CN111728030B - Sucrose-free yogurt with immunity improving function and long shelf life at normal temperature and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of foods, and discloses a normal-temperature sucrose-free yogurt with a long shelf life for improving immunity and a preparation method thereof. The normal-temperature long-shelf-life sucrose-free yoghurt for improving immunity comprises raw milk or milk powder and an immunity-improving functional component; the immunity enhancing functional ingredient at least comprises lactobacillus helveticus and/or mutants thereof. The yoghurt is prepared by fermenting cow milk or reconstituted milk through lactobacillus helveticus, and is compounded with various natural functional components for improving immunity, so that the immunity of a human body can be improved after long-term eating; in addition, the lactobacillus helveticus WH2580 still has the immunity enhancing effect in the inactivated state, so that the yoghourt can be normally stored under the normal temperature condition, does not need refrigeration, and has long shelf life.

Description

Sucrose-free yogurt with immunity improving function and long shelf life at normal temperature and preparation method thereof

Technical Field

The invention relates to the technical field of foods, in particular to a normal-temperature sucrose-free yoghurt with long shelf life for improving immunity and a preparation method thereof.

Background

The autumn and winter seasons are the time period of the disease, and various epidemic diseases such as influenza begin to be abused, and children and old people with lower immunity are more ill disaster areas. The so-called "immunity" is the best doctor. The measures such as wearing a mask, washing hands and the like can only prevent external viruses from invading, and the immunity of the patient is the only force the patient can depend on once the patient is infected without special medicine.

Cow milk is nutritious and contains various unique components capable of regulating immune function, such as immunoglobulin, lactoferrin and various amino acids. The lysozyme and lactoperoxidase contained in the dairy product can play an anti-infective role in the intestines, activate an immune system, strengthen 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 deal of literature reports that probiotics can improve human immunity, such as lactobacillus reuteri can colonize in intestinal tracts, improve intestinal flora distribution, antagonize harmful bacteria, grow and improve host immunity. Meanwhile, lactobacillus reuteri can produce 'reuterin', and can widely inhibit the growth of gram-negative bacteria, gram-positive bacteria, yeast, fungi, protozoa and the like; lactobacillus rhamnosus LGG can increase secretion of intestinal mucosa IgA, and tissue pathogenic bacteria infect epithelial cells to enhance immunity; lactobacillus plantarum Lp9 and Lp91 can secrete anti-inflammatory cytokines in LPS-induced HT-29 cells, regulating immune function. A German study also shows that D-phenyllactic acid produced by lactobacillus fermentation can activate G protein receptor HCA3 of human immune system, cause chemotaxis of neutrophils and promote immunity. Brazil researchers found that acetate produced by lactic acid bacteria fermentation stimulated human GPR43-1 type interferon response against respiratory syncytial virus.

Propolis, ganoderma extract, wall-broken Ganoderma spore powder, beta-glucan, herba Dendrobii extract, herba Sambuci Williamsii, and acerola cherry etc. are generally considered to be rich in substances which are helpful for immunity, such as flavone, polyphenol, ganoderic acid, fungus polysaccharide, mucopolysaccharide, anthocyanin, vitamin C, etc.

When the cow milk capable of improving the human immunity is fermented by probiotics capable of improving the human immunity, and is supplemented with the functional components capable of improving the human immunity, the three-strength combination and the synergy are combined, so that a good immunity enhancing effect can be achieved.

At present, yogurt capable of enhancing immunity is declared on the market as shown in fig. 2, and probiotics used by the yogurt only have the function of adjusting immunity in a living state, so that the yogurt can be stored in a refrigerating 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 carbon sources to maintain the activity of the strain. The refrigerated storage represents that the product has harsh requirements on storage, logistics and sales environments, and the intermediate cost is high; the product is easy to expire due to the short shelf life, so that waste is caused; white granulated sugar is one of the leading causes of obesity in this century, and is now widely subject to consumer and industry problems. But the normal temperature long shelf life (3-6 months) sucrose-free yoghurt capable of improving immunity is still a market blank at present.

Disclosure of Invention

In order to solve the technical problems, the invention provides the normal-temperature sucrose-free yoghurt with long shelf life for improving the immunity and the preparation method thereof. The yogurt can effectively improve immunity, can be stored normally 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 improved immunity and long shelf life at normal temperature comprises raw milk or milk powder and effective components for improving immunity; the immunity enhancing functional ingredient at least comprises lactobacillus helveticus and/or mutants thereof;

the lactobacillus helveticus is named WHH2580, and the microorganism deposit number is: CGMCC No.18730, and the microorganism classification is named as Lactobacillus helveticus Lactobacillus helveticus; the lactobacillus helveticus has a 16s rRNA gene sequence shown as 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 bacterial strain screened from yoghourt collected from Xinjiang Nalatin red mountain furrows, and proved by animal experiments, the probiotic bacterial strain has excellent adhesion performance, can smoothly reach intestinal tracts, can adhere to intestinal epithelial cells, and plays a probiotic effect; the strain can improve in vitro spleen lymphocyte proliferation and secretion of interleukin IL-12 by spleen lymphocytes, and promote 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 remarkably improve the organ index of mice, promote the proliferation of spleen lymphocytes and improve the activity of NK cells. Therefore, the lactobacillus helveticus WH2580 has good immunity enhancing effect. More importantly, the strain still has better immunity enhancing function after inactivation. In addition, in the animal experiment process, the bad states such as death, listlessness, 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 adhesion characteristics: the method has the advantages that the method shows excellent adhesion capability in a human colon cancer epithelial cell HT-29 cell model test, the number of single-cell adhesion living bacteria reaches 15.69+/-5.94, the number of single-cell adhesion non-living bacteria reaches 11.59 +/-4.02, and the adhesion capability is obviously better than that of positive control strains lactobacillus rhamnosus LGG and Tian Zhu LcS. The strain can adhere to intestinal epithelial cells to exert physiological effects. Although adhesion is not a prerequisite for the bacterial strain to possess probiotic properties, the interaction of probiotics with the intestinal mucosa is considered critical for a number of reasons. Binding to the intestinal mucosa may extend the time that the probiotic bacterial strain is present in the intestine. This interaction with the mucosa brings the probiotics into close contact with the intestinal immune system, giving it a better opportunity to modulate the immune response. It can also fight intestinal pathogens by limiting its ability to reside in the intestine.

