CN104774890A - Paenibacillus sp. bacteriocin crude extract as well as preparation method and application thereof - Google Patents
Paenibacillus sp. bacteriocin crude extract as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a paenibacillus sp. bacteriocin crude extract as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) inoculating paenibacillus sp. into a culture medium for culturing the paenibacillus sp., shocking and carrying out fermentation cultivation to obtain fermentation liquor, and centrifuging the obtained fermentation liquor so as to take liquid supernatant; and (2) adding ammonium sulfate to the obtained liquid supernatant, centrifuging and collecting precipitates which are separated out when the saturation of the ammonium sulfate is 30-70%, dissolving the obtained precipitates and then dialyzing, and carrying out freeze drying to obtain the paenibacillus sp. bacteriocin crude extract. Raw materials for preparing the paenibacillus sp. bacteriocin crude extract are bran, wide in source and low in cost, and a fermentative strain is a paenibacillus sp. single strain; and the technology facilitates the standardization of product quality and the cost control of industrialized mass production. The paenibacillus sp. bacteriocin crude extract can be directly applied to food production, thus achieving the purposes of inhibiting the proliferation of putrefying bacteria and prolonging the shelf period of food and reducing the application cost.
Description
Technical field
The invention belongs to biological technical field, be specifically related to Bacillus species bacteriocin crude extract and its preparation method and application.
Background technology
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.Although the bacteriocin (Nisin) that exploitation obtains at present has possessed high anticorrosion with antibacterial using value, but by the characteristic (stable in properties under low pH condition of Nisin itself, pH>7.0 bacteriostatic activity is lost gradually) impact, the scope of its application receives great limitation, secondly, the acquisition of current bacteriocin is all from chemosynthesis substratum, this type of medium component is various, formula is complicated, process for preparation is loaded down with trivial details has potential food safety risk, moreover, the cost preparing bacteriocin is very high, the source of bacteriocin is relatively limited, the problems referred to above limit the development and utilization of bacteriocin to a great extent.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly for existing bacteriocin poor stability, preparation cost is high, source is not enough, fermentation medium components is various, formula is complicated, and process for preparation is loaded down with trivial details and have the present situation that there is potential food safety risk, provides Bacillus species bacteriocin crude extract and its preparation method and application.
The present inventor finds series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain gained supernatant after bran mass fermentation, after being redissolved by ammonium sulfate precipitation, the centrifugal precipitation obtained, the series bacillus crude extract with strong bacteriostatic activity can be obtained through dialysis, lyophilize, thus complete the present invention.
For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: the preparation method of a Bacillus species bacteriocin crude extract, and described preparation method comprises the following steps:
(1) be inoculated in by series bacillus (Paenibacillus sp.) in the substratum cultivating series bacillus, concussion fermentation culture obtains fermented liquid, by gained fermented liquid centrifuging and taking supernatant liquor;
(2) in step (1) gained supernatant liquor, add ammonium sulfate, the precipitation separated out when collected by centrifugation ammonium sulfate saturation ratio is 30 ~ 70%, dialyse after gained resolution of precipitate, lyophilize, to obtain final product.
Wherein series bacillus (Paenibacillus sp.) is inoculated in the substratum cultivating series bacillus by step (1), and concussion fermentation culture obtains fermented liquid, by gained fermented liquid centrifuging and taking supernatant liquor.The series bacillus that wherein said series bacillus (Paenibacillus sp.) is this area routine, preferably for fermentation produces the series bacillus of bacteriocin, be more preferably series bacillus (Paenibacillus sp.), its 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.
The substratum of wherein said foster series bacillus is the substratum of this area routine, and described substratum is preferably bran mass, is more preferably Testa Tritici substratum, and the preparation method of described bran mass is the preparation method of this area routine.The formula of described bran mass is better comprises wheat bran and water, and wherein the content of wheat bran is preferably 1 ~ 5%, and be more preferably 3%, surplus is water, and described per-cent is mass percent.
The inoculum size of wherein said series bacillus is preferably 1 ~ 5%, be more preferably 2 ~ 4%, be optimally 3%, described per-cent is volume percent, the leavening temperature of described series bacillus is preferably 25 ~ 37 DEG C, it is more preferably 28 DEG C ~ 35 DEG C, be 30 DEG C best, the time of described fermentation is preferably 48 ~ 96 hours, being more preferably 60 ~ 78 hours, is 72 hours best, and the speed of described concussion fermentation culture is preferably 100 ~ 300rpm, being more preferably 150 ~ 200rpm, is 180rpm best.
