阅读说明：本技术 一种油茶专用释磷型生物菌肥的制备方法 (Preparation method of special phosphorus-release biological bacterial fertilizer for oil tea ) 是由 陈华 廖德胜 郑传奇 许俊波 于 2021-08-03 设计创作，主要内容包括：本发明公开了一种油茶专用释磷型生物菌肥的制备方法,其特征在于,包括以下步骤：1)取食用菌废菌棒和小麦秸秆粉碎过筛后混合均匀,然后加水预湿,得到预湿混合料；2)向所述预湿混合料中加入发酵菌剂,然后加入尿素调节碳氮比,在充分混匀后进行第一阶段发酵；3)取步骤2)所得物料加入菌炭混合物和干燥鸡蛋壳粉充分混匀,然后进行第二阶段发酵；4)取步骤3)所得物料,调整水分至含水量为25％～35wt％,然后过筛即得。本发明的一种油茶专用释磷型微生物菌肥,含有能分解无机磷的菌株,能够将土壤中结合态的磷元素释放出来,变成游离的磷元素,达到提高土壤中有效磷的作用,进而促进作物吸收土壤中的磷素营养。(The invention discloses a preparation method of a special phosphorus-release biological bacterial fertilizer for oil tea, which is characterized by comprising the following steps of: 1) crushing and sieving the waste edible fungus sticks and the wheat straws, uniformly mixing, and adding water for pre-wetting to obtain a pre-wetted mixture; 2) adding a fermentation microbial inoculum into the pre-wet mixture, then adding urea to adjust the carbon-nitrogen ratio, and performing first-stage fermentation after fully and uniformly mixing; 3) adding the material obtained in the step 2) into the fungus charcoal mixture and the dried egg shell powder, fully and uniformly mixing, and then performing second-stage fermentation; 4) taking the material obtained in the step 3), adjusting the water content to 25-35 wt%, and then sieving to obtain the product. The phosphorus-release microbial fertilizer special for the oil tea contains strains capable of decomposing inorganic phosphorus, can release combined phosphorus elements in soil into free phosphorus elements, achieves the effect of improving available phosphorus in the soil, and further promotes crops to absorb phosphorus nutrition in the soil.)

1. A preparation method of a special phosphorus-release biological bacterial fertilizer for oil tea, which is characterized by comprising the following steps:

1) crushing and sieving the waste edible fungus sticks and the wheat straws, uniformly mixing, and adding water for pre-wetting to obtain a pre-wetted mixture;

2) adding a fermentation microbial inoculum into the pre-wet mixture, then adding urea to adjust the carbon-nitrogen ratio, and performing first-stage fermentation after fully and uniformly mixing;

3) adding the material obtained in the step 2) into the fungus charcoal mixture and the dried egg shell powder, fully and uniformly mixing, and then performing second-stage fermentation;

4) taking the material obtained in the step 3), adjusting the water content to 25-35 wt%, and then sieving to obtain the product.

2. The method of claim 1, wherein: in the step 2), the first-stage fermentation is high-temperature compost fermentation, the width of the compost is 1-2 m, the height of the compost is 1-1.8 m, the fermentation temperature is 40-75 ℃, the fermentation time is 15-35 days, at least 3-10 times of turning over the material is carried out during the fermentation, and the thoroughly decomposed material is spread out and cooled to the room temperature.

3. The method of claim 1, wherein: in the step 3), the fermentation temperature of the second stage of fermentation is 22-37 ℃, the fermentation humidity is 50-60%, and the fermentation time is 6-12 days.

4. The method according to claim 1, wherein in step 3), the charcoal mixture is prepared by: inoculating an inorganic phosphate solubilizing functional strain into a first PKO liquid culture medium, culturing for 3-10 days at 28 ℃ to obtain a functional strain, adding the functional strain into a second PKO liquid culture medium, uniformly mixing to obtain a strain solution, adding the strain solution into the charcoal powder until the charcoal powder is fully wetted, and standing for 30-100 min.

5. The method of claim 4, wherein: the inorganic phosphate solubilizing bacteria functional strain is native indigenous bacteria of a tea-oil tree forest.

6. The method of claim 4, wherein: in the preparation of the charcoal mixture, the functional strains account for 3-6 parts by weight, the second PKO liquid culture medium accounts for 130-160 parts by weight, and the biochar accounts for 60-90 parts by weight.

