CN101974601A - Preparation method of R-polysaccharide fermentation liquor and preparation method of composite preservative of R-polysaccharide fermentation liquor - Google Patents
Preparation method of R-polysaccharide fermentation liquor and preparation method of composite preservative of R-polysaccharide fermentation liquor Download PDFInfo
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Abstract
The invention discloses to a preparation method of R-polysaccharide fermentation liquor and a preparation method of a composite preservative of the R-polysaccharide fermentation liquor, relating to a preparation of fermentation liquor and a preparation method of a composite preservative of the fermentation liquor, which solves the problems of complex preparation technology of the traditional R-polysaccharide preparation, and high production cost and low yield of the preparation of a preservative of the traditional R-polysaccharide preparation. The preparation method of the R-polysaccharide fermentation liquor comprises the following steps of activating bacillus subtilis, streptococcus thermophilus and brewers' yeast; collecting and washing after being centrifuged; fermenting and cultivating the thalluses after mixing; and purifying the fermentation liquor. The preparation method of the preservative is as follows: mixing nisin, natamycin, propionic acid, antioxidant of bamboo-leaf, chitosan, ethylene diamine tetraacetic acid (EDTA), cellulose, ethanol, sorbitol and oxygen-enriched active water; and mixing the mixed liquor and the R-polysaccharide fermentation liquor to be reacted. The preparation of the R-polysaccharide fermentation liquor has the advantages of simple technology, no three-waste generation, no pollution discharge and no peculiar smell. The R-polysaccharide fermentation liquor is prepared into a composite preservative, the production cost is ** yuan/ton, and the yield is high.
Description
Technical field
The present invention relates to the preparation method of a kind of preparation of fermentation liquid and compound preservative thereof.
Background technology
The R-polysaccharide is an antiseptics for natural food, its bactericidal ability is extensive, effect is fine, antifungal mechanism is to have set up enzyme biosynthesizing and active adjusting pattern, forms micropore on the microbial cell film, causes membrane permeability to increase and energy system destruction, thereby can promptly kill microorganism in the food, and a small amount of microorganism in the food suppressed, avoid its breeding, thereby reach the purpose of anti-corrosive fresh-keeping.
At present, the complicated process of preparation of R-polysaccharide needs in the production through Technologies such as a large amount of blowdown reaction treatment, and often uses the chemosynthesis raw material, easily produces peculiar smell; There is production cost height (up to 20000 yuan/ton) in the preparation of its sanitas, yields poorly, and promptly with the investment cost is quantitatively to produce to adjust, and invests 1,000 ten thousand yuan, only can build 10000 tons/year production line.
Summary of the invention
The present invention is the complicated process of preparation that has the R-polysaccharide now in order to solve, and there is the problem that production cost is high and yield poorly in the preparation of its sanitas, and a kind of preparation of R-polysaccharide fermentation liquid and the preparation method of compound preservative thereof are provided.
The preparation method of R-polysaccharide fermentation liquid carries out according to the following steps: one, respectively be inoculated in substratum by 5% inoculum size subtilis, thermophilus streptococcus and cereuisiae fermentum, in 30 ℃ of constant incubators, cultivate 48h, get the activation bacterial strain of three kinds of bacterium, three kinds of thalline are collected in centrifugal respectively back, and the phosphate buffered saline buffer washing of employing sterilization is 3 times then; Two, three kinds of thalline after will washing mix than 1: 1: 1 by thalline quantity, be inoculated in then in the fermentor tank that fermention medium is housed, be that 30 ± 1 ℃, shaking table speed are the condition bottom fermentation 48h of 120r/min at leavening temperature, fermented liquid; Three, after fermented liquid is purified, promptly finish the preparation of R-polysaccharide fermentation liquid.
The method for preparing R-polysaccharide fermentation liquid compound preservative is carried out according to the following steps: one, by volume per-cent mixes the oxygen enrichment active water of 1.5% nisin, 1.45% tennecetin, 4.5% propionic acid, 6.2% Antioxidant of bamboo leaves, 3.35% chitosan, 3.5% EDTA, 0.5% Mierocrystalline cellulose, 0.8% ethanol, 0.4% Sorbitol Powder and 77.8%, obtains mixed solution; Two, by volume per-cent mixes the R-polysaccharide fermentation liquid of 75% mixed solution and 25%, at 55 ℃ of reaction 1.5h down, promptly finishes the preparation of R-polysaccharide fermentation liquid compound preservative.
