CN109022317B - Preparation method of clostridium butyricum powder - Google Patents
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Abstract
The invention belongs to the technical field of preparation of bacterium powder, and particularly relates to a preparation method of clostridium butyricum bacterium powder. And (3) carrying out spray drying on the clostridium butyricum fermentation liquor, wherein the inlet temperature of the spray drying is 150-180 ℃. The preparation method of the clostridium butyricum powder has the advantages of low production cost, strong process controllability, high viable count of the obtained clostridium butyricum powder, good normal-temperature storage stability of products and important value in promoting the market of clostridium butyricum.
Description
Technical Field
The invention belongs to the technical field of preparation of bacterium powder, and particularly relates to a preparation method of clostridium butyricum bacterium powder.
Background
Clostridium butyricum (Clostridium butyricum) is also known as Clostridium butyricum, Clostridium butyricum. The clostridium butyricum is a gram-positive bacterium, is obligate anaerobe, has a straight rod shape or a microbend, and has a spore endogenic; the bacterial colony is irregular round, white or milk white and has acid odor; the suitable growth temperature is 25-37 ℃, and the suitable growth pH is 4.0-9.8; can utilize various carbon sources such as glucose, sucrose, fructose, maltose, starch, cellulose and the like, and the fermentation product is mainly butyric acid. The clostridium butyricum belongs to one of normal flora in human and animal intestinal tracts, and has the probiotic characteristics of regulating intestinal micro-ecological balance, enhancing immunity, generating probiotic substances and the like.
Clostridium butyricum has the following probiotic properties:
1) regulating intestinal microecological balance: microecological balance refers to the dynamic balance of a physiological combination of normal microbial flora and its host ecological environment during long term evolution. The clostridium butyricum can promote the proliferation of beneficial intestinal flora and inhibit the growth of partial pathogenic bacteria, thereby maintaining the steady state of the intestinal flora and being beneficial to the health of the intestinal tract. The clostridium butyricum can produce amylase and cellulase, and the amylase can hydrolyze starch and cellulose in intestinal tracts to produce small molecular sugar for the growth and utilization of probiotics such as bifidobacterium and the like. The clostridium butyricum fermentation liquor extract is added into a culture medium of lactobacillus acidophilus, bifidobacterium and streptococcus faecalis, so that the number of live bacteria in an experimental group is far higher than that in a control group, the clostridium butyricum can promote the growth of the lactobacillus acidophilus, the bifidobacterium and the streptococcus faecalis, and the same conclusion is drawn through experimental researches such as jupiti and the like. The clostridium butyricum can effectively inhibit the growth of intestinal pathogenic bacteria, experiments such as old autumn red and the like find that escherichia coli ACCC10080, Shigella ACCC01690, salmonella SM0807 and clostridium butyricum are mixed and cultured, after 24 hours, compared with the independent culture of pathogenic bacteria, the escherichia coli is reduced by 4 orders of magnitude, the Shigella is reduced by 3 orders of magnitude, and the salmonella is reduced by 5 orders of magnitude, which indicates that the clostridium butyricum can effectively inhibit the growth of the escherichia coli ACCC10080, the Shigella ACCC01690 and the Salmonella SM 0807. Zhangbo and other researches find that the Clostridium butyricum O3131 can obviously inhibit enterohemorrhagic Escherichia coli, Shigella dysenteriae, Salmonella choleraesuis and Vibrio choleraesuis. Compared with single culture, enterohemorrhagic Escherichia coli is reduced by 4 orders of magnitude, shigella dysenteriae is reduced by 4 orders of magnitude, salmonella choleraesuis is reduced by 7 orders of magnitude, and the number of live vibrio choleraesuis almost zero. For the inhibition effect of animal intestinal pathogenic bacteria, the antagonism effect of clostridium butyricum RH2 and pig Escherichia coli C83902, chicken Escherichia coli C83851, and chicken Salmonella pullorum Sg9 is discussed by Tangbaoying and the like, and the viable count of the pathogenic bacteria can be reduced to 0 after the culture is finished by adjusting the viable count proportion. Experiments such as Shebuigui and the like show that the clostridium butyricum B1 has obvious inhibition effect on porcine escherichia coli K88, K99 and O139, staphylococcus aureus and salmonella enteritidis O4 Hi.
