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Title:
PRODUCTION OF FERMENTED MILK PRODUCTS FROM NATURAL ISOLATE STARTER CULTURES
Document Type and Number:
WIPO Patent Application WO/2021/080537
Kind Code:
A1
Abstract:
With the invention, it is possible to commercialize Streptococcus thermophilus and Lactobacillus bulgaricus bacteria, which are naturally found in local village yogurts in Anatolia, after isolation and purification, and with a process designed for the use of starter cultures containing different combinations of these bacteria, creamy yogurt, homogenized yogurt, fermented milk products such as yogurt and ayran with different fat/dry matter contents are produced. With the invention, by using completely natural isolates isolated from yogurt taken from the local villages of Anatolia, which does not have any modifications; a sustainable method has been developed that can be applied industrially within the scope of mass production and is suitable for the standard structure and taste that consumers in our country want to see in yogurt. With the invention, natural fermented milk products are produced with starter cultures known as yeast/living yogurt cultures in colloquial language and TGK Nutrition and Health Declarations Regulation.

Inventors:
ŞAHİNER MEHMET (TR)
Application Number:
PCT/TR2020/050956
Publication Date:
April 29, 2021
Filing Date:
October 19, 2020
Export Citation:
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Assignee:
PANAGRO TARIM HAYVANCILIK GIDA SAN VE TIC A S (TR)
International Classes:
A23C9/123; C12N1/20; C12R1/225; C12R1/46
Attorney, Agent or Firm:
YALCINER, Ugur G. (YALCINER PATENT & CONSULTING LTD.) (TR)
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Claims:
CLAIMS

1. Starter culture comprising natural lactic acid bacteria, characterized in that said lactic acid bacteria comprises at least one isolate Lactobacillus delbrueckii subsp. bulgaricus selected from the following group; i. Lactobacillus delbrueckii subsp. bulgaricus PNGR _B1 MN791118 represented by 16S rRNA gene sequence SEQ ID NO.l, Lactobacillus delbrueckii subsp. bulgaricus PNGR_B2 MN809269 represented by 16S rRNA gene sequence SEQ ID N0.2, Lactobacillus delbrueckii subsp. bulgaricus PNGR _B6 MN792802 represented by 16S rRNA gene sequence SEQ ID NO.3, Lactobacillus delbrueckii subsp. bulgaricus PNGR_B8 MN811207 represented by 16S rRNA gene sequence SEQ ID NO.4, Lactobacillus delbrueckii subsp. bulgaricus PNGR_B14 MN812281 represented by 16S rRNA gene sequence SEQ ID NO.5 ile temsil edilen and Lactobacillus delbrueckii subsp. bulgaricus PNGR_B36 MN812658 represented by 16S rRNA gene sequence SEQ ID NO.6 and at least one isolate Streptococcus thermophilus selected from the following group; ii. Streptococcus thermophilus PNGR_K1 MN812674 represented by 16S rRNA gene sequence SEQ ID NO.7, Streptococcus thermophilus PNGR_K2 MN812707 represented by 16S rRNA gene sequence SEQ ID NO.8, Streptococcus thermophilus PNGR_K5 MN814043 represented by 16S rRNA gene sequence SEQ ID NO.9, Streptococcus thermophilus PNGR_K13 MN814030 represented by 16S rRNA gene sequence SEQ ID NO.10, Streptococcus thermophilus thermophilus PNGR_K31 MN814032 represented by 16S rRNA gene sequence SEQ ID NO.11 and Streptococcus thermophilus PNGR_K55 MN812789 represented by 16S rRNA gene sequence SEQ ID NO.12.

2. A fermented milk product, characterized by comprising a starter culture comprising natural lactic acid bacteria according to claim 1.

3. A fermented milk product according to claim to 2, characterized by comprising isolat Q Lactobacillus delbrueckii subsp. bulgaricus PNGR_B1 MN791118 and at least one isolate Streptococcus thermophilus selected from the group consisting of PNGR K1 MN812674, PNGR _K2 MN812707, PNGR _K5 MN814043, PNGR K13 MN814030, PNGR K31 MN814032, PNGR_K55 MN812789. 4. A fermented milk product according to claim to 2, comprising isolate Lactobacillus delbrueckii subsp. bulgaricus PNGR_B2 MN809269 and at least one isolate

Streptococcus thermophilus selected from the group consisting of PNGR_K1 MN812674, PNGR K2 MN812707, PNGRJC5 MN814043, PNGR_K13 MN814030, PNGR K31 MN814032, PNGR_K55 MN812789.

