CN115702909A

CN115702909A - Sargassum fusiforme fermentation mixture and preparation method and application thereof

Info

Publication number: CN115702909A
Application number: CN202110935105.6A
Authority: CN
Inventors: 崔永日; 刘佳; 李洪福; 王雷
Original assignee: Qingdao Kangmaichen Biotechnology Co ltd
Current assignee: Qingdao Kangmaichen Biotechnology Co ltd
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2023-02-17
Anticipated expiration: 2041-08-16
Also published as: CN115702909B

Abstract

The invention relates to the technical field of fermentation, and particularly relates to a sargassum fusiforme fermentation mixture and a preparation method and application thereof. The preparation method comprises the following steps: decocting Cyrtymenia Sparsa in water to remove arsenic, and removing arsenic with citric acid; washing the arsenic-removed sargassum fusiforme to neutral pH, mixing with water, pulping, and adjusting the pH value to 6.0-7.0; mixing the obtained Cyrtymenia Sparsa serous fluid with glucose, and sterilizing; inoculating probiotics into the sargassum fusiforme slurry for fermentation, wherein the probiotics are lactobacillus plantarum and/or lactobacillus rhamnosus; centrifuging the fermentation product, and collecting supernatant to obtain Sargassum fusiforme fermentation mixture. The fermentation time of the sargassum fusiforme fermentation mixture is 24-72 h, so that the fermentation time is greatly shortened, and the efficiency is improved; the sargassum fusiforme fermentation mixture has remarkable anti-oxidation and anti-inflammatory effects; the sargassum fusiforme fermentation mixture has an extremely obvious growth inhibition effect on liver cancer cell HEPG2, stomach cancer cell SGC7901 and intestinal cancer cell LOVO.

Description

Sargassum fusiforme fermentation mixture and preparation method and application thereof

Technical Field

The invention relates to the technical field of fermentation, and particularly relates to a sargassum fusiforme fermentation mixture and a preparation method and application thereof.

Background

Hizikia fusiforme (Hizikia fusifarme) is a precious marine algae plant, and is described in books such as compendium of materia Medica and used as a medicine. Along with the improvement of the living standard of people, the sargassum fusiforme is gradually known by people as a new health-care food, the sargassum fusiforme food is consistent with the trend of modern people advocating nature and the trend of people returning dietary structures to plant type foods, has good development prospect and wide market, and the edible and medicinal values of the sargassum fusiforme are more and more valued by people. The sargassum fusiforme is rich in polysaccharide, dietary fiber, vitamins and mineral substances; contains 14 important trace elements required by human body, and the lead content is far lower than that of other seaweeds; the protein mass fraction is 9.63% -15.09%, and the protein mass fraction contains 17 important amino acids (including 8 essential amino acids), and the mass fraction is 0.19% -1.81%. The research shows that Ca, K, mg, fe, zn, I, amino acid, vitamin and the like in the mineral substances of the sargassum fusiforme can promote the growth and development of the tested animals to different degrees.

The sargassum fusiforme contains various functional components and has various active functions, metabolites and secondary metabolites of the lactobacillus can be generated by utilizing the fermentation of the lactobacillus, and the active function of the sargassum fusiforme is strengthened, so that a mixture with better efficacy is obtained, and a foundation is laid for further development of functional food raw materials.

The patent of application No. 201710856642.5 discloses a primary processing method of sargassum fusiforme, which comprises the steps of adding lactic acid bacteria and saccharomycete powder for fermentation, wherein the comprehensive fermentation time is 30min, the relative fermentation time is short, the fermentation is insufficient, and the purpose is to remove the fishy smell of the sargassum fusiforme. The patent of application number 201810256204.X discloses a lactobacillus plantarum and application thereof in deodorization of sargassum fusiforme, wherein the fermentation time is 10-30 days, and the fermentation time is long.

Disclosure of Invention

In view of this, the invention provides a sargassum fusiforme fermented mixture, a preparation method and an application thereof. The sargassum fusiforme fermentation mixture has short fermentation time and has remarkable antioxidant, anti-inflammatory and anticancer effects.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of a sargassum fusiforme fermentation mixture, which comprises the following steps:

step (1), boiling sargassum fusiforme with water to remove arsenic;

step (2) carrying out citric acid dearsenification on the sargassum fusiforme obtained in the step (1);

washing the arsenic-removed sargassum fusiforme to neutral pH, mixing with water and pulping;

step (4), adjusting the pH value of the sargassum fusiforme slurry to 6.0-7.0;

step (5), mixing the sargassum fusiforme serous fluid obtained in the step (4) with glucose, and sterilizing;

step (6) inoculating probiotics into the sargassum fusiforme slurry obtained in the step (5) for fermentation, wherein the probiotics are lactobacillus plantarum and/or lactobacillus rhamnosus;

and (7) centrifuging the fermentation product, and taking supernatant to obtain a sargassum fusiforme fermentation mixture.

