CN102041237A - Bacillus amyloliquefaciens D4 and method for producing chymosin by fermentation of same - Google Patents
Bacillus amyloliquefaciens D4 and method for producing chymosin by fermentation of same Download PDFInfo
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- CN102041237A CN102041237A CN2010100053091A CN201010005309A CN102041237A CN 102041237 A CN102041237 A CN 102041237A CN 2010100053091 A CN2010100053091 A CN 2010100053091A CN 201010005309 A CN201010005309 A CN 201010005309A CN 102041237 A CN102041237 A CN 102041237A
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Abstract
The invention discloses bacillus amyloliquefaciens D4 and a method for producing chymosin by fermentation of the same. The strain is screened from yak pasturing environmental soil of Gannan Tibetan Autonomous Prefecture of Gansu Province by adopting a casein culture medium, the chymosin can be obtained by deep fermentation of shake flask liquid, and the activity of the chymosin can reach 2,310.2SU/ml. The chymosin can be used for producing cheese and casein.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to bacillus amyloliquefaciens D4 bacterial strain and prepare the method for rennin with it.
Background technology
Rennin is a kind of key enzyme in the cheese making process, and it mainly acts on is the peptide bond of narrow spectrum shearing casein Phe 105-Met 106, forms stable secondary κ-casein and hydrophilic PROVON 190; When total κ-casein be hydrolyzed about 85% the time, at Ca
2+Have the breast that forms grumeleuse or solidify by the chemical bond that forms at the casein glue intergranular down, thereby the casein micelle precipitates.Produce fast-developing requirement in order to satisfy China's cheese and casein food grade, solve the supply contradiction of rennin, the research and development of rennin have been become the task of top priority of Dairy industry.The Department of Science and Technology of National Development and Reform Commission classifies the development of rennin as project that national 863 Program first develops and country's " the Seventh Five-Year Plan ", " eight or five ", " 95 " key research projects.Rennin mainly contains three kinds of sources, is respectively animal rennet, plant rennin and microbial rennet.
Most popular in the animal rennet is calf rennet.Have curdled milk effect preferably when adopting calf rennet to produce cheese, it is higher that the cheese that produces has yield rate, do not produce advantages such as bitter taste.5,000 ten thousand calves will be slaughtered every year in the whole world according to statistics, to obtain rennin.Along with the cheese industrial expansion, the rennin demand constantly increases, and supply falls short of demand causes price to rise steadily for calf rennet.Plant rennin (proteolytic enzyme) is distributed in the positions such as fruit, leaf, stem and root of plants such as pawpaw, Fructus Fici, pineapple, pumpkin and ginger.The proteolysis power of most of plant proteases in the cheese production process is strong excessively, and easily cheese produces bitter taste, and the finished product yield is lower, and quality is also soft slightly.
Because it is short that microorganism has growth cycle, advantages such as fast growth, growth conditions are easy to control, and production cost is low, since Arima in 1964 found that first Mucor pusillus (Mucor microus) can produce the proteolytic enzyme of curdled milk, microbial rennet became the emphasis of rennin research.The microorganism of the producing lab ferment of up to the present, discovering has streptomyces (Streptomyces), Micromonospora (Micromonospora), the actinomadura (Actinomadura) in the actinomycetes; Head mold in the fungi (Rhizopus), Mucor racemosus (Mucor racemosu), easy break wool mould (Mucorfuagilis), rice black wool mould (Mucornichei), parasitic inner seat shell bacterium (Endothiaparasitisa), grain vaccine (Endothiaparasitica); Subtilis in the bacterium (Bacillus subtilis) and bacillus natto (Bacillus subtilis natto).At present on the industrial production many with the rice black wool fungi fermentation such as mould and aspergillus niger produce rennin, exist the spawn culture time long, mycelia easily is damaged, fermentation period long, complex manufacturing and tunning are difficult for shortcomings such as extraction.
A bacillus amyloliquefaciens D4 bacterial strain involved in the present invention, have that nutritional requirement is low, volume is little, breeding is fast, tunning is easy to extraction separation, fermentation period is short and rennin produces advantages such as ability is strong, and the rennin of its fermentative production is suitable for cheese and casein food grade production.
Summary of the invention
The purpose of this invention is to provide a kind of new bacterial strain that can produce the bacillus amyloliquefaciens D4 of high vigor rennin.
Another object of the present invention provides a kind of method with the bacillus amyloliquefaciens fermenting and producing rennin.
Bacillus amyloliquefaciens D4 bacterial strain among the present invention screens from the yak grazing habit soil of Tibetan Autonomous Prefecture of Gannan, Gansu Province and obtains, classification called after bacillus amyloliquefaciens (Bacillus amyloliquefaciens) D4, CGMCC No:3290.