(2) Has good acid resistance and bile salt resistance: in the environment with the pH value of 3.0, the survival of the bacteria is not affected significantly by incubation for 2 hours, and the number of the living bacteria is reduced by 0.9 order of magnitude after 3 hours; pepsin has no obvious effect on the viability of the strain in 2 hours, and the number of living strains in 3 hours is reduced by 0.7 order of magnitude; 3h reduction by 3.6 orders of magnitude in 0.5% bile salt system. The acid resistance and the bile salt resistance are good, so that the feed can better pass through the stomach and the duodenum and smoothly reach the intestinal tract, thereby better playing a probiotic role; the good acid resistance enables the yoghurt to be suitable for a wider pH range in the fermentation process of preparing the yoghurt.

(3) In an in vitro cell test, the proliferation of in vitro spleen lymphocytes can be promoted, the multiplication factor of the inducer without the inducer is 1.912+/-0.087, and the multiplication factor of the inducer is 1.533+/-0.109. Can promote the secretion of interleukin IL-12 by spleen lymphocytes in vitro; the IL-12/IL-10 ratio was not significantly different from the Lactobacillus rhamnosus LGG. In an in vitro cell test, the human colon cancer epithelial cell HT-29 can be promoted to secrete interleukin IL-8; the secreted IL-8 was stimulated significantly higher than the commercial strain LGG.

(4) Has good inhibition effect on staphylococcus aureus, salmonella paratyphi B, escherichia coli, shigella flexneri and micrococcus luteus, so that pathogenic bacteria in intestinal tracts can be well inhibited in the intestinal tracts, intestinal flora is regulated, and the probiotic effect is exerted.

(5) Both living and non-living subjects have an enhanced immune function: the concentration of live strain is 2×10 ⁸ CFU/d, concentration of fire-extinguishing strain taken 2X 10 ⁸ CFU/d can significantly improve the organ index of mice, promote the proliferation of spleen lymphocytes and improve the activity of NK cells. This allows the probiotics in the yoghurt of the invention to exist in a non-living form, so that the yoghurt can have a longer shelf life at normal temperature without refrigeration.

Preferably, the yoghurt further comprises sugar alcohols, starch, stabilizers, sweeteners.

Preferably, the yoghurt comprises, by mass, 40% -85% of raw milk or 5-15% of milk powder, 0.001-0.020% of lactobacillus helveticus and/or a mutant thereof, 0.01-10.00% of an immunity-improving functional ingredient except lactobacillus helveticus and/or a mutant 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%.

The mass percentage refers to the percentage of the total mass of the yoghurt.

Preferably, the milk powder is one or more of full cream milk powder, defatted milk powder, whey protein powder and full milk protein powder.

Preferably, the immunity enhancing functional ingredient further comprises one or more of propolis, ganoderma lucidum extract, wall-broken ganoderma lucidum spore powder, beta-glucan, dendrobium candidum 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 carboxymethyl cellulose, 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, alitame and neotame.

The method for preparing the normal-temperature long-shelf-life sucrose-free yoghurt for improving the immunity comprises the following steps of:

scheme a:

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

(2) Inoculating and fermenting: adding lactobacillus helveticus and/or mutants thereof into the sterilization cooling feed liquid obtained in the step (1), stirring and mixing uniformly, and standing and fermenting at constant temperature to obtain a yoghurt base material;

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

(4) And (3) secondary batching: cooling the yoghurt base material obtained in the step (2), and then stirring and demulsifying; adding the glue solution obtained in the step (3), adding the immunity-improving functional components except lactobacillus helveticus and/or the mutant thereof, sugar alcohol, sweetener and starch in the formula dosage, mixing and stirring, and fixing the volume by water to obtain 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 feed liquid in the step (5) to obtain the normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity; or sterilizing the homogenized feed liquid in the step (5) by UHT, hot filling or sterile cold filling, and sealing to obtain the normal-temperature long-shelf-life sucrose-free yoghurt with improved immunity;

scheme B:

1) Preparing milk: heating water, adding milk powder with the formula amount into the water, stirring or shearing, standing and hydrating to obtain milk;

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

3) Inoculating and fermenting: adding lactobacillus helveticus and/or mutants thereof into the sterilization cooling feed liquid obtained in the step 2), stirring and mixing uniformly, and standing and fermenting at constant temperature to obtain a yoghurt base material;

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

5) And (3) secondary batching: cooling the yoghurt base obtained in the step 3), and then stirring to demulsify. Adding the glue solution obtained in the step 4), adding the immunity improving functional components except lactobacillus helveticus and/or the mutant thereof, sugar alcohol, sweetener and starch in the formula dosage, mixing and stirring, and fixing the volume by water to obtain 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 feed liquid in the step 6) to obtain the normal-temperature sucrose-free yogurt with long shelf life and improved immunity; or sterilizing the homogenized feed liquid in the step 6) by UHT, hot filling or sterile cold filling, and sealing to obtain the normal-temperature long-shelf-life sucrose-free yogurt with improved immunity.

The invention prepares the yoghourt through pretreatment, homogenization, sterilization, fermentation, re-sterilization and filling of raw milk or milk powder and various raw and auxiliary materials, can be normally stored under normal temperature conditions, does not need refrigeration, and has a shelf life of 150-180 days; meanwhile, the functional components are kept to the maximum extent and are not damaged in the processing process, so that the functionality of the product is ensured.

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

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

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

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

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

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

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

Preferably, in step (1) or step 2), the temperature is cooled to 35-45 ℃.

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

Preferably, in the step (2) or the step 3), the number of viable bacteria in the fermentation process is not less than 1×10 ⁸ cfu/mL。

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

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 to 40 minutes.

Preferably, in step (3) or step 4), the temperature is cooled to 30 ℃ or lower.

Preferably, in step (4) or step 5), the temperature is cooled to 15 ℃ or lower.