Preferably, in the step (1), the water adding amount for water boiling dearsenification is 5 to 30 times of the weight of the dry vegetables, the temperature for water boiling dearsenification is 50 to 100 ℃, and the time for water boiling dearsenification is 10 to 30min.

Preferably, in the step (1), the water adding amount of the water boiling dearsenification is 15 times of the weight of the dry vegetables, the temperature of the water boiling dearsenification is 90 ℃, and the time of the water boiling dearsenification is 15min.

Preferably, in the step (2), the water adding amount of the citric acid for dearsenification is 5 to 30 times of the weight of the dried vegetables, the mass percentage concentration of the citric acid is 0.2 to 0.8 percent, the temperature of the citric acid for dearsenification is 50 to 100 ℃, and the time of the citric acid for dearsenification is 1 to 5 hours.

Preferably, the water adding amount of the citric acid for dearsenification is 20 times of the weight of the dried vegetables, the mass percentage concentration of the citric acid is 0.4 percent, the temperature of the citric acid for dearsenification is 60 ℃, and the time of the citric acid for dearsenification is 130min.

Preferably, in the step (3), the mass ratio of the dearsenified sargassum fusiforme to water is 1: (3-5).

Preferably, in the step (3), the mass ratio of the arsenic-removed sargassum fusiforme to the water is 1:4.

preferably, the step (4) adjusts the pH of the sargassum fusiforme slurry to 6.5.

Preferably, in the step (5), the mass volume percentage of the glucose and the sargassum fusiforme serous fluid is 1-3%.

Preferably, in the step (5), the mass volume percentage of the glucose and the sargassum fusiforme serous fluid is 2%.

Preferably, the temperature for sterilization is 110 to 118 ℃ and the time is 8 to 30min.

Preferably, in the step (6), the inoculation amount of the lactobacillus plantarum or lactobacillus rhamnosus is 0.4-10%.

Preferably, in the step (6), the inoculation amount of the lactobacillus plantarum or lactobacillus rhamnosus is 4-6%.

In the specific embodiment provided by the invention, in the step (6), the inoculation amount of the lactobacillus plantarum or lactobacillus rhamnosus is 5%.

Preferably, in the step (6), the fermentation temperature is 20-40 ℃, aeration is carried out, and the fermentation time is 24-72 h.

Preferably, in the step (6), the fermentation temperature is 37 ℃, aeration is carried out, and the fermentation time is 48h.

Preferably, in step (7), the rotation speed of the centrifugation is 3000 to 5000rm for 1 to 5min.

Preferably, in step (7), the rotation speed of the centrifugation is 4000rm, and the time is 3min.

The invention also provides a sargassum fusiforme fermentation mixture prepared by the preparation method.

The invention also provides application of the sargassum fusiforme fermentation mixture in preparing products with antioxidant, anti-inflammatory and anticancer effects.

Preferably, the anticancer effect is any one or more of anti-liver cancer, anti-stomach cancer and anti-intestinal cancer.

The invention provides a sargassum fusiforme fermentation mixture and a preparation method and application thereof. The preparation method comprises the following steps: decocting Cyrtymenia Sparsa in water to remove arsenic, and removing arsenic with citric acid; washing the arsenic-removed sargassum fusiforme to neutral pH, mixing with water, pulping, and adjusting the pH value to 6.0-7.0; mixing the obtained Cyrtymenia Sparsa serous fluid with glucose, and sterilizing; inoculating probiotics into the sargassum fusiforme slurry for fermentation, wherein the probiotics are lactobacillus plantarum and/or lactobacillus rhamnosus; centrifuging the fermentation product, and collecting supernatant to obtain Sargassum fusiforme fermentation mixture. The invention has the technical effects that:

the fermentation time of the sargassum fusiforme fermentation mixture is 24-72 h, so that the fermentation time is greatly shortened, and the efficiency is improved;

the sargassum fusiforme fermentation mixture has remarkable anti-oxidation and anti-inflammatory effects;

the sargassum fusiforme fermentation mixture prepared by fermenting lactobacillus rhamnosus has very obvious growth inhibition effect on liver cancer cell HEPG 2;

the sargassum fusiforme fermentation mixture prepared by combined fermentation of lactobacillus plantarum and lactobacillus rhamnosus has an extremely obvious growth inhibition effect on intestinal cancer cells LOVO.

Drawings

FIG. 1 shows the determination of DPPH radical scavenging activity;

FIG. 2 shows the determination of hydrogen peroxide scavenging activity;

FIG. 3 shows toxicity assay of Cyrtymenia Sparsa fermentation supernatant on Vero cells;

FIG. 4 shows the pair of supernatants from the fermentation of Hizikia fusiforme H ₂ O ₂ Measurement of induced protection;

FIG. 5 shows the pair of fermentation supernatants of Hizikia fusiforme H ₂ O ₂ Measurement of protective effects of induced cytotoxicity;

FIG. 6 shows the measurement of anti-inflammatory action of mouse macrophage by sargassum fusiforme fermentation supernatant;

FIG. 7 shows the measurement of mouse macrophage cell activity by sargassum fusiforme fermentation supernatant;

FIG. 8 shows the anticancer experiment (liver cancer cell HEPG 2) of sargassum fusiforme fermentation product;

FIG. 9 shows an anticancer experiment (lung cancer cell A549) using fermentation product of Hizikia fusiforme;

FIG. 10 shows the anticancer experiment (breast cancer cells MCF 7) of the fermentation product of Hizikia fusiforme;

FIG. 11 shows Hizikia fusiforme fermentation product anticancer experiment (gastric cancer cell SGC 7901);

FIG. 12 shows anticancer experiment (intestinal cancer cell LOVO) of fermentation product of Hizikia fusiforme.

Detailed Description

The invention discloses a sargassum fusiforme fermentation mixture and a preparation method and application thereof, and a person skilled in the art can realize the fermentation by properly improving process parameters by referring to the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The sargassum fusiforme fermentation mixture, the preparation method and the raw materials or auxiliary materials used in the application can be purchased from the market.

The invention is further illustrated by the following examples:

example 1

The preparation method of the fermented sargassum fusiforme comprises the following steps:

(1) water boiling for dearsenification: the water amount is 15 times of the weight of the dried vegetable, and the temperature is 90 ℃ and 15min.

(2) Removing arsenic from citric acid: adding water 20 times of the dried vegetable, citric acid 0.4%,60 deg.C, and 130min.

(3) Pulping: washing the boiled sargassum fusiforme subjected to arsenic removal by citric acid with clear water until the pH is neutral, draining, and mixing the sargassum fusiforme with the water according to the mass ratio: water =1:4 beating is carried out.

(4) Adjusting the pH value: adjusting pH of the Hizikia fusiforme slurry to 6.5% with 20% KOH solution.

(5) Adding 2% glucose (W/V) to the processed Cyrtymenia Sparsa slurry.

(6) And (3) sterilization: 115 ℃ for 30min.

(7) The strain is preserved in a freeze-drying tube or a glycerol tube, activation is needed before inoculation, and a culture medium suitable for strain growth is adopted as an activation culture medium. The inoculation strain comprises 1 or two of lactobacillus plantarum and lactobacillus rhamnosus. The inoculum size was 5% for each strain.

(8) And (3) fermentation control: the temperature is 37 ℃, compressed air is used as aeration measure, and fermentation is carried out for 48h.

(9) Centrifuging at 4000r/min for 3min after fermentation is finished, and taking the supernatant to obtain the fermented sargassum fusiforme supernatant.

Test example 1 verification of arsenic removal Effect

1. Experimental materials and experimental methods:

1 test materials:

dried sargassum fusiforme: cultivated in the county of the Wenzhou city of Zhejiang province, royle of November, caught and dried

Citric acid (food grade): weifang Yingxuan industry Co Ltd

2, experimental equipment:

a precision blast drying oven;

an atomic fluorescence spectrometer;

an atomic absorption spectrophotometer;

a water bath constant temperature oscillator;

a high speed drug pulverizer;

an electronic analytical balance.

3, experimental method:

3.1 sample treatment of Cyrtymenia Sparsa:

cyrtymenia Sparsa is processed by the following 5 methods to obtain No. 1-5 samples.

(1) Sample No.:

the Cyrtymenia Sparsa is not treated and pulverized.

(2) Sample No.:

weighing Cyrtymenia Sparsa raw material, adding 15 times of water, stirring at 90 deg.C for 15min, draining, oven drying at 105 deg.C for 5 hr, and grinding into powder.

(3) Sample No.:

the first step is as follows: weighing sargassum fusiforme raw materials, adding water with the weight 15 times that of the sargassum fusiforme raw materials, keeping the temperature at 90 ℃, stirring for 15min, draining, collecting sargassum fusiforme for further processing;

the second step is that: adding 20 times of water, adding citric acid to 0.6% (W/V), stirring at 50 deg.C for 2 hr, washing with clear water until pH is neutral, and draining. Oven drying at 105 deg.C for 5 hr, and grinding into powder.

(4) Sample No.:

weighing Cyrtymenia Sparsa raw material, adding 40 times of water, adding citric acid to 0.6% (W/V), stirring at 50 deg.C for 2 hr, draining, oven drying at 105 deg.C for 5 hr, and grinding into powder.

(5) Sample No.:

the first step is as follows: weighing raw materials of sargassum fusiforme, adding water with the weight being 40 times of the raw materials, adding citric acid to 0.6% (W/V), keeping the temperature at 50 ℃, stirring for 2 hours, draining, collecting sargassum fusiforme, and further processing;

the second step is that: adding 20 times of water, adding citric acid to 0.6% (W/V), stirring at 50 deg.C for 2 hr, washing with clear water until pH is neutral, draining, oven drying at 105 deg.C for 5 hr, and grinding.

3.2 Total arsenic determination method:

measured according to the method specified in GB 5009.11: hydride generation atomic fluorescence spectrometry (wet digestion).

2. Results of the experiment

The 5 samples were measured for total arsenic by the national standard method, and the experimental results are shown in the following table:

TABLE 1

Numbering	Arsenic (mg/kg)	Residual rate
			1	92.34	100％
2	37.16	40％
			3	14.02	15％
4	29.90	32％
			5	21.16	23％

* Based on the dry weight of Cyrtymenia Sparsa (water is subtracted)

According to researches, the total arsenic content of the untreated sargassum fusiforme raw material is 92.36mg/kg, the total arsenic content is 37.16mg/kg after the one-step water boiling method is carried out, and the total arsenic removal rate is 60 percent; after the treatment of the citric acid aqueous solution, the total arsenic content is reduced to 14.02mg/kg, and the total arsenic removal rate reaches 85 percent.

After the raw material of the sargassum fusiforme is treated by the citric acid solution in one step, the total arsenic content is 29.90mg/kg, and the total arsenic removal rate is 68 percent; after the second citric acid solution treatment, the total arsenic content is 21.16mg/kg, and the total arsenic removal rate is 77%.

The research result shows that: the water treatment in the first step and the citric acid solution treatment in the second step have the best effect of removing the total arsenic in the sargassum fusiforme, the arsenic removal rate reaches 85 percent, and the total arsenic content is 14.02mg/kg.

Test example 2 determination of organic acids and amino acids of Hizikia fusiforme fermentation mixture

Measuring the content of organic acid and amino acid in the supernatant of unfermented Cyrtymenia Sparsa and fermented Cyrtymenia Sparsa. The results are as follows:

TABLE 2

TABLE 3

The results show that the nutrient content is improved obviously after fermentation, and particularly, the content of valine is increased by nearly 182 times, the content of leucine is increased by nearly 803 times, and the content of proline is increased by nearly 481 times.

Test example 3 research on antioxidant and anti-inflammatory efficacy of fermented mixture of Hizikia fusiforme prepared by fermenting Lactobacillus plantarum and/or Lactobacillus rhamnosus

1. The efficacy experiment step is as follows:

1. determination of DPPH radical scavenging Activity

The DPPH radical scavenging activity of the samples was determined colorimetrically.

A mixed reaction containing 100. Mu.L of DPPH solution (0.4 mM) and 100. Mu.L of the sample was performed, and the mixture solution was allowed to stand at room temperature for 30 minutes. After standing, the absorbance was measured at 517nm using an ELISA plate reader (Olympus, japan).

2. Determination of Hydrogen peroxide scavenging Activity

The hydrogen peroxide scavenging activity of the samples was determined colorimetrically.

A solution containing 50. Mu.L of 0.1M phosphate buffer (pH 5) was sequentially added to a 96-well plate, and 50. Mu.L each of the sample solutions at different concentrations was mixed with 10. Mu.L of 10mM hydrogen peroxide. After the plate was allowed to stand at 37 ℃ for 5 minutes, 15. Mu.L of 1.25mM ABTS and 15. Mu.L of peroxidase were added to the plate, and allowed to stand at 37 ℃ for 10 minutes. After standing, absorbance at 405nm was measured using an ELISA plate reader (Olympus, japan).

3. Toxicity assay for vero cells

Vero cells were seeded and cultured for 24 hours. Samples were treated with 10. Mu.L to final concentrations of 25, 50, 100 and 200. Mu.g/mL and incubated at 37 ℃ for 24 hours. Cell viability was assessed using MTT assay.

4. For Vero cell H ₂ O ₂ Determination of the protective Effect induced

Vero cells were seeded and cultured for 24 hours. Treatment with 10. Mu.L of sample to final concentrations of 25, 50 and 100. Mu.g/mL and incubation at 37 ℃ CFor 24 hours. H is to be ₂ O ₂ (1 mM) was added to the cells, followed by culture for 1 hour. Finally, DCFH-DA (500. Mu.g/mL) was added to the cells and measured using a fluorescent microplate reader.

5. To H ₂ O ₂ Determination of protective effects of induced cytotoxicity

Vero cells were seeded and cultured for 24 hours. Samples were treated with 10. Mu.L to final concentrations of 25, 50 and 100. Mu.g/mL and incubated at 37 ℃ for 24 hours. H is to be ₂ O ₂ (1 mM) was added to the cells, followed by culture for 24 hours. Cell viability was measured by MTT assay.