5. A fermented milk product according to claim to 2, comprising isolate Lactobacillus delbrueckii subsp. bulgaricus PNGR_B6 MN792802 and at least one isolate

Streptococcus thermophilus selected from the group consisting of PNGR_K1 MN812674, PNGR K2 MN812707, PNGRJC5 MN814043, PNGR K13 MN814030, PNGR K31 MN814032, PNGRJC55 MN812789.

6 A fermented milk product according to claim to 2, comprising isolate Lactobacillus delbrueckii subsp. bulgaricus PNGR_B8 MN811207 and at least one isolate

Streptococcus thermophilus selected from the group consisting of PNGR_K1

MN812674, PNGR K2 MN812707, PNGRJC5 MN814043, PNGR_K13 MN814030, PNGR K31 MN814032, PNGR_K55 MN812789.

7. A fermented milk product according to claim to 2, comprising isolate Lactobacillus delbrueckii subsp. bulgaricus PNGR_B14 MN812281 and at least one isolate

Streptococcus thermophilus selected from the group consisting of PNGR_K1

MN812674, PNGR K2 MN812707, PNGRJC5 MN814043, PNGR K13 MN814030, PNGR K31 MN814032, PNGR K55 MN812789 .

8 A fermented milk product according to claim to 2, comprising isolate Lactobacillus delbrueckii subsp. bulgaricus PNGR_B36 MN812658 and at least one isolate Streptococcus thermophilus selected from the group consisting of PNGR_K1

MN812674, PNGR K2 MN812707, PNGRJC5 MN814043, PNGR_K13 MN814030, PNGR K31 MN814032, PNGR K55 MN812789 .

9. A fermented milk product, which is yogurt according to claim 2.

10. A fermented milk product, which is ayran according to claim 2.

11. Yogurt according to claim 9, wherein the acetaldehyde concentration is in the range of 8-14 ppm.

12. A method of producing a fermented milk product, comprising the following process steps of: i. Regenerating bacteria mentioned in claim 1 and inoculating at the ratio of 1-3% into media and pre-propagating thereof, ii. Preparation of the intermediate culture by inoculating the pre-propagated culture separately into the intermediate culture medium such that the bacteria are present as 2- 5% by volume (1-2.5% Bacillus, 1-2.5% Cocci), iii. Inoculation of the intermediate culture into pasteurized and standardized milk at a rate of 2-3% by volume.

13. A method according to claim 12, wherein a hepa filter is used in the steps of propagation and inoculation of bacteria.

Description:
PRODUCTION OF FERMENTED MILK PRODUCTS FROM NATURAL ISOLATE

STARTER CULTURES

Technical Field

The invention is related to the method of producing fermented milk products, such as yogurt, ayran (yogurt drink), etc. with the starter culture combinations that are created by taking into account the final product structure and the determined aroma profiles of lactic acid bacteria purified by isolating from local village yogurts (from home-made yogurts supplied from different regions) in Anatolia.

Known State of Art (Prior Art)

The use of milk and milk products is chronologically very old. Yogurt known as fermented milk product has been used for many years. According to the standard 1330 of the Turkish Standards Institute; yogurt was defined as “a fermented milk product which is obtained as a result of lactic acid fermentation of milk, complying with raw milk or pasteurized milk standards, preferably homogenized milk with the effect of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus and it contains living yogurt cultures”.

With the fermentation of lactic acid bacteria, milk products (cheese, yogurt, butter, kefir, kumiss, etc.) are provided with a unique aroma, smell and structure. Formulations formed by microorganisms composed of lactic acid bacteria or combinations of microorganisms are called "starter cultures". In fermentation, aroma and/or flavor components, such as product specific lactic acid, acetaldehyde, etc. are formed by adding starter cultures to milk. Thus, fermented milk products are obtained. Today, in various geographical regions around the world, yogurt production, ayran production, etc., production of fermented milk products are carried out with starter cultures prepared with different strains of lactic acid bacteria to appeal to taste of that region.

Lactic acid bacteria are used as starter culture in making yogurt. Lactic acid bacteria are characterized in that they form mainly lactic acid (90%) by hydrolyzing lactose in milk in a homofermentative way. Lactic acid bacteria used in traditional fermented foods are Lactobacillus, Lactococcus, Tetragonococcus, Vagococcus, Weis sella, Streptococcus, Leuconostoc, Aerococcus, Oenococcus and Pediococcus genus bacteria included in Streptococcaceae and Lactobacillaceae families. Methods based on Polymerase Chain Reaction (PCR) using primers targeting 16S rRNA and/or 23 S rRNA genes in identification of lactic acid bacteria are used in identification of Lactobacillus species.

