CN105265994A - Method effectively improving heat stability of Nisin - Google Patents
Method effectively improving heat stability of Nisin Download PDFInfo
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- CN105265994A CN105265994A CN201510703135.9A CN201510703135A CN105265994A CN 105265994 A CN105265994 A CN 105265994A CN 201510703135 A CN201510703135 A CN 201510703135A CN 105265994 A CN105265994 A CN 105265994A
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- 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 invention relates to a method effectively improving heat stability of Nisin. 0.05-4.0% (g/100ml) of calcium salt, 0.02-0.1% (g/100ml) hydroxypropyl-beta cyclodextrin, 0.25-0.8% (g/100ml) bean oil and 0.01-0.1% (g/100ml) of polyethylene glycol as protective substance are added at the beginning, during and at the end of fermentation; Nisin emulsion is finally obtained after homogeneous emulsification; or further drying is performed to form dry powder of the Nisin emulsion; and the heat stability of Nisin is improved in a non-low acid environment. The Nisin emulsion and the dry powder of the Nisin emulsion is prepared by the method, so that the heat stability of Nisin is effectively improved; an application scope of Nisin can be extended; Nisin can be applied in non-low acid food; and the heat stability during high-temperature sterilization is improved.
Description
Technical field
The invention belongs to biological preservative production technical field, relate to the antibacterial preservative of a kind of Nisin, no matter be applied to acid food environment, still be applied in neutrality or meta-alkalescence food product environment, under same food high temperature sterilization, all effectively can improve its heat endurance of Nisin, make it still have the method for effective bacteriostatic activity.
Background technology
Nisin is as one of biological preservative comparatively green and safe in global range, and it not only has good bacteriostasis, and it can be absorbed by the protease digestion of human body and degradation, has good biocompatibility.At present, Nisin all has range of application and development trend comparatively widely in food and medicine field.But Nisin is after same food carries out the PROCESS FOR TREATMENT such as high temperature or HTHP, and the heat endurance of Nisin has part and reduces.Particularly pH be more than or equal to 6 slant acidity, neutrality or meta-alkalescence food substrate environment in, Nisin heat endurance significantly reduces.When pH is more than 4, particularly under heating condition, its decomposition rate is in aqueous accelerated, and heat endurance reduces: as when pH equals 5.0, lose 40% vigor after sterilizing; When pH equals 6.8, after sterilizing, lose 90% vigor.
This is mainly because amino acid unsaturated in Nisin amino acid sequence is as DHB2, the imperfection of DHA5 and DHA33 makes its biologically active along with ambient temperature, pH change waits and heavy losses, at low ph values, DHA double bond and hydrone be combined with each other, form corresponding amino-compound and ketone acid, do not affect its bacteriostatic activity, but along with the increase of pH value, in the basic conditions, along with affinity substance is in conjunction with unsaturated amino acid whose double bond, result in the intermolecular polymerization of Nisin, thus make the reduction of its solubility and bacteriostatic activity, if temperature raises, the loss of activity of Nisin is more serious.The microcapsule embedded technology of many research application, embeds Nisin in microencapsulation, plays the effect of anticorrisive agent slowly-releasing, resist external high temperature high pressure and enzymolysis simultaneously.But the more difficult industrialization of its method.By retrieval, find that the patent publication us relevant to present patent application mainly contains:
The patent No. is: ZL201310337300.4 " a kind of nisin composite biological preservative and preparation method thereof " its protection is selected and is: utilize shitosan, AOB, calgon, natamycin, allicin, sodium ascorbate, maltodextrin and NaCl to play synergistic protective effect to Nisin, overcome simple nisin narrow antimicrobial spectrum and the dependent limitation of low ph value.But its antibacterial synergy feature is realized by the Multiple components acting in conjunction of Compositional antiseptic agent, and not improve antimicrobial spectrum and the pH range of application of nisin self, the scope of restraining fungi of Nisin self and pH range of application do not improve.Such as utilize natamycin, AOB etc. to improve the antibacterial broad spectrum activity of its compound preservative, utilize self pH such as natamycin feature applied widely to make up the narrower feature of the Nisin scope of application.And this patent emphasis utilizes calcium salt etc. to improve Nisin self heat endurance, expand the pH range of application of Nisin self, make Nisin self have wider pH range of application, higher heat endurance can be had in non-low sour environment.
