CN104988099A - Method for producing lactic acid bacteria agent and bacteriocin by using salted vegetable wastewater - Google Patents
Method for producing lactic acid bacteria agent and bacteriocin by using salted vegetable wastewater Download PDFInfo
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Abstract
The present invention discloses a method for producing lactic acid bacteria agent and bacteriocin by using salted vegetable wastewater. The method comprises the production steps of pretreatment, centrifugal separation, blending fermentation, preparation of lactic acid bacteria agent and recovery of bacteriocin. The invention has simple operation, low production costs, environmental protection, low energy consumption, high added value, and important economic value and environmental value. The invention has the beneficial effects that: yield of lactobacillus cell is high, and the obtained lactic acid bacteria has significant inhibition effect on Escherichia coli, Salmonella enterica, Staphylococcus aureus and Listeria monocytogenes bacteria, and shows more significant inhibitory effect in an acidic environment.
Description
Technical field
The present invention relates to the preparation method of agent of lactic acid bacteria and bacteriocin, particularly relate to a kind of method utilizing salted vegetable waste water to produce agent of lactic acid bacteria and bacteriocin.
Background technology
Pickles Gu Cheng Potamogeton, refers to the vegetables by fermentation in order to long storage time.In general, as long as the vegetables of fibre-enrich or fruit, pickles can be made, such as Caulis et Folium Brassicae capitatae, Chinese cabbage, Radix Dauci Sativae, white turnip, garlic, verdant, gherkin, onion, cabbage etc.Vegetables are after pickled and seasoning, and have kind of a special local flavor, a lot of people can be used as a kind of common garnishes and eat.The main production process of pickles is as follows: by the fresh vegetables (radish of collecting, catsup and pickled vegetables, asparagus lettuce etc.) first carry out cleaning (meeting generating portion waste water), clean up rear feeding salt marsh pond dipping (meeting generating portion waste water, i.e. salt accumulated water), beet slicer is sent into after about dipping 10 ~ 20h, vegetables are cut into block or thread according to different requirements, then evaporation plant dehydration (meeting generating portion waste water is sent into, i.e. de-salted water), various vegetables after dehydration stir and add various condiment, be distributed into bag, then vacuum-pumping and sealing, cleaning outer packaging (meeting generating portion brine waste), can to case warehouse-in.
Use the salt solution of lower concentration, or carry out pickled various fresh and tender vegetables with a small amount of salt, then through lactobacillus-fermented, make a kind of acidulated curing food and pickles, as long as lactic acid content reaches certain concentration, and make product completely cut off air, just can reach the object of storage for a long time.Often process 1 ton of catsup and pickled vegetables product and can produce 1.5 ~ 2 tons of salted vegetable waste water, according to China's catsup and pickled vegetables 5,000,000 tons/year calculating, China's salted vegetable wastewater discharge reaches 1,000 ten thousand tons/year.Containing a large amount of compositions such as salt, organic acid, carbohydrate, protein, amino acid, pectin, bacteriocin in salted vegetable waste water.Due to high pollution (COD, BOD, SS) and the supersalinity of pickle wastewater, treatment process is can not adopt biological degradation, the materializing strategy technique that flocculation sediment etc. is general can not be adopted.Current enterprise in most cases directly by first for salted vegetable waste water thin up, then discharges after Sewage treatment systems process.This treatment process not only causes the waste of resource, and dilution process method also increases the cost of enterprise's sewage disposal greatly.The more important thing is, the way of the possibility Interference Detection data such as dilution discharge clearly forbidden by Environmental Protection in China method relevant provision.Therefore, the recycling realizing salted vegetable waste water is extremely urgent.
Application number is that the Chinese patent application of CN200810046482.9 discloses a kind of salting pickled vegetable brine recovery technique, and this technology, by brine concentration post crystallization, reclaims salt and plant amino acid.But energy consumption is higher, added value of product is not high problem that the method exists.In addition, in pickled water, many other constituents are underutilized as functional components such as carbohydrate, protein, pectin, bacteriocins, and the discharge of this constituents enters Sewage treatment systems, increase wastewater treatment difficulty, be also the waste of high added value resource simultaneously.
