CN113355254A

CN113355254A - Formula and application of starter protective agent

Info

Publication number: CN113355254A
Application number: CN202010311191.9A
Authority: CN
Inventors: 林仲翼; 徐葭蓁; 王迺诒; 赖进此
Original assignee: Food Industry Research and Development Institute
Current assignee: Food Industry Research and Development Institute
Priority date: 2020-03-06
Filing date: 2020-04-20
Publication date: 2021-09-07
Anticipated expiration: 2040-04-20
Also published as: TWI737210B; CN113355254B; TW202134423A

Abstract

The invention relates to a formula of a starter protective agent and application thereof. A starter protectant formulation comprising: 1.125 +/-10 to 4.5 +/-10 weight percent of trehalose; 2.25 +/-10 to 4.5 +/-10 weight percent of skimmed milk powder; 1.125 +/-10% to 2.25 +/-10% by weight of mannitol; 0.0225 +/-10% to 0.1125 +/-10% by weight of vitamin C; 0.225 plus or minus 10 percent to 1.125 plus or minus 10 percent of sodium glutethionine by weight; and 0.225 +/-10% to 1.125 +/-10% of glycerol by weight.

Description

Formula and application of starter protective agent

Technical Field

The invention relates to a starter (starter) protectant formula, in particular to a starter protectant formula which is suitable for gram-positive bacteria and gram-negative bacteria simultaneously.

Background

Since the number of bacteria of natural microorganisms is usually not as large as that required for practical use, and it is not easy to preserve the microorganisms, and the microorganisms are often stimulated by the environment or mutated after many generations of culture, they are often used and circulated in the form of "starters" which are hot air-dried or freeze-dried. In the food industry, for example, natural bacteria cannot be used directly when fermentation is required by microorganisms in the process because of the large operation volume. Usually, domesticated starter is used to perform propagation in advance, and the subsequent process is performed after sufficient bacteria number is reached. Practical industrial applications include, for example, industries that require yeast as a raw material, such as: beer fermentation and bread baking industries. In addition, as applied to the agricultural biological agent industry, the biological agent products produced are also required to have a high and stably maintained viable cell count, and therefore, the method is also suitable for the first propagation with a starter. Therefore, after the development of the characteristic potential strains, a starter formula is needed, which can achieve the purposes of high stability and high viable count, thereby increasing the application flexibility in the process. Meanwhile, the thalli can be sold in a starter form, so that the speed and the diversity of product innovation are increased.

CN106244459A discloses a dry pseudomonas powder and a preparation method thereof, wherein the dry pseudomonas powder comprises pseudomonas and a protective agent, wherein the protective agent comprises skimmed milk powder, mannitol, sodium glutamate or glycine, corn starch and trehalose.

CN107828683A discloses a lactobacillus plantarum freeze-dried powder for prolonging the shelf life of yogurt and a preparation method thereof, which is characterized in that lactobacillus plantarum freeze-dried powder is prepared by mixing lactobacillus plantarum strains and an emulsion protective agent and freeze-drying, wherein the lactobacillus plantarum strains are stored in China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC number 10453. The preparation method comprises the steps of strain activation, strain propagation, fermentation culture and freeze-drying. The lactobacillus plantarum freeze-dried powder has an inhibiting effect on main putrefying mold and yeast in the fermented dairy product.

The existing starter protective agent formula has the defect of low viable count during culture, and particularly, the viable count is greatly reduced when the starter protective agent formula is used after a period of time after preparation is finished.

Disclosure of Invention

The main object of the present invention is to provide a starter protectant formulation that can improve the disadvantages of low viable count and survival rate, and in particular a starter protectant formulation with improved stability, which still has comparable viable count after a period of storage compared to the starter protectant formulation just prepared.

The invention aims to provide a formula of a starter protective agent, which comprises the following components:

1.125 +/-10% by weight of trehalose;

2.25 plus or minus 10 percent of skimmed milk powder;

1.125 plus or minus 10 percent of mannitol;

0.0225 plus or minus 10% by weight of vitamin C;

0.225 plus or minus 10 percent of sodium glutethinate; and

0.225 + -10% parts by weight of glycerol.

Other preferred embodiments of the present invention include, but are not limited to, those described in the following claims.

The starter protectant formulation of the present invention has the feature of improved viable count and survival rate whether used for the culture of gram-positive or gram-negative bacteria.

