SINGLE-BREED DAIRY AND CHEESE PRODUCTS OF GREEK INDIGENEOUS SHEEP AND GOAT BREEDS

申请号 EP16386009.1 申请日 2016-06-17 公开(公告)号 EP3108751A1 公开(公告)日 2016-12-28
申请人 Skoufos, Ioannis; Tzora, Athina; Anagnostopoulos, Athanasios; Tsangaris, George; 发明人 Skoufos, Ioannis; Tzora, Athina; Anagnostopoulos, Athanasios; Tsangaris, George;
摘要 The present invention refers to the production of dairy and cheese products (e.g. yogurt, cheese etc.) through processing milk that originates from a single breed of goats or sheep, thus introducing the term "single-breed products" (SBP) or "designated breed of origin" (DBO) products. The exclusive characteristic of these products is that they definitely, and to the fullest extent, reflect/designate the special features of milk of the animal breed they originate from.
权利要求 A dairy product characterized by the fact that milk used for its production is exclusively from a single, phylogenetically and/or genetically and/or molecularly defined breed of milk-producing animal.A cheese product characterized by the fact that milk used for its production is exclusively from a single, phylogenetically and/or genetically and/or molecularly defined breed of milk-producing animal.A product, which according to claims 1 and/or 2, is characterized by the fact that milk used for its production is exclusively from a single sheep breed, e.g. milk from the Chios sheep breed, or the mountain Epirus breed (Boutsko or Boutsiko), or the Karagkouniko breed, or the Frizarta breed (Arta), or the Kalarytiko breed, or the Agrinio breed, or the Argos breed, or the Thrace breed, or the Karistos breed, or the Lesvos breed, or the Pilio breed, or the Serres breed, or the Sarakatsaniko breed, or the Sfakia breed, or the Florina breed etc. or from genetic amelioration (crossbreeding, genetic selection etc.) of the above breeds.A product, which according to claims 1 and/or 2, is characterized by the fact that milk used for its production is exclusively from a single goat breed e.g. goat milk from the Capra prisca breed etc., or from genetic amelioration (crossbreeding, genetic selection etc.) of the above breeds.A dairy product, which according to claims 1, 3 and/or 4, is bottled milk or pasteurized milk, or skim milk, or enriched milk, or milk treated by any other method (such as microfiltration, high pasteurization etc.) or sour milk, or milk butter, or colostrum etc.A dairy product, which according to the claims 1, 3 and/or 4, is a result of milk fermentation (zymosis).A dairy product, which according to claim 6, is yogurt, or yogurt-type, or dessert-type yogurt of any kind (dessert by addition of sweeteners and/or colors and/or fruits and/or fruit products or with low lipids and/or enriched yogurt etc.) or ice cream.A dairy product, which according to claims 1, 3 and/or 4 is of ice-cream type.A dairy product, which according to claims 7 and 8 is of yogurt-type ice cream.A cheese product, which according to claims 2, 3 and/or 4, is produced by fermentation of milk with rennet.A product, which according to claim 10 is of cheese type (fresh cheese, soft cheese, cream cheese, mizithra cheese, anthotiro cheese, white cheese, hard cheese, semi-hard cheese etc.).A product which, according to claim 10, is produced by further fermentation and/or maturation (and/or any other additional processing steps) and/or thermal treatment and/or placed in molds and/or undergoes maturation.A product which, according to claim 10, is characterized by the fact that rennet, used for fermentation, originates from the same animal breed as the milk used as first material.A dairy or cheese product characterized by the fact that its production is performed by processing and/or mixture of two or more milk types originating from phylogenetically and/or genetically and/or molecularly defined milk-producing animals.
说明书全文

B. FIELD OF THE INVENTION

The present invention refers to the production of dairy and cheese products (e.g. yogurt, cheese etc.) through processing milk that originates from a single breed of goats or sheep, thus introducing the term "single-breed products" (SBP) or "designated breed of origin" (DBO) products. The exclusive characteristic of these products is that they definitely, and to the fullest extent, reflect/designate the special features of milk of the animal breed they originate from.