The patent No. is: " a kind of nisin microemulsion " of ZL201410016022.7; its protection is selected and is: utilize the compositions such as ready-mixed oil, triton x-100,95% ethanol, Tween 80, softex kw and pure water; after ultrasonic echography and high-pressure homogeneous process; improve the dissolubility of nisin; obtain the microemulsion with better stability; this microemulsion has the performance of good suppression bacillus subtilis, and Gram-negative bacteria can also be suppressed as Escherichia coli and staphylococcus aureus.This patent is given prominence to the key points and is improved the dissolubility of Nisin, and improves its antimicrobial spectrum.And this patent focuses on the applicability of Nisin under different pH environment of raising, be the heat endurance under non-low sour environment especially.
The patent No. is: " a kind of nano-antibacterial emulsion containing peppermint oil and preparation method thereof " of 201110407688.1, mainly utilizes the components such as peppermint oil, median chain triglyceride oil, emulsifying agent, water to be prepared from.This patent mainly overcomes the creaming phenomenon caused because peppermint oil density is low, and prepares stable nanoemulsions system by high-pressure homogeneous, increases fungistatic effect, Shelf-life.And this patent is by soya-bean oil, polyethylene glycol and high pressure homogenization effect, improve the dissolubility of Nisin and itself and the compatibility containing lipid food simultaneously.Meanwhile, more focus on the heat endurance of Nisin, improve its heat endurance in high-temperature cooking or sterilization process, to improve its practical application in food.
Summary of the invention
The object of the invention is for problem existing in above technology, it is a kind of thermally-stabilised good to provide, with low cost, the preparation method of the Nisin emulsion of the high heat of high pressure can be born, Nisin emulsion prepared by this law and dry powder antimicrobial stability good, thermally-stabilised good, can greatly meet low acidity or neutral food retains bacteriostatic agent Antibacterial Activity under high-temperature sterilization condition, thus the requirement of Shelf-life.
The technical scheme that the present invention realizes object is as follows:
A method for effective raising Nisin heat endurance, by the culture medium before Nisin fermentation, or in culture medium in sweat, or adds calcium salt in the zymotic fluid obtained after fermentation ends, and then obtains Nisin emulsion through emulsification treatment.
And described calcium salt is one or more mixture in calcium chloride, calcium carbonate, calcium lactate.
And, add in zymotic fluid last handling process and add soya-bean oil and polyethylene glycol.
A preparation method for the condensed emulsified liquid of the Nisin that heat endurance is high, step is as follows: (1) after fermentation ends, regulates zymotic fluid pH to be 2.0 ~ 2.5, afterwards zymotic fluid is warming up to 75 ~ 90 DEG C with HCl, continues 20 ~ 30min, is cooled to room temperature rapidly afterwards;
(2) the zymotic fluid (1) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, the microfiltration membranes being 0.45 μm by first filtrate aperture again carries out micro-filtration process, the ultrafiltration system utilizing molecular cut off to be 1KDa the cleaner liquid after micro-filtration carries out ultrafiltration to Nisin and removes small-molecule substance, obtains the Nisin trapped fluid after purifying;
By the Nisin trapped fluid after ultrafiltration purification through rolling NF membrane, nanofiltration desalination, concentrated, obtain Nisin concentrate;
(4) in the Nisin concentrate obtained after nanofiltration, add the calcium salt that concentration is 0.05 ~ 4.0% (g/100ml), under room temperature 25 DEG C of conditions, keep rotating speed 200 ~ 400r/min, 10 ~ 20min, after mixing, regulate concentrate pH value to 3 ~ 6 with NaOH;
(5) in the concentrate after (4) processing to step, add 0.02 ~ 0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 ~ 0.8% (g/100ml) soya-bean oil, 0.01 ~ 0.1% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 20 ~ 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved;
Described calcium salt is one or more mixture in calcium chloride, calcium carbonate, calcium lactate.