Drawings

FIG. 1 shows the viable cell count of a Pseudomonas putida starter cultured at 30 ℃ for 72 hours, which was prepared for each protective solution group in Table 1 of example 1 of the present invention.

FIGS. 2 to 4 show the viable cell counts of

yeast cells

21849, 20405 and 22745, respectively, prepared from the respective protective solutions in Table 1 of example 1 of the present invention, after culturing at 25 ℃ for 72 hours.

FIG. 5 shows the viable cell count of a Pseudomonas putida starter prepared for each of the protective solutions in tables 2 and 3 of comparative example 1 after culturing at 30 ℃ for 72 hours.

FIG. 6 shows viable cell counts of

yeast cells

21849, 20405 and 22745 starting materials prepared from the respective protective solutions in Table 2 and Table 3 of comparative example 1 after culturing at 25 ℃ for 72 hours.

FIG. 7 shows the survival rate of Pseudomonas putida starter cultured at 30 ℃ for 72 hours, prepared for each protection solution group in Table 1 of example 1 of the present invention.

FIGS. 8 to 10 show the survival rates of the

starters

21849, 20405 and 22745 of yeast strains prepared from the respective protective solutions in Table 1 of example 1 of the present invention after culturing at 25 ℃ for 72 hours, respectively.

FIG. 11 shows the survival rate of Pseudomonas putida starter cultured at 30 ℃ for 72 hours, prepared for each of the protective solution groups in Table 2 and Table 3 of comparative example 1.

FIG. 12 shows the survival rates of the starters for

yeast

21849, 20405 and 22745 prepared in each of the protective solutions in Table 2 and Table 3 of comparative example 1 after culturing at 25 ℃ for 72 hours.

FIG. 13 shows the number of viable bacteria of a Pseudomonas putida starter prepared from each of the protective solutions in Table 1 of example 1 of the present invention and cultured at 30 ℃ for 72 hours after being stored at 25 ℃ and 54 ℃ for 14 days.

Detailed Description

The present invention and its improvements will be further understood by the following examples and comparative examples. The following materials and instruments were used in the following examples and comparative examples.

Material

Defatted milk powder	MERCK
		Casein protein	MERCK
Glycerol	SIGMA
		Vitamin C	SIGMA
Trehalose	SIGMA
		Sucrose	SIGMA
Bran amino acid sodium salt (monosodium glutamate)	All-taste food
		Span
60	SIGMA
		YM Broth	Gibico

Formula of Pseu F agar culture medium

Tryptone 1%	MERCK
			1% of trypsinization protein	MERCK
Dipotassium hydrogen phosphate (K)₂HPO₄) 0.15%	SIGMA
		Magnesium sulfate (MgSO)₄) 0.15%	SIGMA
Agar (Agar)	SIGMA

Instrument for measuring the position of a moving object

Pseudomonas putida culture method

Selected pseudomonas putida YLW01 (Pseudomonas putida) Are experimental strains. YLW01 was cultured in a medium containing 1% Molasses, 1% MSG, 1.5 g/L K₂HPO₄With 1.5 g/L MgSO₄ ^.7H₂O medium (50 mL medium/250 mL Erlenmeyer flask) was cultured at 30 ℃ and 150 rpm for 42 hr. After completion of the incubation, the cells were centrifuged at 8000 rpm for 10 minutes at 4 ℃, the supernatant was discarded, washed with water and centrifuged again, and this washing process was repeated twice. And taking out the centrifuged bacterial sludge and storing the bacterial sludge in a refrigerator.

Culture method of yeast

Yeasts 21849, 20405 and 22745 are selected as experimental strains. Yeasts 21849, 20405 and 22745 were cultured in YM broth (Becton Dickinson and Company) (50 mL medium/250 mL Erlenmeyer flask) and cultured at 25 ℃ and 250 rpm for 48 hr. After completion of the incubation, the cells were centrifuged at 8000 rpm for 10 minutes at 4 ℃, the supernatant was discarded, washed with water and centrifuged again, and this washing process was repeated twice. And taking out the centrifuged bacterial sludge and storing the bacterial sludge in a refrigerator.

Formulation of protective agent/preparation of protective liquid

The protective solution was prepared by dissolving the protective agent formulation shown in table 1 in 22.5 mL of water, and after stirring for 30 minutes until the solution was uniform, pH was adjusted to 7 or 8 using the following phosphate solution (adjusted from alkaline to acidic) or glycine-sodium hydroxide buffer (adjusted from acidic to alkaline).

