CN114847350A - Milk powder special for conditioning gastrointestinal tract and preparation method thereof - Google Patents

Abstract

The invention provides milk powder special for conditioning gastrointestinal tracts and a preparation method thereof, wherein different dosage groups of the milk powder special for conditioning the gastrointestinal tracts, which is prepared from 81.33% of milk solid addition amount, 6% of oligosaccharide, 10% of linoleic acid and 2.67% of maltodextrin, have good effects on relaxing the bowels of mice, and the effects on relaxing the bowels of medium and high dosage groups are more obvious. And when the addition amount of oligosaccharide is 6g/100g, the acid production capacity of bifidobacterium and lactobacillus acidophilus is best, and when the ratio of milk powder to whey protein powder is 9:1, the growth condition of bifidobacterium is best.

Description

Milk powder special for conditioning gastrointestinal tract and preparation method thereof

Technical Field

The invention belongs to the technical field of milk powder, and particularly relates to milk powder special for conditioning gastrointestinal tracts and a preparation method thereof.

Background

At present, functional milk powder is added with a lot of functional factors, which commonly comprise saccharides, dietary cellulose, polypeptide, vegetable protein, prebiotics and probiotics. The milk powder has the characteristic of balanced and comprehensive nutritional elements, and the prebiotics or the prebiotics combined with unsaturated fatty acids are additionally added in the milk powder, so that the milk powder is a new direction for developing functional dairy products for assisting in improving the functions of the stomach and the intestinal tract and stabilizing the intestinal flora, and is a new hotspot of dairy product manufacturers in China.

Oligosaccharides and unsaturated fatty acids have been widely used in products such as modified milk powders, milk beverages, and the like. It is generally believed that the functional oligosaccharide can specifically induce the growth of probiotics in the intestinal tract, and has the effects of promoting intestinal peristalsis and moistening the intestines. The fructo-oligosaccharide has the function of regulating intestinal flora. At present, the compound is used as a food ingredient or an auxiliary material, and is generally applied to dairy products and specific nutritional health-care functional foods. Xylo-oligosaccharide is one of the good functional factors for the proliferation of probiotics. Unsaturated fatty acids are also commonly added functional factors, and among them, linoleic acid has the function of regulating intestinal flora and is widely used as a food nutritional supplement.

The invention researches the influence of the milk powder special for conditioning the gastrointestinal tract on the intestinal tract probiotic effect by measuring the change condition of the mouse intestinal tract flora and the gastrointestinal tract regulation function, so as to provide a certain reference value for fully utilizing the functional oligosaccharide and the unsaturated fatty acid, developing the functional milk powder and developing a new product.

Disclosure of Invention

Aiming at the problems, the invention provides milk powder special for conditioning gastrointestinal tracts and a preparation method thereof.

The technical scheme adopted by the invention is as follows:

the milk powder special for conditioning gastrointestinal tract comprises the following components in percentage by weight: 74-82% of milk solid, 1.2-6% of oligosaccharide, 6-12% of linoleic acid and 2-3.5% of maltose.

Preferably, the following weight percentage is adopted: 81.33% of milk solid, 6% of oligosaccharide, 10% of linoleic acid and 2.67% of maltodextrin.

Preferably, the milk solids comprise milk powder and whey protein powder, and the milk powder and the whey protein powder are mixed according to the ratio of 1: 9-9: 1.

Preferably, the milk powder and the whey protein powder are mixed according to a ratio of 9: 1.

Preferably, the oligosaccharide comprises fructo-oligosaccharide and xylo-oligosaccharide, and the fructo-oligosaccharide and the xylo-oligosaccharide are mixed according to a ratio of 7: 3-9: 1.

Preferably, the fructo-oligosaccharide is mixed with xylo-oligosaccharide according to a ratio of 7: 3.

A preparation method of milk powder special for conditioning gastrointestinal tract comprises the following steps:

step 1: carrying out spray drying on the raw milk to obtain spray-dried whole milk powder;

step 2: pouring the whole milk powder obtained in the step 1, the whey protein powder, the fructo-oligosaccharide, the xylo-oligosaccharide, the linoleic acid and the maltodextrin into a mixing instrument according to a certain proportion and mixing uniformly.

The invention has the beneficial effects that:

1. different dosages of the milk powder special for conditioning the gastrointestinal tract, which is prepared according to the milk solid adding amount of 81.33%, the oligosaccharide of 6%, the linoleic acid of 10% and the maltodextrin of 2.67%, have good effects on the effects of moistening the intestines and relaxing the bowels of mice, and the effects of moistening the intestines and relaxing the bowels of medium and high dosages of groups are more obvious. And when the addition amount of oligosaccharide is 6g/100g, the acid production capacity of bifidobacterium and lactobacillus acidophilus is best, and when the ratio of milk powder to whey protein powder is 9:1, the growth condition of bifidobacterium is best.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a morphological diagram of a colony of Lactobacillus acidophilus 336636 of the present invention;

FIG. 2 is a colony morphology of Bifidobacterium animalis subsp lactis 192320 in accordance with the present invention;

FIG. 3 shows the effect of oligosaccharide on the OD value of the in vitro milk powder fermentation broth of Bifidobacterium animalis in different ratios;

FIG. 4 shows the actual effect of oligosaccharide on the OD value of the Lactobacillus acidophilus in vitro milk powder fermentation broth;

FIG. 5 shows the effect of different amounts of compounded oligosaccharides on the viable count of Lactobacillus acidophilus;

FIG. 6 is a graph showing the effect of different milk solids ratios on the viable count of bifidobacteria in animals;

FIG. 7 shows the effect of different milk solids ratios on the viable count of Lactobacillus acidophilus.

