阅读说明：本技术 一种发酵型保健奶醋及其制备方法 (Fermented health-care milk vinegar and preparation method thereof ) 是由 郭润芳 于宏伟 姚妞妞 于 2021-03-01 设计创作，主要内容包括：本发明公开了一种发酵型保健奶醋饮品的制备方法,为两阶段发酵,一次发酵为酵母菌和乳酸菌的混菌厌氧发酵产酒精阶段；二次发酵为醋酸菌的好氧发酵产醋酸阶段。本发明所使用的奶醋发酵菌株Saccharomyces cerevisiae S1,30℃发酵22h后,酒精产量保持稳定,保证了发酵过程和发酵产品的稳定性。发酵菌株植物乳杆菌Lactobacillus plantarum RS13,37℃培养24h,能够产胞外粗多糖95.33mg/g,赋予了奶醋抗氧化保健功能；另外乳酸发酵产生了乳酸为主的有机酸,可以抑制腐败菌和致病菌的生长。第一阶段采用酵母菌和乳酸菌混菌发酵,二者不仅可以协同生长,而且代谢产物酒精和有机酸之间可以发生酯化反应,对改善奶醋的风味和香气发挥了重要作用。(The invention discloses a preparation method of a fermentation type health care milk vinegar beverage, which comprises two-stage fermentation, wherein one-time fermentation is a mixed bacteria anaerobic fermentation alcohol production stage of saccharomycetes and lactic acid bacteria; the secondary fermentation is an aerobic fermentation acetic acid production stage of acetic acid bacteria. The milk vinegar fermentation strain Saccharomyces cerevisiae S1 used in the invention can keep stable alcohol yield after being fermented for 22 hours at 30 ℃, and ensures the stability of the fermentation process and the fermentation product. The Lactobacillus plantarum RS13 is a fermentation strain, and can produce 95.33mg/g extracellular crude polysaccharide after being cultured for 24h at 37 ℃, so that the milk vinegar has an antioxidant health-care function; in addition, lactic acid fermentation generates organic acid mainly comprising lactic acid, and can inhibit the growth of spoilage bacteria and pathogenic bacteria. In the first stage, the yeast and the lactobacillus are mixed for fermentation, the yeast and the lactobacillus can cooperatively grow, and esterification reaction can occur between the metabolite alcohol and the organic acid, so that the method plays an important role in improving the flavor and aroma of the milk vinegar.)

1. A preparation method of a fermented health-care milk vinegar beverage is characterized by comprising the following steps:

(1) strain activation and seed liquid preparation: respectively placing Saccharomyces cerevisiae S1, Lactobacillus plantarum RS13 and acetic acid bacteria Shanghai brewing No. 1.01 stored at 4 ℃ in YPD, MRS and LB broth culture media, shake culturing Saccharomyces cerevisiae and acetic acid bacteria at 30 ℃ for 24h, and standing culturing Lactobacillus plantarum at 37 ℃ for 24 h;

(2) raw material treatment: filtering fresh milk with four layers of gauze to remove impurities and foreign matters, adding white sugar in an amount of 15% of the fresh milk, stirring to dissolve, sterilizing at 65 deg.C for 30min, cooling to room temperature, and fermenting;

(3) alcohol fermentation: fermenting with mixed bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum at an inoculation ratio of 2:1 and an inoculation amount of 4% for the two bacteria at 30 deg.C for 8h to obtain fermented juice with an ethanol content of 5-6;

(4) acetic acid fermentation: performing acetic fermentation on the basis of alcoholic fermentation, wherein the inoculation amount of acetic acid bacteria is 5%, the fermentation time is 22h, and the liquid loading amount is 40%;

(5) blending after fermentation: taking fermentation total acid not less than 8g/kg as a fermentation end point, adding stevioside and pectin after fermentation is finished, and homogenizing under 20 MPa;

(6) killing and sterilizing by using a bar: and (3) keeping the homogenized milk vinegar at 65 ℃ for 30min, carrying out pasteurization, and cooling to room temperature to obtain a milk vinegar finished product.

2. The method for preparing a fermented health milk vinegar beverage according to claim 1, wherein the saccharomyces cerevisiae in the step (3) is s.cerevisiae S1, and the lactobacillus plantarum is l.plantarum RS 13.

