CN110684675B

CN110684675B - Blakeslea trispora fermentation method and product thereof

Info

Publication number: CN110684675B
Application number: CN201911102570.0A
Authority: CN
Inventors: 李翔宇; 陆姝欢; 汪志明; 余超; 杨艳红
Original assignee: Cabio Biotech Wuhan Co Ltd
Current assignee: Cabio Biotech Wuhan Co Ltd
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2023-08-08
Anticipated expiration: 2039-11-12
Also published as: CN110684675A

Abstract

The invention relates to a method for fermenting Blakeslea trispora and a product thereof. The invention creatively replaces the traditional culture medium components with a large amount of rapeseed meal with excellent quality and low cost, and utilizes the rapeseed meal to ferment the Blakeslea trispora to produce the carotenoid, so that not only is the byproduct of the rapeseed meal fully and efficiently utilized, but also the manufacturing cost of the carotenoid is reduced, the production level and the product quality of the carotenoid are improved, and a new strategy is provided for the production and the manufacture of the carotenoid.

Description

Blakeslea trispora fermentation method and product thereof

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a Blakeslea trispora fermentation method and a product thereof.

Background

Beta-carotene is a fat-soluble unsaturated aliphatic hydrocarbon, a typical representative of the carotenoid family, and is the most ubiquitous and stable natural pigment in nature, widely found in green and yellow vegetables and fruits. Its molecular formula is C ₄₀ H ₅₆ The molecular weight is 536.88, the appearance is dark red to dark red, the oxidation-reduction reaction is easy to occur due to instability to oxygen, heat and light, the oxidation-reduction reaction is not easy to dissolve in water, acid and alkali, the oxidation-reduction reaction is easy to dissolve in chloroform, benzene and vegetable oil, and the oxidation-reduction reaction is slightly soluble in ethanol and diethyl ether. In addition to being used as a colorant, beta-carotene has various biological activities and is a medicinal effective ingredient closely related to human health. It has been found that, because the molecular formula of beta-carotene contains 2 beta-ionone rings and 4 isoprene side chains, two vitamin A molecules are produced after middle cleavage and are absorbed by human bodyCan be used as precursor of vitamin A after being collected; in addition, the unsaturated structure contained in the molecule has stronger antioxidant activity and free radical scavenging capability, can effectively prevent, delay and treat certain diseases, especially cancers, and can also improve the immune function of organisms. Therefore, it has been widely used in the industries of foods, medicines, cosmetics and health products. There are two major classes of chemically synthesized beta-carotene and natural beta-carotene from sources. At present, most of beta-carotene (more than 95%) is a chemical synthesis product, and the beta-carotene of natural sources is mainly obtained by taking Dunaliella salina or Blakeslea trispora as raw materials for fermentation.

The Blakeslea trispora belongs to the order Mucor, family Leucomycetaceae, is single-cell lower fungi, can realize large-scale industrial production at present, and is a heterozonic zygomycete, comprising two monosomic strains of positive bacteria B.trispora (+) and negative bacteria B.trispora (-). The mycelium forms of the positive strain and the negative strain have no obvious difference, and both the positive strain and the negative strain can independently and asexually reproduce to form sporangia and sporangia spores. The main components of the culture medium of the Blakeslea trispora in the current fermentation production are glucose, soybean meal, corn steep liquor dry powder or yeast extract and inorganic salt. There are also some reports on the production of beta-carotene.

CN104561211a discloses a method for improving beta-carotene production by blakeslea trispora using organic acids: 1) Spreading the bacterial solutions of Blakeslea trispora ATCC 14271 (+) and 14272 (-) on a corn meal agar medium for culture; two rings of positive bacteria and negative bacteria are respectively picked from ATCC 14271 (+) and 14272 (-) strain plates, inoculated into conical flasks containing a seed culture medium for culture, two seed solutions are mixed, then the mixed seed solutions are inoculated into the conical flasks containing a fermentation culture medium, organic acid is added for culture, after fermentation is finished, thalli are harvested, filtered and washed, and wet thalli are dried to constant weight, so that dry cell weight is obtained. Simple and easy operation, low cost and high beta-carotene content.

CN101870668A discloses a process for preparing beta-carotene from a fermentation broth of blakeslea trispora, comprising: filtering the Blakeslea trispora fermentation broth to obtain wet mycelia; vacuum drying wet mycelium to obtain dry mycelium with water content less than 10%; grinding and crushing dry mycelium; extracting the sieved dry mycelium with dichloromethane with 15-20 times of the dry mycelium, evaporating and concentrating the extract until the beta-carotene concentration is not lower than 50000 mug/ml, preserving heat, and filtering to obtain wet crude product of beta-carotene crystal; dissolving the wet crude product with dichloromethane, filtering the solution, evaporating and concentrating until the concentration of beta-carotene is not lower than 50000 mug/ml, and then preserving heat, filtering and drying in vacuum to obtain beta-carotene crystals.

Rapeseed meal is a byproduct of rapeseed oil extraction, is one of very good protein sources, and can be processed to obtain about 65% of rapeseed oil, so that the yield is huge. At present, the rapeseed meal is mainly used as plant fertilizer or is added as ruminant or freshwater fish culture feed according to a small proportion, but has larger limitation in application, and mainly comes from the fact that common rapeseed meal contains more anti-nutritional factors and serious protein denaturation, so that the use effect of the common rapeseed meal is affected.

At present, related reports of fermenting rapeseed cake by utilizing microorganisms exist, and the relative protein content can be improved while the anti-nutritional factors are reduced, for example, CN101434982B discloses a method for preparing rapeseed active peptide by solid-state fermentation of microorganisms, wherein the rapeseed cake obtained by a squeezing or leaching oil preparation process is crushed, and single strain with protease production capability is inoculated into the crushed rapeseed cake for solid-state fermentation; extracting the solid-state fermented culture medium with water, separating and removing residues to obtain the rapeseed bioactive peptide, which is a low-cost method suitable for industrialized mass preparation of the rapeseed bioactive peptide. For example, CN101492708A discloses a method for preparing rapeseed peptide with specific biological activity by mixed bacteria solid state fermentation, wherein one of bacillus subtilis, lactobacillus or candida utilis is compounded with Mucor elegans or Aspergillus usamii to prepare a starter; inoculating the starter to crushed rapeseed meal, and performing solid state fermentation; extracting the culture medium after solid state fermentation to obtain the rapeseed peptide. The prior art provides an effective means for improving the value of the rapeseed meal, and most of the prior art is solid state fermentation to obtain fermentation products of the rapeseed meal.

