CN107019043A - A kind of Lactobacillus plantarum acidified milk of laetarius volemus polysaccharide and preparation method thereof - Google Patents

Abstract

The invention discloses a lactobacillus plantarum fermented milk with succulent Lactobacillus polysaccharides and a preparation method thereof. The fermented milk, succulent Lactobacillus polysaccharide and plantaractobacillus starter are mixed evenly and then fermented, and fermented to produce milk containing succulent lactobacillus Fermented milk of succulent milk polysaccharide; the fermented milk is also subjected to homogenization and disinfection pretreatment, or the fermented milk is mixed with succulent milk polysaccharide and then subjected to homogenization and disinfection pretreatment. The obtained succulent Lactobacillus polysaccharide fermented milk has good rheological properties, water holding capacity, moderate sweet and sour, rich in essential amino acids and free amino acids, etc., and the number of viable lactic acid bacteria is as high as 10 ¹⁰ CFU/mL; Rich, good taste, color, taste and other sensory properties are in line with the indicators in the national standard GB19302‑2010 for fermented milk.

Description

A kind of lactobacillus plantarum fermented milk of succulent milk mushroom polysaccharide and preparation method thereof

technical field

The invention belongs to the technical field of lactic acid bacteria and dairy processing, and in particular relates to a polysaccharide fermented milk capable of improving human immunity and a preparation method thereof.

Background technique

Studies have confirmed that fungal polysaccharides have various physiological functions such as regulating immunity, lowering blood fat, anti-aging and anti-tumor, and are highly valued by people and have great development value. Because of its excellent taste and nutritional content, fermented milk is considered to be one of the best carriers for adding functional ingredients and realizing product functions. Lactic acid bacteria have extremely important physiological functions in the human body, and their quantity affects human health and even human life span. The important physiological functions of lactic acid bacteria in the human body are mainly realized by the four types of substances produced by them: organic acids, bacteriocins, bacterial surface components and special enzymes. The physiological functions of lactic acid bacteria mainly include: (1) giving food a good flavor and improving the nutritional value of food; (2) promoting the secretion of gastric juice, promoting gastrointestinal peristalsis, preventing indigestion and habitual constipation; (3) resisting foreign bacteria (4) enhance the body's immunity, inhibit the growth and metastasis of tumors; (5) reduce the content of cholesterol in serum and prevent the occurrence of cardiovascular diseases . Because fermented milk contains live probiotics or lactic acid bacteria and active ingredients, it plays an important role in improving human health, and has received great attention in recent years. According to literature reports, fungal polysaccharides can be applied to fermented milk. For example, the polysaccharides of Flammulina velutipes, black fungus polysaccharides, Agaricus blazei polysaccharides, lentinan and Yunzhi polysaccharides are applied to fermented milk. The results show that fungal polysaccharides can promote the growth of lactic acid bacteria, improve the fermentation The speed of producing lactic acid, reducing the fermentation cycle, can also increase the number of viable lactic acid bacteria.

Succulent milk mushroom (Lactarius volemus Fr.), commonly known as red milk mushroom, milk mushroom, milk mushroom, is an edible wild fungus, but the extraction of active polysaccharides, the evaluation of active components and the effect of polysaccharides on fermented milk The application in it has never been reported.

Contents of the invention

Aiming at the unknown immune activity of succulent milk mushroom polysaccharide and the present situation of its application in fermented milk, the present invention provides succulent milk mushroom polysaccharide fermented milk with lactobacillus plantarum capable of improving immunity and a preparation method thereof.

The present invention adopts following technical scheme:

Through in-depth research on the above-mentioned problems of fermented milk additives and unstable texture, it was found that adding succulent Lactobacillus polysaccharides in the preparation process of fermented milk can make it act as a prebiotic factor in the fermentation process. Compared with succulent milk mushroom polysaccharide, it can significantly enhance the total number of viable bacteria in fermented milk and the stability of texture without affecting the inherent flavor of fermented milk. Therefore, the present invention improves the process at the same time on the basis of optimizing the formula: adding succulent milk polysaccharides for fermentation, promoting the growth and reproduction of the starter, prolonging the storage period of fermented milk, and preparing fermented milk with unique flavor, delicate texture and smooth taste. Dairy products, reducing whey precipitation.

A preparation method of Lactobacillus plantarum fermented milk containing succulent Lactobacillus polysaccharides, mixing milk liquid for fermentation with succulent Lactobacillus polysaccharides and plant Lactobacillus starter, mixing them evenly and then fermenting, and making succulent Lactobacillus polysaccharides after fermentation The fermented milk; the fermented milk is also subjected to homogenization and disinfection pretreatment, or the fermented milk is mixed with succulent milk polysaccharide and then subjected to homogenization and disinfection pretreatment.

The preparation method of the succulent milk mushroom polysaccharide is as follows: firstly pretreat the succulent milk mushroom raw material to obtain the succulent milk mushroom fine powder, and then extract the polysaccharide with water after degreasing; then deproteinize; use 95% ethanol Precipitate the polysaccharide, wash the polysaccharide with 75% ethanol in the obtained concentrated solution, and freeze-dry it in vacuum to prepare lactanan from succulent mushroom.