The Nisin concentrated dried powder that heat endurance is high, the condensed emulsified liquid of Nisin claim 4 obtained is dry, and preparation Nisin emulsification dry powder, the dry method adopted is spraying dry, fluidized drying or freeze drying.
The preparation method of the condensed emulsified liquid of the Nisin that a kind of heat endurance is high, step is as follows: (1) before fermentation, 0.05 ~ 4.0% (g/100ml) calcium salt is added in fermentation medium, after mixing, through high-temperature sterilization, switching 1% ~ 6% seed liquor, 30 ~ 37 DEG C of quiescent culture 8 ~ 15h;
(2) after fermentation ends, regulate zymotic fluid pH to be 2.0 ~ 2.5 with HCl, afterwards zymotic fluid is warming up to 75 ~ 90 DEG C, continue 20 ~ 30min, be cooled to room temperature rapidly afterwards;
(3) the zymotic fluid (2) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, then the microfiltration membranes being 0.45 μm by first filtrate aperture carries out micro-filtration process, obtains the cleaner liquid after micro-filtration;
(4) cleaner liquid pH to 3 ~ 6 after the micro-filtration using NaOH regulating step (3) to obtain;
(5) in the concentrate after (4) processing to step, add 0.02 ~ 0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 ~ 0.8% (g/100ml) soya-bean oil, 0.01 ~ 0.1% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 20 ~ 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved;
Described calcium salt is one or more mixture in calcium chloride, calcium carbonate, calcium lactate.
The Nisin concentrated dried powder that heat endurance is high, condensed emulsified for Nisin obtained above liquid is dry, and preparation Nisin emulsification dry powder, the dry method adopted is spraying dry, fluidized drying or freeze drying.
The preparation method of the condensed emulsified liquid of the Nisin that a kind of heat endurance is high, step is as follows: (1) 1 ~ 14h during the fermentation, in fermentation medium, add 0.05 ~ 4.0% (g/100ml) calcium salt, after mixing, continue quiescent culture to fermentation ends;
(2) the zymotic fluid (1) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, the microfiltration membranes being 0.45 μm by first filtrate aperture again carries out micro-filtration process, the ultrafiltration system utilizing molecular cut off to be 1KDa the cleaner liquid after micro-filtration carries out ultrafiltration to Nisin and removes small-molecule substance, obtains the Nisin trapped fluid after purifying;
(3) the zymotic fluid (2) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and not tolerant, obtain just filtrate, then the microfiltration membranes being 0.45 μm by first filtrate aperture carries out micro-filtration process, obtains the cleaner liquid after micro-filtration;
(4) in the concentrate after (3) processing to step, add 0.02 ~ 0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 ~ 0.8% (g/100ml) soya-bean oil, 0.01 ~ 0.1% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 20 ~ 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is improved;
Described calcium salt is one or more mixture in calcium chloride, calcium carbonate, calcium lactate.
The Nisin concentrated dried powder that heat endurance is high, the condensed emulsified liquid of Nisin claim 8 obtained is dry, and preparation Nisin emulsification dry powder, the dry method adopted is spraying dry, fluidized drying or freeze drying.
The advantage that the present invention obtains and beneficial effect are:
The present invention researches and develops a kind of range of application fully can expanding Nisin in actual applications, improves Nisin in pH heat endurance through high-temperature sterilization in 4 ~ 8 is interval, and can play the effect of long-acting bacteriostatic in food.Utilize calcium ion applying pH environment in 4 ~ 8 intervals to Nisin, play the protective effect of heat endurance, the Nisin of prepared crosslinked calcium ion can overcome the limited unfavorable factor of Nisin range of application.Meanwhile, utilize soya-bean oil and polyethylene glycol, strengthen solubility and the emulsibility of Nisin, it is made to be uniformly distributed in food, in food processing process, after its high-temperature sterilization and high-temperature bake, make Nisin while constantly discharging, himself heat endurance is less by temperatures involved.
