Method for realizing high-density fermentation of lactic acid bacteria and simultaneously producing calcium lactate by using shell powder

Abstract

The invention provides a preparation method for realizing high-density fermentation of lactic acid bacteria and simultaneously producing calcium lactate by using shell powder, and belongs to the field of microbial fermentation production. By using the shell powder and utilizing waste of the shell powder, the problem of too low pH caused by lactic acid production during high-glucose fermentation of lactic acid bacteria can be solved, acid produced in the lactic acid bacteria fermentation process is neutralized, the growth density of lactic acid bacteria is improved, a large amount of calcium lactate can be produced, and the utilization additional value of the shell powder is improved; and high-density fermentation of lactic acid bacteria and simultaneous production of calcium lactate are realized. Multiple purposes of efficiently utilizing the shell powder, producing high-yield lactic acid bacteria, producing calcium lactate with high added value and the like are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of high-density culture of lactic acid bacteria, and in particular to a method for realizing high-density fermentation of lactic acid bacteria and simultaneously producing calcium lactate by using shell powder. Background Art

[0002] Lactic acid bacteria is a general term for a class of non-pathogenic Gram-positive bacteria that can ferment carbohydrates and produce lactic acid as the terminal metabolite. Lactic acid bacteria are a class of acid-resistant bacteria that can use glucose-like substances for fermentation to produce lactic acid. It is widely distributed in nature, mainly existing in some fermented foods and the human gastrointestinal tract. It is a class of probiotic bacteria and is considered to be the safest strain of bacteria. It has been proven to have significant health functions for the human body in many aspects. Lactic acid bacteria are widely used in industrial fields such as food, medicine, and feed, and are closely related to human health and production and life. In addition, lactic acid bacteria have antibacterial effects and have also attracted much attention in the field of food preservatives.

[0003] With the continuous development of the market economy and the continuous improvement of people's living standards, their needs are also changing all the time. Obtaining the highest bacterial concentration and its product benefits at the lowest cost has become the focus of the entire production and fermentation industry.

[0004] High-density culture refers to the use of certain culture techniques and equipment to cultivate bacteria, which significantly increases the density of bacteria compared to conventional culture, thereby increasing the productivity of the target product. In the modern fermentation industry, high-density culture technology is widely used in the culture process of lactic acid bacteria. Its ultimate goal is to use new fermentation technology, based on the proliferation dynamics, nutrition and metabolism of lactic acid bacteria, through some feeding operations, constant pH fermentation and other technologies, to achieve the purpose of high-density culture on the basis of saving energy and reducing production costs. Generally, the fermentation density of bacteria is increased by optimizing the carbon-nitrogen ratio of the culture medium, the culture temperature, and the pH value of the culture medium. Most lactic acid bacteria are anaerobic or facultative anaerobic, and their anti-oxidant stress mechanism is imperfect. The reactive oxygen present in the environment and generated by the metabolic process of the strain itself seriously threatens the survival of the strain. Therefore, the high-density culture of lactic acid bacteria is generally carried out under anaerobic conditions. The cell density (OD 600 ) is higher than that cultured under anaerobic conditions, but the bacterial activity (number of live bacteria) is much lower than that under anaerobic conditions. In the later stage of fermentation culture, the number of live bacteria often shows a downward trend.

[0005] Chinese patent CN113234656A discloses a high-density fermentation process for fermenting lactic acid bacteria, which belongs to the field of lactic acid bacteria culture technology. The activated Lactobacillus is placed in a high-density fermentation culture tank, which contains 20-30 parts of oligosaccharides, 10-15 parts of jujube extract, 6-15 parts of beef powder, 5-10 parts of yeast powder, and 10-15 parts of soy protein isolate by weight. Adding jujube extract to the lactic acid bacteria culture tank can have multiple biological activities such as anti-oxidation, hypoglycemic, anti-aging, anti-inflammatory, anti-viral, and anti-tumor, which can effectively help lactic acid bacteria improve the activity of fungi and facilitate the improvement of lactic acid bacteria survival rate; however, it is necessary to continuously add nutrient solution during the culture process to facilitate the fermentation cycle of fungi.

