DK154321B - ADDITIVE TO FOOD AND BEVERAGES FOR PROVIDING FOOD BIFIDO BACTERIES THEREOF AND PROCEDURE FOR MAKING THE SAME AND BIFIGO BACTERIA FOR EXPELLENCE - Google Patents
ADDITIVE TO FOOD AND BEVERAGES FOR PROVIDING FOOD BIFIDO BACTERIES THEREOF AND PROCEDURE FOR MAKING THE SAME AND BIFIGO BACTERIA FOR EXPELLENCE Download PDFInfo
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- DK154321B DK154321B DK590276A DK590276A DK154321B DK 154321 B DK154321 B DK 154321B DK 590276 A DK590276 A DK 590276A DK 590276 A DK590276 A DK 590276A DK 154321 B DK154321 B DK 154321B
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
- A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
- A23G9/36—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
- A23G9/363—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
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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
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/02—Making cheese curd
- A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
- A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
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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
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1232—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt in powdered, granulated or dried solid form
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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
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- 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
- A23C2260/00—Particular aspects or types of dairy products
- A23C2260/15—Frozen dairy products
- A23C2260/152—Frozen fermented milk products, e.g. frozen yoghurt or yoghurt ice cream; Frozen milk products containing living microorganisms
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- Inorganic Chemistry (AREA)
- Dairy Products (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
DK 154321BDK 154321B
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Opfindelsen angår et tilsætningsmiddel til levnedsmidler og drikkevarer til tilvejebringelse af levedygtige bifido-bakterier heri.The invention relates to a food and beverage additive for providing viable bifido bacteria therein.
Opfindelsen angår endvidere en fremgangsmåde til fremstilling af 5 dette tilsætningsmiddel.The invention further relates to a process for preparing this additive.
Bifido-bakterier er kendt som de dominerende bakterier i et brysternæret barns tarmflora. Det er kendt om bifido-bakterier, at de har forskellige fysiologiske egenskaber, såsom evnen til at: 10 a) undertrykke forrådnelsesbakterier, b) inhibere dannelsen af toksiske aminer, c) fremme fordøjelsen af kvindemælk-kasein som følge af virkningen af phosphorproteinphosphatase, og 15 d) inhibere væksten af patogene bakterier i forbindelse med nedsættelse af pH-værdien i tarmene som følge af dannelse af mælkesyre, eddikesyre, myresyre, o.l.Bifido bacteria are known as the dominant bacteria in the intestinal flora of a breast-fed child. Bifido bacteria are known to have various physiological properties, such as the ability to: a) suppress rotten bacteria, b) inhibit the formation of toxic amines, c) promote digestion of female milk casein due to the action of phosphorus protein phosphatase, and D) inhibiting the growth of pathogenic bacteria in connection with the reduction of intestinal pH due to the formation of lactic acid, acetic acid, formic acid, etc.
Et spædbarn, som er kunstigt ernæret, har i tarmfloraen meget få 20 bifido-bakterier, hvilke, som tidligere nævnt, har forskellige virkninger med hensyn til at holde tarmene sunde. Dette anses for at være årsagen til, at et kunstigt ernæret spædbarn er mere modtageligt over for tarmlidelser end et brysternæret spædbarn. Med henblik på at bringe tarmfloraen hos et kunstigt ernæret spædbarn til at 25 svare til tarmfloraen hos et brysternæret spædbarn har man derfor forsøgsvis fremstillet modificeret mælkepulver til spædbørn indeholdende bifido-bakterier som erstatning for kvindemælk.An artificially nourished infant has very few 20 bifido bacteria in the gut flora, which, as mentioned earlier, have different effects in keeping the gut healthy. This is considered to be the reason why an artificially nourished infant is more susceptible to bowel disorders than a breast-fed infant. Therefore, in order to bring the intestinal flora of an artificially nourished infant to correspond to the intestinal flora of a breast-fed infant, modified milk powder for infants containing bifido bacteria has been experimentally prepared as a substitute for breast milk.
Imidlertid har der været knyttet en række vanskeligheder til den 30 sædvanlige fremgangsmåde til dyrkning af bifido-bakterier i et medium, såsom mælk, o.l., og denne fremgangsmåde er derfor ikke blevet bragt til udførelse i praksis i kommerciel målestok. Vanskelighederne (sammenlignet med fremgangsmåden for mælkesyrebakterier, som er almindeligt anvendt til forarbejdning af mælk) består i: 35 a) at betingelserne for dyrkning skal være strengt anaerobe, b) at næringskravene til dyrkningen er komplicerede og strenge, og bakterierne formerer sig derfor ikke i etHowever, a number of difficulties have been associated with the usual method of growing bifido bacteria in a medium such as milk, and the like, and this method has therefore not been put into practice on a commercial scale. The difficulties (compared to the method for lactic acid bacteria commonly used in milk processing) consist of: 35 a) the conditions for cultivation must be strictly anaerobic, b) the nutritional requirements for cultivation are complicated and stringent and the bacteria do not multiply in one
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2 rent mælkemiljø, som ikke indeholder noget vækstfremmende stof, såsom gærekstrakt o.l., og c) at det er vanskeligt at holde bakterierne i live i lang tid ved en lav pH-værdi, såsom i et sædvanligt 5 fermenteret, mælkeprodukt som følge af, at bifido- bakteriernes syreresistens er lav.2 pure milk environment which does not contain any growth promoter such as yeast extract, and c) it is difficult to keep the bacteria alive for a long time at a low pH such as in a usual fermented milk product due to the fact that The acid resistance of the bifido bacteria is low.
Hvis der til et mælkemedium tilsættes et stof, som fremmer væksten af bifido-bakterier, kan bifido-bakterierne dyrkes under aerobe 10 forhold. Imidlertid har produktet fra en sådan dyrkning ofte forringet smag og aroma. Ud over denne ulempe er væsktfremmende stoffer sædvanligvis dyre og anvendes derfor ikke i praksis.If a substance is added to a milk medium which promotes the growth of bifido bacteria, the bifido bacteria can be grown under aerobic conditions. However, the product of such cultivation has often impaired taste and aroma. In addition to this disadvantage, liquid promoters are usually expensive and are therefore not used in practice.
De ovennævnte ulemper gælder bl.a. for de fremgangsmåder til frem-15 stilling af surmælksprodukter og spædbarnsernæringsprodukter, som de kendes fra DE offentliggørelsesskrift nr. 1.952.361 og 6B patentskrift nr. 831.797 henholdsvis DE offentliggørelsesskrift nr. 2.057.906.The above-mentioned disadvantages include for the processes for making sour milk products and infant nutritional products as known from DE Publication No. 1,952,361 and 6B patent specification 831.797 and DE Publication No. 2,057,790, respectively.
20 I forbindelse med studiet og dyrkningen af bifido-bakterier har opfinderne til nærværende opfindelse imidlertid opdaget nye stammer af bifido-bakterier med særlige egenskaber, hvilke bifido-bakterier kan opformeres aktivt under aerobe forhold i et rent mælkemedium, som ikke indeholder væsktfremmende stoffer eller reducerende midler.However, in connection with the study and cultivation of bifido bacteria, the inventors of the present invention have discovered new strains of bifido bacteria with special properties which bifido bacteria can be actively propagated under aerobic conditions in a pure milk medium containing no liquid promoters or reducing agents. agents.
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Baseret på denne opdagelse tilvejebringes der derfor ved nærværende opfindelse et tilsætningsmiddel til levnedsmidler og drikkevarer til tilvejebringelse af levedygtige bifido-bakterier heri, hvilket tilsætningsmiddel er ejendommeligt ved, at det er fremstillet ved 30 aerob propagering af en bakteriekultur af Bifidobacterium bifidum YIT-4005 (dep.nr. FERM-P 3372) og/eller Bifidobacterium bifidum YIT-4002 (dep.nr. FERM-P 3371) i et rent mælkemedium.Therefore, based on this discovery, there is provided by the present invention a food and beverage additive for providing viable bifido bacteria, which additive is characterized by being produced by aerobic propagation of a bacterial culture of Bifidobacterium bifidum YIT-4005 (dep. No. FERM-P 3372) and / or Bifidobacterium bifidum YIT-4002 (Dep. No. FERM-P 3371) in a pure milk medium.