7. The method of claim 4, wherein: the porosity of the biochar is more than 0.5; the methylene blue adsorption value of the biochar is more than 16.

8. The method of claim 1, wherein: in the step 2), the fermentation inoculant comprises lactic acid bacteria, bacillus subtilis, ralstonia solanacearum and bacillus amyloliquefaciens; according to the parts by weight, the weight ratio of the lactic acid bacteria to the bacillus subtilis to the bacillus verrucosus and the bacillus amyloliquefaciens is 1-3: 2-6: 2-6: 1-3; the weight ratio of the starter to the pre-wet mixture is 1: 3000 to 6000.

9. The method of claim 1, wherein: in the step 2), the carbon-nitrogen ratio is 20-30: 1.

10. the method of claim 1, wherein: in the step 1), the mass ratio of the waste edible fungus sticks to the wheat straws to the water is 5-30: 0.5-1.5: 10-40; the pre-wetting time is 6-12 hours.

Technical Field

The invention relates to the technical field of organic fertilizer preparation, in particular to a preparation method of a phosphorus-release biological bacterial fertilizer special for oil tea.

Background

Camellia oleifera, also known as tea tree, camellia oleifera and white tea. The tea oil is also a high-quality edible oil, can be used as lubricating oil and antirust oil for industry, and has high practical value. However, the growth period of the camellia oleifera is long, and the aim of continuous and high yield can be achieved only by maintaining the soil fertility.

Soil in southwest areas of China is mostly 'phosphorus-deficient soil', and the lack of phosphorus elements is not because the phosphorus elements do not exist in the soil, but the lack of effective phosphorus elements which can be directly absorbed and utilized by plants. One of the main reasons for this phenomenon is the unreasonable use of chemical fertilizers, and only about 20% of phosphorus fertilizers used in farming activities are absorbed and utilized by crops in the current year, and the remaining part of phosphorus elements are firmly combined with soil particles under the action of moisture to form combined phosphorus, which causes soil problems such as soil hardening and the like, thereby causing phosphorus deficiency in soil.

The microbial fertilizer can improve the physical and chemical properties of soil, regulate plant growth, increase crop yield and is friendly to ecological environment. The popularization and application of the microbial fertilizer are important measures for promoting the upgrade of the oil tea industry in China. The traditional agriculture uses a large amount of repeated phosphate fertilizers to provide enough phosphorus for plants, so that the production cost is increased, and serious surface source pollution such as arsenic is brought, and the microbial fertilizer promotes the dissolution of the fertilizer and indissolvable phosphorus nutrients in soil by the life activities of microbes, increases soil organic matters, improves the phosphorus supply capacity of the soil, and improves the utilization efficiency of the phosphate fertilizers. Therefore, a phosphorus-release biological bacterial fertilizer applicable to oil tea has become a necessary requirement for the efficient ecological development of the oil tea industry.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a preparation method of a phosphorus-release biological bacterial fertilizer special for oil tea.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a special phosphorus-release biological bacterial fertilizer for oil tea, which is characterized by comprising the following steps:

1) crushing waste edible fungus sticks and wheat straws, sieving, uniformly mixing, and adding water for pre-wetting to obtain a pre-wetted mixture;

2) adding a fermentation microbial inoculum into the pre-wet mixture, then adding urea to adjust the carbon-nitrogen ratio, and performing first-stage fermentation after fully and uniformly mixing;

3) adding the material obtained in the step 2) into the fungus charcoal mixture and the dried egg shell powder (providing an inorganic phosphorus source) to be fully and uniformly mixed, and then performing second-stage fermentation;

4) taking the material obtained in the step 3), adjusting the water content to 25-35 wt%, and then sieving to obtain the product.

Further, in the step 1), the mass ratio of the waste edible fungus sticks to the wheat straws to the water is 5-30: 0.5-1.5: 10-40; the pre-wetting time is 6-12 hours.

Further, in the step 2), the first-stage fermentation is high-temperature compost fermentation, the width of the compost is 1-2 m, the height of the compost is 1-1.8 m, the fermentation temperature is 40-75 ℃, the fermentation time is 15-35 days, the materials are stirred at least 3-10 times during the fermentation, and the thoroughly decomposed materials are spread and cooled to room temperature.

Further, in the step 2), the leavening agent comprises lactic acid bacteria, bacillus subtilis, ralstonia solanacearum and bacillus amyloliquefaciens.

