CN109609586A - A kind of method for counting colonies of fermented feed - Google Patents
A kind of method for counting colonies of fermented feed Download PDFInfo
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- CN109609586A CN109609586A CN201811586633.XA CN201811586633A CN109609586A CN 109609586 A CN109609586 A CN 109609586A CN 201811586633 A CN201811586633 A CN 201811586633A CN 109609586 A CN109609586 A CN 109609586A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
Abstract
The present invention relates to the methods that lactic acid bacteria, bacillus and the saccharomycete in a kind of pair of fermented feed are counted, the method is counted on the MRS solid medium containing Natamycin, YPD culture medium and LB culture medium containing streptomysin and penicillin respectively by the sample after diluting fermented feed, the quantity of lactic acid bacteria, bacillus and saccharomycete in fermented feed can more accurately be counted, the count accuracy of the single microorganism in fermented feed is improved, and easy to operate.
Description
Technical field
The present invention relates to microorganism count field, in particular to in fermented feed lactic acid bacteria, bacillus and
The method that saccharomycete is counted.
Background technique
Fermented feed be under manual control condition, by the metabolic activity of microorganism itself, by vegetalitas, animality and
Anti-nutritional factors in mineral substance is decomposed or conversion, what generation can more be acted on by livestock and poultry feeding, digestion and absorption and nonhazardous
Feedstuff.Studies have shown that feed passes through fermentation process, its nutrient absorption level can be improved, may be deposited in its raw material of degrading
Toxin, and promote growth, maintain microecological balance in animal body, enhancing immunity of organisms, these effects such as prevent and cure diseases
Also available embodiment.
Wherein, microorganism plays a key role in this fermentation process.Therefore, the quality of fermented feed is evaluated
One of index is exactly the viable count in fermented feed.The beneficial bacteriums such as saccharomycete, bacillus and lactic acid bacteria in fermented feed
Quantity is extremely more, and harmful bacteria (such as Escherichia coli, salmonella and staphylococcus aureus) quantity is extremely low, is no more than 10cfu/
g.Therefore, saccharomycete, bacillus and the bacterium number of lactic acid bacteria detected in fermented feed can then indicate viable count therein.It is existing
Feed in total number of bacteria method of counting, be only capable of calculating total number of bacteria, and usually have biggish detection error.If
The strain not belonged to is counted respectively, then need to combine biochemistry detection, this method is time-consuming and laborious, and existing feed mark
Similar product, can not be carried out unified comparison by the accuracy for still guaranteeing bacterium colony counting in standard without unified effective method of counting.
The method of plate culture count is one of saccharomycete, bacillus and the common method of lactic acid bacteria detected in feed.By
In gemma high-output stress-resistance, and bacterium colony is easily grown in other culture mediums, influences the counting of lactic acid bacteria and saccharomycete, therefore existing
Detection method (such as National Standard Method [1] GBT13093-2006) can not accurately be counted, and the accuracy of the value is directly closed
It is the quality to fermented feed.Therefore, it is imperative to develop a kind of new method of counting.
Summary of the invention
The object of the present invention is to provide lactic acid bacteria, bacillus and the saccharomycete in a kind of pair of fermented feed accurately to carry out
The method of counting, wherein the described method includes:
1) it takes fermented feed and feed suspension is made with sterile saline according to w/v 1:8-1:10；
2) suspension is successively subjected to 10 times of gradient dilutions with sterile saline, obtaining extension rate is 104、105With
106Dilution；
3) taking the extension rate is 104、105With 106Dilution and be separately added into culture dish, it is described to being added
The YPD culture medium containing 10-50ppm streptomysin and 10-25ppm penicillin is poured into each culture dish of dilution and is mixed
It closes, obtains bacteria-containing YPD plate, saccharomycete is counted after -48h for 24 hours is cultivated at 28 DEG C -32 DEG C；Meanwhile taking equivalent
The extension rate is 104、105With 106Dilution be heated to 80 DEG C -85 DEG C and be separately added into culture dish, to being added
It states and pours into LB culture medium in each culture dish of dilution and mixed, obtain bacteria-containing LB plate, cultivated at 28 DEG C -32 DEG C
Bacillus is counted after -48h for 24 hours；In addition, taking the extension rate of equivalent is 104、105With 106Dilution, point
It is not applied on the plate of the MRS culture medium containing 100-500ppm Natamycin, after -48h for 24 hours is cultivated at 36 DEG C -38 DEG C
Lactic acid bacteria is counted.