6. Determination of anti-inflammatory action of mouse macrophage and cell activity thereof

Mouse macrophages were inoculated and cultured for 24 hours. The samples were treated with 10. Mu.L to final concentrations of 25, 50 and 100. Mu.g/mL. Lipopolysaccharide (1. Mu.g/mL) was added after 1 hour. After 24 hours, 100 μ L of the experimental sample was transferred to a 96-well plate and 100 μ L Griess reagent (1% sulfonamide and 0.1% naphthyl ethylenediamine dihydrochloride in 2.5% phosphoric acid) was added. After 10 minutes, the absorbance was read on a microplate reader at a wavelength of 540 nm. In addition, 100. Mu.L of MTT solution (2 mg/mL) was added to the remaining cells. And cultured for 3 hours. Cell viability was measured by MTT assay.

2. The result of the detection

1. Measurement of DPPH radical scavenging Activity (FIG. 1)

2. Determination of Hydrogen peroxide scavenging Activity (FIG. 2)

3. Toxicity assay for African Green monkey Kidney cells (FIG. 3)

4. To H ₂ O ₂ Determination of the protective Effect induced (FIG. 4)

5. To H ₂ O ₂ Determination of protective Effect of induced cytotoxicity (FIG. 5)

6. Measurement of anti-inflammatory action of mouse macrophages and their cell viability (FIGS. 6, 7).

3. Results and discussion

(1) After the sargassum fusiforme is fermented, when the concentration of a test sample is 100 mug/mL, the DPPH free radical clearance of the fermented sample is obviously higher than that of an unfermented sample, the hydrogen peroxide clearance of the sample after fermentation at each concentration is higher than that of the unfermented sample, and the fermented sargassum fusiforme sample has good antioxidant activity.

(2) Neither the unfermented nor the fermented samples of sargassum fusiforme showed toxicity to Vero cells.

(3) By H ₂ O ₂ After induction, the cell activity is reduced, the intracellular ROS level is reduced and the cell activity is increased after sample drying, which shows that the non-fermented sample and the fermented sample of the sargassum fusiforme have strong antioxidant activity, and the fermented sample has stronger antioxidant activity than the non-fermented sample.

(4) The cell activity is reduced and the inflammatory factor NO is generated after the LPS induction, the intracellular NO level is reduced and the cell activity is increased after the sample is dried, which shows that the non-fermented sample and the fermented sample of the sargassum fusiforme have strong anti-inflammatory activity, and the anti-inflammatory activity of the fermented sample is stronger than that of the non-fermented sample.

Test example 4 anticancer efficacy study

1 experimental materials:

dried sargassum fusiforme: produced in the county of the first sargassum fusiforme in Wenzhou city of Zhejiang province, captured in November and dried in the sun;

citric acid (food grade);

MRS broth;

tryptone;

soaking yeast into powder;

and (3) probiotics: lactobacillus plantarum, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus fermentum;

DMEM high-glucose medium: 500mL, hyclone;

RPMI 1640 culture solution: 500mL, hyclone;

F12K culture solution: 500mL, hyclone;

AB double antibody: millipore;

FBS (fetal bovine serum): gibco;

MTT:Aolarbio,ultrapure grade 1g；

liver cancer cell HEPG2 cell;

lung cancer cell a549 cell;

breast cancer cells MCF7 cells;

gastric cancer cell SGC7901 cell;

intestinal cancer cell LOVO cell.

2, experimental equipment:

a precision blast drying oven;

an atomic fluorescence spectrometer;

an atomic absorption spectrophotometer;

a water bath constant temperature oscillator;

a high speed drug pulverizer;

an electronic analytical balance;

beating machine;

a vertical pressure steam sterilization pot;

a biochemical incubator;

a carbon dioxide incubator.

3 experimental methods and results:

3.1 sample treatment of Cyrtymenia Sparsa:

the first step is as follows: weighing raw materials of sargassum fusiforme, adding water with the weight being 20 times of that of the raw materials, keeping the temperature and stirring for 15min at 90 ℃, draining, collecting the sargassum fusiforme and further processing;

the second step is that: adding 20 times of water, adding citric acid to 0.6% (W/V), stirring at 50 deg.C for 2 hr, washing with clear water until pH is neutral, and draining.

The third step: the above two steps are processed to obtain the Sargassum fusiforme wet vegetable, and the steps are as follows: water (W/V) =1:4, pulping.

The fourth step: the pH of the Hizikia fusiforme slurry was adjusted to 6.5 with a 20% (W/V) KOH solution.

