阅读说明：本技术 一种仔猪专用功能性发酵酸奶及其制备方法 (Functional fermented yoghourt special for piglets and preparation method thereof ) 是由 苏勇 卢志阳 朱崇淼 于 2020-12-15 设计创作，主要内容包括：本发明公开了一种仔猪专用功能性发酵酸奶及其制备方法,包括如下重量份的原料：奶粉20-30份、蔗糖6-10份、寡果糖1-3份、低聚半乳糖1-3份、复合酸化剂0.25-0.5份和乳酸菌组合培养液5-8份。本发明的仔猪专用功能性发酵酸奶,通过特定的组份配方,使其富含蛋白质、有机酸、益生菌、益生元等营养性及抗腹泻物质,具有适口性好、pH值低、防病抗病等特点,能够降低胃内pH值,促进激活胃蛋白酶原,调节肠道微生态平衡,预防仔猪腹泻,促进仔猪生长。本发明的制备方法流程简单,易于操作,便于实际生产。(The invention discloses a functional fermented yoghourt special for piglets and a preparation method thereof, and the functional fermented yoghourt comprises the following raw materials in parts by weight: 20-30 parts of milk powder, 6-10 parts of cane sugar, 1-3 parts of oligofructose, 1-3 parts of galacto-oligosaccharide, 0.25-0.5 part of compound acidifier and 5-8 parts of lactobacillus combined culture solution. The functional fermented yoghourt specially used for the piglets is rich in nutritional and anti-diarrhea substances such as protein, organic acid, probiotics, prebiotics and the like through a specific component formula, has the characteristics of good palatability, low pH value, disease prevention and resistance and the like, and can reduce the pH value in the stomach, promote the activation of pepsinogen, adjust the intestinal microecological balance, prevent the diarrhea of the piglets and promote the growth of the piglets. The preparation method of the invention has simple flow, easy operation and convenient actual production.)

1. The functional fermented yoghourt special for the piglets is characterized by comprising the following raw materials in parts by weight: 20-30 parts of milk powder, 6-10 parts of cane sugar, 1-3 parts of oligofructose, 1-3 parts of galacto-oligosaccharide, 0.25-0.5 part of compound acidifier and 5-8 parts of lactobacillus combined culture solution.

2. The functional fermented yoghurt special for piglets according to claim 1, wherein the compound acidifier comprises lactic acid, caprylic acid, citric acid and calcium formate; the pH of the fermented yogurt is 3.5-4.0.

3. The functional fermented yogurt specially used for piglets according to claim 1, wherein the lactic acid bacteria in the lactic acid bacteria culture solution comprise enterococcus faecalis, Lactobacillus animalis and enterococcus hirae; viable bacteria content of lactic acid bacteria>10⁹CFU/ml。

4. The functional fermented yogurt special for piglets according to claim 1, wherein the fermented yogurt has a crude protein content of 3-4% and a dry matter content of 22-25%.

5. The preparation method of the functional fermented yoghourt special for the piglets as claimed in claim 1, which is characterized by comprising the following steps:

(1) culturing enterococcus faecalis, animal lactobacillus and enterococcus hirae at constant temperature at normal temperature;

(2) weighing 20-30 parts of milk powder, 6-10 parts of cane sugar, 1-3 parts of fructo-oligosaccharide and 1-3 parts of galacto-oligosaccharide according to parts by weight, adding water, heating and stirring to obtain a stock solution;

(3) sterilizing the stock solution;

(4) cooling the sterilized stock solution to 35-45 deg.C for inoculation, adding cultured enterococcus faecalis, Lactobacillus animalis, and enterococcus hirae into the stock solution, and sealing and fermenting in a constant temperature incubator;

(5) adding 0.25-0.5 part of compound acidifier into fermented yogurt, and adjusting pH to 3.5-4.0;

(6) and (4) carrying out aseptic packaging on the yoghourt with the adjusted pH value, and refrigerating at low temperature.

