CN115786407A - Method for producing bisabolone by using fermentation inulin raffinate - Google Patents

Abstract

The invention belongs to the technical field of inulin fermentation and relates to a method for producing bisabolene from fermented inulin raffinate, which comprises the following steps: 1) pretreating the inulin raffinate for standby; 2) taking the pretreated Mix the inulin raffinate and water at a mass ratio of 1:20, calculate the inoculation amount based on 3% of the inulin raffinate dosage, first inoculate Saccharomyces cerevisiae, and ferment at 26°C to 30°C for 12h to 14h , and then continue to insert Lactobacillus, after 20h to 24h of fermentation, the fermented product containing β-bisabolene is obtained, Lactobacillus includes Lactobacillus plantarum and Lactobacillus reuteri, Saccharomyces cerevisiae: Lactobacillus plantarum: Milk of reuteri Bacillus = 1:1:1. According to the method provided by the invention, the content of β-bisabolene is high, and at the same time, the content of protein, soluble sugar and cellulose in the product is increased, and the nutritional level of the feed is improved.

Description

A method for producing bisabolene from fermented inulin extract

technical field

The invention belongs to the technical field of inulin fermentation and relates to a method for producing bisabolene from fermented inulin raffinate.

Background technique

Bisabolene is a colorless oily liquid with a molecular formula of C ₁₅ H ₂₄ . Bisabolene is a good antioxidant with anti-itch and anti-inflammatory functions. It is widely used in daily cosmetics and in health care. At the same time, it also has warm woody, citrus, floral, fruity, green, and sweet balsamic aromas. Bisabolene exists in many natural essential oils, and it is one of the main components of bisabolene. Commercial bisabolene is mostly a mixture of α-, β- and γ-isomers. Existing bisabolene can be obtained by dehydration of nerolidol. When farnesene is prepared, a large amount of bisabolene, a by-product, usually exists, but the content is low.

The remaining inulin raffinate after extracting inulin from Jerusalem artichoke is a by-product of the inulin industry, which is generally used as a common feed; however, the inulin raffinate is rich in protein, pectin, unextracted dietary fiber and a large number of available components. The use of feed can not give full play to the role of inulin raffinate, resulting in waste of resources and loss of economic benefits and ecological and environmental benefits. Therefore, finding a new way to realize the resource utilization of inulin raffinate is the concern of the industry focus.

Contents of the invention

The invention provides a method for producing bisabolene by using fermented inulin raffinate. The content of β-bisabolene is high, and at the same time, the content of protein, soluble sugar and cellulose in the fermentation product is increased, and the nutritional level of feed is improved. .

In order to achieve the above object, the technical scheme adopted in the present invention is:

A method for producing bisabolene by fermenting inulin raffinate, comprising the following steps:

1) pretreatment of inulin raffinate, set aside;

2) Take the pretreated inulin raffinate and mix it with water according to the mass ratio of 1:20, calculate the inoculation amount according to 3% of the amount of inulin raffinate, first insert Saccharomyces cerevisiae, at 26℃～30℃ After 12h-14h of fermentation under the condition, the lactobacillus is continued to be inserted, and after 20h-24h of fermentation, the fermented product containing β-bisabolene is obtained.

Further, the lactobacillus includes Lactobacillus plantarum and Lactobacillus reuteri.

Further, the Saccharomyces cerevisiae:Lactobacillus plantarum:Lactobacillus reuteri=1:1:1.

Further, in the step 1), the pretreatment is to dry the inulin raffinate; the drying temperature is 40°C-60°C, and the drying time is 6h-9h.

Further, in the fermented product of step 2), the relative content of β-bisabolene is 5.19%.

Application of a fermentation bacterium in the production of β-bisabolene from inulin raffinate.

Further, the fermenting bacteria include Saccharomyces cerevisiae and Lactobacillus, and the Lactobacillus includes Lactobacillus plantarum and Lactobacillus reuteri.

Further, the ratio of Saccharomyces cerevisiae, Lactobacillus plantarum and Lactobacillus reuteri is 1:1:1.

The beneficial effects of the present invention are:

1. The present invention utilizes fermentation bacteria to produce β-bisabolene from the inulin raffinate. The fermentation bacteria include Saccharomyces cerevisiae and Lactobacillus, and the Lactobacillus includes Lactobacillus plantarum and Lactobacillus reuteri. The role of inulin raffinate can save resources, realize economic benefits and ecological and environmental protection benefits, and provide a new way for resource utilization of inulin raffinate.