En anden udførelsesform for tilsætningsmidlet ifølge opfindelsen er 35 ejendommelig ved, at tilsætningsmidlet foreligger i tør form.Another embodiment of the additive according to the invention is characterized in that the additive is in dry form.
Med opfindelsen tilvejebringes også en fremgangsmåde til fremstilling af tilsætningsmidlet ifølge opfindelsen, hvilken fremgangsmåde er ejendommelig ved, at en bakteriekultur af Bifidobacterium bifidumThe invention also provides a process for preparing the additive of the invention, characterized in that a bacterial culture of Bifidobacterium bifidum
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3 YIT-4005 (dep.nr. FERM-P 3372) og/eller Bifidobacterium bifidum YIT-4002 (dep.nr. FERM-P 3371) propageres aerobt i et rent mælkemedium.3 YIT-4005 (dep. No. FERM-P 3372) and / or Bifidobacterium bifidum YIT-4002 (dep. No. FERM-P 3371) is aerobically propagated in a pure milk medium.
5 Endelig tilvejebringes der med opfindelsen hidtil ukendte Bifido-bakterier til udøvelse af fremgangsmåden ifølge opfindelsen, hvilke Bifido-bakterier er ejendommelige ved, at det er Bifidobacterium bifidum YIT-4005 (deponeringsnummer FERM-P 3372) og/eller Bifidobacterium bifi dum YIT-4002 (deponeringsnummer FERM-P 3371).Finally, the invention provides novel Bifido bacteria for carrying out the process of the invention, which Bifido bacteria are characterized by being Bifidobacterium bifidum YIT-4005 (landfill number FERM-P 3372) and / or Bifidobacterium bifum YIT-4002 (deposit number FERM-P 3371).
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Den foreliggende opfindelse vil blive forklaret nærmere i det følgende.The present invention will be explained in more detail below.
Først vil forskellige egenskaber af Bifidobacterium bifidum YIT-4002 15 og YIT-4005, som anvendes i forbindelse med den foreliggende opfindelse, blive beskrevet nedenfor.First, various properties of Bifidobacterium bifidum YIT-4002 and YIT-4005 used in the present invention will be described below.
Den nye stamme "Bifidobacterium bifidum YIT-4002" blev isoleret ved den successive overføringsdyrkning af bifidobakterier udtaget fra 20 fæces fra et sundt brysternæret spædbarn og har følgende egenskaber: (1) Væsentligste bakteriologiske egenskaber.The new strain "Bifidobacterium bifidum YIT-4002" was isolated by the successive transfer culture of bifidobacteria taken from 20 faeces from a healthy breast-fed infant and has the following properties: (1) Essential bacteriological properties.
Denne stamme er en gram-positiv, ikke-sporedannende bacille (0,5 x 25 2-3 μ) og har granuler med affinitet til methylenblåt på cellens inderside. Ved mikroskopisk undersøgelse findes, at bakterien har forgrenede kanter med polymorf form, såsom Y-form, V-form eller bøjning. En koloni dannet ved dobbeltlagsplademetoden har en cylindrisk, konveks eller linseformet facon, og en koloni dannet ved 30 bunden af pladen har en variabel facon. Ved dyrkning i anaerobt VL-G væskemedium i 72 timer blev mol forhol det mellem dannet eddikesyre og mælkesyre 1,6 ± 0,5 (eddikesyre/mælkesyre). Den således dannede mælkesyre i førnævnte væskeformige medium undersøgtes med hensyn til optisk rotation, og resultaterne var som følger: 35This strain is a gram-positive, non-spore forming bacilli (0.5 x 25 2-3 µ) and has granules with affinity for methylene blue on the inside of the cell. By microscopic examination it is found that the bacterium has branched edges with polymorphic shape, such as Y shape, V shape or bend. A colony formed by the double-layer plate method has a cylindrical, convex or lens-shaped shape, and a colony formed at the bottom of the plate has a variable shape. When cultured in anaerobic VL-G liquid medium for 72 hours, the mole ratio of acetic acid to lactic acid formed was 1.6 ± 0.5 (acetic acid / lactic acid). The lactic acid thus formed in the aforementioned liquid medium was examined for optical rotation and the results were as follows:
Specifik rotationsaktivitet: -0,038Specific rotational activity: -0.038
Optisk aktivitet: L (+) 76% + DL 24%Optical activity: L (+) 76% + DL 24%
Resultaterne af nedenstående prøver var følgende: Catalase (-),The results of the tests below were as follows: Catalase (-),
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4 carbondioxidgasdannelse (-), cysteinmælkekoaguleringsaktivitet (+), gelatinehydrolyse (-), nitratreducerende aktivitet (-), indoldannelse (-) og hydrogensulfiddannelse (-).4 carbon dioxide gas formation (-), cysteine milk coagulation activity (+), gelatin hydrolysis (-), nitrate reducing activity (-), indole formation (-) and hydrogen sulfide formation (-).
5 Ovenstående resultater opfylder definitionen af Bifidobacterium-slægten.5 The above results meet the definition of the Bifidobacterium genus.
(2) Optimal propagennostemperatur oa pH.(2) Optimum propageno temperature and pH.
10 Optimal propageringstemperatur: 36-38°COptimal propagation temperature: 36-38 ° C
Bakterierne formerer sig ikke ved en temperatur på under 25“C eller over 45eC.The bacteria do not multiply at a temperature below 25 ° C or above 45 ° C.
15 Optimal propagerings-pH: 6-7Optimal Propagation pH: 6-7
Bakterierne formerer sig ikke ved en pH-værdi under 5,0.The bacteria do not multiply at a pH below 5.0.
(3) Sukkerfermenteri na.(3) Sugar fermentation na.
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De bakterier, der anvendes ifølge opfindelsen, har en positiv fermenteringsreaktion med hensyn til følgende sukkerarter: glucose, fructose, lactose og galactose.The bacteria used in the invention have a positive fermentation reaction for the following sugars: glucose, fructose, lactose and galactose.
25 Bakterierne har negativ fermenteringsreaktion med hensyn til følgende sukkerarter: arabinose, xylose, salicin, mannose, mannitol, melezitose, cellobiose, sorbitol, i nul i n, trehalose, rhamnose, maltose, ribose og sorbose.The bacteria have a negative fermentation reaction for the following sugars: arabinose, xylose, salicin, mannose, mannitol, melezitose, cellobiose, sorbitol, zero, n, trehalose, rhamnose, maltose, ribose and sorbose.
30 Dette fermenteringsmønster svarer fuldstændigt til fermenteringsmønsteret for en bifidobakterie standardstamme (Bifidobacterium bifidum E 319).30 This fermentation pattern is completely similar to the fermentation pattern of a standard bifidobacteria bacterium (Bifidobacterium bifidum E 319).
(4) Oxvaenfølsomhed oo nærinaskrav.(4) Oxen sensitivity and proximity requirements.
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De bakterier, der anvendes ifølge opfindelsen, inkuberedes i et antal på 10 -10 /ml og dyrkedes aerobt ved 37°C i et reduceret skummetmælksmedium (mælketørstofindhold 12%, der ikke indeholdt noget vækstfremmende stof), men som var oxygenmættet (ca. 6,5 ppm).The bacteria used according to the invention were incubated in a number of 10 -10 / ml and cultured aerobically at 37 ° C in a reduced skimmed milk medium (milk solids content 12% containing no growth promoter) but oxygen-saturated (about 6 , 5 ppm).
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Populationen nåede op på over 109/ml ved 24 timers inkubation.The population reached over 109 / ml by 24 hour incubation.
