CN112175858B - Solid culture medium for lactobacillus plantarum fermentation, lactobacillus plantarum solid microbial inoculum and preparation method and application thereof - Google Patents
Solid culture medium for lactobacillus plantarum fermentation, lactobacillus plantarum solid microbial inoculum and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biological inocula, and discloses a solid culture medium for lactobacillus plantarum fermentation, a lactobacillus plantarum solid inocula, and a preparation method and application thereof. The solid culture medium for lactobacillus plantarum fermentation is prepared from raw materials including a starch raw material crushed product, starch residues, carbonate, yeast extract powder and saccharides. The invention also provides a preparation method of the lactobacillus plantarum solid microbial inoculum, which comprises the following steps: inoculating lactobacillus plantarum strain into the solid culture medium for fermentation. The solid culture medium is used for producing the lactobacillus plantarum solid microbial inoculum by a solid fermentation method, the prepared lactobacillus plantarum solid microbial inoculum is full and strong, the effective viable count is more than 500 hundred million cfu/g, and the variation coefficient of the mixing uniformity is less than 5%; the invention has the advantages of few operation steps, low rate of mixed bacteria, improved product quality, saved equipment and energy consumption, and greatly reduced waste water generation, thereby reducing the environmental burden.
Description
Technical Field
The invention relates to the field of microbial agents, and particularly relates to a solid culture medium for lactobacillus plantarum fermentation, a lactobacillus plantarum solid microbial agent, and a preparation method and application thereof.
Background
With the popularization of the application range of fermented feed, the improvement of the yield of the fermented feed is urgent, however, the preparation of the liquid microbial inoculum becomes a key factor for restricting the yield of the fermented feed due to the complex preparation process and short storage period of the liquid microbial inoculum. The solid microbial inoculum has long storage period, is convenient to transport and store, can solve the limitations of production equipment, technology, manpower and the like, and becomes an important factor for stabilizing and improving the product quality and the yield, so the application of the solid microbial inoculum to fermented feed is a breakthrough development, and the development of the solid microbial inoculum has wide market potential.
The preparation of the solid microbial inoculum at present generally comprises the steps of preparing seed liquid, collecting thalli, adding a carrier, drying and the like, but the operation flow is complex, and the problems of waste water generation, low effective viable count of the microbial inoculum, high variation coefficient of mixing uniformity, easy infectious microbes and the like exist in the preparation process.
Disclosure of Invention
The invention aims to overcome the problems in the prior art, and provides a solid culture medium for lactobacillus plantarum fermentation and a method for preparing a lactobacillus plantarum solid microbial inoculum by adopting the culture medium, so that the operation process is less, wastewater is not generated basically, and equipment and cost are saved; the prepared lactobacillus plantarum solid microbial inoculum has the purposes of high effective viable count, low product mixed bacteria rate and probiotic mixing uniformity variation coefficient of less than 5 percent.
In order to achieve the above objects, a first aspect of the present invention provides a solid medium for lactobacillus plantarum fermentation, wherein the solid medium is prepared from the following raw materials: pulverized starchy material, starch residue, carbonate, yeast extract powder and saccharide.
The second aspect of the invention provides a preparation method of a lactobacillus plantarum solid microbial inoculum, which comprises the following steps: the lactobacillus plantarum strain was inoculated into the solid medium as described above for fermentation.
Preferably, the method of vaccination comprises: and mixing the seed solution of the lactobacillus plantarum with water to obtain a seed inoculation solution, and then uniformly mixing the seed inoculation solution with the solid culture medium to obtain a fermentation starting material.
Preferably, the inoculation liquid is mixed with the solid culture medium in a spraying manner.
The third aspect of the invention provides the lactobacillus plantarum solid microbial inoculum prepared by the preparation method of the lactobacillus plantarum solid microbial inoculum.
The fourth aspect of the invention provides the application of the lactobacillus plantarum solid microbial inoculum in preparation of animal feed.