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

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

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

(1) The yoghurt is prepared by fermenting cow milk or reconstituted milk through lactobacillus helveticus WHH2580 and/or mutants thereof, and can be compounded with various natural functional components for improving the immunity, so that the immunity of a human body can be improved after long-term eating;

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

(3) The yoghurt is prepared by pretreatment, homogenization, sterilization, fermentation, re-sterilization and filling of raw milk or milk powder and various raw and auxiliary materials, can be normally stored under normal temperature conditions, does not need refrigeration, and has a shelf life of 150-180 days; meanwhile, the functional components are kept to the maximum extent and are not damaged in the processing process, so that the functionality of the product is ensured;

(4) The yogurt disclosed by the invention is free from adding sucrose, can be drunk by various people in daily life, and particularly has the advantages of high acceptability, high safety and no side effect, and has important efficacy value and social value for people with low autoimmune power and limited sugar intake (diabetes, obesity and the like) and people who are not loving cold food.

Drawings

FIG. 1 is a flow chart of a method for preparing sucrose-free yogurt with enhanced immunity and long shelf life at normal temperature;

FIG. 2 is a schematic flow chart of the preparation process of yoghurt capable of enhancing immunity in the prior art;

FIG. 3 is a schematic flow chart of the preparation of the normal temperature long shelf life sucrose-free yoghurt for improving immunity;

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

FIG. 5 is a graph showing the results of the experiment for transformation of spleen lymphocytes from mice of each group in example 4;

FIG. 6 is a graph of NK cell activity in each group of mice in example 4;

FIG. 7 shows the morphology of Lactobacillus helveticus WH2580 observed under a microscope;

FIG. 8 is a graph showing the effect of Lactobacillus helveticus WH2580 on adhesion of live, non-live, commercial strains LGG and Lcs to HT-29 cells;

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

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

FIG. 11 is a graph showing the spleen lymphocyte transformation values for each group of BALB/c mice in the animal test of example 15;

FIG. 12 is a graph showing NK cell activity in each group of BALB/c mice in example 15.

Detailed Description

The invention is further described below with reference to examples.

General examples

The normal-temperature long-shelf-life sucrose-free yoghurt 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 a mutant thereof, 0.01-10.00% of immunity-improving efficacy components except lactobacillus helveticus and/or a mutant 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%.

The sucrose-free yoghurt with the normal temperature and long shelf life for improving the immunity is prepared by the following steps (the preparation flow is shown in figure 1, and the sketch is shown in figure 3):

scheme a:

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

said sterilization is carried out under sterilization conditions conventional in the art, such as pasteurization;

(2) Inoculating and fermenting: adding Lactobacillus helveticus and/or its mutant into the sterilized cooling liquid obtained in step (1), stirring, mixing, standing at 35-45deg.C in constant temperature incubator for fermenting for 4-16 hr, and viable count not less than 1×10 ⁸ cfu/mL to obtain a yoghurt base material;

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

(4) And (3) secondary batching: cooling the yoghurt base material obtained in the step (2) to below 15 ℃, and then stirring and demulsification; adding the glue solution obtained in the step (3), adding the immunity-improving functional components except lactobacillus helveticus and/or the mutant thereof, sugar alcohol, sweetener and starch in the formula dosage, mixing and stirring, and using ultrapure water to reach 1000mL to obtain feed liquid;

(5) And (3) secondary homogenization: homogenizing the feed liquid obtained in the step (4), wherein the homogenizing pressure is 0-8/5-30MPa;

(6) Filling and sterilizing: filling the homogenized feed liquid in the step (5) into an HDPE packaging container, sealing, pasteurizing and cooling to obtain the normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity; or sterilizing the homogenized feed liquid in the step (5) by UHT, hot filling or sterile cold filling into a PET packaging container, and sealing to obtain the normal-temperature long-shelf-life sucrose-free yogurt with improved immunity;

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

Scheme B:

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

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

said sterilization is carried out under sterilization conditions conventional in the art, such as pasteurization;

3) Inoculating and fermenting: adding Lactobacillus helveticus and/or its mutant into the sterilized cooling liquid obtained in step 2), stirring, fermenting at 35-45deg.C for 4-16 hr in a constant temperature incubator, and keeping viable count at 1×10 or more ⁸ cfu/mL to obtain a yoghurt base material;

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

5) And (3) secondary batching: cooling the yoghurt base material obtained in the step 3) to below 15 ℃, and then stirring and demulsification; adding the glue solution obtained in the step 4), adding the immunity improving functional components except lactobacillus helveticus and/or the mutant thereof, sugar alcohol, sweetener and starch in the formula dosage, mixing and stirring, and using ultrapure water to reach 1000mL to obtain feed liquid;

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

7) Filling and sterilizing: filling the homogenized feed liquid in the step 6) into an HDPE packaging container, sealing, pasteurizing and cooling to obtain the normal-temperature sucrose-free yogurt with long shelf life and improved immunity; or sterilizing the homogenized feed liquid in the step 6) by UHT, hot filling or sterile cold filling into a PET packaging container, and sealing to obtain the normal-temperature long-shelf-life sucrose-free yogurt for improving immunity;

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

Example 1

The components of the embodiment consist of whole milk powder, xylitol, concentrated elderberry juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, sucralose, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each component is 1000 milliliters in total: 100 g of whole milk powder, 20 g of xylitol, 0.1 g of sucralose, 5 g of concentrated elder raspberry juice, 5 g of hydroxypropyl distarch phosphate, 5 g of sodium carboxymethyl cellulose, 0.2 g of gellan gum, 0.02 g of Lactobacillus helveticus WHH-2580 and the balance of water.

The preparation steps of this example are as follows:

1) Preparing milk: weighing 300g of ultrapure water, preheating to 55 ℃, adding full-cream milk powder with the formula dosage into the ultrapure water, shearing for 15min at medium speed, and standing for hydration for 20min;

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

3) Inoculating and fermenting: adding Lactobacillus helveticus WHH-2580 into the sterilized cooling liquid obtained in step 2), stirring, fermenting at 42deg.C for 6 hr in a constant temperature incubator, and sterilizing with viable count of 1×10 or more ⁸ cfu/mL to obtain a yoghurt base material;

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

5) And (3) secondary batching: cooling the yoghurt base material obtained in the step 3) to below 15 ℃, and then stirring and demulsification; adding the glue solution obtained in the step 4), adding sugar alcohol, sweetener, starch and immunity improving functional components except lactobacillus helveticus and/or mutant thereof in the formula dosage, mixing and stirring, and using ultrapure water to reach 1000mL to obtain feed liquid;

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

7) Filling and sterilizing: filling the homogenized feed liquid in the step 6) into a HDPE packaging container, sealing, pasteurizing at 87 ℃ for 18min, and cooling to obtain the sucrose-free yogurt with normal temperature and long shelf life, wherein the immunity of the yogurt can be improved.