6. The method for preparing functional fermented yogurt specially used for piglets according to claim 5, wherein in the step (1), the temperature for constant-temperature culture is any temperature of 36-38 ℃, and the culture time is 20-24 h.

7. The method for preparing functional fermented yogurt specially used for piglets according to claim 5, wherein in the step (2), the heating and stirring temperature is 55-65 ℃ and the stirring time is 25-30 min.

8. The method for preparing functional fermented yogurt specially used for piglets according to claim 5, wherein in the step (3), the sterilization treatment is pasteurization treatment.

9. The method for preparing functional fermented yogurt specially used for piglets according to claim 5, wherein in the step (4), the inoculation amount of the inoculation is 6% -10%.

10. The method for preparing functional fermented yoghourt for piglets according to claim 5, wherein in the step (4), the temperature of the constant temperature incubator is any temperature of 36-38 ℃.

Technical Field

The invention relates to a biological fermented feed, in particular to a functional fermented yoghourt special for piglets.

Background

Since the birth of antibiotics, the antibiotics play an important role in disease resistance and disease prevention, and the wide use of the feed antibiotics in animal husbandry promotes the rapid and stable development of animal husbandry in China. However, with the development of intensive animal husbandry production, the disadvantages of antibiotic addition are gradually exposed, the drug resistance of harmful bacteria is stronger and stronger, and the antibiotic residues in animal products also harm human health along the food chain. The use of feed antibiotics has been declared banned for a long time in many developed countries in the world, and medicinal feed additives are generally banned in China in 7 months and 1 day in 2020. In this context, the search for feed products that can replace feeding antibiotics is a hot spot of concern for many scholars today.

Disclosure of Invention

The purpose of the invention is as follows: the first purpose of the invention is to provide a functional fermented yoghourt which can replace feeding antibiotics and can prevent piglet diarrhea; the second purpose of the invention is to provide a preparation method of the fermented yoghourt.

The technical scheme is as follows: the functional fermented yoghourt special for the piglets comprises the following raw materials in parts by weight: 20-30 parts of milk powder, 6-10 parts of cane sugar, 1-3 parts of oligofructose, 1-3 parts of galacto-oligosaccharide, 0.25-0.5 part of compound acidifier and 5-8 parts of lactobacillus combined culture solution.

Further, the compound acidifier comprises lactic acid, caprylic acid, citric acid and calcium formate; the pH of the fermented yogurt is 3.5-4.0.

Further, the lactic acid bacteria in the lactic acid bacteria culture solution comprise enterococcus faecalis, lactobacillus animalis and enterococcus hirae; content of lactic acid bacteria>10⁹CFU/ml。

Further, the content of crude protein in the fermented yoghourt is 3% -4%, and the content of dry matters is 22% -25%.

The preparation method of the functional fermented yoghourt special for the piglets comprises the following steps:

(1) culturing enterococcus faecalis, animal lactobacillus and enterococcus hirae at constant temperature at normal temperature;

(2) weighing 20-30 parts of milk powder, 6-10 parts of cane sugar, 1-3 parts of fructo-oligosaccharide and 1-3 parts of galacto-oligosaccharide according to parts by weight, adding water, heating and stirring to obtain a stock solution;

(3) sterilizing the stock solution;

(4) cooling the sterilized stock solution to 35-45 deg.C for inoculation, adding cultured enterococcus faecalis, Lactobacillus animalis, and enterococcus hirae into the stock solution, and sealing and fermenting in a constant temperature incubator;

(5) adding 0.25-0.5 part of compound acidifier into fermented yogurt, and adjusting pH to 3.5-4.0;

(6) and (4) carrying out aseptic packaging on the yoghourt with the adjusted pH value, and refrigerating at low temperature.

Further, in the step (1), the temperature of the constant-temperature culture is any temperature of 36-38 ℃, and the culture time is 20-24 h.

