CN103820379B - Meat proteins zymolyte is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt - Google Patents
Meat proteins zymolyte is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt Download PDFInfo
- Publication number
- CN103820379B CN103820379B CN201410056354.8A CN201410056354A CN103820379B CN 103820379 B CN103820379 B CN 103820379B CN 201410056354 A CN201410056354 A CN 201410056354A CN 103820379 B CN103820379 B CN 103820379B
- Authority
- CN
- China
- Prior art keywords
- zymolyte
- milk
- enzymolysis
- acid bacteria
- proliferation function
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses the experimental technique of a kind of meat proteins zymolyte to separating lactic acid bacterium proliferation function in Yoghourt, by being separated to thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacterium casei four kinds of milk-acid bacterias from Yoghourt, after using neutral protease and Sumizyme MP enzymolysis pork and chicken respectively, pass through OD
622value compares the cultivation effect drawing milk-acid bacteria, and show that the proliferation function of zymolyte on four kinds of milk-acid bacterias all has impact, enzymolysis time is 6h, when zymolyte addition is 3%, the strongest to milk-acid bacteria proliferation function; The zymolyte of Sumizyme MP is better to the cultivation effect of milk-acid bacteria than the zymolyte of neutral protease; The cultivation effect of zymolyte to thermophilus streptococcus is best, the poorest to the cultivation effect of lactobacterium casei.The present invention is for improving milk-acid bacteria multiplication capacity, and the shortening production cycle provides theoretical basis, and the nutritive health-care food for development of meat protein zymolyte has established theoretical basis.
Description
Technical field
The invention belongs to milk-acid bacteria multiplication technique field, particularly relate to the experimental technique of a kind of meat proteins zymolyte to separating lactic acid bacterium proliferation function in Yoghourt.
Background technology
At present, along with people are to the attention of health, Yoghourt improves intestine microenvironment as a kind of nutritive health-care food because having, promote the wriggling of stomach and intestine, the effects such as the growth of spoilage microorganisms in enteron aisle are suppressed more and more to be subject to the favor of human consumer, and the bacterial classification in Yoghourt is at important roles such as regulating intestinal canal functions, in Yoghourt, main bacteria seed is thermophilus streptococcus and lactobacillus bulgaricus, Lactobacterium acidophilum is also had in some Yoghourt, lactobacterium casei and this several probiotic bacterium of bifidus bacillus, be referred to as milk-acid bacteria, meat proteins zymolyte or polypeptide are because having different physiological roles also one of novel healthy food becoming research and development in recent years.
Milk-acid bacteria refers to the bacterium utilizing fermentable carbohydrate to produce a large amount of lactic acid, and in Gram-positive, being morphologically divided into bacillus and coccus, is facultative anaerobic bacteria, and milk-acid bacteria is profitable strain main in our enteron aisle, has a lot of health-care effect to human body:
(1) balance of intestinal microflora is improved, the balance improving intestinal microflora is one of function of finding the earliest of milk-acid bacteria, milk-acid bacteria can breed after entering enteron aisle in ramp, its metabolite produced maintains intestinal microecology colony balance by antagonistic action, milk-acid bacteria generally produces two class materials: one is some nutritive ingredients and enzyme system, comprise the multivitamin that fully can be absorbed by human body, amino acid, lactose, the specific enzymes system of lactic acid and polysaccharide etc. and various VITAMIN etc., they can promote the secretion of Host Digestion enzyme and GI wriggling, promote digesting and assimilating and the generation of preventing constipation of food, two is antimicrobial substances, eliminates the existence of pathogenic bacteria with Ant agonism, as hydrogen peroxide has obvious bacteriostatic action, the organic acid such as lactic acid and acetic acid can reduce the colon bacillus that enteron aisle local ph is antibacterial or kill in enteron aisle, many milk-acid bacterias can bacteriocinogeny in food matrix, can suppress spoilage organism or pathogenic bacterium etc., and milk-acid bacteria can also prevent pathogenic bacteria in intestinal epithelial cells surface attachment, field planting invade intestinal cell, and this mechanism has person for " adhesion resistance ",
(2) cholesterol level in serum is reduced, high cholesterol level means the generation of excessive risk cardiovascular disorder, a large amount of clinical experiments both at home and abroad confirm, milk-acid bacteria has the effect reducing cholesterol in various degree, Gilliland [7] is by the research to the decreasing cholesterol effect of enteron aisle Bacterium lacticum, proposing milk-acid bacteria is due to cholate of degrading in process of growth, promote that the katabolism of cholesterol reduces the viewpoint of cholesterol level, Zhao Li Jun, neat oecoeclades falcata, Chen Yourong is reducing in the research of cholesterol effect to enteron aisle milk-acid bacteria, filter out a lot of strain and there is the lactic bacterium strains of reduction courage with alcohol, Xiao Linlin and Dong Mingsheng filters out a strain Tibet cheese milk-acid bacteria, experimental result shows, it significantly can reduce total cholesterol (TC) in hyperlipidemic mice serum, triglyceride (TG) concentration,
(3) resistance to compression effect, hypertension is another important factor causing heart disease, hypertension mainly angiotensin converting enzyme (ACE) causes indirectly, it has been generally acknowledged that lactobacterium helveticus can produce stronger ACE inhibitory substance, thus reduces high pressure and the low pressure of hyperpietic;
(4) anticancer effect, the anticancer effect of milk-acid bacteria is mainly because it has the activity of anti-mutation, the antimutagenic activity of milk-acid bacteria is because milk-acid bacteria itself and meta-bolites thereof are to can be that carcinogenic hydrolase plays restraining effect by the carcinogenic substance precursor conversion in food, experiment shows, the Yoghourt being fed with Lactobacterium acidophilum to mouse can extend the time that cancer cells occurs, the Yoghourt drunk containing Lactobacterium acidophilum can reduce the disease index of cancer;
