CN113462504A

CN113462504A - Apple wine preparation process

Info

Publication number: CN113462504A
Application number: CN202110756861.2A
Authority: CN
Inventors: 王周利; 明巧营; 蔡瑞; 岳田利; 高振鹏; 袁亚宏
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2021-10-01
Anticipated expiration: 2041-07-05
Also published as: CN113462504B

Abstract

The invention discloses a preparation process of cider. The scheme disclosed comprises the steps of taking apple juice with excessive patulin content as a raw material, fermenting by 1027 saccharomyces cerevisiae to prepare the cider, and degrading and adsorbing patulin by the 1027 saccharomyces cerevisiae in the fermentation process. The preparation process of the invention selects the bacterial strain which can degrade patulin for fermentation, simultaneously degrades the patulin in the raw materials, and the degraded product meets the requirement of food safety, thus having outstanding contribution to the processing and utilization of the rotten apples and the polluted apples.

Description

Apple wine preparation process

Technical Field

The invention relates to the technical field of food microorganisms, in particular to a preparation process of cider capable of efficiently degrading patulin in fermented cider.

Background

Patulin (also called patulin, chemical formula C)₇H₆O₄154 relative molecular mass) is a toxic fungal metabolite, which is readily soluble in water, chloroform, acetone, ethanolAnd ethyl acetate, slightly soluble in diethyl ether and benzene, and insoluble in petroleum ether. Because the patulin is easily dissolved in water and is stable in an acid medium, the patulin is difficult to remove in the processing process of fruits and vegetables, and the residual quantity of the patulin in fruit products is large. Recent researches indicate that patulin is widely polluted in fruits such as apples, pears, hawthorns and the like and products thereof. It is a neurotoxin, has carcinogenic, mutagenic and embryotoxic effects, and is a serious health hazard. The limit index of the patulin in the apples, the hawthorns and the products thereof is 50 mug/kg, which is specified in GB1497494 'Limit sanitary Standard of the patulin in the apples and the hawthorns products', and is consistent with GB27612011 'Limit of mycotoxin in foods', and the investigation result shows that the contamination rate of the patulin in rotten apples is 40%, and the contamination of the apples is serious. The detection of patulin in commercially available apple products shows that 35% of patulin exceeds the standard, the highest value can reach 167 mu g/L, and rotten and deteriorated fruits cannot be completely eliminated, so that the processing link is entered, the detection rate of the patulin in fruit juice is high, huge economic loss is brought to the apple industry, and serious threats are also formed to the health and the safety of people.

Disclosure of Invention

Aiming at the defects or shortcomings of the prior art, the invention provides a preparation process of cider.

Therefore, the preparation process of the cider provided by the invention comprises the following steps: apple juice with excessive patulin content is used as a raw material, 1027 saccharomyces cerevisiae is used for fermentation to prepare the cider, and the 1027 saccharomyces cerevisiae has degradation and adsorption effects on the patulin in the fermentation process.

Further, the preparation process comprises fermenting with 1027 Saccharomyces cerevisiae, transferring, aging and clarifying to obtain cider, wherein the 1027 Saccharomyces cerevisiae has degradation and adsorption effects on patulin.

Preferably, the 1027 s.cerevisiae is cultured by induction with apple juice containing patulin.

Specifically, the content of patulin in the apple juice is more than or equal to 50 mu g/L.

Specifically, the content of patulin in the apple juice is more than or equal to 50 mu g/L and less than or equal to 200 mu g/mL.

The preparation process of the invention adopts the bacterial strain which can degrade the patulin for fermentation, and simultaneously degrades the patulin in the raw materials, and the degraded product meets the requirement of food safety. The method has outstanding contribution to the expansion of the processing and utilization of rotten apples and polluted apples.

Drawings

FIG. 1 shows the variation of patulin content in various stages for different starting concentrations of 150. mu.g/mL (a) and 200. mu.g/mL (b) of different strains in fermented cider.

FIG. 2 shows the removal of patulin by the different strains at the main fermentation stage of cider at initial concentrations of 150. mu.g/mL (a) and 200. mu.g/mL (b).

FIG. 3 shows the fermentation capacities of different yeasts at initial concentrations of 150. mu.g/mL and 200. mu.g/mL: (a) the change of the alcoholic strength when the toxin concentration is 150 mug/mL, (b) the change of the alcoholic strength when the toxin concentration is 200 mug/mL; (c) change in soluble solids at a toxin concentration of 150. mu.g/mL, and (d) change in soluble solids at a toxin concentration of 200. mu.g/mL.