We's ratio juris is, calcium ion combines with the aspartic acid in Nisin molecular peptide and residue glutamic acid base side chain, can revise the interaction between albumen, and the stability of conformation.Under the condition of pH3.0, albumen mainly presents protonated, can hinder the combination of protein and calcium ion, keeps the original conformation of protein.In the basic conditions, along with the raising of heat treatment temperature, calcium ion and protein bound combination can cause the change of protein conformation, thus the heat endurance of Nisin gets a promotion.
Nisin emulsion prepared by the present invention and dry powder thereof can directly add in food to be processed together, does not affect food flavor or food later stage fermentation process.Moreover, the maximum advantage of the present invention is that Nisin emulsion and dry powder thereof are in the environment of slant acidity, neutrality and meta-alkalescence, and after HTHP heat treatment, still have higher bacteriostatic activity, heat endurance is more lasting.Through 121 DEG C, after 0.1MPa sterilizing 20min, under the condition of pH4 ~ 8, Nisin emulsion improves 10 ~ 15 times compared with the bacteriostatic activity of free state Nisin, thus protects the heat endurance of Nisin; In addition, Nisin emulsion uses in fermented food processing also can not affect food microorganisms ferment effect, and procedure is simplified, practical.
Accompanying drawing explanation
Fig. 1 is the comparison diagram that Nisin that embodiment 1 is added with protectant Nisin emulsion and free state carries out tiring.
Fig. 2 a is the Nisin concentration liquid of free state, through 121 DEG C, and schematic diagram after 20min heat treatment;
Fig. 2 b be embodiment 1 in the Nisin concentrate being added with protective substance, through 121 DEG C, schematic diagram after 20min heat treatment;
Fig. 2 c is after in Fig. 2 b, right side sample is through emulsification, the view of Nisin emulsion.
Detailed description of the invention
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.
Embodiment 1:
A method for effective raising Nisin heat endurance, step is as follows:
(1) after fermentation ends, regulate zymotic fluid pH2.0 ~ 2.5 with HCl, afterwards zymotic fluid is warming up to 75 ~ 90 DEG C, continue 20 ~ 30min, be cooled to room temperature rapidly afterwards;
(2) the zymotic fluid (1) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and not tolerant, obtain just filtrate, the microfiltration membranes being 0.45 μm by first filtrate aperture again carries out micro-filtration process, the ultrafiltration system utilizing molecular cut off to be 1KDa the cleaner liquid after micro-filtration carries out ultrafiltration to Nisin and removes small-molecule substance, obtains the Nisin trapped fluid after purifying;
By the Nisin trapped fluid after ultrafiltration purification through rolling NF membrane, nanofiltration desalination, concentrated, obtain Nisin concentrate, membrane filtration pressure nanofiltration desalination concentrates pressure: 0.45Mpa;
(4) add the calcium chloride that concentration is 0.1 ~ 1.0% (g/100ml) in the Nisin concentrate obtained after nanofiltration, mixing, under room temperature 25 DEG C of conditions, keep rotating speed 200 ~ 400r/min, 10 ~ 20min, after mixing, regulates concentrate pH value to 3 ~ 6 with NaOH;
(5) in the concentrate after (4) processing to step, add 0.02 ~ 0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 ~ 0.8% (g/100ml) soya-bean oil, 0.01 ~ 0.1% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 20 ~ 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved.
