WO2023116302A1 - 一种木糖母液联产赤藓糖醇和阿拉伯糖的方法 - Google Patents

一种木糖母液联产赤藓糖醇和阿拉伯糖的方法 Download PDF

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WO2023116302A1
WO2023116302A1 PCT/CN2022/133112 CN2022133112W WO2023116302A1 WO 2023116302 A1 WO2023116302 A1 WO 2023116302A1 CN 2022133112 W CN2022133112 W CN 2022133112W WO 2023116302 A1 WO2023116302 A1 WO 2023116302A1
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erythritol
glucose
xylose
mother liquor
arabinose
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PCT/CN2022/133112
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French (fr)
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吴爱娟
罗家星
胡昌辉
杨铭乾
廖承军
郑毅
方顺成
李勉
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浙江华康药业股份有限公司
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Priority to EP22909618.5A priority Critical patent/EP4299578A4/en
Priority to JP2023549915A priority patent/JP2024505107A/ja
Publication of WO2023116302A1 publication Critical patent/WO2023116302A1/zh
Priority to US18/354,044 priority patent/US11866756B2/en

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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
    • C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
    • C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/02—Monosaccharides
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces

Definitions

  • the invention belongs to the technical field of utilization of xylose mother liquor, in particular to a method for co-producing erythritol and arabinose from xylose mother liquor.
  • xylose mother liquor In the process of producing xylitol, a large amount of xylose mother liquor is produced.
  • the xylose mother liquor has high miscellaneous sugar content and is often sold as a by-product for making caramel coloring, etc., with low added value.
  • the content of xylose in the xylose mother liquor is 40%-60%, glucose 10%-20%, arabinose 15%-20%, mannose 0-10%, galactose 0-5%.
  • There are many methods for processing and utilizing xylose mother liquor mainly extracting xylose and arabinose therein. In order to reduce the difficulty of extraction and separation, bacteria or yeast are used for fermentation, and glucose or galactose is consumed as a carbon source for bacterial growth, thereby improving the extraction efficiency of xylose and arabinose.
  • the patent of publication number CN112094956A uses Saccharomyces cerevisiae to ferment xylose mother liquor continuously, consumes glucose, and utilizes chromatography to separate and purify xylose and arabinose.
  • the patent of notification number CN101705253B utilizes secondary fermentation to convert xylose in the mother liquor into xylitol and purify arabinose at the same time.
  • Patent No. CN102603814B also uses yeast fermentation to remove glucose and galactose in xylose mother liquor diluted to 20%, to obtain xylose and arabinose serum.
  • the patent of notification number CN101857523B uses fermentation to remove glucose and galactose in the mother liquor, decolorizes, separates, concentrates, and hydrogenates the filtered clear liquid to prepare xylitol and arabitol, and then separates the two components by chromatography and crystallizes to obtain two finished product.
  • the patent of notification number CN102952165B also uses fermentation to remove glucose and galactose, and then separates arabinose crystals according to different crystallization characteristics.
  • the patent of Publication No. CN109504733A uses Pichia pastoris and Aureobasidium pullulans to ferment mixed bacteria to produce erythritol.
  • the carbon source uses xylose mother liquor, the concentration of erythritol is very low, only about 40g/L.
  • Most of the patented technologies mentioned above are to remove glucose or galactose through bacterial strain fermentation, further separate xylose and arabinose, and improve their purity.
  • bacteria Due to the high sugar content in the xylose mother liquor, bacteria are generally not suitable for growth.
  • High-osmosis-resistant yeast is often used for high-aerobic fermentation, which requires a lot of compressed air.
  • the diluted mother liquor has a large amount and all glucose After utilization, more bacterial cells are required, multiple expansions are required, and the fermentation time is 48 hours or longer.
  • the technical problem to be solved by the present invention is to provide a method for co-producing erythritol and arabinose from xylose mother liquor, reduce the utilization cost of xylose mother liquor, and improve the utilization value of glucose while realizing the separation of xylose and arabinose.
  • the value-added product erythritol is produced by yeast fermentation, and the yield of erythritol is increased.
  • the present invention is achieved in that a method for co-producing erythritol and arabinose from xylose mother liquor is provided, comprising the steps of:
  • Step 1 after the xylose mother liquor is subjected to the first chromatographic separation treatment of the simulated moving bed, the xylose extract with high xylose component content and the xylose raffinate with high glucose component content are respectively obtained, and the xylose extract is concentrated and Xylose crystals are obtained after crystallization treatment.
  • Step 2 Concentrate the xylose raffinate to a solid content of 30% to 50%, and a glucose content of 9% to 14%, and then mix it with liquid glucose or crystalline glucose to obtain a glucose mixture.
  • Glucose content is 40% ⁇ 50%.
  • Step 3 inoculate the pre-prepared Yarrowia lipolytica seed liquid into the fermentation medium of the fermenter, and at the same time add the glucose mixed liquid in step 2 to ferment to obtain a fermented liquid, the glucose content of the fermented liquid is ⁇ 0.3%; ferment The fermentation filtrate is obtained after the liquid is filtered, and the fermentation filtrate is successively decolorized, separated, concentrated, centrifuged and crystallized to obtain erythritol crystals and erythritol centrifuged mother liquor respectively.
  • Step 4 After the second chromatographic separation treatment of the erythritol centrifugal mother liquor, the erythritol extract with high erythritol component content and the erythritol raffinate with high arabinose component content were obtained respectively.
  • the erythritol extract is mixed with the fermentation filtrate in step 3, and the erythritol raffinate is sequentially decolorized, separated, concentrated, and crystallized to obtain arabinose crystals.
  • the method for co-producing erythritol and arabinose from xylose mother liquor of the present invention uses the first chromatography to separate xylose to obtain extract and raffinate, and the extract is used to prepare crystalline xylose,
  • the raffinate is blended with liquid glucose or crystal glucose, and Yarrowia lipolytica, which is resistant to hypertonicity and high conversion rate, is used to ferment and produce erythritol, and then use the low solubility and easy crystallization characteristics of erythritol to conduct centrifugal crystallization first Erythritol crystals are obtained by processing, and the centrifuged mother liquor of erythritol is separated by a second chromatography to obtain a raffinate with high arabinose content, and then arabinose crystals are prepared.
  • the present invention realizes the high-efficiency utilization of xylose mother liquor, while obtaining xylose and arabinose, utilizes glucose to produce erythritol with higher added value, and at the same time mixes with liquid glucose or crystal glucose, on the one hand can improve
  • the content of glucose in the culture medium increases the yield of erythritol in each batch of fermentation.
  • it improves the utilization rate of mother liquor in each batch of fermentation.
  • the ratio of xylose mother liquor raffinate to fermentation broth is used to prepare erythritol.
  • the concentration of erythritol is above 156g/L, and the conversion rate is above 52%.
  • the invention also reduces the fermentation cost, increases the added value of the xylose mother liquor, and increases economic benefits.
  • Fig. 1 is the principle schematic diagram of the method for co-producing erythritol and arabinose from xylose mother liquor of the present invention.
  • the preferred embodiment of the method for the co-production of xylose mother liquor of the present invention erythritol and arabinose comprises the following steps:
  • Step 1 after the xylose mother liquor is subjected to the first chromatographic separation treatment of the simulated moving bed, the xylose extract with high xylose component content and the xylose raffinate with high glucose component content are respectively obtained, and the xylose extract is concentrated and Xylose crystals are obtained after crystallization treatment.
  • Step 2 Concentrate the xylose raffinate to a solid content of 30% to 50% (g/100mL, the following descriptions are the same), and a glucose content of 9% to 14%, and then mix it with liquid glucose or crystal glucose to obtain The glucose mixed solution, in the glucose mixed solution, the glucose content is 40%-50%.
  • Step 3 inoculate the pre-prepared Yarrowia lipolytica seed liquid into the fermentation medium of the fermenter, and at the same time add the glucose mixed liquid in step 2 to ferment to obtain a fermented liquid, the glucose content of the fermented liquid is ⁇ 0.3%; ferment The fermentation filtrate is obtained after the liquid is filtered, and the fermentation filtrate is successively decolorized, separated, concentrated, centrifuged and crystallized to obtain erythritol crystals and erythritol centrifuged mother liquor respectively.
  • Step 4 After the second chromatographic separation treatment of the erythritol centrifugal mother liquor, the erythritol extract with high erythritol component content and the erythritol raffinate with high arabinose component content were obtained respectively solution, the erythritol extract is mixed with the fermentation filtrate in step 3, the erythritol extract is recycled, and the erythritol raffinate is sequentially decolorized, separated, concentrated, and crystallized to obtain Arabica sugar crystals.
  • the fermentation medium is configured according to the following proportions: glucose content 25%-32%, yeast extract 0.5%-1%, corn steep liquor dry powder 0.3%-0.8%, magnesium sulfate 0.03%-0.08% , ammonium citrate 0.2% to 0.8% and dipotassium hydrogen phosphate 0.02% to 0.05%.
  • the pre-prepared Yarrowia lipolytica seed solution is prepared as follows: insert the Yarrowia lipolytica strain into the test tube slant and cultivate it to obtain the test tube slant seed, and prepare the test tube slant in the test tube Seed medium, test tube slant seed medium is configured according to the following proportions: glucose 20%-25%, yeast extract 0.8%-1.5% and agar 1.5%-2%.
  • the pre-prepared Yarrowia lipolytica seed liquid is prepared according to the following method: the Yarrowia lipolytica strain is inserted into an eggplant-shaped bottle slant and cultivated to obtain eggplant-shaped slanted seeds,
  • the eggplant-shaped slant seed medium is prepared in advance, and the eggplant-shaped slant seed medium is configured according to the following proportions: 20%-25% of glucose, 0.8%-1.5% of yeast extract and 1.5%-2% of agar.
  • the pre-prepared Yarrowia lipolytica seed liquid is prepared according to the following method: the Yarrowia lipolytica strain is inserted into a shake flask and cultured to obtain a shake flask seed liquid, and the shake flask seed liquid is prepared in advance.
  • the shake flask seed culture medium is configured according to the following proportions: 20%-25% of glucose, 0.8%-1.5% of yeast extract, 0.03%-0.08% of magnesium sulfate and 0.2%-0.7% of ammonium citrate.
  • the pre-prepared Yarrowia lipolytica seed liquid is prepared according to the following method: the Yarrowia lipolytica strain is inserted into a fermenter for cultivation to obtain a fermenter seed liquid, and the fermenter is prepared in advance Fermenter seed medium, fermenter seed medium is configured according to the following proportions: glucose 25% to 30%, yeast extract 0.5% to 1.0%, peptone 0.3% to 0.8%, magnesium sulfate 0.03% to 0.08%, and ammonium citrate 0.2% % to 0.8%, the inoculum size is 5% to 10%, the initial pH of the fermentation is 6.0 to 7.0, the sterilization temperature of the fermenter seed medium is 115°C to 121°C, and the sterilization time is 20min to 30min.
  • the xylose mother liquor is subjected to the first chromatographic separation treatment to obtain a xylose-containing extract, the extract is concentrated to a solid content of 80%, evaporated and crystallized to obtain crystalline xylose.
  • the proportion of each component in the raffinate is 25%-32% of glucose, 18%-25% of arabinose, and 5%-15% of galactose; It is 12%, and it is proportioned with crystal glucose so that the glucose content of the mixed solution is 45%, and the preparation system is 100L.
  • other auxiliary materials in the fermentation medium formula except glucose are added in proportion, sterilized, and set aside.
  • Fermentation system 70L a total of 3 batches, using the method of process feeding, the specific operation is as follows:
  • the initial volume of fermentation is 40L
  • the medium is prepared according to the above-mentioned fermentation medium formula
  • the initial glucose content in the medium is 18%.
  • Shake flask seeds were fermented in 500mL and 5L shake flasks respectively, and the filling volume was 10% of the total volume, and cultured for 20h to 24h.
  • the bacterial density in the 5L shake flask was 18 to 25 (at a wavelength of 600nm, referred to as OD value) to the fermenter
  • the inoculum size is 8%
  • the fermentation temperature is 30°C
  • the rotation speed is 200rpm-400rpm
  • the dissolved oxygen is 20%-30%
  • the ventilation rate is 1.5Nm 3 /h.
  • the concentration of erythritol is all above 156g/L, and the conversion rate is more than or equal to 52.2%.
  • the fermented liquid is filtered through a ceramic membrane to obtain a supernatant, which is decolorized and separated, concentrated to a solid content of 68%, cooled and crystallized at a rate of 5°C/h, and centrifuged after 20 hours to obtain erythritol crystals .
  • the centrifuged mother liquor is subjected to the second chromatographic separation treatment of the simulated moving bed to obtain erythritol and arabinose components, and the erythritol component is returned to the filtered supernatant to increase the production of erythritol, and the purity of the arabinose component reaches 75%. , concentrated, crystallized, and centrifuged to obtain arabinose crystals.
  • the acquisition of raffinate and the proportion of each component are the same as in Example 1. Concentrate the raffinate to a solid content of 50%, wherein the glucose content is 14.3%, and the liquid glucose is concentrated to a glucose content of 65%, and the two are mixed in a volume ratio of 4:6 so that the glucose content of the mixed solution is 45%.
  • the system is 1000L, and at the same time, other auxiliary materials in the fermentation medium formula except glucose are added in proportion, sterilized, and set aside.
  • the fermentation system is 700L, and there are 3 batches in total.
  • the method of process feeding is adopted.
  • the specific operation is as follows:
  • the initial volume of fermentation is 400L.
  • the culture medium is formulated according to the above-mentioned fermentation medium, and the initial glucose content in the culture medium is 18.7%.
  • the concentration of erythritol is all above 160g/L, and the conversion rate is ⁇ 53.9%.
  • the fermented liquid was filtered through a ceramic membrane to obtain a supernatant, which was decolorized and separated, concentrated to a solid content of 65%, cooled and crystallized at a rate of 6°C/h, and centrifuged after 16 hours to obtain erythritol crystals.
  • the centrifuged mother liquor is subjected to the second chromatographic separation treatment of the simulated moving bed to obtain the erythritol and arabinose components.
  • the erythritol component is returned to the filtered supernatant, and the arabinose component has a purity of 77%.
  • concentration crystallized, and centrifuged to obtain arabinose crystals.
  • Example 2 The acquisition of raffinate and the proportion of each component are the same as in Example 1 and Example 2. Concentrate the raffinate to a solid content of 48%, wherein the glucose content is 13.7%, add 550L, and then add 380kg of crystalline glucose monohydrate and tap water, the glucose content of the mixed solution is 42%, the preparation system is 1000L, and at the same time add in proportion Other auxiliary materials in the fermentation medium formula other than glucose are sterilized and set aside.
  • the fermentation system is 700L, and there are 3 batches in total.
  • the method of process feeding is adopted.
  • the specific operation is as follows:
  • the initial volume of fermentation is 400L, and the medium is prepared according to the above-mentioned fermentation medium formula, and the initial glucose content in the medium is 20%, and it is prepared for sterilization and set aside.
  • the continuous feeding process keeps the glucose content in the fermentation liquid at 15% to 18%.
  • the glucose content in the fermentation liquid is less than 0.3%, the fermentation is stopped, and the total glucose content in the fermentation system is 30%.
  • the concentration of erythritol is all above 159g/L, and the conversion rate is more than or equal to 53.3%.
  • the fermented liquid was filtered through a ceramic membrane to obtain a supernatant, which was decolorized and separated, concentrated to a solid content of 67%, cooled and crystallized at a rate of 6°C/h, and centrifuged after 18 hours to obtain erythritol crystals.
  • the centrifuged mother liquor is subjected to the second chromatographic separation treatment of the simulated moving bed to obtain the erythritol and arabinose components.
  • the erythritol component is returned to the filtered supernatant, and the arabinose component has a purity of 78%.
  • concentration crystallized, and centrifuged to obtain arabinose crystals.
  • the chromatographic raffinate with solid content of 30-40% is mixed with crystal glucose, liquid glucose and glucose monohydrate, all of them are configured as the initial fermentation medium, and the initial glucose content is 21%, except that the fed-batch fermentation process is not carried out
  • other processes adopt the 70L fermentation system and fermentation control of Example 1, and directly ferment.
  • the erythritol is 64.5g/L, and the conversion rate is 30.0%.

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Abstract

本发明涉及一种木糖母液联产赤藓糖醇和阿拉伯糖的方法，利用第一色谱分离出木糖，得到提取液和提余液，提取液用于制备结晶木糖，提余液与液体葡萄糖或晶体葡萄糖进行调配，利用耐高渗、转化率高的解脂耶氏酵母发酵生产赤藓糖醇，再利用赤藓糖醇溶解度低，易结晶特性，先进行离心结晶处理得到赤藓糖醇晶体，赤藓糖醇的离心母液经第二色谱分离得到阿拉伯糖含量高的提余液，然后再制备得到阿拉伯糖晶体。本发明实现了木糖母液的高效利用，在得到木糖和阿拉伯糖的同时，利用其中葡萄糖生产附加值更高的赤藓糖醇，降低了发酵成本，增加了经济效益。

Description

一种木糖母液联产赤藓糖醇和阿拉伯糖的方法 技术领域
本发明属于木糖母液利用技术领域，特别涉及一种木糖母液联产赤藓糖醇和阿拉伯糖的方法。
背景技术
在生产木糖醇过程中，产生大量的木糖母液，木糖母液中杂糖含量高，常常作为副产物销售用于制作焦糖色素等，附加值低。木糖母液中木糖含量在40％～60％，葡萄糖10％～20％，阿拉伯糖15％～20％，甘露糖0～10％，半乳糖0～5％。关于木糖母液的处理和利用的方法屡见不鲜，主要是提取其中的木糖和阿拉伯糖。为了降低提取分离难度，利用细菌或酵母菌进行发酵，葡萄糖或半乳糖作为菌体生长的碳源被消耗完，从而提高木糖和阿拉伯糖的提取效率。
公开号CN112094956A的专利利用酿酒酵母连续发酵木糖母液，消耗掉葡萄糖，利用色谱分离提纯木糖和阿拉伯糖。公告号CN101705253B的专利利用二次发酵将母液中木糖变成木糖醇，同时提纯阿拉伯糖。公告号CN102603814B的专利同样的是利用酵母发酵除去稀释至20％的木糖母液中的葡萄糖和半乳糖，得到木糖和阿拉伯糖清液。公告号CN101857523B的专利利用发酵除去母液中葡萄糖和半乳糖，将过滤清液经过脱色、离交、浓缩，进行氢化，制备木糖醇和阿拉伯糖醇，后经色谱分离两组分，结晶得到两种成品。公告号CN102952165B的专利也是利用发酵除去葡萄糖和半乳糖，再根据结晶特性的不同，分离出阿拉伯糖的晶体。公开号CN109504733A的专利采用毕赤酵母和出芽短梗霉菌混合菌发酵，生产赤藓糖醇，虽然碳源使用的是木糖母液，但赤藓糖醇浓度很低，仅40g/L左右。以上所述的专利技术多数是通过菌株发酵，除去葡萄糖或半乳糖，进一步分离木糖和阿拉伯糖，提高其纯度。但都存在一定的局限性，由于木糖母液中糖度高，细菌一般不适合生长，多用耐高渗透酵母进行高好氧发酵，对压缩空气的需求大，同时稀释后的母液量大，葡萄糖全部利用完需要菌体量更多，需进行多次扩培，发酵时长在48h或更长，虽然能达到提纯的目的，但能源消耗，生产周期及成本则大大增加。
发明内容
本发明所要解决的技术问题在于，提供一种木糖母液联产赤藓糖醇和阿拉伯糖的方法，降低木糖母液利用成本，在实现分离木糖和阿拉伯糖的同时，提高葡萄糖的利用价值，利用酵母发酵生产附加价值产物赤藓糖醇，提高赤藓糖醇的产量。
本发明是这样实现的，提供一种木糖母液联产赤藓糖醇和阿拉伯糖的方法，包括如下步骤：
步骤一、木糖母液经模拟移动床第一色谱分离处理后分别得到木糖组分含量高的木糖提取液和葡萄糖组分含量高的木糖提余液，将木糖提取液进行浓缩和结晶处理后得到木糖晶体。
步骤二、将木糖提余液进行浓缩至固形物含量30％～50％，葡萄糖含量为9％～14％，再与液体葡萄糖或晶体葡萄糖进行调配得到葡萄糖混合液，在葡萄糖混合液中，葡萄糖含量为40％～50％。
步骤三、将预先制备的解脂耶氏酵母种子液接种到发酵罐的发酵培养基中，同时加入步骤二的葡萄糖混合液后进行发酵，得到发酵液，发酵液的葡萄糖含量＜0.3％；发酵液过滤后得到发酵滤液，发酵滤液依次经过脱色、离交、浓缩、离心及结晶处理后分别得到赤藓糖醇晶体和赤藓糖醇离心母液。
步骤四、赤藓糖醇离心母液经过模拟移动床第二色谱分离处理后分别得到赤藓糖醇组分含量高的赤藓糖醇提取液和阿拉伯糖组分含量高的赤藓糖醇提余液，将赤藓糖醇提取液与步骤三中的发酵滤液混合，将赤藓糖醇提余液依次进行脱色、离交、浓缩、结晶处理后得到阿拉伯糖晶体。
与现有技术相比，本发明的木糖母液联产赤藓糖醇和阿拉伯糖的方法，利用第一色谱分离出木糖，得到提取液和提余液，提取液用于制备结晶木糖，提余液与液体葡萄糖或晶体葡萄糖进行调配，利用耐高渗、转化率高的解脂耶氏酵母发酵生产赤藓糖醇，再利用赤藓糖醇溶解度低，易结晶特性，先进行离心结晶处理得到赤藓糖醇晶体，赤藓糖醇的离心母液经第二色谱分离得到阿拉伯糖含量高的提余液，然后再制备得到阿拉伯糖晶体。本发明实现了木糖母液的高效利用，在得到木糖和阿拉伯糖的同时，利用其中葡萄糖生产附加值更高的赤藓糖醇，同时通过与液体葡萄糖或晶体葡萄糖进行混合，一方面可以提高培养基中葡萄糖含量，提高了每批发酵中赤藓糖醇产量，另一方面提高每批发酵中母液的利用率，总体利用木糖母液提余液配比发酵液发酵制备赤藓糖醇，赤藓糖醇的浓度在156g/L以上，转化率在52％以上。本发明还降低了发酵成本，提高了木糖母液的附加值，增加了经济效益。
附图说明
图1为本发明木糖母液联产赤藓糖醇和阿拉伯糖的方法的原理示意图。
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
请参照图1所示，本发明木糖母液联产赤藓糖醇和阿拉伯糖的方法的较佳实施例，包括 如下步骤：
步骤一、木糖母液经模拟移动床第一色谱分离处理后分别得到木糖组分含量高的木糖提取液和葡萄糖组分含量高的木糖提余液，将木糖提取液进行浓缩和结晶处理后得到木糖晶体。
步骤二、将木糖提余液进行浓缩至固形物含量30％～50％(g/100mL，以下描述皆相同)，葡萄糖含量为9％～14％，再与液体葡萄糖或晶体葡萄糖进行调配得到葡萄糖混合液，在葡萄糖混合液中，葡萄糖含量为40％～50％。
步骤三、将预先制备的解脂耶氏酵母种子液接种到发酵罐的发酵培养基中，同时加入步骤二的葡萄糖混合液后进行发酵，得到发酵液，发酵液的葡萄糖含量＜0.3％；发酵液过滤后得到发酵滤液，发酵滤液依次经过脱色、离交、浓缩、离心及结晶处理后分别得到赤藓糖醇晶体和赤藓糖醇离心母液。
步骤四、赤藓糖醇离心母液经过模拟移动床第二色谱分离处理后分别得到赤藓糖醇组分含量高的赤藓糖醇提取液和阿拉伯糖组分含量高的赤藓糖醇提余液，将赤藓糖醇提取液与步骤三中的发酵滤液混合，赤藓糖醇提取液被回收利用，将赤藓糖醇提余液依次进行脱色、离交、浓缩、结晶处理后得到阿拉伯糖晶体。
具体地，在步骤三中，所述发酵培养基按以下比例配置：葡萄糖含量25％～32％、酵母膏0.5％～1％、玉米浆干粉0.3％～0.8％、硫酸镁0.03％～0.08％、柠檬酸铵0.2％～0.8％和磷酸氢二钾0.02％～0.05％。
具体地，在步骤三中，所述预先制备的解脂耶氏酵母种子液按照如下方法制备：将解脂耶氏酵母菌株接入试管斜面上培养得到试管斜面种子，在试管内预先准备试管斜面种子培养基，试管斜面种子培养基按以下比例配置：葡萄糖20％～25％、酵母膏0.8％～1.5％和琼脂1.5％～2％。
具体地，在步骤三中，所述预先制备的解脂耶氏酵母种子液按照如下方法制备：将解脂耶氏酵母菌株接入茄形瓶斜面上培养得到茄形斜面种子，在茄形瓶内预先准备茄形斜面种子培养基，茄形斜面种子培养基按以下比例配置：葡萄糖20％～25％、酵母膏0.8％～1.5％和琼脂1.5％～2％。
具体地，在步骤三中，所述预先制备的解脂耶氏酵母种子液按照如下方法制备：将解脂耶氏酵母菌株接入摇瓶中培养得到摇瓶种子液，在摇瓶内预先准备摇瓶种子培养基，摇瓶种子培养基按以下比例配置：葡萄糖20％～25％、酵母膏0.8％～1.5％、硫酸镁0.03％～0.08％和柠檬酸铵0.2％～0.7％。
具体地，在步骤三中，所述预先制备的解脂耶氏酵母种子液按照如下方法制备：将解脂 耶氏酵母菌株接入发酵罐中培养得到发酵罐种子液，在发酵罐内预先准备发酵罐种子培养基，发酵罐种子培养基按以下比例配置：葡萄糖25％～30％、酵母膏0.5％～1.0％、蛋白胨0.3％～0.8％、硫酸镁0.03％～0.08％和柠檬酸铵0.2％～0.8％，接种量5％～10％，发酵初始pH6.0～7.0，发酵罐种子培养基的灭菌温度115℃～121℃，灭菌时间20min～30min。
下面结合具体实施例来进一步说明本发明的方法。
实施例1
本发明的木糖母液联产赤藓糖醇和阿拉伯糖的方法的第一个实施例，包括如下步骤：
木糖母液经第一色谱分离处理后得到含木糖的提取液，提取液浓缩至固形物含量80％，蒸发结晶得到晶体木糖。提余液中各组分占比分别为，葡萄糖25％～32％、阿拉伯糖18％～25％、半乳糖5％～15％；将提余液进行浓缩至固形物40％，其中葡萄糖含量为12％，与晶体葡萄糖进行配比，使混合液的葡萄糖含量在45％，配制体系100L，同时按比例加入除葡萄糖以外发酵培养基配方中其他辅料，进行灭菌，备用。
发酵体系70L，共3批，采用过程补料的方式，具体操作如下：
发酵初始体积40L，培养基按照上述发酵培养基配方，培养基中初始葡萄糖含量为18％，配制好进行灭菌，备用。摇瓶种子分别使用500mL、5L摇瓶发酵，装液量分别为总体积的10％，培养20h～24h，当5L摇瓶中菌密度18～25(在600nm波长下，菌液的吸光值，简称OD值)时转至发酵罐内，接种量为8％，发酵温度30℃，转速为200rpm～400rpm，溶氧20％～30％，通气量为1.5Nm 3/h，待发酵液中菌密度达到35～40，开始补加上述混合糖液至发酵罐体积到70L，连续补料过程保持发酵液中葡萄糖含量在17％～20％，补料完成后，继续发酵，当发酵液中葡萄糖含量＜0.3％时停止发酵。发酵结果如表1所示
表1实施例1的测试结果
  批次一 批次二 批次三
发酵时长/h 102 97 100
赤藓糖醇浓度/(g/L) 156.74 165.40 162.80
转化率/％ 52.2 55.1 54.3
赤藓糖醇浓度均在156g/L以上，转化率≥52.2％。
发酵结束后，发酵液经过陶瓷膜过滤，得到上清液，经脱色、离交，浓缩至固形物含量68％，以5℃/h速度进行降温结晶，20h后经离心得到赤藓糖醇晶体。离心母液经模拟移动床第二色谱分离处理得到赤藓糖醇和阿拉伯糖组分，赤藓糖醇组分回到过滤上清液中，提高赤藓糖醇产量，阿拉伯糖组分纯度达到75％，经过浓缩，结晶，离心得到阿拉伯糖晶体。
实施例2
本发明的木糖母液联产赤藓糖醇和阿拉伯糖的方法的第二个实施例，包括如下步骤：
提余液的获得和各组分占比与实施例1相同。将提余液进行浓缩至固形物50％，其中葡萄糖含量为14.3％，液体葡萄糖浓缩至葡萄糖含量65％，两种按体积4：6比例进行混合，使混合液的葡萄糖含量为45％，配制体系1000L，同时按比例加入除葡萄糖以外发酵培养基配方中其他辅料，进行灭菌，备用。
发酵体系700L，共3批，采用过程补料的方式，具体操作如下：
发酵初始体积400L，培养基按照上述发酵培养基配方，培养基中初始葡萄糖含量为18.7％，配制好进行灭菌，备用。试管斜面种子转接至500mL茄形瓶斜面，30℃，培养4～5天，每瓶中加入80mL无菌水洗下菌苔，分别转入5L摇瓶发酵，30℃培养20h～24h,菌密度18～25，转至两个50L种子罐内，装液量35L，发酵温度30℃，罐压0.1MPa，待菌密度达到20～25，其中一个种子罐内菌种转入1000L发酵罐，转速为180rpm～300rpm，溶氧20％～30％，通气量为16Nm 3/h，待菌密度达到35～40，开始补入上述混合糖液300L，同时加入另一种子罐内新的种子液，连续补料过程保持发酵液中葡萄糖含量在17％～20％，补料完成后，继续发酵，当发酵液中葡萄糖含量＜0.3％时停止发酵，发酵体系中葡萄糖总浓度为30％。发酵结果如下：
  批次一 批次二 批次三
发酵时长/h 104.5 101 106.5
赤藓糖醇浓度/(g/L) 160.71 165.40 162.80
转化率/％ 53.9 55.5 54.6
赤藓糖醇浓度均在160g/L以上，转化率≥53.9％。
发酵结束后，发酵液经过陶瓷膜过滤，得到上清液，经脱色、离交，浓缩至固形物含量65％，以6℃/h速度进行降温结晶，16h后离心得到赤藓糖醇晶体。离心母液经模拟移动床第二色谱分离处理得到赤藓糖醇和阿拉伯糖组分，同样的，赤藓糖醇组分重新回到过滤上清液中，阿拉伯糖组分纯度达到77％，经过浓缩，结晶，离心得到阿拉伯糖晶体。
实施例3
本发明的木糖母液联产赤藓糖醇和阿拉伯糖的方法的第三个实施例，包括如下步骤：
提余液的获得和各组分占比与实施例1、实施例2相同。将提余液进行浓缩至固形物48％，其中葡萄糖含量为13.7％，加入550L，再投入380kg一水结晶葡萄糖和自来水,混合液的葡萄糖含量为42％，配制体系1000L，同时按比例加入除葡萄糖以外发酵培养基配方中其他辅料， 进行灭菌，备用。
发酵体系700L，共3批，采用过程补料的方式，具体操作如下：
发酵初始体积400L，培养基按照上述发酵培养基配方，培养基中初始葡萄糖含量为20％，配制好进行灭菌，备用。试管斜面种子转接至500mL茄形瓶斜面，30℃，培养4～5天，每瓶中加入80mL无菌水洗下菌苔，分别转入5L摇瓶发酵，装液量800mL，30℃培养22h,菌密度18～25，转至两个50L种子罐内，装液量35L，发酵温度30℃，罐压0.1MPa，同样的，待菌密度达到20～25，其中一个种子罐内菌种转入1000L发酵罐，转速为180～300rpm，溶氧20％～30％，通气量为15Nm 3/h，待菌密度达到35～40，开始补入上述混合糖液300L，同时加入另一种子罐内新的种子液，连续补料过程保持发酵液中葡萄糖含量在15％～18％，当发酵液中葡萄糖含量＜0.3％时停止发酵，发酵体系中总葡萄糖含量为30％。发酵结果如下：
  批次一 批次二 批次三
发酵时长/h 99.5 104 102.5
赤藓糖醇浓度/(g/L) 163.57 159.80 165.22
转化率/％ 54.5 53.3 55.1
赤藓糖醇浓度均在159g/L以上，转化率≥53.3％。
发酵结束后，发酵液经过陶瓷膜过滤，得到上清液，经脱色、离交，浓缩至固形物含量67％，以6℃/h速度进行降温结晶，18h后离心得到赤藓糖醇晶体。离心母液经模拟移动床第二色谱分离处理得到赤藓糖醇和阿拉伯糖组分，同样的，赤藓糖醇组分重新回到过滤上清液中，阿拉伯糖组分纯度达到78％，经过浓缩，结晶，离心得到阿拉伯糖晶体。
对比例1
利用固形物30％的色谱提余液直接发酵制备赤藓糖醇，初始葡萄糖含量为7.2％，除不进行补料发酵工艺外，其他过程采用实施例1的70L发酵体系和发酵控制，发酵结束后赤藓糖醇为31.6g/L，转化率43.8％。
对比例2
利用固形物30～40％的色谱提余液与晶体葡萄糖、液体葡萄糖及一水葡萄糖配比后，全部配置成作为初始发酵培养基使用，初始葡萄糖含量为21％，除不进行补料发酵工艺外，其他过程采用实施例1的70L发酵体系和发酵控制，直接发酵，发酵结束后赤藓糖醇为64.5g/L，转化率30.0％。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (6)

  1. 一种木糖母液联产赤藓糖醇和阿拉伯糖的方法，其特征在于，包括如下步骤：
    步骤一、木糖母液经模拟移动床第一色谱分离处理后分别得到木糖组分含量高的木糖提取液和葡萄糖组分含量高的木糖提余液，将木糖提取液进行浓缩和结晶处理后得到木糖晶体；
    步骤二、将木糖提余液进行浓缩至固形物含量30％～50％，葡萄糖含量为9％～14％，再与液体葡萄糖或晶体葡萄糖进行调配得到葡萄糖混合液，在葡萄糖混合液中，葡萄糖含量为40％～50％；
    步骤三、将预先制备的解脂耶氏酵母种子液接种到发酵罐的发酵培养基中，同时加入步骤二的葡萄糖混合液后进行发酵，得到发酵液，发酵液的葡萄糖含量＜0.3％；发酵液过滤后得到发酵滤液，发酵滤液依次经过脱色、离交、浓缩、离心及结晶处理后分别得到赤藓糖醇晶体和赤藓糖醇离心母液；
    步骤四、赤藓糖醇离心母液经过模拟移动床第二色谱分离处理后分别得到赤藓糖醇组分含量高的赤藓糖醇提取液和阿拉伯糖组分含量高的赤藓糖醇提余液，将赤藓糖醇提取液与步骤三中的发酵滤液混合，将赤藓糖醇提余液依次进行脱色、离交、浓缩、结晶处理后得到阿拉伯糖晶体。
  2. 如权利要求1所述的木糖母液联产赤藓糖醇和阿拉伯糖的方法，其特征在于，在步骤三中，所述发酵培养基按以下比例配置：葡萄糖含量25％～32％、酵母膏0.5％～1％、玉米浆干粉0.3％～0.8％、硫酸镁0.03％～0.08％、柠檬酸铵0.2％～0.8％和磷酸氢二钾0.02％～0.05％。
  3. 如权利要求1所述的木糖母液联产赤藓糖醇和阿拉伯糖的方法，其特征在于，在步骤三中，所述预先制备的解脂耶氏酵母种子液按照如下方法制备：将解脂耶氏酵母菌株接入试管斜面上培养得到试管斜面种子，在试管内预先准备试管斜面种子培养基，试管斜面种子培养基按以下比例配置：葡萄糖20％～25％、酵母膏0.8％～1.5％和琼脂1.5％～2％。
  4. 如权利要求1所述的木糖母液联产赤藓糖醇和阿拉伯糖的方法，其特征在于，在步骤三中，所述预先制备的解脂耶氏酵母种子液按照如下方法制备：将解脂耶氏酵母菌株接入茄形瓶斜面上培养得到茄形斜面种子，在茄形瓶内预先准备茄形斜面种子培养基，茄形斜面种子培养基按以下比例配置：葡萄糖20％～25％、酵母膏0.8％～1.5％和琼脂1.5％～2％。
  5. 如权利要求1所述的木糖母液联产赤藓糖醇和阿拉伯糖的方法，其特征在于，在步骤三中，所述预先制备的解脂耶氏酵母种子液按照如下方法制备：将解脂耶氏酵母菌株接入摇瓶中培养得到摇瓶种子液，在摇瓶内预先准备摇瓶种子培养基，摇瓶种子培养基按以下比例配置：葡萄糖20％～25％、酵母膏0.8％～1.5％、硫酸镁0.03％～0.08％和柠檬酸铵0.2％～0.7％。
  6. 如权利要求1所述的木糖母液联产赤藓糖醇和阿拉伯糖的方法，其特征在于，在步骤三中，所述预先制备的解脂耶氏酵母种子液按照如下方法制备：将解脂耶氏酵母菌株接入发 酵罐中培养得到发酵罐种子液，在发酵罐内预先准备发酵罐种子培养基，发酵罐种子培养基按以下比例配置：葡萄糖25％～30％、酵母膏0.5％～1.0％、蛋白胨0.3％～0.8％、硫酸镁0.03％～0.08％和柠檬酸铵0.2％～0.8％，接种量5％～10％，发酵初始pH6.0～7.0，发酵罐种子培养基的灭菌温度115℃～121℃，灭菌时间20min～30min。
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