[0006] At present, the scale of shellfish farming in my country has developed rapidly, and the utilization rate of shells has been low for a long time. This not only causes waste of resources, but also has adverse effects on the environment. Shell is a natural organic-inorganic composite material, the main components of which are 95% CaCO3 and 5% organic matter. Therefore, fermentation by acid-producing bacteria is a possible means of utilizing shell powder waste. On the one hand, when lactic acid bacteria ferment with high sugar, the low pH caused by the production of lactic acid is one of the main reasons affecting the high-density growth of lactic acid bacteria. Therefore, the neutralization of shell powder on acid may be an effective means to improve the fermentation capacity of lactic acid bacteria. On the other hand, the use of lactic acid bacteria to ferment naturally calcium-rich shell powder can produce a large amount of calcium lactate, further increasing the added value of shell powder utilization.

[0007] The high-density fermentation method of lactic acid bacteria reported in the literature is usually carried out by improving the culture medium components and adjusting the fermentation conditions, which cannot achieve multiple purposes such as efficient utilization of shell powder, high-yield lactic acid bacteria, and production of high-value-added calcium lactate. In addition, the scale of shellfish farming in my country has developed rapidly, and the utilization rate of shells has been low for a long time. Not only has it caused a waste of resources, but it has also had an adverse effect on the environment. Therefore, there is an urgent need for an improved method of utilizing shell powder to ferment lactic acid bacteria at a high density and simultaneously produce calcium lactate to achieve multiple purposes such as efficient utilization of shell powder, high-yield lactic acid bacteria, and production of high-value-added calcium lactate. Summary of the invention

[0008] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a technology for realizing high-density fermentation of lactic acid bacteria and producing calcium lactate at the same time by using shell powder. On the one hand, shell powder is used to neutralize the extremely acidic environment caused by the acid production of lactic acid bacteria under high sugar conditions, thereby realizing high-density culture of lactic acid bacteria; on the other hand, a large amount of calcium carbonate in shell powder can be combined with calcium lactate to produce calcium lactate with higher added value (C3H6O3+CaCO3→Ca(C3H5O3)2+CO2+H2O).

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A preparation method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder, comprising the following steps:

[0011] (1) inoculating the activated lactic acid bacteria strain into a lactic acid bacteria culture medium to which shell powder has been added; preferably, selecting a high-quality lactic acid bacteria strain that has vigorous growth and can reach a logarithmic growth phase within 4-8 hours after inoculation for activation;

[0012] (2) Fermentation is carried out at 35-38° C. After the culture is completed, the bacteria are harvested and / or the produced calcium lactate is collected.

[0013] In a specific embodiment, the step of collecting the produced calcium lactate is specifically performed by centrifuging the fermentation broth, discarding the supernatant, and drying the solid precipitate. More specifically, the fermentation broth is centrifuged at 8000 rpm for 5 minutes, the supernatant is discarded, and the solid precipitate is dried at 70°C.

[0014] Preferably, the inoculation amount of the lactic acid bacteria strain in step (1) is 1%-3%, and the amount of glucose added to the lactic acid bacteria culture medium is 1%-15%. More preferably, the amount of glucose added in step (1) is 1%-10%, for example 4%-10%, for example 4%, 5%, 6%, 7%, 8%, 9% or 10%.

[0015] Furthermore, the lactic acid bacteria culture medium in step (1) has a formula of MRS culture medium (grape grain is added according to the above content) in addition to glucose.

[0016] Furthermore, except for glucose, the other ingredients of the MRS culture medium are composed of the following raw materials by weight: peptone 10g, beef extract powder 5g, Tween 80 1mL, K2HPO4 2g, yeast extract powder 4g, triammonium citrate 2g, MgSO4 0.2g, MnSO4 0.05g, sodium acetate 5g, and water 1000mL.

[0017] Furthermore, in the above-mentioned high-density lactic acid bacteria fermentation technology using shell powder, a preferred embodiment is that the shell powder in step (1) can be summer clams.

[0018] Furthermore, in the above-mentioned high-density lactic acid bacteria fermentation technology using shell powder, a preferred solution is that the amount of shell powder added in step (1) is 1%-5%; preferably 2%-4%, for example 3%.

[0019] Furthermore, in the above-mentioned high-density lactic acid bacteria fermentation technology using shell powder, a preferred embodiment is that the fermentation time in step (2) is 24-36 hours.

[0020] The excellent technical effects achieved by the present invention are:

[0021] The preparation method provided by the present invention utilizes shell powder to ferment lactic acid bacteria at a high density and simultaneously produces calcium lactate, which improves the environmental pollution and resource waste caused by the failure to fully utilize shell powder to a certain extent; and neutralizes lactic acid, thereby improving the problem that the pH value of the fermentation liquid is too low due to the large amount of lactic acid produced by lactic acid bacteria under high-sugar conditions, which affects the high-density fermentation of lactic acid bacteria; in addition, the lactic acid in the fermentation liquid combines with the calcium carbonate in the shell powder to produce calcium lactate with higher added value; thereby achieving multiple purposes such as efficient utilization of shell powder, high-yield lactic acid bacteria, and production of high-value-added calcium lactate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The invention is further described in the following non-limiting drawings:

[0023] Figure 1 : Growth of lactic acid bacteria after fermentation with different contents of glucose added to the culture medium.

[0024] Figure 2 : Changes in pH of fermentation broth before and after adding different contents of glucose-shell powder into the culture medium.

[0025] Figure 3 : Changes in colony count in fermentation broth after adding different contents of glucose-shell powder into the culture medium.

[0026] Figure 4 : Changes in lactic acid content in fermentation broth after adding different contents of glucose-shell powder into the culture medium.

[0027] Figure 5 : Changes in glucose content in fermentation broth after adding different amounts of glucose-shell powder into the culture medium.

[0028] Figure 6 : Changes in calcium lactate content in fermentation broth after adding different contents of glucose-shell powder to the culture medium. DETAILED DESCRIPTION

[0029] The invention is now described in the following non-limiting examples.

[0030] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0031] In the present invention, the comparative experiment of different parameters is performed as follows:

[0032] (1) Weigh different proportions of shell powder-glucose and add them to the MRS solution from which glucose has been removed. The specific formula is 10 g of peptone, 5 g of beef extract powder, 1 mL of Tween 80, 2 g of K2HPO4, 4 g of yeast extract powder, 2 g of triammonium citrate, 0.2 g of MgSO4, 0.05 g of MnSO4, 5 g of sodium acetate, and 1000 mL of water. The shell powder-glucose addition ratio is shown in Table 1;

[0033] (2) MRS culture medium with different proportions of shell powder and glucose was mixed evenly and sterilized by high pressure steam at 115°C and 1.1 KPa for 20 min;

[0034] (3) Inoculate the lactic acid bacteria seed solution into the sterilized MRS medium containing shell powder and glucose in different proportions at a 1% inoculation rate, and culture at 37° C. for 24-36 h. The culture time is the same;

[0035] (4) The OD600, colony count, pH value, glucose-lactic acid content and calcium lactate content of the fermentation broth were measured.

[0036] Table 1 Shell powder-glucose addition ratio

[0037]

[0038] Use a pH meter to measure the pH of the bacterial solution. Use a calibrated pH meter, rinse the probe repeatedly with distilled water before use and dry it with paper. Shake the bacterial solution thoroughly, insert the clean pH meter probe into the bacterial solution, and wait for the reading to stabilize. The reading on the pH meter screen is the pH of the bacterial solution.

[0039] Example 1: Determination of colony count using a spectrophotometer

[0040] Take 3mL of the fermented bacterial solution, centrifuge at 4000rpm for 2min, add to an equal amount of PBS buffer, and homogenize to make the colonies in the suspension evenly distributed. Set the wavelength with a spectrophotometer, generally 600nm. Take 3mL of sterile PBS buffer and measure its absorbance with a spectrophotometer. Use this as the base value to perform blank calibration on the sample. Use the same method to measure the absorbance of each sample and subtract the absorbance obtained after blank calibration.

[0041] The results are visible Figure 1 The growth of lactic acid bacteria after fermentation with different glucose contents in the culture medium increased with OD 600 With the increase of glucose addition, when the glucose addition amount was 5%, OD 600 =10.12, reaching the highest value.

[0042] Example 2: Measurement of total bacterial count

[0043] The total number of colonies is determined by the plate counting method. Take 100 μL of sample and dilute it 10 times with physiological saline, then draw 10 μL into solid MRS culture medium and spread it on the plate with a spreader. After culturing at 37°C for 48 hours, count the colonies visible to the naked eye. The colonies are required to be in the effective dilution gradient of 30-300, and the number of colonies in the original sample is calculated according to the dilution multiple.

[0044] The experimental results can be found in Figure 3 It can be seen that the colony count in the fermentation broth after adding different proportions of glucose-shell powder to the culture medium changes. When the glucose addition exceeds 3%, the colony count in the fermentation broth of the shell powder addition group is significantly higher than that of the control group. Among them, when the glucose addition is 4% and the shell powder addition is 1%, the colony count reaches a peak of 17.38*10 9 , which is 4.35 times the colony count of the control group*10 9 4 times of that.

[0045] Example 3: Determination of pH of bacterial solution

[0046] The changes in the pH of the fermentation liquid before and after fermentation with different contents of glucose-shell powder in the culture medium can be seen in Figure 2 When no shell powder is added, when the amount of glucose added exceeds 2%, the pH is about 3.5. The addition of shell powder at different contents can improve the pH of the fermented liquid after fermentation. When the amount of shell powder added is 3%, the pH is about 6 when the amount of glucose added is 2%. When the amount of glucose added is 3%, the pH is about 5.7. When the amount of glucose added is 4%, the pH is about 5.5. It can be seen that the addition of shell powder can effectively neutralize lactic acid and improve the problem of lactic acid bacteria producing a large amount of lactic acid under high sugar conditions, resulting in too low pH of the fermented liquid, which affects the high-density fermentation of lactic acid bacteria.

[0047] Example 4: Determination of glucose and lactic acid content in bacterial culture

[0048] Determination by SBA-40 glucose-lactic acid biosensor analyzer. Centrifuge 1ml of bacterial solution for 5min (8000rpm / min, 4℃), and take the supernatant for testing. Use the calibrated SBA-40 glucose-lactic acid biosensor analyzer for determination, wash the injection needle with the sample for 3 consecutive times, rotate the needle rod, accurately draw 25μl of the sample to be tested, wipe off the liquid outside the needle, clamp the needle rod with filter paper, and quickly wipe the needle in the direction of the needle; when the screen displays "Sample test, please enter the sample", quickly inject the sample into the reaction pool; after the reaction is completed, the result on the screen is the measured value of glucose and lactic acid content.

[0049] Results from Figure 4It can be seen that after adding a certain proportion of shell powder to the culture medium, the lactic acid content in the fermentation liquid decreased with the addition of shell powder, and the lactic acid content decreased most significantly when the shell powder addition was 3%. Therefore, we can speculate that the addition of shell powder neutralized the pH decrease caused by the large amount of lactic acid produced by lactic acid bacteria under high sugar conditions, thereby affecting the colony count and calcium lactate production in the fermentation liquid after fermentation.

[0050] Depend on Figure 5 It can be seen that since the addition of shell powder can increase the number of colonies in the fermentation liquid and increase the consumption of glucose in the fermentation liquid, the addition of shell powder in different proportions can reduce the glucose content in the fermentation liquid. The most obvious is that when the glucose addition amount is 3%, 4%, and 5%, the glucose content in the fermentation liquid of the control group is 575, 538, and 529 mg / dl. When the shell powder addition amount is 3%, and the glucose addition amount is 3%, 4%, and 5%, the glucose content in the fermentation liquid is only 38, 49, and 56 mg / dl. Among them, 10dl is 1L.

[0051] Example 5: Determination of calcium lactate

[0052] Take the fermentation liquid and centrifuge it at 8000rpm for 5min, discard the supernatant, and dry the solid precipitate at 70℃. Weigh about 0.3g of the dried sample to an accuracy of 0.0002g, dissolve it in 50mL of water with hydrochloric acid solution, add 15mL of disodium ethylenediaminetetraacetic acid standard titration solution dropwise while stirring, add 5mL of sodium hydroxide solution and 0.1g of calcium reagent sodium carboxylate indicator, and titrate with disodium ethylenediaminetetraacetic acid standard titration solution until a blue color appears as the end point.

[0053] The results are as follows Figure 6 As shown in the figure, after adding different amounts of glucose-shell powder to the culture medium, the content of calcium lactate in the fermentation broth increased. When the amount of shell powder added was 3% and the amount of glucose added was 10%, the content of calcium lactate in the fermentation broth was the highest, which was 40.5g / L. This shows that the lactic acid in the fermentation broth combines with the calcium carbonate in the shell powder to produce calcium lactate with higher added value.

Claims

1. A method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder, comprising the following steps: (1) inoculating the activated lactic acid bacteria strain into a lactic acid bacteria culture medium to which shell powder has been added; preferably, selecting a high-quality lactic acid bacteria strain that has vigorous growth and can reach the logarithmic growth phase within 4-8 hours after inoculation for activation; (2) Fermentation is carried out at 35-38° C. After the fermentation is completed, the bacteria are harvested and / or the produced calcium lactate is collected.

2. The method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder as claimed in claim 1, characterized in that: The step of collecting the produced calcium lactate is specifically performed by centrifuging the fermentation liquid, discarding the supernatant, and drying the solid precipitate; more specifically, the fermentation liquid is centrifuged at 8000 rpm for 5 minutes, the supernatant is discarded, and the solid precipitate is dried at 70°C.

3. The method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder according to claim 1 or 2, characterized in that: The inoculation amount of the lactic acid bacteria in step (1) is 1%-3%.

4. The method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder according to any one of claims 1 to 3, characterized in that: The amount of glucose added to the lactic acid bacteria culture medium is 1%-15%.

5. The method for preparing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder according to claim 4, characterized in that: The amount of glucose added in step (1) is 1%-10%, further 4%-10%, for example 4%, 5%, 6%, 7%, 8%, 9% or 10%.

6. The method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder according to any one of claims 1 to 5, characterized in that: The lactic acid bacteria culture medium in step (1) is prepared according to the formula of MRS culture medium except glucose.

7. The method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder according to claim 6, characterized in that: Except for glucose, the other ingredients of the MRS culture medium are composed of the following raw materials by weight: peptone 10g, beef extract powder 5g, Tween 80 1mL, K2HPO4 2g, yeast extract powder 4g, triammonium citrate 2g, MgSO4 0.2g, MnSO4 0.05g, sodium acetate 5g, and water 1000mL.

8. The method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder according to any one of claims 1 to 7, characterized in that: The shell powder in step (1) is shellfish.

9. The method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder according to any one of claims 1 to 8, characterized in that: The amount of shell powder added in step (1) is 1%-5%; preferably 2%-4%, for example 3%.

10. The method for producing calcium lactate by high-density fermentation of lactic acid bacteria using shell powder according to any one of claims 1 to 9, characterized in that: The fermentation time in step (2) is 24-36 hours.

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