The reference numbers are as follows:

1. a leaf shroud; 2. the outer wall of the leaf shroud; 3. grid section; 4. a flange step; 5. the wall surface of the casing; 6. a smooth curve.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.

The embodiment provides milk powder special for conditioning gastrointestinal tract, which is prepared from the following components in percentage by weight: 74-82% of milk solid, 1.2-6% of oligosaccharide, 6-12% of linoleic acid and 2-3.5% of maltose.

The milk solid comprises milk powder and whey protein powder, and the milk powder and the whey protein powder are mixed according to the ratio of 1: 9-9: 1.

The oligosaccharide comprises fructo-oligosaccharide and xylo-oligosaccharide, and the fructo-oligosaccharide and the xylo-oligosaccharide are mixed according to the ratio of 7: 3-9: 1.

A preparation method of milk powder special for conditioning gastrointestinal tract comprises the following steps:

step 1: carrying out spray drying on the raw milk to obtain spray-dried whole milk powder;

step 2: pouring the whole milk powder obtained in the step 1, the whey protein powder, the fructo-oligosaccharide, the xylo-oligosaccharide, the linoleic acid and the maltodextrin into a mixing instrument according to a certain proportion and mixing uniformly.

In the following, the influence of different mixing ratio addition amounts of oligosaccharides in milk powder on the intestinal tract probiotic function is researched through the determination of the growth condition of oligosaccharides on probiotics in vitro experiments, and the best compound ratio to the lactobacillus fermentation condition is researched through taking milk solids with different compound ratios as nutritional factors. Selecting the optimal compound ratio of oligosaccharide and milk solid.

Milk solid, total oligosaccharide and conjugated linoleic acid microcapsule powder are used as main raw materials, sensory evaluation is used as an index, and the optimal formula of the milk powder for conditioning gastrointestinal tracts is determined by combining a single factor with an orthogonal test.

The influence of the milk powder special for gastrointestinal tract conditioning on the intestinal tract probiotic effect is explored by measuring the change condition of the mouse intestinal tract flora and the gastrointestinal tract regulation function.

1. Apparatus and device

1.1 Experimental strains

Lactobacillus acidophilus (Lactobacillus acidophilus.336636) and Bifidobacterium animalis (Bifidobacterium animalis subsp.192320) are from North Na Biotech, Inc.

1.2 Experimental materials

Table 1 main test materials

Table 1 Main test materials

1.3 Experimental reagents

TABLE 2 reagents purchased primarily

Table 2 Main reagents purchased

1.4 Main instruments and devices

TABLE 3 Main instruments

Table 1-3 Main instruments

2. Experimental methods

2.1 Experimental animals

72 KM mice, all female, Xinjiang medical university laboratory animal center, license number: SYXK (NEW) 2021-.

2.2 Strain activation

The freeze-drying tube is opened in a safety cabinet, lactobacillus acidophilus and bifidobacterium animalis are inoculated in a self-prepared MRS culture medium in an inoculation amount of 3 percent, and the mixture is subjected to anaerobic culture in an anaerobic bag at 37 ℃ for 24 hours, and the operation is repeated.

2.3 determination of OD value

The glucose in the conventional MRS culture medium is replaced by oligosaccharide to prepare the MRS-oligosaccharide culture medium. Keeping the total addition of fructo-oligosaccharide and xylo-oligosaccharide in the culture medium unchanged, adjusting different compounding ratios, setting the culture medium taking glucose as a carbon source as a control, inoculating lactobacillus acidophilus and bifidobacterium animalis in each culture medium in an inoculation amount of 3%, taking out the culture medium to observe the proliferation condition after culturing at 37 ℃ in an anaerobic environment for 24h, measuring OD values of different proportioning groups, determining the optimal oligosaccharide mixing ratio, and continuously measuring the absorbance (A) at 600nm after 48h of the culture medium. Calculating the probiotic index PI values of the two strains according to the following formula:

wherein PP0 and PP48 are respectively the absorbance of probiotics cultured for 0h and 48h (carbon source is oligosaccharide); PG0 and PG48 are respectively absorbance of probiotics cultured for 0h and 48h when the carbon source is glucose; PN0 and PN48 show that the probiotics are cultured in a sugar-free medium for 0h and 48h respectively.

