阅读说明：本技术 一种甘蔗糖蜜预处理后生物转化制备低聚果糖的方法 (Method for preparing fructo-oligosaccharide through biotransformation after cane molasses pretreatment ) 是由 李坚斌 李元 李凯 雷光鸿 刘继栋 于 2021-11-16 设计创作，主要内容包括：本发明公开了一种甘蔗糖蜜预处理后生物转化制备低聚果糖的方法,以甘蔗糖蜜为原料,依次经预处理、生物转化、纯化、灭酶、喷雾干燥后制取,其中生物转化以黑曲霉作为生产菌株,甘蔗糖蜜替代原始发酵培养基。通过本方法实现了甘蔗糖蜜的高附加值利用,降低了低聚果糖的制备成本的同时,提高了原有低聚果糖纯度,生物转化后滤出来的黑曲霉菌菌丝体可以重复利用。该操作工艺简便易行,制备周期短,所得产物活性优良,低聚果糖纯度高。(The invention discloses a method for preparing fructo-oligosaccharide by bioconversion after cane molasses pretreatment, which is prepared by taking cane molasses as a raw material and sequentially carrying out pretreatment, biotransformation, purification, enzyme deactivation and spray drying, wherein aspergillus niger is used as a production strain for bioconversion, and the cane molasses replaces an original fermentation culture medium. The method realizes high value-added utilization of cane molasses, reduces the preparation cost of fructo-oligosaccharide, improves the purity of the original fructo-oligosaccharide, and can repeatedly utilize the aspergillus niger mycelia filtered after biotransformation. The operation process is simple and easy to implement, the preparation period is short, the obtained product has excellent activity, and the purity of the fructo-oligosaccharide is high.)

1. A method for preparing fructo-oligosaccharide is characterized in that cane molasses is used as a raw material, and the fructo-oligosaccharide is prepared by pretreatment and biotransformation in turn; the biological transformation takes aspergillus niger as a production strain; the pretreatment is carried out according to the following operations: diluting cane molasses with water, adjusting the pH value to 3.0-4.0 by using phosphoric acid, uniformly stirring, centrifuging, taking supernatant, adding lime milk to the pH value of 7.0-8.0, heating to 45-50 ℃, uniformly stirring to obtain a regulating solution, adding polyacrylamide into the regulating solution, uniformly stirring, centrifuging, taking supernatant, adjusting the pH value to 5.5-6.0 by using hydrochloric acid, standing, and taking supernatant, namely the pretreatment solution.

2. The method for preparing fructooligosaccharide according to claim 1, wherein the bioconversion is carried out by: inoculating aspergillus niger strains into a molasses fermentation medium for culturing to obtain an aspergillus niger whole-cell solution; and (3) filtering the aspergillus niger whole-cell solution by using filter cloth, washing, performing suction filtration to obtain aspergillus niger mycelia, putting the aspergillus niger mycelia into a reaction tank containing pretreatment liquid, and stirring for 12-24 hours to obtain reaction liquid.

3. The method for preparing fructooligosaccharide according to claim 1, further comprising the steps of:

(3) and (3) purification: cooling the reaction liquid to 40-45 ℃, adding glucose oxidase and catalase, then adding calcium carbonate to adjust the concentration of calcium ions in the solution to 5% -6%, ventilating and stirring for 9-12 h, and then filtering to obtain aspergillus niger mycelia and a purified liquid;

(4) enzyme deactivation: heating the purified liquid to 90-98 ℃ to inactivate enzyme for 10-15 min to obtain an enzyme inactivation liquid;

(5) spray drying: and cooling the enzyme-killed liquid to 70-80 ℃, decoloring, filtering, and finally spray-drying to obtain the fructo-oligosaccharide composite powder.

4. The method for preparing fructo-oligosaccharide according to claim 1, wherein the cane molasses is diluted with water to give cane molasses diluted with water to a brix of 40-45 °; the lime milk is 6-10% in mass concentration; the polyacrylamide is regulating solution with the mass concentration of one thousandth and the addition amount of 6 mg-10 mg/kg.