Through the technical scheme, the solid culture medium for lactobacillus plantarum fermentation is used for producing the lactobacillus plantarum solid microbial inoculum through a solid fermentation method, so that the microbial inoculum preparation process is simplified, the equipment and energy consumption are reduced, the generation of wastewater is reduced, and the environmental burden is reduced. The lactobacillus plantarum solid microbial inoculum prepared by the method has the advantages of higher effective viable count, fuller and stronger thallus, low mixed bacteria rate and probiotic mixing uniformity variation coefficient of less than 5%.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In the present invention, the pulverized product of a starchy material refers to a product obtained by directly pulverizing a starchy material, such as a pulverized product of a seed or a rhizome containing starch. For example, corn flour refers to a product obtained by directly pulverizing corn seeds, and sweet potato flour refers to a product obtained by directly pulverizing sweet potato roots.
In the invention, the starch residue refers to a byproduct or leftover obtained in the process of producing starch from a starchy raw material. For example, corn grits refer to by-products or offal from the production of corn starch.
In a first aspect, the present invention provides a solid medium for lactobacillus plantarum fermentation, wherein the solid medium is prepared from the following raw materials: pulverized starchy material, starch residue, carbonate, yeast extract powder and saccharide.
In the present invention, the mass ratio of the pulverized product of the starchy material, the starch residue, the carbonate, the yeast extract powder and the saccharide can be selected from a wide range. Preferably, in the raw materials for preparing the solid culture medium, the mass ratio of the starchy raw material crushed product, the starch residue, the carbonate, the yeast extract powder and the saccharide can be (20-30): (20-30): (1-10): (1-6): (1-5); more preferably, the mass ratio of the starchy raw material crushed product, the starch residue, the carbonate, the yeast extract powder and the saccharides is (23-27): (23-27): (3-7): (3-5): (2-4).
In the present invention, the starchy material may be conventional various high starch-containing materials, such as but not limited to one or more selected from wheat, corn, barley, sweet potato, and combinations thereof, more preferably corn.
In the present invention, the raw material for preparing the starch residue may be conventional various raw materials with high starch content, for example, but not limited to, one or more selected from wheat, corn, barley, sweet potato, potato and combinations thereof, and more preferably corn.
The inventor finds that the starchy raw material and the raw material for preparing the starch residue are corn, and the solid-state fermentation is beneficial to the growth of thalli, particularly, the growth speed is high, the thalli are stout, the mixed bacteria rate is lower, the number of finally obtained effective live bacteria is more, and the variation coefficient of the mixing uniformity of the probiotics is smaller.
In the present invention, the carbonate may be various conventional carbonates, for example, but not limited to, one or more selected from magnesium carbonate, calcium carbonate, sodium carbonate and a combination thereof, and is more preferably calcium carbonate.
In the present invention, the saccharide may be any of various saccharides conventionally used for lactobacillus plantarum culture, for example, but not limited to, one or more selected from sucrose, glucose, fructose, maltose and a combination thereof, and further preferably glucose.
In the present invention, the solid medium may further include water, and the water may be added in any form, for example, directly to the solid medium, mixed with the lactobacillus plantarum strain, and added to the solid medium, or a combination of both methods. When the water is added directly to the solid medium, the water may be sterile water or sterile water; when the water is mixed with the lactobacillus plantarum thalli, the water is sterile water. The amount of the water added is based on the degree of wetting of the solid medium, and specifically, the water may be 20 to 40 parts by weight with respect to 100 parts by weight of the solid medium.
In the present invention, the method for preparing the solid medium is not particularly limited, and can be prepared using a method that is conventional in the art. Preferably, the components of the solid medium are uniformly mixed together, the order of addition of the components is not particularly limited, and the solid medium may be either aqueous or non-aqueous, and then subjected to a sterilization treatment. In the present invention, the solid medium may be sterilized or sterilized by a means conventional in the art, and for example, dry heat sterilization, ozone sterilization, moist heat sterilization, irradiation sterilization, ultra-high pressure sterilization, microwave sterilization, and the like may be mentioned. After the sterilization is finished, the lactobacillus plantarum strain is inoculated into the solid culture medium when the solid culture medium reaches the condition of being inoculated. In the case of moist heat sterilization, inoculation may be carried out after the temperature is lowered to room temperature after sterilization.
The second aspect of the invention provides a preparation method of a lactobacillus plantarum solid microbial inoculum, which comprises the following steps: inoculating lactobacillus plantarum strain into the solid culture medium for fermentation.
In the present invention, the activated lactobacillus plantarum may be inoculated into the solid medium as described above by means of a technique that is conventional in the art. Preferably, the method of inoculation may comprise: and mixing the seed solution of the lactobacillus plantarum with water to obtain a inoculation solution, and then uniformly mixing the inoculation solution with the solid culture medium to obtain a fermentation starting material. Preferably, the inoculation liquid is mixed with the solid culture medium in a spraying manner.
In the present invention, the amount of the Lactobacillus plantarum strain can be selected within a wide range, preferably 1X 10, per gram of the solid medium 5 -1×10 9 cfu, more preferably 1X 10 6 -1× 10 8 cfu。
In the present invention, the lactobacillus plantarum strain may be obtained by means of a conventional technique in the art, and may be, for example, a seed solution obtained by liquid fermentation, a cell collected from the seed solution, or a bacterial suspension obtained by diluting the collected cell to a certain concentration.
In the present invention, conditions for performing fermentation in the solid medium, such as fermentation temperature and fermentation time, etc., may not be particularly limited and may be selected within the conditions conventional in the art. Preferably, during the fermentation, the temperature of the fermentation is 28-38 ℃, more preferably 33-36 ℃; the fermentation time is 2-8 days, more preferably 3-7 days.
In the present invention, the lactobacillus plantarum may be fermented in a manner conventional in the art, and for example, may be anaerobic fermentation or facultative anaerobic fermentation. Preferably, the fermentation mode is anaerobic fermentation.
Preferably, the anaerobic fermentation environment can be obtained by filling the fermentation starting material into a fermentation bag, exhausting gas, and sealing by welding.
Wherein, the vacuum packaging machine can be adopted to automatically pump out the air in the fermentation bag for exhausting, and the welding of the bag is completed after the preset vacuum degree is reached.
In the present invention, the method may further comprise drying the fermentation product, wherein the drying process may be performed by a conventional technique in the art, such as drying at room temperature or freeze drying. Preferably, the drying treatment may be a freeze-drying treatment. The freeze-drying process may be carried out by any means conventional in the art, for example, by freeze-drying with a freeze-dryer. Preferably, the freeze-drying conditions comprise: the temperature is-40 deg.C to-60 deg.C, and the time is 60-150min.
In the present invention, in order to further improve the shelf life of the resulting inoculant and reduce the probiotic mixing uniformity coefficient of variation, the method further comprises mixing the fermentation product with a protective agent, which may be a protective agent conventional in the art, such as glycerol and/or trehalose, prior to freeze-drying. Preferably, the protective agent may be glycerol.
Preferably, the protective agent is glycerol, and the glycerol may be used in an amount of 3 to 10 parts by weight, and more preferably 4 to 6 parts by weight, relative to 100 parts by weight of the fermentation product.
In the present invention, the method may further comprise subjecting the fermentation product to a pulverization treatment, which may be carried out by a means conventional in the art, for example, a pulverization treatment using a solid pulverizer. The fermentation product may be subjected to a single or multiple pulverization treatments as required.
Preferably, the particle size of the fermentation product after the drying and pulverizing treatment may be less than 2mm, preferably 0.001-1mm.
In the present invention, in order to obtain a more uniform fermentation product, the pulverized material may be sieved, and then the oversize product may be pulverized again to obtain a desired particle size, and then the pulverized oversize product powder may be mixed with the undersize product. Wherein, the oversize product refers to the fermentation product in the sieve after sieving, and the undersize product refers to the fermentation product which is sieved because the particle size is smaller than the sieve pore after sieving. The size of the screen is selected so that the undersize can reach the desired particle size, preferably the screen size is 10-100 mesh, more preferably 16-25 mesh. The sieving treatment can be combined with the steps of freeze-drying, crushing and the like, and can be carried out once or for multiple times so as to achieve the level with lower variation coefficient of mixing uniformity.
In a third aspect, the invention also provides a lactobacillus plantarum solid microbial inoculum prepared by the method.
In a fourth aspect, the invention also provides an application of the lactobacillus plantarum solid microbial inoculum in fermented feed.
Examples
The following detailed description is presented in conjunction with specific examples. The advantages and features of the present invention will become more apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents, equipment and the like used in the following examples are commercially available unless otherwise specified.