In the prior art, the production of cultures/bacteria used in milk products designed to meet the desired parameters in the final product, such as taste, aroma, texture, etc. is known. Although the use of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus bacteria in yogurt production by isolation is known in the prior art, there is a need for a sustainable method which can be applied industrially within the scope of mass production using completely natural isolates that has not been modified, by being isolated from yogurts taken from the local villages of Anatolia and which is suitable for the standard structure and taste that consumers in our country want to see in yogurt.

In the prior art, comparative analyzes were made on yogurts produced using Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus bacteria and yogurts made using only commercial culture.

The cultures tried in 3 different combinations in yogurt production are as follows;

1) Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus bacteria isolated from local village yogurts of Anatolia were used by mixing with commercial cultures in different proportions.

2) Only commercial culture was used.

3) Only isolated pure strains Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus isolated from local village yogurts of Anatolia were used.

Tests were carried out on yogurts produced with the above combinations and the viscosity of yogurt produced using only isolated pure strains in the prior art was found to be slightly higher than the others and said sample was not liked by the panelists as a result of the sensory evaluation. As can also be understood from this comparative analysis, in the state of the art, the standard structure and taste that consumers in our country want to see in yogurt cannot be achieved with products containing starter cultures using only isolated pure strains. For this reason, it is known that most of the producers prefer commercial cultures where mutation and/or recombination mechanisms are used in fermented milk products production.

In order for lactic acid bacteria to be used as starter culture, their industrial properties should be well defined. The methods used in commercial starter culture production are very important in terms of production and quality. The commercially available commercial cultures are produced in the liquid, powder and frozen form depending on the production methods.

There is a bacteriophage risk in all production methods using lactic acid bacteria. However, it is known that starter culture producers with their own know-how use mutation and/or recombination mechanisms on bacteria in order to make commercial starter cultures resistant to bacteriophage risk and to create structure and taste alternatives in fermented milk products to meet consumers' expectations.

It is known that the aroma profiles and structure of imported commercial cultures are different from purified starter cultures. These commercial cultures are offered for sale as alternatives in order to provide the structure and aroma suitable for expectations by being imported to producers of milk products in our country. However, the products produced in this way cannot meet the structure and aroma suitable for the traditional taste of consumers in Turkey.

There is no above-mentioned commercial starter culture production can meet the industrial needs in our country. So, there is an external dependence on starter cultures. There is a need for natural starter cultures with suitable combinations for sustainable production of fermented milk product varieties, such as yogurt, ayran, etc. which have local and traditional tastes/flavor suitable for taste of the consumers in our country without external dependence. There is a need to develop standardized industrial production methods in order to increase their diversity by determining the genotypes of microorganisms in local fermented foods, to maintain their continuity by preservation and to obtain products with aroma/flavor profiles and structure specific to our milk products produced by natural methods in the past. Summary and Aims of the Invention

With the invention, a process designed for these cultures that are ready for use after isolation and purification of lactic acid bacteria found naturally in local village yogurts in Anatolia or can be commercialized is carried out for production of fermented products, such as creamy/skimmed yogurt, ayran with different ingredients, such as protein, fat, dry substance, etc. Natural fermented milk products are produced with starter cultures known as yeast/living yogurt cultures in colloquial language and TGK Nutrition and Health Declarations Regulation.

The most important aim of the invention is to reduce foreign dependency by producing inputs such as starter culture, yeast, etc. that we are dependent on outside in our country by our own means. In addition, with the invention, it is aimed to produce unique products with a structure and taste suitable for Turkish culture with culture and yeast isolated and purified from local yogurts of Anatolia.

With the invention, bacteria whose genotypes are isolated and purified under controlled conditions are used, and these bacteria are backed up and propagated in the laboratory and on an industrial scale. By standardizing the processes in which these bacteria are used, it is possible to produce 100% natural yogurt and ayran efficiently at the same quality and sustainably every time.

Also, unlike the products which were not appreciated in the prior art due to the use of isolated pure cultures; with the invention, the products suitable for tastes of Turkey can be produced only by the process made suitable for production and the combinations of appropriate combinations. At the same time, by means of the natural isolate bacteria used in the invention, a sustainable production can be achieved by repeated use of the produced products.

The invention ensures that all process steps in production are carried out in the most sterile way by means of the hepa filter system used in the production method. In this way, it is possible to produce 100% natural fermented milk products by making an effective application in order to avoid impurity/contamination problems in cultures.