Example 2

The components of the embodiment consist of whole milk powder, xylitol, concentrated elderberry juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, acesulfame potassium, aspartame, lucid ganoderma extract, dendrobium candidum extract, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each component is 1000 milliliters in total: 105 g of whole milk powder, 15 g of xylitol, 1 g of elderberry concentrated juice, 10 g of hydroxypropyl distarch phosphate, 6 g of sodium carboxymethyl cellulose, 0.25 g of gellan gum, 0.1 g of acesulfame potassium, 0.1 g of aspartame, 2 g of lucid ganoderma extract, 2 g of dendrobium candidum extract, 0.05 g of lactobacillus helveticus WHH-2580 and the balance of water.

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

Example 3

The components of the embodiment comprise whole milk powder, maltitol, acetylated distarch phosphate, pectin, soybean polysaccharide, stevioside, propolis, broken ganoderma lucidum spore powder, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each component is 1000 milliliters 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, 0.07 g of lactobacillus helveticus WHH-2580 and the balance of water.

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

Example 4

The components of the embodiment consist of whole milk powder, xylitol, concentrated elderberry juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, acesulfame potassium, aspartame, lucid ganoderma extract, dendrobium candidum extract, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each component is 1000 milliliters in total: 105 g of whole milk powder, 15 g of xylitol, 1 g of elderberry concentrated juice, 10 g of hydroxypropyl distarch phosphate, 6 g of sodium carboxymethyl cellulose, 0.25 g of gellan gum, 0.1 g of acesulfame potassium, 0.1 g of aspartame, 2 g of lucid ganoderma extract, 2 g of dendrobium candidum extract, 0.05 g of lactobacillus helveticus WHH-2580 and the balance of water.

The preparation steps of this example are as follows:

1) Preparing milk: weighing 300g of ultrapure water, preheating to 50 ℃, adding full-cream milk powder with the formula dosage into the ultrapure water, shearing for 15min at medium speed, and standing for hydration for 20min;

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

3) Inoculating and fermenting: adding Lactobacillus helveticus WHH-2580 into the sterilized cooling liquid obtained in step 2), stirring, fermenting at 37deg.C in a constant temperature incubator for 16 hr, and sterilizing with viable count of 1×10 or more ⁸ cfu/mL to obtain a yoghurt base material;

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

5) And (3) secondary batching: cooling the yoghurt base material obtained in the step 3) to below 15 ℃, and then stirring and demulsification; adding the cooled stabilizer, adding sugar alcohol, sweetener, starch and immunity enhancing functional components except lactobacillus helveticus and/or its mutant, mixing, stirring, and adding ultrapure water to 1000mL to obtain feed liquid;

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

7) Filling and sterilizing: and (3) sterilizing the homogenized feed liquid in the step (6) by UHT at 121 ℃ for 15 seconds, filling the sterilized feed liquid into a PET packaging container, and sealing the packaging container to obtain the normal-temperature sucrose-free yogurt with long shelf life, wherein the immunity of the yogurt can be improved.

Example 5

The components of the embodiment comprise whole milk powder, maltitol, acetylated distarch phosphate, pectin, soybean polysaccharide, stevioside, propolis, broken ganoderma lucidum spore powder, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each component is 1000 milliliters 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, 0.07 g of lactobacillus helveticus WHH-2580 and the balance of water.

The preparation method of this example was the same as that of example 4.

Example 6

The components of the embodiment consist of whole milk powder, xylitol, concentrated elderberry juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, sucralose, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each component is 1000 milliliters in total: 100 g of whole milk powder, 20 g of xylitol, 0.1 g of sucralose, 5 g of concentrated elder raspberry juice, 5 g of hydroxypropyl distarch phosphate, 5 g of sodium carboxymethyl cellulose, 0.2 g of gellan gum, 0.02 g of Lactobacillus helveticus WHH-2580 and the balance of water.

The preparation method of this example was the same as that of example 4.

Example 7

The components of the embodiment comprise raw milk, xylitol, concentrated elderberry juice, hydroxypropyl distarch phosphate, sodium carboxymethyl cellulose, gellan gum, sucralose, lactobacillus helveticus WHH-2580 and purified water, wherein the weight of each component is 1000 milliliters in total: 700 g of raw milk, 20 g of xylitol, 0.1 g of sucralose, 5 g of concentrated elderberry juice, 5 g of hydroxypropyl distarch phosphate, 5 g of sodium carboxymethyl cellulose, 0.2 g of gellan gum, 0.02 g of Lactobacillus helveticus WHH-2580 and the balance of water.

The preparation steps of this example are as follows:

1) And (3) milk collection: cooling the raw milk which is qualified by inspection to 4 ℃ and collecting the raw milk into a milk storage tank;

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

3) Inoculating and fermenting: adding Lactobacillus helveticus WHH-2580 into the sterilized cooling liquid obtained in step 2), stirring, fermenting at 40deg.C for 8 hr, and sterilizing with viable count of 1×10 or more ⁸ cfu/mL to obtain a yoghurt base material;

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

5) And (3) secondary batching: cooling the yoghurt base material obtained in the step 3) to below 15 ℃, and then stirring and demulsification; adding the cooled stabilizer, adding sugar alcohol, sweetener, starch and immunity enhancing functional components except lactobacillus helveticus and/or its mutant, mixing, stirring, and adding ultrapure water to 1000mL to obtain feed liquid;

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

7) Filling and sterilizing: filling the homogenized feed liquid in the step 6) into a HDPE packaging container, sealing, pasteurizing at 87 ℃ for 18min, and cooling to obtain the sucrose-free yogurt with normal temperature and long shelf life, wherein the immunity of the yogurt can be improved.

Example 8: functional test of the yoghurt obtained by the invention

Mice are fed with the yoghurt obtained in the embodiment 1 of the invention, and after a period of time, cyclophosphamide is injected as an immunosuppressant to observe the effect of the product on improving the immunity of the mice. Before sterilization, the number of the lactobacillus helveticus WHH-2580 obtained by the invention can reach 1 multiplied by 10 ⁸ cfu/mL。

Inbred BALB/C male mice (6-8 weeks old, 16-20 g) were randomized into 4 groups with a 5 day adaptation period of 44, using a randomized group design: blank (CK), model (M), model+yogurt 5-fold (Y5) and model+yogurt 10-fold (Y10) groups each 11. The 5-time yoghurt group and the 10-time yoghurt group respectively simulate normal people (60 kg body weight) to take 200g and 400g of the normal-temperature long-shelf-life sucrose-free yoghurt capable of improving the immunity.

The animal feeding keeps the ambient temperature at 21+/-2 ℃, the humidity at 30-70%, the illumination is alternated for 12 hours, the animals can drink water and take feed freely. The padding is changed every three days, and water and feed are added in time. The subjects were administered continuously for 33 days. The molding drug used was cyclophosphamide (50 mg/kg, intraperitoneal injection, two consecutive days), and mice were sacrificed 5 days after molding, and thymus/body weight ratio was measured, and spleen lymphocyte transformation (MTT method) experiment and NK cell activity measurement were performed.

Grouping condition:

group 1 blank (n=11): gastric lavage water;

group 2 model group (n=11): pouring stomach water and molding cyclophosphamide;

group 3 modeling + yogurt 5-fold group (n=11): filling stomach 5 times of yoghourt 0.2 ml/d, and molding cyclophosphamide;

group 4 modeling + yogurt 10-fold group (n=11): and (3) pouring 10 times of yoghourt of 0.2 ml/d into the stomach, and molding cyclophosphamide.

Thymus index: as shown in fig. 4, the thymus/body weight ratio was significantly lower in the model group than in the blank group (P < 0.05), and there was no significant difference between the other model groups compared to the model group.

Mouse spleen lymphocyte transformation experiment: as shown in FIG. 5, the modeling groups were significantly worse (P < 0.05) than the model groups, indicating that each experimental group promoted proliferation of spleen lymphocytes in immunocompromised mice.

NK cell Activity assay: as shown in fig. 6, the NK cell activity of the model group was significantly lower than that of the blank group (P < 0.05). NK cell activity was significantly higher for the 5-fold and 10-fold yogurts than for the model group (P < 0.05).

According to the judging method of the result in the function evaluation method of the health care product, under the condition that an immune function hypofunction model is established, the total number of blood leucocytes, the cell immune function (a mouse spleen lymphocyte transformation experiment, a delayed type allergy experiment), the humoral immune function (antibody generation cell detection, serum hemolysin measurement), the mononuclear-macrophage function (a mouse carbon clearance experiment, a mouse peritoneal macrophage phagocytosis fluorescent microsphere experiment), the NK cell activity and the test result in any two aspects are positive, and the tested sample is judged to have the effect of enhancing the immunity of the immunocompetent person. In the animal experiment and the mouse spleen lymphocyte transformation experiment, OD values of 5 times of yoghourt groups and 10 times of yoghourt groups are obviously higher than those of a model group, and the result is positive. In the NK cell activity index, the NK cell activity of the 5-time yoghurt group and the 10-time yoghurt group is obviously higher than that of the model group, and the result is positive. Therefore, the 5-time yoghurt and the 10-time yoghurt are judged to have the function of enhancing the immunity of the mice with low immunity.

Example 9

The strain provided by the invention is identified as belonging to lactobacillus helveticus (Lactobacillus helveticus), and is named WH2580, and the microorganism preservation number is: CGMCC No.18730.

The strain provided by the invention is obtained by screening from yoghurt collected by red mountain ditches in Sinkiang Nalatin.

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

morphological features: the growth state in MRS agar medium is: the growth form of the strain in MRS agar culture medium is light milky colony, semitransparent, round, rough in surface, irregular in edge and flat. Gram staining was typically positive and cells were observed under a microscope to be long rod-like, flagellum free, sporulation free, and motionless (shown in fig. 7).

Culturing characteristics: the optimal growth temperature is 37 ℃, and the plant 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: the 16s rRNA gene sequence was sequenced, and the obtained results were subjected to homology alignment analysis with NCBI GenBank database, and the results showed that the strain was Lactobacillus helveticus (Lactobacillus helveticus).

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

CCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGC GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGA GATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACC GGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGT CTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTG GTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGT TAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTG CAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTC CTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTA GCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATC AGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTA AGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCA AGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCGTCCCCAGCGTCATTACCGAAGTAAATCTGCTGGTTCTGCTCGCTCGACTTGC

example 10: strain tolerance

Acid and bile salt resistance of lactobacillus helveticus WHH2580 strain:

acid resistance 1: the second-generation activated strain WH 2580 was centrifuged at 20 mL/tube and washed twice with PBS at pH 7.2. The bacterial sludge was placed in 20mL sterile PBS pH3.0, PBS+0.3% pepsin pH3.0 and PBS pH7.2, respectively, and each system was replicated in duplicate. And (3) placing the sample at 37 ℃ and sampling at 0h, 1h, 2h and 3h respectively, and counting viable bacteria.

The acid resistance test results show that: pH3.0 had no significant effect on the viability of the bacteria at 2h, 3h viable count was reduced by 0.9 orders of magnitude. Pepsin pH3.0 had no significant effect on the viability of the bacteria at 2h, with a 0.7 order of magnitude reduction in the number of viable bacteria at 3 h. The above results indicate that the bacterium has excellent acid resistance.

2 bile salt resistance: the second-generation activated strain WH 2580 was centrifuged at 20 mL/tube and washed twice with PBS at pH 7.2. Bacterial sludge was placed in 20mL of sterile PBS+0.1%Trypsin pH8.0, PBS+0.3% BS pH8.0, PBS+0.5% BS pH8.0 and PBS pH7.2, respectively, and each system was replicated in duplicate. And (3) placing the sample at 37 ℃ and sampling at 0h, 1h, 2h and 3h respectively, and counting viable bacteria.

The results of the bile salt tolerance experiment show that: 3h in 0.1% Trypsin system was reduced by 0.8 orders of magnitude, 3h in 0.3% BS system was reduced by 2.8 orders of magnitude, and 3h in 0.5% BS system was reduced by 3.6 orders of magnitude. The results show that the strain has good cholate resistance.

Example 11: bacterial strain adhesion

The probiotics colonize the intestinal epithelial cells of the host through adhesion, gradually form a biological film and generate various metabolites, thereby forming a natural protective barrier and generating various physiological effects for the host. Thus, adhesion and colonization of probiotics on host intestinal epithelial cells is a prerequisite for their probiotic action.

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

HT-29 cells were transferred to the third generation, centrifuged at 1000rpm for 5min after digestion with 0.25% pancreatin, and resuspended in 5mL DMEM medium containing 10% foetal calf serum (100U/mL penicillin, 100mg/mL streptomycin) until the cells were in single cell suspension. Appropriate amount of cells were counted by using a hemocytometer, and diluted to a cell suspension concentration of 1X 10 in DMEM medium (penicillin 100U/mL, streptomycin 100 mg/mL) containing 10% fetal bovine serum ⁶ 1mL of cells/mL was inoculated into 12-well cell culture plate on which cell slide had been placed, and incubated at 37℃with 5% CO ₂ Culturing for 2 days.

After WH 2580 strain (lactobacillus rhamnosus LGG and generation Tian Zhu LcS are positive control strains) is subjected to second-generation activation, 10mL of bacterial liquid is taken, 4200g is centrifugated for 10min at room temperature to collect bacterial cells, 10mL of DMEM complete medium containing 10% fetal bovine serum is used for culturing (without adding diabody) and resuspension is carried out, and the bacterial concentration is regulated to 2X 10 ⁸ CFU/mL. 5mL of strain regulating solution is taken for inactivation for 5min at 95 ℃. 1mL of each of the strain-adjusting solution and the inactivated strain-adjusting solution was inoculated into a 12-well cell culture plate on which a cell slide had been placed, each3 in parallel with CO ₂ Incubate for 2h at 37℃in incubator.

After the incubation was completed, the culture broth was slowly aspirated, washed 3 times with PBS and fixed with 100% methanol for 8min. Taking out the cell climbing tablet, standing for 20min, and sealing the tablet with neutral resin after gram staining. Observations were made under an optical microscope.

Adhesion test results are shown in the following table.

The effect of Lactobacillus helveticus WH2580 viable, non-viable, commercial strains LGG and Lcs on HT-29 cell adhesion is shown in FIG. 8.

The adhesion capacity of the lactobacillus helveticus WH2580 viable bacteria and non-viable bacteria is obviously higher than that of positive control strains lactobacillus rhamnosus LGG and field-substituting strain LcS. The adhesion rate of the non-living bacteria of the Lactobacillus helveticus WH2580 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 number of bacteria among HT-29 cells is less, and most of the non-living bacteria are tightly adhered on the cells.

Example 12: bacterial strain resistance

Sensitivity to antibiotics of lactobacillus helveticus WHH2580 strain:

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

The antibiotic formulation concentrations were 0.5ug/mL, 1ug/mL, 2ug/mL, 4ug/mL, 8ug/mL, 16ug/mL, 32ug/mL, 64ug/mL, 128ug/mL, 256ug/mL, the actual effect concentrations were 0.125ug/mL, 0.25ug/mL, 0.5ug/mL, 1ug/mL, 2ug/mL, 4ug/mL, 8ug/mL, 16ug/mL, 32ug/mL, 64ug/mL.

Adding 100 μl of culture medium into each well of sterile 96-well polystyrene plate, adding 50 μl of antibiotic diluent (ug/mL) into each well according to the concentration of 2-11 columns, adding 50 μl of bacterial suspension (final concentration of bacterial liquid is 105-106 CFU/mL) into each well of 1-11 columns, sealing, placing in a 37 ℃ incubator for 24-48h,OD625 was visually observed and measured. Wherein lactobacillus rhamnosus LGG and generation Tian Zhu LcS are positive control strains.

Name of the name	1	2	3	4	5	6	7	8	9	10	11	12
													Penicillin	Sterile solvents	0.5	1	2	4	8	16	32	64	128	256	Sterile water
Ampicillin	Sterile solvents	0.5	1	2	4	8	16	32	64	128	256	Sterile water
													Imipenem	Sterile solvents	0.5	1	2	4	8	16	32	64	128	256	Sterile water
Meropenem	Sterile solvents	0.5	1	2	4	8	16	32	64	128	256	Sterile water
													Vancomycin	Sterile solvents	0.5	1	2	4	8	16	32	64	128	256	Sterile water
Daptomycin	Sterile solvents	0.5	1	2	4	8	16	32	64	128	256	Sterile water
													Erythromycin	Sterile solvents	0.5	1	2	4	8	16	32	64	128	256	Sterile water
Clindamycin	Sterile solvents	0.5	1	2	4	8	16	32	64	128	256	Sterile water
													Linezolid	Sterile solvents	0.5	1	2	4	8	16	32	64	128	256	Sterile water

The results of the experiments are shown in the following Table, MIC values ug/mL.

Based on the criterion of sensitivity of the CLSI M4 strain to antibiotics, the following conclusion was reached. WH 2580 strain is resistant to Daptomycin and Clindamycin, neutral to Erythromycin, and sensitive to Penicillin, ampicillin, imipenem, meropenem, vancomycin and Linezolid. LGG strain is resistant to Imipenem, meropenem, vancomycin, daptomycin, clindamycin, neutral to Erythromycin, and sensitive to Penicillin, ampicillin and Linezolid. The LcS strain is resistant to Vancomycin and Daptomycin, neutral to Imipenem, meropenem and erythromycins, and sensitive to Penicillin, ampicillin, clindamycin and Linezolid.

Example 13: bacterial strain inhibiting pathogenic bacteria

Lactobacillus helveticus WH2580 strain bacteriostasis experiment:

sterilized 1.5% plain agar was previously dispensed onto sterile petri dishes. Placing oxford cup. Sterilizing 1% agar MRS culture medium or NB culture medium, inoculating at below 50deg.C for 10 ⁶ -10 ⁷ Five indicator bacteria (Escherichia coli, staphylococcus aureus, shigella flexneri, salmonella paratyphi B, micrococcus luteus) at CFU/mL concentration were mixed with shaking and poured onto the previously poured oxford agar medium. After the culture medium is cooled and solidified, the oxford cup is pulled out. 100uL of the corresponding bacterial solution (WH2580) and positive control (10 g/mL polymyxin B or 1% nisin) were added to different wells, and the wells were placed in a refrigerator at 4℃for 7h for diffusion. After incubation at 37℃for 16h, the size of the zone of inhibition was observed and recorded.

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

Experimental results show that WH 2580 strain has inhibition effect on 5 pathogenic bacteria, has no obvious difference on inhibition effect on salmonella paratyphi B, escherichia coli and shigella flexneri and 10g/mL polymyxin B, has no obvious difference on inhibition effect on staphylococcus aureus and 1% nisin, and is slightly lower than other control strains.

Example 14: the strain has immunity enhancing function

1. In vitro spleen lymphocyte proliferation:

killing mice, aseptically taking spleen, cutting spleen with ophthalmic scissors, grinding with sterile glass syringe core, and passing through 200 mesh metalAfter filtration through a screen, cells were harvested by centrifugation at 1000rpm at 4℃for 5min in sterile Hank's solution containing 10% fetal bovine serum. Adding 5 times of cell volume of cell lysate, lysing for 5min, adding 10% fetal bovine serum containing sterile Hank's solution, stopping lysing, centrifuging at 1000rpm at 4deg.C for 5min, and removing supernatant. Washing with RPMI-1640 medium containing 10% fetal bovine serum, 1000rpm, and centrifuging at 4deg.C for 5min. The pellet was resuspended in 5mL of RPMI-1640 medium containing 10% fetal bovine serum. Cell count, and cell concentration was adjusted to 5X 10 ⁶ cells/mL。

Centrifuging lactobacillus activated bacteria liquid at 4000rpm at room temperature for 10min to remove supernatant, washing twice with PBS, adding appropriate amount of RPMI-1640 medium containing 10% foetal calf serum, and adjusting OD ₆₀₀ To 0.50 + -0.10. Cell suspensions were added to 96-well cell culture plates, 3 replicates per sample. A zeroing group (cell culture medium), a blank group (cell suspension+cell culture medium, inducer group (cell suspension+10. Mu.g/mL Con A), a lactic acid bacteria group (cell suspension+10. Mu.g/mL Con A+bacterial suspension) were set, and incubated at 37℃for 72 hours in a carbon dioxide incubator, 20. Mu.L of MTT solution (2.5 mg/mL) was added to each well, after development at 37℃for 4 hours, centrifugation was performed at 1500rpm at 4℃for 10 minutes, the supernatant was aspirated, and 100. Mu.L of DMSO was added thereto, and absorbance was measured at 490nm using an microplate reader.

Data analysis: probiotics which significantly promote spleen lymphocyte proliferation, both with and without induction by Con a, were used as preferred strains.

The results are shown in the following table, where the multiplication factor of lactobacillus helveticus WHH2580 spleen lymphocytes was higher than that of the two commercial strains, with and without induction by Con a.

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2 in vitro spleen lymphocyte cytokine IL-10 and IL-12 assays

Cell sampling, erythrocyte lysis, washing, cell counting and probiotics culture are the same as the in vitro spleen lymphocyte proliferation step. The cell suspension was added to 96-well cell culture plates, each treatment was repeated 5 times, and the treatment was divided into zeroing groups (100. Mu.L of cell culture medium), blank groups (1X 10) ⁷ cell suspension 100. Mu.l, cell culture medium 100. Mu.l, cell/mL, probiotic group (1X 10) ⁷ cell suspension 100. Mu.L, 1X 10 in cells/mL ⁸ cfu/mL of bacterial suspension 100. Mu.L), 5% CO at 37 ℃ ₂ Culturing in an incubator for 24 hours. 1500 Centrifugation at rpm for 10min, the supernatant was aspirated, filtered through a 0.22 μm filter, and the IL-10 and IL-12 content was determined using BD kit.

Strains with a high IL-12/IL-10 ratio can be used as immunoregulatory potential strains.

The results are shown in the following table, where the ratio of Lactobacillus helveticus WH2580 IL-12/IL-10 is close to that of Lactobacillus rhamnosus LGG, and is not significantly inferior.

3 in vitro determination of human colon cancer cell HT-29 cytokine IL-8

Digesting human colon cancer cells HT-29 which grow and fuse to 70% -80%, and regulating cell concentration to 1 x 10 ⁶ The cells/holes are connected into 24-hole plates for culture at 37 ℃ and liquid exchange is carried out after 24 hours. Treated with 20ng/mL LPS for 6 hours and then added with 8X 10 ⁸ After 18 hours of treatment of CFU/mL bacterial liquid, 3000r/min and 5min are centrifuged to obtain supernatant, and the content of interleukin IL-8 is detected by using a corresponding ELISA kit.

The strain secreting high IL-8 can be used as an immunoregulatory 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 WH2580 IL-8 is higher than the commercial strain LGG.

Example 15: animal test

Inbred BALB/C male mice (6-8 weeks old, 16-20 g) were randomly grouped into 8 groups of 12 mice each with a 5 day adaptation period, using a random grouping design. The animal feeding keeps the ambient temperature at 21+/-2 ℃ and the humidity at 30-70%, the illumination is alternated for 12 hours, the animals can drink water and take feed freely. The padding is changed every three days, and water and feed are added in time. The grouping is as follows:

control group 1: blank control group, lavage water;

control group 2: model control group, lavage water + intraperitoneal injection of 50mg/kg cyclophosphamide for molding;

Control group 3: positive medicine group, lavage 40mg/kg levamisole hydrochloride + intraperitoneal injection 50mg/kg cyclophosphamide mould;

experiment group 1: the strain bacterial suspension of the invention is used for stomach irrigation, and the stomach irrigation dosage is 2-10 ⁸ CFU/d；

Experiment group 2: the strain inactivated bacterial suspension is used for lavage, and the dosage for lavage is 2 x 10 ⁸ CFU/d；

Wherein the inactivated bacteria are treated at 80deg.C for 10 min. The presence of the viable bacteria was confirmed by plate culture.

The test was performed using immunocompromised model animals. The subjects were administered continuously for 38 days, and administration of immunosuppressant was started after day 32. The body weight was weighed once a week before the inhibitor, and once a day after the inhibitor. The detection is started on the 5 th day of intraperitoneal injection of immunosuppressant, and the detection indexes include organ ratio, spleen lymphocyte proliferation and NK cell activity.

The experimental results are as follows:

the body weight results are shown in FIG. 9. The trend of body weight change was similar for each experimental group except for the blank, and body weight was reduced on the first day after injection of the inhibitor, followed by gradual recovery.

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

The proliferation results of the spleen lymphocytes of the mice are shown in FIG. 11. The OD value of the ultra-low dose group, the medium dose group and the high dose group is obviously higher than that of the inactivated bacteria group of the model group and is obviously higher than that of the model group. The WHH2580 ultra-low dose, low, medium, high dose and inactivated bacteria can obviously promote the proliferation of spleen lymphocytes of immunocompromised mice.

The results of the determination of NK cell activity in mice are shown in FIG. 12. The NK cell activity of the medium-dose group is remarkably higher than that of the model group, and the NK cell activity of the inactivated bacteria group is remarkably higher than that of the model group. The results show that the NK cell activity of the immunocompromised mice can be obviously improved by the dosage and the inactivated bacteria in WH 2580.

According to the health product function evaluation method, under the condition that an immune function hypofunction model is established, the total number of blood leucocytes, the cellular immune function (a mouse spleen lymphocyte transformation experiment, a delayed type allergy experiment), the humoral immune function (an antibody generation cell detection, a serum hemolysin measurement), the mononuclear-macrophage function (a mouse carbon clearance experiment, a mouse peritoneal macrophage phagocytosis fluorescent microsphere experiment) and NK cell activity are positive, and the tested sample is judged to have the effect of enhancing the immunity of a person with the immune hypofunction. The WH 2580 strain has positive test results on the aspects of cellular immune function and NK cell activity, and can be judged that the WH 2580 strain has the effect of enhancing the immunity of immunocompromised persons.

The WH 2580 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 unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Sequence listing

<110> Hangzhou child haha technology Co., ltd

Hangzhou Wahaha Group Co.,Ltd.

<120> sucrose-free yogurt with immunity improving function and long shelf life at normal temperature 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. The sucrose-free yoghurt with the normal temperature and long shelf life for improving the immunity is characterized by comprising raw milk or milk powder and an immunity-improving functional component; the immunity improving functional ingredient at least comprises lactobacillus helveticus;

the lactobacillus helveticus is named WHH2580, and the microorganism deposit number is: CGMCC No.18730, the microorganism classification is named as Lactobacillus helveticus Lactobacillus helveticusThe method comprises the steps of carrying out a first treatment on the surface of the The lactobacillus helveticus has a 16s rRNA gene sequence shown as SEQ ID NO. 1.

2. The sucrose-free yogurt of claim 1, further comprising sugar alcohols, starches, stabilizers, sweeteners.

3. The sucrose-free yogurt with the normal temperature and long shelf life for improving immunity according to claim 2, wherein the yogurt comprises, by mass, 40% -85% of raw milk or 5% -15% of milk powder, 0.001% -0.020% of lactobacillus helveticus, 0.01% -10.00% of immunity-improving efficacy components except lactobacillus helveticus, 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%.

4. A sucrose-free yogurt of claim 2 or 3 having a long shelf life at ambient temperature for enhancing immunity, characterized in that:

the milk powder is one or more of full-fat milk powder, defatted milk powder, whey protein powder and full-milk protein powder; and/or

The immunity improving functional ingredient also comprises one or more of propolis, ganoderma lucidum extract, wall-broken ganoderma lucidum spore powder, beta-glucan, dendrobium candidum extract, elderberry and acerola cherry; and/or

The sugar alcohol is one or more of xylitol, maltitol, erythritol, sorbitol, isomaltulose alcohol 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 carboxymethyl cellulose, 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, alitame and neotame.

5. A method for preparing the sucrose-free yogurt with enhanced immunity and long shelf life at normal temperature according to any one of claims 2 to 4, comprising the steps of:

scheme a:

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

(2) Inoculating and fermenting: adding lactobacillus helveticus into the sterilization cooling feed liquid obtained in the step (1), stirring and mixing uniformly, standing and fermenting at constant temperature to obtain a yoghurt base material;

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

(4) And (3) secondary batching: cooling the yoghurt base material obtained in the step (2), and then stirring and demulsifying; adding the glue solution obtained in the step (3), adding the immunity improving functional components except lactobacillus helveticus, sugar alcohol, sweetener and starch in the formula dosage, mixing and stirring, and fixing the volume by water 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 feed liquid in the step (5) to obtain the normal-temperature long-shelf-life sucrose-free yoghourt capable of improving the immunity; or sterilizing the homogenized feed liquid in the step (5) by UHT, hot filling or sterile cold filling, and sealing to obtain the normal-temperature long-shelf-life sucrose-free yoghurt with improved immunity;

scheme B:

1) Preparing milk: heating water, adding milk powder with the formula amount into the water, stirring or shearing, standing and hydrating to obtain milk;

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

3) Inoculating and fermenting: adding lactobacillus helveticus into the sterilization cooling feed liquid obtained in the step 2), stirring and mixing uniformly, and standing and fermenting at constant temperature to obtain a yoghurt base material;

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

5) And (3) secondary batching: cooling the yoghurt base material obtained in the step 3), and then stirring and demulsifying; adding the glue solution obtained in the step 4), adding the immunity improving functional components except lactobacillus helveticus, sugar alcohol, sweetener and starch in the formula dosage, mixing and stirring, and fixing the volume by water 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 feed liquid in the step 6) to obtain the normal-temperature sucrose-free yogurt with long shelf life and improved immunity; or sterilizing the homogenized feed liquid in the step 6) by UHT, hot filling or sterile cold filling, and sealing to obtain the normal-temperature long-shelf-life sucrose-free yogurt with improved immunity.

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

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

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

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

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

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

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

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

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

In step (1) or step 2), cooling to a temperature of 35-45 ℃.

8. The method for preparing the sucrose-free yogurt with the immunity-enhancing normal temperature and long shelf life of 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 4-16h; and/or

In the step (2) or the step (3), the number of viable bacteria is more than or equal to 1 multiplied by 10 in the fermentation process ⁸ cfu/mL。

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

In the step (3) or the step 4), the mass of the water is 200-400g; 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 20-40min; and/or

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

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

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

In the step (4) or the step (5), water is used for fixing the volume to 1000mL; and/or

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

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