CN111728030A - Normal-temperature long-shelf-life sucrose-free yoghourt capable of improving immunity and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of food, and discloses normal-temperature long-shelf-life sucrose-free yoghourt capable of improving immunity and a preparation method thereof. The normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity comprises raw milk or milk powder and functional components capable of improving the immunity; the functional component for improving immunity at least comprises lactobacillus helveticus and/or a mutant thereof. The yoghourt is prepared by fermenting cow milk or reconstituted milk with lactobacillus helveticus, is compounded with various natural functional components for improving the immunity, and can improve the immunity of a human body after being eaten for a long time; in addition, the lactobacillus helveticus WHH2580 used in the invention still has the function of enhancing immunity in an inactivated state, so that the yoghurt can be normally stored at normal temperature without refrigeration and has long shelf life.

Description

Normal-temperature long-shelf-life sucrose-free yoghourt capable of improving immunity and preparation method thereof

Technical Field

The invention relates to the technical field of food, in particular to normal-temperature long-shelf-life sucrose-free yoghourt capable of improving immunity and a preparation method thereof.

Background

The season of autumn and winter is a period with more diseases every year, various epidemic diseases including influenza and the like begin to be too heavily abused, and children and old people with lower immunity are more serious areas of diseases. It is the "immunity" that is the best doctor. The measures of wearing a mask, frequently washing hands and the like can only prevent external virus invasion, and the immunity of the patient is the only strength on which the patient can rely once the patient is infected without specific medicines.

The milk is rich in nutrition, and contains multiple unique components capable of regulating immune function, such as immunoglobulin, lactoferrin and multiple amino acids. The lysozyme and lactoperoxidase contained in the dairy product can play an anti-infection role in the intestines, activate an immune system, enhance the activity of immune cells or improve the resistance of organisms to pathogenic bacteria, and protect the immune function of the bodies.

At present, a great number of documents report that probiotics can improve human immunity, for example, lactobacillus reuteri can be colonized in intestinal tracts, improve the distribution of intestinal flora, antagonize harmful bacteria, grow and improve host immunity. Meanwhile, the lactobacillus reuteri can generate 'reuterin', and can widely inhibit the growth of gram-negative bacteria, gram-positive bacteria, yeast, fungi, pathogenic protozoa and the like; the lactobacillus rhamnosus LGG can increase the secretory volume of IgA of intestinal mucosa, and the pathogenic bacteria of tissues infect epithelial cells to enhance the immunity; the Lactobacillus plantarum Lp9 and Lp91 can secrete anti-inflammatory cytokines in HT-29 cells induced by LPS and regulate immune functions. German research also shows that D-phenyllactic acid produced by fermentation of lactic acid bacteria can activate the G protein receptor HCA3 of human immune system, induce chemotaxis of neutrophil, and improve immunity. Brazil researchers found that acetate produced by lactic acid bacteria fermentation could stimulate the human GPR43-1 type interferon response against respiratory syncytial virus.

Propolis, ganoderma lucidum extract, wall-broken ganoderma lucidum spore powder, beta-glucan, dendrobium officinale extract, elderberry, acerola cherry and other food raw materials are generally considered to be rich in substances which are helpful to the immune function, such as flavone, polyphenol, ganoderic acid, fungus polysaccharide, mucopolysaccharide, anthocyanin, vitamin C and the like.

When the cow milk capable of improving the human immunity is fermented by probiotics capable of improving the human immunity, and is supplemented with an effective component capable of improving the human immunity, the three components are combined, the synergistic effect is achieved, and the effect of enhancing the immunity can be achieved well.

At present, as shown in fig. 2, the yogurt which is declared to enhance immunity in the market has the function of regulating immunity only when probiotics used are in a survival state, so that the yogurt can be stored only in a refrigeration mode, the shelf life is short (generally 15-21 days), and sugar substances such as white granulated sugar and the like are required to be added as a carbon source to maintain the activity of strains. The refrigeration storage means that the product has strict requirements on the storage, logistics and sale environments and has higher intermediate cost; the short shelf life leads to easy overdue of the product and waste; the white granulated sugar is required to be one of the intentions of obesity caused by the century, and is widely suffered by consumers and industries at present. At present, the normal-temperature long-shelf-life (3-6 months) sucrose-free yoghourt capable of improving immunity is still a market blank.

Disclosure of Invention

In order to solve the technical problems, the invention provides the sucrose-free yoghourt with the normal temperature and the long shelf life and the preparation method thereof, which can improve the immunity. The yogurt can effectively improve immunity, can be normally stored at normal temperature, does not need refrigeration, and has long shelf life.

The specific technical scheme of the invention is as follows:

a sucrose-free yogurt with long shelf life at normal temperature for improving immunity comprises raw milk or milk powder, and functional components for improving immunity; the functional component for improving immunity at least comprises lactobacillus helveticus and/or a mutant thereof;

the lactobacillus helveticus is named as WHH2580, and is preserved in the common microorganism center of the china culture collection and management committee for culture collection of microorganisms in 2019, month 10 and 23, and the microorganism preservation numbers are as follows: CGMCC No.18730, and the microorganism classification is named as Lactobacillus helveticus; the Lactobacillus helveticus has a 16s rRNA gene sequence shown in SEQ ID NO. 1; the mutant is obtained by carrying out mutagenesis, domestication, gene recombination or natural mutation on the lactobacillus helveticus.

The lactobacillus helveticus WHH2580 used in the invention is a probiotic strain screened from the yoghourt collected from the Hongshan ditch of Xinjiang nalat, and animal experiments prove that the lactobacillus helveticus strain has excellent adhesion performance, can smoothly reach the intestinal tract and is adhered to the epithelial cells of the intestinal tract to play a probiotic effect; the strain can improve in-vitro spleen lymphocyte proliferation and interleukin IL-12 secretion of spleen lymphocytes, and promote the secretion of interleukin IL-8 by human colon cancer epithelial cells HT-29; in addition, in an animal model with low immunity, the strain can also obviously improve the organ index of a mouse, promote the proliferation of splenic lymphocytes and improve the activity of NK cells. Therefore, the lactobacillus helveticus WHH2580 has a good effect of enhancing immunity. More importantly, the strain still has better immune function enhancement after inactivation. In addition, in the animal experiment process, the adverse states of death, lassitude, inappetence and the like of the experimental animal are not found, which indicates that the strain has higher safety.

Specifically, the lactobacillus helveticus WHH2580 has the following advantages:

(1) both living and non-living bodies have excellent adhesive properties: the cell adhesion performance of the strain is remarkably superior to that of a positive control strain of Lactobacillus rhamnosus LGG and a field strain LcS, and the number of single-cell adhesion living bacteria reaches 15.69 +/-5.94, and the number of single-cell adhesion non-living bacteria reaches 11.59 +/-4.02. The strain can adhere to intestinal epithelial cells to exert physiological effects. Although adhesion is not a prerequisite for the strains to possess probiotic properties, the interaction of probiotics with the intestinal mucosa is considered to be of critical importance for a number of reasons. Binding to the intestinal mucosa may prolong the time that the probiotic bacterial strain is present in the intestine. This interaction with the mucosa brings the probiotic bacteria into close contact with the intestinal immune system, giving it a better chance to modulate the immune response. It can also protect against enteric pathogens by limiting its ability to reside in the intestine.

(2) The acid-resistant and bile salt-resistant composite material has the following characteristics: under the environment of pH3.0, the survival of the strain is not obviously affected after 2h of incubation, and the viable count is reduced by 0.9 order of magnitude after 3 h; the pepsin has no obvious influence on the viability of the bacillus within 2 hours, and the viable count is reduced by 0.7 orders of magnitude within 3 hours; 3.6 orders of magnitude reduction in 0.5% bile salt system over 3 h. The food has good acid resistance and bile salt resistance, so that the food can better pass through the stomach and duodenum and smoothly reach the intestinal tract, thereby better playing the probiotic role; the good acid resistance enables the yogurt to adapt to a wider pH range in the fermentation process of preparing yogurt.

(3) In vitro cell test, it can promote proliferation of splenic lymphocyte in vitro, and the fold increase of no inducer is 1.912 + -0.087, and the fold increase of inducer is 1.533 + -0.109. Can promote spleen lymphocytes in vitro to secrete interleukin IL-12; the IL-12/IL-10 ratio was not significantly different from that of Lactobacillus rhamnosus LGG. In an in vitro cell test, the interleukin IL-8 secretion of the human colon cancer epithelial cells HT-29 can be promoted; IL-8, which stimulates secretion, is significantly higher than that of the commercial strain LGG.