FIG. 4 is a graph showing the effect of yeast activity on patulin content; (a) the effect of different time periods on the degradation rate of patulin, and (b) the effect of dead and viable cells on the patulin content, respectively.

FIG. 5 shows the effect of yeast 1027 cell metabolites and toxin-induced metabolites on patulin (patulin); (a) is a metabolite of a normal strain, and (b) is a metabolite after induction.

FIG. 6 shows the analysis of the products of Saccharomyces cerevisiae 1027 by biodegradation; (a) in the main fermentation stage, the patulin is not completely degraded; (b) is the biodegradation of patulin by Saccharomyces cerevisiae 1027.

Detailed Description

Unless otherwise indicated, the terms or methods herein are understood or implemented using established methods as recognized by one of ordinary skill in the relevant art.

The apple wine is prepared by fermenting apple juice with patulin content exceeding relevant standards as a raw material by 1027 saccharomycetes, and the saccharomycetes have degradation and adsorption effects on the patulin in the preparation process. The apple juice containing patulin is generally juice extracted from rotten apples, or juice extracted from toxin-contaminated apples, or juice extracted from rotten apples or contaminated apples mixed with good fruits. The overproof is measured by whether the minimum content of the patulin in the apple or apple juice specified in the relevant standards of enterprises, industries, countries or internationally is exceeded or not, in particular in terms of the fact that the content of the patulin meets the food safety requirement. The minimum detection limit for patulin in apple juice currently recognized in the industry is 50 μ g/L.

According to the preparation scheme of the invention, the technical personnel in the field adopt the conventional experimental method to optimally select the related parameters or technical means such as the concentration, the temperature, the pH value, the duration and the like of the substances in the process so as to obtain the cider wine which meets the quality requirement and is safe for food and prepared by the invention.

The following are examples provided by the inventors to further illustrate the invention.

Example 1:

in this example, cider processing was carried out by continuously culturing the strains shown in Table 1 in YPD medium for two generations at 28 ℃ and 180rpm, and three groups of strains were set up, respectively: before fermentation, adding standard substances of the patulin into the apple juice until the concentrations are respectively 0 mug/mL, 150 mug/mL and 200 mug/mL, wherein the specific processing technology of each group is as follows:

fermentation: inoculating second-generation yeast into apple juice, and fermenting for 48h to obtain seed liquid; inoculating the seed liquid into 23-degree Brix apple juice for fermentation, wherein the fermentation conditions are as follows: fermenting at 21 deg.C and pH of 3.5 for 14 days; sampling once in 24h in the fermentation process, and respectively measuring the soluble solid, the alcoholic strength and the toxin change;

reladling: filtering the fermentation liquid after fermentation to remove yeast by using sterile gauze and a conical flask, standing and fermenting at 21 ℃, and continuously pouring for 3 days until the solution is basically clear;

and (3) aging: performing ultrasonic aging at 280W and 20 deg.C for 20min by ultrasonic cleaning machine;

clarification: adding 4% chitosan suspension, mixing, standing in a thermostat at 21 deg.C for 24 hr, centrifuging supernatant at 4000r/min for 15min to obtain cider.

TABLE 1 sources of different Yeast strains

The physical and chemical indexes in the process are determined by referring to GBT 15038-; high Performance Liquid Chromatography (HPLC) for detecting patulin; the aroma components of the fruit wine are detected and identified by gas chromatography-mass spectrometry (GC-MS). The results of the physical and chemical index measurements are shown in Table 2.

TABLE 2 other physicochemical indices after fermentation of cider

And (4) analyzing results: the influence of the brewing process on the content of patulin and the influence of toxin on yeast are shown in figures 1, 2 and 3. As shown in fig. 1, at an initial concentration of 150 μ g/mL, the clarification stage showed the best removal capacity with a ratio of 34.56%, followed by the main fermentation stage (31.20%), the decanting (29.96%) and the accelerated aging stage (4.28%); at an initial concentration of 200. mu.g/mL, the effect on patulin content at each stage is as follows: clarification (34.19%) > tank-relayer (33.84%) > main fermentation (29.29%) > accelerated ageing (2.68%), the clarification stage showed the best capacity for adsorption of patulin and the accelerated ageing stage showed the worst efficiency in two fermented ciders of different concentrations (150 μ g/mL and 200 μ g/mL); at low concentrations of 150. mu.g/mL, the removal rate is higher in the main fermentation stage than in the inversion stage, whereas at high concentrations (200. mu.g/mL), the removal rate is lower in the main fermentation stage than in the inversion stage, which may be related to degradation or adsorption of yeast cells during the main fermentation.

As shown in FIG. 2, when the toxin concentration was 150. mu.g/mL, the three strains (1027, HYJ-1 and 1870) showed good patulin content control, with a 38.2-49.1% reduction in toxin content; secondly, the content of the patulin in the fermented apple juice can be reduced by 26.2 to 32.4 percent through the fermentation of H1, 1845, YN6 and WSL21 strains; 1917 and H2 the reduction rate of patulin content is 20.8% and 19.4%, respectively; when the concentration was increased to 200. mu.g/mL, two strains (1027 and 1870) showed better control of patulin content, with a decrease in content (45.2-45.7%), the other 6 strains showed a reduction in patulin of 20.5-32.8%, while strain (1845) was the least performing strain.

Production of alcohol content is an accumulation process, as shown in fig. 3(a and b), five yeast strains (1027, WSL21, H1, 1870, 1917) have better fermentation performance and alcohol productivity, while others are not suitable for fermentation of cider; as shown in fig. 3(c and d), the soluble solids content of the strain decreased rapidly in the initial stage, and then the trend slowed down and gradually leveled off; among them, three strains (H2, YN6, 1845) had weak fermentation and sugar conversion abilities, a soluble solid content of 18.0 to 20.7 ° Brix, and an alcohol content of 1.2 to 2.1%, and although strain HYJ-1 showed a high sugar consumption, the soluble solid content in the cider sample was low, but the alcohol content was only 3.9%, indicating that sugar was mainly used for strain growth, not for alcohol conversion; for strains WSL21, 1917 and 1845, the soluble solids content was lower and the alcohol content was higher in the cider samples with 200 μ g/mL patulin, indicating that high concentrations of patulin may affect the growth and metabolism of these strains.

As shown by the results shown in the figure, it is known that the comprehensive level of patulin removal capability and fermentation performance of the 1027 strain is higher than that of other strains; meanwhile, in the whole brewing process, each stage has influence on the content of patulin, wherein the effect of the clarification stage is most obvious.

Example 2:

in this example, the seed culture (the blood cell count plate ensures a bacterial suspension concentration of 1X 10⁸CFU/mL) was completed and divided into two groups for treatment：

(1)1027 live cell group, i.e., normal cultured yeast cells;

(2)1027 dead cell group: heating 1027 yeast suspension in 100 deg.C boiling water for 30min to kill thallus to obtain dead cells;

the same treatment was performed for the above two groups: 0.2mL of 1X 10 concentration YPD medium was added to each flask⁸CFU live yeast cell or heat killed yeast cell, adding equal amount of sterile water as blank control under the same condition, adding 4mg patulin standard substance into the culture solution, adjusting its initial concentration to 200 μ g/mL, 28 deg.C, shake culturing at 180rpm for 24h, and sampling every 3h from 6 h; HPLC detection of patulin content in samples (see: MacDonald S, Long M, Gilbert J, Felgueras I. liquid chromatographic method for determination of patulin in clear and close apple juice; collagen test. J. AOAC int. 2000Nov-Dec; 83(6):1387-94.PMID:11128142.)

As shown in fig. 4 (a, b), which shows the effect of treatment time on the content of patulin and their overall change in the process, respectively, the highest removal rate (50.2%) was obtained by live cell fermentation and started to decrease at about 38h, and further extension of the fermentation time revealed that the reduction rate of the content of patulin decreased; on the other hand, inactivated yeast cells only removed 17.3% of patulin in apple juice; the results show that the decrease in patulin content is determined by both biodegradation and adsorption.

Example 3:

the example is divided into three groups of schemes:

(1) normal intracellular group (E-ext) and extracellular group (I-ext), when preparing seed solution, not carrying out toxin induction, after culturing for 24h at 28 ℃, 180rpm in a shaking table, 7000 Xg centrifuging for 5min, separating supernatant and thalli, and simultaneously washing and centrifuging for 3 times by using sterile normal saline to ensure that cleaner cell thalli and all supernatant are obtained, wherein the obtained supernatant is the extracellular group (I-ext); the obtained cells were ground and pulverized with liquid nitrogen, and the pulverized yeast cell fragments were extracted with ethyl acetate, shaken for 5min, left to stand for 3min, and extracted repeatedly 3 times, at which time the obtained mixture was a normal intracellular group of cells (E-ext).

(2) Patulin induces intracellular group (I-P-ext): when preparing the seed solution, toxin standard was added to the seed solution to a concentration of 200. mu.g/mL, and all the other operations were identical to those of the normal intracellular group (E-ext).

Three groups are 10mL, and after the preparation is completed respectively, sterile water with the same amount is added under the same condition to be used as a blank control; adding a certain amount of standard patulin, adjusting the initial concentration to 200 μ g/mL, shaking at 28 deg.C and 180rpm for 24h, sampling every 3h from 6h, centrifuging at 7000 Xg for 5min, filtering with 0.22 μm filter membrane, storing in a refrigerator at-20 deg.C, and determining the content of patulin by HPLC.

As shown in fig. 5(a), at 36h, the removal rate of patulin was maximized in all three groups, and the degradation rates of the intracellular metabolite toxin group (I-ext) and the toxin-inducing group (I-P-ext) were about 6.3% and 11.4% of the patulin, respectively, while the degradation rate of the extracellular extract was only 5.2%; when the time is too long, the degradation rate of the toxin begins to decrease, which indicates that the activity of the intracellular and extracellular extracts can be reduced due to the too long time;

as shown in FIG. 5(b), I-ext and I-P-ext can degrade trypsin by about 55% and 86%, respectively, while the extracellular extract (E-ext) degrades by about 38% at 60h, with significant differences in the degradation of patulin by Saccharomyces cerevisiae 1027 intracellular and extracellular extracts. The results suggest that during fermentation of yeast cells, degradation of patulin is possible by a combination of intracellular and extracellular enzymes, with intracellular enzymes playing a more important role than extracellular enzymes.

The results show that: yeast strain 1027 mainly biodegrades the toxin of the penicillium patatinum through the action of intracellular enzymes of living cells, and after toxin induction, the action effect of the intracellular enzymes of the yeast strains is more obvious, which shows that the concentration of low-concentration patulin can be used as an antigen to promote the expression of related genes of saccharomyces cerevisiae 1027 to be up-regulated.

Example 4:

example 200. mu.L of apple juice medium at a concentration of 1X 10 was added to a flask containing 20mL of apple juice medium⁸cells/mL of a seed medium of Saccharomyces cerevisiae 1027 strain not induced by toxin, and under the same conditions, an equal amount of sterile water is added as a blank; adding a certain amount of patulin standard substance, adjusting the initial concentration of the patulin standard substance to 200 mug/mL, culturing for 7 days, and sampling to be tested.

As shown in fig. 6. To infer the possible structure of the degradation products, they were detected using LC-MS in positive ion mode with patulin ([ M H ]]⁺M/z 155.03) the major characteristic fragment in mass spectrometry is m/z 137.11 (missing one molecule of H)₂O), 111.06 (loss of one molecule of CO)₂) M/z 127.06 and 99.07 (continuous loss of one molecule of CO). The main characteristic fragments of the degradation products are: in positive ion mode, the fragment is m/z 157.0 by itself, and the other major characteristic fragment is m/z 139.09 (loss of one molecule of H)₂O), 113.07 (loss of one molecule of CO)₂) There is also a fragmentation characteristic 127.06 as with patulin, and in conjunction with the relevant reference, the material is presumed to be E-ascladiol.

The results show that: 1027 Yeast strains remove Patulin mainly by degradation, and compared with the literature (Tannous, J., Snini, S.P., Khoury, R.E., Canlet, C., Pinton, P., Lippi, Y., Alassane-Kpmibi, I., Gauthier, T., Khoury, A.E., & Atoui, A. (2017). Patulin transformation products and last intermediates in biochemical pathway, E-and Z-ascariol, ore not toxin to human cells, archives of geneticology, 91(6), 2455), no new substances are produced.

Example 5:

in the embodiment, 80 Kunming mice of five weeks are selected and fed with rod-shaped standard daily ration, water is freely drunk, the room temperature is maintained at about 23 ℃, the relative humidity is 40-60%, the feeding environment is kept for 12h illumination, after adaptive feeding is carried out for one week, the mice are divided into 8 groups, the number of male and female groups is equal, and the grouping conditions are shown in table 2.

The mice are fed with normal drinking water every day, the initial weight of the mice is weighed when the mice are not gazed, the mice are continuously gazed for four weeks (2.56mg/kg bw) according to grouping conditions, the medicines are given every 24 hours, the appearance, the body state, the growth and the activity of the mice are observed every day during the gazing period, and the poisoning expression and the death condition are recorded. And (3) weighing the body weight, taking blood from eyeballs, collecting serum, liver, kidney, intestinal tract and the like to detect various indexes after the experiment is finished. The strain used in this safety evaluation was Saccharomyces cerevisiae 1027 strain and was not toxin-induced.

TABLE 3 mouse grouping

The difference between the fermentation intervention group and the patulin poisoning group is: the patulin poisoning group is not added with yeast for fermentation, but only diluted apple juice; the fermentation pretreatment group is added with yeast.

As shown in table 4, the body weight was generally increased as the feeding time increased, and the body weight was substantially normally increased before the feeding (D1), for one week (D8), but it was found that the body weight of the mice in the low toxin group (150 μ g/mL) began to decrease from 36.47g to 35.23g between days 14 and 21, as compared with the control group; the weight of the high-concentration toxin group (200 mug/mL) also decreases, and the decrease trend is more obvious from 38.05g to 35.3 g; during the period, the weight of the yeast dry group is slowly reduced from 39.99g to 38.73g (150 mu g/mL yeast dry group) and 37.51g to 35.52g (200 mu g/mL yeast dry group). This indicates that the toxin also affected the body weight of the mice and that the effect was reduced after yeast fermentation intervention, indicating that the degradation products are less toxic than the toxin. As shown in tables 5 and 6, in the toxin groups at different doses, the mice were roughly dissected, and the presence or absence of lesions in the organs of the mice was observed, and the spleen, kidney, liver and heart were weighed to calculate the body-to-body ratio.

Comparing the biochemical indexes of the liver and the kidney of each group of mice shows that the following results can be obtained only by the toxin group: glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), Malondialdehyde (MDA) and Lactate Dehydrogenase (LDH) in the liver all rise remarkably; glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) are reduced; urea Nitrogen (BUN) in the kidney rises dramatically; after biodegradation of yeast, all indexes are obviously changed, but the indexes belong to a normal range.

All the biochemical indexes are detected by adopting Nanjing to build a test box of a bioengineering institute, wherein the formula of each biochemical index is as follows:

tissue GSH-PX enzyme activity ═ non-enzyme tube OD value)/(standard tube OD value-blank tube OD value) × standard tube concentration (20umol/l) × dilution factor (5)/reaction time/(sample volume × sample protein content)

MDA content (nmol/mgprot) ═ (determination OD value-control OD value)/(standard OD value-blank OD value) × standard concentration (10 nmol/ml)/protein concentration of sample to be tested (mgprot/ml)

SOD activity (U/ml) ═ control OD value-measured OD value)/control OD value/50% × total reaction solution volume/sample volume × dilution multiple before sample test

Urea Nitrogen (BUN) (mg/dl) ═ urea (mmol/L) × 2.8

LDH is detected according to a lactic acid substrate method, ALT is detected according to an alanine substrate method, AST is detected according to an aspartic acid substrate method; all detected by purchasing Wuhan Severe Biotech limited kit. The results are combined to show that the toxicity of the product is obviously reduced after the yeast degrades the toxin.

Claims

1. A preparation process of cider is characterized by comprising the following steps: apple juice with excessive patulin content is used as a raw material, 1027 saccharomyces cerevisiae is used for fermentation to prepare the cider, and the 1027 saccharomyces cerevisiae has degradation and adsorption effects on the patulin in the fermentation process.

2. The cider wine making process of claim 1, wherein the making process comprises fermenting with 1027 saccharomyces cerevisiae, followed by tank inversion, aging and clarification to obtain cider wine, and during the making process, the 1027 saccharomyces cerevisiae degrades and adsorbs patulin.

3. The cider wine preparation process of claim 1 or 2, wherein the 1027 s.cerevisiae is induced and cultured in cider juice containing patulin.

4. The cider wine preparation process of claim 1 or 2, wherein the cider juice has a patulin content of 50 μ g/L or more.

5. The cider wine preparation process of claim 1 or 2, wherein the cider juice has a patulin content of 50 μ g/L or more and 200 μ g/mL or less.