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WO2018079758A1 - Procédé de production d'un produit laitier fermenté et composition contenant une enzyme - Google Patents

Procédé de production d'un produit laitier fermenté et composition contenant une enzyme Download PDF

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Publication number
WO2018079758A1
WO2018079758A1 PCT/JP2017/039035 JP2017039035W WO2018079758A1 WO 2018079758 A1 WO2018079758 A1 WO 2018079758A1 JP 2017039035 W JP2017039035 W JP 2017039035W WO 2018079758 A1 WO2018079758 A1 WO 2018079758A1
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Prior art keywords
protease
bifidobacteria
neutral
milk
lactase
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PCT/JP2017/039035
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English (en)
Japanese (ja)
Inventor
佐藤　智子
敦子 品田
吉川　潤
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Godo Shusei KK
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Godo Shusei KK
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Priority to JP2018547810A priority Critical patent/JP7401970B2/ja
Publication of WO2018079758A1 publication Critical patent/WO2018079758A1/fr
Anticipated expiration legal-status Critical
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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 OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

Definitions

  • the present invention relates to a method for producing a fermented dairy product using lactic acid bacteria and bifidobacteria, and an enzyme-containing composition that can be preferably used therefor.
  • lactic acid bacteria and bifidobacteria constituting the human intestinal flora have beneficial effects in the intestine.
  • the growth rates of lactic acid bacteria and bifidobacteria are slower than other bacteria that make up the intestinal flora. Therefore, an invention for selectively growing lactic acid bacteria and bifidobacteria in the intestine has been proposed (see, for example, Patent Document 1).
  • bifidobacteria have a slow growth rate
  • an invention for specifically growing bifidobacteria has also been proposed (see, for example, Patent Document 2).
  • An object of the present invention is to provide an enzyme-containing composition and a method for producing a fermented milk product that can increase and maintain the viable count of bifidobacteria in a fermented milk product by a simple method.
  • the present invention has solved the problems of the present invention by having the following technical configuration.
  • An enzyme-containing composition comprising a neutral lactase and a protease that specifically degrades ⁇ -casein, wherein the activity of the protease per unit (NLU) of the neutral lactase is 0.
  • An enzyme-containing composition wherein the composition is from 01 to 100 units (PU).
  • the enzyme-containing composition according to (1), wherein the activity of the neutral lactase is 0.1 NLU / g or more.
  • a method for producing a fermented milk product in which a first step of mixing raw material milk, lactic acid bacteria, and bifidobacteria and a second step of fermenting raw material milk are performed, wherein the second step includes Before completion, the raw milk is subjected to a step of adding a protease that degrades ⁇ -casein (protease addition step) and a step of adding neutral lactase (lactase addition step).
  • a method for producing a fermented milk product wherein the activity of the protease per NLU) is 0.01 or more and 100 units (PU) or less.
  • an enzyme-containing composition and a method for producing a fermented milk product that can increase and maintain the viable count of bifidobacteria in a fermented milk product by a simple method.
  • the present invention includes a first step of mixing raw milk, lactic acid bacteria, and bifidobacteria, A second step of fermenting raw milk; A method for producing fermented dairy products, wherein Before the completion of the second step, a method for producing a fermented milk product is characterized in that a step of adding a protease that specifically degrades ⁇ -casein (protease addition step) is performed on the raw milk.
  • protease addition step before the second step is completed, it is possible to reduce the molecular weight of ⁇ -casein contained in the raw milk by an enzymatic reaction.
  • the protease addition step the amount of ⁇ -casein contained in the raw milk is decreased, and the peptide or free amino acid that is a ⁇ -casein degradation product is increased.
  • Lactic acid bacteria and bifidobacteria have poor ability to degrade ⁇ -casein, so it is difficult to incorporate amino acids constituting ⁇ -casein during fermentation, but they can incorporate peptides or free amino acids that are ⁇ -casein degradation products. Is possible.
  • the lactic acid bacterium and bifidobacteria can be in an environment in which peptides or free amino acids derived from ⁇ -casein can easily be taken in, and the number of lactic acid bacteria and bifidobacteria cells increases. To do. As a result, the secretion amount of lactic acid and acetic acid also increases and the pH decreases.
  • the end of the second step is synonymous with the end of the fermentation step.
  • the 1st process should just mix raw material milk, lactic acid bacteria, and bifidobacteria, and the order to add is not restrict
  • raw milk When raw milk is mainly used, lactic acid bacteria and bifidobacteria may be added to the raw milk.
