JPH0777554B2 - Yeast cells for feed - Google Patents

Description

TECHNICAL FIELD The present invention relates to a feed yeast cell, and more particularly to a feed yeast cell for livestock having a rumen.

(Prior Art) It is well known to use L-lysine (hereinafter abbreviated as lysine) as a feed additive which is nutritionally and physiologically effective for livestock. However, in the case of ruminant feed, even if lysine alone is used as a feed additive, lysine is decomposed in the rumen by the action of microorganisms and protozoa that live in the rumen of the ruminant. It has a drawback that lysine cannot be efficiently ingested by ruminant animals. Therefore, it has often been attempted to make lysine insoluble and thereby prevent it from being decomposed by microorganisms, but the method is limited to the artificial means of coating lysine with a material derived from a natural product or a synthetic polymer. It was

On the other hand, the use of yeast cells as a feed for livestock is widely practiced. Conventionally used yeast cells have a relatively high lysine content among microorganisms including bacteria and fungi. However, even with such yeast cells, the content of lysine per dry weight of the cells is about 5% in terms of lysine hydrochloride, so that it has a drawback that lysine cannot be efficiently ingested by ruminant animals.

(Problems to be Solved by the Present Invention) A problem to be solved by the present invention is a drawback that conventional yeast cells for feed have had, that is, low lysine content and that lysine alone was used as a feed additive. It is an object of the present invention to provide a new yeast cell for feed which solves the problem that lysine is decomposed in the rumen of the ruminant and is not efficiently ingested by the ruminant.

(Means for Solving Problems) As a result of intensive studies to solve the above problems, the present inventors have conducted (a) culturing yeast in a medium containing a lysine precursor, or ( B) By imparting lysine analog resistance to yeast as an advantageous property for improving lysine production capacity, or by using (a) and (b) together, lysine will be 10% or more in terms of lysine hydrochloride based on dry cell weight. We succeeded in obtaining yeast cells containing. In addition, a McDough gel buffer solution (NaHCO ₃ : 7.43 g / l, Na ₂ HPO ₄ · 12H ₂ O: 7.00 g / l, used as a rumen model for ruminant animals of this yeast cell)
NaCl: 0.34g / l, KCl: 0.43g / l, MgCl ₂・ 6H ₂ O: 0.10g / l, CaCl
₂ : 0.05g / l, pH6.8), the lysine eluted from the yeast cells into the McDough gel buffer solution when kept at 39 ° C for 12 hours was converted to lysine hydrochloride in the yeast cells. It was found for the first time that yeast cells, which contained less than 10% of the total amount of lysine contained, were extremely effective as feed for livestock, especially those with rumen. The present invention has been made based on these findings.

That is, the present invention is a yeast cell containing 10% or more of L-lysine based on the dry cell weight in terms of L-lysine hydrochloride, and the yeast cell is suspended in a McDough gel buffer solution.
A diet in which L-lysine eluted from the yeast cells into a McDough gel buffer solution when kept at 12 ° C for 12 hours is 10% or less of the total amount of L-lysine contained in the yeast cells in terms of L-lysine hydrochloride. For yeast cells.

The yeast used in the present invention is Sacchromyc (Sacchromyc).
es), Candida (genus Candida), Torulopsis (T)
orulopsis genus, Hansenula genus, Pichia genus, Endomycopsis genus,
It is a yeast belonging to the genus Debaryomyces, the genus Kluyveromyces, and the like.

The following microorganisms can be mentioned as specific examples.

Endomycopsis chodati
i) AJ 4275, FERM-P 1816 Candida pelliculusa A
J 4553, IFO 0707 Debaryomyces klocker
i) AJ 4196 IFO 0036 Kluyveromyces polypo
rus) AJ 4278 IFO 0996 Torulopsis magnoliae
AJ 14521 CBS 166 Saccharomyces cerevisi
ae) AJ 14590 ATCC 26615 Pichia fermentans AJ 41
45 ATCC 9768 Hansenula anomala AJ 41
66 FERM-P 3759 As a first means for obtaining yeast cells having a lysine content of 10% or more in terms of lysine hydrochloride based on the dry weight of the cells in the present invention, the above-mentioned yeast is added with a precursor for lysine biosynthesis. The culture may be performed aerobically in the defined medium.