(5) immunoregulation effect, milk-acid bacteria can improve the immunocompetence of body, mainly because milk-acid bacteria and meta-bolites thereof come activating macrophage and " NK " cytoactive by induction generation Interferon, rabbit and cytokinin, thus performance immunoregulation effect, simultaneously, the meta-bolitess such as the lactic acid that milk-acid bacteria produces in growth metabolism process, acetic acid and hydrogen peroxide, inhibit growth that is corrupt in intestines and other unwanted bacterias, decrease carcinogenic substance and other toxicants such as indoles, toxic amine, indole that these unwanted bacterias produce;
(6) antioxidant effect, the people such as Verena study and find that milk-acid bacteria can reduce the oxidative damage of people's colon cell (HT29) DNA, thus stop the generation of colorectal carcinoma, but, to the research of milk-acid bacteria anti-oxidant activity and imperfection, there are some researches show that Lactobacterium acidophilum has the ability of anti peroxidation of lipid and scavenging hydroxyl, concrete Antioxidation Mechanism is also unclear, and most of investigator thinks may have some oxidation resistant enzyme materials (comprising superoxide dismutase and nadh oxidase etc.) to make it have anti-oxidant activity in milk-acid bacteria;
Protein be hydrolyzed under the effect of enzyme the peptide class that obtains and more the product such as small-molecular-weight amino acid be called protein zymolyte, meat proteins zymolyte or polypeptide are the one in protein zymolyte, it is obtained through being suitably hydrolyzed by meat proteins, because protein zymolyte or polypeptide have than amino acid or the higher nourishing function of protein and nutritive value, become the focus of research in recent years, meat proteins zymolyte or polypeptide mainly contain following a few dot characteristics:
(1) hypoallergenic, retains the nutritive substance of former albumen, and be more easily absorbed by the body utilization;
(2) character such as the solvability of protein in processing and utilization process, emulsifying property, gelation, whipability are improved;
(3) flavor is more abundant, outstanding, improves the local flavor of food;
(4) many physiological functions, as hypotensive, promote that gi tract are suffered from diarrhoea to the absorption of calcium and preventing, improve the metabolism of lipid, strengthening immunity, promotion enterogastric peristalsis, protection stomach mucous membrane prevent stomach ulcer, prevents hyperosteogeny etc.;
Due to these characteristics, meat proteins zymolyte or polypeptide as novel foodstuff additive, for the production of the nutritional fortification of senior seasonings, food, the base-material of functional foodstuff and be widely used in foodstuffs industry produce in.
Domestic and international protein zymolyte is to the influence research of milk-acid bacteria proliferation function:
Zhang Qingli research shows casein Sumizyme MP, neutral protease, trypsinase and flavor protease hydrolysis 12h and has stronger growth-promoting activity with the zymolyte that papain hydrolysis 8h obtains to milk-acid bacteria, hydrophilic amino acid in Hydrolysates of Casein can better promote that milk-acid bacteria breeds, the growth-promoting activity of sulfur-containing amino acid on milk-acid bacteria does not then affect significantly, Oliveira, the research such as Lucas shows in Yoghourt, add the propagation that Hydrolysates of Casein obviously can promote thermophilus streptococcus, but it is very little to other milk-acid bacteria proliferative effect, Bouhallab etc. report caseic trypsin digestion thing and have proliferation function to LactococcuslactissubsplactisCNRZ1076, the speed of growth of this milk-acid bacteria can be improved twice by the zymolyte that molecular weight is less than 3kDa section,
Qu Dujuan, Zhao Mouming research shows: (1) whey-protein zymolyte does not change the growth curve of milk-acid bacteria in Yoghourt; (2) whey-protein zymolyte significantly can promote the propagation of thermophilus streptococcus and Lactobacterium acidophilum, does not make significant difference to the propagation of lactobacillus bulgaricus; (3) whey-protein zymolyte can improve the stability of thermophilus streptococcus in preservation term, and reduce the stability of lactobacillus bulgaricus in preservation term, thus reduce the rear acidifying of Yoghourt, in a word, whey-protein zymolyte can significantly improve the propagation of milk-acid bacteria;
Fan Xiuhua, when people's researchs such as He Xinyi show that soybean protein hydrolysis polypeptide addition is 3%, viable count of lactobacillus reaches the highest, during fermentation termination, viable count of lactobacillus is 1.3 times of control group viable count, result shows that soybean polypeptide obviously can promote the propagation of milk-acid bacteria, the polypeptide that lactobacter growth can be provided required and amino acid
Zhang Zhi, Zhu Hongliang, the people such as button grand Yu research shows that the corn peptide of interpolation 1.2% can promote the propagation of Lactis In Yoghurt, and Yoghourt fermentation Time transfer receiver shortens 1h according to group, and the viable count of milk-acid bacteria can be above standard content in Yoghourt;
The people such as Li Ke, Yang Xiuhua, Hu Lin research shows that bone protein of pig zymolyte is remarkable to the proliferation function of lactobacterium helveticus, to the cultivation effect of lactobacillus bulgaricus and Lactobacterium acidophilum a little less than Hydrolysates of Casein, is significantly higher than soy peptone and fish meal protein peptone;
Milk-acid bacteria breaks down proteins ability own is more weak, growth fraction is slower, add different protein digestion things and polypeptide required for lactobacter growth and total free aminoacids can be provided, thus promote the propagation of milk-acid bacteria, improve the viable count of milk-acid bacteria, shorten fermentation time, in addition, protein digestion thing can also improve the viable count of preservation term milk-acid bacteria, improve the quality of Yoghourt, the factor affecting milk-acid bacteria propagation is study hotspot in recent years in milk-acid bacteria, major part research is that the zymolyte of some protein or polypeptide are on the impact of milk-acid bacteria proliferation function, as corn peptide, soybean polypeptide, Hydrolysates of Casein, whey-protein zymolyte and bone protein of pig zymolyte etc., but not yet there is research meat proteins zymolyte to the impact of milk-acid bacteria proliferation function.