C. BACKGROUND OF THE INVENTION

Milk and milk products, are indispensible components of human nutrition for they are rich in proteins, trace-minerals and minerals that are of fundamental importance for proper growth and health of the human organism.

Milk used for the production of cheese and dairy products must always be of the highest quality and this is achieved, only if milk-producing animals are healthy and milk itself is thoroughly examined for both its chemical and microbiological composition.

Milk collected from mastitis-infected animals or animals under antibiotic treatment is not further processed for human consumption. For the production dairy and cheese products, milk undergoes pasteurization process, through heating, in order to remove all pathogens and microorganisms such as brucella. Phosphatase testing is the defining criterion for a successful disinfection process. In following steps, depending on the end-product, milk is processed (e.g. skimming) or fermented with microbe cultures, in order to obtain fermentation products (yogurt, sour milk) or cheese products (white cheese, cream cheese, hard cheese, fresh cheese etc.), using appropriate technologies for each specific type of product.

Milk used for the production of cheese and dairy products is mostly of cow, sheep and goat origin. Depending on the desired end-product each milk type can be processed alone or through mixing of several milk types, under certain ratios. For example with regard to yogurt production, cow or sheep milk is used, but for feta cheese production according to the European product specification either sheep milk alone, or mixture of sheep (up to 70%) and goat (up to 30%) milk can be used.

Currently, there exist no dairy and/or cheese products that their production is based on the sole use of milk from a single breed of dairy animals.

The present patent application, thus, refers to a novel category of dairy and cheese products [termed: single-breed products (SBP), or designated breed origin (DBO)] possessing the dominant characteristic that the raw material used, milk, originates from a single breed of milk-producing animal.

Under this notion, it is evident that single-breed dairy products designate to the fullest extent the specific milk characteristics of each animal breed that are otherwise suppressed/left unexploited, due to milk mixing and current production techniques.

D. DESCRIPTION OF THE INVENTION

Single-breed dairy and cheese products (produced form milk of single goat or sheep breed) were unanalyzed by state-of-the-art, cutting-edge technologies; proteomics [the separation of protein by two-dimensional electrophoresis followed by protein identification by MALDI-TOF Mass Spectrometry (MS)] as well as nano-liquid chromatography-MS/MS analysis.

By application of the above technologies on milk and cheese of the indigenous Chios breed, 187 proteins were identified, while analysis resulted in 344 protein identifications for the Epirus mountain breed of Boutsko.

Analysis of Karagouniko breed milk/cheese resulted in identification of 226 proteins, and for milk/cheese of the indigenous goat breed of Capra prisca 217 proteins were identified

Comparative study of the results revealed that 187 proteins are exclusively expressed in milk/cheese of Chios breed, compared to the other indigenous sheep breeds. In the milk of Boutsko breed 344 proteins are exclusively expressed in comparison to other breeds, while 118 proteins are exclusively present in milk of the Karagouniko breed. Further, 163 proteins are exclusively expressed in milk of the indigenous goat breed of Capra prisca, compared to other breeds.

Milk used for the production of cheese and dairy products must always be of the highest quality and this is achieved, only if milk-producing animals are healthy and milk is thoroughly examined regarding both its chemical and microbiological composition. Optionally, in certain cases, microorganisms are used in order to provide special taste in the end-product, as well as in helping its maturation process.

For the production of single-breed dairy products, milk originating exclusively from a single breed of milk-producing animal (sheep or goat) is first pasteurized in 71-73°C for 15 seconds or in 65°C for 25 minutes. Secondly, depending on the end-product milk can be packed or be further processed (skimming), or frozen for the production of ice cream. For production of yogurt, milk is either pasteurized or boiled to 82-85°C, and following, after cooling to 42-45°C, it is fermented for 2-3 hours with yogurt yeast or a small yogurt quantity.