2) Enhancing the immunity of the organism: the clostridium butyricum can enhance the immunity of animal organisms and promote the growth of animals. The test of Liu pavilion is to explore the influence of clostridium butyricum on the serum immunity index of the young cocks for eggs, and the result shows that the addition of clostridium butyricum in the feed enables the content of immunoglobulin G (IgG) and serum complement C3 in the serum of the young cocks for eggs to be obviously higher than that of a control group, and the average weight of the young cocks for eggs in an experimental group is obviously improved compared with that of the control group. The heat inactivated clostridium butyricum vaccine can activate macrophages and natural killer cells, and the IgA content in the serum of an organism is obviously improved after a certain amount of the vaccine is ingested. The addition of clostridium butyricum in the feed for weaned pigs can improve the content of IgG and C3 in the serum of the piglets, and is similar to the experimental results of Liutinting and the like. When the clostridium butyricum is used for feeding broiler chickens infected by escherichia coli K88, the content of IgA, IgM, IgY, C3 and C4 in blood serum of the broiler chickens is improved compared with that of a control group, which shows that the clostridium butyricum has positive influence on the immune function of the broiler chickens infected by the escherichia coli.
3) Production of probiotic substances: the main product of clostridium butyricum fermentation is butyric acid which is a short-chain fatty acid and plays an important role in maintaining the shape and the function of the colonic epithelial cells. The clostridium butyricum can generate trace organic substances such as B vitamins, VK and the like, is beneficial to maintaining normal physiological functions of human and animals, researches prove that the clostridium butyricum can promote VE absorption, and the VE absorption promoter with the main component of the clostridium butyricum can obviously increase the VE content in the serum of an experimental group. Clostridium butyricum can produce various enzymes, such as amylase, cellulase, pectinase and the like, and the enzymes can decompose saccharides which cannot be directly absorbed in intestinal tracts and produce glucose and the like which are easy to absorb for organisms and other beneficial bacteria in the intestinal tracts. A large amount of gas, such as CO, is also produced during the fermentation of Clostridium butyricum2、H2Etc. wherein H2Widely used in medical research, and proves that the medicine has the functions of resisting oxidation, resisting inflammation, inhibiting apoptosis, etc.
Currently, clostridium butyricum is mainly divided into clinical applications and animal applications. The clostridium butyricum microecological preparation is clinically used for treating diarrhea and enteritis, assisting in treating hand-foot-and-mouth diseases and preventing colorectal tumor. In the treatment of infant pneumonia, the antibiotic is used and the clostridium butyricum dual-combination bacterial powder is orally taken, so that the occurrence of antibiotic-associated diarrhea can be obviously reduced. The effect of the combination of the clostridium butyricum and the mesalazine tablet on treating the ulcerative colitis is more definite than that of the single use of the mesalazine tablet. The clinical curative effect is obviously improved by the conventional therapy for treating rotavirus enteritis and the adjuvant therapy of the infant amino acid combined clostridium butyricum viable bacteria preparation. The clostridium butyricum can effectively inhibit pathogenic bacteria such as shigella dysenteriae, enterohemorrhagic escherichia coli and the like, promotes the growth of probiotics such as bifidobacterium, lactobacillus acidophilus and the like, and has an important effect on maintaining the stable state of intestinal microbial flora, so that the clostridium butyricum has an obvious curative effect on treating diarrhea, enteritis and the like in clinic. Clostridium butyricum combined with ribavirin and interferon therapyThe hand-foot-and-mouth disease of children has obvious effect, and the butyric acid produced by fermentation can promote the repair of intestinal epithelial cells and effectively prevent virus invasion because the activity of macrophages can be enhanced. Clostridium butyricum used in flos Myricae Rubrae etc. acts on SW-480 cell of colon cancer, 1.5 × 107The clostridium butyricum of cfu/mL acts for 48 hours, the colon cancer cells show apoptosis signs, the activity of caspase-3 and caspase-9 is enhanced, the Bax expression of two proteins of the main mitochondrion apoptosis is up-regulated, the Bcl-2 expression is down-regulated, and the tumor cells are subjected to apoptosis. In the aspect of animal application, the clostridium butyricum is used as a feed additive to be applied to poultry, livestock and aquaculture, and can improve the production performance of animals, enhance the resistance of the animals, improve the quality of animal products and save the feeding cost. The clostridium butyricum with the concentration of 0.9 percent is added into the feed of the argyi Yijia broiler (AA broiler) such as the Liulegting, the intestinal villus length of the broiler in an experimental group is increased, which means that the intestinal epithelial cell number of the broiler is increased, and the capability of digesting and absorbing nutrient substances is enhanced, so that the daily weight gain of the broiler is improved, and the death and culling rate and the incidence of the broiler in the experimental group are obviously reduced. The Liuting finds that the average daily gain of piglets can be remarkably improved by the compound feed additive of glutamine and clostridium butyricum; enhancing superoxide dismutase activity and total antioxidant capacity in serum; c3 and C4 levels are increased, and the immunity of piglets is enhanced; relieving intestinal villus atrophy and crypt hyperplasia; improve cecal colony structure. The clostridium butyricum can improve the quantity of bifidobacteria in the middle intestine and the rear intestine of the miichthys miiuy, reduce the quantity of escherichia coli, reduce the quantity of acinetobacter, brevibacterium and enteroaerobacter in the rear intestine, effectively improve the intestinal flora environment of the miichthys miiuy and is beneficial to the health of the miichthys miiuy.