Advantageous effects of the invention are as follows: this method is suitable for carrying out lactic acid bacteria, bacillus and saccharomycete quasi-
It really counts, the count accuracy of the single microorganism in mixed fermentation feed can be improved, and easy to operate.
Detailed description of the invention
Fig. 1 be lactic acid bacteria on MRS culture medium under the microscope and the form that observes by the naked eye.
Fig. 2 be bacillus on LB culture medium under the microscope and the form that observes by the naked eye.
Fig. 3 be saccharomycete on YPD culture medium under the microscope and the form that observes by the naked eye.
Fig. 4 A- Fig. 4 C respectively illustrate in embodiment 1 for counting according to the method for the present invention and existing national standard
The photo of the form of lactic acid bacteria, bacillus and the saccharomycete on each plate that method obtains after cultivation.
Fig. 5 A- Fig. 5 C respectively illustrate in embodiment 2 for counting according to the method for the present invention and existing national standard
The photo of the form of lactic acid bacteria, bacillus and the saccharomycete on each plate that method obtains after cultivation.
Specific embodiment
" lactic acid bacteria " as described herein refers to the bacterium that lactic acid can be generated using glucose or lactose fermentation, for which
It is grown on lactobacillus bacteria (MRS) culture medium with specificity.After lactobacter growth is metabolized out lactic acid, since pH value drops
Low, general microorganism is not easy to grow on the culture medium, thus MRS culture medium has certain specificity to lactic acid bacteria.
As shown in Fig. 1 right figure, usually on MRS culture medium, after cultivating 24-48h at 37 DEG C, the lactic acid bacteria bacterium colony of growth has following special
Sign: colony diameter is 1~3mm, rounded, protrusion, and micro white moistens, neat in edge.100 it can be seen from Fig. 1 left figure
Under the oil mirror visual field again, lactic acid bacteria is presented below as form: rod-short, and cell is in pairs or chaining arranges, both ends blunt circle, no gemma,
Gram-positive, 0.5 μm~1 μm of diameter.
" bacillus " as described herein refers to the rod-shaped or spherical bacteria that can form gemma (endospore), cultivates in LB
It is grown on base.As shown in Fig. 2 right figure, after cultivating 24-48h usually at 30 DEG C, the Bacillus colonies of growth have following special
Sign: single bacterium colony rough surface is opaque, and dirty white or yellowish, there is apparent brood-gemma in the irregular center in edge, and leads to
Normal bacterium colony joins together, and covers entire culture medium.It can be seen from Fig. 2 left figure under 100 times of the oil mirror visual field, bacillus
Be presented below as form: rod-short arranges in pairs, is in splayed, has end raw or central spore.
" saccharomycete " as described herein is a kind of unicellular fungi, in the yeast extract peptone glucose to it with specificity
(YPD) it is grown on agar medium.As shown in Fig. 3 right figure, after cultivating 24-48h usually at 30 DEG C, the saccharomycete bacterium colony of growth
It has the feature that bacterium colony is smooth, wet, sticky, is easy to provoke, bacterium colony is homogeneous, front and back sides and edge, central part
Color is all very uniform, and bacterium colony is mostly milky.It can be seen from Fig. 3 left figure under 40 times of field of microscope, saccharomycete is presented
Following form: oval or rhizoma nelumbinis shape, 2-6 μm of cell width, 5-20 μm of length.
It is usually sent out simultaneously using lactic acid bacteria, bacillus and saccharomycete in the fermentation process of solid state fermentation feed
Ferment.Count to the bacterium colony of each Pseudomonas in fermented feed is one of the index of quality for evaluating fermented feed.Therefore, it counts
Number results it is accurate whether for evaluation fermented feed quality for it is extremely important.