  • the method of mixing raw material milk, lactic acid bacteria, and bifidobacteria is not particularly limited.
  • the raw milk may be mixed for a short time so that lactic acid bacteria and bifidobacteria are distributed almost uniformly. After the substantially uniform distribution, lactic acid bacteria and bifidobacteria may be distributed on the bottom surface of the container filled with raw material milk.
  • bifidobacteria do not have high oxygen resistance, it is preferable to shorten the mixing time. Lowering the mixing speed is preferable because the amount of dissolved oxygen in the raw material milk can be lowered. When the temperature of the raw milk is increased, the amount of dissolved oxygen can be decreased. It is also important to be careful not to foam when stirring. In addition, a deaeration operation and an aeration operation with an inert gas such as nitrogen are effective for reducing and removing dissolved oxygen.
  • the second step is a step of fermenting the raw milk with lactic acid bacteria and bifidobacteria.
  • the fermentation temperature in the second step may be a temperature at which lactic acid bacteria and bifidobacteria grow.
  • the fermentation temperature varies depending on the lactic acid bacteria and bifidobacteria used, it is preferably in the range of 20 ° C. to 50 ° C., more preferably 25 ° C. to 45 ° C. If it is less than the lower limit, fermentation tends to be delayed, and it may be difficult to obtain a fermented dairy product excellent in economic efficiency. If it exceeds the upper limit value, there is a problem of lactic acid bacteria and bifidobacteria or bifidobacteria death.
  • the fermentation time in the second step depends on the types of lactic acid bacteria and bifidobacteria used and the fermentation temperature, but is preferably in the range of 1 to 48 hours.
  • the fermentation time is preferably 1 hour to 24 hours, more preferably 1 hour to 10 hours.
  • the fermented dairy product according to the present invention has two functions: (1) isoelectric precipitation of casein (isoelectric point 4.6) and (2) ⁇ -casein and ⁇ -casein curdling action by ⁇ -casein degradation.
  • isoelectric precipitation of casein so it begins to harden at about pH 5.5 and hardens at pH 5.0 or lower.
  • the fermentation time usually takes 5 hours or more. Since the fermented milk according to the present invention involves the above two (1) and (2), it begins to harden at pH 6.0 and hardens at pH 5.5.
  • the fermented milk according to the present invention can achieve shortening of the fermentation time as compared with normal fermented milk. If the fermentation time is less than the lower limit, fermentation does not proceed sufficiently, and it may be difficult to obtain desired fermented milk. If the fermentation time exceeds the upper limit, the production cost may increase and the quality of the finished fermented milk may deteriorate.
  • the protease addition step is preferably performed at one or more timings selected before the first step, substantially simultaneously with the first step or after the first step. It is also possible to carry out at two or more timings. After the protease addition step, ⁇ -casein contained in the raw milk is degraded (protease reaction).
  • protease addition step before the first step or substantially simultaneously with the first step.
  • Peptides or free amino acids generated by the degradation of ⁇ -casein can be easily taken into the lactic acid bacterium or the bifidobacteria.
  • a neutral protease When a neutral protease is used in producing a fermented dairy product, the neutral protease is gradually deactivated due to a decrease in pH accompanying the progress of fermentation. That is, the degradation rate of ⁇ -casein contained in raw material milk by neutral protease gradually decreases as fermentation progresses.
  • the action of such a neutral protease can provide an environment in which bifidobacteria can easily take in peptides or free amino acids derived from ⁇ -casein in raw milk in the coexistence of lactic acid bacteria and bifidobacteria. It increases the number of bifidobacteria in the product.
  • by deactivating the protease it is possible to prevent the deterioration of the quality of the fermented milk product after the end of fermentation by suppressing an excessive reaction by the protease.
  • substantially simultaneous means a relative change depending on the growth rate of the lactic acid bacteria and bifidobacteria used, and means that at least one of the lactic acid bacteria or bifidobacteria used corresponds to the induction period.
  • the temperature of the protease reaction on the raw material milk is preferably 0 ° C. to 60 ° C., more preferably 5 to 55 ° C. If the temperature of the protease reaction is lower than the lower limit, the decomposition of ⁇ -casein may be insufficient. In order to solve this, it is necessary to lengthen the reaction time of the protease, so that it is difficult to efficiently produce a fermented milk product. If the temperature of the protease reaction exceeds the upper limit value, the protease tends to be inactivated, and there is a risk that the decomposition of ⁇ -casein will be insufficient. In the coexistence of lactic acid bacteria and bifidobacteria, it becomes difficult to increase bifidobacteria.
