CN102329747B - Culture medium and culture method for high-density culture of Staphylococcus xylosus A2 - Google Patents
Culture medium and culture method for high-density culture of Staphylococcus xylosus A2 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种木糖葡萄球菌A2的高密度培养方法及培养过程中所涉及的培养基。本发明的菌株是从发酵风干肠中提取并分离得到的，经鉴定确定为是一株木糖葡萄球菌(Staphylococcus xylosus)，命名为木糖葡萄球菌A2(Staphylococcus xylosusA2)可用于发酵肉制品，本发明进一步提供了该菌种的高密度培养方法，包括增殖培养基和发酵工艺条件优化控制，以及该菌株的高密度浓缩菌泥的制备方法。通过本发明的培养方法可以得到2.07×1011cfu/g以上活菌数的木糖葡萄球菌的高密度培养物，为进一步利用该菌株制备直投式木糖葡萄球菌发酵剂奠定了基础。The invention discloses a high-density culture method of staphylococcus xylosus A2 and a culture medium involved in the culture process. The bacterial strain of the present invention is extracted and separated from fermented air-dried sausage, and is determined to be a strain of Staphylococcus xylosus (Staphylococcus xylosus) after identification. The invention further provides a high-density culture method of the strain, including optimization control of proliferation medium and fermentation process conditions, and a preparation method of the high-density concentrated bacteria sludge of the strain. Through the cultivation method of the invention, a high-density culture of Staphylococcus xylosus with a viable count of more than 2.07×10 11 cfu/g can be obtained, which lays a foundation for further utilizing the strain to prepare a direct-throwing Staphylococcus xylosus starter.
Description
Technical field
The present invention relates to a kind of method for culturing microbes, particularly the high-density cultivation method of staphylococcus xylosus.Belong to the microorganism culturing technical field.
Background technology
Staphylococcus xylosus (Staphylococcus xylosus) is crucial a kind of bacterial classification in the fermented meat prods; Schleifer Kloos described this bacterium first in 1976 be a kind of CN-S (CNS) (Advances in Microbial Physiology; 1976,13:245-292.).This bacterium extensively be present in multiple traditional meat product (Clinical Laboratory magazine .2001,19 (4): 226.), the overwhelming majority can produce proteolytic enzyme (Applied microbiology; 2002,92 (1): 158-164.) and lipase (Food Microbiology, 2002; 19:441-449.), have gentle protein and steatolysis ability, produce some short chain fatty acids, small peptide and amino acid (Meat Science through metabolism; 2006,74:281-288.), give meat product flavour (foodstuffs industry science and technology; 2008,11 (29): 153-155.), and do not produce like harmful material (Meat Science such as biogenic amine, tyrasamine, phenylethylamine, spermine; 2006,73:559-564.).This bacterium also because of its high nitrate reductase activity (Meat Science, 2007,75:696-708.) and catalase activity (Food science; 2010,31 (17): 388-391.), have the peculiar smell of inhibition and produce; Stable color and the effect of removing superoxide, this bacterium can produce bacteriocin simultaneously, and pathogenic bacterium such as listeria bacteria and false pseudomonas bacillus are had certain restraining effect (Meat science; 2001,59 (3): 267-276.), can prolong the fermented meat prods shelf-lives.At present both at home and abroad report focuses mostly on aspect the effect of in fermented meat prods, bringing into play staphylococcus xylosus, and is also few to its report as the high-density culturing condition of throw type leaven.
Fermented meat prods is meant under nature or manual control condition; The fermentative action of utilizing mikrobe or enzyme to produce makes raw meat that a series of biological chemistries variations and physical change take place; Generation has flavour, color and luster and quality, and has the meat product than long storage life.In recent years, Along with people's strengthens the demand of high-quality meat product gradually, and the solution traditional zymotic meat product process-cycle is long, productive rate is low, the problem of high its large-scale production of restriction of production cost has crucial meaning.
Characteristics such as throw type leaven has the viable bacteria content height, inoculum size is few, fermentative activity is strong, preservation term is long, easy to use; Can make fermented product produce more convenient, the more stable (Food science of quality product; 2006; 27 (8): 191-197.); And avoided the traditional zymotic agent to produce because of middle succeeding transfer culture (processes such as expansion propagation that comprise actication of culture, seed fermentation agent, middle starter, productive leavening agent) that the strain fermentation vigor is degenerated and situation such as living contaminants (Food Science and Technology, 2004,15:67-78.).Throw type leaven can directly be bought to the professional production manufacturer production, has reduced the investment in bacterial classification workshop, has simplified technology, has improved labour productivity.
