CN110777095A - Lactobacillus agent suitable for straw micro-storage and preparation process thereof - Google Patents
Lactobacillus agent suitable for straw micro-storage and preparation process thereof Download PDFInfo
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- 239000010902 straw Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 241000186660 Lactobacillus Species 0.000 title claims abstract description 19
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 19
- 239000003795 chemical substances by application Substances 0.000 title claims description 6
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- 238000004108 freeze drying Methods 0.000 claims abstract description 27
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- 238000000034 method Methods 0.000 claims abstract description 23
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 21
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 21
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 21
- 239000003223 protective agent Substances 0.000 claims abstract description 19
- 241000194031 Enterococcus faecium Species 0.000 claims abstract description 18
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- 239000007787 solid Substances 0.000 claims abstract description 5
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- 235000014655 lactic acid Nutrition 0.000 claims description 15
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- 238000012360 testing method Methods 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
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- 239000002609 medium Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
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- 239000004220 glutamic acid Substances 0.000 claims description 5
- 235000011187 glycerol Nutrition 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
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- 238000007710 freezing Methods 0.000 description 20
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
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- 235000015278 beef Nutrition 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
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- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
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- 239000011702 manganese sulphate Substances 0.000 description 1
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Abstract
The invention discloses a lactobacillus microbial inoculum suitable for straw micro-storage and a preparation process thereof, belonging to the technical field of straw micro-storage in livestock feed. Respectively fermenting and culturing lactobacillus plantarum and enterococcus faecium, and centrifugally collecting bacterial sludge at a high speed, wherein the collected bacterial sludge is mixed according to the ratio of viable count of 2: 1.5-2; the method comprises the following steps of (1) taking skimmed pure milk with the solid content of non-fat milk being more than or equal to 8.5% as a medium to be mixed with a protective agent to form a composite protective agent, and fully mixing the composite protective agent and mixed bacterial sludge of 2 kinds of bacteria according to the volume ratio of 1-1.5: 1; subpackaging the mixed bacterial mud liquid into 2000ml of jellyIn the dry plate, each freeze-drying plate is filled with 800-1000ml of bacterial sludge liquid, pre-frozen for 2h at minus 30 to minus 40 ℃, and then transferred into a freeze dryer and set with specific technological parameters for freeze-drying. The freeze-dried powder prepared by the invention has the survival rate of 90.09 percent and the effective viable count of 4.80 multiplied by 10
11CFU/g。
Description
Technical Field
The invention belongs to the technical field of straw micro-storage in livestock feed, and particularly relates to a lactic acid bacteria microbial inoculum suitable for straw micro-storage and a preparation process thereof.
Background
The micro-storage is a storage mode which converts fermentation substrates in raw materials into acidic substances such as lactic acid and the like through the proliferation of lactic acid bacteria, maintains an acidic anaerobic environment and is beneficial to the long-term storage of micro-storage crops. The micro-storage feed has yellow green color, acid and fragrant smell, softness and juiciness and good palatability, is widely applied to the production of animal husbandry, and becomes an important high-quality coarse feed source particularly in the feeding of ruminants. Compared with the method for airing the forage grass into the hay, the forage grass micro-storage can greatly shorten the time from harvesting to storage, avoid forage grass loss caused by bad weather and furthest preserve the nutrient components of the green forage grass.
At present, about 4 million tons of corn straws can be generated in Heilongjiang province every year, local farmers cannot effectively utilize the straws, the corn straws are mostly burnt or discarded at will, and the treatment mode can cause serious environmental pollution (such as frequent haze) and resource waste. Meanwhile, Heilongjiang is also a big province of livestock raising, and only cattle have about 5 million heads, but the amount of straw which is fed is not only insufficient, but also the quality is not good.