Embodiment 2:
Present embodiment and detailed description of the invention 1 are unlike the addition manner of calcium chloride
Initial in fermentation, add 0.1 ~ 1.0% (g/100ml) calcium chloride, after mixing, switching 1% ~ 6% seed liquor, 30 ~ 37 DEG C of quiescent culture 8 ~ 15h.In the purification process process of calcium chloride zymotic fluid, first filter cloth or membrane filtration is adopted to remove large particulate matter, thalline and not tolerant, obtain just filtrate, then the microfiltration membranes being 0.45 μm by first filtrate aperture carries out micro-filtration process, do not carry out ultrafiltration and nanofiltration desalination operates.Other step and parameter identical with embodiment 1.Through (5) processing with embodiment 1 step, finally obtain Nisin emulsion.
Embodiment 3:
Present embodiment and detailed description of the invention 1,2 unlike: the addition manner of calcium chloride is:
3rd ~ 14h during the fermentation, adds 0.1 ~ 1.0% (g/100ml) calcium chloride in fermentation medium, after mixing, continues quiescent culture to fermentation ends.Other step and parameter identical with embodiment 2.
Embodiment 4:
Present embodiment and embodiment 1,2,3 add 0.5% ~ 4.0% calcium carbonate unlike in fermentation medium, the interpolation time can be 0h (fermentation is initially added) ~ 9h (ferment middle or middle and later periods add), shake up rapidly after interpolation, quiescent culture, treats fermentation ends.Other step and parameter and embodiment 2,3 identical.
Embodiment 5:
The interpolation of the calcium lactate that present embodiment is different from embodiment 1: add 0.05% ~ 0.12% calcium lactate powder in Nisin concentrate, mixing, under room temperature 25 DEG C of conditions, keep rotating speed 200 ~ 400r/min, 10 ~ 20min, after mixing, pH3 ~ 6 regulating zymotic fluid different respectively with NaOH, other step and parameter identical with embodiment 1.
Embodiment 6:
Present embodiment and detailed description of the invention 2,3,4 unlike, 0.05% ~ 0.08% calcium lactate is added in fermentation medium, the interpolation time can be 0h (fermentation is initially added) ~ 12h (ferment middle or middle and later periods add), shake up rapidly after interpolation, quiescent culture, treats fermentation ends.Other step and parameter and embodiment 2,3,4 identical.
Embodiment 7:
Present embodiment and embodiment 1 unlike, by Nisin emulsion, it carries out drying, other step and parameter identical with embodiment 1.
According to the guidance of above-mentioned embodiment, provide following specific embodiment.
Embodiment 1 (detailed description of the invention 1):
A method for effective raising Nisin heat endurance, step is as follows:
(1) after fermentation ends, regulate zymotic fluid pH2.0 with 10mol/LHCl, afterwards zymotic fluid is warming up to 90 DEG C, continue 30min, be cooled to room temperature rapidly afterwards;
(2) the zymotic fluid (1) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, the microfiltration membranes being 0.45 μm by first filtrate aperture again carries out micro-filtration process, the ultrafiltration system utilizing molecular cut off to be 1KDa the cleaner liquid after micro-filtration carries out ultrafiltration to Nisin and removes small-molecule substance, obtains the Nisin trapped fluid after purifying;
By the Nisin trapped fluid after ultrafiltration purification through rolling NF membrane, nanofiltration desalination, concentrated, obtain Nisin concentrate, membrane filtration pressure nanofiltration desalination concentrates pressure: 0.45Mpa;
(4) add the calcium chloride powder that concentration is 0.1% (g/100ml) in the Nisin concentrate obtained after nanofiltration, mixing, under room temperature 25 DEG C of conditions, keep rotating speed 400r/min, 20min, after mixing, regulates concentrate pH value to 3.8 ~ 4.0 with 5mol/LNaOH;
(5) in the concentrate after (4) processing to step, add 0.05% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.5% (g/100ml) soya-bean oil, 0.06% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved;
The Nisin emulsion that this formula is obtained, under pH5.8 condition, (is heated to 68 ~ 70 DEG C through pasteurization, and keep this temperature 30min, be cooled to 4 DEG C rapidly later) adopt agar diffusion method to measure it and tire, compared with free state Nisin, its bacteriostatic activity improves 2.5 ~ 5 times.