The present invention has optimized the culture medium prescription of staphylococcus xylosus; And its culture condition and high density cultures enrichment condition studied; Be intended to improve staphylococcus xylosus viable bacteria density in the culture, lay the foundation for further utilizing its preparation direct-throwing starter for fermentative meat.
Summary of the invention
The object of the present invention is to provide the optimization multiplication culture based formulas of staphylococcus xylosus A2 (Staphylococcus xylosus A2).
Another object of the present invention is to provide the staphylococcus xylosus A2 optimization cultural method of (Staphylococcus xylosus A2).
Another object of the present invention is to provide the enriching method of the high-density culture degree of staphylococcus xylosus A2 (Staphylococcus xylosus A2).
Described staphylococcus xylosus A2 (Staphylococcus xylosus A2) is that the inventor extracts the also bacterial classification of isolation identification from fermenting air-dry sausage.Bacterial classification (strain) A2 is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; The address is institute of microbiology of the Chinese Academy of Sciences in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Its culture presevation is numbered: CGMCC No.5049; Preservation date is on July 8th, 2011, the classification called after wood sugar glucose coccus (Staphylococcus xylosus) of bacterial classification;
The objective of the invention is to realize through following technical scheme:
At first, the invention provides a kind of substratum that is used for the high-density culture of staphylococcus xylosus A2, it is characterized in that said culture medium preparation comprises following steps:
(1) take by weighing following raw material respectively: 0.5 weight part lactose, 2 weight parts show peptone, 0.2 weight part yeast extract paste, 5 weight part mushroom juices, 7.5 weight part NaCl;
(2) above-mentioned raw materials is dissolved in the 1000 weight part zero(ppm) water, stirs, re-use neutralizing agent its pH value is adjusted to 7.2-7.8, be loaded in the triangular flask, sterilized 20 minutes down for 121 ℃ in temperature again, promptly obtain described substratum.
A preferred embodiment of the invention; Described mushroom juice can prepare according to following method: get new fresh mushroom 500g cleaning and chopping; Add an amount of zero(ppm) water and boil 30min; Cooling back is smashed to pieces in tissue mashing machine, is filtered, and filtrating adding distil water after centrifugal complements to 500mL, obtains mushroom juice.
A preferred embodiment of the invention uses neutralizing agent that the pH value of substratum is adjusted to 7.5.
Secondly, the present invention relates to the high-density cultivation method of a kind of staphylococcus xylosus A2, it is characterized in that this method steps is following:
A. actication of culture: the staphylococcus xylosus A2 (Staphylococcus xylosus A2) of freezing preservation is inoculated in the MSA medium slant; Under 32 ℃ of temperature, be cultured to and bacterium colony occurs; And then the picking lawn inserts another inclined-plane from the inclined-plane, bacterium colony appears and after, repeat top-operation; Switching is three times continuously, makes viable count reach 10
7-10
8Cfu/mL promptly gets activated spawn;
Described staphylococcus xylosus A2 (Staphylococcus xylosus A2) is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its culture presevation is numbered: CGMCC No.5049;
B. culture medium preparation:
Take by weighing following raw material at first, respectively: 0.5 weight part lactose, 2 weight parts show peptone, 0.2 weight part yeast extract paste, 5 weight part mushroom juices, 7.5 weight part NaCl;
Then, above-mentioned raw materials is dissolved in the 1000 weight part zero(ppm) water, stirs, re-use neutralizing agent its pH value is adjusted to 7.2-7.8, be loaded in the triangular flask, sterilized 20 minutes down for 121 ℃ in temperature again, obtain described substratum like this;
C. the preparation of strain fermentating liquid:
To use described substratum weight; The activation staphylococcus xylosus A2 bacterial classification that step a is obtained is inoculated in the substratum that step b obtains according to the 2%-4% of substratum weight; At temperature 30-34 ℃; Under the shaking table speed 120-150rpm/min condition, cultivate and obtained staphylococcus xylosus A2 strain fermentating liquid in 15-20 hour;
D. fermentation:
In the substratum weight of using, according to the 2%-4% of substratum weight the staphylococcus xylosus A2 strain fermentating liquid that step c obtains is inoculated in the substratum that step b obtains, under temperature 32-35 ℃, shook shaker fermentation 17 hours;
E. stop fermentation:
After reaching said fermentation time, triangular flask is placed 4 ℃ of stored refrigerated, stop fermentation, obtain staphylococcus xylosus A2 viable count and reach 3 * 10
9The staphylococcus xylosus high density fermentation liquid that cfu/mL is above.