For the production of single-breed cheese products, milk originating exclusively from a single breed of milk-producing animal is first pasteurized in 71-73°C for 15 seconds or in 65°C for 25 minutes and then is coagulated with specific amount of rennet in certain temperature, depending on cheese type and cheese quality. In order to sustain coagulation, before the addition of rennet, milk is cooled and then a special lactic acid culture of microorganisms is added to the mixture (1-2% v/v, depending on cheese type). This maturation step usually lasts for 10 minutes, while optionally 8-10 grams of calcium chloride diluted in water or 20 ml of calcium chloride (40% v/v), are added to 100 kg or liters of milk. Further, for soft cheese production milk is coagulated at 28-30°C for 60 minutes, and regarding semi-hard cheese, milk is coagulated with rennet at 32-35°C for 30-50 minutes. Milk originating exclusively from a single animal breed can be used in cheese production without any prior pasteurization process, as long as milk has been tested to be of highest quality and pathogen-free. Furthermore, coagulation of milk originating exclusively from a single animal breed can be performed by using rennet from same animal breed as the one milk originates from. In this specific case lactic acid culture can be avoided.

Further, depending on cheese type end-product, custard-like mass undergoes relevant treatment. In the case of soft cheese production following coagulation, the curd is cut into small pieces in order to separate the liquid (whey), then put to molds (different shapes and sizes) and drained for relevant time periods, in order to achieve the desired final texture and humidity of the product.

Thus, in the case of white cheese production, the curd is cut in cubes 15-20 mm in size and is being softly stirred. Then it is left to rest (approximately for 10 minutes) and put in molds with small holes in order to drain. Following sufficient drainage and cheese is solidified, the mold is taken off and cheese is cut in pieces that are out to a special surface and salted at different time intervals, for 6-12 hours. Cheese pieces are salted on both sides and each side is salted twice. Cheese is left on the specific table for few more days in order for fermentation to begin. Optionally, following salting procedure cheese can be stored in containers in 16-18°C, and containers cannot be sealed until after 15-20 days. When fermentation commences, containers are filled with brine 8-10%. Following completion of fermentation containers are sealed and stored at 4°C.

For production of hard cheese, the curd is cut and pieces are stirred while being re-heated at 42-50°C for 35-40 minutes. After removal of whey, the curd is cut to pieces depending on cheese type (heads, sticks etc.) and put into specific molds under pressure. Pressure is gradually increased for 8-24 hours, while molds are constantly moved in order for curd to be void of liquids. Next, when curd is taken off pressure, it is placed in maturation chamber at 16-18°C under 85-90% humidity and salted for predefined time periods, depending on cheese type. Alternatively, the curd can be salted in brine 20-22% at 10-12°C for 3-4 days. Cheese, then, is left to undergo maturation and at the end of this step, cheese is covered with a paraffin layer and kept in freezing conditions.

By implementing the above procedures, single-breed cheese, or single-breed white cheese has been produced, by using milk, exclusively from phylogenetically and/or genetically and/or molecularly defined sheep and/or goats of the following breeds: (i) Boutsko breed (mountain breed of Epirus), (ii) Frizarta breed (Arta) and (iii) Capra prisca goat breed (indigenous greek goat breed).

Furthermore, by implementing the above procedures, single-breed hard cheese has been produced, by using milk, exclusively from phylogenetically and/or genetically and/or molecularly defined sheep and/or goats of the following breeds: (i) Boutsko breed (mountain breed of Epirus), (ii) Frizarta breed (Arta) and (iii) Capra prisca goat breed (indigenous greek goat breed).

In order to investigate the characteristics of each type of single-breed cheese, cheese samples were analyzed by means of proteomics. In greater detail, a small cheese sample from each cheese type is rehydrated for 8-24 hours in urea/thiourea buffer and then sample is homogenized both mechanically and by ultrasound dissociation. The mixture is then centrifuged and following centrifugation the protein content is quantitatively measured in the supernatant. Next, 5 mg of total protein from the supernatant are reduced and alkylated via specific buffers and are subjected to digestion by trypsin for 6-8 hours. Finally, proteins are identified by specific software, analyzing the peptides produced in the previous step of tryptic digestion. Through analysis of the results, as can be seen in Table I, 46 proteins are exclusively present in goat cheese of Capra prisca breed, 52 proteins are exclusively present in cheese from sheep of the Boutsko breed and 37 proteins are exclusively present in cheese from sheep of the Frizarta breed.