In one embodiment, the present invention provides lactic acid bacteria, bacillus and the yeast in a kind of pair of fermented feed
The method that bacterium is counted, wherein the described method includes:
1) it takes fermented feed and feed suspension is made with sterile saline according to w/v 1:8-1:10；
2) suspension is successively subjected to 10 times of gradient dilutions with sterile saline, obtaining extension rate is 104、105With
106Dilution；
3) taking the extension rate is 104、105With 106Dilution and be separately added into culture dish, it is described to being added
The YPD culture medium containing 10-50ppm streptomysin and 10-25ppm penicillin is poured into each culture dish of dilution and is mixed
It closes, obtains bacteria-containing YPD plate, saccharomycete is counted after -48h for 24 hours is cultivated at 28 DEG C -32 DEG C；Meanwhile taking equivalent
The extension rate is 104、105With 106Dilution be heated to 80 DEG C -85 DEG C and be separately added into culture dish, to being added
It states and pours into LB culture medium in each culture dish of dilution and mixed, obtain bacteria-containing LB plate, cultivated at 28 DEG C -32 DEG C
Bacillus is counted after -48h for 24 hours；In addition, taking the extension rate of equivalent is 104、105With 106Dilution, point
It is not applied on the plate of the MRS culture medium containing 100-500ppm Natamycin, after -48h for 24 hours is cultivated at 36 DEG C -38 DEG C
Lactic acid bacteria is counted.
Wherein, MRS culture medium, YPD culture medium used in the above method and LB culture medium are known in the art
Common culture medium, the record that is available commercially or can be found in for example following document are prepared: Chen Tianshou chief editor, " micro- life
The manufacture and application of object culture medium ", Chinese agriculture publishing house, June nineteen ninety-five the 1st edition；Horse finds pleasure in good equal chief editor, " microculture
Base application manual ", Jilin science tech publishing house, March the 1st edition in 2006, but not limited to this.
In addition, prepare feed suspension and 10 times it is diluted operation be routine operation well known to those skilled in the art.
" pouring into " described herein is that have it is well known in the art that the preparation of definition is for cultivating the plate of bacterium
Then method, operation can add specified culture medium, quickly mix for first a certain amount of bacterium solution is poured into culture dish
Uniformly, it after culture medium solidification, bacterium plate will be contained will be put into incubator and cultivate, and finally visually observe counting.
" coating " described herein is with it is well known in the art that definition is used to cultivate the one of bacterium on plate
Then kind method, operation use spreading rod or coating to be added to a certain amount of bacterium solution on the surface of specified solid medium
Bacterium solution is uniformly smeared in solid culture primary surface and is opened by ring, is put into incubator and is cultivated, and counting is finally visually observed.
In a preferred embodiment, in step 1), by the fermented feed according to the sterile life of w/v 1:9
The feed suspension is made in reason salt water.
In one preferred embodiment, in step 3), the concentration of the Natamycin is 500ppm.Another excellent
In the embodiment of choosing, the concentration of the streptomysin is 50ppm.In another preferred embodiment, the penicillin it is dense
Degree is 10ppm.
It in a preferred embodiment, is 10 by the extension rate in step 3)4、105With 106Dilution heating
To 85 DEG C.In further preferred embodiment, the heating carries out 2-10min, preferably 5min.
In one preferred embodiment, in step 3), the YPD plate is cultivated at 30 DEG C.Another
In one preferred embodiment, the LB plate is cultivated at 30 DEG C.It, will be described in another preferred embodiment
The plate of MRS culture medium is cultivated at 37 DEG C.
In a preferred embodiment, in step 3), the model that the clump count being observed visually is in 30-300 is chosen
LB plate in enclosing counts bacillus.For example, can be further by the equipment auxiliary counting such as microscope.