Summary of the invention
The object of the embodiment of the present invention is the experimental technique providing a kind of meat proteins zymolyte to separating lactic acid bacterium proliferation function in Yoghourt, is intended to solve existingly lack the problem that affect experimental technique of meat proteins zymolyte on milk-acid bacteria proliferation function.
The embodiment of the present invention realizes like this, a kind of meat proteins zymolyte is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt, this meat proteins zymolyte is cultivated by differential medium the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt, gram stain microscopy is separated thermophilus streptococcus with biochemical test qualification from Yoghourt, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacterium casei four kinds of milk-acid bacterias, after using neutral protease and Sumizyme MP enzymolysis pork and chicken respectively, by 2h, 4h, the zymolyte of 6h enzymolysis time is with different 1%, 2%, the addition of 3% adds in the MRS liquid nutrient medium of four kinds of milk-acid bacterias, after 37 DEG C of constant temperature culture 72h, pass through OD
622value compares the cultivation effect drawing milk-acid bacteria.
Further, the proliferation function of zymolyte on thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacterium casei four kinds of milk-acid bacterias all has impact, and enzymolysis time is 6h, when zymolyte addition is 3%, the strongest to milk-acid bacteria proliferation function; The zymolyte of Sumizyme MP is better to the cultivation effect of milk-acid bacteria than the zymolyte of neutral protease; The cultivation effect of zymolyte to thermophilus streptococcus is best, the poorest to the cultivation effect of lactobacterium casei.
Further, thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacterium casei four kinds of lactic acid bacteria culturers be separated and purifying comprises the following steps:
Step one, draws 1mL to sterile test tube with Sterile pipette by Yoghourt, adds stroke-physiological saline solution 9mL in Bechtop, after abundant mixing, draw 1mL bacterium liquid and be placed in another sterile test tube, then add 9mL stroke-physiological saline solution, dilution like this 4 times, 4 test tubes are designated as 10 respectively in order
-1, 10
-2, 10
-3, 10
-4four gradients;
Step 2, from 4 test tubes, get 0.2mL bacterium liquid respectively coat MRS solid medium plate, maltose-MRS substratum plate, acidifying MRS substratum plate, each gradient do 3 parallel, it is for subsequent use that dilution bacterium liquid is placed in 4 DEG C of refrigerators, and plate to be put in constant incubator 37 DEG C and cultivated 48h;
Step 3, observes the colony characteristics that maltose-MRS substratum plate grows, is connected to 6 kinds of sugar-fermenting pipes, carries out biochemical identification to colonies typical after carrying out gramstaining and microscopy;
Step 4, observes the colony characteristics of the bacterium colony that acidifying MRS substratum plate grows, is connected to 6 kinds of sugar-fermenting pipes, carries out biochemical identification to colonies typical after carrying out gramstaining and microscopy;
Step 5, observes the colony characteristics of the bacterium colony that MRS solid medium plate grows, carries out gramstaining and microscopy respectively to several colonies typical, microscopy is streptococcic bacterium colony repeatedly plate streaking, carry out purifying, microscopy is that the bacterium colony of bacillus is connected to 6 kinds of sugar-fermenting pipes, carries out biochemical identification.
Further, the enzymolysis of pork and chicken comprises the following steps:
Step one, respectively gets six parts by pork and chicken, and every part of 10g is placed in 50mL beaker, and 70 DEG C of hot water rinse after meat blanching with clear water again, are placed in boiling water and boil 25min, and temperature is down to chopping mortar porphyrize after normal temperature;
Step 2, each beaker adds 20mL distilled water, and namely meat water is than being 1:2, and regulate the pH of three portions of chicken and three portions of porks to be 7 with 1mol/LNaOH solution, the pH of other three portions of chicken and three portions of porks is 10;
Step 3, to pH be 7 six portions of chicken and pork sample in add neutral protease 0.2g respectively, to pH be 10 six portions of chicken and pork sample in add Sumizyme MP 0.2g respectively;
Step 4, all chicken and pork sample are all placed in 50 DEG C of thermostat water baths and are incubated enzymolysis, respectively take out two chicken zymolytes of different enzyme enzymolysis when 2h, 4h, 6h, two pork hydrolysate go out enzyme 20min in 95 DEG C of water-baths, go out after enzyme, zymolyte is centrifugal 20min under 4000r/min, gets supernatant enzymolysis solution for subsequent use.
Further, different zymolyte on the method that bacterial classification proliferation function affects is:
Zymolyte (supernatant enzymolysis solution) is made an addition in MRS liquid nutrient medium with 1%, 2%, 3% respectively, stroke-physiological saline solution makes an addition in MRS liquid nutrient medium as blank using 1%, 2%, 3% simultaneously, add in the MRS liquid nutrient medium of zymolyte (supernatant enzymolysis solution) from often kind of bacterium access of the single bacterium colony of MRS solid medium inoculating needle picking equivalent respectively, be placed in 37 DEG C of constant incubators and cultivate 72h, take stroke-physiological saline solution as blank, under 622nm wavelength, measure substratum OD value.
Meat proteins zymolyte provided by the invention is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt, by being separated thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacterium casei four kinds of milk-acid bacterias from Yoghourt, after using neutral protease and Sumizyme MP enzymolysis pork and chicken respectively, pass through OD
622value compares the cultivation effect drawing milk-acid bacteria, and show that the proliferation function of zymolyte on four kinds of milk-acid bacterias all has impact, enzymolysis time is 6h, when zymolyte addition is 3%, the strongest to milk-acid bacteria proliferation function; The zymolyte of Sumizyme MP is better to the cultivation effect of milk-acid bacteria than the zymolyte of neutral protease; The cultivation effect of zymolyte to thermophilus streptococcus is best, the poorest to the cultivation effect of lactobacterium casei.The present invention is affected milk-acid bacteria proliferation function by research meat proteins zymolyte, for improving milk-acid bacteria multiplication capacity, the shortening production cycle provides theoretical basis, also through the hydrolysis of human enzymes after can eating for furtheing investigate meat proteins, understand the direction that the impact of zymolyte on intestinal microflora proliferation function provides research, thus established theoretical basis for the nutritive health-care food of development of meat protein zymolyte, compensate for and existingly lack the blank that affect experimental technique of meat proteins zymolyte on milk-acid bacteria proliferation function.