The preparation of the succulent lactose polysaccharide comprises the following steps:

(1) Raw material pretreatment: washing the succulent milk mushroom, drying it to remove the stalk, grinding the powder with the fruit body part, and then passing through a 150-200 mesh sieve to obtain the succulent milk mushroom fine powder;

(2) Degreasing: add absolute ethanol and petroleum ether to the fine powder of succulent milk mushroom, reflux at 85°C to 95°C for 3h to 4h, and suction filter to obtain the filter residue;

(3) Extract polysaccharides with water: add distilled water to the filter residue, soak for 1h-2h, then 100W ultrasonic treatment for 10min-20min, extract in a water bath at 90°C-95°C for 3h-5h; filter with 150-200 mesh Filter the bag, collect the extract and concentrate it with a rotary evaporator to obtain a 15- to 25-fold concentrated succulent milk polysaccharide concentrate;

(4) Deproteinization treatment: mix the succulent lactose polysaccharide concentrate with 1/3 to 1/5 times the volume of Sevag reagent, let it stand for 20 to 40 minutes, then separate the layers, then centrifuge at low temperature to obtain the upper layer extract, and use distilled water Dilute and remove the organic solvent with a rotary evaporator to obtain the deproteinized succulent lactose polysaccharide concentrate I;

(5) Precipitate polysaccharides with 95% ethanol: Add 2 to 4 times the volume of pre-cooled 95% ethanol to the succulent lactobacillus polysaccharide concentrate I, place it at 1°C to 4°C for 10h to 20h, and then centrifuge at low temperature The flocculent precipitate is obtained, which is the succulent Lactobacillus polysaccharide concentrate II;

(6) Wash the polysaccharide with 75% ethanol: wash the succulent Lactarius polysaccharide concentrate II with 75% ethanol three times, redissolve in water and centrifuge at low temperature to remove insoluble matter, and vacuum freeze-dry the supernatant after pre-freezing , to obtain the succulent milk mushroom polysaccharide powder.

The content of the succulent milk polysaccharide is 0.05%-0.15%.

The fermentation temperature is 35°C-43°C.

The added succulent lactose polysaccharide is prepared into a solution first, and is treated with a membrane.

The fermented milk consists of:

The reconstituted milk of whole milk powder is prepared in proportion with sucrose and stabilizer;

Or the reconstituted milk of skimmed milk powder and cream, sucrose and stabilizer are prepared in proportion;

Or fresh milk, sucrose, and stabilizers are prepared in proportion.

The preparation of plant lactobacillus starter comprises the following steps:

(1) MRS liquid medium is used to activate the freeze-dried bacterial powder of Lactobacillus plantarum for 2 to 3 times to obtain activated bacterial liquid;

(2) Inoculate the activated bacteria liquid into the milk liquid for activation for activation treatment to obtain a Lactobacillus plantarum starter.

The milk for activation is mixed with 10.0%-12.5% whole milk powder, 7.5%-8.5% sucrose, 0.0005%-0.002% manganese gluconate, 0.5%-2.0% soybean polypeptide, etc., and homogenized and disinfected.

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

(1) The succulent milk mushroom polysaccharide adopted in the present invention is a pure natural substance, has good immune activity, is stable in fermented milk, can withstand pasteurization intensity, and will not affect the shelf life and mouthfeel of fermented milk.

(2) Compared with the fermented milk without adding polysaccharide, the polysaccharide fermented milk of the present invention has good rheological properties, water holding capacity, moderate sweet and sour, rich in essential amino acids and free amino acids, etc., and the number of viable lactic acid bacteria It is as high as 10 ¹⁰ CFU/mL; the aroma components are relatively strong, the taste is good, and the sensory properties such as color and taste are in line with the indicators in the national standard GB19302-2010 for fermented milk.

(3) The raw material of fermented milk of the present invention is abundant, can be the reconstituted milk of whole milk powder and fermented milk raw materials such as sucrose, stabilizing agent to prepare in proportion, also can be the reconstituted milk of skimmed milk powder and cream and sucrose, Fermented milk raw materials such as stabilizer are prepared in proportion, and fresh milk, sucrose, stabilizer and other fermented milk raw materials can also be prepared in proportion.

(4) The production process is simple and easy to control, the taste is delicate, the aroma is strong, the number of viable lactic acid bacteria is high, the texture is good, the viscosity is suitable, the water holding capacity is good, and there is no whey precipitation.

Description of drawings

Fig. 1 Pathological slices of livers of mice in normal group (CK), CP injury group (CP ^- ), CP injury positive group (CP ⁺ ) and polysaccharide medium dose group (MP) after ink injection.

Figure 2 is the shear scanning rheological characteristic curve of Lactobacillus plantarum fermented milk with succulent Lactobacillus polysaccharide, Figure 2a is the change of apparent viscosity with shear rate, and Figure 2b is the change of shear stress and shear rate.

Fig. 3 is the frequency scanning rheological characteristic curve of succulent milk mushroom polysaccharide Lactobacillus plantarum fermented milk, wherein G' is the elastic modulus, and G" is the viscous modulus.

detailed description

The present invention will be described in further detail below in conjunction with the examples, but the scope of protection claimed by the present invention is not limited thereto.

