CN112175932A - Microbial inoculum carrier, preparation method and application thereof, and preparation method of lactobacillus solid microbial inoculum - Google Patents
Microbial inoculum carrier, preparation method and application thereof, and preparation method of lactobacillus solid microbial inoculum Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, and discloses a microbial inoculum carrier, a preparation method and application thereof, and a preparation method of a lactic acid bacteria solid microbial inoculum. The microbial inoculum carrier contains calcium carbonate, crude fiber raw material powder, starch and montmorillonite. The lactobacillus solid microbial inoculum comprises a microbial inoculum carrier, lactobacillus and a microbial inoculum protective agent. Compared with the prior art, the microbial inoculum carrier has nutrition slow release performance, the prepared solid microbial inoculum of the lactic acid bacteria can enable the lactic acid bacteria to be slowly proliferated in the early stage of storage due to the metabolic feedback effect of alkaline substances in the microbial inoculum carrier on high-concentration lactic acid and the protective performance of a bacteria protective agent, and the number of bacteria is not lower than an initial value after 6 months of storage. Meanwhile, fermentation liquor is completely utilized in the preparation process of the microbial inoculum, so that the green and high-efficiency production of the lactic acid bacteria microbial inoculum is realized.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a microbial agent carrier, a preparation method and application thereof, and a preparation method of a lactic acid bacteria solid microbial agent.
Background
At present, most of the countries pay attention to the research on the lactic acid bacteria, and the lactic acid bacteria not only play a role in food production, disease prevention, biological preservation and the like, but also have wide application in animal production, and particularly have the most application in fermented feed. The lactobacillus fermented feed can generate the special fermented sour flavor, can improve the palatability of animals to the feed, improve the production performance of the animals, maintain the flora balance in intestinal tracts of the animals, has certain immunoregulation activity, and has important significance in animal production.
With the application and the requirement of the fermented feed industry, the yield of the lactobacillus fermented feed is certainly increased, but the liquid seed liquid has complex preparation process and short storage period; the freeze-dried microbial inoculum has long storage period, but the supernatant fluid of centrifugal separation is wasted, so that not only is waste water generated, but also high-concentration lactic acid in the waste water is lost. Therefore, the development of the green and efficient lactobacillus solid microbial inoculum has wide market potential.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a microbial inoculum carrier, a preparation method and application thereof and a preparation method of a solid lactic acid bacteria agent.
In order to achieve the above object, an aspect of the present invention provides an inoculant carrier, wherein the inoculant carrier comprises calcium carbonate, crude fiber raw material powder, starch and montmorillonite.
The second aspect of the present invention provides a method for preparing the microbial inoculum carrier, which comprises: mixing calcium carbonate, crude fiber raw material powder, starch and montmorillonite to obtain the microbial inoculum carrier;
preferably, the method comprises the steps of:
1) uniformly mixing calcium carbonate and coarse fiber raw material powder to obtain a first mixture, wherein the pH value of the first mixture is 8.0-9.0;
2) uniformly mixing the first mixture with montmorillonite to obtain a second mixture;
3) and uniformly mixing the second mixture with starch to obtain the microbial inoculum carrier.
The third aspect of the invention provides the application of the microbial inoculum carrier or the microbial inoculum carrier prepared by the preparation method in the preparation of a lactic acid bacteria solid microbial inoculum.
The fourth aspect of the invention provides a preparation method of a solid lactobacillus preparation, which comprises the following steps: mixing lactobacillus and a thallus protective agent to obtain a mixed microbial inoculum; and then mixing the mixed microbial inoculum with the microbial inoculum carrier to obtain the lactobacillus solid microbial inoculum.
Compared with the prior art, the microbial inoculum carrier has nutrition slow release performance, the prepared lactic acid bacteria solid microbial inoculum can enable lactic acid bacteria to slowly proliferate in the early storage period due to the metabolic feedback effect of alkaline substances in the microbial inoculum carrier on high-concentration lactic acid and the protective performance of a bacteria protective agent, and the number of bacteria is not lower than an initial value after 6 months of storage. Meanwhile, fermentation liquor is completely utilized in the preparation process of the microbial inoculum, so that the generation of waste water is greatly reduced, and the green and efficient production of the solid microbial inoculum of the lactic acid bacteria is realized.