Further, in the step (2), the heating and stirring temperature is 55-65 ℃, and the stirring time is 25-30 min.

Further, in the step (3), the sterilization process is a pasteurization process.

Further, in the step (4), the inoculation amount of the inoculation is 6-10%.

Further, in the step (4), the temperature of the constant temperature incubator is any temperature of 36-38 ℃.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:

(1) the functional fermented yoghourt specially used for the piglets is rich in nutritional and anti-diarrhea substances such as protein, organic acid, probiotics, prebiotics and the like through a specific component formula, has the characteristics of good palatability, low pH value, disease prevention and resistance and the like, and can reduce the pH value in the stomach, promote the activation of pepsinogen, prevent the diarrhea of the piglets and promote the growth of the piglets.

(2) The lactic acid bacteria and the oligosaccharide prebiotics can also improve the immunity of piglets and reduce the diarrhea rate of the piglets, thereby reducing the morbidity and mortality of the piglets, preventing diseases and improving the growth performance of the piglets, thereby playing a role in replacing feed antibiotics.

(3) The preparation method of the fermented yoghourt has simple process, is easy to operate and is convenient for actual production.

Detailed Description

The technical solution of the present invention is further explained below.

The functional fermented yoghourt special for the piglets comprises the following raw materials in parts by weight: 20-30 parts of milk powder, 6-10 parts of cane sugar, 1-3 parts of fructo-oligosaccharide, 1-3 parts of galacto-oligosaccharide, 0.25-0.5 part of compound acidifier and 5-8 parts of lactic acid bacteria culture solution. The lactic acid bacteria in the lactic acid bacteria culture solution comprise enterococcus faecalis, animal lactobacillus and enterococcus hirae; content of lactic acid bacteria>10⁹CFU/ml. The compound acidifier comprises lactic acid, caprylic acid, citric acid and calcium formate; the pH of the fermented yogurt is 3.5-4.0.

The preparation method of the functional fermented yoghourt special for the piglets comprises the following steps:

(1) preparing a bacterial liquid: preparing MRS broth culture medium, sterilizing at 121 deg.C in autoclave, cooling to 35-45 deg.C, inoculating enterococcus faecalis, Lactobacillus animalis, and enterococcus hirae, respectively, culturing at 37 deg.C for 24 hr to obtain viable bacteria number of 10⁹CFU/ml of stock solution.

(2) Preparing stock solution: weighing baked 20-30% of milk powder, 6-10% of cane sugar, 1-3% of fructo-oligosaccharide and 1-3% of galacto-oligosaccharide according to the weight percentage, filling the mixture into a container with a certain amount of deionized water, slowly heating the mixture to 55-65 ℃, and uniformly stirring the mixture by using a glass rod to prepare the stock solution.

(3) And (3) sterilization: heating at 65 deg.C for 25-30min to pasteurize the stock solution.

(4) Inoculating and fermenting: and cooling the sterilized stock solution to 35-45 ℃ for inoculation, wherein the volume ratio of the three bacteria is 2:1:2, and inoculating the bacteria solution according to the volume ratio of 5-8%. Sucking enterococcus faecalis, animal lactobacillus and enterococcus hirae into the stock solution with a pipette gun, shaking gently, and fermenting in a 37 deg.C constant temperature incubator for 18-24 h.

(5) Adjusting the pH value: adding 0.25-0.5% of compound acidifier into fermented yogurt, and slightly shaking to mix acidifier. Adjusting the pH value to 3.5-4.0.

(6) Adding a stabilizer: adding 0.3-0.5% sodium benzoate to prolong storage time.

(7) Packaging and storing: aseptically packaging the pH-adjusted yogurt, and refrigerating at 4 deg.C.