Unlike yogurt production methods performed in the laboratory environment and/or at home in the prior art, which are not suitable for mass and standard production, the inventive method of producing fermented milk products from natural isolate starter cultures makes it possible to produce standard products each time with the process designed for only this process. Definitions of Drawings Illustrating the Invention

The figures which were prepared in order to better understand the yogurt production method developed with the invention are explained below in flow chart format. Figure 1: Natural Isolate Replication Starter Culture Laboratory Flow Chart Figure 2: Natural Isolate Replication Starter Culture Production Flow Chart Figure 3: Production Flow Chart for Homogenized Yogurt with Natural Isolate Replication Starter Culture

Figure 4: Flow Chart of Ayran Production with Natural Isolate Replication Starter Culture Figure 5: Natural Isolate Streptococcus thermophilus Microscope Image Figure 6: Natural Isolate Lactobacillus bulgaricus Microscope Image

Definitions of Elements/Pieces/Parts Forming the Invention KHT Tank: Culture Preparation Tank

TKM Control: Total Solid Matter Control YKM-FAT Cont: Fat-Free Solid Matter - Fat Control Holder: Starter Culture Stock Tank

M: Natural Isolate Replication Starter Culture Production Flow Chart P: Pre-pasteurization Production Flow Chart

K: Cocci ( Streptococcus thermophilus)

B: Bacillus (Lactobacillus bulgaricus)

Detailed Description of the Invention

The invention is a natural yogurt production method with combinations created on aroma profiles determined by isolating and purifying lactic acid bacteria naturally found in the microbiota of homemade yogurts unique to our country (Anatolia). Streptococcus thermophilus (S. thermophilus) and Lactobacillus bulgaricus (L. bulgaricus ) lactic acid bacteria strains isolated from yogurt samples collected from Anatolia were confirmed by molecular analysis (16 S rRNA, 23 S rRNA, ITS, full genome etc. sequence analysis methods). The inventive the method of producing fermented milk products from natural isolate starter cultures includes the steps performed in the culture laboratory and the large-scale automated process steps in which these steps are transferred to the production line.

Firstly, the process of invention includes the step of regeneration (development, growth, propagation, etc.) and storage by backing-up of the following lactic acid bacteria, which have been isolated and whose purity has been verified, using medium, yeast extract, milk powder or pasteurized milk under appropriate conditions in the culture laboratory:

Lactobacillus bulgaricus, Bacillus: PNGR Bl (Bl), PNGR B2 (B2), PNGR B6 (B6), PNGR B8 (B8), PNGR B14 (B14), PNGR B36 (B36)

Streptococcus thermophilus , Cocci: PNGR Kl (Kl), PNGR K2 (K2), PNGR K5 (K5), PNGR K13 (K13), PNGR K31 (K31), PNGR K55 (K55). Then, live cultures/yeast with suitable combinations for industrial production are prepared based on determined parameters, such as temperature, pH, acidity, time, amount etc. in the laboratory. These cultures are made ready for use in the process by bringing them to the desired dry material in the laboratory and pre-propagation in appropriate pasteurized media (intermediate culture) (Figure 1). Then, the intermediate cultures with suitable combinations, which were pre-propagated in the previous step, are made ready for use in production by large-scale propagation (culture stock) (Figure 2).

In the last stage, the production process of fermented milk products such as yogurt and ayran is completed by infusing the bacteria that are propagated on a large scale in the specified ratio and/or amount to the standardized, concentrated/non-concentrated pasteurized milk.

With the invention, creamy/skimmed yogurt, homogenized yoghurt, fermented milk products with different fat/dry matter contents can be produced. The steps of the invention performed in the culture laboratory (Figure 1) and the steps performed on the production line (Figure 2, Figure 3, Figure 4) are described in detail below.