A preferred embodiment of the invention, described neutralizing agent are 1mol/L NaOH solution.
A preferred embodiment of the invention, the pH value through interpolation neutralizing agent adjusting fermentation media among the said step b is 7.5.
A preferred embodiment of the invention, the volume of fermentation shake flask liquid amount accounts for 90% (450mL/500mL) of triangular flask volume in the described triangular flask.
A preferred embodiment of the invention, the staphylococcus xylosus A2 strain fermentating liquid that step c obtains are seeded in the said substratum that step b obtains, and 32 ℃ of temperature, bottle speed of shaking is under the 130rpm/min condition, ferments 17 hours.
A preferred embodiment of the invention; Described mushroom juice can prepare according to following method: get new fresh mushroom 500g cleaning and chopping; Add an amount of zero(ppm) water and boil 30min; Cooling back is smashed to pieces in tissue mashing machine, is filtered, and filtrating adding distil water after centrifugal complements to 500mL, obtains mushroom juice.
Further; The invention still further relates to the preparation method of the concentrated bacterium mud of a kind of staphylococcus xylosus (Staphylococcus xylosus A2) high-density; It is characterized in that: with the staphylococcus xylosus A2 high density fermentation liquid of method noted earlier preparation under 0～4 ℃; Under the 6000rpm/min condition centrifugal 30 minutes, obtain viable count and reach 2.06 * 10
11The high-density of the staphylococcus xylosus A2 that cfu/mL is above concentrates bacterium mud.
The staphylococcus xylosus A2 high density cultures that is prepared by above method can be used for preparing starter for fermentative meat, especially can be used for the preparation of direct-throwing staphylococcus xylosus starter.
Embodiment
Below in conjunction with specific embodiment the present invention is further specified, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Related material in the specific embodiment of the invention: new fresh mushroom is bought from market; Lactose, show that peptone, yeast extract paste, sodium-chlor etc. all buy the extensive and profound in meaning true tumor technology ltd from Beijing.
Related bacterial classification in the specific embodiment of the invention: staphylococcus xylosus A2; For the inventor extracts the also bacterial classification of isolation identification from fermenting air-dry sausage; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, its culture presevation is numbered: CGMCC No.5049.
Embodiment 1
The high-density cultivation method of a kind of staphylococcus xylosus A2 may further comprise the steps:
A. actication of culture: the staphylococcus xylosus A2 (Staphylococcus xylosus A2) of freezing preservation is inoculated in the MSA medium slant; Under 32 ℃ of temperature, be cultured to and bacterium colony occurs; And then the picking lawn inserts another inclined-plane from the inclined-plane, bacterium colony appears and after, repeat top-operation; Switching is three times continuously, makes viable count reach 10
7-10
8Cfu/mL promptly gets activated spawn;
Described staphylococcus xylosus A2 (Staphylococcus xylosus A2) is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its culture presevation is numbered: CGMCC No.5049;
B. culture medium preparation:
Take by weighing following raw material at first, respectively: 0.5g part lactose, 2g show peptone, 0.2g yeast extract paste, 5g mushroom juice, 7.5g NaCl;
Then, above-mentioned raw materials is dissolved in the 1000g zero(ppm) water, stirs; Re-use 1mol/L NaOH solution its pH value is adjusted to 7.2, be loaded in the triangular flask, the fermentation shake flask liquid amount in the triangular flask is 90%; Sterilized 20 minutes down for 121 ℃ in temperature again, obtain described substratum like this;
C. the preparation of strain fermentating liquid:
To use described substratum weight; The activation staphylococcus xylosus A2 bacterial classification that step a is obtained is inoculated in the substratum that step b obtains according to 2% of substratum weight; 30 ℃ of temperature; Under the shaking table speed 120rpm/min condition, cultivate and obtained staphylococcus xylosus A2 strain fermentating liquid in 20 hours;
D. fermentation:
To use described substratum weight, the said staphylococcus xylosus A2 strain fermentating liquid that step c is obtained is inoculated in the substratum that step b obtains according to 4% of substratum weight, under 35 ℃ of temperature, shakes shaker fermentation 17 hours;
E. stop fermentation:
After reaching said fermentation time, triangular flask is placed 4 ℃ of stored refrigerated, stop fermentation, obtain staphylococcus xylosus A2 viable count and reach 3 * 10
9The staphylococcus xylosus high density fermentation liquid that cfu/mL is above.