The above results safely lead to the conclusion that dairy and cheese products originating from milk of exclusively one breed of phylogenetically and/or genetically and/or molecularly defined milk-producing animals (sheep or goats), present and designate special milk characteristics that are tightly correlated to each specific breed of animal.

<b>TABLE 1</b>

Carpa prisca

Boutsko (Epirus mountain breed)

Frizarta (Arta breed)

Protein Symbol

Protein Name

Protein Symbol

Protein Name

Protein Symbol

Protein Name

MUC15

Mucin-15

CATB

Cathepsin B

CNNM4

Metal transporter CNNM4

NUCB1

Nucleobindin-1

CTHL1

Cathelicidin-1

PININ

Pinin

URP2

Fermitin family homolog 3

CERU

Ceruloplasmin

SNX29

Sorting nexin-29

HSP7C

Heat shock cognate 71 kDa protein

LMNA

Prelamin-A/C

TGFB2

Transforming growth factor beta-2

MYBPP

MYCBP-associated protein

IRS1

Insulin receptor substrate 1

SEMG2

Semenogelin-2

OSTN

Osteocrin

CCPG1

Cell cycle progression protein 1

SCTR

Secretin receptor

NSF1C

NSFL1 cofactor p47

CADH1

Cadherin-1

ST18

Suppression of tumorigenicity 18 protein

ADAM2

Disintegrin and metalloproteinase domain-containing protein 2

CALR

Calreticulin

NUAK1

NUAK family SNF1-like kinase 1

AGRIN

Agrin

SPA34

Serpin A3-4

PRAX

Periaxin

DLGP5

Disks large-associated protein 5

IFM2

Interferon-induced transmembrane protein 2

TLR2

Toll-like receptor 2

STXB3

Syntaxin-binding protein 3

FGFP1

Fibroblast growth factor-binding protein 1

TKN4

Tachykinin-4

ANGT

Angiotensinogen

SCG1

Secretogranin-1

CD36

Platelet glycoprotein 4

MMP23

Matrix metalloproteinase-23

TYB10

Thymosin beta-10

RET4

Retinol-binding protein 4

KI13A

Kinesin-like protein KIF13A

UBP25

Ubiquitin carboxyl-terminal hydrolase 25

FABP6

Gastrotropin

PIBF1

Progesterone-induced-blocking factor 1

MGP

Matrix Gla protein

AP2D

Transcription factor AP-2-delta

PLXA1

Plexin-A1

PDC6I

Programmed cell death 6-interacting protein

CLH2

Clathrin heavy chain 2

FAS

Fatty acid synthase

TRIP4

Activating signal cointegrator 1

CTND1

Catenin delta-1

FAT2

Protocadherin Fat 2

TXD12

Thioredoxin domain-containing protein 12

EFNB1

Ephrin-B1

ANR26

Ankyrin repeat domain-containing protein 26

CAN10

Calpain-10

GKN1

Gastrokine-1

AQP6

Aquaporin-6

DEFB1

Beta-defensin 1

IFRD1

Interferon-related developmental regulator 1

DYH10

Dynein heavy chain 10, axonemal

DYH6

Dynein heavy chain 6, axonemal

SYNE1

Nesprin-1

EDF1

Endothelial differentiation-related factor 1

DYH7

Dynein heavy chain 7, axonemal

TEN3

Teneurin-3

FBF

Fas-binding factor 1

DYH9

Dynein heavy chain 9, axonemal

EPN2

Epsin-2

FSIP2

Fibrous sheath-interacting protein 2

LMOD2

Leiomodin-2

FGFR4

Fibroblast growth factor receptor 4

MOES

Moesin

M3K15

Mitogen-activated protein kinase kinase kinase 15

LRP2

Low-density lipoprotein receptor-related protein 2

NBEL2

Neurobeachin-like protein 2

O10J4

Olfactory receptor 10J4

NEST

Nestin

OR4KD

Olfactory receptor 4K13

O5112

Olfactory receptor 5112

PTHR

Parathyroid hormone-related protein (Fragment)