Further, the weight ratio of the lactic acid bacteria to the bacillus subtilis to the bacillus verrucosus and the bacillus amyloliquefaciens is (1-3): 2-6: 2-6: 1-3; the weight ratio of the starter to the pre-wet mixture is 1: 3000 to 6000.

Further, in the step 2), the carbon-nitrogen ratio is 20-30: 1.

further, in the step 3), the fermentation temperature of the second stage of fermentation is 22-37 ℃, the fermentation humidity is 50-60%, and the fermentation time is 6-12 days.

Wherein, two-step fermentation is carried out in order to ensure the survival of functional strains. The first fermentation needs high-temperature compost fermentation, and aims to ferment and decompose materials such as waste bacterium sticks (organic fertilizer materials can be used after being fully decomposed, otherwise seedlings can be burned). At this time, if the functional strain is added, the functional strain is killed under high temperature and anaerobic conditions. Therefore, the second stage fermentation is needed, the temperature is controlled within the range suitable for the growth of the strains, and functional strains are added to ensure the survival rate of the strains.

Further, in step 3), the preparation method of the charcoal mixture comprises the following steps: inoculating an inorganic phosphate solubilizing functional strain into a first PKO liquid culture medium, culturing for 3-10 days at 28 ℃ to obtain a functional strain, adding the functional strain into a second PKO liquid culture medium, uniformly mixing to obtain a strain solution, adding the strain solution into the charcoal powder until the charcoal powder is fully wetted, and standing for 30-100 min.

The charcoal has a loose and porous structure, so that the functional strains are mixed with the charcoal firstly to provide habitat for the functional strains, and the survival rate and the quality guarantee period of the functional strains are improved. In addition, the biochar is a good soil conditioner, and has the effects of retaining water, improving soil acidity and alkalinity, adsorbing toxic and harmful heavy metals and the like. Standing for 30-100min to allow the charcoal to fully absorb the bacterial liquid and allow the functional strains to fully enter the charcoal aperture.

In addition, the PKO medium comprises 20 parts of glucose, 3 parts of tricalcium phosphate (acidic), 0.5 part of NaCl, MgSO₄·7H₂0.1 part of O, 0.2 part of KCl, 0.5 part of yeast extract and MnSO₄·4H₂0.002 part of O and FeSO₄·7H₂0.002 part of O.

Further, the porosity of the biochar is more than 0.5, and the methylene blue adsorption value is more than 16, so that the functional strains can have enough habitats; the biochar needs to be screened by a 100-mesh sieve so as to meet the requirement of the appearance and shape of the fertilizer.

Further, the inorganic phosphate solubilizing bacteria functional strain is a native strain of the tea-oil tree forest and is obtained by separating collected tea-oil tree forest soil.

The inorganic phosphate solubilizing bacteria functional strain is separated from the oil-tea camellia forest soil environment, belongs to the original indigenous strain of the oil-tea camellia forest, has stronger adaptability after being applied, can quickly and durably survive and play a phosphate solubilizing function

Further, in the preparation of the charcoal mixture, the functional strains account for 3-6 parts by weight, the newly-prepared PKO liquid culture medium accounts for 130-160 parts by weight, and the biochar accounts for 60-90 parts by weight.

The invention has the beneficial effects that:

1. the main raw material of the invention is the waste mushroom stick in the production of edible mushrooms, which can consume a large amount of the waste resource and has low cost. Meanwhile, waste is changed into valuable, the environment is protected, and the burden of environmental pollution is reduced.

2. The bacterial fertilizer obtained by the invention has beneficial functional strains capable of decomposing phosphorus elements in soil, can obviously increase the content of available phosphorus in the soil, and solves the problems of soil hardening, phosphorus deficiency and the like of the tea-oil tree forest.

3. The phosphorus-release biological bacterial fertilizer special for the oil tea prepared by the process of the invention contains 1.0 multiplied by 10 effective viable count⁸CFU/kg can obviously improve the population abundance of beneficial microorganisms in the oil tea field.

Detailed Description

The technical solutions of the present invention are described in further detail below, but the scope of the present invention is not limited to the following.

Example 1

The method for preparing the phosphorus-release biological bacterial fertilizer special for the oil tea comprises the following specific steps:

(1) crushing the waste edible fungus sticks and the wheat straws by a crusher, and sieving by a 50-mesh sieve for later use;

(2) mixing the waste edible fungus sticks with the wheat straws, uniformly mixing, adding water, and pre-wetting for 6 hours. Wherein, useless fungus stick: wheat straw: the water ratio is 10: 1: 10;

(3) fermentation in the first stage: adding a pre-wetted mixture into a commercially available EM (effective microorganism) leavening agent, wherein the mass ratio of the pre-wetted mixture to the commercially available EM leavening agent is 3000: 1, then adding urea to adjust the carbon-nitrogen ratio to 20: 1, fully and uniformly mixing, and then performing first-stage fermentation, wherein the width of a stockpile is 0.8m, and the height of the stockpile is 1.3 m. The indexes such as maximum temperature, turnover number and rotten days are measured at the 0 th, 6 th, 12 th, 18 th, 24 th, 30 th, 36 th, 42 th, 48 th, 54 th, 60 th, 66 th and 72 th days of fermentation, respectively. After the materials are thoroughly decomposed, spreading the materials to reduce the temperature to room temperature for later use; (rule for recording the number of turns: turning the material once the temperature of the material exceeds 50 degrees.)

(4) And (3) functional strain culture: inoculating the inorganic phosphorus-decomposing functional strain into a PKO liquid culture medium, and carrying out shake flask culture at 28 ℃ for 7 days (the PKO liquid culture medium is sterilized at 121 ℃, and the inorganic phosphorus-decomposing functional strain is separated from the oil-tea camellia forest soil environment);

(5) pretreatment of functional strains: adding 5 parts of cultured strains into 155 parts of fresh PKO liquid culture medium which is newly configured, uniformly mixing, slowly pouring 160 parts of strain solution into 80 parts of charcoal powder until the charcoal is fully wetted, and standing for 30min to obtain a charcoal mixture;

(6) and (3) second-stage fermentation: and (3) adding the fungus charcoal mixture pretreated in the step (5) into the fermented and decomposed material in the step (3), fully and uniformly mixing, adding 2 parts of egg shell powder, spreading and fermenting in a ventilated room, controlling the thickness of the material to be 6cm, controlling the indoor humidity to be 60%, controlling the temperature to be 28 ℃, and sampling and determining the effective viable count in the 0 th, 3 rd, 6 th, 9 th, 12 th, 15 th and 18 th days of experiments respectively. During which the material was turned every 6 d.

(7) And after the second stage of fermentation is finished, adjusting the water content of the materials to 25-35%, and then sieving to obtain the fertilizer.

Example 2

The preparation method of the biological bacterial fertilizer comprises the following specific steps:

(1) crushing the waste edible fungus sticks and the wheat straws by a crusher, and sieving by a 50-mesh sieve for later use;

(2) mixing the waste edible fungus sticks with the wheat straws, uniformly mixing, adding water, and pre-wetting for 6 hours. Wherein, useless fungus stick: wheat straw: the water ratio is 10: 1: 10;

(3) fermentation in the first stage: adding a pre-wetted mixture into a self-made leavening agent, wherein the mass ratio of the pre-wetted mixture to the self-made leavening agent is 3000: 1, then adding urea to adjust the carbon-nitrogen ratio to 20: 1, fully and uniformly mixing, and then carrying out first-stage fermentation, wherein the stacking width is 0.8m, and the height is 1. The indexes such as maximum temperature, turnover number and rotten days are measured at the 0 th, 6 th, 12 th, 18 th, 24 th, 30 th, 36 th, 42 th, 48 th, 54 th, 60 th, 66 th and 72 th days of fermentation, respectively. Spreading the material to reduce the temperature to room temperature for later use after the material is thoroughly decomposed, (the leaven comprises 2 parts of lactobacillus, 3 parts of bacillus subtilis, 3 parts of ralstonia solanacearum and 3 parts of bacillus amyloliquefaciens by weight part), (the record rule of the turning times is that once the material temperature exceeds 50 ℃, the material needs to be turned over)

(4) And (3) functional strain culture: inoculating inorganic phosphorus-decomposing functional strains into a PKO liquid culture medium, and carrying out shake flask culture at 28 ℃ for 7 days, (wherein the inorganic phosphorus-decomposing functional strains are separated from the oil-tea camellia forest soil environment);

(5) pretreatment of functional strains: adding 5 parts of cultured strains into 155 parts of fresh PKO liquid culture medium which is newly configured, uniformly mixing, slowly pouring 160 parts of strain solution into 80 parts of charcoal powder until the charcoal is fully wetted, and standing for 30min to obtain a charcoal mixture;

(6) and (3) second-stage fermentation: and (3) adding the fungus charcoal mixture pretreated in the step (5) into the fermented and decomposed material in the step (3), fully and uniformly mixing, adding 2 parts of egg shell powder, spreading and fermenting in a ventilated room, controlling the thickness of the material to be 8cm, controlling the indoor humidity to be 60%, controlling the temperature to be 28 ℃, and sampling and determining the effective viable count in the 0 th, 3 th, 6 th, 9 th, 12 th, 15 th and 18 th days of experiments respectively.

Turning the materials once every 6d in the period;

(7) and after the second stage of fermentation is finished, adjusting the water content of the materials to 25-35%, and then sieving to obtain the fertilizer.

Example 3

The preparation method of the biological bacterial fertilizer comprises the following specific steps:

(1) crushing the waste edible fungus sticks and the wheat straws by a crusher, and sieving by a 50-mesh sieve for later use;

(2) mixing the waste edible fungus sticks with the wheat straws, uniformly mixing, adding water, and pre-wetting for 12 hours. Wherein, useless fungus stick: straw: the water ratio is 20: 1: 20;

(3) fermentation in the first stage: adding a pre-wetted mixture into a self-made leavening agent, wherein the mass ratio of the pre-wetted mixture to the leavening agent is 5000: 1, then adding urea to adjust the carbon-nitrogen ratio to 25: 1, fully and uniformly mixing, and then carrying out first-stage fermentation, wherein the width of a stockpile is 1.2m, and the height of the stockpile is 1.5 m. The indexes such as maximum temperature, turnover number, and decomposing days were measured at 0 th, 6 th, 12 th, 18 th, 24 th, 30 th, 36 th, 42 th, 48 th, 54 th, 60 th, 66 th, and 72 th days of fermentation, respectively. Spreading the materials to reduce the temperature to room temperature for later use after the materials are thoroughly decomposed, (the leaven comprises 2 parts of lactobacillus, 5 parts of bacillus subtilis, 5 parts of ralstonia solanacearum and 3 parts of bacillus amyloliquefaciens by weight part), (the record rule of the turning times is that once the temperature of the materials exceeds 50 ℃, the materials need to be turned over)

(4) And (3) functional strain culture: inoculating the inorganic phosphorus-decomposing functional strain into a PKO liquid culture medium, and carrying out shake flask culture at 28 ℃ for 7 days (the PKO liquid culture medium is sterilized at 121 ℃, and the inorganic phosphorus-decomposing functional strain is separated from the oil-tea camellia forest soil environment);

(5) pretreatment of functional strains: adding 5 parts of cultured strains into 155 parts of fresh PKO liquid culture medium which is newly configured, uniformly mixing, slowly pouring 160 parts of strain solution into 80 parts of charcoal powder until the charcoal is fully wetted, and standing for 60min to obtain a charcoal mixture;

(6) and (3) second-stage fermentation: and (3) adding the fungus charcoal mixture pretreated in the step (5) into the fermented and decomposed material in the step (3), fully and uniformly mixing, adding 2 parts of egg shell powder, spreading and fermenting in a ventilated room, controlling the thickness of the material to be 10cm, controlling the indoor humidity to be 60%, controlling the temperature to be 28 ℃, and sampling and determining the effective viable count in the 0 th, 3 th, 6 th, 9 th, 12 th, 15 th and 18 th days of experiments respectively. During which the material was turned every 6 d.

(7) And after the fermentation is finished, adjusting the water content of the material to 25-35%, and then sieving to obtain the fertilizer.

