CN113549583A - Complex microbial inoculant, application of complex microbial inoculant and method for preparing quinoa fermented milk by using complex microbial inoculant - Google Patents
Complex microbial inoculant, application of complex microbial inoculant and method for preparing quinoa fermented milk by using complex microbial inoculant Download PDFInfo
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- CN113549583A CN113549583A CN202110978149.7A CN202110978149A CN113549583A CN 113549583 A CN113549583 A CN 113549583A CN 202110978149 A CN202110978149 A CN 202110978149A CN 113549583 A CN113549583 A CN 113549583A
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
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- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
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- A23V2400/11—Lactobacillus
- A23V2400/113—Acidophilus
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Abstract
The invention discloses a complex microbial inoculant, application of the complex microbial inoculant and a method for preparing quinoa fermented milk by using the complex microbial inoculant, and belongs to the technical field of health-care food. The composite microbial inoculum is prepared by mixing lactobacillus plantarum and lactobacillus acidophilus in a mass ratio of 1-3: 1-3; the method for preparing the quinoa fermented milk by using the complex microbial inoculum comprises the following steps: aging quinoa, and pulping to obtain quinoa mash; mixing quinoa mash with xylitol and reconstituted milk, heating and homogenizing, sterilizing, cooling, then inoculating a compound microbial inoculum, and fermenting to obtain the quinoa fermented milk. The quinoa fermented milk prepared by the invention has excellent oxidation resistance and capability of inhibiting related digestive enzymes, has a good in vitro auxiliary blood sugar reducing function, and can meet the eating requirements of diabetes patients.
Description
Technical Field
The invention relates to the technical field of health-care food, and in particular relates to a compound microbial inoculum, application of the compound microbial inoculum and a method for preparing quinoa fermented milk by using the compound microbial inoculum.
Background
Lactic Acid Bacteria (LAB) are commonly used probiotics in fermented foods and beverages, as well as food supplements for humans or animals, and are of a wide variety and widely present in the intestinal tract of humans and animals, in food. Lactic acid bacteria positively affect the host by improving and stabilizing the intestinal microflora, which adhere to intestinal epithelial cells, and prevent the development of harmful microorganisms by producing antibacterial substances such as organic acids and bacteriocins. They can also enhance non-specific cellular immune responses by activating macrophages, Natural Killer (NK) cells, and releasing various cytokines. Furthermore, they can improve the intestinal mucosal immune system by increasing the number of IgA (+) cells. Lactobacillus (Lactobacillus) plays an important role in the treatment of many diseases, including diarrhea, gastroenteritis, inflammatory bowel disease, hyperglycemia, and chronic metabolic syndrome directly associated with impaired immune function. The lactobacillus fermented food mainly has the following functions: (1) special taste, aroma and texture can be obtained by fermenting different food substrates; (2) the purpose of preserving food is achieved by metabolizing lactic acid, alcohol, acetic acid and inhibiting putrefying bacteria through alkaline fermentation; (3) the product is biologically enriched by metabolizing proteins, essential amino acids, fatty acids. Different strains have different forms and physiological characteristics.
The coarse cereal fermented milk is a dairy product which has special flavor and is rich in probiotic activity and is prepared by taking coarse cereals and raw milk or reconstituted milk as main materials, performing pasteurization, and inoculating lactic acid bacteria for fermentation. The coarse cereals are rich in dietary fibers, resistant starch and other digestion-resistant substances, can control the weight, cannot cause the rapid rise of the blood sugar concentration in a body after being eaten, and belong to food raw materials with low glycemic index (low GI). Secondly, the cereal food contains rich plant active ingredients, such as phenolic compounds, saponins, phytoestrogens and the like, and can effectively prevent diabetes, cancers, cardiovascular and cerebrovascular chronic diseases and the like after being eaten frequently. The coarse cereal fermented milk has important significance for human dietary balance, and has more research value than common fermented milk.
Quinoa is considered as a vegetable food suitable for all people, can be used as a main dietary choice for consumers with strong health consciousness, vegetarians, athletes and other people, and can replace meat products and milk products to provide protein for human bodies in many places because of higher protein utilization rate and balanced amino acid composition proportion compared with most coarse cereal crops. The quinoa grain contains 7.47-22.08% of protein, wherein the quinoa grain mainly comprises 11S-type globulin and 2S albumin, which respectively account for 37% and 35% of total protein, and also contains a small amount of prolamin. The quinoa amino acid composition proportion is closer to the amino acid proportion required by human bodies, and is rich in lysine (5.1-6.4%), methionine (0.4-1.0%) and histidine required by special people. The quinoa seed has carbohydrate content equivalent to that of other coarse cereals, the main component is starch, the dry matter of the starch is about 30-70%, and the characteristics of the quinoa powder are greatly dependent on the composition and the properties of the quinoa starch. The quinoa starch has small particles, the proportion of amylose to amylopectin is lower, the proportion of amylose to amylopectin is 1:3, and the amylopectin has a large amount of short chains and super-long chains, so the quinoa starch is widely applied to the food industry and other industries, such as the preparation of Pickering emulsion. Quinoa oil can be used as a skin moisturizer in cosmetics because it is a powerful complex of antioxidant and anti-inflammatory activity due to the strong combination of naturally essential fatty acids and vitamin E in quinoa, helps to restore the barrier function of the skin epidermis, and prevents the premature aging phenomenon by increasing collagen and elastin. The quinoa total dietary fiber content is about 13.4%, wherein the soluble dietary fiber content is about 2.4%, and the insoluble dietary fiber content is about 11.0%. The quinoa dietary fiber has strong water absorption and expansion capacity, the volume is rapidly increased after cooking, the satiety of the organism can be improved after the quinoa dietary fiber is ingested, the food intake is reduced, the weight control is facilitated, and in addition, the quinoa dietary fiber can influence the cardiovascular disease prevention, the glucose metabolism and lipid metabolism regulation and the anti-tumor capacity. Tests of feeding mice prove that the blood sugar and blood fat of the body can be greatly reduced by eating the quinoa for a long time, and the quinoa belongs to low-sugar and low-calorie food, so that the quinoa can be used as a main dietary choice for three-high people, obese people and diabetic patients. Chenopodium quinoa is a gluten-free food, can treat celiac disease patients, and provides various nutritional foods for people with gluten allergy (2% of adults and 5% of children).