Accompanying drawing explanation
Fig. 1 is that the meat proteins zymolyte that provides of the embodiment of the present invention is to the experimental technique schema of separating lactic acid bacterium proliferation function in Yoghourt;
Fig. 2 is that the chicken neutral protease enzymolysis thing that the embodiment of the present invention provides affects schematic diagram to thermophilus streptococcus proliferation function;
Fig. 3 is that the chicken Sumizyme MP zymolyte that the embodiment of the present invention provides affects schematic diagram to thermophilus streptococcus proliferation function;
Fig. 4 is that the pork neutral protease enzymolysis thing that the embodiment of the present invention provides affects schematic diagram to thermophilus streptococcus proliferation function;
Fig. 5 is that the pork Sumizyme MP zymolyte that the embodiment of the present invention provides affects schematic diagram to thermophilus streptococcus proliferation function;
Fig. 6 is that the zymolyte of the enzymolysis 6h that the embodiment of the present invention provides affects schematic diagram to thermophilus streptococcus proliferation function;
Fig. 7 is that the chicken neutral protease enzymolysis thing that the embodiment of the present invention provides affects schematic diagram to lactobacillus bulgaricus proliferation function;
Fig. 8 is that the chicken Sumizyme MP zymolyte that the embodiment of the present invention provides affects schematic diagram to lactobacillus bulgaricus proliferation function;
Fig. 9 is that the pork neutral protease enzymolysis thing that the embodiment of the present invention provides affects schematic diagram to lactobacillus bulgaricus proliferation function;
Figure 10 is that the pork Sumizyme MP zymolyte that the embodiment of the present invention provides affects schematic diagram to lactobacillus bulgaricus proliferation function;
Figure 11 is that the zymolyte of the enzymolysis 6h that the embodiment of the present invention provides affects schematic diagram to lactobacillus bulgaricus proliferation function;
Figure 12 is that the chicken neutral protease enzymolysis thing that the embodiment of the present invention provides affects schematic diagram to Lactobacterium acidophilum proliferation function;
Figure 13 is that the chicken Sumizyme MP zymolyte that the embodiment of the present invention provides affects schematic diagram to Lactobacterium acidophilum proliferation function;
Figure 14 is that the pork neutral protease enzymolysis thing that the embodiment of the present invention provides affects schematic diagram to Lactobacterium acidophilum proliferation function;
Figure 15 is that the pork Sumizyme MP zymolyte that the embodiment of the present invention provides affects schematic diagram to Lactobacterium acidophilum proliferation function;
Figure 16 is that the zymolyte of the enzymolysis 6h that the embodiment of the present invention provides affects schematic diagram to Lactobacterium acidophilum proliferation function;
Figure 17 is that the chicken neutral protease enzymolysis thing that the embodiment of the present invention provides affects schematic diagram to lactobacterium casei proliferation function;
Figure 18 is that the chicken Sumizyme MP zymolyte that the embodiment of the present invention provides affects schematic diagram to lactobacterium casei proliferation function;
Figure 19 is that the pork neutral protease enzymolysis thing that the embodiment of the present invention provides affects schematic diagram to lactobacterium casei proliferation function;
Figure 20 is that the pork Sumizyme MP zymolyte that the embodiment of the present invention provides affects schematic diagram to lactobacterium casei proliferation function;
Figure 21 is that the zymolyte of the enzymolysis 6h that the embodiment of the present invention provides affects schematic diagram to lactobacterium casei proliferation function;
Figure 22 is that the chicken neutral protease enzymolysis thing of the enzymolysis 6h that the embodiment of the present invention provides affects schematic diagram to four kinds of milk-acid bacteria proliferation functions;
Figure 23 is that the chicken Sumizyme MP zymolyte of the enzymolysis 6h that the embodiment of the present invention provides affects schematic diagram to four kinds of milk-acid bacteria proliferation functions;
Figure 24 is that the pork neutral protease enzymolysis thing of the enzymolysis 6h that the embodiment of the present invention provides affects schematic diagram to four kinds of milk-acid bacteria proliferation functions;
Figure 25 is that the pork Sumizyme MP zymolyte of the enzymolysis 6h that the embodiment of the present invention provides affects schematic diagram to four kinds of milk-acid bacteria proliferation functions.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
The meat proteins zymolyte of the embodiment of the present invention comprises the following steps the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt:
S101: cultivated by differential medium, the qualification of gram stain microscopy and biochemical test is separated thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacterium casei four kinds of milk-acid bacterias from Yoghourt, uses neutral protease and Sumizyme MP enzymolysis pork and chicken respectively;
S102: the zymolyte (supernatant enzymolysis solution) of different enzymolysis time (2h, 4h, 6h) is added in the MRS liquid nutrient medium of four kinds of milk-acid bacterias with different additions (1%, 2%, 3%), after 37 DEG C of constant temperature culture 72h, passes through OD
622value compares the cultivation effect drawing milk-acid bacteria.
Result of the present invention shows, the proliferation function of zymolyte on four kinds of milk-acid bacterias all has impact, and enzymolysis time is 6h, when zymolyte addition is 3%, the strongest to milk-acid bacteria proliferation function; The zymolyte of Sumizyme MP is better to the cultivation effect of milk-acid bacteria than the zymolyte of neutral protease; The cultivation effect of zymolyte to thermophilus streptococcus is best, the poorest to the cultivation effect of lactobacterium casei.