2. When the present invention is fermenting, first insert Saccharomyces cerevisiae for fermentation, then insert Lactobacillus for fermentation, the original purpose of inoculating bacteria in batches is to prevent Lactobacillus from producing a large amount of lactic acid in the fermentation process, thereby affecting the growth of Saccharomyces cerevisiae; Inoculation in batches can improve the fermentation effect of inulin raffinate, and the relative content of β-bisabolene is 5.19%, which is relatively high; at the same time, the content of protein, soluble sugar and cellulose in the fermentation products are all increased. Fermentation products are used as feed, and the nutritional level is improved.

Description of drawings

Fig. 1 is the state of the inulin raffinate under different fermentation schemes when fermenting 36h;

Figure 2 is the total number of colonies of the inulin raffinate at a dilution of 10 ⁴ ;

Fig. 3 is the measurement result of pH value under different fermentation time lengths of inulin extract;

Figure 4 shows the color change of inulin raffinate under different fermentation times.

Detailed ways

The present invention will be described in detail in conjunction with the accompanying drawings and embodiments.

The invention provides a fermentation bacterium which can be used to produce β-bisabolene from inulin raffinate.

Fermentation bacteria include Saccharomyces cerevisiae and Lactobacillus, and Lactobacillus includes Lactobacillus plantarum and Lactobacillus reuteri.

In the present invention, the ratio of Saccharomyces cerevisiae, Lactobacillus plantarum and Lactobacillus reuteri is 1:1:1.

The method for producing bisabolene by fermented inulin raffinate provided by the invention comprises the following steps:

1) pretreatment of inulin raffinate, set aside;

In step 1), the pretreatment is to dry the inulin raffinate; the drying temperature is 40°C-60°C, and the drying time is 6h-9h. Drying is carried out in a well-ventilated place, and the drying temperature should not be too high. High temperature will easily cause the inulin extraction residue to change color.

2) Take the pretreated inulin raffinate and mix it with water according to the mass ratio of 1:20, calculate the inoculation amount according to 3% of the amount of inulin raffinate, first insert Saccharomyces cerevisiae, at 26℃～30℃ After 12h-14h of fermentation under the condition, the lactobacillus is continued to be inserted, and after 20h-24h of fermentation, the fermented product containing β-bisabolene is obtained.

In the fermentation method provided by the present invention, the original purpose of batch inoculation is to prevent Lactobacillus from producing a large amount of lactic acid during the fermentation process, thereby affecting the growth of Saccharomyces cerevisiae, Saccharomyces cerevisiae and Lactobacillus can improve the effect of fermentation and increase the β in the fermentation product - Bisabolene content, the relative content of β-bisabolene can reach 5.19%.

The production process of β-bisabolene of the present invention is illustrated below with specific examples.

At the time of implementation, the bacteria involved were purchased from the market.

Unless otherwise specified, the related operations adopted belong to the conventional operation method in this field.

Example 1

The fermentation bacteria provided in this embodiment include Saccharomyces cerevisiae and Lactobacillus, and the Lactobacillus includes Lactobacillus plantarum and Lactobacillus reuteri.

In this embodiment, the ratio of Saccharomyces cerevisiae, Lactobacillus plantarum and Lactobacillus reuteri is 1:1:1.

The method for producing bisabolene by fermented inulin raffinate provided by the invention comprises the following steps:

1) pretreatment of inulin raffinate, set aside;

In step 1), the pretreatment is to dry the inulin raffinate; the drying temperature is 55° C., and the drying time is 6 hours.

2) Take the pretreated inulin raffinate and mix it with water according to the mass ratio of 1:20, calculate according to the amount of inulin raffinate, inoculate the amount of 3%, first inoculate Saccharomyces cerevisiae, under the condition of 28 ℃ After 12 hours of fermentation, the lactobacillus was further inserted, and after 24 hours of fermentation, a fermented product containing β-bisabolene was obtained.

Example 2

The fermentation bacteria provided in this embodiment include Saccharomyces cerevisiae and Lactobacillus, and the Lactobacillus includes Lactobacillus plantarum and Lactobacillus reuteri.

In this embodiment, the ratio of Saccharomyces cerevisiae, Lactobacillus plantarum and Lactobacillus reuteri is 1:1:1.

The method for producing bisabolene by fermented inulin raffinate provided by the invention comprises the following steps:

1) pretreatment of inulin raffinate, set aside;

In step 1), the pretreatment is to dry the inulin raffinate; the drying temperature is 40° C., and the drying time is 9 hours.