2.4 determination of viable count of Bifidobacterium animalis and Lactobacillus acidophilus

Milk powder-oligosaccharide liquid medium: milk powder 50g, oligosaccharide (fructo-oligosaccharide, xylo-oligosaccharide): 10g of distilled water 1L. Taking milk powder added with different oligosaccharide mixing ratios as a culture medium, setting a carbon source as glucose as a control group, respectively inoculating two strains of bacteria which are activated in advance into the milk powder culture medium with different oligosaccharide mixing ratios in an inoculation amount of 3%, culturing for 24h, performing gradient dilution, selecting a gradient with convenient counting for gradient dilution coating, counting grown bacterial colonies, and selecting a proportioning group with the best proliferation condition as an optimal group.

2.5 determination of the pH value

Taking milk powder added with different oligosaccharide mixing ratios as a culture medium, taking a milk powder culture medium with a carbon source of glucose as a control group, after the culture medium is sterilized, respectively inoculating the two strains of bacteria which are activated in advance into the milk powder culture medium with different oligosaccharide mixing ratios in an inoculation amount of 3%, culturing for 24h, and taking out bacteria liquid to measure the pH values of different components.

2.6 determination of influence of addition amount of oligosaccharide in formula milk powder on viable counts of two common probiotics

The method comprises the steps of rejuvenating strains before an experiment to ensure and improve the activity of the strains, changing the total addition amount of oligosaccharides by referring to the optimal mixing ratio of the oligosaccharides obtained in the experiment, setting 12 groups of different addition amounts with the maximum addition amount within the range specified in the national standard GB 14880-2012, respectively inoculating two strains after rejuvenation into different components in an inoculation amount of 3%, culturing for 24 hours, diluting and coating, observing and counting grown colonies, and selecting the component with the best proliferation condition.

2.7 determination of influence of culture medium prepared from milk powder and whey protein powder in different proportions on viable count of two common probiotics

Milk powder whey protein powder-liquid culture medium: milk powder whey protein powder 50g in total (milk powder whey protein powder 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1), glucose: 10g of distilled water 1L. Setting 9 groups of different mixing proportions, taking the second rejuvenated liquid culture medium, inoculating the liquid culture medium into culture media with different components according to the inoculation amount of 3%, culturing for 24 hours at 37 ℃ in an anaerobic environment, and taking out for gradient dilution and coating.

2.8 formula principle of milk powder

According to the scientific research report (2021) of Chinese resident dietary guidelines, 300g of milk and milk products are taken every day, so that the mass of the special milk powder is set to be 30g (the special milk powder can have the same nutritional value with milk when the compounding ratio of the milk powder is 1:8, which is equivalent to 270g of milk taken every day), and less than 30g of the special milk powder is supplemented by maltodextrin.

2.9 Single factor and orthogonal design of experiments

According to the preparation principle, the design of a single-factor test (in terms of special milk powder 30 g)

1 content of Whole milk powder and whey protein

2 oligofructose, oligoxylose

Orthogonal experiment factor level meter

Orthogonal experimental design scheme

2.10 animal grouping and modeling

72 mice were randomly divided into 6 groups: blank group, model group, positive control group, low dose group, medium dose group, high dose group, 12 per group.

The experimental animal modeling method is slightly modified by referring to methods such as a square circle and the like. Blank group (0.9% physiological saline), model group (0.9% physiological saline +9.38mg/kg BW loperamide hydrochloride), positive control group (1.25g/kg BW commercial milk powder +9.38mg/kg BW loperamide hydrochloride), low dose group (0.625g/kg BW self-made special milk powder +9.38mg/kg BW loperamide hydrochloride), medium dose group (1.25g/kg BW self-made special milk powder +9.38mg/kg BW loperamide hydrochloride), high dose group (2.5g/kg BW self-made special milk powder +9.38mg/kg BW loperamide hydrochloride).

The blank group and the model group are given with 0.9 percent of normal saline, and the positive control group, the low, medium and high milk powder dosage groups are respectively calculated according to 2.5 times, 1.25 times, 2.5 times and 5 times of the recommended amount of human bodies, namely 1.25g/kg BW, 0.625g/kg BW, 1.25g/kg BW and 2.5g/kg BW are drenched with corresponding dosage of milk powder for continuous drenching for 14 d. At 8d, the rest groups except the blank group were infused with loperamide hydrochloride 9.38mg/kg BW twice a day and the drug was administered for 7d continuously while molding.

(1) General conditions mice activity status, hair color change and weight change were observed daily in experimental and blank groups. The body weight of each animal was weighed and recorded during the experiment, and the mass changes of the blanks and other groups were calculated and compared.

(2) After 7d of safety evaluation of the special milk powder on tissue and organs of the mice, two mice of the mice before the experiment, the mice of the blank group, the mice of the low dose group, the mice of the medium dose group and the mice of the high dose group are taken at random, the animals are killed through spinal dislocation, the abdominal cavity is opened to take out the small intestine of each mouse, and then tissue section observation is carried out.