Sædvanlige bi fidus-bakterier formerede sig næsten ikke under samme forhold som bekrevet ovenfor. Populationen var uændret eller 5 formindsket (se nedenstående eksempel 2).Usual bee scam bacteria hardly propagated under the same conditions as described above. The population was unchanged or decreased (see Example 2 below).
Som ovenfor beskrevet svarer de bakterier, der anvendes ifølge opfindelsen, fuldstændigt til Bifidobacterium bifidum med undtagelse af, at bakterierne ifølge opfindelsen formerer sig godt i et rent 10 mælkemedium under aerobe forhold, hvor sædvanlige bifidobakterier ikke kan formere sig. I lyset af disse egenskaber identificeredes bakteriestammen som en variant af Bifidobacterium bifidum og betegnedes "Bifidobacterium bifidum YIT-4002". Denne identifikation skete ifølge Bargey's Manual fra 1974). Den nye stamme deponeredes i 15 FERMENTATION RESEARCH INSTITUTE, Agency of Industrial Science & Technology, no. 8-1, Inage Higashi-5-chome, Chiba-shi, Chiba-ken,As described above, the bacteria used according to the invention are completely similar to Bifidobacterium bifidum except that the bacteria according to the invention propagate well in a pure milk medium under aerobic conditions where the usual bifidobacteria cannot reproduce. In view of these characteristics, the bacterial strain was identified as a variant of Bifidobacterium bifidum and designated "Bifidobacterium bifidum YIT-4002". This identification was done according to Bargey's Manual of 1974). The new strain was deposited in 15 FERMENTATION RESEARCH INSTITUTE, Agency of Industrial Science & Technology, no. 8-1, Inage Higashi-5-chome, Chiba-shi, Chiba-ken,
Japan. Deponeringsnummeret er FERM-P nr. 3371.Japan. The deposit number is FERM-P No. 3371.
Den nye stamme YIT-4005 i soleredes fra fæces af et sundt brysternæ-20 ret spædbarn ved adskillige ganges behandling i en pufferopløsning med lav pH-værdi.The new strain YIT-4005 was soldered from the faeces of a healthy breast-fed infant by several times of treatment in a low pH buffer solution.
(1) Taxonomiske egenskaber.(1) Taxonomic properties.
25 Denne stamme er en gram-positiv, ikke-sporedannende bacille og har intracellulære granuler med affinitet til methylenblåt. Mikroskopisk undersøgelse viste, at bakterierne er polymorfe med tofligede kanter med Y-form eller bøjning. En koloni dannet ved dobbeltlagsplademetoden er cylindrisk, konveks eller linseformet, og en koloni 30 dannet ved pladens bund har en variabel form. Ved dyrkning i et ikke-fedtholdigt mælkemedium (mælketørstofindhold 12%) i 72 timer bliver mol forholdet mellem dannet eddikesyre og mælkesyre 1,7 ± 0,5 (eddi kesyre/mælkesyre).This strain is a gram-positive, non-spore forming bacilli and has intracellular granules with affinity for methylene blue. Microscopic examination revealed that the bacteria are polymorphic with two-sided Y-shaped or bending edges. A colony formed by the bilayer plate method is cylindrical, convex or lens shaped, and a colony 30 formed at the base of the plate has a variable shape. When grown in a non-fat milk medium (milk solids content 12%) for 72 hours, the mole ratio of acetic acid to lactic acid formed is 1.7 ± 0.5 (acetic acid / lactic acid).
35 Resultaterne af nedenstående prøver var som følger: catalaseakti-vitet (-), mælkekoaguleringsaktivitet (+), gelatinehydrolyse (-), nitratreducerende aktivitet (-), indo!produktion (-) og hydrogensul f iddannelse (-).The results of the tests below were as follows: catalase activity (-), milk coagulation activity (+), gelatin hydrolysis (-), nitrate reducing activity (-), indole production (-) and hydrogen sulfide formation (-).
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De bakterier, der anvendes ifølge opfindelsen, har en positiv fermenteringsreaktion over for følgende sukkerarter: glucose, fructose, lactose og galactose.The bacteria used in the invention have a positive fermentation reaction to the following sugars: glucose, fructose, lactose and galactose.
5 Bakterierne har en negativ fermenteringsreaktion over for følgende sukkerarter: arabinose, xylose, salicin, mannose, mannitol, melezi-tose, cellobiose, sorbitol, inulin, trehalose, rhamnose, maltose, ribose og sorbose.The bacteria have a negative fermentation reaction to the following sugars: arabinose, xylose, salicin, mannose, mannitol, melezotic, cellobiose, sorbitol, inulin, trehalose, rhamnose, maltose, ribose and sorbose.
10 Ud fra ovenstående egenskaber svarer den bakteriestamme, der anvendes ifølge opfindelsen, til Bifidobacterium bifidum ifølge Bargey's Manual fra 1974 men klassificeres som en variant af Bifidobacterium bi f i dum, som følge af, at den har nye egenskaber, som de kendte bifidobakterier ikke besidder, og denne variant betegnedes "Bifido-15 bacterium bifidum YIT-4005".From the above characteristics, the bacterial strain used in the invention is similar to Bifidobacterium bifidum according to Bargey's Manual of 1974 but is classified as a variant of Bifidobacterium bi-fum because it has new properties that the known bifidobacteria do not possess. and this variant was designated "Bifido-bacterium bifidum YIT-4005".
(2) Vækstbetingelser.(2) Growth conditions.
Temperatur: 25-45°C (Optimum 36-38°C) 20 pH: 5-7 (optimum pH 6-7) (3) Syreresi stens.Temperature: 25-45 ° C (Optimum 36-38 ° C) 20 pH: 5-7 (Optimum pH 6-7) (3) Acid resistance.
Stammen, der anvendes ifølge opfindelsen, og en standardstamme 25 (Bifidobacterium bifidum E 319, som i det følgende er betegnet "standardstamme") dyrkedes respektivt i et anaerobt VL-G væskemedium i 48 timer, og de dyrkede bakterier opsamledes aseptisk ved centrifugering. Vaskede cellesuspensioner (OD 660 m/i = 1,5) sattes til pufferopløsninger (pH 6,0: 1/100 M phosphorsyre, pH 5,0-4,0: 30 1/100 M eddikesyre) med forskellige pH-værdier som vist i tabel 1.The strain used according to the invention and a standard strain 25 (Bifidobacterium bifidum E 319, hereinafter referred to as "standard strain") were cultured respectively in an anaerobic VL-G liquid medium for 48 hours, and the cultured bacteria were collected aseptically by centrifugation. Washed cell suspensions (OD 660 m / i = 1.5) were added to buffer solutions (pH 6.0: 1/100 M phosphoric acid, pH 5.0-4.0: 30 1/100 M acetic acid) with various pH values as shown in Table 1.
De fremkomne opløsninger blev holdt ved 5°C til bestemmelse af bifidobakteriernes levedygtighed med tiden.The resulting solutions were maintained at 5 ° C to determine the viability of the bifidobacteria over time.
Som vist i tabel 1 faldt optællingerne af levende bifidobakterier 35 hurtigt med faldende pH-værdi (specielt under 4,6). På den anden side var optællingerne af levende bifidobakterier fra stammen YIT-4005 meget stabile, selv ved lave pH-værdier nær 4,0, og bakterierne ifølge opfindelsen viste sig derved at have høj syreresistens (tallene i tabel 1 angiver optællinger af levende bakterier pr. ml).As shown in Table 1, the counts of live bifidobacteria 35 decreased rapidly with decreasing pH (especially below 4.6). On the other hand, the counts of live bifidobacteria from strain YIT-4005 were very stable even at low pH values near 4.0, and the bacteria according to the invention were found to have high acid resistance (the figures in Table 1 indicate counts of live bacteria per ml).