Application number is the preparation method that the Chinese patent application of CN201010279240.1 discloses a kind of salted vegetable concentrated solution, salted vegetable waste water is prepared concentrated solution after concentrated, again concentrated solution is used in fermented product, effectively make use of the nutritive ingredient in salted vegetable waste water.But the method still exists the not high defect of added value of product, and the salted vegetable concentrated solution material composition of recycling is complicated, and quality is unstable, and its subsequent applications has great limitation.
Summary of the invention
The object of the invention is to the shortcoming overcoming prior art, a kind of method utilizing salted vegetable waste water to produce agent of lactic acid bacteria and bacteriocin is provided, the method can realize the comprehensive utilization of vegetables salting zymolysis waste water, can produce the bacteriocin of high added value, agent of lactic acid bacteria product.
Object of the present invention is achieved through the following technical solutions: a kind of method utilizing salted vegetable waste water to produce agent of lactic acid bacteria and bacteriocin, and it comprises the following steps:
S1. by the salted vegetable wastewater collection of fermenting-ripening in adjustment settling tank in, precipitation 24 ~ 48h, then the filtering net at the bottom of described adjustment settling tank carries out coarse filtration, described sieve meshes is 20 ~ 40 orders, salting zymolysis cell suspending liquid is obtained after active carbon deodorizing, the salinity of described salted vegetable waste water is 1 ~ 9%, with the reducing sugar of glucose meter for 0.01 ~ 10%, in the centrifugal 9 ~ 11min rear solution of 4000rpm with the total nitrogen content of proteinometer for 0.01 ~ 10%, non-salt soluble solid content is 0.01 ~ 10%, organoleptic requirements is without rancid, free from extraneous odour, without black change, with without mould colored epistasis,
S2. described fermented cells suspension is placed in the centrifugal 1 ~ 5min of whizzer, its rotating speed is 4000 ~ 8000rpm, obtains clear liquor;
S3. glucose, yeast extract and calcium carbonate are joined in the clear liquor that step S2 obtains and allocate, gained mixed liquid is sterilization 10 ~ 30min at 100 ~ 150 DEG C, described yeast extract comprises yeast powder and yeast extract paste, described mixed liquid with the reducing sugar massfraction of glucose meter for 1.0 ~ 3.0%, with the total nitrogen content of proteinometer for 0.5 ~ 3.0%;
S4. by the mixed liquid after step S3 sterilization according to 1 ~ 5% inoculum size inoculation plant lactobacillus ferment, calcium carbonate or white lime is adopted to regulate fermented liquid pH to 5.0 ~ 7.0 in fermenting process, control temperature is 25 ~ 35 DEG C, as Lactic Acid from Fermentation Broth mycetocyte concentration OD
600value keeps balance 2 ~ 4d when reaching 1.0 ~ 2.0;
S5. the fermented liquid after being balanced by step S4 is placed in the centrifugal 1 ~ 5min of whizzer, and its rotating speed is 20000 ~ 40000rpm, and collecting precipitation obtains lactic-acid bacteria cells, and the supernatant liquor of separation is for subsequent use;
S6. in described lactic-acid bacteria cells, add the trehalose of 5 ~ 10%, mix rear freeze-drying, obtain agent of lactic acid bacteria;
S7. step S5 is separated the supernatant liquor that obtains adopt mass concentration be 40 ~ 60% saturated ammonium sulphate solution precipitate, by throw out through ultrafiltration membrance filter, collect filtrate and carry out lyophilize and obtain bacteriocin lab.
The present invention has following beneficial effect:
(1) heterogeneity that the present invention is directed in salted vegetable waste water produces agent of lactic acid bacteria and bacteriocin, adopt microbial technique to carry out transforming and realize recycling, decrease quantity discharged and the intractability of salted vegetable waste water, in environment protection, there is important value;
(2) present invention achieves the comprehensive utilization of vegetables salting zymolysis waste water, produce the bacteriocin of high added value, agent of lactic acid bacteria product, achieve less energy-consumption and high added value, there is important economic worth;
(3) bacteriocin lab that the present invention obtains has significant restraining effect to colon bacillus, intestines Salmonellas, streptococcus aureus, single listeria spp that increases, and has a extensive future;
(4), under sour environment, bacteriocin fungistatic effect of the present invention can be further strengthened;
(5) the present invention is simple, convenient, and cost is low, is applicable to industrialization scale operation.