In the following examples and comparative examples:
lactobacillus plantarum (Lactobacillus plantarum CGMCC 15013, china general microbiological culture Collection center)
First-stage seed culture medium: peptone 1 wt%, beef powder 1 wt%, yeast extract 5.5 wt%, magnesium sulfate 0.01 wt%, sodium acetate 0.5 wt%, triammonium citrate 0.2 wt%, dipotassium hydrogen phosphate 0.2 wt%, manganese sulfate 0.005 wt%, tween 80.1 wt%, glucose 2 wt%, and water in balance, and adjusting the pH to 7.2 + -0.2.
Secondary seed culture medium: 4 wt% of yeast extract powder, 0.02 wt% of magnesium sulfate, 0.5 wt% of sodium acetate, 0.2 wt% of triammonium citrate, 0.2 wt% of dipotassium hydrogen phosphate, 0.005 wt% of manganese sulfate, 0.1 wt% of Tween 80,3 wt% of glucose and the balance of water, and the pH value is adjusted to 7.2 +/-0.2.
Expanding culture medium: 2.5 percent by weight of corn steep liquor, 0.8 percent by weight of molasses, 1.5 percent by weight of glucose, 0.5 percent by weight of sodium acetate, 0.02 percent by weight of magnesium sulfate, 0.005 percent by weight of manganese sulfate, 0.6 percent by weight of beef extract, 0.2 percent by weight of dipotassium hydrogen phosphate, 0.2 percent by weight of triammonium citrate, 0.10 percent by weight of Tween and the balance of water, wherein the pH value is 6.2 +/-0.2.
The Coefficient of Variation (CV), also known as the dispersion coefficient, is defined as the ratio of the standard deviation to the average, i.e., CV = standard deviation divided by average.
Coefficient of variation of mixing uniformity: expressed as coefficient of variation CV%, i.e., CV% = standard deviation ÷ mean × 100%, the larger the CV, the worse the mixing uniformity. At least 10 representative samples are taken from each batch of feed, and the effective viable count of the lactobacillus plantarum is determined in each sample and is respectively X 1 、X 2 、X 3 、 X 4 、X 5 、X 6 、X 7 、X 8 、X 9 、X 10 And calculating the average value X and the standard deviation S (refer to GB/T5918-2008 'determination of feed product mixing uniformity').
The mixed bacteria rate is the percentage of the total bacteria number of the mixed bacteria number in the lactobacillus plantarum solid microbial inoculum, wherein bacteria except the target bacteria are regarded as the mixed bacteria.
The effective viable count refers to the number of lactobacillus plantarum in lactobacillus plantarum solid microbial inoculum, and the determination method refers to GB 4789.35-2016 lactic acid bacteria inspection for food safety national standard food microbiology inspection.
The quality guarantee period refers to the time that the effective viable count still meets the standard requirement after the product is placed under certain conditions. In the embodiment of the invention, the effective viable count is more than 200 hundred million, namely in the shelf life.
Preparation example
This preparation example was used to prepare a Lactobacillus plantarum seed liquid used in the following examples
Sterilizing the first-stage seed culture medium at 121 deg.C for 20min, and cooling to room temperature; and (3) sucking 0.5 vol% of lactobacillus plantarum bacterial liquid from a freezing storage tube, inoculating the lactobacillus plantarum bacterial liquid into 200ml of the primary seed culture medium, and performing static culture at 35 ℃ for 15h to obtain primary seed liquid. The number of colonies was 100 hundred million cfu/ml.
Sterilizing the secondary seed culture medium at 121 deg.C for 20min, and cooling to room temperature; and (3) sucking 2 volume percent of bacterial liquid from the primary seed liquid, inoculating the bacterial liquid into a secondary seed culture medium, stirring once every 5 hours at the rotating speed of 10rpm, and culturing at 35 ℃ for 18 hours to obtain a secondary seed liquid. The number of colonies was 200 hundred million cfu/ml.
Sterilizing the culture medium at 121 deg.C for 20min, and cooling to room temperature; and (3) absorbing 5 volume percent of bacterial liquid from the secondary seed liquid, inoculating the bacterial liquid into an expanding culture medium, and standing and culturing for 18 hours at 30 ℃ to obtain fermentation liquid. The number of colonies was 200 hundred million cfu/ml.
Example 1
This example is provided to illustrate the lactobacillus plantarum solid preparation and the method for its preparation.