The strong acid environment of the stomach is needed for the probiotics to play a role in the intestinal tract, and the key for the probiotics to play a curative effect is to ensure that a high number of viable bacteria are still kept after entering the intestinal tract. The probiotic preparation mainly comprises a liquid preparation and a solid preparation, wherein the solid preparation can be divided into: powder, granules, capsules, tablets and the like. The liquid preparation generally has the defects of short shelf life, inconvenient transportation, easy pollution and the like, while the solid preparation has long shelf life, high viable count, easy transportation and carrying and convenient use. The common preparation method of the probiotic powder in the solid dosage form comprises the following steps: natural drying, low-temperature drying, vacuum freeze drying and spray drying. Spray drying refers to a drying technique in which a liquid raw material is dispersed into fine droplets by an atomizer, and the solvent component of the droplets is rapidly evaporated in a drying medium to form dry powder. The spray drying technology is characterized in that: the drying time is short, the damage of the effective components in the product is less, and the drying process is simple. Compared with other methods for preparing probiotic bacteria powder, the spray drying method has unique advantages. Although the spray drying method is a relatively common process, how to provide a preparation process of bacterial powder with high viable bacterial count and good stability aiming at different effective components, such as clostridium butyricum liquid products with poor stability, is still a problem to be solved.
Disclosure of Invention
The invention aims to provide a preparation method of clostridium butyricum bacterial powder with high number of viable bacteria.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a preparation method of clostridium butyricum bacterial powder comprises the step of carrying out spray drying on clostridium butyricum fermentation liquor, wherein the inlet temperature of the spray drying is 150-180 ℃.
Further, the liquid fermentation liquor is pretreated before spray drying, and the pretreatment is centrifugal separation.
Further, controlling centrifugal separation parameters, wherein the rotating speed is 3000-5000 r/min during centrifugation, and centrifuging for 5-30 min; preferably 4000 r/min, and centrifuging for 10 min.
Preferably, after centrifugal separation, the supernatant is discarded, sterile liquid is supplemented, and then a protective agent is added for spray drying.
The protective agent is preferably a composition of sodium alginate, maltodextrin, chitosan oligosaccharide, calcium lactate, sodium carboxymethyl cellulose and silicon dioxide, wherein the addition amount of the maltodextrin is 5-30% of the mass of the fermentation broth, the addition amount of the sodium alginate is 5-30% of the mass of the fermentation broth, the addition amount of the chitosan oligosaccharide is 1-10% of the mass of the fermentation broth, the addition amount of the calcium lactate is 1-10% of the mass of the fermentation broth, the addition amount of the sodium carboxymethyl cellulose is 0.05-0.5% of the mass of the fermentation broth, and the addition amount of the silicon dioxide is 0.05-0.5% of the mass of the fermentation broth.
More preferably, the adding amount of sodium alginate is 10% of the mass of the fermentation broth, the adding amount of maltodextrin is 10% of the mass of the fermentation broth, the adding amount of chitosan oligosaccharide is 2% of the mass of the fermentation broth, the adding amount of calcium lactate is 1% of the mass of the fermentation broth, the adding amount of sodium carboxymethyl cellulose is 0.2% of the mass of the fermentation broth, and the adding amount of silicon dioxide is 0.1% of the mass of the fermentation broth.
Further, the fermentation broth is concentrated after centrifugal separation, and then spray-dried.
Specifically, after the fermentation liquor is centrifugally separated, the supernatant is discarded, and sterile water with the volume not more than the volume of the original fermentation liquor is added for concentration.
The concentration factor is preferably not more than 5, and more preferably the concentration factor is 3.
The invention adopts a spray drying method to prepare the bacterial powder. Researches show that the inlet temperature of the sprayer and the treatment mode of fermentation liquor have great influence on the improvement of the number of viable bacteria in the bacterial powder. Only in the aspect of processing the fermentation liquor, whether the fermentation liquor is subjected to centrifugal processing, centrifugal processing parameters, the concentration multiple of the fermentation liquor, whether a protective agent is added, the type and the addition amount of the protective agent and the like all influence the number of viable bacteria in the prepared bacterial powder. The invention has the innovation points of determining the influence factors of the number of the viable bacteria and comprehensively researching the factors, and provides the preparation method of the clostridium butyricum bacterial powder.
Compared with the prior art, the invention has the following advantages:
the preparation method of the clostridium butyricum powder has the advantages of low production cost, strong process controllability, high viable count of the obtained clostridium butyricum powder, good normal-temperature storage stability of products and important value in promoting market application of clostridium butyricum.
Detailed Description
The technical solution of the present invention is illustrated by the following specific examples, but the scope of the present invention is not limited thereto:
the clostridium butyricum fermentation broth used in the present invention was purchased from lilium huinanensis biotechnology limited. A BUCHI B-290 small spray dryer is adopted, and the inlet temperature can be adjusted between 150 ℃ and 200 ℃.
EXAMPLE 1 Effect of inlet temperature
Regulating the inlet temperature of spray drying, directly spray drying the untreated clostridium butyricum fermentation liquor, and obtaining the original bacterial liquid viable count of 5.7 multiplied by 108 cfu/mL, carrying out plate counting on the obtained bacterial powder by using an iron sulfite (a plate counting mode is adopted below), and carrying out plate counting again after standing at 40 ℃ for 10 days. The results are shown in table 1:
TABLE 1 Effect of inlet temperature
Note: viable count 1 is viable count obtained by immediately detecting the spray-dried bacterial powder, namely viable count; the viable count 2 is the viable count after being placed at 40 ℃ for 10 d; the viable count of 3 is the viable count of 3 months after being placed at normal temperature; the mass yield is 100 mL of the mass of the bacterial powder obtained by spraying the fermentation stock solution; the viable bacteria yield is the ratio of the number of viable bacteria in the bacterial powder to the number of viable bacteria in the fermentation stock solution; the survival rate is the ratio of the viable count of the bacteria powder after storage to the viable count of the bacteria powder just after spraying.
Example 2 influence of centrifugal pretreatment of the bacterial liquid
Taking 100 mL of clostridium butyricum fermentation liquor, centrifuging for 10 min under the condition of 4000 rmp, taking bacterial sludge, reducing the bacterial sludge by using isometric sterile water, and adding 20% sodium alginate into treated bacterial suspension; and (3) directly adding 20% sodium alginate into 100 mL of fermentation liquor as a control, and comparing the viable count and the stability of the spray-dried bacterial powder. The inlet temperature of the sprayer during spray drying is 180 ℃.
Three batches of fermentation liquor are tested simultaneously, and the viable count of the bacterial liquid in batch 1 is 7.2 multiplied by 108 cfu/mL, viable count of bacterial liquid 5.7X 10 in batch 28 cfu/mL batch, batch 3 bacterial liquid viable count 2.7X 108 cfu/mL. The results are shown in table 2:
note: the viable count 1 is the number of detected viable cells; the viable count 2 is the viable count after being placed at 40 ℃ for 10 d; the viable count of 3 is the viable count of 3 months after being placed at normal temperature; the mass yield is 100 mL of the mass of the bacterial powder obtained by spraying the fermentation stock solution; the viable bacteria yield is the ratio of the number of viable bacteria in the bacterial powder to the number of viable bacteria in the fermentation stock solution; the survival rate is the ratio of the viable count of the bacteria powder after storage to the viable count of the bacteria powder just after spraying.
The results show that after the fermentation liquor is treated, the strain powder is stable in performance and good in batch stability after being placed at 40 ℃ for 10 days and three months at normal temperature. The difference between the number of the live bacteria after being placed at 40 ℃ for 10 d and the number of the live bacteria after being placed at normal temperature for three months can be found to be small through analyzing data, so that the number of the live bacteria when being placed at 40 ℃ for 10 d can be detected in a later test to represent the stability of the bacterial powder treated by the corresponding method, and meanwhile, the test period is shortened.
Example 3 Effect of centrifugation conditions
Respectively taking 10 mL of fermentation liquor, centrifuging under different conditions in Table 3, discarding the supernatant, adding sterile water to 10 mL, uniformly mixing, and detecting the viable count content in the bacterial suspension, wherein the results are shown in Table 3:
the result shows that the residual quantity of the viable bacteria in the bacterial sludge is the highest when the rotating speed is 4000 rpm and the centrifugation time is 10 min.
EXAMPLE 4 Effect of protective Agents
Taking 100 mL of clostridium butyricum fermentation liquor, centrifuging for 10 min under the condition of 4000 rmp, taking bacterial sludge, reducing the bacterial sludge by using isometric sterile water, and adding different protective agents in the table 4 into treated bacterial suspension, wherein the addition amount of the protective agents is 20 percent of the mass of the fermentation liquor; spray drying was then carried out (sprayer inlet temperature 180 ℃) with the results shown in table 4:
note: the viable count 1 is the number of detected viable cells; the viable count 2 is the viable count after being placed at 40 ℃ for 10 d; the mass yield is 100 mL of the mass of the bacterial powder obtained by spraying the fermentation stock solution; the viable bacteria yield is the ratio of the number of viable bacteria in the bacterial powder to the number of viable bacteria in the fermentation stock solution; the survival rate is the ratio of the viable count of the bacteria powder after storage to the viable count of the bacteria powder just after spraying.
The bacterial powder with fructo-oligosaccharide as a protective agent is seriously agglomerated after being stored for 10 days at 40 ℃, the sucrose, the galacto-oligosaccharide, the inulin and the corn starch are slightly agglomerated, and the bacterial powder obtained by the protective agent has lower viable bacteria quantity and stability. When sodium alginate is used as a protective agent, the bacterial powder has the highest viable count and good stability; when maltodextrin is used as a protective agent, the stability of the fungus powder is the best; the stability of the skimmed milk powder is poor; the viable count and stability of gum arabic are both less than ideal. (survival after high temperature storage to characterize stability).
Sodium alginate and maltodextrin in different proportions are used as protective agents, other parameters are as shown in the table 4, the result is shown in the table 5, and the dosage of the protective agent refers to the ratio of the mass of the protective agent to the mass of fermentation liquor:
note: h: sodium alginate M: maltodextrin
The viable count 1 is the number of detected viable cells; the viable count 2 is the viable count after being placed at 40 ℃ for 10 d; the mass yield is 100 mL of the mass of the bacterial powder obtained by spraying the fermentation stock solution; the viable bacteria yield is the ratio of the number of viable bacteria in the bacterial powder to the number of viable bacteria in the fermentation stock solution; the survival rate is the ratio of the viable count of the bacteria powder after storage to the viable count of the bacteria powder just after spraying.
The experimental result of combining the two protective agents which are used independently can show that when the dosage of the sodium alginate is reduced, the number of the viable bacteria in the bacterial powder is reduced, when the dosage of the maltodextrin is increased, the survival rate is increased, and when the dosages of the two protective agents are the same, namely 10%, the number of the viable bacteria and the survival rate both reach a relatively ideal state.
The amounts of sodium alginate and maltodextrin were 10% of the mass of the fermentation broth, and the contents of chitosan oligosaccharide, calcium lactate, sodium carboxymethylcellulose and silicon dioxide as other components are shown in table 6.
When 2% of chitosan oligosaccharide, 1% of calcium lactate, 0.2% of sodium carboxymethylcellulose and 0.1% of silicon dioxide are selected, the viable bacteria yield and survival rate are the best.
Example 5 Effect of fermentation broth concentration
Taking clostridium butyricum fermentation liquor, wherein the viable count of the fermentation liquor is 5.70 multiplied by 108 cfu/mL, centrifuging for 10 min under 4000 rmp, taking bacterial sludge, adding sterile water, and adjusting the concentration multiple of the clostridium butyricum fermentation liquor by adding the amount of the sterile water (for example, concentrating 3 times is to obtain a concentrated bacterial suspension by adding 100 mL of sterile water into the bacterial sludge obtained after centrifuging 300 mL of fermentation liquor). Adding a protective agent into the treated bacterial suspension, and then carrying out spray drying; the protective agent is: 10% of sodium alginate, 10% of maltodextrin, 2% of chitosan oligosaccharide, 1% of calcium lactate, 0.2% of sodium carboxymethyl cellulose and 0.1% of silicon dioxide, wherein the inlet temperature of a sprayer is 180 ℃. The results are shown in Table 7:
note: the viable count 1 is the number of detected viable cells; the viable count 2 is the viable count after being placed at 40 ℃ for 10 d; the viable count of 3 is the viable count of 3 months after being placed at normal temperature; the mass yield is 100 mL of the mass of the bacterial powder obtained by spraying the fermentation stock solution; the viable bacteria yield is the ratio of the number of viable bacteria in the bacterial powder to the number of viable bacteria in the fermentation stock solution; the survival rate is the ratio of the viable count of the bacteria powder after storage to the viable count of the bacteria powder just after spraying.
The results show that the viable count of the fermentation liquor after concentration is greatly improved, but the mass yield is reduced along with the increase of the concentration factor, and then the viable bacteriaThe yield is reduced, the survival rate difference is obvious under the condition of three concentration times, the survival rate of the obtained bacterial powder is reduced within 10 percent when the bacterial powder is concentrated by 3 times, and the number of the viable bacteria in the bacterial powder obtained under the condition can reach 1.0 multiplied by 1010cfu/g。
Claims (2)
1. Clostridium butyricum (A. butyricum)Clostridium butyricum) The preparation method of the bacterial powder is characterized in that the clostridium butyricum fermentation liquor is subjected to spray drying, and the inlet temperature of the spray drying is 180 ℃; adding a protective agent during spray drying, wherein the protective agent is a composition of maltodextrin, sodium alginate, chitosan oligosaccharide, calcium lactate, sodium carboxymethylcellulose and silicon dioxide, the adding amount of the sodium alginate is 10% of the mass of the fermentation liquor, the adding amount of the maltodextrin is 10% of the mass of the fermentation liquor, the adding amount of the chitosan oligosaccharide is 2% of the mass of the fermentation liquor, the adding amount of the calcium lactate is 1% of the mass of the fermentation liquor, the adding amount of the sodium carboxymethylcellulose is 0.2% of the mass of the fermentation liquor, and the adding amount of the silicon dioxide is 0.1% of the mass of the fermentation liquor; pretreating fermentation liquor before spray drying, wherein the pretreatment is centrifugal separation; and (3) concentrating the fermentation liquor after centrifugal separation, and then performing spray drying, wherein the concentration multiple is 3.
2. The method for producing Clostridium butyricum bacterial powder according to claim 1, wherein the rotation speed is 3000 to 5000 r/min during the centrifugal separation, and the centrifugal treatment is performed for 5 to 30 min.
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| CN110591944A (en) * | 2019-09-06 | 2019-12-20 | 吉林大学 | Excellent clostridium butyricum for preventing ulcerative colitis |
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| CN111876346A (en) * | 2020-07-07 | 2020-11-03 | 武汉微康益生菌研究院有限公司 | Bacillus coagulans spray drying protective agent and spray drying method using protective agent |
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| CN109161471B (en) * | 2017-11-15 | 2019-08-30 | 浙江惠嘉生物科技股份有限公司 | A kind of fermentation process of Miyarisan |
| CN108392635B (en) * | 2018-01-24 | 2021-04-27 | 东莞波顿香料有限公司 | Essence microcapsule and preparation method and application thereof |
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| CN110959752A (en) * | 2019-12-18 | 2020-04-07 | 湖北华扬科技发展有限公司 | High-stability microecological compound preparation and preparation method and application thereof |
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