Table 1: formula (g) of protective agent/protective solution (g/vol%)

Preparing a glycine-sodium hydroxide buffer solution:

50 mL of an aqueous glycine solution (0.2M) and 8.8 mL of an aqueous sodium hydroxide solution (0.2M) were mixed uniformly and then the mixture was quantified to 200 mL.

Preparing a phosphate solution:

87.7 mL of an aqueous solution of sodium dihydrogenphosphate (0.2M) and 12.3 mL of an aqueous solution of sodium monohydrogenphosphate (0.2M) were mixed uniformly and then the mixture was quantified to 200 mL.

Example 1

Preparation of bottle opener

Taking the prepared bacterial sludge out of the refrigerator, mixing 7.5 g of bacterial sludge with each protective solution in the table 1 respectively, stirring for 30 minutes until the bacterial sludge is uniformly mixed, pre-freezing at the temperature of minus 20 ℃, and freeze-drying to obtain the starter.

1 g of the freshly prepared freeze-dried starter is taken to be redissolved in 10 mL of water, serial dilution with proper multiplying power is carried out, 100 mu L of starter dilution is taken to be smeared on a Pseu F culture medium, and colony counting is carried out after culture is carried out at 30 ℃ for 72 hours.

Comparative example 1

The starter was prepared according to the above-mentioned formulations of CN106244459 and CN 107828683. 22.5 mL of the protective solutions shown in tables 2 and 3 were mixed with 7.5 g of the prepared bacterial sludge, respectively, and the mixture was stirred for 30 minutes until the mixture was uniform, then prefreezed at-20 ℃ and freeze-dried to obtain a starter of comparative example 1.

TABLE 2 CN106244459 protective solution (g/vol%)

Group of	Trehalose (%)	Skimmed milk powder (%)	Mannitol (%)	Yeast powder (%)	Monosodium glutamate (%)	Span60(%)	Glycine (%)	Corn starch (%)
									CN106244459-1	1	25	0.5	1	3	2	3	0.1
CN106244459-2	5	12	5	3	0	1	15	2

TABLE 3 CN107828683 protective solution (g/vol%)

Group of	Trehalose (%)	Skimmed milk powder (%)	Glycerol (%)	Mannitol (%)	Sucrose (%)	Ascorbic acid	Water (W)
								CN107828683	6	8	1	2	4	0.5	78.5

Results

FIG. 1 shows the viable cell count of a Pseudomonas putida starter cultured at 30 ℃ for 72 hours, which was prepared for each protection solution group in Table 1 of example 1. In FIG. 1, it was found that the viable count of Pseudomonas putida was maintained at 10⁹CFU/g above, the protective agent of the invention has good protective effect. It is composed ofThe highest viable count of the Chinese and Japanese medicinal herbs in group 2 reaches 6.3x10⁹ CFU/g。

FIGS. 2 to 4 show the viable cell counts of

yeast

21849, 20405 and 22745 starters prepared from the respective protective solutions in Table 1 of example 1 after culturing at 25 ℃ for 72 hours, respectively. The viable count of

yeast

21849, 20405 and 22745 is 3.44 × 10⁹CFU/g (FIG. 2 group 5), 3.75x10⁹CFU/g (FIG. 3 group 7) and 5x10⁹CFU/g (FIG. 4, group 3).

FIG. 5 shows the viable cell count of a Pseudomonas putida starter prepared for each of the protective solutions in tables 2 and 3 of comparative example 1 after culturing at 30 ℃ for 72 hours. In FIG. 5, the viable count of Pseudomonas putida was found to be 5.8X10 respectively⁷、8.8x10⁸And 2.6x10⁸ CFU/g。

FIG. 6 shows viable cell counts of

yeast cells

21849, 20405 and 22745 starting materials prepared from the respective protective solutions in Table 2 and Table 3 of comparative example 1 after culturing at 25 ℃ for 72 hours. The culture effect is not good, and the number of most viable bacteria is 10⁸CFU/g, the highest viable count group is yeast 22745 and uses CN106244459-2 formula, reaching 1.99x10⁹ CFU/g。

FIG. 7 shows the survival rate of Pseudomonas putida starter cultured at 30 ℃ for 72 hours, prepared for each protection solution group in Table 1 of example 1. The survival rate calculation method is as follows:

survival (%) = [ viable cell count after lyophilization (CFU)/number of cells added before lyophilization (CFU) ] + 100%

In FIG. 7, it was found that the viable count of P.putida was maintained at 15% or more, with the highest survival rate of about 78% in group 2. The survival performance of each group in fig. 7 is similar to the viable count performance of fig. 1.