And (3) uniformly mixing 250g of corn flour, 250g of corn starch residue, 50g of calcium carbonate, 40g of yeast extract powder and 30g of glucose, and sterilizing in an autoclave at 121 ℃ for 20min to obtain the dry component of the solid culture medium. And taking 50ml of diluted secondary seed liquid, adding 330ml of sterile water, uniformly mixing, spraying into the dry components of the solid culture medium, and continuously and uniformly mixing to obtain a fermentation initiator. Wherein the secondary seed liquid is added in an amount such that the viable count in the solid culture medium is 1 × 10 7 cfu/g; filling the fermentation starting material into a fermentation bag, exhausting, welding and sealing, and fermenting at 35 deg.C for 6 days.
Adding 50g of glycerol into the lkg of fermented materials, uniformly mixing, freeze-drying at-50 ℃ for 120min, crushing, sieving with a 20-mesh sieve, repeating the steps of freeze-drying, crushing and sieving on oversize products, and finally, sieving all the materials with the 20-mesh sieve to obtain the solid microbial inoculum.
The effective viable count, the mixed bacteria rate, the variation Coefficient (CV) of the mixing uniformity and the shelf life of the solid microbial inoculum are measured, and the results are shown in table 1.
Example 2
This example is provided to illustrate the lactobacillus plantarum solid preparation and the method for its preparation.
Uniformly mixing 270g of corn flour, 230g of corn starch residue, 70g of calcium carbonate, 30g of yeast extract powder and 20g of glucose, and sterilizing in an autoclave at 121 ℃ for 20min to obtain a solid culture medium dry component; taking 50mL of diluted secondary seed liquid, adding 330mL of sterile water, uniformly mixing, spraying into the dry components of the solid culture medium, and continuously mixingAnd uniformly mixing to obtain a fermentation starting material. Wherein the secondary seed liquid is added in an amount such that the viable count in the solid culture medium is 1 × 10 6 cfu/g; filling the fermentation starting material into a fermentation bag, exhausting, welding and sealing, and fermenting at 35 deg.C for 6 days.
Adding 60g of glycerol into the lkg of fermented materials, uniformly mixing, freeze-drying at-60 ℃ for 90min, crushing, sieving by a 16-mesh sieve, repeating the steps of freeze-drying, crushing and sieving on oversize products, and finally, enabling all the materials to pass through the 16-mesh sieve to obtain the solid microbial inoculum.
The effective viable count, the mixed bacteria rate, the variation Coefficient (CV) of the mixing uniformity and the shelf life of the solid microbial inoculum are measured, and the results are shown in table 1.
Example 3
This example is provided to illustrate the lactobacillus plantarum solid preparation and the method for its preparation.
Uniformly mixing 230g of corn flour, 270g of corn starch residue, 30g of calcium carbonate, 50g of yeast extract powder and 40g of glucose, and sterilizing in an autoclave at 121 ℃ for 20min to obtain a dry component of a solid culture medium; and taking 50ml of diluted secondary seed liquid, adding 330ml of sterile water, uniformly mixing, spraying into the dry components of the solid culture medium, and continuously and uniformly mixing to obtain a fermentation initiator. Wherein the secondary seed liquid is added in an amount such that the viable count in the solid culture medium is 1 × 10 8 cfu/g; filling the fermentation starting material into a fermentation bag, exhausting, welding and sealing, and fermenting at 35 deg.C for 6 days.
Adding 40g of glycerol into the lkg of fermented materials, uniformly mixing, freeze-drying at the temperature of-40 ℃ for 150min, crushing, sieving with a 25-mesh sieve, repeating the steps of freeze-drying, crushing and sieving on oversize products, and finally, sieving all the materials with the 25-mesh sieve to obtain the solid microbial inoculum.
The effective viable count, the mixed bacteria rate, the variation Coefficient (CV) of the mixing uniformity and the shelf life of the solid microbial inoculum are measured, and the results are shown in table 1.
Example 4
This example is provided to illustrate the lactobacillus plantarum solid bacterial agent and the preparation method thereof according to the present invention.