Example 1: bacteriostatic test of compound acidifier

The compound acidifier is a mixture of lactic acid, caprylic acid, citric acid, calcium formate and tween, and has good bacteriostatic action on escherichia coli and salmonella, wherein the lactic acid accounts for 28-40%, the caprylic acid accounts for 8-20%, the citric acid accounts for 8-14%, the calcium formate accounts for 10-22%, and the tween accounts for 23-28%. The different mixture ratio combinations of the acidifier are divided into 7 test groups and 1 control group. The compounding ratio level of the acidifier, the uniform test design table and the acidifier test design table are respectively shown in tables 1, 2 and 3.

(1) Preparing the lactic acid, the caprylic acid, the citric acid and the calcium formate into a bacteriostatic mixture according to the proportion, dissolving the bacteriostatic mixture in 0.5(v/v) absolute ethyl alcohol, and then adding sterile liquid of 0.5% (w/v) tween-80. A mixed liquid level of 0.5(v/v) absolute ethyl alcohol and 0.5% (w/v) Tween-80 was used as a control group (the sterile liquid was proved to have no bacteriostasis by preliminary experiments).

(2) Culturing an indicator bacterium solution of escherichia coli and salmonella: inoculating Escherichia coli and Salmonella to sterilized nutrient broth culture medium, and culturing at 37 deg.C for 24 hr in shaking table to obtain 10⁷CFU/ml of the indicator bacteria solution.

(3) And (3) flattening: sterilizing nutrient agar culture medium, pouring 8-10ml agar into the culture dish to solidify, and standing at room temperature for 12 hr to disperse water.

(4) Inoculating a semi-solid culture medium: 10ml of the suspension (1X 10) was aspirated by a sterile pipette⁷CFU/ml) was added to 90ml of a medium (final concentration of bacterial suspension 1X 10) which had been sterilized and cooled to about 50 deg.C⁶CFU/ml), shaking, subpackaging into test tubes, each containing about 10-15ml of culture medium, pouring into a plate, solidifying (10-15 min), taking out Oxford cup with sterile forceps, and making into small hole;

(5) sample adding: the sample to be tested was added to the well using a pipette, 100. mu.l of each well, diffused at 4 ℃ for 1 hour, and then incubated in a 37 ℃ incubator for 12-16 hours (overnight). The diameter of the zone of inhibition was measured with a ruler using a cross method. And performing three parallel repetitions on each group, and calculating the average value of the diameters of the inhibition zones.

TABLE 1 acidifier acid ratio (%, m/m)

TABLE 2 Uniform test design Table

TABLE 3 acidifier test design Table (%, m/m)

TABLE 4 acidifier bacteriostatic effect table

As can be seen from Table 4, the lactic acid, the octanoic acid, the citric acid and the calcium formate which are combined as the composite acidifier have obvious inhibition effects on the diameters of inhibition zones of escherichia coli and salmonella of more than 15mm, wherein the 6 th group of acidifiers has the most obvious inhibition effect, and therefore, the acidifier combination is used as the optimal acidifier combination of the product.

Example 2 isolation of porcine lactic acid bacteria and screening of combinations of fermentation Agents

(1) Separating pig source lactobacillusSeparation: fresh piglet feces are soaked in physiological saline, the soaking solution is diluted and coated on an MRS culture medium, the MRS culture medium is placed in a constant temperature incubator at 37 ℃ for constant temperature culture for 24 hours, and single colonies with different forms are picked for purification culture and sequencing. Four different lactic acid bacteria were isolated, respectively: enterococcus faecalis, Lactobacillus animalis, enterococcus hirae, and Lactobacillus johnsonii. Determination of the OD of the lactic acid bacteria for 24h₆₀₀The values, the change of the pH value and the acid resistance are as follows:

TABLE 5 lactic acid bacteria OD₆₀₀Change in value

TABLE 6 pH Change of lactic acid bacteria

TABLE 7 survival rate of lactic acid bacteria at different pH values

From the above table, it can be seen that four strains of lactic acid bacteria growth performance enterococcus faecalis > lactobacillus johnsonii > lactobacillus animalis > enterococcus hirae; enterococcus faecalis, lactobacillus johnsonii has the lowest acidity and the best acid production performance; the lactobacillus johnsonii and enterococcus hirae have the best acid resistance.