Example 1

Extraction of polysaccharide from succulent milk mushroom

Wash the succulent milk mushroom, dry it to remove the stalk, grind it with the fruit body part, and then pass it through a 200-mesh sieve to obtain the succulent milk mushroom fine powder. :30 After soaking for 1.5h, use 100W ultrasonic treatment for 15min, extract in 95°C water bath for 4h, filter with 200 mesh filter bag, collect the extract, concentrate to 20 times in 55°C rotary evaporator, then add 1/5 Double the volume of Sevag reagent (the volume ratio of n-butanol:chloroform is 1:4) to deproteinize, centrifuge at 5000r/min for 5min at low temperature to obtain the upper layer extract, dilute with an appropriate amount of distilled water, and remove it with a rotary evaporator For organic reagents, add 3 times the volume of 95% ethanol to the succulent milk polysaccharide concentrate I, place it at 4°C for 20 hours, centrifuge at 5000r/min for 5 minutes to obtain the succulent milk polysaccharide concentrate II, and then wash with 75% ethanol The succulent milk mushroom polysaccharide concentrate II is counted 3 times, redissolved in water and centrifuged at 5000r/min for 5 minutes at low temperature to remove insoluble matter, and then the supernatant is pre-frozen and vacuum freeze-dried to obtain more succulent milk mushroom polysaccharide Powder III was stored in a sealed bag at 4°C, and the polysaccharide yield was 2.18% (by dry weight).

Example 2

Evaluation of the Immunity-enhancing Effect of Succulent Lactarius Polysaccharides in Vivo

(1) Grouping of mice: 72 SPF-grade inbred Balb/c mice aged 7-8 weeks, weighing 18-22 g, were randomly divided into 6 groups according to the weight of the mice. The detailed grouping information is shown in Table 1. And through SPSS17.0 software for significant difference analysis, it shows that there is no significant difference between groups. All mice were reared by professionals, 4/cage. Under the conditions of a temperature of 22-24°C and a humidity of 50%-60%, the light is 12 hours and the darkness is 12 hours a day. During the experiment, the mice were free to drink water and eat, and the mice in each experimental group were gavaged at 9:00 a.m. every day, and the amount of gavage was 200mL/(kg bw d). The CP ^- and CP ⁺ groups were 7 days in advance. Cyclophosphamide (abbreviated as CP), an immune system damage drug, was injected intraperitoneally into mice, and 30 mg/(kg bw) of CP was injected every day for 7 consecutive days, and 80 mg/(kg bw) of CP was injected on the 25th day.

Table 1 Group information table of mice

(2) Determination of various immune indicators

①Determination of delayed hypersensitivity reaction: After feeding for 25 days, each mouse was injected intraperitoneally with 0.2 mL of 2% SRBC sheep red blood cells. After 5 days of immunization, the mice were weighed and the thickness of the left hind foot was measured (H ₀ ), inject 20 μl of 20% SRBC sheep red blood cells at the measurement site, and measure the thickness (H) of the left hind paw _at the same site 24 hours later. .

②Measurement of carbon clearance index: After the measurement on the 30th day, 33% Indian ink was injected into the tail vein, the injection volume was 0.1mL/(kg·bw), and 20μl of blood was collected from the inner canthal vein 2min and 10min after the injection, respectively. And immediately added to the EP tube filled with 2mL0.1% Na ₂ CO ₃ , measured OD _600nm and calculated the corresponding phagocytosis rate K=(lgOD ₁ -lgOD ₂ )/(t ₂ -t ₁ ) and phagocytosis index a= (body weight×K ^1/3 )/(liver weight+spleen weight), the carbon clearance index is the phagocytosis index a.

③Measurement of organ coefficient: At the end of the experiment, all mice were fasted overnight, the eyes were removed to get the whole blood of the mice, and then the mice were killed by neck dislocation, the liver, spleen, and thymus were separated, and the blood stains on the surface of the organs were washed with normal saline. Blot dry with filter paper and weigh. Organ coefficients include liver coefficient, spleen coefficient and thymus coefficient. The calculation formula is: organ coefficient=organ mass (mg)/body weight (g). For the organs of the mice subjected to carbon clearance, the colors of the 6 groups were all dark red, and the Indian ink had not been metabolized, and the CP ^- compared with the dose group, the color state was darker and the metabolism was worse. In the three polysaccharide dosage groups of LP, MP and HP and the CK group, it can be judged that the polysaccharide liquid of Lactarius succulent has certain immune efficacy. After calculation, the results of organ coefficients of mice in each group are shown in Table 2. 3 polysaccharide dosage groups such as LP, MP and HP compared with CK,

The body weight of the mice and three immune organ coefficients including liver coefficient, spleen coefficient ^and thymus coefficient were significantly increased, which was statistically significant (p<0.05). The coefficient decreased obviously, which verified that the immune injury model of mice had been established. Compared with CP ⁺ and CP ^- , the immunity of mice recovered slightly, indicating that the crude polysaccharide of Lactarius succulent has the effect of improving immunity.

Table 2 Body weight (g) and immune organ coefficient (M±SD, n=12) of mice in each group at the 4th week (d28)

Note: organ coefficient = organ weight/body weight, the unit is mg/g. Different symbols (a～d) indicate that there are significant differences between the data in the same column

(p<0.05); significant differences were analyzed using Dunan's analysis in SPSS 17.0.

As shown in Table 3, at the 4th week, the mice were subjected to paw and plantar hypersensitivity swelling (DTH) and carbon clearance test. Compared with CK, the three polysaccharide dosage groups such as LP, MP and HP, the mice's paw and plantar hypersensitivity The symptoms of swelling were more obvious, and at the same time, the ability of macrophages to remove carbon particles (phagocytosis index a) was also stronger ^, and it was statistically significant (p<0.05). The degree and function of macrophages were far inferior to those in the CK group, which verified the establishment of the mouse immune injury model; CP ^- compared with CP ⁺ , the immunity of the mice was slightly restored, indicating that the crude polysaccharide of Lactarius succulent has the effect of improving immunity.