Application experiment:
Respectively Nisin emulsion and free state Nisin are applied to cheese to prepare, long 40 days of the cheese normal-temperature shelf life of the cheese ratio interpolation free state Nisin of interpolation Nisin emulsion, does not affect cheese mouthfeel simultaneously.Under the condition of 4 DEG C of refrigerations, carry out contrast test, its former shelf life is longer than the latter 90 days, does not affect cheese mouthfeel simultaneously.
Embodiment 2 (according to detailed description of the invention 2,3):
In lactalbumin fermentation medium add 1.0% (g/100ml) calcium chloride, the interpolation time be standing for fermentation to 6h time, shake up rapidly after interpolation, quiescent culture, treat fermentation ends;
(2) after fermentation ends, regulate zymotic fluid pH2.5 with 10mol/LHCl, afterwards zymotic fluid is warming up to 85 DEG C, continue 30min, be cooled to room temperature rapidly afterwards;
(3) the zymotic fluid (2) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, the microfiltration membranes being 0.45 μm by first filtrate aperture again carries out micro-filtration process, obtain Nisin trapped fluid, the pH regulating Nisin trapped fluid with 5mol/LNaOH is 4.0;
(4) in the trapped fluid after step (3) process, add 0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25% (g/100ml) soya-bean oil, 0.02% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 25min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved.
Application experiment: respectively Nisin emulsion and free Nisin equivalent are made an addition in the non-acid seafood sauce of less salt, through 121 DEG C of high-temperature sterilization 20min, measure its fungistatic effect; Its former shelf life, than long 2 months of the latter, does not affect the mouthfeel of hoisin sauce simultaneously.
Embodiment 3 (according to detailed description of the invention 4):
(1) in culture medium, add 2.5% (g/100ml) calcium carbonate, the interpolation time can be 0h, shakes up rapidly after interpolation, and quiescent culture treats fermentation ends;
(2) after fermentation ends, regulate zymotic fluid pH2.0 with 10mol/LHCl, afterwards zymotic fluid is warming up to 80 DEG C, continue 30min, be cooled to room temperature rapidly afterwards;
(3) the zymotic fluid (2) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, then the microfiltration membranes being 0.45 μm by first filtrate aperture carries out micro-filtration process, obtains Nisin trapped fluid;
(4) in the trapped fluid after step (3) process, add 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, utilize high-speed homogenization machine, low temperature stirs 20 ~ 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved.
Application experiment: make an addition in fruit and milk drink by Nisin emulsion and free Nisin equivalent respectively, through 121 DEG C of high-temperature sterilization 20min, measure its fungistatic effect, its former shelf life, than long 2 months of the latter, does not affect the mouthfeel of fruit milk drink simultaneously.
Embodiment 4 (according to detailed description of the invention five):
(1) after fermentation ends, regulate zymotic fluid pH2.0 ~ 2.15 with 10mol/LHCl, afterwards zymotic fluid is warming up to 90 DEG C, continue 30min, be cooled to room temperature rapidly afterwards;
With embodiment 1 step (2);
With embodiment 1 step (3);
(4) in Nisin concentrate, add 0.08 ~ 1% (g/100ml) calcium lactate powder, under room temperature 25 DEG C of conditions, keep rotating speed 350r/min, 20min, after mixing, the pH5.0 regulating zymotic fluid different respectively with 5mol/LNaOH;
(5) in the concentrate after (4) processing to step, add 0.08% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.35% (g/100ml) soya-bean oil, 0.06% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved.
Application experiment: Nisin emulsion and free Nisin equivalent are made an addition in children AD calcium milk respectively, through 121 DEG C of high-temperature sterilization 20min, measure its fungistatic effect, its former shelf life, than long 2.5 months of the latter, does not affect the mouthfeel of fruit milk drink simultaneously.
