CN103820379A - Experiment method of proliferation effect of meat protein zymolytes on separation lactic acid bacteria in sour milk - Google Patents
Experiment method of proliferation effect of meat protein zymolytes on separation lactic acid bacteria in sour milk Download PDFInfo
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Abstract
The invention discloses an experiment method of proliferation effect of meat protein zymolytes on separation lactic acid bacteria in sour milk. The sour milk is separated to obtain four types of lactic acid bacteria of streptococcus thermophilus, lactobacillus bulgaricus, lactobacillus acidophilus and lactobacillus casei, a neutral protease and an alkaline protease are respectively used for enzymolysis of pork and chicken, the proliferation effect of the lactic acid bacteria can be obtained by the comparison of OD622 value, the comparison of OD622 value shows that the zymolytes have influences on the proliferation effect of the four types of lactic acid bacteria, when the enzymolysis time is 6h, and the zymolyte additive amount is 3%, the proliferation effect on the lactic acid bacteria is strongest; the alkaline protease zymolyte has a better proliferation effect on the lactic acid bacteria than the neutral protease zymolyte, the zymolytes have a best proliferation effect on the streptococcus thermophilus, and have a worst proliferation effect on the lactobacillus casei. The experiment method provides a theoretical basis for improvement of the proliferation ability of the lactic acid bacteria and shortening of the production cycle, and settles a theoretical basis for development of nutritious and health food of the meat protein zymolytes.
Description
Technical field
The invention belongs to milk-acid bacteria multiplication technique field, relate in particular 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 healthy attention, Yoghourt as a kind of nutritive health-care food because thering is the intestine microenvironment of improvement, promote the wriggling of stomach and intestine, the effects such as the growth of the interior spoilage microorganisms of inhibition enteron aisle are more and more subject to human consumer's favor, and 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, in some Yoghourt, also has Lactobacterium acidophilum, these several probiotic bacteriums of lactobacterium casei and bifidus bacillus, be referred to as milk-acid bacteria, meat proteins zymolyte or polypeptide also become one of novel healthy food of research and development in recent years because having different physiological roles.
Milk-acid bacteria refers to the bacterium that utilizes fermentable carbohydrate to produce a large amount of lactic acid, is Gram-positive, is divided into bacillus and coccus in form, is facultative anaerobic bacteria, and milk-acid bacteria is profitable strain main in our enteron aisle, and human body is had to a lot of health-care effects:
(1) improve the balance of enteric microorganism flora, the balance of improving enteric microorganism flora is one of function of finding the earliest of milk-acid bacteria, milk-acid bacteria is entering after enteron aisle and can ramp breed, the metabolite of its generation maintains intestinal microecology colony balance by antagonistic action, the general two class materials that produce of milk-acid bacteria: the one, some nutritive ingredients and enzyme system, comprise the multivitamin that can fully 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 secretion and the GI wriggling of Host Digestion enzyme, promote the also generation of preventing constipation of digesting and assimilating of food, the 2nd, antimicrobial substance, eliminates existing of pathogenic bacteria with Ant agonism, as hydrogen peroxide has obvious bacteriostatic action, it is antibacterial or kill the colon bacillus in enteron aisle that the organic acid such as lactic acid and acetic acid can reduce enteron aisle local pH value, 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 and invade intestinal cell, and this mechanism has person for " adhesion resistance ",
(2) reduce the cholesterol level in serum, 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 that reduces in various degree cholesterol, Gilliland[7] by the research of the decreasing cholesterol effect to enteron aisle Bacterium lacticum, having proposed milk-acid bacteria is due to the cholate of degrading in process of growth, promote the katabolism of cholesterol to reduce the viewpoint of cholesterol level, Zhao Li Jun, neat oecoeclades falcata, Chen Yourong is in to the research of enteron aisle milk-acid bacteria reduction cholesterol effect, filter out a lot of strains and there is the lactic bacterium strains of reduction courage with alcohol, Xiao Linlin and Dong Mingsheng filter out a strain Tibet cheese milk-acid bacteria, experimental result shows, it can significantly reduce total cholesterol in hyperlipidemia mice serum (TC), triglyceride (TG) concentration,
(3) resistance to compression effect, hypertension is another important factor that causes heart disease, hypertension is mainly that angiotensin converting enzyme (ACE) causes indirectly, it has been generally acknowledged that lactobacterium helveticus can produce stronger ACE inhibitory substance, thereby reduces hyperpietic's high pressure and low pressure;
(4) anticancer effect, the anticancer effect of milk-acid bacteria is mainly the activity due to it with 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 that is fed with Lactobacterium acidophilum to mouse can extend the time that cancer cells occurs, drinks the Yoghourt that contains Lactobacterium acidophilum and can reduce the disease index of cancer;
(5) immunoregulation effect, milk-acid bacteria can be improved the immunocompetence of body, mainly because milk-acid bacteria and meta-bolites thereof can produce Interferon, rabbit and cytokinin comes activating macrophage and " NK " cytoactive by induction, thereby performance immunoregulation effect, simultaneously, the meta-bolitess such as lactic acid, acetic acid and hydrogen peroxide that milk-acid bacteria produces in growth metabolism process, suppress the growth of corrupt in intestines and other unwanted bacterias, reduced carcinogenic substance and other toxicants such as the indoles that these unwanted bacterias produce, toxic amine, indole;
(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, thereby stop the generation of colorectal carcinoma, but, research to milk-acid bacteria anti-oxidant activity imperfection, there are some researches show that Lactobacterium acidophilum has the ability of anti peroxidation of lipid and removing hydroxy radical qiao, concrete Antioxidation Mechanism is also unclear, and most of investigator thinks in milk-acid bacteria may have some oxidation resistant enzyme materials (comprising superoxide dismutase and nadh oxidase etc.) to make it have anti-oxidant activity;
Protein under the effect of enzyme, be hydrolyzed the peptide class that obtains and more the product such as small molecules amount amino acid be called protein zymolyte, meat proteins zymolyte or polypeptide are the one in protein zymolyte, it is to be obtained through being suitably hydrolyzed by meat proteins, because protein zymolyte or polypeptide have than amino acid or higher nourishing function and the nutritive value of protein, become in recent years the focus of research, meat proteins zymolyte or polypeptide mainly contain following a few dot characteristics:
(1) hypoallergenic, retains the nutritive substance of former albumen, and utilization is more easily absorbed by the body;
(2) improve the character such as the solvability of protein in processing and utilization process, emulsifying property, gelation, whipability;
(3) be taste and more enrich, give prominence to, improve the local flavor of food;
(4) many physiological functions, as hypotensive, promote the absorption of gi tract to calcium and prevent diarrhoea, the metabolism, strengthening immunity, promotion enterogastric peristalsis, the protection stomach mucous membrane that improve lipid prevent stomach ulcer, prevents hyperosteogeny etc.;
Due to these characteristics, meat proteins zymolyte or polypeptide be as novel foodstuff additive, is widely used in during foodstuffs industry produces for the production of the base-material of the nutritional fortification of senior seasonings, food, functional foodstuff.
The impact research of protein zymolyte on milk-acid bacteria proliferation function both at home and abroad:
Zhang Qingli research shows that Sumizyme MP for casein, neutral protease, trypsinase and flavor protease hydrolysis 12h and the zymolyte obtaining with papain hydrolysis 8h have stronger growth-promoting activity to milk-acid bacteria, hydrophilic amino acid in Hydrolysates of Casein can better promote milk-acid bacteria propagation, sulfur-containing amino acid is on not significant impact of the growth-promoting activity of milk-acid bacteria, Oliveira, the researchs such as Lucas show in Yoghourt, to add Hydrolysates of Casein and can obviously promote the propagation of thermophilus streptococcus, but very little on other milk-acid bacteria propagation impacts, Bouhallab etc. have reported that caseic trypsin digestion thing has proliferation function to Lactococcus lactis subsp lactisCNRZ1076, the zymolyte that molecular weight is less than 3kDa section can improve twice by the speed of growth of this milk-acid bacteria,
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 can significantly promote the propagation of thermophilus streptococcus and Lactobacterium acidophilum, and the propagation of lactobacillus bulgaricus is not made significant difference; (3) whey-protein zymolyte can improve the stability of thermophilus streptococcus in preservation term, reduces the stability of lactobacillus bulgaricus in preservation term, thereby has reduced the rear acidifying of Yoghourt, and 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%, it is the highest that viable count of lactobacillus has reached, when fermentation termination, viable count of lactobacillus is 1.3 times of control group viable count, result shows that soybean polypeptide can obviously promote the propagation of milk-acid bacteria, the essential polypeptide of lactobacter growth and amino acid can be provided
Zhang Zhi, Zhu Hongliang, the people such as grand Yu of button research shows to add 1.2% corn peptide and can promote the propagation of Lactis In Yoghurt, and the Yoghourt fermentation time shortens 1h than control group, the viable count of milk-acid bacteria the content in Yoghourt that can be above standard;
Li Ke, Yang Xiuhua, people's researchs such as Hu Lin show that bone protein of pig zymolyte is remarkable to the proliferation function of lactobacterium helveticus, and the cultivation effect of lactobacillus bulgaricus and Lactobacterium acidophilum, a little less than Hydrolysates of Casein, is significantly higher than to soy peptone and fish meal protein peptone;
The protein capacity of decomposition of milk-acid bacteria own is more weak, growth fraction is slower, add different protein digestion things the needed polypeptide of lactobacter growth and total free aminoacids can be provided, thereby 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 that affects milk-acid bacteria propagation is the study hotspot aspect milk-acid bacteria in recent years, most of research is zymolyte or the impact of polypeptide on milk-acid bacteria proliferation function of some protein, as corn peptide, soybean polypeptide, Hydrolysates of Casein, whey-protein zymolyte and bone protein of pig zymolyte etc., but not yet there is the impact of research meat proteins zymolyte on milk-acid bacteria proliferation function.
Summary of the invention
The object of the embodiment of the present invention is to provide the experimental technique of a kind of meat proteins zymolyte to separating lactic acid bacterium proliferation function in Yoghourt, is intended to solve the existing problem that affect experimental technique of meat proteins zymolyte on milk-acid bacteria proliferation function that lack.
The embodiment of the present invention is to realize like this, the experimental technique of a kind of meat proteins zymolyte to 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 identified and from Yoghourt, is separated thermophilus streptococcus with biochemical test, lactobacillus bulgaricus, four kinds of milk-acid bacterias of Lactobacterium acidophilum and lactobacterium casei, use respectively after neutral protease and Sumizyme MP enzymolysis pork and chicken, by 2h, 4h, the zymolyte of 6h enzymolysis time is with different 1%, 2%, 3% addition adds in the MRS liquid nutrient medium of four kinds of milk-acid bacterias, after 37 ℃ of constant temperature culture 72h, pass through OD
622value relatively draws the cultivation effect of milk-acid bacteria.
Further, zymolyte all has impact to the proliferation function of thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and four kinds of milk-acid bacterias of lactobacterium casei, 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; Zymolyte is best to the cultivation effect of thermophilus streptococcus, the poorest to the cultivation effect of lactobacterium casei.
Further, thermophilus streptococcus, lactobacillus bulgaricus, four kinds of lactic acid bacteria culturers of Lactobacterium acidophilum and lactobacterium casei separate and purifying comprises the following steps:
Step 5, the colony characteristics of the bacterium colony growing on observation MRS solid medium plate, carries out respectively gramstaining and microscopy to several colonies typicals, microscopy is repeatedly plate streaking of streptococcic bacterium colony, carry out purifying, the bacterium colony that microscopy is 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:
In step 3 is 7 to pH six portions of chicken and pork sample, add respectively neutral protease 0.2g, in be 10 to pH six portions of chicken and pork sample, add respectively Sumizyme MP 0.2g;
Further, different zymolytes on the method for bacterial classification proliferation function impact are:
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) with every kind of bacterium access of the single bacterium colony of inoculating needle picking equivalent from MRS solid medium respectively, be placed in 37 ℃ of constant incubators and cultivate 72h, take stroke-physiological saline solution as blank, under 622nm wavelength, measure substratum OD value.
The experimental technique of meat proteins zymolyte provided by the invention to separating lactic acid bacterium proliferation function in Yoghourt, by separate thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and four kinds of milk-acid bacterias of lactobacterium casei from Yoghourt, use respectively after neutral protease and Sumizyme MP enzymolysis pork and chicken, pass through OD
622value relatively draws the cultivation effect of milk-acid bacteria, shows that zymolyte all has impact to the proliferation function of four kinds of milk-acid bacterias, 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; Zymolyte is best to the cultivation effect of thermophilus streptococcus, the poorest to the cultivation effect of lactobacterium casei.The present invention affects 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 can be the hydrolysis through human body endoenzyme after further investigation meat proteins eats, understanding zymolyte provides the direction of research on the impact of intestinal microflora proliferation function, thereby establish theoretical basis for the nutritive health-care food of development of meat protein zymolyte, made up the existing blank that affect experimental technique of meat proteins zymolyte on milk-acid bacteria proliferation function that lack.
Accompanying drawing explanation
Fig. 1 is the experimental technique schema of the meat proteins zymolyte that provides of the embodiment of the present invention to 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 provides of the embodiment of the present invention 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 provides of the embodiment of the present invention 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 provides of the embodiment of the present invention 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 provides of the embodiment of the present invention affects schematic diagram to lactobacterium casei proliferation function;
Figure 22 is the chicken neutral protease enzymolysis thing of the enzymolysis 6h that provides of the embodiment of the present invention schematic diagram that affects on four kinds of milk-acid bacteria proliferation functions;
Figure 23 is the chicken Sumizyme MP zymolyte of the enzymolysis 6h that provides of the embodiment of the present invention schematic diagram that affects on four kinds of milk-acid bacteria proliferation functions;
Figure 24 is the pork neutral protease enzymolysis thing of the enzymolysis 6h that provides of the embodiment of the present invention schematic diagram that affects on four kinds of milk-acid bacteria proliferation functions;
Figure 25 is the pork Sumizyme MP zymolyte of the enzymolysis 6h that provides of the embodiment of the present invention schematic diagram that affects on four kinds of milk-acid bacteria proliferation functions.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, 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, is 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: identify and separate thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and four kinds of milk-acid bacterias of lactobacterium casei from Yoghourt by differential medium cultivation, gram stain microscopy and biochemical test, use respectively neutral protease and Sumizyme MP enzymolysis pork and chicken;
S102: the zymolyte of different enzymolysis times (2h, 4h, 6h) (supernatant enzymolysis solution) is added in the MRS liquid nutrient medium of four kinds of milk-acid bacterias with different addition (1%, 2%, 3%), after 37 ℃ of constant temperature culture 72h, pass through OD
622value relatively draws the cultivation effect of milk-acid bacteria.
Result of the present invention shows, zymolyte all has impact to the proliferation function of four kinds of milk-acid bacterias, 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; Zymolyte is best to the cultivation effect of thermophilus streptococcus, 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:
By buying Yoghourt in Bechtop with aseptic pipette, extract 1mL to sterile test tube, add stroke-physiological saline solution 9mL, after fully mixing, be placed in another sterile test tube from wherein drawing 1mL bacterium liquid, add again 9mL stroke-physiological saline solution, so dilution 4 times, 4 test tubes are designated as 10 respectively in order
-1, 10
-2, 10
-3, 10
-4four gradients.From 4 test tubes, get respectively 0.2mL bacterium liquid and 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 ℃ of refrigerators, and plate is put 37 ℃ of cultivation 48h in constant incubator.
Observe the colony characteristics growing on maltose-MRS substratum plate, colonies typical is carried out being connected to 6 kinds of sugar-fermenting pipes after gramstaining and microscopy, carry out biochemical identification.
The colony characteristics of observing the bacterium colony growing on acidifying MRS substratum plate, carries out being connected to 6 kinds of sugar-fermenting pipes after gramstaining and microscopy to colonies typical, carries out biochemical identification.
Observe the colony characteristics of the bacterium colony growing on MRS solid medium plate, several colonies typicals are carried out respectively to gramstaining and microscopy, and microscopy is repeatedly plate streaking of streptococcic bacterium colony, 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 the chicken that are purchased from market are respectively got to six parts, and every part of 10g is placed in 50mL beaker, and 70 ℃ of left and right hot water, by rinsing with clear water after meat blanching 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 meat water is than being 1:2, and regulating the pH of three portions of chicken and three portions of porks with 1mol/LNaOH solution is 7, and the pH of other three portions of chicken and three portions of porks is 10.
The 3rd step, enzymolysis and the enzyme that goes out:
In be 7 to pH six portions of chicken and pork sample, add respectively neutral protease 0.2g, in be 10 to pH six portions of chicken and pork sample, add respectively Sumizyme MP 0.2g.All chicken and pork sample are all placed in 50 ℃ of thermostat water baths and are incubated enzymolysis, each two chicken zymolytes, two pork zymolytes that take out different enzyme enzymolysis enzyme 20min that goes out in 95 ℃ 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.
The 4th step, different zymolytes affect 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) with every kind of bacterium access of the single bacterium colony of inoculating needle picking equivalent from MRS solid medium respectively, be placed in 37 ℃ 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 is processed with Excel software.
3, results and analysis:
3.1 strain separating and purifying
The colonial morphology of the milk-acid bacteria separating in Yoghourt, as shown in table 1.
The colonial morphology of the milk-acid bacteria separating 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; The colony colour of lactobacterium casei and bacterium colony mode of appearance 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 little.
Microscopic morphology after the milk-acid bacteria gramstaining being 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 cells 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 separating in table 2 Yoghourt
Five kinds of bacillus biochemical tests, as shown in table 3:
Table 3 biochemical test result
Note: "+" positive reaction; "-" negative reaction; " ± " is weak positive reaction
Every kind of bacterium carbohydrate difference of utilizing of can fermenting as can be seen from Table 3, except variation bacterial classification 1, all the other four kinds of bacterium all can utilize lactose; Variation bacterial classification 1 and unfermentable maltose and the sucrose of utilizing of lactobacillus bulgaricus; 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 the impact of different zymolytes on thermophilus streptococcus proliferation function
Can be found out by Fig. 2 to Fig. 6, the kind of hydrolysis time, enzymolysis solution addition and the zymolyte of pork (chicken) is different from streptococcus thermophilus fermentation liquid OD
622value impact is different, and enzymolysis solution addition is identical, when hydrolysis time is 6h, and OD
622value is maximum; Hydrolysis time is identical, when enzymolysis solution addition is 3%, and OD
622value is 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 that adds chicken protein enzyme zymolyte
622be worth, wherein add the substratum OD of pork Sumizyme MP zymolyte
622value is maximum;
Can be found out by Fig. 7 to Figure 11, the kind of enzymolysis time, enzymolysis solution addition and the zymolyte of pork (chicken) is different from fermentation using lactobacillus bulgaricus liquid OD
622value impact is different, and enzymolysis solution addition is identical, when enzymolysis time is 6h, and OD
622value is maximum; Enzymolysis time is identical, when enzymolysis solution addition is 3%, and OD
622value is 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 between four kinds of zymolytes, the cultivation effect difference to lactobacillus bulgaricus is little, in the time that enzymolysis solution addition is 1% and 2%, adds the substratum OD of pork Sumizyme MP zymolyte
622value is maximum, and 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
622it is maximum that value reaches.
3.2.2 the impact on Lactobacterium acidophilum proliferation function with zymolyte
Can be found out by Figure 12 to Figure 16, the kind of enzymolysis time, enzymolysis solution addition and the zymolyte of pork (chicken) is different from Lactobacterium acidophilum fermented liquid OD
622value impact is different, and enzymolysis solution addition is identical, when enzymolysis time is 6h, and OD
622value is maximum; Enzymolysis time is identical, when enzymolysis solution addition is 3%, and OD
622value is 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 that adds pork protein enzyme zymolyte
622be worth, wherein add the OD of the substratum of chicken Sumizyme MP zymolyte
622value is maximum;
Can be found out by Figure 17 to Figure 21, the kind of enzymolysis time, enzymolysis solution addition and the zymolyte of pork (chicken) is different from lactobacterium casei fermented liquid OD
622value impact is different, and enzymolysis solution addition is identical, when enzymolysis time is 6h, and OD
622value is maximum; Enzymolysis time is identical, when enzymolysis solution addition is 3%, and OD
622value is 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 that adds chicken protein enzyme zymolyte
622be worth, wherein add the substratum OD of pork Sumizyme MP zymolyte
622value is maximum;
Can be found out the OD of thermophilus streptococcus by Figure 22 to Figure 25
622difference is maximum, is secondly lactobacillus bulgaricus, lactobacterium casei OD
622difference minimum.
3.2.3 the impact of zymolyte of the same race on four kinds of milk-acid bacteria proliferation functions
From commercially available yoghourt, be separated to altogether six kinds of bacterium, respectively thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum, lactobacterium casei, variation bacterial classification 1 and variation bacterial classification 2, all identify 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 sterilizing, also be likely in separation and purification and fermenting process, be subject to the impact of ambient conditions and cause the variation of bacterial classification;
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 are more, polypeptide and free aminoacid content that milk-acid bacteria can absorb are many, thereby somatomedin content increases the propagation that promotes 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, when the meat proteins enzymolysis solution of enzymolysis 6h joins in MRS liquid nutrient medium with 3% addition, best to milk-acid bacteria proliferation function; When soybean protein hydrolysis polypeptide addition is 3%, it is the highest that viable count of lactobacillus has reached, and when 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, propagation impact on bacterial classification is also different, the meat proteins enzymolysis product of Sumizyme MP is best on the propagation impact of milk-acid bacteria, shows that 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 milk-acid bacteria propagation better, and sulfur-containing amino acid is on not significant impact of the growth-promoting activity of milk-acid bacteria; Bouhal lab research shows that casein obtains zymolyte that molecular weight is less than 3kDa section and significantly improve the proliferation function of Lactococcus lactis subsp lactis CNRZ1076 through trypsin digestion;
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 can significantly promote the propagation of thermophilus streptococcus and Lactobacterium acidophilum, and the propagation of lactobacillus bulgaricus is not made significant difference; Bone protein of pig zymolyte is remarkable to the proliferation function of lactobacterium helveticus;
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 which molecular weight section the most obvious to the cultivation effect of milk-acid bacteria; (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 enzymes, such as stomach en-, and the impact of the enzymolysis product of real simulation meat in stomach on milk-acid bacteria proliferation function more; (4) difference of casein, whey-protein and meat proteins that can comparative studies the same enzyme same hydrolysis degree to milk-acid bacteria cultivation effect, wishes, by further research, finally can develop a kind of nutritive health-care food of meat proteins zymolyte,
In commercially available yoghourt, there is certain viable bacteria, therefrom can be separated to the milk-acid bacteria of the interpolation marking in allocation sheet, when the enzymolysis solution of enzymolysis 6h joins in MRS liquid nutrient medium with 3% addition, best to milk-acid bacteria cultivation effect, wherein the zymolyte after Sumizyme MP enzymolysis meat proteins is better than the zymolyte after neutral protease enzymolysis meat proteins to milk-acid bacteria cultivation effect, zymolyte is best to the cultivation effect of thermophilus streptococcus, 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 modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (5)
1. the meat proteins zymolyte experimental technique to 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 are identified the thermophilus streptococcus being separated to from Yoghourt, lactobacillus bulgaricus, four kinds of milk-acid bacterias of Lactobacterium acidophilum and lactobacterium casei, use respectively after neutral protease and Sumizyme MP enzymolysis pork and chicken, by 2h, 4h, the zymolyte of 6h enzymolysis time is with different 1%, 2%, 3% addition adds in the MRS liquid nutrient medium of four kinds of milk-acid bacterias, after 37 ℃ of constant temperature culture 72h, pass through OD
622value relatively draws the cultivation effect of milk-acid bacteria.
2. the experimental technique of meat proteins zymolyte as claimed in claim 1 to separating lactic acid bacterium proliferation function in Yoghourt, it is characterized in that, zymolyte all has impact to the proliferation function of thermophilus streptococcus, lactobacillus bulgaricus, Lactobacterium acidophilum and four kinds of milk-acid bacterias of lactobacterium casei, 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; Zymolyte is best to the cultivation effect of thermophilus streptococcus, the poorest to the cultivation effect of lactobacterium casei.
3. the experimental technique of meat proteins zymolyte as claimed in claim 1 to separating lactic acid bacterium proliferation function in Yoghourt, it is characterized in that, thermophilus streptococcus, lactobacillus bulgaricus, four kinds of lactic acid bacteria culturers of Lactobacterium acidophilum and lactobacterium casei separate and purifying comprises the following steps:
Step 1, by Yoghourt in Bechtop with aseptic pipette, extract 1mL to sterile test tube, add stroke-physiological saline solution 9mL, after fully mixing, draw 1mL bacterium liquid and be placed in another sterile test tube, then add 9mL stroke-physiological saline solution, so dilution 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 respectively 0.2mL bacterium liquid and 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 ℃ of refrigerators, plate is put in constant incubator 37 ℃ and is cultivated 48h;
Step 3, observes the colony characteristics growing on maltose-MRS substratum plate, and colonies typical is carried out being connected to 6 kinds of sugar-fermenting pipes after gramstaining and microscopy, carries out biochemical identification;
Step 4, the colony characteristics of the bacterium colony growing on observation acidifying MRS substratum plate, carries out being connected to 6 kinds of sugar-fermenting pipes after gramstaining and microscopy to colonies typical, carries out biochemical identification;
Step 5, the colony characteristics of the bacterium colony growing on observation MRS solid medium plate, carries out respectively gramstaining and microscopy to several colonies typicals, microscopy is repeatedly plate streaking of streptococcic bacterium colony, carry out purifying, the bacterium colony that microscopy is bacillus is connected to 6 kinds of sugar-fermenting pipes, carries out biochemical identification.
4. the experimental technique of meat proteins zymolyte as claimed in claim 1 to separating lactic acid bacterium proliferation function in Yoghourt, is characterized in that, the enzymolysis of pork and chicken comprises the following steps:
Step 1, respectively gets six parts by pork and chicken, and every part of 10g is placed in 50mL beaker, and 70 ℃ of hot water, by rinsing with clear water after meat blanching 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 meat water is than being 1:2, and regulating the pH of three portions of chicken and three portions of porks with 1mol/LNaOH solution is 7, and the pH of other 3 portions of chicken and 3 portions of porks is 10;
In step 3 is 7 to pH six portions of chicken and pork sample, add respectively neutral protease 0.2g, in be 10 to pH six portions of chicken and pork sample, add respectively Sumizyme MP 0.2g;
Step 4, all chicken and pork sample are all placed in 50 ℃ of thermostat water baths and are incubated enzymolysis, each two chicken zymolytes, two pork zymolytes that take out different enzyme enzymolysis enzyme 20min that goes out in 95 ℃ 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.
5. the experimental technique of meat proteins zymolyte as claimed in claim 1 to separating lactic acid bacterium proliferation function in Yoghourt, is characterized in that, different zymolytes on the method for bacterial classification proliferation function impact are:
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 with every kind of bacterium access of the single bacterium colony of inoculating needle picking equivalent from MRS solid medium respectively, be placed in 37 ℃ of constant incubators and cultivate 72h, take stroke-physiological saline solution as blank, under 622nm wavelength, measure substratum OD value.
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| KR102675379B1 (en) * | 2023-02-23 | 2024-06-14 | 대구한의대학교산학협력단 | A food composition for improving muscle strength, antioxidation or gut microbiota environment comprising chicken breast protein hydrolysate as an active ingredient |
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