2.11 influence of milk powder specially used for gastrointestinal tract conditioning on intestinal flora of loperamide-induced constipation mice

After 14d, fresh mouse feces were collected by pressing and abdominal massage into sterile EP tubes, and feces were snap frozen in liquid nitrogen and stored in a-80 ℃ freezer. Three samples were randomly taken from each group and sent to Shanghai Bionics GmbH by dry ice delivery. 16S microbial classification and sequencing were performed. Providing sample DNA and performing 16S rDNA amplification on a V3-V4 region, wherein the primer sequences are as follows: 341F: CCTACGGGNGGCWGCAG, 805R: GACTACHVGGGTATCTAATCC are provided. The purified amplification products were ligated to a sequencer to build a sequencing database and sequenced on the Illumina platform.

2.12 mouse defecation test

After 2.11 experiments, the mice in each group were fasted but normally drunk water for 12h, except for the blank group, the mice in each other group were administrated loperamide hydrochloride (9.38mg/kg BW) for 30min and then administrated with 0.4% Evans blue (10ml/kg BW) and started timing, and each experimental mouse was individually fed with free food and water, and the time of the first defecation, the time of the first blue defecation and the total number of the defecation granules of 6h were observed and recorded.

2.13 intestinal propulsion experiment in mice

The experiment refers to the intestinal propulsion method of the Beeffrageena efflorescens et al. After 1.2.6, the mice in each group were fasted but normally drunk water for 12h, except for the blank group, the mice in each other group were drenched with 0.4% Evans blue (10ml/kg BW)30min after the drenched with loperamide hydrochloride (9.38mg/kg BW), and after twenty minutes, the mice in each group were sacrificed. The abdominal cavity was opened and the intestine from the pylorus to the ileocecal region was collected. The intestinal tract was gently straightened, the total length of the small intestine was measured, and the rate of evans blue propulsion in the small intestine of mice was calculated.

Evans blue propulsion probability (%) ═ evans blue propulsion length (cm)/total small intestine length (cm) × 100

2.13 data processing

The experimental data analysis was performed using SPSS 26 and Excel 2016, and the results are indicated by "". P <0.05 indicates statistically significant differences.

3. Results and analysis

3.1 Strain activation

As shown in fig. 1, the colony morphology of lactobacillus acidophilus 336636: the size is 1-2mm, and the shape is as follows: round, white in color, flat in crown, thick in texture.

As shown in fig. 2, bifidobacterium animalis subsp lactis 192320 colony morphology: the size is 2mm, and the shape is as follows: round, milky white in color, flat in degree of swelling, and easy to pick up when wet.

3.3 Effect of oligosaccharides with different mixing ratios on OD values of two probiotic in-vitro fermentation solutions

The effect of the different mixing ratios of fructo-oligosaccharide (FOS) and xylo-oligosaccharide (XOS) on the OD value of the fermentation broth inoculated with bifidobacteria is shown in figure 3, and the OD values detected by the oligosaccharide adding proportioning groups are obviously improved compared with the OD value detected by the control group, which indicates that the bifidobacteria can utilize the compound oligosaccharide for fermentation and growth. When the mixing ratio is 7:3, the OD value of the bifidobacterium is 1.632A, the bacterial concentration is the highest, then 8:2, the OD value is 1.615A, and the difference with the 7:3 ratio group is not obvious (P is more than 0.05), which shows that for the bifidobacterium, the effect of the oligosaccharide fermentation with the mixing ratio of 7:3 is similar to that of the mixing ratio of 8: 2.

The influence of different mixing ratios of FOS and XOS on the OD value of the lactobacillus acidophilus inoculated fermentation liquid is known, the OD values measured by all mixing forms are obviously higher than that of a glucose control group (P is less than 0.05), and the fact that the oligosaccharide is added in the mixing form to obviously promote the growth of lactobacillus acidophilus (P is less than 0.05) shows that the proliferation effect of the compound oligosaccharide on lactobacillus acidophilus is better than that of single oligosaccharide. Compared with xylo-oligosaccharide, fructo-oligosaccharide has better proliferation effect on lactobacillus acidophilus. As shown in FIG. 4, the OD reached a maximum of 1.422 when the mixing ratio was 7:3, which is significantly higher than that of the other ratio groups (P <0.05), indicating that the growth efficiency was the best for Lactobacillus acidophilus when the mixing ratio reached 7: 3.

The calculated probiotic index PI values of the two strains after fermentation for 48 hours in a sugar-free MRS culture medium respectively with the optimal compound oligosaccharide as a unique carbon source and glucose as a unique carbon source are shown in the following table.

TABLE 7 PI values of fermentation broths after 48h of two strains culture

The two strains of lactic acid bacteria have different utilization degrees of the compound oligosaccharide. The PI values of the two lactobacillus fermented compound oligosaccharides reach over 0.6, which shows that the compound oligosaccharides have good proliferation effect. Compared with two strains of bacteria, the PI value of the lactobacillus acidophilus is larger than that of the bifidobacterium animalis, the lactobacillus acidophilus has stronger fermentation capacity than that of the bifidobacterium animalis under the condition, the two lactic acid bacteria are fermented by a culture medium with the same carbon source, and the lactobacillus acidophilus has better proliferation effect compared with other lactic acid bacteria. The compound oligosaccharide lactobacillus acidophilus has stronger utilization capability. The reason may be that different strains secrete different glycosidases and different strains are good at hydrolyzing oligosaccharides with different chain lengths.