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.
The invention provides a microbial inoculum carrier, which contains calcium carbonate, crude fiber raw material powder, starch and montmorillonite.
In the invention, the preservation period of the solid microbial inoculum can be effectively prolonged by preparing the microbial inoculum carrier by combining the components and applying the carrier to preservation of the microbial inoculum, and the content of each component in the microbial inoculum carrier can be selected in a wide range. Preferably, the content of the calcium carbonate is not higher than 20 parts by weight, the content of the crude fiber raw material powder is not higher than 90 parts by weight, the content of the montmorillonite powder is not higher than 20 parts by weight, and the content of the starch is 10-30 parts by weight relative to 100 parts by weight of the microbial inoculum carrier in dry weight. Further preferably, the content of the calcium carbonate is 5-10 parts by weight, the content of the coarse fiber raw material powder is 50-80 parts by weight, the content of the montmorillonite powder is 5-15 parts by weight, and the content of the starch is 10-25 parts by weight, relative to 100 parts by weight of a dry weight of the microbial inoculum carrier. In a preferred case, the shelf life of the prepared solid can be further extended.
In the present invention, the calcium carbonate may be various calcium carbonates conventionally used in the art, preferably light calcium carbonate, the purity of which may be selected within a wide range, for example, may be 90% or more. The calcium carbonate can be obtained from the market, for example, the calcium carbonate can be purchased from Henan coke and used as the special light calcium carbonate for edible fungi.
In the present invention, the kind of the raw material powder for crude fiber may be selected from a wide range, and may be a powder of a raw material having a high crude fiber content, which is conventionally used in the art, for example, at least one of rice hull powder, corncob powder, peanut hull powder, wheat-germ powder, and bran; preferably, the crude fiber raw material powder is rice hull powder and/or corncob powder. When the crude fiber raw material powder is rice husk powder and corn cob powder, the weight ratio of the rice husk powder to the corn cob powder can be 1 (0.5-1.5).
Wherein, the rice hull powder can be rice hull powder which is conventionally used in the field, and the particle size can be 40-100 meshes. The rice hull powder may be obtained by pulverizing rice hulls, or may be commercially available, for example, ultrafine rice hull powder available from stone house gold ear agricultural product trade company ltd.
Wherein, the corncob meal can be the corncob meal which is conventionally used in the field, and the particle size of the corncob meal can be 10-100 meshes. The corncob powder can be obtained by crushing corncobs, can also be obtained by commercial sale, and can be the corncob powder which is purchased from Henan province and is 30-60 meshes of Fuquan refractories. Generally, the corncob meal is a weakly acidic material.
In the present invention, the starch may be various starches conventionally used in the art, for example, corn starch, wheat starch, sweet potato starch, etc., and preferably corn starch. The corn starch may be a corn starch conventionally used in the art, and may be commercially available, for example, a food corn starch available from yuzu bioenergy (elm) ltd. Typically, the corn starch is also a weakly acidic material.
In the present invention, the montmorillonite may be montmorillonite conventionally used in the art, and is preferably a feed grade montmorillonite powder, which is commercially available, for example, may be a fodder grade montmorillonite powder available from the chemical company of loddigege of inner Mongolia.
In the present invention, the pH of the microbial inoculum carrier can be selected within a wide range. The inventor of the invention finds that when the pH value of the microbial inoculum carrier is 5.5-7.5, the shelf life of the solid microbial inoculum of lactic acid bacteria prepared by using the microbial inoculum carrier can be further prolonged.
In another aspect, the present invention provides a method for preparing the microbial inoculum carrier, which comprises: and mixing calcium carbonate, crude fiber raw material powder, starch and montmorillonite to obtain the microbial inoculum carrier.
In the present invention, the types and contents of calcium carbonate, crude fiber raw material powder, starch and montmorillonite involved in the preparation method of the microbial inoculum carrier are described in detail in the first aspect of the present invention, and are not described herein again.