The screening method of bacillus amyloliquefaciens is to gather Tibetan Autonomous Prefecture of Gannan Grazing Yak living environment pedotheque, and sample is sieved, remove foreign material such as chad, grass after, take by weighing 1g and in the 99mL stroke-physiological saline solution, be diluted to 10
-3～10
-7, draw 1mL and coat on the casein plate culture medium, to cultivate down at 29,33,37 ℃ respectively, the bacterium colony that the selective precipitation circle is big, the hydrolysis circle the is little separation of further ruling is till separation obtains pure strain.The primary dcreening operation bacterial strain is carried out shake flask fermentation cultivate,, obtain bacterial strain by comparing the milk-curdling activity size.
The casein substratum is formed: the casein substratum is formed: peptone 2.5g/L, glucose 10g/L, yeast extract paste 1g/L, casein food grade 10g/L, agar 20g/L, skimmed milk 50g/L, pH value 7.0.
Have following biological characteristics according to bacillus amyloliquefaciens D4 of the present invention (CGMCC 3290):
1. morphological specificity: the result is positive, shaft-like for bacterial strain D4 gramstaining, and single arrangement forms gemma, and gemma is a central spore, the gemma oval, and sporangiocyst expands, and bacterial strain D4 gramstaining and stereoscan photograph are seen Fig. 1.
2. cultural characteristic:
Bacterial strain D4 is 37 ℃ of constant temperature culture 48h on the casein flat board, and bacterium colony is irregular, white, and projection is not obvious, the no mucus in bacterium colony surface.Bacterial strain D4 bacterium colony photo is seen Fig. 2.
3. physiological and biochemical property:
4. genetics feature (bacterial strain 16srRNA sequence): the present invention has surveyed the 16srRNA sequence of bacterial strain D4, and is as follows, and total length is 1425bp, and the accession number in GenBank is GQ918136, and referral web site is
Http:// www.ncbi.nlm.nih.gov/nuccore/261286617, can carry out sequence retrieval.
1 acgctggcgg?cgtgcctaat?acatgcaagt?cgagcggaca?gatgggagct?tgctccctga
61 tgttagcggc?ggacgggtga?gtaacacgtg?ggtaacctgc?ctgtaagact?gggataactc
121 cgggaaaccg?gggctaatac?cggatgcttg?tttgaaccgc?atggttcaaa?cataaaaggt
181 ggcttcggct?accacttaca?gatggacccg?cggcgcatta?gctagttggt?gaggtaacgg
241 ctcaccaagg?cgacgatgcg?tagccgacct?gagagggtga?tcggccacac?tgggactgag
301 acacggccca?gactcctacg?ggaggcagca?gtagggaatc?ttccgcaatg?gacgaaagtc
361 tgacggagca?acgccgcgtg?agtgatgaag?gttttcggat?cgtaaagctc?tgttgttagg
421 gaagaacaag?tgccgttcaa?atagggcggc?accttgacgg?tacctaacca?gaaagccacg
481 gctaactacg?tgccagcagc?cgcggtaata?cgtaggtggc?aagcgttgtc?cggaattatt
541 gggcgtaaag?ggctcgcagg?cggtttctta?agtctgatgt?gaaagccccc?ggctcaaccg
601 gggagggtca?ttggaaactg?gggaacttga?gtgcagaaga?ggagagtgga?attccacgtg
661 tagcggtgaa?atgcgtagag?atgtggagga?acaccagtgg?cgaaggcgac?tctctggtct
721 gtaactgacg?ctgaggagcg?aaagcgtggg?gagcgaacag?gattagatac?cctggtagtc
781 cacgccgtaa?acgatgagtg?ctaagtgtta?gggggtttcc?gccccttagt?gctgcagcta
841 acgcattaag?cactccgcct?ggggagtacg?gtcgcaagac?tgaaactcaa?aggaattgac
901 gggggcccgc?acaagcggtg?gagcatgtgg?tttaattcga?agcaacgcga?agaaccttac
961 caggtcttga?catcctctga?caatcctaga?gataggacgt?ccccttcggg?ggcagagtga
1021?caggtggtgc?atggttgtcg?tcagctcgtg?tcgtgagatg?ttgggttaag?tcccgcaacg
1081?agcgcaaccc?ttgatcttag?ttgccagcat?tcagttgggc?actctaaggt?gactgccggt
1141?gacaaaccgg?aggaaggtgg?ggatgacgtc?aaatcatcat?gccccttatg?acctgggcta
1201?cacacgtgct?acaatgggca?gaacaaaggg?cagcgaaacc?gcgaggttaa?gccaatccca
1261?caaatctgtt?ctcagttcgg?atcgcagtct?gcaactcgac?tgcgtgaagc?tggaatcgct
1321?agtaatcgcg?gatcagcatg?ccgcggtgaa?tacgttcccg?ggccttgtac?acaccgcccg
1381?tcacaccacg?agagtttgta?acacccgaag?tcggtgttgg?taacc
The D4 that makes up according to 16S rDNA sequence homology sees Fig. 3 with relevant bacterial strain systematic evolution tree, analyzes into by evolutionary tree and one has verified that this bacterium is a bacillus amyloliquefaciens.