(2) The combined screening test of the starter is as follows

Fermenting with a single strain: four lactic acid bacteria were inoculated into each of the sterilized raw milk, and fermented in the above-described embodiment, and the number of viable bacteria and the change in pH after fermentation were measured. The results are shown in Table 8

TABLE 8 fermentation results of individual strains

Fermenting by two strains: two strains with the highest survival rate when the pH is 3 are adopted, and the lactobacillus animalis and the enterococcus hirae are used as fermenting agents to research the lactobacillus animalis: the influence of the enterococcus hirae inoculation ratio (1: 1, 1:2, 1: 3, 2:1 and 3: 1) on the pH value and the viable count. The results of the two strain fermentation tests are shown in Table 9.

TABLE 9 results of fermentation test with two strains

Fermenting by three strains: and performing orthogonal test by using two strains with the highest survival rate when the pH value is 3 and three strains of enterococcus faecalis, lactobacillus animalis and enterococcus hirae as fermenting agents. The orthogonal test design is shown in Table 10, and the three-strain fermentation results are shown in Table 11.

TABLE 10 orthogonal test design Table (v/v)

TABLE 11 results of fermentation test with two strains

The conclusion can be drawn from the above table that the mixed fermentation effect of the three strains is better than that of single-strain and double-strain fermentation. The pH value difference of all experimental fermentation end points is not large, the mixed fermentation effect of the three strains is the best, and the number of viable bacteria is the most. Three-strain mixed fermentation combination 5: the ratio of enterococcus faecalis to lactobacillus animalis to enterococcus hirae is 2:1:2, the effect is optimal, and the combination is used as the optimal leavening agent.

EXAMPLE 3 screening of optimal oligosaccharide combinations

Different proportions of oligofructose were studied with a total amount of oligosaccharide added of 2%: the galacto-oligosaccharides (1: 1, 1:2, 1: 3, 2:1, 3: 1) had an effect on the number of viable bacteria and pH of the fermentation results, and the optimum oligosaccharide addition ratio was determined, and the results are shown in Table 12.

TABLE 12 fermentation results for different oligosaccharide ratios

The fructooligosaccharides are prepared by the following steps: the optimal ratio of galactooligosaccharides (1: 1) was determined and the effect of different amounts of oligosaccharides (1%, 2%, 3%) on the fermentation results was investigated and the results are shown in Table 13.

TABLE 13 fermentation results with different oligosaccharide addition amounts

As can be seen from the table, the ratio of oligofructose to galactooligosaccharide was 1:1, and the most viable fermentation bacteria were found at an addition amount of 2%, resulting in the best effect.

Example 4 Effect of acidulants on pH and viable count of fermented yogurt

Based on the embodiments 1-3, acidulants with different concentrations were added to the fermented yogurt, and the pH value and viable count of the yogurt were measured, the results are shown in Table 14.

TABLE 14 pH change and viable cell rate before and after adding acidifying agent

The results show that the addition of 0.25-0.5% of acidifying agent can reduce the pH value of the yoghourt to about 4.0 and has higher viable count.

Example 5 Effect of storage time on viable fermented yogurt bacteria and pH

TABLE 15 Effect of storage time on viable fermented yogurt bacteria (CFU/ml) and pH

Fermented yogurt was prepared according to the above 1-5, and the fermentation was stored in a refrigerator at 4 deg.C, and the viable count and pH of the yogurt were measured at 0, 7, 14, 21, 28, and 35d, and the results are shown in Table 15. As can be seen from the table, the fermented yoghourt is stable in viable count after being refrigerated for 0-21 days at 4 ℃, the pH value is reduced by 21-35 days, and the viable count is obviously reduced, so that the yoghourt has the shelf life of being stored for 21 days at 4 ℃ in a dark place.