Specific precursors include α-aminoadipic acid, 2-oxoadipic acid, α-ketoglutaric acid, oxalglutaric acid, homocitric acid, homoisocitric acid, α-N-succinyl-2-aminoadipic acid, α- Examples include N-succinyl lysine, saccharobin, aminocaprolactam, glutamic acid, aspartic acid, alanine, valine and glycine. These precursors may be added from the beginning of the culture, may be added during the culture, or may be added continuously or batchwise.

As the components of the medium other than these precursors, any medium can be used as long as it appropriately contains nutrients such as carbon sources, nitrogen sources, inorganic salts, other amino acids and nucleic acid vitamins that can be assimilated by yeast. Carbon sources include hydrocarbons (n-paraffin, kerosene, light oil, etc.), organic acids (acetic acid, butyric acid, etc.), carbohydrates (glucose, sucrose, molasses, etc.)
Any carbon source that can be assimilated by the yeast, such as molasses etc.), fatty acids (palmitic acid etc.), alcohols (ethanol etc.) can be used alone or in combination.
As the nitrogen source, ammonium sulfate, ammonium salt, ammonium nitrate, phosphorus phosphorus, etc., as well as urea, ammonium vinegar, yeast extract, corn steep liquid, meat extract,
Peptone and the like can be used alone or in combination. As the inorganic salts, monopotassium phosphate, magnesium sulfate, zinc nitrate, ferrous sulfate, etc. are used. In addition, the addition of trace metals such as aluminum, manganese, copper, and boron may give favorable results. Other amino acids, nucleic acids, vitamins, nutrients of organic acids, production promoters and the like may be added.

Culturing is carried out under aerobic conditions, usually at 20-40 ° C. and pH 3-9 for 2-5 days. At this time, since the pH of the culture solution varies, if necessary, calcium carbonate, sodium hydroxide, ammonia water, ammonia gas, hydrochloric acid, sulfuric acid, or the like is used to adjust the pH to a suitable value. Further, intermittently or continuously adding a solution containing an organic acid such as acetic acid or a salt thereof and a carbon source metabolizable by the yeast while adjusting the pH also accumulates lysine in the cells. It is extremely effective for

The yeast cells cultivated by the above-mentioned method are yeast cells containing 10% or more of L-lysine per dry cell weight in terms of L-lysine hydrochloride, and the yeast cells were buffered by McDough gel. L which is suspended in a liquid and is incubated at 39 ° C for 12 hours to elute from the yeast cells into a McDough gel buffer
-Lysine is 10% or less of the total amount of L-lysine contained in this yeast cell in terms of L-lysine hydrochloride.

Lysine content in terms of lysine hydrochloride per dry cell weight
As another means for obtaining 10% or more yeast cells, there is a means for imparting the property of making yeast resistant to lysine analogs.

In the present invention, the lysine analog is a compound having a chemical structure similar to that of lysine, and the compound inhibits the growth of yeast and restores the growth by adding lysine. Specific lysine analogs include S- (β aminoethyl) -L-cysteine and δ-
Hydrolysine, DL-4,5-transdehydrolysine, DL- (amino-3) cyclohexylalanine, diaminopimelic acid, 6-aminocaproic acid, DL-N-6-
Acetyllysine, DL-N-2-acetyllysine, ornithine, cataverine, lysine methyl ester, DL-2-aminoadipic acid, pipecolic acid, dipicolinic acid, canavalin, oxolysine and the like. The following is a specific method for collecting yeast that has been made resistant to a lysine analog.