(4) The compound preparation has good inhibition effect on staphylococcus aureus, salmonella paratyphi B, escherichia coli, shigella flexneri and micrococcus luteus, so that the compound preparation can inhibit pathogenic bacteria in intestinal tracts well, regulate intestinal flora and play a probiotic role.

(5) The living and non-living body have immunity enhancing effect, and the administration concentration of the living strain is 2 × 10⁸CFU/d, concentration of fire extinguishing strain taking 2 × 10⁸CFU/d can obviously improve the organ index of mice, promote the proliferation of splenic lymphocytes and improve the activity of NK cells. This can be doneThe probiotics in the yoghourt can exist in a non-living form, so that the yoghourt can have a longer shelf life at normal temperature without refrigeration.

Preferably, the yoghourt also comprises sugar alcohol, starch, a stabilizer and a sweetener.

Preferably, the yoghourt comprises 40-85% of raw milk or 5-15% of milk powder, 0.001-0.020% of lactobacillus helveticus and/or mutants thereof, 0.01-10.00% of components with the function of improving immunity except lactobacillus helveticus and/or mutants thereof, 0.5-5.0% of sugar alcohol, 0.1-5.0% of starch, 0.01-1.00% of stabilizing agent, 0.001-0.050% of sweetening agent and the balance of water in percentage by mass; the sum of all the raw materials is 100 percent.

The mass percentage is the percentage of the total mass of the yoghourt.

Preferably, the milk powder is one or more of whole milk powder, skim milk powder, whey protein powder and whole milk protein powder.

Preferably, the functional components for improving immunity further comprise one or more of propolis, ganoderma lucidum extract, wall-broken ganoderma lucidum spore powder, beta-glucan, dendrobium officinale extract, elderberry and acerola cherry.

Preferably, the sugar alcohol is one or more of xylitol, maltitol, erythritol, sorbitol, isomalt and lactitol.

Preferably, the starch is one or more of physical starch, acetate starch, sodium starch octenyl succinate, hydroxypropyl distarch phosphate, phosphate distarch, acid-treated starch, oxidized hydroxypropyl starch, acetylated distarch phosphate and acetylated distarch adipate.

Preferably, the stabilizer is one or more of sodium carboxymethylcellulose, pectin, soybean polysaccharide, agar, sodium caseinate, propylene glycol alginate, gelatin, gellan gum, guar gum and xanthan gum.

Preferably, the sweetener is one or more of acesulfame potassium, aspartame, sucralose, stevioside, mogroside, isomaltulose, edmuntin and neotame.

A method for preparing the sucrose-free yoghourt with the normal temperature and the long shelf life and the function of improving the immunity comprises the following steps:

scheme A:

(1) homogenizing and sterilizing: preheating raw milk, homogenizing, sterilizing, and cooling to obtain sterilized cooling feed liquid;

(2) inoculating and fermenting: adding lactobacillus helveticus and/or a mutant thereof into the sterilized cooling liquid obtained in the step (1), uniformly stirring, standing at a constant temperature, and fermenting to obtain a yogurt base material;

(3) preparing glue solution: preheating water, adding a formula amount of stabilizer into the water, shearing or stirring, and cooling to obtain a glue solution;

(4) secondary burdening: cooling the yogurt base material obtained in the step (2), and then stirring and demulsifying; adding the glue solution obtained in the step (3), adding the components with the formula dosage, sugar alcohol, sweetening agent and starch for improving the immunity except the lactobacillus helveticus and/or the mutant thereof, mixing and stirring, and adding water to a constant volume to obtain a feed liquid;

(5) and (3) secondary homogenization: homogenizing the feed liquid obtained in the step (4);

(6) filling and sterilizing: filling, sealing, pasteurizing and cooling the homogenized material liquid in the step (5) to obtain the normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity; or (5) carrying out UHT sterilization on the homogenized material liquid, carrying out hot filling or aseptic cold filling, and sealing to obtain the sucrose-free yoghourt with the immunity improving function and the long shelf life at normal temperature;

scheme B:

1) preparing milk: heating water, adding the formula amount of milk powder into the water, stirring or shearing, and standing for hydration to obtain milk;

2) homogenizing and sterilizing: preheating the milk obtained in the step 1), homogenizing, sterilizing and cooling to obtain a sterilized cooling feed liquid;

3) inoculating and fermenting: adding lactobacillus helveticus and/or a mutant thereof into the sterilized cooling liquid obtained in the step 2), uniformly stirring, standing at a constant temperature, and fermenting to obtain a yogurt base material;

4) preparing glue solution: preheating water, adding a formula amount of stabilizer into the water, shearing or stirring, and cooling to obtain a glue solution;

5) secondary burdening: cooling the yogurt base material obtained in the step 3), and then stirring and demulsifying. Adding the glue solution obtained in the step 4), adding the components with the formula dosage, sugar alcohol, sweetener and starch for improving the immunity except the lactobacillus helveticus and/or the mutant thereof, mixing and stirring, and adding water to a constant volume to obtain a feed liquid;

6) and (3) secondary homogenization: homogenizing the feed liquid obtained in the step 5);

7) filling and sterilizing: filling, sealing, pasteurizing and cooling the homogenized material liquid obtained in the step 6) to obtain the normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity; or carrying out UHT sterilization on the homogenized material liquid in the step 6), carrying out hot filling or aseptic cold filling, and sealing to obtain the sucrose-free yoghourt with the immunity improving function and the long shelf life at normal temperature.

The yoghourt prepared by pretreating, homogenizing, sterilizing, fermenting, re-sterilizing and filling the raw milk or milk powder and various raw and auxiliary materials can be normally stored at normal temperature without refrigeration, and the quality guarantee period can reach 150-180 days; meanwhile, the functional components are furthest kept from being damaged in the processing process, so that the functionality of the product is ensured.

Preferably, in step 1), the mass of the water is 200-400 g.

Preferably, in step 1), the water is heated to a temperature of 45-60 ℃.

Preferably, in step 1), the stirring or shearing time is 15 to 30 min.

Preferably, in the step 1), the standing hydration time is 20-40 min.

Preferably, in step (1) or step 2), the preheating is carried out to a temperature of 60 to 65 ℃.

Preferably, in the step (1) or the step 2), the homogenization pressure is 0 to 8/5 to 30 MPa.

Preferably, in the step (1) or the step 2), the sterilization temperature is 90-97 ℃ and the time is 3-10 min.

Preferably, in step (1) or step 2), the cooling is carried out to a temperature of 35 to 45 ℃.

Preferably, in the step (2) or the step 3), the fermentation temperature is 35-45 ℃ and the fermentation time is 4-16 h.

Preferably, in the step (2) or the step 3), the number of viable bacteria is more than or equal to 1 × 10 during the fermentation process⁸cfu/mL。

Preferably, in the step (3) or the step 4), the mass of the water is 200-400 g.

Preferably, in step (3) or step 4), the water is preheated to a temperature of 50-60 ℃.

Preferably, in step (3) or step 4), the shearing or stirring time is 20-40 min.

Preferably, in step (3) or step 4), the cooling is carried out to a temperature of 30 ℃ or lower.

Preferably, in step (4) or step 5), the cooling is carried out to a temperature of 15 ℃ or lower.

Preferably, in step (4) or step 5), the volume is set to 1000mL with water.

Preferably, in the step (5) or 6), the homogenization pressure is 0 to 8/5 to 30 MPa.

Compared with the prior art, the invention has the following advantages:

(1) the yoghourt is prepared by fermenting cow milk or reconstituted milk with lactobacillus helveticus WHH2580 and/or mutants thereof, is compounded with various natural functional components for improving the immunity, and can improve the immunity of a human body after being eaten for a long time;

(2) the lactobacillus helveticus WHH-2580 used in the formula of the yoghurt disclosed by the invention is a probiotic strain with an immunity-enhancing function, and is characterized in that the lactobacillus helveticus WHH-2580 still has an immunity-enhancing function in an inactivated state, so that the yoghurt disclosed by the invention can be pasteurized or UHT sterilized and has the characteristic of being preserved at normal temperature;

(3) the yoghourt is prepared by pretreating, homogenizing, sterilizing, fermenting, sterilizing and filling raw milk or milk powder and various raw and auxiliary materials, can be normally stored at normal temperature without refrigeration, and has a shelf life of up to 150-180 days; meanwhile, the functional components are furthest kept from being damaged in the processing process, so that the functionality of the product is ensured;

(4) the yoghourt disclosed by the invention is free of sucrose, can be drunk by various people in daily life, particularly people (diabetes, obesity and the like) with low autoimmunity and limited sugar intake and people who do not like cold food, has the advantages of high acceptability, high safety and no side effect, and has important efficacy value and social value.