Before described series bacillus CGMCC No.8333 is inoculated in substratum 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) for add ammonium sulfate in step (1) gained supernatant liquor, and the precipitation separated out when collected by centrifugation ammonium sulfate saturation ratio is 30 ~ 70%, dialyse after gained resolution of precipitate, lyophilize, to obtain final product.
Wherein said ammonium sulfate saturation ratio is preferably 30 ~ 70%, be more preferably 40 ~ 60%, centrifugal speed is preferably 8, 000 ~ 15, 000g, be more preferably 10, 000 ~ 12, 000g, the centrifugal time is preferably 5 ~ 15 minutes, it is more preferably 8 ~ 10 minutes, centrifugal temperature is preferably 4 DEG C ~ 8 DEG C, it is more preferably 5 DEG C ~ 7 DEG C, the molecular weight cut-off of the dialysis membrane that described dialysis uses is preferably 800Da ~ 1200Da, be more preferably 1000Da, dialysis time is preferably 1 ~ 3 day, it is more preferably 2 days, the preferred solvents ground of wherein said dissolving is water, be more preferably distilled water.Preferably also comprise in dialysis procedure and change water step, the number of times changing water is preferably 2 ~ 4 times/day.When the technical parameter value of described centrifugal condition or dialysis condition is outside request protection domain of the present invention, the bacteriostatic activity of gained series bacillus bacteriocin crude extract can be made to produce and significantly to reduce.
Wherein said lyophilize is the freeze drying technology of this area routine, as long as by removal of solvents, can obtain the series bacillus bacteriocin crude extract with bacteriostatic activity.
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: preparation method's gained series bacillus bacteriocin crude extract as described herein.
The technology contents that in the preparation method of series bacillus bacteriocin crude extract of the present invention, described in each step, the preferable range of technical characteristic is corresponding to preparation method mentioned above is completely the same, specifically refers to technology contents mentioned above, repeats no more herein.
For solving the problems of the technologies described above, three of the technical scheme that the present invention takes is: series bacillus bacteriocin crude extract is preparing the application in functional foodstuff as described herein.
Series bacillus bacteriocin crude extract of the present invention can be widely used in preparing various food preservatives, in functional foodstuff or functional feed.Described application is preferably for prepare the application in food preservatives.
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, the invention solves current bacteriocin to originate narrow present situation, in a creative way series bacillus is applied to the preparation technology of bacteriocin.
2, the preparation of bacteriocin all depends on chemosynthesis substratum at present, and the research of using bran mass to prepare bacteriocin was not yet reported, bran mass is applied to the preparation of bacteriocin by the present invention first as a kind of natural fermention medium, thus its product has higher food safety.
3, the raw material for the preparation of bacteriocin crude extract of the present invention is wheat bran, and its source is wide, cost is low, and fermented bacterium is series bacillus single culture, and aforesaid combination is conducive to the stdn of product quality and the cost control of industrial mass production.
4, wheat bran is a kind of byproduct of wheat processing, therefore, has higher food safety with the chemosynthesis substratum that it is the standby bacteriocin of base-material fermentation adopts than ordinary method.
5, compared with conventional bacteria element Nisin, the bacteriocin crude extract character adopting the present invention to prepare is more stable, and bacteriostatic activity is significantly higher than Nisin especially in neutral conditions, therefore, has the using value more excellent than Nisin.
6, the bacteriocin crude extract adopting the present invention to prepare can be directly used in the production of food, thus reaches the propagation suppressing spoilage organism, extends the object of Food Shelf-life, and then reduces the cost of application.
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, is analytical reagent, buys from traditional Chinese medicines group.
The preparation of embodiment 1 series bacillus bacteriocin crude extract 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.
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 bought 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, and makes its bacterial concentration be about 10
7cfu/mL, injects 45 DEG C of aseptic LB solid mediums with the indicator liquid of the ratio of volume ratio 1:150 absorption dilution, is fully down flat plate rapidly after mixing, 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.