Embodiment
Below in conjunction with specific embodiment to being described in detail herein.Experimental method used in following embodiments is such as without spy
Different explanation, is conventional method.The materials, reagents and the like used in the following examples, unless otherwise specified, commercially
It obtains.Unless otherwise instructed, whole reagents are purchased from (Beijing extensive and profound in meaning star biotechnology Co., Ltd).
Reagent used in embodiment:
2.5g/L Natamycin prefabricated solution: weighing 0.5g Natamycin, and 200mL distilled water is added to mix, and 121 DEG C of sterilizings 15~
20min is spare.
10g/L ampicillin mother liquor: weighing 10g ampicillin, adds 1L distilled water to mix, through 0.22 μm of sterile film
It filters spare.
10g/L streptomycin stock solution: weighing 10g streptomysin, and 1L distilled water is added to mix, and the non-velum filteration through 0.22 μm is standby
With.
The MRS culture medium of the Natamycin containing 500ppm: 65.25g MRS culture medium, distilled water are settled to 800mL, and 121 DEG C
Sterilize 15~20min, when it is cooled to 45 DEG C~50 DEG C, 200mL2.5g/L Natamycin prefabricated solution is added, makes Natamycin
Final concentration of 500ppm, be sufficiently mixed.
LB culture medium: 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar, distilled water are settled to 1L, and 121
DEG C sterilizing 15~20min.
The YPD solid medium of penicillin containing 10ppm and 50ppm streptomysin: 10g yeast powder, 20g peptone, 20g grape
Sugar, 10g agar, distilled water are settled to 1L, 121 DEG C of 15~20min of sterilizing, are cooled to 45~50 DEG C to it, it is green that 1mL ammonia benzyl is added
Mycin mother liquor and 5mL streptomycin stock solution, making the final concentration of penicillin and streptomysin is respectively 10ppm and 50ppm.
Fermented feed 1:10% (w/v) corn starch residue, 2% (w/v) molasses, 24% (w/v) DDS, 14% (w/v) lemon
Sour grain, 6% (w/v) corn flour, 2% (w/v) DDGS, 40% (w/v) maize peel, 2% (w/v) seed liquor are (to lactic acid bacteria, gemma
Bacillus, saccharomycete, which conventionally activate, to be made), after stirring and evenly mixing, it is placed in 30 DEG C of fermentation 72h-96h.
Fermented feed 2:23% (w/v) sugar residue, 4% (w/v) molasses, 11% (w/v) maize peel, 38% (w/v) the plumule dregs of rice,
2% (w/v) glutamic acid slag, 20% (w/v) distilled water, 2% (w/v) seed liquor (to lactic acid bacteria, bacillus, saccharomycete according to
Conventional method activation is made), after stirring and evenly mixing, it is placed in 30 DEG C of fermentation 72h-96h.
Embodiment 1: the counting of three kinds of strains in fermented feed 1
It weighs 10g fermented feed 1 and is added into 90mL sterile saline and mix, obtain 10-1Sample suspension,
The sample suspension for drawing 1mL carries out 10 times of dilutions using sterile saline, successively obtains dilution 102、103、104、105、106
Sample diluting liquid again.
Drawing 1mL extension rate respectively is 104、105、106Sample diluting liquid and be added separately to 3 diameters be 90mm
The center (the sample diluting liquid setting three for each concentration repeats, totally 9 plates) of sterilized petri dishes, is added into each plate
The MRS culture medium for the Natamycin containing 500ppm that 50 DEG C of 20mL, mixes well, cooling until being inverted in 37 after its solidification completely
About 48h is cultivated in DEG C constant incubator, takes out plate when naked eyes can clearly be seen and separate clear bacterium colony.
Drawing 1mL extension rate respectively is 104、105、106Sample diluting liquid 5min is heated at 85 DEG C, then will be upper
The sample diluting liquid stated is separately added into the center that 3 diameters are 90mm sterilized petri dishes and (sets for the sample diluting liquid of each concentration
Three repetitions are set, totally 9 plates), 50 DEG C of 20mL of LB culture medium is added into each plate, mixes well, it is cooling until its solidification
It after completely, is inverted in 30 DEG C of constant incubators and cultivates about 48h, by plate when naked eyes can clearly be seen and separate clear bacterium colony
It takes out.The plate for selecting macroscopic clump count to be between 30-300 is counted.
Drawing 1mL extension rate respectively is 104、105、106Sample diluting liquid and be added separately to streptomysin containing 50ppm
With in 3 YPD solid medium plates of 10ppm penicillin (the sample diluting liquid setting three for each concentration repeats, totally 9
A plate), media surface is spread evenly across with spreading rod and is done to micro-, is inverted in 30 DEG C of constant incubators and is cultivated about 48h, to
Naked eyes can clearly be seen and take out plate when separating clear bacterium colony.
It is visually observed using microscope (Axio Lab A1) auxiliary to lactic acid bacteria (100 times of oil mirrors), (100 times of bacillus
Oil mirror), saccharomycete (40 times) counted.Most of colony diameter as shown in Fig. 4 A- Fig. 4 C, on MRS culture medium plate
For 1-3mm, rounded, raised, micro white, wet, neat in edge, these bacterium colonies are lactic acid bacteria bacterium colonies；Wherein there is a small amount of bacterium colony
Rough surface, opaque, dirt is white or yellowish, edge is irregular, there is apparent brood-gemma in center, these bacterium colonies are gemma bars
Bacterium bacterium colony.Bacillus has brood-gemma, can distinguish over lactic acid bacteria, assist through microscopy, and the viable count of lactic acid bacteria is 1.8 ×
108Cfu/mL (Fig. 4 A left figure).Major part on LB culture medium plate is bacillus, and viable count is 2.3 × 108cfu/
ML (Fig. 4 B left figure).Bacterium colony on YPD solid medium plate is all smooth, wet, sticky, is easy to provoke, homogeneous,
The color at front and back sides and edge, central part is uniform, mostly milky, is saccharomycete bacterium colony, without other varied bacteria growings, through microscopy
Auxiliary, the viable count of saccharomycete are 2.4 × 108Cfu/mL (Fig. 4 C left figure).
Using National Standard Method [1] GBT13093-2006 to lactic acid bacteria, bacillus and the yeast in same sample diluting liquid
Bacterium is counted, as a result as shown in Fig. 4 A- Fig. 4 C, in MRS culture medium grown on flat dishes saccharomycete and bacillus and sternly
Ghost image rings the counting (Fig. 4 A right figure) of lactic acid bacteria；And on LB culture medium plate, bacillus joins together and covers entire
Thus culture medium can not count (Fig. 4 B right figure)；In YPD solid medium grown on flat dishes bacillus and seriously affect
The counting of saccharomycete can not effectively be counted (Fig. 4 C right figure).Method of the present invention and National Standard Method [1] will be used
The result and colonial morphology remittance that GBT13093-2006 counts lactic acid bacteria, bacillus and the saccharomycete in fermented feed 1
Always in table 1.
The count results of three kinds of strains in 1 fermented feed 1 of table
It can be seen that using method of counting of the invention can reduce lactic acid bacteria, bacillus and saccharomycete to each other it
Between the influence that counts, can be convenient for more accurately carrying out bacterium colony counting.
Embodiment 2: the counting of three kinds of bacterial strains in fermented feed 2
In addition to the fermented feed 1 in embodiment 1 is substituted for fermented feed 2, using method same as Example 1 to cream
Sour bacterium, bacillus and saccharomycete are counted.
As a result as shown in figures 5a-5c, most of bacterium colony on MRS culture medium plate is lactic acid bacteria bacterium colony, and viable count is
1.0×108Cfu/mL (Fig. 5 A left figure).Most of bacterium colony on LB culture medium plate is Bacillus colonies, viable count
It is 3.0 × 108Cfu/mL (Fig. 5 B left figure).Bacterium colony on YPD solid medium plate is saccharomycete bacterium colony, viable count entirely
It is 1.6 × 108Cfu/mL (Fig. 5 C left figure).
