CN114350526A - Method for improving monascus pigment tone - Google Patents
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- 241000228347 Monascus <ascomycete fungus> Species 0.000 title claims abstract description 75
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 83
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- 150000003751 zinc Chemical class 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 6
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229960001763 zinc sulfate Drugs 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 235000005074 zinc chloride Nutrition 0.000 claims description 3
- 239000011592 zinc chloride Substances 0.000 claims description 3
- -1 polyoxyethylene lauryl ether Polymers 0.000 claims 5
- 229920000259 polyoxyethylene lauryl ether Polymers 0.000 claims 5
- 239000001963 growth medium Substances 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 22
- 229940051841 polyoxyethylene ether Drugs 0.000 abstract description 21
- 229920000056 polyoxyethylene ether Polymers 0.000 abstract description 21
- LQZZUXJYWNFBMV-UHFFFAOYSA-N dodecan-1-ol Chemical compound CCCCCCCCCCCCO LQZZUXJYWNFBMV-UHFFFAOYSA-N 0.000 abstract description 19
- SFNALCNOMXIBKG-UHFFFAOYSA-N ethylene glycol monododecyl ether Chemical compound CCCCCCCCCCCCOCCO SFNALCNOMXIBKG-UHFFFAOYSA-N 0.000 abstract description 10
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- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical group CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 4
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- 241000233866 Fungi Species 0.000 description 1
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Abstract
本发明涉及生物发酵技术领域，提供了一种提高红曲色素色调的方法包括：将处于对数生长期的红曲菌种子液、月桂醇聚氧乙烯醚和发酵培养基混合，进行发酵培养，得到红曲色素。本发明选用的月桂醇聚氧乙烯醚浊点温度接近发酵温度，促进了发酵体系浊点系统的形成；同时由于其亲水性较强，显示出强烈的有益增溶，从而进一步促进了胶束分子在水溶液中的稳定性；最终在上述原理的共同作用下，提高了对红曲色素萃取能力，从而提高了红曲色素色调。实验结果表明，利用本发明提供的技术方案，红曲色素中红、橙、黄三色素的胞外色价(U/mL)分别为126.1、103.5、99.6，色调为0.79。The invention relates to the technical field of biological fermentation, and provides a method for improving the color tone of Monascus pigment, comprising: mixing the Monascus seed liquid in logarithmic growth phase, laureth and fermentation medium, and fermenting and culturing, Obtain Monascus pigment. The cloud point temperature of the lauryl alcohol polyoxyethylene ether selected in the present invention is close to the fermentation temperature, which promotes the formation of the cloud point system of the fermentation system; at the same time, due to its strong hydrophilicity, it shows strong beneficial solubilization, thereby further promoting the micelle The stability of the molecule in the aqueous solution; finally, under the joint action of the above principles, the extraction ability of the Monascus pigment is improved, thereby improving the color of the Monascus pigment. The experimental results show that, using the technical solution provided by the present invention, the extracellular color values (U/mL) of red, orange and yellow pigments in monascus pigments are 126.1, 103.5 and 99.6 respectively, and the hue is 0.79.
Description
技术领域technical field
本发明涉及生物发酵技术领域，尤其涉及一种提高红曲色素色调的方法。The invention relates to the technical field of biological fermentation, in particular to a method for improving the color tone of Monascus pigment.
背景技术Background technique
红曲色素是一种由红曲霉属的丝状真菌经发酵而成的优质的天然食用色素，其在食品添加剂领域的需求越来越大。目前红曲色素的生产方法主要是液态发酵法，但是传统的发酵过程中，菌体细胞内产物的高浓度所导致的负反馈抑制，致使不但色素产量低，而且色素色调也低，同时发酵结束后，细胞内色素分离纯化，需要消耗大量的有机溶剂。Monascus pigment is a high-quality natural food pigment fermented by filamentous fungi of the genus Monascus, and its demand in the field of food additives is increasing. At present, the production method of Monascus pigment is mainly liquid fermentation method. However, in the traditional fermentation process, the negative feedback inhibition caused by the high concentration of the product in the bacterial cell leads to not only low pigment output, but also low pigment tone, and the fermentation ends at the same time. After the separation and purification of intracellular pigments, a large amount of organic solvent needs to be consumed.
双液相偶联萃取发酵又称“挤奶发酵”，是目前解决上述问题的有效方法，具体是指通过向发酵培养基中添加非离子表面活性剂，伴随着浊点系统形成，发酵体系自动形成非离子表面活性剂胶束富集的聚集相和胶束很少的稀相，发酵过程中疏水性红曲色素从胞内萃取到胞外并大量富集在聚集相，从而解除胞内产物反馈抑制，增加色素产量；另外胞内色素被大量分泌到胞外，减少了有机溶剂的用量。Double-liquid phase coupling extraction fermentation, also known as "milking fermentation", is an effective method to solve the above problems. The non-ionic surfactant micelle-enriched aggregate phase and the dilute phase with few micelles are formed. During the fermentation process, the hydrophobic Monascus pigment is extracted from the intracellular to the extracellular and abundantly enriched in the aggregated phase, thereby releasing intracellular products. Feedback inhibition increases the production of pigments; in addition, intracellular pigments are secreted into the extracellular space, reducing the amount of organic solvents.
现有技术中利用双液相偶联萃取发酵时，通常采用的萃取剂为Span80，虽然其相比传统发酵工艺可以提高红曲色素的产量和色素色调，但是其色素色调还是相对较低。In the prior art, when using double-liquid phase coupling extraction and fermentation, the commonly used extractant is Span80. Although it can improve the yield and color tone of Monascus pigment compared with the traditional fermentation process, its color tone is still relatively low.
发明内容SUMMARY OF THE INVENTION
鉴于此，本发明的目的在于提供一种提高红曲色素色调的方法，包括：In view of this, the object of the present invention is to provide a kind of method that improves monascus pigment hue, comprising:
将处于对数生长期的红曲菌种子液、月桂醇聚氧乙烯醚和发酵培养基混合，进行发酵培养，得到红曲色素。The Monascus seed solution in logarithmic growth phase, lauryl alcohol polyoxyethylene ether and fermentation medium are mixed to carry out fermentation culture to obtain Monascus pigment.
优选地，所述红曲菌种子液和发酵培养基的体积比为6～12％。Preferably, the volume ratio of the Monascus seed solution to the fermentation medium is 6-12%.
优选地，所述红曲菌种子液和发酵培养基的体积比为9～11％。Preferably, the volume ratio of the Monascus seed solution to the fermentation medium is 9-11%.
优选地，所述红曲菌种子液和发酵培养基的体积比为10％。Preferably, the volume ratio of the Monascus seed solution and the fermentation medium is 10%.
优选地，所述月桂醇聚氧乙烯醚的质量和发酵培养基的体积比为30～34g/L。Preferably, the mass ratio of the lauryl alcohol polyoxyethylene ether to the volume of the fermentation medium is 30-34 g/L.
优选地，所述月桂醇聚氧乙烯醚的质量和发酵培养基的体积比为31～33g/L。Preferably, the mass ratio of the lauryl alcohol polyoxyethylene ether to the volume of the fermentation medium is 31-33 g/L.
优选地，所述月桂醇聚氧乙烯醚的质量和发酵培养基的体积比为32g/L。Preferably, the mass ratio of the lauryl alcohol polyoxyethylene ether to the volume of the fermentation medium is 32 g/L.
优选地，所述处于对数生长期的红曲菌种子液、月桂醇聚氧乙烯醚和发酵培养基的混合完成后，加入水溶性锌盐，进行发酵培养，得到红曲色素。Preferably, after the Monascus seed solution in the logarithmic growth phase, the laureth polyoxyethylene ether and the fermentation medium are mixed, water-soluble zinc salt is added to carry out fermentation culture to obtain Monascus pigment.
优选地，所述水溶性锌盐包括硝酸锌、氯化锌和硫酸锌中的至少一种。Preferably, the water-soluble zinc salt includes at least one of zinc nitrate, zinc chloride and zinc sulfate.
优选地，所述水溶性锌盐的物质的量和发酵培养基中水的体积比为3.4×10-3～1×10-2mol/L。Preferably, the ratio of the amount of the water-soluble zinc salt to the volume of water in the fermentation medium is 3.4×10 -3 to 1×10 -2 mol/L.
本发明提供了一种提高红曲色素色调的方法，包括：将处于对数生长期的红曲菌种子液、月桂醇聚氧乙烯醚和发酵培养基混合，进行发酵培养，得到红曲色素。本发明通过选用月桂醇聚氧乙烯醚作为萃取剂，其原理在于月桂醇聚氧乙烯醚浊点温度(42℃)最接近发酵温度(30℃)，且同时具有较高的HLB值(HLB＝16.9)。浊点的高低决定发酵体系的浊点系统的形成，而萃取发酵过程必须伴随着浊点系统的形成。HLB表征非离子表面活性剂的亲水亲油平衡值，HLB值越高(HLB>13)，非离子表面活性剂的亲水性越强。当HLB值介于15和18之间时，非离子表面活性剂显示出强烈的有益增溶。因此，HLB值越高，非离子表面活性剂在溶液中的胶束分子的稳定性越好。只有这两个条件同时满足时，非离子表面活性剂才能表现出较强的红曲色素萃取能力，从而提高色调。实验结果表明，利用本发明提供的技术方案，红曲色素中红、橙、黄三色素的胞外色价分别为126.1U/mL、103.5U/mL、99.6U/mL，色调为0.79，优于同等条件下以Span80为萃取剂时，红、橙、黄三色素的胞外色价分别为87.4U/mL、73.9U/mL、63.3U/mL，色调为0.72。The invention provides a method for improving the color tone of Monascus pigment, comprising: mixing the Monascus seed liquid in logarithmic growth phase, lauryl alcohol polyoxyethylene ether and fermentation medium, and fermenting and culturing to obtain the Monascus pigment. The present invention selects lauryl alcohol polyoxyethylene ether as the extractant, the principle is that the lauryl alcohol polyoxyethylene ether cloud point temperature (42°C) is closest to the fermentation temperature (30°C), and at the same time has a higher HLB value (HLB= 16.9). The level of the cloud point determines the formation of the cloud point system of the fermentation system, and the extraction and fermentation process must be accompanied by the formation of the cloud point system. HLB characterizes the hydrophilic-lipophilic balance value of non-ionic surfactants. The higher the HLB value (HLB>13), the stronger the hydrophilicity of non-ionic surfactants. When the HLB value is between 15 and 18, nonionic surfactants show strong beneficial solubilization. Therefore, the higher the HLB value, the better the stability of the micellar molecules of the nonionic surfactant in solution. Only when these two conditions are satisfied at the same time, the non-ionic surfactant can show a strong extraction ability of Monascus pigment, thereby improving the color tone. The experimental results show that by using the technical solution provided by the present invention, the extracellular color values of red, orange and yellow pigments in Monascus pigments are 126.1U/mL, 103.5U/mL and 99.6U/mL respectively, and the color tone is 0.79, which is excellent. Under the same conditions, when Span80 was used as the extractant, the extracellular color values of red, orange and yellow pigments were 87.4U/mL, 73.9U/mL and 63.3U/mL, respectively, and the hue was 0.72.
具体实施方式Detailed ways
本发明提供了一种提高红曲色素色调的方法，包括：The invention provides a method for improving the color tone of Monascus, comprising:
将处于对数生长期的红曲菌种子液、月桂醇聚氧乙烯醚和发酵培养基混合，进行发酵培养，得到红曲色素。The Monascus seed solution in logarithmic growth phase, lauryl alcohol polyoxyethylene ether and fermentation medium are mixed to carry out fermentation culture to obtain Monascus pigment.
在本发明中，所述处于对数生长期的红曲菌种子液优选由紫红曲霉菌株依次经过活化和种子培养得到。在本发明中，所述处于对数生长期的红曲菌种子液中的红曲菌以几何级数恒定快速增殖，此时期的细菌大小、形态、染色性、生理活动等都处于典型状态。In the present invention, the Monascus seed solution in the logarithmic growth phase is preferably obtained from a Monascus purpura strain through activation and seed culture in sequence. In the present invention, the Monascus in the Monascus seed solution in the logarithmic growth phase proliferates constantly and rapidly in a geometric progression, and the size, shape, dyeability, physiological activity, etc. of the bacteria in this period are all in a typical state.
本发明对所述紫红曲霉菌株的种类没有特殊规定，能够产生红曲色素的紫红曲霉菌株均可。本发明对所述紫红曲霉菌株的来源没有特殊规定，采用常规市售的紫红曲霉菌株即可。在本发明实施例中，所述紫红曲霉菌株优选为紫红曲霉菌株NJ1，所述紫红曲霉菌株NJ1为常规市售。The present invention has no special provisions on the types of the Monascus purpureus strains, and any Monascus purpureus strains capable of producing monascus pigments may be used. The present invention has no special provisions on the source of the Monascus purpureus strain, and conventional commercially available Monascus purpureus strains may be used. In the embodiment of the present invention, the Monascus purpureus strain is preferably the Monascus purpureus strain NJ1, and the Monascus purpureus strain NJ1 is commercially available.
本发明对所述活化的活化培养基没有特殊规定，采用本领域技术人员熟知的活化培养基即可。在本发明实施例中，所述活化的培养基优选为马铃薯葡萄糖琼脂培养基，简称PDA培养基。本发明对所述PDA培养基的组成没有特殊规定，采用本领域技术人员熟知的PDA培养基即可。在本发明中，所述活化培养基提供菌种活化所需要的营养物质。The present invention has no special provisions on the activated activation medium, and an activated medium well known to those skilled in the art can be used. In the embodiment of the present invention, the activated medium is preferably potato dextrose agar medium, PDA medium for short. The present invention has no special provisions on the composition of the PDA medium, and the PDA medium well known to those skilled in the art can be used. In the present invention, the activation medium provides nutrients required for the activation of bacterial species.
本发明对所述活化的方式没有特殊规定，采用本领域技术人员熟知的活化方式，对所述紫红曲霉菌株进行活化培养即可。在本发明实施例中，所述活化优选为将紫红曲霉NJ1菌株于PDA平板培养基上划线，在30℃恒温培养箱中活化菌种3代，每代培养3天。本发明通过活化，将保藏状态的菌种放入活化培养基中培养，逐级扩大培养得到纯而壮的培养物，即获得活力旺盛的、接种数量足够的培养物。The present invention does not specifically stipulate the activation method, and the activation method well known to those skilled in the art can be used to activate and cultivate the Monascus purpureus strain. In the embodiment of the present invention, the activation is preferably performed by streaking the NJ1 strain of Monascus purpurea on a PDA plate medium, and activating the strain in a 30°C constant temperature incubator for 3 generations, and culturing each generation for 3 days. In the present invention, by activating, the bacteria in the preserved state are cultured in an activated medium, and the culture is expanded step by step to obtain a pure and strong culture, that is, a culture with vigorous vitality and sufficient inoculation quantity is obtained.
本发明对所述种子培养的种子培养基没有特殊规定，采用本领域技术人员熟知的紫红曲霉菌株的种子培养基即可。在本发明实施例中，所述种子培养基优选为每升水中加入葡萄糖30g、黄豆粉15g、甘油70g、蛋白胨10g、MgSO4·7H2O1g、KH2PO4 2g、NaNO3 2g。在本发明中，所述种子培养基提供紫红曲霉菌株扩大培养时所需要的营养物质。The present invention has no special provisions on the seed medium for the seed culture, and the seed medium of the strain of Monascus purpureus well-known to those skilled in the art can be used. In the embodiment of the present invention, the seed medium is preferably 30 g of glucose, 15 g of soybean powder, 70 g of glycerol, 10 g of peptone, 1 g of MgSO 4 ·7H 2 O 1 g, 2 g of KH 2 PO 4 , and 2 g of NaNO 3 per liter of water. In the present invention, the seed medium provides nutrients required for the expansion of the Monascus purpureus strain.
本发明对所述种子培养的培养方式没有特殊规定，采用本领域技术人员熟知的种子培养方式，对所述紫红曲霉菌株进行种子培养即可。在本发明实施例中，所述种子培养优选为将活化好的紫红曲霉菌株接种到种子液，置于温度为30℃，转速为200r/min的摇床中培养48h。本发明通过种子培养，实现对紫红曲霉菌株逐级扩大培养，进而获得以几何级数恒定快速增殖的红曲菌。处于对数生长的红曲菌和种子培养基，即称为处于对数生长期的红曲菌种子液。The present invention does not have special provisions on the cultivation method of the seed cultivation, and the seed cultivation method well known to those skilled in the art can be used to cultivate the Monascus purpureus strain. In the embodiment of the present invention, the seed culture is preferably by inoculating the activated Monascus purpureus strain into the seed solution, and placing it in a shaker with a temperature of 30° C. and a rotation speed of 200 r/min for 48 hours. The invention realizes the step-by-step expansion and cultivation of Monascus purpureus strains through seed culture, and further obtains Monascus spp. with constant and rapid proliferation in geometric progression. Monascus and seed medium in logarithmic growth are called Monascus seed solution in logarithmic growth phase.
本发明对所述发酵培养的发酵培养基没有特殊规定，采用本领域技术人员熟知的紫红曲霉菌株进行发酵获得红曲色素的发酵培养基即可。在本发明实施例中，所述发酵培养基优选为每升水中加入大米粉20g、甘油90g、蛋白胨10g、MgSO4·7H2O 1g、KH2PO4 2.5g、NaNO3 5 g。在本发明中，所述发酵培养基提供微生物发酵时所需要的原料。The present invention does not have special provisions on the fermentation medium of the fermentation culture, and the fermentation medium of Monascus pigment can be obtained by fermenting a strain of Monascus purpureus well-known to those skilled in the art. In the embodiment of the present invention, the fermentation medium is preferably 20 g of rice flour, 90 g of glycerol, 10 g of peptone, 1 g of MgSO 4 ·7H 2 O 1 g, 2.5 g of KH 2 PO 4 , and 5 g of NaNO 3 per liter of water. In the present invention, the fermentation medium provides the raw materials required for the fermentation of microorganisms.
在本发明中，所述红曲菌种子液和发酵培养基的体积比优选为6～12％，更优选为9～11％，最优选为10％。本发明将所述红曲菌种子液和发酵培养基的用量控制在上述范围，得到的红曲色素色调最好。In the present invention, the volume ratio of the Monascus seed solution and the fermentation medium is preferably 6-12%, more preferably 9-11%, and most preferably 10%. In the present invention, the dosage of the Monascus seed liquid and fermentation medium is controlled within the above range, and the obtained Monascus pigment has the best color tone.
本发明对所述月桂醇聚氧乙烯醚的来源没有特殊规定，采用本领域技术人员熟知的常规市售产品即可。在本发明中，所述月桂醇聚氧乙烯醚的质量和发酵培养基的体积比优选为30～34g/L，更优选为31～33g/L，最优选为32g/L。本发明将所述月桂醇聚氧乙烯醚的用量控制在上述范围，得到的红曲色素的产量和色调最好。本发明通过向发酵培养基中添加非离子表面活性剂月桂醇聚氧乙烯醚，伴随着浊点系统形成，发酵体系自动形成非离子表面活性剂胶束富集的聚集相和胶束很少的稀相，发酵过程中疏水性红曲色素从胞内萃取到胞外并大量富集在聚集相，从而解除胞内产物反馈抑制，增加色素产量。The present invention has no special provisions on the source of the lauryl alcohol polyoxyethylene ether, and conventional commercial products well known to those skilled in the art can be used. In the present invention, the mass ratio of the lauryl alcohol polyoxyethylene ether to the volume of the fermentation medium is preferably 30-34 g/L, more preferably 31-33 g/L, and most preferably 32 g/L. In the present invention, the amount of the lauryl alcohol polyoxyethylene ether is controlled within the above-mentioned range, and the yield and color tone of the obtained Monascus pigment are the best. In the present invention, by adding the nonionic surfactant lauryl alcohol polyoxyethylene ether into the fermentation medium, along with the formation of the cloud point system, the fermentation system automatically forms a nonionic surfactant micelle-enriched aggregated phase and a small amount of micelles. In the dilute phase, during the fermentation process, the hydrophobic Monascus pigment is extracted from the intracellular to the extracellular and enriched in the aggregated phase, thereby releasing the feedback inhibition of the intracellular product and increasing the pigment production.
本发明对所述处于对数生长期的红曲菌种子液、月桂醇聚氧乙烯醚和发酵培养基的混合方式没有特殊规定，采用本领域技术人员熟知的混合方式，将上述物质混合均匀即可。The present invention has no special provisions on the mixing method of the Monascus seed liquid, lauryl alcohol polyoxyethylene ether and fermentation medium in the logarithmic growth phase. The mixing method well-known to those skilled in the art is adopted to mix the above-mentioned substances evenly, that is, Can.
本发明将所述处于对数生长期的红曲菌种子液、月桂醇聚氧乙烯醚和发酵培养基混合后，直接进行发酵培养得到红曲色素。本发明对所述发酵培养的培养方式没有特殊规定，采用本领域技术人员熟知的发酵方式，利用紫红曲霉菌株将发酵培养基原料通过微生物发酵获得目标发酵产品红曲色素即可。In the present invention, the Monascus seed solution in the logarithmic growth phase, the lauryl alcohol polyoxyethylene ether and the fermentation medium are mixed, and then directly fermented and cultured to obtain the Monascus pigment. The present invention has no special provisions on the culture method of the fermentation culture, and the fermentation method well-known to those skilled in the art is adopted, and the target fermentation product Monascus pigment can be obtained by microbial fermentation of the fermentation medium raw material by using Monascus purpura strain.
发酵完成后，本发明优选对所述发酵后的溶液体系依次进行离心和过滤得到红曲色素。After the fermentation is completed, the present invention preferably performs centrifugation and filtration on the fermented solution system in sequence to obtain Monascus pigment.
本发明对所述离心的方式没有特殊规定，采用本领域技术人员熟知的离心方式将溶液中不溶性杂质除去即可。The present invention has no special provisions on the centrifugation method, and the insoluble impurities in the solution can be removed by the centrifugal method well known to those skilled in the art.
本发明对所述过滤的方式没有特殊规定，采用本领域技术人员熟知的过滤方式，将离心后溶液中不溶性物质分离出去即可。The present invention does not specifically stipulate the filtration method, and the filtration method well-known to those skilled in the art can be used to separate out the insoluble substances in the solution after centrifugation.
本发明提供了一种提高红曲色素色调的方法，包括：将处于对数生长期的红曲菌种子液、月桂醇聚氧乙烯醚和发酵培养基混合，进行发酵培养，得到红曲色素。本发明通过选用月桂醇聚氧乙烯醚作为萃取剂，获得的红曲色素具有较高的色调。本发明选用的月桂醇聚氧乙烯醚浊点温度接近发酵温度，促进了发酵体系浊点系统的形成；同时由于其亲水性较强，显示出强烈的有益增溶，从而进一步促进了胶束分子在水溶液中的稳定性；最终在上述原理的共同作用下，提高了对红曲色素萃取能力，从而提高了红曲色素色调。The invention provides a method for improving the color tone of Monascus pigment, comprising: mixing the Monascus seed liquid in logarithmic growth phase, lauryl alcohol polyoxyethylene ether and fermentation medium, and fermenting and culturing to obtain the Monascus pigment. In the present invention, by selecting lauryl alcohol polyoxyethylene ether as the extracting agent, the obtained Monascus pigment has higher color tone. The cloud point temperature of the lauryl alcohol polyoxyethylene ether selected in the present invention is close to the fermentation temperature, which promotes the formation of the cloud point system of the fermentation system; at the same time, due to its strong hydrophilicity, it shows strong beneficial solubilization, thereby further promoting the micelle The stability of the molecule in the aqueous solution; finally, under the joint action of the above principles, the extraction ability of the Monascus pigment is improved, thereby improving the color of the Monascus pigment.
本发明优选在所述处于对数生长期的红曲菌种子液、月桂醇聚氧乙烯醚和发酵培养基的混合完成后，加入水溶性锌盐，进行发酵培养，得到红曲色素。本发明通过在发酵培养基中加入水溶性锌盐，进一步提高了红曲色素的色调。在本发明中，所述水溶性锌盐中的Zn2+对红曲霉产色素具有促进作用，且作用程度较大，对不同的菌种影响不同，部分菌种可以达到100％以上。Zn2+具有最优的促进作用。In the present invention, preferably, after the Monascus seed solution in the logarithmic growth phase, the laureth polyoxyethylene ether and the fermentation medium are mixed, water-soluble zinc salt is added to carry out fermentation culture to obtain Monascus pigment. The present invention further improves the color tone of Monascus pigment by adding water-soluble zinc salt into the fermentation medium. In the present invention, Zn 2+ in the water-soluble zinc salt has a promoting effect on the production of pigment by Monascus, and the effect is large, and has different effects on different strains, and some strains can reach more than 100%. Zn 2+ has the best promoting effect.
在本发明中，所述水溶性锌盐优选包括硝酸锌、氯化锌和硫酸锌中的至少一种，更优选为硫酸锌。在本发明中，所述硫酸锌不但可以提供锌离子，而且硫酸锌中的硫元素还可以提供微生物生长过程中所需要的硫。In the present invention, the water-soluble zinc salt preferably includes at least one of zinc nitrate, zinc chloride and zinc sulfate, more preferably zinc sulfate. In the present invention, the zinc sulfate can not only provide zinc ions, but also the sulfur element in the zinc sulfate can also provide the sulfur required during the growth of microorganisms.
在本发明中，所述锌盐的物质的量和发酵培养基中水的体积比优选为3.4×10-3～1×10-2mol/L，更优选为5×10-3～8×10-3mol/L。本发明将所述锌盐的用量控制在上述范围，得到的红曲色素的色调更好。In the present invention, the ratio of the amount of the zinc salt to the volume of water in the fermentation medium is preferably 3.4×10 -3 to 1×10 -2 mol/L, more preferably 5×10 -3 to 8× 10 -3 mol/L. In the present invention, the dosage of the zinc salt is controlled within the above range, and the color tone of the obtained Monascus pigment is better.
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
色价(U/mL)＝OD值×稀释倍数(其中OD值是指分光光度计分别在410nm、465nm、505nm波长下测定的红曲色素稀释一定倍数的OD值)；其中，红色素色价(U/mL)＝OD(505nm)×稀释倍数；橙色素色价(U/mL)＝OD(465nm)×稀释倍数；黄色素色价(U/mL)＝OD(410nm)×稀释倍数。Color value (U/mL)=OD value×dilution factor (wherein OD value refers to the OD value of Monascus pigment diluted by a certain multiple measured by spectrophotometer at wavelengths of 410nm, 465nm, and 505nm respectively); (U/mL)=OD(505nm)×dilution factor; orange pigment value (U/mL)=OD(465nm)×dilution factor; yellow pigment value (U/mL)=OD(410nm)×dilution factor.
色调＝黄色素色价/红色素色价，色调大于1，黄色素多于红色素，反之红色素多于黄色素。Hue = yellow color value/red color value, if the color tone is greater than 1, the yellow pigment is more than the red pigment, and vice versa, the red pigment is more than the yellow pigment.
实施例1Example 1
实验菌种：产红曲色素的菌株NJ1(常规市售)Experimental strain: Monascus pigment-producing strain NJ1 (commonly available)
步骤(1)菌种活化Step (1) Strain activation
将零下80℃保藏的紫红曲霉NJ1菌株于PDA平板培养基上划线，在30℃恒温培养箱中活化菌种3代，每代培养3天，得到活化好的菌株。The Monascus purpureus NJ1 strain stored at minus 80°C was streaked on the PDA plate medium, and the strain was activated in a 30°C constant temperature incubator for 3 generations, and each generation was cultured for 3 days to obtain an activated strain.
步骤(2)种子培养Step (2) Seed Cultivation
种子培养基组成(g/L)：每升水中加入葡萄糖30g、黄豆粉15g、甘油70g、蛋白胨10g、MgSO4·7H2O1g、KH2PO4 2g、NaNO3 2g。Composition of seed medium (g/L): 30 g of glucose, 15 g of soybean powder, 70 g of glycerol, 10 g of peptone, 1 g of MgSO 4 ·7H 2 O1 g, 2 g of KH 2 PO 4 , and 2 g of NaNO 3 were added per liter of water.
将步骤(1)得到的活化好的菌株接种到种子液(装液量50mL/250mL)，种子液在30℃、200r/min的摇床中培养48h，得到对数生长期的红曲菌种子液。The activated strain obtained in step (1) is inoculated into the seed solution (filling volume of 50mL/250mL), and the seed solution is cultured in a shaker at 30°C and 200r/min for 48h to obtain Monascus seeds in logarithmic growth phase. liquid.
步骤(3)发酵培养Step (3) Fermentation Culture
发酵液培养基(g/L)：每升水中加入大米粉20g、甘油90g、蛋白胨10g、MgSO4·7H2O1g、KH2PO4 2.5g、NaNO3 5g。Fermentation broth medium (g/L): 20 g of rice flour, 90 g of glycerol, 10 g of peptone, 1 g of MgSO 4 ·7H 2 O 1 g, 2.5 g of KH 2 PO 4 , and 5 g of NaNO 3 were added per liter of water.
将步骤(2)得到的对数生长期的红曲菌种子液5mL接入装有50mL发酵培养基的250mL三角瓶中，同时在发酵培养基中加入非离子表面活性剂月桂醇聚氧乙烯醚Brij35(每升发酵培养基中加入32g月桂醇聚氧乙烯醚)；在转速为150r/min，温度25℃的条件下摇瓶培养10天，发酵结束后，取发酵液，12000r/min离心，上清液中可得胞外色素即红曲色素。The 5mL of the Monascus seed liquid in the logarithmic growth phase obtained in step (2) is inserted into the 250mL conical flask that 50mL fermentation medium is housed in, and the nonionic surfactant laureth is added in the fermentation medium simultaneously. Brij35 (add 32g lauryl alcohol polyoxyethylene ether per liter of fermentation medium); under the condition of rotating speed of 150r/min and temperature of 25°C, shake flask culture for 10 days, after fermentation, take the fermentation broth, centrifuge at 12000r/min, The extracellular pigment, monascus pigment, can be obtained in the supernatant.
步骤(4)色价测试，Step (4) color value test,
将步骤(3)得到的红曲色素稀释180倍，进行紫外分光光度计测量。测量结果：发酵液中的红、橙、黄三种色素胞外产量分别为118.3U/mL、94.2U/mL、89.5U/mL，计算得红曲色素的色调为0.76，红多于黄。The monascus pigment obtained in step (3) was diluted 180 times and measured by an ultraviolet spectrophotometer. Measurement results: The extracellular yields of red, orange, and yellow pigments in the fermentation broth were 118.3U/mL, 94.2U/mL, and 89.5U/mL, respectively. The calculated hue of Monascus pigment was 0.76, with more red than yellow.
实施例2Example 2
操作与实施例1相同，区别仅在于在配制发酵液时，发酵液中添加2g/L的ZnSO4·7H2O(即每升水中加入6.96×10-3mol的ZnSO4·7H2O)。测量结果：发酵液中的红、橙、黄三种色素胞外产量分别为126.1U/mL、103.5U/mL、99.6U/mL，色调为0.79，红多于黄。The operation is the same as in Example 1, except that when preparing the fermentation broth, 2g/L of ZnSO 4 ·7H 2 O is added to the fermentation broth (that is, 6.96×10 -3 mol of ZnSO 4 ·7H 2 O is added per liter of water) . Measurement results: The extracellular yields of red, orange, and yellow pigments in the fermentation broth were 126.1 U/mL, 103.5 U/mL, and 99.6 U/mL, respectively, and the hue was 0.79, with more red than yellow.
对比例1Comparative Example 1
操作与实施例1相同，区别仅在于不添加非离子表面活性剂。测量结果：发酵液中的红、橙、黄三种色素胞外产量分别为52.2U/mL、43.7U/mL、36.2U/mL，色调为0.69，红多于黄。The operation was the same as in Example 1, except that no nonionic surfactant was added. Measurement results: The extracellular yields of red, orange, and yellow pigments in the fermentation broth were 52.2U/mL, 43.7U/mL, and 36.2U/mL, respectively, and the hue was 0.69, with more red than yellow.
对比例2：Comparative Example 2:
操作与实施例1相同，区别仅在于添加非离子表面活性剂由月桂醇聚氧乙烯醚Brij35改为Span80。测量结果：发酵液中的红、橙、黄三种色素胞外产量分别为87.4U/mL、73.9U/mL、63.3U/mL，色调为0.72，红多于黄。The operation is the same as in Example 1, except that the nonionic surfactant added is changed from Brij35 to Span80. Measurement results: The extracellular yields of red, orange, and yellow pigments in the fermentation broth were 87.4U/mL, 73.9U/mL, and 63.3U/mL, respectively, and the hue was 0.72, with more red than yellow.
通过实施例1以及对比例1～2可以看出：在发酵液中加入非离子表面活性剂相比未加入非离子表面活性剂，色调提高10.1％；添加Brij35较添加Span80色调提高5.6％。From Example 1 and Comparative Examples 1 to 2, it can be seen that adding non-ionic surfactant to the fermentation broth increases the color tone by 10.1% compared to adding no non-ionic surfactant; adding Brij35 increases the color tone by 5.6% compared to adding Span80.
通过实施例2以及对比例1～2可以看出：在发酵液中同时加入Brij35和锌盐相比不加非离子表面活性剂色调提高14.5％，较加入Brij35不加锌盐，色调提高3.9％。From Example 2 and Comparative Examples 1 to 2, it can be seen that adding Brij35 and zinc salts to the fermentation broth at the same time increases the color tone by 14.5% compared to adding Brij35 without the addition of nonionic surfactants, and increases the color tone by 3.9% compared to adding Brij35 without adding zinc salts .
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0469704A1 (en) * | 1990-08-03 | 1992-02-05 | Uop | Water-soluble colored pigments from monascorubrin and rubropunctatin as food colorants |
| CN104946718A (en) * | 2014-03-26 | 2015-09-30 | 江南大学 | Double-liquid-phase fermentation method of red koji citraurin by coupled in-situ fermentation-extraction |
| CN104946699A (en) * | 2014-03-26 | 2015-09-30 | 江南大学 | Double-liquid-phase fermentation method of monascus yellow pigment by coupled in-situ fermentation-extraction |
| CN106755171A (en) * | 2017-02-09 | 2017-05-31 | 江南大学 | A kind of method for promoting red yeast rice strains liquid fermentation to produce red yeast rice citraurin |
| CN108251457A (en) * | 2017-12-23 | 2018-07-06 | 北京工商大学 | A kind of composite extractant and the method for promoting monascorubin production using composite extractant |
| CN112646842A (en) * | 2021-02-02 | 2021-04-13 | 广东天益生物科技有限公司 | Water-soluble monascus yellow pigment with high hue and color value and preparation method and application thereof |
-
2022
- 2022-01-14 CN CN202210040184.9A patent/CN114350526A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0469704A1 (en) * | 1990-08-03 | 1992-02-05 | Uop | Water-soluble colored pigments from monascorubrin and rubropunctatin as food colorants |
| CN104946718A (en) * | 2014-03-26 | 2015-09-30 | 江南大学 | Double-liquid-phase fermentation method of red koji citraurin by coupled in-situ fermentation-extraction |
| CN104946699A (en) * | 2014-03-26 | 2015-09-30 | 江南大学 | Double-liquid-phase fermentation method of monascus yellow pigment by coupled in-situ fermentation-extraction |
| CN106755171A (en) * | 2017-02-09 | 2017-05-31 | 江南大学 | A kind of method for promoting red yeast rice strains liquid fermentation to produce red yeast rice citraurin |
| CN108251457A (en) * | 2017-12-23 | 2018-07-06 | 北京工商大学 | A kind of composite extractant and the method for promoting monascorubin production using composite extractant |
| CN112646842A (en) * | 2021-02-02 | 2021-04-13 | 广东天益生物科技有限公司 | Water-soluble monascus yellow pigment with high hue and color value and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| XUELIAN YANG ET AL.: ""Effect of nonionic surfactant Brij 35 on morphology, cloud point, and pigment stability in Monascus extractive fermentation"", 《J SCI FOOD AGRIC》 * |
| XUELIAN YANG ET QAL.: ""Effects of nonionic surfactants on pigment excretion and cell morphology in extractive fermentation of Monascus sp. NJ1"", 《J SCI FOOD AGRIC》 * |
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