h obtain fermented product, by above-mentioned fermented product in 92 DEG C of oven dry, obtain biological fermentation wheat bran.Wherein, the solid-liquid ratio of bran mass is 1:1.4, and described solid-liquid ratio is the mass ratio of wheat bran and water.
3, the detection of biological fermentation wheat bran bacteriostatic activity
Detection method as described in Example 1, detected result is: be respectively 14mm, 18mm and 15mm to streptococcus aureus, micrococcus luteus and single antibacterial circle diameter increasing listeria spp generation, as can be seen here, the biological fermentation wheat bran of gained of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The preparation of embodiment 6 biological fermentation wheat bran and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of biological fermentation wheat bran
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic bran mass with 3% (volume percent) inoculum size, 33 DEG C of aerobic cultivation 96h obtain fermented product, by above-mentioned fermented product in 95 DEG C of oven dry, obtain biological fermentation wheat bran.Wherein, the solid-liquid ratio of bran mass is 1:1.1, and described solid-liquid ratio is the mass ratio of wheat bran and water.
3, the detection of biological fermentation wheat bran bacteriostatic activity
Detection method as described in Example 1, detected result is: be respectively 15mm, 17mm and 13mm to streptococcus aureus, micrococcus luteus and single antibacterial circle diameter increasing listeria spp generation, as can be seen here, the biological fermentation wheat bran of gained of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The stability of the biological fermentation wheat bran bacteriostatic activity under effect example 1 normal temperature preservation condition
Biological fermentation wheat bran A, B and C of being prepared by embodiment 1-3 are distributed into aseptic aluminium foil bag, normal temperature (25 DEG C) is preserved after 6 months and 12 months and is taken out, add the fermentation wheat bran solution that sterile distilled water is mixed with 50mg/mL, after mixing, 15,000rpm is centrifugal, and 10min gets supernatant, and measure its bacteriostatic activity, result is as shown in the table.
The stability of biological fermentation wheat bran bacteriostatic activity under table 4 normal temperature preservation condition
As shown in Table 4, the biological fermentation wheat bran of all tests, after normal temperature preserves 12 months, stably maintains streptococcus aureus (Staphylococcus aureus), micrococcus luteus (Micrococcusluteus), singly increases listeria spp (Listeria monocytogenes) bacteriostatic activity.
The stability of biological fermentation wheat bran bacteriostatic activity under effect example 2 condition of different pH
Biological fermentation wheat bran A, B and C prepared by embodiment 1-3 add respectively pH=3,5,7, the 0.2M phosphate buffer soln of 9 is mixed with the biological fermentation wheat bran solution of 50mg/mL, after mixing, 15,000rpm is centrifugal, and 10min gets supernatant, obtain testing sample 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, the bacteriostatic activity of each group sample is as shown in the table:
The bacteriostatic activity of biological fermentation wheat bran under table 5 condition of different pH
As shown in Table 5, the fungistatic effect of biological fermentation wheat bran under condition of different pH is highly stable, and therefore, biological fermentation wheat bran prepared by the present invention can still keep good bacteriostatic activity in acid digestion liquid in animal body.
The stability of the biological fermentation wheat bran bacteriostatic activity after effect example 3 ferment treatment
The present invention additionally uses trypsin E.C.3.4.21.4, buy from Sigma), stomach en-(E.C.3.4.23.1, buy from Sigma), lipase (E.C.3.1.1.3 buys from Sigma), Proteinase K (E.C.3.4.21.64, buy from Sigma) process enzymes different in the next in-vitro simulated animal body of method of biological fermentation wheat bran to the impact of biological fermentation wheat bran bacteriostatic activity, concrete operations are as follows:
The 0.2M phosphate buffer soln of corresponding pH is prepared according to the optimal reaction pH of different enzyme, to biological fermentation wheat bran A prepared by embodiment 1-3, B and C add respectively above-mentioned different pH 0.2M phosphate buffer soln be mixed with the fermentation wheat bran solution of 50mg/mL, after mixing, 15, 000rpm is centrifugal, and 10min gets supernatant, corresponding enzyme is dissolved in the supernatant liquor of different pH, the ultimate density of enzyme is made to be 10mg/ml, mixed solution is incubated 2h in 37 DEG C of water-baths, regulate pH to neutral, measure the bacteriostatic activity of each sample group, and compare with the bacteriostatic activity before ferment treatment, result is as shown in the table:
The bacteriostatic activity of table 6 ferment treatment artifact fermentation wheat bran compares
As shown in Table 6, biological fermentation wheat bran is after different ferment treatment, and its fungistatic effect is still stablized, and therefore, biological fermentation wheat bran prepared by the present invention can keep good bacteriostatic activity in digestive ferment in animal body.
