CN112779306B - Method for screening optimal proportion of calcium carbonate from fermentation liquor and application thereof - Google Patents
Method for screening optimal proportion of calcium carbonate from fermentation liquor and application thereof Download PDFInfo
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 title claims abstract description 150
- 230000004151 fermentation Effects 0.000 title claims abstract description 84
- 238000000855 fermentation Methods 0.000 title claims abstract description 84
- 229910000019 calcium carbonate Inorganic materials 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000012216 screening Methods 0.000 title claims abstract description 14
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims abstract description 96
- 235000006408 oxalic acid Nutrition 0.000 claims abstract description 32
- 239000000706 filtrate Substances 0.000 claims abstract description 18
- 229930194936 Tylosin Natural products 0.000 claims abstract description 13
- 239000004182 Tylosin Substances 0.000 claims abstract description 13
- WBPYTXDJUQJLPQ-VMXQISHHSA-N tylosin Chemical compound O([C@@H]1[C@@H](C)O[C@H]([C@@H]([C@H]1N(C)C)O)O[C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 WBPYTXDJUQJLPQ-VMXQISHHSA-N 0.000 claims abstract description 13
- 229960004059 tylosin Drugs 0.000 claims abstract description 13
- 235000019375 tylosin Nutrition 0.000 claims abstract description 13
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- 238000001556 precipitation Methods 0.000 claims abstract description 7
- 238000004448 titration Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 9
- 238000000967 suction filtration Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 abstract description 4
- 238000003828 vacuum filtration Methods 0.000 abstract description 4
- 229950007634 kitasamycin Drugs 0.000 abstract 1
- XYJOGTQLTFNMQG-KJHBSLKPSA-N leucomycin V Chemical compound CO[C@H]1[C@H](O)CC(=O)O[C@H](C)C\C=C\C=C\[C@H](O)[C@H](C)C[C@H](CC=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](N(C)C)[C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1 XYJOGTQLTFNMQG-KJHBSLKPSA-N 0.000 abstract 1
- 239000002244 precipitate Substances 0.000 abstract 1
- 235000010633 broth Nutrition 0.000 description 34
- 239000002994 raw material Substances 0.000 description 20
- 239000002609 medium Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 7
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 5
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 229910001424 calcium ion Inorganic materials 0.000 description 4
- 238000012805 post-processing Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229920002261 Corn starch Polymers 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 3
- 235000019764 Soybean Meal Nutrition 0.000 description 3
- 241000209140 Triticum Species 0.000 description 3
- 235000021307 Triticum Nutrition 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000008120 corn starch Substances 0.000 description 3
- QYDYPVFESGNLHU-UHFFFAOYSA-N elaidic acid methyl ester Natural products CCCCCCCCC=CCCCCCCCC(=O)OC QYDYPVFESGNLHU-UHFFFAOYSA-N 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000011565 manganese chloride Substances 0.000 description 3
- 235000002867 manganese chloride Nutrition 0.000 description 3
- 229940099607 manganese chloride Drugs 0.000 description 3
- 235000012054 meals Nutrition 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- QYDYPVFESGNLHU-KHPPLWFESA-N methyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC QYDYPVFESGNLHU-KHPPLWFESA-N 0.000 description 3
- 229940073769 methyl oleate Drugs 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000004455 soybean meal Substances 0.000 description 3
- 235000012424 soybean oil Nutrition 0.000 description 3
- 239000003549 soybean oil Substances 0.000 description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 description 3
- 229960001763 zinc sulfate Drugs 0.000 description 3
- 235000019733 Fish meal Nutrition 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 239000002285 corn oil Substances 0.000 description 2
- 235000005687 corn oil Nutrition 0.000 description 2
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 2
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 2
- 235000019838 diammonium phosphate Nutrition 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000004467 fishmeal Substances 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- RRIWRJBSCGCBID-UHFFFAOYSA-L nickel sulfate hexahydrate Chemical compound O.O.O.O.O.O.[Ni+2].[O-]S([O-])(=O)=O RRIWRJBSCGCBID-UHFFFAOYSA-L 0.000 description 2
- 229940116202 nickel sulfate hexahydrate Drugs 0.000 description 2
- 239000006174 pH buffer Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/16—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using titration
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Abstract
Description
技术领域technical field
本发明属于抗生素药物技术领域，具体涉及一种从发酵液中筛选碳酸钙最佳配比的方法及其用途。The invention belongs to the technical field of antibiotic medicines, and in particular relates to a method for screening the optimum ratio of calcium carbonate from fermentation broth and use thereof.