In the invention, the microbial inoculum carrier of the invention can be prepared by directly mixing the components, and the preservation period of the prepared solid microbial inoculum can be effectively prolonged by applying the microbial inoculum carrier to preservation of the microbial inoculum. The steps include:
1) uniformly mixing calcium carbonate and coarse fiber raw material powder to obtain a first mixture, wherein the pH value of the first mixture is 8-9;
2) uniformly mixing the first mixture with montmorillonite to obtain a second mixture;
3) and uniformly mixing the second mixture with starch to obtain the microbial inoculum carrier.
In the invention, all the components of the microbial inoculum carrier are solid powder, and the mixture obtained after mixing also exists in the form of solid powder, so that the solid powder cannot be directly measured by using a method for measuring the pH of the liquid in the field. Therefore, in the present invention, the pH measurement method of the solid powder may be: after 5g of the solid powder was added to 45ml of water and stirred uniformly, the pH of the supernatant was measured using a pH meter, and the pH of the supernatant was measured as the pH of the solid powder.
In the present invention, in step 1), the pH after uniformly mixing the calcium carbonate and the raw fiber material powder may not be in the range of 8 to 9, and in this case, it is necessary to adjust the pH using an acid agent and/or an alkali agent so that the pH of the first mixture is in the range of 8 to 9. The acid agent may be an acid conventionally used in the art, and may be at least one of lactic acid, citric acid, oxalic acid, or hydrochloric acid, preferably lactic acid. The alkaline agent may be an alkali conventionally used in the art, and may be, for example, at least one of calcium oxide, calcium hydroxide or sodium hydroxide, preferably calcium oxide. The acid agent and the alkali agent may be optionally mixed with calcium carbonate and/or the raw fiber material powder, and preferably, the alkali agent may be mixed with calcium carbonate first.
In the invention, in the step 3), the pH value of the microbial inoculum carrier can be selected in a wide range, and the shelf life of the solid microbial inoculum can be further prolonged when the microbial inoculum carrier is preferably controlled to be in a range of 5.5-7.5. Generally, the pH of the microbial inoculum carrier obtained by directly and uniformly mixing the second mixture and the starch is within the range of 5.5-7.5. If the pH is not within the range of 5.5 to 7.5, the pH can be adjusted to be within the range of 5.5 to 7.5 by using an acidic agent and/or an alkaline agent. The acid agent may be an acid conventionally used in the art, and may be at least one of lactic acid, citric acid, oxalic acid, or hydrochloric acid, preferably lactic acid. The alkaline agent may be an alkali conventionally used in the art, and may be, for example, at least one of calcium oxide, calcium hydroxide or sodium hydroxide, preferably calcium oxide.
The inventor of the invention finds that when the pH adjusting range of each step is in the preferable range, the mixed bacteria in the raw materials can be effectively inhibited or killed, the storage period of the microbial inoculum is prolonged, and the step of sterilizing the prepared microbial inoculum carrier is avoided; when the pH value is adjusted to exceed the range, the storage period is shortened or the microbial inoculum has more mixed bacteria, which causes the adverse result.
The invention further provides the application of the microbial inoculum carrier or the microbial inoculum carrier prepared by the method in the preparation of the solid microbial inoculum of lactic acid bacteria.
The invention also provides a preparation method of the lactobacillus solid microbial inoculum, which comprises the following steps: mixing lactobacillus and a thallus protective agent to obtain a mixed microbial inoculum; and then mixing the mixed microbial inoculum with the microbial inoculum carrier to obtain the lactobacillus solid microbial inoculum.
In the present invention, the lactic acid bacteria species may be of the kind conventionally used in the art, i.e., any spore-free gram-positive bacterium, preferably lactobacillus plantarum, in which lactic acid is the main product of the fermentation of sugars. The Lactobacillus plantarum can inhibit the growth of putrefying bacteria in intestinal tract, promote the proliferation of bifidobacteria in intestinal tract, and improve the intestinal environment, thereby being beneficial to health. The lactobacillus plantarum can produce vitamins VK, VB6, VB12, RIBOFLAVIN, folic acid and the like required by a human body in the intestinal tract, and can inhibit and destroy vitamin B1 enzymes so as to keep the vitamin B group in the intestinal tract stable.
In the invention, the lactic acid bacteria can be fermentation liquor of the lactic acid bacteria or thallus suspension of the lactic acid bacteria, and the storage period of the solid microbial inoculum can be effectively prolonged by preparing the lactic acid bacteria and other components into microbial inoculum carriers and applying the microbial inoculum carriers to the storage of the microbial inoculum.
