阅读说明：本技术 一种高活性发酵乳杆菌grx08发酵液的制备方法及其应用 (Preparation method and application of high-activity lactobacillus fermentum grx08 fermentation broth ) 是由 顾瑞霞 刘洋 陈大卫 张辰臣 瞿恒贤 刘潇潇 于 2021-08-30 设计创作，主要内容包括：本发明涉及一种高活性发酵乳杆菌grx08发酵液的制备方法及其应用,在木瓜蛋白酶酶解乳清蛋白水解液中添加复合低聚糖,搅拌均匀后,加入发酵乳杆菌grx08发酵剂发酵后,制备得到高活性发酵乳杆菌grx08发酵液。基于木瓜蛋白酶酶解乳清蛋白水解液与组分包括0.20%-0.40%w/w低聚果糖、0.3%-0.50%w/w低聚半乳糖、0.3%-0.5%w/w菊粉、0.6%-0.8%w/w水苏糖、0.02%-0.05%w/w低聚木糖的复合低聚糖结合,加入具有降血脂、降血压、降胆固醇等优良性能的发酵乳杆菌grx08制备发酵液。本发明可用于制备具有高活菌型合生元发酵乳、功能食品、药品等系列产品。(The invention relates to a preparation method and application of a high-activity lactobacillus fermentum grx08 fermentation broth, wherein the high-activity lactobacillus fermentum grx08 fermentation broth is prepared by adding composite oligosaccharide into papain enzymatic hydrolysis whey protein hydrolysate, uniformly stirring, adding a lactobacillus fermentum grx08 leaven, and fermenting. Based on the combination of papain enzymolysis whey protein hydrolysate and composite oligosaccharide with the components including 0.20-0.40% w/w fructo-oligosaccharide, 0.3-0.50% w/w galacto-oligosaccharide, 0.3-0.5% w/w inulin, 0.6-0.8% w/w stachyose and 0.02-0.05% w/w xylo-oligosaccharide, lactobacillus fermentum grx08 with excellent performances of reducing blood fat, blood pressure and cholesterol is added to prepare the fermentation broth. The invention can be used for preparing high viable bacteria synbiotic fermented milk, functional food, medicines and other series products.)

1. A preparation method of high-activity lactobacillus fermentum grx08 fermentation liquor is characterized in that:

adding composite oligosaccharide into the papain enzymolysis whey protein hydrolysate, mixing uniformly, filtering, homogenizing, performing heat treatment, cooling, adding a fermentation agent of lactobacillus fermentum grx08, and fermenting to obtain the high-activity lactobacillus fermentum grx08 fermentation liquid.

2. The method for preparing fermentation broth of lactobacillus fermentum grx08 with high activity according to claim 1, wherein: adding a sterilized aqueous solution in which a stabilizer and an emulsifier are dissolved into the high-activity lactobacillus fermentum grx08 fermentation liquid, and stirring, homogenizing and refrigerating to obtain the high-activity lactobacillus fermentum grx08 beverage.

3. The method for preparing fermentation broth of lactobacillus fermentum grx08 with high activity according to claim 1, wherein: the papain enzymolysis lactalbumin hydrolysate is prepared by the following steps:

adding 5000U g of papain 1000-^-1Carrying out enzymolysis at 55-65 ℃ for 2-3 h, centrifuging after enzymolysis is finished, taking supernate, and inactivating enzyme at 105 ℃/5min to obtain papain enzymolysis lactalbumin hydrolysate.

4. The method for preparing fermentation broth of lactobacillus fermentum grx08 with high activity according to claim 1, wherein: the composite oligosaccharide component comprises 0.20-0.40% w/w fructo-oligosaccharide, 0.3-0.50% w/w galacto-oligosaccharide, 0.3-0.5% w/w inulin, 0.6-0.8% w/w stachyose and 0.02-0.05% w/w xylo-oligosaccharide.

5. The method for preparing fermentation broth of lactobacillus fermentum grx08 with high activity according to claim 1, wherein: when the fermentation is carried out by adding the fermentation agent of the lactobacillus fermentum grx08, the inoculation amount of the fermentation agent of the lactobacillus fermentum grx08 is 2.0% -4.0%, and the fermentation is carried out at 37 ℃ for 18-24 h to prepare the high-activity lactobacillus fermentum grx08 fermentation liquid.

6. The method for preparing fermentation broth of lactobacillus fermentum grx08 with high activity according to claim 1, wherein: the preparation method of the lactobacillus ferments grx08 comprises the following steps:

inoculating a strain of 1 generation of lactobacillus fermentum grx08 into an MRS liquid culture medium according to the inoculation amount of 2-4%, and culturing in an incubator at 37 ℃ for 18h to obtain a lactobacillus fermentum grx 08;

when the MRS liquid culture medium is prepared, 10g of soybean peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 801ml of tween, 2g of sodium dihydrogen phosphate, 5g of anhydrous sodium acetate, 2g of citric acid triamine, 0.02g of manganese sulfate and 0.1g of magnesium sulfate are subjected to constant volume to 1L with distilled water, the pH value is adjusted to 6.2, and the MRS liquid culture medium is obtained after sterilization at 121 ℃ for 15 min.

7. The method for preparing fermentation broth of lactobacillus fermentum grx08 with high activity according to claim 1, wherein: adding compound oligosaccharide into the papain enzymolysis whey protein hydrolysate, uniformly mixing, filtering, homogenizing under 20Mpa, performing heat treatment at 95 ℃ for 5-10 min, cooling to 37 ℃, and adding lactobacillus ferments grx08 for fermentation.

8. The method for preparing fermentation broth of lactobacillus fermentum grx08 with high activity according to claim 2, wherein: after fermentation with the fermentation agent of the lactobacillus fermentum grx08, dissolving a sterilized water solution of a stabilizer and an emulsifier, stirring, homogenizing, filling into a sales container, and refrigerating at 4 ℃ for 24h to obtain the high-activity lactobacillus fermentum grx08 fermentation liquid beverage.

9. The method for preparing fermentation broth of lactobacillus fermentum grx08 with high activity according to claim 1, wherein: said lactobacillus fermentum grx08(Lactobacillus fermentum) And (3) in 2013, 6, 9, and in China general microbiological culture Collection center (CGMCC), addresses: the microbial research institute of the Chinese academy of sciences, No. 3 Xilu-Beijing, Chaoyang, Beijing area, Beijing province, and the accession number is GCMCC NO: 7695.

10. the high-activity lactobacillus fermentum grx08 fermentation broth prepared by the preparation method of the high-activity lactobacillus fermentum grx08 fermentation broth is used for preparing fermented milk, functional food and medicines with high viable bacteria.

Technical Field

The invention relates to a preparation method and application of high-activity lactobacillus fermentum grx08 fermentation broth, and belongs to the field of functional foods.

Technical Field

The whey protein powder is a byproduct in the processing process of cheese and casein, and is cheap and easy to obtain. The whey protein comprises beta-lactoglobulin, alpha-lactalbumin, cow milk serum albumin, immunoglobulin, lactoferrin and the like, is rich in essential amino acid, has low contents of fat, cholesterol and lactose, has the characteristic of a large number of functional components capable of improving absorption, and has extremely high nutritional value.

Functional oligosaccharides are not available to the digestive tract and pathogenic bacteria, and only in the large intestine, they act as proliferation factors and food sources for probiotics and positively affect the gastrointestinal tract of the host. Among them, inulin, fructo-oligosaccharide, galacto-oligosaccharide, stachyose and xylo-oligosaccharide are common functional oligosaccharides and widely used as prebiotics.

The regression rotation design method can carry out systematic research on all factors, has the characteristics of small test scale, simple and convenient calculation, elimination of correlation among regression coefficients and the like, can avoid large errors caused by different positions of test points in a factor space in a regression orthogonal test, and has wide attention in the field of biological growth condition optimization.

The lactobacillus can regulate normal flora of gastrointestinal tract, maintain microecological balance, reduce serum cholesterol, control endotoxin, and inhibit growth and reproduction of putrefying bacteria and generation of putrefying products in intestinal tract. However, many prebiotic products on the market have unstable lactobacillus content and poor survival rate, and scholars at home and abroad do a lot of work to maintain the normal number of lactobacillus in human intestinal tract and prolong the survival rate of lactobacillus, wherein adding probiotic factors such as oligosaccharide into the body to promote the proliferation of probiotics is a mature method.

Disclosure of Invention

The invention aims to provide a preparation method and application of a high-activity lactobacillus fermentum grx08 fermentation liquid aiming at the defects of the prior art.

The purpose of the invention is realized as follows: a preparation method of high-activity lactobacillus fermentum grx08 fermentation liquor is characterized in that:

adding composite oligosaccharide into the papain enzymolysis whey protein hydrolysate, mixing uniformly, filtering, homogenizing, performing heat treatment, cooling, adding a fermentation agent of lactobacillus fermentum grx08, and fermenting to obtain the high-activity lactobacillus fermentum grx08 fermentation liquid.

Adding a sterilized aqueous solution in which a stabilizer and an emulsifier are dissolved into the high-activity lactobacillus fermentum grx08 fermentation liquid, and stirring, homogenizing and refrigerating to obtain the high-activity lactobacillus fermentum grx08 beverage.

The papain enzymolysis lactalbumin hydrolysate is prepared by the following steps:

adding 5000U g of papain 1000-^-1Carrying out enzymolysis at 55-65 ℃ for 2-3 h, centrifuging after enzymolysis is finished, taking supernate, and inactivating enzyme at 105 ℃/5min to obtain papain enzymolysis lactalbumin hydrolysate.

The composite oligosaccharide component comprises 0.20-0.40% w/w fructo-oligosaccharide, 0.3-0.50% w/w galacto-oligosaccharide, 0.3-0.5% w/w inulin, 0.6-0.8% w/w stachyose and 0.02-0.05% w/w xylo-oligosaccharide.

When the fermentation is carried out by adding the fermentation agent of the lactobacillus fermentum grx08, the inoculation amount of the fermentation agent of the lactobacillus fermentum grx08 is 2.0% -4.0%, and the fermentation is carried out at 37 ℃ for 18-24 h to prepare the high-activity lactobacillus fermentum grx08 fermentation liquid.

The preparation method of the lactobacillus ferments grx08 comprises the following steps:

inoculating a strain of 1 generation of lactobacillus fermentum grx08 into an MRS liquid culture medium according to the inoculation amount of 2-4%, and culturing in an incubator at 37 ℃ for 18h to obtain a lactobacillus fermentum grx 08;

when the MRS liquid culture medium is prepared, 10g of soybean peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 801ml of tween, 2g of sodium dihydrogen phosphate, 5g of anhydrous sodium acetate, 2g of citric acid triamine, 0.02g of manganese sulfate and 0.1g of magnesium sulfate are subjected to constant volume to 1L with distilled water, the pH value is adjusted to 6.2, and the MRS liquid culture medium is obtained after sterilization at 121 ℃ for 15 min.

Adding compound oligosaccharide into the papain enzymolysis whey protein hydrolysate, uniformly mixing, filtering, homogenizing under 20Mpa, performing heat treatment at 95 ℃ for 5-10 min, cooling to 37 ℃, and adding lactobacillus ferments grx08 for fermentation.

After fermentation with the fermentation agent of the lactobacillus fermentum grx08, dissolving a sterilized water solution of a stabilizer and an emulsifier, stirring, homogenizing, filling into a sales container, and refrigerating at 4 ℃ for 24h to obtain the high-activity lactobacillus fermentum grx08 fermentation liquid beverage.

Said Lactobacillus fermentum grx08(Lactobacillus fermentum) And (3) in 2013, 6, 9, and in China general microbiological culture Collection center (CGMCC), addresses: the microbial research institute of the Chinese academy of sciences, No. 3 Xilu-Beijing, Chaoyang, Beijing area, Beijing province, and the accession number is GCMCC NO: 7695.

the high-activity lactobacillus fermentum grx08 fermentation broth prepared by the preparation method of the high-activity lactobacillus fermentum grx08 fermentation broth is used for preparing fermented milk, functional food and medicines with high viable bacteria.

The method is advanced and scientific, and the preparation method and the application of the high-activity lactobacillus fermentum grx08 fermentation broth provided by the invention are characterized in that the compound oligosaccharide is added into papain enzymatic hydrolysis whey protein hydrolysate, after uniform stirring, the lactobacillus fermentum grx08 is added for fermentation, and the high-activity lactobacillus fermentum grx08 fermentation broth is prepared. Based on the combination of papain enzymolysis whey protein hydrolysate and composite oligosaccharide with the components including 0.20-0.40% w/w fructo-oligosaccharide, 0.3-0.50% w/w galacto-oligosaccharide, 0.3-0.5% w/w inulin, 0.6-0.8% w/w stachyose and 0.02-0.05% w/w xylo-oligosaccharide, lactobacillus fermentum grx08 with excellent performances of reducing blood fat, blood pressure and cholesterol is added to prepare the fermentation broth.

Lactobacillus fermentum grx08 (used in the present invention)Lactobacillus fermentum) And (3) in 2013, 6, 9, and in China general microbiological culture Collection center (CGMCC), addresses: the microbial research institute of the Chinese academy of sciences, No. 3 Xilu-Beijing, Chaoyang, Beijing area, Beijing province, and the accession number is GCMCC NO: 7695.

in the steps, the method comprises the following steps:

1. preparing a leavening agent;

inoculating the strain of 1 generation of lactobacillus fermentum grx08 into MRS liquid culture medium according to the inoculum size of 2-4%, and culturing in an incubator at 37 ℃ for 18 h.

2. Preparing and treating enzymolysis lactalbumin hydrolysate;

the enzymolysis lactalbumin hydrolysate for preparing the fermentation liquor comprises the following components: adding 5000U g of papain 1000-^-1Carrying out enzymolysis at 55-65 ℃ for 2-3 h, centrifuging after enzymolysis is finished, taking supernate, and inactivating enzyme at 105 ℃/5 min.

3. The mixed oligosaccharide of the fermentation liquor comprises the following components: 0.20 to 0.40 percent of fructo-oligosaccharide, 0.3 to 0.50 percent of galacto-oligosaccharide, 0.3 to 0.5 percent of inulin, 0.6 to 0.8 percent of stachyose and 0.02 to 0.05 percent of xylo-oligosaccharide.

4. Preparing fermentation liquor;

adding mixed oligosaccharide into the enzymatic whey protein hydrolysate, performing heat treatment, adding a fermentation agent of lactobacillus fermentum grx08 with the inoculum size of 2.0-4.0%, and fermenting at 37 ℃ for 18-24 h to prepare the microbial agent.

In conclusion, the invention is prepared by adding composite oligosaccharide on the basis of enzymolysis of whey protein hydrolysate by papain, adding lactobacillus fermentum grx08 with excellent performances of reducing blood fat, blood pressure, cholesterol and the like after conventional treatment, and fermenting. The invention can be used for preparing high viable bacteria synbiotic fermented milk, functional food, medicines and other series products.

The lactobacillus plays an important role in guaranteeing the quality of the food and improving the quality of the food. Research and development for improving and maintaining the viable count of the lactic acid bacteria are always paid extensive attention, and the utilization of oligosaccharide combination to improve the growth characteristics of the lactic acid bacteria brings huge economic benefits to the dairy product market.

Detailed Description

The invention is further illustrated by the following examples.

Example 1:

a preparation process of synbiotics solidified type fermented milk for improving viable count by using composite oligosaccharide combination comprises the following specific steps:

uniformly mixing papain enzymolysis whey protein hydrolysate with 0.28% of fructo-oligosaccharide, 0.4% of galacto-oligosaccharide, 0.4% of inulin, 0.8% of stachyose and 0.05% of xylo-oligosaccharide, filtering, homogenizing under 20Mpa, carrying out heat treatment at 95 ℃ for 5-10 min, cooling to about 37 ℃, inoculating 3% of lactobacillus fermentum grx08 starter, fermenting at 37 ℃ for 18h, and refrigerating at 4 ℃ for 24h to obtain the high-activity lactobacillus fermentum grx08 fermentation liquid.

Example 2:

uniformly mixing papain enzymolysis whey protein hydrolysate with 0.30% of fructo-oligosaccharide, 0.5% of galacto-oligosaccharide, 0.3% of inulin, 0.7% of stachyose and 0.03% of xylo-oligosaccharide, filtering, homogenizing under 20Mpa, carrying out heat treatment at 95 ℃ for 5-10 min, cooling to about 37 ℃, inoculating 3% of lactobacillus fermentum grx08 starter, fermenting at 37 ℃ for 18h to obtain fermentation liquor, stirring the fermentation liquor, adding sterilized aqueous solution dissolved with a stabilizer and an emulsifier, wherein the aqueous solution is 3 times of the fermentation liquor, homogenizing, filling into a sales container, and refrigerating at 4 ℃ for 24h to obtain the high-activity lactobacillus fermentum grx08 beverage. The viable count of Lactobacillus fermentum grx08 in the beverage is 1.23 × 10⁹CFU/mL。

In examples 1 and 2:

the preparation method of the papain enzymatic hydrolysis whey protein hydrolysate comprises the following steps:

adding 5000U g of papain 1000-^-1Carrying out enzymolysis at 55-65 ℃ for 2-3 h, centrifuging after enzymolysis is finished, taking supernate, and inactivating enzyme at 105 ℃/5min to obtain papain enzymolysis lactalbumin hydrolysate.

The preparation method of the lactobacillus ferments grx08 comprises the following steps: inoculating a strain 1 generation of lactobacillus fermentum grx08 in an MRS liquid culture medium according to an inoculation amount of 2-4%, and culturing in an incubator at 37 ℃ for 18h to obtain the lactobacillus fermentum grx08 strain;

when the MRS liquid culture medium is prepared, adding 1L of distilled water into 10g of soybean peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 801ml of tween, 2g of sodium dihydrogen phosphate, 5g of anhydrous sodium acetate, 2g of citric acid triamine, 0.02g of manganese sulfate and 0.1g of magnesium sulfate, adjusting the pH to about 6.2, and sterilizing at 121 ℃ for 15min to obtain the MRS liquid culture medium.

The present invention is not limited to the above-described embodiments, and any variations, modifications, and alterations that may occur to one skilled in the art without departing from the spirit of the invention are intended to be within the scope of the invention.