Embodiment 2
The high-density cultivation method of a kind of staphylococcus xylosus A2 is characterized in that, may further comprise the steps:
A. actication of culture: the staphylococcus xylosus A2 (Staphylococcus xylosus A2) of freezing preservation is inoculated in the MSA medium slant; Under 32 ℃ of temperature, be cultured to and bacterium colony occurs; And then the picking lawn inserts another inclined-plane from the inclined-plane, bacterium colony appears and after, repeat top-operation; Switching is three times continuously, makes viable count reach 10
7-10
8Cfu/mL promptly gets activated spawn;
Described staphylococcus xylosus A2 (Staphylococcus xylosus A2) is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its culture presevation is numbered: CGMCC No.5049;
B. culture medium preparation:
Take by weighing following raw material at first, respectively: 0.5g lactose, 2g show peptone, 0.2g yeast extract paste, 5g mushroom juice, 7.5g NaCl;
Then, above-mentioned raw materials is dissolved in the 1000g zero(ppm) water, stirs, re-use 1mol/L NaOH solution its pH value is adjusted to 7.5, be loaded in the triangular flask, sterilized 20 minutes down for 121 ℃ in temperature again, obtain described substratum like this;
C. the preparation of strain fermentating liquid:
In the substratum weight of using; The activation staphylococcus xylosus A2 bacterial classification that step a is obtained is inoculated in the substratum that step b obtains according to 4% of substratum weight; 32 ℃ of temperature, under the shaking table speed 130rpm/min condition, cultivate and obtained staphylococcus xylosus A2 strain fermentating liquid in 17 hours;
D. fermentation:
In the substratum weight of using, the staphylococcus xylosus A2 strain fermentating liquid that step c is obtained is inoculated in the substratum that step b obtains according to substratum 2%, under 34 ℃ of temperature, shakes shaker fermentation 17 hours;
E. stop fermentation:
After reaching said fermentation time, triangular flask is placed 4 ℃ of stored refrigerated, stop fermentation, obtain staphylococcus xylosus A2 viable count and reach 3 * 10
9The staphylococcus xylosus high density fermentation liquid that cfu/mL is above.
The staphylococcus xylosus A2 high density fermentation liquid that obtains at 4 ℃, under the 6000rpm/min condition centrifugal 30 minutes, is obtained viable count and reaches 2.06 * 10
11The above staphylococcus xylosus A2 high-density of cfu/mL concentrates bacterium mud.
The condition optimizing of the high-density cultivation method of experimental example 1 staphylococcus xylosus A2
The present invention relates to the high-density cultivation method of a kind of staphylococcus xylosus A2.
The high-density cultivation method step of said staphylococcus xylosus A2 is following:
The selection of 1 carbon source
Studied and added the liquid nutrient medium that nine kinds of different carbon sources are processed, inoculated the good staphylococcus xylosus A2 of activation respectively, 32 ℃ of constant temperature culture 17 hours detect the bacteria suspension viable count, measure the pH value, observe the enriching effect of every kind of carbon source to it.1 result shows according to subordinate list, but staphylococcus xylosus A2 equal normal growth and carry out metabolism and produce acid in adding the substratum of 9 kinds of carbon sources.Enriching effect is significantly higher than and adds other single-factor carbon source enriching effects (P＜0.05) in the substratum of interpolation starch; It is also better that the substratum that adds dextrin, lactose, semi-lactosi and trehalose adds the enriching effect of other single-factor substratum; And difference not significantly (P＞0.05) each other; Staphylococcus xylosus A2 utilizes the carbon source metabolism to produce lactic acid in process of growth, and the pH value of solution value is reduced, and crosses when the pH value of solution value and thalline " self-dissolving " phenomenon will take place when low and suppress to increase; Can find out the good group of enriching effect behind growth metabolism 17h by table 1, pH value descend not significantly (P＞0.05) in the nutrient solution.Make substratum be stratification state because of starch and dextrin become pasty state behind high-temperature sterilization, take all factors into consideration the working service of Financial cost, suitability for industrialized production fermentor tank and the factors such as concentration and separation of thalline, so choose the optimum carbon source that lactose is staphylococcus xylosus A2.
The different carbon sources of table 1 are to the influence of staphylococcus xylosus A2 thalli growth
Annotate: the same row of letter representation compare in the table, the identical difference not remarkable (P＞0.05) of then representing of letter, and difference is then represented significant difference (P＜0.05)
The selection of 2 nitrogenous sources
Studied and added the liquid nutrient medium that 11 kinds of different nitrogen sources are processed; Table 2 is the result show; But staphylococcus xylosus A2 tests in used 11 kinds of nitrogenous source growth mediums all normal growth and carries out metabolism at this, and in 1% urea medium, grow is suppressed, the thalline viable count minimizing of fermentation back.Add the organic nitrogen source substratum and add inorganic nitrogen-sourced substratum enriching effect significant difference (P＜0.05); After adding inorganic nitrogen-sourced substratum fermentation; The pH value does not have obviously reduction; Enriching effect is not obvious even do not have an enriching effect, and this possibly be because be difficult to utilize inorganic nitrogen-sourced fermentation and acid in the staphylococcus xylosus growth metabolism process, and part is inorganic nitrogen-sourced to its growth even restraining effect is arranged; Add enriching effect that acid hydrolyzed casein and the substratum that shows peptone add other single-factor substratum and compare and analyze significant difference (P＜0.05), but difference not significantly (P＞0.05) each other.Consider in conjunction with the Financial cost factor, choose and show that peptone is the optimum nitrogen source of staphylococcus xylosus A2.
Table 2 different nitrogen sources is to the influence of staphylococcus xylosus A2 thalli growth
Annotate: the same row of letter representation compare in the table, the identical difference not remarkable (P＞0.05) of then representing of letter, and difference is then represented significant difference (P＜0.05)
The selection of 3 nutritional factor
Studied and added the liquid nutrient medium that eight kinds of Different Nutrition factors are processed.Show according to table 3, but staphylococcus xylosus A2 tests in used 8 kinds of nutritional factor growth mediums all normal growth and carries out metabolism at this.Add in mushroom juice, lime carbonate, flat mushroom juice, Tomato juice and the yeast extract medium; Staphylococcus xylosus A2 enriching effect significantly is better than the enriching effect (P＜0.05) that adds other nutritional factor; Contain growth factors such as the VITAMINs that promotes staphylococcus xylosus, amino acid in mushroom juice, flat mushroom juice, Tomato juice and the yeast extract paste, remarkable to the enriching effect effect; Bacterial classification for fermentation and acid; Add in the fermented liquid lime carbonate can in the thalli growth breeding in the acid that produces to guarantee that the medium pH value can noticeable change; Somatic cells increases better; But lime carbonate can be deposited on container bottom and make substratum be stratification state in culturing process, is unfavorable for the concentration and separation of the working service and the thalline of suitability for industrialized production fermentor tank, so lime carbonate is not added in follow-up test.Homology in conjunction with nutritional factor is taken all factors into consideration, and chooses mushroom juice and yeast extract paste for being the best nutritional factor of staphylococcus xylosus A2.
The influence of table 3 Different Nutrition factor pair staphylococcus xylosus A2 thalli growth
Annotate: the same row of letter representation compare in the table, the identical difference not remarkable (P＞0.05) of then representing of letter, and difference is then represented significant difference (P＜0.05)
4 proliferated culture medium determination of formula
Consider the interaction that possibly have combination and cooperation between several kinds of MFs, thereby the more tangible several kinds of MFs of cultivation effect in single factor experiment (lactose, show peptone, mushroom juice and yeast extract paste) are adopted L
9(3
4) the best of optimization of orthogonal test staphylococcus xylosus A2 increases bacterium and cultivate.Table 4 is the result show, in four factors, is B＞C＞D＞A in proper order to the influence of staphylococcus xylosus A2 viable bacteria proliferative amount; Explain that influencing the topmost factor that staphylococcus xylosus A2 grows is to show peptone; Other 3 factors are mushroom juice, yeast extract paste and lactose successively, and nitrogenous source is the principal element of staphylococcus xylosus A2 growth, and staphylococcus xylosus A2 propagation rapidly in the high nutrient solution of nitrogenous source content; And carbon source is little to this effect, confirms that therefore the optimum multiplication medium proportioning is A
1B
3C
1D
3, promptly 0.5% lactose, 2% shows peptone, 5% mushroom juice and 0.2% yeast extract paste.