Table 3 Detection results of DTH and carbon clearance index of mice in each group at the 4th week (d28) (M±SD, n=12)

Note: The degree of allergic swelling of the foot and plantar is expressed by DTH, and the unit is mm; the carbon clearance index refers to the phagocytosis index a. Note: Different letters (a～f) indicate significant differences between data in the same column (p<0.05); significant differences were analyzed using Dunan’s analysis in SPSS 17.0.

④ Whole blood analysis: The whole blood of the mouse was immediately sent to the Experimental Animal Center of Sun Yat-sen University East Campus for 18 automatic whole blood analysis, focusing on the number of lymphocytes and white blood cells, the number of lymphocytes and white blood cells in the whole blood of mice As shown in Table 4, the proportion of lymphocytes in the three polysaccharide dosage groups of LP, MP and HP was significantly higher than that of other groups, which was comparable to that of the normal group, and the number of white blood cells and lymphocytes of LP were significantly higher than that of other groups ( p<0.05). And CP ^- compared with the normal group, the number of white blood cells and ^lymphocytes in mice decreased, which also verified the establishment of the immune injury model in mice; in addition, compared with CP- ^, the immune indicators of mice were slightly lower There was recovery, between CP- ^and CK, indicating that lactan succulent exhibited an immune-enhancing effect. The number of white blood cells and lymphocytes in CP ⁺ was also significantly higher than that in CP ^- , which indicated that when mice were attacked by antigens, polysaccharides could significantly enhance the cellular immune function of mice, and the effect of low dose was the most significant.

Table 4 The whole blood test results of mice in each group at the 4th week (d28) (M±SD, n=12)

Note: Different letters (a～e) indicate that there is a significant difference between the data in the same column (p<0.05); the significant difference adopts the Dunan's analysis method in SPSS17.0. WBC means the number of white blood cells, the unit is 10 ⁹ /L; LYM means the number of lymphocytes, the unit is 10 ⁹ /L; LYM% means the percentage of lymphocytes in the whole blood cells.

⑤ Pathological section analysis of organs: The mouse organs that had been preliminarily fixed with polyformaldehyde were immediately sent to the Experimental Animal Center of Sun Yat-Sen University East Campus for hematoxylin-eosin staining, and the livers of mice injected with ink were observed under a microscope Normal tissue morphology and lesion tissue structure morphology, pathological section results are shown in Figure 1, in which Figure 1A shows that 5-10% of Kupffer cells that phagocytize pigment and participate in immune regulation can be seen around the hepatic sinusoid of the normal group (CK), 10 ~20% of hepatic nuclei vacuolization caused by inflammation can represent the degree of liver cell damage; 10-20% of Kupffer cells and 10-20% of hepatic sinusoids in the CP injury group (CP ^- ) can be seen Nuclei are vacuolated (Fig. 1B); 10-20% of pigment-phagocytic Kupffer cells, 10-20% of hepatic nuclei and 5-10% of hepatic cytoplasm can be seen around the hepatic sinusoids of CP ⁺ (Fig. 1C); 20-30% of pigment-phagocytic Kupffer cells, 10-20% of hepatocyte nuclei vacuoles, and 1-5% of hepatocyte plasma vacuoles can be seen around the hepatic sinusoids of the medium-dose polysaccharide group (MP) (Fig. 1D). The number of Kupffer cells in MP was significantly higher than that in other groups, and the degree of vacuolization of hepatic cytoplasm was the lowest, and the degree of vacuolization of hepatic cells in CP ⁺ was also lower than that in CP ^- , indicating that succulent lactose polysaccharides are beneficial to the formation of hepatic macrophages. Activated, participates in the scavenging reaction of carbon foreign matter and has a certain repairing effect on liver cell damage.

The unremoved carbon particles in the liver slices were integrated by imageplus-pro software, and the results are shown in Table 5. Obviously, the proportion of 0.60% of CP ^- unremoved carbon particles is slightly greater than 0.50% of CP ⁺ , and MP is less than CP ⁺ and CK. It further illustrates that polysaccharides can enhance the function of hepatic macrophages in mice.

Table 5 Integral results of liver carbon particle distribution of mice in each group

Note: Different letters (a～d) indicate significant differences between data in the same column (p<0.05); significant differences are analyzed using Dunan’s analysis in SPSS 17.0

Example 3

Application of succulent milk polysaccharide in fermented milk

The first step of fermentation agent preparation: resuspend Lactobacillus plantarum (DMDL 9010) in 1mL sterile water, which was preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on August 19, 2011, address: Chaoyang, Beijing District Datun Road, the preservation number is CGMCC NO.5172) freeze-dried powder, and then all transferred to MRS liquid medium, cultivated at 37°C for 16h, and then transferred to fresh MRS with 5% to 10% inoculum Activation in liquid medium for 3 times, centrifugation at 4000r/min at low temperature to collect the thallus, and transfer all of them to milk for activation and culture for 2 to 3 times at 37°C to obtain Lactobacillus plantarum starter; The milk liquid for activation is prepared by mixing 12.5% skimmed milk powder, 8.5% sucrose, 0.0005%-0.002% manganese gluconate and 0.5%-2.0% soybean polypeptide, homogenized and sterilized.