Many facts show that after being fermented by a micro-fermentation method, the corn straws become coarse feed which contains acid and fragrant smell, is lovely eaten by livestock and is easy to digest. Such a treatment method not only solves the above problems to a certain extent, but also brings about great economic benefits. The lactic acid bacteria have important functions in the process of micro-storage fermentation, and can directly influence the quality of the micro-storage feed preparation. Because the quantity of lactobacillus carried by the corn straw is less, the lactobacillus cannot play a role in the micro-storage fermentation process, and the preparation of the micro-storage feed can be seriously influenced, so that the high-quality micro-storage feed can be ensured to be produced only by adding the lactobacillus additionally. Lactic acid bacteria are micro-storage microbial additives which are popularized in recent years, and have the main functions of adjusting the microbial composition in micro-storage materials, quickly becoming dominant flora, competitively inhibiting the growth of harmful microorganisms, quickly converting limited sugar content and obviously reducing the pH value of micro-storage, thereby effectively improving the quality of micro-storage feed and having larger potential and wide development prospect.
The micro-storage lactic acid bacteria additive sold in the market at present is normal-temperature lactic acid bacteria, the optimal growth temperature of the micro-storage lactic acid bacteria is generally higher than 15 ℃, when the temperature is lower than the optimal growth temperature, the micro-storage lactic acid bacteria additive can slow the growth and metabolism, cause the delay and even stagnation of fermentation, and is not suitable for the environmental temperature of the preparation of the corn straw micro-storage feed in Heilongjiang province. And the large-area construction of fermentation workshops or the provision of continuous heating systems has large investment, which is not in accordance with the actual development situation.
Moreover, most of the lactic acid bacteria are supplied to the market in the form of freeze-dried powder, but the lactic acid bacteria are easy to fade and inactivate when being directly subjected to freeze drying, and the stability of the viable bacteria is low. At present, the research and development of micro-storage lactic acid bacteria mainly focuses on single strains such as lactobacillus plantarum and the like, and the freeze-dried powder has low effective viable count and low survival rate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a Lactobacillus microbial inoculum suitable for straw micro-storage, which is composed of freeze-dried mixed powder of Lactobacillus plantarum LP-1522 and enterococcus faecium Ef0518, wherein the Lactobacillus plantarum strain LP-1522 is preserved in the general microbiological center of China Committee for culture Collection of microorganisms (CGMCC) No.10516 in 2015, 2 months and 4 days, and the Lactobacillus plantarum strain is classified and named as Lactobacillus plantarum; the Enterococcus faecium strain Ef0518 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 2018, 05 and 31, the preservation number is CGMCC No.15833, and the taxonomic name is Enterococcus faecalis; the lactobacillus plantarum strain and the enterococcus faecium strain are both deposited at No. 3 of Xilu No.1 on North Chen of the Korean-Yang region in Beijing, and the institute of microbiology of Chinese academy of sciences.
The invention also provides a preparation process of the lactobacillus microbial inoculum suitable for straw micro-storage, which comprises the following steps:
(1) respectively carrying out large-scale culture on lactobacillus plantarum LP-1522 and enterococcus faecium Ef0518 by using a fermentation tank;
(2) separating the 2 kinds of bacteria culture solution by using a centrifugal machine, and collecting bacteria to form bacteria mud;
(3) mixing the lactobacillus plantarum bacterial sludge and the enterococcus faecium bacterial sludge according to the ratio of viable count of 2: 1.5-2;
(4) the method comprises the following steps of (1) taking skimmed pure milk with the solid content of non-fat milk being more than or equal to 8.5% as a medium to be mixed with a protective agent to form a composite protective agent, and fully mixing the composite protective agent and mixed bacterial sludge of 2 kinds of bacteria according to the volume ratio of 1-1.5: 1;
(5) the uniformly mixed bacterial sludge liquid is subpackaged into 2000ml of freeze-drying trays, each freeze-drying tray is subpackaged with 800-;
the components of the composite protective agent except the skimmed milk and the final concentration thereof are respectively 0.4-0.6% Vc, 3.0-5.0% glycerin, 7.0-9.0% sucrose and 4.0-6.0% glutamic acid, and the PH value is adjusted to 7.5;
the freeze-drying technological parameters of the freeze-dryer are as follows: 30min at minus 30 ℃, 1h at minus 20 ℃, 20h at minus 10 ℃, 2.5h at minus 5 ℃, 2.5h at 0 ℃, 1h at 5 ℃, 1h at 10 ℃, 1h at 15 ℃ and 4h at 25 ℃;
further, the large-scale culture of the lactobacillus plantarum in the step (1) comprises the following steps:
the pilot plant was cultured in a 500L fermenter containing 350L of MRS as the initial medium at 0.5Kgf/cm
2Sterilizing for 30min, inoculating when the temperature of the culture medium is lower than 38 ℃, keeping the inoculum size at 0.5%, carrying out stationary culture at 37 ℃, stirring for 5min every 4h for the first 16h in the culture process, stirring once every 2h later, feeding materials for 16h and 24h 2 in the culture process until the total sugar mass concentration is 8.0%, synchronously and directly supplementing the mixed nitrogen source mass concentration to 8.0%, adjusting the pH to 6.0-6.5 by using sodium hydroxide for three times in the culture process, and finishing the culture at 40-41 h.