The fifth step: respectively treating the sargassum fusiforme serosity processed in the fourth step according to the following methods:

3.1.1 sterilizing at 115 deg.C for 30min without adding nutrient components;

3.1.2 adding 2% glucose (W/V), subpackaging with 100 mL/bottle, and sterilizing at 115 deg.C for 30min;

3.1.3 adding 0.5% yeast powder (W/V), subpackaging 100 mL/bottle, sterilizing at 115 deg.C for 30min;

3.1.4 adding 1% peptone (W/V), packaging into 100 mL/bottle, and sterilizing at 115 deg.C for 30min;

3.1.5 Add (W/V) 2% glucose, 0.5% yeast powder, 1% peptone, 100 mL/bottle, sterilize at 115 ℃ for 30min.

3.2 fermentation of Hizikia fusiforme

3.2.1 Probiotics activation

Weighing MRS broth culture medium according to the formula, dissolving in water, subpackaging according to 40 mL/bottle, sterilizing at 121 ℃ for 20min, taking out the preserved strain glycerin tube, inoculating to the MRS broth culture medium according to 1% (V/V), and performing activated culture in a biochemical incubator at 37 ℃ for 18h.

3.2.2 screening of Medium in Hizikia fusiforme serum

After the seed liquid is activated, inoculating the seed liquid with 5 percent of seed liquid (V/V) into the sargassum fusiforme serous fluid treated according to the method in 3.1, fermenting for 48 hours at 37 ℃, and detecting the pH value of the fermentation liquid, wherein the results are shown in the following table:

TABLE 4

Processing method	Bacterial strain	Inoculation amount (W/V)	pH after inoculation	pH at the end of fermentation
					3.1.1	Lactobacillus plantarum	5％	4.8	4.8
3.1.2	Lactobacillus plantarum	5％	4.8	3.5
					3.1.3	Lactobacillus plantarum	5％	4.8	4.8
3.1.4	Lactobacillus plantarum	5％	4.8	4.8
					3.1.5	Lactobacillus plantarum	5％	4.8	3.0

As shown in the above table, when Lactobacillus plantarum is just inoculated and not fermented, the pH is reduced from 6.5 to 4.8, since the Lactobacillus plantarum seed solution is acidic, inoculation of 5% (V/V) changes the pH of the Hizikia fusiformis slurry, and the pH at the end of fermentation of the three samples, i.e., 3.1.1, 3.1.3 and 3.1.4, is the same as that at the time of inoculation and is 4.8, indicating that Lactobacillus plantarum does not grow in the three samples. However, only the sample No. 3.1.2 to which 2% glucose was added and the sample No. 3.1.5 to which the rich medium was added showed a significant decrease in pH, i.e., 3.5 and 3.0, respectively, and thus it was found that Lactobacillus plantarum grew normally when 2% glucose was added to the Hizikia fusiformis serum.

Therefore, sargassum fusiforme was treated according to the method of 3.1.2 (i.e., the following method), and further studies on probiotic fermentation were conducted.

the second step: adding 20 times of water, adding citric acid to 0.6% (W/V), stirring at 50 deg.C for 2 hr, washing with clear water until pH is neutral, and draining.

The fourth step: adjusting pH of the Hizikia fusiforme slurry to 6.5 with 20% (W/V) KOH solution.

The fifth step: adding 2% glucose (W/V) into the processed Cyrtymenia Sparsa slurry, packaging into 40 mL/bottle, and sterilizing at 115 deg.C for 30min.

3.2.3 screening of Hizikia fusiforme fermentation Strain

Cyrtymenia Sparsa was treated according to the treatment method of 3.1.2, inoculated in the following manner, and fermented at 37 ℃ for 48 hours, with the results shown in the following table:

TABLE 5

The lactic acid bacteria fermentation consumes glucose and produces acid components such as lactic acid, so that the reduction of the pH value and the reduction of the concentration of the glucose can indicate the growth condition of the probiotics, and the results show that:

(1) The lactobacillus plantarum, the lactobacillus rhamnosus, the lactobacillus acidophilus and the lactobacillus fermentum can grow, and the growth condition of the lactobacillus acidophilus is poorer than that of other strains;

(2) After the fermentation of the two bacteria, the glucose concentration is greatly reduced, the growth condition of the probiotics is better than that of the single bacteria, and the probiotics generate mutual promotion effect;

(3) The lactobacillus acidophilus is fermented in a single bacterium, the pH value is 3.8, the glucose concentration is 20.0g/L, and the lactobacillus acidophilus does not grow well.