1. THE STEPS PERFORMED IN THE CULTURE LABORATORY In Figure 1, Natural Isolate Replication Starter Culture Laboratory Flow Chart is shown. In the production of Natural Isolate Replication Starter Culture in the laboratory, stock Streptococcus thermophilus and Lactobacillus bulgaricus bacterial cultures taken from -86°C are inoculated into Ml 7 or MRS broth medium sterilized for 15 minutes at 121 °C at a rate of 1-2% by volume. Incubation conditions are 18-24 hours at 42°C, storage conditions are 2 days at 2-4°C. Broth media sterilized at 121°C for 15 minutes during the transition to stock culture, are mixed with sterile glycerol. Stock cultures are prepared by adding the bacteria that propagate after incubation at a rate of 2-3% by volume to the medium with 40% by volume glycerol and can be stored at -86°C for 12 months. After the stock is taken, the remaining propagated bacteria are inoculated at a rate of 1-2% by volume into these media prepared after sterilization at 95°C for 15 minutes for intermediate culture. The total dry matter value (TKM) of the media prepared for intermediate culture should be in the range of 5-7% by weight. The incubation conditions of bacteria inoculated into media are ~6 hours at 42°C. When the pH is ~4.6, it is conditioned under room conditions for 10-15 minutes, and then it is cooled at 4-6°C and stored as an intermediate (bulk) culture at 4-6°C for 2-4 days.

The process steps specified in the Natural Isolate Replication Starter Culture Laboratory flow chart are detailed below.

Preparation of Appropriate Media

After the isolation, identification and purification of lactic acid bacteria from the collected yogurt samples, the appropriate nutrient medium/medium is prepared to be used primarily to regenerate and develop the bacteria. M17 medium is used to regenerate and develop Streptococcus thermophilus bacteria belonging to lactic acid bacteria strains from stock cultures stored at -86°C. In order to prepare the said medium, powdered M17 broth is weighed to 42.5 grams (g) and mixed in an Erlenmeyer flask by adding 1000 milliliters (ml)/l liter (L) liter of distilled water. Then it is sterilized in autoclave at 121 °C for 15 minutes. MRS agar is used to regenerate and develop Lactobacillus bulgaricus bacteria, which is another lactic acid bacteria strain, from stock cultures stored at -86°C. In order to prepare the said medium, powdered MRS broth is weighed to 52.2 g and mixed in an Erlenmeyer flask by adding 1 liter of distilled water. Then it is sterilized in autoclave at 121°C for 15 minutes.

Regeneration and Pre-propagation of Bacteria from Stock Culture

In order to regenerate S. thermophilus cultures which was previously prepared as stock culture and have microscope image in Figure 4 and L. bulgaricus cultures with microscope image in Figure 5, firstly MRS and Ml 7 media are prepared as mentioned. After the prepared media are sterilized in autoclave at 121°C for 15 minutes, the media coming out of the autoclave are cooled under room conditions. Before starting inoculation, the laminar air cabinet is sterilized under ultraviolet (UV) light for 20 minutes. From stock cultures taken at -86°C, S. thermophilus is inoculated into Ml 7 medium and L. bulgaricus is inoculated into MRS medium in aseptic conditions at a rate of 1-3% by volume. Then, the inoculated broth media are allowed to incubate at 42°C for 18-24 hours so that the bacteria can grow in the medium homogeneously. The end of the incubation is decided according to the turbidity in the medium. The regenerated cultures are stored at +4°C for preparation of intermediate culture.

Preparation of Stock Culture

After preparation of the medium (media), S. thermophilus and L. bulgaricus bacteria are provided as stock cultures for later use.

Preparation of stock cultures of bacteria consists of the following process steps:

• Preparation of 60 ml of MRS and Ml 7 broth media according to the procedures mentioned above,

• Preparation of 40 ml glycerol separately for both broths and sterilization of the glycerol at 121 °C for 15 minutes,

• After sterilization, preparation of glycerol-broth stock media with the glycerol final concentration of 40% by volume with the broth medium,

• Before starting inoculation, sterilization of the laminar air cabinet under ultraviolet (UV) light for 20 minutes,

• From stock cultures taken at -86°C, inoculation of S. thermophilus into Ml 7 medium in aseptic conditions at a rate of 1-3% by volume,

• From stock cultures taken at -86°C, inoculation of L. bulgaricus into MRS medium in aseptic conditions at a rate of 1-3% by volume,

• Allowing inoculated broth media to incubate for 18-24 hours at 42°C,

• Observing the growth of bacteria,

• Preparing a stock culture by combining an equal volume of the cultures growing to have final concentration of 20% by volume and glycerol-broth medium prepared to be 40% by volume in eppendorf tubes previously sterilized at 121°C for 15 minutes,

• Storing the stock culture at -86°C.

Preparation of Intermediate Culture

Cultures that have been regenerated and pre-propagated in the laboratory environment in a suitable medium (MRS or Ml 7) are reproduced on a larger scale and used in the intermediate step, thus ensuring that the final product is creamy and homogenized yogurt.

