CN106912602B - Lactobacillus acidophilus fermented milk containing succulent lactarius polysaccharide and preparation method of lactobacillus acidophilus fermented milk - Google Patents
Lactobacillus acidophilus fermented milk containing succulent lactarius polysaccharide and preparation method of lactobacillus acidophilus fermented milk Download PDFInfo
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Abstract
The invention discloses lactobacillus acidophilus fermented milk of succulent lactarius polysaccharide and a preparation method thereof, milk for fermentation, the succulent lactarius polysaccharide and lactobacillus acidophilus starter are mixed uniformly and then fermented, and fermented milk containing the succulent lactarius polysaccharide is prepared after fermentation; the milk for fermentation is also subjected to homogenization and disinfection pretreatment, or the milk for fermentation is mixed with the lactarius hatsudake tanaka polysaccharide and then subjected to homogenization and disinfection pretreatment. The obtained succulent lactarius polysaccharide fermented milk has good rheological property, proper water retention capacity, moderate sour and sweet, and abundant essential amino acids and free amino acids, and the viable count of lactobacillus is as high as 1010CFU/mL; the aroma components are rich, the taste is good, and the sensory properties such as color and luster, taste and the like all accord with various indexes in the national standard GB19302-2010 of the fermented milk.
Description
Technical Field
The invention belongs to the technical field of lactobacillus and dairy processing, and particularly relates to polysaccharide fermented milk capable of improving human immunity and a preparation method thereof.
Background
Researches have proved that the fungus polysaccharide has multiple physiological functions of regulating immunity, reducing blood fat, resisting aging, resisting tumor and the like, is deeply valued by people and has great development value. The fermented milk is considered to be one of the best carriers for adding functional components and realizing the functions of products due to the excellent taste and nutrient components. Lactic acid bacteria have vital physiological functions in human body, and the number of the lactic acid bacteria affects human health and even human life. The important physiological functions of lactic acid bacteria in human body are mainly realized by 4 kinds of substances, namely organic acid, bacteriocin, bacterial surface components and special enzyme systems generated by the lactic acid bacteria. The physiological functions of lactic acid bacteria mainly include: (1) endows the food with good flavor and improves the nutritional value of the food; (2) promoting gastric secretion, promoting gastrointestinal peristalsis, and preventing and treating dyspepsia and habitual constipation; (3) can resist the invasion of external bacteria, inhibit the growth and reproduction of pathogenic bacteria, and maintain the balance of intestinal flora; (4) enhancing immunity, and inhibiting tumor growth and metastasis; (5) reducing cholesterol content in blood serum, and preventing cardiovascular diseases. Fermented milk contains live probiotics or lactic acid bacteria and active ingredients, and therefore has an important role in improving human health, and has received great attention in recent years. According to the literature, the fungal polysaccharide can be applied to fermented milk, such as flammulina velutipes polysaccharide, black fungus polysaccharide, agaricus blazei murill polysaccharide, lentinan and coriolus versicolor polysaccharide, and the results show that the fungal polysaccharide has the effects of promoting the growth of lactic acid bacteria, improving the speed of producing lactic acid by fermentation, reducing the fermentation period and increasing the number of viable bacteria of the lactic acid bacteria.
Lactarius serohilus Fr, commonly known as Lactarius volemus Fr, Lactarius edulis, and Lactarius edulis are edible wild fungi, and extraction of active polysaccharides, evaluation of active ingredients, and application of polysaccharides in fermented milk have not been reported.
Disclosure of Invention
The invention provides lactobacillus acidophilus fermented milk with lactobacillus polysaccharide capable of improving immunity and a preparation method thereof, aiming at the current situation that the lactobacillus polysaccharide has unclear immunocompetence and is applied to fermented milk.
The invention adopts the following technical scheme:
as a result of intensive studies on the above-mentioned problems of the fermented milk additives, the unstable texture, and the like, it has been found that the addition of the lactarius deliciosus polysaccharide in the process of preparing the fermented milk serves as a prebiotic factor to act during the fermentation process, and the total viable count and the stability of the texture in the fermented milk can be significantly enhanced without affecting the intrinsic flavor of the fermented milk, as compared with the case where the lactarius deliciosus polysaccharide is not added. Therefore, the invention improves the process on the basis of optimizing the formula: the lactarius deliciosus polysaccharide is added for fermentation, so that the growth and the propagation of a leavening agent are promoted, the storage life of the preserved fermented milk is prolonged, the fermented milk product with unique flavor, fine texture and smooth mouthfeel is prepared, and the whey precipitation is reduced.
A method for preparing Lactobacillus acidophilus fermented milk containing succulent Lactarius polysaccharide comprises mixing milk for fermentation with succulent Lactarius polysaccharide and Lactobacillus acidophilus starter, fermenting, and fermenting to obtain fermented milk containing succulent Lactarius polysaccharide; the milk for fermentation is also subjected to homogenization and disinfection pretreatment, or the milk for fermentation is mixed with the lactarius hatsudake tanaka polysaccharide and then subjected to homogenization and disinfection pretreatment.