背景技术Background technique
发酵培养基中碳酸钙的主要作用为pH缓冲剂，可以中和一些有机酸和无机酸，以保证整个体系pH的稳定，中和后钙将会以离子的状态存在。为了保持发酵过程中pH的稳定性，发酵培养基的配比中常常会加入过量的pH缓冲剂碳酸钙。但是过多的钙离子又会对发酵液的后处理带来麻烦，目前，发酵结束后发酵液的预处理中经常使用草酸除去钙离子，多余的碳酸钙在发酵液预处理时会与草酸反应，然后随着菌渣一起被过滤除去。由此可见，多余的碳酸钙需要投加足量的草酸才能清除，大幅度提高了发酵液的预处理成本，残留的钙还有可能会给后续的产品后处理带来麻烦，甚至增加成品的残渣比例。The main function of calcium carbonate in the fermentation medium is a pH buffer, which can neutralize some organic and inorganic acids to ensure the stability of the pH of the entire system. After neutralization, calcium will exist in the state of ions. In order to maintain the stability of pH during the fermentation process, excess pH buffer calcium carbonate is often added to the ratio of the fermentation medium. However, too much calcium ions will bring trouble to the post-processing of the fermentation broth. At present, oxalic acid is often used to remove calcium ions in the pretreatment of the fermentation broth after fermentation, and the excess calcium carbonate will react with oxalic acid during the pretreatment of the fermentation broth. , and then removed by filtration along with the bacterial residue. It can be seen that the excess calcium carbonate needs to be removed by adding a sufficient amount of oxalic acid, which greatly increases the pretreatment cost of the fermentation broth. Residue ratio.
如何确定发酵培养基中碳酸钙的最佳配比以达到提高发酵产品质量，降低发酵成本，是一项很有意义的研究。现有技术中为了确定发酵液中碳酸钙的最佳添加量，需要采用正交实验进行优化，一般是改变碳酸钙的添加量进行一系列的平行试验，根据发酵结果来确定发酵配比，整个过程耗时较长，成本较高，并且结果仅仅能适用于该种特定的发酵液。如果能开发一种操作简单、准确率高、耗时更短、适用性更广的确定发酵液中碳酸钙最佳配比的方法具有重要的应用价值。How to determine the optimal ratio of calcium carbonate in the fermentation medium to improve the quality of fermented products and reduce the cost of fermentation is a very meaningful research. In the prior art, in order to determine the optimum addition amount of calcium carbonate in the fermentation broth, it is necessary to use an orthogonal experiment to optimize, generally changing the addition amount of calcium carbonate to carry out a series of parallel experiments, and determining the fermentation ratio according to the fermentation results. The process is time-consuming, expensive, and the results are only applicable to this specific fermentation broth. It would be of great application value to develop a method for determining the optimal ratio of calcium carbonate in the fermentation broth with simple operation, high accuracy, shorter time-consuming and wider applicability.
发明内容SUMMARY OF THE INVENTION
为解决上述现有技术中存在的问题，本发明提供一种从发酵液中筛选碳酸钙最佳配比的方法，该方法可以简单快速确定发酵液中的碳酸钙的最佳配比，得到的发酵液明显的降低碳酸钙的投加量，同时减少后续发酵液预处理中草酸的使用量，为后续的发酵液深度处理以及成品的质量提高带来帮助。本发明还提供两种应用上述方法制备的发酵液。In order to solve the problems existing in the above-mentioned prior art, the present invention provides a method for screening the optimum proportion of calcium carbonate from fermentation broth, which can simply and quickly determine the optimum proportion of calcium carbonate in the fermentation broth, and the obtained The fermentation broth obviously reduces the dosage of calcium carbonate, and at the same time reduces the usage of oxalic acid in the subsequent fermentation broth pretreatment, which is helpful for the subsequent advanced treatment of the fermentation broth and the improvement of the quality of the finished product. The present invention also provides two fermentation broths prepared by the above method.