Further, the large-scale culture of enterococcus faecium in the step (1) comprises the following steps:
the pilot plant test was carried out in a 500L fermenter with a capacity of 350L, MRS medium with a capacity of 350L, at 0.5kgf/cm
2Sterilizing under steam pressure for 30min, inoculating with 0.5% inoculum size when the temperature of the culture medium is reduced to below 40 deg.C, controlling temperature to 36-38 deg.C, standing for culturing, adjusting pH to 6.0-6.8 with 30% sodium hydroxide for 16 hr and 24 hr respectively, and culturing for 32 hr.
Further, the 2 bacterial culture solutions in the step (2) are respectively separated by a tube centrifuge at the rotating speed of 16000r/min, and bacterial sludge is respectively collected.
Further, the composite protective agent in the step (4) is fully mixed with the mixed bacterial sludge of 2 kinds of bacteria according to the volume ratio of 1: 1.
Further, 900ml of bacterial sludge liquid is filled in each freeze-drying tray in the step (5).
Further, the final concentration of each component of the compound protective agent except the skim milk is 0.5% Vc, 4.0% glycerin, 8.0% sucrose and 5.0% glutamic acid, and the pH value is adjusted to 7.5.
Compared with the prior art, the invention has the following beneficial effects:
the preparation process of the lactic acid bacteria agent provided by the invention adopts the steps of mixing lactobacillus plantarum LP-1522 bacterial mud and enterococcus faecium Ef0518 bacterial mud according to a certain proportion to be used as strains, adding a specific composite antifreezing agent and adjusting the pH value, and preparing the freeze-dried powder with higher thallus survival rate and higher effective viable count by pre-freezing and freeze-drying by a freeze-dryer with specific parameters, simplifies the operation steps, and does not need to prepare the freeze-dried powder respectively and then mix the freeze-dried powder.
Drawings
FIG. 1 is a flow chart of the preparation of a lactobacillus preparation.
Detailed Description
Example 1
1. The large-scale culture of the lactobacillus plantarum LP-1522 fermentation tank comprises the following steps:
the MRS culture medium comprises the following components: 10.0g of peptone, 5.0g of beef extract powder, 20.0g of glucose, 4.0g of yeast extract powder and sodium acetate (CH)
3COONa·3H
2O)5.0g, dipotassium hydrogen phosphate (K)
2HPO
4·3H
2O)2.0g, magnesium sulfate (MgSO)
4·7H
2O)0.58g, diammonium citrate 2.0g, Tween 801.0ml, manganese sulfate (MnSO)
4·H
2O)0.25g, distilled water 1000ml, pH 6.2-6.6.
The pilot plant was cultured in a 500L fermenter containing 350L of MRS as the initial medium at 0.5Kgf/cm
2Sterilizing for 30min, inoculating at a culture medium temperature below 38 deg.C, inoculating at 0.5%, standing at 37 deg.C, stirring for 5min every 4h for the first 16h, stirring once every 2h later, feeding to total sugar mass concentration of 8.0% for 16h and 24h 2 times, and synchronously and directly supplementing mixed nitrogen source to mass concentration of 8.0%Percent, sodium hydroxide is used for three times to adjust the pH value to 6.0-6.5 in the culture process, and the culture is finished when the culture lasts for 40-41 hours. Separating the culture solution by a tube centrifuge at 16000r/min, and collecting bacterial sludge. Under the process conditions, the test results of four batches (as shown in Table 1) show that the number of viable bacteria in the fermentation liquor is 5.8 multiplied by 10
9CFU/ml, average yield of thallus 0.63%, residual reducing sugar 1.55%.