Mixing 300g corn flour, 200g corn starch residue, 10g calcium carbonate, 60g yeast extract powder and 50g glucoseMixing, sterilizing at 121 deg.C for 20min in autoclave to obtain solid culture medium dry component; and taking 50ml of diluted secondary seed liquid, adding 330ml of sterile water, uniformly mixing, spraying into the dry components of the solid culture medium, and continuously and uniformly mixing to obtain a fermentation initiator. Wherein the secondary seed liquid is added in an amount such that the viable count in the solid culture medium is 1 × 10 5 cfu/g; filling the fermentation starting material into a fermentation bag, exhausting, welding and sealing, and fermenting at 35 deg.C for 6 days.
Adding 30g of glycerol into the lkg of fermented materials, uniformly mixing, freeze-drying at the temperature of minus 40 ℃ for 150min, crushing, sieving by a 10-mesh sieve, repeating the steps of freeze-drying, crushing and sieving on oversize products, and finally, sieving all the materials by the 10-mesh sieve to obtain the solid microbial inoculum.
The effective viable count, the mixed bacteria rate, the variation Coefficient (CV) of the mixing uniformity and the quality guarantee period of the solid microbial inoculum are measured, and the results are shown in table 1.
Example 5
This example is provided to illustrate the lactobacillus plantarum solid preparation and the method for its preparation.
Uniformly mixing 200g of corn flour, 300g of corn starch residue, 100g of calcium carbonate, 10g of yeast extract powder and 10g of glucose; sterilizing in autoclave at 121 deg.C for 20min to obtain solid culture medium dry component. And taking 50ml of diluted secondary seed liquid, adding 330ml of sterile water, uniformly mixing, spraying into the dry components of the solid culture medium, and continuously and uniformly mixing to obtain a fermentation initiator. Wherein the secondary seed liquid is added in an amount such that the viable count in the solid culture medium is 1 × 10 9 cfu/g; the fermentation starting material is filled into a fermentation bag, sealed by welding after exhausting, and fermented for 6 days at 35 ℃.
Adding 100g of glycerol into the lkg of fermented materials, uniformly mixing, freeze-drying at-60 ℃ for 60min, crushing, sieving by using a 100-mesh sieve, repeating the steps of freeze-drying, crushing and sieving on oversize products, and finally passing all the materials through the 100-mesh sieve to obtain the solid microbial inoculum.
The effective viable count, the mixed bacteria rate, the variation Coefficient (CV) of the mixing uniformity and the shelf life of the solid microbial inoculum are measured, and the results are shown in table 1.
Example 6
This example is provided to illustrate the lactobacillus plantarum solid bacterial agent and the preparation method thereof according to the present invention.
Example 6-1: this example was carried out as in example 1, except that the corn flour was replaced by an equal amount of wheat flour and the corn starch residue was replaced by an equal amount of wheat starch residue.
Example 6-2: this example was carried out as in example 1, except that the corn flour was replaced by an equal amount of wheat flour.
Examples 6 to 3: this example was carried out as in example 1, except that the corn starch residue was replaced with an equal amount of wheat starch residue.
The effective viable count, the mixed bacteria rate, the variation Coefficient (CV) of the mixing uniformity and the shelf life of the solid microbial inoculum are measured, and the results are shown in table 1.
Comparative example 1
This comparative example is used to illustrate the effect of solid medium composition on lactobacillus plantarum solid inoculum.
Comparative examples 1-1: the conditions of this comparative example were the same as example 1, except that the solid medium contained 500g of corn flour, 50g of calcium carbonate, 40g of yeast extract powder, and 30g of glucose.
Comparative examples 1 to 2: the conditions of this comparative example were the same as example 1 except that the solid medium contained 500g of corn starch residue, 50g of calcium carbonate, 40g of yeast extract powder, and 30g of glucose.
Comparative examples 1 to 3: the conditions of this comparative example were the same as example 1, except that the solid medium contained 270g of corn flour, 270g of corn starch residue, 40g of yeast extract powder, and 40g of glucose.
Comparative examples 1 to 4: the conditions of this comparative example were the same as example 1 except that the solid medium contained 260g of corn flour, 260g of corn starch residue, 60g of calcium carbonate, and 40g of glucose.
And (3) determining the effective viable count, the mixed bacteria rate, the mixing uniformity Coefficient of Variation (CV) and the shelf life of the solid microbial inoculum. The results are shown in Table 1.
Comparative example 2
This comparative example is used to illustrate the preparation of a lactobacillus plantarum solid inoculum using a liquid fermentation process.