Example 4

The preparation method of the biological bacterial fertilizer comprises the following specific steps:

(1) crushing the waste edible fungus sticks and the wheat straws by a crusher, and sieving by a 50-mesh sieve for later use;

(2) mixing the waste edible fungus sticks with the wheat straws, uniformly mixing, adding water, and pre-wetting for 24 hours. Wherein, useless fungus stick: straw: the water ratio is 30: 1: 40;

(3) fermentation in the first stage: adding a self-made leavening agent into the pre-wetted mixture, wherein the mass ratio of the pre-wetted mixture to the leavening agent is 6000: 1, then adding urea to adjust the carbon-nitrogen ratio to 30: 1, fully and uniformly mixing, and then carrying out first-stage fermentation, wherein the width of a stockpile is 1.5m, and the height of the stockpile is 1.8 m. The indexes such as maximum temperature, turnover number, and decomposing days were measured at 0 th, 6 th, 12 th, 18 th, 24 th, 30 th, 36 th, 42 th, 48 th, 54 th, 60 th, 66 th, and 72 th days of fermentation, respectively. Spreading the materials to reduce the temperature to room temperature for later use after the materials are thoroughly decomposed, (the leaven comprises 3 parts of lactic acid bacteria, 5 parts of bacillus subtilis, 5 parts of ralstonia solanacearum and 3 parts of bacillus amyloliquefaciens by weight part), (the record rule of the turning times is that once the temperature of the materials exceeds 50 ℃, the materials need to be turned over)

(4) And (3) functional strain culture: inoculating the inorganic phosphorus-decomposing functional strain into a PKO liquid culture medium, and carrying out shake flask culture at 28 ℃ for 7 days (the PKO liquid culture medium is sterilized at 121 ℃, and the inorganic phosphorus-decomposing functional strain is separated from the oil-tea camellia forest soil environment;

(5) pretreatment of functional strains: adding 5 parts of cultured strains into 155 parts of fresh PKO liquid culture medium which is newly configured, uniformly mixing, slowly pouring 160 parts of strain solution into 80 parts of charcoal powder until the charcoal is fully wetted, and standing for 90min to obtain a charcoal mixture;

(6) and (3) second-stage fermentation: and (3) adding the fungus charcoal mixture pretreated in the step (5) into the fermented and decomposed material in the step (3), fully and uniformly mixing, adding 2 parts of egg shell powder, spreading and fermenting in a ventilated room, controlling the indoor humidity to be 60% and the temperature to be 28 ℃, and sampling and determining the number of effective viable bacteria in the 0 th, 3 rd, 6 th, 9 th, 12 th, 15 th and 18 th days of experiments respectively. Turning the materials once every 6d in the period;

(7) and after the second stage of fermentation is finished, adjusting the water content of the materials to 25-35%, and then sieving to obtain the fertilizer.

Experimental example 1

The data of the embodiments 1 to 4 are summarized, and the indexes of the obtained fertilizer are detected according to the national standard NY525-2012, and the test results are shown in tables 1 and 2 (whether the table meets the national standard only contains material indexes, but does not contain effective viable bacteria number indexes).

TABLE 1 indexes of the first stage fermentation materials

As can be seen from Table 1: under the condition of the same dosage, the maximum fermentation temperature of the commercially available microbial inoculum is only 48 ℃, and the fermentation temperature of the self-made microbial inoculum can reach 56 ℃, so that the self-made microbial inoculum is presumed to be more suitable for fermentation of organic fertilizers taking waste bacteria sticks as main raw materials. Under the same conditions of the fermentation inoculum, the fermentation temperature of the example 4 is the highest, but the decomposing days are longer than that of the example 3, and the turning times are the least as compared with the example 3. The materials obtained in the fermentation tests of the 4 embodiments all meet the specification of each index of the industrial standard NY 525-2015, and each index of the embodiment 3 is optimal. From the viewpoint of production cost and various indexes, the optimal fermentation conditions in example 3 are more suitable.

TABLE 2 indexes of the second stage fermentation materials

The second stage fermentation aims at planting functional strains and ensuring the effective viable count. Under the condition that various fermentation conditions are consistent, the key factor influencing the viable count is the thickness of the stockpile. As is clear from Table 2, in example 1, the maximum viable cell count was 6X 10 at 16d of fermentation⁷CFU/kg, the effective viable count begins to decrease after 16 days; example 2 maximum viable count of 7X 10 at fermentation 12d⁷CFU/kg, the effective viable count begins to decrease after 12 days; example 3 maximum viable count was 1.2X 10 at fermentation time 9d⁸CFU/kg, the effective viable count begins to decrease after 9 days; example 4 the maximum viable count reached at 9d of fermentation, 1.3X 10⁸CFU/kg, the effective viable count began to decrease after 9 days. It can be assumed that the second stage fermentation is optimized for the fermentation conditions of example 4.、

The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.