3.3 cell efficacy experiments

And 3.2.3, carrying out cell efficacy experiments on a plurality of samples in the step of screening the sargassum fusiforme fermentation strains, and verifying the growth inhibition effect of the sargassum fusiforme on lung cancer cells (A549), liver cancer cells (HEPG 2), breast cancer cells (MCF-9), intestinal cancer cells (LOVO) and stomach cancer cells (SGC-7901) after the sargassum fusiforme is fermented by probiotics, thereby providing scientific basis for further utilization of the sargassum fusiforme. The anti-cancer effect of a fermentation product prepared by fermenting lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus fermentum and lactobacillus plantarum + lactobacillus rhamnosus is researched aiming at liver cancer cells (HEPG 2) and lung cancer cells (A549); the anticancer efficacy of the fermentation products prepared by fermenting lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus fermentum, lactobacillus plantarum + lactobacillus rhamnosus was studied against breast cancer cells (MCF-9), colon cancer cells (LOVO), and stomach cancer cells (SGC-7901).

3.3.1 reagent preparation

3.3.1.1 preparation of the culture Medium

DMEM culture solution is added with 1% double antibody and 15% FBS fetal bovine serum, fully mixed and placed in a refrigerator at 4 ℃ for standby.

RPMI 1640 culture medium was added with 1% double antibody and 10% FBS fetal bovine serum, mixed well and placed in a refrigerator at 4 ℃ for use.

The F12K culture medium was mixed with 1% double antibody and 10% FBS fetal bovine serum, mixed well and placed in a refrigerator at 4 ℃ for use.

3.3.1.2MTT formulation

Dissolving MTT in PBS to 2.5mg/mL, performing ultrasonic assisted dissolution, filtering and sterilizing with 0.45um filter membrane, wrapping with tinfoil paper, and storing at 4 deg.C in dark place.

3.3.1.3 pharmaceutical formulation

Vacuum concentrating the fermented Cyrtymenia Sparsa samples of 6 different methods in 3.3.2 to dry, preparing into 5mg/mL solution with 3.3.1.1 prepared culture medium, and filtering with 0.45um filter membrane for sterilization.

3.3.2 cell viability assay

Cell viability is measured according to the MTT method, also known as MTT colorimetric method, which is a method for detecting cell survival and growth. The detection principle is succinate dehydrogenase in mitochondria of living cells, which can reduce exogenous MTT into water-insoluble blue-purple crystalline formazan and deposit the formazan in the cells, while dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured by enzyme linked immunosorbent assay (ELISA) at 540 or 720nm wavelength, which can indirectly reflect the number of living cells. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number. The method is widely used for activity detection of some bioactive factors, large-scale screening of anti-tumor drugs, cytotoxicity test, tumor radiosensitivity determination and the like.

3.3.2.1 cell activation

The cells required for the experiment were inoculated into the medium and placed in a carbon dioxide incubator (37 ℃,5% CO) ₂ ) Culturing and subculturing according to the growth condition of the cells until the activity of the cells returns to normal.

3.3.2.2 plank

Inoculating the cultured cells into 96-well plates, adding the cell suspension 100 ul/well, controlling the cell concentration (1 ten thousand/well), placing in a carbon dioxide incubator (37 deg.C, 5% CO) ₂ ) And culturing for 24h.

The cultured 96-well plate was taken out from the incubator, the culture medium was removed by vacuum aspiration, blank wells (medium only) and drug addition wells (drug solution treated by 3.3.1.3) were provided in the 96-well plate, 200. Mu.l of the set drug solution was added to each well, and the plate was placed in a carbon dioxide incubator (37 ℃ C., 5% CO) ₂ ) And culturing for 24h.

3.3.2.3 detection

The cultured 96-well plates were removed, 50ul of MTT solution was added to each well, and the resulting mixture was placed in a carbon dioxide incubator (37 ℃,5% CO) ₂ ) And culturing for 3h.

Taking out the 96-well plate, absorbing and removing supernatant in the well, adding DMSO (dimethyl sulfoxide AR), fully shaking to enable the well to become purple, putting the 96-well plate into an enzyme-labeling instrument for detection at 540nm, reading an absorbance value, and calculating the survival rate of cells.

3.3.3 cell assay results and discussion

The effect of 3.3.3.1 sargassum fusiforme on the survival rate of HEPG2 cells before and after fermentation is shown in FIG. 8.

After the sargassum fusiforme is fermented by lactobacillus rhamnosus, when the cell experiment concentration is more than 4.6mg/mL, the sargassum fusiforme has an extremely obvious growth inhibition effect on liver cancer cell HEPG2, wherein when the cell experiment concentration is 4.6mg/mL, the survival rate of the HEPG2 cell is 78.76%; when the cell experiment concentration is 9.2mg/mL, the survival rate of the HEPG2 cells is 64.08%. And the other strain samples have no obvious inhibition effect on HEPG2 cells within the cell experiment concentration range of 9.2 mg/mL.

The effect of 3.3.3.2 sargassum fusiforme on the survival rate of A549 cells before and after fermentation is shown in FIG. 9.

The hizikia fusiforme unfermented sample and the probiotic fermented sample have no obvious growth inhibition effect on the lung cancer cell A549 within the range of cell experiment concentration of 9.2 mg/mL.