FIGS. 8 to 10 show the survival rates of

yeast

21849, 20405 and 22745 starters prepared from the respective protective solutions in Table 1 of example 1 after culturing at 25 ℃ for 72 hours, respectively. The survival rates of

yeast

21849, 20405 and 22745 were 53.22% (fig. 8, group 3), 47.2% (fig. 9, group 3) and 47.41% (fig. 10, group 3), respectively.

FIG. 11 shows the survival rate of Pseudomonas putida starter cultured at 30 ℃ for 72 hours, prepared for each of the protective solution groups in Table 2 and Table 3 of comparative example 1. The survival rates for P.putida were found to be 0.07%, 1.02% and 0.3% in FIG. 11, respectively.

FIG. 12 shows the survival rates of the starters for

yeast

21849, 20405 and 22745 prepared in each of the protective solutions in Table 2 and Table 3 of comparative example 1 after culturing at 25 ℃ for 72 hours. FIG. 12 shows the survival rate of the same trend as that of FIG. 6, which is about 2%, wherein the highest group is CN106244459-1 cultured in yeast 20405.

As can be seen from FIGS. 1 to 12, the tests of the formulations of the present invention and the former formulations with P.putida and 3 yeasts (21849, 20405 and 22745) revealed that the viable count and survival rate of the formulations of the present invention were significantly improved compared to the former formulations CN106244459-1, CN106244459-2 and CN 107828683. The unexpected enhancement effect of the invention is proved.

Example 2: freeze-dried bottle opener stability test

The freeze-dried starter prepared in example 1 was stored at 25 ℃ and 54 ℃ for 14 days, 1 g of the starter was redissolved in 10 mL of water, and serially diluted at an appropriate ratio, 100. mu.L of a dilution of Pseudomonas putida starter was applied to a Pseu F medium, and cultured at 30 ℃ for 72 hours, followed by colony counting.

The results are shown in fig. 13, which also shows the viable count of the freshly prepared freeze-dried starter. FIG. 13 shows that the viable cell counts of most formulation groups are significantly reduced and the maximum reduction is achieved in group 2 after culturing the Pseudomonas putida starter prepared from each of the protection solution groups in Table 1 of example 1 of the present invention at 30 ℃ for 72 hours after storage at 25 ℃ for 14 days, but the viable cell counts are still maintained at 2X 10⁸CFU/g. Group 4 had the smallest descending extent and the number of viable bacteria reached 1.2 x10⁹CFU/g, and maintained survival above 87% (not shown). From the results of the culture after storage at 54 ℃ for 14 days in FIG. 13, it was found that the number of viable bacteria was greatly decreased, and the number of viable bacteria in most groups was decreased to 10⁷Below CFU/g,

only groups

3, 4 and 7 remain at 10⁷CFU/g or more, and the highest viable count of group 7 is 6.2 x10⁷CFU/g. Jelly of the inventionThe dried starter has good stability, and can maintain 2 x10 after being stored for 14 days at 25 deg.C⁸The viable count above CFU/g is only about one log lower than that of the freeze-dried starter which is just prepared.

Claims

1. A starter protectant formulation comprising:

1.125 +/-10 to 4.5 +/-10 weight percent of trehalose;

2.25 +/-10 to 4.5 +/-10 weight percent of skimmed milk powder;

1.125 +/-10% to 2.25 +/-10% by weight of mannitol;

0.0225 +/-10% to 0.1125 +/-10% by weight of vitamin C;

0.225 plus or minus 10 percent to 1.125 plus or minus 10 percent of sodium glutethionine by weight; and

0.225 +/-10% to 1.125 +/-10% by weight of glycerol.

2. The starter protector formulation of claim 1, further comprising gram positive bacteria.

3. The starter protectant formulation of claim 2, wherein the gram-positive bacteria is a yeast.

4. The starter protector formulation of claim 1, further comprising gram negative bacteria.

5. A starter protector formulation as claimed in claim 4, wherein the gram-negative bacterium is Pseudomonas putida (Pseudomonas putida).