5. The method for preparing fructo-oligosaccharide according to claim 1, wherein the molasses fermentation medium is prepared from the following components: pretreatment liquid: 25g/L, yeast extract: 15g/L, MgSO₄·7H₂O：0.5g/L，KCL：0.5g/L，NaNO₃：2g/L，K₂HPO₄: 5 g/L; the inoculation amount is 1 multiplied by 10⁸1 x 10 per liter¹⁰The culture conditions are 28-32 ℃, the pH is 5.0-6.0, the rotating speed is 150-200 r/min, and the time is 36-60 h; the biotransformation is carried out under the conditions that the addition amount of the aspergillus niger mycelia is 80-100 g/L, the pH is 5.0-6.0, the temperature is 50-60 ℃, and the stirring is carried out for 12-24 hours.

6. The method for preparing fructooligosaccharide according to claim 1, wherein the glucose oxidase and catalase are added in an amount of glucose oxidase: 1500-2000U/kg filtrate and catalase: 16000-20000U/kg filtrate; the addition amount of the calcium carbonate is 100-120 g/kg of filtrate.

7. The method for preparing fructo-oligosaccharide according to claim 1, wherein the enzyme deactivation is carried out by heating the solution to 90-100 ℃ for 10-15 min; the spray drying conditions are as follows: the inlet air temperature is 140-150 ℃, and the feed concentration is 20-30%.

Technical Field

The invention relates to the field of fructo-oligosaccharide preparation by molasses, and particularly relates to a method for preparing fructo-oligosaccharide by biotransformation after cane molasses pretreatment.

Background

Cane molasses is a dark brown, viscous and semi-flowing liquid as a by-product in the sugar production process of sugarcane. Usually, the sugar cane molasses contains 40-60% (w/w) of sugar, less than 20% (w/w) of water, and also contains a certain amount of amino acid, vitamins and inorganic Salt (SO)₄ ^2～、Ca²⁺、Na⁺Etc.), and some color formers such as caramel, melanin, and colloids, etc. Compared with glucose and other carbon sources, cane molasses containing a large amount of sugar is a cheap raw material, has a good development prospect, and can be used for producing biological products with high added values through biotransformation. However, at present, cane molasses is simply used for feed production or industrial ethanol production, and these low-value applications are likely to cause environmental pollution and waste of resources. Therefore, the search for the efficient high-added-value utilization of the cane molasses becomes a key problem to be solved urgently in the whole application field of the cane molasses.

Fructo-oligosaccharide is an important functional prebiotic, has the effects of promoting the growth of beneficial bacteria in the digestive tract, adsorbing pathogenic bacteria in the intestinal tract, improving the utilization rate of feed and the like, and is widely applied to the fields of food, medicine, animal feed and the like. However, the main production method of fructo-oligosaccharide in the market at present is to convert sucrose as raw material by fructosyltransferase, and then to separate, concentrate and crystallize. In the transformation process, fructosyltransferase is inhibited by glucose, so that the transformation rate of fructo-oligosaccharide is only about 80%, and the production cost is high due to the influence of the price of sucrose. Therefore, the method has great practical significance for preparing the fructo-oligosaccharide by using the cane molasses as the raw material and further improving the purity and the conversion rate of the fructo-oligosaccharide.

Bioconversion, also known as whole cell catalysis, is a controlled enzymatic fermentation process based on enzyme production applications, using whole cells as biocatalysts, without the need for cumbersome enzyme purification from microbial cells: the enzyme is protected in natural cells for biological catalysis, so that higher production efficiency can be obtained, sucrose can easily react with the enzyme through external cells, and the product is quickly released; meanwhile, the cells can be easily reused, an expensive fixing process is not needed, and the method has a good application prospect.

Disclosure of Invention

In order to solve the problems in the background art, the invention provides a method for preparing fructo-oligosaccharide by biological conversion after sugar cane molasses pretreatment. Glucose oxidase and catalase are adopted to remove the byproduct glucose, so that the purity and the conversion rate of the fructo-oligosaccharide are further improved.

In order to solve the technical problems, the invention adopts the technical scheme that:

a method for preparing fructo-oligosaccharide uses cane molasses as raw material, and prepares the fructo-oligosaccharide after pretreatment and biotransformation in sequence; the biological transformation takes aspergillus niger as a production strain; the pretreatment is carried out according to the following operations: diluting cane molasses with water, adjusting the pH value to 3.0-4.0 by using phosphoric acid, uniformly stirring, centrifuging, taking supernatant, adding lime milk to the pH value of 7.0-8.0, heating to 45-50 ℃, uniformly stirring to obtain a regulating solution, adding polyacrylamide into the regulating solution, uniformly stirring, centrifuging, taking supernatant, adjusting the pH value to 5.5-6.0 by using hydrochloric acid, standing, and taking supernatant, namely the pretreatment solution.

