CN111100881A

CN111100881A - Method for producing ethanol by fermenting starch raw material

Info

Publication number: CN111100881A
Application number: CN202010085066.0A
Authority: CN
Inventors: 佟毅; 修志龙; 李义; 李凡; 李伟豪; 孙振江; 陈博; 刘胜; 李永恒; 牟英; 刘辉; 罗虎; 王小艳
Original assignee: Guangxi Cofco Bio Energy Co ltd; Dalian University of Technology; Jilin COFCO Bio Chemical Co Ltd
Current assignee: Guangxi Cofco Bio Energy Co ltd; Dalian University of Technology; Jilin COFCO Bio Chemical Co Ltd
Priority date: 2020-02-10
Filing date: 2020-02-10
Publication date: 2020-05-05

Abstract

The invention relates to the field of yeast fermentation, and discloses a method for producing ethanol by fermenting a starch raw material. The method comprises inoculating yeast into liquid culture medium for ethanol fermentation; wherein the liquid culture medium comprises a starch raw material and rice hull powder, and the weight ratio of the starch raw material to the starch raw material is 100; the using amount of the rice hull powder in the liquid culture medium is 3-25 parts by weight. The invention preferably adopts a raw material method for ethanol fermentation. The method of the invention can improve the culture temperature of the yeast, namely improve the heat resistance of the yeast, so that the yeast can grow and metabolize at high efficiency at high temperature (37-39 ℃), thereby reducing the cost of the ethanol production process.

Description

Method for producing ethanol by fermenting starch raw material

Technical Field

The invention relates to the field of yeast fermentation, in particular to a method for producing ethanol by fermenting starch raw materials.

Background

In the actual production, the ethanol fermentation needs to keep a lower temperature (28-32 ℃), the yeast growth can release a large amount of heat in the fermentation process, if the temperature is not reduced and the temperature can exceed the tolerant temperature of the yeast, generally can not exceed 34 ℃, the fermentation tank is cooled by using circulating cooling water, the working principle is that return water from the fermentation tank enters the top of a water cooling tower to be sprayed downwards, and the return water is used as cooling water for the fermentation tank after being evaporated, radiated and cooled on the surface of a filler in the water cooling tower. This process directly correlates the temperature of the fermenter cooling water with the ambient temperature, typically only 2-3 ℃ below ambient temperature. However, in summer, the temperature of outdoor water can even reach 40 ℃, and cooling water needs to be treated at low temperature to ensure that fermentation is normally carried out, which undoubtedly increases the production cost.

The high-temperature fermentation production process in summer faces the high-temperature challenge, so the high-temperature fermentation production of ethanol is a technical problem which needs to be solved urgently. Researchers also do a lot of work to realize high-temperature fermentation for ethanol production, wherein one of the main ideas is to select and breed high-temperature resistant yeast, and the method is divided into a traditional breeding method and a modern breeding method. The traditional breeding method comprises high-temperature domestication, natural breeding, mutation breeding and the like, but the method has the advantages of large workload, low success rate, unstable characters and easy degradation of passage; modern breeding methods include protoplast fusion techniques and gene recombination techniques, among others, which allow targeted manipulation of genes to achieve expression of heat-resistant traits. Besides improving heat resistance from the strain, another idea is to construct a microenvironment to help the strain resist high temperature, and an immobilized cell technology is common. However, these techniques have many problems, such as harsh conditions, complex process, high cost, and the like.

Disclosure of Invention

The invention aims to overcome the problems of complexity, high cost and the like of the prior art for improving the heat resistance of yeast, and provides an ethanol fermentation method which can improve the heat resistance of yeast, enables the yeast to grow and metabolize at a higher temperature (37-39 ℃), is simple and has low cost.

In order to achieve the above objects, the present invention provides a method for producing ethanol by fermenting starch feedstock, comprising inoculating yeast into a liquid medium to perform ethanol fermentation;

wherein the liquid culture medium comprises a starch raw material and rice hull powder, and the using amount of the rice hull powder in the liquid culture medium is 3-25 parts by weight relative to 100 parts by weight of the starch raw material.