The R-polysaccharide fermentation liquid adopts subtilis, thermophilus streptococcus and cereuisiae fermentum comprehensively to cultivate the remix fermentation to make among the present invention, technology is simple, do not need through Technologies such as a large amount of blowdown reaction treatment, there is not the chemosynthesis raw material in process of production, a small amount of substratum residue is arranged during the fermentation, residual quantity is that the 0.75%(of finished product is 100 kilograms of fermented liquids, and 0.75 kilogram of residue is arranged); Do not produce the three wastes, not blowdown, free from extraneous odour in the how molten production process of biology, various production jars are closed low temperature fermentation and configuration, and hygienic condition are clean and tidy, the pollution-free source of room air, and the purge tank water reclaims, and discharging is zero.
R-polysaccharide fermentation liquid of the present invention is orange-yellow liquid, leaf of bamboo fragrant slightly, in the pH value is 3～8, can and not lose efficacy through 180 ℃/5min sterilization, water-soluble, ethanol, sulfuric acid etc. add 1% in grease, stir, dissolving does not separate fully, easily resolved into carbohydrate by the albumen lysozyme in the digestive tube after edible, so its edible safety is very high; Simultaneously aspergillus niger, Penicillium citrinum, yeast saccharomyces cerevisiae, intestinal bacteria, subtilis, streptococcus aureus etc. are tried bacterium, the fungistatic effect of R-polysaccharide generally is higher than potassium sorbate, especially the strongest to the yeast, mould, the bacteriological action that cause food spoilage, the effective concentration of its mould fungus inhibition is 0.005～0.1%, general usual amounts is 0.1～0.2%, and the effective concentration that suppresses bacterium is 0.001～0.1%.
The R-polysaccharide fermentation liquid is prepared into compound preservative among the present invention, and production cost is 15000 yuan/ton, and the sale price of market is 40000 yuan/ton, and the market profit margin is big; The output height is quantitatively to produce to adjust with the investment cost, invests 1,000 ten thousand yuan and can build 20000 tons/year production line; Practical effect is good, and it is higher 12 times than effective quality guaranteed period of Chemical Preservative Sodium Benzoate to add same dosage in food, higher 8 times than effective quality guaranteed period of potassium sorbate.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the preparation method of present embodiment R-polysaccharide fermentation liquid carries out according to the following steps: one, respectively be inoculated in substratum by 5% inoculum size subtilis, thermophilus streptococcus and cereuisiae fermentum, in 30 ℃ of constant incubators, cultivate 48h, get the activation bacterial strain of three kinds of bacterium, three kinds of thalline are collected in centrifugal respectively back, and the phosphate buffered saline buffer washing of employing sterilization is 3 times then; Two, three kinds of thalline after will washing mix than 1: 1: 1 by thalline quantity, be inoculated in then in the fermentor tank that fermention medium is housed, be that 30 ± 1 ℃, shaking table speed are the condition bottom fermentation 48h of 120r/min at leavening temperature, fermented liquid; Three, after fermented liquid is purified, promptly finish the preparation of R-polysaccharide fermentation liquid.
Present embodiment gained R-polysaccharide fermentation liquid detects:
Toxicity: No. the 06477th, Hubei Province disease control (2009) searching
1. the R-polysaccharide fermentation liquid, chmice acute per os toxicity test MTD＞20ml/kg(BW);
2. in the observation period of 14d, animal does not see tangible acute poisoning reaction, does not also have animal dead, puts to death animal when experiment finishes, and carries out gross anatomy, and each internal organs shows no obvious abnormalities;
3. belong to nontoxic level.
Quality standard: accord with Q/HFC01-2010.
The Oranoleptic indicator:The Oranoleptic indicator of product should meet the regulation of table 1.
Table 1
| Project | Index |
| Smell | Has inherent smell free from extraneous odour own |
| Color and luster | Orange-yellow |
| Proterties | Transparent liquid |
| Impurity | The visible impurity of no naked eyes, no foreign matter |
Physical and chemical index:The physical and chemical index of product should meet the regulation of table 2.
Table 2
| Project | Index |
| Vegetable polysaccharides (IU/mg)＞ | 800 |
| Protein (in Du mg/L) 〉= | 1.2 |
| Relative density | 1.020±0.001 |
The pollutent index:The pollutent index of product should meet the regulation of table 3.