  • the protease reaction time for the raw milk is preferably 0.5 to 48 hours, more preferably 1 to 40 hours.
  • the neutral protease is gradually deactivated as the pH of the raw material milk is lowered, so that the protease reaction time is shorter than that in the second step. If the time for the protease reaction is less than the lower limit, the decomposition of ⁇ -casein tends to be insufficient. If the protease reaction time exceeds the upper limit, there is a problem of an increase in production cost.
  • the pH of the protease reaction on the raw milk is preferably 2.0 to 10.0, more preferably 2.5 to 9.0, and particularly preferably 3.0 to 8.0.
  • the pH of the protease reaction is preferably 5.0 to 8.0. If it is less than the lower limit value or more than the upper limit value, the protease tends to be inactivated, and there is a possibility that the decomposition of ⁇ -casein will be insufficient.
  • the pH of the raw milk decreases.
  • the protease is a neutral protease, it is inactivated as the pH of the raw milk decreases.
  • the fermented dairy product according to the present invention is a fermented dairy product containing lactic acid bacteria, bifidobacteria, and protease, wherein the number of lactic acid bacteria is 1 ⁇ 10 4 to 1 ⁇ 10 10 per mL.
  • the number of the bifidobacteria is in the range of 1 ⁇ 10 4 to 1 ⁇ 10 12 per 1 mL.
  • the number of lactic acid bacteria and the number of bifidobacteria contained in the fermented milk product can be within the above ranges.
  • the number of lactic acid bacteria and bifidobacteria contained in the fermented milk product means the number of viable bacteria.
  • the protease contained in the fermented milk product is a neutral protease.
  • the neutral protease is preferably present in an inactivated state in the fermented milk product. Even when neutral protease is inactivated, its protein structure is maintained. Therefore, it is possible to confirm whether neutral protease is present in the fermented milk product by performing electrophoresis on the fermented milk product itself or the concentrated fermented milk product. It is also possible to estimate the amino acid sequence from the specific band obtained after electrophoresis. The sequence of the neutral protease varies depending on its origin, but since it is known, the presence of the protease can be confirmed from the electrophoresis result and its amino acid sequence.
  • the enzyme to be combined is preferably one or more selected from neutral lactase, glucose isomerase, glucose oxidase, transglutaminase, lipase, cellulase, amylase, inulinase, laccase, and peroxidase.
  • the pH of the fermented milk product is preferably 6.0 or less, and more preferably pH 5.0 or less.
  • the lower limit value of the pH of the fermented milk product is preferably 3.0 or more, and more preferably 3.5 or more. If the pH of the fermented dairy product exceeds the upper limit, there is a problem that curdling is insufficient. If the pH of the fermented dairy product is too low, the acidity becomes too strong, and the balance of taste tends to be impaired.
  • the ⁇ -casein degradation rate of the fermented dairy product is preferably 50% or more, more preferably 70% or more, and even more preferably 90% or more.
  • the upper limit is not limited, but is 100%, for example.
  • the degradation rate of ⁇ -casein is represented by a value obtained by dividing 100 by the value obtained by dividing the amount of ⁇ -casein before fermentation by the amount of ⁇ -casein after fermentation.
  • the ⁇ -casein degradation rate is a decrease in the amount of ⁇ -casein, (1) the amount of ⁇ -casein degradation by the added protease, (2) the amount of ⁇ -casein degradation by the protease of the lactic acid bacteria and bifidobacteria, (3) Total amount of ⁇ -casein taken up by lactic acid bacteria and bifidobacteria.
  • the degradation rate of ⁇ -casein of fermented milk products (including lactic acid bacteria and bifidobacteria) that have not undergone a protease addition step is the sum of (2) and (3) above, but lactic acid bacteria and bifidobacteria are (2) and (3 ) Is almost zero, so it is almost zero%.
  • the rate of ⁇ -casein degradation of the fermented milk product by the protease can be increased. If the ⁇ -casein degradation rate is less than the lower limit, bifidobacteria may not sufficiently grow in fermented dairy products containing lactic acid bacteria and bifidobacteria.
  • the amount of ⁇ -casein can be calculated by performing electrophoresis of raw milk or fermented dairy products and taking in the shade of the ⁇ -casein band.
  • the ⁇ -casein band of ⁇ -casein contained in the fermented milk that has undergone the protease addition step substantially disappears when subjected to electrophoresis by the method described below. That is, what is necessary is just to apply 10 microliters per lane what was prepared by diluting the obtained fermented milk product 20 times with ultrapure water, mixing with the sample buffer for SDS-PAGE 1: 1, and boiling for 5 minutes.