3.3 Effect of oligosaccharides with different mixing ratios on viable count of two probiotics

The influence of oligosaccharide with different mixing ratios on the viable count of bifidobacterium is known, and the viable count is different when the oligosaccharide mixing ratios in the milk powder are different. The number of viable bacteria of the matching group added with the oligosaccharide is obviously higher than that of the control group not added with the oligosaccharide (P is less than 0.05), and the result simultaneously shows that the oligosaccharide has a certain promotion effect on the growth of the bifidobacterium. It is shown that FOS and XOS are added into milk powder in a mixed form to promote the growth and reproduction of bifidobacteria. When the mixing ratio is 7:3, the viable count reaches the highest value, namely 8.03 multiplied by 107cfu/ml, and is obviously higher than that of a control group and other proportioning groups (P < 0.05).

The effect of adding FOS and XOS in different mixing ratios to the lactobacillus acidophilus can be known. The number of live bacteria in the group added with oligosaccharide in different mixing ratios is obviously higher than that in the control group (P <0.05) which does not add oligosaccharide carbon source as glucose, which indicates that the oligosaccharide added into the milk powder in a mixing mode can also promote the growth of lactobacillus acidophilus. Wherein, when the ratio is 7:3, the viable count of the lactobacillus acidophilus reaches the highest value, namely 5.58 multiplied by 107cfu/ml, and secondly 9:1, and no significant difference exists between the two groups (P is more than 0.05).

3.4 Effect of oligosaccharides in different mixing ratios on pH

The influence of the mixing ratio of different oligosaccharides added into the milk powder on the pH value of the inoculated bifidobacterium is known, the pH values among different oligosaccharide proportioning groups are different, and the pH values of all the milk powder added with the oligosaccharides are obviously lower than that of a control group without the oligosaccharides (P < 0.05). The bifidobacterium can ferment to produce acid by using the compound oligosaccharide, and the acid production is obviously greater than that of a milk powder control group. At a ratio of 7:3, the lowest pH was found to be 4.19, which was a 6.05% reduction compared to the control. At a ratio of 8:2, the pH was 4.21, which was reduced by 5.61% compared to the control group, and there was no significant difference between the two groups (P >0.05), indicating that the acid production of bifidobacterium was similar using the mixture ratio of 7:3 and 8:2 oligosaccharides, and was significantly better than that of the other group (P < 0.05).

The influence of the different oligosaccharide mixing ratios added to the milk powder on the pH value of lactobacillus acidophilus is known, and the pH value of the milk powder added with the mixed oligosaccharide is obviously lower than that of a control group without the oligosaccharide (P < 0.05). At a 7:3 mixing ratio, the pH was at least 4.39, with a 5.59% difference in pH reduction compared to the control (P < 0.05). The second is the 8:2 ratio group, the pH result is 4.41, and the difference is significantly reduced by 5.16% compared with the control group (P < 0.05). At a ratio of 9:1, pH 4.40, three matched groups were significantly lower than the other matched groups (P <0.05), but there were no significant differences between the three groups (P > 0.05). The growth effect is best when the mixing ratio of GOS to FOS in the formula milk powder is 7:3, 8:2 and 9: 1.

3.5 influence of different oligosaccharide addition amounts in milk powder on viable count of probiotic

The different addition amounts of oligosaccharide in the milk powder have different influences on the intestinal probiotic function, the optimal ratio of the oligosaccharide is determined according to the results, the total addition amount of the oligosaccharide is changed, and the proliferation condition of the viable count is determined by taking the milk powder culture medium without the oligosaccharide as a control group. The influence results of different total oligosaccharide addition amounts on bifidobacteria show that the viable count of the bifidobacteria in the oligosaccharide milk powder added group is all obviously higher than that in the control group (P < 0.05). With the increase of the addition amount of oligosaccharide, the number of live bifidobacteria tends to increase gradually. When the total addition amount is 6g/100g, the colony number reaches the highest 1.62 multiplied by 108cfu/ml, then 5g/100g and 5.5g/100g dose groups, and the three dose groups have no significant difference (P >0.05) and are all significantly higher than other dose groups (P < 0.05).

The counting result of the number of live bacteria of lactobacillus acidophilus added with different dosages of mixed oligosaccharide is shown in figure 5, the growth effects of different total oligosaccharide adding dosages on lactobacillus acidophilus are different and are all obviously superior to those of a control group (P <0.05) without adding oligosaccharide, and the fact that the addition of the mixed oligosaccharide in the milk powder plays a role in promoting the growth and the reproduction of lactobacillus acidophilus is shown. With the increase of the additive amount, the number of the viable bacteria of the lactobacillus acidophilus is increased. When the additive amount is 6g/100g, the growth effect of the lactobacillus acidophilus can reach 7.2 x 107cfu/ml optimally, and is obviously higher than that of a control group and other several proportioning groups (P < 0.05). When the addition amount is 1g/100g, the minimum viable count is 5.87 multiplied by 107cfu/ml, which is obviously lower than other proportioning groups (P is less than 0.05), the analysis reason is probably that the carbon source is insufficient, and the proliferation of bifidobacterium and lactobacillus can not be well promoted.