Table 3
| Project | Index |
| Vitriol is (with SO 4 2-Count), %≤ | 0.04 |
| Arsenic (in As), mg/L≤ | 0.1 |
| Heavy metal (in Pb), mg/L≤ | 0.3 |
Microbiological indicator:The microbiological indicator of product should meet the regulation of table 4.
Table 4
| Project | Index |
| Total number of bacterial colony, cfu/ml≤ | 1000 |
| Coliform, MPN/100ml≤ | 30 |
| Pathogenic bacterium (referring to Salmonellas, Shigellae, streptococcus aureus, Hemolytic streptococcus) | Must not detect |
Performance:
Present embodiment gained R-polysaccharide fermentation liquid is safe and harmless to human body, in digestive tube, be degraded to the normal composition of food, do not influence the human body alimentary canal flora, do not influence the use of medicinal antibiotic, the bactericidal ability is strong, have and very strong kill and suppress yeast, mould, the effect of bacterium (inhibited equally) to fungi, test of many times shows through China National Academy of Food ﹠ Fermentation Industries, under addition and the identical situation of experiment condition, to aspergillus niger, Penicillium citrinum, yeast saccharomyces cerevisiae, intestinal bacteria, subtilis, streptococcus aureuses etc. are tried bacterium, the fungistatic effect of R-polysaccharide generally is higher than potassium sorbate, especially to causing the yeast of food spoilage, mould, bacteriological action is the strongest, the effective concentration of its mould fungus inhibition is 0.005～0.1%, general usual amounts is 0.1～0.2%, and the effective concentration that suppresses bacterium is 0.001～0.1%.
Scope of restraining fungi:
Mainly kill and suppress object and be: Rhodopseudomonas, micrococcus sp, bacillus, each genus of enterobacteriaceae, Vibrio, Halobacterium, mould, lactobacillus, flavus, intestinal bacteria, general Bacillus proteus, lactobacterium casei, Lactobacillus plantarum, aerobic spore-bearing bacilli, Gram-negative bacteria, gram-positive microorganism, Clostridium botulinum, Shigellae, Salmonellas, streptococcus aureus, Hemolytic streptococcus etc.; As seen the scope of restraining fungi of present embodiment gained R-polysaccharide fermentation liquid is wide.
Range of application:
Acid-basicity to food is stable, adapts to the food of various potential of hydrogen.Pig beef product, fish product, the sauerkraut of pickling and not pickling, jam product, greengrocery, seasoning class, candied fruit filling material class, drink class, soybean product, metric system category and starch cake class, addition must not surpass 1% in milk preparation; As seen the usage quantity of present embodiment gained R-polysaccharide fermentation liquid is little, applied range.
Embodiment two: what present embodiment and embodiment one were different is that subtilis is bought from Chinese agriculture microbial strains preservation center (ACCC) in the step 1, and culture presevation is numbered 01185, and preservation date is on February 25th, 2002.Other step and parameter are identical with embodiment one.
Embodiment three: what present embodiment was different with embodiment one or two is that thermophilus streptococcus is bought from Chinese agriculture microbial strains preservation center (ACCC) in the step 1, and culture presevation is numbered 10213, and preservation date is on June 20th, 2006.Other step and parameter are identical with embodiment one or two.
Embodiment four: what present embodiment was different with one of embodiment one to three is that cereuisiae fermentum is bought from Chinese agriculture microbial strains preservation center (ACCC) in the step 1, and culture presevation is numbered 21136, and preservation date is on June 16th, 2000.Other step and parameter are identical with one of embodiment one to three.
Embodiment five: present embodiment is different with one of embodiment one to four is substratum in the step 1, and the substratum of the activation bacterium liquid of inoculation subtilis is by the glucose of 100g, the CaCO of 20g
3, the yeast extract of 10g and 15g agar add distilled water and be settled to 1000mL and form; The potassium primary phosphate of 7 water magnesium sulfates of the sodium acetate of the glucose of the yeast extract paste of the casein peptone of the substratum 10g of inoculation thermophilus streptococcus activation bacterium liquid, the extractum carnis of 10g, 5g, the diammonium hydrogen citrate of 2g, 5g, the tween 80 of 1mL, 5g, the dipotassium hydrogen phosphate of 2g, 0.2g, the agar of 15g, 2g and the beer liquid of 5g add distilled water and be settled to 1000mL to be formed; The agar of the manganous sulfate of the dipotassium hydrogen phosphate of the peptone of the substratum 3.5g of inoculation cereuisiae fermentum activation bacterium liquid, the yeast extract of 3g and 2g, 7 water magnesium sulfates of 1g, 1g, the glucose of 20g, 15g adds distilled water and is settled to 1000mL to be formed.Other step and parameter are identical with one of embodiment one to four.