Accompanying drawing explanation
Fig. 1 is that bacteriocin prepared by the present invention contrasts figure with Nisin to streptococcus aureus fungistatic effect;
Fig. 2 is process flow sheet of the present invention;
In Fig. 1, A is bacteriocin (100mg/ml) prepared by the present invention, and B is Nisin (100mg/ml), r is Oxford cup size (8mm), and R is antibacterial circle diameter.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention will be further described, and as shown in Figure 2, but protection scope of the present invention is not limited to Fig. 2 and the following stated to integral production technique of the present invention.
Embodiment 1: a kind of method utilizing salted vegetable waste water to produce agent of lactic acid bacteria and bacteriocin, it comprises the following steps:
S1. by the salted vegetable wastewater collection of fermenting-ripening in adjustment settling tank in, precipitation 24h, then through regulating filtering net at the bottom of settling tank to carry out coarse filtration, described sieve meshes is 20 orders, remove the dish leaf in salted vegetable waste water stoste, dish slag, the insoluble impuritiess such as sandstone, salting zymolysis cell suspending liquid is obtained after active carbon deodorizing, the salinity of described salted vegetable waste water is 1%, with the reducing sugar of glucose meter for 0.01%, in the centrifugal 9min rear solution of 4000rpm with the total nitrogen content of proteinometer for 3%, non-salt soluble solid content is 0.01%, organoleptic requirements is without rancid, free from extraneous odour, without black change, with without mould colored epistasis,
S2. salting zymolysis cell suspending liquid is adopted the centrifuge 2min of 4000rpm centrifugal force, remove insoluble impurities, obtain clear liquor;
S3. glucose, yeast extract and calcium carbonate are joined in the clear liquor that step S2 obtains and allocate, gained mixed liquid is sterilization 10min at 100 DEG C, reducing sugar 1.0% (with glucose meter) and total nitrogen content (with proteinometer) 2.2% in described mixed liquid;
S4. by the mixed liquid after step S3 sterilization according to 1% inoculum size inoculation plant lactobacillus ferment, adopt calcium carbonate or white lime to regulate fermented liquid pH to 6.0 in fermenting process, control temperature is 25 DEG C, as Lactic Acid from Fermentation Broth mycetocyte concentration OD
600value keeps balance 2d when reaching 1.0, and whole fermenting process terminates, and lactic-acid bacteria cells yield is 1.21%;
S5. the fermented liquid obtained by step S4 adopts the centrifuge 2min of 20000rpm centrifugal force, and be separated and obtain lactic-acid bacteria cells, the supernatant liquor of separation is for subsequent use;
S6. add the trehalose of 5% in the lactic-acid bacteria cells obtained to step S5, mix rear freeze-drying, obtain agent of lactic acid bacteria;
S7. by be separated in step S5 the supernatant liquor that obtains adopt mass concentration be 40% saturated ammonium sulphate solution precipitate, by throw out through ultrafiltration membrance filter process, collect filtrate and carry out lyophilize and obtain bacteriocin lab.
Embodiment 2: a kind of method utilizing salted vegetable waste water to produce agent of lactic acid bacteria and bacteriocin, it comprises the following steps:
S1. by the salted vegetable wastewater collection of fermenting-ripening in adjustment settling tank in, precipitation 30h, then through regulating filtering net at the bottom of settling tank to carry out coarse filtration, described sieve meshes is 25 orders, remove the dish leaf in salted vegetable waste water stoste, dish slag, the insoluble impuritiess such as sandstone, salting zymolysis cell suspending liquid is obtained after active carbon deodorizing, the salinity of described salted vegetable waste water is 3%, with the reducing sugar of glucose meter for 3%, in the centrifugal 10min rear solution of 4000rpm with the total nitrogen content of proteinometer for 0.01%, non-salt soluble solid content is 3%, organoleptic requirements is without rancid, free from extraneous odour, without black change, with without mould colored epistasis,
S2. salting zymolysis cell suspending liquid is adopted the centrifuge 1min of 5000rpm centrifugal force, remove insoluble impurities, obtain clear liquor;
S3. glucose, yeast extract and calcium carbonate are joined in the clear liquor that step S2 obtains and allocate, gained mixed liquid is sterilization 15min at 110 DEG C, reducing sugar 2.2% (with glucose meter) and total nitrogen content (with proteinometer) 1.1% in described mixed liquid;
S4. by the mixed liquid after step S3 sterilization according to 2% inoculum size inoculation plant lactobacillus ferment, adopt calcium carbonate or white lime to regulate fermented liquid pH to 7.0 in fermenting process, control temperature is 30 DEG C, as Lactic Acid from Fermentation Broth mycetocyte concentration OD
600value reaches 1.25 and keeps balance 2.5d, and whole fermenting process terminates, and lactic-acid bacteria cells yield is 1.37%;
S5. the fermented liquid obtained by step S4 adopts the centrifuge 3min of 25000rpm centrifugal force, and be separated and obtain lactic-acid bacteria cells, the supernatant liquor of separation is for subsequent use;
S6. add the trehalose of 7.5% in the lactic-acid bacteria cells obtained to step S5, mix rear freeze-drying, obtain agent of lactic acid bacteria;
S7. by be separated in step S5 the supernatant liquor that obtains adopt mass concentration be 60% saturated ammonium sulphate solution precipitate, by throw out through ultrafiltration membrance filter process, collect filtrate and carry out lyophilize and obtain bacteriocin lab.