In the present invention, it is preferable that the lactic acid bacteria is a fermentation broth of lactic acid bacteria, and the storage life of the prepared solid can be further prolonged. When the lactobacillus solid microbial inoculum prepared under the condition is prepared into feed, the lactic acid (salt) in the fermentation liquor can be added into the feed, so that the pH value in the stomach can be reduced, the effects of activating digestive enzymes and improving the digestion capability of amino acids are achieved, and the growth of intestinal epithelium is benefited; and can also be used as a preservative for feed and a microbial stabilizer for by-products of feed, grain and meat processing products.
In the present invention, the suspension of the lactic acid bacteria may be a suspension obtained by centrifugally collecting and fermenting lactic acid bacteria and resuspending the lactic acid bacteria in a solution such as physiological saline or PBS buffer.
The specific preparation method and parameters of the lactobacillus fermentation liquid can be controlled according to the conventional mode in the field, for example, the lactobacillus fermentation liquid can be obtained by strain activation and propagation. The culture medium used for the activation and propagation culture of lactic acid bacteria may be a culture medium of lactic acid bacteria conventionally used in the art, such as MRS broth. The lactobacillus strain can be inoculated into a shake flask containing MRS broth for activation culture, wherein the shake flask culture can be performed in 1-3 grades, and the skilled person can make specific selection according to actual conditions. After activation, the activated seed solution can be inoculated into a fermentation tank for propagation so as to obtain the lactobacillus fermentation liquor, and the propagation conditions can be as follows: the inoculation amount is 0.15-0.25 vol%, the temperature is 34-37.8 ℃, the stirring speed is 80-120rpm, and the culture time is 12-14 hours.
In the present invention, the cytoprotective agent may be a conventional cytoprotective agent used in the art, and may be one or more of skim milk powder, trehalose, sodium glutamate, and sorbitol, for example. The skim milk powder, trehalose, sodium glutamate and sorbitol are commercially available. The inventor of the invention finds that the shelf life of the lactobacillus solid microbial inoculum is the best when the thallus protective agent is a mixture of skim milk powder, trehalose and sorbitol.
In the present invention, the mixing method of the lactic acid bacteria and the microbial cell protective agent is not particularly limited, and it is only necessary to wrap the lactic acid bacteria with the microbial cell protective agent. The inventor of the invention finds that the lactobacillus and the thallus protective agent are mixed and then kept for 30-120min, so that the thallus can be uniformly wrapped by the protective agent, and the aim of protecting the thallus is fulfilled.
In the present invention, the mass ratio of the lactic acid bacteria to the cell protecting agent can be selected from a wide range, and preferably, the cell protecting agent is 5 to 30 parts by weight based on 100 parts by weight of the lactic acid bacteria fermentation liquid or cell suspension.
In the present invention, the mass ratio of the mixed microbial inoculum to the microbial inoculum carrier can be selected in a wide range, and preferably, the microbial inoculum carrier can be 100-500 parts by weight relative to 100 parts by weight of the mixed microbial inoculum.
In the invention, the lactobacillus solid inoculum can be further processed by the conventional technical means in the field, such as drying treatment, preferably, the drying temperature is 32-40 ℃; the drying time is 0.5-2 h.
Through the preferred adding sequence and adding proportion, the invention obtains the microbial inoculum carrier for preparing the solid microbial inoculum of the lactic acid bacteria and the solid microbial inoculum of the lactic acid bacteria, so that the shelf life of the solid microbial inoculum of the lactic acid bacteria is further prolonged, and the bacteria can be proliferated at the initial stage of storage. And the operation is simple in the whole process, the generation of waste water is greatly reduced, and the green and efficient production of the lactobacillus solid microbial inoculum is realized.
