CN105265994B - A method of effectively improving Nisin thermal stability - Google Patents
A method of effectively improving Nisin thermal stability Download PDFInfo
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- CN105265994B CN105265994B CN201510703135.9A CN201510703135A CN105265994B CN 105265994 B CN105265994 B CN 105265994B CN 201510703135 A CN201510703135 A CN 201510703135A CN 105265994 B CN105265994 B CN 105265994B
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
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Abstract
The present invention relates to a kind of methods for effectively improving Nisin thermal stability; by in initial or fermentation process of fermenting or after fermentation; add protective substance 0.05~4.0% (g/100ml) calcium salt, 0.02~0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin; 0.25~0.8% (g/100ml) soya-bean oil and 0.01~0.1% (g/100ml) polyethylene glycol; Nisin emulsion is finally obtained after emulsifying or is further dried to obtain the dry powder of Nisin emulsion, is improved its thermal stability under non-low in acidity environment.Nisin emulsion and its dry powder prepared by the present invention, not only effectively improve the thermal stability of Nisin, but also can expand the application range of Nisin, allow to be applied in non-low acidity food, thermal stability when improving through high-temperature sterilization.
Description
Technical field
The invention belongs to biological preservative production technical fields, are related to the antibacterial preservative of Nisin a kind of, no matter apply
In acid food environment, or it is applied in neutral or meta-alkalescence food product environment, in the case where same food high temperature sterilizes, can effectively mentions
Its thermal stability of high Nisin, making it still has the method for effective bacteriostatic activity.
Background technique
For Nisin as one of more green and biological preservative of safety in global range, it not only has good suppression
Bacterium effect, and it can be absorbed by the protease digestion and degradation of human body, have good bio-compatible
Property.Currently, Nisin has relatively broad application range and development trend in food and medicine field.However, Nisin is same
After food carries out the process such as high temperature or high temperature and pressure, the thermal stability of Nisin has part reduction.Especially it is greater than in pH
In the food substrate environment of slant acidity, neutrality or meta-alkalescence equal to 6, Nisin thermal stability is greatly reduced.When pH is more than 4,
Especially under heating condition, its decomposition rate in aqueous solution is accelerated, and thermal stability reduces: such as when pH is equal to 5.0, sterilizing
After lose 40% vigor；When pH is equal to 6.8,90% vigor is lost after sterilizing.
This is mainly due to amino acid such as DHB2 unsaturated in Nisin amino acid sequence, the imperfections of DHA5 and DHA33
So that its bioactivity with ambient temperature, pH change etc. and heavy losses, at low ph values, DHA double bond and hydrone are mutual
In conjunction with forming corresponding amino-compound and ketone acid, its bacteriostatic activity not influenced, but with the increase of pH value, in alkaline condition
Under, as affinity substance combines the double bond of unsaturated amino acid, the intermolecular polymerization of Nisin is resulted in, so that
The reduction of its solubility and bacteriostatic activity, if temperature increases, the loss of activity of Nisin is more serious.Micro- glue is applied in many researchs
Capsule embedding techniques, Nisin is embedded into microencapsulation, plays the effect of preservative sustained release, while resisting external high temperature high pressure
And enzymolysis.But the more difficult industrialization of its method.By retrieval, patent publication us master relevant to present patent application is found
Have:
A kind of Patent No.: " nisin composite biological preservative and its preparation side of ZL201310337300.4
Its protection point of method " is: utilizing chitosan, bamboo leaf antioxidant, calgon, natamycin, allicin, ascorbic acid
Sodium, maltodextrin and NaCl play synergistic protective effect to Nisin, overcome simple nisin narrow antimicrobial spectrum and
The limitation of low ph value dependence.But its antibacterial synergy feature is realized by the Multiple components collective effect of Compositional antiseptic agent
, and the antimicrobial spectrum and pH application range of nisin itself is not improved, the scope of restraining fungi and pH application model of Nisin itself
It encloses and is not improved.Such as the antibacterial broad spectrum activity of its compound preservative is improved using natamycin, bamboo leaf antioxidant etc., benefit
With feature itself pH such as natamycin applied widely come the characteristics of making up Nisin narrow scope of application.And this patent emphasis
Nisin itself thermal stability is improved using calcium salt etc., expands the pH application range of Nisin itself, there is Nisin itself wider
PH application range, can in non-low in acidity environment thermal stability with higher.
Patent No.: a kind of " the nisin microemulsion " of ZL201410016022.7, protection point are: utilizing
The ingredients such as ready-mixed oil, triton x-100,95% ethyl alcohol, Tween 80, cetyl trimethylammonium bromide and pure water, through ultrasound
After wave ultrasound and high-pressure homogeneous processing, the dissolubility of nisin is improved, obtains the microemulsion with better stability,
The microemulsion has the good performance for inhibiting bacillus subtilis, additionally it is possible to inhibit Gram-negative bacteria such as Escherichia coli and gold
Staphylococcus aureus.This patent, which is given prominence to the key points, improves the dissolubility of Nisin, and improves its antimicrobial spectrum.And this patent with emphasis on
It is especially the thermal stability under non-low in acidity environment in applicability of the Nisin under different pH environment of raising.
Patent No.: 201110407688.1 " a kind of nano-antibacterial emulsion and preparation method thereof containing peppermint oil ",
It is main that the components such as peppermint oil, median chain triglyceride oil, emulsifier, water is utilized to be prepared.This patent mainly overcome due to
Creaming phenomenon caused by peppermint oil density is low, and stable nanoemulsions system is prepared by high-pressure homogeneous, increase
Add fungistatic effect, Shelf-life.And this patent is acted on by soya-bean oil, polyethylene glycol and high pressure homogenization, is improved simultaneously
The dissolubility of Nisin and itself and the compatibility containing lipid food.At the same time, the thermal stability for more focusing on Nisin, mentions
Its high thermal stability in high-temperature cooking or sterilization process, to improve its practical application in food.
Summary of the invention
Aiming at the problem that the purpose of the present invention is in the presence of the above technology, it is good to provide a kind of thermostabilization, low in cost,
It can bear the preparation method of the Nisin emulsion of high pressure high fever, the Nisin emulsion and its antibacterial stabilization of dry powder of this law preparation
Property is good, thermostabilization is good, can meet low in acidity or neutral food significantly and retain bacteriostatic agent Antibacterial Activity under the conditions of high-temperature sterilization,
To the requirement of Shelf-life.
The present invention realizes that the technical solution of purpose is as follows:
A method of Nisin thermal stability is effectively improved, by the culture medium before Nisin ferments, or was fermented
Calcium salt is added in culture medium in journey, or in the fermentation liquid that obtains after fermentation, then obtains Nisin cream through emulsification treatment again
Change liquid.
Moreover, the mixture that the calcium salt is calcium chloride, one or more of in calcium carbonate, calcium lactate.
Moreover, addition soya-bean oil and polyethylene glycol are added in fermentation liquid last handling process.
A kind of preparation method of the condensed emulsified liquid of the Nisin that thermal stability is high, steps are as follows: (1) to after fermentation, use
It is 2.0~2.5 that HCl, which adjusts fermentation liquid pH, and fermentation liquid is warming up to 75~90 DEG C later, continues 20~30min, cold rapidly later
But to room temperature；
By (1) fermentation liquid that step is cooled to room temperature first use filter cloth or film be removed by filtration large particulate matter, thallus and
Insoluble matter obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, by the filtering after micro-filtration
Liquid carries out ultrafiltration to Nisin using the ultrafiltration system that molecular cut off is 1KDa and removes small-molecule substance, obtains after purification
Nisin trapped fluid；
(3) the Nisin trapped fluid after ultrafiltration purification is passed through into rolling nanofiltration membrane, nanofiltration desalination, concentration obtain Nisin concentration
Liquid；
(4) the calcium salt that addition concentration is 0.05~4.0% (g/100ml) into the Nisin concentrate obtained after nanofiltration,
Under the conditions of 25 DEG C of room temperature, 200~400r/min of revolving speed, 10~20min are kept, after mixing, adjusts concentrate pH with NaOH
It is worth to 3~6；
(5) 0.02~0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 are added into step (4) treated concentrate
~0.8% (g/100ml) soya-bean oil, 0.01~0.1% (g/100ml) polyethylene glycol, using high-speed homogenization machine, low temperature stirring 20~
30min recycles high pressure homogenisers to carry out emulsifying, finally obtains the Nisin emulsion for improving thermal stability；
The calcium salt is calcium chloride, calcium carbonate, mixture one or more of in calcium lactate.
A kind of Nisin concentrated dried powder that thermal stability is high, the condensed emulsified liquid of the Nisin that claim 4 is obtained is dry, system
Standby Nisin emulsifies dry powder, and the dry method used is spray drying, fluidized drying or freeze-drying.
A kind of preparation method of the condensed emulsified liquid of the Nisin that thermal stability is high, steps are as follows: (1) before fermentation, to fermentation
0.05~4.0% (g/100ml) calcium salt is added in culture medium, after mixing, through high-temperature sterilization, 1%~6% seed of transferring
Liquid, 30~37 DEG C of 8~15h of stationary culture；
(2) to after fermentation, be 2.0~2.5 with HCl adjusting fermentation liquid pH, fermentation liquid is warming up to 75~90 later
DEG C, continue 20~30min, is rapidly cooled to room temperature later；
By (2) fermentation liquid that step is cooled to room temperature first use filter cloth or film be removed by filtration large particulate matter, thallus and
Insoluble matter obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, the filter after obtaining micro-filtration
Clear liquid；
(4) cleaner liquid pH to 3~6 after the micro-filtration obtained with NaOH regulating step (3)；
(5) 0.02~0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 are added into step (4) treated concentrate
~0.8% (g/100ml) soya-bean oil, 0.01~0.1% (g/100ml) polyethylene glycol, using high-speed homogenization machine, low temperature stirring 20~
30min recycles high pressure homogenisers to carry out emulsifying, finally obtains the Nisin emulsion for improving thermal stability；
The calcium salt is calcium chloride, calcium carbonate, mixture one or more of in calcium lactate.
A kind of Nisin concentrated dried powder that thermal stability is high, the condensed emulsified liquid of Nisin obtained above is dry, preparation
Nisin emulsifies dry powder, and the dry method used is spray drying, fluidized drying or freeze-drying.
A kind of preparation method of the condensed emulsified liquid of the Nisin that thermal stability is high, steps are as follows: (1) 1 during the fermentation
~14h adds 0.05~4.0% (g/100ml) calcium salt into fermentation medium, after mixing, continues stationary culture to hair
Ferment terminates；
By (1) fermentation liquid that step is cooled to room temperature first use filter cloth or film be removed by filtration large particulate matter, thallus and
Insoluble matter obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, by the filtering after micro-filtration
Liquid carries out ultrafiltration to Nisin using the ultrafiltration system that molecular cut off is 1KDa and removes small-molecule substance, obtains after purification
Nisin trapped fluid；
By (2) fermentation liquid that step is cooled to room temperature first use filter cloth or film be removed by filtration large particulate matter, thallus and
Insoluble substance obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, the filter after obtaining micro-filtration
Clear liquid；
(4) 0.02~0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 are added into step (3) treated concentrate
~0.8% (g/100ml) soya-bean oil, 0.01~0.1% (g/100ml) polyethylene glycol, using high-speed homogenization machine, low temperature stirring 20~
30min recycles high pressure homogenisers to carry out emulsifying, is improved the Nisin emulsion of thermal stability；
The calcium salt is calcium chloride, calcium carbonate, mixture one or more of in calcium lactate.
A kind of Nisin concentrated dried powder that thermal stability is high, the condensed emulsified liquid of the Nisin that claim 8 is obtained is dry, system
Standby Nisin emulsifies dry powder, and the dry method used is spray drying, fluidized drying or freeze-drying.
The advantages of present invention obtains and beneficial effect are:
The present invention researches and develops a kind of application range that can sufficiently expand Nisin in practical applications, improves Nisin and exists in pH
Through the thermal stability of high-temperature sterilization in 4~8 sections, and long-acting bacteriostatic can be played the role of in food.Utilize calcium ion pair
Nisin in 4~8 sections, plays the protective effect of thermal stability in application pH environment, prepared crosslinking calcium ion
The unfavorable factor that Nisin can overcome Nisin application range limited.At the same time, soya-bean oil and polyethylene glycol, enhancing are utilized
The solubility and emulsibility of Nisin, is uniformly distributed in it in food, in food processing process, high-temperature sterilization and height
After temperature is baked and banked up with earth, make Nisin while continuous release, its own thermal stability is influenced smaller by high temperature.
The principle of this method is, calcium ion in Nisin molecular peptide aspartic acid and residue glutamic acid base side chain mutually tie
It closes, the interaction between albumen and its stability of conformation can be modified.Under conditions of pH3.0, matter is mainly presented in albumen
Son can hinder the combination of protein and calcium ion, keep the original conformation of protein.Under alkaline condition, at heat
The raising of temperature is managed, calcium ion and protein bound combination can cause the variation of protein conformation, so that the thermal stability of Nisin obtains
To promotion.
Nisin emulsion and its dry powder prepared by the present invention can be directly appended to process together in food, not influence to eat
Object flavor or food later stage fermentation process.Moreover, the best advantage is that Nisin emulsion and its dry powder are inclined
In acid, neutral and meta-alkalescence environment, after high temperature and pressure is heat-treated, still bacteriostatic activity with higher, heat are steady
It is qualitative relatively lasting.Through 121 DEG C, 0.1MPa sterilizes after 20min, and under the conditions of pH4~8, Nisin emulsion is compared with free state Nisin's
Bacteriostatic activity improves 10~15 times, to protect the thermal stability of Nisin；In addition, Nisin emulsion adds in fermented food
Using also will not influence food microorganisms ferment effect in work, so that processing program is simplified, it is practical.
Detailed description of the invention
Fig. 1 is the comparison diagram that embodiment 1 carries out potency added with the Nisin of protectant Nisin emulsion and free state.
Fig. 2 a is the Nisin concentration liquid of free state, by 121 DEG C, schematic diagram after 20min heat treatment；
Fig. 2 b is embodiment 1 in the Nisin concentrate added with protective substance, through 121 DEG C, after 20min heat treatment
Schematic diagram；
Fig. 2 c is that right side sample is the status diagram of Nisin emulsion after Over emulsfication in Fig. 2 b.
Specific embodiment
Below by specific embodiment, the invention will be further described, and it is not limit that following embodiment, which is descriptive,
Qualitatively, this does not limit the scope of protection of the present invention.
Embodiment 1:
A method of Nisin thermal stability being effectively improved, steps are as follows:
(1) to after fermentation, adjust fermentation liquid pH 2.0~2.5 with HCl, fermentation liquid is warming up to 75~90 DEG C later,
Continue 20~30min, is rapidly cooled to room temperature later；
By (1) fermentation liquid that step is cooled to room temperature first use filter cloth or film be removed by filtration large particulate matter, thallus and
Insoluble substance obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, by the filtering after micro-filtration
Liquid carries out ultrafiltration to Nisin using the ultrafiltration system that molecular cut off is 1KDa and removes small-molecule substance, obtains after purification
Nisin trapped fluid；
(3) the Nisin trapped fluid after ultrafiltration purification is passed through into rolling nanofiltration membrane, nanofiltration desalination, concentration obtain Nisin concentration
Pressure: 0.45Mpa is concentrated in liquid, film filter pressure nanofiltration desalination；
(4) the calcium chloride that addition concentration is 0.1~1.0% (g/100ml) into the Nisin concentrate obtained after nanofiltration, is mixed
It is even, under the conditions of 25 DEG C of room temperature, 200~400r/min of revolving speed, 10~20min are kept, after mixing, is adjusted and is concentrated with NaOH
Liquid pH value is to 3~6；
(5) 0.02~0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 are added into step (4) treated concentrate
~0.8% (g/100ml) soya-bean oil, 0.01~0.1% (g/100ml) polyethylene glycol, using high-speed homogenization machine, low temperature stirring 20~
30min recycles high pressure homogenisers to carry out emulsifying, finally obtains the Nisin emulsion for improving thermal stability.
Embodiment 2:
Present embodiment is unlike specific embodiment 1: the addition manner of calcium chloride
It is initial in fermentation, 0.1~1.0% (g/100ml) calcium chloride is added, after mixing, 1%~6% seed of transferring
Liquid, 30~37 DEG C of 8~15h of stationary culture.During the purification process of calcium chloride fermentation liquid, crosses and filter out using filter cloth or film first
Fall large particulate matter, thallus and insoluble substance, obtain primary filtrate, then primary filtrate is carried out with the microfiltration membranes that aperture is 0.45 μm micro-
Filter processing, operates without ultrafiltration and nanofiltration desalination.Other steps and parameter are identical as embodiment 1.Passing through same embodiment party
(5) 1 step of formula is handled, final to obtain Nisin emulsion.
Embodiment 3:
Present embodiment is unlike specific embodiment 1,2: the addition manner of calcium chloride are as follows:
3~14h during the fermentation adds 0.1~1.0% (g/100ml) calcium chloride into fermentation medium, mixes
After closing uniformly, continue stationary culture to fermentation ends.Other steps and parameter are identical as embodiment 2.
Embodiment 4:
Present embodiment adds 0.5%~4.0% carbonic acid unlike embodiment 1,2,3 into fermentation medium
Calcium, addition time can be 0h (the initial addition of fermentation)~9h (ferment middle or middle and later periods addition), shake up rapidly, stand after addition
Culture, to fermentation ends.Other steps and parameter are identical as embodiment 2,3.
Embodiment 5:
The addition of the present embodiment calcium lactate different from embodiment 1: into Nisin concentrate add 0.05%~
0.12% calcium lactate powder mixes, and under the conditions of 25 DEG C of room temperature, keeps 200~400r/min of revolving speed, 10~20min, mixing is equal
After even, different pH3~6 of fermentation liquid are adjusted respectively with NaOH, other steps and parameter are identical as embodiment 1.
Embodiment 6:
Present embodiment unlike specific embodiment 2,3,4, into fermentation medium add 0.05%~
0.08% calcium lactate, the addition time can be 0h (the initial addition of fermentation)~12h (ferment middle or middle and later periods addition), fast after addition
Speed shakes up, stationary culture, to fermentation ends.Other steps and parameter are identical as embodiment 2,3,4.
Embodiment 7:
Present embodiment is unlike embodiment 1, and by Nisin emulsion, it is dried, other steps and parameter
It is identical as embodiment 1.
According to the guidance of above embodiment, following specific embodiments are provided.
Embodiment 1 (specific embodiment 1):
A method of Nisin thermal stability being effectively improved, steps are as follows:
(1) to after fermentation, adjust fermentation liquid pH2.0 with 10mol/LHCl, fermentation liquid is warming up to 90 DEG C later, is held
Continuous 30min, is rapidly cooled to room temperature later；
By (1) fermentation liquid that step is cooled to room temperature first use filter cloth or film be removed by filtration large particulate matter, thallus and
Insoluble matter obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, by the filtering after micro-filtration
Liquid carries out ultrafiltration to Nisin using the ultrafiltration system that molecular cut off is 1KDa and removes small-molecule substance, obtains after purification
Nisin trapped fluid；
(3) the Nisin trapped fluid after ultrafiltration purification is passed through into rolling nanofiltration membrane, nanofiltration desalination, concentration obtain Nisin concentration
Pressure: 0.45Mpa is concentrated in liquid, film filter pressure nanofiltration desalination；
(4) the calcium chloride powder that addition concentration is 0.1% (g/100ml) into the Nisin concentrate obtained after nanofiltration, is mixed
It is even, under the conditions of 25 DEG C of room temperature, revolving speed 400r/min, 20min are kept, after mixing, adjusts concentrate with 5mol/LNaOH
PH value is to 3.8~4.0；
(5) 0.05% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.5% (g/ are added into step (4) treated concentrate
100ml) soya-bean oil, 0.06% (g/100ml) polyethylene glycol, using high-speed homogenization machine, low temperature stirs 30min, recycles high pressure equal
Matter device carries out emulsifying, finally obtains the Nisin emulsion for improving thermal stability；
This is formulated Nisin emulsion obtained and (is heated to 68~70 DEG C through pasteurization under the conditions of pH5.8, and protects
This temperature 30min is held, later rapid cooling is to 4 DEG C) its potency is measured using agar diffusion method, compared with free state Nisin, suppression
Bacterium activity improves 2.5~5 times.
Application experiment:
Nisin emulsion and free state Nisin are prepared applied to cheese respectively, add the cheese ratio of Nisin emulsion
The cheese normal-temperature shelf life for adding free state Nisin is 40 days long, while not influencing cheese mouthfeel.Under conditions of 4 DEG C of refrigeration into
Row comparative test, the former shelf life is 90 days longer than the latter, while not influencing cheese mouthfeel.
Embodiment 2 (according to specific embodiment 2,3):
(1) 1.0% (g/100ml) calcium chloride is added into lactalbumin fermentation medium, and the addition time is to be left to ferment extremely
It when 6h, is shaken up rapidly after addition, stationary culture, to fermentation ends；
(2) to after fermentation, adjust fermentation liquid pH 2.5 with 10mol/L HCl, fermentation liquid is warming up to 85 DEG C later,
Continue 30min, is rapidly cooled to room temperature later；
By (2) fermentation liquid that step is cooled to room temperature first use filter cloth or film be removed by filtration large particulate matter, thallus and
Insoluble matter obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, obtains Nisin retention
Liquid is 4.0 with the pH that 5mol/LNaOH adjusts Nisin trapped fluid；
(4) 0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25% (g/ are added into step (3) treated trapped fluid
100ml) soya-bean oil, 0.02% (g/100ml) polyethylene glycol, using high-speed homogenization machine, low temperature stirs 25min, recycles high pressure equal
Matter device carries out emulsifying, finally obtains the Nisin emulsion for improving thermal stability.
Application experiment: Nisin emulsion and free Nisin equivalent are made an addition into the non-acid seafood sauce of less salt respectively
In, by 121 DEG C of high-temperature sterilization 20min, measure its fungistatic effect；Its former shelf life is 2 months longer than the latter, while not influencing
The mouthfeel of hoisin sauce.
Embodiment 3 (according to specific embodiment 4):
(1) 2.5% (g/100ml) calcium carbonate is added into culture medium, and the addition time can be 0h, shake up rapidly after addition, quiet
Culture is set, to fermentation ends；
(2) to after fermentation, adjust fermentation liquid pH 2.0 with 10mol/L HCl, fermentation liquid is warming up to 80 DEG C later,
Continue 30min, is rapidly cooled to room temperature later；
By (2) fermentation liquid that step is cooled to room temperature first use filter cloth or film be removed by filtration large particulate matter, thallus and
Insoluble matter obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, obtains Nisin retention
Liquid；
(4) added into step (3) treated trapped fluid 0.04% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25~
0.8% (g/100ml) soya-bean oil, 0.01~0.1% (g/100ml) polyethylene glycol, using high-speed homogenization machine, low temperature stirring 20~
30min recycles high pressure homogenisers to carry out emulsifying, finally obtains the Nisin emulsion for improving thermal stability.
Application experiment: respectively making an addition to Nisin emulsion and free Nisin equivalent in fruit and milk drink, by 121 DEG C of height
Temperature sterilizing 20min, measures its fungistatic effect, the former shelf life is 2 months longer than the latter, while not influencing the mouthfeel of fruit milk drink.
Embodiment 4 (according to specific embodiment five):
(1) to after fermentation, adjust fermentation liquid pH 2.0~2.15 with 10mol/L HCl, be later warming up to fermentation liquid
90 DEG C, continues 30min, be rapidly cooled to room temperature later；
With the step of embodiment 1 (2)；
With the step of embodiment 1 (3)；
(4) 0.08~1% (g/100ml) calcium lactate powder is added into Nisin concentrate, under the conditions of 25 DEG C of room temperature, is protected
Revolving speed 350r/min, 20min are held, after mixing, adjusts the different pH5.0 of fermentation liquid respectively with 5mol/LNaOH；
(5) 0.08% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.35% (g/ are added into step (4) treated concentrate
100ml) soya-bean oil, 0.06% (g/100ml) polyethylene glycol, using high-speed homogenization machine, low temperature stirs 30min, recycles high pressure equal
Matter device carries out emulsifying, finally obtains the Nisin emulsion for improving thermal stability.
Application experiment: Nisin emulsion and free Nisin equivalent are made an addition in children's AD calcium milk respectively, by 121 DEG C
High-temperature sterilization 20min measures its fungistatic effect, the former shelf life is 2.5 months longer than the latter, while not influencing the mouth of fruit milk drink
Sense.
Embodiment 5 (according to specific embodiment six):
(1) 0.08% (g/100ml) calcium lactate is added into fermentation medium, when the addition time is standing for fermentation 7h, addition
It shakes up rapidly afterwards, stationary culture, to fermentation ends；
(2) to after fermentation, adjust fermentation liquid pH 2.4~2.5 with 10mol/L HCl, be later warming up to fermentation liquid
90 DEG C, continues 20min, be rapidly cooled to room temperature later；
By (2) fermentation liquid that step is cooled to room temperature first use filter cloth or film be removed by filtration large particulate matter, thallus and
Insoluble matter obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, obtains Nisin retention
Liquid；
(4) 0.03~0.04% (g/100ml) hydroxy propyl-Beta ring of addition paste into step (3) treated Nisin trapped fluid
Essence, 0.45% (g/100ml) soya-bean oil, 0.1% (g/100ml) polyethylene glycol, using high-speed homogenization machine, low temperature stirs 25min, then
Emulsifying is carried out using high pressure homogenisers, finally obtains the Nisin emulsion for improving thermal stability.
(5) application experiment: Nisin emulsion and free Nisin equivalent are made an addition in nonacid butter respectively, by 121
After DEG C high-temperature sterilization 20min, filling and sealing, cryo-conservation, and results of regular determination its fungistatic effect is verified, the former shelf life
It is 3.5 months longer than the latter.
Embodiment 6 (according to specific embodiment seven):
According to 1 step of embodiment, Nisin emulsion is prepared, is spray-dried to obtain the dry of Nisin emulsion
Powder, the dry powder of Nisin emulsion made from the method use agar diffusion method under the conditions of pH4 after 121 DEG C of heating 20min
Its potency is measured, compared with free state Nisin, bacteriostatic activity improves 3.5 times；Under the conditions of pH6,5 times are improved；Under the conditions of pH7, mention
It is 3.2 times high；Under the conditions of pH8,1 times is improved.
Application experiment: it is non-acid that the dry powder of Nisin emulsion and free Nisin dry powder equivalent, which are made an addition to pH, respectively
In the sauce of canned tunas, and the flesh of fish is impregnated wherein, soaking time 10min is surveyed by 121 DEG C of high-temperature sterilization 20min
Its fixed fungistatic effect, the former shelf life is 30 days longer than the latter, while not influencing can mouthfeel.
It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, above-described embodiment and explanation
It is merely illustrated the principles of the invention described in book, under the premise of not departing from the spirit and scope of the present invention, the present invention is also had
Various changes and modifications, these changes and improvements are fallen into scope of the claimed invention.
Claims (4)
1. a kind of preparation method of the condensed emulsified liquid of the Nisin that thermal stability is high, it is characterised in that: steps are as follows: (1) wait ferment
After, adjusting fermentation liquid pH with HCl is 2.0~2.5, fermentation liquid is warming up to 75~90 DEG C later, continues 20~30min,
It is rapidly cooled to room temperature later；
(2) first use filter cloth or film to be removed by filtration large particulate matter, thallus and insoluble (1) fermentation liquid that step is cooled to room temperature
Object obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, by the cleaner liquid benefit after micro-filtration
The ultrafiltration system for being 1KDa with molecular cut off carries out ultrafiltration removal small-molecule substance to Nisin, and the Nisin obtained after purification is cut
Stay liquid；
(3) the Nisin trapped fluid after ultrafiltration purification is passed through into rolling nanofiltration membrane, nanofiltration desalination, concentration obtain Nisin concentrate；
Into the Nisin concentrate obtained after nanofiltration addition 0.05~4.0% calcium salt, described 0.05~4.0% with g/
100ml meter keeps 200~400r/min of revolving speed, 10~20min, after mixing, with NaOH tune under the conditions of 25 DEG C of room temperature
Concentrate pH value is saved to 3~6；
(5) added into step (4) treated concentrate 0.02~0.1% hydroxy propyl-Beta cyclodextrin, 0.25~0.8% soya-bean oil,
0.01~0.1% polyethylene glycol, using high-speed homogenization machine, low temperature stirs 20~30min, and high pressure homogenisers is recycled to carry out homogeneous
Emulsification finally obtains the Nisin emulsion for improving thermal stability；Described 0.02~0.1%, 0.25~0.8% and 0.01~
0.1% in terms of g/100ml；
The calcium salt is calcium chloride, calcium carbonate, mixture one or more of in calcium lactate.
2. a kind of Nisin concentrated dried powder that thermal stability is high, it is characterised in that: the Nisin for obtaining claim 1 is condensed emulsified
Liquid is dry, and preparation Nisin emulsifies dry powder, and the dry method used is spray drying, fluidized drying or freeze-drying.
3. a kind of preparation method of the condensed emulsified liquid of the Nisin that thermal stability is high, it is characterised in that: steps are as follows: (1) fermenting
Before, 0.05~4.0% calcium salt is added into fermentation medium, after mixing, through high-temperature sterilization, 1%~6% seed of transferring
Liquid, 30~37 DEG C of 8~15h of stationary culture；Described 0.05~4.0% in terms of g/100ml；
(2) to after fermentation, be 2.0~2.5 with HCl adjusting fermentation liquid pH, fermentation liquid is warming up to 75~90 DEG C later, is held
Continuous 20~30min, is rapidly cooled to room temperature later；
(3) first use filter cloth or film to be removed by filtration large particulate matter, thallus and insoluble (2) fermentation liquid that step is cooled to room temperature
Object obtains primary filtrate, then primary filtrate is carried out micro-filtration processing with the microfiltration membranes that aperture is 0.45 μm, the filtering after obtaining micro-filtration
Liquid；
(4) cleaner liquid pH to 3~6 after the micro-filtration obtained with NaOH regulating step (3)；
(5) added into step (4) treated concentrate 0.02~0.1% hydroxy propyl-Beta cyclodextrin, 0.25~0.8% soya-bean oil,
0.01~0.1% polyethylene glycol, using high-speed homogenization machine, low temperature stirs 20~30min, and high pressure homogenisers is recycled to carry out homogeneous
Emulsification finally obtains the Nisin emulsion for improving thermal stability；Described 0.02~0.1%, 0.25~0.8% and 0.01~
0.1% in terms of g/100ml；
The calcium salt is calcium chloride, calcium carbonate, mixture one or more of in calcium lactate.
4. a kind of Nisin concentrated dried powder that thermal stability is high, it is characterised in that: the Nisin for obtaining claim 3 is condensed emulsified
Liquid is dry, and preparation Nisin emulsifies dry powder, and the dry method used is spray drying, fluidized drying or freeze-drying.
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