Comparative example 2-1: taking the fermentation liquor obtained by the spread culture in the preparation example, carrying out centrifugal separation to obtain bacterial sludge with the water content of 90%, adding 620g of corn flour, and uniformly mixing to obtain the final effective viable count of 500 hundred million cfu/g.
Adding 5 wt% of glycerol based on the weight of the mixed material, uniformly mixing, freeze-drying at-50 ℃ for 120min, crushing, sieving with a 20-mesh sieve, repeating the steps of freeze-drying, crushing and sieving on oversize products, and finally, enabling all the materials to pass through the 20-mesh sieve to obtain the solid microbial inoculum.
Comparative examples 2 to 2: the conditions of this comparative example were the same as comparative example 2-1 except that 620g of corn flour was replaced with 250g of corn flour, 250g of corn starch residue, 50g of calcium carbonate, 40g of yeast extract powder and 30g of glucose, resulting in a final effective viable count of 500 hundred million cfu/g.
And (3) determining the effective viable count, the mixed bacteria rate, the mixing uniformity Coefficient of Variation (CV) and the shelf life of the solid microbial inoculum. The results are shown in Table 1.
TABLE 1
| Number of | Effective viable count/cfu/g | Miscellaneous rate/% | Coefficient of Variation (CV)/% | Shelf life/month |
| Example 1 | 550 hundred million | 0.005 | 3.5％ | 15 |
| Example 2 | 520 hundred million of | 0.005 | 3.3％ | 15 |
| Example 3 | 530 hundred million | 0.002 | 3.5％ | 15 |
| Example 4 | 390 hundred million | 0.01 | 4.0％ | 12 |
| Example 5 | 380 hundred million | 0.01 | 3.9％ | 12 |
| Example 6-1 | 320 hundred million | 0.01 | 5.4％ | 10 |
| Example 6 to 2 | 330 hundred million | 0.01 | 5.3％ | 10 |
| Examples 6 to 3 | 360 hundred million (million) | 0.01 | 5.5％ | 10 |
| Comparative examples 1 to 1 | 230 hundred million | 0.01 | 9.5％ | 6 |
| Comparative examples 1 to 2 | 240 hundred million | 0.01 | 8.8％ | 7 |
| Comparative examples 1 to 3 | 230 hundred million | 0.01 | 9.0％ | 7 |
| Comparative examples 1 to 4 | 260 hundred million | 0.01 | 9.5％ | 6 |
| Comparative example 2 to 1 | 450 hundred million | 0.50 | 22％ | 4 |
| Comparative examples 2 to 2 | 480 hundred million (million) | 0.40 | 20％ | 4 |
According to the experimental results of the examples and the comparative example 1 in the table 1, the lactobacillus plantarum solid microbial inoculum prepared by using the solid culture medium disclosed by the invention has the advantages of higher effective viable count, lower infectious microbe rate, lower mixing uniformity variation coefficient and longer shelf life. According to microscopic examination results, when corn is used as the starchy raw material, the thalli are full and strong.
Compared with the solid microbial inoculum obtained by physically mixing bacterial sludge and a solid culture medium, the experimental results of the embodiment 1 and the embodiment 2 show that the solid microbial inoculum obtained by using the solid fermentation method has the advantages of lower rate of mixed bacteria, obviously reduced coefficient of variation of mixing uniformity and longer shelf life of products.
As can be seen by comparing the results of examples 1-3 and examples 4-5, when the components for preparing the solid culture medium of the present invention are used in the preferred amounts, the effective viable count of the prepared lactobacillus plantarum solid microbial inoculum is higher, the rate of mixed bacteria is lower, and the shelf life is further prolonged.
Comparing the results of example 1 and example 6, it can be seen that when the starchy material and the starch residue material for preparing the solid medium of the present invention are corn, the prepared lactobacillus plantarum solid bacterial agent has the advantages of higher effective viable count, lower infectious microbe rate, lower variation coefficient of mixing uniformity, and longer shelf life. According to microscopic examination results, when corn is used as the starchy raw material, the thalli are full and strong.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (21)
1. A solid culture medium for lactobacillus plantarum fermentation is prepared from the following raw materials (1) or (2):
(1) Starchy raw material crushed product, starch residue, carbonate, yeast extract powder and sugar;
(2) Starchy raw material crushed product, starch residue, carbonate, yeast extract powder, sugar and water;
in the preparation raw materials of the solid culture medium, the mass ratio of the starchy raw material crushed product, the starch residue, the carbonate, the yeast extract powder and the saccharides is (20-30): (20-30): (1-10): (1-6): (1-5);
the starchy raw material is corn; the raw material for preparing the starch residue is corn; the carbonate is calcium carbonate; the saccharide is glucose.
