CN102286397A - Polymicrobic fermentation agent and polymicrobic fermentation milk product for preventing atherosclerosis and production method thereof - Google Patents
Polymicrobic fermentation agent and polymicrobic fermentation milk product for preventing atherosclerosis and production method thereof Download PDFInfo
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Abstract
The invention relates to a polymicrobic fermentation agent and a polymicrobic fermentation milk product for preventing atherosclerosis and a production method thereof. The polymicrobic fermentation milk product is prepared by the following steps of : step 1, heating proper quantity of milk or skimmed milk to 50-60 DEG C; homogenizing, carrying out pasteurization, cooling to 40-45 DEG C, adding proper quantity of the polymicrobic fermentation agent, and fermenting for 6-8 hours under a constant temperature of 40-45 DEG C; step 2, after fermentation, maturing for 72-120 hours under the temperature of 18-23 DEG C; step 3, uniformly mixing for 1.5-2 hours, extracting fat, and boiling the rest of the mixture for 1-2 hours under the temperature of 50-60 DEG C; and step 4, separating whey: carrying out sterilization and filter cloth filtration under the temperature of 18-23 DEG C so as to respectively obtain cheese and the whey, wherein the whey is the polymicrobic fermentation milk product. The invention is simple to operate and has a simple production method, the obtained polymicrobic fermentation milk product has rich nutrients, and contains probiotics, ACE inhibitory peptide, antioxidant peptide, and the like, has the functions of preventing the atherosclerosis, adjusting blood fat, preventing inflammatory, antioxidation, and the like, can adjust the intestinal tract bacteria colony to be balanced, and can delay senility.
Description
Technical field
The invention belongs to the technical field of mixed culture fermentation and dairy products thereof and application, be a kind of mixed culture fermentation agent and be used for antiatherogenic mixed culture fermentation dairy products and production method thereof.
Background technology
(Atherosclerosis, AS) as human life's first killer's cardiovascular and cerebrovascular disease, current serious is threatening human life and health to atherosclerosis.China's annual kainogenesis cerebral apoplexy case is about 3,000,000, myocardial infarction 600,000 people, and the cardiovascular and cerebrovascular disease death toll is about 2,600,000, on average per hour dead 300 people.More alarmingly be, cardiovascular and cerebrovascular disease morbidity and dead crowd present rejuvenation trend day by day, and generation myocardial infarction and cerebral apoplexy is of common occurrence about 30 years old, and crowd's hypertension morbidity is about 14% more than 15 years old, increases by one times before 30 years.In close relations between above-mentioned disease, intricate.And atherosclerosis is the main pathological basis of many cardiovascular and cerebrovascular disease, and therefore, preventing and treating atherosclerosis is the essence strategy measure that prevents and treats cardiovascular and cerebrovascular disease.
The Antiatherosclerosis medicine of the classics of Ying Yonging mainly contains lipid regulating agent, antioxidant, polyene fatty acid class, antiplatelet drug and protection arterial endothelium medicine etc. clinically.Lipid regulating agent commonly used mainly contains Statins, the special class of shellfish, cholic acid binding resin class, nicotinic acid etc., can improve hdl level and play therapeutic action by reduction blood cholesterol, triglyceride, low-density lipoprotein white level in various degree.But clinical epidemiology discovers that the prolonged application fibrate can increase the mortality ratio of non-coronary artery disease, though cholic acid binding resin class reduces the mortality ratio of coronary artery disease, general mortality rate does not reduce.Statins is widely-used since the eighties in 20th century, makes treatment of atherosclerosis obtain major progress.Yet there is certain limit in the benefit of bringing by independent reduction LDL cholesterol levels, and from statins self, it is its fatal shortcoming that rhabdomyolysis takes place.
Summary of the invention
The invention provides a kind of mixed culture fermentation agent and be used for antiatherogenic mixed culture fermentation dairy products and production method thereof,, make it have atherosclerosis, regulating blood fat, anti-inflammatory, effect such as anti-oxidant because of its unique manufacture craft.
One of technical scheme of the present invention realizes by following measure: a kind of mixed culture fermentation agent, it obtains by following parts by weight of raw materials fermentation, the weight ratio of zymophyte and milk or skimmed milk be 1: 20 to 30, zymophyte is short lactobacillus (Lactobacillus brevis), plant lactobacillus (Lactobacillus plantarum), lactobacterium helveticus (Lactobacillus.helveticus), lactobacillus kefiranofaciens (Lactobacillus kefianofaciens), lactobacterium casei (Lactobacillus casei), Lactobacillus farciminis (Lactobacillus farciminis), Lactococcus lactis (Lactococcus lactic), monospore yeast saccharomyces cerevisiae (Saccharomyces unisporus), Issatchenkia orientalis (Issatchenkia orientalis), the weight ratio between each bacterial classification be 0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:1.0 to 1.2:0.2 to 0.4:0.2 to 0.4.
One of technique scheme can further be optimized or/and select as follows:
Above-mentioned mixed culture fermentation agent obtains by following step: at first after the milk or the pasteurize of skimmed milk process with requirement, next is cooled to 42 ℃ to the 45 ℃ zymophytes that add requirement, obtains the mixed culture fermentation agent in 8 hours to 12 hours at 40 ℃ to 45 ℃ constant temperature bottom fermentations then.
At least stir twice in the time of 5 hours to 12 hours at above-mentioned constant temperature bottom fermentation.
Two of technical scheme of the present invention realizes by following measure: a kind of production method of above-mentioned mixed culture fermentation agent, it is undertaken by following step: at first after the milk or the pasteurize of skimmed milk process with requirement, next is cooled to 42 ℃ to the 45 ℃ zymophytes that add requirement, obtains the mixed culture fermentation agent in 8 hours to 12 hours at 40 ℃ to 45 ℃ constant temperature bottom fermentations then.
Two of technique scheme can further be optimized or/and select as follows:
At least stir twice in the time of 5 hours to 12 hours at above-mentioned constant temperature bottom fermentation.
Above-mentioned milk or skimmed milk through the operating parameters of pasteurize are: heat 90 ℃ to 95 ℃, be incubated sterilization in 10 minutes to 15 minutes.
Three of technical scheme of the present invention realizes by following measure: a kind of antiatherogenic mixed culture fermentation dairy products that are used for that utilize above-mentioned mixed culture fermentation agent, it obtains by following fermenting raw materials, adds the above-mentioned mixed culture fermentation agent that makes in 3% to 5% ratio of milk or skimmed milk gross weight.
Three of technique scheme can further be optimized or/and select as follows:
Above-mentioned mixed culture fermentation dairy products obtain by following step: the first step, at first the milk of requirement or skimmed milk being heated to 50 ℃ to 60 ℃ carries out behind the homogeneous through pasteurize, next is cooled to 40 ℃ to the 45 ℃ mixed culture fermentation agent that add requirements, then 40 ℃ to 45 ℃ constant temperature bottom fermentations 6 hours to 8 hours; In second step, after the fermentation ends, maturation is 72 hours to 120 hours under 18 ℃ to 23 ℃ constant temperature; The 3rd step, at the uniform velocity stirred 1.5 hours to 2 hours, extracting fat, remainder boiled 1 hour to 2 hours at 50 ℃ to 60 ℃; The 4th step, sepg whey: 18 ℃ under 23 ℃, filter through the sterilization filter cloth, obtain cheese and whey respectively, this whey is the mixed culture fermentation dairy products.
The four-way of technical scheme of the present invention crosses that following measure realizes: a kind of production method of above-mentioned mixed culture fermentation dairy products, it is undertaken by following step: the first step, at first the milk of requirement or skimmed milk being heated to 50 ℃ to 60 ℃ carries out behind the homogeneous through pasteurize, next is cooled to 40 ℃ to the 45 ℃ mixed culture fermentation agent that add requirements, then 40 ℃ to 45 ℃ constant temperature bottom fermentations 6 hours to 8 hours; In second step, after the fermentation ends, maturation is 72 hours to 120 hours under 18 ℃ to 23 ℃ constant temperature; The 3rd step, at the uniform velocity stirred 1.5 hours to 2 hours, extracting fat, remainder boiled 1 hour to 2 hours at 50 ℃ to 60 ℃; The 4th step, sepg whey: 18 ℃ under 23 ℃, filter through the sterilization filter cloth, obtain cheese and whey respectively, this whey is the mixed culture fermentation dairy products.
Four of technique scheme can further be optimized or/and select as follows:
Above-mentioned milk or skimmed milk through the operating parameters of pasteurize are: heat 90 ℃ to 95 ℃, be incubated sterilization in 10 minutes to 15 minutes.
The present invention is easy to operate, production method is simple, gained mixed culture fermentation dairy products are nutritious, contain compositions such as profitable probliotics, ace inhibitory peptide, anti-oxidation peptide, have atherosclerosis, regulating blood fat, anti-inflammatory, effect such as anti-oxidant, can strengthen people's physique and overall activity, delay people's aging.
Description of drawings
Accompanying drawing 1 is the enlarged view of normal group coronary artery 10 times of microscopicallies.
Accompanying drawing 2 is the enlarged view of normal group coronary artery 40 times of microscopicallies.
Accompanying drawing 3 is the enlarged view of model group 10 times of microscopicallies.
Accompanying drawing 4 is the enlarged view of model group 40 times of microscopicallies.
Accompanying drawing 5 is the enlarged view of Simvastatin group 10 times of microscopicallies.
Accompanying drawing 6 is the enlarged view of Simvastatin group 40 times of microscopicallies.
Accompanying drawing 7 is the enlarged view of TFCW small dose group 10 times of microscopicallies.
Accompanying drawing 8 is the enlarged view of TFCW small dose group 40 times of microscopicallies.
Accompanying drawing 9 be among the TFCW dosage group at the enlarged view of 10 times of microscopicallies.
Accompanying drawing 10 be among the TFCW dosage group at the enlarged view of 40 times of microscopicallies.
Accompanying drawing 11 is that normal group coronary artery ICAM-1 is at microscopically enlarged view doubly.
Accompanying drawing 12 is the enlarged view of normal group coronary artery ICAM-1 40 times of microscopicallies.
Accompanying drawing 13 is that model group coronary artery ICAM-1 is at microscopically enlarged view doubly.
Accompanying drawing 14 is the enlarged view of model group coronary artery ICAM-1 40 times of microscopicallies.
Accompanying drawing 15 is the enlarged view of Simvastatin group coronary artery ICA M-1 10 times of microscopicallies.
Accompanying drawing 16 is the enlarged view of Simvastatin group coronary artery ICA M-1 40 times of microscopicallies.
Accompanying drawing 17 is the enlarged view of TFCW small dose group ICAM-1 10 times of microscopicallies.
Accompanying drawing 18 is the enlarged view of TFCW small dose group ICAM-1 40 times of microscopicallies.
Accompanying drawing 19 be among the TFCW dosage group ICAM-1 at the enlarged view of 10 times of microscopicallies.
Accompanying drawing 20 be among the TFCW dosage group ICAM-1 at the enlarged view of 40 times of microscopicallies.
Embodiment
The present invention is not subjected to the restriction of following embodiment, can determine concrete embodiment according to the technical scheme and the practical situation of the invention described above.
Below by embodiment the present invention is further described:
Embodiment 1, this mixed culture fermentation agent obtains by following parts by weight of raw materials fermentation, the weight ratio of zymophyte and milk or skimmed milk be 1: 20 to 30, zymophyte is short lactobacillus (Lactobacillus brevis), plant lactobacillus (Lactobacillus plantarum), lactobacterium helveticus (Lactobacillus.helveticus), lactobacillus kefiranofaciens (Lactobacillus kefianofaciens), lactobacterium casei (Lactobacillus casei), Lactobacillus farciminis (Lactobacillus farciminis), Lactococcus lactis (Lactococcus lactic), monospore yeast saccharomyces cerevisiae (Saccharomyces unisporus), Issatchenkia orientalis (Issatchenkia orientalis), the weight ratio between each bacterial classification be 0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:1.0 to 1.2:0.2 to 0.4:0.2 to 0.4.
Embodiment 2, this mixed culture fermentation agent obtains by following parts by weight of raw materials fermentation, the weight ratio of zymophyte and milk or skimmed milk be 1: 20 to 30, zymophyte is short lactobacillus (Lactobacillus brevis), plant lactobacillus (Lactobacillus plantarum), lactobacterium helveticus (Lactobacillus.helveticus), lactobacillus kefiranofaciens (Lactobacillus kefianofaciens), lactobacterium casei (Lactobacillus casei), Lactobacillus farciminis (Lactobacillus farciminis), Lactococcus lactis (Lactococcus lactic), monospore yeast saccharomyces cerevisiae (Saccharomyces unisporus), Issatchenkia orientalis (Issatchenkia orientalis), the weight ratio between each bacterial classification are 0.8 or 1.0:0.8 or 1.0:0.8 or 1.0:0.8 or 1.0:0.8 or 1.0:0.8 or 1.0:1.0 or 1.2:0.2 or 0.4:0.2 or 0.4.
Embodiment 3, be with the difference of embodiment 1 and embodiment 2: the mixed culture fermentation agent of embodiment 3 obtains by following step: at first after the milk or the pasteurize of skimmed milk process with requirement, next is cooled to 42 ℃ to the 45 ℃ zymophytes that add requirement, obtains the mixed culture fermentation agent in 8 hours to 12 hours at 40 ℃ to 45 ℃ constant temperature bottom fermentations then.
Embodiment 4, be with the difference of embodiment 1 and embodiment 2: the mixed culture fermentation agent of embodiment 4 obtains by following step: at first after the milk or the pasteurize of skimmed milk process with requirement, next is cooled to 42 ℃ to the 45 ℃ zymophytes that add requirement, obtains the mixed culture fermentation agent in 8 hours to 12 hours at 40 ℃ to 45 ℃ constant temperature bottom fermentations then.
Embodiment 5, and be with the difference of embodiment 1 to embodiment 4: embodiment 4 stirs twice in the time of 5 hours to 12 hours at least at the constant temperature bottom fermentation.
Embodiment 6, and this is used for antiatherogenic mixed culture fermentation dairy products and obtains by following fermenting raw materials, add the mixed culture fermentation agent that the foregoing description 1 to embodiment 5 makes in 3% to 5% ratio of milk or skimmed milk gross weight.
Embodiment 7, and this is used for antiatherogenic mixed culture fermentation dairy products and obtains by following fermenting raw materials, add the mixed culture fermentation agent that the foregoing description 1 to embodiment 5 makes in 3% or 5% ratio of milk or skimmed milk gross weight.
Embodiment 8, be with the difference of embodiment 6 and embodiment 7: the mixed culture fermentation dairy products of embodiment 8 obtain by following step: the first step, at first the milk of requirement or skimmed milk being heated to 50 ℃ to 60 ℃ carries out behind the homogeneous through pasteurize, next is cooled to 40 ℃ to the 45 ℃ mixed culture fermentation agent that add requirements, then 40 ℃ to 45 ℃ constant temperature bottom fermentations 6 hours to 8 hours; In second step, after the fermentation ends, maturation is 72 hours to 120 hours under 18 ℃ to 23 ℃ constant temperature; The 3rd step, at the uniform velocity stirred 1.5 hours to 2 hours, extracting fat, remainder boiled 1 hour to 2 hours at 50 ℃ to 60 ℃; The 4th step, sepg whey: 18 ℃ under 23 ℃, filter through the sterilization filter cloth, obtain cheese and whey respectively, this whey is the mixed culture fermentation dairy products.
Embodiment 9, are with the difference of embodiment 1 to embodiment 8: the milk of embodiment 9 or skimmed milk through the operating parameters of pasteurize are: heat 90 ℃ to 95 ℃, be incubated sterilization in 10 minutes to 15 minutes.
Embodiment 10, are with the difference of embodiment 1 to embodiment 8: the milk of embodiment 10 or skimmed milk through the operating parameters of pasteurize are: heat 90 ℃ or 95 ℃, be incubated 10 minutes or sterilization in 15 minutes.
In the present invention: % per-cent all is weight percentage.
The test and the pharmacodynamics test of the nourishing function composition of the foregoing description gained mixed culture fermentation dairy products of the present invention are as follows:
Following testing data is the average data of the foregoing description gained mixed culture fermentation dairy products test of the present invention.
The analysis of the nourishing function composition of mixed culture fermentation dairy products of the present invention
1.1 nutritive ingredient
1.1.1 detection method and foundation
Protein is measured with kjeldahl apparatus, detects foundation: GB/T5413.1.3-1997;
Ash content is pressed GB/ T5009.4-2003 and is measured;
Moisture is pressed GB/T5009.3-2003 and is measured;
Lactose is pressed GB/T5413.5-1997 and is measured;
Press the content that GB/T5413.22-1997 measures phosphorus
Mineral substance is pressed the content that GB/ T5009.14-2003 measures zinc with Avanta-PM type atomic absorption spectrophotometer; Press the content that GB/T5009.90-2003 measures iron and magnesium; Press GB/T5009.91,93-2003 measures the content of calcium and selenium; Press the content that GB/ T5009.91-2003 measures potassium and sodium
Amino acids is pressed GB/T5009-2003 with automatic analyzer for amino acids and is measured;
Lipid acid is pressed GB/T17377-1998 with Sigma 115 type gas chromatographs and is measured.
Vitamins is measured ascorbic content with 1525 pump series of high efficiency liquid chromatographs by GB/T6195-1986; Press the content that GB/T5009.82-2003 measures vitamin ae.
1.1.2 result
1.1.2.1 total protein and 17 seed amino acid assays
Measurement result shows: total protein content is 1.72% in the mixed culture fermentation dairy products of the present invention, total amino acid content is 0.734%, the amino acid (containing the essential Histidine of infant) that wherein contains 9 kinds of needed by human, content is 0.296%, account for the ratio 40.33% of total amino acid, close with human milk (43.0%) (seeing Table 1-1-2-1).In each amino acid, for the highest, being 0.47% with the content of Serine, is L-glutamic acid secondly, and content is 0.14%.In addition, proline(Pro) (0.091%), Methionin (0.056%), leucine (0.047%), aspartic acid (0.046%) content are also higher.
2.1.2.2 lipid acid is formed and content in the mixed culture fermentation dairy products of the present invention
Fat constitutes in the mixed culture fermentation dairy products of the present invention: saturated resin acid (palmitinic acid, stearic acid) content is 38.7%, unsaturated fatty acid content is 61.3%, and wherein, monounsaturated fatty acids (erucic acid does not detect) is up to 46.0%, polyunsaturated fatty acid is 15.3%, the results are shown in Table 2-1-2-2.
2.1.2.3 carbohydrate content in the mixed culture fermentation dairy products of the present invention
Main carbohydrate-lactose-content is 5.58% in the mixed culture fermentation dairy products of the present invention, is higher than milk, and is close with the sheep breast.
2.1.2.4 mixed culture fermentation dairy products mineral of the present invention is formed and content
Mixed culture fermentation dairy products mineral content of the present invention is abundant, wherein the potassium element content to the human body beneficial is the highest, be 246.3 mg/100g, secondly be calcium, content is 200.2mg/100g, and magnesium 18.7 mg/100g are significantly higher than other newborn classes, zinc is 0.09 mg/100g, and to also have selenium to detect in mixed culture fermentation dairy products of the present invention be 1.5 μ g.The results are shown in Table 2-1-2-4.
2.1.2.5 VITAMIN is formed and content in the mixed culture fermentation dairy products of the present invention
In the mixed culture fermentation dairy products of the present invention in the main vitamins content, vitamins C and vitamins B
2Content is higher, but is lower than other newborn class, the results are shown in Table 2-1-2-5.
2.1.3.1 the result shows
Measurement result shows: be rich in multiple nutritional components in the mixed culture fermentation dairy products of the present invention, total protein and aminoacids content are abundant, as total protein content is 1.72%, in 18 seed amino acids, the amino acid of 9 kinds of needed by human (containing the essential Histidine of infant) content is 0.30%, accounts for the ratio 40.33% of total amino acid; Content with Serine in each amino acid is the highest, is 0.47g/100g, is 0.14 g/100g for L-glutamic acid secondly.
2.1.3.2 in fatty acid analysis, monounsaturated fatty acids is up to 46.0% in the mixed culture fermentation dairy products of the present invention
Recent many studies confirm that, the ability of the single unsaturated fatty acids of cis aspect reducing cholesterol is identical with polyunsaturated fatty acid (PUFA), has various biological effects such as regulating blood fat effect, decreasing cholesterol effect, reduction oxidative stress.Monounsaturated fatty acids content is very high in the mixed culture fermentation dairy products of the present invention, and serine content is also higher, to a kind of good selection of may can yet be regarded as of cardiovascular and cerebrovascular diseases such as control senile dementia, hyperlipidemia.
2.1.3.3 lactose-content in the mixed culture fermentation dairy products of the present invention
Lactose-content is in the mixed culture fermentation dairy products of the present invention: 5.58%, be higher than milk, and close with the sheep breast.Lactose is regulated intestinal environment and is had good efficacy for the absorption that promotes calcium.
2.2 probiotic bacterium
Detection method
Zymophyte in the mixed culture fermentation dairy products of the present invention is carried out separation and purification, and combining form then, physio-biochemical characteristics and 16SrDNA, 16SrRNA, 26SrDNAD1/D2 district gene sequencing method are identified.
The result: being separated to two kinds of valuable microorganism one classes from mixed culture fermentation dairy products of the present invention is milk-acid bacteria, and another kind of is yeast; Ownership is respectively: short lactobacillus (Lactobacillus brevis), plant lactobacillus (Lactobacillus plantarum), lactobacterium helveticus (Lactobacillus.helveticus) lactobacillus kefiranofaciens (Lactobacillus kefianofaciens), lactobacterium casei (Lactobacillus casei) and Lactobacillus farciminis (Lactobacillus farciminis), Lactococcus lactis (Lactococcus lactic); Monospore yeast saccharomyces cerevisiae (Saccharomyces unisporus) and Issatchenkia orientalis (Issatchenkia orientalis).
2.3 ace inhibitory peptide, anti-oxidation peptide
Method: by angiotensin-converting enzyme (ACE), 2 to mixed culture fermentation dairy products of the present invention, the bitter diazanyl free radical (DPPH) of 2-phenylbenzene-1-, hydroxy radical qiao (OH) and ultra-oxygen anion free radical (O
2 -) external removing ability measure.
The result: studies confirm that mixed culture fermentation dairy products of the present invention by zymogenic extracellular protease, the hydrolytic action of peptase, the peptide fragment that has physiologically active in the milk-protein discharges, and for example ace inhibitory peptide, anti-oxidation peptide etc. see table 2-3 for details.
Conclusion: ace inhibitory peptide is the peptide material that a class has hypotensive activity, belong to competitive inhibitor, they to the avidity of ACE active region greater than angiotensin and bradykinin avidity to ACE, and they in case and the combination of ACE active region, just be difficult to discharge, thereby the nervous plain I of line artery is converted into angiotensin and bradykinin is decomposed into the inactivation fragment, reaches antihypertensive function.Hypertension is to cause one of atherosclerotic Hazard Factor, and mixed culture fermentation dairy products of the present invention can atherosclerosis.Mixed culture fermentation dairy products total antioxidation function of the present invention is that the people who makes longer-term take mixed culture fermentation dairy products of the present invention can build up health and overall activity, delays senility by the synergistic result of each antioxidant composition in the mixed culture fermentation dairy products of the present invention.
3. mixed culture fermentation dairy products pharmacodynamics test of the present invention
3.1 mixed culture fermentation dairy products regulating blood fat of the present invention, study of anti-atherogenic effect
3.1.1 materials and methods
3.1.1.1 laboratory animal and reagent
50 of healthy male new zealand rabbits, body weight (2 ± 0.05) kg, 3 monthly ages are available from Xinjiang Medicine University's Experimental Animal Center (conformity certification number: the new 2003-0001 of SYXK).Cholesterol (analytical pure), Sodium cholic acid are all purchased the extensive and profound in meaning star biotechnology responsibility company limited in Beijing, and propylthiouracil is purchased in Shanghai chemical reagents corporation, Simvastatin (Hangzhou Mo Shadong pharmaceutical Co. Ltd).The ICAM-1 test kit be ADL(No.:QRCT-3103223EIA UTL) product, the quantitative elisa kit for detecting of VCAM-1 is purchased in Senxiong Science ﹠ Technology Industry Co., Ltd., Shanghai, does experiment with the mixed culture fermentation dairy products of the embodiment of the invention 1 to embodiment 10 gained.
3.1.1.2 the foundation of new zealand rabbit Atherosclerosis Model
50 of healthy new zealand rabbits, male and female half and half about body weight 2kg, are divided into normal group (feed and raise the plain particles feed), dosage group in moving congee model group, positive controls, mixed culture fermentation dairy products small dose group of the present invention and the mixed culture fermentation dairy products of the present invention at random.Except that normal group, all the other each groups adopt high fat granulated feed.After high fat granulated feed is fed 8w, normal group and moving congee group compare to pure water, experimental group gives mixed culture fermentation dairy products of the present invention, be of the ratio table calculating of the consumption of mixed culture fermentation dairy products of the present invention according to the dose,equivalent of humans and animals body surface area conversion, middle dosage group gives mixed culture fermentation dairy products 50mgkg-1 of the present invention, small dose group gives mixed culture fermentation dairy products 25mgkg-1 of the present invention, and positive controls gives Simvastatin 20mgkg-1, altogether 28d.Omnidistance experiment 87d irritates 6h behind the stomach for the last time, Sodital anesthesia new zealand rabbit, and abdominal aortic cannulation is got blood, measures various indexs.
3.1.1.3 the mensuration of various indexs
3.1.1.3.1 the omnidistance experiment of blood fat and liver tissue homogenate's lipid the 87th day, Sodital anesthesia new zealand rabbit, abdominal aortic cannulation is got blood, get right lobe of liver 1g, 4 ℃ of homogenate of making 10 % with 0.9% physiological saline, centrifugal 15min leaves and takes supernatant liquor, measure the content of serum and liver tissue homogenate's total cholesterol (Tc), triglyceride level (TG), high density lipoprotein cholesterol (HDL-C) with the Beckman automatic biochemical analyzer, serum low-density LP cholesterol (LDL-C) is by converting.
3.1.1.3.2 body weight and liver coefficient and C-reactive protein feed give high lipid food before, respectively weigh once behind the 12w and before the anesthesia, calculate rabbit body weight gain situation, put to death rabbit after, take by weighing liver weight, the liver coefficient is a liver weight and the ratio of rabbit body weight.Nephelometry (rate method) rate scattering method is used Beckman Coulter Image and is measured the high quick C-reactive protein content of serum.
3.1.1.3.3 the omnidistance experiment of ICAM and VCAM Determination on content the 87th day, Sodital anesthesia new zealand rabbit, abdominal aortic cannulation is got blood, and centrifugal 15min leaves and takes serum, and the mensuration of VCAM-1 adopts double-antibody sandwich ABC-ELISA method.Combine with monoclonal antibody with anti-people sVCAM-1, add biotinylated anti-people sVCAM-1 antibody, form immunocomplex and connect onboard, the Streptavidin of horseradish peroxidase mark combines with vitamin H, adds enzyme substrates OPD, occur yellow, add stop buffer sulfuric acid, darken, survey the OD value at the 492nm place, sVCAM-1 concentration is directly proportional with the OD value, can obtain sVCAM-1 concentration in the sample by the drawing standard curve.Quantitatively require to measure its content according to enzyme joint inspection test kit
3.1.1.3.4 paraffin section-20 ℃ acetone fixed 5 min of immunohistochemistry arterial tissue, carry out tissue staining according to immunohistochemistry ABC method then, one anti-anti-rabbit VCAM-1 polyclonal antibody (Wuhan Boster Biological Technology Co., Ltd. provides, dilution in 1: 200) that adopts.With the DAB colour developing, the phenodin lining dyes, and examines under a microscope after the mounting.Other establishes the PBS negative control group.There are pale brown look or brown granular shape material in after birth or endochylema place, are latticed or the positive cell of ring-type precipitation person.Positive cell percentage ratio counting is carried out in (10 * 40) optional 5 visuals field under powerful microscope, averages.
3.1.2 statistical procedures
Data with
Expression, mean at first carries out homogeneity test of variance between each group, satisfies homoscedasticity as the result, then adopts the one-way analysis of variance or the t method of inspection, as not satisfying homoscedasticity, then adopts order Transformation Tests method.All processes is all undertaken by the SPSS11.5 statistical analysis software, has the boundary of statistical significance as difference with P＜0.05.
3.1.3. experimental result:
3.1.3.1. mixed culture fermentation dairy products of the present invention are to the influence of experimental atherosclerosis rabbit blood fat
Moving congee model group is compared with normal group, and serum cholesterol value, triglyceride level value and low-density lipoprotein white level raise.In the mixed culture fermentation dairy products of the present invention, cholesterol (Tc) level of small dose group and Simvastatin group (20 mgkg-1) reduces, the LDL-C level reduces; The TG level reduces.In moving congee model group, Simvastatin group, mixed culture fermentation dairy products small dose group of the present invention, the mixed culture fermentation dairy products of the present invention the dosage group and with the normal group HDL-C that more all can raise, difference has statistical significance, the results are shown in Table 3-1-3-1.
3.1.3.2 mixed culture fermentation dairy products of the present invention are to the influence of experimental atherosclerosis rabbit body weight, liver exponential sum c reactive protein.
Compare with normal group, moving congee model group c reactive protein content raises, and mixed culture fermentation dairy products 25 mgkg-1 of the present invention, 50 mgkg-1 group and Simvastatin group significantly reduce.Liver index, body weight not statistically significant the results are shown in Table 3-1-3-2.
3.1.3.3 influence to experimental atherosclerosis rabbit arterial pathology
Visual inspection sees that the normal rabbits aortic tunica intima is smooth, does not see that atheromatous plaque forms.Microscopically normal group (Fig. 1, Fig. 2) vessel wall three-decker is clear, and inner membrance is smooth complete, the smooth muscle cell marshalling.Moving congee model group (Fig. 3, Fig. 4) rabbit vessel wall diffusivity protuberance, prominent inner membrance is imperfect to tube chamber, and has atheromatous plaque to form.Aortic tunica intima thickens particularly evident, all visible a large amount of foam cells of inner membrance and middle film and a spot of fibroblast, smooth muscle cell arrangement disorder.Most of coronary artery forms typical atherosclerotic lesion, and cardiac muscular tissue's lipidosis is more serious.Simvastatin group (Fig. 5, Fig. 6) inner membrance and the equal visible foam cell of middle film, but do not see that atheromatous plaque forms, two dosage groups of mixed culture fermentation dairy products of the present invention (Fig. 7 to Figure 10) inner membrance is complete, the visible small amount of foam cell of inner membrance and middle film, but do not see that atheromatous plaque forms.
3.1.3.4 immunohistochemistry detects
Normal group (Figure 11, Figure 12) the rabbit coronary artery inner membrance is smooth, the endotheliocyte endochylema is not seen xanthochromia, moving congee model group (Figure 13, Figure 14) inner membrance endotheliocyte and smooth muscle cell endochylema have obvious brown yellow granule, the per-cent of positive cell area is (30.12 ± 9.95) %, the positive controls arterial endothelium has a small amount of ICAM-1 to express, the per-cent of positive cell area is (2.77 ± 0.53) %, mixed culture fermentation dairy products of the present invention are little, the visible endothelial cell membrane dyeing of middle dosage group (Figure 17-20), brown yellow granule seldom in the endotheliocyte endochylema, mainly outside endotheliocyte, express, the per-cent of positive cell area is for being (6.46 ± 1.55) % respectively, (2.49 ± 0.53) %, each group of moving congee model group and administration is compared difference all significance (P＜0.01).ICAM-1 and VCAM-1 quantitatively then require to measure according to enzyme joint inspection test kit, the results are shown in Table 3-1-3-4 and Figure 11 to Figure 20.
By giving rabbit 12w high fat diet, serum TC, TG, LDL-C content and CRP level significantly raise in this experiment, successfully set up atherosclerosis new zealand rabbit model.Experimental data is mutually attached with bibliographical information.This experimental result shows: mixed culture fermentation dairy products of the present invention can obviously reduce serum TC, TG, LDL-C content, and rising HDL-C level obviously reduces change of serum C RP level.
Mixed culture fermentation dairy products of the present invention can obviously reduce serum I CAM-1 and VCAM-1 content, the expression that the as seen moving congee model group ICAM-1 of immunohistochemical methods result all has at cell, endotheliocyte and the smooth muscle cell of rabbit coronary artery patch intralesional.When atheromatous plaque came off, ICAM-1 expressed at chamber face endothelium and is minimum level or disappearance, mainly is expressed as the master with smooth muscle cell, and administration is respectively organized VCAM-1 and expressed at most at endothelial cell surface, and is less in the cytoplasm.
Histopathology result also as seen, mixed culture fermentation dairy products two dosage groups of the present invention, endotheliocyte is still complete, visible small amount of foam cell, atherosclerotic plaque obviously reduces, the vessel wall fibrosis is light, inflammatory cell is few.
Among the TFCE, small dose group has regulating blood fat and reduces adhesion molecule expression, points out mixed culture fermentation dairy products anti-experimental character atherosclerosis of the present invention effect relevant with its protection of ecs effect and anti-inflammatory action.
3.2 cell protection of ecs effect
3.2.1 materials and methods
3.2.1.1 laboratory animal and reagent
50 of healthy male SD rats, body weight (200 ± 10) g, 3 monthly ages are available from Xinjiang Medicine University's Experimental Animal Center (conformity certification number: the new 2003-0001 of SYXK).
With the direct administration of mixed culture fermentation dairy products of the present invention.
3.2.1.2 the foundation of the atherosis model of rat artery
The foundation of SD big white mouse Atherosclerosis Model: 50 of healthy SD big white mouse, male and female half and half, about body weight 200g, 3 monthly ages, be divided into normal group (feed and raise the plain particles feed) at random, the heavy dose of group of dosage group and mixed culture fermentation dairy products of the present invention in moving congee model group, positive controls, mixed culture fermentation dairy products small dose group of the present invention, the mixed culture fermentation dairy products of the present invention.Except that normal group, all the other are respectively organized and adopt high lipoprotein emulsion to irritate stomach. and after high lipoprotein emulsion filling stomach began a week, disposable celiac was injected vitamins D simultaneously
3600,000 IUkg-1.The high lipoprotein emulsion of feeding is after 4 weeks, normal group and moving congee group compare to pure water, experimental group gives mixed culture fermentation dairy products of the present invention, be of the ratio table calculating of the consumption of mixed culture fermentation dairy products of the present invention according to the dose,equivalent of humans and animals body surface area conversion, heavy dose of group gives mixed culture fermentation dairy products 100mgkg-1 of the present invention, middle dosage group gives mixed culture fermentation dairy products 50mgkg-1 of the present invention, small dose group gives mixed culture fermentation dairy products 25mgkg-1g of the present invention, positive controls gives Simvastatin 20mgkg-1g, and every afternoon, gastric infusion was total to 28d altogether.Omnidistance experiment 57d irritates 6h behind the stomach for the last time, the Sodital anesthetized rat, and the carotid artery intubate is got blood, measures various indexs.
3.2.1.3 the mensuration of various indexs
TC, TG, HDL-C, LDL-C; Measured by radioimmunoassay endothelin (ET), thromboxane B2 (TXB
2), tumour necrosis factor (TNF-α), interleukin 8 (IL-8)
3.2.2 experimental result:
3.2.2.1 mixed culture fermentation dairy products of the present invention are to the influence of experimental atherosclerosis rat fat
Moving congee model group is compared with normal group, and serum cholesterol value, triacylglycerol value and low-density lipoprotein white level raise.Cholesterol (TCH) level of large, medium and small dosage group of mixed culture fermentation dairy products of the present invention and Simvastatin group (20mg/kg) reduces, and the LDL-C level reduces; The TG level reduces, and moving congee model group and normal group and each administration group all do not have influence to hdl level, and difference does not have statistical significance, the results are shown in Table 3-2-2-1.
3.2.2.2 mixed culture fermentation dairy products of the present invention are to the influence of experimental atherosclerosis rat endothelial function.
Endothelin (ET), tumor necrosis factor alpha (TNF-α) and interleukin 8 inflammatory factors such as (IL-8) all are higher than normal group in the moving congee model group, the Simvastatin group can significantly reduce the level of ET, TNF-α, IL-8, mixed culture fermentation dairy products small dose group of the present invention can significantly reduce the level of ET, IL-8, the heavy dose of group of mixed culture fermentation dairy products of the present invention can significantly reduce the level of IL-8 and TNF-α, and the dosage group can significantly reduce the level of ET, IL-8 in the mixed culture fermentation dairy products of the present invention.Mixed culture fermentation dairy products small dose group of the present invention is better than the Simvastatin group reducing effect aspect the ET.Each dosage group of mixed culture fermentation dairy products of the present invention does not have statistical significance to TXB2.The results are shown in Table 3-2-2-2.
Experimental results show that the feeding cholesterol can cause hyperlipidaemia, the injured blood vessel endothelium, endothelial injury or serum cholesterol level be too high to cause typical atheromatous lesions.This experiment is by high fat diet and injection megavitamin D
3, cause endothelial injury, successfully set up the atherosclerotic rat model.Experimental data is mutually attached with bibliographical information.This experimental result shows: mixed culture fermentation dairy products of the present invention can obviously reduce serum T ch, TG, LDL-C content, and rising HDL-C level helps that cholesterol is delivered to liver metabolism and decomposes, and suppresses cell and takes in LDL-C, alleviates AS.Mixed culture fermentation dairy products group of the present invention and positive control drug Simvastatin are relatively having superiority aspect reduction serum cholesterol, the reduction c reactive protein content.
3.3 anti-inflammatory, antioxygenation
3.3.1 materials and methods
Animal: 100 of healthy male SD rats, body weight (200 ± 10) g, 3 monthly ages are available from Xinjiang Medicine University's Experimental Animal Center (conformity certification number: the new 2003-0001 of SYXK).
Medicine: acetylsalicylic acid (Western Regions, Xinjiang medicine company limited-liability company), Ibuprofen BP/EP (Hubei encyclopaedia Heng Di pharmaceutcal corporation, Ltd), mixed culture fermentation dairy products of the present invention (the mixed culture fermentation dairy products of the present invention from embodiments of the invention 1 to embodiment 10 gained are TFCW).
Carrageenin causes mouse foot swelling Preparation of model: 50 mouse are divided into 4 groups, saline control group, acetylsalicylic acid positive controls (15mgkg-1), little, the middle dosage group of TFCW (25,50 mgkg-1) at random.Each treated animal gastric infusion every day, 7d altogether.1h causes inflammation at every mouse right hind subcutaneous injection 1% carrageenin suspension 0.11mL after the last administration, and survey right foot swelling degree (the scorching metapedes Zhou Jing of paw swelling=cause-cause scorching front foot Zhou Jing) with vernier callipers causing scorching back 1h, 2h, 3h and 4h respectively, calculate inhibiting rate.
The swollen Preparation of model that forms of rat granuloma: 40 rats are divided into 4 groups, 10 every group at random.Each treated animal vetanarcol anesthesia, implant cotton balls (each cotton balls weighs 20 mg, behind the autoclaving, 60 ℃ of baking boxs oven dry) under the aseptic technique in the rat oxter, grouping and administration are the same, each treated animal is distinguished gastric infusion then, continuous 10d, d11, the vetanarcol anesthetized rat, take out cotton balls, peel off granulation tissue, it is roasting to constant weight to put 60 ℃ of baking boxs.Granuloma dry weight=implantation granuloma induced by implantation of cotton pellets weight-implantation cotton balls weight.Calculate granuloma according to following formula and form inhibiting rate.Inhibiting rate P%=salt solution group granuloma induced by implantation of cotton pellets weight (mg)-administration group granuloma induced by implantation of cotton pellets weight (mg)/salt solution group granuloma induced by implantation of cotton pellets weight (mg) * 100%.
Carrageenin causes the preparation of rat air sac models of synovitis: 40 rats are divided 4 groups: dosage group among physiological saline control group, Ibuprofen BP/EP group, TFCW small dose group, the TFCW.Organize rat back subcutaneous injection through filter filtered air 20ml in experiment d1 at each, make rat back form air bag, treat that d3 injects at former air bag place through filter filtered air 20ml again, respectively organize rat the same day simultaneously and begin gastric infusion respectively, promptly give physiological saline 1ml/100g, Ibuprofen BP/EP group 15mgkg-1, little, the middle dosage group 25 of TFCW, 50mgkg-1, successive administration 7d.Experiment d8 is after each group rat back subcutaneous injection is through filter filtered air 10ml, and injection 2% carrageenin 2ml causes inflammation in air bag again.After causing scorching 24h, adopt venous blood, and rat is put to death, use Hank ' s liquid 1ml lavation air bag, collect irrigating solution, centrifugal, collect supernatant liquor respectively, for every index determining.WBC measures row routine smear method white blood cell count(WBC) and observes 3～5 10 * 40 visuals field continuously in the transudate, averages; Adopt nephelometry (rate method), the rate scattering method is used Beckman Coulter Immage and is measured Serum hs-CRP content.
The mensuration of antioxygenation: utilize above-mentioned carrageenin to cause rat air bag synovitis chronic inflammation model, after causing scorching 24h, adopt venous blood, and rat put to death, measure the test kit explanation according to Total antioxidant capacity, adopt the total Total antioxidant capacity of colorimetric method for determining, the mensuration of mda (MDA) adopts the TBA method.
3.3.2 result
3.3.2.1 mixed culture fermentation dairy products on Carrageenan of the present invention causes the influence of mouse foot swelling
Compare with the physiological saline group, little, middle dosage group of mixed culture fermentation dairy products of the present invention and acetylsalicylic acid group (except acetylsalicylic acid group cause scorching back 2 h), causing inflammation back 1h, 2h, each time period of 3h, 4h, the swelling degree all obviously alleviates, the paw swelling of mixed culture fermentation dairy products small dose group particularly of the present invention significantly alleviates, and the results are shown in Table 3-3-2-1.
3.3.2.2 to the swollen influence that forms of rat granuloma
Compare with the physiological saline group, the granuloma induced by implantation of cotton pellets dry weight of little, middle dosage group of mixed culture fermentation dairy products of the present invention and Ibuprofen BP/EP group all significantly reduces, and each group of administration does not relatively have significant difference with the Ibuprofen BP/EP group.The Ibuprofen BP/EP group is 24.17% to the inhibiting rate that granuloma forms, and little, the middle dosage group of mixed culture fermentation dairy products of the present invention is respectively 17.11% and 17.18% to the inhibiting rate that granuloma forms.Compare with the physiological saline group, little, middle dosage group of mixed culture fermentation dairy products of the present invention and Ibuprofen BP/EP group are to the influence of rat body weight, and not statistically significant the results are shown in Table 3-3-2-2.
3.3.2.3 mixed culture fermentation dairy products on Carrageenan of the present invention causes the influence of the rat air sac models of synovitis transudate scale of construction, sees Table 3-3-2-3.
With the salt solution group relatively, Ibuprofen BP/EP group and mixed culture fermentation dairy products dosage group of the present invention be little, in, heavy dose of group on Carrageenan transudate scale of construction of causing rat air bag synovitis has remarkable restraining effect, difference has statistical significance.
3.5 mixed culture fermentation dairy products on Carrageenan of the present invention causes the influence of hs-CRP in the rat air bag synovitis serum, total oxidation capacity and MDA.
Compare with the salt solution group, Ibuprofen BP/EP group, mixed culture fermentation dairy products of the present invention low dose and middle dosage group have remarkable restraining effect to CRP in the air bag synovitis rat blood serum, and difference has statistical significance.
Compare mixed culture fermentation dairy products 50mg.kg of the present invention with model group
-1Group significantly improves carrageenin and causes Total antioxidant capacity in the serum of air bag synovitis rat, difference have statistical significance (
PValue is 0.047), positive group Ibuprofen BP/EP group to Total antioxidant capacity in the serum do not have obvious influence (
PValue is 0.231).
With model group relatively, in the mixed culture fermentation dairy products of the present invention, the small dose group on Carrageenan content that causes MDA in the rat blood serum of air bag synovitis has remarkable restraining effect, difference have statistical significance (
PValue is respectively 0.019,0.048), but each the group between the difference not statistically significant (
P0.05).Positive control Ibuprofen BP/EP group then to the content of MDA do not have obvious influence (
PValue is respectively 0.197).The results are shown in Table 3-5.
Claims (10)
1. mixed culture fermentation agent, it is characterized in that obtaining by following parts by weight of raw materials fermentation, the weight ratio of zymophyte and milk or skimmed milk be 1: 20 to 30, zymophyte is short lactobacillus, plant lactobacillus, lactobacterium helveticus, lactobacillus kefiranofaciens, lactobacterium casei, Lactobacillus farciminis, Lactococcus lactis, monospore yeast saccharomyces cerevisiae, Issatchenkia orientalis, the weight ratio between each bacterial classification be 0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:0.8 to 1.0:1.0 to 1.2:0.2 to 0.4:0.2 to 0.4.
2. mixed culture fermentation agent according to claim 1, it is characterized in that obtaining: at first after the milk or the pasteurize of skimmed milk process with requirement by following step, next is cooled to 42 ℃ to the 45 ℃ zymophytes that add requirement, obtains the mixed culture fermentation agent in 8 hours to 12 hours at 40 ℃ to 45 ℃ constant temperature bottom fermentations then.
3. plant mixed culture fermentation agent according to claim 2, it is characterized in that the constant temperature bottom fermentation stirs twice in the time of 5 hours to 12 hours at least.
4. the production method of a mixed culture fermentation agent according to claim 1, it is characterized in that being undertaken: at first after the milk or the pasteurize of skimmed milk process with requirement by following step, next is cooled to 42 ℃ to the 45 ℃ zymophytes that add requirement, obtains the mixed culture fermentation agent in 8 hours to 12 hours at 40 ℃ to 45 ℃ constant temperature bottom fermentations then.
5. the production method of mixed culture fermentation agent according to claim 4 is characterized in that the constant temperature bottom fermentation stirs twice in the time of 5 hours to 12 hours at least.
6. according to the production method of claim 4 or 5 described mixed culture fermentation agent, it is characterized in that milk or skimmed milk through the operating parameters of pasteurize are: heat 90 ℃ to 95 ℃, be incubated sterilization in 10 minutes to 15 minutes.
7. antiatherogenic mixed culture fermentation dairy products that are used for that utilize claim 1 or 2 or 3 described mixed culture fermentation agent, it is characterized in that obtaining, add the above-mentioned mixed culture fermentation agent that makes in 3% to 5% ratio of milk or skimmed milk gross weight by following fermenting raw materials.
8. mixed culture fermentation dairy products according to claim 7, it is characterized in that obtaining: the first step by following step, at first the milk of requirement or skimmed milk being heated to 50 ℃ to 60 ℃ carries out behind the homogeneous through pasteurize, next is cooled to 40 ℃ to the 45 ℃ mixed culture fermentation agent that add requirements, then 40 ℃ to 45 ℃ constant temperature bottom fermentations 6 hours to 8 hours; In second step, after the fermentation ends, maturation is 72 hours to 120 hours under 18 ℃ to 23 ℃ constant temperature; The 3rd step, at the uniform velocity stirred 1.5 hours to 2 hours, extracting fat, remainder boiled 1 hour to 2 hours at 50 ℃ to 60 ℃; The 4th step, sepg whey: 18 ℃ under 23 ℃, filter through the sterilization filter cloth, obtain cheese and whey respectively, this whey is the mixed culture fermentation dairy products.
9. the production method of mixed culture fermentation dairy products according to claim 7, it is characterized in that being undertaken: the first step by following step, at first the milk of requirement or skimmed milk being heated to 50 ℃ to 60 ℃ carries out behind the homogeneous through pasteurize, next is cooled to 40 ℃ to the 45 ℃ mixed culture fermentation agent that add requirements, then 40 ℃ to 45 ℃ constant temperature bottom fermentations 6 hours to 8 hours; In second step, after the fermentation ends, maturation is 72 hours to 120 hours under 18 ℃ to 23 ℃ constant temperature; The 3rd step, at the uniform velocity stirred 1.5 hours to 2 hours, extracting fat, remainder boiled 1 hour to 2 hours at 50 ℃ to 60 ℃; The 4th step, sepg whey: 18 ℃ under 23 ℃, filter through the sterilization filter cloth, obtain cheese and whey respectively, this whey is the mixed culture fermentation dairy products.
10. the production method of mixed culture fermentation dairy products according to claim 9 is characterized in that milk or skimmed milk through the operating parameters of pasteurize are: heat 90 ℃ to 95 ℃, be incubated sterilization in 10 minutes to 15 minutes.
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