Table 4 staphylococcus xylosus A2 fermention medium optimizing components orthogonal experiments
The selection of 5 initial pH value of medium
Studied staphylococcus xylosus A2 and transferred the growing state in the 6.0-8.0 scope at the initial pH of substratum, table 5 is the result show, the substratum initial value is bigger to the influence of bacterial strain thalline growth pattern.Initial pH value of medium is that 7.5 enriching effect is apparently higher than other groups (P＜0.05).Staphylococcus xylosus utilizes growth substance fermentations such as carbon source, nitrogenous source to produce lactic acid; Reduction along with pH value in the accumulation of lactic acid, the nutrient solution; Can suppress the normal growth metabolism of staphylococcus xylosus thalline, therefore initial pH value is lower than in 7.5 the nutrient solution, and staphylococcus xylosus A2 propagation is slow.But initial pH is too high, also can produce restraining effect to the growth of staphylococcus xylosus thalline not, so definite staphylococcus xylosus A2 proliferated culture medium original ph is 7.5.
Table 5 initial pH value of medium is to the influence of staphylococcus xylosus A2 thalli growth
Annotate: the identical difference not remarkable (P＞0.05) of then representing of letter in the table, difference is then represented significant difference (P＜0.05)
6 shake the selection of bottled liquid measure
Studied staphylococcus xylosus A2 at substratum by the growing state in liquid amount (being sub-packed in the 500mL triangular flask) scope of 60%-100%; Table 6 is the result show; It is bigger to the influence of bacterial strain thalline growth pattern to shake bottled liquid measure; Wherein liquid amount is that the substratum of 90% (450mL/500mL) is obvious than other liquid amount enriching effects; Significant difference (P＜0.05), this possibly be because mostly staphylococcus xylosus is aerobic bacteria or facultative anaerobe, what of liquid amount can influence the dissolved oxygen amount in the liquid nutrient medium; Too much or very few oxygen content all is unfavorable for its growing multiplication, can find out that through test 90% (450mL/500mL) shakes bottled liquid measure for the best of staphylococcus xylosus A2.
Table 6 liquid amount is to the influence of staphylococcus xylosus A2 thalli growth
Annotate: the identical difference not remarkable (P＞0.05) of then representing of letter in the table, difference is then represented significant difference (P＜0.05)
The invention still further relates to the enriching method of the high density cultures of a kind of staphylococcus xylosus A2, it is characterized in that as follows:
Study the high-density enrichment condition that staphylococcus xylosus A2 nutrient solution carries out under 6 groups of centrifugal conditions, shown in subordinate list 7, during the thalline enrichment, selected appropriate parameter of noncentricity (revolution, time) to be very important.In general, centrifugation time is long more, and centrifugal revolution is high more, and is big more to the physical abuse of thalline, and centrifugal back thalline survival rate is low more.But select the slow speed short period of time centrifugally can make the thalline enrichment incomplete again, the part thalline also is present in the supernatant.Take all factors into consideration above two aspects and real cost and to the working service of whizzer, 6000rpm is adopted in this test, 30min is as the centrifugal condition of staphylococcus xylosus A2 thalline enrichment.
The different centrifugal conditions of table 7 are to the influence of staphylococcus xylosus A2 thalli growth
Annotate: the same row of letter representation relatively in the table; The identical difference not remarkable (P＞0.05) of then representing of letter, difference is then represented lactobacterium casei viable bacteria density (cfu/g) in the bacterium mud of the centrifugal back of significant difference (P＜0.05)=centrifugal back total viable count (cfu)/centrifugal back bacterium mud gross weight (g)
The above is merely the preferred embodiments of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and in spirit that claim of the present invention limited and scope, can carry out many changes to it, revise, even equivalence change, but all will fall in protection scope of the present invention.