The second step is the preparation of milk liquid for fermentation and mixing with polysaccharide: add 0.00%, 0.05%, 0.10%, 0.15% of 12.5% whole milk powder, 8.5% sucrose, and 0.1% stabilizer powder according to the mass volume ratio After the succulent Lactobacillus polysaccharide powder is mixed evenly, preheat to 60°C and homogenize at 18Mpa, sterilize at 95°C for 5 minutes, then cool down to 43°C, and set aside;

The third step of inoculation and fermentation: adding 10% Lactobacillus plantarum starter into the milk for fermentation, fermenting at 43°C, and stopping the fermentation when the titrated acidity reaches 75°T to obtain succulent Lactobacillus polysaccharide fermented milk .

The fourth step: evaluation of various indicators of succulent milk mushroom polysaccharide fermented milk

(1) The sensory quality evaluation is carried out in accordance with the requirements of the national standard for fermented milk GB19302-2010. The specific evaluation items are shown in Table 6. The total score is 100 points, of which the flavor index and taste index each account for 30 points, and the appearance index has a full score of 15 points , the full score of quality index is 25 points. Each indicator is divided into three scoring segments according to the degree of acceptability: not very acceptable, generally acceptable, and like to accept. The score distribution is shown in Table 6. The scoring results are shown in Table 7. From the perspective of flavor, taste, appearance and texture, the fermented milk with 0.10% polysaccharide content is better, and the fermented milk with 0.15% polysaccharide has a darker color, which completely deviates from the original color of fermented milk. Fermented milk with a polysaccharide content of 0.05% is also acceptable.

Table 6 Sensory evaluation indexes of fermented milk

Table 7 Effects of added amount of succulent milk polysaccharides on the sensory properties of fermented milk

Note: Different letters (a～d) indicate significant differences between the data in the same column (p<0.05); the significant differences were analyzed by Dunan’s in SPSS17.0.

(2) Evaluation of physical and chemical indicators of succulent milk mushroom polysaccharide fermented milk Determination of pH, water holding capacity, free amino acid, viable count of lactic acid bacteria, and rheological properties of fermented milk after fermentation

The influence of different pH polysaccharide content on the pH of fermented milk is shown in Table 8. It can be seen that with the increase of polysaccharide concentration, the pH of fermented milk also decreases, which shows that adding polysaccharide can promote the acid production ability of Lactobacillus plantarum. It may be related to the fact that polysaccharides can promote the growth of lactic acid bacteria.

Weigh the mass of the 15mL EP tube holding water and record it as W ₀ , then weigh 10g of each yogurt sample into the EP tube, centrifuge at 5000r/min for 5min, discard the whey and invert the EP tube for 10min, weigh the total mass and record for W. Take the average value of 3 parallel measurements, water holding capacity=(WW ₀ )/10×100%. The effects of different polysaccharide contents on the water holding capacity of fermented milk are shown in Table 8, from which it can be seen that the addition of polysaccharides will not affect the water holding capacity of fermented milk, which is about 99.0%.

Table 8 Effect of polysaccharide content on pH, water holding capacity and viable count of Lactobacillus plantarum fermented milk

The number of viable lactic acid bacteria is taken out and mixed with 1 mL of the fermented milk sample prepared above and 9 mL of sterilized normal saline, that is, the dilution factor is 10 ¹ , and then 1 mL of the previous diluted sample is mixed with 9 mL of sterilized normal saline to obtain the diluted For samples with multiples of 10 ² , and so on, dilute to 10 ¹⁰ times, take 1mL of diluted fermented milk in a plate, then pour MRS solid medium to cover the surface of the bacterial liquid, and place it at 37°C for 24 hours after constant temperature incubation. Colony count. The effect of polysaccharide content on Lactobacillus plantarum fermented milk is shown in Table 8. With the increase of polysaccharide content, the number of viable lactic acid bacteria is increasing. It is confirmed again that succulent Lactobacillus polysaccharides can promote the growth and reproduction of Lactobacillus plantarum. When the polysaccharide content is 0.15%. , the maximum number of live bacteria is 1.60×10 ¹¹ CFU/mL, and the fermented milk of the four formulas all meet the requirements of the national standard GB19302-2010 for fermented milk.

Free amino acid Weigh 10g of yogurt and mix it with 5mL of distilled water, stir well, and then refrigerate and centrifuge at 4500r/min for 5min, take 4mL of the supernatant and mix it with 1mL of 15% sulfosalicylic acid solution, and place it at 4°C for more than 6h. Then centrifuge at 10000r/min for 15min, take the supernatant, repeat the centrifugation once, determine the nitrogen content of the sample by the Kjeldahl method, and then dilute the sample until the nitrogen content is between 0.6% and 1.0%. After filtering with a 0.22 μm filter membrane, put it into a sample bottle, use a C18 column, a quaternary pump, UV detection, an OPA automatic derivatization device, and an automatic sample injection analysis. The impact of polysaccharide content on free amino acids in Lactobacillus plantarum fermented milk is shown in Table 9. When the polysaccharide content was 0.05%, 0.10% and 0.15%, the net content of essential amino acids increased by 1870.6 and 1803.22 respectively compared with the control group (without polysaccharide). and 1851.17nmol/L, each essential amino acid content increased significantly (p<0.05). Compared with the control group, the total free amino acid content also increased, respectively 3669.05, 3477.02 and 3578.40nmol/L. It was shown again that succulent Lactobacillus polysaccharide can promote the growth and reproduction of Lactobacillus plantarum, thereby producing more essential amino acids and free amino acids, and improving the biological potency of fermented milk.