The complicated chronic disease diabetes is the 11 th death reason worldwide, and at present, no method for completely curing diabetes exists, and the diabetes can be treated only by thinking of improving the sensitivity of human islet cells, reducing insulin resistance, reducing postprandial blood sugar and the like. At present, probiotics with anti-diabetes potential such as lactic acid bacteria and the like are applied to inoculation of natural products to further improve anti-diabetes activity of the natural products, researches prove that probiotic fermented products can be used as functional food for supplementing or assisting in treating diabetes, but the anti-oxidation and auxiliary blood sugar reduction effects of coarse cereal fermented milk prepared by using the existing lactic acid bacteria fermenting agent are not ideal, and a quinoa fermented milk health-care food with excellent auxiliary blood sugar reduction effect is not available in the market. Therefore, the quinoa fermented milk which has a good auxiliary blood sugar reduction effect and can meet the eating requirements of diabetes patients is very necessary to prepare.
Disclosure of Invention
The invention aims to provide a complex microbial inoculant, application and a method for preparing quinoa fermented milk by using the complex microbial inoculant, so as to solve the problems in the prior art, ensure that the quinoa fermented milk has a good auxiliary blood sugar reduction effect, and can meet the edible requirements of diabetes patients.
In order to achieve the purpose, the invention provides the following scheme:
one of the technical schemes of the invention is to provide a composite microbial inoculum, wherein the composite microbial inoculum is prepared by mixing lactobacillus plantarum and lactobacillus acidophilus in a mass ratio of 1-3: 1-3.
Further, the composite microbial inoculum is a composite microbial inoculum with the mass ratio of lactobacillus plantarum to lactobacillus acidophilus being 2: 1.
One of the technical schemes of the invention is to provide application of the complex microbial inoculum in preparation of fermented milk.
Further, the fermented milk is quinoa fermented milk.
The third technical scheme of the invention is to provide a method for preparing quinoa fermented milk by using the complex microbial inoculum, which comprises the following steps:
aging quinoa, and pulping to obtain quinoa mash;
mixing the quinoa wheat pulp with xylitol and reconstituted milk, and heating and homogenizing the obtained mixture to obtain a homogenized mixture;
and sterilizing and cooling the homogenized mixture, inoculating the compound microbial inoculum, and fermenting to obtain the quinoa fermented milk.
Further, the pulping material-water ratio is 1g:5 mL.
Furthermore, the addition amount of the quinoa wheat pulp is 20-40% of the mass of the mixture.
Further, the addition amount of the xylitol is 3-7% of the mass of the mixture.
Further, the heating temperature is 70 ℃; the homogenizing pressure is 20MPa, and the homogenizing time is 10 min.
Further, the inoculation amount of the composite microbial inoculum is 2-4%.
Further, the inoculation amount of the composite microbial inoculum is 3%.
Further, the fermentation temperature is 36-40 ℃, and the fermentation time is 6-8 h.
Further, the fermentation temperature is 38 ℃, and the fermentation time is 8 h.
The invention discloses the following technical effects:
the quinoa fermented milk prepared by the compound microbial inoculum has basic nutritional ingredients meeting the requirements of diabetes patients, and microbial indexes meeting the standards.
The cane sugar-free quinoa fermented milk prepared by the invention has DPPH free radical clearance rate and ABTS+Radical scavenging, hydroxyl radical scavenging, Fe3+The reduction capacity, the alpha-glucosidase inhibition rate and the alpha-amylase inhibition rate are all higher than those of common fermented milk, the fermented milk has better oxidation resistance and relevant digestive enzyme inhibition capacity, has good in vitro auxiliary blood sugar reducing function, and can meet the eating requirements of diabetes patients.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a lactic acid bacteria growth curve;
FIG. 2 shows the results of tolerance of lactic acid bacteria at different pH values, wherein A is the results of tolerance of lactic acid bacteria at pH 2.0 and B is the results of tolerance of lactic acid bacteria at pH 3.0;
FIG. 3 shows the results of the tolerance of the bile salts of lactic acid bacteria, wherein, the graph A shows the results of the tolerance of lactic acid bacteria at 0.3% of the bile salts, and the graph B shows the results of the tolerance of lactic acid bacteria at 0.5% of the bile salts;
FIG. 4 is a graph showing the effect of the ratio of the composite starter on the pH and viable count of fermented milk;
FIG. 5 shows the effect of xylitol addition on the quality of fermented milk;
FIG. 6 shows the effect of the addition of quinoa pulp on the SOD activity and sensory score of fermented milk;
FIG. 7 shows the effect of lactobacillus inoculation on fermented milk SOD activity and sensory score;
FIG. 8 is a graph showing the effect of fermentation temperature on fermented milk SOD activity and sensory score;
FIG. 9 is a graph showing the effect of fermentation time on fermented milk SOD activity and sensory score;
FIG. 10 shows the change in DPPH radical scavenging efficiency of fermented milk at different concentrations;
FIG. 11 shows fermented milk ABTS at different concentrations+A change in free radical clearance;
FIG. 12 shows the change of the clearance rate of hydroxyl radicals of fermented milk at different concentrations;
FIG. 13 shows fermented milk Fe at different concentrations3+A change in reducing power;
FIG. 14 shows the alpha-glucosidase inhibition rate changes of fermented milk at different concentrations;
FIG. 15 shows the variation of the alpha-amylase inhibition of fermented milks at different concentrations.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Materials and reagents used in the present invention:
chenopodium quinoa willd, Qinghai plateau red chenopodium quinoa willd; skimmed milk powder, black dragon river double city nestle ltd; lactobacillus Acidophilus (LA), Lactobacillus Plantarum (LP), Lactobacillus Rhamnosus (LR), Lactobacillus paracasei (LCP), northeast university of agriculture bacterial pool; sodium hydroxide, hydrochloric acid, ferric chloride, taurocholate, potassium ferricyanide, phenolphthalein, Hengxing chemical reagent manufacturing Co., Ltd; SOD activity detection kit, hydroxyl radical test kit, Nanjing to build a bioengineering research institute; 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH), 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), Sigma company, usa; alpha-glucosidase, p-nitrophenol-alpha-D-glucopyranoside (PNPG) solution, alpha-amylase, Shanghai-derived leaf Biotech, Inc.