The method of using bacillus amyloliquefaciens D4 bacterial strain of the present invention to make rennin comprises seed culture and liquid submerged fermentation step:
(1) the seed culture based component is: extractum carnis 3g/L, peptone 10～12g/L, sodium-chlor 5g/L, pH value 7.0; Culture condition: incubation time is 13 hours, 37 ℃ of temperature, shaking speed 200～240r/min;
(2) the liquid submerged fermentation substratum is formed by percentage to the quality: the wheat bran leach liquor is 10～18%, glucose 3～8%, skimming milk 2%, Sodium phosphate dibasic 0.2%, casein food grade 0.1%, sal epsom 0.10%, surplus are distilled water;
Fermentation condition: inoculum size is 3%, fermentation time 48 hours, and 37 ℃ of temperature, shaking speed are 170～180r/min;
Used wheat bran leach liquor preparation method is: 10～18g wheat bran is joined in the 100ml water, boil 10min, filter the back and supply 100ml with distilled water, obtain 10～18% wheat bran leach liquors.
The rennin zymologic property is as follows:
(1) the suitableeest curdling temperature is 70 ℃;
More than (2) 40 ℃ after the thermal treatment milk-clotting activity in various degree loss is arranged, milk-curdling activity forfeiture behind 70 ℃ of thermal treatment 10min;
(3) to be 5～8 o'clock milk-clotting activities strengthen with the reduction of newborn pH value the pH value, and pH is that 7 o'clock rennins are the most stable;
(4) Ca
2+, Fe
2+, Fe
3+, Mn
2+, Mg
2+, Al
3+Enzymic activity there is certain activation; Li
2+, Na
+, Gu
2+, Zn
2+Enzymic activity there is restraining effect.
A standard enzyme unit alive (1SU) is defined as: under 35 ℃, the enzyme amount that 40min solidifies 1mL 100g/L skimming milk is defined as a Soxhlet unit (Soxhlet Unit).
The enzyme activity determination method: adopt the Arima method, get the skimming milk of 5mL 100g/L, be incubated 5min down at 35 ℃, add the 0.5mL crude enzyme liquid, mix rapidly, accurate recording is from adding enzyme liquid to the solid time of curdling (s).
Wherein t is the curdled milk time, and D is an extension rate.
The relative enzyme work of the rennin that bacterial strain of the present invention makes under the differing temps sees Table 1 and Fig. 4, pH sees Table 2 to the influence of milk-curdling activity, the relative enzyme work of rennin liquid saw Table 3 when different pH handled, different metal ion pair rennin effect of vigor sees Table 4, Different Ca 2+ concentration sees Table 5 to the rennin effect of vigor, by above chart as can be known the zymologic property of this rennin be suitable for cheese and casein food grade production, can be used as the substitute of calf rennet.
The relative enzyme of rennin is lived during table 1 treatment of different temperature
Table 2pH is to the influence of milk-curdling activity
The relative enzyme of rennin liquid was lived when the different pH of table 3 handled
Table 4 different metal ion pair rennin effect of vigor
Table 5 Different Ca
2+Concentration is to the rennin effect of vigor
The present invention makes rennin with bacterial strain, and enzyme activity reaches 2310.2SU/ml, can be used for cheese and casein food grade production.
Bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) D4 on September 23rd, 2009 in Chinese microorganism strain management committee common micro-organisms center (Beijing) preservation, and receive preservation registration number CGMCC3290.
Description of drawings
Fig. 1 is bacillus amyloliquefaciens thalli morphology figure of the present invention;
Fig. 2 is bacillus amyloliquefaciens colonial morphology figure of the present invention;
Fig. 3 is the figure according to bacillus amyloliquefaciens D4 of the present invention with the relevant bacterial strain systematic evolution tree of 16S rDNA sequence homology structure;
Fig. 4 is the relative enzyme of the rennin that bacterial strain of the present invention makes under differing temps figure alive.
Embodiment
The following examples can further specify the present invention, but do not limit the present invention in any way.
Embodiment all adopts bacillus amyloliquefaciens (Bacillus amyloliquefaciens) D4 as original strain.