Embodiment six: present embodiment is different with one of embodiment one to five be in the step 1 centrifugal be enlarged culturing liquid with the enlarged culturing liquid of subtilis and thermophilus streptococcus respectively with the centrifugal 30min of the rotating speed of 4000r/min, the enlarged culturing liquid of cereuisiae fermentum is with the centrifugal 10min of the rotating speed of 4000r/min.Other step and parameter are identical with one of embodiment one to five.
Embodiment seven: present embodiment is different with one of embodiment one to six be in the step 2 fermention medium by the yeast extract paste of 10g, the casein of 10g, the ammonium sulfate of 10g, the dipotassium hydrogen phosphate of 2g, the sal epsom of 0.5g, the CaCO of 20g
3, the glucose of 10g and 5g extractum carnis mixed fermentation liquid add distilled water and be settled to 1000mL and form.Other step and parameter are identical with one of embodiment one to six.
Embodiment eight: present embodiment prepares the method for R-polysaccharide fermentation liquid compound preservative and carries out according to the following steps: one, by volume per-cent mixes the oxygen enrichment active water of 1.5% nisin, 1.45% tennecetin, 4.5% propionic acid, 6.2% Antioxidant of bamboo leaves, 3.35% chitosan, 3.5% EDTA, 0.5% Mierocrystalline cellulose, 0.8% ethanol, 0.4% Sorbitol Powder and 77.8%, obtains mixed solution; Two, by volume per-cent mixes the R-polysaccharide fermentation liquid of 75% mixed solution and 25%, at 55 ℃ of reaction 1.5h down, promptly finishes the preparation of R-polysaccharide fermentation liquid compound preservative.
EDTA in the present embodiment step 1 (disodium ethylene diamine tetraacetate), Mierocrystalline cellulose and ethanol are the auxiliary agents of having stipulated to allow in the food-processing use in GB2760-2007.
Sorbitol Powder uses as emulsifying agent in the present embodiment step 1, is the localized emulsifying agent of standard, stablizer in GB2760-2007.
Present embodiment prepares gained R-polysaccharide fermentation liquid compound preservative, and it is higher 12 times than effective quality guaranteed period of Chemical Preservative Sodium Benzoate to add same dosage in food, higher 8 times than effective quality guaranteed period of potassium sorbate.
Present embodiment prepares gained R-polysaccharide fermentation liquid compound preservative and detects:
Toxicity: No. the 06477th, Hubei Province disease control (2009) searching
1. R-polysaccharide fermentation liquid compound preservative, chmice acute per os toxicity test MTD＞20ml/kg(BW);
2. in the observation period of 14d, animal does not see tangible acute poisoning reaction, does not also have animal dead, puts to death animal when experiment finishes, and carries out gross anatomy, and each internal organs shows no obvious abnormalities;
3. belong to nontoxic level.
Embodiment nine: present embodiment and embodiment eight are different is that CNS number of nisin in the step 1 is 17.019; The CNS of tennecetin number is 10.007; The CNS of propionic acid number is 17.029,17.006 or 17.005; The CNS of Antioxidant of bamboo leaves number is 04.019; The CNS of chitosan number is 20.026; The CNS of Sorbitol Powder number is 19.006.Other step and parameter are identical with embodiment eight.
Claims (9)
1. the preparation method of a R-polysaccharide fermentation liquid, the preparation method who it is characterized in that the R-polysaccharide fermentation liquid carries out according to the following steps: one, respectively be inoculated in substratum by 5% inoculum size subtilis, thermophilus streptococcus and cereuisiae fermentum, in 30 ℃ of constant incubators, cultivate 48h, get the activation bacterial strain of three kinds of bacterium, three kinds of thalline are collected in centrifugal respectively back, and the phosphate buffered saline buffer washing of employing sterilization is 3 times then; Two, three kinds of thalline after will washing mix than 1: 1: 1 by thalline quantity, be inoculated in then in the fermentor tank that fermention medium is housed, be that 30 ± 1 ℃, shaking table speed are the condition bottom fermentation 48h of 120r/min at leavening temperature, fermented liquid; Three, after fermented liquid is purified, promptly finish the preparation of R-polysaccharide fermentation liquid.
2. the preparation method of a kind of R-polysaccharide fermentation liquid according to claim 1, it is characterized in that subtilis is bought from Chinese agriculture microbial strains preservation center (ACCC) in the step 1, culture presevation is numbered 01185, and preservation date is on February 25th, 2002.
3. the preparation method of a kind of R-polysaccharide fermentation liquid according to claim 1, it is characterized in that thermophilus streptococcus is bought from Chinese agriculture microbial strains preservation center (ACCC) in the step 1, culture presevation is numbered 10213, and preservation date is on June 20th, 2006.
4. the preparation method of a kind of R-polysaccharide fermentation liquid according to claim 1 is characterized in that cereuisiae fermentum is bought from Chinese agriculture microbial strains preservation center (ACCC) in the step 1, and culture presevation is numbered 21136, and preservation date is on June 16th, 2000.
5. the preparation method of a kind of R-polysaccharide fermentation liquid according to claim 1 is characterized in that the substratum in the step 1, and the substratum of the activation bacterium liquid of inoculation subtilis is by the glucose of 100g, the CaCO of 20g
3, the yeast extract of 10g and 15g agar add distilled water and be settled to 1000mL and form; The potassium primary phosphate of 7 water magnesium sulfates of the sodium acetate of the glucose of the yeast extract paste of the casein peptone of the substratum 10g of inoculation thermophilus streptococcus activation bacterium liquid, the extractum carnis of 10g, 5g, the diammonium hydrogen citrate of 2g, 5g, the tween 80 of 1mL, 5g, the dipotassium hydrogen phosphate of 2g, 0.2g, the agar of 15g, 2g and the beer liquid of 5g add distilled water and be settled to 1000mL to be formed; The agar of the manganous sulfate of the dipotassium hydrogen phosphate of the peptone of the substratum 3.5g of inoculation cereuisiae fermentum activation bacterium liquid, the yeast extract of 3g and 2g, 7 water magnesium sulfates of 1g, 1g, the glucose of 20g, 15g adds distilled water and is settled to 1000mL to be formed.
6. the preparation method of a kind of R-polysaccharide fermentation liquid according to claim 1, it is characterized in that in the step 1 centrifugal be enlarged culturing liquid with the enlarged culturing liquid of subtilis and thermophilus streptococcus respectively with the centrifugal 30min of the rotating speed of 4000r/min, the enlarged culturing liquid of cereuisiae fermentum is with the centrifugal 10min of the rotating speed of 4000r/min.
7. the preparation method of a kind of R-polysaccharide fermentation liquid according to claim 1 is characterized in that fermention medium is by the yeast extract paste of 10g, the casein of 10g, the ammonium sulfate of 10g, the dipotassium hydrogen phosphate of 2g, the sal epsom of 0.5g, the CaCO of 20g in the step 2
3, the glucose of 10g and 5g extractum carnis mixed fermentation liquid add distilled water and be settled to 1000mL and form.
8. the method for preparing the compound preservative of R-polysaccharide fermentation liquid as claimed in claim 1, it is characterized in that the method for preparing R-polysaccharide fermentation liquid compound preservative carries out according to the following steps: one, by volume per-cent mixes the oxygen enrichment active water of 1.5% nisin, 1.45% tennecetin, 4.5% propionic acid, 6.2% Antioxidant of bamboo leaves, 3.35% chitosan, 3.5% EDTA, 0.5% Mierocrystalline cellulose, 0.8% ethanol, 0.4% Sorbitol Powder and 77.8%, obtains mixed solution; Two, by volume per-cent mixes the R-polysaccharide fermentation liquid of 75% mixed solution and 25%, at 55 ℃ of reaction 1.5h down, promptly finishes the preparation of R-polysaccharide fermentation liquid compound preservative.
9. the method for preparing R-polysaccharide fermentation liquid compound preservative according to claim 8, CNS number of it is characterized in that nisin in the step 1 is 17.019; The CNS of tennecetin number is 10.007; The CNS of propionic acid number is 17.029,17.006 or 17.005; The CNS of Antioxidant of bamboo leaves number is 04.019; The CNS of chitosan number is 20.026; The CNS of Sorbitol Powder number is 19.006.
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Application publication date: 20110216 |