OSBP2

Oxysterol-binding protein 2

052L1

Olfactory receptor 52L1

CETN2

Centrin-2

PALD

Paladin

RDH11

Retinol dehydrogenase 11

CLHC1

Clathrin heavy chain linker domain-containing protein 1

ZN674

Zinc finger protein 674

SAP

Prosaposin

CRFR2

Corticotropin-releasing factor receptor 2

ZN880

Zinc finger protein 880

SYPL1

Synaptophysin-like protein 1

FGRL1

Fibroblast growth factor receptor-like 1

GUC2D

Olfactory guanylyl cyclase GC-D

TEKT5

Tektin-5

HYCCI

Hyccin

ICA1L

Islet cell autoantigen 1-like protein

TITIN

Titin

ITSN2

Intersectin-2

NF1

Neurofibromin

CADH6

Cadherin-6

NPYSR

Neuropeptide Y receptor type 5

PLST

Plastin-3

DOK1

Docking protein 1

SPG20

Spartin

AL1A1

Retinal dehydrogenase 1

FNBP1

Formin-binding protein 1

TR134

Taste receptor type 2 member 134

APOE

Apolipoprotein E

SPRL1

SPARC-like protein 1

ARBK2

Beta-adrenergic receptor kinase 2

CATD

Cathepsin D (Fragment)

CASPC

Caspase-12

CP2R1

Vitamin D 25-hydroxylase

GANAB

Neutral alpha-glucosidase AB

EZRI

Ezrin

HSP7E

Heat shock 70 kDa protein 14

GELS

Gelsolin

KLH41

Kelch-like protein 41

IFNA4

Interferon alpha-4

MRP7

Multidrug resistance-associated protein 7

IL1B

Interleukin-1 beta

SAA2

Serum amyloid A-2 protein

JPH3

Junctophilin-3

T2EB

General transcription factor IIE subunit 2

MNS1

Meiosis-specific nuclear structural protein 1

UBR4

E3 ubiquitin-protein ligase UBR4

MUC1

Mucin-1

[NUP37

Nucleoporin Nup37

PLEC

Plectin (Fragment)

PPR18

Phostensin

SDF2

Stromal cell-derived factor 2

TRFM

Melanotransferrin

VTNC

Vitronectin

E. ADVANTAGES OF THE CURRENT INVENTION

Currently dairy and cheese products are produced mainly by use of cow, sheep or goat milk. Depending on the end-product, cow, sheep or goat milk are used alone or in mixture, under certain ratios. However, while for most products, the animal species that milk originates from is described, never does milk description includes the animal breed. Currently, due to progress and advancement in scientific technologies, it has been found that milk originating form certain breeds of milk-producing animals that have been phylogenetically and/or genetically and/or molecularly investigated, has definite properties that are in tight junction with the characteristics of its breed of origin. Therefore, milk from each animal breed has contents (proteins, metabolites, lipids etc.) differing in their concentration from breed to breed. Certain proteins, lipids, and metabolites may be present in one breed of animals, while absent from another, or existing under different quantities in each breed. These specific biological factors give SBP special features (in tight correlation with the animal breed), that are responsible for SBP bioactive properties, and therefore for health benefits (anti-hypertensive, lipid regulation, anti-athyatheromatic and anticancer) for the human organism. It can, thus, be said that by the dairy product and cheese production procedure followed so far, the special features of milk of each animal breed are left suppressed in the final product and therefore are unexploited.

The present invention refers to the production of a series of dairy and cheese products based on processing milk that originates exclusively from a phylogenetically and/or genetically and/or molecularly defined animal breed, thus unraveling the superior milk features that would otherwise be suppressed.