Comparative example 1
By the inoculum size in embodiment 3, culture temperature, fermentation time, culture base-material liquor ratio and bake out temperature adjust one by one, obtain the biological fermentation wheat bran prepared with next group different methods.Shown in the fungistatic effect following table of each group of biological fermentation wheat bran.
Table 7 different methods prepares the fungistatic effect of gained biological fermentation wheat bran
Can draw from the result shown in table 7, by inoculum size in the preparation method of described biological fermentation wheat bran, culture temperature, time fermentation time, culture base-material liquor ratio and bake out temperature are adjusted to outside the present invention, the fungistatic effect of gained biological fermentation wheat bran significantly reduces.
Comparative example 2
Method described in reference example 3, compare the fungistatic effect of the biological fermentation wheat bran prepared by series bacillus (Paenibacillus sp.) CGMCCNo.8333, plant lactobacillus (L.plantarum) ATCC 14917 (buying from ATCC) and lactobacterium casei (L.casei) ATCC 393 (buying from ATCC), concrete operations are as follows:
1, materials and methods
The preparation of seed (fermented bacterium):
The preparation of series bacillus CGMCC No.8333 seed is with embodiment 1.
The preparation of plant lactobacillus and lactobacterium casei seed: the lyophilized powder of plant lactobacillus ATCC 14917 and lactobacterium casei ATCC 393 is dissolved with a small amount of sterile distilled water, getting a ring with transfering loop lines on MRS solid medium (buying from Merck Co. Germany), 37 DEG C of Anaerobic culturel 24h take out, 1mL MRS liquid (buying from buying from Merck Co. Germany) is put into transfering loop picking list bacterium colony, vortex oscillator is used to be dispersed in liquid nutrient medium by bacterium colony, 37 DEG C of Anaerobic culturel 24h take out, 50mL MRS liquid is inoculated in 2% (volume percent) inoculum size, after 37 DEG C of cultivation 24h, culture 9, centrifugal 10 minutes of 000rpm, supernatant discarded, after thalline sterile distilled water washs 2 times, suspend with the sterile distilled water of former volume of culture, obtain the seed fermented.
Indicator strain, indicator liquid, fermention medium, the preparation of instruction flat board and the detection method of bacteriostatic activity are with embodiment 1.
2, the preparation of biological fermentation wheat bran bacteriostatic activity
Each bacterial strain is inoculated in aseptic bran mass with 2% (volume percent) inoculum size, cultivate (plant lactobacillus and lactobacterium casei 37 DEG C of Anaerobic culturel respectively, series bacillus 30 DEG C of aerobic cultivations) 96h obtains fermented product, by above-mentioned fermented product in 90 DEG C of oven dry, obtain the biological fermentation wheat bran that different strains is produced.Wherein, the solid-liquid ratio of bran mass is 1:1.3, and described solid-liquid ratio is the mass ratio of wheat bran and water.
3, the detection of biological fermentation wheat bran bacteriostatic activity
Add to biological fermentation wheat bran the fermentation wheat bran solution that sterile distilled water is mixed with 50mg/mL, after mixing, 15,000rpm is centrifugal, and 10min gets supernatant, measures its bacteriostatic activity.Result is as shown in the table:
The fungistatic effect of biological fermentation wheat bran prepared by table 8 different strains
As shown in Table 8, the fungistatic effect of the biological fermentation wheat bran prepared by series bacillus is significantly higher than other bacterial strains, and therefore, fermenting to wheat bran with series bacillus is the preferred method that preparation has bacteriostatic action biological fermentation wheat bran.
Comparative example 3
To having in the biological fermentation wheat bran C of bacteriostatic action of preparing of method described in embodiment 3 add pH=3,5,7, the 0.2M phosphate buffer soln of 9, be mixed with the biological fermentation wheat bran solution of 50mg/mL, after mixing, 15,000rpm is centrifugal, and 10min gets supernatant, obtains testing sample group S-3, S-5, S-7, S-9.
Select the general woods (Nisaplin of bacteriocin product Nysa conventional in the market simultaneously, buy from Danisco A/S BJ Rep Office) be contrast, be dissolved in pH=3,5,7, in the 0.2M phosphate buffer soln of 9, obtain control group N-3, N-5, N-7, N-9 that concentration is 50mg/mL, the fungistatic effect of each group sample is as shown in the table.