一种从发酵液中筛选碳酸钙最佳配比的方法，包括以下步骤：A method for screening the best proportioning ratio of calcium carbonate from fermentation broth, comprising the following steps:
(1)将生产终点的发酵液经过滤获得洁净的滤液；(1) the fermentation broth of the production end is filtered to obtain a clean filtrate;
(2)配制草酸溶液用于滤液的滴定，同时计算发酵配方中所添加碳酸钙完全除去所需草酸量的理论值；(2) prepare oxalic acid solution for the titration of filtrate, calculate the theoretical value that added calcium carbonate in the fermentation formula completely removes the required amount of oxalic acid;
(3)按照理论值依次进行递减添加不同量的草酸，所得溶液再次真空抽滤，抽滤后的滤液再次加入适量草酸溶液，放置至少2小时，观察有无沉淀产生；(3) according to the theoretical value, successively add different amounts of oxalic acid in decreasing order, the obtained solution is vacuum filtered again, and the filtrate after the suction filtration is again added with an appropriate amount of oxalic acid solution, placed for at least 2 hours, and it is observed whether there is precipitation;
(4)根据沉淀产生的情况计算碳酸钙的实际利用量，再根据所述实际利用量确定碳酸钙的最佳配比。(4) Calculate the actual utilization amount of calcium carbonate according to the situation of precipitation, and then determine the optimal proportion of calcium carbonate according to the actual utilization amount.
优选地，所述发酵液为吉他霉素发酵液或泰乐菌素发酵液。Preferably, the fermentation broth is guitarmycin fermentation broth or tylosin fermentation broth.
作为优选，所述的草酸溶液的浓度为5～15％。进一步优选为10％。Preferably, the concentration of the oxalic acid solution is 5-15%. More preferably, it is 10%.
上述筛选碳酸钙最佳配比的方法中：In the above-mentioned method of screening the best proportioning calcium carbonate:
步骤(1)中，所述发酵液选用发酵终点的放罐发酵液。过滤方法采用滤纸过滤或真空抽滤。In step (1), the fermented liquid is selected from the fermented liquid at the end of the fermentation. The filtration method adopts filter paper filtration or vacuum filtration.
步骤(2)中，所述草酸溶液中草酸应完全溶解，并根据草酸的溶解度与温度的对应关系进行溶液的配制。In step (2), the oxalic acid should be completely dissolved in the oxalic acid solution, and the solution is prepared according to the corresponding relationship between the solubility of the oxalic acid and the temperature.
步骤(3)中，添加实验量草酸后的溶液应根据产品的工艺情况进行过滤。过滤后的滤液需先澄清，再加入适量草酸溶液，放置观察。再次添加草酸溶液后，放置至少2小时，以提高筛选结论的准确性。In step (3), the solution after adding the experimental amount of oxalic acid should be filtered according to the technological conditions of the product. The filtered filtrate needs to be clarified first, then an appropriate amount of oxalic acid solution is added and placed for observation. After adding the oxalic acid solution again, let it stand for at least 2 hours to improve the accuracy of the screening conclusion.
步骤(4)中，考虑到碳酸钙可能会参与发酵中其它的合成反应，实验量的碳酸钙添加值应比实验得出的略多，作为优选，所述碳酸钙的最佳配比高于计算所得的碳酸钙的实际利用量。作为进一步的优选，步骤(4)中，所述碳酸钙的最佳配比为计算所得的碳酸钙的实际利用量的1.45～1.85倍。通过发酵培养的对比实验发现，当碳酸钙的用量在计算所得的碳酸钙的实际利用量的1.45～1.85倍时，可降低产品后处理的工序和成本，并且得到的发酵液具有更高的生物效价。而当碳酸钙的用量进一步降低时，则会影响发酵过程，导致产品的生物效价偏低，因此碳酸钙的用量也不易过低。In step (4), considering that calcium carbonate may participate in other synthetic reactions in the fermentation, the calcium carbonate addition value of experimental amount should be slightly more than the experimental draw, as preferably, the optimum ratio of described calcium carbonate is higher than Calculate the actual utilization of calcium carbonate obtained. As a further preference, in step (4), the optimal proportion of the calcium carbonate is 1.45-1.85 times the actual utilization amount of the calculated calcium carbonate. Through the comparative experiment of fermentation culture, it is found that when the amount of calcium carbonate is 1.45 to 1.85 times the actual utilization amount of calcium carbonate obtained by calculation, the process and cost of post-processing of the product can be reduced, and the obtained fermentation broth has higher biological potency. When the amount of calcium carbonate is further reduced, it will affect the fermentation process, resulting in a low biological titer of the product, so the amount of calcium carbonate is not easy to be too low.