Embodiment 1
Scrape from the inclined-plane and to get two ring bacterial classifications, be inoculated in the 250ml triangular flask that the 70ml seed culture medium is housed and carry out seed culture.Condition is: incubation time is 13 hours, 37 ℃ of temperature, shaking speed 240r/min.Insert in the 250ml triangular flask that the 50ml fermention medium is housed with 3% inoculum size again and carry out fermentation culture.Condition is: fermentation time 48 hours, 37 ℃ of temperature, shaking speed are 180r/min, can obtain the fermented liquid of enzyme 2310.2SU/ml alive.
The seed culture based component is: extractum carnis 3g/L, peptone 10g/L, NaCl 5g/L, pH value 7.0;
The liquid submerged fermentation substratum is formed by percentage to the quality: the wheat bran leach liquor is 18%, glucose 3%, skimming milk 2%, Sodium phosphate dibasic 0.2%, casein food grade 0.1%, sal epsom 0.10%, surplus are distilled water;
Used wheat bran leach liquor preparation method is, wheat bran is boiled 10min in 100ml water, filters the back and supplies 100ml with distilled water, obtains the wheat bran leach liquor.
Embodiment 2
Scrape from the inclined-plane and to get two ring bacterial classifications, be inoculated in the 250ml triangular flask that the 70ml seed culture medium is housed and carry out seed culture.Condition is: incubation time is 13 hours, 37 ℃ of temperature, shaking speed 220r/min.Insert in the 250ml triangular flask that the 50ml fermention medium is housed with 3% inoculum size again and carry out fermentation culture.Condition is: fermentation time 48 hours, 37 ℃ of temperature, shaking speed are 175r/min, can obtain the fermented liquid of enzyme 2308.5SU/ml alive.
The seed culture based component is: extractum carnis 3g/L, peptone 11g/L, sodium-chlor 5g/L, pH7.0;
The liquid submerged fermentation substratum is formed by percentage to the quality: the wheat bran leach liquor is 14%, glucose 5%, skimming milk 2%, Sodium phosphate dibasic 0.2%, casein food grade 0.1%, sal epsom 0.10%, surplus are distilled water.Wheat bran leach liquor preparation method is with embodiment 1.
Embodiment 3
Scrape from the inclined-plane and to get two ring bacterial classifications, be inoculated in the 250ml triangular flask that the 70ml seed culture medium is housed and carry out seed culture.Condition is: incubation time is 13 hours, 37 ℃ of temperature, shaking speed 200r/min.Insert in the 250ml triangular flask that the 50ml fermention medium is housed with 3% inoculum size again and carry out fermentation culture.Condition is: fermentation time 48 hours, 37 ℃ of temperature, shaking speed are 170r/min, can obtain the fermented liquid of enzyme 2214.7SU/ml alive.
The seed culture based component is: extractum carnis 3g/L, peptone 12g/L, sodium-chlor 5g/L, pH7.0;
The liquid submerged fermentation medium component: the wheat bran leach liquor is 10%, glucose 8%, skimming milk 2%, Sodium phosphate dibasic 0.2%, casein food grade 0.1%, sal epsom 0.10%, surplus are distilled water.Wheat bran leach liquor preparation method is with embodiment 1.
Claims (3)
1. a bacillus amyloliquefaciens D4 (CGMCC 3290) has the ability of producing lab ferment.
2. the method with the bacillus amyloliquefaciens fermenting and producing rennin is characterized in that: cultivate bacillus amyloliquefaciens D4 (CGMCC 3290) in substratum, make rennin through seed culture and liquid submerged fermentation.
3. according to the method for claim 2, it is characterized in that:
The seed culture based component is extractum carnis 3g/L, peptone 10～12g/L, sodium-chlor 5g/L, pH value 7.0; Culture condition: incubation time is 13 hours, 37 ℃ of temperature, shaking speed 200～240r/min;
The liquid submerged fermentation substratum is formed by percentage to the quality: the wheat bran leach liquor is 10～18%, glucose 3～8%, skimming milk 2%, Sodium phosphate dibasic 0.2%, casein food grade 0.1%, sal epsom 0.10%, surplus are distilled water;
Fermentation condition: inoculum size is 3%, fermentation time 48 hours, and 37 ℃ of temperature, shaking speed are 170～180r/min;
Used wheat bran leach liquor preparation method is: 10～18g wheat bran is joined in the 100ml water, boil 10min, filter the back and supply 100ml with distilled water, obtain 10～18% wheat bran leach liquors.
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| CN103451128B (en) * | 2013-07-12 | 2015-07-08 | 江南大学 | Bacillus amyloliquefaciens and application thereof in preventing and controlling peach bleeding disease |
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| CN104694424A (en) * | 2015-02-12 | 2015-06-10 | 江西师范大学 | Bacillus amyloliquefaciens separated from fermented soya beans and used for producing protease |
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