  • ⁇ -casein can be detected in fermented milk products that have not undergone a protease addition step. Therefore, by confirming ⁇ -casein contained in the fermented milk product by electrophoresis, it can be estimated whether or not the protease addition step has been performed.
  • the bifidobacteria may not sufficiently proliferate in the fermented milk product containing lactic acid bacteria and bifidobacteria.
  • the fact that the protease contained in the fermented milk product is inactivated means that the protease activity measurement method described later is performed at each fermentation time point, and becomes below the lower limit of detection.
  • lactase addition step Before the second step is completed, it is preferable to perform a step of adding neutral lactase to the raw milk (lactase addition step).
  • the lactase addition step is preferably performed at one or more timings selected before the first step, substantially simultaneously with the first step, or after the first step. It is also possible to carry out at two or more timings. After the lactase addition step, lactose contained in the raw milk is decomposed (lactase reaction).
  • lactase addition step before the first step or substantially simultaneously with the first step.
  • Glucose and galactose produced by the decomposition of lactose can be assimilated by the lactic acid bacterium or the bifidobacteria.
  • the decomposition rate of lactose contained in raw material milk by neutral lactase gradually decreases as fermentation progresses.
  • Such neutral lactase action can provide an environment in which bifidobacteria can easily assimilate saccharides in raw milk in the coexistence of lactic acid bacteria and bifidobacteria, and the number of bifidobacteria in fermented milk products can be reduced.
  • the fact that the neutral lactase contained in the fermented dairy product is inactivated means that the lactase activity measurement method described later is performed at each fermentation time point, and is below the lower limit of detection.
  • the proliferation effect of bifidobacteria can be further increased by performing the protease reaction and the lactase reaction on the raw milk. Both the protease reaction and the lactase reaction are preferably performed substantially simultaneously.
  • the ratio of neutral lactase activity (NLU / g) to protease activity (PU / g) is preferably in the range of 0.01 to 100, more preferably in the range of 0.05 to 80. More preferably, it is within the range of 0.1 to 50.
  • the reason why the ratio of enzyme activity is used in this way is that the activity of each enzyme is different at the time of production and use, but the ratio is constant. If this ratio is less than the lower limit, the protease reaction may not be sufficiently performed, so that it is difficult to sufficiently obtain the growth effect of bifidobacteria. If this ratio exceeds the upper limit value, the protease is liable to decompose neutral lactase, which is not preferable.
  • the activity of the protease is in the range of 0.01 to 100 PU / g as a final concentration when added to the raw milk. If the concentration is less than the lower limit, the protease reaction may not be sufficiently performed, and thus it is difficult to sufficiently obtain the effect of bifidobacteria growth in the above system. Even if it exceeds the upper limit, the growth effect of bifidobacteria does not change, so it is not economical.
  • the activity of the neutral lactase is preferably in the range of 0.1 to 50 NLU / g at the final concentration when added to the raw milk. If it is less than the lower limit, the lactase reaction may not be sufficiently performed, and thus it is difficult to sufficiently obtain the growth effect of bifidobacteria in the above system. If it exceeds the upper limit value, the amount of lactose decomposition by the neutral lactase reaction does not change so much, and the bifidobacteria growth effect does not increase, which is not economical.
  • the enzyme-containing composition according to the present invention can be preferably used in the method for producing a fermented dairy product according to the present invention.
  • the present invention is an enzyme-containing composition comprising a protease that specifically degrades ⁇ -casein and a neutral lactase, The ratio of the neutral lactase activity (NLU / g) to the protease activity (PU / g) is in the range of 0.01-100.
  • the ratio is more preferably in the range of 0.05 to 80, and still more preferably in the range of 0.1 to 50.
  • the reason why the ratio of enzyme activity is used in this way is that the activity of each enzyme is different at the time of production and use, but the ratio is constant. If this ratio is less than the lower limit value, the lactase reaction may not be sufficiently performed or the protease may decompose the neutral lactase. Therefore, it is difficult to sufficiently obtain the bifidobacteria growth effect in the above system. . If this ratio exceeds the upper limit value, the protease reaction is not sufficiently performed, and there is a possibility that the risk is likely to occur.
  • the activity of the protease in the enzyme-containing composition of the present invention is preferably in the range of 0.01 to 13000000 PU / g.
  • the upper limit is more preferably 7000000 PU / g, and further preferably 5000000 PU / g.
  • a solid protease is easy to maintain in a highly active state.
  • the protease activity in the enzyme-containing composition may be prepared by dissolving and diluting in various solvents when added to milk. If it is less than the lower limit, the protease reaction after addition to the raw material milk may not be sufficiently performed, so that it is difficult to sufficiently obtain the bifidobacteria growth effect in the above system.
  • the activity of the protease in the enzyme-containing composition when added to the raw milk is preferably in the range of 0.01 to 100 PU / g at the final concentration. Even if it exceeds the upper limit, the growth effect of bifidobacteria does not change, so it is not economical.
  • the activity of neutral lactase in the enzyme-containing composition of the present invention may be higher than the final concentration in milk, and is preferably in the range of 0.1 to 50000 NLU / g.
  • the upper limit is more preferably 10,000 NLU / g, and further preferably 5000 NLU / g.
  • the neutral lactase activity in the enzyme-containing composition may be prepared for use when added to milk. If it is less than the lower limit, the lactase reaction after addition to the raw material milk may not be sufficiently performed, so that it is difficult to sufficiently obtain the bifidobacteria growth effect in the above system.
  • the activity of neutral lactase in the enzyme-containing composition when added to the raw milk is preferably in the range of 0.1 to 50 NLU / g at the final concentration. If it exceeds the upper limit value, the amount of lactose decomposition by the neutral lactase reaction does not change so much, and the bifidobacteria growth effect does not increase, which is not economical.
  • the enzyme-containing composition of the present invention may be solid or liquid. In consideration of handleability, it is preferably a liquid.
  • a protease and lactase may be mixed in advance, or a protease and lactase may be mixed at the time of use. Even if it mixes protease and lactase in raw milk, since the bifidobacteria growth effect is acquired, this is also included in an enzyme-containing composition.
  • the pH of the enzyme-containing composition of the present invention is preferably in the range of 5.0 to 8.5. Within this pH range, both protease and neutral lactase are active.
  • the present invention is a fermented milk product comprising lactic acid bacteria, bifidobacteria, protease that specifically degrades ⁇ -casein, and neutral lactase, A fermented milk product characterized in that the protease and the neutral lactase are inactivated.
  • the fermented milk product may be any product as long as it undergoes a fermentation process in which the milk raw material is fermented with lactic acid bacteria.
  • fermented milk, sour cream, quark, fermented milk beverage, cheese, fresh cheese and the like can be mentioned.
  • Fermented milk may be any of a hard type, a soft type, and a drink type.
  • the present invention can be preferably used particularly for fermented milk.
  • any milk containing lactose can be used.
  • animal milk such as cow's milk, sheep milk or goat milk, breast milk or powdered milk obtained by drying these can be used alone or in combination.
  • raw milk is obtained by adding lactose and water to these.
  • the raw material milk is preferably 90% by mass or more, preferably 95% by mass or more, and more preferably 98% by mass or more.
  • milk until completion of fermentation is referred to as raw material milk
  • milk after completion of fermentation is referred to as fermented milk or fermented milk product.
  • the raw milk can be sterilized as long as it can sterilize the microorganisms present in the raw milk and other components described below, and the sterilization method is not limited.
  • sterilization methods a method of performing ultra-high temperature for a short time (several seconds), a method of performing high temperature for a relatively short time (several minutes), a method of performing low temperature (tens of degrees) for a long time (several minutes to several tens of minutes) Is mentioned.
  • lactic acid bacteria examples include microorganisms belonging to the genus Lactococcus, the genus Lactobacillus, and the genus Streptococcus.
  • Lactococcus lactis Lactococcus lactis subspice cremoris, Lactobacillus casei, Lactobacillus casei Lactobacillus rhamnosus), Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus brevis delbrucil
  • Bulgaricus Streptococcus thermophilus (Streptococcus thermophilus).
  • These lactic acid bacteria may be used alone or in combination of two or more.
  • a narrow gull that uses Lactobacillus debrucecii subsp.
  • the amount of lactic acid bacteria added to the raw milk is preferably 1 ⁇ 10 4 to 1 ⁇ 10 10 per mL. If it is less than the lower limit, there is a problem of a decrease in fermentation rate. If it exceeds the upper limit, there is a problem of an increase in production cost.
  • bifidobacteria examples include microorganisms belonging to the genus Bifidobacterium.
  • Bifidobacterium adolecentis Bifidobacterium bifidum, Bifidobacterium longumbium fistum bispidum, -Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium animalis ssp. Lactis.
