CN116590185B - Application of lactobacillus rhamnosus and saccharomycetes in co-fermentation to improvement of pea meal characteristics - Google Patents
Application of lactobacillus rhamnosus and saccharomycetes in co-fermentation to improvement of pea meal characteristics Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/05—Mashed or comminuted pulses or legumes; Products made therefrom
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
- A23L31/10—Yeasts or derivatives thereof
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12R2001/225—Lactobacillus
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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Abstract
The invention discloses an application of lactobacillus rhamnosus and saccharomycetes in co-fermentation to improve pea meal characteristics, wherein the lactobacillus rhamnosus (Lactobacillus rhamnosus) is named L08 and is preserved in China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC) at the year 11 and 8 of 2022, and the preservation number of the lactobacillus rhamnosus is: CGMCC No.26088. The lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 have a mutually-beneficial symbiotic relationship, and the two are subjected to co-fermentation, so that the content of branched chain amino acid and aromatic amino acid is increased, the content of aldol furan type undesirable compounds is reduced, the composition of the flavor substances of the ester acid type compounds is increased, the gelatinization speed of pea flour is reduced, the emulsion stability and the foam stability of the pea flour are improved, and the partial physicochemical properties of the pea flour are improved, so that theoretical basis is provided for the peas as high-quality protein sources and application of the peas.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an application of lactobacillus rhamnosus and saccharomycetes in co-fermentation to improve pea meal characteristics.
Background
Peas as the world's second soybean crop have attracted considerable attention because of their low sensitization, high nutritional value, availability, and low cost, however, pea proteins, like other vegetable proteins, have limited application in the food industry due to their beany flavor and lower functional properties. In addition to the off-flavor effects, lipid oxidized secondary metabolites can react with pea proteins, resulting in loss of essential amino acids and structural changes of proteins, thus resulting in loss of functionality, while also limiting their use in the food processing field due to relatively poor physicochemical properties such as solubility and emulsifying properties. At present, research at home and abroad focuses on the processing of fertilizer feeds, pea starch, pea protein and the like, and the comprehensive utilization of resources and the deep processing technology of the fertilizer are still to be studied deeply.
It has been found that lactic fermentation can significantly alter the aroma components of lupins, which undergo concomitant biochemical changes during fermentation, such as organic degradation, thereby forming a more intense aroma profile, while metabolic enzymes and metabolites released during fermentation can affect protein function. After lactobacillus plantarum is used for fermenting pea protein for 11 hours, the hydrolysis degree can reach 13.5%, the hydrophobicity is improved along with the prolongation of fermentation time under the condition of pH 4, and the foamability and the foam stability are also improved. These prior studies have shown that fermentation can improve the characteristics of different legume products, leading to their use as functional food ingredients. Fermentation has been widely used to improve the organoleptic properties of different cereal and legume products.
Yeasts are often used for fermenting food and beverages, especially commercial Saccharomyces cerevisiae, for its reliability, rapidity and specificity, exclusively for wine fermentation. Saccharomyces cerevisiae has enzymatic activities such as aldol dehydrogenases that reduce off-flavors in soybeans by degrading aldehydes and alcohols. In addition, the addition of yeast can add new descriptions such as banana flavor, apricot flavor. The lactobacillus rhamnosus has the functions of improving immunity, balancing intestinal flora, treating diarrhea, eliminating toxin and the like as probiotics, and is excellent in the aspects of dairy products, drinks, health-care foods and the like. The lactobacillus rhamnosus L08 and the saccharomyces cerevisiae have a mutually-beneficial symbiotic relationship, and the two are fermented together, so that the peculiar smell compounds of the pea powder are reduced, the flavor components of the pea powder are enriched, the gelatinization speed of the pea powder is reduced, the emulsion stability, the foam stability and the like of the pea powder are improved, and the partial physicochemical properties of the pea powder are improved.
The microbiota are used to ferment plant-based products, with potential synergistic effects between them, which contribute to the quality of the fermented product. Lactobacillus plantarum and lactobacillus rhamnosus have the capability of fermenting by utilizing plant-based components, the influence of strain characteristics and fermentation conditions on volatile compounds of pea powder is explored, most peculiar smell compounds in pea powder are degraded through a lactobacillus rhamnosus L08 and saccharomyces cerevisiae co-fermentation process, the flavor substance composition is enriched, the functional properties are also improved, the method has important significance for improving the integral flavor quality of pea products, theoretical support is provided for developing high-quality pea products with controllable flavor, scientific basis is provided for applying peas as high-quality protein sources and recycling comprehensive utilization of peas, and the industrialized development of peas is promoted to meet industry requirements and consumer expectations.
Disclosure of Invention
The invention aims at solving the problems existing in the prior art, overcomes the defects of the prior art, and designs an application of lactobacillus rhamnosus and saccharomycetes in co-fermentation to improve the pea meal characteristics.
The invention provides a strain with a preservation number of: lactobacillus rhamnosus L08 (Lactobacillus rhamnosus L) of CGMCC No.26088.
Furthermore, the invention takes the activity and the flavor of the strain as key indexes, screens out lactobacillus rhamnosus L08 which can grow well in pea matrixes, carries out co-fermentation with saccharomyces cerevisiae, and optimizes the conditions of strain proportion, inoculum size, fermentation temperature, fermentation time and the like; analyzing the change of the physicochemical properties of pea meal before and after fermentation, and providing theoretical reference for improving the functional properties and processing characteristics of the pea meal; the SPME-GC-MS technology is used for dynamically monitoring the content and the composition of amino acids in a combined mode, changes of flavor components in the fermentation process are researched, and theoretical basis is provided for reducing the peculiar smell of pea meal and the application of the pea meal serving as a functional food ingredient component.
In order to achieve the above object, the present invention provides the following technical solutions:
1) Preparation of MRS Medium and YPD Medium.
2) Preparation of pea fermentation liquor: a certain amount of pea powder is taken and put into a 50mL conical flask for sterilization for 15min at 115 ℃, and after cooling to room temperature, sterilized distilled water with the concentration of 20% (w/v) is added.
3) Activation of the strain: thawing lactobacillus rhamnosus and Saccharomyces cerevisiae at room temperature, and respectively inoculating to MRS and YPD culture medium for 3 generations.
4) Growth curve of strain: inoculating activated 3-generation lactobacillus rhamnosus L08 to an MRS culture medium, culturing at 37 ℃ for 24 hours, sampling and diluting every 2 hours, then coating the diluted sample on the MRS solid culture medium, culturing at 37 ℃ for 48 hours, and counting; the activated 3-generation Saccharomyces cerevisiae M31 is inoculated in YPD medium, cultured for 24 hours at 28 ℃, sampled and diluted every 2 hours, then coated in YPD solid medium, and counted after culturing for 48 hours at 28 ℃.
5) Inoculating: the activated lactobacillus rhamnosus and the saccharomyces cerevisiae M31 are inoculated into a conical flask containing 20mL of pea solution for culture.
Preferably, in the foregoing step 1), the preparation method of the MRS liquid medium includes: mixing 20g of glucose, 10g of tryptone, 5g of peptone, 5g of yeast powder, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1g of tween, 5g of beef extract and 1L of distilled water in a container, and shaking the container until the solvent is dissolved to obtain an MRS liquid culture medium; the preparation method of the MRS solid culture medium comprises the following steps: adding 2% -3% agar into MRS liquid culture medium to obtain MRS solid culture medium. Preferably, the sterilization condition is 121 ℃ for 20min.
Preferably, in the foregoing step 1), the preparation method of the YPD liquid medium comprises: mixing 20g of glucose, 20g of peptone, 10g of yeast extract and 1L of distilled water in a container, and shaking the container until the solvent is dissolved to obtain YPD liquid culture medium; the preparation method of the YPD solid culture medium comprises the following steps: adding 2% -3% agar into YPD liquid culture medium to obtain YPD solid culture medium. Preferably, the sterilization condition is 121 ℃ for 15min.
Preferably, in the foregoing steps 3) to 5), the co-fermentation conditions of lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31 are: the ratio of the strains is 1:1, the inoculum size is 3-10%, the fermentation temperature is 30-35 ℃ and the fermentation time is 10-14h; more preferably, the ratio of the inoculated strain is 1:1 (v/v), the inoculation amount is 3% (v/v), the fermentation temperature is 32 ℃, and the fermentation time is 12 hours.
The invention has the advantages that: the invention screens and obtains lactobacillus rhamnosus L08 (Lactobacillus rhamnosus L08) which can mutually promote with saccharomyces cerevisiae M31 to improve partial physicochemical properties of pea meal. Compared with independent fermentation of lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31, the co-fermentation increases the substrate utilization rate, improves the emulsion stability and foam stability of pea powder, reduces the gelatinization speed of pea powder, and has a stable system. And the two are fermented together to increase the content of branched chain amino acid and aromatic amino acid, reduce the content of aldol furan type bad compounds, increase the composition of the flavor substances of the ester acid type compounds, and the pea meal has better flavor after the co-fermentation, thereby providing a theoretical basis for the pea meal as a functional base material component, the pea as a high-quality protein source and the application thereof.
Drawings
FIG. 1 is a technical scheme for co-fermentation of Lactobacillus rhamnosus L08 with Saccharomyces cerevisiae M31 in a pea matrix;
FIG. 2 shows the growth curves of Lactobacillus rhamnosus L08 and Saccharomyces cerevisiae M31 when they are co-fermented in a pea matrix;
FIG. 3 is the effect of Lactobacillus rhamnosus L08 to Saccharomyces cerevisiae M31 strain ratio on pea juice; a) -the effect of strain proportion on biomass; b) -influence of strain ratio on pea fermentation broth pH and titrating acidity; c) -fermenting sample electronic nose response radar graphs at different strain ratios; d) -sensory scores of the fermented samples at different strain ratios.
FIG. 4 is a graph showing the effect of Lactobacillus rhamnosus L08 and Saccharomyces cerevisiae M31 strain inoculum size on pea liquid; a) -effect of inoculum size on biomass; b) -effect of inoculum size on pea fermentation broth pH and titrating acidity; c) -electronic nose response radar plots for different inoculum size fermentation samples; d) -different inoculum size fermented sample sensory scores.
FIG. 5 shows the effect of fermentation temperature on pea juice when lactobacillus rhamnosus L08 and Saccharomyces cerevisiae M31 are co-fermented; a) -the effect of fermentation temperature on biomass; b) -effect of fermentation temperature on pea fermentation broth pH and titrating acidity; c) -fermenting sample electronic nose response radar maps at different temperatures; d) -sensory scores of the fermented samples at different temperatures.
FIG. 6 shows the effect of fermentation time on pea juice when lactobacillus rhamnosus L08 is co-fermented with Saccharomyces cerevisiae M31; a) -the effect of fermentation time on biomass; b) -effect of fermentation time on pea fermentation broth pH and titrating acidity; c) -a radar chart of the electronic nose response of the fermented samples at different times; d) -sensory scores of different time fermented samples.
FIG. 7 is the effect of Lactobacillus rhamnosus L08 co-fermentation with Saccharomyces cerevisiae M31 on pea particle size distribution; a) Influence of lactobacillus rhamnosus L08 fermentation alone on pea particle size distribution; b) Influence of lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31 co-fermentation on pea particle size distribution; c) Influence of Saccharomyces cerevisiae M31 fermentation alone on pea particle size distribution.
FIG. 8 shows the effect of co-fermentation of Lactobacillus rhamnosus L08 with Saccharomyces cerevisiae M31 on zeta potential of pea flour; a refers to lactobacillus rhamnosus L08 which is fermented singly; b refers to co-fermentation of lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31; c means that Saccharomyces cerevisiae M31 is fermented alone; the following is the same. Different lowercase letters indicate that the samples of the same strain were significantly different at different times, P <0.05; different capital letters indicate that the difference between different treatment groups at the same time is significant, P <0.05, the following.
FIG. 9 is the effect of lactobacillus rhamnosus L08 co-fermentation with Saccharomyces cerevisiae M31 on pea meal solubility; a) -solubility of pea flour at pH 4; b) -solubility of pea flour at pH 7.
FIG. 10 shows the degree of hydrolysis of pea flour during co-fermentation of Lactobacillus rhamnosus L08 with Saccharomyces cerevisiae M31;
FIG. 11 is the effect of lactobacillus rhamnosus L08 co-fermentation with Saccharomyces cerevisiae M31 on the emulsion stability of pea meal;
FIG. 12 is the effect of Lactobacillus rhamnosus L08 co-fermentation with Saccharomyces cerevisiae M31 on the foam stability of pea meal;
fig. 13 is a cluster heat map of pea meal after co-fermentation of lactobacillus rhamnosus L08 with saccharomyces cerevisiae M31.
Wherein, in fig. 3-6, different lowercase letters indicate that lactobacillus rhamnosus L08 differs significantly in biomass, P <0.05; different capital letters indicate that the difference in Saccharomyces cerevisiae M31 biomass was significant, with P <0.05.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
The present invention includes materials and apparatus as set forth in tables 1-4 below:
TABLE 1 MRS Medium
Note that: preparing a solid culture medium, adding 2% -3% agar, and sterilizing at 121 ℃ for 20min.
TABLE 2 YPD Medium
Note that: adding 2% -3% agar when preparing solid culture medium, and sterilizing at 121deg.C for 15min.
TABLE 3 principal materials and reagents
Note that: the above reagents are all analytically pure except as specified.
TABLE 4 Main instruments and apparatus
The Saccharomyces cerevisiae M31 described in the examples is commercially available from the company Bettshi self-brewing machine, inc. of the tobacco stand Di.
Example 1
1. Strain activation and growth curves
(1) Activation of lactobacillus rhamnosus L08
Lactobacillus rhamnosus L08 (Lactobacillus rhamnosus L) is obtained through screening, and is preserved in China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC) at the address of 2022, 11 and 8: the Beijing city, the Korean region, north Chen Xili No. 1, 3, the strain deposit number is: CGMCC No.26088.
Lactobacillus rhamnosus L08 was inoculated in 3% (v/v) inoculum size to MRS medium for 3 passages of activation after thawing at room temperature.
(2) Saccharomyces cerevisiae M31 separation and purification
Proliferation culture: and inoculating the purchased saccharomyces cerevisiae M31 into YPD culture medium to culture at 28 ℃ until bacterial liquid becomes turbid. Then, the cells were inoculated in YPD medium at a 3% (v/v) inoculum size and cultured at 28℃for 24 hours.
And (3) separating and purifying: repeatedly streaking and separating the bacterial liquid on YPD solid culture medium to obtain pure culture, observing single bacterial colony by microscopic examination, preserving bacterial colony conforming to the characteristics, and storing in a refrigerator at-20 ℃.
Activating: the isolated and purified yeast was inoculated in an amount of 3% (v/v) to YPD medium for 3 generations of activation.
(3) Growth curve
The lactobacillus rhamnosus L08 which is activated for 3 generations is inoculated into an MRS culture medium with an inoculum size of 3% (v/v), is cultured for 24 hours at 37 ℃, is sampled and diluted every 2 hours, is coated on the MRS solid culture medium, and is counted after being cultured for 48 hours at 37 ℃.
The 3-generation activated Saccharomyces cerevisiae M31 was inoculated to YPD medium at an inoculum size of 3% (v/v), cultured at 28℃for 24 hours, diluted by sampling every 2 hours, and then spread on YPD solid medium, and after culturing at 28℃for 48 hours, counted.
2. Co-fermentation of lactobacillus rhamnosus L08 and Saccharomyces cerevisiae M31
The lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 are inoculated into a conical flask containing 20mL of pea solution at a total inoculum size of 3% (v/v), the ratio of inoculated strains is 1:1 (v/v), the lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 are cultivated for 12 hours at a constant temperature of 37 ℃, biomass, acid production, flavor and sensory scores in pea matrixes are observed, and subsequent tests are carried out.
3. Biomass determination
100. Mu.L of pea fermentation broth was diluted with sterilized physiological saline gradient (10 -4 、10 -5 、10 -6 、10 -7 ) After that, lactobacillus rhamnosus L08 is coated with MRS solid culture medium when being fermented singly, and is cultured for 48 hours at 37 ℃; coating YPD solid culture medium during the single fermentation of Saccharomyces cerevisiae M31, and culturing at 28deg.C for 48 hr; lactobacillus rhamnosus L08 was co-fermented with modified MC agar medium and Saccharomyces cerevisiae M31 with Bengalia agar medium, and biomass was expressed in terms of lg CFU/mL.
4. Lactobacillus rhamnosus L08 and Saccharomyces cerevisiae M31 co-fermentation condition optimization
The optimal conditions of co-fermentation of lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31 are that biomass, pH value, titrated acidity and flavor are used as evaluation indexes, a single factor test is carried out by adopting a plate colony counting method, a pH meter, an acid-base titration method, an electronic nose and sensory evaluation, the influence of the strain proportion of lactobacillus rhamnosus and saccharomyces cerevisiae (2:1, 1.5:1, 1:1.5, 1:2), inoculum size (2%, 3%, 4%, 5%, 6%), fermentation temperature (28 ℃, 30 ℃, 32 ℃, 35 ℃,37 ℃) and fermentation time (6 h, 12h, 24h, 48 h) on the biomass, the pH value, titrated acidity and flavor is examined, and the effects are observed and recorded after culture.
5. Determination of physical Properties
The lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 are independently fermented to serve as a control, particle size, zeta potential, solubility, microstructure, thermal property and gelatinization property are used as evaluation indexes, and the influence of the co-fermentation of the lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 on the physical properties of pea powder is studied.
(1) Particle size distribution and zeta potential measurement
A certain amount of the freeze-dried sample was weighed and dissolved in ultrapure water, stirred at room temperature for 2 hours, and centrifuged at 8000rpm for 15 minutes to remove insoluble precipitate. Particle size distribution and zeta potential of soluble proteins in the sample were determined using a particle size and potential analyzer.
(2) Solubility determination
A certain amount of the lyophilized sample was weighed and dissolved in ultrapure water, stirred at room temperature for 2 hours, and centrifuged at 8000rpm for 15 minutes. Protein content was determined using BCA kit and standard curves were drawn using Bovine Serum Albumin (BSA) as standard. Protein solubility is expressed as the percentage of supernatant protein content to total protein content.
(3) Microstructure determination
And placing the dried sample on an aluminum platform by using a conductive adhesive tape, then placing the sample platform in a gold plating instrument, taking out the object platform after 20min, and observing by using a tungsten filament scanning electron microscope under the magnification of 500 times, wherein the accelerating voltage is 5KV.
(4) Measurement of thermal Properties
3.5mg of pea meal was placed in an aluminum pan dedicated to DSC, 8. Mu.L of distilled water was added and compacted. The differential calorimeter was equilibrated at 20deg.C for 3min, raised to 140deg.C at 10deg.C/min, lowered to 30deg.C at 30deg.C/min, and thermal characteristics were recorded.
(5) Determination of the gelatinization Property
Accurately weighing 3.00g of pea powder, putting into an RVA container, adding 25mL of deionized water, stirring uniformly, heating to 50 ℃ for 1min, heating to 95 ℃ for 2.5min within 3.75min, and then cooling to 50 ℃ for 2min within 3.75 min. The 10s rotation speed before the test was 960rpm/min, and the rotation speed after the test was 160rpm/min, and the characteristic value of gelatinization was recorded.
6. Determination of chemical Properties
The lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 are independently fermented to serve as a control, the degree of hydrolysis, the emulsifying property, the emulsifying stability, the foamability and the foam stability are used as evaluation indexes, and the influence of the co-fermentation of the lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 on the chemical characteristics of the pea powder is studied.
(1) Measurement of degree of hydrolysis
The method for measuring the Degree of Hydrolysis (DH) was measured according to the o-phthalaldehyde (OPA) method.
1) Standard curve determination
0mg, 2.5mg, 5mg, 10mg, 20mg and 40mg serine are respectively weighed and placed in different beakers, deionized water is added for dissolution, serine standard liquids with different concentrations are prepared, the light absorption value is measured at 340nm, and a standard curve is drawn.
2) Sample measurement
1mL of fermented pea liquid was taken and diluted 10-fold. After mixing 400. Mu.L of the sample with 3mL of OPA in an EP tube, the mixture was allowed to stand for 2min at 340nm to measure the absorbance, and deionized water was used instead of the blank. DH is defined as the percentage of cleaved peptide bonds and the degree of hydrolysis of the sample is calculated according to the following formula.
Wherein:
ODsample: sample OD value;
ODblank: blank sample OD value;
ODstandard: standard OD value;
x: sample amount;
p: protein content in the sample.
Wherein:
α＝0.970，β＝0.342；
h: the number of hydrolytic bonds;
htot: the total number of peptide bonds per protein equivalent depends on the starting amino acid composition.
(2) Determination of emulsifying Property and emulsion stability
Weighing a certain amount of freeze-dried sample, dissolving in deionized water, mixing 20mL of sample solution with 5mL of peanut oil, emulsifying at 10000rpm/min under a high-speed homogenizer for 2min, and immediately sucking 50 μL of milk from the bottom
The emulsion was diluted 100-fold with 0.1% (w/v) SDS solution, and the emulsifying property (EAI) was calculated as follows, with 0.1% SDS solution as a blank:
wherein:
T：2.303；
n: dilution factor 100;
c: protein concentration before emulsion formation (g/mL);
u: volume fraction of oil 0.25;
a0: absorbance measured at 500nm after 5s of shaking.
Emulsion Stability (ESI) was calculated as follows:
wherein: a10: absorbance measured at 500nm after 10min of rest.
(3) Determination of foamability and foam stability
A certain amount of the freeze-dried sample is weighed and dissolved in deionized water. Taking a certain volume of sample solution, recording the height V0, homogenizing for 2min at 10000rpm/min under a high-speed homogenizer, recording the height V1, standing for 30min, and recording the height V30 again. The Foaming Capacity (FC) and Foam Stability (FS) formulas are as follows:
7. flavour determination
The lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 are independently fermented to serve as a control, amino acid and volatile components are used as evaluation indexes, and the influence of the lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 fermentation on the flavor of pea meal is studied.
(1) PH determination
5.0mL of pea solution fermented to a certain time is taken in a test tube and directly measured by a pH meter.
(2) Titration acidity determination
Reference is made to the method for determining acidity in GB 5009.239-2016.
(3) Electronic nose measurement
2mL of pea fermentation broth was placed in a10 mL special bottle and the sample was tested at 25℃using an electronic nose.
(4) Establishment of sensory evaluation
10 persons with food professional background are invited to participate in sensory evaluation by taking color, aroma, tissue state and overall acceptability as sensory evaluation indexes, and the full scale is 100 minutes.
(5) Amino acid content determination
The amino acid content was determined by means of acid hydrolysis. Accurately weighing 0.50g of pea meal, placing in a10 mL hydrolysis tube, adding 10mL of HCl (6N), hydrolyzing at 110 ℃ for 22h, cooling to room temperature, filtering the hydrolyzed solution to a 50mL volumetric flask, and shaking uniformly after the volume is fixed by deionized water. Accurately sucking 1.0mL of filtrate into a pear-shaped bottle, drying at 50 ℃ under reduced pressure by using a rotary evaporator, dissolving the dried residue with 1mL of deionized water, drying under reduced pressure until the residue is evaporated, adding 2mL of sodium citrate (pH 2.2) buffer solution into the bottle to fully dissolve the residue, uniformly mixing the solution, filtering the solution with a 0.22 mu m filter membrane, and transferring the solution to an instrument sample injection bottle to obtain a sample measuring solution.
The column temperature is 57 ℃, the reaction temperature is 135 ℃, the sample injection amount is 20 mu L, and the detection wavelengths are 570nm and 440nm. The amino acid concentration in the sample was calculated by the external standard method by peak area.
(6) Volatile component determination
1) SPME extraction of sample volatile components
Accurately weighing 1.00g of pea powder, placing into a 15mL sample bottle, adding 1mL of NaCl (20%) solution, placing into a magnetic stirrer for sealing, balancing for 5min, inserting an aged extraction head for adsorption for 45min, then analyzing for 5min at a sample inlet, and heating the sample bottle in a water bath at 60 ℃ of a magnetic stirrer. The extraction head was aged at 250 ℃ for 30min at the gas chromatograph inlet before each use.
2) Instrument conditions
Gas chromatography conditions: the column type is DB-5MS capillary column (30.0mX0.25mm, 0.25 μm), the temperature of the sample inlet is 250 ℃, high purity He is used as carrier gas, the flow rate is 1.0mL/min, and sample injection is not split; the temperature program was 45℃to 80℃for 2min, then 5℃to 230℃for 8min.
Mass spectrometry conditions: ionization mode EI, ionization voltage 70eV, ion source temperature 230 ℃, scanning mass range 40-400m/z, scanning mode Scan.
3) Compound identification and analysis
The unknown compounds were matched to a mass spectrum library (NIST 17-1) by computer search and compared to a series of standard alkanes (C8-C40) and the volatile components were determined based on their CAS numbers and molecular structures, in combination with literature reports. And calculating the relative percentage content of each substance by adopting a peak area normalization method.
8. Data processing and statistical analysis
Each set of test data was repeated three times and the results were expressed as mean ± standard deviation, and the results were analyzed for significance using SPSS Statistics21 software, plotted against Excel 2016 and Origin 2018, and P <0.05 was considered significant. The electronic nose data adopts WinMuster 1.6.2 to carry out principal component analysis and linear discriminant analysis.
9. Conclusion analysis
(1) The lactobacillus rhamnosus L08 can be used as a potential fermentation strain of pea meal, the lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31 can mutually promote the growth of the lactobacillus rhamnosus L08 and the saccharomyces cerevisiae M31, and the pea meal has good flavor when the lactobacillus rhamnosus L08 and the saccharomyces cerevisiae are co-fermented.
(2) The optimal conditions for co-fermentation of lactobacillus rhamnosus L08 and Saccharomyces cerevisiae M31 are as follows: the ratio of the inoculated strain is 1:1 (v/v), the inoculum size is 3% (v/v), the fermentation temperature is 32 ℃, and the fermentation time is 12h.
(3) Co-fermentation improves the physical properties of the pea meal fraction. Compared with lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31 which are fermented independently, the co-fermented system is stable, and the gelatinization speed of pea powder is reduced.
(4) Co-fermentation improves the chemical properties of the pea meal fraction. The co-fermentation can increase the substrate utilization rate and improve the emulsion stability and foam stability of the pea powder.
(5) Co-fermentation improves pea meal flavor. The flavor substance composition of pea flour is enriched by increasing the content of branched chain amino acid and aromatic amino acid, reducing the content of undesirable compounds such as aldol furans and increasing the content of compound types such as ester acids, and the co-fermentation generates more flavor compound types.
In a word, lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31 are fermented together, so that the processing characteristics and partial functional characteristics of pea meal are improved, the flavor substance composition of the pea meal is improved, and theoretical basis is provided for the pea meal serving as a functional base material component, the pea serving as a high-quality protein source and application thereof.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.
Claims (8)
1. The lactobacillus rhamnosus is characterized in that the lactobacillus rhamnosus (Lactobacillus rhamnosus) is named as L08, and is preserved in China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC) in 2022 and 11-8 days, and the strain preservation number is: CGMCC No.26088.
2. A composition for improving the characteristics of pea meal, comprising lactobacillus rhamnosus L08 according to claim 1 and saccharomyces cerevisiae M31.
3. The method for preparing the composition according to claim 2, wherein the co-fermentation conditions of lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31 are as follows: the ratio of the strains is 1:1, the inoculation amount is 3-10%, the fermentation temperature is 30-35 ℃, and the fermentation time is 10-14h.
4. Use of lactobacillus rhamnosus according to claim 1 or a composition according to claim 2 for improving pea meal properties.
5. The use according to claim 4, wherein said improving pea meal characteristics comprises: the pea flour has the advantages of peculiar smell compounds, pea flour gelatinization speed, emulsion stability and foam stability.
6. The use according to claim 4 or 5, characterized in that the whole pea particles after fermentation of lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31 are moved in the direction of high particle size, exhibiting a more pronounced bimodal distribution; the minimum zeta-potential absolute value change of the co-fermented pea powder is 2.07 and mV, and the co-fermented system is stable; the co-fermentation increases the gelatinization temperature by 2.00 ℃ and reduces the gelatinization speed of the pea flour.
7. The use according to claim 4 or 5, characterized in that the foamability of the co-fermented pea flour of lactobacillus rhamnosus L08 with saccharomyces cerevisiae M31 is slightly reduced; and the emulsion stability and the foam stability are increased.
8. The use according to claim 4 or 5, wherein the total amino acid content, the aromatic amino acid content of the sample co-fermented with lactobacillus rhamnosus L08 and saccharomyces cerevisiae M31 are both increased.
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| CN116590185A (en) | 2023-08-15 |
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