The method of the invention is adopted for ethanol fermentation, can improve the culture temperature of the yeast, namely improve the heat resistance of the yeast, and lead the yeast to grow and metabolize at higher efficiency under high temperature (37-39 ℃), thereby not needing to use cooling water to strictly control the temperature of the system in the culture process, reducing the cost of the ethanol production process, and having simple method, low cost and industrialization suitability.

Drawings

FIG. 1 is a graph showing the mass loss in shake flask culture in the cases of examples 1-1 to 1-4, in which the amounts of rice hull powder added are different;

FIG. 2 is a graph showing the effect of different amounts of powdered rice hull added on pH, glucose concentration and ethanol concentration during ethanol production by fermentation in examples 2-1 to 2-3.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention provides a method for producing ethanol by fermenting starch raw materials, which comprises the steps of inoculating saccharomycetes into a liquid culture medium for ethanol fermentation;

The inventor of the invention finds that when the yeast is cultured by adding the rice hull powder into the liquid culture medium, the heat resistance of the yeast can be improved, so that the yeast can be fermented at 37-39 ℃ to produce ethanol, thereby reducing energy consumption, shortening fermentation time, improving fermentation efficiency and having low cost. Moreover, the inventor of the invention also finds that in the process of research and development, under the condition that the yeast can normally ferment within the range of 37-39 ℃, the fermentation effect and efficiency at 39 ℃ are higher than those at 37 ℃, such as higher ethanol yield and starch conversion rate.

In the present invention, the amount of the rice hull powder used in the liquid medium is 7 to 22 parts by weight, for example, 7, 8, 10, 12, 14, 16, 18, 20, 22 parts by weight and a range between any two points, based on 100 parts by weight of the starch material, and in the above preferable case, the yeast cell density and the ethanol production effect by fermentation can be improved in the case of further improving the heat resistance of yeast (allowing yeast to produce ethanol by fermentation at 39 ℃).

In the invention, the rice hull powder refers to a product obtained by crushing rice hulls, the particle size of the rice hull powder can be selected in a wide range, and preferably, the particle size of the rice hull powder is 12-80 meshes, namely, the rice hull powder is undersize with 12-80 meshes. In the preferable case, the yeast cell density and the ethanol production effect by fermentation can be improved under the condition of improving the heat resistance of the yeast, and the grain size of the rice hull powder has little influence on the fermentation result.

In the present invention, the species of the yeast may not be particularly limited, and the yeast conventionally used in the art may be any yeast, preferably saccharomyces cerevisiae, such as high temperature resistant active dry yeast produced by angel yeast.

In the invention, the liquid culture medium comprises a starch raw material and rice hull powder. Preferably, the starch material is selected from at least one of rice flour, millet flour, corn flour, sweet potato flour and tapioca flour. It will be appreciated that the starch feedstock, without further purification treatment, contains not only starch feedstock but also other substances such as proteins and lipids.

In the present invention, the source of the rice flour may not be particularly limited, and the rice flour may be obtained by directly pulverizing rice or rice flour provided from rice flour as a raw material. It should be understood that when the liquid medium is formulated in the form of rice flour pulverized with rice, rice hull powder is also simultaneously provided.

In the present invention, it is further preferred that the starch material is rice flour.

In the present invention, the particle size of the starch raw material can be selected in a wide range, and preferably, the particle size of the starch raw material is 20 to 100 meshes, that is, the starch raw material is undersize with 20 to 100 meshes. In such a case, higher fermentation efficiency and higher utilization rate of raw materials can be obtained.

In the present invention, the liquid medium preferably further comprises a nitrogen source; more preferably, the nitrogen source is selected from at least one of yeast extract, peptone, corn steep liquor, urea, ammonium sulfate, ammonium chloride and ammonia water. Preferably, the nitrogen source is an inorganic nitrogen source such as at least one of urea, ammonium sulfate, ammonium chloride and aqueous ammonia, and further preferably urea. The effect of ethanol production is better when urea is used as the nitrogen source.

In the present invention, in order to improve the raw material utilization rate, it is preferable that the liquid medium further comprises an enzyme preparation, which may be an enzyme preparation conventionally used in the art, and it is preferable that the enzyme preparation is selected from amylase and at least one of protease, cellulase, hemicellulase, pectinase. It is to be understood that the enzyme preparation comprises at least amylase.

In the present invention, the kind of the protease may not be particularly limited as long as it can hydrolyze protein, and preferably, the protease is an acid protease.

Amylases, which are a generic term for a class of enzymes capable of breaking down starch glycosidic bonds, generally include α -amylase, β -amylase, saccharifying enzyme, and isoamylase.

α -amylase, also known as starch 1, 4-dextrinase, liquefying enzyme, which can randomly and irregularly cleave α -1, 4-glycosidic bonds within the starch chain to hydrolyze the starch into maltose, oligosaccharides containing 6 glucose units, and oligosaccharides with branched chains, is commonly used as starch liquefaction, and can comprise α -amylase conventionally used in the art, such as high temperature resistant α -amylase available from Novitin.

β -amylase, also known as starch 1, 4-maltosidase, cleaves the 1, 4-glycosidic bond from the non-reducing end of the starch molecule to form maltose.

The saccharifying enzyme is also called starch α -1, 4-glucosidase, the saccharifying enzyme acts on the non-reducing end of starch molecule, takes glucose as unit, acts on α -1, 4-glycosidic bond in starch molecule in sequence to generate glucose, the product of saccharifying enzyme acts on amylopectin contains glucose and oligosaccharide with α -1, 6-glycosidic bond, the product of saccharifying enzyme acts on amylose is almost all glucose, the saccharifying enzyme can be the saccharifying enzyme of Novicin company.

Isoamylase, also known as starch α -1, 6-glucosidase, branching enzyme, acts on α -1, 6-glycosidic bonds at the branching point of the amylopectin molecule to cleave the entire side chain of the amylopectin into amylose.

It will be appreciated that the skilled person can select a suitable enzyme or combination of enzymes for the enzymatic hydrolysis (liquefaction and saccharification) of the starch feedstock as desired.

In the present invention, preferably, the enzyme preparation includes a liquefying enzyme, a saccharifying enzyme, and an acid protease.

In the present invention, the content of each component in the liquid medium can be selected within a wide range, and preferably, the nitrogen source is used in an amount of 0.1 to 0.5 parts by weight and the liquefying enzyme is used in an amount of 10 to 120U/g of the starch raw material, relative to 100 parts by weight of the starch raw material in the liquid medium; the dosage of the saccharifying enzyme is 10-120U/g starch raw material; the dosage of the acid protease is 50-500U/g of starch raw material; the amount of water is 130-1100 parts by weight.

Further preferably, in the liquid medium, the amount of the starch raw material is adjusted to 100 parts by weight; the dosage of the nitrogen source is 0.1-0.3 weight part, and the dosage of the liquefying enzyme is 30-80U/g starch raw material; the dosage of the saccharifying enzyme is 30-80U/g starch raw material; the dosage of the acidic protease is 150-300U/g starch raw material; the amount of water is 150-400 parts by weight. Within the above preferable range, the fermentation efficiency can be further improved.

In the present invention, the yeast inoculation form may be a form conventional in the art, and preferably, the yeast is inoculated in the form of a seed liquid.

In the present invention, the seed density of the yeast seed solution may be selected within a wide range. Preferably, the yeast seed liquid has a seed density OD₅₆₀>1, more preferably > 2, still more preferably 2.2 to 9.5. Within the preferred range, the efficiency and effect of ethanol production by fermentation of starch feedstock can be improved.

In the present invention, the inoculation amount of the yeast seed solution can be selected from a wide range, and preferably, the inoculation amount is 2-10 vol%, such as 2, 4, 6, 8, 10 vol% or any value between any two values, and more preferably 2-6 vol%.

In the present invention, the conditions for the fermentation may be conditions conventional in the art including: introducing air for 6-10h at the beginning of fermentation, wherein the air flow is 0.01-0.1vvm, the pH is 4-6, and the fermentation time is 24-50 h. More preferably, the fermentation is started by introducing air for 6-8h, and the fermentation time is 24-40 h.

In the present invention, the fermentation temperature may be a temperature conventionally used in the art, and the fermentation process can also be performed at 37 ℃ or higher in the presence of the medium of the present invention, and preferably, the fermentation temperature further includes 37 to 39 ℃, such as any temperature between 37.2 ℃, 37.4 ℃, 37.6 ℃, 37.8 ℃, 38 ℃, 38.2 ℃, 38.4 ℃, 38.6 ℃, 38.8 ℃, 39 ℃ and two points.

In the present invention, the fermentation process is accompanied by stirring, and the stirring rate can be adjusted according to practical situations, for example, the stirring speed in the 5L fermentation tank can be 180-220 rpm.

In the invention, preferably, the method for ethanol fermentation is one of a step-by-step saccharification fermentation method, a simultaneous saccharification fermentation method and a raw meal method. More preferably, the method of ethanol fermentation is a raw meal method. Under the condition that the method is a raw material method, the production cost can be greatly reduced and the time of the process for producing the ethanol by fermentation can be shortened while the higher fermentation efficiency is ensured.

Wherein, the step saccharification fermentation method, namely the SHF method, means that saccharification and fermentation are separately reacted in two containers. In a preferred embodiment of the present invention, the SHF process comprises: mixing a starch raw material with water for size mixing to obtain size; then sequentially liquefying and saccharifying the slurry to obtain saccharified liquid; and inoculating saccharomycetes into the saccharified liquid for fermentation.

The SSF method, which is a simultaneous saccharification and fermentation method, is a method in which saccharification and fermentation are performed simultaneously. In a preferred embodiment of the present invention, the SSF process comprises: mixing a starch raw material with water for size mixing to obtain size; then liquefying the slurry to obtain liquefied liquid; and (3) inoculating saccharomycetes into the liquefied liquid and adding saccharifying enzyme for fermentation.

The raw material method is a method for directly adding liquefying enzyme and saccharifying enzyme into a fermentation tank and inoculating the liquefying enzyme and the saccharifying enzyme into the fermentation tank for fermentation without liquefying and saccharifying slurry obtained after starch raw material size mixing.

In the present invention, the operating conditions and enzymes used in the SHF and the SSF may be those conventionally used in the art, and the amount of the enzyme may be selected from a wide range.

In a preferred embodiment of the present invention, the fractional saccharification and fermentation process comprises: mixing and pulping the rice flour and water in the presence of a liquefying enzyme to obtain a powder pulp, wherein the concentration of the rice flour in the powder pulp is 90-400 g/L. Liquefying the powder slurry, wherein the liquefying temperature is 80-90 ℃, the consumption of the liquefying enzyme is 35-75U/g of rice flour, and the liquefying time is 0.5-2 h; cooling the liquefied mash to 40-60 ℃, adding saccharifying enzyme for saccharifying for 0.5-2h to obtain the saccharified mash. Cooling the mash to 30-39 ℃, and adding rice hull powder, protease and an optional inorganic nitrogen source into the mash to obtain a fermentation culture medium. And (3) inoculating yeast seed liquid into the fermentation culture medium, and culturing for 24-40 h.

In a preferred embodiment of the present invention, the simultaneous saccharification and fermentation process comprises: mixing rice flour and water in the presence of liquefying enzyme, and pulping to obtain powder pulp, wherein the concentration of rice flour is 90-400 g/L. Liquefying the powder slurry, wherein the liquefying temperature is 80-90 ℃, the consumption of the liquefying enzyme is 35-75U/g of rice flour, and the liquefying time is 0.5-2 h; cooling the liquefied mash to 30-39 ℃, and adding rice hull powder, saccharifying enzyme, protease and optional inorganic nitrogen source into the liquefied mash to obtain a fermentation culture medium. And (3) inoculating yeast seed liquid into the fermentation culture medium, and culturing for 24-40 h.

In a preferred embodiment of the present invention, the raw meal process comprises: mixing rice flour and water, and making into powder slurry, wherein the concentration of rice flour is 90-400 g/L. Adding rice hull powder, liquefying enzyme, saccharifying enzyme, protease and optional inorganic nitrogen source into the slurry to obtain a fermentation culture medium. And (3) inoculating yeast seed liquid into the fermentation culture medium, and culturing for 24-40 h.

In the present invention, preferably, the process for producing ethanol by high-temperature fermentation using yeast is batch fermentation.

The present invention will be described in detail below by way of examples.

In the following examples, the yeast is high temperature resistant high activity dry yeast for brewing wine from Angel Yeast, and can be activated by adding water for 10 min. Experiments show that in a fermentation experiment using glucose as a carbon source, yeast can only tolerate fermentation at 35 ℃, and cannot perform ethanol fermentation at the temperature of over 37 ℃.

The preparation method of the yeast liquid stored at low temperature comprises the following steps: adding 1g dry yeast into 200mL sterile water, shaking, taking 2mL after 10min, adding into 200mLYPD culture medium, culturing at 28 deg.C for 9h, adding 20% glycerol (v/v), and storing at-70 deg.C. When in use, the yeast solution is melted at room temperature to obtain the yeast solution.

The liquefying enzyme is high temperature resistant α -amylase from Novoxin

The enzyme activity is 12300U/mL. The saccharifying enzyme is purchased from Novoxin

The enzyme activity is 13060U/mL. The protease is an acidic protease from Solebao

The enzyme activity is 50000U/g.

Seed Medium (YPD Medium) (g/L): 10 parts of yeast extract, 20 parts of peptone, 20 parts of glucose and 20 parts of glucose solution are independently sterilized at 115 ℃ for 20min, the other components of the culture medium are sterilized at 121 ℃ for 20min, and the components are mixed in a sterile operating platform for use.

The paddy rice used in the following examples and comparative examples was purchased from Hunan province, the variety was Xiangzai long-grained nonglutinous rice No. 31, the whole growth period was 107 days, and the paddy rice was stored in a dry environment for 4 years and was aged grains. Husking rice to obtain 77.3% rice grains, pulverizing, sieving with 40 mesh sieve, and using the sieved rice flour as fermentation substrate; 22.7% of the rice hulls are crushed, wherein the 40-mesh sieve accounts for 18.9% of the crushed rice hulls, and the non-sieved rice hulls account for 81.1%.

The rice hull powder used in the following examples and comparative examples refers to undersize obtained by pulverizing rice hulls and sieving through a 12-mesh sieve.

In the following examples, Excel and Origin 9.0 were used to process data. Estimating a fermentation process according to the bubble escape speed of the tail gas absorption bottle and the glucose content in the fermentation liquid, detecting a liquid phase after the fermentation is finished, and calculating the ethanol yield, the starch utilization rate and the production intensity to evaluate the fermentation efficiency (ethanol recovered from tail gas is merged into ethanol for quality calculation), wherein the calculation formula is as follows:

example 1

This example illustrates the process for producing ethanol according to the present invention

100mL of the starch slurry was prepared in a 250mL Erlenmeyer flask, and the rice flour concentration was 94 g/L. Liquefying enzyme (added amount is 65.4U/g rice flour), saccharifying enzyme (added amount is 69.5U/g rice flour), acid protease (added amount is 228U/g rice flour) and urea (added amount is 1.94mg/g rice flour) are added to prepare a liquid culture medium.

Adjusting initial pH of the culture medium to 5 with sulfuric acid, inoculating the yeast liquid stored at low temperature into the above conical flask at an inoculation amount of 2% (v/v), introducing air for 8h at the initial fermentation temperature of 39 ℃, and periodically weighing at a shaking table rotation speed of 170rpm, and recording the mass loss (g) of the shake flask, wherein the result is shown in FIG. 1.

Wherein, in example 1-1, rice hull powder was added to the liquid medium so that the content of the rice hull powder was 5.6% by weight compared to the rice hull powder;

example 1-2 rice hull powder was added to the liquid medium such that the content of rice hull powder was 10.8 wt% compared to rice hull powder;

example 1-3 rice hull powder was added to the liquid medium such that the content of rice hull powder was 16.1 wt% compared to rice hull powder;

in examples 1 to 4, rice hull powder was added to the liquid medium so that the content of the rice hull powder was 21.4% by weight.

As can be seen from FIG. 1, yeast containing 5.6 wt% of rice hull powder hardly ferments at 39 ℃ and loses little mass, mainly due to water evaporation, about 3.05g at 84 h. In the three examples in which 10.8 to 21.4 wt% of rice hull powder was added, there was a significant mass loss, indicating that the yeast released CO by the ethanol metabolism with glucose₂。

The mass loss gradually increased with the increase of the rice hull adding proportion, and the mass loss was stable around 100h (not shown in the figure), and the fermentation was finished. In example 1-2 in which 10.8 wt% of rice hull powder was added, the yeast could perform ethanol fermentation against high temperature stress with a final mass loss of 13.5 g; examples 1-3 and 1-4, in which 16.1 wt% and 21.4 wt% of rice hull powder was added, were similar in fermentation results, and the mass loss was 18.9g and 20.1g, respectively, and the difference was not large, indicating that there was no more excellent effect of adding too much rice hull.

Example 2

This example illustrates the method of producing ethanol by fermentation of yeast cells (raw meal method) according to the present invention

Example 2-1: inoculating the yeast liquid stored at low temperature into a triangular flask containing 100mL of seed culture medium at an inoculation amount of 2% (v/v), culturing at 37 deg.C and a shaking table rotation speed of 170rpm for 10h to obtain OD₅₆₀The seed liquid was 8.36.

Weighing a certain amount of rice flour, uniformly stirring the rice flour and 50 ℃ tap water in a 2L beaker to enable the substrate concentration to be 332g/L, obtaining a flour slurry, transferring the flour slurry into a 5L mechanical stirring fermentation tank, and adding liquefying enzyme (the adding amount of the rice flour is 65.4U/g), saccharifying enzyme (the adding amount of the rice flour is 69.5U/g), acid protease (the adding amount of the rice flour is 228U/g) and urea (the adding amount of the rice flour is 1.94 mg/g) into the flour slurry to prepare a fermentation culture medium. Rice hull powder was additionally added so that the rice hull powder content was 8.6% by weight of the rice flour, and the pH was adjusted to 5 with sulfuric acid.

Inoculating the seed liquid into a fermentation culture medium with an inoculation amount of 5% (v/v), stirring at 200rpm, ventilating for 8h at the initial fermentation stage with a ventilation amount of 0.05vvm and a temperature of 39 deg.C, adjusting pH during fermentation, connecting a tail gas absorption bottle, and absorbing CO escaped from the fermentation process with distilled water₂Sampling the carried ethanol at regular time, finishing fermentation after 24h, and showing the variation curve of the pH, glucose and ethanol concentration of the fermentation liquid in the fermentation process as shown in figure 2.

The ethanol yield, starch utilization and production strength were recorded and calculated and the results are shown in table 1.

Example 2-2: the yeast culture and ethanol production experiment by fermentation were carried out as described in example 2-1, except that rice hull powder was added to the fermentation medium in an amount of 7.1 wt% based on the rice flour, and the change profiles of the pH, glucose and ethanol concentration of the fermentation broth during the fermentation are shown in FIG. 2.

The ethanol concentration, the ethanol yield, the starch utilization rate and the production intensity are detected and calculated, and the results are shown in table 1.

Examples 2 to 3: the yeast culture and the ethanol production process by fermentation were carried out as described in example 2-1, except that rice hull powder was added to the fermentation medium in an amount of 5.6 wt% of the rice flour. The graph of the change in pH, glucose content and ethanol content during fermentation is shown in FIG. 2.

As can be seen from the results of example 2, the fermented mash of examples 2-3 was free of ethanol production at 39 ℃ and as a raw material fermentation, the glucose concentration in the mash was from the initial 22.6g/L to 70.5g/L for 8h and the final glucose concentration was as high as 102.3g/L, indicating that the yeast did not undergo ethanol metabolism and produced a large amount of acid on the contrary when exposed to a high temperature environment, and the pH profile also indicates that the enzymes in the early stage of inoculation act on the starch feedstock, releasing various substances in the cells, raising the pH a little and rapidly lowering the pH to 3.6 after 8 h. The pH was not changed much in examples 2-1 and 2-2 by fermentation under the same conditions, and the minimum pH was about 4.71. However, the rice husk addition in different degrees has different influences on the yeast fermentation, and the examples of adding 7.1 percent and 8.6 percent of rice husk powder end the fermentation within 24 hours, but the ethanol concentration in the fermentation mash of the former is 50.6g/L, and the ethanol concentration of the latter can reach 81.6 g/L.

Example 3

The yeast culture and ethanol production experiments by fermentation were carried out as described in examples 2-3, except that the fermentation temperature was 37 ℃ and the fermentation was terminated after 32 hours. The ethanol concentration, the ethanol yield, the starch utilization rate and the production intensity are detected and calculated, and the results are shown in table 1.

Compared with rice flour, the added rice hull powder accounts for 5.6 wt%, and in the fermentation medium, the yeast is difficult to grow and ferment at 39 ℃ but can tolerate the high temperature of 37 ℃ to ferment and produce ethanol.

Example 4

This example illustrates the method for producing ethanol by fermentation of yeast cells (simultaneous saccharification and fermentation method) according to the present invention

The yeast was cultured in the same manner as in example 2-1 to obtain a seed liquid.

Weighing a certain amount of rice flour, mixing with tap water at 50 ℃ and liquefying enzyme of 65.4U/g rice flour, stirring uniformly in a 2L beaker, and adjusting pH to 5 to obtain powder slurry, wherein the concentration of the rice flour is 332 g/L. Liquefying the powder slurry, wherein the liquefying temperature is 85 ℃, and the liquefying time is 1 h; cooling the liquefied mash to 39 ℃, and adding saccharifying enzyme (69.5U/g rice flour), acid protease (228U/g rice flour) and urea (1.94 mg/g rice flour) to obtain a fermentation culture medium, wherein the content of rice hull powder in the fermentation culture medium is 5.6 wt% relative to the rice flour.

Inoculating the seed liquid into a fermentation culture medium with an inoculation amount of 5% (v/v), stirring at 200rpm, ventilating for 8h at the initial fermentation stage with a ventilation amount of 0.05vvm, a temperature of 37 deg.C, an initial pH of 5, adjusting pH during fermentation, connecting a tail gas absorption bottle, and absorbing CO escaped from the fermentation process with distilled water₂And (5) sampling carried ethanol at regular time, and finishing fermentation for 28 hours.

Example 5

This example illustrates the method for producing ethanol by fermentation of yeast (step-by-step saccharification fermentation method) according to the present invention

Weighing a certain amount of rice flour, mixing with tap water at 50 ℃ and liquefying enzyme of 65.4U/g rice flour, stirring uniformly in a 2L beaker, and adjusting pH to 5 to obtain powder slurry, wherein the concentration of the rice flour is 332 g/L. Liquefying the powder slurry, wherein the liquefying temperature is 85 ℃, and the liquefying time is 1 h; cooling the liquefied mash to 50 ℃, adding saccharifying enzyme (69.5U/g rice flour) and saccharifying for 1h to obtain the saccharified mash. And (3) cooling the mash to 30 ℃, and adding acid protease (with the dosage of 228U/g rice flour) and urea (with the addition of 1.94mg/g rice flour) into the mash to obtain a fermentation culture medium. In the fermentation medium, the content of rice hull powder was 5.6 wt% with respect to rice flour.

Inoculating the seed liquid into a fermentation culture medium with an inoculation amount of 5% (v/v), stirring at 200rpm, ventilating for 8h at the initial fermentation stage with a ventilation amount of 0.05vvm, a temperature of 37 deg.C, an initial pH of 5, adjusting pH during fermentation, connecting a tail gas absorption bottle, and absorbing CO escaped from the fermentation process with distilled water₂And (5) sampling carried ethanol at regular time, and finishing fermentation for 24 hours.

Example 6

The yeast cultures and ethanol production experiments by fermentation were performed as described in example 3, except that urea was not added to the fermentation medium, and an equal amount of ammonium sulfate was added as an inorganic nitrogen source.

Example 7

Yeast cultures and fermentation ethanol production experiments were performed as described in example 3, except that the substrate concentration in the fermentation medium was 379 g/L.

TABLE 1

As can be seen from the data in Table 1, the addition of rice hulls to the fermentation medium is beneficial to improving the heat resistance of the yeast, enabling it to ferment rice flour at high temperatures to produce ethanol.

Experimental examples 3, 4 and 5 show that it is feasible to produce ethanol by fermenting rice flour at high temperature by a raw meal method, a simultaneous saccharification and fermentation method and a step-by-step saccharification and fermentation method.

Experimental examples 5, 6 and 7 show that the fermentation medium using urea as an inorganic nitrogen source and having a substrate concentration of 332g/L has a better fermentation effect.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. A method for producing ethanol by fermenting starch raw materials is characterized by comprising the steps of inoculating saccharomycetes into a liquid culture medium for ethanol fermentation;

2. The method according to claim 1, wherein the rice hull powder is used in the liquid medium in an amount of 7-22 parts by weight relative to 100 parts by weight of the starch feedstock.

3. The method of claim 1 or 2, wherein the starch feedstock is selected from at least one of rice flour, millet flour, corn flour, sweet potato flour, and tapioca flour;

preferably, the starch material is rice flour.

4. The method of claim 1, wherein the liquid medium further comprises a nitrogen source;

preferably, the nitrogen source is selected from at least one of yeast extract, peptone, corn steep liquor, urea, ammonium sulfate, ammonium chloride and ammonia water, more preferably urea.

5. The method of claims 1-4, wherein the liquid medium further comprises an enzyme preparation selected from the group consisting of amylase and at least one of acid protease, cellulase, hemicellulase, pectinase;

preferably, the enzyme preparation comprises a liquefying enzyme, a saccharifying enzyme and an acid protease.

6. The method according to claims 4 and 5, wherein the nitrogen source is used in an amount of 0.1-0.5 parts by weight and the liquefying enzyme is used in an amount of 10-120U/g of starch raw material, relative to 100 parts by weight of starch raw material; the dosage of the saccharifying enzyme is 10-120U/g starch raw material; the dosage of the acid protease is 50-500U/g of starch raw material; the amount of water is 130-1100 parts by weight.

7. The method according to claim 6, wherein the nitrogen source is used in an amount of 0.1 to 0.3 parts by weight and the liquefying enzyme is used in an amount of 30 to 80U/g of the starch raw material, relative to 100 parts by weight of the starch raw material, in the liquid medium; the dosage of the saccharifying enzyme is 30-80U/g starch raw material; the dosage of the acidic protease is 150-300U/g starch raw material; the amount of water is 140-400 parts by weight.

8. The method according to claims 1-7, wherein the yeast is inoculated in the form of a seed liquid having a seed density OD₅₆₀>1, the inoculation amount is 2-10 volume percent.

9. The method of any one of claims 1-8, wherein the ethanol fermentation conditions comprise: introducing air for 6-10h at the beginning of fermentation, wherein the air flow is 0.01-0.1vvm, the pH is 4-6, and the fermentation time is 24-50 h;

preferably, the fermentation temperature comprises 37-39 ℃.

10. The method of any one of claims 1-9, wherein the ethanol fermentation process is one of a step saccharification fermentation process, a simultaneous saccharification fermentation process, and a raw meal process;

preferably, the method of ethanol fermentation is a raw meal method.