The present invention will be described in detail below by way of examples. In the following examples and comparative examples:
the calcium carbonate is light calcium carbonate and is purchased from Sanyao chemical company Limited, Jiaozuo, Henan;
the rice hull powder is purchased from Shijia village gold ear agricultural product trade Co., Ltd;
the corncob powder is purchased from Henan Fuquan refractory;
the corn starch is purchased from middle grain bioenergy (elm) ltd;
the montmorillonite is feed-grade montmorillonite, and is purchased from Nemontage Ivory chemical Co Ltd;
the skim milk powder is purchased from Wandashan;
the trehalose is purchased from a japanese forest source;
the sodium glutamate is purchased from plum blossom biotechnology group, ltd;
the sorbitol is purchased from Henan Qianjin commerce and trade Co., Ltd;
adjusting the pH of the material by using calcium oxide and lactic acid;
the lactobacillus strain is plant lactobacillus (number is BC00171) from strain preservation center of Chinese food nutrition and health institute, and a plurality of identical freezing and storing tubes are prepared for later use;
commercially available MRS broth: purchased from beijing land bridge technology, llc under the designation CM 187.
The OD value was measured using a spectrophotometer (shanghai precision scientific instruments ltd., No. 070708040154).
The reagents and materials used in the following examples and comparative examples are all conventional types unless otherwise specified.
The determination method of the viable count (meeting GB 4789.35-2016 food safety national standard food microbiology inspection lactobacillus inspection): the formula of the culture medium is MRS broth culture medium, except that 1.5-2 wt% of agar is added into the culture medium to prepare solid plate culture medium, a certain amount of microbial solid microbial inoculum is diluted to a proper multiple by using normal saline within a specified time and then coated on a plate, inverted culture is carried out for 48 hours at 35-37 ℃, the number of bacterial colonies is counted, and the number of viable bacteria is calculated.
The mixed bacteria rate is the proportion of the mixed bacteria quantity in the solid microbial inoculum to the total bacteria quantity, and can be obtained by calculating through a dilution coating plate method.
Preparation example 1
This preparation example is illustrative of the method for preparing a lactic acid bacterium fermentation broth
1) Activating strains: the lactobacillus plantarum stored in the ultra-low temperature refrigerator is naturally thawed 0.5h in advance, and inoculated into MRS broth culture medium, wherein the inoculation amount is 0.2 vol%.
The culture conditions are as follows: standing and culturing at 35 ℃. And (5) ending the shaking of the first-class seeds when the OD value is 6-7 and the pH value is 4.0.
2) And (3) secondary shake flask fermentation: the culture medium is MRS broth culture medium, and the shake flask inoculation amount is as follows: 0.2 volume%;
the culture conditions are as follows: standing and culturing at 35 ℃.
The OD value is 12-15, the pH value is about 5.0, and the shaking of the second-level seeds is finished.
3) Expanding culture tank fermentation
The culture medium is MRS broth culture medium, and the inoculation amount is 0.2 volume percent; the culture conditions were static culture at 37 ℃.
OD value is 16-18, pH is about 4.6, the fermentation in the culture expanding tank is finished, and the number of viable bacteria in the fermentation liquid is 1.0 x 1011-1.0*1012cfu/ml。
Example 1
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
1) Uniformly mixing 8g of calcium carbonate and 67g of rice hull powder, and measuring and adjusting the pH value to 8.5 to obtain a first mixture;
2) uniformly mixing the first mixture with 10g of feed-grade montmorillonite powder to obtain a second mixture;
3) and uniformly mixing the second mixture with 15g of corn starch, and measuring and adjusting the pH value to 7 to obtain the microbial inoculum carrier.
4) And (3) directly adding 10g of skim milk powder into 50g of lactobacillus fermentation liquor, and mixing for 60min to obtain the mixed microbial inoculum.
5) And adding the microbial inoculum carrier into the mixed microbial inoculum, mixing, and drying in an oven at the drying temperature of 35 ℃ for 1h to obtain the lactic acid bacteria solid microbial inoculum A1.
Example 2
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
1) Uniformly mixing 10g of calcium carbonate and 50g of corncob meal, and measuring and adjusting the pH value to 9.0 to obtain a first mixture;
2) uniformly mixing the first mixture with 15g of feed-grade montmorillonite to obtain a second mixture;
3) and uniformly mixing the second mixture with 25g of corn starch to obtain a microbial inoculum carrier, and measuring and adjusting the pH value to 7.5.
4) And (3) directly adding 20g of sorbitol into 80g of lactobacillus fermentation liquor, and mixing for 30min to obtain the mixed microbial inoculum.