(Collection method) The strains shown in the column of parent strain in Table 1 were used as Bact Yeast Nitrogen Base (Di
fco) 6.7 g / l, glucose 20 g / l, pH 6.0 liquid medium was shake-cultured at 30 ° C. for 2 days. The obtained bacterial cells were washed twice with 0.067M phosphate buffer, and then a bacterial suspension was prepared so that the bacterial count was adjusted to 10 ⁸ cells / ml. DL- (amino-
3) Bactto yeast nitrogen base (Bacto Ye) containing cyclohexylalanine, DL-4,5-transhydrolysine, S- (β-aminoethyl) -L-cysteine or δ-hydroxylysine at 0.001-1.0 g / dl each.
ast Nitrogen Base) (manufactured by Difco) 6.7 g / l, glucose 2
1 ml of the above-mentioned bacterial suspension was smeared on each agar plate medium of 0 g / l, agar 20 g / l, pH 5-6. These flat plates were placed at a position of 35 cm under an ultraviolet light source (15 W ultraviolet light, wavelength 253 angstrom) and irradiated with ultraviolet light for 40 seconds. The plate subjected to this ultraviolet irradiation treatment was kept warm at 30 ° C. for 4 days, and colonies appearing on the plate were collected.

The obtained colonies were designated as Bacto Yeast Nitrogen Base (Difco) 1
The cells were cultured in a liquid medium consisting of 3.4 g / l, glucose 40 g / l, pH 6.0 for 2 days, and the culture was washed with 0.067 M, pH 6.0 phosphate buffer. After this, 0.1 g of yeast cells, converted to dry cell weight, was suspended in 5 ml of 0.067 M, pH 6.0 phosphate buffer, and the temperature was adjusted to 100 ° C.
Incubate for 10 minutes, extract the lysine in the cells, quantify the lysine in the extract, and convert the lysine in the cells to lysine hydrochloride conversion.
Contains 10% or more and 39 in McDough gel buffer
A strain was selected in which the elution rate of lysine in the cells when kept at 12 ° C for 12 hours in the buffer was 10% or less.

Table 1 shows parent strains, mutant strains, and resistant drug names of the mutant strains.

In order to obtain yeast cells having a lysine content of 10% or more in terms of lysine hydrochloride based on the dry weight of the cells by culturing the yeast having resistance to the lysine analogue in the present invention, the precursor for lysine biosynthesis described above was added. The culture may be performed using the culture medium or a non-addition medium under the above-mentioned culture conditions.

The yeast cells for feed in the present invention refer to the culture itself, the dried culture, the separated bacterial cells obtained by separating only the bacterial cells from the culture, and the dried product of the isolated bacterial cells. Further, an inorganic substance which does not harm ruminants such as calcium carbonate may be added to these.

The feed yeast cell of the present invention may be administered to livestock alone or in a mixture with other feed.

When mixed with other feeds and fed, they can be mixed at any ratio.

Example 1 Preparation of Lysine-Containing Yeast Cells by Precursor Addition Method Glucose: 50 g / l, ammonium sulfate 3.8 g / l, corn steep liquid: 3.4%
(V / v), magnesium sulfate heptahydrate: 1 g / l, potassium dihydrogen phosphate: 1 g / l, L-α-aminoadipic acid or L-α-ketoadipic acid was added at 5 g / l The yeasts shown in Table 1 were cultured at 30 ° C for 2 days. After culturing,
The bacterial cells were collected, suspended in 6N hydrochloric acid, and then stored in a vacuum sealed tube at 110 ° C for 24 hours.
After hydrolysis for a period of time, the content of lysine contained in the cells was quantified. The results are shown in Table 2.

Example 2 Preparation of Lysine-Containing Yeast Cells Using Lysine Analog-Resistant Strains Each of the yeasts shown in Table 3 in the basal medium and L-α-aminoadipic acid-added medium shown in Example 1 was subjected to the same conditions as in Example 1. After culturing, the content of lysine contained in the cells was quantified. The results are shown in Table 3.

Example 3 Dissolution of intracellular lysine into McDough gel buffer solution McDough gel buffer (NaHCO ₃ : 7.43 g / l, Na ₂ HPO ₄ · 12H ₂ O: 7.00 g / l) which is a rumen animal rumen model , NaC
l: 0.34g / l, KCl: 0.43g / l, MgCl 2 · 6H 2 O: 0.10g / l, CaCl 2:
0.05 g / l, pH 6.8) 200 ml of the lysine-containing yeast cells shown in Examples 1 and 2 were suspended to 1 g in terms of lysine hydrochloride, and incubated at 39 ° C for 12 hours with shaking. The elution rate of lysine eluted from the yeast cells into the buffer was determined by centrifugation and quantifying the concentration of lysine in the supernatant. The results are shown in Table 4.

Example 4 Persistence of lysine in yeast cell in rumen juice Lumen juice was collected from a rumen fistula device test cow that was fed a concentrated diet and a roughage twice a day (morning and evening), and filtered with a triple gauze, 80 ml thereof and 80 ml of the McDough gel buffer solution used in Example 3 were mixed, transferred to a reaction tube equipped with a Bunsen valve, and containing lysine prepared by the precursor addition method or culture of a lysine analog-resistant strain shown in Example 1 or 2. Yeast was added so as to be 400 mg in terms of lysine hydrochloride, kept at pH 6.8 and 39 ° C under nitrogen gas aeration for 24 hours, then collected by centrifugation, and the lysine content remaining in the cells was quantified and remained. I asked for the rate. As a control, 400 mg of lysine hydrochloride crystals were added instead of yeast cells, and the residual rate was calculated. The results are shown in Table 5.

Example 5 Change in Blood Lysine Concentration after Administration of Lysine-Containing Yeast Cells Saccharomyce containing 18% of lysine shown in Example (2) in the rumen from fistula of cows fitted with lumen fistula
s cerevisiae FERM-P8205 (AJ14629) was administered at 50 g in terms of lysine hydrochloride, and after administration, blood was collected from the jugular vein over time, and 3 ml of plasma was added with 3 ml of 10% sulfosalicylic acid and mixed. Then, the mixture was centrifuged, and the amount of free lysine in the supernatant was quantified, and the change in lysine concentration in blood was examined. As an indicator, polyethylene glycol was administered at the same time. As a control, an experiment was conducted in which 50 g of lysine hydrochloride was administered. The results are shown in Table 6.

As shown in Table 6, the yeast cells of the present invention increased the concentration of lysine in blood as compared with lysine hydrochloride used as a control, and thus were difficult to be decomposed in the rumen of ruminant animals, and thus, after the fourth stomach. It is shown that lysine is transferred to the blood from the inside of cells by being digested and absorbed by.

Example 6 Twenty-four Holstein steers having a body weight of about 230 kg were prepared, and these eight cows were divided into three experimental groups A, B, and C, respectively. For the cows of experimental section A, the feed having the composition shown in A of Table 7 was used, for the cows of experimental section B, the feed of the composition shown in B of Table 7, and for the cows of experimental section C, Table 7 5 kg of the feed of the composition was fed twice a day at 8:00 am and 6:00 pm per animal once.
The water supply was free intake. The body weight of cows was measured every 30 days, and the average body weight of cows in each experimental group was calculated. This result is the eighth
Shown in the table.

As shown in Table 8, the cows of experimental group C fed with the feed containing the yeast cells of the present invention showed a significant increase in body weight as compared with the cows of experimental groups A and B. Yeast cells are excellent as feed additives for livestock, especially ruminants.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area C12R 1:72) (C12N 1/16 C12R 1:78) (C12N 1/16 C12R 1:84) (C12N 1/16 C12R 1:85) (C12N 1/16 C12R 1:88) microbial accession number FERM P-8206 microbial accession number FERM P-8207 microbial accession number FERM P-8208 microbial accession number FERM P -8209 microbial accession number FERM P-8217

Claims (1)

[Claims] 1. An Endomycopsis genus, a Candida genus, a Debaryomyces genus, a Kliberomyces genus, a Tollopsis genus,
A yeast cell belonging to the genus Saccharomyces, Pichia or Hansenula, containing 10% or more and 40% or less of L-lysine in terms of L-lysine hydrochloride in terms of dry cell weight, and the yeast cell is McDough gel ( L-lysine eluted from the yeast cells suspended in the McDugall) buffer solution and incubated at 39 ° C for 12 hours into McDugall buffer solution was converted into L-lysine hydrochloride as L-lysine hydrochloride. A yeast cell for feed which is 10% or less of the total amount of L-lysine contained.