SU707320A1 - Method of preparing nisin - Google Patents

Description

one

This invention relates to the microbiological industry, namely the production of nisin used in the food industry.

There is a known method of cultivating Nisin by growing the producer in a nutrient medium based on whey, followed by extraction of the target product from microbial cells, separation of the extract, its concentration, salting out, filtration and drying 1.

However, the drug obtained in a known manner, unstable during storage.

The purpose of the invention is to stabilize the activity of nisin during storage.

This is achieved in that the producer grows on a medium containing whey and extract of fodder yeast grown on plant waste, the purified culture liquid is concentrated with sorbital C .20, filtered through perlite filter, the resulting ni paste. Zin-perlite is dissolved in a 0.02 N solution of HCI, then re-filtered and the concentrate is dried in the presence of sodium chloride by spraying on sodium caseinate at a ratio of 1-2: 2.3-1.3.

In addition, the cultivation is carried out at a pH of 6.8-7.0 ...

And in order to utilize fermentation wastes, the precipitate after separation of the culture liquid is hydrolyzed with pectofoethidine P10X or amylchlorin and introduced into the nutrient medium as a nitrogen source during re-fermentation.

The method is carried out as follows.

Prepare feed extract

5 yeast. To do this, 250 g of dry fodder yeast are filled with 2.5 liters of warm tap water, drawn for 2 hours, boiled over low heat for 15 minutes, filtered and sterilized in an autoclave.

0 at O-, 7 atm 20 min. 18-20% of the extract is added to the medium until the content of 60-70 mg /% of amine nitrogen in the medium.

Waste hydrolyzate is prepared as follows.

five

Waste (biomass, milk proteins, with perlite) is obtained by filtration through perlite culture liquid of the previous fermentation, the output from 20 l is 500-600 g. Waste is diluted with tap water at an Ratio of 1: 5, thoroughly mixed until homogeneous and heated. ; bwyll, the resulting pH is 2.5-3.0 cf this corresponds to the optimum effect of pectofoethyne Dyne P10X; in case of amilorine, the optimum pH is 4.5-7.0. The enzyme is added. in the amount of 0.20-0.25% to the hydrolysable volume (fermene: gee 1 its§1 is dissolved in a small volume of water). Hydrolyzate is carried out for 24-30 hours with chlorine form antiseptic - "l 5 ml / l. At the end of the hydrolysis, the mixture is boiled for 20-30 minutes until the odor of chloroform disappears; the solid phase is removed by filtration or centrifugation; hydrolyzed sterilize at 0.7 atm. 20 min. 10-12% 1 $ of the hydrolyzate is added to the medium up to the L content of an LO-70 mg /% amine nitrogen medium.

Example 1: To 18 L of MOUTHFLOW, add 4L (20%) of extract of yeast feed obtained 20

as described. The medium is sterilized at 20 minutes, cooled to 28–30 ° C, the pH is adjusted to 6.8–7.0, and 3% of inoculum of the 18–20 hour age is set at 55% StrvBactis. At 28–30 ° C, the pH of the medium is adjusted to pH tj, 8–7.0 with the introduction of 40% alkali (NapH i or KOH). Fermentation is quenched at the time of slowing the acidity shift, automatically registrated by the equipment; -

 rum. The duration of fermentation is 12-1 bch .. The activity of the culture fluid reaches 4-6 thousand units / ml, or 100-150 MKS / ml.35

Beginning before the second fermentation of pi: tatyllrn from the reefer of the milk jar with 10-12% of waste hydrolyzate (from the 1st and each subsequent fermentation) obtained by the specified method. The culture fluid 1/2 and all subsequent fermentations are processed by operations. Thus, the culture fluid containing 100 µg / ml nisin (total activity .. 2.10 µg) is acidified with 18% HCP to a pH of 2.2-2.5 and heated in within 3-5 minutes to 95-100 0, cooled to. and filtered through perlite. The solid phase (waste) is directed to a guide. " roliz. The volume of filtrate is 20 l, active -. 97 μg / ml (total activity. J 1.94-10 μg), 97% feed. The resulting filtrate with an activity of 97 µg / ml is subjected to foaming at pH 4.0-4.5 with C-20 sorbital 0.03%. Volume of pen-

2l, activity 920.1 µg / ml, a total of 1.84-10 µg of yield into the foam from the filtrate of the foamy bottom product, dry Nace (25%), pH 2.2-2.5. the antibiotic is suction filtered through perlite, SeparateHiftyio pasta ni-1 yerigr1gSh18

rty in 0.02 n. solution Ysv, and again jj

l i ii 707320

subjected to vacuum filtration and washing. The volume of the obtained liquid concentrate is 400 ml, the activity is 4425 μg / ml, the total activity is 1.77. “10 μg, the yield from the foam is 96% or.90.7% of the filtrate, the solids content is 3%. :.