  • BB-12 strain manufactured by Christian Hansen
  • Lactis is preferable because it has acid resistance.
  • the BB-12 strain has oxygen resistance to some extent, it is easy to use in the production of fermented milk products.
  • the amount of bifidobacteria added to the raw milk is preferably 1 ⁇ 10 4 to 1 ⁇ 10 12 per mL. If it is less than the lower limit, there is a problem of insufficient number of bacteria. If it exceeds the upper limit, there is a problem of an increase in manufacturing cost.
  • the ratio of the number of bifidobacteria and lactic acid bacteria added to the raw milk is preferably in the range of 0.01 to 100,000, more preferably in the range of 0.1 to 10,000, and in the range of 1-1000. More preferably it is. If it is less than the lower limit, the number of bifidobacteria after fermentation may be difficult to increase. Exceeding the upper limit is not preferable because the number of bifidobacteria after fermentation increases, but the production cost tends to increase.
  • the protease that specifically degrades ⁇ -casein is preferably a neutral protease. If it is a neutral protease, the neutral protease is gradually deactivated due to a decrease in pH accompanying the progress of fermentation, and the neutral protease in the fermented milk product is deactivated. Is easy to keep constant.
  • the action pH of the neutral protease is preferably 5.5 to 9.0.
  • the protease that specifically degrades ⁇ -casein may be either endo-type or exo-type.
  • An endo-type protease is preferable.
  • An endo-type protease that specifically degrades ⁇ -casein is, for example, a neutral protease derived from the genus Penibacillus. Endo-type protease degrades amino acids from the inside of the ⁇ -casein molecule, so that it is difficult to produce a bitter taste peptide.
  • proteases proteinases, endopeptidases and exoproteases
  • lactic acid bacteria and bifidobacteria proteinsases, endopeptidases and exoproteases
  • proteases possessed by lactic acid bacteria and bifidobacteria have a weak ability to degrade ⁇ -casein, it is necessary to add a protease that specifically degrades ⁇ -casein to obtain the effects of the present invention.
  • chymosin is an endo-type protease, it is expensive and unfavorable because it is derived from calves.
  • the Penibacillus-derived neutral protease capable of obtaining the effects of the present invention has the following properties. (1) produced by bacteria belonging to the genus Penibacillus, (2) Decomposes ⁇ -casein and hemoglobin in the neutral range; (3) The optimum pH is 7.0 to 8.0, (4) a neutral protease that is stable at pH 5.5 to 9.0, (5) It operates at 20 to 75 ° C, and the optimum temperature is 55 ° C. (6) The molecular weight by electrophoresis is estimated to be 32,000-34,000 Da. The genus Penibacillus was formerly classified as the genus Bacillus, but has recently been reclassified and proposed as a new genus.
  • Proteases having the above properties can be obtained from another species including Penibacillus polymixer or Penibacillus sp. Re-identified from Bacillus polymixer. Although it may be displayed as a Bacillus polymixer depending on documents etc., it is synonymous.
  • the above-mentioned neutral protease derived from the genus Penibacillus has the following properties in detail.
  • ⁇ -casein and ⁇ -casein show almost no proteolytic action even in the neutral pH range.
  • C Optimum pH and stable pH range a) Optimum pH: The optimum pH for the proteolytic action on ⁇ -casein is 7.0 to 8.0. B Stable pH range: Extremely stable in the range of 5.5 to 9.0.
  • D Range of optimum temperature of action The temperature is in the range of 20 ° C to 75 ° C, and the optimum temperature is 50 to 60 ° C. The optimum temperature is 55 ° C.
  • E Deactivation conditions due to pH, temperature, etc. At pH 3.0 or lower and pH 10.0 or higher, the activity is completely lost. Further, it is completely deactivated by heat treatment at 65 ° C.
  • the amount of protease added is preferably in the range of 0.001 to 100 protease units / g, more preferably 0.005 to 50 protease units / g as the final concentration added to the raw material milk. It is more preferably 0.01 to 20 protease units / g, and particularly preferably 0.1 to 10 protease units / g.
  • “protease unit” may be abbreviated as “PU”. Below the lower limit, sufficient ⁇ -casein-derived peptides and free amino acids cannot be obtained, and the effect of increasing bifidobacteria is difficult to obtain. Even if the value exceeds the upper limit, the amount of ⁇ -casein is limited, so that no effect is obtained.
  • Neutral lactase has an action of degrading lactose into galactose and glucose.
