Culture medium for lactic acid bacteria prepared from chinese cabbage wastes

专利摘要:
PURPOSE: A culture medium for lactic acid bacteria prepared from Chinese cabbage wastes is provided, thereby effectively treating Chinese cabbage wastes which may cause environmental pollution, and mass-producing lactic acid bacteria. CONSTITUTION: A method for producing a culture medium for lactic acid bacteria prepared from Chinese cabbage wastes comprises the steps of: pulverizing Chinese cabbage wastes with a pulverizer; adding 0.5 to 3 parts by weight of egg white into 100 parts by weight of pulverized Chinese cabbage wastes; heating the mixture to 80 deg. C and cooling it to room temperature; and filtering the mixture with a membrane filter, wherein the lactic acid bacteria is Leuconostoc gasicomitatum.
公开号:KR20040033593A
申请号:KR1020020062743
申请日:2002-10-15
公开日:2004-04-28
发明作者:박현근；전인철
申请人:박현근；전인철；
IPC主号:

专利说明:

Culture Medium for Lactic Acid Bacteria Prepared from Chinese Cabbage Wastes
[7] The present invention relates to a culture medium for lactic acid bacteria prepared using cabbage waste generated as a large amount of waste during kimchi production. More specifically, the present invention relates to a method for obtaining a large amount of useful lactic acid bacteria, in particular, Lukonostock spiny comitatum, using a medium prepared using a large amount of Chinese cabbage waste generated during kimchi production.
[8] Kimchi, our traditional food, is not only one of Koreans' favorite foods, but it is also widely distributed internationally, and exports are increasing rapidly. Chinese cabbage, which is the main raw material for making kimchi, accounts for about 90% by weight of the whole raw material, and a large part of it is disposed of as waste. In particular, the Kimchi plant and the fruit and vegetable market, where a large amount of cabbage waste is generated, are spending considerable effort, time, and money in collecting and processing cabbage waste.
[9] Organic wastes, including Chinese cabbage waste, currently make up a significant portion of the total waste, are easily decaying, causing odors, leaking sewage, causing difficulties in treatment, and causing environmental problems. Therefore, research on the treatment method of such organic waste is actively progressing.
[10] To date, most of Chinese cabbage waste is reclaimed and rely on soil microorganisms, but there is a limit to landfill. Chinese cabbage contains more than 80% of moisture, so decay develops rapidly, and odor is generated. Inflows to this water system may cause eutrophication. Accordingly, other treatment methods have been sought to replace the landfill method.
[11] The present inventors have developed a culture medium for lactic acid bacteria that can produce a large amount of kimchi lactic acid bacteria biomass while simultaneously processing the Chinese cabbage waste.
[1] Figure 1 is a graph showing the pH change of kimchi solution measured at 24 hours interval in the process of fermenting cabbage waste for 21 days at 15 ℃.
[2] Figure 2 is a graph showing the change in the acidity of kimchi solution measured at intervals of 24 hours in the process of fermenting cabbage waste at 15 ℃ for 21 days.
[3] Figure 3 is a graph showing the change in the number of lactic acid bacteria cells in kimchi solution measured at 24 hours in the process of fermenting cabbage waste for 21 days at 15 ℃.
[4] 4 is a graph showing the growth curve of the JN3 strain isolated from kimchi solution according to the present invention, incubated at 25 ℃ using a Chinese cabbage medium was measured by the absorbance for each time was converted into cell number.
[5] 5 is a growth curve showing the change in cell number of JN3 calculated from the growth curve of FIG.
[6] 6 is a block diagram of a continuous culture fermenter consisting of a fermenter, a mixer, a medium and a culture medium reservoir, and a pH adjusting device.
[12] In the present invention, prior to producing a culture medium using the Chinese cabbage waste, firstly fermented kimchi using the Chinese cabbage waste to produce lactic acid bacteria.
[13] A. Kimchi Fermentation for Isolation of Kimchi Lactic Acid Bacteria
[14] The fermentation process of Chinese cabbage waste uses a conventional kimchi soaking process. Materials used are 100 parts by weight of Chinese cabbage waste, 1 to 3 parts by weight of red pepper powder, garlic, and leeks, 2 to 5 parts by weight of onion, and 0.01 to 0.1 parts by weight of tyrosine. Chinese cabbage is cut into 3 to 5 cm in length, and the remaining ingredients are ground to a suitable size, mixed together, and mixed with 30 to 70 parts by weight of brine at a concentration of 2 to 5%. The mixture is fermented in a polyethylene or polypropylene material reactor for 15 to 25 days while maintaining a temperature of 13 to 17 ° C. Tyrosine in the material serves to selectively grow only the specific lactic acid bacteria among the lactic acid bacteria growing during the fermentation process.
[15] According to the present invention, it is preferable to ferment kimchi using cabbage waste from the viewpoint of resource recycling and environmental friendliness. However, since the Lukonosstock spiny comitatum bacteria to be obtained in the present invention are lactic acid bacteria obtained during the kimchi fermentation process, the same results can be obtained by fermenting kimchi in the same manner as above using general cabbage instead of cabbage waste.
[16] B. Isolation and Identification of Lactic Acid Bacteria
[17] Kimchi solution is collected from fermented kimchi 24 hours in the above process and diluted 10 times with sterile distilled water to inoculate the diluted solution in MRS-BPB (de Man, Rgosa and Sharpe-bromophenol blue) medium and smear. Colonies produced by incubating at 15 ° C. for about 48 hours are separated from the colony by pure inoculation using pure inoculation needles and purely cultured in MRS (de Man, Rgosa and Sharpe) slope medium. The isolates are preserved by passage in MRS slope medium every 15 days and the strains are identified.
[18] C. Preparation of Chinese Cabbage Medium
[19] In order to produce the cabbage medium using the cabbage waste, the cabbage waste is pulverized with a grinder to separate the liquid. After heating to 80 ℃ to remove the solids in the cabbage juice mixed 0.5 to 3 parts by weight of egg white with a flocculant to 100 parts by weight cabbage juice and then cooled to room temperature. The cooled cabbage juice is filtered through a gauze and the filtrate is filtered through a filter paper. The filtrate is sterilized by membrane filtration (Φ, 0.45㎛) and used as a medium.
[20] D. Growth of Lactic Acid Bacteria in Chinese Cabbage Medium
[21] The strain isolated in step A is cultured in the cabbage medium prepared in step C to check the growth, select the highest growth rate lactic acid bacteria, and then culture to obtain a large amount of lactic acid bacteria biomass.
[22] The invention can be better understood by the following examples, which are intended to illustrate the invention and are not intended to limit the scope of protection defined by the appended claims.
[23] Example
[24] Identification of Lactobacillus
[25] 100 g of Chinese cabbage waste, 1.5 g of red pepper powder, garlic, and green onion, 1.5 g of onion, 3 g of onion, and 0.05 g of tyrosine were prepared and mixed with 50 g of 2.5% brine. Chinese cabbage waste was cut to 3cm in length, and red pepper powder, garlic, green onion, and onion were ground to a suitable size using a grinder. The mixture was fermented for 21 days at 15 ° C. temperature in a polypropylene reactor.
[26] During the fermentation process, the pH, acidity, and lactic acid cell number of kimchi were measured at 24 hour intervals.
[27] pH was measured using a pH meter (ORION 920A), the results are shown in FIG. The pH was increased temporarily up to 24 hours after kimchi immersion, and then drastically decreased for 5 days to maintain pH 3.6 to 4.2 thereafter.
[28] Acidity was measured in terms of lactic acid by titration with 0.1 N-NaOH, and the results are shown in FIG. 2. Unlike pH, acidity increased up to 7 days and maintained 0.6-0.7%.
[29] Lactobacillus cell count was measured by viable cell count. After diluting the sample (kimchi solution) by 10-fold dilution with sterile distilled water, 0.1 ml dilution was inoculated into MRS-BPB medium, which is a culture medium for lactic acid bacteria, and then cultured at 15 ° C. for 48 hours. The colonies formed in the medium were counted and converted into cell numbers, and the results are shown in FIG. 3. The number of cells increased rapidly until day 4, indicating 10 ¹² cells / ml, and thereafter, a wave phenomenon.
[30] In order to separate the kimchi lactic acid bacteria, colonies having different forms and colors of colonies generated in MRS-BPB medium were inoculated into MRS slope medium by pure separation using an inoculation needle. Every 15 days thereafter, subcultured to MRS slope medium was used. A total of 17 strains were isolated.
[31] Preparation of Chinese Cabbage Medium
[32] Apart from the above process, in order to prepare the cabbage medium, to make a cabbage juice by grinding the cabbage with a grinder, in order to remove the solids in the juice, the cabbage juice is heated to 80 ℃ and 100g cabbage juice egg white 1g as a flocculant Were mixed and cooled to room temperature. The cooled cabbage juice was filtered through gauze and the filtrate was again filtered through filter paper (Whatman NO. 1 USA). The filtrate was sterilized by membrane filtration (Φ, 0.45㎛) was used as a medium.
[33] Culture of Lactic Acid Bacteria
[34] 17 kinds of lactic acid bacteria isolated above were cultured in the prepared cabbage medium. Among them, 4 kinds of lactic acid bacteria with high growth rate were isolated, which were designated as JN1 to JN4. All four strains were cocci, and gram staining, dextran formation in sucrose medium, and gas production test in MRS medium were all positive. In the growth test by temperature, all growth was performed at 5-37 ° C., but growth was inhibited in 1% NaCl medium and 2.5% NaCl medium, respectively. Taken together, these isolates corresponded to mesophilic bacteria in terms of growth temperature, were grown at low temperature (5 ℃), and growth was inhibited as NaCl concentration increased. The morphology and ecological characteristics of each strain are shown in Table 1.
[35] characteristicJN1JN2JN3JN4 Cell morphologycoccuscoccuscoccuscoccus Gram dyeing++++ Dextran formed from sucrose++++ Gas produced from MRS medium++++ Growth at 5 ° C++++ Growth at 15 ° C++++ Growth at 25 ° C++++ Growth at 37 ° C++++ Growth at 0% NaCl++++ Growth in 1% NaCl(+)(+)(+)(+) Growth at 2.5% NaCl----
[36] +: Positive,-: negative, (+): weak positive
[37] Determination of Biochemical Properties of Strains
[38] In order to measure the biochemical properties of the strain, the sugar fermentation test was carried out using a multi-test system. MRS medium was used as a medium, and instead of glucose, 19 sugars to be tested were sterilized using membrane filtration to add a final concentration of 1% and used as a test medium. After inoculating the strain into the medium and incubated at 25 ° C. for 2 days, the presence or absence of fermentation was determined using a complex pH indicator. The results are shown in Table 2.
[39] The isolates fermented L-arabinose, melibiose, raffinose, ribose, D-xylose, maltose, sucrose, fructose and melezitose, while galactose, lactose, mannitol, salicycin, rhamnose and sorbitol fermented. I could not let you. The above results were found to be highly related to Leuconostoc gasicomitatum.
[40] For more accurate identification, it was identified by PCR (polymerase chain reaction) method using the Lukonostach species specific primers, and as a result, it was confirmed that it is Lukonostek chycomattum. Luconotstock Kashikomitum was first discovered in 2000 and has not yet been reported for its specific use. However, it is a useful lactic acid bacterium that is useful for probiotics (animal feed additives), dressing agents (for human medicines), health supplements (for health foods), And cosmetic additives.
[41] characteristicJN1JN2JN3JN4L.mL.gL.cL.lL.ga Acid from L-Arabinose+++++++++ Acid produced from cellobiose+-++D++-+ Acid produced from galactose----+-++ND Acid produced from lactose----D--+- Acid produced from mannitol----D++-- Acid produced from melibiose++++D+--+ Acid produced from raffinose++++D+--+ Acid produced from ribose+++-++--+ Acid produced from salicycin----D++-- Acid produced from trehalose+-+++++-+ Acid produced from di-xylose++++D+--+ Acid produced from maltose++++ND+NDND+ Acid produced from mannose--+-NDNDNDND+ Acid produced from sucrose++++NDNDNDND+ Acid Produced from Fructose+++-NDNDNDND+ Acid from Rhamnose----NDNDNDND- Acid produced from gluconate-+--ND-+-ND Acid Produced from Melezitose++++NDNDNDND- Acid produced from sorbitol----NDNDNDND-
[42] JN1-4: Isolated Strains
[43] Lm: Lukonotstock mesenteroroids sp. Mesenteroids ,
[44] Lg: Lukonot Stock Geladium
[45] Lc: Lukonotstock Citrium
[46] Ll: Lukonostact lactis
[47] L.ga: Lukonok Stark Komitatum
[48] +: 90% or more strains are positive;
[49] D: 89-11% of the strains are positive;
[50] -: 90% or more strains are negative;
[51] ND: not detected.
[52] Biomass Measurement in Chinese Cabbage Medium
[53] After culturing the strain isolated from the cabbage medium for 3 days at 25 ℃ was measured for absorbance at 550nm using a spectrophotometer, the biomass was measured by dry weight after filtration of 20ml culture medium and dried at 105 ℃ for 4 hours. The results are shown in Table 3. As a result, absorbance and dry weight were irrelevant. This is judged to be due to the difference in the shape and size of the cells, the dry weight was the highest in JN3.
[54] JN1JN2JN3JN4 Absorbance (550nm)0.670.530.540.73 Dry weight (g / L)0.3550.3600.4200.395
[55] The growth curve of JN3 with the highest dry weight was measured. Growth curve measurement was incubated at 25 ℃ using cabbage medium to measure the absorbance by time and converted into cell number. Correlation between absorbance and cell number is shown in FIG. 5, and the growth curve of Lukonstock Takkomitatum (JN3) is shown in FIG. 6.
[56] Obtaining the growth rate constant (K) from the growth curve is as follows.
[57]
[58] ( X _t is the number of cells / ml, t is the time)
[59] Since K = 1 / t _gen , t _gen = 1 / 0.77 = 1.3 (hours).
[60] ( T _gen is the doubling time.)
[61] Therefore, the specific growth rate (μ) is 0.693 × K, so μ is 0.53.
[62] Continuous culture fermenter
[63] Continuous culture fermenter for cultivating kimchi according to the present invention is divided into a fermenter and a mixer, a medium and a culture medium reservoir, a pH adjusting device, the schematic diagram of the fermenter is shown in FIG.
[64] 1) Fermenter and Mixer
[65] The fermenter uses a 1 L container of sterile glass. A magnetic stirrer was used to mix the medium, the culture medium, and the pH control solution, and a pH electrode for pH measurement was installed.
[66] 2) Medium and culture reservoir
[67] A sterilizable container was also used for the medium and the culture reservoir, and a peristaltic pump was installed to quantitatively flow the medium and the culture medium.
[68] 3) pH controller
[69] In order to prevent the pH from being lowered by the lactic acid produced by lactic acid bacteria growth and inhibiting the growth, a pH control device was installed. When the pH is less than 6.5, a peristaltic pump (2) is installed to operate 0.1N-NaOH. Injection.
[70] 4) flow rate of the medium
[71] The flow rate of the medium flowing into the fermenter from the reservoir of the medium was 1.3 hours for the doubling time of the Lukonstock Kashikomitatum, and the capacity of the fermenter was 1 L, so that the flow rate was 13 ml / min.
[72] The present invention has the effect of providing a culture medium capable of mass production of high value-added lactic acid bacteria biomass using cabbage waste. The strain that can be cultured in the bulk in the medium is Lukonostach spycomitatum. According to the present invention, a large amount of lactic acid bacteria biomass can be obtained while treating a large amount of cabbage waste, which has been a problem of waste treatment.

权利要求:
Claims (2)
[1" claim-type="Currently amended] The cabbage waste is pulverized by a grinder, and mixed with 0.5 to 3 parts by weight of egg white to 100 parts by weight of the crushed cabbage waste, the temperature is raised to 80 ℃, cooled to room temperature and produced by lactic acid bacteria culture medium.
[2" claim-type="Currently amended] According to claim 1, wherein the lactic acid bacteria is lactic acid bacteria culture medium, characterized in that Lukonostak chycomitatum.

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同族专利:

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公开号 | 公开日

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KR100453376B1|2004-10-15|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2002-10-15|Application filed by 박현근, 전인철

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2002-10-15|Priority to KR20020062743A

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2004-04-28|Publication of KR20040033593A

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2004-10-15|Application granted

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2004-10-15|Publication of KR100453376B1

优先权:

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申请号 | 申请日 | 专利标题

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KR20020062743A|KR100453376B1|2002-10-15|2002-10-15|Culture Medium for Lactic Acid Bacteria Prepared from Chinese Cabbage Wastes|