3.6 influence of different milk solid mixing proportions in the milk powder special for gastrointestinal tract conditioning on the viable count of probiotics

The effect of different mixing ratios of milk solids on the viable count of bifidobacteria is shown in fig. 6, with different mixing ratios of milk solids and different measured viable counts. In the set proportioning group, when the proportion of the milk powder is continuously improved and the proportion of the whey protein powder is continuously reduced, the number of the live bacteria shows an ascending trend, and when the proportion of the milk powder and the whey protein powder reaches 9:1, the number of the live bacteria reaches 2.79 multiplied by 108cfu/ml to the maximum and is far higher than that of other proportioning groups (P <0.05), which indicates that the milk solid proportion is most beneficial to the growth and proliferation of bifidobacteria when the proportion of the milk solid is 9: 1.

The effect of different mixing ratios of milk solids on the viable count of lactobacillus acidophilus is shown in fig. 7, where the mixing ratios of milk solids are different and the measured viable counts are also different. When the ratio is 9:1, the viable count of the bifidobacteria reaches 1.25 multiplied by 108cfu/ml to the maximum and is far higher than that of other proportioning groups (P <0.05), and when the ratio is 6:4, the viable count of the bifidobacteria is 9.75 multiplied by 107cfu/ml, and the two proportioning groups have obvious difference (P <0.05), which indicates that the milk powder and the whey protein powder ratio reaches 9:1, and is most beneficial to the proliferation of the lactobacillus acidophilus.

3.7 one-way and orthogonal test

The optimum addition amount of milk solids is 24g, the optimum addition amount of oligosaccharides is 1.8g, and the optimum addition amount of linoleic acid is 3.15 g.

The formula of the milk powder special for relaxing bowel is as follows: the adding amount of each component is 81.33% of milk solid adding amount, 6% of oligosaccharide, 10% of linoleic acid and 2.67% of maltodextrin respectively according to 30 g.

3.8 measurement of gastrointestinal tract regulatory function and changes of mouse intestinal flora

(1) In the whole experiment, the mouse is normal in basic state, and has no abnormality in activity and hair color. The weight of the mice has no significant difference (P is more than 0.05) among mice in each group before the experiment, the blank group after the experiment for 7d has no significant difference (P is more than 0.05) compared with other groups, which shows that the stomach-perfused normal saline and the special milk powder for gastrointestinal tract conditioning have no influence on the weight of the mice, the blank group after the experiment for 14d has significant difference (P is less than 0.05) with a model group, a positive control group and a low-dose group, which shows that the loperamide hydrochloride has certain influence on the weight of the mice, and the loperamide hydrochloride can slow down the weight growth speed of the mice from the weight growth value. This conclusion is consistent with the conclusion that Huang Xiao Li et al, in the study of building and blank groups, have statistical differences in body weight, fecal water content and oro-anal transit time. In addition, in the experimental process, the shape of the feces of the mouse is normal, no constipation, diarrhea and other adverse symptoms appear, and the special milk powder for conditioning the gastrointestinal tract, which is compounded with the functional oligosaccharide and the conjugated linoleic acid, has no adverse effect on the growth condition of the mouse.

(2) Tissue section observation shows that the small intestine wall goblet cells of the mice in each dose group are complete, and the blank group and each dose group have no obvious difference, which indicates that the special milk powder added with the two oligosaccharides and the conjugated linoleic acid has no damage to the small intestine of the mice.

(3) Compared with the blank group, the time for defecation of the first mouse grain and the time for defecation of the blue stool of the first mouse grain of the model group are obviously increased (P is less than 0.05), and the total number of the first mouse grains is obviously reduced (P is less than 0.05), which indicates that the constipation model is successfully modeled. Compared with a model group, the positive control group has no obvious difference between the first defecation time and the number of defecation granules in 6 hours (P is more than 0.05), which shows that the commercial milk powder has a certain gastrointestinal tract conditioning effect but is not obvious, and the positive control group and the low dose group have no obvious difference between the first defecation time, the blue defecation time and the total number of defecation granules in 6 hours, which shows that the effect of the low dose special milk powder is similar to that of the commercial milk powder. The self-made milk powder special for conditioning the gastrointestinal tract has the effects of remarkably promoting the first defecation time and the first defecation time of constipation mice and is dose-dependent. The dosage of the milk powder special for conditioning the gastrointestinal tract is increased, so that the effect of promoting defecation of mice is increased. The experimental result is similar to the experimental result of researching the influence of the prebiotics milk powder on the defecation of rats. While there was no significant difference in the number of 6h faecal particles between the different dose groups (P >0.05), the analytical reasons may differ from stool size, and stool hardness and water content.