5) And adding the microbial inoculum carrier into the mixed microbial inoculum, mixing, and drying in an oven at the drying temperature of 32 ℃ for 2h to obtain the lactic acid bacteria solid microbial inoculum A2.
Example 3
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
1) Uniformly mixing 5g of calcium carbonate and 80g of rice hull powder, and measuring and adjusting the pH value to 8.0 to obtain a first mixture;
2) uniformly mixing the first mixture with 5g of feed-grade montmorillonite to obtain a second mixture;
3) and (3) uniformly mixing the second mixture with 10g of corn starch to obtain a microbial inoculum carrier, and measuring and adjusting the pH value to 6.5.
4) And (3) directly adding 1g of trehalose into 20g of lactobacillus fermentation liquor, and mixing for 120min to obtain the mixed microbial inoculum.
5) And adding the microbial inoculum carrier into the mixed microbial inoculum, mixing, and drying in an oven at the drying temperature of 36 ℃ for 0.5h to obtain the lactic acid bacteria solid microbial inoculum A3.
Example 4
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
Example 4-1: the lactic acid bacteria solid preparation A4-1 was prepared according to the method described in example 1, except that the initial amounts of the components in the solid preparation were: 2.5g calcium carbonate, 85g rice hull powder, 2.5g feed grade montmorillonite, 10g corn starch, 50g lactobacillus fermentation broth and 10g skim milk powder.
Example 4-2: the lactic acid bacteria solid preparation A4-2 was prepared according to the method described in example 1, except that the initial amounts of the components in the solid preparation were: 20g of calcium carbonate, 30g of rice hull powder, 20g of feed grade montmorillonite, 30g of corn starch, 50g of lactic acid bacteria fermentation liquor and 10g of skim milk powder.
Example 5
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
Example 5-1: a lactic acid bacterium solid preparation A5-1 was prepared according to the method described in example 1, except that the rice husk powder was replaced with a mixture of equal mass of rice husk powder and corn cob powder at a weight ratio of 1: 1.
Example 5-2: a lactic acid bacterium solid preparation A5-2 was prepared according to the method described in example 1, except that the corn starch was replaced with wheat starch of equal weight.
Example 6
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
Example 6-1: a solid lactic acid bacterium A6-1 was prepared according to the method described in example 1, except that the pH of the bacterium carrier was 5.
Example 6-2: a solid lactic acid bacterium A6-2 was prepared according to the method described in example 1, except that the pH of the bacterium carrier was 8.
Example 7
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
Example 7-1: a solid preparation of lactic acid bacterium A7-1 was prepared according to the method described in example 1, except that the pH of the first mixture was 7.5.
Example 7-2: a solid preparation of lactic acid bacterium A7-2 was prepared according to the method described in example 1, except that the pH of the first mixture was 9.5.
Example 8
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
Example 8-1: a lactic acid bacterium solid preparation A8-1 was prepared according to the method described in example 1, except that calcium carbonate, rice hull powder, feed-grade montmorillonite and corn starch were added and mixed at the same time to prepare a preparation carrier.
Example 8-2: the solid lactic acid bacteria preparation A8-2 was prepared according to the method described in example 1, except that calcium carbonate, rice hull powder, feed grade montmorillonite, corn starch, fermentation broth and skim milk powder were added and mixed at the same time to prepare the solid lactic acid bacteria preparation.
Examples 8 to 3: the lactic acid bacteria solid preparation A8-3 was prepared according to the method described in example 1, except that calcium carbonate and feed-grade montmorillonite were added first, and then rice hull powder, corn starch, fermentation broth, and skim milk powder were added.
Example 9
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
The lactic acid bacteria solid preparation A9 was prepared according to the method described in example 1, except that skim milk powder was added directly to the lactic acid bacteria fermentation broth and mixed for 10 min.
Example 10
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
A solid lactic acid bacteria preparation A10 was prepared according to the method described in example 1, except that the skim milk powder was replaced with equal mass of the bacterial protective agent, wherein the bacterial protective agent was skim milk powder 6g, trehalose 3g and sorbitol 1 g.