For the preparation of intermediate culture medium, TW 60 Commercial Medium, which is the common medium for both bacterial strains and 10% by volume Skim Milk Powder - 1-2% by volume Yeast Extract are used. Media are prepared so that the total dry matter (TKM) is in the range of 5-7%.

For intermediate culture, TW 60 Commercial Medium is weighed in autoclave bottles to 60 grams (6%) per 1 liter and distilled water is added and mixed. The prepared TW 60 Commercial Media is sterilized by autoclaving at 95°C for 15 minutes.

As an alternative to TW 60 Commercial Medium, 10% by volume skimmed milk powder for intermediate culture medium is weighed in autoclave bottles and distilled water is added according to the liter of the autoclave bottle. 1% by volume yeast extract is added onto 10% skimmed milk powder, which is dissolved homogeneously, and is sterilized by autoclaving for 10 minutes at 110°C.

Preparation of intermediate cultures from bacteria whose pre-propagation has been completed after the preparation of suitable media includes the following process steps.

• Before starting inoculation, sterilization of the laminar air cabinet under ultraviolet (UV) light for 20 minutes,

• Bringing the intermediate culture medium to room temperature,

• Inoculating into the intermediate culture medium separately (1-2.5% Bacillus, 1-2.5% Cocci) from the cultures that are regenerated in appropriate broth medium (MRS or Ml 7) to be in total ratio of 2-5% by volume (v/v),

• Allowing incubated intermediate media to incubate for 3-6 hours at 42°C, Monitoring the pH of the cultures in the incubation with intervals of 30 minutes, . Observing that the biomass deposits at the bottom in TW 60 medium towards the end of the incubation,

• Ending the incubation so that the final pH is of 4.60,

• Bringing the intermediate cultures to room temperature and keeping them at +4°C under refrigerator conditions.

Yogurt Test (Structure, Taste and Aroma)

Testing how the propagated intermediate cultures will create structure, taste and aroma in yogurt includes the following process steps.

• Autoclaving the bottles at 121 °C for 15 minutes for testing,

• Before starting testing, sterilization of the laminar air cabinet under ultraviolet (UV) light for 20 minutes,

• Adding pasteurized milk to sterile autoclave bottles in an aseptic environment,

• Inoculating the prepared intermediate cultures into milk with the help of automatic pipette, representing the actual production (1-1.5% Bacillus, 1-1.5% Cocci) that will contain 2-3% bacteria by volume (v/v) in the final product,

• Allowing intermediate cultures inoculated into milk to incubate for 3-5 hours at 42°C, Monitoring the pH of the milks in the incubation with intervals of 30 minutes,

• When the pH at the end of the incubation reaches 4.60, yogurt production is completed.

Construction of Aroma Profiles in Starter Cultures and Yogurts Gas Chromatography is used to determine and quantitatively calculate the aroma substances of starter cultures and obtained yogurts. For this, Headspace method is used. The yogurt aroma is formed with the presence of Acetaldehyde. FID detector is used to determine aroma substances and their amounts. Agilent HP-5 (30m; 0.25 mm; 0.25 pm) column is used for separating aroma substances.

Determination of Acetaldehyde Amount

10 g of intermediate culture or yogurt sample is weighed into 20 ml headspace vials and tightly closed with a 20 mm aluminum lid. The sample is treated at 80DC for 20 minutes to ensure volatile substances occur. GC/FID conditions are as follows:

• Injector temperature is 250DC, carrier gas helium is in constant flow mode and flow rate is lml/min, furnace temperature is initially at 35°C, after 6 minutes it is increased from 35°C to 250°C at 30DC/min and kept at 250°C for 3 minutes. The total measurement takes 16.17 minutes. FID surface is fixed at 300°C.

• Stock acetaldehyde solution is prepared in distilled water at 10000 mg/L (ppm).

• Standard solutions are prepared by selecting 6 different calibration points. These are 1 pi, 2 pi, 5 pi, 10 pi, 20 pi and 40 pi stock solutions. The calibration curve is drawn using these 6 points in Least-square regression. The R2 value is determined using the linear calibration curve.

Table 1: Acetaldehyde Amounts in Starter Cultures and Yogurts

Without limiting the above combinations, each combination of Cocci and Bacillus bacteria can be applied as a starter culture with at least one Cocci and at least one Bacillus. The bacteria from the Cocci-Bacillus starter culture pairs constituting yogurts with determined acetaldehyde amounts in the preferred proportions are grown in the next stages and dosed into pasteurized milk, and yogurt is produced on a large scale.