3. The method for preparing a fermented health-care milk vinegar beverage according to claim 1, wherein the addition amount of the inulin in the step (5) is 0.15%, and the addition amount of the pectin is 0.2%.

Technical Field

The invention belongs to the technical field of dairy product processing, and particularly relates to fermented health-care milk vinegar and a preparation method thereof.

Background

Milk is one of the oldest natural beverages and is known as white blood. The milk contains rich nutrient substances such as protein, fat, vitamins, mineral substances and the like, and the milk protein contains all amino acids necessary for human bodies; milk fat is mostly short-chain and medium-chain fatty acid; the ratio of mineral substances such as potassium, phosphorus, calcium and the like is reasonable, so that the milk is a nutrient health-care food accepted by various countries in the world. At present, main processed products of cow milk comprise pure milk, yoghourt, milk beverage, milk slices, cheese and the like, while milk vinegar products on the market are extremely rare.

Compared with the prepared milk vinegar, the fermented milk vinegar not only utilizes the biotransformation of microorganisms to macromolecular nutrient substances to generate micromolecular nutrition which is easier to be absorbed by human bodies, but also the zymophyte can generate functional active substances, thereby greatly enhancing the nutrition and health care functions of the product. Therefore, the milk vinegar is a functional health-care beverage with great market potential, which combines the nutrition, flavor and health-care functions of milk, health-care vinegar and functional active substances, can meet the multi-level requirements of people on taste, nutrition, health care and the like, and has very important significance for the development of health-care food industry and the body health of people.

Disclosure of Invention

The invention aims to provide an antioxidant milk vinegar health drink which is prepared by taking cow milk as a raw material and performing a two-stage fermentation process by utilizing active yeast, EPS-rich lactic acid bacteria and acetobacter.

The preparation process of the fermented milk vinegar comprises two-stage fermentation, wherein one-time fermentation is a mixed bacteria anaerobic fermentation alcohol production stage of saccharomycetes and lactic acid bacteria; the secondary fermentation is an aerobic fermentation acetic acid production stage of acetic acid bacteria. The invention selects the strains suitable for fermenting the milk vinegar: the saccharomyces cerevisiae is S.cerevisiae S1, sucrose 100-150g of raw materials are added into 1L of milk, fermentation is carried out at 30 ℃, and the alcohol yield is kept to be less than 8g/100 g; the lactobacillus is lactobacillus plantarum RS13, and the screening and identification of the strain are disclosed in the literature (separation and identification of the dominant strain in the fermentation process of raspberry enzyme; Wedongdong, Helmann, Yin-Yang-Na, hong Wei, Guo-Lu-Yang, Chun-Bei university of agriculture), and the extracellular crude polysaccharide yield is 95.33 mg/g; the acetobacter has strong acid production capacity and the optimal growth temperature is 30 ℃.

The invention discloses a preparation method of a fermented health-care milk vinegar beverage, which comprises the following steps:

(1) strain activation and seed liquid preparation: respectively placing Saccharomyces cerevisiae, Lactobacillus plantarum and acetic acid bacteria stored at 4 ℃ in YPD, MRS and LB broth culture media, shake culturing Saccharomyces cerevisiae and acetic acid bacteria at 30 ℃ for 24h, and standing culturing Lactobacillus plantarum at 37 ℃ for 24 h;

(2) raw material treatment: filtering fresh milk with four layers of gauze to remove impurities and foreign matters, adding white sugar in an amount of 15% of the fresh milk, stirring to dissolve, sterilizing at 65 deg.C for 30min, cooling to room temperature, and fermenting;

(3) alcohol fermentation: fermenting with mixed bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum at an inoculation ratio of 2:1 and an inoculation amount of 4% for the two bacteria at 30 deg.C for 8h to obtain fermented juice with an ethanol content of 5-6;

(4) acetic acid fermentation: performing acetic fermentation on the basis of alcoholic fermentation, wherein the inoculation amount of acetic acid bacteria is 5%, the fermentation time is 22h, and the liquid loading amount is 40%;

(5) blending after fermentation: taking fermentation total acid not less than 8g/kg as a fermentation end point, adding stevioside and pectin after fermentation is finished, and homogenizing under 20 MPa;

(6) killing and sterilizing by using a bar: and (3) keeping the homogenized milk vinegar at 65 ℃ for 30min, carrying out pasteurization, and cooling to room temperature to obtain a milk vinegar finished product.

