CN113308402A

CN113308402A - Probiotic high-density fermentation composition for relieving side effects of multi-kinase inhibitor

Info

Publication number: CN113308402A
Application number: CN202110571640.8A
Authority: CN
Inventors: 徐浩宇; 梁文; 李明松
Original assignee: Jiangsu Hengkang Bio Tech Co ltd
Current assignee: Jiangsu Hengkang Bio Tech Co ltd
Priority date: 2021-05-25
Filing date: 2021-05-25
Publication date: 2021-08-27

Abstract

The invention relates to the field of functional probiotic development, and discloses a probiotic high-density fermentation composition for relieving the side effect of a multi-kinase inhibitor in order to solve the problems of low live bacteria amount and low vitamin B6 content of the conventional probiotic fermentation composition. The probiotic high-density fermentation composition has high live bacteria content and high vitamin B6 content, and the beverage obtained by fermentation applied to the probiotic fermented beverage has good acceptability and has certain effect of relieving the side effect of the multi-kinase inhibitor.

Description

Probiotic high-density fermentation composition for relieving side effects of multi-kinase inhibitor

Technical Field

The invention relates to the field of functional probiotic development, in particular to product development of probiotic multi-strain microecology for relieving side effects of a multi-kinase inhibitor.

Background

Multi-kinase inhibitors (MKIs) are a new class of multi-target antineoplastic drugs that have proven effective in renal cell carcinoma, hepatocellular carcinoma, and gastrointestinal stromal tumors in clinical studies and have been approved by the U.S. food and drug administration for the treatment of these tumors. However, the drug-related toxic reactions, including hand-foot skin reaction (HFSR), limit the clinical application of the drug, and have serious effects on the physiological, psychological and social activities of patients, and the severe patients cause the dosage of the drug to be reduced and the therapeutic effect to be weakened. The pathogenesis of HFSR is not clear, MKIS may damage the capillaries, and when the hands and feet are subjected to direct stress such as walking, washing hands or other daily activities, the damaged vessels are again subjected to mechanical damage such as stress, thus now accompanied by inflammation and vesicular HFSR.

At present, HFSR relieving schemes comprise aloe emulsion, emollient, urea ointment and the like, vitamin B6, vitamin E, glucocorticoid and the like, but in the long-term treatment process of patients, the intestinal absorption effect is poor, so that the drug absorption is poor, and HFSR cannot be well treated. At present, more domestic documents show that probiotics can be combined with B vitamins, for example, CN201911133644.7 is derived from Lactobacillus plantarum Bama06 with high vitamin B yield ability in Himala cantonensis and application thereof, CN202010472345.2 is a preparation method and application of vitamin B-rich fermented milk, and CN202010756524.9 is derived from vitamin B12 lactic acid bacteria and application thereof, and the researches provide a better scheme for treating FHSR, and by using the high-yield vitamin B6 type probiotics, on one hand, the intestinal tract of a patient is improved, the absorption of nutrient components in the intestinal tract is promoted, and on the other hand, vitamin B6 is generated to improve HFSR. The existing probiotic fermentation composition has the problems of low live bacteria amount and low vitamin B6 content, and does not have the effect of relieving the side effect of the multi-kinase inhibitor.

Disclosure of Invention

The invention aims to develop a probiotic high-density fermented composition, which can improve the intestinal tract of a patient, promote the absorption of nutrient components in the intestinal tract and produce B vitamins to improve HFSR.

In order to achieve the purpose, the invention adopts the following technical scheme.

A probiotic high-density fermentation composition comprises basic fermentation substrate, saccharified barley bud liquid, acid-base regulator and probiotic.

The basic fermentation substrate comprises the following components in percentage by mass and volume:

yeast extract: 2g/L-6g/L

Hydrolyzing soybean protein: 5g/L-10g/L

Isomalt: 30g/L-60g/L

Sodium citrate: 3g/L-7g/L

Xanthan gum: 1g/L-3g/L

Magnesium sulfate: 30mg/L-50mg/L

Manganese sulfate: 20mg/L-50mg/L

The preparation method of the saccharified barley bud liquid comprises the following steps: uniformly mixing 5% of barley sprout powder with water, heating to 68 ℃, keeping the temperature for 3h, removing residues, starting a heating program of 1 ℃/min, heating from 68 ℃ to 95 ℃, and keeping the temperature for 90min to obtain a saccharified barley sprout liquid, wherein the content of primary sugar is 90-120 mg/mL;

the pH regulator is 30g/L sodium hydroxide.

The probiotics comprise lactobacillus delbrueckii subsp bulgaricus, lactobacillus fermentum, lactobacillus paracasei, lactobacillus casei, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus rhamnosus and lactobacillus lactis subsp lactis.

The high-density fermentation process parameters are as follows:

the fermentation temperature is 37 ℃;

the fermentation speed is 30 r/min;

and (3) pH control: from the beginning of fermentation, when the pH value is reduced to 5.0, an acid-base regulator is used for maintaining the pH value at 5 +/-0.3 until the fermentation is finished;

adding sequence to the saccharified barley bud liquid: 160ml/L is added at the beginning of fermentation; starting fermentation at 8h, feeding 30ml/L/h of feed per hour until the fermentation is finished;

the fermentation time is 24 h.

Preferably, the yeast extract is used for removing carboxyl and sulfur-containing amino acid monomers to reduce the delicate flavor;

preferably, the hydrolyzed soy protein is amino acid monomer and polypeptide with larger hydrophobicity, so that the bitter taste is reduced;

preferably, the saccharified barley bud liquid is saccharified barley bud liquid with reducing sugar content of more than 100 mg/mL;

preferably, the basic fermentation substrate comprises 4g/L yeast extract, 6g/L hydrolyzed soybean protein, 50g/L isomalt, 160g/L saccharified barley malt extract, 5g/L sodium citrate, 2g/L xanthan gum, 40mg/L magnesium sulfate and 30mg/L manganese sulfate;

preferably, the Lactobacillus delbrueckii subspecies bulgaricus in the probiotics is a strain screened and trained by Jiangsu Hengkang probiotic research center, is numbered as HKMCC2020028, is numbered as AHK028, has the capacity of high-yield vitamin B, and has the yield of 221 mg/L.

Preferably, the lactobacillus fermentum among the probiotics is a screened and trained strain of Jiangsu Hengkang probiotic research center, the strain is numbered HKMCC2020002, the strain is AHK002, the lactobacillus fermentum has the capacity of high-yield vitamin B, and the yield is 187 mg/L.

Preferably, the lactobacillus paracasei in the probiotics is a screened and trained strain of Jiangsu Hengkang probiotic research center, the strain is numbered HKMCC2020005, the strain is AHK005, the lactobacillus paracasei has the capacity of high-yield vitamin B, and the yield is 166 mg/L.

Preferably, the lactobacillus casei in the probiotics is a screened and trained strain of Jiangsu Hengkang probiotic research center, the number of the strain is HKMCC2020016, the strain number is AHK0016, the lactobacillus casei has the capacity of high-yield vitamin B, and the yield is 190 mg/L.

Preferably, the lactobacillus acidophilus in the probiotics is a screened and trained strain of Jiangsu Hengkang probiotic research center, the number of the strain is HKMCC2020015, the strain number is AHK015, the lactobacillus acidophilus has the capacity of producing vitamin B with high yield, and the yield is 163 mg/L.

Preferably, the lactobacillus plantarum in the probiotics is a screened and trained strain of Jiangsu Hengkang probiotic research center, the number of the strain is HKMCC2020009, the strain number is TG-95, the lactobacillus plantarum has the capacity of high vitamin B yield, and the yield is 107 mg/L.

Preferably, the lactobacillus rhamnosus in the probiotics is a strain screened and trained by Jiangsu Hengkang probiotic research center, the strain is numbered HKMCC2020026, the strain is A-4, the capacity of high-yield vitamin B is realized, and the yield is 115 mg/L.

Preferably, lactococcus lactis subspecies lactis in the probiotics is a screened and trained strain of Jiangsu Hengkang probiotic research center, is numbered as HKMCC2020027, is numbered as AHK027, has the capacity of high-yield vitamin B, and has the yield of 137 mg/L.

The preparation method of the probiotic high-density fermented composition comprises the following steps:

step 1: preparing a basic fermentation substrate as a primary and secondary seed activation culture medium, respectively inoculating 8 strains of probiotic glycerol into the primary seed activation culture medium according to a proportion of 1%, standing and activating at 37 ℃ for 24h to serve as primary activation strain liquid; respectively inoculating 8 strains of probiotic primary activated strain liquid into a secondary seed activation culture medium according to the proportion of 1%, standing and activating at 37 ℃ for 24h to serve as secondary activated strain liquid;

step 2: uniformly mixing 5% of barley sprout powder with water, heating to 68 ℃, keeping the temperature for 3h, removing slag, starting a heating program of 1 ℃/min, heating from 68 ℃ to 95 ℃, and keeping the temperature for 90min to obtain a saccharified barley sprout liquid;

and step 3: preparing a basic fermentation substrate according to a formula, respectively inoculating 8 strains of probiotics according to a proportion of 0.1%, feeding 160ml/L of saccharified barley bud liquid, controlling the temperature at 37 ℃ and the rotating speed at 30r/min, and starting fermentation;

and 4, step 4: when the fermentation pH is reduced to below 5.0, controlling the pH to be within the range of 5.0 +/-0.3 by using 30g/L sodium hydroxide until the fermentation is finished; and (4) feeding the saccharified barley bud liquid at the speed of 30ml/L/h after the fermentation is carried out for 8 hours until the fermentation is finished.

The probiotic high-density fermentation composition consists of a basic fermentation substrate, saccharified barley bud liquid, an acid-base regulator and probiotics, and the amount of the obtained viable bacteria is 1 multiplied by 10 through high-density fermentation¹⁰More than cfu/ml, and the content of vitamin B6 is more than 200 mg/ml; the beverage obtained by fermentation applied to the probiotic fermented beverage has good acceptability and reaches the expectation of optimizing the process.

Clinical effects prove that the probiotic high-density fermented composition has the effect of remarkably relieving HFSR symptoms of patients taking MKIS.

The invention has the following advantages:

1. the viable bacteria content of the probiotic high-density fermented composition can reach 1 × 10¹⁰cfu/ml；

2. The content of vitamin B6 in the probiotic high-density fermented composition can reach 200 mg/mL;

3. the probiotic high-density fermented composition live bacteria can improve the intestinal health of patients, and relieve HFSR symptoms of patients taking MKIS through vitamin B6.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.

The implementation materials and the detection method of the case all meet the requirements of the national relevant standards.

Example 1: screening of probiotic strains with high vitamin B6 yield

20 gastric acid-resistant strains are selected from Jiangsu Hengkang strain management center (HKMCC), 1% of glycerol strains are inoculated in MRS culture media respectively, the cells are activated for 24 hours at 37 ℃, and supernatant is collected after centrifugation. Detecting the content of vitamin B6 by high performance liquid chromatography, and selecting strains with yield of 100mg/L as fermentation strains, as shown in Table 1, including: lactobacillus fermentum AHK002, lactobacillus paracasei AHK005, lactobacillus rhamnosus A-4, lactobacillus acidophilus AHK015, lactobacillus casei AHK016, lactobacillus delbrueckii subspecies bulgaricus AHK025, lactobacillus plantarum TG-95, and lactobacillus lactis subspecies lactis AHK 027.

TABLE 1

Example 2: selection of Nitrogen Source

Respectively inoculating 8 secondary activated seed solutions of probiotics by using 4g/L yeast extract, 50g/L isomaltitol, 200g/L glucose, 5g/L sodium citrate and 2g/L xanthan gum according to the proportion of 0.1%, placing in an environment at 37 ℃, stirring at the speed of 90r/min, culturing for 24h, and then checking the amount of the viable bacteria according to GB 4789.35-2016.

On the basis of example 2, comparative examples 1-3 were set for the addition of the nitrogen source, and comparative example 1 added 4g/L of yeast extract and 6g/L of soy protein hydrolysate as the nitrogen source compared to example 2; comparative example 24 g/L yeast extract and 5g/L soy protein peptide were added as nitrogen source; comparative example 3 yeast extract 4g/L and beef extract 5g/L were added as nitrogen source;

example 2 and comparative examples 1 to 3 carbon source addition and examination of the amount of live bacteria are shown in Table 2:

TABLE 2

As can be seen from Table 1, the amount of viable bacteria tested in comparative example 1 was the largest, and thus the addition of 4g/L yeast extract and 6g/L soy protein hydrolysate as the nitrogen source in comparative example 1 was the optimum nitrogen source formulation.

Example 3: selection of carbon sources

The method comprises the steps of taking 4g/L yeast extract, 6g/L hydrolyzed soybean protein, 50g/L isomaltitol, 5g/L sodium citrate and 2g/L xanthan gum as basic raw materials, adding 200g/L glucose as a carbon source, inoculating 8 probiotic secondary activated seed solutions in a fermentation volume of 100ml according to a proportion of 0.1%, placing the seed solutions in an environment with the temperature of 37 ℃, stirring at a speed of 90r/min, culturing for 24 hours, and then checking the viable bacteria amount according to GB 4789.35-2016.

On the basis of example 3, comparative examples 4-5 were set for the case of carbon source addition, and comparative example 4 added 160g/L of the saccharified barley malt solution as a carbon source as compared with example 3; comparative example 5 sucrose was added as a carbon source at 100 g/L;

example 3 and comparative examples 4 to 5 carbon source addition and examination of the amount of live bacteria are shown in Table 3:

TABLE 3

Carbon source	glucose/(g/L)	Saccharified barley bud liquid/(g/L)	sucrose/(g/L)	Viable bacteria/(cfu/ml)
					Example 3	200	——	——	4.1×10⁸
Comparative example 4	——	160	——	4.8×10⁹
					Comparative example 5	——	——	100	4.7×10⁸

As can be seen from Table 3, the amount of viable bacteria examined in comparative example 4 was the largest, and therefore 160g/L of the malted barley malt solution added in comparative example 4 as a carbon source was formulated as an optimum carbon source.

Example 4 selection of trace elements

4g/L yeast extract, 6g/L hydrolyzed soybean protein, 160g/L saccharified barley bud liquid, 50g/L isomaltitol, 5g/L sodium citrate and 2g/L xanthan gum are taken as basic raw materials, 2g/L dipotassium hydrogen phosphate is added to be taken as salt ions, 8 strains of secondary activated seed liquid of probiotics are respectively inoculated according to the proportion of 0.1 percent in 100ml fermentation volume, the secondary activated seed liquid is placed in an environment at 37 ℃, the secondary activated seed liquid is stirred at the speed of 90r/min, and the viable bacteria amount is tested according to GB4789.35-2016 after the secondary activated seed liquid is cultured for 24 h.

On the basis of example 4, comparative examples 6 to 8 were set for the case of addition of trace elements, and in comparison with example 4, comparative example 6 added 0.04g/L of magnesium sulfate as a trace element; comparative example 7 adding 0.03g/L manganese sulfate as a trace element; comparative example 8 magnesium sulfate of 0.04g/L and manganese sulfate of 0.03g/L were added as trace elements.

The trace element addition and the amount of live bacteria were examined as in Table 4 for example 4 and comparative examples 6 to 8:

TABLE 4

Trace elements	Dipotassium hydrogen phosphate/(g/L)	Magnesium sulfate/(g/L)	Manganese sulfate/(g/L)	Viable bacteria/(cfu/ml)
					Example 4	2	——	——	1.2×10⁹
Comparative example 6	——	0.04	——	2.8×10⁹
					Comparative example 7	——	——	0.03	4.4×10⁹
Comparative example 8	——	0.04	0.03	6.9×10⁹

As can be seen from Table 4, the amount of viable bacteria examined in comparative example 8 was the largest, and therefore 0.04g/L magnesium sulfate and 0.03g/L manganese sulfate in comparative example 8 were the optimum trace element formulations.

Example 5: selection of fermentation pH control value

Preparing 15L fermentation substrate by using 4g/L yeast extract, 6g/L hydrolyzed soybean protein, 160g/L saccharified barley bud liquid, 50g/L isomaltitol, 5g/L sodium citrate, 2g/L xanthan gum, 40mg/L magnesium sulfate and 30mg/L manganese sulfate as basic raw materials, respectively inoculating 8 secondary activated seed liquids of probiotic bacteria in a 20L fermentation tank according to the proportion of 0.1%, starting fermentation at 37 ℃ and 90r/min, controlling the pH to be 4.5 by using 30g/L sodium hydroxide during the fermentation, and testing the viable bacteria amount according to GB4789.35-2016 after 24 hours of fermentation.

On the basis of example 5, comparative examples 6 to 8 were set for pH control value, and the pH control value of comparative example 9 was set to 5 as compared with example 5; the pH control value of comparative example 10 was set to 4.

The pH control and viable bacteria tested in example 5 and comparative examples 9-10 are shown in Table 5:

TABLE 5

pH control value	pH value	Viable bacteria/(cfu/ml)
			Example 5	4.5	7.6×10⁹
Comparative example 9	5	9.1×10⁹
			Comparative example 10	4	7.1×10⁹

As is clear from Table 5, the viable cell count in comparative example 9 was the largest, and therefore pH control value 5 in comparative example 9 was the preferable pH control value.

Example 6: selection of mode for supplementing saccharified barley bud liquid in fermentation

Taking 4g/L yeast extract, 6g/L hydrolyzed soybean protein, 160g/L saccharified barley bud liquid, 50g/L isomaltitol, 5g/L sodium citrate, 2g/L xanthan gum, 40mg/L magnesium sulfate and 30mg/L manganese sulfate as basic raw materials, preparing 15L fermentation substrate, respectively inoculating 8 secondary activated seed liquids of probiotics in a 20L fermentation tank according to the proportion of 0.1%, starting fermentation at 37 ℃ and 90r/min, controlling the pH to be 5 by using 30g/L sodium hydroxide during the fermentation, not supplementing the saccharified barley bud liquid in the whole process, and testing the viable bacteria amount according to GB4789.35-2016 after fermenting for 24 hours.

On the basis of example 6, comparative examples 11-13 are set for a mode of supplementing the saccharified barley bud liquid in the fermentation, and compared with example 6, comparative example 11 is supplemented with the saccharified barley bud liquid at a speed of 30mg/L/h after 8 hours of fermentation until the end of the fermentation; compared with example 6, comparative example 12 supplemented the saccharified barley sprout liquid at a rate of 50mg/L/h after fermenting for 8h until the end of fermentation; in comparison with example 6, comparative example 13 supplemented the saccharified barley malt liquid at a rate of 10mg/L/h throughout the fermentation until the end of the fermentation.

The modes of supplying glycated barley malt and the viable bacteria measured in example 6 and comparative examples 11 to 13 are shown in Table 6:

TABLE 6

Saccharification barley bud liquid supplementing mode	Replenishment method	Viable bacteria/(cfu/ml)
			Example 6	No supplement in the whole fermentation process	9.3×10⁹
Comparative example 11	After fermenting for 8h, supplementing at 30mg/L/h	1.9×10⁹
			Comparative example 12	After fermenting for 8h, supplementing at a rate of 50mg/L/h	1.3×10¹⁰
Comparative example 13	The whole fermentation process is supplemented at the speed of 10mg/L/h	1.0×10¹⁰

As is clear from Table 6, comparative example 11 has the highest viable cell count, and thus comparative example 9 has a preferred mode of supplementing the malt extract of the saccharified barley at a rate of 30mg/L/h after 8 hours of fermentation until the end of fermentation.

Example 7

Preparing 15L fermentation substrate by using 4g/L yeast extract, 6g/L hydrolyzed soybean protein, 160g/L saccharified barley bud liquid, 50g/L isomaltitol, 5g/L sodium citrate, 2g/L xanthan gum, 40mg/L magnesium sulfate and 30mg/L manganese sulfate as basic raw materials, respectively inoculating 8 strains of secondary activated seed liquid of probiotic bacteria in a 20L fermentation tank according to the proportion of 0.1%, starting fermentation at 37 ℃ and 90r/min, controlling the pH to be 5 by using 30g/L sodium hydroxide during the fermentation, supplementing the saccharified barley bud liquid at the speed of 30mg/L/h after 8 hours of fermentation, and finishing the fermentation after 24 hours. Adding appropriate amount of fruit essence and sucralose, and aseptically packaging into 50ml glass bottles.

Taste evaluation was performed on 50 volunteers on a 10 point scale, and the results are listed in table 7:

TABLE 7

Item	Score the points
		Color	9.2
Smell(s)	9.5
		Acidity of the solution	8.9
Sweetness level	8.5

As can be seen from Table 7, the product has an evaluation score of 8.5 points or more, indicating that the product has a certain acceptability. And sampling and detecting live bacteria according to GB4789.35-2016, and the result shows that the live bacteria of the pilot sample is 1.9 x 10¹⁰cfu/ml, which reaches the expectation of optimizing the process.

Experimental example 1: clinical research on treatment of moderate and severe hand-foot skin reaction caused by multi-target kinase inhibitor by probiotic high-density fermentation composition and vitamin B6

The probiotics in the probiotic high-density fermented composition can improve the intestinal health of a patient suffering from HFSR, promote the absorption of nutrient components, and simultaneously, the probiotics in the composition can produce a large amount of vitamin B6, so that the HFSR symptom of the patient can be relieved to a certain extent.

1.1 preparation method of probiotic high-density fermentation composition

1.2 detailed description of the invention

1.2.1 inclusion criteria

Patients are admitted to the oncology department at the affiliated college medical college of science and technology of Huazhong from 6 months to 1 month of 2021 in 2020 under the following conditions:

1) age 18 or more, male and female;

2) taking anticancer drug and treating tumor with HFSR above II degree for more than 6 weeks (42 days);

3) expected survival time of at least 6 months;

4) if HFSR is being treated by various methods prior to enrollment, it is required to be ineffective for the treatment method used, and it is required to discontinue the use of the drug for treating HFSR for at least 1 week prior to enrollment;

5) an ECOG score of less than or equal to 2;

6) the disease history of important visceral organs such as the center of gravity, lung, liver, kidney and the like is not strict;

7) signing an informed consent;

8) the patient compliance is good;

9) during the research observation period, other foods or beverages containing probiotics cannot be drunk, if the foods or beverages are necessary to be drunk, the dosage and the type need to be consistent with the living habits before the foods or beverages are put into groups, and the intake of other probiotic foods cannot be increased.

The following conditions are met and excluded:

1) pregnant or lactating women;

2) in 3 weeks, the patients who had taken antibiotics or medicines or foods containing other probiotics;

3) patients who have multiple organ tumors or have severe uncontrolled medical illness or acute infection at the same time;

4) patients were differently intended to stop the original oral medication during the trial;

5) patients were unable to understand the purpose of the study or did not agree to the requirements of the study;

6) lack of legal capacity or restricted legal capacity;

7) researchers judge that compliance is poor and cannot strictly execute the protocol;

8) any history, at the discretion of the investigator, may interfere with the outcome of the trial or increase patient risk.

A total of 10 patients reached cohort conditions during this period, with 6 males and 4 females; the age is 33-58 years, and the average age is 47 years; the course of HFSR is 6-18 months, and the average is 12 months; numbering is done from 1 to 10 according to the time of entry. The patient details are listed in table 8.

TABLE 8

1.2.2 methods of treatment

Randomly administering 200mg of vitamin B6 per day to 10 persons, and orally administering 50mL of probiotic high-density fermented composition three times per day; patient symptoms were observed and recorded at week 2, week 5 and week 7 follow-up with a 6 week treatment cycle.

1.3 results of clinical study

The treatment results of 10 patients in the group during the treatment period are shown in table 9, and it can be known that 10 patients in the group have different improvements of the disease conditions by orally taking 200mg of vitamin B6 and 50mL of probiotic high-density fermented composition three times a day, wherein 5 patients have obvious curative effect, and 5 patients are effective, which indicates that the orally taken probiotic fermented composition and vitamin B6 have certain curative effect on relieving HFSR.

TABLE 9

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. A probiotic high-density fermentation composition is characterized by comprising a basic fermentation substrate, saccharified barley bud liquid, an acid-base regulator and probiotics.

2. The probiotic high-density fermented composition according to claim 1, wherein the basic fermentation substrate comprises the following components in percentage by mass and volume:

yeast extract: 2g/L-6g/L

Hydrolyzing soybean protein: 5g/L-10g/L

Isomalt: 30g/L-60g/L

Sodium citrate: 3g/L-7g/L

Xanthan gum: 1g/L-3g/L

Magnesium sulfate: 30mg/L-50mg/L

Manganese sulfate: 20mg/L-50 mg/L.

3. The probiotic high-density fermented composition according to claim 1, wherein the preparation method of the saccharified barley malt liquor comprises the following steps: mixing 5% of barley sprout powder with water uniformly, heating to 68 deg.C, keeping the temperature for 3h, removing residue, starting a heating program of 1 deg.C/min, heating from 68 deg.C to 95 deg.C, and keeping the temperature for 90min to obtain saccharified barley sprout liquid with raw sugar content of 90-120 mg/mL.

4. The probiotic high-density fermented composition according to claim 1, wherein the probiotic bacteria include Lactobacillus delbrueckii subsp.

5. The probiotic high-density fermented composition according to claim 1, wherein the high-density fermentation is characterized by the following parameters:

the fermentation temperature is 37 ℃;

the fermentation speed is 30 r/min;

the fermentation time is 24 h.

6. The probiotic high-density fermented composition according to claim 2, wherein the yeast extract is used for removing carboxyl-and sulfur-containing amino acid monomers to reduce the umami taste; the hydrolyzed soybean protein is amino acid monomer and polypeptide with larger hydrophobicity, and reduces bitter taste; the saccharified barley bud liquid is saccharified barley bud liquid with reducing sugar content of more than 100 mg/mL.

7. The probiotic high-density fermented composition according to claim 4, wherein 8 probiotics are characterized by the following:

the Lactobacillus delbrueckii subspecies bulgaricus in the probiotics is a strain screened and trained by Jiangsu Hengkang probiotic research center, is numbered as HKMCC2020028, has AHK028 strain number, has the capacity of high-yield vitamin B, and has the yield of 221 mg/L;

the lactobacillus fermentum in the probiotics is a strain screened and trained by Jiangsu Hengkang probiotics research center, the number of the strain is HKMCC2020002, the strain number is AHK002, the lactobacillus fermentum has the capacity of high-yield vitamin B, and the yield is 187 mg/L;

the lactobacillus paracasei in the probiotics is a strain screened and trained by Jiangsu Hengkang probiotic research center, is numbered as HKMCC2020005, is numbered as AHK005, has the capacity of high-yield vitamin B, and has the yield of 166 mg/L;

the lactobacillus casei in the probiotics is a screened and trained strain of Jiangsu Hengkang probiotics research center, the number of the strain is HKMCC2020016, the strain number is AHK0016, the lactobacillus casei has the capacity of high-yield vitamin B, and the yield is 190 mg/L;

the lactobacillus acidophilus in the probiotics is a screened and trained strain of Jiangsu Hengkang probiotics research center, the number of the strain is HKMCC2020015, the strain number is AHK015, the lactobacillus acidophilus has the capacity of high-yield vitamin B, and the yield is 163 mg/L;

the lactobacillus plantarum in the probiotics is a screened and trained strain of Jiangsu Hengkang probiotic research center, the number of the strain is HKMCC2020009, the strain number is TG-95, the lactobacillus plantarum has the capacity of high-yield vitamin B, and the yield is 107 mg/L;

the lactobacillus rhamnosus in the probiotics is a strain screened and trained by Jiangsu Hengkang probiotic research center, is numbered as HKMCC2020026, has the strain number of A-4, has the capacity of high-yield vitamin B and has the yield of 115 mg/L;

the lactococcus lactis subspecies lactis in the probiotics is a screened and trained strain of Jiangsu Hengkang probiotic research center, is numbered as HKMCC2020027, has AHK027 strain number, has the capacity of high-yield vitamin B, and has the yield of 137 mg/L.

8. The process for the preparation of a probiotic fermented composition at high density according to any of claims 1 to 7, characterized in that it comprises the following steps: