CN115595332A - Method for preparing tremella fermentation liquor by combining compound enzyme with microbial fermentation - Google Patents

Method for preparing tremella fermentation liquor by combining compound enzyme with microbial fermentation Download PDF

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CN115595332A
CN115595332A CN202211368709.8A CN202211368709A CN115595332A CN 115595332 A CN115595332 A CN 115595332A CN 202211368709 A CN202211368709 A CN 202211368709A CN 115595332 A CN115595332 A CN 115595332A
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tremella
mass
fermentation
polysaccharide
performing enzymolysis
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周婵
黄燕
周颢
王斐嘉
陆文莹
陆跃乐
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Hangzhou Jiajiale Biotechnology Co ltd
Guangdong Zhiya Shijia Biotechnology Co ltd
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Abstract

The invention provides a method for preparing tremella fermentation liquor by combining complex enzyme with microbial fermentation, and belongs to the technical field of biological fermentation. The method comprises the following steps: (1) Pulverizing dried Tremella into powder, adding into purified water, and adjusting pH to 4-5 with citric acid; (2) Adding pectinase, and performing enzymolysis at 50-60 deg.C for 1-3 hr; (3) Continuously adding acidic cellulase, and performing enzymolysis at 50-60 deg.C for 1-3 hr; (4) Continuously adding neutral protease, and performing enzymolysis at 50-60 deg.C for 1-3 hr; (5) Continuously adding the composite bacteria, and fermenting at 25-40 deg.C for 130-150 hr. The tremella fermentation liquid prepared by the method has low viscosity, high polysaccharide concentration, excellent yield and low molecular weight of the polysaccharide, and is convenient for organisms to absorb and utilize.

Description

Method for preparing tremella fermentation liquor by combining compound enzyme with microbial fermentation
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a method for preparing tremella fermentation liquor by combining complex enzyme with microbial fermentation.
Background
Tremella fuciformis is taken as the traditional edible fungus in China and is a food deeply loved by the masses of people all the time. The tremella contains rich nutrients, protein, fat, amino acids and minerals, and has special health function.
The protein accounts for 6-10% of the weight of the dry tremella, the tremella contains 17 amino acids, namely valine, proline, serine, arginine, glycine, lysine, alanine, threonine, leucine, isoleucine, tyrosine, phenylalanine, glutamic acid, cystine, aspartic acid, methionine, histidine and the like, wherein the content of the glutamic acid is the highest, the content of the aspartic acid is the next, 7 amino acids of 8 amino acids necessary for a human body can be provided by the tremella, and the tremella is a good protein source.
Tremella contains about 0.6% -1.28% of fat, wherein unsaturated fatty acid accounts for about 75% of total fatty acid, and linoleic acid is main ingredient. Tremella contains 4.0% -5.44% of inorganic salts, such as S, P, ca, fe, mg and Na. Tremella contains multiple vitamins, such as VB1, VB2, VC and VD, and also contains a certain amount of cellulose.
The tremella fermentation liquor contains organic compounds such as protein, amino acid and the like, wherein the content of tremella polysaccharide is outstanding, so the extraction of tremella functional components is usually based on indexes such as extraction rate, concentration, molecular weight and the like of tremella polysaccharide.
In the current common methods for extracting the tremella polysaccharide, the hot water method has long extraction time and low yield, the acid and alkali extraction method is easy to destroy the three-dimensional optical active structure of the tremella polysaccharide, and the enzyme separation is a research trend. However, the enzyme separation method has some defects which are difficult to overcome, for example, in the research on extracting tremella polysaccharide by enzyme method (Linyuye, etc., food and fermentation industry, no.1 in 1995), the yield of tremella polysaccharide is only 16.3%, and the molecular weight of the polysaccharide is as large as 71000-78000, which is not favorable for absorption and utilization; in the preparation process and research progress of tremella polysaccharide (Liu Ji et al, biological industry technology, 9 months in 2019), it is mentioned that the yield of tremella polysaccharide in several researches is 19.08-40.57%, and the production efficiency is low; in the exploration and analysis of the optimal extraction process conditions of the tremella polysaccharide (Sun Chenyu, agricultural product processing, 4 th 4 months in 2018), the mass concentration of the tremella polysaccharide is only 0.191mg/ml at most; although the concentration of tremella in the research on submerged fermentation conditions of tremella (Wu Da kang et al, food science, 2002, vol.23, no. 1) reaches 5.5mg/ml, a large amount of thickening agent and culture medium are required to be added, the preparation method is complex, and the production cost is high.
In view of the above, it is urgently needed to develop a tremella fermentation broth extraction method with simple process, good yield, high concentration and low cost.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for preparing tremella fermentation liquor by combining complex enzyme with microorganism fermentation. The tremella fuciformis fermentation liquor prepared by the method is low in viscosity, high in polysaccharide concentration, excellent in yield, low in molecular weight of polysaccharide and convenient for absorption and utilization of organisms.
The technical scheme of the invention is as follows:
a method for preparing tremella fermentation liquor by combining compound enzyme with microbial fermentation comprises the following steps:
(1) Pulverizing dried Tremella into powder, adding into purified water, and adjusting pH to 4-5 with citric acid;
(2) Adding pectinase, and performing enzymolysis at 50-60 deg.C for 1-3 hr;
(3) Continuously adding acidic cellulase, and performing enzymolysis at 50-60 deg.C for 1-3 hr;
(4) Continuously adding neutral protease, and performing enzymolysis at 50-60 deg.C for 1-3 hr;
(5) Continuously adding the compound bacteria, and fermenting at 25-40 deg.C for 130-150 hr.
Preferably, the water content of the dried tremella in the step (1) is less than 15%; pulverizing dried Tremella into powder, and sieving with at least 150 mesh sieve.
Preferably, the mass volume ratio of the dried tremella and the purified water in the step (1) is 1g:40-60ml.
Preferably, the mass of the pectinase in the step (2) is 0.08-0.12% of the mass of the dried tremella.
Preferably, the mass of the acid cellulose in the step (3) is 0.08-0.12% of the mass of the dry tremella.
Preferably, the mass of the neutral protease in the step (4) is 0.08-0.12% of the mass of dry tremella.
Preferably, the composite bacteria in the step (5) are mixed strains of lactobacillus plantarum, lactobacillus delbrueckii subsp bulgaricus and streptococcus thermophilus, and the mass ratio of the lactobacillus plantarum to the lactobacillus delbrueckii subsp bulgaricus to the streptococcus thermophilus is 1:1.8-2.2:1.8-2.2.
Preferably, the mass of the compound bacteria in the step (5) is 1-1.5% of the mass of the dry tremella.
The beneficial technical effects of the invention are as follows:
1. the invention is different from the prior art that several enzymes are added at one time, but three enzymes are added step by step. Through a large number of experiments of the inventor, the pectinase acts on pectin on the cell wall of the tremella to enable the cell to be broken more easily to release intracellular polysaccharide; further cellulase action and cell wall action, so that macromolecular polysaccharide on the cell wall is changed into micromolecular polysaccharide; finally, the protease changes the protein in the cell into small molecular polysaccharide and even amino acid.
2. The viscosity of the tremella fermentation liquid obtained by the invention is 25.5 mPas at the lowest, and the fermentation liquid with reduced viscosity is convenient for subsequent feed liquid separation in the industrial aspect; the obtained polysaccharide has a concentration of 4.57mg/ml at most, and the polysaccharide has a molecular weight less than 10000 and accounts for more than 70%, so that the polysaccharide is convenient for absorption and utilization by organisms; the yield of the invention is up to 76.17 percent, which is 1.5 to 2 times of the prior art.
3. After the complex agent is fermented, the complex agent can be directly utilized only by filtering and deslagging. The tremella fermentation liquor contains not only tremella polysaccharide, but also various amino acids, minerals and vitamins, has wide application, is edible, and can also be used as cosmetic raw materials and the like.
Drawings
FIG. 1 is a graph showing the molecular weight distribution of polysaccharides in a fermentation broth prepared in example 1;
FIG. 2 is a graph showing the viscosity and polysaccharide content of the fermentation broth obtained in example 2 at different fermentation temperatures;
FIG. 3 is a graph showing the viscosity and polysaccharide content of fermentation broth obtained in example 3 at different feed-to-liquid ratios;
FIG. 4 is a graph showing the viscosity and polysaccharide content of the fermentation broth obtained in example 4 at different fermentation times.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the pectinase is food grade pectinase, activity of which is 6 ten thousand U/mL, prepared by Shandong-Long-Kete enzyme preparation GmbH; the acidic cellulase is food-grade acidic cellulase prepared by Shandong Kete enzyme preparation GmbH, and the activity is 20 ten thousand U/mL; the neutral protease is food-grade neutral protease with activity of 5 ten thousand U/mL, prepared from Shandong Kete enzyme preparation GmbH.
In the used composite strains, the lactobacillus plantarum adopts Harvest LB-1 of Kehansen company, and the product number is 3580179; the mixed strain of the Lactobacillus delbrueckii subspecies bulgaricus and the Streptococcus thermophilus, namely a mixed strain of DuPont nutritional food ingredient (Beijing) Limited company is named as lactic acid strain TS-H2084LYO 200DCU with the product number of 61033903, and the mass ratio of the Lactobacillus delbrueckii subspecies bulgaricus and the Streptococcus thermophilus in the mixed strain is 1:1.
example 1:
taking 40g of dried tremella powder, adding 2000mL of purified water, mixing, adding a proper amount of citric acid, and adjusting the pH value to 4.5; adding 0.1% of pectinase, and performing enzymolysis at 55 deg.C for 2h; 0.1% of acid cellulase, and carrying out enzymolysis for 2h at 55 ℃; neutral protease 0.1%, enzymolysis at 55 deg.C for 2h; after the enzymolysis is finished, the enzymolysis liquid is taken to detect the polysaccharide content, the polysaccharide molecular weight and the viscosity, and the detection result is shown in the following table 1. Wherein the water content of the used dried tremella is less than 15%, and the tremella is ground into powder and then sieved by a 150-mesh sieve.
Adding 0.5g of composite bacteria into the obtained enzymolysis liquid, and fermenting at 30 ℃ for 144h. After fermentation, the fermentation broth is taken to detect the polysaccharide content, the polysaccharide molecular weight and the viscosity, and the detection results are shown in the following table 1.
The molecular weight distribution of the polysaccharide in the fermentation broth is shown in FIG. 1, from which it can be seen that the molecular weight of the product becomes smaller, and the molecular weight distribution is below 1 ten thousand, mainly at 9000.
Comparative example 1:
0.5g of Lactobacillus plantarum was added to the enzymatic hydrolysate of example 1, and fermented at 30 ℃ for 144 hours. After fermentation, the fermentation broth is taken to detect the polysaccharide content, the polysaccharide molecular weight and the viscosity, and the detection results are shown in the following table 1. The strain of the lactobacillus plantarum is obtained from the national strain collection and management center, and the preservation number is as follows: CGMCC1.12732.
TABLE 1
Figure BDA0003924144160000051
Example 2:
0.5g of complex bacteria was added to the enzymatic hydrolysate in example 1, and then fermented at 10 ℃,20 ℃, 30 ℃, 40 ℃ and 50 ℃ for 144 hours, respectively, and the experimental results are shown in FIG. 2. As can be seen from FIG. 2, the polysaccharide content reached the highest value at a fermentation temperature of 30 ℃ and the solution viscosity was the lowest at this point.
Example 3:
the fermentation material liquid ratio (namely, the mass volume ratio of the tremella to the water) in example 1 is respectively set as 1: 30. 1: 40. 1: 50. 1: 60. 1:70, the pH value is 4.5, 0.5g of composite bacteria is added after the enzymolysis of the composite enzyme preparation, the mixture is fermented for 144 hours at the temperature of 30 ℃, and the experimental result is shown in figure 3. As can be seen from fig. 3, at a fermentation broth to liquid ratio of 1.
Example 4:
0.5g of complex bacteria was added to the enzymatic hydrolysate in example 1, and the mixture was fermented at 30 ℃ for 96h, 120h, 144h, 168h, and 192h, respectively, and the experimental results are shown in FIG. 4. As can be seen from FIG. 4, the polysaccharide content was highest at 144h of fermentation time, and the viscosity did not change much overall.
While the embodiments of the invention have been described in detail, it is not intended to limit the invention to the exact construction and operation illustrated and described, and it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention.

Claims (8)

1. A method for preparing tremella fermentation liquor by combining compound enzyme with microbial fermentation is characterized by comprising the following steps:
(1) Pulverizing dried Tremella into powder, adding into purified water, and adjusting pH to 4-5 with citric acid;
(2) Adding pectinase, and performing enzymolysis at 50-60 deg.C for 1-3 hr;
(3) Continuously adding acidic cellulase, and performing enzymolysis at 50-60 deg.C for 1-3 hr;
(4) Continuously adding neutral protease, and performing enzymolysis at 50-60 deg.C for 1-3 hr;
(5) Continuously adding the composite bacteria, and fermenting at 25-40 deg.C for 130-150 hr.
2. The method according to claim 1, wherein the water content of the dried tremella of step (1) is less than 15%; pulverizing dried Tremella into powder, and sieving with at least 150 mesh sieve.
3. The method according to claim 1, wherein the mass-to-volume ratio of the dried tremella and the purified water in step (1) is 1g:40-60ml.
4. The method according to claim 1, wherein the mass of the pectinase in the step (2) is 0.08-0.12% of the mass of the dry tremella.
5. The method according to claim 1, wherein the mass of the acidic cellulose in the step (3) is 0.08-0.12% of the mass of dry tremella.
6. The method according to claim 1, wherein the mass of the neutral protease in the step (4) is 0.08-0.12% of the mass of dry tremella.
7. The method according to claim 1, wherein the complex bacteria in step (5) are mixed bacteria of Lactobacillus plantarum, lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus in a mass ratio of 1:1.8-2.2:1.8-2.2.
8. The method according to claim 1, wherein the mass of the complex bacteria in the step (5) is 1-1.5% of the mass of the dry tremella.
CN202211368709.8A 2022-07-28 2022-11-03 Method for preparing tremella fermentation liquor by combining compound enzyme with microbial fermentation Pending CN115595332A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116268382A (en) * 2023-05-20 2023-06-23 广东泰升药业有限公司 Tremella Chen Pihua glue and preparation method thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103356716A (en) * 2013-07-18 2013-10-23 吴炳新 Fermented composition of multiple probiotics and composite medicinal and edible fungi, and preparation method and application thereof
CN104248646A (en) * 2013-06-27 2014-12-31 瑞普（天津）生物药业有限公司 Preparation method of fungus sporophore fermented by lactic acid bacteria
CN104739896A (en) * 2015-03-06 2015-07-01 吉林省现代中药工程研究中心有限公司 Composite fungal intercellular polysaccharide composition with liver protecting function and preparation method
CN107509901A (en) * 2017-08-16 2017-12-26 大连工业大学 The method for preparing flavor edible mushroom juice using leavening is compounded
CN112941128A (en) * 2021-02-24 2021-06-11 青岛农业大学 Method for extracting tremella polysaccharide from tremella

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104248646A (en) * 2013-06-27 2014-12-31 瑞普（天津）生物药业有限公司 Preparation method of fungus sporophore fermented by lactic acid bacteria
CN103356716A (en) * 2013-07-18 2013-10-23 吴炳新 Fermented composition of multiple probiotics and composite medicinal and edible fungi, and preparation method and application thereof
CN104739896A (en) * 2015-03-06 2015-07-01 吉林省现代中药工程研究中心有限公司 Composite fungal intercellular polysaccharide composition with liver protecting function and preparation method
CN107509901A (en) * 2017-08-16 2017-12-26 大连工业大学 The method for preparing flavor edible mushroom juice using leavening is compounded
CN112941128A (en) * 2021-02-24 2021-06-11 青岛农业大学 Method for extracting tremella polysaccharide from tremella

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116268382A (en) * 2023-05-20 2023-06-23 广东泰升药业有限公司 Tremella Chen Pihua glue and preparation method thereof

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