6. A starter protector formulation as claimed in any one of claims 1 to 5 comprising:

1.125 +/-5 to 4.5 +/-5 weight percent of trehalose;

2.25 +/-5 to 4.5 +/-5 weight percent of skimmed milk powder;

1.125 +/-5 to 2.25 +/-5 weight percent of mannitol;

0.0225 + -5% to 0.1125 + -5% by weight of vitamin C;

0.225 plus or minus 5 percent to 1.125 plus or minus 5 percent of sodium glutethinate in weight portion; and

0.225 +/-5% to 1.125 +/-5% by weight of glycerol.

7. The starter protector formulation of claim 6 comprising a group selected from the group consisting of groups 1 to 8:

group 1:

1.125 parts by weight of trehalose;

2.25 parts by weight of skimmed milk powder;

1.125 parts by weight of mannitol;

0.0225 parts by weight of vitamin C;

0.225 parts by weight of sodium glutethinate; and

0.225 parts by weight of glycerol;

group 2:

1.125 parts by weight of trehalose;

2.25 parts by weight of skimmed milk powder;

2.25 parts by weight of mannitol;

0.1125 parts by weight of vitamin C;

1.125 parts by weight of sodium glutethinate; and

1.125 parts by weight of glycerol;

group 3:

4.5 parts by weight of trehalose;

4.5 parts by weight of skimmed milk powder;

1.125 parts by weight of mannitol;

0.0225 parts by weight of vitamin C;

1.125 parts by weight of sodium glutethinate; and

1.125 parts by weight of glycerol;

group 4:

4.5 parts by weight of trehalose;

4.5 parts by weight of skimmed milk powder;

2.25 parts by weight of mannitol;

0.1125 parts by weight of vitamin C;

0.225 parts by weight of sodium glutethinate; and

0.225 parts by weight of glycerol;

group 5:

1.125 parts by weight of trehalose;

4.5 parts by weight of skimmed milk powder;

1.125 parts by weight of mannitol;

0.1125 parts by weight of vitamin C;

0.225 parts by weight of sodium glutethinate; and

1.125 parts by weight of glycerol;

group 6:

1.125 parts by weight of trehalose;

4.5 parts by weight of skimmed milk powder;

2.25 parts by weight of mannitol;

0.0225 parts by weight of vitamin C;

1.125 parts by weight of sodium glutethinate; and

0.225 parts by weight of glycerol;

group 7:

4.5 parts by weight of trehalose;

2.25 parts by weight of skimmed milk powder;

1.125 parts by weight of mannitol;

0.1125 parts by weight of vitamin C;

1.125 parts by weight of sodium glutethinate; and

0.225 parts by weight of glycerol;

group 8:

4.5 parts by weight of trehalose;

2.25 parts by weight of skimmed milk powder;

2.25 parts by weight of mannitol;

0.0225 parts by weight of vitamin C;

0.225 parts by weight of sodium glutethinate; and

1.125 parts by weight of glycerol.

8. A starter protector formulation as claimed in any one of claims 1 to 5 which is in powder form.

9. A starter protector formulation as claimed in claim 1 which consists of:

1.125 +/-10 to 4.5 +/-10 weight percent of trehalose;

2.25 +/-10 to 4.5 +/-10 weight percent of skimmed milk powder;

1.125 +/-10% to 2.25 +/-10% by weight of mannitol;

0.0225 +/-10% to 0.1125 +/-10% by weight of vitamin C;

0.225 plus or minus 10 percent to 1.125 plus or minus 10 percent of sodium glutethionine by weight;

0.225 plus or minus 10 percent to 1.125 plus or minus 10 percent of glycerol by weight; and

inevitable impurities contained in the above components after purification.

10. The starter protectant formulation of any of claims 1-5, wherein the skimmed milk powder is less than 4.5 parts by weight and the mannitol is less than 2.25 parts by weight.

11. Use of a starter protectant formulation according to claim 1 in the culture of gram-positive bacteria.

12. The use according to claim 11, wherein the gram-positive bacterium is a yeast.

13. Use of a starter protectant formulation according to claim 1 in the culture of gram-negative bacteria.

14. The use of claim 13, wherein the gram-negative bacterium is pseudomonas putida (c), (d) and d) b) anPseudomonas putida)。