The preparation method of the succulent lactarius polysaccharide comprises the following steps: firstly, pretreating a lactarius deliciosus raw material to obtain lactarius deliciosus fine powder, degreasing the lactarius deliciosus fine powder, and then extracting polysaccharide by using water; then protein removal treatment is carried out; precipitating polysaccharide with 95% ethanol, washing the obtained concentrated solution with 75% ethanol, and vacuum freeze drying to obtain Lactarius Polyphyllus polysaccharide.
The preparation method of the lactarius deliciosus polysaccharide comprises the following steps:
(1) pretreatment of raw materials: washing the lactarius deliciosus, drying and removing the stalk of the lactarius deliciosus, grinding the sporocarp part into powder, and then sieving the powder by a sieve of 150 meshes to 200 meshes to obtain the lactarius deliciosus fine powder;
(2) degreasing: adding absolute ethyl alcohol and petroleum ether into the lactarius deliciosus fine powder, refluxing for 3-4 h at 85-95 ℃, and performing suction filtration to obtain filter residue;
(3) polysaccharide extraction with water: adding distilled water into the filter residue, soaking for 1-2 h, performing 100W ultrasonic treatment for 10-20 min, and leaching for 3-5 h under the water bath condition of 90-95 ℃; filtering with a filter bag of 150-200 meshes, collecting the leaching liquor, and concentrating with a rotary evaporator to obtain concentrated succulent lactarius mushroom polysaccharide solution of 15-25 times;
(4) deproteinization treatment: mixing the lactarius deliciosus polysaccharide concentrated solution with a Sevag reagent with the volume being 1/3-1/5 times, standing for 20-40 min, layering, centrifuging at low temperature to obtain an upper-layer leaching solution, diluting with distilled water, and removing an organic solvent by using a rotary evaporator to obtain a deproteinized lactarius deliciosus polysaccharide concentrated solution I;
(5) polysaccharide precipitation with 95% ethanol: adding 2-4 times volume of precooled 95% ethanol into the lactarius deliciosus polysaccharide concentrated solution I, standing for 10-20 h at 1-4 ℃, and then carrying out low-temperature centrifugation to obtain flocculent precipitate which is a lactarius deliciosus polysaccharide concentrated solution II;
(6) the polysaccharide was washed with 75% ethanol: washing the lactarius deliciosus polysaccharide concentrated solution II with 75% ethanol for 3 times, dissolving in water again, centrifuging at low temperature to remove insoluble substances, pre-freezing the supernatant, and vacuum freeze-drying to obtain lactarius deliciosus polysaccharide powder.
The content of the lactarius deliciosus polysaccharide is 0.05-0.15%, and the preferable content is 0.1%.
The fermentation temperature is 35-43 ℃.
The lactarius hatsudake tanaka polysaccharide solution is added with membrane treatment.
The milk for fermentation comprises the following components:
the reconstituted milk of the whole milk powder is prepared by sucrose and a stabilizer according to a proportion;
or reconstituted milk of skimmed milk powder and cream, sucrose and stabilizer in proportion;
or the fresh milk, the cane sugar and the stabilizer are prepared according to the proportion.
The preparation of Lactobacillus acidophilus (LAP 1.1878) leaven comprises the following steps:
(1) performing lactobacillus acidophilus freeze-dried powder activation culture for 2-3 times by adopting an MRS liquid culture medium to obtain activated bacterium liquid;
(2) inoculating the activated bacteria liquid into the activated milk liquid for activation treatment to obtain the lactobacillus acidophilus starter.
The milk for activation is prepared by mixing 10.0-12.5% of whole milk powder and 7.5-8.5% of cane sugar, homogenizing and sterilizing.
Compared with the prior art, the invention has the following advantages:
(1) the succulent lactarius polysaccharide adopted by the invention is a pure natural substance, has good immunocompetence, has stable property in fermented milk, can resist pasteurization intensity, and does not influence the quality guarantee period and the taste of the fermented milk.
(2) Compared with fermented milk without the added polysaccharide, the polysaccharide fermented milk has good rheological property, moderate water retention capacity and sour-sweet taste, contains abundant essential amino acid, free amino acid and the like, and the viable count of the lactic acid bacteria is as high as 1010CFU/mL; the aroma components are rich, the taste is good, and the sensory properties such as color and luster, taste and the like all accord with various indexes in the national standard GB19302-2010 of the fermented milk.
(3) The fermented milk has rich raw materials, can be prepared by proportionally mixing reconstituted milk of whole milk powder, cane sugar, a stabilizer and other fermented milk raw materials, can also be prepared by proportionally mixing reconstituted milk of skim milk powder and cream, cane sugar, a stabilizer and other fermented milk raw materials, and can also be prepared by proportionally mixing fresh milk, cane sugar, a stabilizer and other fermented milk raw materials.
(4) The production process is simple and easy to control, the taste is fine and smooth, the aroma is strong, the viable count of the lactobacillus is high, the texture is better, the viscosity is proper, the water holding capacity is better, and no whey is separated out.
Drawings
FIG. 1 is a shear scan rheological profile of a Lactobacillus acidophilus fermented milk of the Polyporus polysaccharide, FIG. 1a is the change of apparent viscosity with shear rate, and FIG. 1b is the change of shear rate with shear stress.
FIG. 2 is a frequency-scanning rheological profile of a Lactobacillus acidophilus fermented milk of the Polyporus polysaccharide.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the invention to be claimed is not limited thereto.
Example 1
Extraction of polysaccharide from lactarius hatsudake tanaka
Washing Lactarius deliciosus, oven drying, removing stalk, pulverizing fruiting body, sieving with 200 mesh sieve to obtain Lactarius deliciosus fine powder, defatting at 90 deg.C for 3.5 hr, soaking at a material-liquid ratio of 1:30 for 1.5 hr, treating with 100W ultrasonic wave for 15min, leaching in 95 deg.C water bath for 4 hr, filtering with 200 mesh filter bag, collecting leaching solution, concentrating to 20 times at 55 deg.C rotary evaporator, adding 1/5 times volume of Sevag reagent (n-butanol: chloroform volume ratio of 1:4) for deproteinization, centrifuging at 5000rpm for 5min at low temperature to obtain upper layer leaching solution, diluting with appropriate amount of distilled water, removing organic reagent with rotary evaporator, adding 3 times volume of 95% ethanol into Lactarius deliciosus polysaccharide concentrate I, standing at 4 deg.C for 20 hr, centrifuging at 5000rpm for 5min to obtain Lactarius deliciosus polysaccharide concentrate II, washing with 75% ethanol for 3 times, redissolving in water, centrifuging at 5000rpm for 5min at low temperature to remove insoluble substances, prefreezing the supernatant, and vacuum freeze drying to obtain Lactarius Polyporus powder III, storing in a sealed bag at 4 deg.C, with polysaccharide yield of 2.18% (based on dry weight).
Example 2
Evaluation of immunity-improving effect of lactarius deliciosus polysaccharide in vivo
(1) Grouping mice: 72 SPF-level inbred Balb/c mice with age of 7-8 weeks, weight of 18-22 g, are randomly divided into 6 groups according to the weight of the mice, and detailed grouping information is shown in Table 1. And the SPSS17.0 software is used for analyzing the significance difference, and the significance difference between groups is not shown. All mice were housed by professionals, 4 per cage. Under the conditions of temperature of 22-24 ℃ and humidity of 50-60%, the light is irradiated for 12 hours and the darkness is 12 hours every day. During the experiment, mice were free to drink and eat, 9 a.m. each day: 00 the gavage is carried out on the mice of each experimental group, the gavage amount is 200mL/(kg bw d), CP-And CP+The mice were injected with cyclophosphamide (abbreviated as CP) for immune system injury in the abdominal cavity 7 days earlier, 30 mg/kg bw of CP per day, 7 days continuously, and 80 mg/kg bw of CP every 25 days.
TABLE 1 mouse grouping information Table
(2) Determination of various immunological indices
① delayed allergy determination, after 25 days of feeding, each mouse was injected intraperitoneally with 0.2mL of 2% SRBC sheep red blood cells, and 5 days after immunization, the mice were weighed and the left hind paw plantar thickness was measured (H)0) 20 μ l of 20% SRBC sheep red blood cells were injected at the measurement site, and the thickness (H) of the plantar region of the left hind foot of the same site was measured 24H later, and the difference between the thickness of the plantar region of the foot before and after injection was expressed as DTH ═ H-H0In mm.
② determination of carbon clearance index, after the measurement on day 30, tail vein injecting 33% India ink with injection amount of 0.1mL/(kg · bw), respectively after 2min and 10min, collecting 20 μ l of blood from inner canthus vein, and immediately adding into the container2mL0.1％Na2CO3In EP tube of (1), OD is measured600nmAnd calculating the corresponding phagocytic rate K ═ lgOD1-lgOD2)/(t2-t1) And phagocytosis index a ═ (body weight K)1/3) /(liver weight + spleen weight), the carbon clearance index is the phagocytic index a.
③ viscera coefficient measurement, at the termination of experiment, all mice are fasted overnight, the eyeball is removed to take whole blood, then the mice are killed by dislocation of neck, liver, spleen and thymus are separated, the surface bloodiness of viscera is washed by physiological saline and is sucked by filter paper for weighing, the viscera coefficient refers to the liver coefficient, spleen coefficient and thymus coefficient, the formula is calculated as viscera weight (mg)/body weight (g), for viscera of carbon clearance test mice, the color of 6 groups is dark red, India ink is not metabolized, CP can be observed by naked eye-Compared with the dosage group, the color state is darker, the metabolism condition is worse than that of the CK group and the 3 polysaccharide dosage groups such as LP, MP, HP and the like, and the polysaccharide liquid of the lactarius hatsudake tanaka can be judged to have certain immunity efficacy. The results of obtaining the organ coefficients of the mice of each group by calculation are shown in table 2. Compared with CK, the 3 polysaccharide dose groups of LP, MP, HP and the like have obvious improvement on the body weight and the liver coefficient, the spleen coefficient, the thymus coefficient and other 3 immune organ coefficients of the mouse, and have statistical significance (p is less than 0.05), and CP-Compared with CK, the body weight and 3 immune organ coefficients of the mouse are obviously reduced, which verifies that CP is established by the immune model of the mouse-And CP+Compared with the prior art, the immunity of the mice is slightly recovered, which shows that the polysaccharide of the lactarius hatsudake tanaka has the function of improving the immunity.
Table 2 body weight (g) and immune organ coefficients (M ± SD, n ═ 12) for each group of mice at week 4 (d28)
Note that the organ coefficient is the organ weight/body weight in mg/g. The different symbols (a-d) indicate that the data in the same row have significant differences (p < 0.05); significant differences were analyzed by Dunan's analysis in SPSS 17.0.
As shown in Table 3, mice were treated at week 4When the experiments of allergic swelling of foot soles (DTH) and carbon clearance are carried out, compared with CK, the 3 polysaccharide dose groups of LP, MP, HP and the like have more obvious symptoms of allergic swelling of foot soles of mice, meanwhile, the macrophage capacity (phagocytosis index a) of removing carbon granules is stronger, and the CP has statistical significance (p is less than 0.05), and-compared with CK, the degree of plantar swelling and macrophage function of the mice are far inferior to those of the CK group, and the establishment of an immune injury model of the mice is verified; CP (CP)-And CP+Compared with the prior art, the immunity of the mice is slightly recovered, which shows that the polysaccharide of the lactarius hatsudake tanaka has the function of improving the immunity.
Table 3 week 4 (d28) mice DTH and clearance index test results (M + -SD, n-12)
The degree of allergic swelling of the plantar area of the foot is expressed by DTH and is in mm; the carbon clearance index refers to the phagocytic index a. Note: different letters (a-f) indicate significant differences between the same column of data (p < 0.05); significant differences were analyzed by Dunan's analysis in SPSS 17.0.
④ Whole blood analysis, the method comprises automatically analyzing eighteen whole blood items by sending whole blood of mouse to the center of the eastern school district of Zhongshan university, and analyzing the lymphocyte and leukocyte amount in the whole blood of mouse, wherein the lymphocyte amount and leukocyte amount in the whole blood of mouse are shown in Table 4, the lymphocyte ratio of the group with 3 polysaccharide doses such as LP, MP and HP is obviously higher than that in other groups, and the lymphocyte amount and lymphocyte amount in the whole blood of mouse are equivalent to those in normal group, and the leukocyte amount and lymphocyte amount in LP are obviously higher than those in other groups (p <0.05), and CP is higher than that in other groups (p <0.05)-Compared with a normal group, the number of white blood cells and the number of lymphocytes of the mice are reduced, and the establishment of an immune injury model of the mice is verified; in addition, CP+And CP-In contrast, the mouse had a slightly restored immune index, which is between CP-And CK, indicating that the polysaccharide of the succulent lactarius shows the immune enhancement effect. CP (CP)+The white blood cell number and the lymphocyte number are both obviously higher than that of CP-This shows that when the mice are attacked by antigen, the polysaccharide can obviously enhance the cellular immune function of the mice and the promotion effect of low dose is most obvious.
Table 4 week 4 (d28) mice whole blood test results (M ± SD, n ═ 12)
Note that different letters (a-e) indicate significant differences (p) between the same column of data<0.05); significant differences were analyzed by Dunan's analysis in SPSS 17.0. WBC represents the number of leukocytes and has a unit of 109Per liter; LYM represents the number of lymphocytes in 109Per liter; LYM% represents the percentage of lymphocytes in whole blood cells.
Example 3
Application of lactarius hatsudake tanaka polysaccharide in fermented milk
First-step starter preparation: 1mL of sterile water is used for resuspending Lactobacillus acidophilus (with the number of CGMCC 1.1878, Lactobacillus acidophilus 1.1878, preserved by China general microbiological culture Collection center, and the address: No. 3 of Western No. 1 of Suh West district, Beijing, the rising district, and zip code: 100101) freeze-dried powder, then all the freeze-dried powder is transferred into an MRS liquid culture medium, the culture is carried out for 16h at 37 ℃, then the culture is transferred into a fresh MRS liquid culture medium for activation for 3 times by 5 to 10 percent of inoculation amount, the bacteria are centrifugally collected at the low temperature of 4000rpm, and all the bacteria are transferred into activating milk liquid for activation culture for 2 to 3 times at 37 ℃, thus obtaining the Lactobacillus acidophilus fermentation agent; the milk for activation is prepared by mixing 12.5% of whole milk powder and 8.5% of cane sugar and then sterilizing.
The second step is that milk for fermentation is prepared and mixed with polysaccharide: adding 12.5% of whole milk powder, 8.5% of cane sugar and 0.1% of stabilizer powder according to the mass-volume ratio, uniformly mixing with 0.00%, 0.05%, 0.10% and 0.15% of lactarius hatsudake polysaccharide powder, preheating to 60 ℃, homogenizing under 18MPa, sterilizing at 95 ℃ for 5min, and cooling to 43 ℃ for later use;
thirdly, inoculation and fermentation: adding lactobacillus acidophilus starter with the volume ratio of 10% into the milk for fermentation, fermenting at 43 ℃, and stopping fermentation when the titrated acidity reaches 75 DEG T to obtain the lactobacillus plantarum fermented milk.
Fourth, evaluating the indexes of the polysaccharide fermented milk
(1) The sensory quality evaluation is carried out according to the requirements of national standards GB19302-2010 of the fermented milk, the specific evaluation items are shown in Table 5, the total score is 100, wherein the flavor index and the taste index respectively account for 30 points, the appearance index is fully 15 points, and the texture index is fully 25 points. Each index is divided into three sections according to the acceptable degree: less accepted, generally accepted, and liked accepted, with the score distribution shown in table 5. The results are shown in Table 6, and it is found that the fermented milk containing 0.10% of polysaccharide is preferable in terms of flavor, texture, appearance and texture, and the color of the fermented milk containing 0.15% of polysaccharide is somewhat darker and completely deviates from the original color of the fermented milk. Fermented milks with a polysaccharide content of 0.05% are also acceptable.
TABLE 5 sensory evaluation index of fermented milk
TABLE 6 Effect of the addition of Lactarius Polyphyllus polysaccharide on the sensory properties of fermented milks
Note: different letters (a-g) indicate significant differences between the same column of data (p < 0.05); significant differences were analyzed by Dunan's analysis in SPSS 17.0.
(2) Evaluation of physicochemical indexes of fermented milk containing Lactarius deliciosus polysaccharide for measuring pH, water holding capacity, free amino acids, viable count of lactobacillus, and rheological properties of fermented milk after fermentation
The effect of the different polysaccharide contents on the pH of the fermented milk is shown in Table 7, and it can be seen that the pH of the fermented milk decreases with increasing polysaccharide concentration, which indicates that the addition of the polysaccharide can promote the acid production capacity of Lactobacillus acidophilus, which may be related to the growth of lactic acid bacteria.
The weight of an EP tube weighing 15mL of water holding capacity was recorded as W0Respectively weighing 10g of each yoghourt sample in an EP tube, centrifuging for 5min at 5000rpm, abandoning whey, inverting the EP tube for 10min, and weighing the total mass as W. Parallel measurement for 3 times, average value, water holding capacity (W-W)0) The influence of different polysaccharide contents on the water holding capacity of the fermented milk is shown in table 7, and it can be seen that the addition of the polysaccharide does not affect the water holding capacity of the fermented milk, and the polysaccharide contents are all around 99.0%.
TABLE 7 influence of polysaccharide content on pH, Water holding Capacity, viable count of Lactobacillus acidophilus fermented milk
Viable count of lactic acid bacteria 1mL of the fermented milk sample prepared as described above was mixed with 9mL of sterilized physiological saline, i.e., the dilution ratio was 101Sequentially mixing 1mL of the previous diluted sample with 9mL of sterilized normal saline to obtain the product with the dilution multiple of 102The sample of (2), and so on, diluted to 1010Taking 1mL of diluted fermented milk in a plate, pouring an MRS solid culture medium to cover the surface of a bacterial liquid, placing the plate at the constant temperature of 37 ℃ for 24 hours, and then carrying out bacterial colony counting, wherein the influence of the polysaccharide content on lactobacillus acidophilus of the fermented milk is shown in Table 7, the viable count of the lactobacillus is increased along with the increase of the polysaccharide content, and the fact that the lactobacillus succulent polysaccharide can promote the growth and the propagation of the lactobacillus acidophilus is proved again, and when the polysaccharide content is 0.15%, the maximum viable count is 2.14 × 1010The fermented milk with the CFU/mL and 4 formulas meets the requirements of the national standard GB19302-2010 of fermented milk.
Weighing 10g of yoghourt and 5mL of distilled water, mixing the yoghourt and the distilled water, fully stirring, carrying out refrigerated centrifugation at 4500rpm for 5min, taking 4mL of supernatant and 1mL of 15% sulfosalicylic acid solution, mixing the mixture at 4 ℃, standing for more than 6h, then centrifuging at 10000rpm for 15min, taking the supernatant, repeatedly centrifuging once, determining the nitrogen content of a sample by a Kjeldahl method, and diluting the sample until the nitrogen content is 0.6-1.0%. Filtering with 0.22 μm filter membrane, loading into sample bottle, and analyzing with C18 column, quaternary pump, ultraviolet detection, OPA automatic derivatization device, and automatic sample introduction. The effect of polysaccharide content on free amino acids in fermented milk of Lactobacillus acidophilus is shown in Table 8. when the polysaccharide content is 0.05% and 0.10%, the content of essential amino acids is increased by 5.56% and 6.02% respectively compared with the control group (without polysaccharide), and the content of valine is increased mainly from 12.56nmol/L to 22.31nmol/L and 31.02 nmol/L. The total free amino acid content was also increased compared to the control group, 547.96nmol/L and 545.97nmol/L, respectively. The lactobacillus plantarum polysaccharide is shown to be capable of promoting growth and propagation of lactobacillus acidophilus, so that more essential amino acids and free amino acids are produced, and the biological value of the fermented milk is improved.
TABLE 8 influence of polysaccharides on 17 free amino acids (M + -SD) (nmol/L) in Lactobacillus acidophilus fermented milk
Rheological property 5mL of fermented milk is placed on an objective table of a Haake rheometer, a proper program is selected to measure parameters such as deformation resistance, viscosity and the like of a sample, after each group of programs is finished, a probe is opened, the sample on the probe and the objective table is carefully processed, a sample is replaced, all measured data are stored, and origin8.5 is applied to analyze and process the measured data. The effect of polysaccharide content on the rheological properties of lactobacillus acidophilus fermented milk is shown in fig. 1 and fig. 2. It is clear that as the shear rate increases, both the apparent viscosity (fig. 1a) and the shear stress tend to decrease, and the apparent viscosities of the fermented milks of the respective groups do not differ much. When the shear rate is controlled at 20s-1In the above three cases, the addition of polysaccharide can improve the fermented milkWhen the polysaccharide content is 0.15%, the apparent viscosity and shear stress are the highest and change slowly, and the polysaccharide can increase the activity of lactic acid bacteria and improve the texture of fermented milk (fig. 1 b). FIG. 2 shows the results of frequency scanning and shear scanning of Lactobacillus acidophilus fermented milk, wherein G 'and G' respectively represent the elastic modulus and viscous modulus, which can respectively characterize the deformation resistance and viscosity of the fermented milk. As can be seen from FIG. 2, the lactobacillus acidophilus fermented milk with the added amount of the polysaccharide of 0.10% has the best deformation resistance, the largest viscosity and the best crosslinking condition, which may be caused by the polysaccharide promoting lactic acid bacteria to generate a large amount of extracellular polysaccharide, thereby improving the crosslinking structure of the fermented milk. In summary, the addition amount of the polysaccharide lactobacillus polysaccharide in the lactobacillus acidophilus fermented milk is recommended to be 0.50-0.10%.
Example 4
Polyporus lactis polysaccharide extraction and application in fermented milk
Firstly, washing the lactarius deliciosus, drying and removing the handle of the lactarius deliciosus, grinding the sporocarp part into powder, then sieving the powder by a 150-mesh sieve to obtain lactarius deliciosus fine powder, refluxing the powder for 4 hours at 85 ℃, degreasing the powder, soaking the powder for 2 hours according to the material-liquid ratio of 1:30, carrying out ultrasonic treatment for 10 minutes by 100W, leaching the powder in a water bath at 90 ℃ for 5 hours, filtering the powder by a filter bag with 150 meshes, collecting leaching liquor, concentrating the leaching liquor to 15 times in a rotary evaporator at 55 ℃, adding a Sevag reagent (n-butyl alcohol: chloroform, in a volume ratio of 1:4) in an 1/3 times, centrifuging the solution for 5 minutes at 5000rpm at low temperature to obtain an upper layer leaching liquor, diluting the upper layer leaching liquor by proper amount of distilled water, removing an organic reagent by the rotary evaporator, adding 95% ethanol in an amount of 2 times in volume into the lactarius deliciosus polysaccharide concentrated solution I, standing the solution for 18 hours at 4 ℃, centrifuging the solution for 5 minutes at 5000rpm to obtain a, redissolving in water, centrifuging at 5000rpm for 5min at low temperature to remove insoluble substances, pre-freezing the supernatant, and vacuum freeze drying to obtain Lactarius Polyporus polysaccharide powder III, storing in a sealed bag at 4 deg.C, with polysaccharide yield of 2.01% (based on dry weight).
The second step of preparing a leaven and mixing the leaven with polysaccharide: resuspending lactobacillus acidophilus freeze-dried powder by 1mL of sterile water, then completely transferring the freeze-dried powder into an MRS liquid culture medium, culturing for 16-20 h at 37 ℃, transferring the freeze-dried powder into a fresh MRS liquid culture medium for activation for 3 times by 5-10% of inoculation amount, centrifugally collecting thalli at a low temperature of 4000rpm, and completely transferring the thalli into milk for fermentation to perform activation culture for 2-3 times at 35 ℃ to obtain a lactobacillus acidophilus starter; the milk for activation is prepared by mixing 12.5% of whole milk powder and 8.5% of cane sugar and then sterilizing.
The third step is that milk for fermentation is prepared: adding 0.05% of lactarius deliciosus polysaccharide powder III into fresh milk, 8.5% of cane sugar and 0.1% of stabilizer powder according to the mass-volume ratio, uniformly mixing, preheating to 60 ℃, homogenizing under 18Mpa, sterilizing at 95 ℃ for 5min, and cooling to 35 ℃ for later use;
fourth, inoculation and fermentation, namely adding lactobacillus acidophilus starter with the volume ratio of 5 percent into the milk for fermentation, fermenting at 35 ℃, stopping fermentation when the titrated acidity reaches 75 degrees, and obtaining the lactobacillus plantarum fermented milk, wherein the pH, the water holding capacity and the viable count at the fermentation end are respectively 3.85 percent, 99.10 percent and 1.56 × 1010CFU/mL, the taste quality meets the requirement. And (3) demulsifying the solidified fermented milk obtained by the steps by using a peristaltic pump to obtain the stirred fermented milk.
Example 5
Polyporus lactis polysaccharide extraction and application in fermented milk
Firstly, washing the lactarius deliciosus, drying and removing the handle of the lactarius deliciosus, grinding the sporocarp part into powder, then sieving the powder by a 150-mesh sieve to obtain lactarius deliciosus fine powder, refluxing the powder for 3 hours at 95 ℃, degreasing the powder, soaking the powder for 1 hour according to the material-liquid ratio of 1:30, carrying out ultrasonic treatment for 20 minutes by 100W, leaching the powder in a water bath at 93 ℃ for 3.5 hours, filtering the powder by a 200-mesh filter bag, collecting the leaching solution, concentrating the leaching solution to 25 times in a rotary evaporator at 55 ℃, adding a Sevag reagent (the volume ratio of n-butyl alcohol to chloroform is 1:4) with 1/4 times of volume, centrifuging the solution for 5 minutes at 5000rpm at low temperature to obtain an upper layer leaching solution, diluting the upper layer leaching solution by a proper amount of distilled water, removing an organic reagent by the rotary evaporator, adding 95% ethanol with 4 times of volume into the lactarius deliciosus polysaccharide concentrated solution I, standing the solution for 15 hours at 3 ℃, centrifuging the solution for 5 minutes at 5000rpm to obtain a lacta, redissolving in water, centrifuging at 5000rpm for 5min at low temperature to remove insoluble substances, pre-freezing the supernatant, and vacuum freeze drying to obtain Lactarius Polyporus powder III, storing in a sealed bag at 4 deg.C, with polysaccharide yield of 2.15% (based on dry weight).
The second step of starter preparation: resuspending lactobacillus acidophilus freeze-dried powder by 1mL of sterile water, then completely transferring the freeze-dried powder into an MRS liquid culture medium, culturing for 16-20 h at 37 ℃, transferring the freeze-dried powder into a fresh MRS liquid culture medium for activation for 3 times by 5-10% of inoculation amount, centrifugally collecting thalli at a low temperature of 4000rpm, and completely transferring the thalli into milk for fermentation to perform activation culture for 2-3 times at 35 ℃ to obtain a lactobacillus acidophilus starter; the milk for activation is prepared by mixing 10.0% of whole milk powder and 7.5% of cane sugar and then sterilizing.
Thirdly, preparing milk for fermentation and mixing the milk with polysaccharide: adding 0.05% of lactarius deliciosus polysaccharide powder III into fresh milk, 8.5% of cane sugar and 0.1% of stabilizer powder according to the mass-volume ratio, uniformly mixing, preheating to 60 ℃, homogenizing under 18Mpa, sterilizing at 95 ℃ for 5min, and cooling to 37 ℃ for later use;
fourth, inoculation and fermentation, namely adding lactobacillus acidophilus starter accounting for 5 percent of the volume ratio into the milk for fermentation, fermenting at 37 ℃, stopping fermentation when the titrated acidity reaches 75 degrees, and obtaining the lactobacillus plantarum fermented milk, wherein the pH, the water holding capacity and the viable count at the fermentation end are respectively 3.91, 99.03 percent and 1.47 × 1010CFU/mL, the taste quality meets the requirement. And (3) demulsifying the solidified fermented milk obtained by the steps by using a peristaltic pump to obtain the stirred fermented milk.
Example 6
Polyporus lactis polysaccharide extraction and application in fermented milk
Firstly, washing the lactarius deliciosus, drying and removing the handle of the lactarius deliciosus, grinding the sporocarp part into powder, then sieving the powder by a 180-mesh sieve to obtain lactarius deliciosus fine powder, refluxing the powder for 3.5 hours at 90 ℃, degreasing the powder, soaking the powder for 2 hours according to the material-liquid ratio of 1:30, treating the powder by 100W ultrasonic waves for 18 minutes, leaching the powder for 3 hours in a water bath at 95 ℃, filtering the powder by a 200-mesh filter bag, collecting the leaching solution, concentrating the leaching solution to 20 times in a rotary evaporator at 55 ℃, adding a Sevag reagent (the volume ratio of n-butyl alcohol to chloroform is 1:4) with 1/3 times of volume, centrifuging the solution for 5 minutes at 5000rpm at low temperature to obtain an upper layer leaching solution, diluting the upper layer leaching solution by a proper amount of distilled water, removing an organic reagent by the rotary evaporator, adding 95% ethanol with the volume of 3.5 times of volume into the lactarius deliciosus polysaccharide concentrated solution I, standing the solution for 15 hours at 4 ℃, centrifuging the solution for 5 minutes at 5000rpm to, redissolving in water, centrifuging at 5000rpm for 5min at low temperature to remove insoluble substances, pre-freezing the supernatant, and vacuum freeze drying to obtain Lactarius Polyporus polysaccharide powder III, storing in a sealed bag at 4 deg.C, with polysaccharide yield of 2.05% (based on dry weight).
The second step of starter preparation: resuspending lactobacillus acidophilus freeze-dried powder by 1mL of sterile water, then completely transferring the freeze-dried powder into an MRS liquid culture medium, culturing for 16-20 h at 37 ℃, transferring the freeze-dried powder into a fresh MRS liquid culture medium for activation for 3 times by 5-10% of inoculation amount, centrifugally collecting thalli at a low temperature of 4000rpm, and completely transferring the thalli into milk for fermentation to perform activation culture for 2-3 times at 41 ℃ to obtain a lactobacillus acidophilus starter; the milk for activation is prepared by mixing 11.0% of whole milk powder and 8.0% of cane sugar and then sterilizing.
Thirdly, preparing milk for fermentation and mixing the milk with polysaccharide: adding 0.10% succulent lactarius polysaccharide powder III into 8.5% skimmed milk powder, 3.0% cream, 7.5% sucrose and 0.1% stabilizer powder according to mass-volume ratio, mixing, preheating to 60 deg.C, homogenizing under 18Mpa, sterilizing at 95 deg.C for 5min, and cooling to 41 deg.C for use;
fourth, inoculation and fermentation, namely adding lactobacillus acidophilus starter accounting for 5 percent of the volume ratio into the milk for fermentation, fermenting at 41 ℃, stopping fermentation when the titrated acidity reaches 75 degrees, and obtaining the lactobacillus plantarum fermented milk, wherein the pH, the water holding capacity and the viable count at the fermentation end are respectively 3.88, 99.20 percent and 1.35 × 1010CFU/mL, the taste quality meets the requirement. And (3) demulsifying the solidified fermented milk obtained by the steps by using a peristaltic pump to obtain the stirred fermented milk.
Claims (10)
1. A preparation method of lactobacillus acidophilus fermented milk of succulent lactarius polysaccharide is characterized in that milk for fermentation, the succulent lactarius polysaccharide and lactobacillus acidophilus starter are uniformly mixed and then fermented, and fermented to prepare fermented milk containing the succulent lactarius polysaccharide; the milk for fermentation is further subjected to homogenization and disinfection pretreatment, or the milk for fermentation is mixed with lactarius hatsudake tanaka polysaccharide and then subjected to homogenization and disinfection pretreatment, and the Lactobacillus acidophilus starter is Lactobacillus acidophilus (Lactobacillus acidophilus)1.1878 with the serial number of CGMCC 1.1878.
2. The method for preparing lactarius deliciosus polysaccharide according to claim 1, wherein the method for preparing lactarius deliciosus polysaccharide comprises the following steps: firstly, pretreating a lactarius deliciosus raw material to obtain lactarius deliciosus fine powder, degreasing the lactarius deliciosus fine powder, and then extracting polysaccharide by using water; then protein removal treatment is carried out; precipitating polysaccharide with 95% ethanol, washing the obtained concentrated solution with 75% ethanol, and vacuum freeze drying to obtain Lactarius Polyphyllus polysaccharide.
3. The method for preparing according to claim 2, wherein the preparation of the lactarius hatsudake polysaccharides comprises the following steps:
(1) pretreatment of raw materials: washing the lactarius deliciosus, drying and removing the stalk of the lactarius deliciosus, grinding the sporocarp part into powder, and then sieving the powder by a sieve of 150 meshes to 200 meshes to obtain the lactarius deliciosus fine powder;
(2) degreasing: adding absolute ethyl alcohol and petroleum ether into the lactarius deliciosus fine powder, refluxing for 3-4 h at 85-95 ℃, and performing suction filtration to obtain filter residue;
(3) polysaccharide extraction with water: adding distilled water into the filter residue, soaking for 1-2 h, performing 100W ultrasonic treatment for 10-20 min, and leaching for 3-5 h under the water bath condition of 90-95 ℃; filtering with a filter bag of 150-200 meshes, collecting the leaching liquor, and concentrating with a rotary evaporator to obtain concentrated succulent lactarius mushroom polysaccharide solution of 15-25 times;
(4) deproteinization treatment: mixing the lactarius deliciosus polysaccharide concentrated solution with a Sevag reagent with the volume being 1/3-1/5 times, standing for 20-40 min, layering, centrifuging at low temperature to obtain an upper-layer leaching solution, diluting with distilled water, and removing an organic solvent by using a rotary evaporator to obtain a deproteinized lactarius deliciosus polysaccharide concentrated solution I;
(5) polysaccharide precipitation with 95% ethanol: adding 2-4 times volume of precooled 95% ethanol into the lactarius deliciosus polysaccharide concentrated solution I, standing for 10-20 h at 1-4 ℃, and then carrying out low-temperature centrifugation to obtain flocculent precipitate which is a lactarius deliciosus polysaccharide concentrated solution II;
(6) the polysaccharide was washed with 75% ethanol: washing the lactarius deliciosus polysaccharide concentrated solution II with 75% ethanol for 3 times, dissolving in water again, centrifuging at low temperature to remove insoluble substances, pre-freezing the supernatant, and vacuum freeze-drying to obtain lactarius deliciosus polysaccharide powder.
4. The method according to claim 1, 2 or 3, wherein the content of the lactarius hatsudake polysaccharides is 0.05-0.15%.
5. The method according to claim 4, wherein the content of the lactarius deliciosus polysaccharide is 0.1%.
6. The method according to claim 1, 2 or 3, wherein the fermentation temperature is 35 ℃ to 43 ℃.
7. The method according to claim 1, 2 or 3, wherein the milk for fermentation consists of:
the reconstituted milk of the whole milk powder is prepared by sucrose and a stabilizer according to a proportion;
or reconstituted milk of skimmed milk powder and cream, sucrose and stabilizer in proportion;
or the fresh milk, the cane sugar and the stabilizer are prepared according to the proportion.
8. The method according to claim 1, 2 or 3, wherein the Lactobacillus acidophilus starter culture is prepared by a method comprising the following steps:
(1) performing lactobacillus acidophilus freeze-dried powder activation culture for 2-3 times by adopting an MRS liquid culture medium to obtain activated bacterium liquid;
(2) inoculating the activated bacteria liquid into the activated milk liquid for activation treatment to obtain the lactobacillus acidophilus starter.
9. The method according to claim 8, wherein the activating milk is prepared by mixing 10.0-12.5% of whole milk powder and 7.5-8.5% of sucrose, homogenizing and sterilizing.
10. Lactobacillus acidophilus fermented milk of lactarius succulent polysaccharides prepared by the method according to any of claims 1 to 9.
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