Table 9 compares with the fungistatic effect of Conventional bacteria element product
Table 9 shows the fungistatic effect of biological fermentation wheat bran under condition of different pH prepared by conventional bacteriocin goods and the present invention, the data obtained shows, under the pH condition of the general woods of Nysa more than neutrality or neutrality, its bacteriostatic activity starts to lose, and biological fermentation wheat bran prepared by the present invention still remains stable bacteriostasis under the same conditions, as can be seen here, biological fermentation wheat bran prepared by the present invention has more outstanding antimicrobial stability, this characteristic has overthrown the limitation that existing bacteriocin goods can only be applied to the food processing field under meta-acid environment, therefore, biological fermentation wheat bran prepared by the present invention has more wide range of application and higher using value in the application of feed.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. have a preparation method for the biological fermentation wheat bran of bacteriostatic action, it is characterized in that, described preparation method comprises the following steps:
(1) be inoculated in bran mass by series bacillus (Paenibacillus sp.), aerobic cultivation obtains fermented product;
(2) step (1) gained fermented product is dried and be get final product.
2. preparation method as claimed in claim 1, it is characterized in that, the deposit number of step (1) described series bacillus is CGMCC No.8333.
3. preparation method as claimed in claim 1, it is characterized in that, the solid-liquid ratio of step (1) described bran mass is 1:1 ~ 1:1.5, and described solid-liquid ratio is the mass ratio of wheat bran and water.
4. preparation method as claimed in claim 1, it is characterized in that, the inoculum size of the series bacillus described in step (1) is 1 ~ 5%, and described per-cent is volume percent, the temperature of described aerobic cultivation is 25 DEG C ~ 37 DEG C, and the time of described aerobic cultivation is 72 hours ~ 120 hours.
5. preparation method as claimed in claim 1, it is characterized in that, the temperature of the oven dry described in step (2) is 80 DEG C ~ 100 DEG C.
6. preparation method as claimed in claim 1, it is characterized in that, the inoculum size of the series bacillus described in step (1) is 2 ~ 4%, and described per-cent is volume percent, the temperature of described aerobic cultivation is 30 DEG C ~ 35 DEG C, and the time of described aerobic cultivation is 96 hours ~ 118 hours.
7. preparation method as claimed in claim 1, it is characterized in that, the inoculum size of the series bacillus described in step (1) is 3%, and described per-cent is volume percent, the temperature of described aerobic cultivation is 33 DEG C, and the time of described aerobic cultivation is 98 hours.
8. preparation method as claimed in claim 1, it is characterized in that, the temperature of the oven dry described in step (2) is 85 DEG C ~ 95 DEG C.
9. the preparation method's gained as described in any one of claim 1 ~ 8 has the biological fermentation wheat bran of bacteriostatic action.
10. the biological fermentation wheat bran as claimed in claim 9 with bacteriostatic action is preparing the application in performance animal feed.
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Cited By (5)
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| CN105285700A (en) * | 2015-09-23 | 2016-02-03 | 浙江恒乐粮食有限公司 | Biologic modifying method of bran for producing whole wheat flour |
| CN106701610A (en) * | 2015-11-18 | 2017-05-24 | 光明乳业股份有限公司 | Paenibacillus polymyxa, as well as culture method and application thereof |
| CN106978457A (en) * | 2017-04-24 | 2017-07-25 | 光明乳业股份有限公司 | A kind of antibiotic FusaricidinA preparation method |
| CN107033222A (en) * | 2017-04-24 | 2017-08-11 | 光明乳业股份有限公司 | A kind of antibacterial material Bovisin and preparation method thereof |
| CN108728497A (en) * | 2017-04-24 | 2018-11-02 | 光明乳业股份有限公司 | A kind of streptococcus mutans inhibitor and preparation method thereof |
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| CN103740618A (en) * | 2013-12-31 | 2014-04-23 | 光明乳业股份有限公司 | Novel bacillus-like strain as well as culture method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105285700A (en) * | 2015-09-23 | 2016-02-03 | 浙江恒乐粮食有限公司 | Biologic modifying method of bran for producing whole wheat flour |
| CN106701610A (en) * | 2015-11-18 | 2017-05-24 | 光明乳业股份有限公司 | Paenibacillus polymyxa, as well as culture method and application thereof |
| CN106978457A (en) * | 2017-04-24 | 2017-07-25 | 光明乳业股份有限公司 | A kind of antibiotic FusaricidinA preparation method |
| CN107033222A (en) * | 2017-04-24 | 2017-08-11 | 光明乳业股份有限公司 | A kind of antibacterial material Bovisin and preparation method thereof |
| CN108728497A (en) * | 2017-04-24 | 2018-11-02 | 光明乳业股份有限公司 | A kind of streptococcus mutans inhibitor and preparation method thereof |
| CN107033222B (en) * | 2017-04-24 | 2021-02-19 | 光明乳业股份有限公司 | Antibacterial substance Bovisin and preparation method thereof |
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| CN104762350B (en) | 2020-09-15 |
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