2. The solid medium according to claim 1, wherein the mass ratio of the starchy raw material crushed product to the starch residue to the carbonate to the yeast extract powder to the saccharides is (23-27): (23-27): (3-7): (3-5): (2-4).
3. A preparation method of a lactobacillus plantarum solid microbial inoculum is characterized by comprising the following steps: inoculating lactobacillus plantarum into the solid medium of claim 1 or 2 for fermentation.
4. The method of claim 3, wherein the method of seeding comprises: and mixing the seed solution of the lactobacillus plantarum with water to obtain a seed inoculation solution, and then uniformly mixing the seed inoculation solution with the solid culture medium to obtain a fermentation starting material.
5. The method of claim 4, wherein the inoculum solution is mixed with the solid medium by spraying.
6. The method of claim 3, wherein the amount of lactobacillus plantarum inoculated is 1 x 10 per gram of solid culture medium 5 -1×10 9 cfu。
7. The method of claim 3, wherein the amount of lactobacillus plantarum inoculated is 1 x 10 per gram of solid culture medium 6 -1×10 8 cfu。
8. The method of claim 3, wherein the fermentation conditions comprise: the fermentation temperature is 28-38 ℃; the fermentation time is 2-8 days.
9. The method of claim 3, wherein the fermentation conditions comprise: the fermentation temperature is 33-36 ℃; the fermentation time is 3-7 days.
10. The method of claim 3, wherein the fermentation is an anaerobic fermentation or a facultative anaerobic fermentation.
11. The method of claim 3, further comprising drying and comminuting the fermentation product.
12. The method of claim 11, wherein the drying method is ambient temperature drying or freeze drying.
13. The method of claim 11, wherein the drying is by freeze drying.
14. The method according to claim 12 or 13, wherein the freeze-drying temperature is-40 to-60 ℃ and the time is 60 to 150min.
15. The method of claim 12 or 13, wherein the fermentation product is mixed with a protectant prior to lyophilization.
16. The method of claim 15, wherein the protectant is one or more of glycerol, trehalose, and combinations thereof.
17. The method of claim 15, wherein the protective agent is glycerol.
18. The method according to claim 16 or 17, wherein the glycerol is used in an amount of 3 to 10 parts by weight relative to 100 parts by weight of the fermentation product.
19. The method according to claim 16 or 17, wherein the glycerol is used in an amount of 4 to 6 parts by weight relative to 100 parts by weight of the fermentation product.
20. A lactobacillus plantarum solid inoculant produced by the method of any one of claims 3 to 19.
21. Use of the lactobacillus plantarum solid bacterial agent according to claim 20 for the preparation of animal feed.
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| KR20180051025A (en) * | 2016-11-08 | 2018-05-16 | 김혜미 | Method for manufacturing the composition of improved Lactic acid bacteria feed additive and Lactic acid bacteria manufactured by the same |
| CN108048381A (en) * | 2018-02-28 | 2018-05-18 | 苏州昆蓝生物科技有限公司 | A kind of preparation method of high-activity lactic acid bacteria solid formulation |
| CN109423467A (en) * | 2017-12-29 | 2019-03-05 | 吉林中粮生化有限公司 | A kind of lactobacillus plantarum of lactic acid high yield and its purposes in food and field of fodder |
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| KR20180051025A (en) * | 2016-11-08 | 2018-05-16 | 김혜미 | Method for manufacturing the composition of improved Lactic acid bacteria feed additive and Lactic acid bacteria manufactured by the same |
| CN109423467A (en) * | 2017-12-29 | 2019-03-05 | 吉林中粮生化有限公司 | A kind of lactobacillus plantarum of lactic acid high yield and its purposes in food and field of fodder |
| CN108048381A (en) * | 2018-02-28 | 2018-05-18 | 苏州昆蓝生物科技有限公司 | A kind of preparation method of high-activity lactic acid bacteria solid formulation |
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