Preparation process and flavor analysis of Chinese chestnut probiotic plant-based fermented milk

Abstract

The invention discloses a preparation process and flavor analysis of a Chinese chestnut probiotic plant-based fermented milk beverage. The preparation process comprises the following steps: unfreezing shelled raw Chinese chestnuts which are frozen and preserved at-20 DEG C at normal temperature, slicing, baking, crushing, sieving, gelatinizing, liquefying, saccharifying, adding protein liquid, uniformly mixing, sterilizing, inoculating bacteria and fermenting to obtain a finished product. Eight common probiotics for milk beverage fermentation are screened, compound strains with unique flavor and strong oxidation resistance, namely lactobacillus rhamnosus and lactobacillus casei, are obtained by taking flavor and probiotic characteristics as indexes, and the fermented milk prepared by fermentation under the condition is strong in flavor, uniform in tissue, fine, smooth, sour and sweet in mouth feel and relatively strong in oxidation resistance. Through headspace-gas chromatography-ion mobility spectrometry (GC-IMS) identification, 36 volatile substances, including 10 alcohol substances, 9 ketone substances, 5 ester substances, 4 aldehyde substances, 2 pinene substances, 1 acid substance and 5 other substances, are identified compared with a milk beverage without inoculated fermentation.

Description

Technical Field

[0001] The invention relates to the technical field of preparation technology of fermented milk, and specifically to a preparation technology and flavor analysis of chestnut probiotic plant-based fermented milk. Background Art

[0002] Chestnut, commonly known as chestnut, is a plant of the Fagaceae family and Castanea genus. The chestnut planting area in Qinhuangdao City is 1.22 million mu, with an annual output of 65,000 tons. Chestnut is rich in nutrients, containing starch, protein, minerals and various vitamins. In addition, chestnut is also rich in active ingredients such as polyphenols and polysaccharides, which generally have antioxidant, anti-tumor and antibacterial effects. Chestnut kernels have a sweet and sticky taste and a unique aroma, but they are not resistant to storage and are prone to mold and rot, resulting in a waste of resources. Although chestnut powder, chestnut beverages, sugar-fried chestnuts, chestnut sauce and other products have been developed on the market, primary products dominated by sugar-fried chestnuts still have an absolute advantage. Therefore, it is urgent to develop deep-processed chestnut products to improve resource utilization and chestnut added value.

[0003] Probiotics are active microorganisms that can improve and enhance the balance of human intestinal flora, and have a good influence and promoting effect on the overall health of the human body and the development of physiological mechanisms. The use of probiotics and other biological fermentation technologies to develop nutritious and multifunctional fermented milk beverages can not only extend the shelf life of chestnut kernels but also inhibit the growth of other fungi, and enable the beverage to obtain better fermentation flavor and higher nutritional value. At the same time, the use of probiotic lactic acid bacteria microbial fermentation technology can not only optimize the flavor of chestnuts, but also improve the flavor of chestnuts, promote the health of the human digestive system, increase the nutritional value of chestnuts, and enhance the body's immunity. Therefore, in order to improve the utilization rate of chestnut resources and the added value of products, it is of great significance to develop a chestnut probiotic plant-based fermented milk beverage. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation process and flavor analysis of chestnut probiotic plant-based fermented milk to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a preparation process of a chestnut probiotic plant-based fermented milk beverage,

[0006] The production process of chestnut probiotic plant-based fermented milk beverage is as follows: shelled raw chestnuts frozen and stored at -20°C are thawed at room temperature → sliced ​​→ baked → crushed → sieved → gelatinized → liquefied → saccharified → added with protein solution and mixed evenly → sterilized → inoculated and fermented → finished product;

[0007] Baking: Place the cut chestnuts in the oven, then set the baking temperature to 50 degrees Celsius and the baking time to 2.5 hours to ensure that they are evenly heated and achieve the ideal baking effect;

[0008] Sieve: Pass the crushed chestnut powder through a 200-mesh sieve and take the chestnut powder under the sieve;

[0009] Gelatinization: Mix 200-mesh chestnut powder and drinking water in a ratio of 1:9 and place in a magnetic stirrer, stirring at 90°C for 10 min;

[0010] Liquefaction: add 0.25% liquid volume of α-amylase to the gelatinization solution and stir with a magnetic stirrer at 70°C for 2h;

[0011] Saccharification: add 0.25% liquid volume of glucose amylase to the saccharification solution and stir with a magnetic stirrer at 60°C for 2.5h;

[0012] Preparation of protein liquid: walnut protein and drinking water are mixed evenly in a ratio of 1:19;

[0013] Sterilization: The mixed fermentation liquid is sterilized at 80℃ for 15min;

[0014] Inoculation and fermentation: The sterilized fermentation liquid is cooled to room temperature, and the activated Lactobacillus rhamnosus and Lactobacillus casei are inoculated in a volume ratio of 1:1, and the two bacteria are added and mixed and fermented;

[0015] The fermentation temperature is 37°C, the fermentation time is 20h, the inoculation amount is 10% (based on the mass of chestnut and walnut protein slurry), and the ratio of chestnut to protein powder is 6:4 (volume ratio). The fermented milk produced under these conditions has a strong aroma, uniform texture, delicate sweet and sour taste, and strong antioxidant properties.

[0016] Preferably, a fermented chestnut probiotic plant-based milk beverage is screened out from 8 probiotics (LR-Lactobacillus rhamnosus, LC-Lactobacillus casei, LP-Lactobacillus plantarum, L.P95-Lactobacillus plantarum, L.F04-fermented mucus Lactobacillus, L.C17-Lactobacillus casei, LF-fermented mucus Lactobacillus, L.F90-Lactobacillus plantarum), among which the milk beverage fermented by LR-Lactobacillus rhamnosus has strong oxidizing properties, and the milk beverage fermented by LF-fermented mucus Lactobacillus has more flavor substances. After combining the two strains, a composite strain with strong oxidizing properties and more flavor substances is obtained.

[0017] Preferably, the flavor analysis of the chestnut probiotic plant-based fermented milk beverage is used for the flavor analysis of the chestnut probiotic plant-based fermented milk beverage prepared according to claim 1, characterized in that the ratio of saccharified liquid to protein liquid (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2), inoculation amount (6%, 7%, 8%, 9%, 10%, 11%, 12%), fermentation time (4h, 8h, 12h, 16h, 20h, 24h, 28h), and fermentation temperature (35°C, 36°C, 37°C, 38°C, 39°C, 40°C) are used as investigation factors, and sensory evaluation is used as the evaluation index to conduct a single factor experiment to determine the optimal fermentation conditions.

[0018] The electronic nose was used for initial screening, the volatile flavor substances of the product were determined by GC-MS technology, and the non-volatile flavor substances were determined by LC-MS technology.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. Establishment of chestnut pretreatment and sensory evaluation method: Determine the conditions for chestnut starch liquefaction and saccharification, and establish the sensory evaluation standard of chestnut probiotic plant-based fermented milk beverage from the perspective of physical and chemical properties, taste, flavor, and sourness and sweetness;

[0021] 2. Screening and combination optimization of bacterial strains: Fermented chestnut probiotic plant-based milk beverage was screened out from 8 probiotics (LR-Lactobacillus rhamnosus, LC-Lactobacillus casei, LP-Lactobacillus plantarum, L.P95-Lactobacillus plantarum, L.F04-fermented mucus Lactobacillus, L.C17-Lactobacillus casei, LF-fermented mucus Lactobacillus, L.F90-Lactobacillus plantarum). Among them, the milk beverage fermented by LR-Lactobacillus rhamnosus has strong oxidizing properties, and the milk beverage fermented by LC-Lactobacillus casei has more flavor substances. After combining the two strains, a composite strain with strong oxidizing properties and more flavor substances was obtained.

[0022] 3. Optimization of the fermentation process of chestnut probiotic plant-based fermented milk beverage. Using chestnut and walnut protein powder as the basic raw materials, optimize the base material formula of chestnut probiotic plant-based fermented milk beverage. Secondly, using composite strains as the starter strains, orthogonal technology is used to optimize the fermentation process parameters (fermentation time, temperature, inoculation amount) to develop chestnut probiotic plant-based fermented milk beverage processing technology;

[0023] 4. Determination of the physical and chemical properties (reducing sugar content, pH, titratable acidity, antioxidant capacity, and total bacterial count) of chestnut probiotic plant-based fermented milk beverages, and analysis of the fermentation flavor of composite strains using electronic nose technology and GC-IMS;

[0024] 5. Compared with the unfermented milk beverage, a total of 36 volatile substances were identified, including 10 alcohols, 9 ketones, 5 esters, 4 aldehydes, 2 pinenes, 1 acid, and 5 other substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Preparation process flow chart;

[0026] Figure 2 Schematic diagram of electronic nose assay of fermented milk with different strains;

[0027] Figure 3 Determination of antioxidant activity of fermentation of different strains;

[0028] Figure 4 Schematic diagram of the effect of chestnut juice and protein ratio on the sensory quality of milk beverages;

[0029] Figure 5 Schematic diagram of the effect of inoculum size on the sensory quality of milk beverages;

[0030] Figure 6 Schematic diagram of the effect of fermentation temperature on the sensory quality of milk beverages;

[0031] Figure 7 Schematic diagram of the effect of fermentation time on the sensory quality of milk beverages;

[0032] Figure 8 Schematic diagram of the two-dimensional profiles of chestnut probiotic plant-based fermented milk beverages with different treatments;

[0033] Fig. 9 Schematic diagram of fingerprints of chestnut probiotic plant-based fermented milk beverages with different treatments;

[0034] Fig.10 Heat map of LC-MS determination of composite strains and single strain fermentation. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The terms "first", "second", etc. in the specification and claims of the present application are used to distinguish similar objects, and are not used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable under appropriate circumstances, so that the embodiments of the present application can be implemented in an order other than those illustrated or described here, and the objects distinguished by "first", "second", etc. are generally of one type, and the number of objects is not limited. For example, the first object can be one or more. In addition, "and / or" in the specification and claims represents at least one of the connected objects, and the character " / " generally indicates that the objects associated with each other are in an "or" relationship.

[0037] In the description of the present invention, it should be understood that the terms "center", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0038] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "set" should be understood in a broad sense, for example, they can be fixedly connected or set, or detachably connected or set, or integrally connected or set. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0039] See also Figures 1 to 8 , the present invention provides a technical solution:

[0040] Example 1 Preparation process of chestnut probiotic plant-based fermented milk

[0041] The production process of chestnut probiotic plant-based fermented milk beverage is as follows: shelled raw chestnuts frozen and stored at -20°C are thawed at room temperature → sliced ​​→ baked → crushed → sieved → gelatinized → liquefied → saccharified → added with protein solution and mixed evenly → sterilized → inoculated and fermented → finished product;

[0042] Baking: Place the cut chestnuts in the oven, then set the baking temperature to 50 degrees Celsius and the baking time to 2.5 hours to ensure that they are evenly heated and achieve the ideal baking effect;

[0043] Sieve: Pass the crushed chestnut powder through a 200-mesh sieve and take the chestnut powder under the sieve;

[0044] Gelatinization: Mix 200-mesh chestnut powder and drinking water in a ratio of 1:9 and place in a magnetic stirrer, stirring at 90°C for 10 min;

[0045] Liquefaction: add 0.25% liquid volume of α-amylase to the gelatinization solution and stir with a magnetic stirrer at 70°C for 2h;

[0046] Saccharification: add 0.25% liquid volume of glucose amylase to the saccharification solution and stir with a magnetic stirrer at 60°C for 2.5h;

[0047] Preparation of protein liquid: walnut protein and drinking water are mixed evenly in a ratio of 1:19;

[0048] Sterilization: The mixed fermentation liquid is sterilized at 80℃ for 15min;

[0049] Inoculation and fermentation: The sterilized fermentation liquid is cooled to room temperature, and the activated Lactobacillus rhamnosus and Lactobacillus casei are inoculated in a volume ratio of 1:1. After adding the two bacteria, mix and ferment.

[0050] Example 2 Determination of fermentation process

[0051] 1) Screening and combination optimization of bacterial strains: Fermented chestnut probiotic plant-based milk beverage was screened out from 8 probiotics (LR-Lactobacillus rhamnosus, LC-Lactobacillus casei, LP-Lactobacillus plantarum, L.P95-Lactobacillus plantarum, L.F04-fermented mucus Lactobacillus, L.C17-Lactobacillus casei, LF-fermented mucus Lactobacillus, L.F90-Lactobacillus plantarum). Among them, the milk beverage fermented by LR-Lactobacillus rhamnosus has strong oxidizing properties, and the milk beverage fermented by LC-Lactobacillus casei has more flavor substances. After combining the two strains, a composite strain with strong oxidizing properties and more flavor substances was obtained.

[0052] 2) With the ratio of saccharification liquid to protein liquid (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2), inoculation amount (6%, 7%, 8%, 9%, 10%, 11%, 12%), fermentation time (4h, 8h, 12h, 16h, 20h, 24h, 28h), fermentation temperature (35℃, 36℃, 37℃, 38℃, 39℃, 40℃) as investigation factors and sensory evaluation as the evaluation index, a single factor experiment was carried out to determine the optimal fermentation conditions.

[0053] Example 3 Establishment of sensory evaluation

[0054] The sensory evaluation method of chestnut probiotic plant-based fermented milk beverage was slightly modified by referring to "GB19302-2010 National Food Safety Standard Fermented Milk". Ten boys and ten girls were randomly selected to form a 20-member professional evaluation team to conduct sensory evaluation on the taste, flavor, sourness and sweetness, and color of the prepared chestnut probiotic plant-based fermented milk beverage. The sensory scoring results were collected and statistically analyzed. The sensory evaluation standards of chestnut probiotic plant-based fermented milk beverage are shown in Table 1.

[0055] Table 1 Sensory evaluation standards for fermented milk

[0056]

[0057]

[0058] Example 4 Antioxidant Determination

[0059] 1) Determination of DPPH free radical scavenging rate:

[0060] Take 1 ml of methanol and 4 ml of DPPH-methanol solution (0.1 mmol / L), mix them in a dark place for 30 min, and measure the absorbance A0 at 517 nm. Take 1 ml of sample solution and 4 ml of DPPH-methanol solution (0.1 mmol / L), mix them in a dark place for 30 min, and measure the absorbance Ai at 517 nm.

[0061]

[0062] 2) FRAP antioxidant assay:

[0063] The total antioxidant activity of the reaction system was determined by referring to the Rumpf FRAP method and modified, and used according to the instructions of the T-AOC kit.

[0064] Example 5 Determination of reducing sugar and chromaticity

[0065] 1) The reducing sugar content in rice milk fermented with lactic acid bacteria was determined by 3,5-dinitrosalicylic acid (DNS) method. The absorbance value was used as the horizontal axis (X) and the glucose content was used as the vertical axis (Y). The reducing sugar standard curve was drawn, and the regression equation was Y = (0.65552 + 0.0065) × 2 × 100 (R2 = 0.9996). Weigh 2g of fermented milk beverage sample, place it in a conical flask, add 28mL of distilled water, and extract it in a 50℃ water bath for 30min. After taking it out, cool it to room temperature, filter and collect the extract. Take 0.2mL of the extract, put it in an EP tube, add 0.15mL DNS, vortex mix it, place it in a boiling water bath and heat it for 5min, take it out and immediately put it in a cold water stream to cool it, and then dilute it to 1mL with distilled water. The group of 0.2 mL of distilled water plus 0.15 mL of DNS was used as the blank control, and the absorbance was measured at a wavelength of 540 nm. The measurement was repeated 3 times and the reducing sugar content was calculated by substituting into the standard curve.

[0066] 2) The color of the chestnut probiotic plant-based fermented milk beverage was measured using a colorimeter.

[0067] Example 6 Analysis of flavor

[0068] 1) Electronic nose measurement: Take 5 ml of sample solution and add it to a 20 ml injection bottle, tighten the bottle cap and leave it for 30 minutes. Detection procedure: preparation time 5 seconds, cleaning time 60 seconds, injection detection time 120 seconds.

[0069] Table 2 Electronic nose sensors corresponding to sensitive chemical types

[0070]

[0071] 2) GC-IMS determination:

[0072] Referring to Tan's method, 1 mL of sample was taken and placed in a 20 mL headspace bottle. After incubation at 80 °C for 15 minutes, 500 μL was injected, the analysis time was 20 min, the chromatographic column type was MXT-5, 15 mL, 0.53 mm ID, 1 um FT, the column temperature was 60 °C, the carrier gas / drift gas was high-purity N2 (99.999%), the IMS temperature was 45 °C, the injection volume was 500 ul, the incubation time was 15 min, the incubation temperature was 80 °C, the injection needle temperature was 85 °C, and the incubation speed was 500 rpm.

[0073] Data processing: The experiments were repeated three times, and the results were expressed as mean ± standard deviation. Origin2024 software was used to draw the graphs, and IBM SPSS Statistics26 was used to perform univariate significance analysis on the experimental data. P < 0.05 indicated a significant difference.

[0074] 3) LC-MS determination: Sample pretreatment, take the sample and mix it, take 200ul into a 1.5EP tube, add 600ul methanol acetonitrile mixture (1:1 / V), vortex for 60S, centrifuge at 4°C, 17000g, 15min, take the supernatant into a new mLEP tube, 35°C, vacuum centrifuge to dry, add 100ul 50% methanol water to the sample to dissolve, ultrasonicate for 10 minutes, centrifuge at 20°C, 17000g, 15min, take 60ul supernatant into a sample injection bottle;

[0075] Instrument parameter settings:

[0076] Mobile phase conditions: column temperature 40°C, sample volume 5 μl; positive ion mode: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile, negative ion mode: A: water (2 mM ammonium acetate); B: acetonitrile;

[0077] Table 1 Results and analysis of orthogonal experiment of chestnut plant-based probiotic fermented milk beverage

[0078]

[0079]

[0080] Table 2 Analysis of variance of orthogonal test

[0081]

[0082]

[0083] Note: ** indicates extremely significant effect, P<0.01, * indicates significant effect, P<0.05, and - indicates no significant effect.

[0084] Table 3 Physical and chemical properties of plant-based fermented milk

[0085]

[0086] Table 4 Microbiological indicators of plant-based fermented milk beverages

[0087]

[0088]

[0089] Table 5 Antioxidant indexes of plant-based fermented milk beverages

[0090]

[0091] Table 6 Chromaticity index of plant-based fermented milk beverages

[0092]

[0093] Table 7 List of compounds identified by gas phase ion mobility spectrometry

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] It should be noted that, in this article, the terms "comprise", "include" or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise one..." do not exclude the presence of other identical elements in the process, method, article or device including the element. In addition, it should be pointed out that the scope of the methods and devices in the embodiments of the present application is not limited to performing functions in the order shown or discussed, and may also include performing functions in a substantially simultaneous manner or in reverse order according to the functions involved, for example, the described method may be performed in an order different from that described, and various steps may also be added, omitted, or combined. In addition, the features described with reference to certain examples may be combined in other examples.

[0102] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A preparation process of a chestnut probiotic plant-based fermented milk beverage, characterized in that: The shelled raw chestnuts stored in -20℃ are thawed at room temperature → sliced ​​→ baked → crushed → sieved → gelatinized → liquefied → saccharified → added with protein solution and mixed evenly → sterilized → inoculated and fermented → finished product.

2. A starter for preparing a chestnut probiotic plant-based fermented milk beverage is characterized by: A composite strain starter with strong oxidizing properties and more flavor substances was screened out from 8 probiotics (LR-Lactobacillus rhamnosus, LC-Lactobacillus casei, LP-Lactobacillus plantarum, L.P95-Lactobacillus plantarum, L.F04-Lactobacillus mucilaginosus, L.C17-Lactobacillus casei, LF-Lactobacillus mucilaginosus, L.F90-Lactobacillus plantarum).

3. An optimal fermentation process condition for a chestnut probiotic plant-based milk beverage, characterized in that: The optimal fermentation conditions were determined by single factor and orthogonal experiments using the ratio of saccharification liquid to protein liquid (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2), inoculation amount (6%, 7%, 8%, 9%, 10%, 11%, 12%), fermentation time (4h, 8h, 12h, 16h, 20h, 24h, 28h) and fermentation temperature (35℃, 36℃, 37℃, 38℃, 39℃, 40℃) as investigation factors and sensory evaluation as evaluation index.

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