EXAMPLE 2. To 18 d of dairy milk is added. 2.1 l of the enzymatic hydrolyzate of waste obtained by the above method, the sterilized medium is cooled to 28-30 s, the pH is adjusted to 6.8-7.0 and inoculated with 3-5% inoculum 18-20 hours of age Str. gactis.

Cultivate at a steady-state avchonatically controlled acidity of the medium within 6, 8 -7.0 by introducing 40% alkali (NaOH or KOH). Fermentation is completed at the moment of slowing the acidity shift, automatically; Fermentation time 12-16 hours. Activity k. G. 150 µg, total activity 3 10 °. mcg After filtration through filter-perlite and washing the precipitate 1: while with a pH of 2.3, the volume of the filtrate is 20 liters with an activity of 145 μg. . the total activity of the filtrate is 2.9, the yield of activity from the culture fluid is 97%. A single-stage foaming results in 2 l of a frothy product with l40 μg —J / wn activity; total foam capacity 2.8010 µg: After salting out with dry NaCf, filtration is carried out through perlite; Sol. The cut-off pass of nisin with perlite is filtered and filtered to obtain a volume of Cl concentrate in an amount of 400 ml with an activity of 2.78-10 µg, which is 96% of the filtrate. The content of SUCHI. Substances is 2.5%. We take into account losses of overestimate - (8-10%), i.e., 2.78-10 - p, 28-.10 2 ,.

To obtain the amount of powder 2,5010: 20 (standard activity according to TU) 125 g should be obtained, of which 10 g falls on the dry weight (2.5%) 125 g - GO t - 115 g. General, the filler should be 115 g are given, i.e. 57.2 g of NaCe and so are sodium caseinate (115: 2 57.2), NaC is added to 400 ml of nisin concentrate — K and sprayed onto 57.2 g of protein in the fluidized bed. A dry preparation is obtained 125 g with an activity of 20 µg, MH in mg of dyxorp substance, which makes up 86% of the activity of the filtrate,

The table shows the activity of the drug nisin when stored in a refrigerator (4 -) for a year, (determined by diffusion into agar with a test culture of BacvcoaguPans I 15 ,. Method error 10-12%),

.Offering is a way to use insufficiently and non-deficient enzymes, the medium is 5-6 times cheaper than the known one, the resulting preparation has a standard activity of 20 µg / ml of dry matter and

Activity, u / mg dry drug

Shelf life in months OH 3 J 6 D 9 D 12

1804 + 80 800 + 64 792 + 52 784 + 56 800 + 76

2820 + 52 804 + 56 792 + 84 820 + 72 820 + 68

31040 76 976 + 76 936 + 96 984 + 88 984 + 84

Claims (3)

1. The method of obtaining nisin by growing the producer in a nutrient medium based on whey, followed by extraction of the target product from microbial cells, separation of the extract, its end by spraying, salting out, filtering and drying, so that that, in order to stabilize the activity of nisin during storage, the producer is grown on medium containing whey and an extract of fodder yeast grown on plant waste, purified cultural liquid is concentrated with sorbital -20, filtration is carried out through a filter perlite, the resulting paste nisin-pearlite was dissolved in 0.02 and. HCf solution, then repeated: st; abilen during storage for 2 pts. . -, ii-A-S, -., Slf filtering and the concentrate is dried in the presence of sodium chloride by spraying on sodium caeoynat at a ratio of 1-25 2.3-1.3. 2. The method according to claim 1, about tl and h ayusch and the fact that the extraction is carried out at pH 6.8-7, .0. 3. The method according to claim 1, characterized in that, in order to utilize the waste of fermentation, the residue after separation of the I-1: green and 6th liquid is subjected to hydrolysis with pectofoelectric P10X or amilorizin and introduced into the nutrient medium as a source nitrogen when pO: Ytornoy fermentation. Sources of information taken into account when ek; 1. USSR patent number 42751l cl. C 12 D 9/12. 1976.