本发明还提供一种利用上述筛选碳酸钙最佳配比的方法确定的吉他霉素发酵液，以重量百分数计，轻质碳酸钙的比例为0.21～0.26％。The present invention also provides a guitarmycin fermentation broth determined by the above-mentioned method for screening the optimum proportion of calcium carbonate, and the proportion of light calcium carbonate is 0.21-0.26% in weight percentage.
进一步的，具体包括以下重量分数的组份：Further, specifically include the following components by weight:
本发明还提供一种吉他霉素的生产方法，采用上述吉他霉素发酵液进行发酵生产。The present invention also provides a method for producing guitarmycin, which adopts the above-mentioned guitarmycin fermentation liquid for fermentation production.
本发明还提供一种利用上述筛选碳酸钙最佳配比的方法确定的泰乐菌素发酵液，以重量百分数计，轻质碳酸钙的比例为0.19～0.21％。The present invention also provides a tylosin fermentation broth determined by the above-mentioned method for screening the optimum proportion of calcium carbonate, in which the proportion of light calcium carbonate is 0.19-0.21% in weight percentage.
进一步地，具体包括以下重量分数的组份：Further, specifically include the following components by weight:
本发明还提供一种泰乐菌素的生产方法，包括以下步骤：采用上述泰乐菌素发酵液进行发酵生产。The present invention also provides a method for producing tylosin, which comprises the following steps: using the above-mentioned tylosin fermentation liquid for fermentation production.
本发明采用发酵液直接过滤后的滤液用草酸进行滴定，以确定发酵培养基中碳酸钙的利用情况；同时筛选后的碳酸钙进行发酵培养的生物效价对比试验，以确定碳酸钙的发酵最佳配比。In the present invention, the filtrate directly filtered from the fermentation broth is titrated with oxalic acid to determine the utilization of calcium carbonate in the fermentation medium; at the same time, the screened calcium carbonate is subjected to a biological titer comparison test of fermentation culture to determine the best fermentation rate of calcium carbonate. best ratio.
与现有技术相比，本发明的有益效果为：本发明采用碳酸钙的利用情况进行其配比的优选，与发酵培养基中所采用基础培养基的正交优化实验方案不同，该方法对碳酸钙等个别离子的使用将会更加准确。因为碳酸钙在配方中主要用于缓冲溶液的pH，其过多的使用不会带来代谢产物的增多，反而过量的碳酸钙会给产品的后处理增加成本和不必要的麻烦。本发明的筛选方法，提高了碳酸钙的添加精度，降低了产品后处理的工序和成本，并且终点发酵液的生物效价得到了一定程度的提高。Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention adopts the utilization of calcium carbonate to optimize its proportion, which is different from the orthogonal optimization experimental scheme of the basal medium adopted in the fermentation medium. The use of individual ions such as calcium carbonate will be more accurate. Because calcium carbonate is mainly used to buffer the pH of the solution in the formula, its excessive use will not bring about the increase of metabolites, but excessive calcium carbonate will increase the cost and unnecessary trouble for the post-processing of the product. The screening method of the invention improves the addition precision of calcium carbonate, reduces the process and cost of post-processing of the product, and improves the biological titer of the end-point fermentation broth to a certain extent.
具体实施方式Detailed ways
下面结合实施例，更具体地说明本发明的内容。应当理解，本发明的实施并不局限于下面的实施例，对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。The content of the present invention will be described in more detail below with reference to the embodiments. It should be understood that the implementation of the present invention is not limited to the following examples, and any modifications and/or changes made to the present invention will fall within the protection scope of the present invention.
分别通过草酸滴定法测定终点发酵液中吉他霉素和泰乐菌素碳酸钙的利用情况，具体分述如下：The utilization of guitarmycin and tylosin calcium carbonate in the end-point fermentation broth was determined by oxalic acid titration, and the details are as follows:
1、吉他霉素的碳酸钙利用评估1. Evaluation of calcium carbonate utilization of guitarmycin
取吉他霉素发酵液直接过滤，收集滤液，每次取滤液100ml，加入实验量的配制好的10％的草酸溶液(草酸溶液6.6ml为理论所添加除去钙离子的最大值)，之后用10％氢氧化钠回调pH＝3.1，再次将发酵液真空抽滤，过滤后的滤液再次加入草酸，放置2小时，观察有无沉淀产生，具体情况如下：Get the guitarmycin fermentation broth and filter directly, collect the filtrate, take 100ml of the filtrate each time, add the prepared 10% oxalic acid solution of the experimental amount (6.6ml of the oxalic acid solution is the theoretically added maximum value of removing calcium ions), then use 10 % sodium hydroxide was adjusted to pH=3.1, the fermentation broth was vacuum filtered again, oxalic acid was added to the filtered filtrate again, and it was placed for 2 hours to observe whether there was precipitation. The details are as follows:
从上面连续三批实验来看，都表明发酵培养基中投加的碳酸钙有一定的过量，大约过量1/2，吉他霉素原始配方中碳酸钙的添加量为0.31％，实际利用量约为0.14％，考虑到碳酸钙可能会参与发酵代谢的其它反应，同时考虑到生产上异常时会影响到pH的波动，导致产品的生物效价偏低，本实验以0.26％(为实际利用量的1.85倍)和0.31％分别进行试验。From the above three consecutive batches of experiments, it is shown that there is a certain excess of calcium carbonate in the fermentation medium, about 1/2 of the excess. It is 0.14%, considering that calcium carbonate may participate in other reactions of fermentation metabolism, and considering that abnormal production will affect the pH fluctuation, resulting in a low biological titer of the product, this experiment takes 0.26% (as the actual utilization amount). 1.85 times) and 0.31% were tested respectively.
2、泰乐菌素的碳酸钙利用评估2. Evaluation of calcium carbonate utilization of tylosin
取泰乐菌素发酵液直接真空抽滤，收集滤液，每次取滤液100ml，加入实验量的配制好的10％的草酸溶液(草酸溶液2.7ml为理论所添加除去钙离子的最大值)，再次将发酵液真空抽滤，过滤后的滤液再次加入草酸，放置2小时，观察有无沉淀产生，具体情况见下：Take the tylosin fermentation liquid and directly vacuum filtration, collect the filtrate, take 100ml of the filtrate each time, add the prepared 10% oxalic acid solution of the experimental amount (2.7ml of the oxalic acid solution is the theoretically added maximum value for removing calcium ions), The fermentation broth was vacuum filtered again, and oxalic acid was added to the filtered filtrate again, and the filtrate was placed for 2 hours to observe whether there was precipitation. The details are as follows:
从上面连续三批实验来看，都表明发酵培养基中投加的碳酸钙有一定的过量，至少过量1/3，泰乐菌素原始配方中碳酸钙的添加量为0.22％，实际利用量约为0.13％，本实验以0.19％(约为实际利用量的1.46倍)和0.22％分别进行试验。From the above three consecutive batches of experiments, it is shown that there is a certain excess of calcium carbonate in the fermentation medium, at least 1/3 of the excess. About 0.13%, this experiment was carried out with 0.19% (about 1.46 times of the actual utilization amount) and 0.22% respectively.
实施例1：Example 1:
吉他霉素发酵培养基中实验组和对照组主要在于轻质碳酸钙的比例不同，分别为0.26％和0.31％。其它物质的配比为：The proportion of light calcium carbonate in the test group and the control group in the guitarmycin fermentation medium was different, which were 0.26% and 0.31%, respectively. The proportions of other substances are:
将发酵终点的发酵液分别进行生物效价检测，实验组与对照组分别为10850u/ml和10784u/ml。The fermentation broth at the end of fermentation was tested for biological titer, the experimental group and the control group were 10850u/ml and 10784u/ml, respectively.
实施例2：Example 2:
吉他霉素发酵培养基中实验组和对照组主要在于轻质碳酸钙的比例不同，分别为0.19％(约为实际利用量的1.36倍)和0.31％。其它物质的配比为：The ratio of light calcium carbonate in the test group and the control group in the guitarmycin fermentation medium was different, which were 0.19% (about 1.36 times of the actual utilization) and 0.31%, respectively. The proportions of other substances are:
将发酵终点的发酵液分别进行生物效价检测，实验组与对照组分别为8159u/ml和10893u/ml。The fermentation broth at the end of the fermentation was tested for biological titer, the experimental group and the control group were 8159u/ml and 10893u/ml, respectively.
实施例2的结果表明，当将轻质碳酸钙的比例控制在实际利用量的1.36倍，会导致产品的生物效价大幅度降低，可能原因在于碳酸钙会参与发酵代谢的其它反应，因此，并不能完全按照实际消耗量来确定轻质碳酸钙的比例。The result of embodiment 2 shows that when the ratio of light calcium carbonate is controlled at 1.36 times of the actual utilization amount, the biological titer of the product can be greatly reduced, and the possible reason is that calcium carbonate can participate in other reactions of fermentation metabolism. Therefore, The proportion of light calcium carbonate cannot be determined completely according to the actual consumption.
实施例3：Example 3:
吉他霉素发酵培养基中实验组和对照组主要在于轻质碳酸钙的比例不同，分别为0.21％(约为实际利用量的1.46倍)和0.31％。其它物质的配比为：The ratio of light calcium carbonate in the test group and the control group in the guitarmycin fermentation medium was different, which were 0.21% (about 1.46 times of the actual utilization) and 0.31%, respectively. The proportions of other substances are:
将发酵终点的发酵液分别进行生物效价检测，实验组与对照组分别为10865u/ml和10776u/ml。The fermentation broth at the end of fermentation was tested for biological titer, the experimental group and the control group were 10865u/ml and 10776u/ml respectively.
实施例3的结果表明，当将轻质碳酸钙的比例增加至实际利用量的1.46倍，可以较好地保证发酵水平。The results of Example 3 show that when the proportion of light calcium carbonate is increased to 1.46 times the actual utilization amount, the fermentation level can be better guaranteed.
实施例4：Example 4:
泰乐菌素发酵培养基中实验组和对照组主要在于轻质碳酸钙的比例不同，分别为0.19％和0.22％。其它物质的配比为：In the tylosin fermentation medium, the experimental group and the control group mainly have different proportions of light calcium carbonate, which are 0.19% and 0.22%, respectively. The proportions of other substances are:
将发酵终点的发酵液分别进行生物效价检测，实验组与对照组分别为10293u/ml和10231u/ml。The fermentation broth at the end of fermentation was tested for biological titer, the experimental group and the control group were 10293u/ml and 10231u/ml, respectively.
实施例5：Example 5:
泰乐菌素发酵培养基中实验组和对照组主要在于轻质碳酸钙的比例不同，分别为0.18％(约为实际利用量的1.38倍)和0.22％。其它物质的配比为：In the tylosin fermentation medium, the experimental group and the control group mainly have different proportions of light calcium carbonate, which are 0.18% (about 1.38 times of the actual utilization) and 0.22%, respectively. The proportions of other substances are:
将发酵终点的发酵液分别进行生物效价检测，实验组与对照组分别为8131u/ml和9983u/ml。The fermentation broth at the end of fermentation was tested for biological titer, the experimental group and the control group were 8131u/ml and 9983u/ml respectively.
实施例5的结果表明，当将轻质碳酸钙的比例控制在实际利用量的1.36倍，会导致产品泰乐菌素的生物效价大幅度降低，可能原因在于碳酸钙会参与发酵代谢的其它反应，因此，并不能完全按照实际利用量来确定轻质碳酸钙的比例。The results of Example 5 show that when the ratio of light calcium carbonate is controlled at 1.36 times of the actual utilization, the biological titer of the product tylosin can be greatly reduced, and the possible reason is that calcium carbonate can participate in other fermentation and metabolism. Therefore, the proportion of light calcium carbonate cannot be determined completely according to the actual utilization amount.