  • Neutral lactase is sometimes called ⁇ -galactosidase.
  • Neutral lactase includes bacteria, yeast, and fungi. Among these, neutral lactase having an optimum pH for neutrality can be used.
  • Neutral lactase may be used alone or in combination of two or more.
  • neutral lactase examples include neutral lactase derived from Kluyveromyces lactis or Kluyveromyces fragilis, Kluyveromyces marxianus, Lanceis neutral lactase derived from circulans) is preferred.
  • the neutral lactase derived from Kluyveromyces includes neutral lactase derived from Kluyveromyces lactis as well as Kluyveromyces fungus itself.
  • the optimum pH for activity is 6.0 to 7.5 and the deactivation pH is 5.5 to 4.0. Since the pH of fermented milk products is 5.0 or less, when using neutral lactase, the neutral lactase contained in fermented milk products is in the inactivated state.
  • the amount of neutral lactase added is preferably in the range of 0.001 to 100 units / g, more preferably 0.01 to 50 units / g, as the final concentration added to the raw milk. It is more preferably 0.05 to 40 units / g, and particularly preferably 0.1 to 30 units / g.
  • “unit” may be abbreviated as “NLU”. If it is less than the lower limit, sufficient lactose decomposition cannot be obtained, and the effect of increasing bifidobacteria is difficult to obtain. Above the upper limit, lactose decomposition proceeds rapidly, so that it is difficult to obtain the effect of increasing bifidobacteria.
  • Sugars such as glucose and galactose other than lactose, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors, and the like can be added to raw milk or fermented milk products. When these materials are added, they may be added after sterilization treatment in advance, or may be sterilized after addition to raw material milk.
  • the enzyme-containing composition of the present invention preferably contains an aggregation inhibitor that prevents protein aggregation.
  • an aggregation inhibitor that prevents protein aggregation.
  • a surfactant (2) a protective agent having an action of covering the surface of a protein, and (3) a metal ion having a salt solubility effect or a salt thereof can be used.
  • a metal ion having a salt solubility effect or a salt thereof can be used.
  • These aggregation inhibitors may be used alone, or different types of aggregation inhibitors may be used in combination.
  • the enzyme-containing composition of the present invention may contain various components as necessary. Specific examples include metal salts that contribute to the stabilization of lactase and protease, various sugars, ascorbic acid, glycerin, and the like, starch, dextrin, and inorganic salts having a buffering action for improving usability. be able to.
  • neutral lactase was GODO-YNL2 manufactured by Godo Shusei Co., Ltd. unless otherwise specified.
  • Bifidobacteria can be obtained from Bifidobacterium animalis ssp. lactis BB12 strain was used.
  • the neutral protease (neutral metalloprotease) derived from the genus Penibacillus used in the following examples was prepared by the following method. The supernatant obtained by inoculating Paenibacillus polymyxa NBRC 15309 strain in Difco's Nutrient liquid medium and culturing at 30 ° C. for 2 days at 10,000 g for 10 minutes was desalted and concentrated by ultrafiltration.
  • the neutral protease had the properties (a) to (g) described above.
  • the addition amount (enzyme amount) of the neutral metalloprotease can be calculated as an enzyme activity per enzyme weight based on an enzyme amount of 1.0 PU defined by the following method. Add 1 mL of enzyme diluent to 0.6% aqueous casein solution (pH 7.5, 50 mM Tris-HCl buffer containing 2 mM calcium acetate), react at 30 ° C.
  • Neutral lactase activity was measured using the published FCC, 4th edition, July 1, 1996, pages 801-802 / lactase (neutral) ( ⁇ -galactosidase) activity.
  • the number of bifidobacteria was measured by mixing 0.1 mL of raw milk before fermentation and the prepared fermented milk appropriately diluted with physiological saline in a TOS propionate agar medium manufactured by Eiken Chemical Co., Ltd. The number of bacteria after daily anaerobic culture was measured.
  • fermented milk not subjected to neutral protease treatment was obtained as follows.
  • Commercially available skim milk manufactured by Morinaga Milk Industry Co., Ltd.
  • commercially available milk manufactured by Meiji Dairies Co., Ltd.
  • the raw milk was cooled to 43 ° C.
  • lactic acid bacteria and bifidobacteria were added in the same manner as in Reference Example 1 and fermented at 43 ° C. for 4 hours to obtain fermented milk of Comparative Example 1.
  • 100 mg of the starter was suspended in 1,000 ⁇ L of sterilized water, and 100 ⁇ L (Final 10 mg / 100 g) was added to the Erlenmeyer flask. After adding the starter and the enzyme, the mixture was stirred for several minutes while being kept at 43 ° C, dispensed from each Erlenmeyer flask into 6 centrifuge tubes of about 13 mL / 15 mL, and fermented at 43 ° C. The centrifuge tube was sampled over time, and pH measurement and sample preparation for SDS-PAGE were performed. The sample for SDS-PAGE was prepared by diluting the obtained fermented milk 20 times with ultrapure water, mixing 1: 1 with the sample buffer for SDS-PAGE, and boiling for 5 minutes.
  • Reference Example 5 Fermented milk of Reference Example 5 was obtained in the same manner as Reference Example 1 except that the final concentration of neutral protease was 0.5 PU / g.
  • Comparative Example 2 A fermented milk of Comparative Example 2 that was not subjected to neutral protease treatment was obtained in the same manner as in Comparative Example 1, except that it was performed simultaneously with Reference Example 5.
  • Reference Example 5 in which neutral protease treatment was performed, the BB-12 strain showed good growth during fermentation.
  • Examples 1 to 3 Commercially available skim milk (manufactured by Morinaga Milk Industry Co., Ltd.) was mixed and dissolved in 2 parts by mass and commercially available milk (manufactured by Meiji Dairies Co., Ltd.) in 98 parts by mass, and sterilized at 100 ° C. for 5 minutes.
  • the raw milk is cooled to 43 ° C.
  • the final protease is 0.1 to 4.0 PU / g) neutral protease, 0.05 part by mass (final concentration 2.5 NLU / g) neutral lactase and lactic acid bacteria ( YF-L812 strain) 0.1 mg / raw material milk 1 mL and bifidobacteria 0.05 mg / raw material milk 1 mL were added and fermented at 43 ° C. for 4 hours to obtain fermented milk of Examples 1 to 3 (neutral protease, Neutral lactase added simultaneously).
  • Comparative Example 3 A fermented milk of Comparative Example 3 which was not subjected to neutral protease treatment and neutral lactase treatment was obtained in the same manner as in Comparative Example 1 except that Examples 1 to 3 were performed simultaneously.
  • Example 2 and Comparative Example 3 showed good growth during fermentation.
  • the difference between Example 2 and Comparative Example 3 at the end of fermentation was approximately 298%, and the difference between Example 3 and Comparative Example 3 was 598%.
  • the concentration of neutral protease increased, the number of bifidobacteria increased. Since the pH after fermentation tended to decrease as the neutral protease was increased, it was suggested that the fermentation time can be shortened by the present invention.
  • Example 4 Fermented milk of Example 4 was obtained in the same manner as Example 1 except that neutral protease was added so that the final concentration was 0.5 PU / g (neutral addition of neutral protease and neutral lactase).
  • Comparative Example 4 A fermented milk of Comparative Example 4 was obtained in the same manner as in Comparative Example 1 except that neutral protease treatment and neutral lactase treatment were not performed, except that Example 4 was performed simultaneously.
  • Example 4 in which protease treatment and neutral lactase treatment were performed, the BB-12 strain showed good growth during fermentation. The difference between Example 4 and Comparative Example 4 at the end of fermentation was approximately 219%.
  • neutral lactase and neutral protease act on milk are different. That is, neutral lactase degrades lactose into glucose and galactose, and the neutral protease used in this example specifically degrades ⁇ -casein.
  • Bifidobacteria are slower to assimilate saccharides including lactose than lactic acid bacteria. By supplying glucose and galactose beyond that of lactic acid bacteria, it is presumed that Bifidobacteria will easily grow. Bifidobacteria and lactic acid bacteria have almost no ability to degrade ⁇ -casein.

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Abstract

La présente invention concerne une composition renfermant une enzyme et qui est capable d'augmenter et de maintenir le nombre de cellules revivifiables de bactéries bifidus dans des produits laitiers fermentés par un procédé simple ; et un procédé de production d'un produit laitier fermenté. La présente invention concerne une composition renfermant une enzyme comprenant une lactase neutre et une protéase qui dégrade spécifiquement la caséine κ, ladite composition étant caractérisée en ce que l'activité de la protéase par unité d'activité (NLU) de la lactase neutre est de 0,01 à 100 unités (PU). Il est préférable que l'activité de la protéase soit d'au moins 0,01 PU/g.
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CN114806906B (zh) * 2022-05-31 2023-02-21 江南大学 一株产中性乳糖酶的乳酸克鲁维酵母

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