Embodiment 3: a kind of method utilizing salted vegetable waste water to produce agent of lactic acid bacteria and bacteriocin, it comprises the following steps:
S1. by the salted vegetable wastewater collection of fermenting-ripening in adjustment settling tank in, precipitation 36h, then through regulating filtering net at the bottom of settling tank to carry out coarse filtration, described sieve meshes is 30 orders, remove the dish leaf in salted vegetable waste water stoste, dish slag, the insoluble impuritiess such as sandstone, salting zymolysis cell suspending liquid is obtained after active carbon deodorizing, the salinity of described salted vegetable waste water is 5%, with the reducing sugar of glucose meter for 5%, in the centrifugal 11min rear solution of 4000rpm with the total nitrogen content of proteinometer for 7%, non-salt soluble solid content is 5%, organoleptic requirements is without rancid, free from extraneous odour, without black change, with without mould colored epistasis,
S2. salting zymolysis cell suspending liquid is adopted the centrifuge 3min of 6000rpm centrifugal force, remove insoluble impurities, obtain clear liquor;
S3. glucose, yeast extract and calcium carbonate are joined in the clear liquor that step S2 obtains and allocate, gained mixed liquid is sterilization 20min at 125 DEG C, reducing sugar 1.5% (with glucose meter) and total nitrogen content (with proteinometer) 0.5% in described mixed liquid;
S4. by the mixed liquid after step S3 sterilization according to 3% inoculum size inoculation plant lactobacillus ferment, adopt calcium carbonate or white lime to regulate fermented liquid pH to 5.5 in fermenting process, control temperature is 35 DEG C, as Lactic Acid from Fermentation Broth mycetocyte concentration OD
600value reaches 1.5 and keeps balance 3d, and whole fermenting process terminates, and lactic-acid bacteria cells yield is 1.42%;
S5. the fermented liquid obtained by step S4 adopts the centrifuge 1min of 30000rpm centrifugal force, and be separated and obtain lactic-acid bacteria cells, the supernatant liquor of separation is for subsequent use;
S6. add the trehalose of 6% in the lactic-acid bacteria cells obtained to step S5, mix rear freeze-drying, obtain agent of lactic acid bacteria;
S7. by be separated in step S5 the supernatant liquor that obtains adopt mass concentration be 50% saturated ammonium sulphate solution precipitate, by throw out through ultrafiltration membrance filter process, collect filtrate and carry out lyophilize and obtain bacteriocin lab.