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).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 series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic fermention medium with 5% (volume percent) inoculum size, 25 DEG C, 100rpm shakes fermentation culture 48h and obtains fermented liquid, by above-mentioned fermented liquid 4 DEG C, 8, 000g is centrifugal, and 15min gets supernatant, slowly ammonium sulfate is added in supernatant liquor, again 8, the precipitation separated out when 000g centrifugal 15min collection ammonium sulfate saturation ratio is 30 ~ 60%, the dialysis tubing that this precipitation is 1000Da with the rear interception of distilled water redissolution is dialysed 1 day, change water every day 4 times, to dialyse solution lyophilize in back pkt., obtain bacteriocin crude extract A.Wherein, the formula of fermention medium is: Testa Tritici 5%, and surplus is water, and described per-cent is mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Bacteriocin crude extract A is dissolved the aqueous solution being mixed with 50mg/mL with sterile distilled water, measure its bacteriostatic activity.Result is as shown in the table:
The fungistatic effect of table 1 series bacillus bacteriocin crude extract
As can be seen from Table 1, series bacillus bacteriocin crude extract A acts on streptococcus aureus (Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), singly increase the antibacterial circle diameter that listeria spp (Listeria monocytogenes) produces is respectively 13mm, 16mm and 13mm, as can be seen here, gained series bacillus bacteriocin crude extract A of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The preparation of embodiment 2 series bacillus bacteriocin crude extract and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic fermention medium with 1% (volume percent) inoculum size, 37 DEG C, 300rpm shakes fermentation culture 96h and obtains fermented liquid, by above-mentioned fermented liquid 8 DEG C, 10, 000g is centrifugal, and 10min gets supernatant, slowly ammonium sulfate is added in supernatant liquor, again 10, the precipitation separated out when 000g centrifugal 10min collection ammonium sulfate saturation ratio is 50 ~ 70%, the dialysis tubing that this precipitation is 1000Da with the rear interception of distilled water redissolution is dialysed 1 day, change water every day 4 times, to dialyse solution lyophilize in back pkt., obtain bacteriocin crude extract B.Wherein, the formula of fermention medium is: Testa Tritici 1%, and surplus is water, and described per-cent is mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Bacteriocin crude extract B is dissolved the aqueous solution being mixed with 50mg/mL with sterile distilled water, measure its bacteriostatic activity.Result is as shown in the table:
The fungistatic effect of table 2 series bacillus bacteriocin crude extract
As can be seen from Table 2, series bacillus bacteriocin crude extract B acts on streptococcus aureus (Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), singly increase the antibacterial circle diameter that listeria spp (Listeria monocytogenes) produces is respectively 9mm, 12mm and 10mm, as can be seen here, gained series bacillus bacteriocin crude extract B of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The preparation of embodiment 3 series bacillus bacteriocin crude extract and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic substratum with 2% (volume percent) inoculum size, 30 DEG C, 180rpm shakes fermentation culture 72h and obtains fermented liquid, by above-mentioned fermented liquid, 4 DEG C, 15, 000g is centrifugal, and 5min gets supernatant, slowly ammonium sulfate is added in supernatant liquor, again 15, the precipitation separated out when 000g centrifugal 5min collection ammonium sulfate saturation ratio is 40 ~ 60%, the dialysis tubing that this precipitation is 1000Da with the rear interception of distilled water redissolution is dialysed 2 days, change water every day 3 times, to dialyse solution lyophilize in back pkt., obtain bacteriocin crude extract C.Wherein, the formula of fermention medium is: Testa Tritici 3%, and surplus is water, and described per-cent is mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Bacteriocin crude extract C is dissolved the aqueous solution being mixed with 50mg/mL with sterile distilled water, measure its bacteriostatic activity.Result is as shown in the table:
The fungistatic effect of table 3 series bacillus bacteriocin crude extract
As can be seen from Table 3, series bacillus bacteriocin crude extract C acts on streptococcus aureus (Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), singly increase the antibacterial circle diameter that listeria spp (Listeria monocytogenes) produces is respectively 15mm, 20mm and 16mm, as can be seen here, gained series bacillus bacteriocin crude extract C of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The preparation of embodiment 4 series bacillus bacteriocin crude extract and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic substratum with 4% (volume percent) inoculum size, 28 DEG C, 150rpm shakes fermentation culture 60h and obtains fermented liquid, by above-mentioned fermented liquid 5 DEG C, 12, 000g is centrifugal, and 8min gets supernatant, slowly ammonium sulfate is added in supernatant liquor, again 15, the precipitation separated out when 000g centrifugal 5min collection ammonium sulfate saturation ratio is 30 ~ 70%, the dialysis tubing that this precipitation is 1000Da with the rear interception of distilled water redissolution is dialysed 3 days, change water every day 3 times, to dialyse solution lyophilize in back pkt., obtain bacteriocin crude extract.Wherein, the formula of fermention medium is: Testa Tritici 3%, and surplus is water, and described per-cent is mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Gained series bacillus bacteriocin crude extract is dissolved the aqueous solution being mixed with 50mg/mL with sterile distilled water, measure its bacteriostatic activity.The antibacterial result of gained is: to streptococcus aureus (Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), singly increase antibacterial circle diameter 11mm, 14mm and 12mm respectively that listeria spp (Listeria monocytogenes) produces, as can be seen here, the bacteriocin crude extract of gained of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
The preparation of embodiment 5 series bacillus bacteriocin crude extract and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated in aseptic substratum with 4% (volume percent) inoculum size, 35 DEG C, 200rpm shakes fermentation culture 78h and obtains fermented liquid, by above-mentioned fermented liquid 7 DEG C, 12, 000g is centrifugal, and 8min gets supernatant, slowly ammonium sulfate is added in supernatant liquor, again 15, the precipitation separated out when 000g centrifugal 5min collection ammonium sulfate saturation ratio is 30 ~ 70%, the dialysis tubing that this precipitation is 1000Da with the rear interception of distilled water redissolution is dialysed 3 days, change water every day 3 times, to dialyse solution lyophilize in back pkt., obtain bacteriocin crude extract.Wherein, the formula of fermention medium is: Testa Tritici 3%, and surplus is water, and described per-cent is mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Gained series bacillus bacteriocin crude extract is dissolved the aqueous solution being mixed with 50mg/mL with sterile distilled water, measure its bacteriostatic activity.The antibacterial result of gained is: to streptococcus aureus (Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), singly increase antibacterial circle diameter 11mm, 13mm and 10mm respectively that listeria spp (Listeria monocytogenes) produces, as can be seen here, the bacteriocin crude extract of gained of the present invention has significant fungistatic effect to above-mentioned gram-positive microorganism.
Comparative example 1
By the inoculum size in embodiment 3, culture temperature, fermentation time, the speed of fermentation concussion, wheat bran concentration, ammonium sulfate saturation ratio and dialysis time adjust one by one, obtain the bacteriocin crude extract prepared with next group different methods.After each group of series bacillus bacteriocin crude extract is reduced to the volume before freeze-drying, shown in its fungistatic effect following table measured.
Table 4 different methods prepares the fungistatic effect of gained series bacillus bacteriocin crude extract
Can draw from the result shown in table 4, by inoculum size in the preparation method of described series bacillus bacteriocin crude extract, culture temperature, fermentation time, the speed of fermentation concussion, wheat bran concentration, time ammonium sulfate saturation ratio and dialysis time are adjusted to outside the present invention, the fungistatic effect of gained series bacillus bacteriocin crude extract significantly reduces.
Comparative example 2
The series bacillus bacteriocin crude extract C prepared with method described in embodiment 3 is for testing sample, select the general woods (Nisaplin of bacteriocin product Nysa conventional in the market simultaneously, buy from Danisco A/S BJ Rep Office) be contrast, the general woods of Nysa is traditional antibacterial product of commercialization, core effective constituent is wherein nisin Nisin, also has other compositions such as sodium-chlor in addition.Series bacillus bacteriocin crude extract C and the general standing forest of Nysa are not dissolved in pH=3,5,7, in the 0.2M phosphate buffer soln of 9, obtain testing sample group S-3 that concentration is 50mg/mL, S-5, S-7, S-9 and control group N-3, N-5, N-7, N-9, the fungistatic effect of each group sample is as shown in the table.
Table 5 compares with the fungistatic effect of Conventional bacteria element product
Table 5 shows the fungistatic effect of series bacillus bacteriocin crude extract under condition of different pH prepared by conventional bacteriocin goods and the present invention, data show, under the pH condition of the general woods of Nysa more than neutrality or neutrality, its bacteriostatic activity starts to lose, and series bacillus bacteriocin crude extract prepared by the present invention still remains stable bacteriostasis under the same conditions, as can be seen here, series bacillus bacteriocin crude extract 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, series bacillus bacteriocin crude extract applicable food-processing scope prepared by the present invention will be wider than existing bacteriocin goods.
Effect example 1
By traditional technology make sausage (making method is drawn from following document: Yuan Qiuping. the application of nisin in meat product [J]. foodstuffs industry science and technology, 1998 (4): 27-28.), wherein, only Sodium Nitrite is added in the sausage of control group 1, addition is 0.15g/kg, Sodium Nitrite and the general woods of Conventional bacteria cellulose product Nysa is added in the sausage of control group 2, addition is respectively 0.04g/kg and 0.4g/kg, the bacteriocin crude extract C that nitrite and embodiment 3 prepare is with the addition of in the sausage of sample sets, addition is respectively 0.04g/kg and 0.3g/kg, mensuration total number of bacterial colony after each sausage sample making completes, result is as shown in the table.
The fungistatic effect of series bacillus bacteriocin crude extract in table 6 sausage maker skill
As shown in Table 6, in the manufacture craft of sausage, add the series bacillus bacteriocin crude extract prepared by the present invention, fungistatic effect in its finished product and existing bacteriocin product similar, and the color of sausage does not significantly change, as can be seen here, with the complete alternative existing bacteriocin product of the series bacillus bacteriocin crude extract prepared by the present invention various anticorrosion, antibacterial etc. in application.
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. the preparation method of a Bacillus species bacteriocin crude extract, it is characterized in that, described preparation method comprises the following steps:
(1) be inoculated in by series bacillus (Paenibacillus sp.) in the substratum cultivating series bacillus, concussion fermentation culture obtains fermented liquid, by gained fermented liquid centrifuging and taking supernatant liquor;
(2) in step (1) gained supernatant liquor, add ammonium sulfate, the precipitation separated out when collected by centrifugation ammonium sulfate saturation ratio is 30 ~ 70%, dialyse after gained resolution of precipitate, lyophilize, to obtain final product.
2. preparation method as claimed in claim 1, it is characterized in that, the deposit number of step (1) described series bacillus (Paenibacillus sp.) is CGMCC No.8333.
3. preparation method as claimed in claim 1, it is characterized in that, the formula of the substratum of the described cultivation series bacillus of step (1) is bran mass, and the formula of described bran mass is: wheat bran 1 ~ 5%, surplus is water, and described per-cent is mass percent.
4. preparation method as claimed in claim 1, it is characterized in that, the inoculum size of step (1) described series bacillus is 1 ~ 5%, described per-cent is volume percent, the temperature of described fermentation is 25 DEG C ~ 37 DEG C, the speed of described concussion is 100 ~ 300rpm, and the time of described fermentation is 48 ~ 96 hours.
5. preparation method as claimed in claim 1, it is characterized in that, the inoculum size of step (1) described series bacillus is 2 ~ 4%, described per-cent is volume percent, the temperature of described fermentation is 28 DEG C ~ 35 DEG C, the speed of described concussion is 150 ~ 200rpm, and the time of described fermentation is 60 ~ 78 hours.
6. preparation method as claimed in claim 1, it is characterized in that, the described centrifugal speed of step (2) is 8,000 ~ 15,000g, and the centrifugal time is 5 ~ 15 minutes, and centrifugal temperature is 4 DEG C ~ 8 DEG C.
7. preparation method as claimed in claim 1, it is characterized in that, the described centrifugal speed of step (2) is 10,000 ~ 12,000g, and the centrifugal time is 8 ~ 10 minutes, and centrifugal temperature is 5 DEG C ~ 7 DEG C.
8. preparation method as claimed in claim 1, is characterized in that, the molecular weight cut-off of the dialysis membrane that the described dialysis of step (2) uses is 800Da ~ 1200Da, and dialysis time is 1 ~ 3 day.
9. preparation method's gained series bacillus bacteriocin crude extract as described in any one of claim 1 ~ 8.
10. series bacillus bacteriocin crude extract is preparing the application in functional foodstuff as claimed in claim 9.
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| 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 |
| CN108441533A (en) * | 2018-03-23 | 2018-08-24 | 北京中特养生物技术研究所有限公司 | The method that bacteriocin is extracted from lactic acid bacteria |
| 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 |
| CN110898211A (en) * | 2019-12-02 | 2020-03-24 | 福建省闽东水产研究所 | Antibacterial composition for inhibiting pseudosciaena crocea visceral white spot pathogenic bacteria in vitro and preparation method thereof |
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| 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 |
| 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 |
| CN108441533A (en) * | 2018-03-23 | 2018-08-24 | 北京中特养生物技术研究所有限公司 | The method that bacteriocin is extracted from lactic acid bacteria |
| CN110898211A (en) * | 2019-12-02 | 2020-03-24 | 福建省闽东水产研究所 | Antibacterial composition for inhibiting pseudosciaena crocea visceral white spot pathogenic bacteria in vitro and preparation method thereof |
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| CN104774890B (en) | 2018-12-14 |
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