Embodiment 5 (according to detailed description of the invention six):
(1) in fermentation medium, add 0.08% (g/100ml) calcium lactate, when the interpolation time is standing for fermentation 7h, shake up rapidly after interpolation, quiescent culture, treat fermentation ends;
(2) after fermentation ends, regulate zymotic fluid pH2.4 ~ 2.5 with 10mol/LHCl, afterwards zymotic fluid is warming up to 90 DEG C, continue 20min, be cooled to room temperature rapidly afterwards;
(3) the zymotic fluid (2) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, then the microfiltration membranes being 0.45 μm by first filtrate aperture carries out micro-filtration process, obtains Nisin trapped fluid;
(4) in the Nisin trapped fluid after (3) processing to step, add 0.03 ~ 0.04% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.45% (g/100ml) soya-bean oil, 0.1% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 25min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved.
(5) application experiment: respectively Nisin emulsion and free Nisin equivalent are made an addition in nonacid butter, after 121 DEG C of high-temperature sterilization 20min, filling and sealing, Cord blood, and its fungistatic effect of results of regular determination, empirical tests, its former shelf life was than long 3.5 months of the latter.
Embodiment 6 (according to detailed description of the invention seven):
According to embodiment 1 step, prepare Nisin emulsion, carry out the dry powder that spraying dry obtains Nisin emulsion, the dry powder of the Nisin emulsion that the method obtains, under pH4 condition, after 121 DEG C of heating 20min, adopt agar diffusion method to measure it tire, compared with free state Nisin, its bacteriostatic activity improves 3.5 times; Under pH6 condition, improve 5 times; Under pH7 condition, improve 3.2 times; Under pH8 condition, improve 1 times.
Application experiment: respectively the dry powder of Nisin emulsion and free Nisin dry powder equivalent being made an addition to pH is in the sauce of non-acid canned tunas, and the flesh of fish is soaked wherein, soak time is 10min, through 121 DEG C of high-temperature sterilization 20min, measure its fungistatic effect, its the former shelf life is longer than the latter 30 days, does not affect can mouthfeel simultaneously.
The technical staff of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and description just illustrates principle of the present invention; do not departing under the spirit and scope of the present invention prerequisite; the present invention also has various changes and modifications, and these changes and improvements fall in the claimed scope of the invention.
Claims (9)
1. one kind is effectively improved the method for Nisin heat endurance, it is characterized in that: by the culture medium before Nisin fermentation, or in culture medium in sweat, or add calcium salt in the zymotic fluid obtained after fermentation ends, and then obtain Nisin emulsion through emulsification treatment.
2. the method for effective raising Nisin heat endurance according to claim 1, is characterized in that: described calcium salt is one or more mixture in calcium chloride, calcium carbonate, calcium lactate.
3. the method for effective raising Nisin heat endurance according to claim 1, is characterized in that: add in zymotic fluid last handling process and add soya-bean oil and polyethylene glycol.
4. the preparation method of the condensed emulsified liquid of Nisin that a heat endurance is high, it is characterized in that: step is as follows: (1) after fermentation ends, regulate zymotic fluid pH to be 2.0 ~ 2.5 with HCl, afterwards zymotic fluid is warming up to 75 ~ 90 DEG C, continue 20 ~ 30min, be cooled to room temperature rapidly afterwards;
(2) the zymotic fluid (1) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, the microfiltration membranes being 0.45 μm by first filtrate aperture again carries out micro-filtration process, the ultrafiltration system utilizing molecular cut off to be 1KDa the cleaner liquid after micro-filtration carries out ultrafiltration to Nisin and removes small-molecule substance, obtains the Nisin trapped fluid after purifying;
By the Nisin trapped fluid after ultrafiltration purification through rolling NF membrane, nanofiltration desalination, concentrated, obtain Nisin concentrate;
(4) in the Nisin concentrate obtained after nanofiltration, add the calcium salt that concentration is 0.05 ~ 4.0% (g/100ml), under room temperature 25 DEG C of conditions, keep rotating speed 200 ~ 400r/min, 10 ~ 20min, after mixing, regulate concentrate pH value to 3 ~ 6 with NaOH;
(5) in the concentrate after (4) processing to step, add 0.02 ~ 0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 ~ 0.8% (g/100ml) soya-bean oil, 0.01 ~ 0.1% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 20 ~ 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved;
Described calcium salt is one or more mixture in calcium chloride, calcium carbonate, calcium lactate.