In conjunction with following specific embodiment, the present invention is described further:
1, materials and methods:
Material:
Method: the preparation of substratum
MRS solid medium;
MRS liquid nutrient medium;
MRS improved formulations (sugar-fermenting substratum, i.e. sugar-fermenting pipe);
Maltose-MRS substratum;
Acidifying MRS substratum;
The first step, the separation of bacterial classification and purifying:
The Yoghourt of buying is drawn 1mL to sterile test tube with Sterile pipette in Bechtop, add stroke-physiological saline solution 9mL, after abundant mixing, another sterile test tube is placed in from wherein drawing 1mL bacterium liquid, add 9mL stroke-physiological saline solution again, dilution like this 4 times, 4 test tubes are designated as 10 respectively in order
-1, 10
-2, 10
-3, 10
-4four gradients.From 4 test tube, get 0.2mL bacterium liquid respectively coat MRS solid medium plate, maltose-MRS substratum plate, acidifying MRS substratum plate, each gradient do 3 parallel.It is for subsequent use that dilution bacterium liquid is placed in 4 DEG C of refrigerators, and 37 DEG C of cultivation 48h in constant incubator put by plate.
Observe the colony characteristics that maltose-MRS substratum plate grows, be connected to 6 kinds of sugar-fermenting pipes after gramstaining and microscopy are carried out to colonies typical, carry out biochemical identification.
Observe the colony characteristics of the bacterium colony that acidifying MRS substratum plate grows, be connected to 6 kinds of sugar-fermenting pipes after gramstaining and microscopy are carried out to colonies typical, carry out biochemical identification.
Observe the colony characteristics of the bacterium colony that MRS solid medium plate grows, carry out gramstaining and microscopy respectively to several colonies typical, microscopy is streptococcic bacterium colony repeatedly plate streaking, carries out purifying, microscopy is that the bacterium colony of bacillus is connected to 6 kinds of sugar-fermenting pipes, carries out biochemical identification.
Second step, the enzymolysis of meat:
The pre-treatment of meat:
The pork and chicken that are purchased from market are respectively got six parts, and every part of 10g is placed in 50mL beaker, and about 70 DEG C hot water rinse after meat blanching with clear water again, are placed in boiling water and boil 25min, and temperature is down to chopping mortar porphyrize after normal temperature;
Each beaker adds 20mL distilled water, and namely meat water is than being 1:2, and regulate the pH of three portions of chicken and three portions of porks to be 7 with 1mol/LNaOH solution, the pH of other three portions of chicken and three portions of porks is 10.
3rd step, enzymolysis and the enzyme that goes out:
To pH be 7 six portions of chicken and pork sample in add neutral protease 0.2g respectively, to pH be 10 six portions of chicken and pork sample in add Sumizyme MP 0.2g respectively.All chicken and pork sample are all placed in 50 DEG C of thermostat water baths and are incubated enzymolysis, respectively take out two chicken zymolytes of different enzyme enzymolysis, two pork hydrolysate go out enzyme 20min in 95 DEG C of water-baths when 2h, 4h, 6h.Go out after enzyme, zymolyte is centrifugal 20min under 4000r/min, gets supernatant enzymolysis solution for subsequent use.
4th step, different zymolyte affects bacterial classification proliferation function:
Zymolyte (supernatant enzymolysis solution) is made an addition in MRS liquid nutrient medium with 1%, 2%, 3% respectively, stroke-physiological saline solution makes an addition in MRS liquid nutrient medium as blank using 1%, 2%, 3% simultaneously, add in the MRS liquid nutrient medium of zymolyte (supernatant enzymolysis solution) from often kind of bacterium access of the single bacterium colony of MRS solid medium inoculating needle picking equivalent respectively, be placed in 37 DEG C of constant incubators and cultivate 72h, take stroke-physiological saline solution as blank, under 622nm wavelength, measure substratum OD value.
2, statistical study: data acquisition Excel software processes.
3, results and analysis:
3.1 strain separating and purifying
The colonial morphology of the milk-acid bacteria be separated in Yoghourt, as shown in table 1.
The colonial morphology of the milk-acid bacteria be separated in table 1 Yoghourt
As shown in Table 1, the colony characteristics of variation bacterial classification 1 and 2 is obviously different from other four kinds of bacterium; Colony colour and the bacterium colony mode of appearance of lactobacterium casei are different from lactobacillus bulgaricus, Lactobacterium acidophilum and thermophilus streptococcus; Lactobacterium acidophilum bacterium colony is moistening, and deep layer is transparent, different with thermophilus streptococcus colony characteristics from lactobacillus bulgaricus, lactobacterium casei; Lactobacillus bulgaricus and the difference of thermophilus streptococcus colony characteristics are not quite.
Microscopic morphology after the milk-acid bacteria gramstaining be separated to, as shown in table 2.
As can be seen from Table 2, except variation bacterial classification 2, all the other five kinds of bacterium are gram positive bacterium, and in five kinds of bacillus, variation bacterial classification 1 cell is the shortest, exists mainly with chain; Lactobacillus bulgaricus, Lactobacterium acidophilum and variation bacterial classification 2 cell are rod-short, and mainly with single existence, minority becomes two and occurs; Lactobacterium casei is elongated rod shape, is obviously different from other four kinds of bacillus.
Microscopic morphology after the milk-acid bacteria gramstaining be separated in table 2 Yoghourt
Five kinds of bacillus biochemical tests, as shown in table 3:
Table 3 biochemical results
Note: "+" is positive reaction; "-" is negative reaction; " ± " is weak positive reaction
Can the ferment carbohydrate that utilizes of often kind of bacterium is different as can be seen from Table 3, and except variation bacterial classification 1, all the other four kinds of bacterium all can utilize lactose; Variation bacterial classification 1 and lactobacillus bulgaricus is unfermentable utilizes maltose and sucrose; Five kinds of bacterium all can utilize fructose; Only have lactobacterium casei to ferment and utilize N.F,USP MANNITOL.
3.2 different zymolytes affect milk-acid bacteria proliferation function
3.2.1 different zymolyte is on the impact of thermophilus streptococcus proliferation function
As can be seen from Fig. 2 to Fig. 6, the hydrolysis time of pork (chicken), enzymolysis solution addition are different from the kind of zymolyte streptococcus thermophilus fermentation liquid OD
622value impact is different, and enzymolysis solution addition is identical, when hydrolysis time is 6h, and OD
622be worth maximum; Hydrolysis time is identical, when enzymolysis solution addition is 3%, and OD
622be worth maximum; Add the substratum OD of the enzymolysis solution of enzymolysis 6h
622value is all than the OD of the substratum of blank group
622value is large, adds the substratum OD of pork protein enzyme zymolyte
622value is all greater than the substratum OD adding chicken protein enzyme zymolyte
622value, wherein adds the substratum OD of pork Sumizyme MP zymolyte
622be worth maximum;
As can be seen from Fig. 7 to Figure 11, the enzymolysis time of pork (chicken), enzymolysis solution addition are different from the kind of zymolyte fermentation using lactobacillus bulgaricus liquid OD
622value impact is different, and enzymolysis solution addition is identical, when enzymolysis time is 6h, and OD
622be worth maximum; Enzymolysis time is identical, when enzymolysis solution addition is 3%, and OD
622be worth maximum; When enzymolysis time is 6h, add the substratum OD of enzymolysis solution
622value is all than the OD of the substratum of blank group
622value is large, but little to the cultivation effect difference of lactobacillus bulgaricus between four kinds of zymolytes, when enzymolysis solution addition is 1% and 2%, adds the substratum OD of pork Sumizyme MP zymolyte
622be worth maximum, absorbancy is in rising trend, and when enzymolysis solution addition is 2%-3%, absorbancy tends towards stability, and adds the substratum OD of chicken Sumizyme MP zymolyte
622value still presents ascendant trend, when enzymolysis solution addition is 3%, and OD
622value reaches maximum.
3.2.2 with the impact of zymolyte on Lactobacterium acidophilum proliferation function
As can be seen from Figure 12 to Figure 16, the enzymolysis time of pork (chicken), enzymolysis solution addition are different from the kind of zymolyte Lactobacterium acidophilum fermented liquid OD
622value impact is different, and enzymolysis solution addition is identical, when enzymolysis time is 6h, and OD
622be worth maximum; Enzymolysis time is identical, when enzymolysis solution addition is 3%, and OD
622be worth maximum; Add the OD of the substratum of the enzymolysis solution of enzymolysis 6h
622value is all than the OD of the substratum of blank group
622value is large, adds the substratum OD of chicken protein enzyme zymolyte
622value is all greater than the substratum OD adding pork protein enzyme zymolyte
622value, wherein adds the OD of the substratum of chicken Sumizyme MP zymolyte
622be worth maximum;
As can be seen from Figure 17 to Figure 21, the enzymolysis time of pork (chicken), enzymolysis solution addition are different from the kind of zymolyte lactobacterium casei fermented liquid OD
622value impact is different, and enzymolysis solution addition is identical, when enzymolysis time is 6h, and OD
622be worth maximum; Enzymolysis time is identical, when enzymolysis solution addition is 3%, and OD
622be worth maximum; Add the OD of the substratum of the enzymolysis solution of enzymolysis 6h
622value is all than the OD of the substratum of blank group
622value is large, adds the substratum OD of pork protein enzyme zymolyte
622value is all greater than the substratum OD adding chicken protein enzyme zymolyte
622value, wherein adds the substratum OD of pork Sumizyme MP zymolyte
622be worth maximum;
As can be seen from Figure 22 to Figure 25, the OD of thermophilus streptococcus
622difference is maximum, is secondly lactobacillus bulgaricus, lactobacterium casei OD
622difference is minimum.
3.2.3 zymolyte of the same race is on the impact of four kinds of milk-acid bacteria proliferation functions
Six kinds of bacterium are separated to altogether from commercially available yoghourt, thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum, lactobacterium casei, variation bacterial classification 1 and variation bacterial classification 2 respectively, all identified by gramstaining and microscopy and biochemical test, but variation bacterial classification 1 and 2 fails to identify strain name, not killed miscellaneous bacteria when these two kinds variation bacterial classifications may be raw dairy sterilizings, also be likely in separation and purification and fermenting process, cause the variation of bacterial classification by the impact of ambient conditions;
Protein zymolyte has certain proliferation function to four kinds of milk-acid bacterias, when enzymolysis solution addition is identical, hydrolysis time is longer, protein digestion is more thorough, polypeptide and free aminoacid content more, polypeptide and the free aminoacid content of the utilization of milk-acid bacteria Absorbable rod are many, and somatomedin content increases thus promotes the propagation of milk-acid bacteria; When enzymolysis time is identical, enzymolysis solution addition is more, and polypeptide and free aminoacid content are also more, is conducive to milk-acid bacteria propagation, the meat proteins enzymolysis solution of enzymolysis 6h with 3% addition join in MRS liquid nutrient medium time, best to milk-acid bacteria proliferation function; When soybean protein hydrolysis polypeptide addition is 3%, viable count of lactobacillus reaches the highest, and during fermentation termination, viable count of lactobacillus is 1.3 times of control group viable count; The corn peptide of interpolation 1.2% can promote the propagation of Lactis In Yoghurt;
In enzymolysis product, polypeptide fragment is different with total free aminoacids, also be different to the proliferative effect of bacterial classification, the proliferative effect of meat proteins enzymolysis product to milk-acid bacteria of Sumizyme MP is best, shows that the polypeptide that meat proteins obtains after Sumizyme MP enzymolysis and total free aminoacids can promote the propagation of milk-acid bacteria better; Zhang Qingli research shows that the hydrophilic amino acid in Hydrolysates of Casein can promote that milk-acid bacteria breeds better, and the growth-promoting activity of sulfur-containing amino acid on milk-acid bacteria does not then affect significantly; Bouhallab research shows that casein obtains through trypsin digestion the proliferation function that zymolyte that molecular weight is less than 3kDa section significantly improves LactococcuslactissubsplactisCNRZ1076;
The polypeptide chain that different strain can absorb is different with amino acid, therefore zymolyte is also different to the cultivation effect of different strain, meat proteins zymolyte is best to thermophilus streptococcus cultivation effect, is secondly lactobacillus bulgaricus, the poorest to the proliferation function effect of lactobacterium casei; Whey-protein zymolyte significantly can promote the propagation of thermophilus streptococcus and Lactobacterium acidophilum, does not make significant difference to the propagation of lactobacillus bulgaricus; The proliferation function of bone protein of pig zymolyte to lactobacterium helveticus is remarkable;
The present invention preliminary proof meat protein zymolyte can promote the propagation of milk-acid bacteria, use for reference the method that forefathers study casein, whey-protein and bone protein of pig zymolyte, next step can do further research from the following aspects: the hydrolysis degree of (1) quantitative examination meat proteins, analyze the molecular weight after meat proteolysis, study the cultivation effect of which molecular weight section to milk-acid bacteria the most obvious; (2) enzymolysis time of proteolytic enzyme can be extended, the addition level of zymolyte increases, and determines the best enzymolysis time and the zymolyte addition that promote milk-acid bacteria propagation; (3) multiselect carries out the enzymolysis of meat with several enzyme, such as stomach en-, and the enzymolysis product of real simulation meat in stomach is on the impact of milk-acid bacteria proliferation function more; (4) can the casein of comparative studies the same enzyme same hydrolysis degree, whey-protein and meat proteins to the difference of milk-acid bacteria cultivation effect, wish by further research, finally can develop a kind of nutritive health-care food of meat proteins zymolyte,
Certain viable bacteria is had in commercially available yoghourt, therefrom can be separated to the milk-acid bacteria of the interpolation marked in allocation sheet, the enzymolysis solution of enzymolysis 6h with 3% addition join in MRS liquid nutrient medium time, best to milk-acid bacteria cultivation effect, zymolyte wherein after Sumizyme MP enzymolysis meat proteins is better than the zymolyte after neutral protease enzymolysis meat proteins to milk-acid bacteria cultivation effect, the cultivation effect of zymolyte to thermophilus streptococcus is best, the poorest to the cultivation effect of lactobacterium casei.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. a meat proteins zymolyte is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt, it is characterized in that, this meat proteins zymolyte is cultivated by differential medium the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt, gram stain microscopy and biochemical test identify the thermophilus streptococcus be separated to from Yoghourt, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacterium casei four kinds of milk-acid bacterias, after using neutral protease and Sumizyme MP enzymolysis pork and chicken respectively, by 2h, 4h, the zymolyte of 6h enzymolysis time is with different 1%, 2%, the addition of 3% adds in the MRS liquid nutrient medium of four kinds of milk-acid bacterias, after 37 DEG C of constant temperature culture 72h, pass through OD
622value compares the cultivation effect drawing milk-acid bacteria, the enzymolysis of pork and chicken comprises the following steps:
Step one, respectively gets six parts by pork and chicken, and every part of 10g is placed in 50mL beaker, and 70 DEG C of hot water rinse after meat blanching with clear water again, are placed in boiling water and boil 25min, and temperature is down to chopping mortar porphyrize after normal temperature;
Step 2, each beaker adds 20mL distilled water, and namely meat water is than being 1:2, and regulate the pH of three portions of chicken and three portions of porks to be 7 with 1mol/LNaOH solution, the pH of other 3 portions of chicken and 3 portions of porks is 10;
Step 3, to pH be 7 six portions of chicken and pork sample in add neutral protease 0.2g respectively, to pH be 10 six portions of chicken and pork sample in add Sumizyme MP 0.2g respectively;
Step 4, all chicken and pork sample are all placed in 50 DEG C of thermostat water baths and are incubated enzymolysis, respectively take out two chicken zymolytes of different enzyme enzymolysis when 2h, 4h, 6h, two pork hydrolysate go out enzyme 20min in 95 DEG C of water-baths, go out after enzyme, zymolyte is centrifugal 20min under 4000r/min, gets supernatant enzymolysis solution for subsequent use.
2. meat proteins zymolyte as claimed in claim 1 is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt, it is characterized in that, the proliferation function of zymolyte on thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacterium casei four kinds of milk-acid bacterias all has impact, enzymolysis time is 6h, when zymolyte addition is 3%, the strongest to milk-acid bacteria proliferation function; The zymolyte of Sumizyme MP is better to the cultivation effect of milk-acid bacteria than the zymolyte of neutral protease; The cultivation effect of zymolyte to thermophilus streptococcus is best, the poorest to the cultivation effect of lactobacterium casei.
3. meat proteins zymolyte as claimed in claim 1 is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt, it is characterized in that, thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and lactobacterium casei four kinds of lactic acid bacteria culturers be separated and purifying comprises the following steps:
Step one, draws 1mL to sterile test tube with Sterile pipette by Yoghourt, adds stroke-physiological saline solution 9mL in Bechtop, after abundant mixing, draw 1mL bacterium liquid and be placed in another sterile test tube, then add 9mL stroke-physiological saline solution, dilution like this 4 times, 4 test tubes are designated as 10 respectively in order
-1, 10
-2, 10
-3, 10
-4four gradients;
Step 2, from 4 test tubes, get 0.2mL bacterium liquid respectively coat MRS solid medium plate, maltose-MRS substratum plate, acidifying MRS substratum plate, each gradient do 3 parallel, it is for subsequent use that dilution bacterium liquid is placed in 4 DEG C of refrigerators, and plate to be put in constant incubator 37 DEG C and cultivated 48h;
Step 3, observes the colony characteristics that maltose-MRS substratum plate grows, is connected to 6 kinds of sugar-fermenting pipes, carries out biochemical identification to colonies typical after carrying out gramstaining and microscopy;
Step 4, observes the colony characteristics of the bacterium colony that acidifying MRS substratum plate grows, is connected to 6 kinds of sugar-fermenting pipes, carries out biochemical identification to colonies typical after carrying out gramstaining and microscopy;
Step 5, observes the colony characteristics of the bacterium colony that MRS solid medium plate grows, carries out gramstaining and microscopy respectively to several colonies typical, microscopy is streptococcic bacterium colony repeatedly plate streaking, carry out purifying, microscopy is that the bacterium colony of bacillus is connected to 6 kinds of sugar-fermenting pipes, carries out biochemical identification.
4. meat proteins zymolyte as claimed in claim 1 is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt, and it is characterized in that, different zymolyte on the method that bacterial classification proliferation function affects is:
Zymolyte is made an addition in MRS liquid nutrient medium with 1%, 2%, 3% respectively, zymolyte is supernatant enzymolysis solution, stroke-physiological saline solution makes an addition in MRS liquid nutrient medium as blank using 1%, 2%, 3% simultaneously, add in the MRS liquid nutrient medium of zymolyte from often kind of bacterium access of the single bacterium colony of MRS solid medium inoculating needle picking equivalent respectively, be placed in 37 DEG C of constant incubators and cultivate 72h, take stroke-physiological saline solution as blank, under 622nm wavelength, measure substratum OD value.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410056354.8A CN103820379B (en) | 2014-02-19 | 2014-02-19 | Meat proteins zymolyte is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410056354.8A CN103820379B (en) | 2014-02-19 | 2014-02-19 | Meat proteins zymolyte is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103820379A CN103820379A (en) | 2014-05-28 |
| CN103820379B true CN103820379B (en) | 2016-04-06 |
Family
ID=50755673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410056354.8A Active CN103820379B (en) | 2014-02-19 | 2014-02-19 | Meat proteins zymolyte is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103820379B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104186921B (en) * | 2014-09-23 | 2017-03-08 | 福州大学 | A kind of method of modifying of wheat gluten protein and the application in Yoghourt product |
-
2014
- 2014-02-19 CN CN201410056354.8A patent/CN103820379B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| 猪骨蛋白酶解物对乳酸菌增殖作用研究;李珂等;《中国酿造》;20091015(第10期);全文 * |
| 蛋白水解物促乳酸菌增殖及高密度培养体系研究;白凤翎;《中国博士学位论文全文数据库》;20100915(第9期);第1页摘要、第26页 * |
| 蛋白酶解物对酸奶发酵剂增殖的促进作用;支潇;《中国优秀硕士学位论文全文数据库》;20130315(第3期);全文 * |
| 酪蛋白活性肽对乳酸菌生长代谢及酸乳发酵影响的研究;张清丽;《中国博士学位论文全文数据库》;20111115(第11期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103820379A (en) | 2014-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102181376B (en) | Bacillus subtilis for simultaneously degrading zearalenone and cellulose and application thereof | |
| CN102876614B (en) | Bacillus licheniformis and application of bacillus licheniformis | |
| CN101338283B (en) | Lactobacillus casei and applications thereof in solid-state fermentation | |
| CN105062933A (en) | Lactobacillus reuteri and application thereof | |
| CN106754619A (en) | It is a kind of that the method that traditional Chinese medicinal components promote growth of probiotics is added in grain culture medium | |
| CN104472879A (en) | Tea residue feed additive and preparation method thereof | |
| CN110106119B (en) | Lactobacillus rhamnosus M9 separated from breast milk and application thereof | |
| CN104651268A (en) | Lactobacillus plantarum and application thereof | |
| CN107198076A (en) | A kind of bacillus coagulans probiotics normal temperature viable bacteria beverage and preparation method thereof | |
| CN109315586A (en) | A kind of preparation method and application of ruminant fermentation of Chinese herbal medicine biological feedstuff | |
| CN105400727A (en) | Lactobacillus paracasei with antioxidant activity and application thereof | |
| CN110607255B (en) | Preparation method and application of lactobacillus delbrueckii and direct vat set lactobacillus delbrueckii starter | |
| CN107974421A (en) | A kind of lactobacillus acidophilus and its screening technique and application, a kind of microbial inoculum | |
| CN113604404B (en) | Bacillus coagulans YSF17 and application thereof | |
| CN105661230A (en) | Probiotic functional beverage and probiotic food prepared from Lens culinaris by fermentation | |
| CN107151638A (en) | One plant improvement liver function Lactobacillus plantarum ZY001 and its application in acidified milk | |
| CN108220343B (en) | Fermentation method for improving calcium conversion rate and oxidation resistance in sheep bone powder enzymatic hydrolysate | |
| CN116286468A (en) | Lactobacillus mucilaginosus LF-ONLLY with antioxidant function and application thereof in fermented food | |
| CN103952336A (en) | Bacillus licheniformis-Bacillus subtilis-Lactobacillus casei preparation and preparation method thereof | |
| CN102199565A (en) | Enterococcus faecium and method for producing equol by using Enterococcus faecium and application of Enterococcus faecium | |
| CN104365858B (en) | A kind of preparation method of fermented type ginkgo peanut milk drink | |
| CN103271155A (en) | Method for increasing viable count of probiotics | |
| CN102093966B (en) | Mink-derived Lactobacillus plantarum strain MDL1118 and application thereof | |
| CN103820379B (en) | Meat proteins zymolyte is to the experimental technique of separating lactic acid bacterium proliferation function in Yoghourt | |
| CN112813006B (en) | Lactobacillus plantarum and application thereof in fermentation of fruit and vegetable juice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |