CN109868251A - One plant of Staphylococcus carnosus B1-2 and its application with color development effect - Google Patents
One plant of Staphylococcus carnosus B1-2 and its application with color development effect Download PDFInfo
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Abstract
The Staphylococcus carnosus B1-2 for having color development effect the present invention relates to one plant and its application, belong to food fermentation technical field.The bacterial strain B1-2, classification naming be Staphylococcus carnosus (Staphylococcus camosus), it has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, preservation date on March 11st, 2019, deposit number CGMCC No.17325.It is applied to and prepares leavening, realizes color-protecting function to substitute nitrite.Staphylococcus carnosus B1-2 provided by the invention with color development effect can replace color development effect of the nitrite in meat products, be added in salami to realize and ferment without nitrite.
Description
Technical field
The Staphylococcus carnosus B1-2 for having color development effect the present invention relates to one plant and its application belong to food fermentation technology neck
Domain.
Background technique
Meat products color embodies the freshness and superiority and inferiority quality of meat products, is the important indicator for measuring meat products quality,
It is also the important evidence for influencing consumer's selection.The meat of fresh cutting is in aubergine, but on its myoglobins the 6th on iron atom
Coordinate bond, which is easy to the generation oxymyoglobin in conjunction with oxygen, becomes pink, continues to aoxidize, and ferrous iron switchs to ferric iron, raw
At the metmyoglobin of brown.The formation of the bad color of meat products is mainly related with the accumulation of metmyoglobin.Nitrous acid
Salt can form nitrous acid in acid condition, and then disproportionated reaction occurs and is converted to nitric oxide (NO).Nitric oxide and flesh are red
Albumen reaction generates nitrous myoglobins.Therefore the addition of nitrite contributes to form characteristic butcher's meat color.But it is sub-
Nitrate decomposition product is reacted with secondary amine in meat can produce nitrosamine compound, and some of compounds have carcinogenic, teratogenesis
With cause mutagenicity.
In recent years, the concern and attention of people have been caused about the safety problem of nitrite, intake in vivo is excessive sub-
Nitrate can cause acute toxicity to be poisoned, and the ability for making red blood cell lose oxygen carrying leads to histanoxia, generate Nausea and vomiting, head
The symptoms such as dizzy, serious person can be because of respiratory failure death；In addition, the nitrosamine that nitrite and secondary amine reaction generate carcinogenicity draws
More and more concerns are played, safety issue limits its application in food.
Myoglobins red derivative is converted by the metmyoglobin of brown now concerning using microbe fermentation method
The color protecting method for reaching color development effect is gradually widely studied.Staphylococcus is used for fermentation meat product, can not only improve meat system
The characteristic of product absorption easy to digest improves flavor, and the staphylococcus of catalase-positive can decompose other microbial metabolisms
The H of middle generation2O2, inhibit fat oxidation, and the formation of color can be influenced by nitrate reductase and reduction pH, play hair
Color effect, for substitution nitrous acid, color development effect provides possibility in meat products.
Summary of the invention
The purpose of the present invention is overcoming above-mentioned shortcoming, provide one plant of Staphylococcus carnosus B1-2 with color development effect and
It is applied, and be can be applied in salami, is played the role of preferable color development.
Technical solution of the present invention, one plant of Staphylococcus carnosus B1-2 with color development effect, has been preserved in China Microbiological
Culture presevation administration committee common micro-organisms center CGMCC, address section, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China
Institute of microbiology, institute, classification naming be Staphylococcus carnosus (Staphylococcus camosus), preservation date 2019 3
The moon 11, deposit number CGMCC No.17325.
Staphylococcus carnosus B1-2 in the present invention has following biological property:
(1) morphological feature: Staphylococcus carnosus B1-2 is gram-positive bacteria, spherical or slightly oval, pairs of or single form
In the presence of atrichia cannot move.Without brood cell.
(2) colonial morphology it is rounded, it is smooth, have protrusion, illuminated light.
Staphylococcus carnosus B1-2 of the invention can play the role of preferable color development when no nitrite adds, and be real
The substitution of nitrite chromogenic effect provides possible in existing salami.
The application of the Staphylococcus carnosus B1-2 bacterial strain, is applied to and prepares leavening, to substitute nitrite realization
Color-protecting function.
Further, the leavening is applied to ferment in salami.
Further, the fermentation is the fermentation of no nitrite.
Further, the leavening preparation process is as follows: by Staphylococcus carnosus (staphylococcus carnosus)
B1-2 ferments for 24 hours in 37 DEG C of condition MRS culture solutions, and the bacteria suspension that culture finishes aseptically is centrifuged, is delivered directly
Formula liquid starter；Or the thallus for obtaining centrifugation is freeze-dried by addition cryoprotector and freeze-dried type leavening is made,
Inoculum concentration in salami is 0.01%-0.05%.
Further, the freeze-dried type leavening preparation process is as follows: the thallus of centrifugation acquisition is collected in freeze-drying plate,
Cryoprotector is added, 10-14h in -75 ~ -85 DEG C of refrigerators is placed in, with the dry 60-80h of vacuum freeze drier, passes through freezing
Dry obtained freeze-dried type leavening.
Further, centrifugal condition is 3-5 DEG C, and 7500-8500 rpm is centrifuged 8-12min.
Further, the cryoprotector adds 18%-22% skimmed milk power in terms of thallus volume, 2%-3% glucose and
1% sucrose.
Beneficial effects of the present invention: the Staphylococcus carnosus B1-2 provided by the invention with color development effect can replace nitrous
Color development effect of the hydrochlorate in meat products, is added in salami to realize and ferment without nitrite.
Biological material specimens preservation: one plant of Staphylococcus carnosus B1-2 with color development effect has been preserved in China Microbiological
Culture presevation administration committee common micro-organisms center CGMCC, address: section, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China
Institute of microbiology, institute, classification naming be Staphylococcus carnosus (Staphylococcus camosus), preservation date 2019 3
The moon 11, deposit number CGMCC No.17325.
Detailed description of the invention
Fig. 1 bacterial strain MRS(MbFe ()) culture solution 450nm-750nm within the scope of spectral scan result.
Fig. 2 bacterial strain MRS(MbFe ()) culture solution culture 20h in metmyoglobin changes of contents.
Fig. 3 bacterial strain MRS(MbFe ()) culture solution culture 20h each myoglobins derivative changes of contents.
Each bacterial strain MRS(MbFe of Fig. 4 ()) culture solution culture 20h transmission chromatic value.
Free Radical Signal ESR map in Fig. 5 strain cultured solution.
Chromatic value a-value variation in Fig. 6 Sa rummy fermentation process in 21d.
Specific embodiment
1 bacterial strain of embodiment converts metmyoglobin process trace
(1) experimental design
The MRS Liquid Culture containing metmyoglobin Staphylococcus carnosus B1-2 somatic cells being seeded in Anaerobic culturel bottle
In base (inflated with nitrogen deoxygenation adds methanol induction NOS to express), surface adds one layer of paraffin oil, and 37 DEG C of constant temperature cultures take culture 18-22h
Medium centrifugal (10000 × g, 5min, 4 DEG C), take supernatant carry out spectroscopy analysis and chromatic value measurement etc..
The content calculation formula of each myoglobins derivative: for the MRS(Met-Mb to inoculating strain) in culture solution
Each myoglobins derivative is quantified, by uv-vis spectra analyzer to several specific wavelengths of myoglobins derivative
Absorbance value (the A at place525nm, A545nm, A565nm, A572nm) be measured, the content calculation formula of each myoglobins derivative is such as
Under:
TMb/(mg/g)=-0.166A572nm+0.086A565nm+0.088A545nm+0.099A525nm；
MbO2/%=(0.882R1-1.267R2+0.809R3-0.361)×100；
Met-Mb/%=(-2.514R1+0.777R2+0.800R3+1.098)×100；
Mb/%=(0.369R1+1.140R2-0.941R3+0.015)×100；
R1、R2、R3It is absorbance ratio A respectively572nm/A525nm, A565nm/A525nm, A545nm/A525nm。
TMb is total myoglobins.
(2) the uv-vis spectra scanning of culture solution: the color of myoglobins depends primarily on haem iron atom the 6th
The bonding state of a dentate, and the difference of the 6th dentate, corresponding difference myoglobins derivative is at each wavelength
Absorbance value it is also different, therefore can by measure metmyoglobin solution characteristic absorption peak, analyze bacterial strain to high-speed rail
The conversion process of myoglobins.
By Staphylococcus carnosus B1-2 strain inoculated into the MRS fluid nutrient medium containing metmyoglobin, 37 DEG C of temperature
Under, 18h is cultivated in anaerobism bottle (inflated with nitrogen deoxygenation), is carried out uv-vis spectra after centrifugation in 450-750nm wave-length coverage and is swept
It retouches, measures the characteristic absorption peak of corresponding strain cultured solution, specific data are as shown in table 1.
Table 1
(3) the transmission chromatic value measurement of culture solution: by Staphylococcus carnosus B1-2 strain inoculated to the MRS containing metmyoglobin
18h(37 DEG C of Staphylococcus carnosus B1-2, anaerobism are cultivated in fluid nutrient medium) it is centrifuged afterwards, take supernatant to be added in quartz colorimetric utensil
(5000 rpm, 4 DEG C, 10 min), using the MRS fluid nutrient medium for containing metmyoglobin of non-inoculating strain as control,
Measure transmission the chromatic value L-value, a-value, b-value of culture solution.
(4) bacterial strain identifies metmyoglobin converted product in culture solution: Staphylococcus carnosus B1-2 strain inoculated is arrived
(5000 are centrifuged after culture 18h in MRS fluid nutrient medium (without containing manganese ion, 37 DEG C, anaerobism) containing metmyoglobin
Rpm, 4 DEG C, 10 min), take 0.2mL supernatant to be added in capillary, carry out ESR spectrum scanning.Testing conditions
It is as follows: microwave power 4mW；Frequency modulation and width 100kHz, 1mT, temperature 150k, minute 8min.
(5) result and analysis: experiment has chosen Staphylococcus carnosus B1-2, measures in the MRS training containing metmyoglobin
Support the generation of nitrosomyoglobin in base.By visually observing, inoculated Staphylococcus carnosus B1-2's contains metmyoglobin
Culture medium cerise is converted by brown within the 8-18h period, this is because bacterial strain makes the metmyoglobin of brown
(MbFe (III)) is changed into cerise (MbFe (II) O2, MbFe (II) NO), and it is high not to be inoculated with containing for Staphylococcus carnosus B1-2
The culture medium of iron myoglobins maintains original brown during strain culturing.
From fig. 1, it can be seen that the spectral scan result of Staphylococcus carnosus B1-2 group can be seen that the spy of two metmyoglobins
Levying peak reduces at significant (505nm and the place 625nm), and the absorption peak of nitrosomyoglobin increase it is more apparent (548nm with
579nm).
As seen from Figure 2 in each strain cultured solution content of the metmyoglobin in the 20h of culture variation, bacterium
In the culture solution of strain B1-2, the content of metmyoglobin reduces obvious.And from the figure 3, it may be seen that positive controls master
Wanting converted product is de-oxygenated myoglobin (MbFe (II)), and de-oxygenated myoglobin has also accounted for higher in the converted product of B1-2
Ratio, it is preliminary to infer the B1-2 myoglobins (MbFe (II) that metmyoglobin (MbFe (III)) reduction generates in culture solution
O2) Partial Conversion generates de-oxygenated myoglobin (MbFe (II)).Analyzing reason may be low oxygen under anaerobic culture environment
Pressure can promote the reduction of metmyoglobin, generate de-oxygenated myoglobin and oxymyoglobin, the NO that microbial metabolism generates
Further it is translated into nitrosomyoglobin (MbFe (II) NO).
Metmyoglobin does not convert in the experiment of non-microbe inoculation, therefore the conversion of visible metmyoglobin
Rely on the microorganism of our inoculations.From the MRS meat soup that the ESR scanning optical spectrum of Fig. 5 MRS strain cultured solution can obtain bacterial strain B1-2
Detect the signal of selenite, it was demonstrated that bacterial strain converts metmyoglobin and generates nitrosomyoglobin.ESR detection
Experimental result in g value be that can be calculated by known microwave frequency and field strength.It is detected in ESR experimental result
G value (g1=2.0182, g2=2.0081, g3=1.9988, g4=1.9872) and result of study before in map at appearance
It is closely similar, show that the substance of the signal of detection is same substance, i.e. nitrosomyoglobin.The ESR of the sample detected
Hyperfine splitting has occurred in g value (g1=2.0182, g2=2.0081, g3=1.9988, g4=1.9872) in signal, it was demonstrated that sample
Nitrosomyoglobin is in glassy state.
Application of the 2 color development bacterial strain of embodiment in salami
(1) preparation of freeze-dried type leavening:
A, cell centrifugation and drying: the bacteria suspension finished will be cultivated and be aseptically centrifuged (4 DEG C, 8000rpm, 10 min).
Thallus is collected after centrifugation in freeze-drying plate, adds cryoprotector (20% skimmed milk power, 2.5% glucose, 1% sucrose), sets
The 12h in -80 DEG C of refrigerators, with vacuum freeze drier drying time about 72h.
B, viable count measures: taking the freeze-dried powder made to be dissolved with physiological saline, using gradient dilution inverted plate method.It takes
The bacteria suspension of appropriate dilutions multiple is inoculated on MRS solid medium, is counted after 37 DEG C of culture 48h, and bacterium in freeze-dried vaccine powder is calculated
The survival rate of strain.
(2) production of salami:
Bacterial strain: Staphylococcus carnosus (staphylococcus carnosus) B1-2.Formula: with pork (pig tenterloin: pig back fat=7:
It 3) is 100% meter, glucose 0.2%, sucrose 0.3%, salt 6%, (nitrite 100mg/kg) is sodium ascorbate 0.05%, compound
Phosphate 0.3%.
Fermented Sausages fermentation process temperature and its humidity value control are as shown in table 2 below:
Table 2
(3) chromatic value changes during salami fermenting-ripening
A, different fermentations agent influences Sa rummy chromatic value: Sa rummy ferments initial stage (0-7d), while measuring the surface of Fermented Sausages
Chromatic value and section chromatic value take five points as test point, measure the chromatic value of Sa rummy, take intermediate three worth average values
As the chromatic value of the sample, the Sa rummy chromatic value difference of analysis inoculation different fermentations agent.
B, the Sa rummy chromatic value for being inoculated with different fermentations agent changes over time: during Sa rummy fermenting-ripening (0-21d),
The surface chromatic value and section chromatic value for measuring Fermented Sausages simultaneously take five points as test point, measure the chromatic value of Sa rummy,
Chromatic value of the intermediate three worth average values as the sample is taken, the Sa rummy chromatic value of analysis inoculation different fermentations agent is at any time
Changing rule.
(4) result and analysis: fermentation intestinal fermentation 0 day, 3 days, 7 days, 21 days when take respectively the fresh-cut face of Fermented Sausages into
The measurement of row chromatic value a-value, as a result as shown in Figure 6.In entirely fermentation phase nitrite group (additive amount 60ppm) and inoculation
Bacterial strain B1-2 group chromatic value is all remarkably higher than the salami (p<0.05) of spontaneous fermentation without significant difference (p>0.05).
Show that the color development action substitution in meat products provides possibility to bacterial strain B1-2 for nitrous acid.
Claims (8)
1. one plant of Staphylococcus carnosus B1-2 with color development effect, it is general to be preserved in China Committee for Culture Collection of Microorganisms
Logical microorganism center CGMCC, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, classification
Be named as Staphylococcus carnosus (Staphylococcus camosus), preservation date on March 11st, 2019, deposit number CGMCC
No.17325。
2. the application of Staphylococcus carnosus B1-2 bacterial strain described in claim 1, it is characterised in that: it is applied to and prepares leavening,
Color-protecting function is realized to substitute nitrite.
3. the application of Staphylococcus carnosus B1-2 bacterial strain as claimed in claim 2, it is characterised in that: the leavening is applied to Sa
It ferments in rummy sausage.
4. the application of Staphylococcus carnosus B1-2 bacterial strain as claimed in claim 3, it is characterised in that: the fermentation is no nitrite
Fermentation.
5. the application of Staphylococcus carnosus B1-2 bacterial strain as claimed in claim 2, it is characterised in that the leavening preparation process is such as
Under: by Staphylococcus carnosus (staphylococcus carnosus) B1-2 ferments for 24 hours in 37 DEG C of condition MRS culture solutions, will train
It supports the bacteria suspension finished to be aseptically centrifuged, obtains direct putting type liquid starter；Or the thallus that centrifugation obtains is passed through and is added
Add cryoprotector to be freeze-dried and freeze-dried type leavening is made；Its inoculum concentration in salami is 0.01%-0.05%.
6. the application of Staphylococcus carnosus B1-2 bacterial strain as claimed in claim 5, it is characterised in that the freeze-dried type leavening preparation
Process is as follows: collecting the thallus of centrifugation acquisition in freeze-drying plate, adds cryoprotector, be placed in -75 ~ -85 DEG C of refrigerators
Freeze-dried type leavening is made by being freeze-dried with the dry 60-80h of vacuum freeze drier in 10-14h.
7. the application of Staphylococcus carnosus B1-2 bacterial strain as claimed in claim 6, it is characterised in that: centrifugal condition is 3-5 DEG C,
7500-8500 rpm is centrifuged 8-12min.
8. the application of Staphylococcus carnosus B1-2 bacterial strain as claimed in claim 6, it is characterised in that: the cryoprotector is with thallus
Stereometer add 18%-22% skimmed milk power, 2%-3% glucose and 1% sucrose.
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| CN115005385A (en) * | 2022-06-14 | 2022-09-06 | 北京工商大学 | Low-salt compound curing agent for ham and preparation method and using method thereof |
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