The biotransformation is carried out as follows: inoculating aspergillus niger strains into a molasses fermentation medium for culturing to obtain an aspergillus niger whole-cell solution; and (3) filtering the aspergillus niger whole-cell solution by using filter cloth, washing, performing suction filtration to obtain aspergillus niger mycelia, putting the aspergillus niger mycelia into a reaction tank containing pretreatment liquid, and stirring for 12-24 hours to obtain reaction liquid.

The method for preparing the fructo-oligosaccharide further comprises the following steps:

(3) and (3) purification: cooling the reaction liquid to 40-45 ℃, adding glucose oxidase and catalase, then adding calcium carbonate to adjust the concentration of calcium ions in the solution to 5% -6%, ventilating and stirring for 9-12 h, and then filtering to obtain aspergillus niger mycelia and a purified liquid;

(4) enzyme deactivation: heating the purified liquid to 90-98 ℃ to inactivate enzyme for 10-15 min to obtain an enzyme inactivation liquid;

(5) spray drying: and cooling the enzyme-killed liquid to 70-80 ℃, decoloring, filtering, and finally spray-drying to obtain the fructo-oligosaccharide composite powder.

The cane molasses is diluted by adding water, and the cane molasses is diluted by adding water to the brix of 40-45 degrees; the lime milk is 6-10% in mass concentration; the polyacrylamide is regulating solution with the mass concentration of one thousandth and the addition amount of 6 mg-10 mg/kg.

The molasses fermentation medium is prepared from the following components: pretreatment liquid: 25g/L, yeast extract: 15g/L, MgSO₄·7H₂O：0.5g/L，KCL：0.5g/L，NaNO₃：2g/L，K₂HPO₄: 5 g/L; the inoculation amount is 1 multiplied by 10⁸1 x 10 per liter¹⁰The culture conditions are 28-32 ℃, the pH is 5.0-6.0, the rotating speed is 150-200 r/min, and the time is 36-60 h; the biotransformation is carried out under the conditions that the addition amount of the aspergillus niger mycelia is 80-100 g/L, the pH is 5.0-6.0, the temperature is 50-60 ℃, and the stirring is carried out for 12-24 hours.

The addition amount of the glucose oxidase and the catalase is glucose oxidase: 1500-2000U/kg filtrate and catalase: 16000-20000U/kg filtrate; the addition amount of the calcium carbonate is 100-120 g/kg of filtrate.

The enzyme deactivation is to heat the solution to 90-100 ℃ and keep the temperature for 10-15 min; the spray drying conditions are as follows: the inlet air temperature is 140-150 ℃, and the feed concentration is 20-30%.

The invention has the following beneficial effects:

according to the method in the national standard GB/T23528-2009 fructo-oligosaccharide, the content of the fructo-oligosaccharide in FOS syrup (purified liquid) is detected by high pressure chromatography, the content of the fructo-oligosaccharide reaches above 209.01g/L, the conversion efficiency is high, and the peak emergence time of kestose, kestotetrasaccharide and kestopentasaccharide is 12.340min, 15.537min and 19.197min respectively. The resulting syrup had a Fructooligosaccharide (FOS) content of up to 221.36g/L, relative percentage 42.91%.

Because the adopted cane molasses is converted, the cane molasses is used as a byproduct in the sugar production process of the sugarcane, and compared with the method for preparing fructo-oligosaccharide by using the cane sugar as a raw material, the method can effectively reduce the preparation cost and realize the high added value utilization of the cane molasses.

Meanwhile, the method discovers that the aspergillus niger strains are cultured by taking cane molasses as a culture medium, the enzyme activity in the mycelia can be improved after fermentation optimization, the production cost is reduced, the aspergillus niger mycelia can be reused, and the utilization rate is high.

Drawings

FIG. 1 is a process flow diagram of the present invention.

FIG. 2 is a FOS standard liquid chromatogram.

FIG. 3 is a liquid chromatogram of the FOS product versus the standard.

Detailed Description

The invention is further illustrated by the following specific examples. Each of the following examples requires the initial culture of an Aspergillus niger strain, ATCC 20611, purchased from American type culture Collection, U.S. Florida. The culture process of the aspergillus niger strain is as follows:

culturing aspergillus niger strains: inoculating the aspergillus niger strains preserved on the inclined plane of a Potato Dextrose Agar (PDA) culture medium into a yeast maltose culture medium (YM) culture medium, and culturing for 24-48 h at 28-32 ℃.

Example 1

The embodiment is an embodiment of a method for preparing fructo-oligosaccharide by biological transformation after sugar cane molasses pretreatment, and the specific steps are as follows:

(1) pretreatment of cane molasses: diluting 1L of cane molasses with water to 45 ° Bx, adjusting pH to 3.0 with phosphoric acid, stirring, centrifuging to obtain supernatant, adding lime milk to pH 8.0, heating to 50 deg.C, adding 10mg/kg polyacrylamide (one thousandth of concentration), stirring, centrifuging to obtain supernatant, adjusting pH to 6.0 with hydrochloric acid, and standing;

(2) and (3) biotransformation: inoculating Aspergillus niger strain to molasses fermentation medium, and culturing with the inoculum size of 1 × 10⁸Culturing the Aspergillus niger cells per liter for 60 hours at the conditions of 28 ℃, pH 5.0-6.0 and rotation speed of 150r/min to obtain the Aspergillus niger whole-cell solution. Wherein the molasses fermentation medium is pretreated by a pretreatment solution25g/L, yeast extract 15g/L, MgSO₄·7H₂O 0.5g/L，KCL 0.5g/L，NaNO₃2 g/L，K₂HPO₄5g/L preparation; filtering the cultured Aspergillus niger whole-cell solution by using filter cloth, washing, performing suction filtration to obtain Aspergillus niger mycelia, adding the Aspergillus niger mycelia into a reaction tank containing pretreatment liquid according to the addition of 80g/L, and stirring for 12h at 50 ℃ and pH value of 5.0 to obtain reaction liquid;

(3) and (3) purification: cooling the reaction liquid to 40 ℃, adding 1500U/kg glucose oxidase and 16000U/kg catalase, adjusting the calcium ion solubility of the solution to 5.0% by using 100g/kg calcium carbonate, ventilating and stirring for 9h, removing by-products and purifying fructooligosaccharides, and filtering to obtain aspergillus niger mycelia and a purified liquid, wherein the aspergillus niger mycelia can be continuously used for biotransformation;

(4) enzyme deactivation: heating the purified solution to 90 deg.C for inactivating enzyme, and keeping the temperature for 10min to obtain enzyme-inactivated solution;

(5) spray drying: cooling the enzyme-inactivating liquid to 80 ℃, decolorizing and filtering by active carbon, and finally spray drying to obtain the fructo-oligosaccharide composite powder. The spray drying conditions were: the inlet air temperature is 140 ℃, and the feed concentration is 30%.

According to the detection of a high performance liquid method in the fructo-oligosaccharide in the national standard GB/T23528-2009, the total content of the fructo-oligosaccharide (FOS) in the syrup (purified liquid) obtained in the example reaches 209.01g/L, the relative percentage is 40.51%, and the water content of the composite powder is 6.46%.

Example 2

The embodiment is another embodiment of a method for preparing fructo-oligosaccharide by biological transformation after sugar cane molasses pretreatment, and the specific steps are as follows:

(1) pretreatment of cane molasses: diluting 1L of cane molasses with water to 42 ° Bx, adjusting pH to 3.5 with phosphoric acid, stirring, centrifuging to obtain supernatant, adding lime milk to pH 7.0, heating to 48 deg.C, adding 8mg/kg polyacrylamide (one thousandth of concentration), stirring, centrifuging to obtain supernatant, adjusting pH to 5.8 with hydrochloric acid, and standing;

(2) and (3) biotransformation: inoculating aspergillus niger strain into molasses fermentation mediumCulturing with an inoculum size of 1 × 10⁹Culturing at 30 deg.C, pH 5.5 and rotation speed of 180r/min for 48 hr to obtain Aspergillus niger whole cell solution. Wherein the molasses fermentation medium is prepared from pretreatment liquid 25g/L, yeast extract 15g/L, and MgSO₄·7H₂O0.5g/L，KCL 0.5g/L，NaNO₃2 g/L，K₂HPO₄5g/L preparation; filtering the cultured Aspergillus niger whole-cell solution by using filter cloth, washing, performing suction filtration to obtain Aspergillus niger mycelia, adding the Aspergillus niger mycelia into a reaction tank containing pretreatment liquid according to the addition of 90g/L, and stirring for 18h at 55 ℃ and pH value of 6.0 to obtain reaction liquid;

(3) and (3) purification: cooling the reaction liquid to 45 ℃, adding 1800U/kg glucose oxidase and 18000U/kg catalase, adjusting the calcium ion solubility of the solution to 6.0% by using 110g/kg calcium carbonate, stirring for 10 hours in a ventilating way, removing by-products and purifying fructo-oligosaccharide, and filtering to obtain aspergillus niger mycelia and a purified liquid, wherein the aspergillus niger mycelia can be continuously used for biotransformation;

(4) enzyme deactivation: heating the purified solution to 95 deg.C for inactivating enzyme, and keeping the temperature for 12min to obtain enzyme-inactivated solution;

(5) spray drying: cooling the enzyme-inactivating liquid to 75 ℃, decolorizing and filtering by active carbon, and finally spray drying to obtain the fructo-oligosaccharide composite powder. The spray drying conditions were: the inlet air temperature is 145 ℃, and the feed concentration is 25%.

According to the detection of a high performance liquid method in the fructo-oligosaccharide of the national standard GB/T23528-2009, the total content of the fructo-oligosaccharide (FOS) in the obtained syrup (purified liquid) reaches 216.02g/L, the relative percentage is 41.87%, and the water content of the composite powder is 5.37%.

Example 3

The embodiment is another embodiment of a method for preparing fructo-oligosaccharide by biological transformation after sugar cane molasses pretreatment, and the specific steps are as follows:

(1) pretreatment of cane molasses: diluting 1L of cane molasses with water to 40 ° Bx, adjusting pH to 4.0 with phosphoric acid, stirring, centrifuging to obtain supernatant, adding lime milk to pH 8.0, heating to 45 deg.C, adding 6mg/kg polyacrylamide (one thousandth of concentration), stirring, centrifuging to obtain supernatant, adjusting pH to 5.5 with hydrochloric acid, and standing;

(2) and (3) biotransformation: inoculating Aspergillus niger strain to molasses fermentation medium, and culturing with the inoculum size of 1 × 10¹⁰Culturing at 32 deg.C, pH 6.0, and rotation speed of 200r/min for 36 hr to obtain Aspergillus niger whole cell solution. Wherein the molasses fermentation medium is prepared from pretreatment liquid 25g/L, yeast extract 15g/L, and MgSO₄·7H₂O 0.5g/L，KCL 0.5g/L，NaNO₃2 g/L，K₂HPO₄5g/L preparation; filtering the cultured Aspergillus niger whole-cell solution by using filter cloth, washing, performing suction filtration to obtain Aspergillus niger mycelia, adding the Aspergillus niger mycelia into a reaction tank containing pretreatment liquid according to the addition of 100g/L, and stirring for 24h at 55 ℃ and pH value of 6.0 to obtain reaction liquid;

(3) and (3) purification: cooling the reaction liquid to 45 ℃, adding 2000U/kg glucose oxidase and 20000U/kg catalase, adjusting the calcium ion solubility of the solution to 5.5% by using 120g/kg calcium carbonate, stirring for 12h under ventilation, removing by-products and purifying fructo-oligosaccharide, and filtering to obtain aspergillus niger mycelia and a purified liquid; aspergillus niger mycelia can be used for biotransformation continuously;

(4) enzyme deactivation: heating the purified solution to 98 deg.C for inactivating enzyme, and keeping the temperature for 15min to obtain enzyme-inactivated solution;

(5) spray drying: cooling the enzyme-inactivating liquid to 70 ℃, decolorizing and filtering by active carbon, and finally spray drying to obtain the fructo-oligosaccharide composite powder. The spray drying conditions were: the inlet air temperature is 150 ℃, and the feed concentration is 20%.

According to the detection of a high performance liquid method in the fructo-oligosaccharide of the national standard GB/T23528-2009, the total content of the fructo-oligosaccharide (FOS) in the obtained syrup (purified liquid) reaches 221.36g/L, the relative percentage is 42.91%, and the water content of the composite powder is 4.96%.

The fructo-oligosaccharide composite powder prepared by the method can be widely applied to feed as a feed additive.