Given the vast geographical dispersion of rural rearing for animal breeds (e.g. Chios breed animals are reared in Western Macedonia, Boutsko breed animals are reared in Epirus and Thessaly, Skopelos breed animals are reared in Magnesia and Sporades), the production of SBP is independent of the rearing region as well as the geographic origin of milk from each breed.

The present invention describes a novel category of dairy and cheese products [termed: single-breed products (SBP), or designated breed of origin (DBO) products], designed to include a single type of milk originating from one phylogenetically and/or genetically and/or molecularly defined animal breed. Therefore, the special milk features of each animal breed separately, that are now blocked due to current dairy and cheese product engineering (milk mixing), are being revealed.

Z. DETAILED DESCRIPTION OF AN APPLICATION

a) Production of single-breed feta cheese from sheep milk of Boutsiko or Boutsko breed (mountain breed of Epirus).

Milk is first tested and verified, by application of mass spectrometry, to confirm its breed of origin (Boutsko) and its specific characteristics.

The single-breed mass spectrometry-verified milk is then pasteurized in 71-73°C for 15 seconds, or in 65°C for 25 minutes. Next milk is refrigerated and a lactic acid culture is added (1-2% v/v, depending on cheese type). This maturation step lasts for 10 minutes, while optionally 8-12 grams of calcium chloride diluted in water or 20 ml of calcium chloride (40% v/v), are added to 100 kg or liters of milk.

Next, for the production of feta, white and soft cheese, milk is coagulated with certain amount of rennet in 28-30°C for 55 minutes. Besides using commercially available rennet, milk coagulation can be performed with rennet from the same animal breed as the one milk originates from.

Following, the curd is cut in 15-20 mm size cubes and is softly stirred. Then is left to rest (approximately for 10 minutes) and put in molds with holes in order to drain, for 5-6 hours depending on the season. When cheese is drained enough turned solid, the mold is taken off, and cheese is cut into pieces.

Cheese pieces are placed on a special table and are periodically salted by thick salt for 6-12 hours. Cheese pieces are salted in such manner that each side is salted at least two times.

Cheese is left on the special table for few more days in order for fermentation to begin, while daily turned on each side. Optionally following salting procedure cheese can be stored in containers in 16-18°C, and these can be sealed after 15-20 days at 16-18°C. When fermentation commences, containers are filled with brine 8-10%. Following completion of fermentation containers are sealed and stored at 4°C.

b) Production of single-breed hard cheese from sheep milk of Boutsiko or Boutsko breed (mountain breed of Epirus).

Milk is first tested and verified, by application of mass spectrometry, to confirm its breed of origin (Boutsko) and its specific characteristics.

The single-breed mass spectrometry-verified milk is then pasteurized in 71-73°C for 15 seconds, or in 65°C for 25 minutes. Next milk is refrigerated and a lactic acid culture is added (1-2% v/v, depending on cheese type). This maturation step lasts for 10 minutes, while optionally 8-12 grams of calcium chloride diluted in water or 20 ml of calcium chloride (40% v/v), are added to 100 kg or liters of milk.

Next, milk is coagulated with certain amount of rennet in 32-38°C for 30-50 minutes. Besides using commercially available rennet, milk coagulation can be performed with rennet from the same animal breed (Mputsko breed) as the one milk originates from.

The curd is cut into pieces and pieces are constantly stirred while being reheated at 42-50°C for 35-40 minutes. Then, after removal of whey, the curd is cut to pieces depending on cheese type (heads, sticks etc.) and into specific molds, under pressure. Pressure is gradually increased for 8-24 hours, while molds are constantly moved in order for curd to be void of liquids. Next, when curd is taken off pressure, it is placed in a maturation chamber at 16-18°C under 85-90% humidity and is being salted for predefined time periods, depending on end-product cheese type. Alternatively, the curd can be salted in brine 20-22% at 10-12°C for 3-4 days. Cheese, then, is left to undergo maturation and at the end of this step, cheese is covered with paraffin or other protective layer and is kept under freezing conditions..

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