In the step (5), the addition amount of the inulin is 0.15 percent, and the addition amount of the pectin is 0.2 percent.

In the invention, alcohol content is measured by an alcohol meter after alcohol aqueous solution is obtained by distillation; measuring acidity by an acid-base titration method; the sugar degree is measured by using a handheld sugar meter; sensory evaluation and physical and chemical index measurement refer to GB/T15038-2006.

The milk vinegar product prepared by the technical scheme has the following quality characteristics:

(1) sensory indexes are as follows: the milk vinegar is milky in color, has milk fragrance and acetic acid taste, and tastes sour, sweet and refreshing;

(2) physical and chemical indexes are that the total acid TA is 8.18g/kg, the total sugar content: 6.9%, total solids TSS: 20.88 Brix.

(3) The microbial index is that the total number of bacterial colonies (cfu/mL) is less than or equal to 100; the coliform group (MPN/100mL) is less than or equal to 3; pathogen, not detected.

Has the advantages that: (1) according to the milk vinegar fermentation strain S.cerevisiae S1 provided by the invention, 150g of sucrose 100-one is added into 1L of milk, and after fermentation is carried out for 22h at 30 ℃, the alcohol yield is kept stable and is always kept at <8g/100g, so that the stability of the fermentation process and the fermentation product is ensured.

(2) The lactobacillus plantarum L.plantarum RS13 of the milk vinegar fermentation strain provided by the invention is cultured for 24 hours at 37 ℃, can produce 95.33mg/g of extracellular crude polysaccharide, and endows milk vinegar with an antioxidant health-care function; in addition, lactic acid fermentation generates organic acid mainly comprising lactic acid, so that the growth of putrefying bacteria and pathogenic bacteria can be inhibited, and the quality of the product is ensured.

(3) In the fermentation process provided by the invention, the yeast and the lactobacillus are mixed for fermentation in the first stage, the yeast and the lactobacillus can cooperatively grow, and esterification reaction can occur between the metabolite alcohol and the organic acid, so that the method plays an important role in improving the flavor and aroma of the milk vinegar.

Drawings

FIG. 1 is a graph showing the variation of total sugar content and alcohol content of products in cow milk with different sucrose addition amounts;

FIG. 2 is a diagram of the screening of exopolysaccharide producing strains of lactic acid bacteria;

FIG. 3 is a graph showing the influence of the addition of stevioside on sensory scores and sugar-acid ratio of fermented milk vinegar;

FIG. 4 is a graph comparing the oxidation resistance before and after milk fermentation.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Example 1 selection of Yeast and Acetobacter

1. Milk vinegar fermentation process flow

2. Yeast screening and alcohol generation kinetics suitable for milk vinegar fermentation

Respectively selecting yeasts stored on a slant, inoculating the yeasts into an YPD broth culture medium, culturing at 30 ℃ for 12h for activation, then inoculating the yeasts into a milk substrate according to the inoculation amount of 2%, culturing at 30 ℃ for 48h, and according to indexes such as color, taste, tissue state, flavor, alcohol content and the like in table 1, the yeast S.cerevisiae S1 strain is the most suitable strain, and then carrying out the next step of kinetic test.

TABLE 1 sensory analysis score and ethanol production

Inoculating the activated 24h S.cerevisiae S1 and bacterial liquid into cow milk with different initial sucrose addition amounts (0, 50, 100, 150g/L) according to 2% inoculation amount, and performing anaerobic fermentation at 30 ℃. Sampling every 8h from 0h, and determining the substrate sucrose content of the saccharomycetes and the volume fraction of the product alcohol in the fermented milk. As shown in FIG. 1, the total sugar content of the substrate in the fermentation process gradually decreased with the increase of the fermentation time, while the alcohol content gradually increased, indicating that the substrate sugar was consumed and converted into alcohol. Adding 100-150g/L sucrose, the alcohol yield is not changed greatly, the alcohol content is maintained below 8% after fermenting for 40h, and the sucrose addition is determined to be 150 g/L.

3. Content of extracellular polysaccharide of lactobacillus

Selecting the test lactobacillus growing on the inclined plane, inoculating the test lactobacillus in a liquid MRS culture medium, culturing at 37 ℃ for 24h for activation, transferring the test lactobacillus into 100mL of liquid MRS according to the proportion of 2%, and culturing at 37 ℃ for 24 h. The fermentation liquor is centrifuged for 5min at 2000r/min at room temperature. Pouring out the supernatant, adding 45% phenol aqueous solution into the lower layer of thallus (the 45% phenol is easy to separate in a heating state and needs to be shaken up each time the phenol is added into the sample), and enabling the solid-to-liquid ratio to be 1: 30. heating with microwave oven for 5min, leaching at 60 deg.C for 60min, and concentrating under reduced pressure at 75 deg.C. Adding 95% ethanol with 5 times volume of the concentrated solution for alcohol precipitation. The mixture was allowed to stand at 4 ℃ overnight. And pouring out the supernatant, collecting the precipitate, drying for 60min by a metal bath, and weighing the dry weight to obtain the extracellular polysaccharide. As a result, as shown in fig. 2, the lactobacillus plantarum RS13 strain produced the highest level of exopolysaccharides, and thus was determined to be a suitable fermentation strain.

EXAMPLE 2 optimization of fermentation conditions of milk Vinegar

1. Optimization of alcoholic fermentation conditions in the first stage of milk vinegar fermentation

On the basis of determining that 150g/L of sucrose is added into a milk substrate and the fermentation temperature is 30 ℃, three-factor three-level Design and experiments are carried out by using software Design-expert 8.0.6 according to three factors of a Box-Benhnken Design (BBD) Design method on the compound proportion of strains influencing the yield and the sense of alcohol, the inoculation amount and the fermentation time, and the results of response surface test Design and response values are shown in tables 2-4.

TABLE 2 fermentation substrate Box-Benhnken center combination design table

TABLE 3 fermentation substrate response surface test design and results

TABLE 4 regression model analysis of variance and significance

Performing multiple fitting regression analysis according to the data, and respectively establishing regression models of the alcohol content (Y1) and the sensory score (Y2) on the bacterial liquid proportion (A), the bacterial inoculation amount (B) and the fermentation time (C), wherein the regression equations are as follows: y1 ═ 5.06+0.22A +0.36B +0.84C-0.050AB-0.60AC-0.075BC-0.52A²-1.49B²-0.093C²

Y2＝87.00+1.00A-4.88B-11.13C+6.00AB+4.5AC-1.75BC-9.63A²-4.87B²-1.38C²

Regression equation determination coefficient R1²＝0.9272，R2²＝0.9305。

According to analysis of variance and equation, whether the alcohol content or the sensory response value, the model is expressed as P<0.01, indicating that the model is extremely significant and that the term P is misleading>0.05, the mismatching item is not obvious, so the alcohol content and the sensory prediction model have reasonable structure. In addition, A²And B²The influence on Y1 is obvious, the nonlinear relation of fermentation conditions to response values is shown, and FC can be known by analyzing the F value of each parameter>FB>FA, therefore the magnitude of the effect of each parameter on the first stage fermentation is in turn: time of fermentation>Bacterial inoculation amount>And (4) proportioning the strains.

The optimal fermentation conditions of the first stage of response surface result prediction are as follows: the yeast, lactobacillus plantarum is 2:1, the mixed inoculation amount is 4.08%, the fermentation time is 8.12h, the alcohol content can reach 5.50%, and the sensory score can be 84.14. In consideration of actual operation, fermentation condition parameters were adjusted to yeast: the ratio of lactobacillus plantarum to 2:1, the mixed inoculation amount of 4 percent and the fermentation time of 8 hours, the alcohol content of the obtained fermented juice is 5.68, the sensory score is 86, the alcohol content is 3.3 percent different from the predicted result, the sensory score is 2.2 percent different from the predicted value, and the result shows that the actual operation result is close to the predicted value.

2. Optimization of acetic acid fermentation conditions in the second stage of milk vinegar fermentation

On the basis of the first stage fermentation, three-factor three-level Design and experiments are carried out by using software Design-expert 8.0.6 according to three factors of liquid loading, inoculation amount and fermentation time which affect the yield and sense of acetic acid by a Box-Benhnken Design (BBD) Design method, and the results of response surface experiment Design and response values are shown in tables 5-7.

TABLE 5 fermentation conditions Box-Benhnken center combination design table

TABLE 6 fermentation substrate response surface test design and results

Performing multiple fitting regression analysis on the data by using software, and respectively establishing regression models of total acid content (Y1) and sensory score (Y2) on the bacterial load (A), fermentation time (B) and liquid loading (C), wherein the regression equations are as follows:

Y1＝+8.24+0.31A+0.94B-0.22C-0.086AB-0.33AC-0.094BC-1.32A²-0.28B²-0.32C²

Y2＝+89.00+1.75A+5.00B-4.25C-5.00AB+0.50AC+0.50BC-9.50A²-5.00B²-1.50C²

system of determination of regression equationNumber R1²＝0.9163，R2²＝0.9084

TABLE 7 regression model analysis of variance and significance

According to analysis of variance and equation, whether the alcohol content or the sensory response value, the model is expressed as P<0.01, indicating that the model is extremely significant and that the term P is misleading>0.05, the mismatching item is not obvious, so the alcohol content and the sensory prediction model have reasonable structure. In addition, A²And B²The influence on Y1 is obvious, the nonlinear relation of fermentation conditions to response values is shown, and FB can be known by analyzing F values of various parameters>FC>FA, therefore, the influence of each parameter on the fermented milk vinegar is as follows: time of fermentation>Liquid loading amount>And (4) inoculating bacterial quantity.

The optimal prediction condition for obtaining the fermented milk vinegar according to the response surface result is as follows: the inoculation amount of acetic acid bacteria is 5.01%, the fermentation time is 22.06h, the liquid loading amount is 40%, the total acid can be predicted to reach 8.43g/kg, the sensory score can be 90.03, and the feasibility is realized. In order to facilitate the practical operation, the fermentation condition parameters are set to 5 percent of acetic acid bacteria inoculation amount, 22 hours of fermentation time and 40 percent of liquid loading amount, so that the total acid in the fermented milk vinegar is 8.182g/kg, and the sensory score is 88. Three replicates were designed to verify the feasibility and validity of the predicted conditions. The difference between the total acid content of the result and the predicted result is only 2.9 percent, the difference between the sensory score and the predicted value is 2.3 percent, and the result shows that the actual operation result is close to the predicted value.

Example 3 formulation

After fermentation is finished, blending the fermented product, mainly adjusting the sugar-acid ratio and the stability. As shown in FIG. 3, the sensory score was 88 or more when the amount of stevioside added was 0.15 to 0.20%, indicating that the flavor was good and the texture was acceptable, and the amount of stevioside added was 0.15% in total.

The effect of the addition amounts of the four stabilizers CMC-Na, guar gum, xanthan gum and pectin on the stability of the fermented milk vinegar is shown in table 8. The addition of 0.2% of pectin in the milk vinegar is beneficial to the improvement of the product quality in consideration of water retention and sense.

TABLE 8 Effect of stabilizers on the quality of fermented milk Vinegar

Example 4 product Property testing

1. Physical and chemical indexes of product

(1) Sensory indexes are as follows: the milk vinegar is milky in color, has milk fragrance and acetic acid taste, and tastes sour, sweet and refreshing;

(2) physical and chemical indexes are that the total acid TA is 8.18g/kg, the total sugar content: 6.9%, total solids TSS: 20.88 Brix.

(3) The microbial index is that the total number of bacterial colonies (cfu/mL) is less than or equal to 100; the coliform group (MPN/100mL) is less than or equal to 3; pathogen, not detected.

2. Antioxidation property of milk vinegar

As shown in figure 4, the total antioxidant capacity of the cow milk is remarkably enhanced (P is less than 0.01) after two fermentation processes, wherein the total reducing capacity of the fermented milk vinegar is improved by 1.71 times than that of the unfermented milk, the ABTS free radical scavenging capacity is improved by 0.49 times, and the DPPH free radical scavenging capacity is improved by 0.62 times.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.