Example 11
This example is provided to illustrate the preparation method of the solid lactic acid bacteria agent of the present invention
A solid preparation A11 of lactic acid bacteria was prepared by following the procedure described in example 1, except that the fermentation broth of lactic acid bacteria was replaced with a suspension of the cells of lactic acid bacteria.
Wherein the preparation method of the thallus suspension comprises the steps of carrying out centrifugal treatment on the fermentation liquor obtained in the preparation example 1 at 5000rpm to obtain bacterial sludge; the bacterial sludge was resuspended with physiological saline, and the pH of the bacterial suspension was adjusted to about 4.6 with citric acid so that the pH and bacterial density of the obtained bacterial suspension were consistent with those of the fermentation broth obtained in preparation example 1.
Comparative example 1
This comparative example is used to illustrate the preparation method of a reference lactic acid bacteria solid inoculum
Comparative example 1-1: the lactic acid bacteria solid preparation D1-1 was prepared according to the method described in example 1, except that calcium sulfate of equal mass was used instead of precipitated calcium carbonate.
Comparative examples 1 to 2: the lactic acid bacteria solid preparation D1-2 was prepared as described in example 1, except that talc powder of equal mass was used instead of feed-grade montmorillonite powder.
Comparative example 2
This comparative example is used to illustrate the preparation method of a reference lactic acid bacteria solid inoculum
Comparative example 2-1: a lactic acid bacteria solid preparation D2-1 was prepared according to the method described in example 1, except that calcium carbonate was replaced with an equal mass of montmorillonite powder, i.e., the total weight of feed-grade montmorillonite powder was 15 g.
Comparative examples 2 to 2: a lactic acid bacterium solid preparation D2-2 was prepared according to the method described in example 1, except that the montmorillonite powder was replaced with calcium carbonate of equal mass, i.e., the total weight of calcium carbonate was 15 g.
Comparative examples 2 to 3: a lactic acid bacterium solid preparation D2-3 was prepared according to the method described in example 1, except that corn starch was replaced with equal mass of rice hull powder, i.e., the total weight of the rice hull powder was 85 g.
Comparative example 3
This comparative example is used to illustrate the preparation method of a reference lactic acid bacteria solid inoculum
Centrifuging the fermentation liquor to collect bacterial sludge, suspending 100g of bacterial sludge in 70g of sterile water containing 30g of skim milk powder, placing the suspended solution in a freeze-drying tray, pre-freezing for 2h at-20 ℃, and then placing in a vacuum freeze-drying machine for freeze-drying for about 24h at-86 ℃ to obtain the lactobacillus solid microbial inoculum D3.
Test example
The rate of mixed bacteria and the number of viable bacteria in the solid microbial inoculum prepared by the example 1-the example 11 and the comparative example 1-the comparative example 3 are measured; the microbial inoculum is stored at room temperature, and the viable count of the lactic acid bacteria solid microbial inoculum is measured when the microbial inoculum is stored for 2 months, 4 months, 6 months and 8 months.
Specific results are shown in table 1.
TABLE 1
According to the table 1, as can be seen from the comparison of the data of the embodiment and the comparative example, the quality guarantee period of the prepared lactobacillus solid microbial inoculum is remarkably prolonged by adopting the microbial inoculum carrier of the invention and controlling the conditions in the preparation process of the microbial inoculum carrier, and the mixed microbial inoculum rate of the preserved lactobacillus solid microbial inoculum is also greatly reduced.
As can be seen from comparison of examples 1 to 8, under the preferred conditions, namely the preferred carrier components and proportions according to the invention, and the use of the microbial inoculum carrier prepared according to the specific addition sequence of the invention to prepare the solid microbial inoculum of lactic acid bacteria, the microbial inoculum rate can be further reduced, the shelf life can be prolonged, the microbial inoculum rate can be controlled within 0.2%, the shelf life can reach more than 8 months, and the product quality can be improved.
The data in examples 1, 9 and 10 show that the preferred cell protectant and the preferred mixing time of the present invention can further improve product quality.
As can be seen by comparing the data of example 1 and example 11, even though citric acid is used to adjust the pH of the bacterial suspension to a level consistent with that of the fermentation broth and to ensure consistent cell density, the shelf life of the obtained solid lactic acid bacteria preparation is still significantly different from that of the solid lactic acid bacteria preparation prepared from the fermentation broth, possibly due to the fact that lactic acid and other components in the fermentation broth provide a better and suitable storage environment for the lactic acid bacteria.