Claims (7)
1. substratum that is used for staphylococcus xylosus (Staphylococcus xylosus) A2 culture presevation number for the high-density culture of CGMCC No.5049 is characterized in that said culture medium preparation comprises following steps:
Take by weighing following raw material at first, respectively: 0.5 weight part lactose, 2 weight parts show peptone, 0.2 weight part yeast extract paste, 5 weight part mushroom juices, 7.5 weight part NaCl;
Described mushroom juice prepares by following method: get new fresh mushroom 500g cleaning and chopping, add an amount of zero(ppm) water and boil 30min, cooling back is smashed to pieces in tissue mashing machine, is filtered, and filtrating adding distil water after centrifugal complements to 500mL, obtains mushroom juice;
Then, above-mentioned raw materials is dissolved in the 1000 weight part zero(ppm) water, stirs, re-use neutralizing agent its pH value is adjusted to 7.2-7.8, be loaded in the triangular flask, sterilized 20 minutes down for 121 ℃ in temperature again, promptly obtain described substratum.
2. substratum according to claim 1 is characterized in that using neutralizing agent that its pH value is adjusted to 7.5.
3. the high-density cultivation method of a staphylococcus xylosus A2 is characterized in that, may further comprise the steps:
A. actication of culture: the staphylococcus xylosus A2 (Staphylococcus xylosus A2) of freezing preservation is inoculated in the MSA medium slant; Under 32 ℃ of temperature, be cultured to and bacterium colony occurs; And then the picking lawn inserts another inclined-plane from the inclined-plane, bacterium colony appears and after, repeat top-operation; Switching is three times continuously, makes viable count reach 10
7-10
8Cfu/mL promptly gets activated spawn;
Described staphylococcus xylosus A2 (Staphylococcus xylosus A2) is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its culture presevation is numbered: CGMCC No.5049;
B. culture medium preparation:
Take by weighing following raw material at first, respectively: 0.5 weight part lactose, 2 weight parts show peptone, 0.2 weight part yeast extract paste, 5 weight part mushroom juices, 7.5 weight part NaCl;
Described mushroom juice prepares by following method: get new fresh mushroom 500g cleaning and chopping, add an amount of zero(ppm) water and boil 30min, cooling back is smashed to pieces in tissue mashing machine, is filtered, and filtrating adding distil water after centrifugal complements to 500mL, obtains mushroom juice;
Then; Above-mentioned raw materials is dissolved in the 1000 weight part zero(ppm) water, stirs, re-use neutralizing agent its pH value is adjusted to 7.2-7.8; Be loaded in the triangular flask; The volume of the fermentation shake flask liquid amount in the triangular flask accounts for 90% of triangular flask volume, sterilizes 20 minutes down for 121 ℃ in temperature again, obtains described substratum like this;
C. the preparation of strain fermentating liquid:
To use described substratum weight; The activation staphylococcus xylosus A2 bacterial classification that step a is obtained is inoculated in the substratum that step b obtains according to the 2%-4% of substratum weight; At temperature 30-34 ℃; Under the shaking table speed 120-150rpm/min condition, cultivate and obtained staphylococcus xylosus A2 strain fermentating liquid in 15-20 hour;
D. fermentation:
In the substratum weight of using, according to the 2%-4% of substratum weight the staphylococcus xylosus A2 strain fermentating liquid that step c obtains is inoculated in the substratum that step b obtains, under temperature 32-35 ℃, shook shaker fermentation 17 hours;
E. stop fermentation:
After reaching said fermentation time, triangular flask is placed 4 ℃ of stored refrigerated, stop fermentation, obtain staphylococcus xylosus A2 viable count and reach 3 * 10
9The staphylococcus xylosus high density fermentation liquid that cfu/mL is above.
4. method according to claim 3 is characterized in that the pH value through interpolation neutralizing agent adjusting fermentation media is 7.5 among the said step b; Neutralizing agent is a 1mol/L NaOH solution among the said step b.
5. method according to claim 3 is characterized in that said staphylococcus xylosus A2 strain fermentating liquid is seeded in the substratum that step b obtains, and 32 ℃ of temperature, bottle speed of shaking is under the 130rpm/min condition, ferments 17 hours.
6. method according to claim 3 is characterized in that further comprising with the staphylococcus xylosus A2 high density fermentation liquid that obtains under 0 ~ 4 ℃, under the 6000rpm/min condition centrifugal 30 minutes, promptly obtains viable count and reaches 2.06 * 10
11The above staphylococcus xylosus A2 high-density of cfu/mL concentrates bacterium mud.
7. the application of staphylococcus xylosus A2 high density cultures in the preparation starter for fermentative meat for preparing by each described method of claim 3-6.
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| 贾士芳等.肉制品发酵剂木糖葡萄球菌J23 发酵条件的研究.《肉类工业》.2006,(第11期),34-36. |
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