Using National Standard Method [1] GBT13093-2006 to lactic acid bacteria, bacillus and the yeast in same sample diluting liquid
Bacterium is counted, as a result, in MRS culture medium grown on flat dishes saccharomycete and bacillus and seriously affects the meter of lactic acid bacteria
Number, clump count mostly can not count (Fig. 5 A right figure)；And on LB culture medium plate, bacillus joins together and covers entire training
Base is supported, (Fig. 5 B right figure) thus can not be counted；In YPD solid medium grown on flat dishes bacillus and influence saccharomycete
Counting, due to the interference of bacillus, bacterium colony joins together, clump count mostly can not count (Fig. 5 C right figure).It will be using the present invention
The method and National Standard Method [1] GBT13093-2006 are to lactic acid bacteria, the bacillus in the sample diluting liquid of fermented feed 2
The result and colonial morphology counted with saccharomycete is summarised in table 2.The counting knot of three kinds of bacterial strains in 2 fermented feed 2 of table
Fruit
It can be seen that using method of counting of the invention can reduce lactic acid bacteria, bacillus and saccharomycete to each other it
Between the influence that counts, can be convenient for more accurately carrying out bacterium colony counting.And the influence of miscellaneous bacteria is avoided, it is more convenient to operate.
In the case where being fully described the present invention, for those of ordinary skills obviously
It is that many changes and improvements can be carried out in the case where not departing from the spirit or scope of invention as described herein.
Claims (10)
1. the method that lactic acid bacteria, bacillus and the saccharomycete in a kind of pair of fermented feed are counted, wherein the method packet
It includes:
1) it takes fermented feed and feed suspension is made with sterile saline according to w/v 1:8-1:10；
2) suspension is successively subjected to 10 times of gradient dilutions with sterile saline, obtaining extension rate is 104、105With 106
Dilution；
3) taking the extension rate is 104、105With 106Dilution and be separately added into culture dish, to the dilution has been added
The YPD culture medium containing 10-50ppm streptomysin and 10-25ppm penicillin is poured into each culture dish of liquid and is mixed, and is obtained
Bacteria-containing YPD plate is obtained, saccharomycete is counted after cultivating 24-48h at 28 DEG C -32 DEG C；Meanwhile taking the described dilute of equivalent
Releasing multiple is 104、105With 106Dilution be heated to 80 DEG C -85 DEG C and be separately added into culture dish, to the dilution has been added
LB culture medium is poured into each culture dish of liquid and is mixed, and is obtained bacteria-containing LB plate, is cultivated 24-48h at 28 DEG C -32 DEG C
Bacillus is counted afterwards；In addition, taking the extension rate of equivalent is 104、105With 106Dilution, be respectively coated
To the plate of the MRS culture medium containing 100-500ppm Natamycin, to lactic acid bacteria after culture 24-48h at 36 DEG C -38 DEG C
It is counted.
2. the fermented feed is used according to w/v 1:9 the method for claim 1, wherein in step 1)
The feed suspension is made in sterile saline.
3. method according to claim 1 or 2, wherein in step 3), the concentration of the Natamycin is 500ppm.
4. method as claimed in any one of claims 1-3, wherein in step 3), the concentration of the streptomysin is
50ppm。
5. such as method of any of claims 1-4, wherein in step 3), the concentration of the penicillin is
10ppm。
6. method according to any one of claims 1 to 5, wherein in step 3), by the YPD plate at 30 DEG C into
Row culture.
7. such as method of any of claims 1-6, wherein be 10 by the extension rate in step 3)4、105
With 106Dilution be heated to 85 DEG C；Preferably, the heating carries out 2-10min, preferably 5min.
8. such as method of any of claims 1-7, wherein in step 3), by the LB plate at 30 DEG C into
Row culture.
9. such as method of any of claims 1-8, wherein in step 3), the plate of the MRS culture medium is existed
It is cultivated at 37 DEG C.
10. method as claimed in any one of claims 1-9 wherein, wherein in step 3), choose the clump count being observed visually
LB plate in 30-300 ranges counts bacillus.
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