2. THE STEPS PERFORMED IN THE PRODUCTION LINE 2.1. Propagation of Natural Isolate Replication Starter Cultures

At this stage, cultures composed of bacteria whose regeneration and pre-propagation were completed in the laboratory environment and stored as intermediate cultures are propagated on a large scale and stored as stock cultures to be used in the production of homogenized yogurt.

In Figure 2, Natural Isolate Replication Starter Culture Production Flow Chart is shown. Natural Isolate Replication Starter Culture Production is disclosed in detail below. Media filling is started in the production line, continuous media feeding is made to ensure the continuity of the process, and the system receives input controlled water into the Culture Preparation Tank (KHT) according to the amount written.

The following process or operations are performed in a sterile environment with hepa filter (200 m).

The media are added to KHT after water intake. After checking that the TKM value is in the range of 5-7% by weight, the pasteurization phase is started.

Pasteurization conditions are 95±2°C and 3000± 500 dm 3 /h. Pasteurized TW60 (5-7%) is transferred from the culture preparation tank to the culture stock tank (Holder). Culture stock tanks stand by for 20±5 minutes at 95°C with the mixer closed. After ~20 minutes, the system automatically turns on the mixer and ice water, reduces the tank temperature up to 43±1°C, cools the TW60 (5-7%) medium and makes it ready for the addition of bacterial cultures.

After the connection points are sterilized on the culture tanks, the culture tubes pre-propagated in the laboratory are attached. After the culture tubes coded as K (cocci) and B (bacillus) are connected to the tanks according to the recipe, pasteurized TW60 (6%) and bacterial cultures are mixed. The valve on the line with the tube connected to the tank in the system opens automatically and the dosing of the culture into the medium continues for 20 minutes while the tank mixer is running. After 20 minutes, the automatic mixer and ice water turn off. Bacteria are incubated at 43±1°C and bacteria propagate. pH control is done automatically, when the pH value reaches 4.70, the crushing step of the tanks is approved and the cultures are cooled at pH -4.20, 4±2’°C for -3 hours and stored in the culture stock tank at 4-6°C for 2 days.

2.2. Production of Homogenized Yogurt with Natural Isolate Replication Starter Culture

At this stage, cultures consisting of bacteria that have been grown on a large scale and stored as stock culture are used in the production of homogenized yogurt.

In Figure 3, Production Flow Chart for Homogenized Yogurt with Natural Isolate Replication Starter Culture is shown. Production of Homogenized Yogurt with Natural Isolate Replication Starter Culture is disclosed in detail below.

Pre-pasteurization: All raw milk incoming to the plant is cooled down to 4-6°C through filters after input control and transferred to raw milk tanks. After it is standardized and pre-pasteurized in accordance with the milk fat ratio standards to be processed through filters, it is stored in pasteurized stock tanks at 4-6°C. This stage is a common process for all raw milk entering the plant. After pasteurized stock tanks, they are processed according to the relevant product flow chart.

As shown in Figure 3, the pre-pasteurized standardized milk evaporation conditions are concentrated to 59 ±1°C, -0.7- -0.8 bars, then the milk cooled to 4-6°C with the help of ice water is transferred to the pasteurized stock tank at 4-6°C for a maximum of 18 hours.

After YKM-Fat control, foreign vapors and odors are removed from the milk by removing the air at 78±2°C,-0.4- -0.6 bar de-aeration conditions. Afterwards, the homogenization process is applied to the milk to be processed in yogurt at 200 bar 70±2°C conditions in order to ensure a homogeneous distribution of milk fat in the whole mass.

Immediately after homogenization, pasteurization is applied to milk for 5 minutes ± 30 seconds at 90±2°C, 15000± 1000 dm 3 /hr conditions. With this pasteurization process, besides the strengthening of the milk structure, pathogens are destroyed and a more suitable environment is provided for the yogurt culture to work, so if the pasteurization temperature is not suitable, the pasteurization stage is returned. After pasteurization, milk is cooled to 4-6°C with the help of ice water. In the previous step (2.1. Inter-Propagation Natural Isolate Replication Starter Cultures), the cultures (M), whose large-scale propagation has been completed and stored in the culture stock tank, are dosed to pasteurized and cooled milk at 1- 2%, the final product should contain at least 10 8 kob/ml bacteria. Milk (product), where bacteria cultures are dosed, fills the pasteurized yogurt stock tank.