Compared with the data of the embodiment 1 and the comparative example 3, the lactobacillus solid microbial inoculum prepared by the method has longer shelf life, and the fermentation liquor is fully utilized, so that the generation of waste water is greatly reduced, and the green and efficient production of the lactobacillus solid microbial inoculum is realized.
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 each of the specific features, are combined in any suitable manner. The invention is not described in detail in order to avoid unnecessary repetition. Such simple modifications and combinations should be considered within the scope of the present disclosure as well.
Claims (10)
1. The microbial inoculum carrier is characterized by comprising calcium carbonate, crude fiber raw material powder, starch and montmorillonite.
2. The microbial inoculum carrier of claim 1, wherein the calcium carbonate content is not higher than 20 parts by weight, the crude fiber raw material powder content is not higher than 90 parts by weight, the montmorillonite powder content is not higher than 20 parts by weight, and the starch content is 10-30 parts by weight, relative to 100 parts by weight of the microbial inoculum carrier on a dry weight basis.
3. The microbial inoculum carrier of claim 1, wherein the calcium carbonate is 5-10 parts by weight, the crude fiber raw material powder is 50-80 parts by weight, the montmorillonite powder is 5-15 parts by weight, and the starch is 10-25 parts by weight, relative to 100 parts by weight of the microbial inoculum carrier on a dry weight basis.
4. The microbial inoculant carrier of any one of claims 1 to 3 wherein the calcium carbonate is precipitated calcium carbonate; and/or
The crude fiber raw material powder is at least one of rice hull powder, corn cob powder, peanut hull powder, wheat and mango powder and bran; and/or
The starch is at least one of corn starch, wheat starch and sweet potato starch; and/or
The montmorillonite powder is feed-grade montmorillonite powder.
5. The microbial inoculant carrier of any one of claims 1 to 4 wherein the pH of the microbial inoculant carrier is between 5.5 and 7.5.
6. The method for preparing the microbial agent carrier according to any one of claims 1 to 5, comprising: mixing calcium carbonate, crude fiber raw material powder, starch and montmorillonite to obtain the microbial inoculum carrier;
preferably, the method comprises the steps of:
1) uniformly mixing calcium carbonate and coarse fiber raw material powder to obtain a first mixture, wherein the pH value of the first mixture is 8-9;
2) uniformly mixing the first mixture with montmorillonite to obtain a second mixture;
3) uniformly mixing the second mixture with starch to obtain the microbial inoculum carrier;
preferably, the pH of the microbial inoculum carrier is 5.5-7.5.
7. Use of the microbial agent carrier of any one of claims 1 to 5 or the microbial agent carrier prepared by the preparation method of claim 6 in preparation of a lactic acid bacteria solid microbial agent.
8. A preparation method of a solid lactobacillus preparation is characterized by comprising the following steps: mixing lactobacillus and a thallus protective agent to obtain a mixed microbial inoculum; then mixing the mixed microbial inoculum with the microbial inoculum carrier of any one of claims 1 to 5 or the microbial inoculum carrier prepared by the preparation method of claim 6 to obtain the lactobacillus solid microbial inoculum;
preferably, the lactic acid bacteria and the thallus protective agent are mixed and then kept for 30-120 min;
preferably, the thallus protective agent is one or more of skimmed milk powder, trehalose, sodium glutamate and sorbitol;
preferably, the microbial inoculum carrier is used in an amount of 100-500 parts by weight relative to 100 parts by weight of the mixed microbial inoculum.
9. The method according to claim 8, wherein the lactic acid bacterium is a fermentation broth of a lactic acid bacterium; the lactic acid bacteria are preferably lactobacillus plantarum;
preferably, the cell protectant is used in an amount of 5-30 parts by weight, based on 100 parts by weight of the lactic acid bacteria fermentation broth.
10. The method of claim 8 or 9, wherein the method further comprises: drying the lactobacillus solid microbial inoculum, preferably, the drying temperature is 32-40 ℃; the drying time is 0.5-2 h.
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