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Tabel 1Table 1
OISLAND
Stamme Dage 5 fiiL (*) 0_ 3_ 5_ 7_ 10_ 6.0 V 4,7xl08 3,8xl08 3,7xl08 4,0xl08 3,0xl08 S 4,5xl08 3,lxl08 6,8xl07 4,2xl07 2,lxl07 5.0 V 5,0xl08 3,8xl08 2,4xl08 3,0xl08 3,3xl08 10 S 4,9xl08 3,lxl08 5,5xl07 l,9xl07 2,lxl06 4,6 V 4,2xl08 5,lxl08 4,2xl08 2,0xl08 9,3xl07 S 4,5xl08 Ι,ΟχΙΟ7 3,6xl05 4,0xl03 <102 15 4,3 V 4,6xl08 4,0xl08 2,lxl08 6,9xl07 3,0xl07 S 4,2xl08 3,9xl06 2,lxl04 <102 <102 4.1 V 4,7xl08 9,5xl07 3,4xl07 5,6xl06 4,3xl05 S 4,5xl08 4,lxlO4 Ι,ΟχΙΟ3 <102 <102 20 4,0 V 5,0xl08 7,0xl06 8,0xl05 2,8xl05 2,9xl04 S 4,9xl08 l,2xl03 <102 <102 <102 (*) V: Bifidobacterium bifidum YIT-4005 25 S: Bifidobacterium bifidum E 319.Tribal Days 5 fiL (*) 0_ 3_ 5_ 7_ 10_ 6.0 V 4.7xl08 3.8xl08 3.7xl08 4.0xl08 3.0xl08 S 4.5xl08 3, lxl08 6.8xl07 4.2xl07 2, lxl07 5.0 V 5.0xl08 3.8x108 2.4x108 3.0x108 3.3x108 S 4.9x108 3, lxl08 5.5x107 l, 9xl07 2, lxl06 4.6 V 4.2xl08 5, lxl08 4.2xl08 2.0xl08 9.3xl07 S 4 , 5x108 Ι, ΟχΙΟ7 3.6x105 4.0x103 <102 15 4.3 V 4.6x108 4.0x108 2, lx108 6.9x107 3.0x107 S 4.2x108 3.9x106 2, lx104 <102 <102 4.1 V 4 , 7x108 9.5x107 3.4x107 5.6x106 4.3x105 S 4.5x108 4, lx104 Ι, ΟχΙΟ3 <102 <102 20 4.0 V 5.0x108 7.0x106 8.0x105 2.8x105 2.9x104 S 4 , 9x108 l, 2x103 <102 <102 <102 (*) V: Bifidobacterium bifidum YIT-4005 S: Bifidobacterium bifidum E 319.
Samme tendens sås i mælkekulturen. De forskellige stammer af bifidobacterium dyrkedes som angivet i tabel 2 i et ikke-fedtholdigt mælkemedium (mælketørstofindhold 10%) indeholdende 0,3% gærekstrakt, 30 indtil pH-værdien for kulturerne nåede de i tabellen angivne værdi er. Kulturerne afkøledes derefter hurtigt og blev holdt ved 5eC til bestemmelse af bifidobakteriernes levedygtighed med tiden.The same trend was seen in the milk culture. The various strains of bifidobacterium were grown as indicated in Table 2 in a non-fat milk medium (milk solids content 10%) containing 0.3% yeast extract, until the pH of the cultures reached the value given in the table. The cultures were then cooled rapidly and maintained at 5 ° C to determine the viability of the bifidobacteria over time.
Tabel 2 35Table 2 35
Dagedays
Stamme pH (*) 0_ 3_ 5_ 7_ 10_ 4,61 V 4,lxlO9 3,lxlO9 2,0xl09 7,6xl08 4,3xl08 DK 154321 B j 8 4.61 S 6,8xl08 2,0xl07 4,3xl06 5,3xl05 l,6xl04 A 3,3xl09 5,6xl08 l,5xl07 4,3xl06 2,lxl05 4.61 B l,5xl09 2,7xl08 5,3xl07 9,6xl06 7,7xl06 5 C 8,8xl08 6,2xl07 2,5xl06 5,lxl05 2,9xl04 j D 4,3xl09 7,lxl08 7,9xl07 6,3xl06 4,4xl05 4.21 V l,8xl09 7,6xl08 9,8xl08 7,0xl07 2,4xl06 S 2,5xl08 2,0xl06 4,5xl04 <102 <102 10 A 9,9xl08 7,5x1O6 8,2xl05 2,3xl03 <102 4.21 B 4,2xl09 2,5xl07 4,9xl05 2,4xl03 <102 C 2,lxl09 l,5xl07 8,7xl04 <102 <102 D 7,7xl08 2,lxl07 6,3xl05 l,5xl04 <102 15 (*) V: Bifidobacterium bifidum YIT-4005 S: Bifidobacterium bifidum E319 A: Bifidobacterium bifidum B: Bifidobacterium bifidum YIT-4002 20 C: Bifidobacterium lonaum standardstamme D: Bifidobacterium lonaum (4) Vækst under aerobe forhold.Strain pH (*) 0_ 3_ 5_ 7_ 10_ 4.61 V 4, lxlO9 3, lxlO9 2.0xl09 7.6xl08 4.3xl08 DK 154321 B j 8 4.61 S 6.8xl08 2.0xl07 4.3xl06 5.3xl05 l, 6x104 A 3.3x109 5.6x108 l, 5xl07 4.3xl06 2, lxl05 4.61 B l, 5xl09 2.7xl08 5.3xl07 9.6xl06 7.7xl06 5 C 8.8xl08 6.2xl07 2.5xl06 5, lxl05 2 9x104 j D 4.3xl09 7, lxl08 7.9xl07 6.3xl06 4.4xl05 4.21 V l, 8xl09 7.6xl08 9.8xl08 7.0xl07 2.4xl06 S 2.5xl08 2.0xl06 4.5xl04 <102 <102 10 A 9.9x108 7.5x106 8.2x105 2.3x103 <102 4.21 B 4.2x109 2.5x107 4.9x105 2.4x103 <102 C 2, x109 l, 5x107 8.7x104 <102 <102 D 7.7x108 2 (x) V: Bifidobacterium bifidum YIT-4005 S: Bifidobacterium bifidum E319 A: Bifidobacterium bifidum B: Bifidobacterium bifidum YIT-4002 20 C: Bifidobacterium lonaum Growth under aerobic conditions.
2525
Et ikke-fedtholdigt mælkemedium (mælketørstofindhold 12%) steriliseredes ved opvarmning og afkøledes derefter til 37°C under gennem-ledning af luft gennem mediet. Stammen YIT-4005, der anvendes ifølge opfindelsen, og nogle af de i Tabel 2 viste sammenligningsstammer 30 dyrkedes hver for sig i det ovenfor fremstillede medium ved 37eC.A non-fat milk medium (milk solids content 12%) was sterilized by heating and then cooled to 37 ° C while passing air through the medium. The strain YIT-4005 used according to the invention and some of the comparative strains shown in Table 2 were grown separately in the medium prepared above at 37 ° C.
Ændringen i optællingerne af levende bakterier og surheden af kulturen (antal ml 0,1N natriumhydroxid nødvendig til neutralisering af 10 ml af kulturen) under dyrkningen er vist i fig. 3. Betegnelserne i fig. 3 angiver det samme som i Tabel 2.The change in the counts of live bacteria and the acidity of the culture (number of ml of 0.1N sodium hydroxide needed to neutralize 10 ml of the culture) during cultivation is shown in fig. 3. The designations of FIG. 3 indicates the same as in Table 2.
3535
Som det ses af figuren, formerede bakterien YIT-4005, der anvendes g ifølge opfindelsen, sig og nåede op på 10 levende celler/ml ellerAs seen in the figure, the bacterium YIT-4005 used in accordance with the invention propagated to reach 10 living cells / ml or
7 R7 R
derover fra begyndelsespopulationen på 10-10 celler/ml i løbet af 24 timer, medens standardstammen ikke formerede sig under dissemoreover from the initial population of 10-10 cells / ml over 24 hours, while the standard strain did not propagate below these
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5 9 forhold.5 9 conditions.
Denne formenngstendens af bakterier, der anvendes ifølge opfindelsen, var stort set den samme som under anaerobe forhold.This propagation tendency of bacteria used according to the invention was substantially the same as under anaerobic conditions.
Den nye stamme ifølge opfindelsen, "Bifidobacterium bifidum YIT-4005" deponeredes i FERMENTATION RESEARCH INSTITUTE, Agency of Industrial Science & Technology, no. 8-1, Inage Higashi-5-chome, Chibashi, Chiba-ken, Japan. Deponeringsnummeret er FERM-P nr. 3372.The new strain of the invention, "Bifidobacterium bifidum YIT-4005" was deposited in the FERMENTATION RESEARCH INSTITUTE, Agency of Industrial Science & Technology, no. 8-1, Inage Higashi-5-chome, Chibashi, Chiba-ken, Japan. The deposit number is FERM-P No. 3372.
1010
Bifidobacterium bifidum YIT-4002 har omtrent samme egenskaber som Bifidobacterium bifidum YIT-4005, undtagen hvad angår syreresistens.Bifidobacterium bifidum YIT-4002 has about the same properties as Bifidobacterium bifidum YIT-4005, except in terms of acid resistance.
Skønt syreresistens ikke altid er påkrævet, alt efter produkternes 15 brug, er det til mange formål ønskeligt at populationen af levende 4 bifidobakterier er mindst 10 /ml efter 7 dages opbevaring ved pH 4,0 under de betingelser, som er anvendt ved tilvejebringelsen af de i ovenstående Tabel 1 viste data. Stammen YIT-4005 opfylder denne betingelse.Although acid resistance is not always required, depending on the use of the products, it is desirable for many purposes that the population of live 4 bifidobacteria be at least 10 / ml after 7 days of storage at pH 4.0 under the conditions used to provide the data shown in Table 1 above. The strain YIT-4005 meets this condition.
2020
Som tidligere nævnt er fordelene ved at anvende de nye bifidobak-teriestammer følgende: (a) de dyrkes let uden anvendelse af særlige dyrkningsbetingelser, 25 dvs. at de opformeres let under aerobe forhold uden tilsætning af vækstfremmende stoffer, (b) problemet med, at aromaen forringes af vækstfremmende tilsætningsstoffer løses, og modningen af det dyrkede produkt er enkel, 30 (c) som følge af, at det dyrkede produkt ifølge opfindelsen ikke indeholder tilsætningsstoffer, såsom vækstfremmende stoffer, er det ønskeligt som tilsætningsmiddel til spædbørnsmad, og 35 (d) de nye stammer er stort set oxygenresistente, og de næringskrav, der stilles til dyrkningen, er enkle.As previously mentioned, the advantages of using the new bifidobacteria strains are as follows: (a) they are readily grown without the application of particular culture conditions, i.e. that they are readily propagated under aerobic conditions without the addition of growth promoters, (b) the problem of the deterioration of the flavor by growth promoting additives is solved and the maturation of the cultivated product is simple, (c) as a result of the cultivated product of the invention does not contain additives, such as growth promoters, it is desirable as an infant food additive, and (35) the new strains are largely oxygen resistant and the nutritional requirements of the culture are simple.
Som følge heraf bevarer det dyrkede produkt (eller forarbejdede dyrkede produkt) den høje levedygtighed af bakterierne under opbe-As a result, the cultivated product (or processed cultivated product) maintains the high viability of the bacteria during storage.
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10 varing og kan tilsættes til levnedsmidler og drikkevarer uden væsentlig nedsættelse af bakteriernes levedygtighed.10 foods and can be added to foods and beverages without significantly reducing the viability of the bacteria.
Ved at anvende de nye bifidobakteriestammer YIT-4002 og 4005 er det 5 derfor blevet muligt at-gennemføre en effektiv dyrkning, som ikke kunne gennemføres ved anvendelse af de sædvanlige kendte bifido-bakterier og at fremstille levnedsmiddel- og drikkevaretilsætningsstoffer indeholdende levende bifidobakterier.Therefore, by using the new bifidobacteria strains YIT-4002 and 4005, it has become possible to carry out efficient cultivation which could not be carried out using the usual known bifido bacteria and to produce food and beverage additives containing live bifidobacteria.
10 Ved dyrkningen af de bifidobakterier, der anvendes ifølge opfindelsen, er det muligt at anvende aerobe forhold, som anvendes ved dyrkningen af sædvanlige mælkesyrebakterier, og som følge heraf er det muligt at gennemføre en blandet dyrkning af bifidobakterierne sammen med mælkesyrebakterier. Reducerende midler og vækstfremmende 15 stoffer, som er absolut nødvendige ved dyrkningen af sædvanlige bifidobakterier, er ik^ nødvendige ved dyrkningen af de nye bifido-bakteriestammer. Et vækstfremmende middel kan imidlertid anvendes ved fremgangsmåden ifølge opfindelsen til vækststimulering i et sådant omfang, at brugen af det dyrkede produkt ikke hindres.In the cultivation of the bifidobacteria used according to the invention, it is possible to use aerobic conditions which are used in the cultivation of usual lactic acid bacteria, and as a result it is possible to carry out a mixed culture of the bifidobacteria together with lactic acid bacteria. Reducing agents and growth promoters, which are absolutely necessary in the cultivation of conventional bifidobacteria, are not necessary in the cultivation of the new bifido bacterial strains. However, a growth promoter can be used in the method of the invention for growth stimulation to such an extent that the use of the cultivated product is not hindered.
2020
De mælkekulturmedier, som anvendes til udøvelse af opfindelsen, er et lactoseholdigt medium, såsom genfortyndet mælk, mælkevalle, helmælk, skummetmælk og lignende fremstillet ud fra disse bestanddele.The milk culture media used in the practice of the invention is a lactose-containing medium such as reconstituted milk, milk whey, whole milk, skimmed milk and the like made from these ingredients.
2525
Under sædvanlige dyrkningsforhold når populationen af levende bifidobakterier et maksimum i løbet af 18-24 timer, og pH-værdien af kulturen falder som følge af syreproduktionen under dyrkningen.Under normal culture conditions, the population of live bifidobacteria reaches a maximum within 18-24 hours and the pH of the culture decreases as a result of acid production during cultivation.
30 Hvis dyrkningen fortsættes yderligere, begynder populationen af levende bifidobakterier at falde, men syreproduktionen fortsætter endnu et stykke tid, og syren bliver mættet. Følgelig bringes dyrkningen til ophør på et passende tidspunkt, der afhænger af brugen af det dyrkede produkt. For bakterier af stammen YIT-4005 35 begynder eddikesyreproduktionen i begyndelsesstadiet af dyrkningen.30 If cultivation is continued further, the population of live bifidobacteria begins to decline, but acid production continues for a while and the acid becomes saturated. Accordingly, the cultivation is terminated at an appropriate time, which depends on the use of the cultivated product. For bacteria of the strain YIT-4005 35, acetic acid production begins at the beginning stage of cultivation.
Mol forholdet mellem eddikesyre og mælkesyre forbliver stort set konstant under dyrkningen og er 1,7 ± 0,5 (eddikesyre/mælkesyre). Så længe der anvendes levende bifidobakterier, må populationen af levende bifidobakterier være mindst /m\. Ved anvendelse afThe molar ratio of acetic acid to lactic acid remains largely constant during cultivation and is 1.7 ± 0.5 (acetic acid / lactic acid). As long as live bifidobacteria are used, the population of live bifidobacteria must be at least / m \. Using
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11 dyrkningsmetoden ifølge opfindelsen opnås let en population af g levende biiffi dobakterier af stammen YIT-4005 på mindst 10 /ml.In the method of cultivation according to the invention, a population of g live bivalve tuberculosis of the strain YIT-4005 of at least 10 / ml is readily obtained.
Det dyrkede produkt ifølge opfindelsen kan anvendes som til sæt-5 ningsmiddell til levnedsmidler og drikkevarer. F.eks. kan det dyrkede produkt nffiølge opfindelsen blandes med sødestoffer, frugtsaft, aromastoffer, vand og lignende. Omhyggeligt frysetørret eller spraytørrett materiale af det dyrkede produkt ifølge opfindelsen g indeholder 10 eller derover levende bifidobakterier pr. gram 10 tørvægt. .Det således tørrede, pulveriserede materiale er let at behandle og kan derfor anvendes til andet end ovennævnte anvendelser. For stammen YIT-4005 indeholder det dyrkede produkt et stort antal levende bifidobakterier med en særdeles god overlevelsesevne samt disses mælkemetaboliter, navnlig en blanding af organiske 15 syrer, som overvejende består af eddikesyre og mælkesyre, og det dyrkede produkt og det tørrede materiale heraf kan anvendes i forbindelse med levnedsmidler og drikkevarer med en pH-værdi på 4-7, og som ikke opvarmes til bifidobakteriernes drabstemperatur, såsom mælk, kondenseret mælk, mælkepulver, råfløde, mælk fermenteret med 20 mælkesyrebakterier, iscreme, smør, ost, grøntsagssaft, frugtsaft, sojabønnemælk, fermenteret søjabønnemælk, mayonnaise, dressing, ketchup, dej, syltetøj, fiskemel og andre specialiteter, såsom babymad og diætmad. Produktet, der indeholder de levende bifido-bakterier, blandes med disse levnedsmidler og drikkevarer 12 timer 25 inden servering eller kan iblandes under fremstillingsprocessen for disse levnedsmidler og drikkevarer, hvorved der tilvejebringes levnedsmidler og drikkevarer indeholdende levende bifidobakterier og med en behagelig aroma. Det tørrede produkt kan blandes med gærpulver, Chlorellapul ver, sukker og lignende efter ønske, eller 30 blandingen kan bringes på tabletform. Det kan også blandes med medikamenter, såsom fordøjelsesfremmende midler.The cultivated product of the invention can be used as a food additive and beverage additive. Eg. For example, the cultivated product of the invention may be blended with sweeteners, fruit juices, flavors, water and the like. Carefully freeze-dried or spray-dried material of the cultivated product of the invention g contains 10 or more live bifidobacteria per grams 10 dry weight. The powdered material thus dried is easy to treat and can therefore be used for other than the above applications. For the strain YIT-4005, the cultured product contains a large number of live bifidobacteria with a very good survivability and their milk metabolites, in particular a mixture of organic acids consisting mainly of acetic acid and lactic acid, and the cultured product and its dried material can be used. in the case of foods and beverages having a pH value of 4-7 and not heated to the killing temperature of bifidobacteria such as milk, condensed milk, milk powder, raw cream, milk fermented with 20 lactic acid bacteria, ice cream, butter, cheese juice, fruit juice, soybean milk, fermented bean milk, mayonnaise, dressing, ketchup, dough, jam, fishmeal and other specialties such as baby food and diet food. The product containing the live bifido bacteria is mixed with these foods and beverages 12 hours before serving or can be mixed during the manufacturing process for these foods and beverages to provide food and beverages containing live bifidobacteria and with a pleasant aroma. The dried product may be mixed with yeast powder, Chlorella powder, sugar and the like as desired, or the mixture may be tableted. It can also be mixed with drugs such as digestive agents.
Produktet ifølge opfindelsen anvendes som tilsætningsstof til levnedsmidler og drikkevarer og medikamenter i en sådan mængde, at 35 der opnås mindst 10^ levende bifidobakterier pr. gram (eller ml) slutprodukt, f.eks. 0,1-5% på tørvægtsbasis.The product of the invention is used as an additive for food and beverages and medicaments in an amount such that at least 10 5 live bifidobacteria are obtained. grams (or ml) of final product, e.g. 0.1-5% on a dry weight basis.
Opfindelsen vil blive belyst nærmere ved hjælp af de efterfølgende eksempler og under henvisning til tegningen, hvor:The invention will be further illustrated by the following examples and with reference to the drawings, in which:
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12 figur 1 viser forholdet mellem dyrkningstid og population af levende bifidobakterier, aciditet og pH ifølge eksempel 1, figur 2 viser forholdet mellem dyrkningstid og population af levende 5 bifidobakterier, aciditet og pH ifølge eksempel 2, og figur 3 viser forholdet mellem dyrkningstid, population af levende bifidobakterier og aciditet ved en anden aerob dyrkning som omtalt under punkt 4, side 8.Figure 1 shows the ratio of culture time to population of live bifidobacteria, acidity and pH of Example 1, Figure 2 shows the ratio of culture time to population of live 5 bifidobacteria, acidity and pH of Example 2, and Figure 3 shows the ratio of culture time to population of live bifidobacteria and acidity in another aerobic culture as discussed in section 4, page 8.
10 I eksemplerne måltes antallet af levende bakterier ifølge en éndimensional diffusionsmetode under anvendelse af et modificeret Rogosa-dyrkningsmedium, og "aciditeten" angiver den mængde 0,IN natriumhydroxid i ml, som er nødvendig til at neutralisere dyrk-15 ningsopløsningen. "Variant 1" og "Variant 2" i eksemplerne betegner de nye stammer ifølge opfindelsen, d.v.s. "Bifidobacterium bifidum YIT-4002" henholdsvis "Bifidobacterium bifidum YIT-4005".In the examples, the number of live bacteria was measured according to a one-dimensional diffusion method using a modified Rogosa culture medium, and the "acidity" indicates the amount of 0.1 N sodium hydroxide in ml needed to neutralize the culture solution. "Variant 1" and "Variant 2" in the Examples denote the novel strains of the invention, i.e. "Bifidobacterium bifidum YIT-4002" and "Bifidobacterium bifidum YIT-4005" respectively.
Eksempel 1 20 "Variant 1" og tre standardstammer af typiske bi fidusbakterier (sammenligningseksempler) dyrkedes anaerobt.Example 1 20 "Variant 1" and three standard strains of typical beeswax bacteria (comparative examples) were grown anaerobically.
Podekultur fremstilledes ved at dyrke de ønskede bakterier i et 25 modificeret Rogosa-medium (der tilsattes 1 ml pyrudruesyre og 0,2 g reduceret glutathion pr. 1 væskeformigt medium uden agar) og suspendere de opsamlede og under sterile forhold vaskede, dyrkede bakterier i en puffer-opløsning i form af 1/100 M phosphat (pH 6,8) til tilvejebringelse af en suspension (OD 660 nm = 1,5).Graft culture was prepared by growing the desired bacteria in a modified Rogosa medium (1 ml of pyruvic acid and 0.2 g of reduced glutathione per 1 liquid medium without agar) was added and suspending the collected and cultured washed bacteria in a buffer solution in the form of 1/100 M phosphate (pH 6.8) to provide a suspension (OD 660 nm = 1.5).
3030
Det til hovedpropageringen anvendte medium var reduceret ikke-fedtholdigt mælk (mælketørstofindhold 12%). Mediet steriliseredes og anbragtes i en anaerob beholder, hvis atmosfære var erstattet af en blanding af 95% nitrogengas og 5% carbondioxidgas til frembringelse 35 af den anaerobe tilstand. Mængden af opløst oxygen i mediet var 0,1 ppm ved dyrkningens begyndelse.The medium used for the main propagation was reduced non-fat milk (milk solids content 12%). The medium was sterilized and placed in an anaerobic vessel whose atmosphere was replaced by a mixture of 95% nitrogen gas and 5% carbon dioxide gas to produce the anaerobic state. The amount of dissolved oxygen in the medium was 0.1 ppm at the beginning of cultivation.
Fig. 1 viser sammenhængen mellem dyrkningstiden og variationerne i population af levende bifidobakterier, surhed og pH. For "Variant 1"FIG. Figure 1 shows the relationship between culture time and variations in population of live bifidobacteria, acidity and pH. For "Variant 1"
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13 g nåeds populationen af levende bifidobakteriiær 6,5 x 10 /ml 24 timer efter inokulering, og under forøgelse af populationen af levende bifidobakterier dannedes en betydelig mængde syre, hvorved mediets pH-værdi sænkedes. På den anden side kunne der kun spores en ringe 5 grad af formering af sammenligningsstammearne, og dette viser, at sammenligningsstammerne ikke formerer sig ii væsentlig grad i et rent ikke-fedtholdigt mælkemedium, selv under amaerobe forhold.13 g reached the live bifidobacterial population 6.5 x 10 10 / ml 24 hours after inoculation, and with increasing the live bifidobacteria population, a significant amount of acid was formed, thereby lowering the pH of the medium. On the other hand, only a slight degree of reproduction of the comparative strains could be detected, and this shows that the comparison strains do not propagate to a significant degree in a pure non-fat milk medium, even under amaerobic conditions.
Eksempel 2 10Example 2 10
Aerob dyrkning gennemførtes under anvendelse af samme podekultur og -medium som i Eksempel 1. Efter sterilisation af mediet blev oxygengas ledt gennem mediet til mætning af dette med oxygen (opløst oxygenmængde =6,5 ppm).Aerobic culture was performed using the same seed culture and medium as in Example 1. After sterilization of the medium, oxygen gas was passed through the medium to saturate it with oxygen (dissolved oxygen amount = 6.5 ppm).
1515
Resultaterne af dyrkningen er vist på fig. £. "Variant l·' propagerer på samme måde som ved ovennævnte anaerobe (dyrkning, og populationenThe results of the culture are shown in FIG. £. "Variant II propagates in the same way as for the above anaerobic culture and population
af levende bifidobakterier nåede 5,0 x 10 //ml 24 timer efter inokulering. På den anden side sporedes ingen 'Væsentlig propagering for 20 sammenligningsstammerne, men i visse tilfælde faldt populationen af levende bifidobakterier endog straks efter inokuleringen.of live bifidobacteria reached 5.0 x 10 6 / ml 24 hours after inoculation. On the other hand, no significant propagation was detected for the 20 strains of comparison, but in some cases the population of live bifidobacteria even decreased immediately after inoculation.
Eksempel 3 25 I dette eksempel studeredes forskellige betingelser for dyrkning af "Variant 1" ifølge opfindelsen.Example 3 In this example various conditions for cultivating "Variant 1" according to the invention were studied.
(I) Forhold mellem mængden af igangsætter og propagering.(I) Relationship between the amount of initiator and propagation.
30 Igangsætter fremstilledes ved dyrkning i reduceret, ikke-fedtholdigt mælkemedium (mælketørstofindhold 8¾) i 20 timer og opbevaredes ved lav temperatur i tre dage. Den således fremstillede igangsætter tilsattes til reduceret, ikke-fedtholdig mælk i forskellige mængder mellem 1 og 10% og dyrkedes aerobt. Når igangsætteren var tilsat i 35 en mængde på op til 3%, forøgedes mængden af syredannelse i forhold til forøgelsen i den tilsatte igangsættermængde. Også når igangsætteren ti Isattes i en mængde på over 3%, var mængden af syredannelse imidlertid stort set den samme. Ved et pilot-forsøg vedrørende mængden af tilsat igangsætter viste det sig, at 1-2% igangsætter er 1430 Starters were prepared by growing in reduced, non-fat milk medium (milk solids content 8¾) for 20 hours and stored at low temperature for three days. The starter thus prepared was added to reduced, non-fat milk in various amounts between 1 and 10% and cultured aerobically. When the starter was added in an amount of up to 3%, the amount of acid formation increased in proportion to the increase in the added starter amount. However, even when the initiator ten Isattes in an amount greater than 3%, the amount of acid formation was essentially the same. In a pilot study regarding the amount of added starter it was found that 1-2% starter is 14
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tilstrækkelig. Ifølge de sædvanlige beretninger er igangsætteren almindeligvis blevet anvendt i en mængde på 5-10%. For at fremstille en så stor mængde igangsætter er en igangsætter til fremstilling af "igangsætteren" nødvendig.sufficient. According to the usual reports, the initiator has generally been used in an amount of 5-10%. In order to produce such a large amount of starter, a starter for producing the "starter" is required.
5 (II) Forhold mellem temperaturen og progageringen af kulturen.5 (II) Relationship between temperature and propagation of culture.
Reduceret, ikke-fedtholdig mælk (mælketørstofindhold 16%) fremstilledes, og mediet steriliseredes derefter. Det steriliserede medium 10 afkøledes derefter til 37eC under passage gennem steriliseret luft.Reduced, non-fat milk (milk solids content 16%) was prepared and the medium was then sterilized. The sterilized medium 10 was then cooled to 37 ° C while passing through sterilized air.
2,0% af den ovenfor fremstillede igangsætter inokuleredes i mediet og dyrkedes ved henholdsvis 30eC, 34eC, 37®C, 40eC og 43°C. Propagering af kulturen ved 37®C var bedst, og i kulturen ved 34®C og 40®C var propageringen god, medens propageringerne i kulturerne ved 15 30®C og 43®C var begrænset betrageligt.2.0% of the initiator prepared above was inoculated into the medium and grown at 30 ° C, 34 ° C, 37 ° C, 40 ° C and 43 ° C, respectively. Propagation of the culture at 37 ° C was best, and in the culture at 34 ° C and 40 ° C, propagation was good, while propagation in the cultures at 15 30 ° C and 43 ° C was limited.
(III) Forhold mellem opretholdelse af populationen af levende bifidobakterier og pH.(III) Relationships between maintaining the population of live bifidobacteria and pH.
20 "Variant 1" inokuleredes i ikke-fedtholdige mælkemedier (mælketørstof indhold 12%) og dyrkedes, indtil pH-værdien for medierne nåede henholdsvis 5,0, 4,6, 4,3 og 4,1. De fire medier afkøledes hurtigt til 5®C og blev holdt herpå ved bestemmelse af variationerne i populationen af levende bifidobakterier med tiden. Resultaterne er 25 angivet i nedenstående Tabel 3 (disse talværdier er populationen af levende bifidobakterier pr. ml dyrkningsmedium).20 "Variant 1" was inoculated into non-fat milk media (milk solids content 12%) and grown until the media pH reached 5.0, 4.6, 4.3 and 4.1, respectively. The four media were rapidly cooled to 5 ° C and maintained thereon by determining the variation in the population of live bifidobacteria over time. The results are given in Table 3 below (these numerical values are the population of live bifidobacteria per ml of culture medium).
30 3530 35
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1515
Tabel 3Table 3
Dage g pH Surihed 0 3 5 7 10 5.0 9„© 4,4xl09 3,2xl09 9,6xl08 4,2xl08 6,8xl07 4,6 9.,S 5,4xl09 7,2xl08 2,3xl08 8,0xl07 l,2xl07 4,3 11.,:5 3,0xl09 1,7χ107 5,7xl05 2,6xl03 Ι,δχΙΟ1 4.1 13,,4 6,0xl08 8,5xl05 4,7xl03 dO1 <10* 15 Faldet i populationen af levende bifidobakterier med tiden varierer med mediets pH-værdi. I tilfælde af, at mediet har en pH-værdi på 4,3 eller derunder, falder populationen af levende bifidobakterier hurtigt med tiden, medens populationen af levende bifidobakterier ikke formindskes så hurtigt i tilfælde af, at mediet har en pH-værdi 20 på 4,6 eller derover. Ovenstående prøve er blevet beskrevet i forbindelse med reduceret, ikke-fedtholdig mælk, men samme resultater som ovenfor opnåedes i forbindelse med helmælk, ikke-fedtholdig mælk og blandet mælk heraf.Days of pH Acidity 0 3 5 7 10 5.0 9 9 © © 4.4x109 3.2x109 9.6x108 4.2x108 6.8x107 4.6 9., S 5.4x109 7.2x108 2.3x108 8.0x107 l, 2x107 4.3 11.: 5 3.0x109 1.7χ107 5.7x105 2.6x103 Ι, δχΙΟ1 4.1 13,, 4 6.0x108 8.5x105 4.7x103 dO1 <10 * 15 Decreased in the population of live bifidobacteria over time varies with the pH of the medium. In the event that the medium has a pH of 4.3 or less, the population of live bifidobacteria decreases rapidly with time, while the population of live bifidobacteria does not decrease as rapidly in case the medium has a pH of 20 , 6 or above. The above test has been described in connection with reduced, non-fat milk, but the same results as above were obtained in whole milk, non-fat milk and mixed milk thereof.
2g Eksempel 42g Example 4
Til fremstilling af et medium opløstes 100 g skummetmælkspulver i 900 ml vand. Efter sterilisation inokuleredes mediet med 2% af igangsætterkul turen af "Variant 2" og inokuleredes ved 37°C i 24 g 30 timer. Det således dyrkede medium havde 2,8 x 10 levende bifido-bakterier/ml, en surhed på 10,5 og et molforhold mellem eddikesyre og mælkesyre på 1,7.To prepare a medium, 100 g of skim milk powder was dissolved in 900 ml of water. After sterilization, the medium was inoculated with 2% of the starter culture of "Variant 2" and inoculated at 37 ° C for 24 g for 30 hours. The medium so grown had 2.8 x 10 live bifido bacteria / ml, an acidity of 10.5 and a mole ratio of acetic acid to lactic acid of 1.7.
Kulturen blev nedfrosset direkte og tørredes i ca. 7 timer under 3g reduceret tryk ved 2 mm Hg eller mindre. Derved fremkom 105 g tørret materiale indeholdende 5,5 x 109 levende bifidobakterier/g.The culture was frozen directly and dried for approx. 7 hours below 3g reduced pressure at 2 mm Hg or less. There were thus obtained 105 g of dried material containing 5.5 x 10 9 live bifidobacteria / g.
Eksempel 5 3% Af igangsætterkulturen af "Variant 2" inokuleredes i et medium 16Example 5 3% of the initiator culture of "Variant 2" was inoculated into a medium 16
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med en pH-værdi på 6,8 og indeholdende 3% gærekstraktpulver, 5% majsstøbevæske, 0,5% citronsyre, 0,5% ammoniumsulfat, 1% lactose og 0,03% cysteinhydrochlorid. Dyrkningen gennemførtes ved 37°C i 72 timer. Kulturen centrifugeredes, og de opsamlede bakterier suspen-5 deredes i vand indeholdende 5% skummetmælksproteasehydrolysater, 5% sukker og 1% C-vitamin. Suspensionen frysetørredes derefter, hvorved der fremkom et tørret bakteriepulver.with a pH of 6.8 and containing 3% yeast extract powder, 5% corn casting liquid, 0.5% citric acid, 0.5% ammonium sulfate, 1% lactose and 0.03% cysteine hydrochloride. The culture was carried out at 37 ° C for 72 hours. The culture was centrifuged and the collected bacteria were suspended in water containing 5% skimmed milk protease hydrolysates, 5% sugar and 1% vitamin C. The suspension was then lyophilized to give a dried bacterial powder.
10 Eksempel 6Example 6
Til fremstilling af 150 liter udgangsmateriale til iscreme opløstes 7 kg råfløde (fedtindhold 40%), 20 kg sødet kondenseret helmælk, 64 1 mælk, 2 kg skummetmælkspulver, 6 kg sukker og 0,3 kg stabilisator 15 i vand. Blandingen varmesterili seredes derefter, homogeniseredes og afkøledes til en temperatur på 40°C eller derunder.To prepare 150 liters of ice cream starting material, 7 kg of raw cream (fat content 40%), 20 kg of sweetened condensed whole milk, 64 1 of milk, 2 kg of skimmed milk powder, 6 kg of sugar and 0.3 kg of stabilizer 15 were dissolved in water. The mixture was then sterilized, homogenized and cooled to a temperature of 40 ° C or below.
Det i ovenstående eksempel 5 fremstillede tørre bakteriepulver sattes til blandingen i en mængde på 1%. Blandingen hældtes i kopper 20 og bragtes til at størkne ved -20°C, hvorved der fremstilledesThe dry bacterial powder prepared in the above Example 5 was added to the mixture in an amount of 1%. The mixture was poured into cups 20 and allowed to solidify at -20 ° C to prepare
OISLAND
iscreme indeholdende 3,0 x 10 levende bifidobakterier/ml. Optællingerne faldt til 2,2 x 10^/ml i løbet af 3. måneds opbevaring.ice cream containing 3.0 x 10 5 live bifidobacteria / ml. The counts dropped to 2.2 x 10 6 / ml during the 3 month storage.
25 30 3525 30 35
Claims (4)
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29576 | 1976-01-01 | ||
| JP29576A JPS5283975A (en) | 1976-01-01 | 1976-01-01 | Method of producing fermented milk product containg living cell of bifidus strain |
| JP29676A JPS5283974A (en) | 1976-01-01 | 1976-01-01 | Incubation mixture of milk containing living cell of bifidus strain and method of producing same |
| JP29676 | 1976-01-01 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| DK590276A DK590276A (en) | 1977-07-02 |
| DK154321B true DK154321B (en) | 1988-11-07 |
| DK154321C DK154321C (en) | 1989-06-12 |
Family
ID=26333246
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK590276A DK154321C (en) | 1976-01-01 | 1976-12-30 | ADDITIVE FOR FOOD AND BEVERAGES FOR PROVIDING FOOD BIFIDO BACTERIES THEREOF AND PROCEDURE FOR PRODUCING THE SAME AND BIFIGO BACTERIA FOR EXPELLENCE |
Country Status (4)
| Country | Link |
|---|---|
| DK (1) | DK154321C (en) |
| GB (1) | GB1535109A (en) |
| HK (1) | HK53279A (en) |
| MY (1) | MY8000028A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2159834B (en) * | 1984-05-01 | 1988-02-10 | Keith Thomas | Substrate containing microorganisms which can degrade it |
| US20100247712A1 (en) * | 2009-03-26 | 2010-09-30 | Rudolph Marvin J | Food compositions compromising dried probiotics, methods of manufacture and uses thereof |
| CN108782758B (en) * | 2018-06-22 | 2022-03-08 | 浙江忠梦昌健康科技有限公司 | Fermented synbiotic goat milk powder and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB831797A (en) * | 1955-05-28 | 1960-03-30 | Adam Schmidt Burbach | Process of preparing milk products such as sour milk by use of starter cultures |
| DE1952361A1 (en) * | 1969-10-17 | 1971-04-29 | Rudolf Hinterwaldner | Yoghurt prodn with bifdus bacteria added - to souring bacteria |
| DE2057906A1 (en) * | 1970-11-25 | 1972-06-15 | Rolf Dr Schuler | Preacidulated milk for infants - without the risk of acidosis |
-
1976
- 1976-12-29 GB GB5425976A patent/GB1535109A/en not_active Expired
- 1976-12-30 DK DK590276A patent/DK154321C/en not_active IP Right Cessation
-
1979
- 1979-08-02 HK HK53279A patent/HK53279A/en unknown
-
1980
- 1980-12-30 MY MY8000028A patent/MY8000028A/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB831797A (en) * | 1955-05-28 | 1960-03-30 | Adam Schmidt Burbach | Process of preparing milk products such as sour milk by use of starter cultures |
| DE1952361A1 (en) * | 1969-10-17 | 1971-04-29 | Rudolf Hinterwaldner | Yoghurt prodn with bifdus bacteria added - to souring bacteria |
| DE2057906A1 (en) * | 1970-11-25 | 1972-06-15 | Rolf Dr Schuler | Preacidulated milk for infants - without the risk of acidosis |
Also Published As
| Publication number | Publication date |
|---|---|
| HK53279A (en) | 1979-08-10 |
| MY8000028A (en) | 1980-12-31 |
| DK590276A (en) | 1977-07-02 |
| DK154321C (en) | 1989-06-12 |
| GB1535109A (en) | 1978-12-06 |
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