Table 9 Effects of polysaccharides on 17 free amino acids in Lactobacillus plantarum fermented milk (M±SD) (nmol/L)

Rheological properties Take 5mL of fermented milk on the stage of the Haake rheometer, select the appropriate program to measure the parameters such as the anti-deformation ability and viscosity of the sample, and after each set of procedures, open the probe and carefully dispose of the probe And the sample on the stage, replace the sample, save all the measurement data, and use origin8.5 to analyze and process it. The effect of polysaccharide content on the rheological properties of Lactobacillus plantarum fermented milk is shown in Figure 2 and Figure 3. It can be seen that with the increase of the shear rate, the apparent viscosity (Figure 2a) and the shear stress both showed a decreasing trend, and the apparent viscosity of fermented milk in each group was not significantly different. When the shear rate is controlled above 20s ^-1 , the addition of polysaccharide can increase the shear stress of fermented milk (Fig. 2b). When the content of polysaccharide is 0.15%, the apparent viscosity and shear stress are the highest and change slowly. Polysaccharides can increase the activity of lactic acid bacteria and improve the texture of fermented milk. Figure 3 shows the measurement results of Lactobacillus plantarum fermented milk after frequency scanning. G' and G" respectively represent elastic modulus and viscous modulus, which can respectively characterize the deformation resistance and viscosity of fermented milk. As can be seen from Figure 3, polysaccharide The addition of 0.15% Lactobacillus plantarum fermented milk had the best deformation resistance, the highest viscosity, and the best cross-linking situation, which may be due to the fact that polysaccharides promote the production of large amounts of exopolysaccharides by lactic acid bacteria, thereby improving the cross-linking structure of fermented milk. Based on the above, in Lactobacillus plantarum fermented milk, it is recommended that the amount of the polysaccharide Lactobacillus polysaccharide added be higher than 0.15% or lower than 0.05%.

Example 4

Extraction of Polysaccharide from Lactarius succulent and Its Application in Fermented Milk

The first step is to wash the succulent milk mushroom, dry it to remove the stalk, grind it with the fruit body part, and then pass it through a 150-mesh sieve to obtain the succulent milk mushroom fine powder. After defatting at 85°C for 4 hours, press the feed After soaking for 2 hours at a ratio of 1:30, use 100W ultrasonic treatment for 10 minutes, extract in a 90°C water bath for 5 hours, filter through a 150-mesh filter bag, collect the extract, concentrate it in a rotary evaporator at 55°C to 15 times, and then add 1/ 3 times the volume of Sevag reagent (the volume ratio of n-butanol:chloroform is 1:4) to deproteinize, centrifuge at 5000r/min for 5min at low temperature to obtain the upper layer extract, dilute with an appropriate amount of distilled water, and then use a rotary evaporator Remove organic reagents, add 2 times the volume of 95% ethanol to the succulent milk polysaccharide concentrate I, place it at 4°C for 18 hours, centrifuge at 5000r/min for 5 minutes to obtain the succulent milk polysaccharide concentrate II, and then use 75% ethanol Wash the succulent milk mushroom polysaccharide concentrate II for 2 to 3 times, redissolve it in water and centrifuge at 5000r/min for 5 minutes at low temperature to remove insoluble matter, then pre-freeze the supernatant and vacuum freeze-dry it to make it juicier Lactobacillus polysaccharide powder III was stored in a sealed bag at 4°C, and the polysaccharide yield was 2.01% (by dry weight).

The second step of starter preparation and mixing with polysaccharides: resuspend the lyophilized powder of Lactobacillus plantarum with 1 mL of sterile water, then transfer all of them to MRS liquid medium, culture at 37°C for 16h~20h, and then add 5%~ 10% of the inoculum was transferred to fresh MRS liquid medium for activation 3 times, and the bacterial cells were collected by centrifugation at 4000r/min at low temperature, and all of them were transferred to milk for fermentation and activated for 2 to 3 times at 35°C. The Lactobacillus plantarum starter is obtained; the milk liquid for activation is sterilized after mixing 12.5% whole milk powder, 8.5% sucrose, 0.002% manganese gluconate, 0.5% soybean polypeptide and other raw materials.

The third step is milk preparation for fermentation: fresh milk, 8.5% sucrose, 0.1% stabilizer powder, and 0.05% succulent lactobacillus polysaccharide powder III are mixed according to the mass volume ratio, and then preheated to 60°C at 18Mpa Homogenize, and sterilize at 95°C for 5 minutes, then cool down to 35°C, and set aside;

The fourth step is inoculation and fermentation: adding 5% Lactobacillus plantarum starter into the milk for fermentation, fermenting at 35°C, and stopping the fermentation when the titrated acidity reaches 75°, to obtain succulent Lactobacillus polysaccharide fermented milk, The pH, water holding capacity and viable count at the end of fermentation were 3.82, 98.79% and 8.70×10 ¹⁰ CFU/mL, respectively, and the taste quality met the requirements. If the coagulated fermented milk obtained through the above steps is demulsified by a peristaltic pump, it will be agitated fermented milk.