5. the Nisin concentrated dried powder that heat endurance is high, is characterized in that: the condensed emulsified liquid of Nisin claim 4 obtained is dry, and preparation Nisin emulsification dry powder, the dry method adopted is spraying dry, fluidized drying or freeze drying.
6. the preparation method of the condensed emulsified liquid of Nisin that a heat endurance is high, it is characterized in that: step is as follows: (1) before fermentation, 0.05 ~ 4.0% (g/100ml) calcium salt is added in fermentation medium, after mixing, through high-temperature sterilization, switching 1% ~ 6% seed liquor, 30 ~ 37 DEG C of quiescent culture 8 ~ 15h;
(2) after fermentation ends, regulate zymotic fluid pH to be 2.0 ~ 2.5 with HCl, afterwards zymotic fluid is warming up to 75 ~ 90 DEG C, continue 20 ~ 30min, be cooled to room temperature rapidly afterwards;
(3) the zymotic fluid (2) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, then the microfiltration membranes being 0.45 μm by first filtrate aperture carries out micro-filtration process, obtains the cleaner liquid after micro-filtration;
(4) cleaner liquid pH to 3 ~ 6 after the micro-filtration using NaOH regulating step (3) to obtain;
(5) in the concentrate after (4) processing to step, add 0.02 ~ 0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 ~ 0.8% (g/100ml) soya-bean oil, 0.01 ~ 0.1% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 20 ~ 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is finally improved;
Described calcium salt is one or more mixture in calcium chloride, calcium carbonate, calcium lactate.
7. the Nisin concentrated dried powder that heat endurance is high, is characterized in that: the condensed emulsified liquid of Nisin claim 6 obtained is dry, and preparation Nisin emulsification dry powder, the dry method adopted is spraying dry, fluidized drying or freeze drying.
8. the preparation method of the condensed emulsified liquid of Nisin that a heat endurance is high, it is characterized in that: step is as follows: (1) 1 ~ 14h during the fermentation, in fermentation medium, add 0.05 ~ 4.0% (g/100ml) calcium salt, after mixing, continue quiescent culture to fermentation ends;
(2) the zymotic fluid (1) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and insoluble matter, obtain just filtrate, the microfiltration membranes being 0.45 μm by first filtrate aperture again carries out micro-filtration process, the ultrafiltration system utilizing molecular cut off to be 1KDa the cleaner liquid after micro-filtration carries out ultrafiltration to Nisin and removes small-molecule substance, obtains the Nisin trapped fluid after purifying;
(3) the zymotic fluid (2) step being cooled to room temperature first adopts filter cloth or membrane filtration to remove large particulate matter, thalline and not tolerant, obtain just filtrate, then the microfiltration membranes being 0.45 μm by first filtrate aperture carries out micro-filtration process, obtains the cleaner liquid after micro-filtration;
(4) in the concentrate after (3) processing to step, add 0.02 ~ 0.1% (g/100ml) hydroxy propyl-Beta cyclodextrin, 0.25 ~ 0.8% (g/100ml) soya-bean oil, 0.01 ~ 0.1% (g/100ml) polyethylene glycol, utilize high-speed homogenization machine, low temperature stirs 20 ~ 30min, recycling high pressure homogenisers carries out emulsifying, the Nisin emulsion of the heat endurance that is improved;
Described calcium salt is one or more mixture in calcium chloride, calcium carbonate, calcium lactate.
9. the Nisin concentrated dried powder that heat endurance is high, is characterized in that: the condensed emulsified liquid of Nisin claim 8 obtained is dry, and preparation Nisin emulsification dry powder, the dry method adopted is spraying dry, fluidized drying or freeze drying.
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