3.3.3.3 effects on cell survival of Breast cancer cells (MCF 7) before and after fermentation of Hizikia fusiforme are shown in FIG. 10.

And (4) conclusion: the unfermented sargassum fusiforme has certain effect of inhibiting the growth of breast cancer cells, and the inhibition effect of various schemes after fermentation is weakened.

3.3.3.4 sargassum fusiforme before and after fermentation had an effect on the cell survival rate of gastric cancer cells (SGC 7901) as shown in FIG. 11.

And (4) conclusion: has no inhibiting effect on gastric cancer cells before and after fermentation.

3.3.3.5 Hizikia fusiforme affects survival rate of intestinal cancer cells (LOVO) before and after fermentation as shown in FIG. 12.

And (4) conclusion: the lactobacillus rhamnosus and lactobacillus plantarum are mixed and fermented, an obvious dose-effect relationship exists in the inhibition of intestinal cancer cells, and the higher the dose is, the better the inhibition effect is.

3.3.4 discussion of results

(1) After the sargassum fusiforme is fermented by lactobacillus rhamnosus, when the cell experiment concentration is more than 4.6mg/mL, the sargassum fusiforme has an extremely obvious growth inhibition effect on liver cancer cell HEPG2, wherein when the cell experiment concentration is 4.6mg/mL, the survival rate of the HEPG2 cell is 78.76%; when the cell experiment concentration is 9.2mg/mL, the survival rate of the HEPG2 cells is 64.08%;

(2) The sargassum fusiforme is mixed and fermented by lactobacillus rhamnosus and lactobacillus plantarum, has extremely obvious growth inhibition effect on intestinal cancer cells LOVO when the cell concentration is 9.2mg/mL, has obvious dose-effect relationship, and has better inhibition effect when the dosage is higher.

(3) The unfermented sargassum fusiforme has certain effect of inhibiting the growth of breast cancer cells (MCF 7), wherein the survival rate of the MCF7 cells is 72.02% when the cell experiment concentration is 4.6 mg/mL; when the cell experiment concentration is 9.2mg/mL, the survival rate of MCF7 cells is 70.93%, and the inhibition effect of other schemes after fermentation is weakened;

(4) The single-strain fermentation or mixed-strain fermentation of other strains of sargassum fusiforme has no effect of inhibiting the growth of cancer cells.

Research of numerous scholars at home and abroad shows that the micromolecule fucoidan has the effect of inhibiting the growth of specific cancer cells, the research shows that the sargassum fusiforme fermented by lactobacillus rhamnosus has the effect of inhibiting the growth of liver cancer cells, the unfermented sargassum fusiforme has the effect of inhibiting the growth of breast cancer cells (MCF 7), and the other strains of sargassum fusiforme do not have the effect of inhibiting the growth of cancer cells in unfermented, single-strain fermentation or mixed-strain fermentation. This is probably because sargassum fusiforme is rich in nutrients, contains a substance that promotes cell growth and a substance that inhibits cancer cell growth, and results are shown above when the effect of promoting cell growth dominates.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of sargassum fusiforme fermentation mixture is characterized by comprising the following steps:

step (1), boiling sargassum fusiforme with water to remove arsenic;

step (4), adjusting the pH value of the sargassum fusiforme slurry to 6.0-7.0;

2. The preparation method according to claim 1, wherein in the step (1), the water adding amount for water boiling dearsenification is 5-30 times of the weight of the dried vegetables, the temperature for water boiling dearsenification is 50-100 ℃, and the time for water boiling dearsenification is 10-30 min.

3. The preparation method according to claim 1, wherein in the step (2), the water addition amount of the citric acid for dearsenification is 5-30 times of the weight of the dried vegetables, the mass percentage concentration of the citric acid is 0.2-0.8%, the temperature of the citric acid for dearsenification is 50-100 ℃, and the time of the citric acid for dearsenification is 1-5 hours.

4. The method as set forth in claim 1, wherein in the step (3), the mass ratio of the dearsenified sargassum fusiforme to water is 1: (3-5).

5. The method according to claim 1, wherein the mass volume percentage of the glucose to the Hizikia fusiforme slurry in the step (5) is 1-3%.

6. The method according to claim 1, wherein the lactobacillus plantarum or lactobacillus rhamnosus inoculation amount in step (6) is 0.4-10%.

7. The process according to any one of claims 1 to 6, wherein in the step (6), the fermentation temperature is 20 to 40 ℃, aeration is carried out, and the fermentation time is 24 to 72 hours.

8. A fermented mixture of Hizikia fusiforme prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the sargassum fusiforme fermentation mixture according to claim 8 for preparing products with antioxidant, anti-inflammatory and anticancer effects.

10. The use according to claim 9, wherein the anticancer effect is any one or two or more of anti-liver cancer, anti-stomach cancer and anti-intestinal cancer.