Activating the lactobacillus strain:
inoculating the bacterium liquid preserved by the glycerol into a sterilized MRS liquid culture medium, shaking up, placing in a constant-temperature incubator at 37 ℃ for culturing for 24h, selecting the bacterium liquid in an aseptic operation table, inoculating into an MRS solid culture medium by adopting a plate marking method, placing in the constant-temperature incubator at 37 ℃ for culturing for 24h, continuously carrying out passage for 3 times, fully activating the bacterium liquid, selecting a bacterium colony with better growth of a slant culture medium, inoculating into the MRS liquid culture medium for culturing for 24h at 37 ℃, and preserving in a refrigerator at 4 ℃ for later use.
Example 1
(1) Preparing quinoa slurry: selecting full-grain and uniform-color quinoa wheat, repeatedly cleaning for 2 times, aging in a steamer at 100 deg.C for 40min, pulping at 10000 rpm according to a material-water ratio of 1:5(g/mL) for 5min to thoroughly grind, sieving with a 200-mesh sieve to obtain quinoa wheat pulp, and storing in a refrigerator at 4 deg.C for use.
(2) Preparing reconstituted milk: adding 25g of skimmed milk powder into 180mL of 70 ℃ distilled water, and mixing uniformly.
(3) Homogenizing: adding 30% quinoa wheat pulp and 5% xylitol into the reconstituted milk, preheating to 70 deg.C, mixing well, and homogenizing under 20MPa for 10 min.
(4) And (3) sterilization: sterilizing at 95 deg.C for 10min by autoclaving.
(5) And cooling the sterilized mixture to 25 ℃, inoculating 3% of compound leaven (LP: LA mass ratio is 2:1), and fermenting at 38 ℃ for 8 hours to obtain the quinoa fermented milk.
The SOD activity of the prepared quinoa fermented milk is 241.17U/mL.
Example 2
(1) Preparing quinoa slurry: selecting full-grain and uniform-color quinoa wheat, repeatedly cleaning for 2 times, aging in a steamer at 100 deg.C for 50min, pulping at 10000 rpm according to a material-water ratio of 1:5(g/mL) for 5min to thoroughly grind, sieving with a 200-mesh sieve to obtain quinoa wheat pulp, and storing in a refrigerator at 4 deg.C for use.
(2) Preparing reconstituted milk: adding 25g of skimmed milk powder into 180mL of 70 ℃ distilled water, and mixing uniformly.
(3) Homogenizing: adding 20% Chenopodium quinoa linn wheat pulp and 3% xylitol into the reconstituted milk, preheating to 70 deg.C, mixing, and homogenizing under 20MPa for 10 min.
(4) And (3) sterilization: sterilizing at 95 deg.C for 10min by autoclaving.
(5) And cooling the sterilized mixture to 25 ℃, inoculating 2% of compound leaven (LP: LA mass ratio is 1:1), and fermenting at 36 ℃ for 7 hours to obtain the quinoa fermented milk.
Example 3
(1) Preparing quinoa slurry: selecting full-grain and uniform-color quinoa wheat, repeatedly cleaning for 2 times, aging in a steamer at 100 deg.C for 45min, pulping at 10000 rpm according to a material-water ratio of 1:5(g/mL) for 5min to thoroughly grind, sieving with a 200-mesh sieve to obtain quinoa wheat pulp, and storing in a refrigerator at 4 deg.C for use.
(2) Preparing reconstituted milk: adding 25g of skimmed milk powder into 180mL of 70 ℃ distilled water, and mixing uniformly.
(3) Homogenizing: adding 40% quinoa wheat pulp and 7% xylitol into the reconstituted milk, preheating to 70 deg.C, mixing well, and homogenizing under 20MPa for 10 min.
(4) And (3) sterilization: sterilizing at 95 deg.C for 10min by autoclaving.
(5) And cooling the sterilized mixture to 25 ℃, inoculating 4% of compound leaven (LP: LA mass ratio is 1:2), and fermenting at 40 ℃ for 6 hours to obtain the quinoa fermented milk.
Example 4 lactic acid bacteria growth curves
Inoculating the 4 strains of lactobacillus into MRS liquid culture medium, taking MRS liquid culture medium without inoculated lactobacillus as blank control, taking quantitative and uniformly mixed fermentation liquor every 6h, and measuring OD600The values, results are shown in FIG. 1.
FIG. 1 shows that the growth rate of most strains is reduced in about 24 hours, the growth conditions are in a transition from a logarithmic phase to a stationary phase, when the fermentation time is 30 hours, the growth rate is close to 0, the change of the quantity of thalli is small, namely, the growth process of most strains enters a stationary phase in 30 hours, the quantity of thalli of a strain LCP is rapidly increased between 12 hours and 30 hours, the growth logarithmic phase is in the range, compared with other strains, the logarithmic phase time of the strain LCP is long, and the strains LP and LA are rapidly increased between 0 hours and 6 hours, which indicates that the lag phase of the two strains is short, the strains can rapidly enter a growth and propagation phase and are always in a high viable count state.
Example 5 lactic acid bacteria are resistant to pH 2.0, pH 3.0 acidity
The lactobacillus is inoculated in MRS liquid culture medium with pH adjusted to 2.0 and 3.0 by HCl respectively, and cultured at constant temperature of 37 ℃ for 0h, 1h, 2h and 3h, and then gradient dilution coating is carried out to determine the viable count, and the result is shown in figure 2, wherein, the graph A is the tolerance result of the lactobacillus at pH 2.0, and the graph B is the tolerance result of the lactobacillus at pH 3.0.
Example 6 lactic acid bacteria resistant to 0.3%, 0.5% bile salts
The lactic acid bacteria were inoculated into MRS liquid medium with 0.3% and 0.5% of bile salt concentration, and cultured in a 37 ℃ incubator at constant temperature for 0h, 1h, 2h, and 4h, and then gradient dilution coating was performed to determine viable count, as shown in FIG. 3, wherein graph A shows the tolerance of lactic acid bacteria at 0.3% bile salt, and graph B shows the tolerance of lactic acid bacteria at 0.5% bile salt.
The human small intestine bile salt concentration is normally about 0.3%, the bile salt can destroy the structure of the cell membrane of the lactic acid bacteria and damage deoxyribonucleic acid of the bacteria, so that the growth is inhibited or the bacteria are gradually killed, the lactic acid bacteria need to adapt to the special gastrointestinal environment in order to fully exert the physiological activity, and therefore the tolerance capability of the bacterial strain to acid and bile salt is one of necessary conditions for evaluating whether the bacterial strain has probiotic characteristics.
Under the condition that the concentration of bile salt in an MRS liquid culture medium is 0.3%, the strains have different bile salt tolerance, the number of viable bacteria is reduced in different degrees along with the prolonging of time, when the time is 1-4 h, the change of the number of the bacteria tends to be gentle, the lactobacillus bacteria adapt to the growth environment, when the fermentation time is 4h, the number of viable bacteria of a strain LP is the largest, and the best tolerance of the strain LP is seen when the concentration of the bile salt is 0.3%.
Under the condition that the concentration of the bile salt in the MRS liquid culture medium is 0.5%, the concentration of the bile salt is increased, the growth conditions of 4 different strains are obviously different, the viable count of 4 lactic acid bacteria is reduced to a greater extent along with the time, finally, when the fermentation time is 4 hours, the number of the viable bacteria of the strain LP is the largest, the tolerance of the strain LP with the concentration of the bile salt of 0.5% is the best, and the result is basically consistent with that with the concentration of the bile salt of 0.3%.
The experimental result shows that the bile salt resistance of the 4 lactic acid bacterial strains are sequentially LP, LCP, LA and LR from large to small.
Example 7 Effect of Complex starter ratio on pH and viable count of fermented milk
LP and LA are selected as fermentation strains, composite leavening agents with different proportions (LP: LA is 1:3, 1:2, 1:1, 2:1 and 3:1) are arranged, and the influence of the proportion of the composite leavening agents on the pH value and the viable count of the fermented milk is analyzed, and the result is shown in figure 4.
As can be seen from FIG. 4, the pH of the fermented milk gradually decreased with the increase of the LP inoculation ratio. Analysis reason the acid production amount of LP is larger, and the acidity of the fermented milk is higher when the inoculation proportion is larger. When LP and LA are compounded in a ratio of 2:1, the viable count in the fermented milk can reach 6.54 multiplied by 109CFU/mL, then decreased, considering possibly too high acidity of the internal environment of the fermented milk, resulting in a certain inhibition of the growth and propagation of the strainsThe preparation method reduces the number of viable bacteria of the strain, and the acidity of the fermented milk is too high, so that the fermented milk is not acceptable to consumers.
Example 8 Effect of xylitol addition on sensory evaluation of fermented milks
Xylitol (3%, 4%, 5%, 6%, 7%) in different contents was added, and the fermented milk was subjected to comprehensive sensory evaluation such as color, smell, taste, texture and the like, and the effect of the xylitol addition amount on the quality of the fermented milk is shown in fig. 5.
The sensory score of the fermented milk showed a tendency to increase first and then decrease as the addition amount of xylitol was increased. Too little xylitol is added, the fermented milk is too acid, the fermentation effect is poor, and the tissue state is unstable. And the addition amount of xylitol is too much, the mouthfeel of the fermented milk is sweet, and the sensory score is reduced.
Example 9 Effect of Chenopodium quinoa pulp addition amount on fermented milk SOD Activity and sensory score
Setting the inoculation amount to be 3%, the fermentation temperature to be 38 ℃, the fermentation time to be 8h, taking the addition amount of the quinoa slurry to be 10%, 20%, 30%, 40% and 50% in sequence, standing for 2h at 4 ℃ to stop fermentation, and measuring SOD activity and sensory score after the fermentation is finished, wherein the result is shown in figure 6.
Chenopodium quinoa L.is fermented and metabolized by lactobacillus to generate multifunctional enzymes such as superoxide dismutase (SOD), lipase, protease, etc., wherein SOD is one of the most important antioxidant enzymes, and can cause superoxide anion free radical to be disproportionated in organism, thereby reducing concentration of free metal ions in cells and relieving H2O2The damage of (2) can be directly used for measuring the oxidation resistance of the organism. When the addition amount of the quinoa pulp is 30%, the fermented milk SOD activity and the sensory score are both the highest, which is probably because the carbohydrate in the mixed liquid just promotes the growth and the reproduction of the lactic acid bacteria. Quinoa is rich in active substances such as phenols, flavonoids and SOD, and can remarkably inhibit lipid peroxidation and eliminate O2-The ability of free radicals; when the quinoa slurry is too much, the raw materials are difficult to mix uniformly, so that the lactobacillus fermentation is insufficient, the antioxidant enzyme system is damaged, and the fermented milk SOD activity is reduced.
Example 10 Effect of lactic acid bacteria inoculum size on fermented milk SOD Activity and sensory score
Setting the addition amount of quinoa at 30%, fermentation temperature at 38 deg.C, and fermentation time at 8h, sequentially taking 1%, 2%, 3%, 4%, and 5% of inoculation amount, standing at 4 deg.C for 2h to stop fermentation, and determining SOD activity and sensory score after fermentation is finished, with the result shown in FIG. 7.
As can be seen from FIG. 7, the SOD activity and sensory score of the fermented milk both increased and then decreased with increasing amounts of lactobacillus. When the inoculation amount of the lactic acid bacteria is 3%, the SOD activity and the sensory score of the fermented milk are both highest, and when the inoculation amount is less, the fermented milk easily pollutes mixed bacteria to form an environment which is not beneficial to the proliferation and the growth of the bacteria, and the milk curd state of the fermented milk is poor, and the tissue is unclear. If the inoculation amount exceeds 3%, the pH value of the fermented milk is changed due to over fermentation, and the bacteria are in an environment which is not beneficial to the propagation and growth of the bacteria due to peracid and over-alkali, so that the growth of the lactic acid bacteria enters the decay period in advance. The chemical nature of SOD is protein, and the regular structure in the molecule is easily influenced by physical or chemical factors such as pH, temperature and the like to cause hydrogen bond breakage and further damage to the spatial structure, so that the SOD activity is reduced.
Example 11 Effect of fermentation temperature on SOD Activity and sensory scores of fermented milks
Setting the addition amount of quinoa at 30%, the inoculation amount at 3%, the fermentation time at 8h, standing at 34 deg.C, 36 deg.C, 38 deg.C, 40 deg.C, 42 deg.C, and 4 deg.C for 2h to stop fermentation, and determining SOD activity and sensory score after fermentation is finished, the result is shown in FIG. 8.
As can be seen from fig. 8, the SOD activity and sensory score of the fermented milk both increased and then decreased with increasing fermentation temperature, and were the highest at 38 ℃. This may be due to the fact that the fermentation temperature is too low to facilitate the growth of lactic acid bacteria, resulting in a slow fermentation rate, insufficient metabolites, a decreased SOD activity and a slow formation of the fermented milk tissue state. According to the enzymatic reaction kinetics, the temperature is increased, the reaction speed is accelerated, and the respiratory intensity is increased, so that the microbial reproduction and metabolism are accelerated. However, the temperature rise will increase the enzyme inactivation speed and shorten the fermentation period, the lactobacillus thallus is aged and autolyzed in advance, the activity of the metabolite enzyme is affected, and the fermented milk has no milk flavor and the quality is reduced.
Example 12 Effect of fermentation time on fermented milk SOD Activity and sensory score
Setting the addition amount of quinoa at 30%, the inoculation amount at 3%, the fermentation temperature at 38 deg.C, sequentially taking 6h, 8h, 10h, 12h, and 14h for fermentation time, standing at 4 deg.C for 2h to stop fermentation, and determining SOD activity and sensory score after fermentation is finished, with the result shown in FIG. 9.
As can be seen from FIG. 9, the SOD activity and sensory score of fermented milk both increased and then decreased with increasing fermentation time. The fermentation time is 8h, the SOD activity and sensory score of the fermented milk are highest, and at the moment, the fermented milk has mellow and positive smell, slightly has quinoa fragrance and is proper in taste. This is because if the fermentation time is too short, the growth and propagation of lactic acid bacteria are insufficient, the accumulation of metabolites is insufficient, and the enzyme activity is low. If the time is too long, nutrient substances required by growth and reproduction of the lactobacillus are insufficient, the growth of the lactobacillus enters a decline period in advance, the death number of thalli is increased, metabolic wastes in the fermented milk are gradually accumulated, the activity of SOD is rapidly reduced, the acid yield of the fermented milk is too high, the sour-sweet ratio of the fermented milk is unbalanced, the flavor of the fermented milk is seriously influenced, the tissue of the fermented milk is flocculent, and the taste quality is reduced.
The sensory score of the invention is the average score of 10 professional sensory evaluators as the sensory score, and the scoring standard is shown in table 1:
TABLE 1 sensory Scoring criteria
EXAMPLE 13DPPH radical scavenging assay
Taking 2mL of the diluted fermented milk with different concentrations and 2mL of 0.02mg/mL DPPH-ethanol solution, mixing uniformly, keeping out of the sun for 30min, centrifuging the sample for 10min under the condition of 5000r/min, and measuring the absorbance value of the sample at 517nm, wherein the calculation formula is as follows:
A1-absorbance of DPPH solution in the presence of the sample;
A2-absorbance of the sample when mixed with absolute ethanol;
A0absorbance of DPPH solution without sample.
The DPPH free radical clearance change of the fermented milk with different concentrations is shown in figure 10, the DPPH clearance of the sucrose-free quinoa fermented milk is obviously higher than that of common fermented milk, the DPPH clearance is respectively 86.23 +/-2.35% and 76.81 +/-3.12% at the highest, the DPPH clearance of the fermented milk added with quinoa pulp is improved by nearly 10%, which shows that quinoa in the sucrose-free quinoa fermented milk plays an important antioxidation role, because quinoa is rich in active substances such as polysaccharide, polyphenol and the like, Vollmannov's research proves that the contents of phenolic substances and rutin in buckwheat, amaranth and quinoa directly influence the antioxidation capability of quinoa, and the DPPH clearance is stronger when the ratio of free phenol to bound phenol is larger. After the quinoa is fermented by the lactic acid bacteria, the content of polyphenol compounds such as quercetin and vanillic acid is increased, and the synergistic effect is generated with the self inoxidizability of the lactic acid bacteria, so that the DPPH free radical scavenging capability of the fermented milk is further improved.
Example 14ABTS+Determination of radical scavenging Rate
Referring to the method of Zhengjin bear et al, ABTS solution is prepared. Taking 0.5mL of the fermented milk diluted to different concentrations, adding 3.5mL of ABTS solution, dark reacting for 6min in a dark place, and measuring the absorbance value (734nm) of a sample, wherein the calculation formula is as follows:
A0absorbance when 0.5mL of distilled water and 3.5mL of ABTS solution are mixed;
A1-absorbance of 0.5mL sample when mixed with 3.5mL ABTS solution;
A2absorbance when 0.5mL of sample was mixed with 3.5mL of absolute ethanol.
Fermented milk ABTS with different concentrations+The change of the free radical clearance is shown in FIG. 11, and the ABTS of the sugar-free quinoa fermented milk can be seen+The clearance rate is obviously higher than that of common fermented milk, and the pair ABTS of the fermented milk+The clearance ability of the fermented milk is enhanced along with the increase of the concentration, and the final ABTS of the saccharose-free quinoa fermented milk and the common fermented milk+The clearance rates are 71.32 + -2.32% and 52.96 + -1.95%, respectively, and fermented milk ABTS containing quinoa wheat pulp is added+The clearance rate is improved by nearly 20 percent, which shows that the addition of the quinoa wheat pulp can obviously improve the ABTS of the fermented milk+The cleaning ability of (1).
EXAMPLE 15 determination of hydroxyl (. OH) radical scavenging Rate
The hydroxyl radical clearance of the fermented milk is measured by using the hydroxyl radical test kit, the change of the hydroxyl radical clearance of the fermented milk with different concentrations is shown in figure 12, the hydroxyl radical clearance of the sugar-free quinoa fermented milk is obviously higher than that of the common fermented milk, the in-vitro hydroxyl radical inhibition capacity of the sugar-free quinoa fermented milk and the common fermented milk is stably increased along with the increase of the concentration of the fermented milk, the hydroxyl radical clearance of the sugar-free quinoa fermented milk and the hydroxyl radical inhibition capacity of the common fermented milk are 48.19 +/-2.69% and 63.27 +/-2.01%, the hydroxyl radical clearance of the fermented milk added with quinoa pulp is improved by nearly 15%, and the hydroxyl radical clearance of the fermented milk added with quinoa pulp can be obviously improved.
Example 16Fe3+Reduction force measurement
Reference is made to the method of the poplar shunie to prepare the FRAP reagent. 1mL of the fermented milk sample was mixed with 5mL of FRAP reagent, left to stand at 37 ℃ for 10min, and the absorbance value was measured at 593nm by a linear regression equation of standard solution with y being 0.0009x +0.0767 (R)20.9914) sample Fe was calculated3+Reduction power (mmol/L). Fermented milk Fe with different concentrations3+The change in reducing power is shown in FIG. 13. The ferric iron reducing ability can evaluate the total oxidation resistance of the material, and the material with strong reducing ability contains abundant electron donor, so that Fe3+The reduction ability can be used as the most effective method for detecting whether the sample contains the electron donor, or Fe if the electron donor is contained3+Reduction to Fe2+So that the color of the solution is changed. As can be seen from FIG. 13, Fe in the sucrose-free quinoa fermented milk3+Good reduction abilityThe Fe content of the two types of fermented milk is higher than that of common fermented milk, and the Fe content in vitro of the two types of fermented milk is higher with the continuous increase of the concentration of the fermented milk3+The reducing capability is steadily increased, and finally, when the concentration reaches 100 percent, the reducing capability and the reducing capability are Fe3+The reducing power is respectively 0.48 plus or minus 0.02mmol/L and 0.29 plus or minus 0.03mmol/L, which proves that the addition of quinoa wheat pulp can obviously improve the Fe content of the fermented milk3+Reducing power. It has been shown that Fe3+The reducing power is related to the content of free phenol in the substances, and the fermentation can promote the release of the bonded phthalein component in the quinoa, so that the fermented quinoa can enable the fermented milk to have higher Fe3+Reducing power.
Example 17 alpha-glucosidase inhibition
The measurement was carried out by the glucose oxidase method. Taking 50 μ L of sample solution, adding 100 μ L of PBS buffer (pH 6.8) with concentration of 0.1mol/L, adding 100 μ L of PNPG solution with concentration of 20mmol/L, mixing, water bathing at 37 deg.C for 20min, adding 60 μ L of alpha-glucosidase solution with concentration of 20U/mL, continuing water bathing for 10min, adding 100 μ L of Na with concentration of 1mol/L2CO3And (3) as a reaction termination solution, measuring the light absorption value of the termination solution at 405nm, wherein a PBS solution (pH is 6.8) of 0.1mol/L is used as a blank control of the alpha-glucosidase solution and a sample to be measured, and the alpha-glucosidase inhibition rate is calculated according to the following calculation formula:
a-solution containing no sample to be tested but containing alpha-glucosidase;
b-does not contain the sample to be detected and the alpha-glucosidase solution;
c, containing a sample to be detected and an alpha-glucosidase solution;
d, the solution containing the solution to be detected but not containing the alpha-glucosidase.
The alpha-glucosidase inhibition rate of the fermented milk with different concentrations is shown in fig. 14, as can be seen from fig. 14, the alpha-glucosidase inhibition rate of the fermented milk is in positive correlation with the concentration of the fermented milk, the alpha-glucosidase inhibition rate of the quinoa-free fermented milk is obviously higher than that of common fermented milk, when the concentration of the fermented milk is 20% -60%, the inhibition rate is rapidly increased and is basically stable within the range of 60% -100%, the inhibition rates of the alpha-glucosidase inhibition rate and the alpha-glucosidase inhibition rate are respectively 46.33% and 27.79%, the alpha-glucosidase inhibition rate of the fermented milk added with quinoa pulp is improved by nearly 20%, and compared with common fermented milk, the quinoa-free fermented milk has stronger alpha-glucosidase activity inhibition capability.
Example 18 alpha-Amylase inhibition
According to the method of the reference literature, 2mL of a sample solution to be tested is taken, 2mL of an alpha-amylase solution (prepared by using a phosphate buffer solution with a pH value of 7.0) with a concentration of 2mg/mL is added, the reaction is carried out at 37 ℃ for 20min, 2mL of soluble starch (mass fraction of 1%) is added, the reaction is carried out in a water bath at 37 ℃ for 15min, an iodine solution is added for color development, the light absorption value is measured at 660nm, and the alpha-amylase inhibition rate is calculated according to the following calculation formula:
a-does not contain a sample to be detected and an alpha-amylase solution;
b, not containing the sample to be detected but containing alpha-amylase solution;
c, containing the sample to be detected but not containing the alpha-amylase solution;
d, containing the sample to be detected and the alpha-amylase solution.
The alpha-amylase inhibition rates of the fermented milk with different concentrations are changed as shown in figure 15, the alpha-amylase inhibition rates of the fermented milk and the concentrations of the fermented milk are in a dependency relationship, the alpha-amylase inhibition rates of the fermented milk without the cane sugar and the quinoa are also obviously higher than those of the fermented milk without the cane sugar and the quinoa, the alpha-amylase inhibition rates of the fermented milk without the cane sugar and the quinoa are respectively 56.50% and 31.29%, the alpha-amylase inhibition rates of the fermented milk with the quinoa pulp are improved by nearly 25%, and the fermented milk without the cane sugar and the quinoa pulp has stronger alpha-amylase activity inhibition capability compared with the fermented milk without the cane sugar and the quinoa pulp.
Example 19 sucrose-free quinoa fermented milk basic Nutrition ingredients
The results of the measurement of the basic nutritional ingredients of the fermented milk are shown in table 2, and it can be seen from table 2 that the protein, fat, non-fat milk solid and energy in the sucrose-free quinoa fermented milk are slightly lower than those of the common fermented milk, because the common fermented milk uses whole milk powder as the raw material, the quinoa serum is added into the raw material of the sucrose-free quinoa fermented milk to replace part of skim milk, and the xylitol is used to replace sucrose, so that the protein, fat and non-fat milk solid contents and energy in the sucrose-free quinoa fermented milk are slightly lower.
TABLE 2 measurement results of basic nutrient components of fermented milk
Example 20 comparison of pH, acidity, viscosity, and Water holding percentage of sucrose-free quinoa fermented milk and plain fermented milk
TABLE 3 measurement results of physicochemical indexes of fermented milk
As can be seen from table 3, the acidity of the sucrose-free quinoa fermented milk is lower than that of the common fermented milk, while the pH is opposite, because quinoa pulp is added into the sucrose-free quinoa fermented milk, the proportion of reconstituted milk in the raw material is low, the lactose content of the reconstituted milk is reduced overall, and the lactic acid bacteria fermentation mechanism is that lactic acid bacteria utilize lactose in the raw material to generate lactic acid, so the acidity of the final product is reduced, and the pH is increased; the principle of viscosity formation is that saccharides in a lactobacillus fermentation raw material generate extracellular polysaccharide, wherein the extracellular polysaccharide comprises capsular polysaccharide and mucopolysaccharide with adhesiveness, the water holding capacity is formed because fat is broken into tiny fat globules through homogenization and is uniformly distributed on a fermentation substrate, so that the adsorption surface area of the polysaccharide and protein is increased, the casein structure is changed to generate aggregation of a gel network, and the sucrose-free quinoa fermented milk takes skim milk powder as the substrate and xylitol to replace sucrose, so that the contents of saccharides and fat are lower than those of common fermented milk, the viscosity and the water holding capacity of the product are reduced, and the viscosity and the water holding capacity of the fermented milk are reduced due to the reduction of the protein content, while the quinoa belongs to high-protein coarse cereals, so the viscosity and the water holding capacity of the sucrose-free quinoa fermented milk taking skim milk powder as the substrate are slightly lower than those of common fermented milk.
EXAMPLE 20 color measurement
The color of the fermented milk was measured using a color difference meter, and the data were read and recorded after 30 seconds, and the results of the color measurement of the fermented milk are shown in table 4.
TABLE 4 color measurement results of fermented milks
The L value is a brightness parameter, the larger the value is, the brighter the sample is, the a value and the b value are color parameters, the a value is green and red, the larger the + a value is, the more red the color of the tested sample is, and the larger the a value is, the more green the color of the tested sample is. The b value is yellow and blue, the more the + b value is, the more yellow the color of the tested sample is, and the more blue the color of the tested sample is. The L value of the quinoa fermented milk without cane sugar is lower than that of the common fermented milk, and the a value and the b value are opposite, which is probably because the red quinoa pulp or reducing sugar degradation products in the raw materials and polyphenol compounds are added, the color value is increased, and the brightness is reduced.
Example 21 fermented milk microbiological assay
And (3) carrying out microorganism index determination on the cane sugar-free quinoa fermented milk, wherein as shown in table 5, the number of lactic acid bacteria of the cane sugar-free quinoa fermented milk is higher than that of common fermented milk, pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella and the like are not detected, the sanitation index is qualified, and the requirements of national standards are met.
TABLE 5 results of measurement of microbial indicators
The results show that the cane sugar-free quinoa fermented milk prepared by the invention has DPPH free radical clearance rate and ABTS+Radical scavenging, hydroxyl radical scavenging, Fe3+The reducing capacity, the alpha-glucosidase inhibition rate and the alpha-amylase inhibition rate are all higher than those of common fermented milk, which shows that the cane sugar-free quinoa fermented milk has better oxidation resistance and the capability of inhibiting related digestive enzymes, and has good function of assisting in reducing blood sugar in vitro.
Example 22
On the basis of the embodiment 1, the quinoa is further processed, and the specific operations are as follows:
(1) preparing quinoa slurry: selecting red quinoa with plump seeds and uniform color, repeatedly cleaning for 2 times, baking for 20min at 150 ℃, cooling the quinoa to room temperature, soaking for 0.5min in 20g/L sodium carbonate aqueous solution, then soaking for 20s in 5 g/L-cysteine hydrochloride solution, washing with clear water, pulping according to a material-water ratio of 1:5(g/mL) until the mixture is fully ground uniformly, sieving with a 200-mesh sieve to obtain quinoa pulp, and storing in a refrigerator at 4 ℃ for later use.
(2) Preparing reconstituted milk: adding 25g of skimmed milk powder into 180mL of 70 ℃ distilled water, and mixing uniformly.
(3) Homogenizing: adding 30% quinoa wheat pulp and 5% xylitol into the reconstituted milk, preheating to 70 deg.C, mixing well, and homogenizing under 20MPa for 10 min.
(4) And (3) sterilization: sterilizing at 95 deg.C for 10min by autoclaving.
(5) And cooling the sterilized mixture, inoculating 3% of compound leaven (LP: LA mass ratio is 2:1), and fermenting at 38 ℃ for 8h to obtain the quinoa fermented milk.
After the chenopodium quinoa willd is soaked by the sodium carbonate aqueous solution and the L-cysteine hydrochloride solution in sequence, the basic nutrient components and the color of the prepared chenopodium quinoa willd fermented milk are equivalent to those of the fermented milk prepared in the embodiment 1, obvious change is not generated, and the microbial index measurement result also meets the standard requirement.
The quinoa fermented milk prepared by the embodiment has improved antioxidant capacity and related digestive enzyme inhibition capacity, and the SOD activity of the quinoa fermented milk is 258.62U/mL; DPPH free radical clearance rate of 86.23 percent and ABTS+71.32% of free radical clearance rate, 48.19% of hydroxyl free radical clearance rate and Fe3+The reduction capacity is 0.48mmol/L, the alpha-glucosidase inhibition rate is 46.33 percent, and the alpha-amylase inhibition rate is 56.50 percent.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. The composite microbial inoculum is characterized by being prepared from lactobacillus plantarum and lactobacillus acidophilus in a mass ratio of 1-3: 1-3.
2. The composite bacterial agent of claim 1, wherein the composite bacterial agent is a composite bacterial agent with a mass ratio of lactobacillus plantarum to lactobacillus acidophilus of 2: 1.
3. Use of the complex microbial agent of claim 1 or 2 for preparing fermented milk.
4. A method for preparing quinoa fermented milk by using the complex microbial inoculant of claim 1 or 2, wherein the method comprises the following steps of:
aging quinoa, and pulping to obtain quinoa mash;
mixing the quinoa wheat pulp with xylitol and reconstituted milk, and heating and homogenizing the obtained mixture to obtain a homogenized mixture;
and sterilizing and cooling the homogenized mixture, inoculating the compound microbial inoculum, and fermenting to obtain the quinoa fermented milk.
5. The method of claim 4, wherein said quinoa mash is added in an amount of 20-40% by mass of said mixture.
6. The method according to claim 4, wherein the xylitol is added in an amount of 3-7% by mass of the mixture.
7. The method of claim 4, wherein the heating is at a temperature of 70 ℃; the homogenizing pressure is 20MPa, and the homogenizing time is 10 min.
8. The method according to claim 4, wherein the inoculation amount of the complex microbial inoculum is 2-4%.
9. The method according to claim 4, wherein the inoculation amount of the complex microbial inoculum is 3%.
10. The method according to claim 4, wherein the fermentation temperature is 36-40 ℃ and the fermentation time is 6-8 h.
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