TABLE 1500L fermenter production test results
2. The large-scale culture of the enterococcus faecium Ef0518 fermentation tank comprises the following steps:
the pilot plant test was carried out in a 500L fermenter with a capacity of 350L, MRS medium with a capacity of 350L, at 0.5kgf/cm
2Sterilizing under steam pressure for 30min, inoculating with 0.5% inoculum size when the temperature of the culture medium is reduced to below 40 deg.C, controlling temperature to 36-38 deg.C, standing for culturing, adjusting pH to 6.0-6.8 with 30% sodium hydroxide for 16 hr and 24 hr respectively, and culturing for 32 hr.
3. The production of freeze-dried powder products is mainly carried out by depending on fermentation equipment, thallus separation equipment and freeze-drying equipment, the large-scale culture of lactobacillus plantarum and enterococcus faecium is already finished, and a fermentation tank with the volume of 500L is utilized to successfully produce a high-density liquid microbial inoculum. The continuous centrifugation is carried out by a high-speed tubular centrifuge at the rotation speed of 10000r/min and the flow rate of 150L/hr (table 2), the recovery rates of the collected thalli, the lactobacillus plantarum and the enterococcus faecium respectively reach 88.63 percent and 89.30 percent, the water content of the collected bacterial sludge is below 65 percent, the yield of the thalli is respectively 0.63 percent and 0.61 percent, and the method lays a foundation for the freeze-drying of the thalli.
TABLE 2 production test results of bacterial body (bacterial sludge) collection in 500L fermentation tank
4. Pre-freezing of thallus and freeze-drying by freeze-dryer
Mixing the lactobacillus plantarum bacterial sludge and the enterococcus faecium bacterial sludge according to the ratio of viable count of 2:1.5-2, mixing the degreased pure milk with the solid content of non-fat milk being more than or equal to 8.5% as a medium with a protective agent to form a composite protective agent, and fully mixing the composite protective agent and the mixed bacterial sludge of 2 bacteria according to the volume ratio of 1-1.5: 1; and (3) subpackaging the uniformly mixed bacterial sludge liquid into 2000ml of freeze-drying discs, subpackaging 800 plus 1000ml of bacterial sludge liquid into each freeze-drying disc, pre-freezing for 2h at-30 to-40 ℃, and then transferring into a freeze dryer for freeze-drying.
The final concentration of each component of the compound protective agent except the skimmed milk is 0.5% Vc, 4.0% glycerol, 8.0% sucrose and 5.0% glutamic acid, and the PH value is adjusted to 7.5.
Freezing has a certain destructive effect on cells and living bodies, the mechanism of the freezing is very complex, and no unified theory exists at present, but the freezing is mainly caused by mechanical effect and solute effect. The freezing process of the biomass starts with the freezing of pure water, the growth of ice crystals gradually causes the concentration of the electrolyte, the solidification of the eutectic mixture follows, and finally the complete change to solid. The mechanical effect is caused by mechanical force generated by the growth of ice crystals inside and outside cells, particularly, the influence on life bodies with cell membranes is large, and generally, the cell membranes are easy to break when the ice crystals are larger, so that the cells die; the ice crystal is small, and the mechanical damage to the cell membrane is small. The ice crystals produced by slow freezing are larger, and the ice crystals produced by fast freezing are smaller. In this regard, rapid freezing has less effect on cells, and slow freezing more easily causes cell death. Before freeze drying, the product needs to be put into a proper container for pre-freezing, and then sublimation drying can be carried out. The pre-freezing process is not only to protect the main properties of the substance from change, but also to obtain a product with a reasonable structure after freezing to facilitate the sublimation of water. Therefore, pre-freezing of the sample is necessary. Although the liquid sample in the freeze-drying bottle can be frozen under the pre-freezing condition, the freezing time is correspondingly prolonged along with the increase of the temperature, the actual freeze-drying effect is greatly different, and the appearance form of the sample is extremely poor at the temperature of-5 ℃; the material sample has defects at the temperature of minus 20 ℃; the material sample is neat at minus 30 ℃ and minus 40 ℃, which is a more suitable pre-freezing condition.
Freeze drying is achieved by sublimation of ice, the rate of water vapor overflowing from the material layer determines the freeze drying rate, the resistance to water vapor overflow comes from many aspects, such as product type, components, concentration, protective agents and the like, and the thickness of the material layer is certainly one of important influencing factors, theoretically, the thinner the material stacking thickness is, the faster the heat and mass transfer is, and the shorter the drying time is. The liquid filling amount in the freeze-drying bottle with the same specification determines the thickness of a material layer during drying, and correspondingly influences the freeze-drying speed.
After pre-freezing, lyophilization was performed according to the lyophilizer process parameters shown in table 3.
TABLE 3 Freeze dryer Process parameters
| Segment of | Run time | Setting temperature of |
| 1 | 30min | -30 |
| 2 | 60min | -20 |
| 3 | 20hr | -10 |
| 4 | 2h30min | -5 |
| 5 | 2h30min | 0 |
| 6 | 60min | 5 |
| 7 | 60min | 10 |
| 8 | 60min | 15 |
| 9 | 4h | 25 |
| Predicted total time | 33hr 30min |
5. Inspection results of lyophilized powder products
The production test of the freeze-dried powder product is carried out by adopting the established process flow and process parameters. The total effective viable count is detected by semisolid agar test tube method, the survival rate of the thallus before and after freeze-drying reaches 90.09%, and the effective viable count is 4.80 multiplied by 10
11CFU/g, the sanitation index of the product reaches the requirement of the feed sanitation standard (GB 13078).
TABLE 4 test results of lyophilized powder product according to feed hygiene Standard (GB 13078)
| Serial number | Measurement items | Allowance (minimum) ① | Actual measurement results |
| 1 | The rate of mixed bacteria% | ≤1.0 | -- |
| 2 | Aflatoxins B 1μg/kg | ≤10.0 | ≤0.1 |
| 3 | Arsenic (in terms of total As) mg/kg | ≤2.0 | ≤0.04 |
| 4 | Lead (in terms of Pb) mg/kg | ≤5.0 | 0.02 |
| 5 | Mercury (in Hg) mg/kg | ≤0.1 | 0.01 |
| 6 | Cadmium (in Cd) mg/kg | ≤0.5 | 0.01 |
| 7 | Coliform bacteria/kg | ≤1.0×10 5 | Not detected out |
| 8 | Total number of moulds/kg | ≤2.0×10 7 | Not detected out |
| 9 | Salmonella | Cannot be detected | Not detected out |
⑴ standard for sanitary fodder (GB 13078) does not provide clear indexes for microbial fodder additives, ⑵ in the table "standard requirement" lists the minimum value in the standard, ⑶ standard, and "when the ratio of concentrated fodder and additive premixed fodder is different from the standard remark, the allowable amount of sanitary indexes can be converted".
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solution of the present invention is defined by the claims.
Claims (9)
1. The lactobacillus agent suitable for micro-storage of straws is characterized in that the lactobacillus agent is mixed freeze-dried powder of lactobacillus plantarum and enterococcus faecium, strains of the lactobacillus plantarum are preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) 2, 4 and 2015, and the preservation number is CGMCC No. 10516; the enterococcus faecium strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 05 and 31, with the preservation number of CGMCC No. 15833; the lactobacillus plantarum strain and the enterococcus faecium strain are both deposited at No. 3 of Xilu No.1 on North Chen of the Korean-Yang region in Beijing, and the institute of microbiology of Chinese academy of sciences.
2. The lactobacillus preparation suitable for straw micro-storage according to claim 1, wherein the effective viable count of the lactobacillus preparation is 4.80 x 10
11CFU/g。
3. The lactic acid bacteria microbial inoculum suitable for straw micro-storage according to claim 1 or 2, characterized in that the preparation process of the lactic acid bacteria microbial inoculum comprises the following steps:
(1) respectively carrying out large-scale culture on lactobacillus plantarum and enterococcus faecium by using a fermentation tank;
(2) separating the 2 kinds of bacteria culture solution by using a centrifugal machine, and collecting bacteria to form bacteria mud;
(3) mixing the lactobacillus plantarum bacterial sludge and the enterococcus faecium bacterial sludge according to the ratio of viable count of 2: 1.5-2;
(4) the method comprises the following steps of (1) taking skimmed pure milk with the solid content of non-fat milk being more than or equal to 8.5% as a medium to be mixed with a protective agent to form a composite protective agent, and fully mixing the composite protective agent and mixed bacterial sludge of 2 kinds of bacteria according to the volume ratio of 1-1.5: 1;
(5) the uniformly mixed bacterial sludge liquid is subpackaged into 2000ml of freeze-drying trays, each freeze-drying tray is subpackaged with 800-;
the components of the composite protective agent except the skimmed milk and the final concentration thereof are respectively 0.4-0.6% Vc, 3.0-5.0% glycerin, 7.0-9.0% sucrose and 4.0-6.0% glutamic acid, and the PH value is adjusted to 7.5;
the freeze-drying technological parameters of the freeze-dryer are as follows: 30min at minus 30 ℃, 1h at minus 20 ℃, 20h at minus 10 ℃, 2.5h at minus 5 ℃, 2.5h at 0 ℃, 1h at 5 ℃, 1h at 10 ℃, 1h at 15 ℃ and 4h at 25 ℃.
4. A lactobacillus preparation suitable for straw micro-storage according to claim 3, wherein the large-scale culture of Lactobacillus plantarum in the step (1) comprises the following steps:
the pilot plant was cultured in a 500L fermenter containing 350L of MRS as the initial medium at 0.5Kgf/cm
2Sterilizing for 30min, inoculating when the temperature of the culture medium is lower than 38 ℃, keeping the inoculum size at 0.5%, carrying out stationary culture at 37 ℃, stirring for 5min every 4h for the first 16h in the culture process, stirring once every 2h later, feeding materials for 16h and 24h 2 in the culture process until the total sugar mass concentration is 8.0%, synchronously and directly supplementing the mixed nitrogen source mass concentration to 8.0%, adjusting the pH to 6.0-6.5 by using sodium hydroxide for three times in the culture process, and finishing the culture at 40-41 h.
5. The lactobacillus preparation suitable for straw micro-storage according to claim 3, wherein the large-scale culture of enterococcus faecium in the step (1) comprises the following steps:
the pilot plant test was carried out in a 500L fermenter with a capacity of 350L, MRS medium with a capacity of 350L, at 0.5kgf/cm
2Sterilizing under steam pressure for 30min, inoculating with 0.5% inoculum size when the temperature of the culture medium is reduced to below 40 deg.C, controlling temperature to 36-38 deg.C, standing for culturing, adjusting pH to 6.0-6.8 with 30% sodium hydroxide for 16 hr and 24 hr respectively, and culturing for 32 hr.
6. A lactobacillus preparation suitable for straw micro-storage according to claim 3, wherein the 2 kinds of bacteria culture solution in the step (2) are separated by a tube centrifuge respectively, the rotating speed is 16000r/min, and bacterial sludge is collected respectively.
7. A lactobacillus preparation suitable for straw micro-storage according to claim 3, wherein the composite protectant in step (4) is fully mixed with the mixed bacterial sludge of 2 bacteria according to the volume ratio of 1: 1.
8. A lactobacillus preparation suitable for straw micro-storage according to claim 3, wherein 900ml of bacterial sludge liquid is filled in each freeze-drying tray in the step (5).
9. A lactobacillus preparation suitable for straw micro-storage according to claim 3, wherein the final concentrations of the components of the compound protective agent except skimmed milk are 0.5% Vc, 4.0% glycerol, 8.0% sucrose and 5.0% glutamic acid respectively, and the pH value is adjusted to 7.5.
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