After YKM-Fat control, the product passing through the hepa filter system is heated to 43±2°C. The product, which is filled in packages at the same temperature, passes through dating, coding and metal detector and they are placed on pallets. Then, the product is taken to incubation chambers at 43±2°C for ~4 hours in the range of pH 4.30-4.85. If the pH value of the acidity-checked product is not suitable, it returns to the incubation stage. The product with suitable pH value is sent to rapid cooling at 4°C and stored at 4-6°C in order to stop the incubation. Products that are found suitable as a result of quality control after storage are shipped at 4-6°C. 2.3. Production of Ayran with Natural Isolate Replication Starter Culture

Ayran (full-fat), whose detailed description is given below, is a fermented milk product which is prepared by adding water to the yogurt and adding Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus cultures which are microbial yogurt cultures to dry matter-adjusted milk and in which salt (sea/lake) can be added to.

At this stage, in 2.1. Inter-Propagation of Natural Isolate Replication Starter Cultures, the cultures consisting of bacteria that are grown on a large scale and stored as stock cultures are used in ayran production.

In Figure 4, Production Flow Chart for Ayran with Natural Isolate Replication Starter Culture is shown. Production of Ayran with Natural Isolate Replication Starter Culture is disclosed in detail below.

The pre-pasteurized, standardized milk is subjected to a de-aeration process at 78±2°C,-0.4- -0.6 bar conditions, followed by homogenization at 200 bar, 70±2°C conditions.

Immediately after homogenization, pasteurization is applied for 5 minutes ± 30 seconds at 90±2°C, 15000± 1000 dm 3 /hr conditions. With this pasteurization process, besides the strengthening of the milk structure, pathogens are destroyed and a more suitable environment is provided for the ayran culture to work, so the pasteurization temperature is controlled, and if it is not suitable, the pasteurization stage is returned.

After pasteurization, the milk is cooled down to an incubation temperature of 43 ± 2°C. In the previous step (2.1. Inter-Propagation Natural Isolate Replication Starter Cultures), the cultures (M), whose large-scale propagation has been completed and stored in the culture stock tank, are dosed to pasteurized and cooled milk at 1-2% by volume, and ayran is incubated at an incubation temperature of 43 ± 2°C between pH 4.30-4.50 in the incubation tank.

After the incubation, ayran is lysed, the milk cooled to 4-6°C is transferred to the ayran storage tank for maximum 18 hours at 4-6°C. pH, salt and fat of pasteurized water, salt-added ayran is controlled. Then, it is filled into the ayran glass/bottle in the filling machine with hepa filter and the product is palletized. Products that are found suitable as a result of quality control after storage at 4-6°C are shipped at 4-6°C. The storage conditions and shelf life of the obtained product is 30 days at 4-10°C. Physical and Chemical Properties of Ayran Produced with Natural Isolate Replication Starter Cultures

. Fat-Free Dry Matter, % (m/m): 5.8 - 6.2 . Fat, % (m/m): 1.8 - 2.1 . Acidity, % (m/m) (as lactic acid): 0.5- 1.0 . Viscosity (sec / 100 mL): Min.22 . Salt, %: Max.1

. Milk Protein (m/m), %: Min.2 pH: Min.4,20

PANAGRO TARIM HAYVANCILIK GIDA SAN. VE TIC. A.§. Address: Ka§inham Yeni Mahallesi Sardunya Sokak No:l Meram/KONYA Natural Isolate Starter Culture 16S Gene Sequence identities (IDENT)

Culture Code: B1 (PNGR_B1) Lactobacillus delbrueckii subsp. bulgaricus, identity %99.78 Culture Code: B2 (PNGR_B2) Lactobacillus delbrueckii subsp. bulgaricus identity % 100.00 Culture Code: B6 (PNGR_B6) Lactobacillus delbrueckii subsp. bulgaricus identity % 100.00 Culture Code: B8 (PNGR_B8) Lactobacillus delbrueckii subsp. bulgaricus identity %99.80 Culture Code: B14 (PNGR_B14) Lactobacillus delbrueckii subsp. bulgaricus identity %99.80 Culture Code: B36 (PNGR_B36) Lactobacillus delbrueckii subsp. bulgaricus identity %99.90 Culture Code: K1 (PNGR_K1) Streptococcus thermophilus identity % 100.00 Culture Code: K2 (PNGR_K2) Streptococcus thermophilus identity %99.89 Culture Code: K5 (PNGR_K5) Streptococcus thermophilus identity %99.83 Culture Code: K13 (PNGR_K13) Streptococcus thermophilus identity %99.90 Culture Code: K31 (PNGR_K31) Streptococcus thermophilus identity % 100.00 Culture Code: K55 (PNGR_K55) Streptococcus thermophilus identity % 100.00