(4) The advancing rate of the model group Evans blue is obviously different from that of the blank group Evans blue (P is less than 0.05), which shows that the constipation model of the mouse is successfully established under the action of the loperamide hydrochloride. After the mice are perfused with the gastrointestinal tract conditioning special milk powder 14d with different dosages, the ink propulsion rate has no obvious difference (P is less than 0.05) between the positive control group and the model group, and the evans blue propulsion rate of the mice with the low, medium and high dosages is obviously increased (P is less than 0.05) compared with the model group, which shows that the gastrointestinal tract conditioning special milk powder can promote the intestinal tract peristalsis of the mice, and the evans blue propulsion rate is also increased along with the increase of the dosage. The commercial milk powder has no obvious effect. The conclusion is consistent with the result of researching the influence of different oligosaccharide addition amounts in milk powder on the bowel relaxing and bowel advancing of mice by Liu Meng et al. The milk powder containing oligosaccharide and linoleic acid specially for conditioning gastrointestinal tract is proved to be capable of promoting and enhancing the propulsive force of mouse small intestine. Whereas the low and medium dose groups exhibited similar rates of propulsion.

(5) The composition of the mouse fecal flora is analyzed by adopting a 16S rDNA microbial classification sequencing technology, and the result shows that at the phylum level, the mouse intestinal flora mainly comprises bacteroides, Firmicutes and Proteobacteria, and at the phylum level, the most obvious change of the composition of the fecal flora is the increase of the abundance of Lactobacillus; the abundance of the blank group and the model group is only 7.84 percent and 7.34 percent, while the abundance of the Lactobacillus in the high-dose group reaches 29.3 percent, thereby proving the influence of loperamide medicine on the mouse flora and simultaneously showing that the gastrointestinal tract conditioning milk powder can achieve the effect of conditioning the gastrointestinal tract by probiotic beneficial bacteria in intestinal tracts.

Discussion 4

4.1 determination of oligosaccharide mixing ratio in milk powder

In the experiment, the optimal mixing ratio of fructo-oligosaccharide and xylo-oligosaccharide is determined by measuring the OD value, the pH value and the viable count of lactic acid bacteria, and in the measurement of the OD value, the result of the OD value of bifidobacterium shows that the FOS and XOS grow better when the mixing ratio is 7:3 and 8:2, and the FOS and XOS grow better and are respectively improved by 12.32 percent and 11.15 percent compared with a control group, and the difference is obvious (P is less than 0.05). In the determination of the OD value of the lactobacillus acidophilus, when the ratio is 7:3, the colony concentration is higher and is obviously higher than that of other proportioning groups (P is less than 0.05), which shows that the OD value of the lactobacillus acidophilus can be obviously improved after the composite oligosaccharide optimized culture medium carbon source is added into the culture medium. Researchers find that the growth speed of the lactobacillus plantarum can be obvious by adding the compound oligosaccharide into the dairy product. When the utilization conditions of the two probiotics on the compound oligosaccharide are comprehensively compared, the growth promotion effect on the lactic acid bacteria is the best when the ratio is 7: 3.

In the lactobacillus acidophilus test, the growth reaches the best effect at 7:3, the ratio group of 9:1 and the ratio group of 7:3 have no obvious difference (P is more than 0.05), the utilization effect is similar when the ratio of lactobacillus acidophilus to oligosaccharide is 7:3 and 9:1, and the optimal oligosaccharide mixing ratio is 7:3 in comprehensive consideration.

The prebiotics can not be well digested and absorbed by human body, but can promote the growth of probiotics in human intestinal tract, when the probiotics in the intestinal tract proliferate, a large amount of short chain fatty acid can be generated, so that the pH value is reduced, and the optimum ratio of oligosaccharide is further determined according to the determination of the pH value of milk powder added with different oligosaccharide mixing ratios. Both probiotics can utilize the mixed FOS and XOS in the formula to ferment to produce acid. In the determination of the bifidobacterium, when the ratio is 7:3 and 8:2, the pH value is lowest and no obvious difference exists, which shows that the bifidobacterium has better utilization effect on the mixing ratio of the two oligosaccharides. In the pH value measurement of lactobacillus acidophilus, the pH values at 7:3, 8:2 and 9:1 are not significantly different (P <0.05), but are all significantly lower than those of other proportioning groups (P < 0.05). Considering two probiotics, the ratio of 7:3 is optimal for promoting the fermentation acid production capability of the two bacteria. In the measurement of OD value, viable count and p H value, it is shown that the growth promoting effect on the two kinds of bacteria is best when the oligosaccharide mixing ratio is 7: 3. Therefore, the oligosaccharide mixing ratio in the milk powder is finally determined to be 7: 3. Compared with bifidobacterium, lactobacillus acidophilus can better utilize compound oligosaccharide in the probiotic designated mapping.

4.2 Effect of oligosaccharide addition in milk powder on viable count of probiotic

In the determination of viable count, the formula milk powder added with oligosaccharide can effectively promote the growth of lactic acid bacteria, and the viable counts of the two bacteria are gradually increased along with the increase of the addition amount of oligosaccharide. In the determination of the bifidobacterium, when the addition amount is 6g/100g, 5.5g/100g and 5g/100g, the viable count is obviously higher than that of other dosage groups and a control group (P <0.05), and no obvious difference exists among the three groups (P > 0.05). In the determination of viable count of Lactobacillus acidophilus, the highest viable count was obtained when the amount of Lactobacillus acidophilus was added at 6g/100 g. By combining two probiotics, the addition amount of 6g/100g of two oligosaccharides has better promotion effect on the growth of bifidobacterium and lactobacillus acidophilus. Meanwhile, the number of the bacteria of the lactic acid bacteria is correspondingly increased along with the increase of the carbon source content of the culture medium.

4.3 Effect of milk solids ratio in milk powder on viable count of Probiotics

In vitro fermentation tests are carried out to study milk solids in different mixing ratios in milk powder, and in the determination of the viable count of bifidobacteria, when the ratio of the milk powder to the whey protein powder is 9:1, the growth condition of bifidobacteria is the best. In the determination of the viable count of lactobacillus acidophilus, when the milk solid ratio is 9:1 and 6:4, the milk solid ratio has no significant difference, but is significantly higher than other ratio groups (P < 0.05). And finally determining the mixing ratio of the milk powder and the whey protein powder to be 9:1 by integrating the growth conditions of the two bacteria.

4.4 Effect on intestinal flora

A large number of researches show that linoleic acid has potential functions of regulating intestinal flora and influencing intestinal microecology. The functional oligosaccharide can regulate intestinal flora in a rat body and promote gastrointestinal peristalsis by drenching the functional oligosaccharide to the rat body. The test researches the influence of the compound oligosaccharide and the conjugated linoleic acid in the milk powder special for conditioning the gastrointestinal tract on the intestinal tract probiotic function by measuring the intestinal tract flora and the gastrointestinal tract conditioning effect of the mice. The experimental result shows that the milk powder special for gastrointestinal tract conditioning can shorten the first-particle defecation time of the mouse, increase the Evans blue propulsion rate, improve the constipation symptom of the mouse, regulate the intestinal flora of the mouse, does not influence the goblet cells of the intestinal wall of the mouse, and has the effect of relaxing bowel.

5 conclusion

The experiment researches the influence of different addition amounts and different mixing ratios of fructo-oligosaccharide and xylo-oligosaccharide on probiotic functions in formula milk powder by measuring the growth conditions of bifidobacterium and lactobacillus acidophilus, determines that the optimal mixing ratio of the fructo-oligosaccharide and the xylo-oligosaccharide is 7:3, the addition amount of total oligosaccharide in the formula milk powder is 6g/100g, and the optimal mixing ratio of the milk powder and lactalbumin is 9:1 by taking the OD values of two probiotics, the number of viable bacteria and the pH value as indexes, and determines the content of the oligosaccharide and the milk solid in the final formula milk powder of the experiment.

The formula of the milk powder special for relaxing bowel is as follows: the adding amount of each component is 81.33% of milk solid adding amount, 6% of oligosaccharide, 10% of linoleic acid and 2.67% of maltodextrin respectively according to 30 g.

Different dosage groups of the milk powder special for gastrointestinal tract conditioning have good effect on the effect of relaxing the bowel of mice, the effect of relaxing the bowel of medium and high dosage groups is more obvious, the feasibility of relaxing the bowel by adding compound oligosaccharide and unsaturated fatty acid into the milk powder is verified, and the research result provides a certain theoretical support for the application and development of the oligosaccharide and the unsaturated fatty acid in functional milk powder.

The above description is only for the purpose of illustrating the technical solutions of the present invention and not for the purpose of limiting the same, and other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (7)

1. The milk powder special for conditioning gastrointestinal tract is characterized by comprising the following components in percentage by weight: 74-82% of milk solid, 1.2-6% of oligosaccharide, 6-12% of linoleic acid and 2-3.5% of maltose.

2. The milk powder special for conditioning gastrointestinal tract as claimed in claim 1, is characterized by comprising the following components in percentage by weight: 81.33% of milk solid, 6% of oligosaccharide, 10% of linoleic acid and 2.67% of maltodextrin.

3. The special milk powder for conditioning gastrointestinal tract as claimed in claim 1, wherein the milk solids comprise milk powder and whey protein powder, and the milk powder and the whey protein powder are mixed according to a ratio of 1: 9-9: 1.

4. The special milk powder for conditioning gastrointestinal tract as claimed in claim 3, wherein the milk powder and the whey protein powder are mixed according to a ratio of 9: 1.

5. The special milk powder for conditioning gastrointestinal tract as claimed in claim 1, wherein the oligosaccharide comprises fructo-oligosaccharide and xylo-oligosaccharide, and the fructo-oligosaccharide and the xylo-oligosaccharide are mixed according to a ratio of 7: 3-9: 1.

6. The special milk powder for conditioning gastrointestinal tract as claimed in claim 5, wherein the fructo-oligosaccharide and the xylo-oligosaccharide are mixed according to a ratio of 7: 3.

7. The preparation method of the milk powder special for conditioning the gastrointestinal tract is characterized by comprising the following steps:

step 1: carrying out spray drying on the raw milk to obtain spray-dried whole milk powder;

step 2: pouring the whole milk powder obtained in the step 1, the whey protein powder, the fructo-oligosaccharide, the xylo-oligosaccharide, the linoleic acid and the maltodextrin into a mixing instrument according to a certain proportion and mixing uniformly.

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