2) Take the pretreated inulin raffinate and mix it with water according to the mass ratio of 1:20, calculate according to the amount of inulin raffinate, inoculate the amount of 3%, first insert Saccharomyces cerevisiae, at 26°C After 14 hours of fermentation, the lactobacillus was continued to be inserted, and after 20 hours of fermentation, a fermented product containing β-bisabolene was obtained.

Example 3

The fermentation bacteria provided in this embodiment include Saccharomyces cerevisiae and Lactobacillus, and the Lactobacillus includes Lactobacillus plantarum and Lactobacillus reuteri.

In this embodiment, the ratio of Saccharomyces cerevisiae, Lactobacillus plantarum and Lactobacillus reuteri is 1:1:1.

The method for producing bisabolene by fermented inulin raffinate provided by the invention comprises the following steps:

1) pretreatment of inulin raffinate, set aside;

In step 1), the pretreatment is to dry the inulin raffinate; the drying temperature is 60° C., and the drying time is 6 hours.

2) Take the pretreated inulin raffinate and mix it with water according to the mass ratio of 1:20, calculate according to the amount of inulin raffinate, inoculate the amount of 3%, first insert Saccharomyces cerevisiae, at 30 ℃ After 12 hours of fermentation, the lactobacillus was further inserted, and after 24 hours of fermentation, a fermented product containing β-bisabolene was obtained.

Comparative example 1

In the comparative example, the fermentation bacteria include Saccharomyces cerevisiae and Lactobacillus, and the Lactobacillus includes Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus rhamnosus.

In the comparative example, the strain ratio is Saccharomyces cerevisiae:Lactobacillus plantarum:Lactobacillus reuteri:Lactobacillus rhamnosus:Lactobacillus rhamnosus=1:1:1:1:1.

The fermentation process in this comparative example is: uniformly mix the dried inulin raffinate with water at a mass ratio of 1:20, first insert Saccharomyces cerevisiae into it, and the inoculation amount is 3% of the inulin raffinate According to the calculation, after fermenting at 28° C. for 12 hours, adding lactobacillus, and fermenting at 28° C. for 24 hours, a fermentation product is obtained.

Comparative example 2

In this comparative example, the fermentation bacteria include Lactobacillus, and the Lactobacillus includes Lactobacillus plantarum and Lactobacillus reuteri.

In this comparative example, the strain ratio is Lactobacillus plantarum: Lactobacillus reuteri=1:1.

The fermentation process in this comparative example is: uniformly mix the dried inulin extract and water at a mass ratio of 1:20, and centrifuge and filter the seed liquid of Lactobacillus plantarum and Lactobacillus reuteri to obtain the bacterial liquid Stand-by, the amount of bacteria inoculated is calculated according to the amount of inulin raffinate 3%, Lactobacillus plantarum and Lactobacillus reuteri are inoculated, fermented at 28° C. for 36 hours, and then measured for various indexes.

Further, the production method of β-bisabolene of the present invention is verified through experiments.

test 1

Experiment with the fermentation process.

1. Sample

The fermentation product of Example 1, the fermentation product of Comparative Example 1 and the fermentation product of Comparative Example 2.

2. Test process:

Take the fermentation products of three samples respectively, and observe the state of the products, and the growth of the flora at the same time, the results are shown in Fig. 1, wherein, (a1) (b1) (c1) are respectively comparative example 1, embodiment 1 and comparative example 2 (a2) (b2) (c2) are the flora photos of Comparative Example 1, Example 1 and Comparative Example 2 respectively.

It can be seen from Figure 1 that the color of the inulin extraction residue after fermentation is yellowish, and there is little difference between different fermentation schemes; as can be seen in the photos of the flora, the amount of bacteria in Comparative Example 2 is more, indicating that the addition of yeast is more effective for lactic acid bacteria There will be a certain inhibitory effect on the growth of comparative example 1 and embodiment 1. In addition, the difference in the figure between comparative example 1 and embodiment 1 is not significant, indicating that the increase of bacterial species can not play a synergistic growth effect on the product, only under the effect of the bacteria in embodiment 1 , β-bisabolene is produced in the fermentation product of inulin raffinate.

test 2

1. Sample

The fermentation product of Example 1, the fermentation product of Comparative Example 1 and the fermentation product of Comparative Example 2.

2. Test process:

The fermentation products of three samples were respectively taken, diluted 104, and the total number of colonies was determined, and the results are shown in Figure 2.

It can be seen from Figure 2 that in the fermented feed, the yeast in sample 1 grows the most vigorously. It can be obtained that lactic acid bacteria will affect the growth and reproduction of yeast. The possible reason is that lactic acid bacteria produce lactic acid during the fermentation process, which reduces the pH of the environment. value, thereby inhibiting the growth of yeast. However, the addition of yeast will not affect the growth and reproduction of lactic acid bacteria, and may even promote it to a certain extent.

Test 3

During the fermentation process of Example 1, use a pH meter to measure the pH of the fermentation product at 12h, 24h, and 36h of fermentation, and the results are shown in Figure 3; use a colorimeter to measure at 12h, 24h, and 36h of fermentation, and the results are as follows Shown in Fig. 4, in Fig. 4, (A) is fermentation time and L ^* value change trend, (B) is fermentation time and a ^* value change trend; (C) is fermentation time and b ^* value change trend; (D ) is the variation trend of fermentation time and ΔE value.

It can be seen from Figure 3 that with the increase of fermentation time, the pH value of the fermentation product shows a downward trend, which may be caused by the growth of lactic acid bacteria and acid production.

It can be seen from Figure 4 that as the fermentation time increases, the L ^* value and ΔE value gradually decrease, the b ^* value increases, and the a ^* value first decreases and then increases. The possible reason is that yeast and lactic acid bacteria are consuming Material produced colored matter simultaneously, caused the color of inulin raffinate to change; From ΔE value value change, it can be seen that the color change of inulin raffinate in the fermentation process is still comparatively obvious, illustrates that in the present invention The fermentation speed of strain collocation is faster.

Test 4

The fermented product of Example 1, the fermented product of Comparative Example 1 and the fermented product of Comparative Example 2 were respectively taken, and the dry matter content thereof was measured, and the results are shown in Table 1.

Table 1 Dry matter content in the fermentation product of inulin raffinate

Dry matter content (unit %) Comparative example 1 0.047 Example 1 0.043 Comparative example 2 0.048

As can be seen from Table 1, the different fermentation results are not large for the dry matter content of the inulin extraction residue, but compared with the blank (Table 2), the content of proteins and other substances has increased, indicating that in the fermentation process, the Jerusalem artichoke extract Some of the dry matter in the residue is transformed after being utilized by bacteria, thereby increasing the content of certain nutrients.

Test 5

The fermentation product of Example 1, the fermentation product of Comparative Example 1 and the fermentation product of Comparative Example 2 were respectively taken, and the contents of lactic acid, protein, soluble sugar and cellulose were determined, and the results are shown in Table 2. At the same time, the unfermented inulin extract was used as the blank group for comparison.

(1) The determination of lactic acid is carried out according to the method of GB 1886.173-2016 "National Food Safety Standard Food Additive Lactic Acid".

(2) After the fermentation product is freeze-dried, the protein is determined using a fully automatic Kjeldahl nitrogen analyzer.

(3) After freeze-drying the fermented product, weigh the dry sample and bathe it in ethanol several times to obtain an extract for determining the content of soluble sugar.

(4) Determination of cellulose by anthrone sulfate method.

The specific measurement process is: after the fermentation product is freeze-dried, the dry sample is weighed, and the soluble sugar is removed in multiple water baths with ethanol, and then the starch is removed with low-concentration sodium hypochlorite, and then washed with NaOH solution, hot distilled water, and acetone. , to get rid of impurities such as protein, pectin, lipids, add 60% sulfuric acid to the precipitate after washing, stir evenly, place it in a refrigerator at 4°C for hydrolysis for more than 12 hours, and dilute to volume in a 25ml volumetric flask after repeated extraction. The cellulose content was determined by the anthrone sulfate method.

Table 2 Protein content in fermented products

Mass fraction of lactic acid content fiber content Soluble sugar content protein content Comparative example 1 0.013 0.0312 0.2905 0.1745 Example 1 0.010 0.0395 0.3550 0.1703 Comparative example 2 0.010 0.0259 0.3004 0.1701 blank 0.008 0.0145 0.7616 0.1158

It can be seen from Table 2 that as the fermentation proceeds, the lactic acid and protein contents in the inulin raffinate increase to varying degrees, and the soluble sugar content decreases, indicating that the inulin raffinate was consumed during the feed fermentation process. Part of the soluble sugar in the product, but the content of cellulose has no obvious change, indicating that the growth and reproduction of the bacteria does not use cellulose, and in the fermentation product of this example, the content of fiber, protein and soluble sugar is relatively high , When the fermentation product is used as feed, it can improve the nutritional level of the feed.

Test 6

The flavor compounds of fermentation products were determined by gas chromatography-mass spectrometry. Specifically, headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used for determination.

1. Sample:

The fermentation product of Example 1, the fermentation product of Comparative Example 1 and the fermentation product of Comparative Example 2; unfermented inulin raffinate.

2. The test process is:

Headspace solid-phase microextraction (SPME) method: Take 5mL of each sample and put it into a 15mL sample bottle, keep it on the heating platform at 50°C for 10min, insert the SPME extraction head into the bottle, and keep it about 1.5cm away from the sample liquid surface distance, the magnetic stirring speed was 100r/min, and the extraction was carried out at 50°C for 30min.

GC-MS parameter conditions: capillary column DB-WAX (60mx0.25mmx0.25um), inlet temperature 250°C, SPME inserted into the injection hole for desorption for 3min; initial temperature 40°C, hold for 2min, at a rate of 5°C/min Raise the temperature to 80°C, then raise the temperature to 180°C at 8°C/min, then rise to 240°C at 15°C/min, and keep for 8 minutes; the flow rate of carrier gas (He) is 1.0mL/min, splitless. The mass spectrometer interface temperature is 250°C, the ion source temperature is 200°C, the electron energy is 70eV, and the mass scanning range is 33-450amu.

Use the HP Chemworkstation software to analyze the data against the NIST library, make a preliminary identification through the library, and then conduct qualitative analysis in combination with the mass spectrum, retention time, and retention index of the chemical components, and use the area regression method for relative quantitative analysis. The results are shown in the table 3 shown.

Volatile matter content in the fermentation product of table 3

The results in Table 3 show that Example 1 produced β-bisabolene during the fermentation process, and this substance is generally a sesquiterpene isolated from Paeoniae officinalis, which is an antitumor agent and can be used in the research of breast cancer , but in the blank sample (unfermented inulin raffinate), the fermentation products of Comparative Example 1 and Comparative Example 2, no β-bisabolene was detected.

In addition, butyric acid that can be used as a feed preservative and electrolyte balance agent is also produced in Example 1, and the nutritional level of the fermentation product as feed is improved.

Experiment 7 sensory evaluation

The fermentation products of Example 1, the fermentation products of Comparative Example 1 and the fermentation products of Comparative Example 2 were all evaluated according to the standards in Table 4 for the sensory properties of the fermentation products, and the evaluation results are shown in Table 5.

Table 4 Odor, color, texture scoring basis (DLG)

Table 5 Sensory evaluation results of fermentation products

As can be seen from Table 5, the sensory evaluation of the product after fermentation in this embodiment is good.

Claims (8)

1. A method for producing bisabolene by using fermentation inulin raffinate is characterized by comprising the following steps:

1) Pretreating inulin extract residues for later use;

2) Taking the inulin raffinate after pretreatment and water according to the ratio of 1:20, calculating the bacterial inoculation amount according to 3 percent of the inulin raffinate consumption, firstly inoculating saccharomyces cerevisiae, fermenting for 12-14 h at 26-30 ℃, then continuously inoculating lactobacillus, and fermenting for 20-24 h to obtain the fermentation product containing beta-bisabolene.

2. The method of claim 1, wherein the lactobacillus comprises lactobacillus plantarum and lactobacillus reuteri.

3. The method of claim 2, wherein the saccharomyces cerevisiae: the lactobacillus plantarum: lactobacillus reuteri =1:1:1.

4. the method for producing bisabolone using fermented inulin raffinate according to claim 3, wherein in the step 1), the pre-treatment is drying the inulin raffinate; the drying temperature is 40-60 ℃, and the drying time is 6-9 h.

5. The method for producing bisabolene using fermented inulin raffinate according to claim 4, wherein the relative content of β -bisabolene in the fermented product of step 2) is 5.19%.

6. An application of zymophyte in the production of beta-bisabolene from inulin raffinate.

7. The use of claim 6, wherein the fermentation bacteria comprise Saccharomyces cerevisiae and Lactobacillus comprising Lactobacillus plantarum and Lactobacillus reuteri.

8. The use according to claim 7, wherein the ratio of Saccharomyces cerevisiae, lactobacillus plantarum and Lactobacillus reuteri is 1:1:1.

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