Example 5

Extraction of Polysaccharide from Lactarius succulent and Its Application in Fermented Milk

The first step is to wash the succulent milk mushroom, dry it to remove the stalk, grind it with the fruit body part, and then pass it through a 150-mesh sieve to obtain the succulent milk mushroom fine powder. After defatting at 95°C for 3 hours, press the feed After soaking for 1 hour at a ratio of 1:30, use 100W ultrasonic treatment for 20 minutes, extract in a 93°C water bath for 3.5 hours, filter through a 200-mesh filter bag, collect the extract, concentrate to 25 times in a 55°C rotary evaporator, and then add 1 /4 times the volume of Sevag reagent (the volume ratio of n-butanol:chloroform is 1:4) to deproteinize, centrifuge at 5000r/min for 5min at low temperature to obtain the upper layer extract, dilute with an appropriate amount of distilled water, and then use rotary evaporation To remove organic reagents, add 4 times the volume of 95% ethanol to the succulent milk polysaccharide concentrate I, place it at 3°C for 15 hours, centrifuge at 5000r/min for 5 minutes to obtain the succulent milk polysaccharide concentrate II, and then use 75% Wash the succulent milk mushroom polysaccharide concentrate II with ethanol for 2 to 3 times, redissolve it in water and centrifuge at 5000r/min for 5min at low temperature to remove insoluble matter, then pre-freeze the supernatant and vacuum freeze-dry it. Lactobacillus polysaccharide powder III was stored in a sealed bag at 4°C, and the polysaccharide yield was 2.15% (by dry weight).

The second step of starter preparation: resuspend the lyophilized powder of Lactobacillus plantarum in 1 mL of sterile water, then transfer all of it to MRS liquid medium, culture at 37°C for 16h-20h, and then inoculum with 5%-10% Transfer to fresh MRS liquid medium for activation 3 times, collect the bacteria by centrifugation at 4000r/min at low temperature, and transfer all of them to fermented milk, activate and culture at 35°C for 2 to 3 times to obtain vegetable milk Bacillus starter: the milk liquid for activation is formed by mixing uniformly raw materials such as 10.0% whole milk powder, 7.5% sucrose, 0.0005% manganese gluconate, and 2.0% soybean polypeptide, and then sterilized.

The third step is the preparation of milk liquid for fermentation and mixing with polysaccharide: fresh milk, 8.5% sucrose, 0.1% stabilizer powder, 0.05% milky mushroom polysaccharide powder III according to the mass volume ratio, mix evenly, and preheat to 60°C Homogenize at 18Mpa, sterilize at 95°C for 5 minutes, cool down to 37°C, and set aside;

The fourth step is inoculation and fermentation: adding 5% Lactobacillus plantarum starter into the milk for fermentation, fermenting at 37°C, and stopping the fermentation when the titrated acidity reaches 75°, to obtain succulent Lactobacillus polysaccharide fermented milk, The pH, water holding capacity and viable count at the end of fermentation were 3.85, 99.03% and 9.30×10 ¹⁰ CFU/mL, respectively, and the taste quality met the requirements. If the coagulated fermented milk obtained through the above steps is demulsified by a peristaltic pump, it will be agitated fermented milk.

Example 6

Extraction of Polysaccharide from Lactarius succulent and Its Application in Fermented Milk

The first step is to wash the succulent milk mushroom, dry it to remove the stalk, grind it with the fruit body part, and then pass it through a 180-mesh sieve to obtain the succulent milk mushroom fine powder. After defatting at 90°C for 3.5 hours, press the After soaking for 2 hours at a liquid ratio of 1:30, use 100W ultrasonic treatment for 18 minutes, extract in a 95°C water bath for 3 hours, filter through a 200-mesh filter bag, collect the extract, concentrate it to 20 times in a 55°C rotary evaporator, and then add 1 /3 times the volume of Sevag reagent (the volume ratio of n-butanol:chloroform is 1:4) to deproteinize, centrifuge at 5000r/min for 5min at low temperature to obtain the upper layer extract, dilute with an appropriate amount of distilled water, and then use rotary evaporation To remove organic reagents, add 3.5 times the volume of 95% ethanol to the succulent lactopolysaccharide concentrate I, place it at 4°C for 15 hours, centrifuge at 5000r/min for 5min to obtain the succulent lactopolysaccharide concentrate II, and then use 75% Wash the succulent milk mushroom polysaccharide concentrate II with ethanol for 2 to 3 times, redissolve it in water and centrifuge at 5000r/min for 5min at low temperature to remove insoluble matter, then pre-freeze the supernatant and vacuum freeze-dry it. Lactobacillus polysaccharide powder III was stored in a sealed bag at 4°C, and the polysaccharide yield was 2.05% (by dry weight).

The second step of starter preparation: resuspend the lyophilized powder of Lactobacillus plantarum in 1 mL of sterile water, then transfer all of it to MRS liquid medium, culture at 37°C for 16h-20h, and then inoculum with 5%-10% Transfer to fresh MRS liquid medium for activation 3 times, collect the bacteria by centrifugation at 4000r/min at low temperature, and transfer all of them to fermented milk, activate and culture at 41°C for 2 to 3 times to obtain vegetable milk Bacillus starter: the milk liquid for activation is sterilized after mixing 11.0% whole milk powder, 8.0% sucrose, 0.001% manganese gluconate, 1.5% soybean polypeptide and other raw materials. The third step is milk preparation for fermentation and mixing with polysaccharide: 8.5% skimmed milk powder, 3.0% cream, 7.5% sucrose, 0.1% stabilizer powder, add 0.10% succulent milk mushroom polysaccharide powder III by mass volume ratio After mixing evenly, preheat to 60°C and homogenize at 18Mpa, sterilize at 95°C for 5 minutes, then cool down to 41°C, and set aside;

The fourth step is inoculation and fermentation: adding 5% Lactobacillus plantarum starter into the milk for fermentation, fermenting at 41°C, and stopping the fermentation when the titrated acidity reaches 75°, to obtain succulent Lactobacillus polysaccharide fermented milk, The pH, water holding capacity and viable count at the end of fermentation were 3.79, 99.11% and 1.46×10 ¹¹ CFU/mL, respectively, and the taste quality met the requirements. If the coagulated fermented milk obtained through the above steps is demulsified by a peristaltic pump, it will be agitated fermented milk.