实施例6：Example 6:
泰乐菌素发酵培养基中实验组和对照组主要在于轻质碳酸钙的比例不同，分别为0.21％(约为约为实际利用量的1.61倍)和0.22％。其它物质的配比为：In the tylosin fermentation medium, the experimental group and the control group mainly have different proportions of light calcium carbonate, which are 0.21% (about 1.61 times of the actual utilization) and 0.22%, respectively. The proportions of other substances are:
将发酵终点的发酵液分别进行生物效价检测，实验组与对照组分别为10654u/ml和10569u/ml。The fermentation broth at the end of the fermentation was tested for biological titer, the experimental group and the control group were 10654u/ml and 10569u/ml, respectively.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1443851A (en) * | 2002-03-13 | 2003-09-24 | 宁夏多维药业有限公司 | Method for producing tylosin by using corn as main raw material |
| CN101037723A (en) * | 2007-03-22 | 2007-09-19 | 广东富远稀土新材料股份有限公司 | Carbonate deposition method of crystal type heavy rare earth |
| CN102419325A (en) * | 2011-07-27 | 2012-04-18 | 中国科学院青海盐湖研究所 | Method for measuring strontium calcium barium in strontium carbonate |
| CN103074402A (en) * | 2013-02-05 | 2013-05-01 | 宁夏泰瑞制药股份有限公司 | Culture medium for producing tylosin through fermentation of streptomyces fradiae and fermentation method |
| CN103484514A (en) * | 2013-09-25 | 2014-01-01 | 宁夏泰瑞制药股份有限公司 | Culture medium for producing tylosin by fermenting streptomyces fradiae, and fermenting method |
| CN105063121A (en) * | 2015-01-20 | 2015-11-18 | 河北圣雪大成制药有限责任公司 | Fermentation culture medium for improving spectinomycin yield, and optimization method thereof |
| CN106591403A (en) * | 2015-10-14 | 2017-04-26 | 山东东药药业股份有限公司 | Method for improving fermentation level of kitasamycin |
| CN106591402A (en) * | 2015-10-14 | 2017-04-26 | 山东东药药业股份有限公司 | Tylosin fermentation formula |
| CN106609287A (en) * | 2015-10-22 | 2017-05-03 | 山东东药药业股份有限公司 | Kitasamycin fermenting culture medium |
| CN108315376A (en) * | 2018-04-19 | 2018-07-24 | 齐鲁制药（内蒙古）有限公司 | A kind of fermented nutritive liquid and fermentation process improving tylosin broth quality |
| CN111733202A (en) * | 2020-06-30 | 2020-10-02 | 宁夏泰益欣生物科技有限公司 | Mixed fermentation method of tylosin |
-
2021
- 2021-01-26 CN CN202110104721.7A patent/CN112779306B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1443851A (en) * | 2002-03-13 | 2003-09-24 | 宁夏多维药业有限公司 | Method for producing tylosin by using corn as main raw material |
| CN101037723A (en) * | 2007-03-22 | 2007-09-19 | 广东富远稀土新材料股份有限公司 | Carbonate deposition method of crystal type heavy rare earth |
| CN102419325A (en) * | 2011-07-27 | 2012-04-18 | 中国科学院青海盐湖研究所 | Method for measuring strontium calcium barium in strontium carbonate |
| CN103074402A (en) * | 2013-02-05 | 2013-05-01 | 宁夏泰瑞制药股份有限公司 | Culture medium for producing tylosin through fermentation of streptomyces fradiae and fermentation method |
| CN103484514A (en) * | 2013-09-25 | 2014-01-01 | 宁夏泰瑞制药股份有限公司 | Culture medium for producing tylosin by fermenting streptomyces fradiae, and fermenting method |
| CN105063121A (en) * | 2015-01-20 | 2015-11-18 | 河北圣雪大成制药有限责任公司 | Fermentation culture medium for improving spectinomycin yield, and optimization method thereof |
| CN106591403A (en) * | 2015-10-14 | 2017-04-26 | 山东东药药业股份有限公司 | Method for improving fermentation level of kitasamycin |
| CN106591402A (en) * | 2015-10-14 | 2017-04-26 | 山东东药药业股份有限公司 | Tylosin fermentation formula |
| CN106609287A (en) * | 2015-10-22 | 2017-05-03 | 山东东药药业股份有限公司 | Kitasamycin fermenting culture medium |
| CN108315376A (en) * | 2018-04-19 | 2018-07-24 | 齐鲁制药（内蒙古）有限公司 | A kind of fermented nutritive liquid and fermentation process improving tylosin broth quality |
| CN111733202A (en) * | 2020-06-30 | 2020-10-02 | 宁夏泰益欣生物科技有限公司 | Mixed fermentation method of tylosin |
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