Drawings

FIG. 1 is a flow chart of a method for preparing the sucrose-free yogurt with long shelf life at normal temperature for enhancing immunity according to the present invention;

FIG. 2 is a schematic diagram of a prior art process for preparing a yogurt with enhanced immunity;

FIG. 3 is a simplified diagram of a process for preparing the sucrose-free yogurt with long shelf life at room temperature for enhancing immunity according to the present invention;

FIG. 4 is a graph of thymus index of each group of mice in example 4;

FIG. 5 is a graph showing the results of experiments on spleen lymphocyte transformation in each group of mice in example 4;

FIG. 6 is a graph showing NK cell activities of various groups of mice in example 4;

FIG. 7 shows the morphology of Lactobacillus helveticus WHH2580 observed under a microscope;

FIG. 8 is a graph showing the effect of cell adhesion between viable, nonviable and commercial strains of Lactobacillus helveticus WHH2580, LGG and Lcs, and HT-29;

FIG. 9 is a graph showing the body weight change of BALB/c mice in each group in the animal test of example 15;

FIG. 10 is a graph showing organ ratios of BALB/c mice in each group in the animal test of example 15;

FIG. 11 is a graph showing spleen lymphocyte transformation numbers of groups of BALB/c mice in the animal test of example 15;

FIG. 12 is a graph showing NK cell activities of groups of BALB/c mice in example 15.

Detailed Description

The present invention will be further described with reference to the following examples.

General examples

A normal temperature long-shelf-life sucrose-free yoghourt for improving immunity comprises, by mass, 40% -85% of raw milk or 5-15% of milk powder, 0.001-0.020% of Lactobacillus helveticus and/or mutants thereof, 0.01-10.00% of components for improving immunity except the Lactobacillus helveticus and/or mutants thereof, 0.5-5.0% of sugar alcohol, 0.1-5.0% of starch, 0.01-1.00% of stabilizer, 0.001-0.050% of sweetener and the balance of water; the sum of all the raw materials is 100 percent.

The normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity is prepared through the following steps (the preparation process is shown as figure 1, and the schematic diagram is shown as figure 3):

scheme A:

(1) homogenizing and sterilizing: preheating raw milk to 60-65 ℃, and homogenizing under the homogenizing pressure of 0-8/5-30 MPa; sterilizing at 90-97 deg.C for 3-10min, and cooling to 35-45 deg.C to obtain sterilized cooling liquid;

the sterilization is the same as the conventional sterilization conditions in the field, such as pasteurization treatment;

(2) inoculating and fermenting, namely adding the Lactobacillus helveticus and/or the mutant thereof into the sterilized cooling feed liquid obtained in the step (1), uniformly stirring, placing in a constant-temperature incubator at 35-45 ℃, standing and fermenting for 4-16h, wherein the viable count is not less than 1 × 10⁸cfu/mL to obtain the yoghourt base material;

(3) preparing glue solution: weighing 200g-400g of ultrapure water, preheating to 50-60 ℃, adding the stabilizer with the formula amount into the ultrapure water, shearing or stirring for 20-40min, and cooling to below 30 ℃ to obtain glue solution;

(4) secondary burdening: cooling the yogurt base material obtained in the step (2) to below 15 ℃, and then stirring for demulsification; addition step

(3) Adding the components with the immunity improving effect except the lactobacillus helveticus and/or the mutant thereof, sugar alcohol, a sweetening agent and starch into the glue solution obtained in the step (1), mixing and stirring, and fixing the volume to 1000mL by using ultrapure water to obtain a feed liquid;

(5) and (3) secondary homogenization: homogenizing the feed liquid obtained in the step (4) at the pressure of 0-8/5-30 MPa;

(6) filling and sterilizing: filling the homogenized material liquid obtained in the step (5) into an HDPE material packaging container, sealing, and performing pasteurization and cooling to obtain the sucrose-free yoghourt with the immunity improving function and the long shelf life at normal temperature; or (5) carrying out UHT sterilization on the homogenized material liquid, carrying out hot filling or aseptic cold filling into a PET material packaging container, and sealing to obtain the normal-temperature sucrose-free yoghourt with long shelf life and improved immunity;

the pasteurization and UHT sterilization are the same as conventional sterilization conditions in the art.

Scheme B:

1) preparing milk: weighing 200 and 400g of ultrapure water, heating to 45-60 ℃, adding the milk powder with the formula amount into the ultrapure water, stirring or shearing for 15-30min, and standing for hydration for 20-40 min;

2) homogenizing and sterilizing: preheating the milk obtained in the step 1) to 60-65 ℃, and then homogenizing under the homogenizing pressure of 0-8/5-30 MPa; sterilizing at 90-97 deg.C for 3-10min, and cooling to 35-45 deg.C to obtain sterilized cooling liquid;

the sterilization is the same as the conventional sterilization conditions in the field, such as pasteurization treatment;

3) inoculating and fermenting, namely adding the Lactobacillus helveticus and/or the mutant thereof into the sterilized cooling feed liquid obtained in the step 2), uniformly stirring, placing in a constant-temperature incubator at 35-45 ℃, standing and fermenting for 4-16h, wherein the viable count is not less than 1 × 10⁸cfu/mL to obtain the yoghourt base material;

4) preparing glue solution: weighing 200g-400g of ultrapure water, preheating to 50-60 ℃, adding the stabilizer with the formula amount into the ultrapure water, shearing or stirring for 20-40min, and cooling to below 30 ℃ to obtain glue solution;

5) secondary burdening: cooling the yogurt base material obtained in the step 3) to below 15 ℃, and then stirring for demulsification; adding the glue solution obtained in the step 4), adding the components with the formula dosage, sugar alcohol, sweetener and starch for improving the immunity except the lactobacillus helveticus and/or the mutant thereof, mixing and stirring, and fixing the volume to 1000mL by using ultrapure water to obtain a feed liquid;

6) and (3) secondary homogenization: homogenizing the feed liquid obtained in the step 5), wherein the homogenizing pressure is 0-8/5-30 MPa;

7) filling and sterilizing: filling the homogenized material liquid obtained in the step 6) into an HDPE material packaging container, sealing, and performing pasteurization and cooling to obtain the sucrose-free yoghourt with the immunity improving function and the long shelf life at normal temperature; or carrying out UHT sterilization on the homogenized material liquid in the step 6), then carrying out hot filling or aseptic cold filling on the material liquid into a PET material packaging container, and sealing the container to obtain the sucrose-free yoghourt with the immunity improving function and the long shelf life at normal temperature;

the pasteurization and UHT sterilization are the same as conventional sterilization conditions in the art.

Example 1

The ingredients of the present example consist of whole milk powder, xylitol, elderberry concentrated juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, sucralose, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each ingredient is calculated by total amount of 1000 ml: 100 g of whole milk powder, 20g of xylitol, 0.1 g of sucralose, 5 g of elderberry concentrated juice, 5 g of hydroxypropyl distarch phosphate, 5 g of sodium carboxymethyl cellulose, 0.2 g of gellan gum, 0. 25800.02 g of lactobacillus helveticus and the balance of water.

The preparation procedure of this example is as follows:

1) preparing milk: weighing 300g of ultrapure water, preheating to 55 ℃, adding the whole milk powder with the formula amount into the ultrapure water, shearing at a medium speed for 15min, and standing for hydration for 20 min;

2) homogenizing and sterilizing: preheating the milk obtained in the step 1) to 65 ℃, and homogenizing under 5/20 Mpa; sterilizing at 95-96 deg.C for 5min, and cooling to 42 + -1 deg.C to obtain sterilized cooling liquid;

3) inoculating and fermenting, namely adding lactobacillus helveticus WHH-2580 into the sterilized cooling feed liquid obtained in the step 2), uniformly stirring, standing and fermenting for 6 hours at 42 ℃ in a constant-temperature incubator, wherein the viable count is more than or equal to 1 × 10⁸cfu/mL to obtain the yoghourt base material;

4) preparing glue solution: weighing 300g of ultrapure water, heating to 55 ℃, adding the stabilizer with the formula amount into the ultrapure water, shearing at a medium speed for 20min, and cooling to below 30 ℃ to obtain a glue solution;

5) secondary burdening: cooling the yogurt base material obtained in the step 3) to below 15 ℃, and then stirring for demulsification; adding the glue solution obtained in the step 4), adding sugar alcohol, a sweetening agent, starch and components for improving the immunity except the lactobacillus helveticus and/or the mutant thereof according to the formula dosage, mixing and stirring, and fixing the volume to 1000mL by using ultrapure water to obtain a feed liquid;

6) and (3) secondary homogenization: homogenizing the yogurt base material obtained in the step 5), wherein the homogenizing pressure is 0/5 MPa;

7) filling and sterilizing: filling the homogenized material liquid obtained in the step 6) into an HDPE material packaging container, sealing, performing pasteurization at 87 ℃ for 18min, and cooling to obtain the sucrose-free yoghourt with long shelf life at normal temperature and capable of improving immunity.

Example 2

The ingredients of the present example consist of whole milk powder, xylitol, elderberry concentrated juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, acesulfame potassium, aspartame, ganoderma lucidum extract, dendrobium officinale extract, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each ingredient is calculated by total amount of 1000 ml: 105 g of whole milk powder, 15 g of xylitol, 1 g of elderberry concentrated juice, 10g of hydroxypropyl distarch phosphate, 6 g of sodium carboxymethylcellulose, 0.25 g of gellan gum, 0.1 g of acesulfame, 0.1 g of aspartame, 2 g of ganoderma lucidum extract, 2 g of dendrobium officinale extract, WHH-25800.05 g of lactobacillus helveticus and the balance of water.

The preparation method of this example is the same as example 1.

Example 3

The ingredients of the present example consist of whole milk powder, maltitol, acetylated distarch phosphate, pectin, soybean polysaccharide, stevioside, propolis, wall-broken ganoderma lucidum spore powder, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each ingredient is 1000ml in total: 105 g of milk powder, 40 g of maltitol, 5 g of acetylated distarch phosphate, 5 g of pectin, 2 g of soybean polysaccharide, 0.2 g of stevioside, 2 g of propolis, 2 g of wall-broken ganoderma lucidum spore powder, WHH-25800.07 g of lactobacillus helveticus and the balance of water.

The preparation method of this example is the same as example 1.

Example 4

The ingredients of the present example consist of whole milk powder, xylitol, elderberry concentrated juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, acesulfame potassium, aspartame, ganoderma lucidum extract, dendrobium officinale extract, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each ingredient is calculated by total amount of 1000 ml: 105 g of whole milk powder, 15 g of xylitol, 1 g of elderberry concentrated juice, 10g of hydroxypropyl distarch phosphate, 6 g of sodium carboxymethylcellulose, 0.25 g of gellan gum, 0.1 g of acesulfame, 0.1 g of aspartame, 2 g of ganoderma lucidum extract, 2 g of dendrobium officinale extract, WHH-25800.05 g of lactobacillus helveticus and the balance of water.

The preparation procedure of this example is as follows:

1) preparing milk: weighing 300g of ultrapure water, preheating to 50 ℃, adding the whole milk powder with the formula amount into the ultrapure water, shearing at a medium speed for 15min, and standing for hydration for 20 min;

2) homogenizing and sterilizing: preheating the milk obtained in the step 1) to 60 ℃, and then homogenizing at 5/20 MPa; sterilizing at 95-96 deg.C for 5min, and cooling to 37 + -1 deg.C to obtain sterilized cooling liquid;

3) inoculating and fermenting, namely adding lactobacillus helveticus WHH-2580 into the sterilized cooling feed liquid obtained in the step 2), uniformly stirring, placing in a constant-temperature incubator at 37 ℃, standing and fermenting for 16h, wherein the viable count is more than or equal to 1 × 10⁸cfu/mL to obtain the yoghourt base material;

4) preparing glue solution: weighing 300g of ultrapure water, heating to 55 ℃, adding the stabilizer with the formula amount into the ultrapure water, shearing at a medium speed for 20min, and cooling to below 30 ℃ to obtain a glue solution;

5) secondary burdening: cooling the yogurt base material obtained in the step 3) to below 15 ℃, and then stirring for demulsification; adding the cooled stabilizer, adding sugar alcohol, sweetener, starch and components for improving immunity except lactobacillus helveticus and/or mutants thereof according to the formula dosage, mixing and stirring, and adding ultrapure water to a constant volume of 1000mL to obtain a feed liquid;

6) and (3) secondary homogenization: homogenizing the yogurt base material obtained in the step 5), wherein the homogenizing pressure is 0/5 Mpa;

7) filling and sterilizing: sterilizing the homogenized material liquid in the step 6) by UHT at 121 ℃ for 15s, filling into a PET material packaging container, and sealing to obtain the sucrose-free yoghourt with long shelf life at normal temperature and capable of improving immunity.

Example 5

The ingredients of the present example consist of whole milk powder, maltitol, acetylated distarch phosphate, pectin, soybean polysaccharide, stevioside, propolis, wall-broken ganoderma lucidum spore powder, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each ingredient is 1000ml in total: 105 g of milk powder, 40 g of maltitol, 5 g of acetylated distarch phosphate, 5 g of pectin, 2 g of soybean polysaccharide, 0.2 g of stevioside, 2 g of propolis, 2 g of wall-broken ganoderma lucidum spore powder, WHH-25800.07 g of lactobacillus helveticus and the balance of water.

The preparation method of this example is the same as example 4.

Example 6

The ingredients of the present example consist of whole milk powder, xylitol, elderberry concentrated juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, sucralose, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each ingredient is calculated by total amount of 1000 ml: 100 g of whole milk powder, 20g of xylitol, 0.1 g of sucralose, 5 g of elderberry concentrated juice, 5 g of hydroxypropyl distarch phosphate, 5 g of sodium carboxymethyl cellulose, 0.2 g of gellan gum, 0. 25800.02 g of lactobacillus helveticus and the balance of water.

The preparation method of this example is the same as example 4.

Example 7

The ingredients of the present example consist of raw milk, xylitol, elderberry concentrated juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, sucralose, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each ingredient is in a total amount of 1000 ml: 700 g of raw milk, 20g of xylitol, 0.1 g of sucralose, 5 g of elderberry concentrated juice, 5 g of hydroxypropyl distarch phosphate, 5 g of sodium carboxymethyl cellulose, 0.2 g of gellan gum, 0. 25800.02 g of lactobacillus helveticus and the balance of water.

The preparation procedure of this example is as follows:

1) milk recovery: cooling the qualified raw milk to 4 ℃ and putting the cooled raw milk into a milk storage tank;

2) homogenizing and sterilizing: preheating the milk obtained in the step 1) to 65 ℃, and homogenizing under 5/20 MPa; sterilizing at 95-96 deg.C for 8min, and cooling to 40 + -1 deg.C to obtain sterilized cooling liquid;

3) inoculating and fermenting, namely adding lactobacillus helveticus WHH-2580 into the sterilized cooling feed liquid obtained in the step 2), uniformly stirring, placing in a constant-temperature incubator at 40 ℃, standing and fermenting for 8 hours, wherein the viable count is more than or equal to 1 × 10⁸cfu/mL to obtain the yoghourt base material;

4) preparing glue solution: weighing 250g of ultrapure water, heating to 55 ℃, adding the stabilizer with the formula amount into the ultrapure water, shearing at a medium speed for 20min, and cooling to below 30 ℃ to obtain a glue solution;

5) secondary burdening: cooling the yogurt base material obtained in the step 3) to below 15 ℃, and then stirring for demulsification; adding the cooled stabilizer, adding sugar alcohol, sweetener, starch and components for improving immunity except lactobacillus helveticus and/or mutants thereof according to the formula dosage, mixing and stirring, and adding ultrapure water to a constant volume of 1000mL to obtain a feed liquid;

6) and (3) secondary homogenization: homogenizing the yogurt base material obtained in the step 5), wherein the homogenizing pressure is 0/5 Mpa;

7) filling and sterilizing: filling the homogenized material liquid obtained in the step 6) into an HDPE material packaging container, sealing, performing pasteurization at 87 ℃ for 18min, and cooling to obtain the sucrose-free yoghourt with long shelf life at normal temperature and capable of improving immunity.

Example 8: functionality testing of yoghurts obtained according to the invention

The yoghourt obtained in the embodiment 1 of the invention is fed to mice, and after a period of time, cyclophosphamide is injected to be used as an immunosuppressant to observe the improvement effect of the product on the mouse immunity, the number of lactobacillus helveticus WHH-2580 can reach 1 × 10 before the yoghourt obtained in the invention is sterilized⁸cfu/mL。

Adopting a random grouping design, 44 inbred line BALB/C male mice (6-8 weeks old, 16-20g) are subjected to an adaptation period of 5 days and are randomly divided into 4 groups: 11 blank control groups (CK), model groups (M), a molding + yoghourt 5 times group (Y5) and a molding + yoghourt 10 times group (Y10) respectively. The 5-time yoghourt group and the 10-time yoghourt group respectively simulate that a normal person (60kg of body weight) takes 200g and 400g of the sucrose-free yoghourt with normal temperature and long shelf life, which can improve the immunity.

Keeping the environment temperature of the animal breeding at 21 +/-2 ℃, the humidity at 30-70%, illuminating for 12h alternately, freely drinking water and freely taking the feed. Changing padding every three days, and adding water and feed in time. Subjects were administered for 33 consecutive days. The molding drug used was cyclophosphamide (50mg/kg, intraperitoneal injection, two consecutive days), and the mice were sacrificed 5 days after molding, and thymus/body weight ratio measurement, mouse spleen lymphocyte transformation (MTT method) experiment and NK cell activity measurement were performed.

Grouping condition:

group 1 blank (n ═ 11): irrigating stomach water;

group 2 model group (n ═ 11): perfusing stomach water, and molding cyclophosphamide;

group 3 molding + 5 times of yogurt (n is 11): intragastric administration of 5 times of 0.2 ml/d of yoghourt, and molding by cyclophosphamide;

group 4 molding +10 times of yogurt (n is 11): and (3) intragastric administration of 10 times of 0.2 ml/d of yoghourt, and molding by using cyclophosphamide.

Thymus index: as shown in fig. 4, the thymus/body weight ratio of the model group was significantly lower than that of the blank control group (P <0.05), and the remaining model groups were not significantly different from the model group.

Mouse spleen lymphocyte transformation experiments: as shown in FIG. 5, the difference (P <0.05) between the model group and the model group was significant, indicating that the experimental groups can promote the proliferation of splenic lymphocytes of immunocompromised mice.

Determination of NK cell Activity: as shown in fig. 6, the NK cell activity of the model group was significantly lower than that of the blank control group (P < 0.05). The NK cell activity of 5 times of yoghurt and 10 times of yoghurt is obviously higher than that of the model group (P < 0.05).

According to the method for judging the result in the method for evaluating the function of the health-care product, under the condition that an immune function reduction model is established, the total number of blood leucocytes, the cellular immune function (a mouse spleen lymphocyte transformation experiment, a delayed type allergic reaction experiment), the humoral immune function (antibody-producing cell detection, serum hemolysin determination), the mononuclear-macrophage function (a mouse clearance experiment, a mouse abdominal cavity macrophage phagocytosis fluorescent microsphere experiment) and the NK cell activity are positive, and the test result is judged to be positive in any two aspects, so that the test sample has the effect of enhancing the immunity of a person with low immune function. In the animal experiment and the mouse spleen lymphocyte transformation experiment, the OD values of the 5-time yoghourt group and the 10-time yoghourt group are obviously higher than those of the model group, and the result is positive. In the NK cell activity indexes, the NK cell activities of 5 times of yogurt groups and 10 times of yogurt groups are obviously higher than those of model groups, and the result is positive. Therefore, 5 times of yoghourt and 10 times of yoghourt have the function of enhancing the immunity of mice with low immunity.

Example 9

The strain provided by the invention belongs to Lactobacillus helveticus (Lactobacillus helveticus) through identification, is named as WHH2580, is preserved in the China general microbiological culture Collection center at 23/10 th 2019, and has the microbiological preservation number as follows: CGMCC No. 18730.

The strain provided by the invention is obtained by screening yogurt collected from the Hongshan ditch of Xinjiang Nalati.

The biological properties of lactobacillus helveticus 2580 of the present invention are as follows:

morphological characteristics: the growth state in MRS agar medium is: the growth form of the bacterial colony in the MRS agar culture medium is light milky colony, and the bacterial colony is semitransparent, round, rough in surface, irregular in edge and flat. Gram staining was typically positive, and cells were observed microscopically to be long-rod, nonfilaginous, non-sporulating, and non-motile (FIG. 7).

The culture characteristics are as follows: the optimal growth temperature is 37 ℃, and the culture medium is facultative anaerobic and grows in MRS culture medium.

Physiological characteristics: using the API 50CHL system, WHH2580 strain can utilize 6 carbon sources: d-glucose, D-fructose, D-mannose, N-acetylglucosamine, D-lactose and D-trehalose.

Biological identification: and (3) sequencing a 16s rRNA gene sequence, and performing homology comparison analysis on the obtained result and an NCBI GenBank database to show that the strain is Lactobacillus helveticus (Lactobacillus helveticus).

The gene sequence is shown as SEQ ID NO. 1:

CCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGC GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCG ATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGA GATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAG CCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCG TTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGT CTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTG GTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCC GTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGT TAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCAT GGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTG CAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCT ACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTC CTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGC CCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTA GCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTC ACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATC AGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGT GTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTA AGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACG CCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCA AGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGC GTCCCCAGCGTCATTACCGAAGTAAATCTGCTGGTTCTGCTCGCTCGACTTGC

example 10: tolerance of strains

Acid and bile salt resistance of the lactobacillus helveticus WHH2580 strain:

1 acid resistance: the second generation of WHH2580 strain activated bacteria solution is centrifuged at 20 mL/tube, and washed twice with PBS (pH 7.2). The bacterial sludge was placed in 20mL sterile PBS pH3.0, PBS + 0.3% peptin pH3.0, and PBS pH7.2, respectively, with two replicates per system. Sampling at 37 deg.C for 0h, 1h, 2h and 3h respectively, and counting viable bacteria.

The acid resistance experiment results show that: the pH of 3.0 has no obvious influence on the viability of the bacteria within 2h, and the viable count is reduced by 0.9 order of magnitude within 3 h. The pepsin pH3.0 has no obvious influence on the viability of the bacteria within 2h, and the viable count within 3h is reduced by 0.7 order of magnitude. The above results indicate that the bacterium has excellent acid resistance.

2, bile salt resistance: the second generation of WHH2580 bacterial strain after activation is centrifuged in 20 mL/tube and washed twice with PBS (pH 7.2PBS). The bacterial sludge was placed in 20mL sterile PBS + 0.1% Trypsin ph8.0, PBS + 0.3% BS ph8.0, PBS + 0.5% bsph8.0, and PBS ph7.2, respectively, with two replicates for each system. Sampling at 37 deg.C for 0h, 1h, 2h and 3h respectively, and counting viable bacteria.

The results of the bile salt resistance experiment show that: 3h in a 0.1% Trypsin system is reduced by 0.8 magnitude order, 3h in a 0.3% BS system is reduced by 2.8 magnitude order, and 3h in a 0.5% BS system is reduced by 3.6 magnitude order. The results show that the bacterium has good bile salt resistance.

Example 11: adhesion of strains

The probiotics colonize epithelial cells of a host intestine through adhesion, gradually form a biological membrane and produce various metabolites, so that a natural protective barrier is formed, and various physiological effects are produced on the host. Thus, the adherence and colonization of the host intestinal epithelial cells by probiotics is a prerequisite for their probiotic action.

HT-29 cells are used as cell strains for adhesion tests, and the specific experimental method is as follows:

HT-29 cells were passed to the third generation, digested with 0.25% trypsin and centrifuged at 1000rpm for 5min, and resuspended in 5mL of DMEM medium containing 10% fetal bovine serum (100U/mL penicillin, 100mg/mL streptomycin) until the cells appearedTaking a proper amount of cells, counting the cells by using a blood cell counting plate, and diluting the cells by using a DMEM medium (containing penicillin 100U/mL and streptomycin 100mg/mL) containing 10% fetal calf serum until the concentration of the cell suspension is 1 × 10⁶cells/mL, 1mL was inoculated into a 12-well cell culture plate containing a cell slide at 37 ℃ with 5% CO₂The culture was carried out for 2 days.

After the WHH2580 strain (LGG of lactobacillus rhamnosus and the Dysidema strain LcS are positive control strains) is activated for the second generation, 10mL of bacterial liquid is taken, 4200g of centrifugal force is carried out for 10min at room temperature, the bacterial body is collected, 10mL of DMEM complete culture medium containing 10% fetal calf serum is used for culture (double antibody is not added), and the bacterial concentration is adjusted to 2 × 10⁸CFU/mL. 5mL of strain adjustment solution is inactivated for 5min at 95 ℃. Inoculating 1mL of each of the strain adjusting solution and the inactivated strain adjusting solution into 12-well cell culture plate containing cell slide, 3 of each of the solutions in parallel to CO₂Incubate at 37 ℃ for 2h in an incubator.

After incubation, the culture medium was slowly aspirated, washed 3 times with PBS, and fixed with 100% methanol for 8 min. Taking out the cell slide, standing for 20min, gram staining, and sealing with neutral resin. Observation was performed under an optical microscope.

The results of the adhesion experiments are given in the table below.

The effect of cell adhesion between live, non-live and commercial strains LGG and Lcs of Lactobacillus helveticus WHH2580 and HT-29 is shown in FIG. 8.

The adhesion capability of the live bacteria and the non-live bacteria of the lactobacillus helveticus WHH2580 is obviously higher than that of the positive control strains of the lactobacillus rhamnosus LGG and the genealogical strain LcS. The adhesion rate of non-living bacteria of the lactobacillus helveticus WHH2580 is slightly lower than that of the living bacteria, but the adhesion effect of the non-living bacteria is better than that of the living bacteria, the thallus among HT-29 cells is less, and most of the non-living bacteria are tightly adhered to the cells.

Example 12: resistance of strains

Sensitivity of lactobacillus helveticus WHH2580 strain antibiotics:

the sensitivity of probiotics to antibiotics is an important indicator for testing the safety of strains. In order to test the safety of the WHH2580 strain, the following 9 antibiotics are selected for testing, and the sensitivity of the WHH2580 strain to the 9 antibiotics is tested, and the specific experimental design is as follows.

The antibiotic preparation concentration is 0.5ug/mL, 1ug/mL, 2ug/mL, 4ug/mL, 8ug/mL, 16ug/mL, 32ug/mL, 64ug/mL, 128ug/mL, 256ug/mL, and the actual concentration is 0.125ug/mL, 0.25ug/mL, 0.5ug/mL, 1ug/mL, 2ug/mL, 4ug/mL, 8ug/mL, 16ug/mL, 32ug/mL, 64 ug/mL.

100. mu.L of the medium was added to each well of a sterile 96-well polystyrene plate, 50. mu.L of the antibiotic dilution (ug/mL) was added to each well in columns 2-11, as indicated by the following table concentration, and 50. mu.L of the bacterial suspension (final concentration of 105. sup. th and 106. sup. th CFU/mL) was added to each well in columns 1-11, which were sealed and placed in a 37 ℃ incubator for 24-48h, and visually observed and measured for OD 625. Wherein the Lactobacillus rhamnosus LGG and the Cistanchis strain LcS are positive control strains.

Name (R)	1	2	3	4	5	6	7	8	9	10	11	12
													Penicillin	Sterile solvent	0.5	1	2	4	8	16	32	64	128	256	Sterile water
Ampicillin	Sterile solvent	0.5	1	2	4	8	16	32	64	128	256	Sterile water
													Imipenem	Sterile solvent	0.5	1	2	4	8	16	32	64	128	256	Sterile water
Meropenem	Sterile solvent	0.5	1	2	4	8	16	32	64	128	256	Sterile water
													Vancomycin	Sterile solvent	0.5	1	2	4	8	16	32	64	128	256	Sterile water
Daptomycin	Sterile solvent	0.5	1	2	4	8	16	32	64	128	256	Sterile water
													Erythromycin	Sterile solvent	0.5	1	2	4	8	16	32	64	128	256	Sterile water
Clindamycin	Sterile solvent	0.5	1	2	4	8	16	32	64	128	256	Sterile water
													Linezolid	Sterile solvent	0.5	1	2	4	8	16	32	64	128	256	Sterile water

The experimental results are shown in the following table, MIC values ug/mL.

The following conclusions were drawn on the basis of the criteria for determining the sensitivity of the CLSI M4 strain to antibiotics. The WHH2580 strain is resistant to Daptomycin and Clindamycin, neutral to Erythromycin erythorycin, and sensitive to Penicillin, Ampicillin, Imipenem, Meropeenem, Vancomycin, and Linezolid. LGG strains are resistant to Imipenem, Meroperem, Vancomycin, Daptomycin, Clindamycin, neutral to Erythromycin Erythromycin, sensitive to Penicillin, Ampicillin and Linezolid. LcS strains are resistant to Vancomycin and Daptomycin, neutral to Imipenem, Meroperem and Erythromycin, and sensitive to Penicillin, Ampicillin, Clindamycin and Linezolid.

Example 13: bacterial strain for inhibiting pathogenic bacteria

Lactobacillus helveticus WHH2580 strain bacteriostasis experiment:

sterilized 1.5% agar was dispensed in sterile petri dishes. The oxford cup is placed. Sterilizing 1% agar MRS culture medium or NB culture medium, inoculating 10 at 50 deg.C below⁶-10⁷Five indicator bacteria (Escherichia coli, staphylococcus aureus, Shigella flexneri, Salmonella paratyphi B and Micrococcus luteus) with the concentration of CFU/mL are shaken, mixed uniformly and poured on the previously poured vegetarian agar medium with an Oxford cup. And (5) after the culture medium is cooled and solidified, pulling out the oxford cup. 100uL of corresponding bacteria liquid (WHH2580) and positive control (10g/mL polymyxin B or 1% Nisin) are added into different pores respectively, and the mixture is placed into a refrigerator at 4 ℃ for diffusion for 7 hours. After culturing at 37 ℃ for 16h, observing and recording the size of the inhibition zone.

The experimental results are shown in the following table, and the numerical values in the table are the size cm of the inhibition zone.

The experimental result shows that the WHH2580 strain has an inhibiting effect on 5 pathogenic bacteria, the inhibiting effect on salmonella paratyphi B, escherichia coli and shigella flexneri is not obviously different from that of 10g/mL polymyxin B, and the inhibiting effect on staphylococcus aureus is not obviously different from that of 1% Nisin, and is slightly lower than that of other control strains.

Example 14: the strain has the function of enhancing immunity

1. In vitro splenic lymphocyte proliferation:

killing mouse, taking spleen aseptically, cutting spleen with ophthalmologic scissors, crushing with aseptic glass injector core, filtering with 200 mesh metal screen, adding sterile Hank's solution containing 10% fetal calf serum, centrifuging at 1000rpm and 4 deg.C for 5min, harvesting cells, adding cell lysate 5 times of cell volume, lysing for 5min, adding sterile Hank's solution containing 10% fetal calf serum to stop lysing, centrifuging at 1000rpm and 4 deg.C for 5min to remove supernatant, washing with RPMI-1640 medium containing 10% fetal calf serum, centrifuging at 1000rpm and 4 deg.C for 5min, washing twice, resuspending in 5mL RPMI-1640 medium containing 10% fetal calf serum, counting cells, adjusting cell concentration to 5 × 10⁶cells/mL。

Centrifuging lactobacillus activated bacteria liquid at 4000rpm at room temperature for 10min to remove supernatant, washing with PBS twice, adding appropriate amount of RPMI-1640 culture medium containing 10% fetal calf serum, and adjusting OD₆₀₀To 0.50. + -. 0.10. The cell suspension was added to a 96-well cell culture plate, 3 replicates per sample. Setting a zero-adjustment group (cell culture medium), a blank control group (cell suspension + cell culture medium, inducer group (cell suspension +10 mu g/mL Con A), and lactobacillus group (cell suspension +10 mu g/mL Con A + bacterial suspension), placing in a carbon dioxide incubator at 37 ℃ for culturing for 72h, adding 20 mu L MTT solution (2.5mg/mL) into each well, developing for 4h at 37 ℃, centrifuging at 1500rpm and 4 ℃ for 10min, sucking supernatant, adding 100 mu L DMSO, and using a microplate reader at 490nAnd measuring the absorbance value at m.

And (3) data analysis: probiotic bacteria that significantly promote splenic lymphocyte proliferation both with and without Con a induction are preferred strains.

Results as shown in the table below, the proliferation fold of splenic lymphocytes of lactobacillus helveticus WHH2580 was higher than that of the two commercial strains with and without Con a induction.

2 in vitro spleen lymphokine IL-10 and IL-12 assay

The steps of cell selection, erythrocyte lysis, washing, cell counting and probiotic culture are the same as the steps of in-vitro splenic lymphocyte proliferation, the cell suspension is added into a 96-well cell culture plate, each treatment is repeated for 5 times, and the cell suspension is divided into a zero adjustment group (cell culture medium is 100 mu L) and a blank control group (1 × 10)⁷cell/mL 100. mu.l of cell suspension, cell culture medium 100. mu.l), probiotic group (1 × 10)⁷ cell suspension 100. mu.L, 1 × 10 of cells/mL⁸cfu/mL bacterial suspension 100. mu.L), 5% CO at 37 ℃₂Culturing in an incubator for 24 h. Centrifuging at 1500rpm for 10min, sucking supernatant, filtering with 0.22 μm filter membrane, and determining IL-10 and IL-12 content with BD kit.

Strains with high IL-12/IL-10 ratios can be used as immunomodulatory potential strains.

The results are shown in the table below, and the ratio of L.helveticus WHH2580 IL-12/IL-10 is close to that of L.rhamnosus LGG, and is not significantly poor.

3 in vitro human Colon cancer cell HT-29 cytokine IL-8 assay

Digesting the human colon cancer cells HT-29 grown and fused to 70% -80%, adjusting the cell concentration, 1 x 10⁶And (4) inoculating the cell/hole into a 24-hole plate, culturing at 37 ℃, and changing the liquid after 24 hours. Adding 20ng/mL LPS for 6 hr, and adding 8 x 10⁸After the CFU/mL bacterial liquid is treated for 18 hours, centrifuging at 3000r/min for 5minObtaining the supernatant, and detecting the content of interleukin IL-8 by using a corresponding ELISA kit.

The strain with high IL-8 secretion can be used as an immune regulation potential strain.

The results are shown in the following table.

Strain numbering	IL-8 concentration (ng/mL)
		Lactobacillus rhamnosus LGG	2981±95
Lactobacillus helveticus WHH2580	4021±145

Lactobacillus helveticus WHH2580 IL-8 is higher than commercial strain LGG.

Example 15: animal testing

A random grouping design is adopted, 192 inbred line BALB/C male mice (6-8 weeks old, 16-20g) are divided into 8 groups at random, and each group is 12, with an adaptation period of 5 days. Keeping the environment temperature of 21 + -2 deg.C and humidity of 30-70% for animal feeding, and freely drinking water and taking feed after 12h illumination alternation. Changing padding every three days, and adding water and feed in time. The grouping is as follows:

control group 1: performing intragastric administration on a blank control group;

control group 2: a model control group is subjected to stomach irrigation and 50mg/kg cyclophosphamide intraperitoneal injection for molding;

control group 3: a positive drug group, 40mg/kg of levamisole hydrochloride by intragastric administration and 50mg/kg of cyclophosphamide by intraperitoneal injection for molding;

experimental group 1: the bacterial strain suspension of the invention is perfused with stomach, and the perfusing dosage is 2 x 10⁸CFU/d；

Experimental group 2: the bacterial strain suspension of the invention is used for intragastric administration, and the intragastric administration dosage is 2 x 10⁸CFU/d；

Wherein the inactivated bacteria is treated at 80 deg.C for 10 min. The existence of no viable bacteria is confirmed by plate culture.

The test is carried out by adopting a model animal with low immune function. Subjects were administered for 38 consecutive days and administration of immunosuppressive agents was started after day 32. Body weight was weighed once a week before inhibitor and once a day after inhibitor. The detection is started on the 5 th day after the intraperitoneal injection of the immunosuppressant, and the detection indexes comprise organ ratio, spleen lymphocyte proliferation and NK cell activity.

The experimental results are as follows:

the body weight results are shown in figure 9. The body weight change trend was similar in each experimental group except for the blank control, and the body weight decreased on the first day of inhibitor injection and gradually recovered thereafter.

The results of organ comparison are shown in FIG. 10. The thymus/body weight ratio and spleen/body weight ratio of the model group were significantly lower than those of the control group. The thymus/body weight ratio of the low dose group and the positive drug group was significantly higher than that of the model group (P <0.05), and that of the high dose group and the dead bacteria group was significantly higher than that of the model group (P < 0.001). Spleen/body weight ratios were significantly higher in the low dose, medium dose, dead bacteria and positive drug groups than in the model group (P < 0.05).

The proliferation results of splenic lymphocytes of mice are shown in FIG. 11. The OD values of the ultra-low dose group, the medium dose group and the high dose group are obviously higher than those of the inactivated bacteria group of the model group, and are extremely obviously higher than those of the model group. The WHH2580 can obviously promote spleen lymphocyte proliferation of mice with low immunity by virtue of ultralow dose, low dose, medium dose and high dose and inactivated bacteria.

The results of the measurement of NK cell activity of mice are shown in FIG. 12. The activity of NK cells of the medium-dose group is remarkably higher than that of the model group, and the activity of NK cells of the inactivated bacterial group is remarkably higher than that of the model group. The results show that the dose and the inactivated bacteria in the WHH2580 can obviously improve the activity of NK cells of the mice with low immunity.

According to the health product function evaluation method, under the establishment condition of an immune function low model, the total number of blood leucocytes, the cellular immune function (a mouse spleen lymphocyte transformation experiment, a delayed type allergic reaction experiment), the humoral immune function (an antibody-producing cell detection, a serum hemolysin determination), the mononuclear-macrophage function (a mouse carbon clearance experiment, a mouse abdominal cavity macrophage phagocytosis fluorescent microsphere experiment) and the NK cell activity are determined to be positive, and the test result is positive, so that the tested sample has the function of enhancing the immunity of a person with low immune function. The test result of the WHH2580 strain is positive in both the cellular immune function and the NK cell activity, and the WHH2580 strain can be judged to have the effect of enhancing the immunity of a person with low immune function.

The WHH2580 inactivated strain can also improve the immunity of the organism by improving and regulating the organ index, nonspecific immunity and specific immune response of the organism.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Sequence listing

<110> Hangzhou baby Haha science Co Ltd

Hangzhou Wahaha Group Co.,Ltd.

<120> normal-temperature long-shelf-life sucrose-free yoghourt capable of improving immunity and preparation method thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1425

<212>DNA

<213> Lactobacillus helveticus (Lactobacillus helveticus)

<400>1

ccttcccgaa ggttaggcca ccggctttgg gcattgcaga cttccatggt gtgacgggcg 60

gtgtgtacaa ggcccgggaa cgtattcacc gcggcgttct gatccgcgat tactagcgat 120

tccagcttcg tgcagtcgag ttgcagactg cagtccgaac tgagaacagc tttcagagat 180

tcgcttgcct tcgcaggctc gcttctcgtt gtactgtcca ttgtagcacg tgtgtagccc 240

aggtcataag gggcatgatg acttgacgtc atccccacct tcctccggtt tatcaccggc 300

agtctcatta gagtgcccaa cttaatgctg gcaactaata acaagggttg cgctcgttgc 360

gggacttaac ccaacatctc acgacacgag ctgacgacag ccatgcacca cctgtcttag 420

cgtccccgaa gggaactcct aatctcttag gatggcacta gatgtcaaga cctggtaagg 480

ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat 540

tcctttgagt ttcaaccttg cggtcgtact ccccaggcgg agtgcttaat gcgttagctg 600

cagcactgag aggcggaaac ctcccaacac ttagcactca tcgtttacgg catggactac 660

cagggtatct aatcctgttc gctacccatg ctttcgagcc tcagcgtcag ttgcagacca 720

gagagtcgcc ttcgccactg gtgttcttcc atatatctac gcattccacc gctacacatg 780

gagttccact ctcctcttct gcactcaaga aaaacagttt ccgatgcagt tcctcggtta 840

agccgagggc tttcacatca gacttattct tccgcctgcg ctcgctttac gcccaataaa 900

tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt tagccgtgac 960

tttctggttg attaccgtca aataaaggcc agttactacc tctatccttc ttcaccaaca 1020

acagagcttt acgatccgaa aaccttcttc actcacgcgg cgttgctcca tcagacttgc 1080

gtccattgtg gaagattccc tactgctgcc tcccgtagga gtttgggccg tgtctcagtc 1140

ccaatgtggc cgatcagtct ctcaactcgg ctatgcatca ttgccttggt aagccgttac 1200

cttaccaact agctaatgca ccgcggggcc atcccatagc gacagcttac gccgcctttt 1260

ataagctgat catgcgatct gctttcttat ccggtattag cacctgtttc caagtggtat 1320

cccagactat ggggcaggtt ccccacgtgt tactcaccca tccgccgctc gcgtccccag 1380

cgtcattacc gaagtaaatc tgctggttct gctcgctcga cttgc 1425

Claims (10)

1. A normal-temperature long-shelf-life sucrose-free yoghourt capable of improving immunity is characterized by comprising raw milk or milk powder and functional components capable of improving immunity; the immunity-improving functional component at least comprises lactobacillus helveticus and/or a mutant thereof;

the lactobacillus helveticus is named as WHH2580, and is preserved in the common microorganism center of the china culture collection and management committee for culture collection of microorganisms in 2019, month 10 and 23, and the microorganism preservation numbers are as follows: CGMCC No.18730, and the classification name of microorganisms is Lactobacillus helveticusLactobacillus helveticus(ii) a The Lactobacillus helveticus has a 16s rRNA gene sequence shown in SEQ ID NO. 1; the mutant is obtained by carrying out mutagenesis, domestication, gene recombination or natural mutation on the lactobacillus helveticus.

2. The normal-temperature long-shelf-life sucrose-free yogurt for improving immunity of claim 1, further comprising sugar alcohol, starch, stabilizer, sweetener.

3. The normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity as claimed in claim 2, which comprises, by mass, 40% -85% of raw milk or 5-15% of milk powder, 0.001-0.020% of lactobacillus helveticus and/or mutants thereof, 0.01-10.00% of an immunity-improving effective component excluding lactobacillus helveticus and/or mutants thereof, 0.5-5.0% of sugar alcohol, 0.1-5.0% of starch, 0.01-1.00% of a stabilizer, 0.001-0.050% of a sweetener, and the balance of water; the sum of all the raw materials is 100 percent.

4. The normal-temperature long-shelf-life sucrose-free yogurt for improving immunity of claim 2 or 3, characterized in that:

the milk powder is one or more of whole milk powder, skim milk powder, whey protein powder and whole milk protein powder; and/or

The functional components for improving immunity also comprise one or more of propolis, Ganoderma extract, wall-broken Ganoderma spore powder, beta-dextran, herba Dendrobii extract, herba Sambuci Williamsii, and acerola cherry; and/or

The sugar alcohol is one or more of xylitol, maltitol, erythritol, sorbitol, isomaltitol and lactitol; and/or

The starch is one or more of physical starch, acetate starch, sodium starch octenyl succinate, hydroxypropyl distarch phosphate, phosphate distarch, acid-treated starch, oxidized hydroxypropyl starch, acetylated distarch phosphate and acetylated distarch adipate; and/or

The stabilizer is one or more of sodium carboxymethylcellulose, pectin, soybean polysaccharide, agar, sodium caseinate, propylene glycol alginate, gelatin, gellan gum, guar gum and xanthan gum; and/or

The sweetener is one or more of acesulfame potassium, aspartame, sucralose, stevioside, mogroside, isomaltulose, edmuntin and neotame.

5. A method for preparing the sucrose-free yogurt with the normal temperature and the long shelf life for improving the immunity according to any one of claims 2 to 4, which is characterized by comprising the following steps:

scheme A:

(1) homogenizing and sterilizing: preheating raw milk, homogenizing, sterilizing, and cooling to obtain sterilized cooling feed liquid;

(2) inoculating and fermenting: adding lactobacillus helveticus and/or a mutant thereof into the sterilized cooling liquid obtained in the step (1), uniformly stirring, standing at a constant temperature, and fermenting to obtain a yogurt base material;

(3) preparing glue solution: preheating water, adding a formula amount of stabilizer into the water, shearing or stirring, and cooling to obtain a glue solution;

(4) secondary burdening: cooling the yogurt base material obtained in the step (2), and then stirring and demulsifying; adding the glue solution obtained in the step (3), adding the components with the formula dosage, sugar alcohol, sweetener and starch for improving the immunity except the lactobacillus helveticus and/or the mutant thereof, mixing and stirring, and adding water to a constant volume to obtain a feed liquid;

(5) and (3) secondary homogenization: homogenizing the feed liquid obtained in the step (4);

(6) filling and sterilizing: filling, sealing, pasteurizing and cooling the homogenized material liquid in the step (5) to obtain the normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity; or (5) carrying out UHT sterilization on the homogenized material liquid, carrying out hot filling or aseptic cold filling, and sealing to obtain the sucrose-free yoghourt with the immunity improving function and the long shelf life at normal temperature;

scheme B:

1) preparing milk: heating water, adding the formula amount of milk powder into the water, stirring or shearing, and standing for hydration to obtain milk;

2) homogenizing and sterilizing: preheating the milk obtained in the step 1), homogenizing, sterilizing and cooling to obtain a sterilized cooling feed liquid;

3) inoculating and fermenting: adding lactobacillus helveticus and/or a mutant thereof into the sterilized cooling liquid obtained in the step 2), uniformly stirring, standing at a constant temperature, and fermenting to obtain a yogurt base material;

4) preparing glue solution: preheating water, adding a formula amount of stabilizer into the water, shearing or stirring, and cooling to obtain a glue solution;

5) secondary burdening: cooling the yogurt base material obtained in the step 3), and then stirring and demulsifying; adding the glue solution obtained in the step 4), adding the components with the formula dosage, sugar alcohol, sweetener and starch for improving the immunity except the lactobacillus helveticus and/or the mutant thereof, mixing and stirring, and adding water to a constant volume to obtain a feed liquid;

6) and (3) secondary homogenization: homogenizing the feed liquid obtained in the step 5);

7) filling and sterilizing: filling, sealing, pasteurizing and cooling the homogenized material liquid obtained in the step 6) to obtain the normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity; or carrying out UHT sterilization on the homogenized material liquid in the step 6), carrying out hot filling or aseptic cold filling, and sealing to obtain the sucrose-free yoghourt with the immunity improving function and the long shelf life at normal temperature.

6. The method for preparing the sucrose-free yogurt with the normal temperature and the long shelf life for improving the immunity according to claim 5, wherein the method comprises the following steps:

in the step 1), the mass of the water is 200-400 g; and/or

In the step 1), water is heated to 45-60 ℃; and/or

In the step 1), the stirring or shearing time is 15-30 min; and/or

In the step 1), standing and hydrating for 20-40 min.

7. The method for preparing the sucrose-free yogurt with the normal temperature and the long shelf life for improving the immunity according to claim 5, wherein the method comprises the following steps:

in the step (1) or the step 2), preheating to the temperature of 60-65 ℃; and/or

In the step (1) or the step 2), the homogenizing pressure is 0-8/5-30 MPa; and/or

In the step (1) or the step 2), the sterilization temperature is 90-97 ℃, and the time is 3-10 min; and/or

In the step (1) or the step 2), cooling is carried out until the temperature is 35-45 ℃.

8. The method for preparing the sucrose-free yogurt with the normal temperature and the long shelf life for improving the immunity according to claim 5, wherein the method comprises the following steps:

in the step (2) or the step (3), the fermentation temperature is 35-45 ℃ and the fermentation time is 4-16 h; and/or

In the step (2) or the step 3), the number of viable bacteria is more than or equal to 1 × 10 in the fermentation process⁸cfu/mL。

9. The method for preparing the sucrose-free yogurt with the normal temperature and the long shelf life for improving the immunity according to claim 5, wherein the method comprises the following steps:

in the step (3) or the step 4), the mass of the water is 200-400 g; and/or

In the step (3) or the step 4), water is preheated to 50-60 ℃; and/or

In the step (3) or the step (4), the shearing or stirring time is 20-40 min; and/or

In the step (3) or the step (4), the temperature is cooled to be below 30 ℃.

10. The method for preparing the sucrose-free yogurt with the normal temperature and the long shelf life for improving the immunity according to claim 5, wherein the method comprises the following steps:

in the step (4) or the step (5), cooling to the temperature below 15 ℃; and/or

In the step (4) or the step 5), the volume is adjusted to 1000mL by using water; and/or

In the step (5) or the step 6), the homogenizing pressure is 0-8/5-30 MPa.

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