The prior art has limited strategies for preparing natural beta-carotene, and therefore it is of great interest to develop a new low-cost, easy-to-handle strategy for preparing natural beta-carotene.

Disclosure of Invention

The current application of the rapeseed meal is limited, firstly, the utilization of protein by microorganisms is influenced due to the high content of cellulose in the rapeseed meal; secondly, because the rapeseed meal contains about 2.2 to 4.4 percent of phytic acid component, the phytic acid has strong chelating cationic effect, can form complex with elements such as metal ions iron, zinc, calcium, magnesium, potassium and some proteins, and greatly reduces the bioavailability of the elements and the biological titer of the proteins; in addition, the glucosinolates and phenols contained in the rapeseed meal have adverse effects on the fermentation of microorganisms.

Aiming at the defects of the prior art, the invention aims to provide a Blakeslea trispora fermentation method and a product thereof. The fermentation method creatively replaces the traditional culture medium components such as yeast extract, corn steep liquor dry powder and the like with a large amount of rapeseed meal with good quality and low cost, and ferments and produces carotenoid by using Blakeslea trispora, so that not only is the byproduct of the large amount of rapeseed meal with good quality and low cost fully utilized, but also the manufacturing cost of the carotenoid is reduced, and a new strategy is provided for the production and manufacturing of the carotenoid.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

In one aspect, the invention provides a method for fermenting Blakeslea trispora, which comprises the following steps: the carotenoid is obtained by fermenting the Blakeslea trispora with rapeseed meal as the main component of a fermentation medium.

In general, the common fermentation organic nitrogen sources used when the Blakeslea trispora is used as a raw material for fermentation to obtain the carotenoid mainly comprise yeast extract, corn steep liquor dry powder and other traditional nitrogen sources, and the invention creatively replaces a large amount of rapeseed meal with excellent quality and low cost with traditional culture medium components, and the Blakeslea trispora is used for fermentation to produce the carotenoid, so that not only is the byproduct of the rapeseed meal fully and efficiently utilized, but also the manufacturing cost of the carotenoid is reduced, the production level and the product quality of the carotenoid are improved, and a new strategy is provided for the production and manufacture of the carotenoid.

Preferably, the method comprises the steps of:

(1) Performing slant culture on Blakeslea trispora;

(2) Culturing the Blakeslea trispora obtained in the step (1) in a seed culture medium added with rapeseed meal to obtain Blakeslea trispora seed liquid;

(3) Fermenting and culturing the Blakeslea trispora seed solution obtained in the step (2) in a fermentation medium added with rapeseed meal to obtain a fermentation liquid;

(4) And (3) collecting thalli in the fermentation broth obtained in the step (3), drying the thalli, breaking walls, and carrying out extraction and desolventizing treatment to obtain carotenoid. Preferably, the rapeseed meal is the rapeseed meal subjected to enzymolysis treatment.

The rapeseed meal can be subjected to enzymolysis to generate peptides with higher physiological activity, and the method is more beneficial to the fermentation of Blakeslea trispora by utilizing the rapeseed meal in the later stage, so that the rapeseed meal is subjected to the enzymatic hydrolysis pretreatment before being subjected to the fermentation treatment.

Preferably, the enzyme is a protease, which is any one or a combination of at least two of alkaline protease, neutral protease or acid protease, preferably neutral protease. The combination of at least two of the above-mentioned compounds, such as a combination of alkaline protease and acid protease, a combination of neutral protease and alkaline protease, etc., is not described herein.

Preferably, when the acid protease and the alkaline protease are combined to hydrolyze the rapeseed meal, the enzyme adding sequence is that the alkaline protease is added first and then the acid protease is added.

Preferably, different types of proteases are chosen for pH adjustment according to their nature.

Preferably, the neutral protease is added in an amount of 1500-4500U/g per gram of rapeseed meal, e.g., 1500U/g, 2000U/g, 2500U/g, 3000U/g, 3500U/g, 4000U/g, 4500U/g, etc.

Preferably, the neutral protease hydrolyzes rapeseed meal at a pH value of 6-7.5, e.g. ph=6, ph=6.5, ph=7 or ph=7.5, etc.

Preferably, the neutral protease hydrolyzes rapeseed meal at a temperature of 30-50 ℃, for example 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ or the like.

Preferably, the neutral protease hydrolyzes the rapeseed meal for a period of time ranging from 1 to 4 hours, for example 1 hour, 2 hours, 3 hours or 4 hours, etc.

Preferably, the preliminary enzymatic hydrolysis is performed with cellulases and/or xylanases, preferably cellulases, prior to treatment with proteases.

Preferably, the xylanase is added in an amount of 10-450U/g per gram of rapeseed meal, the optimum temperature is 35-50 ℃, and the optimum pH is 4.5-5.5.

Preferably, the temperature of the cellulase for hydrolyzing rapeseed meal is 40-60 ℃, such as 40 ℃, 45 ℃, 48 ℃, 50 ℃, 55 ℃, 60 ℃, or the like.

Preferably, the cellulase hydrolyzes rapeseed meal at a pH of 3.5-6.0, e.g. 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0, etc.

Preferably, the cellulase hydrolyzes the rapeseed meal for a period of time ranging from 1 to 2 hours, such as 1 hour, 1.2 hours, 1.5 hours, 1.8 hours, 2 hours, etc.

Preferably, the cellulase is added in an amount of 10 to 300U/g, for example 10U/g, 20U/g, 50U/g, 80U/g, 100U/g, 150U/g, 180U/g, 200U/g, 250U/g or 300U/g, etc., preferably 10 to 150U/g, per gram of rapeseed meal.

Preferably, the mass percentage of the small peptide in the rapeseed meal obtained by the enzymatic hydrolysis pretreatment is not less than 10%, for example, 10%, 15%, 20%, 25%, 30%, 40% or 50%, etc.

Preferably, the mass percentage of the small peptide in the rapeseed meal obtained by the enzymatic hydrolysis pretreatment is not less than 18%.

The small peptide refers to a small molecular protein with molecular weight below 10000 daltons. The small peptides have higher solubility than the crude protein and are more readily available to the mold than the crude protein in the rapeseed meal.

The temperature, the pH value, the time and the addition amount which are defined during enzymolysis are all important factors influencing the enzymolysis effect of the rapeseed meal, and the proteolytic treatment effect is better only under the cooperation of the numerical range, so that the content of the carotenoid prepared by fermentation is higher.

Preferably, the rapeseed meal is a rapeseed meal subjected to phytase hydrolysis.

Because the phytic acid component exists in the rapeseed meal, the phytic acid content is about 2.2% -4.4%, the phytic acid has strong chelating cationic effect and can form a complex with elements such as metal ions iron, zinc, calcium, magnesium, potassium and some proteins, the bioavailability of the elements and the biological titer of the proteins are greatly reduced, so that excessive phytic acid can greatly influence the growth metabolism of the Blakeslea trispora to block the fermentation process of the Blakeslea trispora, but a proper amount of phytic acid is found to promote the accumulation of carotenoids by the Blakeslea trispora, and the phytic acid can participate in the metabolism, so that the preferred scheme provided herein is to degrade the phytic acid component in the rapeseed meal before the fermentation treatment of the rapeseed meal so as to promote the fermentation of the Blakeslea trispora.

Preferably, the temperature of the hydrolysis is 40-60 ℃, e.g., 40 ℃, 45 ℃, 48 ℃, 50 ℃, 55 ℃, 60 ℃, or the like.

Preferably, the pH of the hydrolysis is 3.5-6.0, e.g. 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0, etc.

Preferably, the time of the hydrolysis is 1-3.5 hours, such as 1h, 1.2h, 1.5h, 1.7h, 1.8h, 2h, or 3.5h, etc.

Preferably, the time of the hydrolysis is 1-2 hours, such as 1 hour, 1.5 hours, 2 hours, etc.

Preferably, the phytase is added in an amount of 2-35U/g, e.g. 2U/g, 3U/g, 5U/g, 10U/g, 15U/g, 20U/g, 25U/g, 30U/g or 35U/g etc., preferably 2-10U/g, calculated per gram of rapeseed meal.

The temperature, the pH value, the time and the addition amount which are defined when the phytase is hydrolyzed are all important factors influencing the hydrolysis effect of the phytic acid, and the treatment effect of the phytic acid is better only under the cooperation of the numerical range, so that the content of the carotenoid prepared by fermentation is higher.

Preferably, the hydrolysis treatment of the phytase may be performed simultaneously with the hydrolysis treatment of the cellulase.

Preferably, the phytase is added in an amount of 2-10U/g (e.g., 2U/g, 3U/g, 5U/g, 10U/g, etc.) per gram of rapeseed meal; the cellulase is added in an amount of 10 to 150U/g (e.g., 10U/g, 20U/g, 50U/g, 80U/g, 100U/g, 150U/g, etc.) per gram of rapeseed meal.

Preferably, the hydrolysis reaction pH is 3.5-6.0, such as 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0, etc.

Preferably, the hydrolysis time is 1-2 hours, such as 1 hour, 1.5 hours, 2 hours, etc.

The invention surprisingly discovers that the cellulase and the phytase play a more remarkable role in degrading phytic acid when being used for simultaneously preprocessing the rapeseed meal, namely, the cellulase and the phytase produce a synergistic effect, and the invention is particularly characterized in that the content of phytic acid in the rapeseed meal can be reduced more quickly under the same condition, the content of small peptide after the protease hydrolysis can be promoted to be higher, and when the addition amount of the cellulase is 10-150U/g and the addition amount of the phytase is 2-10U/g, the cellulase and the phytase have the best synergistic effect, and meanwhile, the rapeseed meal obtained by hydrolysis under the condition is beneficial to the accumulation of products of the Blakeslea trispora.

The phytic acid content in the rapeseed meal can be reduced by adopting any one of the following modes or the mode of being cooperated with the technical scheme of the invention: extracting phytic acid under acidic conditions; or EDTA, sodium chloride or calcium chloride is added before extraction to break the complex between protein and phytic acid to further reduce the phytic acid content. Of course, the invention can also adopt other treatment means to remove other non-beneficial components in the rapeseed dregs, such as adsorption of the active carbon to remove the glucoside, alcohol washing to remove phenol, and the like.

Preferably, the pH between the different enzymatic hydrolysis steps can be adjusted to the appropriate conditions with pH adjusting agents such as hydrochloric acid, sodium hydroxide, citric acid, etc., depending on the nature of the different enzymes.

Preferably, the stirring operation is performed during the enzyme treatment.

Preferably, the solid-to-liquid ratio of the enzyme-treated system is controlled to be 6-30%, and in order to highlight the point of the invention, the solid-to-liquid ratio in the embodiment of the invention is 20%.

The rapeseed meal after hydrolysis can be further applied after drying, or quantitative hydrolysate can be directly put into fermentation culture for use.

In the invention, the method for carrying out slant culture on the Blakeslea trispora spores in the step (1) comprises the following steps: and (3) taking suspension of Blakeslea trispora positive bacteria and Blakeslea trispora negative bacteria spores, respectively coating the suspension on a PDA slant culture medium, and culturing at constant temperature.

The PDA culture medium is prepared by the following steps: cutting potato into 1cm square, adding deionized water, boiling for 30min, cooling, filtering with four layers of gauze, collecting the clear liquid, adding glucose and agar powder, wherein the total content of glucose and agar powder is 20g/L, the content of agar powder is 25g/L, and the content of peeled potato is 200g/L.

Preferably, the temperature of the constant temperature culture is 20-30 ℃, for example 20 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, or the like.

Preferably, the incubation is for a period of 5-7 days, for example 5, 6 or 7 days.

In the invention, the seed culture method in the step (2) comprises the following steps: taking the positive strain and the negative strain of the Blakeslea trispora obtained in the step (1), and respectively inoculating the positive strain and the negative strain into a seed culture medium for culture.

Preferably, the seed medium comprises glucose, corn starch, rapeseed meal, potassium dihydrogen phosphate and magnesium sulfate.

Preferably, the concentration of the rapeseed meal is 30-50g/L.

Preferably, the seed culture medium comprises 5-15g/L glucose, 25-35g/L corn starch, 30-50g/L rapeseed meal, 0.5-2g/L monopotassium phosphate and 0.05-0.2g/L magnesium sulfate.

The concentration of the rapeseed meal during seed culture needs to be specifically selected within the range of 30-50g/L, and the concentration is lower than that, so that nitrogen sources are insufficient, the growth of thalli is limited, the biomass is lower, and the subsequent expansion culture is limited. Above this concentration, the nitrogen source is excessive, which has an effect on the subsequent ability of the cell product to accumulate.

The glucose concentration may be 5g/L, 6g/L, 8g/L, 9g/L, 10g/L, 12g/L, 13g/L, 14g/L, 15g/L, etc.

The corn starch concentration may be 25g/L, 26g/L, 27g/L, 28g/L, 29g/L, 30g/L, 31g/L, 33g/L, 35g/L, etc.

The concentration of the rapeseed meal can be 30g/L, 35g/L, 36g/L, 38g/L, 39g/L, 40g/L, 41g/L, 42g/L, 44g/L, 45g/L, 50g/L or the like.

The concentration of the potassium dihydrogen phosphate can be 0.5g/L, 0.6g/L, 0.8g/L, 1.0g/L, 1.2g/L, 1.5g/L, 2g/L, etc.

The concentration of the magnesium sulfate may be 0.05g/L, 0.1g/L, 0.12g/L, 0.15g/L, 0.2g/L, or the like.

Preferably, the temperature of the culture is 20-30 ℃, e.g., 20 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, or the like.

Preferably, the time of the cultivation is 40-50h, e.g., 40h, 41h, 42h, 43h, 44h, 45h, 46h, 48h, 50h, etc.

The cultivation may be carried out in a shaker, a seed tank, for example at a rotational speed of 100-200r/min, for example 100r/min, 120r/min, 150r/min, 160r/min, 180r/min or 200r/min, etc.

Preferably, the pH of the culture environment is 6.5-7.5, e.g., 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, etc.

In the invention, the fermentation culture method in the step (3) comprises the following steps: and (3) mixing the positive bacterial seed liquid of the Blakeslea trispora and the negative bacterial seed liquid of the Blakeslea trispora obtained in the step (2), and inoculating the mixed seed liquid into a fermentation medium for culturing.

Preferably, the mass ratio of the Blakeslea trispora positive strain seed liquid to the Blakeslea trispora negative strain seed liquid is 1 (0.5-2), such as 1:2, 1:1 or 1:0.5.

Preferably, the volume ratio of the mixed solution of the Blakeslea trispora positive strain seed solution and the Blakeslea trispora negative strain seed solution to the fermentation medium is 1 (8-10), for example, 1:8, 1:8.5, 1:9, 1:9.5, 1:10 or the like.

Preferably, the fermentation medium comprises glucose, corn starch, rapeseed meal, soybean meal, potassium dihydrogen phosphate and magnesium sulfate.

Preferably, the concentration of the rapeseed meal is 30-60g/L.

Preferably, the fermentation medium comprises 15-25g/L glucose, 35-45g/L corn starch, 30-60g/L rapeseed meal, 35-45g/L soybean meal, 0.5-2g/L potassium dihydrogen phosphate and 0.05-0.2g/L magnesium sulfate.

The concentration of the rapeseed meal during fermentation culture needs to be specifically selected within the range of 30-60g/L, and if the addition amount of the rapeseed meal is lower than the concentration range, the nitrogen source is insufficient, the growth of bacteria is limited, and if the addition amount of the rapeseed meal is higher than the concentration range, the nitrogen source is excessive, the metabolism of bacteria is inhibited, and the accumulation of products is influenced.

The glucose concentration may be 15g/L, 16g/L, 18g/L, 19g/L, 20g/L, 21g/L, 22g/L, 24g/L, 25g/L, etc.

The corn starch concentration may be 35g/L, 36g/L, 38g/L, 39g/L, 40g/L, 41g/L, 42g/L, 44g/L, 45g/L, etc.

The concentration of the rapeseed meal can be 30g/L, 32g/L, 35g/L, 40g/L, 45g/L, 50g/L, 52g/L, 55g/L, 60g/L or the like.

The concentration of the soybean cake powder can be 35g/L, 36g/L, 38g/L, 39g/L, 40g/L, 41g/L, 42g/L, 44g/L or 45g/L, etc.

Preferably, the temperature of the fermentation culture is 20-30 ℃, e.g., 20 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, or the like.

Preferably, the fermentation culture is performed for a period of time ranging from 100 to 150 hours, for example, 100 hours, 110 hours, 115 hours, 120 hours, 125 hours, 130 hours, 135 hours, 140 hours, 150 hours, etc.

Preferably, the pH of the fermentation culture environment is in the range of 6.5-7.5, e.g., 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, etc.

Preferably, the fermentation stirring speed is 100-200r/min, for example 100r/min, 120r/min, 150r/min, 160r/min, 180r/min or 200r/min, etc.

The conditions of temperature, time and the like related to the slant culture, the seed culture and the fermentation culture are mutually matched, and the carotenoid content obtained by final preparation can be highest only under the synergistic combination of the conditions.

In addition, in the present invention, the extractant used in the extraction in the step (4) includes ethyl acetate, butyl acetate or isobutyl acetate.

After the desolventizing is finished, the carotenoid can be purified and crystallized.

As a preferred technical scheme of the invention, the method for preparing carotenoid by fermenting rapeseed meal specifically comprises the following steps:

(1) Carrying out enzymolysis treatment on the rapeseed dregs to obtain a rapeseed dreg treated product, wherein the content of small peptide is not less than 10%;

(2) Respectively coating the suspension of the Blakeslea trispora positive bacteria and Blakeslea trispora negative bacteria spores on a PDA slant culture medium, and culturing at a constant temperature of 20-30 ℃ for 5-7 days to obtain Blakeslea trispora positive strains and Blakeslea trispora negative strains;

(3) Respectively inoculating the positive strain and the negative strain of the Blakeslea trispora obtained in the step (2) into a seed culture medium added with rapeseed meal, and culturing for 40-50 hours at 20-30 ℃ and pH value of 6.5-7.5 to obtain a positive strain seed liquid and a negative strain seed liquid of the Blakeslea trispora, wherein the content of the rapeseed meal in the seed culture medium is 30-50g/L;

(4) Mixing the positive strain seed liquid and the negative strain seed liquid of the Blakeslea trispora obtained in the step (3) according to the mass ratio of 1:2-1:0.5, inoculating the mixture into a fermentation medium added with rapeseed meal, and culturing the mixture for 100-150h at the temperature of 20-30 ℃ at the pH value of 6.5-7.5 to obtain fermentation liquor, wherein the content of the rapeseed meal in the fermentation medium is 30-60g/L;

(5) And (3) after collecting thalli in the fermentation broth obtained in the step (4), drying, breaking walls of the thalli, and carrying out extraction and desolventizing treatment to obtain carotenoid.

Preferably, the carotenoid is lycopene and/or beta-carotene.

In another aspect, the invention provides a carotenoid prepared by a Blakeslea trispora fermentation process as described above.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively replaces the traditional culture medium components with a large amount of rapeseed meal with excellent quality and low cost, utilizes the rapeseed meal to ferment the Blakeslea trispora to produce carotenoid, establishes a complete fermentation system of the Blakeslea trispora, not only makes full and efficient use of the byproduct of the rapeseed meal, reduces the manufacturing cost of the carotenoid, improves the production level and the product quality of the carotenoid, but also provides a new strategy for the production and manufacture of the carotenoid.

Detailed Description

In order to further describe the technical means adopted by the present invention and the effects thereof, the following describes the technical scheme of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

Blakeslea trispora can produce beta-carotene and can also obtain lycopene by adding a blocking agent in the fermentation process, and the invention aims to better show the invention, and the examples are listed by taking the beta-carotene as an example, but the invention aims not to be limited by the type of the product.

The Blakeslea trispora adopted in the embodiment is a mutant strain obtained by self-mutagenesis of the applicant, and is preserved in China Center for Type Culture Collection (CCTCC) in 8 months and 8 days of 2014, wherein the preservation addresses are China, the university of Wuhan and the university of Wuhan, and the preservation number is: blakeslea trispora BT7251 (+) CCTCC M2014378; blakeslea trispora BT7603 (-) CCTCC M2014379.

Example 1

The present embodiment provides a method for producing beta-carotene, the method comprising the steps of:

(1) Respectively coating the suspension of the Blakeslea trispora positive bacteria and Blakeslea trispora negative bacteria spores on a PDA slant culture medium, and culturing at a constant temperature of 25 ℃ for 6 days to obtain Blakeslea trispora positive strains and Blakeslea trispora negative strains;

(2) Taking the positive strain and the negative strain of the Blakeslea trispora obtained in the step (1), respectively inoculating the positive strain and the negative strain into a seed culture medium, and culturing for 48 hours at 25 ℃ under shaking of a shaker with the pH value of 7.0 and 180r/min to obtain positive strain seed liquid and negative strain seed liquid of the Blakeslea trispora; the seed culture medium is as follows: 10g/L glucose, 30g/L corn starch, 40g/L rapeseed meal, 1g/L monopotassium phosphate and 0.1g/L magnesium sulfate.

(3) Mixing the positive strain seed liquid and the negative strain seed liquid of the Blakeslea trispora obtained in the step (2) according to the mass ratio of 1:1, inoculating the mixture into a fermentation medium, and culturing the mixture for 120 hours at 25 ℃ and pH value of 7.0 to obtain fermentation liquor; the fermentation medium comprises 20g/L glucose, 40g/L corn starch, 50g/L rapeseed meal, 40g/L soybean meal, 1g/L potassium dihydrogen phosphate and 0.1g/L magnesium sulfate.

(4) And (3) collecting thalli in the fermentation broth obtained in the step (3), drying, breaking walls of the thalli, and performing extraction and desolventizing treatment by ethyl acetate to obtain the beta-carotene.

Example 2

The present example provides a process for the preparation of beta-carotene, which process is as follows:

(1) The rapeseed meal is subjected to hydrolytic pretreatment for 2 hours in an environment with the pH value of 5 at 50 ℃, and the cellulose addition amount is 50U/g; then the pH value is adjusted to 7, the temperature of the feed liquid is reduced to 40 ℃, neutral protease is added for hydrolysis for 2 hours, the addition amount is 4000U/g, and the treated rapeseed meal is obtained after the hydrolysis is finished and is used as the additive component of each culture medium;

(2) The following steps refer to the method in the embodiment 1, namely, taking positive Blakeslea trispora fungus and negative Blakeslea trispora fungus spore suspension, sequentially carrying out slant culture, seed culture and fermentation culture, fermenting for 120 hours to obtain fermentation liquor, collecting thalli, drying and breaking walls of the thalli, and carrying out extraction and desolventizing treatment by ethyl acetate to obtain the beta-carotene.

Example 3

(1) Hydrolyzing and pre-treating the rapeseed meal with phytase for 2 hours in an environment with pH value of 5 at 50 ℃, wherein the adding amount of the phytase is 5U/g, and obtaining the treated rapeseed meal after the hydrolysis is finished as an adding component of each culture medium;

(2) The following steps refer to the method in example 1, namely, taking positive Blakeslea trispora and negative Blakeslea trispora spore suspensions, and sequentially carrying out slant culture, seed culture and fermentation culture, and fermenting for 120 hours to obtain fermentation liquor. And then collecting thalli, drying and breaking walls of the thalli, and extracting and desolventizing the thalli by ethyl acetate to obtain the beta-carotene.

Example 4

(1) Hydrolyzing and pre-treating the rapeseed meal with phytase and cellulase at 50 ℃ under the condition that the pH value is 5 for 2 hours, wherein the adding amount of the phytase is 5U/g, and the adding amount of the cellulase is 50U/g; regulating pH to 7, cooling to 40deg.C, adding neutral protease for hydrolysis, wherein the addition amount of neutral protease is 4000U/g, and hydrolyzing for 2 hr to obtain treated rapeseed cake as additive component of each culture medium;

(2) Respectively coating the suspension of the Blakeslea trispora positive bacteria and Blakeslea trispora negative bacteria spores on a PDA slant culture medium, and culturing at a constant temperature of 25 ℃ for 6 days to obtain Blakeslea trispora positive strains and Blakeslea trispora negative strains;

(3) Taking rapeseed meal as a nitrogen source, taking the positive strain and the negative strain of the Blakeslea trispora obtained in the step (2), respectively inoculating the positive strain and the negative strain of the Blakeslea trispora into a seed culture medium, and culturing for 48 hours at 25 ℃ under the shaking of a shaking table with the pH value of 7.0 and 180r/min to obtain positive strain seed liquid and negative strain seed liquid of the Blakeslea trispora; the seed culture medium is as follows: 10g/L glucose, 30g/L corn starch, 40g/L rapeseed meal, 1g/L monopotassium phosphate and 0.1g/L magnesium sulfate.

(4) Taking rapeseed meal as a nitrogen source, mixing the positive strain seed liquid and the negative strain seed liquid of the Blakeslea trispora obtained in the step (3) according to the mass ratio of 1:1, inoculating the mixture into a fermentation medium, and culturing the mixture at 25 ℃ for 120 hours at the pH value of 7.0 to obtain fermentation liquor; the fermentation medium comprises 20g/L glucose, 40g/L corn starch, 50g/L rapeseed meal, 40g/L soybean meal, 1g/L potassium dihydrogen phosphate and 0.1g/L magnesium sulfate.

(5) And (3) after collecting thalli in the fermentation broth obtained in the step (4), drying and breaking walls of the thalli, and performing extraction and desolventizing treatment by ethyl acetate to obtain the beta-carotene.

Example 5

(1) Carrying out hydrolytic pretreatment on the rapeseed meal with phytase at 50 ℃ under the condition that the pH value is 5 for 2 hours, wherein the adding amount of the phytase is 5U/g; regulating pH to 7, cooling to 40deg.C, adding neutral protease for hydrolysis, wherein the addition amount of neutral protease is 4000U/g, and hydrolyzing for 2 hr to obtain treated rapeseed cake as additive component of each culture medium;

(2) Respectively coating the suspension of the Blakeslea trispora positive bacteria and Blakeslea trispora negative bacteria spores on a PDA slant culture medium, and culturing at a constant temperature of 20 ℃ for 7 days to obtain Blakeslea trispora positive strains and Blakeslea trispora negative strains;

(3) Taking rapeseed meal as a nitrogen source, taking the positive strain and the negative strain of the Blakeslea trispora obtained in the step (2), respectively inoculating the positive strain and the negative strain of the Blakeslea trispora into a seed culture medium, and culturing for 50 hours at 20 ℃ under the shaking of a shaking table with the pH value of 7.5 and 200r/min to obtain positive strain seed liquid and negative strain seed liquid of the Blakeslea trispora; the seed culture medium is as follows: glucose 5g/L, corn starch 25g/L, rapeseed meal 35g/L, monopotassium phosphate 0.5g/L and magnesium sulfate 0.05g/L.

(4) Taking rapeseed meal as a nitrogen source, mixing the positive strain seed liquid and the negative strain seed liquid of the Blakeslea trispora obtained in the step (3) according to the mass ratio of 1:2, inoculating the mixture into a fermentation medium, and culturing the mixture at 20 ℃ for 120 hours at the pH value of 7.5 to obtain fermentation liquor; the fermentation medium comprises 15g/L of glucose, 35g/L of corn starch, 45g/L of rapeseed meal, 35g/L of soybean meal, 0.5g/L of monopotassium phosphate and 0.05g/L of magnesium sulfate.

Example 6

(1) Hydrolyzing and pre-treating the rapeseed meal with phytase and cellulase at 60 ℃ for 1h in an environment with the pH value of 5.5, wherein the adding amount of the phytase is 10U/g, and the adding amount of the cellulase is 150U/g; regulating pH to 6, cooling to 30deg.C, adding neutral protease for hydrolysis, wherein the addition amount of neutral protease is 1500U/g, and hydrolyzing for 4 hr to obtain treated rapeseed meal as additive component of each culture medium;

(2) Respectively coating the suspension of the Blakeslea trispora positive bacteria and Blakeslea trispora negative bacteria spores on a PDA slant culture medium, and culturing at a constant temperature of 30 ℃ for 5 days to obtain Blakeslea trispora positive strains and Blakeslea trispora negative strains;

(3) Taking rapeseed meal as a nitrogen source, taking the positive strain and the negative strain of the Blakeslea trispora obtained in the step (2), respectively inoculating the positive strain and the negative strain of the Blakeslea trispora into a seed culture medium, and culturing the positive strain and the negative strain of the Blakeslea trispora for 40h at 30 ℃ under the shaking of a shaking table with the pH value of 6.5 and a shaking table with the pH value of 100r/min to obtain positive strain seed liquid and negative strain seed liquid of the Blakeslea trispora; the seed culture medium is as follows: 15g/L glucose, 35g/L corn starch, 45g/L rapeseed meal, 2g/L monopotassium phosphate and 0.2g/L magnesium sulfate.

(4) Taking rapeseed meal as a nitrogen source, mixing the positive strain seed liquid and the negative strain seed liquid of the Blakeslea trispora obtained in the step (3) according to the mass ratio of 2:1, inoculating the mixture into a fermentation medium, and culturing the mixture at 30 ℃ for 120 hours at the pH value of 6.5 to obtain fermentation liquor; the fermentation medium comprises 25g/L of glucose, 45g/L of corn starch, 55g/L of rapeseed meal, 45g/L of soybean cake powder, 2g/L of monopotassium phosphate and 0.2g/L of magnesium sulfate.

(5) And (3) after collecting thalli in the fermentation broth obtained in the step (4), drying and breaking walls of the thalli, and extracting and desolventizing the thalli by ethyl acetate to obtain the beta-carotene.

Example 7

(1) Hydrolyzing and pre-treating rapeseed meal with phytase and cellulase at 40 ℃ for 2 hours in an environment with the pH value of 4.5, wherein the adding amount of the phytase is 2U/g, the adding amount of the cellulase is 10U/g, the pH value is regulated to 7.5, the temperature of the feed liquid is reduced to 45 ℃, neutral protease is added for hydrolysis, the adding amount of the neutral protease is 4500U/g, and the hydrolysis is carried out for 1 hour, so that the treated rapeseed meal is obtained and is used as an additive component of each culture medium;

Example 8

The present example provides a process for the preparation of beta-carotene, which process is as follows: the difference from example 4 is only that "40 g/L of rapeseed meal" in step (3) was replaced with "50 g/L of rapeseed meal"; in the step (4), the '50 g/L of rapeseed meal' is replaced by the '60 g/L of rapeseed meal', and other conditions are unchanged.

Example 9

The present example provides a process for the preparation of beta-carotene, which process is as follows: the difference from example 4 is only that "40 g/L of rapeseed meal" in step (3) was replaced with "20 g/L of rapeseed meal"; in the step (4), the '50 g/L of rapeseed meal' is replaced by the '30 g/L of rapeseed meal', and other conditions are unchanged.

Example 10

This example provides a process for preparing β -carotene which differs from example 4 only in that the amount of neutral protease added in step (1) is 500U/g, all other conditions being unchanged.

Example 11

This example provides a process for the preparation of beta-carotene which differs from example 4 only in that the "cellulase" in step (1) is replaced by a "xylanase", all other conditions being unchanged.

Example 12

The present example provides a method for producing β -carotene, which is different from example 4 in that in step (1), rapeseed meal is subjected to hydrolysis pretreatment with phytase and cellulase at 50 ℃ in an environment with a pH value of 5 for 2 hours, the addition amount of phytase is 15U/g, and the addition amount of cellulase is 50U/g; regulating pH to 7, cooling to 40deg.C, adding neutral protease for hydrolysis, wherein the addition amount of neutral protease is 4000U/g, hydrolyzing for 2 hr, and drying to obtain treated rapeseed cake as additive component of each culture medium;

Other fermentation conditions are unchanged.

Comparative example 1

This comparative example provides a process for preparing beta-carotene which differs from example 4 only in that the "rapeseed meal" in step (3) (4) is replaced entirely by "yeast extract", all other conditions being unchanged.

Comparative example 2

This comparative example provides a process for preparing beta-carotene which differs from example 4 only in that the "rapeseed meal" in step (3) (4) is replaced entirely by "corn steep liquor dry powder", all other conditions being unchanged.

Evaluation experiment 1:

the phytic acid degradation rate and the content of small peptides of the rapeseed meal in examples 1 to 7 and examples 10 to 12 were measured. Wherein, the method for measuring phytic acid is measured according to GB 17406-1998; the detection method of the small peptide adopts trichloroacetic acid soluble protein to detect small molecular protein with molecular weight below 10000 dalton: accurately weighing 1.0g (accurate to 0.001 g) of a sample, adding 15% trichloroacetic acid (TCA) solution for dissolution, fixing the volume to 50mL, uniformly mixing and standing for 5min, filtering, removing primary filtrate, taking the filtrate as standby liquid, and then measuring according to the method for measuring the peptide content of annex B in GB/T22492-2008 to obtain the peptide content. The vegetable seed meal sources in the examples are common commercial untreated rapeseed meal, wherein the phytic acid content in the rapeseed meal is 3.6%, and the results after treatment are shown in table 1:

TABLE 1

	The degradation rate of phytic acid is%	Small peptides%
			Example 1	-	0.25
Example 2	0.5	10.07
			Example 3	46.8	0.26
Example 4	72.6	24.26
			Example 5	46.5	11.67
Example 6	79.8	18.35
			Example 7	63.4	21.42
Example 10	68.7	8.90
			Example 11	63.2	15.68
Example 12	84.1	22.40

Evaluation experiment 2:

determination of the yield and content of beta-carotene in the cells was performed on the desolventized fermentation broths prepared in examples 1 to 12 and comparative examples 1 to 2.

The method for detecting the content of beta-carotene in the thalli comprises the following steps:

accurately weighing the sample, repeatedly extracting with chloroform after breaking the wall until the sample is completely colorless, and fixing the volume with cyclohexane.

Absorbance was measured at 455nm using an ultraviolet spectrophotometer.

The beta-carotene content was calculated as follows, and the calculation results are shown in Table 1:

wherein: a-absorbance value of the sample to be tested;

n-dilution fold;

m-weighing the sample;

percent absorbance of 2500-beta-carotene in cyclohexane

The measurement of the yield of the beta-carotene refers to national standard GB8821-2011, and the result is shown in Table 2 (DCW refers to the mass of dry thalli in each liter of fermentation broth, and is obtained by sampling, drying and weighing, BC content refers to the percentage of the beta-carotene in the dry thalli, and BC yield refers to the mass of the beta-carotene in each liter of fermentation broth):

TABLE 2

According to the data in tables 1 and 2: example 1 shows that when the non-pretreated rapeseed meal is directly used, the Blakeslea trispora can be used as a nitrogen source for fermentation to produce beta-carotene, but the non-pretreated rapeseed meal is used as a culture medium component, so that the Blakeslea trispora biomass and the beta-carotene content are low.

Although the biomass of the pretreated rapeseed meal is slightly reduced compared with that of the traditional culture medium components, the pretreated rapeseed meal has a promoting effect on the accumulation of beta-carotene products, and the whole beta-carotene yield can be improved by 10%. And the degradation rate of phytic acid in the rapeseed meal is not positively related to the yield of beta-carotene, and when the degradation rate is 60-80% and the phytic acid content is about 0.8% -1.31%, the degradation rate is preferentially fermented under other conditions, and the accumulated content of beta-carotene in the Blakeslea trispora is higher.

The increased content of rapeseed meal, while able to increase biomass, is not conducive to accumulation of beta-carotene; the content of the rapeseed meal is reduced, so that the growth of mould is limited, and the biomass is lower.

The applicant states that the above examples are shake flask fermentations, and the culture medium system can be extended during the amplification culture, and the invention is described by the above examples as a method for fermenting Blakeslea trispora and its products, but the invention is not limited to the above examples, i.e. it does not mean that the invention must be practiced by relying on the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. A method for fermenting Blakeslea trispora, which is characterized in that the method comprises the following steps: the rapeseed meal is used as the main component of a fermentation medium, and carotenoid is obtained through Blakeslea trispora fermentation;

the rapeseed meal is the rapeseed meal subjected to enzymolysis treatment by neutral protease, and is subjected to preliminary hydrolysis by adopting cellulase and phytase before being subjected to enzymolysis treatment by the neutral protease;

the addition amount of the cellulase is 10-150U/g calculated by each gram of rapeseed meal; the temperature of the cellulase for hydrolyzing the rapeseed meal is 40-60 ℃, the pH value is 3.5-6.0, and the time is 1-2 h;

The addition amount of the phytase is 2-10U/g calculated by each gram of rapeseed meal; the temperature of the phytase hydrolysis is 40-60 ℃, the pH value is 3.5-6.0, and the time is 1-3.5 h;

the addition amount of the neutral protease is 1500-4500U/g calculated by each gram of rapeseed meal; the pH value of the neutral proteinase hydrolyzed rapeseed meal is 6-7.5, the temperature is 30-50 ℃ and the time is 1-4 h.

2. The method of fermentation of b.trispora according to claim 1, characterized in that it comprises the steps of:

(1) Performing slant culture on Blakeslea trispora;

(4) And (3) collecting thalli in the fermentation broth obtained in the step (3), drying the thalli, breaking walls, and carrying out extraction and desolventizing treatment to obtain carotenoid.

3. The method for fermenting Blakeslea trispora as in claim 1, wherein the mass percentage of small peptides in rapeseed meal obtained through enzymatic hydrolysis pretreatment is not less than 10%.

4. The method for fermenting Blakeslea trispora as in claim 1, wherein the mass percentage of small peptides in rapeseed meal obtained through enzymatic hydrolysis pretreatment is not less than 18%.

5. The method of fermentation of b.trispora according to claim 1, wherein the phytase is hydrolysed for a time period of 1-2 h.

6. The method of claim 1, wherein the hydrolysis of phytase is performed simultaneously with the hydrolysis of cellulase.

7. The process for fermentation of Blakeslea trispora as in claim 6, characterized in that the phytase is added in an amount of 2-10U/g per gram of rapeseed meal; the addition amount of the cellulase is 10-150U/g calculated by each gram of rapeseed meal.

8. The process for fermentation of Blakeslea trispora as in claim 6, wherein the hydrolysis treatment has a pH of 3.5 to 6.0.

9. The process for fermentation of Blakeslea trispora as in claim 6, wherein the temperature of the hydrolysis treatment is between 40 and 60 ℃.

10. The method of fermentation of Blakeslea trispora as in claim 6, wherein the hydrolysis treatment is carried out for a period of time ranging from 1 to 2 h.

11. The method of fermentation of b.trispora according to claim 2, wherein the method of seed culture of step (2) is: taking the positive strain and the negative strain of the Blakeslea trispora obtained in the step (1), and respectively inoculating the positive strain and the negative strain into a seed culture medium for culture.

12. The method of fermentation of b.trispora according to claim 11, wherein the seed medium comprises glucose, corn starch, rapeseed meal, potassium dihydrogen phosphate and magnesium sulfate.

13. The process for fermentation of b.trispora according to claim 12, wherein the concentration of rapeseed meal is 30-50 g/L.

14. The method of claim 11, wherein the seed medium comprises glucose 5-15 g/L, corn starch 25-35 g/L, rapeseed meal 30-50 g/L, potassium dihydrogen phosphate 0.5-2 g/L, and magnesium sulfate 0.05-0.2 g/L.

15. The process for fermentation of b.trispora according to claim 11, wherein the temperature of the cultivation is 20-30 ℃.

16. The method of fermentation of b.trispora according to claim 11, wherein the time of the cultivation is 40-50 h.

17. The process for fermentation of b.trispora according to claim 11, wherein the pH of the environment of the culture is between 6.5 and 7.5.

18. The method for fermenting the Blakeslea trispora according to claim 2, wherein the method for fermenting and culturing in the step (3) is as follows: and (3) mixing the positive bacterial seed liquid of the Blakeslea trispora and the negative bacterial seed liquid of the Blakeslea trispora obtained in the step (2), and inoculating the mixed seed liquid into a fermentation medium for culturing.

19. The method for fermenting Blakeslea trispora as in claim 18, wherein the mass ratio of Blakeslea trispora positive seed liquid to Blakeslea trispora negative seed liquid is 1 (0.5-2).

20. The method for fermenting Blakeslea trispora as in claim 18, wherein the volume ratio of the mixed solution of Blakeslea trispora positive seed solution and Blakeslea trispora negative seed solution to the fermentation medium is 1 (8-10).

21. The method of claim 18, wherein the fermentation medium comprises glucose, corn starch, rapeseed meal, soybean meal, potassium dihydrogen phosphate, and magnesium sulfate.

22. The process for fermentation of b.trispora according to claim 21, wherein the concentration of rapeseed meal is 30-60 g/L.

23. The method of claim 18, wherein the fermentation medium comprises 15-25 g g/L glucose, 35-45 g g/L corn starch, 30-60 g g/L rapeseed meal, 35-45 g g/L soybean meal, 0.5-2 g g/L potassium dihydrogen phosphate, and 0.05-0.2 g g/L magnesium sulfate.

24. The process for fermentation of b.trispora according to claim 18, wherein the temperature of the fermentation culture is 20-30 ℃.

25. The method of fermentation of b.trispora according to claim 18, wherein the fermentation culture is performed for a period of time ranging from 100 to 150 h.

26. The process for fermentation of b.trispora according to claim 18, wherein the pH of the environment of the fermentation culture is between 6.5 and 7.5.

27. The process for fermentation of b.trispora according to claim 2, wherein the extractant used in the extraction in step (4) comprises ethyl acetate, butyl acetate or isobutyl acetate.