Embodiment 4: a kind of method utilizing salted vegetable waste water to produce agent of lactic acid bacteria and bacteriocin, it comprises the following steps:
S1. by the salted vegetable wastewater collection of fermenting-ripening in adjustment settling tank in, precipitation 42h, then through regulating filtering net at the bottom of settling tank to carry out coarse filtration, described sieve meshes is 35 orders, remove the dish leaf in salted vegetable waste water stoste, dish slag, the insoluble impuritiess such as sandstone, salting zymolysis cell suspending liquid is obtained after active carbon deodorizing, the salinity of described salted vegetable waste water is 7%, with the reducing sugar of glucose meter for 7%, in the centrifugal 9min rear solution of 4000rpm with the total nitrogen content of proteinometer for 10%, non-salt soluble solid content is 7%, organoleptic requirements is without rancid, free from extraneous odour, without black change, with without mould colored epistasis,
S2. salting zymolysis cell suspending liquid is adopted the centrifuge 4min of 7000rpm centrifugal force, remove insoluble impurities, obtain clear liquor;
S3. glucose, yeast extract and calcium carbonate are joined in the clear liquor that step S2 obtains and allocate, gained mixed liquid is sterilization 25min at 135 DEG C, reducing sugar 3.0% (with glucose meter) and total nitrogen content (with proteinometer) 1.75% in described mixed liquid;
S4. by the mixed liquid after step S3 sterilization according to 4% inoculum size inoculation plant lactobacillus ferment, adopt calcium carbonate or white lime to regulate fermented liquid pH to 5.0 in fermenting process, control temperature is 32.5 DEG C, as Lactic Acid from Fermentation Broth mycetocyte concentration OD
600value reaches 1.75 and keeps balance 3.5d, and whole fermenting process terminates, and lactic-acid bacteria cells yield is 1.32%;
S5. the fermented liquid obtained by step S4 adopts the centrifuge 5min of 35000rpm centrifugal force, and be separated and obtain lactic-acid bacteria cells, the supernatant liquor of separation is for subsequent use;
S6. in lactic-acid bacteria cells, add the trehalose of 8%, mix rear freeze-drying, obtain agent of lactic acid bacteria;
S7. by be separated in step S5 the supernatant liquor that obtains adopt mass concentration be 45% saturated ammonium sulphate solution precipitate, by throw out through ultrafiltration membrance filter process, collect filtrate and carry out lyophilize and obtain bacteriocin lab.
Embodiment 5: a kind of method utilizing salted vegetable waste water to produce agent of lactic acid bacteria and bacteriocin, it comprises the following steps:
S1. by the salted vegetable wastewater collection of fermenting-ripening in adjustment settling tank in, precipitation 48h, then through regulating filtering net at the bottom of settling tank to carry out coarse filtration, described sieve meshes is 40 orders, remove the dish leaf in salted vegetable waste water stoste, dish slag, the insoluble impuritiess such as sandstone, salting zymolysis cell suspending liquid is obtained after active carbon deodorizing, the salinity of described salted vegetable waste water is 9%, with the reducing sugar of glucose meter for 10%, in the centrifugal 11min rear solution of 4000rpm with the total nitrogen content of proteinometer for 5%, non-salt soluble solid content is 10%, organoleptic requirements is without rancid, free from extraneous odour, without black change, with without mould colored epistasis,
S2. salting zymolysis cell suspending liquid is adopted the centrifuge 5min of 8000rpm centrifugal force, remove insoluble impurities, obtain clear liquor;
S3. glucose, yeast extract and calcium carbonate are joined in the clear liquor that step S2 obtains and allocate, gained mixed liquid is sterilization 30min at 150 DEG C, reducing sugar 2.5% (with glucose meter) and total nitrogen content (with proteinometer) 3.0% in described mixed liquid;
S4. by the mixed liquid after step S3 sterilization according to 5% inoculum size inoculation plant lactobacillus ferment, adopt calcium carbonate or white lime to regulate fermented liquid pH to 6.5 in fermenting process, control temperature is 27.5 DEG C, as Lactic Acid from Fermentation Broth mycetocyte concentration OD
600value reaches 2.0 and keeps balance 4d, and whole fermenting process terminates, and lactic-acid bacteria cells yield is 1.36%;
S5. the fermented liquid obtained by step S4 adopts the centrifuge 4min of 40000rpm centrifugal force, and be separated and obtain lactic-acid bacteria cells, the supernatant liquor of separation is for subsequent use;
S6. in lactic-acid bacteria cells, add the trehalose of 10%, mix rear freeze-drying, obtain agent of lactic acid bacteria;
S7. by be separated in step S5 the supernatant liquor that obtains adopt mass concentration be 55% saturated ammonium sulphate solution precipitate, by throw out through ultrafiltration membrance filter process, collect filtrate and carry out lyophilize and obtain bacteriocin lab.
Below by way of description of test beneficial effect of the present invention:
1, lactic-acid bacteria cells yield determination
(1) determination object: the lactic-acid bacteria cells yield of embodiment 1 ~ 5;
(2) measuring method: centrifugal separation;
(3) measurement result: experimental result is in table 1.
The lactic-acid bacteria cells yield table of table 1 embodiment 1 ~ 5 gained
| Title | Lactic-acid bacteria cells yield (%) |
| Embodiment 1 | 1.21 |
| Embodiment 2 | 1.37 |
| Embodiment 3 | 1.42 |
| Embodiment 4 | 1.32 |
| Embodiment 5 | 1.36 |
2, fungistatic effect measures
(1) determination object: the bacteriocin prepared by embodiment 1 ~ 5 and the fungistatic effect of Nisin;
(2) measuring method: adopt Odontothrips loti to detect bacteriocin prepared by embodiment 1 ~ 5 to colon bacillus, the fungistatic effect singly increasing listeria spp, streptococcus aureus and intestines Salmonellas, and with Nisin in contrast;
The preparation of test solution to be measured and mensuration: be heated to by sterilized nutrient agar and melt completely, be poured in culture dish, every ware 15ml (lower floor), treats that it solidifies.In addition, add the bacteriocin after freeze-drying described in step S7 of the present invention after the PDA substratum of thawing is cooled to 50 DEG C, be dissolved as the solution of 100mg/ml, the substratum 5ml being mixed with bacterium is added to (upper strata) to be solidified on the substratum solidified.Directly vertically put Oxford cup (the circular tubule of internal diameter 6mm, external diameter 8mm, high 10mm, volume 240 μ L) with aseptic technique in media surface, pressurize gently, make it contact tight with substratum, in cup, add measuring samples.Fill it up with rearmounted 37 DEG C and cultivate observations after 16 ~ 18h, measure antibacterial circle diameter with chi.
(3) measurement result: experimental result is shown in Fig. 1 and table 2.
Bacteriocin prepared by table 2 the present invention and Nisin fungistatic effect synopsis
Fig. 1 and table 2 shows, bacteriocin of the present invention all has significant restraining effect to above-mentioned four kinds of pathogenic bacterium, and the restraining effect of Nisin to colon bacillus and intestines Salmonellas is more weak.
3, pH and temperature measure the impact of bacteriocin fungistatic effect of the present invention
Activity and the fungistatic effect thereof of bacteriocin lab restrict by many factors, as temperature, pH etc.Now investigation pH and temperature are on the impact of the bacteriocin fungistatic effect prepared by the present invention.
(1) determination object: the bacteriocin prepared by embodiment 1 ~ 5;
(2) measuring method: adopt Odontothrips loti to detect bacteriocin prepared by embodiment 1 ~ 5 to colon bacillus, the fungistatic effect singly increasing listeria spp, streptococcus aureus and intestines Salmonellas;
The preparation of test solution to be measured and mensuration: be heated to by sterilized nutrient agar and melt completely, be poured in culture dish, every ware 15ml (lower floor), treats that it solidifies.In addition, add the bacteriocin after freeze-drying described in step S7 of the present invention after the PDA substratum of thawing is cooled to 50 DEG C, be dissolved as the solution of 100mg/ml, the substratum 5ml being mixed with bacterium is added to (upper strata) to be solidified on the substratum solidified.Directly vertically put Oxford cup (the circular tubule of internal diameter 6mm, external diameter 8mm, high 10mm, volume 240 μ L) with aseptic technique in media surface, pressurize gently, make it contact tight with substratum, in cup, add measuring samples.Fill it up with rearmounted 37 DEG C and cultivate observations after 16 ~ 18h, measure antibacterial circle diameter with chi.
(3) measurement result: experimental result is in table 3.
The bacteriocin fungistatic effect synopsis that table 3pH and temperature are prepared the present invention
Table 3 illustrates, when pH value becomes 4.0, bacteriocin fungistatic effect prepared by the present invention obtains reinforcement, and utilizes 100 DEG C to carry out the activity of 5min thermal treatment to bacteriocin prepared by the present invention to have no significant effect.
The plant lactobacillus that the present invention inoculates is SICC1.10 plant lactobacillus, purchased from Microbial resources platform DSMZ of Sichuan Province, is derived from pickles bacterial classification, is adapted at fermenting in salted vegetable fermentation, MRS can be utilized to cultivate and produce its seed liquor.
Claims (5)
1. utilize salted vegetable waste water to produce a method for agent of lactic acid bacteria and bacteriocin, it is characterized in that, it comprises the following steps:
S1. by the salted vegetable wastewater collection of fermenting-ripening in adjustment settling tank in, precipitation 24 ~ 48 h, the filtering net then at the bottom of described adjustment settling tank carries out coarse filtration, obtains salting zymolysis cell suspending liquid after active carbon deodorizing;
S2. described fermented cells suspension is placed in centrifugal 1 ~ 5 min of whizzer, its rotating speed is 4000 ~ 8000 rpm, obtains clear liquor;
S3. glucose, yeast extract and calcium carbonate are joined in the clear liquor that step S2 obtains and allocate, gained mixed liquid is sterilization 10 ~ 30 min at 100 ~ 150 DEG C;
S4. by the mixed liquid after step S3 sterilization according to 1 ~ 5% inoculum size inoculation plant lactobacillus ferment, calcium carbonate or white lime is adopted to regulate fermented liquid pH to 5.0 ~ 7.0 in fermenting process, control temperature is 25 ~ 35 DEG C, as Lactic Acid from Fermentation Broth mycetocyte concentration OD
600value keeps balance 2 ~ 4 d when reaching 1.0 ~ 2.0;
S5. the fermented liquid after being balanced by step S4 is placed in centrifugal 1 ~ 5 min of whizzer, and its rotating speed is 20000 ~ 40000 rpm, and collecting precipitation obtains lactic-acid bacteria cells, and the supernatant liquor of separation is for subsequent use;
S6. in described lactic-acid bacteria cells, add the trehalose of 5 ~ 10%, mix rear freeze-drying, obtain agent of lactic acid bacteria;
S7. step S5 is separated the supernatant liquor that obtains adopt mass concentration be 40 ~ 60% saturated ammonium sulphate solution precipitate, by throw out through ultrafiltration membrance filter, collect filtrate and carry out lyophilize and obtain bacteriocin lab.
2. the salted vegetable waste water that utilizes according to claim 1 produces agent of lactic acid bacteria and bacteriocin method, it is characterized in that: the salinity of described salted vegetable waste water is 1 ~ 9%, with the reducing sugar of glucose meter for 0.01 ~ 10%, in the centrifugal 9 ~ 11min rear solution of 4000 rpm with the total nitrogen content of proteinometer for 0.01 ~ 10%, non-salt soluble solid content is 0.01 ~ 10%, and organoleptic requirements is without rancid, free from extraneous odour, without black change with without mould colored epistasis.
3. the salted vegetable waste water that utilizes according to claim 1 produces agent of lactic acid bacteria and bacteriocin method, it is characterized in that: the mesh of described filtering net is 20 ~ 40 orders.
4. the salted vegetable waste water that utilizes according to claim 1 produces agent of lactic acid bacteria and bacteriocin method, it is characterized in that: mixed liquid described in step S3 with the reducing sugar massfraction of glucose meter for 1.0 ~ 3.0%, with the total nitrogen content of proteinometer for 0.5 ~ 3.0%.
5. the salted vegetable waste water that utilizes according to claim 1 produces agent of lactic acid bacteria and bacteriocin method, it is characterized in that: described yeast extract comprises yeast powder and yeast extract paste.
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| CN105495483A (en) * | 2015-12-29 | 2016-04-20 | 四川东坡中国泡菜产业技术研究院 | Method for making bacteriostatic-agent dry powder with pickle brine |
| CN113772878A (en) * | 2021-08-10 | 2021-12-10 | 云南立达尔生物科技有限公司 | Treatment method and application of marigold fermentation wastewater |
| CN113973848A (en) * | 2021-10-29 | 2022-01-28 | 辽宁省农业科学院 | Preparation method and application of pickled Chinese cabbage fermentation waste liquid bacteriostatic agent |
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| CN105495483A (en) * | 2015-12-29 | 2016-04-20 | 四川东坡中国泡菜产业技术研究院 | Method for making bacteriostatic-agent dry powder with pickle brine |
| CN113772878A (en) * | 2021-08-10 | 2021-12-10 | 云南立达尔生物科技有限公司 | Treatment method and application of marigold fermentation wastewater |
| CN113973848A (en) * | 2021-10-29 | 2022-01-28 | 辽宁省农业科学院 | Preparation method and application of pickled Chinese cabbage fermentation waste liquid bacteriostatic agent |
| CN113973848B (en) * | 2021-10-29 | 2023-02-03 | 辽宁省农业科学院 | Preparation method and application of pickled Chinese cabbage fermentation waste liquid bacteriostatic agent |
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