Claims (10)

1. a kind of preparation method of the Lactobacillus plantarum acidified milk of laetarius volemus polysaccharide, it is characterised in that by fermentation milk with Laetarius volemus polysaccharide and lactobacillus plantarum ferment, are well mixed after fermentation, the fermentation of the polysaccharide containing laetarius volemus are made after fermentation Breast；The fermentation is passed through again after also being mixed with milk through homogeneous and sterilization pretreatment, or fermentation with milk with laetarius volemus polysaccharide Homogeneous and sterilization are pre-processed.

2. preparation method according to claim 1, it is characterised in that the preparation method of the laetarius volemus polysaccharide is：First Laetarius volemus raw material pre-process to obtain laetarius volemus fine powder, Polyose extraction is carried out after degreasing, then with water；After through de- Protein processing；With 95% ethanol precipitation polysaccharide, gained concentrate 75% ethanol washing polysaccharide and vacuum freeze drying are made Laetarius volemus polysaccharide.

3. preparation method according to claim 2, it is characterised in that the preparation of the laetarius volemus polysaccharide includes following step Suddenly：

(1) pre-treatment of raw material：Wash laetarius volemus, be baked to handle, with fructification part be milled, then cross 150 mesh~ 200 eye mesh screens, obtain laetarius volemus fine powder；

(2) degreasing：Absolute ethyl alcohol and petroleum ether are added into laetarius volemus fine powder, flow back 3h~4h, suction filtration at 85 DEG C~95 DEG C Obtain filter residue；

(3) Polyose extraction is carried out with water：Added into filter residue after distilled water, immersion 1h~2h, 100W ultrasonications 10min~ 20min, in extraction 3h~5h under 90 DEG C~95 DEG C of water bath condition；With the mesh sock filtration of 150 mesh~200, collect leaching liquor and use Rotary Evaporators are concentrated, and obtain the laetarius volemus polysaccharide concentrate of 15 times~25 times concentrations；

(4) deproteinization is handled：Laetarius volemus polysaccharide concentrate is mixed with the Sevag reagents of 1/3~1/5 times of volume, stood It is layered after 20min~40min, then low-temperature centrifugation obtains upper strata leaching liquor, is removed again with Rotary Evaporators with distilled water diluting organic Solvent, obtains Deproteinated laetarius volemus polysaccharide concentrate I；

(5) with 95% ethanol precipitation polysaccharide：In laetarius volemus polysaccharide concentrate I, 2 times~4 times second of volume precooling 95% are added Alcohol, in placing 10h~20h at 1 DEG C~4 DEG C, then carries out low-temperature centrifugation and obtains flocculent deposit, be laetarius volemus polysaccharide concentrate II；

(6) with 75% ethanol washing polysaccharide：Laetarius volemus polysaccharide concentrate II is carried out after 3 washings with 75% ethanol, again It is dissolved in after water low-temperature centrifugation removes insoluble matter, supernatant pre-freeze and carries out vacuum freeze drying, obtains many saccharin of laetarius volemus Powder processed.

4. the preparation method according to claim 1 or 2 or 3, it is characterised in that the addition of the laetarius volemus polysaccharide is 0.05%~0.15%.

5. preparation method according to claim 4, it is characterised in that the fermentation temperature is 35 DEG C~43 DEG C.

6. preparation method according to claim 5, it is characterised in that the laetarius volemus polysaccharide of addition is first configured to solution, And through film process.

7. the preparation method according to claim 1 or 2 or 3, it is characterised in that the fermentation milk, which is constituted, is：

The recovery milk of whole-fat milk powder is formulated in proportion with sucrose, stabilizer；

Or skimmed milk power and the recovery milk of dilute cream are formulated in proportion with sucrose, stabilizer；

Or fresh milk is formulated in proportion with sucrose, stabilizer.

8. the preparation method according to claim 1 or 2 or 3, it is characterised in that the preparation of the lactobacillus plantarum ferment Method is comprised the following steps：

(1) Lactobacillus plantarum is carried out using MRS fluid nutrient mediums and freezes bacterium powder activation culture 2~3 times, obtain activation bacterium solution；

(2) activation bacterium solution is inoculated in activation process in activation milk, obtains lactobacillus plantarum ferment.

9. preparation method according to claim 8, it is characterised in that the activation milk is 10.0%~12.5% Skimmed milk powder, 7.5%~8.5% sucrose, 0.0005%~0.002% manganese gluconate, 0.5%~2.0% soybean are more After peptide mixing, formed through homogeneous and sterilization.

10. the Lactobacillus plantarum acidified milk of laetarius volemus polysaccharide prepared by claim 1~9 any one methods described.